Efficient reductive desymmetrization of bulky 1,3-cyclodiketones enabled by structure-guided directed evolution of a carbonyl reductase

Reductive desymmetrization of 2,2-disubstituted prochiral 1,3-cyclodiketones to 2,2-disubstituted-3-hydroxycycloketones is a highly desired transformation for the construction of complex molecules with multiple chiral centres, but the generation of a single stereoisomer is difficult and an extremely challenging task in organic chemistry. In this study, by using ethyl secodione as the model substrate and an engineered carbonyl reductase from Ralstonia sp. as the biocatalyst, we realized the efficient reductive desymmetrization of 2,2-disubstituted cyclodiketones to give essentially one single stereoisomer. The mutant enzyme F12 (I91V/I187S/I188L/Q191N/F205A) showed an 183-fold enhancement of enzyme activity and outstanding stereoselectivity towards most of the tested prochiral 1,3-cyclodiketones. Crystal structural analysis and molecular dynamics studies reveal the molecular basis for activity improvement and the stereoselectivity control mechanism. Our results show that by altering the active site conformation populations (particularly the position of an alpha-helix) to properly accommodate the larger substrate and co-factor for catalysis, this challenging synthetic problem can ultimately be addressed.