Directed Evolution of Class I Aldolases for Cooperative Photobiocatalytic Enantioselective Radical α-Alkylation of Aldehydes

By harnessing the synergy between enzymes and photoredox catalysts, cooperative photobiocatalysis has recently emerged as a promising strategy for developing stereoselective radical reactions. While various cofactor-dependent enzymes have been repurposed, the use of cofactor-independent enzymes in such cooperative catalysis without requiring expensive cofactors remains rare. Herein, we report the successful repurposing of class I aldolases, a prominent family of naturally occurring, cofactor-independent enzymes, to catalyze unnatural radical α-alkylation of aldehydes in a highly enantioselective fashion. Through directed evolution of <i>Escherichia coli</i> 2-deoxy-d-ribose-5-phosphate aldolase (<i>Ec</i>DERA), we developed an effective radical alkylase bearing five mutations and inverted π-facial selectivity relative to wild-type <i>Ec</i>DERA, allowing a range of aldehydes to couple with α-iodoesters, α-iodoketones, and α-iodonitriles with excellent enantiocontrol. This study represents the first demonstration of leveraging the nucleophilic enamine intermediate in class I aldolases for radical-mediated stereoselective C-C bond formation. Mechanistic investigations suggested that when irradiated at 440 nm, cooperative catalysis with an exogenous Ir photocatalyst more effectively induces enzymatic enamine radical activity than charge-transfer complex photochemistry. Together, these findings underscore the potential of class I aldolases to enable general and stereoselective new-to-nature radical transformations.