[A structural and thermodynamic analysis of novatrone and flavin mononucleotide heteroassociation in aqueous solution by 1H NMR spectroscopy].

A heteroassociation of antitumor antibiotic novatrone (NOV) and flavin mononucleotide (FMN) in aqueous solution was studied by one- and two-dimentional 1H NMR spectroscopy (500 MHz) to elucidate the molecular mechanism of the possible combined action of the antibiotic and vitamin. The equilibrium reaction constants, induced proton chemical shifts, and the thermodynamic parameters (deltaH and deltaS) of the NOV and FMN heteroassociation were determined from the concentration and temperature dependences of proton chemical shifts of the aromatic molecules. The most favorable structure of the 1 : 1 NOV-FMN complex was determined by both the method of molecular mechanics (X-PLOR software) and the induced proton chemical shifts of the molecules. An analysis of the results suggests that the NOV-FMN intermolecular complexes are mainly stabilized by stacking interactions of their aromatic chromophores. An additional stabilization is possible due to intermolecular hydrogen bonds. It was concluded that the aromatic molecules of vitamins, in particular, FMN, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solutions, which could result in a modulation of their medical and biological action.

Hetero-association of anticancer antibiotics in aqueous solution: NMR and molecular mechanics analysis.

In order to investigate the effect on combinations of aromatic antibiotics used in chemotherapy, the hetero-association of the antitumour antibiotics actinomycin D (AMD) with daunomycin (DAU) or novatrone (NOV) has been studied by the methods of 1D- and 2D 500 MHz 1H-NMR spectroscopy and molecular mechanics calculations. The experimental concentration and temperature dependences of the proton chemical shifts of mixtures of the aromatic drugs have been analyzed in terms of a modified statistical-thermodynamical model of hetero-association to give the equilibrium reaction constants, the thermodynamical parameters (deltaH, deltaS) of hetero-association of AMD with DAU or NOV and the limiting values of proton chemical shifts of the molecules in the hetero-complexes. The most favorable averaged structures of the 1:1 DAU-AMD and NOV-AMD hetero-association complexes have been determined using both the limiting values of proton chemical shifts of the molecules and molecular mechanics methods (X-PLOR software). The results show that intermolecular complexes between DAU-AMD and NOV-AMD are mainly stabilized by stacking interactions of the aromatic chromophores, although the DAU-AMD hetero-complex has additional stabilization, which may be explained by an intermolecular hydrogen bond between a carbonyl group of ring C of DAU and the NH group of D-Val of the pentapeptide side chain ring of AMD. The relative content of each type of molecular complex in the mixed solution has been calculated at different values of the ratio (r) of the initial concentrations of DAU and AMD. It is found that the contributions of hetero-complexes to the general equilibrium in solution are predominant at quite different values of r, viz. at r>12 for AMD with NOV and at r>2 for AMD with DAU, compared to r>0.3 for the DAU-NOV system observed previously. It is concluded that anticancer drugs have quite different affinities for formation of hetero-complexes with other aromatic antibiotics in aqueous solution, which may need to be taken into consideration for their use in combination chemotherapy.

[Structural analysis of the complex of phenoxazone antibiotic actinocyl-bis-(2-deoxytetranucleotide 5'-d(TpGpCpA) by the methods of two dimensional 1H-NMR-spectroscopy and molecular mechanics].

The spatial structures of intercalated complexes of synthetic phenoxazone antibiotic actinocyl-bis-(2-dimethylaminoethyl) amide with self-complementary deoxytetranucleotide 5'-d(TpGpCpA) have been investigated. Analysis has been made using two-dimensional NMR (2D-NOESY) data in aqueous solution and molecular mechanics simulation. Distinctive features of the conformation of drug-DNA complexes have been determined at two possible orientations of the chromophore of phenoxazone antibiotic at the intercalation site.

[1H NMR analysis of the complex formation of aromatic molecules of antibiotic and vitamin in aqueous solution: heteroassociation of actinomycin D and flavin mononucleotide].

The molecular mechanism of the combined action of antibiotic and vitamin was studied by NMR spectroscopy. The heteroassociation of the antitumor antibiotic actinomycin D and flavin mononucleotide was investigated as a function of concentration and temperature by 500 MHz 1H NMR spectroscopy. The equilibrium association constant, the thermodynamic parameters (deltaH, deltaS) of heteroassociation of actinomycin D with flavin mononucleotide, and the limiting values of proton chemical shifts in the heterocomplex were determined from the concentration and temperature dependences of proton chemical shifts of molecules. The most favorable structure of the 1:1 actinomycin D-flavin mononucleotide heteroassociation complex was determined using both the molecular mechanics methods (X-PLOR software) and the limiting values of proton chemical shifts of the molecules. In the calculated structure, the planes of the chromophores of actinomycin D and flavin mononucleotide molecules in the 1:1 heterocomplex are parallel and separated from each other by a distance of about 0.34 nm. At the same time, there is a probability of formation of intermolecular hydrogen bonds in the calculated structure of 1:1 actinomycin D-flavin mononucleotide complex. The analysis of the results obtained suggests that aromatic molecules of vitamins, e.g., flavin mononucleotide, can form energetically favorable heterocomplexes with aromatic antitumor antibiotics in aqueous solution, modulating thereby the efficacy of their medical and biological action.

[Distinctive features of the self-association of deoxyhexanucleotide 5'-D(GpCpApTpGpC) in aqueous solution: 1H NMR analysis]. / Osobennosti protsessa samoassotsiatsii molecul dezoksigeksanukleotida 5'-D(GpCpApTpGpC) v vodnom rastvore: 1H-IAMR-analiz.

The self-association of self-complementary deoxyhexanucleotide d(GCATGC) was investigated in aqueous salt solution. Homonuclear 1H NMR correlation spectroscopy (2D-TOCSY and 2D-NOESY) was used for complete assignments of nonexchangeable protons of the hexamer. The equilibrium reaction constants and thermodynamical parameters of duplex d(GCATGC)2 formation were determined from experimental concentration and temperature dependences of proton chemical shifts of the deoxyhexanucleotide. Distinctive features of the concentration dependences in the range of small concentrations at relatively low temperatures of solution enable one to assume that one single-stranded hexamer sequence forms a compact structure (similar to a hairpin) in aqueous solution. A possible spatial hairpin structure of the hexamer was proposed. Comparative analysis of the experimental and theoretical (using the "nearest neighbor" model) thermodynamical parameters of duplex formation was made.

[A stochastic model for the NMR analysis of the heteroassociation of biologically active aromatic molecules]. / Stokhasticheskaia model' dlia IaMR-analiza geteroassotsiatsii biologicheski aktivnykh aromaticheskikh molekul.

A stochastic model for the NMR analysis of the heteroassociation of two aromatic compounds was developed, which takes into account all physically possible reactions of association of molecules in solution. Expressions for calculating the experimentally observed proton chemical shift were obtained in the general form, and an algorithm for calculating the parameters of heteroassociation using the stochastic model was proposed. The effects of limitations of the basic and general models, as compared with the stochastic model, on the model parameters of the heteroassociation of various biologically active aromatic molecules was analyzed. It was shown that the basic model can be used with a sufficient degree of accuracy for systems with a relatively small contribution of heteroassociation reactions to the total dynamic equilibrium in solution, whereas the general model describes satisfactorily the parameters of heteroassociation practically for all systems studied.

[1H-NMR analysis of the heteroassociation of caffeine with the antibiotic actinocyl-bis(3-dimethylaminopropylamine) in aqueous solution]. / 1H-IaMP-analiz geteroassotsiatsii kofeina s antibiotikom aktinotsil-bis(3-dimetilaminopropilaminom) v vodnom rastvore.

The heteroassociation of caffeine (CAF) and the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) (ACT) was studied in aqueous solution by one- and two-dimensional 1H NMR spectroscopy at 500 MHz. The equilibrium reaction constants, thermodynamic parameters (delta H and delta S) of ACT heteroassociation with CAF, the limiting values of proton chemical shifts of their molecules in the heteroassociation complex, and the spatial structure of the ACT-CAF complex were determined from the experimental dependences of proton chemical shifts of the aromatic molecules on concentration and temperature. The parameters of CAF heteroassociation with the phenoxazone antibiotic actinomycin D and its synthetic analogue ACT were comparatively analyzed and conclusions were made on the crucial role of stacking interactions of the chromophores of CAF and the phenoxazone antibiotics in the formation of the heterocomplexes in aqueous solution.

[NMR study of complex formation of aromatic ligands with heptadeoxynucleotide 5'-d(GCGAAGC) forming stable hairpin structure in aqueous solution]. / IaMR-analiz kompleksoobrazovaniia aromaticheskikh ligandov s dezoksigeptanukleotidom 5'd(GCGAAGC), obrazuiushchim stabil'nuiu shpilechnuiu strukturu v vodnom rastvore.

Complex formation of hairpin-producing heptadeoxynucleotide 5'-d(GCGAAGC) with aromatic molecules: acridine dye proflavine and anthracycline antibiotic daunomycin was studied by one-dimensional 1H NMR and two-dimensional correlation 1H-1H (2M-TOCSY, 2M-NOESY), 1H-31P (2M-HMBC) NMR spectroscopy (500 and 600 MHz) in aqueous solution. Concentration and temperature dependences for the chemical shifts of ligand protons were measured, molecular models of equilibrium in solution were developed, and equilibrium thermodynamic parameters for the formation of intercalation complexes were calculated. Spatial structures of dye and antibiotic complexes with the heptamer hairpin were constructed on the basis of 2M-NOE data and the calculated values of limiting chemical shifts of ligand protons.

[1H-NMR analysis of self association of an antibiotic actinocyl-bis(3-dimethylaminopropylamine) in water solutions]. / 1H-IaMR-analiz samoassotsiatsii antibiotika aktinotsil-bis(3-dimetilaminopropilamina) v vodnom rastvore.

The self-association of the synthetic antibiotic actinocyl-bis(3-dimethylaminopropylamine) was studied in aqueous solution by one- and two-dimensional 1H NMR spectroscopy at 500 MHz. The two-dimensional homonuclear correlation NMR techniques (TOCSY and ROESY) were used to completely assign all the proton signals of the antibiotic and to quantitatively analyze the mutual arrangement of the antibiotic molecules in their aggregates. The concentration and temperature dependences of proton chemical shifts were used to determine the equilibrium constants and the thermodynamic parameters (delta H and delta S) of the self-association, as well as the limiting values of proton chemical shifts in associates. The experimental results were analyzed using both the indefinite noncooperative and cooperative models of the molecular self-association. The calculated value of the cooperativity coefficient (sigma approximately 1.1) for our synthetic antibiotic confirmed a substantially lower anticooperative effect at the aggregation of its molecules in comparison with that of the antitumor antibiotic actinomycin D (sigma approximately 1.5). We calculated the most favorable structure of the dimeric associate of the synthetic antibiotic in aqueous solution and found that, like in the actinomycin D dimer, the antiparallel orientation of the phenoxazone chromophore planes of interacting molecules is characteristic of its dimer. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2002, vol. 28, no. 4; see also http://www.maik.ru.

[Thermodynamic analysis of interaction of mitoxantrone with deoxytetranucleotide 5'-d(TpGpCpA) in the water solution based on 1H-NMR spectrophotometry]. / Termodinamicheskii analiz vzaimodeistviia antibiotika mitoksantrona s dezoksitetranukleotidom 5'-d(TpGpCpA) v vodnom rastvore po dannym 1H-IaMR-spektroskopii.

The complex formation of the antibiotic mitoxantrone (novantrone) with the deoxytetranucleotide 5'-d(TpGpCpA) in an aqueous salt solution was studied by one- and two-dimensional (2D-TOSCY and 2D-NOESY) 1H NMR spectroscopy (500 MHz). Concentration and temperature dependence of proton chemical shifts of molecules were measured. On the basis of these data, the equilibrium constants of the reaction, the relative content of various complexes as a function of concentration and temperature, the limiting values of chemical shifts of novantrone in complexes, and the thermodynamic parameters delta H and delta S of complex formation of molecules were calculated. It was concluded that the attachment sites for novantrone are pyrimidine-purine nucleotide sequences, sites d(TG) and d(CA) of the tetranucleotide duplex. The analysis of the thermodynamic parameters of the complex formation suggests that intermolecular hydrogen bonds and electrostatic interactions of the aminoalkyl chains of novantrone with the duplex d(TpGpCpA)2 play an important role in the stabilization of complexes 1:2 and 2:2. The results were compared with those obtained earlier for typical intercalators of ethidium bromide and daunomycin under identical experimental conditions.

[Analysis of complex formation of daunomycin with the deoxyhexanucleotide 5'-d(TpApCpGpTpA) in an aqueous solution from NMR-spectroscopy data]. / Analiz kompleksoobrazovaniia daunomitsina s dezoksigeksanukleotidom 5'-d(TpApCpGpTpA) v vodom rastvore po dannym IaMR-spektroskopii.

Self-association of hexadeoxynucleotide 5'-d(TpApCpGpTpA) and its complexation with antitumor antibiotic daunomycin were studied by one- and two-dimensional homonuclear 1H NMR spectroscopy and heteronuclear 1H-31P NMR spectroscopy in water-salt solution. The concentration and temperature dependences of proton chemical shifts of the hexadeoxynucleotide and the ligand were measured, and equilibrium constants and thermodynamic parameters of corresponding reactions were calculated on this basis using models for the formation of hexadeoxynucleotide duplex and its complex with the antibiotic. The spatial structure of daunomycin-d(TACGTA)2 complex in solution was calculated using X-PLOR software on the basis of 2D NOE spectral data and the limit values of proton chemical shifts of the ligand. Comparative analysis of different intermolecular interactions in sequence-specific binding of the antibiotic to the DNA fragment was carried out.

Hetero-association of caffeine and aromatic drugs and their competitive binding with a DNA oligomer.

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.

[Study of the complex formation of daunomycin with deoxytetranucleotides with bases of differing sequence in an aqueous solution by 1H-NMR spectroscopy]. / Issledovanie kompleksoobrazovaniia daunomitsina s dezoksitetranukleotidami razlichnoi posledovatel'nosti osnovanii v vodnom rastvore metodom 1H-IaMR-spektroskopii.

The complex formation of the antibiotic daunomycin with deoxytetranucleotides of different base sequence in the chain, 5'-d(GpCpGpC), 5'-d(CpGpCpG), and 5'-d(TpGpCpA) in aqueous salt solution was studied by 1D and 2D (2M-TOCSY and 2M-NOESY) 1H-NMR spectroscopy. Concentration and temperature dependences of proton chemical shifts of molecules were measured. Based on these dependences, reaction equilibrium constants, relative content of various complexes depending on concentration and temperature, limiting values of chemical shifts of protons of daunomycin incorporated in various complexes, and the thermodynamic parameters delta H and delta S of complex formation were calculated. The analysis of the results enables the conclusion that the sites of predominant intercalation of daunomycin are triplet nucleotide sequences, the binding sites of the antibiotic with three consecutive GC pairs in the tetranucleotide duplex being more preferential. Daunomycin exhibits no sequence specificity upon binding to the single-stranded deoxynucleotide sequence. From the calculated values of induced chemical shifts of daunomycin protons and 2M-NOE data, the most probable spatial structures of complexes (1:2) of the antibiotic with deoxytetranucleotides were constructed. The binding of the second daunomycin molecule to both the single-stranded and duplex form of tetramers is of pronounced anticooperative mode, which is explained by the presence in the antibiotic of a positively charged amino sugar residue, which poses considerable steric constraints for the insertion of the second antibiotic molecule into the short tetranucleotide sequence. The results were compared with the data obtained under identical experimental conditions for typical intercalators proflavine and ethidium bromide.

NMR investigation of the complexation of daunomycin with deoxytetranucleotides of different base sequence in aqueous solution.

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.

[1H-NMR analysis of heteroassociation of caffeine with antibiotic actinomycin D in the aqueous solution]. / 1H-IaMR analiz geteroassotsiatsii kofeina s antibiotikom aktinomitsinom D v vodnom rastvore.

The molecular basis of the action of caffeine as a complex forming agent, an interceptor of aromatic drugs intercalating into DNA was studied by the example of the an anticancer antibiotic actinomycin D examined. The hetero-association of caffeine and actionomycin D was studied by one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Concentration and temperature dependences of the proton chemical shifts of molecules in aqueous solution were measured. The equilibrium reaction constant of hetero-association of caffeine with actinomycin D (K = 246 +/- 48 M-1), the limiting chemical shifts of caffeine protons in complexes were determined. The most favourable structure of the 1:1 caffeine-actinomycin D hetero-complex in aqueous solution was constructed using the calculated values of the induced proton chemical shifts of molecules and the quantum-mechanical iso-shielding curves for caffeine and actinomycin D. The thermo-dynamical parameters of the hetero-complex formation between caffeine and actinomycin D were also determined. The structural and thermo-dynamical analysis showed that dispersive forces and hydrophobic interactions play the major role in hetero-association of caffeine and actinomycin D in aqueous-salt solution. The relative content of different complexes in mixed solutions containing caffeine and actinomycin D was calculated and distinctive features of the dynamic equilibrium of caffeine-actinomycin D hetero-associates were revealed as a function of concentration and temperature. It is concluded that hetero-association of caffeine and actinomycin D molecules a lowers the effective concentration of the drug in solution and hence the pharmacological activity of actinomycin D.

[Study of self-association of molecules of deoxyhexanucleotides 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in water solutions by NMR]. / IaMR-issledovanie samoassotsiatsii molekul dezoksigeksanukleotidov 5'-d(CpGpTpApCpG) i 5'-d(CpGpCpGpCpG) v vodnom rastvore.

The self-association of deoxyribohexanucleoside pentaphosphates 5'-d(CpGpTpApCpG) and 5'-d(CpGpCpGpCpG) in aqueous salt solutions was studied by 1 D- and 2 D homonuclear PMR and heteronuclear 1H-31P-spectroscopies. Signals from nonexchangeable protons of hexamers in NMR spectra were assigned using the available 2M-TOCSY, 2M-NOESY, and 1H-31P-(HMBS) spectra. The dependences of proton chemical shifts of deoxyhexanucleotides on concentration and temperature were measured. In terms of the two-states model (monomer-duplex), constants and thermodynamic parameters of self-association of hexamer molecules in solution were obtained based on these dependences. The values obtained correlate well with theoretical values calculated using the model of the "nearest neighbor" for the formation of duplexes of sequences d(CGTACG) and d(CGCGCG).

Role of crown-like side chains in the biological activity of substituted-phenoxazone drugs.

The antitumour activity of a number of synthetic crown-ether analogues of actinomycin D (AMD) was investigated in order to test the role of side chains that can complex metal cations. The AMD analogues consisted of two series of phenoxazone derivatives substituted with either benzo-15-crown-5 or benzo-18-crown-6 and with different lengths of spacers between the crown groups and the phenoxazone chromophore. The biological activities of the synthetic compounds were investigated by examination of drug-induced apoptosis and cell cycle perturbations in a human leukemia MOLT-3 cell line by flow cytometry. A compound with dimethylaminopropyl side chains on the phenoxazone chromophore was used as a control; this molecule was shown to intercalate into DNA by UV-visible spectroscopy and was found to have considerable cytotoxic activity in the 1-9 microM concentration range. Compounds with five-membered crown-ether side chains showed biological activity comparable with the control drug, whereas increasing the length of the spacers between the crown groups and the phenoxazone chromophore reduced the cytotoxic effect of the drugs. Compounds with six-membered crown-ether side chains reduced stabilization of the DNA double helical structure and abolished biological activity. Cell cycle alterations were observed only in drug systems which demonstrated cytotoxic activity. Cell cycle regulation was found to be sensitive to minor modifications (elongation of the spacer by one methylene group) in the side chains of the benzo-15-crown-5 derivatives, indicating that such series of synthetic drugs may serve as useful probes for investigation of cell cycle regulation processes.

[1H-NMR study of complex formation between the aromatic dye ethidium bromide and the single-stranded noncomplementary deoxytetranucleotide 5'-d(CpTpGpA) in aqueous solution]. / Issledovaniie kompleksoobrazovaniia aromaticheskogo krasitelia bromistogo etidiia s odnotsepochechnym nekomplementarnym dezoksi tetranucleotidom 5'-d(CpTpGpA) v vodnom rastvore metodom 1H IaMR -spektroskopii.

Complex formation between the aromatic dye ethidium bromide (3,8-diamino-6-phenyl-5-ethylphenanthridine) and the single-stranded noncomplementary deoxytetranucleotide 5'-d(CpTpGpA) in aqueous solution was studied by one- and two-dimensional 1H NMR spectroscopy (500 and 600 MHz). Complete assignments of proton signals from the deoxytetranucleotide 5'-d(CTGA) were made using 2D-TOCSY and 2D-NOESY spectra. The concentration dependences of proton chemical shifts of the tetranucleotide were measured at T = 298 K to determine the self-association constants of tetranucleotide molecules in solution. Due to a small probability of tetranucleotide duplex formation, the complexing of dye molecules with the monomer of the tetramer plays the main role in the equilibrium of the complex in solution, which makes it possible to analyze the specificity of interactions of the aromatic ligand with the single-stranded DNA. Different schemes of complex formation were examined, and equilibrium constants for the reactions and the limiting chemical shifts of dye protons in different complexes were determined. The relative content of different types of complexes was analyzed, and specific features of the dynamic equilibrium were revealed as a function of the dye-tetranucleotide concentration ratio. The analysis indicates a sequence-specific binding of ethidium bromide to the single-stranded oligonucleotide. The structures of 1:1 complexes of the dye with the single strand of the tetranucleotide that correspond to two most probable orientations of the dye chromophore in the pyrimidine-purine T-G-site of the tetramer base sequence were constructed using the calculated values of induced chemical shifts of ethidium bromide protons.

1H-NMR determination of the thermodynamics of drug complexation with single-stranded and double-stranded oligonucleotides in solution: ethidium bromide complexation with the deoxytetranucleotides 5'-d(ApCpGpT), 5'-d(ApGpCpT), and 5'-d(TpGpCpA).

The thermodynamical parameters (free energy, enthalpy, and entropy) of complex formation between ethidium bromide and single-stranded and double-stranded tetranucleotides of different base sequence [5-d(TpGpCpA), 5-d(ApCpGpT), and 5-d(ApGpCpT) have been determined from the temperature dependencies of 500 MHz proton nmr chemical shifts. The analysis enables the contributions to be differentiated for the formation of different types of complexes (1:1, 2:1, 1.2 and 2:2) in aqueous solution. The results have been interpreted in terms of the main types of intermolecular interactions responsible for formation of the different complexes; van der Waals and electrostatic interactions are important for formation of complexes of ethidium bromide with single-stranded tetranucleotides, whereas van der Waals and hydrophobic interactions play a significant role in the binding of the dye to the tetramer duplexes.