New conformational constraints in isotopically (13C) enriched oligosaccharides.

Multidimensional heteronuclear NMR studies have been applied to the resonance assignment and conformational analysis of 13C-enriched Neu5Acalpha2-3Galbeta1-4Glc. It is demonstrated that three-dimensional ROESY-HSQC experiments provide through-space distance restraints which cannot be observed with conventional homonuclear 1H techniques due to resonance overlap. In particular, connectivities demonstrating the existence of the "anti" conformation about the Galbeta1-4Glc glycosidic linkage are unambiguously observed. It is shown that 13C isotopic enrichment of the trisaccharide at a level >95% enables straightforward measurement of trans-glycosidic 1H-13C and 13C-13C coupling constants and a Karplus-type relation is derived for the latter. In total 15 conformational restraints were obtained for the trisaccharide in aqueous solution, all of which were in excellent agreement with theoretical parameters computed from a 5 ns molecular dynamics simulation of the glycan.

Structure of a glycoconjugate in solution and in complex with an antibody Fv fragment.

By use of heteronuclear (13C, 1H) NMR methods, the three-dimensional structure and dynamics of the glycoconjugate estrone-3-glucuronide (E3G) uniformly 13C enriched in the glucuronic acid moiety has been probed both in free solution and in association with an anti-E3G antibody single-chain Fv fragment. The glycan is found to exist in multiple conformations in free solution, with particularly large torsional fluctuations about the glycosidic linkage psi. Resonance assignments and distance restraints for the glycoconjugate in the bound state were obtained from heteronuclear proton-carbon-carbon-proton-COSY and isotope-edited NOESY techniques, respectively. Quantitation of the NOE data with a full-relaxation matrix approach showed that the antibody selects a conformation from the solution repertoire which does not correspond with either of the two lowest energy conformations of the free glycan, and the internal energy of the glycan in the bound state is estimated to be at most approximately 15 kcal/mol higher than the global minimum energy conformation. The glucuronide moiety undergoes a stacking interaction with an aromatic ring in the binding site, and both ring-current shifts and nuclear Overhauser effects computed from the predicted bound-state conformation are in good agreement with experiment. The bound-state conformation is also in good agreement with preliminary x-ray data on a related complex.

Terminal beta-linked tyvelose creates unique epitopes in Trichinella spiralis glycan antigens.

Indirect evidence that the immunodominant N-glycans of the parasite, Trichinella spiralis are capped by novel beta-linked 3,6-dideoxy-D-arabinohexopyranosyl residues (tyvelose, Tyv) was obtained from immunochemical assays employing monoclonal antibodies and synthetic oligosaccharides. Three of four previously characterized monoclonal antibodies generated from the lymphocytes of T.spiralis infected rats bind BSA glycoconjugates bearing the synthetic epitope beta-D-Tyvp(1-->3)-beta-D-GalNAcp but not to the corresponding alpha-D-Tyvp(1-->3)-beta-D-GalNAcp-BSA glycoconjugate. Monosaccharide and disaccharide glycoside inhibition data mirrors the results of the direct binding experiments. The branched tetrasaccharide beta-D-Tyv(1-->3)-beta-D-GalNAcp(1-->4)[alpha-L-Fucp(1 -->3)] beta-D-GalNAcp is the most active synthetic oligosaccharide inhibitor for all four monoclonal antibodies studied, while the corresponding alpha-D-Tyv containing tetrasaccharide and the core trisaccharide beta-D-GalNAcp(1-->4)[alpha-L-Fucp(1-->3)]beta-D-GlcNAcp+ ++ are inactive. The exceptional inhibitory activity of the disaccharide beta-D-Tyvp(1-->3)-beta-D-GalNAcp with one mAb (18H) compared to that of the branched tetrasaccharide beta-D-Tyvp(1-->3)-beta-D-GalNAcp(1-->4)[alpha-L-Fucp( 1-->3)]-beta-D-GlcNAcp is indicative of the presence of linear, nonfucosylated glycan epitopes (beta-D-Tyvp(1-->3)-beta-D-GalNAcp(1-->4) beta-D-GlcNAcp) that lack a fucose residue in one arm of the antigenic, tetra-antennary N-glycan. This observation supports earlier FAB-mass spectrometry evidence for the existence of tetra-antennary, core fucosylated glycans that lack a fucose residue on one of their antennae.

Synthesis of alpha- and beta-linked tyvelose epitopes of the Trichinella spiralis glycan: 2-acetamido-2-deoxy-3-O-(3,6-dideoxy-D-arabino-hexopyranosyl)-beta -D-galactopyranosides.

The anomeric configuration of tyvelose, 3,6-dideoxy-D-arabino-hexopyranose, in the recently discovered glycan epitopes of the parasite Trichinella spiralis has not been established. Two 2-(trimethylsilyl)ethyl disaccharide glycosides, alpha- and beta-Tyv-(1-->3)-beta-D-GalNAc (4 and 5), have been synthesized to provide model compounds that, together with the methyl 3,6-dideoxy-alpha- and beta-D-arabino-hexopyranosides (2 and 3), aid the determination of the anomeric configuration of tyvelose residues in the parasite glycan, either indirectly by immunochemical inhibition data or directly by the technique of 1H NMR spectroscopy. Methyl 3,6-dideoxy-beta-D-arabino-hexopyranoside (3) was synthesized from methyl 2,3-anhydro-4,6-O-benzylidene-beta-D-mannopyranoside (9) by a method previously used for the alpha anomer 2. Benzylation of 2 provided a route to the glycosyl donor, 2,4-di-O-benzyl-3,6-dideoxy-alpha-D-arabino-hexopyranosyl chloride (30), that reacted with the selectively protected 2-acetamido-2-deoxy-D-galactopyranoside alcohol 18 in the presence of an insoluble silver zeolite catalyst to give the alpha- and beta-linked disaccharides 31 and 32. Glycosylation of the related 2-acetamido-2-deoxy-D-galactopyranoside alcohol 27 by 30 under similar conditions provided disaccharides 33 and 34 containing a tether. Deprotection of the saccharide and derivatization of the tether with 1,2-diaminoethane provided amide derivatives 35 and 36 suitable for the preparation of neoglycoconjugate antigens. Complete 1H and 13C NMR chemical shifts of the deprotected disaccharides and monosaccharides are reported.