Histidine (His) bears a uniquely electron-deficient imidazole side chain and plays essential roles in protein interactions and enzyme-catalyzed processes. Modification of His C2 position offers a useful method to fine-tune histidine residues of proteins for their structural and functional study. Due to the moderately nucleophilic imidazole group, the chemoselective modification of histidine in proteins remains particularly challenging. Herein, we report a highly efficient method for the semisynthesis of chitin-binding protein21 (CBP21) bearing various groups at the C2 position of His28. A combination of modern radical-mediated C–H alkylation and recombinant protein engineering offers a powerful strategy to de... More