Tetrasaccharide Fragment Research Articles

Fragments Synthesis of A. baumannii ATCC 17961 O-Antigen

Abstract Acinetobacter baumannii can cause many diseases including septicemia, pneumonia, meningitis, soft tissue, and urinary tract infections. Herein, we described the synthesis of one trisaccharide and two tetrasaccharide fragments derived from A. baumannii ATCC 17961 O-antigen that can be used for screening novel glyco-epitopes and for developing a synthetic carbohydrate-based vaccine against A. baumannii infection. The overall yields for the synthesis of the desired trisaccharide 1, tetrasaccharide 2, and tetrasaccharide 3 are 26.8% (8 steps), 21.6% (9 steps), and 24.5% (6 steps), respectively.

Klebsiella pneumoniae K2 capsular polysaccharide degradation by a bacteriophage depolymerase does not require trimer formation.

Generating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.

Synthesis of the branched tetrasaccharide fragment of saccharomicin B

A synthesis of the branched tetrasaccharide fragment of saccharomicin B through a [2+2] strategy is reported. Stereoselective coupling of a saccharosamine ortho-alkynyl benzoate to a digitoxose thioglycoside acceptor afforded the Sac-11-Digi-10 donor in 50% yield. This latter compound was then united with our previously reported Fuc-8-Eva-9 disaccharide under AgPF6 promotion to afford the target tetrasaccharide in 20% yield. The resulting tetrasaccharide can serve as a linchpin for assembling the target molecule.

Synthesis of the Tetrasaccharide Glycone Part of Tetrocarcin A.

A de novo synthesis of the tetrasaccharide fragment of tetrocarcin A is described. The key feature of this approach is highlighted by the regio- and diastereoselective Pd-catalyzed hydroalkoxylation of ene-alkoxyallenes with an unprotected l-digitoxose glycoside. The subsequent reaction with digitoxal in combination with chemoselective hydrogenation generated the target molecule.

Synthesis of the Branched Tetrasaccharide Fragment of Saccharomicin A.

A synthesis of the branched tetrasaccharide fragment of saccharomicin A using 1-OTBS donors to stereoselectively synthesize both α- and β-linked disaccharides is reported. The disaccharides were united using BSP/Tf2O to afford the tetrasaccharide fragment as a single α-anomer in 72% yield. This branched tetrasaccharide fragment can be used as donor and acceptor species to synthesize larger fragments of saccharomicin A.

Stereoselective Synthesis of a Tetrasaccharide Fragment from Rhamnogalacturonan II Side Chain A

Comprehensive SummaryAn efficient synthesis of a complex tetrasaccharide fragment 1 structurally related to rhamnogalacturonan II side chain A has been accomplished through a stepwise glycosylation strategy. Challenges involved in the synthesis include the facile construction of the sterically crowded l‐fucopyranose core and the stereoselective formation of two 1,2‐cis‐glycosidic linkages. The 3,4‐disubstituted l‐fucopyranoside structure was successfully constructed through a ‘counterclockwise’ glycosylation sequence, namely, the less reactive axial 4‐OH group of the central fucose unit was glycosylated first, then the 3‐OH. Besides, a 2‐pyridinecarbonyl‐assisted α‐d‐xylosylation was developed to synthesize the α‐d‐xylopyranosidic linkage and a 3,4‐O‐benzoyl‐controlled α‐l‐galactosylation reaction was used for the stereoselective synthesis of the corresponding α‐l‐galactopyranosidic linkage.

Chemical Synthesis and Evaluation of Exopolysaccharide Fragments Produced by Leuconostoc mesenteroides Strain NTM048.

Exopolysaccharides (EPSs) occur widely in natural products made by bacteria, fungi and algae. Some EPSs have intriguing biological properties such as anticancer and immunomodulatory activities. Our group has recently found that EPSs generated from Leuconostoc mesenteroides ssp. mesenteroides strain NTM048 (NTM048 EPS) enhanced a production of mucosal immunoglobulin A (IgA) of mouse. Herein, we described the synthesis and evaluation of the tetrasaccharide fragments of NTM048 EPS to obtain information about the molecular mechanism responsible for the IgA-inducing activity.

Synthesis of a Glycosylphosphatidylinositol (GPI) Fragment as a Potential Substrate for Mannoprotein Transglycosidases

AbstractA glycophosphatidylinositol tetrasaccharide fragment was synthesized to mimic the core features of primary model, that of Saccharomyces cerevisiae. The salient feature of this approach is centered on the quick access to various α-1,2- and α-1,6-mannosyl and α-1,4-glycosyl linkages by using simple glycosylation and protective-group techniques. 1D and 2D-J-resolved NMR spectroscopy was used to verify the α-configuration of the new linkages. The tetrasaccharides obtained in this work are useful for examining fungal cell-wall glycoprotein cross-linking by transglycosidase enzymes for antifungal drug development.

Defining Polysaccharide-Specific Antibody Targets against Vibrio cholerae O139 in Humans following O139 Cholera and following Vaccination with a Commercial Bivalent Oral Cholera Vaccine, and Evaluation of Conjugate Vaccines Targeting O139.

ABSTRACTCholera caused by Vibrio cholerae O139 could reemerge, and proactive development of an effective O139 vaccine would be prudent. To define immunoreactive and potentially immunogenic carbohydrate targets of Vibrio cholerae O139, we assessed immunoreactivities of various O-specific polysaccharide (OSP)-related saccharides with plasma from humans hospitalized with cholera caused by O139, comparing responses to those induced in recipients of a commercial oral whole-cell killed bivalent (O1 and O139) cholera vaccine (WC-O1/O139). We also assessed conjugate vaccines containing selected subsets of these saccharides for their ability to induce protective immunity using a mouse model of cholera. We found that patients with wild-type O139 cholera develop IgM, IgA, and IgG immune responses against O139 OSP and many of its fragments, but we were able to detect only a moderate IgM response to purified O139 OSP-core, and none to its fragments, in immunologically naive recipients of WC-O1/O139. We found that immunoreactivity of O139-specific polysaccharides with antibodies elicited by wild-type infection markedly increase when saccharides contain colitose and phosphate residues, that a synthetic terminal tetrasaccharide fragment of OSP is more immunoreactive and protectively immunogenic than complete OSP, that native OSP-core is a better protective immunogen than the synthetic OSP lacking core, and that functional vibriocidal activity of antibodies predicts in vivo protection in our model but depends on capsule thickness. Our results suggest that O139 OSP-specific responses are not prominent following vaccination with a currently available oral cholera vaccine in immunologically naive humans and that vaccines targeting V. cholerae O139 should be based on native OSP-core or terminal tetrasaccharide.IMPORTANCE Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP). Little is known about immunity to O139 OSP. In this study, we used synthetic fragments of the O139 OSP to define immune responses to OSP in humans recovering from cholera caused by V. cholerae O139, compared these responses to those induced by the available O139 vaccine, and evaluated O139 fragments in next-generation conjugate vaccines. We found that the terminal tetrasaccharide of O139 is a primary immune target but that the currently available bivalent cholera vaccine poorly induces an anti-O139 OSP response in immunologically naive individuals.

Acinetobacter baumannii K106 and K112: Two Structurally and Genetically Related 6-Deoxy-l-talose-Containing Capsular Polysaccharides.

Whole genome sequences of two Acinetobacter baumannii clinical isolates, 48-1789 and MAR24, revealed that they carry the KL106 and KL112 capsular polysaccharide (CPS) biosynthesis gene clusters, respectively, at the chromosomal K locus. The KL106 and KL112 gene clusters are related to the previously described KL11 and KL83 gene clusters, sharing genes for the synthesis of l-rhamnose (l-Rhap) and 6-deoxy-l-talose (l-6dTalp). CPS material isolated from 48-1789 and MAR24 was studied by sugar analysis and Smith degradation along with one- and two-dimensional 1H and 13C NMR spectroscopy. The structures of K106 and K112 oligosaccharide repeats (K units) l-6dTalp-(1→3)-D-GlcpNAc tetrasaccharide fragment share the responsible genes in the respective gene clusters. The K106 and K83 CPSs also have the same linkage between K units. The KL112 cluster includes an additional glycosyltransferase gene, Gtr183, and the K112 unit includes α l-Rhap side chain that is not found in the K106 structure. K112 further differs in the linkage between K units formed by the Wzy polymerase, and a different wzy gene is found in KL112. However, though both KL106 and KL112 share the atr8 acetyltransferase gene with KL83, only K83 is acetylated.

Stereoselective Synthesis of 2-Azido-2-deoxy-β-d-mannosides via Cs2CO3-Mediated Anomeric O-Alkylation with Primary Triflates: Synthesis of a Tetrasaccharide Fragment of Micrococcus luteus Teichuronic Acid.

Cesium carbonate-mediated anomeric O-alkylation of various protected 2-azido-2-deoxy-d-mannoses with primary triflate electrophiles afforded corresponding 2-azido-2-deoxy-β-mannosides in good yields and excellent anomeric selectivity. In addition, 1,3-dibromo-5,5-dimethylhydantoin was found to be the optimal oxidant for preparation of those 2-azido-2-deoxy-d-mannoses from their corresponding thioglycosides. The utilization of this method was demonstrated in the synthesis of a tetrasaccharide fragment of Micrococcus luteus teichuronic acid containing N-acetyl-β-d-mannosaminuronic acid (ManNAcA).

Unusual Outcome of Glycosylation: Hydrogen‐Bond Mediated Control of Stereoselectivity by N‐Trifluoroacetyl Group?

During the synthesis of protected tetrasaccharide fragment of the repeating unit of polysaccharide of Azospirillum brasilense sp7 we found that glycosylation with a mixture of protected N‐trifluoroacetyl (TFA)‐protected glucosamine N‐phenyltrifluoroacetimidate and the corresponding 2‐trifluoromethyl‐oxazoline led to 1,2‐cis‐linked glycoside along with the expected 1,2‐trans‐linked isomer. This fact looks quite unusual bearing in mind that both N‐TFA‐containing glycosyl donor and 2‐trifluoromethyl‐oxazoline are usually expected to favor the formation of 1,2‐trans‐glycosides. Formation of 1,2‐cis‐glycoside was explained by hydrogen‐bond mediated complexation of hydrogen atom of NHTFA at C‐2 of oxacarbenium ion derived from glycosyl donor and oxygen atom of hydroxy group of the incoming nucleophile. Conversely, glycosylations with N‐phthaloyl protected glycosyl donors resulted in exclusive formation of 1,2‐trans‐isomer presumably due to a significant steric effect and the absence of additional hydrogen‐bonding sites of N‐phthaloyl group.

Reagent-Controlled α-Selective Dehydrative Glycosylation of 2,6-Dideoxy Sugars: Construction of the Arugomycin Tetrasaccharide.

The first synthesis of the tetrasaccharide fragment of the anthracycline natural product Arugomycin is described. A reagent controlled dehydrative glycosylation method involving cyclopropenium activation was utilized to synthesize the α-linkages with complete anomeric selectivity. The synthesis was completed in 20 total steps, and in 2.5% overall yield with a longest linear sequence of 15 steps.

Stereoselective Synthesis of a Tetrasaccharide Fragment from Cellulosome Produced by Clostridium thermocellum

AbstractAn efficient synthesis of a structurally congested tetrasaccharide portion from the cellulosome produced by Clostridium thermocellum has been accomplished through a stepwise glycosylation strategy. The key steps for the synthesis include the stereocontrolled construction of a range of challenging 1,2‐cis glycosidic linkages. The 5‐O‐(2‐quinolinecarbonyl) substituted D‐galactofuranosyl thioglycoside 2, 4,6‐O‐benzylidene protected 2‐azido‐2‐deoxyglucose thioglycoside 3 c, and 4,6‐O‐benzylidene protected D‐galactopyranosyl thioglycoside 4 were employed as glycosyl donors, respectively, for the high‐yielding and stereoselective formation of the corresponding 1,2‐cis‐α‐Galf, α‐GlcNAc, and α‐Galp linkages.

Stereoselective synthesis of a branched α-decaglucan.

The first and convergent synthesis of a branched Arca subcrenata Lischke α-decaglucan containing all of the α-(1 → 3), α-(1 → 4), and α-(1 → 6) glycosyl linkages was efficiently achieved. The tri- and tetrasaccharide fragments and fully protected decasaccharide were assembled in a one-pot manner with excellent α-stereoselectivity, which was secured by the synergistic α-directing effects of the TolSCl/AgOTf catalysis system and the remote participation effect or steric β-shielding of functionalized groups at the donor 6-O-position. Low substrate concentration was revealed to favor the α-stereochemical outcome of glycosylations between bulkier building blocks. The synthetic approach established here would be very useful for the preparation of more complex α-glucans containing different types of glycosidic linkages and branched architectures.

The alginate lyase from Isoptericola halotolerans CGMCC 5336 as a new tool for the production of alginate oligosaccharides with guluronic acid as reducing end

Alginate oligosaccharides are depolymerization products of alginate by enzymatic degradation or physicochemical treatments. Alginate lyase has received great interests due to its use in oligosaccharide preparation. The substrate specificity of alginate lyase directly determines the type and degree of polymerization of alginate oligosaccharides. In this paper, the degradation products of Alginate lyase from Isoptericola halotolerans CGMCC 5336 were size-fractionated by gel filtration chromatography and the fractions were characterized by TLC, HPLC, MS and 1H NMR spectroscopy. The Mw values for these fractions were 352, 528, 704 Da which represented di-, tri-, and tetrasaccharide fragments, respectively. The 1H NMR revealed that the non-reducing ends of these oligosaccharides were 4-deoxy-L-erythro-hex-4-enepyranosyluronate and the reducing ends of these oligosaccharides were all guluronic acid. Therefore, it is believed that the alginate lyase produced by Isoptericola halotolerans CGMCC 5336 was capable of performing β elimination on guluronic acid residue so that low molecular weight oligosaccharides with guluronic acid on the reducing end could be obtained in this way.

Structural Analysis of Heparin-Derived 3-O-Sulfated Tetrasaccharides: Antithrombin Binding Site Variants

Heparin is a polysaccharide that is widely used as an anticoagulant drug. The mechanism for heparin’s anticoagulant activity is primarily through its interaction with a serine protease inhibitor, antithrombin III (AT), that enhances its ability to inactivate blood coagulation serine proteases, including thrombin (factor IIa) and factor Xa. The AT-binding site in the heparin is one of the most well-studied carbohydrate-protein binding sites and its structure is the basis for the synthesis of the heparin pentasaccharide drug, fondaparinux. Despite our understanding of the structural requirements for the heparin pentasaccharide AT-binding site, there is a lack of data on the natural variability of these binding sites in heparins extracted from animal tissues. The present work provides a detailed study on the structural variants of the tetrasaccharide fragments of this binding site afforded following treatment of a heparin with heparin lyase II. The 5 most commonly observed tetrasaccharide fragments of the AT-binding site are fully characterized, and a method for their quantification in heparin and low-molecular-weight heparin products is described.

Preparation and immunogenicity of gold glyco-nanoparticles as antipneumococcal vaccine model.

Nanotechnology-based fully synthetic carbohydrate vaccines are promising alternatives to classic polysaccharide/protein conjugate vaccines. We have prepared gold glyco-nanoparticles (GNP) bearing two synthetic carbohydrate antigens related to serotypes 19F and 14 of Streptococcus pneumoniae and evaluated their immunogenicity in vivo. A tetrasaccharide fragment of serotype 14 (Tetra-14), a trisaccharide fragment of serotype 19F (Tri-19F), a T-helper peptide and d -glucose were loaded onto GNP in different ratios. Mice immunization showed that the concomitant presence of Tri-19F and Tetra-14 on the same nanoparticle critically enhanced the titers of specific IgG antibodies toward type 14 polysaccharide compared with GNP exclusively displaying Tetra-14, while no IgG antibodies against type 19F polysaccharide were elicited. This work is a step forward toward synthetic nanosystems combining carbohydrate antigens and immunogenic peptides as potential carbohydrate-based vaccines.

The Interaction of Heparin Tetrasaccharides with Chemokine CCL5 Is Modulated by Sulfation Pattern and pH

Interactions between chemokines such as CCL5 and glycosaminoglycans (GAGs) are essential for creating haptotactic gradients to guide the migration of leukocytes into inflammatory sites, and the GAGs that interact with CCL5 with the highest affinity are heparan sulfates/heparin. The interaction between CCL5 and its receptor on monocytes, CCR1, is mediated through residues Arg-17 and -47 in CCL5, which overlap with the GAG-binding (44)RKNR(47) "BBXB" motifs. Here we report that heparin and tetrasaccharide fragments of heparin are able to inhibit CCL5-CCR1 binding, with IC50 values showing strong dependence on the pattern and extent of sulfation. Modeling of the CCL5-tetrasaccharide complexes suggested that interactions between specific sulfate and carboxylate groups of heparin and residues Arg-17 and -47 of the protein are essential for strong inhibition; tetrasaccharides lacking the specific sulfation pattern were found to preferentially bind CCL5 in positions less favorable for inhibition of the interaction with CCR1. Simulations of a 12-mer heparin fragment bound to CCL5 indicated that the oligosaccharide preferred to interact simultaneously with both (44)RKNR(47) motifs in the CCL5 homodimer and engaged residues Arg-47 and -17 from both chains. Direct engagement of these residues by the longer heparin oligosaccharide provides a rationalization for its effectiveness as an inhibitor of CCL5-CCR1 interaction. In this mode, histidine (His-23) may contribute to CCL5-GAG interactions when the pH drops just below neutral, as occurs during inflammation. Additionally, an examination of the contribution of pH to modulating CCL5-heparin interactions suggested a need for careful interpretation of experimental results when experiments are performed under non-physiological conditions.

NMR characterization of the binding properties and conformation of glycosaminoglycans interacting with interleukin-10.

The cytokine interleukin-10 (IL-10) is an important regulator of the host immune system with both pro- and anti-inflammatory functions. Glycosaminoglycans (GAGs) play a decisive role in the biology of many growth factors, e.g., for receptor binding or protection from proteolytic degradation. GAGs of the extracellular matrix inhibit IL-10 signaling, however, the molecular mechanism is so far unknown. Here, we studied the interaction between GAGs and IL-10 using a combination of nuclear magnetic resonance (NMR) spectroscopy and computer simulations. The binding region of a set of heparin and chondroitin sulfate GAG disaccharides with varying sulfation pattern were determined by saturation transfer difference (STD) NMR spectroscopy. From the initial growth rate of the STD amplification factor binding affinities were determined and KD values in the low millimolar to micromolar range were obtained. We observed the highest binding affinity to IL-10 with fully sulfated heparin; however, a hyaluronan hexasaccharide did not exhibit binding, which suggests that GAG sulfation is necessary for interaction with IL-10. For octasaccharides or longer GAGs, a cooperative binding behavior was observed, which could indicate simultaneous interaction with both dimer subunits of IL-10. Finally, structural information about the bound GAG was exemplarily obtained for a heparin tetrasaccharide fragment (ΔUA,2S-GlcNS,6S-IdoA,2S-GlcNS,6S) using transferred NOESY experiments, proton-proton scalar couplings and molecular dynamics simulations. The overall backbone conformation is only slightly changed in the presence of IL-10 and the conformational equilibrium between (1)C4 chair and (2)So skew-boat structure of the internal iduronic acid residue is preserved.