Chemical Research in Toxicology ARTICLE Figure 2. 15N HSQC spectra of the 15N-labeled DNA KWKK conjugates at diﬀerent temperatures at pH 5.3: (A) at 10 C, (B) at 20 C, (C) at 30 C, and (D) at 40 C. X7 N2 f N2H correlations were assigned to (a) N2-aldehyde 3, (b) N2-aldehydrol 4, (c) DNA interstrand cross-link 5, (d) DNA KWKK conjugates 8, and (e) unassignable, might be arising from DNA KWKK conjugates 9, 10, or a side-chain byproduct. N4H2, X7 N2H f HR1, X7 N2H f HR2, X7 N2H f Hβ, and X7 N2H f A8 H2 NOEs (Figure 4). Pyrimidopurinone 10 lacks an imino proton. The observation of the X7 N1H resonance proved that these two DNA KWKK conjugates were diastereomeric carbinolamines 8. This conclusion was also consistent with the chemical shifts of the Hγ protons. Pyrimidopurinone 10 has a chemical structure similar to that of γ-OH-PdG and the pyr- imidopurinone dG-dG cross-link. Compared with their Hγ protons,17,38 40 the Hγ resonances of the two DNA KWKK conjugates were shifted signiﬁcantly upﬁeld. Thermal Melting Studies. The thermal melting of the DNA KWKK conjugates was monitored using UV spectrosco- py at pH 7 in 100 mM NaCl (Figure 5). The DNA KWKK conjugates exhibited a melting temperature (Tm) of 62 ( 1 C. Compared with the unmodified DNA (Tm: 60 C) and the DNA containing the γ-OH-PdG adduct (Tm: 48 C), the formation of these DNA KWKK conjugates stabilized the DNA. When the denatured DNA duplex containing the DNA KWKK conjugates was reannealed, and the melting experiment repeated, a melting transition corresponding to DNA containing the γ-OH-PdG adduct was observed together with a weaker melting transition corresponding to the DNA KWKK conju- gates. Further thermal cycling showed that the melting transition of the DNA KWKK conjugates became weaker in the third melting cycle and disappeared in the fourth. Thus, the DNA KWKK conjugates reversed when the DNA was denatured. NMR experiments agreed with this conclusion. When the 13C-labeled DNA KWKK conjugates were denatured at 85 C for 30 min and reannealed, the Cγ f Hγ correlations of stereoisomeric carbinolamine conjugates 8 disappeared in the 13C HSQC spectrum, and simultaneously, the Cγ f Hγ correlations of N2-propylaldehyde 3 and N2-propanoaldehydrol 4 appeared. The lifetimes of the carbinolamine DNA KWKK conjugates 8 in DNA were evaluated by 13C HSQC NMR using a 13C- labeled sample (Figure 3). Immediately after isolation, two strong Cγ f Hγ correlations were observed. They became weaker as time elapsed. The minor carbinolamine diastereomer was unobservable after 2 weeks and the major after 4 weeks. They slowly converted to the unconjugated N2-propylaldehyde 3 (its Cγ f Hγ correlation not shown in Figure 3), N2-propa- noaldehydrol 4, and DNA interstrand cross-links 5. Thus, the stereoisomeric carbinolamine DNA KWKK conjugates have longer lifetimes than do the DPCs induced by formaldehyde whose half-life times are less than 1 day in vitro,10 and their lifetimes are comparable to the lifetime of DPC induced by malondialdehyde.41 NMR Resonance Assignments. Exchangeable protons of the major DNA KWKK conjugate were assigned by comparing the 15N HSQC filtered NOESY spectrum with the nonisotope- edited NOESY spectrum (Figure 4). The X7 N1H imino resonance of the major DNA KWKK conjugate broadened at 30 C. Notably, it still exhibited a weak X7 N1H f C18 N4H2 NOE in acidic solution (Figure 4A). This assignment was supported by NOEs including C18 N4H2 f C18 N4H1, C18 N 4 H2 f C18 H5, C18 N 4 H1 f C18 H5, and C18 H5 f C18 H6 1127 dx.doi.org/10.1021/tx200113n |Chem. Res. Toxicol. 2011, 24, 1123–1133