1H-NMR analysis of heteroassociation of caffeine with antibiotic actinomycin D in the aqueous solution

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.     

1H-NMR analysis of self association of an antibiotic actinocyl-bis(3-dimethylaminopropylamine) in water solutions

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.     

1H-NMR studies on the self-association of chloroquine in aqueous solution

The concentration dependences of 1H-NMR chemical shifts and spin-lattice relaxation rates were measured for chloroquine in aqueous solution. The weak self-association constant was evaluated according to a dimerization equilibrium with the formation of self-stacked adducts (Kd = 4.52 +/- 0.68 l mol-1). The motional correlation times were evaluated for the monomer and the dimer by measuring intramolecular dipolar cross-relaxation rates of aromatic vicinal protons (tau cm = 0.06 ns and tau cd = 0.26 ns). The geometry of the stacked dimer was elucidated by measuring intermolecular dipolar cross-relaxation rates and interpreted in terms of partial superposition of quinoline moieties.     

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.     

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.     

Heteroassociation of Caffeine with the Antibiotic Actinocyl- bis(3-dimethylaminopropylamine) in Aqueous Solution: 1H NMR Analysis

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 (H and 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.     

Thermodynamic analysis of interaction of mitoxantrone with deoxytetranucleotide 5'-d(TpGpCpA) in the water solution based on 1H-NMR spectrophotometry

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.     

Heteroassociation of antibiotic norfloxacin with aromatic vitamins in aqueous solution

Heteroassociation of antibacterial antibiotic norfloxacin with aromatic vitamins nicotinamide and flavin mononucleotide in aqueous solution was studied by ¹H NMR spectroscopy (500 MHz). Equilibrium constants, induced proton chemical shifts, and thermodynamic parameters (ΔH, ΔS) for the reactions of heteroassociation of the molecules were determined on the basis of the concentration and temperature dependences of proton chemical shifts for interacting aromatic molecules. The analysis of the results obtained indicates the formation of heterocomplexes between vitamin molecules and norfloxacin owing to stacking interactions between aromatic chromophores and additional intermolecular hydrogen bonding in norfloxacin-nicotinamide. The most probable spatial structures of 1:1 norfloxacin-flavin mononucleotide and norfloxacin-nicotinamide heterocomplexes were determined by molecular modeling methods using X-PLOR software on the basis of analysis of induced proton chemical shifts.     

Heteroassociation of antibiotic norfloxacin with aromatic vitamins in aqueous solution

The heteroassociation of the antibacterial antibiotic norfloxacin with aromatic vitamins nicotineamide and flavin mononucleotide in aqueous solution has been studied by 1H NMR spectroscopy (503 MHz). Equilibrium constants, induced proton chemical shifts, and the thermodynamic parameters (deltaH, deltaS) of the heteroassociation of molecules were determined from the concentration and temperature dependences of chemical shifts of protons of interacting aromatic molecules. An analysis of the results indicates the formation of heterocomplexes between the molecules of the vitamins and norfloxacin, which is caused by stacking interactions between aromatic chromophores and an additional intermolecular hydrogen bond in the norfloxacin-nicotinamide system. Based on the analysis of induced chemical shifts of protons of molecules, the most probable spatial structures 1:1 of norfloxacin-flavin mononucleitide and norfloxacin-nicotinamide heterocomolexes were determined by the methods of molecular modeling using the X-PLOR program.     

Study of the complex formation of daunomycin with deoxytetranucleotides with bases of differing sequence in an aqueous solution by 1H-NMR spectroscopy

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.     

The Self-Association of Antibiotic Actinocyl-bis(3-dimethylaminopropylamine) in Aqueous Solution: A 1H NMR Analysis

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 (H and 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 ( 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 ( 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.     

Study of the spatial structure of the duplex d(pTGTTTGGC) d(pCCAAAC)A in aqueous solution by methods of uni- and two-dimensional (1)H-NMR spectroscopy and organic molecular mechanics

Structure of the complementary complex d(pTGTTTGGC) d(pCCAAAC)A in the aqueous solution has been investigated by one- and two-dimensional 1H NMR spectroscopy. The resonances of nonexchangeable protons of bases as well as methyl and deoxyribose 1', 2'a, 2'b, 3' and 4' protons have been assigned by means of two-dimensional J-correlated spectroscopy (COSY) and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY). Using one-dimensional NOE measurements, 62 interproton distances (intranucleotide: (H6/H8)i--(H1')t, (H6/H8)i--(H2'a)i, (H1')i--(H2'a)i, (H1')i--(H2'b)i; internucleotide: (H6/H8)i--(H1')i-1, (H6/H8)i--(H2'a)i-1, (H6/H8)i--(H2'b)i-1, (H5/CH3)i--(H6/H8)i-1, (H5/CH3)i--(H2'a/H2'b)i-1) have been determined for nearest-neighbour protons. Spin-coupling constant values for some sugar protons have been obtained from COSY spectra. The restrained molecular mechanics calculations have yielded the possible solution structures of duplex fitting the experimental set of interproton distances and coupling constants.     

1H-NMR assignment and secondary structure of human insulin-like growth factor-I (IGF-I) in solution.

Human insulin-like growth factor-I (IGF-I) was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for all the backbone protons and almost all of the sidechain protons of the total 70 amino acid residues, using sequence-specific assignment procedures. The secondary structure elements of human IGF-I were identified by investigation of the sequential and medium range NOEs as a preliminary step in determining the three-dimensional structure of this protein by means of distance geometry calculations. The typical NOEs of d alpha beta(i,i + 3) and d alpha N(i,i + 3), as well as the successive strong NOEs of dNN connectivities and slowly exchanging amide protons confirmed the presence of three helical segments corresponding to the sequence regions, Ala8-Cys18, Gly42-Cys48, and Leu54-Cys61, and the existence of a beta-turn in the Gly19-Gly22 region. Our results definitely indicate that the secondary structure of human IGF-I in solution is consistent with that of insulin in the crystalline state.     

Thermodynamical model of mixed aggregation of ligands with caffeine in aqueous solution. Part II

A statistical-thermodynamical model of mixed association in which one component's self-association is unlimited while the second component does not self-aggregate is described. The model was tested with 4',6-diamidino-2-phenyl-indole-dihydrochloride (DAPI) and ethidium bromide (EB) using light absorption spectroscopy and calorimetry. The system is controlled by two parameters, which represent self-aggregation 'neighborhood' association constant KCC and mixed 'neighborhood' association constant KAC. Calculated, using this model, KAC = 58.2 +/- 1 M-1, KAC = 64.6 +/- 2 M-1 for DAPI and EB, respectively, are in good agreement with known values of stacking interactions. The titration microcalorimetric measurement of DAPI-CAF interaction delta H = -11.1 +/- 0.4 kcal/mol is also consistent with this type of reaction. The structures of the stacking complexes were also confirmed by semi-empirical molecular modeling in the presence of water. The data indicate that CAF forms stacking complexes with DAPI and EB, thus effectively lowering the concentration of the free ligands in the solution, and therefore, CAF can be used to modulate aromatic compound activity.     

The complete chirospectroscopic signature of the peptide 3(10)-helix in aqueous solution

We synthesized by solution methods a water-soluble, terminally blocked heptapeptide based on five markedly helicogenic, C(alpha)-tetrasubstituted alpha-amino acids C(alpha)-methyl-L-norvalines and two strongly hydrophilic 2-amino-3-[1-(1,4,7-triazacyclononane)]-L-propanoic acid residues at positions 2 and 5. A Fourier transform infrared absorption and NMR analysis in deuterated chloroform and aqueous solutions of the heptapeptide and two side-chain protected synthetic precursors confirmed our working hypothesis that all oligomers are folded in the 3(10)-helical conformation. Based on these findings, we exploited this heptapeptide as a chiral reference compound for detailed electronic CD, vibrational CD, and Raman optical activity characterizations of the 3(10)-helix in aqueous solution.     

Distinctive features of the self-association of deoxyhexanucleotide 5'-D(GpCpApTpGpC) in aqueous solution: 1H NMR analysis

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.     

Conformational analysis of a segment in bacterioopsin by two-dimensional (1)H-NMR spectroscopy]

The spatial structure of a synthetic peptide, an analogue of the membrane spanning segment B (residues 34-65) of bacterioopsin from Halobacterium halobium, has been refined. Backbone torsion angles were derived from intensities of short-range interproton NOEs. These, together with a complete set of the NOEs integral intensities formed the basis for the three-dimensional structure refinement by the energy minimization with consideration of NOE penalty functions. Analysis indicates the right-handed alpha-helical conformation of segment B extending from Asp-38 to Tyr-64 with a kink of the helical axis (27 degrees) at Pro-50. The most stable region with an average root-mean-square deviation of 0.43 A between the backbone atoms includes residues 42-60 in six energy refined structures. The N-terminal part of segment B (residues 34-37) has no ordered conformation. The inferred structure is in close agreement with the electron cryomicroscopy structure of bacteriorhodopsin, differing from it in conformations of most of the side chains.     

Conformational studies of the copolymer poly(L-lysine,L-tyrosine) 1:1 in aqueous solution

The absorption and circular dichroism spectra of the 1:1 copolymer (L -Lys, L -Tyr)_n have been investigated in aqueous solutions at pH ranging from 3 to 13. The spectral patterns indicate that the fully charged polympholyte assumes a nonperiodic conformation on the acid and basic sides of the isoelectric point. At pH ranging from 9.2 to 11.6, the polymer is largely ordered and takes mostly an antiparallel β-structure as is shown by the infrared spectra in D₂O solutions. Moreover, the rotational strength of the L_a transition of tyrosyl is independent of the polymer conformation, whereas that of the L_b transition is strongly sensitive to it.     

Study of the structure of the duplex (Phn-NH(CH2)2NH)pd(CCAAACA) .pd(TGTTTGGC) with covalently bound 10-(2-hydroxyethyl)phenazine in an aqueous solution by 2D-1H-NMR spectroscopy]

The spatial structure of duplex (Phn-NH(CH2)2NH)pd(CCAAACA).pd(TGTTTGGC) having a N-(2-oxyethyl)-phenazinium residue covalently linked with the 5'-terminal phosphate of the heptanucleotide was studied by means of one- and two-dimensional 1H-NMR spectroscopy. The resonances of phenazinium protons, ethylenediamine linker protons, as well as, oligonucleotide H5/H6/H8/CH3 base protons and H1',H2'a, H2'b, H3', H4' deoxyribose protons have been assigned by means of 1H-COSY, 1H-NOESY and 1H-13C-COSY. The presence of the phenazine residue in duplex causes an additional imino proton signal of the terminal (G-7).(C-1) base pair, suggesting a higher stability of the duplex (Phn-NH(CH2)2NH)pd(CCAAACA).pd(TGTTTGGC) as compared to the unmodified duplex pd(CCAAACA).pd(TGTTTGGC). Analysis of NOE interactions between protons of the dye and the oligonucleotides show the phenazinium polycyclic system to intercalate between G-7 and C-8 residues of the octanucleotide.