Directed Evolution Enables Dynamic Control of Transient Intermediates for Anti-Markovnikov Wacker-Tsuji-Type Oxidation of Aliphatic Alkenes

Controlling the dynamic behavior of highly reactive fleeting intermediates is an intriguing concept to generate selectivity in competing reaction pathways. Here we report the directed evolution of a P450 enzyme for anti-Markovnikov-selective Wacker-Tsuji-type oxidation of aliphatic alkenes. This reaction is a long-standing challenge in catalysis due to its unfavorable energetics and competing reactions. The evolved aMOx-A enzyme performs anti-Markovnikov oxidation of aliphatic alkenes with several hundred turnovers and possesses kinetic parameters comparable to those of average natural enzymes. The biocatalyst guides an oxo-transfer process through multiple competing reactions, including bifurcating reaction pathways that originate after a rate-limiting transition state. Chemoselectivity of aMOx-A arises from controlling substrate conformations as well as dynamics of reactive intermediates. This includes accessing and controlling a fleeting carbocation intermediate that does not correspond to a minimum on the potential energy surface. Engineering of aMOx-A highlights how directed enzyme evolution optimizes unique catalytic processes that have largely eluded efficient catalysis.