Chemical Research in Toxicology Scheme 2. Numbering Scheme of the Carbinolamine N2-dG:N-Lys DNA γ-Hydroxytrimethylene Tether was Isotope-Edited (In Red Color) ARTICLE KWKK Conjugates in Which the X7 N2 or Cγ of the dimension and 2048 real data in the t2 dimension, and were zero-filled during processing to create a matrix of 1024 1024 real points. 15N HSQC experiments21 23 were performed with WATERGATE water suppression.20,24 The spectra were recorded with 256 real data in the t1 dimension and 1024 real data in the t2 dimension and were zero filled to create a matrix of 512 256 real points. The 2D 15N NOESY-HSQC pulse program was modified from the corresponding 3D pulse program25,26 with 128 points centered at 81 ppm. The water signal was suppressed using the WATERGATE method. The mixing time was 250 ms. The spectrum was recorded with 512 real data in the t1 dimension and 2048 real data in the t2 dimension, and was zero-filled during processing to create a 1024 512 matrix. 13C HSQC experi- ments were carried out using standard 1H-detected pulse programs with States-TPPI phase cycling.21 23 The spectra were recorded with 256 real data in the t1 dimension and 1024 real data in the t2 dimension, and were zero filled to create a matrix of 512 256 real points. The lifetimes of the peptide conjugates were determined with the 13C-labeled sample using 13C HSQC NMR. The sample was incubated for one month at 37 C in 10 mM NaH2PO4, 100 mM NaCl, and 50 μM EDTA (pH 7.0). Thermal Melting Studies. Melting temperatures of the DNA KWKK conjugates were measured in 10 mM NaH2PO4, 100 mM NaCl, and 50 μM EDTA (pH 7.0) by UV spectroscopy at 260 nm. The strand concentration was 10 μM. The thermal scan proceeded from 15 to 85 C. The melting temperatures were calculated by differentiating the absorbance profiles. Molecular Modeling. Starting structures were created from refined structures of the fully reduced trimethylene DNA peptide conjugate,18 using the program INSIGHT II (Accelrys, Inc., San Diego, CA). The partial charges of the γ-hydroxytrimethylene tether were obtained from density function theory (DFT) calculations using a neutral total charge, utilizing the B3LYP/6-31G* basis set and the program Gaussian.27 Potential energy minimization calculations were conducted with the program AMBER28 and the parm99 force field. The pairwise generalized Born (GB) model29,30 was used to simulate implicit waters. The cutoff radius for nonbonding interactions was 18 Å. A 1000- iteration potential energy minimization was performed, using the conjugate gradient algorithm. Molecular Dynamics Simulations. The simulations were per- formed in explicit water using the AMBER force field with the particle mesh Ewald (PME) method.31,32 The potential energy-minimized structures of the R- and S-carbinolamine DNA KWKK conjugates were used as the starting structures. The conjugates were surrounded by a 10.0 Å TIP3P water box in each direction. A total of 20 Naþ ions were added to neutralize the duplex. Bond lengths involving hydrogen were fixed using the SHAKE algorithm.33 The cutoff radius for nonbonded interactions was 10 Å. The conjugate was first energy-minimized for 2,500 iterations. The MD simulation was then carried out with constant volume at 300 K for 10,000 iterations with an integrator time of 1 fs. The MD simulation at constant pressure was performed at 300 K for 5 ns with an integrator time of 2 fs. The PTRAJ program from the AMBER package28 [v. 10] was used to analyze the trajectories. The rmsd values of the trajectories were referenced to the starting structures. A distance of less than 3.5 Å and an angle of greater than 120 between the potential hydrogen donor and acceptor were used as criteria for hydrogen bond formation. Helicoidal analysis of the backbone was carried out using CURVES.34,35 ’ RESULTS Synthesis of DNA KWKK Conjugates. The intramolecular formation of N2-dG:N2-dG DNA interstrand cross-links (5,6) competes with the bimolecular association of the KWKK peptide with the DNA (Scheme 1). To minimize the formation of interstrand DNA cross-links, excess KWKK was used. To avoid producing DNA KWKK conjugates resulting from the reaction with the amino groups of the lysine side chains, the reaction was carried out at pH 7.0 such that the lysine side chains were protonated.36 Using a 15N-labeled sample, the reaction was monitored by 15N HSQC NMR (Figure 1). Two X7 N2 f N2H correlations arising from the DNA KWKK conjugate 8 became stronger as time elapsed. Simultaneously, the X7 N2 f 2 N H correlations of other γ-OH-PdG derivatives became weaker and disappeared. The intensities of the three cross-peaks arising from the natural abundance of the excess KWKK amides remained constant. At 5 C, the reaction approached equilibrium after 6 weeks. After 8 weeks, the excess peptide was removed by gel filtration to facilitate the analyses of the DNA KWKK conjugate. A total of less than 10% of nonconjugated oligodeox- ynucleotides and DNA interstrand cross-links was also observed 1125 dx.doi.org/10.1021/tx200113n |Chem. Res. Toxicol. 2011, 24, 1123–1133