Retrosynthetic Design of Dinuclear Copper Enzymes for Azide–Alkyne Cycloaddition via Clickable Noncanonical Amino Acids

Copper-catalyzed azide–alkyne cycloaddition (CuAAC) has enabled numerous synthetic and biological applications, driven by advances in the synthesis and optimization of copper-binding ligands. However, to the best of our knowledge, no bottom-up protein-based ligands have been specifically developed to catalyze this reaction. Here, we present a retrosynthetic protein design that leverages the introduction, duplication, and diversification of metal-chelating amino acid residues via a clickable noncanonical amino acid and CuAAC-mediated post-translational modification. A naturally occurring homodimer, dTDP-4-keto-6-deoxy-D-hexulose 3,5-epimerase, was engineered to structurally mimic the molecular framework of well-known CuAAC ligands, featuring multidentate triazole-containing motifs with four nitrogen donor atoms capable of accommodating two copper-binding sites. Remarkably, one protein construct R79TP exhibits CuAAC activity toward exogenous alkyne and azide substrates at rates exceeding that of a benchmark ligand, likely via a dinuclear mechanism. This work highlights the potential of genetically encoded precursors for multidentate ligand in proteins, expands the molecular complexity achievable in metalloenzyme engineering, and provides mechanistic insights and potential for copper-mediated bioorthogonal catalysis.