A Novel Head-to-Head Conformer of d(GpG) Cross-linked by Pt: New Light on the Conformation of Such Cross-links Formed by Pt Anticancer Drugs

Abstract

The critical DNA lesion accounting for the anticancer activity of cis-PtCl2(NH3)2 and its analogues [cis-PtX2A2: A2 = a diamine or two amines, X2 = anionic leaving ligand(s)] is an unusual intrastrand cis-Pt(d(GpG))A2 cross-link with Pt linking N7's of adjacent guanines (G). The only known cross-link form with two anti G's, HH1, has head-to-head (HH) bases. We provide NMR, HPLC, and mass spectral evidence for a second, distinct HH cis-Pt(d(GpG))A2 cross-link conformer, HH2, in BipPt(d(GpG)) (Bip = 2,2‘-bipiperidine, where the coordinated Bip has R, S, S, and R configurations at the asymmetric N, C, C, and N chelate ring atoms). The HH1 and HH2 BipPt(d(GpG)) conformers are formed both kinetically and thermodynamically in comparable amounts. The NMR results showed for both BipPt(d(GpG)) conformers that the bases were anti, anti HH, the 5‘-G sugar pucker was N, and the 3‘-G sugar pucker was S. The major difference between the HH1 and HH2 conformers is the propagation direction of the phosphodiester linkage. Molecular modeling calculations with NMR restraints on the HH1 and HH2 conformers indicate comparable energies and no unusual features that should have precluded prediction of the existence of the HH2 conformer. Calculations led to similar conclusions for the cis-Pt(d(GpG))(NH3)2 HH2 conformer. During the two decades of intense interest in this cross-link, this new form has gone unrecognized, although published results have suggested the presence of unknown conformers. Our results in this first report of a second anti, anti HH d(GpG) adduct place an entirely different perspective on the conformational diversity of cis-Pt(d(GpG))(NH3)2 in solution. Although the new HH2 conformation is unlikely to exist in a duplex at low temperature, the new form may be important in mutational events, in duplex breathing, or in duplex interactions with DNA damage recognition proteins and repair enzymes. Finally, the spectral features, especially the H8 NMR signals, of HH1 d(GpG) species in single strands and in duplexes are typically very different, results attributed to differences in both extent and direction of base canting. Bip is an example of a chirality controlling chelate (CCC) ligand that can influence canting. The HH1 conformer of BipPt(d(GpG)) is the first single-stranded species that has key spectral characteristics very similar to those of a typical duplex cross-linked species. Thus, even the HH1 conformer of BipPt(d(GpG)) is an unusual species.