Tuning an Imine Reductase for the Asymmetric Synthesis of Azacycloalkylamines by Concise Structure-Guided Engineering

Jun Zhang, Daohong Liao, Rongchang Chen, Fangfang Zhu, Yaqing Ma, Lei Gao,Ge Qu, Chengsen Cui, Zhoutong Sun,* Xiaoguang Lei,* and Shu-Shan Gao*
Angew. Chem. Int. Ed. 2022, 61, e202201908.

Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm, and a significant increase in the e.e. value from 78% (R) to >99% (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.

Jun Zhang, Daohong Liao, Rongchang Chen, Fangfang Zhu, Yaqing Ma, Lei Gao,Ge Qu, Chengsen Cui, Zhoutong Sun,* Xiaoguang Lei,* and Shu-Shan Gao*

Angew. Chem. Int. Ed. 2022, 61, e202201908.
 

Although imine reductases (IREDs) are emerging as attractive reductive aminases (RedAms), their substrate scope is still narrow, and rational engineering is rare. Focusing on hydrogen bond reorganization and cavity expansion, a concise strategy combining rational cavity design, combinatorial active-site saturation test (CAST), and thermostability engineering was designed, that transformed the weakly active IR-G36 into a variant M5 with superior performance for the synthesis of (R)-3-benzylamino-1-Boc-piperidine, with a 4193-fold improvement in catalytic efficiency, a 16.2 °C improvement in Tm, and a significant increase in the e.e. value from 78% (R) to >99% (R). M5 exhibits broad substrate scope for the synthesis of diverse azacycloalkylamines, and the reaction was demonstrated on a hectogram-scale under industrially relevant conditions. Our study provides a compelling example of the preparation of versatile and efficient IREDs, with exciting opportunities in medicinal and process chemistry as well as synthetic biology.