Intermolecular C−C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent 'ene'-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, αchloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.

Biocatalysis has revolutionized chemical synthesis, providing sustainable methods for preparing various organic molecules. In enzyme-mediated organic synthesis, most reactions involve molecules operating from their ground states. Over the past 25 years, there has been an increased interest in enzymatic processes that utilize electronically excited states accessed through photoexcitation. These photobiocatalytic processes involve a diverse array of reaction mechanisms that are complementary to one another. This comprehensive review will describe the state-of-the-art strategies in photobiocatalysis for organic synthesis until December 2022. Apart from reviewing the relevant literature, a central goal of this review is to delineate the mechanistic differences between the general strategies employed in the field. We will organize this review based on the relationship between the photochemical step and the enzymatic transformations. The review will include mechanistic studies, substrate scopes, and protein optimization strategies. By clearly defining mechanisticallydistinct strategies in photobiocatalytic chemistry, we hope to illuminate future synthetic opportunities in the area.

Substituted arenes are ubiquitous in molecules with medicinal functions, making their synthesis a critical consideration when designing synthetic routes. Regioselective C–H functionalization reactions are attractive for preparing alkylated arenes; however, the selectivity of existing methods is modest and primarily governed by the substrate’s electronic properties. Here, we demonstrate a biocatalyst-controlled method for the regioselective alkylation of electron-rich and electron-deficient heteroarenes. Starting from an unselective “ene”-reductase (ERED) (GluER-T36A), we evolved a variant that selectively alkylates the C4 position of indole, an elusive position using prior technologies. Mechanistic studies across the evolutionary series indicate that changes to the protein active site alter the electronic character of the charge transfer (CT) complex responsible for radical formation. This resulted in a variant with a significant degree of ground-state CT in the CT complex. Mechanistic studies on a C2-selective ERED suggest that the evolution of GluER-T36A helps disfavor a competing mechanistic pathway. Additional protein engineering campaigns were carried out for a C8-selective quinoline alkylation. This study highlights the opportunity to use enzymes for regioselective radical reactions, where small molecule catalysts struggle to alter selectivity.