Uncovering Hydroxynitrile Lyase Variants with Promiscuous Diastereoselective Nitroaldolase Activity toward the Highly Stereocontrolled Synthesis of Anti β-Nitroalcohols

Diastereoselective Henry reaction (DHR) is an effective direct C–C bond ligation transformation that enables the synthesis of β-nitroalcohols having two contiguous chiral centers. Despite significant applications of optically active β-nitroalcohol diastereomers as key chiral intermediates for the synthesis of important pharmaceuticals and biologically active molecules, the biocatalytic asymmetric Henry reaction remains underdeveloped. Here, we show thatArabidopsis thaliana hydroxynitrile lyase (AtHNL) variants with single amino acid substitution, Y14C and Y14A, empower promiscuous DHR with a broad synthetic scope toward different aldehydes and nitroalkane and high enantio- and diastereoselectivity (up to >99% ee, >99% de, >99% ic, and ∼3470 total turnover number) in the production of diverse anti (1R,2S)-β-nitroalcohols. Y14C displayed a remarkable >250-fold increase in catalytic efficiency to that of the wild-type toward stereoselective nitroethane addition to benzaldehyde. A gram-scale biocatalysis was illustrated using readily available benzaldehyde and nitroethane followed by a one-step chemical reduction of the product to prepare l-norephedrine, a therapeutic, with >99% ee and de. The origin of the anti diastereoselectivity was probed using computational studies and isotope labeling experiments. This study describes the uncovering of variants providing a simple, sustainable, and economical biocatalytic platform for the synthesis of a repertoire of anti-β-nitroalcohol diastereomers of structural diversity with excellent stereocontrol.