Ribosomal Synthesis of Macrocyclic Peptides in Vitro and in Vivo 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 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.
Department of Chemistry, University of Rochester, Hutchinson Hall, Rochester, New York 14627, United States.
John R
JR
Frost
Department of Chemistry, University of Rochester, Hutchinson Hall, Rochester, New York 14627, United States.
Nicholas T
NT
Jacob
Department of Chemistry, University of Rochester, Hutchinson Hall, Rochester, New York 14627, United States.
Louis J
LJ
Papa
Department of Chemistry, University of Rochester, Hutchinson Hall, Rochester, New York 14627, United States.
Andrew E
AE
Owens
Department of Chemistry, University of Rochester, Hutchinson Hall, Rochester, New York 14627, United States.
Rudi
R
Fasan
D000586:Amination; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D003500:Cyclization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047028:Macrocyclic Compounds / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D047028:Macrocyclic Compounds; D019151:Peptide Library; D010455:Peptides; D010456:Peptides, Cyclic; D013438:Sulfhydryl Compounds; D019809:Streptavidin; D000604:Amino Acyl-tRNA Synthetases