A new class of ammonium ylid for [2,3]-sigmatropic rearrangement reactions: ene-endo-spiro ylids

The first examples of sigmatropic rearrangements of ene-endo-spirocyclic, tetrahydropyridine-derived ammonium ylids are reported. Thus, spiro[6.7]-ylids rearrange primarily by a [2,3]-pathway, whereas the analogous [6.6]-ylids rearrange by [1,2]- and [2,3]-mechanisms in roughly equal proportions. This method serves as a rapid entry to the core of a range of alkaloids bearing a pyrrolo[1,2-a]azepine or octahydroindolizidine nucleus.

Preparation of transition metal complexes of bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (C7H9CO2H) and X-ray crystal structure of [Cu2?(±)-endo-μ-O2CC7H9?4 (CH3OH)2]·2CH3OH

Metathesis reactions were used to prepare a range of dicopper(II), monocopper(I), diruthenium(II, III), dimolybdenum(II,II) and dirhodium(II,II) complexes of either racemic or resolved forms of endo- and exo-bicyclo[2.2.1]hept-5-ene-2-carboxylic acid (C7H9CO2H). The X-ray crystal structure of [Cu2{(±)-endo-μ-O2CC7H9}4(CH3OH)2]·2CH3OH shows the two copper(II) ions bridged by two (+)-endo-bicyclo[2.2.1]hept-5-ene-2-carboxylate anions and two (−)-endo-bicyclo[2.2.1]hept-5-ene-2-carboxylate anions. Methanol molecules occupy the two trans axial sites, and there are also two methanol molecules hydrogen bonded to opposite carboxyl oxygens.

Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects.

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant inhibitors of fungal endopolygalacturonases (PGs) that belong to the superfamily of Leu-rich repeat proteins. We have characterized the full complement of pgip genes in the bean (Phaseolus vulgaris) genotype BAT93. This comprises four clustered members that span a 50-kb region and, based on their similarity, form two pairs (Pvpgip1/Pvpgip2 and Pvpgip3/Pvpgip4). Characterization of the encoded products revealed both partial redundancy and subfunctionalization against fungal-derived PGs. Notably, the pair PvPGIP3/PvPGIP4 also inhibited PGs of two mirid bugs (Lygus rugulipennis and Adelphocoris lineolatus). Characterization of Pvpgip genes of Pinto bean showed variations limited to single synonymous substitutions or small deletions. A three-amino acid deletion encompassing a residue previously identified as crucial for recognition of PG of Fusarium moniliforme was responsible for the inability of BAT93 PvPGIP2 to inhibit this enzyme. Consistent with the large variations observed in the promoter sequences, reverse transcription-PCR expression analysis revealed that the different family members differentially respond to elicitors, wounding, and salicylic acid. We conclude that both biochemical and regulatory redundancy and subfunctionalization of pgip genes are important for the adaptation of plants to pathogenic fungi and phytophagous insects.

The use of culture-independent tools to characterize bacteria in endo-tracheal aspirates from pre-term infants at risk of bronchopulmonary dysplasia

Although premature infants are increasingly surviving the neonatal period, up to one-third develop bronchopulmonary dysplasia (BPD). Despite evidence that bacterial colonization of the neonatal respiratory tract by certain bacteria may be a risk factor in BPD development, little is known about the role these bacteria play. The aim of this study was to investigate the use of culture-independent molecular profiling methodologies to identify potential etiological agents in neonatal airway secretions. This study used terminal restriction fragment length polymorphism (T-RFLP) and clone sequence analyses to characterize bacterial species in endo-tracheal (ET) aspirates from eight intubated pre-term infants. A wide range of different bacteria was identified in the samples. Forty-seven T-RF band lengths were resolved in the sample set, with a range of 0-15 separate species in each patient. Clone sequence analyses confirmed the identity of individual species detected by T-RFLP. We speculate that the identification of known opportunistic pathogens including S. aureus, Enterobacter sp., Moraxella catarrhalis, Pseudomonas aeruginosa and Streptococcus sp., within the airways of pre-term infants, might be causally related to the subsequent development of BPD. Further, we suggest that culture-independent techniques, such as T-RFLP, hold important potential for the characterization of neonatal conditions, such as BPD.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-beta-1,3-glucanase from Thermotoga petrophila

1,3-beta-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal beta-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90 degrees C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Síntese e enriquecimento enantiomérico via catálise enzimática do sistema 5-bromo-12-oxa-pentaciclo[6.2.16,9.12,7.12,10]dodeca-4-eno-3-endo-ol e derivados. RMN de 1H e 13C: correlação de parâmetros teóricos e experimentais

Partindo de ciclopentadieno, ciclohexadieno, p-benzoquinona e 2,5-dibromo-pbenzoquinona, os adutos 1, 5, 30 e 31 foram sintetizados. Os adutos 1, 5 e 30 foram utilizados como produtos de partida para a síntese de 13 (treze) novos compostos, em sua maioria com potenciais características para apresentarem atividade biológica inibidora de glicosidases e reguladora da liberação de Insulina no sangue. O aduto 31 é inédito na literatura até o momento. Cinco novas propostas de mecanismos são apresentadas. Os álcoois racêmicos 6 e 29 foram submetidos a reações de transesterificação catalisadas por lipase de Pseudomonas cepacia em diferentes preparações e seus enantiômeros separados com enantiosseletividade (E) maior que 100 em todos os casos. Este processo resultou, também, na obtenção dos respectivos acetatos 43 e 44 enantiomericamente puros e com excelentes rendimentos químicos. Os compostos 6, 29 e 34 depois de terem suas estruturas moleculares resolvidas através dos métodos espectroscópicos de rotina, tiveram suas estruturas moleculares calculadas pelo método ab initio e por Funcionais de Densidade. As geometrias otimizadas foram submetidas ao método GIAO para o cálculo dos tensores de blindagem magnética isotrópica. Estes cálculos mostraram-se eficazes na descrição dos deslocamentos químicos da maioria dos átomos, incluindo os dos anéis ciclopropanos presentes nas estruturas moleculares de cada composto. Algumas dificuldades foram encontradas para a descrição do sistema vinílico halogenado dos álcoois 6 e 29. Foram utilizadas moléculas modelo para verificar a extensão de tais dificuldades.

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.

Studies on productivity and characterization of polygalacturonase from Aspergillus giganteus submerged culture using citrus pectin and orange waste

Polygalacturonases are part of the group of enzymes involved in pectin degradation. The aim of this work was to investigate some of the factors affecting polygalacturonase production by an Aspergillus giganteus strain and to characterize this pectinolytic activity. Several carbon sources, both pure substances and natural substrates, were tested in standing cultures, and the best results were obtained with orange bagasse and purified citrus pectin. on citrus pectin as sole carbon source, the highest extracellular activity (9.5 U/ml and 40.6 U/mg protein) was obtained in 4.5-day-old cultures shaken at 120 rpm, pH 3.5 and 30 degrees C, while on orange bagasse, the highest extracellular activity (48.5 U/ml and 78.3 U/mg protein) was obtained in 3.5-day-old cultures shaken at 120 rpm, pH 6.0 and 30 degrees C. Optimal polygalacturonase activity was observed in assays conducted at pH 5.5-6.5 and 55-60 degrees C. The activity showed good thermal stability, with half-lives of 90 and 30 min when incubated at 55 and 60 degrees C, respectively. High stability was observed from pH 4.5 to 8.5; more than 90% of the activity remained after 24 h in this pH range.

Production and characterization of polygalacturonase from thermophilic Thermoascus aurantiacus on submerged fermentation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Screening of bacterial strains for pectinolytic activity: characterization of the polygalacturonase produced by Bacillus sp

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Purification and characterization of an exo-polygalacturonase produced by Penicillium viridicatum RFC3 in solid-state fermentation

The pectinolytic enzyme obtained from Penicillium viridicatum RFC by solid-state fermentation was purified to homogeneity by pretreatment with kaolin (40 mg mL(-1) ) and ultrafiltration. followed by chromatography on a Sephadex G50 column. The apparent molecular weight of the enzyme was 24 kDa. Maximal activity occurred at pH 6.0 and at 60 degrees C. The enzyme proved to be an exo-polygalacturonase, releasing galacturonic acid by hydrolysis of highly esterified pectin. The presence of 10 mM Ba2+ increased the enzyme activity by 96% and its thermal stability by 30%. besides increasing its stability at acid pH. The apparent K-m with apple pectin as substrate was 1.82 mg mL(-1) and the V-max was 81 mu mol min(-1). (c) 2007 Elsevier Ltd. All rights reserved.

Pectinolytic enzyme production by Bacillus species and their potential application on juice extraction

Five Bacillus strains isolated from decaying vegetable material were cultivated on wheat bran and endo-polygalacturonases, exo-polygalacturonase and pectin lyase activities in the crude enzymatic solution obtained were determined. Highest activity was observed for all enzymes when fermentation was carried out at 28 degreesC, the highest activity values were obtained after 120 h of cultivation for exo-PG and after 48 h for endo-PG and PL. The use of the enzymatic solution for treatment of fruits and vegetable mash afforded a high juice extraction and a pulp with good pressing characteristics.

Stimulation of polygalacturonase production in an immobilized system by Aspergillus sp.: effect of pectin and glucose

The synthesis of polygalacturonases (PG) is known to be influenced by Aspergillus growth conditions, namely, environmental factors and pectin content in the cultivation medium containing a mixed carbon source. Optimal conditions were attained at a temperature of 30 A degrees C and an initial pH of 4.5. PG activity (3.29 and 2.48 U/mL) was determined after a two-day culture of Aspergillus sp. HC1 and Aspergillus sp. CC1, respectively, in a basic medium containing 2% citrus pectin as the sole carbon source. The addition of glucose (2% w/v) to the basic medium led to a 2-fold increase in PG production. However, enzyme synthesis was repressed when a higher concentration of glucose was used in the medium containing the mixed carbon source. Spores from the two fungi were immobilized in a 3% Ca-alginate system and the mechanical strength of the gel beads allowed the use of this process system 6-fold longer (288 h) than the free culture. In the Aspergillus sp. CC1 immobilized system, PG production increased nearly 10-fold in the medium with 2% glucose added (5.95 U/mL) in comparison to the medium without sugar (0.55 U/mL). The results demonstrate that a different response in activity was produced by free and entrapped spore systems. PG production remained approximately constant throughout the six 48 h cycles in the medium containing citrus pectin (2% w/v) as the sole carbon source.

Sistemas para liberação colônica: Uma alternativa para a veiculação de fármacos para efeito local ou sistêmico

Problems related to the systemic administration of drugs, such as biodistribution, difficulty of targeting, necessity of high doses to achieve adequate levels of the drug in specific sites, toxicity, and undesirable side effects have lead to the development of systems able to direct the drug to specific sites in the body. Among the possible organs to the targeting of drugs, the colon can be used for local and systemic therapies. By developing such systems some models have been tested, using pH dependent release, release controlled by enzymatic degradation, time controlled release systems and pressure controlled release systems. This review presents an overview of the colonic release of drugs and the strategies used to achieve such targeting.