title: On the mechanism of degradation of oxytocin and its analogues in aqueous solution.
abstract: Oxytocin (OT) is a cyclic nonapeptide containing one internal disulfide bond between its Cys(1) and Cys(6) 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-Cys(1)]OT, a series of N-alkylated OT analogues, [[(13)C3,(15) N]Cys(1)]OT, and OT where each sulfur atom was systematically replaced by either methylene, (34)S, or Se. The peptides were incubated at 40°C and the degradation products studied by HPLC, LCMS, and (13)C-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 Cys(6). Based on the MS and (13)C-NMR data of the products from degradation of [[(13)C3,(15)N]Cys(1)]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 Cys(6) residues and by one more, non-reducible, linkage resulting from an aldol-type condensation between the two N-terminal pyruvoyl groups.
authors.affiliation: Ferring Research Institute, 4245 Sorrento Valley Blvd, San Diego, CA 92121.
authors.forename: Kazimierz
authors.initials: K
authors.lastname: Wiśniewski
authors.forename: Jens
authors.initials: J
authors.lastname: Finnman
authors.forename: Marion
authors.initials: M
authors.lastname: Flipo
authors.forename: Robert
authors.initials: R
authors.lastname: Galyean
authors.forename: Claudio D
authors.initials: CD
authors.lastname: Schteingart
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D010121:Oxytocin / Q000031:analogs & derivatives
chemical_list: D004220:Disulfides; D010121:Oxytocin
keywords: OT analogues; mechanism of degradation; oxytocin; stability in solution; β-elimination