Flipping the Substrate Creates a Highly Selective Halohydrin Dehalogenase for the Synthesis of Chiral 4-Aryl-2-oxazolidinones from Readily Available Epoxides

Chiral oxazolidinones are a class of important heterocyclic compounds in pharmaceutical chemistry due to their biological activity. Halohydrin dehalogenase-catalyzed epoxide ring-opening reaction with cyanate offers an attractive approach to the synthesis of chiral oxazolidinones, but the α/β-regioselectivity and stereoselectivity are still un-addressed issues. In this study, a unique halohydrin dehalogenase (AbHheG) was found to have high activity and α/β-regioselectivity toward the ring opening of racemic styrene oxide with cyanate but with poor stereoselectivity (E < 3). By reshaping the substrate-binding site of AbHheG, a variant Y15M/N182S was obtained with excellent α/β-regioselectivity and stereoselectivity. The variant showed E > 200 for 9 of the 13 tested styrene oxides. Since (R)-4-aryl-2-oxazolidinones were easily separated from (R)-styrene oxides, both (R)- and (S)-4-aryl-2-oxazolidinones could be readily prepared. Crystallographic and enzyme–substrate docking analysis showed that flipping of the substrate in the binding site resulted in the R-configuration substrate being away from the catalytic triad in the mutant, which was responsible for the enhanced α/β-regioselectivity and stereoselectivity. This work has demonstrated that halohydrin dehalogenase is a useful biocatalyst for the synthesis of both enantiomers of 4-aryl-2-oxazolidinones from readily available racemic styrene oxides. The structural and computational studies provide a guidance for further engineering of halohydrin dehalogenases to control the α/β-regioselectivity and stereoselectivity for the efficient synthesis of the desired optically pure 4- or 5-substituted 2-oxazolidinones.