微生物转化法制备普瑞巴林手性中间体

发布时间：2018-01-22 07:43

  本文关键词： 普瑞巴林 (S)-2-羧乙基-3-氰基-5-甲基已酸乙酯 假单胞菌 对映选择性 静息细胞　出处：《浙江大学》2011年硕士论文　论文类型：学位论文

【摘要】：普瑞巴林(Pregabalin),(S)-3-氨甲基-5-甲基己酸,是一种用于抗神经痛和癫痫症的辅助治疗的手性药物,其国际市场需求呈现较快增长之势。开展生物催化法制备普瑞巴林的研究,筛选拆分制备普瑞巴林的微生物,具有重要的应用前景和价值。 (S)-2-羧乙基-3-氰基-5-甲基己酸乙酯((S)-CCMAE)是合成普瑞巴林的关键手性中间体。本文首先以外消旋体2-羧乙基-3-氰基-5-甲基己酸乙酯(CCMAE)为底物,从土壤中筛选得到一株具有自主知识产权的能够对映选择性水解(R)-CCMAE的假单胞菌(Pseudomonas) CGMCC No.4184。对其产酶条件及培养基进行了优化,使酶活从15.92 U/L提高到33.43 U/L。随后对假单胞菌CGMCCNo.4184静息细胞水解拆分底物的反应条件进行了研究,确定了较优反应条件为：30～35℃,pH 7.5-8.5。并成功利用微生物静息细胞转化法制备了ee值达99.8%的(S)-CCMAE,这与目前报道的生物拆分工艺均有所不同。研究还发现Tween-80和TritonX-100可明显提高对映选择率E值和酶活。最后利用海藻酸钠-PVA复合载体法制备了假单胞菌CGMCC No.4184的固定化细胞,对其技术参数和反应条件作了优化,并利用所制得的固定化细胞制备ee值99%以上的(S)-CCMAE。该固定化细胞能够较好地进行重复利用。 本研究中筛选获得的高选择性R型水解酶可能是一种新酶,后续的基因克隆和表达、酶学性质研究工作正在进行中,为进一步改善酶的催化性能和拓展应用范围打下了良好的基础。
[Abstract]:Pregabalinine is a chiral drug used in the adjuvant treatment of neuralgia and epilepsy. The research on the preparation of PreBahrain by biocatalysis and the screening and resolution of microbes in the preparation of PreBahrain have important application prospect and value. Sh 2 carboxyethyl 3 cyano 5 methyl hexanoic acid ethyl Ester (S0. CCMAE). It is a key chiral intermediate for the synthesis of PreBahrain. Firstly, the racem-2-carboxyethyl-3-cyano-5-methyl-caproate ethyl ester (CCMAE) was used as the substrate. A Pseudomonas pseudomonas strain with independent intellectual property rights capable of enantioselective hydrolysis of RCMAE was obtained from soil. CGMCC No. 4184. the conditions of producing enzyme and the medium were optimized. The enzyme activity was increased from 15.92 U / L to 33.43 UL. The reaction conditions of CGMCCNo.4184 resting cell hydrolysis and resolution of substrate were studied. The optimum reaction conditions were determined as follows: 1: 30 ~ 35 鈩，

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