Yury E. Tsvetkov

5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in bacterial glycopolymers: chemistry and biochemistry.

Publisher Summary This chapter provides an overview of the chemistry of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids in bacterial glycopolymers. Ald-2-ulosonic acids are the important components of natural glycoconjugates. Sialic acids—namely, N - and O -acyl derivatives of 5-amino-3,5-dideoxy- D -glycero- D -galacto-non-2-ulosonic acid (neuraminic acid, Neu), generally occur in glycoconjugates of vertebrates and play a significant role in their recognition, regulation, and protection. A deamino analogue of neuraminic acid—3-deoxy- D -glycero- D -galacto-non-2-ulosonic acid (Kdn)—has also been found in a variety of animal tissues. 3-Deoxy- D - manno -oct-2-ulosonic acid (Kdo) is an essential component of lipopolysaccharides (LPSs) of Gram-negative bacteria that functions to link the carbohydrate portion to the lipid moiety. In rare cases, Kdo in LPS is replaced with a 3-hydroxylated analogue— D -glycero- D -talo-oct-2-ulosonic acid. The chapter focuses on the occurrence and characterization of derivatives of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids and presents experimental approaches that are used to identify them and to elucidate the structures of the bacterial polysaccharides that contain the nonulosonic acids. It also presents the recent data on the biosynthesis of these sugars and discusses their role in immune recognition.

Synthetic β-(1→6)-Linked N-Acetylated and Nonacetylated Oligoglucosamines Used To Produce Conjugate Vaccines for Bacterial Pathogens

ABSTRACT Vaccines for pathogens usually target strain-specific surface antigens or toxins, and rarely is there broad antigenic specificity extending across multiple species. Protective antibodies for bacteria are usually specific for surface or capsular antigens. β-(1→6)-Poly-N-acetyl-d-glucosamine (PNAG) is a surface polysaccharide produced by many pathogens, including Staphylococcus aureus, Escherichia coli, Yersinia pestis, Bordetella pertussis, Acinetobacter baumannii, and others. Protective antibodies to PNAG are elicited when a deacetylated glycoform (deacetylated PNAG [dPNAG]; <30% acetate) is used in conjugate vaccines, whereas highly acetylated PNAG does not induce such antibodies. Chemical derivation of dPNAG from native PNAG is imprecise, so we synthesized both β-(1→6)-d-glucosamine (GlcNH2) and β-(1→6)-d-N-acetylglucosamine (GlcNAc) oligosaccharides with linkers on the reducing termini that could be activated to produce sulfhydryl groups for conjugation to bromoacetyl groups introduced onto carrier proteins. Synthetic 5-mer GlcNH2 (5GlcNH2) or 9GlcNH2 conjugated to tetanus toxoid (TT) elicited mouse antibodies that mediated opsonic killing of multiple S. aureus strains, while the antibodies that were produced in response to 5GlcNAc- or 9GlcNAc-TT did not mediate opsonic killing. Rabbit antibodies to 9GlcNH2-TT bound to PNAG and dPNAG antigens, mediated killing of S. aureus and E. coli, and protected against S. aureus skin abscesses and lethal E. coli peritonitis. Chemical synthesis of a series of oligoglucosamine ligands with defined differences in N acetylation allowed us to identify a conjugate vaccine formulation that generated protective immune responses to two of the most challenging bacterial pathogens. This vaccine could potentially be used to engender protective immunity to the broad range of pathogens that produce surface PNAG.

Antibody to a conserved antigenic target is protective against diverse prokaryotic and eukaryotic pathogens

Significance Poly-N-acetylglucosamine (PNAG) has been identified as a conserved surface polysaccharide produced by major bacterial, fungal, and protozoal parasites, including malarial sporozoites and blood-stage forms, which can all be targeted for vaccination using this single antigen. Surface carbohydrates are among the most successful vaccines against human microbial pathogens but have tremendous variability that complicates vaccine development. The species of bacteria, fungi, and protozoa shown here to produce PNAG lack an identifiable genetic locus for this antigen’s biosynthetic proteins based on known loci, indicative of a possible evolutionary convergent acquisition of PNAG synthesis with potential important significance for microbial biology. Microbial capsular antigens are effective vaccines but are chemically and immunologically diverse, resulting in a major barrier to their use against multiple pathogens. A β-(1→6)–linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule is synthesized by four proteins encoded in genetic loci designated intercellular adhesion in Staphylococcus aureus or polyglucosamine in selected Gram-negative bacterial pathogens. We report that many microbial pathogens lacking an identifiable intercellular adhesion or polyglucosamine locus produce PNAG, including Gram-positive, Gram-negative, and fungal pathogens, as well as protozoa, e.g., Trichomonas vaginalis, Plasmodium berghei, and sporozoites and blood-stage forms of Plasmodium falciparum. Natural antibody to PNAG is common in humans and animals and binds primarily to the highly acetylated glycoform of PNAG but is not protective against infection due to lack of deposition of complement opsonins. Polyclonal animal antibody raised to deacetylated glycoforms of PNAG and a fully human IgG1 monoclonal antibody that both bind to native and deacetylated glycoforms of PNAG mediated complement-dependent opsonic or bactericidal killing and protected mice against local and/or systemic infections by Streptococcus pyogenes, Streptococcus pneumoniae, Listeria monocytogenes, Neisseria meningitidis serogroup B, Candida albicans, and P. berghei ANKA, and against colonic pathology in a model of infectious colitis. PNAG is also a capsular polysaccharide for Neisseria gonorrhoeae and nontypable Hemophilus influenzae, and protects cells from environmental stress. Vaccination targeting PNAG could contribute to immunity against serious and diverse prokaryotic and eukaryotic pathogens, and the conserved production of PNAG suggests that it is a critical factor in microbial biology.

Synthesis and NMR spectroscopy of nine stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids

Derivatives of 5,7-diamino-3,5,7,9-tetradeoxynon-2-ulosonic acids are essential constituents of some bacterial polysaccharides and glycoproteins. In order to establish reliably the configuration of the natural sugars, nine stereoisomeric 5,7-diacetamido-3,5,7,9-tetradeoxynon-2-ulosonic acids were synthesized, including di-N-acetyl-legionaminic and -pseudaminic acids (the D-glycero-D-galacto and L-glycero-L-manno isomers, respectively) and their isomers at C-4, C-5, C-7, and C-8 having the L-glycero-D-galacto, D-glycero-D-talo, L-glycero-D-talo, D-glycero-L-altro, L-glycero-L-altro, D-glycero-L-manno, and L-glycero-L-gluco configurations. Synthesis was performed by condensation of 2,4-diacetamido-2,4,6-trideoxy-L-gulose, -D-mannose, -D-talose, and -L-allose with oxalacetic acid under basic conditions, the reaction of the last two precursors being accompanied by epimerisation at C-2. The 1H and 13C NMR data of the synthetic compounds are discussed. Acetylated methyl esters of the C-7 and C-8 isomeric nonulosonic acids were prepared and used for analysis of the side-chain conformation by NMR spectroscopy.

Synthesis and Molecular Recognition Studies of the HNK-1 Trisaccharide and Related Oligosaccharides. The Specificity of Monoclonal Anti-HNK-1 Antibodies as Assessed by Surface Plasmon Resonance and STD NMR

The human natural killer cell carbohydrate, HNK-1, plays function-conducive roles in peripheral nerve regeneration and synaptic plasticity. It is also the target of autoantibodies in polyneuropathies. It is thus important to synthesize structurally related HNK-1 carbohydrates for optimizing its function-conducive roles, and for diagnosis and neutralization of autoantibodies in the fatal Guillain-Barré syndrome. As a first step toward these goals, we have synthesized several HNK-1 carbohydrate derivatives to assess the specificity of monoclonal HNK-1 antibodies from rodents: 2-aminoethyl glycosides of selectively O-sulfated trisaccharide corresponding to the HNK-1 antigen, its nonsulfated analogue, and modified structures containing 3-O-fucosyl or 6-O-sulfo substituents in the N-acetylglucosamine residues. These were converted, together with several related oligosaccharides, into biotin-tagged probes to analyze the precise carbohydrate specificity of two anti-HNK-1 antibodies by surface plasmon resonance that revealed a crucial role of the glucuronic acid in antibody binding. The contribution of the different oligosaccharide moieties in the interaction was shown by saturation transfer difference (STD) NMR of the complex consisting of the HNK-1 pentasaccharide and the HNK-1 412 antibody.

Synthesis of 1,2-trans-disaccharides via sugar thio-orthoesters

Abstract The reaction of sugar 1,2-thio-orthoesters in the d - gluco , d - galacto , d - manno , and l - rhamno series with primary and secondary trityl ethers of monosaccharides, in the presence of triphenylmethylium perchlorate as catalyst, affords, stereospecifically, derivatives of 1,2- trans -disaccharides in good yields. 4-Trityl ethers of benzyl 2-acetamido-3,6-di- O -acetyl-2-deoxy-α- d -glucopyranoside and methyl 2,3,6-tri- O -benzoyl-α- d -galactopyranoside exhibit low reactivity in glycosylation by thio-orthoesters. A reaction scheme for the glycosylation is discussed.

Synthesis and identification in bacterial lipopolysaccharides of 5,7-diacetamido-3,5,7,9-tetradeoxy-d-glycero-d-galacto- and -d-glycero-d-talo-non-2-ulosonic acids

5,7-Diacetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto- and -D-glycero-D-talo-non-2-ulosonic acids were synthesized by condensation of 2,4-diacetamido-2,4,6-trideoxy-D-mannose with oxalacetic acid. Comparison of the 1H and 13C NMR data and the specific optical rotation values of these monosaccharides and the corresponding L-glycero-D-galacto and L-glycero-D-talo isomers synthesized earlier [Tsvetkov, Y. E.; Shashkov, A. S.; Knirel, Y. A.; Backinowsky, L. V.; Zähringer, U. Mendeleev Commun. 2000, 90-92] with data of the natural compounds enabled the identification in bacterial lipopolysaccharides of derivatives of 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic (legionaminic) acid and epimers of legionaminic acid at C-4 and C-8.

Synthesis of Multivalent Carbohydrate-Centered Glycoclusters as Nanomolar Ligands of the Bacterial Lectin LecA from Pseudomonas aeruginosa

A family of fifteen glycoclusters based on a cyclic oligo-(1→6)-β-D-glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme-linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub-micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.

Synthesis of a common polysaccharide antigen of Pseudomonas aeruginosa as the 6-aminohexyl glycoside

The synthesis is described of a tritylated 1,2-O-cyanoethylidene derivative (3) of the trisaccharide alpha-D-Rha-(1----2)-alpha-D-Rha-(1----3)-D-Rha. Triphenylmethylium perchlorate-catalysed polycondensation of 3 in the presence of 6-phthalimidohexyl 2,4-di-O-benzoyl-3-O-trityl-alpha-D-rhamnopyranoside followed by deprotection afforded the 6-aminohexyl glycoside of a D-rhamnan corresponding to a common polysaccharide antigen of Pseudomonas aeruginosa.

Selectin Receptors 4: Synthesis of Tetrasaccharides Sialyl Lewis A and Sialyl Lewis X Containing A Spacer Group1,2

ABSTRACT Synthesis of two isomeric tetrasaccharides, namely Neu5Acα(2→3)Galβ(1→3)[Fucα(1→4)GlcNAcβ (sLea) and Neu5Acα(2→3)Galβ(1→4)[Fucα(1→3)]GlcNAcβ (sLex) as 3-aminopropyl glycosides is described. Preparation of these compounds was performed by sialylation of selectively protected trisaccharides Lea and Lex which contain three unsubstituted OH groups at positions 2, 3 and 4 of Gal residue. Glycosylation of Lex trisaccharide with ethylthio sialoside under promotion by NIS and TfOH in acetonitrile was effective and regio- and stereoselective to give sLex derivative in 81% yield. In contrast, sialylation of the Lca acceptor was accompanied by a variety of undesirable by-processes, namely. N-thioethylation of the GlcNAc residue, β-sialylation, and lactonisation. In order to improve the yield of sLca tetrasaccharide the glycosylation of Lea acceptor by sialyl donors of ethyl and phenyl thioglycoside (promoted by NIS-TfOH or NBS-Bu4NBr), xanthate (promotion by NIS-TfOH mixture or MeOTf) and phosphite (promote...