7,7-dimetoxi-1,4,5,6-tetraclorobiciclo[2.2.1]heptan-5-en-2-endo-ol : um versátil reagente na síntese de 1,3-aminoálcoois e de ciclopentanóides quirais altamente funcionalizados

Este trabalho relata a síntese de uma série de novos ligantes quirais (+) e (-)-syn-1,3-aminoálcoois derivados do norbornano. Através da reação de transesterificação enzimática com a lípase da Candida rugosa em acetato de vinila do álcool racêmico 7,7-dimetoxi-1,4,5,6-tetraclorobiciclo[2.2.1]heptan-5-en-2-ol, (±)-3, foram obtidos os álcoois quirais (+)-3 e (-)-3 (Esquema 1). Através da reação de redução e descloração destes álcoois com Na0/NH3/etanol foram obtidos os respectivos álcoois (+)-4 e (-)-4 (Esquema 2). Os álcoois quirais (+)-4 e (-)-4 foram utilizados como produtos de partida para a síntese dos 1,3-aminoálcoois quirais (+)-9 e (-)-9 em 5 etapas. Deste modo, a partir destes aminoálcoois (9), foi possível sintetizar 12 novos compostos (Esquema 2), todos inétidos na literatura. Os 1,3-aminoálcoois 10, 11, 13, 14 e 15 foram empregados como catalisadores quirais na adição enantiosseletiva de ZnEt2 ao benzaldeído. Excelentes rendimentos e excessos enantioméricos (até 91%) foram obtidos. A relação entre a configuração absoluta do 1-fenilpropanol com a configuração do carbono ligado ao grupo hidroxila dos ligantes foi estudada e, de acordo, com a enantiosseletividade observada foi sugerido um mecanismo para a reação Os produtos com esqueleto ciclopentila são importantes compostos com potencial atividade biológica, fazendo parte da estrutura de prostaglandinas, agentes antitumorais e inibidores da glicosidase. Portanto, nós decidimos usar o acetato clorado quiral 2 para preparar ciclopentanóides quirais altamente funcionalizados. Para isso, o acetato clorado quiral 2 foi submetido à oxidação usando uma quantidade catalítica de RuCl3 anidro na presença de NaIO4 obtendo-se a dicetona 16 (Esquema 3). A dicetona 16 foi clivada com H2O2 em meio alcalino fornecendo os diácidos 17a e 17b, que foram esterificados in situ com excesso de CH2N2 para fornecer uma mistura do hidroxi e acetoxi diéster 18 e 19, respectivamente. A redução da mistura 18 e 19 ou da mistura 17a e 17b com BH3.THF fornece a lactona 20 com excelentes rendimentos.

Immobilization of lipases and assay in continuous fixed bed reactor

Lipases are versatile enzymes regarding the range of reactions they catalyse and substrates on which they act. They are as well important as catalyst in organic synthesis. Their immobilization on appropriate supports confer them greater stability besides the possibility of operating in continuous reactors. In order to explore these abilities, the reactions involving hydrolysis of p-nitrophenyl acetate (PNPA) and transesterification of PNPA with n-butanol were chosen. Lipases from two different sources were assayed, namely: microbial (Candida rugosa, CRL, Sigma Type VII) and pancreatic (PPL, Sigma, Type 11). Two immobilization methods were also used, namely: 1) adsorption, using as support the following silica derivatives (150-300μm e 450μ): phenyl, epoxy, amino and without derivation, and 2) covalent binding, using glutaraldehyde as binding agent and silica amino as support. This later method led to better results. Hydrolytic activity was 6.1 U/gsupport for CRL and 0.97U/gsupport for PPL, and of transesterification, 2,8U/gsupport for CRL and 1,9U/gsupport for PPL. Stability of the immobilized enzyme as a function of temperature was evaluated for CRL at 40°C and 50°C and for PPL at 32°C and 40°C. The assays were initially carried out batchwise, both for soluble and immobilized enzymes, aiming to the obtention of parameters for the continues reactor. Lipases immobilized by covalent binding were used in the assays of operacional stability in continuos reactors. For PPL in aqueous medium, at 32°C, and CRL in organic medium at 40°C, both operating continuously, no significant loss of activity was detected along the analysis period of 17 days. In the case of CRL in aqueous medium at 40°C there was a loss of activity around 40% after 18 days. For PPL in organic medium at 40°C the loss was 33% after 20 days. Compairing both sources with each other, very different results were obtained. Higher activitiy was found for CRL, both for hydrolysis and for transesterification reactions, with higher stability in organic medium. PPL showed lower activity as well as higher stability in aqueous medium. The immobilization method by covalent binding showed to be the most appropriate. Immobilized lipases are therefore relatively stable both in aqueous and organic medium.

Oral microbial colonization in children with sickle cell anaemia under long-term prophylaxis with penicillin

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Influence of the use of Aliquat 336 in the immobilization procedure in sol-gel of lipase from Bacillus sp ITP-001

Aliquat 336, a liquid hydrophobic material, was used at different concentrations (0.5-3.0%, w/v) as an additive in the preparation of encapsulated lipase from Bacillus sp. ITP-001 on sol-gel silica matrices using tetraethoxysilane (TEOS) as the precursor. The resulting hydrophobic matrices and immobilized lipases were characterized with regard to specific surface area (BET method), adsorption-desorption isotherms, pore volume (Vp) and size (dp) by nitrogen adsorption (BJH method) and scanning electron microscopy (SEM). The catalytic activities and the corresponding coupling yields were assayed in the hydrolysis of olive oil. In comparison with pure silica matrices, the immobilization process in the presence of Aliquat 336 decreased the values for specific surface area and increased the values for pore specific volume (Vp) and mean pore diameter (dp). This behavior may be related to the partial adsorption of the enzyme on the external surface of the hydrophobic matrix as indicated by scanning electron microscopy. Aliquat 336 concentrations in the range from 0.5 to 1.5% (w/v) provided immobilized derivatives with higher coupling yields and better substrate affinity. The highest coupling yield (Y-A = 71%) was obtained for the immobilized enzyme prepared in the presence of 1.5% Aliquat which gave the following morphological properties: specific surface area = 183 m(2)/g, pore specific volume (Vp) = 0.36 cc/g and mean pore diameter (dp)= 91 angstrom. (c) 2012 Elsevier B.V. All rights reserved.

Lipase-catalyzed kinetic resolution of beta-borylated carboxylic esters

The kinetic resolution of chiral beta-borylated carboxylic esters via lipase-catalyzed hydrolysis and transesterification reactions was studied. The enantioselective hydrolysis catalyzed by CAL-B furnished the beta-borylated carboxylic acid with reasonable enantiomeric excess (62% ee), while both methyl and ethyl beta-borylated carboxylic esters were recovered with excellent ee (>99%). Meanwhile, the transesterification reaction of beta-borylated carboxylic esters and several alcohols, catalyzed by CAL-B, only indicated a high selectivity when ethanol and methyl-(beta-pinacolylboronate)-butanoate were used as substrates, which gave ethyl-(beta-pinacolylboronate)-butanoate with >99% ee. (C) 2012 Elsevier Ltd. All rights reserved.

Evaluation of immobilized lipases on poly-hydroxybutyrate beads to catalyze biodiesel synthesis

Five microbial lipase preparations from several sources were immobilized by hydrophobic adsorption on small or large poly-hydroxybutyrate (PHB) beads and the effect of the support particle size on the biocatalyst activity was assessed in the hydrolysis of olive oil, esterification of butyric acid with butanol and transesterification of babassu oil (Orbignya sp.) with ethanol. The catalytic activity of the immobilized lipases in both olive oil hydrolysis and biodiesel synthesis was influenced by the particle size of PHB and lipase source. In the esterification reaction such influence was not observed. Geobacillus thermocatenulatus lipase (BTL2) was considered to be inadequate to catalyze biodiesel synthesis, but displayed high esterification activity. Butyl butyrate synthesis catalyzed by BTL2 immobilized on small PHB beads gave the highest yield (approximate to 90 mmol L-1). In biodiesel synthesis, the catalytic activity of the immobilized lipases was significantly increased in comparison to the free lipases. Full conversion of babassu oil into ethyl esters was achieved at 72 h in the presence of Pseudozyma antarctica type B (CALB), Thermomyces lanuginosus lipase (Lipex (R) 100L) immobilized on either small or large PHB beads and Pseudomonas fluorescens (PFL) immobilized on large PHB beads. The latter preparation presented the highest productivity (40.9 mg of ethyl esters mg(-1) immobilized protein h(-1)). (C) 2012 Elsevier B.V. All rights reserved.

Atividade antimicrobiana e antioxidante de Licania rigida e Turnera ulmifolia

Introduction: Licania rigida Benth and Turnera ulmifolia Linn. var. elegans are species of semi-arid regional plants used in the treatment of various diseases. Objectives: The purpose of this study was chemically characterize the extracts and fractions and investigate the antimicrobial and antioxidant potential. Methods: For chemical analysis, were performed spectrophotometric quantification of the total phenolic and characterization of the extracts by chromatographic analysis. Evaluation of antioxidant activity was done by determining the radical scavenging capacity DPPH •. Antimicrobial activity was evaluated by agar diffusion, broth microdilutionand time-kill assays. Results: The extracts and fractions L. rigid and T. ulmifolia showed a high phenolic content, the presence of flavonoids, which were determined as chemical markers. It was observed that the extracts of both species performed as sequestering agents in the trial of antioxidant activity in vitro. The L. rigida extract was the only active front strains of S. aureus 33591 (methicillin-resistant), S. aureus 29213, S. epidermidis 12228, and also against the yeast, Candida albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Candida rugosa, Candida krusei eTrichosporon asahii. Conclusions: Based on these results it is possibly affirm the antioxidant and antimicrobial activity of L. rigida and attributed the presence of polyphenolic flavonoid like responsible.

Optimization of lipase production from yeasts strains isolated from olive mil wastewater

Dissertação submetida à Universidade de Lisboa, Faculdade de Ciências para a obtenção do Grau de Mestre em Microbiologia Aplicada.

Enantioselective transesterification catalysis by Candida antarctica lipase immobilized on superparamagnetic nanoparticles

Lipase B from Candida antarctica can be directly immobilized onto functionalized superparamagnetic nanoparticles, preserving its enzymatic activity in the enantioselective transesterification of secondary alcohols, with excellent results in terms of enantiomeric discrimination. The immobilized enzyme can be easily recovered with a magnet, allowing its reuse with negligible loss of activity. (C) 2009 Elsevier Ltd. All rights reserved

Genetic relatedness of commensal strains of Candida albicans carried in the oral cavity of patients' dental prosthesis users in Brazil

The aim of this study is to describe the degree of yeast-colonization in diabetic and hemodialysed-users of dental prostheses. Individuals (306) were examined using an oral rinse technique in order to evaluate the incidence of yeast-carriage, and genotype of C. albicans. Yeasts were isolated from 68.4% (91/133) individual's dental prostheses users. Dental prostheses were found to be a significant factor for the yeast colonization (P < 0.05). Overall, the intensity of carriage was higher in diabetic patients as compared with health and hemodialysed individuals (P < 0.05). The isolation rates were: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). Ready-To-Go RAPD Analysis Beads were used and primer OPJ 6 distinguished the C. albicans isolates found in prostheses users. All the isolates were grouped into 11 RAPD profiles in four main clusters and, the average S (AB) for the entire collection of 47 C. albicans isolates were 0.779 +/- 0.178. Over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses, independently of the host's clinical condition, probably provides the necessary conditions for these strains to gain a growth-specific advantage over others.

Isolation of Candida spp. from mastitic bovine milk in Brazil

The purpose of this study was to isolate yeast (Candida) from the quarter milk of cow udders from 37 dairy farms in Brazil and to identify the different species involved in mastitis. The samples were collected between October 2002 and February 2003. Two-hundred-and-sixty milk samples from cows with clinical and subclinical mastitis were examined. Milk samples were plated onto Blood agar, Mac Conkey agar and Sabouraud dextrose agar. Forty-five (17.3%) samples were positive for the genus Candida. The Candida species isolated were C. krusei (44.5%), C. rugosa (24.5%), C. albicans (8.9%), C. guilliermondii (8.9%), and others (13.2%). We also isolated Escherichia coli (26.5%), coagulase-positive Staphylococcus (25.0%), Streptococcus spp. (8.1%), Enterobacter spp. (8.1%), and other fungi (8.1%), among others.

Efetividade de vernizes fotopolimerizáveis experimentais na redução da hidrofobicidade e adesão de Candida albicans em uma resina para base de prótese

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Effect Of Daily Use Of An Enzymatic Denture Cleanser On Candida Albicans Biofilms Formed On Polyamide And Poly(methyl Methacrylate) Resins: An In Vitro Study.

Candida biofilms on denture surfaces are substantially reduced after a single immersion in denture cleanser. However, whether this effect is maintained when dentures are immersed in cleanser daily is unclear. The purpose of this study was to evaluate the effect of the daily use of enzymatic cleanser on Candida albicans biofilms on denture base materials. The surfaces of polyamide and poly(methyl methacrylate) resin specimens (n=54) were standardized and divided into 12 groups (n=9 per group), according to study factors (material type, treatment type, and periods of treatment). Candida albicans biofilms were allowed to form over 72 hours, after which the specimens were treated with enzymatic cleanser once daily for 1, 4, or 7 days. Thereafter, residual biofilm was ultrasonically removed and analyzed for viable cells (colony forming units/mm(2)) and enzymatic activity (phospholipase, aspartyl-protease, and hemolysin). Factors that interfered with the response variables were analyzed by 3-way ANOVA with the Holm-Sidak multiple comparison method (α=.05). Polyamide resin presented more viable cells of Candida albicans (P<.001) for both the evaluated treatment types and periods. Although enzymatic cleansing significantly (P<.001) reduced viable cells, daily use did not maintain this reduction (P<.001). Phospholipase activity significantly increased with time (P<.001) for both materials and treatments. However, poly(methyl methacrylate) based resin (P<.001) and enzymatic cleansing treatment (P<.001) contributed to lower phospholipase activity. Aspartyl-protease and hemolysin activities were not influenced by study factors (P>.05). Although daily use of an enzymatic cleanser reduced the number of viable cells and phospholipase activity, this treatment was not effective against residual biofilm over time.

Bay 41-2272 Activates Host Defence Against Local And Disseminated Candida Albicans Infections.

In our previous study, we have found that 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]-pyrimidin-4-ylamine (BAY 41-2272), a guanylate cyclase agonist, activates human monocytes and the THP-1 cell line to produce the superoxide anion, increasing in vitro microbicidal activity, suggesting that this drug can be used to modulate immune functioning in primary immunodeficiency patients. In the present work, we investigated the potential of the in vivo administration of BAY 41-2272 for the treatment of Candida albicans and Staphylococcus aureus infections introduced via intraperitoneal and subcutaneous inoculation. We found that intraperitoneal treatment with BAY 41-2272 markedly increased macrophage-dependent cell influx to the peritoneum in addition to macrophage functions, such as spreading, zymosan particle phagocytosis and nitric oxide and phorbol myristate acetate-stimulated hydrogen peroxide production. Treatment with BAY 41-2272 was highly effective in reducing the death rate due to intraperitoneal inoculation of C. albicans, but not S. aureus. However, we found that in vitro stimulation of peritoneal macrophages with BAY 41-2272 markedly increased microbicidal activities against both pathogens. Our results show that the prevention of death by the treatment of C. albicans-infected mice with BAY 41-2272 might occur primarily by the modulation of the host immune response through macrophage activation.

Candida albicans in patients with oronasal communication and obturator prostheses

Patients using obturator prostheses often present denture-induced stomatitis. In order to detect the presence of oral Candida albicans in patients with oronasal communications and to evaluate the effectiveness of a topical antifungal treatment, cytological smears obtained from the buccal and palatal mucosa of 10 adult patients, and from the nasal acrylic surface of their obturator prostheses were examined. A therapeutic protocol comprising the use of oral nystatin (Mycostatin®) and prosthesis disinfection with sodium hypochlorite was prescribed for all patients. Seven patients were positive for C. albicans in the mucosa, with 1 negative result for the prosthetic surface in this group of patients. Post-treatment evaluation revealed the absence of C. albicans on prosthesis surface and on the oral mucosa of all patients. The severity of the candidal infection was significantly higher in the palatal mucosa than in the buccal mucosa, but similar in the palatal mucosa and prosthesis surface, indicating that the mucosa underlying the prosthesis is more susceptible to infection. The therapeutic protocol was effective in all cases, which emphasizes the need for denture disinfection in order to avoid reinfection of the mucosa.