Nitrogen-15 Nuclear Magnetic Resonance Spectroscopy of 15N-Labeled Nucleotides

Oxidative damage to DNA is an established source of genomic instability. In this paper, we describe the synthesis and characterization of several pyrimidine deoxynucleoside oxidation damage products, enriched with stable isotopes. These products include the 2'-deoxynucleoside derivatives of 5-(hydroxymethyl)uracil, 5-formyluracil, 5-hydroxyuracil, 5-(hydroxymethyl)cytosine, 5-formylcytosine, and 5-hydroxycytosine. The common precursor is 2'-deoxy-2"-deutero[1,3-15N]uridine. Additional stable isotopes are added during functional group conversions. Characterization of these derivatives includes mass spectrometry and 1H and 15N NMR spectroscopy. Proton and nitrogen NMR studies reported here allow an examination of the influence of the modification on sugar conformation and tautomeric equilibrium, properties likely to be important in understanding the biological consequences of these DNA damage products.

Section: Resultssupporting

confidence: 92%

Synthesis and Characterization of Isotopically Enriched Pyrimidine Deoxynucleoside Oxidation Damage Products

LaFrancois

Fujimoto

Sowers

1998

“…It is significant that the Nl chemical shifts of 8-OH-dG and 8-OH-G are essentially the same as for dG and G, both of which are reported to exist in the 6-keto form (30). This is substantiated by the observation of a sharp doublet for the Nl resonance of G at 148.2 ppm (Figure 1), and by previous studies (19,22,31). Furthermore, the shift of the Nl resonance of 1-methylguanosine is reported to be 143 ppm, which is in excellent agreement with that (148 ppm) of dG or G, once the estimated inductive effect (+3.3 ppm) from the methyl group is considered (23).…”

Section: Resultssupporting

confidence: 77%

“…Likewise, it can be concluded that large changes in the electronegativity of the C8 substituent, including both strong electron donating and electron withdrawing groups, do not alter the tautomerism of this class of modified nucleosides. We also note from published chemical shift data (19,22,31) that Nl is insensitive to changes in the sugar portion (C2,-hydroxyl or -deoxy and C3'or C5'-phosphates) of G or dG, as is expected from our analysis.…”

Section: Resultssupporting

confidence: 72%

“…This establishes that a proton is attached to N7 and that exchange is minimal. The N7 chemical shift lies in the range of urea-type nitrogens and exhibits a large upfield shift due to the 8-keto group (+138 ppm) compared to the parent compound, G, indicating a conversion from a pyridine-type nitrogen to a pyrrole-type nitrogen (Figure 1) (31). The N9 resonance exhibits a moderate upfield shift (+26 ppm) compared to G, due to its conversion to an amide type nitrogen.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Nitrogen-15 nuclear magnetic resonance studies on the tautomerism of 8-hydroxy-2'-deoxyguanosine, 8-hydroxyguanosine, and other C8-substituted guanine nucleosides

Cho¹,

Kadlubar²,

Culp³

et al. 1990

The favored tautomeric and ionic structures were examined for the oxidative DNA damage adduct 8-hydroxy-2'-deoxyguanosine and its RNA analogue 8-hydroxyguanosine by 15N NMR spectroscopy. In addition, 15N chemical shifts and coupling constants from 13 different guanine nucleosides, including a wide variety of C8 substitutions (OH, SH, Br, OCH2C6H5, OCH3, SCH3, and SO2CH3), have been analyzed with respect to their tautomeric structures. A -98.5-Hz proton-nitrogen coupling constant observed for the N7 resonance of 8-hydroxyguanosine in dimethyl sulfoxide was evidence for 8-keto substitution, which is contrary to the structure implied by the generally used nomenclature. The pH dependence of 15N NMR spectra of 8-hydroxyguanosine in aqueous solution showed downfield shifts of the N1 and N7 resonances that were greater than 50 ppm, which indicated the conversion from a neutral 6,8-diketo to a 6-enolate-8-keto (pKa1 = 8.6) and finally to a 6,8-dienolate structure (pKa2 = 11.7). There was no evidence of an 8-enol substituent in the absence of ionization. It is proposed that the syn conformation of these oxidized bases in duplex DNA and RNA can be further stabilized by abnormal hydrogen bonding or mispairing that involves N7-H. The combined data show that 15N NMR is a sensitive probe to examine tautomerism of the guanine ring system. The analysis indicates that the change from a single to a double bond for the C8 substituent, and the accompanying removal of the normal double bond between N7 and C8 on the imidazole ring system, has no detectable effect on the tautomerism at the N1-O6 site of the pyrimidine ring system for both the 8-keto and 8-thio substitutions. In addition, large differences in electronegativity of the C8 substituents do not alter the N1-O6 tautomerism.

“…Both the 6,8-dienol and the 6-keto-8-enol tautomers would have an acidic pKa resulting from protonation of N7. The N3 of the purine ring in 2,-deoxyguanosine does not readily undergo protonation in acid in comparison to the pyridine-like N7 (20); however, protonation of Nl in the 6- "The parentheses indicate a shoulder 6 Taken from ref 24. or point of inflection.…”

Section: Methodsmentioning

confidence: 99%

Structural and conformational analyses of 8-hydroxy-2'-deoxyguanosine

Culp¹,

Cho²,

Kadlubar³

et al. 1989

161

124

Oxidative DNA damage has been shown to involve formation of 8-hydroxy-2'-deoxyguanosine, which may serve as a mispairing lesion during cellular DNA replication. In order to assess the mutagenic potential of this DNA adduct, we examined the possible occurrence of several tautomeric forms and of different base conformations about the deoxyribose. Several spectroscopic and electronic absorption techniques were employed and showed structural changes occurring over a broad pH range. Two pKa's near pH 8 and 12 were observed by pH-solvent partitioning experiments, ultraviolet absorption spectral analyses, and 13C NMR spectroscopic methods. The presence of two pKa's suggested the formation of a dianion, with the second being formed in strong alkali. A comparison of ultraviolet absorption band features of 8-hydroxy-2'-deoxyguanosine with that of different C6,C8-diketo or enol derivatives supported a C8-keto assignment and also provided evidence that this DNA adduct contains a C6-keto group at physiological pH. 13C NMR data showed marked chemical shifts at C6 in solutions of pH 7.4-9.3, indicating the location of the first ionization. Increasing basicity produced further shifts at C5 and C8, indicating the C8 position for the second ionization. Multiple infrared bands were observed in the carbonyl region of the neutral compound, but only a single carbonyl band at 1692 cm-1 remained at pH 9.1, implying a keto group at C8. Determination of the chemical shifts and the nuclear Overhauser enhancements of the N1 and N7 resonances in the proton-decoupled 15N NMR spectrum indicated that both nitrogens were indeed protonated at neutral pH.(ABSTRACT TRUNCATED AT 250 WORDS)