Synthesis of heptapeptides and analysis of sequence by tandem ion trap mass spectrometry

Chenxi Jia 1 , Wei Qi 1 , Zhimin He 1 , Haoming Yang 1 , and Bin Qiao 1
  • 1 Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin, 300072, People’s Republic of China


Two heptapeptides have been prepared by Fmoc methodology using Wang resin as solid support. For attachment of the first amino acid, several coupling systems were evaluated, and DIC/DMAP system could give yields of >99% and low levels of racemization. The selection of scavenger combination to deprotect side chains revealed that H2O/p-cresol was good at scavenging trityl and 1,2-ethanedithiol was highly efficient for scavenging t-butyl. Through shortening the preactivation time to 5 min, the racemization which occurred during formation of amide bonds coupled by HBTU was minimized. The crude peptides were characterized by RP-HPLC and MS, and sequenced by MS/MS to acquire reliable amino acid sequence information.

If the inline PDF is not rendering correctly, you can download the PDF file here.

  • [1] S.A. Kates and F. Albericio: Solid-Phase Synthesis, Marcel Dekker, Inc., New York, 2000.

  • [2] R.B. Merrifield: “Solid phase peptide synthesis. I. The synthesis of a tetrapeptide”, J. Am. Chem. Soc., Vol. 85, (1963), pp. 2149–2154.

  • [3] W.C. Chan and P.D. White: Fmoc Solid Phase Peptide Synthesis. A Practical Approach, Oxford University Press, Inc., New York, 2000.

  • [4] Z.G. Zhang, H.B. Cai, L. Bai and S.L. He: “A comparison between the yield of the attachment of Boc-AA-O−Cs+ to Merrifeild resin and of Fmoc-AA-OH to Wang resin”, Chinese J. Org. Chem., Vol. 20, (2000), pp. 419–423.

  • [5] A. Eric, R.C. Linda and C.S. Robert: “Peptide synthesis: Part 10. Use of pentafluorophenyl esters of fluorenylmethoxycarbonylamino acids in solid phase peptide synthesis”, Tetrahedron, Vol. 44, (1988), pp. 843–857.

  • [6] Y.H. Ye, T.H. Li and X.H. Jiang: “DEPBT as an efficient coupling reagent for amide bond formation with remarkable resistance to racemization”, Biopolymers, Vol. 80, (2005), pp. 172–178.

  • [7] M. Kunishima, A. Kitao, C. Kawachi, Y. Watanabe, S. Iguchi, K. Hioki and S. Tani: “A racemization test in peptide synthesis using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM)”, Chem. Pharm. Bull., Vol. 50, (2002), pp. 549–550.

  • [8] M.P. Souza, M.F.M. Tavares and M.T.M. Miranda: “Racemization in stepwise solid-phase peptide synthesis at elevated temperatures”, Tetrahedron, Vol. 60, (2004), pp. 4671–4681.

  • [9] Y.M. Angell, J. Alsina, F. Albericio and G. Barany: “Practical protocols for stepwise solid-phase synthesis of cysteine-containing peptides”, J. Peptide Res., Vol. 60, (2002), pp. 292–299.

  • [10] A.D. Fenza, M. Tancredi, C. Galoppini and P. Rovero: “Racemization studies of Fmoc-Ser(tBu)-OH during stepwise continuous-flow solid-phase peptide synthesis”, Tetrahedron Lett. Vol. 39, (1998), pp. 8529–8532.

  • [11] T. Tedeschi, R. Corradini, R. Marchelli, A. Pushl and P.E. Nielsen: “Racemization of chiral PNAs during solid-phase synthesis: effect of the coupling conditions on enantiomeric purity”, Tetrahedron: Asymmetry, Vol. 13, (2002), pp. 1629–1636.

  • [12] R. Corradini, S. Sforza, A. Dossena, G. Palla, R. Rocchi, F. Filira, F. Nastri and R. Marchelli: “Epimerization of peptide nucleic acids analogs during solid-phase synthesis: optimization of the coupling conditions for increasing the optical purity”, J. Chem. Soc., Perkin Trans., Vol. 20, (2001), pp. 2690–2696.

  • [13] D.N.W. Van, S. Yuval and F. Albericio: “Cu(OBt)2 and Cu(OAt)2, copper(II)-based racemization suppressors ready for use in fully automated solid-phase peptide synthesis”, J. Peptide Sci. Vol. 7, (2001), pp. 115–120.

  • [14] L.A. Carpino, A. El-Faham and F. Albericio: “Racemization studies during solid-phase peptide synthesis using azabenzotriazole-based coupling reagents”, Tetrahedron Lett., Vol. 35, (1994), pp. 2279–2282.

  • [15] Y.X. Han, F. Albericio and G. Barany: “Occurrence and minimization of cysteine racemization during stepwise solid-phase peptide synthesis”, J. Org. Chem., Vol. 62, (1997), pp. 4307–4312

  • [16] M.T.W. Hiern: HPLC of Proteins, Peptides and polynucleotides, VCR, New York, 1991.

  • [17] A.D. Fenza and P. Rovero: “Assessment of new 6-Cl-HOBt based coupling reagents for peptide synthesis. Part 2: Racemization studies”, Lett. Peptide Sci., Vol. 9, (2002), pp. 125–129

  • [18] W. Qi, C.X. Jia, Z.M. He and B. Qiao: “Analysis of racemization products of synthetic heptapeptide by reversed phase high performance liquid chromatography/mass spectrometry”, Chinese J. Anal. Chem., submitted for publication.

  • [19] V.H. Wysocki, K.A. Resing, Q.F. Zhang and G.L. Cheng: “Mass spectrometry of peptides and proteins”, Methods, Vol 35, (2005), pp. 211–222.

  • [20] B.M. Souza, M.R. Marques, D.M. Tomazela, M.N. Eberlin, M.A. Mendes and M.S. Palma: “Mass spectrometric characterization of two novel inflammatory peptides from the venom of the social wasp Polybia paulista”, Rapid Commun. Mass Specrom., Vol. 18, (2004), pp. 1095–1102.

  • [21] Y.S. Zou and X.Z. Qian: “Test of the amino group of amino acid and polypeptides on solid phase”, Prog. Biochem. Biophys., Vol. 14, (1978), pp. 12–14.

  • [22] A. Grandas, X. Jorba, E. Giralt and E. Pedroso: “Anchoring of Fmoc-amino acids to hydroxylmethyl resins”, Int. J. Peptide Protein Res., Vol. 33, (1989), pp. 386–390.


Journal + Issues