RESUMEN
The molecular basis of the marked structure-activity relationship for a homologous series of DNA-binding phenoxazone drugs (ActII-ActIV) has been investigated by NMR spectroscopy and molecular mechanics. The spatial structures of the complexes between the drugs and a model deoxytetranucleotide, 5'-d(TpGpCpA), have been determined by molecular mechanics methods using homonuclear (1)H-(1)H 2D-NOESY and heteronuclear (1)H-(31)P (HMBC) NMR spectroscopic data. Observed intermolecular NOE contacts and equilibrium binding studies confirm that the binding affinity of the synthetic phenoxazone derivatives with d(TGCA) decreases with an increase in the number of CH(2) groups in the dimethylaminoalkyl side chains, i.e., ActII > ActIII > ActIV, in agreement with the observed biological activity of these compounds. Molecular mechanics calculations of the spatial structures of the intercalated complexes of ActII-ActIV with d(TGCA) indicate that the different binding constants of the phenoxazone derivatives with the DNA oligomer are due to the different degrees of intercalation of the chromophore and the different steric arrangements of aminoalkyl side chains in the minor groove of the tetramer duplex; this results in different distances between the negatively-charged phosphates of the DNA duplex and the terminal positively-charged N(CH(3))(2) groups of the side chains.
Asunto(s)
ADN/química , Sustancias Intercalantes/metabolismo , Espectroscopía de Resonancia Magnética , Oxazinas/metabolismo , Sitios de Unión , ADN/metabolismo , Sustancias Intercalantes/química , Imagen por Resonancia Magnética , Modelos Moleculares , Conformación Molecular , Oxazinas/química , Relación Estructura-Actividad , TermodinámicaRESUMEN
The self-association of thiazine dye, Methylene Blue (MB), and its hetero-association with Caffeine (CAF), were studied in aqueous solution by means of spectrophotometry in the visible range of spectrum. Concentration and temperature dependences of molar absorption of the interacting molecules were used to analyse dynamic equilibrium in solution in terms of two-component model of molecular hetero-association. The magnitudes of equilibrium dimerization and hetero-association constants as well as thermodynamic parameters, enthalpy and entropy, were determined. The calculation of the fraction of different types of associates in the mixed solution, containing Methylene Blue and Caffeine, was done. It was concluded that the hetero-association of Methylene Blue and Caffeine molecules results in lower effective concentration of the dye in solution, which may account for the alteration of its biological activity.
Asunto(s)
Cafeína/química , Azul de Metileno/química , Tiazinas/química , Agua , Soluciones , Espectrofotometría , TermodinámicaRESUMEN
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.
Asunto(s)
Antibióticos Antineoplásicos/metabolismo , ADN/metabolismo , Daunorrubicina/metabolismo , Nucleótidos/metabolismo , Agua/metabolismo , Sitios de Unión , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Modelos Teóricos , Temperatura , TermodinámicaRESUMEN
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.