Chemical Synthesis of Ubiquitin Chains

Hosahalli P. Hemantha1, Somasekhar Bondalapati1, Sumeet K. Singh1, Ashraf Brik1

1 Schulich Faculty of Chemistry, Technion—Israel Institute of Technology, Haifa, Israel
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch150099
Online Posting Date:  December, 2015
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Chemical synthesis of complex biomolecules such as proteins is a challenging adventure, yet rewarding in driving various biochemical and biophysical research activities. Over the years, the refinement of peptide synthesis and invention of ligation methodologies have led to the successful synthesis of several complex protein targets. Ubiquitin bioconjugates, which are being studied intensively by many groups due to their involvement in numerous biological processes, represent a fine example where chemistry is greatly aiding these studies. In this article, we describe the synthetic routes and strategies to prepare different ubiquitin analogs with desired modifications, as well as di‐ubiquitin chains. © 2015 by John Wiley & Sons, Inc.

Keywords: ubiquitin; ubiquitin chains; SPPS; ligation; desulfurization

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Synthesis of Ub Building Blocks
  • Alternate Protocol 1: One‐Pot Ligation Coupled with Desulfurization
  • Alternate Protocol 2: Ub‐MPAA Thioester
  • Alternate Protocol 3: Ub‐Hydrazide as a Facile Tunable Handle
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Synthesis of Ub Building Blocks

  • Knorr Amide MBHA resin (solid support, loading 0.27 mmol/g; e.g., Advanced Chemtech)
  • Dichloromethane (DCM; LR grade, 99.5%)
  • Anhydrous dichloromethane (anhydrous DCM; e.g., Sigma‐Aldrich, ≥99.8%)
  • N,N‐dimethylformamide (DMF, Bio‐analytical grade, 99.8%)
  • Piperidine (peptide synthesis grade, 99.5%)
  • 1‐Hydroxybenzotriazole (HOBt, e.g., GL‐Biochem)
  • 9‐Fluorenylmethyloxycarbonyl (Fmoc)‐protected amino acids (e.g., GL‐Biochem):
    • Fmoc‐Gly‐OH
    • Fmoc‐Ala‐OH
    • Fmoc‐Arg(Pbf)‐OH
    • Fmoc‐Asn(Trt)‐OH
    • Fmoc‐Asp(OtBu)‐OH
    • Fmoc‐Gln(Trt)‐OH
    • Fmoc‐Glu(OtBu)‐OH
    • Fmoc‐His(Trt)‐OH
    • Fmoc‐Ile‐OH
    • Fmoc‐Leu‐OH
    • Fmoc‐Lys(Boc)‐OH
    • Fmoc‐Phe‐OH
    • Fmoc‐Pro‐OH
    • Fmoc‐Ser(tBu)‐OH
    • Fmoc‐Thr(tBu)‐OH
    • Fmoc‐Tyr(tBu)‐OH
    • Fmoc‐Val‐OH
    • Boc‐Nle‐OH
    • Fmoc‐Ile‐Thr(ΨMe,Mepro)‐OH
    • Fmoc‐Leu‐Ser(ΨMe,Mepro)‐OH for Ile13‐Thr14 and Leu56‐Ser57 positions
    • Protected dipeptide Fmoc‐Asp (OtBu)‐(Dmb)‐Gly‐OH for Asp52‐Gly53
    • Fmoc‐Dbz‐OH
    • Fmoc‐Cys‐(2‐nitrobenzyl)‐OH
  • 2‐(7‐Aza‐1 H‐benzotriazole‐1yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HATU, e.g., Luxembourg Bio‐tech)
  • N,N‐diisopropylethylamine (DIEA, e.g., Merck, 98%)
  • Peptide cleavage cocktail (see recipe)
  • Diethyl ether (e.g., J.T. Baker)
  • Acetonitrile (ACN, HPLC grade)
  • Liquid N 2
  • HPLC buffer A: Milli‐Q water containing 0.1% (v/v) trifluoroacetic acid (TFA; HPLC grade, 99.5%)
  • HPLC buffer B: acetonitrile (HPLC grade) with 0.1% TFA (HPLC grade, 99.5%)
  • Methoxyamine hydrochloride (MeONH 2·HCl; e.g., Alfa Aesar, 98%)
  • 2‐(1 H‐Benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HBTU, e.g., Luxembourg Bio‐tech)
  • Allylchloroformate (e.g., Sigma‐Aldrich, 97%)
  • Tetrakis(triphenylphosphine) palladium (0) (Pd(PPh 3) 4; e.g., Chem‐Impex International, 99.89%)
  • Phenylsilane (PhSiH3; e.g., Acros Organics, 97%)
  • p‐nitrophenylchloroformate (e.g., Alfa Aesar, 97%)
  • Methanol (MeOH, HPLC grade)
  • Ligation buffer (see recipe)
  • Methyl 3‐mercaptopropionate (MMP; e.g., Alfa Aesar, 98%)
  • Hydrazine hydrate [N 2H 4·H 2O; 80% (v/v) in H 2O; e.g., Sigma‐Aldrich]
  • Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP; e.g., Apollo Scientific Ltd.)
  • 2‐(6‐Chloro‐1 H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethylaminiumhexafluorophosphate (HCTU, e.g., Luxembourg Bio‐tech)
  • 2,4,6‐trimethylpyridine (collidine; e.g., Sigma‐Aldrich)
  • 2‐nitrobenzenesulfonyl chloride (e.g., Alfa Aesar, 98%)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; e.g., Alfa Aesar, 98%)
  • Methyl‐4‐nitrobenzenesulfonate (e.g., Aldrich, 99%)
  • β‐mercaptoethanol (e.g., Aldrich, 98%)
  • Ascorbic acid (vitamin C; e.g., Chem‐Impex International, 99.75%)
  • 3‐mercaptopropionic acid (MPA; e.g., Alfa Aesar, 99%)
  • Concentrated (32%) HCl
  • 4‐mercaptophenylacetic acid (MPAA; e.g., Chem‐Impex International, 99.67%)
  • Sodium hydroxide (NaOH, 98%)
  • Argon gas
  • Trifluroacetic acid (TFA, HPLC grade, 99.5%)
  • 2,2′‐Azobis[2‐(2‐imidazolin‐2‐yl)propane]dihydrochloride (VA‐044, water soluble azo initiator; e.g., Wako, 97%)
  • tert‐Butylthiol (tBu‐SH; e.g., JK chemicals, 98.5%)
  • 5‐, 10‐ and 20‐ml fritted solid‐phase extraction (SPE) tubes (e.g., Torviq)
  • Screw‐capped glass vials: 7‐ and 20‐ml glass scintillation vials with caps
  • Automated solid‐phase peptide synthesizer (CSBio;
  • Preparative and analytical HPLC systems, equipped with UV detector (Thermo Scientific; also see Josic and Kovac, )
  • Liquid chromatograph−mass spectrometer (LC‐MS; LCQ Fleet Ion Trap; Thermo Scientific; also see Zhang et al., )
  • Preparative HPLC column: C18/C4 300 Å, 10 μm, 250 × 21.20 mm (e.g., Waters)
  • Analytical HPLC column: C18/C4 300 Å, 3.5 μm, 150 × 4.6 mm (e.g., Waters)
  • Centrifuge [accommodating 15 and 50 ml tubes and −20°C cooling and capable of 23,545 × g]
  • Lyophilizer (e.g., FreeZone Plus 2.5 liter benchtop freeze dry system; Labconco)
  • Platform shaker (e.g., Unimax 1010, Heidolph)
  • 1‐ to 2‐ml siliconized low‐retention microcentrifuge tubes
  • Syringe filters (PVDF membrane, 0.45 μm)
  • UV‐vis reaction chamber with 350 nm lamp (e.g., Rayonet, model RPR 200)
  • Additional reagents and equipment for HPLC (Josic and Kovac, ) and mass spectrometric analysis (Zhang et al., ) of peptides
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Literature Cited

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