Synthesis of di- to hexasaccharide 1,2-linked beta-mannopyranan oligomers, a terminal S-linked tetrasaccharide congener and the corresponding BSA glycoconjugates.

Homo oligomers of (1-->2)-beta-D-mannopyranosyl residues have been synthesized in order to study the unique immunological properties of the cell wall mannan of C. albicans. p-Chlorobenzyl-protected ulosyl bromide (2) in combination with the sterically hindered, participating solvent, pivaloyl nitrile, facilitated a new approach for the synthesis of these unique homooligomers ranging from disaccharide up to hexasaccharide. The glycosyl donor 2 demonstrates high diastereoselectivity over both the glycosylation and subsequent reduction step and minimizes the number of protecting group manipulations necessary for the synthesis. Congeners of the (1-->2)-beta-D-mannotetraose were synthesized containing a terminal S-linked (1-->2)-beta-D-mannopyranosyl residue. Deprotection of these compounds afforded the propyl glycosides as well as oligomers with amino terminated aglyconic tethers. The tethers were generated from the oligosaccharide allyl glycosides by photoaddition with 2-aminoethanethiol. The functionalized haptens were coupled to BSA via squarate conjugation, and the degree of incorporation was established by TOF mass spectrometry.

Glycan mimicry as a basis for novel anti-infective drugs.

The idea of using carbohydrate-based drugs to prevent attachment of microbial pathogens to host tissues has been around for about three decades. This concept evolved from the observation that many pathogenic microbes bind to complex carbohydrate sequences on the surface of host cells. It stands to reason, therefore, that analogs of the carbohydrate sequences pathogens bind to could be used to competitively inhibit these interactions, thereby preventing microbial damage to the host. This article will summarize some of the recent advances in developing such carbohydrate-based anti-infective drugs.

Mild oxidative one-pot allyl group cleavage.

[reaction: see text]. A new one-pot method is described for the removal of O- and N-allyl protecting groups under oxidative conditions at near neutral pH. The allyl group undergoes hydroxylation and subsequent periodate scission of the vicinal diol, followed by repetition of this reaction sequence on the enolic form of the aldehyde intermediate.

The observation of multivalent complexes of Shiga-like toxin with globotriaoside and the determination of their stoichiometry by nanoelectrospray Fourier-transform ion cyclotron resonance mass spectrometry.

We show by nanoelectrospray ionization (nanoES) Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS) that it is possible to observe oligosaccharide-protein complexes with dissociation constants in the millimolar range, such as P(k) trisaccharide (globotriaoside) complexed with the Shiga-like toxin (SLT) of pathogenic E. coli. It is further demonstrated that nanoES/FT-ICR MS is an exquisite method to study quantitative aspects of the association of mono- and polyvalent oligosaccharide ligands with multimeric proteins, such as the SLTs. At increasing trisaccharide:protein ratios it was shown that the B(5 )toxin subunit complexes with 5 P(k) trisaccharides and only after all 5 copies of site 2 are essentially filled do any of the remaining 10 receptor sites become occupied. From the distribution of bound P(k)'s at the five binding sites, it was possible to establish association constants for each of the five sites and to confirm that binding occurs noncooperatively, the association constants for each site are identical and that compared to site 1, site 2 exhibits a tenfold higher affinity for the globotriaoside synthetic ligand 1. The facile identification of the occupancy of binding sites represents information that is not readily available by other techniques. This sensitive and rapid estimation of association constants for protein-ligand complexes, which are free of unpredictable secondary effects that plague enzyme linked assays, is likely to find wide application.

Synthesis of a beta1,2-mannopyranosyl tetrasaccharide found in the phosphomannan antigen of Candida albicans.

The synthesis of a portion of the challenging beta1,2-mannosyl polymer found in the cell walls of Candida albicans was undertaken to develop a conjugate vaccine against C. albicans and to facilitate NMR conformational studies of this unique polysaccharide. The novel approach to the synthesis of tetrasaccharide 1 employed the modified ulosyl bromide 11 as the glycosyl donor which provided high diastereoselectivity. A participating solvent as well as p-chlorobenzyl protection facilitated the new approach.

Efficient synthesis of 3, 6-dideoxy-beta-D-arabino-hexopyranosyl-terminated LacdiNac glycan chains of the Trichinella spiralis parasite.

The synthesis of a linear trisaccharide epitope of the Trichinella spiralis N-linked glycan, in a form amenable to glycoconjugate formation, is reported. The trisaccharide contains the synthetically challenging LacdiNAc [beta-GalpNAc(1-->4)-beta-GlcpNAc] element, as well as a terminal 3,6-dideoxy-beta-D-arabino-hexopyranose (tyvelose) residue. An orthogonal protection strategy is described, which permits the protection and manipulation of three amino groups present in the disaccharide beta-GalNAc(1-->4)-beta-GlcNAc and the tether used to prepare neoglycoconjugates. The beta-linked dideoxyhexose was generated in excellent yield by the introduction of the dideoxyhexose unit as a beta-D-ribo-hexopyranoside (paratose) followed by an oxidation-reduction sequence to generate the beta-D-arabino configuration in high diastereomeric excess. The required dideoxyhexose donor was synthesized in a series of high-yielding steps from glucose utilizing the p-methoxyphenyl glycoside.

Shiga-like toxins are neutralized by tailored multivalent carbohydrate ligands.

The diseases caused by Shiga and cholera toxins account for the loss of millions of lives each year. Both belong to the clinically significant subset of bacterial AB5 toxins consisting of an enzymatically active A subunit that gains entry to susceptible mammalian cells after oligosaccharide recognition by the B5 homopentamer. Therapies might target the obligatory oligosaccharide-toxin recognition event, but the low intrinsic affinity of carbohydrate-protein interactions hampers the development of low-molecular-weight inhibitors. The toxins circumvent low affinity by binding simultaneously to five or more cell-surface carbohydrates. Here we demonstrate the use of the crystal structure of the B5 subunit of Escherichia coli O157:H7 Shiga-like toxin I (SLT-I) in complex with an analogue of its carbohydrate receptor to design an oligovalent, water-soluble carbohydrate ligand (named STARFISH), with subnanomolar inhibitory activity. The in vitro inhibitory activity is 1-10-million-fold higher than that of univalent ligands and is by far the highest molar activity of any inhibitor yet reported for Shiga-like toxins I and II. Crystallography of the STARFISH/Shiga-like toxin I complex explains this activity. Two trisaccharide receptors at the tips of each of five spacer arms simultaneously engage all five B subunits of two toxin molecules.

Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses.

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.

Molecular basis for the lack of mimicry of Brucella polysaccharide antigens by Ab2gamma antibodies.

The crystal structure of the complex of an anti-Id Fab with an Fab specific for a Brucella polysaccharide antigen has previously been reported (Evans et al., 1994, J. Mol. Biol. 241, 691-705). To complement this study, the binding characteristics and immunological properties of this Ab2 and two others raised with a second anti-Brucella antibody were investigated, including quantitative kinetic measurements by surface plasmon resonance. The affinities of the Fabs from the Ab2s for the Ab1s were three orders of magnitude greater than those estimated for the antigen, but the Ab2s failed to induce antigen-binding Ab3s, that is, they were of the Ab2gamma type. The avidities of the Ab1s for antigen were however within one order of magnitude of their avidities for Ab2. Tests of 16 other anti-Brucella polysaccharide antibodies showed that the two idiotopes were not present in them, and in confirmation of the lack of a dominant idiotope, N-terminal sequencing of their H and L chains showed a wide variety of V genes were employed in the immune response to the Brucella polysaccharides. The failure of the Ab2 to induce antigen-reactive Ab3 thus appears to be due to neither intrinsic affinity nor idiotope frequency, but arises instead from structural reasons, for example, the incomplete penetration of the Ab2 into the binding-site cleft of the Ab1. The surface topography of polysaccharide antigens and their binding-sites thus appears to be especially difficult for Ab2s to mimic and will restrict their routine use as surrogates for T-cell independent polysaccharide antigens.

Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli.

The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.

Crystal state and solution conformation of the B blood group trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)]-beta-D-Galp-OCH3.

The crystal structure of the human B blood group related trisaccharide alpha-L-Fucp-(1-->2)-[alpha-D-Galp]-(1-->3)-beta-D-Galp-OCH3 (1) has been determined. The solution structure of 1 was studied by two-dimensional NMR techniques at 600 MHz in D2O solution and the conformational properties were analyzed in terms of the torsional angles phiH and psiH, derived from 3JCH coupling constants, and 10 inter-residue proton-proton distances. 3JCH could be accurately measured by a recently introduced two-dimensional heteronuclear correlation experiment (EXSIDE). The nuclear Overhauser enhancement-derived distances and the calculated torsion angles were compared with the same information available from the crystal structure. The agreement is excellent, indicating that the trisaccharide adopts a restricted conformation in solution, which was also predicted by the Hard Sphere Exo-Anomeric forcefield. The data of 1 are complemented by NMR studies of the closely related alpha-L-Fucp-(1-->2)-[6-deoxy-alpha-D-Galp]-(1-->3)-beta-D-Galp O-(CH2 )7CH3 trisaccharide (2).

The synthesis of 16-mercaptohexadecanyl glycosides for biosensor applications.

The Pk trisaccharide and the central disaccharide element of asialo GM1 activated as their trichloroacetimidates were each used to glycosylate 16-(p-toluensulfonyloxy) hexadecanol 1. Displacement of the tosyl group by thiocyanate followed by sodium borohydride reduction and saponification afforded oligosaccharide 16-mercaptohexadecanyl glycosides that were isolated as the corresponding disulfides 6 and 17 unless oxygen was rigorously excluded from the solvents used for work-up. Dithiothreitol reduction of disulfides and subsequent isolation under an inert atmosphere with degassed solvents gave the thiols 7 and 18. Chemisorption of omega-glycosyl alkanethiols and alkanethiols onto gold electrodes produces self-assembled monolayers that can act as amperometric biosensors for the detection of proteins that bind to the immobilized oligosaccharide epitope.

Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes.

The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 --> Gly, Gly-235 --> Ser, Leu-266 --> Met, and Gly-268 --> Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase. To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time. A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the kcat being 5-fold higher for the A donor. Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 --> Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the kcat and increased specificity. The increases observed in kcat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

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.

Fucosyltransferase-catalyzed formation of L-galactosylated Lewis structures.

The Lewis (alpha 1-3/4) fucosyltransferase isolated from human milk could be used for preparative fucosylations of the disaccharide acceptors Gal(beta 1-3)GlcNAc(beta 1-O)R (at position OH-4) and Gal(beta 1-4)GlcNAc(beta 1-O)R (at position OH-3) [R = (CH2)8COOMe]. As donors GDP-L-Gal and deoxygenated derivatives were used to lead to a series of novel modified trisaccharides of the Lewis(a) and the Lewis(x) type, respectively.

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.

Synthesis and conformational studies of the tyvelose capped, Lewis-x like tetrasaccharide epitope of Trichinella spiralis.

Chemical synthesis and high resolution NMR studies are reported for the tetrasaccharide epitope and constituent structures that occur as terminal elements of Trichinella spiralis cell surface glycans. The 2-(trimethylsilyl)ethyl and methyl glycosides of Lewis-x type trisaccharides, in which beta GalNAc replaces beta Gal were synthesized from monosaccharide synthons utilizing thioglycoside and halide-ion glycosylation methods. The unique 3,6-dideoxy-beta-D-arabinohexopyranosyl residue that caps the structure was introduced to selectively protected 2-(trimethylsilyl)ethyl and methyl trisaccharide glycosides by utilizing an insoluble silver zeolite catalyst and a glycosyl halide. In separate reactions not only were the principal targets obtained but also the corresponding alpha-linked tetrasaccharides. A comparison of the NMR spectra of the methyl tetrasaccharide glycosides showed that at the site of the alpha-linked tyvelose structure, specific GalNAc resonances (C-1, C-2, C-3) possess uncharacteristically wide 13C (8-21 Hz) and 1H lines. The beta-linked tetrasaccharide glycoside, that represents the native parasite epitope, exhibited only narrow line widths (3 Hz, 13C). Since NOE derived distance constraints for the alpha-linked tyvelose structure were not consistent with the existence of unusual glycosidic conformers, the origin of the limited number of wide lines was attributed to local rigidity in the GalNAc residue, at the site of tyvelose glycosylation.

Analysis by surface plasmon resonance of the influence of valence on the ligand binding affinity and kinetics of an anti-carbohydrate antibody.

The kinetics of ligand binding by Se155-4, an antibody specific for the Salmonella serogroup B O-polysaccharide, were studied by surface plasmon resonance. Because trace amounts of oligomers in Fab and single-chain antibody variable domain (scFv) preparations resulted in biphasic binding profiles that were difficult to analyze, all kinetic measurements were performed on purified monomeric fragments and, for certain mutant scFv, dimeric forms. Results obtained with monomeric forms indicated that the relatively low affinity of the antibody was due to rapid dissociation (koff approximately 0.25 s-1). Dimeric forms generally showed off-rates that were approximately 20-fold slower and a 5-fold increase in association rate constants to approximately 2 x 10(5) M-1 s-1. Although the association phases for scFv dimers showed good curve fitting to a one component interaction model, the dissociation phases were biphasic, presumably because the availability and accessibility of sites on the antigen always leads to some monovalent attachment. The fast off-rate for dimers was the same as the monomer off-rate. Se155-4 IgG off-rates were very similar to those observed for scFv dimer, whereas the onrate was the same as that obtained with Fab and scFv monomer.