Sulfated Carbohydrate Research Articles

Automated solution-phase syntheses of alpha 1 → 2, 1 → 3 type rhamnans and rhamnan sulfate fragments

Rhamnan and rhamnan sulfate are naturally occurring carbohydrates that have important biological functions and possible therapeutic applications, but studies are limited to the microheterogeneous mixtures from natural sources. This work reports the first synthesis of any sulfated rhamnan fragments and successful automation of the process with a recently developed automated solution-phase approach using N-iodosuccinimide/trimethylsilyl triflate (NIS/TMSOTf) promotor and levulinoyl ester deprotection conditions. The automated solution-phase activation/deprotection approach was initially able to create alpha 1 → 2, 1 → 3 type rhamnan di- and trisaccharide in moderate yields. Once these targets were achieved, a process to use SO3•pyridine complex in DMF for sulfation compatible with an automated solution-phase liquid handling system was developed and successfully applied to carbohydrate sulfation to create two rhamnan sulfate fragments with differing monosulfation patterns.

Structures and Bioactivities of Six New Triterpene Glycosides, Psolusosides E, F, G, H, H1, and I and the Corrected Structure of Psolusoside B from the Sea Cucumber Psolus fabricii.

Seven sulfated triterpene glycosides, psolusosides B (1), E (2), F (3), G (4), H (5), H1 (6), and I (7), along with earlier known psolusoside A and colochiroside D have been isolated from the sea cucumber Psolus fabricii collected in the Sea of Okhotsk. Herein, the structure of psolusoside B (1), elucidated by us in 1989 as a monosulfated tetraoside, has been revised with application of modern NMR and particularly MS data and proved to be a disulfated tetraoside. The structures of other glycosides were elucidated by 2D NMR spectroscopy and HR-ESI mass-spectrometry. Psolusosides E (2), F (3), and G (4) contain holostane aglycones identical to each other and differ in their sugar compositions and the quantity and position of sulfate groups in linear tetrasaccharide carbohydrate moieties. Psolusosides H (5) and H1 (6) are characterized by an unusual sulfated trisaccharide carbohydrate moiety with the glucose as the second sugar unit. Psolusoside I (7) has an unprecedented branched tetrasaccharide disulfated carbohydrate moiety with the xylose unit in the second position of the chain. The cytotoxic activities of the compounds 2–7 against several mouse cell lines—ascite form of Ehrlich carcinoma, neuroblastoma Neuro 2A, normal epithelial JB-6 cells, and erythrocytes—were quite different, at that hemolytic effects of the tested compounds were higher than their cytotoxicity against other cells, especially against the ascites of Ehrlich carcinoma. Interestingly, psolusoside G (4) was not cytotoxic against normal JB-6 cells but demonstrated high activity against Neuro 2A cells. The cytotoxic activity against human colorectal adenocarcinoma HT-29 cells and the influence on the colony formation and growth of HT-29 cells of compounds 1–3, 5–7 and psolusoside A was checked. The highest inhibitory activities were demonstrated by psolusosides E (2) and F (3).

Conformational changes in common monosaccharides caused by per-O-sulfation

Abstract Polysulfated carbohydrates play an important role in many biological processes because of their ability to bind to various protein receptors such as different growth factors, blood coagulation factors, adhesion lectins etc. Precise information about spatial organization of sulfated derivatives is of high demand for molecular modelling of such interactions as well as for understanding of the mechanism of pyranoside-into-furanoside rearrangement. In this review we summarize the changes recently revealed for the conformations of common pyranosides and furanosides upon total O-sulfation which were studied by means of NMR spectroscopy as well as molecular modelling. It was found that pentoses, being more flexible, undergo complete conformational chair inversion. Meanwhile, for hexoses the situation strongly depends on the monosaccharide configuration. Conformational changes are most pronounced in gluco-compounds though quantum chemical calculations helped to establish that no complete chair inversion occurred. In furanosides distortions of two types were observed: either the ring conformation or the conformation of the side chain changed. The presented data may be used for the analysis of chemical, physical and biological properties of sulfated carbohydrates.

Use of sulfated cellulose membrane adsorbers for chromatographic purification of cell cultured-derived influenza A and B viruses

Influenza virus particles can be purified by pseudo-affinity chromatography using sulfated carbohydrate matrices. In this study, we compared the binding capacity and the purification performance of two bead-based resins and one membrane adsorber for three influenza virus strains (H1N1, H3N2 and B) produced in MDCK suspension cells in a chemically defined medium. The dynamic binding capacity for the sulfated cellulose membrane adsorbers was consistently higher than for the resins (8 to 22-fold). Confocal microscopy demonstrated, that while virus particles uniformly covered the complete surface of the membrane, the virus particles only bound to the external surface of the sulfated beads. Overall, recovery of virus varied between 66% and 81%. Total protein and DNA removal were >74% and >96%, respectively. Statistically significant differences in the purification performance were more often observed between strains than between matrices. In particular, H3N2 influenza virus particles could only be purified using the sulfated membrane adsorbers. Due to the higher operating flow rate and binding capacity, the productivity with the membrane adsorbers was on average 25-times higher than with the resins.

Diarylborinic Acid-Catalyzed, Site-Selective Sulfation of Carbohydrate Derivatives.

Sulfated carbohydrates have been implicated in diverse biological processes, with the position and extent of sulfation of a glycoside often playing important roles in determining the affinity and specificity of its binding to a biomolecular partner. Methods for the site-selective introduction of sulfate groups to carbohydrates are thus of interest. Here, we describe the development of a diarylborinic acid-catalyzed protocol for selective sulfation of pyranoside derivatives at the equatorial position of a cis-1,2-diol group. This method, which employs the sulfur trioxide-trimethylamine complex as the electrophile, has been employed for installation of a sulfate group at the 3-position of a range of galacto- and mannopyranosides, including substrates having a free primary OH group. By using a full equivalent of the diarylborinic acid, selective syntheses of more complex monosulfated glycosides, namely, a 3'-sulfolactose derivative and 3'-sulfo-β-galactosylceramide, have been accomplished. Preliminary kinetics experiments suggested that the catalyst resting state is a tetracoordinate diarylborinic ester that reacts with the SO3 complex in the turnover-limiting step. Catalyst inhibition by the pyranoside sulfate product and trialkylamine byproduct of the reaction was demonstrated.

Fucoidan : Manfaat dalam Industri Peternakan

Research of interest marine algae and seagrasses have been renewed, because they are considered to be promising resources of functional ingredients in the development of novel products. Fucoidans are a class of fucose-rich sulfated carbohydrates found in brown marine algae and echinoderms, and more recently identified in seagrasses. Chemical compositions of most fucoidans are complex, mainly being composed of fucose and sulfate. They also contain other monosaccharides like mannose, galactose, glucose, xylose and uronic acids, even acetyl groups and protein and it’s have potential therapeutic properties, including anti-viral activity. Currently, fucoidans are available for use in therapeutic and dietary supplements for inclusion in pet, livestock and aquaculture feed supplements. This review summarizes experimental evidence indicating that Fucoidan can improve the quality of feed ingredients for livestock, pets and aquaculture to improve health.

Structure-based design of nucleoside-derived analogues as sulfotransferase inhibitors.

Sulfotransferases (STs) catalyse the transfer of a sulfonyl group (‘sulfation’) from the enzyme co-factor 3′-phosphoadenosine 5′-phosphosulfate (PAPS) to a variety of biomolecules. Tyrosine sulfation of proteins and carbohydrate sulfation play a crucial role in many protein–protein interactions and cell signalling pathways in the extracellular matrix. This is catalysed by several membrane-bound STs, including tyrosylprotein sulfotransferase 1 (TPST1) and heparan sulfate 2-O-sulfotransferase (HS2ST1). Recently, involvement of these enzymes and their post-translational modifications in a growing number of disease areas has been reported, including inflammation, cancer and Alzheimer's disease. Despite their growing importance, the development of small molecules to probe the biological effect of TPST and carbohydrate ST inhibition remains in its infancy. We have used a structure-based approach and molecular docking to design a library of adenosine 3′,5′-diphosphate (PAP) and PAPS mimetics based upon 2′-deoxyadenosine and using 2′-deoxy-PAP as a benchmark. The use of allyl groups as masked methyl esters was exploited in the synthesis of PAP-mimetics, and click chemistry was employed for the divergent synthesis of a series of PAPS-mimetics. A suite of in vitro assays employing TPST1 and HS2ST, and a kinase counter screen, were used to evaluate inhibitory parameters and relative specificity for the STs.

New tools for evaluating protein tyrosine sulfation and carbohydrate sulfation.

Sulfation is a common modification of extracelluar glycans and tyrosine residues on proteins, which is important in many signalling pathways and interactions. Existing methods for studying sulfotransferases, the enzymes that catalyse sulfation, are cumbersome and low-throughput. Recent studies published in the Biochemical Journal have repurposed established biochemical assays from the kinase field and applied them to the characterisation of sulfotransferases. Biochemical screening of a library of kinase inhibitors revealed that compounds that target RAF kinases may also be repurposed to inhibit sulfotransferases. Together with the available structures of sulfotransferases, these studies open the door to the development of chemical tools to probe the biological functions of these important enzymes.

New tools for carbohydrate sulfation analysis: heparan sulfate 2-O-sulfotransferase (HS2ST) is a target for small-molecule protein kinase inhibitors.

Sulfation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulfate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulfotransferases, including HS 2-O-sulfotransferase (HS2ST), which transfers sulfate from the cofactor PAPS (3′-phosphoadenosine 5′-phosphosulfate) to the 2-O position of α-l-iduronate in the maturing polysaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulfation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In the present paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalysed oligosaccharide sulfation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set, to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell-permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with the present study, we demonstrated that tyrosyl protein sulfotranferases are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small-molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulfation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.

Electrochemical Behavior and Detection of Sulfated Sucrose at a Liquid|Organogel Microinterface Array.

The electrochemical behavior and detection of sulfated carbohydrates were investigated at an array of microinterfaces between two immiscible electrolyte solutions where the organic phase was gelled. It was found that the electrochemical signal was dependent on the organic phase electrolyte cation. Cyclic voltammetry (CV) of sucrose octasulfate (SOS) with bis(triphenylphosphoranylidene)ammonium BTPPA+ as the organic phase cation did not provide a response to a 10 μM SOS concentration. However, when the organic phase cation was tetradodecylammonium TDDA+, a distinct peak was present in the CV at ca. -0.47 V, indicative of a desorption process following adsorption during the preceding scan. This detection peak shifted to ca. -0.28 V when tridodecylmethylammonium TDMA+ was the organic phase cation, indicating an increased binding strength between this alkylammonium cation and SOS. By combining electroadsorption with TDMA+ as the organic phase electrolyte cation, detection limits of 0.064 μM SOS in 10 mM LiCl and 0.16 μM in a synthetic urine aqueous phase were achieved. The detection limit was improved to 0.036 μM SOS (10 mM LiCl) when the electroadsorption time was increased to 180 s, indicating the analytical capability for the detection of SOS and related sugars by ion-transfer adsorptive stripping voltammetry.

Inhibition of Homophilic Interactions and Ligand Binding of the Receptor for Advanced Glycation End Products by Heparin and Heparin-Related Carbohydrate Structures.

Background: Heparin and heparin-related sulphated carbohydrates inhibit ligand binding of the receptor for advanced glycation end products (RAGE). Here, we have studied the ability of heparin to inhibit homophilic interactions of RAGE in living cells and studied how heparin related structures interfere with RAGE–ligand interactions. Methods: Homophilic interactions of RAGE were studied with bead aggregation and living cell protein-fragment complementation assays. Ligand binding was analyzed with microwell binding and chromatographic assays. Cell surface advanced glycation end product binding to RAGE was studied using PC3 cell adhesion assay. Results: Homophilic binding of RAGE was mediated by V1- and modulated by C2-domain in bead aggregation assay. Dimerisation of RAGE on the living cell surface was inhibited by heparin. Sulphated K5 carbohydrate fragments inhibited RAGE binding to amyloid β-peptide and HMGB1. The inhibition was dependent on the level of sulfation and the length of the carbohydrate backbone. α-d-Glucopyranosiduronic acid (glycyrrhizin) inhibited RAGE binding to advanced glycation end products in PC3 cell adhesion and protein binding assays. Further, glycyrrhizin inhibited HMGB1 and HMGB1 A-box binding to heparin. Conclusions: Our results show that K5 polysaccharides and glycyrrhizin are promising candidates for RAGE targeting drug development.

Identification of internally sialylated carbohydrate tumor marker candidates, including Sda/CAD antigens, by focused glycomic analyses utilizing the substrate specificity of neuraminidase.

In our previous study, 14 sulfated carbohydrate tumor marker candidates were identified by focused glycomic analyses. Here, glycomic analyses focused on internally sialylated glycans to identify novel marker candidates. Internally sialylated glycans were enriched by digestion of pyridylaminated glycans prepared from sera with α-neuraminidase from Salmonella typhimurium, which did not cleave sialic acids linked to internal residues, followed by anion-exchange chromatography. Next, internally sialylated O-glycan profiles were constructed using two types of high performance liquid chromatography, which were compared between 20 healthy controls and 11 patients with gastric cancer and 9 patients with pancreatic cancer. In all, 17 marker candidates were identified. The structures of glycan candidates were precisely analyzed using enzymatic digestion, glycan synthesis, 2D mapping and mass spectrometry. Among 17 candidates, one was STn, and the other 16 comprised 10 core1, 1 core2 and 5 core3 glycans. The various structures included a α2,6-sialylated reducing terminal GalNAc and α2,6-sialylated type1 N-acetyl-lactosamine. Eight candidates possessed the Sda/CAD antigen. The levels of these candidate glycans in sera from all 40 subjects were quantified using a selected reaction monitoring assay and found to be elevated in at least one or more patients. Although the serum levels of each candidate glycan varied between patients, those candidates having the same backbone or determinant, such as core3 backbone and core1 structures with extended type1 N-acetyl-lactosamine, displayed similar patterns of elevation. These results suggest that analysis of multiple markers may be an effective means of diagnosing various cancers.

In vitro investigations on the effects of semi-synthetic, sulphated carbohydrates on the immune status of cultured common carp (Cyprinus carpio) leucocytes

The rapid emergence of drug resistance, unfavourable immunosuppression and mounting evidence to suggest the deleterious accumulation of drug breakdown residues within animal tissues has driven a strong desire to move away from these current methods of disease control. Some natural products such as β-glucan, which are extracted from, for example, plants and fungi, are able to modulate the immune system and increase protection against diseases. However, these products are heterogeneous and their effects can be variable thus limiting their applicability and reliability. Carbohydrates were modified via chemical sulphation and these semi-synthetic, sulphated carbohydrates analysed for their immunological activity utilising carp pronephric cells and a carp leucocyte cell line (CLC). A sulphated β(1,4)-glucan, methyl hydroxyethyl cellulose sulphate (MHCS), demonstrated a stimulatory effect on fish immune cells. MHCS induced a range of bioactive effects in carp leucocyte cells whilst not affecting cell viability when cells were exposed for 24 h at concentrations of 1–150 μgml−1. MHCS stimulated the innate immune system where a significant increase in respiratory burst activity was observed at concentrations 25–250 μgml−1 in comparison to control (sterile water), cellulose ether, MacroGard® and zymosan. Also, under in mock bacterial and viral infection conditions i.e. Lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (Poly(I:C)), MHCS enhanced the immune responses of pronephric cells by stimulating the respiratory burst activity at concentrations 50 and 150 μgml−1. MHCS also enhanced the expression of cytokines including interleukin 1 beta (IL1β), tumor necrosis factor alpha 1 and 2 (TNFα 1,2), interferons alpha 2 (IFN α2) and inducible nitric oxide synthase (iNOS) in carp pronephric cells. It is proposed that this new semi-synthetic carbohydrate is a potential candidate for the development of a new generation of immunostimulants and adjuvants for use in vaccination strategies in aquaculture.

Cladolosides I1, I2, J1, K1, K2 and L1, monosulfated triterpene glycosides with new carbohydrate chains from the sea cucumber Cladolabes schmeltzii

Six new monosulfated triterpene glycosides, cladolosides I1 (1), I2 (2), J1 (3), K1 (4), K2 (5) and L1 (6) were isolated from the tropical Indo-West Pacific sea cucumber Cladolabes schmeltzii (Cladolabinae, Sclerodactylidae, Dendrochirotida). Structures of these glycosides were elucidated by 2D NMR spectroscopy and mass spectrometry. Four new types of carbohydrate chains have been found in 1–6. Cladolosides of the groups I and J are characterized by pentasaccharide carbohydrate chains sulfated at a terminal 3-O-methylglucose residue and branched by the position 4 of the first xylose residue, but differing from each other in the lengths of the main and side carbohydrate chains. Cladolosides of the groups K and L contain hexasaccharide chains with different positions of a sulfated terminal 3-O-methylglucose residue (as the fourth or the sixth monosaccharide residue). Sulfated hexasaccharide carbohydrate chains were found in the sea cucumbers glycosides for the first time. A pentasaccharide carbohydrate chain of cladoloside J1 (3) having a disaccharide moiety of glucose and a sulfated 3-O-methylglucose linked to the first xylose residue in a linear trisaccharide fragment is also unusual. All substances studied demonstrated strong or moderate hemolytic and cytotoxic effects.

Correction: Identification and Characterization of Sulfated Carbohydrate-Binding Protein from Lactobacillus reuteri

[This corrects the article DOI: 10.1371/journal.pone.0083703.].

IRMPD Spectroscopy Sheds New (Infrared) Light on the Sulfate Pattern of Carbohydrates.

IR spectroscopy of gas-phase ions is proposed to resolve positional isomers of sulfated carbohydrates. Mass spectrometric fingerprints and gas-phase vibrational spectra in the near and mid-IR regions were obtained for sulfated monosaccharides, yielding unambiguous signatures of sulfated isomers. We report the first systematic exploration of the biologically relevant but notoriously challenging deprotonated state in the near IR region. Remarkably, anions displayed very atypical vibrational profiles, which challenge the well-established DFT (Density Functionnal Theory) modeling. The proposed approach was used to elucidate the sulfate patterns in glycosaminoglycans, a ubiquitous class of mammalian carbohydrates, which is regarded as a major challenge in carbohydrate structural analysis. Isomeric glycosaminoglycan disaccharides from heparin and chondroitin sources were resolved, highlighting the potential of infrared multiple photon dissociation spectroscopy as a novel structural tool for carbohydrates.

Composition and characteristics of continuous enzymatic hydrolysis products from Kappaphycus striatum

The oligosaccharide κ-carrageenan is sulfated carbohydrate with various bioactivities. In order to prepare this functional oligosaccharide directly from the red alga Kappaphycus striatum, a continuous enzymatic hydrolysis method was used to digest K. striatum. The yield of oligosaccharide (product KS-3) reached 43.26 g per 100 g of dry algae, and the product was identified as high-purity sulfated oligosaccharides mainly composed of κ-carrageenan hexasaccharide ([A-G4S]3) and octasaccharide ([A-G4S]4). The by-products generated during the preparation of oligosaccharide were also collected. The yield of insoluble product (KS-1) was 14.46 g per 100 g of dry algae, it was rich in protein (63.23%) and the content of essential amino acid was close to 40 g per 100 g of dry protein. The yield of soluble high-molecular weight product (KS-2) was 18.04 g per 100 g of dry algae, it was mainly composed of soluble dietary fibers (69.49%) and showed perfect expansion characteristics. We describe here a novel method for the preparation of oligosaccharides directly from seaweed, which is also beneficial for the comprehensive utilization of seaweed.

Compositional and structural characteristics of sulfated polysaccharide from Enteromorpha prolifera

Polysaccharide from Enteromorpha prolifera (PE) has been reported to have biological activities such as anticancer, antioxidant and immunomodulatory activities, but the structural characteristics of PE remain unclear. In this study, the composition of PE was characterized using chemical methods, IR spectroscopy and HPLC. PE was identified as a sulfated polysaccharide mainly composed of rhamnose (Rha), glucuronic (GlcUA), xylose (Xyl) at a molar ratio of 3.2: 1.1: 1, and the average molecular weight was 620.3kDa. An enzymatic hydrolysis method was used to obtain three carbohydrate products. The MS and NMR results of these residues revealed that the backbone of PE consisted of D-GlcUAp-α-(1→4)-3-sulfate-l-Rha p-β-(1→4)-d-Xyl p-β-(1→4)-3-sulfate-l-Rha p units. In this study, specific carbohydrate residues rich in sulfated rhamnose were prepared from E. prolifera. The structural determination of these sulfated carbohydrates may contribute to future studies on the relationship between structure and bioactivities.

Automated Glycan Assembly of Oligo-N-Acetyllactosamine and Keratan Sulfate Probes to Study Virus-Glycan Interactions

Summary Oligo- N -acetyllactosamine (LacNAc) and keratan sulfate (KS) glycans exert crucial functions in disease-relevant processes, including cancer formation, inflammation, and viral infection. To facilitate structure-activity studies with these glycans, we established a universal strategy to synthesize linear and branched LacNAc as well as differentially sulfated KS oligosaccharides by automated glycan assembly. We synthesized oligosaccharides as long as hexamers by combining four monosaccharide building blocks. Key to the strategy was installing three orthogonal protection groups, 9-fluorenylmethoxycarbonyl (Fmoc), levulinoyl (Lev) ester, and 2-naphthylmethyl (Nap) ether, which were selectively removed from a common oligosaccharide precursor for differential sulfation. Microarrays presenting the synthetic oligosaccharides revealed a specific interaction between a disulfated KS tetrasaccharide and the adeno-associated virus AAVrh10 gene-therapy vector, which was further corroborated by surface plasmon resonance studies. Thus, KS represents a novel receptor candidate for AAVrh10. These insights could have implications for cell-type-specific gene-delivery approaches.

Various sulfated carbohydrate tumor marker candidates identified by focused glycomic analyses.

Glycomic analysis focused on sulfated O-glycans was performed to identify novel serum carbohydrate tumor markers. Sulfated glycans were enriched by α-neuraminidase digestion of pyridylaminated glycans prepared from sera, followed by anion exchange chromatography. Sulfated O-glycan profiles were constructed by two types of high performance liquid chromatography separation. Comparison of the profiles from 20 healthy controls with those of 11 gastric and 9 pancreatic cancer patients identified 14 marker candidates. The structures of these candidates were precisely analyzed using various methods including enzymatic digestion and mass spectrometry. The candidates comprised 9 core1 and 5 core2 glycans. All these candidates were monosulfated, and 11 were also mono- or difucosylated, and included various determinants such as 6-sulfo type2 lactosamine, 6-sulfo Lewis X, 6-sulfo Lewis Y, 3'-sulfo type1 lactosamine and 3'-sulfo Lewis A. Furthermore, among the core1 glycans, five candidates displayed a type1 and type2 lactosamine hybrid backbone. The levels of these candidate glycans in the sera from all 40 subjects were quantified using a selected reaction monitoring assay. These analyses revealed: (i) the levels of all candidates were elevated in sera of at least one or more patients; (ii) core1 candidates having type1-type2 hybrid backbones with 6-sulfo Lewis X, 6-sulfo type2 lactosamine or 3'-sulfo Lewis A were elevated in sera of variety of patients; and (iii) levels of the candidates varied widely among patients, suggesting analysis of multiple candidates will be an effective means of screening various cancers. To fully evaluate the clinical utility of these candidates, a further verification study is required.