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.     

Synthesis of a library of allyl alpha-L-arabinofuranosyl-alpha- or beta-D-xylopyranosides; route to higher oligomers

Six isomeric disaccharides allyl 2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl-alpha-d-xylopyranosides and beta-d-xylopyranosides were synthetized by the stereoselective glycosylation of pure allyl alpha- or beta-d-xylopyranosides with 1-O-acetyl-2,3,5-tri-O-benzoyl-l-arabinofuranose as donor, catalyzed with BF(3).Et(2)O in DCM. Regio- and stereoselective glycosylation with excess of donor furnished almost exclusively the trisaccharides allyl 2,3-di-O-(2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl)-alpha- or beta-d-xylopyranosides. Extension of the reaction to the triol beta-d-xylopyranosyl-(1-->4)-1,2,3-tri-O-acetyl-alpha-d-xylopyranose, obtained from the 4-hydroxyl penta-O-acetyl-alpha-xylobiose, gave in the same manner the tetrasaccharide [2,3-di-O-(2,3,5-tri-O-benzoyl-alpha-l-arabinofuranosyl)-beta-d-xylopyranosyl]-(1-->4)-1,2,3-tri-O-acetyl-alpha-d-xylopyranose. The protocol described herein should offer the possibility to produce branched oligosaccharides with a 2,3-di-O-(alpha-l-Ara(f))-beta-d-Xyl(p) block unit at the terminal non-reducing end.     

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.     

Alternansucrase acceptor reactions with D-tagatose and L-glucose

Alternansucrase (EC 2.4.1.140) is a d-glucansucrase that synthesizes an alternating alpha-(1-->3), (1-->6)-linked d-glucan from sucrose. It also synthesizes oligosaccharides via d-glucopyranosyl transfer to various acceptor sugars. Two of the more efficient monosaccharide acceptors are D-tagatose and L-glucose. In the presence of d-tagatose, alternansucrase produced the disaccharide alpha-d-glucopyranosyl-(1-->1)-beta-D-tagatopyranose via glucosyl transfer. This disaccharide is analogous to trehalulose. We were unable to isolate a disaccharide product from L-glucose, but the trisaccharide alpha-D-glucopyranosyl-(1-->6)-alpha-d-glucopyranosyl-(1-->4)-l-glucose was isolated and identified. This is analogous to panose, one of the structural units of pullulan, in which the reducing-end D-glucose residue has been replaced by its L-enantiomer. The putative L-glucose disaccharide product, produced by glucoamylase hydrolysis of the trisaccharide, was found to be an acceptor for alternansucrase. The disaccharide, alpha-D-glucopyranosyl-(1-->4)-L-glucose, was a better acceptor than maltose, previously the best known acceptor for alternansucrase. A structure comparison of alpha-D-glucopyranosyl-(1-->4)-L-glucose and maltose was performed through computer modeling to identify common features, which may be important in acceptor affinity by alternansucrase.     

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).     

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 (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.     

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.     

Substrate specificity of the alpha-L-arabinofuranosidase from Rhizomucor pusillus HHT-1

The alpha-L-arabinofuranosidase (AF) from the fungus Rhizomucor pusillus HHT-1 released arabinose at appreciable rates from (1-->5)-alpha-L-arabinofuranooligosaccharides, sugar beet arabinan and debranched arabinan. This enzyme preferentially hydrolyzed the terminal arabinofuranosyl residue [alpha-(1-->5)-linked] of the arabinan backbone rather than the arabinosyl side chain [alpha-(1-->3)-linked residues]. The enzyme-hydrolyzed arabinan reacted at and debranched the arabinan almost at the same rate, and the degree of conversion for both cases was 65%. Methylation analysis of arabinan showed that the arabinosyl-linkage proportions were 2:2:2:1, respectively, for (1-->5)-Araf, T-Araf, (1-->3, 5)-Araf and (1-->3)-Araf, while the ratios for the AF-digested arabinan shifted to 3:1:2:1. Enzyme digestion resulted in an increase in the proportion of (1-->5)-linked arabinose and a decrease in the proportion of terminal arabinose indicated this AF cleaved the terminal arabinosyl residue of the arabinan back bone [alpha-(1-->5)-linked residues]. Peak assignments in the 13C NMR spectra also confirmed this linkage composition of four kinds of arabinose residues. Both 1H and 13C NMR spectra are dominated by signals of the alpha-anomeric configuration of the arabinofuranosyl moieties. No signals were recorded for arabinopyranosyl moieties in the NMR spectra. Methylation and NMR analysis of native and AF-digested arabinan revealed that this alpha-L-arabinofuranosidase can only hydrolyse alpha-L-arabinofuranosyl residues of arabinan.     

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.     

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'.     

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.     

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.     

Solution properties of an alpha-(1-->3)-D-glucan from Lentinus edodes and its sulfated derivatives

A water-insoluble alpha-(1-->3)-D-glucan (A) from Lentinus edodes was fractionated into 13 fractions in dimethyl sulfoxide containing 0.25 M lithium chloride (0.25 M LiCl-Me(2)SO). Five fractions were treated with sulfur trioxide-pyridine complex at 25 degrees C to synthesize water-soluble sulfated derivatives (S-A). The weight-average molecular weights, M(w), and intrinsic viscosities [eta], of the samples A and S-A were determined by multi-angler laser light scattering (MALLS), and viscosity. The M(w) dependence of [eta] and of the radius of gyration (z)(1/2), was found to be represented approximately by [eta]=4.9 x 10(-2) M(w)(0.67) (cm(3) g(-1)), and (z)(1/2)=4.8 x 10(-2) M(w)(0.54) (nm) for the alpha-glucan in 0.25 M LiCl-Me(2)SO in the M(w) range from 7.24 x 10(4) to 4.21 x 10(5), and by [eta]=6.8 x 10(-4) M(w) 1.06 (cm(3) g(-1)), and (z)(1/2)=9.4 x 10(-4) M(w)(0.92) (nm) for the sulfated alpha-glucan in aqueous 0.5 M NaCl in the M(w) range from 5.92 x 10(4) to 1.42 x 10(5) at 25 degrees C. The results indicate that the alpha-(1-->3)-D-glucan exists as a flexible chain in 0.25 M LiCl-Me(2)SO, and its sulfated derivative in 0.5 M NaCl aqueous has stiffer chains than the original. (13)C NMR indicated that intramolecular hydrogen bonding occurred in the sulfated alpha-glucan, causing the observed chain stiffness.     

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.     

The antileishmanial activity assessment of unusual flavonoids from Kalanchoe pinnata

The importance of flavonoids for the antileishmanial activity of Kalanchoe pinnata was previously demonstrated by the isolation of quercitrin, a potent antileishmanial flavonoid. In the present study, the aqueous leaf extract from the medicinal plant K. pinnata (Crassulaceae) afforded a kaempferol di-glycoside, named kapinnatoside, identified as kaempferol 3-O-alpha-L-arabinopyranosyl (1-->2) alpha-L-rhamnopyranoside (1). In addition, two unusual flavonol and flavone glycosides already reported, quercetin 3-O-alpha-L-arabinopyranosyl (1-->2) alpha-L-rhamnopyranoside (2) and 4',5-dihydroxy-3',8-dimethoxyflavone 7-O-beta-D-glucopyranoside (3), have been isolated. Their structures were determined via analyses of mono and bi-dimensional (1)H and (13)C NMR spectroscopic experiments and HR-MALDI mass spectra. Because of its restricted occurrence and its abundance in K. pinnata, flavonoid (2) may be a chemical marker for this plant species of high therapeutic potential. The three flavonoids were tested separately against Leishmania amazonenis amastigotes in comparison with quercitrin, quercetin and afzelin. The quercetin aglycone - type structure, as well as a rhamnosyl unit linked at C-3, seem to be important for antileishmanial activity.     

Synthesis of 2-[(2-pyridyl)amino]ethyl beta-D-lactosaminide and evaluation of its acceptor ability for sialyltransferase: a comparison with 4-methylumbelliferyl and dansyl beta-D-lactosaminide

We reported the synthesis of beta-D-lactosaminide with a 2-aminopyridyl group that is linked to a glycosyl tether at the reducing end. This fluorescent disaccharide acts as an acceptor for both alpha-(2-->6)- and alpha-(2-->3)-sialyltransferases. In addition, the acceptor ability of this disaccharide was evaluated and compared with that of beta-D-lactosaminide having a dansyl or a 4-methylumbelliferyl group.     

1,5-Anhydro-D-fructose; a versatile chiral building block: biochemistry and chemistry

There is a steadily increasing need to expand sustainable resources, and carbohydrates are anticipated to play an important role in this respect, both for bulk and fine chemical preparation. The enzyme alpha-(1-->4)-glucan lyase degrades starch to 1,5-anhydro-D-fructose. This compound, which has three different functional properties, a prochiral center together with a permanent pyran ring, renders it a potential chiral building block for the synthesis of valuable and potentially biologically active compounds. 1,5-Anhydro-D-fructose is found in natural materials as a degradation product of alpha-(1-->4)-glucans. The occurrence of lyases and the metabolism of 1,5-anhydro-D-fructose are reviewed in the biological part of this article. In the chemical part, the elucidated structure of 1,5-anhydro-D-fructose will be presented together with simple stereoselective conversions into hydroxy/amino 1,5-anhydro hexitols and a nojirimycin analogue. Synthesis of 6-O-acylated derivatives of 1,5-anhydro-D-fructose substituted with long fatty acid residues is carried out using commercially available enzymes. Those reactions lead to compounds with potential emulsifying properties. The use of protected derivatives of 1,5-anhydro-D-fructose for the synthesis of natural products is likewise reviewed. The potential utilization of this chemical building block is far from being exhausted. Since 1,5-anhydro-D-fructose now is accessible in larger amounts through a simple-enzyme catalyzed degradation of starch by alpha-(1-->4)-glucan lyase, the application of 1,5-anhydro-D-fructose may be considered a valuable contribution to the utilization of carbohydrates as the most abundant resource of sustainable raw materials.     

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.     

Golgi endomannosidase inhibitor, alpha-D-glucopyranosyl-(1 --> 3)-1-deoxymannojirimycin: a five-step synthesis from maltulose and examples of N-modified derivatives

Acid-catalysed O-acetylation of D-maltulose furnished the corresponding per-O-acetylated fructopyranose derivative that, after in situ deprotection at O-2 by reaction with triphenylphosphane dibromide, gave open-chain 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl-(1 --> 4)-1,3,5-tri-O-acetyl-6-bromo-6-deoxy-D-fructose. Standard deprotection employing sodium methoxide in methanol at -30 degrees C, followed by treatment of the resulting free 6-bromodeoxymaltulose with sodium azide in N,N-dimethylformamide, allowed access to 6-azidodeoxymaltulose. Hydrogenation over Pearlman's catalyst, accompanied by intramolecular reductive amination, yielded the desired title compound. This route allows access to preparative quantities and to a range of novel analogues with improved biostability.