Reversing the Enantioselectivity of Enzymatic Dynamic Kinetic Asymmetric Transformations in the Synthesis of Fused Lactones

The rational design of one ketoreductase into stereocomplementary variants for controlling the stereoselectivity of bulky chiral molecules bearing contiguous stereocenters is highly desirable and challenging. Herein, we report protein engineering of ketoreductase from Chryseobacterium sp. CA49 (ChKRED20) through targeted mutagenesis of only two key residues (Y188 and H145) located in the enzyme pocket, achieving the precise stereocontrol over the synthesis of tricyclic fused lactones (highest reversing enantioselectivity from >99:1 e.r. to <1:99 e.r.). Notably, both kinetic resolution asymmetric reduction (KR-AR) and dynamic kinetic asymmetric transformation (DyKAT) were observed in this system. In the KR-AR process, ChKRED20 variants exclusively convert (R)- or (S)-keto esters to corresponding enantio- and diastereoenriched (R,S)- or (S,R)-cis-lactones and deliver leftover (S)- or (R)-keto esters. On the contrary, in the DyKAT process, unreactive configurations of substrates undergo efficient equilibration via an enolization through protonation–deprotonation in enzymes. Computational studies are also conducted to get insight into the origin of stereoselectivity and enantioselectivity.