Engineered Artificial Carboligase-Catalyzed Stereoselective Michael Addition of Nitroalkane to Cyclohexadienone

Enzyme-catalyzed enantioselective desymmetrization is a versatile synthetic approach to accessing chiral molecules, particularly those containing quaternary carbons. Achiral γ,γ-disubstituted cyclohexadienones possess great potential for desymmetrization transformations, given their easy preparation and the high significance of the resulting chiral cyclohexenones. Nevertheless, enzyme-catalyzed enantioselective desymmetrization of cyclohexadienones has just emerged very recently and has been restricted to proceed via either the C–H bond formation or intramolecular C–C bond formation mode. Herein, we report unprecedented enzyme-catalyzed intermolecular C–C bond formation on cyclohexadienone. Enabled by directed evolution, artificial carboligases were evolved to efficiently catalyze the Michael addition of nitroalkane to cyclohexadienones, furnishing chiral cyclohexenones bearing vicinal quaternary and tertiary stereocenters with high conversions (up to 96%), isolated yields (up to 74%), and stereoselectivities (up to 26:1 dr and 97:3 er). The synthetic capability of the current strategy was further showcased by semipreparative scale synthesis and product diversification. Protein crystallographic data and related biochemical studies provided mechanistic insights for effective catalysis, including the possible roles the introduced mutations (L131A, N135A, and F184A) played in improving activity and stereoselectivity for the synthesis of the Michael addition products (chiral cyclohexenones). This work expands the repertoire of enzyme-catalyzed enantioselective desymmetrization and highlights the prospect of applying artificial carboligases to catalyze valuable stereoselective C–C bond formation reactions.