Synthetic Oligosaccharides Research Articles

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods.

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly. Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

Development of an Efficacious, Semisynthetic Glycoconjugate Vaccine Candidate against Streptococcus pneumoniae Serotype 1.

Infections with Streptococcus pneumoniae are a major health burden. Glycoconjugate vaccines based on capsular polysaccharides (CPSs) successfully protect from infection, but not all pneumococcal serotypes are covered with equal potency. Marketed glycoconjugate vaccines induce low levels of functional antibodies against the highly invasive serotype 1 (ST1), presumably due to the obscuring of protective epitopes during chemical activation and conjugation to carrier proteins. Synthetic oligosaccharide antigens can be designed to carry linkers for site-selective protein conjugation while keeping protective epitopes intact. Here, we developed an efficacious semisynthetic ST1 glycoconjugate vaccine candidate. A panel of synthetic oligosaccharides served to reveal a critical role of the rare aminosugar, 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (d-AAT), for ST1 immune recognition. A monovalent ST1 trisaccharide carrying d-AAT at the nonreducing end induced a strong antibacterial immune response in rabbits and outperformed the ST1 component of the multivalent blockbuster vaccine Prevenar 13, paving the way for a more efficacious vaccine.

Novel mouse monoclonal antibodies specifically recognize Aspergillus fumigatus galactomannan.

A panel of specific monoclonal antibodies (mAbs) against synthetic pentasaccharide β-D-Galf-(1→5)-[β-D-Galf-(1→5)]3-α-D-Manp, structurally related to Aspergillus fumigatus galactomannan, was generated using mice immunized with synthetic pentasaccharide-BSA conjugate and by hybridoma technology. Two selected mAbs, 7B8 and 8G4, could bind with the initial pentasaccharide with affinity constants of approximately 5.3 nM and 6.4 nM, respectively, based on surface plasmon resonance-based biosensor assay. The glycoarray, built from a series of synthetic oligosaccharide derivatives representing different galactomannan fragments, demonstrated that mAb 8G4 could effectively recognize the parental pentasaccharide while mAb 7B8 recognizes its constituting trisaccharide parts. Immunofluorescence studies showed that both 7B8 and 8G4 could stain A. fumigatus cells in culture efficiently, but not the mutant strain lacking galactomannan. In addition, confocal microscopy demonstrated that Candida albicans, Bifidobacterium longum, Lactobacillus plantarum, and numerous gram-positive and gram-negative bacteria were not labeled by mAbs 7B8 and 8G4. The generated mAbs can be considered promising for the development of a new specific enzyme-linked assay for detection of A. fumigatus, which is highly demanded for medical and environmental controls.

Analyzing Xyloglucan Endotransglycosylases by Incorporating Synthetic Oligosaccharides into Plant Cell Walls.

The plant cell wall is a cellular exoskeleton consisting predominantly of a complex polysaccharide network that defines the shape of cells. During growth, this network can be loosened through the action of xyloglucan endotransglycosylases (XETs), glycoside hydrolases that "cut and paste" xyloglucan polysaccharides through a transglycosylation process. We have analyzed cohorts of XETs in different plant species to evaluate the substrate specificities of xyloglucan acceptors by using a set of synthetic oligosaccharides obtained by automated glycan assembly. The ability of XETs to incorporate the oligosaccharides into polysaccharides printed as microarrays and into stem sections of Arabidopsis thaliana, beans, and peas was assessed. We found that single xylose substitutions are sufficient for transfer, and xylosylation of the terminal glucose residue is not required by XETs, independent of plant species. To obtain information on the potential xylosylation pattern of the natural acceptor of XETs, that is, the nonreducing end of xyloglucan, we further tested the activity of xyloglucan xylosyl transferase (XXT) 2 on the synthetic xyloglucan oligosaccharides. These data shed light on inconsistencies between previous studies towards determining the acceptor substrate specificities of XETs and have important implications for further understanding plant cell wall polysaccharide synthesis and remodeling.

Identification of the Minimal Glycotope of Streptococcus pneumoniae 7F Capsular Polysaccharide using Synthetic Oligosaccharides.

Streptococcus pneumoniae causes life-threatening diseases including meningitis, pneumonia and sepsis. Existing glycoconjugate vaccines based on purified capsular polysaccharides are widely used and help to prevent millions of deaths every year. Herein, the total syntheses of oligosaccharides resembling portions of the S. pneumoniae serotype 7F (ST7F) capsular polysaccharide repeating unit are reported. To define minimal glycan epitopes, glycan microarrays containing the synthetic oligosaccharides were used to screen human reference serum and revealed that both side chains of the ST7F play a key role in antigen recognition. The identification of protective minimal epitopes is vital to design efficient semi- and fully-synthetic glycoconjugate vaccines.

Study of the Carbohydrate Specificity of Antibodies Against Aspergillus fumigatus Using the Library of Synthetic Mycoantigens

The carbohydrate specificity of antibodies obtained by immunizing laboratory animals with immunogens prepared from the Aspergillus fumigatus cell wall was analyzed using the library of biotinylated synthetic oligosaccharides. It has been shown that the main part of antipolysaccharide antibodies recognizes galactomannan in the studied immunogens with preference to its fragments containing more than two β-(1→5)-linked galactofuranosyl units. The data obtained can form the basis for the development of enzyme immunoassay for the detection of this dangerous fungal pathogen.

Synthesis of oligosaccharides related to galactomannans from Aspergillus fumigatus and their NMR spectral data.

The synthesis of model oligosaccharides related to antigenic galactomannans of the dangerous fungal pathogen Aspergillus fumigatus has been performed employing pyranoside-into-furanoside (PIF) rearrangement and controlled O(5) → O(6) benzoyl migration as key synthetic methods. The prepared compounds along with some previously synthesized oligosaccharides were studied by NMR spectroscopy with the full assignment of 1H and 13C signals and the determination of 13C NMR glycosylation effects. The obtained NMR database on 13C NMR chemical shifts for oligosaccharides representing galactomannan fragments forms the basis for further structural analysis of galactomannan related polysaccharides by a non-destructive approach based on the calculation of the 13C NMR spectra of polysaccharides by additive schemes.

Synthetic Oligosaccharide Libraries and Microarray Technology: A Powerful Combination for the Success of Current Glycosaminoglycan Interactomics.

Glycosaminoglycans (GAGs) are extracellular matrix and/or cell-surface sulfated glycans crucial to the regulation of various signaling proteins, the functions of which are essential in many pathophysiological systems. Because structural heterogeneity is high in GAG chains and purification is difficult, the use of structurally defined GAG oligosaccharides from natural sources as molecular models in both biophysical and pharmacological assays is limited. To overcome this obstacle, GAG-like oligosaccharides of well-defined structures are currently being synthesized by chemical and/or enzymatic means in many research groups around the world. These synthetic GAG oligosaccharides serve as useful molecular tools in studies of GAG-protein interactions. In this review, besides discussing the commonest routes used for the synthesis of GAG oligosaccharides, we also survey some libraries of these synthetic models currently available for research and discuss their activities in interaction studies with functional proteins, especially through the microarray approach.

Branched Pectic Galactan in Phloem-Sieve-Element Cell Walls: Implications for Cell Mechanics.

A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus x giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a β-1,6-galactosyl substitution of β-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear β-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.

Sequencing the oligosaccharide pool in the low molecular weight heparin dalteparin with offline HPLC and ESI–MS/MS

Low molecular weight heparins (LMWHs) are widely used anticoagulant drugs. The composition and sequence of LMWH oligosaccharides determine their safety and efficacy. The short oligosaccharide pool in LMWHs undergoes more depolymerization reactions than the longer chains and is the most sensitive indicator of the manufacturing process. Electrospray ionization tandem mass spectrometry (ESI–MS/MS) has been demonstrated as a powerful tool to sequence synthetic heparin oligosaccharide but never been applied to analyze complicated mixture like LMWHs. We established an offline strong anion exchange (SAX)-high performance liquid chromatography (HPLC) and ESI–MS/MS approach to sequence the short oligosaccharides of dalteparin sodium. With the help of in-house developed MS/MS interpretation software, the sequences of 18 representative species ranging from tetrasaccharide to octasaccharide were obtained. Interestingly, we found a novel 2,3-disulfated hexauronic acid structure and reconfirmed it by complementary heparinase digestion and LC–MS/MS analysis. This approach provides straightforward and in-depth insight to the structure of LMWHs and the reaction mechanism of heparin depolymerization.

A Streptococcus pneumoniae Type 2 Oligosaccharide Glycoconjugate Elicits Opsonic Antibodies and Is Protective in an Animal Model of Invasive Pneumococcal Disease.

Invasive pneumococcal diseases (IPDs) remain the leading cause of vaccine-preventable childhood death, even though highly effective pneumococcal conjugate vaccines (PCVs) are used in national immunization programs in many developing countries. Licensed PCVs currently cover only 13 of the over 90 serotypes of Streptococcus pneumoniae (Sp), so nonvaccine serotypes are a major obstacle to the effective control of IPD. Sp serotype 2 (ST2) is such a nonvaccine serotype that is the main cause of IPD in many countries, including Nepal, Bangladesh, and Guatemala. Glycoconjugate vaccines based on synthetic oligosaccharides instead of isolated polysaccharides offer an attractive alternative to the traditional process for PCV development. To prevent the IPDs caused by ST2, we identified an effective ST2 neoglycoconjugate vaccine candidate that was identified using a medicinal chemistry approach. Glycan microarrays containing a series of synthetic glycans resembling portions of the ST2 capsular polysaccharide (CPS) repeating unit were used to screen human and rabbit sera and identify epitope hits. Synthetic hexasaccharide 2, resembling one repeating unit (RU) of ST2 CPS, emerged as a hit from the glycan array screens. Vaccination with neoglycoconjugates consisting of hexasaccharide 2 coupled to carrier protein CRM197 stimulates a T-cell-dependent B-cell response that induced CPS-specific opsonic antibodies in mice, resulting in killing of encapsulated bacteria by phagocytic activity. Subcutaneous immunization with neoglycoconjugate protected mice from transnasal challenge with the highly virulent ST2 strain NCTC 7466 by reducing the bacterial load in lung tissue and blood.

Semisynthetic glycoconjugate vaccine candidate against Streptococcus pneumoniae serotype 5

Glycoconjugate vaccines based on isolated capsular polysaccharide (CPS) save millions of lives annually by preventing invasive pneumococcal disease caused by Streptococcus pneumoniae Some components of the S. pneumoniae glycoconjugate vaccine Prevnar13 that contains CPS antigens from 13 serotypes undergo modifications or degradation during isolation and conjugation, resulting in production problems and lower efficacy. We illustrate how stable, synthetic oligosaccharide analogs of labile CPS induce a specific protective immune response against native CPS using S. pneumoniae serotype 5 (ST-5), a problematic CPS component of Prevnar13. The rare aminosugar l-PneuNAc and a branched l-FucNAc present in the natural repeating unit (RU) are essential for antibody recognition and avidity. The epitope responsible for specificity differs from the part of the antigen that is stabilized by chemical modification. Glycoconjugates containing stable, monovalent synthetic oligosaccharide analogs of ST-5 CPS RU induced long-term memory and protective immune responses in rabbits superior to those elicited by the ST-5 CPS component in multivalent Prevnar13.

A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision.

Modernization of Enoxaparin Molecular Weight Determination Using Homogeneous Standards

Enoxaparin is a low-molecular weight heparin used to treat thrombotic disorders. Following the fatal contamination of the heparin supply chain in 2007–2008, the U.S. Pharmacopeia (USP) and U.S. Food and Drug Administration (FDA) have worked extensively to modernize the unfractionated heparin and enoxaparin monographs. As a result, the determination of molecular weight (MW) has been added to the monograph as a measure to strengthen the quality testing and to increase the protection of the global supply of this life-saving drug. The current USP calibrant materials used for enoxaparin MW determination are composed of a mixture of oligosaccharides; however, they are difficult to reproduce as the calibrants have ill-defined structures due to the heterogeneity of the heparin parent material. To address this issue, we describe a promising approach consisting of a predictive computational model built from a library of chemoenzymatically synthesized heparin oligosaccharides for enoxaparin MW determination. Here, we demonstrate that this test can be performed with greater efficiency by coupling synthetic oligosaccharides with the power of computational modeling. Our approach is expected to improve the MW measurement for enoxaparin.

Study of Antigenic Activity of Experimental Samples of the Conjugated Vaccines Based on Synthetic Oligosaccharide Ligands and CRM197 Carrier Protein against <i>Candida albicans, Aspergillus fumigatus</i> and <i>Pseudomonas aeruginosa</i>

By now there is no effective treatment for a number of socially significant bacterial and fungal diseases. β-(1→3)-glucans are the principal components of the cell wall of fungi, including Candida albicans, Aspergillusfumigatus and other. At the same time, β-(1→3)-glucans are absent in mammals and man, that makes them promising components of carbohydrate-protein conjugated vaccines for the prevention and treatment of fungal infections. Alginic acid, constructed of β-(1→ 4)-linked mannuronic acid are extracellular polysaccharides produced by Pseudomonas aeruginosa. The protein CRM197 is a non toxic derivative of diphtheria toxin, which is widely used as a safe carrier in conjugated vaccines. The purpose of this study was to investigate the antigenic activity of experimental samples of the conjugated vaccines based on synthetic oligosaccharide ligands and CRM197 carrier protein in the competitive enzyme-linked immunosorbent assay. Two-time immunization Balb/c mice with experimental samples of the conjugated vaccines induced the formation of high titers of specific antibodies. High antibody’s avidity to their oligosaccharide ligands was shown in competitive ELISA. These data suggest the relevance of further preclinical trials of the conjugated antifungal vaccine against Candida and Aspergillus and antibacterial vaccine against Pseudomonas.

Oligosaccharide ligand tuning in design of third generation carbohydrate pneumococcal vaccines

Abstract Streptococcus pneumoniae can cause many types of dangerous infectious diseases such as otitis media, pneumonia, meningitis and others that are more common in the very young and very old age. Available to date commercial vaccines based on capsular polysaccharides of S. pneumoniae of clinically important strains (first generation carbohydrate vaccines) and conjugated vaccines based on these polysaccharides (second generation carbohydrate vaccines) have certain limitations in protective efficiency. However, the efficiency of vaccines can be increased by the use of third generation vaccines based on synthetic oligosaccharide ligands representing in their structures the protective epitopes of capsular polysaccharides. The proper choice of an optimal oligosaccharide ligand is the most important step in the design of third generation carbohydrate vaccines. Herein we overview our works on the synthesis of three oligosaccharides corresponding to one, “one and a half” and two repeating units of S. pneumoniae type 14 capsular polysaccharide, immunogenic conjugates thereof and comparative immunological study of their conjugates with bovine serum albumin, which was used as a model protein carrier. The ability of obtained products to raise antibodies specific to capsular polysaccharide and homologous oligosaccharides, the induction of phagocytosis by immune antisera and active protection of immunized animals from S. pneumoniae type 14 infection were evaluated. On the basis of the results obtained tetrasaccharide comprising the repeating unit of S. pneumoniae type 14 capsular polysaccharide is an optimal carbohydrate ligand to be used as a part of the third generation carbohydrate pneumococcal vaccine.

Neoglycoconjugate of Tetrasaccharide Representing One Repeating Unit of the Streptococcus pneumoniae Type 14 Capsular Polysaccharide Induces the Production of Opsonizing IgG1 Antibodies and Possesses the Highest Protective Activity As Compared to Hexa- and Octasaccharide Conjugates.

Identifying protective synthetic oligosaccharide (OS) epitopes of Streptococcus pneumoniae capsular polysaccharides (CPs) is an indispensable step in the development of third-generation carbohydrate pneumococcal vaccines. Synthetic tetra-, hexa-, and octasaccharide structurally related to CP of S. pneumoniae type 14 were coupled to bovine serum albumin (BSA), adjuvanted with aluminum hydroxide, and tested for their immunogenicity in mice upon intraperitoneal prime-boost immunizations. Injections of the conjugates induced production of opsonizing anti-OS IgG1 antibodies (Abs). Immunization with the tetra- and octasaccharide conjugates stimulated the highest titers of the specific Abs. Further, the tetrasaccharide ligand demonstrated the highest ability to bind OS and CP Abs. Murine immune sera developed against tetra- and octasaccharide conjugates promoted pathogen opsonization to a higher degree than antisera against conjugated hexasaccharide. For the first time, the protective activities of these glycoconjugates were demonstrated in mouse model of generalized pneumococcal infections. The tetrasaccharide conjugate possessed the highest protective activities. Conversely, the octasaccharide conjugate had lower protective activities and the lowest one showed the hexasaccharide conjugate. Sera against all of the glycoconjugates passively protected naive mice from pneumococcal infections. Given that the BSA-tetrasaccharide induced the most abundant yield of specific Abs and the best protective activity, this OS may be regarded as the most promising candidate for the development of conjugated vaccines against S. pneumoniae type 14 infections.

Probing cleavage promiscuity of heparinase III towards chemoenzymatically synthetic heparan sulfate oligosaccharides

An insightful investigation into specificity of bacterial heparinase III has been intriguingly difficult due to heterogeneity of polymeric substrates. Herein, we chemoenzymatically synthesized a tailored library of HS oligosaccharides as substrates. A ∼15-fold reactivity difference to heparinase III was found between trisaccharides bearing different primary cleavage sites. Variable glucosamine modification decreased reactivity of trisaccharides by >20-fold compared with their counterpart primary substrates, while iduronate-containing secondary linkage showed slightly less sensitivity. The 2-sulfated iduronate residue extremely reduced reactivity to its adjacent primary site at reducing end of oligosaccharides, but showed marginal influence on the non-reducing site. Moreover, oligosaccharide susceptibility to digestion was size-dependent and had an obvious preference for the internal linkages over those near to non-reducing/reducing ends. Surface plasmon resonance revealed cleavage promiscuity attributed to different affinities or incorrect binding of substrates to the enzyme. The attractive information on heparinase III will be valuable in characterizing heparin and HS.

A semisynthetic Streptococcus pneumoniae serotype 8 glycoconjugate vaccine.

Glycoconjugate vaccines based on capsular polysaccharides (CPSs) of pathogenic bacteria such as Streptococcus pneumoniae successfully protect from disease but suffer from incomplete coverage, are troublesome to manufacture from isolated CPSs, and lack efficacy against certain serotypes. Defined, synthetic oligosaccharides are an attractive alternative to isolated CPSs but require the identification of immunogenic and protective oligosaccharide antigens. We describe a medicinal chemistry strategy based on a combination of automated glycan assembly (AGA), glycan microarray-based monoclonal antibody (mAb) reverse engineering, and immunological evaluation in vivo to uncover a protective glycan epitope (glycotope) for S. pneumoniae serotype 8 (ST8). All four tetrasaccharide frameshifts of ST8 CPS were prepared by AGA and used in glycan microarray experiments to identify the glycotopes recognized by antibodies against ST8. One tetrasaccharide frameshift that was preferentially recognized by a protective, CPS-directed mAb was conjugated to the carrier protein CRM197. Immunization of mice with this semisynthetic glycoconjugate followed by generation and characterization of a protective mAb identified protective and nonprotective glycotopes. Immunization of rabbits with semisynthetic ST8 glycoconjugates containing protective glycotopes induced an antibacterial immune response. Coformulation of ST8 glycoconjugates with the marketed 13-valent glycoconjugate vaccine Prevnar 13 yielded a potent 14-valent S. pneumoniae vaccine. Our strategy presents a facile approach to develop efficient semisynthetic glycoconjugate vaccines.

Mixed-Linkage Glucan Oligosaccharides Produced by Automated Glycan Assembly Serve as Tools To Determine the Substrate Specificity of Lichenase.

The mixed-linkage (1→3),(1→4)-d-glucan (MLG) specific glycosyl hydrolase lichenase is an important biochemical tool for the structural characterization of MLGs. It holds potential for application in the brewery, animal feed, and biofuel industries. Several defined MLG oligosaccharides obtained by automated glycan assembly are used to analyze the substrate specificities of Bacillus subtilis lichenase. Two glucose building blocks (BBs), equipped with a temporary fluorenylmethyloxycarbonyl chloride (Fmoc) protecting group in the C-3 or C-4 position, served to assemble different oligosaccharides by using an automated oligosaccharide synthesizer. Light-induced cleavage of the glycan products from the solid support followed by global deprotection provided seven MLG oligosaccharides of different length and connectivity. After incubation of the MLG oligosaccharides with lichenase, the digestion products were analyzed by HPLC-MS. These digestion experiments provided insights into the enzyme's active site that is in line with other recent evidence suggesting that the substrate specificity of lichenases has to be reconsidered. These results demonstrate that synthetic MLG oligosaccharides are useful tools to analyze mixed-linkage β-glucanases.