Synthesis of l- and d-Ubiquitin by One-Pot Ligation and Metal-Free Desulfurization

Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one‐pot native chemical ligation and metal‐free desulfurization for chemical protein synthesis. This one‐pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X‐ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9‐fluorenylmethoxycarbonyl (Fmoc) solid‐phase peptide synthesis coupled with a one‐pot ligation–desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies. Making links: An efficient one‐pot ligation‐desulfurization strategy to synthesize proteins has been developed (see scheme). Key to this technical advance is the use of a new thiol additive, methyl thioglycolate, in the ligation process.