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

ACS Chem Biol. 2015 Aug 21;10(8):1805-16. doi: 10.1021/acschembio.5b00119. Epub 2015 May 15.


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 in vitro 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 (KD = 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Amination
  • Amino Acid Sequence
  • Amino Acids / chemistry
  • Amino Acids / genetics
  • Amino Acids / metabolism*
  • Amino Acyl-tRNA Synthetases / metabolism
  • Cyclization
  • Escherichia coli / chemistry
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Macrocyclic Compounds / chemistry
  • Macrocyclic Compounds / metabolism*
  • Molecular Sequence Data
  • Peptide Library
  • Peptides / chemistry
  • Peptides / genetics
  • Peptides / metabolism*
  • Peptides, Cyclic / chemistry
  • Ribosomes / chemistry
  • Ribosomes / genetics
  • Ribosomes / metabolism*
  • Streptavidin / metabolism
  • Sulfhydryl Compounds / chemistry
  • Sulfhydryl Compounds / metabolism


  • Amino Acids
  • Macrocyclic Compounds
  • Peptide Library
  • Peptides
  • Peptides, Cyclic
  • Sulfhydryl Compounds
  • Streptavidin
  • Amino Acyl-tRNA Synthetases