Protein engineering of carbonyl reductase for asymmetric synthesis of ticagrelor precursor (1S)-2-chloro-1-(3,4-difluorophenyl)ethanol

(1S)--2-chloro-1-(3,4-difluorophenyl)ethanol((S)-CFPL) is the key chiral intermediate of antiplatelet inhibitor ticagrelor. An NADPH-dependent carbonyl reductase KmCR from Kluyveromyces marxianus ZJB14056 displayed enantioselective reduction activity toward 2-chloro-1-(3,4-difluorophenyl)ethanone (CFPO) with moderate stereoselectivity (ee(p) =80.3%, S). Key residues controlling the stereoselectivity of KmCR were identified, guided by our previous research; saturation mutation and combinatorial mutation were performed to enhance the stereoselectivity of KmCR toward CFPO, generating mutant KmCR-A129W/V239N (KmCR-W-2) with strict enantioselectivity (ee(p )> 99.7%, S). Next, a triple mutant KmCR-A129W/V239N/V268A (KmCR-W-3, eep > 99.7%, S) with 83 % increase in catalytic activity was obtained by regional error-prone PCR (epPCR). The enantiose-lective reduction of (S)-CFPL was achieved by using KmCR coupled with the glucose dehydrogenase EsGDH from Exiguobacterium sibiricum to recycle NADPH. The best mutant KmCR-W-3 was able to transform 30 g/L CFPO completely with strict enantioselectivity (ee(p) > 99.7%, S, yield=95.5%). Moreover, the substrate scope of KmCR was extended. Although there is much space for improvement in the catalytic performance of KmCR-W3, our work enriches the knowledge of carbonyl reductase's structure and function relationships.

Automated summary

This study reports a carbonyl reductase capable of enantioselective reduction of 2-chloro-1-(3,4-difluorophenyl)ethanone and improves its stereoselectivity and catalytic activity through mutation and PCR techniques. Furthermore, the synthesis of the key chiral intermediate of the antiplatelet inhibitor ticagrelor was successfully achieved by coupling with other enzymes, and the substrate scope of the carbonyl reductase was expanded.