NMR Investigation of the Complexation of Daunomycin with Deoxytetranucleotides of Different Base Sequence in Aqueous Solution

Abstract

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.

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.…”

“…[18]. The presence of large groups in the structure of an aromatic molecule, say, the phenol ring in Ethidium Bromide, results in an increase of σ up to the unity and in the case of the anthracycline antibiotics, Daunomycin and Nogalomycin?, up to 1.3 and higher [21]. Taking these factors into consideration indicates that the σ ≈ 1 for Norfloxacin must result from some mutual compensation of the contribution to cooperativity from the increased hydrophobicity of the molecule and an anticooperative contribution to the formation aggregates from the piperazine ring side chain in the NOR structure.…”

“…The scheme of dynamic equilibrium reactions of NOR and TGCA in solution is the same as in previous work [12,21]; viz. formation of 1:1, 1:2, 2:1 and 2:2 complexes of NOR (X) with the single-(N) and double-stranded (N 2 ) forms of the DNA oligomer in solution with the corresponding complexation constants, K 11 , K 12 , K 21 , K 22 respectively, and the dimerization constant for the tetramer, K N (reaction 5b):…”

“…Normally, the complexation of aromatic drug molecules with short DNA sequences occurs with a binding constant of 10 3 -10 5 M -1 [12,21], which is greater than the self-association constants for the drug by at least an order of magnitude. Hence, a simple dimerization of NOR is sufficient for such analysis rather than the indefinite self-association given above,.…”