Reprogramming Glutathione-Binding Proteins into Artificial Photoenzymes by Engineered Glutathione-Type Cofactors

Energy transfer (EnT) photocatalysis provides access to excited-state reactivity beyond thermal processes, yet highly enantioselective cycloadditions of sulfonamide-containing substrates remain challenging. Here we report a cofactor engineering strategy that reprograms glutathione S-transferases (GSTs) into artificial photoenzymes for enantioselective intramolecular [2 + 2] photocycloadditions to furnish chiral bicyclic sultam scaffolds. A photoactive glutathione-derived cofactor bearing a benzophenone photosensitizer (GS-Bp1) is prepared and reversibly assembled within GST scaffolds through native cofactor-like molecule-protein interactions. Screening and directed evolution of human GST A1-1 yield an optimized artificial photoenzyme that delivers a range of bicyclic sultams with moderate to excellent yields and enantioselectivities (up to 99% ee and up to 480 TONs), surpassing small-molecule benzophenone photocatalysts by more than 2 orders of magnitude in efficiency. Combined spectroscopic and computational studies reveal that directed evolution enhances stereocontrol by rebalancing π-π stacking and dispersion interactions in the transition state. Together with our previous NAD+-derived artificial cofactor systems, this work establishes cofactor engineering as a general strategy for reprogramming native protein families using canonical cofactors or cofactor-like molecules, enabling enantioselective photochemical transformations beyond those found in nature.