The Directed Evolution of Halogenases for Late-Stage Functionalization of Bioactive Molecules

Halogenation is a ubiquitous and vital process in the development of pharmaceuticals; an estimated quarter of all pharmaceuticals and agrochemicals are halogenated, and halogenation plays an integral role in the synthesis of many more. Despite this, the prevalent methods of halogenation suffer from a lack of site-selectivity and harsh reaction conditions, making the production of halogenated drug candidates difficult and environmentally unfriendly. To this end I have been working on the development of enzyme catalysts which perform site-selective halogenations of drug candidates in environmentally sustainable conditions. My preliminary work focused on improving the expression of natural halogenating enzymes in E. coli, particularly one called RebH, and exploring the scope and selectivity of wild-type RebH on a range of substrates on the preparative scale. My work now is focused on the directed evolution of RebH to further enhance its activity and expand its substrate scope with two primary goals: first, the halogenation of biologically active substrates, with a focus on bacterial biofilm inhibitors, in order to alter their biological properties; and second, the enantioselective halogenation of prochiral symmetrical compounds. Through the introduction of mutations in the genetic code for RebH, a variety of slightly altered RebH mutants are obtained and screened for changes in activity. Improved variants are characterized and further altered, thus recapitulating the process of evolution at a much accelerated pace in our laboratory. This research will both elucidate the role various mutations have in altering the activity of RebH and produce RebH variants useful for the development of novel drug candidates.