Chemical Synthesis of Fucosylated Chondroitin Sulfate Tetrasaccharide with Fucosyl Branch at the 6-OH of GalNAc Residue.

Fucosylated chondroitin sulfate is a unique glycosaminoglycan isolated from sea cucumbers, with excellent anticoagulant activity. The fucosyl branch in FCS is generally located at the 3-OH of D-glucuronic acid but, recently, a novel structure with α-L-fucose linked to the 6-OH of N-acetyl-galactosamine has been found. Here, using functionalized monosaccharide building blocks, we prepared novel FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and 3-OH of GlcA. In the synthesis, the protective group strategy of selective O-sulfation, as well as stereoselective glycosylation, was established, which enabled the efficient synthesis of the specific tetrasaccharide compounds. This research enriches knowledge on the structural types of FCS oligosaccharides and facilitates the exploration of the structure-activity relationship in the future.

Nuclear magnetic resonance-based structural elucidation of novel marine glycans and derived oligosaccharides.

Marine glycans of defined structures are unique representatives among all kinds of structurally complex glycans endowed with important biological actions. Besides their unique biological properties, these marine sugars also enable advanced structure-activity relationship (SAR) studies given their distinct and defined structures. However, the natural high molecular weights (MWs) of these marine polysaccharides, sometimes even bigger than 100 kDa, pose a problem in many biophysical and analytical studies. Hence, the preparation of low MW oligosaccharides becomes a strategy to overcome the problem. Regardless of the polymeric or oligomeric lengths of these molecules, structural elucidation is mandatory for SAR studies. For this, nuclear magnetic resonance (NMR) spectroscopy plays a pivotal role. Here, we revisit the NMR-based structural elucidation of a series of marine sulfated poly/oligosaccharides discovered in our laboratory within the last 2 years. This set of structures includes the α-glucan extracted from the bivalve Marcia hiantina; the two sulfated galactans extracted from the red alga Botryocladia occidentalis; the fucosylated chondroitin sulfate isolated from the sea cucumber Pentacta pygmaea; the oligosaccharides produced from the fucosylated chondroitin sulfates from this sea cucumber species and from another species, Holothuria floridana; and the sulfated fucan from this later species. Specific 1H and 13C chemical shifts, generated by various 1D and 2D homonuclear and heteronuclear NMR spectra, are exploited as the primary source of information in the structural elucidation of these marine glycans.

Anti-SARS-CoV-2 and anticoagulant properties of Pentacta pygmaea fucosylated chondroitin sulfate depend on high molecular weight structures.

Fucosylated chondroitin sulfate (FucCS) is a unique marine glycosaminoglycan that exhibits diverse biological functions, including antiviral and anticoagulant activity. In previous work, the FucCS derived from Pentacta pygmaea (PpFucCS) showed moderate anticoagulant effect but high inhibitory activity against the Wuhan strain of severe acute respiratory syndrome coronavirus (SARS-CoV-2). In this study, we perform free-radical depolymerization of PpFucCS by the copper-based Fenton method to generate low molecular weight (MW) oligosaccharides. PpFucCS oligosaccharides were structurally analyzed by 1H nuclear magnetic resonance spectroscopy and were used to conduct structure-activity relationship studies regarding their effects against SARS-CoV-2 and clotting. Anticoagulant properties were measured by activated partial thromboplastin time, protease (factors Xa and IIa) inhibition by serine protease inhibitors (antithrombin [AT] and heparin cofactor II [HCII]), and competitive surface plasmon resonance (SPR) assay using AT, HCII, and IIa. Anti-SARS-CoV-2 properties were measured by the concentration-response inhibitory curves of HEK-293T-human angiotensin-converting enzyme-2 cells infected with a baculovirus pseudotyped SARS-CoV-2 Delta variant spike (S)-protein and competitive SPR assays using multiple S-proteins (Wuhan, N501Y [Alpha], K417T/E484K/N501Y [Gamma], L542R [Delta], and Omicron [BA.2 subvariant]). Cytotoxicity of native PpFucCS and oligosaccharides was also assessed. The PpFucCS-derived oligosaccharide fraction of the highest MW showed great anti-SARS-CoV-2 Delta activity and reduced anticoagulant properties. Results have indicated no cytotoxicity and MW dependency on both anti-SARS-CoV-2 and anticoagulant effects of PpFucCS, as both actions were reduced accordingly to the MW decrease of PpFucCS. Our results demonstrate that the high-MW structures of PpFucCS is a key structural element to achieve the maximal anti-SARS-CoV-2 and anticoagulant effects.

Structural and kinetic analyses of holothurian sulfated glycans suggest potential treatment for SARS-CoV-2 infection.

Certain sulfated glycans, including those from marine sources, can show potential effects against SARS-CoV-2. Here, a new fucosylated chondroitin sulfate (FucCS) from the sea cucumber Pentacta pygmaea (PpFucCS) (MW ∼10-60 kDa) was isolated and structurally characterized by NMR. PpFucCS is composed of {→3)-ß-GalNAcX-(1→4)-ß-GlcA-[(3→1)Y]-(1→}, where X = 4S (80%), 6S (10%) or nonsulfated (10%), Y = α-Fuc2,4S (40%), α-Fuc2,4S-(1→4)-α-Fuc (30%), or α-Fuc4S (30%), and S = SO3-. The anti-SARS-CoV-2 activity of PpFucCS and those of the FucCS and sulfated fucan isolated from Isostichopus badionotus (IbFucCS and IbSF) were compared with that of heparin. IC50 values demonstrated the activity of the three holothurian sulfated glycans to be ∼12 times more efficient than heparin, with no cytotoxic effects. The dissociation constant (KD) values obtained by surface plasmon resonance of the wildtype SARS-CoV-2 spike (S)-protein receptor-binding domain (RBD) and N501Y mutant RBD in interactions with the heparin-immobilized sensor chip were 94 and 1.8 × 103 nM, respectively. Competitive surface plasmon resonance inhibition analysis of PpFucCS, IbFucCS, and IbSF against heparin binding to wildtype S-protein showed IC50 values (in the nanomolar range) 6, 25, and 6 times more efficient than heparin, respectively. Data from computational simulations suggest an influence of the sulfation patterns of the Fuc units on hydrogen bonding with GlcA and that conformational change of some of the oligosaccharide structures occurs upon S-protein RBD binding. Compared with heparin, negligible anticoagulant action was observed for IbSF. Our results suggest that IbSF may represent a promising molecule for future investigations against SARS-CoV-2.

Inhibition of SARS-CoV-2 wild-type (Wuhan-Hu-1) and Delta (B.1.617.2) strains by marine sulfated glycans.

The Coronavirus disease pandemic has steered the global therapeutic research efforts toward the discovery of potential anti-severe acute respiratory syndrome coronavirus (SARS-CoV-2) molecules. The role of the viral spike glycoprotein (S-protein) has been clearly established in SARS-CoV-2 infection through its capacity to bind to the host cell surface heparan sulfate proteoglycan (HSPG) and angiotensin-converting enzyme-2. The antiviral strategies targeting these 2 virus receptors are currently under intense investigation. However, the rapid evolution of the SARS-CoV-2 genome has resulted in numerous mutations in the S-protein posing a significant challenge for the design of S-protein-targeted inhibitors. As an example, the 2 key mutations in the S-protein receptor-binding domain (RBD), L452R, and T478K in the SARS-CoV-2 Delta variant (B.1.617.2) confer tighter binding to the host epithelial cells. Marine sulfated glycans (MSGs) demonstrate excellent inhibitory activity against SARS-CoV-2 via competitive disruption of the S-protein RBD-HSPG interactions and thus have the potential to be developed into effective prophylactic and therapeutic molecules. In this study, 7 different MSGs were evaluated for their anti-SARS-CoV-2 activity in a virus entry assay utilizing a SARS-CoV-2 pseudovirus coated with S-protein of the wild-type (Wuhan-Hu-1) or the Delta (B.1.617.2) strain. Although all tested MSGs showed strong inhibitory activity against both strains, no correlations between MSG structural features and virus inhibition could be drawn. Nevertheless, the current study provides evidence for the maintenance of inhibitory activity of MSGs against evolving SARS-CoV-2 strains.

Branched Chondroitin Sulfate Oligosaccharides Derived from the Sea Cucumber Acaudina molpadioides Stimulate Neurite Outgrowth.

Fucosylated chondroitin sulfate (FCS) from the sea cucumber Acaudina molpadioides (FCSAm) is the first one that was reported to be branched by disaccharide GalNAc-(α1,2)-Fuc3S4S (15%) and sulfated Fuc (85%). Here, four size-homogenous fractions, and seven oligosaccharides, were separated from its ß-eliminative depolymerized products. Detailed NMR spectroscopic and MS analyses revealed the oligomers as hexa-, hepta-, octa-, and nonasaccharide, which further confirmed the precise structure of native FCSAm: it was composed of the CS-E-like backbone with a full content of sulfation at O-4 and O-6 of GalNAc in the disaccharide repeating unit, and the branches consisting of sulfated fucose (Fuc4S and Fuc2S4S) and heterodisaccharide [GalNAc-(α1,2)-Fuc3S4S]. Pharmacologically, FCSAm and its depolymerized derivatives, including fractions and oligosaccharides, showed potent neurite outgrowth-promoting activity in a chain length-dependent manner. A comparison of analyses among oligosaccharides revealed that the sulfate pattern of the Fuc branches, instead of the heterodisaccharide, could affect the promotion intensity. Fuc2S4S and the saccharide length endowed the neurite outgrowth stimulation activity most.

Prospects for the Use of Marine Sulfated Fucose-Rich Polysaccharides in Treatment and Prevention of COVID-19 and Post-COVID-19 Syndrome.

Symptoms of the new coronavirus infection that appeared in 2019 (COVID-19) range from low fever and fatigue to acute pneumonia and multiple organ failure. The clinical picture of COVID-19 is heterogeneous and involves most physiological systems; therefore, drugs with a wide spectrum of mechanism of action are required. The choice of the treatment strategy for post-COVID-19 syndrome is still a challenge to be resolved. Polysaccharides with a high fucose content derived from seaweed and marine animals can form the basis for the subsequent development of promising agents for the treatment of COVID-19 and post-COVID-19 syndrome. This class of biopolymers is characterized by a variety of biological activities, including antiviral, antithrombotic, anticoagulant, hemo-stimulating, anti-inflammatory and immune-regulatory. Low molecular weight derivatives of these polysaccharides, as well as synthetic oligosaccharides with a sufficient amount and sulfation type may be considered as the most promising compounds due to their better bioavailability, which undoubtedly increases their therapeutic potential.

Pharmacological Activities of Sulfated Fucose-Rich Polysaccharides after Oral Administration: Perspectives for the Development of New Carbohydrate-Based Drugs.

Marine organisms are a source of active biomolecules with immense therapeutic and nutraceutical potential. Sulfated fucose-rich polysaccharides are present in large quantities in these organisms with important pharmacological effects in several biological systems. These polysaccharides include sulfated fucan (as fucoidan) and fucosylated chondroitin sulfate. The development of these polysaccharides as new drugs involves several important steps, among them, demonstration of the effectiveness of these compounds after oral administration. The oral route is the more practical, comfortable and preferred by patients for long-term treatments. In the past 20 years, reports of various pharmacological effects of these polysaccharides orally administered in several animal experimental models and some trials in humans have sparked the possibility for the development of drugs based on sulfated polysaccharides and/or the use of these marine organisms as functional food. This review focuses on the main pharmacological effects of sulfated fucose-rich polysaccharides, with an emphasis on the antidislipidemic, immunomodulatory, antitumor, hypoglycemic and hemostatic effects.

Marine Antithrombotics.

Thrombosis remains a prime reason of mortality worldwide. With the available antithrombotic drugs, bleeding remains the major downside of current treatments. This raises a clinical concern for all patients undergoing antithrombotic therapy. Novel antithrombotics from marine sources offer a promising therapeutic alternative to this pathology. However, for any potential new molecule to be introduced as a real alternative to existing drugs, the exhibition of comparable anticoagulant potential with minimal off-target effects must be achieved. The relevance of marine antithrombotics, particularly sulfated polysaccharides, is largely due to their unique mechanisms of action and lack of bleeding. There have been many investigations in the field and, in recent years, results have confirmed the role of potential marine molecules as alternative antithrombotics. Nonetheless, further clinical studies are required. This review covers the core of the data available so far regarding the science of marine molecules with potential medical applications to treat thrombosis. After a general discussion about the major biochemical steps involved in this pathology, we discuss the key structural and biomedical aspects of marine molecules of both low and high molecular weight endowed with antithrombotic/anticoagulant properties.

(Semi)-Synthetic Fucosylated Chondroitin Sulfate Oligo- and Polysaccharides.

Fucosylated chondroitin sulfate (fCS) is a glycosaminoglycan (GAG) polysaccharide with a unique structure, displaying a backbone composed of alternating N-acetyl-d-galactosamine (GalNAc) and d-glucuronic acid (GlcA) units on which l-fucose (Fuc) branches are installed. fCS shows several potential biomedical applications, with the anticoagulant activity standing as the most promising and widely investigated one. Natural fCS polysaccharides extracted from marine organisms (Echinoidea, Holothuroidea) present some advantages over a largely employed antithrombotic drug such as heparin, but some adverse effects as well as a frequently found structural heterogeneity hamper its development as a new drug. To circumvent these drawbacks, several efforts have been made in the last decade to obtain synthetic and semi-synthetic fCS oligosaccharides and low molecular weight polysaccharides. In this Review we have for the first time collected these reports together, dividing them in two topics: (i) total syntheses of fCS oligosaccharides and (ii) semi-synthetic approaches to fCS oligosaccharides and low molecular weight polysaccharides as well as glycoclusters displaying multiple copies of fCS species.

Fucosylated Chondroitin Sulfates from the Sea Cucumbers Paracaudina chilensis and Holothuria hilla: Structures and Anticoagulant Activity.

Fucosylated chondroitin sulfates (FCSs) PC and HH were isolated from the sea cucumbers Paracaudina chilensis and Holothuria hilla, respectively. The purification of the polysaccharides was carried out by anion-exchange chromatography on a DEAE-Sephacel column. The structural characterization of the polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of nondestructive NMR spectroscopic methods. Both polysaccharides were shown to contain a chondroitin core [→3)-ß-d-GalNAc (N-acethyl galactosamine)-(1→4)-ß-d-GlcA (glucuronic acid)-(1→]n, bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in their pattern of sulfation: PC contained Fuc2S4S and Fuc4S in a ratio of 2:1, whereas HH included Fuc2S4S, Fuc3S4S, and Fuc4S in a ratio of 1.5:1:1. Moreover, some GalNAc residues in HH were found to contain an unusual disaccharide branch Fuc4S-(1→2)-Fuc3S4S-(1→ at O-6. Sulfated GalNAc4S6S and GalNAc4S units were found in a ratio of 3:2 in PC and 2:1 in HH. Both polysaccharides demonstrated significant anticoagulant activity in a clotting time assay, which is connected with the ability of these FCSs to potentiate the inhibition of thrombin and factor Xa in the presence of anti-thrombin III (ATIII) and with the direct inhibition of thrombin in the absence of any cofactors.

A Study for the Access to a Semi-synthetic Regioisomer of Natural Fucosylated Chondroitin Sulfate with Fucosyl Branches on N-acetyl-Galactosamine Units.

Fucosylated chondroitin sulfate (fCS) is a glycosaminoglycan found up to now exclusively in the body wall of sea cucumbers. It shows several interesting activities, with the anticoagulant and antithrombotic as the most attractive ones. Its different mechanism of action on the blood coagulation cascade with respect to heparin and the retention of its activity by oral administration make fCS a very promising anticoagulant drug candidate for heparin replacement. Nonetheless, its typically heterogeneous structure, the detection of some adverse effects and the preference for new drugs not sourced from animal tissues, explain how mandatory is to open an access to safer and less heterogeneous non-natural fCS species. Here we contribute to this aim by investigating a suitable chemical strategy to obtain a regioisomer of the natural fCS polysaccharide, with sulfated l-fucosyl branches placed at position O-6 of N-acetyl-d-galactosamine (GalNAc) units instead of O-3 of d-glucuronic acid (GlcA) ones, as in natural fCSs. This strategy is based on the structural modification of a microbial sourced chondroitin polysaccharide by regioselective insertion of fucosyl branches and sulfate groups on its polymeric structure. A preliminary in vitro evaluation of the anticoagulant activity of three of such semi-synthetic fCS analogues is also reported.

Galactosaminoglycans: Medical Applications and Drawbacks.

Galactosaminoglycans (GalAGs) are sulfated glycans composed of alternating N-acetylgalactosamine and uronic acid units. Uronic acid epimerization, sulfation patterns and fucosylation are modifications observed on these molecules. GalAGs have been extensively studied and exploited because of their multiple biomedical functions. Chondroitin sulfates (CSs), the main representative family of GalAGs, have been used in alternative therapy of joint pain/inflammation and osteoarthritis. The relatively novel fucosylated chondroitin sulfate (FCS), commonly found in sea cucumbers, has been screened in multiple systems in addition to its widely studied anticoagulant action. Biomedical properties of GalAGs are directly dependent on the sugar composition, presence or lack of fucose branches, as well as sulfation patterns. Although research interest in GalAGs has increased considerably over the three last decades, perhaps motivated by the parallel progress of glycomics, serious questions concerning the effectiveness and potential side effects of GalAGs have recently been raised. Doubts have centered particularly on the beneficial functions of CS-based therapeutic supplements and the potential harmful effects of FCS as similarly observed for oversulfated chondroitin sulfate, as a contaminant of heparin. Unexpected components were also detected in CS-based pharmaceutical preparations. This review therefore aims to offer a discussion on (1) the current and potential therapeutic applications of GalAGs, including those of unique features extracted from marine sources, and (2) the potential drawbacks of this class of molecules when applied to medicine.

Structural elucidation of fucosylated chondroitin sulfates from sea cucumber using FTICR-MS/MS.

Fucosylated chondroitin sulfates are complex polysaccharides extracted from sea cucumber. They have been extensively studied for their anticoagulant properties and have been implicated in other biological activities. While nuclear magnetic resonance spectroscopy has been used to extensively characterize fucosylated chondroitin sulfate oligomers, we herein report the first detailed mass characterization of fucosylated chondroitin sulfate using high-resolution Fourier transform ion cyclotron resonance mass spectrometry. The two species of fucosylated chondroitin sulfates considered for this work include Pearsonothuria graeffei (FCS-Pg) and Isostichopus badionotus (FCS-Ib). Fucosylated chondroitin sulfate oligosaccharides were prepared by N-deacetylation-deaminative cleavage of the two fucosylated chondroitin sulfates and purified by repeated gel filtration. Accurate mass measurements obtained from electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry measurements confirmed the oligomeric nature of these two fucosylated chondroitin sulfate oligosaccharides with each trisaccharide repeating unit averaging four sulfates per trisaccharide. Collision-induced dissociation of efficiently deprotonated molecular ions through Na/H+ exchange proved useful in providing structurally relevant glycosidic and cross-ring product ions, capable of assigning the sulfate modifications on the fucosylated chondroitin sulfate oligomers. Careful examination of the tandem mass spectrometry of both species deferring in the positions of sulfate groups on the fucose residue (FCS-Pg-3,4- OS) and (FCS-Ib-2,4- OS) revealed cross-ring products 0,2Aαf and 2,4X2αf which were diagnostic for (FCS-Pg-3,4- OS) and 0,2X2αf diagnostic for (FCS-Ib-2,4- OS). Mass spectrometry and tandem mass spectrometry data acquired for both species varying in oligomer length (dp3-dp15) are presented.

Structure and Anti-Inflammatory Activity of a New Unusual Fucosylated Chondroitin Sulfate from Cucumaria djakonovi.

Fucosylated chondroitin sulfate CD was isolated from the sea cucumber Cucumaria djakonovi collected from the Avachinsky Gulf of the eastern coast of Kamchatka. Structural characterization of CD was performed using a series of non-destructive NMR spectroscopic procedures. The polysaccharide was shown to contain a chondroitin core [→3)-ß-d-GalNAc-(1→4)-ß-d-GlcA-(1→]n where about 60% of GlcA residues were 3-O-fucosylated, while another part of GlcA units did not contain any substituents. The presence of unsubstituted both at O-2 and O-3 glucuronic acid residues in a structure of holothurian chondroitin sulfate is unusual and has not been reported previously. Three different fucosyl branches Fucp2S4S, Fucp3S4S and Fucp4S were found in the ratio of 2:1:1. The GalNAc units were mono- or disulfated at positions 4 and 6. Anti-inflammatory activity of CD was assessed on a model of acute peritoneal inflammation in rats. About 45% inhibition was found for CD, while a structurally related linear chondroitin sulfate SS from cartilage of the fish Salmo salar demonstrated only 31% inhibition, indicating that the presence of sulfated fucosyl branches is essential for anti-inflammatory effect of chondroitin sulfates of marine origin.

Pharmacological Potential of Sea Cucumbers.

This review presents a detailed analysis of published research data focused on the pharmacological activity exerted by biologically active compounds isolated from sea cucumbers belonging to the class of Holothuroidea, phylum Echinodermata. The review contains descriptions of the structure, physico-chemical properties and pharmacological effects of these active substances. Particular attention is given to compounds with anticoagulant, antithrombotic, antioxidant, anticancer, anti-infectious, immune-stimulating and anti-ACE (angiotensin converting enzyme) activities as well as to the substances exerting a regulating influence on lipid and carbohydrate metabolism. All these compounds may be considered as prototypes for development of new pharmaceutical substances and medicines.

Synthesis of Fucosylated Chondroitin Sulfate Nonasaccharide as a Novel Anticoagulant Targeting Intrinsic Factor Xase Complex.

Fucosylated chondroitin sulfate (FuCS) is a structurally distinct glycosaminoglycan, and its oligosaccharides exhibit excellent anticoagulant activity with lower risks of adverse effects and bleeding. Herein we report a facile approach to the synthesis of FuCS hexa- and nonasaccharides on the basis of the enzymatic degradation of chondroitin over 12 linear steps. As compared with a clinical low-molecular-weight heparin drug (enoxaparin), the nonasaccharide synthesized in this study displayed similar APTT activity and selective intrinsic factor Xase complex inhibitory activity ((12.9±0.83) nm) by binding to factor IXa with high affinity, thus offering promise for the development of new anticoagulant agents targeting the intrinsic coagulation pathway.

Fucoidan and Fucosylated Chondroitin Sulfate Stimulate Hematopoiesis in Cyclophosphamide-Induced Mice.

Application of cytostatics in cancer patients' chemotherapy results in a number of side effects, including the inhibition of various parts of hematopoiesis. Two sulfated polysaccharides, fucoidan from the seaweed Chordaria flagelliformis (PS-Fuc) and fucosylated chondroitin sulfate from the sea cucumber Massinium magnum (PS-FCS), were studied as stimulators of hematopoiesis after cyclophosphamide immunosuppression in mice. Recombinant granulocyte colony-stimulating factor (r G-CSF) was applied as a reference. Both tested polysaccharides PS-Fuc and PS-FCS have a similar activity to r G-CSF, causing pronounced neutropoiesis stimulation in animals with myelosuppression induced by cyclophosphamide (CPh). Moreover, these compounds are also capable to enhance thrombopoiesis and erythropoiesis. It should be noted that PS-FCS demonstrated a greater activity than r G-CSF. The results indicate the perspective of further studies of PS-Fuc and PS-FCS, since these compounds can be considered as potentially promising stimulators of hematopoiesis. Such drugs are in demand for the accompanying treatment of cancer patients who suffer from hematological toxicity during chemo and/or radiation therapy.

Structure and biological activity of a fucosylated chondroitin sulfate from the sea cucumber Cucumaria japonica.

A fucosylated chondroitin sulfate (FCS) was isolated from the body wall of Pacific sea cucumber Cucumaria japonicaby extraction in the presence of papain followed by Cetavlon precipitation and anion-exchange chromatography. FCS was shown to contain D-GalNAc, D-GlcA, L-Fuc and sulfate in molar proportions of about 1:1:1:4.5. Structure of FCS was elucidated using NMR spectroscopy and methylation analysis of the native polysaccharide and products of its desulfation and carboxyl reduction. The polysaccharide was shown to contain a typical chondroitin core → 3)-ß-D-GalNAc-(1 → 4)-ß-D-GlcA-(1 →. Sulfate groups in this core occupy O-4 and the majority of O-6 of GalNAc. Fucosyl branches are represented by 3,4- and 2,4-disulfated units in a ratio of 4:1 and are linked to O-3 of GlcA. In addition, ∼ 33% of GlcA are 3-O-sulfated, and hence, the presence of short fucooligosaccharide chains side by side with monofucosyl branches cannot be excluded. FCS was shown to inhibit platelets aggregation in vitro mediated by collagen and ristocetin, but not adenosine diphosphate, and demonstrated significant anticoagulant activity, which is connected with its ability to enhance inhibition of thrombin and factor Xa by antithrombin III, as well as to influence von Willebrand factor activity. The latest property significantly distinguished FCS from low-molecular-weight heparin.

Depolymerization of Fucosylated Chondroitin Sulfate with a Modified Fenton-System and Anticoagulant Activity of the Resulting Fragments.

Fucosylated chondroitin sulfate (fCS) from sea cucumber Isostichopus badionotus (fCS-Ib) with a chondroitin sulfate type E (CSE) backbone and 2,4-O-sulfo fucose branches has shown excellent anticoagulant activity although has also show severe adverse effects. Depolymerization represents an effective method to diminish this polysaccharide's side effects. The present study reports a modified controlled Fenton system for degradation of fCS-Ib and the anticoagulant activity of the resulting fragments. Monosaccharides and nuclear magnetic resonance (NMR) analysis of the resulting fragments indicate that no significant chemical changes in the backbone of fCS-Ib and no loss of sulfate groups take place during depolymerization. A reduction in the molecular weight of fCS-Ib should result in a dramatic decrease in prolonging activated partial thromboplastin time and thrombin time. A decrease in the inhibition of thrombin (FIIa) by antithromin III (AT III) and heparin cofactor II (HCII), and the slight decrease of the inhibition of factor X activity, results in a significant increase of anti-factor Xa (FXa)/anti-FIIa activity ratio. The modified free-radical depolymerization method enables preparation of glycosaminoglycan (GAG) oligosaccharides suitable for investigation of clinical anticoagulant application.