Characterization of the Macroscopic and Microscopic Acid‒Base Chemistry of the Native Disulfide and Reduced Dithiol Forms of Oxytocin, Arginine-Vasopressin, and Related Peptides

Béla Noszál, Wei Guo, Dallas L. Rabenstein

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Acid-base properties of the native disulfide and reduced dithiol forms of oxytocin, arginine-vasopressin, tocinoic acid, pressinoic acid, and tocinamide were studied by 500-MHz 1H NMR-pH titrations. Cysteine methyl ester and cysteinylglycine served as model compounds for the N-terminal cysteine-1 residue of the reduced oligopeptides and were studied by UV, 1H NMR, and pHmetry. The resonances for 1H nuclei in the various peptides and the model compounds undergo the expected downfield shift upon protonation of adjacent basic sites, with the exception of the resonances for the δ CH2 protons of the proline-7 residues of oxytocin and arginine-vasopressin, which are shifted significantly upfield, presumably reflecting conformational changes. The interacting basicities of the amino and thiolate groups of the cysteine-1 residues of the reduced dithiol forms of the five peptides and the two model compounds are characterized in terms of protonation microconstants. All other basic groups of the peptides (tyrosine phenolate, cysteine-6 thiolate of the reduced peptides, cysteine-6 carboxylate of the reduced and disulfide forms of tocinoic acid and pressinoic acid, and cysteine-1 amino of the disulfide forms of the peptides) bind protons in isolated pH ranges or are separated by several bonds from other basic groups, and their basicities are characterized in terms of group constants. Analogous groups in the various peptides show somewhat different basicities, depending on the adjacent residues. However, the basicity of the cysteine-6 thiolate of the reduced peptides covers a remarkably wide range. Specifically, the protonation constant of the cysteine-6 thiolate of the reduced forms of tocinoic acid and pressinoic acid is about 1.8 log K units larger than that of the reduced forms of oxytocin and arginine-vasopressin, due to the significantly different electron-withdrawing effects of the adjacent carboxylate and peptide groups, while the protonation constant of the cysteine-6 thiolate group of the reduced form of tocinamide is 0.6 log K units larger than that of oxytocin, even though the covalent environments are the same up to four bonds removed. Using the microscopic and group protonation constants, the probabilities of both thiol groups being in the ionized form were calculated as a function of pD for the dithiol forms of the five peptides. The results show that, at physiological pH, intramolecular disulfide bond formation via thiolate anions is predicted to be more favorable for the nonapeptides oxytocin and arginine-vasopressin than for tocinamide, which in turn is more favorable than for tocinoic acid and pressinoic acid.

Original languageEnglish
Pages (from-to)2327-2334
Number of pages8
JournalJournal of Organic Chemistry
Issue number8
Publication statusPublished - Apr 1 1992


ASJC Scopus subject areas

  • Organic Chemistry

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