Sulfated Sugars Research Articles

Sugar-based anionic surfactants: synthesis and micelle formation of sodium methyl 2-acylamido-2-deoxy-6- O-sulfo- d-glucopyranosides

The following sequence of reactions has been employed to synthesize the title anionic surfactants: where R=C 7H 15 ( 3a); C 11H 23 ( 3b); and C 15H 31 ( 3c), respectively, and Py refers to pyridine. Aggregation of the surfactants synthesized (predominantly α anomers) in water was studied at 40 °C by conductivity measurements. Increasing the chain length of R decreases the critical micelle concentration (CMC) and the degree of counter-ion dissociation. The dependence of the Gibbs free energy of micellization and CMC on the length of R is similar to other ionic surfactants, but the head-group, i.e., the sulfated sugar moiety is less hydrophilic than the structurally related group (OCH 2CH 2) 2OSO 3 −Na +, most probably because of intermolecular H-bonding in the micellar pseudo-phase

Reconstitution of Prion Infectivity from Solubilized Protease-resistant PrP and Nonprotein Components of Prion Rods

The scrapie isoform of the prion protein, PrP(Sc), is the only identified component of the infectious prion, an agent causing neurodegenerative diseases such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Following proteolysis, PrP(Sc) is trimmed to a fragment designated PrP 27-30. Both PrP(Sc) and PrP 27-30 molecules tend to aggregate and precipitate as amyloid rods when membranes from prion-infected brain are extracted with detergents. Although prion rods were also shown to contain lipids and sugar polymers, no physiological role has yet been attributed to these molecules. In this work, we show that prion infectivity can be reconstituted by combining Me(2)SO-solubilized PrP 27-30, which at best contained low prion infectivity, with nonprotein components of prion rods (heavy fraction after deproteination, originating from a scrapie-infected hamster brain), which did not present any infectivity. Whereas heparanase digestion of the heavy fraction after deproteination (originating from a scrapie-infected hamster brain), before its combination with solubilized PrP 27-30, considerably reduced the reconstitution of infectivity, preliminary results suggest that infectivity can be greatly increased by combining nonaggregated protease-resistant PrP with heparan sulfate, a known component of amyloid plaques in the brain. We submit that whereas PrP 27-30 is probably the obligatory template for the conversion of PrP(C) to PrP(Sc), sulfated sugar polymers may play an important role in the pathogenesis of prion diseases.

Carbohydrate sulfation effects on growth of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a key player in the pathology and morbidity of cystic fibrosis. Chronic obstructive pulmonary disease, which results from the most common and severe mutations in this genetic disorder, typically includes chronic infection with P. aeruginosa which, even with rugged antibiotic and physical therapy regimens, is rarely eradicated. It is not known whether the increased oligosaccharide sulfation characteristic of cystic fibrosis tracheobronchial mucins plays a role in the survival of P. aeruginosa in the airway. In this study, sulfated monosaccharides were synthesized and tested for their effects on the growth of clinical isolates and laboratory strains of this organism when supplied as the sole carbon source in vitro. Carbohydrate sulfation was observed to reduce, but not prohibit, growth of P. aeruginosa on carbohydrates normally utilized in their nonsulfated form. The various sulfated sugars employed as the sole carbon source gave characteristic and consistent growth profiles and maximum growth values across the strains tested. P. aeruginosa isolates from patients with cystic fibrosis often express a mucoid phenotype, which is thought to contribute to their ability to survive in harsh conditions. Carbohydrate sulfation effects on growth did not differ significantly between mucoid and nonmucoid strains. These results suggest that the additional sulfation of tracheobronchial mucin documented in cystic fibrosis may in fact contribute to the mucin's resistance to utilization by P. aeruginosa and potentially other pathogens, providing an additional level of host protection, and limiting the available nutrient pool and thereby bacterial growth.

Engineering of an FGF-proteoglycan fusion protein with heparin-independent, mitogenic activity.

In the absence of heparan sulfate (HS) on the surface of target cells, or free heparin (HP) in the vicinity of their receptors, fibroblast growth factor (FGF) family members cannot exert their biological activity and are easily damaged by proteolysis. This limits the utility of FGFs in a variety of applications including treatment of surgical, burn, and periodontal tissue wounds, gastric ulcers, segmental bony defects, ligament and spinal cord injury. Here we describe an FGF analog engineered to overcome this limitation by fusing FGF-1 with HS proteoglycan (PG) core protein. The fusion protein (PG-FGF-1), which was expressed in Chinese hamster ovary cells and collected from the conditioned medium, possessed both HS and chondroitin sulfate sugar chains. After fractionation, intact PG-FGF-1 proteins with little affinity to immobilized HP and high-level HS modification, but not their heparitinase or heparinase digests, exerted mitogenic activity independent of exogenous HP toward HS-free Ba/F3 transfectants expressing FGF receptor. Although PG-FGF-1 was resistant to tryptic digestion, its physiological degradation with a combination of heparitinase and trypsin augmented its mitogenic activity toward human endothelial cells. The same treatment abolished the activity of simple FGF-1 protein. By constructing a biologically active proteoglycan-FGF-1 fusion protein, we have demonstrated an approach that may prove effective for engineering not only FGF family members, but other HP-binding molecules as well.

Docking of Glycosaminoglycans to Heparin-Binding Proteins: Validation for aFGF, bFGF, and Antithrombin and Application to IL-8

Heparin−protein interactions play an important role in many steps of the immune system. Here, we evaluated the search capabilities of three widely used programsGRID, DOCK, and AutoDockfor heparin binding sites. Because of the weak surface complementarity and the high charge density of the sulfated sugar chain, the docking of heparin to its protein partners presents a challenging task for computational docking. Our protocols were tested on antithrombin and acidic and basic fibroblast growth factor, the only three proteins for which structures of their complexes with heparin are available. With all three programs, the heparin binding site for these test cases was determined correctly. We then used these protocols to predict the heparin binding site on Interleukin-8, a chemokine with a central role in the human immune response. The results indicate that His18, Lys20, Arg60, Lys64, and Arg68 in interleukin-8 bind to heparin.

The structure and function of a foot-and-mouth disease virus-oligosaccharide receptor complex.

Heparan sulfate has an important role in cell entry by foot-and-mouth disease virus (FMDV). We find that subtype O1 FMDV binds this glycosaminoglycan with a high affinity by immobilizing a specific highly abundant motif of sulfated sugars. The binding site is a shallow depression on the virion surface, located at the junction of the three major capsid proteins, VP1, VP2 and VP3. Two pre-formed sulfate-binding sites control receptor specificity. Residue 56 of VP3, an arginine in this virus, is critical to this recognition, forming a key component of both sites. This residue is a histidine in field isolates of the virus, switching to an arginine in adaptation to tissue culture, forming the high affinity heparan sulfate-binding site. We postulate that this site is a conserved feature of FMDVs, such that in the infected animal there is a biological advantage to low affinity, or more selective, interactions with glycosaminoglycan receptors.

A Toxoplasma Lectin-like Activity Specific for Sulfated Polysaccharides Is Involved in Host Cell Infection

Toxoplasma gondii is one of the most widespread parasites of humans and animals. The parasite has a remarkable ability to invade a broad range of cells within its mammalian hosts by mechanisms that are poorly understood at the molecular level. This broad host cell specificity suggests that adhesion should involve the recognition of ubiquitous surface-exposed host molecules or, alternatively, the presence of various parasite attachment molecules able to recognize different host cell receptors. We have discovered a sugar-binding activity (lectin) in tachyzoites of T. gondii that plays a role in vitro in erythrocyte agglutination and infection of human fibroblasts and epithelial cells. The ability to agglutinate erythrocytes can be reversed by a variety of soluble glycoconjugates, of which heparin, fucoidan, and dextran sulfate were the most effective. Interestingly, infectivity of tachyzoites for human foreskin fibroblasts, cells that are commonly used to grow T. gondii in vitro, was increased by low concentrations of the sulfated glycoconjugates that inhibited hemagglutination activity (i.e. dextran sulfate and fucoidan) whereas high concentrations inhibited parasite infection. Furthermore, inhibition of glycosaminoglycan biosynthesis and sulfation on the host cells reduced Toxoplasma infectivity. Finally, Toxoplasma tachyzoites showed a reduced ability to infect epithelial cell mutants deficient in the biosynthesis of surface proteoglycans. The probable identity of the hemagglutinin(s) was investigated by 1) direct binding of red blood cells to filter blots of Toxoplasma proteins separated by polyacrylamide gel electrophoresis, and 2) binding of metabolically labeled parasite proteins to fixed mammalian cells. Three parasite bands were thus identified as candidate adhesins. These results suggest that attachment of T. gondii to its target cell is mediated by parasite lectins and that sulfated sugars on the surface of host cells may function as a key parasite receptor.

糖鎖リガンドの分子モデリングに基づく非糖リガンドのデザイン セレクチン阻害剤を例に

A design of non-carbohydrate selectin blockers based on a carbohydrate compound is described. We first investigated the mode of interaction between selectin and its carbohydrate ligand, GSC-150. GSC-150 has a sulfated sugar unit and a branched long alkyl chain (B-30). From a molecular dynamics simulation of the complex of GSC-150 with E-selectin in water, we found the essential three functional groups of GSC-150 necessary for binding toward selectin: namely, the negatively-charged sulfate, the fucose, and B-30. Especially, B-30 would play an important role in the tight hydrophobic interaction with selectin. The lactose unit of GSC-150 may serve as a scaffold that keeps those functional groups to their proper orientation. Next we searched for another scaffold, and found that the modified Ser-Glu dipeptides could be good surrogates. They are about 50 to 100-fold more potent blocker than GSC-150. A detailed analysis of their binding mode suggested that the dipeptide backbone could adopt type-II β-turn conformation, a minor subtype of β-turn, in their bound state. This type-II β-turn would be necessary to fix the critical functional groups in the dipeptides to their proper orientation.

Endocytosis of lutropin by Leydig cells through a pathway distinct from the high-affinity receptor

In porcine Leydig cells in primary culture, 95% of the internalization of [ 125I]porcine lutropin ([ 125I]pLH, which bears sulfated GalNAc) could not be ascribed to the high-affinity LH receptor (LHR). In contrast, >40% of [ 125I]human choriogonadotropin (hCG, with sialylated sugar chains) uptake was performed by the LHR itself. When the LHR was down-regulated by excess unlabeled hormone, the LHR-independent incorporation of [ 125I]pLH could be inhibited in a dose-dependent fashion by sulfated polysaccharides such as fucoidan or chondroitin-(4 or 6)-sulfate, but not by other polyanionic compounds, nor by sulfated chondroitin disaccharides. Endocytosis occurred through a clathrin-dependent pathway and was inhibited by low temperature, endocytosis inhibitors, increased ionic strength, or by EDTA and dithiothreitol. Taken together, these results suggest that a Leydig cell membrane protein (possibly a lectin, or a glycosaminoglycan receptor) could perform specific LH clearance in the testis via recognition of its sulfated sugars.

Identification and characterization of heparin binding regions of the Fim2 subunit of Bordetella pertussis.

Bordetella pertussis fimbriae bind to sulfated sugars such as heparin through the major subunit Fim2. The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins. Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin. The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine. These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required. Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity. Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides. A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars. A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved. The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant. Since B. pertussis fimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed.

Beta-Carboline alkaloids as matrices for UV-matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in positive and negative ion modes. Analysis of proteins of high molecular mass, and of cyclic and acyclic oligosaccharides.

We report that commercially available beta-carbolines (nor-harmane (9H-pyrido[3,4-b]indole), harmane (1-methyl-9H-pyrido[3,4-b]indole), harmine (7-methoxy-1-methyl-9H-pyrido[3,4-b]indole), harmol (1-methyl-9H-pyrido[3,4-b]indol-7-ol), harmaline (3,4-dihydro-7-methoxy-1-methyl-9H-pyrido[3,4-b]indole) and harmalol (3,4-dihydro-1-methyl-9H-pyrido[3,4-b]indol-7-ol)), are useful MALDI matrices at 337 nm, for cyclic oligosaccharides (cyclodextrins, range 972-1290 Da), acyclic oligosaccharides (range 342-828 Da) and high molecular mass proteins (range 23,290-66,525 Da) in both positive and negative modes. This was investigated by using time-of-flight (TOF) mass spectrometers of different sensitivities, equipped with and without pulse extraction facilities. A comparison with conventional matrices for carbohydrates (DHB and DHB/HIC) indicates that beta-carbolines provide the same level of sensitivity and resolution in the positive mode, but offer the advantage of high levels of sensitivity and resolution in the negative mode. Harmaline has been found to be specially effective for the analysis of high-mass proteins in both modes, and also exhibits excellent experimental reproducibility of the results owing to the homogeneous crystallization of the analyte-matrix mixture over the entire sample surface area. Harmane and nor-harmane are both excellent matrices for high-mass proteins also. As MALDI matrices, beta-carbolines permit measurement of sulfated sugars in the negative ion mode as ([M-H]), and of neutral sugars and proteins as both [M + H]+ and [M-H]- in appropriate modes.

Convenient synthetic approach towards regioselectively sulfated sugars using limpet and abalone sulfatase-catalyzed desulfation

3,6-Disulfated p-nitrophenyl β-D-galactopyranoside 3 and 3′,6′-disulfated p-nitrophenyl β-lactoside 7, which were regioselectively obtained via organotin methodology, were subjected to enzymatic desulfation: limpet and abalone sulfatases (EC 3.1.6.1) hydrolyzed regioselectively the 3- and 3′-sulfate moieties of 3 and 7 to afford 6-monosulfated galactopyranoside 4 and 6′-monosulfated lactoside 8, respectively.

Charge-based interactions of mammalian sperm with oocytes: inhibition of fertilization of mouse oocytes by ligands of macrophage scavenger receptor(s)

Objective: To determine whether anionic ligands for the macrophage scavenger receptor inhibit the fertilization of mouse oocytes by mouse spermatozoa. Design: In vitro study of sperm binding and two-cell embryo formation in the presence of scavenger receptor ligands. Sperm-oocyte interaction may be mediated by sulfated sugars. In this study, we tested other nonsulfated anionic ligands for the scavenger receptor for their ability to affect fertilization. The only common feature of these ligands is their anionic nature. Setting: Oocytes and sperm from mice were used. Main Outcome Measure(s): Binding of sperm to oocytes and subsequent formation of two cell embryos were determined. Result(s): Fucoidin, polyinosinic acid, oxidized low-density lipoprotein, acetyl low-density lipoprotein, and malondialdehyde-modified LDL inhibited the binding and fertilization of mouse sperm to mouse oocytes. Addition of fresh sperm to oocytes previously treated with sperm in the presence of these agents restored the binding and fertilization. Conclusion(s): These results show that charge-based interactions analogous to the interactions of the scavenger receptor with its ligands may play an important role in mammalian fertilization.

Expeditious synthesis of monosulfated thio-linked disaccharides

J. A. Calvo-Asín, F. G. Calvo-Flores, J. M. Exposito-López, F. Hernández-Mateo, J. J. García-López, J. Isac-García, F. Santoyo-González and A. Vargas-Berenguel, J. Chem. Soc., Perkin Trans. 1, 1997, 1079 DOI: 10.1039/A701345K

Pertussis antigens that abrogate bacterial adherence and elicit immunity.

Infectious disease processes follow the initial steps of adherence of the organism to host tissues and subsequent colonization of the target tissues that can occur through specific adhesion-receptor systems. Bordetella pertussis, the human pathogen that causes whooping cough, has evolved a genetically controlled system whereby adhesins are expressed when they enter the human host. Two adhesins, filamentous hemagglutinin (FHA) and pertactin, mediate the adherence of the bacterium to eukaryotic cells through varied attachment mechanisms, including lectin-like binding sites that interact with sulfated sugars on cell surface glycoconjugates and the ARG-GLY-ASP binding sequence, which recognizes a family of integrins found on the cell surface. The differential expression of relevant receptors by various eukaryotic cells likely plays a role in the pathogenesis and immune response to the bacterium by the host, directing the organism to specific cell types and to specific tissue sites. Substantial evidence exists that the B. pertussis adhesins, FHA and pertactin, elicit immune responses that are protective in animal models for the disease, including serum antibody production and local immune responses in the respiratory tract following nasal administration of encapsulated antigens. Both of these adhesins are components of new acellular pertussis vaccines that have proven safe and highly effective for prevention of serious disease in infants.

Sulfated malto-oligosaccharides bind to basic FGF, inhibit endothelial cell proliferation, and disrupt endothelial cell tube formation

The interaction of basic FGF (bFGF) with heparin, heparan sulfate and related sugars can potentiate or antagonize bFGF activity, depending on the size of the saccharide used. Oligosaccharides based on heparin structures, as small as six sugar residues, have been demonstrated to bind to bFGF and block its activity, while larger structures (> 10 sugar residues) tend to potentiate bFGF. In this study we have synthesized a series of compounds designed to test the requirements of size and sulfation for binding of oligosaccharides to bFGF. These oligosaccharides are not derived from heparin, but rather, are linear chains of glucose linked alpha 1-4 (malto-oligosaccharides) that have been chemically sulfated. In addition to bFGF binding, these compounds were tested for their ability to block basic functions of endothelial cells that are known to be mediated, at least in part, by bFGF. We report that the ability of sulfated malto-oligosaccharides to block binding of bFGF to heparan sulfate was dependent on the size (at least a tetrasaccharide is required), and the degree of sulfation. The activity profile in the bFGF ELISA closely correlated with the ability of these compounds to block REEC or HMVEC tube formation on Matrigel. There was a similar relationship of size and sulfation to the ability of the sulfated malto-oligosaccharides to inhibit endothelial cell growth for most human and rat EC types tested. The single exception was REEC cell growth. One isolate of these cells was stimulated by sulfated malto-oligosaccharides rather than inhibited by them, while a second isolate was neither stimulated nor inhibited. This stimulation showed no correlation with inhibition of bFGF binding in the ELISA assay, suggesting that growth of this cell type was probably not dependent on bFGF. Compounds derived from this series of sulfated, malto-oligosaccharides have the potential to function as bFGF antagonists, are relatively easy to produce, and possess relatively low anticoagulant properties.

The major fimbrial subunit of Bordetella pertussis binds to sulfated sugars.

Bordetella pertussis fimbriae are composed of major and minor subunits, and recently it was shown that the minor fimbrial subunit binds to Vla-5, a receptor located on monocytes (W. Hazenbos, C. Geuijen, B. van den Berg, F. Mooi, and R. van Furth, J. Infect. Dis. 171:924-929, 1995). Here we present evidence that the major subunits bind to sulfated sugars, which are ubiquitous in the respiratory tract. Binding was observed to chondroitin sulfate, heparan sulfate, and dextran sulfate but not to dextran. Removal of the minor subunit from fimbriae did not significantly affect binding to sulfated sugars, indicating that the major subunit alone is sufficient for this binding. Fimbriae were also able to bind HEp-2 cells, which are known to display glycoconjugates on their surface. This binding was not dependent on the presence of the minor subunit. However, binding was dependent on the sulfation state of the glycoconjugates, since inhibition of the sulfation resulted in a significant reduction of fimbria binding. The specificity of fimbria binding was further characterized by using heparan sulfate-derived disaccharides in inhibition assays. Two disaccharides were highly effective inhibitors, and it was observed that both the degree of sulfation and the arrangement of the sulfate groups on the disaccharides were important for binding to fimbriae. B. pertussis bacteria also bound to sulfated sugars and HEp-2 cells, and analysis of B. pertussis mutants indicated that both filamentous hemagglutinin and fimbriae were required for this binding. A host protein present in the extracellular matrix, fibronectin, has binding activities similar to those of B. pertussis fimbriae, binding to both Vla-5 and sulfated sugars. Two regions in the major fimbrial subunit were identified which showed similarity with fibronectin peptides which bind to sulfated sugars. Thus, B. pertussis fimbriae exemplify molecular mimicry and may co-opt host processes by mimicking natural ligand-receptor interactions.

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids.

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le(a)- and Le(x)-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higher m/z values than the precursor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.

Determination of alkyl sulfonic acids in pharmaceuticals by ion chromatography

Ion chromatography (IC) using chemically suppressed conductivity detection was developed to separate and detect methane- through octanesulfonic acids. Using a gradient borate-acetonitrile mobile phase system on a Dionex PAX-500 column, one is able to resolve the alkylsulfonic acids from most matrix interferents. Resolution was determined for all acid pairs from methane- through octanesulfonic acid. A specific application is described using a SARASEP AN1 column with a borate mobile phase for the assay of methanesulfonic acid (MSA) in a sulfated sugar compound.under pharmaceutical investigation. MSA is one constituent used in the synthesis of this sulfated sugar compound. The average method recovery for MSA over the six levels investigated was 102.9% and the average method recovery for sulfate was 92.3%. Method reproducibility was determined over a two-day period with a single sample to be 7.5% and 14% for MSA and sulfate, respectively. The IC method was determined to have a linear response for MSA and sulfate over the working range investigated. The limit of quantification was 0.004% and 0.02% (w/w) for MSA and sulfate, respectively. The limit of detection was 0.0005% and 0.001% for MSA and sulfate, respectively.

A New Approach to the Characterization of Sulfated and Sialyl Lewis-Type Glycosphingolipids Using Positive-Ion FABMS, ESIMS, and CID-MS/MS.

Positive-ion fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry have been used in the structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated and sialyl sugars, and provided information on the sugar chain sequences. The positive-ion tandem FABMS of sulfated Le-type glycosphingolipids gave the daughter ions produced by the elimination of (fucose-H2O) unit from the fragment ions containing the non-reducing end. On the other hand, the CID-MS/MS of sialyl analogs showed the characteristic ions derived from the loss of (N-acetylneuraminic acid-H2O) unit, in addition to the elimination of (fucose-H2O) unit. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the daughter ions useful for structural studies appeared in the higher mass region than the precursor owing to a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogs.