Phosphorylation of levan by microwave-assisted synthesis enhanced anticancer ability.

Levan is an exopolysaccharide produced by Bacillus licheniformis (strain FRI MY-55) that shows promising pharmacological activity. Phosphorylation is a chemical modification that can increase the biological and antioxidant properties of levan. In this study, levan was phosphorylated by microwave-assisted synthesis to achieve a degree of substitution of 0.29. The hydroxyl radical scavenging activity of microwave-assisted phosphorylated levan (microwave P) increased significantly (6-fold) over native levan; this activity was only slightly lower than vitamin C. Other free radical scavenging and reducing power tests revealed that Microwave P activity was increased by 30-40%. Microwave P inhibited the proliferation of HCT-116 and A549 cancer cell lines more readily than native levan with an IC50 of 1.03 mg/mL and 1.38 mg/mL for HCT-116 and A549 cells, respectively. Cells treated with native levan and its derivatives remained in the sub-G1 phase according to cell cycle analysis, whereas Microwave P treatment increased the proportion of cells undergoing apoptosis. Furthermore, Microwave P effectively upregulated pro-apoptosis marker Bax and downregulated anti-apoptosis marker Bcl-2, in addition to inducing the expression of caspase-9 and caspase-3. These findings show that levan phosphorylated via microwave-assisted synthesis showed increased antioxidant and antitumor activity over native levan or levan phosphorylated via traditional long-term heating. In particular, Microwave P possesses antiproliferative activity and can induce apoptosis through mitochondrial pathways in cancerous cells.

Influence of acid depolymerization parameters on levan molar mass distribution and its utilization by bacteria.

Levan is a fructan composed of ß -(2, 6) linkages in its main chain. Its health properties, especially its prebiotic potential can be partially modified by changing its molar mass distribution. Given that native levan is rarely fermented by probiotic bacteria, especially lactic acid bacteria (LAB), levanoligosaccharides (LOS) were produced by mild acid hydrolysis. The response surface methodology (RSM) was applied to determine the optimum parameters for depolymerization. Gel permeation chromatography (GPC) was used to characterize the LOS produced and to show the differences between inulin and levan. The prebiotic potential of four fractions of LOS with different molar mass distributions was investigated. MRS (Mann Rogosa Sharpe) medium supplemented with the LOS were inoculated with bacterial strains and growth was monitored by measuring the turbidity of the cultures. The utilization of oligofructans was also confirmed by RP-UHPLC-UV-ESI-MS (liquid chromatography coupled with mass spectrometry) measurements of LOS derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP). It was observed that the degree of polymerization of LOS has an influence on the growth of the tested bacteria.

Biosynthesis of levan from sucrose using a thermostable levansucrase from Lactobacillus reuteri LTH5448.

Levan, a versatile fructan, possesses promising prospects for application in the food and pharmaceutical industries and in many other fields due to the superior physicochemical properties and physiological functions of this fructan. In this study, a thermostable levansucrase was identified from Lactobacillus reuteri LTH5448. This enzyme shared higher identity with reported inulosucrases than levansucrases but, unexpectedly, yielded a levan-type fructan. Structural analysis of the produced polysaccharide revealed that the glycosidic bonds were ß-(2, 6) linkages, and the average molecular weight was 3.924×107Da under optimized conditions. The optimal pH and temperature for levan formation were 6.0 and 35°C, respectively. The total activity was enhanced to 135% in the presence of Ca2+. After optimizing the production of levan, 13U/g sucrose of levansucrase and 500g/L sucrose were utilized to obtain 183g/L levan after a 12-h reaction.

Bacillus amyloliquefaciens levansucrase-catalyzed the synthesis of fructooligosaccharides, oligolevan and levan in maple syrup-based reaction systems.

Maple syrups with selected degree Brix (°Bx) (15, 30, 60) were investigated as reaction systems for levansucrase from Bacillus amyloliquefaciens. The enzymatic conversion of sucrose present in the maple syrup and the production of the transfructosylation products were assessed over a time course of 48h. At 30°C, the use of maple syrup 30°Bx led to the highest levansucrase activity (427.53µmol/mg protein/min), while maple syrup 66°Bx led to the highest converted sucrose concentration (1.53M). In maple syrup 30°Bx, oligolevans (1080%). In maple syrup 66°Bx, the most abundant products were oligolevans at 30°C and levans (DP≥30) at 8°C. The acceptor specificity study revealed the ability of B. amyloliquefaciens levansucrase to synthesize a variety of hetero-fructooligosaccharides (FOSs) in maple syrups 15°Bx and 30°Bx enriched with various disaccharides, with lactose being the preferred fructosyl acceptor. The current study is the first to investigate maple-syrup-based reaction systems for the synthesis of FOSs/oligolevans/levans.

Stereocontrolled synthesis of four diastereomeric C-aryl manno- and talofuranosides.

In a chiral-pool synthesis starting from D-mannono-1,4-lactone 1a, the four diastereomeric C-aryl furanosides (1S,4R)-4a, (1S,4S)-4b, (1R,4R)-4c, and (1R,4S)-4d were obtained in a stereocontrolled manner. The key steps of the synthetic pathway comprise a stereoselective reduction of the diastereomeric hemiketals (4R)-2a and (4S)-2b as well as a stereospecific cycloetherification of the resulting diols (1R,4R)-5a, (1S,4R)-5c, and (1S,4S)-5d. This ring closure which led to the desired C-glycosides was achieved by a Mitsunobu reaction or by preparing the 1-O-benzoyl-4-O-methylsulfonyl derivative 7 which was then treated with sodium methoxide. Final hydrolysis of the 5,6-O-isopropylidene protecting group led to the diastereomeric diols (1S,4R)-4a, (1S,4S)-4b, (1R,4R)-4c, and (1R,4S)-4d, representing versatile building blocks for further synthetic transformations.

Partial purification of a Bacillus licheniformis levansucrase producing levan with antitumor activity.

The extracellular fructosyltransferase (FTase) of a novel strain of Bacillus licheniformis capable of producing fructooligosaccharides (FOS) and a polysaccharide type levan was obtained and partially purified. The purification was achieved by ammonium sulfate precipitation, DEAE cellulose and gel filtration chromatographies. The enzyme was partially purified as determined by SDS-PAGE, and the specific activity reached was 67.5, representing a purification factor of 177 and yield of 40%. Levan was isolated from the cultures of B. licheniformis. The levan was composed mainly of fructose residues when analyzed by TLC after acid hydrolysis and NMR analysis. In a previous study, the levan produced exhibited a hypoglycemiant activity. The present paper deals with the study of the antitumor and anti-cytotoxic effect of levan produced by B. licheniformis strain. In the in vitro antitumor activity test of levan against some tumor cell lines, relatively the significantly high activity was observed against the HepG(2).

Sulfonated cyclofructan 6 based stationary phase for hydrophilic interaction chromatography.

A stationary phase composed of silica-bonded sulfonated cyclofructan 6 (SCF6) was synthesized and evaluated for hydrophilic interaction chromatography (HILIC). The separation of a large variety of polar compounds was evaluated on different versions of the stationary phase and compared with the same separations obtained with commercially available HILIC columns. The new columns successfully separate polar and hydrophilic compounds including ß blockers, xanthines, salicylic acid related compounds, nucleic acid bases, nucleosides, maltooligosaccharides, water soluble vitamins and amino acids. The separation conditions were optimized by changing the composition and the pH of the mobile phase. The dependence of analyte retention on temperature was studied using van't Hoff plots. The newly synthesized stationary phase showed broad applicability for HILIC mode separations.

Synthesis of C-furanosides from a D-glucal-derived cyclopropane through a ring-expansion/ring-contraction sequence.

gem-Dibromocyclopropane 1, prepared from tri-O-benzyl-D-glucal, undergoes thermal and silver-promoted ring expansion in the presence of alcohols to give substituted oxepines. With further heating, ring contraction to highly substituted tetrahydrofurans follows. These represent C-furanosides, potentially useful as precursors to C-nucleosides and other carbohydrate mimics.

Evaluation of supplementation of sucrose medium on the synthesis of Zymomonas mobilis bio-products / Avaliação da suplementação do meio de sacarose na formação de bio–produtos por Zymomonas mobilis

The effect of the variables pantothenic acid, yeast extract and sodium chloride, as well as the cell permeabilization technique, were investigated on the formation of levan, ethanol, sorbitol and biomass of Zymomonas mobilis, using a 24-1 fraction factorial design. Cell growth was determined by turbidimetry at 605 nm, relating it to a biomass with a dry weight calibration curve. Reducing sugars were quantified according to Somogyi and Nelson. Total sugars were quantified by the phenol-sulfuric acid method, sorbitol by HPLC and ethanol. The levan produced was precipitated by the addition of absolute ethanol and quantified in fructose units. In levan biosynthesis, the variable that had the largest contribution was cell condition. The results suggested that the factors that most affected biomass and ethanol formation were sodium chloride concentration and cell condition that affected negatively on production. For sorbitol, the variable that had a significant effect was permeabilization, which decreased its synthesis. Studies to amplify the range of established factors would be important.

A influência das variáveis: ácido pantotênico, extrato de levedura, cloreto de sódio, e a técnica de permeabilização celular foram investigadas na formação de levana, sorbitol, etanol e biomassa de Zymomonas mobilis utilizando um delineamento estatístico fatorial fracionado 24-1. A biomassa foi determinada por turbidimetria, Os açúcares redutores foram quantificados por Somogy e Nelson, açúcar total por Fenol Sulfúrico, sorbitol por HPLC e etanol por micro-destilação. A levana produzida foi precipitada com etanol absoluto e determinada como unidade de frutose. Na biossíntese de levana, a variável que mais contribuiu foi a condição celular. Os resultados sugerem que, para a formação da biomassa e etanol, os fatores que mais interferiram foram a concentração de cloreto de sódio e a condição celular que influencia negativamente a produção. Para o sorbitol, a variável que teve efeito significativo foi a permeabilização celular que atuou diminuindo a sua síntese. Estudos que ampliam a faixa de variação dos fatores estabelecidos são interessantes.

Development of new HPLC chiral stationary phases based on native and derivatized cyclofructans.

An unusual class of chiral selectors, cyclofructans, is introduced for the first time as bonded chiral stationary phases. Compared to native cyclofructans (CFs), which have rather limited capabilities as chiral selectors, aliphatic- and aromatic-functionalized CF6s possess unique and very different enantiomeric selectivities. Indeed, they are shown to separate a very broad range of racemic compounds. In particular, aliphatic-derivatized CF6s with a low substitution degree baseline separate all tested chiral primary amines. It appears that partial derivatization on the CF6 molecule disrupts the molecular internal hydrogen bonding, thereby making the core of the molecule more accessible. In contrast, highly aromatic-functionalized CF6 stationary phases lose most of the enantioselective capabilities toward primary amines, however they gain broad selectivity for most other types of analytes. This class of stationary phases also demonstrates high "loadability" and therefore has great potential for preparative separations. The variations in enantiomeric selectivity often can be correlated with distinct structural features of the selector. The separations occur predominantly in the presence of organic solvents.

Synthesis and examination of sulfated cyclofructans as a novel class of chiral selectors for CE.

Cyclofructans (CFs) are a class of cyclic oligosaccharides with a crown ether skeleton. No enantioseparations have previously been reported using this class of chiral oligosaccharides in chromatography or electrophoresis. CFs and their sulfated derivatives were examined as chiral selectors using CE. The native CFs showed no enantioselectivity toward any tested compounds, while the sulfated CFs showed exceptional selectivity toward many cationic analytes, including primary, secondary, and tertiary amines and amino acids. Enantiomeric resolution factors as high as 15.4 were achieved within short analysis times (generally below 10 min). The effect of buffer type, buffer concentration, buffer pH, chiral selector concentration and organic modifier concentration was examined and optimized.

Ocorrência e biossíntese de frutooligossacarídeos em banana / Occurrence and biosynthesis of fructooligosaccharides in banana

A banana tem sido comumente indicada como uma boa fonte de frutooligossacarídeos (FOS), que são considerados componentes funcionais de alimentos. Contudo, diferenças significantes em suas quantidades têm sido referidas na literatura. Portanto, uma parte do trabalho foi destinada à identificação e quantificação de FOS durante o amadurecimento de cultivares de bananas pertencentes aos grupos genômicos mais comumente cultivados no Brasil. Considerando as diferencas de cultivar, estágio do amadurecimento e metodologia usada para análise de FOS, os conteúdos dos acúcares foram analisados por cromatografia líquida de alta performance (HPAEC-PAD) e cromatografia a gás (CG-MS). Uma pesquisa inicial entre oito cultivares no estágio maduro, mostrou acúmulo de 1-cestose, primeiro membro da série de FOS, em todas elas (quantidades entre 297 e 1600 ´MUg/g M. S´). A nistose, o segundo membro, foi detectado somente no cultivar Prata. Com bases nestes dados, foram escolhidas cinco cultivares, para que fossem analisadas durantes todo o amadurecimento. Os resultados mostraram uma forte correlação entre a síntese de 1-cestose e um nível específico de sacarose (~200mg/g M.S)...

Synthesis of levan in water-miscible organic solvents.

The synthesis of levan using a levansucrase from a strain of Bacillus subtilis was studied in the presence of the water-miscible solvents: acetone, acetonitrile and 2-methyl-2-propanol (2M2P). It was found that while the enzyme activity is only slightly affected by acetone and acetonitrile, 2M2P has an activating effect increasing the total activity 35% in 40-50% (v/v) 2M2P solutions at 30 degrees C. The enzyme is highly stable in water at 30 degrees C; however, incubation in the presence of 15 and 50% (v/v) 2M2P reduced the half-life time to 23.6 and 1.8 days, respectively. This effect is reversed in 83% 2M2P, where a half-life time of 11.8 days is observed. The presence of 2M2P in the system increases the transfer/hydrolysis ratio of levansucrase. As the reaction proceeds with 10% (w/v) sucrose in 50/50 water/2M2P sucrose is converted to levan and an aqueous two-phase system (2M2P/Levan) is formed and more sucrose can be added in a fed batch mode. It is shown that high molecular weight levan is obtained as an hydrogel and may be easily recovered from the reaction medium. However, when high initial sucrose concentrations (40% (w/v) in 50/50 water/2M2P) are used, an aqueous two-phase system (2M2P/sucrose) is induce, where the synthesized levan has a similar molecular weight distribution as in water and remains in solution.

Chemical syntheses of inulin and levan structures.

A fructofuranosyl thiglycoside donor, ethyl 6-O-acetyl-3-O-benzyl-1,4-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-2-thio-beta-D-fructofuranoside (11), designed to yield stereospecifically beta-linkages and also to allow subsequent elongation in the 6- and/or 1-positions, was prepared and used in syntheses of levan and inulin structures. DMTST-promoted glycosylation between 11 (1.3 mol equiv) and methyl beta-D-fructofuranoside 6-OH and 1-OH acceptors (3 and 6) gave stereospecifically the protected methyl levanobioside 12 and inulinobioside 17 in excellent yields (80 and 86%), respectively. Protecting group manipulations on these afforded new disaccharide 6'-OH and 1'-OH acceptors (13 and 19), which were coupled again with donor 11 (1.0 mol equiv) to yield methyl levanotrioside 14 and inulinotrioside 20 in high yields, 65 and 67%, respectively. These were transformed into new acceptors and also fully deprotected to afford the methyl glycosides of levanotriose and inulinotriose, all structures that have earlier not been accessible by chemical synthesis.

Synthesis of fructans by fructosyltransferase from the tuberous roots of Viguiera discolor (Asteraceae)

Sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyl-transferase (FFT) activities from crude extracts of tuberous roots of Viguiera discolor growing in a preserved area of cerrado were analyzed in 1995-1996. SST activity was characterized by the synthesis of 1-kestose from sucrose and FFT activity by the production of nystose from 1-kestose. The highest fructan-synthesizing activity was observed during early dormancy (autumn), when both (SST and FFT) activities were high. The increase in synthetic activity seemed to start during the fruiting phase in the summer, when SST activity was higher than in spring. During winter and at the beginning of sprouting, both activities declined. The in vitro synthesis of high molecular mass fructans from sucrose by enzymatic preparations from tuberous roots collected in summer showed that long incubations of up to 288 h produced consistently longer polymers which resembled those found in vivo with respect to chromatographic profiles.