3′-Intercalation of a N²-dG 1R-trans-anti-Benzo[c]phenanthrene DNA Adduct in an Iterated (CG)₃ Repeat

The conformation of the 1R,2S,3R,4S-benzo[c]phenanthrene-N²-dG adduct, arising from trans opening of the (+)-1S,2R,3R,4S-anti-benzo[c]phenanthrene diol epoxide, was examined in 5′- d(ATCGCXCGGCATG)-3′·5′-d(CATGCCGCGCGAT)-3′, where X = 1R,2S,3R,4S-B[c]P-N²-dG. This duplex, derived from the hisD3052 frameshift tester strain of Salmonella typhimurium, contains a (CG)₃ iterated repeat, a hotspot for frameshift mutagenesis. NMR experiments showed a disconnection in sequential NOE connectivity between X⁴ and C⁵, and in the complementary strand, they showed another disconnection between G¹⁸ and C¹⁹. In the imino region of the ¹H NMR spectrum, a resonance was observed at the adducted base pair X⁴·C¹⁹. The X⁴ N1H and G¹⁸ N1H resonances shifted upfield as compared to the other guanine imino proton resonances. NOEs were observed between X⁴ N1H and C¹⁹ N⁴H and between C⁵ N⁴H and G¹⁸ N1H, indicating that base pairs X⁴·C¹⁹ and C⁵·G¹⁸ maintained Watson−Crick hydrogen bonding. No NOE connectivity was observed between X⁴ and G¹⁸ in the imino region of the spectrum. Chemical shift perturbations of greater than 0.1 ppm were localized at nucleotides X⁴ and C⁵ in the modified strand and G¹⁸ and C¹⁹ in the complementary strand. A total of 13 NOEs between the protons of the 1R-B[c]Ph moiety and the DNA were observed between B[c]Ph and major groove aromatic or amine protons at base pairs X⁴·C¹⁹ and 3′-neighbor C⁵·G¹⁸. Structural refinement was achieved using molecular dynamics calculations restrained by interproton distances and torsion angle restraints obtained from NMR data. The B[c]Ph moiety intercalated on the 3′-face of the X⁴·C¹⁹ base pair such that the terminal ring of 1R-B[c]Ph threaded the duplex and faced into the major groove. The torsion angle α′ [X⁴]−N3−C2−N2−B[c]Ph]−C1 was calculated to be −177°, maintaining an orientation in which the X⁴ exocyclic amine remained in plane with the purine. The torsion angle β′ [X⁴]−C2−N2−[B[c]Ph]−C1−C2 was calculated to be 75°. This value governed the 3′-orientation of the B[c]Ph moiety with respect to X⁴. The helical rise between base pairs X⁴·C¹⁹ and C⁵·G¹⁸ increased and resulted in unwinding of the right-handed helix. The aromatic rings of the B[c]Ph moiety were below the Watson−Crick hydrogen-bonding face of the modified base pair X⁴·C¹⁹. The B[c]Ph moiety was stacked above nucleotide G¹⁸, in the complementary strand.