醇脱氢酶TbSADH半理性设计影响合成重要医药中间体(S)-(4-氯苯基)吡啶-2-甲醇的研究

(S)-2-(4-Chlorophenyl)pyridylmethanol, an important medical intermediate in the synthesis of antihistamine drugs S-Carbinoxamine and Bepotastine besilate, is mainly synthesized by chemical methods which has resulted in lots of environmental problems. Since the poorly catalytic efficiency and stereoselectivity of reported biocatalysts cannot be applied to pharmaceutical synthesis, in this project, a new biocatalytic reaction intends to be designed which would convert achiral ketone to (S)-2-(4-Chlorophenyl)pyridylmethanol by an engineered alcohol dehydrogenase TbSADH that has been chosen due to its high thermostability. Assisted by molecular docking and molecular dynamic(MD) simulation based on X-ray structure, the key residues lining the binding pocket of TbSADH were chosen and subjected to engineering by Triple code saturation mutagenesis (TCSM) to accept and efficiently catalyze the substrate 4-chlorophenyl-2-pyridyl ketone to high ee chiral alcohol. X-ray crystal structures and MD simulation studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme and provide theoretical and practical guidance for same type of enzyme reaction and engineering.

(S)-(4-氯苯基)吡啶-2-甲醇是重要医药中间体，可用来合成抗组胺类药物卡比沙明和苯磺酸贝他斯汀。目前主要是由高污染的化学法合成。虽然有生物法研究，但其催化效率和立体选择远达不到用于药物合成的要求。本研究拟采用半理性设计定向进化新技术对耐热醇脱氢酶TbSADH进行改造，基于其晶体结构，通过分子模拟和对接分析，对TbSADH底物结合口袋氨基酸残基进行分析，寻找阻碍其催化前手性酮底物的位阻氨基酸和影响手性选择的关键残基，采用三密码子饱和突变方法（TCSM）对其进行设计和改造，使其接受二芳基类底物4-氯苯基-2-吡啶基-甲酮，不对称高效合成高ee值手性醇产物。通过突变体晶体结构解析和分子动力学模拟，解析底物结合口袋重塑与催化效率和立体选择之间的分子机制，为同类型催化反应和酶分子改造研究提供理论和实践指导。

(S)-(4-氯苯基)吡啶-2-甲醇是重要医药中间体，可以用来合成抗组胺类药物卡比沙明和苯磺酸贝他斯汀等。但目前主要是化学法合成，污染大，劳动保护要求高。虽然有生物法在研究，但其催化效率和立体选择性远远达不到用于药物合成的要求。本研究将采用半理性设计等定向进化新技术对耐热醇脱氢酶TbSADH进行改造，基于其晶体结构，通过分子动力学模拟和分子对接分析对工业感兴趣用酶嗜热醇脱氢酶TbSADH进行改造，建立“small but smart”高质量突变体文库及虚拟突变体筛选技术，有效减少筛选工作量，构建和筛选可以不对称催化还原目的底物(4-氯苯基)(2-吡啶基)甲酮为(S)-(4-氯苯基)吡啶-2-甲醇的高性能突变体，可实现(S)-型产物ee值>99%，转化率98%，具有一定的工业应用前景。

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