Ruthenium-Catalyzed One-pot Peptide Ligation.

The chemical synthesis of proteins consists of three steps: synthesis of peptide segments, native chemical ligation, and desulfurization. The native chemical ligation is repeated to grow polypeptides, but at each step, deprotection and purification also had to be repeated. To address these issues, by repeating ruthenium-catalyzed fast deprotection of the cysteine terminus of the peptide segment and slow inactivation of the catalyst by thiophenol step by step, we were able to achieve one-pot, repeated native chemical ligation without purification steps. In this method, the ruthenium catalyst rapidly removes the Alloc group for cysteine protection and is slowly deactivated by 4-mercaptophenylacetic acid, which is added to promote peptide ligation and to remove the allyl group from the ruthenium complex. By using this chemical reaction, we have chemically prepared epigenetically modified proteins such as histone protein H1.2, and the protocol is described here.