Synthesis of 8‐Oxoguanosine Phosphoramidite and Its Incorporation into Oligoribonucleotides

Yosuke Taniguchi1, Yohei Koga1, Shigeki Sasaki1

1 Graduate School of Pharmaceutical Sciences, Kyushu University, Maidashi, Higashi‐ku, Fukuoka
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.58
DOI:  10.1002/0471142700.nc0458s56
Online Posting Date:  March, 2014
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Abstract

The detailed synthetic protocol for preparation of the phosphoramidite of an oxidatively damaged ribonucleotide, 8‐oxoguanosine (8‐oxo‐G), and its incorporation into RNA are described. The O6‐ and N7‐bisdiphenylcarbamoyl‐protected 8‐oxoguanosine phosphoramidite was synthesized as a new phosphoramidite precursor unit for the synthesis of RNA. It was successfully incorporated into the RNA sequences, and the synthesized RNAs were completely deprotected with 28% aqueous ammonia solution at 55°C for 24 hr. After purification using HPLC, they were identified by MALDI‐TOF mass measurement. The base‐pairing properties showed that 8‐oxo‐G forms base pairs not only with rC or dC in anti‐conformation, but also with rA in syn conformation within the RNA duplexes or RNA/DNA heteroduplexes. Curr. Protoc. Nucleic Acid Chem. 56:4.58.1‐4.58.10. © 2014 by John Wiley & Sons, Inc.

Keywords: 8‐oxoguanosine; oxidatively damaged ribonucleotide; base pairing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of the 8‐Oxoguanosine Phosphoramidite
  • Basic Protocol 2: Synthesis, Purification, and Base‐Pairing Properties of Oligoribonucleotides Containing 8‐Oxoguanosine
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of the 8‐Oxoguanosine Phosphoramidite

  Materials
  • 8‐Hydroxyguanosine (8‐oxoguanosine, 1; Wako Pure Chemical Industries, Ltd., purity >95%)
  • Trimethylsilyl chloride (TMSCl)
  • Dry pyridine
  • Dry dichloromethane (CH 2Cl 2)
  • Argon source
  • Phenoxyacetyl chloride (PacCl)
  • Chloroform (CHCl 3)
  • 28% (v/v) aqueous ammonia (Nacalai Tesque, cat. no. 02511‐05)
  • Methanol (MeOH)
  • N,N‐dimethyl‐4‐aminopyridine (DMAP)
  • 4,4′‐dimethoxytrityl chloride (DMTrCl)
  • Ethyl acetate (EtOAc)
  • Anhydrous Na 2SO 4
  • Silica gel Kanto 60N (spherical, neutral, 63 to 210 μm, Kanto Chemical)
  • Diisopropylethylamine
  • Diphenylcarbamoyl chloride (DPCCl)
  • Hexane
  • Saturated sodium hydrogen carbonate solution (NaHCO 3 solution)
  • Silica gel CHROMATOREX FL60D (spherical, neutral, 62 μm Fuji Silysia Chemical Co.)
  • di‐n‐Butyltin dichloride (Bu 2SnCl 2)
  • (Triisopropylsiloxy)methyl chloride (TOMCl; Aldrich, cat. no. 91415)
  • Diethyl ether
  • Dry acetonitrile (CH 3CN; appendix 2A)
  • Diisopropylammonium 1H‐tetrazolide (see recipe)
  • N,N,N′,N′‐tetraisopropylphosphoramidite (Aldrich, cat. no. 305995)
  • 30‐, 50‐, and 100‐mL round‐bottom flasks
  • Silica gel TLC plate Kiselgel 60F 254 (0.2 mm, Merck)
  • Rotary evaporator
  • Filter paper
  • Büchner funnel
  • Vacuum source
  • 100‐ and 200‐mL separatory funnel
  • 1, 2, and 3 × 30–cm chromatography columns
  • Oil bath
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis, Purification, and Base‐Pairing Properties of Oligoribonucleotides Containing 8‐Oxoguanosine

  Materials
  • 8‐Oxoguanosine phosphoramidite (6; see protocol 1)
  • Dry acetonitrile (CH 3CN; appendix 2A)
  • 28% (v/v) aqueous ammonia (Nacalai Tesque, cat. no. 02511-05)
  • 99.5% ethanol (Nacalai Tesque, cat. no. 14713‐95)
  • N‐methyl‐2‐pyrrolidone
  • Triethylamine
  • Triethylamine trihydrofluoride
  • 0.1 M triethylammonium acetate (TEAA) buffer, pH 7.0
  • 3‐Hydroxypicolinic acid (3‐HPA)
  • Diammonium citrate
  • 10 mM sodium phosphate at pH 7.0 ( appendix 2A) containing 100 mM NaCl
  • 0.25 M 5‐benzylmercapto‐1H‐tetrazole in CH 3CN (Glen Research, cat. no. 30‐3170)
  • Complementary RNAs and DNAs (Genenet Co. Ltd.)
  • nS‐8 oligonucleotide synthesizer (Gene Design Inc.)
  • 0.2 μmol CPG column (controlled pore glass, Glen Research)
  • 1.5‐mL screw‐cap tubes (Maruemu Corp., MV vial V‐15)
  • SpeedVac evaporator
  • HPLC column, Waters: X‐bridge C18 5 μm, 10 × 100‐mm (also see Josic and Kovac, )
  • Additional reagents and equipment for solid‐phase oligonucleotide synthesis (Chapter 3), RP‐HPLC (Josic and Kovac, ), spectrophotometric quantitation of nucleic acids (unit 10.3), MALDI‐TOF mass spectrometry (unit 10.1), and melting curve analysis (unit 7.3)
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Figures

Videos

Literature Cited

Literature Cited
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