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. 2016 Jan 15;81(2):351-9.
doi: 10.1021/acs.joc.5b02180. Epub 2015 Nov 24.

V体育安卓版 - pH-Dependent Equilibrium between 5-Guanidinohydantoin and Iminoallantoin Affects Nucleotide Insertion Opposite the DNA Lesion

Judy Zhu  1 , Aaron M Fleming  1 , Anita M Orendt  1   2 , Cynthia J Burrows  1
Affiliations

"V体育安卓版" pH-Dependent Equilibrium between 5-Guanidinohydantoin and Iminoallantoin Affects Nucleotide Insertion Opposite the DNA Lesion

Judy Zhu (VSports app下载) et al. J Org Chem. .

Abstract

Four-electron oxidation of 2'-deoxyguanosine (dG) yields 5-guanidinohydantoin (dGh) as a product. Previously, we hypothesized that dGh could isomerize to iminoallantoin (dIa) via a mechanism similar to the isomerization of allantoin. The isomerization reaction was monitored by HPLC and found to be pH dependent with a transition pH = 10. 1 in which dGh was favored at low pH and dIa was favored at high pH. The structures for these isomers were confirmed by UV-vis, MS, and (1)H and (13)C NMR. Additionally, the UV-vis and NMR experimental results are supported by density functional theory calculations. A mechanism is proposed to support the pH dependency of the isomerization reaction. Next, we noted the hydantoin ring of dGh mimics thymine, while the iminohydantoin ring of dIa mimics cytosine; consequently, a dGh/dIa site was synthesized in a DNA template strand, and standing start primer extension studies were conducted with Klenow fragment exo(-). The dATP/dGTP insertion ratio opposite the dGh/dIa site as a function of pH was evaluated from pH 6. 5-9. 0. At pH 6 VSports手机版. 5, only dATP was inserted, but as the pH increased to 9. 0, the amount of dGTP insertion steadily increased. This observation supports dGh to dIa isomerization in DNA with a transition pH of ∼8. 2. .

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Conflict of interest statement

Conflict of Interest

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Analysis of the pH-dependent equilibrium between dGh and dIa via HPLC analysis. (A) Sample Hypercarb HPLC trace showing the elution profile for dGh and dIa after incubation at pH 11.5. (B) Plot of the pH-Dependent HPLC analysis of dGh and dIa. Relative abundances of the nucleosides were quantified by integrating the area under the elution peaks and normalization by each compounds’ computed extinction coefficient at 240 nm.
Figure 2
Figure 2
Experimental vs. calculated UV-vis spectra for dGh and dIa. The experimental spectra were recorded on the nucleosides during HPLC separation. (A) Comparison of dGh to the TD-DFT simulated UV-vis spectra for Gh with the ribosyl group replaced with a methyl and the addition of 7 explicit waters. The theoretical spectrum was blue shifted by 13 nm to match the experimental spectrum (shown by the dashed arrow). (B) Comparison of dIa to the TD-DFT simulated UV-vis spectrum for Ia with the ribosyl group replaced with a methyl and the addition of 10 explicit waters. The theoretical spectrum was blue shifted by 10 nm to match the experimental spectrum (shown by the dashed arrow). Details of these calculations are described in the text.
Figure 3
Figure 3
Comparison of 13C-NMR chemical shifts experimentally determined at pH 6.0 and 10.5 vs. computed values for dGh and dIa. (A) The scheme to illustrate the interconversion between the diastereomers of dGh or dIa, and the numbering for both compounds.(B) The Δ ppm observed for each carbon atom when the pH 6.0 experimental values were compared to the dGh and dIa theoretical values obtained by DFT calculations as described in the text. (C) The Δ ppm observed for each carbon atom when the pH 10.5 experimental values were compared to the dGh and dIa theoretical values. Comparisons to the R diastereomers are on the left and those to the S diastereomers are on the right.
Figure 4
Figure 4
Line drawings for the dGh•dA and dIa•dG base pairs to illustrate the proposed mimickry of dT by dGh and dC by dIa.
Figure 5
Figure 5
The pH-dependent insertion of dATP or dGTP opposite a dGh/dIa site or dSp site via the DNA polymerase Kf exo−. (A) Plot for the pH-dependent insertion opposite dGh/dIa. (B) Plots for the pH-dependent insertion opposite dSp.
Scheme 1
Scheme 1
Oxidation pathways of dG leading to dOG, the diastereotopic dSp or dGh lesions, a seven-membered ring product (1) observed during oxidations in organic solvents, as well as isomerization of dGh to its constitutional isomer dIa compared to the analogous isomerization of allantoin.
Scheme 2
Scheme 2
Proposed mechanism for the pH-dependent interconversion between the dGh and dIa constitutional isomers (A) and related heterocycles (B, C).
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