Four different types of new two-dimensional (2D) NMR techniques have been used to determine unambiguous and ,3C spectral assignments for (5'-deoxyadenosyl)cobalamin (coenzyme Bl2, M, 1580,6.5-mg sample in 0.35 mL of 2H20). Two-dimensional homonuclear Hartmann-Hahn spectroscopy in combination with 2D spin-locked NOE spectroscopy was used to assign the resonances of all nonexchangeable protons. Sensitivity enhanced -detected 2D multiple-quantum NMR then provided the resonance assignments for all protonated 13C nuclei. The resonance assignments for nonprotonated carbons were determined via *H-13C multiple-bond, multiple-quantum spectroscopy. This experiment also confirmed independently our and 13C assignments made with the other methods. The relative intensity of long-range *H-13C correlations is related to the magnitude of the /CH coupling involved and therefore provides qualitative structural information. Despite the careful application of classical ,3C NMR assignment techniques in recent reports on coenzyme B12, we found that nearly one-third of the 13C resonances had been assigned erroneously.

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.

N7−Pt−N7 d(GpG) intrastrand cross-link adducts are formed in DNA by the anticancer drug, cisplatin. By creating adducts with slow dynamic motion, we have identified a new abundant conformer with the guanine bases in a head-to-tail (HT) arrangement and with both sugars in the N pucker of A-form DNA instead of the S pucker of B-form DNA. Both features are unprecedented for such cross-links. The HT form is one of two abundant thermodynamic and kinetic products formed by addition of d(GpG) to [(S,R,R,S)-BipPt(H2O)2]2+ (Bip = 2,2‘-bipiperidine with S, R, R, and S configurations at the asymmetric N, C, C, and N chelate ring atoms). The second form has the common head-to-head conformation (HH1) with the backbone propagating in the normal direction, both G's anti and with N and S puckers for the 5‘- and 3‘-G residues, respectively. This form has a typical NMR shift pattern:  upfield 5‘-G H8, downfield 3‘-G H8, and downfield 31P NMR signal. In contrast, the HT (S,R,R,S)-BipPt(d(GpG)) form has several unusual or unique NMR spectral features, including pronounced upfield shifts of both G H8 signals, unusually shifted 5‘-G H3‘ and 3‘-G H2‘ signals, and an unexpectedly upfield-shifted 31P NMR signal. A strong 3‘-G H8−H1‘ NOE cross-peak, the absence of an H8−H8 NOE cross-peak, and H1‘ couplings establish that this form is an HT conformer with a syn 3‘-G, an anti 5‘-G, and both sugars having mainly N pucker. The HT base orientation in cross-links introduces chirality, and two conformers, ΔHT1 and ΛHT2, are possible with normal and opposite directions of backbone propagation, respectively. NMR-restrained molecular mechanics and dynamics calculations show that the ΔHT1 conformer has the lower energy. Of some interest, rules in the literature cannot account for the G H8 shifts of any BipPt(d(GpG)) form reported by us here and previously. We advance new rules that allow successful G H8 shift predictions for these cross-links and also for cisplatin oligonucleotide adducts; these rules are consistent with the solid-state structure of cis-Pt(NH3)2(d(pGpG)) (Sherman, S. E.; Gibson, D.; Wang, A. H.-J.; Lippard, S. J. J. Am. Chem. Soc. 1988, 110, 7368−7381), but they conflict with widely held interpretations of cis-Pt(NH3)2(d(GpG)) solution structure and dynamics. Finally, our results suggest that, in the future, the effect of the carrier ligands on the HH vs HT cross-link conformation must be considered in drug design.

Chirality-controlling chelate (CCC) ligands are a class of chiral diamine ligands with one or two chiral secondary amine ligating groups. Analogues of platinum anticancer agents containing CCC ligands exhibit unusual steric and dynamic features. In this study NMR and CD methods were used to evaluate the influence of the N9 substituent in guanine derivatives (G) on conformer distribution in one class of (CCC)PtG(2)() complexes. We employed the CCC ligand, N,N'-dimethyl-2,3-diaminobutane [Me(2)()DAB with S,R,R,S or R,S,S,R configurations at the four asymmetric centers, N, C, C, and N]. For each Me(2)()DABPtG(2) complex, the presence of four G H8 signals demonstrated formation of all three possible atropisomers: DeltaHT (head-to-tail), LambdaHT, and HH (head-to-head). Different G ligands (5'-GMP, 3'-GMP, 1-MeGuo, Guo, or 9-EtG) were chosen to assess the effect of the N9 substituent on the relative stability and spectral properties of the atropisomers. The conformations of the atropisomers of Me(2)()DABPtG(2) were determined from CD spectra and from NOE cross-peaks (assigned via COSY spectra) between G H8 signals and those for the Me(2)()DAB protons. Regardless of the N9 substituent, the major form was HT. However, this form had the opposite chirality, LambdaHT and DeltaHT, and base tilt direction, left- and right-handed, respectively, for the S,R,R,S and R,S,S,R configurations of the Me(2)()DAB ligand. Thus, the chirality of the CCCligand, not hydrogen bonding, is the most important determinant of conformation. For each Me(2)()DABPtG(2) complex, the tilt direction of all three atropisomers is the same and, except for 5'-GMP, the order of abundance was major HT > minor HT > HH. For 5'-GMP, the HH atropisomer was three times as abundant as the minor HT species, suggesting that phosphate-NH(Me(2)()DAB) hydrogen bonds could be present since such bonding is possible only for the 5'-GMP derivatives. However, if such phosphate-NH hydrogen bonds exist, they are weak since the percentage of the major HT form of 5'-GMP complexes is similar and indeed can be smaller compared to this percentage for complexes with other G's. The CD spectra of all (S,R,R,S)-Me(2)()DABPtG(2) complexes were similar and opposite to those of all (R,S,S,R)-Me(2)()DABPtG(2) complexes, indicating the CD signature is characteristic of the dominant HT conformer, which has a chirality dictated by the chirality of the CCC ligand and not the N9 substituent.

Features of cisplatin-type anticancer drug adducts with nucleic acids and their constituents are clouded because they exist as a fluxional mixture of conformers. Retro-model adducts containing the specially designed chiral diamine ligand, Bip = 2,2'-bipiperidine, are dramatically less fluxional. Conformers of BipPtG(2) adducts with R,S,S,R and S,R,R,S asymmetric centers at the N, C, C, and N Bip chelate ring atoms and G = 5'-GMP, 5'-dGMP, 3'-GMP, or 9-ethylguanine are amenable to separate characterization. All possible BipPtG(2) atropisomers (one head-to-head (HH) and Delta and Lambda head-to-tail (HT) atropisomers) were observed by NMR spectroscopy. At equilibrium at low pH, one HT atropisomer dominates. CD spectra, G H8 chemical shifts, and low-pH equilibria of BipPtG(2) and Me(2)()DABPtG(2) (Me(2)()DAB = N,N '-dimethyl-2,3-diaminobutane) are similar when the chelate ring atoms have the same stereochemistries; thus, Bip and Me(2)()DAB are termed chirality-controlling chelates (CCC) since these ligands dictate the absolute conformation of the major HT rotamer. In each case, the HT conformer that cannot form G O6-NH(CCC) hydrogen bonds was dominant, and the G H8 chemical shift indicated that this conformer had less tilted bases, allowing favorable base-base dipole-dipole interactions. For both the R,S,S,R and S,R,R,S Bip chiralities of the BipPt(3'-GMP)(2) complexes, the percentage of DeltaHT rotamer increased near pH 7, a probable consequence of phosphate-cis-G hydrogen bonds accompanied by favorable dipole interactions of less tilted bases. For the 5'-GMP complexes, these factors favor the LambdaHT rotamer near pH 7. When G has a 5'-phosphate group, rotamer distribution is also influenced by phosphate-NH(Bip) hydrogen bonds. At high pH, the nature and/or strength of interactions such as G dipole-G dipole interactions and G O6-NH(Bip) and phosphate-cis-G hydrogen bonding are altered by G N(1)H deprotonation. The features of the complexes at high pH can be largely explained as arising from the net result of these interactions. This information from retro models with a CCC ligand lays the foundation for understanding and evaluating the properties of the highly dynamic adducts of anticancer drugs.

Guanine O6 to carrier ligand hydrogen bonding is a central feature of many hypotheses advanced to explain the anticancer activity of cis-type anticancer drugs, cis-PtA(2)X(2) (A(2) = diamine or two amines). Early structural evidence suggested that cis-Pt(NH(3))(2)(d(GpG)) (the cross-link model for the key cisplatin-DNA adduct) and other cis-PtA(2)(d(GpG)) adducts exist exclusively or mainly as the HH1 conformer with head-to-head (HH) bases. The dynamic motion of the d(GpG) in these adducts is too rapid to permit definitive characterization of both the conformation and the H-bonding. Hence, we use retro models having A(2) ligands designed to slow the motion. Here, we employ Me(2)ppz (N,N'-dimethylpiperazine), which lacks NH groups. Me(2)ppz is unique in having sp(3) N-methyl groups directly in the coordination plane, allowing the coexistence of multiple conformers but hindering dynamic motion in Me(2)ppzPt(d(GpG)) and Me(2)ppzPt(GpG) retro models. Dynamic processes are decreased enough in Me(2)ppzPt(d(GpG)) to permit HPLC separation of three abundant forms. After HPLC separation, the three re-equilibrate, proving that the three forms must be conformers and that Me(2)ppz has little influence on conformer distribution. This marks the first reported characterization of three abundant conformers for one cis-PtA(2)(d(GpG)) adduct. From NMR evidence, the Me(2)ppzPt(d(GpG)) HH1 conformer has uncanted bases. Another conformer, one of two recently discovered conformer types, has head-to-tail (HT) bases with Delta chirality. For this Delta HT1 form, several lines of evidence establish that the dinucleotide moieties have essentially identical structures in d(GpG) (and GpG) adducts of Me(2)ppzPt and other cis-PtA(2) complexes. For example, the shifts of the highly structure-sensitive G H8 NMR signals are almost identical for the Delta HT1 form of all adducts. In previous models, the stabilization of the Delta HT1 form could be attributed to G O6 H-bonding to A(2) NH groups. Such H-bonds are not possible for Me(2)ppz. The unambiguous conclusions are that G O6 H-bonding is weak and that neither canting nor H-bonding is essential in HH forms. These two features are present in almost all other small models but are essentially absent in the cross-link base pair (bp) step in duplexes. We conclude from our work that the forces favoring canting and H-bonding are weak, and we hypothesize that steric effects within the Lippard bp step adjacent to this cross-link bp step easily overcome these forces.

DNA bases in the three-base-pair (3bp) region of duplexes with the two major lesions of cisplatin (cis-PtCl(2)(NH(3))(2)) with DNA, namely d(XGG) and d(XAG) ( = N7-platinated base), differ in their relative positions by as much as approximately 3.5 A in structures in the literature. Such large differences impede drug design and assessments of the effects of protein binding on DNA structure. One recent and several past structures based on NMR-restrained molecular dynamics (RMD) differ significantly from the reported X-ray structure of an HMG-bound XGG 16-mer DNA duplex (Ohndorf, U.-M.; Rould, M. A.; He, Q.; Pabo, C. O.; Lippard, S. J. Nature 1999, 399, 708). This 16-mer structure has several significant novel and unique features (e.g., a bp step with large positive shift and slide). Hypothesizing that novel structural features in the XGG or XAG region of duplexes elude discovery by NMR methods (especially because of the flexible nature of the 3bp region), we studied an oligomer with only G.C bp's in the XGGY site by NMR methods for the first time. This 9-mer gave a 5'-G N1H signal with a normal shift and intensity and showed clear NOE cross-peaks to C NHb and NHe. We assigned for the first time (13)C NMR signals of a duplex with a GG lesion. These data, by adding NMR-based criteria to those inherent in NOESY and COSY data, have more specifically defined the structural features that should be present in an acceptable model. In particular, our data indicated that the sugar of the X residue has an N pucker and that the GG cross-link should have a structure similar to the original X-ray structure of cis-Pt(NH(3))(2)(d(pGpG)) (Sherman S. E.; Gibson, D.; Wang, A. H.-J.; Lippard, S. J. J. Am. Chem. Soc. 1988, 110, 7368). With these restrictions added to NOE restraints, an acceptable model was obtained only when we started our modeling with the 16-mer structural features. The new X-ray/NMR-based model accounted for the NOESY data better than NOE-based models, was very similar in structure to the 16-mer, and differed from solely NOE-based models. We conclude that all XGG and XAG (X = C or T) duplexes undoubtedly have structures similar to those of the 16-mer and our model. Thus, protein binding does not change greatly the structure of the 3bp region. The structure of this region can now be used in understanding structure-activity relationships needed in the design of new carrier ligands for improving Pt anticancer drug activity.

No abstract