N2-guanyl and N6-adenyl arylation of chicken erythrocyte DNA by the ultimate carcinogen of 4-nitroquinoline 1-oxide.

Great quantities of chicken erythrocyte DNA with high levels of modification were obtained in vitro by reaction with 4-acetoxyaminoquinoline 1-oxide, a model ultimate carcinogen of 4-nitroquinoline 1-oxide. After enzymatic hydrolysis of the modified DNA, the three main adducts were separated and isolated by semipreparative high performance liquid chromatography. These three adducts were already characterized in vivo and in vitro in our previous work (S. Galiègue-Zouitina et al., Cancer Res., 45: 520-525, 1985). The structure of one of them was previously identified as N-(deoxyguanosin-8-yl)-4-aminoquinoline 1-oxide (B. Bailleul et al., Cancer Res., 41: 4559-4565, 1981). In this paper we have identified by mass spectroscopy and nuclear magnetic resonance the structures of the two other main adducts as 3-(deoxyguanosin-N2-yl)-4-aminoquinoline 1-oxide and 3-(deoxyadenosin-N6-yl)-4-aminoquinoline 1-oxide, respectively.

Quasielastic neutron scattering measurements of fast local translational diffusion of lipid molecules in phospholipid bilayers.

Quasielastic incoherent neutron scattering has been used to investigate the rate of local translational diffusion of lipid molecules in phospholipid bilayers of dipalmitoyl-phosphatidylcholine. The measured translational diffusion constants (4 x 10(-10) m2 s-1 at 63 degrees C and 1.4 x 10(-11) m2 s-1 at 30 degrees C) are considerably faster than those deduced using other less direct methods, but are in agreement with those measured in soap-water lyotropic liquid crystals, and with calculated values. This disagreement is attributed to differences in the time and distance scales characterising the various measurements. Quasielastic neutron scattering experiments observe fast motions over molecular distances, whereas other methods tend to measure a rate of diffusion which is averaged over macroscopic distances, and may thus contain contributions from long distance slow diffusive motions such as diffusion between the bilayers.

Tryptamine-adenosine 5'-monophosphate interactions as studied by nuclear magnetic resonance and relaxation.

The conformation of tryptamine-adenosine 5'-monosphate and of their 1:1 complex in neutral aqueous solution at 297 K has been investigated by proton NMR and relaxation. The dependences of the proton chemical shift as a function of the tryptamine and AMP concentrations yield an association constant of 6.5 +/- 0.5 1 . mol-1. The reorientation correlation time of the complex tau R = (2.5 +/- 0.1) . 10(-10) s has been determined from the deuteron and ESR linewidth measurements on specifically labelled AMP. The proton longitudinal relaxation shows that the adenine and indole rings are head-to-head stacked 0.31 +/- 0.01 nm apart as confirmed by proton chemical shift measurements. In this complex, the AMP ribose ring takes the 3'-endo (N) conformation and the orientation of the adenine base is anti, whereas the tryptamine aminoethyl residue, in the gauche conformation, is most likely bound to the phosphate by coulombic interactions.

Isolation and two-dimensional 1H-NMR of peptide [1-24] of dog serum albumin and studies of its complexation with copper and nickel by NMR and CD spectroscopy.

The NH2-terminal peptide fragment [1-24] of dog serum albumin was obtained by controlled peptic digestion of the protein. The peptide was purified to homogeneity by gel filtration and ion-exchange chromatography. The NMR assignments of the protons of the individual amino acid residues were made by using two-dimensional correlation matrix, spin-decoupling experiments and analysis of the titration curves. The polypeptide itself has a random-coil conformation. There is a conformational change as a function of pH, but it does not arise from any direct involvement of the amino acid side chains. Complexation of the peptide fragment with Ni(II) and Cu(II) has been investigated by NMR and CD. The Ni(II) complex is in slow exchange with the free ligand on the NMR time scale. The complexation involves the alpha-NH2, three deprotonated amide nitrogens of Ala-2, Tyr-3 and Lys-4 residues. The phenolate oxygen of Tyr-3 is not involved in the metal binding; however, an interaction between the aromatic ring and the metal ion is likely. The CD results of Cu(II)-binding to this peptide suggest that the complexation takes place from the terminal NH2 and step by step to three deprotonated amide nitrogens. There is no major conformational change of the peptide fragment upon complexation.

A nuclear magnetic resonance study of the interactions of antimalarial drugs with porphyrins.

Haematins (hydroxyferriprotoporphyrin IX) constitute a possible receptor for antimalarial drugs such as chloroquine or quinine. This paper reports the study of the interactions of these two molecules with two tetrapyrrole (haematin and uroporphyrin I) by 1H-NMR spectroscopy. This method provided us with the geometry of the interactions in aqueous medium. The interaction consists of a close stacking of the porphyrin ring and the quinoleine moiety of the drugs. Using a porphyrin ring current model it was possible to reach the spatial relationships of the interacting species. It was concluded that hydrophobic forces play a key role in the interaction. The porphyrin plane can accommodate wide structural variations of the interacting species, leading to a weak specificity. The consequences on the mode of action of antimalarial drugs are discussed.

Solution structure determination by NMR spectroscopy of a synthetic peptide corresponding to a putative amphipathic alpha-helix of spiralin: resonance assignment, distance geometry and simulated annealing.

Spiralin is the major protein of the plasma membrane of several spiroplasmas. Neither the function of this protein nor the crystallographic structure is known. Analysis of the primary structure of spiralin from Spiroplasma melliferum BC3 suggests the presence of an amphipathic peptide in the 143-162 region (Chevalier, C., Saillard, C. and Bové, J.M. (1990) J. Bacteriol. 172, 6090-6097). The structure of a synthetic peptide, H2N-L-N-A-V-N-T-Y-A-T-L-A-K-A-V-L-D-A-I-Q-N-amide, corresponding to this fragment has been examined by 1H-NMR spectroscopy. This 20 amino acid peptide adopts a random coil structure in solution, but the addition of trifluoroethanol stabilizes a structure exhibiting alpha-helical character. The 1H-NMR spectrum has been fully assigned in CF3CD2OD/H2O (30:70, v/v) and the three-dimensional structure has been elucidated using NMR-derived distance information. The calculated structures have been obtained by dynamical simulated annealing or distance geometry followed by simulated annealing. Both sets of structures have been energy-minimized using CHARMm potential. The resulting structures are very similar in terms of constraint violations and energies. It is demonstrated that whereas the first three residues exhibit a large flexibility, the remaining sequence is helical.

Estimation of the location of natural alpha-tocopherol in lipid bilayers by 13C-NMR spectroscopy.

Natural, 2R,4R',8R'-alpha-tocopherol (vitamin E), labelled selectively with 13C in the methyl group at position 5, was incorporated into unilamellar vesicles of egg phosphatidylcholine. The vesicles are impermeable to the shift reagent Pr3+ and, in the presence of this reagent, separate 13C resonances due to labelled alpha-tocopherol in the outer and inner monolayers could be observed with relative intensities, 2:1. Subsequent addition of the relaxation reagent Gd2+ causes broadening and greatly shortened spin-lattice relaxation times for the resonance due to alpha-tocopherol in the outer monolayer only. These data confirm that alpha-tocopherol is located in both halves of the bilayers with its more hydrophilic chroman moiety very near the lipid-water interface, and indicate that the methyl group at position 5 of the alpha-tocopherol in the inner monolayer must be at least 40 A from the aqueous interface of the outer monolayer.

Pharmacological in vitro evaluation of new substance P-cyclodextrin derivatives designed to drug targeting towards NK1-receptor bearing cells.

Some biological properties of new bifunctional conjugates designed for drug targeting were evaluated through in vitro experiments. Eight peptidylcyclodextrin compounds were used, which correspond to modified beta- or gamma-cyclodextrin (CD) grafted on neuropeptide substance P (SP) or a shorter derivative (SP(4-11)). Using anti-SP and anti-CD antibodies as molecular probes, we showed that the main structural features of the two moieties of these adducts were preserved. Binding experiments, using CHO cells expressing the human SP-specific NK1 receptor, demonstrated the functionality of all peptidylcyclodextrin derivatives, which exhibited IC50 values in a 10(-9)-10(-7) M range. All compounds were able to induce a pharmacological response, triggering phosphatidylinositol turnover with EC50 values in the same range as the natural ligand. Moreover, autoradiography analysis of rat spinal corn sections proved that [125I]SP binding was dose-dependently displaced by one selected compound (a gamma-CD-SP), showing a similar affinity of this adduct for the rat neurokinin 1 receptor. Our observations demonstrate that these peptidylcyclodextrins efficiently target NK1 receptor-expressing cells.

[Interaction of chloroquine with ferriprotophorphyrin IX. Nuclear magnetic resonance study]. / Interactions de la chloroquine avcc la ferriprotoporphyrine IX. Etude par résonance magnétique nucléaire.

Chloroquine is still the antimalarial drug which is the most utilized. Nevertheless the molecular mode of action of this drug is not very well understood. When mouse erythrocytes injected with Plasmodium berghei are exposed to chloroquine, the first biochemical event is rapid accumulation of the drug. This process is energy dependent, saturable and competitively inhibited by drugs of the same therapeutic class (Quinine, Amodiaquine, Mefloquine). Receptors for chloroquine have been proposed for the process of accumulation. The nature of the chloroquine receptor is presently the subject of debates. The latest hypothesis proposed by Chou and coll. [12], is that ferriprotoporphyrin IX, formed by the degradation of hemoglobin by the parasite, binds to chloroquine with a dissociation constant of 3.5.10(-9) M. We studied here the molecular interactions between these two species by Proton Nuclear Magnetic Resonance in order to elucidate the nature and the geometry of were undertaken. The perturbations of the NMR spectra of chloroquine (10(-2) M) induced by addition of hematin or hemin were measured. Two types of measures were undertaken. The first study carried out in organic solvent (DMSO) has shown that the interaction occurred between the acidic functions of hemin and the side-chain nitrogen of chloroquine. The iron atom was not implicated in this process. The second study carried out in aqueous medium (phosphate buffer; 0.1 M; pH = 7) allowed us to demonstrate that chloroquine is able to intercalate into a polymer of hematin. The quinoleic nucleus of chloroquine was intercalated between two dimers of hematin as shown by the broadening of the signal of the quinoleic protons due to very large increase in the correlation time. Finally it was shown that chloroquine is associated as a dimer in aqueous medium by hydrophobic interactions. The association constant is 5.5 M-1.

(1)H NMR Study of the solution structure of sarafotoxin-S6b.

Sarafotoxin-S6b has been synthesized and studied by (1)H NMR in 50 50 acetonitrile/water mixture. All spin systems were identified and assigned with the aid of 2D experiments. On the basis of these data, a 3D structure of sarafotoxin is proposed and compared to that of [Nle(7)]endothelin obtained in the same conditions. From this study, it appeared that sarafotoxin-S6b and [Nle(7)]endothelin roughly share the same 3D structure, the main differences being located in the 4-7 loop bearing the sequence variation.

Skin compatibility of cyclodextrins and their derivatives: a comparative assessment using a corneoxenometry bioassay.

Few studies have been performed to assess the risk of skin damage by cyclodextrins (CD) and they have yielded contradictory results. The present study was conducted using the corneoxenometry bioassay on human stratum corneum to compare the skin compatibility of CD currently used in pharmaceutical preparations (betaCD, gammaCD, Rameb, Dimeb, Trimeb, HP-betaCD and HP-gammaCD) and that of new amphiphilic CD derivatives, namely, the phospholipidyl-CD (DMPE-Dimeb and DMPE-Trimeb). All the tested CD were well tolerated by the stratum corneum at a concentration of 5%. However, inter-individual reactivity was larger for DMPE-Dimeb, suggesting a more aggressive trend for this compound. Cutaneous Index of Mildness values obtained confirm that Dimeb is able to extract some skin components and shows that DMPE-Dimeb performs similarly.

Nuclear magnetic resonance investigation of the stoichiometries in beta-cyclodextrin:steroid inclusion complexes.

Nuclear magnetic resonance at high field has been used to investigate the stoichiometries of pharmacologically important beta-cyclodextrin:steroid complexes. This technique provides evidence for the existence of true inclusion complexes and allows unequivocal determination of the stoichiometry for complexes obtained in the solid state, as well as in solution. The present data clarify previously published determinations of the stoichiometry of these complexes and show the influence of the nature and position of functional groups on the molecular ratio in the complex. The observed behavior can be rationalized using representative steroids and allows prediction of the strength of inclusion and the most probable stoichiometry.

High-field nuclear magnetic resonance techniques for the investigation of a beta-cyclodextrin:indomethacin inclusion complex.

The inclusion complex of indomethacin sodium salt in beta-cyclodextrin has been studied by proton NMR at high magnetic field. The continuous variation technique was used to evidence the formation of a soluble 1:1 complex in aqueous solution at physiological pH. The effective association constant was determined by the Benesi-Hildebrand procedure to be 760 M-1 at 303 K. This technique requires NMR measurements in the presence of a very large excess of one of the complex components and, since both beta-cyclodextrin and the sodium salt of indomethacin are sparingly soluble in water, NMR spectrometers operating at very high magnetic fields were used. Besides the effective association constant, the Benesi-Hildebrand approach allows a precise determination of all NMR parameters of the pure inclusion complex which may be used for a complete analysis of the geometry of this complex in solution.

Molecular modeling of beta-cyclodextrin complexes with nootropic drugs.

The geometry and structural features of the inclusion complexes of beta-cyclodextrin (beta-CD) with the chiral antiamnesic drugs (+/-)-1-benzyl-4-hydroxymethylpyrrolidin-2-one (WEB-1868). (+/-)-1-benzenesulfonyl-5-ethoxypyrrolidin-2-one (RU-35929), and (+/-)-1-(3-pyridinlysulfonyl)-5-ethoxypyrrolidin-2-one (RU-47010) were studied by the molecular modeling method (MacroModel interactive computer program). Docking procedures yielded the most stable complexes, which showed the aromatic ring of the guests inside the cavity and the pyrrolidinone ring out from the side of the beta-CD secondary hydroxyl groups. The binding energies were essentially due to hydrogen-bonded structures involving the C=O group of the guests. Selective interactions allowed chiral discrimination, and accordingly, separate beta-CD complexes of the R and S enantiomers of each guest compound were studied. The almost round beta-CD structure, in all the cases, assumed an elliptic shape on passing from the isolated molecule to the docked complex. The optimized structures and conformations of beta-CD and its inclusion compounds showed acceptable general agreement with information from proton nuclear magnetic resonance studies.

Physicochemical characterization of different batches of ethylated beta-cyclodextrins.

Different ethylated beta-cyclodextrin (Et-beta-CD) derivatives were obtained by various synthetic routes and their chemical structures and physical properties were elucidated. The aqueous solubility studies were carried out at 25 and 37 degrees C. A gas phase chromatography analysis, with head space extraction, was developed to detect the presence of residual solvents in the dry preparations. Electrospray ionization mass spectrometry allowed the determination of the average degree of substitution and the molecular mass of the Et-beta-CDs. Nuclear magnetic resonance analysis was used to elucidate the relationship between the solubility behavior and substituted positions of ethyl groups on the CD glucopyranose units. Finally, this paper deals with different physico-chemical methods used to fully characterize the different batches of Et-beta-CD to correlate the data obtained with the pharmacotechnical behavior of these cyclodextrins.

Synthetic pyridopurines derived from food pyrolysis products: intercalation, interactions with membranes, cyclodextrin complexation, and biological mitogenic properties.

Crucial conditions for the pharmacological use of active compounds are their ability to cross the biological barriers and reach their intracellular target. In the case of two antiviral pyridopurine derivatives, 1 and 2, this included essentially the membranes and the nucleic acids. Thus the interactions of 1 and 2 with model membranes and oligonucleotides were studied using NMR spectroscopy. It was found that these hydrophobic molecules can be incorporated into the model membranes at the terminal methyl group level, inducing dynamic perturbations in the bilayer. In the presence of the synthetic oligonucleotide ACATGT, both molecules can intercalate aspecifically in AT and GC systems. Inclusion complexes of 1 and 2 beta-cyclodextrins with a 1:1 stoichiometry, were also prepared. This led to to propose two galenic forms 1 and 2, i.e. included in phospholipid vesicles in the form of a beta-cyclodextrin complex

Pharmacokinetic analysis of 6-monoamino-beta-cyclodextrin after intravenous or oral administration to rats using a specific enzyme immunoassay.

We have developed a highly sensitive enzyme immunoassay for 6-monoamino-beta-CD (mono(6-amino-6-deoxy)cyclomaltoheptaose) and its parent compound (beta-CD) with a detection limit in the 100 pg/mL range. The polyclonal antibodies obtained are highly specific for the beta-cyclodextrin core and do not recognize other cyclic cyclodextrins (i.e., alpha- and gamma-CD) or linear analogues. This enzyme immunoassay can be used to quantify 6-monoamino-beta-CD in rat urine and plasma. Using this immunoassay, we have evaluated the main pharmacokinetic parameters of 6-monoamino-beta-CD after iv administration to the rat of a 25 mg/kg dose. Since this method is strictly specific to the native beta-CD form, we have demonstrated that the molecule rapidly disappeared from plasma but is probably distributed in the tissues. The urinary route appears as the predominant way of elimination since almost all the administered drug is recovered in urine. Finally, analysis of the same molecule after oral administration to the rat (25 mg/kg) demonstrates low plasma levels and that about 1% of the administered dose is excreted in urine. These experiments demonstrate the high stability of the beta-CD core irrespective of the method of administration. This immunological method could provide relevant information on the fate of beta-CD and some derivatives for drug delivery using different modes of administration (oral, parenteral, transmucosal, or dermal).

A comparative 1H-n.m.r. study of MurNAc-L-Ala-D-iGln (MDP) and its analogue murabutide: evidence for a structure involving two successive beta-turns in MDP.

The active principle, MurNAc-L-Ala-D-iGln (MDP), of complete Freund's adjuvant and its analogue, MurNAc-L-Ala-D-Gln-OnBu (murabutide), which express immunomodulatory as well as other biological properties, have been studied by 2D-1H-n.m.r. spectroscopy at 500 MHz. The results suggest the presence in MDP of two successive turns involving the MurNAc-L-Ala and L-Ala-D-iGln moieties, respectively, whereas only the former turn persists in murabutide. This turn mimics the type II beta-turn found in L-D depsipeptides, whereas the other is a typical type II beta-turn for L-D peptides.

A sensitive and specific enzyme immunoassay for cyclomaltoheptaose and some derivatives.

Antibodies were raised against cyclomaltoheptaose (beta-cyclodextrin), providing a highly sensitive enzyme immunoassay for beta-cyclodextrin and some derivatives with a detection limit of 120 pg/mL. Investigations of cross-reactivities with a wide variety of linear and cyclic maltooligosaccharides demonstrate that the antibodies are highly specific for cyclomaltoheptaose and a number of derivatives. The epitope is probably located on the secondary hydroxyl groups of the rim side. This enzyme immunoassay is shown to be suitable to detect cyclomaltoheptaose in urine and in plasma.

Conformation of the pentasaccharide corresponding to the binding site of heparin for antithrombin III.

The conformation in solution of the pentasaccharide methyl glycoside (As-G-A*-Is-AM; 1), which represents the binding site of heparin for Antithrombin III, has been investigated using molecular mechanics and 1H-n.m.r. spectroscopy. The pentasaccharide has a rather rigid (As-G-A*) and a more flexible (Is-AM) region. A simplified model of 1, comprising two conformations, corresponding to the 1C4 and the 2S0 forms of the iduronate residue, and modified at the G-A* glycosidic linkage with respect to the energy minimum, reproduces most of the observed 3J values and n.O.e. enhancements. The possible role in the binding to Antithrombin III of a low-energy conformer, not observed in solution, is discussed.