Different Degrees Of Sulfation Research Articles

Highly sulfated K5 Escherichia coli polysaccharide derivatives inhibit respiratory syncytial virus infectivity in cell lines and human tracheal-bronchial histocultures.

Respiratory syncytial virus (RSV) exploits cell surface heparan sulfate proteoglycans (HSPGs) as attachment receptors. The interaction between RSV and HSPGs thus presents an attractive target for the development of novel inhibitors of RSV infection. In this study, selective chemical modification of the Escherichia coli K5 capsular polysaccharide was used to generate a collection of sulfated K5 derivatives with a backbone structure that mimics the heparin/heparan sulfate biosynthetic precursor. The screening of a series of N-sulfated (K5-NS), O-sulfated (K5-OS), and N,O-sulfated (K5-N,OS) derivatives with different degrees of sulfation revealed the highly sulfated K5 derivatives K5-N,OS(H) and K5-OS(H) to be inhibitors of RSV. Their 50% inhibitory concentrations were between 1.07 nM and 3.81 nM in two different cell lines, and no evidence of cytotoxicity was observed. Inhibition of RSV infection was maintained in binding and attachment assays but not in preattachment assays. Moreover, antiviral activity was also evident when the K5 derivatives were added postinfection, both in cell-to-cell spread and viral yield reduction assays. Finally, both K5-N,OS(H) and K5-OS(H) prevented RSV infection in human-derived tracheal/bronchial epithelial cells cultured to form a pseudostratified, highly differentiated model of the epithelial tissue of the human respiratory tract. Together, these features put K5-N,OS(H) and K5-OS(H) forward as attractive candidates for further development as RSV inhibitors.

Structure of a homofructosan from Saussurea costus and anti-complementary activity of its sulfated derivatives

A homogeneous water-soluble polysaccharide APS-W1, (2→1)-β-d-fructofuranosan, with an average molecular weight of 3.9kDa, was isolated and characterized from the roots of Saussurea costus. Five sulfated derivatives of APS-W1 with different degrees of sulfation were prepared and they showed strong inhibitory effect on the complement activation through the classical pathway (CP50: 2.2–18.9μg/mL; 8.3μg/mL for heparin) and alternative pathway (AP50: 11.4–115.8μg/mL; 89.2μg/mL for heparin). Mechanism studies by using complement-depleted sera indicated that sulfated derivatives with different positions of sulfation targeted to different complement proteins. Meanwhile the sulfated derivatives have limited anticoagulant effect based on re-calcification time and thrombin time. These results suggested that the sulfated derivatives prepared from APS-W1 could be promising potential complement inhibitors for the treatment of diseases caused by an over-activated complement system.

Small synthetic hyaluronan disaccharides afford neuroprotection in brain ischemia-related models

High molecular weight (HMW) glycosaminoglycanes of the extracellular matrix have been implicated in tissue repair. The aim of this study was to evaluate if small synthetic hyaluronan disaccharides with different degrees of sulfation (methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-O-sulfo-α-d-glucopyranoside, sodium salt (di0S), methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-6-di-O-sulfo-α-d-glucopyranoside, disodium salt (di6S) and methyl 2-acetamido-2-deoxy-3-O-(β-d-glucopyranosyluronic acid)-4,6-di-O-sulfo-α-d-glucopyranoside, trisodium salt (di4,6S)) could improve cell survival in in vitro and in vivo brain ischemia-related models. Rat hippocampal slices subjected to oxygen and glucose deprivation and a photothrombotic stroke model in mice were used. The three hyaluran disaccharides, incubated during the oxygen and glucose deprivation (15min) and re-oxygenation periods (120min), reduced cell death of hippocampal slices measured as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction, being the most potent di4,6S; in contrast, high molecular hyaluronan was ineffective. The protective actions of di4,6S against oxygen and glucose deprivation were related to activation of the PI3K/Akt survival pathway, reduction of p65 translocation to the nucleus, inhibition of inducible nitric oxide oxidase induction and reactive oxygen species production, and to an increase in glutathione levels. Administered 1h post-stroke, di4,6S reduced cerebral infarct size and improved motor activity in the beam walk test. In conclusion, di4,6S affords neuroprotection in in vitro and in vivo models of ischemic neuronal damage. Our results suggest that its neuroprotective effect could be exerted through its capability to reduce oxidative stress during ischemia. Its small molecular size makes it a more potential druggable drug to target the brain as compared with its HMW parent compound hyaluronan.

Improvement of the digestibility of sulfated hyaluronans by bovine testicular hyaluronidase: a UV spectroscopic and mass spectrometric study.

Glycosaminoglycans (GAGs) such as hyaluronan (HA) and chondroitin sulfate (CS) are important, natural polysaccharides which occur in biological (connective) tissues and have various biotechnological and medical applications. Additionally, there is increasing evidence that chemically (over)sulfated GAGs possess promising properties and are useful as implant coatings. Unfortunately, a detailed characterization of these GAGs is challenging: although mass spectrometry (MS) is one of the most powerful tools to elucidate the structures of (poly)saccharides, MS is not applicable to high mass polysaccharides, but characteristic oligosaccharides are needed. These oligosaccharides are normally generated by enzymatic digestion. However, chemically modified (particularly sulfated) GAGs are extremely refractive to enzymatic digestion. This study focuses on the investigation of the digestibility of GAGs with different degrees of sulfation by bovine testicular hyaluronidase (BTH). It will be shown by using an adapted spectrophotometric assay that all investigated GAGs can be basically digested if the reaction conditions are carefully adjusted. However, the oligosaccharide yield correlates reciprocally with the number of sulfate residues per polymer repeating unit. Finally, matrix-laser desorption and ionization (MALDI) MS will be used to study the released oligosaccharides and their sulfation patterns.

Catalytic synthesis and antioxidant activity of sulfated polysaccharide from Momordica charantia L.

Sulfated derivatives of polysaccharide from Momordica charantia L. (MCPS) with different degree of sulfation (DS) were synthesized by chlorosulfonic acid method with ionic liquids as solvent. Fourier transform infrared spectra and 13C nuclear magnetic resonance spectra indicated that C-6 substitution was predominant in MCPS compared with the C-2 position. Compared with the native polysaccharide from Momordica charantia L. (MCP), MCPS exhibited more excellent antioxidant activities in vitro, which indicated that sulfated modification could enhance antioxidant activities of MCP. Furthermore, high DS and moderate molecular weight could improve the antioxidant activities of polysaccharide.

Sulfation of Agrocybe chaxingu polysaccharides can enhance the immune response in broiler chicks

SUMMARY In this study, our objective was to compare the effects of sulfated Agrocybe chaxingu polysaccharide (saCP) and nonsulfated aCP (naCP) on lymphocyte proliferation in vitro and immune-enhancing activity in broiler chicks. In the in vitro test, 3 saCP with different degrees of sulfation—saCP0.83, saCP1.32, and saCP 1.79—were used to study the peripheral lymphocyte proliferation using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay with naCP as the control. On the basis of this test, saCP 1.32 and saCP 1.79 were selected for the in vivo test. In the in vivo test, 300 one-day-old chicks were randomly divided into 5 groups (negative control, vaccine control, naCP, saCP 1.32, and saCP 1.79). Chicks in the vaccine control and aCP groups were vaccinated with newcastle disease (nD) vaccine at 10 d of age, with a booster dose at 21 d of age. at the time of the first vaccination, the chicks in the 3 aCP groups were injected with naCP, saCP 1.32, and saCP 1.79, whereas those in the vaccine control group were injected with 0.5 ml of physiological saline once a day for 3 successive days. In comparison to naCP, saCP 1.32 and saCP 1.79 significantly increased the relative weight of the thymus and spleen at 22 d of age, enhanced serum nD antibody titers at 16, 22, 28, 34, and 42 d of age, and enhanced splenic t- and B-lymphocyte proliferation at 22 and 28 d of age. Furthermore, saCP 1.32 and saCP 1.79 could induce significant augmented levels of interferon-γ and Il-6. Sulfation could enhance both cellular and humoral immunity in broiler chicks on a degrees of sulfation basis. the saCP 1.79 exerted the best efficacy and would be expected to be immunopotentiator molecule during nD vaccination in broilers.

Influence of degree of sulfation on the rheology of cellulose nanocrystal suspensions

The rheology and microstructure of two different cellulose nanocrystals (CNC) samples possessing different degrees of sulfation are studied over a broad concentration range of 1 to 15 wt%. CNC suspensions are isotropic at low concentration and experience two different transitions as concentration increases. First, they form chiral nematic liquid crystals above a first critical concentration where the samples exhibit a fingerprint texture and the viscosity profile shows a three-region behavior, typical of liquid crystals. By further increasing the concentration, CNC suspensions form gels above a second critical concentration, where the viscosity profile shows a single shear-thinning behavior over the whole range of shear rates investigated. It has been found that the degree of sulfation of CNC particles has a significant effect on the critical concentrations at which transitions from isotropic to liquid crystal and liquid crystal to gel occur. Rheological properties and microstructure of these suspensions have been studied using polarized optical microscopy combined with rheometry.

Molecular Mass Characterization of Glycosaminoglycans with Different Degrees of Sulfation in Bioengineered Heparin Process by Size Exclusion Chromatography

Different degrees of glycosaminoglycan sulfation result in their different charge densities. The charge density differences impact their migration behavior in polyacrylamide gel electrophoresis and size exclusion chromatography, two of the most common methods for determining relative molecular masses of polysaccharides. In this study, we investigated the feasibility of using commercially available heparin oligosaccharides as calibrants for measuring the relative molecular masses of intermediates in a bioengineered heparin process that have different levels of sulfation. A size exclusion chromatography method was established that eliminates this charge density effect and allows the determination of relative molecular mass using a single calibration curve with heparin oligosaccharides calibrants. This is accomplished by overcoming the electrostatic interaction between the glycosaminoglycans and size exclusion chromatography stationary phase using high ionic strength mobile phase.

Semi‐Synthetic Polysaccharide Sulfates as Anticoagulant Coatings for PET, 1 – Cellulose Sulfate

In the present study, blood-compatible PET surfaces were prepared by coating with anticoagulant cellulose sulfates that were synthesized homogeneously in ionic liquids. The adsorption behavior of polysaccharides on PET films was investigated using QCM-D. It was demonstrated that pre-coating with different amino-group-containing polysaccharides improves the affinity toward cellulose sulfate. Moreover, the effect of different degrees of sulfation on the adsorption process was evaluated. Based on these results, several layer-by-layer coated PET foils were prepared that showed significantly improved blood compatibility compared to the initial untreated material.

Synthesis of sulfated pectins and their anticoagulant activity

The following pectins were sulfated: bergenan BC (the pectin of Bergenia crassifolia L), lemnan LM (the pectin of Lemna minor L), and galacturonan as a backbone of pectins. Pyridine monomethyl sulfate, pyridine sulfotrioxide, and chlorosulfonic acid were used as reagents for sulfation. Chlorosulfonic acid proved to be the optimal reagent for sulfation of galacturonan and other pectins. Galacturonan and pectin derivatives with different degrees of sulfation were synthesized and their anticoagulant activities were shown to depend on the quantity of sulfate groups in the pectin macromolecules.

Sulfated modification can enhance the immune-enhancing activity of lycium barbarum polysaccharides

In test in vitro, four sulfated lycium barbarum polysaccharides (sLBPSs) with different degrees of sulfation (DS), sLBPS 0.7, sLBPS 1.1, sLBPS 1.5 and sLBPS 1.9, were added into cultured chicken peripheral lymphocytes and the changes of lymphocytes proliferation were compared by MTT assay taking the non-modified LBPS as control. Two sLBPSs with better efficacy, sLBPS 1.5 and sLBPS 1.9 were selected. In test in vivo, one hundred 14-day-old chickens were averagely divided into five groups randomly. The chickens except blank control group were vaccinated with Newcastle disease vaccine, repeated vaccination at 28 days old. At the same time of the first vaccination, the chickens in three experimental groups were injected with 0.5 mL of sLBPS 1.5, sLBPS 1.9 and LBPS at 4 mg mL −1, in vaccination control group, with 0.5 mL of physiological saline, once a day for three successive days. On days 7, 14, 21 and 28 after the first vaccination, the changes of peripheral lymphocytes proliferation and serum HI antibody titer were determined. The result showed that two sLBPSs could significantly promote lymphocytes proliferation and enhance serum antibody titer. These results indicated that sulfated modification could enhance the immune-enhancing activity of LBPS, which there was a certain relativity with the DS of sulfated polysaccharide. sLBPS 1.5 possessed the best efficacy and would be expected as the component drug of a new-type immunopotentiator.

Sequential Enrichment of Sulfated Glycans by Strong Anion-Exchange Chromatography Prior to Mass Spectrometric Measurements

Structural characterization of sulfated glycans through mass spectrometry (MS) has been often limited by their low abundance in biological materials and inefficient ionization in the positive-ion mode. Here, we describe a microscale method for sequentially enriching sulfated glycans according to their degree of sulfation. This method is based on modifying the binding ability of strong anion-exchange material through the use of different sodium acetate concentrations, thus enabling fairly selective binding and a subsequent elution of different glycans according to their degree of sulfation. Before this enrichment, the negative charge on the sialic acid, which is commonly associated with such glycans, was eliminated through permethylation that is used to enhance the positive-ion mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS) signal for all glycans. This enrichment approach minimizes competitive ionization between sulfated and neutral glycans, as well as that between sulfated species with a different degree of sulfation. The described method was initially optimized using sulfated oligosaccharide standards, while its potential has been verified for the sulfated N-glycans originated from the bovine thyroid-stimulating hormone (bTSH), a glycoprotein possessing mono- and disulfated N-glycans. This enhancement of the MALDI-MS signal facilitates analysis of some otherwise undetected components.

Sulfation of (1→3)-β- d-glucan from the fruiting bodies of Russula virescens and antitumor activities of the modifiers

A water-insoluble (1→3)-β- d-glucan isolated from the fresh fruiting bodies of Russula virescens was sulfated using sulfur trioxide-pyridine complex as reagent in dimethyl sulfoxide. Depending on the reaction conditions, the products showed different degrees of sulfation (DS) ranging from 0.17 to 1.17 and different weight average molecular weights (M ws) ranging from 2.5 × 10 4 to 1.2 × 10 5 Da. Moreover, the antitumor activities of the five sulfated derivatives against Sarcoma 180 tumor cell were tested both in vitro and in vivo. The results indicated that the native (1→3)-β- d-glucan did not show antitumor activity, while the sulfated derivatives exhibited enhanced antitumor activities. This study demonstrated that DS and M w could influence the antitumor activities of the sulfated derivatives.

Kinetic Analysis of Heparin and Glucan Sulfates Binding to P-Selectin and Its Impact on the General Understanding of Selectin Inhibition

P-Selectin, expressed on activated endothelial cells and platelets, is a high kinetic adhesion receptor involved in leukocyte rolling of the inflammatory response, or in tumor cell binding in the course of metastasis. Thus, P-selectin inhibition is a promising therapeutic target. The anti-inflammatory and anti-metastatic activities of heparin have partly been related to the inhibition of P-selectin binding. Here we apply a quartz crystal microbalance (QCM) biosensor to determine the kinetic constants of heparin and other sulfated polysaccharides binding to immobilized P-selectin. Binding kinetics of the derivatives were correlated with their inhibitory capacity in a P-selectin cell rolling assay. Three commercial heparins differ in cell rolling inhibition and display slightly different affinities (KD 1.21 x 10(-6) M to 5.86 x 10(-7) M). Inhibitory capacity appears to be mainly driven by a slow off-rate from the receptor (2.27 x 10(-3) s-1 to 1.23 x 10(-3) s-1). To correlate the impact of binding kinetics on inhibitory capacity structurally, we analyzed six semisynthetic glucan sulfates. They display different degrees of sulfation (DS), which has a strong influence on inhibitory activity. Kinetic data illustrate that the inhibitory capacity correlates excellently with the off-rate of these polysaccharides (R = 0.99), while the association (on-rate) affects activity to a lesser extent. In general, the consideration of binding kinetics sheds new light on the mechanism of selectin inhibition. A much slower dissociation of the inhibitors from the receptor than the physiological ligands is key for inhibitory capacity. Structurally, highly charged compounds with a slow off-rate, such as heparin or glucan sulfates, appear as potent candidates for P-selectin inhibition.

Combined removal of SO2 and no using sol‐gel‐derived copper oxide coated alumina sorbents/catalysts

The present paper reports experimental results on the removal of sulfur dioxide and nitrogen oxide from simulated flue gas using a copper oxide coated on alumina sorbent/catalyst prepared by the sol‐gel method. Selective catalytic reduction of nitric oxide by ammonia over sol‐gel derived CuO/y‐Al2O3 sorbents/catalysts with different degrees of sulfation was studied in a fixed‐bed packed reactor. The optimum temperature for NO reduction was found at 350°C for both fresh and sulfated catalysts. The properties for simultaneous removal of SO2 and NO by the sol‐gel‐derived CuO/γ‐Al2O3 sorbents were studied using simulated dry flue gas. The optimum operating temperature for the combined deSO2/deNO operations was identified at 350°C. At the space velocity of 5200 h‐1 and 350°C, a fixed‐bed reactor packed with the 7.9 wt% CuO/γ‐Al2O3 sorbent prepared by the sol‐gel method offers SO2 Sorption capacity of 2.3 mmol g‐1 and NO conversion of 92% with a dry simulated flue gas as the feed. Under these experimental conditions, the sol‐gel derived sorbents/catalysts have comparable efficiency for removal of SO2 and NOX as their commercial counterparts. The significantly higher crush strength of the sol‐gel derived sorbents/catalysts make them very promising for their use in the copper oxide process for combined removal of SO2 and NOx from flue gas in a single unit operation.

Structural investigation of chondroitin/dermatan sulfate oligosaccharides from human skin fibroblast decorin.

Hybrid chondroitin/dermatan sulfate (CS/DS) glycosaminoglycan chains, derived from decorin secreted by human skin fibroblasts, were shown to interact with FGF-2, as did oligosaccharides derived therefrom by chondroitin B lyase digestion. In a first attempt to identify the biologically active sequence, a novel protocol for structural analysis of enzyme-resistant oligosaccharides larger than standard trisulfated hexasaccharides was developed. The method bases on capillary electrophoresis (CE) for separating oversulfated species in offline combination with nanoelectrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoESI-QTOF-MS/MS) in the negative ion mode. Under optimized CE and ESI-MS conditions, up to 12-mer oligosaccharides with different degrees of sulfation were identified. A novel tandem MS protocol (CID-VE) was applied to elucidate the structure of a previously undescribed pentasulfated CS/DS hexasaccharide, Delta-4,5-IdoAGalNAc[GlcAGalNAc]2(5S). In this molecular species, detected as a triply charged ion at m/z 511.38, three sulfates are found in the IdoAGalNAcGlcA moiety offering two structural variants: one containing sulfated IdoA together with a disulfated GalNAc moiety and in the other one both uronic acids, that is, GlcA and IdoA and the amino sugar each carry a sulfate ester group.

Chemical modification, characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides

A natural lacquer polysaccharide with complex branches was separated into two fractions, LPH (MW 16.9×10 4) and LPL (MW 6.85×10 4). Results of 13C NMR and FT-IR indicated they had the same structure. The treatment of LPL with sodium periodate led to a partial cut-off of side chains with 4- O-methyl-- d-glucuronic acid in the terminal. These polysaccharides were sulfated in the presence of Py·SO 3/DMSO. Depending on the reaction conditions, the products showed a different degree of sulfation (DS) ranging from 0.57 to 1.57 and different molecular weights ranging from 1.71×10 4 to 3.49×10 4. FT-IR analysis showed the equatorial primary OH at O-6 and the axial secondary OH at O-4 were sulfated. Activated partial thromboplastin time (APTT), prothrombin time and thrombin time (TT) assays showed the sulfated polysaccharides could prolong APTT and TT, but not TP. These activities strongly depended on the DS, the molecular weights (MW) and the branching structure of polysaccharides. DS of above 0.8 was essential for anticoagulant activity. The anticoagulant activity increased with the DS and the molecular weights. The molecular weights played a more important role. The branching structure of polysaccharides increased the activities. In our studies, the sulfated polysaccharides with the DS of 1.15 and the highest MW of 3.49×10 4 had the best blood anticoagulant activities.

Effects of calcium ions on the interactions between antithrombin and factor Xa mediated by variously sulfated, semisynthetic low-molecular-weight heparins.

A homogeneous set of low-molecular weight heparins, chemically modified to yield different degrees of sulfation, were investigated for their ability to interfere with the antithrombin (AT)-factor Xa (FXa) interaction process in the presence or absence of physiological concentrations of calcium ions. The heparin-AT dissociation constants were not appreciably affected by the presence of the metal ion, whereas the catalytic process was strongly dependent on Ca 2+. Our data suggest that AT binding to heparin represents the main factor driving the FXa inhibition process. In addition, the presence of the metal ion is likely to mask favorable AT- heparin ionic contacts occurring with the highly sulfated material. These results help in assessing proper structure-activity relationships for glycosaminoglycans, a multitarget family of biologically active compounds.

Development of SPC-ELISA: A New Assay Principle for the Study of Sulfated Polysaccharide–Protein Interactions

A sulfated polysaccharide-coating enzyme-linked immunosorbent assay (SPC-ELISA), a new screening assay for the study of interactions between sulfated polysaccharides and proteins, has been developed. Fibrinogen was used as representative for the protein. A microplate is coated with the sulfated polysaccharide to be tested and then incubated with various concentrations of fibrinogen. The bound fibrinogen is quantified by ELISA technique. The assay has been optimized with respect to coating procedure, incubation times, antibody concentrations, and detection conditions. Its capacity was demonstrated using three different sulfated polysaccharides: heparin, a sulfated glucuronogalactan extracted from a red algae, and a semisynthetic xanthan sulfate. Furthermore, the fibrinogen binding of semisynthetic laminarin sulfates with different degrees of sulfation showed good correlation with their anticoagulant effect as measured by fibrinogen clotting time. The intraassay as well as the interassay variations were lower than 8%. The binding properties observed in the SPC-ELISA correlated well with those found utilizing conventional gel permeation chromatography and fibrinogen affinity gel electrophoresis. Compared to these methods, the SPC-ELISA has several advantages: It is more rapid and far easier to perform, allows high throughput screening, and is suitable for automation. Furthermore, it is inexpensive, highly sensitive, and reproducible and has no special equipment requirements. Finally, the method represents the basis for multiple variations with regard to the target proteins as well as the detection methods.

A wide diversity of sulfated polysaccharides are synthesized by different species of marine sponges

Sulfated polysaccharides were extracted from four species of marine sponges by exhaustive papain digestion. These compounds were purified by anion-exchange and gel-filtration chromatography. Analysis of the purified polysaccharides revealed a species-specific variation in their chemical composition and also in their molecular masses. In the species Aplysina fulva we found a sulfated glucan with a glycogen-like structure. The other three species contained sulfated polysaccharides with variable proportions of galactose, fucose, arabinose and hexuronic acid and also with different degrees of sulfation. Although the complex nature of these polysaccharides did not allow complete structure determination, we detected the occurrence of 4-sulfated residues of fucose and arabinose in the species Dysidea fragilis. The biological role of these sulfated polysaccharides requires further investigation. They may be involved in the species-specific aggregation of sponge cells and/or in the structural integrity of sponge, resembling the proteoglycans of mammalian connective tissues.