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Optimization of the chemical synthesis of oxytocin
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Mojtaba Bijani   , Hossein Attar , Hooman Kaghazian *  |
| Pasteur Institute of Iran, Tehran, Iran |
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Abstract: (11 Views) |
Background and Aim: Oxytocin is a cyclic nonapeptide containing a single intramolecular disulfide bond and plays a key regulatory role in physiological processes, including uterine contraction, lactation, and hypothalamic pituitary adrenal axis activity. Due to its structural sensitivity and short half-life, achieving highly pure and pharmaceutically acceptable synthetic oxytocin requires rigorous and optimized peptide synthesis strategies. The aim of this study was to optimize the solid-phase chemical synthesis of oxytocin and to evaluate the physicochemical quality attributes of the final product.
Methods: Peptide assembly was performed on Rink amide resin using HATU as the coupling reagent and N,N-diisopropylethylamine as the organic base. Following chain elongation, the peptide was cleaved from the resin using a trifluoroacetic acid/triethylsilane/water mixture. The intramolecular disulfide bond between Cys¹ and Cys⁶ was formed under controlled alkaline conditions at pH≈8. Crude material was purified by preparative reversed-phase chromatography. Final analyses included analytical RP-HPLC, electrospray ionization mass spectrometry, pH measurement, endotoxin testing, and visual inspection.
Results: The synthesized peptide exhibited a purity of 99.6%, a single sharp chromatographic peak, and a molecular mass fully consistent with the theoretical value. Mass spectrometry showed a dominant ion at m/z 1007.2 ([M+H]⁺). The measured pH of a 1 mg/mL solution was 5.2 ± 0.1. Endotoxin levels were below 0.25 EU/mL, confirming microbiological safety and the absence of pyrogenic contaminants. Collectively, these findings demonstrate that the optimized process produced oxytocin with correct disulfide cyclization, structural integrity, and pharmaceutical-grade purity.
Conclusion: Stepwise optimization of the solid-phase synthesis, specifically adjusting the activator/base ratio, extending coupling times, and employing mild controlled oxidation, resulted in high-purity oxytocin with a yield of 69.8% and accurate molecular mass. Shortening the total process time from 80 to 68 hours indicates improved efficiency without compromising quality. The final protocol represents a refined and reliable method with reproducibility and scalability suitable for research and semi-industrial peptide production.
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| Keywords: Optimization, Protein synthesis, Solid phase, Oxytocin hormone |
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Full-Text [PDF 709 kb]
(8 Downloads)
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Type of Study: Original Research |
Subject:
Endocrine
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