树干毕赤酵母不对称还原合成光学纯托伐普坦  

Asymmetric reduction synthesis of optically pure tolvaptan by Pichia stipites

作　　者：韩孟楠 杜洁[1] 王旭明 张芳华 张超 裴朝红 张红蕾[1] 李玮[1] HAN Mengnan;DU Jie;WANG Xuming;ZHANG Fanghua;ZHANG Chao;PEI Chaohong;ZHANG Honglei;LI Wei(College of Chemistry and Environmental Sciences,Laboratory of Medicinal Chemistry and Molecular Diagnosis of the Ministry of Education,Hebei University,Baoding 071002,China)

机构地区：[1]河北大学化学与环境科学学院,药物化学与分子诊断教育部重点实验室,保定071002

出　　处：《应用与环境生物学报》2020年第1期160-166,共7页Chinese Journal of Applied and Environmental Biology

基　　金：2014年河北省高等学校高层次人才科学研究项目(GCC2014013);河北大学自然科学研究计划项目(3333112);2016河北省自然科学基金专项(B2016201031);河北大学研宄生创新资助项目(hbu2019ss007)资助。

摘　　要：外消旋体托伐普坦是治疗重度低钠血症和等容量性低钠血症的关键药物,而光学纯托伐普坦在动物体内具备不同的药代性能,目前尚未有微生物全细胞催化托伐普坦前手性酮(PK1)还原生成光学纯托伐普坦的报道.拟建立微生物合成光学纯托伐普坦的催化体系,对催化过程中所使用的菌种、有机溶剂进行选择,并对有机相与水相比例、菌体浓度、底物浓度和转化时间进行优化.结果显示:本试验选取的5种酵母菌均可将PK1催化还原为S型为主的托伐普坦,选定其中具有较高转化率和e.e.值的树干毕赤酵母作为转化菌株.从多种不同log P值的有机相中筛选出大豆油作为两相催化系统的有机相.通过优化发现两相体系中大豆油与水相的体积比为1:9时为最佳油水比例.当菌体浓度为1.0 g/mL,底物浓度为10.0 mg/mL时转化率显著提高.同时,发现全细胞两相催化体系中产物的积累与时间呈线性关系,并在72 h转化率达到最大值.在以上最佳催化条件下全细胞的转化率可达74.5%,产物e.e.为99.0%.本研究建立的大豆油-水的两相催化体系是一种还原催化难溶性前手性酮底物生成光学纯手性醇类化合物的有效方法.Racemic tolvaptan is a key drug in the treatment of severe hyponatremia and isovolumic hyponatremia,while enantiopure tolvaptan has different drug metabolic properties in animals.Presently,there are no reports of microbial whole-cell catalytic reduction of prochiral ketone(PK1)to optically pure tolvaptan.This study proposes to establish a catalytic system for the synthesis of optically pure tolvaptan by microorganisms.The strains and organic solvents in the catalytic system were selected.The ratio of organic solvents to water,the concentration of bacterial cells,the concentration of substrates,and the conversion time were optimized.All five tested strains exhibited catalytic activities on the reduction of PK1 to S-tolvaptan.Pichia stipites with high conversion rate and ee value were selected for further research.Soybean oil is considered as the best solvent in a two-phase system for various organic phases with different log P values.It was found that a ratio of 1:9 for organic phase to aqueous phase was the most efficient for bioconversion in a two-phase system.When the concentration of the whole cell was 1.0 g/mL and the substrate concentration was 10.0 mg/mL,the conversion rate significantly increased.Furthermore,the accumulation of products in the whole-cell two-phase system showed a linear relationship with time.The conversion efficiency reached the maximum value at 72 h.Under the optimum catalytic conditions,the bioconversion efficiency of the whole cell reached 74.5%with an ee value of 99.0%.The soybean oil-water two-phase system is an effective method for catalyzing and reducing insoluble prochiral ketone substrates into valuable chiral alcohols.

关 键 词：树干毕赤酵母 不对称还原 两相催化 光学纯托伐普坦 

分 类 号：R915[医药卫生—微生物与生化药学]