Isolation and Characterization of Diazoate Intermediate upon Nitrous Acid and Nitric Oxide Treatment of 2‘-Deoxycytidine

• Published in: Biochemistry (Easton) Vol. 38; no. 22; pp. 7151 - 7158
• Main Authors: Suzuki, Toshinori, Nakamura, Takanori, Yamada, Masaki, Ide, Hiroshi, Kanaori, Kenji, Tajima, Kunihiko, Morii, Takashi, Makino, Keisuke
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.06.1999
• Subjects: Deoxycytidine - chemistry; Diazonium Compounds - chemistry; Diazonium Compounds - isolation & purification; DNA Glycosylases; Drug Stability; Kinetics; Mutagens - chemistry; N-Glycosyl Hydrolases - chemistry; Nitric Oxide - chemistry; Nitrous Acid - chemistry; Nuclear Magnetic Resonance, Biomolecular; Nucleic Acid Heteroduplexes - chemistry; Nucleic Acid Precursors - chemistry; Nucleic Acid Precursors - isolation & purification; Pyrimidines - isolation & purification; Substrate Specificity; Uracil-DNA Glycosidase
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi982803t

The intermediate produced from dCyd by HNO2 and NO treatments was isolated and characterized. When 10 mM dCyd was treated with 100 mM NaNO2 in 1.0 M acetate buffer (pH 3.7) at 37 °C, a previously unidentified product was formed. By spectrometric measurements, the product was identified as a diazoate derivative of dCyd, 1-(β-d-2‘-deoxyribofuranosyl)-2-oxopyrimidine-4-diazoate. The time course of the concentration change of the diazoate showed a profile characteristic of a reaction intermediate, and the maximum yield was 37 μM at the reaction time of 25 min. Up to the reaction time of 10 min, the diazoate concentration was greater than that of dUrd, a deamination product of dCyd. Addition of thiocyanate increased the yield of the diazoate in HNO2 treatment, whereas addition of ascorbate decreased the yield. When 10 mM dCyd in 100 mM phosphate buffer was treated with NO at 37 °C under aerobic conditions holding the pH (7.2−7.6), the diazoate was also generated. The yield of the diazoate was higher than that of dUrd up to 15 mmol of NO absorption. At pH 3.7 and 37 °C, the diazoate was converted to dUrd with the first-order rate constant k = 4.8 × 10-4 s-1 (t 1/2 = 24 min). Under physiological conditions (pH 7.4, 37 °C), however, it was fairly stable (k = 5.8 × 10-7 s-1, t 1/2 = 330 h). In both cases, the diazoate was converted to dUrd exclusively and no other intermediates were detected by HPLC analysis. Uracil-DNA glycosylase did not remove the diazoate residue from an oligodeoxynucleotide containing this damage, [d(T6DT5), D = the diazoate]. The T m value of a duplex containing the diazoate, d(T6DT5)·d(A5GA6), was much lower than that of a duplex containing a correct C:G base pair, d(T6CT5)·d(A5GA6). These results show that the diazoate is generated as a stable intermediate in the reactions of dCyd with HNO2 and NO and that the major product is the diazoate but not dUrd in the initial stage of the reactions. Thus, once formed in vivo, the diazoate persists for long time in DNA and may act as a major cytotoxic and/or genotoxic lesion with biologically relevant doses of HNO2 and NO.