ENGINEERING OF A KETOREDUCTASE TO PERFORM RADICAL DEHALOGENATION AND CHIRAL HYDROGEN ATOM TRANSFER

Abstract

Radical-mediated reactions are ubiquitous in the synthetic literature. Yet, despite their synthetic utility, methods for rendering these types of transformations asymmetric are rare. In this project, Lactobacillus Kefiri Alcohol Dehydrogenase, a nicotinamide-dependent ketoreductase, was engineered to perform a radical dehalogenation and chiral hydrogen atom transfer from its NADPH cofactor to ∝-halolactones, when irradiated with visible light. Site-saturated mutagenesis of active-site residues was used to expand the small-binding pocket of the wild-type ketoreductase. After three rounds of engineering, a variant was discovered to perform the light-induced dehalogenation in 72% yield and 96:4 er, favoring the formation of the (R)-enantiomer. This project reports the development of the first enzyme with a known protein sequence to perform a stereoselective light-induced radical reaction, providing scientists with an enzyme to be further developed for other synthetically useful dehalogenation reactions. Furthermore, this enzyme may also enable the performance of other enantioselective radical reactions.