{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T14:07:01Z","timestamp":1788876421510,"version":"build-2803163510"},"reference-count":23,"publisher":"American Chemical Society (ACS)","issue":"35","license":[{"start":{"date-parts":[[2026,8,27]],"date-time":"2026-08-27T00:00:00Z","timestamp":1787788800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100004945","name":"Nankai University","doi-asserted-by":"publisher","award":["24NKSYJS09"],"award-info":[{"award-number":["24NKSYJS09"]}],"id":[{"id":"10.13039\/501100004945","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Tianjin Administration for Market Regulation","doi-asserted-by":"publisher","award":["2023-W14"],"award-info":[{"award-number":["2023-W14"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Tianjin Administration for Market Regulation","doi-asserted-by":"publisher","award":["2023-W36"],"award-info":[{"award-number":["2023-W36"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Tianjin Administration for Market Regulation","doi-asserted-by":"publisher","award":["2023-W42"],"award-info":[{"award-number":["2023-W42"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/na","name":"Tianjin Administration for Market Regulation","doi-asserted-by":"publisher","award":["2025-W35"],"award-info":[{"award-number":["2025-W35"]}],"id":[{"id":"10.13039\/na","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,8]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Desmopressin, a synthetic analog of vasopressin widely used in the treatment of diabetes insipidus and bleeding disorders, is a representative disulfide-containing peptide therapeutic prepared by Fmoc-based solid-phase peptide synthesis (SPPS). Its unique sequence architecture, particularly the spatial proximity between the N-terminal mercaptopropionic acid residue Mpa(1) and the Tyr(2) residue, provides a distinct structural environment for investigating sequence-dependent side reactions during peptide manufacturing. The tert-butyl (t-Bu) protecting group is widely employed for side-chain protection in Fmoc-based solid-phase peptide synthesis (SPPS). However, under certain reaction conditions, the highly reactive t-Bu+ carbocation generated during acidic deprotection can participate in nonspecific alkylation, leading to challenging process-related impurities. In this study, we investigated a unique and highly site-selective +56 Da impurity observed during side-chain deprotection. High-resolution MS\/MS analysis unambiguously identified this species as a site-selective product arising from the migration of t-Bu from the Tyr(2) side chain to the thiol group of Mpa(1). Based on the structural features of desmopressin, in particular, the spatial proximity between Tyr(2) and Mpa(1), we propose that the formation of this impurity most plausibly proceeds via a vicinal-thiol-induced intramolecular migration mechanism, which competes significantly with the conventional diffusion-controlled carbocation intermediate intermolecular pathway. Strengthening the scavenger conditions failed to eliminate this impurity, further supporting the intramolecular migration mechanism induced by vicinal thiol. This finding accounts for the observed site selectivity that cannot be explained by the conventional carbocation intermediate pathway alone. Guided by these mechanistic insights, we re-evaluated the necessity of Tyr side-chain protection and proposed a \u201csource-elimination\u201d strategy. Experimental validation demonstrated that the use of unprotected Fmoc-Tyr-OH (free phenolic hydroxyl) completely suppressed the formation of the t-Bu migration impurity and improved the crude peptide purity to 93.5%. Overall, this study elucidates the formation mechanism of t-Bu migration impurities and provides a broadly applicable, mechanism-driven strategy for process optimization in solid-phase peptide synthesis. While this study focuses on the desmopressin system, the findings highlight a proximity-driven reactivity pattern that may be relevant to other thiol-containing peptide sequences.<\/jats:p>","DOI":"10.1021\/acsomega.6c07280","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T17:27:52Z","timestamp":1788283672000},"page":"53469-53476","source":"Crossref","is-referenced-by-count":0,"title":["Tyr\u2013Mpa Proximity-Induced Atypical\n                    <i>tert<\/i>\n                    -Butyl\nIntramolecular Migration in Desmopressin: Mechanistic\nElucidation of a Site-Selective Impurity and Its Suppression Strategy"],"prefix":"10.1021","volume":"11","author":[{"given":"Xin","family":"Lu","sequence":"first","affiliation":[{"name":"Tianjin Institute for Drug Control , No. 98, Guizhou Road , ,","place":["Tianjin, China, 300070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fangfang","family":"Yuan","sequence":"additional","affiliation":[{"name":"Tianjin Institute for Drug Control , No. 98, Guizhou Road , ,","place":["Tianjin, China, 300070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaojie","family":"Han","sequence":"additional","affiliation":[{"name":"Tianjin Institute for Drug Control , No. 98, Guizhou Road , ,","place":["Tianjin, China, 300070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tingting","family":"Qin","sequence":"additional","affiliation":[{"name":"Tianjin Institute for Drug Control , No. 98, Guizhou Road , ,","place":["Tianjin, China, 300070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qianqian","family":"Wang","sequence":"additional","affiliation":[{"name":"Nankai University , , ,","place":["Tianjin, China, 300350"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zengli","family":"Li","sequence":"additional","affiliation":[{"name":"Anhui Anke Biotechnology (Group) Co., Ltd , No. 9 Haiguan Road , , ,","place":["Hefei, Anhui Province, China, 230088"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chuanjun","family":"Ge","sequence":"additional","affiliation":[{"name":"Anhui Anke Biotechnology (Group) Co., Ltd , No. 9 Haiguan Road , , ,","place":["Hefei, Anhui Province, China, 230088"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wenqing","family":"Shi","sequence":"additional","affiliation":[{"name":"Anhui Anke Biotechnology (Group) Co., Ltd , No. 9 Haiguan Road , , ,","place":["Hefei, Anhui Province, China, 230088"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haijiao","family":"Bai","sequence":"additional","affiliation":[{"name":"Tianjin Institute for Drug Control , No. 98, Guizhou Road , ,","place":["Tianjin, China, 300070"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-5268-0490","authenticated-orcid":true,"given":"Pei","family":"Wang","sequence":"additional","affiliation":[{"name":"Tianjin Institute for Drug Control , No. 98, Guizhou Road , ,","place":["Tianjin, China, 300070"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,27]]},"reference":[{"key":"2026090809102133600_cit1","doi-asserted-by":"publisher","first-page":"4","DOI":"10.1002\/psc.2836","article-title":"Advances in Fmoc solid-phase peptide\nsynthesis","volume":"22","author":"Behrendt","year":"2016","journal-title":"J. Pept. Sci."},{"key":"2026090809102133600_cit2","doi-asserted-by":"publisher","first-page":"2455","DOI":"10.1021\/cr800323s","article-title":"Amino Acid-Protecting\nGroups","volume":"109","author":"Isidro-Llobet","year":"2009","journal-title":"Chem. Rev."},{"key":"2026090809102133600_cit3","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1016\/j.jpba.2014.06.012","article-title":"Related impurities in\npeptide medicines","volume":"101","author":"D\u2019Hondt","year":"2014","journal-title":"J. Pharm. Biomed. Anal."},{"key":"2026090809102133600_cit4","doi-asserted-by":"publisher","first-page":"270","DOI":"10.1021\/acs.oprd.4c00386","article-title":"Therapeutic Peptides\nControl Strategy: Perspective on Current Industry Practices","volume":"29","author":"Pack","year":"2025","journal-title":"Org. Process Res. Dev."},{"key":"2026090809102133600_cit5","doi-asserted-by":"publisher","first-page":"996","DOI":"10.1039\/C9GC03982A","article-title":"Greening Fmoc\/tBu\nsolid-phase peptide synthesis","volume":"22","author":"Al Musaimi","year":"2020","journal-title":"Green Chem."},{"key":"2026090809102133600_cit6","doi-asserted-by":"publisher","first-page":"256","DOI":"10.1016\/j.jpha.2015.02.001","article-title":"Identification, synthesis\nand characterization of process related impurities of benidipine hydrochloride,\nstress-testing\/stability studies and HPLC\/UPLC method validations","volume":"5","author":"Bellur Atici","year":"2015","journal-title":"J. Pharm. Anal."},{"key":"2026090809102133600_cit7","doi-asserted-by":"publisher","first-page":"217","DOI":"10.1021\/co300153c","article-title":"Handles for Fmoc Solid-Phase Synthesis\nof Protected Peptides","volume":"15","author":"Go\u0301ngora-Beni\u0301tez","year":"2013","journal-title":"ACS Comb. Sci."},{"key":"2026090809102133600_cit8","doi-asserted-by":"publisher","first-page":"1357","DOI":"10.1007\/s00726-013-1471-7","article-title":"Side reactions in the\nSPPS of Cys-containing peptides","volume":"44","author":"Stathopoulos","year":"2013","journal-title":"Amino Acids"},{"key":"2026090809102133600_cit9","doi-asserted-by":"publisher","first-page":"5441","DOI":"10.1016\/S0040-4039(00)79116-7","article-title":"Improved efficiency\nand selectivity in peptide-synthesis: use of triethylsilane as a carbocation\nscavenger in deprotection of t-Bu esters and tert-butoxycarbonyl-protected sites","volume":"33","author":"Mehta","year":"1992","journal-title":"Tetrahedron Lett."},{"key":"2026090809102133600_cit10","doi-asserted-by":"publisher","first-page":"26","DOI":"10.1021\/acs.oprd.3c00425","article-title":"Ready to Use\nCysteine Thiol Protecting Groups in SPPS, A Practical Overview","volume":"28","author":"Chakraborty","year":"2024","journal-title":"Org. Process Res. Dev."},{"key":"2026090809102133600_cit11","doi-asserted-by":"publisher","first-page":"196","DOI":"10.1016\/j.jpha.2013.11.002","article-title":"Highly sensitive assay\nfor the determination of therapeutic peptide desmopressin in human\nplasma by UPLC-MS\/MS","volume":"7","author":"Gudlawar","year":"2017","journal-title":"J. Pharm. Anal."},{"key":"2026090809102133600_cit12","doi-asserted-by":"publisher","first-page":"697","DOI":"10.1002\/psc.1167","article-title":"Quality specifications\nfor peptide drugs: a regulatory-pharmaceutical approach","volume":"15","author":"Vergote","year":"2009","journal-title":"J. Pept. Sci."},{"key":"2026090809102133600_cit13","doi-asserted-by":"publisher","first-page":"17774","DOI":"10.1021\/ja805074d","article-title":"Sequence-Scrambling\nFragmentation Pathways of Protonated Peptides","volume":"130","author":"Bleiholder","year":"2008","journal-title":"J. Am. Chem. Soc."},{"key":"2026090809102133600_cit14","doi-asserted-by":"publisher","first-page":"1853","DOI":"10.1021\/ja310221t","article-title":"Quantification of the\nEffect of Conformational Restriction on Supramolecular Effective Molarities","volume":"135","author":"Adams","year":"2013","journal-title":"J. Am. Chem. Soc."},{"key":"2026090809102133600_cit15","doi-asserted-by":"publisher","first-page":"7147","DOI":"10.1021\/jp301812j","article-title":"Hard and\nSoft Acids and Bases: Structure and Process","volume":"116","author":"Reed","year":"2012","journal-title":"J. Phys. Chem. A"},{"key":"2026090809102133600_cit16","doi-asserted-by":"publisher","first-page":"691","DOI":"10.1021\/acs.oprd.4c00443","article-title":"TFA Cleavage Strategy\nfor Mitigation of S-tButylated Cys-Peptide Formation in Solid-Phase\nPeptide Synthesis","volume":"29","author":"Mthembu","year":"2025","journal-title":"Org. Process Res. Dev."},{"key":"2026090809102133600_cit17","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1111\/j.1399-3011.1991.tb00755.x","article-title":"Cyclization Of Disulfide-Containing\nPeptides In Solid-Phase Synthesis","volume":"37","author":"Albericio","year":"1991","journal-title":"Int. J. Pept.\nProtein Res."},{"key":"2026090809102133600_cit18","doi-asserted-by":"publisher","first-page":"606","DOI":"10.1039\/B701677H","article-title":"Amide bond formation:\nbeyond the myth of coupling reagents","volume":"38","author":"Valeur","year":"2009","journal-title":"Chem.\nSoc. Rev."},{"key":"2026090809102133600_cit19","doi-asserted-by":"publisher","first-page":"10929","DOI":"10.1039\/D4GC03209H","article-title":"Solid phase peptide\nsynthesis using side-chain unprotected arginine and histidine with\nOxyma Pure\/TBEC in green solvents","volume":"26","author":"Fantoni","year":"2024","journal-title":"Green Chem."},{"key":"2026090809102133600_cit20","doi-asserted-by":"publisher","first-page":"194","DOI":"10.1111\/j.1399-3011.1998.tb01216.x","article-title":"Fmoc\/Trt-amino\nacids: comparison\nto Fmoc\/tBu-amino acids in peptide synthesis","volume":"51","author":"Barlos","year":"1998","journal-title":"J. Pept. Res."},{"key":"2026090809102133600_cit21","doi-asserted-by":"publisher","first-page":"1520","DOI":"10.1021\/acs.oprd.2c00083","article-title":"Side-Chain\nUnprotected Fmoc-Arg\/His\/Tyr-OH\nCouplings and Their Application in Solid-Phase Peptide Synthesis through\na Minimal-Protection\/Green Chemistry Strategy","volume":"26","author":"Yang","year":"2022","journal-title":"Org. Process Res. Dev."},{"key":"2026090809102133600_cit22","doi-asserted-by":"publisher","first-page":"11362","DOI":"10.1021\/acs.joc.2c00828","article-title":"Water-Based\nSolid-Phase\nPeptide Synthesis without Hydroxy Side Chain Protection","volume":"87","author":"Hojo","year":"2022","journal-title":"J. Org. Chem."},{"key":"2026090809102133600_cit23","doi-asserted-by":"publisher","DOI":"10.1016\/j.rechem.2025.102664","article-title":"Changing permanent protecting groups\nfor avoiding tert-butylation in solid-phase peptide synthesis of somatostatinamide","volume":"18","author":"Gamal","year":"2025","journal-title":"Res. Chem."}],"container-title":["ACS Omega"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acsomega.6c07280","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/acsodf\/article-pdf\/11\/35\/53469\/67483568\/acsomega.6c07280.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/acsodf\/article-pdf\/11\/35\/53469\/67483568\/acsomega.6c07280.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T13:10:31Z","timestamp":1788873031000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/acsodf\/article\/11\/35\/53469\/5336655\/Tyr-Mpa-Proximity-Induced-Atypical-tert-Butyl"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,27]]},"references-count":23,"journal-issue":{"issue":"35","published-online":{"date-parts":[[2026,8,27]]},"published-print":{"date-parts":[[2026,9,8]]}},"URL":"https:\/\/doi.org\/10.1021\/acsomega.6c07280","relation":{},"ISSN":["2470-1343"],"issn-type":[{"value":"2470-1343","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,27]]}}}