Isolation and characterization of diazoate intermediate upon nitrous acid and nitric oxide treatment of 2 '-deoxycytidine

T. Suzuki et al., Isolation and characterization of diazoate intermediate upon nitrous acid and nitric oxide treatment of 2 '-deoxycytidine, BIOCHEM, 38(22), 1999, pp. 7151-7158
Citations number
Categorie Soggetti
Biochemistry & Biophysics
Journal title
ISSN journal
0006-2960 → ACNP
Year of publication
7151 - 7158
SICI code
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 a cetate buffer (pH 3.7) at 37 degrees C, a previously unidentified product w as formed. By spectrometric measurements, the product was identified as a d iazoate derivative of dCyd, 1-(beta-D-2'-deoxyribofuranosyl)-2-oxopyrimidin e-4-diazoate. The time course of the concentration change of the diazoate s howed a profile characteristic of a reaction intermediate, and the maximum yield was 37 mu M at the reaction time of 25 min. Up to the reaction time o f 10 min, the diazoate concentration was greater than that of dUrd, a deami nation product of dCyd. Addition of thiocyanate increased the yield of the diazoate in HNO2 treatment, whereas addition of ascorbate decreased the yie ld. When 10 mM dCyd in 100 mM phosphate buffer was treated with NO at 37 de grees 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 degrees C, the diazoate was c onverted to dUrd with the first-order rate constant k = 4.8 x 10(-4) s(-1) (t(1/2) = 24 min). Under physiological conditions (pH 7.4, 37 degrees C), h owever, it was fairly stable (k = 5.8 x 10(-7) s(-1), t(1/2) = 330 h), In b oth cases, the diazoate was converted to dUrd exclusively and no other inte rmediates were detected by HPLC analysis. Uracil-DNA glycosylase did not re move the diazoate residue from an oligodeoxynucleotide containing this dama ge, [d(T6DT5), D = the diazoate]. The T-m value of a duplex containing the diazoate, d(T6DT5) . d(A(5)GA(6)), was much lower than that of a duplex con taining a correct C:G base pair, d(T6CT5). d(A(5)GA(6)). These results show that the diazoate is generated as a stable intermediate in the reactions o f 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.