Síntese química e avaliação das propriedades antibacterianas e antiparasitárias de análogos de peptídeos antimicrobianos de anuros

Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2015.

Phenolic compounds and antioxidant activity of hydroalcoholic extracts of wild and cultivated murtilla (Ugni molinae Turcz.)

Over the last decade a considerable increase in the number of studies addressing the use of antioxidants from natural sources has led to the identification and understanding of the potential mechanisms of biologically active components. This results from the fact that they can be used to replace synthetic antioxidants commonly used in food. Murtilla (Ugni molinae Turcz) is a native berry grown in Chile, and in the present study, the phenolic composition and antioxidant activity of its fruits were studied. Hydroalcoholic extracts of dehydrated fruits from two genotypes of murtilla (Ugni molinae Turcz.) were produced. Extracts of wild murtilla and 14-4 genotype fruits had 19.35 and 40.28mg GAE/g for Total Phenolic Compounds, 76.48, and 134.35μmol TEAC/g for DPPH, and 157.04 and 293.99 μmol TEAC/g for ABTS, respectively. Components such as quercetin, epicatechin, and gallic, benzoic and hydrocaffeic acids were identified by CG/MS analysis. All of them showed antioxidant activity. Therefore, it is possible to say that the hydroalcoholic extracts of murtilla have antioxidant potential to be used in lipidic food.

Synthesis of new bioactive β-lactam compounds

New biologically active β-lactams were designed and synthesized, developing novel antibiotics and enzymatic inhibitors directed toward specific targets. Within a work directed to the synthesis of mimetics for RGD (Arg-Gly-Asp) sequence able to interact with αvβ3 and α5β1-type integrins, new activators were developed and their Structure-Activity Relationships (SAR) analysis deepened, enhancing their activity range towards the α4β1 isoform. Moreover, to synthesize novel compounds active both against bacterial infections and pulmonary conditions of cystic fibrosis patients, new β-lactam candidates were studied. Among the abundant library of β-lactams prepared, mainly with antioxidant and antibacterial double activities, it was identified a single lead to be pharmacologically tested in vivo. Its synthesis was optimized up to the gram-scale, and pretreatment method and HPLC-MS/MS analytical protocol for sub-nanomolar quantifications were developed. Furthermore, replacement of acetoxy group in 4-acetoxy-azetidinone derivatives was studied with different nucleophiles and in aqueous media. A phosphate group was introduced and the reactivity exploited using different hydroxyapatites, obtaining biomaterials with multiple biological activities. Following the same kind of reactivity, a small series of molecules with a β-lactam and retinoic hybrid structure was synthesized as epigenetic regulators. Interacting with HDACs, two compounds were respectively identified as an inhibitor of cell proliferation and a differentiating agent on steam cells. Additionally, in collaboration with Professor L. De Cola at ISIS, University of Strasbourg, some new photochemically active β-lactam Pt (II) complexes were designed and synthesized to be used as bioprobes or theranostics. Finally, it was set up and optimized the preparation of new chiral proline-derived α-aminonitriles through an enantioselective Strecker reaction, and it was developed a chemo-enzymatic oxidative method for converting alcohols to aldehydes or acid in a selective manner, and amines to relative aldehydes, amides or imines. Moreover, enzymes and other green chemistry methodologies were used to prepare Active Pharmaceutical Ingredients (APIs).

Development of tandem C-H borylation/functionalization procedures for late stage functionalization of compounds

The development of procedures for the iridium catalyzed C-H borylation of 1-aryl pyrazolopyrimidines and 1-aryl indazoles is reported. Investigation on the activity of the catalyst revealed the combination of an iridium (I) precursor and tetramethylphenantroline as the best catalytic system. Moreover, the procedures are regioselective resulting in the selective borylation of different C-H bonds within the substrates. The application of C-H borylation to late stage functionalization is demonstrated: a biologically active compound in AstraZeneca's project underwent tandem borylation/oxidation reaction, in order to obtain a functionalized product containing an OH group.

A review of the potential role of tumour-promoting compounds produced by Lyngbya majuscula in marine turtle fibropapillomatosis

Harmful algal blooms (HABs) have increased in abundance and severity in recent decades. Whereas the implications for human impacts and intoxication resulting from blooms have been extensively studied, the ecological implications of these microalgae are less well understood. Many HAB species produce biologically active, secondary metabolites and the fate of these toxins through the foodweb is generally not well understood unless it culminates in extensive fish mortalities or human poisonings. This review focusses on one HAB species, the cyanobacterium Lyngbya majuscula, and presents a hypothetical role for its involvement in fibro-papillornatosis (FP), a neoplastic disease of marine turtles. FP is expressed as benign tumours that grow both internally and externally on marine turtles, preventing vision, movement and organ function. The aetiology of FP is currently not conclusively understood, but virus material has been associated with tumours and previous studies have suggested a role for naturally produced tumour promoters. In this review, we present a hypothesis regarding the involvement of L. majuscula in FP, either through direct intoxication and action of tumour-promoting compounds or indirectly by causing seagrass loss and compromised immune function, thus leaving the turtles more susceptible to disease.

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Investigation of the antiproliferative properties of tumour promoting phorbol esters and related compounds

Tumour promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) exert a multitude of biological effects on many cellular systems, many of which are believed to be mediated via the activation of the enzyme protein kinase C (PKC). TPA and other biologically active phorbol esters inhibited the proliferation of the A549 human lung carcinoma cell line. However, after 5-6 days culture in the continued presence of the phorbol ester cells began to proliferate at a rate similar to that of untreated cells. Resistance to TPA was lost following subculturing, although subculture in the presence of 10 nM TPA for more than 9 weeks resulted in a more resistant phenotype. The selection of a TPA-resistant subpopulation was not responsible for the observed resistance. The antiproliferative properties of other PKC activators were investigated. Mezerein induced the same antiproliferative effects as TPA but synthetic diacylglycerols (DAGs), the presumed physiological ligands of PKC, exerted only a non-specific cytotoxic influence on growth. Bryostatins 1 and 2 were able to induce transient growth arrest of A549 cells in a manner similar to phorbol esters at nanomolar concentrations, but at higher concentrations blocked both their own antiproliferative action and also that of phorbol esters and mezerein. Fourteen compounds synthesized to mimic features of the phorbol ester pharmacophore and/or DAGs did not mimic the antiproliferative properties of TPA in A549 cells and exerted only a DAG-like non-specific cytotoxicity at high concentrations. The subcellular distribution and activity of PKC was determined following partial purification by non-denaturing polyacrylamide gel electrophoresis. Treatment with TPA, mezerein or bryostatins resulted in a concentration-dependent shift of PKC activity from the cytosol to cellular membranes within 30 min. Significant translocation was not observed on treatment with DAGs. Chronic exposure of cells to TPA caused a time- and concentration dependent down-regulation of functional PKC activity. A complete loss of PKC activity was also observed on treatment with growth-inhibitory concentrations of bryostatins. No PKC activity was detected in cells resistant to the growth-inhibitory influence of TPA. Measurement of intracellular Ca2+ concentrations using A549 cells cultured on Cytodex 1 microcarrier beads revealed that TPA, mezerein and the bryostatins induced a similar rapid rise in intracellular Ca2+ levels.

Antileishmanial Activity of the Hydroalcoholic Extract of Miconia langsdorffii, Isolated Compounds, and Semi-Synthetic Derivatives

The in vitro activity of the crude hydroalcoholic extract of the aerial parts of Miconia langsdorffii Cogn. was evaluated against the promastigote forms of L. amazonensis, the causative agent of cutaneous leishmaniasis in humans. The bioassay-guided fractionation of this extract led to identification of the triterpenes ursolic acid and oleanolic acid as the major compounds in the fraction that displayed the highest activity. Several ursolic acid semi-synthetic derivatives were prepared, to find out whether more active compounds could be obtained. Among these ursolic acid-derived substances, the C-28 methyl ester derivative exhibited the best antileishmanial activity.

Isolation and analysis of bioactive isoflavonoids and chalcone from a new type of Brazilian propolis

Activity-directed fractionation and purification processes were employed to identify isoflavonoids with antioxidant and antimicrobial activities from Brazilian red propolis. Crude propolis was extracted with ethanol (80%. v/v) and fractioned by liquid-liquid extraction technique using hexane and chloroform. Since chloroform fraction showed strong antioxidant and antimicrobial activities it was purified and isolated using various chromatographic techniques. Comparing our spectral data (UV, NMR, and mass spectrometry) with values found in the literature, we identified two bioactive isoflavonoids (vestitol and neovestitol), together with one chalcone (isoliquiritigenin). Vestitol presented higher antioxidant activity against beta-carotene consumption than neovestitol. The antimicrobial activity of these three compounds against Staphylococcus aureus, Streptococcus mutans, and Actinomyces naeslundii was evaluated and we concluded that isoliquiritigenin was the most active one with lower MIC, ranging from 15.6 to 62.5 mu g/mL. Our results showed that Brazilian red propolis has biologically active isoflavonoids that may be used as a mild antioxidant and antimicrobial for food preservation. (C) 2010 Elsevier B.V. All rights reserved.

Multilabeled classification approach to find a plaint source for terpenoids

Recently, we have built a classification model that is capable of assigning a given sesquiterpene lactone (STL) into exactly one tribe of the plant family Asteraceae from which the STL has been isolated. Although many plant species are able to biosynthesize a set of peculiar compounds, the occurrence of the same secondary metabolites in more than one tribe of Asteraceae is frequent. Building on our previous work, in this paper, we explore the possibility of assigning an STL to more than one tribe (class) simultaneously. When an object may belong to more than one class simultaneously, it is called multilabeled. In this work, we present a general overview of the techniques available to examine multilabeled data. The problem of evaluating the performance of a multilabeled classifier is discussed. Two particular multilabeled classification methods-cross-training with support vector machines (ct-SVM) and multilabeled k-nearest neighbors (M-L-kNN)were applied to the classification of the STLs into seven tribes from the plant family Asteraceae. The results are compared to a single-label classification and are analyzed from a chemotaxonomic point of view. The multilabeled approach allowed us to (1) model the reality as closely as possible, (2) improve our understanding of the relationship between the secondary metabolite profiles of different Asteraceae tribes, and (3) significantly decrease the number of plant sources to be considered for finding a certain STL. The presented classification models are useful for the targeted collection of plants with the objective of finding plant sources of natural compounds that are biologically active or possess other specific properties of interest.

Cyanogenic polymorphism in Eucalyptus polyanthemos Schauer subsp vestita L. Johnson and K. Hill (Myrtaceae)

Plant cyanogenesis, the release of cyanide from endogenous cyanide-containing compounds, is an effective herbivore deterrent. This paper characterises cyanogenesis in the Australian tree Eucalyptus polyanthemos Schauer subsp. vestita L. Johnson and K. Hill for the first time. The cyanogenic glucoside prunasin ((R)-mandelonitrile beta-D-glucoside) was determined to be the only cyanogenic compound in E. polyanthemos foliage. Two natural populations of E. polyanthernos showed quantitative variation in foliar prumasin concentration, varying from zero (i.e. acyanogenic) to 2.07 mg CN g(-1) dry weight in one population and from 0.17 to 1.98 mg CN g(-1) dry weight in the other. No significant difference was detected between the populations with respect to the mean prunasin concentration or the degree of variation in foliar prunasin, despite significant differences in foliar nitrogen. Variation between individuals was also observed with respect to the capacity of foliage to catabolise prunasin to form cyanide. Moreover, variation in this capacity generally correlated with the amount of prunasin in the tissue, suggesting genetic linkage between prunasin and beta-glucosidase. (C) 2002 Elsevier Science Ltd. All rights reserved.

Purification and structural characterization of compounds isolated from Cystoseira abies-marina

6th Spanish-Portuguese-Japanese Organic Chemistry Symposium, Lisboa, de 18 a 20 de Julho de 2012 (Poster Communication).

Valorização de subprodutos da vinha e do vinho: impacto do tempo e métodos de preservação nas suas características

Os polifenóis constituem um grupo biologicamente relevante de compostos naturais, que têm gerado um crescente interesse por parte dos consumidores e das indústrias alimentar, farmacêutica e de cosméticos. Os compostos fenólicos exibem uma vasta gama de propriedades fisiológicas, tais como anti-alérgica, anti-aterogénica, anti-inflamatória, anti-microbiana, antioxidante, anti-trombótica, possuindo efeitos cardio-protetores e vasodilatadores. Por conseguinte, o interesse na obtenção deste compostos ativos de uma forma fácil, barata e rápida tem vindo a aumentar. Uma das fontes de polifenóis são os subprodutos agrícolas, tais como os produtos derivados do vinho e/ou processamento de uvas. As folhas, os caules e os resíduos obtidos durante o processo de produção do vinho têm sido subutilizados, apesar de representarem uma boa fonte de compostos antioxidantes e bioativos. Portugal é um dos mais importantes países produtores de vinho, onde todos os anos são geradas várias toneladas de subprodutos associados a esta indústria. A extração, caracterização, quantificação e avaliação da atividade antioxidante de compostos bioativos extraídos destes subprodutos são, pois, tarefas importantes a fim de avaliar o potencial de utilização destes produtos como novas fontes de compostos antioxidantes. O objetivo deste trabalho foi quantificar e avaliar os teores de polifenóis de extratos preparados a partir de folhas e caules de videira, e bagaço, engaço e grainha de uva. Os parâmetros experimentais da extração foram otimizados, incluindo a proporção de solvente de extração (água/etanol), a temperatura e o tempo de extração. A quantificação dos polifenóis (como fenóis totais e flavonoides) foi efetuada por espetrofotometria de UV/Vis. Verificou-se que a melhor razão de solventes extratores foi de 50/50 (v/v) em água/etanol para todos os subprodutos estudados. No caso dos subprodutos folhas e caules, as condições ideais de extração foram obtidas quando se usou uma temperatura de 40ºC e um tempo de extração de 30 min. Para o bagaço, a temperatura ideal foi de 55ºC durante 5 min. Para o engaço e grainha a utilização de uma temperatura de 40ºC durante 5 min e 30 min, respetivamente, revelou-se o ideal para se obter os compostos. Com este trabalho conclui-se que os subprodutos da vinha e do vinho efetivamente possuem compostos de elevado valor e com capacidade antioxidante.

The removal of xenobiotic compounds from wastewater through the use of biological processes and advanced oxidation technologies

Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Química e Bioquímica pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Photochemical synthesis and functional transformations of bicyclic vinyl aziridines

Aziridines, a class of organic compounds containing a three membered heterocycle with a nitrogen atom, are extremely valuable molecules in organic and medicinal chemistry. They are frequently used as versatile precursors in the synthesis of natural products, and many biologically active molecules possess the aziridine moiety. The reactivity of aziridines has been studied, for example, in ring-opening reactions with thiols. However, not much interest seems to be given to reactions of aziridines in aqueous media, despite the numberless advantages of using water as solvent in organic chemistry. The nucleophilic ring-opening reaction of aziridines in aqueous media was here explored. Following the Kaplan aziridine synthetic methodology, in which pyridinium salts undergo a photochemical transformation to give bicyclic vinyl aziridines, new aziridines were synthetized. Their nucleophilic ring-opening reaction in water under physiological conditions was investigated and a range of sulphur, nitrogen, carbon and oxygen nucleophiles tested. Thiols, anilines and azide proved to be good nucleophiles to react with the aziridines, giving the ring-opening product in moderate to good yields. The best results were obtained with thiols, more specifically with cysteine-derived nucleophiles. Preliminary results show that these bicyclic vinyl aziridines can modify calcitonin, a peptide containing two cysteine amino acids residues, grating them the potential to be used in bioconjugation as ligands to cysteine-containing proteins, or even as enzyme inhibitors of, for example, cysteine proteases. Additionally, exploratory investigations suggest that the separation of both enantiomers of the bicyclic vinyl aziridine can be performed by taking advantage of an enzymatic methodology for the resolution of racemic secondary alcohols. Both enantiomers would be highly valuable as precursors in the synthesis of enantiomerically pure molecules, as no other method is currently reported for their separation.