固相合成抗凝血药比伐卢定及合成条件的优化  

Solid-phase synthesis of bivalirudin and its method optimization

作　　者：沈忱[1] 刘建宁[1] 王莹[1] 邓华玲[1] 唐艳春[1] 

机构地区：[1]南京大学分子医学研究所,江苏南京210093

出　　处：《南京医科大学学报（自然科学版）》2010年第9期1282-1286,共5页Journal of Nanjing Medical University(Natural Sciences)

基　　金：南京大学人才引进项目资助

摘　　要：目的:研究使用Wang树脂与Fmoc保护策略固相合成抗凝血药比伐卢定(bivalirudin)的实验方法。方法:比伐卢定的合成经过了逐步偶联、TFA裂解、脱保护、HPLC纯化等一系列步骤。为了优化反应条件,选择了不同氨基酸取代度的树脂(0.29、0.48、0.59mmol/g)和不同的缩合试剂(HBTU、DCC、HATU)进行试验。结果:成功得到了纯度为98%的比伐卢定。活性测定表明,不同方法合成的比伐卢定均具有与标准样品相当的抗凝活性,并且显示:①氨基酸取代度为0.3～0.4mmol/g的Wang树脂可以得到产率及纯度均较高的产品,取代度太大(>0.5mmol/g)得到的产品副产物多、纯度低;②用HBTU/HOBt为缩合试剂具有操作方便、成本经济、合成效率高等优点。根据以上实验结果选择优化条件,用5gWang树脂合成克级单位的比伐卢定,得到的粗品产率可达84%,纯度可达68%以上。结论:通过固相合成的方法,考察了不同氨基酸取代度、不同缩合试剂及规模大小对合成比伐卢定的产率、纯度及活性的影响,对比伐卢定的大量合成具有一定的指导作用。Objective：To study the solid-phase synthesis of the thrombin inhibitor bivalirudin using Wang Resin and Fmoc-protection strategy.Methods：Bivalirudin was synthesized through the process of coupling reaction,TFA cleavage /deprotection and HPLC purification.Different Fmoc-Leu substitution resins（0.29 mmol/g,0.48 mmol/g,0.59 mmol/g） and different coupling reagents（HBTU,DCC,HATU） were used to synthesize the bivalirudin.Results：The purity of bivalirudin could be 〉98%.The bioactive assay showed that the synthetic bivalirudin had the same extent anticoagulant activity as the standard sample.The results showed：① the high Fmoc-Leu substitution resin（〉0.5 mmol /g） results in the low purity product,which was also difficult to purify;the substitution 0.30.4 mmol/g of Fmoc-Leu-Wang Resin was recommended;②using HBTU/HOBt as the coupling reagent had the advantages of convenient operation,economic and high efficiency.Following the results above,the optimized condition was used to synthesize gram grade of the Bivalirudin using 5 g Fmoc-Leu-Wang Resin.The crude product with the 84% total yield and 68% purity was obtained.Conclusion：The solid-phase synthesis method was used to evaluate the effect of different Fmoc-Leu substitution resins,different coupling reagents and different synthesis size on the total yield,purity and activity of bivalirudin.This research has instruction for the synthesis of bivalirudin in large amount.

关 键 词：凝血酶 固相合成 比伐卢定 缩合试剂 取代度 

分 类 号：Q516[生物学—生物化学]