Improved Fmoc Solid-Phase Peptide Synthesis of Oxytocin with High Bioactivity

Abstract

We described here the synthesis of oxytocin by an improved Fmoc solid-phase peptide synthesis (SPPS) method with a Rink-Amide resin as the solid support, HBTU as the coupling reagent, Fmoc-protected amino acids as the building blocks, and piperazine for Fmoc removal as a substitute for the standard reagent piperidine. Unlike previously reported syntheses, the removal of the S-Acm protecting group of Cys and cyclization forming the disulfide bond were carried out by using iodine on the resin with the fully protected peptide chains. Finally, a crude oxytocin with a purity of 92% was obtained by simultaneous cleavage of the peptide chains from the resin and removal of all side-chain protecting groups with trifluoroacetic acid containing the scavengers (yield 85%). The crude peptide was purified by using preparative RP-HPLC to obtain oxytocin (high purity 99.3%) with a bioactivity of 588 IU/mg, the highest reported so far in the literature. This investigation provides a contribution in efforts for the large-scale synthesis of oxytocin in high purity under mild conditions with iodine for on-resin disulfide bond formation and a substitute for the standard Fmoc-deprotecting reagent piperidine, a controlled substance.

Key words

oxytocin - Fmoc solid-phase peptide synthesis - on-resin cyclization - disulfide bond formation by iodine - piperazine

• References

• 1 Robinson DA. Wei F. Wang GD. Li P. Kim SJ. Vogt SK. Muglia LJ. Zhuo M. J. Physiol. 2002; 540: 593
• 2a Honnebier MB. Figueroa JP. Rivier J. Vale W. Nathanielsz PW. J. Dev. Physiol. 1989; 12: 225
• 2b Shukovski L. Reprod., Fertil. Dev. 1992; 4: 99
• 2c Gurrieri F. Neri G. BMC Med. 2009; 7: 1
• 2d Sasayama D. Hattori K. Teraishi T. Hori H. Ota M. Yoshida S. Arima K. Higuchi T. Amano N. Kunugi H. Schizophr. Res. 2012; 139: 201
• 2e McGregor IS. Bowen MT. Horm. Behav. 2012; 61: 331
• 2f Seng J. Miller J. Sperlich M. van de Ven Cosmas JM. Brown S. Carter CS. Liberzon I. J. Trauma Dissociation 2013; 14: 40
• 2g Poulin MJ. Holman EA. Horm. Behav. 2013; 63: 510
• 2h Calcagnoli F. de Boer SF. Althaus M. den Boer JA. Koolhaas JM. Psychopharmacology (Heidelberg, Ger.) 2013; 229: 639
• 2i Peltola MikkoJ. Yrttiaho S. Puura K. Leppanen JukkaM. Proverbio AliceM. Mononen N. Lehtimaki T. Emotion 2014; 14: 469
• 3 du Vigneaud V. Ressler C. Trippett S. J. Biol. Chem. 1953; 205: 949
• 4 Vigneaud VD. Ressler C. Swan CJ. M. Roberts CW. Katsoyannis PG. Gordon S. J. Am. Chem. Soc. 1953; 75: 4879
• 5 Bodanszky M. du Vigneaud V. J. Am. Chem. Soc. 1959; 81: 2504
• 6a Bodanszky M. du Vigneaud V. J. Am. Chem. Soc. 1959; 81: 5688
• 6b Sakakibara S. Nobuhara Y. Shimonishi Y. Kiyoi R. Bull. Chem. Soc. Jpn. 1965; 38: 120
• 7 Vigneaud VD. Schneider CH. Stouffer JE. Murti VV. S. Aroskar JP. Winestock G. J. Am. Chem. Soc. 1962; 84: 409
• 8 Beyerman HC. Bontekoe JS. Koch AC. Recl. Trav. Chim. Pays-Bas. 1959; 78: 935
• 9 du Vigneaud V. Winestock G. Murti VV. S. Hope DB. Kimbrough RD. J. Biol. Chem. 1960; 235: 64
• 10 Ives DA. J. Can. J. Chem. 1968; 46: 2318
• 11 Photaki I. J. Am. Chem. Soc. 1966; 88: 2292
• 12 Fujii N. Otaka A. Funakoshi S. Bessho K. Watanabe T. Akaji K. Yajima H. Chem. Pharm. Bull. 1987; 35: 2339
• 13 Akaji K. Tatsumi T. Yoshida M. Kimura T. Fujiwara Y. Kiso Y. J. Chem. Soc., Chem. Commun. 1991; 3: 167
• 14a Manning M. J. Am. Chem. Soc. 1968; 90: 1348
• 14b Spatola AF. Cornelius DA. Hruby VJ. Blomquist AT. J. Org. Chem. 1974; 39: 2207
• 15a Takashima H. Du Vigneaud V. Merrifield RB. J. Am. Chem. Soc. 1968; 90: 1323
• 15b Baxter JW. M. Manning M. Sawyer WH. Biochemistry 1969; 8: 3592
• 15c Manning M. Coy E. Sawyer WH. Biochemistry 1970; 9: 3925
• 16 Live DH. Agosta WC. Cowburn D. J. Org. Chem. 1977; 42: 3556
• 17 Shih H. J. Org. Chem. 1993; 58: 3003
• 18 Albericio F. Hammer RP. García-Echeverría C. Molins MA. Chang JL. Munson MC. Pons M. Giralt E. Barany G. Int. J. Pept. Protein Res. 1991; 37: 402
• 19 Li ST. Zhao HL. Gao Y. Wang XQ. Wang LY. Chinese J. Phar. 2015; 01: 7
• 20a Barlos K. Gatos D. Pept. Sci. 1999; 51: 266
• 20b Chan W. White PD. Fmoc Solid Phase Peptide Synthesis: A Practical Approach. 1st ed Oxford University Press; Oxford: 2000
• 20c Hughes AB. Amino Acids, Peptides and Proteins in Organic Chemistry, V3: Building Blocks, Catalysis and Coupling Chemistry. Wiley-VCH; Weinheim: 2011
• 21 Sarin VK. Kent SB.H. Tan JP. Merrifield RB. Anal. Biochem. 1981; 117: 147
• 22 Ralhan K. KrishnaKumar VG. Gupta S. RSC Adv. 2015; 5: 104417
• 23 EDQM, European Pharmacopoeia 9.0. 2017, 3250