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


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

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Synthesis and Purification of Hydrogen‐Bond Surrogate α‐Helices
  • Commentary
  • Literature Cited
  • Figures
PDF or HTML at Wiley Online Library


Basic Protocol 1:

  • 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
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Arkin, M.R. and Wells, J.A. 2004. Small‐molecule inhibitors of protein‐protein interactions: Progressing towards the dream. Nat. Rev. Drug. Discov. 3:301‐317.
  Azzarito, V., Long, K., Murphy, N.S., and Wilson, A.J. 2013. Inhibition of α‐helix‐mediated protein‐protein interactions using designed molecules. Nat. Chem. 5:161‐173.
  Bergey, C.M., Watkins, A.M., and Arora, P.S. 2013. HippDB: A database of readily targeted helical protein‐protein interactions. Bioinformatics 29:2806‐2807.
  Bullock, B.N., Jochim, A.L., and Arora, P.S. 2011. Assessing helical protein interfaces for inhibitor design. J. Am. Chem. Soc. 133:14220‐14223.
  Chan, W.C. and White, P.D. 2000. Fmoc Solid Phase Peptide Synthesis : A Practical Approach. Oxford University Press, New York.
  Chapman, R.N. and Arora, P.S. 2006. Optimized synthesis of hydrogen‐bond surrogate helices: Surprising effects of microwave heating on the activity of Grubbs catalysts. Org. Lett. 8:5825‐5828.
  Chapman, R.N., Dimartino, G., and Arora, P.S. 2004. A highly stable short α‐helix constrained by a main‐chain hydrogen‐bond surrogate. J. Am. Chem. Soc. 126:12252‐12253.
  Coin, I., Beyermann, M., and Bienert, M. 2007. Solid‐phase peptide synthesis: From standard procedures to the synthesis of difficult sequences. Nat. Protoc. 2:3247‐3256.
  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.
  Henchey, L.K., Kushal, S., Dubey, R., Chapman, R.N., Olenyuk, B.Z., and Arora, P.S. 2010a. Inhibition of hypoxia inducible factor 1—Transcription coactivator interaction by a hydrogen bond surrogate alpha‐helix. J. Am. Chem. Soc. 132:941‐943.
  Henchey, L.K., Porter, J.R., Ghosh, I., and Arora, P.S. 2010b. High specificity in protein recognition by hydrogen‐bond‐surrogate alpha‐helices: Selective inhibition of the p53/MDM2 complex. ChemBioChem 11:2104‐2107.
  Jochim, A.L. and Arora, P.S. 2009. Assessment of helical interfaces in protein‐protein interactions. Mol. BioSyst. 5:924‐926.
  Jochim, A.L. and Arora, P.S. 2010. Systematic analysis of helical protein interfaces reveals targets for synthetic inhibitors. ACS Chem. Biol. 5:919‐923.
  Jones, S. and Thornton, J.M. 1995. Protein‐protein interactions: A review of protein dimer structures. Prog. Biophys. Mol. Biol. 63:31‐65.
  Kaiser, E., Colescott, R.L., Bossinger, C.D., and Cook, P.I. 1970. Color test for detection of free terminal amino groups in the solid‐phase synthesis of peptides. Anal. Biochem. 34:595‐598.
  Koes, D.R. and Camacho, C.J. 2012. PocketQuery: Protein‐protein interaction inhibitor starting points from protein‐protein interaction structure. Nucleic Acids Res. 40:W387‐392.
  Kortemme, T. and Baker, D. 2002. A simple physical model for binding energy hot spots in protein‐protein complexes. Proc. Natl. Acad. Sci. U.S.A. 99:14116‐14121.
  Kortemme, T., Kim, D.E., and Baker, D. 2004. Computational alanine scanning of protein‐protein interfaces. Sci. STKE 2004:pl2.
  Kushal, S., Lao, B.B., Henchey, L.K., Dubey, R., Mesallati, H., Traaseth, N.J., Olenyuk, B.Z., and Arora, P.S. 2013. Protein domain mimetics as in vivo modulators of hypoxia‐inducible factor signaling. Proc. Natl. Acad. Sci. U.S.A. 110:15602‐15607.
  Liu, J., Wang, D., Zheng, Q., Lu, M., and Arora, P.S. 2008. Atomic structure of a short α‐helix stabilized by a main chain hydrogen‐bond surrogate. J. Am. Chem. Soc. 130:4334‐4337.
  Mahon, A.B., Miller, S.E., Joy, S.T., and Arora, P.S. 2012. Rational Design Strategies for Developing Synthetic Inhibitors of Helical Protein Interfaces Protein‐Protein Interactions, vol. 8 (M.D. Wendt, ed.) pp. 197‐230. Springer, Berlin, Heidelberg.
  Patgiri, A., Jochim, A.L., and Arora, P.S. 2008. A hydrogen bond surrogate approach for stabilization of short peptide sequences in alpha‐helical conformation. Acc. Chem. Res. 41:1289‐1300.
  Patgiri, A., Menzenski, M.Z., Mahon, A.B., and Arora, P.S. 2010a. Solid‐phase synthesis of short α‐helices stabilized by the hydrogen bond surrogate approach. Nat. Protoc. 5:1857‐1865.
  Patgiri, A., Witten, M.R., and Arora, P.S. 2010b. Solid phase synthesis of hydrogen bond surrogate derived alpha‐helices: Resolving the case of a difficult amide coupling. Org. Biomol. Chem. 8:1773‐1776.
  Patgiri, A., Yadav, K.K., Arora, P.S., and Bar‐Sagi, D. 2011. An orthosteric inhibitor of the Ras‐Sos interaction. Nat. Chem. Biol. 7:585‐587.
  Patgiri, A., Joy, S.T., and Arora, P.S. 2012. Nucleation effects in peptide foldamers. J. Am. Chem. Soc. 134:11495‐11502.
  Raj, M., Bullock, B.N., and Arora, P.S. 2013. Plucking the high hanging fruit: A systematic approach for targeting protein‐protein interactions. Bioorg. Med. Chem. 21:4051‐4057.
  Vojkovsky, T. 1995. Detection of secondary‐amines on solid‐phase. Peptide Res. 8:236‐237.
  Wang, D., Liao, W., and Arora, P.S. 2005. Enhanced metabolic stability and protein‐binding properties of artificial α helices derived from a hydrogen‐bond surrogate: Application to Bcl‐xL. Angew. Chem. Int. Ed. 44:6525‐6529.
  Wang, D., Chen, K., Dimartino, G., and Arora, P.S. 2006a. Nucleation and stability of hydrogen‐bond surrogate‐based alpha‐helices. Org. Biomol. Chem. 4:4074‐4081.
  Wang, D., Chen, K., Kulp, J.L. III, and Arora, P.S. 2006b. Evaluation of biologically relevant short alpha‐helices stabilized by a main‐chain hydrogen‐bond surrogate. J. Am. Chem. Soc. 128:9248‐9256.
  Wells, J.A. and McClendon, C.L. 2007. Reaching for high‐hanging fruit in drug discovery at protein‐protein interfaces. Nature 450:1001‐1009.
PDF or HTML at Wiley Online Library