Tommy Iversen

Antigenic determinants of Salmonella serogroups A and D1.Synthesis of trisaccharide glycosides for use as artificial antigens

The disaccharide glycoside 8-methoxycarbonyloctyl 4,6-O-cyclohexylidine-2-O-(tetra-O-benzyl-alpha-D-galactopyranosyl)-alpha-D-manno pyranoside (7) was used as a common intermediate to the trisaccharide determinants of both Salmonella serogroups A and D1. Acetalation of 8-methoxycarbonyloctyl alpha-D-mannopyranoside provided the 4,6-acetal derivative, which was selectively benzoylated to give the partially protected mannoside 4. Reaction of 4 with tetra-O-benzyl-alpha-D-galacto-pyranosyl chloride afforded the fully protected disaccharide, which, after transesterification, gave the selectively blocked, disaccharide glycoside (7). Addition of tyvelose by way of its 2,4-di-O-benzoyl-3,6-dideoxy-alpha-D-arabino-hexopyranosyl chloride derivative gave the blocked trisacchride determinant of Salmonella serogroup D1. 2,4,-Di-O-benzyl-3,6-dideoxy-alpha-D-ribo-hexopyranosyl chloride reacted with 7 to provide, after removal of blocking groups, the paratose-containing determinant of serogroup A.

The conformation of Salmonella O-antigenic oligosaccharides of serogroups A, B, and D1 inferred from 1H- and 13C-nuclear magnetic resonance spectroscopy☆

The conformational model derived by the HSEA calculation method was used to interpret the n.m.r. data for solutions of oligosaccharides corresponding to the Salmonella serogroups A, B, and D1 antigenic determinants. The favored conformer, derived by calculation, accounted for the observed, chemical-shift changes and accurately predicted the existence and magnitude of inter-ring proton n.O.e.s. Extensive proton-density and compression of proton, Van der Waals radii were correlated with deshielding of specific proton-resonances. The model of lipopolysaccharide conformation accounts for the known antigenic properties of Salmonella O-antigens.

The conformation of Salmonella O-antigenic polysaccharide chains of serogroups A, B, and D1 predicted by semi-empirical, hard-sphere (HSEA) calculations

The preferred conformation of the tetrasaccharide repeating units of Salmonella Serogroups A, B, and D1 have been calculated. The semiempirical calculations used the Hard-Sphere, Exo-Anomeric (HSEA) approach to derive a conformational model, which could be used to assist the interpretation of conformations significantly populated in aqueous solution. The calculated model was extended to include a pentasaccharide repeating unit bearing an alpha-D-glucopyranose-branch point. The 3,6-dideoxyhexose to D-mannose linkage was shown to possess a steep energy surface with a minimum, which results in good exposure of the dideoxyhexose O-2 and O-4 atoms. Stereochemical changes involving the equatorial or axial disposition of these atoms are the distinguishing structural features of the A, B, and D1 serogroups. A lipophilic surface involving the 6-deoxy groups of the dideoxy-D-hexose and L-rhamnose residues was identified, and the possible implications of these features in antigenic determinants is discussed. The preferred conformation predicted by the HSEA method correlates with the known antigenic specificities of polysaccharides belonging to these Salmonella serogroups.

Acid-catalysed benzylation and allylation by alkyl trichloroacetimidates

Benzyl and allyl trichloroacetimidate (1) and (2) are convenient reagents for the O-alkylation of hydroxy groups under mildly acidic conditions, which are compatible with imide, ester, and acetal protecting groups. The base-catalyzed addition of benzyl alcohol or allyl alcohol to trichloroacetonitrile provides a simple synthesis of these imidates, but published methods for the recovery of related molecules by distillation leads to variable amounts of a rearranged product, N-alkyl trichloroacetamide. A modified procedure, suitable for the large scale synthesis of (1) and (2) without the need for a distillation step, is reported. The introduction of benzyl and allyl ethers to a variety of carbohydrate derivatives illustrates the potential of these reagents.

Synthesis of the trisaccharide moiety of gangliotriosylceramide (asialo GM2)

The synthesis of the trisaccharide methyl glycoside beta-D-GalNAc-(1----4)-beta-D-Gal-(1----4)-beta-D-Glc-OMe, which corresponds to the carbohydrate portion of gangliotriosylceramide (asialo GM2), was accomplished by the reaction of 4-O-acetyl-3,6-di-O-benzoyl-2-deoxy-2-phthalimido-D-galactopyranosyl bromide (18) with a benzylated derivative of methyl 4-O-beta-D-galactopyranosyl-beta-D-glucopyranoside. Comparative studies with a 6-benzyl ether and a 6-benzoate revealed that the substituent at O-6 is crucial to the outcome of glycosylations at O-4, the ether derivative being much the more reactive. tert-Butyl 4-O-acetyl-3,6-di-O-benzoyl-2-deoxy-2-phthalimido-D-galactopyranoside, which was readily converted into the corresponding bromide 18, was obtained from the gluco derivative via a single-step, crown ether-assisted epimerization.

Benzyl trichloroacetimidate, a versatile reagent for acid-catalysed benzylation of hydroxy-groups

Benzyl trichloroacetimidate reacts with hydroxyl-groups under acid catalysis to give the corresponding benzyl etehrs in good yield; acid-labile protecting groups such as acetals are able to survive the reaction conditions.

Synthesis of streptococcal Groups A, C and variant-A antigenic determinants

The antigenic determinants of the cell wall polysaccharides belonging to the β-haemolytic Streptococci Groups A, A-variant, and C have been synthesized as the glycosides of 8-methoxycarbonyloctanol. In this form they may be used to generate artificial antigens and immunoabsorbents. The terminal disaccharide, 3-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-L-rhamnopyranoside, of the Group A polysaccharide was synthesized by a Konigs–Knorr reaction between 8-methoxycarbonyloctyl 2,4-di-O-benzoyl-α-L-rhamnopyranoside (1) and 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl bromide (2) which gave the antigenic determinant (6) after removal of the blocking groups. Similarly, addition of 3,4,6-tri-O-acetyl-2-azido-2-deoxy-β-D-galactopyranosyl chloride (3) to (1) gave a disaccharide (7). The suitably blocked benzylidene acetal (11) was treated with the glactopyranosyl chloride (3) to yield the trisaccharide (12). The deblocked trisaccharide, O-(2-acetamido-2-deoxy-α-D-galactopyranosyl)-(1→3)-O-(2-acetamido-2-deoxy-α-D-galactopyranosyl)-(1→3)-α-L-rhamnopyranoside (14), is similar to the determinant of the Group C streptococcal cell wall and is also related to the Forssman antigen. The previously synthesized disaccharide glycoside, 8-methoxycarbonyloctyl 3,4-di-O-benzyl-2O-(2,4-di-O-benzoyl-α-L-rhamnopyranosyl)-α-L-rhamnopyranoside (15) was subjected to sequential chain-extension reactions with 2-O-acetyl-3,4-di-O-benzyl-α-L-rhamnopyranosyl chloride (4) to give the trisaccharide (16) and from this the slectively blocked precursor (17) from which the tetrasaccharide (18) was formed by reaction with (4). The tetrasaccharide glycoside O-(α-L-rhamnopyranosyl)-(1→2)-O-(α-L-rhamnopyranosyl)-(1→3)-O-(α-L-rhamnopyranosyl)-(1→2)-α-L-rhamnopyranoside (19) mimics the core structure of Groups A and C streptococcal polysaccharides and is identical to the sequence of the variant-A cell-wall polysaccharide.