Chemical Synthesis of Hydrocarbon‐Stapled Peptides for Protein Interaction Research and Therapeutic Targeting

Gregory H. Bird1, W. Christian Crannell1, Loren D. Walensky1

1 Dana‐Farber Cancer Institute and Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110042
Online Posting Date:  September, 2011
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Abstract

The peptide α‐helix represents one of nature's most featured protein shapes and is employed in a diversity of protein architectures, from the cytoskeletal infrastructure to the most intimate contact points between crucial signaling proteins. By installing an all‐hydrocarbon crosslink into native sequences, the shape and biological activity of natural peptide α‐helices can be recapitulated, yielding a chemical toolbox that can be used both to interrogate the protein interactome and to modulate interaction networks for potential therapeutic benefit. Here, current methodology for synthesizing stabilized α‐helices (SAH) corresponding to key protein interaction domains is described. A stepwise approach is taken for the production of crosslinking non‐natural amino acids, incorporation of the residues into peptide templates, and application of ruthenium‐catalyzed ring‐closing metathesis to generate hydrocarbon‐stapled peptides. Through facile derivatization and functionalization steps, SAHs can be tailored for a broad range of applications in biochemical, structural, proteomic, cellular, and in vivo studies. Curr. Protoc. Chem. Biol. 3:99‐117 © 2011 by John Wiley & Sons, Inc.

Keywords: α‐helix; peptide; hydrocarbon stapling; olefin metathesis; photoreactive; protein interaction; targeting

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Asymmetric Synthesis of Stapling Amino Acids via Benzylprolylaminobenzophenone
  • Basic Protocol 2: Synthesis and Derivatization of Hydrocarbon‐Stapled Peptides
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Asymmetric Synthesis of Stapling Amino Acids via Benzylprolylaminobenzophenone

  Materials
  • Nitrogen source
  • Potassium hydroxide pellets (KOH)
  • Isopropanol, HPLC grade
  • D‐Proline
  • Benzyl chloride, anhydrous
  • Hydrochloric acid (HCl)
  • Chloroform, HPLC grade (CHCl 3)
  • Magnesium sulfate powder, anhydrous (MgSO 4)
  • Acetone, HPLC grade
  • Dichloromethane, reagent grade and anhydrous (DCM)
  • 2 M thionyl chloride (SOCl 2, anhydrous) in DCM
  • o‐Aminobenzophenone
  • Saturated sodium carbonate solution
  • Brine
  • Racemic alanine
  • Nickel (II) nitrate hexahydrate (Ni(NO 3) 2⋅6H 2O)
  • Methanol, reagent grade (MeOH)
  • Acetic acid (AcOH)
  • Sodium iodide (NaI)
  • 8‐Bromo‐1‐octene
  • Ethyl acetate, reagent grade (EtOAc)
  • Hexanes, reagent grade
  • Dimethylformamide, anhydrous (DMF)
  • Celite
  • Acetonitrile (ACN)
  • Trifluoroacetic acid (TFA)
  • 9‐Fluorenylmethoxycarbonyl‐N‐hydroxysuccinimide (Fmoc‐OSu)
  • 500‐ml three‐neck round‐bottom flasks
  • Glass stoppers
  • Addition funnels
  • Medium porosity Buchner funnels
  • Syringes
  • 50‐, 250‐, 500‐, and 1000‐ml separatory funnels
  • 250‐ml two‐neck round‐bottom flasks
  • Reflux condensers
  • 500‐ and 1000‐ml Erlenmeyer flasks
  • Rubber septa
  • 250‐ml round‐bottom flask
  • 43‐g reversed‐phase (C18) MPLC column
  • Additional reagents and equipment for column chromatography

Basic Protocol 2: Synthesis and Derivatization of Hydrocarbon‐Stapled Peptides

  Materials
  • Fmoc‐4‐benzoyl‐L‐phenylalanine (Fmoc‐Bpa, Advanced ChemTech, optional)
  • Rink amide AM resin (200‐400 mesh, EMD Biosciences)
  • Fmoc‐protected amino acids: stapling amino acids (see protocol 1) and natural amino acids (Advanced ChemTech, EMD Biosciences)
  • N‐Methylpyrrolidinone (NMP, Aldrich; anhydrous, 99.5% for reagent solutions; ReagentPlus, 99%, for washing)
  • Deprotection solution: 20% (v/v) piperidine in NMP
  • N,N‐Dimethylformamide (DMF, Aldrich; HPLC grade for washing)
  • 2‐(6‐Chloro‐1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethylaminium hexafluorophosphate (HCTU, Peptides International)
  • Diisopropylethylamine (DIPEA)
  • Trifluoroacetic acid (TFA)
  • Triisopropylsilane (TIS)
  • Diethyl ether (ACS grade)
  • Hexanes (technical grade)
  • Acetonitrile (HPLC grade)
  • Grubbs catalyst, first generation: benzylidene‐bis(tricyclohexylphosphine) dichlororuthenium (Sigma‐Aldrich/Fluka)
  • 1,2‐Dichloroethane (DCE)
  • Fluorescein isothiocyanate isomer I (FITC, Sigma, ≥90%, optional)
  • D‐Biotin‐OSu (optional)
  • β‐Alanine or short PEG linker (e.g., Fmoc‐NH‐(PEG) n‐COOH, n = 1‐5; optional)
  • Ethanedithiol
  • Formic acid
  • Methanol (MeOH, reagent grade, 99%)
  • Acetic anhydride (Ac 2O)
  • S‐(2,2,5,5‐Tetramethyl‐2,5‐dihydro‐1H‐pyrrol‐3‐yl)methyl methanesulfonothioate (MTSL, optional)
  • Dimethyl sulfoxide (DMSO, optional)
  • Automated solid‐phase peptide synthesizer
  • High‐performance liquid chromatography/mass spectrometer (LC/MS)
  • Tabletop centrifuge
  • Lyophilizer
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Figures

Videos

Literature Cited

Literature Cited
   Bechinger, B. 1997. Structure and functions of channel‐forming peptides: Magainins, cecropins, melittin and alamethicin. J. Membr. Biol. 156:197‐211.
   Belokon, Y., Tararov, V., Maleev, V., Savel'eva, T., and Ryzhov, M. 1998. Improved procedures for the synthesis of (S)‐2‐[N‐(N′‐benzylprolyl)amino]benzophenone (BPB) and Ni(II) complexes of Schiff's bases derived from BPB and amino acids. Tetrahedron‐Asymmetry 9:4249‐4252.
   Bernal, F., Wade, M., Godes, M., Davis, T.N., Whitehead, D.G., Kung, A.L., Wahl, G.M., and Walensky, L.D. 2010. A stapled p53 helix overcomes HDMX‐mediated suppression of p53. Cancer Cell 18:411‐422.
   Bird, G.H., Bernal, F., Pitter, K., and Walensky, L.D. 2008. Synthesis and biophysical characterization of stabilized alpha‐helices of BCL‐2 domains. Methods Enzymol. 446:369‐386.
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   Gavathiotis, E., Reyna, D.E., Davis, M.L., Bird, G.H., and Walensky, L.D. 2010. BH3‐triggered structural reorganization drives the activation of proapoptotic BAX. Mol. Cell 40:481‐492.
   Henchey, L.K., Jochim, A.L., and Arora, P.S. 2008. Contemporary strategies for the stabilization of peptides in the alpha‐helical conformation. Curr. Opin. Chem. Biol. 12:692‐697.
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   Walensky, L.D., Pitter, K., Morash, J., Oh, K.J., Barbuto, S., Fisher, J., Smith, E., Verdine, G.L., and Korsmeyer, S.J. 2006. A stapled BID BH3 helix directly binds and activates BAX. Mol. Cell 24:199‐210.
   Williams, R.M. and Im, M.N. 1991. Asymmetric synthesis of monosubstituted and alpha, alpha‐disubstituted amino acids via diastereoselective glycine enolate alkylations. J. Am. Chem. Soc. 113:9276‐9286.
   Williams, R.M., Sinclair, P.J., Zhai, D., and Chen, D. 1988. Practical asymmetric syntheses of alpha‐amino acids through carbon‐carbon bond constructions on electrophilic glycine templates. J. Am. Chem. Soc. 110:1547‐1557.
   Williams, R.M., Sinclair, P.J., DeMong, D.E., Chen, D., and Zhai, D. 2003. Asymmetric synthesis of N‐tert‐butoxycarbonyl alpha‐amino acids: Synthesis of (5S, 6R)‐4‐tert‐butoxycarbonyl‐5,6‐diphenylmorpholin‐2‐one. Org. Synth. 80:18‐30.
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