On the mechanism of degradation of oxytocin and its analogues in aqueous solution

• Published in: Biopolymers Vol. 100; no. 4; pp. 408 - 421
• Main Authors: Wiśniewski, Kazimierz, Finnman, Jens, Flipo, Marion, Galyean, Robert, Schteingart, Claudio D.
• Format: Journal Article
• Language: English
• Published: United States Blackwell Publishing Ltd 01.07.2013
• Subjects: Amino Acid Sequence; Chromatography, High Pressure Liquid; Disulfides - chemistry; Magnetic Resonance Spectroscopy; mechanism of degradation; OT analogues; oxytocin; Oxytocin - analogs & derivatives; stability in solution; β-elimination; oxytocin; β-elimination; stability in solution; OT analogues; mechanism of degradation
• ISSN: 0006-3525; 1097-0282
• DOI: 10.1002/bip.22260

ABSTRACT Oxytocin (OT) is a cyclic nonapeptide containing one internal disulfide bond between its Cys1 and Cys6 residues. Although OT is one of the most commonly used peptidic drugs, the mechanism of its degradation in aqueous solution and the identity of its degradants have not been fully elucidated. To investigate the pathways and products of OT degradation in slightly acidic to neutral solutions, we prepared the peptides: OT, [d‐Cys1]OT, a series of N‐alkylated OT analogues, [[13C3,15N]Cys1]OT, and OT where each sulfur atom was systematically replaced by either methylene, 34S, or Se. The peptides were incubated at 40°C and the degradation products studied by HPLC, LCMS, and 13C‐NMR. Our findings suggest that the degradation begins with β‐elimination of the disulfide linkage to form a putative intermediate linear peptide containing an S‐thiocysteine (a persulfide) in position 6 and a dehydroalanine in position 1. This intermediate persulfide appears to donate a sulfur atom to an intact OT molecule to form OT trisulfide and higher monomeric polysulfides, while the dehydroalanine residue is hydrolyzed with loss of the N‐terminal amino group to yield a linear N‐pyruvoylated octapeptide containing a reduced Cys6. Based on the MS and 13C‐NMR data of the products from degradation of [[13C3,15N]Cys1]OT, we postulate that the ultimate degradation products of OT are dimers composed of two pyruvoylated octapeptides held together by one disulfide bridge between the two Cys6 residues and by one more, non‐reducible, linkage resulting from an aldol‐type condensation between the two N‐terminal pyruvoyl groups. © 2013 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 408–421, 2013.