Tyrosinase-Catalyzed Peptide Stapling Using para-Amino Phenylalanine and Tyrosine Anchoring Residues

Peptide stapling techniques have historically relied on metal-catalyzed chemical reactions, with no examples using enzymes. Here, inspired by tyrosinase-mediated oxidation, we describe the efficient side-chain to side-chain coupling of p-amino phenylalanine (Z) and tyrosine (Y) amino acids using a commercially available tyrosinase. Stapling reactions between the i, i+3 to i, i+7 positions were all performed, proceeding in good conversion and under mild conditions compatible with various side chains, functional motifs and ring sizes, with the Z-Y product found to be more stable and obtained in a higher yield than the Y-Z product. Z-Y stapled versions of ER, MC4 and GPR54 binding peptides exhibited higher serum stability, helical content and binding affinity than their linear counterparts, proving the utility of our method to synthesize biologically significant peptides. The tyrosinase-catalyzed Z-Y peptide stapling technique expands the scope of available stapling techniques, and is proposed to develop stapled peptide drug candidates.