反应详情

温度

45 °C

2-Amino-6-propylthio-9H-purine (0.29 g 1.4 mmoles) and 2',3'-dideoxy-3'-fluorouridine (0.39 g, 1.7 mmoles) were suspended in potassium phosphate buffer (10 mM) 90 ml), pH 6.8, containing 0.04% potassium azide. Purified purine nucleoside phosphorylase (370 I.U., PNP) and thymidine phosphorylase (TPase) (Krenitsky et al., Biochemistry, 20, 3615 (1981) and U.S. Pat. No. 4,381,444) adsorbed on to DEAE-cellulose were added to The reaction mixture and the suspension stirred at 45° C. After 2 days, additional PNP (370 I.U.) and TPase adsorbed onto DEAE-cellulose was added. The next day the reaction was filtered to remove the immobilized protein and fresh PNP (2700 I.U.) and TPase (1000 I.U.) was added. After 4 days, additional free PNP (2700 I.U.) and TPase (1000 I.U.) was added to the reaction mixture. After 3 days, PNP (5400 I.U.) and TPase (2000 I.U.) were added. After a total of 14 days, solvent was removed in vacuo and the residue was flash chromatographed on silica gel (2.5×23 cm) using CH2Cl2 :MeOH (97:3). Product-containing fractions were pooled and rechromatographed on a second silica gel column (2.5×20 cm) using CHCl3 :MeOH (199:1). Solvent was removed and lyophilization yielded 2-amino-6-propylthio-9-(2,3-dideoxy-3-fluoro-β-D-erythro-pentofuranosyl)-9H-purine (0.14 g).

1. additionwere added to The reaction mixture
2. additionwas added
3. filtrationThe next day the reaction was filtered
4. customto remove the immobilized protein and fresh PNP (2700 I.U.) and TPase (1000 I.U.)
5. additionwas added
6. waitAfter 4 days
7. additionadditional free PNP (2700 I.U.) and TPase (1000 I.U.) was added to the reaction mixture
8. waitAfter 3 days
9. additionPNP (5400 I.U.) and TPase (2000 I.U.) were added
10. customAfter a total of 14 days, solvent was removed in vacuo
11. customchromatographed on silica gel (2.5×23 cm)
12. customrechromatographed on a second silica gel column (2.5×20 cm)
13. customSolvent was removed