M. Sameiro T. Gonçalves

Bifunctionalised long-wavelength fluorescent probes for biological applications

Fundacao para a Ciencia e Tecnologia (FCT, Portugal) - financial support to the NMR portuguese network (PTNMR, Bruker Avance III 400-Univ. Minho), FCT and FEDER (European Fund for Regional Development)-COMPETE-QREN-EU - financial support to the Research Centre, CQ/UM [PEst-C/QUI/UI0686/2011 (FCOMP-01-0124-FEDER-022716)].

Application of benzo[a]phenoxazinium chlorides in antimicrobial photodynamic therapy of Candida albicans biofilms

The use of Antimicrobial Photodynamic Therapy (APDT) as a new approach to treat localized Candida infections is an emerging and promising field nowadays. The aim of this study was to verify the efficacy of photodynamic therapy using two new benzo[a]phenoxazinium photosensitizers against Candida albicans biofilms: N-(5-(3-hydroxypropylamino)-10-methyl-9H-benzo[a]phenoxazin-9-ylidene)ethanaminium chloride (FSc) and N-(5-(11-hydroxyundecylamino)-10-methyl-9H-benzo[a]phenoxazin-9-ylidene)ethanaminium chloride (FSd). The photodynamic activity of dyes against C. albicans biofilms was evaluated by incubating biofilms with dyes in the range of 100-300 μM for 3 or 18 h followed by illumination at 12 or 36 J cm(-2), using a xenon arc lamp (600 ± 2 nm). A total photoinactivation of C. albicans biofilm cells was achieved using 300 μM of FSc with 18 h of incubation, followed by illumination at 36 J cm(-2). Contrarily, FSd had insignificant effect on biofilms inactivation by APDT. The higher uptake of FSc than FSd dye by biofilms during the dark incubation may explain the greater photodynamic effectiveness achieved with FSc. The results obtained stresses out the FSc-mediated APDT potential use to treat C. albicans infections.

Ultrasound promoted synthesis of Nile Blue derivatives.

Ultrasound irradiation was used for the first time towards the synthesis of new Nile Blue related benzo[a]phenoxazinium chlorides possessing isopentylamino, (2-cyclohexylethyl)amino and phenethylamino groups at 5-position of the heterocyclic system. The efficacy of sonochemistry was investigated with some of our earlier reported synthesis of benzo[a]phenoxazinium chlorides. This newer protocol proved competent in terms of reaction times and enhanced yields. Photophysical studies carried out in ethanol, water and simulated physiological conditions, revealed that emission maxima occurred in the range 644-656 nm, with high fluorescent quantum yields. Other attractive feature exhibited by these materials includes good thermal stability. These properties might be useful in the development of fluorescent probes for biotechnology.

Synthesis of naphtho[2,3-a]phenoxazinium chlorides: Structure–activity relationships of these heterocycles and benzo[a]phenoxazinium chlorides as new antimicrobials

Synthesised functionalised naphtho[2,3-a]phenoxazinium chlorides revealed great fluorescence with maximum emission wavelengths between 630 and 676 nm, in ethanol and water at physiological pH. Naphtho[2,3-a]phenoxazines, as well as a series of benzo[a]phenoxazines, were evaluated against Saccharomyces cerevisiae, in a broth microdilution assay. This family of compounds exhibited antifungal activity depending both on the substituents of the heterocycle nucleus as well as on its size. The best activities were obtained for four-ring systems, and particularly for 5,9-diaminobenzo[a]phenoxazines with R=Me, R(1)=H and R(2)=Et. As for R(3) substitution, the greatest efficiency was obtained for R(3)=(CH(2))(3)Cl, with a MIC of 3.75 microM. The linkage of different amino acids to the functional group of the 5-amino position of diaminobenzo[a]phenoxazinium moiety resulted in compounds with diverse antimicrobial efficiencies, depending on the polar character of the amino acid, on its linkage position and on the size of the alkyl chain linker.

Synthesis of novel derivatives of 4-amino-3,5-dicyanopyrazole

Diazotisation of substituted arylamines followed by reaction with malononitrile gave substituted arylazomalononitriles. Cyclisation of these intermediates with chloroacetonitrile and triethylamine, as base, gave the corresponding new aminodicyanopyrazoles, in 22–80% yields.

Synthesis and light triggered release of catecholamines from pyrenylmethyl carbamate cages

A series of catecholamines (dopamine, norepinephrine, tyramine and octopamine) and their corresponding amino acid precursors in the biosynthetic pathway, L-phenylalanine, L-tyrosine and L-3,4-dihydroxyphenylalanine (DOPA), were reacted with 1-hydroxymethylpyrene in the presence of N,N′-carbonyldiimidazole (CDI). Photolysis studies on the corresponding carbamate conjugates were carried out under irradiation at different wavelengths (250, 300, and 350 nm), followed by HPLC/UV and 1H NMR, with the aim of evaluating the applicability of the N-pyrenylmethoxycarbonyl group as a photolabile protecting group for the amino function of neuroactive amines and amino acids. It was found that the carbamate bond between the catecholamine and the pyrenylmethoxycarbonyl unit cleaved readily with the results obtained by irradiation at 350 nm being promising for practical applications.

Aminobenzocoumarinylmethyl esters as photoactive precursors for the release of butyric acid

The evaluation of the photorelease of a carboxylic acid drug, using butyric acid as a representative model, was carried out by using 7-amino-4-chloromethyl-2-oxo-2H-naphtho[1,2-b]pyran, an aminobenzocoumarin, and its mono- and di-methylated or ethylated derivatives. This study was intended to improve the release of butyric acid from benzocoumarins by the addition of an amino group to the heterocycle by applying the knowledge of second-generation coumarinylmethyl-based photoremovable protecting groups. Photolysis studies were performed on the resultant ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80 : 20 solutions as solvent. The data obtained showed that these new fluorescent aminobenzocoumarins are superior to all the previously tested benzocoumarins with the same or different ring fusions. The photophysics of the compounds was characterised by both steady state and time-resolved methods.

Light triggering of 5-aminolevulinic acid from fused coumarin ester cages

The evaluation of the photorelease of 5-aminolevulinic acid (5-ALA), a small molecule which has considerable interest in the area of medicine as a photosensitizer prodrug in PDT and cosmetic treatments, and in agriculture as a herbicide/insecticide, was carried out by using a series of fused coumarin derivatives with different substituents and ring fusions in the preparation of 5-ALA photosensitive ester cages, in order to tune the photophysical and photolytic properties of the resulting conjugates. This study was intended to assess the viability of the photorelease of 5-ALA from lipophilic conjugates since it has hydrophilic character, does not penetrate efficiently through the skin or cell membranes and appropriate derivatisation can enhance its lipophilicity and its application scope in biological environment. Photolytic studies were performed on the ester cages by irradiation in a photochemical reactor at 254, 300, 350 and 419 nm, using methanol/HEPES buffer 80 : 20 solutions as solvent. The data obtained confirmed the suitability of the tested photosensitive moieties for the release of 5-aminolevulinic acid at the various wavelengths of irradiation. As well as the photolysis, the photophysics of the compounds was characterised by both steady state and time-resolved methods which uncovered the presence of different fluorescing species. Additionally, an on–off experiment was carried out by using two excitation sources (cleave and probe) to enable fluorescence to follow the kinetics of the process and to ascertain optical control over the bond scission.

Fluorescent probes based on side-chain chlorinated benzo[a]phenoxazinium chlorides: Studies of interaction with DNA.

The interaction of DNA with six water soluble benzo[a]phenoxazinium chlorides mono- or di-substituted with 3-chloropropyl groups at the O and N of 2- and 9-positions, along with methyl, hydroxyl and amine terminal groups at 5-positions, was investigated by photophysical techniques. The results indicated that almost all compounds intercalated in DNA base pairs at phosphate to dye ratio higher than 5. At lower values of this ratio, electrostatic binding mode with DNA was observed. Groove binding was detected mainly for the benzo[a]phenoxazinium dye with NH2·HBr terminal. The set of six benzo[a]phenoxazinium chlorides proved successful to label the migrating DNA in agarose gel electrophoresis assays. These finding proves the ability of these benzo[a]phenoxazinium dyes to strongly interact with DNA.

Antiproliferative Activity of 2,3,6,7-Tetrahydro-1H-benzo[a]quinolizino[1,9-hi]phenoxazin-14(5H)-iminium Chloride Derivatives

PTNMR, Bruker Avance III 400-Univ. Minho, PEst-C/QUI/UI0686/2013 (FCOMP-01-0124-FEDER-037302), PEst-OE/BIA/UI4050/2014, SFRH/BPD/62881/2009