Chemo-enzymatic synthesis of 3′-O,4′-C-methylene-linked α-L-arabinonucleosides

Abstract

Lipozyme® TL IM catalyzed the diastereoselective acetylation of C-4′-hydroxymethyl over the other three hydroxyl groups of C-4′-hydroxymethyl-β-D-xylofuranosylnucleosides using vinyl acetate as acetyl donor to afford the corresponding C-4′-acetoxymethylnucleosides in 88 to 95% yields. The developed biocatalytic methodology has been successfully used for the efficient and environmentally friendly synthesis of 3′-O,4′-C-methylene-linked α-L-arabinofuranosylnucleosides from enzymatically monoacetylated nucleosides in 63 to 79% overall yields. The screening of vinyl esters of different alkyl chain lengths, i.e. vinyl acetate, vinyl propanoate and vinyl butanoate as acylating agent for biocatalytic diastereoselective acylation of the C-4′-hydroxymethyl group of tetrahydroxy-β-D-xylofuranosylthymine revealed that the rate of butanoylation and propanoylation is 2.0 and 1.5 times faster than that of acetylation, respectively.

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Introduction

The chemical modification in the sugar moiety of oligonucleotides to restrict the furanose ring to flipping in the desired fashion has been proven to enhance the hybridization energy with complimentary DNA/RNA strands to attain better biological activities, in particular antisence/antigene activities.^1–4 Various structural changes to the sugar moiety of nucleosides have been introduced over time in a consecutive manner to add more and better biological traits to existing candidates, but the best known is the bicyclic bridging between 2′-O and 4′-C atoms, known as locked nucleic acid monomers (Fig. 1).^5–9

Fig. 1 Various possible isomers of LNA and ONA.

The biocatalytic transformation of nucleosides has allowed the production of various pharmacologically active compounds in last few decades.^10,11 Recently, our research group has demonstrated Lipozyme® TL IM mediated synthesis of 3′-azido/-amino-xylobicyclonucleosides.¹² We herein report the chemo-enzymatic synthesis of novel 3′-O,4′-C-methylene-linked α-L-arabinofuranosylnucleosides, i.e. monomer of α-L-arabino-oxetanonucleic acids. These nucleoside monomers are similar to the previously reported α-L-ribo-oxetano nucleic acid (ONA) monomer but with inverted stereochemistry at C-2′ position as present in RNA (Fig. 1).^9a In one of the crucial steps of the synthesis of 3′-O,4′-C-methylene-α-L-arabino-oxetanonucleosides, a lipase from Thermomyces lanuginosus immobilized on silica (Lipozyme® TL IM) has been used for diastereoselective modification of one hydroxyl out of four hydroxyl groups present in C-4′-hydroxymethyl-β-D-xylofuranosylnucleosides, which makes the whole synthesis green, efficient and environment friendly.

Results and discussion

The synthesis of key precursors C-4′-hydroxymethyl-β-D-xylofuranosylnucleosides (4a–d) of the targeted α-L-arabino-oxetanonucleosides were successfully achieved in three steps, via acetolysis of 4-C-hydroxymethyl-1,2-O-isopropylidine-α-D-xylofuranose¹³ (1) to an anomeric mixture of pentaacetoxyfuranosides 2a–2b,¹⁴ which on Vorbrüggen's nucleoside coupling¹⁵ with thymine, uracil, cytosine and adenine afforded C-4′-acetoxymethyl-2′,3′,5′-tri-O-acetyl-β-D-xylofuranosylnucleosides 3a–d in an overall yields of 70 to 85%. The hydrolysis of tetra-O-acetylated nucleosides 3a–d with aqueous-methanolic potassium carbonate solution afforded the desired precursor nucleosides 4a–d in 75 to 93% yields (Scheme 1).

Scheme 1 Synthesis of C-4′-hydroxymethyl-β-D-xylofuranosyl-nucleosides 4a–d. Reagents and conditions (% yield): (i) Ac₂O, AcOH, conc. H₂SO₄ (100 : 10 : 0.1), 0–30 °C, 12 h (94%); (ii) nucleobases, N,O-bis(trimethylsilyl)acetamide, TMS-triflate, acetonitrile or dichloroethane (for C^Bz & A^Bz), 80 °C, 4–10 h (75–90%); (iii) K₂CO₃ in CH₃OH : H₂O, rt, 4–6 h (75–93%).

Two different lipases,¹⁶ viz. Candida antarctica lipase-B immobilized on polyacrylate (Lewatit), commonly known as Novozyme®-435 and Thermomyces lanuginosus lipase immobilized on silica, commonly known as Lipozyme® TL IM were screened for the selective acetylation of C-4′-hydroxymethyl over the other three hydroxyl groups present in tetrahydroxy nucleosides 4a–d in an incubator shaker in five different organic solvents, i.e. tetrahydrofuran (THF), acetonitrile (MeCN), diisopropyl ether (DIPE), dimethyl sulphoxide (DMSO) and N,N-dimethyl formamide (DMF) using vinyl acetate or acetic anhydride as acetyl donor at 40, 45 and 50 °C and at 200 rpm. Among the two lipases, Lipozyme® TL IM (50% w/w of the substrate) and vinyl acetate in THF or vinyl acetate in MeCN at 45 °C showed exclusive selectivity towards acetylation on C-4′-hydroxymethyl over the other hydroxyl groups in nucleoside 4a to afford C-4′-acetoxymethyl-β-D-xylofuranosylthymine (5a) in 95% and 72% yields, respectively (Scheme 2). The Lipozyme® TL IM and acetic anhydride in THF or in MeCN at 45 °C showed appreciable conversion, but there was no selectivity in the acetylation reaction resulting in the formation of mixture of products. Although, there was appreciable conversion in the acetylayion reaction of tetrahydroxy nucleoside 4a with Novozyme®-435 using vinyl acetate or acetic anhydride either in THF or in MeCN, all these reactions led to the formation of mixture of products, therefore the enzyme was non-selective (see Table in ESI†).

Scheme 2 Lipozyme® TL IM catalyzed acetylation study on tetrahydroxy nucleosides 4a–d.

Thus, in the biocatalytic reaction, a solution of compounds 4a–d and vinyl acetate in THF was incubated with Lipozyme® TL IM (50% w/w) at 45 °C and 200 rpm in an incubator shaker for 12–16 h. On completion, the reaction was quenched by filtering off the enzyme, and solvent was removed under reduced pressure to afford monoacetylated compounds, i.e. C-4′-acetoxymethyl-β-D-xylofuranosylnucleosides (5a–d) in 88–95% yields. The lipase-mediated selective acetylation reaction was successfully scaled up to 6.0 g scale under the same condition for all four tetrahydroxy nucleosides 4a–d. Lipozyme® TL IM was used for five cycles to acetylate nucleosides 4a–d and was found to be equally diastereoselective for all these cycles, except minor enhancement in the reaction time. The acetylation reaction carried out on nucleosides 4a–d in the absence of Lipozyme® TL IM did not yield any product.

The synthesis of targeted oxetanonucleosides, i.e. 3′-O,4′-C-methylene-α-L-arabinofuranosylthymine (7a), 3′-O,4′-C-methylene-α-L-arabinofuranosyluracil (7b), 3′-O,4′-C-methylene-α-L-arabinofuranosylcytosine (7c) and 3′-O,4′-C-methylene-α-L-arabinofuranosyladenine (7d) were successfully achieved in two steps from the monoacetylated nucleosides 5a–d in overall yields from 63 to 79%. Thus, the mesylation of the lone primary hydroxyl group in monoacetylated nucleosides 5a–d with methanesulphonyl chloride in pyridine at low temperature led to the formation of 5′-O-mesylated nucleosides 6a–d in 70–82% yields, which on treatment with 2 M NaOH solution in water–dioxane mixture (1 : 1) resulted in the oxetane ring formation due to the displacement of mesyloxy group by appropriately placed C-3′-hydroxyl group together with hydrolysis of the C-4′-acetoxymethyl group to afford α-L-arabino-oxetanonucleosides 7a–d in 87–96% yields (Scheme 3).

Scheme 3 Synthesis of α-L-arabino-oxetanonucleosides 7a–d. Reagents and conditions (% yield): (i) MsCl, Py, −10 °C, 4–5 h (70–82%); (ii) 2 M NaOH, water : dioxane (1 : 1), 0–25 °C, 1–2 h (+NH₄OH only for 6c & 6d, 30 h) (87–96%).

The diastereoselective preference of Lipozyme® TL IM for the acetylation of C-4′-hydroxylmethyl in tetrahydroxy β-D-xylofuranosylnucleosides 4a–d was further confirmed by this transformation of biocatalytically obtained monoacetylated products 5a–d to the corresponding oxetanonucleosides 7a–d. The selective enzymatic acetylation of the other primary hydroxyl group at C-5′-position would have led to the preferential formation of 2′-O,4′-C-methylene-β-D-xylofuranosyl nucleosides after same set of reaction steps.¹⁴

The Lipozyme® TL IM mediated comparative acyl donor efficiency of vinyl-acetate, -propanoate and -butanoate was investigated under established condition for diastereoselective acylation of C-4′-hydroxymethyl group of tetrahydroxy β-D-xylofuranosylthymine 4a. It was observed that the selectivity of lipase mediated acetylation, propanoylation and butanoylation with vinyl-acetate, -propanoate and -butanoate remains the same, i.e. C-4′-hydroxymethyl group is exclusively acylated over other hydroxyl groups in compound 4a. However the reaction time for the acylation reaction of C-4′-hydroxymethyl group of nucleoside 4a decreases with the increase in acyl chain length, i.e. butanoylation and propanoylation with vinyl-butanoate and -propanoate were 2 times and 1.5 times faster than acetylation reaction with vinyl acetate. This may be due to the increase in the lipophilicity of the acyl donating agent, which is in accordance with the Kazlauskas rule¹⁷ (Table 1).

Table 1 Lipozyme® TL IM catalyzed acylation of 4a with vinyl esters in THF at 45 °C

Entry	Acylating agent	Time (h)	Product	Yield (%)
1	Vinyl acetate	12	5a	95
2	Vinyl propionate	8	8a	90
3	Vinyl butyrate	6	8b	95

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The structures of all synthesized compounds 1, 2a–2b, 3a–d, 4a–d, 5a–d, 6a–d, 7a–d, 8a and 8b were unambiguously established on the basis of their spectral data (¹H-, ¹³C-NMR, ¹H–¹H COSY, ¹H–¹³C HMQC, ¹H–¹³C HMBC spectra, IR spectra and HRMS) analysis. The structure of known compounds 1, 2a–2b, 3a, 3b, 3d and 4a were further confirmed by comparing their physical and spectral data with those reported in the literature.^14,18

Conclusions

Lipozyme® TL IM showed exclusive selectively for the acylation of C-4′-hydroxymethyl group of C-4′-hydroxymethyl-β-D-xylofuranosylnucleosides. The developed biocatalytic methodology has been used for the synthesis of 3′-O,4′-C-methylene-α-L-arabinofuranosylthymine, -uracil, -cytosine and -adenine in high yields. It has been observed that the rate of acylation of C-4′-hydroxymethyl group in tetrahydroxy β-D-xylofuranosylnucleosides increases with the increase in chain length of vinyl esters. The selective biocatalytic acetylation reaction for synthesis of monoacetylated nucleosides was optimized successfully up to 6.0 g scale. The developed methodology can find application for synthesis of different oxetano nucleosides in efficient way.

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 at USIC, University of Delhi. The chemical shift values are on δ scale and the coupling constants (J) are in Hz. HRMS were recorded using Quattro II (Micromass). The optical rotations were measured on Rudolph Autopol II automatic polarimeter using light of 546 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 5% alcoholic H₂SO₄. Silica gel (100–200 mesh) was used for column chromatography. All chemicals and solvents were used as obtained without further purification. The lipase Novozyme®-435 was purchased from Sigma-Aldrich Chemical Company, USA and Lipozyme® TL IM was obtained as a gift from Novozyme A/S Denmark.

Synthesis of C-4′-acetoxymethyl-2′,3′,5′-tri-O-acetyl-β-D-xylofuranosyl-N⁴-benzoylcytosine (3c)

To the stirred solution of penta-O-acetylated sugar derivatives 2a–2b (2.0 g, 5.12 mmol) and N⁴-benzoylcytosine (1.65 g, 7.69 mmol) in anhydrous 1,2-dichloroethane (30 mL) N,O-bis(trimethylsilyl)acetamide (7.60 mL, 30.76 mmol) was added dropwise. The reaction mixture was stirred under reflux for 1 h and then cooled to 0 °C. To the cooled reaction mixture trimethylsilyltrifluoromethane sulfonate (3.49 mL, 19.2 mmol) was added dropwise under stirring and the solution was reflux for 8 h. The reaction was quenched with a cold saturated aqueous solution of NaHCO₃ (80 mL) and extracted with CHCl₃ (3 × 100 mL). The combined organic phase was washed with brine solution (2 × 100 mL) and organic layer was dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using MeOH–CHCl₃ as eluent to afford the titled nucleoside 3c as a white solid (2.25 g) in 80% yield. R_f = 0.4 (10% MeOH in CHCl₃); mp 125–129 °C; [α]²⁹_D = −42.11 (c = 0.1, MeOH); ¹H NMR (CDCl₃, 400 MHz): δ 8.81 (1H, brs), 8.03 (1H, d, J = 7.6 Hz), 7.83 (2H, d, J = 8.0 Hz), 7.56–7.43 (4H, m), 6.26 (1H, d, J = 5.2 Hz), 5.50 (1H, d, J = 5.6 Hz), 5.38 (1H, t, J = 5.2 Hz), 4.53 (1H, d, J = 12.4 Hz), 4.31–4.21 (2H, m), 4.01 (1H, d, J = 11.6 Hz), 2.09 (6H, s), 2.07 (3H, s), 2.03 (3H, s); ¹³C NMR (CDCl₃, 100.6 MHz): δ 170.16, 169.84, 169.33, 162.47, 143.49, 133.35, 132.88, 129.09, 127.60, 97.36, 86.97, 84.51, 79.14, 75.08, 63.61, 62.10, 20.91, 20.78, 20.70 and 20.54; IR (KBr) ν_max: 1752, 1486, 1054 and 707 cm⁻¹; HRMS (ESI): found m/z 546.1743 ([M + H]⁺), calcd for [C₂₅H₂₇N₃O₁₁ + H]⁺ 546.1718.

General procedure for the synthesis of C-4′-hydroxymethyl-β-D-xylofuranosylnucleosides (4a–d)

Solution of acetylated nucleosides 3a–d (1.0 mmol) and potassium carbonate (0.5 equiv.) in methanol–water mixture (2 : 1, 20 mL) was stirred at 0 °C for 4–6 h. After completion of reaction, solvents were removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using MeOH in CHCl₃ as eluent to afford nucleosides 4a–d in 75–93% yields.

C-4′-Hydroxymethyl-β-D-xylofuranosylthymine (4a). It was obtained as a white solid (0.31 g), in 93% yield. Its physical and spectroscopic data (¹H, ¹³C, IR and HRMS) was found to be identical with the data reported in the literature.¹⁴

C-4′-Hydroxymethyl-β-D-xylofuranosyluracil (4b). It was obtained as a white solid (0.25 g) in 90% yield. R_f = 0.4 (30% MeOH in CHCl₃); mp: 120–123 °C; [α]²⁹_D = −47.18 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.29 (1H, brs), 8.08 (1H, d, J = 8.4 Hz), 5.76 (1H, d, J = 6.0 Hz), 5.69 (1H, d, J = 8.4 Hz), 5.58 (1H, d, J = 4.8 Hz), 5.38 (1H, d, J = 4.8 Hz), 4.91 (1H, t, J = 6.4 Hz), 4.70 (1H, t, J = 5.2 Hz), 4.12–4.09 (2H, m), 3.59 (1H, dd, J = 5.2 and 11.4 Hz), 3.41–3.16 (3H, m); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 163.14, 151.11, 141.05, 102.18, 86.35, 85.40, 78.51, 74.73, 62.34, 61.38; IR (KBr) ν_max: 3358, 2947, 1689 and 1011 cm⁻¹; HRMS (ESI): found m/z 275.0874 ([M + H]⁺), calcd for [C₁₀H₁₄N₂O₇ + H]⁺ 275.0874.

C-4′-Hydroxymethyl-β-D-xylofuranosyl-N⁴-benzoylcytosine (4c). It was obtained as a white solid (0.29 g) in 75% yield. R_f = 0.5 (25% MeOH in CHCl₃); mp: 145–148 °C; [α]²⁹_D = −39.45 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.31 (1H, brs), 8.49 (1H, d, J = 7.6 Hz), 8.03 (2H, d, J = 7.6 Hz), 7.64 (1H, t, J = 7.6 Hz), 7.53 (2H, t, J = 7.6 Hz), 7.36 (1H, d, J = 6.8 Hz), 5.90 (1H, d, J = 5.2 Hz), 5.71 (1H, d, J = 4.8 Hz), 5.38 (1H, brs), 4.95 (1H, brs), 4.74 (1H, brs), 4.19–4.15 (2H, m), 3.70 (1H, d, J = 9.2 Hz), 3.55–3.47 (3H, m); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 167.76, 163.05, 154.72, 145.80, 133.24, 132.80, 128.50, 96.46, 88.86, 88.19, 80.60, 75.49, 61.98 and 61.11; IR (thin film) ν_max: 3419, 1647, 1487, 1259 and 786 cm⁻¹; HRMS (ESI): found m/z 378.1295 ([M + H]⁺), calcd for [C₁₇H₁₉N₃O₇ + H]⁺ 378.1296.

C-4′-Hydroxymethyl-β-D-xylofuranosyl-N⁶-benzoyladenine (4d). It was obtained as a light yellow solid (0.32 g) in 78% yield. R_f = 0.5 (25% MeOH in CHCl₃); mp: 115–119 °C; [α]²⁹_D = −39.45 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.23 (1H, brs), 8.77 (2H, s), 8.06 (2H, d, J = 7.6 Hz), 7.66 (1H, t, J = 7.2 Hz), 7.56 (2H, t, J = 7.6 Hz), 5.99 (1H, d, J = 6.8 Hz), 5.79 (1H, brs), 5.50 (1H, brs), 4.97 (1H, brs), 4.82–4.75 (2H, m), 4.30 (1H, d, J = 7.6 Hz), 3.72 (1H, d, J = 11.6 Hz), 3.52–3.42 (3H, m); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 165.80, 152.48, 151.61, 150.36, 143.29, 133.36, 132.50, 128.51, 125.61, 87.07, 86.05, 78.86, 75.02, 62.18 and 61.66; IR (thin film) ν_max: 3336, 1698, 1411, 1056 and 797 cm⁻¹; HRMS (ESI): found m/z 402.1409 ([M + H]⁺), calcd for [C₁₈H₁₉N₅O₆ + H]⁺ 402.1408.

General procedure for the synthesis of C-4′-acetoxymethyl-β-D-xylofuranosyl nucleosides (5a–d)

To a solution of C-4′-hydroxymethyl-β-D-xylofuranosyl nucleosides (4a–d, 1.0 mmol) and vinyl acetate (1.2 mmol) in THF (25 mL), Lipozyme® TL IM (50% w/w of the substrate) was added in one lot. The reaction mixture was stirred at 45 °C in an incubator shaker for 12–16 h and the progress of the reaction was monitored by TLC. On completion, the reaction was quenched by filtering off the Lipozyme® TL IM, the solvent was removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using MeOH in CHCl₃ to afford the mono acetylated nucleosides 5a–d in 88–95% yields.

C-4′-Acetoxymethyl-β-D-xylofuranosylthymine (5a). It was obtained as white solid (0.31 g) in 95% yield. R_f = 0.5 (25% MeOH in CHCl₃); mp: 147–149 °C; [α]³⁰_D = −34.89 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.34 (1H, brs), 7.86 (1H, s), 5.78 (1H, d, J = 7.2 Hz), 5.67 (1H, d, J = 5.2 Hz), 5.60 (1H, d, J = 5.2 Hz), 4.89 (1H, t, J = 5.2 Hz), 4.17–3.98 (4H, m), 3.63 (1H, dd, J = 5.2 and 11.6 Hz), 3.53–3.48 (1H, m), 2.05 (3H, s), 1.78 (3H, s); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 170.20, 163.69, 150.94, 136.53, 109.57, 85.45, 84.12, 77.59, 75.55, 64.38, 60.81, 20.67 and 12.32; IR (KBr) ν_max: 3339, 2949, 1670 and 1050 cm⁻¹; HRMS (ESI): found m/z 331.1136 ([M + H]⁺), calcd for [C₁₃H₁₈N₂O₈ + H]⁺ 331.1136.

C-4′-Acetoxymethyl-β-D-xylofuranosyluracil (5b). It was obtained as a white solid (0.28 g) in 89% yield. R_f = 0.4 (25% MeOH in CHCl₃); mp: 155–157 °C; [α]³⁰_D = −29.54 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.34 (1H, brs), 7.99 (1H, d, J = 8.4 Hz), 5.79 (1H, d, J = 6.0 Hz), 5.73–5.69 (2H, m), 5.63 (1H, d, J = 5.2 Hz), 4.88 (1H, t, J = 5.2 Hz), 4.15–4.07 (3H, m), 4.01 (1H, t, J = 5.6 Hz), 3.65–3.61 (1H, m), 3.51–3.47 (1H, m), 2.05 (3H, s); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 170.24, 163.11, 150.90, 141.06, 102.21, 85.95, 84.71, 78.10, 75.49, 64.22, 60.65 and 20.72; IR (thin film) ν_max: 3430, 1654 and 1052 cm⁻¹; HRMS (ESI): found m/z 317.0979 ([M + H]⁺), calcd for [C₁₂H₁₆N₂O₈ + H]⁺ 317.0979.

C-4′-Acetoxymethyl-β-D-xylofuranosyl-N⁴-benzoylcytosine (5c). It was obtained as a white solid (0.38 g) in 90% yield. R_f = 0.5 (30% MeOH in CHCl₃); mp: 156–159 °C; [α]³²_D = −37.23 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.28 (1H, brs), 8.36 (1H, d, J = 7.6 Hz), 8.00 (2H, d, J = 7.6 Hz), 7.61 (1H, t, J = 7.6 Hz), 7.50 (2H, t, J = 7.6 Hz), 7.34 (1H, s), 5.88 (1H, d, J = 4.4 Hz), 5.85 (1H, d, J = 4.4 Hz), 5.57 (1H, d, J = 5.6 Hz), 4.87 (1H, t, J = 5.6 Hz), 4.21–4.11 (3H, m), 4.00 (1H, t, J = 4.8 Hz), 3.73–3.69 (1H, m), 3.61 (1H, dd, J = 5.2 and 11.4 Hz), 2.05 (3H, s); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 170.21, 167.36, 163.03, 154.97, 145.94, 133.19, 132.80, 128.49, 96.41, 89.52, 86.81, 80.14, 75.86, 63.65, 60.21 and 20.74; IR (thin film) ν_max: 3428, 2836, 1657 and 1027 cm⁻¹; HRMS (ESI): found m/z 420.1401 ([M + H]⁺), calcd for [C₁₉H₂₁N₃O₈ + H]⁺ 420.1401.

C-4′-Acetoxymethyl-β-D-xylofuranosyl-N⁶-benzoyladenine (5d). It was obtained as a light yellow solid (0.39 g) in 88% yield. R_f = 0.5 (20% MeOH in CHCl₃); mp: 123–126 °C; [α]³⁰_D = −27.76 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.23 (1H, brs), 8.77 (1H, s), 8.73 (1H, s), 8.05 (2H, d, J = 7.6 Hz), 7.64 (1H, t, J = 7.6 Hz), 7.55 (2H, t, J = 7.6 Hz), 6.00 (1H, d, J = 6.0 Hz), 5.91 (1H, d, J = 5.6 Hz), 5.74 (1H, d, J = 5.6 Hz), 4.91 (1H, brs), 4.81–4.78 (1H, m), 4.19–4.14 (3H, m), 3.77–3.74 (1H, m), 3.62–3.58 (1H, m), 2.08 (3H, s); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 170.66, 165.57, 152.51, 151.73, 150.48, 143.06, 133.13, 132.39, 128.52, 125.72, 86.30, 85.21, 78.22, 75.47, 64.27, 61.17 and 20.76; IR (KBr) ν_max: 3366, 1741, 1704, 1249 and 1043 cm⁻¹; HRMS (ESI): found m/z 444.1514 ([M + H]⁺), calcd for [C₂₀H₂₁N₅O₇ + H]⁺ 444.1514.

General protocol for the synthesis of C-4′-acetoxymethyl-β-D-xylofuranosyl nucleosides (5a–d) for 6.0 g scale

To a solution of C-4′-hydroxymethyl-β-D-xylofuranosyl nucleosides 4a–d (6.0 g, 20.8/21.9/15.9/15.0 mmol, respectively) and vinyl acetate (27.0/28.4/20.6/19.4 mmol, respectively) in THF (200 mL), Lipozyme® TL IM (50% w/w of the substrate) was added in one lot. The reaction mixture was stirred at 45 °C in an incubator shaker for 17–22 h. On completion, the reaction was quenched by filtering off the enzyme, the solvent was removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using MeOH in CHCl₃ to afford the mono acetylated nucleosides 5a–d (93/86/89/85% yields, respectively). We found that the yield of the reactions with 1.0 mmol of the substrate is slightly higher than that of the 6.0 g scale.

General procedure for the synthesis of C-4′-acetoxymethyl-5′-methanesulfonyloxy-β-D-xylofuranosylnucleosides (6a–d)

A solution of nucleosides 5a–d (1.0 mmol) and methanesulphonyl chloride (1.5 mmol) in anhydrous pyridine (10 mL) was stirred at −10 °C under nitrogen atmosphere for 4–5 h. On completion, the reaction mixture was poured over 10% hydrochloric acid solution (ice-cold) and the product was extracted with EtOAc (3 × 50 mL). The combined organic phase was washed with saturated NaHCO₃ (2 × 100 mL) solution and dried over anhydrous Na₂SO₄. The solvent was removed under reduced pressure and residue thus obtained was purified by silica gel column chromatography using MeOH in CHCl₃ as gradient solvent to afford the mesylated nucleosides 6a–d in 70–82% yields.

C-4′-Acetoxymethyl-5′-methanesulfonyloxy-β-D-xylofuranosylthymine (6a). It was obtained as a white solid (0.33 g) in 82% yield. R_f = 0.6 (15% MeOH in CHCl₃); mp: 166–169 °C; [α]³⁰_D = −34.12 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.40 (1H, brs), 7.62 (1H, s), 6.02 (1H, d, J = 4.8 Hz), 5.83 (2H, d, J = 5.6 Hz), 4.44 (1H, d, J = 10.8 Hz), 4.27 (1H, d, J = 10.4 Hz), 4.20–4.11 (4H, m), 3.23 (3H, s), 2.08 (3H, s), 1.79 (3H, s); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 170.06, 163.58, 150.86, 135.87, 110.23, 85.34, 81.45, 76.54, 75.53, 69.38, 63.80, 36.54, 20.60 and 12.09; IR (KBr) ν_max: 3360, 2947, 1701, 1366 and 1051 cm⁻¹; HRMS (ESI): found m/z 409.0911 ([M + H]⁺), calcd for [C₁₄H₂₀N₂O₁₀S + H]⁺ 409.0911.

C-4′-Acetoxymethyl-5′-methanesulfonyloxy-β-D-xylofuranosyluracil (6b). It was obtained as a white solid (0.31 g) in 78% yield. R_f = 0.5 (15% MeOH in CHCl₃); mp: 145–148 °C; [α]³⁰_D = −34.65 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.42 (1H, brs), 7.76 (1H, d, J = 7.6 Hz), 6.05 (1H, d, J = 4.4 Hz), 5.93 (1H, d, J = 4.4 Hz), 5.84 (1H, d, J = 6.0 Hz), 5.69 (1H, d, J = 7.6 Hz), 4.41 (1H, d, J = 10.4 Hz), 4.31 (1H, d, J = 10.4 Hz), 4.21–4.09 (4H, m), 3.21 (3H, s), 2.07 (3H, s); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 170.04, 162.93, 150.76, 140.68, 102.45, 86.70, 82.35, 77.44, 75.69, 68.82, 63.35, 36.59 and 20.61; IR (KBr) ν_max: 3428, 1689, 1387 and 1017 cm⁻¹; HRMS (ESI): found m/z 395.0755 ([M + H]⁺), calcd for [C₁₃H₁₈N₂O₁₀S + H]⁺ 395.0755.

C-4′-Acetoxymethyl-5′-methanesulfonyloxy-β-D-xylofuranosyl-N⁴-benzoylcytosine (6c). It was obtained as a white solid (0.38 g) in 76% yield. R_f = 0.6 (20% MeOH in CHCl₃); mp: 195–198 °C; [α]³⁰_D = −29.54 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.32 (1H, brs), 8.22 (1H, d, J = 7.6 Hz), 8.01 (2H, d, J = 7.6 Hz), 7.63 (1H, t, J = 8.0 Hz), 7.52 (2H, t, J = 7.6 Hz), 7.39 (1H, s), 6.09 (1H, d, J = 4.8 Hz), 6.00 (1H, d, J = 4.8 Hz), 5.92 (1H, d, J = 4.8 Hz), 4.53 (1H, d, J = 10.8 Hz), 4.43 (1H, d, J = 10.8 Hz), 4.28–4.19 (3H, m), 4.12 (1H, t, J = 4.0 Hz), 3.25 (3H, s), 2.09 (3H, s); ¹³C NMR (CDCl₃, 100.6 MHz): δ 170.92, 166.71, 163.11, 156.42, 145.37, 133.35, 132.74, 128.97, 128.04, 97.70, 93.54, 86.77, 81.84, 76.84, 67.50, 63.20, 37.63 and 20.94; IR (KBr) ν_max: 3447, 1646, 1112 and 618 cm⁻¹; HRMS (ESI): found m/z 498.1177 ([M + H]⁺), calcd for [C₂₀H₂₃N₃O₁₀S + H]⁺ 498.1177.

C-4′-Acetoxymethyl-5′-methanesulfonyloxy-β-D-xylofuranosyl-N⁶-benzoyladenine (6d). It was obtained as a light yellow solid (0.37 g) in 70% yield. R_f = 0.6 (20% MeOH in CHCl₃); mp: 137–139 °C; [α]³⁰_D = −32.14 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.27 (1H, brs), 8.79 (1H, s), 8.72 (1H, s), 8.05 (2H, d, J = 7.6 Hz), 7.66 (1H, t, J = 8.0 Hz), 7.56 (2H, t, J = 7.6 Hz), 6.17 (1H, d, J = 5.2 Hz), 6.09–6.05 (2H, m), 4.89–4.85 (1H, m), 4.64 (1H, d, J = 10.8 Hz), 4.40 (1H, d, J = 10.8 Hz), 4.32–4.26 (2H, m), 4.21 (1H, d, J = 12.0 Hz), 3.19 (3H, s), 2.12 (3H, s); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 170.10, 165.64, 152.43, 151.91, 150.62, 149.62, 142.97, 133.28, 132.53, 128.51, 125.68, 85.97, 82.59, 77.36, 75.85, 69.48, 63.46, 36.58 and 20.67; IR (thin film) ν_max: 3423, 1664, 1383 and 1008 cm⁻¹; HRMS (ESI): found m/z 522.1289 ([M + H]⁺), calcd for [C₂₁H₂₃N₅O₉S + H]⁺ 522.1289.

General procedure for the synthesis of 3′-O,4′-C-methylene-α-L-arabinofuranosyl nucleosides (7a & 7b)

A solution of mesylated nucleosides 6a and 6b (1.0 mmol) and 2 M NaOH in dioxane/water mixture (1 : 1, 15 mL) was stirred at 0–25 °C for 1–2 h. On completion of the reaction (analytical TLC), the solvents were removed under reduced pressure. The residue thus obtained was purified by silica gel column chromatography using MeOH in CHCl₃ as a gradient solvent to afford bicyclic nucleosides 7a and 7b in 96 and 92% yields, respectively.

3′-O,4′-C-Methylene-α-L-arabinofuranosylthymine (7a). It was obtained as a white solid (0.26 g) in 96% yield. R_f = 0.5 (15% MeOH in CHCl₃); mp: 156–160 °C; [α]³⁰_D = −24.32 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.41 (1H, brs), 7.81 (1H, s), 6.15 (1H, d, J = 3.6 Hz), 5.80 (1H, d, J = 4.4 Hz), 5.22 (1H, t, J = 6.0 Hz), 4.90 (1H, s), 4.61 (1H, d, J = 7.6 Hz), 4.53 (1H, t, J = 4.4 Hz), 4.20 (1H, d, J = 7.2 Hz), 3.54 (2H, d, J = 5.6 Hz), 1.82 (3H, s); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 163.71, 150.92, 136.52, 110.21, 93.74, 90.98, 89.01, 79.03, 75.47, 61.01 and 12.26; IR (thin film) ν_max: 3436, 1654, and 1015 cm⁻¹; HRMS (ESI): found m/z 271.0925 ([M + H]⁺), calcd for [C₁₁H₁₄N₂O₆ + H]⁺ 271.0925.

3′-O,4′-C-Methylene-α-L-arabinofuranosyluracil (7b). It was obtained as a white solid (0.24 g) in 92% yield. R_f = 0.4 (15% MeOH in CHCl₃); mp: 125–129 °C; [α]³⁰_D = −23.24 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.38 (1H, brs), 7.96 (1H, d, J = 8.4 Hz), 6.16 (1H, d, J = 3.2 Hz), 5.82 (1H, d, J = 2.8 Hz), 5.72 (1H, d, J = 8.0 Hz), 5.22 (1H, t, J = 4.4 Hz), 4.90 (1H, s), 4.62 (1H, d, J = 7.6 Hz), 4.50 (1H, s), 4.13 (1H, d, J = 7.6 Hz), 3.52 (2H, d, J = 4.4 Hz); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 163.07, 150.90, 141.20, 102.62, 94.38, 90.97, 89.85, 78.92, 75.48 and 60.90; IR (KBr) ν_max: 3341, 1688 and 1030 cm⁻¹; HRMS (ESI): found m/z 257.0768 ([M + H]⁺), calcd for [C₁₀H₁₂N₂O₆ + H]⁺ 257.0768.

General procedure for the synthesis of 3′-O,4′-C-methylene-α-L-arabinofuranosyl nucleosides (7c & 7d)

A solution of mesylated nucleosides 6c and 6d (1.0 mmol) in dioxane–water mixture (1 : 1, 12 mL) were added 2 M NaOH (6 mL) and NH₄OH (10 mL) respectively at 0 °C and the reaction was stirred at 25 °C for 30 h. On completion of the reaction (analytical TLC), the solvents were removed under reduced pressure. The residue thus obtained was purified by silica gel column chromatography using MeOH in CHCl₃ as a gradient solvent to afford bicyclic nucleosides 7c and 7d in 87 and 90% yields, respectively.

3′-O,4′-C-Methylene-α-L-arabinofuranosylcytosine (7c). It was obtained as a white solid (0.23 g) in 87% yield. R_f = 0.4 (30% MeOH in CHCl₃); mp: 152–154 °C; [α]³⁰_D = −39.73 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 7.91 (1H, d, J = 6.8 Hz), 7.30 (1H, brs), 7.20 (1H, brs), 6.21 (1H, d, J = 2.8 Hz), 5.79 (1H, d, J = 7.6 Hz), 5.73 (1H, d, J = 4.4 Hz), 5.21 (1H, t, J = 5.6 Hz), 4.87 (1H, s), 4.62 (1H, d, J = 6.8 Hz), 4.40 (1H, s), 4.12 (1H, d, J = 6.8 Hz), 3.50 (2H, d, J = 5.2 Hz); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 165.58, 155.46, 141.62, 95.47, 94.78, 91.21, 89.81, 79.29, 75.44 and 60.93; IR (KBr) ν_max: 3416, 1648, 1188 and 1050 cm⁻¹; HRMS (ESI): found m/z 256.0927 ([M + H]⁺), calcd for [C₁₀H₁₃N₃O₅ + H]⁺ 256.0928.

3′-O,4′-C-Methylene-α-L-arabinofuranosyladenine (7d). It was obtained as a white solid (0.25 g) in 90% yield. R_f = 0.5 (30% MeOH in CHCl₃); mp: 238–241 °C; [α]³⁰_D = −26.37 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 8.39 (1H, s), 8.18 (1H, s), 7.32 (2H, brs), 6.32 (1H, s), 5.91 (1H, d, J = 2.0 Hz), 5.24 (1H, t, J = 5.2 Hz), 5.02 (2H, s), 4.68 (1H, d, J = 6.8 Hz), 4.00 (1H, d, J = 6.8 Hz), 3.56–3.51 (2H, m); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 156.06, 152.86, 149.82, 138.92, 118.62, 92.24, 90.84, 90.39, 77.88, 76.24 and 60.86; IR (thin film) ν_max: 3417, 1646 and 1015 cm⁻¹; HRMS (ESI): found m/z 280.1040 ([M + H]⁺), calcd for [C₁₁H₁₃N₅O₄ + H]⁺ 280.1042.

General procedure for the synthesis of C-4′-acyloxymethyl-β-D-xylofuranosyl nucleosides (8a & 8b)

To solution of C-4′-hydroxymethyl-β-D-xylofuranosylthymine (4a, 1.0 mmol) and vinyl-propionate/vinyl-butanoate (1.2 mmol) in THF (25 mL), Lipozyme® TL IM (50% w/w of the substrate) was added. The reaction mixture was stirred at 45 °C in an incubator shaker for 6–8 h and the progress of the reaction was monitored by TLC. On completion, the reaction was quenched by filtering off the Lipozyme® TL IM, the solvent was removed under reduced pressure and the residue thus obtained was purified by silica gel column chromatography using MeOH in CHCl₃ as a gradient solvent to afford the monoacylated nucleosides 8a and 8b in 90 and 95% yields, respectively.

C-4′-Propanoyloxymethyl-β-D-xylofuranosylthymine (8a). It was obtained as a white solid (0.31 g) in 90% yield. R_f = 0.5 (25% MeOH in CHCl₃); mp: 165–168 °C; [α]³⁰_D = −29.50 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.31 (1H, brs), 7.83 (1H, s), 5.77 (1H, d, J = 6.8 Hz), 5.65 (1H, d, J = 5.2 Hz), 5.58 (1H, d, J = 4.8 Hz), 4.86 (1H, t, J = 4.4 Hz), 4.17–3.96 (4H, m), 3.64–3.60 (1H, m), 3.49 (1H, dd, J = 5.2 and 11.6 Hz), 2.35 (2H, q_t, J = 14.8 Hz), 1.77 (3H, s), 1.04 (3H, t, J = 6.8 Hz); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 173.44, 163.74, 150.99, 136.66, 109.62, 85.36, 84.12, 77.50, 75.50, 64.33, 60.92, 26.87, 12.35 and 9.05; IR (KBr) ν_max: 3456, 1743 and 1071 cm⁻¹; HRMS (ESI): found m/z 345.1292 ([M + H]⁺), calcd for [C₁₄H₂₀N₂O₈ + H]⁺ 345.1292.

C-4′-Butanoyloxymethyl-β-D-xylofuranosylthymine (8b). It was obtained as a white solid (0.34 g) in 95% yield. R_f = 0.4 (20% MeOH in CHCl₃); mp: 173–175 °C; [α]³⁰_D = −24.67 (c = 0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz): δ 11.30 (1H, brs), 7.83 (1H, s), 5.76 (1H, d, J = 6.8 Hz), 5.65 (1H, d, J = 4.4 Hz), 5.57 (1H, d, J = 5.2 Hz), 4.86 (1H, t, J = 5.6 Hz), 4.17–3.95 (4H, m), 3.64–3.60 (1H, m), 3.50–3.46 (1H, m), 2.31 (2H, t, J = 7.6 Hz), 1.77 (3H, s), 1.60–1.51 (2H, m), 0.88 (3H, t, J = 7.6 Hz); ¹³C NMR (DMSO-d₆, 100.6 MHz): δ 172.59, 163.72, 150.96, 136.65, 109.61, 85.38, 84.10, 77.52, 75.48, 64.21, 60.92, 35.40, 17.96, 13.48 and 12.35; IR (KBr) ν_max: 3456, 1733 and 1007 cm⁻¹; HRMS (ESI): found m/z 359.1430 ([M + H]⁺), calcd for [C₁₅H₂₂N₂O₈ + H]⁺ 359.1449.

Acknowledgements

We are grateful to the University of Delhi and DU-DST Purse Grant for the financial support. R. K. and M. K. thank UGC and CSIR, New Delhi for providing JRF and SRF research fellowship. We thank USIC-CIF, University of Delhi for providing instrumentation facility.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹H and ¹³C NMR spectra of new compounds. See DOI: 10.1039/c6ra17218k

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