Vccines Based on Synthetic Glycoconjugates:? Our studies are aimed at designing and developing bacterial vaccines. Human bacterial pathogens may have surface exposed saccharides serving as virulence factors and as protective antigens. Such saccharides include capsular polysaccharides (CPS), lipopolysaccharides (LPS) and cell wall polysaccharides and may vary in size and complexity. We are studying synthetic chemical approaches to bacterial surface-exposed saccharide structures that can be used to elicit serum IgG antibodies against these organisms.? ? Synthetic Vaccine Against Shigella dysenteriae Type 1:? Endemic and epidemic shigellosis, an acute invasive disease of the lower intestine, afflicts millions of people worldwide with an estimated one million fatalities annually at a low infectious dose. Our approach to vaccine development against Shigella is based on the hypothesis that serum IgG antibodies to the O-specific polysaccharide (O-SP) domains of the lipopolysaccharides (LPS) of these organisms confer immunity to this pathogen. The synthetic oligosaccharides corresponding to the tetrasaccharide repeating unit of the O-SP of S. dysenteriae type 1 covalently linked to human serum albumin elicited O-SP-specific IgG in mice (PNAS, 1999;96:5194). The antibody levels were a function of both the saccharide chain length and their loading on the protein. These synthetic saccharide conjugates elicited significantly higher levels of IgG anti O-SP than conjugates prepared with the O-SP from the bacteria. We evaluated the influence of the non-reducing terminal monosaccharide on the serum antibody response to these conjugates. Synthetic oligosaccharides were prepared composed of hexa- to trideca-saccharide fragments of the native O-SP, having one of the four monosaccharide residues that constitute the repeating unit at their termini, and bound to BSA by a single-point attachment. The conjugates contained an average of 19 saccharide chains per BSA. The synthetic oligosaccharides inhibited the binding of serum raised against whole bacteria to its LPS to a similar extent but less than the native O-SP. The highest anti-LPS levels were elicited by conjugates having N-acetylglucosamine (10-mer) or galactose residues (7- and 11-mers) at their non-reducing termini. To test the effect of different carriers, hexa- to hexadecamer fragments of the O-SP were bound to diphtheria toxoid at an average of 7-8 copies. No significant differences were found at this level. Based on these experiments clinical trials are planned.? ? As part of our synthetic vaccine project we synthesized a tripalmitoyl cysteine derivative (Pal3Cys) that has been shown to have adjuvant properties in synthetic glycoconjugates containing covalently linked Pal3Cys. Our derivative is equipped with a linker that will allow its covalent binding to proteins under mild conditions. These experiments are underway.? ? Borrelia burgdorferi expresses a unique glycolipid on its surface composed of galactosyl-glycerol having palmitoly and oleoyl groups at the primary and secondary hydroxyl groups. Because of its surface location on the bacterium, this unusual glycolipid may be a candidate for vaccine development. The natural glycolipid is slightly immunogenic and it is likely that its immunogenicity may be enhanced by covalent attachment to an immunogenic protein. We have studied chemical synthesis of this glycolipid in a form that allows its covalent attachment to proteins. Starting from commercially available R and S isopropylidene glycerols, we prepared in a multi step synthesis an advanced derivative featuring a latent aldehydo group at the end of the palmitoyl moiety. Current experiments are aimed at conjugating the lipid to carrier proteins using the reverse micelle technique.? ? Synthetic Organic Methods Development:? The success of oligosaccharide synthesis depends, to a large extent on the availability of versatile O-protecting groups that are stable under a variety of conditions, can chemo-selectively be removed by mild treatment and that allow easy purification and separation from the other components of the reaction mixture. In order to improve the efficiency of intermediate separation, we have tested the applicability of fluorinated protecting groups. These moieties render the compounds amenable to purification using fluorinated adsorbents that selectively bind fluorine-containing intermediates while not adsorbing those that contain conventional protecting groups. We have prepared selectively protected glucose derivatives that feature a fluorinated protecting group at their primary position. This intermediate was coupled to non-fluorinated glucose derivatives. The products were isolated in a highly efficient manner using the principle of fluorous adsorption. Based on these explorations, we are planning to use these techniques for the synthesis of oligosaccharides to be used as vaccine components.? ? A benzyl group is one of the most frequently used protecting groups in oligosaccharide synthesis. Despite its use for decades, a mild method for its introduction is still missing. We have prepared two potentially useful benzylating reagents, namely benzyl N-phenyltrifluoroacetimidate and S-benzyl-mercaptobenzoxazole and are currently evaluating their benzyl-donor ability.
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