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.