Photosensitizer Repositioning Affords an Enantiocomplementary Enzyme for [2 + 2]‐Cycloadditions

The combination of genetic code expansion and directed evolution has recently given rise to enantioselective photoenzymes for [2 + 2]-cycloadditions of quinolone and indole derivatives. However, the enzymes reported to date only allow access to one enantiomeric series of the strained cyclobutane products. Here, guided by a crystal structure of our previously engineered enzyme EnT1.3, we show how judicious repositioning of the genetically programmed benzophenone photosensitizer affords an enantiocomplementary [2 + 2]-cyclase, CEnT1.0. Following directed evolution, a proficient and oxygen-tolerant photoenzyme (CEnT1.4) emerged that promotes [2 + 2]-cycloadditions of a quinolone derivative with exquisite enantiocontrol (99% e.e.) and substantially enhanced regioselectivity compared with EnT1.3 (r.r. 62:1 vs. 9:1). Structural analysis of CEnT1.4, coupled with molecular dynamic simulations, reveals a well-sculpted active site pocket that pre-organises the substrate for regio- and enantioselective catalysis. This study highlights the versatility offered by genetically programmed (photo)catalytic elements when developing enzymes for altered stereochemical outcomes.