Synthesis and Application of Peptide Dendrimers As Protein Mimetics

James P. Tam1, Jane C. Spetzler1

1 Vanderbilt University School of Medicine, Nashville
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 18.5
DOI:  10.1002/0471140864.ps1805s17
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The use of peptides to mimic a portion of a protein structure is a challenging and powerful tool in the discovery of new drugs. In native proteins, discontinuous bioactive peptide surfaces are held together in a particular conformation by the structural rigidity of the protein. Approaches to mimicking a structural surface center on bringing the potential peptide sequences together by assembling the peptide chains on a template. These templates can be flexible dendrimeric or cyclic peptides as well as more rigid organic molecules. The Multiple Antigen Peptide (MAP) system represents 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. A variation of this procedure, the cyclic Multiple Antigen Peptide (cMAP) approach, is also presented here. Having branched multiple closed‐chain architectures, the cMAP system is often a superior approach for protein mimetics because the multiple constrained peptides can mimic bioactive conformations. Whether to select this approach over MAP depends on the properties of the peptides, but usually if the peptides are too small to adopt a stable conformation on their own, incorporation of a cyclic structure may be necessary. MAPs have been applied to areas of study such as inhibitors, artificial proteins, affinity purifications, and intracellular transport.

     
 
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
  • Literature Cited
  • 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 18.5.1; 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‐mercaptoethanol (2‐ME)
  • 10% (v/v) acetic acid in water
  • Automated peptide synthesizer (e.g., Perkin‐Elmer Applied Biosystems #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‐alanine 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
  • recipeCleavage solution (see recipe), 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 #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 (DCC) in DCM (DCC/DCM)
  • recipeSulfhydryl reducing solution (see recipe)
  • Nitrogen source
  • 1 M Tris⋅Cl
  • 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 11.6), 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 2E)
  • 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 11.6)
  • Additional reagents and equipment for solid‐phase peptide synthesis using Boc chemistry ( protocol 1) or Fmoc chemistry ( protocol 2), and purification of peptides by semipreparative RP‐HPLC (unit 11.6)

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 Basic Protocols 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 (Basic Protocols protocol 11, protocol 22, or protocol 33, or protocol 2)
  • 0.1 M potassium phosphate, pH 7.0 ( appendix 2E)
  • 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 #125‐0062)
  • Guard column (75‐mm length × 7.5‐mm i.d.; Bio‐Rad #125‐0061)
  • Additional reagents and equipment for HPLC (unit 11.6)

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) TFA/5% (v/v) triisopropylsilane (TIS; Aldrich) in DCM
  • Cleavage solution: 92.5/2.5/2.5/2.5 (v/v) trifluoroacetic acid (TFA)/triisopropyl silane (TIS) (Aldrich)/thioanisole (Aldrich)/H 2O, prepared fresh
  • 0.01 M sodium phosphate buffer, pH 6.8 ( appendix 2E)
  • Sodium periodate (Aldrich)
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP; Calbiochem‐Novabiochem)
  • 10 mM sodium phosphate, pH 6.8 ( appendix 2E)
  • 10 mM sodium acetate buffer, pH 4.2 ( appendix 2E)
  • 70% (v/v) formic acid in H 2O
  • Additional reagents and equipment for peptide synthesis ( protocol 2 and Table 18.5.2), semipreparative HPLC (unit 11.6), and assay of free sulfhydryls with Ellman's reagent (unit 18.3)

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) 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 2E)
  • 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 2E)
  • Tetravalent MAP core containing aldehyde groups (see protocol 4)
  • Acetic acid
  • Additional reagents and equipment for peptide synthesis (see protocol 2 and Table 18.5.2), semipreparative and preparative HPLC (unit 11.6), and mass spectrometry (Chapter 16),

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
  • 20 mM Na 2HPO 4/10mM citric acid buffer, pH 7.5
  • Tris(2‐carboxyethyl)phosphine, hydrochloride (TCEP; Calbiochem‐Novabiochem)
  • 0.2 M sodium phosphate buffer, pH 7.4 ( appendix 2E)/10 M ethylenediaminetetraacetic acid (EDTA; Aldrich)
  • Argon source
  • Tetravalent chloroacetyl(lysinyl) core matrix (see protocol 3)
  • Dimethylformamide (DMF)
  • Additional reagents and equipment for peptide synthesis (see protocol 1), preparative RP‐HPLC (unit 11.6), and mass spectrometry (Chapter 16)

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% TFA in H 2O, pH 2
  • Sodium acetate, solid
  • Dithiothreitol (DTT; Aldrich)
  • Additional reagents and equipment for peptide synthesis (see protocol 1 and Table 18.5.1) and RP‐HPLC (unit 11.6)

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 recipeacetal resin)
  • Cleavage solution: 91:3:3:3 (v/v) TFA/H 2O/thioanisole/anisole
  • NaIO 4
  • 0.01 M sodium phosphate buffer, pH 5.5, 5.7 and 6 ( appendix 2E)
  • Tributyl phosphine (Bu 3P)
  • Isopropyl alcohol
  • Additional reagents and equipment for peptide synthesis (see protocol 22 and Table 18.5.2) and RP‐HPLC (unit 11.6)

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

  Materials
  • Fmoc‐Lys(Mtt)
  • 1% (v/v) TFA/5% (v/v) triisopropylsilane (TIS; Aldrich) in DCM
  • Boc‐Ser(tBu)
  • Cleavage solution: 92.5/2.5/2.5/2.5 (v/v) TFA/TIS/thioanisol/H 2O
  • Sodium periodate
  • 0.01 M sodium phosphate buffer, pH 6.8 ( appendix 2E)
  • 0.01 M sodium acetate buffer, pH 4.2 ( appendix 2E)
  • Tris(2‐carboxyethyl)phosphine, hydrochloride (TCEP; Calbiochem‐Novabiochem)
  • Additional reagents and equipment for peptide synthesis (see protocol 2 and Table 18.5.2), RP‐HPLC (unit 11.6), and mass spectrometry (Chapter 16)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

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.
   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 (Lond.) 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(6):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 (ed B. Gutte) 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