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High-resolution electrospray mass spectra (MS and MS/MS CID) of positive ions of a series of protonated, ammoniated, and metallated molecules of cyclic N-substituted oligo-&beta, (1&rarr, 6)-D-glucosamines differing in cycle size and N-acyl substituents were registered and interpreted. It was shown that the main type of fragmentation is a cleavage of glycosidic bonds of a cycle, and in some cases fragmentation of amide side chains is possible. If labile fragments in substituents (e.g., carbohydrate chains) are present, a decay of the cycle and an elimination of labile fragments are of comparable possibility. It was found that in some cases rearrangements with loss of an internal carbohydrate residue (IRL), or an internal part of a side chain, are feasible.

A linear tetramer of β-(1 → 6)-linked 3-azido-3-deoxy- d -allose containing glycosyl donor and glycosyl acceptor functions in the terminal monosaccharide units was prepared starting from 3-azido-3-deoxy-1,2:5,6-di-O-isopropylidene-α- d -allofuranose. Cyclization of the linear tetramer under glycosylation conditions afforded the corresponding cyclic tetrasaccharide in 77% yield; its deprotection and reduction of the azido groups resulted in the formation of the cyclic tetramer of 3-amino-3-deoxy- d -allose with axial amino groups, a potential scaffold for the synthesis of tetravalent functional clusters.

The disaccharide β-d-mannopyranosyl-(1 → 2)-d-mannopyranose obtained by chemical cleavage and enzymatic dephosphorylation of biotechnologically available phosphomannan was transformed over six steps into a biotinylated probe suitable for assessment of carbohydrate specificity of antibodies induced by yeast cell wall preparations.

3-Aminopropyl glycosides of a series of linear β-(1 → 3)-linked D-glucooligosaccharides containing from 3 to 13 monosaccharide units were efficiently prepared. The synthetic scheme featured highly regioselective glycosylation of 4,6-O-benzylidene-protected 2,3-diol glycosyl acceptors with a disaccharide thioglycoside donor bearing chloroacetyl groups at O-2' and -3' as a temporary protection of the diol system. Iteration of the deprotection and glycosylation steps afforded the series of the title oligoglucosides differing in length by two monosaccharide units. A novel procedure for selective removal of acetyl groups in the presence of benzoyl ones consisting in a brief treatment with a large excess of hydrazine hydrate has been proposed.

3-Aminopropyl β-(1 → 6)- d -glucotetraoside has been synthesized from 3-benzyloxycarbonylaminopropanol and 6- O -acetyl-2,3,4-tri- O -benzoyl- d -glucopyranosyl trichloroacetimidate by successive attachment of one monosaccharide unit in total yield of 22%. Free aminopropyl glycoside was converted into a biotin derivative that can be used for controlled immobilization of the oligosaccharide on streptavidin-coated ELISA plates and for tracing carbohydrate binding molecules.

The immunobiological efficacy of synthetically prepared mannooligosaccharides and a glucooligosaccharide mimicking the structure of Candida albicans cell wall glycans was assessed in vivo and in vitro to exploit immune responses. The exposure of mice splenocytes to BSA-based conjugates of synthetic oligomannosides and oligoglucoside revealed intense influence on T-cell subset polarization. The conjugates biased the immune responses towards Th1 and Th17 with respect to the prevalence of interferon-gamma (IFN-γ) and interleukin (IL)-17 (IL-17) over IL-4 and IL-10 levels. The inflammatory activity of the conjugates has been evaluated based on the induction of pro-inflammatory cytokines. Postvaccination, antimannooligosaccharide and antiglucooligosaccharide antisera were subjected to an evaluation of the structure-immunomodulation activity relationship. Clinical isolates of C. albicans CCY 29-3-32 and C. albicans CCY 29-3-164 were applied to study interactions between Candida cells and anti-oligosaccharide antibodies. In situ recognition of parietal oligomannosyl and oligoglucosyl sequences in C. albicans cell wall by the antisera raised against BSA-based conjugates of synthetic oligomannosides and oligoglucoside revealed the effective recognition of specific distribution of natural oligosaccharide sequences in the cell wall of C. albicans serotype A. With respect to these results, it can be concluded that new, synthetically prepared oligosaccharides mimicking Candida cell wall structures represent prospective immunobiologically effective components for further immunopharmacologically relevant Candida vaccine design.

A conjugate of a synthetic hexasaccharide fragment of the Streptococcus pneumoniae type 14 capsular polysaccharide with bovine serum albumin (BSA) has been prepared. The antigenic activity and specificity of this conjugate are comparable with those of natural antigens of S. pneumoniae type 14. The data suggest that the resulting synthetic conjugate can be used as a coating antigen in an experimental test system (based on enzyme immunoassay) for evaluating the antigenic activity and specificity of synthetic oligosaccharide ligands and for testing specimens of natural capsular polysaccharides and immune sera.

The synthesis was described of branched glucooctaoside bearing the β-(1 → 3)-glucotrioside side chain at O-6 of the second (from the reducing end) monosaccharide unit of the linear β-(1 → 3)-glucopentaoside core.

Oligosaccharides have a variety of versatile biological effects, but unlike peptides and oligonucleotides, investigation of the roles of oligosaccharides is not easy. Since biosynthesis of oligosaccharides does not comply with direct genetic control, their isolation from natural sources and biotechnological preparation are complicated, due to the heterogeneous composition of raw carbohydrates. Oligosaccharide synthesis is needed for the establishment or confirmation of the structure of natural glycocompounds. Also, synthetically prepared, defined oligosaccharides and their derivatives are becoming increasingly important tools for many biological and immunological research projects. The key step of oligosaccharide synthesis involves glycosylation, a reaction that builds glycosidic bonds. Usually, construction of 1,2-trans-bonds is easy, and therefore, this reaction can even be included into automated syntheses. At this time, installation of the 1,2-cis-bond remains simultaneously frustrating and compelling. In our and other laboratories, a strategy of α-selective (1,2-cis) glycosylation, relying on remote anchimeric assistance with acyl groups, is studied. The state of the art in this work is summarized in this review.

Cystic fibrosis (CF) is a congenital disease caused by a mutation in a gene responsible for the synthesis of a membrane protein called the cystic fibrosis transmembrane conductance regulator (CFTR). Resistance to Pseudomonas aeruginosa infection is closely related to the biological properties of CFTR; however, these properties have not been clearly linked to the known role of CFTR as a chloride and bicarbonate ion channel. Indeed, data indicate that CFTR is an epithelial cell receptor for P. aeruginosa, with CFTR binding to the oligosaccharide of the outer core region of the bacterial lipopolysaccharide (LPS), of which two distinct glycoforms have been identified. Binding leads to effective innate immunity to clear this pathogen in individuals with wild-type CFTR. To reveal the molecular basis of elimination of the bacterium through this interaction, the synthesis of pentasaccharides corresponding to both glycoforms of the outer core region of P. aeruginosa LPS was undertaken. Here we report the synthesis of the glycoform II. Like glycoform I, it was prepared as three pentasaccharides bearing naturally occurring N-alanyl and N-acetyl substituents in the galactosamine moiety as well as unnatural N-acetylalanine to reveal the role of the amino group in the alanyl substituent. Key features of the synthesis were two α-glucosylations with glucosyl donors bearing α-stereodirecting acyl groups at O-6 and/or O-3 and high-yielding reduction of the azido group followed by N-acylation and final O-debenzylation.

The driving force for the constant improvement and development of synthetic methodologies in carbohydrate chemistry is the importance of natural oligosaccharide chains in numerous biological phenomena such as cell growth, differentiation, adhesion, etc. Here, we report our syntheses of the spacer-armed oligosaccharides of sialylated lacto- and neo- lacto-, globo-, ganglio-, and sulfoglucuronylparagloboside-series, which include new rationally designed synthetic blocks, efficient solutions for the stereoselective construction of glycosidic bonds, and novel protection group strategies.

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.

The stereodirecting effect of a 3- O -acetyl protecting group, which is potentially capable of the remote anchimeric participation, and other protecting groups in 2- O -benzyl glucosyl donors with flexible and rigid conformations has been investigated. To this aim, an array of N -phenyltrifluoroacetimidoyl and sulfoxide donors bearing either 3- O -acetyl or 3- O -benzyl groups in combination with 4,6-di- O -benzyl, 6- O -acyl-4- O -benzyl, or 4,6- O -benzylidene protecting groups was prepared. The conformationally flexible 3-O-acetylated glucosyl donor protected at other positions with O -benzyl groups demonstrated very low or no α-stereoselectivity upon glycosylation of primary or secondary acceptors. On the contrary, 3,6-di-O-acylated glucosyl donors proved to be highly α-stereoselective as well as the donor having a single potentially participating acetyl group at O-6. The 3,6-di-O-acylated donor was shown to be the best α-glucosylating block for the primary acceptor, whereas the best α-selectivity of glycosylation of the secondary acceptor was achieved with the 6-O-acylated donor. Glycosylation of the secondary acceptor with the conformationally constrained 3- O -acetyl-4,6- O -benzylidene-protected donor displayed under standard conditions (−35 °C) even lower α-selectivity as compared to the 3- O -benzyl analogue. However, increasing the reaction temperature essentially raised the α-stereoselectivities of glycosylation with both 3- O -acetyl and 3- O -benzyl donors and made them almost equal. The stereodirecting effects of protecting groups observed for N -phenyltrifluoroacetimidoyl donors were also generally proven for sulfoxide donors.

A broad spectrum of carbohydrate-derived azides can be efficiently reduced by DTT in the presence or absence of base to the corresponding amines.

Several studies have established the potential efficacy of humoral immunity, primarily mannan-specific antibodies, in host protection against major fungal pathogen Candida albicans. In this study, we analysed humoral immune response induced by immunization with BSA-based conjugates bearing synthetic α-1,6-branched oligomannosides (pentamannosides (M5) or hexamannosides (M6)) mimicking antigenic sequences of Candida cell wall mannan. We analysed the ability of antibodies prepared by immunization to recognize relevant antigenic determinants in mannan polysaccharide structure and in C. albicans yeast and hyphal morphoforms. M6-BSA conjugate induced markedly higher levels of mannan-specific IgG compared with M5-BSA conjugate. In contrast to M5-BSA conjugate, M6-BSA conjugate induced immunoglobulin isotype class switch from IgM to IgG, as revealed also from ELISPOT analysis. Immunization-induced antibodies showed higher reactivity with hyphal form of C. albicans cells. The reduced immunogenicity of M5-BSA conjugate seems to be related to branching point location at terminal non-reducing end in comparison with M6-BSA oligomannoside with branching point at non-terminal location. Candidacidal activity assay revealed different capacity of sera prepared by immunization with M5-BSA and M6-BSA conjugates to improve candidacidal activity of polymorphonuclear leucocytes. Limited capacity of α-1,6-branched oligomannoside – BSA conjugates to induce antibodies significantly enhancing candidacidal activity of polymorphonuclear leucocytes – was presumably related to absence of antibodies with strong reactivity to corresponding antigenic determinants in natural cell wall mannan and with reduced ability to activate complement. The study documented markedly structure-dependent immunogenicity and limited capacity of branched α-mannooligosides conjugates to induce production of potentially protective antibodies.

The conformational behavior of a series of linear and cyclic oligo-(1→6)-β- d -glucosamines and their N-acetylated derivatives, which are related to fragments of natural poly- N -acetylglucosamine, was studied by theoretical molecular modeling and experimental determination of transglycosidic vicinal coupling constants 3 J C,H and 3 J H,H . Molecular dynamics simulations were performed under several types of conditions varying in the consideration of ionization of amino groups, solvent effect, and temperature. Neural network clustering and asphericity calculations were performed on the basis of molecular dynamics data. It was shown that disaccharide fragments in the studied linear oligosaccharides were not rigid, and tended to have several conformers, thus determining the overall twisted shape with helical elements. In addition, it was found that the behavior of C5–C6 bond depended significantly upon the simulation conditions. The cyclic di-, tri-, and tetrasaccharides mostly had symmetrical ring-shaped conformations. The larger cycles tended to adopt more complicated shapes, and the conformational behavior of their disaccharide fragments was close to that in the linear oligosaccharides.

A series of five 3-acetamidopropyl β-glycosides of nona-β-(1→6)-glucosamines containing two N-acetylglucosamine residues separated by a different number of glucosamine units with free amino groups have been synthesized using a convergent blockwise approach. Oxazoline glycosylation was used to introduce N-acetylglucosamine residues. These nonasaccharides are structurally related to the poly-N-acetylglucosamine (PNAG) extracellular polysaccharide of Staphylococcus aureus and can be used as models for biochemical and immunological studies.

Synthesis of Neu5Acα(2−3)Galβ(1−4)Glc and Neu5Acα(2−8)Neu5Ac derivatives 12α and 14α, from lactose derivative 6 and 2,3-dehydro-Neu5Ac derivative 7, respectively, with anchimerically assisted Neu5Ac donor 3e, 3f, or 3g has been studied. Reaction of halogenose 3e, having a 3-phenoxythiocarbonyloxy moiety as the assisting group, afforded with 6 and 7 in the presence of equimolar amounts of AgOTf as promoter α-sialosides 11α and 13α which were readily deoxygenated to afford 12α and 14α, respectively. Reaction of phosphite 3f, having a 3-bromo substituent as the potentially anchimeric assisting group, furnished in the presence of catalytic amounts of TMSOTf with 6 as acceptor α-sialoside 15α and with 7 as acceptor β-sialoside 17β, which gave on debromination 12α and 14β, respectively. Reaction of readily available phosphite 3g, having a 3-thiobenzoyloxy group as the anchimeric assisting group, afforded with 6 and 7 in the presence of catalytic amounts of TMSOTf in very high yields α-linked sialosides 21α and ...

Uridine 5′-diphosphate α-D-galactofuranose, which is likely to be a biochemical donor of D-galactofuranosyl residues in Nature, is synthesized from α-D-galactofuranosyl phosphate and uridine 5′-monophosphate.

A set of phosphodisaccharides, structural analogues of the β-D-galactosyl-(1→4)-α-D-mannosyl phosphate 1, are synthesized using the Koenigs–Knorr method for the glycosylation reactions and the glycosyl hydrogenphosphonate method for phosphorylation. The compounds were tested as acceptor substrates/putative inhibitors for the Leishmania α-D-mannosylphosphate transferase.

The effect of Candida cell wall mannan-derived alpha-oligomannoside structural components on the modulation of the immune system and their role in protective immunity are studied here. Semi-synthetic alpha-mannoside-bovine serum albumin conjugates were used for immunization of rabbits. Dimeric alpha-mannoside, representing Candida antigenic factor 1, was used as a model of linear alpha-mannoside, and pentameric alpha-mannoside was used as a model of branched oligomannoside side chain structure. The induction of humoral immune response and the functionality of the serum tested by induction of peripheral blood leukocyte (PBL) candidacidal activity are documented. Anti-Candida albicans serotype B immunoglobulins (IgG and IgM) levels were higher than anti-serotype A following immunization with both conjugates. Dimer-conjugate postimmunization sera evidently enhanced C. albicans killing activity of PBLs in candidacidal assay. The study shows the importance of alpha-mannoside structures in perspective anti-Candida vaccine with a broad spectrum of effectiveness.

The synthesis of a pentasaccharide representing the glycoform I, which is one of two naturally occurring glycoforms of the outer core of Pseudomonas aeruginosa lipopolysaccharide, and its analogues, differing in the N-substituent in the galactosamine unit, is reported. The main features of the synthetic scheme included the assembly of the pentasaccharide backbone by successive introduction of monosaccharide units, the use of glucosyl donors with specific location of acyl protecting groups capable of the remote anchimeric participation for highly stereoselective α-glucosylation, and efficient reduction of the azido group allowing high-yielding transformation of the intermediary azido pentasaccharide into final products.

The 3-aminopropyl glycoside of a heptasaccharide fragment of the cell wall mannan from Candida guilliermondii 18 , which corresponds to the antigenic Factor 9, has been synthesized by a convergent approach based on glycosylation of a tetrasaccharide acceptor with a trisaccharide donor as the key step to give a protected heptasaccharide 17 . Subsequent two-step deprotection of 17 afforded the heptamannoside 18 , which was then conjugated with BSA using the squarate procedure.

A series of 3-beta-acetamidopropyl oligo-beta-(1-->6)-glucosamines consisting of 5, 7, 9 and 11 glucosamine residues, and a series of corresponding per-N-acetylated derivatives were synthesized using a convergent blockwise approach. These compounds represent fragments of a bacterial surface polysaccharide produced by numerous bacterial pathogens, including Staphylococcus aureus, and will be used as models for its biochemical and immunological properties.

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 1 H and 13 C 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.

Reaction of 8-O-unprotected Neu5Ac-1,7-lactone derivatives 3a–c, which were readily obtained from Neu5Ac, gave with sialyl donor 4 exclusively β(2–8)-linked disaccharides 5a–c in good yields. The lactone ring in disaccharides 5a–c was readily cleaved, thus 5c afforded Neu5Acβ(2–8)Neu5Ac derivative 6.

Reaction du O-benzyl-4 O-benzylidene-1,2(R) β-L-rhamnopyranose avec le bromure de desoxy-2 phtalimido-2 tri-O-acetyl-3,4,6 α-D-glucopyranosyle

Deacylation of acetylated and benzoylated sugar 1,2-thio-orthoesters bearing an S -aromatic residue with methanolic sodium methoxide is accompanied by inter- and intra-molecular nucleophilic substitution of the arylthio group with formation of the corresponding bi- and tri-cyclic orthoesters. Stereochemical aspects and a possible mechanism for this reaction are discussed. Free arylthio-orthoesters were obtained by performing the deacylation with methanolic sodium methoxide in pyridine.

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.

Background Constructs composed of cell wall mannan-derived moieties conjugated to immunogenic proteins could be promising agents for induction of protective anti- Candida immune responses. Methods This report is focused on the cellular immune response differences induced by BSA-based conjugates bearing synthetic α-1,6-branched oligomannosides. For monitoring of the immune responses following active immunization we evaluated changes in the frequencies of T and B lymphocytes and their activation status in the blood and spleen. We compared the immunization-induced changes of co-stimulatory molecules CD80 and CD86 expression on blood neutrophils and Th1/Th2 polarization of the immune response based on IFN-γ, TNF-α (pro-Th1), IL-4, and IL-10 (pro-Th2) cytokines levels and induction of IL-17. Results The results pointed out a comparable effect of the conjugates on the modulation of T and B lymphocytes frequencies in blood and spleen. Both conjugates induced upregulation of CD25 surface antigen on CD4 + T lymphocytes, independently on the structural differences of oligosaccharides. The differences in structure of oligomannoside antigens or conjugate constructs were reflected in the increase of co-stimulatory molecules CD80 and CD86 expression on neutrophils, and in induced cytokine response. M5–BSA conjugate induced only a slight increase in CD80 expression but a significant increase in IFN-γ, TNF-α, and IL-10. M6–BSA conjugate induced a significant increase of CD80 expression and increase of TNF-α, IL-4, and IL-10. Conclusion Obtained data demonstrate the importance of cellular immune response analysis for investigation of immunomodulatory properties of oligomannoside–protein conjugates.