Abstract
The title peptide, oxytocin trisulfide (3), was required on a multi-100 mg scale, as part of an analytical method validation process for the clinically and commercially important product “Oxytocin Injection,” which is a sterile isotonic injectable formulation of oxytocin (2). We report here that previous chemistry from our academic laboratory can be successfully scaled up in the biotechnology sector to indeed provide pure 3 (overall yield 6–7%, > 95% purity). The scaled-up synthesis also gave rise, somewhat unexpectedly, to modest levels of oxytocin tetrasulfide (4), and even higher homologues. Protocols for synthesis, purification, and characterization [by HPLC, mass spectrometry and amino acid analysis] are described and discussed, along with possible mechanistic explanations for our findings.
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Notes
Information from the United States of America Food and Drug Administration (FDA) website. See https://labels.fda.gov/ in general, and specifically https://www.accessdata.fda.gov/drugsatfda_docs/label/2014/018261s031lbl.pdf (PITOCIN label).
Further biological indications of the hormone, as yet not harnessed clinically, have been reviewed (Magon and Kalra 2011).
In the nomenclature of contemporary organosulfur chemistry, compounds with two are more linearly connected sulfurs are referred to as (poly)sulfanes (suffix “ane”), but the “ide” suffix is so widely entrenched historically that we continue to name the range of peptide derivatives described throughout this article as di-, tri-, tetra-, etc., -sulfides.
The AAA protocol used adheres to guidance from the United States Pharmacopeia (USP) for quantitation of peptide and protein concentration following hydrolysis to constituent amino acids; USP < 1052>.
Two reviewers correctly pointed out an apparent inconsistency between the distribution of products in the crude material and the isolated yields after purification. We believe that the isolation of pure oxytocin (disulfide) (2) was highly efficient [and matches the expected amount], whereas the isolation of pure trisulfide (3) was not nearly as efficient because we placed a premium on obtaining highly pure material over maximizing the yield. It is also possible that in the leadup to purification, some of the 3 that had been produced may have lost a sulfur atom, hence raising the yield of 2 while lowering the yield of 3.
The requirement for highly dilute concentrations during literature solution cyclizations should be contrasted to the effective concentration for the cyclization method described in this paper, which can be estimated as two orders of magnitude higher, i.e., 7 to 10 mm.
Higher polysulfides are almost surely present, albeit in diminishing amounts. For the present work, we provide strong support for the presence of oxytocin pentasulfide, based on UPLC/QTOF-MS of the crude product mixture.
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Acknowledgements
We thank Teva Parenteral Medicines, Inc. for sponsorship of this research. We are grateful to Brandon C. Smith of Biosyn for help in retrieving data from a dozen years ago and for insightful current discussions, and appreciate the contributions of Kristin M. Coari and Yi Z. Wang to experiments aimed at the efficient creation of compound 6. Finally, we thank Kaeli Hammer for assistance with graphics.
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Experimental design and supervision, RPH and GB; Organosulfur chemistry, PTG and AMS; Peptide synthesis, KD and RPH; Peptide purification and analytical characterization, MAB and RPH; UPLC/ESI-MS, JJD; Writing, GB and RPH; Background and literature review, RPH, AB, and GB. All authors have read and agreed to the published version of the manuscript.
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None of the authors have competing interests that are relevant to the content of this article. None of the authors have any relevant financial interests to disclose. In the early 2010s, although not overlapping with the duration of the sponsored support to his research program at the University of Minnesota, GB consulted for Teva Parenteral Medicines, and received appropriate remuneration.
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Hammer, R.P., Butrie, M.A., Davidson, K. et al. Scaled-up Synthesis and Characterization of Oxytocin Trisulfide. Int J Pept Res Ther 30, 5 (2024). https://doi.org/10.1007/s10989-023-10580-9
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DOI: https://doi.org/10.1007/s10989-023-10580-9