Copper-Catalyzed Azide–Alkyne Cycloaddition on Live M13 Bacteriophage for Expanding the Molecular Diversity of Phage-Displayed Peptide Libraries

Phage display is a powerful platform for ligand evolution, but conventional phage display libraries are confined to the twenty canonical amino acids, greatly limiting the chemical space that these libraries can be used to explore. Here we present an approach to expand the molecular diversity of phage-displayed peptides that combines unnatural amino acid mutagenesis with chemical post-translational modification. By incorporating azide-functionalized unnatural amino acids into phage-displayed peptides and applying optimized, phage-compatible conditions for copper-catalyzed azide–alkyne cycloaddition, we achieve quantitative and selective peptide modification with a series of alkyne-functionalized small molecules. This approach provides a general platform for constructing chemically augmented phage-displayed libraries with broad utility in ligand discovery.