Carbohydrate‐Oligonucleotide Conjugates

Gwladys Pourceau1, Albert Meyer1, Jean‐Jacques Vasseur1, François Morvan1

1 Institut des Biomolécules Max Mousseron, CNRS, Université Montpellier 1, Université Montpellier 2, Montpellier, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.38
DOI:  10.1002/0471142700.nc0438s39
Online Posting Date:  December, 2009
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This unit describes a strategy for attaching two mannose and two galactose residues to an oligonucleotide. This conjugation can be performed at the 5′‐end of the oligonucleotide sequence, using modified phosphoramidites. First, the oligonucleotide scaffold is synthesized on solid support using a DNA synthesizer, with commercially available and modified phosphoramidites. After the first “click” reaction with a galactosylated azide derivative on solid support, the bromine atoms are replaced with azides and a second click reaction is performed with propargylated mannose either on solid support or in solution. Additionally, using a monoalkynated solid support, the conjugation with carbohydrate residues can be performed at the 3′‐end of the oligonucleotide according to a similar protocol. Curr. Protoc. Nucleic Acid Chem. 39:4.38.1‐4.38.25. © 2009 by John Wiley & Sons, Inc.

Keywords: carbohydrate; oligonucleotide; click chemistry; conjugate; galactose; mannose

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Table of Contents

  • Introduction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:

  • 1,1,1‐Tris(hydroxymethyl)ethane
  • Anhydrous pyridine (dried over CaH 2 and distilled)
  • Argon
  • Methanol (MeOH)
  • 4,4′‐Dimethoxytrityl chloride
  • Sulfuric acid stain solution [10% (v/v) sulfuric acid in ethanol]
  • Anhydrous methylene chloride (CH 2Cl 2; dried over P 2O 5 and distilled)
  • Saturated aqueous sodium bicarbonate solution
  • Solid anhydrous sodium sulfate
  • Toluene
  • Silica gel (0.04 to 0.06 nm)
  • Triethylamine (Et 3N)
  • Acetone
  • Anhydrous acetonitrile (dried over CaH 2 and distilled)
  • Anhydrous tetrahydrofuran (THF; dried over alumina)
  • 60% (w/v) sodium hydride in oil
  • 80% (w/v) propargyl bromide in toluene
  • Cyclohexane
  • Ethyl acetate (EtOAc)
  • Sodium iodide
  • 1,6‐Dibromohexane
  • Diisopropylammonium tetrazolide (DIAT)
  • 2‐cyanoethyl N,N,N′,N′‐tetraisopropylphosphordiamidite
  • Molybde stain solution (see unit 3.16)
  • 50‐mL and 500‐mL round‐bottom flasks equipped with stir bar and CaCl 2 guard
  • TLC silica plates (0.20‐mm silica gel 60 with fluorescent indicator UV 254; Macherey‐Nagel)
  • 254‐nm UV lamp
  • Rotary evaporator
  • Vacuum pump
  • 250‐mL separatory funnel
  • Cotton
  • 2.5‐cm and 5‐cm diameter chromatography columns
  • Additional reagents and equipment thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)
NOTE: For each step, starting materials, solvents, and glassware must be as dry as possible to obtain high yields.

Basic Protocol 2:

  • 1,4‐cyclohexanedimethanol (mixture of cis and trans)
  • 4‐dimethylaminopyridine (DMAP)
  • Anhydrous pyridine
  • Anhydrous methylene chloride
  • p‐Toluenesulfonyl chloride
  • Anhydrous triethylamine (dried over CaH 2 and distilled)
  • Argon
  • Sulfuric acid stain solution
  • Saturated aqueous sodium bicarbonate solution
  • Methylene chloride (CH 2Cl 2)
  • Solid anhydrous sodium sulfate
  • Silica gel (0.04 to 0.06 nm)
  • Methanol
  • N,N‐dimethylformamide (DMF)
  • Sodium iodide
  • Sodium azide
  • Vanillin stain solution (see recipe)
  • Saturated aqueous sodium chloride solution (brine)
  • Ethyl acetate
  • Cyclohexane
  • Peracetylated D‐galactose
  • Anhydrous acetonitrile
  • Boron trifluoride etherate
  • 50‐mL round‐bottom flask equipped with stir bar and CaCl 2 guard
  • TLC silica plates (0.20 mm silica gel 60 with fluorescent indicator UV 254, Macherey‐Nagel)
  • 254‐nm UV lamp
  • 250‐mL separatory funnel
  • Cotton
  • Rotary evaporator
  • Vacuum pump
  • 2.5‐cm diameter chromatography column
  • Vacuum evaporator
  • 100‐mL round‐bottom flask equipped with stir bar and condenser
  • Oil bath and heater
  • Additional reagents and equipment for performing thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 3:

  • Peracetylated D‐mannose
  • Propargyl alcohol
  • Petroleum ether (Ep)

Basic Protocol 4:

  • Long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å, 80 to 120 mesh, amino group, 80 to 90 µmol/g)
  • 3% trichloroacetic acid (TCA) in methylene chloride (commercial deblocking solution)
  • Anhydrous methylene chloride
  • Phosphorous oxide (P 2O 5)
  • Anhydrous pyridine
  • Succinic anhydride
  • 4‐dimethylaminopyridine (DMAP)
  • 1‐O‐(4,4′‐dimethoxytrityl)‐2‐propargyloxymethyl‐2‐methyl‐1,3‐propanediol ( S.3)
  • N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide (DEC)
  • Anhydrous triethylamine
  • Pentachlorophenol
  • Anhydrous piperidine (dried over CaH 2 and distilled)
  • Acetic anhydride in dry pyridine/THF 10/10/80 v/v (commercial Cap A solution)
  • 10% (w/v) N‐methylimidazole in dry THF (commercial Cap B solution)
  • 0.1 M p‐toluenesulfonic acid solution in acetonitrile
  • Frits (porosity 3)
  • Vacuum desiccator
  • Sealed tube
  • Shaker
  • 10‐mL volumetric flasks
  • Spectrophotometer
  • Additional reagents and equipment for automated oligonucleotide synthesis and the trityl assay ( appendix 3C)

Basic Protocol 5:

  • Reagents recommended for automated solid‐phase oligonucleotide synthesis:
    • Standard 2‐cyanoethyl deoxyribonucleoside phosphoramidites (T, CBz, ABz, and GiBu)
    • Activator: 5‐Benzylthio‐1H‐tetrazole (BMT)
    • Oxidation solution: 0.1 M iodine in 70:10:20 (v/v/v) THF/pyridine/water
  • Extra dry acetonitrile
  • Cap A solution: 10:10:80 (v/v/v) acetic anhydride in dry pyridine/THF
  • Cap B solution: 10% N‐methylimidazole in dry THF
  • Deblocking solution: 3% dichloroacetic acid (DCA) in dry methylene chloride
  • Commercial thymidine‐derived solid support (Link technologies, CPG 500 Å, L=61 µmol/g)
  • Phosphoramidites S.5 and S.6 ( protocol 1)
  • Galactopyranoside S.10 ( protocol 2)
  • Methanol
  • Copper sulfate (CuSO 4)
  • Sodium ascorbate (Na Asc)
  • Degassed water
  • Dry argon gas cylinder
  • Dry nitrogen
  • Sodium azide (NaN 3)
  • Sodium iodide (NaI)
  • Dimethylformamide (DMF)
  • Methylene chloride
  • Mannopyranoside S.11 ( protocol 3)
  • Concentrated aqueous ammonia solution (30%)
  • 0.05 M triethylammonium acetate, pH 7
  • ABI 394 DNA Synthesizer (Applied Biosystems)
  • Empty synthesis columns, TWIST style (Glen Research)
  • Initiator microwave synthesizer (Biotage)
  • 0.2‐ to 0.5‐mL microwave vials (equipped with Teflon septa, micro stir bar, and aluminum cap)
  • 0.2‐ to 0.5‐mL vial adapters
  • Crimper
  • 0.5‐ and 2‐mL microcentrifuge tubes
  • 0.5‐ to 10‐µL, 5‐ to 40‐µL, 40‐ to 200‐µL, and 200‐ to 1000‐µL micropipettors with tips
  • 2‐mL syringes
  • Ultrasound bath
  • 65°C oven
  • Sealed vials (with Teflon septa and screw top) for HPLC samples preparations
  • 55°C dry bath
  • Speed vacuum
  • Vacuum desiccator
  • Reverse‐phase C 18 (5 µm) column (150 × 4.6–mm; for analyses)
  • Reverse‐phase C 18 (15 µm) column (300 × 7.8–mm; for analyses)
  • NAP 10 column
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C), synthesis yield (unit 10.3), MALDI‐TOF mass spectrometry (unit 10.1), and reversed‐phase HPLC chromatography (unit 10.5)

Basic Protocol 6:

  • Solid support S.12 ( protocol 4)
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Literature Cited

Literature Cited
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