Directed Evolution of a Ketone Synthase for Efficient and Highly Selective Functionalization of Internal Alkenes by Accessing Reactive Carbocation Intermediates

The direct regioselective oxidation of internal alkenes to ketones could simplify synthetic routes and solve a longstanding challenge in synthesis. This reaction is of particular importance because ketones are predominant moieties in valuable products as well as crucial intermediates in synthesis. Here we report the directed evolution of a ketone synthase that oxidizes internal alkenes directly to ketones with several thousand turnovers. The evolved ketone synthase benefits from more than a dozen crucial mutations, most of them distal to the active site. Computational analysis reveals that all these mutations collaborate to facilitate the formation of a highly reactive carbocation intermediate by generating a confined, rigid and preorganized active site through an enhanced dynamical network. The evolved ketone synthase fully exploits a catalytic cycle that has largely eluded small molecule catalysis and consequently enables various challenging functionalization reactions of internal alkenes. This includes the first catalytic, enantioselective oxidation of internal alkenes to ketones, as well as the formal asymmetric hydration and hydroamination of unactivated internal alkenes in combination with other biocatalysts.