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

Seung Pil Pack¹, Keisuke Makino²

¹Korea University, Jochiwon, Chungnam, Korea
²Kyoto 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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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)
HNO₂ solution (see recipe)
5 M sodium hydroxide (NaOH) solution
200 mM sodium phosphate buffer, pH 7.4
CH₃CN, 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 (CH₂Cl₂), anhydrous (synthesis-grade and HPLC-grade)
Sodium sulfate (Na₂SO₄), 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 (NaHCO₃) 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)
C₁₈ columns:
- For collection of dOxo (S.2): COSMOSIL C₁₈-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 Basic Protocol 1)
Acetonitrile (CH₃CN), anhydrous, DNA-synthesis-grade (ABI Biosystems)
CH₂Cl₂, anhydrous (DCM), DNA-synthesis-grade (ABI Biosystems)
3% dichloroacetic acid in CH₂Cl₂ (DCA/DCM, DNA synthesis–grade; ABI Biosystems)
Activator: 0.45% tetrazole in CH₃CN (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 I₂ in THF/pyridine/H₂O (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
C₁₈ 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

Figure 4.39.1

Conversion of dGuo (S.1) to dimethoxytrityl-protected phophoramidite of dOxo (DMT-dOxo-amidite; S.4) Reproduced from Pack et al. (2005b) with permission from Oxford University Press.

View Image
Figure 4.39.2

NP-HPLC chromatogram of dimethoxytrityl-protected phophoramidite of dOxo (DMT-dOxo-amidite; S.4). NP-HPLC conditions: isocratic elution with 65:35:0.33 EtOAc/CH₂Cl₂/MeOH; 1 mL/min flow rate; Ultron VX-SIL (150 × 4.6–mm, 5-µm) column; ambient temperature; 11.86 and 12.79 min (diastereoisomer) detection. The NMR data is provided in Table 4.39.1. Reproduced from Pack et al. (2005b) with permission from Oxford University Press.

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Figure 4.39.3

Conformational change of Oxa base: (A) open-ring form of dOxo-NH₃, (B) ring opening/closure of dOxo depending on pH. Reproduced from Pack et al. (2005b) with permission from Oxford University Press.

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Figure 4.39.4

RP-HPLC separation of detritylated 5-mer Oxa-ODN (5¢-GCOAT-3¢) purified by a Poly-Pak cartridge (A), and DNA monomer, dCyd, dGuo, dThd, dOxo, and dAdo formed by the digestion of the oligomer with nuclease P1 and alkaline phosphatase (B). HPLC conditions: 100 mM TEAA solution (pH 7.0) with a gradient of CH₃CN [for A, 7.6% (0 min) to 14% (40 min) and for B, 0% (0 min) to 20% (20 min)] mobile phase; 1 mL/min with flow rate; Ultron VX-ODS columns (150 × 4.6–mm, 5-µm) (see Basic Protocol 2). Reproduced from Pack et al. (2005b) with permission from Oxford University Press.

View Image
Figure 4.39.5

Oxanine, a novel deaminated product from guanine via NO- or HNO₂-induced nitrosative oxidation. Reproduced from Suzuki et al. (1996) with permission from the American Chemical Society.

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Figure 4.39.6

Recognition of Oxa in DNA by restriction endonucleases (A) and incorporation efficiencies of normal bases opposite Oxa in the chain elongation using E. coli DNA polymerase I Kf (B). Reproduced from Pack et al. (2007b) with permission from Taylor & Francis.

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Figure 4.39.7

Adduct formation of Oxa in DNA with amine derivatives. 25X/C, substrate are a mixture of ³²P-labeled 25X (5¢-CATCGATAGCATCCTXCCTTCTCTC-3¢; X = O or G) and its complementary oligomer. Reproduced from Nakano et al. (2003). This research was originally published in the Journal of Biological Chemistry. 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. 278:25264-25272. Copyright by the American Society for Biochemistry and Molecular Biology.

View Image
Figure 4.39.8

O-acylisourea formation of Oxa (A) and its application for DNA probe immobilization on an amine-functionalized surface (B). Reproduced from Pack et al. (2007a) with permission from Oxford University Press.

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Literature Cited

Literature Cited
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	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.