Practical Synthesis of 4 -Thioribonucleosides Starting from D-Ribose

UNIT 14.12

Noriaki Minakawa1 and Akira Matsuda2 1

Graduate School of Pharmaceutical Sciences, The University of Tokushima, Tokushima, Japan 2 Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

A practical synthesis of 4 -thioribonucleosides, i.e., 4 -thiouridine, -cytidine, -adenosine, and -guanosine, which are versatile units for nucleic acids–based therapeutics, is described. Large-scale synthesis of 4-thiosugar starting from Dribose was achieved (33%) in eight steps and with only three chromatographic purifications. After the appropriate chemical conversion of the 4-thiosugar, the resulting sulfoxide was subjected to the Pummerer reaction in the presence of silylated nucleobases. In reactions with silylated pyrimidine bases, the desired 4 -thioribonucleoside derivatives were obtained in good yield and β-selectively. On the other hand, N-7 isomers were obtained mainly in the Pummerer reaction with purine bases under the same conditions. However, the desired N-9 isomers were obtained in moderate yields when the reaction mixtures were subsequently heated under reflux. As a result, effective synthesis of 4 -thioribonucleosides C 2014 by John Wiley & Sons, Inc. was accomplished.  Keywords: D-ribose r 4-thiosugar r sulfoxide r Pummerer reaction r 4 thioribonucleoside

How to cite this article: Minakawa, N. and Matsuda, A. 2014. Practical Synthesis of 4 -Thioribonucleosides Starting from D-Ribose. Curr. Protoc. Nucleic Acid Chem. 59:14.12.1-14.12.19. doi: 10.1002/0471142700.nc1412s59

INTRODUCTION Development of chemically modified nucleoside derivatives is pertinent because of the availability of nucleic-acids-based therapeutics including antisense, short-interfering RNA (siRNA) strategies, and aptamers isolated by SELEX technology. Among the nucleoside derivatives developed thus far, 4 -thioribonucleosides, in which the furanose ring oxygen is replaced by a sulfur atom, are attractive since the resulting 4 -thioribonucleic acid (4 -thioRNA) shows high nuclease resistance and thermal stability (Bellon et al., 1993; Leydier et al., 1995). However, no practical synthetic method has been reported (Bobek et al., 1975; Tiwari et al., 1994). Because of the aforementioned research backgrounds, the practical synthesis of 4 -thioribonucleosides was deemed to be necessary and the synthesis of desired compounds was achieved via a large-scale synthesis of 4thiosugar (Minakawa et al., 2003) and a stereoselective condensation with nucleobases using the Pummerer reaction (Naka et al., 2000). The resulting 4 -thioribonucleosides are currently utilized not only for 4 -thioRNA-based therapeutics (Hoshika et al., 2004, 2005, 2007; Kato et al., 2005; Minakawa et al., 2008; Takahashi et al., 2009, 2012, 2013; Kikuchi et al., 2013), but also for 4 -thioDNA-based therapeutics (Inoue et al., 2006, 2007; Kojima et al., 2013), which is described in detail in the 4 -thioribonucleosides synthetic protocols. Current Protocols in Nucleic Acid Chemistry 14.12.1-14.12.19, December 2014 Published online December 2014 in Wiley Online Library (wileyonlinelibrary.com). doi: 10.1002/0471142700.nc1412s59 C 2014 John Wiley & Sons, Inc. Copyright 

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HO D-ribose

PMBO

O

O

OAllyl

HO

OH

PMBO

1

PMBO OAllyl OPMB

PMBO

2

PMBO

S OPMB

OPMB

OH

OPMB 4

PMBO Br

PMBO

7

8

PMBO

OPMB

PMBO Br

S

PMBO

OH

OH

3

+

PMBO PMBO

PMBO

O

OMs

PMBO

OPMB 6

OMs

OPMB 5

O HO HO

OH 9

Figure 14.12.1

O

S

O

S

TIPDS O

OH 10

O

S

TIPDS O

ODMBz 11

S

TIPDS O

ODMBz 12

Schema for the synthesis of 4-thiosugar starting from D-ribose.

Since 4 -thioribonucleosides have an unnatural sugar, condensation of nucleobases with an appropriate 4-thiosugar is required for their preparation. At first, D-ribose was converted into 1,4-anhydro-4-thio-D-ribitol (9) in eight steps via two consecutive SN 2 reactions. Then, 9 was converted into the corresponding sulfoxide (see Basic Protocol 1). The Pummerer reaction in the presence of silylated nucleobases, followed by appropriate chemical conversions, afforded 4 -thioribonucleosides (see Basic Protocols 2 and 3). BASIC PROTOCOL 1

PREPARATION OF 4-THIOSUGAR To conduct the Pummerer reaction, 1,4-anhydro-4-thio-D-ribitol (9) is needed. The synthetic tactics presented here involve a combination of allyl and p-methoxybenzyl (PMB) protecting groups, and two consecutive SN 2 reactions of D-ribose (Dyson et al., 1992; Leydier et al., 1995) (Fig. 14.12.1). 2,3,5-Tri-O-p-methoxybenzyl-D-ribitol (4) was first prepared in four steps without choromatographic separation of intermediates 1, 2, and 3. After treatment of 4 with methanesulfonyl chloride in pyridine, the resulting dimesylate 5 was heated under reflux in methyl ethyl ketone in the presence of lithium bromide, followed by treatment with sodium sulfide to give 7. Deprotection of PMB groups afforded the desired 9. To conduct the stereoselective Pummerer reaction, 9 was converted into the sulfoxide 12, which has a 2,4-dimethoxybenzoyl group on its 2-position for β-selective coupling with nucleobases. NOTE: In the large-scale synthesis, 1, 2, 3, and 5 were not isolated by a chromatographic purification. A small amount of 2 and 3 were purified for analytical data. Although 2 was obtained as a mixture of diastereomers arising from an anomeric position, their stereochemistries were not determined and named them as isomer A and isomer B in their 1 H NMR spectra. ˚ NOTE: Dry solvent means a commercially available, high-grade solvent stored with 4A molecular sieves, while anhydrous solvent means a solvent distilled from an appropriate ˚ molecular sieves. drying agent and stored with 4A NOTE: 2,4-Dimethoxybenzoyl chloride was prepared from 2,4-dimethoxybenzic acid and thionyl chloride immediately prior to the reaction.

Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

Materials D-Ribose,

98% (TCI) dried well with P2 O5 at 50°C using a vacuum oil pump Allyl alcohol, 99% (Kanto Chemical)

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Sulfuric acid (H2 SO4 ), 96% (Kanto Chemical) Sodium bicarbonate (NaHCO3 ), 99% (Junsei Chemical) Celite pad (Kanto Chemical) Methanol (MeOH), 99.5% (Wako Pure Chemical) Sodium hydride (NaH), 60% dispersion in mineral oil (Kanto Chemical) ˚ Dry tetrahydrofuran (THF), 99.5% (Wako Pure Chemical), stored with 4 A molecular sieves ˚ Dry dimethylformamide (DMF), 99.5% (Wako Pure Chemical), stored with 4 A molecular sieves p-Methoxybenzyl chloride (PMBCl), 95% (Wako Pure Chemical) Ammoniun chloride (NH4 Cl), 98.5%, saturated aqueous (Wako Pure Chemical) Ethyl acetate (AcOEt), 99% (Junsei Chemical) Anhydrous sodium sulfite (Na2 SO4 ), 99% (Kanto Chemical) Chloroform, 99% (Junsei Chemical) Palladium chloride (PdCl2 ), 99% (Wako Pure Chemical) Oxygen, 99.5% Sodium borohydride (NaBH4 ), 95% (Wako Pure Chemical) Merck silica gel 60 F254 , 0.063 to 0.2 mm, 70 to 230 mesh (Kanto Chemical) Anhydrous pyridine, 99% (Wako Pure Chemical) distilled from KOH and stored ˚ molecular sieves with 4 A Argon Methanesulfonyl chloride (MsCl), 98% (Kanto Chemical) Toluene ˚ molecular Dry methyl ethyl ketone, 99.5% (Kishida Chemical) stored with 4 A sieves Lithium bromide, 95% (Kishida Chemical) dried well with P2 O5 at 50°C using a vacuum oil pump Sodium sulfide nonahydrate, 98% (Wako Pure Chemical) Anhydrous dichloromethane, 98% (Junsei Chemical) distilled from P2 O5 and ˚ molecular sieves stored with 4 A Trifluoroacetic acid, 99% (TCI) 1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane, 99.5% (Shin-Etsu Chemical) 2,4-Dimethoxybenzoic acid, 98% (TCI) Thionyl chloride, 95% (Wako Pure Chemical) ˚ molecular sieves (Wako Pure Chemical) 4A 500-, 1-, 2-, and 5-L round-bottom flasks Magnetic stir plate and magnetic stir bars Vacuum oil pump TLC plates 3-L three-neck flask attached to a bubbler 1-L flasks 150- and 500-mL dropping funnels Chromatography columns, 10 × 15–cm Glass rods Prepare diol 4 Protection with an allyl group 1. Dissolve 60.0 g (0.4 mol) of D-ribose in 1.8 L allyl alcohol in a 5-L round-bottom flask and add 6.4 mL sulfuric acid (0.12 mol) at 0°C. Stir mixture with a mechanical stirrer overnight at room temperature. Biologically Active Nucleosides

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2. Quench the reaction by adding sodium bicarbonate until the reaction mixture is neutralized. Filter the solids through a Celite pad and wash the Celite pad with 500 mL MeOH. 3. Concentrate the combined filtrate and washings, and dry the residue well using a vacuum oil pump. 4. Characterize the product by TLC. 1-O-Allyl D-ribose (1). Rf : 0.63 (1:4 v/v MeOH/CHCl3 ).

Protect with PMB groups 5. Add 64 g (1.6 mol) NaH in 700 mL dry THF in a 3-L three-neck flask attached to a bubbler. 6. Dissolve crude 1 (calculated as 0.4 mol) in 300 mL dry DMF in a 1-L flask and pour the solution into a 500-mL dropping funnel attached to the 3-L three-neck flask (in step 5). 7. Add the dry DMF solution of 1 dropwise to the THF suspension of NaH over 3 hr at 0°C in an ice bath. Stir the whole mixture for 4 hr at room temperature. 8. Cool the whole mixture to 0°C in an ice bath. 9. Pour 190 mL PMBCl (1.4 mol) into the 500-mL dropping funnel attached to the 3-L three-neck flask, and add about one-third of PMBCl dropwise (10 mL/15 min) to the mixture at 0°C. CAUTION: A violent evolution of hydrogen sometimes takes place if one adds all of the PMBCl at 0°C.

10. Remove the ice bath and stir reaction mixture for 1 hr at room temperature. 11. Add the remaining PMBCl dropwise (10 mL/15 min) to the reaction mixture at room temperature to keep the reaction nonviolent. Stir the whole mixture for >24 hr at room temperature. 12. Quench the reaction carefully (monitor evolution of hydrogen from active NaH) by adding saturated aqueous NH4 Cl at 0°C. Dilute the reaction mixture with AcOEt (2000 mL). Wash two times with H2 O (700 mL each time), followed by brine (700 mL). 13. Dry organic layer with anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness. 14. Characterize the product by TLC and 1 H NMR.

Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

1-O-Allyl 2,3,5-tri-O-p-methoxybenzyl-D-ribose (2). Rf : 0.63 (1:1 v/v EtOAc/hexane). 1 H NMR of isomer A (CDCl3 ) δ: 7.30–7.18 (m, 6 H), 6.87–6.81 (m, 6 H), 5.81 (dddd, 1 H, J = 17.3, 10.3, 6.2, 5.5 Hz), 5.20 (dd, 1 H, J = 17.3, 1.2 Hz), 5.13 (dd, 1 H, J = 10.3, 1.2 Hz), 5.02 (s, 1 H), 4.61–4.31 (m, 6 H), 4.29 (ddd, 1 H, J = 6.7, 6.2, 3.8 Hz), 4.15 (dd, 1 H, J = 12.9, 5.5 Hz), 3.98 (dd, 1 H, J = 6.7, 4.7 Hz), 3.90 (dd, 1 H, J = 12.9, 6.2 Hz), 3.85 (d, 1 H, J = 4.7 Hz), 3.80, 3.79, 3.78 (each s, each 3 H), 3.56 (dd, 1 H, J = 10.6, 3.8 Hz), 3.46 (dd, 1 H, J = 10.6, 6.2 Hz). 1 H NMR of isomer B (CDCl3 ) δ: 7.26–7.12 (m, 6 H), 6.85–6.79 (m, 6 H), 5.98 (dddd, 1 H, J = 17.3, 10.3, 6.5, 5.9 Hz), 5.32 (dd, 1 H, J = 17.3, 1.3 Hz), 5.18 (dd, 1 H, J = 10.3, 1.3 Hz), 5.02 (d, 1 H, J = 4.1 Hz), 4.71–4.33 (m, 6 H), 4.27 (m, 1 H, J = 13.2, 4.1 Hz), 4.21 (m, 1 H), 4.13 (dd, 1 H, J = 13.2, 6.7 Hz), 3.81, 3.80, 3.79 (each s, each 3 H), 3.74 (m, 2 H), 3.38 (dd, 1 H, J = 10.6, 3.8 Hz), 3.31 (dd, 1 H, J = 10.6, 4.4 Hz).

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Deprotection of allyl group 15. Dissolve crude 2 (calculated as 0.4 mol) in a mixture of CHCl3 /H2 O (1.2 L/800 mL) in a 5-L round-bottom flask and add 21 g PdCl2 (0.12 mol). Stir the two-phase solution vigorously with a mechanical stirrer for 24 hr at 50°C under an O2 atmosphere (attach a balloon with O2 ). 16. Filter the solids through a Celite pad, and wash the Celite pad with AcOEt (1000 mL). 17. Evaporate organic solvent in vacuo and dilute with AcOEt (2000 mL). Wash two times with H2 O (700 mL each time), followed by brine (700 mL). 18. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness. 19. Characterize the product by TLC and 1 H NMR. 2,3,5-Tri-O-p-methoxybenzyl-D-ribose (3). Rf : 0.50 (1:1 v/v AcOEt/hexane). 1 H NMR (CDCl3 ) δ: 7.30–7.16 (m, 6 H), 6.87–6.82 (m, 6 H), 5.28–5.23 (m, 1 H), 4.63–4.08 (m, 8 H), 3.80–3.78 (m, 9 H), 3.60 (m, 0.5 H), 3.41 (m, 2.5 H).

Reduction with NaBH4 20. Dissolve crude 3 (calculated as 0.4 mol) in 800 mL MeOH in a 3-L round-bottom flask and add 30 g NaBH4 (0.8 mol) portion-wise (add 5 g every 5 min) at 0°C. Stir reaction mixture 1.5 hr at room temperature. 21. Evaporate solvent in vacuo, and coevaporate three times with MeOH (150 mL each time). Dilute with AcOEt (1800 mL). Wash two times with H2 O (600 mL each time), followed by brine (600 mL). 22. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate solvent to dryness. 23. Absorb the crude products on silica gel and purify by column chromatography (φ = 10 × 15–cm) using AcOEt/hexane (1:5 to 1:1) as the solvent. 24. Combine the fractions containing the product 4. Evaporate the solvent and dry the residue using a vacuum oil pump. 25. Characterize the product by TLC and 1 H NMR. 2,3,5-Tri-O-p-methoxybenzyl-D-ribitol (4). Yield of a colorless oil from D-ribose 162.4 g (79%). Rf : 0.32 (3:2 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 7.25–7.15 (m, 6 H), 6.88– 6.82 (m, 6 H), 4.66–4.40 (m, 6 H), 3.95 (m, 1 H), 3.78 (m, 11 H), 3.72 (m, 2 H), 3.54 (m, 2 H), 2.67 (br s, 1 H), 2.37 (br s, 1 H).

Prepare 4-thiosugar 7 Mesylation with MsCl 26. Dissolve 162 g (0.32 mol) of 4 in 900 mL anhydrous pyridine in a 2-L round-bottom flask with an argon balloon and add 122 mL MsCl (1.6 mol) at 0°C. 27. Stir the reaction mixture for 30 min at 0°C under argon atmosphere. 28. Quench the reaction by adding ice. 29. Evaporate the solvent in vacuo, and dilute with AcOEt (2000 mL). Wash with H2 O (700 mL), three times with saturated aqueous NaHCO3 (700 mL each time), and brine (700 mL). 30. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness, and coevaporate three times with toluene (50 mL).

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31. Characterize the product by TLC. 1,4-O-Dimesyl-2,3,5-tri-O-p-methoxybenzyl-D-ribitol (5). Rf : 0.66 (2:1 v/v EtOAc/ hexane).

Bromination with LiBr 32. Dissolve crude 5 (calculated as 0.32 mol) in 1 L dry methyl ethyl ketone in a 2-L round-bottom flask with an argon balloon and add 278 g LiBr (3.2 mol). Stir the reaction mixture for 12 hr under reflux. 33. Cool the reaction mixture to room temperature, and dilute with AcOEt (2000 mL). Wash two times with H2 O (700 mL each time) and brine (700 mL). 34. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness. 35. Characterize the product by TLC. 2,5-Dibromo-2,5-dideoxy-1,3,4-tri-O-p-methoxybenzyl-l-arabinitol (6). Rf : 0.63 (2:3 v/v EtOAc/hexane).

Cyclization with sodium sulfide 36. Dissolve crude 6 (calculated as 0.32 mol) in 1 L dry DMF in a 2-L round-bottom flask and add 92.2 g sodium sulfide nonahydrate (0.38 mol). Stir reaction mixture for 30 min at 100°C. 37. Cool the reaction mixture to room temperature, and dilute with AcOEt (2000 mL). Wash three times with H2 O (700 mL each time) and brine (700 mL). 38. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness. 39. Absorb the crude products on silica gel and purify by column chromatography (φ = 10 × 12 cm) using AcOEt/hexane (1:10 to 1:1) as the solvent. 40. Combine the fractions containing the products. Evaporate the solvent and dry the residue using a vacuum oil pump to give a mixture of 7 and 8 (7:1). 41. Crystalize the mixture from hexane/AcOEt to give pure 7. 42. Characterize the product by TLC and 1 H NMR. 1,4-Anhydro-2,3,5-tri-O-p-methoxybenzyl-4-thio-D-ribitol (7). Yield of white crystals from 4 69.1 g (42%). Rf : 0.25 (1:2 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 7.25– 7.19 (m, 6 H), 6.87–6.81 (m, 6 H), 4.55–4.39 (m, 6 H), 3.97 (ddd, 1 H, J = 7.0, 5.6, 3.8 Hz), 3.90 (dd, 1 H, J = 3.8, 4.1 Hz), 3.81 (s, 9 H), 3.63 (m, 1 H), 3.43 (m, 2 H), 2.99 (dd, 1 H, J = 7.0, 10.6 Hz), 2.84 (dd, 1 H, J = 5.6, 10.6 Hz).

Deprotection of PMB groups 43. Dissolve 69 g (135 mmol) of 7 in 560 mL dry CH2 Cl2 in a 2-L round-bottom flask and add 140 mL TFA at room temperature. Stir the reaction mixture for 2 hr at room temperature. 44. Evaporate the solvent in vacuo, and coevaporate five times with MeOH (50 mL each time). Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

45. Filter the resulting precipitates and wash with MeOH (200 mL). Evaporate the solvent to dryness.

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46. Absorb the crude products on silica gel and purify by column chromatography (φ = 10 × 10–cm) using MeOH in CHCl3 (2% to 10%) as the solvent. 47. Combine the fractions containing the product. Evaporate the solvent and dry the residue using a vacuum oil pump. 48. Characterize the product by TLC and 1 H NMR. 1,4-Anhydro-4-thio-D-ribitol (9). Yield of a yellow oil 18.7 g (92%). Rf : 0.30 (1:5 v/v MeOH/CHCl3 ). 1 H NMR (DMSO-d6 ) δ: 4.85 (brs, 3 H), 4.04 (dd, 1 H, J = 5.6, 9.0 Hz), 3.76 (t, 1 H, J = 3.9 Hz), 3.57 (dd, 1 H, J = 6.3, 11.0 Hz), 3.31 (dd, 1H, J = 7.3, 11.0 Hz), 3.16 (dd, 1 H, J = 6.3, 11.9 Hz), 2.76 (dd, 1 H, J = 5.4, 10.5 Hz), 2.59 (dd, 1 H, J = 5.9, 10.2 Hz).

Prepare selenoxide 12 Protection with TIPDS group 49. Dissolve 18.5 g (123 mmol) of 9 in 200 mL anhydrous pyridine in a 500-mL round-bottom flask with an argon balloon and add a solution of TIPDSCl (41 mL, 129 mmol) in anhydrous pyridine (46 mL) dropwise over 1 hr at 0°C using a 150-mL dropping funnel. 50. Stir reaction mixture for 5 hr at room temperature under an argon atmosphere. 51. Quench reaction by adding ice. 52. Evaporate the solvent in vacuo, and dilute with AcOEt (500 mL). Wash with saturated aqueous NH4 Cl (200 mL), saturated aqueous NaHCO3 (200 mL), and brine (200 mL). 53. Dry over the organic layer with anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness, and coevaporate three times with toluene (20 mL each time). 54. Absorb the crude products on silica gel and purify by column chromatography (φ = 10 × 10–cm) using AcOEt/hexane (1:50 to 1:5) as the solvent. 55. Combine the fractions containing the product. Evaporate the solvent and dry the residue using a vacuum oil pump. 56. Characterize the product by TLC and 1 H NMR. 1,4-Anhydro-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4-thio-D-ribitol (10). Yield of a pale yellow oil 36.7 g (76%). Rf : 0.78 (1:2 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 4.33 (ddd, 1 H, J = 1.6, 0.8, 4.0 Hz), 4.24 (dd, 1 H, J = 4.0, 8.3 Hz), 4.05 (dd, 1 H, J = 3.2, 12.3 Hz), 3.92 (dd, 1 H, J = 4.4, 12.3 Hz), 3.50 (ddd, 1 H, J = 8.3, 3.2, 4.4 Hz), 3.04 (dd, 1 H, J = 1.6, 12.3 Hz), 2.86 (dd, 1 H, 0.8, 12.3 Hz), 2.67 (s, 1 H), 1.12–1.05 (m, 28 H).

Preparation of DMBzCl 57. Dissolve 51 g (280 mmol) of 2,4-dimethoxybenzoic acid in 180 mL anhydrous CH2 Cl2 in a 500-mL round-bottom flask with an argon balloon and add 43 mL thionyl chloride (562 mmol). Stir the reaction mixture for several hours (26 hr) under reflux. 58. Pick an aliquot of reaction mixture and measure the 1 H NMR in CDCl3 to check the reaction progress (Compare the integration values of methoxy signals appearing at 3.90 and 3.89 ppm for the desired DMBzCl and 4.05 and 3.88 ppm for 2,4dimethoxybenzoic acid. Stop the reaction when its ratio becomes >9:1).

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59. Evaporate the solvent in vacuo. Add 100 mL hexane to the residue, and stir with glass rod to give white precipitate. 60. Collect the resulting precipitate by filtration, and wash with hexane. 61. Dry the white solid using a vacuum oil pump, and use it without further purification. 62. Characterize the product by 1 H NMR. 2,4-Dimethoxybenzoyl chloride. 1 H NMR (CDCl3 ) δ: 8.16 (d, 1 H, J = 9.0 Hz), 6.55 (dd, 1 H, J = 2.5, 9.0 Hz), 6.45 (d, 1 H, J = 2.5 Hz), 3.90 (s, 3H), 3.89 (s, 3H).

Protection with DMBz group 63. Dissolve 36.6 g (93.3 mmol) of 10 in 300 mL anhydrous pyridine in a 1-L roundbottom flask with an argon balloon and add 28 g DMBzCl (140 mmol) at 0°C. 64. Stir the reaction mixture for 15 hr at room temperature under an argon atmosphere. 65. Quench the reaction by adding ice. 66. Evaporate the solvent in vacuo, and dilute with AcOEt (500 mL). Wash with H2 O (200 mL), saturated aqueous NaHCO3 (200 mL), and brine (200 mL). 67. Dry over the organic layer with anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness, and coevaporate three times with toluene (20 mL each time). 68. Absorb the crude products on silica gel and purify by column chromatography (φ = 10 × 10–cm) using AcOEt/hexane (1:8 to 1:6) as the solvent. 69. Combine the fractions containing the product. Evaporate the solvent and dry the residue using a vacuum oil pump. 70. Characterize the product by TLC and 1 H NMR. 1,4-Anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3diyl)-4-thio-D-ribitol (11). Yield of a yellow oil 51.7 g (99%). Rf : 0.45 (1:5 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 7.88 (d, 1 H J = 9.5 Hz), 6.49 (m, 2 H), 5.71 (dd, 1 H, J = 4.8, 4.0 Hz), 4.33 (dd, 1 H, J = 4.0, 9.5 Hz), 4.11 (dd, 1 H, J = 2.8, 12.3 Hz), 3.96 (dd, 1 H, J = 3.2, 12.3 Hz), 3.88, 3.85 (each s, each 3 H,), 3.66 (ddd, 1 H, J = 9.5, 2.8, 3.2 Hz), 3.21 (dd, 1 H, J = 4.8, 12.7 Hz), 2.89 (d, 1 H, J = 12.7 Hz), 1.13–0.96 (m, 28 H).

Oxidation with O2 71. Dissolve 25.7 g (46.2 mmol) of 11 in 300 mL dry CH2 Cl2 in a 1-L round-bottom flask, which was cooled to –78°C. 72. Bubble O3 gas for 4 hr at –78°C. 73. Bubble argon gas for 30 min to remove an excess O3 gas at the same temperature. 74. Evaporate the solvent in vacuo. 75. Absorb the crude products on silica gel and purify by column chromatography (φ = 5 × 10−cm) using AcOEt/hexane (1:2 to 2:1) as the solvent. 76. Combine the fractions containing the product. Evaporate the solvent and dry the residue using a vacuum oil pump. Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

77. Characterize the product by TLC and 1 H NMR. 1,4-Anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3diyl)-4-sulfinyl-D-ribitol (12). Yield of a yellow oil 22.5 g (85% as a mixture of

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diastereomers). Rf : 0.17 (1:1 v/v EtOAc/hexane). 1 H NMR of R-isomer (CDCl3 ) δ: 7.92 (d, 1 H, J = 8.3 Hz), 6.49 (m, 2 H), 5.79 (dd, 1 H, J = 5.4, 3.6 Hz), 4.59 (d, 1 H, J = 12.8 Hz), 4.22 (dd, 1 H, J = 2.8, 12.8 Hz), 4.12 (dd, 1 H, J = 3.6, 12.0 Hz), 3.88, 3.86 (each s, each 3 H), 3.57 (dd, 1 H, J = 5.4, 15.5 Hz), 3.49 (dd, 1 H, J = 12.0, 2.8 Hz), 2.89 (d, 1 H, J = 15.5 Hz), 1.11–0.94 (m, 28 H). 1 H NMR of S-isomer (CDCl3 ) δ: 7.76 (d, 1 H, J = 9.3 Hz), 6.44 (m, 2 H), 5.96 (ddd, 1 H, J = 5.4, 0.9, 3.9 Hz), 5.36 (dd, 1 H, J = 3.9, 10.3 Hz), 4.49 (dd, 1 H, J = 3.6, 12.8 Hz), 4.43 (dd, 1 H, J = 4.6, 12.8 Hz), 3.83, 3.82 (each s, each 3 H), 3.70 (dd, 1 H, J = 0.9, 15.0 Hz), 3.05 (dd, 1 H, J = 5.4, 15.0 Hz), 3.01 (ddd, 1 H, J = 10.3, 3.6, 4.6 Hz), 1.08–0.87 (m, 28 H).

PREPARATION OF 4 -THIOPYRIMIDINE NUCLEOSIDES

BASIC PROTOCOL 2

4 -thioribonucleosides

To prepare the desired β-selectively, introduction of an acyl protecting group on the 2-position of the 4-thiosugar derivative, a precursor of condensation with nucleobases, is needed (see Fig. 14.12.2). In the classical thioglycosydation using a corresponding 1-acetoxy-4-thiosugar, the reactivity of the 4-thiosugar derivative is low (Naka et al., 2000), which is explained in terms of the so-called armed-disarmed principle (Mootoo et al., 1988). In addition, the stereocontrol in the thioglycosydation was unsatisfactory even with the assistance of the neighboring acyl protecting group (Bobek et al., 1975; Tiwari et al., 1994). Contrary to the classical thioglycosydation, the Pummerer reaction between the sulfoxide 12 and silyated pyrimidine bases proceeded smoothly to give 4 -thiopyrimidine nucleosides β-selectively with the assistance of the DMBz group (Naka et al., 1999, 2000). NOTE: All glassware and equipment should be dried well prior to the Pummerer reaction.

Materials Anhydrous triethylamine, 99% (Et3 N, Kishida Chemical) distilled from CaH prior to the reaction Trimethylsilyl trifluoromethanesulfonate (TMSOTf), 98% (TCI) Uracil, 99% (Wako Pure Chemical) dried well with P2 O5 at 50°C using a vacuum oil pump Anhydrous toluene, 99% (Kishida Chemical) distilled from P2 O5 and stored with ˚ molecular sieves 4A Argon

O

O

NH

NH O TIPDS O

O O

S

TIPDS O

O

N S

ODMBz

HO

NHBz

N

N

N

Figure 14.12.2

O

ODMBz 16

HO

HO

HO

N

ODMBz 17

Schema for the synthesis of 4 -thiopyrimidine nucleosides.

OH

NH2 N

O

S

HO

O

N S

15

NHBz

S

TIPDS O

ODMBz

NH

14

12

O

O

N S

HO

13

ODMBz

O

HO

N

O

S

HO

OH 18

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Anhydrous dichloromethane (CH2 Cl2 ), 98% (Junsei Chemical) distilled from P2 O5 ˚ molecular sieves and stored with 4 A 1,4-Anhydro-2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3tetraisopropyldisiloxane-1,3-diyl)-4-sulfinyl-D-ribitol (12, see Basic Protocol 1) Ethyl acetate (AcOEt), 99% (Junsei Chemical) Saturated aqueous sodium bicarbonate (NaHCO3 ) Anhydrous sodium sulfite (Na2 SO4 ), 99% (Kanto Chemical) Silica gel 60, 0.063 to 0.2 mm, 70 to 230 mesh (Kanto Chemical) TLC plates with Merck silica gel 60 F254 Hexane Methanol (MeOH), 99.5% (Wako Pure Chemical) Ammonium fluoride (NH4 F), 97% (Wako Pure Chemical) Methanolic ammonia (NH3 /MeOH), saturated NH3 at 0°C Ammonia gas (NH3 ), 99.9% (Shikoku Acetylene) Chloroform (CHCl3 ) N4 -benzoylcytosine, 97% (TCI) dried well with P2 O5 at 50°C using a vacuum oil pump 30-mL, 500-mL and 1-L round-bottom flasks Magnetic stir plate and magnetic bars Cannula Rotary evaporator Chromatography columns (φ = 5 × 10, φ = 2 × 11, and φ = 5 × 18) Vacuum oil pump Prepare 4 -thiouridine Pummerer reaction of 12 with uracil 1. Add 14.5 mL anhydrous Et3 N (104 mmol) and 38 mL TMSOTf (208 mmol) to a suspension of 5.8 g uracil (52 mmol) in 144 mL anhydrous toluene in a 500-mL round-bottom flask with an argon balloon. Stir reaction mixture at room temperature under argon atmosphere until a two-phase, clear solution is obtained. 2. Add 72 mL anhydrous CH2 Cl2 to the solution, which will give a one-phase, clear solution. 3. Add entire solution to a solution of 14.9 g of 12 (26 mmol) in 144 mL anhydrous CH2 Cl2 in a 1-L round-bottom flask with an argon balloon dropwise over 3 hr via a cannula at room temperature. 4. Add an additional 14.5 mL of anhydrous Et3 N (104 mmol) in 72 mL anhydrous toluene via a cannula at room temperature. Stir entire mixture for 2 hr at room temperature under an argon atmosphere. 5. Quench the reaction by adding ice. 6. Evaporate solvent in vacuo, and dilute with AcOEt (400 mL). Wash with H2 O (250 mL), three times with saturated aqueous NaHCO3 (250 mL each time), and brine (250 mL). 7. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness. Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

8. Absorb the crude products on a silica gel and purify by column chromatography (φ = 5 × 10−cm) using AcOEt/hexane (1:3 to 1:1) as the solvent.

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9. Combine the fractions containing the product. Evaporate the solvent and dry the residue using a vacuum oil pump. 10. Characterize the product by TLC and 1 H NMR. 1-[2-O-(2,4-Dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4thio-β-D-ribofuranosyl]uracil (13). Yield of a white foam 13.8 g (78%). Rf : 0.49 (2:1 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 8.82 (br s, 1 H), 8.15 (d, 1H, J = 8.1 Hz), 7.85 (d, 1 H, J = 8.6 Hz), 6.50 (m, 2 H), 6.00 (s, 1 H), 5.74 (d, 1 H, J = 8.1 Hz), 5.61 (d, 1 H, J = 3.7 Hz), 4.45 (dd, 1 H, J = 3.7, 9.5 Hz), 4.16 (dd, 1 H, J = 2.7, 12.7 Hz), 4.07 (d, 1 H, J = 12.7 Hz), 3.87, 3.86 (each s, each 3 H), 3.73 (dd, 1 H, J = 9.5, 2.7 Hz), 1.15–0.90 (m, 28 H).

Deprotection of protecting groups to give 4 -thiouridine (15) 11. Dissolve 460 mg of 13 (0.69 mmol) in 10 mL MeOH in a 30-mL round-bottom flask and add 510 mg NH4 F (13.8 mmol). Stir reaction mixture for 12 hr under reflux. 12. Evaporate solvent in vacuo to give crude 14. 13. Add 20 mL NH3 /MeOH (saturated at 0°C), and allow mixture to stand for 24 hr at room temperature. Evaporate solvent in vacuo. 14. Absorb the crude products on silica gel and purify by column chromatography (φ = 2 × 11) using MeOH in CHCl3 (5% to 25%) as the solvent. 15. Combine fractions containing the product. Evaporate solvent and dry the residue using a vacuum oil pump. 16. Characterize product by TLC and 1 H NMR. 1-(4-Thio-β-D-ribofuranosyl)uracil (15). Yield of a pale brown foam 152 mg (85%). Rf : 0.46 (1:5 v/v MeOH/CHCl3 ). 1 H NMR (DMSO-d6 ): δ: 11.09 (br s, 1 H), 7.98 (d, 1 H, J = 8.2 Hz), 5.88 (d, 1 H, , J = 7.4 Hz), 5.68 (d, 1 H, J = 8.2 Hz), 5.46 (d, 1 H, J = 6.1 Hz), 5.24 (d, 1 H, J = 4.2 Hz), 5.16 (t, 1 H, J = 5.3 Hz), 4.13 (ddd, 1 H, J = 6.1, 7.4, 3.5 Hz), 4.00 (ddd, 1 H, J = 4.2, 3.5, 3.0 Hz), 3.61 (ddd, 1 H, J = 5.3, 6.6, 11.4 Hz), 3.53 (ddd, 1 H, J = 5.3, 5.4, 11.4 Hz), 3.18 (ddd, 1 H, J = 3.0, 6.6, 5.4 Hz).

Pummerer reaction of 12 with N4 -benzoylcytosine 17. Add 9.3 mL anhydrous Et3 N (67 mmol) and 49 mL TMSOTf (268 mmol) to a suspension of 14.4 g N4 -benzoylcytosine (67 mmol) in 350 mL anhydrous toluene in a 1-L round-bottom flask with an argon balloon. Stir reaction mixture at room temperature under argon atmosphere until a two-phase, clear solution is obtained. 18. Add 200 mL anhydrous CH2 Cl2 to the solution, which will give a one-phase, clear solution. 19. Add entire solution to a solution of 25.6 g of 12 (44.7 mmol) in 200 mL anhydrous CH2 Cl2 in a 1-L round-bottom flask with an argon balloon dropwise over 3 hr via a cannula at room temperature. 20. Add an additional 28 mL of anhydrous Et3 N (201 mmol) in 100 mL anhydrous toluene via a cannula at room temperature. Stir mixture for 1 hr room temperature under an argon atmosphere. 21. Quench reaction by adding ice. 22. Evaporate the solvent in vacuo, and dilute with AcOEt (800 mL). Wash with H2 O (400 mL), three times with saturated aqueous NaHCO3 (400 mL each time), and brine (400 mL).

Biologically Active Nucleosides

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23. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness. 24. Absorb crude products on silica gel and purify by column chromatography (φ = 5 × 18−cm) using AcOEt/hexane (1:3 to 1:1) as the solvent. 25. Combine fractions containing the product. Evaporate solvent and dry the residue using a vacuum oil pump. 26. Characterize the product by TLC and 1 H NMR. 1-[2-O-(2,4-Dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4thio-β-D-ribofuranosyl]-N4 -benzoylcytosine (16). Yield of a white foam 22.9 g (67%). Rf : 0.17 (1:1 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 9.44 (brs, 1H), 8.57 (d, 1H, J = 7.6 Hz), 7.82 (d, 1H, J = 8.7 Hz), 5.66 (d, 1H, J = 3.7 Hz), 4.40 (dd, 1H, J = 3.7, 9.5 Hz), 4.14 (dd, 1H, J = 3.0, 12.8 Hz), 4.06 (d, 1H, J = 12.8 Hz), 3.83 (s, 6H), 3.74 (dd, 1H, J = 9.5, 3.0 Hz),2.24 (s, 3H), 1.14-0.87 (m, 28H).

Deprotection of protecting groups to give 4 -thiocytidine (18) 27. Dissolve 351 mg of 16 (0.46 mmol) in 10 mL MeOH in a 30-mL round-bottom flask and add 340 mg NH4 F (9.2 mmol). Stir reaction mixture for 12 hr under reflux. 28. Evaporate solvent in vacuo to give crude 17. 29. Add 20 mL NH3 /MeOH (saturated at 0°C), and allow mixture to stand for 24 hr at room temperature. Evaporate solvent in vacuo. 30. Absorb the crude products on silica gel and purify by column chromatography (φ = 2 × 10−cm) using MeOH in CHCl3 (5% to 25%) as the solvent. 31. Combine fractions containing the product. Evaporate solvent and dry the residue using a vacuum oil pump. 32. Characterize the product by TLC and 1 H NMR. 1-(4-Thio-β-D-ribofuranosyl)cytosine (18). Yield of a pale brown foam 75 mg (63%). Rf : 0.36 (1:1 v/v MeOH/CHCl3 ). 1 H NMR (DMSO-d6 ) δ: 7.94 (d, 1 H, J = 7.5 Hz), 7.16 and 7.12 (each br s, each 1 H), 5.92 (d, 1 H, J = 6.6 Hz), 5.78 (d, 1 H, J = 7.5 Hz), 5.30 (d, 1 H, J = 6.1 Hz), 5.14 (d, 1 H, J = 4.2 Hz), 5.10 (t, 1 H, J = 5.1 Hz), 4.04 (ddd, 1 H, J = 6.1, 6.6, 3.6 Hz), 3.96 (ddd, 1 H, J = 4.2, 3.6, 3.7 Hz), 3.61 (ddd, 1 H, J = 5.1, 6.3, 11.3 Hz), 3.53 (ddd, 1 H, J = 5.1, 5.6, 11.3 Hz), 3.19 (ddd, 1 H, J = 3.7, 6.3, 5.6 Hz). BASIC PROTOCOL 3

Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

PREPARATION OF 4 -THIOPURINE NUCLEOSIDES In contrast to reactions with pyrimidine bases, those with purine bases are more complex due to the possible formation of regioisomers, i.e., N-7 and the desired N-9 isomers (Fig. 14.12.3). In addition, screening of suitable purine bases for the Pummerer reaction is also required. Accordingly, the Pummerer reaction was attempted in the presence of various purine bases such as adenine, N6 -benzoyladenine, 6-chloropurine, and hypoxanthine. Among the nucleobases examined, the reaction with 6-chloropurine gave the simplest result, i.e., two separable compounds were obtained in 43% (N-7 isomer) and 3% (N-9 isomer) yields. Since the kinetic N-7 isomer is expected to rearrange into the N-9 isomer (Vorbr¨uggen and Bennua, 1981; Boryski, 1996), the Pummerer reaction was conducted first at room temperature then under reflux conditions to give the desired N-9 isomer as a major product. For the synthesis of 4 -thioguanosine, the Pummerer reaction with 2-amino-6-chloropurine was the best and the desired N-9 isomer was obtained under the same reaction conditions. NOTE: All glassware and equipment should be dried well prior to the Pummerer reaction.

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O

O

S

O

+

Cl ODMBz

N

N

HO

S

TIPDS O

N

N

N

19

S

TIPDS O

N

N

TIPDS O

O

N

HO

ODMBz

NH2

N

12

O

S

TIPDS O

N

N

N

+

Cl

N

O

ODMBz 23

O

S MeO O O

N

S

HO

OH 22

Cl N

N

NH2

HO

ODMBz 24

TIPDS O

N

N

HO

ODMBz

N

S

TIPDS O

N

21

20

N

N

N

S

Cl ODMBz

NH2

Cl

Cl N

N

N

O

N

S

HO

N

N NH2

HO

ODMBz 25

N S

HO

NH N

NH2

OH 26

OMe

CH2CN

27

Figure 14.12.3

Schema for the synthesis of 4 -thiopurine nucleosides.

NOTE: As described in Critical Parameters and Troubleshooting, formation of 27 together with N-7 and N-9 isomers is sometimes observed in the Pummerer reaction with purine bases. TLC and 1 H-NMR spectrum of 1,2-O-[nitrilemethyl-(2,4dimethoxyphenyl)]methylidene-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-4thio-α-D-ribofuranose (27) are below: Rf : 0.87 (1:1 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 7.73 (d, 1 H, J = 8.6 Hz), 6.47 (m, 2 H), 6.08 (d, 1 H, J = 5.5 Hz), 5.12 (dd, 1 H, J = 4.0, 5.5 Hz), 4.10 (dd, 1 H, J = 4.0, 9.9 Hz), 3.98 (dd, 1 H, J = 2.7, 12.7 Hz), 3.86, 3.82 (each s, each 3 H), 3.72 (d, 1 H, J = 12.7 Hz), 3.26, 3.09 (each d, each 1 H, J = 16.6 Hz), 3.04 (d, 1 H, J = 9.9 Hz), 1.14–0.96 (m, 28 H).

Materials Anhydrous triethylamine, 99% (Et3 N, Kishida Chemical) distilled from CaH prior to the reaction Trimethylsilyl trifluoromethanesulfonate (TMSOTf), 98% (TCI) 6-Chloropurine, 98% (TCI) dried well with P2 O5 at 50°C using a vacuum oil pump Anhydrous acetonitrile (CH3 CN), 98% (Wako Pure Chemical) distilled from P2 O5 ˚ molecular sieves and stored with 4 A Anhydrous 1,2-dichloroethane, 99.5% (ClCH2 CH2 Cl, Nacalai Tesque) distilled ˚ molecular sieves from P2 O5 and stored with 4 A Argon Ethyl acetate (AcOEt), 99% (Junsei Chemical) Saturated aqueous sodium bicarbonate (NaHCO3 ) Brine Anhydrous sodium sulfite (Na2 SO4 ), 99% (Kanto Chemical) Hexane Silica gel 60, 0.063 to 0.2 mm, 70 to 230 mesh (Kanto Chemical) Dry tetrahydrofuran (THF) Acetic acid, 99.7% (AcOH, Wako Pure Chemical) Tetrabutylammonium fluoride (TBAF), 1 M THF solution (TCI) Acetone, 99.5% (Wako Pure Chemical) Ethanolic ammonia (NH3 /EtOH), saturated NH3 at 0°C MeOH (Wako Pure Chemical) Chloroform (CHCl3 ) 2-Amino-6-chloropurine, 98% (Wako Pure Chemical) dried well with P2 O5 at 50°C using a vacuum oil pump

Biologically Active Nucleosides

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TLC plates with Merck silica gel 60 F254 2-Mercaptoethanol, 95% (Wako Pure Chemical) Sodium methoxide (NaOMe), 28% Hydrochloric acid, 37% (Kanto Chemical) 20-, 30-, and 500-mL round-bottom flasks Magnetic heat plate and bars 1-L two-neck flasks Cannula Rotary evaporator Chromatography columns (φ = 5 × 12−cm; φ = 1 × 6−cm; φ = 1 × 5−cm; φ = 5 × 10−cm) Vacuum oil pump 50-mL steel container Perform Pummerer reaction of 12 with 6-chloropurine 1. Add 3.8 mL Et3 N (27.3 mmol) and 10.7 mL TMSOTf (59.2 mmol) to a suspension of 5.6 g of 6-chloropurine (36.4 mmol) in 180 mL anhydrous CH3 CN and 90 mL anhydrous 1,2-dichloroethane in a 500-mL round-bottom flask with an argon balloon. Stir reaction mixture at room temperature under argon atmosphere until a clear solution is obtained. 2. Add entire solution to a solution of 5.2 g of 12 (9.1 mmol) in 90 mL anhydrous 1,2-dichloroethane in a 1-L two-neck flask with an argon balloon dropwise over 5 hr via a cannula at room temperature. 3. Add an additional 3.8 mL of Et3 N (27.3 mmol) in 45 mL anhydrous ClCH2 CH2 Cl dropwise over 3 hr via a cannula at room temperature. Heat entire mixture for 6 to 12 hr at 85°C under an argon atmosphere by monitoring isomerization from N-7 isomer 19 to N-9 isomer 20 by TLC. 4. Quench the reaction by adding ice. 5. Evaporate the solvent in vacuo, and dilute with AcOEt (400 mL). Wash with H2 O (250 mL), three times with saturated aqueous NaHCO3 (250 mL each time), and brine (250 mL). 6. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate solvent to dryness. 7. Absorb the crude products on silica gel and purify by column chromatography (φ = 5 × 12−cm) using AcOEt/hexane (1:3 to 1:1) as solvent. 8. Combine fractions containing the product. Evaporate the solvent and dry the residue using a vacuum oil pump to give 19 and desired 20. 9. Characterize the product by TLC and 1 H NMR. 6-Chloro-9-[2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3diyl)-4-thio-β-D-ribofuranosyl]purine (20). Yield of a white foam 2.49 g (39%). Rf : 0.59 (1:1 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 8.76 (s, 1 H), 8.59 (s, 1 H), 7.93 (d, 1 H, J = 8.8 Hz), 5.96 (m, 2H), 6.07 (s, 1 H), 5.84 (d, 1 H, J = 3.9 Hz), 4.96 (dd, 1 H, J = 3.9, 9.8 Hz), 4.56 (dd, 1 H, J = 2.9, 12.9 Hz), 4.12 (d, 1 H, J = 12.9 Hz), 3.90 (s, 3 H), 3.88 (s, 3 H), 3.86 (dd, 1 H, J = 9.8, 2.9 Hz), 1.16–0.89 (m, 28 H). Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

Chloro-7-[2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3-tetraisopropyldisiloxane-1,3diyl)-4-thio-β-D-ribofuranosyl]purine (19). Yield of a white foam 1.56 g (24%). Rf : 0.47 (1:1 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 9.11, 8.88 (each s, each 1 H), 7.82 (d, 1 H, J = 8.4 Hz), 6.45 (m, 2 H), 6.41 (s, 1 H), 5.83 (d, 1 H, J = 3.2 Hz), 4.60 (dd, 1 H,

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J = 3.2, 9.2 Hz), 4.19 (dd, 1 H, J = 3.2, 13.0 Hz), 4.10 (d, 1 H, J = 13.0 Hz), 3.86, 3.84 (each s, each 3 H), 3.82 (m, 1 H), 1.17–0.87 (m, 28 H).

Deprotect TIPDS group 10. Dissolve 52 mg of 20 (0.07 mmol) in 2 mL dry THF in a 20-mL round-bottom flask, and add 9 μL AcOH (0.15 mmol) and 150 μL TBAF (0.15 mmol). Stir the reaction mixture for 10 min at room temperature. 11. Evaporate the solvent in vacuo. 12. Pack a φ = 1 × 6–cm glass column with silica gel in AcOEt, and purify the residue by chromatography on silica gel using acetone/AcOEt (0:1 to 1:0) as the solvent. 13. Combine fractions containing the product. Evaporate solvent and dry the residue using a vacuum oil pump. 14. Characterize the product by TLC and 1 H NMR. 6-Chloro-9-[2-O-(2,4-dimethoxybenzoyl)-4-thio-β-D-ribofuranosyl]purine (21). Yield of a white solid 34 mg (99%). Rf : 0.63 (1:4 v/v MeOH/CHCl3 ). 1 H NMR (CDCl3 ) δ: 8.81, 8.34 (each s, each 1 H), 7.73 (d, 1 H, J = 8.6 Hz), 6.50 (m, 2 H), 6.27 (d, 1 H, J = 6.3 Hz), 6.23 (dd, 1 H, J = 6.3, 9.8 Hz), 4.81 (dd, 1 H, J = 9.8, 3.7 Hz), 4.27 (m, 1 H), 4.15 (m, 1 H, J = 3.2, 12.0 Hz), 4.01 (m, 1 H, J = 2.9, 12.0 Hz), 3.88, 3.85 (each s, each 3 H), 3.79 (ddd, 1 H, J = 3.7, 3.2, 2.9 Hz), 3.20 (d, 1 H, J = 3.2 Hz).

Convert to 4 -thioadenosine (22) 15. Dissolve 37 mg of 21 (0.08 mmol) in 5 mL NH3 /EtOH (saturated at 0°C), and heat mixture for 24 hr at 100°C in a 50-mL steel container. 16. Evaporate the solvent in vacuo. 17. Absorb crude products on a silica gel and purify by column chromatography (φ = 1 × 5−cm) using MeOH in CHCl3 (5% to 30%) as the solvent. 18. Combine fractions containing the product. Evaporate solvent and dry the residue using a vacuum oil pump. 19. Characterize the product by TLC and 1 H NMR. 1-(4-Thio-β-D-ribofuranosyl)adenine (22). Yield of a white solid 18 mg (83%). Rf : 0.12 (1:5 v/v MeOH/CHCl3 ). 1 H NMR (DMSO-d6 ) δ: 8.42, 8.12 (each s, each 1 H), 7.27 (br s, 2 H), 6.59 (d, 1 H, J = 6.6 Hz), 5.57 (d, 1 H, J = 6.1 Hz), 5.35 (d, 1 H, J = 4.6 Hz), 5.23 (t, 1 H, J = 5.6 Hz), 4.63 (ddd, 1 H, J = 6.1, 6.6, 3.4 Hz), 4.17 (ddd, 1 H, J = 4.6, 3.4, 3.4 Hz), 3.76 (ddd, 1 H, J = 5.6, 6.6, 11.2 Hz), 3.59 (ddd, 1 H, J = 5.6, 5.9, 11.2 Hz), 3.29 (ddd, 1 H, J = 3.4, 6.6, 5.9 Hz).

Perform Pummerer reaction of 12 with 2-amino-6-chloropurine 20. Add 3.3 mL Et3 N (23.8 mmol) and 9.2 mL TMSOTf (51 mmol) to a suspension of 2.3 g of 2-amino-6-chloropurine (13.6 mmol) in 140 mL anhydrous CH3 CN and 70 mL anhydrous 1,2-dichloroethane in a 500-mL round-bottom flask with an argon balloon. Stir reaction mixture at room temperature under argon atmosphere until clear solution is obtained. 21. Add entire solution to a solution of 3.9 g of 12 (6.8 mmol) in 70 mL anhydrous 1,2-dichloroethane in a 1-L two-neck flask with an argon balloon dropwise over 5 hr via a cannula at room temperature. 22. Add an additional 3.3 mL of Et3 N (23.8 mmol) in 35 mL anhydrous ClCH2 CH2 Cl dropwise over 3 hr via a cannula at room temperature. Heat entire mixture for 6 to 12 hr at 85°C under an argon atmosphere, monitoring isomerization from N-7 isomer 23 to N-9 isomer 24 by TLC.

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23. Quench reaction by adding ice. 24. Evaporate solvent in vacuo, and dilute with AcOEt (300 mL). Wash with H2 O (150 mL), three times with saturated aqueous NaHCO3 (150 mL each time), and brine (150 mL). 25. Dry organic layer over anhydrous Na2 SO4 , and filter off the drying agent. Evaporate the solvent to dryness. 26. Absorb the crude products on silica gel and purify by column chromatography (φ = 5 × 10−cm) using AcOEt/hexane (1:3 to 2:1) as the solvent. 27. Combine fractions containing the product. Evaporate solvent and dry the residue using a vacuum oil pump to give 23 and the desired 24. 28. Characterize the product by TLC and 1 H NMR. 2-Amino-6-chloro-9-[2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-D-ribofuranosyl]purine (24). Yield of a white foam 2.24 g (45%). Rf : 0.45 (1:1 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 8.25 (s, 1 H), 7.87 (d, 1 H, J = 8.4 Hz), 6.49 (m, 2 H), 5.84 (s, 1 H), 5.77 (d, 1 H, J = 3.6 Hz), 5.29 (br s, 2 H), 4.71 (dd, 1 H, J = 3.6, 9.5 Hz), 4.17 (dd, 1 H, J = 2.9, 12.6 Hz), 4.08 (d, 1 H, J = 12.6), 3.87 (s, 3 H), 3.84 (s, 3 H), 3.79 (m, 1 H), 1.13-0.86 (m, 28 H). 2-Amino-6-chloro-7-[2-O-(2,4-dimethoxybenzoyl)-3,5-O-(1,1,3,3tetraisopropyldisiloxane-1,3-diyl)-4-thio-β-D-ribofuranosyl]purine (23). Yield of a white foam 1.13 g (23%). Rf : 0.17 (1:1 v/v EtOAc/hexane). 1 H NMR (CDCl3 ) δ: 9.11, 8.88 (each s, each 1 H), 7.82 (d, 1 H, J = 8.4 Hz), 6.45 (m, 2 H), 6.41 (s, 1 H), 5.83 (d, 1 H, J = 3.2 Hz), 4.60 (dd, 1 H, J = 3.2, 9.2 Hz), 4.19 (dd, 1 H, J = 3.2, 13.0 Hz), 4.10 (d, 1 H, J = 13.0 Hz), 3.86, 3.84 (each s, each 3 H), 3.82 (m, 1 H), 1.17–0.87 (m, 28 H).

Deprotect TIPDS group 29. Dissolve 127 mg of 24 (0.18 mmol) in 10 mL dry THF in a 30-mL round-bottom flask, and add 20 μL AcOH (0.36 mmol) and 360 μL TBAF (0.36 mmol). Stir reaction mixture for 10 min at room temperature. 30. Evaporate solvent in vacuo. 31. Absorb the crude products on silica gel and purify by column chromatography (φ = 1 × 5−cm) using acetone/AcOEt (0:1 to 1:0) as the solvent. 32. Combine fractions containing the product. Evaporate solvent and dry the residue using a vacuum oil pump. 33. Characterize the product by TLC and 1 H NMR. 2-Amino-6-chloro-9-[2-O-(2,4-dimethoxybenzoyl)-4-thio-β-D-ribofuranosyl]purine (25). Yield of a white solid 84 mg (99%). Rf : 0.60 (1:5 v/v MeOH/CHCl3 ). 1 H NMR (DMSO-d6 ) δ: 8.55 (s, 1 H), 7.66 (d, 1 H, J = 9.3 Hz), 7.04 (br s, 2 H), 6.54 (m, 2 H), 6.14 (d, 1 H, J = 7.3 Hz), 5.75 (m, 2 H), 5.32 (br s, 1 H), 4.57 (m, 1 H), 3.86 (m, 1 H), 3.79 (s, 3 H), 3.72 (m, 1 H), 3.64 (s, 3 H), 3.43 (m, 1 H).

Convert to 4 -thioguanosine (26) 34. Dissolve 80 mg of 25 (0.17 mmol) in 10 mL MeOH in a 20-mL round-bottom flask, and add 47 μL of 2-mercaptoethanol (0.66 mmol) and 130 μL NaOMe (28% MeOH solution). Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

35. Heat the reaction mixture for 24 hr under reflux. 36. Neutralize with 1 N HCl, and evaporate solvent in vacuo. 37. Dissolve the residue in 10 mL H2 O, and wash with AcOEt (5 mL).

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38. Evaporate the aqueous layer to dryness, and crystalize the residue from H2 O. 39. Characterize the product by TLC and 1 H NMR. 1-(4-Thio-β-D-ribofuranosyl)guanosine (26). Yield of pale brown crystals 28 mg (55%). Rf : 0.03 (1:3 v/v MeOH/CHCl3 ). 1 H NMR (DMSO-d6 ) δ: 10.64 (br s, 1 H), 8.02 (s, 1 H), 6.50 (br s, 2 H), 5.65 (d, 1 H, J = 6.8 Hz), 5.48 (d, 1 H, J = 5.6 Hz), 5.28 (m, 1 H), 5.15 (t, 1 H, J = 5.4 Hz), 4.46 (ddd, 1 H, J = 5.6, 6.8, 3.4 Hz), 4.15 (m, 1 H), 3.72 (ddd, 1 H, J = 5.4, 7.1, 11.4 Hz), 3.53 (ddd, 1 H, J = 5.4, 5.9, 11.4 Hz), 3.24 (m, 1 H).

COMMENTARY Background Information Preparation of 4-thiosugar 4 -Thioribonucleosides are versatile units for not only 4 -thioRNA synthesis, but also for the preparation of 2 -deoxy and 2 -modified 4 thionucleoside derivatives (Inoue et al., 2005; Takahashi et al., 2009). Therefore, practical and large-scale synthesis of the 4-thiosugar is needed. Thus far, several groups reported its synthesis (Altenbach et al., 1997; Halila et al., 2002); however, no efficient and practical method applicable to large-scale synthesis was reported. For the first generation synthesis of the desired 4-thiosugar 9, 1-O-methyl 2,3,5-tri-Obenzyl-D-ribose (Barker and Fletcher, 1961) was used. However, a difficult reaction (BCl3 in CH2 Cl2 , < –90°C) was required for the deprotection of the benzyl groups in the last step. In addition, the Mitsunobu reaction, which is unsuitable for large-scale synthesis, was used for the inversion of the hydroxyl group (Naka et al., 2000). The former drawback was solved by using 1-O-allyl 2,3,5-tri-O-pmethoxybenzyl-D-ribose (2) rather than the 1O-methyl 2,3,5-tri-O-benzyl-D-ribose. Thus, the O-allyl group was selectively removed by treatment with PdCl2 in a mixture of CHCl3 H2 O under an O2 atmosphere (Mereyala and Reddy Lingannagaru, 1997). In addition, the PMB groups of 7 were easily removed by treatment with TFA at room temperature. The latter drawback was solved by an alternative SN 2 reaction, namely treatment of 4 with MsCl, followed by LiBr (Minakawa et al., 2002). The resulting 6 afforded the desired 7. Accordingly, a practical and large-scale synthesis of 4-thiosugar 7 was accomplished (33%) in eight steps with only three chromatographic purifications starting from D-ribose. To subject the coupling reaction with nucleobases, 7 has to be converted into an appropriate structure. Since the Pummerer reaction was used for the synthesis of 4 thioribonucleosides (see Basic Protocols 2 and 3), 7 was converted into the corresponding sulfoxide 12. In the oxidation of 11, 12 was

a diastereomeric mixture, i.e, 12-S and 12R. When the separated 12-R was subjected to the Pummerer reaction, 13 was obtained in good yield, while the reaction with 12-S was very poor (Naka et al., 2000). Thus, oxidation with ozone in CH2 Cl2 at −78°C, in which 12 was obtained with >16:1 R/S ratio, was adopted. Alternatively, oxidation using a Di Furia–Modena oxidation is also available to give predominantly 12-R (Dande et al., 2006). Pummerer reaction of 12 in the presence of nucleobases Since 4 -thioribonucleosides have an unnatural sugar, condensation of nucleobases with an appropritate 4-thiosugar is required for their preparation. Thus far, three types of reactions to give 4 -thioribonucleosides are known: (1) classical thioglycosidation with a 1-acetoxy-4thiosugar, (2) Pummerer reaction with a sufoxide, and (3) electrophilic glycosidation with 4-thiofuranoid glycal (Haraguchi et al., 2002). Among them, the method using the Pummerer reaction appears to be the most effective due to a higher reactivity of the corresponding glycosyl donor (Pummerer reaction versus classical thioglycosidation) and milder reaction conditions (Pummerer reaction versus electrophilic glycosidation with a 4-thiofuranoid glycal). To prepare the desired compounds βselectively, assistance from the neighboring C-2 acetoxy group is needed. However, the stereocontrol in the reaction with 4-thiosugar derivatives is unsatisfactory, unlike the normal glycosidation with ribofuranose derivatives (Bobek et al., 1975; Tiwari et al., 1994). A DMBz group on the 2-position was revealed to be the most effective for β-selective condensation (Naka et al., 1999). As described above, the reactions with purine bases are more complex due to the formation of regioisomers, i.e., N-7 and the desired N-9 isomers, than those with pyrimidine bases (Nishizono et al., 2008; Haraguchi et al., 2009). Accordingly, optimization of reaction conditions such as the purine base utilized, solvent, and reaction temperature is absolutely necessary. In the case presented in

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this unit, the Pummerer reaction, which used 6-chloropurine and 2-amino-6-chloropurine in a mixture of CH3 CN and ClCH2 CH2 Cl under reflux, afforded the best results to give the desired N-9 isomers.

Critical Parameters and Troubleshooting

Practical Synthesis of 4 Thioribonucleosides Starting from D-Ribose

Preparation of various compounds requires prior experience with standard chemical laboratory techniques such as the slow addition of chemicals using a dropping funnel or a cannula, solvent distillation, evaporation, extraction, TLC, and chromatography. Characterization of the products demands knowledge of 1 H NMR. General laboratory safety is also of primary concern when hazardous materials are involved. Especially, careful operation is required in the chemical conversion of 1 to 2, since a violent evolution of hydrogen takes place in the large-scale synthesis if one adds all of the PMBCl at once, then the reaction mixture warms to room temperature. In the synthesis of 7 from 4, formation of 8 was observed together with 7. This could be attributed to further substitution of 6 by the bromonium ion, and it is difficult to eliminate this over reaction on a large scale. Although 7 and 8 were obtained as an inseparable mixture, the undesired 8 was obtained as an oil, while the desired 7 was obtained as crystals. Therefore, the undesired 8 was removed by crystallization to give pure 7. To minimize the formation of 8, the reaction to give 6 should be quenched immediately after the consumption of the monobrominated product by TLC analysis, which is an intermediate of the reaction from 5 to 6. Unlike the Pummerer reaction with pyrimidine bases, those with purine bases require personal handling techniques as well as dryness and freshness of chemicals and solvents. One may face a problem of formation of 27 together with coupling products (see Basic Protocol 3). To avoid such a problem, the solution of Et3 N in anhydrous ClCH2 CH2 Cl should be added slowly. Another possible problem may be the difficulty to accomplish isomerization of the N-7 isomer (19 or 23) to the desired N-9 isomer (20 or 24). This problem is not solved by extending the heating time, which oppositely decreases the yields of the desired N-9 isomers. Rather, quench the reaction once to isolate the N-7 isomer as a major product, and subject it again under the conditions of the Pummerer reaction to give the N-9 isomer. Practice the reaction using 1 to 2 mmol of 12 prior to scaling up the experiment.

Time Considerations Large-scale synthesis of 9 starting from can be accomplished in 4 weeks. An additional 1 week is required to prepare 12, a common intermediate of the Pummerer reaction. The synthesis of free 4 thioribonucleosides (15, 18, 22, and 25) from 12 requires 3 days each. D-ribose

Literature Cited Altenbach, H.J., Brauer, D.J., and Merhof, G.F. 1997. Synthesis of 1-deoxy-4-thio-Dribose starting from thiophene-2-carboxylic acid. Tetrahedron 53:6019-6026. Barker, R. and Fletcher, H.G. 1961. 2,3,5-Tri-Obenzyl-D-ribosyl and -L-arabinosyl Bromides. J. Org. Chem. 26:4605-4609. Bellon, L., Barascut, J.L., Maury, G., Divita, G., Goody, R., and Imbach, J.L. 1993. 4 -Thio-oligo-beta-D-ribonucleotides: Synthesis of beta-4 -thio-oligouridylates, nuclease resistance, base pairing properties, and interaction with HIV-1 reverse transcriptase. Nucleic Acids Res. 21:1587-1593. Bobek, M., Bloch, A., Parthasarathy, R., and Whistler, R.L. 1975. Synthesis and biological activity of 5-fluoro-4 -thiouridine and some related nucleosides. J. Med. Chem. 18:784-787. Boryski, J. 1996. Transglycosylation reactions of purine nucleosides. A review. Nucleosides Nucleotides 15:771-791. Dande, P., Prakash, T.P., Sioufi, N., Gaus, H., Jarres, R., Berdeja, A., Swayze, E.E., Griffey, R.H., and Bhat, B. 2006. Improving RNA interference in mammalian cells by 4 -thio-modified small interfering RNA (siRNA): Effect on siRNA activity and nuclease stability when used in combination with 2 -O-alkyl modifications. J. Med. Chem. 49:1624-1634. Dyson, M.R., Coe, P.L., and Walker, R.T. 1992. An improved synthesis of benzyl 3,5-di-O-benzyl2-deoxy-1,4-dithio-D-erythro-pentofuranoside, an intermediate in the synthesis of 4 thionucleosides. Carbohydr. Res. 216:237-248. Halila, S., Benazza, M., and Demailly, G. 2002. New synthesis of alditol thiaheterocycles via ring closure of vicinal bis-cyclic thionocarbonates of alditols. Tetrahedron Lett. 43:815-818. Haraguchi, K., Takahashi, H., Shiina, N., Horii, C., Yoshimura, Y., Nishikawa, A., Sasakura, E., Nakamura, K.T., and Tanaka, H. 2002. Stereoselective synthesis of the beta-anomer of 4 thionucleosides based on electrophilic glycosidation to 4-thiofuranoid glycals. J. Org. Chem. 67:5919-5927. Haraguchi, K., Matsui, H., Takami, S., and Tanaka, H. 2009. Additive Pummerer reaction of 3,5O-(di-tert-butyl)silylene-4-thiofuranoid glycal: A high-yield and beta-selective entry to 4 thioribonucleosides. J. Org. Chem. 74:26162619. Hoshika, S., Minakawa, N., and Matsuda, A. 2004. Synthesis and physical

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and physiological properties of 4 -thioRNA: Application to post-modification of RNA aptamer toward NF-kappaB. Nucleic Acids Res. 32:3815-3825. Hoshika, S., Minakawa, N., Kamiya, H., Harashima, H., and Matsuda, A. 2005. RNA interference induced by siRNAs modified with 4 -thioribonucleosides in cultured mammalian cells. FEBS Lett. 579:3115-3118. Hoshika, S., Minakawa, N., Shionoya, A., Imada, K., Ogawa, N., and Matsuda, A. 2007. Study of modification pattern-RNAi activity relationships by using siRNAs modified with 4 thioribonucleosides. Chem. Biochem. 8:21332138. Inoue, N., Kaga, D., Minakawa, N., and Matsuda, A. 2005. Practical synthesis of 2 -deoxy-4 thioribonucleosides: Substrates for the synthesis of 4 -thioDNA. J. Org. Chem. 70:8597-8600. Inoue, N., Minakawa, N., and Matsuda, A. 2006. Synthesis and properties of 4 -ThioDNA: Unexpected RNA-like behavior of 4 -ThioDNA. Nucleic Acids Res. 34:3476-3483. Inoue, N., Shionoya, A., Minakawa, N., Kawakami, A., Ogawa, N., and Matsuda, A. 2007. Amplification of 4 -thioDNA in the presence of 4 thio-dTTP and 4 -thio-dCTP, and 4 -thioDNAdirected transcription in vitro and in mammalian cells. J. Am. Chem. Soc. 129:15424-15425. Kato, Y., Minakawa, N., Komatsu, Y., Kamiya, H., Ogawa, N., Harashima, H., and Matsuda, A. 2005. New NTP analogs: The synthesis of 4 thioUTP and 4 -thioCTP and their utility for SELEX. Nucleic Acids Res. 33:2942-2951. Kikuchi, Y., Yamazaki, N., Tarashima, N., Furukawa, K., Takiguchi, Y., Itoh, K., and Minakawa, N. 2013. Gene suppression via U1 small nuclear RNA interference (U1i) machinery using oligonucleotides containing 2 -modified-4 thionucleosides. Bioorg. Med. Chem. 21:52925296. Kojima, T., Furukawa, K., Maruyama, H., Inoue, N., Tarashima, N., Matsuda, A., and Minakawa, N. 2013. PCR amplification of 4 -thioDNA using 2 -deoxy-4 -thionucleoside 5 -triphosphates. ACS Synth. Biol. 2:529-536. Leydier, C., Bellon, L., Barascut, J.L., and Imbach, J.L. 1995. 4 -Thio-β-D-oligoribonucleotides: Nuclease resistance and hydrogen bonding properties. Nucleosides Nucleotides Nucleic Acids 14:1027-1030. Mereyala, H.B. and Reddy Lingannagaru, S. 1997. A study of catalysed Wacker reaction for the deprotection of prop-2-enyl and prop-1-enyl ethers. Tetrahedron 53:17501-17512. Minakawa, N., Kaga, D., Kato, Y., Endo, K., Tanaka, M., Sasaki, T., and Matsuda, A. 2002. Synthesis and structural elucidation of 1-(3-C-

ethynyl-4-thio-β-D-ribofuranosyl)cytosine (4 thioECyd). J. Chem. Soc. Perkin Trans. 1:21822189. Minakawa, N., Kato, Y., Uetake, K., Kaga, D., and Matsuda, A. 2003. An improved large scale synthesis of 1,4-anhydro-4-thio-D-ribitol. Tetrahedron 59:1699-1702. Minakawa, N., Sanji, M., Kato, Y., and Matsuda, A. 2008. Investigations toward the selection of fully-modified 4 -thioRNA aptamers: Optimization of in vitro transcription steps in the presence of 4 -thioNTPs. Bioorg. Med. Chem. 16:94509456. Mootoo, D.R., Konradsson, P., Udodong, U., and Fraser-Reid, B. 1988. Armed and disarmed n-pentenyl glycosides in saccharide couplings leading to oligosaccharides. J. Am. Chem. Soc. 110:5583-5584. Naka, T., Nishizono, N., Minakawa, N., and Matsuda, A. 1999. Nucleosides and nucleotides. 189. Investigation of the stereoselective coupling of thymine with meso-thiolane-3,4-diolL-oxide derivatives via the Pummerer reaction. Tetrahedron Lett. 40:6297-6300. Naka, T., Minakawa, N., Abe, H., Kaga, D., and Matsuda, A. 2000. The stereoselective synthesis of 4 -β-thioribonucleosides via the Pummerer reaction. J. Am. Chem. Soc. 122:7233-7243. Nishizono, N., Soma, K., Baba, R., Machida, M., and Oda, K. 2008. Synthesis of 4 -thiopurine nucleosides using hypervalent iodine compounds. Heterocycles 75:619-634. Takahashi, M., Minakawa, N., and Matsuda, A. 2009. Synthesis and characterization of 2 modified-4 -thioRNA: A comprehensive comparison of nuclease stability. Nucleic Acids Res. 37:1353-1362. Takahashi, M., Nagai, C., Hatakeyama, H., Minakawa, N., Harashima, H., and Matsuda, A. 2012. Intracellular stability of 2 -OMe-4 thioribonucleoside modified siRNA leads to long-term RNAi effect. Nucleic Acids Res. 40:5787-5793. Takahashi, M., Yamada, N., Hatakeyama, H., Murata, M., Sato, Y., Minakawa, N., Harashima, H., and Matsuda, A. 2013. In vitro optimization of 2 -OMe-4 -thioribonucleoside-modified antimicroRNA oligonucleotides and its targeting delivery to mouse liver using a liposomal nanoparticle. Nucleic Acids Res. 41:10659-10667. Tiwari, K.N., Secrist, J.A., and Montgomery, J.A. 1994. Synthesis and biological activity of 4 thionucleosides of 2-chloroadenine. Nucleosides Nucleotides 13:1819-1828. Vorbr¨uggen, H. and Bennua, B. 1981. Nucleoside syntheses, XXV1. A new simplified nucleoside synthesis. Chem. Ber. 114:12791286.

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