Molecular Biotechnology

, Volume 33, Issue 3, pp 239–254 | Cite as

Methods and protocols of modern solid phase peptide synthesis

  • Muriel AmblardEmail author
  • Jean-Alain Fehrentz
  • Jean Martinez
  • Gilles Subra


The purpose of this article is to delineate strategic considerations and provide practical procedures to enable non-experts to synthesize peptides with a reasonable chance of success. This article is not encyclopedic but rather devoted to the Fmoc/tBu approach of solid phase peptide synthesis (SPPS), which is now the most commonly used methodology for the production of peptides. The principles of SPPS with a review of linkers and supports currently employed are presented. Basic concepts for the different steps of SPPS such as anchoring, deprotection, coupling reaction and cleavage are all discussed along with the possible problem of aggregation and side-reactions. Essential protocols for the synthesis of fully deprotected peptides are presented including resin handling, coupling, capping, Fmoc-deprotection, final cleavage and disulfide bridge formation.

Index Entries

Solid phase peptide synthesis (SPPS) resin Fmoc SPPS coupling reagents protecting groups anchoring side reaction 


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  1. 1.
    Merrifield, R. B. (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149–2154.CrossRefGoogle Scholar
  2. 2.
    Bergmann, M. and Zervas, L. (1928) Über katalytische racemisation von aminosäuren und peptiden. Biochem. Z. 203, 280–292.Google Scholar
  3. 3.
    Goodman, M. and Levine, L. (1964) Peptide synthesis via active esters. IV. Racemization and ring-opening reactions of optically active oxazolones. J. Am. Chem. Soc. 86, 2918–2922.CrossRefGoogle Scholar
  4. 4.
    Carpino, L. A. and Han, G. Y. (1972) 9-Fluorenyl-methoxycarbonyl amino-protecting group. J. Org. Chem. 37, 3404–3409.CrossRefGoogle Scholar
  5. 5.
    Forreview Lloyd-Williams, P., Giralt, E. and Albericio F. (eds.) (1997) Chemical Approaches to the Synthesis of Peptides and Proteins, CRCLLC, NY.Google Scholar
  6. 6.
    Czarnik, A. W. (1998) Solid-phase synthesis supports are like solvents. Biotechnol. Bioeng. (Comb. Chem.) 61, 77–79.CrossRefGoogle Scholar
  7. 7.
    Sherrington, D. C. (1998) Preparation, structure and morphology of polymer supports. Chem. Commun. 2275–2286.Google Scholar
  8. 8.
    Rink H. (1987) Solid-phase synthesis of protected peptide fragments using a trialkoxy-diphenyl-methylester resin. Tetrahedron Lett. 28, 3787–3790.CrossRefGoogle Scholar
  9. 9.
    Bernatowicz, M. S., Daniels, S. B. and Köster, H. (1989) A comparison of acid labile linkage agents for the synthesis of peptide C-terminal amides. Tetrahedron Lett. 30, 4645–4648.CrossRefGoogle Scholar
  10. 10.
    Sieber, P. (1987) A new acid-labile anchor group for the solid-phase synthesis of C-terminal peptide amides by the Fmoc method. Tetrahedron Lett. 28, 2107–2110.CrossRefGoogle Scholar
  11. 11.
    Wang, S.-S. (1973) p-Alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments. J. Am. Chem. Soc. 95, 1328–1333.PubMedCrossRefGoogle Scholar
  12. 12.
    Mergler, M., Nyfeler, R., Tanner, R., Gosteli, J., and Grogg, P. (1988) Peptide synthesis by a combination of solid-phase and solution methods II synthesis of fully protected peptide fragments on 2-methoxy-4-alkoxy-benzyl alcohol resin. Tetrahedron Lett. 29, 4009–4012.CrossRefGoogle Scholar
  13. 13.
    Flörsheimer, A. and Riniker, B. (1991). Solid-phase synthesis of peptides with the highly acid-sensitive HMPB linker. in Peptides 1990: Proceedings of the 21st European Peptide Symposium (Giralt, E. and Andreu, D. eds) ESCOM. Lieden, 131–133.Google Scholar
  14. 14.
    Barlos, K., Gatos, D., Kallitsis, J., Papaphotiu, G., Sotiriu, P., Wenqing, Y. and Schäfer, W. (1989) Darstellung geschützter peptid-fragmente unter einsatz substituierter triphenylmethyl-harze. Tetrahedron Lett. 30, 3943–3946.CrossRefGoogle Scholar
  15. 15.
    Albericio, F. (2000) Orthogonal Protecting groups for Nα-amino and C-terminal carboxylic functions in solid-phase peptide synthesis. Biopolymers (Peptide Science) 55, 123–139.CrossRefGoogle Scholar
  16. 16.
    Coste, J., Le Nguyen, D. and Castro, B. (1990) PyBOP®: A new peptide coupling reagent devoid of toxic by-product. Tetrahedron Lett. 31, 205–208.CrossRefGoogle Scholar
  17. 17.
    Knorr, R., Trzeciak, A., Bannwarth, W. and Gillessen, D. (1989) New coupling reagents in peptide chemistry. Tetrahedron Lett. 30, 1927–1930.CrossRefGoogle Scholar
  18. 18.
    Dourtouglou, V., Ziegler, J. C. and Gross, B. (1978) L'hexafluorophosphate de O-benzotriazolyl-N,N-tetramethyluronium: un réactif de couplage peptidique nouveau et efficace. Tetrahedron Lett. 19, 1269–1272.CrossRefGoogle Scholar
  19. 19.
    Carpino, L. A. (1993) 1-Hydroxy-7-azabenzotriazole. An efficient peptide coupling additive. J. Am. Chem. Soc. 115, 4397–4398.CrossRefGoogle Scholar
  20. 20.
    Carpino, L. A., El-Faham, A., Minor, C. A. and Albericio, F. (1994) Advantagenous applications of azabenzotriazole (triazolopyridine)-based coupling reagents to solid-phase peptide synthesis. J. Chem. Soc. Chem. Commun. 201–204.Google Scholar
  21. 21.
    Story S. C. and Aldrich J. V. (1994) Side-product for-mation during cyclization with HBTU on a solid support. Int. J. Pept. Protein Res. 43, 292–296.PubMedCrossRefGoogle Scholar
  22. 22.
    Westall, F. C. and Robinson, A. B. (1970) Solvent modification in Merrifield solid-phase peptide synthesis. J. Org. Chem. 35, 2842–2844.PubMedCrossRefGoogle Scholar
  23. 23.
    Yamashiro, D., Blake, J. and Li, C. M. (1976) The use of trifluoroethanol for improved coupling in solid-phase peptide synthesis. Tetrahedron Letters 17, 1469–1472.CrossRefGoogle Scholar
  24. 24.
    Milton, S. C. and Milton, R. C. (1990) An improved solid-phase synthesis of a difficult-sequence peptide using hexafluoro-2-propanol. Int. J. Pept. Protein Res. 36, 193–196.PubMedCrossRefGoogle Scholar
  25. 25.
    Hendrix, J. C., Halverson, K. J., Jarrett, J. T. and Lansbury, P. T. (1990) A novel solvent system for solid-phase synthesis of protected peptides: the disaggregation of resin-bound antiparallel beta-sheet. J. Org. Chem. 55, 4517–4518.CrossRefGoogle Scholar
  26. 26.
    Hyde, C. B., Johnson, T. and Sheppard, R. C. (1992). Internal Aggregation during Solid Phase Peptide Synthesis. Dimethyl Sulfoxide as a Powerful Dissociating Solvent. J. Chem. Soc. Chem. Commun. 1573–1575.Google Scholar
  27. 27.
    Stewart, J. M. and Klis, W. A. (1990) Peptides, polypeptides and oligonucleotides. Macro-organic reagents and catalysts and biomedical applications, in Innovation and Perspective in solid phase synthesis and related technologies (Epton, R., ed.): Mayflower Worldwide Ltd. Birmingham, pp. 1–9.Google Scholar
  28. 28.
    Zhang, L., Goldhammer, C., Henkel, B., Zühl, F., Panhaus, G., Jung, G. and Bayer, E. (1994) Peptides, proteins and nucleic acids. Biological and biomedical applications, in Innovation and Perspectives in solid phase synthesis (Epton, R., ed.): Mayflower Worldwide Ltd. Birmingham, pp. 711–716.Google Scholar
  29. 29.
    Bedford, J., Hyde C., Johnson, T., Jun, W., Owen, D., Quibell, M. and Sheppard, R. C. (1992) Amino acid structure and “difficult sequences” in solid phase peptide synthesis. Int. J. Pept. Protein Res. 40 300–307.PubMedCrossRefGoogle Scholar
  30. 30.
    Hyde, C., Johnson, T., Owen, D., Quibell, M. and Sheppard, R. C. (1994) Some difficult sequences made easy. A study of interchain association in solid-phase peptide synthesis. Int. J. Pept. Protein Res. 43, 431–440.PubMedCrossRefGoogle Scholar
  31. 31.
    Mutter, M., Nefzi, A., Sato, T., Sun, X., Wahl, F. and Wohr, T. (1995) Pseudo-prolines (psi Pro) for accessing “inaccessible” peptides. Peptide Res. 8, 145–153.Google Scholar
  32. 32.
    Wohr, T., Wahl, F., Nefzi, A., Rohwedder, B., Sato, T., Sun, X. and Mutter, M. (1996) Pseudo-Prolines as a Solubilizing, Structure-Disrupting Protection Technique in Peptide Synthesis. J. Am. Chem. Soc. 118, 9218–9227.CrossRefGoogle Scholar
  33. 33.
    Guichou, J. F., Patiny, L. and Mutter, M. (2002) Pseudo-prolines (Pro): direct insertion of Pro systems into cysteine containing peptides. Tetrahedron Lett. 43, 4389–4390.CrossRefGoogle Scholar
  34. 34.
    Pearson, D. A., Blanchette, M., Baker, M. L. and Guindon, C. A. (1989) Trialkylsilanes as scavengers for the trifluoroacetic acid deblocking of protecting groups in peptide synthesis. Tetrahedron Lett. 30, 2739–2742.CrossRefGoogle Scholar
  35. 35.
    Tam, J. P., Wu, C. R., Liu, W. and Zhang, J. W. (1991) Disulfide bond formation in peptides by dimethyl sulfoxide: scope and applications. J. Am. Chem. Soc. 113, 6657–6662.CrossRefGoogle Scholar
  36. 36.
    Quibell, M., Owen, D., Packman, L. C., Johnson, T. (1994) Suppression of piperidine-mediated side product formation for Asp(OBut)-containing peptides by the use of N-(2-hydroxy-4-methoxybenzyl)(Hmb) back bone amide protection. J. Chem. Soc. Chem. Commun. 20, 2343–2344.CrossRefGoogle Scholar
  37. 37.
    Han, Y., Albericio, F. and Barany, G. (1997) Occurence and minimization of cysteine racemization during stepwise solid-phase peptide synthesis. J. Org. Chem. 62, 4307–4312.PubMedCrossRefGoogle Scholar

Copyright information

© Human Press Inc 2006

Authors and Affiliations

  • Muriel Amblard
    • 1
    Email author
  • Jean-Alain Fehrentz
    • 1
  • Jean Martinez
    • 1
  • Gilles Subra
    • 1
  1. 1.Laboratoire des Amino Acides. Peptides et Protéine UMR-CNRS 5810Faculté de PharmacieMontpellier cedex 5France

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