Synthesis and Application of Peptide Dendrimers As Protein Mimetics

James P. Tam1, Jane C. Spetzler1

1 Vanderbilt University School of Medicine, Nashville, Tennessee
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 9.6
DOI:  10.1002/0471142735.im0906s34
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The multiple antigenic peptide (MAP) is a novel approach to preparing peptide immunogens. The MAP consists of an inner core matrix built up of a large layer of Lys residues and a surface of peptide chains attached to the core matrix. Because of its dendrimeric structure, MAP can be very useful as a template for assembling potential peptide surfaces. Two methods for preparing MAP systems are described in this unit. The direct approach for preparing MAP systems is presented in the first two protocols, including the procedure for b‐butyloxycarbonyl (Boc) chemistry and the procedure for 9 ‐fluorenylmethyloxycarbonyl (Fmoc) chemistry. An indirect approach for preparing MAP systems, in which peptide and core matrix are synthesized separately and conjugated by several ligation methods, is then described. The cMAP approach is also executed using either the direct or indirect approach, but requires an additional cyclization step to constrain the peptides after synthesis. The synthesis of cMAP is described, and the preparation of cyclic peptides is illustrated. A support protocol describes the ninhydrin assay to assess the completeness of the coupling reaction. In most cases, MAP systems can be used directly after simple dialysis or desalting. Some immunological studies, however, require purified MAPs. Additional support protocols describe MAP system purification by dialysis and high‐performance gel‐filtration chromatography.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Direct Boc Synthesis of Map Systems
  • Alternate Protocol 1: Direct Fmoc Solid‐Phase Synthesis of Map Systems
  • Basic Protocol 2: Indirect Boc and Fmoc Synthesis of Map Systems using Thiol Chemistry
  • Basic Protocol 3: Indirect Boc and Fmoc Synthesis of Map Systems using Carbonyl Chemistry
  • Support Protocol 1: Ninhydrin Test
  • Support Protocol 2: Purification of Map System by Dialysis
  • Support Protocol 3: Purification of Map using High‐Performance Gel‐Filtration Chromatography
  • Basic Protocol 4: Direct Synthesis of End–To–Side Chain cMAP
  • Basic Protocol 5: Indirect Fmoc Synthesis of Side Chain‐to‐Side Chain cMAP using Carbonyl Chemistry
  • Basic Protocol 6: Indirect Preparation of End‐to‐End cMAP using Thiol Chemistry
  • Support Protocol 4: Preparing End‐to‐End Cyclic Peptides Via an X‐CYS Ligation Site
  • Support Protocol 5: Preparing End–to–End Cyclic Peptides Via an X‐Thiaproline Ligation Site
  • Support Protocol 6: Preparing End–to–Side Chain Cyclic Peptides Via a Thiazolidine or Oxime Linkage
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Direct Boc Synthesis of Map Systems

  Materials
  • Synthesis reagents (Table 9.6.1; prepared as described in Stewart and Young, ):
  •  Dichloromethane (methylene chloride, DCM)
  •  50% (v/v) trifluoroacetic acid (TFA) in DCM (TFA/DCM)
  •  5% (v/v) diisopropylethylamine (DIEA) in DCM (DIEA/DCM)
  •  1.0 M N,N′‐dicyclohexylcarbodiimide (DCC) in DCM (DCC/DCM)
  • N,N′‐Dimethylformamide (DMF)
  • 0.1 mmol/g Boc‐β‐Ala‐OCH 2‐PAM resin (Bachem California)
  • Boc‐Lys(Boc) (Bachem California)
  • Boc‐amino acids (Boc‐AA), with protected side‐chain groups (Bachem California, Peninsula Laboratories) appropriate for synthesis of the desired peptide antigen, dissolved in dichloromethane (DCM)
  • 10% (v/v) thiophenol in DMF or 10% (v/v) mercaptoethanol (2‐ME)/5% (v/v) DIEA in DMF
  • p‐Cresol
  • p‐Thiocresol
  • Dimethyl sulfide (DMS)
  • Liquid hydrogen fluoride (HF), –78°C
  • 99:1 (v/v) cold diethyl ether/2‐ME
  • 10% (v/v) acetic acid in water
  • Automated peptide synthesizer (e.g., Perkin‐Elmer Applied Biosystems no. ABI430A)
  • Manual reaction vessel (Pierce)
  • Hydrogen fluoride cleavage apparatus (Peptide Institute or Peninsula Laboratories)
  • 50‐ml round‐bottom flask
  • Coarse‐ and fine‐porosity fritted glass funnels
  • 50°C water bath

Alternate Protocol 1: Direct Fmoc Solid‐Phase Synthesis of Map Systems

  Materials
  • Fmoc‐Ala‐Wang resin (p‐alkoxybenzyl alcohol resin preloaded with Fmoc‐Ala at 0.3 to 0.5 mmol alanine/g resin; Bachem California)
  • Fmoc synthesis reagents
  • N,N′‐Dimethylformamide (DMF)
  •  20% (v/v) piperidine in DMF (pip/DMF)
  •  0.5% (w/v) 1‐hydroxybenzotriazole (HOBt) in DMF (HOBt/DMF)
  • Fmoc‐Lys(Fmoc) (Bachem California; Peninsula Laboratories)
  • Acetic anhydride
  • Fmoc‐amino acids (Fmoc‐AA) with protected side‐chain groups (Bachem California; Peninsula Laboratories) appropriate for synthesis of the desired peptide antigen, dissolved in DMT
  • Cleavage solution 90:6:3:1 trifluoroacetic acid (TFA)/thioanisole/1,2‐ethanedithiol (EDT)/anisole (all from Aldrich; prepare fresh and prechill to 0°C)
  • Anhydrous methyl t‐butyl ether, cold
  • Anhydrous diethyl ether
  • 10% (v/v) acetic acid in water
  • Automated peptide synthesizer (e.g., Perkin‐Elmer Applied Biosystems no. ABI430A)
  • Manual reaction vessel (Pierce)
  • Coarse‐ and fine‐porosity fritted glass funnels

Basic Protocol 2: Indirect Boc and Fmoc Synthesis of Map Systems using Thiol Chemistry

  Materials
  • Chloroacetic acid (Aldrich)
  • 1.0 M N,N′‐dicyclohexylcarbodiimide in dichloromethane (DCC/DCM)
  • recipeSulfhydryl reducing solution (see recipe)
  • Nitrogen source
  • 1 M Tris⋅Cl ( appendix 2A)
  • 1 M NaOH
  • 0.1 M NH 4HCO 3 or 10% acetic acid
  • Additional reagents and equipment for solid‐phase peptide synthesis using Boc (see protocol 1) or Fmoc chemistry (see protocol 2), purification of peptides via RP‐HPLC (unit 9.2), dialysis (see protocol 6), and high performance gel‐filtration chromatography (see protocol 7)

Basic Protocol 3: Indirect Boc and Fmoc Synthesis of Map Systems using Carbonyl Chemistry

  Materials
  • recipeBoc‐aminooxyacetic acid (see recipe) or recipeBoc‐succinic acid hydrazide (see recipe)
  • Benzotriazolyl N‐oxytrisdimethylaminophosphonium hexafluorophosphate (BOP) (Richelieu Biotechnologies)
  • Diisopropylethylamine (DIEA; Aldrich)
  • 0.01 M sodium phosphate buffer, pH 7 ( appendix 2A)
  • Sodium periodate (Aldrich)
  • Ethylene glycol (Aldrich)
  • 0.4 M sodium acetate buffer: mix equal volumes 0.4 M sodium acetate and 0.4 M acetic acid
  • Dimethyl sulfoxide (DMSO; Aldrich)
  • Dimethyl formamide (DMF)
  • 0.1 M NaOH or glacial acetic acid
  • Argon source
  • C8 RP‐HPLC column (see unit 9.2)
  • Additional reagents and equipment for solid‐phase peptide synthesis using Boc chemistry (see protocol 1) or Fmoc chemistry (see protocol 2) and for purification of peptides by semipreparative RP‐HPLC (unit 9.2)

Support Protocol 1: Ninhydrin Test

  Materials
  • recipeNinhydrin test reagents: Solution A/B mixture and Solution C (see recipe)
  • 60% (v/v) ethanol in H 2O
  • 0.5 M tetraethylammonium chloride in dichloromethane (DCM)
  • 10 × 75–mm test tubes
  • 100°C heating block
  • Pasteur pipet containing glass wool plug

Support Protocol 2: Purification of Map System by Dialysis

  Materials
  • Crude MAP system (see protocol 1, protocol 32, or protocol 43, or protocol 2)
  • 0.1 M NH 4HCO 3/(NH 4) 2CO 3 (pH 8.0) in 8 M and 2 M urea
  • 1 M acetic acid
  • Dialysis tubing (e.g., Spectra/Por 6, MWCO 1000, Spectrum)

Support Protocol 3: Purification of Map using High‐Performance Gel‐Filtration Chromatography

  Materials
  • 10 mg crude MAP system (see protocol 1, protocol 32, or protocol 43, or protocol 2)
  • 0.1 M potassium phosphate, pH 7.0 ( appendix 2A)
  • Column for high‐performance gel‐filtration chromatography (prepacked Bio‐Sil TSK 250, 300‐mm length × 7.5‐mm i.d.; mol. wt. range for separation: 10 to 300 kDa; Bio‐Rad)
  • Guard column (75‐mm length × 7.5‐mm i.d.; Bio‐Rad)
  • Additional reagents and equipment for HPLC (unit 9.2)

Basic Protocol 4: Direct Synthesis of End–To–Side Chain cMAP

  Materials
  • Fmoc‐Lys[methyltrityl(Mtt)] (Calbiochem‐Novabiochem)
  • Fmoc‐Lys(Fmoc) (Bachem California)
  • Fmoc‐Cys(StBu) (Calbiochem‐Novabiochem)
  • 1% (v/v) trifluoroacetic acid (TFA) 5% (v/v) triisopropylsilane (TIS; Aldrich) in dichloromethane (DCM)
  • Cleavage solution: 92.5:2.5:2.5:2.5 (v/v) TFA/TIS/thioanisole (Aldrich)/H 2O, prepared fresh
  • 0.01 M sodium phosphate buffer, pH 6.8 ( appendix 2A)
  • Sodium periodate (NaIO 4; Aldrich)
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP; Calbiochem‐Novabiochem)
  • 0.01 M sodium acetate buffer, pH 4.2 ( appendix 2A)
  • 70% (v/v) formic acid in H 2O
  • Additional reagents and equipment for peptide synthesis ( protocol 2 and Table 9.6.2), RP‐HPLC (unit 9.2), and assay of free sulfhydryls in peptides (unit 9.4)

Basic Protocol 5: Indirect Fmoc Synthesis of Side Chain‐to‐Side Chain cMAP using Carbonyl Chemistry

  Materials
  • Fmoc‐Lys[methyltrityl(Mtt)] (Calbiochem‐Novabiochem)
  • 1% (v/v) trifluoroacetic acid (TFA)/5% (v/v) triisopropylsilane (TIS; Aldrich) in DCM
  • 20% piperidine in DMF (pip/DMF)
  • Boc‐Ser(tBu; Bachem California)
  • Boc‐(aminooxy)acetic acid (Bachem California)
  • Boc‐Lys(Fmoc; Bachem California)
  • 0.01 M sodium acetate buffer, pH 7 ( appendix 2A)
  • Sodium periodate (NaIO 4; Aldrich)
  • Ethylene glycol
  • 0.1 M NaOH
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP; Calbiochem‐Novabiochem)
  • 0.05 M sodium acetate buffer, pH 6 ( appendix 2A)
  • Tetravalent MAP core containing aldehyde groups (see protocol 4)
  • Acetic acid
  • Additional reagents and equipment for peptide synthesis (see protocol 2 and Table 9.6.2), and RP‐HPLC (unit 9.2)

Basic Protocol 6: Indirect Preparation of End‐to‐End cMAP using Thiol Chemistry

  Materials
  • 3‐Thiopropionic acid (Aldrich)
  • MBHA (p‐methyl benzhydrylamine) or Gly‐PAM resin (Calbiochem‐Novabiochem)
  • 1 mM dithiothreitol (DTT; Aldrich) in distilled water
  • Cysteine methyl ester (Aldrich), neutralized by DIEA in DMF
  • 20 mM Na 2HPO 4/10 mM citric acid buffer, pH 7.5
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP; Calbiochem‐Novabiochem)
  • 0.2 M sodium phosphate buffer, pH 7.4 ( appendix 2A)/10 M ethylenediaminetetraacetic acid (EDTA)
  • Argon source
  • Tetravalent chloroacetyl(lysinyl) core matrix (see protocol 3)
  • Dimethylformamide (DMF)
  • Additional reagents and equipment for peptide synthesis (see protocol 1) and RP‐HPLC (unit 9.2)

Support Protocol 4: Preparing End‐to‐End Cyclic Peptides Via an X‐CYS Ligation Site

  Materials
  • recipeThiobenzhydryl resin for C‐terminal thioacid (see recipe)
  • Boc‐Cys(Npys) (Bachem California)
  • 25% acetonitrile/0.05% trifluoroacetic acid (TFA) in H 2O, pH 2
  • Sodium acetate, solid
  • Dithiothreitol (DTT; Aldrich)
  • Additional reagents and equipment for peptide synthesis (see protocol 1 and Table 9.6.1) and RP‐HPLC (unit 9.2)

Support Protocol 5: Preparing End–to–End Cyclic Peptides Via an X‐Thiaproline Ligation Site

  Materials
  • Fmoc‐Gly‐O‐CH 2‐cyclic‐acetal resin (see recipe for acetal resin)
  • Cleavage solution: 91:3:3:3 (v/v/v/v) TFA/H 2O/thioanisole/anisole
  • Sodium periodate (NaIO 4)
  • 0.01 M sodium phosphate buffer, pH 5.5, 5.7, and 6 ( appendix 2A)
  • Tributyl phosphine (Bu 3P)
  • Isopropyl alcohol
  • Additional reagents and equipment for peptide synthesis (see Alternate Protocol 2 and Table 9.6.2) and RP‐HPLC (unit 9.2)

Support Protocol 6: Preparing End–to–Side Chain Cyclic Peptides Via a Thiazolidine or Oxime Linkage

  Materials
  • Fmoc‐Lys(Mtt)
  • 1% (v/v) trifluoroacetic acid (TFA)/5% (v/v) triisopropylsilane (TIS; Aldrich) in DCM
  • Boc‐Ser(tBu)
  • Cleavage solution: 92.5:2.5:2.5:2.5 (v/v/v/v) TFA/TIS/thioanisole/H 2O
  • Sodium periodate (NaIO 4)
  • 0.01 M sodium phosphate buffer, pH 6.8 ( appendix 2A)
  • 0.01 M sodium acetate buffer, pH 4.2 ( appendix 2A)
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP; Calbiochem‐Novabiochem)
  • Additional reagents and equipment for peptide synthesis (see protocol 2 and Table 9.6.2), and RP‐HPLC (unit 9.2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Amon, R. and Horwitz, R.J. 1992. Synthetic peptides as vaccines. Curr. Opin. Immunol. 4:449‐453.
   Botti, P., Pallin, T.D., and Tam, J.P. 1996. Cyclic peptides from linear unprotected peptide precursors through thiazolidine formation. J. Am. Chem. Soc. 118:10018‐10024.
  Coligan, J.E., Dunn, B.M., Ploegh, H.L., Speicher, D.W., and Wingfield, P.T. (eds.) 1999. Current Protocols in Protein Science. John Wiley & Sons, New York.
   Defoort, J.‐P., Nardelli, B., Huang, W., Ho, D.D., and Tam, J.P. 1992. Macromolecular assemblage in the design of a synthetic AIDS vaccine. Proc. Natl. Acad. Sci. U.S.A. 89:3879‐3883.
   Francis, M.J., Hastings, G.Z., Brown, F., McDermed, J., Lu, Y.A., and Tam, J.P. 1991. Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic site of foot‐and‐mouth disease virus. Immunology 73:249‐254.
   Houghten, R.A., Pinilla, C., Blondelle, S.E., Appel, J.R., Dooley, C.T., and Cuervo, J.H. 1991. Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery. Nature 354(6348):84‐6.
   Lerner, R.A. 1982. Tapping the immunological repertoire to produce antibodies of predetermined specificity. Nature 299:592‐596.
   Liu, C.‐F. and Tam, J.P. 1994. Peptide segment ligation strategy without use of protecting groups. Proc. Natl. Acad. Sci. U.S.A. 91:6584‐6588.
   Liu, C.‐F. and Tam, J.P. 1997. Synthesis of a symmetric branched peptide: Assembly of a cyclic peptide on a small tetraacetate template. Chem. Commun. 1619‐1620.
   Lu, Y.A., Clavijo, P., Galantino, M., Shen, Z.‐Y., Liu, W., and Tam, J.P. 1991. Chemically unambiguous peptide immunogen: Preparation, orientation and antigenicity of purified peptide conjugated to the multiple antigen peptide system. Mol. Immunol. 28:623‐630.
   Merrifield, R.B. 1963. Solid phase synthesis. I. J. Am. Chem. Soc. 85:2149‐2154.
   Mutter, M. and Vuilleumier, S. 1989. A chemical approach to protein design‐template‐assembled synthetic proteins (TASP). Angew. Chem. Int. Ed. Engl. 28:535‐554.
   Nardelli, B., Lu, Y.A., Shim, D.R., Delpierre‐Defoort, C., Profy, A.T., and Tam, J.P. 1992. A chemically defined synthetic vaccine model for HIV‐1. J. Immunol. 148:914‐920.
   Pallin, D.T. and Tam, J.P. 1995. Cyclisation of totally unprotected peptides in aqueous solution by oxime formation. Chem. Commun. 2021‐2022.
   Pallin, T.D. and Tam, J.P. 1996. Assembly of cyclic peptide dendrimers from linear building blocks in aqueous solution. Chem. Commun. 11:1345‐1346.
   Pessi, A., Valmori, D., Migliorni, P., Tougne, C., Bianchi, E., Lambert, P.‐H., Corradin, G., and Giudice, del G. 1991. Lack of H‐2 restriction of Plasmodium falciparum (NANP) sequence as multiple antigen peptide. Eur. J. Immunol. 21:2273‐2276.
   Posnett, D.N., McGrath, H., and Tam, J.P. 1988. A novel method for producing antipeptide antibodies. J. Biol. Chem. 263:1719‐1725.
   Sarin, V.K., Kent, S.B.H., Tam, J.P., and Merrifield, R.B. 1981. Quantitative monitoring of solid‐phase peptide synthesis by the ninhydrin reaction. Anal Chem. 117:147‐157.
   Sasaki, T. and Kaiser, E.T. 1989. Helicrome: Synthesis and enzymatic activity of a designed heme protein. J. Am. Chem. Soc. 111:380‐381.
   Schneider, J.P. and Kelly, J.W. 1995. Templates that induce α‐helical, β‐sheet, and loop conformations. Chem. Rev. 95:2169‐2187.
   Shao, J. and Tam, J.P. 1995. Unprotected peptides as building blocks for the synthesis of peptide dendrimers with oxime, hydrazone and thiazolidine linkages. J. Am. Chem. Soc. 117:3893‐3899.
   Spetzler, J.C. and Tam, J.P. 1996. Self‐assembly of cyclic peptides on a dendrimer: Multiple cyclic antigen peptides. Pept. Res. 9:290‐296.
   Stewart, J.M. and Young, J.D. 1984. Solid Phase Peptide Synthesis, 2nd ed. Pierce Chemical Co., Rockford, Ill.
   Tam, J.P. 1988. Synthetic peptide vaccine design: Synthesis and properties of a high density multiple antigenic peptide system. Proc. Natl. Acad. Sci. U.S.A. 85:5409‐5413.
   Tam, J.P. 1995. Synthesis and applications of branched peptides in immunological methods and vaccines. In Peptides: Synthesis, Structures and Applications (B. Gutte, ed.) pp. 455‐501. Academic Press, San Diego.
   Tam, J.P. 1996. Recent advances in multiple antigen peptides. J. Immunol. Methods 196:17‐32.
   Tam, J.P. and Lu, Y.‐A. 1989. Vaccine engineering: Enhancement of immunogenicity of synthetic peptide vaccine related to hepatitis in chemically defined models consisting of T and B cell epitopes. Proc. Natl. Acad. Sci. U.S.A. 86:9084‐9088.
   Tam, J.P. and Spetzler, J.C. 1997. Multiple antigen peptide system. Methods Enzymol. 289:612‐637.
   Tam, J.P., Clavijo, P., Lu, Y.A., Nussenzweig, R.S., Nussenzweig, V., and Zavala, F. 1990. Incorporation of T and B epitopes of the circumsporozoite protein in a chemically defined synthetic vaccine against malaria. J. Exp. Med. 171:299‐306.
   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.
   Unson, C.G., Erickson, B.W., Richardson, D.G., and Richardson, J.S. 1984. Protein Engineering: Design and synthesis of a protein. Fed. Proc. 43:1837.
   Wrighton, N.C., Balasubramanian, P., Barbone, F.P., Kashyap, A.K., Farrell, F.X., Jolliffe, L.K., Barrett, R., and Dower, W.J. 1997. Increased potency of an erythropoietin peptide mimetic through covalent dimerization. Nat. Biotechnol. 15:1261‐1265.
   Zhang, L. and Tam, J.P. 1997. Synthesis and application of unprotected cyclic peptides as building blocks for peptide dendrimers. J. Am. Chem. Soc. 119:2363‐2370.
Key References
   Fields, G.B. and Noble, R. 1990. Solid phase peptide synthesis utilizing 9‐fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35:161‐214.
  An extensive and useful literature review of Fmoc solid‐phase peptide synthesis.
  Grant, G.A. (ed.) 1992. Synthetic Peptides—A User's Guide. W.H. Freeman, New York.
  A practical book for solid‐phase peptide synthesis which covers synthesis, purification, analysis, and applications of synthetic peptides. References include papers published in 1992.
   Stewart and Young, 1984. See above.
  An excellent laboratory book for solid‐phase peptide synthesis.
   Tam and Spetzler, 1997. See above.
  A practical protocol for preparing MAPs and cMAP using direct and indirect approaches.
   Zhang and Tam, 1997. See above.
  A useful research paper discussing preparation and analysis of cyclic peptides and cMAP.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library