Directed Evolution of a Nonheme Iron Enzyme to Access Chiral α‐Amino Acid Derivatives by 1,3‐Migratory Nitrene C(sp ³ )─H Insertion

Abstract Nonheme iron enzymes are among nature's most versatile catalysts for molecular functionalization. Engineering nonheme enzymes for abiological reactions unlocks new catalytic possibilities beyond the limits of natural evolution. In this work, we engineered a nonheme enzyme, leucoanthocyanidin dioxygenase from Arabidopsis thaliana ( At LDOX), to catalyze an asymmetric 1,3‐migratory nitrene C(sp 3 )─H insertion reaction. Through directed evolution, the final optimized At LDOX_LS variant efficiently delivers a range of chiral α ‐amino acids derivatives with exceptional activity and enantioselectivity (up to 81% yield, 850 total turnover number, and 98:2 enantiomeric ratio). Preliminary mechanistic studies suggest the involvement of radical intermediates for this transformation. This work advances the biocatalytic toolbox for radical involved transformations and broadens the scope of enzymatic migration chemistry.