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.