Mechanistic studies of the reactions of nitrone spin trap PBN with glutathiyl radical

We performed mechanistic studies of the reaction of PBN with the physiologically relevant glutathiyl radical, GS*, formed upon oxidation of the intracellular antioxidant, glutathione, GSH. The scavenging rate constant of GS* by PBN has been measured directly by laser flash photolysis and indirectly by competitive EPR of the spin adduct of PBN and another spin trap, DMPO (5,5-dimethyl-1-pyrroline N-oxide), and was found to be 6.7 x 107 M(-1) s(-1). Reverse decomposition of the paramagnetic PBN-glutathiyl radical adduct to the nitrone and thiyl radical was observed for the first time. The rate constant for the reaction of the monomolecular decomposition of the radical adduct was found to be 1.7 s(-1). Diamagnetic, EPR-invisible products of PBN adduct degradation were studied by 1H NMR and 19F NMR using newly synthesized fluorine-substituted PBN.     

Inclusion complexes of PBN-type nitrone spin traps and their superoxide spin adducts with cyclodextrin derivatives: parallel determination of the association constants by NMR titrations and 2D-EPR simulations

(1)H NMR and electron paramagnetic resonance (EPR) titrations were used to determine the association constants of the complexes of alpha-phenyl-N-tert-butylnitrone (PBN) analogues and their superoxide spin adducts, respectively, with methylated beta-cyclodextrins. A 1:1 stoichiometry for the nitrones with randomly methylated beta-cyclodextrin and 2,6-di-O-methyl-beta-cyclodextrin and 1:1 and 1:2 stoichiometries for the corresponding cyclodextrin-nitroxide complexes were observed. After the superoxide radical spin trapping reaction, EPR titrations afforded the association constants of the corresponding cyclodextrin-nitroxide complexes. Two-dimensional EPR simulations indicated a bimodal inclusion of the nitroxide free radical spin adducts into the cyclodextrins. For all the nitrone-cyclodextrin and nitroxide-cyclodextrin complexes, the association constants were always higher for the nitroxide complexes than for the nitrone complexes. A cooperative system concerning the complexation of the nitroxide spin adduct with a cyclodextrin was evidenced by EPR titrations. The efficiency of the cyclodextrin inclusion technique to trap superoxide and to resist bioreduction by sodium l-ascorbate was also investigated.     

Interaction of Naproxen with Crystalline and Amorphous Methylated β-Cyclodextrin in the Liquid and Solid State

Abstract

Interactions of naproxen (NAP) with amorphous, randomly methylated β-cyclodextrin at a degree of substitution per anhydroglucose unit of 1.8 (RAMEB) and with crystalline heptakis-(2,6-di-O-methyl)-β-cyclodextrin (DIMEB) were studied in aqueous solution and in the solid state using, respectively, phase-solubility analysis (at 25 °C, 37 °C and 47 °C) and differential scanning calorimetry (DSC) supported by X-ray powder diffractometry. RAMEB and DIMEB displayed similar solubilizing and complexing abilities towards NAP, suggesting analogous inclusion modes of the drug in the host cavity in aqueous solution. Differences were instead observed in interactions in the solid state, where the amorphizing capacity of RAMEB toward NAP (evaluated by DSC) was about twice that of DIMEB at each drug-to-carrier ratio. Assuming that inclusion complexation is also involved in solid-state interactions, molecular modelling accounted for the experimental results in terms of structural features of DIMEB, i.e. the particular inwards orientation of O-6-C-8 groups of three alternate glucoses on the primary hydroxyl side which hampers a deep penetration of NAP in the DIMEB cavity in the solid state. On the contrary, no obstruction of the cavity apparently occurs with RAMEB due its noncrystalline state. The aqueous dissolution rate of NAP from NAP-RAMEB and NAP-DIMEB blends containing 0.59, 0.73, 0.85, and 0.92 mass fraction of carrier linearly increased at decreasing drug-to-carrier ratios. The improvement was 5 to 20 times (from powders) and 50 to 200 times (from discs) the dissolution rate of NAP alone for both carrier. Therefore the choice of the amorphous RAMEB in pharmaceutical formulations can be recommended mainly for economic reasons, though the anhydrous and non-hygroscopic nature of crystalline DIMEB might be of particular advantage in case of moisture sensitive formulations.     

Differential scanning calorimetry as an analytical tool in the study of drug-cyclodextrin interactions

The interactions of trimethoprim, sulphadiazine and sulphamethoxazole with natural (a- b-, g- ) and amorphous (RAMEB) or crystalline (DIMEB) methylated b-cyclodextrins were investigated both in aqueous solution (using phase-solubility analysis) and in the solid state (using DSC supported by X-ray analysis). In particular, DSC studies enabled determination of the relative degree of crystallinity of each drug in its physical and ground mixtures with the different cyclodextrins on the basis of the variation of its heat of fusion in comparison with that of the pure drug. In all cases, the host cavity size was a prevalent factor for the inclusion complexation in liquid state. On the contrary, it had a negligible effect on solid-state interactions in terms of drug amorphization. DIMEB and RAMEB exhibited similar performances in aqueous solution, showing that the presence of methyl-groups improved the complexing and solubilizing properties of b-cyclodextrin. However, DSC studies revealed that RAMEB was clearly more active in performing solid-state interactions, i.e. drug amorphization, and as stabilizing agent for the amorphous state brought forth. This revised version was published online in July 2006 with corrections to the Cover Date.     

Methylation of cyclodextrins by phase-transfer catalysis

A new, simple method has been developed for the methylation of cyclodextrins. The reaction proceeds in the heterogeneous phase with dimethyl sulphate, using a solvent in which the original cyclodextrins and the bases used are poorly soluble or insoluble. However, in the presence of phase transfer catalysts, methylation proceeds with good yields. The products are mixtures of randomly methylated cyclodextrins (RAMEB), containing 60–70% of heptakis(2,6-di-O-methyl)--cyclodextrin (DIMEB), 10–15% of heptakis(2,3,6-tri-O-methyl)--cyclodextrin (TRIMEB) and some monomethylated isomers. These methylated products have proved to be excellent detergents; e.g., they are able to significantly increase the water solubility of hydrocortisone, methyltesterone, etc. On repeating methylation twice, the amount of TRIMEB increases, and a pure product (28% yield) can be obtained by crystallisation.Proceedings of the Fourth International Symposium on Cyclodextrins (Ed. O. Huber and J. Szejtli), Munich 1988, p. 113. Kluwer Academic Publishers.Dedicated to Professor Szejtli.     

Gas Chromatography - Mass Spectroscopy (GC/MS) of Single and Double Spin Adducts of PBN and the Hydroxylamines of Corresponding Structure

Abstract

The GC/MS spectra of the methyl adduct of PBN as the aminoxyl and as the corresponding hydroxylamine are reported. Similar results are obtained with higher alkyl analogues. With excess alkyl Grignard the O-alkyl hydroxylamine ether (PBN double adduct) is obtained if the solution is exposed to O₂ in the presence of the alkyl spin adduct of PBN. A mechanism involving radical trapping by the alkyl spin adduct is proposed.     

Dendritic galactosides based on a beta-cyclodextrin core for the construction of site-specific molecular delivery systems: synthesis and molecular recognition studies

In order to evaluate the ability of multivalent glycosides based on a beta-cyclodextrin core as site-specific molecular carriers, a study on both the inclusion complexation behaviour and lectin binding affinity of branched and hyperbranched beta-cyclodextrins is presented. A series of cluster galactosides constructed on beta-cyclodextrin scaffolds containing seven 1-thio-beta-lactose or beta-lactosylamine bound to the macrocyclic core through different spacer arms were synthesised. In addition, the first synthesis of three first-order dendrimers based on a beta-cyclodextrin core containing fourteen 1-thio-beta-D-galactose, 1-thio-beta-lactose and 1-thio-beta-melibiose residues was performed. Calorimetric titrations performed at 25 degrees C in buffered aqueous solution (pH 7.4) gave the affinity constants and the thermodynamic parameters for the complex formation of these beta-cyclodextrin derivatives with guests sodium 8-anilino-1-naphthalenesulfonate (ANS) and 2-naphthalenesulfonate, and lectin from peanut (Arachis hypogaea) (PNA). The persubstitution of the primary face of the beta-cyclodextrin with saccharides led to a slight increase of the binding constant values for the inclusion complexation with ANS relative to the native beta-cyclodextrin. However, the increase of the steric congestion due to the presence of the saccharide residues on the narrow rim of the beta-cyclodextrin may cause a decrease of the binding ability as shown for sodium 2-naphthalenesulfonate. The spacer arms are not passive elements and influence the host binding ability according to their chemical nature. PNA forms soluble cross-linked complexes with cluster galactosides and lactosides scaffolded on beta-cyclodextrin but not with cluster galactopyranosylamines or melibiose. Both, perbranched and hyperbranched beta-cyclodextrins, form stronger complexes with PNA than the monomeric analogues. However, the use of hyperbranched CDs does not contribute to the improvement of the complex stability relative to heptakis-glycocyclodextrin derivatives. Finally, a titration experiment with PNA and a complex formed by a heptakis lactose beta-cyclodextrin derivative with sodium 2-naphthalenesulfonate showed the formation of a soluble cross-linked complex with stronger affinity constant and higher stoichiometry than those observed for the complex formation of PNA with the same heptakis-lactose beta-cyclodextrin derivative, suggesting the formation of a three component complex.     

Reactivity of superoxide radical anion and hydroperoxyl radical with alpha-phenyl-N-tert-butylnitrone (PBN) derivatives

Nitrones have exhibited pharmacological activity against radical-mediated pathophysiological conditions and as analytical reagents for the identification of transient radical species by electron paramagnetic resonance (EPR) spectroscopy. In this work, competitive spin trapping, stopped-flow kinetics, and density functional theory (DFT) were employed to assess and predict the reactivity of O(2)(*-) and HO(2)(*) with various para-substituted alpha-phenyl-N-tert-butylnitrone (PBN) spin traps. Rate constants of O(2)(*-) trapping by nitrones were determined using competitive UV-vis stopped-flow method with phenol red (PR) as probe, while HO(2)(*) trapping rate constants were calculated using competition kinetics with 5,5-dimethylpyrroline N-oxide (DMPO) by employing EPR spectroscopy. The effects of the para substitution on the charge density of the nitronyl-carbon and on the free energies of nitrone reactivity with O(2)(*-) and HO(2)(*) were computationally rationalized at the PCM/B3LYP/6-31+G(d,p)//B3LYP/6-31G(d) level of theory. Theoretical and experimental data show that the rate of O(2)(*-) addition to PBN derivatives is not affected by the polar effect of the substituents. However, the reactivity of HO(2)(*) follows the Hammett equation and is increased as the substituent becomes more electron withdrawing. This supports the conclusion that the nature of HO(2)(*) addition to PBN derivatives is electrophilic, while the addition of O(2)(*-) to PBN-type compounds is only weakly electrophilic.     

The properties and potential uses of cyclodextrin derivatives

The hydroxyl groups can be selectively substituted to control the solubility and the complex forming selectivity of the modified cyclodextrins (CDs). Among the methylated CDs only two well-defined compounds can be taken into consideration: dimethyl- and dimethyl-CD (DIMEB and TRIMEB). In an aqueous solution of DIMEB the solubility of rather insoluble compounds and drugs like steroids, vitamin D₃, lidocaine and hydrocortisone increases. In some cases their stability and bioavailability are also improved. On the other hand, the hydrolysis rate of carmofur, coumarins etc. is retarded by the methylated cyclodextrins with blocked hydroxyl groups. The drug solubilizing capacity of hydroxypropyl-CD (HPBCD) is in most cases lower than that of DIMEB. The degree of substitution (DS) shows no remarkable effect on the solubilizing properties of HPBCD in the case of indomethacin, Dipiridamole etc., but in the case of Tolnaftate the solubility was enhanced by increasing the DS, other examples are shown.     

Comparative study of oxaprozin complexation with natural and chemically-modified cyclodextrins in solution and in the solid state

The complexing, solubilizing and amorphizing abilities toward oxaprozin (a poorly water-soluble anti-inflammatory agent) of some β-cyclodextrin derivatives (hydroxypropyl-βCd, heptakis-2,6-di-O-methyl-βCd (DIMEB) amorphous randomly substituted methyl-βCd (RAMEB) and semi-crystalline methyl-βCd (CRYSMEΒ)) were investigated and compared with those of natural (α-, β-, γ-) cyclodextrins. The role of both the cavity size, the amorphous or crystalline state and the presence and type of substituent on the ability of cyclodextrins in establishing effective interactions with the drug has been evaluated. Equimolar drug-cyclodextrin solid systems were prepared by blending, kneading, co-grinding, sealed-heating, coevaporation, and colyophilization. Drug-carrier interactions were studied in both the liquid and solid state by phase-solubility analysis, differential scanning calorimetry, X-ray powder diffractometry, FT-IR spectroscopy and scanning electron microscopy. βCd showed the best performance among the natural Cds, indicating that its cavity was the most suitable for accommodating the drug molecule. The presence of substituents on the rim of the βCd cavity significantly improved its complexing and solubilizing effectiveness towards the drug, and methylated derivatives were better than the hydroxy-propylated ones The amorphous nature of the partner was also important: among the examined methyl-derivatives, RAMEB proved to be the most effective in performing solid state interactions and in improving drug wettability and dissolution properties.     

Muonium addition to DMPO and PBN sorbed in silica-gel

Nitroxyl radicals were formed by adding the light hydrogen isotope, muonium to the spin traps DMPO (5,5-dimethyl-1-pyrroline-N-oxide) and PBN (N-tert-butyl-alpha-phenylnitrone) sorbed as 30 wt% ethanol solutions in silica-gel; evidence is presented for a specific hydrogen-bonded interaction between the DMPO adduct and the silica surface; longitudinal-field muon spin relaxation measurements (LF-MuSRx) were performed which identified two distinct motional regimes in both samples.     

Spin trapping of superoxide in the presence of beta-cyclodextrins

The trapping of superoxide anion with DMPO and DEPMPO has been carried out in the presence of a methylated beta-cyclodextrin, Me-beta-CD; inclusion of the spin adducts in the cavity of Me-beta-CD resulted in a seven-fold increase of their half-life (t1/2 = 96 min for DEPMPO-superoxide spin adduct) and in their protection towards glutathione peroxidase (Gpx) and also ascrobate anion in the case of DEPMPO.     

Fluorescence anisotropy decay and solvation dynamics in a nanocavity: coumarin 153 in methyl beta-cyclodextrins

Fluorescence anisotropy decay and solvation dynamics of coumarin 153 (C153) are studied in dimethyl beta-cyclodextrin (DIMEB) and trimethyl beta-cyclodextrin (TRIMEB) nanocavity in water. C153 binds to DIMEB and TRIMEB to form both 1:1 and 1:2 (C153:cyclodextrin) complexes. The anisotropy decays of C153 in DIMEB and TRIMEB are found to be biexponential. The fast component of anisotropy decay (approximately 1000 ps) is attributed to the 1:1 complex and the slower one (approximately 2500 ps) to the 1:2 complex. From the components of the anisotropy decay, the length of the 1:1 and 1:2 complexes are estimated. Solvation dynamics of C153 in DIMEB exhibits a very fast (2.4 ps) component (41%) and two slower components of 50 ps (29%) and 1450 ps (30%). Solvation dynamics in TRIMEB is described by three slow components of 10.3 ps (24%), 240 ps (45%), and 2450 ps (31%). Possible origins of the ultraslow components are discussed.     

ESR study of spin-trapping with two glycosylated analogues of PBN able to target cell membrane lectins

The spin trapping behaviour of the two galactosylated nitrones LAMPBN and TA1PBN, both of which are able to target cells through recognition by cell membrane lectins, were widely investigated on a variety of free radicals in aqueous media. The ESR spectra of the more amphiphilic nitrone, TA1PBN, were interpreted in the light of the LAMPBN trapping results. The spin adducts of nitrone TA1PBN, which may be better distributed inside the cell, were surrounded by two distinct environments due to the tensioactive organisation of the trap and gave two different ESR signals for each radical trapped.     

Spectrophotometric and calorimetric titration studies on molecular recognition of camphor and borneol by nucleobase-modified beta-cyclodextrins

A series of modified beta-cyclodextrins with nucleobase substituents, that is, mono(6-ade-6-deoxy)-beta-cyclodextrin (2) and mono(6-ura-6-deoxy)-beta-cyclodextrin (3) as well as mono(6-thy-6-deoxy)-beta-cyclodextrin (4), were selected as molecular receptors to investigate their conformation and inclusion complexation behaviors with some chiral molecules, that is, (+)-camphor, (-)-camphor, (+)-borneol, and (-)-borneol, by spectrophotometric and microcalorimetric titrations in aqueous phosphate buffer solution (pH 7.2) at 298.15 K. Circular dichroism and NMR studies demonstrated that these nucleobase-modified beta-cyclodextrins adopted a co-inclusion mode upon complexation with guest molecules; that is, the originally self-included nucleobase substituents of the host did not move out from the beta-cyclodextrin cavity, but coexisted with guest molecule in the beta-cyclodextrin cavity upon inclusion complexation. Significantly, these nucleobase-modified beta-cyclodextrins efficiently enhanced the molecular binding ability and the chiral recognition ability of native beta-cyclodextrin, displaying enantioselectivity up to 3.7 for (+)-camphor/(-)-camphor pair by 2 and 3.5 for (-)-borneol/(+)-borneol pair by 3. The enhanced molecular/chiral recognition abilities of 2-4 toward (+/-)-camphor were mainly attributed to the increased entropic gains due to the extensive desolvation effects, while the favorable enthalpic gains originating from the good size-fit relationship as well as the hydrogen bond interactions between host and guest result in the enhanced molecular/chiral recognition abilities of 2-4 toward (+/-)-borneol.     

Noncovalent binding of sensitizers for lanthanide(III) luminescence in an EDTA-bis(beta-cyclodextrin) ligand

EDTA-linked beta-cyclodextrin dimer 3 was synthesized from EDTA bis(anhydride) 1 and mono(propylamino)-appended beta-cyclodextrin 2. p-tert-butylbenzoate 5, bound by the beta-cyclodextrin cavities of 3 with an association constant of 10(4) M(-1) in water, acts as a sensitizer for the Eu(III) and Tb(III) complexes of 3. Luminescence spectroscopy, microcalorimetry, and Gd(III)-induced NMR relaxation rate measurements prove that 3 forms a 1:2 complex with 5 and that one of the beta-cyclodextrin-bound sensitizers coordinates to the EDTA-encapsulated Ln(III) ion. The Eu(III) complex of 3 forms strong 1:1 complexes (K approximately 10(7) M(-1)) with bis(propylamido adamantyl)-functionalized biphenyl sensitizers 7 and 8 in water. Both beta-cyclodextrins of 3 are involved in the binding of these guests. The amide functionality adjacent to the biphenyl unit in 7 and 8 coordinates to the EDTA-encapsulated Ln(III) ion. For these biphenyl-based antennae both binding to beta-cyclodextrin and coordination to the Ln(III) center are crucial for efficient sensitization.     

毛细管柱GC/MS/ESR 联用研究自由基PBN加合物

采用硅烷化方法，并用石英毛细管柱气相色谱与双聚焦质谱仪组成的ＧＣ／ＭＳ联用系统，首次成功地分析鉴别了六种自由基的ＰＢＮ加合物，总结了这些自旋加合物的断裂规律。实验结果表明，硅烷化结合ＧＣ／ＭＳ和自旋捕捉ＥＳＲ联用技术是确定活泼自由基的一种有效方法。     

The trapping of a spontaneously "flipped-out" base from double helical nucleic acids by host-guest complexation with beta-cyclodextrin: the intrinsic base-flipping rate constant for DNA and RNA

Beta-cyclodextrin, which forms stable host-guest complexes with purine bases, induces the melting of RNA and DNA duplexes below their normal melting temperatures. Alpha-cyclodextrin, which does not form stable complexes, has no effect on either RNA or DNA. Gamma-cyclodextrin, which forms weaker complexes, has no effect on RNA and a smaller effect than beta-cyclodextrin on DNA. The rate of melting is kinetically first-order in duplex and, above about 20 mM beta-cyclodextrin, is independent of the beta-cyclodextrin concentration with a first-order rate constant, common to both RNA and DNA, of (3.5 +/- 0.5) x 10(-3) s(-1) at 61 degrees C (DNA) and at 50 degrees C (RNA). This is taken to be the rate constant for spontaneous "flipping out" of a base from within the duplex structure of the nucleic acids, the exposed base being rapidly trapped by beta-cyclodextrin. Like beta-cyclodextrin, nucleic acid methyltransferases bind the target base for methylation in a site that requires it to have flipped out of its normal position in the duplex. The spontaneous flip-out rate constant of around 10(-3) s(-1) is near the value of k(cat) for the methyltransferases (ca. 10(-3) to 10(-1) s(-1)). In principle, the enzymes, therefore, need effect little or no catalysis of the flipping-out reaction. Nevertheless, the flip-out rate in enzyme/DNA complexes is much faster. This observation suggests that the in vivo circumstances may differ from in vitro models or that factors other than a simple drive toward higher catalytic power have been influential in the evolution of these enzymes.     

Hydrolysis and Enantiodiscrimination of (R)- and (S)-Oxazepam Hemisuccinate by Methylated β-Cyclodextrins: An NMR Investigation

Partially and exhaustively methylated β-cyclodextrins [(2-methyl)-β-CD (MCD), heptakis-(2,6-di-O-methyl)-β-CD (DIMEB), and heptakis-(2,3,6-tri-O-methyl)-β-CD (TRIMEB)] have been compared in the hydrolysis and enantiodiscrimination of benzodiazepine derivative (R)- or (S)-oxazepam hemisuccinate (OXEMIS), using nuclear magnetic resonance (NMR) spectroscopy as an investigation tool. After 6 h, MCD induced an 11% hydrolysis of OXEMIS, remarkably lower in comparison with underivatized β-CD (48%), whereas no hydrolysis was detected in the presence of DIMEB or TRIMEB after 24 h. DIMEB showed greater ability to differentiate OXEMIS enantiomers in comparison to TRIMEB, by contrast MCD did not produce any splitting of racemic OXEMIS resonances. Both enantiomers of OXEMIS underwent deep inclusion of their phenyl pendant into cyclodextrins cavities from their wider rims, but tighter complexes were formed by DIMEB with respect to TRIMEB.     

Comparative analysis of the full set of methylated β‐cyclodextrins as chiral selectors in capillary electrophoresis

The chiral separation ability of the full library of methylated‐β‐cyclodextrins towards pharmacologically significant racemic drugs including basic compounds was studied by chiral CE. The syntheses of all the methylated, single isomer β‐cyclodextrins were revised and optimized and the aqueous solubility of the derivatives was unambiguously established. The three most relevant commercially available methylated isomeric mixtures were also included in the screening, so a total of ten various methylated CDs were investigated. The effects of the selector concentration on the enantiorecognition properties at acidic pH were investigated. Among the dimethylated β‐cyclodextrins, the heptakis (2,6‐di‐O‐methyl)‐β‐cyclodextrin isomer (2,6‐DIMEB) resulted to be the most versatile chiral selector. Terbutaline was selected as a model compound for the in‐depth investigation of host‐guest enantiodiscrimination ability. The association constants between the two terbutaline enantiomers and 2,6‐DIMEB were determined in order to support that the enantioseparation is driven by differences is host‐guest binding. The migration order of the enantiomers was confirmed by performing spiking experiments with the pure enantiomers. 1D and 2D NMR spectroscopy was applied to the 2,3‐, and 2,6‐DIMEB/terbutaline systems to rationalize at molecular level the different enantioseparation ability of the dimethylated β‐cyclodextrin selectors.