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.     

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-D-Xyl,a part of the common linkage region of a glycosaminoglycan

A practical synthesis of beta-D-GlcA-(1-->3)-beta-D-Gal-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl-(1-->OMe) was achieved by coupling of methyl 2,3,4-tri-O-acetyl-alpha-D-glucopyranosyluronate trichloroacetimidate with a trisaccharide acceptor. The trisaccharide acceptor was obtained by condensation of 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-2,4,6-tri-O-benzoyl-alpha-D-galactopyranosyl trichloroacetimidate with methyl 2,3-di-O-benzoyl-beta-D-xylopyranoside, followed by deallylation. The beta-(1-->3)-linked disaccharide was prepared readily with p-methoxyphenyl 3-O-allyl-2,4,6-tri-O-benzoyl-beta-D-galactopyranoside as the key synthon. The alpha-(1-->3)-linkage was formed in considerable amount with galactose mono- and disaccharide trichloroacetimidate donors with C-2 neighboring group participation.     

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.     

Synthesis of two isomeric pentasaccharides,the possible repeating unit of the beta-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht

Two isomeric pentasaccharides, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp (I) and beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)]-beta-D-Glcp (II), the possible repeating unit of the beta-glucan from the micro fungus Epicoccum nigrum Ehrenb. ex Schlecht, were synthesized as their 4-methoxyphenyl glycosides in a regio- and stereoselective manner. The pentasaccharide I was obtained from 3-O-selective glycosylation of 4-methoxyphenyl 4,6-O-benzylidene-beta-D-glucopyranoside (12) with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (6) followed by acetylation, debenzylidenation, and 6-O-selective glucosylation with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl trichloroacetimidate (1), and then by deprotection. The pentasaccharide II was obtained from 3-O-selective coupling of 12 with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)-2,4-di-O-acetyl-3-O-allyl-alpha-D-glucopyranosyl trichloroacetimidate (10) followed by acetylation, debenzylidenation, and 6-O-selective glycosylation with 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11), and finally by deprotection.     

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.     

Synthesis of beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp-(1-->3)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->3)]-beta-D-GlcpOLauryl, an oligosaccharide with anti-tumor activity

A concise and effective synthesis of lauryl heptasaccharide 17 was achieved from the key intermediates lauryl 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-2,4-di-O-benzoyl-beta-D-glucopyranoside (10) and isopropyl 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-beta-D-glucopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-1-thio-beta-D-glucopyranoside (15). The key trisaccharide glycosyl acceptor 10 was constructed by coupling 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzoyl-alpha-D-glucopyranosyl trichloroacetimidate (3) with lauryl 6-O-acetyl-2,4-di-O-benzoyl-beta-D-glucopyranoside (9), followed by deacetylation. The thioglycoside donor 15 was obtained by condensation of 2,4,6-tri-O-acetyl-3-O-allyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-glucopyranosyl trichloroacetimidate (11) with isopropyl 4,6-O-benzylidene-1-thio-beta-D-glucopyranoside (12), followed by debenzylidenation and acetylation. A bioassay of the inhibition of S180 noumenal tumors showed that lauryl heptasaccharide 17 could be employed as a potential agent for cancer treatment.     

Synthesis and stability assay of 4-methylumbelliferyl (1-->3)-beta-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 --> 3)-beta-D-pentaglucoside 3 are described. The (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. The acid-solubilized (1 --> 3)-beta-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 --> 3)-beta-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 --> 3)-beta-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 --> 3)-beta-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl4) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with beta-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.     

Enzymatic synthesis of a beta-D-galactopyranosyl cyclic tetrasaccharide by beta-galactosidases

The galactosyl transfer reaction to cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->] (CTS) was examined using lactose as a donor and beta-galactosidases from Aspergillus oryzae and Bacillus circulans. The A. oryzae beta-galactosidase produced three galactosyl derivatives of CTS. The main galactosyl derivative produced by the A. oryzae enzyme was identified as 6-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-[beta-D-Galp-(1-->6)]-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. The B. circulans beta-galactosidase also synthesized three galactosyl-transfer products to CTS. The structure of main transgalactosylation product was 3-O-beta-D-galactopyranosyl-CTS, cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[beta-D-Galp-(1-->3)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These results showed that beta-galactosidase transferred galactose directly to the ring glucose residue of CTS.     

Transglycosylation of glycosyl residues to cyclic tetrasaccharide by Bacillus stearothermophilus cyclomaltodextrin glucanotransferase using cyclomaltodextrin as the glycosyl donor

Cyclomaltodextrin glucanotransferase (EC 2.4.1.19, abbreviated as CGTase) derived from Bacillus stearothermophilus produced a series of transfer products from a mixture of cyclomaltohexaose and cyclic tetrasaccharide (cyclo[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], CTS). Of the transfer products, only two components, saccharides A and D, remained and accumulated after digestion with glucoamylase. The total combined yield of the saccharides reached 63.4% of total sugars, and enzymatic and instrumental analyses revealed the structures of both saccharides. Saccharide A was identified as 4-mono-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->], and sachharide D was 4,4'-di-O-alpha-glucosyl-CTS, [-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-[alpha-D-Glcp-(1-->4)]-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->]. These structures led us to conclude that the glycosyltransfer catalyzed by CGTase was specific to the C4-OH of the 6-linked glucopyranosyl residues in CTS.     

Enzymatic synthesis of a novel cyclic pentasaccharide consisting of alpha-D-glucopyranose with 6-alpha-glucosyltransferase and 3-alpha-isomaltosyltransferase

A novel cyclic pentasaccharide (CPS) and a branched cyclic pentasaccharide (6G-CPS) consisting of d-glucopyranose were synthesized with 6-alpha-glucosyltransferase (6GT) and 3-alpha-isomaltosyltransferase (IMT) from Bacillus globisporus N75. The structure of CPS was cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The other, 6G-CPS, had the structure cyclo-[-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-[alpha-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->4)-alpha-D-Glcp-(1-->]. The formation of CPS was presumed to occur after the following four successive reactions: a 6-glucosyltransfer reaction with 6GT, a 4-glucosyltransfer reaction with 6GT, a 3-isomaltosyltransfer reaction with IMT, and a cyclization reaction with IMT.     

Synthesis of galactose-containing analogues of (1-->6)-branched (1-->3)-glucohexaose and its lauryl glycoside

Coupling of the trisaccharide acceptor either 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-5-O-acetyl-1,2-O-isopropylidene-alpha-D-glucofuranose (13) or lauryl 2,4,6-tri-O-acetyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,5-di-O-acetyl-alpha-D-glucopyranoside (15) with the trisaccharide donor 2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-glucopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate (12) gave alpha-linked hexasaccharides 14 and 16, respectively, while coupling of either 13 or 15 with trisaccharide donor 2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->3)-[2,3,4,6-tetra-O-benzoyl-beta-D-galactopyranosyl-(1-->6)]-2,4-di-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate 17 did not afford any hexasaccarides. The analogues of the immunomodulator beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-beta-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp (1) was obtained by deprotection of 14 and 16.     

Efficient synthesis of a heptasaccharide,the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642

alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->3)-[beta-D-Xylp-(1-->2)-][beta-D-Xylp-(1-->4)-]alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-chain lipopolysaccharide produced by Xanthomonas campestris strain 642 was synthesized as its methyl glycoside via 3-O-selective glycosylation of methyl alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (9) with 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (8), followed by dixylosylation with 2,3,4-tri-O-benzoyl-alpha,beta-D-xylopyranosyl trichloroacetimidate (12) and subsequent deacylation.     

An NMR study of the lactonization of alpha-N-acetylneuraminyl-(2 --> 3)-lactose

The composition of the products formed by treatment of commercial alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc (3'-sialyllactose) with glacial acetic acid was investigated by 1H-13C one- and two-dimensional NMR spectroscopy and fast atom bombardment-mass spectrometry. The data confirmed that the major product of the reaction was alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc-(1c --> 2b)-lactone, which reverted to the starting material on standing in aqueous solution at ambient temperature, but for which complete NMR assignments are reported. The NMR data led to the tentative conclusion that the reaction also yielded small amounts of lactose, and alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc-(1c --> 4b)-lactone which was stable in aqueous solution.     

Separation and structural analysis of some saponins from Quillaja saponaria Molina

A fraction of saponins from Quillaja saponaria Molina, QH-B, was fractionated by consecutive separations on three different reverse-phase HPLC systems. Eight compounds were isolated and the structures of these were elucidated mainly by sugar analysis and NMR spectroscopy. The structures consisted of a quillaic acid substituted with two different trisaccharides at C-3, beta-D-Galp-(1-->2)-[alpha-L-Rhap-(1-->3)]-beta-D-GlcpA and beta-D-Galp-(1-->2)-[beta-D-Xylp-(1-->3)]-beta-D-GlcpA, and a tetra- or pentasaccharide at C-28, beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3)]-alpha-L-Rhap-(1--> 2)-beta-D-Fucp and beta-D-Apif-(1-->3)-beta-D-Xylp-(1-->4)-[beta-D-Glcp-(1-->3) ]-alpha-L- Rhap-(1-->2)-beta-D-Fucp. These compounds were further substituted with an acyl group either at O-3 or O-4 of the fucose residue, which is the sugar linked to C-28 of the quillaic acid.     

Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Two hexasaccharides were formed in approximately equal quantities: alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc and alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Just one heptasaccharide was isolated from the reaction mixture, alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. We conclude that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages. The distribution of products may be kinetically determined.     

Structure of the O-specific polysaccharide of the lipopolysaccharide of Azospirillum brasilense Sp245

An O-specific polysaccharide was isolated from the lipopolysaccharide of a plant-growth-promoting bacterium Azospirillum brasilense Sp245 and studied by sugar analyses along with one- and two-dimensional 1H and 13C NMR spectroscopy, including NOESY. The polysaccharide was found to be a new rhamnan with a pentasaccharide repeating unit having the following structure:-->2)-beta-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->2)-alpha-D-Rhap-(1-->     

Type I arabinogalactan contains beta-D-Galp-(1-->3)-beta-D-Galp structural elements

Arabinogalactan type I from potato was partially degraded by endo-galactanase from Aspergillus niger. High-performance anion-exchange chromatography revealed that several of the oligomeric degradation products eluted as double peaks. To investigate the nature of these products, the digest was fractionated by Bio-Gel P2 chromatography. The pool that contained tetramers was treated with a beta-D-Galp-(1-->4)-specific galactosidase from Bifidobacterium adolescentis to obtain a dimer with deviating linkage type, which was further purified by BioGel P2 chromatography. By obtaining all (1)H and (13)C chemical shifts and the presence of intra residual scalar coupling (HMBC) it could be concluded that the dimer contained a beta-(1-->3)-linkage instead of the expected beta-(1-->4)-linkage. Using the same NMR techniques as for the dimer, it was found that the pool of tetramers consisted of the following two galactose tetramers: beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->4)-alpha/beta-Galp-OH and beta-Galp-(1-->4)-beta-Galp-(1-->4)-beta-Galp-(1-->3)-alpha/beta-Galp-OH. The fact that the deviating beta-(1-->3)-linked galactose was found at the reducing end of the dimer showed that this deviating linkage is present within the backbone. The beta-(1-->3)-galactosyl interruption appeared to be a common structural feature of type I arabinogalactans with a frequency ranging from approximately 1 in 160 (potato, soy, citrus) to 1 in 250 (onion).     

Structure and heterogeneity of the O-antigen chain of Salmonella Agona lipopolysaccharide

Lipopolysaccharide of Salmonella Agona smooth-type cells was obtained from bacteria by a hot phenol-water extraction procedure. Mild acid hydrolysis of lipopolysaccharide, followed by gel filtration, yielded the pure O-polysaccharide. Abequose, rhamnose, mannose, galactose and glucose in the molar ratio 0.8 : 1.0 : 1.0 : 1.1 : 0.5 were detected, and their linkages were established. Sugar configurations were determined by gas chromatography. Two repeating units, namely -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-alpha-d-Galp-(1-->and -->2)-[alpha-Abep-(1-->3)-]-alpha-d-Manp-(1-->4)-alpha-l-Rhap-(1-->3)-[alpha-d-Glcp-(1-->4)-]-alpha-d-Galp-(1-->, were deduced from nuclear magnetic resonance studies. The effort to separate them was unsuccessful. An immunochemical test performed by means of Western blotting with anti O12 serum demonstrated that glucose was present in the longer lipopolysaccharide chains, at some distance from the core region.     

Lactones of disialyl lactose: characterisation by NMR and mass spectra

The lactonisation of alpha-Neup5Ac-(2-->8)-alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc (disialyl lactose) was investigated. (1)H and (13)C NMR chemical shifts of disialyl lactose and alpha-Neup5Ac-(2-->8, 1-->9)-alpha-Neup5Ac-(2-->3, 1-->2)-beta-D-Galp-(1-->4)-D-Glc (disialyl lactose-dilactone) were assigned based on 1D and 2D NMR results, including edited HSQC, HSQC-TOSCY and HMBC. The time course of lactonisation was followed by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) with electrospray ionisation (ESI) mass spectrometry (MS) detection. The rate of lactonisation between alpha-(8)Neu5Ac and alpha-(3)Neu5Ac residues (lactonisation at the alpha-(2-->8) linkage) was faster than that of lactonisation between alpha-(3)Neu5Ac and Gal residues (lactonisation at the alpha-(2-->3) linkage). The mass spectra of disialyl lactose, its lactones, alpha-Neup5Ac-(2-->8)-alpha-Neup5Ac (alpha-(2-->8) disialic acid) and alpha-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-D-Glc-lactone (3'-sialyllactose-lactone) showed that the alpha-(2-->8) linkage between Neu5Ac residues is difficult to cleave in the ESI-MS, compared with the alpha-(2-->3) linkage between Neu5Ac and Gal residues.     

Synthesis of two oligosaccharides, the GPI anchor glycans from S. cerevesiae and A. fumigatus

Two oligosaccharides, alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)-alpha-D-Manp-(1-->4)-alpha-D-GlcpNAc (I) and alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->6)-alpha-D-Manp-(1-->4)-alpha-D-GlcpNAc (II), the glycosylphosphatidylinositol (GPI) anchor glycans from S. cerevesiae and A. fumigatus were synthesized as their methyl glycosides in a regio- and stereoselective manner. The pentasaccharide I was obtained from 6-O-selective glycosylation of methyl 2,3-di-O-benzoyl-alpha-D-mannopyranosyl-(1-->4)-2-acetamido-3,6-di-O-benzoyl-2-deoxy-alpha-D-glucopyranoside (8) with 2-O-acetyl-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (9), followed by benzoylation, deacetylation, and mannosylation, and then by deprotection. The hexasaccharide (II) was obtained via condensation of allyl 3,4,6-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->2)-3,4,6-tri-O-benzoyl-alpha-D-mannopyranoside (17) with 2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl-(1-->3)-2,4,6-tri-O-acetyl-alpha-D-mannopyranosyl trichloroacetimidate (16), followed by deallylation, trichloroacetimidation, and coupling with acceptor (8), and finally by deprotection.