Kang, J.; (2011) Native chemical thioesterification: synthesis of peptide and protein thioesters through an N→S acyl shift. Doctoral thesis , UCL (University College London).

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Abstract

The total chemical synthesis of a protein provides atomic-level control over its covalent structure, however polypeptides prepared by solid phase peptide synthesis are limited to approximately fifty amino acid residues. This limitation has been overcome by 'Native Chemical Ligation‘, which involves amide bond formation between two unprotected polypeptides: a peptide with a C-terminal thioester and an N-terminal cysteinyl peptide. Synthesis of the required peptide thioester is difficult, particularly by Fmoc-chemistry. During our studies towards the semisynthesis of erythropoietin, we discovered reaction conditions that reversed Native Chemical Ligation and generated peptide and protein thioesters through an N→S acyl transfer. A peptide with both a Gly-Cys and an Ala-Cys-Pro-glycolate ester sequence was selectively thioesterified between the Gly-Cys sequence upon microwave-heating at 80 °C with 30 % v/v 3-mercaptopropionic acid (MPA), to afford the peptide-Gly-MPA thioester (84 % yield). Recombinant erythropoietin containing 4(Xaa-Cys) sites was also selectively thioesterified between His-Cys and Gly-Cys sequences upon treatment with 20 % MPA at 60 °C to give the peptide-MPA thioesters. His-/Gly-/Cys-Cys sequences were found to be particularly prone to thioesterification but thioester hydrolysis and disulfide bond formation also occurred with MPA, and therefore optimisation studies were conducted using 13C NMR spectroscopy. Sodium 2-mercaptoethanesulfonate (MESNa) was found to be a better thiol because it resulted in 100 % conversion into the peptide thioester and no thioester hydrolysis. The first biological application of this new method was then completed with the total synthesis of 45-amino acid Ser22His human β-defensin 3 (HBD3) for which a more complex 22-amino acid peptide thioester (22 % yield) was synthesised. Folded Ser22His HBD3 was assessed to have bioactivity equivalent to that reported: MBCs against P. aeruginosa = 0.9 μM, S. aureus = 1.8 μM; and optimal chemoattraction concentration = 0.2 nM. Posttranslationally modified native N-linked glycopeptide thioesters were also synthesised using the new thioesterification method, containing protected (20 % yield) or deprotected monosaccharides (64 % yield).

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