反应详情

温度

37 °C

2-Aminopurine (Pacific Chemical Laboratories, 0.3 g, 2.2 mmoles) and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)thymine (C. H. Tann et al., J. Org. Chem., 50:3647, 1985; 0.3 g; 1.2 mmoles) were suspended in 25 ml of 5 mM potassium phosphate buffer, pH 7.0, which contained 0.04% (w/v) potassium azide. Thymidine phosphorylase (12,000 I.U.) (T. A. Krenitsky, et. al, Biochemistry, 20:3615, 1981) was added and the suspension stirred at 37° C. On day 3, 5,500 I,U, of purine nucleoside phosphorylase (T. A. Krenitsky, et al., Biochemistry, 20:3615, 1981 and U.S. Pat. No. 4,381,444) was added. On day 18, the reaction was diluted to 200 ml with 5 mM potassium phosphate buffer, pH 7.0, which contained 0.4% (w/v) potassium azide. On day 26, 0.4 g of 2-aminopurine, 8.000 I.U. of thymidine phosphorylase, and 2,800 I.U. of purine phosphorylase were added. On day 41, 0.2 g of 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)thymidine, 8,000 I.U. of thymidine phosphorylase and 2,800 I.U. of purine nucleoside phosphorylase were added. On day 48, the reaction was diluted to 1,000 ml with 5 mM potassium phosphate buffer, pH 7.0 which contained 0.04% (w/v) potassium azide and 2.0 g of 2-aminopurine, 16,000 I.U. of thymidine phosphorylase and 5,500 I.U. purine nucleoside phosphorylase were added. On day 54, 1 g of 2-aminopurine, 8,000 I.U. of thymidine phosporylase, and 2,800 I.U. of purine nucleoside phosphorylase were added. On day 68, the suspension was evaporated to near dryness. Three volumes of ethanol were added to precipitate the protein and the suspension filtered. The title compound which was contained in the filtrate was purified by chromatography on AG1X2-hydroxide (Bio-Rad) with methanol/water (9/1) as the solvent followed by chromatography on silica gel with acetonitrile/water (95/5) as the solvent. Lyophilisation yielded 0.13 g of the title compound that analysed as a 0.3 hydrate.

1. additionKrenitsky, et. al, Biochemistry, 20:3615, 1981) was added
2. waitOn day 3, 5,500 I,U
3. addition4,381,444) was added
4. waitOn day 18
5. waitOn day 48
6. waitOn day 68
7. customthe suspension was evaporated
8. additionThree volumes of ethanol were added
9. customto precipitate the protein
10. filtrationthe suspension filtered
11. customwas purified by chromatography on AG1X2-hydroxide (Bio-Rad) with methanol/water (9/1) as the solvent