Ribosomal Synthesis of Macrocyclic Peptides <i>in Vitro</i> and <i>in Vivo</i> Mediated by Genetically Encoded Aminothiol Unnatural Amino Acids

A versatile method for orchestrating the formation of side chain-to-tail cyclic peptides from ribosomally derived polypeptide precursors is reported. Upon ribosomal incorporation into intein-containing precursor proteins, designer unnatural amino acids bearing side chain 1,3- or 1,2-aminothiol functionalities are able to promote the cyclization of a downstream target peptide sequence via a C-terminal ligation/ring contraction mechanism. Using this approach, peptide macrocycles of variable size and composition could be generated in a pH-triggered manner <i>in vitro</i> or directly in living bacterial cells. This methodology furnishes a new platform for the creation and screening of genetically encoded libraries of conformationally constrained peptides. This strategy was applied to identify and isolate a low-micromolar streptavidin binder (<i>K</i>_D = 1.1 μM) from a library of cyclic peptides produced in Escherichia coli, thereby illustrating its potential toward aiding the discovery of functional peptide macrocycles