Synthesis of Acetylene‐Substituted Probes with Benzene‐Phosphate Backbones for RNA Labeling

Yoshiaki Kitamura1, Yoshihito Ueno2, Yukio Kitade1

1 Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu, 2 Course of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.59
DOI:  10.1002/0471142700.nc0459s57
Online Posting Date:  June, 2014
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Conversion of dimethyl 5‐aminoisophthalate into the iodoarene via the corresponding diazonium intermediate, followed by Sonogashira coupling with trimethylsilylacetylene afford the alkynylarene, which is reduced with LiAlH4 to give 5‐ethynyl‐1,3‐benzenedimethanol (BE). One hydroxyl group is protected with a 4,4′‐dimethoxytrityl (DMTr) group and subsequently another hydroxyl group is phosphitylated to produce the phosphoramidite. The mono‐DMTr compound is also modified to afford the corresponding succinate, which is then reacted with controlled pore glass (CPG) to provide the solid support. Either the phosphoramidite or the solid support is employed in solid‐phase synthesis of RNA containing BE. RNA oligomers bearing BE rapidly react with 4‐fluorobenzylazide to produce the cycloaddition products in good to excellent yield. Curr. Protoc. Nucleic Acid Chem. 57:4.59.1‐4.59.15. © 2014 by John Wiley & Sons, Inc.

Keywords: alkynes; RNA labeling; Huisgen cycloaddition; click chemistry

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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis, Purification, and Characterization of 5‐Ethynyl‐1,3‐Benzenedimethanol
  • Basic Protocol 2: Synthesis, Purification, and Characterization of RNA Sequences
  • Basic Protocol 3: Rapid, Ligand‐Free, Copper‐Catalyzed, Azide‐Alkyne Cycloaddition
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Synthesis, Purification, and Characterization of 5‐Ethynyl‐1,3‐Benzenedimethanol

  • Dimethyl 5‐aminoisophthalate (2; TCI)
  • Concentrated hydrochloric acid (HCl)
  • Sodium nitrite (NaNO 2)
  • Potassium iodide (KI), ice cold
  • Ethyl acetate (EtOAc)
  • Brine
  • Magnesium sulfate, anhydrous (MgSO 4)
  • n‐Hexane
  • Silica gel (60Å, 63 to 210 mesh; Kanto)
  • Methanol (MeOH)
  • Bis(triphenylphosphine)palladium(II) dichloride (PdCl 2(PPh 3) 2; Aldrich)
  • Copper(I) iodide (CuI)
  • Triphenylphosphine (PPh 3)
  • Tetrahydrofuran, anhydrous (THF)
  • Argon
  • Trimethylsilylacetylene
  • Piperidine
  • Ammonium chloride (NH 4Cl)
  • Sodium sulfate, anhydrous (Na 2SO 4)
  • Lithium aluminum hydride (LiAlH 4)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Potassium sodium tartrate (KNaC 4H 4O 6·4H 2O)
  • Chloroform (CHCl 3)
  • 20‐, 300‐, and 500‐mL round‐bottom flasks
  • Magnetic stirrer and stir bars
  • Pipets
  • 50‐ and 500‐mL separatory funnels
  • 500‐mL Erlenmeyer flasks
  • Glass filter
  • Vacuum
  • 1‐L round‐bottom flasks
  • Rotary evaporator
  • Pasteur pipets
  • 2 × 20–cm and 7.5 × 20–cm chromatography columns
  • 60°C water bath
  • Funnel, oven heated
  • Kiriyama funnel
  • Septa
  • 10‐mL syringes
  • Funnels fitted with cotton

Basic Protocol 2: Synthesis, Purification, and Characterization of RNA Sequences

  • 5‐Ethynyl‐1,3‐benzenedimethanol (1, see protocol 1)
  • Pyridine (Wako)
  • Argon
  • 4,4′‐Dimethoxytritylchloride (DMTrCl)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Ethyl acetate (EtOAc)
  • Brine
  • Sodium sulfate, anhydrous (Na 2SO 4)
  • n‐Hexane
  • Silica gel (60Å, 63 to 210 mesh; Kanto)
  • Tetrahydrofuran, anhydrous (THF)
  • N,N‐Diisopropylethylamine (i‐Pr 2NEt)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (i‐Pr 2NP(Cl)O(CH 2) 2CN)
  • Chloroform (CHCl 3)
  • Succinic anhydride
  • N,N‐Dimethylaminopyridine (DMAP)
  • 1‐Ethyl‐3‐(3‐dimethylaminopropyl)carbodiimide hydrochloride (EDC‐HCl)
  • N,N‐Dimethylformamide (DMF)
  • Aminopropyl controlled pore glass (CPG; GhemGenes)
  • Acetic anhydride (Ac 2O)
  • Ethanol (EtOH)
  • Acetonitrile (MeCN)
  • 70% perchloric acid (HClO 4)
  • 3′‐O‐TBDMS‐protected adenosine phosphoramidite (prepared according to Ueno et al., ):
    • N6‐Benzoyl‐3′‐O‐(tert‐butyldimethylsilyl)‐2′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐5′‐O‐(4,4′‐dimethoxytrityl)adenosine
  • Chemical phosphorylation reagent (CPR; Glen Research):
    • 2‐Cyanoethyl‐N,N‐diisopropyl‐2‐[2‐(4,4′‐dimethoxytrityloxy)ethylsulfonyl]ethyl‐phosphoramidite
  • 2′‐O‐TBDMS‐protected phosphoramidites (Glen Research):
    • 2′‐O‐(tert‐Butyldimethylsilyl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐N6‐phenoxyacetyladenosine
    • 2′‐O‐(tert‐Butyldimethylsilyl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐ 5′‐O‐(4,4′‐dimethoxytrityl)‐N2‐isopropylphenoxyacetylguanosine
    • N4‐Acetyl‐2′‐O‐(tert‐butyldimethylsilyl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐5′‐O‐(4,4′‐dimethoxytrityl)cytidine
    • 2′‐O‐(tert‐Butyldimethylsilyl)‐3′‐O‐[(N,N‐diisopropylamino)(2‐cyanoethyloxy)]phosphinyl‐5′‐O‐(4,4′‐dimethoxytrityl)uridine
  • Reagents for oligonucleotide synthesis:
    • Cap A solution: acetic anhydride in tetrahydrofuran (THF)/pyridine (Glen Research)
    • Cap B solution: 1‐methylimidazole in THF (Glen Research)
    • Oxidation solution: 0.02 M iodine in THF/pyridine/water (Glen Research)
    • 1H‐Tetrazole (prepare 3% solution in MeCN before use)
    • Trichloroacetic acid (prepare 3% solution in CH 2Cl 2 before use)
  • Concentrated ammonium hydroxide (conc. aqueous NH 3)
  • Tetra‐n‐butylammonium fluoride (TBAF; 1 M solution in THF, available from Aldrich)
  • TEAA buffer, pH 7.0 (prepare 2 M solution in MilliQ from triethylamine and acetic acid)
  • 20‐, 50‐, and 100‐mL round‐bottom flasks
  • Magnetic stir plate and bars
  • 50‐mL syringes
  • Rubber septa
  • 50‐ and 100‐mL separatory funnels
  • 50‐ and 100‐mL Erlenmeyer flasks
  • Funnels fitted with cotton
  • Rotary evaporator
  • Vacuum pump
  • Pasteur pipets
  • 2 × 10–cm and 4 × 10–cm chromatography columns
  • Platform shaker
  • Glass filters
  • 25‐mL volumetric flasks
  • Spectrophotometer
  • DNA/RNA synthesizer
  • 1.5‐mL screw‐cap stock tubes
  • Vortexer
  • 55°C heating block/water bath
  • 1.5‐mL microcentrifuge tubes (Eppendorf)
  • Centrifugal evaporator
  • Reversed‐phase C18 column (Sep‐Pak Vac tC18 cartridge, Waters)

Basic Protocol 3: Rapid, Ligand‐Free, Copper‐Catalyzed, Azide‐Alkyne Cycloaddition

  • 4‐Fluorobenzylazide (10; prepared in Kitamura et al., )
  • 5‐Ethynyl‐1,3‐benzenedimethanol (1, see protocol 1)
  • 50% ethanol (EtOH)
  • Sodium L‐ascorbate (Aldrich)
  • Copper (II) sulfate pentahydrate (CuSO 4·5H 2O)
  • Ethyl acetate (EtOAc)
  • Brine
  • Sodium sulfate, anhydrous (Na 2SO 4)
  • Chloroform (CHCl 3)
  • Methanol (MeOH)
  • Silica gel (60Å, 63 to 210 mesh, Kanto)
  • MilliQ water (Millipore)
  • Dimethyl sulfoxide (DMSO)
  • MeCN
  • 0.1 M phosphate buffer, pH 7.0, sterile
  • 20‐mL round‐bottom flasks
  • Magnetic stir plate and bars
  • 50‐mL separatory funnels
  • 50‐mL Erlenmeyer flasks
  • Funnels fitted with cotton
  • Rotary evaporator
  • Vacuum pump
  • Pasteur pipets
  • 2 × 20–cm chromatography columns
  • UV‐VIS spectrophotometer
  • 1.5‐ and 2‐mL microcentrifuge tubes (Eppendorf)
  • Vortexer
  • 1‐mL syringe
  • Membrane filter (DISMIC‐13CP, 0.45 µm, ADVANTEC)
  • Inertsil ODS‐3 HPLC column (GL Sciences)
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Literature Cited

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