Chemical Research in Toxicology ARTICLE Figure 3. 13C HSQC spectra of the 13C labeled DNA KWKK conjugates at pH 7.0: (A) immediately after isolation, (B) at 37 C for 1 week, (C) at 37 C for 2 weeks, and (D) at 37 C for 4 weeks. Cγ f Hγ correlations were assigned to (a) N2-aldehydrol 4, (b) DNA interstrand cross-link 5, (c) major DNA KWKK conjugate 8, and (d) minor DNA KWKK conjugate 8. Other cross-peaks were assigned to the C f H correlations of natural abundance. (Figure 4C). The X7 N1H f C18 N4H2 NOE suggested that the X7 3 C18 base pair maintained Watson Crick hydrogen bonding. No evidence was observed for protonated G 3 C Hoogs- teen base pairing often observed for 1,N2-dG adducts in acidic solution.42 44 Neither the C18 N 4 H1 or C18 N 4 H2 resonances shifted downfield compared to other cytosine amino protons. The nonexchangeable protons were assigned on the basis of the NOE sequential connectivity of the base H6/H8 protons with 20-deoxyribose H10 protons.45,46 Complete sequential NOESY connectivities were observed for both modiﬁed and complementary strands of the major DNA peptide conjugate. The X7 H8 f C6 H10 NOE was weaker than other NOEs of the aromatic protons with their 50-neighboring H10 protons, indicat- ing the X7 3 C18 and C6 3 G19 base pairs underwent small pertur- bations. The chemical shifts of aromatic H6/H8 and sugar H10 protons were compared with those in the corresponding un- modiﬁed oligodeoxynucleotide. Except for C6, X7, and C18 H10 protons, very small chemical shift perturbations were observed, indicating that the major DNA peptide conjugate maintained B-DNA geometry. C6, X7, and C18 H10 protons shifted downﬁeld by 0.26, 0.25, and 0.34 ppm, respectively. These remarkable chemical shift perturbations suggested structural perturbations at base pairs X7 3 C18 and C6 3 G19. The deoxyribose H30 and H20(00) protons were also assigned. Signiﬁcant downﬁeld shifts were not observed for the H20(00) protons, suggesting the nucleotides did not adopt the syn conformation.47 50 Molecular Modeling. The diastereomeric DNA KWKK con- jugates were modeled using the refined structure of the fully reduced trimethylene conjugate18 as a starting structure. The carbinolamine conjugates 8 of either R- or S-configurations were built by adding a hydroxyl group to the Cγ of the tether. Potential energy minimization was carried out to provide model structures. Figure 6 displays the modified region of structures viewed from the minor groove. Consistent with NMR data, the structures main- tained Watson Crick hydrogen bonding at the site of conjuga- tion. The KWKK peptide was located in the minor groove. Molecular Dynamics Simulations. To assess the potential hydrogen bonding interactions of the carbinol hydroxyl group in the DNA KWKK conjugates, a series of MD simulations were carried out in explicit water. Table 1 lists hydrogen bonding occupations of the hydroxyl group by potential hydrogen bond acceptors. For both isomers, the carbinol hydroxyl showed minimal intermolecular hydrogen bonding with water. The occupancy of an intramolecular hydrogen bond between the hydroxyl group and G19 O40 was greatest for the S-configuration of the carbinolamine link 8. The S-isomer also exhibited a low 1128 dx.doi.org/10.1021/tx200113n |Chem. Res. Toxicol. 2011, 24, 1123–1133