Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Structural relation of the antigenic polysaccharides of Escherichia coli O40, Shigella dysenteriae type 9, and E. coli K47

O-polysaccharides were isolated from the lipopolysaccharides of Escherichia coli O40 and Shigella dysenteriae type 9 and studied by chemical analyses along with (1)H and (13)C NMR spectroscopy. The following new structure of the O-polysaccharide of E. coli O40 was established: -->2)-beta-D-Galp-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1--> TheO-polysaccharide structure of S. dysenteriae type 9 established earlier was revised and found to be identical to the reported structure of the capsular polysaccharide of E. coli K47 and to differ from that of the E. coli O40 polysaccharide in the presence of a 3,4-linked pyruvic acid acetal having the (R)-configuration (RPyr): -->2)-beta-D-Galp3,4(RPyr)-(1-->4)-beta-D-Manp-(1-->4)-alpha-D-Galp-(1-->3)-beta-D-GlcpNAc-(1-->     

Synthesis of spacer-equipped phosphorylated di-, tri- and tetrasaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139

The synthesis of oligosaccharide fragments of the O-specific polysaccharide of Vibrio cholerae O139 containing a 4,6-cyclic phosphate galactose residue linked to GlcNAc is described. 8-Azido-3,6-dioxaoctyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, obtained by condensation of 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide and 8-azido-3,6-dioxaoctyl 2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranoside, was converted to 8-azido-3,6-dioxaoctyl 3-O-benzyl-beta-D-galactopyranosyl-(1-->3)-2-acetamido-6-O-benzyl-2-deoxy-beta-D-glucopyranoside (6) by reductive opening of the acetal, followed by deacetylation and selective benzylation. Phosphorylation of 6 furnished two isomeric 4,6-cyclic 2,2,2-trichloroethyl phosphates. Glycosylation of the (S)-phosphate with 2,4-di-O-benzyl-3,6-dideoxy-alpha-L-xylo-hexopyranosyl bromide under halide-assisted conditions gave the desired tetrasaccharide, together with a trisaccharide. Global deprotection and reduction of the azide to an amine was effected by catalytic hydrogenation/hydrogenolysis to give the deprotected tetrasaccharide, which is functionalized for conjugation.     

Structure of the O-polysaccharide of Pseudomonas putida FERM P-18867

The O-polysaccharide of the lipopolysaccharide of Pseudomonas putida FERM P-18867 was found to contain D-mannose and D-rhamnose and have the following structure of the trisaccharide repeating unit:-->2)-alpha-D-Rhap-(1-->3)-alpha-D-Rhap-(1-->3)-beta-D-Manp-(1-->     

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->O(CH(2))(6)NH(2) (1), beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (2), beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (3), and beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (4), representing fragments of the repeating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5-8 were synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk beta-1,4-galactosyltransferase as a catalyst. The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal models.     

Exopolygalacturonate lyase from Thermotoga maritima: cloning,characterization and organic synthesis application

A new exopolygalacturonate lyase (Pel) gene of the hyperthermophilic bacterium Thermotoga maritima was cloned and overexpressed in Escherichia coli cells. A 42 kDa monomeric Pel was shown to undergo N-terminal processing by cleavage at a putative site between alanine and serine residues. The enzyme catalyzes selectively a beta-4,5 elimination at the third galacturonic unit from the reducing end of polygalacturonic acid by producing (4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid)-(1-->4)-(alpha-D-galactopyranosyluronic acid)-(1-->4)-alpha-D-galactopyranuronic acid (3) with a 60% yield. The optimum activity of the enzyme was detected at pH 9.5 and T> or=95 degrees C. The highly thermostable enzyme constitutes a useful catalyst for a simplified synthesis of 4,5-unsaturated trigalacturonic acid 3, a trisaccharide which is extremely difficult to obtain via chemical synthesis.     

Structure of the O-polysaccharide of Providencia stuartii O49

The O-specific polysaccharide chain (O-antigen) of the lipopolysaccharide (LPS) of Providencia stuartii O49 was studied using sugar and methylation analyses along with 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, ROESY, H-detected 1H, 13C HSQC and HMBC experiments. The polysaccharide was found to have the trisaccharide repeating unit with the following structure: -->6)-beta-D-Galp(1-->3)-beta-D-GalpNAc(1-->4)-alpha-D-Galp(1-->     

The structure of the O-specific polysaccharide from Salmonella cerro (serogroup K, O:6,14,18)

The following structure of the Salmonella cerro LPS O-chain repeating unit has been determined using NMR and chemical methods: -->4)-alpha-D-Man(1-->2)-alpha-D-Man(1-->2)-beta-D-Man(1-->3)-alpha-D-GalNAc-(1-->.     

Synthesis of alpha-series ganglioside GM1alpha containing C20-sphingosine

A synthesis of alpha-series ganglioside GM1alpha (III(6)Neu5AcGgOse4Cer) containing C20-sphingosine(d20:1) is described. Glycosylation of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside with the glucosamine donor ethyl 3-O-acetyl-2-deoxy-4,6-O-[(4-methoxyphenyl)methylene]-2-phthalimido-1-thio-beta-D-glucopyranoside furnished a beta-(1-->4)-linked trisaccharide. Reductive cleavage of the p-methoxybenzylidene group followed by intramolecular inversion of its triflate afforded the desired trisaccharide, which was transformed into a trisaccharide acceptor via removal of the phthaloyl and O-acetyl groups followed by N-acetylation. A tetrasaccharide acceptor was obtained by glycosylation of the trisaccharide acceptor with dodecyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-galactopyranoside, followed by removal of the p-methoxybenzyl group. Coupling of the tetrasaccharide acceptor with ethyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-1-thio-5-trichloroacetamido-D-glycero-D-galacto-2-nonulopyranosid)onate and subsequent radical reduction gave the desired GM1alpha saccharide derivative, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-eicosene-1,3-diol after conversion into the imidate.     

Flavonol tetraglycosides and other constituents from leaves of Styphnolobium japonicum (Leguminosae) and related taxa

Two flavonol tetraglycosides comprising a trisaccharide at C-3 and a monosaccharide at C-7 were isolated from the leaves of Styphnolobium japonicum (L.) Schott and characterised as the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranoside-7-O-alpha-rhamnopyranosides of quercetin and kaempferol. The 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside-7-O-alpha-rhamnopyranoside of kaempferol, the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-glucopyranosides of kaempferol and quercetin and the 3-O-alpha-rhamnopyranosyl(1-->2)[alpha-rhamnopyranosyl(1-->6)]-beta-galactopyranoside of kaempferol were also obtained from this species for the first time. Some or all of these flavonol tetra- and triglycosides were detected in 17 of 18 specimens of S. japonicum examined from living and herbarium material, although the most abundant flavonoid in the leaves was generally quercetin 3-O-alpha-rhamnopyranosyl(1-->6)-beta-glucopyranoside (rutin). The triglycosides, but not the tetraglycosides, were detected in herbarium specimens of Styphnolobium burseroides M. Sousa, Rudd & Medrano and Styphnolobium monteviridis M. Sousa & Rudd, but specimens of Styphnolobium affine (Torrey & A. Gray) Walp. contained a different profile of flavonol glycosides. The flavonol tetra- and triglycosides of S. japonicum were also present in leaves of Cladrastis kentukea (Dum. Cours.) Rudd, a representative of a genus placed close to Styphnolobium in current molecular phylogenies. An additional constituent obtained from leaves of Styphnolobium japonicum was identified as the maltol derivative, 3-hydroxy-2-methyl-4H-pyran-4-one 3-O-(4'-O-p-coumaroyl-6'-O-(3-hydroxy-3-methylglutaroyl))-beta-glucopyranoside.     

Conformation of the branched O-specific polysaccharide of Shigella dysenteriae type 2: molecular mechanics calculations show a compact helical structure exposing an epitope which potentially mimics galabiose

Conformational analyses of the branched repeating unit of the O-antigenic polysaccharide of Shigella dysenteriae type 2 have been performed with molecular mechanics MM3. A filtered systematic search on the trisaccharide alpha-D-GalNAc-(1-->3)-[alpha-D-GlcNAc-(1-->4)]-alpha-D-GalNAc forming the branch, shows essentially a single favored conformation. Also, the downstream alpha-D-GalNAc-(1-->4)-alpha-D-Glc linkage is sterically constrained. The alpha-D-Glc-(1-->4)-beta-D-Gal moiety, however, forms a more flexible link region between the branch points, and shows a 90 degrees bend similar to what is known for the galabiose moiety occurring in globo-glycolipids. The calculations indicate that consecutive repeating units in their minimum energy conformation arrange in a helical structure with three repeating units per turn. This helix is very compact and appears to be stabilized by hydrophobic interactions involving the N-acetyl groups at the branch points. Random conformational search suggests the existence of another helical structure with four repeating units per turn. It appears possible that the alpha-D-Glc-(1-->4)-beta-D-Gal moiety, which is exposed on the surface of the helical structures, can evade recognition by the immune system of the host by the mimicry of globo structures.     

Synthesis of a novel glycosphingolipid from the millipede, Parafontaria laminata armigera, and the assembly of its carbohydrate moiety into multivalent structures

A novel glycosphingolipid, beta-D-Manp-(1-->4)-[alpha-L-Fucp-(1-->3)]-beta-D-Glcp-(1-->1)-Cer, found in the millipede, Parafontaria laminata armigera, and multivalent derivatives of its carbohydrate moiety were synthesized. As the key step, the target glycolipid (1) was obtained through an inversion reaction at the 2-position of a beta-glucopyranoside residue yielding a beta-mannopyranoside. In addition, the synthesis of fluorescently labeled trimer and tetramer glycoconjugates (2, 3) was achieved by iterative amide bond formation using a monomer unit (24).     

Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas flavipulchra NCIMB 2033(T)

An acidic polysaccharide was isolated from Pseudoalteromonas flavipulchra type strain NCIMB 2033(T) and found to consist of 6-deoxy-L-talose (L-6dTal), D-galactose and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The identities of the monosaccharides were ascertained by sugar analysis and 1D 1H and 13C NMR spectroscopy in conjunction with 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments, which enabled determination of the following structure of the trisaccharide repeating unit of the polysaccharide:-->3)-alpha-L-6dTalp4Ac-(1-->3)-beta-D-Galp-(1-->7)-alpha-Kdop-(2-->.     

Structural elucidation of the O-antigenic polysaccharide from the enteroaggregative Escherichia coli strain 180/C3 and its immunochemical relationship with E. coli O5 and O65

The structure of the O-antigen polysaccharide (PS) from the enteroaggregative Escherichia coli strain 180/C3 has been determined. Sugar and methylation analysis together with (1)H and (13)C NMR spectroscopy were the main methods used. The PS is composed of tetrasaccharide repeating units with the following structure: -->2)beta-D-Quip3NAc-(1-->3)beta-D-RIBf-(1-->4)beta-D-Galp-(1-->3)alpha-D-GalpNAc-(1-->. Analysis of NMR data indicates that the presented sequence of sugar residues also represents the biological repeating unit of the O-chain. The structure is closely related to that of O-antigen polysaccharide from E. coli O5 and partially to that of E. coli O65. The difference between the O-antigen from the 180/C3 strain and that of E. coli O5 is the linkage to the D-Quip3NAc residue, which in the latter strain is 4-O-substituted. The E. coli O65 O-antigen contains as part of its linear pentasaccharide repeating unit a similar structural element, namely -->4)-beta-d-GalpA-(1-->3)-alpha-D-GlcpNAc-(1-->2)-beta-D-Quip3NAc-(1-->, thereby indicating that a common epitope could be present for the two polysaccharides. Monospecific anti-E. coli O5 rabbit serum did not distinguish between the two positional isomeric structures neither in slide agglutination nor in an indirect enzyme immunoassay. The anti-O65 serum did react with both the 180/C3 and O5 LPS showing a partial cross-reactivity.     

Structure of the O-polysaccharide of Proteus mirabilis CCUG 10701 (OB) classified into a new Proteus serogroup, O74

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus mirabilis CCUG 10701 (OB) and studied by chemical analyses and (1)H and (13)C NMR spectroscopy. The following structure of the tetrasaccharide repeating unit of the polysaccharide was established: --> 3)-beta-D-GlcpNAc6Ac-(1 --> 2)-beta-D-GalpA4Ac-(1--> 3)-alpha-D-GalpNAc-(1 --> 4)-alpha-D-GalpA-(1 -->, where the degree of O-acetylation at position 6 of GlcNAc is approximately 50% and at position 4 of beta-GalA approximately 60%. Based on the unique structure of the O-polysaccharide and serological data, it is proposed to classify P. mirabilis CCUG 10701 (OB) into a new Proteus serogroup, O74.     

Structure of a lactic acid ether-containing and glycerol phosphate-containing O-polysaccharide from Proteus mirabilis O40

An O-polysaccharide was isolated by mild acid hydrolysis from the lipopolysaccharide of Proteus mirabilis O40 and studied by NMR spectroscopy, including 2D 1H, 1H COSY, TOCSY, ROESY, and 1H, 13C HMQC experiments, along with chemical methods. The polysaccharide was found to contain an ether of GlcNAc with lactic acid and glycerol phosphate in the main chain and to have the following structure: --> 3)-beta-D-GlcpNAc4(R-Lac)-(1 --> 3)-alpha-D-Galp-(1 --> 3)-D-Gro-1-P-(O --> 3)-beta-D-GlcpNAc-(1 --> where D-GlcpNAc4(R-Lac) stands for 2-acetamido-4-O-[(R)-1-carboxyethyl]-2-deoxy-D-glucose. This structure is unique among the known structures of the Proteus O-polysaccharides, which is in agreement with the classification of the strain studied into a separate O-serogroup. A serological relatedness of P. mirabilis O40 with some other Proteus strains was revealed and discussed in view of the O-polysaccharide structures.     

Synthesis of pentose-containing disaccharides using a thermostable alpha-L-arabinofuranosidase

To date, the enzymatically-catalysed synthesis of pentose-containing compounds has been limited to the production of oligo-beta-(1-->3) and oligo-beta-(1-->4)-linked xylopyranosides. To our knowledge, no such syntheses have involved arabinofuranose or, indeed, any other sugars in the furanose configuration. In this report, we describe the use of a thermostable alpha-L-arabinofuranosidase for the synthesis of p-nitrophenyl alpha-L-arabinofuranosyl-(1-->2)-alpha-L-arabinofuranoside, p-nitrophenyl beta-D-xylopyranosyl-(1-->2)-beta-D-xylopyranoside, p-nitrophenyl beta-D-xylopyranosyl-(1-->3)-beta-D-xylopyranoside and benzyl alpha-D-xylopyranosyl-(1-->2)-alpha-L-arabinofuranoside. Importantly, this latter compound is synthesised in a highly regiospecific reaction, which leads to the production of a single disaccharide.     

Synthesis of tetra- and pentasaccharides corresponding to the capsular polysaccharide of Streptococcus pneumoniae type 9A&L, 9N and 9A

Two tetrasaccharides, alpha-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp and alpha-D-GlcAp-(1-->3)-alpha-D-Glcp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp (protected form), and a pentasaccharide, alpha-D-Glcp-(1-->4)-alpha-D-GlcAp-(1-->3)-alpha-D-Galp-(1-->3)-beta-D-ManpNAc-(1-->4)-beta-D-Glcp have been synthesised from 2-aminoethyl glycoside trisaccharide acceptors in a linear approach via consecutive alpha-glycosylations. Ethyl thioglycosides were used as glycosyl donors and DMTST in Et(2)O or NIS/TfOH in CH(2)Cl(2) were employed as promoters.     

Development of a coupled spectrophotometric assay for GlfT2, a bifunctional mycobacterial galactofuranosyltransferase

As a key constituent of their protective cell wall all mycobacteria produce a large structural component, the mycolyl-arabinogalactan (mAG) complex, which has at its core a galactan moiety of alternating beta-(1-->5) and beta-(1-->6) galactofuranosyl residues. Galactan biosynthesis is essential for mycobacterial viability and thus inhibitors of the enzymes involved in its assembly are potential drugs for the treatment of mycobacterial diseases, including tuberculosis. Only two galactofuranosyltransferases, GlfT1 and GlfT2, are responsible for the biosynthesis of the entire galactan domain of the mAG and we report here the first high-throughput assay for GlfT2. Successful implementation of the assay required the synthesis of multi-milligram amounts of the donor for the enzyme, UDP-Galf, 1, which was achieved using a chemoenzymatic approach. We also describe an improved expression system for GlfT2, which provides a larger amount of active protein for the assay. Kinetic analysis of 1 and a known trisaccharide acceptor for the enzyme, 2, have been carried out and the apparent K(m) and k(cat) values obtained for the latter are in agreement with those obtained using a previously reported radiochemical assay. The assay has been implemented in 384-well microtiter plates, which will facilitate the screening of large numbers of potential GlfT2 inhibitors, with possible utility as novel anti-TB drugs.     

Synthesis of the O-linked pentasaccharide in glycoproteins of Trypanosoma cruzi and selective sialylation by recombinant trans-sialidase

The mucin-like glycoproteins of Trypanosoma cruzi have novel O-linked oligosaccharides that are acceptors of sialic acid in the trans-sialidase (TcTS) reaction. The transference of sialic acid from host glycoconjugates to the mucins is involved in infection and pathogenesis. The synthesis of the pentasaccharide, beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-beta-D-Galp-(1-->6)-[beta-D-Galf-(1-->4)]-D-GlcpNAc and the corresponding alditol, previously isolated by reductive beta-elimination of the mucins, is described. The key step was the 6-O-glycosylation of a easily accessible derivative of beta-D-Galf-(1-->4)-D-GlcpNAc with a beta-D-Galp-(1-->2)-[beta-D-Galp-(1-->3)]-D-Galp donor using the trichloroacetimidate method. The beta-linkage was diastereoselectively obtained by the nitrile effect. The pentasaccharide is the major oligosaccharide in the mucins of T. cruzi, G strain and presents two terminal beta-D-Galp residues for possible sialylation by TcTS. A preparative sialylation reaction was performed with its benzyl glycoside and the sialylated product was isolated and characterized. NMR spectroscopic analysis showed that selective monosialylation occurred at the terminal (1-->3) linked galactopyranose.