Two novel oligosaccharides isolated from a beverage produced by fermentation of a plant extract

An extract from 50 kinds of fruits and vegetables was fermented to produce a new beverage. Natural fermentation of the extract was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Two new saccharides were found in this fermented beverage. The saccharides were isolated using carbon-Celite column chromatography and preparative high performance liquid chromatography. Gas liquid chromatography analysis of methylated derivatives as well as MALDI-TOF MS and NMR measurements were used for structural confirmation. The (1)H and (13)C NMR signals of each saccharide were assigned using 2D-NMR including COSY, HSQC, HSQC-TOCSY, CH(2)-HSQC-TOCSY, and CT-HMBC experiments. The saccharides were identified as beta-D-fructopyranosyl-(2-->6)-beta-D-glucopyranosyl-(1-->3)-D-glucopyranose and beta-D-fructopyranosyl-(2-->6)-[beta-D-glucopyranosyl-(1-->3)]-D-glucopyranose.     

Kinetic investigation of a new inhibitor for human salivary alpha-amylase

This study is the first report on the effectiveness and specificity of alpha-acarviosinyl-(1-->4)-alpha-D-glucopyranosyl-(1-->6)-D-glucopyranosylidene-spiro-thiohydantoin (PTS-G-TH) inhibitor on the 2-chloro-4-nitrophenyl-4-O-beta-D-galactopyranosyl-maltoside (GalG2CNP) and amylose hydrolysis catalysed by human salivary alpha-amylase (HSA). Synthesis of PTS-G-TH was carried out by transglycosylation using acarbose as donor and glucopyranosylidene-spiro-thiohydantoin (G-TH) as acceptor. This new compound was found to be a much more efficient HSA inhibitor than G-TH. The inhibition is a mixed-noncompetitive type on both substrates and only one molecule of inhibitor binds to the enzyme. Kinetic constants calculated from secondary plots are in micromolar range. Values of K(EI) and K(ESI) are very similar in the presence of GalG2CNP substrate; 0.19 and 0.24 microM, respectively. Significant difference can be found for K(EI) and K(ESI) using amylose as substrate; 8.45 and 0.5 microM, respectively. These values indicate that inhibition is rather uncompetitive than competitive related to amylose hydrolysis.     

Two new flavonol glycosides from Gymnema sylvestre and Euphorbia ebracteolata

Two new flavonol glycosides, namely kaempferol 3-O-beta-D-glucopyranosyl-(1-->4)-alpha-L-rhamnopyranosyl-(1-->6)-beta-D-galactopyranoside (1) and quercetin 3-O-6"-(3-hydroxyl-3-methylglutaryl)-beta-D-glucopyranoside (2), have been isolated from the aerial parts of Gymnema sylvestre and Euphorbia ebracteolata, respectively. Their structures were determined on the basis of chemical and spectroscopic methods.     

Synthesis, (1-->3)-beta-D-glucanase-binding ability, and phytoalexin-elicitor activity of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides

We describe a approach for the synthesis of a mixture of 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. The particular (1-->3)-beta-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method, and it was characterized by FTIR spectroscopy. The acid-solubilized (1-->3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan particles, which were qualitatively analyzed by fluorophore-assisted carbohydrate electrophoresis (FACE). The peracetylated 3-butenyl (1-->3)-beta-D-oligoglucosides were synthesized by treating peracetylated (1-->3)-beta-D-oligoglucosides with the 3-butenyl alcohols and a Lewis acid (SnCl4) catalyst. Epoxidation of the peracetylated 3-butenyl oligoglucosides took place with m-chloroperoxybenzoic acid (m-CPBA). NaOMe in dry methanol was used for the deacetylation of the blocked derivatives, to give the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture in an overall yield of 21%. The sample was analyzed by positive-ion electrospray ionization mass spectrometry (ESIMS). In a 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside-binding (1-->3)-beta-D-glucanase assay, we found that the (1-->3)-beta-D-glucanase was obviously inactivated by the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucosides. At the same time, we found the 3,4-epoxybutyl (1-->3)-beta-D-oligoglucoside mixture was more active as compared to the underivatized oligoglucoside mixture in eliciting phytoalexin accumulation in tobacco cotyledon tissue. Furthermore, it could be kept for a longer time than a (1-->3)-beta-D-oligoglucoside mixture, which indicated it is much more stable than (1-->3)-beta-D-oligoglucosides.     

Bioconversion of ginsenosides Rb(1), Rb(2), Rc and Rd by novel β-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: cloning, expression, and enzyme characterization

A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb₁, Rb₂, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc₁ and ginsenoside F₂, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent K_m and V_max values of 2.9 ± 0.3 mM and 515.4 ± 38.3 μmol min⁻¹ mg of protein⁻¹ against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb₁, Rb₂, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc₁ and F₂ quickly at optimal conditions of pH 5.0 and 37 °C. A little ginsenoside F₂ production from ginsenosides Gyp XVII, C-O, and C-Mc₁ was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.     

Enzymatic synthesis of 4-methylumbelliferyl (1-->3)-beta-D-glucooligosaccharides-new substrates for beta-1,3-1,4-D-glucanase

The transglycosylation reactions catalyzed by beta-1,3-D-glucanases (laminaranases) were used to synthesize a number of 4-methylumbelliferyl (MeUmb) (1-->3)-beta-D-gluco-oligosaccharides having the common structure [beta-D-Glcp-(1-->3)](n)-beta-D-Glcp-MeUmb, where n=1-5. The beta-1,3-D-glucanases used were purified from the culture liquid of Oerskovia sp. and from a homogenate of the marine mollusc Spisula sachalinensis. Laminaran and curdlan were used as (1-->3)-beta-D-glucan donor substrates, while MeUmb-beta-D-glucoside (MeUmbGlcp) was employed as a transglycosylation acceptor. Modification of [beta-D-Glcp-(1-->3)](2)-beta-D-Glcp-MeUmb (MeUmbG(3)) gives 4,6-O-benzylidene-D-glucopyranosyl or 4,6-O-ethylidene-D-glucopyranosyl groups at the non-reducing end of artificial oligosaccharides. The structures of all oligosaccharides obtained were solved by 1H and 13C NMR spectroscopy and electrospray tandem mass spectrometry. The synthetic oligosaccharides were shown to be substrates for a beta-1,3-1,4-D-glucanase from Rhodothermus marinus, which releases MeUmb from beta-di- and beta-triglucosides and from acetal-protected beta-triglucosides. When acting upon substrates with d.p.>3, the enzyme exhibits an endolytic activity, primarily cleaving off MeUmbGlcp and MeUmbG(2).     

Stereoselective synthesis of a model alpha-glycoside of the beta-D-ManNAcp-(1-->4)-d-Glc disaccharide starting from lactose, avoiding the beta-mannosaminylation step

A model isopropyl alpha-glycoside of the beta-d-ManNAc-(1-->4)-d-Glc disaccharide has been prepared from lactose, avoiding the beta-mannosaminylation step. Three complementary approaches involving first the preparation and then the glycosidation of beta-thiophenyl donors of the protected disaccharides, (a) beta-d-ManNAc-(1-->4)-d-Glc, (b) beta-d-TalNAc-(1-->4)-d-Glc and (c) lactose, were compared. The best results were obtained employing a suitably protected lactose donor, and submitting its alpha-isopropyl glycoside to an amination with inversion in position 2' followed by an epimerization at C-4'.     

Sesquiterpene lactone glucosides and alkyl glycosides from the fruit of cumin

From the polar portion of the methanolic extract of cumin (fruit of Cuminum cyminum L.), two sesquiterpenoid glucosides, cuminoside A and B, and two alkyl glycosides were isolated together with five known compounds. Their structures were established as (1S,5S,6S,10S)-10-hydroxyguaia-3,7(11)-dien-12,6-olide beta-D-glucopyranoside, (1R,5R,6S,7S,9S,10R,11R)-1,9-dihydroxyeudesm-3-en-12,6-olide 9-O-beta-D-glucopyranoside, methyl beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside and ethane-1,2-diol 1-O-beta-D-apiofuranosyl-(1-->6)-beta-D-glucopyranoside, respectively.     

Enzymatic synthesis of beta-xylanase substrates: transglycosylation reactions of the beta-xylosidase from Aspergillus sp

A beta-D-xylosidase with molecular mass of 250+/-5 kDa consisting of two identical subunits was purified to homogeneity from a cultural filtrate of Aspergillus sp. The enzyme manifested high transglycosylation activity in transxylosylation with p-nitrophenyl beta-D-xylopyranoside (PNP-X) as substrate, resulting in regio- and stereoselective synthesis of p-nitrophenyl (PNP) beta-(1-->4)-D-xylooligosaccharides with dp 2-7. All transfer products were isolated from the reaction mixtures by HPLC and their structures established by electrospray mass spectrometry and 1H and 13C NMR spectroscopy. The glycosides synthesised, beta-Xyl-1-->(4-beta-Xyl-1-->)(n)4-beta-Xyl-OC6H4NO2-p (n=1-5), were tested as chromogenic substrates for family 10 beta-xylanase from Aspergillus orizae (XynA) and family 11 beta-xylanase I from Trichoderma reesei (XynT) by reversed-phase HPLC and UV-spectroscopy techniques. The action pattern of XynA against the foregoing PNP beta-(1-->4)-D-xylooligosaccharides differed from that of XynT in that the latter released PNP mainly from short PNP xylosides (dp 2-3) while the former liberated PNP from the entire set of substrates synthesised.     

1-O-Acetyl-beta-D-galactopyranose: a novel substrate for the transglycosylation reaction catalyzed by the beta-galactosidase from Penicillium sp

1-O-Acetyl-beta-D-galactopyranose (AcGal), a new substrate for beta-galactosidase, was synthesized in a stereoselective manner by the trichloroacetimidate procedure. Kinetic parameters (K(M) and k(cat)) for the hydrolysis of 1-O-acetyl-beta-D-galactopyranose catalyzed by the beta-D-galactosidase from Penicillium sp. were compared with similar characteristics for a number of natural and synthetic substrates. The value for k(cat) in the hydrolysis of AcGal was three orders of magnitude greater than for other known substrates. The beta-galactosidase hydrolyzes AcGal with retention of anomeric configuration. The transglycosylation activity of the beta-D-galactosidase in the reaction of AcGal and methyl beta-D-galactopyranoside (1) as substrates was investigated by 1H NMR spectroscopy and HPLC techniques. The transglycosylation product using AcGal as a substrate was beta-D-galactopyranosyl-(1-->6)-1-O-acetyl-beta-D-galactopyranose (with a yield of approximately 70%). In the case of 1 as a substrate, the main transglycosylation product was methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside. Methyl beta-D-galactopyranosyl-(1-->3)-beta-D-galactopyranoside was found to be minor product in the latter reaction.     

Simple syntheses of 4-O-glucosylated 1-deoxynojirimycins from maltose and cellobiose

Glucosidase inhibitors alpha-D-glucopyranosyl-(1-->4)-1-deoxynojirimycin and beta-D-glucopyranosyl-(1-->4)-1-deoxynojirimycin were prepared from maltose and cellobiose, respectively, via the corresponding 5,6-eno derivatives, their epoxidation and the subsequent double reductive amination of the resulting 5-uloses. In both cases, the reported route is the first chemical synthesis not based on enzymatic glucosyl transfer.     

Structure of oligosaccharide side chains of an intestinal immune system modulating arabinogalactan isolated from rhizomes of Atractylodes lancea DC

An intestinal immune system modulating arabino-3,6-galactan (ALR-5IIa-1-1) has been found in rhizomes of Atractylodes lancea DC. [Planta Medica 1998, 64, 714-719; Carbohydr. Polyms. 2001, 46, 147-156], however other arabino-3,6-galactans from Larix and Acacia failed to express the modulating activity. Degradation of the galactosyl side chains in Araf-side chain-trimmed ALR-5IIa-1-1 (AF-ALR-5IIa-1-1) with an endo-beta-D-(1-->6)-galactanase remarkably decreased the activity of AF-ALR-5IIa-1-1. Structural analysis indicated that the major endo-beta-D-(1-->6)-galactanase-digestable side chains in ALR-5IIa-1-1 are composed of beta-D-(1-->6)-galactopyranosyl oligosaccharides having d.p. 1-8. Because degradation of the beta-D-(1-->3)-galactan backbone in AF-ALR-5IIa-1-1 also significantly reduced its activity, some of these galactosyl side chains attached to beta-D-(1-->3)-galactan backbone are suggested to be responsible for expression of the activity of ALR-5IIa-1-1.     

alpha-Mannosidase-catalyzed synthesis of a (1-->2)-alpha-D-rhamnodisaccharide derivative

Enzymatic transglycosylation using p-nitrophenyl alpha-D-rhamnopyranoside as the glycosyl donor and 6equiv of ethyl 1-thio-alpha-D-rhamnopyranoside as the glycosyl acceptor yielded a D-rhamnooligosaccharide derivative. The reaction was catalyzed by jack bean alpha-mannosidase in a 1:1 (v/v) mixture of 0.1 M sodium citrate buffer (pH4.5)-MeCN at 25 degrees C. The enzyme exhibited high catalytic activity for the reaction, to afford in 32.1% isolated yield (based on donor substrate) ethyl alpha-D-rhamnopyranosyl-(1-->2)-1-thio-alpha-D-rhamnopyranoside, which is a derivative of the common oligosaccharide unit of the antigenic lipopolysaccharides from Pseudomonas.     

Synthesis of (R)-2,3-epoxypropyl(1-->3)-beta-D-pentaglucoside

The title pentasaccharide was synthesized via a 2+3 strategy. The disaccharide donor, 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (8), was obtained by selective coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranoside with 3-O-acetyl-2-O-benzoyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (4), followed by deallylation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside (12), was prepared by coupling of allyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranoside with 4, followed by deacetylation. Condensation of 8 with 12, followed by epoxidation, and deprotection, gave the target pentaoside.     

Fast and efficient synthesis of a novel homologous series of L-fucosylated trisaccharides using the Helix pomatia alpha-(1-->2)-L-galactosyltransferase

The alpha-(1-->2)-L-galactosyltransferase from the albumen gland of the vineyard snail Helix pomatia exhibits high alpha-(1-->2)-L-fucosyltransferase activity and can be used to transfer L-fucose from GDP-L-fucose to terminal, non-reducing D-galactose residues of an oligosaccharide, thus providing facile access to a range of H-antigen-containing oligosaccharides. The enzymatic glycosylation was applied here on a milligram scale to a series of disaccharide acceptor substrates. Apparently the site of interglycosidic linkage between the terminal and subterminal acceptor sugar units is of little or no consequence. The homologous series of trisaccharides thus produced were fully characterised by NMR analysis of their peracetates.     

Structural analysis of a novel saccharide isolated from fermented beverage of plant extract

Fermented beverage of plant extract was prepared from about 50 kinds of vegetables and fruits. Natural fermentation was carried out mainly by lactic acid bacteria (Leuconostoc spp.) and yeast (Zygosaccharomyces spp. and Pichia spp.). Three kinds of saccharides have been found in this beverage and produced by fermentation. The saccharides isolated from the beverage using carbon-Celite column chromatography and preparative HPLC, were identified as a new saccharide, beta-d-fructopyranosyl-(2-->6)-d-glucopyranose, laminaribiose and maltose by examination of constituted sugars, GLC and GC-MS analyses of methyl derivatives and MALDI-TOF-MS and NMR measurements of the saccharides.     

Synthesis and intramolecular reactions of Tyr-Gly and Tyr-Gly-Gly related 6-O-glucopyranose esters

6-O-(L-Tyrosylglycyl)- and 6-O-(L-tyrosylglycylglycyl)-D-glucopyranose were synthesized by condensation of the pentachlorophenyl esters of the respective di- and tripeptide with fully unprotected D-glucose. The intramolecular reactivity of the sugar conjugates was studied in pyridine-acetic acid and in dry methanol, at various temperatures and for various incubation times. The composition of the incubation mixtures was monitored by a reversed-phase HPLC method that permits simultaneous analysis of the disappearance of the starting material and the appearance of rearrangement and degradation products. To determine the influence of esterification of the peptide carboxy group on its amino group reactivity, parallel experiments were done in which free peptides were, under identical reaction conditions, incubated with D-glucose (molar ratios 1:1 and 1:5). Depending on the starting compound, different types of Amadori products (cyclic and bicyclic form), methyl ester of peptides, and Tyr-Gly-diketopiperazine were obtained.     

The gel-forming polysaccharide of psyllium husk (Plantago ovata Forsk)

The physiologically active, gel-forming fraction of the alkali-extractable polysaccharides of Plantago ovata Forsk seed husk (psyllium seed) and some derived partial hydrolysis products were studied by compositional and methylation analysis and NMR spectroscopy. Resolving the conflicting claims of previous investigators, the material was found to be a neutral arabinoxylan (arabinose 22.6%, xylose 74.6%, molar basis; only traces of other sugars). With about 35% of nonreducing terminal residues, the polysaccharide is highly branched. The data are compatible with a structure consisting of a densely substituted main chain of beta-(1-->4)-linked D-xylopyranosyl residues, some carrying single xylopyranosyl side chains at position 2, others bearing, at position 3, trisaccharide branches having the sequence L-Araf-alpha-(1-->3)-D-Xylp-beta-(1-->3)-l-Araf. The presence of this sequence is supported by methylation and NMR data, and by the isolation of the disaccharide 3-O-beta-D-xylopyranosyl-L-arabinose as a product of partial acid hydrolysis of the polysaccharide.     

Synthesis of beta-hederin and Hederacolchiside A1: triterpenoid saponins bearing a unique cytotoxicity-inducing disaccharide moiety

A facile synthetic approach toward oleanolic acid glycoside bearing alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl moiety, a unique oligosaccharide that strongly induces antitumor activity of oleanane-type triterpenoid saponins, was developed. Based on this approach beta-hederin (oleanolic acid 3-O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranoside) was efficiently prepared from oleanolic acid through stepwise glycosylation in linear eight steps with 52% overall yield, while Hederacolchiside A1 (oleanolic acid 3-O-alpha-L-rhamnopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)]-alpha-L-arabinopyranoside) in linear 13 steps with 20% overall yield.     

Synthesis of beta-D-glucose oligosaccharides from Phytophthora parasitica

The glucohexaose, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-D-Glcp, was synthesized as its allyl glycoside via 3+3 strategy. The trisaccharide donor, 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11), was obtained by 3-selective coupling of isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (2) with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2-O-acetyl-4,6-O-benzylidene-alpha-D-glucopyranosyl trichloroacetimidate (6), followed by hydrolysis, acetylation, dethiolation, and trichloroacetimidation. Meanwhile, the trisaccharide acceptor, allyl 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2-O-acetyl-beta-D-glucopyranosyl-(1-->3)-4,6-di-O-acetyl-2-O-benzoyl-alpha-D-glucopyranoside (14), was prepared by coupling of allyl 4,6-di-O-acetyl-2-O-benzoyl-alpha-D-glucopyranoside (12) with 6, followed by debenzylidenation. Condensation of 14 with 11, followed by deacylation, gave the target hexaoside. A beta-(1-->3)-linked tetrasaccharide 29 was also synthesized with methyl 2-O-benzoyl-4,6-O-benzylidene-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-beta-D-glucopyranoside (25) as the acceptor and acylated beta-(1-->3)-linked disaccharide 21 as the donor.