Solid-phase Synthesis Of Oxytocin And Its Purification

Oxytocin is a cyclic nonapeptide containing a disulfide bond.It is mainly produced in the posterior lobe of the pituitary of humans and mammalian animals.In the 1950s,scientists determined the sequence of this peptide and synthesized it by solution phase synthesis.With the development of peptide synthesis chemistry,the method of synthesizing this peptide has been greatly improved.The complicated and tedious liquid phase synthesis was replaced by a simple solid phase synthesis for the synthesis of long peptide,leading to a higher efficiency of synthesis.In this paper,we synthesized oxytocin by solid phase peptide synthesis.We selected Rink-Amide resin as the solid support,HBTU as a coupling agent and Fmoc protected amino acids for building blocks.Iodine was used to form the disulfide bond of oxytocin on solid support.Finally,we obtained crude oxytocin after cleavage of the peptide from the resin and removal of protecting groups on the amino acid using trifluoroacetic acid.In this project,piperazidine was used for Fmoc deprotection instead of piperidine.Piperazidine is a solid crystal compound,to be easier to transport and handle,more importantly,not a controlled chemical.In the synthesis stage,we used the same procedures for the synthesis of several truncated peptide impurities and analyzed them by ESI-mass spectrometry and RP-HPLC,confirmed that there were no truncated peptide impurities in the oxytocin samples we synthesized.In this study,we optimized the reaction conditions of cyclization on the solid support so that the product had higher purity with simple operation.We studied the effect of several mobile phases on the RP-HPLC purification of the crude samples,and chose phosphate buffer based acetonitrile as the mobile phase system.Finally we prepared pure oxytocin by this method.In the desalting process,we adopted a relatively new technique using reverse phase polymer,which was well tolerant to strong acid and alkaline(stable to strong acid and alkaline),low cost and long lifetime,leading to an economical way of oxytocin production.After lyophilization of oxytocin solution,we measured its potency by chemical method,which met the standard of Chinese Pharmacopoeia.In this paper,a convenient solid-phase synthesis method was used to synthesize oxytocin,which only took 3 days.The disulfide-bond was formed by carrying out the iodine oxidation step directly on solid support to increase the purity,yield of crude product with simple process.The purification process was also studied for crude sample.Our research provided a promising method for synthesis of oxytocin.