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Synthesis 2024; 56(06): 1035-1041
DOI: 10.1055/s-0041-1738440
paper
Emerging Trends in Glycoscience

Chemoenzymatic Synthesis of arabino-Configured Bicyclic Nucleosides

Harbansh Singla
a   Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
,
Jyotirmoy Maity
b   Department of Chemistry, St. Stephen’s College, University of Delhi, Delhi 110 007, India
,
Sandeep Kumar
a   Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
,
Kavita Kavita
a   Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
,
Riya Chaudhary
a   Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
,
Ashok K. Prasad  ∗
a   Bioorganic Laboratory, Department of Chemistry, University of Delhi, Delhi 110 007, India
› Author Affiliations

Harbansh Singla and Kavita thank CSIR, New Delhi for the award of a Senior research fellowship. We are grateful to Institute of Eminence, University of Delhi for providing financial support to strengthen research and development.
› Further Information
 
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Abstract

A convergent route for the synthesis of a new class of bicyclic nucleosides has been developed. The synthetic route to the corresponding arabino-configured uracil and thymine bicyclic nucleosides proceeds in 24 and 27% overall yields, respectively, starting from 1,2,5,6-di-O-isopropylidene-α-d-glucofuranose. This synthetic protocol includes some crucial steps such as Vorbrüggen base coupling and chemo-enzymatic regioselective acetylation of the primary hydroxyl group by using Lipozyme® TL IM where it was found that Lipozyme® TL IM could be recovered and reused for selective acetylation without losing its selectivity.


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Key words

chemo-enzymatic pathway - regioselective monoacetylation - Lipozyme® TL IM - bicyclic nucleosides

Nucleosides are one of the most widely studied compounds. Nucleoside analogues have been a point of interest for chemists and biochemists due to their role in nucleic acid biosynthesis and various other biologically significant processes such as viral replication and division of cells.[1] In the past few decades, a variety of artificial or modified nucleosides have been synthesized by chemists to incorporate them in the antisense oligonucleotides[2] [3] [4] or to screen their biological activities such as antiviral,[5,6] anti-HIV,[7,8] anticancer,[9] [10] antimetabolites,[11] [12] antisense properties,[13] and many more.[14] [15] [16] Conformationally restricted nucleoside analogues with reduced conformational flexibility constituted a major class of these modified nucleosides.[17] The restriction in the conformation of nucleosides was introduced at sugar moiety, either in the form of locked nucleosides[18] [19] [20] or spironucleosides[21] [22] [23] and nucleosides connecting sugar and base moieties.[24] [25] The first total synthesis of naturally occurring herbicidal spironucleosides by Mio and co-workers[26] [27] in 1991 drew the attention of organic chemists towards the synthesis of modified spironucleosides. Wengel et al.[28] [29] synthesized 2′-O,5′-C-methylene linked nucleoside A and bicyclic nucleotide monomer B for their use in antisense therapy as DNA mimics. Paquette et al.[30] synthesized biologically active conformationally restricted nucleosides by introducing a carbocyclic ring at C-4′ position of furanose and thiofuranose ring, respectively.[31] [32] [33] Similarly Dang et al.[34] and Jonckers et al.[35] synthesized C-4′ and C-2′ spironucleosides that showed HCV NS5B polymerase inhibitory activity. In the past few years, our research group has aimed and successfully executed the synthesis of various sugar modified nucleosides following chemical and biochemical pathways. 6′-Methyl-2′-O,4′-C-methylene-α-l-ribofuranosylpyrimidines,[36] α-l-ribofuranosylnucleosides,[37] C-4′-spiro-oxetano-α-l-ribonucleosides[38] were synthesized following chemical pathway, whereas 2′-O,4′-C-methyleneribonucleosides C,[39] C-4′-spiro-oxetanoribonucleosides,[40] homolyxofuranosylpyrimidines D [41] were synthesized following chemoenzymatic pathway. Herein, a facile and efficient methodology has been described for the synthesis of LNA nucleosides 8a,b following a chemoenzymatic pathway (Figure [1]).

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Figure 1 Some representative literature based spiro-nucleosides
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Scheme 1 Retrosynthetic route for the synthesis of titled bicyclic nucleosides
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Scheme 2 Reagents and conditions: (a) B, BSA, TMSOTf, CH3CN, 80 °C; 88% (R = H), 90% (R = CH3) (where B = uracil and thymine); (b) K2CO3, MeOH:H2O (9:1), 25 °C, 1 h; 92% (R = H), 92% (R = CH3); (c) Lipozyme® TL IM, vinyl acetate, MeCN, 45 °C, 1 h; 99% (R = H), 97% (R = CH3); (d) MsCl, Py, 25 °C, 4 h, 90% (R = H), 94% (R = CH3); (e) aq 2 M NaOH, 1,4-dioxane:H2O (1:1), 25 °C, 24 h, 72% (R = H), 74% (R = CH3); (f) Pd(OH)2/C, HCO2H, THF:MeOH (1:1), reflux, 30 min, 82% (R = H), 85% (R = CH3).

First, we chalked out the retrosynthetic pathway for synthesis of targeted bicyclic nucleosides where it was proposed that the targeted bicyclic pyrimidine nucleosides could be synthesized by starting from a commercially available, inexpensive furanoside, that is, 1,2,5,6-di-O-isopropylidine-α-d-glucofuranose. A regioselective enzymatic acetylation reaction step was also envisioned, which would make the process a chemoenzymatic methodology (Scheme [1]).

So, the synthesis of bicyclic nucleosides 8a,b was commenced from 1,2,5,6-di-O-isopropylidine-α-d-glucofuranose (1), which was further converted to (3R,4R,5R)-4-(benzyloxy)-5-((S)-1,2diacetoxyethyl)tetrahydrofuran-2,3-diyl diacetate (2) by following the literature procedure.[42] The coupling reaction of compound 2 with nucleobases, uracil, and thymine under Vorbrüggen glycosylation[43] conditions produced compound 3a and 3b in 88% and 90% yield, respectively, which on subsequent deacetylation under basic condition resulted in the formation of (S)-2-((2R,3S,4R,5R)-3-(benzyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)-2-hydroxyethyl acetate (4a) and (S)-2-((2R,3S,4R,5R)-3-(benzyloxy)-4-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidine-1(2H)-yl)tetrahydrofuran-2-yl)-2-hydroxyethyl acetate (4b) in 92% yield each (Scheme [2]).

Lipases from different sources such as Novozyme® 435 and Lipozyme® TL IM were screened for selective acetylation of the primary hydroxyl group of pyrimidine nucleosides 4a,b in five organic solvents, that is, acetone, acetonitrile, tetrahydrofuran (THF), 2-methyltetrahydrofuran (2-Me-THF) and toluene in an incubator shaker by using vinyl acetate as acetyl donor at different temperatures ranging from 25 °C to 45 °C. It was observed that the selective acetylation of the primary hydroxyl group of compounds 4a,b took place by using Lipozyme® TL IM at 45 °C at 200 rpm in acetonitrile in 99% and 97% yield, respectively (Figure [2]).

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Figure 2 Optimization chart for the selective acetylation of nucleosides 4a,b

Further, monoacetylated nucleosides, that is, (S)-2-((2R,3S,4R,5R)-3-(benzyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)-2-hydroxyethyl acetate (5a) and (S)-2-((2R,3S,4R,5R)-3-(benzyloxy)-4-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)-2-hydroxyethyl acetate (5b) were mesylated using MsCl in pyridine at room temperature to get compounds 6a,b, which on concomitant intramolecular cyclization under alkaline conditions resulted in the formation of benzylated bicyclic nucleosides 7a,b in 72% and 74% yield, respectively. Finally, debenzylation of nucleosides 7a,b was achieved using 20% Pd(OH)2/C/HCO2H to yield the titled bicyclic nucleosides, that is, 1-((1S,3R,4S,7R)-7-hydroxy-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)pyrimidine-2,4(1H,3H)-dione (8a) and 1-((1S,3R,4S,7R)-7-hydroxy-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8b) in 82% and 85% yield, respectively, by following the literature procedure.[41] (Scheme [2]). Herein, yields of the chemical reactions of this series and corresponding C-3′ epimers were compared. Acetolysis and nucleobase coupling reactions had comparable yields for both series, whereas the deacetylation reaction of 3a,b produced the corresponding products 4a,b in lesser yields. Lipozyme® TL IM was found to be an appropriate biocatalyst for the acetylation of nucleosides 4a,b, whereas its C-3′ epimer was a better substrate for Novozyme® 435. Mesylation and benzylated nucleosides formation reaction had comparable yields but the hydrogenation reaction to afford the unprotected moieties 8a,b proceeded in lower yield.

In our earlier studies, the nucleoside monomer of locked-nucleic acid (nucleoside D, Figure [1]) was found to be locked into C3′-exo sugar puckering with pseudorotational phase angle (P = 193.74 and 201.13°), indicating the S-type conformation of the nucleoside.[41] The pseudorotational phase angle (P) calculated[44] for the 1-((1S,3R,4S,7R)-7-hydroxy-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8b) was found to be 196.11° implying C3′-exo sugar puckering and S-type conformation of the nucleoside. The calculated values of dihedral angles (ν0 = –2.45, ν1 = 38.64, ν2 = –55.59, ν3 = 55.59, ν4 = –34.91) confirmed the conformation of the nucleoside 8b to be C3′-exo, that is, S-type conformation. Puckering amplitude (νmax = 57.87), backbone angle (γ = 37.74), as well as the torsion describing the anomeric bond (χ = –177.88), support the inference. The X-ray crystal structure of compound 8b is shown in Figure [3].[45]

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Figure 3 (a) ORTEP diagram of 1-((1S,3R,4S,7R)-7-hydroxy-6-(hydroxy­methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8b) drawn in 30% thermal probability ellipsoids showing the atom numbering scheme; (b) Exhibition of S-type puckering of furanose ring in nucleoside 8b.

The structures of all synthesized compounds 2, 3a,b, 4a,b, 5a,b, 6a,b, 7a,b, 8a,b were confirmed on the basis of their spectral data analysis (IR, 1H NMR, 13C NMR, DEPT-135 NMR) and HRMS data analysis.

Herein, an efficient, ecological, and bio-catalytic pathway has been reported for the synthesis of bicyclic nucleosides 1-((1S,3R,4S,7R)-7-hydroxy-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)pyrimidine-2,4(1H,3H)-dione (8a) and 1-((1S,3R,4S,7R)-7-hydroxy-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8b) starting from 1,2,5,6-di-O-isopropylidine-α-d-glucofuranose in 24% and 27% overall yield, respectively. Lipozyme® TL IM was used for selective acetylation of primary hydroxyl group of (S)-2-((2R,3S,4R,5R)-3-(benzyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)-2-hydroxyethyl acetate (4a) and (S)-2-((2R,3S,4R,5R)-3-(benzyloxy)-4-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)-2-hydroxyethyl acetate (4b) in the cardinal step of this methodology. Structure of one of the titled bicyclic nucleosides 8b was further confirmed by X-ray crystallography, and it was found that the synthesized nucleoside has been locked into S-type sugar puckering. Hence, this work shows the wide spectrum of restricted arabinofuranose conformations attainable for bicyclic nucleoside analogues.

All the commercially available reagents were used without further purification, however, solvents were distilled before use. Reactions were conducted under an atmosphere of N2 when anhydrous solvents were used. Melting points were determined on Büchi M-560 instrument and are uncorrected. The IR spectra were recorded on a PerkinElmer model 2000 FT-IR spectrophotometer by making KBr disc for solid samples and thin film for oils. The 1H and 13C spectra were recorded on a Jeol alpha-400 spectrophotometer 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 optical rotations were measured with Rudolph autopol II automatic polarimeter. The HRMS recording was carried out in ESI mode using a Q-TOF mass spectrometer. Analytical TLCs were performed on pre-coated Merck silica gel 60F254 plates, the spots were detected either using UV light or with 4% alcoholic sulfuric acid. Silica gel (100–200 mesh) was used for column chromatography. Lipases were obtained from Sigma-Aldrich and dried over P2O5 for overnight prior to use.


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Synthesis of (3R,4R,5R)-4-(Benzyloxy)-5-((S)-1,2diacetoxyethyl)tetrahydrofuran-2,3-diyl Diacetate (2)

Ac2O (9.5 mL, 99.8 mmol) and concd H2SO4 (0.053 mL, 0.998 mmol) were added to a stirred solution of 3-O-benzyl-1,2,5,6-di-O-isopropylidine-α-d-allofuranose (1; 3.5 g, 9.98 mmol) in AcOH (57.12 mL, 998 mmol) at 0 °C and the resulting mixture was allowed to stir at 25 °C for 6 h. Upon completion of the reaction (monitored by TLC), the reaction mixture was quenched by the addition of ice-cold H2O (100 mL) and extracted with EtOAc (2 × 100 mL). The combined EtOAc layers were washed with aq NaHCO3 (2 × 100 mL). The EtOAc layer was evaporated under reduced pressure and the crude residue thus obtained was purified by silica gel chromatography using EtOAc in PE (petroleum ether) as gradient solvent to afford an anomeric mixture of allofuranoside 4 (α:β = 1:10 on integration of anomeric protons in 1H NMR spectrum) as a colorless viscous oil; yield: 4.02 g (92%); Rf = 0.45 (30% EtOAc in PE); [α]D 28 +45.19 (c 0.1, MeOH).

IR (KBr): 1755, 1470, 1321, 1200, 1060, 890, 767, 720, 640 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 7.28–7.37 (m, 5 H), 6.13 (s, 1 H), 5.30 (d, J = 4.4 Hz, 1 H), 5.24 (td, J = 6.5, 3.2 Hz, 1 H), 4.58 (d, J = 10.7 Hz, 1 H), 4.43 (d, J = 10.9 Hz, 1 H), 4.36 (dd, J = 12.1, 3.3 Hz, 1 H), 4.31 (dd, J = 7.6, 4.5 Hz, 1 H), 4.19 (dd, J = 7.7, 6.0 Hz, 1 H), 4.07 (dd, J = 12.1, 6.6 Hz, 1 H), 2.11 (s, 3 H), 2.09 (s, 3 H), 2.03 (s, 3 H), 2.03 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 170.8, 170.2, 169.9, 169.0, 137.0, 128.7, 128.6, 128.4, 128.3, 98.5, 80.0, 78.5, 73.6, 73.6, 71.6, 62.8, 21.2, 21.1, 20.9, 20.9.

HRMS (ESI): m/z calcd for C21H26O10 [M + NH4]+: 456.1864; found: 456.1839.


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Synthesis of Nucleosides 3a,b; General Procedure

To the anomeric mixture 2 (1.0 g, 2.28 mmol) and uracil/thymine (3.42 mmol) in anhyd MeCN was added N,O-bis(trimethylsilyl)acetamide (BSA; 2.23 mL, 9.12 mmol) dropwise. The reaction mixture was refluxed for 1 h and then it was cooled to 0 °C followed by dropwise addition of trimethylsilyl trifluoromethanesulfonate (0.70 mL, 3.88 mmol). The resulting solution was again refluxed for 6 h and the progress of the reaction was monitored on TLC. On completion of the reaction, the reaction mixture was cooled to rt and extracted with EtOAc. The combined EtOAc layers were washed with aq NaHCO3 (3 × 100 mL). The excess of solvent was removed, and the crude product thus obtained was purified by silica gel column chromatography using MeOH in CHCl3 as eluent to afford pure nucleosides in 88% and 90% yield, respectively.


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(S)-1-((2R,3R,4R,5R)-4-Acetoxy-3-(benzyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)ethane-1,2-diyl Diacetate (3a)

Colorless viscous oil; yield: 0.98 g (88%); Rf = 0.45 (5% MeOH in CHCl3­); [α]D 28 +32.65 (c 0.1, MeOH).

IR (KBr): 3110, 1760, 1680, 1490, 1365, 1240, 1120, 770, 820, 610, 520 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 8.99 (s, 1 H), 7.30–7.37 (m, 5 H), 7.19 (d, J = 8.2 Hz, 1 H), 5.76 (dd, J = 8.1, 2.2 Hz, 1 H), 5.70 (d, J = 4.0 Hz, 1 H), 5.40 (dd, J = 5.8, 3.8 Hz, 1 H), 5.34 (td, J = 6.6, 3.4 Hz, 1 H), 4.51–4.58 (m, 2 H), 4.38–4.42 (m, 2 H), 4.15 (t, J = 6.0 Hz, 1 H), 4.09 (dd, J = 12.2, 6.6 Hz, 1 H), 2.11 (s, 3 H), 2.05 (s, 3 H), 2.03 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 170.86, 170.31, 170.23, 163.04, 149.9, 141.2, 137.0, 128.8, 128.5, 128.3, 103.3, 91.2, 80.4, 76.6, 73.6, 73.5, 70.5, 62.4, 21.1, 21.0, 20.9.

HRMS (ESI): m/z calcd for C23H27N2O10 [M + H]+: 491.1660; found: 491.1682.


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(S)-1-((2R,3R,4R,5R)-4-Acetoxy-3-(benzyloxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)ethane-1,2-diyl Diacetate (3b)

White solid; yield: 1.03 g (90%); mp 140–142 °C; Rf = 0.50 (5% MeOH in CHCl3); [α]D 28 +42.81 (c 0.1, MeOH).

IR (KBr): 3070, 1740, 1685, 1470, 1385, 1323, 1290, 1180, 1060, 950, 820, 790, 629 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 9.05 (s, 1 H), 7.28–7.37 (m, 5 H), 6.99 (s, 1 H), 5.73 (d, J = 4.3 Hz, 1 H), 5.38 (dd, J = 5.9, 4.1 Hz, 1 H), 5.32–5.35 (m, 1 H), 4.51–4.58 (m, 2 H), 4.38–4.43 (m, 2 H), 4.12–4.15 (m, 1 H), 4.08 (dd, J = 12.1, 6.6 Hz, 1 H), 2.10 (s, 3 H), 2.05 (s, 3 H), 2.03 (s, 3 H), 1.93 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 170.5, 170.1, 169.9, 163.6, 149.9, 136.9, 136.7, 128.5, 128.2, 128.0, 111.6, 90.4, 80.2, 76.4, 73.4, 73.3, 70.3, 62.3, 20.8, 20.7, 20.7, 12.4.

HRMS (ESI): m/z calcd for C24H29N2O10 [M + H]+: 505.1817; found: 505.1827.


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Synthesis of Nucleosides 4a,b; General Procedure

Pyrimidine nucleoside 3a (1.2 g, 2.44 mmol) or 3b (1.2 g, 2.38 mmol) was dissolved in a solvent mixture of MeOH and H2O (9:1). K2CO3 (1.01 g, 7.32 mmol for 3a/0.98 g, 7.14 mmol for 3b) was added to the reaction mixture portionwise at 0 °C and the mixture was allowed to stir at rt for 1 h. On completion of reaction (observed by TLC), the solvent was evaporated under reduced pressure and the crude product was purified by using silica gel column chromatography to afford compound 4a,b, respectively.


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(S)-2-((2R,3S,4R,5R)-3-(Benzyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)-2-hydroxyethyl Acetate (4a)

Colorless viscous oil; yield: 0.68 g (92%); Rf = 0.20 (5% MeOH in CHCl3­); [α]D 28 +48.10 (c 0.1, MeOH).

IR (KBr): 3410, 3220, 3090, 1780, 1695, 1482, 1338, 1305, 1220, 1040, 950, 785, 696 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.37 (s, 1 H), 7.83 (d, J = 8.1 Hz, 1 H), 7.27–7.39 (m, 5 H), 5.85 (d, J = 7.1 Hz, 1 H), 5.67 (d, J = 8.1 Hz, 1 H), 5.50 (d, J = 6.6 Hz, 1 H), 5.27 (d, J = 5.2 Hz, 1 H), 4.71 (t, J = 5.4 Hz, 1 H), 4.59–4.69 (m, 2 H), 4.17–4.22 (m, 1 H), 4.08–4.10 (m, 1 H), 3.96 (d, J = 7.3 Hz, 1 H), 3.62–3.67 (m, 1 H), 3.35 (s, 2 H).

13C NMR (CDCl3, 100.6 MHz): δ = 163.0, 150.9, 140.5, 138.5, 128.2, 127.4, 127.3, 102.1, 86.4, 83.3, 76.5, 73.0, 71.5, 70.9, 62.4.

HRMS (ESI): m/z calcd for C17H21N2O7 [M + H]+: 365.1343; found: 365.1330.


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(S)-2-((2R,3S,4R,5R)-3-(Benzyloxy)-4-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)-2-hydroxyethyl Acetate (4b)

White solid; yield: 0.69 g (92%); mp 110–112 °C; Rf = 0.20 (5% MeOH in CHCl3); [α]D 28 +58.92 (c 0.1, MeOH).

IR (KBr): 3390, 3035, 2958, 1695, 1470, 1385, 1210, 1025, 995, 820, 725, 625 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.32 (s, 1 H), 7.71 (s, 1 H), 7.29–7.40 (m, 5 H), 5.86 (d, J = 7.3 Hz, 1 H), 5.44 (d, J = 6.7 Hz, 1 H), 5.35 (d, J = 5.2 Hz, 1 H), 4.73 (t, J = 5.4 Hz, 1 H), 4.68 (d, J = 12.2 Hz, 1 H), 4.61 (d, J = 12.2 Hz, 1 H), 4.19–4.24 (m, 1 H), 4.08–4.09 (m, 1 H), 3.97 (dd, J = 5.2, 2.1 Hz, 1 H), 3.66–3.68 (m, 1 H), 3.35–3.37 (m, 2 H), 1.79 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 163.7, 151.0, 138.5, 136.1, 128.1, 127.4, 127.3, 109.6, 86.2, 83.2, 76.5, 72.8, 71.5, 70.9, 62.5, 12.2.

HRMS (ESI): m/z calcd for C18H23N2O7 [M + H]+: 379.1500; found: 379.1524.


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Lipase-Assisted Synthesis of Nucleosides 5a,b; General Procedure

Compound 4a 1.0 g (2.74 mmol) or 4b 1.0 g (2.64 mmol) was dissolved in 2-Me-THF (40 mL) and vinyl acetate (2.74 mmol for 4a)/(3.17 mmol for 4b) was added followed by the addition of Lipozyme® TL IM. The reaction mixture was stirred at 45 °C in an incubator shaker for 1 h at 200 rpm. On completion, the reaction was quenched by filtering off the enzyme. Excess of solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography by using MeOH in CHCl3 as eluent to obtain pure nucleoside 5a,b, respectively.


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(S)-2-((2R,3S,4R,5R)-3-(Benzyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-hydroxytetrahydrofuran-2-yl)-2-hydroxyethyl Acetate (5a)

White solid; yield: 1.10 g (99%); mp 177–180 °C; Rf = 0.25 (5% MeOH in CHCl3); [α]D 28 +29.42 (c 0.1, MeOH).

IR (KBr): 3233, 3034, 2497, 1679, 1406, 1368, 1275, 1130, 1093, 900, 756, 643, 551 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.37 (s, 1 H), 7.81 (d, J = 11.7 Hz, 1 H), 7.26–7.40 (m, 5 H), 5.84 (dd, J = 7.0, 3.4 Hz, 1 H), 5.67 (dd, J = 8.0, 3.5 Hz, 1 H), 5.62 (t, J = 4.6 Hz, 1 H), 5.56–5.58 (m, 1 H), 4.70 (dd, J = 12.1, 3.7 Hz, 1 H), 4.60 (dd, J = 12.1, 3.7 Hz, 1 H), 4.25–4.28 (m, 1 H), 3.97–4.03 (m, 3 H), 3.84–3.96 (m, 2 H).

13C NMR (CDCl3, 100.6 MHz): δ = 170.30, 163.0, 150.9, 140.7, 138.4, 128.2, 127.4, 127.4, 102.7, 87.0, 82.7, 76.8, 72.3, 71.1, 68.1, 64.9, 20.7.

HRMS (ESI): m/z calcd for C19H23N2O8 [M + H]+: 407.1449; found: 407.1451.


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(S)-2-((2R,3S,4R,5R)-3-(Benzyloxy)-4-hydroxy-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)-2-hydroxyethyl Acetate (5b)

White solid; yield: 1.07 g (97%); mp 165–167 °C; Rf = 0.35 (5% MeOH in CHCl3); [α]D 28 +47.55 (c 0.1, MeOH).

IR (KBr): 3420, 3157, 3094, 3015, 2909, 1685, 1487, 1390, 1038, 966, 835, 750, 597 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.37 (s, 1 H), 7.66 (s, 1 H), 7.29–7.40 (m, 5 H), 5.84 (d, J = 7.1 Hz, 1 H), 5.68 (d, J = 5.4 Hz, 1 H), 5.54 (d, J = 6.6 Hz, 1 H), 4.70 (d, J = 12.1 Hz, 1 H), 4.61 (d, J = 12.1 Hz, 1 H), 4.25–4.29 (m, 1 H), 4.01–4.05 (m, 2 H), 3.97–3.99 (m, 1 H), 3.95 (d, J = 6.6 Hz, 1 H), 3.90–3.92 (m, 1 H), 2.02 (s, 3 H), 1.79 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 170.3, 163.7, 150.9, 138.4, 136.1, 128.2, 127.4, 127.3, 109.6, 86.7, 82.6, 76.9, 72.4, 71.1, 68.1, 65.0, 20.7, 12.1.

HRMS (ESI): m/z calcd for C20H25N2O8 [M + H]+: 421.1605; found: 421.1607.


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Synthesis of Mesylated Pyrimidine Nucleosides 6a,b; General Procedure

Methanesulfonyl chloride (6.15 mmol for 5a)/(5.92 mmol for 5b) was added to a solution of 5a (1.0 g, 2.64 mmol)/5b (1.0 g, 2.37 mmol) in anhyd pyridine (10 mL) at 0 °C. The reaction mixture was stirred at 25 °C for 4 h. Upon completion of the reaction (monitored by TLC), pyridine was neutralized by adding 10% ice-cold HCl (100 mL). The product was extracted with CHCl3 (2 × 30 mL) and the combined organic layers were washed with aq NaHCO3 (2 × 50 mL). The residue thus obtained was purified by silica gel column chromatography to afford nucleoside 6a,b, respectively.


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(S)-2-((2S,3R,4R,5R)-3-(Benzyloxy)-5-(2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-((methylsulfonyl)oxy)tetrahydrofuran-2-yl)-2-((methylsulfonyl)oxy)ethyl Acetate (6a)

Colorless viscous oil; yield: 1.24 g (90%); Rf = 0.30 (5% MeOH in CHCl3); [α]D 28 +97.06 (c 0.1, MeOH).

IR (KBr): 3127, 2914, 2134, 1732, 1654, 1643, 1355, 1275, 1167, 1020, 981, 729, 720, 568 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.54 (s, 1 H), 7.75 (d, J = 8.1 Hz, 1 H), 7.32–7.41 (m, 5 H), 5.95 (d, J = 3.8 Hz, 1 H), 5.67 (dd, J = 8.0, 2.3 Hz, 1 H), 5.63 (dd, J = 5.7, 3.8 Hz, 1 H), 5.09–5.12 (m, 1 H), 4.67 (d, J = 11.0 Hz, 1 H), 4.59 (d, J = 11.0 Hz, 1 H), 4.52 (t, J = 6.0 Hz, 1 H), 4.33 (dd, J = 12.7, 3.0 Hz, 1 H), 4.16–4.21 (m, 2 H), 3.33 (s, 3 H), 3.21 (s, 3 H), 2.04 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 170.0, 163.1, 150.3, 142.2, 137.1, 128.3, 128.2, 127.9, 102.1, 89.8, 78.9, 77.2, 77.1, 75.0, 71.8, 61.6, 38.2, 37.7, 20.5.

HRMS (ESI): m/z calcd for C21H26N2O12S2Na [M + Na]+: 585.0819; found: 585.0805.


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(S)-2-((2S,3R,4R,5R)-3-(Benzyloxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-4-((methylsulfonyl)oxy)tetrahydrofuran-2-yl)-2-((methylsulfonyl)oxy)ethyl Acetate (6b)

Colorless viscous oil; yield: 1.28 g (94%); Rf = 0.40 (5% MeOH in CHCl3­); [α]D 28 +120.11 (c 0.1, MeOH).

IR (KBr): 3003, 2973, 2341, 1638, 1468, 1236, 1147, 1111, 1029, 932, 831, 700, 650, 570 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.54 (s, 1 H), 7.59 (s, 1 H), 7.32–7.40 (m, 5 H), 5.95 (d, J = 4.1 Hz, 1 H), 5.55–5.58 (m, 1 H), 5.11 (t, J = 7.6 Hz, 1 H), 4.65–4.69 (m, 1 H), 4.60 (d, J = 11.1 Hz, 1 H), 4.51 (q, J = 5.6 Hz, 1 H), 4.35 (dd, J = 12.6, 3.0 Hz, 1 H), 4.17–4.22 (m, 2 H), 3.32 (s, 3 H), 3.22 (s, 3 H), 2.04 (s, 3 H), 1.78 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 170.0, 163.7, 150.4, 137.2, 137.1, 128.4, 128.1, 127.9, 109.9, 88.8, 78.8, 77.2, 77.2, 74.9, 71.8, 61.7, 38.3, 37.8, 20.5, 11.9.

HRMS (ESI): m/z calcd for C22H29N2O12S2 [M + H]+: 577.1156; found: 577.1150.


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Synthesis of Benzylated LNA Monomers 7a,b; General Procedure

To a solution of compound 6a,b (1.76 mmol) in 1,4-dioxane:H2O (1:1, 20 mL) was added aq 2 M NaOH (0.6 mL) at 0 °C and the reaction mixture was stirred for 24 h at 25 °C. On completion of the reaction, AcOH (10 mL) was added to neutralize the reaction mixture. Excess of solvent was co-evaporated with toluene under reduced pressure. The crude product thus obtained was purified by silica gel column chromatography to obtain pure nucleoside 7a,b, respectively.


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1-((1R,3R,4S,7R)-7-(Benzyloxy)-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)pyrimidine-2,4(1H,3H)-dione (7a)

White solid; yield: 0.17 g (72%); mp 180–182 °C; Rf = 0.26 (5% MeOH in CHCl3); [α]D 28 +240.38 (c 0.1, MeOH).

IR (KBr): 3473, 3260, 2981, 1608, 1437, 1317, 1296, 1115, 1007, 825, 723, 549 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.40 (s, 1 H), 7.85 (d, J = 8.1 Hz, 1 H), 7.31–7.38 (m, 5 H), 5.97 (s, 1 H), 5.60 (dd, J = 8.1, 1.9 Hz, 1 H), 4.96 (t, J = 5.6 Hz, 1 H), 4.69 (d, J = 11.7 Hz, 1 H), 4.66 (s, 1 H), 4.62 (d, J = 11.7 Hz, 1 H), 4.57 (d, J = 2.7 Hz, 1 H), 4.44–4.47 (m, 1 H), 4.09 (t, J = 5.4 Hz, 1 H), 3.37 (t, J = 5.4 Hz, 2 H).

13C NMR (CDCl3, 100.6 MHz): δ = 163.2, 150.4, 140.9, 137.6, 128.4, 127.8, 127.7, 100.4, 88.7, 84.0, 80.3, 79.5, 74.2, 71.4, 61.3.

HRMS (ESI): m/z calcd for C17H19N2O6 [M + H]+: 347.1238; found: 347.1240.


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1-((1R,3R,4S,7R)-7-(Benzyloxy)-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (7b)

White solid; yield: 0.18 g (74%); mp 202–205 °C; Rf = 0.32 (5% MeOH in CHCl3); [α]D 28 +272.91 (c 0.1, MeOH).

IR (KBr): 3343, 3093, 2821, 1727, 1607, 1620, 1477, 1317, 1237, 1187, 1014, 939, 850, 710, 592 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.40 (s, 1 H), 7.70 (s, 1 H), 7.31–7.38 (m, 5 H), 5.95 (s, 1 H), 4.96 (t, 1 H), 4.61–4.70 (m, 3 H), 4.58 (s, 1 H), 4.45 (s, 1 H), 4.15 (t, J = 5.4 Hz, 1 H), 3.39 (s, 1 H), 3.37 (s, 1 H), 1.82 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 163.9, 150.4, 137.6, 136.2, 128.4, 127.8, 127.7, 108.1, 86.6, 83.8, 80.3, 79.5, 74.2, 71.4, 61.3, 12.2.

HRMS (ESI): m/z calcd for C18H21N2O6 [M + H]+: 361.1394; found: 361.1398.


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Synthesis of Titled LNA Monomers 8a,b; General Procedure

To a solution of compound 7a,b (0.55 mmol) in anhyd THF:MeOH (9:1, 20 mL), was added Pd(OH)2/C (20 wt%, 0.04 g) and 88% formic acid (0.20 mL, 4.4 mmol). The reaction mixture was refluxed for 30 min. On completion of reaction (checked by TLC), the catalyst was filtered off and washed with excess of MeOH. Excess of solvent was co-evaporated with toluene under reduced pressure. The crude product thus obtained was purified by silica gel column chromatography to obtain nucleoside 8a,b, respectively, in pure form.


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1-((1S,3R,4S,7R)-7-Hydroxy-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)pyrimidine-2,4(1H,3H)-dione (8a)

White solid; yield: 0.13 g (82%); mp 220–222 °C; Rf = 0.32 (10% MeOH in CHCl3); [α]D 28 +298.12 (c 0.1, MeOH).

IR (KBr): 3369, 3053, 2812, 1496, 1395, 1217, 1134, 1099, 1033, 1042, 998, 765, 629, 547 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.39 (s, 1 H), 7.85 (d, J = 8.1 Hz, 1 H), 5.99 (s, 1 H), 5.91 (s, 1 H), 5.60 (d, J = 8.1 Hz, 1 H), 4.95 (s, 1 H), 4.54 (s, 1 H), 4.36 (s, 1 H), 4.23 (s, 1 H), 4.03 (t, J = 5.6 Hz, 1 H), 3.37 (s, 1 H), 3.35 (s, 1 H).

13C NMR (CDCl3, 100.6 MHz): δ = 163.3, 150.5, 141.0, 100.4, 88.7, 84.0, 81.8, 75.7, 73.0, 61.5.

HRMS (ESI): m/z calcd for C10H13N2O6 [M + H]+: 257.0768; found: 257.0770.


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1-((1S,3R,4S,7R)-7-Hydroxy-6-(hydroxymethyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8b)

White solid; yield: 0.14 g (85%); mp 253–255 °C; Rf = 0.35 (10% MeOH in CHCl3); [α]D 28 +304.04 (c 0.1, MeOH).

Recrystallization of the compound 8b was carried out using a solvent mixture of MeOH and CHCl3 (1:1) and allowing the solution for slow evaporation at rt.

IR (KBr): 3135, 3430, 2972, 2861, 1707, 1667, 1602, 1389, 1336, 1331, 1297, 1039, 953, 801, 696, 502 cm–1.

1H NMR (CDCl3, 400 MHz): δ = 11.38 (s, 1 H), 7.69 (s, 1 H), 5.97 (s, 1 H), 5.88 (d, J = 3.4 Hz, 1 H), 4.94 (t, J = 5.8 Hz, 1 H), 4.54 (s, 1 H), 4.35 (s, 1 H), 4.22 (d, J = 3.0 Hz, 1 H), 4.07 (t, J = 5.4 Hz, 1 H), 3.38 (s, 1 H), 3.36 (s, 1 H), 1.81 (s, 3 H).

13C NMR (CDCl3, 100.6 MHz): δ = 163.9, 150.4, 136.4, 108.0, 88.7, 83.9, 81.8, 75.7, 73.0, 61.5, 12.3.

HRMS (ESI): m/z calcd for C11H15N2O6 [M + H]+: 271.0925; found: 271.0930.


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Conflict of Interest

The authors declare no conflict of interest.

Acknowledgment

We are thankful to DST-FIST program, CIF-USIC and Department of Chemistry, University of Delhi for providing the NMR spectral and HRMS recording facilities. Special thanks to guest editor Professor Vinod­ K. Tiwari (IIT BHU, India) for constant support and suggestions throughout the revision of manuscript.

Supporting Information

    Supporting information for this article is available online at https://doi-org.accesdistant.sorbonne-universite.fr/10.1055/s-0041-1738440.
  • Supporting Information

Corresponding Author

Ashok K. Prasad
Bioorganic Laboratory, Department of Chemistry, University of Delhi
Delhi 110 007
India   

Publication History

Received: 07 February 2023

Accepted after revision: 20 April 2023

Article published online:
22 May 2023

© 2023. Thieme. All rights reserved

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Zoom Image
Figure 1 Some representative literature based spiro-nucleosides
Zoom Image
Scheme 1 Retrosynthetic route for the synthesis of titled bicyclic nucleosides
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Scheme 2 Reagents and conditions: (a) B, BSA, TMSOTf, CH3CN, 80 °C; 88% (R = H), 90% (R = CH3) (where B = uracil and thymine); (b) K2CO3, MeOH:H2O (9:1), 25 °C, 1 h; 92% (R = H), 92% (R = CH3); (c) Lipozyme® TL IM, vinyl acetate, MeCN, 45 °C, 1 h; 99% (R = H), 97% (R = CH3); (d) MsCl, Py, 25 °C, 4 h, 90% (R = H), 94% (R = CH3); (e) aq 2 M NaOH, 1,4-dioxane:H2O (1:1), 25 °C, 24 h, 72% (R = H), 74% (R = CH3); (f) Pd(OH)2/C, HCO2H, THF:MeOH (1:1), reflux, 30 min, 82% (R = H), 85% (R = CH3).
Zoom Image
Figure 2 Optimization chart for the selective acetylation of nucleosides 4a,b
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Figure 3 (a) ORTEP diagram of 1-((1S,3R,4S,7R)-7-hydroxy-6-(hydroxy­methyl)-2,5-dioxabicyclo[2.2.1]heptan-3-yl)-5-methylpyrimidine-2,4(1H,3H)-dione (8b) drawn in 30% thermal probability ellipsoids showing the atom numbering scheme; (b) Exhibition of S-type puckering of furanose ring in nucleoside 8b.