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Existing one\u2010pot 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\u2010acetyl cysteine, for selective quenching of arylthiol following NCL, enabling ligation and desulfurization in one\u2010pot without any intermediate purification step. The reagent combination facilitates one\u2010pot reactions by selectively capping aryl thiols in the presence of peptidic cysteine residue, leveraging the increased nucleophilicity of aryl thiols over alkyl thiols. N\u2010acetyl 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\u2010free proteins. Its operational simplicity and broad applicability make this one\u2010pot ligation\u2010desulfurization protocol promising for widespread adoption in synthesizing therapeutic proteins and related biomolecules.<\/jats:p>","DOI":"10.1002\/advs.202510194","type":"journal-article","created":{"date-parts":[[2025,8,25]],"date-time":"2025-08-25T14:45:58Z","timestamp":1756133158000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["An Elegant Method of One\u2010Pot Ligation\u2010Desulfurization for High\u2010Yielding Chemical Protein Synthesis"],"prefix":"10.1002","volume":"12","author":[{"given":"Vishal","family":"Malik","sequence":"first","affiliation":[{"name":"Tata Institute of Fundamental Research Hyderabad  36\/p Gopanpally Hyderabad Telangana 500046 India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Abhisek","family":"Kar","sequence":"additional","affiliation":[{"name":"Tata Institute of Fundamental Research Hyderabad  36\/p Gopanpally Hyderabad Telangana 500046 India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anandh Muthiah","family":"Venkatachalam","sequence":"additional","affiliation":[{"name":"Tata Institute of Fundamental Research Hyderabad  36\/p Gopanpally Hyderabad Telangana 500046 India"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3194-1378","authenticated-orcid":false,"given":"Kalyaneswar","family":"Mandal","sequence":"additional","affiliation":[{"name":"Tata Institute of Fundamental Research Hyderabad  36\/p Gopanpally Hyderabad Telangana 500046 India"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,8,25]]},"reference":[{"key":"e_1_2_7_1_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.8b00712"},{"key":"e_1_2_7_2_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja00897a025"},{"key":"e_1_2_7_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.2318"},{"key":"e_1_2_7_4_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.7973629"},{"key":"e_1_2_7_5_1","doi-asserted-by":"publisher","DOI":"10.1021\/ja058344i"},{"key":"e_1_2_7_6_1","unstructured":"\u201cProtScale Tool: Amino acid composition (%) in the UniProtKB\/Swiss\u2010Prot data bank. \u201dhttps:\/\/web.expasy.org\/protscale\/pscale\/A.A.Swiss\u2010Prot.html. 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