Tyr-Mpa Proximity-Induced Atypical tert-Butyl Intramolecular Migration in Desmopressin: Mechanistic Elucidation of a Site-Selective Impurity and Its Suppression Strategy.

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 "source-elimination" 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.