整细胞酶促合成抗HIV新药D-D4FC

Enzymatic Synthesis of D-D4FC Using Intact Cells

D-D4FC(β-D-2′,3′-双脱氢双脱氧-5-氟胞嘧啶核苷)是一种新型抗HIV病毒的核苷类药物,目前正在美国、法国和德国进行Ⅱ期临床。利用乳酸杆菌提取的粗制N-脱氧核糖转移酶实现了由D4T(β-D-2′,3′-双脱氢双脱氧-胸苷,司他夫定)和5-FC(5-氟胞嘧啶)合成D-D4FC,转化率达到25%。现在,发现利用乳酸杆菌整细胞也可实现此反应,其转化率经过12.5h可达到50%,更有利于可能的工业化连续生产。研究了整细胞催化合成D-D4FC反应中,pH值、缓冲液类型、底物浓度、加菌量、反应时间等条件的影响并进行了优化,探讨了反应中乳酸杆菌整细胞催化的可能机理。

D-D4FC （β-D-2＇ ,3＇-didehydro-2＇ ,3＇-dideoxy-5-fluorocytidine）, a new anti-HIV drug, is on its Phase 11 clinical trials in America, France and Germany. Our lab has synthesized D-D4FC successfully using N-deoxyribosyhransferase from Lactobacinus helveticto catalyzing the ribose transfer from D4T （β-D-2＇, 3＇-unsaturated thymidine） to 5-FC （5-fluorocytidine）. The yield of D-D4FC reached 25 %. We discovered the reaction could also be done by using intact cells. The yield could increase to 50% in 12.5 hours and more convenient to industrial continuous process. In this paper, the conditions including pH, buffer, substrates concentration, cells amount, reaction time and a possible catalytic mechanism were studied and discussed.