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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 anticoagulant bivalirudin using Wang resin and Fmoc protection strategy. Methods: The synthesis of bivalirudin was subjected to a series of steps such as stepwise coupling, TFA cleavage, deprotection and HPLC purification. In order to optimize the reaction conditions, different degrees of amino acid substitution resin (0.29, 0.48, 0.59 mmol / g) and different condensation reagents (HBTU, DCC, HATU) were selected for the experiment. Results: Bivalirudin was successfully obtained at a purity of 98%. The activity assay showed that the bivalirudin synthesized by different methods had anticoagulant activity comparable to that of standard samples and showed that: ① Wang resin with amino acid substitution degree of 0.3 ~ 0.4mmol / g can obtain products with high yield and purity, Substitution degree is too large (> 0.5mmol / g) to get the product by-product, low purity; ② with HBTU / HOBt as a condensing reagent has the advantages of convenient operation, low cost, high synthesis efficiency. Based on the above experimental results, the optimal conditions were selected, and 5gWang resin was used to synthesize bivalirudin of gram level. The yield of crude product was up to 84% and the purity was up to 68%. Conclusion: The effects of different amino acid substitution degree, different condensation reagents and size on the yield, purity and activity of synthetic bivalirudin were investigated by solid-phase synthesis. The large amount of synthesis of bivalirudin has a certain guiding role .