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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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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Basic Protocol 1: Preparation of the 8‐Oxoguanosine Phosphoramidite

  • 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

  • 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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Literature Cited

Literature Cited
  Andreoli, R., Mutti, A., Goldoni, M., Manini, P., Apostoli, P., and De Palma, G. 2011. Reference ranges of urinary biomarkers of oxidized guanine in (2′‐deoxy)ribonucleotides and nucleic acids. Free Radic. Biol. Med. 50:254‐261.
  Chang, Y., Kong, Q., Shan, X., Tian, G., Ilieva, H., Cleveland, D.W., Rothstein, J.D., Borchelt, D.R., Wong, P.C., and Lin, C.L. 2008. Messenger RNA oxidation occurs early in disease pathogenesis and promotes motor neuron degeneration in ALS. PLoS One 3:e2849.
  Henriksen, T., Hillestrom, P.R., Poulsen H.E., and Weimann, A. 2009. Automated method for the direct analysis of 8‐oxo‐guanosine and 8‐oxo‐2′‐deoxyguanosine in human urine using ultraperformance liquid chromatography and tandem mass spectrometry. Free Radic. Biol. Med. 47:629‐635.
  Hofer, T., Badouard, C., Bajak, E., Ravanat, J.L., Mattsson, A., and Cotgreave, I.A. 2005. Biol. Chem. 386:333‐337.
  Hofer, T., Seo, A.Y., Prudencio, M., and Leeuwenburgh, C. 2006. A method to determine RNA and DNA oxidation simultaneously by HPLC‐ECD: Greater RNA than DNA oxidation in rat liver after doxorubicin administration. Biol. Chem. 387:103‐111.
  Josic, D. and Kovac, S. 2010. Reversed‐phase high performance liquid chromatography of proteins. Curr. Protoc. Protein Sci. 61:8.7.1‐8.7.22.
  Kamiya, H., Suzuki, A., Yamaguchi, Y., Handa, H., and Harashima, H. 2009. Incorporation of 8‐hydroxyguanosine (8‐oxo‐7,8‐dihydroguanosine) 5′‐triphosphate by bacterial and human RNA polymerases. Free Radic. Biol. Med. 46:1703‐1707.
  Kim, S.K., Yokoyama, S., Takaku, H., and Moon, B.J. 1998. Oligoribonucleotides containing 8‐oxo‐7,8‐dihydroguanosine and 8‐oxo‐7,8‐dihydro‐2′‐O‐methylguanosine: Synthesis and base pairing properties. Bioorg. Med. Chem. Lett. 8:939‐944.
  Kim, S.K., Lee, S.H., Kwon, O.‐S., and Moon, B.J. 2004. DNA RNA heteroduplex containing 8‐oxo‐7,8‐dihydroguanosine: Base pairing, structures, and thermodynamic stability. J. Biochem. Mol. Biol. 37:657‐662.
  Koga, Y., Taniguchi, Y., and Sasaki, S. 2013. Synthesis of the oligoribonucleotides incorporating 8‐oxo‐guanosine and evaluation of their base pairing properties. Nucleosides Nucleotides Nucleic Acids 32:124‐136.
  Martinet, W., de Meyer, G.R., Herman, A.G., and Kockx, M.M. 2004. Reactive oxygen species induce RNA damage in human atherosclerosis. Eur. J. Clin. Invest. 34:323‐327.
  Nunomura, A., Chiba, S., Kosaka, K., Takeda, A., Castellani, R.J., Smith, M.A., and Perry, G. 2002. Neuronal RNA oxidation is a prominent feature of dementia with Lewy bodies. NeuroReport 13:2035‐2039.
  Nunomura, A., Moreira, P.I., Castellani, R.J., Lee, H., Zhu, X., Smith, M.A., and Perry, G. 2012. Oxidative damage to RNA in aging and neurodegenerative disorders. Neurotox. Res. 22:231‐248.
  Shan, X., Tashiro, H., and Lin, C.‐L. 2003. The identification and characterization of oxidized RNAs in Alzheimer's disease. J. Neurosci. 23:4913‐4921.
  Tanaka, M., Chock, P., and Stadtman, E. 2007. Oxidized messenger RNA induces translation errors. Proc. Nat. Acad. Sci. U.S.A. 104:66‐71.
  Taniguchi, Y., Kawaguchi, R., and Sasaki, S. 2011. Adenosine‐1,3‐diazaphenoxazine derivative for selective base pair formation with 8‐oxo‐2′‐deoxyguanosine in DNA. J. Am. Chem. Soc. 133:7272‐7275.
  Taniguchi, Y., Koga, Y., Fukabori, K., Kawaguchi, R., and Sasaki, S. 2012. OFF‐to‐ON type fluorescent probe for the detection of 8‐oxo‐dG in DNA by the Adap‐masked ODN probe. Bioorg. Med. Chem. Lett. 22:543‐546.
  Zhang, J., Perry, G., Smith, M.A., Robertson, D., Olson, S.J., Graham, D.G., and Montine, T J. 1999. Parkinson's disease is associated with oxidative damage to cytoplasmic DNA and RNA in substantia nigra neurons. Am. J. Pathol. 154:1423‐1429.
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