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Keywords:

  • Coupling;
  • Peptides;
  • Macrocycles;
  • Synthetic methods;
  • Thioester

Abstract

Thioester-mediated peptide coupling reactions are powerful tools in protein synthesis. The fragment coupling occurs extremely fast at ligation sites that contain an N-terminal cysteine residue. This native chemical ligation involves a capture step that affixes the acyl component in the immediate vicinity of the N-terminal amino group. The subsequent intramolecular S,N-acyl transfer step proceeds via an entropically favored five-membered ring intermediate. In this study we investigated whether sequence internal cysteine residues that lead to the formation of macrocyclic intermediates are also able to accelerate the rate of thioester-based fragment couplings. It was the aim to identify distance requirements that enable internal cysteine-mediated ligation reactions to proceed at synthetically useful rates. It was found that appropriately positioned cysteine residues can induce up to 25-fold rate enhancements compared to a cysteine-lacking control peptide. Highest ligation rates and yields were obtained when the internal thiol amino acid was incorporated at the fifth or sixth position from the N-terminus of the C-terminal coupling segment. The findings reveal a correlation between the size of the macrocyclic ring intermediate and the ease of the peptide ligation. Internal cysteine ligation may provide the opportunity to shift amino acid cysteine bonds that are difficult to access through native chemical ligation (such as Pro–Cys bonds) by 4–5 amino acids to the N-terminus.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)