Photoenzymatic Catalysis of Asymmetric Remote Radical Functionalization by Engineered 'Ene'-reductase

Abstract

Photoenzymatic catalysis builds on the strengths of biocatalysis, which include unparalleled stereoselectivity and sustainability, while drawing out modes of non-natural reactivity by the photoexcitation of common enzymes. The asymmetric remote functionalization of unactivated C-H bonds remains elusive, with challenges of thermodynamics and stereoselectivity particularly difficult to overcome. Past studies have shown that the irradiation of ‘ene’-reductases with visible light allows access to enantioselective radical reactions with precise stereocontrol. Given these considerations, this work harnesses photoenzymatic catalysis to catalyze a 1,5-hydrogen atom transfer mediated asymmetric remote functionalization of an α-chloroamide. The proposed three-step mechanism involves an initial photoinduced electron transfer and mesolytic cleavage that oxidizes flavin hydroquinone to its semiquinone form. Next, a 1,5-hydrogen atom transfer accesses the remote stereocenter. In the final step, flavin semiquinone is fully oxidized to flavin quinone while facilitating an enantioselective hydrogen atom transfer to form the enantioenriched product. After rounds of site-saturated mutagenesis and high throughput screening, the mutant OYE2 Trp116Gly/Tyr375Gly was found to give the dehalogenated amide product with a yield of 37% and enantioselectivity of 83 : 17 e.r. Preliminary mechanistic studies support the proposed mechanism.