2,2,2-trichloroethyl propanoate

反応条件: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine (1.0 g, 3.3 mmol) (prepared as described in European Patent Application No. 294 114) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 2 ml of trichloroethyl propionate (Trichloroethyl propionate was synthesized by addition of 19 ml of propionyl chloride (Aldrich) over 30 minutes to 19.1 ml of trichlorethanol (Aldrich) in 40 ml of pyridine at 0° C.). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O. 1H-NMR (200 MHz, CDCl3, 4.74 (s, 2H, Cl3CH2-), 2.49 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.21 (t, 3H, J=7.6 Hz, CH3CH2 CO2-). The reaction was initiated with 0.100 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot No. 38F-0356), which had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C., the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.76 g of the desired product as a white powder: m.p. 124° C.; TLC Rf =0.43 (silica gel; CH2Cl2 :CH3OH (9:1); UVλmax (ε, mM-1 cm-1) at pH 7.0, 278 nm (9.5). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine (1.0 g, 3.3 mmol) (prepared as described in European Patent Application No. 294 114) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 2 ml of trichloroethyl propionate (Trichloroethyl propionate was synthesized by addition of 19 ml of propionyl chloride (Aldrich) over 30 minutes to 19.1 ml of trichlorethanol (Aldrich) in 40 ml of pyridine at 0° C.). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O. 1H-NMR (200 MHz, CDCl3, 4.74 (s, 2H, Cl3CH2-), 2.49 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.21 (t, 3H, J=7.6 Hz, CH3CH2 CO2-). The reaction was initiated with 0.100 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot No. 38F-0356), which had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C., the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.76 g of the desired product as a white powder: m.p. 124° C.; TLC Rf =0.43 (silica gel; CH2Cl2 :CH3OH (9:1); UVλmax (ε, mM-1 cm-1) at pH 7.0, 278 nm (9.5). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine (1.0 g, 3.3 mmol) (prepared as described in European Patent Application No. 294 114) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 2 ml of trichloroethyl propionate (Trichloroethyl propionate was synthesized by addition of 19 ml of propionyl chloride (Aldrich) over 30 minutes to 19.1 ml of trichlorethanol (Aldrich) in 40 ml of pyridine at 0° C.). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O. 1H-NMR (200 MHz, CDCl3, 4.74 (s, 2H, Cl3CH2-), 2.49 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.21 (t, 3H, J=7.6 Hz, CH3CH2 CO2-). The reaction was initiated with 0.100 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot No. 38F-0356), which had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C., the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.76 g of the desired product as a white powder: m.p. 124° C.; TLC Rf =0.43 (silica gel; CH2Cl2 :CH3OH (9:1); UVλmax (ε, mM-1 cm-1) at pH 7.0, 278 nm (9.5). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-b-D-arabinofuranosyl-9H-purine (1.0 g, 3.3 mmol) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 1 ml of trichloroethyl propionate (Trichloroethyl acetate was synthesized as follows:2,2,2-trichloroethanol (19.1 mL, 197.1 mmole) and dry pyridine (40 mL) were placed in a three-neck, round-bottom flask equipped with argon inlet valve, thermometer, dropping funnel, magnetic stirring, and ice/H2O bath. Acetyl chloride (14.5 mL, 199.8 mmole) was placed in the dropping funnel and added over a ten minute period, keeping the temperature below 25° C. while stirring under argon. The resulting product was washed with H2O (2×100 mL), 5% NaHCO3 (2×100 mL), and H2O (2×100 mL). The organic layer was dried over MgSO4, then filtered through Whatman #1 paper, and distilled under vacuum. A middle cut of 5.18 g was the desired material, contaminated with a small amount of acetic acid: 1H-NMR (CDCl3): δ4.73 (s, 2H, CH2O), 2.20 (s, 3H, CH3CO); MS (Cl, CH4): m/z 197 (M+H, C4H5O237Cl3), 195 (M+H, C4H5O237Cl235Cl), 193 (M+H, C4H5O2O35Cl237Cl), 191 (M+H, C4H5O235Cl3), 159 (195-HCl, C4H4O237Cl2), 157 (193-HCl, C4H4O237Cl35Cl), 155 (191-HCl, C4H4O235Cl2); (El): m/z 195 (M+H), 193 (M+H), 191 (M+H), 157 (193-HCl), 155 (191-HCl). Analysis for C4H5Cl3O2 +0.054 mole CH3COOH: C, 25.35; H, 2.70; CI, 54.62. Found: C, 25.57; H, 2.72; Cl, 54.66.). The reaction was initiated with 0.050 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot#38F-0356,), which had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C. an additional 50 mg of subtilisin and 2 mL of trichloroethyl acetate were added to the reaction. After stirring at 40° for an additional 24 hours the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.28 g of the desired product as a white powder. TLC Rf =0.35 (silica gel; CH2Cl2 :CH3OH (9:1)); UV λmax (ε, mM-1 cm-1) at pH 7.0, 279 nm (8.8). 1H-NMR(200 MHz, DMSO-d6): δ7.83 (s, 1H, H8), 6.45 (s, 2H, 2-NH2), 6.14(d, 1H, J=3.7 Hz, H1'), 5.75 (d, 1H, J=4.5 Hz, 2'--OH), 5.65 (d, 1H, J=3.7 Hz, 3'--OH), 4.26(m, 2H, H2' and H3'), 4.07 (m, 2H, H5') 3.94 (s, 3H, --OCH3) 3.92 (m, 1H, H4'), 2.01 (s, 3H, CH3CO2--); MS (Cl) 340 (M+1), 280 (M--CH3CO2). Anal. Calcd. for C14H19N5O6.0.52 H2O: Calcd: C, 44.77; H, 5.22; N, 20.12. Found: C, 44.79; H, 5.21; N, 20.09. was synthesized equipped with argon inlet valve added over a ten minute period the temperature below 25° C. The resulting product was washed with H2O (2×100 mL), 5% NaHCO3 (2×100 mL), and H2O (2×100 mL) The organic layer was dried over MgSO4 filtered through Whatman #1 paper distilled under vacuum were added to the reaction After stirring at 40° for an additional 24 hours the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-b-D-arabinofuranosyl-9H-purine (1.0 g, 3.3 mmol) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 1 ml of trichloroethyl propionate (Trichloroethyl acetate was synthesized as follows:2,2,2-trichloroethanol (19.1 mL, 197.1 mmole) and dry pyridine (40 mL) were placed in a three-neck, round-bottom flask equipped with argon inlet valve, thermometer, dropping funnel, magnetic stirring, and ice/H2O bath. Acetyl chloride (14.5 mL, 199.8 mmole) was placed in the dropping funnel and added over a ten minute period, keeping the temperature below 25° C. while stirring under argon. The resulting product was washed with H2O (2×100 mL), 5% NaHCO3 (2×100 mL), and H2O (2×100 mL). The organic layer was dried over MgSO4, then filtered through Whatman #1 paper, and distilled under vacuum. A middle cut of 5.18 g was the desired material, contaminated with a small amount of acetic acid: 1H-NMR (CDCl3): δ4.73 (s, 2H, CH2O), 2.20 (s, 3H, CH3CO); MS (Cl, CH4): m/z 197 (M+H, C4H5O237Cl3), 195 (M+H, C4H5O237Cl235Cl), 193 (M+H, C4H5O2O35Cl237Cl), 191 (M+H, C4H5O235Cl3), 159 (195-HCl, C4H4O237Cl2), 157 (193-HCl, C4H4O237Cl35Cl), 155 (191-HCl, C4H4O235Cl2); (El): m/z 195 (M+H), 193 (M+H), 191 (M+H), 157 (193-HCl), 155 (191-HCl). Analysis for C4H5Cl3O2 +0.054 mole CH3COOH: C, 25.35; H, 2.70; CI, 54.62. Found: C, 25.57; H, 2.72; Cl, 54.66.). The reaction was initiated with 0.050 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot#38F-0356,), which had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C. an additional 50 mg of subtilisin and 2 mL of trichloroethyl acetate were added to the reaction. After stirring at 40° for an additional 24 hours the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.28 g of the desired product as a white powder. TLC Rf =0.35 (silica gel; CH2Cl2 :CH3OH (9:1)); UV λmax (ε, mM-1 cm-1) at pH 7.0, 279 nm (8.8). 1H-NMR(200 MHz, DMSO-d6): δ7.83 (s, 1H, H8), 6.45 (s, 2H, 2-NH2), 6.14(d, 1H, J=3.7 Hz, H1'), 5.75 (d, 1H, J=4.5 Hz, 2'--OH), 5.65 (d, 1H, J=3.7 Hz, 3'--OH), 4.26(m, 2H, H2' and H3'), 4.07 (m, 2H, H5') 3.94 (s, 3H, --OCH3) 3.92 (m, 1H, H4'), 2.01 (s, 3H, CH3CO2--); MS (Cl) 340 (M+1), 280 (M--CH3CO2). Anal. Calcd. for C14H19N5O6.0.52 H2O: Calcd: C, 44.77; H, 5.22; N, 20.12. Found: C, 44.79; H, 5.21; N, 20.09. was synthesized equipped with argon inlet valve added over a ten minute period the temperature below 25° C. The resulting product was washed with H2O (2×100 mL), 5% NaHCO3 (2×100 mL), and H2O (2×100 mL) The organic layer was dried over MgSO4 filtered through Whatman #1 paper distilled under vacuum were added to the reaction After stirring at 40° for an additional 24 hours the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine (1.0 g, 3.3 mmol) (prepared as described in European Patent Application No. 294 114) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 2 ml of trichloroethyl propionate (Trichloroethyl propionate was synthesized by addition of 19 ml of propionyl chloride (Aldrich) over 30 minutes to 19.1 ml of trichlorethanol (Aldrich) in 40 ml of pyridine at 0° C.). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O. 1H NMR (200 MHz, CDCl3, 4.74 (s, 2H, Cl3CH2-), 2.49 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.21 (t, 3H, J-7.6 Hz, CH3CH2CO2-). The reaction was initiated with 0.100 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot No. 38F-0356), whch had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C., the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.76 g of the desired product as a white powder: m.p. 124° C.; TLC Rf =0.43 (silica gel; CH2Cl2 :CH3OH (9:1); UV λmax (ε, mM-1 cm-1) at pH 7.0, 2.78 nm (9.5). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine (1.0 g, 3.3 mmol) (prepared as described in European Patent Application No. 294 114) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 2 ml of trichloroethyl propionate (Trichloroethyl propionate was synthesized by addition of 19 ml of propionyl chloride (Aldrich) over 30 minutes to 19.1 ml of trichlorethanol (Aldrich) in 40 ml of pyridine at 0° C.). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O. 1H NMR (200 MHz, CDCl3, 4.74 (s, 2H, Cl3CH2-), 2.49 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.21 (t, 3H, J-7.6 Hz, CH3CH2CO2-). The reaction was initiated with 0.100 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot No. 38F-0356), whch had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C., the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.76 g of the desired product as a white powder: m.p. 124° C.; TLC Rf =0.43 (silica gel; CH2Cl2 :CH3OH (9:1); UV λmax (ε, mM-1 cm-1) at pH 7.0, 2.78 nm (9.5). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine (1.0 g, 3.3 mmol) (prepared as described in European Patent Application No. 294 114) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 2 ml of trichloroethyl propionate (Trichloroethyl propionate was synthesized by addition of 19 ml of propionyl chloride (Aldrich) over 30 minutes to 19.1 ml of trichlorethanol (Aldrich) in 40 ml of pyridine at 0° C.). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O. 1H NMR (200 MHz, CDCl3, 4.74 (s, 2H, Cl3CH2-), 2.49 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.21 (t, 3H, J-7.6 Hz, CH3CH2CO2-). The reaction was initiated with 0.100 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot No. 38F-0356), whch had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C., the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.76 g of the desired product as a white powder: m.p. 124° C.; TLC Rf =0.43 (silica gel; CH2Cl2 :CH3OH (9:1); UV λmax (ε, mM-1 cm-1) at pH 7.0, 2.78 nm (9.5). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-b-D-arabinofuranosyl-9 H-purine (1.0 g, 3.3 mmol) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 1 ml of trichloroethyl propionate (Trichloroethyl acetate was synthesized as follows: 2,2,2-trichloroethanol (19.1 mL, 197.1 mmole) and dry pyridine (40 mL) were placed in a three-neck, round-bottom flask equipped with argon inlet valve, thermometer, dropping funnel, magnetic stirring, and ice/H2O bath. Acetyl chloride (14.5 mL, 199.8 mmole) was placed in the dropping funnel and added over a ten minute period, keeping the temperature below 25° C. while stirring under argon. The resulting product was washed with H2O (2×100 mL), 5% NaHCO3 (2×100 mL), and H2O (2×100 mL). The organic layer was dried over MgSO4, then filtered through Whatman #1 paper, and distilled under vacuum. A middle cut of 5.18 g was the desired material, contaminated with a small amount of acetic acid: 1H-NMR (CDCl3): δ4.73 Cs, 2 H, CH2O), 2.20 Cs, 3 H, CH3CO); MS (Cl, CH4): m/z 197 (M+H, C4H5O237Cl3), 195 (M+H, C4H5O237Cl235Cl), 193 (M+H, C4H5O2O35Cl237Cl), 191 (M+H, C4H5O235Cl3), 159 (195-HCl, C4H4O237Cl2), 157 (193-HCl, C4H4O237Cl35Cl), 155 (191-HCl, C4H4O235Cl2); (EI): m/z 195 (M+H), 193 (M+H), 191 (M+H), 157 (193-CHl), 155 (191-HCl). Analysis for C4H5Cl3O2 +0.054 mole CH3COOH: C, 25.35; H, 2.70; Cl, 54.62. Found: C, 25.57; H, 2.72; Cl, 54.66.). The reaction was initiated with 0.050 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot#38F-0356,), which had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C. an additional 50 mg of subtilisin and 2 mL of trichioroethyl acetate were added to the reaction. After stirring at 40° for an additional 24 hours the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.28 g of the desired product as a white powder. TLC Rf =0.35 (silica gel; CH2Cl2 :CH3OH (9:1)); UV λmax (.di-elect cons., mM-1 cm-1) at pH 7.0, 279 nm (8.8). 1H-NMR (200 MHz, DMSO-d6): δ7.83 (s, 1 H, H8), 6.45 (s, 2 H, 2-NH2), 6.14 (d, 1 H, J=3.7 Hz, H1'), 5.75 (d, 1 H, J=4.5 Hz, 2'-OH), 5.65(d, 1 H, J=3.7 Hz, 3'-OH), 4.26 (m, 2 H, H2' and H3'), 4.07 (m, 2 H, H5') 3.94 (s, 3 H, --OCH3) 3.92 (m, 1 H, H4'), 2.01 (s, 3 H, CH3CO2--); MS (Cl) 340 (M+1), 280 (M-CH3CO2). was synthesized equipped with argon inlet valve added over a ten minute period the temperature below 25° C. The resulting product was washed with H2O (2×100 mL), 5% NaHCO3 (2×100 mL), and H2O (2×100 mL) The organic layer was dried over MgSO4 filtered through Whatman #1 paper distilled under vacuum were added to the reaction After stirring at 40° for an additional 24 hours the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine (1.0 g, 3.3 mmol) (prepared as described in European Patent Application No. 294 114) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 2 ml of trichloroethyl propionate (Trichloroethyl propionate was synthesized by addition of 19 ml of propionyl chloride (Aldrich) over 30 minutes to 19.1 ml of trichlorethanol (Aldrich) in 40 ml of pyridine at 0° C.). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O. 1H NMR (200 MHz, CDCl3 , 4.74 (s, 2H, Cl3CH2-), 2.49 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.21 (t, 3H, J=7.6 Hz, CH3CH2 CO2-). The reaction was initiated with 0.100 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot No. 38F-0356), whch had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C., the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.76 g of the desired product as a white powder: m.p. 124° C.; TLC Rf =0.43 (silica gel; CH1Cl2 :CH3OH (9:1)); UV λmax (ε, mM-1 cm-1) at pH 7.0, 278 nm (9.5). 1H NMR (200 MHz, DMSO-d6), δ 7.83(s, 1H, H8, 6.44 (s, 2H, 2-NH2), 6.14 (s, 1H, H1'), 5.75 (d, 1H, J=4.3 Hz, 2'-OH), 5.65 (d, 1H, J=3.5 Hz, 3'-OH), 4.28 (m, 2H, H2' and H3'), 4.08 (m, 2H, H5'), 3.95 (s, 3H, --OCH3), 3.91 (m, 1H, H4 '), 2.32 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.01 (t, 3H, J=7.5 Hz, CH3CH2 CO2-); MS (ci) 354 (M+1), 280 (M--C2H5CO2). Anal. Calcd. for C14H19N5O6.0.46 H2O: Calcd: C, 46.49; H, 5.55; N, 19.36 Found: C, 46.46; H, 5.52; N. 19.45. The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:40.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-(β-D-arabinofuranosyl)-9H-purine (1.0 g, 3.3 mmol) (prepared as described in European Patent Application No. 294 114) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 2 ml of trichloroethyl propionate (Trichloroethyl propionate was synthesized by addition of 19 ml of propionyl chloride (Aldrich) over 30 minutes to 19.1 ml of trichlorethanol (Aldrich) in 40 ml of pyridine at 0° C.). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O. 1H-NMR (200 MHz, CDCl3, 4.74 (s, 2H, Cl3CH2-), 2.49 (q, 2H, J=7.6 Hz, CH3CH2CO2-), 1.21 (t, 3H, J=7.6 Hz, CH3CH2 CO2-). The reaction was initiated with 0.100 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot No. 38F-0356), which had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C., the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.76 g of the desired product as a white powder: m.p. 124° C.; TLC Rf =0.43 (silica gel; CH2Cl2 :CH3OH (9:1); UVλmax (ε, mM-1 cm-1) at pH 7.0, 278 nm (9.5). The product was purified by successive washing with 2×100 ml aliquots of H2O, 5% NaHCO3, and H2O by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: 2-Amino-6-methoxy-9-b-D-arabinofuranosyl-9H-purine (1.0 g, 3.3 mmol) was suspended in 40 ml of pyridine that contained 300 μL of H2O and 1 ml of trichloroethyl propionate (Trichloroethyl acetate was synthesized as follows:2,2,2-trichloroethanol (19.1 mL, 197.1 mmole) and dry pyridine (40 mL) were placed in a three-neck, round-bottom flask equipped with argon inlet valve, thermometer, dropping funnel, magnetic stirring, and ice/H2O bath. Acetyl chloride (14.5 mL, 199.8 mmole) was placed in the dropping funnel and added over a ten minute period, keeping the temperature below 25° C. while stirring under argon. The resulting product was washed with H2O (2×100 mL), 5% NaHCO3 (2×100 mL), and H2O (2×100 mL). The organic layer was dried over MgSO4, then filtered through Whatman #1 paper, and distilled under vacuum. A middle cut of 5.18 g was the desired material, contaminated with a small amount of acetic acid: 1H-NMR (CDCl3): δ4.73 (s, 2H, CH2O), 2.20 (s, 3H, CH3CO); MS (Cl, CH4): m/z 197 (M+H, C4H5O237Cl3), 195 (M+H, C4H5O237Cl235Cl), 193 (M+H, C4H5O2O35Cl237Cl), 191 (M+H, C4H5O235Cl3), 159 (195-HCl, C4H4O237Cl2), 157 (193-HCl, C4H4O237Cl35Cl), 155 (191-HCl, C4H4O235Cl2); (El): m/z 195 (M+H), 193 (M+H), 191 (M+H), 157 (193-HCl), 155 (191-HCl). Analysis for C4H5Cl3O2 +0.054 mole CH3COOH: C, 25.35; H, 2.70; CI, 54.62. Found: C, 25.57; H, 2.72; Cl, 54.66.). The reaction was initiated with 0.050 g of subtilisin (Sigma Chemical Co., St. Louis, Mo., P-5380, lot#38F-0356,), which had been activated by dissolving 1 g of the enzyme in 20 ml of 0.1M potassium phosphate at pH 7.8 and lyophilizing to dryness. After stirring for 23 hours at 40° C. an additional 50 mg of subtilisin and 2 mL of trichloroethyl acetate were added to the reaction. After stirring at 40° for an additional 24 hours the reaction was quenched by filtering off the enzyme and the solvent was removed in vacuo. The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant. Product fractions were pooled and lyophilized from water to yield 0.28 g of the desired product as a white powder. TLC Rf =0.35 (silica gel; CH2Cl2 :CH3OH (9:1)); UV λmax (ε, mM-1 cm-1) at pH 7.0, 279 nm (8.8). 1H-NMR(200 MHz, DMSO-d6): δ7.83 (s, 1H, H8), 6.45 (s, 2H, 2-NH2), 6.14(d, 1H, J=3.7 Hz, H1'), 5.75 (d, 1H, J=4.5 Hz, 2'--OH), 5.65 (d, 1H, J=3.7 Hz, 3'--OH), 4.26(m, 2H, H2' and H3'), 4.07 (m, 2H, H5') 3.94 (s, 3H, --OCH3) 3.92 (m, 1H, H4'), 2.01 (s, 3H, CH3CO2--); MS (Cl) 340 (M+1), 280 (M--CH3CO2). Anal. Calcd. for C14H19N5O6.0.52 H2O: Calcd: C, 44.77; H, 5.22; N, 20.12. Found: C, 44.79; H, 5.21; N, 20.09. was synthesized equipped with argon inlet valve added over a ten minute period the temperature below 25° C. The resulting product was washed with H2O (2×100 mL), 5% NaHCO3 (2×100 mL), and H2O (2×100 mL) The organic layer was dried over MgSO4 filtered through Whatman #1 paper distilled under vacuum were added to the reaction After stirring at 40° for an additional 24 hours the reaction was quenched by filtering off the enzyme the solvent was removed in vacuo The crude product was purified by chromatography on a 4.5×25 cm silica gel column with CH2Cl2 :CH3OH (9:1) as eluant