the development of 
 novel conformationally restricted nucleotides 
 is a vibrant area of research .
efforts are driven by 
 the interesting properties of oligodeoxyribonucleotides ( ons ) modified 
 with classic examples of conformationally restricted nucleotides such 
 as homo - dna , hexitol nucleic acid ( hna ) , cyclohexane nucleic acid ( cena ) , bicyclo dna , tricyclo dna , or locked nucleic acid ( lna ) , which 
 is also known as bridged nucleic acid ( bna ) .
ons comprising these building blocks display high affinity toward 
 complementary dna / rna often due to reduced entropic binding penalties and are accordingly in high demand for a wide 
 range of nucleic acid targeting applications in molecular biology , 
 biotechnology , and pharmaceutical science .
their use as rna - targeting antisense oligonucleotides to decrease 
 gene expression is a particularly prominent example .
lna is an especially interesting member of this compound 
 class 
 because it induces some of the greatest duplex stabilizations observed 
 to date ( figure 1 ) .
modulation 
 of gene expression through lna - mediated targeting of mrna , pre - mrna , 
 or mirna has accelerated gene function studies and led to the development 
 of lna - based drug candidates against diseases of genetic origin .
other applications of lna include its use as an in situ hybridization 
 probe to monitor spatiotemporal expression patterns of mirnas .
many analogues of lna have been synthesized 
 with the aim of further 
 improving the binding affinity / specificity , enzymatic stability and 
 pharmokinetic characteristics of lna .
the vast majority of these efforts have focused on 
 modifying the oxymethylene bridge spanning the c2/c4-positions 
 and/or introducing minor - groove - oriented substituents on the bridge . 

these structural perturbations have resulted in improved enzymatic 
 stability and altered biodistribution and/or toxicity profiles but 
 have generally not resulted in major improvements in binding affinity 
 and specificity .
c5-functionalized pyrimidine dna monomers have 
 also attracted considerable 
 attention , as they enable predictable positioning 
 of functional entities in the major groove of nucleic acid duplexes .
small c5-alkynyl substituents such as propyn-1-yl 
 and 3-aminopropyn-1-yl induce considerable duplex thermostabilization 
 relative to unmodified duplexes , while large hydrophobic substituents 
 typically decrease duplex thermostability .
attachment of polarity - sensitive 
 fluorophores to the c5 position of dna pyrimidine monomers has produced 
 several interesting oligonucleotide probes for structural studies 
 of nucleic acids and applications in nucleic acid diagnostics . in light of the above and our ongoing interest in lna chemistry
, we recently set out to study c5-alkynyl - functionalized lna uridine 
 ( u ) monomers , on the basis of the hypothesis that these monomers will 
 exhibit beneficial properties from both compound classes , i.e. , high 
 affinity toward rna complements and good mismatch discrimination ( lna ) , 
 along with the ability to position blocking groups in the major groove 
 to confer protection against enzymatic degradation ( c5 substituent ) . 

ons modified with small c5-alkynyl - functionalized 
 lna - u monomers display high affinity toward complementary rna and 
 moderate protection against 3-exonucleases , while ons modified 
 with large c5-alkynyl - functionalized lna - u monomers display greatly 
 increased enzymatic stability but decreased rna affinity . in 
 the present article ,
we describe full experimental details concerning 
 the synthesis of 12 different c5-functionalized lna - u phosphoramidites , 
 their incorporation into ons , and the characterization of these modified 
 ons by means of thermal denaturation experiments , analysis of thermodynamic 
 parameters , nuclease stability experiments , and fluorescence spectroscopy . 

the monomers in question were selected to ensure a representation 
 of substituents with different sizes , polarities , linker chemistries , 
 and fluorescence characteristics ( figure 1 ) .
our route to target phosphoramidites 5b  l initiates from lna uridine diol 1 , which is 
 obtained from commercially available diacetone--d - allose 
 in 52% yield ( scheme 1 ) .
c5-iodination of 1 was 
 accomplished through treatment with iodine and ceric ammonium nitrate 
 ( can ) in acetic acid at 80 c for 40 min to afford nucleoside 2 in 87% yield .
prolonged heating and/or higher reaction temperatures 
 result in the formation of nonpolar impurities , which complicate purification 
 and reduce product yield .
terminal alkynes were 
 then coupled with 3 under typical sonogashira conditions to provide c5-alkynyl - functionalized lna uridines 4a  j in 5387% yield .
careful deoxygenation 
 is critical to the outcome of these reactions , as they otherwise do 
 not proceed to completion .
finally , o3-phosphitylation using 
 2-cyanoethyl - n , n-diisopropylchlorophosphoramidite 
 afforded target phosphoramidites 5b  j in 4383% yield .
abbreviations : can , 
 ceric 
 ammonium nitrate ; dmtrcl , 4,4-dimethoxytrityl chloride ; pcl , 
 2-cyanoethyl - n , n - diisopropylchlorophosphoramidite ; 
 dipea , n , n-diisopropylethylamine . in order to obtain c5-triazolyl - functionalized 
 lna uridine phosphoramidites 5k and 5l , 
 c5-ethynyl - functionalized lna uridine 4b ( obtained via 
 standard tbaf - mediated desilylation of 4a ) was reacted 
 with 1-azidopyrene or 1-azidomethylpyrene in a cu(i)-catalyzed 
 azide alkyne huisgen 1,3-dipolar cycloaddition , followed by standard o3-phosphitylation ( scheme 2 ) .
phosphoramidites 5b  l were used in machine - assisted solid - phase dna synthesis 
 ( 0.2 mol scale ) to incorporate monomers k  z into ons .
standard conditions were used except for extended 
 hand - coupling ( generally 15 min with 4,5-dicyanoimidazole or 5-[3,5-bis(trifluoromethyl)phenyl]-1h - tetrazole as activator ) when using 5b 
the composition and purity of all modified ons was ascertained 
 by maldi ms analysis ( table s1 , supporting information ) and ion - pair reversed - phase hplc , respectively .
ons containing 
 a single incorporation in the 5-gtgabatgc context 
 are denoted k1m1 and so on .
the thermostabilities of duplexes between modified 
 ons and complementary 
 dna / rna were evaluated by determining their thermal denaturation temperature 
 ( tm ) in medium salt phosphate buffer ( [ na ] = 110 mm , ph 7.0 ) .
tm s 
 of modified duplexes are discussed relative to tm s of unmodified reference duplexes ( table 1 ) .
tm = change in tm values relative to unmodified 
 reference duplexes d1:d2 ( tm  29.5 c ) , d1:r2 ( tm  27.0 c ) , and d2:r1 ( tm  

tm values were determined 
 as the first - derivative maximum of denaturation curves ( a260 vs t ) recorded in medium salt phosphate 
 buffer ( [ na ] = 110 mm , [ cl ] = 100 mm , 
 ph 7.0 ( nah2po4/na2hpo4 ) ) , using 1.0 m of each strand .
tm values are averages of at least two measurements within 1.0 c ; 
 see figure 1 for structures of monomers . nd 
 = not determined . data for duplexes between
l / k / n / q / s - modified ons and complementary 
 rna has been previously reported in ref ( 22 ) .
as 
 anticipated , ons modified with one or two conventional lna - t 
 monomers form very thermostable duplexes with rna targets in particular 
 ( see tm values for l1l4 , table 1 ) .
interestingly , 
 ons modified with lna monomers featuring small c5-alkynyl moieties 
 generally result in the formation of even more thermostable duplexes 
 ( compare tm values of k / m / n series with those of the l series , table 1 ) .
the effect is most pronounced 
 for ons modified with aminopropynyl - functionalized monomer n , which display increases in tm values 
 of up to + 13 c per modification .
the greater thermostability 
 of duplexes modified with k / m / n monomers is most likely the result of enhanced stacking interactions and , in the case of monomer n , 
 favorable electrostatic interactions and/or hydration in the major 
 groove , in a manner similar to that previously suggested for c5-aminopropynyl - modified 
 dna .
in contrast , duplexes modified with 
 lna monomers that are conjugated 
 to medium - sized hydrophobic c5-alkynyl substituents are less thermostable 
 than the corresponding lna - modified duplexes ( compare tm values of the o / p - series with those of the l series , table 1 ) .
the trend is particularly prominent in dna : rna duplexes , 
 presumably due to a suboptimal fit of the c5-alkynyl substituent in 
 the narrow major groove of a / b - type 
 duplexes .
however , other factors , such as different influences on 
 hydration , can not be ruled out .
the 
 resulting duplexes are , nevertheless , still significantly more stable 
 that the unmodified reference duplexes .
ons modified with lna 
 monomers that are conjugated to long hydrophobic 
 c5-alkynyl substituents display even lower affinity toward their targets 
 ( see tm values of the q / s series , table 1 ) .
it is particularly 
 noteworthy that duplexes involving the doubly modified q4 or s4 do not display transitions above 10 c . 

we hypothesize that 
 interactions between the hydrophobic groups in single - stranded q4 or s4 interfere with duplex formation .
the 
 fact that dna duplexes with interstrand zipper arrangements of two q monomers are rather thermostable supports this hypothesis 
 ( see table s2 , supporting information ) .
similarly , ons modified with lna monomers that are conjugated to 
 large hydrophobic fluorophores generally form very thermolabile duplexes , 
 regardless of whether the fluorophore is attached via an alkynyl or 
 triazoyl linker ( see tm values 
 of the v  z series , table 1 ) . the use of monomers in which the fluorophore is attached to the 
 nucleobase via a short rigid linker , such as in monomers w  y , results in particularly unstable duplexes . 
 once again
, we speculate that these trends reflect a poor fit of the 
 fluorophore in the major groove ; short rigid linkers between the fluorophore 
 and nucleobase moieties may prevent the fluorophore from sampling 
 more suitable conformational space .
interestingly , with the exception 
 of pyrene- and perylene - functionalized w4 and x4 , duplexes entailing the doubly modified b4 ons are 
 considerably more stable than those entailing their singly modified 
 counterparts ( e.g. , compare tm / mod 
 of b4:d1 relative to b2:d1 and b3:d1 , table 1 ) .
similar stabilizing trends have been reported for other 
 densely fluorophore modified duplexes and were attributed to the formation 
 of chromophore arrays in the major groove .
the presence of pyrene excimer signals in the steady - state fluorescence 
 emission spectra of duplexes between v4/y4/z4 and dna / rna complements supports this hypothesis 
 ( figure s3 , supporting information ) .
the tm - based conclusions 
 are largely corroborated through analysis of the thermodynamic parameters 
 for duplex formation , which were derived from thermal denaturation 
 curves through curve fitting .
thus , the 
 formation of duplexes between conventional lna l1l3 and complementary dna or rna is 47 and 813 
 kj / mol more favorable , respectively , in comparison to unmodified reference 
 duplexes ( see g values 
 for l1l3 , table 2 ) . the greater stability of lna - modified duplexes is generally 
 a result of lower enthalpy ( h < 
 0 kj / mol for l2 and l3 , table 2 ) , but entropic stabilization is also observed ( (ts ) < 0 kj / mol 
 for l1 , table 2 ) .
parameters were determined from 
 thermal denaturation curves , which were recorded as described in table 1 .
g , h , and (ts ) are calculated relative 
 to reference duplexes d1:d2 ,
formation of duplexes entailing 
 ons modified with k / m / n monomers , 
 which are conjugated to 
 small and/or relatively polar alkynes , is 17 kj / mol more favorable 
 than formation of the corresponding lna - modified duplexes ( compare 
 g values for the k / m / n series vs l series , 
 table 2 ) .
the additional duplex stabilization 
 is generally enthalpic in origin , which is consistent with improved 
 base stacking due to the extended  surface of the c5-alkynyl - functionalized 
 lna monomers ( compare h values for 
 the k / m / n series vs l series , table 2 ) ; similar trends have been 
 previously reported for c5-propynyl - functionalized dna monomers .
duplexes involving ons modified with monomers o / p / q , which are conjugated to moderately 
 large 
 hydrophobic alkynyl substituents , are 07 kj / mol less favorable 
 than the corresponding lna - modified duplexes ( compare g values for the o / p / q series vs the l series , table 2 ) .
comparison with k - modified duplexes 
 suggests that the hydrophobic substituents counteract the favorable 
 enthalpy of the extended  surfaces ( compare h values for the o / p / q series vs the k series , table 2 ) .
one possible interpretation of this is that the hydrophobic substituents 
 disrupt hydration in the major groove .
dna duplexes modified 
 with c5-cholesterol - functionalized lna monomer s are 
 less stable than the control duplex , whereas duplexes 
 with rna are slightly more stable ( see g values for s1s3 , 
 table 2 ) .
the favorable enthalpic contribution 
 of the alkyne functionality is fully counteracted by low entropy in 
 dna duplexes but only partially counteracted in dna : rna duplexes ( compare 
 h vs (ts ) for s1s3 , table 2 ) .
the 
 binding specificities of centrally modified ons ( b1 series ) 
 were determined by using dna / rna targets with mismatched nucleotides 
 opposite of the modified monomer . as expected , lna - modified 
 on l1 displays improved binding specificity relative 
 to unmodified reference strand d1 , as evidenced by the 
 more pronounced decreases in tm values 
 of mismatched duplexes ( compare tm values for l1 and d1 , table 3 ) .
interestingly , many of the c5-functionalized lna monomers 
 induce additional improvements in binding specificity ( note tm values of k1/m1/n1/o1/p1/q1 , 
 table 3 ) .
it is recognized that nucleotide 
 modifications , which improve target affinity as well as binding specificity , 
 are desirable for nucleic acid targeting applications .
cholesterol - functionalized lna s1 and fluorophore - functionalized lnas v1/w1/x1/y1/z1 display poor discrimination 
 of mismatched dna targets but maintain reasonable specificity against 
 rna targets ( table 3 ) .
these trends are indicative 
 of different binding modes of the pyrene and perylene moieties in 
 dna : dna vs dna : rna duplexes .
intercalation of aromatic units , which 
 is known to stabilize mismatched base pairs , is more favorable in dna : dna than in dna : rna duplexes . for a discussion of the binding specificities 
 of double - modified ons ( b4-series ) ,
see the supporting information ( table s3 ) . for experimental 
 conditions and 
 sequences ,
tm = change in tm value relative 
 to fully matched on : dna or on : rna duplex ( b = a ) .
data for l1/k1/n1/q1/s1 against mismatched rna have been 
 previously published in ref ( 22 ) .
next , we examined the 
 enzymatic stability of select c5-functionalized 
 lnas and reference strands in the presence of snake venom phosphodiesterase 
 ( svpde ) , a 3-exonuclease .
as expected , unmodified d2 is quickly degraded ( > 95% cleavage after 15 min ) , while the singly 
 modified lna l2 offers moderate protection against svpde 
 ( >
ons 
 modified with a single c5-ethynyl- or c5-aminopropynyl - functionalized 
 lna monomer are markedly more resistant toward svpde degradation ( 55% 
 and 35% cleavage of k2 and n2 , respectively , 
 after 2 h ) .
interestingly , ons that are modified with lna monomers 
 conjugated to large hydrophobic substituents are completely inert 
 against svpde - mediated degradation , following a brief period of cleavage 
 ( see degradation profiles for q2 and s2 ; 
 figure 2 ) . m2/o2/p2
also display markedly increased 3-exonuclease resistance 
 ( figure s1 , supporting information ) . as 
 expected ,
these trends are even more pronounced with the doubly modified b4 series ( figure 2 ) .
thus , the data 
 strongly suggest that large hydrophobic c5-alkynyl substituents offer 
 effective protection from enzymatic degradation .
3-exonuclease 
 ( svpde ) degradation of singly ( left , 3-cac bat acg ) and doubly modified ( right , 
 3-cac bab acg ) c5-functionalized lna and reference strands . 

nuclease degradation studies were performed in magnesium buffer ( 50 
 mm tris - hcl , 10 mm mg , ph 9.0 ) by using 3.3 m 
 ons and 0.03 u of svpde .
data depicted in the left panel have been 
 previously reported in ref ( 22 ) .
steady - state 
 fluorescence emission spectra of ons modified with c5-fluorophore - functionalized 
 lna monomers and the corresponding duplexes with complementary or 
 mismatched dna targets were recorded to gain further insight into 
 the binding modes of the fluorophores .
in addition to studying the 
 fluorescence properties of b1 and b4 probes 
 in the presence or absence of matched / mismatched dna / rna ( figures 
 s2 and s3 , supporting information ) , we 
 also studied centrally modified 13-mer ons ( v5z5 series ) and their duplexes with matched / mismatched dna 
 targets ( figure 3 ) .
the thermal denaturation 
 characteristics of these ons ( table s4 , supporting 
 information ) closely follow those of the singly modified 9-mer 
 ons : i.e. ( i ) the corresponding duplexes with dna / rna targets are 
 less stable than unmodified reference duplexes ( only z5-modified duplexes are slightly more stable ) and ( ii ) w5y5 display very poor thermal discrimination 
 of mismatched dna targets , while v5 and z5 display similar binding specificity as the unmodified reference 
 strands .
v / y / z - modified 
 duplexes exhibit emission peaks of varying broadness at 390/402 
 nm ( v ) , 381/398 nm ( y ) , and 376/396/416 
 nm ( z ) , respectively , which are typical emission maxima 
 for electronically isolated pyrene units ( figure 3 ) . as expected for duplexes modified with the 1-ethynylpyrene 
 fluorophore , the duplex between w5 and complementary dna exhibits broad red - shifted emission 
 centered around 465 nm , which is indicative of strong electronic 
 coupling between the pyrene and nucleobase moiety .
interestingly , 
 the emission intensities of pyrene - functionalized ons v5/w5/y5/z5 increase upon binding 
 to complementary dna ( 3.8- , 3.9- , 3.1- , and 
 51-fold increases for v5 , w5 , y5 and z5 , respectively , figure 3 ) .
in contrast , much smaller increases are observed upon hybridization 
 with mismatched dna targets .
the intensity differences are most likely 
 due to different positioning of the pyrene moieties in matched vs 
 mismatched duplexes , in a manner similar to that proposed for the 
 corresponding dna analogues of monomers v / w / y / z .
thus , the pyrene moieties likely point into the nonquenching 
 environment of the major groove in matched duplexes ( nucleobase in anti conformation ) , while they are intercalating into mismatched 
 duplexes leading to nucleobase - mediated quenching of pyrene fluorescence ( nucleobase in syn conformation ) .
regardless of the mechanism , the results strongly 
 suggest that v / w / y / z - modified ons are promising probes for the detection of nucleic acid 
 targets and fluorescent discrimination of single - nucleotide polymorphisms 
 ( snps ) .
steady - state fluorescence emission spectra of single - stranded b5 ons ( 5-cg caa cbc aac gc ) and the corresponding duplexes with fully complementary 
 or singly mismatched dna strands ( mismatched nucleotide opposite of 
 modification is specified ) .
conditions : ex 344 nm 
 ( v5/y5/z5 ) , ex 375 nm 
 ( w5 ) , ex 448 nm ( x5 ) ; t = 5 c . note that different axis scales are used .
duplexes between perylene - functionalized x5 and complementary 
 dna display broad emission maxima at 487 and 517 nm , 
 whereas the emission maxima are red - shifted by 10 nm in mismatched 
 dna duplexes ( figure 3 ) .
the emission intensity 
 of x5 does not change significantly upon binding with 
 complementary dna but is reduced by 3060% upon binding to 
 mismatched targets , presumably due to nucleobase - mediated quenching 
 of intercalating perylene units .
recently , we examined the snp - discriminating 
 properties of v - modified ons and compared them to probes 
 modified with the 
 corresponding dna analogue of monomer v. we found that there are distinct advantages to conjugating 
 the 1-pyrenecarboxamido fluorophore to the c5-position of lna - u , including 
 ( i ) greater increases in fluorescence intensity upon target binding , 
 ( ii ) formation of more brightly fluorescent duplexes , and ( iii ) stricter 
 fluorescent discrimination of dna targets with snp sites .
force field 
 calculations suggested that the extreme pucker of the lna skeleton 
 influences the rotational freedom around the n1c1 
 glycosyl bond due to steric hindrance between h6 and h3 , leading 
 to different positioning and modulated photophysical properties of 
 the c5-fluorophore relative to the analogous dna monomer .
direct comparison of y5 and z5 with the 
 corresponding dna - based probes y5d and z5d(18j ) ( for structures of the dna analogues 
 of v / y / z monomers , see figure 
 s4 in the supporting information ) reveals 
 similar advantages ( figure 4 ) .
thus , the results 
 suggest that conjugation of fluorophores to the c5 position of lna 
 monomers is an effective strategy toward the generation of building 
 blocks with interesting photophysical properties . fluorescence intensity 
 of single - stranded probes ( ssps ) in the 
 presence or absence of complementary or singly mismatched dna / rna 
 strands .
hybridization - induced increases and discrimination factors ( defined 
 as the fluorescence intensity of duplexes with complementary dna / rna 
 divided by the intensity of ssps or duplexes with mismatched dna / rna , 
 respectively ) are given above the corresponding histograms .
intensity 
 recorded at em 382 nm for y5/y5d and em 377 nm for z5/z5d at t = 5 c .
the large increases in fluorescence intensity upon hybridization 
 of z5 with complementary targets prompted us to examine 
 the potential of z - modified ons as hybridization probes in greater detail .
three additional 13-mer ons 
 were therefore prepared in which the nucleotides flanking monomer z were systematically varied ( table s4 , supporting information ) .
although the increases in fluorescence 
 intensities upon hybridization with dna targets are less pronounced 
 ( 417-fold , figure 5 ) and the resulting 
 duplexes are significantly less fluorescent than with z5 , moderate to excellent fluorescent 
 discrimination of mismatched dna targets is observed with all z - modified probes ( discrimination factors from 1.5 to 48 , 
 figure 5 ) . accordingly , z - modified 
 ons constitute an interesting addition to the existing pool of pyrene - based 
 hybridization probes .
fluorescence intensity of single - stranded 
 probes ( ssps ) in the 
 presence or absence of complementary or singly mismatched dna strands . 

hybridization - induced 
 increases and discrimination factors are given above the corresponding 
 histograms .
target : 5-cg caa bzb aac gc , where b = c / a / g / t for on58 , respectively .
the hybridization characteristics and enzymatic 
 stabilities of 
 ons modified with lna uridines can be extensively modulated through 
 conjugation of different entities to the c5 position of the nucleobase . 

monomers that are conjugated to small alkynyl substituents 
 result in significantly greater target affinity and specificity than 
 regular lna monomers .
conjugation of bulky moieties confers complete 
 protection against 3-exonucleases but also decreases target 
 affinity .
ons modified with c5-fluorophore - functionalized lna uridines 
 display improved photophysical characteristics relative to the corresponding 
 dna - based probes , including greater hybridization - induced increases 
 in fluorescence intensity , formation of more brightly fluorescent 
 duplexes , and strict fluorescent discrimination of single - nucleotide 
 polymorphisms .
these properties 
 render c5-functionalized lna as promising building blocks for rna - targeting 
 applications and nucleic acid diagnostics , although concerns regarding 
 the potential toxicity of c5-alkynyl entities must be alleviated prior to biological evaluation .
the present 
 study suggests that it is possible to combine desirable 
 properties from lna ( target affinity / specificity ) and c5-functionalized 
 dna monomers ( positioning of functional entities in the major groove ) 
 into one compound class .
the subsequent article in this issue demonstrates 
 that the properties of ons modified with -l - lna uridines 
 also can be modulated through functionalization of the nucleobase .
we therefore anticipate that c5 functionalization 
 of pyrimidines will serve as a general and synthetically straightforward 
 approach for modulation of pharmocodynamic and pharmacokinetic properties 
 of oligonucleotides modified with lna or other conformationally restricted monomers .
efforts aiming at delineating 
 whether the biophysical properties of lna purines also can be improved 
 through functionalization of the nucleobase are ongoing , and the results 
 from these studies will be reported shortly .
the appropriate carboxylic acid and 1-ethyl-3-(3-dimethyllaminopropyl)carbodiimide 
 hydrochloride ( edchcl ) were added to propargylamine in anhydrous 
 ch2cl2 , and the reaction mixture was stirred 
 under an argon atmosphere until analytical tlc indicated full conversion 
 ( quantities , volumes , reaction time , and temperature are specified 
 below ) . at this point , ch2cl2 ( 40 ml ) was added 
 and the organic phase was washed with 5% aqueous citric acid ( 2  
 20 ml ) and h2o ( 20 ml ) .
the aqueous phase was back - extracted 
 with ch2cl2 ( 25 ml ) , the combined organic layers 
 were concentrated to dryness , and the resulting residue was purified 
 by column chromatography ( 04% meoh in ch2cl2 ) to afford the desired product ( quantities and yields specified 
 below ) .
1-adamantaneacetic acid ( 1.60 g , 8.24 mmol ) , 
 edchcl ( 1.80 g , 9.42 mmol ) , and propargylamine ( 0.60 ml , 9.38 
 mmol ) in anhydrous ch2cl2 ( 30 ml ) were set up , 
 reacted ( 14 h at room temperature ) , and worked up , and the product 
 was purified as described above to afford alkyne ae(43 ) ( 1.40 g , 76% ) as a white solid material : rf = 0.8 ( 10% meoh / ch2cl2 , v / v ) ; maldi - hrms m / z 254.1527 
 ( [ m + na ] , c15h21nona , calcd 254.1515 ) ; h nmr ( cdcl3 )  
 5.56 ( br s , 1h , nh ) , 4.01 ( dd , 2h , j = 5.2 hz , 2.5 
 hz , ch2nh ) , 2.19 ( t , 1h , j = 2.5 hz , hcc ) , 
 1.931.97 ( m , 3h , 3  ch ) , 1.92 ( s , 2h , ch2conh ) , 1.581.70 ( 12h , 6  ch2-ada ) ; c
nmr ( cdcl3 )  170.8 , 80.0 , 71.6 ( hcc ) , 
 51.6 ( ch2conh ) , 42.8 ( ch2-ada ) , 37.0 ( ch2-ada ) , 33.1 , 29.2 ( ch2nh ) , 28.9 ( ch - ada ) .
lauric 
 acid ( dodecanoic acid , 1.60 g , 8.00 mmol ) , edchcl 
 ( 1.80 g , 9.42 mmol ) , and propargylamine ( 0.60 ml , 9.38 mmol ) in anhydrous 
 ch2cl2 ( 30 ml ) were set up , reacted ( 12 h at 
 room temperature ) , and worked up , and the product was purified as 
 described above to afford alkyne af(43 ) ( 1.40 g , 76% ) as a white solid material : rf = 0.8 ; maldi - hrms m / z 260.1978 ( [ m + na ] , c15h27nona , calcd 260.1985 ) ;
h nmr ( cdcl3 )  
 5.66 ( bs , ex , 1h , nh ) , 4.03 ( dd , 2h , j = 5.0 hz , 
 2.5 hz , ch2cch ) , 2.19 ( t , 1h , j = 2.5 hz , hcc ) , 2.16 ( 2d , 2h , j = 7.7 hz , 

ch2ch2co ) , 1.201.30 ( m , 16h , 8  
 ch2 ) , 0.85 ( t , 3h , j = 6.5 hz , ch3 ) ; c nmr ( cdcl3 )  172.9 , 79.9 , 
 71.7 , 36.7 ( ch2co ) , 32.1 ( ch2 ) , 29.8 ( ch2 ) , 29.7 ( ch2 ) , 29.53 ( ch2 ) , 29.47 ( ch2 ) , 29.4 ( ch2cch ) , 25.8 ( ch2ch2co ) , 22.9 , 14.3 ( ch3 ) .
stearic acid ( octadecanoic acid , 1.42 g , 5.00 mmol ) , edchcl 
 ( 1.15 g , 6.00 mmol ) , and propargylamine ( 0.40 ml , 6.25 mmol ) in anhydrous 
 ch2cl2 ( 30 ml ) were set up , reacted ( 12 h at 
 room temperature ) , and worked up , and the product was purified as 
 described above to afford alkyne ag(43 ) ( 1.40 g , 87% ) as a white solid material : rf = 0.1 ( ch2cl2 ) ; fab - hrms m / z 321.3020 ( [ m ] , c21h39no , calcd 321.3032 ) ; h nmr 
 ( cdcl3 )  5.54 ( br s , 1h , ex , nh ) , 4.03 ( dd , 2h , j = 5.5 hz , 2.5 hz , ch2nh ) , 2.20 ( t , 1h , j = 2.5 hz , hcc ) , 2.17 ( t , 2h , j = 7.5 hz , ch2co ) , 1.61 ( quintet , 2h , j = 7.5 hz , ch2ch2co ) , 1.231.27 ( m , 
 28h , 14  ch2 ) , 0.87 ( t , 1h , j = 
 7.0 hz ,
ch3 ) ; c nmr ( cdcl3 )  
 172.7 , 79.7 , 71.5 ( hcc ) , 36.5 ( ch2co ) , 31.9 ( ch2 ) , 29.69 ( ch2 ) , 29.684 ( ch2 ) , 29.677 
 ( ch2 ) , 29.66 ( ch2 ) , 29.65 ( ch2 ) , 
 29.64 ( ch2 ) , 29.59 ( ch2 ) , 29.46 ( ch2 ) , 29.35 ( ch2 ) , 29.31 ( ch2 ) , 29.2 ( ch2 ) , 29.1 ( ch2nh ) , 25.5 ( ch2ch2conh ) , 
 22.7 ( ch2 ) , 14.1 ( ch3 ) . the nmr data are in 
 excellent agreement with literature reports . to a solution of nucleoside 1(23 ) ( 4.00 g , 15.62 mmol ) in glacial acoh 
 ( 150 ml ) were added iodine ( 2.40 g , 9.44 mmol ) and ceric ammonium 
 nitrate ( 4.26 g , 7.77 mmol ) , and the reaction mixture was stirred 
 at 80 c for 40 min . after it was cooled to room temperature , 

the mixture was evaporated to dryness and the resulting residue was 
 suspended in meoh ( 150 ml ) .
the mixture was concentrated and the resulting 
 residue adsorbed on silica gel and purified by column chromatography 
 ( 015% meoh / ch2cl2 , v / v ) to afford nucleoside 2 ( 5.21 g , 87% ) as a white solid material : rf = 0.4 ( 10% meoh / ch2cl2 , v / v ) ; 
 fab - hrms m / z 382.9732 ( [ m + h ] , c10h11in2o6h , calcd 382.9735 ) ; h nmr ( dmso - d6 )  11.69 ( s , 1h , ex , nh ) , 8.13 ( s , 1h , h6 ) , 5.65 
 ( d , 1h , ex , j = 4.5 hz , 3oh ) , 5.40 
 ( s , 1h , h1 ) , 5.27 ( t , 1h , ex , j = 5.4 hz , 
 5oh ) , 4.14 ( s , 1h , h2 ) , 3.91 ( d , 1h , j = 4.5 hz , h3 ) , 3.793.82 ( d , 1h , j = 8.5 hz , h5 ) , 3.683.75 ( m , 2h , h5 ) , 
 3.583.62 ( d , 1h , j = 8.5 hz ,
h5 ) ; c nmr ( dmso - d6 )  160.5 , 
 149.6 , 143.5 ( c6 ) , 88.8 , 86.4 ( c1 ) , 78.5 ( c2 ) , 70.8 
 ( c5 ) , 68.4 , 68.2 ( c3 ) , 55.3 ( c5 ) .
diol 2 ( 5.00 g , 13.0 mmol ) 
 was coevaporated with anhydrous pyridine ( 100 ml ) and redissolved 
 in anhydrous pyridine ( 100 ml ) .
to this was added 4,4-dimethoxytrityl 
 chloride ( dmtr - cl , 5.75 g , 16.9 mmol ) , and the reaction mixture was 
 stirred at room temperature for 16 h , whereupon solvent was evaporated 
 off .
the residue was dissolved in ch2cl2 ( 300 
 ml ) and washed with saturated aqueous nahco3 ( 300 ml ) . 

the aqueous layer was back - extracted with ch2cl2 ( 2  100 ml ) , and the combined organic layer was washed with 
 saturated aqueous nahco3 ( 100 ml ) , dried ( na2so4 ) , evaporated to near dryness , and coevaporated with 
 toluene / absolute etoh ( 100 ml , 1/2 , v / v ) .
the resulting residue was 
 purified by column chromatography ( 05% meoh in ch2cl2 , v / v ) to afford key intermediate 3 ( 7.52 
 g , 82% ) as a slightly yellow solid material : rf = 0.5 ( 5% meoh in ch2cl2 , v / v ) ; fab - hrms m / z 684.0977 ( [ m ] , c31h29in2o8 , calcd 684.0969 ) ; h nmr ( dmso - d6 )  11.74 
 ( s , 1h , ex , nh ) , 7.96 ( s , 1h , h6 ) ,
7.237.45 ( m , 9h , ar ) , 6.91 
 ( d , 4h , j = 8.5 hz ,
ar ) , 5.70 ( d , 1h , ex , j = 4.5 hz , 3oh ) , 5.44 ( s , 1h , h1 ) , 
 4.24 ( s , 1h , h2 ) , 4.07 ( d , 1h , j = 4.5 hz , 
 h3 ) , 3.743.76 ( m , 8h , 2  och3 , 2 
  h5 ) , 3.393.42 ( d , 1h , j = 
 11.0 hz , h5 ) , 3.283.31 ( d , 1h , j = 
 11.0 hz , h5 , overlap with h2o ) ; c 
 nmr ( dmso - d6 )  160.5 , 158.1 , 158.0 , 
 149.7 , 144.6 , 142.7 ( c6 ) , 135.3 , 135.2 , 129.7 ( ar ) , 129.6 ( ar ) , 127.9 
 ( ar ) , 127.5 ( ar ) , 126.6 ( ar ) , 113.3 ( ar ) , 87.5 , 86.9 ( c1 ) , 
 85.6 , 78.8 ( c2 ) , 71.3 ( c5 ) , 69.4 ( c3 ) , 68.9 , 
 58.9 ( c5 ) , 55.0 ( ch3o ) . key intermediate 3 , 
 pd(pph3)4 , cui , and alkyne were added to anhydrous 
 dmf ( quantities and volumes specified below ) , and the reaction chamber 
 was degassed and placed under an argon atmosphere . to this
was added 
 et3n , and the reaction mixture was stirred in the dark 
 until analytical tlc indicated full conversion of the starting material 
 ( reaction time and temperature specified below ) , whereupon solvents 
 were evaporated off .
the resulting residue was taken in up in etoac 
 ( 100 ml ) and washed with brine ( 2  50 ml ) and saturated aqueous 
 nahco3 ( 50 ml ) .
the combined aqueous phase was back - extracted 
 with etoac ( 100 ml ) , the combined organic phase dried ( na2so4 ) and evaporated to dryness , and the resulting residue 
 purified by column chromatography ( 05% meoh in ch2cl2 ( v / v ) to afford the desired product .
nucleoside 3 ( 0.68 g , 1.00 
 mmol ) , pd(pph3)4 ( 120 mg , 0.10 mmol ) , cui ( 40 
 mg , 0.20 mmol ) , trimethylsilylacetylene ( 294 mg , 0.42 ml , 3.00 mmol ) , 
 and et3n ( 0.60 ml , 4.27 mmol ) in dmf ( 10 ml ) were reacted 
 as described in the representative sonogashira protocol , and the mixture 
 was stirred stirred at room temperature for 12 h. after workup and 
 purification , nucleoside 4a ( 0.56 g , 85% ) was obtained 
 as a brown solid material .
rf = 0.5 ( 5% 
 meoh in ch2cl2 , v / v ) ; esi - hrms m / z 677.2297 ( [ m + na ] , c36h38n2o8sina , calcd 
 677.2290 ) ; h nmr ( dmso - d6 ) 
  11.69 ( s , 1h , ex , nh ) , 7.86 ( s , 1h , h6 ) , 7.207.45 
 ( m , 9h , ar ) , 6.89 ( d , 4h , j = 8.5 hz ,
ar ) , 5.72 ( d , 
 1h , ex , j = 4.5 hz , 3oh ) , 5.39 ( s , 
 1h , h1 ) , 4.28 ( s , 1h , h2 ) , 4.08 ( d , 1h , j = 4.5 hz , h3 ) , 3.753.80 ( m , 2h , 2  h5 ) , 
 3.73 ( s , 6h , 2  och3 ) , 3.403.43 ( d , 1h , j = 11.0 hz , h5 ) , 3.303.33 ( d , 1h , j = 11.0 hz , h5 , overlap with h2o signal ) , 
 0.04 ( s , 9h , me3si ) ; c nmr ( dmso - d6 )  161.5 , 158.05 , 158.03 , 148.9 , 144.6 , 
 142.5 ( c6 ) , 135.4 , 135.2 , 129.7 ( ar ) , 129.5 ( ar ) , 127.8 ( ar ) , 127.6 
 ( ar ) , 126.6 ( ar ) , 113.2 ( ar ) , 97.9 , 97.5 , 96.9 , 87.7 , 87.1 ( c1 ) , 
 85.5 , 78.6 ( c2 ) , 71.3 ( c5 ) , 69.5 ( c3 ) , 58.9 
 ( c5 ) , 55.0 ( ch3o ) , 0.47 ( me3si ) . to a solution of nucleoside 4a ( 0.53 g , 0.81 mmol ) in thf ( 20 ml ) was added tetrabutylammonium 
 fluoride in thf ( tbaf , 1 m , 1.2 ml , 1.2 mmol ) , and the reaction mixture 
 was stirred at room temperature for 2 h. etoac ( 50 ml ) was added and 
 the organic phase washed with brine ( 2  30 ml ) and h2o ( 30 ml ) .
the combined organic phase was dried ( na2so4 ) and evaporated to dryness and the resulting residue purified by 
 column chromatography ( 05% meoh in ch2cl2 , v / v ) to afford nucleoside 4b ( 0.37 g , 78% ) as a lightly 
 brown solid material : rf = 0.4 ( 5% meoh / ch2cl2 ,
v / v ) ; esi - hrms m / z 621.1666 ( [ m + k ] , c33h30n2o8k , calcd 621.1634 ) ; h nmr ( dmso - d6 )  11.70 
 ( s , 1h , ex , nh ) , 7.88 ( s , 1h , h6 ) , 7.217.45
( m , 9h , ar ) , 6.886.92 
 ( m , 4h , ar ) , 5.70 ( d , 1h , ex , j = 4.5 hz , 3oh ) , 
 5.45 ( s , 1h , h1 ) , 4.27 ( s , 1h , h2 ) , 4.05 ( d , 1h , j = 4.5 hz , h3 ) , 3.95 ( s , 1h , ch ) , 3.77 ( s , 2h , 
 2  h5 ) , 3.75 ( s , 6h , 2  ch3o ) , 3.433.45 
 ( d , 1h , j = 11.0 hz , h5 ) , 3.283.31 
 ( d , 1h , j = 11.0 hz , h5 , overlap with h2o signal ) ; c nmr ( dmso - d6 )  161.7 , 158.1 , 149.0 , 144.6 , 142.2 ( c6 ) , 135.3 , 135.2 , 
 129.7 ( ar ) , 129.6 ( ar ) , 127.9 ( ar ) , 127.5 ( ar ) , 126.7 ( ar ) , 113.3 
 ( ar ) , 97.2 , 87.5 , 86.9 ( c1 ) , 85.7 , 83.6 , 78.9 ( c2 ) , 
 76.2 , 71.4 ( c5 ) , 69.4 ( c3 ) , 59.0 ( c5 ) , 55.0 
 ( ch3o ) .
nucleoside 3 ( 0.50 g , 0.73 
 mmol ) , pd(pph3)4 ( 90 mg , 0.07 mmol ) , cui ( 30 
 mg , 0.14 mmol ) , prop-2-ynyl benzoate ( 180 
 mg , 1.12 mmol ) , and et3n ( 0.40 ml , 2.84 mmol ) in dmf ( 10 
 ml ) were reacted as described in the representative sonogashira protocol , 
 and the reaction mixture was stirred at room temperature for 12 h. 
 after workup and purification , nucleoside 4c ( 0.37 g , 
 70% ) was obtained as a light brown solid material : rf = 0.5 ( 5% meoh in ch2cl2 , v / v ) ; 
 esi - hrms m / z 739.2289 ( [ m + na ] , c41h36n2o10na , 
 calcd 739.2262 ) ; h nmr ( dmso - d6 )  11.72 ( s , 1h , ex , nh ) , 7.917.93 ( d , 2h , j = 7.7 hz , bzortho ) , 7.89 ( s , 1h , h6 ) , 7.657.70 
 ( t , 1h , j = 7.7 hz , bzpara ) , 7.497.54 
 ( t , 2h , j = 7.7 hz , bzmeta ) , 7.427.46 
 ( d , 2h , j = 8.5 hz , dmtr ) , 7.287.34 ( m , 6h , 
 dmtr ) , 7.187.22 ( t , 1h , j = 7.5 hz , dmtr ) , 
 6.876.91 ( 2d , 4h , j = 9.0 hz , dmtr ) , 5.74 
 ( d , 1h , ex , j = 4.5 hz , 3oh ) , 5.42 
 ( s , 1h , h1 ) , 4.934.97 ( d , 1h , j = 
 16.0 hz , ch2obz ) , 4.864.90 ( d , 1h , j = 16.0 hz , ch2obz ) , 4.25 ( s , 1h , h2 ) , 4.10 ( d , 
 1h , j = 4.5 hz , h3 ) , 3.783.82 ( d , 
 1h , j = 8.0 hz , h5 ) , 3.753.76 ( d , 
 1h , j = 8.0 hz , h5 ) , 3.71 ( s , 6h , 2  
 ch3o ) , 3.533.56 ( d , 1h , j = 11.0 
 hz , h5 ) , 3.273.30 ( d , 1h , j = 11.0 
 hz , h5 , overlap with h2o ) ; c nmr ( dmso - d6 )  164.9 , 161.6 , 158.1 , 158.0 , 149.0 , 
 144.6 , 142.7 ( c6 ) , 135.4 , 135.0 , 133.5 ( cbzpara ) , 129.7 
 ( dmtr ) , 129.6 ( dmtr ) , 129.2 ( cbzortho ) , 129.0 , 128.7 ( cbzmeta ) , 127.8 ( dmtr ) , 127.5 ( dmtr ) , 126.6 ( dmtr ) , 113.20 ( dmtr ) , 
 113.17 ( dmtr ) , 96.8 , 87.6 , 86.9 ( c1 ) , 86.3 , 85.6 , 79.2 , 78.7 
 ( c2 ) , 71.3 ( c5 ) , 69.4 ( c3 ) , 58.7 ( c5 ) , 
 54.9 ( ch3o ) , 53.1 ( ch2obz ) .
nucleoside 3 ( 0.50 g , 0.73 
 mmol ) , pd(pph3)4 ( 90 mg , 0.07 mmol ) , cui ( 30 
 mg , 0.14 mmol ) , 2,2,2-trifluoro - n-(prop-2-ynyl)acetamide ( 180 mg , 1.46 mmol ) , and et3n ( 0.40 
 ml , 2.84 mmol ) in dmf ( 10 ml ) were reacted as described in the representative 
 sonogashira protocol , and the mixture was stirred at room temperature 
 for 12 h. after workup and purification , nucleoside 4d ( 0.41 g , 80% ) was obtained as a brown solid material : rf = 0.5 ( 5% meoh in ch2cl2 , v / v ) ; 
 esi - hrms m / z 714.2247 ( [ m+li ] , c36h32f3n3o9li , calcd 714.2245 ) ; h nmr ( dmso - d6 )  11.69 ( s , 1h , ex , nh(u ) ) , 9.95 ( t , 
 1h , ex , j = 5.5 hz , nhch2 ) , 7.78 ( s , 1h , 
 h6 ) , 7.227.46 ( m , 9h , ar ) , 6.90 ( dd , 4h , j = 9.0 hz , 3.5 hz , ar ) , 5.73 ( d , 1h , ex , j = 4.5 
 hz , 3oh ) , 5.41 ( s , 1h , h1 ) , 4.25 ( s , 1h , h2 ) , 
 3.974.10 ( m , 3h , h3 , ch2nh ) , 3.793.83 
 ( 2d , 2h , j = 8.0 hz , 2  h5 ) , 3.74 ( s , 
 6h , 2  ch3o ) , 3.563.58 ( d , 1h , j = 11.0 hz , h5 ) , 3.263.28 ( d , 1h , j = 11.0 hz , h5 ) ; c nmr ( dmso - d6 )  161.6 , 158.11 , 158.06 , 155.9 ( q , j = 36.1 hz , cocf3 ) , 149.0 , 144.6 , 142.1 ( c6 ) , 135.4 , 134.9 , 
 129.8 ( ar ) , 129.6 ( ar ) , 127.8 ( ar ) , 127.5 ( ar ) , 126.6 ( ar ) , 115.6 
 ( q , j = 287 hz , cf3 ) , 113.22 ( ar ) , 113.20 
 ( ar ) , 97.2 , 87.6 , 87.2 , 86.9 ( c1 ) , 85.6 , 78.7 ( c2 ) , 
 75.4 , 71.3 ( c5 ) , 69.6 ( c3 ) , 59.1 ( c5 ) , 55.0 
 ( ch3o ) , 29.4 ( ch2nh ) ; f ( dmso - d6 , 470 mhz )  74.7 .
nucleoside 3 ( 0.50 g , 0.73 
 mmol ) , pd(pph3)4 ( 90 mg , 0.05 mmol ) , cui ( 30 
 mg , 0.10 mmol ) , n-(prop-2-ynyl)-1-adamantaneacetamide 
 ( 220 mg , 1.00 mmol ) , and et3n ( 0.40 ml , 2.84 mmol ) in dmf 
 ( 10 ml ) were reacted as described in the representative sonogashira 
 protocol , and the mixture was stirred at room temperature for 12 h. 
 after workup and purification , nucleoside 4e ( 0.43 g , 
 76% ) was obtained as a white solid material : rf = 0.5 ( 5% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 810.3330 ( [ m + na ] , c46h49n3o9na , calcd 810.3361 ) ; h nmr ( dmso - d6 )  11.66 ( s , 1h , ex , nh(u ) ) , 8.00 ( t , 1h , ex , j = 5.5 hz , nhco ) , 7.75 ( s , 1h , h6 ) , 7.427.45 ( m , 
 2h , ar ) , 7.237.34 ( m , 7h , ar ) , 6.896.92 ( 2d , 4h , j = 9.0 hz , ar ) , 5.72 ( d , 1h , ex , j = 4.5 
 hz , 3oh ) , 5.42 ( s , 1h , h1 ) , 4.24 ( s , 1h , h2 ) , 
 4.01 ( d , 1h , j = 4.5 hz , h3 ) , 3.873.93 
 ( dd , 1h , j = 17.5 hz , 5.5 hz , ch2nhco ) , 
 3.823.87 ( dd , 1h , j = 17.5 hz , 5.5 hz , ch2nhco ) , 3.783.82 ( 2d , 2h , j = 8.2 
 hz , h5 ) , 3.75 ( s , 3h , ch3o ) , 3.74 ( s , 3h , ch3o ) , 3.543.56 ( d , 1h , j = 11.0 hz , 

h5 ) , 3.273.30 ( d , 1h , j = 11.0 hz , 
 h5 , overlap with h2o ) , 1.821.87 ( m , 5h , 
 3 ada - ch / ch2conh ) , 1.531.63 ( m , 12h , 6  
 ada - ch2 ) ; c nmr ( dmso - d6 )  169.6 , 161.7 , 158.12 , 158.07 , 149.0 , 144.7 , 141.5 
 ( c6 ) , 135.4 , 134.9 , 129.8 ( ar ) , 129.6 ( ar ) , 127.9 ( ar ) , 127.5 ( ar ) , 
 126.7 ( ar ) , 113.24 ( ar ) , 113.23 ( ar ) , 97.7 , 89.6 , 87.5 , 86.9 ( c1 ) , 
 85.6 , 78.8 ( c2 ) , 74.2 , 71.4 ( c5 ) , 69.6 ( c3 ) , 
 59.1 ( c5 ) , 54.9 ( ch3o ) , 49.5 ( ch2conh ) , 
 42.0 ( ada - ch2 ) , 36.4 ( ada - ch2 ) , 32.3 , 28.4 ( ch2nhco ) , 28.0 ( ada - ch ) .
nucleoside 3 ( 200 mg , 0.29 
 mmol ) , pd(pph3)4 ( 34 mg , 0.03 mmol ) , cui ( 11 
 mg , 0.06 mmol ) , n-(prop-2-ynyl)lauroylamide ( 110 
 mg , 0.44 mmol ) , and et3n ( 0.18 ml , 1.29 mmol ) in dmf ( 3 
 ml ) were reacted as described in the representative sonogashira protocol , 
 and the mixture was stirred at room temperature for 15 h. after workup 
 and purification , nucleoside 4f ( 202 mg , 87% ) was obtained 
 as a white solid material : rf = 0.2 ( 5% 
 meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 816.3835 ( [ m + na ] , c46h55n3o9na , calcd 
 816.3831 ) ; h nmr ( dmso - d6 ) 
  11.67 ( s , 1h , ex , nh(u ) ) , 8.08 ( t , 1h , j = 
 5.4 hz , nhch2 ) , 7.76 ( s , 1h , h6 ) , 7.227.46 ( m , 
 9h , ar ) , 6.876.96 ( m , 4h , ar ) , 5.72 ( d , 1h , ex , j = 4.7 hz , 3oh ) , 5.42 ( s , 1h , h1 ) , 4.25 ( s , 
 1h , h2 ) , 4.03 ( d , 1h , j = 4.7 hz , h3 ) , 
 3.883.94 ( dd , 1h , j = 12.4 hz , 5.4 hz , ch2nhco ) , 3.813.88 ( dd , 1h , j = 12.4 
 hz , 5.4 hz , ch2nhco ) , 3.783.82 ( 2d , 2h , j = 8.0 hz , h5 ) , 3.75 ( s , 6h , 2  ch3o ) , 3.553.57 ( d , 1h , j = 10.9 hz , h5 ) , 
 3.273.30 ( d , 1h , j = 10.9 hz , h5 , 
 overlap with h2o ) , 2.05 ( t , 2h , j = 7.4 
 hz , ch2conh ) , 1.431.48 ( m , 2h , ch2ch2conh ) , 1.191.28 ( m , 16h , 8  ch2 ) , 
 0.85 ( t , 3h , j = 7.0 hz ,
ch3 ) ; c nmr ( dmso )  171.7 , 161.7 , 158.11 , 158.06 , 149.0 , 144.6 , 
 141.6 ( c6 ) , 135.4 , 134.9 , 129.8 ( ar ) , 129.6 ( ar ) , 127.9 ( ar ) , 127.5 
 ( ar ) , 126.6 ( ar ) , 113.22 ( ar ) , 113.21 ( ar ) , 97.7 , 89.5 , 87.5 , 86.9 
 ( c1 ) , 85.6 , 78.8 ( c2 ) , 74.3 , 71.4 ( c5 ) , 69.6 
 ( c3 ) , 59.0 ( c5 ) , 55.0 ( ch3o ) , 35.0 ( ch2conh ) , 31.2 ( ch2 ) , 28.94 ( ch2 ) , 28.92 
 ( ch2 ) , 28.8 ( ch2 ) , 28.7 ( ch2 ) , 28.63 
 ( ch2 ) , 28.60 ( ch2 ) , 28.52 ( ch2nhco ) , 
 25.0 ( ch2ch2conh ) , 22.0 ( ch2ch3 ) , 13.9 ( ch3 ) .
nucleoside 3 ( 0.34 g , 0.50 
 mmol ) , pd(pph3)4 ( 60 mg , 0.05 mmol ) , cui ( 20 
 mg , 0.10 mmol ) , n-(prop-2-ynyl)stearamide ( 0.28 g , 
 1.00 mmol ) , and et3n ( 0.30 ml , 2.13 mmol ) in dmf ( 10 ml ) 
 were reacted as described in the representative sonogashira protocol , 
 and the mixture was stirred at 40 c for 6 h. after workup and 
 purification , nucleoside 4 g ( 0.29 g , 68% ) was obtained 
 as a brown solid material , which was used in the next step without 
 further purification : rf = 0.5 ( 5% meoh 
 in ch2cl2 , v / v ) ; fab - hrms m / z 877.4844 ( [ m ] , c52h67n3o9 , calcd 877.4877 ) ; h nmr ( cdcl3 )  9.45 ( br s , 1h , ex , nh(u ) ) , 
 8.05 ( s , 1h , h6 ) , 7.227.50 ( m , 9h , ar ) , 6.856.89 ( dd , 
 4h , j = 9.0 hz , 1.5 hz , ar ) , 5.565.59 ( m , 
 2h , 1 ex , h1 , nhch2 ) , 4.53 ( s , 1h , h2 ) , 
 4.29 ( s , 1h , h3 ) , 3.784.01 ( m , 10h , 2  h5 , 
 ch2nh , 2  ch3o ) , 3.533.57 ( d , 
 1h , j = 11.0 hz , h5 ) , 3.493.52 ( d , 
 1h , j = 11.0 hz , h5 ) , 3.35 ( br s , 1h , ex , 
 3oh ) , 1.851.89 ( m , 2h , ch2conh ) , 
 1.441.51 ( m , 2h , ch2ch2conh ) , 1.231.28 
 ( m , 28h , 14  ch2 ) , 0.89 ( t , 3h , j = 6.5 hz , ch3 ) ; c nmr ( cdcl3 ) 
  172.7 , 162.1 , 158.69 , 158.67 , 148.6 , 144.6 , 141.9 ( c6 ) , 135.5 , 
 135.4 , 130.02 ( ar ) , 130.01 ( ar ) , 128.1 ( ar ) , 128.0 ( ar ) , 127.0 ( ar ) , 
 113.45 ( ar ) , 113.43 ( ar ) , 99.1 , 89.9 , 88.4 , 87.4 ( c1 ) , 86.6 , 
 79.1 ( c2 ) , 74.2 , 71.8 ( c5 ) , 70.5 ( c3 ) , 58.5 
 ( c5 ) , 55.3 ( ch3o ) , 36.1 ( ch2conh ) , 31.9 
 ( ch2 ) , 29.9 ( ch2nh ) , 29.69 ( ch2 ) , 
 29.68 ( ch2 ) , 29.66 ( ch2 ) , 29.6 ( ch2 ) , 29.5 ( ch2 ) , 29.4 ( ch2 ) , 29.34 ( ch2 ) , 29.33 ( ch2 ) , 25.4 ( ch2ch2conh ) , 
 22.7 ( ch2 ) , 14.1 ( ch3 ) .
a small impurity of 
 silicon grease was observed at 1 ppm in the c 
 nmr .
nucleoside 3 ( 0.34 g , 0.50 
 mmol ) , pd(pph3)4 ( 60 mg , 0.05 mmol ) , cui ( 20 
 mg , 0.10 mmol ) , cholesterylprop-2-ynylcarbamate ( 0.47 g , 1.00 mmol ) , and et3n ( 0.30 ml , 2.13 
 mmol ) in dmf ( 8 ml ) were reacted as described in the representative 
 sonogashira protocol , and the mixture was stirred at room temperature 
 for 12 h. after workup and purification , nucleoside 4h ( 0.27 g , 53% ) was obtained as a brown solid material , which was 
 used in the next step without further purification : rf = 0.5 ( 5% meoh in ch2cl2 , v / v ) ; 
 fab - hrms m / z 1046.5560 ( [ m + na ] , c62h77n3o10na , calcd 1046.5507 ) ; h nmr ( cdcl3 )  
 9.40 ( br s , 1h , ex , nh(u ) ) , 7.97 ( s , 1h , h6 ) , 7.227.49 ( m , 
 9h , ar ) , 6.87 ( d , 4h , j = 9.0 hz , ar ) , 5.58 ( s , 1h , 
 h1 ) , 5.34 ( d , 1h , j = 5.0 hz ,
hc = c - chol ) , 
 4.96 ( bs , 1h , ex , nhch2 ) , 4.53 ( s , 1h , h2 ) , 4.404.47 
 ( m , 1h , hc - o - chol ) 4.23 ( bs , 1h , h3 ) , 3.833.98 ( m , 
 4h , 2  h5 , ch2nh ) , 3.80 ( s , 3h , ch3o ) , 3.79 ( s , 6h , 2  ch3o ) , 3.583.61 ( d , 
 1h , j = 11.0 hz , h5 ) , 3.513.53 ( d , 
 1h , j = 11.0 hz , h5 ) , 3.27 ( bs , 1h , ex , 3-oh ) , 
 0.872.29 ( m , 40 h , chol ) , 0.69 ( s , 3h , ch3-chol ) ; c nmr ( cdcl3 )  162.1 , 158.63 , 158.60 , 155.5 , 
 148.7 , 144.5 , 141.8 ( c6 ) , 139.8 , 135.44 , 135.39 , 130.0 ( ar ) , 128.1 
 ( ar ) , 128.0 ( ar ) , 127.0 ( ar ) , 122.5 ( = ch , chol ) , 113.4 ( ar ) , 
 99.2 , 90.1 , 88.4 , 87.4 ( c1 ) , 86.6 , 79.1 ( c2 ) , 74.7 
 ( och - chol ) , 74.1 , 71.9 ( c5 ) , 70.6 ( c3 ) , 58.6 ( c5 ) , 
 56.7 ( ch - chol ) , 56.2 ( ch - chol ) , 55.2 ( ch3o ) , 50.0 ( ch - chol ) , 
 42.3 , 39.8 ( ch2-chol ) , 39.5 ( ch2-chol ) , 38.5 
 ( ch2-chol ) , 37.0 ( ch2-chol ) , 36.5 , 36.2 ( ch2-chol ) , 35.8 ( ch - chol ) , 31.9 ( ch - chol / ch2nh ) , 28.2 
 ( ch2-chol ) , 28.1 ( ch2-chol ) , 28.0 ( ch - chol ) , 
 24.3 ( ch2-chol ) , 23.8 ( ch2-chol ) , 22.8 ( ch3-chol ) , 22.5 ( ch3-chol ) , 21.0 ( ch2-chol ) , 
 19.3 ( ch3-chol ) , 18.7 ( ch3-chol ) , 11.8 ( ch3-chol ) .
signals at 41.4 , 29.0 , 22.6 , 20.4 , 19.4 , 14.3 , and 
 11.4 ppm , presumably arising from a small contamination of unreacted 
 cholesterylprop-2-ynylcarbamate , were also observed in the c nmr spectrum .
nucleoside 3 ( 0.34 g , 0.50 
 mmol ) , pd(pph3)4 ( 60 mg , 0.05 mmol ) , cui ( 20 
 mg , 0.10 mmol ) , 1-ethynylpyrene ( 0.28 
 g , 1.00 mmol ) , and et3n ( 0.30 ml , 2.84 mmol ) in dmf ( 10 
 ml ) were reacted as described in the representative sonogashira protocol , 
 and the mixture was stirred at room temperature for 12 h. following 
 workup and purification , nucleoside 4i ( 0.31 g , 80% ) 
 was obtained as a slightly yellow solid , which was used in the next 
 step without further purification : rf = 
 0.5 ( 5% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 805.2554 ( [ m + na ] , c49h38n2o8na , calcd 805.2520 ) ; h nmr ( dmso - d6 )  11.89 ( s , 1h , ex , nh ) , 8.35 ( d , 1h , j = 8.5 hz ,
ar ) , 8.318.34 ( d , 1h , j = 8.5 
 hz , ar ) , 8.258.28 ( d , 1h , j = 8.0 hz ,
ar ) , 
 8.148.23 ( m , 4h , h6 , ar ) , 8.098.12 ( ap t , 1h , j = 8.0 hz ,
ar ) , 7.497.50 
 ( m , 2h , ar ) , 7.337.38 ( m , 4h , ar ) , 7.267.30 ( ap t , 
 2h , j = 7.5 hz ,
ar ) , 6.786.85 ( m , 4h , ar ) , 5.78 ( d , 
 1h , ex , j = 4.5 hz , 3-oh ) , 5.53 ( s , 1h , h1 ) , 
 4.34 ( s , 1h , h2 ) , 4.24 ( d , 1h , j = 4.5 hz , 

h5 ) , 3.433.52 ( m , 7h , och3 , h5 ) , 
 3.383.42 ( d , 1h , j = 11.0 hz , h5 ) ; c nmr ( dmso - d6 )  161.7 , 
 158.02 , 157.95 , 149.1 , 144.4 , 141.4 ( c6 ) , 135.4 , 130.8 , 130.7 , 130.6 , 
 130.4 , 129.6 ( ar ) , 129.5 ( ar ) , 128.8 ( ar ) , 128.2 ( ar ) , 128.1 ( ar ) , 
 127.8 ( ar ) , 127.7 ( ar ) , 127.1 ( ar ) , 126.63 ( ar ) , 126.58 ( ar ) , 125.7 
 ( ar ) , 125.6 ( ar ) , 124.8 ( ar ) , 124.5 ( ar ) , 123.5 , 123.3 , 116.9 , 113.17 
 ( ar ) , 113.16 ( ar ) , 98.2 , 91.3 , 88.2 , 87.8 , 87.2 ( c1 ) , 85.6 , 
 78.8 ( c2 ) , 71.4 ( c5 ) , 69.4 ( c3 ) , 58.7 ( c5 ) , 
 54.7 ( och3 ) , 54.6 ( och3 ) . a trace of pyridine 
 was observed in the c nmr .
nucleoside 3 ( 0.50 g , 0.73 
 mmol ) , pd(pph3)4 ( 90 mg , 0.07 mmol ) , cui ( 30 
 mg , 0.14 mmol ) , 3-ethynylperylene ( 0.27 
 g , 1.00 mmol ) , and et3n ( 0.40 mmol , 2.84 mmol ) in dmf ( 10 
 ml ) were reacted as described in the representative sonogashira protocol , 
 and the mixture was stirred at room temperature for 12 h. after workup 
 and purification , nucleoside 4j ( 0.49 g , 80% ) was obtained 
 as a brown solid material : rf = 0.5 ( 5% 
 meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 855.2675 ( [ m + na ] , c53h40n2o8na , calcd 
 855.2677 ) ; h nmr ( dmso - d6 ) 
  11.86 ( s , 1h , ex , nh ) , 8.348.40 ( m , 3h , ar ) , 8.22 
 ( d , 1h , j = 8.0 hz ,
ar ) , 8.13 ( s , 1h , h6 ) , 8.02 ( d , 
 1h , j = 8.5 hz ,
ar ) , 7.797.85 ( m , 2h , ar ) , 
 7.55 ( dt , 2h , j = 8.0 hz , 2.0 hz , ar ) , 7.487.51 
 ( m , 2h , ar ) , 7.287.37 ( m , 7h , ar ) , 7.22 ( d , 1h , j = 8.0 hz ,
ar ) , 7.097.12 ( t , 1h , j = 7.5 
 hz , ar ) , 6.836.88 ( m , 4h , ar ) , 5.77 ( d , 1h , ex , j = 4.5 hz , 3-oh ) , 5.51 ( s , 1h , h1 ) , 4.33 ( s , 1h , 
 h2 ) , 4.22 ( d , 1h , j = 4.5 hz , h3 ) , 
 3.773.83 ( m , 2h , 2  h5 ) , 3.59 ( s , 3h , och3 ) , 3.56 ( s , 3h , och3 ) , 3.483.51 ( d , 1h , j = 11.0 hz , h5 ) , 3.353.39
( d , 1h , j = 11.0 hz , h5 ) ; c nmr ( dmso - d6 )  161.7 , 158.05 , 158.00 , 149.0 , 144.4 , 
 141.3 ( c6 ) , 135.43 , 135.37 , 134.2 , 133.6 , 130.9 , 130.7 , 130.4 ( ar ) , 
 130.1 , 129.9 , 129.6 ( ar ) , 129.5 ( ar ) , 128.5 ( ar ) , 128.2 ( ar ) , 127.9 
 ( ar ) , 127.7 ( ar ) , 127.60 , 127.57 , 127.47 ( ar ) , 127.0 ( ar ) , 126.9 ( ar ) , 
 126.7 ( ar ) , 125.7 ( ar ) , 121.5 ( ar ) , 121.2 , 121.1 ( ar ) , 120.0 ( ar ) , 
 119.4 , 113.2 ( ar ) , 98.1 , 90.9 , 88.5 , 87.7 , 87.1 ( c1 ) , 85.6 , 
 78.8 ( c2 ) , 71.4 ( c5 ) , 69.4 ( c3 ) , 58.7 ( c5 ) , 
 54.80 ( ch3o ) , 54.75 ( ch3o ) . to a solution of nucleoside 4b ( 0.25 g , 0.36 mmol ) and 1-pyrenyl azide ( 110 mg , 0.45 mmol ) in thf / h2o / t - buoh 
 ( 10 ml , 3/1/1 , v
/ v / v ) were added aqueous sodium ascorbate ( 1 m , 0.70 
 ml , 0.70 mmol ) and aqueous cuso4 ( 7.5% , w / v , 0.65 ml , 0.19 
 mmol ) .
the solution was stirred at room temperature for 2 h , whereupon 
 it was taken up in etoac ( 50 ml ) and brine ( 50 ml ) .
the layers were 
 separated , and the organic phase was washed with saturated aqueous 
 nahco3 ( 50 ml ) .
the combined aqueous phase was back - extracted 
 with etoac ( 50 ml ) .
the combined organic phase was then dried ( na2so4 ) and evaporated to dryness and the resulting 
 residue purified by column chromatography ( 075% etoac in petroleum 
 ether , v / v ) to afford nucleoside 4k ( 230 mg , 76% ) as 
 a slightly yellow solid material : rf = 
 0.4 ( 70% etoac in petroleum ether , v / v ) ; esi - hrms m / z 848.2712 ( [ m + na ] , c49h39n5o8na , calcd 
 848.2691 ) ; h nmr ( dmso - d6 ) 
  11.85 ( s , 1h , ex , nh ) , 8.84 ( s , 1h , h - tz ) , 8.58 ( s , 1h , h6 ) , 
 8.48 ( d , 1h , j = 8.0 hz ,
ar ) , 8.358.38 ( d , 1h , j = 9.0 hz , ar ) , 8.328.35 ( d , 1h , j = 9.0 
 hz , ar ) , 8.30 ( d , 1h , j = 9.0 hz , ar ) , 8.168.20 
 ( overlapping d and t , 2h , ar ) , 7.79 ( d , 1h , j = 8.0 
 hz , ar ) , 7.457.48 ( d , 2h , j = 7.5 hz ,
ar ) , 
 7.287.39 ( m , 6h , ar ) , 7.177.20 ( t , 1h , j = 7.5 hz ,
ar ) , 6.886.93 ( 2d , 4h , j = 9.0 
 hz , ar ) , 5.79 ( d , 1h , ex , j = 4.5 hz , 3-oh ) , 
 5.64 ( s , 1h , h1 ) , 4.43 ( s , 1h , h2 ) , 4.13 ( d , 1h , j = 4.5 hz , h3 ) , 3.943.97 ( d , 1h , j = 9.0 hz , h5 ) , 3.863.90 ( d , 1h , j = 9.0 hz , h5 ) , 3.681 ( s , 3h , ch3o ) , 
 3.675 ( s , 3h , ch3o ) , 3.573.60 ( d , 1h , j = 11.0 hz , h5 ) ,
3.343.37 ( m , 1h , j = 11.0 hz , h5 ) ; c nmr ( dmso - d6 )  161.3 , 158.05 , 158.02 , 149.4 , 144.6 , 139.4 , 
 135.3 , 135.2 , 135.0 ( c6 ) , 131.6 , 130.6 , 130.14 , 130.09 , 129.7 ( ar ) , 
 129.6 ( ar ) , 129.5 ( ar ) , 128.7 ( ar ) , 127.8 ( ar ) , 127.6 ( ar ) , 127.0 
 ( ar ) , 126.6 ( ar ) , 126.5 ( ar ) , 126.1 ( ar ) , 125.3 , 125.1 ( ar ) , 124.8 
 ( ch - tz ) , 124.0 , 123.7 ( ar ) , 123.3 , 120.9 ( ar ) , 113.24 ( ar ) , 113.19 
 ( ar ) , 104.3 , 87.6 , 87.2 ( c1 ) , 85.6 , 79.0 ( c2 ) , 71.5 
 ( c5 ) , 70.0 ( c3 ) , 59.5 ( c5 ) , 54.9 ( ch3o ) . to a solution of nucleoside 4b ( 0.33 g , 0.56 mmol ) and 1-azidomethylpyrene ( 200 mg , 0.78 mmol ) in thf / h2o / t - buoh 
 ( 10 ml , 3/1/1 , v
/ v / v ) were added aqueous sodium ascorbate ( 1 m , 1.00 
 ml , 1.00 mmol ) and aqueous cuso4 ( 7.5% , w / v , 1.00 ml , 0.30 
 mmol ) .
the reaction mixture was stirred at room temperature for 2 
 h , whereupon it was taken up in etoac ( 50 ml ) and brine ( 50 ml ) .
the 
 layers were separated , and the organic phase was washed with saturated 
 aqueous nahco3 ( 50 ml ) .
the combined aqueous phase was 
 back - extracted with etoac ( 50 ml ) .
the combined organic phase was 
 dried ( na2so4 ) and evaporated to dryness and 
 the resulting residue purified by column chromatography ( 075% 
 etoac in petroleum ether , v / v ) to afford nucleoside 4l ( 0.43 g , 91% ) as a slightly yellow solid material : rf = 0.4 ( 70% etoac in petroleum ether ,
v / v ) ; esi - hrms m / z 862.2869 ( [ m + na ] , c50h41n5o8na , calcd 862.2847 ) ; h nmr ( dmso - d6 )  11.70 ( s , 1h , ex , nh ) , 8.56 ( d , 1h , j = 9.0 hz , ar ) , 8.44 ( s , 1h , h - tz ) , 8.298.36 ( m , 5h , h6 , 
 ar ) , 8.208.23 ( d , 1h , j = 9.0 hz , ar ) , 8.178.20 
 ( d , 1h , j = 9.0 hz , ar ) , 8.098.12 ( t , 1h , j = 8.0 hz ,
ar ) , 7.227.33 
 ( m , 6h , ar ) , 7.097.12 ( t , 1h , j = 7.5 hz , 

ar ) , 6.40 ( s , 2h , ch2py ) , 5.69 ( d , 1h , ex , j = 4.5 hz , 3-oh ) , 
 5.55 ( s , 1h , h1 ) , 4.32 ( s , 1h , h2 ) , 3.913.96 
 ( m , 2h , h3 , h5 ) , 3.813.84 ( d , 1h , j = 8.0 hz , h5 ) , 3.70 ( s , 3h , ch3o ) , 
 3.68 ( s , 3h , ch3o ) , 3.503.54 ( d , 1h , j = 11.0 hz , h5 ) , 3.253.29
( d , 1h , j = 11.0 hz , h5 ) ; c nmr ( dmso - d6 )  161.2 , 158.1 , 158.0 , 149.2 , 144.7 , 138.9 , 135.1 , 
 134.2 ( c6 ) , 131.0 , 130.7 , 130.1 , 129.7 ( ar ) , 129.6 ( ar ) , 129.1 , 128.4 , 
 128.2 ( ar ) , 127.8 ( ar ) , 127.6 ( ar ) , 127.5 ( ar ) , 127.2 ( ar ) , 126.52 
 ( ar ) , 126.45 ( ar ) , 125.7 ( ar ) , 125.5 ( ar ) , 125.0 ( ar ) , 124.0 , 123.7 , 
 122.7 ( ar ) , 122.4 ( ch - tz ) , 113.3 ( ar ) , 113.2 ( ar ) , 104.5 , 87.5 , 87.1 
 ( c1 ) , 85.6 , 79.0 ( c2 ) , 71.6 ( c5 ) , 70.0 ( c3 ) , 
 59.8 ( c5 ) , 54.9 ( ch3o ) , 54.8 ( ch3o ) , 
 50.7 ( ch2py ) .
alcohols 5b  l were dried by coevaporation 
 with anhydrous 1,2-dichloroethane and dissolved in anhydrous ch2cl2 . to this
were added anhydrous n , n-diisopropylethylamine ( dipea ) and 2-cyanoethyl - n , n - diisopropylchlorophosphoramidite ( pcl - reagent ) 
 ( quantities and volumes specified below ) , and the reaction mixture 
 was stirred at room temperature until analytical tlc indicated complete 
 conversion ( 2 h unless otherwise mentioned ) .
unless otherwise mentioned , 
 the reaction mixture was diluted with ch2cl2 ( 25 ml ) and washed with aqueous nahco3 ( 2  10 ml ) , 
 the combined aqueous phases were back - extracted with ch2cl2 ( 2  10 ml ) , and the combined organic phases 
 were dried ( na2so4 ) and evaporated to dryness . 

regardless of the workup procedure , the resulting residue was purified 
 by silica gel column chromatography ( typically 04% meoh / ch2cl2 , v / v ) and subsequent trituration from ch2cl2 and petroleum ether to provide the target phosphoramidites .
nucleoside 4b ( 0.34 g 0.58 
 mmol ) , dipea ( 0.50 ml , 2.90 mmol ) , pcl - reagent ( 0.20 ml , 0.87 mmol ) , 
 and anhydrous ch2cl2 ( 10 ml ) were mixed , reacted , 
 worked up , and purified as described in the representative protocol 
 to provide nucleoside 5b ( 0.38 g , 83% ) as a white foam : rf = 0.5 ( 2% meoh in ch2cl2 ,
v / v ) ; esi - hrms m / z 805.2973 ( [ m 
 + na ] , c42h47n4o9pna , calcd 805.2958 ) ; p nmr 
 ( cdcl3 )  149.8 , 149.3 .
nucleoside 4c ( 0.30 g , 0.42 
 mmol ) , dipea ( 300 l , 1.67 mmol ) , pcl - reagent ( 121 l , 
 0.54 mmol ) , and anhydrous ch2cl2 ( 5 ml ) were 
 mixed and reacted ( 5 h ) as described above . at this point the reaction 
 mixture was concentrated to one - third volume and diluted with diethyl 
 ether ( 100 ml ) and the organic phase sequentially washed with h2o ( 35 ml ) , h2o / dmf ( 70 ml , 1/1 , v / v ) , h2o ( 35 ml ) , and brine ( 35 ml ) . the organic phase was evaporated to 
 dryness and the resulting residue purified as described in the representative 
 protocol to provide nucleoside 5c ( 150 mg , 43% ) as a 
 white foam : rf = 0.6 ( 5% meoh in ch2cl2 , v / v ) ; esi - hrms m / z 939.3356 ( [ m + na ] , c50h53n4o11pna , calcd 939.3341 ) ; p nmr ( cdcl3 )  149.8 , 149.3 .
nucleoside 4d ( 0.37 g 0.52 
 mmol ) , dipea ( 0.44 ml , 2.52 mmol ) , pcl - reagent ( 0.18 ml , 0.78 mmol ) , 
 and anhydrous ch2cl2 ( 10 ml ) were mixed , reacted , 
 worked up , and purified as described in the representative protocol 
 to provide nucleoside 5d ( 0.39 g , 80% ) as a white foam : rf = 0.5 ( 2% meoh in ch2cl2 , v / v ) ; esi - hrms m / z 930.3068 ( [ m 
 + na ] , c45h49f3n5o10pna , calcd 930.3061 ) ; p nmr ( cdcl3 )  149.7 , 149.1 .
nucleoside 4e ( 204 mg , 0.26 
 mmol ) , dipea ( 184 l , 1.06 mmol ) , and pcl - reagent ( 106 l , 
 0.48 mmol ) in anhydrous ch2cl2 ( 4 ml ) were mixed 
 and reacted as described in the representative protocol .
at this point , 
 ice - cold etoh ( 1 ml ) was added and the solvents were evaporated off . 
 purification as described in the representative protocol provided 
 nucleoside 4e ( 190 mg , 74% ) as a slightly yellow foam : rf = 0.4 ( 5% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 1010.4408 
 ( [ m + na ] , c55h66n5o10pna , calcd 1010.4440 ) ; p nmr 
 ( cdcl3 )  149.8 , 149.2 . a minor impurity at 14 
 ppm was observed .
nucleoside 4f ( 175 mg , 0.22 
 mmol ) , dipea ( 154 l , 0.88 mmol ) , n - methylimidazole 
 ( 14 l , 0.18 mmol ) , pcl - reagent ( 75 l , 0.33 mmol ) , and 
 anhydrous ch2cl2 ( 3 ml ) were mixed and reacted 
 ( 2.5 h ) . the solvents were evaporated off , and the resulting residue 
 was purified as described in the representative protocol to provide 
 nucleoside 5f ( 183 mg , 83% ) as a white foam : rf = 0.4 ( 4% meoh in ch2cl2 , v / v ) ; maldi - hrms m / z 1016.4983 
 ( [ m + na ] , c55h72n5o10pna , calcd 1016.4909 ) ; p nmr 
 ( cdcl3 )  149.8 , 149.2 .
nucleoside 4 g ( 0.25 g , 0.28 
 mmol ) , dipea ( 0.24 ml , 1.37 mmol ) , pcl - reagent 0.10 ml , 0.42 mmol ) , 
 and anhydrous ch2cl2 ( 10 ml ) were mixed , reacted , 
 worked up , and purified as described in the representative protocol 
 to provide nucleoside 5 g ( 180 mg , 60% ) as a white foam : rf = 0.5 ( 2% meoh in ch2cl2 , v / v ) ; esi - hrms m / z 1100.5836 
 ( [ m + na ] , c61h84n5o10pna , calcd 1100.5848 ) ; p nmr 
 ( cdcl3 )  149.8 , 149.2 .
nucleoside 4h ( 240 mg , 0.23 
 mmol ) , dipea ( 0.19 ml , 1.08 mmol ) , pcl - reagent ( 0.08 ml , 0.34 mmol ) , 
 and anhydrous ch2cl2 ( 10 ml ) were mixed , reacted , 
 worked up , and purified as described in the representative protocol 
 to provide nucleoside 5h ( 190 mg , 66% ) as a white foam : rf = 0.5 ( 2% meoh in ch2cl2 , v / v ) ; esi - hrms m / z 1246.6571 
 ( [ m + na ] , c71h94n5o11pna , calcd 1246.6579 ) ; p nmr 
 ( cdcl3 )  149.8 , 149.3 .
nucleoside 4i ( 0.20 g , 0.26 
 mmol ) , dipea ( 225 l , 1.28 mmol ) , pcl - reagent ( 114 l , 
 0.51 mmol ) , and anhydrous ch2cl2 ( 4 ml ) were 
 mixed and reacted ( 4.5 h ) as described in the representative protocol . 

solvents were evaporated off , and the resulting residue was purified 
 as described in the representative protocol to provide nucleoside 5i ( 188 mg , 75% ) as a pale yellow foam : rf = 0.5 ( 5% meoh in ch2cl2 , v / v ) ; 
 maldi - hrms m / z 1005.3661 ( [ m + na ] , c58h55n4o9pna , calcd 1005.3606 ) ; p nmr ( cdcl3 ) 
  149.7 , 149.3 .
nucleoside 4j ( 0.47 g , 0.56 
 mmol ) , dipea ( 0.40 ml , 2.26 mmol ) , pcl - reagent ( 165 l , 0.73 
 mmol ) , and anhydrous ch2cl2 ( 4 ml ) were mixed , 
 reacted ( 3 h ) , worked up , and purified as described in the representative 
 protocol to provide nucleoside 5j ( 0.43 g , 78% ) as a 
 yellow foam : rf = 0.4 ( 4% meoh in ch2cl2 , v / v ) ; esi - hrms m / z 1055.3751 ( [ m + na ] , c62h57n4o9pna , calcd 1055.3763 ) ; p nmr ( cdcl3 )  149.7 , 149.3 nucleoside 4k ( 180 mg , 0.22 
 mmol ) , dipea ( 160 l , 0.88 mmol ) , pcl - reagent ( 63 l , 
 0.28 mmol ) , and anhydrous ch2cl2 ( 1.5 ml ) were 
 mixed and reacted ( 2.5 h ) as described in the representative protocol . 
 at this point
, the reaction mixture was diluted with etoac ( 20 ml ) 
 and washed with h2o ( 2  25 ml ) .
the organic phase 
 was dried ( na2so4 ) and evaporated to dryness 
 and the resulting residue purified as described in the representative 
 protocol to provide nucleoside 5k ( 184 mg , 82% ) as a 
 white foam : rf = 0.7 ( 5% meoh in ch2cl2 , v / v ) ; esi - hrms m / z 1048.3789 ( [ m + na ] , c58h56n7o9pna , calcd 1048.3769 ) ; p nmr ( cdcl3 )  149.8 , 149.1 .
nucleoside 4l ( 0.41 g , 0.49 
 mmol ) , dipea ( 345 l , 1.95 mmol ) , pcl - reagent ( 165 l , 
 0.73 mmol ) , and anhydrous ch2cl2 ( 5 ml ) were 
 mixed , reacted ( 2.5 h ) , worked up , and purified as described in the 
 representative protocol to provide nucleoside 5l ( 190 
 mg , 40% ) as a white foam : rf = 0.5 ( 3% 
 meoh in ch2cl2 , v / v ) ; esi - hrms m / z 1062.3909 ( [ m + na ] , c59h58n7o9pna , calcd 
 1062.3934 ) ; p nmr ( cdcl3 )  149.6 , 149.1 .
l14 , k14 , n14 , q14 , s14 , and v5 were prepared and characterized with 
 respect to identity ( maldi - ms ) and purity ( > 80% , ion - pair reverse - phase 
 hplc ) in previous studies .
all of the other ons 
 were synthesized , worked up , purified , and characterized essentially 
 as previously described .
briefly , ons 
 were synthesized on a 0.2 mol scale using an automated dna 
 synthesizer and long chain alkyl amine controlled pore glass columns 
 with a pore size of 500  .
the following 
 hand - coupling conditions were employed to incorporate monomers k  z into ons , which generally resulted 
 in coupling yields in excess of 95% ( coupling time ; activator ; phosphoramidite 
 solvent ) : monomers k / l / m / n / o / q / s / w / z ( 15 min ; 0.25 m 4,5-dicyanoimidazole in ch3cn ; ch3cn ) , monomer p ( 15 min ; 0.25 m 4,5-dicyanoimidazole 
 in ch3cn ; ch2cl2 ) , monomer v ( 30 min ; 0.25 m 4,5-dicyanoimidazole in ch3cn ; 
 ch2cl2 ) , and monomers x / y / z ( 15 min ; 0.25 m 5-[3,5-bis(trifluoromethyl)phenyl]-1h - tetrazole in ch3cn ; ch2cl2 ) .
ons were cleaved from the solid 
 support and protecting groups removed through treatment with concentrated 
 aqueous ammonia ( 55 c , 24 h ) .
ons were purified by ion - pair 
 reverse - phase hplc ( xterra ms c18 column ) using a gradient of 0.05 
 m triethylammonium acetate in water and 25% water in ch3cn , followed by detritylation ( 80% aqueous acoh ) and precipitation 
 ( naoac / naclo4/acetone , 18 c for 1216 
 h ) .
the identity of all synthesized ons was verified by maldi ms analysis 
 ( table s1 , supporting information ) recorded 
 in positive ion mode on a quadrupole time - of - flight tandem mass spectrometer 
 equipped with a maldi source .
purity ( > 80% ) was verified by ion - pair 
 reverse - phase hplc running in analytical mode .
thermal denaturation 
 temperatures and steady - state fluorescence emission spectra were determined 
 essentially as previously described .
briefly , 
 thermal denaturation temperatures were determined as the maximum of 
 the first derivative of the thermal denaturation curve ( a260 vs t ) recorded in medium salt buffer 
 ( tm buffer : 110 mm nacl , 0.1 mm edta , 
 ph adjusted with 10 mm na2hpo4/nah2po4 ) . a temperature ramp of 0.5 c / min was used in 
 all experiments .
reported thermal denaturation temperatures are an 
 average of at least two experiments within 1.0 c .
thermodynamic parameters for duplex formation were determined through 
 baseline fitting of denaturation curves ( vant hoff analysis ) 
 using software provided with the uv / vis spectrometer .
bimolecular 
 reactions , two - state melting behavior , and a heat capacity change 
 of cp = 0 upon hybridization were 
 assumed .
a minimum of two experimental denaturation curves were each 
 analyzed to minimize errors arising from baseline choice .
3-exonuclease degradation studies were performed 
 by observing 
 the change in absorbance at 260 nm and 37 c as a function of 
 time for a solution of ons ( 3.3 m ) in magnesium buffer ( 600 
 l , 50 mm tris - hcl , 10 mm mgcl2 , ph 9.0 ) to which 
 svpde ( snake venom phosphodiesterase ) dissolved in h2o 
 was added ( 12 l , 0.52 g , 0.03 u ) .
steady - state 
 fluorescence emission spectra were recorded using 
 the same buffers and on concentrations ( 1.0 m ) as in thermal 
 denaturation studies .
deoxygenation was deliberately 
 not applied to the samples , since the scope of the work was to determine 
 fluorescence under aerated conditions prevailing in bioassays .
steady - state 
 fluorescence emission spectra were obtained as an average of five 
 scans using ex 344 nm for v / y / z - modified 
 ons , ex 375 nm for w - modified ons , 
 ex 448 nm for x - modified ons , excitation 
 slit 5.0 nm , emission slit 5.0 nm , and a scan speed of 600 nm / min .
 oligonucleotides modified with conformationally 
 restricted nucleotides 
 such as locked nucleic acid ( lna ) monomers are used extensively in 
 molecular biology and medicinal chemistry to modulate gene expression 
 at the rna level . 
 major efforts have been devoted to the design of 
 lna derivatives that induce even higher binding affinity and specificity , 
 greater enzymatic stability , and more desirable pharmacokinetic profiles . 
 
 most of this work has focused on modifications of lna s oxymethylene 
 bridge . here , we describe an alternative approach for modulation of 
 the properties of lna : i.e. , through functionalization of lna nucleobases . 
 twelve structurally diverse c5-functionalized lna uridine ( u ) phosphoramidites 
 were synthesized and incorporated into oligodeoxyribonucleotides ( ons ) , 
 which were then characterized with respect to thermal denaturation , 
 enzymatic stability , and fluorescence properties . 
 ons modified with 
 monomers that are conjugated to small alkynes display significantly 
 improved target affinity , binding specificity , and protection against 
 3-exonucleases relative to regular lna . in contrast , ons modified 
 with monomers that are conjugated to bulky hydrophobic alkynes display 
 lower target affinity yet much greater 3-exonuclease resistance . 
 
 ons modified with c5-fluorophore - functionalized lna - u monomers enable 
 fluorescent discrimination of targets with single nucleotide polymorphisms 
 ( snps ) . in concert , 
 these properties render c5-functionalized lna 
 as a promising class of building blocks for rna - targeting applications 
 and nucleic acid diagnostics . 
Introduction
Results and Discussion
Conclusion
Experimental
Section