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 sp. (DERA ) toward the heteroaryl aldehydes through a rational protein engineering approach. We successfully performed iterative saturation mutagenesis of DERA , resulting in two enantiocomplementary variants, viz., ( )-selective DERA -S185G and ( )-selective DERA -T12I/S185A, with enhanced catalytic efficiencies and enantioselectivities. Remarkably, the natural enantioselectivity of DERA 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 ( )- and ( )-enantiomers of heteroaryl aldols were isolated with high yields (up to 99%) and excellent enantiopurities (up to 99:1 e.r.).