Synthesis of the tetrasaccharide alpha-D-Glcp-(1-->3)-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp recognized by calreticulin/calnexin

The title compound as its methyl glycoside was efficiently synthesized using a block synthesis approach. Halide-assisted glycosidations between 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl iodide and ethyl 2-O-acetyl-4,6-di-O-benzyl-1-thio-alpha-D-mannopyranoside using triphenylphosphine oxide as promoter yielded, with complete alpha-selectivity, a disaccharide building block in high yield. The perbenzylated derivative of this proved to be an excellent donor affording 88% of the protected target tetrasaccharide in an NIS/AgOTf-promoted coupling to a known methyl dimannoside acceptor. Deprotection through catalytic hydrogenolysis then gave the target compound in 47% overall yield.     

Convergent synthesis of the pentasaccharide repeating unit of the O-antigenic polysaccharide of enterohaemorrhagic Escherichia coli O113

An acidic pentasaccharide repeating unit corresponding to the O-antigenic polysaccharide of enterohaemorrhagic Escherichia coli O113 as its p-methoxyphenyl glycoside has been synthesized in a convergent manner by adopting a [3+2] block glycosylation strategy. During the synthetic endeavor a one-pot reaction condition for stereoselective glycosylation and protecting group manipulation has been applied. All glycosylation steps are highly stereoselective with good to excellent yield.     

Synthesis of beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-[beta-L-Xylp-(1-->4)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap,the repeating unit of the O-antigen produced by Pseudomonas solanacearum ICMP 7942

An efficient synthesis of beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-[beta-L-Xylp-(1-->4)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap, the repeating unit of the O-antigen produced by Pseudomonas solanacearum ICMP 7942 and its isomer beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->4)-[beta-L-Xylp-(1-->2)]-alpha-L-Rhap-(1-->3)-alpha-L-Rhap was achieved via sequential assembly of the building blocks, allyl 2,3-O-isopropylidene-alpha-L-rhamnopyranoside (2), allyl 3,4-O-isopropylidene-alpha-L-rhamnopyranoside (3), allyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside (6), 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate (7), and 2,3,4-tri-O-benzoyl-beta-L-xylopyranosyl trichloroacetimidate (12). The process was carried out in a regio- and stereoselective manner using glycosyl trichloroacetimidates as donors and unprotected or partially protected rhamnopyranosides as acceptors in the presence of a catalytic amount of trimethylsilyl trifluoromethanesulfonate (TMSOTf).     

Synthesis of O-alpha-D-mannopyranosyl-(1 leads to 2)-O-alpha-D-mannopyranosyl-(1 leads to 2)-D-mannose, the repeating unit of the O8-antigen of Escherichia coli

The trisaccharide α- d-Man p-(1→2)-α- d-Man p-(1→2)- d-Man was synthesised by using a stepwise method. A key reaction in the preparation of the intermediates was the selective hydrogenolysis of mannopyranoside derivatives having both dioxane- and dioxolane-type benzylidene acetals, resulting in the exclusive cleavage of the former acetal rings.     

Convergent synthesis of the tetrasaccharide repeating unit corresponding to the O-antigen of the verotoxin-producing Escherichia coli O176

A convergent synthesis of the tetrasaccharide repeating unit of the O-antigen of the verotoxin producing E. coli O176 has been achieved in excellent yield adopting a [2+2] block glycosylation strategy. The β-D-mannosidic moiety of the tetrasaccharide was prepared from β-D-glucoside and α-D-galactosamine moiety was derived from D-galactal. The tetrasaccharide was synthesized as its 2-trimethylsilylethyl glycoside in excellent yield. All intermediate steps are high yielding.     

Synthesis of 8-azidooctyl glycoside derivatives of the O-chain repeating unit of Escherichia coli O9a lipopolysaccharide and a methylated analog

Described is the synthesis of 8-azidooctyl glycoside derivatives of the Escherichia coli serotype O9a O-chain tetrasaccharide repeating unit and the terminal tetrasaccharide motif in this polysaccharide, which contains a methyl group on O-3 of the distal mannopyranose residue. The assembly of these compounds involved the sequential addition of monosaccharide residues from the reducing to the nonreducing end of the molecule using glycosyl trichloroacetimidate donors. Both compounds were initially prepared as p-methoxyphenyl glycosides, which were converted to the corresponding 8-azidooctyl derivatives at a late stage in the synthesis.     

Identification of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-d-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->)

A structural characterization of bound water molecules in the cyclic tetrasaccharide, cyclo-{-->6}-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->), was carried out by NMR spectroscopy. H-1', 2'-OH, H-3', and 4'-OH of the 3-O-glycosylated residue and H-1 of the 6-O-glycosylated residue were found to cross-relax with protons of bound waters using the double-pulsed field-gradient spin-echo ROESY experiment. In the crystal structure, one water molecule is located in the center of the plate, and its temperature factor is very low, indicating that this water molecule is an intrinsic component.     

Synthesis of the pentasaccharide repeating unit of Escherichia coli O128 antigen

A pentasaccharide, 4-methoxyphenyl 2-acetamido-2-deoxy-β-d-galactopyranosyl-(1→4)-α-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-β-d-galactopyranosyl-(1→6)-[α-l-fucopyranosyl-(1→2)]-β-d-galactopyranoside (1), representing the repeating unit of Escherichia coli O128 antigen, was successfully prepared in 23% overall yield via a convergent ‘2+3’ glycosylation strategy.     

Synthesis of a hexasaccharide fragment of group E streptococci polysaccharide and the tetrasaccharide repeating unit of E. coli O7:K98:H6

Syntheses of a hexasaccharide, the dimer of the repeating unit of the group E streptococci polysaccharide, and a tetrasaccharide, the repeating unit of the E. coli O7:K98:H6, were achieved by constructing alternate alpha-L-(1-->2)- and alpha-L-(1-->3)-linked L-rhamnopyranose backbones and substituting with beta-linked D-glucopyranose side chains for the former, and a D-glucopyranosyluronate branch for the latter, respectively, at O-2 of the L-rhamnose ring.     

A practical synthesis of alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp, an O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c

An O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-[alpha-D-Glcp-(1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp was synthesized as its methyl glycoside. Acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenization and benzoylation gave allyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (3), and subsequent deacetylation of 3 with CH(3)COCl-MeOH gave the monosaccharide acceptor 4. Condensation of isopropyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (6) with 4 selectively afforded the alpha-(1-->3)-linked disaccharide 7. Condensation of 7 with the (1-->3)-linked disaccharide donor 9, followed by deallylation and trichloroacetimidation, afforded the tetrasaccharide donor 12. Coupling of 12 with disaccharide acceptor 13, followed by debenzylation and deacylation, furnished the target heterohexasaccharide 16.     

Synthesis of spacer-equipped di-, tri-, and the tetrasaccharide fragments of the deacetylated O-PS1 of Citrobacter gillenii O9a,9b, and a related pentasaccharide

The title rhamnooligosaccharides [alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, alpha-D-Rhap4NAc-(1-->2)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe, and alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->2)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-(1-->3)-alpha-D-Rhap4NAc-1-O-(CH(2))(5)COOMe] were synthesized in a stepwise fashion from 5-methoxycarbonylpentyl 4-azido-4,6-dideoxy-2-O-benzyl-alpha-D-mannopyranoside and orthogonally protected 1-thioglycoside glycosyl donors. The amorphous, final products were fully characterized as corresponding per-O-acetyl derivatives.     

Synthesis of a D-rhamnose branched tetrasaccharide, repeating unit of the O-chain from Pseudomonas syringae pv. Syringae (cerasi) 435

The first synthesis of a d-rhamnose branched tetrasaccharide, corresponding to the repeating unit of the O-chain from Pseudomonas syringae pv. cerasi 435, as methyl glycoside is reported. The approach used is based on the synthesis of an opportune building-block, that is the methyl 3-O-allyl-4-O-benzoyl-alpha-D-rhamnopyranoside, which was then converted into both a glycosyl acceptor and two different protected glycosyl trichloroacetimidate donors. Successive couplings of these three compounds afforded the target oligosaccharide. The reported synthesis is also useful to perform the oligomerization of the repeating unit.     

Solution structure of a pentasaccharide representing the repeating unit of the O-antigen polysaccharide from Escherichia coli O142: NMR spectroscopy and molecular simulation studies

Conformational studies have been performed of a pentasaccharide derived from the O-polysaccharide from Escherichia coli O142. The polymer was selectively degraded by anhydrous hydrogen fluoride and reduced to yield an oligosaccharide model of its repeating unit, which in the branching region consists of four aminosugars. A comparison of (1)H and (13)C chemical shifts between the pentasaccharide and the polymer showed only minor differences, except where the cleavage had taken place, indicating that the oligomer is a good model of the repeating unit. Langevin dynamics and molecular dynamics simulations with explicit water molecules were carried out to sample the conformational space of the pentasaccharide. For the glycosidic linkages between the hexopyranoside residues, small but significant changes were observed between the simulation techniques. One-dimensional (1D) (1)H,(1)H double pulsed field gradient spin echo (DPFGSE) transverse rotating-frame Overhauser effect spectroscopy (T-ROESY) experiments were performed, and homonuclear cross-relaxation rates were obtained. Subsequently, a comparison of interproton distances from NMR experiment and the two simulation approaches showed that in all cases the use of explicit water in the simulations resulted in better agreement. Hydrogen-bond analysis of the trajectories from the molecular dynamics simulation revealed interresidue interactions to be important as a cluster of different hydrogen bonds and as a distinct highly populated hydrogen bond. NMR data are consistent with the presence of hydrogen bonding within the model of the repeating unit.     

Generation of Escherichia coli O9a Serotype, a Subtype of E. coli O9, by Transfer of the wb* Gene Cluster of Klebsiella O3 into E. coli via Recombination

Genetic characterization of the wb* gene in a series of Escherichia coli and Klebsiella strains possessing the mannose homopolymer as the O-specific polysaccharide was carried out. The partial nucleotide sequences and PCR-restriction fragment length polymorphism analysis suggested that E. coli serotype O9a, a subtype of E. coli O9, might have been generated by the insertion of the Klebsiella O3 wb* gene into a certain E. coli strain.     

First synthesis of beta-D-Galf-(1-->3)-D-Galp--the repeating unit of the backbone structure of the O-antigenic polysaccharide present in the lipopolysaccharide (LPS) of the genus Klebsiella

beta-D-Galactofuranosyl-(1-->3)-D-galactopyranose (1), the repeating unit of the backbone structure of the O-antigenic polysaccharide present in the lipopolysaccharide (LPS) of the genus Klebsiella, has been efficiently synthesized using 1,2:5,6-di-O-isopropylidine-alpha-D-galactofuranose (3) as the glycosyl acceptor and 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranosyl trichloroacetimidate (6) as the glycosyl donor with TMSOTf as catalyst by the well-known Schmidt glycosylation method. The preparation of 3 was improved by increasing the ratio of DMF to acetone and employing a solid-supported catalyst.     

Synthesis of divalent beta-(1-->6)-branched (1-->3)-glucohexaose and trivalent beta-(1-->6)-branched (1-->3)-glucotriose

Hexaose, beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-alpha-D-Glcp-(1-->3)-beta-D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp, based dimers were synthesized by twofold glycosidation of the hexaosyl trichloroacetimidate with hexylene 1,6-diol, diethylene glycol and triethylene glycol, respectively. Meanwhile, a triose, beta-1D-Glcp-(1-->3)-[beta-D-Glcp-(1-->6)]-beta-D-Glcp, based trimer was obtained by glycosidation of the triosyl trichloroacetimidate with a glycerol-derived triol scaffold.     

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 an L-rhamnose tetrasaccharide, the common and major structure of the repeating unit of the O-antigenic polysaccharide of a strain of Klebsiella pneumoniae and Pseudomonas holci.

A tetrasaccharide, alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-L-Rhap, the common and major structure of the repeating unit of the O-antigenic polysaccharide of a strain of Klebsiella pneumoniae and Pseudomonas holci was synthesized as its methyl and octyl glycosides. Selective 3-O-glycosylation of allyl alpha-L-rhamnopyranoside with 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl trichloroacetimidate gave allyl 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-alpha-L-rhamnopyranoside (3). Benzoylation, deallylation, and trichloroacetimidation afforded 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl trichloroacetimidate (6). Self condensation of 3,4-di-O-benzoyl-beta-L-rhamnopyranose 1,2-methyl orthoester or 1,2-octyl orthoester gave methyl or octyl 2-O-acetyl-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl-(1-->2)-3,4-di-O-benzoyl-alpha-L-rhamnopyranoside (16 or 17), and subsequent selective deacetylation gave the disaccharide acceptor (18 or 19). Coupling of 6 with 18 (or 19), followed by deacylation in ammonia-saturated methanol, produced the target tetrasacharide.     

Synthesis of the 'primer-adaptor' trisaccharide moiety of Escherichia coli O8, O9, and O9a lipopolysaccharide

Described is the synthesis of the trisaccharide alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-beta-D-GlcpNAcO(CH2)8N3, the glycan portion of which corresponds to the 'adaptor-primer' moiety linking the O-chain and core oligosaccharide in the lipopolysaccharide of several Escherichia coli and Klebsiella pneumoniae serotypes. This report represents the first synthesis of this trisaccharide motif, and in the route involved, a key step is a [2+1] coupling of a protected Manp-(1-->3)-alpha-D-Manp glycosyl donor with a GlcpNAc acceptor. The azido group was included in the target to facilitate future preparation of neoglycoconjugates.     

Synthesis and biological activities of octyl 2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-beta-L-fucopyranoside

Octyl 2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-alpha-L-fucopyranosyl-(1-->3)-2-O-sulfo-alpha-L-fucopyranosyl-(1-->4)-2,3-di-O-sulfo-beta-L-fucopyranoside, a fucosyl pentasaccharide with a regular structure resembling the repeating unit of a natural sulfated fucan, was chemically synthesized using a convergent '2+3' strategy. Regioselective 3-O-silylation of beta-thiofucopyranoside and AgOTf-catalyzed glycosylation of the protected glycosyl trichloroacetimidate facilitated a one-pot trisaccharide synthesis. The synthesized target compound showed good antitumor activity in vivo, and promising anticoagulant activity in vitro.