We develop strategies that enable enzymes to catalyze reactions that are currently unknown in nature.

Non-native photoenzymes have enabled a myriad of asymmetric bond-forming events that are otherwise challenging or currently impossible with small molecule catalysis. These reactions require enzymes with cofactors that are strong absorbers in the visible region with reasonably long-lived excited states, such as flavin and nicotinamide. However, there exists a substantial chromophoric cofactor “dark space” where no known photoenzymatic activity has been characterized. Increased knowledge of the photophysics of the cofactors in the “dark space” would increase the types of bonds that photoenzymes can form by accessing new excited state intermediates in enzyme classes with divergent reactivities and selectivities. Here, we establish pyridoxal 5’-phosphate (PLP) as a photoenzymatic cofactor by leveraging the excited state quinonoid intermediate as a potent single-electron reductant. We overcome the poor photophysical properties of the native quinonoid intermediate by employing non-native benzyl amine substrates and exploiting Förster resonance energy transfer mechanism from an exogenous photosensitizer to access the quinonoid excited state. This redox neutral approach enables an asymmetric radical-radical cross-coupling between benzyl amines and reductive radical precursors through concomitant generation and localization of a radical pair in an enzyme active site—overcoming the typical challenges associated with this reaction by removing the necessity for radical sorting and the persistent radical effect. The emergent photoexcited intermediates of PLP identified in this work greatly expands the potential avenues for valuable bond forming events by PLP-dependent enzymes.

Asymmetric radical alkene hydroamination offers a potentially powerful strategy to form enantioenriched, saturated N-heterocycles, but these reactions have proved challenging to render stereoselective. Here, we report an engineered flavin enzyme that catalyzes the formation of chiral pyrrolidines with high yield and enantioselectivity. Development of this transformation addresses a long-standing limitation in flavin photobiocatalysis: the short lifetime of the photoexcited flavin quinone has previously made it challenging to leverage this species as a photooxidant. We report that the photophysical properties of a flavin enzyme can be tuned via rational mutagenesis to obtain a variant with a long-lived excited state, and that this enzyme can be further engineered to create an efficient catalyst for asymmetric radical hydroaminations. Moreover, this work introduces two mechanistic discoveries that can be applied broadly in photobiocatalysis: (1) an exogeneous cophotocatalyst can serve as a photoprotectant for the enzymatic cofactor and (2) enantiospecific termination of chiral radical intermediates provides a mechanism to achieve stereocontrol over previously challenging classes of transformations.

We describe a photoenzymatic hydroalkylation reaction that enables the efficient and stereocontrolled synthesis of aryl glutarimide precursors—chemically and configurationally robust entry points to bioactive agents for targeted protein degradation. Screening of flavin-dependent “ene”-reductases identified GluER HA_rac, a G. oxydans variant, as an efficient and substrate-tolerant catalyst, granting access to >30 (hetero)aryl glutarimide precursors. A directed evolution campaign then furnished a hexamutant, GluER HA_ent, that delivers the products in up to 93:7 enantiomeric ratio. Mechanistic experiments revealed a pathway that departs from the hydrogen atom transfer mechanism previously established for related systems, proceeding instead via radical–polar crossover followed by enantioselective proton transfer from an active-site tyrosine residue. Collectively, these studies establish a biocatalytic platform for advancing the synthesis and diversification of glutarimide-containing degraders.