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In this paper, we report the use of thiocholine in chemical protein synthesis, including NCL-based peptide ligation and metal-free desulfurization. Evaluation of thiocholine peptide thioester in terms of NCL and hydrolysis kinetics revealed its practical utility, which was comparable to that of other alkyl thioesters. Importantly, thiocholine showed better reactivity as a thiol additive in desulfurization, which is often used in chemical protein synthesis to convert Cys residues to more abundant Ala residues. Finally, we achieved chemical synthesis of two differently methylated histone H3 proteins via one-pot NCL and desulfurization with thiocholine.<\/jats:p>","DOI":"10.3390\/molecules28093655","type":"journal-article","created":{"date-parts":[[2023,4,24]],"date-time":"2023-04-24T02:31:05Z","timestamp":1682303465000},"page":"3655","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Thiocholine-Mediated One-Pot Peptide Ligation and Desulfurization"],"prefix":"10.3390","volume":"28","author":[{"given":"Sae","family":"Suzuki","sequence":"first","affiliation":[{"name":"Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuya","family":"Nakajima","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Naoki","family":"Kamo","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akihisa","family":"Osakabe","sequence":"additional","affiliation":[{"name":"Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7418-6237","authenticated-orcid":false,"given":"Akimitsu","family":"Okamoto","sequence":"additional","affiliation":[{"name":"Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gosuke","family":"Hayashi","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Murakami","sequence":"additional","affiliation":[{"name":"Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan"},{"name":"Institute of Nano-Life-Systems, Institutes of Innovation for Future Society, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8603, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,4,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"776","DOI":"10.1126\/science.7973629","article-title":"Synthesis of Proteins by Native Chemical Ligation","volume":"266","author":"Dawson","year":"1994","journal-title":"Science"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"7328","DOI":"10.1021\/acs.chemrev.8b00712","article-title":"Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations","volume":"119","author":"Agouridas","year":"2019","journal-title":"Chem. 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