Enantioselective Synthesis of α-Hydroxy-β-Amino Ketones via Engineered ThDP-Dependent Enzyme Catalysis

Abstract

α-Hydroxy-β-amino ketones are key structural motifs in many bioactive compounds, yet their asymmetric synthesis remains challenging. Although numerous approaches for accessing simple amino alcohols are well established, methods specifically targeting the construction of the α-hydroxy-β-amino ketones motif have been rarely explored. Herein, we report a green biocatalytic approach to access these scaffolds via cross-benzoin condensation of α-amino aldehydes and aryl aldehydes using an engineered ThDP-dependent enzyme. Through protein engineering of benzaldehyde lyase from Pseudomonas fluorescens (PfBAL), the variant A28G was identified that efficiently catalyzes this transformation. Compared with the wild-type enzyme, which gave only 33% yield of the desired product along with 49% self-benzoin byproduct, the A28G mutant significantly improved the cross-coupling efficiency while reducing the homocoupling side reaction to just 3%. Under optimized aqueous conditions, the PfBAL A28G catalyst accommodated a broad range of aryl aldehydes bearing electron-withdrawing or electron-donating substituents, as well as heteroaromatic (furan, thiophene) and disubstituted derivatives, delivering the corresponding α-hydroxy-β-amino ketones in up to 98% isolated yields with excellent enantioselectivities (up to >99% ee). The practical utility of this method was demonstrated by gram-scale synthesis (1.13 g of product 3a) with 81% yield and retaining 96% ee. Molecular dynamics simulations revealed that the A28G mutation shortens the attack distance between the Breslow intermediate and the α-amino aldehyde acceptor from 6.4 Å to 4.5 Å, accounting for the enhanced reactivity. Moreover, differential binding of the product enantiomers to the active site explains the strict R-configuration control. This work not only provides a green, atom-economic, and highly stereoselective route to enantioenriched α-hydroxy-β-amino ketones under mild aqueous conditions but also expands the synthetic repertoire of ThDP-dependent enzymes to nitrogen-functionalized aldehyde acceptors, opening new avenues for biocatalytic C–C bond formation in pharmaceutical chemistry and natural product synthesis.

Keywords

Biocatalysis / Asymmetric synthesis / ThDP-dependent enzymes / Benzoin reaction / α-Hydroxy-β-amino ketones / Cross-benzoin reaction / Protein engineering / Stereoselectivity

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