Zwitterionic Polysaccharide Research Articles

Total synthesis of a structurally complex zwitterionic hexasaccharide repeating unit of polysaccharide B from Bacteroides fragilis via one-pot glycosylation

Zwitterionic polysaccharides (ZPSs) present on the surface of a common gut commensal Bacteroides fragilis are endowed with unique immunological properties as they can directly bind to T-cells in the absence of protein conjugation. ZPSs are therefore considered to be potential antigens for the development of totally carbohydrate-based vaccines. Herein, we disclose the first total synthesis of a highly branched phosphorylated zwitterionic capsular polysaccharide repeating unit of Bacteroides fragilis. The hexasaccharide repeating unit bearing six different monosaccharides comprises three 1,2-cis-glycosidic linkages, a challenging 1,2-trans linkage in D-QuipNAc-β-(1→4)-D-Gal motif, and a 2-aminoethyl phosphonate appendage. The synthesis of target ZPS was accomplished utilizing an expeditious, highly stereoselective and convergent (1 + 2 + 2 + 1) one-pot glycosylation strategy. The striking features include efficient synthesis of rare deoxy amino sugars D- and L-quinovosamine, stereoselective installation of three 1,2-cis glycosidic linkages, glycosylation of D-quinovosamine donor with a sterically crowded, poorly reactive 4-OH galactose moiety, as well as late stage phosphorylation.

Reductive amination of oxidized hydroxypropyl cellulose with ω-aminoalkanoic acids as an efficient route to zwitterionic derivatives

Zwitterionic polymers, with their equal amounts of cationic and anionic functional groups, have found widespread utility including as non-fouling coatings, hydrogel materials, stabilizers, antifreeze materials, and drug carriers. Polysaccharide-derived zwitterionic polymers are attractive because of their sustainable origin, potential for lower toxicity, and possible biodegradability, but previous methods for synthesis of zwitterionic polysaccharide derivatives have been limited in terms of flexibility and attainable degree of substitution (DS) of charged entities. We report herein successful design and synthesis of zwitterionic polysaccharide derivatives, in this case based on cellulose, by reductive amination of oxidized 2-hydroxypropyl cellulose (Ox-HPC) with ω-aminoalkanoic acids. Reductive amination products could be readily obtained with DS(cation) (= DS(anion)) up to 1.6. Adduct hydrophilic/hydrophobic balance (amphiphilicity) can be influenced by selecting the appropriate chain length of the ω-aminoalkanoic acid. This strategy is shown to produce a range of amphiphilic, water-soluble, moderately high glass transition temperature (Tg) polysaccharide derivatives in just a couple of efficient steps from commercially available building blocks. The adducts were evaluated as crystallization inhibitors. They are strong inhibitors of crystallization even for the challenging, poorly soluble, fast-crystallizing prostate cancer drug enzalutamide, as supported by surface tension and Flory–Huggins interaction parameter results.

Synthetic Zwitterionic Streptococcus pneumoniae Type 1 Oligosaccharides Carrying Labile O‐Acetyl Esters

AbstractWe herein report the first total synthesis of the Streptococcus pneumoniae serotype 1 (Sp1) oligosaccharide, a unique zwitterionic capsular polysaccharide carrying labile O‐acetyl esters. The target oligosaccharides, featuring rare α‐2,4‐diamino‐2,4,6‐trideoxy galactose (AAT) and α‐galacturonic acids, were assembled up to the 9‐mer level, in a highly stereoselective manner using trisaccharide building blocks. The lability of the O‐acetyl esters imposed a careful deprotection scheme to prevent migration and hydrolysis. The migration was investigated in detail at various pD values using NMR spectroscopy, to show that migration and hydrolysis of the C‐3‐O‐acetyl esters readily takes place under neutral conditions. Structural investigation showed the oligomers to adopt a right‐handed helical structure with the acetyl esters exposed on the periphery of the helix in close proximity of the neighboring AAT residues, thereby imposing conformational restrictions on the AATα1‐4GalA(3OAc) glycosidic linkages, supporting the helical shape of the polysaccharide, that has been proposed to be critical for its unique biological activity.

Synthetic Zwitterionic Streptococcus pneumoniae Type 1 Oligosaccharides Carrying Labile O-Acetyl Esters.

We herein report the first total synthesis of the Streptococcus pneumoniae serotype 1 (Sp1) oligosaccharide, a unique zwitterionic capsular polysaccharide carrying labile O-acetyl esters. The target oligosaccharides, featuring rare α-2,4-diamino-2,4,6-trideoxy galactose (AAT) and α-galacturonic acids, were assembled up to the 9-mer level, in a highly stereoselective manner using trisaccharide building blocks. The lability of the O-acetyl esters imposed a careful deprotection scheme to prevent migration and hydrolysis. The migration was investigated in detail at various pD values using NMR spectroscopy, to show that migration and hydrolysis of the C-3-O-acetyl esters readily takes place under neutral conditions. Structural investigation showed the oligomers to adopt a right-handed helical structure with the acetyl esters exposed on the periphery of the helix in close proximity of the neighboring AAT residues, thereby imposing conformational restrictions on the AATα1-4GalA(3OAc) glycosidic linkages, supporting the helical shape of the polysaccharide, that has been proposed to be critical for its unique biological activity.

Total Synthesis of the Repeating Unit of Bacteroides fragilis Zwitterionic Polysaccharide A1.

Zwitterionic polysaccharides isolated from commensal bacteria are endowed with unique immunological properties and are emerging as immunotherapeutic agents as well as vaccine carriers. Reported herein is a total synthesis of the repeating unit of Bacteroides fragilis zwitterionic polysaccharide A1 (PS A1). The structurally complex tetrasaccharide unit contains a rare sugar 2-acetamido-4-amino-2,4,6-trideoxy-d-galactose (AAT) and two consecutive 1,2-cis glycosidic linkages. The repeating unit was efficiently assembled by rapid synthesis of d-galactosamine and AAT building blocks from cheap and abundant d-mannose via a one-pot SN2 displacement of 2,4-bistriflates and installation of all of the glycosidic bonds in a highly stereoselective manner. The total synthesis involves a longest linear sequence of 17 steps with 3.47% overall yield.

Highly antifouling double network hydrogel based on poly(sulfobetaine methacrylate) and sodium alginate with great toughness

Hydrogels with good antifouling and mechanical properties as well as biocompatibility have great application potential in the field of biomedicine. In this paper, a newly double network (DN) hydrogel was prepared based on zwitterionic material sulfobetaine methacrylate (SBMA) and natural polysaccharide, sodium alginate (SA). The PSBMA network is covalently crosslinked while the SA network is ionically crosslinked by Ca2+. The hybrid crosslinked double network structure endows the DN hydrogel with excellent mechanical properties (E = 0.19 ± 0.01 MPa, σ = 0.73 ± 0.03 MPa), fast self-recovery ability as well as excellent fatigue resistance. Moreover, the results show that the PSBMA/SA-Ca2+ DN hydrogel is biocompatible and resists the absorption of non-specific proteins and adhesion of microorganisms, such as cells and algae, exhibiting outstanding antifouling properties. These unique characteristics of PSBMA/SA-Ca2+ DN hydrogel make it a promising candidate for biomedical application, such as artificial connective tissues, implantable devices, and underwater equipment.

Capsular Polysaccharide From Bacteroides fragilis Protects Against Ulcerative Colitis in an Undegraded Form.

The prominent human symbiont Bacteroides fragilis protects animals from intestinal diseases, such as ulcerative colitis, and its capsular polysaccharide plays a key role in reducing inflammation. B. fragilis strain ZY-312 was isolated from the feces of a healthy breast-fed infant, and the zwitterionic capsular polysaccharide zwitterionic polysaccharide, TP2, was extracted. In rats with 2,4-dinitrobenzenesulfonic acid (DNBS)-induced enteritis, TP2 at an optimal dose of 2.5 mg/kg could significantly alleviate enteritis and reduced the degree of intestinal adhesions, the intestinal ulcer area, and the incidence of ulcers in rats. To understand the underlying mechanism, TP2 was labeled with Fluorescein isothiocyanate and orally administered at a dose of 2.5 mg/kg in rats. TP2 was mainly distributed in the cecum and colorectum, but it was not detected in the blood and other organs except that a compound with a molecular weight greater than that of TP2-FITC was found in liver tissue. During the absorption, distribution, metabolism, and excretion, TP2 was indigestible. These results were further confirmed by investigation in the simulated gastric, intestinal fluid, and colonic fluid with fecal microbiota in vitro, where TP2 remained unaltered at different time points. Furthermore, flora composition was analyzed in simulated colonic fluid with TP2 added and it was found that TP2 increased the abundance of Faecalibacterium, Enterococcus romboutsia, and Ruminococcaceae, whereas the abundance of the phylum Proteobacteria represented by Sutterella, Desulfovibrio, and Enterobacteriaceae was decreased. However, the amount of short-chain fatty acids in the simulated colonic fluid was not changed by intestinal flora post-TP2 addition. In conclusion, these findings confirmed that TP2, a capsular polysaccharide of B. fragilis, protects against ulcerative colitis in an undegraded form.

Synthesis of an Aminooxy Derivative of the GM3 Antigen and Its Application in Oxime Ligation.

The anomeric aminooxy GM3 trisaccharide cancer antigen (Neu5Acα2,3Galβ1,4Glcβ-ONH2) has been chemically synthesized using a linear glycosylation approach. The key step involves a highly α(2,3)-stereoselective sialylation to a galactose acceptor. The Neu5Acα2,3Gal intermediate was functionalized as a donor for a [2 + 1] glycosylation, including a glucose acceptor that featured an O-succinimidyl group on the reducing end as an aminooxy precursor. The fully deprotected anomeric aminooxy GM3 trisaccharide was then conjugated to the immunologically relevant zwitterionic polysaccharide PS A1 via an oxime link.

Synthesis of glycoimmunogen Tn-Thr-PS A1 via hydrazone bond and stability optimization of PS A1 monosaccharide mimics under vaccine development conditions

Previously, our group constructed several immunogens utilizing oxime linkage to conjugate a T-cell stimulatory zwitterionic polysaccharide PS A1 and tumor associated carbohydrate antigens (TACAs) in acetate buffer. Here, a semi-synthetic immunogen was synthesized using hydrazone conjugation between PS A1 and a glycopeptide hydrazide (α-D-GalNAc-L-Thr-NH-NH2) with an excellent loading in PBS buffer. To get robust immune response, the retention of zwitterionic character of PS A1 under vaccine construction conditions is essential. In this regard, the stability of embedded pyruvate acetal moiety in tetrasaccharide repeating unit of PS A1 can validate the retention of the dual charges. Therefore, rather than utilizing this highly immunogenic PS A1 fully, stability studies were performed with synthetic 1-thiophenyl-4,6-O-pyruvate acetal-D-galactopyranose in varying acetate buffer pHs and time intervals. Furthermore, 1-propyl-D-galactofuranose was synthesized to mimick the D-Galf of PS A1 to examine regioselective hydrazone and oxime formation with α-D-GalNAc-L-Thr-NH-NH2 and α-D-GalNAc-ONH2 moieties respectively.

Glycoconjugate vaccines: some observations on carrier and production methods

ABSTRACTGlycoconjugate vaccines use protein carriers to improve the immune response to polysaccharide antigens. The protein component allows the vaccine to interact with T cells, providing a stronger and longer-lasting immune response than a polysaccharide interacting with B cells alone. Whilst in theory the mere presence of a protein component in a vaccine should be sufficient to improve vaccine efficacy, the extent of improvement varies. In the present review, a comparison of the performances of vaccines developed with and without a protein carrier are presented. The usefulness of analytical tools for macromolecular integrity assays, in particular nuclear magnetic resonance, circular dichroism, analytical ultracentrifugation and SEC coupled to multi-angle light scattering (MALS) is indicated. Although we focus mainly on bacterial capsular polysaccharide-protein vaccines, some consideration is also given to research on experimental cancer vaccines using zwitterionic polysaccharides which, unusually for polysaccharides, are able to invoke T-cell responses and have been used in the development of potential all-polysaccharide-based cancer vaccines.A general trend of improved immunogenicity for glycoconjugate vaccines is described. Since the immunogenicity of a vaccine will also depend on carrier protein type and the way in which it has been linked to polysaccharide, the effects of different carrier proteins and production methods are also reviewed. We suggest that, in general, there is no single best carrier for use in glycoconjugate vaccines. This indicates that the choice of carrier protein is optimally made on a case-by-case basis, based on what generates the best immune response and can be produced safely in each individual case.Abbreviations: AUC: analytical ultracentrifugation; BSA: bovine serum albumin; CD: circular dichroism spectroscopy; CPS: capsular polysaccharide; CRM197: Cross Reactive Material 197; DT: diphtheria toxoid; Hib: Haemophilius influenzae type b; MALS: multi-angle light scattering; Men: Neisseria menigitidis; MHC-II: major histocompatibility complex class II; NMR: nuclear magnetic resonance spectroscopy; OMP: outer membrane protein; PRP: polyribosyl ribitol phosphate; PSA: Polysaccharide A1; Sa: Salmonella; St.: Streptococcus; SEC: size exclusion chromatography; Sta: Staphylococcus; TT: tetanus toxoid; ZPS: zwitterionic polysaccharide(s).

Total Synthesis of Zwitterionic Tetrasaccharide Repeating Unit from Bacteroides fragilis ATCC 25285/NCTC 9343 Capsular Polysaccharide PS A1 with Alternating Charges on Adjacent Monosaccharides.

The tetrasaccharide repeating unit of zwitterionic polysaccharide A1 (PS A1) from Bacteroides fragilis ATCC 25285/NCTC 9343 has been synthesized using a linear glycosylation approach. One key step includes an α(1,4)-stereoselective [2 + 1] glycosylation of a 2,4,6-trideoxy-2-acetamido-4-amino-d-Gal p (AAT) donor with a poorly reactive axial C4-OH disaccharide acceptor. Mild acid-mediated deacetylation and a challenging [3 + 1] glycosylation are also highlighted. The strategy is inclusive of a single-pot, three-step deprotection affording PS A1 with alternating charges on adjacent monosaccharide units.

Finding a needle in a haystack: Bacteroides fragilis polysaccharide A as the archetypical symbiosis factor.

Starting from birth, all animals develop a symbiotic relationship with their resident microorganisms that benefits both the microbe and the host. Recent advances in technology have substantially improved our ability to direct research toward the identification of important microbial species that affect host physiology. The identification of specific commensal molecules from these microbes and their mechanisms of action is still in its early stages. Polysaccharide A (PSA) of Bacteroides fragilis is the archetypical example of a commensal molecule that can modulate the host immune system in health and disease. This zwitterionic polysaccharide has a critical impact on the development of the mammalian immune system and also on the stimulation of interleukin 10-producing CD4+ T cells; consequently, PSA confers benefits to the host with regard to experimental autoimmune, inflammatory, and infectious diseases. In this review, we summarize the current understanding of the immunomodulatory effects of B.fragilis PSA and discuss these effects as a novel immunological paradigm. In particular, we discuss recent advances in our understanding of the unique functional mechanisms of this molecule and its therapeutic potential, and we review the recent literature in the field of microbiome research aimed at discovering new commensal products and their immunomodulatory potential.

Synthesis of building blocks for an iterative approach towards oligomers of theStreptococcus pneumoniaetype 1 zwitterionic capsular polysaccharide repeating unit

Zwitterionic capsular polysaccharide extracts, ∼8 kDa in mass, from Streptococcus pneumoniae type 1 (Spt1) have shown unique T-cell activating properties. Oligomers of the trisaccharide repeating unit of the Spt1 capsular polysaccharide [→3)-4-NH2-α-d-QuipNAc-(1→4)-α-d-GalpA-(1→3)-α-d-GalpA-(1-]nof defined length are needed to further investigate this response. An approach towards iteratively extendable trisaccharide building blocks of the zwitterionic capsular polysaccharides of Spt1 is described. Key elements include the comparison of pre-glycosylation oxidation and post-glycosylation oxidation approaches using thioglycoside donors to the target trisaccharide, the optimisation of the post-glycosylation oxidation approach, and the conversion of the trisaccharide to building blocks tailored for iterative glycosylation. The construction and evaluation of stereotunable 2-N-3-O-oxazolidinone donors for the common bacterial 2-acetamido-4-amino-2,4,6-trideoxy-α-d-galactopyranoside motif is also described, as is a key intermediate for their efficient synthesis.

Dendritic cell-specific Mgat2 knockout mice show antigen presentation defects but reveal an unexpected CD11c expression pattern.

Zwitterionic polysaccharide antigens such as polysaccharide A (PSA) from Bacteroides fragilis have been shown to activate CD4+ T cells upon presentation by class II major histocompatibility complex (MHCII) on professional antigen presenting cells. For T cell recognition and activation, high affinity binding between MHCII and PSA is required, and complex N-glycans on conserved MHCII asparagine residues play a central role in controlling this interaction. By truncating these glycans in a myeloid-specific knockout of Mgat2, created using the LyzM-CRE mouse (M-cKO), we previously reported defects in PSA responses in vivo. Unfortunately, the M-cKO also showed a propensity to develop common variable immunodeficiencywith autoimmune hemolytic anemia features. Here, we describe a novel murine model in which Mgat2 was targeted for ablation using the dendritic cell (DC)-specific CD11c-CRE-GFP strain in order to develop a more specific and robust in vivo model of PSA presentation defects (DC-cKO). This study shows that Mgat2 deficient DCs from DC-cKO mice show ablation of PSA presentation and downstream T cell activation in vitro. However, the CD11c promoter was unexpectedly active and triggered Mgat2 deletion within multiple hematopoietic lineages, showed remarkably poor penetrance within native DC populations, and produced almost undetectable levels of green fluorescent proteinsignal. These findings show that the CD11c promoter is not DC-specific, and extreme care should be taken in the interpretation of data using any mouse created using the CD11c-CRE model.

Immunological evaluation of the entirely carbohydrate-based Thomsen-Friedenreich - PS B conjugate.

PS B, a naturally occurring CD4(+) T-cell simulating zwitterionic polysaccharide from Bacteroides fragilis ATCC 25285/NCTC 9343, was conjugated with aminooxy Thomsen Friedenreich (TF or T) [α-d-Gal-(1,3)-β-d-GalNAc-ONH2] tumor antigen. Immunization in Jax C57BL/6, followed by ELISA revealed IgM and IgG antibody TF specificity. FACS data noted preferential binding to TF-laced MCF-7 cells but not to HCT-116 cells.

Potlatch in the Gut - Immunomodulatory Molecules of Potential Therapeutic Benefit from the Commensal Microbiota

Mutualism between members of the gut microbiota and their mammalian host offers several advantages to both. As revealed by recent research, the co-evolution of specific microbial species with their particular mammalian host directs the maturation of the host’s immune system in a way that enables it to respond to infectious or inflammatory challenges appropriately. In addition, changes in microbial species with the host’s age, sex, diet, and other variable conditions allow beneficial adjustments. The effect of the gut microbiota on the immune system is not limited to gut tissue but also extends to peripheral sites. Thus the gut microenvironment can be viewed as a specialized immunologic sensor responsible for maintaining the general health of the host. Very little is known, however, about the ability of specific molecules from the commensal microbiota to effect changes in the host’s immune system. Molecules from the human gut commensal Bacteroides fragilis have been studied more extensively than molecules from other commensal microorganisms and can be described as “torch bearers” in the field. Two broad types of B. fragilis molecules have been determined to be immunomodulatory: polysaccharides and sphingolipids. Polysaccharide A (PSA), a zwitterionic capsular polysaccharide, is the best studied with regard to its presentation by the major histocompatibility class II (MHCII) pathway, its stimulation via the Toll-like receptor 2 (TLR2) pathway, its role in the maturation of the host’s immune system, and its immunoprotective role in murine models of colitis, multiple sclerosis, and surgical fibrosis. Our recent studies have shown that antigen-presenting cells of a particular subset i.e., plasmacytoid dendritic cells (PDCs)- are necessary for mediation of PSA’s immunoprotective function in murine models of chemically induced colitis and antigen-induced multiple sclerosis. PDCs are stimulated by PSA in a TLR2-dependent manner; this pathway had not previously been recognized in such cells. Molecules on antigenpresenting cells known for cognate interaction with CD 4+ T cells, such as

Synthesis of orthogonally protected bacterial, rare-sugar and D-glycosamine building blocks

Bacterial glycoconjugates comprise atypical deoxy amino sugars that are not present on the human cell surface, making them good targets for drug discovery and carbohydrate-based vaccine development. Unfortunately, they cannot be isolated with sufficient purity in acceptable amounts, and therefore chemical synthesis is a crucial step toward the development of these products. Here we describe a detailed protocol for the synthesis of orthogonally protected bacterial deoxy amino hexopyranoside (2,4-diacetamido-2,4,6-trideoxyhexose (DATDH), D-bacillosamine, D-fucosamine, and 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (AAT)), D-glucosamine and D-galactosamine building blocks starting from β-D-thiophenylmannoside. Readily available β-D-thiophenylmannoside was first converted into the corresponding 2,4-diols via deoxygenation or silylation at C6, followed by O3 acylation. The 2,4-diols were converted into 2,4-bis-trifluoromethanesulfonates, which underwent highly regioselective, one-pot, double-serial and double-parallel displacements by azide, phthalimide, acetate and nitrite ions as nucleophiles. Thus, D-rhamnosyl- and D-mannosyl 2,4-diols can be efficiently transformed into various rare sugars and D-galactosamine, respectively, as orthogonally protected thioglycoside building blocks on a gram scale in 1-2 d, in 54-85% overall yields, after a single chromatographic purification. This would otherwise take 1-2 weeks. D-Glucosamine building blocks can be prepared from β-D-thiophenylmannoside in four steps via C2 displacement of triflates by azide in 2 d and in 66-70% overall yields. These procedures have been applied to the synthesis of L-serine-linked trisaccharide of Neisseria meningitidis and a rare disaccharide fragment of the zwitterionic polysaccharide (ZPS) A1 (ZPS A1) of Bacteroides fragilis.

Synthesis of a trisaccharide repeat of the zwitterionic Sp1 capsular polysaccharide utilizing 2-azido-4-benzylamino-4N,3-O-carbonyl-2,4,6-trideoxy-d-galactopyranosyl trichloroacetimidate

2-Azido-4-benzylamino-4N,3-O-carbonyl-2,4,6-trideoxy-d-galactopyranosyl trichloroacetimidate 2 conveniently prepared in six steps from 6-deoxy-d-glucal glycosylated a selectively protected α1,3 linked methyl galabioside to afford the trisaccharide skeleton of a repeating unit of the Sp1 zwitterionic capsular polysaccharide. Lithium hydroxide hydrolysis of the 3,4-cyclic carbamate permitted the creation of a 2-acetamido-4-amino-2,4,6-trideoxygalactose residue. Selective cleavage of p-methoxybenzyl ethers by trifluoroacetic acid gave a selectively deprotected trisaccharide with two hydroxymethyl groups that were oxidized by the TEMPO reagent to afford access to trisaccharide glycoside 1 containing 2-acetamido-4-amino-2,4,6-trideoxygalactose and two galacturonic acid residues.

Interaction of the Capsular Polysaccharide A from Bacteroides fragilis with DC-SIGN on Human Dendritic Cells is Necessary for Its Processing and Presentation to T Cells

The zwitterionic capsular polysaccharide A (PSA) of Bacteroides fragilis is the first carbohydrate antigen described to be presented in major histocompatibility complex (MHC) class II for the induction of CD4+ T cell responses. However, the identity of the receptor mediating binding and internalization of PSA in antigen presenting cells remains elusive. C-type lectins are glycan-binding receptors known for their capacity to target ligands for antigen presentation to T cells. Here, we investigated whether C-type lectins were involved in the internalization of PSA and identified dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as the main receptor for PSA on human dendritic cells (DC). The induction of PSA-specific T cell proliferation appeared to be completely dependent on DC-SIGN. These data reveal a crucial role for DC-SIGN in the endocytosis and routing of PSA in human DC for the efficient stimulation of PSA-specific CD4+ T cells.

Orthogonally protected d-galactosamine thioglycoside building blocks via highly regioselective, double serial and double parallel inversions of β-d-thiomannoside

An efficient route for the synthesis of orthogonally protected D-galactosamine thioglycosides via one-pot double serial and double parallel displacements of the D-mannosyl-2,4-bis-trifluoromethanesulfonates by azide and nitrite ions is described. These building blocks were utilized to synthesize the rare disaccharide moiety of the zwitterionic polysaccharide of Bacteroides fragilis and the selectively protected Tn antigen.