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.

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.

To maintain genomic integrity and avoid diseases, the DNA damage response (DDR) not only detects and repairs DNA lesions, but also contributes to the resistance to DNA-damaging chemotherapeutics. Targeting the DDR plays a significant role in drug discovery using the principle of synthetic lethality. The incomplete current knowledge of the DDR encouraged us to develop new strategies to identify and study its components and pathways. Polycarcin V, belonging to the C-aryl glycoside natural products, is a light-activatable DNA intercalating agent which causes DNA damage by forming a covalent [2+2] cycloadduct with thymine residue under 365–450 nm light irradiation in a DNA sequence independent manner. Taking advantage of the light-activatable feature and temporal control of DDR, we designed and synthesized polycarcin V-based bifunctional chemical probes, including one that crosslinks DNA to DNA-binding protein to explore the DDR induced by polycarcin V and uncover novel DNA-protein interactions. Utilizing this chemical probe and ABPP-SILAC, we identified 311 DNA-binding proteins, including known DDR factors and additional proteins that may be of interest in discovering new biology. We validated our approach by showing that our probe could specifically crosslink proteins involved in nucleotide excision repair (NER) that repair bulky DNA adducts. Our studies showed that the [2+2] cycloadduct formed by polycarcin V could indeed be repaired by NER in vivo. As a DNA damaging agent, polycarcin V or its drug-like derivative plus blue light showed promising properties for psoriasis treatment, suggesting that it may itself hold promise for clinic applications.