酰亚胺酶法制备普瑞巴林中间体(R)-3-异丁基戊二酸单酰胺Preparation of pregabalin intermediate (R)-isobutylglutarate monoamide with imidase
蔡青峰,覃文平,杨仲毅,徐龙,汪怡璐,方嘉怡,芦银华
CAI Qingfeng,QIN Wenping,YANG Zhongyi,XU Long,WANG Yilu,FANG Jiayi,LU Yinhua
摘要(Abstract)：
目的 利用重组酰亚胺酶催化3-异丁基戊二酸亚胺(3-isobutylglutaric anhydride, IBI)的去对称化水解，制备普瑞巴林的重要中间体(R)-3-异丁基戊二酸单酰胺[(R)-3-isobutyl glutaric acid monoamide,(R)-IBM]。方法 构建酰亚胺酶BpIH基因工程菌pT67/BL21(DE3),并在摇瓶和3 L发酵罐上进行表达，细胞均质后用于底物IBI的转化反应，提取及精制得到产物(R)-IBM,并测定其色谱纯度、对映体过剩值和相对分子质量。结果与结论酰亚胺酶BpIH得到了较好的表达，3 L发酵罐中的酶活达到了1 880 mU·L~(-1),利用该酶液进行IBI的转化，产物(R)-IBM的质量浓度达到了49.11 g·L~(-1),转化率和提取收率分别达到了90.9%和68.74%,产物的对映体过量值达到了99.96%,该工艺路线已经具备了替代常规酶拆分路线的基础。
Objective To prepare(R)-isobutylglutarate monoamide[(R)-IBM],an important intermediate of pregabalin, by the asymmetric hydrolysis of isobutylglutarate imine(IBI) with recombinant imidase.Methods Recombinant bacteria E.coli pT67/BL21(DE3) of imidase BpIH was constructed and expressed in shake flask and 3 L fermentor.After homogenization, it was used to transform the substrate IBI.The product(R)-IBM was extracted and refined.The chromatographic purity, enantiomeric excess and molecular weight of the product were determined.Results and Conclusions The imidase BpIH was well expressed, and the enzyme activity in 3 L fermentor reached 1 880 mU·L~(-1).The concentration of(R)-IBM reached 49.11 g·L~(-1) in the reaction, the conversion of IBI and extraction yield of(R)-IBM reached 90.9% and 68.74%,respectively.The enantiomeric excess value of product reached 99.96%.This process has the basis to replace the conventional enzyme resolution route.
关键词(KeyWords)：
酰亚胺酶;生物转化;普瑞巴林;去对称化
imidase;biotransformation;pregabalin;desymmetry
基金项目(Foundation):
作者(Author):
蔡青峰,覃文平,杨仲毅,徐龙,汪怡璐,方嘉怡,芦银华
CAI Qingfeng,QIN Wenping,YANG Zhongyi,XU Long,WANG Yilu,FANG Jiayi,LU Yinhua
DOI: 10.14066/j.cnki.cn21-1349/r.2021.0840
参考文献(References)：
- [1] SILVERMAN R B.From basic science to blockbuster drug:the discovery of lyrica [J].Angew Chem Int Ed Engl,2008,47(19):3500-3504.
- [2] CHEN X W.Research on the synthetic process of pregabalin [J].Home Medicine (家庭医药),2020(3):337.
- [3] XIONG F,CHEN L,ZHU Y J,et al.Research advance on the synthetic process of pregabalin [J].Chinese Journal of Synthetic Chemistry(合成化学),2021,29(2):148-165.
- [4] YANG D Y,WANG J,LIU M Y.Application of lipase in the preparation of pregabalin [J].Chinese Journal of Modern Applied Pharmacy (中国现代应用药学),2017,34(3):479-482.
- [5] XU F,CHEN S,XU G,et al.Discovery and expression of a Pseudomonas sp.esterase as a novel biocatalyst for the efficient biosynthesis of a chiral intermediate of pregabalin [J].Biotechnol Bioprocess Eng,2015,20(3):473-487.
- [6] LI E J,TANG X L,WU Z M,et al.Synthesis of (S)-3-cyano-5-methylhexanoic acid catalyzed by recombinant nitrilase [J].Chinese Journal of Bioprocess Engineering (生物加工过程),2019,17(1):105-109.
- [7] CHEN Z,ZHAO J,JIANG S,et al.Recent research advancements on regioselective nitrilase:fundamental and applicative aspects [J].Appl Microbiol Biotechnol,2019,103:6393-6405.
- [8] NOJIRI M,UEKITA K,OHNUKI M,et al.Microbial asymmetric hydrolysis of 3-substituted glutaric acid diamides [J].J Appl Microbiol,2013,115:1127-1133.
- [9] OGAWA J,SOONG C L,HONDA M,et al.Novel metabolic transformation pathway for cyclic imides in Blastobacter sp.strain A17p-4 [J].Appl Environ Microbiol,1996,62:3814-3817.
- [10] OGAWA J,SOONG C L,HONDA M,et al.Imidase,a dihydropyrimidinase-like enzyme involved in the metabolism of cyclic imides [J].Eur J Biochem,1997,243:322-327.
- [11] NOJIRI M,HIBI M,SHIZAWA H,et al.Imidase catalyzing desymmetric imide hydrolysis forming optically active 3-substituted glutaric acid monoamides for the synthesis of gamma-aminobutyric acid (GABA) analogs [J].Appl Microbiol Biotechnol,2015,99:9961-9969.