Ribosomal Incorporation of Fluorosulfonyloxy-

Macrocyclic peptides are emerging as a promising molecular framework in covalent drug development due to their high specificity, affinity, and low toxicity, addressing challenges such as off-target effects and nonspecific binding associated with traditional covalent binders. Although mRNA display technology has advanced the discovery of covalent peptide binders, it has primarily focused on cysteine residues, thereby limiting the diversity of targetable proteins. In this study, we ribosomally incorporated 4-fluorosulfonyloxy-l-phenylalanine (FSY) into macrocyclic peptides, enabling the construction of a diverse covalent macrocyclic peptide library for de novo screening against human α-thrombin and fibroblast activation protein. This led to the identification of novel peptides with covalent binding capabilities and a distinct dissociation profile, which in turn enabled potent inhibitory activities at concentrations within the low nanomolar range. Notably, FSY-bearing macrocyclic peptides conjugated with a radionuclide chelator demonstrated significantly improved tumor-selective uptake and prolonged retention, outperforming their noncovalent counterparts while underscoring their remarkable potential in targeted radionuclide therapy. This study provided a robust and efficient platform for the de novo discovery of FSY-bearing macrocyclic peptides, broadening the scope of covalent drug development for diverse biomedical applications.