Semi-rational engineering an aldo-keto reductase for stereocomplementary reduction of α-keto amide compounds

Enantio-pure alpha-hydroxy amides are valuable intermediates for the synthesis of chiral pharmaceuticals. The asymmetric reduction of alpha-keto amides to generate chiral alpha-hydroxy amides is a difficult and challenging task in biocatalysis. In this study, iolS, an aldo-keto reductase from Bacillus subtilis 168 was exhibited as a potential biocatalyst, which could catalyze the reduction of diaryl alpha-keto amide such as 2-oxo-N, 2-diphenyl-acetamide (ONDPA) with moderate S-selectivity (76.1%, ee) and 60.5% conversion. Through semi-rational engineering, two stereocomplementary variants (I57F/F126L and N21A/F126A) were obtained with ee value of 97.6% (S) and 99.9% (R) toward ONDPA (1a), respectively, delivering chiral alpha-hydroxy amide with > 98% conversions. Moreover, the excellent S- and R-preference variants displayed improved stereoselectivities toward the other alpha-keto amide compounds. Molecular dynamic and docking analysis revealed that the two key residues at 21 and 126 were identified as the switch, which specifically controlled the stereopreference of iolS by regulating the shape of substrate binding pocket as well as the substrate orientation. Our results offer an effective strategy to obtain alpha-hydroxy amides with high optical purity and provide structural insights into altering the stereoselectivity of AKRs.

Automated summary

In this study, iolS from Bacillus subtilis 168 was identified as a potential biocatalyst for the asymmetric reduction of alpha-keto amides to yield chiral alpha-hydroxy amides. Through semi-rational engineering, stereocomplementary variants with high enantioselectivity were obtained, providing an effective strategy for obtaining alpha-hydroxy amides with high optical purity and offering insights into altering the stereoselectivity of AKRs.