This article was downloaded by: [University of Chicago]On: 16 March 2013, At: 07:40Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Nucleosides, Nucleotides and NucleicAcidsPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/lncn20

Effective Synthesis of 3′-Deoxy-3′-Azido Nucleosides for Antiviral andAntisense Ribonucleic Guanidine (RNG)ApplicationsEric R. Samuels a b , Joshua McNary a , Maribel Aguilar a & Ahmed M.Awad aa Chemistry Program, California State University Channel Islands,California, USAb Biology/Biotechnology Program, California State UniversityChannel Islands, California, USAVersion of record first published: 08 Mar 2013.

To cite this article: Eric R. Samuels , Joshua McNary , Maribel Aguilar & Ahmed M. Awad (2013):Effective Synthesis of 3′-Deoxy-3′-Azido Nucleosides for Antiviral and Antisense Ribonucleic Guanidine(RNG) Applications, Nucleosides, Nucleotides and Nucleic Acids, 32:3, 109-123

To link to this article: http://dx.doi.org/10.1080/15257770.2013.766752

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representationthat the contents will be complete or accurate or up to date. The accuracy of anyinstructions, formulae, and drug doses should be independently verified with primarysources. The publisher shall not be liable for any loss, actions, claims, proceedings,demand, or costs or damages whatsoever or howsoever caused arising directly orindirectly in connection with or arising out of the use of this material.

Nucleosides, Nucleotides and Nucleic Acids, 32:109–123, 2013Copyright C© Taylor and Francis Group, LLCISSN: 1525-7770 print / 1532-2335 onlineDOI: 10.1080/15257770.2013.766752

EFFECTIVE SYNTHESIS OF 3′-DEOXY-3′-AZIDO NUCLEOSIDES

FOR ANTIVIRAL AND ANTISENSE RIBONUCLEIC

GUANIDINE (RNG) APPLICATIONS

Eric R. Samuels,1,2 Joshua McNary,1 Maribel Aguilar,1 and Ahmed M. Awad1

1Chemistry Program, California State University Channel Islands, California, USA2Biology/Biotechnology Program, California State University Channel Islands,California, USA

� Two synthetic routes to 3′-deoxy-3′-azido nucleosides are described, one toward the synthesis of3′-deoxy-3′-azidouridine and a second toward 3′-deoxy-3′-azidocytidine. The target compounds mayserve as precursors to provide building blocks for use in automated synthesis of guanidine-linkedRNA analogs (RNG) or oligonucleotide N3′→P5′ phosphoramidates. Moreover, the synthetic ap-proaches are adaptable to the general synthesis of 3′-substituted 3′-deoxynucleosides for developmentof new antiviral drugs.

Keywords Antisense; RNA; ribonucleic guanidine; solid-phase synthesis; nucleoside-3′-azido

INTRODUCTION

Nucleoside analogs and modified nucleic acids possess numerous thera-peutic applications, including antiviral and anticancer activities. Nucleosideanalogs were among the first cytotoxic chemotherapeutic agents used forcancer treatment.[1] Additionally, the first approved antiretroviral treatmentfor HIV, azidothymidine (Figure 1a), is a nucleoside analog, sparking thesearch for similar modified dideoxynucleosides as antiretroviral drugs.[2]

Similarly, modified nucleotides can be used for gene silencing throughantisense technology. Oligonucleotide analogs capable of recognizing andbinding complementary strands of RNA/DNA, with the intention of pre-venting gene expression, are known as antisense/antigene agents.[3] Withrespect to single stranded mRNA silencing, these antisense agents formhybrid duplexes, via Watson-Crick base pairing, with the target mRNA

Received 19 November 2012; accepted 11 January 2013.Funding was provided by the Chemistry Program and Biology/Biotechnology Program at CSU

Channel Islands. We thank Scott Duffer and the University of California Santa Barbara for assistance inobtaining spectral data. We also thank Tobin Streamland for assistance with experimentation.

Address correspondence to Ahmed M. Awad, Chemistry Program, California State University Chan-nel Islands, One University Drive, Camarillo, CA 93012, USA. E-mail: ahmed.awad@csuci.edu

109

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

110 E. R. Samuels et al.

FIGURE 1 Structure of (a) 3′-azidothymidine; (b) Guanidinium linkage in RNG; (c) N3′→P5′ phos-phoramidate RNA.

and thus act by translational arrest or via mRNA degradation through anRNase H mechanism.[4] However, these oligonucleotides must show resis-tance to naturally occurring nucleases that cleave phosphodiester bonds.Nuclease resistance is therefore obtained by chemical modification of theoligonucleotide structure,[5] either by manipulation of the phosphate it-self or replacement of the sugar-phosphate backbone entirely. Replacementof the phosphodiester linkage with neutral linkages, as in PNA, not onlyresists nuclease cleavage, but also presents higher affinity and specificitybecause of its uncharged flexible backbone.[6] The same can be said for neu-trally altered methylphosphonate or even the negatively charged phosphor–othioate.[7,8]

It is therefore not surprising that positively charged oligonucleotideanalogs, such as guanidine-linked oligonucleotides (RNG in Figure 1b),would present a greater binding affinity for the negatively charged DNAor RNA targets.[9–14] Previous studies show that deoxyribonucleic guanidine(DNG) is nuclease resistant,[9,10,12,14] exhibits unprecedented stability,[10,11]

has greater cell permeability due to negatively charged membranes,[12] andbinds specifically to complimentary sequences to form double and triplehelices.[13]

Extensive work has been done toward the synthesis of 3′-, 5′-, and internalmonomers that are required for the solid-phase synthesis of DNG. However,RNG monomers exist only for adenyl and uridyl RNG.[14,15] Recently, we havesynthesized the 3′-terminal monomer for cytidyl RNG.[16] In this article, wereport a highly effective method and efficient separation techniques to at-tain a 5′-terminal monomer that contains 3′-azidocytidine, a new monomerthat may be used in cytidyl RNG oligonucleotide synthesis. This monomercan also be applied for the solid-phase synthesis of N3′→P5′ phosphorami-date oligonucleotides (Figure 1c) where 3′-O-nucleotides are replaced by

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

Synthesis of 3 ′-Deoxy-3 ′-Azido Nucleosides 111

3′-N -nucleotides.[17] In addition, an effective synthetic route to 3′-azido-2′-O-(tert-butyldimethylsilyl)-5′-O-(4-monomethoxytrityl)-3′-deoxyuridine is de-scribed. This latter compound can serve as uridyl RNG precursor. Further-more, the methods described herein can be applied as a convenient startingpoint for other 3′-deoxy-3′-substituted uridine or cytidine for antiviral appli-cations or other therapeutic purposes.

RESULTS AND DISCUSSION

Direct replacement of the 3′-hydroxyl on the ribose sugar with an al-ternative group produces the xylofuranose structure, which is undesiredin RNG synthesis. Therefore, correct protection and careful manipulation isnecessary to fabricate the desired stereoisomer. We have found two syntheticapproaches to produce 3′-deoxy-3′-azidoribonucleosides (Figure 2). Protec-tion of the other hydroxyl groups was also adjusted, for uridine, to makethem compatible with solid-phase RNG synthesis. The first method involvesepimerization of the 3′-oxygen with sodium benzoate on a 5′-O-tritylated-3′-O-mesylated precursor. Isomerization at the 3′ position has been reportedearlier, for instance from 5′-O-benzoylated analogs.[18] Replacement of thebenzoyl group with mesyl followed by direct addition of the azido groupproduces the correct stereoisomer. The second method involves cyclizationof the 3′-hydroxyl and the carbonyl group of the cytosine base with excesspotassium fluoride. Addition of the azido group can then occur directly with

O

OHOH

HO Pyrimidine

O

OH

HO

N

N

NH

O

O

OH

RO

NH

N

O

O

OBz

O

OHN3

HO Pyrimidine

FIGURE 2 Two synthetic approaches to produce 3′-deoxy-3′-azidoribonucleosides.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

112 E. R. Samuels et al.

a very high yield. Both methods offer a straightforward, effective alterna-tive to the previously reported addition of 3′-azido to the ribose sugar.[15]

Furthermore, the routes reported have flexibility and generality and are,therefore, potentially more useful.

Uridine

Previous synthesis of the 5′-terminal monomer for uridyl RNG involvedaddition of imino via hydroxylamine. The imino was then reduced withNaBH4 and AcOH at 5◦C. However, acetic acid was found to solidify atlow temperature, making it difficult to use effectively. After formation ofthe hydroxyamino, multistep reduction and subsequent addition of trifluo-roacetate, then treatment with methanolic ammonia for 5 days at 0◦C wasperformed.[15] We now have found a more convenient method to generatethe 5′-uridyl RNG monomer. This method involves simple protection, inver-sion in configuration, and eventual addition of azide to the 3′-hydroxyl onthe ribose sugar (Scheme 1). The azido can be easily reduced using 10%Pd/C and H2, as previously recorded.[19]

Commercially available uridine was protected with TBDMS on 2′- and5′-hydroxyl groups using the established method.[20] The formed 2′,5′-bis-O-(tert-butyldimethylsilyl)uridine 1 was treated with 2.5 equivalents ofmethanesulfonyl chloride to provide 2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-O-mesyluridine 2. Treatment of 2 with 90% aqueous TFA at 0◦C allowed selec-tive removal of the 5′-TBDMS to afford compound 3,[15,21] which was thentreated with monomethoxytrityl chloride (MMTrCl) in Et3N and CH2Cl2 toyield 4. MMTr protection of the 5′-hydroxyl is suitable for the RNG synthesisbecause it can be selectively removed by brief acid treatment during the syn-thesis.[15] Refluxing with excess sodium benzoate substituted the 3′-O-mesylwith benzoate to produce 1-[3′-O-benzoyl-5′-O-(4-monomethoxytrityl)-β-D-xylofuranosyl]uracil 5. The addition of sodium benzoate with heat alsoremoved the 2′-TBDMS group, which was re-added with 3 equivalents ofTBDMSCl and imidazole to provide 6. Removal of the benzoyl group wasthen performed using NaOMe and the formed compound, 7, was mesylatedto afford 8. Heating with LiN3 allowed simple nucleophilic substitutionof the 3′-O-mesyl with the azide group resulting in the final product3′-azido-2′-O-(tert-butyldimethylsilyl)-5′-O-(4-monomethoxytrityl)-3′-deoxy–uridine 9.

Cytidine

Although cytidyl DNG has been previously synthesized,[19] little atten-tion has been assigned to cytidyl RNG beyond the 3′-terminal monomer.[16]

While DNG cytidine involves a similar synthesis, including mesylation andcyclization, the conditions and isolation methods are quite different. We

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

Synthesis of 3 ′-Deoxy-3 ′-Azido Nucleosides 113

SCHEME 1 Synthesis of 3′-azidouridine: (a) TBDMSCl/AgNO3/Pyridine/THF; (b) MsCl/Et3N/DMAP/CH2Cl2 anhyd.; (c) 90% aq TFA, 0◦C; (d) MMTrCl/Et3N/CH2Cl2 anhyd.; (e)NaOBz/DMF, 130◦C; (f) TBDMSCl/imidazole/pyridine; (g) NaOMe/MeOH; (h) MsCl/Et3N/DMAP/CH2Cl2 anhyd.; (i) LiN3/DMF, 90◦C.

found and optimized an efficient method for the addition of azido to the3′-hydroxyl region of cytidine (Scheme 2). This method can also be used toappend any nucleophilic group to this 3′-hydroxyl region.

Synthesis began with benzoylation of the commercially available cytidineusing the reputable protection method,[22,23] in order to protect the exo-cyclicamine on the nucleobase. The resulting N 4-benzoylcytidine 10 was treatedwith TBDMSCl and AgNO3 as a catalyst to protect the 2′- and 5′-hydroxylgroups, resulting in 11. The 2′,5′-TBDMS protected product was isolatedusing a combination of normal silica gel chromatography and dry columnvacuum chromatography.[24] The dry column vacuum was found to be aquick and efficient alternative to the familiar flash column when isolatingeither a single product from salts, or removing a distinct impurity, such asthe more polar 3′,5′-TBDMS protected cytidine in this case. The purifiedcompound 11 was treated with 2.5 equivalents of methanesulfonyl chlorideto mesylate the 3′-hydroxyl, and the product was purified by dry columnvacuum chromatography to afford 12. Deprotection of the hydroxyl groups

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

114 E. R. Samuels et al.

SCHEME 2 Synthesis of 3′-azidocytidine: (a) (1) BzCl/TMSCl/pyridine; (2) NH4OH (aq); (b)TBDMSCl/AgNO3/Pyridine/THF; (c) MsCl/Et3N/DMAP/CH2Cl2 anhyd.; (d) TBAF/THF; (e) KF(excess)/2-methoxyethanol; (f) LiN3/DMF, 90◦C; (g) (1) BzCl/TMSCl/pyridine; (2) NH4OH (aq)

and removal of TBDMS groups[20,21] was achieved using a five-fold TBAF inTHF to form compound 13. It was found that previous methods of cyclizationwith KF[25] produced little or no anhydro species when using compound 13.However, cyclization of 13 occurred in high yield when exposed to excessanhydrous KF in 2-methoxyethanol at room temperature resulting in the 2,3′-anhydro product 14. Treatment with excess KF also displaced the N 4-benzoylmoiety, perhaps due to free nucleophilic fluorine in solution. Heating the2,3′-anhydro product in the presence of LiN3 allowed for ring-opening anddirect addition of the azide to afford 15. Replacement of benzoyl to theN 4 position on the nucleobase produced the final product N 4-Benzoyl-3′-azidocytidine 16, which can go through further protection and reduction aspreviously described.

CONCLUSION

The two synthetic routes provided are means of conveniently achievingnucleosides with 3′-azido moiety. The final products 9 and 16 can be used as5′-terminal monomers in the synthesis of either RNG or N3′→P5′ phospho-ramidate oligonucleotides for antisense therapy. In addition, synthesis andmanipulation of the xylofuranosyl structure 5 and the cyclized compound14 permits direct addition of nucleophiles to produce nucleosides with de-sired configurations. Accordingly, the methods described can be utilizedin the synthesis of other useful nucleoside analogs, such as antivirals with3′-C -methyl[26] or 3′-fluoro.[27] In conclusion, the synthetic approaches in the

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

Synthesis of 3 ′-Deoxy-3 ′-Azido Nucleosides 115

present work not only establish RNG monomers and several potentially bioac-tive compounds, but also bring an appropriate solution to 3′-isomerizationas a result of nucleophilic addition to the ribose sugar.

EXPERIMENTAL

General Procedure

1H and 13C NMR spectra were recorded on 400 or 500 MHz Varianinstruments, using CDCl3 or DMSO-d6 as solvents. Chemical shifts are re-ported in δ ppm and coupling constants (J ) are given in Hz. Thin-layerchromatography (TLC) was carried out on Silica Gel 60 F-254 pre-coatedplates (Selecto Scientific, Georgia, USA and EMD Millipore, Merck KGaA,Germany) and visualization of the products was performed under UV light.Silica gel used for flash column chromatography was Scientific Silica Gel(particle size 32–63, Selecto Scientific and/or particle size 38–63, What-man International). Silica gel used for dry vacuum chromatography wasFisher Scientific 350–600 mesh (particle size 15–40). ESI mass spectra wererecorded on QSTAR Pulsar Quadrupole/time-of-flight mass spectrometerwith Turbo Ion Spray Ionization Source (Applied Biosystems, Foster city,CA).

2′,5′-bis-O-(tert-Butyldimethylsilyl)uridine (1)

To a suspension of the commercially available uridine (3.66 g, 15 mmol)in anhydrous THF (120 mL) were added pyridine (6.1 mL, 75 mmol) andAgNO3 (5.61 g, 33 mmol). The mixture was stirred under N2 at room tem-perature for 10 min. TBDMSCl (4.97 g, 33 mmol) was added and the reactionmixture was stirred under N2 at room temperature for 24 h. The reactionwas quenched by addition of ethanol (8 mL), and the mixture was filteredthrough Celite, which was then washed twice with 50 mL of ethanol. The fil-trate was concentrated under reduced pressure and the residue was dissolvedin EtOAc, washed with H2O and brine, dried over anhydrous Na2SO4, andconcentrated in vacuum. The crude mixture was purified by silica gel col-umn chromatography (20% ethyl acetate in hexanes) to afford compound 1(4.95 g, 70%). 1H NMR (400 MHz, CDCl3): 8.55 br s, 1H (NH); 7.99 d, 1H,J = 8.3 (H-6); 5.98 d, 1H, J = 4.4 (H-1′); 5.70 dd, 1H, J = 8.3 (H-5); 4.20t, 1H, J = 4.4 (H-2′); 4.14–4.11 m, 2H (H-3′ and H-4′); 4.10–3.81 m, 2H (H-5′); 0.94 s, 9H (SiC(CH3)3); 0.91 s, 9H (SiC(CH3)3); 0.13 s, 6H (Si(CH3)2);0.12 s, 6H (Si(CH3)2).

2′,5′-bis-O-(tert-Butyldimethylsilyl)-3′-O-mesyluridine (2)

To a solution of 2′,5′-bis-O-(tert-butyldimethylsilyl)uridine (4.34 g,9.2 mmol) in dry methylene chloride (150 mL) were added DMAP (0.48 g,

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

116 E. R. Samuels et al.

3.9 mmol) and Et3N (12.9 mL, 92 mmol). The mixture was cooled to 0◦Cfor 15 min. MsCl (1.8 mL, 23 mmol) was added dropwise and the mixturewas stirred under N2 at 0◦C for 2 h. The solvent was removed under re-duced pressure and the residue was dissolved in CH2Cl2, washed with H2Oand brine, dried over anhydrous Na2SO4 and concentrated in vacuum. Thecrude mixture was purified by silica gel column chromatography (25% ethylacetate in hexanes) to afford compound 2 (4.5 g, 89%). HRMS (ESI) m/zcalcd for C22H42N2O8Si2S (M+Na)+ 573.2093, found 573.2118. 1H NMR(400 MHz, CDCl3): 8.33 br s, 1H (NH); 7.86 d, 1H, J = 8.2 (H-6); 6.01 d,1H, J = 5.4 (H-1′); 5.72 d, 1H, J = 8.0 (H-5); 4.96 t, 1H, J = 4.0 (H-3′); 4.40d, 1H, J = 2.0 (H-2′); 4.33 t, 1H, J = 5.2 (H-4′); 3.98–3.80 m, 2H (H-5′);3.10 s, 3H (SCH3); 0.93 s, 9H (SiC(CH3)3); 0.87 s, 9H (SiC(CH3)3); 0.13 s,6H (Si(CH3)2); 0.05 s, 6H (Si(CH3)2).

2′-O-(tert-Butyldimethylsilyl)-3′-O-mesyluridine (3)

A solution of compound 2 (3.4 g) in aqueous trifluoroacetic acid (90%,25 mL) was stirred at 0◦C for 30 min. The solvent was co-evaporated withtoluene (15 mL × 3), and the residue was purified by silica gel columnchromatography (80% ethyl acetate in hexanes) to obtain compound 3(2.5 g, 93%). HRMS (ESI) m/z calcd for C16H28N2O8SiS (M+Na)+ 459.1227,found 459.1235. 1H NMR (400 MHz, DMSO-d6): 11.46 s, 1H (NH); 7.89 d,1H, J = 8.0 (H-6); 5.84 d, 1H, J = 6.6 (H-1′); 5.76 dd, 1H, J = 8.0, 2.0 (H-5);4.96 m, 1H (H-3′); 4.46 m, 1H (H-4′); 4.24 d, 1H, J = 2.2 (H-2′); 3.67 d, 2H J= 2.8 (H-5′); 3.25 s, 3H (SCH3); 0.80 s, 9H (SiC(CH3)3); 0.05 s, 3H (SiCH3);−0.02 s, 3H (SiCH3). 13C NMR (400 MHz, DMSO-d6): 162.8, 150.7, 139.9,102.6, 86.4, 82.7, 79.1, 72.8, 60.2, 31.5, 25.4, 17.6, −5.1, −5.4.

2′-O-(tert-Butyldimethylsilyl)-3′-O-mesyl-5′-O-(4-monomethoxy-

trityl)uridine (4)

To a solution of compound 3 (2.0 g, 4.6 mmol) in dry methylene chloride(100 mL) and Et3N (3.2 mL, 23 mmol) was added MMTrCl (2.0 g, 6.9 mmol).The mixture was stirred under N2 at room temperature for 2 h, then cooledto 0◦C and quenched with MeOH (10 mL). The solvent was removed underreduced pressure and the residue was dissolved in CH2Cl2, washed withH2O and brine, dried over anhydrous Na2SO4 and concentrated in vacuum.The crude mixture was purified by silica gel column chromatography pre-washed with 2% Et3N (40% ethyl acetate in hexanes) to afford compound 4(2.9 g, 89%). HRMS (ESI) m/z calcd for C36H44N2O9SiS (M+Na)+ 731.2429,found 731.2450. 1H NMR (400 MHz, CDCl3): 8.56 br s, 1H (NH); 7.94 d, 1H,J = 8.2 (H-6); 7.32–7.20 m, 12H (ArH); 6.87 d, 2H, J = 9.0 (ArH); 5.94 d,1H, J = 4.0 (H-1′); 5.27 d, 1H, J = 8.2 (H-5); 5.11 t, 1H, J = 5.0 (H-3′); 4.48t, 1H, J = 4.4 (H-4′); 4.39 d, 1H, J = 5.2 (H-2′); 3.79 s, 3H (MMTr-OCH3);

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

Synthesis of 3 ′-Deoxy-3 ′-Azido Nucleosides 117

3.73–3.43 m, 2H (H-5′); 2.91 s, 3H (SCH3); 0.88 s, 9H (SiC(CH3)3); 0.13 s, 6H(Si(CH3)2). 13C NMR (400 MHz, CDCl3): 163.9, 159.2, 150.4, 143.4, 143.3,139.8, 134.1, 130.7, 128.7, 128.6, 128.4, 127.8, 113.6, 102.7, 89.1, 88.0, 80.7,76.1, 74.6, 61.3, 55.5, 38.4, 25.7, 18.2, −4.7, −4.9.

1-[3′-O-Benzoyl-5′-O-(4-monomethoxytrityl)-β-D-xylofuranosyl]-

uracil (5)

Sodium benzoate (9.37 g, 65 mmol) was added to a solution of compound4 (9.2 g, 13 mmol) in anhydrous DMF (300 mL). The reaction mixture wasrefluxed with stirring under N2 at 130◦C for 24 h. After cooling to roomtemperature, the mixture was filtered through Celite, washed with ethanoland the filtrate was concentrated in vacuum. The crude was dissolved inCH2Cl2 (200 mL) and further impurities were removed by filtration. Thefiltrate was concentrated under reduced pressure and the crude mixturewas purified by silica gel column chromatography pre-washed with 2% Et3N(60% ethyl acetate in hexanes) to afford compound 5 (2.9 g, 36%). HRMS(ESI) m/z calcd for C36H32N2O8 (M+Na)+ 643.2050, found 643.2057. 1HNMR (400 MHz, CDCl3): 10.22 br s, 1H (NH); 7.67 d, 1H, J = 8.0 (H-6);7.55 d, 2H, J = 7.4 (BzH); 7.38–7.17 m, 15H (ArH and BzH); 6.74 d, 2H,J = 8.8 (ArH); 5.80 s, 1H (H-1′); 5.53–5.49 m, 2H, (H-5 and H-3′); 4.87 q,1H, J = 3.6 (H-4′); 4.43 s, 1H (H-2′); 3.74 s, 3H (MMTr-OCH3); 3.55 d, 2H, J= 5.2 (H-5′). 13C NMR (400 MHz, CDCl3): 165.1, 164.0, 162.8, 158.8, 151.0,143.9, 140.2, 135.0, 133.9, 130.5, 129.7, 128.9, 128.7, 128.5, 128.0, 127.2,113.3, 101.7, 93.0, 87.3, 82.1, 79.7, 61.0, 55.4.

1-[3′-O-Benzoyl-2′-O-(tert-butyldimethylsilyl)-5′-O-(4-monometh-

oxytrityl)-β-D-xylofuranosyl]uracil (6)

To a solution of compound 5 (1.55 g, 2.5 mmol) in dry pyridine (25 mL)were added imidazole (0.51 g, 7.5 mmol) and tert-butyldimethylsilyl chloride(1.13 g, 7.5 mmol). The reaction mixture was stirred under N2 at roomtemperature for 24 h. The solvent was removed under reduced pressureand the residue was dissolved in EtOAc, washed with H2O and brine, driedover anhydrous Na2SO4 and concentrated in vacuum. The crude mixturewas purified by silica gel column chromatography pre-washed with 2% Et3N(33% ethyl acetate in hexanes) to afford compound 6 (1.5 g, 82%). HRMS(ESI) m/z calcd for C42H46N2O8Si (M+Na)+ 757.2915, found 757.2951. 1HNMR (400 MHz, CDCl3): 7.71 d, 2H, J = 7.4 (BzH); 7.61 d, 1H, J = 8.0(H-6); 7.40–7.18 m, 15H (ArH and BzH); 6.75 d, 2H, J = 8.6 (ArH); 5.75 s,1H (H-1′); 5.44 d, 1H, J = 8.2 (H-5); 5.28 t, 1H, J = 1.6 (H-3′); 4.77 q, 1H,J = 4.0 (H-4′); 4.39 s, 1H (H-2′); 3.74 s, 3H (MMTr-OCH3); 3.55 d, 2H, J =5.2 (H-5′); 0.93 s, 9H (SiC(CH3)3); 0.20 s, 6H (Si(CH3)2).

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

118 E. R. Samuels et al.

1-[2′-O-(tert-Butyldimethylsilyl)-5′-O-(4-monomethoxytrityl)

-β-D-xylofuranosyl]uracil (7)

A saturated solution of NaOMe in MeOH (20 mL) was added to com-pound 6 (1.1 g, 1.5 mmol) and the reaction mixture was stirred under N2

at room temperature for 1 h. Brine (40 mL) was added to the mixture andthe aqueous phase was separated from the formed precipitate by filtration.The filtrate was washed several times with CH2Cl2. The combined organiclayers were dried over anhydrous Na2SO4 and concentrated in vacuum. Thecrude mixture was purified by silica gel column chromatography pre-washedwith 2% Et3N (40% ethyl acetate in hexanes) to afford compound 7 (0.8 g,84%). HRMS (ESI) m/z calcd for C35H42N2O7Si (M+Na)+ 653.2653, found653.2676. 1H NMR (400 MHz, CDCl3): 7.88 d, 1H, J = 8.2 (H-6); 7.43–7.24 m,12H (ArH); 6.88 d, 2H, J = 8.8 (ArH); 5.73 s, 1H (H-1′); 5.53 d, 1H, J =8.2 (H-5); 4.35 q, 1H, J = 3.2 (H-4′); 4.19 s, 1H (H-2′); 4.03 d, 1H, J =2.2 (H-3′); 3.79 s, 3H (MMTr-OCH3); 3.70–3.64 m, 2H (H-5′); 0.87 s, 9H(SiC(CH3)3); 0.17 s, 3H (SiCH3); 0.11 s, 3H (SiCH3). 13C NMR (400 MHz,CDCl3): 163.9, 159.1, 150.4, 143.7, 141.4, 134.7, 130.5, 128.4, 128.3, 127.5,113.6, 101.0, 93.1, 88.0, 82.1, 81.7, 77.6, 62.6, 55.5, 25.8, 18.1, −4.6,−5.0.

1-[2′-O-(tert-Butyldimethylsilyl)-3′-O-mesyl-5′-O-(4-monomethoxytrityl)-β-D-xylofuranosyl]uracil (8)

To a solution of compound 7 (0.50 g, 0.8 mmol) in dry methylenechloride (25 mL) were added DMAP (0.05 g, 0.4 mmol) and Et3N (1.12 mL,8.0 mmol). The mixture was cooled to 0◦C for 15 min. MsCl (0.16 mL,2.0 mmol) was added dropwise and the mixture was stirred under N2 at0◦C for 4 h. The solvent was removed under reduced pressure and theresidue was dissolved in CH2Cl2, washed with H2O and brine, dried overanhydrous Na2SO4 and concentrated in vacuum. The crude mixture waspurified by silica gel column chromatography pre-washed with 2% Et3N(40% ethyl acetate in hexanes) to afford compound 8 (0.45 g, 80%). HRMS(ESI) m/z calcd for C36H44N2O9SiS (M+Na)+ 731.2429, found 731.2409. 1HNMR (400 MHz, CDCl3): 8.42 br s, 1H (NH); 7.46 d, 1H, J = 8.0 (H-6);7.44–7.28 m, 12H (ArH); 6.87 d, 2H, J = 9.0 (ArH); 5.76 d, 1H, J = 1.2(H-1′); 5.54 d, 1H, J = 8.2 (H-5); 4.78 dd, 1H, J = 2.0, 1.6 (H-3′); 4.58 q, 1H,J = 3.6 (H-4′); 4.52 s, 1H (H-2′); 3.79 s, 3H (MMTr-OCH3); 3.65–3.34 m,2H (H-5′); 2.77 s, 3H (SCH3); 0.91 s, 9H (SiC(CH3)3); 0.18 d, J = 2.6, 6H(Si(CH3)2). 13C NMR (400 MHz, CDCl3): 163.5, 159.0, 150.3, 143.8, 143.7,139.8, 134.8, 130.5, 128.6, 128.3, 127.5, 113.5, 101.6, 92.1, 87.6, 82.2, 80.7,79.7, 61.2, 55.5, 38.4, 25.7, 18.0, −4.7, −4.9.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

Synthesis of 3 ′-Deoxy-3 ′-Azido Nucleosides 119

3′-Azido-2′-O-(tert-butyldimethylsilyl)-5′-O-(4-monomethoxy-

trityl)-3′-deoxyuridine (9)

To a solution of compound 8 (0.43 g, 0.6 mmol) in anhydrous DMF(10 mL) was added lithium azide (0.25 g, 5.0 mmol) and the reaction mixturewas heated under N2 at 90◦C for 24 h. The solvent was removed underreduced pressure and the residue was taken in EtOAc, washed with H2Oand brine, dried over anhydrous Na2SO4 and concentrated in vacuum. Thecrude mixture was purified by silica gel column chromatography pre-washedwith 2% Et3N (30% ethyl acetate in hexanes) to afford compound 9 (0.28 g,70%). HRMS (ESI) m/z calcd for C35H41N5O6Si (M+Na)+ 678.2724, found678.2759. 1H NMR (400 MHz, CDCl3): 8.01 d, 1H, J = 8.2 (H-6); 7.41–7.28 m,12H (ArH); 6.88 d, 2H, J = 9.0 (ArH); 5.84 d, 1H, J = 2.4 (H-1′); 5.32 d,1H, J = 8.2 (H-5); 4.47 q, 1H, J = 2.4 (H-3′); 4.18 m, 1H (H-2′); 4.00 m,1H (H-4′); 3.79 s, 3H (MMTr-OCH3); 3.66–3.41 ddd, 2H, J = 2.0, 9.4, 24.8(H-5′); 0.92 s, 9H (SiC(CH3)3); 0.17 s, 6H (Si(CH3)2). 13C NMR (400 MHz,CDCl3): 163.5, 159.1, 150.3, 143.8, 143.6, 140.2, 134.5, 130.6, 128.5, 128.3,127.6, 113.6, 102.3, 90.2, 87.8, 80.7, 77.0, 61.7, 60.3, 55.5, 25.8, 18.2, −4.6,−4.9.

N4-Benzoylcytidine (10)

To a suspension of cytidine (4.86 g, 20 mmol) in anhydrous pyridine(100 mL) was added chlorotrimethylsilane (12.67 mL, 100 mmol) and themixture was stirred under N2 at room temperature for 15 min. Benzoylchloride (4.64 mL, 40 mmol) was added to the mixture and the reactionwas stirred under N2 at room temperature for 3 h. The mixture was cooledto 0◦C, and the reaction was quenched by addition of H2O (20 mL). Afterstirring for 5 min at 0◦C, 28% aqueous NH4OH (40 mL) was added andthe mixture was stirred at room temperature for 30 min. The mixture wasconcentrated under reduced pressure to near dryness and the residue wasstirred with cold H2O (40 mL) for 5 min, filtered, washed with cold H2O(40 mL), then washed twice with methanol (40 mL) and once with diethylether to afford white solid of pure compound 10 (6.1 g, 88%). 1H NMR(500 MHz, DMSO-d6): 11.20 br s, 1H (NH); 8.49 d, 1H, J = 6.8 (H-6);7.99–7.48 m, 5H (H-Bz); 7.32 br s, 1H (H-5); 5.79 d, 1H, J = 3.0 (H-1′); 5.51d, 1H, J = 4.9 (OH-2′); 5.19 t, 1H, J = 4.9 (OH-5′); 5.06 d, 1H, J = 5.9 (OH-3′); 4.00–3.95 m, 2H (H-2′ and H-3′); 3.91–3.89 m, 1H (H-4′); 3.76–3.57 m,2H (H-5′).

N4-Benzoyl-2′,5′-bis-O-(tert-butyldimethylsilyl)cytidine (11)

To a suspension of compound 10 (5.20 g, 15 mmol) in anhydrous THF(120 mL) were added pyridine (6.1 mL, 75 mmol) and AgNO3 (5.61 g,

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

120 E. R. Samuels et al.

33 mmol). The mixture was stirred under N2 at room temperature for 10 min.TBDMSCl (4.97 g, 33 mmol) was added and the reaction mixture was stirredunder N2 at room temperature for 24 h. The reaction was quenched by ad-dition of ethanol (8 mL), and the mixture was filtered through Celite, whichwas then washed twice with 50 mL of ethanol. The filtrate was concentratedunder reduced pressure and the residue was dissolved in EtOAc, washed withH2O and brine, dried over anhydrous Na2SO4, and concentrated in vacuum.The crude mixture was purified by silica gel column chromatography (20%ethyl acetate in hexanes) followed by dry column vacuum chromatographyto afford compound 11 (5.91 g, 69%). 1H NMR (500 MHz, CDCl3): 8.54br s, 1H (NH); 7.92 d, 1H, J = 6.8 (H-6); 7.62–7.50 m, 5H (H-Bz); 7.46br s, 1H (H-5); 5.97 br s, 1H (H-1′); 4.22 m, 1H (H-2′); 4.16–4.10 m, 2H(H-3′ and H-4′); 4.08–3.76 m, 2H (H-5′); 0.96 s, 9H (SiC(CH3)3); 0.93 s, 9H(SiC(CH3)3); 0.16 s, 6H (Si(CH3)2); 0.12 s, 6H (Si(CH3)2).

N4-Benzoyl-2′,5′-bis-O-(tert-butyldimethylsilyl)-3′-O-mesylcy-

tidine (12)

To a solution of N 4-Benzoyl-2′,5′-bis-O-(tert-butyldimethylsilyl)cytidine 11(2.31 g, 4.0 mmol) in dry methylene chloride (40 mL) were added DMAP(0.24 g, 2.0 mmol) and Et3N (5.6 mL, 40 mmol). The mixture was cooled to0◦C for 15 min. Methanesulfonyl chloride (0.77 mL, 10 mmol) was addeddropwise and the mixture was stirred under N2 at 0◦C for 2 h, then at roomtemperature overnight. The solvent was removed under reduced pressureand the residue was dissolved in CH2Cl2, washed with H2O and brine, driedover anhydrous Na2SO4 and concentrated in vacuum. The crude mixturewas purified by dry column vacuum chromatography (50% ethyl acetatein hexanes) to afford compound 12 (2.0 g, 75%) white crystals. HRMS(ESI) m/z calcd for C29H47N3O8Si2S (M+Na)+ 676.2520, found 676.2491.1H NMR (500 MHz, CDCl3): 8.42 br s, 1H (NH); 8.25 d, 1H, J = 6.9 (H-6);7.89–7.61 m, 5H (H-Bz); 7.54 d, 1H, J = 7.8 (H-5); 6.02 d, 1H, J = 2.9 (H-1′);4.98 t, 1H, J = 4.4 (H-3′); 4.45–4.43 m, 2H (H-2′ and H-4′); 4.11–3.91 dd, 2H(H-5′); 3.08 s, 3H (SCH3); 0.98 s, 9H (SiC(CH3)3); 0.91 s, 9H (SiC(CH3)3);0.18 s, 6H (Si(CH3)2); 0.13 s, 6H (Si(CH3)2).

N4-Benzoyl-3′-O-mesylcytidine (13)

To a solution of compound 12 (2.1 g, 3.2 mmol) in anhydrous THF(20 mL), tetra-n-butylammonium fluoride (1M in THF, 11.9 mL, 16.0 mmol)was added dropwise and the mixture was stirred under N2 at room temper-ature for 24 h. The solvent was removed under reduced pressure and theresidue was re-suspended in EtOAc, thoroughly washed with H2O and brine,dried over anhydrous Na2SO4 and concentrated in vacuum to afford whitecrystals of pure compound 13 (1.34 g, 98%). HRMS (ESI) m/z calcd for

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

Synthesis of 3 ′-Deoxy-3 ′-Azido Nucleosides 121

C17H19N3O8S (M+Na)+ 448.0789, found 448.0791. 1H NMR (500 MHz,DMSO-d6): 11.22 br s, 1H (NH); 8.34 br s, 1H (H-6); 8.00–7.48 m, 5H (H-Bz); 7.34 br s, 1H (H-5); 6.12 s, 1H (OH-2′); 5.90 d, 1H, J = 4.9 (H-1′); 5.40t, 1H, J = 4.9 (OH-5′); 4.98 t, 1H, J = 4.9 (H-3′); 4.41 q, 1H, J = 5.4 (H-4′);4.22 q, 1H, J = 4.4 (H-2′); 3.74–3.61 m, 2H (H-5′); 3.25 s, 3H (SCH3).

2,3′-Anhydrocytidine (14)

Excess anhydrous potassium fluoride (4.0 g) was added to a solution ofcompound 13 (1.0 g, 2.3 mmol) in anhydrous 2-methoxyethanol (25 mL).The reaction mixture was stirred under N2 at room temperature for 24 h.The solvent was removed under reduced pressure, and the crude mixturewas purified by silica gel column chromatography (40% MeOH in CH2Cl2)to afford 14 (0.46 g, 87%). HRMS (ESI) m/z calcd for C9H12N3O4 (M+H)+

226.0833, found 226.0828. 1H NMR (500 MHz DMSO-d6) δ (ppm) 7.75 d,1H, J = 7.5 (H-6); 7.25 d, 1H, J = 7.5 (H-5); 5.93 d, 1H, J = 6.5 (H-1′); 5.29t, 1H, J = 5 (H-3′); 4.31 t, 1H, J = 6 (H-4′); 4.11 d, 1H, J = 2.5 (H-2′); 3.61q, 2H, J = 10 (H-5′).

3′-Azidocytidine (15)

To a solution of compound 14 (0.7 g, 3.1 mmol) in anhydrous DMF(10 mL) was added lithium azide (1.28 g, 26 mmol), and the reaction mix-ture was heated under N2 at 90◦C for 24 h. After cooling to room temper-ature, the solvent was removed under reduced pressure and the residuewas dissolved in EtOAc, washed with H2O and brine, dried over anhydrousNa2SO4, and concentrated in vacuum. The crude mixture was purified by dryvacuum silica gel chromatography pre-washed with 5% Et3N (25% MeOH inCH2Cl2) to afford compound 15 (0.85 mg, 100%). HRMS (ESI) m/z calcdfor C9H12N6O4 (M+Na)+ 291.0811, found 291.0818. 1H NMR (500 MHzDMSO-d6) δ (ppm) 7.93 d, 1H, J = 7.5 (H-6); 7.29 d, 1H, J = 7 (H-5); 5.74 s,2H, (NH2); 5.70 d, 1H, J = 7 (H-1′); 5.54 s, 1H (OH-2′); 5.24 s, 1H (OH-5′);4.64 m, 1H (H-3′); 4.21 q, 1H, J = 5.0 (H-4′); 4.16 t, 1H, J = 5.0 (H-2′);3.67–3.63 dd, 2H, J = 16.5 (H-5′).

N4-Benzoyl-3′-azidocytidine (16)

Compound 15 (0.85 g, 3.1 mmol) was suspended in dry pyridine (25 mL)under N2, and chlorotrimethylsilane (1.41 mL, 11.1 mmol) was added. Thereaction mixture was stirred at room temperature for 20 min, then benzoylchloride (0.736 mL, 6.34 mmol) was added dropwise over 15 min, and themixture was stirred at room temperature for 24 h. The reaction mixturewas cooled to 0◦C, and the reaction was quenched by addition of H2O(10 mL). After stirring at 0◦C for 5 min, 28% NH4OH (20 mL) was addedand the mixture was stirred at room temperature for 30 min. The mixture

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

122 E. R. Samuels et al.

was evaporated under reduced pressure, and the residue was dissolved inEtOAc, washed with H2O and brine, dried over anhydrous Na2SO4, andconcentrated in vacuum to afford pure compound 16 (0.73 g, 65%). HRMS(ESI) m/z calcd for C16H16N6O5 (M+Na)+ 395.1075, found 395.1080. 1HNMR (500 MHz DMSO-d6) δ (ppm) 7.95 d, 1H, J = 7.5 (H-6); 7.85–7.41 m,5H (H-Bz); 7.34 d, 1H, J = 7.5 (H-5); 5.69 d, 1H, J = 2 (H-1′); 5.56 s, 1H(OH-2′); 5.35 s, 1H (OH-5′); 5.09 t, 1H, J = 5.0 (H-3′); 4.24 q, 1H, J = 5.5(H-4′); 4.01 q, 1H, J = 7 (H-2′); 3.84–3.71 dd, 2H, (H-5′).

REFERENCES

1. Galmarini, C.M.; Mackey, J.R.; Dumontet, C. Nucleoside analogues and nucleobases in cancer treat-ment. Lancet Oncol. 2002, 3(7), 415–424.

2. De Clercq, E. Antiretroviral drugs. Curr. Opin. Pharmacol. 2010, 10(5), 507–515.3. Uhlmann, E.; Peyman, A. Antisense oligonucleotides: a new therapeutic principle. Chem. Rev. 1990,

90(4), 543–584.4. Opalinska, J.B.; Gewirtz, A.M. Nucleic-acid therapeutics: basic principles and recent applications.

Nat. Rev. Drug Discov. 2002, 1, 503–514.5. Cohen, J.S. Selective anti-gene therapy for cancer: principles and prospects. Tohoku J. Exp Med. 1992,

168(2), 351–359.6. Pellestor, F.; Paulasova, P. The peptide nucleic acids (PNAs), powerful tools for molecular genetics

and cytogenetics. Eur. J. Hum. Genet. 2004, 12(9), 694–700.7. Stein, C.A.; Cohen, J.S. Oligonucleotides as inhibitors of gene expression: a review. Cancer Res. 1988,

48, 2659–2668.8. Stein, C.A.; Cheng, Y.C. Antisense oligonucleotides as therapeutic agents—is the bullet really magi-

cal? Science. 1993, 261(5124), 1004–1012.9. Barawkar, D.A.; Bruice, T.C. Synthesis, biophysical properties, and nuclease resistance properties of

mixed backbone oligodeoxynucleotides containing cationic internucleoside guanidinium linkages:deoxynucleic guanidine/DNA chimeras. Proc. Natl. Acad. Sci. USA. 1998, 95(19), 11047–11052.

10. Dempcy, R.O.; Almarsson, O.; Bruice, T.C. Design and synthesis of deoxynucleic guanidine: a poly-cation analogue of DNA. Proc. Natl. Acad. Sci. USA. 1994, 91(17), 7864–7868.

11. Dempcy, R.O.; Browne, K.A.; Bruice, T.C. Synthesis of the polycation thymidyl DNG, its fidelity inbinding polyanionic DNA/RNA, and the stability and nature of the hybrid complexes. J. Am. Chem.Soc. 1995, 117(22), 6140–6141.

12. Linkletter, B.A.; Szabo, I.E.; Bruice, T.C. Solid-phase synthesis of oligopurine deoxynucleic guanidine(DNG) and analysis of binding with DNA oligomers. Nucleic Acids Res. 2001, 29(11), 2370–2376.

13. Dempcy, R.O.; Browne, K.A.; Bruice, T.C. Synthesis of a thymidyl pentamer of deoxyribonucleicguanidine and binding studies with DNA homopolynucleotides. Proc. Natl. Acad. Sci. USA. 1995,92(13), 6097–6101.

14. Dempcy, R.O.; Luo, J.; Bruice, T.C. Design and synthesis of ribonucleic guanidine: a polycationicanalog of RNA. Proc. Natl. Acad. Sci. USA. 1996, 93(9), 4326–4330.

15. Kojima, N.; Szabo, I.E.; Bruice, T.C. Synthesis of ribonucleic guanidine: replacement of the nega-tive phosphodiester linkages of RNA with positive guanidinium linkages. Tetrahedron. 2002, 58(2),867–879.

16. Awad, A.M.; Collazo, M.J.; Carpio, K.; Flores, C.; Bruice, T.C. A convenient synthesis of the cytidyl3′-terminal monomer for solid-phase synthesis of RNG oligonucleotides. Tetrahedron Lett. 2012, 53,3792–3794.

17. Gryaznov, S.M. Oligonucleotide N3′→P5′ phosphoramidates and thio-phoshoramidates as potentialtherapeutic agents. Chem. Biodivers. 2010, 7, 477–493.

18. Herdewijn, P. Anchimeric assistance of a 5′-O-carbonyl function for inversion of configuration at the3′-carbon atom of 2′-deoxyadenosine. Synthesis of 3′-azido-2′,3′-dideoxyadenosine and 3′-azido-2′,3′-dideoxyinosine. J. Org. Chem. 1988, 53, 5050–5053.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013

Synthesis of 3 ′-Deoxy-3 ′-Azido Nucleosides 123

19. Szabo, I.E.; Bruice, T.C. DNG cytidine: synthesis and binding properties of octameric guanidinium-linked deoxycytidine oligomer. Bioorg. Med. Chem. 2004, 12(15), 4233–4244.

20. Ogilvie, K.K.; Beaucage, S.L.; Schifman, A.L.; Theriault, N.Y.; Sadana, K.L. The synthesis of oligori-bonucleotides. II. The use of silyl protecting groups in nucleoside and nucleotide chemistry. VII.Can. J. Chem. 1978, 56(21), 2768–2780.

21. Crouch, R.D. Selective monodeprotection of bis-silyl ethers. Tetrahedron. 2004, 60, 5833–5871.22. Gait, M.J. in Oligonucleotide Synthesis: A Practical Approach, IRL: Oxford Oxfordshire, Washington, DC,

1984.23. Zhu, X.; Williams, H.J.; Scott, A.I. An improved transient method for the synthesis of N -benzoylated

nucleosides. Synth. Commun. 2003, 33, 1233–1243.24. Pedersen, D.S.; Rosenbohm, C. Dry column vacuum chromatography. Synthesis. 2001, 16, 2431–2434.25. Pierra, C.; Amador, A.; Badaroux, E.; Storer, R.; Gosselin, G. Synthesis of 2′-C-methylcytidine and

2′-C-methyluridine derivatives modified in the 3′-position as potential antiviral agents. Coll. Czech. C.C. 2006, 71, 991–1010.

26. Aljarah, M.; Couturier, S.; Mathe, C.; Perigaud, C. Synthesis of 3′-deoxy-3′-C-methyl nucleosidederivatives. Bioorg. Med. Chem. 2008, 16, 7436–7442.

27. Mentel, R.; Kinder, M.; Wegner, U.; von Janta-Lipinski, M.; Matthes, E. Inhibitory activity of 3′-fluoro-2′ deoxythymidine and related nucleoside analogues against adenoviruses in vitro. AntiviralRes. 1997, 34, 113–119.

Dow

nloa

ded

by [

Uni

vers

ity o

f C

hica

go]

at 0

7:40

16

Mar

ch 2

013