Native chemical ligation, featuring chemoselective reactions between thioester and N-terminal cysteinyl peptides, has served as the most promising coupling methodology for protein synthesis. One limitation of the NCL is that the ligation site requires the cysteinyl residue, which has prompted one to establish a ligation protocol accomplished at non-cysteine sites. Here, we report a coupling method at proline site using 4-mercaptoproline (HSPro) via a sequence of two chemoselective reactions including NCL-like reaction between HSPro and thioester peptides, followed by desulfurization. A dual kinetically-controlled NCL strategy using HSPro combined with N -sulfanylethylanilide (SEAlide) peptide is also described. And this newly developed kinetic reaction allowed three peptide fragments simultaneously presented to be ligated in correct order.
