Evaluation of a di-O-methylated glycan as a potential antigenic target for the serodiagnosis of human toxocariasis.

Serodiagnosis of human toxocariasis is based on the detection of specific IgG antibodies by the enzyme-linked immunosorbent assay (ELISA) using Toxocara larvae excretory-secretory (TES) antigens, but its production is a laborious and time-consuming process being also limited by the availability of adult females of T. canis as source for ova to obtain larvae. Chemical synthesis of the di-O-methylated (DiM) glycan structure found in the TES antigens has provided material for studying the antibody reactivity in a range of mammalian hosts, showing reactivity with human IgM and IgG. In this study, we have evaluated the performance of the DiM glycan against a panel of sera including patients with toxocariasis (n = 60), patients with other helminth infections (n = 75) and healthy individuals (n = 94), showing that DiM is able to detect IgG antibodies with a sensitivity and specificity of 91·7% and 94·7%, respectively, with a very good agreement with the TES antigens (kappa = 0·825). However, cross-reactivity was observed in some sera from patients with ascariasis, hymenolepiasis and fascioliasis. These results show that the DiM glycan could be a promising antigenic tool for the serodiagnosis of human toxocariasis.

Chlamydial lipopolysaccharide.

Chlamydiae are obligatory intracellular parasites which are responsible for various acute and chronic diseases in animals and humans. The outer membrane of the chlamydial cell wall contains a truncated lipopolysaccharide (LPS) antigen, which harbors a group-specific epitope being composed of a trisaccharide of 3-deoxy-D-manno-oct-2-ulosonic (Kdo) residues of the sequence alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo. The chemical structure was established using LPS of recombinant Escherichia coli and Salmonella enterica strains after transformation with a plasmid carrying the gene encoding the multifunctional chlamydial Kdo transferase. Oligosaccharides containing the Kdo region attached to the glucosamine backbone of the lipid A domain have been isolated or prepared by chemical synthesis, converted into neoglycoproteins and their antigenic properties with respect to the definition of cross-reactive and chlamydia-specific epitopes have been determined. The low endotoxic activity of chlamydial LPS is related to the unique structural features of the lipid A, which is highly hydrophobic due to the presence of unusual, long-chain fatty acids.

Analysis of specificity of interaction between synthetic key saccharide components of lipopolysaccharides and monoclonal antibodies to Coxiella burnetii.

Two monoclonal antibodies (MAbs) against the lipopolysaccharides (LPSs) of Coxiella burnetii (C.b.) strains Priscilla and Nine Mile were prepared characterized by their interaction with synthetic glycoconjugates representing parts of LPSs of C.b. in virulent phase. Both MAbs were directed against immunodominant epitopes comprising core constituent of LPSs, Kdo (3-deoxy-alpha-D-manno-2-octulo-pyranosylonic acid). ELISA showed that the anti-Nine Mile MAb 4/11 bound preferably to disaccharides (alpha-Kdo (2 --> 4) alpha-Kdo and alpha-Kdo (2 --> 4) alpha-(5d) Kdo), while the anti-Priscilla MAb 1/4/H bound to all conjugates, though with various intensity. On the other hand, immunoelectron microscopy revealed a positive binding of only one glycoconjugate, namely the trisaccharide alpha-Kdo (2 --> 4) alpha-Kdo (2 --> 4) alpha-Kdo-BSA, to both MAbs. In competitive ELISA (cELISA), the anti-Priscilla MAb 1/4/H distinguished the strains Nine Mile and Priscilla, while the anti Nine Mile MAb 4/11 did not.

In vitro fusion of tissue-derived endosomes and lysosomes.

We investigated the in vitro fusion of different endocytic compartments derived from perfused rat liver, where the cells are assumed to be in their physiological state. Specifically labelled early, late and transcytotic endosomes, as well as lysosomes were tested for their fusion properties. In addition to the expected ATP-dependent fusion between early endosomes, we observed fusion between early and late endosomes with similar efficiency, kinetics and cytosol dependence. Fusion between early endosomes and transcytotic vesicles could not be detected. Prolonged incubation of complementary labelled early endosomes under fusion-supporting conditions followed by Percoll gradient centrifugation revealed the occurrence of fusion product at a dense position, indicating fusion events between light and dense compartments. Incubation of membrane preparations containing avidin-labelled endosomes and biotin-dextran-loaded lysosomes resulted in the formation of avidin-biotin complexes, indicating that fusion between early and late endosomes is followed by fusion with lysosomes. This was verified by colocalization of fluorescently labelled endosomes and lysosomes, as assessed by laser scanning microscopy. Endosome fusion, as well as content mixing between endosomes and lysosomes, were dependent on temperature and ATP, and could be inhibited by N-ethylmaleimide (NEM). The NEM-sensitivity was localised on endosomes and in the cytosol, but not on lysosomes. These observations indicate that early and late endosomes of rat liver exhibit a high fusion competence in vitro, promoting not only homotypic, but also heterotypic fusion.

A monoclonal antibody recognizing the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo of Chlamydophila psittaci 6BC lipopolysaccharide.

A monoclonal antibody (mAb) S45-18 was generated against a synthetic neoglycoconjugate containing the trisaccharide alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo (Kdo, 3-deoxy-D-manno-oct-2-ulopyranosonic acid) which represents a structure of the lipopolysaccharide (LPS) from Chlamydophila psittaci 6BC. The antibody was characterized by binding and inhibition assays in ELISA using: (i) the immunizing antigen and chemically synthesized derivatives thereof; (ii) chlamydial elementary bodies (EB); and (iii) LPS of Chl. psittaci 6BC and Chlamydia trachomatis L2. The specificity was determined in comparison to that of mAb S25-23 recognizing the alphaKdo(2-->8)alphaKdo(2-->4)alphaKdo trisaccharide which represents an epitope shared by all species of the family. MAb S45-18 bound to an epitope of the structure alphaKdo(2-->4)alphaKdo(2-->4)alphaKdo, with lower reactivity with the (2-->8)-(2-->4)-linked analog. Using chlamydial EB or LPS, mAb S45-18 bound preferentially to LPS and EB of Chl. psittaci. Therefore, Chl. psittaci LPS contains, in addition to the known genus-specific epitope, a species-specific epitope.

Synthesis and immunochemical characterization of neoglycoproteins containing epitopes of the inner core region of Pseudomonas aeruginosa RNA group I lipopolysaccharide.

The disaccharide allyl 2-acetamido-2-deoxy-alpha-D-galactopyranosyl-(1-->3)-7-O-carbamoyl-L-glycero-alpha-D-manno-heptopyranoside 5 (GalNAc-cmHep-allyl) was synthesized starting from 1 and 2. Compound 5, cmHep-allyl and the disaccharide cmHep-(1-->3)-Hep-allyl were converted into cysteamine-spacered derivatives and conjugated to bovine serum albumin (BSA) to yield the neoglycoconjugates 7--9, respectively. These conjugates were used to immunize mice and to prepare monoclonal antibodies (mAbs) which were characterized in comparison to mAbs obtained after immunization with heat-killed Pseudomonas aeruginosa strain H4. Two antibodies obtained after immunization with the neoglycoconjugates bound strongly to cmHep-BSA and with lower affinity to cmHep-Hep-BSA but did not bind to GalNAc-cmHep-BSA or to H4 LPS. Another antibody obtained after immunization with heat-killed bacteria bound to LPS and GalNAc-cmHep-BSA but not to cmHep-Hep-BSA or cmHep-BSA

Synthesis of neoglycoproteins containing Kdo epitopes specific for Chlamydophila psittaci lipopolysaccharide.

The oligosaccharides alpha-Kdop-(2-->8)-alpha-Kdop-(2-->6)-beta-D- GlcpNAc-(1-->OAll) 4, alpha-Kdop-(2-->4)-alpha- Kdop-(2-->4)-alpha-Kdop-(2-->6)-beta-D-GlcpNAc-(1-->OAll+ ++) 10, and the branched Kdo tetrasaccharide alpha- Kdop-(2-->4)-[alpha-Kdop-(2-->8)]-alpha-Kdop-(2-->4)-a lpha-Kdop-(2-->OAll) 21 have been prepared using en bloc transfer of Kdo oligosaccharide bromide donors to protected mono- or disaccharide acceptors. Radical addition of cysteamine to the anomeric allyl glycosides afforded good yields of the corresponding 3-(2-aminoethylthio)propyl glycosides 5, 11 and 22. The spacer ligands were activated with thiophosgene and reacted with bovine serum albumin to give the neoglycoconjugates 6, 12 and 23 which were used to prepare solid-phase antigens in enzyme immuno-assays for the characterization of monoclonal antibodies against chlamydial LPS. The data showed that the (2-->8)-linked Kdo disaccharide and the (2-->8)-(2-->4)-linked Kdo trisaccharide portion of the neoglycoconjugate 23 were not available for binding of antibodies which recognize these structures as di- and trisaccharide, respectively.

Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II.

L-Glycero-D-manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L-glycero-D-manno-heptopyranose to Re-LPS and Rd(2)-LPS, respectively. It had been proposed that both reactions involve ADPL-glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferase-deficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-beta-D-manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro.

Occurrence of antibodies against chlamydial lipopolysaccharide in human sera as measured by ELISA using an artificial glycoconjugate antigen.

An artificial glycoconjugate containing, as a ligand, the deacylated carbohydrate backbone of a recombinant Chlamydia-specific lipopolysaccharide was used as a solid-phase antigen in ELISA to measure antibodies against chlamydial LPS. The specificity and reproducibility of the assay was shown by using a panel of prototype monoclonal antibodies representing the spectrum of antibodies also occurring in patient sera. These mAbs recognized Chlamydia-specific epitopes [alpha 2-->8-linked disaccharide of 3-deoxy-D-manno-octulosonic acid (Kdo) or the trisaccharide alpha Kdo-(2-->8)-alpha Kdo-(2-->4)-alpha Kdo] or those shared between chlamydial and Re-type LPS (alpha Kdo, alpha 2-->4-linked Kdo disaccharide). The assay was used to measure IgG, IgA and IgM antibodies against chlamydial LPS in patients with genital or respiratory tract infections. In comparison to the results obtained with sera from blood donors, it became evident that both types of infection result in significant changes in the profile of LPS antibodies.

Synthesis of a trisaccharide of 3-deoxy-D-manno-2-octulopyranosylonic acid (KDO) residues related to the genus-specific lipopolysaccharide epitope of Chlamydia.

The disaccharides, O-(sodium 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosylonate)-(2----8)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate, were prepared via glycosylation of methyl (allyl 4,5,7-tri-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosid)onat e with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-D-manno-2-octulopyranosyl bromide)onate under Helferich and Koenigs-Knorr conditions, respectively. Based on g.l.c.-m.s. data of the alpha- and beta-(2----8)-linked disaccharide derivatives, obtained after carbonyl- and carboxyl-group reduction, followed by methylation, the alpha-anomeric configuration was assigned to the terminal KDO-residue in the KDO-region of Chlamydial lipopolysaccharide. The trisaccharide O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-sodium (allyl 3-deoxy-alpha-D-manno-2-octulopyranosid)onate was obtained via block synthesis using an alpha-(2----8)-linked disaccharide bromide derivative as the glycosyl donor. Copolymerization of the allyl glycosides with acrylamide gave water-soluble macromolecular antigens, suitable for defining epitope specificities of monoclonal antibodies directed against Chlamydial LPS.

Crystal and molecular structure of allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosidonate)-monohydrate.

The alpha-(2-->8)-linked Kdo disaccharide derivative allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosidonate)-monohydrate C19H28O15Na2.H2O, M(r) = 542.32, is orthorhombic, P2(1)2(1)2(1) with a = 9.229(1), b = 12.036(1), c = 21.671(1) A, and Z = 4. The structure was solved by direct methods and refined to R = 0.040 for 2677 observed reflections. The torsion angles about the (2-->8)-glycosidic bond are stabilized by an intramolecular hydrogen bond between the carboxylate group at the anomeric carbon atom of the terminal Kdo residue and the hydroxyl group O-17 of the second Kdo moiety.

Synthesis of neoglycoproteins containing D-glycero-D-talo-oct-2-ulopyranosylonic acid (Ko) ligands corresponding to core units from Burkholderia and Acinetobacter lipopolysaccharide.

Glycal esters of Kdo derivatives were converted into 2,3-anhydro intermediates, which were transformed into D-glycero-D-talo-oct-2-ulopyranosylonic acid (Ko), as well as 3-O- and 4-O-p-nitrobenzoyl-Ko derivatives. The exo-allyl orthoester derivative, methyl [5,7,8-tri-O-acetyl-4-O-(4-nitrobenzoyl)-2,3-O-[(1-exo-allyloxy)-ethylidene]-D-glycero-beta-D-talo-oct-2-ulopyranos]onate, prepared from the 4-O-pNBz-protected Ko derivative, was elaborated into the alpha-Ko allyl ketoside, the reducing disaccharide alpha-Kdop-(2-->4)-Ko and the disaccharide alpha-Kdop-(2-->4)-Kop-(2-->OAll). Conversely, methyl[4,5,7,8-tetra-O-acetyl-3-O-(4-nitrobenzoyl)-alpha-D-glycero-D-talo-2-octulopyranosyl bromide]onate [Carbohydr. Res., 244 (1993) 69-84], was coupled with a Kdo acceptor to give the disaccharide alpha-Kop-(2-->4)-Kdop-(2-->OAll) after orthoester rearrangement and deprotection. The allyl glycosides were treated with cysteamine and converted into neoglycoproteins. The ligands correspond to inner core units from Acinetobacter haemolyticus and Burkholderia cepacia lipopolysaccharides.

GLC-MS of reduced, acetylated, and methylated (2----4)- and (2----8)-linked disaccharides of 3-deoxy-D-manno-octulopyranosonic acid (Kdo).

Synthetic alpha- and beta-(2----4)- and alpha- and beta-(2----8)-linked disaccharides of 3-deoxy-D-manno-octulopyranosonic acid (Kdo), of which the synthesis of the beta-(2----4)-linked compound is described here, were used to develop a simple GLC-MS method for the determination of their anomeric configuration. The GLC and GLC-MS data for the reduced and acetylated or methylated derivatives of the above compounds indicate the alpha-linked synthetic disaccharides to be identical to those isolated from bacterial lipopolysaccharides.

Synthesis of a neoglycoprotein containing the Lewis X analogous trisaccharide beta-D-GalpNAc-(1-->4)[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc.

The trisaccharide allyl glycoside 36 and related disaccharide part structures have been prepared using the 2-trichloroacetamido-2-deoxy-alpha-D-galactopyranosyl trichloroacetimidate derivative 9 as glycosyl donor under promotion with TMSOTf or Sn(OTf)2, respectively, to produce the beta-(1-->4) linkage to suitably protected glucosamine derivatives in fair yields. Fucosylation was effected employing the ethyl 1-thio glycosyl donor 20 in the presence of IDCP. Deprotection of the intermediates afforded the disaccharide allyl glycosides beta-D-GalpNAc-(1-->4)- beta-D-GlcpNAc 13, beta-D-GalpNClAc-(1-->4)-beta-D-GlcpNAc 14, alpha-L-Fucp-(1-->3)-beta-D-GlcpNAc 24, alpha-L-Fucp-(1-->4)-beta-D- GlcpNAc 31 and the branched trisaccharide allyl glycoside beta-D-GalpNAc-(1-->4)[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc 36. The trisaccharide which corresponds to a structural motif occurring in N-glycoprotein glycans from human urokinase, human recombinant protein C, phospholipase A2 as well as O-glycans, was converted into a neoglycoprotein following introduction of a cysteamine-derived spacer group and subsequent activation with thiophosgene.

Synthesis of carboxyl-reduced analogues related to the Chlamydia-specific Kdo trisaccharide epitope.

The disaccharides allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->4)-3-deoxy-a lph a-D- manno-2-octulopyranoside (8), allyl O-(3-deoxy-alpha-D-manno-2-octulopyranosyl)-(2-->8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosidonate) (24), and allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-3-deoxy-a lph a-D- manno-2-octulopyranoside (35), and the trisaccharides allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->4)-3-deoxy-a lph a-D-manno-2-octulopyranoside (13) and allyl O-(3-deoxy-alpha-D-manno-2-octulopyranosyl)-(2-->8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->4)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosidonate) (30) were prepared. The ketosidic linkages were formed in good yields and high stereoselectivity by BF3 . Et2O-catalyzed reaction of the per-O-acetylated 3-deoxy-alpha-D-manno-2-octulopyranosyl fluoride derivative (16) with 8-O-SiButMe2 derivatives 19 and 21. Coupling reactions using the Kdo monosaccharide bromide derivative 4 or the alpha-(2-->8)-linked Kdo disaccharide bromide derivatives 9 and 26 were performed under Helferich conditions in MeCN or MeNO2, respectively. The disaccharide halides were prepared in good overall yields starting from the readily available allyl beta-glycoside of Kdo. The deprotected oligosaccharides correspond to the genus-specific lipopolysaccharide epitope of Chlamydia and part structures thereof, containing the carboxyl-reduced Kdo-residues at the distal and proximal position of the Kdo trisaccharide epitope, respectively.

Synthesis of a tetrasaccharide of the genus-specific lipopolysaccharide epitope of Chlamydia.

Allyl 2-acetamido-2-deoxy-3,4-O-(1,1,3,3-tetraisopropyldisiloxan-1,3- diyl)-beta-D- glucopyranoside was coupled with methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosyl bromide)onate (1) to give a good yield of the alpha-(2----6)-linked disaccharide, isolated after deacetylation and regioselective conversion into the corresponding 7',8'-O-carbonyl or 7',8'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl) derivatives, respectively. Subsequent glycosylation with 1 gave a high yield of the alpha- and beta-(2"----4')-linked trisaccharide derivatives 16 and 18, whereas block synthesis using the alpha-(2----8)-linked Kdo-disaccharide bromide derivative 19 afforded a low yield of the corresponding alpha- and beta-(2"----4')-linked tetrasaccharide derivatives 20 and 22. Removal of the protecting groups furnished the disaccharide allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----6)-2-acetamido -2-deoxy- beta-D-glucopyranoside, the trisaccharide allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----6)-2-acetamido -2-deoxy- beta-D-glucopyranoside, and the tetrasaccharide allyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----8)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----4)-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----6)-2-acetamido -2-deoxy- beta-D-glucopyranoside in high yield. Copolymerization of the allyl glycosides with acrylamide gave artificial polyvalent haptens suitable for defining epitope specificities of monoclonal antibodies directed against Chlamydia lipopolysaccharides.

Synthesis of alternate linear and branched repeating units of the Escherichia coli LP 1092 capsular polysaccharide containing 3-deoxy-alpha-D-manno-2-octulosonic acid (KDO) linked to secondary positions of D-ribose.

The oligosaccharides, methyl 3-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-beta-D-ribofuranosid e, methyl 2-O-beta-D-ribofuranosyl-3-O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-beta-D-ribofuranosid e, and methyl O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2----2)-O-beta-D- ribofuranosyl-(1----2)-beta-D-ribofuranoside were prepared in high purity and good over-all yields. The constitutions of the trisaccharide derivatives correspond to the repeating units of the proposed linear and branched structures of the capsular polysaccharide(s) from Escherichia coli LP 1092. The alpha-KDO-(2----3)-beta-D-Ribf and alpha-KDO-(2----2)-beta-D-Ribf units were synthesized by a modification of the Helferich procedure using methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-2-octulopyranosyl bromide)-onate and appropriate beta-D-ribofuranosyl derivatives. The constitutional and configurational assignments were based on the 250-MHz 1H-n.m.r.-spectra of protected derivatives of the oligosaccharides.

Empirical 13C-n.m.r.-correlations between the Escherichia coli K 13 and LP 1092 capsular polysaccharides and model oligosaccharides containing D-ribose and 3-deoxy-D-manno-2-octulosonic acid.

The proton-decoupled, Fourier-transform, 13C-n.m.r. spectra of the two anomeric sodium (methyl 3-deoxy-7-O-beta-D-ribofuranosyl-alpha- and beta-D-manno-2-octulopyranosid)onates, of the two anomeric sodium [methyl 3-deoxy-7-O-(2-O-beta-D-ribofuranosyl-beta-D-ribofuranosyl)-alpha- or -beta-D-manno-2-octulopyranosid]onates, and of methyl 2-O-beta-D-ribofuranosyl-beta-D-ribofuranoside have been recorded. The constitutions of these compounds correspond to repeating units and partial structures of the capsular polysaccharides from Escherichia coli K 13, K 20, K 23, and LP 1092 strains. The 13C-n.m.r.-line patterns of these oligosaccharide derivatives and the corresponding polysaccharides show striking differences dependent upon the anomeric configurations of the KDO residues. These differences may be used for the identification, by visual or computer-assisted pattern analysis, of the anomeric configurations of KDO-residues in oligo- or poly-saccharides. Thus, it was confirmed that the KDO residues in the K 13, K 20, and K 23 polysaccharides have the beta anomeric configuration, whereas those in the LP 1092 polysaccharide have the alpha anomeric configuration.

Synthesis of allyl O-[sodium(alpha-D-glycero-D-talo-2- octulopyranosyl)onate]-(2-->6)-2-acetamido-2-deoxy-beta-D-glucopyranosi de, a core constituent of the lipopolysaccharide from Acinetobacter calcoaceticus NCTC 10305.

Reaction of methyl 2,6-anhydro-2,3-dideoxy-D-manno-2-octenoate 1 with 3-chloroperoxybenzoic acid gave the 2,3-anhydro derivative 2, which was converted into the per-O-acetylated anomeric methyl glycosides of D-glycero-D-galacto-2-octulopyranosylonic acid in good yield. Subsequent inversion of the configuration at C-3 and deprotection afforded sodium (methyl beta-D-glycero-D-talo-2-octulopyranosid)onate. Alternatively, 2 was transformed into methyl (alpha-D-glycero-D-talo-2- octulopyranosyl bromide(onate derivatives. Reaction with methanol or allyl 2-acetamido-2-deoxy- 3,4-O-(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-beta-D-g lycopyranoside, promoted by silver triflate, gave good yields of the corresponding orthoester derivatives. Me3Si triflate-catalyzed orthoester rearrangement and removal of the protecting groups afforded sodium O-(methyl alpha-D-glycero- D-talo-2-octulopyranosid)onate and the disacchanide, allyl O-[sodium(alpha-D-glycero-D-talo-2- octulopyranosyl)onate]-(2-->6)-2-acetamido-2-deoxy-beta-D-gl ucopyranoside in high yield.

Synthesis and NMR spectroscopic investigation of oligosaccharides containing Kdo and L-glycero-D-manno-heptopyranosyl residues.

The disaccharides O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)-sodium (allyl 3-deoxy-beta-D-manno-2-octulopyranosid)onate (8), O-L-glycero-alpha-D-manno-heptopyranosyl-(1-->7)-sodium (allyl 3-deoxy-beta-D-manno-2-octulopyranosid)onate (12), and O-alpha-D-mannopyranosyl-(1-->7)-sodium (allyl 3-deoxy-beta-D-manno-2-octulopyranosid)onate (21) and the branched trisaccharides O-L-glycero-alpha-D-manno-heptopyranosyl-(1-->7)-[O-(sodium 3-deoxy-alpha- and -beta-D-manno-2-octulopyranosylonate)-(2-->8)]-sodium (allyl 3-deoxy-beta-D-manno-2-octulopyranosid)onate (15 and 16) and O-alpha-D-mannopyranosyl-(1-->7)-[O-(sodium 3-deoxy-alpha-D-manno-2-octulopyranosylonate)-(2-->8)]-sodium (allyl 3-deoxy-beta-D-manno-2-octulopyranosid)onate (24) were prepared. Per-O-acetylated mannopyranosyl or Kdo bromide derivatives were employed for the glycosylation steps under Helferich conditions, whereas the imidate derivative 9 was used for the coupling of the L-glycero-D-manno-heptopyranosyl residues. The oligosaccharides were fully characterized by NMR spectroscopic data. Their structures correspond to an artificial linkage pattern providing a potential cross-reactive epitope for antibodies directed against the inner-core-region of enterobacterial as well as chlamydial lipopolysaccharides.