An Elegant Method of One‐Pot Ligation‐Desulfurization for High‐Yielding Chemical Protein Synthesis

While native chemical ligation (NCL), combined with radical‐mediated desulfurization, has enabled chemical syntheses of a vast array of proteins, the presence of aryl thiols as catalysts in the ligation mixture prevents performing both ligation and desulfurization reactions in one‐pot, as aryl thiols are effective radical scavengers. Existing one‐pot ligation and desulfurization approaches are not ideal as they rely on the use of inefficient alkyl thiols as NCL catalysts. Here an extremely efficient and impressively straightforward method is presented that utilizes bromoacetamide, in conjunction with N‐acetyl cysteine, for selective quenching of arylthiol following NCL, enabling ligation and desulfurization in one‐pot without any intermediate purification step. The reagent combination facilitates one‐pot reactions by selectively capping aryl thiols in the presence of peptidic cysteine residue, leveraging the increased nucleophilicity of aryl thiols over alkyl thiols. N‐acetyl cysteine additionally functions as the alkyl thiol additive for the desulfurization reaction. It is demonstrated the utility of this methodology by synthesizing three different proteins: ubiquitin, collagen, and barstar A. The strategy substantially enhances the efficiency of chemical protein synthesis, especially for cysteine‐free proteins. Its operational simplicity and broad applicability make this one‐pot ligation‐desulfurization protocol promising for widespread adoption in synthesizing therapeutic proteins and related biomolecules.