The novel phase II anticancer drug BBR3464 ([[ trans-PtCl(NH(3))(2)](2)- micro -[ trans-Pt(NH(3))(2)(NH(2)(CH(2))(6)NH(2))(2)]](NO(3))(4)) forms a 1,4-interstrand cross-link adduct with the self-complementary DNA octamer 5'-d(ATG*TACAT)(2)-3', with the two platinum atoms coordinated in the major groove at the N7 positions of guanines that are four base pairs apart on opposite DNA strands. The "central" tetraamine linker [ trans-H(2)N(CH(2))(6)NH(2)Pt(NH(3))(2)NH(2)(CH(2))(6)NH(2)] was located in or close to the minor groove. The adduct was characterized and analyzed by MS, UV and NMR spectroscopy. NMR analysis of the adduct shows strong H8/H1' intraresidue crosspeaks observed for the A1 and A7 resonances, consistent with a syn conformation for these bases which is usually not observed for adenine residues and bases not directly involved in the cross-link in oligonucleotides. The strong intraresidue H8/H1' crosspeak is also observed for G3. Examination of the structure thus reveals unusual cooperative effects unique to this class of anticancer drugs and is the first demonstration of cooperative effects in solution for an anticancer drug. The significant characteristic of the structure is the lack of severe DNA distortion such as a kink, directed bend or significant unwinding of the helices which are characteristic for DNA adducts of mononuclear complexes. This may contribute to the lack of protein recognition of the cross-link by HMG-domain proteins, a biological consequence significantly different from that of mononuclear complexes such as cisplatin. Since DNA is the principal target in vivo for these Pt cross-linking agents, the unique structural perturbations induced by BBR3464 cross-links are likely related to its increased cytotoxicity and antitumor activity as compared to cisplatin ( cis-DDP).

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Combined multidimensional nuclear magnetic resonance spectroscopy and electrospray mass spectrometry was used to analyze the platinated DNA adduct of the phase II anticancer drug [{trans-PtCl(NH 3 ) 2 } 2 -μ-{trans-Pt(NH 3 ) 2 (NH 2 (CH 2 ) 6 NH 2 ) 2 }](NO 3 ) 4 (BBR3464) with [5′-d (ACG*TATACG*T)-3′] 2 . Two 1,2-interstrand cross-links were formed by concomitant binding of two trinuclear moieties to the oligonucleotide. The four DNA-bound platinum atoms coordinated in the major groove at N7 positions of guanines in the 3′ → 3′ direction and the central platinum unit is expected to lie in the DNA minor groove. This is the first report of such a DNA lesion. The melting temperature of the adduct is 76 °C and is 42 °C higher than that of the unplatinated DNA. The sugar residues of the platinated bases are in the N-type conformation and the G9 nucleoside is in the syn orientation, while the G3 nucleoside appears to retain the anti configuration. The secondary structure of DNA was significantly changed upon cross-linking of the two BBR3464 molecules. Base destacking occurs between A1/C2 and C2/G3 and weakened stacking is seen for the C8/G9 and G9/ T10 bases. The lack of Watson-Crick base pairing is also seen for A1-T10 and C2-G9 base pairs, whereas Watson-Crick base pairs in the central sequence of the DNA (T4 → A7) are well maintained. While DNA repair proteins may "see" different platinated adducts as bulky "lesions", the subtle differences involved in base pairing and stacking, as summarized here, may extend to their role as a substrate for repair enzymes. Thus, differences in protein recognition and repair efficiency among the various interstrand cross-links are likely and a subject worthy of detailed exploration.