Application of meta‐ and para‐Phenylenediamine as Enhanced Oxime Ligation Catalysts for Protein Labeling, PEGylation, Immobilization, and Release

Mohammad M. Mahmoodi1, Mohammad Rashidian1, Yi Zhang1, Mark D. Distefano1

1 Department of Chemistry, University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 15.4
DOI:  10.1002/0471140864.ps1504s79
Online Posting Date:  February, 2015
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Abstract

Meta‐ and para‐phenylenediamines have recently been shown to catalyze oxime and hydrazone ligation reactions at rates much faster than aniline, a commonly used catalyst. Here, we demonstrate how these new catalysts can be used in a generally applicable procedure for fluorescent labeling, PEGylation, immobilization, and release of aldehyde‐ and ketone‐ functionalized proteins. The chemical orthogonality of phenylenediamine‐catalyzed oxime ligation versus copper‐catalyzed click reaction has also been harnessed for simultaneous dual labeling of bifunctional proteins containing both aldehyde and alkyne groups in high yield. © 2015 by John Wiley & Sons, Inc.

Keywords: oxime ligation; hydrazone ligation; meta‐phenylenediamine; para‐phenylenediamine; dual protein labeling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Rapid Labeling and PEGylation of Aldehyde‐Functionalized Proteins Using Phenylenediamines as Catalysts
  • Basic Protocol 2: Labeling and PEGylation of Ketone‐Functionalized Proteins Using Phenylenediamines as Catalysts
  • Basic Protocol 3: Protein Immobilization and Release from Agarose Beads Through Oxime Ligation Catalyzed by mPDA OR pPDA
  • Basic Protocol 4: Simultaneous Dual Protein Labeling Using Oxime Ligation and Click Reaction
  • Support Protocol 1: Synthesis of Farnesyl Ketone Diphosphate (Compound 1) and Enzymatic Incorporation into Proteins
  • Support Protocol 2: Synthesis of Compound 2
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Rapid Labeling and PEGylation of Aldehyde‐Functionalized Proteins Using Phenylenediamines as Catalysts

  Materials
  • Aminooxy‐HiLyteFluor488 (Anaspec) or Aminooxy‐PEG (mol. wt. 5000; NOF Corp., http://nofamerica.net/)
  • DMSO or DMF (Aldrich)
  • p‐phenylenediamine (pPDA) or m‐phenylenediamine (mPDA) (both compounds available from Aldrich)
  • 0.3 M and 0.1 M sodium phosphate buffer, pH 7.0 (  )
  • GFP‐aldehyde, prepared as described in Mahmoodi et al. ( )50 mM Tris·Cl, pH 7.5 (  )
  • NAP‐5 or PD‐10 column (Amersham)
  • LC‐MS instrument (Waters Synapt G2 Quadrapole TOF mass spectrometer instrument)
  • MALDI‐MS instrument (Bruker MALDI TOF)
  • Additional reagents and equipment for SDS‐PAGE (unit  ; Gallagher, ) and MALDI‐MS (unit  ; Zhang et al., ; unit  ; Sandoval, ); also see Mahmoodi et al. ( )

Basic Protocol 2: Labeling and PEGylation of Ketone‐Functionalized Proteins Using Phenylenediamines as Catalysts

  Additional Materials (also see protocol 1)
  • GFP‐ketone: prepared in the Distefano laboratory ( ) using methods similar to those employed for the GFP‐aldehyde described in protocol 1, starting with GFP‐CVIA whose preparation is fully described in Mahmoodi et al. ( ); the synthesis procedure for the ketone analog used in this example and subsequent attachment to eGFP‐CVIA is described in protocol 5

Basic Protocol 3: Protein Immobilization and Release from Agarose Beads Through Oxime Ligation Catalyzed by mPDA OR pPDA

  Materials
  • Hydrazide‐functionalized agarose beads (Thermo Scientific)
  • 0.1 M sodium phosphate buffer, pH 7.0 (  )
  • GFP‐aldehyde: prepared in the Distefano laboratory ( ); methods for its preparation are fully described in Mahmoodi et al. ( )
  • Pure, unmodified GFP (Mahmoodi et al., )
  • p‐phenylenediamine (pPDA), m‐phenylenediamine (mPDA), or aniline (compounds available from Aldrich)
  • 1 M KCl in 50 mM Tris·Cl, pH 7.5
  • 50 mM Tris·Cl, pH 7.5 (  )
  • Aminooxy‐HiLyteFluor488 (Anaspec) or Aminooxy‐PEG (mol. wt. 5000; NOF Corp., http://nofamerica.net/)
  • Centrifuge (Beckman‐Coulter)
  • NAP‐5 or PD‐10 column (Amersham)
  • LC‐MS instrument (Waters Synapt G2 Quadrapole TOF mass spectrometer instrument)
  • MALDI‐MS instrument (Bruker MALDI‐TOF)
  • Additional reagents and equipment for SDS‐PAGE (unit  ; Gallagher, ) and MALDI‐MS (unit  ; Zhang et al., ; unit  ; Sandoval, ); also see Mahmoodi et al. ( )

Basic Protocol 4: Simultaneous Dual Protein Labeling Using Oxime Ligation and Click Reaction

  Materials
  • Azido‐TAMRA (Lumiprobe; http://www.lumiprobe.com/)
  • Aminooxy‐PEG (mol. wt. 5,000; NOF Corp., http://nofamerica.net/)
  • Protein: aldehyde‐alkyne‐GFP (prepared as described in Rashidian et al., ); synthesis procedure for the aldehyde‐alkyne‐FPP analog used in this example and subsequent attachment to eGFP‐CVIA is described in protocol 6
  • CuSO 4 (Aldrich)
  • Tris (2‐carboxyethyl)phosphine hydrochloride (TCEP, Aldrich)
  • Tris[(1‐benzyl‐1 H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA, Aldrich)
  • m‐phenylenediamine (mPDA; Aldrich)
  • 1 M sodium phosphate buffer, pH 7.0 (  )
  • 500 mM Tris·Cl, pH 7.5 ( appendix 2E)
  • NAP‐5 or PD‐10 column (GE Healthcare)
  • LC‐MS instrument (Waters Synapt G2 Quadrapole TOF mass spectrometer instrument)
  • MALDI‐MS instrument (Bruker MALDI‐TOF)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher, ) and MALDI‐MS (unit 16.1; Zhang et al., ; unit 16.2; Sandoval, ); also see Mahmoodi et al. ( )

Support Protocol 1: Synthesis of Farnesyl Ketone Diphosphate (Compound 1) and Enzymatic Incorporation into Proteins

  Materials
  • Geraniol (Aldrich)
  • 3,4‐Dihydropyran (Aldrich)
  • Pyridinium p‐toluenesulfonate (PPTS; Aldrich)
  • Dichloromethane (CH 2Cl 2)
  • Diethyl ether (Aldrich)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Silica gel 60
  • Toluene
  • Ethyl acetate (EtOAc)
  • Deuterated chloroform (CDCl 3) as NMR solvent
  • tert‐Butyl hydroperoxide (Aldrich)
  • Selenous acid (H 2SeO 3; Aldrich)
  • Salicylic acid (Aldrich)
  • Sodium bicarbonate (NaHCO 3)
  • 4‐Acetylbenzoic acid (Aldrich)
  • Dimethylaminopyridine (DMAP; Aldrich)
  • 1‐Ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide (EDC)
  • Hydrochloric acid (HCl)
  • Isopropyl alcohol (i‐PrOH)
  • Hexanes (Hex)
  • Trichloroacetonitrile (CCl 3CN)
  • Bis(triethylammonium) hydrogen phosphate (Et 3NH) 2HPO 4
  • Acetonitrile (CH 3CN)
  • Ammonium bicarbonate (NH 4HCO 3)
  • Liquid N 2
  • Deuterium oxide (D 2O; “heavy water”)
  • Sodium pyrophosphate (Na 2H 2P 2O 7)
  • Tris acid and base
  • Magnesium chloride (MgCl 2)
  • Potassium chloride (KCl)
  • Zinc chloride (ZnCl 2)
  • Dithiothreitol (DTT)
  • GFP‐CVIA (Mahmoodi et al., )
  • PFTase stock solution (see Mahmoodi et al., )
  • 50 mM Tris·Cl, pH 7.5 ( appendix 2E)
  • 250‐ml, 100‐ml, and 50‐ml round‐bottom flasks
  • Magnetic stirrer and stir bar
  • Büchi Rotavapor model R‐114 or equivalent rotary evaporator
  • 100‐ and 250‐ml separatory funnels
  • 10 × 2–in. (25.4 × 5.0–cm) chromatography column(s)
  • TLC plates (silica gel 60 F‐254; Merck)
  • Oil bath
  • pH paper
  • Reflux condenser
  • 30°C water bath
  • Addition funnel
  • Büchner funnel
  • 0.45‐μm syringe filter
  • HPLC instrument (for RP‐HPLC): Beckman model 127/166 equipped with a UV detector and a Phenomenex C18 reversed‐phase column (Luna, 10 μm, 10 × 250 mm) with a 5‐cm guard column
  • Electrospray ionization mass spectrometer (ESI‐MS; Bruker BioTOF II)
  • Lyophilization jar and lyophilizer
  • 500‐MHz 1H NMR instrument (Oxford VI‐500 MHz) and NMR tubes
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • 30°C incubator
  • Amicon Centriprep centrifugal filter (MWCO 10,000; Millipore)
  • Centrifuge (Beckman‐Coulter)
  • NAP‐5 or PD‐10 column (GE Healthcare)
  • LC‐MS instrument (Waters Synapt G2 Quadropole TOF mass spectrometer instrument; also see Zhang et al., )
  • Additional reagents and equipment for RP‐HPLC (Josic and Kovac, ) and LC‐MS (Zhang et al., )

Support Protocol 2: Synthesis of Compound 2

  Materials
  • Compound 4 ( protocol 5)
  • Resin‐bound PPh 3 (Aldrich)
  • Tetrabromomethane (CBr 4, Aldrich)
  • Dichloromethane (CH 2Cl 2)
  • 3,5‐Dihydroxy‐benzaldehyde (Ellanova laboratories)
  • Triethylamine (TEA; Aldrich)
  • Ar or N 2 source
  • Trifluoromethanesulfonic anhydride ((CF 3SO 2) 2O, Aldrich)
  • Hydrochloric acid (HCl)
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4)
  • Hexanes (Hex)
  • Ethyl acetate (EtOAc)
  • Deuterated chloroform (CDCl 3)
  • Tetrahydrofuran (THF)
  • Bis(triphenylphosphine)palladium(II) dichloride (Pd(Ph 3) 2Cl 2, Aldrich)
  • Copper (I) iodide (CuI, Aldrich)
  • Ethynyltrimethylsilane (Aldrich)
  • Ammonium chloride (NH 4Cl)
  • Silica gel 60
  • Methanol (MeOH)
  • Lithium hydroxide (LiOH)
  • Diethyl ether (Et 2O; Aldrich)
  • Potassium carbonate (K 2CO 3)
  • Isopropyl alcohol (i‐PrOH)
  • Pyridinium p‐toluenesulfonate (PPTS; Aldrich)
  • Sodium bicarbonate (NaHCO 3)
  • Trichloroacetonitrile (CCl 3CN)
  • Bis(triethylammonium) hydrogen phosphate (Et 3NH) 2HPO 4 (Aldrich)
  • Acetonitrile (CH 3CN)
  • Ammonium bicarbonate (NH 4HCO 3)
  • Liquid N 2
  • Heavy water (D 2O)
  • Sodium pyrophosphate (Na 2H 2P 2O 7)
  • Tris acid and base
  • Magnesium chloride (MgCl 2)
  • Potassium chloride (KCl)
  • Zinc chloride (ZnCl 2)
  • Dithiothreitol (DTT)
  • GFP‐CVIA (Mahmoodi et al., )
  • PFTase stock solution (see Mahmoodi et al., )
  • 50 mM Tris·Cl, pH 7.5 ( appendix 2E)
  • 100‐ml, 25 ml, 10 ml, and 5‐ml round‐bottom flasks
  • Magnetic stirrer and stir bar
  • Sep‐Pak C18 columns (Waters Chromatography)
  • Büchi Rotavapor model R‐114 or equivalent rotary evaporator
  • Septa to fit round‐bottom flasks
  • Syringes
  • Oil bath
  • 10 × 2–in. (25.4 × 5.0–cm) chromatography column(s)
  • TLC plates (silica gel 60 F‐254; Merck)
  • Reflux condenser
  • 100‐ and 250‐ml separatory funnels
  • 30°C water bath
  • Addition funnel
  • Büchner funnel
  • 0.45‐μm syringe filter
  • 30°C incubator
  • HPLC instrument (for RP‐HPLC): Beckman model 127/166 equipped with a UV detector and a Phenomenex C18 column (Luna, 10 μm, 10 × 250 mm) with a 5‐cm guard column
  • Electrospray ionization mass spectrometer (ESI‐MS; Bruker BioTOF II)
  • Lyophilization jars and lyophilizer
  • 500‐MHz 1 H NMR instrument (Oxford VI‐500 MHz) and NMR tubes
  • 15‐ml conical centrifuge tubes (e.g., BD Falcon)
  • Amicon Centriprep centrifugal filter (MWCO 10,000; Millipore)
  • Centrifuge (Beckman‐Coulter)
  • NAP‐5 or PD‐10 columns (GE Healthcare)
  • LC‐MS instrument (Waters Synapt G2 Quadropole TOF mass spectrometer instrument)
  • MALDI‐MS instrument (Bruker MALDI‐TOF)
  • Additional reagents and equipment for RP‐HPLC (Josic and Kovac, ) and LC‐MS (Zhang et al., )
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Literature Cited

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