Thia Zip Reaction for Synthesis of Large Cyclic Peptides:  Mechanisms and Applications

Abstract

This paper describes the mechanism and application of an efficient thia zip cyclization that involves a series of intramolecular rearrangements in a cysteine-rich peptide for the synthesis of large end-to-end cyclic peptides. Key functional groups required in this reaction include an N α-cysteine, a thioester, and at least one internal free thiol in a peptide. The zip reaction is initiated by intramolecular transthioesterification through an internal thiol with the thioester. A thiolactone is formed under ring−chain tautomeric equilibrium that favors ring formation in aqueous buffered solution at pH > 7. Successive ring expansions through thiol−thiolactone exchanges in the direction of the amino terminus lead finally to a large Nα-amino thiolactone which then undergoes a spontaneous and irreversible ring contraction through a sequence-independent S to N acyl isomerization to form an end-to-end lactam. The reversible thiolactone exchanges are sequence-dependent, and the rate-determining steps are shown by rate studies on model peptides. The assistance of internal thiols in reducing the ring sizes and hence the entropy of the thiolactone exchanges correlates well with cyclization rates. Zip-assisted end-to-end cyclizations forming 93- and 99-atom rings through a series of small thiolactone intermediates were 60−200-fold faster under strongly denaturing conditions such as 8 M urea than the corresponding unassisted lactamization. The thia zip reaction has been applied successfully to the synthesis of a 31-amino acid cyclic peptide, the naturally occurring cyclopsychotride that shows the antimicrobial activity. In addition, the thia zip reaction also enables the synthesis of an engineered cyclic 33-amino acid animal defensin by replacing the end-to-end disulfide with a lactam, which retains the antimicrobial activities of the native open-chain form.

Antimicrobial Characterization of Human β-Defensin 3 Derivatives

“…Because mass spectrometric identification of tryptic fragments yielded a chemically identical, two-disulfide-bridged tryptic fragment of 1,846.9 Da, the ambiguity was resolved by subsequent enzymatic digestion with chymotrypsin. All of the common and unique fragments generated by trypsin and chymotrypsin were identified and accounted for by analytical RP-HPLC and ESI-MS. Deprotection and spontaneous oxidation of Cys 11 /Cys 40 or Cys 11 /Cys 41 was achieved through treatment with I 2 at acidic pH according to the published protocols (31). Final products were verified by ESI-MS, yielding an observed mass value of 5,155.5 Da, which is in agreement with the expected value of 5,155.2 Da, calculated based on the average isotope compositions of folded hBD3.…”

Correlations of Cationic Charges with Salt Sensitivity and Microbial Specificity of Cystine-stabilized β-Strand Antimicrobial Peptides

“…The process was assisted by a thia-zip reaction involving a series of thiol-assisted intramolecular rearrangements (30). Ultimately, an N-terminal thiolactone is formed, leading to a spontaneous ring contraction through an S,N-acyl isomerization to form the end-to-end peptide bond.…”

Engineering disulfide bridges to dissect antimicrobial and chemotactic activities of human β-defensin 3

“…Deprotection and spontaneous oxidation of Cys 1 ͞Cys 5 or Cys 1 ͞Cys 6 in species a-f whose disulfide topology had been previously determined were achieved through treatment with I 2 at acidic pH according to the published protocols (33). Final products were verified by ESI-MS, yielding an observed mass value of 5,155.5 Da, in agreement with the expected value of 5,155.2 Da, calculated on the basis of average isotope compositions of fully oxidized hBD3.…”