Tuning Exciton Coupling of Merocyanine Nucleoside Dimers by RNA, DNA and GNA Double Helix Conformations

Abstract Exciton coupling between two or more chromophores in a specific environment is a key mechanism associated with color tuning and modulation of absorption energies. This concept is well exemplified by natural photosynthetic proteins, and can also be achieved in synthetic nucleic acid nanostructures. Here we report the coupling of barbituric acid merocyanine (BAM) nucleoside analogues and show that exciton coupling can be tuned by the double helix conformation. BAM is a nucleobase mimic that was incorporated in the phosphodiester backbone of RNA, DNA and GNA oligonucleotides. Duplexes with different backbone constitutions and geometries afforded different mutual dye arrangements, leading to distinct optical signatures due to competing modes of chromophore organization via electrostatic, dipolar, π–π‐stacking and hydrogen‐bonding interactions. The realized supramolecular motifs include hydrogen‐bonded BAM–adenine base pairs and antiparallel as well as rotationally stacked BAM dimer aggregates with distinct absorption, CD and fluorescence properties.


Materials
All standard chemicals and solvents were purchased from commercial suppliers. Organic solvents were used in pro analysis or for synthesis quality without further purification. Solvents for extraction and column chromatography were purchased in technical quality and distilled prior to use. Dry solvents (dichloromethane, THF, DMF, acetonitrile) were obtained from a solvent purification system (SPS). Nanopure water was obtained from a Sartorius Arium ® pro ultrapure water system. Thin layer chromatography (TLC) was performed silica gel pre-coated aluminum plates (Alugram SIL G/UV254, Macherey-Nagel

Synthesis of phosphoramidite building blocks for incorporation of rBAM, dBAM, gBAM
Scheme S1. Reagents and conditions for the synthesis of the rBAM phosphoramidite building block 8 (see also The synthesis of the rBAM phosphoramidite building block for RNA solid-phase synthesis started with a Silyl-Hilbert-Johnson nucleosidation of 2,4,6-tris(trimethylsilyloxy)pyrimidine S1 (prepared from barbituric acid (BA) with hexamethyldisilazane as described [2] ) to obtain the protected nucleoside S2. After deprotection of the 2', 3' and 5' positions with sodium methoxide, the free BA riboside (5) was coupled in an aldol condensation reaction with aldehyde 7, which was derived from Fischer's indole by Vilsmeier-Haack reaction as previously reported [3] , to give the merocyanine nucleoide rBAM (2). In situ 2',3'-acetal protection of rBAM was followed by 5'-tritylation to give S6. Then, 2'-silyl protection gave derivative S7, which was finally converted to the rBAM phosphoramidite building block 8 by phosphitylation of the 3'-OH group.
Scheme S2. Reagents and conditions for the synthesis of the DNA phosphoramidite building block 9 (see also For the synthesis of the dBAM phosphoramidite building block 9, silylated barbituric acid S1 was reacted with Hoffer's chlorosugar to give an 1:1 anomeric mixture of the desired β-anomer 6 and the respective α-anomer. The two anomers were separated by recrystallization from methanol [4] and the β-anomer was further reacted with the aldehyde 7 to give the protected nucleoside S10. Deprotection of the hydroxyl groups gave dBAM (3), followed by 5'-DMT protection to S12 and phosphitylation to afford the dBAM phosphoramidite building block 9. The synthesis of the gBAM phosphoramidite building block 10 started with an aldol condensation reaction of aldehyde 7 with barbituric acid to give the free chromophore BAM (1). Afterwards, the DMT-protected (R)-glycidol was attached to one of the barbituric acid nitrogen atoms of BAM by a sodium hydride catalyzed epoxide ring opening reaction. The obtained DMT-protected building block S15 was converted to the GNA phosphoramidite building block 10 by phosphitylation.

Synthesis and purification of oligonucleotides
Automated solid phase synthesis was carried using an Applied Biosystems ABI 392 DNA/RNA synthesizer on a 0.6 µmol scale. The following solutions were used: 0.25 M ethylthiotetrazole (ETT) in dry acetonitrile as activator, 3% dichloro acetic acid in 1,2dichloroethane for detritylation of RNA oligonucleotides and 3% trichloro acetic acid in 1,2-dichloroethane for DNA and GNA oligonucleotides, 20 mM iodine in THF/water/pyridine 66/12/22 for oxidation, pyridine/acetic anhydride/THF 10/10/80 as Cap A solution and 16% NMI in THF as Cap B solution. DNA and RNA syntheses were carried out in DMT-off mode while GNA oligonucleotides were synthesized in DMT-on mode to prevent degradation during alkaline deprotection. [7] RNA and DNA oligonucleotides were prepared on commercially available CPG solid supports using 70 mM solutions for standard phosphoramidites and 100 mM solutions for self-made phosphoramidites. Coupling times were 2 min for DNA, and 4 min for RNA and GNA. GNA oligonucleotides were prepared on a 3'phosphate solid support.
RNA oligonucleotides were cleaved from the solid support and deprotected by incubation with aqueous ammonia/abs. ethanol 3/1 overnight at 37 °C. Afterwards, the solid support was filtered off and the solvent was removed in vacuum. The crude samples were redissolved in 1 M TBAF solution in THF (500 µL) and shaken overnight at r. t. After addition of 1 M Tris-HCl pH 8.0 (500 µL), the THF was removed in vacuum and the oligonucleotide samples were desalted by size exclusion chromatography using an Äkta start purification system with three HiTRAP Desalting columns (each 5 mL volume) from GE Healthcare. The desalted oligonucleotides were eluted with water and a flow rate of 2 mL/min. The water was removed under reduced pressure and the samples were redissolved in 600 µL of nanopure water.
DNA oligonucleotides were cleaved from the solid support and deprotected by incubation with 1 mL of aqueous ammonia overnight at 37 °C. After filtration and removal of the solvent in vacuum, the crude oligonucleotides were redissolved in 600 µL of nanopure water.
GNA oligonucleotides were cleaved from the solid support and deprotected by incubation with 1 mL of aqueous ammonia overnight at 37 °C. After filtration and removal of the solvent under reduced pressure, the crude oligonucleotides were dissolved in 600 µL nanopure water. The samples were divided into four portions of 150 µL each and to each portion 150 µL of acetic acid was added. After incubation for 30 min at r. t., 3 M NaCl solution was added to a final NaCl concentration of 300 mM. The crude oligonucleotides were then precipitated with absolute ethanol (70% final concentration) and the dried pellets were redissolved in 600 µL of nanopure water. PAGE purification of the crude oligonucleotides was performed on 300x200x0.7 mm denaturing polyacrylamide gels (20% acrylamide/bisacrylamide 19:1, 7 M urea) with 1 M TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH8.3). Gels were run at 35 W constant power for 2.5 h. The oligonucleotides were visualized by UV shadowing on a TLC plate. After excision from the gel, the gel pieces were soaked in TEN buffer (10 mM Tris-HCl, 0.1 mM EDTA, 300 mM NaCl, pH 8.0) for oligonucleotide extraction. The pure oligonucleotides were precipitated with absolute ethanol (70% final concentration).
Crude as well as pure oligonucleotides were analyzed by anion exchange HPLC. A Dionex DNAPac PA200 column (2x250 mm) with buffer A (25 mM Tris-HCl, 6 M urea, pH 8.0) and buffer B (25 mM Tris, 0.5 M NaClO 4 , 6 M urea, pH8.0) was used for this quality control. Samples were run with a gradient of 0-48% B over 12 column volumes with a flow rate of 0.5 mL/min and UV detection at 260 nm. The oven temperature was set to 60 °C. Pure oligonucleotides were additionally analyzed by HR-ESI mass spectrometry, and the monoisotopic masses were obtained by deconvolution of the raw spectra.

Spectroscopic characterization 4.1 UV/vis spectroscopy
UV/vis absorbance spectra were recorded on a JASCO V-770 spectrophotometer with a PAC-743 cell changer and a Julabo F250 temperature controlling device. 5 µM samples were prepared in 1x phosphate buffer (100 mM NaCl, 10 mM phosphate, pH7.0) and measured in semi-micro quartz cuvettes with a path length of 10 mm. Temperature-dependent spectra were recorded in a temperature range of 20 to 80 °C in steps of 10 °C. Spectra were measured from 200 to 700 nm with a spectral bandwidth of 2 nm. The response was set to 0.06 s. A scan speed of 1000 nm/min with a data interval of 0.5 nm was used.

Thermal UV melting curves
Thermal UV melting curves from 1, 2, 5, 10 and 20 µM samples in phosphate buffer (100 mM NaCl, 10 mM phosphate, pH7.0) were recorded at a VARIAN CARY 100 Bio spectrophotometer equipped with a 6x6 Multicell Block Peltier Series II cell changer and a VARIAN CARY Temperature Controller. For sample concentrations of 1, 2 and 5 µM, 500 µL final sample volumes were prepared and measured in 10 mm semi-micro quartz cuvettes. For sample concentrations of 10 and 20 µM, 300 µL final sample volumes were prepared and measured in 1 mm cuvettes. Prior to measurement, the samples were overlaid with silicon oil to prevent evaporation during heating. Five temperature ramps with a heating/cooling rate of 0.5 °C/min between 10 and 90 °C at 250, 260, 280 and 460 nm were collected. The melting curves were fit to a two-state transition model with upper and lower baselines to determine T m as previously reported, [1b] and thermodynamic parameters were obtained from van't Hoff analysis, plotting 1/T m versus lnc tot .

CD and CPL spectroscopy
CD spectra from 5 µM samples in 1x phosphate buffer (100 mM NaCl, 10 mM phosphate, pH7.0) were recorded on a JASCO P-1020 polarimeter with a sodium light source (λ = 589 nm) in a range between 200 and 600 nm. For the measurements, 10 mm semi-micro quartz cuvettes were used. CPL spectra for 1 µM samples in 1x phosphate buffer (100 mM NaCl, 10 mM phosphate, pH7.0) were recorded on a customized JASCO CPL-300/J-1500 hybrid spectrometer in a range between 600 and 200 nm. 10 mm semi-micro quartz cuvettes were used for measurements.

Fluorescence spectroscopy
Fluorescence spectra were recorded on a Jasco FP-8300 spectrofluorometer equipped with a FCT-817S cell changer and a Julabo F12 temperature control device. 1 µM samples were prepared in 1x phosphate buffer (100 mM NaCl, 10 mM phosphate, pH7.0) and measured in Hellma ultra-micro quartz cuvettes (10x2 mm). Spectra were recorded with an excitation wavelength of 420 nm (measured emission range 440-700 nm) and an emission wavelength of 540 nm (measured excitation range 200-520 nm). Excitation and emission bandwidth were both set to 5 nm. Spectra were recorded with 0.1 s response, a PMT voltage of 400 V with 0.5 nm data interval and a scan speed of 500 nm/min. Absolute fluorescence quantum yield of fluorescein (0.1 µM in 100 mM NaCl, 10 mM phosphate, pH7.0) was conducted with an ILF-835 integrating sphere in a Jasco 6808-H150A liquid cell (1x10x250 mm side length). Spectra were recorded in an emission range between 420 and 750 nm with an excitation wavelength of 435 nm, excitation and emission bandwidths were 2.5 nm, data interval 0.2 nm, scan speed 100 nm/min, response 1 s, PMT voltage 450 V. For relative quantum yield determination, a dilution series (1, 2, 5 µM) of the analyte in 1x phosphate buffer (100 mM NaCl, 10 mM phosphate, pH7.0) was prepared. Fluorescence measurements were conducted in Hellma ultra-micro quartz cuvettes (10x2 mm). spectra were recorded with excitation at 435 nm, emission range 440 to 750 nm, excitation and emission bandwidths 2.5 nm, scan speed 200 nm/min, data interval 0.2 nm, response time 1 s, PMT voltage 300 V. After fluorescence measurement, the samples were transferred into VARIAN semi-micro quartz cuvettes with a path length of 10 mm for absorbance measurements.
Fluorescence lifetimes were determined by TCSPC using a Horiba DeltaFlex spectrometer with a DeltaDiode excitation source (DD-405L ( ex 408 nm) or DD-485L( ex 477 nm), under magic angle conditions (54.7°) of polarization filters. 1 µM samples in 1x phosphate buffer (100 mM NaCl, 10 mM phosphate, pH7.0) were measured in Hellma ultra-micro quartz cuvettes (10×2 mm). The emission bandwidth was adjusted for a stop rate of close to 2%. The photon arrival time was set to 4096 channels (0.026 ns per channel), and data was collected until 10,000 counts had accumulated in the peak channel. The instrument response function was recorded from only buffer by setting the emission monochromator to the peak wavelength of the excitation source. Fluorescence decay curves were analyzed by iterative reconvolution fitting with the instrument response function using the algorithm implemented in Horiba DAS6 (version 6.8).

Simulation of absorption spectra
The absorption spectra for the merocyanine dimer were calculated employing a Holstein-type Hamiltonian [8] with the two-particle approximation [9] following the procedure as previously described. [10] A collinear assignment of the chromophores was assumed with a long-range Coulomb coupling J Coul between the transition dipole moments of the chromophores. First, the monomer spectrum was calculated to adjust the parameters. Accordingly, best agreement with the experimental absorption spectrum of the monomer was obtained with a transition energy of E g = 21400 cm -1 and a vibrational frequency of ω 0 = 1200 cm -1 assuming a one-effective mode. Furthermore, a Huang-Rhys factor of λ 2 = 0.74 has been used, which determines the ratio between the 0-1 and 0-0 absorption bands of the vibronic progression. For the dimer spectrum, a Coulomb coupling of J Coul = 1100 cm -1 has been applied. Furthermore, the Huang-Rhys factor had to be decreased to λ 2 = 0.61 in order to obtain best agreement with the experimental dimer spectrum. The lower Huang-Rhys factor compared to the monomer can be rationalized by the polarizability effect of the adjacent chromophore within the dimer stack, which puts the chromophores closer to the cyanine limit compared to the monomeric chromophore, [11] leading to a lower effective Huang-Rhys factor. Additionally, a "gas-to-crystal" shift of -150 cm -1 was applied to adjust the position of the absorption maximum with the experimental spectrum. For all simulated spectra, a maximum number of 7 vibrational quanta has been used for each chromophore. The absorption spectra were simulated by Here, is the absorption at photon energy and is the transition energy between the ground state with wave function Ѱ and the respective excited state Ѱ . is the band-width with 2 ln 2 being the full width at half height. A value of = 830 cm -1 and = 520 cm -1 has been used for the simulation of the monomer and dimer spectrum, respectively. According to equation (1), Gaussian line shape functions are used to simulate the spectra. In addition, ̂ is the transition dipole moment operator and defined as with the transition dipole moment of the monomeric chromophore. | means that chromophore is electronically excited, whereas | indicates that all chromophores are in the electronic ground state.

Geometry optimization
Geometry optimization for the two geometrical stereoisomers of merocyanine derivative S17 (in which the ribose/deoxyribose/glycidol substituent is abbreviated as a methyl group) have been performed in order to estimate the energy difference between the two isomers. Toward this goal, the structures were optimized at the level of density functional theory employing the long-range correct CAM-B3LYP-D3 [12] functional together with the def2-SVP [13] basis set as implemented in the Gaussian 09 [14] package. Accordingly, the E-isomer is only slightly higher in energy than the respective Z-isomer with an energy difference of 0.70 kJ/mol.
The overlay of the two isomers shows that they have almost equal steric demand and their dipole moments are essentially overlapping. For these reasons the two (experimentally nearly unseparable) isomers are considered as equal in the analysis of the dimer aggregates. Tables   Table S1. Sequences of RNA, DNA and GNA oligonucleotides, isolated yields of purified oligonucleotides, and HR-ESI mass spec data.     Table S2 for sequences). b) Excitation and emission spectra of free BAM nucleobase surrogate (magenta, same as in a) in comparison to single-stranded DNA DS3 containing dBAM, and the duplex D1a, containing adenine opposite to dBAM (see Table S3 for sequences). d) Temperature dependence of folding-induced fluorescence enhancement shows that emission intensity (ex = 420 nm) increases upon cooling below the melting temperature of the duplex, reflecting the rigidifaction of the assembly upon hybridization.   Table S5, for R1, D1, G1 and DS6 the lifetimes are too short and cannot be determined reliably (< 0.1 ns).

Melting curves and van't Hoff plots for UV melting analyses.
For all duplex structures, the hyperchromicity at 260 nm for the final heating ramp and the corresponding van't Hoff plot are shown. The standard deviation for the estimated melting temperatures is ±0.5 °C.