Structural and thermodynamical analysis of molecular complexation by¹H NMR spectroscopy. Intercalation of ethidium bromide with the isomeric deoxytetranucleoside triphosphates 5′-d(GpCpGpC) and 5′-d(CpGpCpG) in aqueous solution

Abstract

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 ...

Complexation of norfloxacin with DNA in the presence of caffeine

“…Special examination of the δ 11 values (Table 3) is required, because the average shielding over all aromatic protons in the 1:1 complex (Δδ 11 =δ m -δ 11 =0.13ppm) is much smaller than that in the 1:2 complex (Δδ 12 =δ m -δ 12 =0.35ppm) and in the NOR-CAF heterocomplex (Δδ h =δ m -δ h =0.29ppm). Moreover, in contrast to the other protons, the H h proton is deshielded moving downfield (to high frequency) on increasing the concentration of TGCA (see Fig.6a), which is also quite different from that observed previously for the three fusedring intercalators [21,23]. This result suggests a non-intercalative mode of binding of NOR with the single-stranded form of the tetramer.…”

“…NOR binds more tightly to the double-stranded form of the oligonucleotide in solution. Even so the complexation parameters for this reaction (K 12 and enthalpy/entropy) are much smaller in absolute value compared with the three-ring intercalators [12,21] but much larger than that calculated above for self-association of NOR and its hetero-association with CAF (Tables 1,2 Tables 1,3), which is known to be a distinctive feature of intercalation [21,23,24], supports this conclusion. Secondly, the decrease in magnitude of the complexation parameters is apparently due to a smaller contribution from π-π stacking arising when the two fused-ring NOR chromophore is intercalated between the adjacent bases along the oligomer sequence.…”

“…Both the derivation of eqn. (6) and the numerical procedure for determination of the unknown quantities is described in detail [23]. The thermodynamical parameters, enthalpies and entropies, of the complexation reactions (5) have been determined from experimental temperature dependences of NOR proton chemical shifts (Fig.6b) using the approach similar to that for the self-and hetero-association, described above.…”

“…On proceeding with the computations the magnitudes of the complexation parameters for the formation of 2:1 (5e) and 2:2 (5f) complexes appeared to be very unreliable, which is due to the relatively small changes in the experimental proton chemical shifts with both concentration and temperature, as compared with three-ring intercalators with the same tetramer [23]. Hence, only the calculated complexation parameters for 1:1 and 1:2 reactions are given in Table 3, which shows that the equilibrium constant for 1:2 complexation is higher than that of 1:1, i.e.…”

Binding of Cationic and Neutral Phenanthridine Intercalators to a DNA Oligomer Is Controlled by Dispersion Energy: Quantum Chemical Calculations and Molecular Mechanics Simulations

“…As a result of these features ethidium has been widely used as a common fluorescence stain and a lot of experimental data on ethidium have been collected over the last three decades. [21][22][23][24][25][29][30][31][32][33] It has been shown experimentally [12] that the intercalation rate of ethidium to DNA is controlled by the insertion of an aromatic ring into the DNA structure. (Earlier, it was suggested that the process was controlled by the diffusion of ethidium toward the DNA.)…”