Valter Stefani

In vitro antimicrobial activity of a new series of 1,4-naphthoquinones

The antibacterial activity of a series of 1,4-naphthoquinones was demonstrated. Disk diffusion tests were carried out against several Gram-positive and Gram-negative bacteria. The compound 5-amino-8-hydroxy-1,4-naphthoquinone was the most effective, presenting inhibition zones measuring 20 mm against staphylococci, streptococci and bacilli at 50 microg/ml. Methicillin-resistant Staphylococcus aureus and several clinical isolates of this bacterium were also inhibited. Naphthazarin, 5-acetamido-8-hydroxy-1,4-naphthoquinone, and 2,3-diamino-1,4-naphthoquinone were the next most active compounds. The minimal inhibitory concentration of the active compounds was determined against S. aureus, ranging from 30 to 125 microg/ml. All compounds presented a minimal bactericidal concentration higher than 500 microg/ml, indicating that their effect was bacteriostatic. The EC50, defined as the drug concentration that produces 50% of maximal effect, was 8 microg/ml for 5-amino-8-hydroxy-1,4-naphthoquinone against S. aureus, S. intermedius, and S. epidermidis. These results indicate an effective in vitro activity of 5-amino-8-hydroxy-1,4-naphthoquinone and encourage further studies for its application in antibiotic therapy.

Synthesis and spectroscopic characterisation of 2-(2'-hydroxyphenyl)benzazole isothiocyanates as new fluorescent probes for proteins

Abstract Three new benzazole isothiocyanate (BzITC) fluorescent dyes, 2-(5′-isothiocyanate-2′-hydroxyphenyl)benzoxazole, 2-(5′-isothiocyanate-2′-hydroxyphenyl)benzimidazole and 2-(5′-isothiocyanate-2′-hydroxyphenyl)oxazole[4,5-b]pyridine, were synthesised, purified until optical purity grade and characterised by elemental analysis, 1 H NMR , IR, UV–VIS and fluorescence spectroscopy. These dyes exhibit an intense fluorescence emission with a large Stokes shift due to an excited state intramolecular proton transfer (ESIPT) mechanism. The BzITCs were also studied for labelling three proteins (bovine serum albumin (BSA), concanavalin-A (con-A) and rabbit immunoglobulin G (rabbit IgG)) and the resulting conjugates presented good and stable fluorescence. A simple assay for detection of these proteins was reported here. The method is based on the direct fluorescence detection of protein-labelled with BzITC fluorophores after polyacrylamide gel electrophoresis and present potential use as fluorescent probes for proteins.

Lipid-core nanocapsules restrained the indomethacin ethyl ester hydrolysis in the gastrointestinal lumen and wall acting as mucoadhesive reservoirs.

The aim of this work was to investigate if the indomethacin ethyl ester (IndOEt) released from lipid-core nanocapsules (NC) is converted into indomethacin (IndOH) in the intestine lumen, intestine wall or after the particles reach the blood stream. NC-IndOEt had monomodal size distribution (242 nm; PDI 0.2) and zeta potential of -11 mV. The everted rat gut sac model showed IndOEt passage of 0.16 micromol m(-2) through the serosal fluid (30 min). From 15 to 120 min, the IndOEt concentrations in the tissue increased from 6.13 to 27.47 micromol m(-2). No IndOH was formed ex vivo. A fluorescent-NC formulation was used to determine the copolymer bioadhesion (0.012 micromol m(-2)). After NC-IndOEt oral administration to rats, IndOEt and IndOH were detected in the gastrointestinal tract (contents and tissues). In the tissues, the IndOEt concentrations decreased from 459 to 5 microg g(-1) after scrapping, demonstrating the NC mucoadhesion. In plasma (peripheric and portal vein), in spleen and liver, exclusively IndOH was detected. In conclusion, after oral dosing of NC-IndOEt, IndOEt is converted into IndOH in the intestinal lumen and wall before reaching the blood stream. The complexity of a living system was not predicted by the ex vivo gut sac model.

The first silica aerogels fluorescent by excited state intramolecular proton transfer mechanism (ESIPT)

Five silyl-functionalized benzazole dyes, fluorescent by excited state intramolecular proton transfer (ESIPT) mechanism, were synthesized by reaction of amino benzazole derivatives with 3-(triethoxysilyl)propyl isocyanate. Fluorescent silica gels were prepared and monolithic aerogels (d ≈ 0.18 g cm−3) were obtained via supercritical CO2 drying of the fluorescent gel. The photophysical behaviour of the dyes and fluorescent silica aerogels was investigated by UV–vis and steady-state fluorescence spectroscopy.

New fluorescent monomers and polymers displaying an intramolecular proton‐transfer mechanism in the electronically excited state (ESIPT), 1. Synthesis of benzazolylvinylene derivatives and its copolymerization with methyl methacrylate (MMA)

Six new fluorescent monomers of the benzazole family were synthesized by the reaction of 2-(5′-amino-2′-hydroxyphenyl)benzazole derivatives and differently functionalized vinylene compounds. The radical copolymerization of the monomers with MMA results in transparent and fluorescent polymers with good optical and thermal properties. These monomers and copolymers emit fluorescence with a large Stokes shift due to the intramolecular proton-transfer mechanism in the electronically excited state (ESIPT).

Fluorescence properties of benzoxazole type dyes entrapped in a silica matrix by the sol-gel method

For the first time, the incorporation of 2,5-bis(benzoxazol-2′-yl)-4-methoxyphenol (BM) in a silica matrix by the sol–gel method has been accomplished, producing transparent and translucent monoliths with fluorescence properties. The intensity and wavelength range of the fluorescence bands F1 (primary structures) and F2 (proton-transferred forms) were extremely sensitive to the presence of solvents and to the dye concentration in the silica pores. The F2 band was observed when the silica pores contained solvent; however, after the solvent evaporation, the interactions of the dye with the silica host hindered the proton-transfer reaction, eliminating the F2 band and indicating the polar and acidic character of this host. Nevertheless, it was observed that the proton-transfer could be recovered after insertion of suitable solvents inside the matrix, like water, ethanol or DMF. Changes in the fluorescence spectra due to the use of different dye concentrations were observed when the monoliths were prepared with ethanol as solvent. During the aging process, the F2 band persists at high dye concentrations, but it disappears for low dye concentrations. The high sensitivity of the proton-transfer reaction of this dye in a particular environment, where the solvent and the host play important roles, suggests the possible application of this dye as a photoprobe for this kind of system.

First hyperpolarizability in proton-transfer benzoxazoles: computer-aided design, synthesis and study of a new model compound

Abstract With regard to second-order nonlinear optics (NLO) applications, a new class of 2-(2′-hydroxyphenyl)benzoxazoles (HBO) was designed for a combination of high first hyperpolarizability, β, with good photothermal stability, in association with a fast excited state intramolecular proton transfer (ESIPT) mechanism. Semi-empirical optimization of molecular structures and ab initio calculations of dipole moments were performed. Clear evidence was found that conditions such as conjugation efficiency and electron donor/acceptor strength cannot be evaluated separately, due to structural changes in molecular spatial distribution. Experimentally, a new fluorescent molecule of the HBO family, 2-(2′-hydroxy-4′-aminophenyl)-6-nitrobenzoxazole (BO6), was synthesized, purified and characterized, including solvent environments of distinct polarities. Hyper-Rayleigh scattering, UV–Vis absorption and emission spectroscopy, differential scanning calorimetry and thermogravimetric analysis of BO6 show a significant β (213.4±25.7×10−30 esu in acetone, at 1064 nm) and thermal stability up to 270 °C. Such results, in this first study of ESIPT dyes for second-order NLO to our best knowledge, indicate that the HBO family well deserves further attention towards promising application materials.

High pressure loading of organic dyes into a silica matrix

Abstract High density, transparent and crack-free compacts of silica gel doped with naphthazarin, quinizarin and rhodamine 6G, were produced by sol–gel synthesis with high pressure at room temperature. Compacts are very stable, being resistent to polishing and leaching by ethanol and acetone. They show optical properties, as absorption and fluorescence, similar to those at dilute solutions, indicating that the dyes are dispersed in the silica matrix as individual molecules. Heat treatment to 150°C for 1 h led to almost no degradation.

Computational study of the geometry and electronic structure of triazolephthalocyanines

Semiempirical molecular orbital methods were used to simulate the molecular structure and electronic spectra of a series of triazolephthalocyanines (Tpcs). All molecular studies are in agreement with an aromatic macrocycle where a hypothetical substitution on both triazole and meso nitrogens of the core causes a partial breakdown in the Goutermans four level model. Minor effects are predicted for the substitution in the isoindole subunit. Comparison between the experimental and theoretical UV–VIS spectra of different metal substituted triazolephthalocyanines is also made.

Genotoxicity of aminohydroxynaphthoquinones in bacteria, yeast, and Chinese hamster lung fibroblast cells.

There are few studies on the biological activity of aminohydroxy derivates of 1,4-naphthoquinone (1,4-NQ) on prokaryotic and eukaryotic cells. We determined the mutagenic activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) and 5-amino-2,8-dihydroxy-1,4-naphthoquinone (ANQ-OH) as compared to the unsubstituted 1,4-NQ in Salmonella/microsome assay. Potential mutagenic and recombinogenic effects and cytotoxicity were analyzed in haploid and diploid cultures of the yeast Saccharomyces cerevisiae. In Salmonella/microsome assay, 1,4-NQ was not mutagenic, whereas aminohydroxynaphthoquinones were weakly mutagenic in TA98 and TA102 strains. In haploid yeast in stationary growth phase (STAT), mutagenic response was only observed for the hom3 locus at the highest dose. In diploid yeast, aminohydroxynaphthoquinones did not induce any recombinogenic events, but 1,4-NQ was shown to be a recombinogenic agent. These results suggest that aminohydroxynaphthoquinones are weak mutagenic agents only in prokaryotic cells. The cytotoxicity of 1,4-NQ in yeast stationary cells was more significant in diploid cells as compared to that observed in haploid cells. However, ANQ and ANQOH were slightly cytotoxic in all treatments. Genotoxicity of these naphthoquinone compounds was also determined in V79 Chinese hamster lung fibroblast cells using standard Comet, as well as modified Comet assay with the bacterial enzymes formamidopyrimidine DNA-glycosylase (FPG) and endonuclease III (ENDOIII). Both 1,4-NQ and ANQ induced pronounced DNA damage in the standard Comet assay. The genotoxic effect of ANQ-OH was observed only at the highest dose. In presence of metabolic activation all substances showed genotoxic effects on V79 cells. Post-treatment of V79 cells with ENDOIII and FPG proteins did not have a significant effect on ANQ-OH-induced oxidative DNA damage as compared to standard alkaline Comet assay. However, all naphthoquinones were genotoxic in V79 cells in the presence of metabolic activation and post-treatment with enzymes, indicating that all compounds induced oxidative DNA damage in V79 cells. Our data suggest that aminohydroxynaphthoquinone pro-oxidant activity, together with their capability of DNA intercalation, have an important role in mutagenic and genotoxic activities.