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NMR spectroscopy has been used to elucidate the molecular basis of the action of caffeine (CAF) on the complexation with DNA of mutagens such as ethidium bromide, propidium iodide, proflavine and acridine orange, and anticancer drugs such as actinomycin D and daunomycin. The hetero-association of CAF and each of the aromatic ligands in 0.1 mol L(-1) phosphate buffer (pD=7.1) has been investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy and analysed in terms of a statistical-thermodynamic model, in which molecules form indefinite aggregates for both self-association and hetero-association. The analysis leads to determination of the equilibrium constants of hetero-association and to the values of the limiting chemical shifts of the heteroassociation of CAF with each of the aromatic molecules. The hetero-association constants between CAF and each of the aromatic drugs/dyes are found to be intermediate in magnitude between those for self-association of CAF and the corresponding drug/dye. The most probable structures of the 1:1 CAF + ligand hetero-association complexes have been determined from the calculated values of the induced limiting chemical shifts of the drug protons. Knowledge of the equilibrium constants for self-association of CAF and the aromatic ligands, for their hetero-association and their complexation with a DNA fragment, the deoxytetranucleotide 5'-d(TpGpCpA), enabled the relative content of each of the CAF-ligand and CAF-ligand-d(TGCA) complexes to be calculated as a function of CAF concentration in mixed solutions. It is concluded that, on addition of CAF to the solution, the decrease in binding of drug or mutagen with DNA is due both to competition for the binding sites by CAF and the aromatic molecules, and to formation of CAF-ligand hetero-association complexes in the mixed solution; the relative importance of each process depends on the drug or mutagen being considered.

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An NMR analysis has been developed for determining the structural and thermodynamical parameters of molecular complexation in solution in situations where there is a multicomponent equilibrium. Using one-dimensional and two-dimensional 500 MHz 'H NMR spectroscopy, the method has been used to investigate complex formation between the phenantridinium dye, ethidium bromide (EB), and self-complementary deoxytetraribonucleoside triphosphates 5'-d(GpCpGpC) and 5'-d(CpGpCpG) in aqueous salt solution. Concentration dependences of proton chemical shifts of the molecules have been measured at constant temperature ( T = 308 K) and the temperature dependence of chemical shifts (283-353 K) measured at constant concentration. Different schemes of complex formation between EB molecules and the tetranucleotides have been examined, successively taking into account various molecular associations in solution, uiz 1 : 1, 1 : 2, 2 : 1 and 2 : 2 complexes. Equilibrium reaction constants and the limiting proton chemical shifts in the complexes have been determined. The relative contributions of the different types of complexes in the equilibrium mixture have been calculated with the 1 : 2 complex preferred for d(GCGC), which has one pyrimidine-purine binding sequence, whereas both the 1 : 2 and 2 : 2 complexes are significant for d(CGCG) which has two CG binding sequences. The induced limiting chemical shifts have been analysed in terms of the structures of the complexes. The results confirm that EB binds preferentially to the CG-sites of the tetranucleotide duplexes and that EB intercalates from the minor groove of the double-helix. These conclusions are supported qualitatively by 2D NOE measurements. The most favourable structures of the 1 : 2 and 2 : 2 dye-tetranucleotide complexes have been constructed using calculated values of induced chemical shifts of EB protons. The results are in good agreement with the structure of the EB-d(CpG), complex derived from X-ray crystallographic measurements. It is also found that EB binds to the single-stranded forms of the tetranucleotides in solution with formation of the 1 : 1 complex significant for d(GCGC) and both the 1 : 1 and 2 : 1 complexes forming for d(CGCG). The analysis shows that it is necessary to define all the reactions in a multi-component equilibrium if observed NMR parameters, which are weighted averages, are to be used to determine reliable structures of molecular complexes in solution.The enthalpies and entropies of complex formation between EB and the tetranucleotides have been determined from the temperature dependence of the 500 MHz proton NMR chemical shifts. The contributions have been determined for the formation of different types of complexes (1 : 1, 2 : 1, 1 : 2 and 2 : 2) in solution. The analysis shows that the observed melting temperatures (T',, which are also weighted averages) in the multi-component system are different from the values calculated for the 1 : 2 and 2 : 2 complexes. Comparison of the thermodynamical parameters has led to further understanding of ...

500 MHz NMR spectroscopy has been used to investigate the complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary deoxytetranucleotides, 5'-d(CGCG), 5'-d(GCGC), 5'-d(TGCA), 5'-d(ACGT) and 5'-d(AGCT), of different base sequence in aqueous salt solution. 2D homonuclear 1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H - 31P NMR spectroscopy (HMBC) have been used for complete assignment of the non-exchangeable protons and the phosphorus resonance signals, respectively, and for a qualitative determination of the preferred binding sites of the drug. Analysis shows that DAU intercalates preferentially into the terminal sites of each of the tetranucleotides and that the aminosugar of the antibiotic is situated in the minor groove of the tetramer duplex, partly eclipsing the third base pair. A quantitative determination of the complexation of DAU with the deoxytetranucleotides has been made using the experimental concentration and temperature dependences of the drug proton chemical shifts; these have been analysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters (enthalpies deltaH, entropies deltaS) of different drug-DNA complexes (1:1, 1:2, 2:1, 2:2) in aqueous solution. It is found that DAU interacts with sites containing three adjacent base pairs but does not show any significant sequence specificity of binding with either single or double-stranded tetranucleotides, in contrast with other intercalating drugs such as proflavine, ethidium bromide and actinomycin D. The most favourable structures of the 1:2 complexes have been derived from the induced limiting proton chemical shifts of the drug in the intercalated complexes with the tetranucleotide duplex, in conjunction with 2D NOE data. It has been found that the conformational parameters of the double helix and the orientation of the DAU chromophore in the intercalated complexes depend on base sequence at the binding site of the tetramer duplexes in aqueous solution.