{"article_id": "PMC5098230", "article_text": ["one of the key challenges \n in the development of novel materials \n is the ability to tune and control their macroscopic physical and \n chemical properties on a molecular level by external stimuli .", "such a degree of control is challenging but crucial for a wide range \n of applications ranging from catalysis to pharmacological and medical \n applications .", "in photoactive materials light absorbed by chromophores can be converted \n into directed functionality , typically by means of photoinduced isomerization \n of nitrogen or carbon containing double bonds and pericylic reactions .", "indeed , e  z isomerization \n results in large amplitude changes in structure as well as chemical \n and physical properties .", "molecular motors based on overcrowded alkenes are particularly \n attractive due to the synthetically tunable properties , such as thermal \n stability , absorption spectra , etc . , and", "therefore are readily applied \n to a wide range of functional systems . however , although the thermally \n activated step in the unidirectional rotary cycle is well understood \n and is now largely predictable , the photochemical properties and especially \n photochemical quantum yields are less well understood .", "hence both \n from a fundamental perspective and in achieving the goal of complete \n molecular design , gaining insight into the excited - state properties \n of light driven molecular - sized machines is essential .", "the overcrowded alkene - based molecular \n rotary motors are composed \n of a central carbon  carbon double bond ( the axle ) embedded \n in an intrinsically chiral environment .", "the unidirectional rotational \n cycle starts with a photoinduced e  z isomerization around the central c = c bond , generating \n a metastable isomer p * ( figure 1 ) , which relaxes thermally to the stable isomer p , which in \n the case of a symmetric stator unit is identical to i. this isomer \n marks half of the rotation cycle and is the starting point for a second \n photoinduced isomerization step that leads again to a metastable isomer . \n", "thermally activated conformational isomerization of this isomer brings \n the system back to its initial state .", "overall , the rotor part has \n then performed a complete rotation with respect to the stator unit \n of the molecular motor .", "although the rotation includes both \n photochemical and thermal steps \n that involve different parts of the motor , the structural evolution \n is largely governed by the excited - state properties of the central \n c = c group , which , in turn , are dictated by the potential energy \n surfaces of the excited states that are accessed upon electronic excitation . \n despite the large number of theoretical and experimental studies that have focused on characterizing \n this process , there are several key questions outstanding including \n identification of the main structural coordinates and the role of \n various electronic states involved in the excited - state dynamics . \n", "time - resolved studies are essential to elucidate these dynamics but \n have thus far focused on probing the change in the electronic structure \n after photoexcitation through uv / vis absorption and fluorescence studies .", "these techniques offer \n a high time resolution but provide only indirect information as to \n how the structure evolves spatially over time . in the present contribution \n we show that this information can be obtained through a combination \n of picosecond time - resolved vibrational spectroscopy of the electronically \n excited states together with quantum - mechanical calculations .", "the setup for the time - resolved infrared measurements has been \n described in detail previously .", "mid - ir \n probe pulses were generated using an amplified ti : sapphire system \n ( spectra - physics hurricane , 600 j , repetition rate 1 khz ) pumping \n a commercial bbo - based opa ( spectra - physics opa-800c ) , the signal \n and idler output of which were difference - frequency - mixed in aggas2 .", "uv pump pulses ( 400 nm , 1 j ) were generated \n by doubling part of the output of the ti : sapphire laser .", "the diameter \n of the uv pump focus was 180200 m ; that of \n the ir probe focus , 150 m . the sample cell with caf2 windows spaced by 500 m was placed in the ir focus . \n to avoid accumulation of the thermally unstable conformer p * during \n the measurements ,", "the pump  probe delay time was scanned by mechanically \n adjusting the beam path of the uv pump .", "the temporal resolution of \n 200 fs has been determined from the full width at half - maximum of \n the pump probe cross - correlation function .", "the pump pulse was chopped \n at 500 hz , and transient spectra were obtained by subtracting nonpumped \n absorption spectra from the pumped absorption spectra , both of which \n were recorded by spectrally dispersing the probe pulses using an oriel \n ms260i spectrograph and measuring the frequency - dependent ir intensity \n using a 2  32 hgcdte detector array ( infrared associates ) .", "compound \n i was available from earlier studies ( see ref ( 44 ) ) . in all experiments", "we \n investigate a 10 mm solution of compound i in deuterated cyclohexane \n ( aldrich , 99.6 atom% d ) .", "for the investigation of the ground - state \n conformers ( including \n vertical excitation energies and forces acting at the franck ", "condon \n point ) quantum mechanical calculations were performed using the turbomole \n suite of programs .", "geometry optimization \n and normal - mode analysis were performed by ( time - dependent ) density \n functional theory ( ( td)-dft ) using the hybrid functional b3lyp . for the geometry optimization the d3 correction \n method of grimme et al .", "the cc - pvdz or aug - cc - pvdz basis \n sets were employed , respectively .", "post - hartree  fock calculations \n were performed on the basis of spin - component scaled second - order \n mller  plesset perturbation theory and the approximate coupled - cluster singles - and - doubles \n model cc2 .", "all perturbation method calculations were performed using \n the resolution of identity ( ri ) approximation and the efficient ricc2 module implemented \n in the turbomole package together with the standard auxiliary basis \n sets .", "the gaussian09 program package was employed to study the excited - state properties \n in more detail and in particular to optimize the structure of the \n dark - state species .", "geometry optimization and normal - mode analysis \n were performed in the framework of td - dft at the b3lyp / cc - pvdz level .", "the structural evolution from the \n initial state i to isomer p * \n ( figure 1 ) was considered first in terms of the spectral properties \n of the initial state and the first metastable product ( p * ) .", "the steady - state \n uv / vis absorption spectra of the two isomers are distinct , reflecting \n a change in the conjugated  system ( figure 1a ) .", "the ftir spectra of the two isomers ( figure 1c ) show that the \n carbon  carbon stretch and ring vibrations couple to a large \n degree as a consequence of the extended  system ; however , dft \n calculations enable us to assign the bands in the 15501650 \n cm range as being mainly associated with \n modes involving the central c = c double bond ( supporting information ) .", "the metastable isomer p * shows a \n shift of these modes to lower frequencies confirming a decrease in \n bond strength .", "comparison of the optimized geometries of the two isomers \n ( i ( p * ) , cc bond length 1.369 ( 1.377 )  ; cc = cc dihedral \n angle 14.1 ( 29.5 ) ) evidence the corresponding lower  \n bond character as well .", "unidirectional motion of the unidirectional \n motor starting from \n the thermally stable conformer ( i ) via the product of the photoinitiated \n step ( p * ) to the thermally stable conformer ( p ) ( optimized geometries \n on the b3lyp+d / aug - cc - pvdz level ) : ( a ) experimental and calculated \n ( light color and dotted line ) uv  vis spectra of conformer i \n and p * ; ( b ) molecular orbitals of the initial conformer ; ( c ) experimental \n and calculated ir spectra ( b3lyp+d / aug - cc - pvdz ) of the initial state \n ( blue ) and final state ( red ) . in both the measured uv / vis absorption \n and ftir spectra at the photostationary state ( pss ) , i and p * are \n present in a 25:75 ratio .", "time - resolved vibrational spectroscopy was employed to achieve the required temporal resolution as well \n as structural sensitivity to elucidate the structural evolution that \n follows photoexcitation with a short uv - pump pulse ( center wavelength \n 400 nm ) .", "the resulting structural evolution was followed by time - resolved \n differential absorption spectra in cyclohexane - d12 with a time resolution of 200 fs ( figure 2 ) .", "the transient ir spectra show , as expected , \n pronounced differences between the ir absorption spectrum of the molecule \n in the initial and excited states .", "a first point of note is that the \n spectrum does not evolve further after ca .", "30 ps , and the difference \n spectrum corresponds perfectly with the difference spectrum obtained \n upon continuous wave excitation ( figure 1 ) ; i.e. , the metastable isomer p * has formed \n fully within the 30 ps after excitation .", "ftir and time - resolved \n infrared spectra of the molecular motor \n in cyclohexane - d12 : ( a ) ftir spectra of \n the thermal stable conformer ( initial , solvent corrected , top ) , uv - ump / ir - probe \n transient infrared spectra ( uv = 400 nm ) ( middle ) , \n and difference ftir spectra between the two ground - state conformers \n of the molecular motor ; ( b ) kinetics of selected bands and corresponding \n results of the global fit ; ( c ) species associated spectra ( sas ) of \n species 1 and calculated ir spectra ( top ) and sas of the pss ( sp3 ) \n together with ftir spectra ( bottom ) .", "negative bands indicate ground - state \n bleaching , and positive bands , species created after excitation . the time - resolved ir spectra obtained \n within the first 30 ps exhibit \n several remarkable features .", "the first and most striking is a broad \n and unstructured absorption over the entire mid - ir range ( 12003000 \n cm ) .", "this absorption , which decays rapidly , is \n particularly prominent at early time delays ( < 5 ps ) .", "individual \n absorption bands with remarkably high integrated intensities in the \n 14001600 cm range are superimposed on \n the broad spectral feature .", "notably , these bands are significantly \n broader than the bleached bands in the transient spectra and bands \n in the ftir spectra .", "the solvent response and the dependence of the \n uv pump intensity ( supporting information ) confirm that these features are intrinsic to the compound and are \n not due to solvent or two - photon processes .", "the width and intensity \n of the broad excited - state absorption bands \n excludes that they arise from vibrational transitions .", "indeed , the \n absorbance is closer to that expected for electronic transitions , \n albeit these typically occur at much higher energies ( in the uv / vis \n range ) .", "the initial state of the compound \n ( i ) shows an absorption band at 390 nm ( figure 1 ) , which is reproduced well by b3lyp / cc - pvdz \n calculations in the gas phase ( 403 nm ; f = 0.428 ) . \n", "the s1  s0 transition has predominantly \n homo  lumo character ( figure 1 ) .", "in contrast to previous assignments , however , we find that this electronic transition \n is not localized on the stator but involves to a significant extent \n excitation from the bonding  orbital of the central double \n bond to the antibonding  * orbital ( figure 1 ) . as a consequence", ", the double bond character \n of this bond is reduced as well as the barrier to rotation around \n this bond . in line with calculations on ethylene and its derivatives , \n", "we find a dark state ( s2 ) associated with the homo1 \n  lumo excitation at slightly higher excitation energies that \n is not directly accessible from the ground state ( figure 1 , table 1 ) . because the homo1 orbital is localized \n on the aromatic stator unit , s2 has a significantly larger \n dipole moment than s1 ( 4.3 vs 2.2 d at the scs - cc2/cc - pvdz \n level ) .", "it should be emphasized that the description of the lower - lying \n electronic states in the compound studied here is thus entirely different \n from ethylene and its derivatives where , as discussed by salem and \n others , for torsion angles of around 90 the two low - lying electronic \n states have either a biradical or zwitterionic character depending \n on the occupation of the two central 2p - orbitals and require multireference \n methods for a proper description . in the present study , in contrast , \n we investigate the dynamics occurring at small torsional angles ( vide infra ) for which the employed single - reference methods \n not only have been validated .", "in addition \n we employ the results of multireference calculations on smaller motor \n molecules as reported by liu and morokuma to make a further comparison with our experimental results .", "for s0 the optimized \n structure on the b3lyp+d / aug - cc - pvdz level was used to calculate the \n vertical excitation energies . for vertical excitation", "they thus strongly suggest that the broad \n and structureless absorption feature observed in the time - resolved \n ir spectra is associated with the electronic s1  \n s2 transition .", "the presence of two close - lying states also \n explains why the induced absorption bands have such high intensities . \n", "our calculations show that the energy gap between the two states strongly \n depends on structural parameters such as the c = c bond length \n that are involved in the 14001600 cm normal \n modes . for these modes", "one therefore expects that their transition \n moments will be enhanced by vibronic coupling as is indeed observed . \n", "the conclusion that the excited - state dynamics of the molecular rotor \n should be described in a multistate model is further supported by \n recent multireference calculations on smaller rotor molecules that \n also find very small energy differences between the s1 and \n s2 states .", "the excited - state \n dynamics were elucidated further using a global \n analysis of the transient spectra , which shows that the spectra can \n be described well with two exponential decay terms with time constants \n of 1.53  0.03 and 12  1 ps .", "we note that our experiments \n have a time resolution of 150200 fs and therefore do not allow \n observation and confirmation of the ultrafast dynamics reported previously \n in fluorescence - upconversion studies .", "species - associated spectra ( sas ) ( supplementary \n figure 2 ) were derived by using a two - step sequential kinetic \n model .", "the sas of the first species shows an overall background \n signal \n with broad and intense features superimposed on it that indicate vibronic \n coupling .", "furthermore , the increase ( decrease ) of absorbance in the \n 1550 ( 1600 ) cm region indicates a substantial \n change in bond character of the central double bond ( figure 2c ) .", "this spectrum was related \n to the structure of this excited - state species using excited - state \n geometry optimizations .", " \n state without any structural constrains associated with the homo1 \n  lumo excitation leads to a stable minimum that can be reached \n from the franck  condon excited geometry by relatively small \n structural changes ( supplementary figure 4 ) , and an excited - state ir absorption spectrum that is in good agreement \n with the sas of species 1 ( figure 2 ) .", "it is notable that for a proper comparison with \n the experimental spectrum , the theoretically predicted stick spectrum \n needs to be convolved with 20 cm gaussian line \n shapes , suggesting that in the  dark  state a wide range \n of structures is sampled .", "our experiments and calculations thus demonstrate \n that excitation and excited - state decay involve two different electronic \n states . in earlier studies , time - resolved fluorescence and visible \n absorption data were interpreted in terms of a decay from a single , \n isolated electronic state decaying to the ground state after structural \n relaxation in the excited state .", "the present data show that the s1 state actually decays to an intermediate , dark electronic \n state , and from there to the ground state . in the sas of the \n second species ( supplementary \n figure", "2 ) the broad background is absent , and bands have widths \n comparable to those in the s0 state .", "the absence of the \n broad background indicates that this species is no longer in an electronically \n excited state but back in the ( vibrationally hot ) electronic ground \n state .", "indeed , the vibrational activity observed in the 1550 cm region follows closely the ir absorption spectrum \n of p*. in addition , repopulation of the original state ( i ) is observed \n also ( i.e. the quantum yield for isomerization is less than unity ) , and the sas of the second species contains contributions \n from both vibrationally hot ground - state i and metastable p * isomers . \n", "vibrational cooling of these isomers leads to the sas of the final \n species , which is in excellent agreement with the steady - state ftir \n difference spectrum of room - temperature i and p*. the present \n experimental and theoretical data allow a complete \n picture of the mechanism by which the molecular rotary motor undergoes \n isomerization after electronic excitation ( figure 3 ) .", "photon absorption brings the motor to \n a  bright  state from which it decays on an ultrafast \n time scale and within the time resolution of the present experiments \n to a different electronically excited  dark  state . \n in line with previous calculations on similar systems ,", "our calculations \n indicate that this is a barrierless process proceeding via a conical \n intersection .", "previously , it was assumed \n that structural evolution on the homo  lumo potential energy \n surface is responsible for the observed ultrafast decay of the fluorescence , \n and ( following commonly accepted models on photoisomerization of carbon ", "carbon \n double bonds ) that it primarily proceeds along torsional and pyramidalization \n coordinates . the present study , in contrast , leads to the conclusion \n that the fluorescence decay is mediated by the passage through the \n conical intersection of the homo  lumo and homo1  \n lumo potential energy surfaces . inspection of the structural changes \n that occur upon geometry relaxation to the minimum on the homo1 \n  lumo potential energy surface show that the central c = c \n bond length increases from 1.381 to 1.461  and that only relatively \n small changes occur in the torsional and pyramidalization coordinates . \n in combination with the forces that act on the molecule at the franck ", "condon \n excited  bright  state ( supplementary \n figure 3 ) , this suggests that motion along the c = c stretch \n coordinate needs to be taken into account as well for understanding \n the decay of the fluorescence .", "proposed multistate dynamics of the photoinduced \n unidirectional \n rotation of the molecular motor starting from conformer i to conformer \n p * ( projected into one dimension ) : dotted arrow , ultrafast process \n reported in previous work ; fast ( red arrow ) and slow process ( blue \n arrow ) observed in the present time - resolved infrared study . ( a ) calculated \n energy profile along the torsion coordinate showing how the energies \n of the electronic states change as the molecules evolves to conformer \n p*. ( b ) schematic representation of the proposed model .", "hence , it can be concluded that \n internal conversion to the electronic \n ground state takes place from the  dark  state .", "the \n decay rate for this process indicates that it is mediated by conical \n intersections between the two states as has indeed been proposed in \n previous theoretical studies .", "these studies \n concluded , however , as well that to reach these intersections a barrier \n needs to be overcome that results from the steric repulsion of the \n methyl group of the rotor unit with the hydrogen of the stator unit . \n", "previous time - resolved fluorescence measurements reported an ultrafast \n damping mode at 113 cm that was speculated to \n arise from motion along a coordinate involving inversion / pyramidalization \n although no direct support could be found for such an assignment .", "interestingly , our calculations find in the \n  dark  state a mode at 119 cm ( b3lyp / cc - pvdz ; \n not scaled , supporting information ) that \n involves motion of the methyl group with respect to the hydrogen of \n the stator unit .", "one might therefore wonder to what extent this mode \n is related to the oscillatory behavior of the fluorescence .", "our observed \n s2 decay constant of 1.5 ps matches the slow component \n of the overall fluorescence decay ( 0.91.5 ps ) .", "our calculation of the excitation energies of \n the  bright  and  dark  states along the \n 119 cm normal mode did not show a conical intersection \n between the two states , supporting the previous explanation of the \n oscillations observed in the fluorescence measurements by the movement of wave packets and suggesting \n a scenario in which a coherent oscillation in the dark s2 state periodically partly repopulates the fluorescent s1 state .", "however , our experimental methods do not provide the time \n resolution to confirm this .", "the photodynamics \n of a unidirectional molecular motor has been \n studied by time - resolved ir spectroscopy in which both vibrational \n and electronic structures are probed . in combination with quantum \n mechanical calculations , the high structural sensitivity and real - time \n character of the technique allows us to understand fundamental aspects \n of the operation mechanism .", "the results show that conversion of photon \n energy into directed motion proceeds on the potential energy surfaces \n of two different electronic states , which is the more important as \n it implies that the conical intersection between the  dark  \n electronically excited state and the ground state determines the efficiency \n of the motor . to rationally tune", "the performance of photoinitiated \n unidirectional rotation thus involves a multicoupled - state optimization \n of nuclear motion ."], "abstract_text": ["<S> controlling the excited - state properties \n of light driven molecular \n machines is crucial to achieving high efficiency and directed functionality . \n a key challenge in achieving control lies in unravelling the complex \n photodynamics and especially in identifying the role played by dark \n states . </S>", "<S> here we use the structure sensitivity and high time resolution \n of uv - pump / ir - probe spectroscopy to build a detailed and comprehensive \n model of the structural evolution of light driven molecular rotors . \n </S>", "<S> the photodynamics of these chiral overcrowded alkene derivatives are \n determined by two close - lying excited electronic states . </S>", "<S> the potential \n energy landscape of these  bright  and  dark  \n </S>", "<S> states gives rise to a broad excited - state electronic absorption band \n over the entire mid - ir range that is probed for the first time and \n modeled by quantum mechanical calculations . </S>", "<S> the transient ir vibrational \n fingerprints observed in our studies allow for an unambiguous identification \n of the identity of the  dark  electronic excited state \n from which the photon s energy is converted into motion , and \n thereby pave the way for tuning the quantum yield of future molecular \n rotors based on this structural motif . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULT AND DISCUSSION", "SUMMARY AND CONCLUSION"], "sections": [["one of the key challenges \n in the development of novel materials \n is the ability to tune and control their macroscopic physical and \n chemical properties on a molecular level by external stimuli .", "such a degree of control is challenging but crucial for a wide range \n of applications ranging from catalysis to pharmacological and medical \n applications .", "in photoactive materials light absorbed by chromophores can be converted \n into directed functionality , typically by means of photoinduced isomerization \n of nitrogen or carbon containing double bonds and pericylic reactions .", "indeed , e  z isomerization \n results in large amplitude changes in structure as well as chemical \n and physical properties .", "molecular motors based on overcrowded alkenes are particularly \n attractive due to the synthetically tunable properties , such as thermal \n stability , absorption spectra , etc . , and", "therefore are readily applied \n to a wide range of functional systems . however , although the thermally \n activated step in the unidirectional rotary cycle is well understood \n and is now largely predictable , the photochemical properties and especially \n photochemical quantum yields are less well understood .", "hence both \n from a fundamental perspective and in achieving the goal of complete \n molecular design , gaining insight into the excited - state properties \n of light driven molecular - sized machines is essential .", "the overcrowded alkene - based molecular \n rotary motors are composed \n of a central carbon  carbon double bond ( the axle ) embedded \n in an intrinsically chiral environment .", "the unidirectional rotational \n cycle starts with a photoinduced e  z isomerization around the central c = c bond , generating \n a metastable isomer p * ( figure 1 ) , which relaxes thermally to the stable isomer p , which in \n the case of a symmetric stator unit is identical to i. this isomer \n marks half of the rotation cycle and is the starting point for a second \n photoinduced isomerization step that leads again to a metastable isomer . \n", "thermally activated conformational isomerization of this isomer brings \n the system back to its initial state .", "overall , the rotor part has \n then performed a complete rotation with respect to the stator unit \n of the molecular motor .", "although the rotation includes both \n photochemical and thermal steps \n that involve different parts of the motor , the structural evolution \n is largely governed by the excited - state properties of the central \n c = c group , which , in turn , are dictated by the potential energy \n surfaces of the excited states that are accessed upon electronic excitation . \n despite the large number of theoretical and experimental studies that have focused on characterizing \n this process , there are several key questions outstanding including \n identification of the main structural coordinates and the role of \n various electronic states involved in the excited - state dynamics . \n", "time - resolved studies are essential to elucidate these dynamics but \n have thus far focused on probing the change in the electronic structure \n after photoexcitation through uv / vis absorption and fluorescence studies .", "these techniques offer \n a high time resolution but provide only indirect information as to \n how the structure evolves spatially over time . in the present contribution \n we show that this information can be obtained through a combination \n of picosecond time - resolved vibrational spectroscopy of the electronically \n excited states together with quantum - mechanical calculations ."], ["the setup for the time - resolved infrared measurements has been \n described in detail previously .", "mid - ir \n probe pulses were generated using an amplified ti : sapphire system \n ( spectra - physics hurricane , 600 j , repetition rate 1 khz ) pumping \n a commercial bbo - based opa ( spectra - physics opa-800c ) , the signal \n and idler output of which were difference - frequency - mixed in aggas2 .", "uv pump pulses ( 400 nm , 1 j ) were generated \n by doubling part of the output of the ti : sapphire laser .", "the diameter \n of the uv pump focus was 180200 m ; that of \n the ir probe focus , 150 m . the sample cell with caf2 windows spaced by 500 m was placed in the ir focus . \n to avoid accumulation of the thermally unstable conformer p * during \n the measurements ,", "the pump  probe delay time was scanned by mechanically \n adjusting the beam path of the uv pump .", "the temporal resolution of \n 200 fs has been determined from the full width at half - maximum of \n the pump probe cross - correlation function .", "the pump pulse was chopped \n at 500 hz , and transient spectra were obtained by subtracting nonpumped \n absorption spectra from the pumped absorption spectra , both of which \n were recorded by spectrally dispersing the probe pulses using an oriel \n ms260i spectrograph and measuring the frequency - dependent ir intensity \n using a 2  32 hgcdte detector array ( infrared associates ) .", "compound \n i was available from earlier studies ( see ref ( 44 ) ) . in all experiments", "we \n investigate a 10 mm solution of compound i in deuterated cyclohexane \n ( aldrich , 99.6 atom% d ) .", "for the investigation of the ground - state \n conformers ( including \n vertical excitation energies and forces acting at the franck ", "condon \n point ) quantum mechanical calculations were performed using the turbomole \n suite of programs .", "geometry optimization \n and normal - mode analysis were performed by ( time - dependent ) density \n functional theory ( ( td)-dft ) using the hybrid functional b3lyp . for the geometry optimization the d3 correction \n method of grimme et al .", "the cc - pvdz or aug - cc - pvdz basis \n sets were employed , respectively .", "post - hartree  fock calculations \n were performed on the basis of spin - component scaled second - order \n mller  plesset perturbation theory and the approximate coupled - cluster singles - and - doubles \n model cc2 .", "all perturbation method calculations were performed using \n the resolution of identity ( ri ) approximation and the efficient ricc2 module implemented \n in the turbomole package together with the standard auxiliary basis \n sets .", "the gaussian09 program package was employed to study the excited - state properties \n in more detail and in particular to optimize the structure of the \n dark - state species .", "geometry optimization and normal - mode analysis \n were performed in the framework of td - dft at the b3lyp / cc - pvdz level ."], ["the structural evolution from the \n initial state i to isomer p * \n ( figure 1 ) was considered first in terms of the spectral properties \n of the initial state and the first metastable product ( p * ) .", "the steady - state \n uv / vis absorption spectra of the two isomers are distinct , reflecting \n a change in the conjugated  system ( figure 1a ) .", "the ftir spectra of the two isomers ( figure 1c ) show that the \n carbon  carbon stretch and ring vibrations couple to a large \n degree as a consequence of the extended  system ; however , dft \n calculations enable us to assign the bands in the 15501650 \n cm range as being mainly associated with \n modes involving the central c = c double bond ( supporting information ) .", "the metastable isomer p * shows a \n shift of these modes to lower frequencies confirming a decrease in \n bond strength .", "comparison of the optimized geometries of the two isomers \n ( i ( p * ) , cc bond length 1.369 ( 1.377 )  ; cc = cc dihedral \n angle 14.1 ( 29.5 ) ) evidence the corresponding lower  \n bond character as well .", "unidirectional motion of the unidirectional \n motor starting from \n the thermally stable conformer ( i ) via the product of the photoinitiated \n step ( p * ) to the thermally stable conformer ( p ) ( optimized geometries \n on the b3lyp+d / aug - cc - pvdz level ) : ( a ) experimental and calculated \n ( light color and dotted line ) uv  vis spectra of conformer i \n and p * ; ( b ) molecular orbitals of the initial conformer ; ( c ) experimental \n and calculated ir spectra ( b3lyp+d / aug - cc - pvdz ) of the initial state \n ( blue ) and final state ( red ) . in both the measured uv / vis absorption \n and ftir spectra at the photostationary state ( pss ) , i and p * are \n present in a 25:75 ratio .", "time - resolved vibrational spectroscopy was employed to achieve the required temporal resolution as well \n as structural sensitivity to elucidate the structural evolution that \n follows photoexcitation with a short uv - pump pulse ( center wavelength \n 400 nm ) .", "the resulting structural evolution was followed by time - resolved \n differential absorption spectra in cyclohexane - d12 with a time resolution of 200 fs ( figure 2 ) .", "the transient ir spectra show , as expected , \n pronounced differences between the ir absorption spectrum of the molecule \n in the initial and excited states .", "a first point of note is that the \n spectrum does not evolve further after ca .", "30 ps , and the difference \n spectrum corresponds perfectly with the difference spectrum obtained \n upon continuous wave excitation ( figure 1 ) ; i.e. , the metastable isomer p * has formed \n fully within the 30 ps after excitation .", "ftir and time - resolved \n infrared spectra of the molecular motor \n in cyclohexane - d12 : ( a ) ftir spectra of \n the thermal stable conformer ( initial , solvent corrected , top ) , uv - ump / ir - probe \n transient infrared spectra ( uv = 400 nm ) ( middle ) , \n and difference ftir spectra between the two ground - state conformers \n of the molecular motor ; ( b ) kinetics of selected bands and corresponding \n results of the global fit ; ( c ) species associated spectra ( sas ) of \n species 1 and calculated ir spectra ( top ) and sas of the pss ( sp3 ) \n together with ftir spectra ( bottom ) .", "negative bands indicate ground - state \n bleaching , and positive bands , species created after excitation . the time - resolved ir spectra obtained \n within the first 30 ps exhibit \n several remarkable features .", "the first and most striking is a broad \n and unstructured absorption over the entire mid - ir range ( 12003000 \n cm ) .", "this absorption , which decays rapidly , is \n particularly prominent at early time delays ( < 5 ps ) .", "individual \n absorption bands with remarkably high integrated intensities in the \n 14001600 cm range are superimposed on \n the broad spectral feature .", "notably , these bands are significantly \n broader than the bleached bands in the transient spectra and bands \n in the ftir spectra .", "the solvent response and the dependence of the \n uv pump intensity ( supporting information ) confirm that these features are intrinsic to the compound and are \n not due to solvent or two - photon processes .", "the width and intensity \n of the broad excited - state absorption bands \n excludes that they arise from vibrational transitions .", "indeed , the \n absorbance is closer to that expected for electronic transitions , \n albeit these typically occur at much higher energies ( in the uv / vis \n range ) .", "the initial state of the compound \n ( i ) shows an absorption band at 390 nm ( figure 1 ) , which is reproduced well by b3lyp / cc - pvdz \n calculations in the gas phase ( 403 nm ; f = 0.428 ) . \n", "the s1  s0 transition has predominantly \n homo  lumo character ( figure 1 ) .", "in contrast to previous assignments , however , we find that this electronic transition \n is not localized on the stator but involves to a significant extent \n excitation from the bonding  orbital of the central double \n bond to the antibonding  * orbital ( figure 1 ) . as a consequence", ", the double bond character \n of this bond is reduced as well as the barrier to rotation around \n this bond . in line with calculations on ethylene and its derivatives , \n", "we find a dark state ( s2 ) associated with the homo1 \n  lumo excitation at slightly higher excitation energies that \n is not directly accessible from the ground state ( figure 1 , table 1 ) . because the homo1 orbital is localized \n on the aromatic stator unit , s2 has a significantly larger \n dipole moment than s1 ( 4.3 vs 2.2 d at the scs - cc2/cc - pvdz \n level ) .", "it should be emphasized that the description of the lower - lying \n electronic states in the compound studied here is thus entirely different \n from ethylene and its derivatives where , as discussed by salem and \n others , for torsion angles of around 90 the two low - lying electronic \n states have either a biradical or zwitterionic character depending \n on the occupation of the two central 2p - orbitals and require multireference \n methods for a proper description . in the present study , in contrast , \n we investigate the dynamics occurring at small torsional angles ( vide infra ) for which the employed single - reference methods \n not only have been validated .", "in addition \n we employ the results of multireference calculations on smaller motor \n molecules as reported by liu and morokuma to make a further comparison with our experimental results .", "for s0 the optimized \n structure on the b3lyp+d / aug - cc - pvdz level was used to calculate the \n vertical excitation energies . for vertical excitation", "they thus strongly suggest that the broad \n and structureless absorption feature observed in the time - resolved \n ir spectra is associated with the electronic s1  \n s2 transition .", "the presence of two close - lying states also \n explains why the induced absorption bands have such high intensities . \n", "our calculations show that the energy gap between the two states strongly \n depends on structural parameters such as the c = c bond length \n that are involved in the 14001600 cm normal \n modes . for these modes", "one therefore expects that their transition \n moments will be enhanced by vibronic coupling as is indeed observed . \n", "the conclusion that the excited - state dynamics of the molecular rotor \n should be described in a multistate model is further supported by \n recent multireference calculations on smaller rotor molecules that \n also find very small energy differences between the s1 and \n s2 states .", "the excited - state \n dynamics were elucidated further using a global \n analysis of the transient spectra , which shows that the spectra can \n be described well with two exponential decay terms with time constants \n of 1.53  0.03 and 12  1 ps .", "we note that our experiments \n have a time resolution of 150200 fs and therefore do not allow \n observation and confirmation of the ultrafast dynamics reported previously \n in fluorescence - upconversion studies .", "species - associated spectra ( sas ) ( supplementary \n figure 2 ) were derived by using a two - step sequential kinetic \n model .", "the sas of the first species shows an overall background \n signal \n with broad and intense features superimposed on it that indicate vibronic \n coupling .", "furthermore , the increase ( decrease ) of absorbance in the \n 1550 ( 1600 ) cm region indicates a substantial \n change in bond character of the central double bond ( figure 2c ) .", "this spectrum was related \n to the structure of this excited - state species using excited - state \n geometry optimizations .", " \n state without any structural constrains associated with the homo1 \n  lumo excitation leads to a stable minimum that can be reached \n from the franck  condon excited geometry by relatively small \n structural changes ( supplementary figure 4 ) , and an excited - state ir absorption spectrum that is in good agreement \n with the sas of species 1 ( figure 2 ) .", "it is notable that for a proper comparison with \n the experimental spectrum , the theoretically predicted stick spectrum \n needs to be convolved with 20 cm gaussian line \n shapes , suggesting that in the  dark  state a wide range \n of structures is sampled .", "our experiments and calculations thus demonstrate \n that excitation and excited - state decay involve two different electronic \n states . in earlier studies , time - resolved fluorescence and visible \n absorption data were interpreted in terms of a decay from a single , \n isolated electronic state decaying to the ground state after structural \n relaxation in the excited state .", "the present data show that the s1 state actually decays to an intermediate , dark electronic \n state , and from there to the ground state . in the sas of the \n second species ( supplementary \n figure", "2 ) the broad background is absent , and bands have widths \n comparable to those in the s0 state .", "the absence of the \n broad background indicates that this species is no longer in an electronically \n excited state but back in the ( vibrationally hot ) electronic ground \n state .", "indeed , the vibrational activity observed in the 1550 cm region follows closely the ir absorption spectrum \n of p*. in addition , repopulation of the original state ( i ) is observed \n also ( i.e. the quantum yield for isomerization is less than unity ) , and the sas of the second species contains contributions \n from both vibrationally hot ground - state i and metastable p * isomers . \n", "vibrational cooling of these isomers leads to the sas of the final \n species , which is in excellent agreement with the steady - state ftir \n difference spectrum of room - temperature i and p*. the present \n experimental and theoretical data allow a complete \n picture of the mechanism by which the molecular rotary motor undergoes \n isomerization after electronic excitation ( figure 3 ) .", "photon absorption brings the motor to \n a  bright  state from which it decays on an ultrafast \n time scale and within the time resolution of the present experiments \n to a different electronically excited  dark  state . \n in line with previous calculations on similar systems ,", "our calculations \n indicate that this is a barrierless process proceeding via a conical \n intersection .", "previously , it was assumed \n that structural evolution on the homo  lumo potential energy \n surface is responsible for the observed ultrafast decay of the fluorescence , \n and ( following commonly accepted models on photoisomerization of carbon ", "carbon \n double bonds ) that it primarily proceeds along torsional and pyramidalization \n coordinates . the present study , in contrast , leads to the conclusion \n that the fluorescence decay is mediated by the passage through the \n conical intersection of the homo  lumo and homo1  \n lumo potential energy surfaces . inspection of the structural changes \n that occur upon geometry relaxation to the minimum on the homo1 \n  lumo potential energy surface show that the central c = c \n bond length increases from 1.381 to 1.461  and that only relatively \n small changes occur in the torsional and pyramidalization coordinates . \n in combination with the forces that act on the molecule at the franck ", "condon \n excited  bright  state ( supplementary \n figure 3 ) , this suggests that motion along the c = c stretch \n coordinate needs to be taken into account as well for understanding \n the decay of the fluorescence .", "proposed multistate dynamics of the photoinduced \n unidirectional \n rotation of the molecular motor starting from conformer i to conformer \n p * ( projected into one dimension ) : dotted arrow , ultrafast process \n reported in previous work ; fast ( red arrow ) and slow process ( blue \n arrow ) observed in the present time - resolved infrared study . ( a ) calculated \n energy profile along the torsion coordinate showing how the energies \n of the electronic states change as the molecules evolves to conformer \n p*. ( b ) schematic representation of the proposed model .", "hence , it can be concluded that \n internal conversion to the electronic \n ground state takes place from the  dark  state .", "the \n decay rate for this process indicates that it is mediated by conical \n intersections between the two states as has indeed been proposed in \n previous theoretical studies .", "these studies \n concluded , however , as well that to reach these intersections a barrier \n needs to be overcome that results from the steric repulsion of the \n methyl group of the rotor unit with the hydrogen of the stator unit . \n", "previous time - resolved fluorescence measurements reported an ultrafast \n damping mode at 113 cm that was speculated to \n arise from motion along a coordinate involving inversion / pyramidalization \n although no direct support could be found for such an assignment .", "interestingly , our calculations find in the \n  dark  state a mode at 119 cm ( b3lyp / cc - pvdz ; \n not scaled , supporting information ) that \n involves motion of the methyl group with respect to the hydrogen of \n the stator unit .", "one might therefore wonder to what extent this mode \n is related to the oscillatory behavior of the fluorescence .", "our observed \n s2 decay constant of 1.5 ps matches the slow component \n of the overall fluorescence decay ( 0.91.5 ps ) .", "our calculation of the excitation energies of \n the  bright  and  dark  states along the \n 119 cm normal mode did not show a conical intersection \n between the two states , supporting the previous explanation of the \n oscillations observed in the fluorescence measurements by the movement of wave packets and suggesting \n a scenario in which a coherent oscillation in the dark s2 state periodically partly repopulates the fluorescent s1 state .", "however , our experimental methods do not provide the time \n resolution to confirm this ."], ["the photodynamics \n of a unidirectional molecular motor has been \n studied by time - resolved ir spectroscopy in which both vibrational \n and electronic structures are probed . in combination with quantum \n mechanical calculations , the high structural sensitivity and real - time \n character of the technique allows us to understand fundamental aspects \n of the operation mechanism .", "the results show that conversion of photon \n energy into directed motion proceeds on the potential energy surfaces \n of two different electronic states , which is the more important as \n it implies that the conical intersection between the  dark  \n electronically excited state and the ground state determines the efficiency \n of the motor . to rationally tune", "the performance of photoinitiated \n unidirectional rotation thus involves a multicoupled - state optimization \n of nuclear motion ."]]}
{"article_id": "PMC3360846", "article_text": ["the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig . ", "1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .", "1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig . ", "1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .", "these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .", "1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .", "the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .", "the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .", "lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .", "the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .", "however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .", "lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case", "gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .", "lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .", "also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .", "unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .", "one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .", "2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation", ". bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .", "the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .  ", "ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .", "they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .", "rajeswaran et al .   solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,", "there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .", "their crystal structures as well as the structure of the native ribosome   are available through the protein data bank ( pdb ) .", "these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .", "two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .", "theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .", "although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,", "no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .", "the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .", "these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .", "the methods applied in the calculations are described in the first section of the paper .", "we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds .", "four structures of clindamycin are available in the protein data bank   ( as of may 2011 ) .", "three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .", "the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome  clindamycin complexes of deinococcus radiodurans   ( pdb code 1jzx ) and haloarcula marismortui   ( pdb code 1yjn ) .", "therefore , based on the known x - ray structures of clindamycin ( fig . ", "2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .", ", we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig . ", "2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .", "the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .", "the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .", "no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called  exp - opt ,  were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .", "to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .", "therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,", "the solvent is described by a dielectric constant , which was set to 80 in our work .", "the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .", "the positions of these point charges were obtained from the x - ray structures of the ribosome ", "the coordinates of residues within 10  of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .", "next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .", "the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .", "nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin  spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .", "shielding constants were calculated using the b3lyp / aug - pcs-1   method and the giao routine [ 2630 ] .", "the chemical shifts of the i - th nuclei were calculated as \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ \\delta ( i ) = \\sigma_i(x ) - \\sigma_i(c ) , $ $ \\end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .", "spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .", "the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .", "the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .", "data were analyzed using gabedit , and the visualizations were carried out with vmd   and xdrawchem .", "the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 . the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol )", "are presented in fig .  5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,", "the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .", "to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .", "in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig .  6 ) . the numerical values for the electron density ( bcp ) and", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions", ". however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure", ", we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .", "as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and", "its chemical shift is lower for the b conformer than for the a conformer of clindamycin .", "the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer", ". however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum", "the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method", "in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 .", "the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol ) are presented in fig . ", "5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize", ", nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig . ", "the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table  4 .", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9  h39c152.3700.01460.0131cp7c17cl28 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .", "bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .", "however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as", "chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group ,", "the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer .", "however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer . this value is large enough to be measured experimentally.fig .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum", "we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .", "internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,", "we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin  spin coupling constants to the conformation of clindamycin .", "the two most stable conformers of clindamycin exhibit c = oh  o intramolecular hydrogen bonds . according to nbo and aim analyses ,", "the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water ."], "abstract_text": ["<S> lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens . </S>", "<S> this group of drugs inhibits the activity of the bacterial ribosome by binding to the 23s rna of the large ribosomal subunit and blocking protein synthesis . </S>", "<S> currently , three x - ray structures of the ribosome in complex with clindamycin are available in the protein data bank , which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin . in this work , we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23s rna . </S>", "<S> we studied three lincosamide antibiotics : clindamycin , lincomycin , and pirlimycin at the b3lyp level with the 6 - 31 g * * basis set . </S>", "<S> the focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties . </S>", "<S> for both functional conformers , we applied natural bond orbital ( nbo ) analysis and the atoms in molecules ( aim ) theory , and calculated the nmr parameters . </S>", "<S> based on the results obtained , we were able to show that the structure with the intramolecular hydrogen bond c = o  h  o is the most stable conformer of clindamycin . </S>", "<S> the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , was found to be the dominant factor influencing the high stability of this conformer.figuremolecular graph of more stable conformer of clindamycin . </S>"], "labels": null, "section_names": ["Introduction", "Computational details", "Results and discussion", "Conformational analysis", "Natural bond analysis", "Atoms in Molecules analysis", "NMR chemical shifts", "Conclusions"], "sections": [["the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig . ", "1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .", "1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig . ", "1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .", "these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .", "1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .", "the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .", "the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .", "lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .", "the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .", "however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .", "lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case", "gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .", "lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .", "also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .", "unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .", "one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .", "2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation", ". bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .", "the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .  ", "ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .", "they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .", "rajeswaran et al .   solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,", "there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .", "their crystal structures as well as the structure of the native ribosome   are available through the protein data bank ( pdb ) .", "these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .", "two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .", "theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .", "although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,", "no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .", "the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .", "these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .", "the methods applied in the calculations are described in the first section of the paper .", "we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds ."], ["four structures of clindamycin are available in the protein data bank   ( as of may 2011 ) .", "three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .", "the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome  clindamycin complexes of deinococcus radiodurans   ( pdb code 1jzx ) and haloarcula marismortui   ( pdb code 1yjn ) .", "therefore , based on the known x - ray structures of clindamycin ( fig . ", "2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .", ", we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig . ", "2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .", "the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .", "the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .", "no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called  exp - opt ,  were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .", "to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .", "therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,", "the solvent is described by a dielectric constant , which was set to 80 in our work .", "the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .", "the positions of these point charges were obtained from the x - ray structures of the ribosome ", "the coordinates of residues within 10  of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .", "next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .", "the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .", "nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin  spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .", "shielding constants were calculated using the b3lyp / aug - pcs-1   method and the giao routine [ 2630 ] .", "the chemical shifts of the i - th nuclei were calculated as \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ \\delta ( i ) = \\sigma_i(x ) - \\sigma_i(c ) , $ $ \\end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .", "spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .", "the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .", "the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .", "data were analyzed using gabedit , and the visualizations were carried out with vmd   and xdrawchem ."], ["the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 . the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol )", "are presented in fig .  5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,", "the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .", "to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .", "in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig .  6 ) . the numerical values for the electron density ( bcp ) and", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions", ". however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure", ", we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .", "as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and", "its chemical shift is lower for the b conformer than for the a conformer of clindamycin .", "the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer", ". however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum"], ["the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method"], ["in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 .", "the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol ) are presented in fig . ", "5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize", ", nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer ."], ["the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig . ", "the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table  4 .", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9  h39c152.3700.01460.0131cp7c17cl28 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .", "bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .", "however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule ."], ["the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as", "chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group ,", "the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer .", "however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer . this value is large enough to be measured experimentally.fig .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum"], ["we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .", "internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,", "we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin  spin coupling constants to the conformation of clindamycin .", "the two most stable conformers of clindamycin exhibit c = oh  o intramolecular hydrogen bonds . according to nbo and aim analyses ,", "the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water ."]]}
{"article_id": "PMC4204277", "article_text": ["graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction .", "firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 .", "then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo", "were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h ,", "the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c .", "blank cu2o was synthesized through the same procedure , except for the addition of go solution .", "xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv .", "the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) .", "uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer .", "pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) .", "photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements ,", "the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode .", "a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system .", "eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz .", "the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz .", "the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants ,", "photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) .", "a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor .", "excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source .", "the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator .", "the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons .", "a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity . for the isotope - tracer experiment ,", "after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min .", "the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc ", "graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction .", "firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 .", "then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo", "were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h ,", "the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c .", "blank cu2o was synthesized through the same procedure , except for the addition of go solution .", "xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv .", "the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) .", "uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer .", "pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) .", "photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements ,", "the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode .", "a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system .", "eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz .", "the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz .", "the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants ,", "photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) .", "a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor .", "excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source .", "the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator .", "the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons .", "a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity .", "after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min .", "the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc ", "as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors"], "abstract_text": ["<S> a facile one - step microwave - assisted chemical method has been successfully used for the synthesis of cu2o / reduced graphene oxide ( rgo ) composites . </S>", "<S> photocatalytic co2 reduction was then investigated on the junction under ambient conditions . </S>", "<S> the rgo coating dramatically increases cu2o activity for co2 photoreduction to result in a nearly six times higher activity than the optimized cu2o and 50 times higher activity than the cu2o / ruox junction in the 20th hour . </S>", "<S> furthermore , an apparent initial quantum yield of approximately 0.34 % at 400 nm has been achieved by the cu2o / rgo junction for co2 photoreduction . </S>", "<S> the photocurrent of the junction is nearly double that of the blank cu2o photocathode . </S>", "<S> the improved activity together with the enhanced stability of cu2o is attributed to the efficient charge separation and transfer to rgo as well as the protection function of rgo , which was proved by xrd , sem , tem , x - ray photoelectron spectroscopy , photo - electrochemical , photoluminescence , and impedance characterizations . </S>", "<S> this study further presents useful information for other photocatalyst modification for efficient co2 reduction without the need for a noble - metal co - catalyst . </S>"], "labels": null, "section_names": ["Experimental Section", "Synthesis of Cu", "Characterization", "Fabrication of film electrodes and electrochemical measurements", "Photocatalytic activity measurements", "Supporting Information"], "sections": [["graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction .", "firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 .", "then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo", "were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h ,", "the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c .", "blank cu2o was synthesized through the same procedure , except for the addition of go solution .", "xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv .", "the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) .", "uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer .", "pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) .", "photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements ,", "the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode .", "a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system .", "eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz .", "the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz .", "the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants ,", "photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) .", "a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor .", "excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source .", "the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator .", "the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons .", "a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity . for the isotope - tracer experiment ,", "after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min .", "the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc "], ["graphene oxide ( go ) solution was synthesized from natural graphite powder by a modification of hummers method.45 cu2o / rgo composites were fabricated by a microwave - assisted hydrothermal reaction .", "firstly , cu(no3)2 was added to a mixture of ethanol and water in the ratio of 64:36 .", "then , a calculated amount of go solution and formic acid ( 3 ml ) were added . for optimization , cu2o / rgo composites with different amounts of rgo", "were also synthesized , which include cu2o/0.25 % rgo , cu2o/0.5 % rgo , and cu2o/1 % rgo , in which x % represents the calculated weight ratio of the go added to cu2o . after stirring for 2 h ,", "the homogeneous solution was heated with stirring in the microwave system at 150 c for 3 h. after the product was cooled to rt , the final product was collected by centrifugation , washed with water five times , and dried at 70 c .", "blank cu2o was synthesized through the same procedure , except for the addition of go solution ."], ["xrd was performed by using a rigaku rint 2100 diffractometer at a voltage of 40 kv .", "the morphologies of the products were characterized by field - emission scanning electron microscopy ( fesem , jeol-6701f ) and tem ( jeol-2010f ) .", "uv / vis spectra were recorded by using a shimadu uv / vis 2550 spectrophotometer .", "pl emission spectra were measured at rt by using a fluorescence spectrophotometer ( f-4500 , hitachi ) ."], ["photocatalyst ( 5 mg ) and nafion solution ( 10 l , 5 wt % ) were dispersed in a water / isopropanol mixture ( 1 ml , 3:1 v / v ) by at least 30 min sonication to form a homogeneous catalyst colloid . for the measurements ,", "the catalyst colloid ( 100 l ) was deposited onto an area of approximately 1 cm of the fto conductive glass to form the working electrode .", "a pt wire was used as a counter electrode , and an ag / agcl electrode was the reference electrode in the three - electrode photo - electrochemical system .", "eis were recorded under an alternating current perturbation signal of 10 mv over the frequency range of 1 mhz to 100 mhz .", "the potential ranged from 1.0 to 0 v with a potential step of 10 mv at a frequency of 1 khz ."], ["the co2 reduction reaction was performed in batches by using a septum - sealed glass chamber with a volume of 120 ml , which was heated at 160 for 1 h prior to measurement . to remove possible trace organic contaminants ,", "photocatalysts were treated at 200 c for 3 h in a tubular furnace under the protection of ar or in air ( denoted cu2o treated in ar or air ) .", "a typical photocatalytic experiment was conducted by using 0.5 g of photocatalysts and 3 ml of deionized water in a co2-purged 120 ml reactor .", "excess ( 0.7 m ) sodium sulfite was added to each batch as a hole scavenger.15 , 46 a 150 w xe lamp ( newport ) was used as a light source .", "the light output was measured by using a newport 1918-r high - performance optical power meter fitted with a newport 918-d calibrated photodetector equipped with an integrated attenuator .", "the reaction product was monitored by periodical sampling of the gas phase from the glass chamber by using a gas - tight syringe and analyzed by gc ( varian gc-450 ) with a thermal conductivity detector ( tcd , connected to a molecular sieve column ) to detect h2 , o2 , and n2 and a flame ionization detector ( fid , connected to a cp - sil 5cb capillary column ) to detect hydrocarbons .", "a methanizer was installed to enable the fid to detect co with 1000 higher sensitivity .", "after the addition of cu2o / rgo ( 0.5 g ) into co2-saturated water ( 10 ml ) , the septum - sealed reactor was purged by ar gas for 10 min .", "the sample was irradiated with a 150 w xe lamp for 30 min , and then 0.5 ml of the reaction product taken from the vessel headspace was analyzed by gc "], ["as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors"]]}
{"article_id": "PMC1852396", "article_text": ["nature evolved a very limited number of chromophores in the different evolutionary branches of the tree of life because of the similar needs of the most diverse organisms for both the detection of the external world and the interaction with it .", "the study of these chromophores and their distribution inside cellular structures is usually based on extractive procedures , followed by biochemical or spectroscopic assays .", "extractive techniques often include disadvantages : they can modify the nature of the components and they are not successful in isolating the chromophores .", "direct investigation by means of microspectrophotometry of on intact samples has the advantage of preserving the integrity of biological structures or substructures .", "a microspectrophotometer is a simple apparatus consisting of a modified microscope that can measure absorption or emission spectra of very small areas inside a cell 1 .", "a high quality microscope is equipped with a polychromator , i.e. a flat field concave grating , which is connected to the slit - shaped exit pupil of a 19 light - guides probe .", "the dispersion image of the exit pupil produced by the polychromator is focused onto a digital slow scan cooled ccd camera .", "the main feature of this instrumentation is the possibility to measure in vivo absorption or emission spectra at the same time on different sub - cellular compartments using extremely low light intensities .", "this set - up represents a better strategy with respect to traditional instruments , since it eliminates errors typical of microspectrophotometry , such as photobleaching , distributional errors , the schwarzschild - villiger effect , and allows in vivo reliable spectroscopic investigations 2 , 3 .", "the photosynthetic and the photoreceptive compartments are the main location of chromophores inside algal cells . absorption and emission spectra measured in vivo on these compartments can provide very precise and accurate information about the spectral range in which chromophore molecules capture photons in their natural environment and their de - excitation pathways 4 .", "we tested our apparatus by measuring absorption spectra on the eyespot and chloroplast of the unicellular alga dunaliella and emission spectra on the photoreceptor and chloroplast of the unicellular alga euglena gracilis .", "cultures . euglena gracilis strain z ( sammlung von algenkulturen gttingen , 1224 - 5/25 ) cells", "were grown axenically in cramer - myers medium 0.025 m in sodium acetate ( ph 6.8 ) 5 ; cultures of dunaliella spp .", "( sammlung von algenkulturen gttingen , 19 - 5 ) were grown axenically in johnson 's medium 6 .", "both cultures were kept under constant temperature ( 24 c ) and continuous illumination ( 2x10 mol photons m sec ) .", "the hardware platform consists of a zeiss axioplan microscope ( zeiss , oberkoken , de ) , equipped with an epifluorescence system , 100x ( n.a .", "the emission spectra were acquired with a combination of a uv - blue filter set ( 8 nm band pass excitation filter , 365 nm ; chromatic beam splitter , 395 nm ; barrier filter , 397 nm ; irradiance 800 w / cm ) , and a blue - violet filter set ( 8 nm band pass excitation filter , 436 nm ; chromatic beam splitter , 460 nm ; barrier filter , 470 nm ; irradiance 1100 w / cm ) .", "77423 oriel , stratford , connecticut , usa ) connected to a schott kl 1500 probe illuminator system ( schott corporation , mainz , de ) was mounted in the back focal plane of the ocular of the microscope ( figure 1 ) .", "this probe consists of an outer bundle of 24 light - guides and a central bundle of 19 light - guides ( figure 2a ) .", "the outer bundle is used for centering the objects , while the central bundle of the probe is used for acquiring transmitted or emitted light .", "the sub - cellular components on which the spectra have to be measured , are placed in the microscope field and finely adjusted .", "the exit pupil of the probe ( figure 2b ) is connected to a flat field imaging concave grating polychromator ( mod .", "52300070 , jobin yvon , longjumenau , france ) that produces a dispersion image of the probe .", "this image is in turn focused onto a digital slow scan cooled ccd camera ( dta discovery ds260e , pisa , italy ) , and it consists of 19 multi - lines strips showing the distribution of light intensity according to the wavelength .", "the microspectophotometer instrumentation possesses a graphical interface that allows the set - up of both the optics and the frame grabber ( scion corporation , frederick , maryland , usa ) and controls the measurements .", "once the instrumental has been set - up , the operator , on the basis of the light guide positions upon the cell , selects the zones of the dispersion image displayed on the top of the graphical layout ( in the layout r stands for reference , and s for sample ) .", "the resulting spectrum is displayed at the bottom of the graphical layout , ( figure 3 ) .", "absorption measurements are based on the comparison of two radiant fluxes density is and ir .", "is results from the interaction of light with the sample ( it is related to absorption cross section of the molecules and the number of absorbing molecules ) 7 , while ir results from the interaction of light with the reference material . therefore , we can consider the absorbance of a sample ( as ) as derived from the measures as follows : this equation is known as the lambert - beer 's law . for the discussion on the theoretical aspects of image formation and the light transmission in microspectrophotometry see barsanti et al .", "absorption spectra were performed on both the eyespot ( screening device ) and the chloroplast ( photosynthetic apparatus ) of the unicellular alga dunaliella .", "the central bundle of the probe was centered on both the structures in the apical portion of the cell ( is ) , in such a way that some light - guides were located outside the cell ; these guides measure ir .", "measurements of emission microspectroscopy are based on the radiant flux density f. this density is given by : where ia is the amount of light absorbed by the chromophores ; f is their fluorescence quantum yield and z represents the fraction of fluorescence collected by the objective , 9 .", "emission spectra were performed on both the photoreceptor and chloroplast of the photosensitive alga euglena gracilis .", "the outer bundle of the probe was used for centering the photoreceptor and the chloroplast in the apical portion of the cell , while the central bundle of the probe was used for acquiring light emitted by the two structures .", "photographs were taken with an olympus camedia c-30303 digital camera ( olympus , tokyo , japan ) mounted onto the microscope .", "the eyespot is recognizable as a the bright orange spot located on the left side of the cell in the apical portion .", "the size of the eyespot is about 3  and the size of the chloroplast is about 10  ; the probe is superimposed on the structures in order to show their relative position ( figures 4b ) .", "this spectrum closely resembles the spectrum obtained by batra and tollin 10 on a suspension of eyespot granules , and the other in vivo spectra previously recorded by strother and wolken 11 , by benedetti et al .", "major peaks are due to lutein whose bands are centered at 410 , 479.5 , and 510 nm and -carotene whose bands are centered at 455.5 , 481.5 , and 510.5 nm ( not shown ) .", "it clearly shows that dunaliella belongs to the green lineage of eukaryotic algae since only chlorophylls a and b and carotenoids are present in this spectrum .", "gaussian bands decomposition of this spectrum is easily explained as a combination of chlorophyll a bands centered at ( 410 , 435 , 444 , 585 , 615 , 626 , 634.5 , 663 , 672 , 678 , 683 , 695 nm , chlorophyll b bands centered at 412 , 428.5 , 445 , 452 , 582 , 594 , 607 , 621.5 , 652 nm , and carotenoids lutein and bands centered at 410 , 479.5 , and 510 nm , ( not shown ) , 14 .", "the photoreceptor is recognizable as a the bright green spot located in the apical portion of the cell .", "the size of the photoreceptor is about 2  and the size of the chloroplast is about 10  ; the probe is superimposed on the structures in order to show their relative position ( figure 6b ) .", "figure 7a shows the emission spectrum of a single photoreceptor under 436 nm excitation light , after 10 seconds of excitation with the 365 nm light .", "gaussian bands decomposition of the emission spectrum reveals 3 bands with different intensity centered at about 500 nm , 525 nm , and 556 nm ( not shown ) .", "the bands obtained by the gaussian decomposition indicate the presence of a mixture of very similar conformers in the photoreceptor , each one capable of photocycling between a non - fluorescent parent species and a fluorescent excited species 15 .", "the presence in the photoreceptor of euglena of a photochromic chromophore , which undergoes light - driven reversible photochromism has been well established by means of digital and fluorescence microscopy 16 .", "the photoreceptor possesses optical bistability , i.e. upon photoexcitation the ground state generates a stable excited state , which can be photochemically driven back to the ground state .", "the 27 kda protein extracted from the photoreceptor shows a similar behavior , the photochromic reaction cycling between two different stable conformers , the parent and the excited conformers 17 .", "figure 7b shows the emission spectrum of the thylakoid compartment of euglena gracilis excited at 463 nm .", "it is the typical emission spectrum of a chloroplast that contains only chlorophyll a and b whose bands are centered at 685 for the photosystem ii and at 725 nm for the photosystem i , 18 .", "set - up of the polychromator - based microspectrophotometer entrance ( a ) and exit ( b ) pupils of the light guide probe layout of the software interface a ) bright field image of dunaliella .", "b ) position of the probe upon dunaliella   absorption spectra of the eyespot ( a ) and a chloroplast ( b ) of dunaliella a ) fluorescent image of euglena gracilis ; b ) position of the probe upon euglena gracilis emission spectra of the photoreceptor ( a ) and a chloroplast ( b ) of euglena gracilis"], "abstract_text": ["<S> a microspectrophotometer is a digital microscope used to measure absorption and fluorescence spectra . in this paper </S>", "<S> we describe a polychromator - based microspectrophotometer that performs in vivo absorption or emission measurements at the same time on different subcellular compartments such as photoreceptive and photosynthetic structures of algal cells . in this system , a flat field imaging concave grating polychromator is connected to the slit - shaped exit pupil of a light - guide probe mounted onto a microscope equipped with an epifluorescence module.the subcellular components , on which the spectra will be measured , are placed in the microscope field and finely adjusted . </S>", "<S> the outer bundle of the probe is used for centering the objects , while the central bundle of the probe , containing 19 light guides , is used for acquiring either transmitted or emitted light ( i.e. fluorescence ) . </S>", "<S> the light transmitted or emitted by the subcellular components is collected by the probe mounted in the back focal plane of the ocular . </S>", "<S> the exit pupil of this probe , connected to a flat field imaging concave grating polychromator , produces a dispersion image that in turn is focused onto a digital slow scan cooled ccd camera . </S>", "<S> absorption and emission spectra of algal subcellular compartments are presented </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results and Discussion", "Figures and Tables"], "sections": [["nature evolved a very limited number of chromophores in the different evolutionary branches of the tree of life because of the similar needs of the most diverse organisms for both the detection of the external world and the interaction with it .", "the study of these chromophores and their distribution inside cellular structures is usually based on extractive procedures , followed by biochemical or spectroscopic assays .", "extractive techniques often include disadvantages : they can modify the nature of the components and they are not successful in isolating the chromophores .", "direct investigation by means of microspectrophotometry of on intact samples has the advantage of preserving the integrity of biological structures or substructures .", "a microspectrophotometer is a simple apparatus consisting of a modified microscope that can measure absorption or emission spectra of very small areas inside a cell 1 .", "a high quality microscope is equipped with a polychromator , i.e. a flat field concave grating , which is connected to the slit - shaped exit pupil of a 19 light - guides probe .", "the dispersion image of the exit pupil produced by the polychromator is focused onto a digital slow scan cooled ccd camera .", "the main feature of this instrumentation is the possibility to measure in vivo absorption or emission spectra at the same time on different sub - cellular compartments using extremely low light intensities .", "this set - up represents a better strategy with respect to traditional instruments , since it eliminates errors typical of microspectrophotometry , such as photobleaching , distributional errors , the schwarzschild - villiger effect , and allows in vivo reliable spectroscopic investigations 2 , 3 .", "the photosynthetic and the photoreceptive compartments are the main location of chromophores inside algal cells . absorption and emission spectra measured in vivo on these compartments can provide very precise and accurate information about the spectral range in which chromophore molecules capture photons in their natural environment and their de - excitation pathways 4 .", "we tested our apparatus by measuring absorption spectra on the eyespot and chloroplast of the unicellular alga dunaliella and emission spectra on the photoreceptor and chloroplast of the unicellular alga euglena gracilis ."], ["cultures . euglena gracilis strain z ( sammlung von algenkulturen gttingen , 1224 - 5/25 ) cells", "were grown axenically in cramer - myers medium 0.025 m in sodium acetate ( ph 6.8 ) 5 ; cultures of dunaliella spp .", "( sammlung von algenkulturen gttingen , 19 - 5 ) were grown axenically in johnson 's medium 6 .", "both cultures were kept under constant temperature ( 24 c ) and continuous illumination ( 2x10 mol photons m sec ) .", "the hardware platform consists of a zeiss axioplan microscope ( zeiss , oberkoken , de ) , equipped with an epifluorescence system , 100x ( n.a .", "the emission spectra were acquired with a combination of a uv - blue filter set ( 8 nm band pass excitation filter , 365 nm ; chromatic beam splitter , 395 nm ; barrier filter , 397 nm ; irradiance 800 w / cm ) , and a blue - violet filter set ( 8 nm band pass excitation filter , 436 nm ; chromatic beam splitter , 460 nm ; barrier filter , 470 nm ; irradiance 1100 w / cm ) .", "77423 oriel , stratford , connecticut , usa ) connected to a schott kl 1500 probe illuminator system ( schott corporation , mainz , de ) was mounted in the back focal plane of the ocular of the microscope ( figure 1 ) .", "this probe consists of an outer bundle of 24 light - guides and a central bundle of 19 light - guides ( figure 2a ) .", "the outer bundle is used for centering the objects , while the central bundle of the probe is used for acquiring transmitted or emitted light .", "the sub - cellular components on which the spectra have to be measured , are placed in the microscope field and finely adjusted .", "the exit pupil of the probe ( figure 2b ) is connected to a flat field imaging concave grating polychromator ( mod .", "52300070 , jobin yvon , longjumenau , france ) that produces a dispersion image of the probe .", "this image is in turn focused onto a digital slow scan cooled ccd camera ( dta discovery ds260e , pisa , italy ) , and it consists of 19 multi - lines strips showing the distribution of light intensity according to the wavelength .", "the microspectophotometer instrumentation possesses a graphical interface that allows the set - up of both the optics and the frame grabber ( scion corporation , frederick , maryland , usa ) and controls the measurements .", "once the instrumental has been set - up , the operator , on the basis of the light guide positions upon the cell , selects the zones of the dispersion image displayed on the top of the graphical layout ( in the layout r stands for reference , and s for sample ) .", "the resulting spectrum is displayed at the bottom of the graphical layout , ( figure 3 ) .", "absorption measurements are based on the comparison of two radiant fluxes density is and ir .", "is results from the interaction of light with the sample ( it is related to absorption cross section of the molecules and the number of absorbing molecules ) 7 , while ir results from the interaction of light with the reference material . therefore , we can consider the absorbance of a sample ( as ) as derived from the measures as follows : this equation is known as the lambert - beer 's law . for the discussion on the theoretical aspects of image formation and the light transmission in microspectrophotometry see barsanti et al .", "absorption spectra were performed on both the eyespot ( screening device ) and the chloroplast ( photosynthetic apparatus ) of the unicellular alga dunaliella .", "the central bundle of the probe was centered on both the structures in the apical portion of the cell ( is ) , in such a way that some light - guides were located outside the cell ; these guides measure ir .", "measurements of emission microspectroscopy are based on the radiant flux density f. this density is given by : where ia is the amount of light absorbed by the chromophores ; f is their fluorescence quantum yield and z represents the fraction of fluorescence collected by the objective , 9 .", "emission spectra were performed on both the photoreceptor and chloroplast of the photosensitive alga euglena gracilis .", "the outer bundle of the probe was used for centering the photoreceptor and the chloroplast in the apical portion of the cell , while the central bundle of the probe was used for acquiring light emitted by the two structures .", "photographs were taken with an olympus camedia c-30303 digital camera ( olympus , tokyo , japan ) mounted onto the microscope ."], ["the eyespot is recognizable as a the bright orange spot located on the left side of the cell in the apical portion .", "the size of the eyespot is about 3  and the size of the chloroplast is about 10  ; the probe is superimposed on the structures in order to show their relative position ( figures 4b ) .", "this spectrum closely resembles the spectrum obtained by batra and tollin 10 on a suspension of eyespot granules , and the other in vivo spectra previously recorded by strother and wolken 11 , by benedetti et al .", "major peaks are due to lutein whose bands are centered at 410 , 479.5 , and 510 nm and -carotene whose bands are centered at 455.5 , 481.5 , and 510.5 nm ( not shown ) .", "it clearly shows that dunaliella belongs to the green lineage of eukaryotic algae since only chlorophylls a and b and carotenoids are present in this spectrum .", "gaussian bands decomposition of this spectrum is easily explained as a combination of chlorophyll a bands centered at ( 410 , 435 , 444 , 585 , 615 , 626 , 634.5 , 663 , 672 , 678 , 683 , 695 nm , chlorophyll b bands centered at 412 , 428.5 , 445 , 452 , 582 , 594 , 607 , 621.5 , 652 nm , and carotenoids lutein and bands centered at 410 , 479.5 , and 510 nm , ( not shown ) , 14 .", "the photoreceptor is recognizable as a the bright green spot located in the apical portion of the cell .", "the size of the photoreceptor is about 2  and the size of the chloroplast is about 10  ; the probe is superimposed on the structures in order to show their relative position ( figure 6b ) .", "figure 7a shows the emission spectrum of a single photoreceptor under 436 nm excitation light , after 10 seconds of excitation with the 365 nm light .", "gaussian bands decomposition of the emission spectrum reveals 3 bands with different intensity centered at about 500 nm , 525 nm , and 556 nm ( not shown ) .", "the bands obtained by the gaussian decomposition indicate the presence of a mixture of very similar conformers in the photoreceptor , each one capable of photocycling between a non - fluorescent parent species and a fluorescent excited species 15 .", "the presence in the photoreceptor of euglena of a photochromic chromophore , which undergoes light - driven reversible photochromism has been well established by means of digital and fluorescence microscopy 16 .", "the photoreceptor possesses optical bistability , i.e. upon photoexcitation the ground state generates a stable excited state , which can be photochemically driven back to the ground state .", "the 27 kda protein extracted from the photoreceptor shows a similar behavior , the photochromic reaction cycling between two different stable conformers , the parent and the excited conformers 17 .", "figure 7b shows the emission spectrum of the thylakoid compartment of euglena gracilis excited at 463 nm .", "it is the typical emission spectrum of a chloroplast that contains only chlorophyll a and b whose bands are centered at 685 for the photosystem ii and at 725 nm for the photosystem i , 18 ."], ["set - up of the polychromator - based microspectrophotometer entrance ( a ) and exit ( b ) pupils of the light guide probe layout of the software interface a ) bright field image of dunaliella .", "b ) position of the probe upon dunaliella   absorption spectra of the eyespot ( a ) and a chloroplast ( b ) of dunaliella a ) fluorescent image of euglena gracilis ; b ) position of the probe upon euglena gracilis emission spectra of the photoreceptor ( a ) and a chloroplast ( b ) of euglena gracilis"]]}
{"article_id": "PMC5098230", "article_text": ["one of the key challenges \n in the development of novel materials \n is the ability to tune and control their macroscopic physical and \n chemical properties on a molecular level by external stimuli .", "such a degree of control is challenging but crucial for a wide range \n of applications ranging from catalysis to pharmacological and medical \n applications .", "in photoactive materials light absorbed by chromophores can be converted \n into directed functionality , typically by means of photoinduced isomerization \n of nitrogen or carbon containing double bonds and pericylic reactions .", "indeed , e  z isomerization \n results in large amplitude changes in structure as well as chemical \n and physical properties .", "molecular motors based on overcrowded alkenes are particularly \n attractive due to the synthetically tunable properties , such as thermal \n stability , absorption spectra , etc . , and", "therefore are readily applied \n to a wide range of functional systems . however , although the thermally \n activated step in the unidirectional rotary cycle is well understood \n and is now largely predictable , the photochemical properties and especially \n photochemical quantum yields are less well understood .", "hence both \n from a fundamental perspective and in achieving the goal of complete \n molecular design , gaining insight into the excited - state properties \n of light driven molecular - sized machines is essential .", "the overcrowded alkene - based molecular \n rotary motors are composed \n of a central carbon  carbon double bond ( the axle ) embedded \n in an intrinsically chiral environment .", "the unidirectional rotational \n cycle starts with a photoinduced e  z isomerization around the central c = c bond , generating \n a metastable isomer p * ( figure 1 ) , which relaxes thermally to the stable isomer p , which in \n the case of a symmetric stator unit is identical to i. this isomer \n marks half of the rotation cycle and is the starting point for a second \n photoinduced isomerization step that leads again to a metastable isomer . \n", "thermally activated conformational isomerization of this isomer brings \n the system back to its initial state .", "overall , the rotor part has \n then performed a complete rotation with respect to the stator unit \n of the molecular motor .", "although the rotation includes both \n photochemical and thermal steps \n that involve different parts of the motor , the structural evolution \n is largely governed by the excited - state properties of the central \n c = c group , which , in turn , are dictated by the potential energy \n surfaces of the excited states that are accessed upon electronic excitation . \n despite the large number of theoretical and experimental studies that have focused on characterizing \n this process , there are several key questions outstanding including \n identification of the main structural coordinates and the role of \n various electronic states involved in the excited - state dynamics . \n", "time - resolved studies are essential to elucidate these dynamics but \n have thus far focused on probing the change in the electronic structure \n after photoexcitation through uv / vis absorption and fluorescence studies .", "these techniques offer \n a high time resolution but provide only indirect information as to \n how the structure evolves spatially over time . in the present contribution \n we show that this information can be obtained through a combination \n of picosecond time - resolved vibrational spectroscopy of the electronically \n excited states together with quantum - mechanical calculations .", "the setup for the time - resolved infrared measurements has been \n described in detail previously .", "mid - ir \n probe pulses were generated using an amplified ti : sapphire system \n ( spectra - physics hurricane , 600 j , repetition rate 1 khz ) pumping \n a commercial bbo - based opa ( spectra - physics opa-800c ) , the signal \n and idler output of which were difference - frequency - mixed in aggas2 .", "uv pump pulses ( 400 nm , 1 j ) were generated \n by doubling part of the output of the ti : sapphire laser .", "the diameter \n of the uv pump focus was 180200 m ; that of \n the ir probe focus , 150 m . the sample cell with caf2 windows spaced by 500 m was placed in the ir focus . \n to avoid accumulation of the thermally unstable conformer p * during \n the measurements ,", "the pump  probe delay time was scanned by mechanically \n adjusting the beam path of the uv pump .", "the temporal resolution of \n 200 fs has been determined from the full width at half - maximum of \n the pump probe cross - correlation function .", "the pump pulse was chopped \n at 500 hz , and transient spectra were obtained by subtracting nonpumped \n absorption spectra from the pumped absorption spectra , both of which \n were recorded by spectrally dispersing the probe pulses using an oriel \n ms260i spectrograph and measuring the frequency - dependent ir intensity \n using a 2  32 hgcdte detector array ( infrared associates ) .", "compound \n i was available from earlier studies ( see ref ( 44 ) ) . in all experiments", "we \n investigate a 10 mm solution of compound i in deuterated cyclohexane \n ( aldrich , 99.6 atom% d ) .", "for the investigation of the ground - state \n conformers ( including \n vertical excitation energies and forces acting at the franck ", "condon \n point ) quantum mechanical calculations were performed using the turbomole \n suite of programs .", "geometry optimization \n and normal - mode analysis were performed by ( time - dependent ) density \n functional theory ( ( td)-dft ) using the hybrid functional b3lyp . for the geometry optimization the d3 correction \n method of grimme et al .", "the cc - pvdz or aug - cc - pvdz basis \n sets were employed , respectively .", "post - hartree  fock calculations \n were performed on the basis of spin - component scaled second - order \n mller  plesset perturbation theory and the approximate coupled - cluster singles - and - doubles \n model cc2 .", "all perturbation method calculations were performed using \n the resolution of identity ( ri ) approximation and the efficient ricc2 module implemented \n in the turbomole package together with the standard auxiliary basis \n sets .", "the gaussian09 program package was employed to study the excited - state properties \n in more detail and in particular to optimize the structure of the \n dark - state species .", "geometry optimization and normal - mode analysis \n were performed in the framework of td - dft at the b3lyp / cc - pvdz level .", "the structural evolution from the \n initial state i to isomer p * \n ( figure 1 ) was considered first in terms of the spectral properties \n of the initial state and the first metastable product ( p * ) .", "the steady - state \n uv / vis absorption spectra of the two isomers are distinct , reflecting \n a change in the conjugated  system ( figure 1a ) .", "the ftir spectra of the two isomers ( figure 1c ) show that the \n carbon  carbon stretch and ring vibrations couple to a large \n degree as a consequence of the extended  system ; however , dft \n calculations enable us to assign the bands in the 15501650 \n cm range as being mainly associated with \n modes involving the central c = c double bond ( supporting information ) .", "the metastable isomer p * shows a \n shift of these modes to lower frequencies confirming a decrease in \n bond strength .", "comparison of the optimized geometries of the two isomers \n ( i ( p * ) , cc bond length 1.369 ( 1.377 )  ; cc = cc dihedral \n angle 14.1 ( 29.5 ) ) evidence the corresponding lower  \n bond character as well .", "unidirectional motion of the unidirectional \n motor starting from \n the thermally stable conformer ( i ) via the product of the photoinitiated \n step ( p * ) to the thermally stable conformer ( p ) ( optimized geometries \n on the b3lyp+d / aug - cc - pvdz level ) : ( a ) experimental and calculated \n ( light color and dotted line ) uv  vis spectra of conformer i \n and p * ; ( b ) molecular orbitals of the initial conformer ; ( c ) experimental \n and calculated ir spectra ( b3lyp+d / aug - cc - pvdz ) of the initial state \n ( blue ) and final state ( red ) . in both the measured uv / vis absorption \n and ftir spectra at the photostationary state ( pss ) , i and p * are \n present in a 25:75 ratio .", "time - resolved vibrational spectroscopy was employed to achieve the required temporal resolution as well \n as structural sensitivity to elucidate the structural evolution that \n follows photoexcitation with a short uv - pump pulse ( center wavelength \n 400 nm ) .", "the resulting structural evolution was followed by time - resolved \n differential absorption spectra in cyclohexane - d12 with a time resolution of 200 fs ( figure 2 ) .", "the transient ir spectra show , as expected , \n pronounced differences between the ir absorption spectrum of the molecule \n in the initial and excited states .", "a first point of note is that the \n spectrum does not evolve further after ca .", "30 ps , and the difference \n spectrum corresponds perfectly with the difference spectrum obtained \n upon continuous wave excitation ( figure 1 ) ; i.e. , the metastable isomer p * has formed \n fully within the 30 ps after excitation .", "ftir and time - resolved \n infrared spectra of the molecular motor \n in cyclohexane - d12 : ( a ) ftir spectra of \n the thermal stable conformer ( initial , solvent corrected , top ) , uv - ump / ir - probe \n transient infrared spectra ( uv = 400 nm ) ( middle ) , \n and difference ftir spectra between the two ground - state conformers \n of the molecular motor ; ( b ) kinetics of selected bands and corresponding \n results of the global fit ; ( c ) species associated spectra ( sas ) of \n species 1 and calculated ir spectra ( top ) and sas of the pss ( sp3 ) \n together with ftir spectra ( bottom ) .", "negative bands indicate ground - state \n bleaching , and positive bands , species created after excitation . the time - resolved ir spectra obtained \n within the first 30 ps exhibit \n several remarkable features .", "the first and most striking is a broad \n and unstructured absorption over the entire mid - ir range ( 12003000 \n cm ) .", "this absorption , which decays rapidly , is \n particularly prominent at early time delays ( < 5 ps ) .", "individual \n absorption bands with remarkably high integrated intensities in the \n 14001600 cm range are superimposed on \n the broad spectral feature .", "notably , these bands are significantly \n broader than the bleached bands in the transient spectra and bands \n in the ftir spectra .", "the solvent response and the dependence of the \n uv pump intensity ( supporting information ) confirm that these features are intrinsic to the compound and are \n not due to solvent or two - photon processes .", "the width and intensity \n of the broad excited - state absorption bands \n excludes that they arise from vibrational transitions .", "indeed , the \n absorbance is closer to that expected for electronic transitions , \n albeit these typically occur at much higher energies ( in the uv / vis \n range ) .", "the initial state of the compound \n ( i ) shows an absorption band at 390 nm ( figure 1 ) , which is reproduced well by b3lyp / cc - pvdz \n calculations in the gas phase ( 403 nm ; f = 0.428 ) . \n", "the s1  s0 transition has predominantly \n homo  lumo character ( figure 1 ) .", "in contrast to previous assignments , however , we find that this electronic transition \n is not localized on the stator but involves to a significant extent \n excitation from the bonding  orbital of the central double \n bond to the antibonding  * orbital ( figure 1 ) . as a consequence", ", the double bond character \n of this bond is reduced as well as the barrier to rotation around \n this bond . in line with calculations on ethylene and its derivatives , \n", "we find a dark state ( s2 ) associated with the homo1 \n  lumo excitation at slightly higher excitation energies that \n is not directly accessible from the ground state ( figure 1 , table 1 ) . because the homo1 orbital is localized \n on the aromatic stator unit , s2 has a significantly larger \n dipole moment than s1 ( 4.3 vs 2.2 d at the scs - cc2/cc - pvdz \n level ) .", "it should be emphasized that the description of the lower - lying \n electronic states in the compound studied here is thus entirely different \n from ethylene and its derivatives where , as discussed by salem and \n others , for torsion angles of around 90 the two low - lying electronic \n states have either a biradical or zwitterionic character depending \n on the occupation of the two central 2p - orbitals and require multireference \n methods for a proper description . in the present study , in contrast , \n we investigate the dynamics occurring at small torsional angles ( vide infra ) for which the employed single - reference methods \n not only have been validated .", "in addition \n we employ the results of multireference calculations on smaller motor \n molecules as reported by liu and morokuma to make a further comparison with our experimental results .", "for s0 the optimized \n structure on the b3lyp+d / aug - cc - pvdz level was used to calculate the \n vertical excitation energies . for vertical excitation", "they thus strongly suggest that the broad \n and structureless absorption feature observed in the time - resolved \n ir spectra is associated with the electronic s1  \n s2 transition .", "the presence of two close - lying states also \n explains why the induced absorption bands have such high intensities . \n", "our calculations show that the energy gap between the two states strongly \n depends on structural parameters such as the c = c bond length \n that are involved in the 14001600 cm normal \n modes . for these modes", "one therefore expects that their transition \n moments will be enhanced by vibronic coupling as is indeed observed . \n", "the conclusion that the excited - state dynamics of the molecular rotor \n should be described in a multistate model is further supported by \n recent multireference calculations on smaller rotor molecules that \n also find very small energy differences between the s1 and \n s2 states .", "the excited - state \n dynamics were elucidated further using a global \n analysis of the transient spectra , which shows that the spectra can \n be described well with two exponential decay terms with time constants \n of 1.53  0.03 and 12  1 ps .", "we note that our experiments \n have a time resolution of 150200 fs and therefore do not allow \n observation and confirmation of the ultrafast dynamics reported previously \n in fluorescence - upconversion studies .", "species - associated spectra ( sas ) ( supplementary \n figure 2 ) were derived by using a two - step sequential kinetic \n model .", "the sas of the first species shows an overall background \n signal \n with broad and intense features superimposed on it that indicate vibronic \n coupling .", "furthermore , the increase ( decrease ) of absorbance in the \n 1550 ( 1600 ) cm region indicates a substantial \n change in bond character of the central double bond ( figure 2c ) .", "this spectrum was related \n to the structure of this excited - state species using excited - state \n geometry optimizations .", " \n state without any structural constrains associated with the homo1 \n  lumo excitation leads to a stable minimum that can be reached \n from the franck  condon excited geometry by relatively small \n structural changes ( supplementary figure 4 ) , and an excited - state ir absorption spectrum that is in good agreement \n with the sas of species 1 ( figure 2 ) .", "it is notable that for a proper comparison with \n the experimental spectrum , the theoretically predicted stick spectrum \n needs to be convolved with 20 cm gaussian line \n shapes , suggesting that in the  dark  state a wide range \n of structures is sampled .", "our experiments and calculations thus demonstrate \n that excitation and excited - state decay involve two different electronic \n states . in earlier studies , time - resolved fluorescence and visible \n absorption data were interpreted in terms of a decay from a single , \n isolated electronic state decaying to the ground state after structural \n relaxation in the excited state .", "the present data show that the s1 state actually decays to an intermediate , dark electronic \n state , and from there to the ground state . in the sas of the \n second species ( supplementary \n figure", "2 ) the broad background is absent , and bands have widths \n comparable to those in the s0 state .", "the absence of the \n broad background indicates that this species is no longer in an electronically \n excited state but back in the ( vibrationally hot ) electronic ground \n state .", "indeed , the vibrational activity observed in the 1550 cm region follows closely the ir absorption spectrum \n of p*. in addition , repopulation of the original state ( i ) is observed \n also ( i.e. the quantum yield for isomerization is less than unity ) , and the sas of the second species contains contributions \n from both vibrationally hot ground - state i and metastable p * isomers . \n", "vibrational cooling of these isomers leads to the sas of the final \n species , which is in excellent agreement with the steady - state ftir \n difference spectrum of room - temperature i and p*. the present \n experimental and theoretical data allow a complete \n picture of the mechanism by which the molecular rotary motor undergoes \n isomerization after electronic excitation ( figure 3 ) .", "photon absorption brings the motor to \n a  bright  state from which it decays on an ultrafast \n time scale and within the time resolution of the present experiments \n to a different electronically excited  dark  state . \n in line with previous calculations on similar systems ,", "our calculations \n indicate that this is a barrierless process proceeding via a conical \n intersection .", "previously , it was assumed \n that structural evolution on the homo  lumo potential energy \n surface is responsible for the observed ultrafast decay of the fluorescence , \n and ( following commonly accepted models on photoisomerization of carbon ", "carbon \n double bonds ) that it primarily proceeds along torsional and pyramidalization \n coordinates . the present study , in contrast , leads to the conclusion \n that the fluorescence decay is mediated by the passage through the \n conical intersection of the homo  lumo and homo1  \n lumo potential energy surfaces . inspection of the structural changes \n that occur upon geometry relaxation to the minimum on the homo1 \n  lumo potential energy surface show that the central c = c \n bond length increases from 1.381 to 1.461  and that only relatively \n small changes occur in the torsional and pyramidalization coordinates . \n in combination with the forces that act on the molecule at the franck ", "condon \n excited  bright  state ( supplementary \n figure 3 ) , this suggests that motion along the c = c stretch \n coordinate needs to be taken into account as well for understanding \n the decay of the fluorescence .", "proposed multistate dynamics of the photoinduced \n unidirectional \n rotation of the molecular motor starting from conformer i to conformer \n p * ( projected into one dimension ) : dotted arrow , ultrafast process \n reported in previous work ; fast ( red arrow ) and slow process ( blue \n arrow ) observed in the present time - resolved infrared study . ( a ) calculated \n energy profile along the torsion coordinate showing how the energies \n of the electronic states change as the molecules evolves to conformer \n p*. ( b ) schematic representation of the proposed model .", "hence , it can be concluded that \n internal conversion to the electronic \n ground state takes place from the  dark  state .", "the \n decay rate for this process indicates that it is mediated by conical \n intersections between the two states as has indeed been proposed in \n previous theoretical studies .", "these studies \n concluded , however , as well that to reach these intersections a barrier \n needs to be overcome that results from the steric repulsion of the \n methyl group of the rotor unit with the hydrogen of the stator unit . \n", "previous time - resolved fluorescence measurements reported an ultrafast \n damping mode at 113 cm that was speculated to \n arise from motion along a coordinate involving inversion / pyramidalization \n although no direct support could be found for such an assignment .", "interestingly , our calculations find in the \n  dark  state a mode at 119 cm ( b3lyp / cc - pvdz ; \n not scaled , supporting information ) that \n involves motion of the methyl group with respect to the hydrogen of \n the stator unit .", "one might therefore wonder to what extent this mode \n is related to the oscillatory behavior of the fluorescence .", "our observed \n s2 decay constant of 1.5 ps matches the slow component \n of the overall fluorescence decay ( 0.91.5 ps ) .", "our calculation of the excitation energies of \n the  bright  and  dark  states along the \n 119 cm normal mode did not show a conical intersection \n between the two states , supporting the previous explanation of the \n oscillations observed in the fluorescence measurements by the movement of wave packets and suggesting \n a scenario in which a coherent oscillation in the dark s2 state periodically partly repopulates the fluorescent s1 state .", "however , our experimental methods do not provide the time \n resolution to confirm this .", "the photodynamics \n of a unidirectional molecular motor has been \n studied by time - resolved ir spectroscopy in which both vibrational \n and electronic structures are probed . in combination with quantum \n mechanical calculations , the high structural sensitivity and real - time \n character of the technique allows us to understand fundamental aspects \n of the operation mechanism .", "the results show that conversion of photon \n energy into directed motion proceeds on the potential energy surfaces \n of two different electronic states , which is the more important as \n it implies that the conical intersection between the  dark  \n electronically excited state and the ground state determines the efficiency \n of the motor . to rationally tune", "the performance of photoinitiated \n unidirectional rotation thus involves a multicoupled - state optimization \n of nuclear motion ."], "abstract_text": ["<S> controlling the excited - state properties \n of light driven molecular \n machines is crucial to achieving high efficiency and directed functionality . \n a key challenge in achieving control lies in unravelling the complex \n photodynamics and especially in identifying the role played by dark \n states . </S>", "<S> here we use the structure sensitivity and high time resolution \n of uv - pump / ir - probe spectroscopy to build a detailed and comprehensive \n model of the structural evolution of light driven molecular rotors . \n </S>", "<S> the photodynamics of these chiral overcrowded alkene derivatives are \n determined by two close - lying excited electronic states . </S>", "<S> the potential \n energy landscape of these  bright  and  dark  \n </S>", "<S> states gives rise to a broad excited - state electronic absorption band \n over the entire mid - ir range that is probed for the first time and \n modeled by quantum mechanical calculations . </S>", "<S> the transient ir vibrational \n fingerprints observed in our studies allow for an unambiguous identification \n of the identity of the  dark  electronic excited state \n from which the photon s energy is converted into motion , and \n thereby pave the way for tuning the quantum yield of future molecular \n rotors based on this structural motif . </S>"], "labels": null, "section_names": ["INTRODUCTION", "METHODS", "RESULT AND DISCUSSION", "SUMMARY AND CONCLUSION"], "sections": [["one of the key challenges \n in the development of novel materials \n is the ability to tune and control their macroscopic physical and \n chemical properties on a molecular level by external stimuli .", "such a degree of control is challenging but crucial for a wide range \n of applications ranging from catalysis to pharmacological and medical \n applications .", "in photoactive materials light absorbed by chromophores can be converted \n into directed functionality , typically by means of photoinduced isomerization \n of nitrogen or carbon containing double bonds and pericylic reactions .", "indeed , e  z isomerization \n results in large amplitude changes in structure as well as chemical \n and physical properties .", "molecular motors based on overcrowded alkenes are particularly \n attractive due to the synthetically tunable properties , such as thermal \n stability , absorption spectra , etc . , and", "therefore are readily applied \n to a wide range of functional systems . however , although the thermally \n activated step in the unidirectional rotary cycle is well understood \n and is now largely predictable , the photochemical properties and especially \n photochemical quantum yields are less well understood .", "hence both \n from a fundamental perspective and in achieving the goal of complete \n molecular design , gaining insight into the excited - state properties \n of light driven molecular - sized machines is essential .", "the overcrowded alkene - based molecular \n rotary motors are composed \n of a central carbon  carbon double bond ( the axle ) embedded \n in an intrinsically chiral environment .", "the unidirectional rotational \n cycle starts with a photoinduced e  z isomerization around the central c = c bond , generating \n a metastable isomer p * ( figure 1 ) , which relaxes thermally to the stable isomer p , which in \n the case of a symmetric stator unit is identical to i. this isomer \n marks half of the rotation cycle and is the starting point for a second \n photoinduced isomerization step that leads again to a metastable isomer . \n", "thermally activated conformational isomerization of this isomer brings \n the system back to its initial state .", "overall , the rotor part has \n then performed a complete rotation with respect to the stator unit \n of the molecular motor .", "although the rotation includes both \n photochemical and thermal steps \n that involve different parts of the motor , the structural evolution \n is largely governed by the excited - state properties of the central \n c = c group , which , in turn , are dictated by the potential energy \n surfaces of the excited states that are accessed upon electronic excitation . \n despite the large number of theoretical and experimental studies that have focused on characterizing \n this process , there are several key questions outstanding including \n identification of the main structural coordinates and the role of \n various electronic states involved in the excited - state dynamics . \n", "time - resolved studies are essential to elucidate these dynamics but \n have thus far focused on probing the change in the electronic structure \n after photoexcitation through uv / vis absorption and fluorescence studies .", "these techniques offer \n a high time resolution but provide only indirect information as to \n how the structure evolves spatially over time . in the present contribution \n we show that this information can be obtained through a combination \n of picosecond time - resolved vibrational spectroscopy of the electronically \n excited states together with quantum - mechanical calculations ."], ["the setup for the time - resolved infrared measurements has been \n described in detail previously .", "mid - ir \n probe pulses were generated using an amplified ti : sapphire system \n ( spectra - physics hurricane , 600 j , repetition rate 1 khz ) pumping \n a commercial bbo - based opa ( spectra - physics opa-800c ) , the signal \n and idler output of which were difference - frequency - mixed in aggas2 .", "uv pump pulses ( 400 nm , 1 j ) were generated \n by doubling part of the output of the ti : sapphire laser .", "the diameter \n of the uv pump focus was 180200 m ; that of \n the ir probe focus , 150 m . the sample cell with caf2 windows spaced by 500 m was placed in the ir focus . \n to avoid accumulation of the thermally unstable conformer p * during \n the measurements ,", "the pump  probe delay time was scanned by mechanically \n adjusting the beam path of the uv pump .", "the temporal resolution of \n 200 fs has been determined from the full width at half - maximum of \n the pump probe cross - correlation function .", "the pump pulse was chopped \n at 500 hz , and transient spectra were obtained by subtracting nonpumped \n absorption spectra from the pumped absorption spectra , both of which \n were recorded by spectrally dispersing the probe pulses using an oriel \n ms260i spectrograph and measuring the frequency - dependent ir intensity \n using a 2  32 hgcdte detector array ( infrared associates ) .", "compound \n i was available from earlier studies ( see ref ( 44 ) ) . in all experiments", "we \n investigate a 10 mm solution of compound i in deuterated cyclohexane \n ( aldrich , 99.6 atom% d ) .", "for the investigation of the ground - state \n conformers ( including \n vertical excitation energies and forces acting at the franck ", "condon \n point ) quantum mechanical calculations were performed using the turbomole \n suite of programs .", "geometry optimization \n and normal - mode analysis were performed by ( time - dependent ) density \n functional theory ( ( td)-dft ) using the hybrid functional b3lyp . for the geometry optimization the d3 correction \n method of grimme et al .", "the cc - pvdz or aug - cc - pvdz basis \n sets were employed , respectively .", "post - hartree  fock calculations \n were performed on the basis of spin - component scaled second - order \n mller  plesset perturbation theory and the approximate coupled - cluster singles - and - doubles \n model cc2 .", "all perturbation method calculations were performed using \n the resolution of identity ( ri ) approximation and the efficient ricc2 module implemented \n in the turbomole package together with the standard auxiliary basis \n sets .", "the gaussian09 program package was employed to study the excited - state properties \n in more detail and in particular to optimize the structure of the \n dark - state species .", "geometry optimization and normal - mode analysis \n were performed in the framework of td - dft at the b3lyp / cc - pvdz level ."], ["the structural evolution from the \n initial state i to isomer p * \n ( figure 1 ) was considered first in terms of the spectral properties \n of the initial state and the first metastable product ( p * ) .", "the steady - state \n uv / vis absorption spectra of the two isomers are distinct , reflecting \n a change in the conjugated  system ( figure 1a ) .", "the ftir spectra of the two isomers ( figure 1c ) show that the \n carbon  carbon stretch and ring vibrations couple to a large \n degree as a consequence of the extended  system ; however , dft \n calculations enable us to assign the bands in the 15501650 \n cm range as being mainly associated with \n modes involving the central c = c double bond ( supporting information ) .", "the metastable isomer p * shows a \n shift of these modes to lower frequencies confirming a decrease in \n bond strength .", "comparison of the optimized geometries of the two isomers \n ( i ( p * ) , cc bond length 1.369 ( 1.377 )  ; cc = cc dihedral \n angle 14.1 ( 29.5 ) ) evidence the corresponding lower  \n bond character as well .", "unidirectional motion of the unidirectional \n motor starting from \n the thermally stable conformer ( i ) via the product of the photoinitiated \n step ( p * ) to the thermally stable conformer ( p ) ( optimized geometries \n on the b3lyp+d / aug - cc - pvdz level ) : ( a ) experimental and calculated \n ( light color and dotted line ) uv  vis spectra of conformer i \n and p * ; ( b ) molecular orbitals of the initial conformer ; ( c ) experimental \n and calculated ir spectra ( b3lyp+d / aug - cc - pvdz ) of the initial state \n ( blue ) and final state ( red ) . in both the measured uv / vis absorption \n and ftir spectra at the photostationary state ( pss ) , i and p * are \n present in a 25:75 ratio .", "time - resolved vibrational spectroscopy was employed to achieve the required temporal resolution as well \n as structural sensitivity to elucidate the structural evolution that \n follows photoexcitation with a short uv - pump pulse ( center wavelength \n 400 nm ) .", "the resulting structural evolution was followed by time - resolved \n differential absorption spectra in cyclohexane - d12 with a time resolution of 200 fs ( figure 2 ) .", "the transient ir spectra show , as expected , \n pronounced differences between the ir absorption spectrum of the molecule \n in the initial and excited states .", "a first point of note is that the \n spectrum does not evolve further after ca .", "30 ps , and the difference \n spectrum corresponds perfectly with the difference spectrum obtained \n upon continuous wave excitation ( figure 1 ) ; i.e. , the metastable isomer p * has formed \n fully within the 30 ps after excitation .", "ftir and time - resolved \n infrared spectra of the molecular motor \n in cyclohexane - d12 : ( a ) ftir spectra of \n the thermal stable conformer ( initial , solvent corrected , top ) , uv - ump / ir - probe \n transient infrared spectra ( uv = 400 nm ) ( middle ) , \n and difference ftir spectra between the two ground - state conformers \n of the molecular motor ; ( b ) kinetics of selected bands and corresponding \n results of the global fit ; ( c ) species associated spectra ( sas ) of \n species 1 and calculated ir spectra ( top ) and sas of the pss ( sp3 ) \n together with ftir spectra ( bottom ) .", "negative bands indicate ground - state \n bleaching , and positive bands , species created after excitation . the time - resolved ir spectra obtained \n within the first 30 ps exhibit \n several remarkable features .", "the first and most striking is a broad \n and unstructured absorption over the entire mid - ir range ( 12003000 \n cm ) .", "this absorption , which decays rapidly , is \n particularly prominent at early time delays ( < 5 ps ) .", "individual \n absorption bands with remarkably high integrated intensities in the \n 14001600 cm range are superimposed on \n the broad spectral feature .", "notably , these bands are significantly \n broader than the bleached bands in the transient spectra and bands \n in the ftir spectra .", "the solvent response and the dependence of the \n uv pump intensity ( supporting information ) confirm that these features are intrinsic to the compound and are \n not due to solvent or two - photon processes .", "the width and intensity \n of the broad excited - state absorption bands \n excludes that they arise from vibrational transitions .", "indeed , the \n absorbance is closer to that expected for electronic transitions , \n albeit these typically occur at much higher energies ( in the uv / vis \n range ) .", "the initial state of the compound \n ( i ) shows an absorption band at 390 nm ( figure 1 ) , which is reproduced well by b3lyp / cc - pvdz \n calculations in the gas phase ( 403 nm ; f = 0.428 ) . \n", "the s1  s0 transition has predominantly \n homo  lumo character ( figure 1 ) .", "in contrast to previous assignments , however , we find that this electronic transition \n is not localized on the stator but involves to a significant extent \n excitation from the bonding  orbital of the central double \n bond to the antibonding  * orbital ( figure 1 ) . as a consequence", ", the double bond character \n of this bond is reduced as well as the barrier to rotation around \n this bond . in line with calculations on ethylene and its derivatives , \n", "we find a dark state ( s2 ) associated with the homo1 \n  lumo excitation at slightly higher excitation energies that \n is not directly accessible from the ground state ( figure 1 , table 1 ) . because the homo1 orbital is localized \n on the aromatic stator unit , s2 has a significantly larger \n dipole moment than s1 ( 4.3 vs 2.2 d at the scs - cc2/cc - pvdz \n level ) .", "it should be emphasized that the description of the lower - lying \n electronic states in the compound studied here is thus entirely different \n from ethylene and its derivatives where , as discussed by salem and \n others , for torsion angles of around 90 the two low - lying electronic \n states have either a biradical or zwitterionic character depending \n on the occupation of the two central 2p - orbitals and require multireference \n methods for a proper description . in the present study , in contrast , \n we investigate the dynamics occurring at small torsional angles ( vide infra ) for which the employed single - reference methods \n not only have been validated .", "in addition \n we employ the results of multireference calculations on smaller motor \n molecules as reported by liu and morokuma to make a further comparison with our experimental results .", "for s0 the optimized \n structure on the b3lyp+d / aug - cc - pvdz level was used to calculate the \n vertical excitation energies . for vertical excitation", "they thus strongly suggest that the broad \n and structureless absorption feature observed in the time - resolved \n ir spectra is associated with the electronic s1  \n s2 transition .", "the presence of two close - lying states also \n explains why the induced absorption bands have such high intensities . \n", "our calculations show that the energy gap between the two states strongly \n depends on structural parameters such as the c = c bond length \n that are involved in the 14001600 cm normal \n modes . for these modes", "one therefore expects that their transition \n moments will be enhanced by vibronic coupling as is indeed observed . \n", "the conclusion that the excited - state dynamics of the molecular rotor \n should be described in a multistate model is further supported by \n recent multireference calculations on smaller rotor molecules that \n also find very small energy differences between the s1 and \n s2 states .", "the excited - state \n dynamics were elucidated further using a global \n analysis of the transient spectra , which shows that the spectra can \n be described well with two exponential decay terms with time constants \n of 1.53  0.03 and 12  1 ps .", "we note that our experiments \n have a time resolution of 150200 fs and therefore do not allow \n observation and confirmation of the ultrafast dynamics reported previously \n in fluorescence - upconversion studies .", "species - associated spectra ( sas ) ( supplementary \n figure 2 ) were derived by using a two - step sequential kinetic \n model .", "the sas of the first species shows an overall background \n signal \n with broad and intense features superimposed on it that indicate vibronic \n coupling .", "furthermore , the increase ( decrease ) of absorbance in the \n 1550 ( 1600 ) cm region indicates a substantial \n change in bond character of the central double bond ( figure 2c ) .", "this spectrum was related \n to the structure of this excited - state species using excited - state \n geometry optimizations .", " \n state without any structural constrains associated with the homo1 \n  lumo excitation leads to a stable minimum that can be reached \n from the franck  condon excited geometry by relatively small \n structural changes ( supplementary figure 4 ) , and an excited - state ir absorption spectrum that is in good agreement \n with the sas of species 1 ( figure 2 ) .", "it is notable that for a proper comparison with \n the experimental spectrum , the theoretically predicted stick spectrum \n needs to be convolved with 20 cm gaussian line \n shapes , suggesting that in the  dark  state a wide range \n of structures is sampled .", "our experiments and calculations thus demonstrate \n that excitation and excited - state decay involve two different electronic \n states . in earlier studies , time - resolved fluorescence and visible \n absorption data were interpreted in terms of a decay from a single , \n isolated electronic state decaying to the ground state after structural \n relaxation in the excited state .", "the present data show that the s1 state actually decays to an intermediate , dark electronic \n state , and from there to the ground state . in the sas of the \n second species ( supplementary \n figure", "2 ) the broad background is absent , and bands have widths \n comparable to those in the s0 state .", "the absence of the \n broad background indicates that this species is no longer in an electronically \n excited state but back in the ( vibrationally hot ) electronic ground \n state .", "indeed , the vibrational activity observed in the 1550 cm region follows closely the ir absorption spectrum \n of p*. in addition , repopulation of the original state ( i ) is observed \n also ( i.e. the quantum yield for isomerization is less than unity ) , and the sas of the second species contains contributions \n from both vibrationally hot ground - state i and metastable p * isomers . \n", "vibrational cooling of these isomers leads to the sas of the final \n species , which is in excellent agreement with the steady - state ftir \n difference spectrum of room - temperature i and p*. the present \n experimental and theoretical data allow a complete \n picture of the mechanism by which the molecular rotary motor undergoes \n isomerization after electronic excitation ( figure 3 ) .", "photon absorption brings the motor to \n a  bright  state from which it decays on an ultrafast \n time scale and within the time resolution of the present experiments \n to a different electronically excited  dark  state . \n in line with previous calculations on similar systems ,", "our calculations \n indicate that this is a barrierless process proceeding via a conical \n intersection .", "previously , it was assumed \n that structural evolution on the homo  lumo potential energy \n surface is responsible for the observed ultrafast decay of the fluorescence , \n and ( following commonly accepted models on photoisomerization of carbon ", "carbon \n double bonds ) that it primarily proceeds along torsional and pyramidalization \n coordinates . the present study , in contrast , leads to the conclusion \n that the fluorescence decay is mediated by the passage through the \n conical intersection of the homo  lumo and homo1  \n lumo potential energy surfaces . inspection of the structural changes \n that occur upon geometry relaxation to the minimum on the homo1 \n  lumo potential energy surface show that the central c = c \n bond length increases from 1.381 to 1.461  and that only relatively \n small changes occur in the torsional and pyramidalization coordinates . \n in combination with the forces that act on the molecule at the franck ", "condon \n excited  bright  state ( supplementary \n figure 3 ) , this suggests that motion along the c = c stretch \n coordinate needs to be taken into account as well for understanding \n the decay of the fluorescence .", "proposed multistate dynamics of the photoinduced \n unidirectional \n rotation of the molecular motor starting from conformer i to conformer \n p * ( projected into one dimension ) : dotted arrow , ultrafast process \n reported in previous work ; fast ( red arrow ) and slow process ( blue \n arrow ) observed in the present time - resolved infrared study . ( a ) calculated \n energy profile along the torsion coordinate showing how the energies \n of the electronic states change as the molecules evolves to conformer \n p*. ( b ) schematic representation of the proposed model .", "hence , it can be concluded that \n internal conversion to the electronic \n ground state takes place from the  dark  state .", "the \n decay rate for this process indicates that it is mediated by conical \n intersections between the two states as has indeed been proposed in \n previous theoretical studies .", "these studies \n concluded , however , as well that to reach these intersections a barrier \n needs to be overcome that results from the steric repulsion of the \n methyl group of the rotor unit with the hydrogen of the stator unit . \n", "previous time - resolved fluorescence measurements reported an ultrafast \n damping mode at 113 cm that was speculated to \n arise from motion along a coordinate involving inversion / pyramidalization \n although no direct support could be found for such an assignment .", "interestingly , our calculations find in the \n  dark  state a mode at 119 cm ( b3lyp / cc - pvdz ; \n not scaled , supporting information ) that \n involves motion of the methyl group with respect to the hydrogen of \n the stator unit .", "one might therefore wonder to what extent this mode \n is related to the oscillatory behavior of the fluorescence .", "our observed \n s2 decay constant of 1.5 ps matches the slow component \n of the overall fluorescence decay ( 0.91.5 ps ) .", "our calculation of the excitation energies of \n the  bright  and  dark  states along the \n 119 cm normal mode did not show a conical intersection \n between the two states , supporting the previous explanation of the \n oscillations observed in the fluorescence measurements by the movement of wave packets and suggesting \n a scenario in which a coherent oscillation in the dark s2 state periodically partly repopulates the fluorescent s1 state .", "however , our experimental methods do not provide the time \n resolution to confirm this ."], ["the photodynamics \n of a unidirectional molecular motor has been \n studied by time - resolved ir spectroscopy in which both vibrational \n and electronic structures are probed . in combination with quantum \n mechanical calculations , the high structural sensitivity and real - time \n character of the technique allows us to understand fundamental aspects \n of the operation mechanism .", "the results show that conversion of photon \n energy into directed motion proceeds on the potential energy surfaces \n of two different electronic states , which is the more important as \n it implies that the conical intersection between the  dark  \n electronically excited state and the ground state determines the efficiency \n of the motor . to rationally tune", "the performance of photoinitiated \n unidirectional rotation thus involves a multicoupled - state optimization \n of nuclear motion ."]]}
{"article_id": "PMC3360846", "article_text": ["the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig . ", "1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .", "1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig . ", "1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .", "these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .", "1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .", "the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .", "the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .", "lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .", "the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .", "however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .", "lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case", "gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .", "lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .", "also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .", "unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .", "one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .", "2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation", ". bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .", "the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .  ", "ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .", "they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .", "rajeswaran et al .   solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,", "there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .", "their crystal structures as well as the structure of the native ribosome   are available through the protein data bank ( pdb ) .", "these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .", "two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .", "theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .", "although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,", "no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .", "the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .", "these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .", "the methods applied in the calculations are described in the first section of the paper .", "we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds .", "four structures of clindamycin are available in the protein data bank   ( as of may 2011 ) .", "three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .", "the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome  clindamycin complexes of deinococcus radiodurans   ( pdb code 1jzx ) and haloarcula marismortui   ( pdb code 1yjn ) .", "therefore , based on the known x - ray structures of clindamycin ( fig . ", "2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .", ", we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig . ", "2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .", "the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .", "the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .", "no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called  exp - opt ,  were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .", "to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .", "therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,", "the solvent is described by a dielectric constant , which was set to 80 in our work .", "the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .", "the positions of these point charges were obtained from the x - ray structures of the ribosome ", "the coordinates of residues within 10  of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .", "next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .", "the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .", "nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin  spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .", "shielding constants were calculated using the b3lyp / aug - pcs-1   method and the giao routine [ 2630 ] .", "the chemical shifts of the i - th nuclei were calculated as \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ \\delta ( i ) = \\sigma_i(x ) - \\sigma_i(c ) , $ $ \\end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .", "spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .", "the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .", "the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .", "data were analyzed using gabedit , and the visualizations were carried out with vmd   and xdrawchem .", "the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 . the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol )", "are presented in fig .  5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,", "the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .", "to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .", "in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig .  6 ) . the numerical values for the electron density ( bcp ) and", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions", ". however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure", ", we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .", "as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and", "its chemical shift is lower for the b conformer than for the a conformer of clindamycin .", "the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer", ". however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum", "the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method", "in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 .", "the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol ) are presented in fig . ", "5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize", ", nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig . ", "the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table  4 .", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9  h39c152.3700.01460.0131cp7c17cl28 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .", "bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .", "however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as", "chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group ,", "the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer .", "however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer . this value is large enough to be measured experimentally.fig .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum", "we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .", "internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,", "we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin  spin coupling constants to the conformation of clindamycin .", "the two most stable conformers of clindamycin exhibit c = oh  o intramolecular hydrogen bonds . according to nbo and aim analyses ,", "the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water ."], "abstract_text": ["<S> lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens . </S>", "<S> this group of drugs inhibits the activity of the bacterial ribosome by binding to the 23s rna of the large ribosomal subunit and blocking protein synthesis . </S>", "<S> currently , three x - ray structures of the ribosome in complex with clindamycin are available in the protein data bank , which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin . in this work , we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23s rna . </S>", "<S> we studied three lincosamide antibiotics : clindamycin , lincomycin , and pirlimycin at the b3lyp level with the 6 - 31 g * * basis set . </S>", "<S> the focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties . </S>", "<S> for both functional conformers , we applied natural bond orbital ( nbo ) analysis and the atoms in molecules ( aim ) theory , and calculated the nmr parameters . </S>", "<S> based on the results obtained , we were able to show that the structure with the intramolecular hydrogen bond c = o  h  o is the most stable conformer of clindamycin . </S>", "<S> the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , was found to be the dominant factor influencing the high stability of this conformer.figuremolecular graph of more stable conformer of clindamycin . </S>"], "labels": null, "section_names": ["Introduction", "Computational details", "Results and discussion", "Conformational analysis", "Natural bond analysis", "Atoms in Molecules analysis", "NMR chemical shifts", "Conclusions"], "sections": [["the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig . ", "1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .", "1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig . ", "1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .", "these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .", "1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .", "the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .", "the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .", "lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .", "the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .", "however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .", "lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case", "gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .", "lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .", "also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .", "unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .", "one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .", "2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation", ". bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .", "the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .  ", "ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .", "they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .", "rajeswaran et al .   solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,", "there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .", "their crystal structures as well as the structure of the native ribosome   are available through the protein data bank ( pdb ) .", "these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .", "two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .", "theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .", "although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,", "no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .", "the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .", "these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .", "the methods applied in the calculations are described in the first section of the paper .", "we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds ."], ["four structures of clindamycin are available in the protein data bank   ( as of may 2011 ) .", "three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .", "the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome  clindamycin complexes of deinococcus radiodurans   ( pdb code 1jzx ) and haloarcula marismortui   ( pdb code 1yjn ) .", "therefore , based on the known x - ray structures of clindamycin ( fig . ", "2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .", "black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .", ", we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig . ", "2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .", "the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .", "the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .", "no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called  exp - opt ,  were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .", "to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .", "therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,", "the solvent is described by a dielectric constant , which was set to 80 in our work .", "the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .", "the positions of these point charges were obtained from the x - ray structures of the ribosome ", "the coordinates of residues within 10  of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .", "next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .", "the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .", "nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin  spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .", "shielding constants were calculated using the b3lyp / aug - pcs-1   method and the giao routine [ 2630 ] .", "the chemical shifts of the i - th nuclei were calculated as \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$ \\delta ( i ) = \\sigma_i(x ) - \\sigma_i(c ) , $ $ \\end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .", "spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .", "the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .", "the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .", "data were analyzed using gabedit , and the visualizations were carried out with vmd   and xdrawchem ."], ["the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 . the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol )", "are presented in fig .  5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,", "the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .", "to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .", "the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .", "in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig .  6 ) . the numerical values for the electron density ( bcp ) and", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions", ". however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .", "the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure", ", we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .", "as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and", "its chemical shift is lower for the b conformer than for the a conformer of clindamycin .", "the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer", ". however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum"], ["the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15  k calculated in the gas phase , taking solvent effects into account , are depicted in table  1 .", "the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table  2 .", "3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures  e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381  zpe323.26323.66332.14332.40305.59305.85  g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20  e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988  zpe323.29323.53332.19331.99305.35305.37  g8.050.00.230.05.320.0 ", "e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224  zpe323.87324.10332.77332.56305.92305.94  g6.580.00.430.05.400.0  e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514  zpe329.99329.71339.94339.56312.95312.74  g5.550.01.070.03.860.0table 2selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15  k ( g , in kcal / mol ) .", "top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .", "bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .", "two dihedrals were kept constant , d(c15c16n2c19 )  =  47.0 and d(c16n2c19c20 ) =  180.0 for conformer a , and d(c15c16n2c19 )  =  119.0 and d(c16n2c19c20 )  =  178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in  and degrees ) for the  exp - opt  structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .", "top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .", "our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o  h , n ", "h , c  h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .", "because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .", "first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0  kcal / mol .", "the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,", "no single conformer is favored at 298.15  k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .", "the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980  ) and the shortest ihb distance , r(c19o9 ", "h39c15 ) ihb distance in the less stable a conformer is 2.370  , and r(c15h39 )  =  0.970  is the most important component of the conformer s stability .", "the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .", "n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .", "the values of the starting dihedrals were different in the a and b conformers , as shown in fig .  2 .", "full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .", "the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig . ", "both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .", "table  1 confirms that solvation has a relatively small effect on the energy difference between conformers .", "the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6  kcal / mol according to the pcm and point - ion models , respectively.fig .", "4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method"], ["in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .", "natural population analysis   is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section", "analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .", "the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .", "such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .", "the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table  3 .", "the selected orbital interactions ( with a stabilizing effect of over 8  kcal / mol ) are presented in fig . ", "5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .", "5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .", "calculations were performed for  exp - opt  structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin  exp - opt  conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor ", "acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2", "is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .", "other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .", "additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .", "the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize", ", nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .", "therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer ."], ["the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .", "it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .", "topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .", "it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .", "however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .", "the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .", "a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .", "a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .", "in other words , generally , the laplacian of  is positive when  is locally reduced , and negative if it is locally concentrated .", "according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch  oc in the a conformer and oh  o = c in the b conformer , and unconventional h - bonds of type oh  x ( x  =  o , cl ) in both conformers ( fig . ", "the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table  4 .", "figure  7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .", "the general shape of the bcp curve is that of an exponential decay , as expected ( see fig . ", "7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .", "the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7  h41c172.3220.01550.0164cp6c19o9  h39c152.3700.01460.0131cp7c17cl28 ", "7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both ", "exp - opt  conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .", "the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .", "the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .", "bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .", "0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .", "however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .", "this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule ."], ["the experimental c nmr chemical shifts of clindamycin fall within the interval 1690  pm .", "the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as", "chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .", "the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185  ppm , (c  h ) close to 70  ppm .", "(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6  ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9 ", "intramolecular hydrogen bond more than the a conformer ( 185.1  ppm ) is stabilized by the c19o9 ", "these chemical shifts have also been shown to depend strongly on the local properties of the electron density .", "the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .", "finally , it is clear that (o ) can be classified into two groups . in the first group ,", "the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin  exp - opt  conformers optimized at the b3lyp/6 - 31 g level in vacuum .", "we used h ( tms )  =  31.50 , c ( tms )  =  182.20 , n ( ch3no2 )  =  167.89 , o ( ch3no2 )  =  389.95 ( + 605 ) , s ( cs2 )  =  1131.94 , and cl ( nacl )  =  151.02 as references", "the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .", "some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7  hz when moving from the a to the b conformer .", "however , more interesting is the jh37o9 sscc transmitted through the c = o  h  o ihb , which is equal to 5.3  hz in the b conformer . this value is large enough to be measured experimentally.fig .", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin ", "spin constants j ( in hz ) for both  exp - opt  conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum"], ["we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .", "internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,", "we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin  spin coupling constants to the conformation of clindamycin .", "the two most stable conformers of clindamycin exhibit c = oh  o intramolecular hydrogen bonds . according to nbo and aim analyses ,", "the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water ."]]}
{"article_id": "PMC4141698", "article_text": ["recently \n we reported on an analysis of the infrared spectrum of \n c2f4 . by observation \n of many combination and difference bands , including many from two c - containing isotopologues , and by comparison with mp2 and \n density - functional calculations , all 12 fundamentals could be assigned , \n although only five are ir active .", "the geometry \n was deduced from ro - vibrational analysis of strong ir active bands \n and by comparison with theoretical calculations .", "however , it is also \n instructive to correlate anharmonic constants with some peculiarities \n of the reactions of c2f4 .", "accordingly , in this \n work more anharmonic constants ( altogether 54 ) are derived from the \n spectra , and a complete set ( 78 ) is calculated .", "the earlier vibrational \n assignments of c2f4 were reported at a time \n when the unusual properties of this molecule were barely known .", "explained this property by positing that the electron attraction \n of fluorine leads to a preference of sp- over sp - type carbon : radical addition converts both \n c atoms from sp to sp hybridization , which \n stabilizes the primary product , lowers the transition state , and thus \n accelerates the reaction .", "alder reactions , for example , \n which was again explained by stabilization of radical intermediates .", "( borden also presented an alternative but related description based \n on delocalization of unpaired radicalic electrons to accepting  * \n orbitals of cf groups . )", "the preference \n for carbon sp hybridization can also be expected to lower \n the force constants for the pyramidalization ( wagging ) vibrations \n ( 7 , 8 ) and/or increase their anharmonicity . \n", "furthermore , torsion ( 4 ) should be facilitated by \n rehybridization and in fact quantum chemistry confirmed this for a \n 90 twist .", "another spectacular property of c2f4 is how \n readily it can dissociate thermally to two cf2 molecules .", "a c = c bond energy of only 2.95 ev is derived , less than that of a typical c  c single \n bond ( 4 ev ) .", "the low dissociation energy is caused by stabilization \n of the resulting two difluorocarbenes by back - bonding from nonbonding \n fluorine electrons to the empty carbon p - orbital .", "this also explains why cf2 has a singlet \n ground state which is more stable than the triplet ( by 2.458 ev , taken \n from the band origin of the phosphorescence spectrum ) . whereas in the previous work", ", only cc stretching is considered \n as the dissociation coordinate , it was argued in ref ( 16 ) that pyramidalization \n must also be involved .", "this idea is supported in the present work \n by the large value found for the anharmonic constant x18 coupling cc stretch with trans pyramidalization .", "the \n magnitude of x18 as compared to the much \n smaller x17 ( coupling cc stretch with \n cis pyramidalization ) also supports a valence - bond analysis of weak \n double bonds , which was reviewed in ref ( 19 ) .", "the samples in natural isotopic abundance were either taken from \n commercial c2f4 ( hoechst ) , that contained a \n trace of the cyclic dimer , or prepared from c2f4br2 + zn in a high - boiling \n alcohol ( diethylene glycol ) ; the comparison allowed for recognizing \n absorptions of impurities .", "a sample enriched in c to \n 50% was prepared from chclf2 by co2 laser isotope \n separation with enriched cf2 as the primary product .", "ir spectra of c2f4 in natural isotopic \n abundance \n and isotopically enriched were recorded between 370 and 4000 cm with 0.2 cm resolution by a perkinelmer \n ftir spectrometer in garching . at the \n synchrotron lab ,", "the isotopically enriched sample was measured at \n moderate 0.1 cm resolution in the far ir region \n from 100 to 600 cm ( capturing the band at 210 \n cm ) , and at high resolution for the ir active \n cf stretch bands , see ref ( 1 ) .", "the present work focuses on anharmonic constants of c2f4 . only where the constants were \n expected to be different for ccf4 and c2f4 ( e.g. , in the case of \n fermi resonance )", "theoretical values of the anharmonic constants xij were obtained from anharmonic \n vibrational \n frequency calculations performed using gaussian 03 revision c02 at the mp2 level using \n dunning - type correlation - consistent ( cc - pvtz ) orbitals .", "the molecular force field at this level of theory \n was shown in the previous study to reproduce the fundamental wavenumbers \n and the isotopic shifts of c2f4 very satisfactorily \n ( for details , see ref ( 1 ) ) , and hence was an appropriate choice for computing xij values .", "the calculations were performed for each of \n the c2f4 , ccf4 , and c2f4 isotopologues .", "the spectra were measured previously and the \n fundamentals extracted \n from them , using a limited number of anharmonic constants ( from combination \n and hot bands ) where necessary . for convenience \n table 1", "lists the fundamentals again . in this \n work we derived a large set of experimental anharmonic constants from \n combination bands and  in particular for the lower - wavenumber \n vibrations  from hot bands , and report a complete set of theoretically \n computed values .", "symmetry types refer to a d2h molecule with z axis along the cc bond and the y axis perpendicular \n to the plane , as used in ref ( 1 ) for easier comparison with the c2v symmetric ccf4 ; the more conventional designation with x axis along the cc bond and z axis perpendicular \n to the plane is given in parentheses .", "revised value ( instead of 210 cm ) , based on a more definite \n assignment of a hot - band structure and x10,10 , see supporting information .", "band origins from high - resolution \n spectra ; low - resolution values are 1338.4 \n and 1187.0 .", "upright : \n experimental values . \n in parentheses : values calculated by mp2/pvtz . where two values or \n a range are given for the experimental data", ", it may indicate ( a ) a \n dependence of the effective xij on the involved levels due to perturbations ( fermi resonances , \n section 4.4 ) or ( b ) an uncertainty due to error \n limits ( e.g. , where broad bands are involved ) or an uncertain assignment . \n", "a preferred value is indicated by bold face . for further details on \n the derivation of each xij and some isotopic data ,", "the anharmonic constants xij were calculated from observed \n combination and difference bands \n by assuming that anharmonic shifts are additive : for binary combination \n bands ( i  j ) and for overtones \n we used1and2with obvious extensions for more complicated \n combinations .", "( the xij values are mostly negative , as usual . ) the left - hand side of these \n equations should be read as  wavenumber of ( name )  . for hot bands ( satellite transitions near i that originate from a thermally populated \n lower level j ) we used3again \n with obvious extensions .", "one can often \n observe sequences of equidistant q branches , corresponding to hot \n bands starting from higher levels mj , or combination of such sequences ( from mj + nk ) .", "equation 3 implies that the distance of a ( first ) hot band from the fundamental \n directly indicates the corresponding anharmonic constant .", "combination \n bands allow for a direct assignment , whereas hot bands must be identified \n by their intensity ( controlled by the boltzmann factor ) . in a number \n of cases", "the supporting information ( si ) compiles the sources for each xij in table s2 , whereas table s1 lists the observed \n bands ( with hot bands ) and their isotopic shifts .", "two examples of \n hot bands , whose shifts are dominated by the large x18 anharmonicity , are shown in figure 1 .", "absorbance spectra demonstrating the large x18 derived shifts for hot - bands originating in 8 .", "the spectra were recorded \n in the garching laboratory , with pressure and path length of 2 bar \n and 10 cm ( for the lower trace of each panel ) , and 140 mbar and 4 \n m ( upper trace ) , respectively . for levels involving more than two quanta of vibration , anharmonic \n shifts", "this helped to decide between different \n assignments of combination bands in a few cases where the harmonic \n sums were similar to each other .", "however , such additivity need not \n apply when the levels involved are subject to fermi resonance , producing \n shifts that depend on the separation of nearby interacting levels . \n", "we use eqs 13 to \n obtain the experimental values , and hence , table 2 reports phenomenological xij , some of which show a spread in the values suggestive of fermi \n resonances ( in particular x18 , x26 , and x77 ) .", "fermi \n resonances can also be recognized if the apparent xij or the hot - band structure depends \n on the isotopologue . for c2f4 ,", "fermi resonances \n were identified ( see section 4.4 and si ) between:5 and 2+6 ( discussed already in ref ( 1)),1 and 5+6 ( indicated by the hot - band structure \n of 1-combinations , which are different in the mixed \n isotopologue),2 and 27 ( causing irregularities \n in overtone levels of 7 , also contributing to the \n large magnitude of x27 and x77).2 and 23 , identified from the \n mp2 calculations .", "5 and 2+6 ( discussed already in ref ( 1 ) ) , 1 and 5+6 ( indicated by the hot - band structure \n of 1-combinations , which are different in the mixed \n isotopologue ) , 2 and 27 ( causing irregularities \n in overtone levels of 7 , also contributing to the \n large magnitude of x27 and x77 ) . 2 and 23 , identified from the \n mp2 calculations .", "the mp2/cc - pvtz calculations \n reproduced the observed wavenumbers \n and isotopic shifts very well . supporting \n the overall vibrational analysis", "exceptions are 15 ( x22 , x28 , x29 , x2,10 , x2,11 , x36 , x3,10 , x45 , x59 , x5,11 , x66 , x68 , x77 , x88 , x10,11 ) out of 54 cases where an observed value is available \n for comparison . some of these will be discussed below .", "as already \n described in the introduction , the peculiar \n reactions of c2f4 have to do \n with cc stretching and carbon pyramidalization . the discussion will \n focus on these coordinates ( sections 4.14.3 ) . for \n a planar geometry , \n", "cc dissociation correlates c2h4 and c2f4 with a pair of carbenes in their lowest triplet states ( figure 2a ) . because \n the energy of forming these products is practically the same for the \n two molecules", ", one could also expect that the potential energy curves \n coincide and the cc stretch force constants are equal .", "in fact , already \n early force - field calculations showed no substantial difference ( 889 \n n / m for c2f4 and \n 940", "furthermore , \n the short cc distance ( 132 pm , which is 1 pm shorter than in ethylene ) and the high cc stretch wavenumber ( 1873.8 cm , compared to 1625.4 cm   for ethylene ) do not point to a weakened bond . \n as early as 1965 , the low dissociation energy of c2f4 was attributed to switching over to a channel producing two \n singlet carbenes and to the stabilization ( by 2.458 ev , see introduction ) of singlet cf2 by back bonding , so that this channel is energetically favored by 4.9 ev .", "this path hence leads over an avoided \n crossing of potential surfaces ( figure 2a ) . \n", "if the avoidance is strong , one could expect an influence at least \n on the anharmonicity x11 ( 1 is dominated by cc stretching ) , even if not on the force \n constant at the bottom of the potential energy well .", "table 2 shows that the magnitude of x11 ( calculated 7.8 cm ) is not unusually \n large for such a high - frequency vibration , though noticeably larger \n than for ethylene ( x22 = 2.3 cm  ) . that the increase \n in magnitude is only moderate", "can be taken as a sign that the avoidance \n along this coordinate is not very strong .", "hence , the back reaction \n ( recombination ) will have a high barrier in planar geometry . whereas \n the measured recombination barrier is not , in fact , zero , it is only \n minor ( 9 kj / mol = 0.09 ev  ) .", "so , \n a lower - energy path must lead around the barrier , out of the drawing \n plane of figure 2a , that is , with distortion \n different from cc stretch .", "potentials for ground - state cc dissociation \n of c2f4 , leading over an avoided crossing to \n two singlet carbenes . \n in ( a ) it is assumed that the one - sided pyramidalization ( q7 + q8 ) begins only after overcoming the barrier , whereas \n ( b ) shows the preferred path , where this distortion is already fully \n developed on the barrier and is preceded by a trans pyramidalization \n ( q8 ) , while the cc distance is steadily increasing .", "it was argued in ref ( 16 ) that recombination of \n two cf2 molecules should \n be easiest if one of them points with its filled n orbital \n to the empty p orbital of the other .", "hence , the dissociation \n path will change from pure cc stretch to include also pyramidalization \n ( figure 2b ) , and the potential should be lowered \n in energy in the direction of q7 ( the coordinate of 7 ) and/or q8 . in turn", "in fact x18 has an unusually large magnitude , although x17 is in the normal range ( table 2 ) .", "( the magnitude of x18 is not \n caused by secondary effects such as fermi resonance , as we shall see \n in section 4.4.3 . )", "we can conclude that on \n stretching the cc bond the molecule first bends toward trans pyramidalization \n ( q8 ) ( figure 2b ) .", "the one - sided \n nonplanar bending ( figure 2 ) is a superposition \n of q7 and q8 .", "the reason why q8 is \n active earlier than q7 can be understood by a valence - bond \n model for weak double bonds . in this model", "several symmetric arrangements \n of carbenes ( a  c \n of scheme 2 ) were considered : whereas with \n two triplet carbenes all arrangements ( a  d ) lead to bonding \n interaction , with two singlet carbenes bonding occurs only in the \n trans - bent case ( c ) ; this corresponds to a double donor ", "one can add that the cis - bent case with singlet carbenes would \n lead only to antibonding ( scheme 2d ) .", "malrieu \n and trinquier derived a criterion for trans - bending ( deriving even \n the angle ) by comparing the standard double - bond energy with the singlet ", "their findings were confirmed by many examples , in particular with \n heavier - element homologues .", "we can apply \n this rule not only to the equilibrium geometry of c2f4 but also to its dissociation path , if we consider in the \n comparison the double - bond energy that is reduced on extension of \n this bond .", "the rule thus predicts that on cc extension c2f4 is distorted from planar geometry to a trans - bent structure , \n and this rationalizes the large magnitude of x18 . in the triplet case ,", "the interaction \n can be bonding in all arrangements . in the singlet case , ( a )", "is antibonding , \n ( b ) nonbonding , ( c ) bonding , ( d ) antibonding .", "more recently , borisov et al . found evidence \n for trans pyramidalization \n on extension of the cc bond also from molecular orbital calculation \n ( at high level : mller  plesset perturbation theory of \n second , third and fourth order ) : the \n authors found that the onset is relatively sudden on cc extension \n to 142 pm and that the distortion lowers the energy appreciably .", "( the \n large x18 anharmonicity is an indication \n that this pyramidalization already begins not far from the s0 minimum .", "in fact , a cc stretch by 10 pm  from 132 to 142 pm ", "is \n just about the vibrational amplitude in the 1 = \n 1 level . )", "although borisov et al . did not find an energy minimum with \n such a distortion , they suggested that it plays a crucial role in \n the low - energy real intermediate found by buravtsev , kolbanovskii \n et al . in thermal reactions of c2f4 and recombination \n of cf2 ( see ref ( 31 ) and literature quoted therein and section 4.3 ) .", "one could suppose that in order to lower the activation \n energy \n for dissociation the only necessary deviation from simple cc stretching \n is distortion toward the 8 coordinate ( q8 ) .", "however , there is experimental \n evidence that the asymmetric pyramidalization ( corresponding to superposition \n of q7 and q8 as in figure 2b ) is already crucial in the transition state : the pre - exponential \n factor found in the rate of thermal c2f4 dissociation \n ( 2.8  10 s  ) is clearly larger than would be typical for \n a simple bond splitting ( 10 s ) .", "this \n corresponds to a raised entropy of activation , such as that which \n happens if an additional degree of freedom is activated in the transition \n state .", "the most plausible candidate is free internal rotation , as \n it can be expected in the asymmetric structure with a single dative \n bond but not in symmetric geometry with double donor  acceptor \n bond .", "this picture is also consistent with a stepwise cleavage of \n the double donator  acceptor bond on c2f4 dissociation and its stepwise formation on cf2 recombination .", "it is worth noting that the molecule with one - sided pyramidalization \n has an electron distribution ( figure 2b ) that \n is zwitterionic and correlates in planar geometry with the 2ag state with two  electrons excited to  * , as \n already pointed out in ref ( 16 ) .", "a fully analogous dissociation path can also be \n expected for the \n other systems considered by malrieu and trinquier .", "in fact , according \n to ref ( 32 ) ground - state \n ( ir induced ) dissociation of diazomethane ( h2cnn ) follows \n a path with a pyramidalized methylene group to produce n2 + singlet ch2 ; the out - of - plane deformation was experimentally \n confirmed . in the backward reaction ,", "n2 points with its \n n electron pair to the empty p orbital of the singlet \n methylene ( see refs ( 32 and 33 ) for a calculated potential in two dimensions , which would look similar \n for c2f4 ) .", "hence , \n the out - of - plane deformation on dissociation of c2f4 is not an isolated phenomenon . applying \n their energetic criterion to the equilibrium geometry of c2f4 , trinquier and malrieu found that this molecule is \n not far from the limit , where it would become nonplanar , and suggested \n that the 8 force constant may be significantly lower \n than that of ethylene .", "this is in fact \n confirmed in the following way : analytical expressions for a valence \n force field model for planar x2y4 were given \n by herzberg . besides the masses mx and my , bond lengths l1 ( for xx ) and l2 ( xy ) and the angle  ( half of yxy ) , both 7 and 8 only depend on the force constant for pyramidalization \n ( k/l2 ) . with equilibrium distances and angles from ref ( 1 ) and i = ( 2ci )", "one derives this force constant from \n 7 and 8 ( with identical results \n for c2f4 and c2f4):4both values should be identical \n in this valence - force field model .", "in fact for ethylene , the value \n is 22.9 n / m , whether derived from 7 or 8 .", "the pyramidalization force constant \n derived from 7 of c2f4 is \n slightly higher than in ethylene ( by 17% ) , but the same quantity derived \n from 8 is only 49% of the ethylene value .", "( of course , \n these values are not quantitatively meaningful , as the discrepancy \n highlights that a pure valence - force field model is not sufficient \n for wagging of c2f4 . )", "so in fact the restoring \n force for the 8 vibration is significantly smaller \n than in ethylene and than that expected on the basis of the same pyramidalization \n force constant as for 7 .", "this is consistent with \n the prediction of the valence - bond model favoring the trans - bent geometry .", "the slightly raised force constant for 7 could \n be due to repulsive nonbonding interaction in cis pyramidalization . \n", "such a geometry would be involved in the hypothetical transition state \n of the diels ", "alder addition of c2f4 to \n butadiene , a reaction that has not been observed .", "it was argued that \n an increased energy associated with this distortion might contribute \n to inhibiting the reaction .", "however , an \n increase in the force constant of only 17% is hardly sufficient to \n support this explanation , and the other effects discussed in ref ( 7 ) , in particular the competing \n fast radical reactions , are probably more important . applying \n again the force - field formulas of herzberg ,", "the force constant for torsion , k/l2 , is found to be 30.3 \n n / m for both ethylene and tetrafluoroethylene .", "that is , near the potential \n minimum the c = c bond in c2f4 behaves \n just like a normal double bond .", "consequently , the torsional barrier \n at a 90 twist ( which is a measure of the  bond strength ) \n would be identical for both molecules ( 2.8 ev ) if the two c  cf2 groups remained planar ; this was confirmed by a high - level \n calculation .", "however , pyramidalization \n lowers this barrier by 0.57 ev ( at mp2 level , consistent with ref ( 35 ) ) .", "if this stabilization is activated early along q4 , \n it could give rise to substantially negative values for x44 and especially for x47 and x48 .", "however , x44 and x47 are actually slightly positive \n ( 0.8 and 0.3 cm ) and x48 has no unusual magnitude ( 0.7 to 1.05 cm ) .", "evidently , the stabilizing effect of pyramidalization becomes \n effective only at high torsional angles .", "the corresponding values \n for ethylene are 2.4 , 8.9 , and 7.2 cm , respectively .", "the nondiagonal anharmonicities xi4 and x4j are \n generally smaller in magnitude than in ethylene . but this may simply \n have to do with the relative vibrational amplitudes , which are much \n larger for torsion of ch2 than of cf2 due to \n the relative masses . in the twisted - pyramidalized structure \n of c2f4 ,", "the triplet ( t1 ) is only \n 0.013 ev lower than the singlet \n ( s0 ) .", "it \n can therefore act as a real intermediate and undergo radical - type \n reactions , further assisted by its low energy that is calculated at \n 2.1 ev .", "this has been a widespread interpretation \n of some peculiarities of c2f4 reactions ( see , \n for example ref ( 7 ) ) .", "however , buravtsev , kolbanovskii et al . found in shock - tube experiments \n ( with c2f4 diluted by ar and adiabatically compressed ) \n an intermediate at much lower energy ( 0.8 to 1 ev ) with uv and visible \n absorption ( 235 and 500 nm ) ( see refs ( 31,36 , and 37 ) and the \n literature quoted there ) .", "they supposed it to be a singlet diradicaloid \n of c2f4 with a geometry corresponding not to \n an energy minimum but to a point on a slope of the potential ( see , \n for example ref ( 31 ) ) .", "however , as these points are not stationary there are too many \n ( nearly ) isoenergetic locations with shifted electronic transitions , \n so that the electronic absorption would be smeared out .", "it was therefore \n suggested that the observed intermediate \n is another low - energy isomer , tetrafluoro - ethylidene cf3cf ( electronic absorption in an ar matrix : ) .", "however , its energy ( 1.9 ev  ) seems to be nearly as high as that of the twisted - pyramidalized \n triplet .", "( in one study the twisted - pyramidalized c2f4 triplet is calculated at 0.90 ev , but computational details are not given , and previous theoretical \n results are not discussed . )", "some more anharmonicities of table 1 have unusually large magnitudes or show a range \n of values and/or deviate from prediction . in many of them", "fermi resonances \n play a role , as will be explained here . in section4.4.3 , however , it is shown that x18 is hardly influenced by such effects and its large magnitude mainly \n results from the overall shape of the potential energy surface .", "fermi resonances mix two nearby levels of the \n same symmetry , which differ by three units in the quantum numbers . \n", "the fermi - induced \n repulsion of the levels depends in a nonlinear way on both the magnitude \n of the fermi interaction parameter and on the original ( i.e. , unperturbed ) \n energetic distance of the levels .", "this separation will , in general , \n change ( a ) on isotopic substitution and ( b ) on adding a quantum of \n another vibration ( i.e. , in analogous combination bands ; similarly \n also in higher overtones , in which case a quantum number dependence \n must also be taken into account ) .", "another method of characterizing fermi resonance interactions relies \n on intensity borrowing caused by the mixing of levels . according \n to the mp2 calculations , the great majority of xij change by 1 cm on isotopic substitution", "the few exceptions are \n listed in table 3 and are compared to experimental \n values . identified ( fr13 ) and", "the interpretation in the line  influenced by  \n is based on the assumption that an xij is affected by such a resonance if at least one \n of the levels i and j is involved in the fermi interaction .", "the table shows that \n the calculated isotopic shift is sometimes \n much larger than in the experiment , for instance x26 , x27 .", "also x56 for c2f4 is probably \n too large , as the two observed bands 5+6+11 and 5+6+12 are close to their harmonic positions \n ( table s1 in si ) .", "it seems that at the \n present level of theory , the calculation does not reliably reproduce \n the fermi resonance induced shifts , at least the stronger ones .", "this \n is not surprising given the highly sensitive and nonlinear dependence \n of fermi shift on the  unperturbed  level separations . \n", "the same deficiency will also affect those xij connected with at least one level ( or a \n combination thereof ) involved in a resonance listed above ( examples \n in figure 3a ) .", "in fact , 10 of the 15 deviations \n listed at the end of section 3 can be attributed \n in this way to the resonances fr1 and fr2 with possible contributions \n of fr3 .", "the exceptions are x36 , x3,10 , x66 , x68 , and x88 . in \n all 42 other cases", "it is worthwhile to consider \n the two strongest fermi resonances , \n fr1 ( 2/27 ) and fr2 ( 5/2+6 ) , in c2f4 in some detail .", "the effect of these resonances \n is summarized in figure 3 . some levels involved \n in fermi resonances and observed transitions . \n", "solid arrows : ir , broken arrows : raman ; double arrows ( with interaction matrix element ) indicate repulsion \n between level pairs .", "the anharmonic shifts involving x77 are only nominal , because the fermi perturbation is \n strong :  6x77  = 18.1 is \n less than 3 times ", "(= 8.2 ) and  4x77  is only \n 9.9 . ( but  2x77  +  4x77  =  6x77  , as is obvious from the level scheme , and the raman \n anharmonic shifts practically coincide with those from the ir spectra . ) \n ( b ) three - level fermi resonance 5/2+6/27+6 and \n their combinations with 9 for c2f4 and c2f4 . \n", "the indicated level energies are from the spectra except those in \n parentheses , which are calculated .", "the broken horizontal lines are \n the estimated unperturbed positions ( see the si ) , and the signed numbers are the calculated shifts .", "the 16.4 cm magnitude of the interaction term ( with its expected dependence \n on quantum numbers ) has been derived \n from band shifts and intensities for a series of levels built on 2/27 for all three isotopologues .", "bands for \n 27+x ( x = 9 , 11 ) borrow their entire intensity from 2+x . the detailed analysis \n is provided in si .", "it \n is also evident that x77 , x27 and x22 are affected by \n the fermi resonance ( and probably other x2i , if the level separations differ ) and that x77 is level dependent because of the varying \n separation .", "the fermi term is almost entirely responsible for the \n + 8.2 cm shift of 27 for c2f4 and those of the heavier isotopologues \n ( table 3 under fr1 ) . in the latter", "the fermi - induced \n shift is increased , because the levels 2 and 27 ( before perturbation ) are closer together than in c2f4 and are practically degenerate in c2f4 .", "the success of these calculations \n shows that the additional mixing of 2 with 23 ( fr3 ) is apparently a weak perturbation at most .", "the \n fermi resonance fr2 between 5 and 2+6 was qualitatively discussed in ref ( 1 ) .", "a more complete understanding \n of the resonance shifts and pattern of intensity borrowing requires \n the three - level scheme shown in figure 3b ; \n this figure also shows combinations with 9 , as they \n are all ir active and were directly observed .", "analysis based on this \n model has enabled the magnitude of the fermi term to be determined \n as 11 cm ; the fr1 term of 16.4 cm was used without change .", "significantly , the model predicts varying \n fr shifts that even differ in sign ( originating from the different \n repulsions in figure 3b ) and can thus account \n for the unusual isotopic shifts observed between c2f4 and c2f4 . \n", "it explains a substantial deviation of the observed isotopic shift \n 5 = 51.9 from the value of 5 = 43.1 \n expected from the teller  redlich product rule , which is valid \n for harmonic oscillators .", "additionally \n the relative intensities of combination bands and their isotopic dependence \n are well predicted .", "it is also remarkable that according to the model \n the largest part of the anharmonicity x26 is due to fermi resonance .", "as described in ref ( 1 ) , the lower symmetry in ccf4 leads to strong mixing of 5 with 9 ( which are nearly degenerate in c2f4 ) .", "the two levels repel each other \n ( even in the harmonic approximation ) and give rise to two infrared - active \n transitions .", "it is sometimes difficult to disentangle them and their \n combination bands from the spectra .", "if the large magnitude of some \n anharmonicities is caused by fermi resonance , a crucial question is \n whether this might also be the case for x18 .", "if so , the conspicuous value of x18 could be largely a matter of the character of the resonance and \n less a consequence of global features of the potential for dissociation , \n which we considered in section 4.1 .", "in fact , \n weak perturbations of the 1 level and its combinations \n by the 1/5+6 fermi resonance are indicated by the slight x18 dependence on the level and on isotopic substitution and \n the change of the hot - band structure of 1 combinations \n in ccf4 , as explained in the si .", "a quantitative estimate of the strength \n of the 1/5+6 interaction can be deduced from the intensities of 5+6 combinations relative to those of the corresponding \n 1 combinations , assuming that the former borrow \n their full strength from the latter .", "the 5+6+12 band ( 2444.3 cm ) \n has 12% the strength of the 1+12 band ( 2428.7 cm ) , see figure 1a .", "similarly , the 5+6+11 shoulder ( 3072 cm ) is < 1% as intense as 1+11 ( 3056.2 cm ) and clearly visible only with longer \n path length than shown in figure 1b .", "from both \n observations , one can estimate a fermi term ( w ) of \n no more than 2.5 cm , causing a shift ( and \n consequent change in x18 ) of < 0.5 cm .", "this is actually an upper bound , because such weak \n bands can also be expected without intensity borrowing .", "hence , fermi \n resonance affects the magnitude of x18 only by a little ( by < 3% ) , and we can maintain the conclusions \n of section 4.1 on the shape of the potential .", "a large set of anharmonic \n constants of c2f4 was derived from spectroscopic \n data , and a complete set was calculated . \n", "most of the calculated and experimental values agree very well , and \n nearly all deviations can be attributed to fermi resonances . the values \n were used to interpret peculiarities of the potential and reactions \n of this molecule .", "the facile thermal cc dissociation of c2f4 is due to curve crossing of the ground state \n ( 1ag ) with \n the two - electron excited zwitterionic 2ag state .", "the crossing \n barely alters the properties of the potential curve ( cut purely as \n a function of cc stretch ) near the equilibrium geometry ; the force \n constant for cc stretching ( 1 in c2f4 , 2 in c2h4 ) is nearly \n identical for the two molecules .", "only a slightly larger magnitude \n of the anharmonicity x11 compared to the \n corresponding x22 of ethylene provides \n a hint of a weakly avoided crossing in planar geometry of c2f4", ". however , the large magnitude of x18 leads us to conclude that the dissociation path bends \n early toward \n q8 , i.e. to trans pyramidalization of the two carbon atoms \n ( figure 2b ) .", "this is rationalized by a valence - bond \n model , which describes the stretched cc bond as a double donor  acceptor \n bond ( scheme 2c ) .", "it is also consistent with \n the tendency of fluorine to induce sp hybridization at \n carbon , although this preference alone would also allow cis pyramidalization , \n q7 , which is not involved early in dissociation .", "on further \n cc stretching , one of the two donor  acceptor bonds breaks , \n and the pyramidalization becomes fully one - sided already in the transition \n state ( figure 2b ) . in this way , free internal \n rotation of the cf2 groups becomes possible , providing \n an additional degree of freedom that rationalizes the high pre - exponential \n factor in the rate constant of dissociation .", "this structure with perpendicular \n arrangement of the two cf2 groups appears very plausible \n for the back reaction .", "thus , one cf2 group approaches with \n its filled n orbital the empty p orbital of the other \n cf2 group ( figure 2b ) .", "the easy \n trans pyramidalization on minor cc stretching is probably also the \n reason that the corresponding 8 force constant is \n lowered , although all other force constants are similar to those in \n ethylene .", "the preference of fluorine for sp hybridization \n at \n carbon has been invoked to explain not only the easy radical addition \n and other reactions , but also the low - lying , twisted - pyramidalized \n triplet , which is nearly isoenergetic with the singlet that has a \n saddle point there .", "this is indicated by x44 , x47 , and x48 , which have no unusual values .", "very recently , \n high - level theoretical studies investigated \n electronic ground and excited states of c2f4 and their dependence on some coordinates , mainly \n to discover the fate of this molecule after electronic excitation \n and make comparisons with a corresponding time - resolved experiment \n ( ref ( 16 ) ) . for the \n ground state , the stabilizing effect of pyramidalization in the twisted \n molecule was confirmed .", "interestingly , the excited molecule enters \n the s1/s0 conical intersection from the 2ag state , where the molecule has a large one - sided pyramidalization , \n a 90 cc twist and a cc bond length ( 1.51  ) similar to \n that of a single bond . in the present work ,", "the transition state for \n s0 dissociation was said to result from avoided crossing \n of the 2ag and 1ag ( i.e. , s0 ) states , \n also with large one - sided pyramidalization but at longer cc distances \n and perhaps without twist", ". it would be interesting to check whether \n these geometrical differences are large enough to generate an excited - state \n barrier , because evidence was found in ref ( 16 ) that cc dissociation does not begin in the excited \n state ."], "abstract_text": ["<S> compared to ethylene and its nonfluorinated \n derivatives , c2f4 is peculiar in many reactions . \n </S>", "<S> it very easily \n adds to radicals and prefers formation of four - membered rings over \n diels  alder reactions . </S>", "<S> this has been rationalized by the preference \n of fluorine for carbon sp3 hybridization , which is possible \n on opening of the double bond . </S>", "<S> another property , the thermal dissociation \n of the c = c bond , has been explained by the stabilization of \n the product ( cf2 ) by back - bonding . </S>", "<S> here , it is attempted \n to correlate such properties with vibrational constants , in particular \n for c = c stretching and twisting and for carbon pyramidalization . \n </S>", "<S> the only force constant found to be lowered compared to ethylene is \n that for trans pyramidalization ( 8 ) , and cc bond \n softening on 8 distortion is indicated by the conspicuously \n large magnitude of anharmonic constant , x18 . </S>", "<S> both observations can be rationalized by a valence - bond model that \n predicts a trans bent structure on weakening the cc bond . </S>", "<S> conclusions \n are drawn about the dissociation path and peculiarities of the potential . \n </S>", "<S> other anharmonicities , both experimental and calculated and some in 12c13cf4 and 13c2f4 , are also discussed . in particular </S>", "<S> some strong fermi \n resonances are identified and their effects accounted for . </S>"], "labels": null, "section_names": ["Introduction", "Methods", "Results", "Discussion", "Conclusion"], "sections": [["recently \n we reported on an analysis of the infrared spectrum of \n c2f4 . by observation \n of many combination and difference bands , including many from two c - containing isotopologues , and by comparison with mp2 and \n density - functional calculations , all 12 fundamentals could be assigned , \n although only five are ir active .", "the geometry \n was deduced from ro - vibrational analysis of strong ir active bands \n and by comparison with theoretical calculations .", "however , it is also \n instructive to correlate anharmonic constants with some peculiarities \n of the reactions of c2f4 .", "accordingly , in this \n work more anharmonic constants ( altogether 54 ) are derived from the \n spectra , and a complete set ( 78 ) is calculated .", "the earlier vibrational \n assignments of c2f4 were reported at a time \n when the unusual properties of this molecule were barely known .", "explained this property by positing that the electron attraction \n of fluorine leads to a preference of sp- over sp - type carbon : radical addition converts both \n c atoms from sp to sp hybridization , which \n stabilizes the primary product , lowers the transition state , and thus \n accelerates the reaction .", "alder reactions , for example , \n which was again explained by stabilization of radical intermediates .", "( borden also presented an alternative but related description based \n on delocalization of unpaired radicalic electrons to accepting  * \n orbitals of cf groups . )", "the preference \n for carbon sp hybridization can also be expected to lower \n the force constants for the pyramidalization ( wagging ) vibrations \n ( 7 , 8 ) and/or increase their anharmonicity . \n", "furthermore , torsion ( 4 ) should be facilitated by \n rehybridization and in fact quantum chemistry confirmed this for a \n 90 twist .", "another spectacular property of c2f4 is how \n readily it can dissociate thermally to two cf2 molecules .", "a c = c bond energy of only 2.95 ev is derived , less than that of a typical c  c single \n bond ( 4 ev ) .", "the low dissociation energy is caused by stabilization \n of the resulting two difluorocarbenes by back - bonding from nonbonding \n fluorine electrons to the empty carbon p - orbital .", "this also explains why cf2 has a singlet \n ground state which is more stable than the triplet ( by 2.458 ev , taken \n from the band origin of the phosphorescence spectrum ) . whereas in the previous work", ", only cc stretching is considered \n as the dissociation coordinate , it was argued in ref ( 16 ) that pyramidalization \n must also be involved .", "this idea is supported in the present work \n by the large value found for the anharmonic constant x18 coupling cc stretch with trans pyramidalization .", "the \n magnitude of x18 as compared to the much \n smaller x17 ( coupling cc stretch with \n cis pyramidalization ) also supports a valence - bond analysis of weak \n double bonds , which was reviewed in ref ( 19 ) ."], ["the samples in natural isotopic abundance were either taken from \n commercial c2f4 ( hoechst ) , that contained a \n trace of the cyclic dimer , or prepared from c2f4br2 + zn in a high - boiling \n alcohol ( diethylene glycol ) ; the comparison allowed for recognizing \n absorptions of impurities .", "a sample enriched in c to \n 50% was prepared from chclf2 by co2 laser isotope \n separation with enriched cf2 as the primary product .", "ir spectra of c2f4 in natural isotopic \n abundance \n and isotopically enriched were recorded between 370 and 4000 cm with 0.2 cm resolution by a perkinelmer \n ftir spectrometer in garching . at the \n synchrotron lab ,", "the isotopically enriched sample was measured at \n moderate 0.1 cm resolution in the far ir region \n from 100 to 600 cm ( capturing the band at 210 \n cm ) , and at high resolution for the ir active \n cf stretch bands , see ref ( 1 ) .", "the present work focuses on anharmonic constants of c2f4 . only where the constants were \n expected to be different for ccf4 and c2f4 ( e.g. , in the case of \n fermi resonance )", "theoretical values of the anharmonic constants xij were obtained from anharmonic \n vibrational \n frequency calculations performed using gaussian 03 revision c02 at the mp2 level using \n dunning - type correlation - consistent ( cc - pvtz ) orbitals .", "the molecular force field at this level of theory \n was shown in the previous study to reproduce the fundamental wavenumbers \n and the isotopic shifts of c2f4 very satisfactorily \n ( for details , see ref ( 1 ) ) , and hence was an appropriate choice for computing xij values .", "the calculations were performed for each of \n the c2f4 , ccf4 , and c2f4 isotopologues ."], ["the spectra were measured previously and the \n fundamentals extracted \n from them , using a limited number of anharmonic constants ( from combination \n and hot bands ) where necessary . for convenience \n table 1", "lists the fundamentals again . in this \n work we derived a large set of experimental anharmonic constants from \n combination bands and  in particular for the lower - wavenumber \n vibrations  from hot bands , and report a complete set of theoretically \n computed values .", "symmetry types refer to a d2h molecule with z axis along the cc bond and the y axis perpendicular \n to the plane , as used in ref ( 1 ) for easier comparison with the c2v symmetric ccf4 ; the more conventional designation with x axis along the cc bond and z axis perpendicular \n to the plane is given in parentheses .", "revised value ( instead of 210 cm ) , based on a more definite \n assignment of a hot - band structure and x10,10 , see supporting information .", "band origins from high - resolution \n spectra ; low - resolution values are 1338.4 \n and 1187.0 .", "upright : \n experimental values . \n in parentheses : values calculated by mp2/pvtz . where two values or \n a range are given for the experimental data", ", it may indicate ( a ) a \n dependence of the effective xij on the involved levels due to perturbations ( fermi resonances , \n section 4.4 ) or ( b ) an uncertainty due to error \n limits ( e.g. , where broad bands are involved ) or an uncertain assignment . \n", "a preferred value is indicated by bold face . for further details on \n the derivation of each xij and some isotopic data ,", "the anharmonic constants xij were calculated from observed \n combination and difference bands \n by assuming that anharmonic shifts are additive : for binary combination \n bands ( i  j ) and for overtones \n we used1and2with obvious extensions for more complicated \n combinations .", "( the xij values are mostly negative , as usual . ) the left - hand side of these \n equations should be read as  wavenumber of ( name )  . for hot bands ( satellite transitions near i that originate from a thermally populated \n lower level j ) we used3again \n with obvious extensions .", "one can often \n observe sequences of equidistant q branches , corresponding to hot \n bands starting from higher levels mj , or combination of such sequences ( from mj + nk ) .", "equation 3 implies that the distance of a ( first ) hot band from the fundamental \n directly indicates the corresponding anharmonic constant .", "combination \n bands allow for a direct assignment , whereas hot bands must be identified \n by their intensity ( controlled by the boltzmann factor ) . in a number \n of cases", "the supporting information ( si ) compiles the sources for each xij in table s2 , whereas table s1 lists the observed \n bands ( with hot bands ) and their isotopic shifts .", "two examples of \n hot bands , whose shifts are dominated by the large x18 anharmonicity , are shown in figure 1 .", "absorbance spectra demonstrating the large x18 derived shifts for hot - bands originating in 8 .", "the spectra were recorded \n in the garching laboratory , with pressure and path length of 2 bar \n and 10 cm ( for the lower trace of each panel ) , and 140 mbar and 4 \n m ( upper trace ) , respectively . for levels involving more than two quanta of vibration , anharmonic \n shifts", "this helped to decide between different \n assignments of combination bands in a few cases where the harmonic \n sums were similar to each other .", "however , such additivity need not \n apply when the levels involved are subject to fermi resonance , producing \n shifts that depend on the separation of nearby interacting levels . \n", "we use eqs 13 to \n obtain the experimental values , and hence , table 2 reports phenomenological xij , some of which show a spread in the values suggestive of fermi \n resonances ( in particular x18 , x26 , and x77 ) .", "fermi \n resonances can also be recognized if the apparent xij or the hot - band structure depends \n on the isotopologue . for c2f4 ,", "fermi resonances \n were identified ( see section 4.4 and si ) between:5 and 2+6 ( discussed already in ref ( 1)),1 and 5+6 ( indicated by the hot - band structure \n of 1-combinations , which are different in the mixed \n isotopologue),2 and 27 ( causing irregularities \n in overtone levels of 7 , also contributing to the \n large magnitude of x27 and x77).2 and 23 , identified from the \n mp2 calculations .", "5 and 2+6 ( discussed already in ref ( 1 ) ) , 1 and 5+6 ( indicated by the hot - band structure \n of 1-combinations , which are different in the mixed \n isotopologue ) , 2 and 27 ( causing irregularities \n in overtone levels of 7 , also contributing to the \n large magnitude of x27 and x77 ) . 2 and 23 , identified from the \n mp2 calculations .", "the mp2/cc - pvtz calculations \n reproduced the observed wavenumbers \n and isotopic shifts very well . supporting \n the overall vibrational analysis", "exceptions are 15 ( x22 , x28 , x29 , x2,10 , x2,11 , x36 , x3,10 , x45 , x59 , x5,11 , x66 , x68 , x77 , x88 , x10,11 ) out of 54 cases where an observed value is available \n for comparison . some of these will be discussed below ."], ["as already \n described in the introduction , the peculiar \n reactions of c2f4 have to do \n with cc stretching and carbon pyramidalization . the discussion will \n focus on these coordinates ( sections 4.14.3 ) . for \n a planar geometry , \n", "cc dissociation correlates c2h4 and c2f4 with a pair of carbenes in their lowest triplet states ( figure 2a ) . because \n the energy of forming these products is practically the same for the \n two molecules", ", one could also expect that the potential energy curves \n coincide and the cc stretch force constants are equal .", "in fact , already \n early force - field calculations showed no substantial difference ( 889 \n n / m for c2f4 and \n 940", "furthermore , \n the short cc distance ( 132 pm , which is 1 pm shorter than in ethylene ) and the high cc stretch wavenumber ( 1873.8 cm , compared to 1625.4 cm   for ethylene ) do not point to a weakened bond . \n as early as 1965 , the low dissociation energy of c2f4 was attributed to switching over to a channel producing two \n singlet carbenes and to the stabilization ( by 2.458 ev , see introduction ) of singlet cf2 by back bonding , so that this channel is energetically favored by 4.9 ev .", "this path hence leads over an avoided \n crossing of potential surfaces ( figure 2a ) . \n", "if the avoidance is strong , one could expect an influence at least \n on the anharmonicity x11 ( 1 is dominated by cc stretching ) , even if not on the force \n constant at the bottom of the potential energy well .", "table 2 shows that the magnitude of x11 ( calculated 7.8 cm ) is not unusually \n large for such a high - frequency vibration , though noticeably larger \n than for ethylene ( x22 = 2.3 cm  ) . that the increase \n in magnitude is only moderate", "can be taken as a sign that the avoidance \n along this coordinate is not very strong .", "hence , the back reaction \n ( recombination ) will have a high barrier in planar geometry . whereas \n the measured recombination barrier is not , in fact , zero , it is only \n minor ( 9 kj / mol = 0.09 ev  ) .", "so , \n a lower - energy path must lead around the barrier , out of the drawing \n plane of figure 2a , that is , with distortion \n different from cc stretch .", "potentials for ground - state cc dissociation \n of c2f4 , leading over an avoided crossing to \n two singlet carbenes . \n in ( a ) it is assumed that the one - sided pyramidalization ( q7 + q8 ) begins only after overcoming the barrier , whereas \n ( b ) shows the preferred path , where this distortion is already fully \n developed on the barrier and is preceded by a trans pyramidalization \n ( q8 ) , while the cc distance is steadily increasing .", "it was argued in ref ( 16 ) that recombination of \n two cf2 molecules should \n be easiest if one of them points with its filled n orbital \n to the empty p orbital of the other .", "hence , the dissociation \n path will change from pure cc stretch to include also pyramidalization \n ( figure 2b ) , and the potential should be lowered \n in energy in the direction of q7 ( the coordinate of 7 ) and/or q8 . in turn", "in fact x18 has an unusually large magnitude , although x17 is in the normal range ( table 2 ) .", "( the magnitude of x18 is not \n caused by secondary effects such as fermi resonance , as we shall see \n in section 4.4.3 . )", "we can conclude that on \n stretching the cc bond the molecule first bends toward trans pyramidalization \n ( q8 ) ( figure 2b ) .", "the one - sided \n nonplanar bending ( figure 2 ) is a superposition \n of q7 and q8 .", "the reason why q8 is \n active earlier than q7 can be understood by a valence - bond \n model for weak double bonds . in this model", "several symmetric arrangements \n of carbenes ( a  c \n of scheme 2 ) were considered : whereas with \n two triplet carbenes all arrangements ( a  d ) lead to bonding \n interaction , with two singlet carbenes bonding occurs only in the \n trans - bent case ( c ) ; this corresponds to a double donor ", "one can add that the cis - bent case with singlet carbenes would \n lead only to antibonding ( scheme 2d ) .", "malrieu \n and trinquier derived a criterion for trans - bending ( deriving even \n the angle ) by comparing the standard double - bond energy with the singlet ", "their findings were confirmed by many examples , in particular with \n heavier - element homologues .", "we can apply \n this rule not only to the equilibrium geometry of c2f4 but also to its dissociation path , if we consider in the \n comparison the double - bond energy that is reduced on extension of \n this bond .", "the rule thus predicts that on cc extension c2f4 is distorted from planar geometry to a trans - bent structure , \n and this rationalizes the large magnitude of x18 . in the triplet case ,", "the interaction \n can be bonding in all arrangements . in the singlet case , ( a )", "is antibonding , \n ( b ) nonbonding , ( c ) bonding , ( d ) antibonding .", "more recently , borisov et al . found evidence \n for trans pyramidalization \n on extension of the cc bond also from molecular orbital calculation \n ( at high level : mller  plesset perturbation theory of \n second , third and fourth order ) : the \n authors found that the onset is relatively sudden on cc extension \n to 142 pm and that the distortion lowers the energy appreciably .", "( the \n large x18 anharmonicity is an indication \n that this pyramidalization already begins not far from the s0 minimum .", "in fact , a cc stretch by 10 pm  from 132 to 142 pm ", "is \n just about the vibrational amplitude in the 1 = \n 1 level . )", "although borisov et al . did not find an energy minimum with \n such a distortion , they suggested that it plays a crucial role in \n the low - energy real intermediate found by buravtsev , kolbanovskii \n et al . in thermal reactions of c2f4 and recombination \n of cf2 ( see ref ( 31 ) and literature quoted therein and section 4.3 ) .", "one could suppose that in order to lower the activation \n energy \n for dissociation the only necessary deviation from simple cc stretching \n is distortion toward the 8 coordinate ( q8 ) .", "however , there is experimental \n evidence that the asymmetric pyramidalization ( corresponding to superposition \n of q7 and q8 as in figure 2b ) is already crucial in the transition state : the pre - exponential \n factor found in the rate of thermal c2f4 dissociation \n ( 2.8  10 s  ) is clearly larger than would be typical for \n a simple bond splitting ( 10 s ) .", "this \n corresponds to a raised entropy of activation , such as that which \n happens if an additional degree of freedom is activated in the transition \n state .", "the most plausible candidate is free internal rotation , as \n it can be expected in the asymmetric structure with a single dative \n bond but not in symmetric geometry with double donor  acceptor \n bond .", "this picture is also consistent with a stepwise cleavage of \n the double donator  acceptor bond on c2f4 dissociation and its stepwise formation on cf2 recombination .", "it is worth noting that the molecule with one - sided pyramidalization \n has an electron distribution ( figure 2b ) that \n is zwitterionic and correlates in planar geometry with the 2ag state with two  electrons excited to  * , as \n already pointed out in ref ( 16 ) .", "a fully analogous dissociation path can also be \n expected for the \n other systems considered by malrieu and trinquier .", "in fact , according \n to ref ( 32 ) ground - state \n ( ir induced ) dissociation of diazomethane ( h2cnn ) follows \n a path with a pyramidalized methylene group to produce n2 + singlet ch2 ; the out - of - plane deformation was experimentally \n confirmed . in the backward reaction ,", "n2 points with its \n n electron pair to the empty p orbital of the singlet \n methylene ( see refs ( 32 and 33 ) for a calculated potential in two dimensions , which would look similar \n for c2f4 ) .", "hence , \n the out - of - plane deformation on dissociation of c2f4 is not an isolated phenomenon . applying \n their energetic criterion to the equilibrium geometry of c2f4 , trinquier and malrieu found that this molecule is \n not far from the limit , where it would become nonplanar , and suggested \n that the 8 force constant may be significantly lower \n than that of ethylene .", "this is in fact \n confirmed in the following way : analytical expressions for a valence \n force field model for planar x2y4 were given \n by herzberg . besides the masses mx and my , bond lengths l1 ( for xx ) and l2 ( xy ) and the angle  ( half of yxy ) , both 7 and 8 only depend on the force constant for pyramidalization \n ( k/l2 ) . with equilibrium distances and angles from ref ( 1 ) and i = ( 2ci )", "one derives this force constant from \n 7 and 8 ( with identical results \n for c2f4 and c2f4):4both values should be identical \n in this valence - force field model .", "in fact for ethylene , the value \n is 22.9 n / m , whether derived from 7 or 8 .", "the pyramidalization force constant \n derived from 7 of c2f4 is \n slightly higher than in ethylene ( by 17% ) , but the same quantity derived \n from 8 is only 49% of the ethylene value .", "( of course , \n these values are not quantitatively meaningful , as the discrepancy \n highlights that a pure valence - force field model is not sufficient \n for wagging of c2f4 . )", "so in fact the restoring \n force for the 8 vibration is significantly smaller \n than in ethylene and than that expected on the basis of the same pyramidalization \n force constant as for 7 .", "this is consistent with \n the prediction of the valence - bond model favoring the trans - bent geometry .", "the slightly raised force constant for 7 could \n be due to repulsive nonbonding interaction in cis pyramidalization . \n", "such a geometry would be involved in the hypothetical transition state \n of the diels ", "alder addition of c2f4 to \n butadiene , a reaction that has not been observed .", "it was argued that \n an increased energy associated with this distortion might contribute \n to inhibiting the reaction .", "however , an \n increase in the force constant of only 17% is hardly sufficient to \n support this explanation , and the other effects discussed in ref ( 7 ) , in particular the competing \n fast radical reactions , are probably more important . applying \n again the force - field formulas of herzberg ,", "the force constant for torsion , k/l2 , is found to be 30.3 \n n / m for both ethylene and tetrafluoroethylene .", "that is , near the potential \n minimum the c = c bond in c2f4 behaves \n just like a normal double bond .", "consequently , the torsional barrier \n at a 90 twist ( which is a measure of the  bond strength ) \n would be identical for both molecules ( 2.8 ev ) if the two c  cf2 groups remained planar ; this was confirmed by a high - level \n calculation .", "however , pyramidalization \n lowers this barrier by 0.57 ev ( at mp2 level , consistent with ref ( 35 ) ) .", "if this stabilization is activated early along q4 , \n it could give rise to substantially negative values for x44 and especially for x47 and x48 .", "however , x44 and x47 are actually slightly positive \n ( 0.8 and 0.3 cm ) and x48 has no unusual magnitude ( 0.7 to 1.05 cm ) .", "evidently , the stabilizing effect of pyramidalization becomes \n effective only at high torsional angles .", "the corresponding values \n for ethylene are 2.4 , 8.9 , and 7.2 cm , respectively .", "the nondiagonal anharmonicities xi4 and x4j are \n generally smaller in magnitude than in ethylene . but this may simply \n have to do with the relative vibrational amplitudes , which are much \n larger for torsion of ch2 than of cf2 due to \n the relative masses . in the twisted - pyramidalized structure \n of c2f4 ,", "the triplet ( t1 ) is only \n 0.013 ev lower than the singlet \n ( s0 ) .", "it \n can therefore act as a real intermediate and undergo radical - type \n reactions , further assisted by its low energy that is calculated at \n 2.1 ev .", "this has been a widespread interpretation \n of some peculiarities of c2f4 reactions ( see , \n for example ref ( 7 ) ) .", "however , buravtsev , kolbanovskii et al . found in shock - tube experiments \n ( with c2f4 diluted by ar and adiabatically compressed ) \n an intermediate at much lower energy ( 0.8 to 1 ev ) with uv and visible \n absorption ( 235 and 500 nm ) ( see refs ( 31,36 , and 37 ) and the \n literature quoted there ) .", "they supposed it to be a singlet diradicaloid \n of c2f4 with a geometry corresponding not to \n an energy minimum but to a point on a slope of the potential ( see , \n for example ref ( 31 ) ) .", "however , as these points are not stationary there are too many \n ( nearly ) isoenergetic locations with shifted electronic transitions , \n so that the electronic absorption would be smeared out .", "it was therefore \n suggested that the observed intermediate \n is another low - energy isomer , tetrafluoro - ethylidene cf3cf ( electronic absorption in an ar matrix : ) .", "however , its energy ( 1.9 ev  ) seems to be nearly as high as that of the twisted - pyramidalized \n triplet .", "( in one study the twisted - pyramidalized c2f4 triplet is calculated at 0.90 ev , but computational details are not given , and previous theoretical \n results are not discussed . )", "some more anharmonicities of table 1 have unusually large magnitudes or show a range \n of values and/or deviate from prediction . in many of them", "fermi resonances \n play a role , as will be explained here . in section4.4.3 , however , it is shown that x18 is hardly influenced by such effects and its large magnitude mainly \n results from the overall shape of the potential energy surface .", "fermi resonances mix two nearby levels of the \n same symmetry , which differ by three units in the quantum numbers . \n", "the fermi - induced \n repulsion of the levels depends in a nonlinear way on both the magnitude \n of the fermi interaction parameter and on the original ( i.e. , unperturbed ) \n energetic distance of the levels .", "this separation will , in general , \n change ( a ) on isotopic substitution and ( b ) on adding a quantum of \n another vibration ( i.e. , in analogous combination bands ; similarly \n also in higher overtones , in which case a quantum number dependence \n must also be taken into account ) .", "another method of characterizing fermi resonance interactions relies \n on intensity borrowing caused by the mixing of levels . according \n to the mp2 calculations , the great majority of xij change by 1 cm on isotopic substitution", "the few exceptions are \n listed in table 3 and are compared to experimental \n values . identified ( fr13 ) and", "the interpretation in the line  influenced by  \n is based on the assumption that an xij is affected by such a resonance if at least one \n of the levels i and j is involved in the fermi interaction .", "the table shows that \n the calculated isotopic shift is sometimes \n much larger than in the experiment , for instance x26 , x27 .", "also x56 for c2f4 is probably \n too large , as the two observed bands 5+6+11 and 5+6+12 are close to their harmonic positions \n ( table s1 in si ) .", "it seems that at the \n present level of theory , the calculation does not reliably reproduce \n the fermi resonance induced shifts , at least the stronger ones .", "this \n is not surprising given the highly sensitive and nonlinear dependence \n of fermi shift on the  unperturbed  level separations . \n", "the same deficiency will also affect those xij connected with at least one level ( or a \n combination thereof ) involved in a resonance listed above ( examples \n in figure 3a ) .", "in fact , 10 of the 15 deviations \n listed at the end of section 3 can be attributed \n in this way to the resonances fr1 and fr2 with possible contributions \n of fr3 .", "the exceptions are x36 , x3,10 , x66 , x68 , and x88 . in \n all 42 other cases", "it is worthwhile to consider \n the two strongest fermi resonances , \n fr1 ( 2/27 ) and fr2 ( 5/2+6 ) , in c2f4 in some detail .", "the effect of these resonances \n is summarized in figure 3 . some levels involved \n in fermi resonances and observed transitions . \n", "solid arrows : ir , broken arrows : raman ; double arrows ( with interaction matrix element ) indicate repulsion \n between level pairs .", "the anharmonic shifts involving x77 are only nominal , because the fermi perturbation is \n strong :  6x77  = 18.1 is \n less than 3 times ", "(= 8.2 ) and  4x77  is only \n 9.9 . ( but  2x77  +  4x77  =  6x77  , as is obvious from the level scheme , and the raman \n anharmonic shifts practically coincide with those from the ir spectra . ) \n ( b ) three - level fermi resonance 5/2+6/27+6 and \n their combinations with 9 for c2f4 and c2f4 . \n", "the indicated level energies are from the spectra except those in \n parentheses , which are calculated .", "the broken horizontal lines are \n the estimated unperturbed positions ( see the si ) , and the signed numbers are the calculated shifts .", "the 16.4 cm magnitude of the interaction term ( with its expected dependence \n on quantum numbers ) has been derived \n from band shifts and intensities for a series of levels built on 2/27 for all three isotopologues .", "bands for \n 27+x ( x = 9 , 11 ) borrow their entire intensity from 2+x . the detailed analysis \n is provided in si .", "it \n is also evident that x77 , x27 and x22 are affected by \n the fermi resonance ( and probably other x2i , if the level separations differ ) and that x77 is level dependent because of the varying \n separation .", "the fermi term is almost entirely responsible for the \n + 8.2 cm shift of 27 for c2f4 and those of the heavier isotopologues \n ( table 3 under fr1 ) . in the latter", "the fermi - induced \n shift is increased , because the levels 2 and 27 ( before perturbation ) are closer together than in c2f4 and are practically degenerate in c2f4 .", "the success of these calculations \n shows that the additional mixing of 2 with 23 ( fr3 ) is apparently a weak perturbation at most .", "the \n fermi resonance fr2 between 5 and 2+6 was qualitatively discussed in ref ( 1 ) .", "a more complete understanding \n of the resonance shifts and pattern of intensity borrowing requires \n the three - level scheme shown in figure 3b ; \n this figure also shows combinations with 9 , as they \n are all ir active and were directly observed .", "analysis based on this \n model has enabled the magnitude of the fermi term to be determined \n as 11 cm ; the fr1 term of 16.4 cm was used without change .", "significantly , the model predicts varying \n fr shifts that even differ in sign ( originating from the different \n repulsions in figure 3b ) and can thus account \n for the unusual isotopic shifts observed between c2f4 and c2f4 . \n", "it explains a substantial deviation of the observed isotopic shift \n 5 = 51.9 from the value of 5 = 43.1 \n expected from the teller  redlich product rule , which is valid \n for harmonic oscillators .", "additionally \n the relative intensities of combination bands and their isotopic dependence \n are well predicted .", "it is also remarkable that according to the model \n the largest part of the anharmonicity x26 is due to fermi resonance .", "as described in ref ( 1 ) , the lower symmetry in ccf4 leads to strong mixing of 5 with 9 ( which are nearly degenerate in c2f4 ) .", "the two levels repel each other \n ( even in the harmonic approximation ) and give rise to two infrared - active \n transitions .", "it is sometimes difficult to disentangle them and their \n combination bands from the spectra .", "if the large magnitude of some \n anharmonicities is caused by fermi resonance , a crucial question is \n whether this might also be the case for x18 .", "if so , the conspicuous value of x18 could be largely a matter of the character of the resonance and \n less a consequence of global features of the potential for dissociation , \n which we considered in section 4.1 .", "in fact , \n weak perturbations of the 1 level and its combinations \n by the 1/5+6 fermi resonance are indicated by the slight x18 dependence on the level and on isotopic substitution and \n the change of the hot - band structure of 1 combinations \n in ccf4 , as explained in the si .", "a quantitative estimate of the strength \n of the 1/5+6 interaction can be deduced from the intensities of 5+6 combinations relative to those of the corresponding \n 1 combinations , assuming that the former borrow \n their full strength from the latter .", "the 5+6+12 band ( 2444.3 cm ) \n has 12% the strength of the 1+12 band ( 2428.7 cm ) , see figure 1a .", "similarly , the 5+6+11 shoulder ( 3072 cm ) is < 1% as intense as 1+11 ( 3056.2 cm ) and clearly visible only with longer \n path length than shown in figure 1b .", "from both \n observations , one can estimate a fermi term ( w ) of \n no more than 2.5 cm , causing a shift ( and \n consequent change in x18 ) of < 0.5 cm .", "this is actually an upper bound , because such weak \n bands can also be expected without intensity borrowing .", "hence , fermi \n resonance affects the magnitude of x18 only by a little ( by < 3% ) , and we can maintain the conclusions \n of section 4.1 on the shape of the potential ."], ["a large set of anharmonic \n constants of c2f4 was derived from spectroscopic \n data , and a complete set was calculated . \n", "most of the calculated and experimental values agree very well , and \n nearly all deviations can be attributed to fermi resonances . the values \n were used to interpret peculiarities of the potential and reactions \n of this molecule .", "the facile thermal cc dissociation of c2f4 is due to curve crossing of the ground state \n ( 1ag ) with \n the two - electron excited zwitterionic 2ag state .", "the crossing \n barely alters the properties of the potential curve ( cut purely as \n a function of cc stretch ) near the equilibrium geometry ; the force \n constant for cc stretching ( 1 in c2f4 , 2 in c2h4 ) is nearly \n identical for the two molecules .", "only a slightly larger magnitude \n of the anharmonicity x11 compared to the \n corresponding x22 of ethylene provides \n a hint of a weakly avoided crossing in planar geometry of c2f4", ". however , the large magnitude of x18 leads us to conclude that the dissociation path bends \n early toward \n q8 , i.e. to trans pyramidalization of the two carbon atoms \n ( figure 2b ) .", "this is rationalized by a valence - bond \n model , which describes the stretched cc bond as a double donor  acceptor \n bond ( scheme 2c ) .", "it is also consistent with \n the tendency of fluorine to induce sp hybridization at \n carbon , although this preference alone would also allow cis pyramidalization , \n q7 , which is not involved early in dissociation .", "on further \n cc stretching , one of the two donor  acceptor bonds breaks , \n and the pyramidalization becomes fully one - sided already in the transition \n state ( figure 2b ) . in this way , free internal \n rotation of the cf2 groups becomes possible , providing \n an additional degree of freedom that rationalizes the high pre - exponential \n factor in the rate constant of dissociation .", "this structure with perpendicular \n arrangement of the two cf2 groups appears very plausible \n for the back reaction .", "thus , one cf2 group approaches with \n its filled n orbital the empty p orbital of the other \n cf2 group ( figure 2b ) .", "the easy \n trans pyramidalization on minor cc stretching is probably also the \n reason that the corresponding 8 force constant is \n lowered , although all other force constants are similar to those in \n ethylene .", "the preference of fluorine for sp hybridization \n at \n carbon has been invoked to explain not only the easy radical addition \n and other reactions , but also the low - lying , twisted - pyramidalized \n triplet , which is nearly isoenergetic with the singlet that has a \n saddle point there .", "this is indicated by x44 , x47 , and x48 , which have no unusual values .", "very recently , \n high - level theoretical studies investigated \n electronic ground and excited states of c2f4 and their dependence on some coordinates , mainly \n to discover the fate of this molecule after electronic excitation \n and make comparisons with a corresponding time - resolved experiment \n ( ref ( 16 ) ) . for the \n ground state , the stabilizing effect of pyramidalization in the twisted \n molecule was confirmed .", "interestingly , the excited molecule enters \n the s1/s0 conical intersection from the 2ag state , where the molecule has a large one - sided pyramidalization , \n a 90 cc twist and a cc bond length ( 1.51  ) similar to \n that of a single bond . in the present work ,", "the transition state for \n s0 dissociation was said to result from avoided crossing \n of the 2ag and 1ag ( i.e. , s0 ) states , \n also with large one - sided pyramidalization but at longer cc distances \n and perhaps without twist", ". it would be interesting to check whether \n these geometrical differences are large enough to generate an excited - state \n barrier , because evidence was found in ref ( 16 ) that cc dissociation does not begin in the excited \n state ."]]}
{"article_id": "PMC3836355", "article_text": ["obesity is a complex heterogeneous disease that is caused by genes , environmental factors , and the interaction between the two .", "obesity is also a multifactorial condition , and many endocrine and inflammatory pathways are involved in its development and in obesity - related diseases .", "excess weight in obesity may come from muscles , bone , fat , and/or body water , but obesity specifically refers to having an abnormally high proportion of total body fat .", "the world health organization defines  overweight  as a body mass index ( bmi ) of 25 or more and  obesity  as a bmi of 30 or more .", "the prevalence of obesity has been stated as being near epidemic size [ 13 , 57 ] , and obesity has been associated with type ii diabetes , hypertension , coronary artery disease , stroke , and many forms of cancer [ 8 , 9 ] .", "therefore , it is important that the underlying pathophysiology of obesity - related diseases is understood .", "obesity results from the combined effects of genes , lifestyle , and the interactions of these factors , and both familial and nonfamilial factors play an important role in its development . a genetic predisposition to obesity", "has been reported as a major risk factor for individuals .   with the increasing prevalence of obesity , studies on candidate genes for obesity", "most obesity - predisposing genes encode the molecular components of physiological systems related to energy balance .", "leptin is a protein product of the ob gene and is expressed and secreted by adipose tissue in amounts proportional to the body weight content ; studies on its receptor have greatly advanced the comprehension of the mechanism for regulating body weight and energy homeostasis .", "the lipostat system , mediated by leptin and its hypothalamic receptor , reduces food intake and increases thermogenesis [ 10 , 12 ] . the leptin ( lep ) and leptin receptor ( lepr ) genes have been evaluated for polymorphisms that could potentially be related to the pathophysiology of obesity and its complications . although the polymorphisms in these genes have been evaluated [ 1315 ] , the association of these polymorphisms with obesity is still controversial", ". therefore , we investigated whether the lep gene g2548a polymorphism and lepr gene 668a / g ( q223r ) polymorphism might be involved in the pathogenesis of obesity .", "this study included 127 obesity patients ( 93 women , 34 men ) and 105 controls ( 62 women , 43 men ) provided from the department of internal medicine , gazi osmanpaa university in tokat , turkey .", "informed consent was in accordance with the study protocol , and all patients and controls signed a written consent form .", "all patients received a complete clinical evaluation , and all individuals in the control group were healthy and were selected by excluding the diagnosis of obesity .", "genomic dna was isolated from white blood cells by a kit procedure ( invitrogen life technologies , carlsbad , ca , usa ) and stored at 20c .", "lep g2548a and lepr 668a / g polymorphisms were analyzed by polymerase chain reaction based restriction fragment length polymorphism ( pcr - based rflp ) methods . the pcr protocol consisted of an initial melting step of 2 ", "min at 94c , followed by 35 cycles of 30  s at 94c , 30  s at 55c ( for lep ) , 30  s at 60c ( for lepr ) , and 30  s at 72c , and a final elongation step of 5  min at 72c .", "amplification was carried out using primers forward 5-ttt cct gta att ttc ccg tga g-3 and reverse 5-aaa gca aag aca ggc ata aaa a-3 for the lep gene and forward 5-tcc tct tta aag cct atc cag tat tt-3 and reverse 5-agc tag caa ata ttt ttg taa gca at-3 for the lepr gene .", "pcr was performed with a 25  l reaction mixture containing 2550  ng/l dna , 1  l of 10  pmol/l of each primer , 1  l of dntp mixture ( 5  mm dntp , 1  l 2.5  mm mgcl2 , 1  u taq dna polymerase ) , 2.5  l 10x pcr buffer ( mg free , invitrogen life technologies , carlsbad , ca , usa ) , and dh2o .", "amplified products were digested with hhai at 37c for lep and mspi at 37c for lepr , and the resulting fragments were separated by 2% agarose gel electrophoresis .", "the fragments were stained with ethidium bromide and visualized through a vilber - lourmat gel quantification and documentation system ( quantum - st4 ; vilber lourmat bp 66 , torcy , france ) .", "analysis of the data was performed using spss 16.0 ( spss , chicago , il , usa ) and openepi info ( http://www.openepi.com ) .", "the frequencies of the alleles and genotypes ( hardy - weinberg equilibrium ) in patients and controls were compared with  analysis , and 95% confidence intervals were calculated .", "a p value less than 0.05 ( two - tailed ) was regarded as statistically significant .", "the mean age and bmi were 44.86  1.51 , 35.45  4.56 and 34.25  15.43 , 21.57  1.89 in patients and control groups , respectively .", "the mean age of obesity patients with bmi > 35  kg / m and bmi < 35  kg / m was 45.01  1.38 and 44.7  1.65 , respectively , while the mean bmi of these patients was 38.68  3.11 ( bmi > 35  kg / m ) and 32.06  1.56 ( bmi < 35  kg / m ) .", "patients and controls were genotyped for both the g2548a polymorphism in the lep gene promoter and the 668a / g polymorphism in the lepr gene .", "the distribution of the lep g2548a and lepr 668a / g polymorphisms of the patients and control groups are presented in tables 2 and 3 .", "we found no statistically significant difference in the genotype frequencies of the lep gene polymorphism in patients and control groups ( p > 0.05 ) .", "the lepr genotypes differed between the obesity patients and controls , but this was not statistically significant after the bonferroni correction ( p = 0.05 ) .  ", "allele frequencies in the lepr gene showed no statistically significant association ( p > 0.05 ) ( table 3 ) . the lepr gene a allele was 61.4% in patients and 69.5% in the control group , while the g allele frequency was 38.5% in patients and 30.4% in the control group . in the combined analysis of the lep and lepr genes ,", "the lep / lepr gg / gg combined genotype was found to increase the risk of obesity compared to the controls ( p < 0.05 ) ( table 4 ) . in the combined genotype analysis based on the mean bmi of obesity patients", ", there was no association of the lep / lepr combined genotype and obesity between patients with a bmi  35  kg / m and patients with a bmi near 30  kg / m ( p > 0.05 ) ( table 4 ) .", "human obesity is a complex trait determined by the interaction of multiple genes and environmental factors .", "obesity may arise as a result of increased energy intake , decreased energy expenditure , or increased partitioning of nutrients into fat , either alone or in combination .", "the prevalence of obesity and being overweight continues to increase worldwide , not only causing serious personal health problems but also imposing a substantial economic burden on societies .", "genetic influences are difficult to elucidate , and identification of the involved genes is not easily achieved .   in the present study", ", we analyzed the frequencies of lep g2548a and lepr 668a / g polymorphisms in obesity patients in a turkish population .", "there was no statistically significant difference between the groups with respect to the lep genotype distribution ( p > 0.05 ) and allele frequencies ( p > 0.05 ) .", "suggested that the lep g2548a variant may influence gene expression of leptin and leptin secretion by adipose tissue .", "noted that the lep g2548a polymorphism may influence a bmi increase by means of its effects on leptin secretion ; however , they identified a significant and independent association between the lep 2548gg carrier status and higher leptin levels .", "an association of the lep g2548a polymorphism and increased bmi was reported in overweight europeans and in taiwanese subjects with obesity   and the combined lep 759c / t and lep g2548a genotype may be a determinant of obesity .", "the results of our study do not support the results of these studies but do support those of other studies that showed no association between the lep g2548a polymorphism and obesity - related phenotypes [ 11 , 14 , 15 , 21 ] .", "we found that lepr genotypes show a difference , but not statistically significant , between obesity patients and controls .", "we attribute this lack of significance to the low number of patients included in our study , but finding obese patients that have no other disease is difficult .", "some researchers have proposed that the polymorphisms of the leptin receptor gene ( especially lepr 668a / g polymorphism ) may contribute to common forms of human obesity [ 11 , 14 , 2224 ] .", "our results with respect to the lepr polymorphism are in agreement with the results of these studies .  ", "our results showed a statistically significant difference between groups with respect to the distribution of the lep / lepr gg / gg combined genotype .", "obesity results from both gene - gene and gene - environment interactions , and in our study we examined the gene - gene interactions of the lep / lepr genes and their link to obesity .", "demonstrated that the haplotype association of the lep g2548a and lepr q223r variants was related to a 58% increase in obesity risk , and they considered the interactions between lep and lepr gene polymorphisms to intensively influence modulation of energy homeostasis . in agreement with the findings of our study ,", "boumaiza et al . reported that the lep g2548a and lepr q223r polymorphisms and haplotype combination were associated with a metabolic syndrome and obesity risk in tunisian subjects .", "the g2548a and 3hvr variants of the lep gene have been noted as being in linkage disequilibrium , and i / g combined genotypes are associated with obesity .", "in addition , the interactions between the polymorphisms of the lep and lepr genes have been shown to increase the risk of non - hodgkin 's lymphoma and influence insulin plasma concentrations and blood pressure levels .", "our findings indicate that the lep g2548a polymorphism is not a relevant obesity marker and that the lepr 668a / g polymorphism may be related to obesity in a turkish population . additionally , the lep / lepr gg / gg combined genotype was found to increase the risk of obesity in patients compared to controls", ". however , the association of these polymorphisms with obesity is still controversial , and further research with larger patient populations is necessary ."], "abstract_text": ["<S> \n objective . obesity is a complex heterogeneous disease that is caused by genes , environmental factors , and the interaction between the two . </S>", "<S> the leptin ( lep ) and leptin receptor ( lepr ) genes have been evaluated for polymorphisms that could potentially be related to the pathophysiology of obesity and its complications . the aim of this study was to investigate the role of lep g2548a and lepr   668a / g polymorphisms in the pathogenesis of obesity . </S>", "<S> subjects . the study included 127 patients with obesity and 105 healthy controls . polymerase chain reaction and restriction fragment length analysis for lep g2548a and lepr </S>", "<S> 668a / g polymorphisms were applied . </S>", "<S> results . </S>", "<S> there was no statistically significant difference in the genotype frequencies of the lep gene polymorphism between patients and control groups ( p > 0.05 ) . </S>", "<S> we found a difference in the lepr genotypes between patients and controls , but this was not statistically significant ( p = 0.05 ) . </S>", "<S> additionally , we found an increased risk of obesity in the lep / lepr gg / gg combined genotype ( p < 0.05 ) . conclusion . </S>", "<S> our findings indicate that the lep g2548a polymorphism is not a relevant obesity marker and that the lepr 668a / g polymorphism may be related to obesity in a turkish population . </S>", "<S> further researches with larger patient population are necessary to ascertain the implications of lep and lepr polymorphisms in obesity . </S>"], "labels": null, "section_names": ["1. Introduction", "2. Materials and Methods", "3. Results", "4. Discussion"], "sections": [["obesity is a complex heterogeneous disease that is caused by genes , environmental factors , and the interaction between the two .", "obesity is also a multifactorial condition , and many endocrine and inflammatory pathways are involved in its development and in obesity - related diseases .", "excess weight in obesity may come from muscles , bone , fat , and/or body water , but obesity specifically refers to having an abnormally high proportion of total body fat .", "the world health organization defines  overweight  as a body mass index ( bmi ) of 25 or more and  obesity  as a bmi of 30 or more .", "the prevalence of obesity has been stated as being near epidemic size [ 13 , 57 ] , and obesity has been associated with type ii diabetes , hypertension , coronary artery disease , stroke , and many forms of cancer [ 8 , 9 ] .", "therefore , it is important that the underlying pathophysiology of obesity - related diseases is understood .", "obesity results from the combined effects of genes , lifestyle , and the interactions of these factors , and both familial and nonfamilial factors play an important role in its development . a genetic predisposition to obesity", "has been reported as a major risk factor for individuals .   with the increasing prevalence of obesity , studies on candidate genes for obesity", "most obesity - predisposing genes encode the molecular components of physiological systems related to energy balance .", "leptin is a protein product of the ob gene and is expressed and secreted by adipose tissue in amounts proportional to the body weight content ; studies on its receptor have greatly advanced the comprehension of the mechanism for regulating body weight and energy homeostasis .", "the lipostat system , mediated by leptin and its hypothalamic receptor , reduces food intake and increases thermogenesis [ 10 , 12 ] . the leptin ( lep ) and leptin receptor ( lepr ) genes have been evaluated for polymorphisms that could potentially be related to the pathophysiology of obesity and its complications . although the polymorphisms in these genes have been evaluated [ 1315 ] , the association of these polymorphisms with obesity is still controversial", ". therefore , we investigated whether the lep gene g2548a polymorphism and lepr gene 668a / g ( q223r ) polymorphism might be involved in the pathogenesis of obesity ."], ["this study included 127 obesity patients ( 93 women , 34 men ) and 105 controls ( 62 women , 43 men ) provided from the department of internal medicine , gazi osmanpaa university in tokat , turkey .", "informed consent was in accordance with the study protocol , and all patients and controls signed a written consent form .", "all patients received a complete clinical evaluation , and all individuals in the control group were healthy and were selected by excluding the diagnosis of obesity .", "genomic dna was isolated from white blood cells by a kit procedure ( invitrogen life technologies , carlsbad , ca , usa ) and stored at 20c .", "lep g2548a and lepr 668a / g polymorphisms were analyzed by polymerase chain reaction based restriction fragment length polymorphism ( pcr - based rflp ) methods . the pcr protocol consisted of an initial melting step of 2 ", "min at 94c , followed by 35 cycles of 30  s at 94c , 30  s at 55c ( for lep ) , 30  s at 60c ( for lepr ) , and 30  s at 72c , and a final elongation step of 5  min at 72c .", "amplification was carried out using primers forward 5-ttt cct gta att ttc ccg tga g-3 and reverse 5-aaa gca aag aca ggc ata aaa a-3 for the lep gene and forward 5-tcc tct tta aag cct atc cag tat tt-3 and reverse 5-agc tag caa ata ttt ttg taa gca at-3 for the lepr gene .", "pcr was performed with a 25  l reaction mixture containing 2550  ng/l dna , 1  l of 10  pmol/l of each primer , 1  l of dntp mixture ( 5  mm dntp , 1  l 2.5  mm mgcl2 , 1  u taq dna polymerase ) , 2.5  l 10x pcr buffer ( mg free , invitrogen life technologies , carlsbad , ca , usa ) , and dh2o .", "amplified products were digested with hhai at 37c for lep and mspi at 37c for lepr , and the resulting fragments were separated by 2% agarose gel electrophoresis .", "the fragments were stained with ethidium bromide and visualized through a vilber - lourmat gel quantification and documentation system ( quantum - st4 ; vilber lourmat bp 66 , torcy , france ) .", "analysis of the data was performed using spss 16.0 ( spss , chicago , il , usa ) and openepi info ( http://www.openepi.com ) .", "the frequencies of the alleles and genotypes ( hardy - weinberg equilibrium ) in patients and controls were compared with  analysis , and 95% confidence intervals were calculated .", "a p value less than 0.05 ( two - tailed ) was regarded as statistically significant ."], ["the mean age and bmi were 44.86  1.51 , 35.45  4.56 and 34.25  15.43 , 21.57  1.89 in patients and control groups , respectively .", "the mean age of obesity patients with bmi > 35  kg / m and bmi < 35  kg / m was 45.01  1.38 and 44.7  1.65 , respectively , while the mean bmi of these patients was 38.68  3.11 ( bmi > 35  kg / m ) and 32.06  1.56 ( bmi < 35  kg / m ) .", "patients and controls were genotyped for both the g2548a polymorphism in the lep gene promoter and the 668a / g polymorphism in the lepr gene .", "the distribution of the lep g2548a and lepr 668a / g polymorphisms of the patients and control groups are presented in tables 2 and 3 .", "we found no statistically significant difference in the genotype frequencies of the lep gene polymorphism in patients and control groups ( p > 0.05 ) .", "the lepr genotypes differed between the obesity patients and controls , but this was not statistically significant after the bonferroni correction ( p = 0.05 ) .  ", "allele frequencies in the lepr gene showed no statistically significant association ( p > 0.05 ) ( table 3 ) . the lepr gene a allele was 61.4% in patients and 69.5% in the control group , while the g allele frequency was 38.5% in patients and 30.4% in the control group . in the combined analysis of the lep and lepr genes ,", "the lep / lepr gg / gg combined genotype was found to increase the risk of obesity compared to the controls ( p < 0.05 ) ( table 4 ) . in the combined genotype analysis based on the mean bmi of obesity patients", ", there was no association of the lep / lepr combined genotype and obesity between patients with a bmi  35  kg / m and patients with a bmi near 30  kg / m ( p > 0.05 ) ( table 4 ) ."], ["human obesity is a complex trait determined by the interaction of multiple genes and environmental factors .", "obesity may arise as a result of increased energy intake , decreased energy expenditure , or increased partitioning of nutrients into fat , either alone or in combination .", "the prevalence of obesity and being overweight continues to increase worldwide , not only causing serious personal health problems but also imposing a substantial economic burden on societies .", "genetic influences are difficult to elucidate , and identification of the involved genes is not easily achieved .   in the present study", ", we analyzed the frequencies of lep g2548a and lepr 668a / g polymorphisms in obesity patients in a turkish population .", "there was no statistically significant difference between the groups with respect to the lep genotype distribution ( p > 0.05 ) and allele frequencies ( p > 0.05 ) .", "suggested that the lep g2548a variant may influence gene expression of leptin and leptin secretion by adipose tissue .", "noted that the lep g2548a polymorphism may influence a bmi increase by means of its effects on leptin secretion ; however , they identified a significant and independent association between the lep 2548gg carrier status and higher leptin levels .", "an association of the lep g2548a polymorphism and increased bmi was reported in overweight europeans and in taiwanese subjects with obesity   and the combined lep 759c / t and lep g2548a genotype may be a determinant of obesity .", "the results of our study do not support the results of these studies but do support those of other studies that showed no association between the lep g2548a polymorphism and obesity - related phenotypes [ 11 , 14 , 15 , 21 ] .", "we found that lepr genotypes show a difference , but not statistically significant , between obesity patients and controls .", "we attribute this lack of significance to the low number of patients included in our study , but finding obese patients that have no other disease is difficult .", "some researchers have proposed that the polymorphisms of the leptin receptor gene ( especially lepr 668a / g polymorphism ) may contribute to common forms of human obesity [ 11 , 14 , 2224 ] .", "our results with respect to the lepr polymorphism are in agreement with the results of these studies .  ", "our results showed a statistically significant difference between groups with respect to the distribution of the lep / lepr gg / gg combined genotype .", "obesity results from both gene - gene and gene - environment interactions , and in our study we examined the gene - gene interactions of the lep / lepr genes and their link to obesity .", "demonstrated that the haplotype association of the lep g2548a and lepr q223r variants was related to a 58% increase in obesity risk , and they considered the interactions between lep and lepr gene polymorphisms to intensively influence modulation of energy homeostasis . in agreement with the findings of our study ,", "boumaiza et al . reported that the lep g2548a and lepr q223r polymorphisms and haplotype combination were associated with a metabolic syndrome and obesity risk in tunisian subjects .", "the g2548a and 3hvr variants of the lep gene have been noted as being in linkage disequilibrium , and i / g combined genotypes are associated with obesity .", "in addition , the interactions between the polymorphisms of the lep and lepr genes have been shown to increase the risk of non - hodgkin 's lymphoma and influence insulin plasma concentrations and blood pressure levels .", "our findings indicate that the lep g2548a polymorphism is not a relevant obesity marker and that the lepr 668a / g polymorphism may be related to obesity in a turkish population . additionally , the lep / lepr gg / gg combined genotype was found to increase the risk of obesity in patients compared to controls", ". however , the association of these polymorphisms with obesity is still controversial , and further research with larger patient populations is necessary ."]]}
{"article_id": "PMC4878812", "article_text": ["in recent years , nanoparticles \n have been used increasingly for \n biomedical applications , including drug or gene delivery , imaging , \n sensing , or photothermal therapy . in particular ,", "gold nanoparticles ( nps ) have been suggested as highly \n useful sensitizing agents in phototherapy due to their unique size \n and shape - dependent optical properties , high absorption coefficients , \n ease of synthesis , biocompatibility , and their ability to hold a variety \n of functional ligands .", "it is well - known that citrate - stabilized gold nps \n are endocytosed by cells and remain in intracellular vesicles .", "moreover , targeting of specific sites inside \n the cell by functionalization of the surface with cell penetrating \n peptides or peptides containing nuclear localization sequences has \n been reported . alternatively , to selectively \n target specific types of cells , gold nps can be modified with suitable \n antibodies .", "this approach has been studied for potential use in photothermal \n cancer therapy , where cancer cells overexpressing human epidermal \n growth factor receptor 2 ( her2 ) or epithelial growth factor receptor \n ( egfr ) were incubated with gold nps conjugated to anti - her2 or anti - egfr antibodies , respectively . \n", "the nps were then irradiated with light within their plasmon resonance \n absorbance band to heat the cells to temperatures leading to cell \n death .", "most interestingly , it has also been demonstrated \n that endocytosis \n of gold nps by cancer cells and subsequent irradiation of such intracellular \n nps can lead to cell death even at irradiation levels that are not \n high enough to cause significant heating .", "this nonthermal \n route to laser - induced cell death has been ascribed to an as yet not \n fully characterized photochemical reaction , although irradiation of \n endocytosed nps was reported to be accompanied by increased levels \n of reactive oxygen species .", "the use of such a photochemical \n mechanism could be of great advantage in situations where different \n types of cells coexist in close vicinity , since it would allow for \n more selective targeting of particular cells , whereas  due to \n the fast diffusion of heat over the relevant length scales ", "photothermally \n induced cell death will affect all cells within the irradiated volume \n more or less indiscriminately , as long as some of them contain nps .", "irradiation of gold nps with continuous wave ( cw ) lamp or laser \n light has been shown to lead to photogeneration of singlet oxygen \n ( o2 ) in vitro , suggesting that this highly reactive species , which is widely used \n in photodynamic therapy , may be involved in \n the photochemical pathway of cell killing by gold nps . in \n vitroo2", "photogeneration by irradiation \n of spherical gold nps with short laser pulses or cw \n laser light at comparable powers and intensities has also been reported . in this study", ", it was suggested that the mechanism \n of singlet oxygen photogeneration may involve  hot  \n electrons , i.e. , the highly excited conduction band electrons which \n upon absorption of a short laser pulse by a np can reach quasi - equilibrated \n energy distributions corresponding to temperatures of several thousand \n degrees .", "this seems somewhat surprising , since cw \n light at the intensities used does not yield  hot ", "here we present new experimental results on the photogeneration \n of singlet oxygen by irradiation of gold nanoparticles with continuous or pulsed laser light , as well as theoretical work pertaining \n to the underlying mechanism(s ) , which so far had not been addressed . \n", "we show that electron temperatures in excess of 2000 c are easily \n achieved in pulsed laser irradiation experiments , whereas cw light \n under similar conditions yields electronic temperatures of at most \n 10 c above room temperature .", "thus , the photogeneration of o2 by gold nps proceeds by different mechanisms \n under different irradiation conditions ; the implications for the further \n development of medical applications of the effect are discussed in \n detail .", "furthermore , we also found that even a moderately thick , but \n dense , ligand layer significantly reduces the efficiency of o2 photogeneration at the np surface , which also has important \n consequences for practical applications .", "citrate - stabilized spherical \n gold nps with 15 and 46 nm diameter were prepared according to the \n turkevich  frens and a seeded growth method , respectively .", "gold nanorods ( nrods ) were synthesized \n using the seeded - growth method reported by dickerson et al . with slight modifications . more details of the \n np preparation are provided in the supporting information .", "nps were characterized using uv  vis spectroscopy \n ( genesys 10 uv ) , differential centrifugal sedimentation ( cps instruments \n dc24000 ) , and transmission electron microscopy ( tem , fei tecnai spirit \n microscope at 120 kv ) ( see figures s1 and s2 in the supporting information ) .", "the uv  vis absorbance spectrum \n of nrods showed a transverse plasmon resonance band at 522 nm , and \n a longitudinal plasmon resonance band at 798 nm and tem revealed nrods \n to have a length of 40 nm and a diameter of 12 nm .", "functionalization \n with thiolated peg ligands or peptides was achieved by overnight incubation \n with excess ligand , followed by repeated centrifugation for excess \n ligand removal .", "the capping ligands used here were peg - oh ( hs-(ch2)11-(eg)4-oh ) , mpeg5000 ( hs-(ch2)2-(eg)n - o - ch3 , average mw 5000 g mol ) and peptide c - tat ( primary \n sequence calnnagrkkrrqrrr ) ; see figure s3 for the structures of the peg ligands .", "o2 was \n detected via the bleaching of 1,3-diphenylisobenzofuran ( dpbf ) , \n which is widely used for this purpose .", "all experiments involving dpbf \n were carried out in the dark . because dpbf is not soluble in neat \n water ,", "all experiments were conducted \n in 50/50 ( v / v ) mixtures of water and ethanol .", "a fresh solution of \n dpbf ( 3.1 mg , 0.115 mm , a412  nm = 2 ) in etoh ( 100 ml ) was kept stirring in the dark .", "a 10 mm quartz \n cuvette and a 3 mm stirrer bar were left in aqua regia ( 1:3 hno3:hcl ) for 15 min and thoroughly rinsed multiple times with \n milli - q water ( mq h2o ) and etoh . in the clean cuvette with \n stirrer bar , either mq h2o ( 600 l ) or np solution \n ( 600 l ) was mixed with the ethanolic dye solution ( 600 l ) . \n", "where appropriate , the np concentration was adjusted prior to mixing \n to yield an absorbance of 0.4 at 532 nm in the final solution .", "the \n cuvette was sealed with an airtight lid and parafilm , and the uv  vis \n absorbance spectrum was recorded ( genesys 10 uv ) .", "the sample was then \n placed on a stirring plate ( in the dark ) , and its absorbance spectrum \n measured every 10 min for 30 min to ensure the solution was stable . for the irradiation experiments ,", "the cuvette containing 1200 l \n of sample solution was placed on a stirring plate in front of the \n laser . for experiments with nps ,", "the cuvette was fitted with heat \n fins using non - silicone heat transfer paste and cooled using a fan . \n", "no significant increase of the cuvette temperature beyond a slight \n warming was observed . for most experiments ,", "the sample was irradiated \n at 1000 mw ( unless stated otherwise ) using a 532 nm continuous - wave \n diode pumped solid state laser ( laser quantum opus 532 ) with a 1/e beam diameter of 1.85 mm ; for some experiments \n the beam was expanded to 8 mm diameter using a lens , as stated explicitly . \n", "the sample was irradiated for 10 min and then removed from the laser \n setup to record the absorbance spectrum ; this was repeated until the \n sample had been irradiated for 60 min in total .", "irradiation of gold \n nrods in the longitudinal plasmon resonance band at 800 nm was performed \n in the same setup , but using a titanium : sapphire laser ( coherent mira \n 900 ) , aligned on the auxiliary cavity , which prevents mode - locking \n and thus provides continuous - wave laser operation at 800 nm with 1000 \n mw power ; the beam was expanded to 3.4 mm using two lenses . pulsed \n laser irradiation was performed in the same setup , but using the second \n harmonic of a q - switched nd : yag laser ( quantel brilliant ) ( 532 nm , \n 5 ns pulse length , 10 hz repetition rate , 3.5 mm beam size , 15 mj \n pulse energy ) . for experiments that included nps , the absorbance \n of dpbf at 412 \n", "nm was calculated by subtracting the np absorbance at 412 nm ( obtained \n from neat np samples ) from the measured sample absorbance .", "the concentration \n of dpbf in the samples always yielded an initial absorbance very close \n to 1 at 412 nm ; for comparative data analysis , the irradiation - time - dependent \n dpbf absorbance at 412 nm was therefore normalized to 1 at zero irradiation \n time .", "1,3-diphenylisobenzofuran \n ( dpbf ) \n readily undergoes a 1,4-cycloaddition on reaction with o2 to form endoperoxides which irreversibly yield 1,2-dibenzoylbenzene . \n", "dpbf strongly absorbs light at 412 nm ( figure 1a ) , but due to the loss of the -system \n of isobenzofuran , the product does not absorb light at this wavelength ; \n it is this loss of absorbance that is used to detect the presence \n of singlet oxygen . here", ", dpbf was chosen \n as singlet oxygen sensor as it has no absorbance at 532 or 800 nm , \n the wavelengths used for laser irradiation of gold nanoparticles in \n their plasmon resonance bands , and no photobleaching of dpbf was expected \n to occur upon irradiation in the absence of nps .", "contrary to this \n expectation , some bleaching was observed under our cw irradiation \n conditions , with the dpbf absorbance decreasing by ca .", "10% upon irradiation \n at 1 w ( 37 w cm ) for 60 min ( figure 1a ) .", "as shown in figure 2a , and described in more detail \n in the supporting information , this photobleaching \n of dpbf has two phases : ( i ) a rapid phase , extending over the first \n 1020 min of irradiation under the conditions used here , and \n ( ii ) a slower phase , which on the time scale investigated appears \n linear with time .", "we found that phase i depends on laser power , beam \n size , and the presence of oxygen , whereas the slope of the time dependent \n bleaching after 20 min , phase ii , is essentially independent of the \n presence of oxygen and of the beam power / intensity in the range used \n here ( see figure s4 for details ) .", "it should \n be noted that similar effects have been reported previously , with \n cw irradiation at 514 nm for 60 min at significantly lower powers \n ( 40 mw ) than employed here , leading to a ca .", "5% decrease of the absorbance \n of dpbf in benzene , although no explanation was suggested in that \n report .", "the oxygen dependence of phase \n i is in agreement with direct photogeneration of singlet oxygen by \n visible light , which has been suggested to be the reason for dpbf \n photobleaching upon irradiation with light at wavelengths above 470 \n nm .", "however , the slower phase ii is not \n affected by removing oxygen from the solution ( see figure s4b and figure 3 ) and hence can not be ascribed to singlet oxygen formation . \n at the present moment , the mechanism of both phases of dpbf photobleaching \n remains unclear , although both effects are highly reproducible and \n bleaching does not occur when the sample is not exposed to light ( see figure s4a ) .", "photobleaching of the dpbf absorbance \n upon cw irradiation at 532 \n nm , 1 w ( 37 w cm ) , in a 50/50 ( v / v ) mixture of \n water and ethanol : ( a ) in the absence of nps and ( b ) in the presence \n of 15 nm citrate - stabilized spherical gold nps . shown", "are absorbance \n spectra taken at intervals of 10 min from before the irradiation up \n to a maximum irradiation time of 60 min ; the arrows indicate the direction \n of change .", "( a ) time dependence of the photobleaching of \n the dpbf absorbance \n at 412 nm , a412(dpbf ) , upon cw irradiation \n at 532 nm , 1 w ( 37 w cm ) , in the absence and presence \n of citrate - stabilized spherical nps with 15 and 46 nm diameter ; shown \n here are the results from several individual experiments ( dashed lines ) \n and the average ( solid lines ) , after subtraction of the np absorbance \n and normalization to 1 at time zero ; see the experimental \n section for details of data treatment and analysis .", "( b ) effect \n of the nps alone , calculated by subtracting the photobleaching effect \n of dpbf in the absence of nps from the results obtained in the presence \n of nps ; the solid lines in ( b ) are linear fits of the data in the \n range 2060 min .", "gradient of the time - dependent dpbf absorbance photobleaching in \n the irradiation time window 2060 min for different samples \n under cw irradiation at 532 nm , 1 w ( 37 w cm ) . \n for experiments in the presence of nps", ", the np concentration was adjusted \n to yield an absorbance of 0.4 at 532 nm .", "experiments for dpbf in the \n absence of nps and in the presence of 15 nm spherical nps were also \n undertaken after bubbling the sample with nitrogen for 10 min , as \n indicated ( + n2 ) .", "the error bars correspond to the standard deviation \n of several repeat experiments , and  indicates \n statistically significant differences with respect to the experiment \n on dpbf only , as determined by the anova f - test at p < 0.001 ; it should be noted that the results for 15 or 46 nm \n spherical nps without nitrogen bubbling or a peg capping layer were \n found to be different to all other results at this statistical significance \n level .", "no repeat experiment was undertaken for irradiation of nanorods \n at 532 nm , but the same result ( no additional bleaching in the presence \n of nanorods ) was obtained for irradiation at 800 nm ( figure s5 ) . in the presence \n of citrate - stabilized spherical gold nps ,", "the photobleaching \n of dpbf upon irradiation at 532 nm , i.e. , within the nanoparticle \n plasmon resonance band , is significantly increased ( figure 1b ) .", "the time dependence of \n the dpbf photobleaching in the absence of nps and in the presence \n of nps with 15 and 46 nm diameter is shown in figure 2a .", "it can be clearly seen that the \n presence of nps leads to a significant increase of the dpbf photobleaching \n effect and that larger nps yield a larger effect although the np absorbance \n was adjusted to be the same for all samples , so that the same amount \n of light was absorbed .", "the additional photobleaching of dpbf caused \n by the presence of nps can be clearly ascribed to the generation of \n reactive oxygen species ( ros ) , since purging of the samples with nitrogen \n removes this effect ( see below ) .", "moreover , previous experiments had \n shown that light irradiation of citrate - stabilized spherical gold \n nps leads to the characteristic luminescence of o2 at 1280 nm and does not result \n in the generation of superoxide , o2 , \n or hydroxyl , oh , radicals , which leads \n us to conclude that the predominant ros species produced here is singlet \n oxygen , o2 . the fact that significant \n dpbf photobleaching occurs even in the absence of nps requires careful \n consideration for the analysis of these data .", "like dpbf photobleaching \n in the absence of nps , the effect in the presence of nps shows two \n phases .", "however , subtraction of the effect observed when only the \n dye is present yields an essentially linear time dependence for the \n additional effect ascribed to the nps on the time scale of the experiment \n ( figure 2b ) , indicating \n that the np - induced photogeneration of o2 is \n essentially constant over our experimental time interval .", "moreover , \n the second phase of direct dpbf photobleaching is largely independent \n of the laser power , at least down to 0.1 w , i.e. , significantly lower \n powers than those used for most of the experiments reported here , \n as shown in detail in the supporting information ( figure s4a ) , and therefore should be independent of the presence \n of nps whose absorbance leads to a decrease of the laser power along \n the beam path , allowing a direct comparison of the results obtained \n in the time frame of 2060 min .", "moreover , this slower phase \n of direct dpbf photobleaching is also independent of the presence \n of oxygen . for these reasons , only data starting at 20 min irradiation \n will be used for quantitative comparisons .", "figure 3 summarizes \n the main results obtained here after np photoexcitation at 532 nm . \n", "it is very obvious that citrate - stabilized spherical gold nps induce \n significantly faster dpbf photobleaching than is observed in their \n absence ( black ) ; this is found for 15 nm gold nanoparticles ( red ) \n but is even more pronounced for larger nps with 46 nm diameter ( blue ) . \n", "increasing the size of the spherical nps from 15 to 46 nm increases \n the np - induced dpbf photobleaching ( i.e. , the additional effect , after \n subtracting the direct dpbf photobleaching effect ) by ca .", "it is interesting to note \n that unlike the direct dpbf photobleaching , which is not affected \n by nitrogen purging , the np - induced additional bleaching is significantly \n reduced by nitrogen purging , almost down to the level of the direct \n dpbf photobleaching effect .", "this strongly supports the conclusion \n that the np - induced effect is caused by the formation of o2 which then leads to dpbf photobleaching .", "furthermore , \n capping the 15 nm gold nps with peg - oh , a moderately large ligand \n ( figure s3 ) , essentially removes the np - induced \n bleaching and reduces the observed effect to the level of the direct \n dpbf photobleaching .", "gold nanorods ( nrods ) with a length of \n 40 nm and a diameter of \n 12 nm , which have two plasmon resonance bands , namely the transverse \n band at 522 nm and the longitudinal one at 798 nm ( figure s1a ) , stabilized with a capping layer consisting of \n a mixture of a peg polymer ( mpeg5000 ) and a peptide ( c - tat ) ( see the experimental section ) , were also investigated .", "irradiation \n at 532 nm , i.e. , in the transverse plasmon resonance band , showed \n no additional dpbf bleaching above the direct dpbf effect ( figure 3 ) .", "irradiation with \n 1 w ( 11 w cm ) cw laser power at 800 nm , i.e. , \n in the longitudinal plasmon resonance band ( figure s5 ) , showed reduced direct dpbf photobleaching compared to \n irradiation at 532 nm , but again no additional photobleaching was \n observed in the presence of nrods .", "these results show that the nrods \n used here , which have a ligand layer consisting of a mixture of a \n peg polymer and a peptide , do not induce the formation of o2 upon laser irradiation , independent of the plasmon \n resonance which is photoexcited .", "irradiation at 532 nm in the \n presence and absence of citrate - stabilized \n spherical gold nps with 15 nm diameter was also investigated using \n laser pulses with a pulse duration of 5 ns ( figure 4 ) . in this case , the uv  vis spectra \n show a slight broadening of the np plasmon resonance band at 520 nm \n during the first 5 min of irradiation , suggesting that some aggregation \n occurs ; after the initial 5 min , however , the nps remain stable .", "analysis \n of the spectra shows that photobleaching of dpbf in the absence of \n gold nps is similar to the results obtained using cw laser irradiation \n at comparable average powers ( figure s4a ) . in the presence of gold nps with 15 nm diameter , on the other \n hand , the np - induced bleaching effect ( i.e.", ", the effect remaining \n after subtracting the direct dpbf photobleaching effect ) which is \n induced by pulsed laser irradiation is larger than that caused by \n cw irradiation by almost 1 order of magnitude , in spite of the significantly \n lower laser power employed during the pulsed irradiation experiments \n ( 0.15 w vs 1 w ) , which leads to a correspondingly lower number of \n absorbed photons ; compare figure 4b ( pulsed irradiation ) with figure 3 ( cw irradiation ) .", "photobleaching of the \n dpbf absorbance upon laser irradiation with \n 5 ns laser pulses at 532 nm , 0.15 w , 10 hz repetition rate ( corresponding \n to a power density of 1.5 w cm and a pulse energy \n density of 0.15 j cm ) .", "( a ) absorbance spectra \n in the presence of citrate - stabilized spherical gold nps with 15 nm \n diameter , taken at intervals of 5 min from before the irradiation \n ( gray ) up to a maximum irradiation time of 30 min ; the arrow indicates \n the direction of change .", "( b ) gradient of the time - dependent dpbf photobleaching \n ( measured at 412 nm ) in the irradiation time window of 2030 \n min in the absence and presence of nps .", "it is straightforward to estimate the quantum yield of np - induced \n dpbf photobleaching from the absorbed laser power and the observed \n rate of absorbance bleach .", "for the citrate - stabilized spherical nps \n with 15 and 46 nm diameter under cw irradiation at 532 nm , this yields \n values of 5  10 and 8  10 , respectively .", "thus , less than one of each 1 million photons absorbed \n by a np leads to the photobleaching of a dpbf molecule under cw irradiation . \n for irradiation of 15 nm nps with 5 ns laser pulses , on the other \n hand ,", "the quantum yield of np - induced dpbf photobleaching is 3.5  \n 10 , i.e. , almost 2 orders of magnitude larger \n than for cw irradiation , but still very small .", "diffusion of \n oxygen over the lifetime of o2 ( approximately 6 s in 50/50 water / ethanol ) covers a distance larger than the average distance \n between dpbf molecules at the concentrations used here , and hence \n it can be concluded that a significant fraction of the photogenerated o2 which escapes from the np surface should be \n detected .", "10 m , which is close to the \n concentration used here , is sufficient to detect 50% of photogenerated o2 .", "thus , the observed \n low quantum yield of dpbf photobleaching indicates a very low quantum \n yield of o2 photogeneration by gold nps ( i.e. , \n number of o2 generated for each photon absorbed ) , \n having values of the order of ca . 10 for cw irradiation \n and ca . 10 for irradiation with nanosecond laser \n pulses .", "our results show that irradiation of citrate - stabilized \n spherical gold nps at 532 nm , i.e. , in their surface plasmon band , \n with pulsed or cw laser light leads to the production of o2 , detected here by monitoring the bleaching of dpbf \n absorbance .", "however , the rate of o2 production \n is much larger when using short laser pulses than when using cw light \n of comparable intensity . whereas ca .", "24% of the dye is bleached after \n only 10 min of irradiation of 15 nm nps with 5 ns laser pulses at \n an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached over this \n time by irradiation of the same nps with cw light at significantly \n higher power ( 1 w ) ( figure 2 ) .", "this difference allows one to draw important conclusions \n on the mechanism of o2 production by irradiation \n of gold nanoparticles .", "irradiation \n of spherical gold nps at 532 nm leads to the excitation of their plasmon \n resonance , which can be described as a coherent oscillatory motion \n of the conduction band electrons ; this oscillation dephases and decays \n on the sub-100 fs time scale , with only a very small radiative contribution , \n so that most of the excitation energy is retained as electronic excitation \n in the form of electron  hole pairs .", "since the photon energy \n is close to the minimum energy required for direct excitation of d - band \n electrons into the conduction band of gold , a minor contribution of \n this excitation mechanism can not be ruled out ; however , d - band holes \n are filled by conduction band electrons on the 10 fs time scale , yielding \n essentially the same outcome as excitation of the plasmon resonance \n band .", "the excited electrons initially \n have a nonthermal energy distribution , and often are referred to as \n  primary hot electrons ", ", although the concept of temperature \n does not strictly apply to such a distribution .", "they rapidly ( within \n less than 500 fs ) equilibrate by electron  electron scattering \n to yield a fermi distribution corresponding to an elevated temperature \n and can then be referred to as  hot electrons  .", "thiol bond dissociation \n at the surface of gold nps and have been \n suggested to be responsible for the creation of o2 by irradiation of gold nps with nanosecond laser pulses .", "they lose their energy on the time scale of \n a few picoseconds by interaction with the lattice ( electron  phonon \n scattering ) with coupling times that are essentially size - independent \n for nps above 10 nm in diameter , although they strongly depend on \n the amount of energy deposited due to the temperature - dependent electronic \n heat capacity .", "because the lattice heat capacity \n is much larger than the electronic heat capacity , this leaves the \n electrons and the lattice in equilibrium at a temperature which is \n significantly lower than the initial electronic temperature ; finally , \n cooling occurs by heat transfer to the solvent and heat diffusion \n on the time scale of 10 to a few 100 ps , strongly depending on np \n size .", "since the dissipation of \n the absorbed energy proceeds on the picosecond time scale , excitation \n with a nanosecond laser pulse yields a highly nonequilibrium situation \n during the duration of the laser pulse where energy is continuously \n deposited into the electronic system and at the same time flows through \n the lattice into the surrounding solvent .", "it is therefore not straightforward \n to predict the temperature of the hot electrons achieved in such experiments . \n", "we used the  two - temperature model  for the electron \n and phonon heat baths coupled to finite - element heat transfer \n and diffusion simulations in the surrounding solvent to estimate this \n temperature ; details of these simulations are given in the supporting information .", "as shown in figure 5 , under our conditions \n the electrons are expected to reach a temperature of 2100 c , \n whereas the lattice reaches temperatures of the order of 1400 c \n and the solvent in the immediate vicinity of the np a temperature \n of about 900 c .", "time - dependent temperatures of the conduction band electrons \n ( red ) , \n lattice ( black ) , and first solvent layer ( blue ) , calculated for our \n experiments using nanosecond - laser pulse excitation ( 15 nm spherical \n nps in 50/50 etoh / water , 5 ns laser pulses with 0.15 j cm intensity , solid lines ) and for the experiments described in ref ( 24 ) ( 40 nm spherical nps in \n 80/20 etoh / water , 7 ns laser pulses with 0.03 j cm intensity , dashed lines ) using the  two - temperature model  \n for the electron and phonon heat baths , coupled to finite - element \n heat transfer and diffusion simulations in the surrounding solvent \n ( see supporting information for details ) ; \n time zero corresponds to the center of the laser pulse .", "a lattice temperature of 1400 c , which is \n above the melting \n temperature for solid gold , albeit present for only a few nanoseconds , \n might be sufficient to cause temporary melting of the nanoparticle , although the pulse intensities used here are \n still below the reported threshold for size reduction of gold nps \n of 15 nm diameter by nanosecond laser pulses .", "nevertheless , a minor effect on the shape and/or size of the nps , \n especially at the upper end of the size distribution , can not be ruled \n out and may be related to the minor np spectral changes observed upon \n irradiation which suggest some aggregation to occur ( see above ) . because \n of the slower heat dissipation around larger nps , these are heated \n to higher temperatures and hence are more likely to fragment , in agreement \n with experimental results on nanosecond - laser pulse induced fragmentation , \n which also show that fragmentation is finished after 5 min under conditions \n similar to the ones used here . since \n our nps were prepared without excess citrate , the resulting increase \n in the ratio of surface area to volume may indeed cause some aggregation . \n", "it also can not be ruled out that the solvent near the np surface temporarily \n forms bubbles , although the pulse intensities \n used here are still below the reported threshold for bubble formation \n by nanosecond pulses for 15 nm nps .", "such \n bubbles could lead to better thermal insulation , thus potentially \n increasing the maximum temperatures , but on the other hand , they might \n prevent oxygen from reaching the np surface , thus reducing the chance \n of o2 formation .", "the formation of o2 from the triplet ground state by interaction with a \n photoexcited sensitizer requires a change of the electron spin and \n hence can not result from dipole ", "dipole ( frster ) interaction , \n but only from dexter - type electron exchange coupling ; the latter can \n be described as simultaneous transfers of an electron from one of \n the 2 * molecular orbitals on oxygen to a photogenerated hole \n on the sensitizer and of an electron with opposite spin from a high - energy \n excited sensitizer level to the same or the other 2 * orbital , \n resulting in the formation of the  or  singlet oxygen state , respectively .", "this mechanism requires significant overlap of the relevant electronic \n wave functions and hence only occurs at short distances of at most \n 10  . since the photoexcited hot electrons have such a short \n lifetime , this reaction can only occur if an oxygen molecule happens \n to be in the vicinity of the np or is temporarily adsorbed to its \n surface at the moment of excitation .", "a similar reaction occurs on \n photoexcited si nanocrystals , although in this case \n the reaction is more efficient than for au nps due to the much longer \n lifetime of the photoexcited excitons in si . thus , the short lifetime \n of hot electron excitation in gold nanoparticles easily explains the \n low quantum yield of o2 photogeneration which \n is observed here .", "the excitation of electrons to temperatures \n exceeding 2000 c means that a significant number have sufficient \n energy to excite an oxygen molecule to the  state , \n which has an energy of 0.98 ev above the  ground \n state ( see figure 6 ) .", "the number of hot electrons available \n at the higher energy  and the number of holes available at the \n lower energy involved in this two - electron exchange reaction  can \n be estimated from the density of states of gold and the fermi distribution , \n as described in more detail in the supporting information .", "for example , for a spherical np with 15 nm diameter \n at an electron temperature of 2100 c , there are 260 electrons \n within an energy interval of 0.1 ev around the  \n state .", "( it should be noted that the relevant energy interval is the \n width of this state for an oxygen in the vicinity or temporarily adsorbed \n onto a np , which is not known , so only relative numbers will be used \n here . )", "thus , the electron temperatures achieved in our experiment \n are sufficient for a significant population of hot electrons and holes \n at the relevant levels .", "schematic diagram showing the population probability f(e ) for a np electron state at energy e near the fermi level , ef , \n under different \n conditions : ( a ) in equilibrium at room temperature , ( b ) at an electron \n temperature of te = 2100 c after \n electron  electron equilibration (  hot electrons  ) , \n and ( c ) immediately after the absorption of photons by single electrons \n (  primary hot electrons  , with population changes highly \n exaggerated to make them visible ) . also shown are the energies of \n the ground - state triplet (  ) and lowest - excited \n singlet state (  ) of oxygen as well as the next \n singlet state (  ) under the assumption that ef is equidistant from the  \n and  energies .", "excitation of an oxygen molecule \n to o2 requires the simultaneous transfer of \n an electron from the oxygen to a hole at the energy of the  state and of a hot electron with the opposite spin and an \n energy at the  ( or  ) level to \n the oxygen molecule .", "the large number of equilibrated hot electrons available \n during \n a nanosecond - laser pulse also rules out the primary hot electrons \n as the main source of o2 photogeneration .", "the \n number of primary hot electrons available during the pulse duration \n can be estimated from the number of photons which are absorbed per \n nanosecond , multiplied by their lifetime , which is less than 500 fs .", "however , these primary hot electrons populate \n np states at energies from the fermi level ef to ef + 2.34 ev ( the energy of \n a photon at 532 nm ) ( see figure 6c ) . for 15 nm nps and excitation with 5 ns laser pulses \n with 0.15 j cm intensity", ", this predicts that not \n more than three primary hot electrons are available within an energy \n interval of 0.1 ev around the  level at any time \n during the laser pulse , assuming that all levels are equally populated \n and taking into account that the density of states for gold has an \n essentially constant value in the relevant energy range around ef .", "this is significantly \n less than the number of hot ( equilibrated ) electrons available at \n the maximum electron temperature , which was estimated to be on the \n order of 260 for the same energy interval ( see above ) .", "thus , we can \n conclude that it is indeed the hot electrons which are responsible \n for the observed o2 photogeneration when using \n short laser pulses for excitation .", "similar results to those \n obtained here using pulsed irradiation \n have recently been reported for spherical gold nps with 40 nm diameter . although a significantly lower laser pulse energy \n density ( 0.03 j cm , compared to 0.15 j cm here )", "was used under otherwise similar experimental \n conditions , slightly faster o2 photogeneration \n was observed in this study , with the dpbf absorbance decreasing by \n ca .", "24% decrease \n observed here in the first 10 min ( figure 4a ) . in this context , it is interesting to \n note that in spite of the lower laser pulse energy density the nps \n are heated to almost the same electron temperature as in our experiments \n ( see figure 5 ) .", "this \n is largely due to ( i ) heat dissipation from larger nps being slower and ( ii ) the use of 80/20 etoh / water as solvent \n in ref ( 24 ) , which \n has slower heat transport than a 50/50 etoh / water mixture .", "an explicit \n calculation of the number of hot electrons available at the energy \n of the oxygen  state for the two experiments is \n given in the supporting information ; together \n with a detailed consideration of all other experimental differences , \n these numbers yield very good agreement between the expected and the \n observed relative o2 photogeneration rates \n ( see the supporting information for details ) . \n", "this provides further support for the conclusion that o2 photogeneration is mediated by the equilibrated hot \n electrons of gold nanoparticles under nanosecond - pulsed laser irradiation .", "compared to the \n effect of pulsed irradiation , the rate of o2 photogeneration is much smaller when using cw light of comparable \n intensity . whereas ca .", "24% of the dye is bleached after only 10 min \n of irradiation of spherical nps with 15 nm diameter with 5 ns laser \n pulses at an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached \n over this time by irradiation of the same nps with cw light at significantly \n higher power ( 1 w ) ( figure 2 ) . in a previous publication , significantly higher rates of dpbf photobleaching had been reported \n under conditions which appear to be similar to the ones used here . \n in this context", ", we note that we also observed such significantly \n higher rates of bleaching , but only when the sample preparation protocol \n described in the experimental section was \n not followed accurately ; for example , the use of dye solution that \n had not been freshly prepared or of a cuvette that had not been cleaned \n thoroughly and rinsed multiple times with mq water or the use of a \n cell that was not sealed during irradiation , leading to some loss \n of ethanol from the solution , all resulted in larger and highly irreproducible \n bleaching of dpbf under cw irradiation , up to levels comparable to \n those reported in ref ( 24 ) , even in the absence of nps . in the following", ", we will show \n that photogeneration of o2 under cw irradiation , \n unlike pulsed irradiation , is mediated by the initially created ", "primary \n hot  electrons ; i.e. , it occurs during the short time during \n which the excited electrons have not yet relaxed to a thermal distribution \n ( see figure 6c ) . under \n the cw irradiation conditions used here ( 1 w , 1.85 mm beam diameter ) , \n a spherical np with 15 nm diameter absorbs photons at an average rate \n of 1.3  10 s , as estimated from \n the absorption cross section and the \n beam intensity .", "this means that after absorption of a photon there \n is enough time for full relaxation and transfer of the photon energy \n into the solvent , which occurs in less than 100 ps , before absorption of the next photon .", "absorption of one \n photon by a 15 nm np yields  hot  electrons at a temperature \n of 10 k above the surrounding after electron  electron equilibration ; \n these  hot  electrons lose their energy by electron ", "phonon \n scattering within a few picoseconds to yield a np whose temperature \n is only 80 mk above the surrounding . neither of these effects is expected \n to yield any significant photochemical effects ; estimates analogous \n to those described above predict that for a spherical 15 nm np with \n an electron temperature of 35 c there are ca .", "2.6  10 electrons \n within an energy interval of 0.1 ev around the oxygen  state and the same number of holes around the  state energy .", "this means that there are on the order of 10 times less hot electrons and 10 times less holes \n available for the photoreaction than under our pulsed laser irradiation \n conditions , which rules out any significant reaction ; this is also \n confirmed by the fact that these population numbers are less than \n a factor 2 larger than those for room temperature , where no o2 is generated in the absence of light .", "this leaves \n only the primary hot electrons , i.e. , those electrons \n that are excited upon absorption of a photon but have not yet equilibrated \n by electron  electron scattering , as potential cause for photogeneration \n of o2 .", "absorption of a single photon can potentially \n excite electrons to energies of up to 2.34 ev above the fermi level . \n", "if one assumes excitation of only one electron by each photon and \n equal excitation probability for all available electrons , as shown \n in figure 6c , on average \n there will be 0.043 electrons within an energy interval of 0.1 ev \n around the oxygen  state , which is approximately \n 5000 times less than during a single laser pulse in the pulsed experiments , \n see above .", "taking into account that the same factor also applies to \n the holes required for the dexter mechanism , but correcting for the \n lifetime of the excitation ( 500 fs for primary hot electrons created \n during cw irradiation , ca .", "3 ns for the hot electron distribution induced \n by a single laser pulse ; see figure s6 ) \n and the repetition rates ( 1.3  10 s for single photon absorption during cw irradiation , 10 s for the pulsed laser irradiation ) , one would predict a rate of o2 photogeneration under our cw irradiation conditions \n which is smaller than that expected for our pulsed irradiation conditions \n by a factor on the order of 10 .", "this is significantly \n closer to the experimental results ( ratio of o2 photogeneration rates under pulsed vs cw irradiation of 1020 ) \n than any estimate based on the equilibrated  hot  electrons \n after the absorption of a single photon .", "the main discrepancy between \n the predicted and observed cw results arises from the assumption of \n direct excitation of single electrons in the above estimate , which \n is not valid for irradiation at 532 nm , i.e. , in the gold np plasmon \n resonance band , since this leads to the coherent excitation of many \n electrons which rapidly dephases without the electrons exchanging \n energy .", "thus , absorption of a single photon yields more than one primary \n hot electron , with the photon s energy distributed over all \n of them . consequently", ", the energy distribution even of the primary \n hot electrons will not extend up to 2.34 ev above ef but will be shifted toward the states nearer the fermi \n level , thus increasing the population of states around the oxygen  energy and hence the yield of o2 .", "a more quantitative estimate of this effect is beyond the scope \n of this paper .", "an alternative possibility for the mechanism \n of o2 photogeneration by gold nps could be \n envisaged , which is \n based on increased direct photoexcitation of oxygen due to the well - known \n local electric field enhancement in the vicinity of metal nps by the \n plasmon electrons .", "however , significant field enhancement extends \n to distances comparable to the dimensions of the nanoparticle , so that this mechanism is in disagreement with \n our observation that a peg - oh capping layer , which has a thickness \n of only 2 nm , completely inhibits o2 photogeneration ( figure 3 ) .", "dexter - type electron exchange coupling , \n on the other hand , is known to be of significance only over distances \n of less than 1 nm , as discussed above , and thus is further supported \n by this observation . in conclusion ,", "cw irradiation is less efficient \n than pulsed laser \n irradiation in photogenerating o2 since the \n ( equilibrated ) hot electrons , which are the main mediator of the photochemistry \n in the case of pulsed irradiation , do not have sufficient energy / temperature \n to drive o2 photogeneration .", "nevertheless , \n our results confirm that cw irradiation of spherical gold nps produces \n detectable amounts of o2 .", "the absence of significant amounts of ( equilibrated ) hot electrons \n means that o2 photogeneration proceeds via \n a different mechanism under cw irradiation compared to pulsed laser \n generation ; the above estimates indicate that it is the primary hot \n electrons , i.e. , the directly photoexcited electrons , which are responsible \n for the photochemistry here and that the photochemical reaction must \n occur before these equilibrate by electron  electron thermalization .", "photodynamic \n therapy holds great promises for medical applications , such as the \n treatment of cancer , because of the ability to selectively affect \n diseased tissue only .", "however , wider use of photodynamic \n cancer therapy is currently prevented by several limitations imposed \n by the available photosensitizers .", "these limitations include toxicity , \n poor stability and photostability , poor selectivity for cancer tissue , \n and the need of using visible light with poor tissue penetration . \n", "all of these limitations , in principle , can be overcome by the use \n of gold nanoparticles , which are nontoxic , have excellent stability \n even under irradiation , can target cancer tissue either passively \n by the enhanced penetration and retention ( epr ) effect or by active targeting , have extinction coefficients \n that are larger than those of dye molecules by several orders of magnitude , \n and can be tuned to absorb in the near - infrared spectral region for \n maximum tissue penetration .", "gold nanoparticles have been reported \n to have three potential modes of operation for inducing cell death \n by irradiation , namely ( i ) hyperthermia , which is based on the rapid \n conversion of the absorbed light energy into heat , ( ii ) np - assisted \n photodynamic therapy , in which the efficiency of a standard sensitizer \n is amplified by the np plasmon field enhancement effect , or ( iii ) \n a direct photochemical mechanism without involvement of a photosensitizer .", "the feasibility of photothermal therapy has been clearly established \n by careful experiments in vitro(1113,15,54 ) and has been shown \n to work in vivo .", "however , it should be noted that most studies reporting successful \n photothermal therapy made no attempt to either confirm significant \n heating or rule out photochemical effects , which means that some of \n these reported results could in fact arise from photochemical rather \n than photothermal effects or from a synergistic combination of photochemical \n and photothermal effects . as has been pointed out recently , another problem with many reports on photoinduced \n hyperthermia using nps is the relatively high light intensity required \n to reach sufficient temperatures , which often were well above the \n generally accepted skin tolerance threshold .", "the presence of \n gold nps can also lead to increased photogeneration \n of o2 by traditional photodynamic sensitizers , which can be ascribed to the local electric field enhancement in \n the vicinity of metal nps by the plasmon electrons , similar to the \n well - studied sers ( surface - enhanced raman spectroscopy ) effect .", "this \n approach might help to alleviate some of the drawbacks of traditional \n photodynamic therapy but does not directly overcome them , since it \n still requires the presence of a sensitizer .", "in fact , the requirement \n for two active components , gold np and sensitizer , which must be colocalized , \n introduces an additional complication , and care must be taken to avoid \n a reduction of the photosensitizer effect due to quenching of its \n excited state by the metal np . in an alternative approach", ", cell death has been demonstrated to \n occur following irradiation of intracellular ( endocytosed ) gold nps \n even at irradiation levels that are not high enough to cause significant \n heating .", "this photochemical effect has been related to the \n observation that irradiation of nps in vitro results \n in the formation of singlet oxygen , which is the active species in traditional \n photodynamic therapy .", "because of the short lifetime of o2 ( 3.4 s in water ) , its action is highly localized  an oxygen molecule only \n diffuses over the length scale of 100 \n nm in this time . for this reason , only intracellular nps", "are expected \n to trigger cell death by the photochemical route , although they may \n initially be located inside endosomes which are known to be broken \n up by nps under cw irradiation . on the \n other hand", ", the localization of the photochemical effects within individual \n cells also means that in situations where different types of cells \n coexist in close vicinity , selective targeting of particular cells \n and minimization of collateral damage should be achievable .", "this is \n different than photothermally induced cell death , which affects all \n cells within the irradiated volume more or less indiscriminately , \n as long as some of them contain nps , because of the fast diffusion \n of heat over the relevant length scales .", "the results of the \n experiments described here provide more insight \n into the direct photochemical mechanism and allow some important conclusions \n to be made for the further development of practical applications . \n", "they show that detectable amounts of o2 are \n generated by irradiation of nps with short laser pulses or cw light \n even in the absence of a photodynamic sensitizer , albeit with low \n quantum yield .", "short laser pulses are significantly more efficient \n at this process , since they can heat a significant fraction of the \n np conduction band electrons to high enough temperatures to excite \n oxygen to the singlet state .", "however , this requires pulse energy densities \n that are well above generally accepted safe levels for the irradiation \n of skin with pulsed laser light . since the effect requires the absorption \n of many photons by a np during one laser pulse , it is highly nonlinear \n with respect to irradiation intensity , and thus it will not be possible \n to compensate lower irradiation levels by longer irradiation times . \n", "furthermore , pulsed irradiation at the required intensities also can \n cause other effects , such as np fragmentation or bubble formation , and it is not clear what consequences \n these effects may have when occurring in tissue .", "although the use \n of femtosecond laser pulses , as compared to the nanosecond pulses \n used here , might alleviate some of these problems to some extent , \n they would require even more sophisticated equipment which may not \n be suitable for a clinical environment .", "the use of cw light , \n on the other hand , is straightforward and \n does not even require a laser but can be achieved with simple lamps . \n in spite of the lower quantum yield of o2 photogeneration \n by nps under cw irradiation which", "is reported here , cell death induced \n by cw light in the presence of nps has been reported in vitro and in vivo .", "although the experiments \n described here , which did not involve any biological material , were \n undertaken at light intensities that are above safe irradiation levels , \n the mechanism of o2 photogeneration by nps \n under cw illumination is shown to be based on the absorption of single \n photons .", "this means that lower light intensities can be compensated \n for by longer irradiation times , allowing one to reduce the intensity \n to safe levels for biological or medical applications without affecting \n the amount of o2 generated .", "o2 photogeneration and cancer cell destruction in vitro and in vivo have indeed been observed upon irradiation \n on the minute time scale at intensities below safe levels and using \n nonlaser light sources .", "the results presented here indicate another design criterion \n which \n needs to be fulfilled for successful implementation of the photochemical \n route of inducing cell death by irradiation of nps , namely that the \n nps must not possess a dense capping layer . even the thin capping \n layer formed by peg - oh , with a thickness of only 2 nm , is sufficient to completely inhibit o2 photogeneration ( figure 3 ) .", "this is in full agreement with the suggestion that \n energy exchange between the np and the oxygen molecule occurs via \n the dexter ( two electron exchange ) mechanism , which is limited to \n distances of less than 1 nm . similar effects have been reported for o2 photogeneration by si nanocrystals , which is \n significantly affected by a thin oxide layer .", "it is important to point out that the assay used here for o2 detection ( bleaching of dpbf ) only reports on singlet \n oxygen found outside the np capping layer ; thus , it can not be ruled \n out that even on nps with a peg - oh capping layer some o2 is photogenerated at the np surface but reacts with \n the np ligands and hence is quenched .", "on the other hand , such singlet \n oxygen would not be of any direct use for practical applications , \n such as the induction of cell death , so that the assay results in \n fact report the effects relevant for such applications .", "uncapped ( citrate ) \n nps are rapidly covered by a protein corona after they have been taken \n up into live cells , but it appears that \n this corona is permeable enough for oxygen to not completely prevent \n singlet oxygen formation , as evidenced by fact that cell death by \n the photochemical route has been observed with such nps ; it may be that because of their size proteins \n are not able to form a capping layer ( corona ) of similar density as \n the smaller ligands used here .", "similarly , a lipid bilayer appears \n to allow oxygen access to the surface of gold nanorods , whereas a dense pentapeptide ( calnn ) capping \n layer on spherical gold nanoparticles is sufficient to suppress the \n photochemical mechanism of cell death .", "it also seems likely that the thick peg capping layer present \n on \n the nanorods used here is the main reason for the absence of o2 photogeneration by nanorods upon irradiation \n in either the transversal or the longitudinal plasmon resonance band \n that was observed here . because of their longitudinal plasmon resonance \n band , which is in the near - ir spectral region with high tissue penetration , \n nanorods are more suitable for practical photodynamic applications \n in tissue .", "however , standard synthesis protocols yield nanorods within \n a bilayer of cytotoxic ctab , so that ligand exchange is required before \n any biological or medical application , and care will need to be taken \n to choose suitable ligands to allow access of oxygen to the nanorod \n surface . potential examples for these are poly(vinylpyrrolidone ) , \n lipid bilayers , or mesoporous silica , all of which have been used \n successfully in experiments showing photochemically induced cell death \n using gold nanorods .", "dpbf has been used successfully to detect \n singlet oxygen that is \n formed when spherical nps are irradiated at 532 nm , either with cw \n or pulsed laser irradiation .", "singlet oxygen generation by pulsed laser \n irradiation has been shown to act via the equilibrated hot electrons \n that can reach temperatures of several thousand degrees during the \n laser pulse ; cw irradiation , on the other hand , can act only via the \n directly excited primary  hot  electrons , which rapidly \n lose their energy by electron ", "electron equilibration , and hence \n is significantly less efficient for the formation of singlet oxygen . \n", "nevertheless , even cw irradiation can produce enough singlet oxygen \n for photodynamic therapy applications and will allow practical applications \n of the effect at safe irradiation levels .", "photodynamic therapy using \n gold nanoparticles will also require careful design of the nanoparticles \n with respect to size , shape , and capping layer and will require internalization \n of the nps , not just attachment to the cell surface , which is sufficient \n for photothermal therapy ."], "abstract_text": ["<S> the \n formation of singlet oxygen by irradiation of gold nanoparticles \n in their plasmon resonance band with continuous or pulsed laser light \n has been investigated . </S>", "<S> citrate - stabilized nanoparticles were found \n to facilitate the photogeneration of singlet oxygen , albeit with low \n quantum yield . </S>", "<S> the reaction caused by pulsed laser irradiation makes \n use of the equilibrated hot electrons that can reach temperatures \n of several thousand degrees during the laser pulse . </S>", "<S> although less \n efficient , continuous irradiation , which acts via the short - lived \n directly excited primary  hot  electrons only , can produce \n enough singlet oxygen for photodynamic cancer therapy and has significant \n advantages for practical applications . </S>", "<S> however , careful design of \n the nanoparticles is needed , since even a moderately thick capping \n layer can completely inhibit singlet oxygen formation . </S>", "<S> moreover , the \n efficiency of the process also depends on the nanoparticle size . </S>"], "labels": null, "section_names": ["Introduction", "Experimental Section", "Results and Discussion", "Conclusions"], "sections": [["in recent years , nanoparticles \n have been used increasingly for \n biomedical applications , including drug or gene delivery , imaging , \n sensing , or photothermal therapy . in particular ,", "gold nanoparticles ( nps ) have been suggested as highly \n useful sensitizing agents in phototherapy due to their unique size \n and shape - dependent optical properties , high absorption coefficients , \n ease of synthesis , biocompatibility , and their ability to hold a variety \n of functional ligands .", "it is well - known that citrate - stabilized gold nps \n are endocytosed by cells and remain in intracellular vesicles .", "moreover , targeting of specific sites inside \n the cell by functionalization of the surface with cell penetrating \n peptides or peptides containing nuclear localization sequences has \n been reported . alternatively , to selectively \n target specific types of cells , gold nps can be modified with suitable \n antibodies .", "this approach has been studied for potential use in photothermal \n cancer therapy , where cancer cells overexpressing human epidermal \n growth factor receptor 2 ( her2 ) or epithelial growth factor receptor \n ( egfr ) were incubated with gold nps conjugated to anti - her2 or anti - egfr antibodies , respectively . \n", "the nps were then irradiated with light within their plasmon resonance \n absorbance band to heat the cells to temperatures leading to cell \n death .", "most interestingly , it has also been demonstrated \n that endocytosis \n of gold nps by cancer cells and subsequent irradiation of such intracellular \n nps can lead to cell death even at irradiation levels that are not \n high enough to cause significant heating .", "this nonthermal \n route to laser - induced cell death has been ascribed to an as yet not \n fully characterized photochemical reaction , although irradiation of \n endocytosed nps was reported to be accompanied by increased levels \n of reactive oxygen species .", "the use of such a photochemical \n mechanism could be of great advantage in situations where different \n types of cells coexist in close vicinity , since it would allow for \n more selective targeting of particular cells , whereas  due to \n the fast diffusion of heat over the relevant length scales ", "photothermally \n induced cell death will affect all cells within the irradiated volume \n more or less indiscriminately , as long as some of them contain nps .", "irradiation of gold nps with continuous wave ( cw ) lamp or laser \n light has been shown to lead to photogeneration of singlet oxygen \n ( o2 ) in vitro , suggesting that this highly reactive species , which is widely used \n in photodynamic therapy , may be involved in \n the photochemical pathway of cell killing by gold nps . in \n vitroo2", "photogeneration by irradiation \n of spherical gold nps with short laser pulses or cw \n laser light at comparable powers and intensities has also been reported . in this study", ", it was suggested that the mechanism \n of singlet oxygen photogeneration may involve  hot  \n electrons , i.e. , the highly excited conduction band electrons which \n upon absorption of a short laser pulse by a np can reach quasi - equilibrated \n energy distributions corresponding to temperatures of several thousand \n degrees .", "this seems somewhat surprising , since cw \n light at the intensities used does not yield  hot ", "here we present new experimental results on the photogeneration \n of singlet oxygen by irradiation of gold nanoparticles with continuous or pulsed laser light , as well as theoretical work pertaining \n to the underlying mechanism(s ) , which so far had not been addressed . \n", "we show that electron temperatures in excess of 2000 c are easily \n achieved in pulsed laser irradiation experiments , whereas cw light \n under similar conditions yields electronic temperatures of at most \n 10 c above room temperature .", "thus , the photogeneration of o2 by gold nps proceeds by different mechanisms \n under different irradiation conditions ; the implications for the further \n development of medical applications of the effect are discussed in \n detail .", "furthermore , we also found that even a moderately thick , but \n dense , ligand layer significantly reduces the efficiency of o2 photogeneration at the np surface , which also has important \n consequences for practical applications ."], ["citrate - stabilized spherical \n gold nps with 15 and 46 nm diameter were prepared according to the \n turkevich  frens and a seeded growth method , respectively .", "gold nanorods ( nrods ) were synthesized \n using the seeded - growth method reported by dickerson et al . with slight modifications . more details of the \n np preparation are provided in the supporting information .", "nps were characterized using uv  vis spectroscopy \n ( genesys 10 uv ) , differential centrifugal sedimentation ( cps instruments \n dc24000 ) , and transmission electron microscopy ( tem , fei tecnai spirit \n microscope at 120 kv ) ( see figures s1 and s2 in the supporting information ) .", "the uv  vis absorbance spectrum \n of nrods showed a transverse plasmon resonance band at 522 nm , and \n a longitudinal plasmon resonance band at 798 nm and tem revealed nrods \n to have a length of 40 nm and a diameter of 12 nm .", "functionalization \n with thiolated peg ligands or peptides was achieved by overnight incubation \n with excess ligand , followed by repeated centrifugation for excess \n ligand removal .", "the capping ligands used here were peg - oh ( hs-(ch2)11-(eg)4-oh ) , mpeg5000 ( hs-(ch2)2-(eg)n - o - ch3 , average mw 5000 g mol ) and peptide c - tat ( primary \n sequence calnnagrkkrrqrrr ) ; see figure s3 for the structures of the peg ligands .", "o2 was \n detected via the bleaching of 1,3-diphenylisobenzofuran ( dpbf ) , \n which is widely used for this purpose .", "all experiments involving dpbf \n were carried out in the dark . because dpbf is not soluble in neat \n water ,", "all experiments were conducted \n in 50/50 ( v / v ) mixtures of water and ethanol .", "a fresh solution of \n dpbf ( 3.1 mg , 0.115 mm , a412  nm = 2 ) in etoh ( 100 ml ) was kept stirring in the dark .", "a 10 mm quartz \n cuvette and a 3 mm stirrer bar were left in aqua regia ( 1:3 hno3:hcl ) for 15 min and thoroughly rinsed multiple times with \n milli - q water ( mq h2o ) and etoh . in the clean cuvette with \n stirrer bar , either mq h2o ( 600 l ) or np solution \n ( 600 l ) was mixed with the ethanolic dye solution ( 600 l ) . \n", "where appropriate , the np concentration was adjusted prior to mixing \n to yield an absorbance of 0.4 at 532 nm in the final solution .", "the \n cuvette was sealed with an airtight lid and parafilm , and the uv  vis \n absorbance spectrum was recorded ( genesys 10 uv ) .", "the sample was then \n placed on a stirring plate ( in the dark ) , and its absorbance spectrum \n measured every 10 min for 30 min to ensure the solution was stable . for the irradiation experiments ,", "the cuvette containing 1200 l \n of sample solution was placed on a stirring plate in front of the \n laser . for experiments with nps ,", "the cuvette was fitted with heat \n fins using non - silicone heat transfer paste and cooled using a fan . \n", "no significant increase of the cuvette temperature beyond a slight \n warming was observed . for most experiments ,", "the sample was irradiated \n at 1000 mw ( unless stated otherwise ) using a 532 nm continuous - wave \n diode pumped solid state laser ( laser quantum opus 532 ) with a 1/e beam diameter of 1.85 mm ; for some experiments \n the beam was expanded to 8 mm diameter using a lens , as stated explicitly . \n", "the sample was irradiated for 10 min and then removed from the laser \n setup to record the absorbance spectrum ; this was repeated until the \n sample had been irradiated for 60 min in total .", "irradiation of gold \n nrods in the longitudinal plasmon resonance band at 800 nm was performed \n in the same setup , but using a titanium : sapphire laser ( coherent mira \n 900 ) , aligned on the auxiliary cavity , which prevents mode - locking \n and thus provides continuous - wave laser operation at 800 nm with 1000 \n mw power ; the beam was expanded to 3.4 mm using two lenses . pulsed \n laser irradiation was performed in the same setup , but using the second \n harmonic of a q - switched nd : yag laser ( quantel brilliant ) ( 532 nm , \n 5 ns pulse length , 10 hz repetition rate , 3.5 mm beam size , 15 mj \n pulse energy ) . for experiments that included nps , the absorbance \n of dpbf at 412 \n", "nm was calculated by subtracting the np absorbance at 412 nm ( obtained \n from neat np samples ) from the measured sample absorbance .", "the concentration \n of dpbf in the samples always yielded an initial absorbance very close \n to 1 at 412 nm ; for comparative data analysis , the irradiation - time - dependent \n dpbf absorbance at 412 nm was therefore normalized to 1 at zero irradiation \n time ."], ["1,3-diphenylisobenzofuran \n ( dpbf ) \n readily undergoes a 1,4-cycloaddition on reaction with o2 to form endoperoxides which irreversibly yield 1,2-dibenzoylbenzene . \n", "dpbf strongly absorbs light at 412 nm ( figure 1a ) , but due to the loss of the -system \n of isobenzofuran , the product does not absorb light at this wavelength ; \n it is this loss of absorbance that is used to detect the presence \n of singlet oxygen . here", ", dpbf was chosen \n as singlet oxygen sensor as it has no absorbance at 532 or 800 nm , \n the wavelengths used for laser irradiation of gold nanoparticles in \n their plasmon resonance bands , and no photobleaching of dpbf was expected \n to occur upon irradiation in the absence of nps .", "contrary to this \n expectation , some bleaching was observed under our cw irradiation \n conditions , with the dpbf absorbance decreasing by ca .", "10% upon irradiation \n at 1 w ( 37 w cm ) for 60 min ( figure 1a ) .", "as shown in figure 2a , and described in more detail \n in the supporting information , this photobleaching \n of dpbf has two phases : ( i ) a rapid phase , extending over the first \n 1020 min of irradiation under the conditions used here , and \n ( ii ) a slower phase , which on the time scale investigated appears \n linear with time .", "we found that phase i depends on laser power , beam \n size , and the presence of oxygen , whereas the slope of the time dependent \n bleaching after 20 min , phase ii , is essentially independent of the \n presence of oxygen and of the beam power / intensity in the range used \n here ( see figure s4 for details ) .", "it should \n be noted that similar effects have been reported previously , with \n cw irradiation at 514 nm for 60 min at significantly lower powers \n ( 40 mw ) than employed here , leading to a ca .", "5% decrease of the absorbance \n of dpbf in benzene , although no explanation was suggested in that \n report .", "the oxygen dependence of phase \n i is in agreement with direct photogeneration of singlet oxygen by \n visible light , which has been suggested to be the reason for dpbf \n photobleaching upon irradiation with light at wavelengths above 470 \n nm .", "however , the slower phase ii is not \n affected by removing oxygen from the solution ( see figure s4b and figure 3 ) and hence can not be ascribed to singlet oxygen formation . \n at the present moment , the mechanism of both phases of dpbf photobleaching \n remains unclear , although both effects are highly reproducible and \n bleaching does not occur when the sample is not exposed to light ( see figure s4a ) .", "photobleaching of the dpbf absorbance \n upon cw irradiation at 532 \n nm , 1 w ( 37 w cm ) , in a 50/50 ( v / v ) mixture of \n water and ethanol : ( a ) in the absence of nps and ( b ) in the presence \n of 15 nm citrate - stabilized spherical gold nps . shown", "are absorbance \n spectra taken at intervals of 10 min from before the irradiation up \n to a maximum irradiation time of 60 min ; the arrows indicate the direction \n of change .", "( a ) time dependence of the photobleaching of \n the dpbf absorbance \n at 412 nm , a412(dpbf ) , upon cw irradiation \n at 532 nm , 1 w ( 37 w cm ) , in the absence and presence \n of citrate - stabilized spherical nps with 15 and 46 nm diameter ; shown \n here are the results from several individual experiments ( dashed lines ) \n and the average ( solid lines ) , after subtraction of the np absorbance \n and normalization to 1 at time zero ; see the experimental \n section for details of data treatment and analysis .", "( b ) effect \n of the nps alone , calculated by subtracting the photobleaching effect \n of dpbf in the absence of nps from the results obtained in the presence \n of nps ; the solid lines in ( b ) are linear fits of the data in the \n range 2060 min .", "gradient of the time - dependent dpbf absorbance photobleaching in \n the irradiation time window 2060 min for different samples \n under cw irradiation at 532 nm , 1 w ( 37 w cm ) . \n for experiments in the presence of nps", ", the np concentration was adjusted \n to yield an absorbance of 0.4 at 532 nm .", "experiments for dpbf in the \n absence of nps and in the presence of 15 nm spherical nps were also \n undertaken after bubbling the sample with nitrogen for 10 min , as \n indicated ( + n2 ) .", "the error bars correspond to the standard deviation \n of several repeat experiments , and  indicates \n statistically significant differences with respect to the experiment \n on dpbf only , as determined by the anova f - test at p < 0.001 ; it should be noted that the results for 15 or 46 nm \n spherical nps without nitrogen bubbling or a peg capping layer were \n found to be different to all other results at this statistical significance \n level .", "no repeat experiment was undertaken for irradiation of nanorods \n at 532 nm , but the same result ( no additional bleaching in the presence \n of nanorods ) was obtained for irradiation at 800 nm ( figure s5 ) . in the presence \n of citrate - stabilized spherical gold nps ,", "the photobleaching \n of dpbf upon irradiation at 532 nm , i.e. , within the nanoparticle \n plasmon resonance band , is significantly increased ( figure 1b ) .", "the time dependence of \n the dpbf photobleaching in the absence of nps and in the presence \n of nps with 15 and 46 nm diameter is shown in figure 2a .", "it can be clearly seen that the \n presence of nps leads to a significant increase of the dpbf photobleaching \n effect and that larger nps yield a larger effect although the np absorbance \n was adjusted to be the same for all samples , so that the same amount \n of light was absorbed .", "the additional photobleaching of dpbf caused \n by the presence of nps can be clearly ascribed to the generation of \n reactive oxygen species ( ros ) , since purging of the samples with nitrogen \n removes this effect ( see below ) .", "moreover , previous experiments had \n shown that light irradiation of citrate - stabilized spherical gold \n nps leads to the characteristic luminescence of o2 at 1280 nm and does not result \n in the generation of superoxide , o2 , \n or hydroxyl , oh , radicals , which leads \n us to conclude that the predominant ros species produced here is singlet \n oxygen , o2 . the fact that significant \n dpbf photobleaching occurs even in the absence of nps requires careful \n consideration for the analysis of these data .", "like dpbf photobleaching \n in the absence of nps , the effect in the presence of nps shows two \n phases .", "however , subtraction of the effect observed when only the \n dye is present yields an essentially linear time dependence for the \n additional effect ascribed to the nps on the time scale of the experiment \n ( figure 2b ) , indicating \n that the np - induced photogeneration of o2 is \n essentially constant over our experimental time interval .", "moreover , \n the second phase of direct dpbf photobleaching is largely independent \n of the laser power , at least down to 0.1 w , i.e. , significantly lower \n powers than those used for most of the experiments reported here , \n as shown in detail in the supporting information ( figure s4a ) , and therefore should be independent of the presence \n of nps whose absorbance leads to a decrease of the laser power along \n the beam path , allowing a direct comparison of the results obtained \n in the time frame of 2060 min .", "moreover , this slower phase \n of direct dpbf photobleaching is also independent of the presence \n of oxygen . for these reasons , only data starting at 20 min irradiation \n will be used for quantitative comparisons .", "figure 3 summarizes \n the main results obtained here after np photoexcitation at 532 nm . \n", "it is very obvious that citrate - stabilized spherical gold nps induce \n significantly faster dpbf photobleaching than is observed in their \n absence ( black ) ; this is found for 15 nm gold nanoparticles ( red ) \n but is even more pronounced for larger nps with 46 nm diameter ( blue ) . \n", "increasing the size of the spherical nps from 15 to 46 nm increases \n the np - induced dpbf photobleaching ( i.e. , the additional effect , after \n subtracting the direct dpbf photobleaching effect ) by ca .", "it is interesting to note \n that unlike the direct dpbf photobleaching , which is not affected \n by nitrogen purging , the np - induced additional bleaching is significantly \n reduced by nitrogen purging , almost down to the level of the direct \n dpbf photobleaching effect .", "this strongly supports the conclusion \n that the np - induced effect is caused by the formation of o2 which then leads to dpbf photobleaching .", "furthermore , \n capping the 15 nm gold nps with peg - oh , a moderately large ligand \n ( figure s3 ) , essentially removes the np - induced \n bleaching and reduces the observed effect to the level of the direct \n dpbf photobleaching .", "gold nanorods ( nrods ) with a length of \n 40 nm and a diameter of \n 12 nm , which have two plasmon resonance bands , namely the transverse \n band at 522 nm and the longitudinal one at 798 nm ( figure s1a ) , stabilized with a capping layer consisting of \n a mixture of a peg polymer ( mpeg5000 ) and a peptide ( c - tat ) ( see the experimental section ) , were also investigated .", "irradiation \n at 532 nm , i.e. , in the transverse plasmon resonance band , showed \n no additional dpbf bleaching above the direct dpbf effect ( figure 3 ) .", "irradiation with \n 1 w ( 11 w cm ) cw laser power at 800 nm , i.e. , \n in the longitudinal plasmon resonance band ( figure s5 ) , showed reduced direct dpbf photobleaching compared to \n irradiation at 532 nm , but again no additional photobleaching was \n observed in the presence of nrods .", "these results show that the nrods \n used here , which have a ligand layer consisting of a mixture of a \n peg polymer and a peptide , do not induce the formation of o2 upon laser irradiation , independent of the plasmon \n resonance which is photoexcited .", "irradiation at 532 nm in the \n presence and absence of citrate - stabilized \n spherical gold nps with 15 nm diameter was also investigated using \n laser pulses with a pulse duration of 5 ns ( figure 4 ) . in this case , the uv  vis spectra \n show a slight broadening of the np plasmon resonance band at 520 nm \n during the first 5 min of irradiation , suggesting that some aggregation \n occurs ; after the initial 5 min , however , the nps remain stable .", "analysis \n of the spectra shows that photobleaching of dpbf in the absence of \n gold nps is similar to the results obtained using cw laser irradiation \n at comparable average powers ( figure s4a ) . in the presence of gold nps with 15 nm diameter , on the other \n hand , the np - induced bleaching effect ( i.e.", ", the effect remaining \n after subtracting the direct dpbf photobleaching effect ) which is \n induced by pulsed laser irradiation is larger than that caused by \n cw irradiation by almost 1 order of magnitude , in spite of the significantly \n lower laser power employed during the pulsed irradiation experiments \n ( 0.15 w vs 1 w ) , which leads to a correspondingly lower number of \n absorbed photons ; compare figure 4b ( pulsed irradiation ) with figure 3 ( cw irradiation ) .", "photobleaching of the \n dpbf absorbance upon laser irradiation with \n 5 ns laser pulses at 532 nm , 0.15 w , 10 hz repetition rate ( corresponding \n to a power density of 1.5 w cm and a pulse energy \n density of 0.15 j cm ) .", "( a ) absorbance spectra \n in the presence of citrate - stabilized spherical gold nps with 15 nm \n diameter , taken at intervals of 5 min from before the irradiation \n ( gray ) up to a maximum irradiation time of 30 min ; the arrow indicates \n the direction of change .", "( b ) gradient of the time - dependent dpbf photobleaching \n ( measured at 412 nm ) in the irradiation time window of 2030 \n min in the absence and presence of nps .", "it is straightforward to estimate the quantum yield of np - induced \n dpbf photobleaching from the absorbed laser power and the observed \n rate of absorbance bleach .", "for the citrate - stabilized spherical nps \n with 15 and 46 nm diameter under cw irradiation at 532 nm , this yields \n values of 5  10 and 8  10 , respectively .", "thus , less than one of each 1 million photons absorbed \n by a np leads to the photobleaching of a dpbf molecule under cw irradiation . \n for irradiation of 15 nm nps with 5 ns laser pulses , on the other \n hand ,", "the quantum yield of np - induced dpbf photobleaching is 3.5  \n 10 , i.e. , almost 2 orders of magnitude larger \n than for cw irradiation , but still very small .", "diffusion of \n oxygen over the lifetime of o2 ( approximately 6 s in 50/50 water / ethanol ) covers a distance larger than the average distance \n between dpbf molecules at the concentrations used here , and hence \n it can be concluded that a significant fraction of the photogenerated o2 which escapes from the np surface should be \n detected .", "10 m , which is close to the \n concentration used here , is sufficient to detect 50% of photogenerated o2 .", "thus , the observed \n low quantum yield of dpbf photobleaching indicates a very low quantum \n yield of o2 photogeneration by gold nps ( i.e. , \n number of o2 generated for each photon absorbed ) , \n having values of the order of ca . 10 for cw irradiation \n and ca . 10 for irradiation with nanosecond laser \n pulses .", "our results show that irradiation of citrate - stabilized \n spherical gold nps at 532 nm , i.e. , in their surface plasmon band , \n with pulsed or cw laser light leads to the production of o2 , detected here by monitoring the bleaching of dpbf \n absorbance .", "however , the rate of o2 production \n is much larger when using short laser pulses than when using cw light \n of comparable intensity . whereas ca .", "24% of the dye is bleached after \n only 10 min of irradiation of 15 nm nps with 5 ns laser pulses at \n an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached over this \n time by irradiation of the same nps with cw light at significantly \n higher power ( 1 w ) ( figure 2 ) .", "this difference allows one to draw important conclusions \n on the mechanism of o2 production by irradiation \n of gold nanoparticles .", "irradiation \n of spherical gold nps at 532 nm leads to the excitation of their plasmon \n resonance , which can be described as a coherent oscillatory motion \n of the conduction band electrons ; this oscillation dephases and decays \n on the sub-100 fs time scale , with only a very small radiative contribution , \n so that most of the excitation energy is retained as electronic excitation \n in the form of electron  hole pairs .", "since the photon energy \n is close to the minimum energy required for direct excitation of d - band \n electrons into the conduction band of gold , a minor contribution of \n this excitation mechanism can not be ruled out ; however , d - band holes \n are filled by conduction band electrons on the 10 fs time scale , yielding \n essentially the same outcome as excitation of the plasmon resonance \n band .", "the excited electrons initially \n have a nonthermal energy distribution , and often are referred to as \n  primary hot electrons ", ", although the concept of temperature \n does not strictly apply to such a distribution .", "they rapidly ( within \n less than 500 fs ) equilibrate by electron  electron scattering \n to yield a fermi distribution corresponding to an elevated temperature \n and can then be referred to as  hot electrons  .", "thiol bond dissociation \n at the surface of gold nps and have been \n suggested to be responsible for the creation of o2 by irradiation of gold nps with nanosecond laser pulses .", "they lose their energy on the time scale of \n a few picoseconds by interaction with the lattice ( electron  phonon \n scattering ) with coupling times that are essentially size - independent \n for nps above 10 nm in diameter , although they strongly depend on \n the amount of energy deposited due to the temperature - dependent electronic \n heat capacity .", "because the lattice heat capacity \n is much larger than the electronic heat capacity , this leaves the \n electrons and the lattice in equilibrium at a temperature which is \n significantly lower than the initial electronic temperature ; finally , \n cooling occurs by heat transfer to the solvent and heat diffusion \n on the time scale of 10 to a few 100 ps , strongly depending on np \n size .", "since the dissipation of \n the absorbed energy proceeds on the picosecond time scale , excitation \n with a nanosecond laser pulse yields a highly nonequilibrium situation \n during the duration of the laser pulse where energy is continuously \n deposited into the electronic system and at the same time flows through \n the lattice into the surrounding solvent .", "it is therefore not straightforward \n to predict the temperature of the hot electrons achieved in such experiments . \n", "we used the  two - temperature model  for the electron \n and phonon heat baths coupled to finite - element heat transfer \n and diffusion simulations in the surrounding solvent to estimate this \n temperature ; details of these simulations are given in the supporting information .", "as shown in figure 5 , under our conditions \n the electrons are expected to reach a temperature of 2100 c , \n whereas the lattice reaches temperatures of the order of 1400 c \n and the solvent in the immediate vicinity of the np a temperature \n of about 900 c .", "time - dependent temperatures of the conduction band electrons \n ( red ) , \n lattice ( black ) , and first solvent layer ( blue ) , calculated for our \n experiments using nanosecond - laser pulse excitation ( 15 nm spherical \n nps in 50/50 etoh / water , 5 ns laser pulses with 0.15 j cm intensity , solid lines ) and for the experiments described in ref ( 24 ) ( 40 nm spherical nps in \n 80/20 etoh / water , 7 ns laser pulses with 0.03 j cm intensity , dashed lines ) using the  two - temperature model  \n for the electron and phonon heat baths , coupled to finite - element \n heat transfer and diffusion simulations in the surrounding solvent \n ( see supporting information for details ) ; \n time zero corresponds to the center of the laser pulse .", "a lattice temperature of 1400 c , which is \n above the melting \n temperature for solid gold , albeit present for only a few nanoseconds , \n might be sufficient to cause temporary melting of the nanoparticle , although the pulse intensities used here are \n still below the reported threshold for size reduction of gold nps \n of 15 nm diameter by nanosecond laser pulses .", "nevertheless , a minor effect on the shape and/or size of the nps , \n especially at the upper end of the size distribution , can not be ruled \n out and may be related to the minor np spectral changes observed upon \n irradiation which suggest some aggregation to occur ( see above ) . because \n of the slower heat dissipation around larger nps , these are heated \n to higher temperatures and hence are more likely to fragment , in agreement \n with experimental results on nanosecond - laser pulse induced fragmentation , \n which also show that fragmentation is finished after 5 min under conditions \n similar to the ones used here . since \n our nps were prepared without excess citrate , the resulting increase \n in the ratio of surface area to volume may indeed cause some aggregation . \n", "it also can not be ruled out that the solvent near the np surface temporarily \n forms bubbles , although the pulse intensities \n used here are still below the reported threshold for bubble formation \n by nanosecond pulses for 15 nm nps .", "such \n bubbles could lead to better thermal insulation , thus potentially \n increasing the maximum temperatures , but on the other hand , they might \n prevent oxygen from reaching the np surface , thus reducing the chance \n of o2 formation .", "the formation of o2 from the triplet ground state by interaction with a \n photoexcited sensitizer requires a change of the electron spin and \n hence can not result from dipole ", "dipole ( frster ) interaction , \n but only from dexter - type electron exchange coupling ; the latter can \n be described as simultaneous transfers of an electron from one of \n the 2 * molecular orbitals on oxygen to a photogenerated hole \n on the sensitizer and of an electron with opposite spin from a high - energy \n excited sensitizer level to the same or the other 2 * orbital , \n resulting in the formation of the  or  singlet oxygen state , respectively .", "this mechanism requires significant overlap of the relevant electronic \n wave functions and hence only occurs at short distances of at most \n 10  . since the photoexcited hot electrons have such a short \n lifetime , this reaction can only occur if an oxygen molecule happens \n to be in the vicinity of the np or is temporarily adsorbed to its \n surface at the moment of excitation .", "a similar reaction occurs on \n photoexcited si nanocrystals , although in this case \n the reaction is more efficient than for au nps due to the much longer \n lifetime of the photoexcited excitons in si . thus , the short lifetime \n of hot electron excitation in gold nanoparticles easily explains the \n low quantum yield of o2 photogeneration which \n is observed here .", "the excitation of electrons to temperatures \n exceeding 2000 c means that a significant number have sufficient \n energy to excite an oxygen molecule to the  state , \n which has an energy of 0.98 ev above the  ground \n state ( see figure 6 ) .", "the number of hot electrons available \n at the higher energy  and the number of holes available at the \n lower energy involved in this two - electron exchange reaction  can \n be estimated from the density of states of gold and the fermi distribution , \n as described in more detail in the supporting information .", "for example , for a spherical np with 15 nm diameter \n at an electron temperature of 2100 c , there are 260 electrons \n within an energy interval of 0.1 ev around the  \n state .", "( it should be noted that the relevant energy interval is the \n width of this state for an oxygen in the vicinity or temporarily adsorbed \n onto a np , which is not known , so only relative numbers will be used \n here . )", "thus , the electron temperatures achieved in our experiment \n are sufficient for a significant population of hot electrons and holes \n at the relevant levels .", "schematic diagram showing the population probability f(e ) for a np electron state at energy e near the fermi level , ef , \n under different \n conditions : ( a ) in equilibrium at room temperature , ( b ) at an electron \n temperature of te = 2100 c after \n electron  electron equilibration (  hot electrons  ) , \n and ( c ) immediately after the absorption of photons by single electrons \n (  primary hot electrons  , with population changes highly \n exaggerated to make them visible ) . also shown are the energies of \n the ground - state triplet (  ) and lowest - excited \n singlet state (  ) of oxygen as well as the next \n singlet state (  ) under the assumption that ef is equidistant from the  \n and  energies .", "excitation of an oxygen molecule \n to o2 requires the simultaneous transfer of \n an electron from the oxygen to a hole at the energy of the  state and of a hot electron with the opposite spin and an \n energy at the  ( or  ) level to \n the oxygen molecule .", "the large number of equilibrated hot electrons available \n during \n a nanosecond - laser pulse also rules out the primary hot electrons \n as the main source of o2 photogeneration .", "the \n number of primary hot electrons available during the pulse duration \n can be estimated from the number of photons which are absorbed per \n nanosecond , multiplied by their lifetime , which is less than 500 fs .", "however , these primary hot electrons populate \n np states at energies from the fermi level ef to ef + 2.34 ev ( the energy of \n a photon at 532 nm ) ( see figure 6c ) . for 15 nm nps and excitation with 5 ns laser pulses \n with 0.15 j cm intensity", ", this predicts that not \n more than three primary hot electrons are available within an energy \n interval of 0.1 ev around the  level at any time \n during the laser pulse , assuming that all levels are equally populated \n and taking into account that the density of states for gold has an \n essentially constant value in the relevant energy range around ef .", "this is significantly \n less than the number of hot ( equilibrated ) electrons available at \n the maximum electron temperature , which was estimated to be on the \n order of 260 for the same energy interval ( see above ) .", "thus , we can \n conclude that it is indeed the hot electrons which are responsible \n for the observed o2 photogeneration when using \n short laser pulses for excitation .", "similar results to those \n obtained here using pulsed irradiation \n have recently been reported for spherical gold nps with 40 nm diameter . although a significantly lower laser pulse energy \n density ( 0.03 j cm , compared to 0.15 j cm here )", "was used under otherwise similar experimental \n conditions , slightly faster o2 photogeneration \n was observed in this study , with the dpbf absorbance decreasing by \n ca .", "24% decrease \n observed here in the first 10 min ( figure 4a ) . in this context , it is interesting to \n note that in spite of the lower laser pulse energy density the nps \n are heated to almost the same electron temperature as in our experiments \n ( see figure 5 ) .", "this \n is largely due to ( i ) heat dissipation from larger nps being slower and ( ii ) the use of 80/20 etoh / water as solvent \n in ref ( 24 ) , which \n has slower heat transport than a 50/50 etoh / water mixture .", "an explicit \n calculation of the number of hot electrons available at the energy \n of the oxygen  state for the two experiments is \n given in the supporting information ; together \n with a detailed consideration of all other experimental differences , \n these numbers yield very good agreement between the expected and the \n observed relative o2 photogeneration rates \n ( see the supporting information for details ) . \n", "this provides further support for the conclusion that o2 photogeneration is mediated by the equilibrated hot \n electrons of gold nanoparticles under nanosecond - pulsed laser irradiation .", "compared to the \n effect of pulsed irradiation , the rate of o2 photogeneration is much smaller when using cw light of comparable \n intensity . whereas ca .", "24% of the dye is bleached after only 10 min \n of irradiation of spherical nps with 15 nm diameter with 5 ns laser \n pulses at an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached \n over this time by irradiation of the same nps with cw light at significantly \n higher power ( 1 w ) ( figure 2 ) . in a previous publication , significantly higher rates of dpbf photobleaching had been reported \n under conditions which appear to be similar to the ones used here . \n in this context", ", we note that we also observed such significantly \n higher rates of bleaching , but only when the sample preparation protocol \n described in the experimental section was \n not followed accurately ; for example , the use of dye solution that \n had not been freshly prepared or of a cuvette that had not been cleaned \n thoroughly and rinsed multiple times with mq water or the use of a \n cell that was not sealed during irradiation , leading to some loss \n of ethanol from the solution , all resulted in larger and highly irreproducible \n bleaching of dpbf under cw irradiation , up to levels comparable to \n those reported in ref ( 24 ) , even in the absence of nps . in the following", ", we will show \n that photogeneration of o2 under cw irradiation , \n unlike pulsed irradiation , is mediated by the initially created ", "primary \n hot  electrons ; i.e. , it occurs during the short time during \n which the excited electrons have not yet relaxed to a thermal distribution \n ( see figure 6c ) . under \n the cw irradiation conditions used here ( 1 w , 1.85 mm beam diameter ) , \n a spherical np with 15 nm diameter absorbs photons at an average rate \n of 1.3  10 s , as estimated from \n the absorption cross section and the \n beam intensity .", "this means that after absorption of a photon there \n is enough time for full relaxation and transfer of the photon energy \n into the solvent , which occurs in less than 100 ps , before absorption of the next photon .", "absorption of one \n photon by a 15 nm np yields  hot  electrons at a temperature \n of 10 k above the surrounding after electron  electron equilibration ; \n these  hot  electrons lose their energy by electron ", "phonon \n scattering within a few picoseconds to yield a np whose temperature \n is only 80 mk above the surrounding . neither of these effects is expected \n to yield any significant photochemical effects ; estimates analogous \n to those described above predict that for a spherical 15 nm np with \n an electron temperature of 35 c there are ca .", "2.6  10 electrons \n within an energy interval of 0.1 ev around the oxygen  state and the same number of holes around the  state energy .", "this means that there are on the order of 10 times less hot electrons and 10 times less holes \n available for the photoreaction than under our pulsed laser irradiation \n conditions , which rules out any significant reaction ; this is also \n confirmed by the fact that these population numbers are less than \n a factor 2 larger than those for room temperature , where no o2 is generated in the absence of light .", "this leaves \n only the primary hot electrons , i.e. , those electrons \n that are excited upon absorption of a photon but have not yet equilibrated \n by electron  electron scattering , as potential cause for photogeneration \n of o2 .", "absorption of a single photon can potentially \n excite electrons to energies of up to 2.34 ev above the fermi level . \n", "if one assumes excitation of only one electron by each photon and \n equal excitation probability for all available electrons , as shown \n in figure 6c , on average \n there will be 0.043 electrons within an energy interval of 0.1 ev \n around the oxygen  state , which is approximately \n 5000 times less than during a single laser pulse in the pulsed experiments , \n see above .", "taking into account that the same factor also applies to \n the holes required for the dexter mechanism , but correcting for the \n lifetime of the excitation ( 500 fs for primary hot electrons created \n during cw irradiation , ca .", "3 ns for the hot electron distribution induced \n by a single laser pulse ; see figure s6 ) \n and the repetition rates ( 1.3  10 s for single photon absorption during cw irradiation , 10 s for the pulsed laser irradiation ) , one would predict a rate of o2 photogeneration under our cw irradiation conditions \n which is smaller than that expected for our pulsed irradiation conditions \n by a factor on the order of 10 .", "this is significantly \n closer to the experimental results ( ratio of o2 photogeneration rates under pulsed vs cw irradiation of 1020 ) \n than any estimate based on the equilibrated  hot  electrons \n after the absorption of a single photon .", "the main discrepancy between \n the predicted and observed cw results arises from the assumption of \n direct excitation of single electrons in the above estimate , which \n is not valid for irradiation at 532 nm , i.e. , in the gold np plasmon \n resonance band , since this leads to the coherent excitation of many \n electrons which rapidly dephases without the electrons exchanging \n energy .", "thus , absorption of a single photon yields more than one primary \n hot electron , with the photon s energy distributed over all \n of them . consequently", ", the energy distribution even of the primary \n hot electrons will not extend up to 2.34 ev above ef but will be shifted toward the states nearer the fermi \n level , thus increasing the population of states around the oxygen  energy and hence the yield of o2 .", "a more quantitative estimate of this effect is beyond the scope \n of this paper .", "an alternative possibility for the mechanism \n of o2 photogeneration by gold nps could be \n envisaged , which is \n based on increased direct photoexcitation of oxygen due to the well - known \n local electric field enhancement in the vicinity of metal nps by the \n plasmon electrons .", "however , significant field enhancement extends \n to distances comparable to the dimensions of the nanoparticle , so that this mechanism is in disagreement with \n our observation that a peg - oh capping layer , which has a thickness \n of only 2 nm , completely inhibits o2 photogeneration ( figure 3 ) .", "dexter - type electron exchange coupling , \n on the other hand , is known to be of significance only over distances \n of less than 1 nm , as discussed above , and thus is further supported \n by this observation . in conclusion ,", "cw irradiation is less efficient \n than pulsed laser \n irradiation in photogenerating o2 since the \n ( equilibrated ) hot electrons , which are the main mediator of the photochemistry \n in the case of pulsed irradiation , do not have sufficient energy / temperature \n to drive o2 photogeneration .", "nevertheless , \n our results confirm that cw irradiation of spherical gold nps produces \n detectable amounts of o2 .", "the absence of significant amounts of ( equilibrated ) hot electrons \n means that o2 photogeneration proceeds via \n a different mechanism under cw irradiation compared to pulsed laser \n generation ; the above estimates indicate that it is the primary hot \n electrons , i.e. , the directly photoexcited electrons , which are responsible \n for the photochemistry here and that the photochemical reaction must \n occur before these equilibrate by electron  electron thermalization .", "photodynamic \n therapy holds great promises for medical applications , such as the \n treatment of cancer , because of the ability to selectively affect \n diseased tissue only .", "however , wider use of photodynamic \n cancer therapy is currently prevented by several limitations imposed \n by the available photosensitizers .", "these limitations include toxicity , \n poor stability and photostability , poor selectivity for cancer tissue , \n and the need of using visible light with poor tissue penetration . \n", "all of these limitations , in principle , can be overcome by the use \n of gold nanoparticles , which are nontoxic , have excellent stability \n even under irradiation , can target cancer tissue either passively \n by the enhanced penetration and retention ( epr ) effect or by active targeting , have extinction coefficients \n that are larger than those of dye molecules by several orders of magnitude , \n and can be tuned to absorb in the near - infrared spectral region for \n maximum tissue penetration .", "gold nanoparticles have been reported \n to have three potential modes of operation for inducing cell death \n by irradiation , namely ( i ) hyperthermia , which is based on the rapid \n conversion of the absorbed light energy into heat , ( ii ) np - assisted \n photodynamic therapy , in which the efficiency of a standard sensitizer \n is amplified by the np plasmon field enhancement effect , or ( iii ) \n a direct photochemical mechanism without involvement of a photosensitizer .", "the feasibility of photothermal therapy has been clearly established \n by careful experiments in vitro(1113,15,54 ) and has been shown \n to work in vivo .", "however , it should be noted that most studies reporting successful \n photothermal therapy made no attempt to either confirm significant \n heating or rule out photochemical effects , which means that some of \n these reported results could in fact arise from photochemical rather \n than photothermal effects or from a synergistic combination of photochemical \n and photothermal effects . as has been pointed out recently , another problem with many reports on photoinduced \n hyperthermia using nps is the relatively high light intensity required \n to reach sufficient temperatures , which often were well above the \n generally accepted skin tolerance threshold .", "the presence of \n gold nps can also lead to increased photogeneration \n of o2 by traditional photodynamic sensitizers , which can be ascribed to the local electric field enhancement in \n the vicinity of metal nps by the plasmon electrons , similar to the \n well - studied sers ( surface - enhanced raman spectroscopy ) effect .", "this \n approach might help to alleviate some of the drawbacks of traditional \n photodynamic therapy but does not directly overcome them , since it \n still requires the presence of a sensitizer .", "in fact , the requirement \n for two active components , gold np and sensitizer , which must be colocalized , \n introduces an additional complication , and care must be taken to avoid \n a reduction of the photosensitizer effect due to quenching of its \n excited state by the metal np . in an alternative approach", ", cell death has been demonstrated to \n occur following irradiation of intracellular ( endocytosed ) gold nps \n even at irradiation levels that are not high enough to cause significant \n heating .", "this photochemical effect has been related to the \n observation that irradiation of nps in vitro results \n in the formation of singlet oxygen , which is the active species in traditional \n photodynamic therapy .", "because of the short lifetime of o2 ( 3.4 s in water ) , its action is highly localized  an oxygen molecule only \n diffuses over the length scale of 100 \n nm in this time . for this reason , only intracellular nps", "are expected \n to trigger cell death by the photochemical route , although they may \n initially be located inside endosomes which are known to be broken \n up by nps under cw irradiation . on the \n other hand", ", the localization of the photochemical effects within individual \n cells also means that in situations where different types of cells \n coexist in close vicinity , selective targeting of particular cells \n and minimization of collateral damage should be achievable .", "this is \n different than photothermally induced cell death , which affects all \n cells within the irradiated volume more or less indiscriminately , \n as long as some of them contain nps , because of the fast diffusion \n of heat over the relevant length scales .", "the results of the \n experiments described here provide more insight \n into the direct photochemical mechanism and allow some important conclusions \n to be made for the further development of practical applications . \n", "they show that detectable amounts of o2 are \n generated by irradiation of nps with short laser pulses or cw light \n even in the absence of a photodynamic sensitizer , albeit with low \n quantum yield .", "short laser pulses are significantly more efficient \n at this process , since they can heat a significant fraction of the \n np conduction band electrons to high enough temperatures to excite \n oxygen to the singlet state .", "however , this requires pulse energy densities \n that are well above generally accepted safe levels for the irradiation \n of skin with pulsed laser light . since the effect requires the absorption \n of many photons by a np during one laser pulse , it is highly nonlinear \n with respect to irradiation intensity , and thus it will not be possible \n to compensate lower irradiation levels by longer irradiation times . \n", "furthermore , pulsed irradiation at the required intensities also can \n cause other effects , such as np fragmentation or bubble formation , and it is not clear what consequences \n these effects may have when occurring in tissue .", "although the use \n of femtosecond laser pulses , as compared to the nanosecond pulses \n used here , might alleviate some of these problems to some extent , \n they would require even more sophisticated equipment which may not \n be suitable for a clinical environment .", "the use of cw light , \n on the other hand , is straightforward and \n does not even require a laser but can be achieved with simple lamps . \n in spite of the lower quantum yield of o2 photogeneration \n by nps under cw irradiation which", "is reported here , cell death induced \n by cw light in the presence of nps has been reported in vitro and in vivo .", "although the experiments \n described here , which did not involve any biological material , were \n undertaken at light intensities that are above safe irradiation levels , \n the mechanism of o2 photogeneration by nps \n under cw illumination is shown to be based on the absorption of single \n photons .", "this means that lower light intensities can be compensated \n for by longer irradiation times , allowing one to reduce the intensity \n to safe levels for biological or medical applications without affecting \n the amount of o2 generated .", "o2 photogeneration and cancer cell destruction in vitro and in vivo have indeed been observed upon irradiation \n on the minute time scale at intensities below safe levels and using \n nonlaser light sources .", "the results presented here indicate another design criterion \n which \n needs to be fulfilled for successful implementation of the photochemical \n route of inducing cell death by irradiation of nps , namely that the \n nps must not possess a dense capping layer . even the thin capping \n layer formed by peg - oh , with a thickness of only 2 nm , is sufficient to completely inhibit o2 photogeneration ( figure 3 ) .", "this is in full agreement with the suggestion that \n energy exchange between the np and the oxygen molecule occurs via \n the dexter ( two electron exchange ) mechanism , which is limited to \n distances of less than 1 nm . similar effects have been reported for o2 photogeneration by si nanocrystals , which is \n significantly affected by a thin oxide layer .", "it is important to point out that the assay used here for o2 detection ( bleaching of dpbf ) only reports on singlet \n oxygen found outside the np capping layer ; thus , it can not be ruled \n out that even on nps with a peg - oh capping layer some o2 is photogenerated at the np surface but reacts with \n the np ligands and hence is quenched .", "on the other hand , such singlet \n oxygen would not be of any direct use for practical applications , \n such as the induction of cell death , so that the assay results in \n fact report the effects relevant for such applications .", "uncapped ( citrate ) \n nps are rapidly covered by a protein corona after they have been taken \n up into live cells , but it appears that \n this corona is permeable enough for oxygen to not completely prevent \n singlet oxygen formation , as evidenced by fact that cell death by \n the photochemical route has been observed with such nps ; it may be that because of their size proteins \n are not able to form a capping layer ( corona ) of similar density as \n the smaller ligands used here .", "similarly , a lipid bilayer appears \n to allow oxygen access to the surface of gold nanorods , whereas a dense pentapeptide ( calnn ) capping \n layer on spherical gold nanoparticles is sufficient to suppress the \n photochemical mechanism of cell death .", "it also seems likely that the thick peg capping layer present \n on \n the nanorods used here is the main reason for the absence of o2 photogeneration by nanorods upon irradiation \n in either the transversal or the longitudinal plasmon resonance band \n that was observed here . because of their longitudinal plasmon resonance \n band , which is in the near - ir spectral region with high tissue penetration , \n nanorods are more suitable for practical photodynamic applications \n in tissue .", "however , standard synthesis protocols yield nanorods within \n a bilayer of cytotoxic ctab , so that ligand exchange is required before \n any biological or medical application , and care will need to be taken \n to choose suitable ligands to allow access of oxygen to the nanorod \n surface . potential examples for these are poly(vinylpyrrolidone ) , \n lipid bilayers , or mesoporous silica , all of which have been used \n successfully in experiments showing photochemically induced cell death \n using gold nanorods ."], ["dpbf has been used successfully to detect \n singlet oxygen that is \n formed when spherical nps are irradiated at 532 nm , either with cw \n or pulsed laser irradiation .", "singlet oxygen generation by pulsed laser \n irradiation has been shown to act via the equilibrated hot electrons \n that can reach temperatures of several thousand degrees during the \n laser pulse ; cw irradiation , on the other hand , can act only via the \n directly excited primary  hot  electrons , which rapidly \n lose their energy by electron ", "electron equilibration , and hence \n is significantly less efficient for the formation of singlet oxygen . \n", "nevertheless , even cw irradiation can produce enough singlet oxygen \n for photodynamic therapy applications and will allow practical applications \n of the effect at safe irradiation levels .", "photodynamic therapy using \n gold nanoparticles will also require careful design of the nanoparticles \n with respect to size , shape , and capping layer and will require internalization \n of the nps , not just attachment to the cell surface , which is sufficient \n for photothermal therapy ."]]}
{"article_id": "PMC4420582", "article_text": ["two types of cnt were investigated , nc7000 from nanocyl and mwnt  sa from sigma  aldrich ( catalog reference 659258 ) , both produced by chemical vapor deposition ( cvd ) .", "nc7000 has a diameter of 710  nm , and mwnt  sa has a diameter of 110170  nm .", "the cnt were functionalized by a 1,3-dipolar cycloaddition reaction of azomethine ylides using a one - pot functionalization procedure described elsewhere13 leading to the formation of pyrrolidine - type groups bonded to the cnt surface .", "the functionalization was carried out at 250  c over 5  h. the unzipping of the cnt was performed using an ultrasonic processor up100h from hielscher , equipped with a sonotrode ms2 .", "cnt suspensions were prepared by mixing functionalized cnt ( 5  mg ) in ethanol ( 8  ml ) .", "a control experiment was conducted using pristine cnt ( 5  mg ) in ethanol ( 8  ml ) .", "the suspensions were centrifuged ( 8000  rpm , 1  h ) to separate the unzipped cnt , and the gnr supernatant solutions were collected .", "these solutions were analyzed by uv - visible spectroscopy on a shimadzu uv-240 1 pc , using 10  mm light path quartz cells .", "the micro - raman analysis was conducted in the backscattering configuration on a jobin yvon hr800 instrument ( horiba , japan ) , using a 1800  l  mm grating and the 532  nm laser line from a nd : yag dpss laser ( ventus , laser quantum , uk ) . a 50x objective ( spot size : 3  m , na=0.75 , olympus , japan )", "was used to focus the laser light onto the sample ( < 0.15  mw  m ) and to collect the backscattered raman radiation to be detected by a peltier cooled ( 223  k ) ccd sensor .", "the spectrometer was operated in the confocal mode , setting the iris to 300  m , while the acquisition time was set to 30  s with 2  accumulations .", "transmission electron microscopy ( tem ) of the gnr mwnt  sa was performed on an energy filtered 200  kv transmission electron microscope hr-(ef)tem ", "tem analysis of the gnr nc7000 was carried out on a titan chemistem 80200  kv probe  cs corrected microscope .", "low - magnification tem and high - resolution tem ( hrtem ) images were acquired with a gatan ultrascan 1000  p camera controlled with digital micrograph software integrated in the microscopes user interface .", "the samples were prepared by adding a drop of the gnr solution onto a lacey carbon cu grid ( 300  mesh , ted pella ) and allowing it to dry under vacuum .", "x - rays diffraction experiments were performed on a panalytical xpert pro xrd system using the cu k1 wavelength of 0.15406  nm from a copper x - ray tube operated at 45  kv and 40  ma .", "a pixcel-3d detector was used , and the scan range was from 4 to 40 in 2. the gnr samples were deposited on glass lamellae by solvent evaporation .", "the unzipping process produces functionalized gnrs with widths equal or larger than 35  nm for gnr nc7000 and 350  nm for gnr mwnt  sa . for this reason , a model of functionalized graphene ( see figure  6 ) , as opposite to finite gnr , was chosen .", "graphene layers and their intermolecular interactions were modelled with the mm3 force field that has been found to give accurate intermolecular structures.17 all the calculations were performed with the tinker molecular mechanics suite18 using three - dimensional periodic boundary conditions.19 the shape and dimensions of unit cells containing five functionalized graphene layers with different concentrations of functional groups were systematically obtained by energy minimization .", "additional data for the characterization for the gnr : thermogravimetric analysis ( tga ) and fourier - transform infrared spectroscopy ( ftir ) , is provided as supporting information .", "as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors ."], "abstract_text": ["<S> graphene nanoribbons ( gnr ) were generated in ethanol solution by unzipping pyrrolidine - functionalized carbon nanotubes under mild conditions . </S>", "<S> evaporation of the solvent resulted in regular few - layer stacks of graphene nanoribbons observed by transmission electron microscopy ( tem ) and x - ray diffraction . </S>", "<S> the experimental interlayer distance ( 0.490.56  nm ) was confirmed by computer modelling ( 0.51  nm ) . </S>", "<S> computer modelling showed that the large interlayer spacing ( compared with graphite ) is due to the presence of the functional groups and depends on their concentration . </S>", "<S> stacked nanoribbons were observed to redissolve upon solvent addition . </S>", "<S> this preparation method could allow the fine - tuning of the interlayer distances by controlling the number and/or the nature of the chemical groups in between the graphene layers . </S>"], "labels": null, "section_names": ["Experimental Section", "Supporting Information"], "sections": [["two types of cnt were investigated , nc7000 from nanocyl and mwnt  sa from sigma  aldrich ( catalog reference 659258 ) , both produced by chemical vapor deposition ( cvd ) .", "nc7000 has a diameter of 710  nm , and mwnt  sa has a diameter of 110170  nm .", "the cnt were functionalized by a 1,3-dipolar cycloaddition reaction of azomethine ylides using a one - pot functionalization procedure described elsewhere13 leading to the formation of pyrrolidine - type groups bonded to the cnt surface .", "the functionalization was carried out at 250  c over 5  h. the unzipping of the cnt was performed using an ultrasonic processor up100h from hielscher , equipped with a sonotrode ms2 .", "cnt suspensions were prepared by mixing functionalized cnt ( 5  mg ) in ethanol ( 8  ml ) .", "a control experiment was conducted using pristine cnt ( 5  mg ) in ethanol ( 8  ml ) .", "the suspensions were centrifuged ( 8000  rpm , 1  h ) to separate the unzipped cnt , and the gnr supernatant solutions were collected .", "these solutions were analyzed by uv - visible spectroscopy on a shimadzu uv-240 1 pc , using 10  mm light path quartz cells .", "the micro - raman analysis was conducted in the backscattering configuration on a jobin yvon hr800 instrument ( horiba , japan ) , using a 1800  l  mm grating and the 532  nm laser line from a nd : yag dpss laser ( ventus , laser quantum , uk ) . a 50x objective ( spot size : 3  m , na=0.75 , olympus , japan )", "was used to focus the laser light onto the sample ( < 0.15  mw  m ) and to collect the backscattered raman radiation to be detected by a peltier cooled ( 223  k ) ccd sensor .", "the spectrometer was operated in the confocal mode , setting the iris to 300  m , while the acquisition time was set to 30  s with 2  accumulations .", "transmission electron microscopy ( tem ) of the gnr mwnt  sa was performed on an energy filtered 200  kv transmission electron microscope hr-(ef)tem ", "tem analysis of the gnr nc7000 was carried out on a titan chemistem 80200  kv probe  cs corrected microscope .", "low - magnification tem and high - resolution tem ( hrtem ) images were acquired with a gatan ultrascan 1000  p camera controlled with digital micrograph software integrated in the microscopes user interface .", "the samples were prepared by adding a drop of the gnr solution onto a lacey carbon cu grid ( 300  mesh , ted pella ) and allowing it to dry under vacuum .", "x - rays diffraction experiments were performed on a panalytical xpert pro xrd system using the cu k1 wavelength of 0.15406  nm from a copper x - ray tube operated at 45  kv and 40  ma .", "a pixcel-3d detector was used , and the scan range was from 4 to 40 in 2. the gnr samples were deposited on glass lamellae by solvent evaporation .", "the unzipping process produces functionalized gnrs with widths equal or larger than 35  nm for gnr nc7000 and 350  nm for gnr mwnt  sa . for this reason , a model of functionalized graphene ( see figure  6 ) , as opposite to finite gnr , was chosen .", "graphene layers and their intermolecular interactions were modelled with the mm3 force field that has been found to give accurate intermolecular structures.17 all the calculations were performed with the tinker molecular mechanics suite18 using three - dimensional periodic boundary conditions.19 the shape and dimensions of unit cells containing five functionalized graphene layers with different concentrations of functional groups were systematically obtained by energy minimization .", "additional data for the characterization for the gnr : thermogravimetric analysis ( tga ) and fourier - transform infrared spectroscopy ( ftir ) , is provided as supporting information ."], ["as a service to our authors and readers , this journal provides supporting information supplied by the authors .", "such materials are peer reviewed and may be re - organized for online delivery , but are not copy - edited or typeset . technical support issues arising from supporting information ( other than missing files ) should be addressed to the authors ."]]}
{"article_id": "PMC4311927", "article_text": ["it has been proposed that solar devices based on thin film technologies and third - generation materials can greatly benefit from plasmonic and photonic structures that can assist with light trapping and absorption .", "one promising strategy is to replace the traditional transparent conducting oxide layer with a semitransparent metallic contact that exhibits a surface plasmon at visible wavelengths .", "structured appropriately , for example , by perforating with an array of sub - wavelength holes , the metal film can provide the high transparency and conductivity needed in a transparent electrical contact while also actively assisting with photon management .", "a recent study demonstrated a high - performing organic photovoltaic cell with a metal contact patterned with an array of sub - wavelength holes , providing the contact with broad featureless transmission throughout the visible spectrum and robust coupling to the semiconductor as a function of incident light angle . despite this progress ,", "a key challenge is to probe and understand the details of the light - matter interactions between the incident photon , the surface plasmon modes of the structured contact , and the absorption properties of the active area .", "we have developed a set of small area , high - brightness spectroscopies that are appropriate for characterizing prototypes of light - harvesting devices utilizing plasmonic contact materials , for understanding the details of light - matter interactions in these systems .", "our primary interest in developing a set of rapid , high - sensitivity characterization tools capable of probing the influence of the perforated metal nanostructure , including pore size and pitch , as well as film thickness on the optical transmission modes , and ultimately the effect on solar cell performance .", "the aim is both to enable rapid screening of test device structures and to design structures capable of providing specific physical insight into coupling between the device active area and the modes of the patterned metal film .", "our strategy utilizes fourier transform ( ft)-based measurements in the solar portion of the optical spectrum , for optimally combining spatial resolution , spectral resolution , speed , and dynamic range .", "ft - based measurements are highly advantageous for characterizing nanoscale photovoltaic materials in the visible and near infrared ( nir ) portions of the electromagnetic spectrum , although they are more typically applied in the infrared . as compared to scanned techniques , ft photocurrent measurements benefit from the multiplex ( fellgett ) advantage because the detectors are not shot - noise - limited , unlike in optical measurements employing low - noise photodetectors ( e.g. raman scattering with a high sensitivity ccd or pmt ) .", "this is particularly important since , unlike dispersive optical measurements ( e.g. transmission , pl , and raman ) , photocurrent multi - channel detection with scanned sources is not typically possible . for measurements of photovoltaic phenomena ,", "the throughput advantage ( jacquinot ) is much less relevant due to the lower required resolution , when compared to vibrational or high - resolution absorption measurements .", "this is particularly true when using a high - brightness light source and a small sample size such that the throughput becomes comparable to a scanned source .", "however , ft - based measurements still typically have more bandwidth capabilities , as there is no need to switch gratings between the visible and the nir .", "the key to the experiment is achieving solar ( or higher ) spectral irradiance levels in a spot size of < 100  m in order to achieve high signal / noise external quantum efficiency ( eqe ) measurements on prototype devices .", "we have recently demonstrated the power of these types of measurements on individual single - walled carbon nanotube field effect transistors , but the benefit is equally applicable to other structures with active areas of the order of ( 100  m ) , such as photonic structures that are difficult and/or time - consuming to fabricate over large areas .", "achieving a high irradiance in a small area requires the use of high radiance sources .", "the alternative  increasing the power without increasing the size of the source or solid angle of radiation  becomes impractical at these size scales , because most of the photon flux would be wasted . in the mid- to far - ir , similar advantages are gained using synchrotron - based sources for microspectroscopy . here ,", "we achieve these advantages in the solar spectral region with an inexpensive benchtop source  a true - cw high - brightness laser - driven light source ( energetiq eq-99 ) .", "this differs significantly from a laser , in that the output radiates incoherently into 4 rather than being highly directional and coherent .", "we have also examined laser supercontiuum sources for the same purpose ( e.g. fianium sc-450 ) , which are able to achieve even higher brightness levels due to the highly collimated output . however , for photocurrent and photovoltaic applications , we have found the laser - driven light source to be a much better choice for several reasons .", "primarily , the pulsed nature of the laser supercontinuum was found to generate small but detectable electrical transients that added significant noise to low - eqe regions of the spectrum .", "since a 200  m core multi - mode fiber is utilized to simplify alignment to the translating microscope optics , this negates the advantage a smaller emitter has in focusing capabilities .", "additionally , we have found it difficult to stay in a low - fluence ( solar - like ) regime with the pulsed laser ; focusing ps pulses with the full bandwidth of the laser has resulted in either saturation ( resulting in signal non - linearity ) or damage to samples and optical detectors , even with significant attenuation of the beam .", "we did not encounter the same issue for similar power levels in a true cw source .", "lastly , the laser - driven light source unit is significantly cheaper than the laser supercontinuum ( ~5    difference ) .", "similarly , other groups have found that despite the higher spectral power density of a supercontinuum source , the sensitivity of absorption - based measurements are higher with the ldls due to superior long - term stability .", "the laser - driven light source output is collimated by an off - axis parabolic mirror ( 1.5 diameter , 2 efl , na  =  0.375 ) and coupled into a commercial ft spectrometer ( bruker v80 ) using the backward input .", "the light is modulated using a broadband caf2 uv  vis - nir beam splitter and the slowest  rapid - scan  setting available on our instrument ( 2.5  khz referenced to the hene line ) .", "the modulated light is sent to an output port of the spectrometer , where the remaining uv portion of the beam ( < 420  nm ) is filtered out .", "the broadband light is focused into a 200  m core multimode fiber used to couple the light into an upright optical microscope ( nikon fn-1 ) using a reflective collimating optic and a set of steering mirrors attached to an epi - illuminator .", "this microscope is designed for a stationary stage , such that electrical probes can be contacted with a sample via micromanipulators .", "as such , the microscope itself sits on a translating stage for sample visualization and beam alignment . the use of an optical fiber for coupling ensures that the beam stays aligned to the microscope even during translation .", "a 5050 beam splitter is used to simultaneously image and excite the device under test . for photoconductivity measurements", ", the photocurrent interferogram is sent through a trans - impedance amplifier ( dl instruments , 1210 ) and an electronic band - pass filter ( stanford research , sr650 ) before being digitized by the spectrometer bench electronics . to correct for the lamp spectrum and the transmission function of the bench and microscope optics , a set of photodiodes with known responsivity curves", "the resulting single - beam spectra from test devices are corrected by these reference spectra , which are collected at the same time as the unknown devices .", "the spot sizes at the output of the microscope are measured using a calibrated beam profiler ( ophir - spiricon ) . for transmission spectra ,", "the sample compartment of the spectrometer is used , resulting in a spot size of 0.75  mm at the focus .", "samples are aligned to the beam using a xyz translating mount and the transmitted light is detected with a pyrolelectric detector ( la - dtgs ) .", "we define arrays of sub - wavelength holes across areas of ( 300  m ) by focused ion beam ( fib ) milling completely through 50  nm thick ag films sputter deposited on glass .", "holes with average diameter of 200  nm are milled using a beam of ga ions ( 0.26  na ) , with dwell times ranging from 2 to 8  ms for hole separations ( pitch ) ranging from 240 to 560  nm , respectively .", "after defining the perforated ag electrode , the subsequent organic photovolatic device fabrication steps include : thermal evaporation of 9  nm thick of moo3 electron - blocking layer ; spin coating ( 700 rpm for 60  s ) the 170  nm thick semiconductor active layer , a 1:1 wt .", "blend of poly(3-hexlythiophene ) : phenyl - c61-butyric acid methyl ester ( p3ht : pcbm ) ( 3 wt .", "% in chlorobenzene ) ; and thermal evaporation of a 200  nm al film , which serves as an electron - collecting ( and hole - blocking ) electrode .", "we match the source , coupling , and focusing optics in order to optimize the throughput and achieve a high - brightness white - light spot .", "the critical quantity is the optical extent or tendue ( g ) of the system that will maximize the light throughput to the sample .", "it is this quantity that needs to be appropriately matched throughout the system to maximize the spectral irradiance , rather than the numerical aperture of the individual optical components .", "because g is proportional to both the area of the source and the solid angle into which it propagates , we aim to match the collimating and focusing optics without introducing a bottleneck ( e.g. the multimode optical fiber ) . the combination of the laser - driven light source and a parabolic mirror results in g ~ 3    10  mm sr , a value well matched to a 0.22 numerical aperture fiber with a 0.2  mm core size ( ~4.5    10  mm sr maximum g ) .", "the effective tendue of the sample is determined by the spot size of the light and the microscope objective used to image and focus the light .", "the numerical aperture of the objective determines the solid angle , but the spot size is not determined by the nominal magnification , which is referenced to a 200 mm tube lens .", "we assume that the reference specifying the magnification value is a 200  mm tube lens . here", ", the value of the objective magnification is now defined by the numerical aperture of the fiber itself and the collimating optic . as a result ,", "the magnification of the objective is considerably smaller than the 10/50  values specified . for the 10  objective ,", "we obtain nearly 1:1 imaging , with the core diameter of the multimode fiber limiting the ultimate spot size .", "for example , focusing the broadband ft - modulated light source with a 10    ( 0.3 numerical aperture ) long - working distance objective yields a measured spot size of ~215  m ( beam profile shown in figure 1(a ) ) . we can achieve a smaller spot size using the 50 objective , for two reasons .", "first , the higher numerical aperture results in additional magnification , and second , the smaller entrance aperture of the higher na objective slightly reduces the collimated beam size ( thereby reducing the effective source size and the tendue out of the fiber ) .", "the net result is a reduction in spot size to ~40  m using the nominal 50    ( 0.45 numerical aperture ) objective . as we discuss below , this small reduction in throughput still results in an overall higher spectral irradiance .", "these larger - than - diffraction - limit spots are due to the necessity of using of a multimode optical fiber to accommodate a large spectral bandwidth . using a single - mode fiber and a monochromatic solid - state laser", ", we can achieve near diffraction - limited spots using these same optics . from the measured spot sizes and the focal length of the objectives", ", we can determine the ntendu of the sample in order to find the overall limiting factor in the optical system .", "in all cases , the ntendue of the 10  objective is higher than that of the source and fiber , meaning that the limiting factor is not the small area of the detector ( i.e. sample ) .", "( a ) beam profile of the image formed using the eq-99 laser - driven light source and a low magnification 10 objective .", "the spot size is approximately 215  m with the 10 and 40  m with the 50  .", "( b ) spectral irradiance in the visible and near infrared with the 10  and 50  objectives compared to the solar air mass 1.5 standard ( astm g-173 ) .", "( the colour version of this figure is included in the online version of the journal . ) with the combination of a high - radiance light source and microscope focusing optics , we can achieve high - spectral irradiances for highly sensitive quantum efficiency measurements on small area devices .", "the spectral irradiance is shown in figure 1(b ) for the output of the ldls microscopy system with the 10 and 50 objectives , along with the solar air mass 1.5 ( astm g173 - 03 ) spectrum for comparison .", "the key metric is that the solar spectral irradiance can be matched or exceeded across the visible and nir , particularly in the visible region of the spectrum , where we achieve in excess of 10 times higher irradiances .", "these numbers can be further improved since a 50:50 beam splitter ( optimized for the visible ) is used to direct light into the microscope objective .", "replacing the dielectric optic with a metallic one would double the output in the visible , but result in even more significant gains in the nir .", "however , for the purposes of this study , the irradiances achieved are more than sufficient for our measurements .", "the overall photon flux achieved is also quite high due to strong focusing of the light beam , exceeding 4.5    10  ms with high spatial resolution .", "we match the source , coupling , and focusing optics in order to optimize the throughput and achieve a high - brightness white - light spot .", "the critical quantity is the optical extent or tendue ( g ) of the system that will maximize the light throughput to the sample .", "it is this quantity that needs to be appropriately matched throughout the system to maximize the spectral irradiance , rather than the numerical aperture of the individual optical components .", "because g is proportional to both the area of the source and the solid angle into which it propagates , we aim to match the collimating and focusing optics without introducing a bottleneck ( e.g. the multimode optical fiber ) . the combination of the laser - driven light source and a parabolic mirror results in g ~ 3    10  mm sr , a value well matched to a 0.22 numerical aperture fiber with a 0.2  mm core size ( ~4.5    10  mm sr maximum g ) .", "the effective tendue of the sample is determined by the spot size of the light and the microscope objective used to image and focus the light .", "the numerical aperture of the objective determines the solid angle , but the spot size is not determined by the nominal magnification , which is referenced to a 200 mm tube lens .", "we assume that the reference specifying the magnification value is a 200  mm tube lens . here", ", the value of the objective magnification is now defined by the numerical aperture of the fiber itself and the collimating optic . as a result ,", "the magnification of the objective is considerably smaller than the 10/50  values specified . for the 10  objective ,", "we obtain nearly 1:1 imaging , with the core diameter of the multimode fiber limiting the ultimate spot size .", "for example , focusing the broadband ft - modulated light source with a 10    ( 0.3 numerical aperture ) long - working distance objective yields a measured spot size of ~215  m ( beam profile shown in figure 1(a ) ) . we can achieve a smaller spot size using the 50 objective , for two reasons .", "first , the higher numerical aperture results in additional magnification , and second , the smaller entrance aperture of the higher na objective slightly reduces the collimated beam size ( thereby reducing the effective source size and the tendue out of the fiber ) .", "the net result is a reduction in spot size to ~40  m using the nominal 50    ( 0.45 numerical aperture ) objective . as we discuss below , this small reduction in throughput still results in an overall higher spectral irradiance .", "these larger - than - diffraction - limit spots are due to the necessity of using of a multimode optical fiber to accommodate a large spectral bandwidth . using a single - mode fiber and a monochromatic solid - state laser", ", we can achieve near diffraction - limited spots using these same optics . from the measured spot sizes and the focal length of the objectives", ", we can determine the ntendu of the sample in order to find the overall limiting factor in the optical system .", "in all cases , the ntendue of the 10  objective is higher than that of the source and fiber , meaning that the limiting factor is not the small area of the detector ( i.e. sample ) .", "( a ) beam profile of the image formed using the eq-99 laser - driven light source and a low magnification 10 objective .", "the spot size is approximately 215  m with the 10 and 40  m with the 50  .", "( b ) spectral irradiance in the visible and near infrared with the 10  and 50  objectives compared to the solar air mass 1.5 standard ( astm g-173 ) .", "( the colour version of this figure is included in the online version of the journal . )", "with the combination of a high - radiance light source and microscope focusing optics , we can achieve high - spectral irradiances for highly sensitive quantum efficiency measurements on small area devices .", "the spectral irradiance is shown in figure 1(b ) for the output of the ldls microscopy system with the 10 and 50 objectives , along with the solar air mass 1.5 ( astm g173 - 03 ) spectrum for comparison .", "the key metric is that the solar spectral irradiance can be matched or exceeded across the visible and nir , particularly in the visible region of the spectrum , where we achieve in excess of 10 times higher irradiances .", "these numbers can be further improved since a 50:50 beam splitter ( optimized for the visible ) is used to direct light into the microscope objective .", "replacing the dielectric optic with a metallic one would double the output in the visible , but result in even more significant gains in the nir .", "however , for the purposes of this study , the irradiances achieved are more than sufficient for our measurements .", "the overall photon flux achieved is also quite high due to strong focusing of the light beam , exceeding 4.5    10  ms with high spatial resolution .", "in order to demonstrate the utility of a technique having simultaneously high spatial resolution , spectral resolution , and high sensitivity , we examined a set of prototype organic photovoltaic devices that employed a nanostructured plasmonic ag thin film as a transparent electrical contact .", "first , we discuss the photophysical properties of the perforated metal films with sub - wavelength apertures , in order to best match the contact s optical properties to the light absorption bands in the organic active area .", "we fabricated the nanostructured electrical contacts by depositing a thin ag film on glass and using a fib tool ( fei helios dualbeam ) to mill square arrays of uniformly sized holes across typical areas of ( 0.3  mm ) ( scanning electron micrograph , figure 2(a ) ) .", "we can fully characterize even these small - area samples because of the high - brightness light source , therefore minimizing the associated fabrication time .", "the advantage of using the fib for fabrication is that the design can be varied ( e.g. adjusting hole size and/or diameter ) without the need for any process re - optimization .", "the variation in an average hole size introduced by our fabrication approach does not significantly affect the optical properties of the array .", "the absolute optical transmission spectra of ag films fully perforated with hole arrays having pitches ranging from 240 to 560  nm on glass show a series of peaks and troughs resulting from the plasmon and diffraction modes in the array ( figure 2(b ) .", "spectra are offset for clarity ) . as predicted from momentum conservation arguments , these mode positions are nearly constant on a wavelength - to - pitch ratio scale .", "( a ) scanning electron micrograph of a silver thin film perforated with a hole array having a pitch of 400  nm .", "( b ) absolute optical transmission spectra of 100  nm thick ag films with a total area of ( 300  m ) on glass vs. the wavelength / pitch ratio , for different pitch values .", "( the colour version of this figure is included in the online version of the journal . )", "the optical transmission properties of these perforated metal films are sensitive to the dielectric interfaces on either side , such that the modes of the film when assembled into solar cell will shift compared to those of the same metal film on glass . in order to understand the metal film transmission properties of our target device structure , we coat the perforated ag film ( 100  nm thick , 200  nm average hole diameter , and 400  nm average pitch ) with a 220  nm thick polymer layer ( microposit s1811 ) that mimics the dielectric constant of the organic semiconductor in the non - resonant regime of the spectrum , but does not exhibit significant absorption at visible wavelengths .", "to best imitate the device structure , we insert a 17  nm thick tio2 layer between the ag and polymer films , because such selective electronic transport layers are essential for organic device operation .", "we use finite difference time domain ( fdtd ) calculations ( lumerical solutions , inc . ) to understand the origin of the primary transmission resonances and their effect on the target photovoltaic device .", "we find reasonably good agreement between the measured ( figure 3(a ) , black ) and calculated ( figure 3(a ) , red ) transmission curves , with a primary maximum in the transmission spectrum at ~1020  nm and a minimum near ~930  nm . we believe the peak broadening in the nir results from hole size inhomogeneities .", "however , the hole pitch uniformity generates a resonance peak that is well matched to the calculation .", "( a ) experimental ( black line ) and fdtd transmission spectrum ( red ) of a glass / perforated ag electrode ( height  =  100  nm , diameter  =  200  nm , pitch  =  400  nm)/tio2 ( h  =  17  nm)/polymer ( 220  nm)/air dielectric stack", ". normalized field intensity distributions , |e|/|e \n 0| for light with ( b )   =  927  nm and ( c )   =  1020  nm . in ( b ) and", "( c ) , the glass spans from z  =  0.2  m to z  =  0  m , the ag mha spans from z  =  0  m to z  =  0.1  m ( and the hole in the ag layer spans from x  =  0.1  m to x  =  0.1  m ) , the tio2 layer spans from z  =  0.1  m to z  =  0.117  m , and the polymer layer spans from z  =  0.117  m to z  =  0.337  m .", "( the colour version of this figure is included in the online version of the journal . )", "our calculations allow us to examine the light intensities of corresponding spatial modes in order to identify the mechanism for light dissipation or transport . in figure 3(b ) and ( c ) , we plot the normalized field intensity |e|/|e \n 0| in the xz plane ( where incident light propagates in the + z direction , downwards in the figure ) .", "this calculation shows that at 927  nm ( figure 3(b ) ) , near the transmission minimum , the field intensity is delocalized throughout the polymer active area , with a minimum under the hole open area .", "this calculated spatial distribution is consistent with that of a wood s anomaly ( evanescent mode ) , which redirects the light propagation due to diffraction .", "in contrast , the normalized field intensity profile |e|/|e \n 0| for a wavelength near the transmission maximum ( 1020  nm ) shows feature characteristics of the near - field enhancement due to surface plasmon modes in the perforated metal film , resulting in  hot spots  of electric field intensity near the edges of the contacts along the metal - polymer layer .", "though one benefit of these plasmon modes is enhanced light transmission through sub - wavelength apertures , there is also an opportunity for modification of the absorption and radiative properties of the active layer via the purcell effect .", "though this subject is beyond the scope of the current manuscript , it does imply that the benefits of perforated metal films as electrical contacts potentially extend beyond their role as passive , light - transmitting elements .", "because the primary features in the transmission spectrum of the perforated metal film result from light - matter interactions at the metal / polymer interface , we can leverage this structure for design of a solar cell transparent electrical contact . in this demonstration , we employ a 35  nm thick ag film ( perforated with a square array of 200  nm diameter holes on a 410  nm pitch ) to couple light into a 160  nm thick active layer of blended organic p3ht : pcbm .", "the device also includes both hole transporting ( 10  nm thick moo3 ) and electron transporting ( 220  nm thick al ) on either side of the organic active layer ( schematic in figure 4(a ) and cross - sectional sem image in figure 4(b ) .", "( a ) device schematic and ( b ) sem cross - section of an organic photovoltaic device with a perforated ag film as transparent contact .", "the high extinction due to absorption by the active layer occurs around 500  nm and is indicated by the arrow .", "( d ) dark ( dot - dash ) and illuminated ( lines ) j    v curves for fully fabricated devices using the ag / polymer materials shown in ( c ) .", "device hole array pitches are : 320  nm ( green ) , 400  nm ( red ) , and 480  nm ( blue ) . also shown", "is the performance of a similar device having an unpatterned 50  nm thick ag electrical contact ( dark : dashed , illuminated : black ) .", "( the colour version of this figure is included in the online version of the journal . ) the optical transmission spectra of organic devices having metal contacts with a hole array of pitches 320 , 400 , and 480  nm ( figure 4(c ) , measured prior to forming the al electrical contact ) contain features in the nir region that are similar to that of the non - resonant model system ( figure 3(a ) ) , but show dramatic differences in the spectral regions where the p3ht : pcbm blend absorbs .", "the primary polymer absorption manifests as dramatic dip around 500  nm ( indicated with an arrow in figure 4(c ) ) .", "the magnitude of the transmission ( < 10% ) is similar to that of the electrode evanescent mode near 900  nm , and is consistent with an active area thickness of ~170  nm . although the primary propagating mode of the perforated ag film ( > 700  nm ) is far from the organic material absorption peak ( at 500  nm ) , higher order surface plasmon modes ( visible at shorter wavelengths in figure 3(a ) ) can nevertheless couple light efficiently into the device structure in order to generate photocurrent .", "for example , the current - voltage ( j    v ) characteristics of fully assembled devices under illumination by 1 sun simulated am 1.5 g radiation display average photocurrent ( j \n sc ) in the range of j \n sc  ~  4  ma / cm ( nearly independent of hole array pitch ) , open circuit voltage , v \n oc \n =  0.58  v , and power conversion efficiencies around ~1% ( figure 4(d ) ) . although conventional transparent contact materials ( e.g. indium tin oxide ) have achieved power conversion efficiencies around ~4% in p3ht : pcbm devices , there are opportunities for considerable improvement by better matching the electrode s propagating mode to the absorption resonance in the organic layer .", "potential approaches to achieving performance improvements include scaling the hole arrays to smaller pitches , or leveraging recent breakthroughs in utilizing nir absorbing polymers   which are better matched to the size of the perforated metal contacts described in this work .", "the high - brightness photocurrent measurement is ideal for acquiring high - sensitivity eqe spectra of these small - area devices ( 300  m ) ( figure 5 ) .", "although in these measurements we are not required to match the lamp spectrum to the solar spectrum , we nevertheless target an overall spectral irradiance comparable to that of solar illumination in order to avoid high - fluence recombination processes .", "the achievable spot size under low magnification is 215  m , well matched to our device area of ( 300  m ) and to the solar irradiance in the active region of the device .", "these measured eqe spectra result from averaging for approximately one minute in total , because the entire spectrum ( from 420 to 2500  nm ) is collected in a single shot .", "ft photocurrent spectra of ( 300  m ) area devices on a ( a ) linear and ( b ) log scale for organic photovoltaic devices with perforated metal film transparent contacts ( hole array pitches of 320  nm ( green ) , 400  nm ( red ) , and 480  nm ( blue ) , along with a reference device ( black ) utilizing an ito transparent contact . on a log scale ,", "weak absorption by ct states can be visualized with over six orders of magnitude dynamic range .", "( the colour version of this figure is included in the online version of the journal . ) the integrated eqe of the organic devices using perforated metal films as transparent electrical contact ( figure 5(a ) , green , red , and blue colors ) is approximately five times lower than a similar organic device having a traditional ito transparent contact ( figure 5(a ) , black ) , due primarily to low - light transmission through the contact in the wavelength range where the organic material absorbs strongly .", "unlike the j    v measurement ( figure 4(d ) ) , the eqe spectrum more clearly demonstrates the effect of changing hole array pitch on the device performance .", "the primary absorption changes shape as a function of pitch and relative to the ito contact ( figure 5(a ) ) , with the most apparent difference being a flattening of the absorption spectra moving towards the uv region , compared to the conventional ito device .", "the high sensitivity of our high - brightness ftpc measurement facilitates detailed investigations of the nir spectral regions , where although the polymer shows weak absorption , the effect of coupling to surface plasmon modes is expected to be the strongest .", "previous high - sensitivity measurements on organic photovoltaic devices have shown that weak absorption characteristics of charge transfer ( ct ) states contribute to the overall photocurrent signal .", "the response of these ct states are readily seen on a log scale of the eqe spectra ( figure 5(b ) ) in both the ito and hole array contact devices .", "although the primary absorption features of the polymer are considerably weaker than the control devices , the ct absorption is not . in fact , structure in the ct absorption can be seen , leading to fluctuations ( particularly in the 480  nm pitch device ) that exceed those of the control .", "work is still on - going to separate the relative contributions of the propagating ( plasmonic ) and evanescent ( wave - guiding ) modes to the change in the overall eqe spectrum , and this will be the subject of future work . here , we stress that these types of studies are enabled by our ability to look at photocurrent spectra with more than six orders of magnitude of sensitivity with high spatial resolution .", "this allows us to quickly prototype and characterize photovoltaic devices employing plasmonic and photonic structures .", "we demonstrate that high - sensitivity photocurrent spectroscopy can be achieved on small - area organic photovoltaic devices with greater than six orders of magnitude dynamic range in the eqe spectra .", "these methods can be used to understand the interactions between the plasmonic modes of sub - wavelength metal hole arrays and an organic absorber .", "there are several factors that must be considered when evaluating performance of these devices , including scattering of light perpendicular to the direction of incidence , enhancement of absorption and radiative rates in near fields via the purcell effect , and photonic confinement of the propagating modes in the fully assembled device stack ."], "abstract_text": ["<S> we present a methodology for probing light - matter interactions in prototype photovoltaic devices consisting of an organic semiconductor active layer with a semitransparent metal electrical contact exhibiting surface plasmon - based enhanced optical transmission . </S>", "<S> we achieve high - spectral irradiance in a spot size of less than 100  m using a high - brightness laser - driven light source and appropriate coupling optics . </S>", "<S> spatially resolved fourier transform photocurrent spectroscopy in the visible and near - infrared spectral regions allows us to measure external quantum efficiency with high sensitivity in small - area devices ( < 1  mm2 ) . </S>", "<S> this allows for rapid fabrication of variable - pitch sub - wavelength hole arrays in metal films for use as transparent electrical contacts , and evaluation of the evanescent and propagating mode coupling to resonances in the active layer . </S>"], "labels": null, "section_names": ["Introduction", "Experimental methods and setup", "High-brightness broadband measurements for solar applications", "Optimizing tendue", "Solar irradiance and photon flux", "Application to organic photovoltaic devices with a transparent, plasmonic-metal electrical contact", "Conclusion"], "sections": [["it has been proposed that solar devices based on thin film technologies and third - generation materials can greatly benefit from plasmonic and photonic structures that can assist with light trapping and absorption .", "one promising strategy is to replace the traditional transparent conducting oxide layer with a semitransparent metallic contact that exhibits a surface plasmon at visible wavelengths .", "structured appropriately , for example , by perforating with an array of sub - wavelength holes , the metal film can provide the high transparency and conductivity needed in a transparent electrical contact while also actively assisting with photon management .", "a recent study demonstrated a high - performing organic photovoltaic cell with a metal contact patterned with an array of sub - wavelength holes , providing the contact with broad featureless transmission throughout the visible spectrum and robust coupling to the semiconductor as a function of incident light angle . despite this progress ,", "a key challenge is to probe and understand the details of the light - matter interactions between the incident photon , the surface plasmon modes of the structured contact , and the absorption properties of the active area .", "we have developed a set of small area , high - brightness spectroscopies that are appropriate for characterizing prototypes of light - harvesting devices utilizing plasmonic contact materials , for understanding the details of light - matter interactions in these systems .", "our primary interest in developing a set of rapid , high - sensitivity characterization tools capable of probing the influence of the perforated metal nanostructure , including pore size and pitch , as well as film thickness on the optical transmission modes , and ultimately the effect on solar cell performance .", "the aim is both to enable rapid screening of test device structures and to design structures capable of providing specific physical insight into coupling between the device active area and the modes of the patterned metal film .", "our strategy utilizes fourier transform ( ft)-based measurements in the solar portion of the optical spectrum , for optimally combining spatial resolution , spectral resolution , speed , and dynamic range .", "ft - based measurements are highly advantageous for characterizing nanoscale photovoltaic materials in the visible and near infrared ( nir ) portions of the electromagnetic spectrum , although they are more typically applied in the infrared . as compared to scanned techniques , ft photocurrent measurements benefit from the multiplex ( fellgett ) advantage because the detectors are not shot - noise - limited , unlike in optical measurements employing low - noise photodetectors ( e.g. raman scattering with a high sensitivity ccd or pmt ) .", "this is particularly important since , unlike dispersive optical measurements ( e.g. transmission , pl , and raman ) , photocurrent multi - channel detection with scanned sources is not typically possible . for measurements of photovoltaic phenomena ,", "the throughput advantage ( jacquinot ) is much less relevant due to the lower required resolution , when compared to vibrational or high - resolution absorption measurements .", "this is particularly true when using a high - brightness light source and a small sample size such that the throughput becomes comparable to a scanned source .", "however , ft - based measurements still typically have more bandwidth capabilities , as there is no need to switch gratings between the visible and the nir ."], ["the key to the experiment is achieving solar ( or higher ) spectral irradiance levels in a spot size of < 100  m in order to achieve high signal / noise external quantum efficiency ( eqe ) measurements on prototype devices .", "we have recently demonstrated the power of these types of measurements on individual single - walled carbon nanotube field effect transistors , but the benefit is equally applicable to other structures with active areas of the order of ( 100  m ) , such as photonic structures that are difficult and/or time - consuming to fabricate over large areas .", "achieving a high irradiance in a small area requires the use of high radiance sources .", "the alternative  increasing the power without increasing the size of the source or solid angle of radiation  becomes impractical at these size scales , because most of the photon flux would be wasted . in the mid- to far - ir , similar advantages are gained using synchrotron - based sources for microspectroscopy . here ,", "we achieve these advantages in the solar spectral region with an inexpensive benchtop source  a true - cw high - brightness laser - driven light source ( energetiq eq-99 ) .", "this differs significantly from a laser , in that the output radiates incoherently into 4 rather than being highly directional and coherent .", "we have also examined laser supercontiuum sources for the same purpose ( e.g. fianium sc-450 ) , which are able to achieve even higher brightness levels due to the highly collimated output . however , for photocurrent and photovoltaic applications , we have found the laser - driven light source to be a much better choice for several reasons .", "primarily , the pulsed nature of the laser supercontinuum was found to generate small but detectable electrical transients that added significant noise to low - eqe regions of the spectrum .", "since a 200  m core multi - mode fiber is utilized to simplify alignment to the translating microscope optics , this negates the advantage a smaller emitter has in focusing capabilities .", "additionally , we have found it difficult to stay in a low - fluence ( solar - like ) regime with the pulsed laser ; focusing ps pulses with the full bandwidth of the laser has resulted in either saturation ( resulting in signal non - linearity ) or damage to samples and optical detectors , even with significant attenuation of the beam .", "we did not encounter the same issue for similar power levels in a true cw source .", "lastly , the laser - driven light source unit is significantly cheaper than the laser supercontinuum ( ~5    difference ) .", "similarly , other groups have found that despite the higher spectral power density of a supercontinuum source , the sensitivity of absorption - based measurements are higher with the ldls due to superior long - term stability .", "the laser - driven light source output is collimated by an off - axis parabolic mirror ( 1.5 diameter , 2 efl , na  =  0.375 ) and coupled into a commercial ft spectrometer ( bruker v80 ) using the backward input .", "the light is modulated using a broadband caf2 uv  vis - nir beam splitter and the slowest  rapid - scan  setting available on our instrument ( 2.5  khz referenced to the hene line ) .", "the modulated light is sent to an output port of the spectrometer , where the remaining uv portion of the beam ( < 420  nm ) is filtered out .", "the broadband light is focused into a 200  m core multimode fiber used to couple the light into an upright optical microscope ( nikon fn-1 ) using a reflective collimating optic and a set of steering mirrors attached to an epi - illuminator .", "this microscope is designed for a stationary stage , such that electrical probes can be contacted with a sample via micromanipulators .", "as such , the microscope itself sits on a translating stage for sample visualization and beam alignment . the use of an optical fiber for coupling ensures that the beam stays aligned to the microscope even during translation .", "a 5050 beam splitter is used to simultaneously image and excite the device under test . for photoconductivity measurements", ", the photocurrent interferogram is sent through a trans - impedance amplifier ( dl instruments , 1210 ) and an electronic band - pass filter ( stanford research , sr650 ) before being digitized by the spectrometer bench electronics . to correct for the lamp spectrum and the transmission function of the bench and microscope optics , a set of photodiodes with known responsivity curves", "the resulting single - beam spectra from test devices are corrected by these reference spectra , which are collected at the same time as the unknown devices .", "the spot sizes at the output of the microscope are measured using a calibrated beam profiler ( ophir - spiricon ) . for transmission spectra ,", "the sample compartment of the spectrometer is used , resulting in a spot size of 0.75  mm at the focus .", "samples are aligned to the beam using a xyz translating mount and the transmitted light is detected with a pyrolelectric detector ( la - dtgs ) .", "we define arrays of sub - wavelength holes across areas of ( 300  m ) by focused ion beam ( fib ) milling completely through 50  nm thick ag films sputter deposited on glass .", "holes with average diameter of 200  nm are milled using a beam of ga ions ( 0.26  na ) , with dwell times ranging from 2 to 8  ms for hole separations ( pitch ) ranging from 240 to 560  nm , respectively .", "after defining the perforated ag electrode , the subsequent organic photovolatic device fabrication steps include : thermal evaporation of 9  nm thick of moo3 electron - blocking layer ; spin coating ( 700 rpm for 60  s ) the 170  nm thick semiconductor active layer , a 1:1 wt .", "blend of poly(3-hexlythiophene ) : phenyl - c61-butyric acid methyl ester ( p3ht : pcbm ) ( 3 wt .", "% in chlorobenzene ) ; and thermal evaporation of a 200  nm al film , which serves as an electron - collecting ( and hole - blocking ) electrode ."], ["we match the source , coupling , and focusing optics in order to optimize the throughput and achieve a high - brightness white - light spot .", "the critical quantity is the optical extent or tendue ( g ) of the system that will maximize the light throughput to the sample .", "it is this quantity that needs to be appropriately matched throughout the system to maximize the spectral irradiance , rather than the numerical aperture of the individual optical components .", "because g is proportional to both the area of the source and the solid angle into which it propagates , we aim to match the collimating and focusing optics without introducing a bottleneck ( e.g. the multimode optical fiber ) . the combination of the laser - driven light source and a parabolic mirror results in g ~ 3    10  mm sr , a value well matched to a 0.22 numerical aperture fiber with a 0.2  mm core size ( ~4.5    10  mm sr maximum g ) .", "the effective tendue of the sample is determined by the spot size of the light and the microscope objective used to image and focus the light .", "the numerical aperture of the objective determines the solid angle , but the spot size is not determined by the nominal magnification , which is referenced to a 200 mm tube lens .", "we assume that the reference specifying the magnification value is a 200  mm tube lens . here", ", the value of the objective magnification is now defined by the numerical aperture of the fiber itself and the collimating optic . as a result ,", "the magnification of the objective is considerably smaller than the 10/50  values specified . for the 10  objective ,", "we obtain nearly 1:1 imaging , with the core diameter of the multimode fiber limiting the ultimate spot size .", "for example , focusing the broadband ft - modulated light source with a 10    ( 0.3 numerical aperture ) long - working distance objective yields a measured spot size of ~215  m ( beam profile shown in figure 1(a ) ) . we can achieve a smaller spot size using the 50 objective , for two reasons .", "first , the higher numerical aperture results in additional magnification , and second , the smaller entrance aperture of the higher na objective slightly reduces the collimated beam size ( thereby reducing the effective source size and the tendue out of the fiber ) .", "the net result is a reduction in spot size to ~40  m using the nominal 50    ( 0.45 numerical aperture ) objective . as we discuss below , this small reduction in throughput still results in an overall higher spectral irradiance .", "these larger - than - diffraction - limit spots are due to the necessity of using of a multimode optical fiber to accommodate a large spectral bandwidth . using a single - mode fiber and a monochromatic solid - state laser", ", we can achieve near diffraction - limited spots using these same optics . from the measured spot sizes and the focal length of the objectives", ", we can determine the ntendu of the sample in order to find the overall limiting factor in the optical system .", "in all cases , the ntendue of the 10  objective is higher than that of the source and fiber , meaning that the limiting factor is not the small area of the detector ( i.e. sample ) .", "( a ) beam profile of the image formed using the eq-99 laser - driven light source and a low magnification 10 objective .", "the spot size is approximately 215  m with the 10 and 40  m with the 50  .", "( b ) spectral irradiance in the visible and near infrared with the 10  and 50  objectives compared to the solar air mass 1.5 standard ( astm g-173 ) .", "( the colour version of this figure is included in the online version of the journal . ) with the combination of a high - radiance light source and microscope focusing optics , we can achieve high - spectral irradiances for highly sensitive quantum efficiency measurements on small area devices .", "the spectral irradiance is shown in figure 1(b ) for the output of the ldls microscopy system with the 10 and 50 objectives , along with the solar air mass 1.5 ( astm g173 - 03 ) spectrum for comparison .", "the key metric is that the solar spectral irradiance can be matched or exceeded across the visible and nir , particularly in the visible region of the spectrum , where we achieve in excess of 10 times higher irradiances .", "these numbers can be further improved since a 50:50 beam splitter ( optimized for the visible ) is used to direct light into the microscope objective .", "replacing the dielectric optic with a metallic one would double the output in the visible , but result in even more significant gains in the nir .", "however , for the purposes of this study , the irradiances achieved are more than sufficient for our measurements .", "the overall photon flux achieved is also quite high due to strong focusing of the light beam , exceeding 4.5    10  ms with high spatial resolution ."], ["we match the source , coupling , and focusing optics in order to optimize the throughput and achieve a high - brightness white - light spot .", "the critical quantity is the optical extent or tendue ( g ) of the system that will maximize the light throughput to the sample .", "it is this quantity that needs to be appropriately matched throughout the system to maximize the spectral irradiance , rather than the numerical aperture of the individual optical components .", "because g is proportional to both the area of the source and the solid angle into which it propagates , we aim to match the collimating and focusing optics without introducing a bottleneck ( e.g. the multimode optical fiber ) . the combination of the laser - driven light source and a parabolic mirror results in g ~ 3    10  mm sr , a value well matched to a 0.22 numerical aperture fiber with a 0.2  mm core size ( ~4.5    10  mm sr maximum g ) .", "the effective tendue of the sample is determined by the spot size of the light and the microscope objective used to image and focus the light .", "the numerical aperture of the objective determines the solid angle , but the spot size is not determined by the nominal magnification , which is referenced to a 200 mm tube lens .", "we assume that the reference specifying the magnification value is a 200  mm tube lens . here", ", the value of the objective magnification is now defined by the numerical aperture of the fiber itself and the collimating optic . as a result ,", "the magnification of the objective is considerably smaller than the 10/50  values specified . for the 10  objective ,", "we obtain nearly 1:1 imaging , with the core diameter of the multimode fiber limiting the ultimate spot size .", "for example , focusing the broadband ft - modulated light source with a 10    ( 0.3 numerical aperture ) long - working distance objective yields a measured spot size of ~215  m ( beam profile shown in figure 1(a ) ) . we can achieve a smaller spot size using the 50 objective , for two reasons .", "first , the higher numerical aperture results in additional magnification , and second , the smaller entrance aperture of the higher na objective slightly reduces the collimated beam size ( thereby reducing the effective source size and the tendue out of the fiber ) .", "the net result is a reduction in spot size to ~40  m using the nominal 50    ( 0.45 numerical aperture ) objective . as we discuss below , this small reduction in throughput still results in an overall higher spectral irradiance .", "these larger - than - diffraction - limit spots are due to the necessity of using of a multimode optical fiber to accommodate a large spectral bandwidth . using a single - mode fiber and a monochromatic solid - state laser", ", we can achieve near diffraction - limited spots using these same optics . from the measured spot sizes and the focal length of the objectives", ", we can determine the ntendu of the sample in order to find the overall limiting factor in the optical system .", "in all cases , the ntendue of the 10  objective is higher than that of the source and fiber , meaning that the limiting factor is not the small area of the detector ( i.e. sample ) .", "( a ) beam profile of the image formed using the eq-99 laser - driven light source and a low magnification 10 objective .", "the spot size is approximately 215  m with the 10 and 40  m with the 50  .", "( b ) spectral irradiance in the visible and near infrared with the 10  and 50  objectives compared to the solar air mass 1.5 standard ( astm g-173 ) .", "( the colour version of this figure is included in the online version of the journal . )"], ["with the combination of a high - radiance light source and microscope focusing optics , we can achieve high - spectral irradiances for highly sensitive quantum efficiency measurements on small area devices .", "the spectral irradiance is shown in figure 1(b ) for the output of the ldls microscopy system with the 10 and 50 objectives , along with the solar air mass 1.5 ( astm g173 - 03 ) spectrum for comparison .", "the key metric is that the solar spectral irradiance can be matched or exceeded across the visible and nir , particularly in the visible region of the spectrum , where we achieve in excess of 10 times higher irradiances .", "these numbers can be further improved since a 50:50 beam splitter ( optimized for the visible ) is used to direct light into the microscope objective .", "replacing the dielectric optic with a metallic one would double the output in the visible , but result in even more significant gains in the nir .", "however , for the purposes of this study , the irradiances achieved are more than sufficient for our measurements .", "the overall photon flux achieved is also quite high due to strong focusing of the light beam , exceeding 4.5    10  ms with high spatial resolution ."], ["in order to demonstrate the utility of a technique having simultaneously high spatial resolution , spectral resolution , and high sensitivity , we examined a set of prototype organic photovoltaic devices that employed a nanostructured plasmonic ag thin film as a transparent electrical contact .", "first , we discuss the photophysical properties of the perforated metal films with sub - wavelength apertures , in order to best match the contact s optical properties to the light absorption bands in the organic active area .", "we fabricated the nanostructured electrical contacts by depositing a thin ag film on glass and using a fib tool ( fei helios dualbeam ) to mill square arrays of uniformly sized holes across typical areas of ( 0.3  mm ) ( scanning electron micrograph , figure 2(a ) ) .", "we can fully characterize even these small - area samples because of the high - brightness light source , therefore minimizing the associated fabrication time .", "the advantage of using the fib for fabrication is that the design can be varied ( e.g. adjusting hole size and/or diameter ) without the need for any process re - optimization .", "the variation in an average hole size introduced by our fabrication approach does not significantly affect the optical properties of the array .", "the absolute optical transmission spectra of ag films fully perforated with hole arrays having pitches ranging from 240 to 560  nm on glass show a series of peaks and troughs resulting from the plasmon and diffraction modes in the array ( figure 2(b ) .", "spectra are offset for clarity ) . as predicted from momentum conservation arguments , these mode positions are nearly constant on a wavelength - to - pitch ratio scale .", "( a ) scanning electron micrograph of a silver thin film perforated with a hole array having a pitch of 400  nm .", "( b ) absolute optical transmission spectra of 100  nm thick ag films with a total area of ( 300  m ) on glass vs. the wavelength / pitch ratio , for different pitch values .", "( the colour version of this figure is included in the online version of the journal . )", "the optical transmission properties of these perforated metal films are sensitive to the dielectric interfaces on either side , such that the modes of the film when assembled into solar cell will shift compared to those of the same metal film on glass . in order to understand the metal film transmission properties of our target device structure , we coat the perforated ag film ( 100  nm thick , 200  nm average hole diameter , and 400  nm average pitch ) with a 220  nm thick polymer layer ( microposit s1811 ) that mimics the dielectric constant of the organic semiconductor in the non - resonant regime of the spectrum , but does not exhibit significant absorption at visible wavelengths .", "to best imitate the device structure , we insert a 17  nm thick tio2 layer between the ag and polymer films , because such selective electronic transport layers are essential for organic device operation .", "we use finite difference time domain ( fdtd ) calculations ( lumerical solutions , inc . ) to understand the origin of the primary transmission resonances and their effect on the target photovoltaic device .", "we find reasonably good agreement between the measured ( figure 3(a ) , black ) and calculated ( figure 3(a ) , red ) transmission curves , with a primary maximum in the transmission spectrum at ~1020  nm and a minimum near ~930  nm . we believe the peak broadening in the nir results from hole size inhomogeneities .", "however , the hole pitch uniformity generates a resonance peak that is well matched to the calculation .", "( a ) experimental ( black line ) and fdtd transmission spectrum ( red ) of a glass / perforated ag electrode ( height  =  100  nm , diameter  =  200  nm , pitch  =  400  nm)/tio2 ( h  =  17  nm)/polymer ( 220  nm)/air dielectric stack", ". normalized field intensity distributions , |e|/|e \n 0| for light with ( b )   =  927  nm and ( c )   =  1020  nm . in ( b ) and", "( c ) , the glass spans from z  =  0.2  m to z  =  0  m , the ag mha spans from z  =  0  m to z  =  0.1  m ( and the hole in the ag layer spans from x  =  0.1  m to x  =  0.1  m ) , the tio2 layer spans from z  =  0.1  m to z  =  0.117  m , and the polymer layer spans from z  =  0.117  m to z  =  0.337  m .", "( the colour version of this figure is included in the online version of the journal . )", "our calculations allow us to examine the light intensities of corresponding spatial modes in order to identify the mechanism for light dissipation or transport . in figure 3(b ) and ( c ) , we plot the normalized field intensity |e|/|e \n 0| in the xz plane ( where incident light propagates in the + z direction , downwards in the figure ) .", "this calculation shows that at 927  nm ( figure 3(b ) ) , near the transmission minimum , the field intensity is delocalized throughout the polymer active area , with a minimum under the hole open area .", "this calculated spatial distribution is consistent with that of a wood s anomaly ( evanescent mode ) , which redirects the light propagation due to diffraction .", "in contrast , the normalized field intensity profile |e|/|e \n 0| for a wavelength near the transmission maximum ( 1020  nm ) shows feature characteristics of the near - field enhancement due to surface plasmon modes in the perforated metal film , resulting in  hot spots  of electric field intensity near the edges of the contacts along the metal - polymer layer .", "though one benefit of these plasmon modes is enhanced light transmission through sub - wavelength apertures , there is also an opportunity for modification of the absorption and radiative properties of the active layer via the purcell effect .", "though this subject is beyond the scope of the current manuscript , it does imply that the benefits of perforated metal films as electrical contacts potentially extend beyond their role as passive , light - transmitting elements .", "because the primary features in the transmission spectrum of the perforated metal film result from light - matter interactions at the metal / polymer interface , we can leverage this structure for design of a solar cell transparent electrical contact . in this demonstration , we employ a 35  nm thick ag film ( perforated with a square array of 200  nm diameter holes on a 410  nm pitch ) to couple light into a 160  nm thick active layer of blended organic p3ht : pcbm .", "the device also includes both hole transporting ( 10  nm thick moo3 ) and electron transporting ( 220  nm thick al ) on either side of the organic active layer ( schematic in figure 4(a ) and cross - sectional sem image in figure 4(b ) .", "( a ) device schematic and ( b ) sem cross - section of an organic photovoltaic device with a perforated ag film as transparent contact .", "the high extinction due to absorption by the active layer occurs around 500  nm and is indicated by the arrow .", "( d ) dark ( dot - dash ) and illuminated ( lines ) j    v curves for fully fabricated devices using the ag / polymer materials shown in ( c ) .", "device hole array pitches are : 320  nm ( green ) , 400  nm ( red ) , and 480  nm ( blue ) . also shown", "is the performance of a similar device having an unpatterned 50  nm thick ag electrical contact ( dark : dashed , illuminated : black ) .", "( the colour version of this figure is included in the online version of the journal . ) the optical transmission spectra of organic devices having metal contacts with a hole array of pitches 320 , 400 , and 480  nm ( figure 4(c ) , measured prior to forming the al electrical contact ) contain features in the nir region that are similar to that of the non - resonant model system ( figure 3(a ) ) , but show dramatic differences in the spectral regions where the p3ht : pcbm blend absorbs .", "the primary polymer absorption manifests as dramatic dip around 500  nm ( indicated with an arrow in figure 4(c ) ) .", "the magnitude of the transmission ( < 10% ) is similar to that of the electrode evanescent mode near 900  nm , and is consistent with an active area thickness of ~170  nm . although the primary propagating mode of the perforated ag film ( > 700  nm ) is far from the organic material absorption peak ( at 500  nm ) , higher order surface plasmon modes ( visible at shorter wavelengths in figure 3(a ) ) can nevertheless couple light efficiently into the device structure in order to generate photocurrent .", "for example , the current - voltage ( j    v ) characteristics of fully assembled devices under illumination by 1 sun simulated am 1.5 g radiation display average photocurrent ( j \n sc ) in the range of j \n sc  ~  4  ma / cm ( nearly independent of hole array pitch ) , open circuit voltage , v \n oc \n =  0.58  v , and power conversion efficiencies around ~1% ( figure 4(d ) ) . although conventional transparent contact materials ( e.g. indium tin oxide ) have achieved power conversion efficiencies around ~4% in p3ht : pcbm devices , there are opportunities for considerable improvement by better matching the electrode s propagating mode to the absorption resonance in the organic layer .", "potential approaches to achieving performance improvements include scaling the hole arrays to smaller pitches , or leveraging recent breakthroughs in utilizing nir absorbing polymers   which are better matched to the size of the perforated metal contacts described in this work .", "the high - brightness photocurrent measurement is ideal for acquiring high - sensitivity eqe spectra of these small - area devices ( 300  m ) ( figure 5 ) .", "although in these measurements we are not required to match the lamp spectrum to the solar spectrum , we nevertheless target an overall spectral irradiance comparable to that of solar illumination in order to avoid high - fluence recombination processes .", "the achievable spot size under low magnification is 215  m , well matched to our device area of ( 300  m ) and to the solar irradiance in the active region of the device .", "these measured eqe spectra result from averaging for approximately one minute in total , because the entire spectrum ( from 420 to 2500  nm ) is collected in a single shot .", "ft photocurrent spectra of ( 300  m ) area devices on a ( a ) linear and ( b ) log scale for organic photovoltaic devices with perforated metal film transparent contacts ( hole array pitches of 320  nm ( green ) , 400  nm ( red ) , and 480  nm ( blue ) , along with a reference device ( black ) utilizing an ito transparent contact . on a log scale ,", "weak absorption by ct states can be visualized with over six orders of magnitude dynamic range .", "( the colour version of this figure is included in the online version of the journal . ) the integrated eqe of the organic devices using perforated metal films as transparent electrical contact ( figure 5(a ) , green , red , and blue colors ) is approximately five times lower than a similar organic device having a traditional ito transparent contact ( figure 5(a ) , black ) , due primarily to low - light transmission through the contact in the wavelength range where the organic material absorbs strongly .", "unlike the j    v measurement ( figure 4(d ) ) , the eqe spectrum more clearly demonstrates the effect of changing hole array pitch on the device performance .", "the primary absorption changes shape as a function of pitch and relative to the ito contact ( figure 5(a ) ) , with the most apparent difference being a flattening of the absorption spectra moving towards the uv region , compared to the conventional ito device .", "the high sensitivity of our high - brightness ftpc measurement facilitates detailed investigations of the nir spectral regions , where although the polymer shows weak absorption , the effect of coupling to surface plasmon modes is expected to be the strongest .", "previous high - sensitivity measurements on organic photovoltaic devices have shown that weak absorption characteristics of charge transfer ( ct ) states contribute to the overall photocurrent signal .", "the response of these ct states are readily seen on a log scale of the eqe spectra ( figure 5(b ) ) in both the ito and hole array contact devices .", "although the primary absorption features of the polymer are considerably weaker than the control devices , the ct absorption is not . in fact , structure in the ct absorption can be seen , leading to fluctuations ( particularly in the 480  nm pitch device ) that exceed those of the control .", "work is still on - going to separate the relative contributions of the propagating ( plasmonic ) and evanescent ( wave - guiding ) modes to the change in the overall eqe spectrum , and this will be the subject of future work . here , we stress that these types of studies are enabled by our ability to look at photocurrent spectra with more than six orders of magnitude of sensitivity with high spatial resolution .", "this allows us to quickly prototype and characterize photovoltaic devices employing plasmonic and photonic structures ."], ["we demonstrate that high - sensitivity photocurrent spectroscopy can be achieved on small - area organic photovoltaic devices with greater than six orders of magnitude dynamic range in the eqe spectra .", "these methods can be used to understand the interactions between the plasmonic modes of sub - wavelength metal hole arrays and an organic absorber .", "there are several factors that must be considered when evaluating performance of these devices , including scattering of light perpendicular to the direction of incidence , enhancement of absorption and radiative rates in near fields via the purcell effect , and photonic confinement of the propagating modes in the fully assembled device stack ."]]}
{"article_id": "PMC3776250", "article_text": ["photonic forces [ 1 , 2 ] are known and understood for more than 100  years . but the discovery that they can be employed to manipulate small objects like colloids , viruses , or quantum dots became possible only after the establishment of strong laser sources in the infrared .", "this enables one to exert and to control forces acting on a small object in the range between 10 to 100  pn with an extraordinary high resolution of 50  fn . by incorporating additional optical systems to determine the position of a microscopic particle in 3d - space with nanometer precision , the novel experimental tool of optical tweezers was invented", "this initiated a manifold of spectacular experiments in biophysics [ 4a  j ] and colloid research .", "it became , for instance , possible to measure the elastic properties of -phage dna   on a single chain level , the rna polymerase was monitored for a single enzyme in action , or the work which has to be done to package bacteriophage dna into a single viral capsule was measured . in colloid physics , microscopic measurements of the pair interaction potential of two charge - stabilized colloids became possible   or direct determination of the cross correlations between two particles in an external potential .", "experiments with optical tweezers on the elastic properties of dna are mainly restricted to -phage dna which has a contour length of 16  m . in the presented article ,", "novel experiments are reported in which the dna under study is systematically varied in its length by genetic engineering .", "this enables one to analyze in great detail the elastic properties of dna and to check how well it can be described by the worm - like chain model   or competing approaches .", "1 ) is based on an inverted microscope ( axiovert s 100 tv , carl zeiss , jena , germany ) which is designed for epi - illumination fluorescence microscopy . for the optical tweezers ,", "a diode pumped nd : yag laser ( 1,064  nm , 1  w , lcs - dtl 322 ; laser 2000 , wessling , germany ) is used which is mounted to the microscope by the base port . the power and the profile of the laser", "a quarter - wave plate is employed to produce circular polarized light to exclude effects due to reflection differences between the p- and s - part of the laser light .", "the beam is expanded and coupled into the back aperture of the microscope objective ( plan - neofluor 1001.30 oil , carl zeiss , jena , germany ) . after passing through the sample cell , the beam is recollimated by a condenser with high numerical aperture .", "a beam splitting mirror and a convex lens images the forward scattered light of the bead in the optical trap onto a 1010  mm quadrant photodiode ( s 5107 , hamamatsu , herrsching am ammersee , germany ) for the electrical position detection . video imaging and the optical position detection is accomplished by a digital camera ( kp  f 120 , hitachi , dsseldorf , germany ) .", "the optical stage can be positioned in three dimensions with nanometer resolution using piezoactuators ( controller : e-710 , stage : p-517.3cd , pi gmbh , karlsruhe , germany ) .", "the sample cell consists of a closed chamber which can be flushed ( inset fig .  1 ) .", "a micropipette ( self - made ) with an inner diameter at the tip of 1  m is inserted into the chamber to hold one bead by capillary action . to determine the position of both beads and its separation image analysis is employed . by measuring and fitting the intensity profile of the two beads ( fig . ", "2 ) , its position relative to each other can be determined with an accuracy of 2  nm . \n fig . ", "1scheme of the experimental set - up . in the enlargement , the sample chamber is shownfig .", "2image analysis of two separate beads : measured ( a , b ) and calculated ( c ) intensity profile using the fit function : \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$i{\\left ( { x , y } \\right)}\\ ; = \\;i_{0 } + { \\sum\\limits_{i = 1,2 } { a_{i } } } { \\left ( { 1 - a_{i } u_{i } } \\right)}e^{{u^{3}_{i } } } $ $ \\end{document } ( 3 ) \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$u_{i } \\ ; = \\;{\\left ( { \\frac{{x - x_{i } } } { { r_{i } } } } \\right)}^{2 } \\ ; + \\;{\\left ( { \\frac{{y - y_{i } } } { { r_{i } } } } \\right)}^{2}$$\\end{document } ( 4 ) where for each colloid ( x \n i , y \n i ) is the center position , r \n i the optical radius , a \n i the amplitude of the profile relative to the background image intensity .", "i \n 0 and a \n i are parameters to consider the dark diffuse ring around the colloid scheme of the experimental set - up . in the enlargement ,", "the sample chamber is shown image analysis of two separate beads : measured ( a , b ) and calculated ( c ) intensity profile using the fit function : \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$i{\\left ( { x , y } \\right)}\\ ; = \\;i_{0 } + { \\sum\\limits_{i = 1,2 } { a_{i } } } { \\left ( { 1 - a_{i } u_{i } } \\right)}e^{{u^{3}_{i } } } $ $ \\end{document } ( 3 ) \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$u_{i } \\ ; = \\;{\\left ( { \\frac{{x - x_{i } } } { { r_{i } } } } \\right)}^{2 } \\ ; + \\;{\\left ( { \\frac{{y - y_{i } } } { { r_{i } } } } \\right)}^{2}$$\\end{document } ( 4 ) where for each colloid ( x \n i ,", "y \n i ) is the center position , r \n i the optical radius , a \n i the amplitude of the profile relative to the background image intensity .", "i \n 0 and a \n i are parameters to consider the dark diffuse ring around the colloid the calibration of the optical trap is based on stokes law f=6 r where  is the viscosity of the medium , r the radius of the bead , and  its velocity relative to the surrounding solution .", "varying the latter between 50 and 1,000  m / s enables one to calibrate the optical forces with an accuracy of 10% . as expected for the power dependence of the force constant of the optical trap , a linear relationship", "the whole experimental set - up is in a temperature controlled ( 1 ) room . due to the fact that the focus of the microscope changes with 1 by about 500  nm ,", "the actual measurements are fully computer - controlled and carried out automatically within sequences of about 23  min .", "afterwards , the z - positions have to be  if necessary  readjusted . \n", "3calibration of the forces of the optical trap based on stokes law calibration of the forces of the optical trap based on stokes law streptavidin - modified polystyrene beads with a diameter of 2  m were purchased from polysciences europe ( eppelheim , germany ) , oligonucleotides provided by metabion ( martinsried , germany ) and mwg ( ebersberg , germany ) .", "nucleic acid purification kits were supplied by qiagen ( hilden , germany ) and anti - dig antibodies were purchased from roche ( penzberg , germany ) .", "all enzymes and standards were from mbi fermentas ( vilnius , lithuania ) and all other chemicals were delivered from sigma ( deisenhofen , germany ) .", "the double stranded dna handles were amplified from the plasmid puc18 in case of the 2,000  bp product , from pet28a+ in case of the 3,000 and 4,000  bp product , and from ptwin1 in case of the 6,000  bp product via standard polymerase chain reactions ( pcr ) .", "the oligonucleotides which were used were primer a ( 5-bio - cca cct gac gtc taa gaa acc-3 ) and primer b ( 5-dig - ggc aac aac gtt gcg caa ac-3 ) for the 2,000  bp dsdna , primer c ( 5-dig - acg gcc tca acc tac tac tg-3 ) and primer d ( 5-bio - gcg ata agt cgt gtc tta cc-3 ) for the 3,000  bp dsdna , primer e ( 5-dig - cag ctt cct ttc ggg ctt tg-3 ) and primer f ( 5-bio - tga ttg ccc gac att atc gc-3 ) for the 4,000  bp dsdna , and primer g ( 5-dig - atg gag gcg gat aaa gtt gc-3 ) and primer h ( 5-bio - gttgaatcaccgcgtaatcg-3 ) for the 6,000  bp dsdna .", "dna - handles were labeled with digoxygenine or biotine at their termini via 5-end modifications of the primers carried out by metabion or mwg ( fig . ", "the distribution of nucleotides in the artificial ds - dna and -dna was proven to be well comparable ( table  1 ) . \n", "fig .  4 \n a the dnas with the lengths 2,000 , 3,000 , 4,000 , and 6,000 ", "b result of the binding assay . in lane 1 and 3 60  fmol of 2,000  bp dna", "lane 2 shows the result of the binding of the 2,000  bp dna to the anti - dig antibody labeled beads .", "lane 4 shows the negative control with carboxylated beads onlytable  1comparison of the distributions of nucleotides for the used dna samplesdnanumber of base pairsdistributionchemically amplified byhandle 2000 bd2,000  bpa : 486  bp ( 24.2%)puc18c : 522  bp ( 26.0%)g : 532  bp ( 26.5%)t : 471  bp ( 23.4%)handle 3000 bd3,000  bpa : 665  bp ( 22.2%)pet28a+c : 858  bp ( 28.6%)g : 829  bp ( 27.6%)t : 648  bp ( 21.6%)handle 4000 bd4,000  bpa : 888  bp ( 22.2%)pet28a+c : 1,097  bp ( 27.4%)g : 1,114  bp ( 27.9%)t : 901  bp ( 22.5%)handle 6000 bd6,000  bpa : 1,474  bp ( 24.6%)ptwin1c : 1,528  bp ( 25.5%)g : 1,642  bp ( 27.4%)t : 1,356  bp ( 22.6%)lambda - dna48,502  bpa : 12,334  bp ( 25.4%)c:11,362  bp ( 23.4%)g : 12,820  bp ( 26.4% ) t : 11,986  bp ( 24.7% ) \n a the dnas with the lengths 2,000 , 3,000 , 4,000 , and 6,000  bp were analyzed on a 1.0 % agarose gel to check their correct size .", "b result of the binding assay . in lane 1 and 3 60  fmol of 2,000  bp dna", "lane 2 shows the result of the binding of the 2,000  bp dna to the anti - dig antibody labeled beads .", "lane 4 shows the negative control with carboxylated beads only comparison of the distributions of nucleotides for the used dna samples the immobilization process was mainly deduced from protocols previously published by nustad et al . ,", "for the production of antidigoxigenin ( anti - dig ) antibody covered polystyrene beads , carboxylated particles with a diameter of 2.0  m were used .", "the beads from a 50  l bead suspension ( 26.5  mg / ml ) were washed twice with 200  l of carbonate buffer ( 100  mm sodium carbonate , 100  mm sodium bicarbonate , ph  9.6 ) and three times with 200  l buffer a ( 20  mm phosphate buffer , ph  4.5 ) . in between beads were collected by centrifugation ( 5  min 13,000g ) .", "after resuspension of the final bead pellet in 50  l buffer a , the surface carboxyl groups were activated by a 4-h incubation at room temperature with 50  l of a 2% edc solution ( ethyl-3-(3-dimethylaminopropyl)-carbodiimide ) in the same buffer .", "particles were washed three times with buffer a. after resuspension in 80  l buffer b ( 200  mm borate buffer , ph  8.5 ) , 10  l of anti - dig antibody solution ( 1  g ab/l ) were added followed by overnight shaking at room temperature . to avert unspecific binding at remaining free reactive groups , the pellet was resuspended in 100  l bsa solution ( 10  mg", "bsa / ml in buffer b ) and incubated under shaking for 30  min at room temperature .", "finally , the particles were resuspended in 20  l pbs ( 136  mm nacl , 2.7  mm kcl , 15.6  mm na2hpo4 , 17.6  mm kh2po4 ) , and stored at 4c . to determine the coupling efficiency 5  l of particle suspension", "were mixed with 50200  ng of a 2,000  bp pcr - amplified dna fragment , carrying a digoxygenine and a biotine modification at each terminus , respectively , in a total volume of 10  l pbs .", "this mixture was then incubated for 15  min at room temperature . as a negative control ,", "the whole suspension was than applied to a 1.5% agarose gel electrophoresis from which the particle bound dna fraction could be estimated ( fig . ", "to establish single dna strands between two colloids , the following procedure turned out to be effective and reliable ( fig . ", "5 ) : a bead with anti - dig antibodies was captured with the optical trap and brought in contact with the micropipette where it was fixed by capillary forces .", "afterwards , a sa - modified bead covered with dna was placed with the optical trap in the immediate neighborhood of the fixed anti - dig - bead so that still a visible distance between the particles was maintained . if a molecular contact was established , a sudden decrease in the fluctuation amplitude of the bead could be observed . after a short incubation time of 1  min ,", "the particles are pulled apart to ensure that a single ds - dna had bound only . \n", "a polystyrene ( ps ) bead modified with anti - dig antibodies is trapped with the optical tweezers ( a ) and fixed by a micropipette due to capillary forces ( b ) . in the next step , a ps bead on which ds - dna is immobilized via biotin ", "streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette ( c ) . binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette", "is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap ( d ) . by measuring the force extension dependence", ", it is ensured that just one ds - dna strand is fixed between the beads assembly of a single ds - dna chain between two colloids .", "a polystyrene ( ps ) bead modified with anti - dig antibodies is trapped with the optical tweezers ( a ) and fixed by a micropipette due to capillary forces ( b ) . in the next step , a ps bead on which ds - dna is immobilized via biotin ", "streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette ( c ) . binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette", "is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap ( d ) . by measuring the force extension dependence", ", it is ensured that just one ds - dna strand is fixed between the beads", "1 ) is based on an inverted microscope ( axiovert s 100 tv , carl zeiss , jena , germany ) which is designed for epi - illumination fluorescence microscopy . for the optical tweezers ,", "a diode pumped nd : yag laser ( 1,064  nm , 1  w , lcs - dtl 322 ; laser 2000 , wessling , germany ) is used which is mounted to the microscope by the base port . the power and the profile of the laser", "a quarter - wave plate is employed to produce circular polarized light to exclude effects due to reflection differences between the p- and s - part of the laser light .", "the beam is expanded and coupled into the back aperture of the microscope objective ( plan - neofluor 1001.30 oil , carl zeiss , jena , germany ) . after passing through the sample cell , the beam is recollimated by a condenser with high numerical aperture .", "a beam splitting mirror and a convex lens images the forward scattered light of the bead in the optical trap onto a 1010  mm quadrant photodiode ( s 5107 , hamamatsu , herrsching am ammersee , germany ) for the electrical position detection . video imaging and the optical position detection is accomplished by a digital camera ( kp  f 120 , hitachi , dsseldorf , germany ) .", "the optical stage can be positioned in three dimensions with nanometer resolution using piezoactuators ( controller : e-710 , stage : p-517.3cd , pi gmbh , karlsruhe , germany ) .", "the sample cell consists of a closed chamber which can be flushed ( inset fig .  1 ) .", "a micropipette ( self - made ) with an inner diameter at the tip of 1  m is inserted into the chamber to hold one bead by capillary action . to determine the position of both beads and its separation image analysis is employed . by measuring and fitting the intensity profile of the two beads ( fig . ", "2 ) , its position relative to each other can be determined with an accuracy of 2  nm . \n fig . ", "1scheme of the experimental set - up . in the enlargement , the sample chamber is shownfig .", "2image analysis of two separate beads : measured ( a , b ) and calculated ( c ) intensity profile using the fit function : \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$i{\\left ( { x , y } \\right)}\\ ; = \\;i_{0 } + { \\sum\\limits_{i = 1,2 } { a_{i } } } { \\left ( { 1 - a_{i } u_{i } } \\right)}e^{{u^{3}_{i } } } $ $ \\end{document } ( 3 ) \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$u_{i } \\ ; = \\;{\\left ( { \\frac{{x - x_{i } } } { { r_{i } } } } \\right)}^{2 } \\ ; + \\;{\\left ( { \\frac{{y - y_{i } } } { { r_{i } } } } \\right)}^{2}$$\\end{document } ( 4 ) where for each colloid ( x \n i , y \n i ) is the center position , r \n i the optical radius , a \n i the amplitude of the profile relative to the background image intensity .", "i \n 0 and a \n i are parameters to consider the dark diffuse ring around the colloid scheme of the experimental set - up . in the enlargement ,", "the sample chamber is shown image analysis of two separate beads : measured ( a , b ) and calculated ( c ) intensity profile using the fit function : \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$i{\\left ( { x , y } \\right)}\\ ; = \\;i_{0 } + { \\sum\\limits_{i = 1,2 } { a_{i } } } { \\left ( { 1 - a_{i } u_{i } } \\right)}e^{{u^{3}_{i } } } $ $ \\end{document } ( 3 ) \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$u_{i } \\ ; = \\;{\\left ( { \\frac{{x - x_{i } } } { { r_{i } } } } \\right)}^{2 } \\ ; + \\;{\\left ( { \\frac{{y - y_{i } } } { { r_{i } } } } \\right)}^{2}$$\\end{document } ( 4 ) where for each colloid ( x \n i ,", "y \n i ) is the center position , r \n i the optical radius , a \n i the amplitude of the profile relative to the background image intensity .", "i \n 0 and a \n i are parameters to consider the dark diffuse ring around the colloid the calibration of the optical trap is based on stokes law f=6 r where  is the viscosity of the medium , r the radius of the bead , and  its velocity relative to the surrounding solution .", "varying the latter between 50 and 1,000  m / s enables one to calibrate the optical forces with an accuracy of 10% . as expected for the power dependence of the force constant of the optical trap , a linear relationship", "the whole experimental set - up is in a temperature controlled ( 1 ) room . due to the fact that the focus of the microscope changes with 1 by about 500  nm ,", "the actual measurements are fully computer - controlled and carried out automatically within sequences of about 23  min .", "afterwards , the z - positions have to be  if necessary  readjusted . \n", "3calibration of the forces of the optical trap based on stokes law calibration of the forces of the optical trap based on stokes law", "streptavidin - modified polystyrene beads with a diameter of 2  m were purchased from polysciences europe ( eppelheim , germany ) , oligonucleotides provided by metabion ( martinsried , germany ) and mwg ( ebersberg , germany ) .", "nucleic acid purification kits were supplied by qiagen ( hilden , germany ) and anti - dig antibodies were purchased from roche ( penzberg , germany ) .", "all enzymes and standards were from mbi fermentas ( vilnius , lithuania ) and all other chemicals were delivered from sigma ( deisenhofen , germany ) .", "the double stranded dna handles were amplified from the plasmid puc18 in case of the 2,000  bp product , from pet28a+ in case of the 3,000 and 4,000  bp product , and from ptwin1 in case of the 6,000  bp product via standard polymerase chain reactions ( pcr ) .", "the oligonucleotides which were used were primer a ( 5-bio - cca cct gac gtc taa gaa acc-3 ) and primer b ( 5-dig - ggc aac aac gtt gcg caa ac-3 ) for the 2,000  bp dsdna , primer c ( 5-dig - acg gcc tca acc tac tac tg-3 ) and primer d ( 5-bio - gcg ata agt cgt gtc tta cc-3 ) for the 3,000  bp dsdna , primer e ( 5-dig - cag ctt cct ttc ggg ctt tg-3 ) and primer f ( 5-bio - tga ttg ccc gac att atc gc-3 ) for the 4,000  bp dsdna , and primer g ( 5-dig - atg gag gcg gat aaa gtt gc-3 ) and primer h ( 5-bio - gttgaatcaccgcgtaatcg-3 ) for the 6,000  bp dsdna .", "dna - handles were labeled with digoxygenine or biotine at their termini via 5-end modifications of the primers carried out by metabion or mwg ( fig . ", "the distribution of nucleotides in the artificial ds - dna and -dna was proven to be well comparable ( table  1 ) . \n", "2,000 , 3,000 , 4,000 , and 6,000  bp were analyzed on a 1.0 % agarose gel to check their correct size .", "b result of the binding assay . in lane 1 and 3 60  fmol of 2,000  bp dna", "lane 2 shows the result of the binding of the 2,000  bp dna to the anti - dig antibody labeled beads .", "lane 4 shows the negative control with carboxylated beads onlytable  1comparison of the distributions of nucleotides for the used dna samplesdnanumber of base pairsdistributionchemically amplified byhandle 2000 bd2,000  bpa : 486  bp ( 24.2%)puc18c : 522  bp ( 26.0%)g : 532  bp ( 26.5%)t : 471  bp ( 23.4%)handle 3000 bd3,000  bpa : 665  bp ( 22.2%)pet28a+c : 858  bp ( 28.6%)g : 829  bp ( 27.6%)t : 648  bp ( 21.6%)handle 4000 bd4,000  bpa : 888  bp ( 22.2%)pet28a+c : 1,097  bp ( 27.4%)g : 1,114  bp ( 27.9%)t : 901  bp ( 22.5%)handle 6000 bd6,000  bpa : 1,474  bp ( 24.6%)ptwin1c : 1,528  bp ( 25.5%)g : 1,642  bp ( 27.4%)t : 1,356  bp ( 22.6%)lambda - dna48,502  bpa : 12,334  bp ( 25.4%)c:11,362  bp ( 23.4%)g : 12,820  bp ( 26.4% ) t : 11,986  bp ( 24.7% ) \n a the dnas with the lengths 2,000 , 3,000 , 4,000 , and 6,000  bp were analyzed on a 1.0 % agarose gel to check their correct size .", "b result of the binding assay . in lane 1 and 3 60  fmol of 2,000  bp dna", "lane 2 shows the result of the binding of the 2,000  bp dna to the anti - dig antibody labeled beads .", "lane 4 shows the negative control with carboxylated beads only comparison of the distributions of nucleotides for the used dna samples the immobilization process was mainly deduced from protocols previously published by nustad et al .", "for the production of antidigoxigenin ( anti - dig ) antibody covered polystyrene beads , carboxylated particles with a diameter of 2.0  m were used .", "the beads from a 50  l bead suspension ( 26.5  mg / ml ) were washed twice with 200  l of carbonate buffer ( 100  mm sodium carbonate , 100  mm sodium bicarbonate , ph  9.6 ) and three times with 200  l buffer a ( 20  mm phosphate buffer , ph  4.5 ) . in between beads were collected by centrifugation ( 5  min 13,000g ) .", "after resuspension of the final bead pellet in 50  l buffer a , the surface carboxyl groups were activated by a 4-h incubation at room temperature with 50  l of a 2% edc solution ( ethyl-3-(3-dimethylaminopropyl)-carbodiimide ) in the same buffer .", "particles were washed three times with buffer a. after resuspension in 80  l buffer b ( 200  mm borate buffer , ph  8.5 ) , 10  l of anti - dig antibody solution ( 1  g ab/l ) were added followed by overnight shaking at room temperature . to avert unspecific binding at remaining free reactive groups , the pellet was resuspended in 100  l bsa solution ( 10  mg bsa / ml in buffer b ) and incubated under shaking for 30  min at room temperature .", "finally , the particles were resuspended in 20  l pbs ( 136  mm nacl , 2.7  mm kcl , 15.6  mm na2hpo4 , 17.6  mm kh2po4 ) , and stored at 4c . to determine the coupling efficiency 5  l of particle suspension", "were mixed with 50200  ng of a 2,000  bp pcr - amplified dna fragment , carrying a digoxygenine and a biotine modification at each terminus , respectively , in a total volume of 10  l pbs .", "this mixture was then incubated for 15  min at room temperature . as a negative control ,", "the whole suspension was than applied to a 1.5% agarose gel electrophoresis from which the particle bound dna fraction could be estimated ( fig . ", "to establish single dna strands between two colloids , the following procedure turned out to be effective and reliable ( fig . ", "5 ) : a bead with anti - dig antibodies was captured with the optical trap and brought in contact with the micropipette where it was fixed by capillary forces .", "afterwards , a sa - modified bead covered with dna was placed with the optical trap in the immediate neighborhood of the fixed anti - dig - bead so that still a visible distance between the particles was maintained . if a molecular contact was established , a sudden decrease in the fluctuation amplitude of the bead could be observed .", "after a short incubation time of 1  min , the particles are pulled apart to ensure that a single ds - dna had bound only . \n", "a polystyrene ( ps ) bead modified with anti - dig antibodies is trapped with the optical tweezers ( a ) and fixed by a micropipette due to capillary forces ( b ) . in the next step , a ps bead on which ds - dna is immobilized via biotin ", "streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette ( c ) . binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette", "is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap ( d ) . by measuring the force extension dependence , it is ensured that just one ds - dna strand is fixed between the beads assembly of a single ds - dna chain between two colloids .", "a polystyrene ( ps ) bead modified with anti - dig antibodies is trapped with the optical tweezers ( a ) and fixed by a micropipette due to capillary forces ( b ) . in the next step , a ps bead on which ds - dna is immobilized via biotin ", "streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette ( c ) . binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette", "is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap ( d ) . by measuring the force extension dependence", ", it is ensured that just one ds - dna strand is fixed between the beads", "the force  extension dependence ( fig .  6 ) of ds - dna of varying lengths between 2,000 to 6,000  bp can be described for forces 10  pn   by the worm - like chain model suggesting for the force f stretching a chain : \n 1\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$f\\ ; = \\;{\\left ( { \\frac{{k_{{\\text{b } } } t } } { { l_{{\\text{p } } } } } } \\right)}\\;{\\left [ { \\frac{1 } { { 4{\\left ( { 1 - \\frac{x } { { l_{{\\text{0 } } } } } } \\right)}^{2 } } } \\ ; - \\;\\frac{1 } { 4}\\ ; + \\;\\frac{x } { { l_{0 } } } } \\right]}$$\\end{document}where lp is the persistence length , l0 the contour length , x the extension , kb is boltzmann s constant , and t the absolute temperature . in principle , l0 is given by the number of base pairs assuming a length of 0.34  nm   per pair .", "but due to the fact that the polystyrene colloid with antidigoxigenin antibodies had an uneven surface ( with 100  nm deviations from the ideal spherical shape ) , the contour length was determined from the fits using eqs .  1 or 2 .", "comparing these values ( table  2 ) with the calculated lengths delivers agreement within the above mentioned absolute uncertainties of 100  nm . \n", "6force - extension dependence for single ds - dna chains of different length as indicated .", "the solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach .", "the experimental uncertainly is 0.5  pntable  2contour lengths as calculated from the number of base pairs assuming 0.34  nm length per base pair and as determined from the fits using the wlc model or the winkler approachnumber of base pairstheoretical contour length l \n 0 ( nm)experimentally determined contour length l \n 0 with wlc fit ( nm)experimentally determined contour length l \n 0 with winkler fit ( nm)2,0006806526543,0001,0209991,0044,0001,3601,3361,3416,0002,0401,9952,004the fits are restricted to forces 10  pn . the experiments are carried out in pbs - puffer with 137  mm nacl concentration .", "the uncertainty of fits as caused by the experimental accuracy of 0.5  pn in the force measurements and 2  nm in the determination of the position of the colloid in the optical trap force - extension dependence for single ds - dna chains of different length as indicated .", "the solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach .", "the experimental uncertainly is 0.5  pn contour lengths as calculated from the number of base pairs assuming 0.34  nm length per base pair and as determined from the fits using the wlc model or the winkler approach the fits are restricted to forces 10  pn .", "the experiments are carried out in pbs - puffer with 137  mm nacl concentration .", "the uncertainty of fits as caused by the experimental accuracy of 0.5  pn in the force measurements and 2  nm in the determination of the position of the colloid in the optical trap the data can be similarly well described by the model of a semiflexible chain of gaussian segments as suggested by winkler : \n 2\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$f = \\frac{{3k_{{\\text{b } } } tx } } { { 2l_{{\\text{o } } } l_{{\\text{p } } } { \\left ( { 1 - \\frac{{x^{2 } } } { { l_{{\\text{o } } } ^{2 } } } } \\right)}^{2 } } } $ $ \\end{document } the major difference between the approach by r. g. winkler and the worm - like chain ( wlc)-model is based on the fact that in the former the magnitude of the tangent vector is not exactly one but only its average value . as a consequence , the contour length is not fixed .", "the persistence lengths which were calculated by applying these models are listed in table  3 .", "the persistence length ranges between 1620  nm ( 5  nm ) for wlc and 2127  nm ( 4  nm ) for the winkler fit .", "the measurements were carried out in pbs - buffer with a high ionic strength ( 137  mm nacl ) . due to the high number of positively charged ions [ na ] ,", "the repulsion between the negatively charged groups of the phosphate backbone of the dna is reduced , which leads to an increase in flexibility and therefore to a decrease of the persistence length . \n", "table  3persistence length as determined from the fits with the wlc model and with winkler s approachnumber of base pairsexperimentally determined persistence length l \n p with wlc fit ( nm ) ( 5)experimentally determined persistence length l \n p with winkler fit ( nm ) ( 4)2,00020273,00016214,00020276,0001926the fits are restricted to forces 10  pn . in all cases , the sample was kept in pbs buffer having 137  mm nacl concentration persistence length as determined from the fits with the wlc model and with winkler s approach the fits are restricted to forces 10  pn . in all cases ,", "the sample was kept in pbs buffer having 137  mm nacl concentration from the wlc - fit for a 4,000-bp long dna in 10  mm nah2po4/na2hpo4 buffer ph  7 a persistence length of ( table  4 ) lp=46  nm is obtained .", "the fit with the winkler model leads to a value of lp=65  nm ( fig . ", "these values are in good agreement with data published previously by wang et al . .", "as expected with increasing salt concentration a pronounced decrease in the persistence length is obtained   ( table  4 ) . \n", "fig .  7force - extension dependence for a single ds - dna chain of 4,000  bp .", "the solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach .", "the experimental uncertainly is 0.5  pntable  4nacl concentration dependence of the persistence length of dna with 4,000  bp as determined with the wlc model and the winkler s approachnacl concentration ( mm)experimentally determined persistence length l \n p with wlc fit ( nm ) ( 5)experimentally determined persistence length l \n p with winkler fit ( nm ) ( 4)204665137 ( pbs)29401502839the fits are restricted to forces 10  pn force - extension dependence for a single ds - dna chain of 4,000  bp .", "the solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach .", "the experimental uncertainly is 0.5  pn nacl concentration dependence of the persistence length of dna with 4,000  bp as determined with the wlc model and the winkler s approach the fits are restricted to forces 10  pn compared to wang et al .", ", the persistence length obtained in our experiments has a more pronounced dependence on the ionic strength of the surrounding media .", "we attribute this discrepancy to the different buffer systems used  an effect well known and discussed in the current literature [ 16 , 18 , 19 ] .", "for the first time measurements of the force - extension dependence of single strands of ds - dna with systematically varying length are carried out .", "it is proven that the worm - like chain model , as well as the approach by r. g. winkler , describes the data well within experimental accuracy ."], "abstract_text": ["<S> optical tweezers are microscopic tools with extraordinary precision in the determination of the position ( 2  nm ) of a colloid ( diameter : 2.0  m ) in 3d - space and in the measurement of small forces in the range between 0.1 and 100  pn ( pn=1012  n ) . </S>", "<S> experiments are reported in which single double - stranded ( ds)-dna chains of different length [ 2,000 base pairs ( bp ) , 3,000 , 4,000 , and 6,000  bp ] are spanned between two colloidal particles by use of appropriate molecular linkers . for the forces applied ( 40  pn ) </S>", "<S> a fully reversible and well reproducible force  extension dependence is found . </S>", "<S> the data can be well described by both the worm - like chain model or by an approach developed by r. g. winkler . for the resulting persistence length , a pronounced dependence on the ionic concentration in the surrounding medium </S>", "<S> is found . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "The optical tweezers set-up", "Preparation", "Establishing a ", "Results and discussion", "Conclusion"], "sections": [["photonic forces [ 1 , 2 ] are known and understood for more than 100  years . but the discovery that they can be employed to manipulate small objects like colloids , viruses , or quantum dots became possible only after the establishment of strong laser sources in the infrared .", "this enables one to exert and to control forces acting on a small object in the range between 10 to 100  pn with an extraordinary high resolution of 50  fn . by incorporating additional optical systems to determine the position of a microscopic particle in 3d - space with nanometer precision , the novel experimental tool of optical tweezers was invented", "this initiated a manifold of spectacular experiments in biophysics [ 4a  j ] and colloid research .", "it became , for instance , possible to measure the elastic properties of -phage dna   on a single chain level , the rna polymerase was monitored for a single enzyme in action , or the work which has to be done to package bacteriophage dna into a single viral capsule was measured . in colloid physics , microscopic measurements of the pair interaction potential of two charge - stabilized colloids became possible   or direct determination of the cross correlations between two particles in an external potential .", "experiments with optical tweezers on the elastic properties of dna are mainly restricted to -phage dna which has a contour length of 16  m . in the presented article ,", "novel experiments are reported in which the dna under study is systematically varied in its length by genetic engineering .", "this enables one to analyze in great detail the elastic properties of dna and to check how well it can be described by the worm - like chain model   or competing approaches ."], ["1 ) is based on an inverted microscope ( axiovert s 100 tv , carl zeiss , jena , germany ) which is designed for epi - illumination fluorescence microscopy . for the optical tweezers ,", "a diode pumped nd : yag laser ( 1,064  nm , 1  w , lcs - dtl 322 ; laser 2000 , wessling , germany ) is used which is mounted to the microscope by the base port . the power and the profile of the laser", "a quarter - wave plate is employed to produce circular polarized light to exclude effects due to reflection differences between the p- and s - part of the laser light .", "the beam is expanded and coupled into the back aperture of the microscope objective ( plan - neofluor 1001.30 oil , carl zeiss , jena , germany ) . after passing through the sample cell , the beam is recollimated by a condenser with high numerical aperture .", "a beam splitting mirror and a convex lens images the forward scattered light of the bead in the optical trap onto a 1010  mm quadrant photodiode ( s 5107 , hamamatsu , herrsching am ammersee , germany ) for the electrical position detection . video imaging and the optical position detection is accomplished by a digital camera ( kp  f 120 , hitachi , dsseldorf , germany ) .", "the optical stage can be positioned in three dimensions with nanometer resolution using piezoactuators ( controller : e-710 , stage : p-517.3cd , pi gmbh , karlsruhe , germany ) .", "the sample cell consists of a closed chamber which can be flushed ( inset fig .  1 ) .", "a micropipette ( self - made ) with an inner diameter at the tip of 1  m is inserted into the chamber to hold one bead by capillary action . to determine the position of both beads and its separation image analysis is employed . by measuring and fitting the intensity profile of the two beads ( fig . ", "2 ) , its position relative to each other can be determined with an accuracy of 2  nm . \n fig . ", "1scheme of the experimental set - up . in the enlargement , the sample chamber is shownfig .", "2image analysis of two separate beads : measured ( a , b ) and calculated ( c ) intensity profile using the fit function : \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$i{\\left ( { x , y } \\right)}\\ ; = \\;i_{0 } + { \\sum\\limits_{i = 1,2 } { a_{i } } } { \\left ( { 1 - a_{i } u_{i } } \\right)}e^{{u^{3}_{i } } } $ $ \\end{document } ( 3 ) \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$u_{i } \\ ; = \\;{\\left ( { \\frac{{x - x_{i } } } { { r_{i } } } } \\right)}^{2 } \\ ; + \\;{\\left ( { \\frac{{y - y_{i } } } { { r_{i } } } } \\right)}^{2}$$\\end{document } ( 4 ) where for each colloid ( x \n i , y \n i ) is the center position , r \n i the optical radius , a \n i the amplitude of the profile relative to the background image intensity .", "i \n 0 and a \n i are parameters to consider the dark diffuse ring around the colloid scheme of the experimental set - up . in the enlargement ,", "the sample chamber is shown image analysis of two separate beads : measured ( a , b ) and calculated ( c ) intensity profile using the fit function : \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$i{\\left ( { x , y } \\right)}\\ ; = \\;i_{0 } + { \\sum\\limits_{i = 1,2 } { a_{i } } } { \\left ( { 1 - a_{i } u_{i } } \\right)}e^{{u^{3}_{i } } } $ $ \\end{document } ( 3 ) \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$u_{i } \\ ; = \\;{\\left ( { \\frac{{x - x_{i } } } { { r_{i } } } } \\right)}^{2 } \\ ; + \\;{\\left ( { \\frac{{y - y_{i } } } { { r_{i } } } } \\right)}^{2}$$\\end{document } ( 4 ) where for each colloid ( x \n i ,", "y \n i ) is the center position , r \n i the optical radius , a \n i the amplitude of the profile relative to the background image intensity .", "i \n 0 and a \n i are parameters to consider the dark diffuse ring around the colloid the calibration of the optical trap is based on stokes law f=6 r where  is the viscosity of the medium , r the radius of the bead , and  its velocity relative to the surrounding solution .", "varying the latter between 50 and 1,000  m / s enables one to calibrate the optical forces with an accuracy of 10% . as expected for the power dependence of the force constant of the optical trap , a linear relationship", "the whole experimental set - up is in a temperature controlled ( 1 ) room . due to the fact that the focus of the microscope changes with 1 by about 500  nm ,", "the actual measurements are fully computer - controlled and carried out automatically within sequences of about 23  min .", "afterwards , the z - positions have to be  if necessary  readjusted . \n", "3calibration of the forces of the optical trap based on stokes law calibration of the forces of the optical trap based on stokes law streptavidin - modified polystyrene beads with a diameter of 2  m were purchased from polysciences europe ( eppelheim , germany ) , oligonucleotides provided by metabion ( martinsried , germany ) and mwg ( ebersberg , germany ) .", "nucleic acid purification kits were supplied by qiagen ( hilden , germany ) and anti - dig antibodies were purchased from roche ( penzberg , germany ) .", "all enzymes and standards were from mbi fermentas ( vilnius , lithuania ) and all other chemicals were delivered from sigma ( deisenhofen , germany ) .", "the double stranded dna handles were amplified from the plasmid puc18 in case of the 2,000  bp product , from pet28a+ in case of the 3,000 and 4,000  bp product , and from ptwin1 in case of the 6,000  bp product via standard polymerase chain reactions ( pcr ) .", "the oligonucleotides which were used were primer a ( 5-bio - cca cct gac gtc taa gaa acc-3 ) and primer b ( 5-dig - ggc aac aac gtt gcg caa ac-3 ) for the 2,000  bp dsdna , primer c ( 5-dig - acg gcc tca acc tac tac tg-3 ) and primer d ( 5-bio - gcg ata agt cgt gtc tta cc-3 ) for the 3,000  bp dsdna , primer e ( 5-dig - cag ctt cct ttc ggg ctt tg-3 ) and primer f ( 5-bio - tga ttg ccc gac att atc gc-3 ) for the 4,000  bp dsdna , and primer g ( 5-dig - atg gag gcg gat aaa gtt gc-3 ) and primer h ( 5-bio - gttgaatcaccgcgtaatcg-3 ) for the 6,000  bp dsdna .", "dna - handles were labeled with digoxygenine or biotine at their termini via 5-end modifications of the primers carried out by metabion or mwg ( fig . ", "the distribution of nucleotides in the artificial ds - dna and -dna was proven to be well comparable ( table  1 ) . \n", "fig .  4 \n a the dnas with the lengths 2,000 , 3,000 , 4,000 , and 6,000 ", "b result of the binding assay . in lane 1 and 3 60  fmol of 2,000  bp dna", "lane 2 shows the result of the binding of the 2,000  bp dna to the anti - dig antibody labeled beads .", "lane 4 shows the negative control with carboxylated beads onlytable  1comparison of the distributions of nucleotides for the used dna samplesdnanumber of base pairsdistributionchemically amplified byhandle 2000 bd2,000  bpa : 486  bp ( 24.2%)puc18c : 522  bp ( 26.0%)g : 532  bp ( 26.5%)t : 471  bp ( 23.4%)handle 3000 bd3,000  bpa : 665  bp ( 22.2%)pet28a+c : 858  bp ( 28.6%)g : 829  bp ( 27.6%)t : 648  bp ( 21.6%)handle 4000 bd4,000  bpa : 888  bp ( 22.2%)pet28a+c : 1,097  bp ( 27.4%)g : 1,114  bp ( 27.9%)t : 901  bp ( 22.5%)handle 6000 bd6,000  bpa : 1,474  bp ( 24.6%)ptwin1c : 1,528  bp ( 25.5%)g : 1,642  bp ( 27.4%)t : 1,356  bp ( 22.6%)lambda - dna48,502  bpa : 12,334  bp ( 25.4%)c:11,362  bp ( 23.4%)g : 12,820  bp ( 26.4% ) t : 11,986  bp ( 24.7% ) \n a the dnas with the lengths 2,000 , 3,000 , 4,000 , and 6,000  bp were analyzed on a 1.0 % agarose gel to check their correct size .", "b result of the binding assay . in lane 1 and 3 60  fmol of 2,000  bp dna", "lane 2 shows the result of the binding of the 2,000  bp dna to the anti - dig antibody labeled beads .", "lane 4 shows the negative control with carboxylated beads only comparison of the distributions of nucleotides for the used dna samples the immobilization process was mainly deduced from protocols previously published by nustad et al . ,", "for the production of antidigoxigenin ( anti - dig ) antibody covered polystyrene beads , carboxylated particles with a diameter of 2.0  m were used .", "the beads from a 50  l bead suspension ( 26.5  mg / ml ) were washed twice with 200  l of carbonate buffer ( 100  mm sodium carbonate , 100  mm sodium bicarbonate , ph  9.6 ) and three times with 200  l buffer a ( 20  mm phosphate buffer , ph  4.5 ) . in between beads were collected by centrifugation ( 5  min 13,000g ) .", "after resuspension of the final bead pellet in 50  l buffer a , the surface carboxyl groups were activated by a 4-h incubation at room temperature with 50  l of a 2% edc solution ( ethyl-3-(3-dimethylaminopropyl)-carbodiimide ) in the same buffer .", "particles were washed three times with buffer a. after resuspension in 80  l buffer b ( 200  mm borate buffer , ph  8.5 ) , 10  l of anti - dig antibody solution ( 1  g ab/l ) were added followed by overnight shaking at room temperature . to avert unspecific binding at remaining free reactive groups , the pellet was resuspended in 100  l bsa solution ( 10  mg", "bsa / ml in buffer b ) and incubated under shaking for 30  min at room temperature .", "finally , the particles were resuspended in 20  l pbs ( 136  mm nacl , 2.7  mm kcl , 15.6  mm na2hpo4 , 17.6  mm kh2po4 ) , and stored at 4c . to determine the coupling efficiency 5  l of particle suspension", "were mixed with 50200  ng of a 2,000  bp pcr - amplified dna fragment , carrying a digoxygenine and a biotine modification at each terminus , respectively , in a total volume of 10  l pbs .", "this mixture was then incubated for 15  min at room temperature . as a negative control ,", "the whole suspension was than applied to a 1.5% agarose gel electrophoresis from which the particle bound dna fraction could be estimated ( fig . ", "to establish single dna strands between two colloids , the following procedure turned out to be effective and reliable ( fig . ", "5 ) : a bead with anti - dig antibodies was captured with the optical trap and brought in contact with the micropipette where it was fixed by capillary forces .", "afterwards , a sa - modified bead covered with dna was placed with the optical trap in the immediate neighborhood of the fixed anti - dig - bead so that still a visible distance between the particles was maintained . if a molecular contact was established , a sudden decrease in the fluctuation amplitude of the bead could be observed . after a short incubation time of 1  min ,", "the particles are pulled apart to ensure that a single ds - dna had bound only . \n", "a polystyrene ( ps ) bead modified with anti - dig antibodies is trapped with the optical tweezers ( a ) and fixed by a micropipette due to capillary forces ( b ) . in the next step , a ps bead on which ds - dna is immobilized via biotin ", "streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette ( c ) . binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette", "is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap ( d ) . by measuring the force extension dependence", ", it is ensured that just one ds - dna strand is fixed between the beads assembly of a single ds - dna chain between two colloids .", "a polystyrene ( ps ) bead modified with anti - dig antibodies is trapped with the optical tweezers ( a ) and fixed by a micropipette due to capillary forces ( b ) . in the next step , a ps bead on which ds - dna is immobilized via biotin ", "streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette ( c ) . binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette", "is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap ( d ) . by measuring the force extension dependence", ", it is ensured that just one ds - dna strand is fixed between the beads"], ["1 ) is based on an inverted microscope ( axiovert s 100 tv , carl zeiss , jena , germany ) which is designed for epi - illumination fluorescence microscopy . for the optical tweezers ,", "a diode pumped nd : yag laser ( 1,064  nm , 1  w , lcs - dtl 322 ; laser 2000 , wessling , germany ) is used which is mounted to the microscope by the base port . the power and the profile of the laser", "a quarter - wave plate is employed to produce circular polarized light to exclude effects due to reflection differences between the p- and s - part of the laser light .", "the beam is expanded and coupled into the back aperture of the microscope objective ( plan - neofluor 1001.30 oil , carl zeiss , jena , germany ) . after passing through the sample cell , the beam is recollimated by a condenser with high numerical aperture .", "a beam splitting mirror and a convex lens images the forward scattered light of the bead in the optical trap onto a 1010  mm quadrant photodiode ( s 5107 , hamamatsu , herrsching am ammersee , germany ) for the electrical position detection . video imaging and the optical position detection is accomplished by a digital camera ( kp  f 120 , hitachi , dsseldorf , germany ) .", "the optical stage can be positioned in three dimensions with nanometer resolution using piezoactuators ( controller : e-710 , stage : p-517.3cd , pi gmbh , karlsruhe , germany ) .", "the sample cell consists of a closed chamber which can be flushed ( inset fig .  1 ) .", "a micropipette ( self - made ) with an inner diameter at the tip of 1  m is inserted into the chamber to hold one bead by capillary action . to determine the position of both beads and its separation image analysis is employed . by measuring and fitting the intensity profile of the two beads ( fig . ", "2 ) , its position relative to each other can be determined with an accuracy of 2  nm . \n fig . ", "1scheme of the experimental set - up . in the enlargement , the sample chamber is shownfig .", "2image analysis of two separate beads : measured ( a , b ) and calculated ( c ) intensity profile using the fit function : \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$i{\\left ( { x , y } \\right)}\\ ; = \\;i_{0 } + { \\sum\\limits_{i = 1,2 } { a_{i } } } { \\left ( { 1 - a_{i } u_{i } } \\right)}e^{{u^{3}_{i } } } $ $ \\end{document } ( 3 ) \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$u_{i } \\ ; = \\;{\\left ( { \\frac{{x - x_{i } } } { { r_{i } } } } \\right)}^{2 } \\ ; + \\;{\\left ( { \\frac{{y - y_{i } } } { { r_{i } } } } \\right)}^{2}$$\\end{document } ( 4 ) where for each colloid ( x \n i , y \n i ) is the center position , r \n i the optical radius , a \n i the amplitude of the profile relative to the background image intensity .", "i \n 0 and a \n i are parameters to consider the dark diffuse ring around the colloid scheme of the experimental set - up . in the enlargement ,", "the sample chamber is shown image analysis of two separate beads : measured ( a , b ) and calculated ( c ) intensity profile using the fit function : \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$i{\\left ( { x , y } \\right)}\\ ; = \\;i_{0 } + { \\sum\\limits_{i = 1,2 } { a_{i } } } { \\left ( { 1 - a_{i } u_{i } } \\right)}e^{{u^{3}_{i } } } $ $ \\end{document } ( 3 ) \\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$u_{i } \\ ; = \\;{\\left ( { \\frac{{x - x_{i } } } { { r_{i } } } } \\right)}^{2 } \\ ; + \\;{\\left ( { \\frac{{y - y_{i } } } { { r_{i } } } } \\right)}^{2}$$\\end{document } ( 4 ) where for each colloid ( x \n i ,", "y \n i ) is the center position , r \n i the optical radius , a \n i the amplitude of the profile relative to the background image intensity .", "i \n 0 and a \n i are parameters to consider the dark diffuse ring around the colloid the calibration of the optical trap is based on stokes law f=6 r where  is the viscosity of the medium , r the radius of the bead , and  its velocity relative to the surrounding solution .", "varying the latter between 50 and 1,000  m / s enables one to calibrate the optical forces with an accuracy of 10% . as expected for the power dependence of the force constant of the optical trap , a linear relationship", "the whole experimental set - up is in a temperature controlled ( 1 ) room . due to the fact that the focus of the microscope changes with 1 by about 500  nm ,", "the actual measurements are fully computer - controlled and carried out automatically within sequences of about 23  min .", "afterwards , the z - positions have to be  if necessary  readjusted . \n", "3calibration of the forces of the optical trap based on stokes law calibration of the forces of the optical trap based on stokes law"], ["streptavidin - modified polystyrene beads with a diameter of 2  m were purchased from polysciences europe ( eppelheim , germany ) , oligonucleotides provided by metabion ( martinsried , germany ) and mwg ( ebersberg , germany ) .", "nucleic acid purification kits were supplied by qiagen ( hilden , germany ) and anti - dig antibodies were purchased from roche ( penzberg , germany ) .", "all enzymes and standards were from mbi fermentas ( vilnius , lithuania ) and all other chemicals were delivered from sigma ( deisenhofen , germany ) .", "the double stranded dna handles were amplified from the plasmid puc18 in case of the 2,000  bp product , from pet28a+ in case of the 3,000 and 4,000  bp product , and from ptwin1 in case of the 6,000  bp product via standard polymerase chain reactions ( pcr ) .", "the oligonucleotides which were used were primer a ( 5-bio - cca cct gac gtc taa gaa acc-3 ) and primer b ( 5-dig - ggc aac aac gtt gcg caa ac-3 ) for the 2,000  bp dsdna , primer c ( 5-dig - acg gcc tca acc tac tac tg-3 ) and primer d ( 5-bio - gcg ata agt cgt gtc tta cc-3 ) for the 3,000  bp dsdna , primer e ( 5-dig - cag ctt cct ttc ggg ctt tg-3 ) and primer f ( 5-bio - tga ttg ccc gac att atc gc-3 ) for the 4,000  bp dsdna , and primer g ( 5-dig - atg gag gcg gat aaa gtt gc-3 ) and primer h ( 5-bio - gttgaatcaccgcgtaatcg-3 ) for the 6,000  bp dsdna .", "dna - handles were labeled with digoxygenine or biotine at their termini via 5-end modifications of the primers carried out by metabion or mwg ( fig . ", "the distribution of nucleotides in the artificial ds - dna and -dna was proven to be well comparable ( table  1 ) . \n", "2,000 , 3,000 , 4,000 , and 6,000  bp were analyzed on a 1.0 % agarose gel to check their correct size .", "b result of the binding assay . in lane 1 and 3 60  fmol of 2,000  bp dna", "lane 2 shows the result of the binding of the 2,000  bp dna to the anti - dig antibody labeled beads .", "lane 4 shows the negative control with carboxylated beads onlytable  1comparison of the distributions of nucleotides for the used dna samplesdnanumber of base pairsdistributionchemically amplified byhandle 2000 bd2,000  bpa : 486  bp ( 24.2%)puc18c : 522  bp ( 26.0%)g : 532  bp ( 26.5%)t : 471  bp ( 23.4%)handle 3000 bd3,000  bpa : 665  bp ( 22.2%)pet28a+c : 858  bp ( 28.6%)g : 829  bp ( 27.6%)t : 648  bp ( 21.6%)handle 4000 bd4,000  bpa : 888  bp ( 22.2%)pet28a+c : 1,097  bp ( 27.4%)g : 1,114  bp ( 27.9%)t : 901  bp ( 22.5%)handle 6000 bd6,000  bpa : 1,474  bp ( 24.6%)ptwin1c : 1,528  bp ( 25.5%)g : 1,642  bp ( 27.4%)t : 1,356  bp ( 22.6%)lambda - dna48,502  bpa : 12,334  bp ( 25.4%)c:11,362  bp ( 23.4%)g : 12,820  bp ( 26.4% ) t : 11,986  bp ( 24.7% ) \n a the dnas with the lengths 2,000 , 3,000 , 4,000 , and 6,000  bp were analyzed on a 1.0 % agarose gel to check their correct size .", "b result of the binding assay . in lane 1 and 3 60  fmol of 2,000  bp dna", "lane 2 shows the result of the binding of the 2,000  bp dna to the anti - dig antibody labeled beads .", "lane 4 shows the negative control with carboxylated beads only comparison of the distributions of nucleotides for the used dna samples the immobilization process was mainly deduced from protocols previously published by nustad et al .", "for the production of antidigoxigenin ( anti - dig ) antibody covered polystyrene beads , carboxylated particles with a diameter of 2.0  m were used .", "the beads from a 50  l bead suspension ( 26.5  mg / ml ) were washed twice with 200  l of carbonate buffer ( 100  mm sodium carbonate , 100  mm sodium bicarbonate , ph  9.6 ) and three times with 200  l buffer a ( 20  mm phosphate buffer , ph  4.5 ) . in between beads were collected by centrifugation ( 5  min 13,000g ) .", "after resuspension of the final bead pellet in 50  l buffer a , the surface carboxyl groups were activated by a 4-h incubation at room temperature with 50  l of a 2% edc solution ( ethyl-3-(3-dimethylaminopropyl)-carbodiimide ) in the same buffer .", "particles were washed three times with buffer a. after resuspension in 80  l buffer b ( 200  mm borate buffer , ph  8.5 ) , 10  l of anti - dig antibody solution ( 1  g ab/l ) were added followed by overnight shaking at room temperature . to avert unspecific binding at remaining free reactive groups , the pellet was resuspended in 100  l bsa solution ( 10  mg bsa / ml in buffer b ) and incubated under shaking for 30  min at room temperature .", "finally , the particles were resuspended in 20  l pbs ( 136  mm nacl , 2.7  mm kcl , 15.6  mm na2hpo4 , 17.6  mm kh2po4 ) , and stored at 4c . to determine the coupling efficiency 5  l of particle suspension", "were mixed with 50200  ng of a 2,000  bp pcr - amplified dna fragment , carrying a digoxygenine and a biotine modification at each terminus , respectively , in a total volume of 10  l pbs .", "this mixture was then incubated for 15  min at room temperature . as a negative control ,", "the whole suspension was than applied to a 1.5% agarose gel electrophoresis from which the particle bound dna fraction could be estimated ( fig . "], ["to establish single dna strands between two colloids , the following procedure turned out to be effective and reliable ( fig . ", "5 ) : a bead with anti - dig antibodies was captured with the optical trap and brought in contact with the micropipette where it was fixed by capillary forces .", "afterwards , a sa - modified bead covered with dna was placed with the optical trap in the immediate neighborhood of the fixed anti - dig - bead so that still a visible distance between the particles was maintained . if a molecular contact was established , a sudden decrease in the fluctuation amplitude of the bead could be observed .", "after a short incubation time of 1  min , the particles are pulled apart to ensure that a single ds - dna had bound only . \n", "a polystyrene ( ps ) bead modified with anti - dig antibodies is trapped with the optical tweezers ( a ) and fixed by a micropipette due to capillary forces ( b ) . in the next step , a ps bead on which ds - dna is immobilized via biotin ", "streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette ( c ) . binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette", "is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap ( d ) . by measuring the force extension dependence , it is ensured that just one ds - dna strand is fixed between the beads assembly of a single ds - dna chain between two colloids .", "a polystyrene ( ps ) bead modified with anti - dig antibodies is trapped with the optical tweezers ( a ) and fixed by a micropipette due to capillary forces ( b ) . in the next step , a ps bead on which ds - dna is immobilized via biotin ", "streptavidin linkers is captured with the optical tweezers and brought in the immediate neighborhood of the colloid held by the micropipette ( c ) . binding between the digoxigenin labeled end of a dna strand with an antidigoxigenin antibody on the surface of the bead held by the micropipette", "is observed as a sudden decrease of the brownian fluctuations of the bead in the optical trap ( d ) . by measuring the force extension dependence", ", it is ensured that just one ds - dna strand is fixed between the beads"], ["the force  extension dependence ( fig .  6 ) of ds - dna of varying lengths between 2,000 to 6,000  bp can be described for forces 10  pn   by the worm - like chain model suggesting for the force f stretching a chain : \n 1\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$f\\ ; = \\;{\\left ( { \\frac{{k_{{\\text{b } } } t } } { { l_{{\\text{p } } } } } } \\right)}\\;{\\left [ { \\frac{1 } { { 4{\\left ( { 1 - \\frac{x } { { l_{{\\text{0 } } } } } } \\right)}^{2 } } } \\ ; - \\;\\frac{1 } { 4}\\ ; + \\;\\frac{x } { { l_{0 } } } } \\right]}$$\\end{document}where lp is the persistence length , l0 the contour length , x the extension , kb is boltzmann s constant , and t the absolute temperature . in principle , l0 is given by the number of base pairs assuming a length of 0.34  nm   per pair .", "but due to the fact that the polystyrene colloid with antidigoxigenin antibodies had an uneven surface ( with 100  nm deviations from the ideal spherical shape ) , the contour length was determined from the fits using eqs .  1 or 2 .", "comparing these values ( table  2 ) with the calculated lengths delivers agreement within the above mentioned absolute uncertainties of 100  nm . \n", "6force - extension dependence for single ds - dna chains of different length as indicated .", "the solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach .", "the experimental uncertainly is 0.5  pntable  2contour lengths as calculated from the number of base pairs assuming 0.34  nm length per base pair and as determined from the fits using the wlc model or the winkler approachnumber of base pairstheoretical contour length l \n 0 ( nm)experimentally determined contour length l \n 0 with wlc fit ( nm)experimentally determined contour length l \n 0 with winkler fit ( nm)2,0006806526543,0001,0209991,0044,0001,3601,3361,3416,0002,0401,9952,004the fits are restricted to forces 10  pn . the experiments are carried out in pbs - puffer with 137  mm nacl concentration .", "the uncertainty of fits as caused by the experimental accuracy of 0.5  pn in the force measurements and 2  nm in the determination of the position of the colloid in the optical trap force - extension dependence for single ds - dna chains of different length as indicated .", "the solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach .", "the experimental uncertainly is 0.5  pn contour lengths as calculated from the number of base pairs assuming 0.34  nm length per base pair and as determined from the fits using the wlc model or the winkler approach the fits are restricted to forces 10  pn .", "the experiments are carried out in pbs - puffer with 137  mm nacl concentration .", "the uncertainty of fits as caused by the experimental accuracy of 0.5  pn in the force measurements and 2  nm in the determination of the position of the colloid in the optical trap the data can be similarly well described by the model of a semiflexible chain of gaussian segments as suggested by winkler : \n 2\\documentclass[12pt]{minimal } \n\t\t\t\t \\usepackage{amsmath } \n\t\t\t\t \\usepackage{wasysym } \n\t\t\t\t \\usepackage{amsfonts } \n\t\t\t\t \\usepackage{amssymb } \n\t\t\t\t \\usepackage{amsbsy } \n\t\t\t\t \\usepackage{mathrsfs } \n\t\t\t\t \\usepackage{upgreek } \n\t\t\t\t \\setlength{\\oddsidemargin}{-69pt } \n\t\t\t\t \\begin{document}$$f = \\frac{{3k_{{\\text{b } } } tx } } { { 2l_{{\\text{o } } } l_{{\\text{p } } } { \\left ( { 1 - \\frac{{x^{2 } } } { { l_{{\\text{o } } } ^{2 } } } } \\right)}^{2 } } } $ $ \\end{document } the major difference between the approach by r. g. winkler and the worm - like chain ( wlc)-model is based on the fact that in the former the magnitude of the tangent vector is not exactly one but only its average value . as a consequence , the contour length is not fixed .", "the persistence lengths which were calculated by applying these models are listed in table  3 .", "the persistence length ranges between 1620  nm ( 5  nm ) for wlc and 2127  nm ( 4  nm ) for the winkler fit .", "the measurements were carried out in pbs - buffer with a high ionic strength ( 137  mm nacl ) . due to the high number of positively charged ions [ na ] ,", "the repulsion between the negatively charged groups of the phosphate backbone of the dna is reduced , which leads to an increase in flexibility and therefore to a decrease of the persistence length . \n", "table  3persistence length as determined from the fits with the wlc model and with winkler s approachnumber of base pairsexperimentally determined persistence length l \n p with wlc fit ( nm ) ( 5)experimentally determined persistence length l \n p with winkler fit ( nm ) ( 4)2,00020273,00016214,00020276,0001926the fits are restricted to forces 10  pn . in all cases , the sample was kept in pbs buffer having 137  mm nacl concentration persistence length as determined from the fits with the wlc model and with winkler s approach the fits are restricted to forces 10  pn . in all cases ,", "the sample was kept in pbs buffer having 137  mm nacl concentration from the wlc - fit for a 4,000-bp long dna in 10  mm nah2po4/na2hpo4 buffer ph  7 a persistence length of ( table  4 ) lp=46  nm is obtained .", "the fit with the winkler model leads to a value of lp=65  nm ( fig . ", "these values are in good agreement with data published previously by wang et al . .", "as expected with increasing salt concentration a pronounced decrease in the persistence length is obtained   ( table  4 ) . \n", "fig .  7force - extension dependence for a single ds - dna chain of 4,000  bp .", "the solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach .", "the experimental uncertainly is 0.5  pntable  4nacl concentration dependence of the persistence length of dna with 4,000  bp as determined with the wlc model and the winkler s approachnacl concentration ( mm)experimentally determined persistence length l \n p with wlc fit ( nm ) ( 5)experimentally determined persistence length l \n p with winkler fit ( nm ) ( 4)204665137 ( pbs)29401502839the fits are restricted to forces 10  pn force - extension dependence for a single ds - dna chain of 4,000  bp .", "the solid line indicates a fit using the worm - like chain model while the dashed line corresponds to winkler s approach .", "the experimental uncertainly is 0.5  pn nacl concentration dependence of the persistence length of dna with 4,000  bp as determined with the wlc model and the winkler s approach the fits are restricted to forces 10  pn compared to wang et al .", ", the persistence length obtained in our experiments has a more pronounced dependence on the ionic strength of the surrounding media .", "we attribute this discrepancy to the different buffer systems used  an effect well known and discussed in the current literature [ 16 , 18 , 19 ] ."], ["for the first time measurements of the force - extension dependence of single strands of ds - dna with systematically varying length are carried out .", "it is proven that the worm - like chain model , as well as the approach by r. g. winkler , describes the data well within experimental accuracy ."]]}
{"article_id": "PMC2893710", "article_text": ["the strong quantum confinement effect of colloidal semiconductor nanocrystals ( ncs ) has made these materials the subject of extensive research [ 1 - 5 ] . in the past decades , binary semiconductor ncs ,", "identified as groups ii  vi and iii  v , have been studied intensively because of their size - dependent photoluminescence ( pl ) covering the spectrum range from ultraviolet to near infrared [ 6 - 10 ] .", "the importance of cds ncs is manifested in their potential application as fluorescent materials with uv  blue pl emission [ 11 - 17 ] .", "the typical synthesis recipes for cds ncs are based on a process that involves the injection of cd - oleic acid ( oa ) into batch reactors .", "nearly , monodispersed cds ncs have been prepared using octadecene ( ode ) as the non - coordinating solvent , and subsequent treatment of these ncs resulted in considerable improvement of pl efficiency . in these cases , the inefficient heat and mass transfer of batch reactors constrained the economical synthesis of cds ncs .", "in order to overcome the energy barrier to form active monomers , a high reaction temperature ( 250300c ) is generally required . meanwhile , the temperature and concentration gradients involved in large reactors lead to poor quality and low process reproducibility .", "furthermore , according to the international commission on illumination chromaticity diagram , the ideal wavelength for blue light is 460480 nm . to achieve cds ncs with blue pl emission", ", large cds ncs with diameters greater than 5.2 nm are required . however , the preparation of these cds ncs is limited by the high reaction temperature and long reaction time , where the advent of ostwald ripening leads to wide size distributions .", "the enhanced heat and mass transfer properties in the microenvironment could provide highly homogeneous conditions for the chemical synthesis of ncs [ 18 - 24 ] .", "compared with the batch reactor , a microreactor offers a controllable way to synthesize ncs continuously in a steady fashion [ 9,20 - 23 ] .", "meanwhile , the incorporation of novel channel structures and flow patterns facilitated the synthesis of monodisperse ncs in an accelerated manner .", "however , the limited research conducted to date on the microfluidic synthesis of cds ncs has been based on aqueous processes , and the resulting ncs generally had wide size distributions and deep - trap luminescence that overwhelmed the visible range [ 19,25 - 27 ] .", "droplet microfluidics has been demonstrated as a powerful tool for achieving efficient mixing , while realizing highly homogeneous environment for the synthesis of monodisperse cds ncs , but its low throughput limits its use to commercial - scale synthesis . in this study ,", "a capillary microreactor was used for the accelerated synthesis of monodisperse cds ncs . in order to obtain high - quality samples , systematic investigations of temperature and residence time requirements", "also , a quantitative investigation of the influence of oa on size distribution and growth kinetics was conducted to achieve the size - controlled synthesis of cds ncs .", "the influence of oa on the reaction kinetics was evidenced by the collected absorption spectra .", "in addition , the high quality of the resulting cds ncs was confirmed by the pl spectra , powder x - ray diffraction ( xrd ) , and high - resolution transmission electron microscopy ( hrtem ) .", "cadmium oxide ( cdo , scr , 99.9% ) , sulfur ( s , scr , 99.5% ) , 1-octadecene ( ode , fisher , 90% ) , oleic acid ( oa , scr , 90% ) , and analytic grade acetone and chloroform ( scr ) were used directly without further processing .", "cds ncs were prepared using a recipe similar to the one previously reported by yu et al . .", "typically , 1 mmol cdo , 4.5 mmol oa , and ode ( 5 ml in total ) were mixed together and heated to 150c under a nitrogen atmosphere for 1 h with vigorous stirring to prepare a clear , yellow , cadmium - precursor solution . meanwhile , a stock solution of sulfur was prepared by dissolving 1 mmol s powder in ode ( 5 ml in total ) at 150c with magnetic stirring for 1.5 h. the microfluidic reactor included a precursor delivery system , a convective micromixer ( 50 l ) , and a length of polytetrafluoroethylene ( ptfe ) capillary ( i d = 462 m , length = 50 cm ) , as shown in fig .", "equal volume solutions of cd and s precursors were delivered by a syringe pump ( harvard 22 , usa ) at the same flow rate , and the precursors were combined and mixed in a magnetic micromixer .", "then , the mixture of precursors was placed in a heated ptfe capillary where nucleation and growth of the ncs occurred at a constant reaction temperature of 220c .", "a thermally stable oil bath was used as a heat source , and the flow rate of the precursors was set at 4.47 ml h to achieve a residence time of 68 s. during the optimization process for oa concentration , various amounts of oa were replaced by ode , while maintaining a constant volume of solution .", "schematic representation of the capillary microfluidic reactor uv  vis absorption spectra were recorded on a cary 50 uv  vis spectrometer ( varian , usa ) at room temperature .", "pl spectra were measured at room temperature with a cary eclipse spectrofluorometer ( varian , usa ) for colloidal solutions with an optical density of less than 0.2 at an excitation wavelength of 330 nm .", "quantum yields ( qy ) of pl was obtained by comparing the integrated pl intensities of the ncs with the organic dye ( rhodamine 6 g ) . to obtain samples for tem and xrd characterization ,", "the formed cds ncs were precipitated by adding acetone into their chloroform solution ; the ncs were isolated and purified by repeated centrifugation and decantation .", "xrd patterns were obtained using a d / max-2550 diffraction meter ( rigaku , japan ) with a cu anode . during the preparation of samples for xrd analysis ,", "the morphologies and dimensions of the as - formed ncs were observed by a jem-2100f hrtem ( jeol , japan ) .", "the samples for hrtem observations were obtained by dipping a carbon  copper grid in a dilute , chloroform - dispersed solution of ncs .", "cadmium oxide ( cdo , scr , 99.9% ) , sulfur ( s , scr , 99.5% ) , 1-octadecene ( ode , fisher , 90% ) , oleic acid ( oa , scr , 90% ) , and analytic grade acetone and chloroform ( scr ) were used directly without further processing .", "cds ncs were prepared using a recipe similar to the one previously reported by yu et al . .", "typically , 1 mmol cdo , 4.5 mmol oa , and ode ( 5 ml in total ) were mixed together and heated to 150c under a nitrogen atmosphere for 1 h with vigorous stirring to prepare a clear , yellow , cadmium - precursor solution . meanwhile , a stock solution of sulfur was prepared by dissolving 1 mmol s powder in ode ( 5 ml in total ) at 150c with magnetic stirring for 1.5 h. the microfluidic reactor included a precursor delivery system , a convective micromixer ( 50 l ) , and a length of polytetrafluoroethylene ( ptfe ) capillary ( i d = 462 m , length = 50 cm ) , as shown in fig .", "equal volume solutions of cd and s precursors were delivered by a syringe pump ( harvard 22 , usa ) at the same flow rate , and the precursors were combined and mixed in a magnetic micromixer . then , the mixture of precursors was placed in a heated ptfe capillary where nucleation and growth of the ncs occurred at a constant reaction temperature of 220c .", "a thermally stable oil bath was used as a heat source , and the flow rate of the precursors was set at 4.47 ml h to achieve a residence time of 68 s. during the optimization process for oa concentration , various amounts of oa were replaced by ode , while maintaining a constant volume of solution .", "uv  vis absorption spectra were recorded on a cary 50 uv  vis spectrometer ( varian , usa ) at room temperature .", "pl spectra were measured at room temperature with a cary eclipse spectrofluorometer ( varian , usa ) for colloidal solutions with an optical density of less than 0.2 at an excitation wavelength of 330 nm .", "quantum yields ( qy ) of pl was obtained by comparing the integrated pl intensities of the ncs with the organic dye ( rhodamine 6 g ) . to obtain samples for tem and xrd characterization ,", "the formed cds ncs were precipitated by adding acetone into their chloroform solution ; the ncs were isolated and purified by repeated centrifugation and decantation .", "xrd patterns were obtained using a d / max-2550 diffraction meter ( rigaku , japan ) with a cu anode . during the preparation of samples for xrd analysis ,", "the morphologies and dimensions of the as - formed ncs were observed by a jem-2100f hrtem ( jeol , japan ) .", "the samples for hrtem observations were obtained by dipping a carbon  copper grid in a dilute , chloroform - dispersed solution of ncs .", "in batch reactions , the long response time for temperature stabilization and the vibration at reaction conditions make it difficult to achieve precise control of the sizes of cds ncs . on the contrary", "furthermore , the enhanced heat and mass transfer in a microreactor greatly improves the concentrations of reactive monomers , resulting in reductions in the reaction temperature and residence time required to form high - quality ncs . in order to achieve a balance between process stability and the reactivity of precursors ,", ", the reaction temperature is an important parameter that determines both the ligand and reaction kinetics .", "low temperatures are insufficient for overcoming the energy barrier for highly reactive monomers , while high temperatures that exceed a threshold lead to the early advent of ostwald ripening . in this study , the reaction temperature was adjusted from 180 to 260c at a constant residence time of 68 s. figure 2 shows the absorption and pl spectra as well as the kinetics data of cds ncs prepared at various reaction temperatures . generally , the sharp band - edge absorbance with a half width at half maximum ( hwhm ) value of 12 nm and several higher transitions are clearly resolved for cds ncs that were synthesized at temperatures above 200c .", "whereas , due to the weak reactivity , the magic - size cluster was observed at 180c , indicated by the appearance of an absorption peak with a wavelength of almost 320 nm .", "in addition , pure band - gap emissions with very narrow full width at half maximum ( fwhm~17 nm ) were observed at 220c .", "the earlier mentioned results clearly point to a narrow size distribution and well - passivated surfaces of the cds ncs . when the temperature was increased from 180 to 240c ,", "a 44-nm redshift in the pl peak was observed , indicating that the growth rate of the ncs improved at the higher temperatures .", "however , impurities in the technical grade ode resulted in the evolution of gas at high reaction temperatures above 240c .", "the presence of the gas decreased the residence time and induced the blueshift of the pl peak . a absorption , b pl spectra , and c relative kinetics of cds ncs prepared at various temperatures at a constant residence time of 68 s ( oa : 15 . 6 vol % ) fwhm of pl is an indirect measure for the size distribution of ncs . in fig .", "2c , it can be seen that fwhm of the ncs tended to decrease when the temperature was changed from 180 to 220c .", "further increases in the temperature led to wider fwhm values . during the diffusion - controlled synthesis of spherical ncs , the burst of nucleation that separated from the growth is the prerequisite for achieving monodisperse products .", "temperatures below 180c can not overcome the energy barrier to form the reactive monomer species , which restricted the burst of nucleation .", "the nucleation in the whole reaction phase led to poor size distribution . with the increase in reaction temperature from 180 to 220c ,", "the continuously increased reactivity of the monomer allowed the burst of nucleation , leading to gradual narrowing of the size distribution .", "the narrowest fwhm was observed at 220c , indicating a balance of nucleation and growth at this temperature , which is a threshold after which the widened fwhm was induced by further increases in temperature .", "the underlying kinetics for the growth of ncs played an important role in the observed fwhm in fig . 2c . for the diffusion - controlled growth of", "spherical ncs , the size(r)-dependent growth rate ( dr / dt ) of ncs has a maximum at a critical radius rcr .", "when r > rcr , the smaller ncs grow faster than the larger ones , resulting in a narrower size distribution . when r < rcr , the smaller ncs grow slower than the larger ones or even dissolve in the solution ( ostwald ripening ) , which led to a broad size distribution of the final products . in our experiment , temperatures above 220c resulted in the rapid depletion of monomers , and the low concentration of monomers led to a larger critical size . as a result", ", large ncs grew from the monomers formed by the dissolution of small ncs , and broad fwhm was observed with the increase in temperature .", "the microfluidic reaction facilitated the process of optimization using a minimal amount of precursors . in this study ,", "the optimization process for the reaction temperature was achieved within an hour , while consuming less than 1 ml of the precursors .", "furthermore , the enhanced heat and mass transfer in the microreactor resulted in a reduction in the temperature required to achieve the synthesis of high - quality ncs . in our work", ", monodisperse cds ncs were synthesized at a fairly low temperature of 220c , which is among the lowest values reported based on other studies using similar recipes .", "the evaluation of residence time was achieved by changing the flow rate from 17.8 to 2.22 ml h. figure 3 compares the absorption and pl spectra , as well as relative kinetic data , of the samples prepared at residence times ranging from 17 to 136 s at a constant reaction temperature of 220c .", "the sharp band - edge absorbance and three higher transitions , as well as band - gap emissions , can be clearly observed , displaying the high quality of the as - formed ncs . as shown in fig .", "3b , the increased residence time induced a 30-nm redshift of the pl peak , elucidating the growth of ncs .", "concerning the fwhm of pl , the optimal residence time was observed to be 68 s when a narrow fwhm of only 17 nm was observed . with the increase in residence time from 17 to 68", "s , the fwhm of pl continuously decreased from 20 to 17 nm , indicating the focusing of the size distribution in this phase , whereas increasing the residence time beyond 68 s resulted in increased fwhm values . in the early reaction stage ,", "the high monomer concentration led to a small rcr , which is smaller than the normal size of the ncs presented in the solution . in this phase", ", small ncs exhibited a more rapid growth rate than large ncs , and the size distribution of the ncs continuously focused .", "meanwhile , the velocity of the cds ncs had a parabolic profile inside the capillary , while induced residence time distribution ( rtd ) for cds ncs .", "numerical simulation and experimental results demonstrated that the rtd effect becomes trivial at low flow rates ( corresponding to long residence time in this study ) . as a result ,", "the prolonged growth time resulted in low monomer concentration , which led to large rcr , and the size of some cds ncs fell below rcr while the advent of ostwald ripening led to increased fwhm of pl for cds ncs . a absorption , b pl spectra , and c relative kinetics of cds ncs synthesized at different residence times ( with a constant reaction temperature of 220c , oa : 15 . 6 vol % ) in this paper , oa was utilized as the ligand for the formation of cationic precursors and passivation of the ncs , while ode was chosen as the non - coordinating solvent .", "size and size distribution can be simply tuned by varying the concentration of ligands in the solution .", "the temporal evolutions of uv  vis spectra at various residence times were recorded at different volume percentages of oa , as shown in fig .", "the discrete reactions were performed under identical conditions , except for the concentration of oa in the reaction mixture . during the variation of volume ratio for oa over a wide range ( 10.551.2 vol % ) and the variation of residence time from 17 to 136 s ,", "monodisperse cds ncs were obtained in different size ranges , which were clearly shown by the observation of a sharp absorption peak ( hwhm~12 nm ) and up to three excitonic transition states . with the increase in residence time and the variety of ligand concentrations ,", "a 64-nm shift of absorption peaks ( from 389 to 453 nm ) was obtained .", "absorption spectra of the samples monitored during the evolution of residence time and oa volume ( oa only in cd side , t = 220c ) the wavelength of the first excitonic absorption peak and the corresponding absorption intensity , as well as hwhm , was utilized to calculate the mean particle diameter and particle number of the obtained cds ncs , using the empirical fitting function reported by yu . in fig . 5", ", the data obtained appear to show a significant influence of oa on the mean size and size distribution of cds ncs . at the same residence time , a high volume percentage of oa resulted in large particle sizes and low particle concentration .", "taking the residence time of 68 s as an example , tuning the volume percentage of oa from 10.5 to 51.2% led to a significant increase in the mean diameter of the ncs from 3.8 to 5.3 nm , accompanied by dramatic decrease in particle concentration from 2.20  10/l to 0.20  10/l .", "since oa restrains the formation of nuclei during the nucleation process , the low particle concentration was observed at high oa concentrations .", "furthermore , the analysis conducted by peng et al .   indicated that the depletion rate of monomers was basically constant after the primal stage of the reactions , even though different initial oa concentrations were used . as a result , for cds ncs that were prepared by utilizing high oa concentrations", ", less nuclei were formed while the depletion rate of the cd - oa monomers kept the same , compared with low oa concentrations .", "the combination of the above two factors led to a rapid growth rate of ncs , resulting in large particle sizes and low particle concentrations . at the same time , the rapid growth negated the annealing process and led to higher concentrations of surface defects , which led to weak pl intensity . a mean diameter and b particle concentration of the cds samples prepared during the evolution of residence time and volume percentage of oa ( oa only in cd side , t = 220c ) in batch reactions , cds ncs prepared using high oa concentrations generally demonstrated wide size distribution", "however , monodisperse ( hwhm~11 nm ) cds ncs can be obtained with a high volume ratio of oa of 51.2% , and the size of the cds ncs were in the range of 4.55.3 nm in the microreaction .", "the efficient mixing and heat transfer , as well as uniform reaction conditions , achieved by the microreactor contributed to the formation of the high - quality products .", "in addition , the narrow diffusion length along the radius of the microchannels created homogeneous reaction conditions for the uniform growth of cds ncs , even at rapid growth rate that was induced by high oa concentrations .", "the hwhm and pl spectra of the cds ncs prepared using various volume percentages of oa are shown in fig .", ", the absorbance of the samples at the excitation wavelength was adjusted to be the same .", "figure 6a shows that a narrower hwhm of approximately 13 nm can be obtained for different oa content , indicating the narrow size distribution of the resulting ncs .", "however , with the increase in the volume ratio of oa , a significant improvement of pl intensity was observed and reached the highest value when applying volume ratio of oa as 15.6 vol % ( fig .", "6b ) , while further increases in the volume ratio resulted in decreased pl intensity . for cds", "ncs prepared using a volume ratio of 51.2% , the deep - trap emission made the samples demonstrate red color under an uv lamp .", "the pl efficiency of the ncs was mainly governed by the surface state of the ncs . the slow growth rate induced by the low oa concentration and the optimal ligand capping at 15.6 vol % resulted in the best pl efficiency , while the improved growth rate that accompanied the increase in oa concentration resulted in increased defects on the surface of ncs , which justified the low pl intensity under high oa concentration above 35 vol % .", "the qy could be improved by the surface capping with zns shell , and this research is under active investigation in our group .", "a hwhm of uv  vis and b pl intensity of cds samples prepared at various volume percentage of oa ( t = 220c , t = 68", "s ) just by varying the volume ratio of oa and residence time , various sizes of high - quality cds ncs ranging from 3.0 to 5.4 nm were obtained at the fairly low reaction temperature of 220c ( fig .", "the band - gap emission was observed when the particle size was smaller than 5 nm .", "therefore , due to the high quality of the as - prepared cds ncs , zns ", "capped cds core  shell structure ncs with excellent blue luminescence efficiency , color purity , and a narrow size distribution can easily be obtained .", "uv  vis absorption and pl spectra of the as - prepared cds ncs with various mean sizes .", "( a absorption intensity , ipl = pl intensity ) figure 8 shows tem and hrtem images of cds ncs , which clearly display the narrow size distribution and fairly spherical morphology of the cds ncs with an average diameter of 5.2 nm . statistical calculations for the 50 dots in a selected regime", "figure 9 shows the xrd spectra of 5.2-nm cds ncs , which exhibited a typical pattern of the zinc - blende structure with three distinct features .", "the first diffraction peak at 2 = 27 corresponded to the ( 111 ) reflection , and the other two features , which appeared at 2 = 44 and 52 , corresponded to the ( 220 ) and ( 311 ) reflections , respectively .", "a tem and b hrtem images of cds ncs ( band - gap absorption of 449 nm with an hwhm value of 11 nm ) synthesized in open air via microreaction xrd pattern of 5.2-nm cds ncs synthesized at 68 s ( oa : 51.2 vol % )", "in conclusion , a capillary microreactor was used for the continuous synthesis of cds ncs , and the influences of reaction temperature and residence time were investigated systematically to obtain the optimal synthesis parameters .", "the enhanced mass and heat transfer involved in microchannels facilitated the rapid synthesis of high - quality cds ncs at the low reaction temperature of 220c .", "the combined effect of rtd and ostwald ripening resulted in the optimal residence time as 68 s. the continuous operation and low sample consumption involved in microreaction allowed an accelerated study of the kinetics involved .", "variations of oa concentration and residence time were demonstrated as an effective way to control the size of cds ncs produced . with an increase in residence time from 17 to 136 s and an increase in the volume percentage of oa from 10.5 to 51.2% , cds ncs in the size range from 3.0 to 5.4 nm", "were obtained , with corresponding pl peak wavelength from 391 to 463 nm . with the careful selection of reaction parameters , a moderate qy as 14.5% was obtained .", "furthermore , excellent size distributions were obtained over a wide range of oa concentrations , and narrow hwhm absorption peaks ( from 11.6 to 14.0 nm ) were maintained during the entire process .", "authors appreciated the financial supports from the nsfc ( 50772036 ) , the focus of scientific and technological research projects ( 109063 ) and the state key laboratory of chemical engineering at ecust ( skl - che-08c09 ) ."], "abstract_text": ["<S> cds - based nanocrystals ( ncs ) have attracted extensive interest due to their potential application as key luminescent materials for blue and white leds . in this research , the continuous synthesis of monodisperse cds ncs was demonstrated utilizing a capillary microreactor . </S>", "<S> the enhanced heat and mass transfer in the microreactor was useful to reduce the reaction temperature and residence time to synthesize monodisperse cds ncs . </S>", "<S> the superior stability of the microreactor and its continuous operation allowed the investigation of synthesis parameters with high efficiency . </S>", "<S> reaction temperature was found to be a key parameter for balancing the reactivity of cds precursors , while residence time was shown to be an important factor that governs the size and size distribution of the cds ncs . </S>", "<S> furthermore , variation of oa concentration was demonstrated to be a facile tuning mechanism for controlling the size of the cds ncs . </S>", "<S> the variation of the volume percentage of oa from 10.5 to 51.2% and the variation of the residence time from 17 to 136 s facilitated the synthesis of monodisperse cds ncs in the size range of 3.05.4 nm , and the ncs produced photoluminescent emissions in the range of 391463 nm . </S>"], "labels": null, "section_names": ["Introduction", "Experimental", "Raw Materials", "Synthesis of CdS NCs", "Apparatus", "Results and Discussion", "Conclusions", "Acknowledgments"], "sections": [["the strong quantum confinement effect of colloidal semiconductor nanocrystals ( ncs ) has made these materials the subject of extensive research [ 1 - 5 ] . in the past decades , binary semiconductor ncs ,", "identified as groups ii  vi and iii  v , have been studied intensively because of their size - dependent photoluminescence ( pl ) covering the spectrum range from ultraviolet to near infrared [ 6 - 10 ] .", "the importance of cds ncs is manifested in their potential application as fluorescent materials with uv  blue pl emission [ 11 - 17 ] .", "the typical synthesis recipes for cds ncs are based on a process that involves the injection of cd - oleic acid ( oa ) into batch reactors .", "nearly , monodispersed cds ncs have been prepared using octadecene ( ode ) as the non - coordinating solvent , and subsequent treatment of these ncs resulted in considerable improvement of pl efficiency . in these cases , the inefficient heat and mass transfer of batch reactors constrained the economical synthesis of cds ncs .", "in order to overcome the energy barrier to form active monomers , a high reaction temperature ( 250300c ) is generally required . meanwhile , the temperature and concentration gradients involved in large reactors lead to poor quality and low process reproducibility .", "furthermore , according to the international commission on illumination chromaticity diagram , the ideal wavelength for blue light is 460480 nm . to achieve cds ncs with blue pl emission", ", large cds ncs with diameters greater than 5.2 nm are required . however , the preparation of these cds ncs is limited by the high reaction temperature and long reaction time , where the advent of ostwald ripening leads to wide size distributions .", "the enhanced heat and mass transfer properties in the microenvironment could provide highly homogeneous conditions for the chemical synthesis of ncs [ 18 - 24 ] .", "compared with the batch reactor , a microreactor offers a controllable way to synthesize ncs continuously in a steady fashion [ 9,20 - 23 ] .", "meanwhile , the incorporation of novel channel structures and flow patterns facilitated the synthesis of monodisperse ncs in an accelerated manner .", "however , the limited research conducted to date on the microfluidic synthesis of cds ncs has been based on aqueous processes , and the resulting ncs generally had wide size distributions and deep - trap luminescence that overwhelmed the visible range [ 19,25 - 27 ] .", "droplet microfluidics has been demonstrated as a powerful tool for achieving efficient mixing , while realizing highly homogeneous environment for the synthesis of monodisperse cds ncs , but its low throughput limits its use to commercial - scale synthesis . in this study ,", "a capillary microreactor was used for the accelerated synthesis of monodisperse cds ncs . in order to obtain high - quality samples , systematic investigations of temperature and residence time requirements", "also , a quantitative investigation of the influence of oa on size distribution and growth kinetics was conducted to achieve the size - controlled synthesis of cds ncs .", "the influence of oa on the reaction kinetics was evidenced by the collected absorption spectra .", "in addition , the high quality of the resulting cds ncs was confirmed by the pl spectra , powder x - ray diffraction ( xrd ) , and high - resolution transmission electron microscopy ( hrtem ) ."], ["cadmium oxide ( cdo , scr , 99.9% ) , sulfur ( s , scr , 99.5% ) , 1-octadecene ( ode , fisher , 90% ) , oleic acid ( oa , scr , 90% ) , and analytic grade acetone and chloroform ( scr ) were used directly without further processing .", "cds ncs were prepared using a recipe similar to the one previously reported by yu et al . .", "typically , 1 mmol cdo , 4.5 mmol oa , and ode ( 5 ml in total ) were mixed together and heated to 150c under a nitrogen atmosphere for 1 h with vigorous stirring to prepare a clear , yellow , cadmium - precursor solution . meanwhile , a stock solution of sulfur was prepared by dissolving 1 mmol s powder in ode ( 5 ml in total ) at 150c with magnetic stirring for 1.5 h. the microfluidic reactor included a precursor delivery system , a convective micromixer ( 50 l ) , and a length of polytetrafluoroethylene ( ptfe ) capillary ( i d = 462 m , length = 50 cm ) , as shown in fig .", "equal volume solutions of cd and s precursors were delivered by a syringe pump ( harvard 22 , usa ) at the same flow rate , and the precursors were combined and mixed in a magnetic micromixer .", "then , the mixture of precursors was placed in a heated ptfe capillary where nucleation and growth of the ncs occurred at a constant reaction temperature of 220c .", "a thermally stable oil bath was used as a heat source , and the flow rate of the precursors was set at 4.47 ml h to achieve a residence time of 68 s. during the optimization process for oa concentration , various amounts of oa were replaced by ode , while maintaining a constant volume of solution .", "schematic representation of the capillary microfluidic reactor uv  vis absorption spectra were recorded on a cary 50 uv  vis spectrometer ( varian , usa ) at room temperature .", "pl spectra were measured at room temperature with a cary eclipse spectrofluorometer ( varian , usa ) for colloidal solutions with an optical density of less than 0.2 at an excitation wavelength of 330 nm .", "quantum yields ( qy ) of pl was obtained by comparing the integrated pl intensities of the ncs with the organic dye ( rhodamine 6 g ) . to obtain samples for tem and xrd characterization ,", "the formed cds ncs were precipitated by adding acetone into their chloroform solution ; the ncs were isolated and purified by repeated centrifugation and decantation .", "xrd patterns were obtained using a d / max-2550 diffraction meter ( rigaku , japan ) with a cu anode . during the preparation of samples for xrd analysis ,", "the morphologies and dimensions of the as - formed ncs were observed by a jem-2100f hrtem ( jeol , japan ) .", "the samples for hrtem observations were obtained by dipping a carbon  copper grid in a dilute , chloroform - dispersed solution of ncs ."], ["cadmium oxide ( cdo , scr , 99.9% ) , sulfur ( s , scr , 99.5% ) , 1-octadecene ( ode , fisher , 90% ) , oleic acid ( oa , scr , 90% ) , and analytic grade acetone and chloroform ( scr ) were used directly without further processing ."], ["cds ncs were prepared using a recipe similar to the one previously reported by yu et al . .", "typically , 1 mmol cdo , 4.5 mmol oa , and ode ( 5 ml in total ) were mixed together and heated to 150c under a nitrogen atmosphere for 1 h with vigorous stirring to prepare a clear , yellow , cadmium - precursor solution . meanwhile , a stock solution of sulfur was prepared by dissolving 1 mmol s powder in ode ( 5 ml in total ) at 150c with magnetic stirring for 1.5 h. the microfluidic reactor included a precursor delivery system , a convective micromixer ( 50 l ) , and a length of polytetrafluoroethylene ( ptfe ) capillary ( i d = 462 m , length = 50 cm ) , as shown in fig .", "equal volume solutions of cd and s precursors were delivered by a syringe pump ( harvard 22 , usa ) at the same flow rate , and the precursors were combined and mixed in a magnetic micromixer . then , the mixture of precursors was placed in a heated ptfe capillary where nucleation and growth of the ncs occurred at a constant reaction temperature of 220c .", "a thermally stable oil bath was used as a heat source , and the flow rate of the precursors was set at 4.47 ml h to achieve a residence time of 68 s. during the optimization process for oa concentration , various amounts of oa were replaced by ode , while maintaining a constant volume of solution ."], ["uv  vis absorption spectra were recorded on a cary 50 uv  vis spectrometer ( varian , usa ) at room temperature .", "pl spectra were measured at room temperature with a cary eclipse spectrofluorometer ( varian , usa ) for colloidal solutions with an optical density of less than 0.2 at an excitation wavelength of 330 nm .", "quantum yields ( qy ) of pl was obtained by comparing the integrated pl intensities of the ncs with the organic dye ( rhodamine 6 g ) . to obtain samples for tem and xrd characterization ,", "the formed cds ncs were precipitated by adding acetone into their chloroform solution ; the ncs were isolated and purified by repeated centrifugation and decantation .", "xrd patterns were obtained using a d / max-2550 diffraction meter ( rigaku , japan ) with a cu anode . during the preparation of samples for xrd analysis ,", "the morphologies and dimensions of the as - formed ncs were observed by a jem-2100f hrtem ( jeol , japan ) .", "the samples for hrtem observations were obtained by dipping a carbon  copper grid in a dilute , chloroform - dispersed solution of ncs ."], ["in batch reactions , the long response time for temperature stabilization and the vibration at reaction conditions make it difficult to achieve precise control of the sizes of cds ncs . on the contrary", "furthermore , the enhanced heat and mass transfer in a microreactor greatly improves the concentrations of reactive monomers , resulting in reductions in the reaction temperature and residence time required to form high - quality ncs . in order to achieve a balance between process stability and the reactivity of precursors ,", ", the reaction temperature is an important parameter that determines both the ligand and reaction kinetics .", "low temperatures are insufficient for overcoming the energy barrier for highly reactive monomers , while high temperatures that exceed a threshold lead to the early advent of ostwald ripening . in this study , the reaction temperature was adjusted from 180 to 260c at a constant residence time of 68 s. figure 2 shows the absorption and pl spectra as well as the kinetics data of cds ncs prepared at various reaction temperatures . generally , the sharp band - edge absorbance with a half width at half maximum ( hwhm ) value of 12 nm and several higher transitions are clearly resolved for cds ncs that were synthesized at temperatures above 200c .", "whereas , due to the weak reactivity , the magic - size cluster was observed at 180c , indicated by the appearance of an absorption peak with a wavelength of almost 320 nm .", "in addition , pure band - gap emissions with very narrow full width at half maximum ( fwhm~17 nm ) were observed at 220c .", "the earlier mentioned results clearly point to a narrow size distribution and well - passivated surfaces of the cds ncs . when the temperature was increased from 180 to 240c ,", "a 44-nm redshift in the pl peak was observed , indicating that the growth rate of the ncs improved at the higher temperatures .", "however , impurities in the technical grade ode resulted in the evolution of gas at high reaction temperatures above 240c .", "the presence of the gas decreased the residence time and induced the blueshift of the pl peak . a absorption , b pl spectra , and c relative kinetics of cds ncs prepared at various temperatures at a constant residence time of 68 s ( oa : 15 . 6 vol % ) fwhm of pl is an indirect measure for the size distribution of ncs . in fig .", "2c , it can be seen that fwhm of the ncs tended to decrease when the temperature was changed from 180 to 220c .", "further increases in the temperature led to wider fwhm values . during the diffusion - controlled synthesis of spherical ncs , the burst of nucleation that separated from the growth is the prerequisite for achieving monodisperse products .", "temperatures below 180c can not overcome the energy barrier to form the reactive monomer species , which restricted the burst of nucleation .", "the nucleation in the whole reaction phase led to poor size distribution . with the increase in reaction temperature from 180 to 220c ,", "the continuously increased reactivity of the monomer allowed the burst of nucleation , leading to gradual narrowing of the size distribution .", "the narrowest fwhm was observed at 220c , indicating a balance of nucleation and growth at this temperature , which is a threshold after which the widened fwhm was induced by further increases in temperature .", "the underlying kinetics for the growth of ncs played an important role in the observed fwhm in fig . 2c . for the diffusion - controlled growth of", "spherical ncs , the size(r)-dependent growth rate ( dr / dt ) of ncs has a maximum at a critical radius rcr .", "when r > rcr , the smaller ncs grow faster than the larger ones , resulting in a narrower size distribution . when r < rcr , the smaller ncs grow slower than the larger ones or even dissolve in the solution ( ostwald ripening ) , which led to a broad size distribution of the final products . in our experiment , temperatures above 220c resulted in the rapid depletion of monomers , and the low concentration of monomers led to a larger critical size . as a result", ", large ncs grew from the monomers formed by the dissolution of small ncs , and broad fwhm was observed with the increase in temperature .", "the microfluidic reaction facilitated the process of optimization using a minimal amount of precursors . in this study ,", "the optimization process for the reaction temperature was achieved within an hour , while consuming less than 1 ml of the precursors .", "furthermore , the enhanced heat and mass transfer in the microreactor resulted in a reduction in the temperature required to achieve the synthesis of high - quality ncs . in our work", ", monodisperse cds ncs were synthesized at a fairly low temperature of 220c , which is among the lowest values reported based on other studies using similar recipes .", "the evaluation of residence time was achieved by changing the flow rate from 17.8 to 2.22 ml h. figure 3 compares the absorption and pl spectra , as well as relative kinetic data , of the samples prepared at residence times ranging from 17 to 136 s at a constant reaction temperature of 220c .", "the sharp band - edge absorbance and three higher transitions , as well as band - gap emissions , can be clearly observed , displaying the high quality of the as - formed ncs . as shown in fig .", "3b , the increased residence time induced a 30-nm redshift of the pl peak , elucidating the growth of ncs .", "concerning the fwhm of pl , the optimal residence time was observed to be 68 s when a narrow fwhm of only 17 nm was observed . with the increase in residence time from 17 to 68", "s , the fwhm of pl continuously decreased from 20 to 17 nm , indicating the focusing of the size distribution in this phase , whereas increasing the residence time beyond 68 s resulted in increased fwhm values . in the early reaction stage ,", "the high monomer concentration led to a small rcr , which is smaller than the normal size of the ncs presented in the solution . in this phase", ", small ncs exhibited a more rapid growth rate than large ncs , and the size distribution of the ncs continuously focused .", "meanwhile , the velocity of the cds ncs had a parabolic profile inside the capillary , while induced residence time distribution ( rtd ) for cds ncs .", "numerical simulation and experimental results demonstrated that the rtd effect becomes trivial at low flow rates ( corresponding to long residence time in this study ) . as a result ,", "the prolonged growth time resulted in low monomer concentration , which led to large rcr , and the size of some cds ncs fell below rcr while the advent of ostwald ripening led to increased fwhm of pl for cds ncs . a absorption , b pl spectra , and c relative kinetics of cds ncs synthesized at different residence times ( with a constant reaction temperature of 220c , oa : 15 . 6 vol % ) in this paper , oa was utilized as the ligand for the formation of cationic precursors and passivation of the ncs , while ode was chosen as the non - coordinating solvent .", "size and size distribution can be simply tuned by varying the concentration of ligands in the solution .", "the temporal evolutions of uv  vis spectra at various residence times were recorded at different volume percentages of oa , as shown in fig .", "the discrete reactions were performed under identical conditions , except for the concentration of oa in the reaction mixture . during the variation of volume ratio for oa over a wide range ( 10.551.2 vol % ) and the variation of residence time from 17 to 136 s ,", "monodisperse cds ncs were obtained in different size ranges , which were clearly shown by the observation of a sharp absorption peak ( hwhm~12 nm ) and up to three excitonic transition states . with the increase in residence time and the variety of ligand concentrations ,", "a 64-nm shift of absorption peaks ( from 389 to 453 nm ) was obtained .", "absorption spectra of the samples monitored during the evolution of residence time and oa volume ( oa only in cd side , t = 220c ) the wavelength of the first excitonic absorption peak and the corresponding absorption intensity , as well as hwhm , was utilized to calculate the mean particle diameter and particle number of the obtained cds ncs , using the empirical fitting function reported by yu . in fig . 5", ", the data obtained appear to show a significant influence of oa on the mean size and size distribution of cds ncs . at the same residence time , a high volume percentage of oa resulted in large particle sizes and low particle concentration .", "taking the residence time of 68 s as an example , tuning the volume percentage of oa from 10.5 to 51.2% led to a significant increase in the mean diameter of the ncs from 3.8 to 5.3 nm , accompanied by dramatic decrease in particle concentration from 2.20  10/l to 0.20  10/l .", "since oa restrains the formation of nuclei during the nucleation process , the low particle concentration was observed at high oa concentrations .", "furthermore , the analysis conducted by peng et al .   indicated that the depletion rate of monomers was basically constant after the primal stage of the reactions , even though different initial oa concentrations were used . as a result , for cds ncs that were prepared by utilizing high oa concentrations", ", less nuclei were formed while the depletion rate of the cd - oa monomers kept the same , compared with low oa concentrations .", "the combination of the above two factors led to a rapid growth rate of ncs , resulting in large particle sizes and low particle concentrations . at the same time , the rapid growth negated the annealing process and led to higher concentrations of surface defects , which led to weak pl intensity . a mean diameter and b particle concentration of the cds samples prepared during the evolution of residence time and volume percentage of oa ( oa only in cd side , t = 220c ) in batch reactions , cds ncs prepared using high oa concentrations generally demonstrated wide size distribution", "however , monodisperse ( hwhm~11 nm ) cds ncs can be obtained with a high volume ratio of oa of 51.2% , and the size of the cds ncs were in the range of 4.55.3 nm in the microreaction .", "the efficient mixing and heat transfer , as well as uniform reaction conditions , achieved by the microreactor contributed to the formation of the high - quality products .", "in addition , the narrow diffusion length along the radius of the microchannels created homogeneous reaction conditions for the uniform growth of cds ncs , even at rapid growth rate that was induced by high oa concentrations .", "the hwhm and pl spectra of the cds ncs prepared using various volume percentages of oa are shown in fig .", ", the absorbance of the samples at the excitation wavelength was adjusted to be the same .", "figure 6a shows that a narrower hwhm of approximately 13 nm can be obtained for different oa content , indicating the narrow size distribution of the resulting ncs .", "however , with the increase in the volume ratio of oa , a significant improvement of pl intensity was observed and reached the highest value when applying volume ratio of oa as 15.6 vol % ( fig .", "6b ) , while further increases in the volume ratio resulted in decreased pl intensity . for cds", "ncs prepared using a volume ratio of 51.2% , the deep - trap emission made the samples demonstrate red color under an uv lamp .", "the pl efficiency of the ncs was mainly governed by the surface state of the ncs . the slow growth rate induced by the low oa concentration and the optimal ligand capping at 15.6 vol % resulted in the best pl efficiency , while the improved growth rate that accompanied the increase in oa concentration resulted in increased defects on the surface of ncs , which justified the low pl intensity under high oa concentration above 35 vol % .", "the qy could be improved by the surface capping with zns shell , and this research is under active investigation in our group .", "a hwhm of uv  vis and b pl intensity of cds samples prepared at various volume percentage of oa ( t = 220c , t = 68", "s ) just by varying the volume ratio of oa and residence time , various sizes of high - quality cds ncs ranging from 3.0 to 5.4 nm were obtained at the fairly low reaction temperature of 220c ( fig .", "the band - gap emission was observed when the particle size was smaller than 5 nm .", "therefore , due to the high quality of the as - prepared cds ncs , zns ", "capped cds core  shell structure ncs with excellent blue luminescence efficiency , color purity , and a narrow size distribution can easily be obtained .", "uv  vis absorption and pl spectra of the as - prepared cds ncs with various mean sizes .", "( a absorption intensity , ipl = pl intensity ) figure 8 shows tem and hrtem images of cds ncs , which clearly display the narrow size distribution and fairly spherical morphology of the cds ncs with an average diameter of 5.2 nm . statistical calculations for the 50 dots in a selected regime", "figure 9 shows the xrd spectra of 5.2-nm cds ncs , which exhibited a typical pattern of the zinc - blende structure with three distinct features .", "the first diffraction peak at 2 = 27 corresponded to the ( 111 ) reflection , and the other two features , which appeared at 2 = 44 and 52 , corresponded to the ( 220 ) and ( 311 ) reflections , respectively .", "a tem and b hrtem images of cds ncs ( band - gap absorption of 449 nm with an hwhm value of 11 nm ) synthesized in open air via microreaction xrd pattern of 5.2-nm cds ncs synthesized at 68 s ( oa : 51.2 vol % )"], ["in conclusion , a capillary microreactor was used for the continuous synthesis of cds ncs , and the influences of reaction temperature and residence time were investigated systematically to obtain the optimal synthesis parameters .", "the enhanced mass and heat transfer involved in microchannels facilitated the rapid synthesis of high - quality cds ncs at the low reaction temperature of 220c .", "the combined effect of rtd and ostwald ripening resulted in the optimal residence time as 68 s. the continuous operation and low sample consumption involved in microreaction allowed an accelerated study of the kinetics involved .", "variations of oa concentration and residence time were demonstrated as an effective way to control the size of cds ncs produced . with an increase in residence time from 17 to 136 s and an increase in the volume percentage of oa from 10.5 to 51.2% , cds ncs in the size range from 3.0 to 5.4 nm", "were obtained , with corresponding pl peak wavelength from 391 to 463 nm . with the careful selection of reaction parameters , a moderate qy as 14.5% was obtained .", "furthermore , excellent size distributions were obtained over a wide range of oa concentrations , and narrow hwhm absorption peaks ( from 11.6 to 14.0 nm ) were maintained during the entire process ."], ["authors appreciated the financial supports from the nsfc ( 50772036 ) , the focus of scientific and technological research projects ( 109063 ) and the state key laboratory of chemical engineering at ecust ( skl - che-08c09 ) ."]]}
{"article_id": "PMC3993925", "article_text": ["lead \n poisoning is one of the most serious environmental health \n hazards , with a particularly acute effect on young children . the u.s .", "centers for disease control and prevention \n estimated that approximately 2.5% of children aged 15 years \n in the u.s . have elevated blood lead levels .", "the percentage of affected children in other countries is expected \n to be even higher .", "lead poisoning causes learning disabilities , behavioral \n problems , and , at very high levels , seizures , coma , and even death .", "the \n mechanism of pb toxification mainly involves binding \n of pb to proteins and the subsequent inhibition of the \n proteins physiological functions in blood and tissues .", "the documented proteins targeted by pb include several zinc enzymes or proteins ( such as -aminolevulinic \n acid dehydratase ( alad ) , acetylcoline esterase , cys2his2  zinc - finger  proteins , and acid phophatases ) and calcium - binding proteins ( calmodulin , calbindin , \n and troponin c ) .", "pb can replace \n zinc and calcium at the oxygen / nitrogen / sulfur - rich active sites of \n these proteins , thereby inhibiting the protein functions by altering \n their coordination chemistry and native structures .", "for example , the \n function of alad , an enzyme involved in the second step of heme biosynthesis , \n is altered by pb binding at the active site via a trigonal \n pyramidal geometry .", "consequently , the hemoglobin synthesis is blocked , \n leading to anemia . to reduce the pb - induced toxicity ,", "organisms have developed various defensive \n mechanisms with species such as metallothioneins ( mts ) , glutathione , \n phytochelatins , and lead - binding proteins ( pbbps , which are non - mt \n acidic proteins that have not been fully characterized ) .", "mts , a class of thiol - rich ( up to 30% of its \n amino acid residues ) , low - molecular - weight proteins whose abundance \n is particularly high in the liver and kidneys of mammals , are perhaps the most important species for lead \n detoxification .", "mts is crucial to a better understanding of the chemical \n stabilities , biological functions , and detoxification mechanism of \n pb  mts .", "however , due to the absence of single crystals of pb  mts , \n information about the pb ", "the coordination and binding stoichiometry between mts and metals \n are dependent on the type of metal ions .", "usually , divalent metal ions , such as cd and zn , are tetrahedrally coordinated by four cysteine sulfurs \n and bind to mts with a stoichiometry of 7:1 .", "it can be complexed by a combination of s , o , n , and p - donor ligands \n with a coordination number ranging from 2 to 9 .", "when pb binds to sulfur - rich proteins , three \n sulfurs in a trigonal pyramidal geometry and the pb 6s lone - pair electrons occupying the axial position ( hemidirected ) \n constitute the coordination sphere . a \n number of complexes containing the pb ", "s3 coordination \n have been observed using spectroscopic and mass spectrometry ( ms ) \n methods . in the presence of biomolecules \n possessing several distinct donor ligands (", "n , o , and s ) , the pb coordination chemistry is diverse and includes formation of pbsxoy and pbsxny .", "owing to the presence of o- and s - donor \n ligands in mts and the unique electronic configuration of pb , coordination of pb with mt is of higher complexity \n than that of zn or cd .", "visible ( uv  vis ) spectra and microcalorimetry \n have suggested that different pb ", "palacios \n et al . demonstrated with electrospray ionization mass spectrometry \n that the metal content in pb ", "mt2 complexes is dependent on \n the solution ph ( neutral or 4.5 ) . using \n extended x - ray absorption fine structure ( exafs ) , vasak et al .", "however , crucial questions such \n as the difference of pb coordination and the chemical \n stability and physiological function of the two pb ", "theoretical \n calculations have been widely \n used for the prediction of structures and spectra of metalloproteins .", "the combination of quantum mechanics and molecular \n mechanics - based hybrid ( qm / mm ) method allows two or more computational \n methods to be performed in a single calculation , making it possible \n to investigate the chemistry of complex systems with high precision . in this context , the oniom ( our own n - layered \n integrated molecular orbital + molecular mechanics ) scheme is a general \n approach because it can combine any number of molecular orbital and \n molecular mechanics methods .", "the oniom \n method has been successfully utilized in the elucidations of structural \n and functional properties of many metalloproteins such as cyt c and \n azurin . in our study ,", "optical methods \n ( uv  vis absorption and circular dichroism ( cd ) spectrometry ) \n and nmr were used in tandem with the two - layer oniom method to investigate \n the effect of ph on the structures of two different pb ", "the differences in the coordination chemistry of the metal \n centers and the protein structures at various ph were deciphered . \n", "we also investigated the chemical stabilities and structural flexibility \n of these two complexes in proteolytic processing to gain insight into \n the lead detoxification process involving mts .", "zn - containing \n mt2 , isolated from rabbit liver , was purchased from hunan lugu biotechnology \n co. ( changsha , china ) .", "individual metal - free domains ( apo-mt2 \n and apo-mt2 ) of both rabbit liver and human mt2s , and the corresponding \n mutants ( d25n - apo-mt2 and d2n - apo-mt2 ) were synthesized \n by shanghai apeptide co. ( shanghai , china ) . all the domains and their \n mutations", "were confirmed by mass spectrometry performed by the vendor , \n and the corresponding purity values ( > 95% ) were determined with \n hplc - ms .", "pb ( 99.1% ) was acquired from isoflex usa ( san \n francisco , \n ca ) .", "lead nitrate , 5,5-dithio - bis-(2-nitrobenzoate ) ( dtnb ) , \n and cathepsin b were purchased from sigma - aldrich ( st .", "deionized water with resistivity \n of 18.2 m cm was collected from a millipore simplicity 185 \n system ( millipore co. , billerica , ma ) .", "all solutions were prepared \n with deionized water and degassed with nitrogen for at least 30 min .", "a 3 kda cutoff \n millipore ( ym-3 ) membrane ( millipore , billerica , ma ) , equilibrated \n with 0.01 mol l hcl , was used to separate zn from zn7mt2 . after spinning at 13  000 \n rpm for 30 min at room temperature in an eppendorf 5417r centrifuge \n ( eppendorf , hamburg , germany )", "complete removal of zn from zn7mt2 \n was confirmed by the disappearance of the characteristic absorption \n of zn7mt2 at 220 nm .", "the apo - mt2 concentration was determined by assaying thiol groups \n with ellman s reagent , dtnb .", "mt2 \n solution was stored in a nitrogen - saturated flask to avoid thiol oxidation \n in apo ", "vis absorption experiments \n were carried out on a uv-2450 \n spectrophotometer ( shimadzu , japan ) in quartz cuvettes ( 1 cm path \n lengths ) .", "cd data were obtained with a jasco-810 spectrophotometer \n ( jasco corporation , japan ) .", "zeta \n potentials of individual apo - mt2 domains or their mutants were measured \n at room temperature in a folded capillary cell with a zetasizer nano \n zs instrument ( malvern instruments , southborough , uk ) .", "pb was dissolved \n with 0.15 m trace metal grade nitric \n acid ( fisher scientific ) at 250 c , and the pb(no3)2 precipitate was collected , dried , and weighed . \n", "appropriate amounts of mt2 ( 250 m ) , kcl ( 10 mm ) , and d2o were mixed under n2 atmosphere .", "both pb7mt2 complexes were prepared by adding pb(no3)2 to obtain an mt2/pb stoichiometry \n of 1:7 at corresponding ph ( 7.0 or 4.0 ) .", "the resultant solutions were \n allowed to incubate for 1 h , and the solution ph was brought up to \n ph 7.0 with koh .", "d2o was added to a final volume of 1 ml , \n and the solution was transferred to an nmr tube .", "all pb nmr spectra were recorded on a bruker advance drx-400 mhz spectrometer \n at 25 c using 60 pulses , a 2 s relaxation delay , and a \n 0.12 s acquisition time ( spectral width of 555.6 khz ) .", "a linear prediction \n was performed to remove the noise , and the real free induction decay \n ( fid ) was determined before data processing .", "after zero - filling , the \n data ( 128  000 data points ) were processed with an exponential \n line broadening of 5 hz using the software topspin nmr .", "the pb nmr chemical shifts are reported downfield from tetramethyllead \n (  = 0 ppm ; toluene ) using 1.0 m pb(no3)2 salt ( fisher ) as an external standard (  = 2990 ppm , \n d2o , 25 c ; relative to tetramethyllead ) . both pb7mt2(i ) and pb7mt2(ii )", "complexes were freshly prepared and diluted in 10 \n mm kcl solution ( ph 7.0 ) at 37 c to desired concentrations . \n", "cathepsin b ( 1.8 ng ) , with a specific activity of 3000 pmol min g , was mixed with 504 \n pmol of pb7mt2(i ) or pb7mt2(ii ) \n ( the final concentration of pb was 57 m ) in 10 \n mm kcl solution ( ph 5.0 ) .", "oniom calculations \n were performed to predict the optimal geometries and electronic absorption \n spectra of pb7mt2 complexes .", "the initial atomic coordinates of pb4mt2 \n and pb3mt2 were taken from the corresponding \n pb  substituted cd4mt2 \n and cd3mt2 , respectively .", "the structures \n of cd4mt2 and cd3mt2 \n were retrieved from the rcsb protein databank ( pdb i d : 1 mrb for the \n -domain and 2 mrb for the -domain ) , in which the absent \n hydrogen atoms were added using gaussview 4.0", ". the protonation states \n of titratable residues ( e.g. , aspartic acid and lysine ) of pb4mt2 and pb3mt2 at \n ph 4.0 and 7.0 were determined using pka values estimated with propka 2.0 .", "the electronic structures of pb4mt2 and \n pb3mt2 were modeled with the inclusion of \n the protein environment , using the two - layer oniom ( qm / mm ) model . \n", "the qm region comprises the active sites of pb4mt2 \n and pb3mt2 , and the mm region contains the \n protein environment ( cf .", "spin unrestricted density functional theory ( dft ) with \n the becke s three - parameter hybrid exchange functional and \n the lee  yang ", "parr correlation functional ( b3lyp ) or \n pure functional bp86 were used for the qm system , and the dunning \n basis set aug - cc - pvtz - pp was used for pb and 6 - 31+g * basis set for \n other atoms for the qm calculations . for \n the mm region , the protein molecule was treated using the universal \n force field ( uff ) .", "an electronic embedding \n scheme was adopted to deal with the electrostatic interactions between \n the qm and mm regions in the qm / mm calculations . on the basis of the optimized geometries ,", "vertical \n excitation energies \n were computed within the oniom scheme by employing the time - dependent \n density functional theory ( tddft ) .", "tddft \n calculations were carried out with the density functional b3lyp and \n bp86 . the trizeta basis set aug - cc - pvtz - pp", "was used for pb , and 6 - 31+g * * \n was used for others .", "the results were transformed via the swizard \n program ( version 4.6 ) into each uv spectrum \n using gaussian functions with half - widths of 3000 cm .", "uv  vis absorption \n and cd spectra were recorded during the titration of rabbit liver \n apo - mt2 with pb at ph 7.0 ( figure 1 ) . for simplicity , we termed the pb ", "mt2(i ) exhibits a characteristic peak centered at 330 \n nm ( figure 1a ) .", "the time - resolved absorbance \n changes ( inset ) indicates that the complexation reaction is fast ( completed \n in less than 10 min ) . during the titration of apo ", "mt2 with \n pb at neutral ph , some pb(oh)2 ( ksp = 1.4  10 ) precipitate was \n formed and affected the absorbance value of pb7mt(i ) \n at 330 nm , as evidenced by the small fluctuation even after 30 min . \n", "the precipitation can be avoided by lowering the solution ph below \n 6.0 . as shown in the inset of figure s1 in the supporting information ,", "the absorbance value of pb7mt(i ) remains stable after the complexation reaction is completed . \n as indicated by the mt2/pb stoichiometry ( figure 1b ) ,", "the maximum binding stoichiometry is 1:7 , consistent \n with results reported from the substitution experiment of zn7mt2 with pb . the \n absorption wavelength ( 330 nm ) and", "extinction coefficients (  \n  3500 m cm ) of pb7mt2(i ) are analogous to values reported for several \n pbs3 complexes .", "the secondary \n structural variation from apo - mt2 to pb7mt2(i ) \n is shown in figure 1c .", "this \n is in contrast with the intense cd peaks of cd7mt2 \n and zn7mt2 between 210 and 290 nm . in zn7mt2 ,", "the characteristic cd band centering at 244 nm is attributed \n to the zn(sr)4 chromophore .", "similarly , the cd bands of \n cd7mt2 at 242 and 262 nm can be attributed to the \n excitation coupling between adjacent pairs of the cd(sr)4 chromophore .", "since the m(sr)4 chromophore leads to the appearance of these cd peaks , the absence of any obvious cd peaks in figure 1c suggests that pb7mt2(i ) adopts \n a different metal coordination geometry from those of cd7mt2 and zn7mt2 , as alluded to in the introduction . \n", "the individual - and -domains display uv  vis \n and cd spectral features ( data not shown ) similar to those of apo - mt2 \n during the titration with pb , indicating that the metal \n centers in the two different domains have similar coordination spheres", ". \n quantitative analysis confirms that the pb / mt2 stoichiometric \n ratios are 4:1 and 3:1 in the - and -domain , respectively .", "time - dependent \n ( a ) uv  vis absorption and ( c ) cd spectra \n in 10 mm kcl solution ( ph 7.0 ) containing 7.20 m rabbit liver \n apo - mt2 and 20 mol equiv of pb .", "( b ) dependence of uv absorption peak \n at 330 nm upon addition of pb to apo - mt2 .", "the \n pb7mt2(ii ) complex displays dramatically different \n uv  vis \n absorption and cd spectra ( figure 2 ) from those \n of pb7mt2(i ) . in the uv  vis absorption spectra , \n two intense peaks at 325 and 375 nm appear , with the latter having \n a shoulder peak at 400 nm . in the cd spectra ( figure 2c ) , a strong envelope with maxima at 240 ( + ) , 265 ( + ) ,", "320 \n (  ) , 350 ( + ) , 370 ( + ) , and 395 (  ) is produced isodichroically \n ( 280 , 340 , and 375 nm ) .", "positions of all cd peaks are invariant with \n the pb / apo  mt2 ratio ( increased stepwise from \n 1:1 to 7:1 ) , but the peak intensity increases with the ratio .", "the \n cd spectra of cd7mt2 have two bands at 240 and \n 260 nm split from the 250 nm band , which corresponds to the conversion \n of isolated cd(sr)4 to the ( sr)3cd  sr ", "thus , the unsplittable \n pb7mt2(ii ) cd peak is indicative of the absence \n of excitation coupling between adjacent chromophores .", "the multiple \n peaks in the uv  vis absorption and cd spectra suggest that \n pb in the pb7mt2(ii ) complex has binding \n modes that are distinctively different from cd in cd7mt2 .", "similar to pb7mt2(i ) , the \n pb / mt2 stoichiometric ratios in the pb7mt2(ii ) \n complex are 4:1 and 3:1 in the - and -domains , respectively . \n", "the uv  vis absorption peaks of pb4mt2(ii ) \n are slightly shifted , with higher intensity than those of pb3mt2(ii ) ( figure 3d ) .", "these \n differences suggest that the metal centers in the two domains have \n different coordination geometries . to pinpoint the ligands responsible \n for coordination of pb in the two different domains at \n neutral and acidic ph", "time - dependent ( a ) uv  vis absorption and ( c ) cd spectra \n in 10 mm kcl solution ( ph 4.5 ) containing apo - mt2 ( 7.20 m ) \n and 20 mol equiv of pb .", "( inset ) the time - resolved absorbance \n changes at 325 and 375 nm , respectively .", "( b ) the dependence of uv \n absorption peaks at 325 and 375 nm on the addition of pb to apo - mt2 .", "structures and electronic \n absorption spectra of the - and \n -domains in ( a , c ) pb7mt2(i ) and ( b , d ) \n pb7mt2(ii ) obtained by experimental and oniom methods . \n in panels a and b , the dark gray spheres are pb , yellow \n spheres are s , red spheres are o , gray spheres are c , and white spheres \n are h atoms", ". the oniom method has been successfully used \n to predict metalloprotein structures . to validate the method for the studies of mt2 structures , structural \n optimization of cd4mt2 and cd3mt2 models", "compared to the nmr \n results , the computed structures of the \n metal clusters display little deviation ( e.g. , the bond lengths have \n a root - mean - square deviation of only 0.040.05  when \n compared to the experimental data ) ( figure s2 and table s1 in supporting information ) .", "therefore , we conclude \n that the oniom method is viable for the studies of the two pb7-mt2 complexes .", "the theoretical calculations were performed \n separately on the - and -domains on the basis that the \n two domains are structurally independent .", "the initial atomic coordinates \n of each domain in pb7mt2 were respectively adopted \n from the pb - substituted cd4mt2 \n and cd3mt2 because a well - established model \n of apo - mt2 is not available .", "the protonation states of titratable \n residues ( e.g. , asp and lys ) at different ph were determined from \n the pka values estimated with propka 2.0 .", "in the two - layer oniom model of pb7mt2(i ) , the \n qm region consists of four pb atoms , and the side chains consist of \n eleven cysteine residues in the -domain . as for the -domain , \n", "the three pb atoms and the side chains of nine cysteine residues constitute \n the qm region .", "figure 3a displays the optimized molecular geometries of \n the - and -domains in pb7mt2(i ) , \n which are quite different from the well - characterized cd7mt2 and zn7mt2 structures . in cd7mt2 and zn7mt2", ", each metal adopts the \n tetrahedral coordination with terminal and bridging thiolates to form \n a metal  ligand six - membered ring in the -domain and \n two fused six - membered rings in the -domain.(cf .", "figure s2 \n in supporting information ) however , all \n seven pb ions in pb7mt2(i ) are trigonally \n coordinated by three cysteine sulfurs ( pb  s3 ) without \n any metal ", "these \n significant differences can be attributed to the pb 6s lone - pair electrons , which disrupt the tetrahedral coordination \n by occupying the axial position with a significant stereochemical \n activity .", "this point is in line with the report by godwin and co - workers , \n who stated that pb ", "s4 is not a preferred coordination \n and that trigonal pyramidal geometry in all - sulfur coordination is \n predominant .", "moreover , due to the pb  s3 coordination , only one bridging cysteine ( pb6s18pb7 \n in the -domain ) remains , preventing a pb ", "according to the corresponding structural parameters \n ( table 1 ) , the average length of the pb ", "s \n bonds is 2.68  , close to the reported exafs value ( 2.65  ) . the electronic absorption spectra ( red line \n curves ) , simulated by tddft using the b3lyp functional , are overlaid \n with the experimental results ( black line curves ) in figure 3c .", "the agreement between the simulation and experimental \n data in the oscillator strengths indicates that the optimized molecular \n geometries of pb4mt2(i ) and pb3mt2(i ) are reasonable .", "detailed analysis of vertical \n excitation energies , oscillator strengths , and molecular orbital contributions \n ( table s3 in supporting information ) indicated \n that four transitions contribute to the absorption peak at 330 nm . \n", "these transitions are homolumo , homo-1lumo , homo-2lumo , \n and homo-1lumo+1 . from the frontier molecular orbitals ( figure \n s3 in supporting information ) , lumo and \n lumo+1 ( the final state of the transitions )", "s bonds , while homo and homo-1 ( the initial states of \n the transitions ) are largely localized at the sulfur atoms .", "therefore , \n the electronic absorption band at 330 nm can be attributed to the \n spb ligand - to - metal charge transfer ( lmct ) .", "in the two - layer oniom model \n of pb7mt2(ii ) , \n we initially assigned the qm region of each domain to be the same \n as that of pb7mt2(i ) .", "however , the optimized molecular \n geometries revealed two unexpected short pb / o distances ( 5.52  \n for pb1 with the carbonyl oxygen of asp2 in the -domain and \n 4.31  for pb6 with the carbonyl oxygen of asp56 in the -domain ) . \n", "these short distances should result from the electrostatic interaction \n between the protein surface ( the mm region ) and the negatively charged \n pb ", "acidic ph conditions , the protein surface is neutral or positively \n charged , causing the mm and qm regions to move closer , thereby shortening \n the distance between the pb ", "moreover , based on the pka values of asp \n ( 4.00 ) , both asp residues are neutral in weakly acidic solution , \n which are more favorable than the negatively charged ( deprontonated ) \n form to the positioning of the negatively charged pb ", "these pb / o distances are close to the sum of the \n van der waals radius of pb and o ( 3.54  ) , indicating the pbo interaction must be \n taken into account .", "we therefore modified the initial qm region in \n each domain by including the respective asp residue .", "as shown \n in figure 3b , the optimized molecular \n geometry of pb7mt2(ii ) shows an entirely different \n coordination sphere from that of pb7mt2(i ) .", "the \n overall coordination sphere includes the trigonal pyramidal pb  s3 mode , the distorted trigonal pyramidal pb ", "s2o1 mode in the -domain , and the distorted quadrilateral \n pyramidal pb  s3o1 in the -domain . \n", "the pb 6s lone pair electrons occupy the \n axial position with a significant stereochemical activity , resulting \n in hemidirectionality in the pb  ligand coordination .", "moreover , due to protonation of the \n peptide side chain , several cysteine sulfurs ( especially in the -domain ) \n are closer to the protein exterior and increase the effective radius \n of the metal center and the pb  s bond length ( cf .", "the loosened structure \n of the metal center renders a higher flexibility to pb7mt2(ii ) .", "the simulated oscillator strengths are in good agreement \n with the experimental data ( figure 3d ) , indicating \n that the optimized molecular geometries of pb4mt2(ii ) \n and pb3mt2(ii ) are reasonable . the small \n deviation between the electronic absorption spectra of the two domains \n ( cf .", "detailed vertical excitation energies and \n molecular orbital contributions ( table s5 and figure s4 in supporting information ) indicate that all three \n bands are primarily associated with the spb lmct .", "note that all of the above calculations are based on b3lyp , a hybrid \n functional to fit data primarily for main - group elements .", "because of the large exact exchange component \n ( 20% ) , b3lyp is known to favor loose electron densities and low transition \n energies . to confirm the reliability of b3lyp , the pure functional \n bp86 , a generalized - gradient approximation ( gga ) class with zero exact \n exchange ,", "is used as a control study on \n the geometry optimization and calculations of the excited states for \n both pb7mt2 complexes .", "the simulated structures \n of both pb7mt2 complexes from bp86 pure functional \n ( table s69 in supporting information ) are similar to those from b3lyp hybrid functional .", "however , the \n spectra from bp86 pure functional display a significant deviation \n from the experimental data ( figure s5 in supporting \n information ) , indicating that the b3lyp hybrid functional is \n a better choice .", "the b3lyp hybrid functional produces better spectral \n accuracy , which can be attributed to its increased amount of hartree ", " local \n minima  \n in the qm optimization . in the qm / mm calculation of biomolecules , \n the vast size of the available configuration space may cause the qm \n optimization to stop at local minima .", "one way to circumvent this problem \n is to use a reliable structure ( e.g. , a structure deduced from nmr \n or x - ray crystallography ) at the beginning of the qm optimization .", "have used the \n x - ray structure of azurin as the initial structure in the qm / mm calculation \n of metal - substituted azurins and obtained noticeable structural changes \n on the active sites when cu in azurin was substituted \n by metal ions such as co , ni , or zn . we adopted this approach \n by using the pb - substituted cd4mt2 \n and cd3mt2 structures deduced from the nmr \n experiments as the initial structures . \n", "the theoretical spectra agree well with the experimental results , \n suggesting a high level of reliability of the calculation and in the \n predicted structures .", "to provide more \n experimental evidence \n to our computational results about the two different pb7mt2 complexes , we conducted pb nmr in solutions \n of the two complexes ( figure 4 ) .", "pb7mt2(i ) displays one pb peak at 5679 ppm , while \n pb7mt2(ii ) exhibit two peaks at 5820 and 4348 ppm . \n thus it is clear that the pb coordination in these two \n complexes are different .", "furthermore , the pb signal \n of pb7mt2(i ) at 5679 ppm is well within the chemical \n shift region ( from 5600 to 5800 ppm ) where pbs3 species \n with the trigonal pyramidal coordination are observed .", "thus we conclude that pbs3 is the binding mode in pb7mt2(ii ) . for pb7mt2(ii ) , the peak \n at 5820 ppm is also assigned to the pbs3 coordination , \n given its close vicinity to the 56005800 ppm region .", "compared \n to the pb7mt2(i ) peak , the shift by 141 ppm can \n be attributed to the changes of the aforementioned pb  s bond \n length and the s  pb  s bond angle in the pbs3 coordination .", "the lower intensity is indicative of the decreased \n number of pbs3 clusters in pb7mt2(ii ) . \n", "the conversion of metal centers in the pbs3 coordination \n to a different binding mode contributes to the appearance of the peak \n at 4348 ppm .", "it is well - known that the nmr signal of pb bound to o - containing ligands is shifted upfield with respect \n to that bound to s - containing ligands .", "pb nmr spectra of ( red ) pb7mt2(i ) , \n ( blue ) pb7mt2(ii ) , and ( black ) pb(no3)2 at ph 7.0 .", "the carbonyl group of asp is the only o - donor ligand in mt2 . \n to \n further verify the formation of the pb ", "o bond in pb7mt2(ii ) , a titration was performed by adding pb into a peptide solution whose asp residue had been mutated with \n asn ( i.e. , d25n - apo-mt2 and d2n - apo-mt2 ) .", "zeta potential \n and cd measurements did not show discernible changes in the surface \n charge and structure of both mutants ( figure s6 and s7 in supporting information ) , confirming the viability \n of these mutants for coordination studies . at neutral ph ,", "both uv  vis \n absorption and cd spectra of the two mutants show features similar \n to those in the same spectra of pb4mt2(i ) \n and pb3mt2(i ) , verifying that asp does not \n participate in the pb coordination in pb7mt2(i ) . \n at acidic ph ( ph < 5 ) ,", "the uv  vis absorption spectra of \n both mutants display only a single peak at 330 nm , and the cd spectra \n did not reveal any changes . while these features are rather different \n from the corresponding spectra of pb4mt2(ii ) \n and pb3mt2(ii ) , they are analogous to those \n of pb4mt2(i ) and pb3mt2(i ) . \n we therefore conclude that asp is involved in the coordination of \n pb in pb7mt2(ii ) . the unique uv", " vis \n absorption spectra of pb7mt2(ii ) , with multiple \n peaks and the presence of a new pb nmr peak at 4348 \n ppm , are well - correlated with the mutational study . another \n piece of evidence for the formation of pb  o bond \n is the transition from a single peak to three peaks in the time - dependent \n spectra of a mixture of pb / apo - mt2 at ph 4.55.0 \n as well as the appearance of multiple peaks in the cd spectra ( cf . \n", "however , because \n of the negative impact of acidity on the formation of the pb  s \n bond ( cf . the proton - releasing process shown in reaction 1 ) and the rearrangement of the peptide chain , the pb ", "therefore , under \n more acidic conditions ( ph < 4.5 ) , the time - dependent peak transition \n disappears , and the spectra show features typical of pb7mt2(ii).1 time dependence \n of ( a ) uv  vis absorbance and ( b ) cd spectra \n in a kcl solution after the addition of 7 mol equiv of pb to an apo - mt ( 7.20 m ) solution at ph 5.0 .", "reaction times \n from bottom to top : 0 , 1 , 3 , 5 , 7 , 10 , 15 , 20 , 25 , 30 , 45 , 60 , 75 , \n 90 , 105 , 120 , 135 , 150 , 175 , and 180 min . to study the acid tolerance of both pb7mt2 complexes , a series of spectrophotometric titrations \n at different ph was performed .", "the corresponding complex was confirmed \n by the appearance of characteristic uv  vis absorption peaks \n of pb7mt2(i ) or pb7mt2(ii ) . \n", "the results show that pb7mt2(i ) is formed above \n ph 5.0 , whereas pb7mt2(ii ) is produced at more \n acidic ph .", "pb7mt2(i ) can be transformed to pb7mt2(ii ) by adjusting the solution ph to 5.0 or lower . \n", "however , once pb7mt2(ii ) is formed , it remains \n stable at neutral ph .", "our demetalation experiments ( figure s8 in supporting information ) indicate that ph 2.5 \n is sufficiently low for the complete removal of pb from \n pb7mt2(i ) , but stripping pb completely \n of pb7mt2(ii ) requires a ph as low as 2.0 . these \n results ( summarized schematically in figure 6 )", "indicate that pb7mt2(ii ) has a higher tolerance \n toward an acidic environment than pb7mt2(i ) has . \n", "moreover , pb7mt2(ii ) remains stable even in the \n presence of apo - mt at neutral ph ( data not shown ) .", "we believe that \n the greater acid tolerance and higher structural stability of pb7mt2(ii ) results from the pb  o bond .", "such a \n finding has a significant implication to the lead detoxification process \n in physiological milieu .", "the pb - inflicted toxicity stems from its tight \n binding \n to a variety of sulfur - rich proteins , such as gata proteins and the \n steroid receptor dna - binding domains .", "mt2 reduces the pb - inflicted toxicity \n by seizing free pb or sequestering pb from \n these proteins to recover the native protein function .", "higher acid \n tolerance and greater structural stability render mt2 a greater power \n in effectively scavenging pb in different environments . \n as shown in reaction 1 ,", "mt2 accompanies the release of h , which \n in turn increases the acidity in a highly localized region ( e.g. , \n in cytosol ) .", "moreover , it has been reported that elevated pb concentrations induce the stress level of various organisms , and \n acidity is also correlated with the stress level .", "we posit that pb7mt2(ii ) is more effective \n than pb7mt2(i ) in lead detoxification , given its \n greater structural stability and acid tolerance .", "elucidation of the metabolism of the pb - mt \n complexes is vital for understanding the lead detoxification by mts . \n", "some reports have suggested that exogenous mt is processed mainly \n by the lysosomal protease , and the rate of mt degradation is dependent \n on the types of metals bound by mts .", "four \n different cathepsins have been identified in lysosomes , and the cysteine \n protease ( cathepsin b , l , and h ) is the principal protease for mt \n degradation .", "variations in the concentrations of the two pb7mt2 \n complexes in the presence of cathepsin b were measured by uv  vis \n absorption spectrometry . in figure 7 , the pb7mt2 concentrations were normalized with respect to \n their initial concentrations . within 120 min , pb7mt2(ii ) \n", "such a \n higher degradation rate can be attributed to the more flexible structure \n of the pb7mt2(ii ) complex .", "cathepsin b is an endopeptidase \n that cleaves internal peptide bonds and favors a large hydrophobic \n side chain in the substrate protein .", "side \n chains on the amino acids dock into the cathepsin s subsites , \n whose interaction with the protein substrate is dependent on the flexibility \n of substrate protein . in mts ,", "the existence \n of bridging cysteine sulfurs compacts the metal center , which dominates \n the protein folding . in pb7mt2(ii ) , due to the lack of bridging cysteine sulfurs , the \n metal center is loosened , which improves the flexibility of the protein . \n moreover , when the pb ", "s3o1 become \n distorted . consequently , pb ions are positioned farther \n from the cysteine sulfurs , and the pb  s bonds are weakened . \n both processes facilitate the conformational adjustment of pb7mt2(ii ) .", "the significantly improved flexibility of \n pb7mt2(ii ) facilitates the protein in the  induced - fit  \n model with cathepsin b. as a result , \n the proteolytic processing of pb7mt2(ii ) is greatly \n accelerated . in living organisms ,", "the acidity in lysosome is about \n 5 , which is sufficiently low to cause the structural conversion from \n pb7mt2(i ) to pb7mt2(ii ) and \n to accelerate the degradation of pb  mt2 complexes .", "time - dependent proteolytic processing of ( black ) pb7mt2(i ) and ( red ) pb7mt2(ii ) by cathepsin \n b. the coordination of pb with individual human apo  mt2 \n or apo  mt3 domains at different ph was also studied by uv  vis \n absorption and cd spectrometry .", "mutational studies revealed \n that the asp residue is also essential for the ph - dependent structural \n variation . in line with the data observed for the rabbit liver mt2 , \n the structure - dependent chemical and biological activities of pb7hmt2(ii ) formed between human mt2 and pb have higher acid tolerance , more coordination stability , and faster \n proteolytic processing than pb7hmt2(i ) ( figure \n s10 in supporting information ) .", "our results \n suggest that there exists a commonality in the pb coordination \n chemistry among mammalian mt2s .", "in this work , the ph - dependent coordination chemistry between pb and mt2 was systematically studied .", "the combination of spectroscopic \n studies and oniom calculations provided a detailed description of \n the two different pb7mt structures .", "the results \n and structures were further verified by pb nmr and mutational \n experiments .", "the similar structural , chemical , and biological properties \n between rabbit liver pb7mt2(ii ) and human pb7mt2(ii ) suggest a commonality in the pb coordination chemistry among mammalian mt2s .", "the higher acid tolerance , \n greater coordination stability , and faster degradation rate of pb7mt2(ii ) have significant implications for the pb detoxification process .", "specifically , mt2 reduces the pb - inflicted toxicity by seizing free pb in the \n cellular milieu or by sequestering pb from pb - inflicted proteins .", "the unique properties of pb7mt2(ii ) \n render mt2 a greater power to effectively scavenge pb in different environments ( e.g. , in a localized acidic cytosol region ) . \n", "moreover , the greater flexibility of pb7mt2(ii ) , \n resulting from the absence of bridging cysteine sulfurs , helps to \n accelerate its processing by lysosomal protease .", "the structural conversion \n from pb7mt2(i ) to pb7mt2(ii ) \n is likely to occur in the acidic environment of lysosome , facilitating \n the effective detoxification and metabolism of pb ."], "abstract_text": ["<S> lead \n is a toxic heavy metal whose detoxification in organisms is \n mainly carried out by its coordination with some metalloproteins such \n as metallothioneins ( mts ) . </S>", "<S> two pb  mt complexes , named as pb7mt2(i ) and pb7mt2(ii ) , form under \n neutral and weakly acidic conditions , respectively . </S>", "<S> however , the structures \n of the two complexes , which are crucial for a better understanding \n of the detoxification mechanism of pb  mts , have not been clearly \n elucidated . in this work , </S>", "<S> coordination of pb2 + with rabbit \n liver apo  mt2 , as well as with the two individual domains ( apomt2 \n and apomt2 ) at different ph , were studied by combined \n spectroscopic ( uv  visible , circular dichroism , and nmr ) and \n computational methods . </S>", "<S> the results showed that in pb7mt2(i ) \n the pb2 + coordination is in the trigonal pyramidal pb  </S>", "<S> s3 mode , whereas the pb7mt2(ii ) complex contains \n mixed trigonal pyramidal pb  </S>", "<S> s3 , distorted trigonal \n pyramidal pb  </S>", "<S> s2o1 , and distorted quadrilateral \n pyramidal pb  </S>", "<S> s3o1 modes . </S>", "<S> the o - donor \n ligand in pb7mt2(ii ) was identified as the carboxyl \n groups of the aspartic acid residues at positions 2 and 56 . </S>", "<S> our studies \n also revealed that pb7mt2(ii ) has a greater acid \n tolerance and coordination stability than pb7mt2(i ) , \n thereby retaining the pb2 + coordination at acidic ph . </S>", "<S> the \n higher flexibility of pb7mt2(ii ) renders it more \n accessible to lysosomal proteolysis than pb7mt2(i ) . \n </S>", "<S> similar spectral features were observed in the coordination of pb2 + by human apo - mt2 , suggesting a commonality among mammalian \n mt2s in the pb2 + coordination chemistry . </S>"], "labels": null, "section_names": ["Introduction", "Experimental Section", "Results and Discussion", "Conclusion"], "sections": [["lead \n poisoning is one of the most serious environmental health \n hazards , with a particularly acute effect on young children . the u.s .", "centers for disease control and prevention \n estimated that approximately 2.5% of children aged 15 years \n in the u.s . have elevated blood lead levels .", "the percentage of affected children in other countries is expected \n to be even higher .", "lead poisoning causes learning disabilities , behavioral \n problems , and , at very high levels , seizures , coma , and even death .", "the \n mechanism of pb toxification mainly involves binding \n of pb to proteins and the subsequent inhibition of the \n proteins physiological functions in blood and tissues .", "the documented proteins targeted by pb include several zinc enzymes or proteins ( such as -aminolevulinic \n acid dehydratase ( alad ) , acetylcoline esterase , cys2his2  zinc - finger  proteins , and acid phophatases ) and calcium - binding proteins ( calmodulin , calbindin , \n and troponin c ) .", "pb can replace \n zinc and calcium at the oxygen / nitrogen / sulfur - rich active sites of \n these proteins , thereby inhibiting the protein functions by altering \n their coordination chemistry and native structures .", "for example , the \n function of alad , an enzyme involved in the second step of heme biosynthesis , \n is altered by pb binding at the active site via a trigonal \n pyramidal geometry .", "consequently , the hemoglobin synthesis is blocked , \n leading to anemia . to reduce the pb - induced toxicity ,", "organisms have developed various defensive \n mechanisms with species such as metallothioneins ( mts ) , glutathione , \n phytochelatins , and lead - binding proteins ( pbbps , which are non - mt \n acidic proteins that have not been fully characterized ) .", "mts , a class of thiol - rich ( up to 30% of its \n amino acid residues ) , low - molecular - weight proteins whose abundance \n is particularly high in the liver and kidneys of mammals , are perhaps the most important species for lead \n detoxification .", "mts is crucial to a better understanding of the chemical \n stabilities , biological functions , and detoxification mechanism of \n pb  mts .", "however , due to the absence of single crystals of pb  mts , \n information about the pb ", "the coordination and binding stoichiometry between mts and metals \n are dependent on the type of metal ions .", "usually , divalent metal ions , such as cd and zn , are tetrahedrally coordinated by four cysteine sulfurs \n and bind to mts with a stoichiometry of 7:1 .", "it can be complexed by a combination of s , o , n , and p - donor ligands \n with a coordination number ranging from 2 to 9 .", "when pb binds to sulfur - rich proteins , three \n sulfurs in a trigonal pyramidal geometry and the pb 6s lone - pair electrons occupying the axial position ( hemidirected ) \n constitute the coordination sphere . a \n number of complexes containing the pb ", "s3 coordination \n have been observed using spectroscopic and mass spectrometry ( ms ) \n methods . in the presence of biomolecules \n possessing several distinct donor ligands (", "n , o , and s ) , the pb coordination chemistry is diverse and includes formation of pbsxoy and pbsxny .", "owing to the presence of o- and s - donor \n ligands in mts and the unique electronic configuration of pb , coordination of pb with mt is of higher complexity \n than that of zn or cd .", "visible ( uv  vis ) spectra and microcalorimetry \n have suggested that different pb ", "palacios \n et al . demonstrated with electrospray ionization mass spectrometry \n that the metal content in pb ", "mt2 complexes is dependent on \n the solution ph ( neutral or 4.5 ) . using \n extended x - ray absorption fine structure ( exafs ) , vasak et al .", "however , crucial questions such \n as the difference of pb coordination and the chemical \n stability and physiological function of the two pb ", "theoretical \n calculations have been widely \n used for the prediction of structures and spectra of metalloproteins .", "the combination of quantum mechanics and molecular \n mechanics - based hybrid ( qm / mm ) method allows two or more computational \n methods to be performed in a single calculation , making it possible \n to investigate the chemistry of complex systems with high precision . in this context , the oniom ( our own n - layered \n integrated molecular orbital + molecular mechanics ) scheme is a general \n approach because it can combine any number of molecular orbital and \n molecular mechanics methods .", "the oniom \n method has been successfully utilized in the elucidations of structural \n and functional properties of many metalloproteins such as cyt c and \n azurin . in our study ,", "optical methods \n ( uv  vis absorption and circular dichroism ( cd ) spectrometry ) \n and nmr were used in tandem with the two - layer oniom method to investigate \n the effect of ph on the structures of two different pb ", "the differences in the coordination chemistry of the metal \n centers and the protein structures at various ph were deciphered . \n", "we also investigated the chemical stabilities and structural flexibility \n of these two complexes in proteolytic processing to gain insight into \n the lead detoxification process involving mts ."], ["zn - containing \n mt2 , isolated from rabbit liver , was purchased from hunan lugu biotechnology \n co. ( changsha , china ) .", "individual metal - free domains ( apo-mt2 \n and apo-mt2 ) of both rabbit liver and human mt2s , and the corresponding \n mutants ( d25n - apo-mt2 and d2n - apo-mt2 ) were synthesized \n by shanghai apeptide co. ( shanghai , china ) . all the domains and their \n mutations", "were confirmed by mass spectrometry performed by the vendor , \n and the corresponding purity values ( > 95% ) were determined with \n hplc - ms .", "pb ( 99.1% ) was acquired from isoflex usa ( san \n francisco , \n ca ) .", "lead nitrate , 5,5-dithio - bis-(2-nitrobenzoate ) ( dtnb ) , \n and cathepsin b were purchased from sigma - aldrich ( st .", "deionized water with resistivity \n of 18.2 m cm was collected from a millipore simplicity 185 \n system ( millipore co. , billerica , ma ) .", "all solutions were prepared \n with deionized water and degassed with nitrogen for at least 30 min .", "a 3 kda cutoff \n millipore ( ym-3 ) membrane ( millipore , billerica , ma ) , equilibrated \n with 0.01 mol l hcl , was used to separate zn from zn7mt2 . after spinning at 13  000 \n rpm for 30 min at room temperature in an eppendorf 5417r centrifuge \n ( eppendorf , hamburg , germany )", "complete removal of zn from zn7mt2 \n was confirmed by the disappearance of the characteristic absorption \n of zn7mt2 at 220 nm .", "the apo - mt2 concentration was determined by assaying thiol groups \n with ellman s reagent , dtnb .", "mt2 \n solution was stored in a nitrogen - saturated flask to avoid thiol oxidation \n in apo ", "vis absorption experiments \n were carried out on a uv-2450 \n spectrophotometer ( shimadzu , japan ) in quartz cuvettes ( 1 cm path \n lengths ) .", "cd data were obtained with a jasco-810 spectrophotometer \n ( jasco corporation , japan ) .", "zeta \n potentials of individual apo - mt2 domains or their mutants were measured \n at room temperature in a folded capillary cell with a zetasizer nano \n zs instrument ( malvern instruments , southborough , uk ) .", "pb was dissolved \n with 0.15 m trace metal grade nitric \n acid ( fisher scientific ) at 250 c , and the pb(no3)2 precipitate was collected , dried , and weighed . \n", "appropriate amounts of mt2 ( 250 m ) , kcl ( 10 mm ) , and d2o were mixed under n2 atmosphere .", "both pb7mt2 complexes were prepared by adding pb(no3)2 to obtain an mt2/pb stoichiometry \n of 1:7 at corresponding ph ( 7.0 or 4.0 ) .", "the resultant solutions were \n allowed to incubate for 1 h , and the solution ph was brought up to \n ph 7.0 with koh .", "d2o was added to a final volume of 1 ml , \n and the solution was transferred to an nmr tube .", "all pb nmr spectra were recorded on a bruker advance drx-400 mhz spectrometer \n at 25 c using 60 pulses , a 2 s relaxation delay , and a \n 0.12 s acquisition time ( spectral width of 555.6 khz ) .", "a linear prediction \n was performed to remove the noise , and the real free induction decay \n ( fid ) was determined before data processing .", "after zero - filling , the \n data ( 128  000 data points ) were processed with an exponential \n line broadening of 5 hz using the software topspin nmr .", "the pb nmr chemical shifts are reported downfield from tetramethyllead \n (  = 0 ppm ; toluene ) using 1.0 m pb(no3)2 salt ( fisher ) as an external standard (  = 2990 ppm , \n d2o , 25 c ; relative to tetramethyllead ) . both pb7mt2(i ) and pb7mt2(ii )", "complexes were freshly prepared and diluted in 10 \n mm kcl solution ( ph 7.0 ) at 37 c to desired concentrations . \n", "cathepsin b ( 1.8 ng ) , with a specific activity of 3000 pmol min g , was mixed with 504 \n pmol of pb7mt2(i ) or pb7mt2(ii ) \n ( the final concentration of pb was 57 m ) in 10 \n mm kcl solution ( ph 5.0 ) .", "oniom calculations \n were performed to predict the optimal geometries and electronic absorption \n spectra of pb7mt2 complexes .", "the initial atomic coordinates of pb4mt2 \n and pb3mt2 were taken from the corresponding \n pb  substituted cd4mt2 \n and cd3mt2 , respectively .", "the structures \n of cd4mt2 and cd3mt2 \n were retrieved from the rcsb protein databank ( pdb i d : 1 mrb for the \n -domain and 2 mrb for the -domain ) , in which the absent \n hydrogen atoms were added using gaussview 4.0", ". the protonation states \n of titratable residues ( e.g. , aspartic acid and lysine ) of pb4mt2 and pb3mt2 at \n ph 4.0 and 7.0 were determined using pka values estimated with propka 2.0 .", "the electronic structures of pb4mt2 and \n pb3mt2 were modeled with the inclusion of \n the protein environment , using the two - layer oniom ( qm / mm ) model . \n", "the qm region comprises the active sites of pb4mt2 \n and pb3mt2 , and the mm region contains the \n protein environment ( cf .", "spin unrestricted density functional theory ( dft ) with \n the becke s three - parameter hybrid exchange functional and \n the lee  yang ", "parr correlation functional ( b3lyp ) or \n pure functional bp86 were used for the qm system , and the dunning \n basis set aug - cc - pvtz - pp was used for pb and 6 - 31+g * basis set for \n other atoms for the qm calculations . for \n the mm region , the protein molecule was treated using the universal \n force field ( uff ) .", "an electronic embedding \n scheme was adopted to deal with the electrostatic interactions between \n the qm and mm regions in the qm / mm calculations . on the basis of the optimized geometries ,", "vertical \n excitation energies \n were computed within the oniom scheme by employing the time - dependent \n density functional theory ( tddft ) .", "tddft \n calculations were carried out with the density functional b3lyp and \n bp86 . the trizeta basis set aug - cc - pvtz - pp", "was used for pb , and 6 - 31+g * * \n was used for others .", "the results were transformed via the swizard \n program ( version 4.6 ) into each uv spectrum \n using gaussian functions with half - widths of 3000 cm ."], ["uv  vis absorption \n and cd spectra were recorded during the titration of rabbit liver \n apo - mt2 with pb at ph 7.0 ( figure 1 ) . for simplicity , we termed the pb ", "mt2(i ) exhibits a characteristic peak centered at 330 \n nm ( figure 1a ) .", "the time - resolved absorbance \n changes ( inset ) indicates that the complexation reaction is fast ( completed \n in less than 10 min ) . during the titration of apo ", "mt2 with \n pb at neutral ph , some pb(oh)2 ( ksp = 1.4  10 ) precipitate was \n formed and affected the absorbance value of pb7mt(i ) \n at 330 nm , as evidenced by the small fluctuation even after 30 min . \n", "the precipitation can be avoided by lowering the solution ph below \n 6.0 . as shown in the inset of figure s1 in the supporting information ,", "the absorbance value of pb7mt(i ) remains stable after the complexation reaction is completed . \n as indicated by the mt2/pb stoichiometry ( figure 1b ) ,", "the maximum binding stoichiometry is 1:7 , consistent \n with results reported from the substitution experiment of zn7mt2 with pb . the \n absorption wavelength ( 330 nm ) and", "extinction coefficients (  \n  3500 m cm ) of pb7mt2(i ) are analogous to values reported for several \n pbs3 complexes .", "the secondary \n structural variation from apo - mt2 to pb7mt2(i ) \n is shown in figure 1c .", "this \n is in contrast with the intense cd peaks of cd7mt2 \n and zn7mt2 between 210 and 290 nm . in zn7mt2 ,", "the characteristic cd band centering at 244 nm is attributed \n to the zn(sr)4 chromophore .", "similarly , the cd bands of \n cd7mt2 at 242 and 262 nm can be attributed to the \n excitation coupling between adjacent pairs of the cd(sr)4 chromophore .", "since the m(sr)4 chromophore leads to the appearance of these cd peaks , the absence of any obvious cd peaks in figure 1c suggests that pb7mt2(i ) adopts \n a different metal coordination geometry from those of cd7mt2 and zn7mt2 , as alluded to in the introduction . \n", "the individual - and -domains display uv  vis \n and cd spectral features ( data not shown ) similar to those of apo - mt2 \n during the titration with pb , indicating that the metal \n centers in the two different domains have similar coordination spheres", ". \n quantitative analysis confirms that the pb / mt2 stoichiometric \n ratios are 4:1 and 3:1 in the - and -domain , respectively .", "time - dependent \n ( a ) uv  vis absorption and ( c ) cd spectra \n in 10 mm kcl solution ( ph 7.0 ) containing 7.20 m rabbit liver \n apo - mt2 and 20 mol equiv of pb .", "( b ) dependence of uv absorption peak \n at 330 nm upon addition of pb to apo - mt2 .", "the \n pb7mt2(ii ) complex displays dramatically different \n uv  vis \n absorption and cd spectra ( figure 2 ) from those \n of pb7mt2(i ) . in the uv  vis absorption spectra , \n two intense peaks at 325 and 375 nm appear , with the latter having \n a shoulder peak at 400 nm . in the cd spectra ( figure 2c ) , a strong envelope with maxima at 240 ( + ) , 265 ( + ) ,", "320 \n (  ) , 350 ( + ) , 370 ( + ) , and 395 (  ) is produced isodichroically \n ( 280 , 340 , and 375 nm ) .", "positions of all cd peaks are invariant with \n the pb / apo  mt2 ratio ( increased stepwise from \n 1:1 to 7:1 ) , but the peak intensity increases with the ratio .", "the \n cd spectra of cd7mt2 have two bands at 240 and \n 260 nm split from the 250 nm band , which corresponds to the conversion \n of isolated cd(sr)4 to the ( sr)3cd  sr ", "thus , the unsplittable \n pb7mt2(ii ) cd peak is indicative of the absence \n of excitation coupling between adjacent chromophores .", "the multiple \n peaks in the uv  vis absorption and cd spectra suggest that \n pb in the pb7mt2(ii ) complex has binding \n modes that are distinctively different from cd in cd7mt2 .", "similar to pb7mt2(i ) , the \n pb / mt2 stoichiometric ratios in the pb7mt2(ii ) \n complex are 4:1 and 3:1 in the - and -domains , respectively . \n", "the uv  vis absorption peaks of pb4mt2(ii ) \n are slightly shifted , with higher intensity than those of pb3mt2(ii ) ( figure 3d ) .", "these \n differences suggest that the metal centers in the two domains have \n different coordination geometries . to pinpoint the ligands responsible \n for coordination of pb in the two different domains at \n neutral and acidic ph", "time - dependent ( a ) uv  vis absorption and ( c ) cd spectra \n in 10 mm kcl solution ( ph 4.5 ) containing apo - mt2 ( 7.20 m ) \n and 20 mol equiv of pb .", "( inset ) the time - resolved absorbance \n changes at 325 and 375 nm , respectively .", "( b ) the dependence of uv \n absorption peaks at 325 and 375 nm on the addition of pb to apo - mt2 .", "structures and electronic \n absorption spectra of the - and \n -domains in ( a , c ) pb7mt2(i ) and ( b , d ) \n pb7mt2(ii ) obtained by experimental and oniom methods . \n in panels a and b , the dark gray spheres are pb , yellow \n spheres are s , red spheres are o , gray spheres are c , and white spheres \n are h atoms", ". the oniom method has been successfully used \n to predict metalloprotein structures . to validate the method for the studies of mt2 structures , structural \n optimization of cd4mt2 and cd3mt2 models", "compared to the nmr \n results , the computed structures of the \n metal clusters display little deviation ( e.g. , the bond lengths have \n a root - mean - square deviation of only 0.040.05  when \n compared to the experimental data ) ( figure s2 and table s1 in supporting information ) .", "therefore , we conclude \n that the oniom method is viable for the studies of the two pb7-mt2 complexes .", "the theoretical calculations were performed \n separately on the - and -domains on the basis that the \n two domains are structurally independent .", "the initial atomic coordinates \n of each domain in pb7mt2 were respectively adopted \n from the pb - substituted cd4mt2 \n and cd3mt2 because a well - established model \n of apo - mt2 is not available .", "the protonation states of titratable \n residues ( e.g. , asp and lys ) at different ph were determined from \n the pka values estimated with propka 2.0 .", "in the two - layer oniom model of pb7mt2(i ) , the \n qm region consists of four pb atoms , and the side chains consist of \n eleven cysteine residues in the -domain . as for the -domain , \n", "the three pb atoms and the side chains of nine cysteine residues constitute \n the qm region .", "figure 3a displays the optimized molecular geometries of \n the - and -domains in pb7mt2(i ) , \n which are quite different from the well - characterized cd7mt2 and zn7mt2 structures . in cd7mt2 and zn7mt2", ", each metal adopts the \n tetrahedral coordination with terminal and bridging thiolates to form \n a metal  ligand six - membered ring in the -domain and \n two fused six - membered rings in the -domain.(cf .", "figure s2 \n in supporting information ) however , all \n seven pb ions in pb7mt2(i ) are trigonally \n coordinated by three cysteine sulfurs ( pb  s3 ) without \n any metal ", "these \n significant differences can be attributed to the pb 6s lone - pair electrons , which disrupt the tetrahedral coordination \n by occupying the axial position with a significant stereochemical \n activity .", "this point is in line with the report by godwin and co - workers , \n who stated that pb ", "s4 is not a preferred coordination \n and that trigonal pyramidal geometry in all - sulfur coordination is \n predominant .", "moreover , due to the pb  s3 coordination , only one bridging cysteine ( pb6s18pb7 \n in the -domain ) remains , preventing a pb ", "according to the corresponding structural parameters \n ( table 1 ) , the average length of the pb ", "s \n bonds is 2.68  , close to the reported exafs value ( 2.65  ) . the electronic absorption spectra ( red line \n curves ) , simulated by tddft using the b3lyp functional , are overlaid \n with the experimental results ( black line curves ) in figure 3c .", "the agreement between the simulation and experimental \n data in the oscillator strengths indicates that the optimized molecular \n geometries of pb4mt2(i ) and pb3mt2(i ) are reasonable .", "detailed analysis of vertical \n excitation energies , oscillator strengths , and molecular orbital contributions \n ( table s3 in supporting information ) indicated \n that four transitions contribute to the absorption peak at 330 nm . \n", "these transitions are homolumo , homo-1lumo , homo-2lumo , \n and homo-1lumo+1 . from the frontier molecular orbitals ( figure \n s3 in supporting information ) , lumo and \n lumo+1 ( the final state of the transitions )", "s bonds , while homo and homo-1 ( the initial states of \n the transitions ) are largely localized at the sulfur atoms .", "therefore , \n the electronic absorption band at 330 nm can be attributed to the \n spb ligand - to - metal charge transfer ( lmct ) .", "in the two - layer oniom model \n of pb7mt2(ii ) , \n we initially assigned the qm region of each domain to be the same \n as that of pb7mt2(i ) .", "however , the optimized molecular \n geometries revealed two unexpected short pb / o distances ( 5.52  \n for pb1 with the carbonyl oxygen of asp2 in the -domain and \n 4.31  for pb6 with the carbonyl oxygen of asp56 in the -domain ) . \n", "these short distances should result from the electrostatic interaction \n between the protein surface ( the mm region ) and the negatively charged \n pb ", "acidic ph conditions , the protein surface is neutral or positively \n charged , causing the mm and qm regions to move closer , thereby shortening \n the distance between the pb ", "moreover , based on the pka values of asp \n ( 4.00 ) , both asp residues are neutral in weakly acidic solution , \n which are more favorable than the negatively charged ( deprontonated ) \n form to the positioning of the negatively charged pb ", "these pb / o distances are close to the sum of the \n van der waals radius of pb and o ( 3.54  ) , indicating the pbo interaction must be \n taken into account .", "we therefore modified the initial qm region in \n each domain by including the respective asp residue .", "as shown \n in figure 3b , the optimized molecular \n geometry of pb7mt2(ii ) shows an entirely different \n coordination sphere from that of pb7mt2(i ) .", "the \n overall coordination sphere includes the trigonal pyramidal pb  s3 mode , the distorted trigonal pyramidal pb ", "s2o1 mode in the -domain , and the distorted quadrilateral \n pyramidal pb  s3o1 in the -domain . \n", "the pb 6s lone pair electrons occupy the \n axial position with a significant stereochemical activity , resulting \n in hemidirectionality in the pb  ligand coordination .", "moreover , due to protonation of the \n peptide side chain , several cysteine sulfurs ( especially in the -domain ) \n are closer to the protein exterior and increase the effective radius \n of the metal center and the pb  s bond length ( cf .", "the loosened structure \n of the metal center renders a higher flexibility to pb7mt2(ii ) .", "the simulated oscillator strengths are in good agreement \n with the experimental data ( figure 3d ) , indicating \n that the optimized molecular geometries of pb4mt2(ii ) \n and pb3mt2(ii ) are reasonable . the small \n deviation between the electronic absorption spectra of the two domains \n ( cf .", "detailed vertical excitation energies and \n molecular orbital contributions ( table s5 and figure s4 in supporting information ) indicate that all three \n bands are primarily associated with the spb lmct .", "note that all of the above calculations are based on b3lyp , a hybrid \n functional to fit data primarily for main - group elements .", "because of the large exact exchange component \n ( 20% ) , b3lyp is known to favor loose electron densities and low transition \n energies . to confirm the reliability of b3lyp , the pure functional \n bp86 , a generalized - gradient approximation ( gga ) class with zero exact \n exchange ,", "is used as a control study on \n the geometry optimization and calculations of the excited states for \n both pb7mt2 complexes .", "the simulated structures \n of both pb7mt2 complexes from bp86 pure functional \n ( table s69 in supporting information ) are similar to those from b3lyp hybrid functional .", "however , the \n spectra from bp86 pure functional display a significant deviation \n from the experimental data ( figure s5 in supporting \n information ) , indicating that the b3lyp hybrid functional is \n a better choice .", "the b3lyp hybrid functional produces better spectral \n accuracy , which can be attributed to its increased amount of hartree ", " local \n minima  \n in the qm optimization . in the qm / mm calculation of biomolecules , \n the vast size of the available configuration space may cause the qm \n optimization to stop at local minima .", "one way to circumvent this problem \n is to use a reliable structure ( e.g. , a structure deduced from nmr \n or x - ray crystallography ) at the beginning of the qm optimization .", "have used the \n x - ray structure of azurin as the initial structure in the qm / mm calculation \n of metal - substituted azurins and obtained noticeable structural changes \n on the active sites when cu in azurin was substituted \n by metal ions such as co , ni , or zn . we adopted this approach \n by using the pb - substituted cd4mt2 \n and cd3mt2 structures deduced from the nmr \n experiments as the initial structures . \n", "the theoretical spectra agree well with the experimental results , \n suggesting a high level of reliability of the calculation and in the \n predicted structures .", "to provide more \n experimental evidence \n to our computational results about the two different pb7mt2 complexes , we conducted pb nmr in solutions \n of the two complexes ( figure 4 ) .", "pb7mt2(i ) displays one pb peak at 5679 ppm , while \n pb7mt2(ii ) exhibit two peaks at 5820 and 4348 ppm . \n thus it is clear that the pb coordination in these two \n complexes are different .", "furthermore , the pb signal \n of pb7mt2(i ) at 5679 ppm is well within the chemical \n shift region ( from 5600 to 5800 ppm ) where pbs3 species \n with the trigonal pyramidal coordination are observed .", "thus we conclude that pbs3 is the binding mode in pb7mt2(ii ) . for pb7mt2(ii ) , the peak \n at 5820 ppm is also assigned to the pbs3 coordination , \n given its close vicinity to the 56005800 ppm region .", "compared \n to the pb7mt2(i ) peak , the shift by 141 ppm can \n be attributed to the changes of the aforementioned pb  s bond \n length and the s  pb  s bond angle in the pbs3 coordination .", "the lower intensity is indicative of the decreased \n number of pbs3 clusters in pb7mt2(ii ) . \n", "the conversion of metal centers in the pbs3 coordination \n to a different binding mode contributes to the appearance of the peak \n at 4348 ppm .", "it is well - known that the nmr signal of pb bound to o - containing ligands is shifted upfield with respect \n to that bound to s - containing ligands .", "pb nmr spectra of ( red ) pb7mt2(i ) , \n ( blue ) pb7mt2(ii ) , and ( black ) pb(no3)2 at ph 7.0 .", "the carbonyl group of asp is the only o - donor ligand in mt2 . \n to \n further verify the formation of the pb ", "o bond in pb7mt2(ii ) , a titration was performed by adding pb into a peptide solution whose asp residue had been mutated with \n asn ( i.e. , d25n - apo-mt2 and d2n - apo-mt2 ) .", "zeta potential \n and cd measurements did not show discernible changes in the surface \n charge and structure of both mutants ( figure s6 and s7 in supporting information ) , confirming the viability \n of these mutants for coordination studies . at neutral ph ,", "both uv  vis \n absorption and cd spectra of the two mutants show features similar \n to those in the same spectra of pb4mt2(i ) \n and pb3mt2(i ) , verifying that asp does not \n participate in the pb coordination in pb7mt2(i ) . \n at acidic ph ( ph < 5 ) ,", "the uv  vis absorption spectra of \n both mutants display only a single peak at 330 nm , and the cd spectra \n did not reveal any changes . while these features are rather different \n from the corresponding spectra of pb4mt2(ii ) \n and pb3mt2(ii ) , they are analogous to those \n of pb4mt2(i ) and pb3mt2(i ) . \n we therefore conclude that asp is involved in the coordination of \n pb in pb7mt2(ii ) . the unique uv", " vis \n absorption spectra of pb7mt2(ii ) , with multiple \n peaks and the presence of a new pb nmr peak at 4348 \n ppm , are well - correlated with the mutational study . another \n piece of evidence for the formation of pb  o bond \n is the transition from a single peak to three peaks in the time - dependent \n spectra of a mixture of pb / apo - mt2 at ph 4.55.0 \n as well as the appearance of multiple peaks in the cd spectra ( cf . \n", "however , because \n of the negative impact of acidity on the formation of the pb  s \n bond ( cf . the proton - releasing process shown in reaction 1 ) and the rearrangement of the peptide chain , the pb ", "therefore , under \n more acidic conditions ( ph < 4.5 ) , the time - dependent peak transition \n disappears , and the spectra show features typical of pb7mt2(ii).1 time dependence \n of ( a ) uv  vis absorbance and ( b ) cd spectra \n in a kcl solution after the addition of 7 mol equiv of pb to an apo - mt ( 7.20 m ) solution at ph 5.0 .", "reaction times \n from bottom to top : 0 , 1 , 3 , 5 , 7 , 10 , 15 , 20 , 25 , 30 , 45 , 60 , 75 , \n 90 , 105 , 120 , 135 , 150 , 175 , and 180 min . to study the acid tolerance of both pb7mt2 complexes , a series of spectrophotometric titrations \n at different ph was performed .", "the corresponding complex was confirmed \n by the appearance of characteristic uv  vis absorption peaks \n of pb7mt2(i ) or pb7mt2(ii ) . \n", "the results show that pb7mt2(i ) is formed above \n ph 5.0 , whereas pb7mt2(ii ) is produced at more \n acidic ph .", "pb7mt2(i ) can be transformed to pb7mt2(ii ) by adjusting the solution ph to 5.0 or lower . \n", "however , once pb7mt2(ii ) is formed , it remains \n stable at neutral ph .", "our demetalation experiments ( figure s8 in supporting information ) indicate that ph 2.5 \n is sufficiently low for the complete removal of pb from \n pb7mt2(i ) , but stripping pb completely \n of pb7mt2(ii ) requires a ph as low as 2.0 . these \n results ( summarized schematically in figure 6 )", "indicate that pb7mt2(ii ) has a higher tolerance \n toward an acidic environment than pb7mt2(i ) has . \n", "moreover , pb7mt2(ii ) remains stable even in the \n presence of apo - mt at neutral ph ( data not shown ) .", "we believe that \n the greater acid tolerance and higher structural stability of pb7mt2(ii ) results from the pb  o bond .", "such a \n finding has a significant implication to the lead detoxification process \n in physiological milieu .", "the pb - inflicted toxicity stems from its tight \n binding \n to a variety of sulfur - rich proteins , such as gata proteins and the \n steroid receptor dna - binding domains .", "mt2 reduces the pb - inflicted toxicity \n by seizing free pb or sequestering pb from \n these proteins to recover the native protein function .", "higher acid \n tolerance and greater structural stability render mt2 a greater power \n in effectively scavenging pb in different environments . \n as shown in reaction 1 ,", "mt2 accompanies the release of h , which \n in turn increases the acidity in a highly localized region ( e.g. , \n in cytosol ) .", "moreover , it has been reported that elevated pb concentrations induce the stress level of various organisms , and \n acidity is also correlated with the stress level .", "we posit that pb7mt2(ii ) is more effective \n than pb7mt2(i ) in lead detoxification , given its \n greater structural stability and acid tolerance .", "elucidation of the metabolism of the pb - mt \n complexes is vital for understanding the lead detoxification by mts . \n", "some reports have suggested that exogenous mt is processed mainly \n by the lysosomal protease , and the rate of mt degradation is dependent \n on the types of metals bound by mts .", "four \n different cathepsins have been identified in lysosomes , and the cysteine \n protease ( cathepsin b , l , and h ) is the principal protease for mt \n degradation .", "variations in the concentrations of the two pb7mt2 \n complexes in the presence of cathepsin b were measured by uv  vis \n absorption spectrometry . in figure 7 , the pb7mt2 concentrations were normalized with respect to \n their initial concentrations . within 120 min , pb7mt2(ii ) \n", "such a \n higher degradation rate can be attributed to the more flexible structure \n of the pb7mt2(ii ) complex .", "cathepsin b is an endopeptidase \n that cleaves internal peptide bonds and favors a large hydrophobic \n side chain in the substrate protein .", "side \n chains on the amino acids dock into the cathepsin s subsites , \n whose interaction with the protein substrate is dependent on the flexibility \n of substrate protein . in mts ,", "the existence \n of bridging cysteine sulfurs compacts the metal center , which dominates \n the protein folding . in pb7mt2(ii ) , due to the lack of bridging cysteine sulfurs , the \n metal center is loosened , which improves the flexibility of the protein . \n moreover , when the pb ", "s3o1 become \n distorted . consequently , pb ions are positioned farther \n from the cysteine sulfurs , and the pb  s bonds are weakened . \n both processes facilitate the conformational adjustment of pb7mt2(ii ) .", "the significantly improved flexibility of \n pb7mt2(ii ) facilitates the protein in the  induced - fit  \n model with cathepsin b. as a result , \n the proteolytic processing of pb7mt2(ii ) is greatly \n accelerated . in living organisms ,", "the acidity in lysosome is about \n 5 , which is sufficiently low to cause the structural conversion from \n pb7mt2(i ) to pb7mt2(ii ) and \n to accelerate the degradation of pb  mt2 complexes .", "time - dependent proteolytic processing of ( black ) pb7mt2(i ) and ( red ) pb7mt2(ii ) by cathepsin \n b. the coordination of pb with individual human apo  mt2 \n or apo  mt3 domains at different ph was also studied by uv  vis \n absorption and cd spectrometry .", "mutational studies revealed \n that the asp residue is also essential for the ph - dependent structural \n variation . in line with the data observed for the rabbit liver mt2 , \n the structure - dependent chemical and biological activities of pb7hmt2(ii ) formed between human mt2 and pb have higher acid tolerance , more coordination stability , and faster \n proteolytic processing than pb7hmt2(i ) ( figure \n s10 in supporting information ) .", "our results \n suggest that there exists a commonality in the pb coordination \n chemistry among mammalian mt2s ."], ["in this work , the ph - dependent coordination chemistry between pb and mt2 was systematically studied .", "the combination of spectroscopic \n studies and oniom calculations provided a detailed description of \n the two different pb7mt structures .", "the results \n and structures were further verified by pb nmr and mutational \n experiments .", "the similar structural , chemical , and biological properties \n between rabbit liver pb7mt2(ii ) and human pb7mt2(ii ) suggest a commonality in the pb coordination chemistry among mammalian mt2s .", "the higher acid tolerance , \n greater coordination stability , and faster degradation rate of pb7mt2(ii ) have significant implications for the pb detoxification process .", "specifically , mt2 reduces the pb - inflicted toxicity by seizing free pb in the \n cellular milieu or by sequestering pb from pb - inflicted proteins .", "the unique properties of pb7mt2(ii ) \n render mt2 a greater power to effectively scavenge pb in different environments ( e.g. , in a localized acidic cytosol region ) . \n", "moreover , the greater flexibility of pb7mt2(ii ) , \n resulting from the absence of bridging cysteine sulfurs , helps to \n accelerate its processing by lysosomal protease .", "the structural conversion \n from pb7mt2(i ) to pb7mt2(ii ) \n is likely to occur in the acidic environment of lysosome , facilitating \n the effective detoxification and metabolism of pb ."]]}
{"article_id": "PMC4471641", "article_text": ["an increased man - made radiation exposure - risk from the use of high - dose imaging modalities such as computed tomography and angiographic suites is now being observed in many health - care centers with over 3.6 million diagnostic examinations performed annually worldwide .", "interventional procedures are performed by cardiologists , radiologists , endovascular surgeons , operation theater staff , etc .", ", due to the well - known benefits in medicine . however , it is crucial for the referring clinician and the interventionalist ( radiologist / cardiologist / clinician in the operation theater ) to assess the potential benefit - risk ratio for various interventional procedures as some of the procedures involve a high radiation exposure due to prolonged fluoroscopic screening .", "all interventional cardiological procedures are invariably performed using dedicated fluoroscopy and angiography suites equipped with either image intensifier ( ii)-based or flat panel detector ( fpd)-based systems .", "the ii - based systems have been used for fluoroscopy for more than two decades . on the other hand ,", "the fpd - based systems for medical imaging emerged in the 1990s initially for two - dimensional ( 2d ) projection x - ray image , and subsequently for a  real - time ", "interventional angiography suites equipped with ii or fpd have the potential to impart high radiation doses to patients if optimization strategies are not well - implemented .", "stringent optimization involves orientation of staff , consistent restriction of frame rates during image acquisition , using low dose settings , judicious use of magnification , etc .", "it is also necessary to understand the potential risks due to radiation from different interventional procedures .", "for this reason , it is necessary that one should be knowledgeable in the magnitude of radiation dose associated with each intervention .", "this can be achieved by measuring real - time doses using devices such as a dose area product ( dap ) meter .", "most of the newer angiography machines are equipped with a dap meter fitted on the collimator assembly of the machine .", "dap is particularly a useful method for assessing and comparing the radiation dose from screening procedures and acts as a surrogate for radiation risk .", "entrance surface dose ( esd ) is also used for measuring radiation doses . from the dose descriptors , it is possible to estimate organ doses as well as effective doses for each procedure .", "radiation doses from interventional procedures have been widely reported in literature , with more emphasis on doses from ii - based systems . however , there are only a few reports on radiation doses from fpd systems as it may be a transition period from ii to fpd for most of the interventional users .", "some patient and phantom - based studies reported in literature state that doses from fpd are higher than ii systems .", "few other studies report that radiation doses from fpd are lower than ii systems . in comparing conventional and digital systems ,", "hence , it is not clear whether fpd imparts lower radiation dose than ii and whether there would still be a need to further optimize doses in the newer fpd systems .", "the purpose of this article is to review and compare radiation doses from ii and fpd - based systems in interventional cardiology in a tertiary referral center that has introduced fpd system recently .", "it is anticipated that this information will be useful for those performing cardiological interventions and for those who are on a transition from ii to fpd .", "cardiovascular interventions were performed using two dedicated catheterization labs , each equipped with philips allura fd10 flat panel system ( netherlands ) .", "the dose area product ( dap ) values for coronary angiography ( cag , n = 222 ) and percutaneous transluminal coronary angioplasty ( ptca , n = 75 ) procedures performed during a one year period 20122013 were prospectively recorded using a built - in calibrated dap meter ( transmission ionization chamber ) .", "the ptca procedure was invariably performed by a senior interventionalist assisted by two junior cardiologists , while the cag was performed either by the senior interventionalist or by junior cardiologists . for a similar comparison of clinical protocols adopted in the institution , dap values from cag and ptca performed using philips integris h3000 and h5000 ii - based systems ( netherlands ) reported earlier", "all the x - ray systems were on periodic qa programs and conformed to the manufacturer 's specifications .", "the ii and fpd systems had low- , normal- , and high - dose settings , respectively , for fluoroscopy .", "these machines incorporated a total filtration of 2.5 mmal with possibility of selecting spectral filters such as 0.1 mm , 0.2 mm , 0.4 mm cu for dose reduction . during the course of the study , low dose setting with 0.4 mm cu filter was invariably selected during fluoroscopy . in the earlier work using ii - based system , a 23-cm image intensifier format ( iif )", "was used during fluoroscopic screening in cag and ptca procedures for tracing the path of the catheter from the region of arterial puncture and to the screening of the cardiac valve region .", "a 17-cm iif was used for the oblique , caudal , cranial , and lateral projections delineating the coronary anatomy . in the fpd system ,", "25 cm fluoro format was used during screening and 20 cm was used for other projections to delineate coronary anatomy .", "a transition from ii to fpd system for a catheterization lab would require adequate justification in terms of radiation dose , image quality , maintenance , and investment .", "it has been reported that the fpds designed specifically for fluoroscopic purposes provide superior image quality and dose efficiency compared to the ii systems , except at the lowest fluoroscopic dose levels .", "prieto et al . , reports that even after upgrading to the fpd from ii , significant increase in patient doses were observed though the fluoroscopic time and number of images remaining the same in both cases during the initial transition period . as only a few studies on radiation doses", "are available for fpd systems ; more work is required on optimization strategies in the fpd systems .", "table 1 shows the dap values and fluoroscopic time duration for ii and fpd systems from the referral center where the study was conducted .", "the dap values for ii - based systems represented in table 1 is from the use of optimized protocol as reported in the previous published article from the same referral center where the study was conducted . prior to optimization in ii systems ,", "the doses were above 50% ; however , it was possible to optimize dose by halving the entrance dose ratesby selecting 0.4 mm cu filtration ( generally recruited in pediatric protocols ) during fluoroscopy .", "selection of 0.4 mm cu filtration did not suffer significant loss of image quality ; however , tube potentials were increased from 80 kvp to 103 kvp during fluoroscopy . in the fpd system ,", "tube potentials ranged from 90 kvp110 kvp when 0.4 mm cu filtration was selected with low dose settings during fluoroscopy . having adhered to the same optimization strategies in both systems , doses were similar owing to the fact that further optimization is warranted in fpd system .", "reported dap values of 31 and 33 gycm from cag with the use of ii and fpd system , respectively .", "they have also reported that the total dap from fluoroscopy and cine for ii and fpd are not significantly different and the image quality from fpd is better than ii in cine mode with no difference in the imaging performance in fluoroscopy mode .", "fluoroscopic time and dose area product values from ii and flat panel detector ( fpd)-based systems from cardiological interventions tables 2 and 3 shows dap values for cag and ptca procedures from various studies in literature .", "the arithmetic mean dap and fluoroscopic time duration using ii system as reported in literature was 39 gycm for 6.6 min and 61.2 gycm for 17 min for cag and ptca respectively [ table 2 ] . with the use of fpd ,", "mean dap and fluoroscopic time duration were 28.4 gycm for 7 min and 61 gycm for 18.05 min for cag and ptca , respectively [ table 3 ] . from tables 2 and 3 , radiation doses from fpd", "were significantly low for cag but were similar for ptca when compared to ii systems .", "it should be noted that time duration for cag and ptca was not available for some reported studies in tables 2 and 3", ". wide variation of doses is observed from these studies which may be attributed to the angiographic system used , time duration of the procedure and work environment .", "doses of the order of 492 gycm were recorded in ptca procedure from fpd system which was higher than the ii systems .", "chida et al . , conducted studies from various ii and fpd system and the average entrance doses of cine angiography and fluoroscopy in fpd systems were not significantly different .", "though fpds possess good detective quantum efficiency , they did not inherently reduce the radiation dose .", "jensen et al . , observed that patient doses from fpd were lower than ii systems ; however , the eye lens doses for radiologists were higher in fpd than in ii due to the use of high filtration and recruitment of high tube potentials . in our study ,", "high tube potentials were recruited when low dose settings involving high filtration were selected , which may have the potential to increase staff doses .", "radiation doses from cardiological interventions performed using image intensifier - based systems radiation doses from cardiological interventions performed using flat panel detector - based systems it is prudent to adopt stringent optimization measures in fpd as the dose may be higher than the ii systems as reported by prieto et al . and", "dose reduction is possible in either ii or fpd systems . during the initial stages of our study ,", "the doses from fpd were similar to the ii system though high filtration was used .", "reports that it is possible to achieve mean dap of 6.2 and 10.4 gycm for cag and ptca procedures performed using ii system .", "they further report that the reduction of doses was by influencing the quality of fellowship training , consistent restriction to mean values of 171 frames per cag , 165 frames per ptca , low - level fluoroscopy , training in the use of fluoroscopy - free blind positioning to the region of interest , restrictions to achieve lower ii entrance dose for adequate image quality . from tables 1 and 3 ,", "the mean dap for cag from fpd were higher than those reported by kuon et al .", "tsapaki et al . , reported the doses from fpd were increased by 35% compared to ii when fluoroscopy levels were changed from low to high mode ; they also recorded a minimum dap value of 6.1 and 14.3 gycm for cag and ptca , respectively , with the use of low dose fluoroscopy settings in fpd .", "dekker et al . , reported that the new generation fpds incorporated with good image processing and noise reduction techniques resulted in reducing patient doses by 43% without compromising image quality and staff doses by 50% during electrophysiological interventions .", "though fpd has reduced entrance dose rates , it does not automatically reduce radiation doses in clinical practice .", "further work is necessary to study the possibilities of dose reduction in fpd so as to be implemented in the clinical set up .", "the patient dose and image quality in any newer modality needs to be permanently monitored and transition from ii to fpd requires careful attention .", "this is a preliminary study as the institution where the study was conducted recently moved from ii to fpd - based systems .", "though radiation doses for cardiological interventions from fpd were similar to the ii - based system achieved after optimization , the advantages of fpd in terms of good image uniformity , improved patient imaging accessibility due to smaller size , absence of geometric distortion / veiling glare or vignetting make the fpd superior to the ii systems . to achieve improved patient dose reduction , it is advisable to strictly adhere to low dose protocols with high filtration in fpd systems .", "in addition , more attention for staff doses is warranted especially for interventionalists when this stringent patient dose reduction is employed .", "it is recommended to follow stringent dose reduction strategies right from the period of initial installation when there is a transition from ii to fpd systems .", "further studies are required to develop dose optimization in fpd , though use of high filtration is already in place ."], "abstract_text": ["<S> flat panel detector ( fpd ) technology in interventional cardiology is on the increase due to its varied advantages compared to the conventional image intensifier ( ii ) systems . </S>", "<S> it is not clear whether fpd imparts lower radiation doses compared to ii systems though a few studies support this finding . </S>", "<S> this study intends to compare radiation doses from ii and fpd systems for coronaryangiography ( cag ) and percutaneous transluminal coronary angioplasty ( ptca ) performed in a tertiary referral center . </S>", "<S> radiation doses were measured using dose area product ( dap ) meter from patients who underwent cag ( n = 222 ) and ptca ( n = 75 ) performed using fpd angiography system . </S>", "<S> the dap values from fpd were compared with earlier reported data using ii systems from the same referral center where the study was conducted . </S>", "<S> the mean dap values from fpd system for cag and ptca were 24.35 and 63.64 gycm2 and those from ii system were 27.71 and 65.44 gycm2 . </S>", "<S> transition from ii to fpd system requires stringent dose optimization strategies right from the initial period of installation . </S>"], "labels": null, "section_names": ["Introduction", "Materials and Methods", "Results and Discussion", "Conclusion"], "sections": [["an increased man - made radiation exposure - risk from the use of high - dose imaging modalities such as computed tomography and angiographic suites is now being observed in many health - care centers with over 3.6 million diagnostic examinations performed annually worldwide .", "interventional procedures are performed by cardiologists , radiologists , endovascular surgeons , operation theater staff , etc .", ", due to the well - known benefits in medicine . however , it is crucial for the referring clinician and the interventionalist ( radiologist / cardiologist / clinician in the operation theater ) to assess the potential benefit - risk ratio for various interventional procedures as some of the procedures involve a high radiation exposure due to prolonged fluoroscopic screening .", "all interventional cardiological procedures are invariably performed using dedicated fluoroscopy and angiography suites equipped with either image intensifier ( ii)-based or flat panel detector ( fpd)-based systems .", "the ii - based systems have been used for fluoroscopy for more than two decades . on the other hand ,", "the fpd - based systems for medical imaging emerged in the 1990s initially for two - dimensional ( 2d ) projection x - ray image , and subsequently for a  real - time ", "interventional angiography suites equipped with ii or fpd have the potential to impart high radiation doses to patients if optimization strategies are not well - implemented .", "stringent optimization involves orientation of staff , consistent restriction of frame rates during image acquisition , using low dose settings , judicious use of magnification , etc .", "it is also necessary to understand the potential risks due to radiation from different interventional procedures .", "for this reason , it is necessary that one should be knowledgeable in the magnitude of radiation dose associated with each intervention .", "this can be achieved by measuring real - time doses using devices such as a dose area product ( dap ) meter .", "most of the newer angiography machines are equipped with a dap meter fitted on the collimator assembly of the machine .", "dap is particularly a useful method for assessing and comparing the radiation dose from screening procedures and acts as a surrogate for radiation risk .", "entrance surface dose ( esd ) is also used for measuring radiation doses . from the dose descriptors , it is possible to estimate organ doses as well as effective doses for each procedure .", "radiation doses from interventional procedures have been widely reported in literature , with more emphasis on doses from ii - based systems . however , there are only a few reports on radiation doses from fpd systems as it may be a transition period from ii to fpd for most of the interventional users .", "some patient and phantom - based studies reported in literature state that doses from fpd are higher than ii systems .", "few other studies report that radiation doses from fpd are lower than ii systems . in comparing conventional and digital systems ,", "hence , it is not clear whether fpd imparts lower radiation dose than ii and whether there would still be a need to further optimize doses in the newer fpd systems .", "the purpose of this article is to review and compare radiation doses from ii and fpd - based systems in interventional cardiology in a tertiary referral center that has introduced fpd system recently .", "it is anticipated that this information will be useful for those performing cardiological interventions and for those who are on a transition from ii to fpd ."], ["cardiovascular interventions were performed using two dedicated catheterization labs , each equipped with philips allura fd10 flat panel system ( netherlands ) .", "the dose area product ( dap ) values for coronary angiography ( cag , n = 222 ) and percutaneous transluminal coronary angioplasty ( ptca , n = 75 ) procedures performed during a one year period 20122013 were prospectively recorded using a built - in calibrated dap meter ( transmission ionization chamber ) .", "the ptca procedure was invariably performed by a senior interventionalist assisted by two junior cardiologists , while the cag was performed either by the senior interventionalist or by junior cardiologists . for a similar comparison of clinical protocols adopted in the institution , dap values from cag and ptca performed using philips integris h3000 and h5000 ii - based systems ( netherlands ) reported earlier", "all the x - ray systems were on periodic qa programs and conformed to the manufacturer 's specifications .", "the ii and fpd systems had low- , normal- , and high - dose settings , respectively , for fluoroscopy .", "these machines incorporated a total filtration of 2.5 mmal with possibility of selecting spectral filters such as 0.1 mm , 0.2 mm , 0.4 mm cu for dose reduction . during the course of the study , low dose setting with 0.4 mm cu filter was invariably selected during fluoroscopy . in the earlier work using ii - based system , a 23-cm image intensifier format ( iif )", "was used during fluoroscopic screening in cag and ptca procedures for tracing the path of the catheter from the region of arterial puncture and to the screening of the cardiac valve region .", "a 17-cm iif was used for the oblique , caudal , cranial , and lateral projections delineating the coronary anatomy . in the fpd system ,", "25 cm fluoro format was used during screening and 20 cm was used for other projections to delineate coronary anatomy ."], ["a transition from ii to fpd system for a catheterization lab would require adequate justification in terms of radiation dose , image quality , maintenance , and investment .", "it has been reported that the fpds designed specifically for fluoroscopic purposes provide superior image quality and dose efficiency compared to the ii systems , except at the lowest fluoroscopic dose levels .", "prieto et al . , reports that even after upgrading to the fpd from ii , significant increase in patient doses were observed though the fluoroscopic time and number of images remaining the same in both cases during the initial transition period . as only a few studies on radiation doses", "are available for fpd systems ; more work is required on optimization strategies in the fpd systems .", "table 1 shows the dap values and fluoroscopic time duration for ii and fpd systems from the referral center where the study was conducted .", "the dap values for ii - based systems represented in table 1 is from the use of optimized protocol as reported in the previous published article from the same referral center where the study was conducted . prior to optimization in ii systems ,", "the doses were above 50% ; however , it was possible to optimize dose by halving the entrance dose ratesby selecting 0.4 mm cu filtration ( generally recruited in pediatric protocols ) during fluoroscopy .", "selection of 0.4 mm cu filtration did not suffer significant loss of image quality ; however , tube potentials were increased from 80 kvp to 103 kvp during fluoroscopy . in the fpd system ,", "tube potentials ranged from 90 kvp110 kvp when 0.4 mm cu filtration was selected with low dose settings during fluoroscopy . having adhered to the same optimization strategies in both systems , doses were similar owing to the fact that further optimization is warranted in fpd system .", "reported dap values of 31 and 33 gycm from cag with the use of ii and fpd system , respectively .", "they have also reported that the total dap from fluoroscopy and cine for ii and fpd are not significantly different and the image quality from fpd is better than ii in cine mode with no difference in the imaging performance in fluoroscopy mode .", "fluoroscopic time and dose area product values from ii and flat panel detector ( fpd)-based systems from cardiological interventions tables 2 and 3 shows dap values for cag and ptca procedures from various studies in literature .", "the arithmetic mean dap and fluoroscopic time duration using ii system as reported in literature was 39 gycm for 6.6 min and 61.2 gycm for 17 min for cag and ptca respectively [ table 2 ] . with the use of fpd ,", "mean dap and fluoroscopic time duration were 28.4 gycm for 7 min and 61 gycm for 18.05 min for cag and ptca , respectively [ table 3 ] . from tables 2 and 3 , radiation doses from fpd", "were significantly low for cag but were similar for ptca when compared to ii systems .", "it should be noted that time duration for cag and ptca was not available for some reported studies in tables 2 and 3", ". wide variation of doses is observed from these studies which may be attributed to the angiographic system used , time duration of the procedure and work environment .", "doses of the order of 492 gycm were recorded in ptca procedure from fpd system which was higher than the ii systems .", "chida et al . , conducted studies from various ii and fpd system and the average entrance doses of cine angiography and fluoroscopy in fpd systems were not significantly different .", "though fpds possess good detective quantum efficiency , they did not inherently reduce the radiation dose .", "jensen et al . , observed that patient doses from fpd were lower than ii systems ; however , the eye lens doses for radiologists were higher in fpd than in ii due to the use of high filtration and recruitment of high tube potentials . in our study ,", "high tube potentials were recruited when low dose settings involving high filtration were selected , which may have the potential to increase staff doses .", "radiation doses from cardiological interventions performed using image intensifier - based systems radiation doses from cardiological interventions performed using flat panel detector - based systems it is prudent to adopt stringent optimization measures in fpd as the dose may be higher than the ii systems as reported by prieto et al . and", "dose reduction is possible in either ii or fpd systems . during the initial stages of our study ,", "the doses from fpd were similar to the ii system though high filtration was used .", "reports that it is possible to achieve mean dap of 6.2 and 10.4 gycm for cag and ptca procedures performed using ii system .", "they further report that the reduction of doses was by influencing the quality of fellowship training , consistent restriction to mean values of 171 frames per cag , 165 frames per ptca , low - level fluoroscopy , training in the use of fluoroscopy - free blind positioning to the region of interest , restrictions to achieve lower ii entrance dose for adequate image quality . from tables 1 and 3 ,", "the mean dap for cag from fpd were higher than those reported by kuon et al .", "tsapaki et al . , reported the doses from fpd were increased by 35% compared to ii when fluoroscopy levels were changed from low to high mode ; they also recorded a minimum dap value of 6.1 and 14.3 gycm for cag and ptca , respectively , with the use of low dose fluoroscopy settings in fpd .", "dekker et al . , reported that the new generation fpds incorporated with good image processing and noise reduction techniques resulted in reducing patient doses by 43% without compromising image quality and staff doses by 50% during electrophysiological interventions .", "though fpd has reduced entrance dose rates , it does not automatically reduce radiation doses in clinical practice .", "further work is necessary to study the possibilities of dose reduction in fpd so as to be implemented in the clinical set up .", "the patient dose and image quality in any newer modality needs to be permanently monitored and transition from ii to fpd requires careful attention ."], ["this is a preliminary study as the institution where the study was conducted recently moved from ii to fpd - based systems .", "though radiation doses for cardiological interventions from fpd were similar to the ii - based system achieved after optimization , the advantages of fpd in terms of good image uniformity , improved patient imaging accessibility due to smaller size , absence of geometric distortion / veiling glare or vignetting make the fpd superior to the ii systems . to achieve improved patient dose reduction , it is advisable to strictly adhere to low dose protocols with high filtration in fpd systems .", "in addition , more attention for staff doses is warranted especially for interventionalists when this stringent patient dose reduction is employed .", "it is recommended to follow stringent dose reduction strategies right from the period of initial installation when there is a transition from ii to fpd systems .", "further studies are required to develop dose optimization in fpd , though use of high filtration is already in place ."]]}
{"article_id": "PMC2756085", "article_text": ["poly(vinyl alcohol ) ( pva ) is a biocompatible polymer with a great variety of biomedical and pharmaceutical applications .", "pva in aqueous solution is able to form chemical or physical gels under a variety of conditions .", "the physical gelation capacity of pva solutions has been well known for a long time ; however , the preparation of hydrogels by repeated freezingthawing cycles has attracted renewed interest .", "the high degree of swelling in water , the rubbery elasticity , the chemical and mechanical stability , the porous fibrilar network structure , and the lack of toxicity makes freezethaw pva hydrogels an attractive polymer matrix for biotechnological applications . to understand the origin of these properties and therefore the bioapplicability of this material ,", "it is necessary to study the physical gelation process produced by the freezingthawing cycles as well as the formed network structure .", "komatsu el al.(13 ) investigated the phase diagram of the water / pva binary system . according to this diagram", ", gelation occurs with or without spinodal decomposition according to the polymer concentration and temperature .", "the process seems to start with a clustering of chains , which is primarily caused by the association of polar groups of the dissolved polymer , followed by polymer crystallization .", "this means that the physical cross - links , which are responsible for the network formation , could be formed by different processes , such as hydrogen bonding , polymer crystallization , and in some cases ( depending on the gelation conditions ) , phase separation.(14 ) in addition , the application of freezingthawing cycles to pva solutions leads to the formation of heterogeneous networks with different morphology and properties as compared with nonfrozen gel systems .", "were the pioneers in the development of this type of polymer cryogels.(7 ) in earlier work , they attribute the gel formation to a partial crystallization of chain segments to microcrystalline structures .", "however , the intensity of the diffraction maximum that corresponds to the 101 reflection is clear for only pva hydrogels with high polymer content .", "for hydrogels with 10 to 15% polymer , solid - state nmr was used to identify and quantify the crystalline phase , although x - ray analysis is also possible.(29 ) most recent works show that gelation by freezethaw processing forms heterogeneous networks of interconnected micro- and macropores .", "willcox et al.,(28 ) in an exhaustive study using transmission electron microscopy ( tem ) images and nmr , showed the formation of networks with rounded pore morphology , fibrillar network morphology , or both depending on the number of cycles as well as aging .", "they attribute the formation of cross - links to a kinetically frustrated crystallization in the first freezing cycle . after this process ,", "subsequent cycles ( or aging processes ) lead to the creation of new ( secondary ) crystallites and the growing of the primary crystals ; however , the mesh spacing slightly changes .", "this fact seems to indicate that the average distance between the primary crystallites that are formed during the first thermal treatment constitutes the main controlling factor for the network structure . in addition", ", auriemma and coworkers in a series of works showed that the porous structure originates from freezing in the first step because of the incomplete crystallization of water .", "the polymer concentration in this unfrozen water phase is higher than that in the original solution , and the polymer network is formed within this microphase because of pva crystallization .", "the cross - link points are therefore constituted of polymer crystallites , and the formed gels could be described by a bicontinuous structure of polymer - rich and polymer - poor regions .", "on the basis of dsc measurements , also inferred the possibility of the existence of covalent cross - links between the polymer chains brought about by the formation of radicals due to strong local shear during water crystal growth.(33 ) in summary , the pva hydrogels obtained by freezingthawing cycles appear to be constituted by a very complicated network structure that is based on different phenomena ( i.e. , crystallization , hydrogen bonding , liquidliquid phase separation , and covalent bonds ) .", "therefore , a complete analysis of this structure requires the use of different characterization techniques .", "however , most of them require the manipulation of the hydrogel sample , provoking changes in the original structure .", "for example , the use of chromatographic techniques or scattering techniques to measure molecular weight or cluster size requires the dilution of the gel(24 ) ( although it was shown that it is possible ) .", "mechanical measurements have the limitation of water evaporation at higher temperatures.(9 ) the cryo - tem technique has the inconvenience of handling difficulties to avoid artifacts.(28 ) for the study of the crystallite content , very useful techniques such as differential scanning calorimetry ( dsc ) as well as x - ray diffraction are challenged by a sensitivity problem in dilute samples ; for this reason , measurements were sometimes made in dry samples.(34 ) in this vein , solid - state nmr is a powerful tool for studying polymer networks , but it has not been extensively used in this field .", "the focus of this work is the use of a low - field nmr spectrometer to analyze the structure of the pva gel in detail as a function of the freezingthawing cycles , checking all of the structural models suggested in the literature . in addition , a melting process that transforms these physical gels into isotropic solutions of pva in water was also studied .", "importantly , we focus on detecting protons so that sensitivity , even at the low concentrations under study , is not a serious issue .", "the use of an inexpensive low - field nmr spectrometer on as - prepared samples to study this complex matrix has the advantage that several of the cited inconveniences in sample characterization are avoided .", "by using different nmr methodologies , we are able to carry out a complete and quantitative study of the complex structure exhibited by the pva gels : multiple - quantum ( mq ) nmr allows us to gain direct access to residual dipolar coupling constants that persist because of the existence of cross - links and other topological constrains . therefore", ", this experimental procedure is a powerful tool for quantifying the gelation process(41 ) and gives us quantitative detail on not only the microstructure(42 ) but also the dynamics(43 ) of the gel , that is , a complete picture of the network structure evolution depending on , for example , the number of freezingthawing cycles .", "in addition , we used pulsed mixed magic - sandwich echo ( mse ) experiments that provide a near - quantitative refocusing of the rigid contribution to the initial part of the free induction decay ( fid)(44 ) and allow for an essentially quantitative determination of the crystallinity . to our knowledge , this is the first time that advanced nmr pulse sequences performed on a low - field spectrometer were applied to the study of the network structure and phase composition of physical hydrogels .", "the sample was > 99% hydrolyzed , had a weight - average molecular weight of 94  000 g / mol , and had syndio- , hetero- , and isotacticities of 17.2 , 54.1 , and 28.7% , respectively .", "pva was dissolved in deuterated water ( with polymer concentrations of 10 and 20% w / w ) at 80 c under continuous stirring to avoid inhomogeneities and local gelation .", "solutions were stored at 80 c overnight ; then , they were cooled to room temperature for 1 h. then , pva solutions were introduced to 10 mm od nmr tubes and were flame sealed to avoid variations in the water content .", "two technical considerations are important at this point : ( i ) the quantity of sample inside the tube has to be 8 mm in height and located in the center of the radio frequency ( rf ) coil to ensure good rf field ( b1 ) homogeneity .", "( ii ) the length of the nmr tube should be as short as possible for good control of the temperature .", "this is important because in this way , the whole tube can be inserted in the probe head , thus avoiding possible temperature gradients due to heat conduction .", "pva hydrogels were obtained by repetition of freezingthawing cycles of solutions inside the sealed tubes that consisted of 1 h of cooling to 32 c and 1 h of thawing at room temperature . after this process", ", samples were immediately inserted in the nmr spectrometer and measured to avoid the effect of aging . in some cases ( specified below ) , the first freezing cycle was extended to 12 h ( overnight ) , and subsequent cycles were 3 h long .", "solid - state h nmr spectroscopy is a powerful and widely used experimental technique in the field of polymer science.(35 ) in recent years , the use of inexpensive and easy - to - use low - field spectrometers has gained the attraction of scientists because it certainly produces quantitative results on polymer structure and dynamics , albeit , of course , without chemical resolution .", "on the basis of different experiments , which will be extensively described in the next sections , applications include quantitative studies on polymer network structure(38 ) and polymer crystallization,(44 ) that is , the main topics of this study . in this work , experiments were carried out on a bruker minispec mq20 spectrometer operating at 0.5 t with 90 pulses of 1.7 s length and a dead time of 12 s .", "mq spectroscopy is one of the most versatile and robust quantitative techniques for investigating not only the structure but also the dynamics of polymer networks.(38 ) in solution , the fast segmental motion of the polymer chains through the different accessible conformations is isotropic and completely averages the dipolar coupling interaction that is typical for solid - state spectra .", "however , constraints , irrespective of their nature ( e.g. , cross - links , entanglements , or chain packing ) , lead to nonisotropic segmental fluctuations and therefore to the persistence of a weak residual dipolar coupling ( dres ) , which is our central nmr observable . in other words ,", "the measured effect relies on the orientation dependence of the fluctuating dipolar coupling tensor with respect to the magnetic field , which can be described by an orientation autocorrelation function of the individual chain segments .", "fast segmental dynamics ( in the range of nanoseconds to microseconds ) lead to a loss of correlation to a plateau value that is related to the existence of preferential local orientation generated by the existence of cross - links .", "in fact , the measurable dres is directly proportional to a local dynamic order parameter of the polymer backbone,(38 ) which is defined as a time average over the fluctuations of the segment - fixed dipolar tensor over the time until the plateau region is reached .", "it connects the experimental observable with the network parameters : \n  here dstat is the segmental averaged dipolar coupling constant in the static limit and k is a rescaling factor that takes into account the fast dynamics ( in the range of picoseconds ) inside the statistical ( kuhn ) segments .", "the nmr observable is related to r , that is , the ratio of the end - to - end vector to its average unperturbed melt state ( r = r/r0 ) , and to n , the number of statistical segments between constraints .", "a variety of nmr experiments was used to detect residual dipolar couplings to monitor gelation processes , and recently , h mq nmr has evolved as the most powerful tool for obtaining a direct measurement of dres and even its distribution .", "details of the pulse sequence as well as the data analysis are already published elsewhere . here", "temperature usually plays an important role in mq measurements(43 ) because as previously mentioned , the order parameter should be obtained as an average of the segmental fluctuations over all of the possible conformations on the experimental time scale ( less than microseconds ) .", "therefore , the temperature is required to be far above the polymer glass - transition temperature tg to ensure that the segmental dynamics is fast enough to sweep out the whole conformational space between topological constraints and to achieve full averaging .", "if this is not the case , then the obtained dres ( and therefore the number of junctions in the network ) will be overestimated.(41 ) in this work , the studied samples are hydrogels ; therefore , the dissolved state of the polymer chain ensures that the segmental dynamics is indeed fast enough to detect the order parameter in the plateau regime , even at room temperature .", "similar to the traditional transverse relaxation experiments , the analyzed result of the mq experiment is a time - dependent but normalized double - quantum ( dq ) signal function , indq , that has the advantage of being independent of any temperature - dependent true relaxation ( decay ) effect .", "it is dominated by the dipolar interactions and independent of the polymer dynamics . for this purpose , the directly measured experimental dq build - up", "curve ( idq ) must be relaxation - corrected by the use of the also measured reference intensity ( iref ) .", "initially , iref contains a signal from not only the dipolar - coupled network chains but also the uncoupled , isotropically mobile components such as sol , dangling chains , and so on .", "the total mq magnetization ( imq ) needed for normalization is the sum of idq and iref but only after subtracting the non - network contributions .", "this fraction has a slower relaxation and can be easily identified ( figure 1a ) . in the studied hydrogels ,", "even though we used deuterated water to dissolve the polymer , we found a non - negligible water signal resulting from actual residual water and exchanged oh protons .", "therefore , we had to identify and subtract two exponential long - time contributions to imq ( i.e. , the noncoupled fraction of polymer chains ( b fraction in eq 2 ) and the solvent signal ( fraction c ) ) , which relax with longer relaxation times ( t2c *  t2b * ) \n  mq nmr build - up and decay data for 10% pva sample after one freezingthawing cycle .", "( a ) as - acquired idq and imq and ( b ) imq ( after extraction of noncoupled contributions ) and normalized dq build - up curves ( idq ) . the solid line in a represents the exponential fit for water subtraction , and the dashed line is the exponential fit representing the subtracted noncoupled polymeric defects . in b ,", "solid and dashed lines represent the fits assuming a gamma distribution of dres and a single residual dipolar constant , respectively .", "( see figure 1b . ) \n  finally , it is important to point out that in rubber networks the distribution effects of dres ( related to different end - to - end separations and polydispersities of network chains ) usually do not play any role.(56 ) therefore , the normalized dq build - up curve of a bulk rubber can be analyzed in the quasi - static limit in terms of a single dres(38 ) however , the complex and heterogeneous pva network structure , in addition to specific effects related to the swollen state of the sample ( which is proven to be heterogeneous in polymer networks(57 ) ) , leads to large deviations from the inverted gaussian shape in the studied pva gels . the actual coupling distribution ( related to a microstructurally heterogeneous distribution of constraints )", "the insert in figure 1b shows the broad distribution of dipolar interactions in pva networks .", "it resembles a gamma distribution and is consistent with observations of very heterogeneous network structures proposed in the literature .", "all analyzed samples showed a similar distribution shape independently of the number of freezingthawing cycles . because regularization analysis is subject to some limitations(42 )", "( further work about this point is in progress ) , we have analyzed all build - up curves under the assumption of the gamma distribution of dipolar couplings that is predicted by the gaussian distribution of end - to - end separations ( but which is screened in bulk elastomers ) upon fitting , the integral is numerically evaluated within the curve fitting environment of the origin software . in using this distribution , we do not want to imply that this distribution is of any significance in the studied structure ; the resulting build - up curves merely describe all of our experimental build - up data very well and have the benefit of not introducing an additional fit parameter , yet they provide a well - defined average residual coupling davg .", "( the width of the gamma distribution is directly related to the average . ) finally , note that the mq analysis pertains to the mobile fraction of the cryogels only ; this fraction always constitutes the major part of the sample ( > 80% ) ; the rest is rigid crystallites , and the signal of which rapidly relaxes and is not observable under the conditions of the mq experiment . according to previous papers , polymer crystallites act as junctions in pva networks obtained by freezingthawing cycles .", "solid - state nmr is a useful method for characterizing polymer crystallinity or phase composition in this type of system .", "the main concept is that the crystalline signal usually decays in the first 20 s of the free - induction decay ; therefore , the signal detected after the dead time ( 12 s in our case ) partially conceals this information .", "nevertheless , in some works , the fraction of protons in a glassy state in pva gels was estimated by monitoring the fid intensity during the first 20 s , which renders these studies qualitative .", "perhaps the most important is the use of solid echoes with different echo delays combined with back extrapolation(64 ) to correct for its inability to refocus the dipolar interactions in a multiple - spin system fully .", "certainly , the best approach is the use of a spectrometer with short ( 1 to 2 s ) dead time.(65 ) the use of an mse was recently proposed(44 ) as a more effective method for refocusing the multiple dipolar interactions that lead to the fast decay of the initial part of the fid , therefore removing the dead - time problem and obtaining a near - quantitative rigid fraction determination in polymer systems . under the same experimental conditions , differences of 40% in the quantity of rigid phase in pva compounds", "figure 2 shows the mse - fids for different total echo duration ( realized by increasing the interpulse spacings ) .", "the signal decay data are well represented by a gaussian function ( parabolic initial decay ) , and its extrapolation to t = 0 allows us to conclude that the signal loss is negligible .", "( it could be estimated to be 2% on the absolute scale , which corresponds to 10% on the relative scale . )", "parabolic ( gaussian ) extrapolation of the maximum mse - refocused fid intensities to t = 0 .", "it indicates only minor losses of rigid signal at the shortest echo time ( 10% on a scale relative to the total crystallinity ) .", "the inserted graph shows mse - refocused fids of 10% pva gel after six cycles measured at different echo times at room temperature ( 304 k ) .", "however , note that even for the mse the refocusing efficiency breaks when there is molecular motion on its time scale . in particular , this may be the case if the observed rigid component is glassy rather than crystalline and close to tg . under any condition irrespective of temperature , however , the typical decay time upon incrementing the echo was never significantly shorter than the ( relatively weak ) decay seen in figure 2 , which is ultimately associated with the breakdown of the pulse sequence efficiency because of higher - order imperfections .", "we thus conclude that all observed rigid signals are associated with crystal - like domains ; otherwise , a significant signal loss upon going through tg would have been observed .", "therefore , mse - fid curves were used to quantify the fraction of fast - decaying rigid polymer present in pva gels .", "the first fast decay ( caused by the rigid polymer fraction ) in the mse - fids ( insert in figure 2 ) has a quasi - gaussian decay , whereas the slower signal decay could be fitted with an exponential function .", "therefore , the first 140 s of the normalized mse - fid curves ( unity signal for zero time ) were fit according to eq 6 in this expression , a is the fraction of detectable rigid phase in the sample and b is an adjustable parameter for a better fit of the fast decay shape . in all of the cases ,", "b  2 ( i.e. , the crystalline fraction is described by a gaussian function ) .", "in addition , to reference the hard polymer phase to the total amount of polymer ( instead of the sample fraction ) , we corrected the a parameter according to the actual fraction of polymer in the sample obtained from saturation recovery experiments ( note again that the samples contain residual water ) on the basis of the fact that water has a much longer t1 relaxation time . from such experiments , and assuming no significant noe transfer between the species on the same time scale , the water content can be obtained from a biexponential fit \n\n where d is the water fraction in the sample and t1w and t1p are the longitudinal relaxation time constant of water and polymer , respectively ( with t1w  t1p ) .", "for example , at t = 304 k , the relaxation times were t1w = 1520 ms and t1p = 60 ms for a network with 10% pva after seven freezingthawing cycles .", "the fast - decaying rigid crystallite signal was not detected in these experiments ( the fid was evaluated at a time beyond 50 s ) such that d is determined relative to the mobile polymer fraction ( i.e. , ( 1  a ) in eq 6 ) .", "finally , the rigid polymer fraction discussed below was obtained according to \n\n obviously , for these corrections , it is essential to use a recycle delay that is long enough to observe the full equilibrium magnetization of the sample ( 5(t1w ) was used as a recycle delay in all experiments , i.e. , 720 s depending on the temperature ) .", "the aim of this work is to study the structure and phase composition of pva cryo - gels by using a low - field nmr spectrometer . to revise the structural models suggested in the literature", ", we will separately analyze the effects of three factors on the pva network structure : ( i ) the number of applied freezingthawing cycles , ( ii ) the temperature and melting process , and ( iii ) the aging process . to achieve this objective , in every point ,", "dq experiments were performed to quantify the proportion of noncoupled network defects , and the molecular weight between cross - links and mse - fid pulse sequence were applied to determine the rigid fraction of the polymer .", "the starting point of our study is the characterization of two isotropic solutions of 10 and 20% ( w / v ) of pva in water", ". the 10% pva solution does not give any detectable dq signal , but for the decay ( iref ) data , two different processes can be identified ( i.e. , the decay of the polymer magnetization and the water relaxation ) .", "this result demonstrates that under these preparation and measurement conditions , the dilute solution can be considered to be nonentangled and above the gelation threshold .", "this is not a trivial result because dq experiments are , in principle , sensitive to transient links ( entanglements or hydrogen bonds ) , and this indeed appears indicated by the 20% pva solution ; under the same preparation conditions , dq experiments did yield a detectable build - up curve . however , this is in our case due to a nondissolved or partially gelled pva subphase . to avoid this problem ,", "20% pva solutions were prepared under more vigorous stirring as well as higher temperatures up to 90 c . in this way , a totally isotropic solution of polymer was obtained according the dq experiments .", "dq experiments were performed on pva samples subject to different numbers of freezingthawing cycles to determine the evolution of noncoupled network defects as well as the network mesh size . in figure 3", ", we can see that after the first freezingthawing cycle , 50% of the polymer chains are coupled and are therefore part of the network , whereas the rest are not linked to it or at least belong to elastically inactive dangling chains or loops .", "the number of pva chains incorporated in the gel network increases with the successive cycles until it reaches a plateau after the sixth cycle , where the network has the minimum defect content .", "this important result shows that independent of the number of freezingthawing cycles applied to a pva solution 25% of the polymer is not coupled and therefore should not be elastically active .", "this is a very significant observation that is usually not taken into consideration when mechanical , dynamic - mechanical , or rheological properties of this material are analyzed .", "urushizaki et al.(66 ) have related the viscoelastic properties of this type of gel to the nonincorporated pva chains , and they have estimated their amount from the difference in weight between the polymer gel before and after immersion in distilled water .", "they found that 10% of the polymer was not incorporated after the first freezingthawing cycle .", "similar results were also indirectly inferred from the measure of intradiffusion coefficients of micellar aggregates of surfactant molecules dissolved in these types of gels.(67 ) obviously , our experimental values are much larger because we detect not only the non - cross - linked chains but also the dangling chains and other nonelastic network defects ( e.g. , loops ) .", "the effect of d2o in measured noncoupled network defects is another important point to take into consideration when this fraction of polymer is analyzed .", "pva monomers have an exchangeable proton ( oh ) , which , in presence of d2o , could be replaced by deuterium that is undetectable by h nmr , whereas the proton will become detectable as water ( hdo ) .", "free water signal ( h2o or hdo ) is subtracted from the long - time decay of the signal because it can be identified by its own longer t2 .", "however , the number of noncoupled network defects could be slightly overestimated because of the protondeuterium exchange .", "variation of noncoupled network defects ( i.e. , non - cross - linked polymer chains , dangling chains , and loops ) as a function of the number of freezingthawing cycles for the studied pva solutions .", "in addition , the decrease in the number of defects is correlated with an increase in dres that is proportional to the network chain order parameter ( eq 1 ) , see figure 4 .", "the formation of cross - links ( independent of their nature ) renders the polymer segmental motion nonisotropic , whereby the chain segments become ordered with respect to the end - to - end distance , and residual dipolar couplings arise .", "according to this basic principle , the increase in dres is directly related to the ( inverse ) length of the chains between the constraints .", "similar to the observation for the noncoupled network defects , the network chain length appears to approach a plateau after cycle number six , yet the tendency is not as clear as the former result . even after 16 cycles , the cross - link density still appears to increase .", "variation of the average residual dipolar couplings ( directly related to 1/n and therefore the cross - link density or the inverse mesh size ) extracted from dq experiments as a function of the number of freezingthawing cycles for the studied pva solutions .", " represents a sample containing 20% pva after 3 h of freezing , and  is from the same sample after 16 h of freezing . with this observation ,", "it is important to note that samples with different polymer concentration appear to exhibit the same behavior during gelation .", "a possible explanation could be that in the framework of a heterogeneous scenario cross - linking occurs via partial crystallization in the nonfrozen parts of the sample ; it simply means that in less concentrated samples more water freezes .", "this fact could explain the dependence of the dimension and shape of pores on polymer concentration and the regimes of cryogenic treatment.(20 ) in this context , it appears to be worthwhile to study the influence of the actual freezing temperature , which should affect the polymer concentration under the conditions at which the crystallites are formed .", "however , the freezing time ( especially in the first cycle when the network is formed ) seems to have some influence in the gel network structure . as is shown in figure 4 ,", "the apparent dipolar coupling constant is very low ( 48 hz ) after 1 h of freezing at 32 c . if the freezing period is increased to 3 h , then the cross - link density of the gel almost doubles ( 82 hz ) , whereas longer freezing ( e.g. , 16 h ) does not lead to further variation", "this may indicate that 1 h of freezing is not long enough to complete the process , and for this reason , further analyses are based on samples with a first freezing cycle of 12 h and subsequent cycles of 3 h each .", "returning to the origin of the pva networks produced via freezingthawing cycles from homogeneous polymer solutions in water , it appears to be well demonstrated by now that during the first freezing cycle , freezing of some water(32 ) increases the concentration of pva in the still unfrozen phase.(31 ) crystallization of pva takes place in this concentrated microphase,(68 ) but it is kinetically frustrated by the gelation of the polymer solution .", "this fact could explain the small size of these primary crystallites ( 5 nm ) , which then act as physical junctions between the amorphous and mobile polymer chains .", "results extracted from the mse - fid curves of the pva gels after the first freezingthawing cycle ( figure 5 ) indicate that 8% of the polymer behaves like a rigid solid .", "this rigid fraction increases with the number of cycles until it reaches a plateau close to cycle number six , just as our other reported network properties .", "the maximum amount of rigid phase estimated by our nmr methodology ( 20% of the total polymer ) is substantially higher than the crystallinity of similar samples deduced by dsc as well as some other h nmr methods .", "variation of the rigid ( crystalline ) polymer fraction extracted from mse - fid curves as a function of the number of freezingthawing cycles for the studied pva solutions . in all cases , the crystallinity of pva cryo - gels obtained in dsc studies is estimated to be 5% ( not taking into account studies of dry networks(34 ) because the structure should be different ) . however , as was pointed out by willcox et al.,(28 ) this magnitude could very well be underestimated because the heat of fusion of the crystals in gels may be substantially lower than the assumed pva bulk value because of their small size and large surface effects ( hydrogen bonds to surrounding water , etc . ) .", "in addition , another important point is the difficulty in measuring small quantities of crystal phase in dilute gels via calorimetric methods in general ; for example , a crystallinity of 8% in a gel with a pva concentration of 20% represents only a sample fraction of 0.016 .", "nevertheless , this difficulty seems to be reduced by the use of microcalorimetry . in this way ,", "similar results were obtained by performing this type of measurement on gels with low pva concentrations,(8 ) although the uncertainties related to the obtained results are still large . for these reasons ,", "the use of solid - state nmr seems to be a better methodology for quantifying the crystallinity in dilute pva gels . however , there are still some discrepancies in the total amount of rigid phase measured by the use of different nmr methodologies .", "ricciardi et al.(18 ) estimated the crystallinity of pva gels after 11 freezingthawing cycles to be 8% by analyzing the first microsecond of the h fid .", "clearly , as was pointed out in the , this experimental procedure falls victim to the dead - time problem .", "depending on the spectrometer , the amount of rigid signal lost during this time could be more or less significant .", "( ref ( 15 ) does not specify the dead time ; therefore , it is not possible to estimate the related error . ) for example , our low - field spectrometer has a dead time of 12 s ; therefore , the fid analysis would lead to an underestimation of the rigid polymer fraction by 50% . by using different c nmr experiments ( direct polarization as well as hc cross polarization ) , willcox et al.(28 )", "established that after the first freezingthawing cycle , pva gel contains a rigid polymer phase of 5  2% , increasing up to 12  4% after cycle number 12 .", "as was pointed out by the authors,(28 ) the relative fraction comparing different samples could be estimated with a high accuracy , but the used methodology ( in particular , the use of the intrinsically nonquantitative cross polarization ) does not give a reliable absolute crystallinity . in conclusion , the key to obtaining the most accurate crystal fraction in these dilute polymer samples is to use a solid - state nmr method that allows us to detect the rigid part fully by refocusing the multispin dipolar interactions with the mse , thus solving the dead - time problem and minimizing the signal loss .", "of course , detecting the more - sensitive and 100% abundant protons instead of the naturally abundant c is an additional advantage , which more than compensates for the use of an intrinsically less - sensitive low - field spectrometer .", "summarizing all results discussed so far , the network structure of pva cryo - gels is very apparently based on the formation of rigid polymer areas ( most probably polymer crystallites ) during the cooling process .", "after the first cycle , almost half ( 8% ) of the final amount ( 20% ) of the rigid phase is already formed . in this stage ,", "subsequent freezingthawing cycles increase the rigid polymer fraction , which has two consequences : first , the noncoupled polymer fraction is further reduced ( i.e. , more pva segments become elastically active ) , and second , the network mesh size is further reduced but only slightly during further freezingthawing cycles .", "therefore , we believe that subsequent freezingthawing cycles not only increase the size of the crystals formed during the first freezing process but also lead to the creation of other more imperfect crystals .", "independent of the polymer concentration of the gel , the melting of smaller and more imperfect secondary crystals takes place at lower temperature and over a large range , leading to a continuous decrease in the detected rigid polymer phase with the temperature ( figure 6 ) . at 7580 c ( in good agreement with", "the main endotherm observed in dsc measurements ) , this quantity drops below the detection limit .", "this should be correlated with the melting of the main ( primary ) crystals ( estimated to 5% of the polymer fraction ) that support the network structure of the gel .", "these results are consistent with dsc measurements because they show an increase in the melting endotherm that corresponds to the initial crystals as well as to the appearance of a second endotherm at lower temperatures when the number of freezingthawing cycles was increased for a given pva solution .", "variation of the rigid polymer fraction ( extracted from mse - fid experiments ) as a function of temperature for pva gels formed after seven freezingthawing cycles . a network with 10% pva was first heated until 324 k (  ) and then kept at room temperature for 12 h before the second heating procedure was performed (  ) .", "a solution with 20% pva was submitted to two different treatments , that is , one freezingthawing cycle and seven freezingthawing cycles , respectively .", "all samples were frozen for 12 h in the first cycle and 3 h in the subsequent cycles .", "the effect of the number of freezingthawing cycles on the crystallinity is easily understandable by comparing the evolution of the rigid polymer fraction of a pva gel obtained after seven cycles with that of a pva gel after only one freezingthawing cycle ( figure 6 ) .", "the onset of melting after only one freezing cycle is estimated to be 50 c , leaving the rigid polymer fraction constant at lower temperatures . therefore , subsequent cycles seem to create more , yet imperfect , secondary crystallite structures . above 50 c ,", "all samples exhibit a similar behavior related to the partial melting of primary crystals or to the melting of less - perfect secondary structures .", "importantly , the temperature above which it is not possible to detect any more signal of rigid polymer remains invariant , yet the final fraction seems to increase slightly with the number of cycles .", "therefore , according to this result , freezingthawing cycles have a direct influence on not only the number of crystals formed during the first freezing process but also the creation of other more imperfect secondary crystallites . to ensure that the results shown in figure 6 are not artifacts produced by different magnetization relaxation at different temperatures that could possibly arise from regions that undergo a glass - to - liquid rather than a melting transition ,", "the evolution of the signal at any given temperature was studied as a function of echo delay , see figure 7 .", "at all of the measured temperatures , similar behavior was found , with a loss of intensity estimated to between 0.7 and 1.8% for the two extreme cases , which corresponds to a constant 10% loss on a relative scale , as is usually found .", "importantly , the drop in overall signal corresponds , within the error margins , to the expected drop due to the curie factor ( temperature - dependent spin polarization ) .", "in addition , a direct check is the control of the total intensity , comparing the pva gels to the corresponding pva solutions ( insert in figure 7 ) .", "it is seen that no significant signal is lost / undetectable ( we observe almost all protons in the sample ) , and from this , it is clear that the loss of intensity caused by the mse in pva gels is at a minimum , with no other ( hidden ) phenomena that could be related to an intermediately mobile ( almost glassy ) rigid fraction . given this experimental evidence , the decay of the rigid h fraction with temperature can be related to only a melting of pva crystallites .", "variation of the initial signal intensity of mse - fids with increasing echo delay at different temperatures for a pva gel containing 20% polymer after one freezingthawing cycle ( 12 h of freezing and 1 h thawing ) .", "the insert presents the mse - fid of the pva gel measured at 304 k with the minimum echo delay ( 0.0022 ms ) as well as the estimated intensity at zero echo time in comparison with the mse - fid of the isotropic solution of the same sample measured under the same conditions after complete melting at 90 c .", "obviously , the temperature - dependent variation of the rigid polymer phase has a direct effect on the network structure , as shown in figure 8 .", "the melting of secondary crystals leads to an almost exponential increase in the number of network defects ( obviously , in a reversible pathway , comparing the phenomenon with the effect of successive freezingthawing cycles ) .", "however , the network mesh size ( as measured by the residual dipolar coupling ) remains almost constant .", "this means that secondary crystallization is very important in the network organization , reducing the number of network defects and therefore increasing the number of polymer segments that are elastically active .", "however , the molecular weight between the constraints appears to be dictated mostly by the primary crystallites formed during the first freezing cycles .", "for this reason , the network is completely destroyed at temperatures of 80 c when the primary crystal phase is molten , leaving no observable dq signal ( i.e. , an isotropic pva solution is formed ) .", "on the side , we note that the differences in the davg values given in figures 4 and 8b , which indicate a 50% decreased mesh size for the samples studied in figure 8 , are related to the increase in the freezing time ( 12 h in the first step and 3 h in the successive cycles instead of 1 h ) used in each cycle , as pointed out above .", "variation of ( a ) the network defect fraction and ( b ) the inverse mesh size ( given by davg measured by mq nmr ) with temperature for the 20% pva gel prepared after seven freezingthawing cycles ( 12 h first freezing cycle , 3 h subsequent cycles ) .", "error bars represent the fitting uncertainty , whereas lines are only guides to the eye .", "these nmr results on the mesh size perfectly agree with the trends observed in studies of mechanical properties reported in the literature . clearly , the elastic behavior of pva gels depends on the number of freezingthawing cycles .", "therefore , the main factor that determines the improvement of the mechanical properties upon further cycles should be associated with the number of elastically active chains .", "the polymer concentration in the original aqueous solution has an important effect on the elastic properties ; that is , the elastic modulus increases .", "however , in the view of our data , this does not appear to have important consequences for the actual network structure ; that is , there are no large variations in the network mesh size .", "therefore , the reported variations are simply related to differences in polymer concentration . in addition ,", "different works demonstrate a slight decrease in the elastic modulus with the temperature , until a major drop occurs at 50 c.(65 ) this is obviously related to the partial melting of secondary and more imperfect crystallites that leads to an increasing amount of network defects ( elastically inactive chains ) . at temperatures close to 80 c ,", "the complete loss of crystallinity essentially liberates all chains , and the system drops below the gel point . in view of the important role of the substantial polymer amount that is not elastically active ( free and dangling chains , loops )", ", it may be worthwhile to reconsider detailed frequency - dependent mechanical measurements , where these components should contribute to an increased loss modulus over the frequency range covering their relaxation .", "the capacity of our investigated systems to be transformed from an isotropic aqueous solution to a physical cryo - gel by the application of freezingthawing cycles , including thermal reversibility back to an isotropic pva solution , is in clear contradiction with the assertion that covalent bonds could form between the polymer chains during gelation . if covalent cross - links were an important contribution to the final gel properties , then residual couplings should persist even above the melting temperature of the crystallites , which is not the case .", "however , the presence of ( rapidly tumbling ) microgels above the main melting point can not be ruled out and is in fact indicated by the relatively high viscosity ( as checked by a simple tilt test ) .", "however , we point out that even the pristine 20% pva solution exhibited some detectable dq signal , indicating extended gel - like structures , unless it was heated to > 90 c", ". therefore , an undetectably small fraction of crystallites ( or extended hydrogen - bonded aggregates ) may always be present , even at > 80 c , and hold the system close to the gel point , depending on the sample and its thermal history . from our perspective", ", permanent bonds can be ruled out as a major factor determining the cryo - gel structure .", "finally , we address aging phenomena that are a part of the data shown in figure 6 and were not commented on until now . after heating a sample containing 10% pva to 50 c and recording the loss of crystallinity , the sample was stored for 12 h , and another measurement was performed at room temperature .", "the amount of rigid polymer fraction after 12 h of storage was again considerably increased , as can be seen in figure 6 .", "yet , this new crystalline fraction melts at rather low temperatures , which again indicates the growth of secondary crystallites .", "similar prominent aging effects have been reported in the literature . for more clear evidence ,", "a sealed sample containing 20% pva was submitted to seven freezingthawing cycles ( with 12 h of freezing in the first cycle ) and was characterized after different storage times at room temperature . according to figure 9 , aging clearly increases the secondary population of imperfect crystals .", "it also increases the amount of elastically active polymer segments but has no significant effect on the network mesh size .", "therefore , aging effects on pva gel properties are again mainly related to an increasing fraction of polymer incorporated into the network structure and not to a decrease in mesh size .", "effect of aging on ( a ) the rigid polymer fraction , ( b ) the noncoupled defects fraction , and ( c ) the average residual dipolar coupling of a gel with 10% pva obtained after seven freezingthawing cycles ( 12 h of freezing for the first cycle and 3 h for the following cycles ) .", "this physical gel is supported by rigid polymer areas ( pva crystallites ) that act as junctions between the remaining mobile and entropically elastic polymer chains and thus determine the network mesh size . in our study ,", "the crystallites are quantitatively detected as a rigidlike fraction of quickly relaxing ( and fully refocused to overcome the dead - time problem ) magnetization , and the mesh size is deduced from mq experiments that measure residual dipolar couplings , the latter arising from constraints on the motion of the mobile chains ( i.e. , cross - links ) that render the segmental motion locally anisotropic .", "the most important crystal fraction forms in the first freezing cycle , and subsequent freezingthawing cycles produce only a slight increase in the initial crystal size as well as induce the formation of more imperfect , secondary crystallites .", "this secondary crystal fraction ( which constitutes around two - thirds of the total crystal phase in samples submitted to a large number of cycles , extended aging , or both ) plays an important role by increasing the number of elastically active polymer chains , but it does not appreciably affect the mesh size .", "pva cryo - hydrogels are shown to be totally thermoreversible ; that is , pva physical networks can be transformed into an isotropic aqueous solution by increasing the temperature . during this process ,", "the more imperfect crystals are molten first , and the number of network defects increases exponentially .", "in contrast , the network mesh size does not undergo significant variations until the primary crystal phase is molten at temperatures of 80 c .", "our experiments did not reveal any evidence of the possible formation of covalent cross - links between pva chains during gelation ; no residual couplings were detected after the melting of this primary crystal fraction .", "independent of the number of cycles , the aging , or the polymer concentration in the original pva solution , a considerable fraction of polymer ( at least 25% ) is not elastically active , which is a result that is not yet not taken into account in the large body of literature addressing the formation and the properties of pva cryo - hydrogels .", "this finding is central to the interpretation and understanding of the elastic properties of this useful material and must be taken into account in a reinterpretation of the origin of the changes of the elastic moduli of pva cryogels .", "finally , it is important that the in - depth study of both processes ( i.e. , the pva gelation by the application of freezingthawing cycles ) as well as the destruction of the network structure by heating were , for the first time , entirely based on the use of an inexpensive low - field solid - state nmr spectrometer .", "we show that the combination of different advanced nmr strategies , that is , mq spectroscopy and component analysis of mse - refocused free - induction decays , is vital for obtaining quantitative information on network structure and phase composition ( crystallinity ) , respectively , to arrive at conclusions that can be matched with information extracted from other diverse techniques .", "in addition , the use of low - field solid - state nmr spectroscopy not only is useful for extracting structural information but also could be a powerful tool for investigating the polymer dynamics of these complex systems ."], "abstract_text": ["<S> the network structure of poly(vinyl alcohol ) ( pva ) hydrogels obtained by freezingthawing cycles was investigated by solid - state 1h low - field nmr spectroscopy . by the application of multiple - quantum nmr experiments , we obtain information about the segmental order parameter , which is directly related to the restrictions on chain motion ( cross - links ) formed upon gelation . </S>", "<S> these measurements indicate that the network mesh size as well as the relative amount of nonelastic defects ( i.e. , non - cross - linked chains , dangling chains , loops ) decrease with the number of freezingthawing cycles but are independent of the polymer concentration . </S>", "<S> the formation of the pva network is accompanied by an increasing fraction of polymer with fast magnetization decay ( 20 s ) . </S>", "<S> the quantitative study of this rigid phase with a specific refocusing pulse sequence shows that it is composed of a primary crystalline polymer phase ( 5% ) , which constitutes the main support of the network structure and determines the mesh size , and a secondary population of more imperfect crystallites , which increase the number of elastic chain segments in the polymer gel but do not affect the average network mesh size appreciably . </S>", "<S> correspondingly , progressive melting of the secondary crystallites with increasing temperature does not affect the network mesh size but only the amount of network defects , and melting of the main pva crystallites at 80 c leads to the destruction of the network gel and the formation of an isotropic pva solution . </S>"], "labels": null, "section_names": ["Introduction", "Experimental Section", "Results and Discussion", "Conclusions"], "sections": [["poly(vinyl alcohol ) ( pva ) is a biocompatible polymer with a great variety of biomedical and pharmaceutical applications .", "pva in aqueous solution is able to form chemical or physical gels under a variety of conditions .", "the physical gelation capacity of pva solutions has been well known for a long time ; however , the preparation of hydrogels by repeated freezingthawing cycles has attracted renewed interest .", "the high degree of swelling in water , the rubbery elasticity , the chemical and mechanical stability , the porous fibrilar network structure , and the lack of toxicity makes freezethaw pva hydrogels an attractive polymer matrix for biotechnological applications . to understand the origin of these properties and therefore the bioapplicability of this material ,", "it is necessary to study the physical gelation process produced by the freezingthawing cycles as well as the formed network structure .", "komatsu el al.(13 ) investigated the phase diagram of the water / pva binary system . according to this diagram", ", gelation occurs with or without spinodal decomposition according to the polymer concentration and temperature .", "the process seems to start with a clustering of chains , which is primarily caused by the association of polar groups of the dissolved polymer , followed by polymer crystallization .", "this means that the physical cross - links , which are responsible for the network formation , could be formed by different processes , such as hydrogen bonding , polymer crystallization , and in some cases ( depending on the gelation conditions ) , phase separation.(14 ) in addition , the application of freezingthawing cycles to pva solutions leads to the formation of heterogeneous networks with different morphology and properties as compared with nonfrozen gel systems .", "were the pioneers in the development of this type of polymer cryogels.(7 ) in earlier work , they attribute the gel formation to a partial crystallization of chain segments to microcrystalline structures .", "however , the intensity of the diffraction maximum that corresponds to the 101 reflection is clear for only pva hydrogels with high polymer content .", "for hydrogels with 10 to 15% polymer , solid - state nmr was used to identify and quantify the crystalline phase , although x - ray analysis is also possible.(29 ) most recent works show that gelation by freezethaw processing forms heterogeneous networks of interconnected micro- and macropores .", "willcox et al.,(28 ) in an exhaustive study using transmission electron microscopy ( tem ) images and nmr , showed the formation of networks with rounded pore morphology , fibrillar network morphology , or both depending on the number of cycles as well as aging .", "they attribute the formation of cross - links to a kinetically frustrated crystallization in the first freezing cycle . after this process ,", "subsequent cycles ( or aging processes ) lead to the creation of new ( secondary ) crystallites and the growing of the primary crystals ; however , the mesh spacing slightly changes .", "this fact seems to indicate that the average distance between the primary crystallites that are formed during the first thermal treatment constitutes the main controlling factor for the network structure . in addition", ", auriemma and coworkers in a series of works showed that the porous structure originates from freezing in the first step because of the incomplete crystallization of water .", "the polymer concentration in this unfrozen water phase is higher than that in the original solution , and the polymer network is formed within this microphase because of pva crystallization .", "the cross - link points are therefore constituted of polymer crystallites , and the formed gels could be described by a bicontinuous structure of polymer - rich and polymer - poor regions .", "on the basis of dsc measurements , also inferred the possibility of the existence of covalent cross - links between the polymer chains brought about by the formation of radicals due to strong local shear during water crystal growth.(33 ) in summary , the pva hydrogels obtained by freezingthawing cycles appear to be constituted by a very complicated network structure that is based on different phenomena ( i.e. , crystallization , hydrogen bonding , liquidliquid phase separation , and covalent bonds ) .", "therefore , a complete analysis of this structure requires the use of different characterization techniques .", "however , most of them require the manipulation of the hydrogel sample , provoking changes in the original structure .", "for example , the use of chromatographic techniques or scattering techniques to measure molecular weight or cluster size requires the dilution of the gel(24 ) ( although it was shown that it is possible ) .", "mechanical measurements have the limitation of water evaporation at higher temperatures.(9 ) the cryo - tem technique has the inconvenience of handling difficulties to avoid artifacts.(28 ) for the study of the crystallite content , very useful techniques such as differential scanning calorimetry ( dsc ) as well as x - ray diffraction are challenged by a sensitivity problem in dilute samples ; for this reason , measurements were sometimes made in dry samples.(34 ) in this vein , solid - state nmr is a powerful tool for studying polymer networks , but it has not been extensively used in this field .", "the focus of this work is the use of a low - field nmr spectrometer to analyze the structure of the pva gel in detail as a function of the freezingthawing cycles , checking all of the structural models suggested in the literature . in addition , a melting process that transforms these physical gels into isotropic solutions of pva in water was also studied .", "importantly , we focus on detecting protons so that sensitivity , even at the low concentrations under study , is not a serious issue .", "the use of an inexpensive low - field nmr spectrometer on as - prepared samples to study this complex matrix has the advantage that several of the cited inconveniences in sample characterization are avoided .", "by using different nmr methodologies , we are able to carry out a complete and quantitative study of the complex structure exhibited by the pva gels : multiple - quantum ( mq ) nmr allows us to gain direct access to residual dipolar coupling constants that persist because of the existence of cross - links and other topological constrains . therefore", ", this experimental procedure is a powerful tool for quantifying the gelation process(41 ) and gives us quantitative detail on not only the microstructure(42 ) but also the dynamics(43 ) of the gel , that is , a complete picture of the network structure evolution depending on , for example , the number of freezingthawing cycles .", "in addition , we used pulsed mixed magic - sandwich echo ( mse ) experiments that provide a near - quantitative refocusing of the rigid contribution to the initial part of the free induction decay ( fid)(44 ) and allow for an essentially quantitative determination of the crystallinity . to our knowledge , this is the first time that advanced nmr pulse sequences performed on a low - field spectrometer were applied to the study of the network structure and phase composition of physical hydrogels ."], ["the sample was > 99% hydrolyzed , had a weight - average molecular weight of 94  000 g / mol , and had syndio- , hetero- , and isotacticities of 17.2 , 54.1 , and 28.7% , respectively .", "pva was dissolved in deuterated water ( with polymer concentrations of 10 and 20% w / w ) at 80 c under continuous stirring to avoid inhomogeneities and local gelation .", "solutions were stored at 80 c overnight ; then , they were cooled to room temperature for 1 h. then , pva solutions were introduced to 10 mm od nmr tubes and were flame sealed to avoid variations in the water content .", "two technical considerations are important at this point : ( i ) the quantity of sample inside the tube has to be 8 mm in height and located in the center of the radio frequency ( rf ) coil to ensure good rf field ( b1 ) homogeneity .", "( ii ) the length of the nmr tube should be as short as possible for good control of the temperature .", "this is important because in this way , the whole tube can be inserted in the probe head , thus avoiding possible temperature gradients due to heat conduction .", "pva hydrogels were obtained by repetition of freezingthawing cycles of solutions inside the sealed tubes that consisted of 1 h of cooling to 32 c and 1 h of thawing at room temperature . after this process", ", samples were immediately inserted in the nmr spectrometer and measured to avoid the effect of aging . in some cases ( specified below ) , the first freezing cycle was extended to 12 h ( overnight ) , and subsequent cycles were 3 h long .", "solid - state h nmr spectroscopy is a powerful and widely used experimental technique in the field of polymer science.(35 ) in recent years , the use of inexpensive and easy - to - use low - field spectrometers has gained the attraction of scientists because it certainly produces quantitative results on polymer structure and dynamics , albeit , of course , without chemical resolution .", "on the basis of different experiments , which will be extensively described in the next sections , applications include quantitative studies on polymer network structure(38 ) and polymer crystallization,(44 ) that is , the main topics of this study . in this work , experiments were carried out on a bruker minispec mq20 spectrometer operating at 0.5 t with 90 pulses of 1.7 s length and a dead time of 12 s .", "mq spectroscopy is one of the most versatile and robust quantitative techniques for investigating not only the structure but also the dynamics of polymer networks.(38 ) in solution , the fast segmental motion of the polymer chains through the different accessible conformations is isotropic and completely averages the dipolar coupling interaction that is typical for solid - state spectra .", "however , constraints , irrespective of their nature ( e.g. , cross - links , entanglements , or chain packing ) , lead to nonisotropic segmental fluctuations and therefore to the persistence of a weak residual dipolar coupling ( dres ) , which is our central nmr observable . in other words ,", "the measured effect relies on the orientation dependence of the fluctuating dipolar coupling tensor with respect to the magnetic field , which can be described by an orientation autocorrelation function of the individual chain segments .", "fast segmental dynamics ( in the range of nanoseconds to microseconds ) lead to a loss of correlation to a plateau value that is related to the existence of preferential local orientation generated by the existence of cross - links .", "in fact , the measurable dres is directly proportional to a local dynamic order parameter of the polymer backbone,(38 ) which is defined as a time average over the fluctuations of the segment - fixed dipolar tensor over the time until the plateau region is reached .", "it connects the experimental observable with the network parameters : \n  here dstat is the segmental averaged dipolar coupling constant in the static limit and k is a rescaling factor that takes into account the fast dynamics ( in the range of picoseconds ) inside the statistical ( kuhn ) segments .", "the nmr observable is related to r , that is , the ratio of the end - to - end vector to its average unperturbed melt state ( r = r/r0 ) , and to n , the number of statistical segments between constraints .", "a variety of nmr experiments was used to detect residual dipolar couplings to monitor gelation processes , and recently , h mq nmr has evolved as the most powerful tool for obtaining a direct measurement of dres and even its distribution .", "details of the pulse sequence as well as the data analysis are already published elsewhere . here", "temperature usually plays an important role in mq measurements(43 ) because as previously mentioned , the order parameter should be obtained as an average of the segmental fluctuations over all of the possible conformations on the experimental time scale ( less than microseconds ) .", "therefore , the temperature is required to be far above the polymer glass - transition temperature tg to ensure that the segmental dynamics is fast enough to sweep out the whole conformational space between topological constraints and to achieve full averaging .", "if this is not the case , then the obtained dres ( and therefore the number of junctions in the network ) will be overestimated.(41 ) in this work , the studied samples are hydrogels ; therefore , the dissolved state of the polymer chain ensures that the segmental dynamics is indeed fast enough to detect the order parameter in the plateau regime , even at room temperature .", "similar to the traditional transverse relaxation experiments , the analyzed result of the mq experiment is a time - dependent but normalized double - quantum ( dq ) signal function , indq , that has the advantage of being independent of any temperature - dependent true relaxation ( decay ) effect .", "it is dominated by the dipolar interactions and independent of the polymer dynamics . for this purpose , the directly measured experimental dq build - up", "curve ( idq ) must be relaxation - corrected by the use of the also measured reference intensity ( iref ) .", "initially , iref contains a signal from not only the dipolar - coupled network chains but also the uncoupled , isotropically mobile components such as sol , dangling chains , and so on .", "the total mq magnetization ( imq ) needed for normalization is the sum of idq and iref but only after subtracting the non - network contributions .", "this fraction has a slower relaxation and can be easily identified ( figure 1a ) . in the studied hydrogels ,", "even though we used deuterated water to dissolve the polymer , we found a non - negligible water signal resulting from actual residual water and exchanged oh protons .", "therefore , we had to identify and subtract two exponential long - time contributions to imq ( i.e. , the noncoupled fraction of polymer chains ( b fraction in eq 2 ) and the solvent signal ( fraction c ) ) , which relax with longer relaxation times ( t2c *  t2b * ) \n  mq nmr build - up and decay data for 10% pva sample after one freezingthawing cycle .", "( a ) as - acquired idq and imq and ( b ) imq ( after extraction of noncoupled contributions ) and normalized dq build - up curves ( idq ) . the solid line in a represents the exponential fit for water subtraction , and the dashed line is the exponential fit representing the subtracted noncoupled polymeric defects . in b ,", "solid and dashed lines represent the fits assuming a gamma distribution of dres and a single residual dipolar constant , respectively .", "( see figure 1b . ) \n  finally , it is important to point out that in rubber networks the distribution effects of dres ( related to different end - to - end separations and polydispersities of network chains ) usually do not play any role.(56 ) therefore , the normalized dq build - up curve of a bulk rubber can be analyzed in the quasi - static limit in terms of a single dres(38 ) however , the complex and heterogeneous pva network structure , in addition to specific effects related to the swollen state of the sample ( which is proven to be heterogeneous in polymer networks(57 ) ) , leads to large deviations from the inverted gaussian shape in the studied pva gels . the actual coupling distribution ( related to a microstructurally heterogeneous distribution of constraints )", "the insert in figure 1b shows the broad distribution of dipolar interactions in pva networks .", "it resembles a gamma distribution and is consistent with observations of very heterogeneous network structures proposed in the literature .", "all analyzed samples showed a similar distribution shape independently of the number of freezingthawing cycles . because regularization analysis is subject to some limitations(42 )", "( further work about this point is in progress ) , we have analyzed all build - up curves under the assumption of the gamma distribution of dipolar couplings that is predicted by the gaussian distribution of end - to - end separations ( but which is screened in bulk elastomers ) upon fitting , the integral is numerically evaluated within the curve fitting environment of the origin software . in using this distribution , we do not want to imply that this distribution is of any significance in the studied structure ; the resulting build - up curves merely describe all of our experimental build - up data very well and have the benefit of not introducing an additional fit parameter , yet they provide a well - defined average residual coupling davg .", "( the width of the gamma distribution is directly related to the average . ) finally , note that the mq analysis pertains to the mobile fraction of the cryogels only ; this fraction always constitutes the major part of the sample ( > 80% ) ; the rest is rigid crystallites , and the signal of which rapidly relaxes and is not observable under the conditions of the mq experiment . according to previous papers , polymer crystallites act as junctions in pva networks obtained by freezingthawing cycles .", "solid - state nmr is a useful method for characterizing polymer crystallinity or phase composition in this type of system .", "the main concept is that the crystalline signal usually decays in the first 20 s of the free - induction decay ; therefore , the signal detected after the dead time ( 12 s in our case ) partially conceals this information .", "nevertheless , in some works , the fraction of protons in a glassy state in pva gels was estimated by monitoring the fid intensity during the first 20 s , which renders these studies qualitative .", "perhaps the most important is the use of solid echoes with different echo delays combined with back extrapolation(64 ) to correct for its inability to refocus the dipolar interactions in a multiple - spin system fully .", "certainly , the best approach is the use of a spectrometer with short ( 1 to 2 s ) dead time.(65 ) the use of an mse was recently proposed(44 ) as a more effective method for refocusing the multiple dipolar interactions that lead to the fast decay of the initial part of the fid , therefore removing the dead - time problem and obtaining a near - quantitative rigid fraction determination in polymer systems . under the same experimental conditions , differences of 40% in the quantity of rigid phase in pva compounds", "figure 2 shows the mse - fids for different total echo duration ( realized by increasing the interpulse spacings ) .", "the signal decay data are well represented by a gaussian function ( parabolic initial decay ) , and its extrapolation to t = 0 allows us to conclude that the signal loss is negligible .", "( it could be estimated to be 2% on the absolute scale , which corresponds to 10% on the relative scale . )", "parabolic ( gaussian ) extrapolation of the maximum mse - refocused fid intensities to t = 0 .", "it indicates only minor losses of rigid signal at the shortest echo time ( 10% on a scale relative to the total crystallinity ) .", "the inserted graph shows mse - refocused fids of 10% pva gel after six cycles measured at different echo times at room temperature ( 304 k ) .", "however , note that even for the mse the refocusing efficiency breaks when there is molecular motion on its time scale . in particular , this may be the case if the observed rigid component is glassy rather than crystalline and close to tg . under any condition irrespective of temperature , however , the typical decay time upon incrementing the echo was never significantly shorter than the ( relatively weak ) decay seen in figure 2 , which is ultimately associated with the breakdown of the pulse sequence efficiency because of higher - order imperfections .", "we thus conclude that all observed rigid signals are associated with crystal - like domains ; otherwise , a significant signal loss upon going through tg would have been observed .", "therefore , mse - fid curves were used to quantify the fraction of fast - decaying rigid polymer present in pva gels .", "the first fast decay ( caused by the rigid polymer fraction ) in the mse - fids ( insert in figure 2 ) has a quasi - gaussian decay , whereas the slower signal decay could be fitted with an exponential function .", "therefore , the first 140 s of the normalized mse - fid curves ( unity signal for zero time ) were fit according to eq 6 in this expression , a is the fraction of detectable rigid phase in the sample and b is an adjustable parameter for a better fit of the fast decay shape . in all of the cases ,", "b  2 ( i.e. , the crystalline fraction is described by a gaussian function ) .", "in addition , to reference the hard polymer phase to the total amount of polymer ( instead of the sample fraction ) , we corrected the a parameter according to the actual fraction of polymer in the sample obtained from saturation recovery experiments ( note again that the samples contain residual water ) on the basis of the fact that water has a much longer t1 relaxation time . from such experiments , and assuming no significant noe transfer between the species on the same time scale , the water content can be obtained from a biexponential fit \n\n where d is the water fraction in the sample and t1w and t1p are the longitudinal relaxation time constant of water and polymer , respectively ( with t1w  t1p ) .", "for example , at t = 304 k , the relaxation times were t1w = 1520 ms and t1p = 60 ms for a network with 10% pva after seven freezingthawing cycles .", "the fast - decaying rigid crystallite signal was not detected in these experiments ( the fid was evaluated at a time beyond 50 s ) such that d is determined relative to the mobile polymer fraction ( i.e. , ( 1  a ) in eq 6 ) .", "finally , the rigid polymer fraction discussed below was obtained according to \n\n obviously , for these corrections , it is essential to use a recycle delay that is long enough to observe the full equilibrium magnetization of the sample ( 5(t1w ) was used as a recycle delay in all experiments , i.e. , 720 s depending on the temperature ) ."], ["the aim of this work is to study the structure and phase composition of pva cryo - gels by using a low - field nmr spectrometer . to revise the structural models suggested in the literature", ", we will separately analyze the effects of three factors on the pva network structure : ( i ) the number of applied freezingthawing cycles , ( ii ) the temperature and melting process , and ( iii ) the aging process . to achieve this objective , in every point ,", "dq experiments were performed to quantify the proportion of noncoupled network defects , and the molecular weight between cross - links and mse - fid pulse sequence were applied to determine the rigid fraction of the polymer .", "the starting point of our study is the characterization of two isotropic solutions of 10 and 20% ( w / v ) of pva in water", ". the 10% pva solution does not give any detectable dq signal , but for the decay ( iref ) data , two different processes can be identified ( i.e. , the decay of the polymer magnetization and the water relaxation ) .", "this result demonstrates that under these preparation and measurement conditions , the dilute solution can be considered to be nonentangled and above the gelation threshold .", "this is not a trivial result because dq experiments are , in principle , sensitive to transient links ( entanglements or hydrogen bonds ) , and this indeed appears indicated by the 20% pva solution ; under the same preparation conditions , dq experiments did yield a detectable build - up curve . however , this is in our case due to a nondissolved or partially gelled pva subphase . to avoid this problem ,", "20% pva solutions were prepared under more vigorous stirring as well as higher temperatures up to 90 c . in this way , a totally isotropic solution of polymer was obtained according the dq experiments .", "dq experiments were performed on pva samples subject to different numbers of freezingthawing cycles to determine the evolution of noncoupled network defects as well as the network mesh size . in figure 3", ", we can see that after the first freezingthawing cycle , 50% of the polymer chains are coupled and are therefore part of the network , whereas the rest are not linked to it or at least belong to elastically inactive dangling chains or loops .", "the number of pva chains incorporated in the gel network increases with the successive cycles until it reaches a plateau after the sixth cycle , where the network has the minimum defect content .", "this important result shows that independent of the number of freezingthawing cycles applied to a pva solution 25% of the polymer is not coupled and therefore should not be elastically active .", "this is a very significant observation that is usually not taken into consideration when mechanical , dynamic - mechanical , or rheological properties of this material are analyzed .", "urushizaki et al.(66 ) have related the viscoelastic properties of this type of gel to the nonincorporated pva chains , and they have estimated their amount from the difference in weight between the polymer gel before and after immersion in distilled water .", "they found that 10% of the polymer was not incorporated after the first freezingthawing cycle .", "similar results were also indirectly inferred from the measure of intradiffusion coefficients of micellar aggregates of surfactant molecules dissolved in these types of gels.(67 ) obviously , our experimental values are much larger because we detect not only the non - cross - linked chains but also the dangling chains and other nonelastic network defects ( e.g. , loops ) .", "the effect of d2o in measured noncoupled network defects is another important point to take into consideration when this fraction of polymer is analyzed .", "pva monomers have an exchangeable proton ( oh ) , which , in presence of d2o , could be replaced by deuterium that is undetectable by h nmr , whereas the proton will become detectable as water ( hdo ) .", "free water signal ( h2o or hdo ) is subtracted from the long - time decay of the signal because it can be identified by its own longer t2 .", "however , the number of noncoupled network defects could be slightly overestimated because of the protondeuterium exchange .", "variation of noncoupled network defects ( i.e. , non - cross - linked polymer chains , dangling chains , and loops ) as a function of the number of freezingthawing cycles for the studied pva solutions .", "in addition , the decrease in the number of defects is correlated with an increase in dres that is proportional to the network chain order parameter ( eq 1 ) , see figure 4 .", "the formation of cross - links ( independent of their nature ) renders the polymer segmental motion nonisotropic , whereby the chain segments become ordered with respect to the end - to - end distance , and residual dipolar couplings arise .", "according to this basic principle , the increase in dres is directly related to the ( inverse ) length of the chains between the constraints .", "similar to the observation for the noncoupled network defects , the network chain length appears to approach a plateau after cycle number six , yet the tendency is not as clear as the former result . even after 16 cycles , the cross - link density still appears to increase .", "variation of the average residual dipolar couplings ( directly related to 1/n and therefore the cross - link density or the inverse mesh size ) extracted from dq experiments as a function of the number of freezingthawing cycles for the studied pva solutions .", " represents a sample containing 20% pva after 3 h of freezing , and  is from the same sample after 16 h of freezing . with this observation ,", "it is important to note that samples with different polymer concentration appear to exhibit the same behavior during gelation .", "a possible explanation could be that in the framework of a heterogeneous scenario cross - linking occurs via partial crystallization in the nonfrozen parts of the sample ; it simply means that in less concentrated samples more water freezes .", "this fact could explain the dependence of the dimension and shape of pores on polymer concentration and the regimes of cryogenic treatment.(20 ) in this context , it appears to be worthwhile to study the influence of the actual freezing temperature , which should affect the polymer concentration under the conditions at which the crystallites are formed .", "however , the freezing time ( especially in the first cycle when the network is formed ) seems to have some influence in the gel network structure . as is shown in figure 4 ,", "the apparent dipolar coupling constant is very low ( 48 hz ) after 1 h of freezing at 32 c . if the freezing period is increased to 3 h , then the cross - link density of the gel almost doubles ( 82 hz ) , whereas longer freezing ( e.g. , 16 h ) does not lead to further variation", "this may indicate that 1 h of freezing is not long enough to complete the process , and for this reason , further analyses are based on samples with a first freezing cycle of 12 h and subsequent cycles of 3 h each .", "returning to the origin of the pva networks produced via freezingthawing cycles from homogeneous polymer solutions in water , it appears to be well demonstrated by now that during the first freezing cycle , freezing of some water(32 ) increases the concentration of pva in the still unfrozen phase.(31 ) crystallization of pva takes place in this concentrated microphase,(68 ) but it is kinetically frustrated by the gelation of the polymer solution .", "this fact could explain the small size of these primary crystallites ( 5 nm ) , which then act as physical junctions between the amorphous and mobile polymer chains .", "results extracted from the mse - fid curves of the pva gels after the first freezingthawing cycle ( figure 5 ) indicate that 8% of the polymer behaves like a rigid solid .", "this rigid fraction increases with the number of cycles until it reaches a plateau close to cycle number six , just as our other reported network properties .", "the maximum amount of rigid phase estimated by our nmr methodology ( 20% of the total polymer ) is substantially higher than the crystallinity of similar samples deduced by dsc as well as some other h nmr methods .", "variation of the rigid ( crystalline ) polymer fraction extracted from mse - fid curves as a function of the number of freezingthawing cycles for the studied pva solutions . in all cases , the crystallinity of pva cryo - gels obtained in dsc studies is estimated to be 5% ( not taking into account studies of dry networks(34 ) because the structure should be different ) . however , as was pointed out by willcox et al.,(28 ) this magnitude could very well be underestimated because the heat of fusion of the crystals in gels may be substantially lower than the assumed pva bulk value because of their small size and large surface effects ( hydrogen bonds to surrounding water , etc . ) .", "in addition , another important point is the difficulty in measuring small quantities of crystal phase in dilute gels via calorimetric methods in general ; for example , a crystallinity of 8% in a gel with a pva concentration of 20% represents only a sample fraction of 0.016 .", "nevertheless , this difficulty seems to be reduced by the use of microcalorimetry . in this way ,", "similar results were obtained by performing this type of measurement on gels with low pva concentrations,(8 ) although the uncertainties related to the obtained results are still large . for these reasons ,", "the use of solid - state nmr seems to be a better methodology for quantifying the crystallinity in dilute pva gels . however , there are still some discrepancies in the total amount of rigid phase measured by the use of different nmr methodologies .", "ricciardi et al.(18 ) estimated the crystallinity of pva gels after 11 freezingthawing cycles to be 8% by analyzing the first microsecond of the h fid .", "clearly , as was pointed out in the , this experimental procedure falls victim to the dead - time problem .", "depending on the spectrometer , the amount of rigid signal lost during this time could be more or less significant .", "( ref ( 15 ) does not specify the dead time ; therefore , it is not possible to estimate the related error . ) for example , our low - field spectrometer has a dead time of 12 s ; therefore , the fid analysis would lead to an underestimation of the rigid polymer fraction by 50% . by using different c nmr experiments ( direct polarization as well as hc cross polarization ) , willcox et al.(28 )", "established that after the first freezingthawing cycle , pva gel contains a rigid polymer phase of 5  2% , increasing up to 12  4% after cycle number 12 .", "as was pointed out by the authors,(28 ) the relative fraction comparing different samples could be estimated with a high accuracy , but the used methodology ( in particular , the use of the intrinsically nonquantitative cross polarization ) does not give a reliable absolute crystallinity . in conclusion , the key to obtaining the most accurate crystal fraction in these dilute polymer samples is to use a solid - state nmr method that allows us to detect the rigid part fully by refocusing the multispin dipolar interactions with the mse , thus solving the dead - time problem and minimizing the signal loss .", "of course , detecting the more - sensitive and 100% abundant protons instead of the naturally abundant c is an additional advantage , which more than compensates for the use of an intrinsically less - sensitive low - field spectrometer .", "summarizing all results discussed so far , the network structure of pva cryo - gels is very apparently based on the formation of rigid polymer areas ( most probably polymer crystallites ) during the cooling process .", "after the first cycle , almost half ( 8% ) of the final amount ( 20% ) of the rigid phase is already formed . in this stage ,", "subsequent freezingthawing cycles increase the rigid polymer fraction , which has two consequences : first , the noncoupled polymer fraction is further reduced ( i.e. , more pva segments become elastically active ) , and second , the network mesh size is further reduced but only slightly during further freezingthawing cycles .", "therefore , we believe that subsequent freezingthawing cycles not only increase the size of the crystals formed during the first freezing process but also lead to the creation of other more imperfect crystals .", "independent of the polymer concentration of the gel , the melting of smaller and more imperfect secondary crystals takes place at lower temperature and over a large range , leading to a continuous decrease in the detected rigid polymer phase with the temperature ( figure 6 ) . at 7580 c ( in good agreement with", "the main endotherm observed in dsc measurements ) , this quantity drops below the detection limit .", "this should be correlated with the melting of the main ( primary ) crystals ( estimated to 5% of the polymer fraction ) that support the network structure of the gel .", "these results are consistent with dsc measurements because they show an increase in the melting endotherm that corresponds to the initial crystals as well as to the appearance of a second endotherm at lower temperatures when the number of freezingthawing cycles was increased for a given pva solution .", "variation of the rigid polymer fraction ( extracted from mse - fid experiments ) as a function of temperature for pva gels formed after seven freezingthawing cycles . a network with 10% pva was first heated until 324 k (  ) and then kept at room temperature for 12 h before the second heating procedure was performed (  ) .", "a solution with 20% pva was submitted to two different treatments , that is , one freezingthawing cycle and seven freezingthawing cycles , respectively .", "all samples were frozen for 12 h in the first cycle and 3 h in the subsequent cycles .", "the effect of the number of freezingthawing cycles on the crystallinity is easily understandable by comparing the evolution of the rigid polymer fraction of a pva gel obtained after seven cycles with that of a pva gel after only one freezingthawing cycle ( figure 6 ) .", "the onset of melting after only one freezing cycle is estimated to be 50 c , leaving the rigid polymer fraction constant at lower temperatures . therefore , subsequent cycles seem to create more , yet imperfect , secondary crystallite structures . above 50 c ,", "all samples exhibit a similar behavior related to the partial melting of primary crystals or to the melting of less - perfect secondary structures .", "importantly , the temperature above which it is not possible to detect any more signal of rigid polymer remains invariant , yet the final fraction seems to increase slightly with the number of cycles .", "therefore , according to this result , freezingthawing cycles have a direct influence on not only the number of crystals formed during the first freezing process but also the creation of other more imperfect secondary crystallites . to ensure that the results shown in figure 6 are not artifacts produced by different magnetization relaxation at different temperatures that could possibly arise from regions that undergo a glass - to - liquid rather than a melting transition ,", "the evolution of the signal at any given temperature was studied as a function of echo delay , see figure 7 .", "at all of the measured temperatures , similar behavior was found , with a loss of intensity estimated to between 0.7 and 1.8% for the two extreme cases , which corresponds to a constant 10% loss on a relative scale , as is usually found .", "importantly , the drop in overall signal corresponds , within the error margins , to the expected drop due to the curie factor ( temperature - dependent spin polarization ) .", "in addition , a direct check is the control of the total intensity , comparing the pva gels to the corresponding pva solutions ( insert in figure 7 ) .", "it is seen that no significant signal is lost / undetectable ( we observe almost all protons in the sample ) , and from this , it is clear that the loss of intensity caused by the mse in pva gels is at a minimum , with no other ( hidden ) phenomena that could be related to an intermediately mobile ( almost glassy ) rigid fraction . given this experimental evidence , the decay of the rigid h fraction with temperature can be related to only a melting of pva crystallites .", "variation of the initial signal intensity of mse - fids with increasing echo delay at different temperatures for a pva gel containing 20% polymer after one freezingthawing cycle ( 12 h of freezing and 1 h thawing ) .", "the insert presents the mse - fid of the pva gel measured at 304 k with the minimum echo delay ( 0.0022 ms ) as well as the estimated intensity at zero echo time in comparison with the mse - fid of the isotropic solution of the same sample measured under the same conditions after complete melting at 90 c .", "obviously , the temperature - dependent variation of the rigid polymer phase has a direct effect on the network structure , as shown in figure 8 .", "the melting of secondary crystals leads to an almost exponential increase in the number of network defects ( obviously , in a reversible pathway , comparing the phenomenon with the effect of successive freezingthawing cycles ) .", "however , the network mesh size ( as measured by the residual dipolar coupling ) remains almost constant .", "this means that secondary crystallization is very important in the network organization , reducing the number of network defects and therefore increasing the number of polymer segments that are elastically active .", "however , the molecular weight between the constraints appears to be dictated mostly by the primary crystallites formed during the first freezing cycles .", "for this reason , the network is completely destroyed at temperatures of 80 c when the primary crystal phase is molten , leaving no observable dq signal ( i.e. , an isotropic pva solution is formed ) .", "on the side , we note that the differences in the davg values given in figures 4 and 8b , which indicate a 50% decreased mesh size for the samples studied in figure 8 , are related to the increase in the freezing time ( 12 h in the first step and 3 h in the successive cycles instead of 1 h ) used in each cycle , as pointed out above .", "variation of ( a ) the network defect fraction and ( b ) the inverse mesh size ( given by davg measured by mq nmr ) with temperature for the 20% pva gel prepared after seven freezingthawing cycles ( 12 h first freezing cycle , 3 h subsequent cycles ) .", "error bars represent the fitting uncertainty , whereas lines are only guides to the eye .", "these nmr results on the mesh size perfectly agree with the trends observed in studies of mechanical properties reported in the literature . clearly , the elastic behavior of pva gels depends on the number of freezingthawing cycles .", "therefore , the main factor that determines the improvement of the mechanical properties upon further cycles should be associated with the number of elastically active chains .", "the polymer concentration in the original aqueous solution has an important effect on the elastic properties ; that is , the elastic modulus increases .", "however , in the view of our data , this does not appear to have important consequences for the actual network structure ; that is , there are no large variations in the network mesh size .", "therefore , the reported variations are simply related to differences in polymer concentration . in addition ,", "different works demonstrate a slight decrease in the elastic modulus with the temperature , until a major drop occurs at 50 c.(65 ) this is obviously related to the partial melting of secondary and more imperfect crystallites that leads to an increasing amount of network defects ( elastically inactive chains ) . at temperatures close to 80 c ,", "the complete loss of crystallinity essentially liberates all chains , and the system drops below the gel point . in view of the important role of the substantial polymer amount that is not elastically active ( free and dangling chains , loops )", ", it may be worthwhile to reconsider detailed frequency - dependent mechanical measurements , where these components should contribute to an increased loss modulus over the frequency range covering their relaxation .", "the capacity of our investigated systems to be transformed from an isotropic aqueous solution to a physical cryo - gel by the application of freezingthawing cycles , including thermal reversibility back to an isotropic pva solution , is in clear contradiction with the assertion that covalent bonds could form between the polymer chains during gelation . if covalent cross - links were an important contribution to the final gel properties , then residual couplings should persist even above the melting temperature of the crystallites , which is not the case .", "however , the presence of ( rapidly tumbling ) microgels above the main melting point can not be ruled out and is in fact indicated by the relatively high viscosity ( as checked by a simple tilt test ) .", "however , we point out that even the pristine 20% pva solution exhibited some detectable dq signal , indicating extended gel - like structures , unless it was heated to > 90 c", ". therefore , an undetectably small fraction of crystallites ( or extended hydrogen - bonded aggregates ) may always be present , even at > 80 c , and hold the system close to the gel point , depending on the sample and its thermal history . from our perspective", ", permanent bonds can be ruled out as a major factor determining the cryo - gel structure .", "finally , we address aging phenomena that are a part of the data shown in figure 6 and were not commented on until now . after heating a sample containing 10% pva to 50 c and recording the loss of crystallinity , the sample was stored for 12 h , and another measurement was performed at room temperature .", "the amount of rigid polymer fraction after 12 h of storage was again considerably increased , as can be seen in figure 6 .", "yet , this new crystalline fraction melts at rather low temperatures , which again indicates the growth of secondary crystallites .", "similar prominent aging effects have been reported in the literature . for more clear evidence ,", "a sealed sample containing 20% pva was submitted to seven freezingthawing cycles ( with 12 h of freezing in the first cycle ) and was characterized after different storage times at room temperature . according to figure 9 , aging clearly increases the secondary population of imperfect crystals .", "it also increases the amount of elastically active polymer segments but has no significant effect on the network mesh size .", "therefore , aging effects on pva gel properties are again mainly related to an increasing fraction of polymer incorporated into the network structure and not to a decrease in mesh size .", "effect of aging on ( a ) the rigid polymer fraction , ( b ) the noncoupled defects fraction , and ( c ) the average residual dipolar coupling of a gel with 10% pva obtained after seven freezingthawing cycles ( 12 h of freezing for the first cycle and 3 h for the following cycles ) ."], ["this physical gel is supported by rigid polymer areas ( pva crystallites ) that act as junctions between the remaining mobile and entropically elastic polymer chains and thus determine the network mesh size . in our study ,", "the crystallites are quantitatively detected as a rigidlike fraction of quickly relaxing ( and fully refocused to overcome the dead - time problem ) magnetization , and the mesh size is deduced from mq experiments that measure residual dipolar couplings , the latter arising from constraints on the motion of the mobile chains ( i.e. , cross - links ) that render the segmental motion locally anisotropic .", "the most important crystal fraction forms in the first freezing cycle , and subsequent freezingthawing cycles produce only a slight increase in the initial crystal size as well as induce the formation of more imperfect , secondary crystallites .", "this secondary crystal fraction ( which constitutes around two - thirds of the total crystal phase in samples submitted to a large number of cycles , extended aging , or both ) plays an important role by increasing the number of elastically active polymer chains , but it does not appreciably affect the mesh size .", "pva cryo - hydrogels are shown to be totally thermoreversible ; that is , pva physical networks can be transformed into an isotropic aqueous solution by increasing the temperature . during this process ,", "the more imperfect crystals are molten first , and the number of network defects increases exponentially .", "in contrast , the network mesh size does not undergo significant variations until the primary crystal phase is molten at temperatures of 80 c .", "our experiments did not reveal any evidence of the possible formation of covalent cross - links between pva chains during gelation ; no residual couplings were detected after the melting of this primary crystal fraction .", "independent of the number of cycles , the aging , or the polymer concentration in the original pva solution , a considerable fraction of polymer ( at least 25% ) is not elastically active , which is a result that is not yet not taken into account in the large body of literature addressing the formation and the properties of pva cryo - hydrogels .", "this finding is central to the interpretation and understanding of the elastic properties of this useful material and must be taken into account in a reinterpretation of the origin of the changes of the elastic moduli of pva cryogels .", "finally , it is important that the in - depth study of both processes ( i.e. , the pva gelation by the application of freezingthawing cycles ) as well as the destruction of the network structure by heating were , for the first time , entirely based on the use of an inexpensive low - field solid - state nmr spectrometer .", "we show that the combination of different advanced nmr strategies , that is , mq spectroscopy and component analysis of mse - refocused free - induction decays , is vital for obtaining quantitative information on network structure and phase composition ( crystallinity ) , respectively , to arrive at conclusions that can be matched with information extracted from other diverse techniques .", "in addition , the use of low - field solid - state nmr spectroscopy not only is useful for extracting structural information but also could be a powerful tool for investigating the polymer dynamics of these complex systems ."]]}
{"article_id": "PMC3185225", "article_text": ["the acridinones represented by imidazo- and triazoloacridinones are a new group of potent antitumor compounds ( cholody et al . , 1990 , 1992 , 1996 ) from which one of the most active derivatives called as c-1305 has been selected for extended preclinical trials and the other one called c-1311 ( for review see mazerska et al", ". , 1998 ) is currently undergoing phase ii clinical trials as drug symadextm ( burger et al .", ", 1996 ; den brok et al . , 2005a , b , c ) .", "imidazoacridinones exhibit in vivo activity against various tumor cell lines as leukemia , melanoma , colon adenocarcinoma , and colon carcinoma .", "triazoloacridinones exhibit in vivo activity against leukemia , murine carcinoma , lung carcinoma , breast carcinoma , and colon carcinoma ( cholody et al .", ", 1990 , 1992 , 1996 ; kusnierczyk et al . , 1994 ; burger et al . ,", "as was previously shown ( skadanowski et al . , 1999 ; lemke et al . , 2004 ; augustin et al . , 2004 , 2006 ; wesierska - gadek et al . , 2004 ; koba and konopa , 2007 ; koba et al . , 2009 ) ,", "cellular dna is important target for the triazoloacridinone drugs , and hence interactions with dna are naturally the crucial point in view of the biological activity of these compounds . in previous article ( skadanowski et al . , 1999 ;", "lemke et al . , 2004 ) , it was indicated that triazoloacridinones inhibit cleavable complexes of topoisomerase ii with dna .", "they inhibit also nucleic acid or protein synthesis induced by g2 block of cell cycle followed by apoptosis ( augustin et al . , 2004 , 2006 ;", "2004 ) , intercalating to dna and binding in minor groove ( koba and konopa , 2007 ; koba et al . , 2009 ) and/or forming of interstrand dna crosslinks ( koba and konopa , 2007 ) .", "in addition , it was shown that intercalation to dna takes place preferentially in guanine triplet regions inducing changes in dna structures ( lemke et al .", ", 2005 ) . for imidazoacridinones , it was demonstrated that intercalation to dna undergoes at physiological condition with parallel stabilization of double - stranded dna and unwinding of supercoiled dna ( burger et al . , 1999 ;", "the intercalative binding mode of acridinone derivatives was also confirmed with the use of molecular - modeling studies ( mazerski and muchniewicz , 2000 ) .", "similar to other dna - binding agents , treatment of tumor cells with imidazoacridinones induces topoisomerase ii - associated dna strand breaks ( skadanowski et al . , 1996 ) , arrests cells in g2 phase , and stimulates apoptosis ( zaffaroni et al . , 2001 ; skwarska et al . , 2007 ) or mitotic catastrophe ( hyzy et al . , 2005 ; skwarska et al . , 2007", "however , after testing imidazoacridinone and triazoloacridinone derivatives , it has been concluded that although the intercalative binding to dna seems to be necessary for their biological activity ( the most active compounds have usually the highest binding affinity ) , it is not sufficient ( some inactive analogs also bind strongly with dna ) ( dziegielewski et al .", "moreover , acridinones undergo enzymatic oxidation , and this reaction is important for their biological activity as intercalation to dna and covalent adducts formation ( dziegielewski and konopa , 1996 ; mazerska et al . , 1999 , 2003 ) .", "in this context , noncovalent interaction of acridinones may help position drug molecules on dna for the covalent reaction . in this article ,", "physicochemical interactions of acridinones with dna were evaluated in view of quantitative structure  activity relationships ( qsar ) .", "multiple regression analysis was used to model relationships between molecular structural descriptors and biological ( antitumor ) activity of molecules , or between molecular structural descriptors and physicochemical interaction of acridinone derivatives with dna .", "the biological data expressed as anti - leukemia p388 activity and parameter describing ability to physicochemical ( noncovalent ) interaction with dna as value of dna - duplexes stabilization were applied in this study .", "the acridinone derivatives examined in this study have been selected to collect analogue compounds differing in chemical structures as well as anticancer activities ( table  1 ) .", "the data of acridinones antitumor activity against p388 leukemia in mice in vivo and expressed as the percentage of increase in survival time of the treated to that of the control mice with p388 leukemia at optimal dose ( ils ) were taken from the literature ( table  1 ) ( cholody et al .", ", 1990 , 1992 ; koba and konopa , 2007 ; mazerska et al . , 1996 ) . the data of physicochemical binding of acridinones to dna ( as values of dna - duplexes stabilization ) , which were expressed as an increase in dna melting temperature in centigrade degrees of ctdna at drug to dna base pairs 0.25  m ratio were taken from the literature ( table  1 ) ( koba and konopa , 2007 ; dziegielewski et al .", ", 2002).table  1chemical structures of acridinones studiedcompoundxnr1r2r3r4r5r6ilstmc-1310c2ch2ch3ch2ch3ohhch3h18515.3c-1311c2ch2ch3ch2ch3ohhhh9313.7c-1330c2ch2ch3ch2ch3och3hhh9611.5c-1415c2ch2ch3ch2ch3hhhh557.2c-1419c2ch2ch3ch2ch3hhhoh278.3c-1558c2ch2ch3ch2ch3c(ch3)3hhh02.4c-1176c2ch3ch3hhhh909.5c-1263c2ch3ch3ohhhh11012.3c-1212c3ch3ch3hhhh2511.5c-1371c3ch3ch3ohhhh1203.5c-1554c5ch2ch3ch2ch3ch3hhh2010.5c-1266c5ch3ch3hhhh109.9c-1492c5ch3ch3ohhhh8513.1c-1233n2ch3ch3hh  h779.1c-1303n2ch3ch3ohh  h10213.1c-1533n2ch3ch3ohch3h108.1c-1567n2ch3ch3c(ch3)3h  h06.8c-1410n2hch2ch3ohh  h787.1c-1296n3ch3ch3ch3h  h1811.5c-1305n3ch3ch3ohh  h16515.1the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosethe increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio chemical structures of acridinones studied the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose the increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio the structure of the tested compounds was studied by molecular modeling using hyperchem 7.5 release software ( kaliszan et al . , 1995 ; ivanciuc , 1996 ) and dragon software ( todeschini et al . , 2000", "the structures of the compounds were first pre - optimized with the molecular mechanics force field ( mm+ ) procedure included in the hyperchem 6.03 ( hypercube ) http://www.hyper.com , and the resulting geometries were further refined by means of the semi - empirical molecular orbitals method am1 using the polak - ribiere algorithm and a gradient norm limit of 0.01  kcal   .", "the following molecular descriptors taken from hyperchem software were considered among quantum and chemical indices : total energy ( te ) , binding energy ( be ) , isolated atomic energy ( iae ) , electron energy ( ee ) , core  core energy ( cce ) , heat flow ( hf ) , energy of the highest occupied molecular orbitals ( e_homo ) , energy of the lowest unoccupied molecular orbitals ( e_lumo ) , and difference between homo and lumo energies referred to as eg  =  energy gap ; ionization energy ( potential ) ( ie ) and electron affinity ( ea ) were calculated as a difference between the hf of positive molecular ion and electrically neutral molecule , and electronegativity ( en ) calculated as average arithmetic potential of ionization and ea .", "in addition , other parameters were used as the value of electron density of atom orbitals from the lowest to the highest ( ed_min and ed_max , respectively ) , the highest positive electron charge on the atoms ( max_pos ) , and the highest negative electron charge on the atoms ( max_neg ) , the difference between the highest positive and negative charge ( delta_q ) , distribution of dipolar moment along x , y , and z axes ( x_dm , y_dm , and z_dm , respectively ) , total dipolar moment ( tdm ) , mean polarizability of molecules ( in atom units ) mp ( mean polarizability ) , energy equal to the length of the wave with the greatest long - wave transfer of electrons , for which the value of oscillator force was different from zero ( el)the value of wave figures were converted to ev  and the value of the most intensive electron transfer ( emax  the maximum value of oscillator force calculated with the use of am1 method  as well as oscillator maximum force used for the transfer ( os_emax ) . moreover ,", "they include the following descriptors : surface area of the molecule available for solvent ( sa ) , molecule volume ( v ) , hydration energy ( he ) , the calculated distribution coefficient logarithm ( logp ) , refraction ( r ) , and polarizability ( p ) . on the other hand ,", "using dragon software , over 1,300 molecular descriptors were calculated and considered for qsar analysis .", "they include molecular parameters from different group and class of descriptors as constitutional , topological , walk and path counts , connectivity indices , information indices , 2d autocorrelations , edge adjacency indices , topological charge indices , eigenvalue - based indices , geometrical , 3d - morse , whim , getaway , functional group counts , atom - centred fragments , charge , molecular properties and other group of descriptors , and describing some properties of compound as geometry , symmetry , topology , electronic , steric or thermodynamic and other properties .", "the definitions of these descriptors are reviewed by todeschini ( todeschini et al . , 2000 ) .", "the statistical qsar analysis was performed with the use of stepwise regression analysis that used statistica 8.0 software ( statsoft , tulsa , ok , usa ) .", "the objective of stepwise regression is to construct a multivariate regression model ( qsar equation ) for a certain property , y , based on several selected explanatory variables . in stepwise regression", ", the first selected explanatory variable has the highest correlation with dependent variable , y. then , explanatory ( independent ) variables are consecutively added to the model in a forward selection procedure .", "a new variable is added to the model if a significant change in residuals of the model can be observed .", "the significance is evaluated using a statistical test , usually f - test ( the value of the f - test of significance , f ) .", "in addition , the multiple correlation coefficients ( r ) , the standard error of estimate ( s ) , and the significance levels of each term and of whole equation ( p ) are calculated for the derived qsar equations . whenever a new variable is included into a model", ", a backward elimination step follows in which an f - test detects the earlier selected variables , which can be removed from the model without any significant change on the level of the residuals .", "stepwise regression is very much popular in qsar studies , since the stepwise procedure is simple and based on the classical multiple linear regression ( mlr ) approach .", "one of the drawbacks of the method is the fact that no optimal variable selection is guaranteed , since the new variables are found based on the previously included variables into the model ( put et al . , 2006 ) .", "during model building , the model fit can be improved proportional to the model complexity .", "therefore , the more the factors are included into the model , the better the model fits the training data .", "usually , the model fit is evaluated by the root mean - squared error ( rmse ) , computed for the training data .", "the determination of the optimal complexity of the model requires an estimation of its predictive ability , to prevent overfitting to the calibration data .", "after all , the main goal of qsar models is to obtain a reasonable prediction of the retention for future samples . to evaluate the prediction by means of an internal validation procedures , cross validation can be used .", "the predictive ability of a model is characterized by the cross - validated root mean - squared error ( rmsecv ) ; test values were calculated with the matlab software ( mathworks , natick , ma , usa ) .", "the rmsecv as values , which quantify the predictive power of the qsar model , were calculated by the leave - one - out method and leave - ten - out method .", "the acridinone derivatives examined in this study have been selected to collect analogue compounds differing in chemical structures as well as anticancer activities ( table  1 ) .", "the data of acridinones antitumor activity against p388 leukemia in mice in vivo and expressed as the percentage of increase in survival time of the treated to that of the control mice with p388 leukemia at optimal dose ( ils ) were taken from the literature ( table  1 ) ( cholody et al .", ", 1990 , 1992 ; koba and konopa , 2007 ; mazerska et al . , 1996 ) . the data of physicochemical binding of acridinones to dna ( as values of dna - duplexes stabilization ) , which were expressed as an increase in dna melting temperature in centigrade degrees of ctdna at drug to dna base pairs 0.25  m ratio were taken from the literature ( table  1 ) ( koba and konopa , 2007 ; dziegielewski et al . , 2002).table  1chemical structures of acridinones studiedcompoundxnr1r2r3r4r5r6ilstmc-1310c2ch2ch3ch2ch3ohhch3h18515.3c-1311c2ch2ch3ch2ch3ohhhh9313.7c-1330c2ch2ch3ch2ch3och3hhh9611.5c-1415c2ch2ch3ch2ch3hhhh557.2c-1419c2ch2ch3ch2ch3hhhoh278.3c-1558c2ch2ch3ch2ch3c(ch3)3hhh02.4c-1176c2ch3ch3hhhh909.5c-1263c2ch3ch3ohhhh11012.3c-1212c3ch3ch3hhhh2511.5c-1371c3ch3ch3ohhhh1203.5c-1554c5ch2ch3ch2ch3ch3hhh2010.5c-1266c5ch3ch3hhhh109.9c-1492c5ch3ch3ohhhh8513.1c-1233n2ch3ch3hh  h779.1c-1303n2ch3ch3ohh  h10213.1c-1533n2ch3ch3ohch3h108.1c-1567n2ch3ch3c(ch3)3h  h06.8c-1410n2hch2ch3ohh  h787.1c-1296n3ch3ch3ch3h  h1811.5c-1305n3ch3ch3ohh  h16515.1the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosethe increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio chemical structures of acridinones studied the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose the increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio", "the structure of the tested compounds was studied by molecular modeling using hyperchem 7.5 release software ( kaliszan et al . , 1995 ; ivanciuc , 1996 ) and dragon software ( todeschini et al . , 2000", "the structures of the compounds were first pre - optimized with the molecular mechanics force field ( mm+ ) procedure included in the hyperchem 6.03 ( hypercube ) http://www.hyper.com , and the resulting geometries were further refined by means of the semi - empirical molecular orbitals method am1 using the polak - ribiere algorithm and a gradient norm limit of 0.01  kcal   .", "the following molecular descriptors taken from hyperchem software were considered among quantum and chemical indices : total energy ( te ) , binding energy ( be ) , isolated atomic energy ( iae ) , electron energy ( ee ) , core  core energy ( cce ) , heat flow ( hf ) , energy of the highest occupied molecular orbitals ( e_homo ) , energy of the lowest unoccupied molecular orbitals ( e_lumo ) , and difference between homo and lumo energies referred to as eg  =  energy gap ; ionization energy ( potential ) ( ie ) and electron affinity ( ea ) were calculated as a difference between the hf of positive molecular ion and electrically neutral molecule , and electronegativity ( en ) calculated as average arithmetic potential of ionization and ea .", "in addition , other parameters were used as the value of electron density of atom orbitals from the lowest to the highest ( ed_min and ed_max , respectively ) , the highest positive electron charge on the atoms ( max_pos ) , and the highest negative electron charge on the atoms ( max_neg ) , the difference between the highest positive and negative charge ( delta_q ) , distribution of dipolar moment along x , y , and z axes ( x_dm , y_dm , and z_dm , respectively ) , total dipolar moment ( tdm ) , mean polarizability of molecules ( in atom units ) mp ( mean polarizability ) , energy equal to the length of the wave with the greatest long - wave transfer of electrons , for which the value of oscillator force was different from zero ( el)the value of wave figures were converted to ev  and the value of the most intensive electron transfer ( emax  the maximum value of oscillator force calculated with the use of am1 method  as well as oscillator maximum force used for the transfer ( os_emax ) . moreover ,", "they include the following descriptors : surface area of the molecule available for solvent ( sa ) , molecule volume ( v ) , hydration energy ( he ) , the calculated distribution coefficient logarithm ( logp ) , refraction ( r ) , and polarizability ( p ) . on the other hand , using dragon software , over 1,300 molecular descriptors were calculated and considered for qsar analysis .", "they include molecular parameters from different group and class of descriptors as constitutional , topological , walk and path counts , connectivity indices , information indices , 2d autocorrelations , edge adjacency indices , topological charge indices , eigenvalue - based indices , geometrical , 3d - morse , whim , getaway , functional group counts , atom - centred fragments , charge , molecular properties and other group of descriptors , and describing some properties of compound as geometry , symmetry , topology , electronic , steric or thermodynamic and other properties .", "the statistical qsar analysis was performed with the use of stepwise regression analysis that used statistica 8.0 software ( statsoft , tulsa , ok , usa ) .", "the objective of stepwise regression is to construct a multivariate regression model ( qsar equation ) for a certain property , y , based on several selected explanatory variables . in stepwise regression ,", "the first selected explanatory variable has the highest correlation with dependent variable , y. then , explanatory ( independent ) variables are consecutively added to the model in a forward selection procedure .", "a new variable is added to the model if a significant change in residuals of the model can be observed .", "the significance is evaluated using a statistical test , usually f - test ( the value of the f - test of significance , f ) .", "in addition , the multiple correlation coefficients ( r ) , the standard error of estimate ( s ) , and the significance levels of each term and of whole equation ( p ) are calculated for the derived qsar equations . whenever a new variable is included into a model", ", a backward elimination step follows in which an f - test detects the earlier selected variables , which can be removed from the model without any significant change on the level of the residuals .", "stepwise regression is very much popular in qsar studies , since the stepwise procedure is simple and based on the classical multiple linear regression ( mlr ) approach .", "one of the drawbacks of the method is the fact that no optimal variable selection is guaranteed , since the new variables are found based on the previously included variables into the model ( put et al . , 2006 ) .", "during model building , the model fit can be improved proportional to the model complexity .", "therefore , the more the factors are included into the model , the better the model fits the training data .", "usually , the model fit is evaluated by the root mean - squared error ( rmse ) , computed for the training data .", "the determination of the optimal complexity of the model requires an estimation of its predictive ability , to prevent overfitting to the calibration data .", "after all , the main goal of qsar models is to obtain a reasonable prediction of the retention for future samples . to evaluate the prediction by means of an internal validation procedures , cross validation can be used .", "the predictive ability of a model is characterized by the cross - validated root mean - squared error ( rmsecv ) ; test values were calculated with the matlab software ( mathworks , natick , ma , usa ) .", "the rmsecv as values , which quantify the predictive power of the qsar model , were calculated by the leave - one - out method and leave - ten - out method .", "the chemical structures of the 20 compounds considered for this study and their antitumor and noncovalent dna - binding activities are presented in table  1 . in this study ,", "qsar analysis using multiple regression method was performed for biological activity data ( ils ) or physicochemical interaction with dna ( tm ) , and non - empirical parameters ( molecular descriptors ) .", "owing to a large number of non - empirical parameters ( over 1,300 ) , treated as independent variables and according to qsar strategy and multi - parameter regression rule in derived multi - parameter regression equation , the number of independent variables must be 56 times less than the number of cases considered in this study . in practice , for obtaining statistically significant equation , one independent variable ( in our case structural descriptor ) falls , generally out of five to maximum six cases considered , in dependent - variable activity ( in our case , activity of acridinones ) . in the research", "done , the data set of 20 acridinone derivatives ( dependent variables ) was taken to qsar analysis , and for this reason the derived qsar equations were maximally limited to four statistically significant independent variables ( structural descriptors ) .", "moreover , correlations were limited to the value of regression coefficient r    0.8 and an additional criterion , considered as relevant to particular independent variables , was established at the significance level p    0.05 .", "the calculated equations are presented in table  2 and characterized by four statistically significant independent variables with a good value of regression coefficient r    0.8 ( r  =  0.9384 and r  =  0.8388 for quantitative structure  antitumor activity relationships and quantitative structure  ability to dna - duplexes stabilization relationships , respectively ) . moreover , all the regression coefficients are highly statistically significant ( p  < ", "0.05 ) as is the whole equation ( p  <  7    10 for quantitative structure  antitumor activity relationships and p  <  9    10 for quantitative structure  ability to dna - duplexes stabilization relationships , respectively ) .", "the values of the multiple correlation coefficient , r ; the standard error of the estimate , s ; and the value of the f - test of significance , f , are also statistically significant.table  2multiple regression qsar equation ( dependent variable  =  k0  +  k1a  +  k2b  +  k3c  +  k4d)dependent variablecoefficients and statistically significant molecular descriptorsstatistical parametersk0k1ak2bk3ck4dr ( r)sfptm97.44    55.096.59    1.50gats7e3.03    0.88i0.64    0.30h-047147.44    83.58mp0.8388 ( 0.7036)2.158.907 ", "  10p  =  1    10p  =  5    10p  =  4    10p  =  5    10p  =  1    10ils88.80    153.448914.33   ", "1225.69g3m36.31    6.02logp4691.69    1227.99g2p4744.01    1451.51g3p0.9384 ( 0.8806)21.0327.659    10p  =  1    10p  =  3    10p  =  2    10p  =  2   ", "10p  =  5    10multiple correlation coefficient ( determination coefficient)standard error of estimatevalue of the f - test of significancesignificance level multiple regression qsar equation ( dependent variable  =  k0  +  k1a  +  k2b  +  k3c  +  k4d ) multiple correlation coefficient ( determination coefficient ) standard error of estimate value of the f - test of significance statistically significant parameters used in qsar analysis ( values of molecular descriptors are presented in the table  3 ) as logarithm of theoretically calculated n - octanol  water partition coefficient ( logp ) from the class of hydrophobic descriptors , 3st component symmetry directional whim index weighted by atomic masses ( g3 m ) , 2st component symmetry directional whim index by atomic polarizabilities ( g2p ) , and 3st component symmetry directional whim directional index / weighted by atomic polarizabilities ( g3p ) from class of whim ( weighted holistic invariant molecular ) descriptors had the influence upon antitumor activity of acridinones .", "the whim descriptors are molecular descriptors based on statistical indices calculated on the projections of the atoms along principal axes .", "they are built in such a way as to capture relevant molecular 3d information regarding molecular size shape , symmetry , and atom distribution with respect to invariant reference frames ( todeschini et al . , 2000 ) . in general , the obtained data indicate that hydrophobic and total molecular symmetry properties are important for antitumor activity of acridinones .", "these observations are in partial agreement with the data obtained by mazerska ( mazerska et al . ,", "however , impact of lipophilicity on the biological activity of these compounds was observed only in the case of derivatives with 8-hydroxyl group , which undergo metabolic activation ( mazerska et al .", "moreover , non - hydroxyl or 9-hydroxyl derivatives also exhibited lipophilic properties , but its effect was not crucial when metabolic activation did not occur .", "relocation of hydroxyl group from position 8 to 9 drastically decreases antitumor activity ( c-1311 8-hydroxyl , c-1419 9-hydroxyl ) .", "in addition , hydrophobic properties of acridinones can play important role in transport and accumulation of these compounds in cells in view of fastening of metabolic activation ( skadanowski et al . ,", "on the other hand , diaminoalkyl side chain has also crucial influence on antitumor activity of acridinones . for compounds without 8-hydroxyl group , the increase in number of carbon atoms between nitrogen atoms from two to three or five ( c-1415 , c-1176 , and c-1233 two ; c-1212 and c-1296 three ; and c-1266 five ) generally decreases antitumor activity of imidazo- and triazoloacridinones . in case of derivatives bearing 8-hydroxyl group , the increase in number of carbon atoms ( c-1311 , c-1263 two , c-1371 three , and c-1492 five ) rather do not augment antitumor activity for imidazoacridinones , while good increase in antitumor activity is observed in case of triazoloacridinones ( c-1303 , c-1410 two , and c-1305 three carbon atoms ) .", "these observations indicate that length and conformation of diaminoalkyl side chain attached to flat aromatic acridinone ring have some influence on proper conformation of acridinone derivatives molecule and is in accordance with earlier modeling studies ( mazerski and muchniewicz , 2000 ) the observations also proved our suggestion that total molecular symmetry properties in context of interaction with dna as intercalation and dna groove binding are crucial for antitumor activity of acridinones.table  3values of molecular descriptors used in qsar analysiscompoundmolecular descriptorsgats7eih-047mpg3mlogpg2pg3pc-13101.073.70130.660.161.980.150.15c-13110.923.06160.660.152.190.150.15c-13301.193.16160.660.152.150.150.15c-14150.902.32140.670.151.160.150.15c-14190.892.01130.660.152.190.150.16c-15582.132.28130.650.150.150.150.15c-11760.942.50160.680.161.120.160.16c-12630.903.34150.670.162.870.160.16c-12121.012.61160.670.161.790.160.16c-13710.942.11150.670.152.820.150.15c-15540.832.66130.660.151.010.150.15c-12660.862.60130.660.150.950.150.16c-14920.863.10150.660.151.970.150.15c-12330.992.99160.680.171.120.170.16c-13030.872.48150.670.162.140.160.16c-15330.911.11150.670.161.780.170.16c-15672.153.53150.660.150.20.150.15c-14100.862.39110.670.162.160.160.16c-12960.943.08190.670.161.060.170.16c-13050.812.44180.670.172.090.160.16 values of molecular descriptors used in qsar analysis on the other hand , statistically significant parameters  values of molecular descriptors are presented in the table  3such as dipole moment ( i ) from class of electronic descriptors , mean atomic polarizability scaled on carbon atom ( mp ) from class of constitutional descriptors , geary autocorrelation - lag 7 weighted by atomic sanderson electronegativities ( gats7e ) from class of 2d autocorrelations descriptors , and h attached to c1(sp3)/co(sp2 ) ( h-047 ) from class of atom - centered fragments descriptors had the influence upon physicochemical ( noncovalent ) dna - duplexes stabilization of acridinone derivatives .", "the obtained data indicate that electronic and topological properties are important for noncovalent dna - duplexes stabilization of these compounds .", "it is known that drug  dna binding induces changes in dna structure and topology and is closely connected with conformation of drug molecule and its electronic and topological properties .", "the presence of a hydroxyl group in position 8 of acridinone ring slightly increases the affinity for dna compared to unsubstituted or alkyl - substituted derivatives , possibly because of additional hydrogen bonds with the dna phosphate backbone .", "as it was mentioned earlier ( mazerski and muchniewicz , 2000 ) , the charged diaminoalkyl side chain of acridinone compounds can interact with dna in the minor groove , in addition to intercalation .", "in addition , some other data ( koba and konopa , 2007 ) indicated that intercalation is not involved in stabilization of secondary structure of dna .", "however , for the biologically non - active compounds , c-1558 and c-1567 , bearing a t - butyl group in position 8 , the tm values were 2.4 and 6.8c , respectively , indicating that stabilization of the dna duplex by these compounds especially by c-1567 probably derived from electrostatic interactions of the side chain with dna and not from its intercalation to dna .", "this means that intercalation to dna is necessary for the biological activity of acridinones via positioning the drug molecules within dna before the covalent reaction and formation of interstrand dna crosslink ( koba and konopa , 2007 ) .", "this also indicated that topological and electronic properties of acridinone derivatives are important for their physicochemical interactions with dna .", "moreover , the molecular modeling studies ( mazerski and muchniewicz , 2000 ) evidenced that when acridinone c-1311 is intercalated between gc , the highly reactive position 8 on acridinone core is in close proximity to nucleophilic n7 position on guanine .", "it is plausible to postulate that drug molecule first intercalates into dna and then , after in situ activation , binds covalently to the neighboring base .", "these observations are compatible with recent findings demonstrating that electrochemically activated c-1311 forms covalent adducts with deoxyguanine ( mazerska et al . , 2003 ) .", "on the other hand , the structure of acridinones suggests that there are at least two possible sites for enzymatic oxidation / activation , which potentially could be involved in the covalent binding to dna .", "one is the diaminoalkyl side chain at position 5 which is necessary for covalent binding of mitoxantrone to dna ( skadanowski and konopa , 2000 ) .", "the other one is the potential quinone  imine group formed by hydroxyl group in position 8 ( 8-oh ) and heterocyclic nitrogen atom in acridinone nucleus ( mazerska et al . , 2003 ) .", "recently proposed mechanism of oxidation involves highly unstable carbocations generated in these two positions ( mazerska et al . , 2003 ) .", "it is suggested that c-1311 carbocations react rapidly with nucleophiles present in the environment , including dna bases forming covalent adducts .", "these observations indicate that topological and electronic properties of acridinone derivatives are also important for their covalent interactions with dna . moreover , the calculated values of ils and tm obtained for other compounds ( table  4 ) and the plots of the experimental data versus the calculated data ( fig . ", "1a  b ) for dna - duplexes stabilization of acridinones expressed as tm ( the increase in dna melting temperature at drug to dna base pairs 0.25  m ratio ) and antitumor activity of acridinones expressed as ils ( survival time of treated to control mice with p388 leukemia at optimal dose ) proved good correlation and predictive potency of proposed qsar models .", "in addition , the rmsecv as value , which quantifies the predictive power of the proposed qsar model , are calculated by the leave - one - out and the leave - ten - out methods and presented in the table  5 .", "the obtained values of rmsecv test ( 22.79 and 22.27 for quantitative structure  antitumor activity relationships as well as 2.39 and 2.41 for quantitative structure  ability to dna - duplexes stabilization relationships ) performed for all the four statistically significant independent variables proved the predictive power of the derived qsar models.table  4values of experimental and calculated data for dna - duplexes stabilization and antitumor activity of acridinonescompoundtm exp.tm calc.ils exp.ils calc.c-131015.312.62.718517213c-131113.713.60.193903c-133011.512.10.696897c-14157.28.81.655532c-14198.38.70.4274316c-15582.42.80.4055c-11769.58.90.6904644c-126312.312.50.21101100c-121211.510.21.3257045c-13713.58.55.01201137c-155410.511.10.6204727c-12669.910.70.81028c-149213.113.50.485823c-12339.110.00.9778811c-130313.110.13.01028319c-15338.15.72.4102313c-15676.86.30.5033c-14107.17.30.278846c-129611.514.02.518315c-130515.112.32.81651705mean value of 1.413the increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratiodifference between experimental and calculated valuesthe percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosefig . ", "1correlation between the experimental data and the calculated data from the derived multiple regression qsar equation for a dna - duplexes stabilization of acridinones expressed as tm ( the increase in dna melting temperature at drug to dna base pairs 0.25  m ratio ) and b antitumor activity of acridinones expressed as ils ( survival time of treated to control mice with p388 leukemia at optimal dose)table  5values of the cross - validated root - mean - square error rmsecv testqsar model for dependent variablermsecv testleave - one - out methodleave - ten - out method12341234tm3.362.532.562.393.442.632.642.41ils53.3942.1028.4822.7954.2342.3528.7422.2714 represents rmsecv test performed only for one , combined two and three , and for all the four significance descriptors in qsar models , respectively . in the case of qsar model , for tm as dependent - variable values ,", "14 were obtained for only gats7e , gats7e combined with i , gats7e combined with i and h-047 ,", "gats7e combined with i , h-047 , and mp descriptors . in the case of qsar model for ils as dependent - variable values , 14 were obtained for only g3 m , g3 m combined with logp , g3 m combined with logp and g2p , and g3 m combined with logp , g2p and g3p descriptors values of experimental and calculated data for dna - duplexes stabilization and antitumor activity of acridinones the increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio difference between experimental and calculated values the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose correlation between the experimental data and the calculated data from the derived multiple regression qsar equation for a dna - duplexes stabilization of acridinones expressed as tm ( the increase in dna melting temperature at drug to dna base pairs 0.25  m ratio ) and b antitumor activity of acridinones expressed as ils ( survival time of treated to control mice with p388 leukemia at optimal dose ) values of the cross - validated root - mean - square error rmsecv test 14 represents rmsecv test performed only for one , combined two and three , and for all the four significance descriptors in qsar models , respectively . in the case of qsar model , for tm as dependent - variable values , 14 were obtained for only gats7e , gats7e combined with i , gats7e combined with i and h-047 , gats7e combined with i , h-047 , and mp descriptors . in the case of qsar model for ils as dependent - variable values , 14 were obtained for only g3 m , g3 m combined with logp , g3 m combined with logp and g2p , and g3 m combined with logp , g2p and g3p descriptors", "statistically significant equations describing structure  antitumor activity relationships and structure  ability to physicochemical ( noncovalent ) interaction with dna relationships in acridinone derivatives group were derived .", "it has been found that hydrophobic and total molecular symmetry properties are important for antitumor activity of acridinone derivatives , and electronic and topological properties are important for physicochemical ( noncovalent ) dna - duplexes stabilization of these compounds ."], "abstract_text": ["<S> the acridinone derivatives with antitumor activity and ability with respect to noncovalent dna binding were investigated for their quantitative structure  activity relationships ( qsar ) . </S>", "<S> multiple regression analysis was used to model relationships between molecular descriptors and antileukemia activity , or between molecular descriptors and dna - duplexes stabilization . </S>", "<S> studies were performed on molecular modeling using hyperchem and dragon computer programs , and molecular geometry optimization using mm+ molecular mechanics and semi - empirical am1 method . </S>", "<S> two multiple regression equations were derived and characterized as good and with statistically significant correlations , r  =  0.9384 and r  =  0.8388 , for quantitative structure  antitumor activity relationships and quantitative structure  ability to dna - duplexes stabilization relationships , respectively . moreover , </S>", "<S> hydrophobic and total molecular symmetry properties are important for antitumor activity of acridinone derivatives , and electronic and topological properties are important for physicochemical ( noncovalent ) dna - duplexes stabilization of these compounds . </S>", "<S> the obtained equations can be used for prediction of acridinone derivatives activity and their ability to noncovalent interaction with dna which , as it was shown earlier , play important role in the antitumor mechanism of action of these compounds . </S>"], "labels": null, "section_names": ["Introduction", "Materials and methods", "Antitumor and physicochemical DNA-binding activity data of acridinones", "Structural parameters", "Statistical analysis", "Results and discussion", "Conclusions"], "sections": [["the acridinones represented by imidazo- and triazoloacridinones are a new group of potent antitumor compounds ( cholody et al . , 1990 , 1992 , 1996 ) from which one of the most active derivatives called as c-1305 has been selected for extended preclinical trials and the other one called c-1311 ( for review see mazerska et al", ". , 1998 ) is currently undergoing phase ii clinical trials as drug symadextm ( burger et al .", ", 1996 ; den brok et al . , 2005a , b , c ) .", "imidazoacridinones exhibit in vivo activity against various tumor cell lines as leukemia , melanoma , colon adenocarcinoma , and colon carcinoma .", "triazoloacridinones exhibit in vivo activity against leukemia , murine carcinoma , lung carcinoma , breast carcinoma , and colon carcinoma ( cholody et al .", ", 1990 , 1992 , 1996 ; kusnierczyk et al . , 1994 ; burger et al . ,", "as was previously shown ( skadanowski et al . , 1999 ; lemke et al . , 2004 ; augustin et al . , 2004 , 2006 ; wesierska - gadek et al . , 2004 ; koba and konopa , 2007 ; koba et al . , 2009 ) ,", "cellular dna is important target for the triazoloacridinone drugs , and hence interactions with dna are naturally the crucial point in view of the biological activity of these compounds . in previous article ( skadanowski et al . , 1999 ;", "lemke et al . , 2004 ) , it was indicated that triazoloacridinones inhibit cleavable complexes of topoisomerase ii with dna .", "they inhibit also nucleic acid or protein synthesis induced by g2 block of cell cycle followed by apoptosis ( augustin et al . , 2004 , 2006 ;", "2004 ) , intercalating to dna and binding in minor groove ( koba and konopa , 2007 ; koba et al . , 2009 ) and/or forming of interstrand dna crosslinks ( koba and konopa , 2007 ) .", "in addition , it was shown that intercalation to dna takes place preferentially in guanine triplet regions inducing changes in dna structures ( lemke et al .", ", 2005 ) . for imidazoacridinones , it was demonstrated that intercalation to dna undergoes at physiological condition with parallel stabilization of double - stranded dna and unwinding of supercoiled dna ( burger et al . , 1999 ;", "the intercalative binding mode of acridinone derivatives was also confirmed with the use of molecular - modeling studies ( mazerski and muchniewicz , 2000 ) .", "similar to other dna - binding agents , treatment of tumor cells with imidazoacridinones induces topoisomerase ii - associated dna strand breaks ( skadanowski et al . , 1996 ) , arrests cells in g2 phase , and stimulates apoptosis ( zaffaroni et al . , 2001 ; skwarska et al . , 2007 ) or mitotic catastrophe ( hyzy et al . , 2005 ; skwarska et al . , 2007", "however , after testing imidazoacridinone and triazoloacridinone derivatives , it has been concluded that although the intercalative binding to dna seems to be necessary for their biological activity ( the most active compounds have usually the highest binding affinity ) , it is not sufficient ( some inactive analogs also bind strongly with dna ) ( dziegielewski et al .", "moreover , acridinones undergo enzymatic oxidation , and this reaction is important for their biological activity as intercalation to dna and covalent adducts formation ( dziegielewski and konopa , 1996 ; mazerska et al . , 1999 , 2003 ) .", "in this context , noncovalent interaction of acridinones may help position drug molecules on dna for the covalent reaction . in this article ,", "physicochemical interactions of acridinones with dna were evaluated in view of quantitative structure  activity relationships ( qsar ) .", "multiple regression analysis was used to model relationships between molecular structural descriptors and biological ( antitumor ) activity of molecules , or between molecular structural descriptors and physicochemical interaction of acridinone derivatives with dna .", "the biological data expressed as anti - leukemia p388 activity and parameter describing ability to physicochemical ( noncovalent ) interaction with dna as value of dna - duplexes stabilization were applied in this study ."], ["the acridinone derivatives examined in this study have been selected to collect analogue compounds differing in chemical structures as well as anticancer activities ( table  1 ) .", "the data of acridinones antitumor activity against p388 leukemia in mice in vivo and expressed as the percentage of increase in survival time of the treated to that of the control mice with p388 leukemia at optimal dose ( ils ) were taken from the literature ( table  1 ) ( cholody et al .", ", 1990 , 1992 ; koba and konopa , 2007 ; mazerska et al . , 1996 ) . the data of physicochemical binding of acridinones to dna ( as values of dna - duplexes stabilization ) , which were expressed as an increase in dna melting temperature in centigrade degrees of ctdna at drug to dna base pairs 0.25  m ratio were taken from the literature ( table  1 ) ( koba and konopa , 2007 ; dziegielewski et al .", ", 2002).table  1chemical structures of acridinones studiedcompoundxnr1r2r3r4r5r6ilstmc-1310c2ch2ch3ch2ch3ohhch3h18515.3c-1311c2ch2ch3ch2ch3ohhhh9313.7c-1330c2ch2ch3ch2ch3och3hhh9611.5c-1415c2ch2ch3ch2ch3hhhh557.2c-1419c2ch2ch3ch2ch3hhhoh278.3c-1558c2ch2ch3ch2ch3c(ch3)3hhh02.4c-1176c2ch3ch3hhhh909.5c-1263c2ch3ch3ohhhh11012.3c-1212c3ch3ch3hhhh2511.5c-1371c3ch3ch3ohhhh1203.5c-1554c5ch2ch3ch2ch3ch3hhh2010.5c-1266c5ch3ch3hhhh109.9c-1492c5ch3ch3ohhhh8513.1c-1233n2ch3ch3hh  h779.1c-1303n2ch3ch3ohh  h10213.1c-1533n2ch3ch3ohch3h108.1c-1567n2ch3ch3c(ch3)3h  h06.8c-1410n2hch2ch3ohh  h787.1c-1296n3ch3ch3ch3h  h1811.5c-1305n3ch3ch3ohh  h16515.1the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosethe increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio chemical structures of acridinones studied the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose the increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio the structure of the tested compounds was studied by molecular modeling using hyperchem 7.5 release software ( kaliszan et al . , 1995 ; ivanciuc , 1996 ) and dragon software ( todeschini et al . , 2000", "the structures of the compounds were first pre - optimized with the molecular mechanics force field ( mm+ ) procedure included in the hyperchem 6.03 ( hypercube ) http://www.hyper.com , and the resulting geometries were further refined by means of the semi - empirical molecular orbitals method am1 using the polak - ribiere algorithm and a gradient norm limit of 0.01  kcal   .", "the following molecular descriptors taken from hyperchem software were considered among quantum and chemical indices : total energy ( te ) , binding energy ( be ) , isolated atomic energy ( iae ) , electron energy ( ee ) , core  core energy ( cce ) , heat flow ( hf ) , energy of the highest occupied molecular orbitals ( e_homo ) , energy of the lowest unoccupied molecular orbitals ( e_lumo ) , and difference between homo and lumo energies referred to as eg  =  energy gap ; ionization energy ( potential ) ( ie ) and electron affinity ( ea ) were calculated as a difference between the hf of positive molecular ion and electrically neutral molecule , and electronegativity ( en ) calculated as average arithmetic potential of ionization and ea .", "in addition , other parameters were used as the value of electron density of atom orbitals from the lowest to the highest ( ed_min and ed_max , respectively ) , the highest positive electron charge on the atoms ( max_pos ) , and the highest negative electron charge on the atoms ( max_neg ) , the difference between the highest positive and negative charge ( delta_q ) , distribution of dipolar moment along x , y , and z axes ( x_dm , y_dm , and z_dm , respectively ) , total dipolar moment ( tdm ) , mean polarizability of molecules ( in atom units ) mp ( mean polarizability ) , energy equal to the length of the wave with the greatest long - wave transfer of electrons , for which the value of oscillator force was different from zero ( el)the value of wave figures were converted to ev  and the value of the most intensive electron transfer ( emax  the maximum value of oscillator force calculated with the use of am1 method  as well as oscillator maximum force used for the transfer ( os_emax ) . moreover ,", "they include the following descriptors : surface area of the molecule available for solvent ( sa ) , molecule volume ( v ) , hydration energy ( he ) , the calculated distribution coefficient logarithm ( logp ) , refraction ( r ) , and polarizability ( p ) . on the other hand ,", "using dragon software , over 1,300 molecular descriptors were calculated and considered for qsar analysis .", "they include molecular parameters from different group and class of descriptors as constitutional , topological , walk and path counts , connectivity indices , information indices , 2d autocorrelations , edge adjacency indices , topological charge indices , eigenvalue - based indices , geometrical , 3d - morse , whim , getaway , functional group counts , atom - centred fragments , charge , molecular properties and other group of descriptors , and describing some properties of compound as geometry , symmetry , topology , electronic , steric or thermodynamic and other properties .", "the definitions of these descriptors are reviewed by todeschini ( todeschini et al . , 2000 ) .", "the statistical qsar analysis was performed with the use of stepwise regression analysis that used statistica 8.0 software ( statsoft , tulsa , ok , usa ) .", "the objective of stepwise regression is to construct a multivariate regression model ( qsar equation ) for a certain property , y , based on several selected explanatory variables . in stepwise regression", ", the first selected explanatory variable has the highest correlation with dependent variable , y. then , explanatory ( independent ) variables are consecutively added to the model in a forward selection procedure .", "a new variable is added to the model if a significant change in residuals of the model can be observed .", "the significance is evaluated using a statistical test , usually f - test ( the value of the f - test of significance , f ) .", "in addition , the multiple correlation coefficients ( r ) , the standard error of estimate ( s ) , and the significance levels of each term and of whole equation ( p ) are calculated for the derived qsar equations . whenever a new variable is included into a model", ", a backward elimination step follows in which an f - test detects the earlier selected variables , which can be removed from the model without any significant change on the level of the residuals .", "stepwise regression is very much popular in qsar studies , since the stepwise procedure is simple and based on the classical multiple linear regression ( mlr ) approach .", "one of the drawbacks of the method is the fact that no optimal variable selection is guaranteed , since the new variables are found based on the previously included variables into the model ( put et al . , 2006 ) .", "during model building , the model fit can be improved proportional to the model complexity .", "therefore , the more the factors are included into the model , the better the model fits the training data .", "usually , the model fit is evaluated by the root mean - squared error ( rmse ) , computed for the training data .", "the determination of the optimal complexity of the model requires an estimation of its predictive ability , to prevent overfitting to the calibration data .", "after all , the main goal of qsar models is to obtain a reasonable prediction of the retention for future samples . to evaluate the prediction by means of an internal validation procedures , cross validation can be used .", "the predictive ability of a model is characterized by the cross - validated root mean - squared error ( rmsecv ) ; test values were calculated with the matlab software ( mathworks , natick , ma , usa ) .", "the rmsecv as values , which quantify the predictive power of the qsar model , were calculated by the leave - one - out method and leave - ten - out method ."], ["the acridinone derivatives examined in this study have been selected to collect analogue compounds differing in chemical structures as well as anticancer activities ( table  1 ) .", "the data of acridinones antitumor activity against p388 leukemia in mice in vivo and expressed as the percentage of increase in survival time of the treated to that of the control mice with p388 leukemia at optimal dose ( ils ) were taken from the literature ( table  1 ) ( cholody et al .", ", 1990 , 1992 ; koba and konopa , 2007 ; mazerska et al . , 1996 ) . the data of physicochemical binding of acridinones to dna ( as values of dna - duplexes stabilization ) , which were expressed as an increase in dna melting temperature in centigrade degrees of ctdna at drug to dna base pairs 0.25  m ratio were taken from the literature ( table  1 ) ( koba and konopa , 2007 ; dziegielewski et al . , 2002).table  1chemical structures of acridinones studiedcompoundxnr1r2r3r4r5r6ilstmc-1310c2ch2ch3ch2ch3ohhch3h18515.3c-1311c2ch2ch3ch2ch3ohhhh9313.7c-1330c2ch2ch3ch2ch3och3hhh9611.5c-1415c2ch2ch3ch2ch3hhhh557.2c-1419c2ch2ch3ch2ch3hhhoh278.3c-1558c2ch2ch3ch2ch3c(ch3)3hhh02.4c-1176c2ch3ch3hhhh909.5c-1263c2ch3ch3ohhhh11012.3c-1212c3ch3ch3hhhh2511.5c-1371c3ch3ch3ohhhh1203.5c-1554c5ch2ch3ch2ch3ch3hhh2010.5c-1266c5ch3ch3hhhh109.9c-1492c5ch3ch3ohhhh8513.1c-1233n2ch3ch3hh  h779.1c-1303n2ch3ch3ohh  h10213.1c-1533n2ch3ch3ohch3h108.1c-1567n2ch3ch3c(ch3)3h  h06.8c-1410n2hch2ch3ohh  h787.1c-1296n3ch3ch3ch3h  h1811.5c-1305n3ch3ch3ohh  h16515.1the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosethe increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio chemical structures of acridinones studied the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose the increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio"], ["the structure of the tested compounds was studied by molecular modeling using hyperchem 7.5 release software ( kaliszan et al . , 1995 ; ivanciuc , 1996 ) and dragon software ( todeschini et al . , 2000", "the structures of the compounds were first pre - optimized with the molecular mechanics force field ( mm+ ) procedure included in the hyperchem 6.03 ( hypercube ) http://www.hyper.com , and the resulting geometries were further refined by means of the semi - empirical molecular orbitals method am1 using the polak - ribiere algorithm and a gradient norm limit of 0.01  kcal   .", "the following molecular descriptors taken from hyperchem software were considered among quantum and chemical indices : total energy ( te ) , binding energy ( be ) , isolated atomic energy ( iae ) , electron energy ( ee ) , core  core energy ( cce ) , heat flow ( hf ) , energy of the highest occupied molecular orbitals ( e_homo ) , energy of the lowest unoccupied molecular orbitals ( e_lumo ) , and difference between homo and lumo energies referred to as eg  =  energy gap ; ionization energy ( potential ) ( ie ) and electron affinity ( ea ) were calculated as a difference between the hf of positive molecular ion and electrically neutral molecule , and electronegativity ( en ) calculated as average arithmetic potential of ionization and ea .", "in addition , other parameters were used as the value of electron density of atom orbitals from the lowest to the highest ( ed_min and ed_max , respectively ) , the highest positive electron charge on the atoms ( max_pos ) , and the highest negative electron charge on the atoms ( max_neg ) , the difference between the highest positive and negative charge ( delta_q ) , distribution of dipolar moment along x , y , and z axes ( x_dm , y_dm , and z_dm , respectively ) , total dipolar moment ( tdm ) , mean polarizability of molecules ( in atom units ) mp ( mean polarizability ) , energy equal to the length of the wave with the greatest long - wave transfer of electrons , for which the value of oscillator force was different from zero ( el)the value of wave figures were converted to ev  and the value of the most intensive electron transfer ( emax  the maximum value of oscillator force calculated with the use of am1 method  as well as oscillator maximum force used for the transfer ( os_emax ) . moreover ,", "they include the following descriptors : surface area of the molecule available for solvent ( sa ) , molecule volume ( v ) , hydration energy ( he ) , the calculated distribution coefficient logarithm ( logp ) , refraction ( r ) , and polarizability ( p ) . on the other hand , using dragon software , over 1,300 molecular descriptors were calculated and considered for qsar analysis .", "they include molecular parameters from different group and class of descriptors as constitutional , topological , walk and path counts , connectivity indices , information indices , 2d autocorrelations , edge adjacency indices , topological charge indices , eigenvalue - based indices , geometrical , 3d - morse , whim , getaway , functional group counts , atom - centred fragments , charge , molecular properties and other group of descriptors , and describing some properties of compound as geometry , symmetry , topology , electronic , steric or thermodynamic and other properties ."], ["the statistical qsar analysis was performed with the use of stepwise regression analysis that used statistica 8.0 software ( statsoft , tulsa , ok , usa ) .", "the objective of stepwise regression is to construct a multivariate regression model ( qsar equation ) for a certain property , y , based on several selected explanatory variables . in stepwise regression ,", "the first selected explanatory variable has the highest correlation with dependent variable , y. then , explanatory ( independent ) variables are consecutively added to the model in a forward selection procedure .", "a new variable is added to the model if a significant change in residuals of the model can be observed .", "the significance is evaluated using a statistical test , usually f - test ( the value of the f - test of significance , f ) .", "in addition , the multiple correlation coefficients ( r ) , the standard error of estimate ( s ) , and the significance levels of each term and of whole equation ( p ) are calculated for the derived qsar equations . whenever a new variable is included into a model", ", a backward elimination step follows in which an f - test detects the earlier selected variables , which can be removed from the model without any significant change on the level of the residuals .", "stepwise regression is very much popular in qsar studies , since the stepwise procedure is simple and based on the classical multiple linear regression ( mlr ) approach .", "one of the drawbacks of the method is the fact that no optimal variable selection is guaranteed , since the new variables are found based on the previously included variables into the model ( put et al . , 2006 ) .", "during model building , the model fit can be improved proportional to the model complexity .", "therefore , the more the factors are included into the model , the better the model fits the training data .", "usually , the model fit is evaluated by the root mean - squared error ( rmse ) , computed for the training data .", "the determination of the optimal complexity of the model requires an estimation of its predictive ability , to prevent overfitting to the calibration data .", "after all , the main goal of qsar models is to obtain a reasonable prediction of the retention for future samples . to evaluate the prediction by means of an internal validation procedures , cross validation can be used .", "the predictive ability of a model is characterized by the cross - validated root mean - squared error ( rmsecv ) ; test values were calculated with the matlab software ( mathworks , natick , ma , usa ) .", "the rmsecv as values , which quantify the predictive power of the qsar model , were calculated by the leave - one - out method and leave - ten - out method ."], ["the chemical structures of the 20 compounds considered for this study and their antitumor and noncovalent dna - binding activities are presented in table  1 . in this study ,", "qsar analysis using multiple regression method was performed for biological activity data ( ils ) or physicochemical interaction with dna ( tm ) , and non - empirical parameters ( molecular descriptors ) .", "owing to a large number of non - empirical parameters ( over 1,300 ) , treated as independent variables and according to qsar strategy and multi - parameter regression rule in derived multi - parameter regression equation , the number of independent variables must be 56 times less than the number of cases considered in this study . in practice , for obtaining statistically significant equation , one independent variable ( in our case structural descriptor ) falls , generally out of five to maximum six cases considered , in dependent - variable activity ( in our case , activity of acridinones ) . in the research", "done , the data set of 20 acridinone derivatives ( dependent variables ) was taken to qsar analysis , and for this reason the derived qsar equations were maximally limited to four statistically significant independent variables ( structural descriptors ) .", "moreover , correlations were limited to the value of regression coefficient r    0.8 and an additional criterion , considered as relevant to particular independent variables , was established at the significance level p    0.05 .", "the calculated equations are presented in table  2 and characterized by four statistically significant independent variables with a good value of regression coefficient r    0.8 ( r  =  0.9384 and r  =  0.8388 for quantitative structure  antitumor activity relationships and quantitative structure  ability to dna - duplexes stabilization relationships , respectively ) . moreover , all the regression coefficients are highly statistically significant ( p  < ", "0.05 ) as is the whole equation ( p  <  7    10 for quantitative structure  antitumor activity relationships and p  <  9    10 for quantitative structure  ability to dna - duplexes stabilization relationships , respectively ) .", "the values of the multiple correlation coefficient , r ; the standard error of the estimate , s ; and the value of the f - test of significance , f , are also statistically significant.table  2multiple regression qsar equation ( dependent variable  =  k0  +  k1a  +  k2b  +  k3c  +  k4d)dependent variablecoefficients and statistically significant molecular descriptorsstatistical parametersk0k1ak2bk3ck4dr ( r)sfptm97.44    55.096.59    1.50gats7e3.03    0.88i0.64    0.30h-047147.44    83.58mp0.8388 ( 0.7036)2.158.907 ", "  10p  =  1    10p  =  5    10p  =  4    10p  =  5    10p  =  1    10ils88.80    153.448914.33   ", "1225.69g3m36.31    6.02logp4691.69    1227.99g2p4744.01    1451.51g3p0.9384 ( 0.8806)21.0327.659    10p  =  1    10p  =  3    10p  =  2    10p  =  2   ", "10p  =  5    10multiple correlation coefficient ( determination coefficient)standard error of estimatevalue of the f - test of significancesignificance level multiple regression qsar equation ( dependent variable  =  k0  +  k1a  +  k2b  +  k3c  +  k4d ) multiple correlation coefficient ( determination coefficient ) standard error of estimate value of the f - test of significance statistically significant parameters used in qsar analysis ( values of molecular descriptors are presented in the table  3 ) as logarithm of theoretically calculated n - octanol  water partition coefficient ( logp ) from the class of hydrophobic descriptors , 3st component symmetry directional whim index weighted by atomic masses ( g3 m ) , 2st component symmetry directional whim index by atomic polarizabilities ( g2p ) , and 3st component symmetry directional whim directional index / weighted by atomic polarizabilities ( g3p ) from class of whim ( weighted holistic invariant molecular ) descriptors had the influence upon antitumor activity of acridinones .", "the whim descriptors are molecular descriptors based on statistical indices calculated on the projections of the atoms along principal axes .", "they are built in such a way as to capture relevant molecular 3d information regarding molecular size shape , symmetry , and atom distribution with respect to invariant reference frames ( todeschini et al . , 2000 ) . in general , the obtained data indicate that hydrophobic and total molecular symmetry properties are important for antitumor activity of acridinones .", "these observations are in partial agreement with the data obtained by mazerska ( mazerska et al . ,", "however , impact of lipophilicity on the biological activity of these compounds was observed only in the case of derivatives with 8-hydroxyl group , which undergo metabolic activation ( mazerska et al .", "moreover , non - hydroxyl or 9-hydroxyl derivatives also exhibited lipophilic properties , but its effect was not crucial when metabolic activation did not occur .", "relocation of hydroxyl group from position 8 to 9 drastically decreases antitumor activity ( c-1311 8-hydroxyl , c-1419 9-hydroxyl ) .", "in addition , hydrophobic properties of acridinones can play important role in transport and accumulation of these compounds in cells in view of fastening of metabolic activation ( skadanowski et al . ,", "on the other hand , diaminoalkyl side chain has also crucial influence on antitumor activity of acridinones . for compounds without 8-hydroxyl group , the increase in number of carbon atoms between nitrogen atoms from two to three or five ( c-1415 , c-1176 , and c-1233 two ; c-1212 and c-1296 three ; and c-1266 five ) generally decreases antitumor activity of imidazo- and triazoloacridinones . in case of derivatives bearing 8-hydroxyl group , the increase in number of carbon atoms ( c-1311 , c-1263 two , c-1371 three , and c-1492 five ) rather do not augment antitumor activity for imidazoacridinones , while good increase in antitumor activity is observed in case of triazoloacridinones ( c-1303 , c-1410 two , and c-1305 three carbon atoms ) .", "these observations indicate that length and conformation of diaminoalkyl side chain attached to flat aromatic acridinone ring have some influence on proper conformation of acridinone derivatives molecule and is in accordance with earlier modeling studies ( mazerski and muchniewicz , 2000 ) the observations also proved our suggestion that total molecular symmetry properties in context of interaction with dna as intercalation and dna groove binding are crucial for antitumor activity of acridinones.table  3values of molecular descriptors used in qsar analysiscompoundmolecular descriptorsgats7eih-047mpg3mlogpg2pg3pc-13101.073.70130.660.161.980.150.15c-13110.923.06160.660.152.190.150.15c-13301.193.16160.660.152.150.150.15c-14150.902.32140.670.151.160.150.15c-14190.892.01130.660.152.190.150.16c-15582.132.28130.650.150.150.150.15c-11760.942.50160.680.161.120.160.16c-12630.903.34150.670.162.870.160.16c-12121.012.61160.670.161.790.160.16c-13710.942.11150.670.152.820.150.15c-15540.832.66130.660.151.010.150.15c-12660.862.60130.660.150.950.150.16c-14920.863.10150.660.151.970.150.15c-12330.992.99160.680.171.120.170.16c-13030.872.48150.670.162.140.160.16c-15330.911.11150.670.161.780.170.16c-15672.153.53150.660.150.20.150.15c-14100.862.39110.670.162.160.160.16c-12960.943.08190.670.161.060.170.16c-13050.812.44180.670.172.090.160.16 values of molecular descriptors used in qsar analysis on the other hand , statistically significant parameters  values of molecular descriptors are presented in the table  3such as dipole moment ( i ) from class of electronic descriptors , mean atomic polarizability scaled on carbon atom ( mp ) from class of constitutional descriptors , geary autocorrelation - lag 7 weighted by atomic sanderson electronegativities ( gats7e ) from class of 2d autocorrelations descriptors , and h attached to c1(sp3)/co(sp2 ) ( h-047 ) from class of atom - centered fragments descriptors had the influence upon physicochemical ( noncovalent ) dna - duplexes stabilization of acridinone derivatives .", "the obtained data indicate that electronic and topological properties are important for noncovalent dna - duplexes stabilization of these compounds .", "it is known that drug  dna binding induces changes in dna structure and topology and is closely connected with conformation of drug molecule and its electronic and topological properties .", "the presence of a hydroxyl group in position 8 of acridinone ring slightly increases the affinity for dna compared to unsubstituted or alkyl - substituted derivatives , possibly because of additional hydrogen bonds with the dna phosphate backbone .", "as it was mentioned earlier ( mazerski and muchniewicz , 2000 ) , the charged diaminoalkyl side chain of acridinone compounds can interact with dna in the minor groove , in addition to intercalation .", "in addition , some other data ( koba and konopa , 2007 ) indicated that intercalation is not involved in stabilization of secondary structure of dna .", "however , for the biologically non - active compounds , c-1558 and c-1567 , bearing a t - butyl group in position 8 , the tm values were 2.4 and 6.8c , respectively , indicating that stabilization of the dna duplex by these compounds especially by c-1567 probably derived from electrostatic interactions of the side chain with dna and not from its intercalation to dna .", "this means that intercalation to dna is necessary for the biological activity of acridinones via positioning the drug molecules within dna before the covalent reaction and formation of interstrand dna crosslink ( koba and konopa , 2007 ) .", "this also indicated that topological and electronic properties of acridinone derivatives are important for their physicochemical interactions with dna .", "moreover , the molecular modeling studies ( mazerski and muchniewicz , 2000 ) evidenced that when acridinone c-1311 is intercalated between gc , the highly reactive position 8 on acridinone core is in close proximity to nucleophilic n7 position on guanine .", "it is plausible to postulate that drug molecule first intercalates into dna and then , after in situ activation , binds covalently to the neighboring base .", "these observations are compatible with recent findings demonstrating that electrochemically activated c-1311 forms covalent adducts with deoxyguanine ( mazerska et al . , 2003 ) .", "on the other hand , the structure of acridinones suggests that there are at least two possible sites for enzymatic oxidation / activation , which potentially could be involved in the covalent binding to dna .", "one is the diaminoalkyl side chain at position 5 which is necessary for covalent binding of mitoxantrone to dna ( skadanowski and konopa , 2000 ) .", "the other one is the potential quinone  imine group formed by hydroxyl group in position 8 ( 8-oh ) and heterocyclic nitrogen atom in acridinone nucleus ( mazerska et al . , 2003 ) .", "recently proposed mechanism of oxidation involves highly unstable carbocations generated in these two positions ( mazerska et al . , 2003 ) .", "it is suggested that c-1311 carbocations react rapidly with nucleophiles present in the environment , including dna bases forming covalent adducts .", "these observations indicate that topological and electronic properties of acridinone derivatives are also important for their covalent interactions with dna . moreover , the calculated values of ils and tm obtained for other compounds ( table  4 ) and the plots of the experimental data versus the calculated data ( fig . ", "1a  b ) for dna - duplexes stabilization of acridinones expressed as tm ( the increase in dna melting temperature at drug to dna base pairs 0.25  m ratio ) and antitumor activity of acridinones expressed as ils ( survival time of treated to control mice with p388 leukemia at optimal dose ) proved good correlation and predictive potency of proposed qsar models .", "in addition , the rmsecv as value , which quantifies the predictive power of the proposed qsar model , are calculated by the leave - one - out and the leave - ten - out methods and presented in the table  5 .", "the obtained values of rmsecv test ( 22.79 and 22.27 for quantitative structure  antitumor activity relationships as well as 2.39 and 2.41 for quantitative structure  ability to dna - duplexes stabilization relationships ) performed for all the four statistically significant independent variables proved the predictive power of the derived qsar models.table  4values of experimental and calculated data for dna - duplexes stabilization and antitumor activity of acridinonescompoundtm exp.tm calc.ils exp.ils calc.c-131015.312.62.718517213c-131113.713.60.193903c-133011.512.10.696897c-14157.28.81.655532c-14198.38.70.4274316c-15582.42.80.4055c-11769.58.90.6904644c-126312.312.50.21101100c-121211.510.21.3257045c-13713.58.55.01201137c-155410.511.10.6204727c-12669.910.70.81028c-149213.113.50.485823c-12339.110.00.9778811c-130313.110.13.01028319c-15338.15.72.4102313c-15676.86.30.5033c-14107.17.30.278846c-129611.514.02.518315c-130515.112.32.81651705mean value of 1.413the increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratiodifference between experimental and calculated valuesthe percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dosefig . ", "1correlation between the experimental data and the calculated data from the derived multiple regression qsar equation for a dna - duplexes stabilization of acridinones expressed as tm ( the increase in dna melting temperature at drug to dna base pairs 0.25  m ratio ) and b antitumor activity of acridinones expressed as ils ( survival time of treated to control mice with p388 leukemia at optimal dose)table  5values of the cross - validated root - mean - square error rmsecv testqsar model for dependent variablermsecv testleave - one - out methodleave - ten - out method12341234tm3.362.532.562.393.442.632.642.41ils53.3942.1028.4822.7954.2342.3528.7422.2714 represents rmsecv test performed only for one , combined two and three , and for all the four significance descriptors in qsar models , respectively . in the case of qsar model , for tm as dependent - variable values ,", "14 were obtained for only gats7e , gats7e combined with i , gats7e combined with i and h-047 ,", "gats7e combined with i , h-047 , and mp descriptors . in the case of qsar model for ils as dependent - variable values , 14 were obtained for only g3 m , g3 m combined with logp , g3 m combined with logp and g2p , and g3 m combined with logp , g2p and g3p descriptors values of experimental and calculated data for dna - duplexes stabilization and antitumor activity of acridinones the increase in dna melting temperature ( expressed in centigrade degrees ) at drug to dna base pairs 0.25  m ratio difference between experimental and calculated values the percentage of increase in survival time of treated to control mice with p388 leukemia at optimal dose correlation between the experimental data and the calculated data from the derived multiple regression qsar equation for a dna - duplexes stabilization of acridinones expressed as tm ( the increase in dna melting temperature at drug to dna base pairs 0.25  m ratio ) and b antitumor activity of acridinones expressed as ils ( survival time of treated to control mice with p388 leukemia at optimal dose ) values of the cross - validated root - mean - square error rmsecv test 14 represents rmsecv test performed only for one , combined two and three , and for all the four significance descriptors in qsar models , respectively . in the case of qsar model , for tm as dependent - variable values , 14 were obtained for only gats7e , gats7e combined with i , gats7e combined with i and h-047 , gats7e combined with i , h-047 , and mp descriptors . in the case of qsar model for ils as dependent - variable values , 14 were obtained for only g3 m , g3 m combined with logp , g3 m combined with logp and g2p , and g3 m combined with logp , g2p and g3p descriptors"], ["statistically significant equations describing structure  antitumor activity relationships and structure  ability to physicochemical ( noncovalent ) interaction with dna relationships in acridinone derivatives group were derived .", "it has been found that hydrophobic and total molecular symmetry properties are important for antitumor activity of acridinone derivatives , and electronic and topological properties are important for physicochemical ( noncovalent ) dna - duplexes stabilization of these compounds ."]]}