Chemical Research in Toxicology ARTICLE Figure 1. 15N HSQC of the reaction of γ-OH-PdG containing oligodeoxynucleotide with the peptide KWKK (1:50, pH 7.0). (A) Immediately after KWKK was added. (B) At 5 C for 2 weeks. (C) At 5 C for 4 weeks. (D) At 5 C for 8 weeks. X7 N2 f N2H correlations were assigned as follows: a, γ-OH-PdG 2; b, N2-aldehyde 3; c, N2-aldehydrol 4; d, DNA KWKK conjugates 8. Other cross-peaks arose from the natural abundances of excess KWKK amides. (Figure 2). These could not be separated from the DNA KWKK conjugate product without causing the dissociation of the DNA KWKK conjugates. The DNA KWKK conjugates were characterized by MALDI-TOF mass spectrometry. NMR Spectroscopy. Figure 2 shows the 15N HSQC spectra of the 15N-labeled DNA KWKK conjugates at different tempera- tures at pH 5.3. The X7 N2 f N2H correlations were broad at 10 C, and they were sharper at higher temperatures. A similar phenomenon was observed for the Cγ f Hγ correlations in the 13C HSQC spectra. This was attributed to aggregation of the DNA KWKK conjugates due to the electrostatic interaction of a positively charged peptide with negatively charged DNA. The spectra at neutral pH and the 1H resonances of the DNA KWKK conjugates were compared with those in acidic and basic solutions. No pH dependence was observed. Satisfac- tory spectra were recorded at pH 5.3, pH 7.0, and pH 8.9 and 25 and 30 C for the exchangeable protons and at 37 C for the nonexchangeable protons. However, some of the exchangeable proton resonances remained broad under these conditions. Spectroscopic Characterization of the DNA KWKK Con- jugate Linkage. Figure 3A displays the isotope-edited 13C HSQC spectrum of the Cγ DNA KWKK conjugates. Two Cγ f Hγ correlations were assigned to the conjugates. They were in equilibrium with N2-dG:N2-dG interstrand cross-links (5,6) and ring-opened species (3,4) derived from the γ-OH-PdG adduct (Scheme 1). Kurtz et al.14 reported that the γ-OH-PdG induced DNA KWKK conjugates were reducible by NaCNBH3, sug- gesting that imine conjugate 9 was present. However, compared with the resonances of imine carbon and hydrogen,37 the resonances of Cγ and Hγ of both DNA KWKK conjugates were located significantly upfield, suggesting that neither of the two NMR-detectable DNA KWKK conjugates was imine 9. Isotope-edited 15N HSQC spectra of the X7 N2 15N-labeled DNA KWKK conjugates are displayed in Figure 2. The X7 N2 f N2H correlations of two DNA KWKK conjugates were not resolved, although the cross-peaks appeared to split at 40 C. Corresponding to the X7 N2 f N2H correlations, two sets of NOEs were observed in the 15N NOESY-HSQC spectrum (Figure 4B). The slightly downﬁeld X7 N2H resonance was assigned to the major DNA KWKK conjugate and the upﬁeld X7 N2H resonance was assigned to the minor. Each conjugate exhibited a strong NOE between X7 N2H and guanine (X7 or G19) N1H imino protons. The NOESY data suggested that all nucleotides of the major DNA KWKK conjugate adopted the anti conformation about the glycosyl bond. In B-DNA, the anti conformation places the neighboring G19 N1H 5.0 6.0 Å from the X7 N2H amino proton. In contrast, the X7 N1H imino proton in carbinolamine 8 is ∼2.0 Å from the X7 N2H amino proton. Therefore, these strong NOEs were assigned to X7 N2H f X7 N1H correlations. This was consistent with the X7 N1H f C18 1126 dx.doi.org/10.1021/tx200113n |Chem. Res. Toxicol. 2011, 24, 1123–1133