Synthesis and anti-HIV properties of new water-soluble bis-functionalized[60]fullerene derivatives

A new series of bis-functionalized fullerene C₆₀ derivatives bearing two or more solubilizing chains have been evaluated for their activity against HIV-1 and HIV-2 strains. Some of the compounds show activity against HIV-1 type in the low micromolar range. The effect of the positions of the addends on the C₆₀ nucleus has been investigated, indicating that only trans-2 isomers possess promising activity. The presence of a quaternary pyrrolidinium nitrogen is essential to increase solubility.     

Stereo- and biochemical profiles of the 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerenes

The 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerene were studied by means of circular dichroism (CD), deuterium labeling, 1H-NMR, molecular-dynamics (MD) calculations, and a lectin-binding assay. The CD spectra of the O-acetylated derivatives allowed clear discrimination of the isomers, while the 1H-NMR spectra, with assistance from deuterium labeling and MD calculations, served to disclose the unique conformation and molecular geometry of each acetylated isomer in chloroform solution. The deprotected 5-6- and 6-6-isomers, which gave colloidal suspensions in aqueous mixtures, displayed marked activity in blocking lectin-induced hemagglutination by concanavalin A.     

Antioxidant action of sugar-pendant C60 fullerenes

The action of C₆₀ fullerene and its derivatives as a radical-scavenging antioxidant has received much attention, but their reactivity toward free radicals and antioxidant capacity have not been well elucidated yet. In the present study, the reactivity of the two types of water-soluble, sugar-pendant C₆₀ fullerenes, C₆₀-1S and C₆₀-2S, toward peroxyl radical and their effect against human plasma lipid peroxidation were measured. The rate constants for the reaction of C₆₀-1S and C₆₀-2S with peroxyl radicals were obtained from their effect on the bleaching of β-carotene in lipid-SDS micelle system as 4.6 × 10³ and 8.0 × 10³ M^?1 s^?1 at 37 °C, respectively. They inhibited the free radical-induced lipid peroxidation in human plasma in a concentration-dependent manner. These results suggest that the sugar-pendant fullerenes C₆₀-1S and C₆₀-2S act as a radical-scavenging antioxidant with the activity similar to the phenolic antioxidants.     

Oxidative properties of photoexcited fullerenes C60 and C60-containing composites in suspension of thymocytes and Ehrlich ascites carcinoma cells

Biological effects of photoexcited in UV/VIS region water-soluble fullerenes C60 and synthesized C60-containing composites after its addition to cell incubation medium are studied. It is shown by EPR method, that in the presence of photoexcited C60 (10(-5) M) reactive oxygen species (ROS) are generated both in water solution and cell suspensions. ROS generation is accelerated if C60 is included into aminopropylaerosyl or antracenaliminopropylaerosyl. Photodynamic damage of Erlich ascites carcinoma cells by photoexcited C60-composites is confirmed by a decrease of cell viability, enhanced content of Schiff base fluorescent products of malonaldehyde as indicator of lipid peroxidation and the decrease in respiratory activity, estimated by MTT-test. No effects of photoexcited C60-composites were detected in thymocytes suspension. The data obtained testity to perspective utility of C60-composites for optimization of photodynamic therapy methods.     

Unusual spectroscopic and electrochemical properties of the 2[4Fe-4S] ferredoxin of Thauera aromatica

A reduced ferredoxin serves as the natural electron donor for key enzymes of the anaerobic aromatic metabolism in the denitrifying bacterium Thauera aromatica. It contains two [4Fe-4S] clusters and belongs to the Chromatium vinosum type of ferredoxins (CvFd) which differ from the "clostridial" type by a six-amino acid insertion between two successive cysteines and a C-terminal alpha-helical amino acid extension. The electrochemical and electron paramagnetic resonance (EPR) spectroscopic properties of both [4Fe-4S] clusters from T. aromatica ferredoxin have been investigated using cyclic voltammetry and multifrequency EPR. Results obtained from cyclic voltammetry revealed the presence of two redox transitions at -431 and -587 mV versus SHE. X-band EPR spectra recorded at potentials where only one cluster was reduced (greater than -500 mV) indicated the presence of a spin mixture of S = (3)/(2) and (5)/(2) spin states of one reduced [4Fe-4S] cluster. No typical S = (1)/(2) EPR signals were observed. At lower potentials (less than -500 mV), the more negative [4Fe-4S] cluster displayed Q-, X-, and S-band EPR spectra at 20 K which were typical of a single S = (1)/(2) low-spin [4Fe-4S] cluster with a g(av) of 1.94. However, when the temperature was decreased stepwise to 4 K, a magnetic interaction between the two clusters gradually became observable as a temperature-dependent splitting of both the S = (1)/(2) and S = (5)/(2) EPR signals. At potentials where both clusters were reduced, additional low-field EPR signals were observed which can only be assigned to spin states with spins of >(5)/(2). The results that were obtained establish that the common typical amino acid sequence features of CvFd-type ferredoxins determine the unusual electrochemical properties of the [4Fe-4S] clusters. The observation of different spin states in T. aromatica ferredoxin is novel among CvFd-type ferredoxins.     

Optical and electrochemical properties and the calculated structure of pentacoordinate aluminum 8-hydroxyquinoline

In this study we report on the characterization of a series of pentacoordinate aluminum 8-hydroxyquinolines, AlQ₂X (X = F, Cl, Br), composed of two 8-hydroxyquinoline (8-HQ) groups and one halogen ligand. These were prepared by reacting 8-hydroxyquinoline and dialkylaluminum halide stoichiometrically. The λ_maxs of absorption and emission were in the range of 385-388 and 515-516 nm, respectively, which were similar to AlQ₃. The molar absorptivity of AlQ₂X is similar regardless of the X group but emission efficiency of AlQ₂X is 2-3 fold higher than that of AlQ₃ when X = F or Br, but not when X = Cl. This result can be attributed to decreased quenching of energy due to a less steric environment by reducing quinolinate content. The overall molecular orbital structures and the absorption spectra of AlQ₂X and AlQ₃ are very similar due to a single quinolinate unit. The value of the potential difference of AlQ₂Xs between anodic and cathodic waves (ΔE = 3.12 V) is close to the estimated HOMO-LUMO energy gap (the optical band gap, ΔE_optical ∼ 1240/λ_max), 3.19 eV. Detailed optical and electrochemical properties of AlQ₂X are discussed.     

Syntheses and biological evaluations of alpha-D-mannosyl [60]fullerenols.

[60]Fullerenols carrying mono- and bis-alpha-D-mannosyl linkages on the surface were prepared via a [3+2]-cycloaddition reaction between 2-azidoethyl alpha-D-mannoside and C(60) followed by polyhydroxylation with aqueous NaOH. Their biological activity was evaluated in terms of binding affinity to lectins by hemagglutination assay and surface plasmon resonance. [60]Fullerenols without the mannosyl linkage caused aggregation of erythrocytes and binding to a beta-D-galactopyranoside specific lectin (RCA(120)). In contrast, mono- and bis-mannosyl fullerenols were found to decrease the activity for both aggregating erythrocytes and binding to RCA(120), and mono-mannosyl fullerenols turned to binding to alpha-D-mannose specific lectin (Con A).     

Optical and magnetic properties of azurin from Pseudomanas aeruginosa]

Optical, fluorescence and EPR spectra of azurin from Pseudomonas aeruginosa are described. Some properties of this protein are found to be similar to those of copper-containing proteins from plants (plastocyanin and plantacyanin). The interaction of ferricyanide with azurin bleached in alkaline media results in the formation of free radicals and an alteration in the shape of the EPR signal of azurin.     

Preparation and supramolecular properties of unadulterated glycosyl liposomes from a bis(alpha-D-mannopyranosyl)-[60]fullerene conjugate

The bis(alpha-D-mannopyranosyl)-[60]fullerene conjugate 3 was prepared by thermal coupling of C60 and either 2-azidoethyl 2,3,4,6-tetra-O-acetyl- or 2,3;4,6-di-O-isopropylidene-alpha-D-mannopyranoside (Scheme). Compound 3 was found to readily self-assemble. Dynamic-light-scattering (DLS) and atomic-force microscopy (AFM) experiments supported that the amphiphilic compound gives rise to nano-sized supramolecular structures during sugar deprotection (Ac-group removal) performed in MeOH/CH2Cl2 solution. Encapsulation studies with an aqueous suspension of 3 showed that the self-assembling structure envelopes Ba2+ and the fluorescent dye Acridine Red during its formation, which indicates that it resembles a bilayer vesicle or an unadulterated liposome with an inner hollow space. In addition to this notable property, the unique molecular geometry of the spatially arranged mannosyl surface residues of 3 gives rise to strong binding of the carbohydrate-recognizing lectin Con A. Hence, the polar amphiphilic end of 3 mimics the structure of 3,6-branched tri-alpha-D-mannoside (6; Fig. 3), a natural ligand of the Con A protein.     

Using C60 fullerenes for photodynamic inactivation of mosquito iridescent viruses

This article describes the photodynamic inactivation of mosquito iridescent virus (MIV) Aedes flavescens in the presence of water-soluble C₆₀ fullerenes. It has been observed that the photodynamic inactivation of MIV for about 1?h reduces the infectious titre of the virus in large wax-moth larvae Galleria mellonella to 4.5 lg ID₅₀/mL. The influence of the C₆₀ concentration on its anti-viral activity was tested in the concentration range from 1 to 0.001?mg/mL. It has been found that C₆₀ is able to inactivate the iridovirus even in low concentrations. Consequently, the findings of this work suggest that photoexcited C₆₀ fullerenes can be successfully used for the inactivation of iridoviruses in biological systems.     

Using C60 fullerenes for photodynamic inactivation of mosquito iridescent viruses

This article describes the photodynamic inactivation of mosquito iridescent virus (MIV) Aedes flavescens in the presence of water-soluble C(60) fullerenes. It has been observed that the photodynamic inactivation of MIV for about 1 h reduces the infectious titre of the virus in large wax-moth larvae Galleria mellonella to 4.5 lg ID(50)/mL. The influence of the C(60) concentration on its anti-viral activity was tested in the concentration range from 1 to 0.001 mg/mL. It has been found that C(60) is able to inactivate the iridovirus even in low concentrations. Consequently, the findings of this work suggest that photoexcited C(60) fullerenes can be successfully used for the inactivation of iridoviruses in biological systems.     

[60]Fullerene displacement from fac-(dihapto-[60]fullerene)(dihapto-1,2-bis-(1,10-phenanthroline) tricarbonyl tungsten(0)

The Lewis bases triphenyl phosphine and tricyclohexyl phosphine (L) displace [60]fullerene (C₆₀) from fac-(η²-C₆₀)(η²-phen)W(CO)₃ (phen=1,10-phenanthroline) to produce fac-(η²-phen)(η¹-L)W(CO)₃. Under flooding conditions, the reactions were first order with respect to fac-(η²-C₆₀)(η²-phen)W(CO)₃. The order with respect to C₆₀ and L depends on the reaction conditions i.e., whether [C₆₀]/[L] ≈ 0 or 0?It [C₆₀]/[L] ≈ 1. Two limiting cases of an interchange displacement of [60]fullerene from fac-(η²-C₆₀)(η²-phen)W(CO)₃, whose relative contributions to the overall mechanism depend on the nature of the solvent, are proposed based on the rate law and on the activation parameters. The mechanism involves an initial [60]fullerene dissociation to produce (i) the electronically unsaturated intermediate (η²-phen)W(CO)₃ for the dissociative displacement and (ii) the solvated intermediate fac-(solvent)(η²-phen)W(CO)₃ for the solvent-assisted [60]fullerene dissociation. The W-C₆₀ bond energy in fac-(η²-C₆₀)(η²-phen)W(CO)₃ was estimated to be in the vicinity of 105 kJ/mol based on the enthalpy of activation of the step where presumably [60]fullerene dissociates from fac-(η²-C₆₀)(η²-phen)W(CO)₃ to produce (η²-phen)W(CO)₃.     

Structure and properties of a ferrocenylpyrimidine-bromanilic acid hydrogen-bonded supramolecular complex

A hydrogen-bonded assembly composed of ferrocenylpyrimidine (FcPM) and bromanilic acid (BA), represented as [FcPM](BA)(acetone)_0.5, was prepared and crystallographically characterized. The asymmetric unit of the crystal contained two crystallographically independent molecules of FcPM and BA, which were alternately connected to form one-dimensional zigzag chains via OH?N hydrogen bonds. The BA molecules were stacked to form one-dimensional columns. No charge transfer was observed between FcPM and BA. Acetone molecules, which were located in channels, were desorbed at 433 K.     

Novel synthesis of [1]-benzothiepino[5,4-b]pyridine-3-carbonitriles and their anti-inflammatory properties

Reaction of 4-arylmethylene-3,4-dihydro-[1]-benzothiepin-5(2H)-ones 1 with malononitrile in the appropriate alcohol in the presence of sodium afforded the 2-alkoxy-4-aryl-5,6-dihydro-[1]-benzothiepino[5,4-b]pyridine-3-carbonitriles 2 and not the isomeric forms [1]-benzothiepino[4,5-c]pyridine-1-carbonitriles 3 in high regioselective manner. The assumed structure of 2 was inferred through independent synthetic reaction of 3,4-dihydro-[1]-benzothiepin-5(2H)-one (4) with ylidenemalononitriles 5 under the same applied reaction conditions and confirmed by single crystal X-ray diffraction studies. However, reaction of 4 with arylidenecyanothioacetamides 6 in refluxing ethanol in the presence of basic catalyst (piperidine or morpholine) does not afford the expected 4-aryl-3-cyano-5,6-dihydro-[1]-benzothiepino[5,4-b]pyridine-2(1H)-thiones 7 and instead 4-aryl-3,5-dicyano-6-thioxo-2(1H)-pyridinethiolate monohydrates were isolated as piperidinium or morpholinium salts 8. On the other hand, reaction of 6 with cyanothioacetamide in the presence of a sufficient amount of basic catalyst yielded exclusively 2-amino-4-aryl-3,5-dicyano-2-pyridinethiolates as piperidinium or morpholinium salts 9. Meanwhile, 7 were prepared through the reaction of 1 with cyanothioacetamide in refluxing ethanol in the presence of a catalytic amount of piperidine. Anti-inflammatory activity screening of the prepared compounds using in vivo acute carrageenan-induced paw oedema in rats exhibited that all the tested compounds possess considerable activity. In addition, few synthesized derivatives reveal remarkable anti-inflammatory properties (2d, k, l) comparable with indomethacin which was used as a reference standard during the pharmacological activity screening studies.     

Structural features of [NiFeSe] and [NiFe] hydrogenases determining their different properties: a computational approach

Hydrogenases are metalloenzymes that catalyze the reversible reaction \textH₂ \leftrightarrows 2\textH ⁺ + 2\texte ^- {\text{H}}_{2} \leftrightarrows 2{\text{H}}^{ + } + 2{\text{e}}^{ - } , being potentially useful in H₂ production or oxidation. [NiFeSe] hydrogenases are a particularly interesting subgroup of the [NiFe] class that exhibit tolerance to O₂ inhibition and produce more H₂ than standard [NiFe] hydrogenases. However, the molecular determinants responsible for these properties remain unknown. Hydrophobic pathways for H₂ diffusion have been identified in [NiFe] hydrogenases, as have proton transfer pathways, but they have never been studied in [NiFeSe] hydrogenases. Our aim was, for the first time, to characterize the H₂ and proton pathways in a [NiFeSe] hydrogenase and compare them with those in a standard [NiFe] hydrogenase. We performed molecular dynamics simulations of H₂ diffusion in the [NiFeSe] hydrogenase from Desulfomicrobium baculatum and extended previous simulations of the [NiFe] hydrogenase from Desulfovibrio gigas (Teixeira et al. in Biophys J 91:2035–2045, 2006). The comparison showed that H₂ density near the active site is much higher in [NiFeSe] hydrogenase, which appears to have an alternative route for the access of H₂ to the active site. We have also determined a possible proton transfer pathway in the [NiFeSe] hydrogenase from D. baculatum using continuum electrostatics and Monte Carlo simulation and compared it with the proton pathway we found in the [NiFe] hydrogenase from D. gigas (Teixeira et al. in Proteins 70:1010–1022, 2008). The residues constituting both proton transfer pathways are considerably different, although in the same region of the protein. These results support the hypothesis that some of the special properties of [NiFeSe] hydrogenases could be related to differences in the H₂ and proton pathways.     

Excited-state properties of chiral [4]helicene cations

The photophysical properties of a series of helicene cations in various solvents have been investigated using stationary and time-resolved spectroscopy. These compounds fluoresce in the near infrared region with a quantum yield ranging between 2 and 20% and a lifetime between 1 and 12 ns, depending of the solvent. No clear solvent dependence could be recognized except for a decrease of fluorescence quantum yield and lifetime with increasing hydrogen-bond donating ability of the solvent. In water, the helicene cations undergo aggregation. This effect manifests itself by the presence of a slow fluorescence decay component, whose amplitude increases with dye concentration, and by a much slower decay of the polarization anisotropy in water compared to an organic solvent of similar viscosity. However, aggregation has essentially no effect on the stationary fluorescence spectrum, whereas relatively small changes can be seen in the absorption spectrum. Analysis of the dependence of aggregation on the dye concentration reveals that the aggregates are mostly dimers and that the aggregation constant is substantially larger for hetero- than homochiral dimers.