Synthesis of 2′‐Deoxyoxanosine from 2′‐Deoxyguanosine, Conversion to Its Phosphoramidite, and Incorporation into Oxanine‐Containing Oligodeoxynucleotides

Seung Pil Pack1, Keisuke Makino2

1 Korea University, Jochiwon, Chungnam, Korea, 2 Kyoto University, Yoshida‐Honmachi, Kyoto, Japan
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.39
DOI:  10.1002/0471142700.nc0439s41
Online Posting Date:  June, 2010
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Abstract

Oxanine (Oxa, O) is one of the damaged bases produced from guanine (G) through nitrosative deamination induced by nitric oxide (NO) or nitrous acid (HNO2). Large‐scale preparation of Oxa‐containing oligodeoxynucleotide (Oxa‐ODN) with the desired base sequence is a prerequisite for exploring detailed properties of Oxa in DNA. This can be accomplished by incubation of G nucleosides with NaNO2 in acetic acid buffer (pH 3.5) to produce Oxa nucleosides (e.g., 2′‐deoxyoxanosine or dOxo), conversion of dOxo to DMT‐dOxo‐amidite by tritylation and conventional phosphoramidation, and subsequent synthesis of Oxa‐ODN. The presence of Oxa in the synthetic ODN is confirmed by enzymatic digestion. Oxa‐ODN is useful for analyzing the biochemical and biophysical properties of Oxa in DNA, which is believed to be involved in NO‐induced genotoxicity and cytotoxicity. In addition, since Oxa possesses the carbodiimide‐activated carboxylate function (O‐acylisourea structure), Oxa‐ODN can be used as a functional DNA oligomer that makes covalent cross‐linkages with amine or amine‐containing biomolecules and amine‐modified solid surfaces. Curr. Protoc. Nucleic Acid Chem. 41:4.39.1‐4.39.20. © 2010 by John Wiley & Sons, Inc.

Keywords: 2′‐deoxyoxanosine; nitrosative deamination; NO‐induced genotoxicity; DMT‐dOxo‐amidite; O‐acylisourea structure

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of 2′‐Deoxyoxanosine Phosphoramidite from 2′‐Deoxyguanosine
  • Basic Protocol 2: Synthesis and Purification of Oxanine‐Containing Oligodeoxynucleotides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of 2′‐Deoxyoxanosine Phosphoramidite from 2′‐Deoxyguanosine

  Materials
  • 2′‐Deoxyguanosine (dGuo; S.1)
  • HNO 2 solution (see recipe)
  • 5 M sodium hydroxide (NaOH) solution
  • 200 mM sodium phosphate buffer, pH 7.4
  • CH 3CN, HPLC‐grade
  • Dry N,N‐dimethylformamide (DMF), anhydrous (synthesis‐grade)
  • Diisopropylethylammonium mesylate (DIEA‐Mes; see recipe)
  • 4‐4′‐Dimethoxytritylchloride (DMTrCl, 95% pure; Wako Pure Chemicals)
  • Imidazole
  • Methanol (MeOH), analytical‐grade
  • Silica gel column:
    • For analysis and collection of DMT‐dOxo‐amidite ( S.4): Ultron VX‐SIL column (for analysis, 150 × 4.6–mm or for preparation, 250 × 20–mm, 5 µm; Shinwa Chemical Industries)
  • Milli‐Q water
  • Dichloromethane (CH 2Cl 2), anhydrous (synthesis‐grade and HPLC‐grade)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Hexane, HPLC‐grade
  • 1H‐Tetrazole
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphorodiamidite (Aldrich)
  • Diisopropylethylamine (DIEA)
  • Ethyl acetate (EtOAc), HPLC‐grade
  • 10% (w/v) sodium hydrogen carbonate (NaHCO 3) solution
  • 100‐, 500‐, and 1000‐mL round‐bottom flasks
  • 45°C incubator containing a magnetic stir plate
  • Rotary evaporator equipped with a water aspirator
  • Filter paper (Advantec no. 2)
  • Millex GS cartridge filters (0.22‐µm; Millipore)
  • RP‐HPLC systems:
    • For collection of dOxo ( S.2): controller (Tosoh PX‐8010), pump (CCPM), and UV detector (UV‐8010) (available from Tosoh)
    • For analysis of dOxo and DMT‐dOxo ( S.3): controller (Tosoh PX‐8020), pump (DP‐8020), temperature controller (CO‐8020), and photo‐diode array detector (PD‐8020) (available from Tosoh)
  • C 18 columns:
    • For collection of dOxo ( S.2): COSMOSIL C 18‐PAQ column (250 × 28–mm, 5‐µm; Nacalai Tesque)
    • For analysis of dOxo: Ultron VX‐ODS column (for analysis, 150 × 4.6–mm, 5‐µm; Shinwa Chemical Industries)
  • Lyophilizer
  • NP‐HPLC system:
    • For analysis and collection of DMT‐dOxo‐amidite ( S.4): controller plus pump (Hitachi L‐6200) and UV detector (L4000) (available from Hitachi)
  • Vacuum oil pump
  • 100‐ and 500‐mL flasks

Basic Protocol 2: Synthesis and Purification of Oxanine‐Containing Oligodeoxynucleotides

  Materials
  • DMT‐dOxo‐amidite ( S.4; see protocol 1)
  • Acetonitrile (CH 3CN), anhydrous, DNA‐synthesis‐grade (ABI Biosystems)
  • CH 2Cl 2, anhydrous (DCM), DNA‐synthesis‐grade (ABI Biosystems)
  • 3% dichloroacetic acid in CH 2Cl 2 (DCA/DCM, DNA synthesis–grade; ABI Biosystems)
  • Activator: 0.45% tetrazole in CH 3CN (Glen Research)
  • Cap mix A: THF/pyridine/phenoxyacetic anhydride (Glen Research)
  • Cap mix B: 1‐methylimidazole in THF (Glen Research)
  • Oxidizing solution: 0.02 M I 2 in THF/pyridine/H 2O (Glen Research)
  • CPG support (0.2‐ or 1‐µmol scale CPG)
  • Phosphoramidites (Glen Research):
    • Ac‐dC‐amidite (acetyl‐protected dC amidite)
    • Pac‐dA‐amidite (phenoxyacetyl‐protected dA amidite)
    • iPr‐Pac‐dG‐amidite (4‐isopropyl‐pheoxyacetyl‐protected dG amidite)
    • dT‐amidite
  • 0.5 M NaOH solution
  • Triethylamine‐acetate buffer (TEAA buffer), pH 7.4: 2 M (for stock) or 100 mM (for HPLC analysis)
  • Milli‐Q water
  • 50 mM sodium acetate buffer, pH 5.8
  • Zinc chloride
  • Nuclease P1
  • Alkaline phosphatase
  • DNA synthesizer (Applied Biosystems 3400 DNA synthesizer)
  • Water aspirator or vacuum oil pump
  • 15‐mL screw‐capped tube
  • 37°C water bath or incubator
  • Poly‐Pak (reverse‐phase cartridge for DNA isolation; Glen Research)
  • Lyophilizer
  • RP‐HPLC system
  • C 18 columns for analysis and collection of dOxo ( S.2) and Oxa‐ODN: 5‐µm Ultron VX‐ODS column—150 × 4.6–mm (for analysis) or 250 × 6–mm (for collection) (Shinwa Chemical Industries)
  • HPLC filter
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Figures

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Literature Cited

Literature Cited
   De Napoli, L., Di Fabio, G., Messere, A., Montesarchio, D., Piccialli, G., and Varra, M. 1998. A new synthesis of oxanosine and 2′‐deoxyoxanosine. Tetrahedron Lett. 39:7397‐7400.
   Glaser, R. and Son, M.S. 1996. Pyrimidine ring opening in the unimolecular dediazoniation of guanine diazonium ion. An ab initio theoretical study of the mechanism of nitrosative guanosine deamination. J. Am. Chem. Soc. 118:10942‐10943.
   Hayakawa, Y. and Kataoka, M. 1998. Facile synthesis of oligodeoxynucleotides via the phosphoramidite method without nucleoside base protection. J. Am. Chem. Soc. 120:12395‐12401.
   Itoh, O., Kuroiwa, S., Atsumi, S., Umezawa, K., Takeuchi, T., and Hori, M. 1989. Induction by guanosine analog oxanosine of reversion toward the normal phenotype of K‐ras‐transformed rat‐kidney cells. Cancer Res. 49:996‐1000.
   Kataoka, M. and Hayakawa, Y. 1999. A conventional method for the synthesis of N‐free 5′‐O‐(p,p′‐dimethoxytrityl)‐2′‐deoxyribonucleodises via the 5′‐O‐selective tritylation of the parent substances. J. Org. Chem. 64:6087‐6089.
   Nakano, T., Terato, H., Asagoshi, K., Masaoka, A., Mukuta, M., Ohyama, Y., Suzuki, T., Makino, K., and Ide, H. 2003. DNA‐protein cross‐link formation mediated by oxanine. A novel genotoxic mechanism of nitric oxide‐induced DNA damage. J. Biol. Chem. 278:25264‐25272.
   Nakano, T., Katafuchi, A., Shimizu, R., Terato, H., Suzuki, T., Tauchi, H., Makino, K., Skorvaga, M., Van Houten, B., and Ide, H. 2005. Repair activity of base and nucleotide excision repair enzymes for guanine lesions induced by nitrosative stress. Nucleic Acids Res. 33:2181‐2191.
   Nakano, T., Morishita, S., Katafuchi, A., Matsubara, M., Horikawa, Y., Terato, H., Salem, A.M.H., Izumi, S., Pack, S.P., Makino, K., and Ide, H. 2007. Nucleotide excision repair and homologous recombination system commit differentially to the repair of DNA‐Protein crosslinks. Mol. Cell 28:147‐158.
   Pack, S.P., Suzuki, T., Ide, H., Kodaki, T., and Makino, K. 2005a. Reaction of NO with nucleic acid bases and its biological implications. Front. Org. Chem. 1:297‐341.
   Pack, S.P., Nonogawa, M., Kodaki, T., and Makino, K. 2005b. Chemical synthesis and thermodynamic characterization of oxanine‐containing oligodeoxynucleotides. Nucleic Acids Res. 33:5771‐5780.
   Pack, S.P., Kamisetty, N.K., Nonogawa, M., Devarayapalli, K.C., Ohtani, K., Yamada, K., Yoshida, Y., Kodaki, T., and Makino, K. 2007a. Direct immobilization of DNA oligomers onto the amine‐functionalized glass surface for DNA microarray fabrication through the activation‐free reaction of oxanine. Nucleic Acids Res. 35:e110.
   Pack, S.P., Doi, A., Nonogawa, M., Kamisetty, N.K., Devarayapalli, K.C., Kodaki, T., and Makino, K. 2007b. Biophysical stability and enzymatic recognition of oxanine in DNA strands. Nucleosides Nucleotides Nucleic Acids 26:1589‐1593.
   Pack, S.P., Doi, A., Kamisetty, N.K., Nonogawa, M., Kodaki, T., and Makino, K. 2007c. Functional reactivity of oxanine: Its biological meanings and biotechnological applications. Nucleic Acids Symp. Ser. 51:53‐54.
   Shimada, N., Yagisawa, N., Naganawa, H., Takita, T., Hamada, M., Takeuchi, T., and Umezawa, H. 1981. Oxanosine, a novel nucleoside from Actinomycetes. J. Antibiot. 34:1216‐1218.
   Suzuki, T., Yamaoka, R., Nishi, M., Ide, H., and Makino, K. 1996. Isolation and characterization of a novel product, 2′‐deoxyoxanosine, from 2′‐deoxyguanosine, oligodeoxyribonucleotide, and calf thymus DNA treated by nitrous acid and nitric oxide. J. Am. Chem. Soc. 118:2515‐2516.
   Suzuki, T., Matsumura, Y., Ide, H., Kanaori, K., Tajima, K., and Makino, K. 1997. Deglycosylation susceptibility and base‐pairing stability of 2′‐deoxyoxanosine in oligodeoxynucleotide. Biochemistry 36:8013‐8019.
   Suzuki, T., Yoshida, M., Yamada, M., Ide, H., Kobayashi, M., Kanaori, K., Tajima, K., and Makino, K. 1998. Misincorporation of 2′‐deoxyoxanosine 5′‐triphosphate by DNA polymerases and its implication for mutagenesis. Biochemistry 37:11592‐11598.
   Suzuki, T., Ide, H., Yamada, M., Endo, N., Kanaori, K., Tajima, K., Morii, T., and Makino, K. 2000a. Formation of 2′‐deoxyoxanosine from 2′‐deoxyguanosine and nitrous acid: Mechanism and intermediates. Nucleic Acids Res. 28:544‐551.
   Suzuki, T., Yamada, M., Ide, H., Kanaori, K., Tajima, K., Morii, T., and Makino, K. 2000b. Identification and characterization of a reaction product of 2′‐deoxyoxanosine with glycine. Chem. Res. Toxicol. 13:227‐230.
   Terato, H., Masaoka, A., Asagoshi, K., Honsho, A., Ohyama, Y., Suzuki, T., Yamada, M., Makino, K., Yamamoto, K., and Ide, H. 2002. Novel repair activities of AlkA (3‐methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid. Nucleic Acids Res. 30:4975‐4984.
   Vongchampa, V., Dong, M., Gingipalli, L., and Dedon, P. 2003. Stability of 2′‐deoxyxanthosine in DNA. Nucleic Acids Res. 31:1045‐1051.
   Wuenschell, G.E., O'Connor, T.R., and Termini, J. 2003. Stability, miscoding potential, and repair of 2′‐deoxyxanthosine in DNA: Implications for nitric oxide‐induced mutagenesis. Biochemistry 42:3608‐3616.
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