Enantiodivergent Synthesis of Both (R)- and (S)-Heteroaryl Aldols by Rationally Engineered Aldolases

Aldolases, especially 2-deoxyribose-5-phosphate aldolase (DERA) enzymes, have been widely employed to access key chiral precursors for various active pharmaceutical ingredients (APIs). This has been enabled by expanding their substrate scope toward non-natural acceptors and donors via protein engineering. In this study, we endeavored to broaden the acceptor substrate scope of DERA from Geobacillus sp. (DERA_Geo) toward the heteroaryl aldehydes through a rational protein engineering approach. We successfully performed iterative saturation mutagenesis of DERA_Geo, resulting in two enantiocomplementary variants, viz., (R)-selective DERA_Geo-S185G and (S)-selective DERA_Geo-T12I/S185A, with enhanced catalytic efficiencies and enantioselectivities. Remarkably, the natural enantioselectivity of DERA_Geo was reversed by a single mutation (S185G). The synthetic applicability of the mutants was demonstrated by conducting aldol reactions on a semipreparative scale, from which both (R)- and (S)-enantiomers of heteroaryl aldols were isolated with high yields (up to 99%) and excellent enantiopurities (up to 99:1 e.r.).