Expanding the scope of N → S acyl transfer in native peptide sequences

Abstract

Understanding the factors that influence N → S acyl transfer in native peptide sequences, and discovery of new reagents that facilitate it, will be key to expanding its scope and applicability. Here, through a study of short model peptides in thioester formation and cyclisation reactions, we demonstrate that a wider variety of Xaa-Cys motifs than originally envisaged are capable of undergoing efficient N → S acyl transfer. We present data for the relative rates of thioester formation and cyclisation for a representative set of amino acids, and show how this expanded scope can be applied to the production of the natural protease inhibitor Sunflower Trypsin Inhibitor-1 (SFTI-1).

Going Round in Circles with N→S Acyl Transfer

“…Head-to-tail peptide cyclisation to afford an "inert" product is an ideal application since, in forming a cyclic product, interference from side-reactions such as thioester hydrolysis are also minimised when cyclisation is rapid. 45 Although acyl transfer reactions utilising cysteine as the acyl transfer facilitator are often described as sluggish, we have found that several short peptides cyclise completely in 6-24 h at 40-50 o C in aqueous buffer. 45 Furthermore the reaction conditions are capable of facilitating effective cyclisation at Ser-Cys, Arg-Cys, Phe-Cys, and Leu-Cys motifs.…”

“…The reason for the increased reactivity of the C-terminal Cys carboxylate may be due to the zwitterionic nature of the intermediate which increases the basicity of the liberated αamino group and presents a barrier to reforming the starting material at acidic pH. 45 Recently the enhanced reactivity of thioester precursors adorned with a C-terminal carboxyl group Scheme 3 Peptide Cα-thioester formation enabled by the cysteinyl prolyl ester (2) 40 and by a single cysteine residue (3). 42 was also observed in the case of N-alkylated cysteine derivatives.…”

“…57 An identical process was adopted to produce variants of 14 residue SFTI-1 which were ultimately tested as inhibitors of Kalikrein 5 (KLK5), a protease involved in skin barrier homeostasis. 45,58 Overall, rather than constituting a severe limitation, the differing responses of Xaa-Cys motifs toward thioloysis and the reduced reactivity at internal Cys residues has in fact proved very beneficial on several occasions. A rather general strategy has been adopted for producing SFTI and cyclotide analogues from linear precursors and it is likely that this approach could be extended to other classes of cyclic peptides and combined with unnatural amino acids using an expanded genetic code, 59 in order to introduce novel functionality.…”

Protected thiol strategies in macromolecular design

“…The method has been used over the years for several applications, going from peptide sequencing [185], the introduction of thiols in polyurethanes for the covalent immobilization of biomolecules [186] to the synthesis of functional microgels [187,188]. Deprotection is mostly done by a nucleophilic addition-elimination reaction, usually via aminolysis, alcoholysis, or hydrolysis.…”

Tissue Kallikrein Inhibitors Based on the Sunflower Trypsin Inhibitor Scaffold – A Potential Therapeutic Intervention for Skin Diseases

“…To make the thioester, we exploited the mechanism of N-S acyl transfer on cysteine (Fig 1, Panel B), which offers a convenient reaction setup and does not require special resins or amino acids. This method gives the best yield of SFTI-1 analogues when the cysteine is preceded by a glycine or histidine residue (“HC”) [30]. Our previous results showed that a histidine instead of glycine substitution on position 10 of the native SFTI (SFTI-1) sequence provided a stronger KLK5 inhibitor [26].…”