Synthesis of Hydrogen‐Bond Surrogate α‐Helices as Inhibitors of Protein‐Protein Interactions

Stephen E. Miller1, Paul F. Thomson1, Paramjit S. Arora1

1 Department of Chemistry, New York University, New York, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130202
Online Posting Date:  June, 2014
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Abstract

The α‐helix is a prevalent secondary structure in proteins and is critical in mediating protein‐protein interactions (PPIs). Peptide mimetics that adopt stable helices have become powerful tools for the modulation of PPIs in vitro and in vivo. Hydrogen‐bond surrogate (HBS) α‐helices utilize a covalent bond in place of an N‐terminal i to i+4 hydrogen bond and have been used to target and disrupt PPIs that become dysregulated in disease states. These compounds have improved conformational stability and cellular uptake as compared to their linear peptide counterparts. The protocol presented here describes current methodology for the synthesis of HBS α‐helical mimetics. The solid‐phase synthesis of HBS helices involves solid‐phase peptide synthesis with three key steps involving incorporation of N‐allyl functionality within the backbone of the peptide, coupling of a secondary amine, and a ring‐closing metathesis step. Curr. Protoc. Chem. Biol. 6:101‐116 © 2014 by John Wiley & Sons, Inc.

Keywords: α‐helix mimetics; hydrogen‐bond surrogate; protein‐protein interactions

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Synthesis and Purification of Hydrogen‐Bond Surrogate α‐Helices
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • HBS peptides (see Strategic Planning)
  • Knorr Amide MBHA resin (solid support, capacity 0.4 mmol/g; Novabiochem)
  • N,N‐dimethylformamide (DMF; Sigma‐Aldrich)
  • 9‐Fluorenylmethyloxycarbonyl (Fmoc)‐protected amino acids (Novabiochem)
  • 1‐Methyl‐2‐pyrrolidinone (NMP; Sigma‐Aldrich)
  • Piperidine (Sigma‐Aldrich)
  • Dichloromethane (DCM; Sigma‐Aldrich)
  • 2‐(1H‐Benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate
  • (HBTU; Novabiochem)
  • N,N’‐diisopropylethylamine (DIEA; Sigma‐Aldrich)
  • Kaiser and chloranil tests (see Kaiser et al., and Vojkovsky, )
  • Ninhydrin (Sigma‐Aldrich)
  • Ethanol
  • Phenol (Sigma‐Aldrich)
  • Potassium cyanide (KCN; Sigma)
  • Pyridine (Sigma)
  • Chloranil (Fluka)
  • Acetaldehyde (Sigma‐Aldrich)
  • Trifluoroacetic acid (TFA; Sigma‐Aldrich)
  • Distilled water
  • Triisopropylsilane (TIPS; Sigma‐Aldrich)
  • Diethyl ether (Sigma‐Aldrich)
  • Acetonitrile (CAN; Fluka)
  • 2‐Nitrobenzenesulfonyl chloride (o‐NsCl; Aldrich)
  • 2,4,6‐Collidine (Sigma‐Aldrich)
  • Methanol (MeOH; Sigma‐Aldrich)
  • Triphenylphosphoine (PPh 3; Fluka)
  • Tris(dibenzylideneacetone)dipalladium(0) [Pd 2(dba) 3; Aldrich]
  • Argon gas
  • Allyl methyl carbonate (Aldrich)
  • Tetrahydrofuran (THF; Sigma‐Aldrich)
  • Sodium diethyldithiocarbamate trihydrate (Sigma‐Aldrich)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Aldrich)
  • 2‐Mercaptoetanol (Aldrich)
  • Nitrogen gas
  • Allyl alcohol (Aldrich)
  • Diisopropyl azodicarboxylate (DIAD; Sigma‐Aldrich)
  • Bromoacetic acid (Aldrich)
  • N,N′‐diisopropylcarbodiimide (DIC; Aldrich)
  • 1‐Hydroxybenzotriazole hydrate (HOBt; Anaspec)
  • Allylamine (Aldrich)
  • 1‐Hydroxy‐7‐azabenzotriazole (HOAt; Genscript)
  • Bis(trichloromethyl) carbonate (Triphosgene; Sigma‐Aldrich)
  • 4‐Pentenoic acid (Aldrich)
  • Hoveyda‐Grubbs II catalyst (Aldrich)
  • Anhydrous 1,2‐dichloroethane (DCE; Sigma‐Aldrich)
  • Fritted solid‐phase extraction (SPE) tubes
  • 25°C incubator
  • CEM Liberty Series Microwave Peptide Synthesizer (CEM), optional
  • Small test tubes
  • Rotary evaporator
  • Liquid chromatograph−mass spectrometer (LC‐MS; Agilent 1100 Series LCMSD)
  • Automatic peptide shaker
  • Vacuum membrane pump
  • Vacuum desiccator
  • 10‐ml microwave reaction tubes with caps (CEM)
  • 1‐ and 5‐ml syringes
  • Magnetic stirrer
  • Magnetic stir bars
  • 20‐ml scintillation vials
  • CEM Discover series microwave reactor with fiber‐optic temperature probe and magnetic stirrer
  • 15‐ml centrifuge tubes
  • Centrifuge
  • Lyophilizer
  • Preparative and analytical HPLC systems, equipped with UV detector and C‐18 reverse phase columns
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Figures

Videos

Literature Cited

Literature Cited
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