Chemo-enzymatic synthesis of bicyclic 3′-azido- and 3′-amino-nucleosides

Abstract

Conformationally locked 3′-azido-3′-deoxythymidine analogues of T, U, A and C containing a 2′-O,4′-C-methylene linked bicyclic furanose moiety has been efficiently synthesized following a greener chemo-enzymatic convergent route. Thus, one of the two diastereotopic hydroxyl groups of 3-azido-3-deoxy-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose has been regioselectively acetylated using Novozyme®-435 in quantitative yield. The selective enzymatic acetylation can be carried out with the same efficiency using Novozyme®-435 for 10 cycles of reaction. The monoacetylated sugar derivative was converted to bicyclic 3′-azidonucleosides in four steps in overall yields of 60 to 68%. It has been demonstrated that 3′-azido-3′-deoxy-2′-O,4′-C-methylenethymidine can easily be converted into 3′-amino-3′-deoxy-2′-O,4′-C-methylenethymidine in 95% yield, which is an important monomer for the synthesis of therapeutically useful sugar-modified oligonucleotides.

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Introduction

Various sugar-modified oligonucleotides have been synthesized with improved pharmaceutical properties but none of the analogues were widely accepted as a Locked Nucleic Acid (LNA).^1,2 LNA is basically modified RNA in which a methylene bridge between 2′-oxygen and 4′-carbon locks the ribose in the 3′-endo conformation (Fig. 1). This leads to the significant enhancement of their hybridization property with the complimentary DNA/RNA strand, which plays a crucial role in the development of nucleic acid based therapeutics and diagnostics.^2–4

Fig. 1 Structure of LNA and bicyclic 3′-azido- & 3′-amino-nucleosides.

The 3′-amino-3′-deoxy-2′-O,4′-C-methyleneribonucleosides (3′-amino-LNA monomers) are important intermediate for N3′-P-O5′ oligonucleotides (ONs) possessing good antisense and antigene activity.⁵ The precursor for 3′-amino-LNA monomers, i.e. 3′-azido-3′-deoxy-2′-O,4′-C-methyleneribonucleosides have been classically synthesized from 3-azido-3-deoxy-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose or its derivatives in 18–41% yields.⁶ All these reported methods either suffer from the poor selectivity between two primary hydroxyl groups of 3-azido ribofuranose sugar or are lengthy due to multiple protection–deprotection steps. The use of lipases for selective protection and de-protection of hydroxyl groups as key step in the total synthesis of important compounds has been well established.⁷ Herein, we report an efficient chemo-enzymatic convergent synthesis of 3′-azido-3′-deoxy-2′-O,4′-C-methyleneribonucleosides 1a–d and conversation of 3′-azido-3′-deoxy-2′-O,4′-C-methylenethymidine (1a) to corresponding 3′-amino derivative 2 in 60–68% and 64% yields, respectively (Fig. 1).

Results and discussion

The starting sugar derivative, 3-azido-3-deoxy-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (3) was prepared from D-glucose by following literature procedure in 44% overall yield.⁸ The regioselective acetylation of C-5 hydroxyl over C-4 hydroxymethyl group was achieved by incubation of sugar derivative 3 with Novozyme®-435 in the presence of vinyl acetate as acetyl donor in toluene to obtain 3-azido-5-acetyl-3-deoxy-4-C-hydroxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (4) in quantitative yield (Scheme 1).⁹ Under optimized conditions, Novozyme®-435 was found efficient and regioselective upto 10 cycles of acetylation on compound 3.

Scheme 1 Chemo-enzymatic synthesis of bicyclic 3′-azido- & 3′-amino-nucleosides. Reagents and conditions (i) Novozyme®-435, toluene, 25–28 °C, 200 rpm, quantitative yield; (ii) TsCl, Py, 0 °C – rt, 95%; (iii) AcOH–Ac₂O–conc. H₂SO₄ (100 : 10 : 0.1), rt, 94%; (iv) nucleobase, N,O-bis(trimethylsilyl)acetamide (BSA), TMSOTf, CH₃CN or 1,2-dichloroethane (for A^BZ only), 80 °C, 80–88%; (v) 2 M NaOH, H₂O : dioxane (1 : 1), NH₄OH (only for 7d), rt, 85–89%; (vi) H₂, Pd/C, EtOAc, rt, 95%.

The tosylation of the lone hydroxyl group in enzymatically mono-acetylated compound 4 afforded 5-O-acetyl-3-azido-3-deoxy-4-C-(p-toluenesulphonyloxymethyl)-1,2-O-isopropylidene-α-D-ribofuranose (5) in 95% yield. The X-ray diffraction analysis of tosylated compound 5 unambiguously established that Novozyme®-435 exclusively acetylates C-5 hydroxyl over C-4 hydroxymethyl group. The ORTEP diagram of compound 5 is shown in Fig. 2 as two crystallographically independent units. The detailed crystallographic data of compound 5 has been deposited in the ESI.†

Fig. 2 ORTEP diagram of compound 5 drawn in 20% thermal probability ellipsoids with atomic numbering scheme showing two crystallographically independent units.

Acetolysis of compound 5 with acetic acid–acetic anhydride–sulphuric acid (100 : 10 : 0.1) afforded a mixture of anomeric azidosugar derivative 6a–b in 94% yield. For the convergent synthesis of bicyclic 3′-azidonucleosides the anomeric mixture 6a–b was used as common glycosyl donor for the Vorbrüggen's coupling reaction¹⁰ with different nucleobases, viz. thymine, uracil, cytosine and 6-N-benzoyladenine in the presence of N,O-bis(trimethylsilyl)acetamide and trimethylsilyltrifluoromethane sulfonate in acetonitrile or 1,2-dichloroethane (only for 6-N-benzoyladenine) resulting in the formation of nucleosides 7a–d in 80–88% yields. Subsequently, deacetylation with concomitant intramolecular 2′-O,4′-C-cyclization in nucleosides 7a–d under alkaline condition afforded 3′-azido-3′-deoxy-2′-O,4′-C-methyleneribonucleosides 1a–d in 85–89% yields. The oligonucleotides having a N3′-P5′ phosphoramidate linkage are well known for showing very high binding affinity with ssDNA and RNA as well as with dsDNA.^6e The selective reduction of azido group of compound 1a was achieved by Pd–C under hydrogen atmosphere in ethyl acetate to afford the amino compound 2 in 95% yield, which is an important precursor for the synthesis of N3′-P5′ oligonucleotides (Scheme 1).

The structure of all synthesized compounds, i.e. 1a–d, 2, 3, 4, 5, 6a–b and 7a–d were unambiguously established on the basis of their spectral data (¹H- and ¹³C- NMR spectra, IR spectra and HRMS) analysis. The structure of known compounds 1a, 1c, 1d, 2, 3, 4 and 5 was further confirmed on the basis of comparison of their physical and spectral data with those reported in the literature.^6,9 The structure of monohydroxy compound 4 obtained by lipase-mediated selective acetylation of one of the two primary hydroxyl groups in dihydroxy compound 3 was confirmed by X-ray crystallographic study of its corresponding tosyl derivative 5.

Experimental section

Melting points were determined on Buchi M-560 instrument and are uncorrected. The IR spectra were recorded on a Perkin-Elmer model 2000 FT-IR spectrometer by making KBr disc for solid samples and thin film for oils. The ¹H- and ¹³C NMR spectra were recorded at Jeol alpha-400 spectrometer at 400 and 100.6 MHz, respectively using TMS as internal standard. The chemical shift values are on δ scale and the coupling constants (J) are in Hz. The mass spectra analyses were done on a microTOF-Q instrument from Bruker Daltonics, Bremen and 6520 Q-TOF instrument from Agilent Technologies. The optical rotations were measured on Rudolph Autopol II automatic polarimeter using light of 589 nm wavelength. Analytical TLCs were performed on precoated Merck silica-gel 60F₂₅₄ plates; the spots were detected either under UV light or by charring with 4% alcoholic H₂SO₄. Silica gel (100–200 mesh) was used for column chromatography. The single crystal X-ray diffraction data was collected on an Oxford Diffraction XCalibur single crystal X-ray instrument having CCD camera [Cu Kα radiation (λ = 1.54184)] at USIC, University of Delhi, Delhi. The lipase Novozyme®-435 was obtained as gift from Novozyme A/S Denmark.

5-O-Acetoxy-1,2-di-O-acetyl-3-azido-3-deoxy-4-C-(p-toluenesulphonyloxymethyl)-α,β-D-ribofuranose (6a–b)

Acetic anhydride (12.72 mL, 135.9 mmol) and concentrated sulphuric acid (0.071 mL, 1.35 mmol) was added to a stirred solution of compound 5 (6.0 g, 13.5 mmol) in acetic acid (78.04 mL, 1359.1 mmol) at 0 °C and mixture was stirred for 5 h. The reaction was quenched by addition of water (150 mL) and extracted with chloroform (3 × 100 mL). The combined organic layer was washed with bicarbonate solution (2 × 100 mL), with cold water (2 × 100 mL) and then dried over sodium sulphate. The excess of solvent was removed under reduced pressure, the residue thus obtained was purified by silica gel column chromatography using ethyl acetate in petroleum ether as gradient solvent system to afford an anomeric mixture (α:β = 1 : 5, based on comparision of integration of anomeric proton) of 6a–b as colourless oil (6.2 g, 94%). R_f = 0.6 (40% ethyl acetate in petroleum ether). IR (thin film) ν_max: 2963, 2121, 1752, 1598, 1437, 1369, 1225, 1178, 1096, 1020, 972, 815 and 666 cm⁻¹;¹H NMR (CDCl₃, 400 MHz): δ 6.34 (J = 4.4 Hz, d, C-1Hα), 6.02 (s, C-1Hβ); Copy of ¹H NMR (CDCl₃, 400 MHz) and ¹³C NMR (CDCl₃, 100.6 MHz) spectra are given in ESI;† HR-ESI-TOF-MS: m/z 508.0996 ([M + Na]⁺), calcd for [C₁₉H₂₃N₃O₁₀S + Na]⁺ 508.0996.

General procedure for the synthesis of nucleosides 7a, 7b and 7c

To the stirred solution of compound 6a–b (1.5 g, 3.0 mmol) and thymine (0.58 g, 4.6 mmol)/uracil (0.51 g, 4.6 mmol) or cytosine (0.51 g, 4.6 mmol) in anhydrous acetonitrile (30 mL) N,O-bis(trimethylsilyl)acetamide (3.05 mL) was added dropwise. The reaction mixture was stirred at reflux for 1 h, and then cooled to 0 °C. In the cooled reaction mixture trimethylsilyltrifluoromethane sulfonate (0.92 mL, 5.2 mmol) was added dropwise under stirring and the solution was heated at 70–80 °C for 4–6 h. The reaction was quenched with a cold saturated aqueous solution of sodium hydrogen carbonate (100 mL) and the compound was extracted with chloroform (3 × 100 mL). The combined organic phase was washed with saturated aqueous solutions of NaHCO₃ (2 × 100 mL) and brine (2 × 50 mL) and was dried over anhydrous Na₂SO₄. The excess of solvent was removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using methanol in chloroform as gradient solvent system to afford nucleosides 7a–c in 80–88% yields.

2′,5′-Di-O-acetyl-3′-azido-3′-deoxy-4′-C-p-toluenesulfonyloxymethylthymidine (7a). It was obtained as off white solid (1.50 g, 88%). R_f = 0.5 (5% methanol in chloroform); M. Pt.: 110–112 °C; [α]²⁹_D = +15.14 (c 0.1, MeOH); ¹H NMR (CDCl₃, 400 MHz): δ 9.07 (1H, brs), 7.81 (2H, d, J = 8.4 Hz), 7.38 (2H, d, J = 8.4 Hz), 7.01 (1H, s), 5.57–5.60 (2H, m), 4.68 (1H, d, J = 6.0 Hz), 4.36 (1H, d, J = 12.0 Hz), 4.10–4.21 (3H, m), 2.46 (3H, s), 2.16 (3H, s), 2.08 (3H, s) and 1.92 (3H, s); ¹³C NMR (CDCl₃, 100.6 MHz): δ 170.0, 169.8, 163.5, 150.0, 145.3, 137.3, 132.3, 130.0, 128.0, 111.7, 90.8, 83.7, 74.4, 67.5, 64.3, 62.8, 21.7, 20.7, 20.4 and 12.4; IR (thin film) ν_max: 3199 (br), 2927, 2120, 1696, 1370, 1228, 1049, 998, 815, 732 and 667 cm⁻¹; HR-ESI-TOF-MS: m/z 574.1213 ([M + Na]⁺), calcd for [C₂₂H₂₅N₅O₁₀S + Na]⁺ 574.1214.

2′,5′-Di-O-acetyl-3′-azido-3′-deoxy-4′-C-p-toluenesulfonyloxymethyluridine (7b). It was obtained as white solid (0.91 g, 83%). R_f = 0.6 (5% methanol in chloroform); M. Pt.: 86–88 °C; [α]³⁰_D = +5.62 (c 0.1, MeOH); ¹H NMR (CDCl₃, 400 MHz): δ 9.24 (1H, brs), 7.81 (2H, d, J = 8.0 Hz), 7.38 (2H, d, J = 8.4 Hz), 7.20 (1H, d, J = 8.4 Hz), 5.75 (1H, dd, J = 2.4 and 8.2 Hz), 5.61–5.56 (2H, m), 4.68 (1H, d, J = 6.4 Hz), 4.35 (1H, d, J = 12.0 Hz), 4.20 (1H, d, J = 11.2 Hz), 4.16–4.11 (2H, m), 2.47 (3H, s), 2.18 (3H, s) and 2.08 (3H, s); ¹³C NMR (CDCl₃, 100.6 MHz): δ 170.2, 169.8, 162.8, 149.9, 145.3, 141.5, 132.3, 129.9, 128.0, 103.2, 91.2, 84.0, 74.5, 67.5, 64.2, 62.8, 21.7, 20.6 and 20.4; IR (thin film) ν_max: 3188 (br), 2922, 2851, 2121, 1752, 1721, 1692, 1461, 1369, 1225, 1177, 1049, 987, 812, 758 and 667 cm⁻¹; HR-ESI-TOF-MS: m/z 538.1237 ([M + H]⁺), calcd for [C₂₁H₂₃N₅O₁₀S + H]⁺ 538.1238.

2′,5′-Di-O-acetyl-3′-azido-3′-deoxy-4′-C-p-toluenesulfonyloxymethylcytidine (7c). It was obtained as white solid (1.42 g, 86%). R_f = 0.3 (10% methanol in chloroform); M. Pt.: 94–96 °C; [α]³⁰_D = +8.29 (c 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 7.80 (2H, d, J = 8.0 Hz), 7.51–7.47 (3H, m), 7.34 (2H, brs), 5.72 (1H, d, J = 7.2 Hz), 5.62 (1H, d, J = 4.4 Hz), 5.53 (1H, dd, J = 4.4 and 6.8 Hz), 4.92 (1H, d, J = 6.4 Hz), 4.12–4.07 (4H, m), 2.41 (3H, s), 2.04 (3H, s) and 1.96 (3H, s); ¹³C NMR (CDCl₃, 100.6 MHz): δ 170.1, 169.9, 166.1, 155.1, 145.2, 143.7, 132.5, 129.9, 128.0, 95.4, 93.8, 84.0, 75.1, 67.9, 64.3, 63.5, 21.7, 20.7 and 20.6; IR (thin film) ν_max: 3344 (br), 2922, 2851, 2121, 1752, 1656, 1483, 1366, 1289, 1225, 1176, 1047, 985, 788 and 667 cm⁻¹; HR-ESI-TOF-MS: m/z 537.1397 ([M + H]⁺), calcd for [C₂₁H₂₄N₆O₉S + H]⁺ 537.1398.

2′,5′-Di-O-acetyl-3′-azido-3′-deoxy-4′-C-p-toluenesulfonyloxymethyl-6-N-benzoyladenosine (7d). To the stirred solution of compound 6a–b (1.5 g, 3.0 mmol) and 6-N-benzoyladenine (1.10 g, 4.6 mmol) in anhydrous dichloroethane (20 mL), N,O-bis(trimethylsilyl)acetamide (4.57 mL, 18.5 mmol) was added dropwise. The reaction mixture was stirred at reflux for 1 h, and then cooled to 0 °C. To the cooled reaction mixture trimethylsilyltrifluoromethane sulfonate (2.10 mL, 11.5 mmol) was added dropwise under stirring and the solution was heated at 70–80 °C for 10–12 h. The reaction was quenched with a cold saturated aqueous solution of sodium hydrogen carbonate (100 mL) and the compound was extracted with chloroform (3 × 100 mL). The combined organic phase was washed with saturated aqueous solutions of NaHCO₃ (2 × 100 mL) and brine (2 × 50 mL), and was dried over anhydrous Na₂SO₄. The excess of solvent was removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using methanol in chloroform as gradient solvent system to afford nucleoside 7d as yellow solid (1.10 g, 80%). R_f = 0.5 (5% methanol in chloroform); M. Pt.: 90–92 °C; [α]²⁸_D =−14.05 (c 0.1, MeOH); ¹H NMR (CDCl₃, 400 MHz): δ 9.17 (1H, brs), 8.78 (1H, s), 8.05 (1H, s), 8.03 (2H, d, J = 7.2 Hz), 7.82 (2H, d, J = 8.4 Hz), 7.62 (1H, t, J = 7.2 Hz), 7.53 (2H, d, J = 8.0 Hz), 7.37 (2H, d, J = 8.0 Hz), 6.17 (1H, dd, J = 5.2 and 6.4 Hz), 6.05 (1H, d, J = 4.4 Hz), 5.01 (1H, d, J = 6.0 Hz), 4.42–4.14 (4H, m), 2.46 (3H, s), 2.16 (3H, s) and 2.02 (3H, s); ¹³C NMR (CDCl₃, 100.6 MHz): δ 169.8, 169.6, 164.6, 152.8, 151.3, 149.8, 145.3, 142.2, 133.3, 132.9, 132.3, 129.9, 128.4, 128.0, 127.9, 123.7, 87.0, 84.1, 74.4, 67.3, 63.7, 62.9, 21.6, 20.6 and 20.3; IR (thin film) ν_max: 3344 (br), 2922, 2851, 2120, 1753, 1599, 1451, 1366, 1219, 1177, 1073, 986, 772, 709 and 668 cm⁻¹; HR-ESI-TOF-MS: m/z 665.1770 ([M + H]⁺), calcd for [C₂₉H₂₈N₈O₉S + H]⁺ 665.1773.

General procedure for the synthesis of bicyclic azidonucleosides 1a–c

To a stirred solution of tosylated nucleosides 7a (0.5 g, 0.91 mmol)/7b (0.5 g, 0.93 mmol) or 7c (0.5 g, 0.93 mmol) in dioxane–water (1 : 1, 4 mL) was added 2 M, NaOH (4 mL) and reaction mixture was stirred at RT for 1–2 hours. On completion (analytical TLC), the reaction mixture was neutralized with acetic acid, the solvent was removed under reduced pressure. The residue thus obtained was purified by silica gel column chromatography using methanol in chloroform as gradient solvent system to afford the locked nucleosides T, U and C 1a–c in 85–89% yields.

3′-Azido-3′-deoxy-2′-O,4′-C-methylenethymidine (1a)^6b,d,e. It was obtained as white solid (0.237 g, 88%). HR-ESI-TOF-MS: m/z 296.0985 ([M + H]⁺), calcd for [C₁₁H₁₃N₅O₅ + H]⁺ 296.0989.

3′-Azido-3′-deoxy-2′-O,4′-C-methyleneuridine (1b). It was obtained as white solid (0.234 g; 89%). R_f=0.3 (10% methanol in chloroform); M. Pt.: 210–212 °C; [α]³¹_D = +129.26 (c 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.37 (1H, brs), 7.70 (1H, d, J = 8.0 Hz), 5.63 (1H, d, J = 8.0 Hz), 5.49 (1H, s), 5.39 (1H, t, J = 6.0 Hz), 4.53 (1H, s), 4.05 (1H, s) and 3.68–3.78 (4H, m); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 163.4, 150.1, 139.0, 100.9, 89.3, 86.4, 78.3, 71.2, 59.9 and 55.8; IR (thin film) ν_max: 3384, 2924, 2852, 2371, 2119, 1686, 1458, 1271, 1105, 1055, 1021, 937, 822, 772 and 714 cm⁻¹; HR-ESI-TOF-MS: m/z 282.0833 ([M + H]⁺), calcd for [C₁₀H₁₁N₅O₅ + H]⁺ 282.0833.

3′-Azido-3′-deoxy-2′-O,4′-C-methylenecytidine (1c)^6a. It was obtained as white solid (0.222 g; 85% yield). HR-ESI-TOF-MS: m/z 281.0993 ([M + H]⁺), calcd for [C₁₀H₁₂N₆O₄ + H]⁺ 281.0993.

3′-Azido-3′-deoxy-2′-O,4′-C-methyleneadenosine (1d)^6c. To a stirred solution of tosylated nucleoside 7d (0.5 g, 0.75 mmol) in dioxane–water (1 : 1, 8 mL) was added 2 M NaOH (8 mL) and NH₄OH (2 mL), and the reaction mixture was stirred at RT for 24 hours. On completion (analytical TLC), the reaction mixture was neutralized with acetic acid, the solvent was removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using methanol in chloroform as eluent to afford the locked nucleoside 1d as yellow solid (0.198 g; 86% yield). HR-ESI-TOF-MS: m/z 305.1104 ([M + H]⁺), calcd for [C₁₁H₁₂N₈O₃ + H]⁺ 305.1105.

3′-Amino-3′-deoxy-2′-O,4′-C-methylenethymidine (2)^6e

The reduction of azido group of bicyclic nucleoside 1a (0.2 g, 0.67 mmol) was achieved by 10% Pd–C (0.02 g) in ethyl acetate solvent (15 mL) under hydrogen atmosphere at RT for 2 hours. On completion, the reaction was quenched by filtering off the Pd–C. Excess solvent was removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using methanol in chloroform as eluent to afford the bicyclic nucleoside 2 as white solid (0.19 g; 95% yield). HR-ESI-TOF-MS: m/z 270.1093 ([M + H]⁺), calcd [C₁₁H₁₅N₃O₅ + H]⁺ 270.1084.

X-ray diffraction study of 5-O-acetoxy-3-azido-3-deoxy-4-C-p-toluenesulphonyloxymethyl-1,2-O-isopropylidene-α-D-ribofuranose (5)

Single crystal suitable for X-ray diffraction was grown by dissolving compound 5 in THF and allowing it to evaporate slowly at room temperature. X-Ray diffraction data was collected on an Oxford Diffraction XCalibur CCD diffractometer with graphite monochromated Cu Kα radiation (λ = 1.54184 Å) at temperature 298 K. The structure was solved by direct methods using SHELXS-97 and refined by full-matrix least-squares method on F² (SHELXL-97).¹¹ All calculations were carried out using the WinGX package of the crystallographic programs.¹² For the molecular graphics, the program DIAMOND-2 (ref. 13) and Mercury¹⁴ was used. Molecular structure have been drawn using ORTEP as software as given in Fig. 2. The selected bond lengths, bond angles, etc. are given in Table 1.

Table 1 Single crystal X-ray diffraction data of compound 5

Compound	5
Empirical formula	C18H23N3O8S
Formula weight	441.45
Temperature	298(2) K
Wavelength	0.71073 Å
Crystal system	Monoclinic
Space group	P21
Unit cell dimensions	a = 9.9038(14) Å, α = 90°
	b = 13.785(3) Å, β = 101.077(15)°
	c = 16.1493(3) Å, γ = 90°
Volume	2163.7(6) Å^3
Z	4
Density (calculated)	1.355 Mg m^−3
Absorption coefficient	0.198 mm^−1
F(000)	928
Crystal size	0.24 × 0.18 × 0.14 mm^3
Theta range for data collection	2.97 to 25.00°
Index ranges	−10 ≤ h ≤ 11, −16 ≤ k ≤ 16, −19 ≤ l ≤ 17
Reflections collected	13 901
Independent reflections	6745 [R(int) = 0.0420]
Completeness to theta = 25.00°	98.4%
Max. and min. transmission	0.9728 and 0.9540
Refinement method	Full-matrix least-squares on F^2
Data/restraints/parameters	6745/1/549
Goodness-of-fit on F^2	0.990
Final R indices [I > 2sigma(I)]	R1 = 0.0545, wR2 = 0.0992
R indices (all data)	R1 = 0.0845, wR2 = 0.1118
Absolute structure parameter	0.05(8)
Largest diff. peak and hole	0.190 and −0.173 e Å^−3
CCDC	995894

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Conclusion

We have successfully demonstrated highly efficient chemo-enzymatic synthesis of 3′-azido-3′-deoxy-2′-O,4′-C-methyleneribonucleosides T, U, C and A. There is an enhancement of yield in chemo-enzymatic synthesis over classical synthesis from 41 to 68% in T, 40 to 64 % in C and 21 to 60 % in case of A. On top of yield enhancement, the lipase can be recovered and reused for 10 cycles of regioselective acetylation reaction on diol precursor. Further, the bicyclic 3′-azidonucleosides can be converted into the corresponding 3′-aminonucleosides in high yield, which is an important monomer for the synthesis of therapeutically useful sugar-modified oligonucleotides.

Acknowledgements

We are grateful to the University of Delhi for providing financial support under DU-DST Purse Grant and under scheme to strengthen research and development. We are also thankful to CIF-USIC University of Delhi, Delhi for providing NMR spectral recording facility. MK and VKS thank CSIR for the award of Junior/Senior Research Fellowships.

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Footnote

† Electronic supplementary information (ESI) available: ¹H and ¹³C NMR spectra of compounds 6a–b, 7a–7d and 1b. CCDC 995894. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra06805j

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