Expanding triazole ligand architectures in artificial metalloenzymes for copper-catalyzed azide−alkyne cycloaddition

Compared to synthetic ligands, metal coordination in proteins relies on a limited set of canonical amino acids, restricting the chemical toolbox of metalloenzyme design. In this study, we expand this repertoire by genetically incorporating, multiplying, and modifying the noncanonical amino acid p -azidophenylalanine using protein symmetry operations and copper-assisted azide-alkyne cycloaddition (CuAAC). This strategy enables the seamless introduction of multiple triazole ligands into a selected protein scaffold, generating diverse copper-binding sites with tunable steric effect and electronic properties. The resulting artificial proteins exhibited distinct stability, copper binding stoichiometries, and catalytic activities in CuAAC, enabling the creation of clickzymes with high catalytic performance in phosphate buffer. These findings broaden the biochemical and inorganic toolkit for ligand design, enhancing the structural and functional diversity of tailor-made artificial metalloenzymes and biocatalysts.