环己胺氧化酶催化的右美沙芬关键中间体绿色合成研究

右美沙芬是全球使用最广泛的非处方中枢性镇咳药，当前我国的右美沙芬主要依赖进口。反应可容许的底物浓度低和反应规模小是环己胺氧化酶催化合成右美沙芬关键中间体（S）-1-（4-甲氧苄基）-1，2，3，4，5，6，7，8-八氢异喹啉（(S)-11）方法的技术瓶颈，而本课题拟解决的关键科学问题：环己胺氧化酶对大位阻底物11的催化活力低下，是产生该技术瓶颈的主要原因。本课题拟通过结合使用基于CAST/ISM、B-FIT的半理性设计手段和基于易错PCR/DNA改组的非理性设计手段定向进化改造环己胺氧化酶CHAOCCH12-C2，获得对11催化活力显著提升的热稳定突变体酶；拟将建立的酶催化方法应用于(S)-11及其类似物的高底物浓度、大规模合成。通过本课题研究，可望在理论上助力阐明环己胺氧化酶的分子作用机制；在应用上为我国自主、高效、经济、绿色生产右美沙芬奠定坚实基础，具有重大经济与社会价值。

Dextromethorphan is the most widely used over-the-counter centrally acting antitussive worldwide. Currently, dextromethorphan in our country is mainly dependent on import. The viability of only low substrate concentration and small reaction scale is the technical bottleneck in the cyclohexylamine oxidase (CHAO)-catalyzed synthesis of (S)-1-(4-methoxybenzyl)-1,2,3,4,5,6,7,8-octahydroisoquinoline ((S)-11), the key synthetic intermediate to dextromethorphan. The low catalytic activity of CHAO towards the sterically demanding substrate 11 is the main reason causing this bottleneck, and it is also the key scientific problem that our program aims to address. In order to obtain a mutant enzyme with much improved catalytic activity towards substrate 11 and with high thermostability, this program aims to engineer the cyclohexylamine oxidase CHAOCCH12-C2 by directed evolution, specifically through the combined use of the semi-rational design approach based on CAST/ISM and B-FIT, and the non-rational design approach based on epPCR and DNA shuffling. The developed enzymatic method will be applied to the synthesis of (S)-11 and its derivatives in a high-substrate-concentration and large-scale manner. In the theoretical aspect, it is hoped that the proposed research can help elucidate the molecular mechanism of cyclohexylamine oxidase. In the application aspect, it is believed that the proposed research will lay a solid foundation to help our country realize its own production of dextromethorphan in an efficient, economic, and green manner, thereby possessing great economic and societal value.