Orientation of the crotonaldehyde-derived N²[3-oxo-1(S)-methyl- propyl]-dG DNA adduct hinders interstrand cross-link formation in the 5′-CpG-3′ sequence

The conformation of the crotonaldehyde-derived N²-[3-oxo-1(S)- methyl-propyl]-dG adduct in the oligodeoxynucleotide 5′-d(G ¹C²T³A⁴G⁵C ⁶X⁷A⁸G⁹T¹⁰C ¹¹C¹²)-3′-5′-d(G¹³G ¹⁴A¹⁵C¹⁶T¹⁷C¹⁸G ¹⁹C²⁰T²¹A²²G²³C ²⁴)-3′, where X = N²-[3-oxo-1(S)-methyl-propyl]-dG, is reported. This adduct arises from opening of the cyclic N²-(S- α-CH₃-γ-OH-1,N²-propano-2′)-dG adduct when placed opposite dC in duplex DNA. This oligodeoxynucleotide contains the 5′-CpG-3′ sequence in which the N²-(R-α-CH ₃-γ-OH-1,N²-propano-2′)-dG but not the N ²-(S-α-CH₃-γ-OH-1,N²-propano- 2′)-dG adduct preferentially formed an interstrand carbinolamine cross-link [Kozekov, I. D., Nechev, L. V., Moseley, M. S., Harris, C. M., Rizzo, C. J., Stone, M. P., and Harris, T. M. (2003) J. Am. Chem. Soc. 125, 50-61; Cho, Y.-J., Wang, H., Kozekov, I. D., Kurtz, A. J., Jacob, J., Voehler, M., Smith, J., Harris, T. M., Lloyd, R. S., Rizzo, C. J., and Stone, M. P. (2006) Chem. Res. Toxicol. 19, 195-208]. Analysis of ¹H NOE data, chemical shift perturbations, and deoxyribose pseudorotations and backbone torsion angles suggested the presence of a stable and ordered DNA conformation at pH 9.3 and 30 °C, with minimal conformational perturbation. The spectral line widths of the adduct protons were comparable to those of the oligodeoxynucleotide, suggesting that the correlation times of these protons were similar to those of the overall duplex. The crotonaldehydic-derived methyl protons showed NOEs in the 5′-direction to C¹⁸ H1′, G¹⁹ H1′, and G¹⁹ H4′ in the complementary strand of the duplex. The aldehyde proton of the adduct exhibited NOEs in the 3′-direction to A ⁸ H1′ and A⁸ H4′ in the modified strand. All of these NOEs involved DNA protons facing the minor groove. Molecular dynamics calculations, restrained by distances and torsion angles derived from the NMR data, revealed that within the minor groove, the aldehyde of the N ²-[3-oxo-1(S)-methyl-propyl]-dG adduct oriented in the 3′-direction, while the 1(S) methyl group oriented in the 5′-direction. This positioned the aldehyde distal to the G¹⁹ exocyclic amine and provided a rationale as to why the N²-(S-α- CH₃-γ-OH-1,N²-propano-2′)-dG adduct generated interstrand cross-links less efficiently than did the N²-(R-α- CH₃-γ-OH-1,N²-propano-2′)-dG adduct.