Introduction to Peptide Synthesis

Maciej Stawikowski1, Gregg B. Fields1

1 Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 18.1
DOI:  10.1002/0471140864.ps1801s69
Online Posting Date:  August, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


A number of synthetic peptides are significant commercial or pharmaceutical products, ranging from the dipeptide sugar substitute aspartame to clinically used hormones such as oxytocin, adrenocorticotropic hormone, and calcitonin. This unit provides an overview of the field of synthetic peptides and proteins. It discusses selecting the solid support and common coupling reagents. Additional information is provided regarding common side reactions and synthesizing modified residues. Curr. Protoc. Protein Sci. 69:18.1.1‐18.1.13. © 2012 by John Wiley & Sons, Inc.

Keywords: peptide; protein; solid‐phase peptide synthesis; coupling reagent; chemoselective ligation; Fmoc–amino acid

PDF or HTML at Wiley Online Library

Table of Contents

  • Development of Solid‐Phase Peptide‐Synthesis Methodology
  • The Solid Support
  • Coupling Reagents
  • Synthesis of Modified Residues and Structures
  • Protein Synthesis
  • Side Reactions
  • Purification and Analysis of Synthetic Peptides
  • Acknowledgement
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

   Albericio, F., Pons, M., Pedroso, E., and Giralt, E. 1989. Comparative study of supports for solid‐phase coupling of protected‐peptide segments. J. Org. Chem. 54:360‐366.
   Albericio, F., Kneib‐Cordonier, N., Biancalana, S., Gera, L., Masada, R.I., Hudson, D., and Barany, G. 1990. Preparation and application of the 5‐(4‐(9‐fluorenylmethyloxycarbonyl)aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid (PAL) handle for the solid‐phase synthesis of C‐terminal peptide amides under mild conditions. J. Org. Chem. 55:3730‐3743.
   Albericio, F., Lloyd‐Williams, P., and Giralt, E. 1997. Convergent solid‐phase peptide synthesis. Methods Enzymol. 289:313‐336.
   Angeletti, R.H., Bonewald, L.F., and Fields, G.B. 1997. Six‐year study of peptide synthesis. Methods Enzymol. 289:697‐717.
   Atherton, E. and Sheppard, R.C. 1987. The fluorenylmethoxycarbonyl amino protecting group. In The Peptides (S. Udenfriend and J. Meienhofer, eds.) pp. 1‐38. Academic Press, New York.
   Ayers, B., Blaschke, U.K., Camarero, J.A., Cotton, G.J., Holford, M., and Muir, T.W. 1999. Introduction of unnatural amino acids into proteins using expressed protein ligation. Peptide Sci. 51:343‐354.
   Barany, G. and Merrifield, R.B. 1979. Solid‐phase peptide synthesis. In The Peptides, Vol. 2 (E. Gross and J. Meienhofer eds.) pp. 1‐284. Academic Press, New York.
   Barlos, K., Gatos, D., Hondrelis, J., Matsoukas, J., Moore, G.J., Schäfer, W., and Sotiriou, P.. 1989a. Darstellung neuer säureempfindlicher Harze vom sek.‐Alkohol‐typ und ihre Anwendung zur Synthese von Peptiden. Liebigs Ann. Chem. 951‐955.
   Barlos, K., Gatos, D., Kallitsis, J., Papaphotiu, G. Sotiriou, P., Wenqing, Y., and Schäfer, W. 1989b. Darstellung geschützter Peptid‐Fragmente unter Einsatz substituierter Triphenylmethyl‐Harze. Tetrahedron Lett. 30:3943‐3946.
   Bergmann, M. and Zervas, L. 1932. Über ein allgemeines Verfahren der Peptid‐synthese. Berichte der Deutschen Chemischen Gesellschaft (A and B Series) 65:1192‐1201.
   Carpino, L.A. and Han, G.Y. 1970. 9‐Fluorenylmethoxycarbonyl function, a new base‐sensitive amino‐protecting group. J. Am. Chem. Soc. 92:5748‐5749.
   Cudic, M. and Burstein, G.D. 2008. Preparation of glycosylated amino acids suitable for Fmoc solid‐phase assembly peptide‐based drug design. In Peptide‐Based Drug Design (L. Otvos, ed.) pp. 187‐208. Humana Press, Totowa, N.J.
   Dawson, P.E., Muir, T.W., Clark‐Lewis, I., and Kent, S.B.H. 1994. Synthesis of proteins by native chemical ligation. Science 266:776‐779.
   Dawson, P.E., Churchill, M.J., Ghadiri, M.R., and Kent, S.B.H. 1997. Modulation of reactivity in native chemical ligation through the use of thiol additives. J. Am. Chem. Soc. 119:4325‐4329.
   du Vigneaud, V.d., Ressler, C., Swan, C.J.M., Roberts, C.W., Katsoyannis, P.G., and Gordon, S. 1953. The synthesis of an octapeptide amide with the hormonal activity of oxytocin. J. Am. Chem. Soc. 75:4879‐4880.
   El‐Faham, A. and Albericio, F. 2011. Peptide coupling reagents, more than a letter soup. Chem. Rev. 111:6557‐6602.
   Felix, A.M., Wang, C.‐T., Heimer, E.P., and Fournier, A. 1988. Applications of BOP reagent in solid phase synthesis. Int. J. Pept. Protein Res. 31:231‐238.
   Fields, C.G. and Fields, G.B. 1994. Solvents for solid‐phase peptide synthesis. Methods Mol. Biol. 35:29‐40.
   Fields, C.G., Fields, G.B., Noble, R.L., and Cross, T.A. 1989. Solid phase peptide synthesis of 15N‐gramicidins A, B, and C and high performance liquid chromatographic purification. Int. J. Pept. Protein Res. 33:298‐303.
   Fields, G.B. (ed.) 1997. Solid‐phase peptide synthesis. Methods in Enzymology, Vol. 289. Academic Press, Orlando, Fla.
   Fields, G.B. and Noble, R.L. 1990. Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35:161‐214.
   Fields, G.B., Otteson, K.M., Fields, C.G., and Noble, R.L. 1990. The versatility of solid phase peptide synthesis. In Innovation and Perspectives in Solid Phase Synthesis (R. Epton, ed.) pp. 241‐260. Solid Phase Conference Coordination, Ltd., Birmingham, U.K.
   Fields, G.B., Lauer‐Fields, J.L., Liu, R.‐q., and Barany, G., 2001. Principles and practice of solid‐phase peptide synthesis. In Synthetic Peptides: A User's Guide, 2nd ed. (G.A. Grant, ed.) pp. 93‐219. W.H. Freeman & Co., New York.
   Fischer, E. and Fourneau, E. 1901. Über einige Derivate des Glykocolls. Berichte der Deutschen Chemischen Gesellschaft 34:2868‐2877.
   Ford, W.T. and Balakrishnan, T. 1981. (13)C‐NMR spectra of cross‐linked poly(styrene‐co‐chloromethylstyrene) gel. Macromolecules 14.
   Guillier, F., Orain, D., and Bradley, M. 2000. Linkers and cleavage strategies in solid‐phase organic synthesis and combinatorial chemistry. Chem. Rev. 100:2091‐2158.
   Kates, S.A., Solé, N.A., Albericio, F., and Barany, G. 1994. Solid‐phase synthesis of cyclic peptides. In Peptides: Design, Synthesis and Biological Activity (C. Basava and G.M. Anantharamaiah, eds.) pp. 39‐57. Birkhauser, Boston.
   King, D.S., Fields, C.G., and Fields, G.B. 1990. A cleavage method which minimizes side reactions following Fmoc solid phase peptide synthesis. Int. J. Pept. Protein Res. 36:255‐266.
   Kitas, E.A., Perich, J.W., Wade, J.D., Johns, R.B., and Tregear, G.W. 1989. FMOC‐polyamide solid phase synthesis of an O‐phosphotyrosine‐containing tridecapeptide. Tetrahedron Lett. 30:6229‐6232.
   Lauer, J.L., Fields, C.G., and Fields, G.B. 1995. Sequence dependence of aspartimide formation during 9‐fluorenylmethoxycarbonyl solid‐phase synthesis. Lett. Peptide Sci. 1:197‐205.
   Live, D. and Kent, S.B.H. 1982. Fundamental aspects of the chemical applications of cross‐linked polymers. In Elastomers and Rubber Elasticity (M.J. Comstock, ed.) pp. 501‐515. American Chemical Society, Washington, D.C.
   Ludwick, A.G., Jelinski, L.W., Live, D., Kintanar, A., and Dumais, J.J. 1986. Association of peptide chains during Merrifield solid‐phase peptide synthesis: A deuterium NMR study. J. Am. Chem. Soc. 108:6493‐6496.
   Lukas, T.J., Prystowsky, M.B., and Erickson, B.W. 1981. Solid‐phase peptide synthesis under continuous‐flow conditions. Proc. Natl. Acad. Sci. U.S.A. 78:2791‐2795.
   Manatt, S.L. et al. 1980. A fluorine‐19 NMR approach for studying Merrifield solid‐phase peptide syntheses. Tetrahedron Lett. 21:1397‐1400.
   Merrifield, B. 1986. Solid phase synthesis. Science 232:341‐347.
   Merrifield, R.B. 1963. Solid phase peptide synthesis I: Synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149‐2154.
   Merrifield, R.B. 1967. New approaches to the chemical synthesis of peptides. Recent Prog. Horm. Res. 23:451‐82.
   Merrifield, R.B., Stewart, J.M., and Jernberg, N. 1966. Instrument for automated synthesis of peptides. Anal. Chem. 38:1905‐1914.
   Muir, T.W., Dawson, P.E., and Kent, S.B.H. 1997. Protein synthesis by chemical ligation of unprotected peptides in aqueous solution. Meth. Enzymol. 289:266‐298.
   Ottinger, E.A., Shekels, L.L., Bernlohr, D.A., and Barany, G. 1993. Synthesis of phosphotyrosine‐containing peptides and their use as substrates for protein tyrosine phosphorylation. Biochemistry 32:4354‐4361.
   Otvös, L., Elekes, I., and Lee, V.M.Y. 1989a. Solid‐phase synthesis of phosphopeptides. Int. J. Pept. Protein Res. 34:129‐133.
   Otvös, L. Jr., Wroblewski, K., Kollat, E., Perczel, A., Hollosi, M., Fasman, G.D., Ertl, H.C., and Thurin, J. 1989b. Coupling strategies in solid‐phase synthesis of glycopeptides. Peptide Res. 2:362‐366.
   Perich, J.W. 1997. Synthesis of phosphopeptides using modern chemical approaches. In Methods in Enzymology (B.F. Gregg, ed.) pp. 245‐266. Academic Press, New York.
   Perich, J.W. and Reynolds, E.C. 1991. Fmoc/solid‐phase synthesis of Tyr(P)‐containing peptides through t‐butyl phosphate protection. Int. J. Pept. Protein Res. 37:572‐575.
   Perich, J.W., Ede, N.J., Eagle, S., and Bray, A.M. 1999. Synthesis of phosphopeptides by the Multipinast method: Evaluation of coupling methods for the incorporation of Fmoc‐Tyr(PO3Bzl,H)‐OH, Fmoc‐Ser(PO3Bzl,H)‐OH and Fmoc‐Thr(PO3Bzl,H)‐OH. Lett. Pept. Sci. 6:91‐97.
   Plaue, S. 1990. Synthesis of cyclic peptides on solid support application to analogs of hemagglutinin of influenza virus. Int. J. Pept. Protein Res. 35:510‐517.
   Pontiroli, A.E. 1998. Peptide hormones: Review of current and emerging uses by nasal delivery. Adv. Drug Deliv. Rev. 29:81‐87.
   Quibell, M., Owen, D., Packman, L.C., and 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) backbone amide protection. J. Chem. Soc. Chem. Commun. 2343‐2344.
   Rich, D.H. and Singh, J. 1979. The carbodiimide method. In The Peptides, Vol. 1 (E. Gross and J. Meienhofer, eds.) pp. 241‐314. Academic Press, New York.
   Sakakibara, S., Shimonishi, Y., Kishida, Y., Okada, M., and Sugihara, H. 1967. Use of anhydrous hydrogen fluoride in peptide synthesis. I. Behavior of various protective groups in anhydrous hydrogen fluoride. Bull. Chem. Soc. Jpn. 40:2164‐2167.
   Saladin, P.M., Zhang, B.D., and Reichert, J.M. 2009. Current trends in the clinical development of peptide therapeutics. Drugs 12:779‐784.
   Sarin, V.K., Kent, S.B.H., and Merrifield, R.B. 1980. Properties of swollen polymer networks: Solvation and swelling of peptide‐containing resins in solid‐phase peptide synthesis. J. Am. Chem. Soc. 102:5463‐5470.
   Shin, Y. Winans, K.A., Backes, B.J., Kent, S.B.H., Ellman, J.A., and Bertozzi, C.R. 1999. Fmoc‐based synthesis of peptide‐thioesters: Applications to the total chemical synthesis of a glycoprotein by native chemical ligation. J. Am. Chem. Soc. 121:11684‐11689.
   Sjölin, P., Elofsson, M., and Kihlberg, J. 1996. Removal of acyl protective groups from glycopeptides:? Base does not epimerize peptide stereocenters, and β‐elimination is slow. J. Org. Chem. 61:560‐565.
   Solé, N.A. and Barany, G. 1992. Optimization of solid‐phase synthesis of [Ala8]‐dynorphin A. J. Org. Chem. 57:5399‐5403.
   Stawikowski, M. and Cudic, P. 2006. A novel strategy for the solid‐phase synthesis of cyclic lipodepsipeptides. Tetrahedron Lett. 47:8587‐8590.
   Tam, J.P. and Lu, Y.‐A. 1995. Coupling difficulty associated with interchain clustering and phase transition in solid phase peptide synthesis. J. Am. Chem. Soc. 117:12058‐12063.
   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.
   Wade, J.D., Bedford, J., Sheppard, R.C., and Tregear, G.W. 1991. DBU as an Na‐deprotecting reagent for the fluorenylmethoxycarbonyl group in continuous flow solid‐phase peptide synthesis. Peptide Res. 4:194‐199.
   Wakamiya, T., Saruta, K., Yasuoka, J.‐i., and Kusumoto, S. 1994. An efficient procedure for solid‐phase synthesis of phosphopeptides by the Fmoc strategy. Chem. Lett. 23:1099‐1102.
   Zardeneta, G., Chen, D., Weintraub, S.T., and Klebe, R.J. 1990. Synthesis of phosphotyrosyl‐containing phosphopeptides by solid‐phase peptide synthesis. Anal. Biochem. 190:340‐347.
Key References
   Atherton, E. and Sheppard, R.C. 1989. Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Oxford.
  An extensive collection of Fmoc‐based synthetic methods and techniques.
   Baasner, B., Hageman, H., and Tatlow, J.C. (eds.). 2004. Houben‐Weyl Methods of Organic Chemistry. Vol 22a‐e, Synthesis of Peptides and Peptidomimetics. Thieme Chemistry, New York.
  A comprehensive description of peptide synthesis methods.
   Barany, G. and Merrifield, R.B. 1979. See above.
  The definitive, comprehensive overview of the solid‐phase method.
   Chan, W.C. and White, P.D. 2000. Fmoc Solid Phase Peptide Synthesis: A Practical Approach. Oxford University Press, New York.
  A contemporary collection of Fmoc‐based synthetic methods and techniques.
   Fields, 1997. See above.
  A collection of SPPS techniques and applications.
PDF or HTML at Wiley Online Library