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Fucan sulfate I (FSI) from the sea cucumber Holothuria fuscopunctata was purified and its structure was clarified based on a bottom-up strategy. The unambiguous structures of a series of oligosaccharides including disaccharides, trisaccharides, and tetrasaccharides, which were released from mild acid hydrolysis of FSI, were identified by one-dimensional (1D)/two-dimensional (2D) nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. All the glycosidic bonds in these oligosaccharides were presented as α1,3 linkages confirmed by correlated signals from their 1H-1H ROESY and 1H-13C HMBC spectra. The structural sequence of these oligosaccharides formed by Fuc2S4S, Fuc2S, and non-sulfated ones (Fuc0S), along with the general structural information of FSI, indicated that the structure of FSI could be elucidated as: [-L-Fuc2S4S-α1,3-L-Fuc(2S)-α1,3-L-Fuc2S-α1,3-L-Fuc0S-α1,3-1-]n. Moreover, the L-Fuc0S-α1,3-L-Fuc2S4S linkage in FSI was susceptible to be cleaved by mild acid hydrolysis. The antioxidant activity assays in vitro showed that FSI and the depolymerized product (dFSI′) had potent activities for superoxide radical scavenging activity with IC50 of 65.71 and 83.72 μg/mL, respectively, while there was no scavenging effect on DPPH, hydroxyl and ABTS radicals. [ABSTRACT FROM AUTHOR]

The elucidation of the precise structure of fucan sulfate is essential for understanding the structure-activity relationship and promoting potential biomedical applications. In this work, the structure of a distinct fucan sulfate fraction V (PmFS in Ref 15 and FSV in Ref 16 → PFV) from Pattalus mollis was investigated using an oligosaccharide mapping approach. Six size-homogeneous fractions were purified from the mild acid hydrolyzed PFV and identified as fucitols, disaccharides and trisaccharides by 1D/2D NMR and MS analysis. Significantly, the sulfation pattern, glycosidic linkages, and sequences of all the oligosaccharides were unambiguously identified. The common 2-desulfation of the reducing end residue of the oligosaccharides was observed. Overall, the backbone of PFV was composed of L-Fuc 2S (major) and L-Fuc 3S (minor) linked by α1,4 glycosidic bonds. Importantly, the branches contain both monosaccharide and disaccharide linked to the backbone by α1,3 glycosidic linkages. Thus, the tentative structure of natural PFV was shown to be {-(R -α1,3)-L-Fuc 2S -α1,4-(L-Fuc 2S/3S -α1,4) x -} n , where R is L-Fuc (2S)4S -α1,3/4-L-Fuc 4S(0S) - or L-Fuc (2S)4S -. Our results provide insight into the heterogeneous structure of the fucan sulfate found in sea cucumbers. Additionally, PFV and its fractions showed strong anticoagulant and anti-iXase activities, which may be related to the distinct structure of PFV. [Display omitted] • A distinct fucan sulfate was investigated using an oligosaccharide mapping approach. • The structures of oligosaccharides were unambiguously identified. • The structure of the mainchain and the branches of PFV has been revised. • PFV displayed strong anticoagulant and anti-iXase activities. [ABSTRACT FROM AUTHOR]

Abstract: A process of depolymerization of a new fucosylated chondroitin sulphate from Thelenata ananas by free-radicals was developed. The fractions with different molecular weights and a narrow molecular weight distribution was obtained. The products were characterised by physicochemical properties, FT-IR spectra and NMR spectra. The results confirm the retention of primary structure of the fractions after the depolymerization. Furthermore, the anticoagulant activity of the fractions was evaluated by the activated partial thromboplastin time (APTT). The APTT activity decreases in proportion to the molecular weight following a logarithmic-like function, and the potentiating of prolongation of APTT activity requires at least an oligosaccharide chain of about 6–8 units (6000–7000Da). Compared to high doses of low molecular weight heparin (LMWH), a more desirable molecular weight fraction (13,950Da) demonstrates significantly lower anticoagulant activity. Thus, the fraction has more potential as an antithrombotic agent with reduced bleeding risk relative to LMWH. [Copyright &y& Elsevier]

Abstract: A process of depolymerization of a new fucosylated chondroitin sulfate from Thelenata ananas by free radicals was developed. The fractions with different molecular weights and narrow molecular weight distribution were obtained. The parameters of the process were investigated by high-performance gel permeation chromatography. The kinetics of the depolymerization of THG by H2O2 was studied, and a possible mechanism was proposed. The results indicated that the levels of final products fragmentation and reproducibility were different depending on the conditions of depolymerization used. The fragmentation of the main chain of THG occurred randomly and obeyed pseudo-first-order kinetics, and produced species with rather narrow and unimodal distribution of molar mass. Chemical compositions of partially depolymerized samples by 1H/13C nuclear magnetic resonance spectroscopy and by elementary analyzer suggested that there was no preferential cleavage of sulfated α-l-fucopyranose side-chain, and chemical composition of products was kept almost unchanged from that of native THG. [Copyright &y& Elsevier]

Abstract: A glycosaminoglycan from sea cucumber Thelenata anana (THG) was isolated as a polymer of molecular weight of around 70kDa. Its low molecular weight derivatives were first prepared by free radical depolymerization with hydrogen peroxide in the presence of copper(II) ion. The parameters of the process were investigated by a high-performance gel permeation chromatography. Analyses of chemical composition and molecular weight distribution indicated that the fragmentation of the main-chain of THG occurred randomly, obeyed pseudo first-order kinetics, and produced species with rather narrow and unimodal distribution of molar mass. The characterization of different molecular weight fractions was investigated by using viscometry and atomic force microscopy (AFM). Analysis of molecular weight and intrinsic viscosity in terms of the known theories for unperturbed wormlike cylinder yielded 1201±110nm−1, 15.3±1.5nm, and 1.5±0.3nm for molar mass per unit contour length M L, persistence length q, and diameter d, respectively. The M L and d values were approximately consistent with those observed by AFM. The present data suggest that THG may dissolve in 0.1M aqueous NaCl as single-stranded helical chains. [Copyright &y& Elsevier]

Fucosylated glycosaminoglycan (FG) from sea cucumbers has been reported to have anticoagulant effects via targeting intrinsic tenase. However, FG from natural source also potentially poses risks due to its FXIIa activation and platelet aggregating effects. Here, we found that the effect of FG on human platelet aggregation depended on both the sulfation pattern and chain length. FGs with higher content of Fuc2S4S and larger molecular weight (≥14 kD) had stronger activity. Both platelet aggregation and P-selectin expression induced by TaFG (an FG from Thelenota ananas) were decreased as the molecular weight reduced. Ticagrelor, aspirin and wortmannin completely blocked the secretion (ADP) but only partially blocked the aggregation induced by TaFG. Tirofiban an αIIbβ3 antagonist however potently inhibited both the secretion and aggregation, with IC50 of 6.01 ± 1.1.97 nM. Furthermore, TaFG could bind to human αIIbβ3 with high affinity, and the affinities of two FGs were paralleled with their activity in platelet aggregation or activation. These results indicated that αIIbβ3 played an important role in TaFG-induced platelet aggregation which was mediated by PI3K, and that platelet secretion was required for the amplification of aggregation. [ABSTRACT FROM AUTHOR]

Fucan sulfates from echinoderm possess characteristic structures and various biological activities. Herein, comprehensive methods including enzymolysis, ion-exchange chromatography and size exclusion chromatography lead to the purification of five fucan sulfates (FSI, FSII, FSIII, FSIV, FSV) from the sea cucumber Pattalus mollis. Chemical composition analysis showed that they were all composed of l -fucose. Their sulfate content was determined by a conductimetric method. The molecular weight (Mw) of FSI, FSII, FSIII, FSIV and FSV were measured as 238.3 kDa, 81.0 kDa, 82.0 kDa, 23.2 kDa and 6.12 kDa, respectively. Detailed NMR spectroscopic analysis revealed that the structural sequence of FSI and FSII was →3)- l -Fuc S -α(1→, where Fuc S were Fuc 2S4S (10%), Fuc 2S (44%), Fuc 0S (10%), Fuc 4S (36%), that of FSIII was →4)- l -Fuc 2S -(α1 → 4)- l -Fuc 2S -(α1 → 4)- l -Fuc 0S/3S -(α1→, where Fuc 0S and Fuc 3S were in equal molar, and that FSIV was →4)- l -Fuc 2S3S -(α1 → 4)- l -Fuc 2S3S -(α1 → 4)- l -Fuc 2S -(α1→4)- l -Fuc 2S -(α1 → 4)- l -Fuc 2S -(α1 → 4)- l -Fuc 2S -(α1 →. This is the first report that such a diversity of fucan sulfates were obtained from the same sea cucumber species. Biological activity showed that FSI, FSII, FSIII and FSIV exhibited potent anticoagulant by prolonging the APTT. Among them, FSII, FSIII and FSIV showed the similar potency, while FSI owned the strongest. Structure-activity relationships analysis showed that molecular weight and sulfation degree should be the crucial factors for the activity. • Five fucan sulfates were purified from sea cucumber Pattalus mollis. • The physicochemical properties of four fucan sulfates were characterized. • Structural unit of FSI and FSIII was →3)- l -Fuc S -α(1→ and →4)- l -Fuc 2S -(α1→4)- l -Fuc 2S -(α1→4)- l -Fuc 0S/3S -(α1→, respectively. • FSIV was composed of an α1,4-linked hexafucose repeating unit. • FSI exhibit strong inhibition of the intrinsic coagulation pathway. [ABSTRACT FROM AUTHOR]

Sulfated polysaccharides from sea cucumber possess unique chemical structure and various biological activities. In this study, four sulfated polysaccharides were purified from the body wall of Holothuria fuscopunctata by anion exchange chromatography and chemical depolymerization. They were identified as sulfated fucan (SFI, SFII), fucosylated chondroitin sulfate (FCS) and sulfated aminoglycan (AG) by physicochemical and structural analyses. The Mw of SFI, SFII, FCS and AG were 470.6, 36.8, 42.6, 39.6 kDa and the sulfate content was 21.40%, 35.86%, 33.70%, 35.70%, respectively. Their primary structures were clarified both by monosaccharide composition and 1D/2D NMR spectroscopy analysis. As a result, the repeating sequences of FCS and SFII were →4)-[L-Fuc 3S4S -(α1 → 3)]-D-GlcA-(β1 → 3)-D-GalNAc 4S6S -(β1 → and →4-L-Fuc-(3SO 3 −)-α1→, respectively. The primary structure of SFI was →3)-L-Fuc 2S4S -(α1 → 4)-L-Fuc-(α1 → 3)-L-Fuc 2S -(α1 → 4)-L-Fuc-(α1→. The sulfated AG was composed of four types of monosaccharides. Their anticoagulant activities were further evaluated in vitro. FCS and AG showed potent anticoagulant activity and intrinsic factor Xase inhibition activity. These results expand the knowledge on the structure types of sulfated polysaccharides from sea cucumber and further illustration of their functionality. • Four sulfated polysaccharides were purified from the sea cucumber H. fuscopunctata. • The physicochemical properties of these polysaccharides were characterized. • The depolymerization methods could facilitate to separate sulfated polysaccharides. • The anticoagulant activities of these polysaccharides were evaluated. [ABSTRACT FROM AUTHOR]

Fucan sulfate (FS) from sea cucumber shows intriguing structure and extensive activities. Here, three homogeneous FS (BaFSI – III) were obtained from Bohadschia argus , followed with physicochemical properties analyses including monosaccharide composition, molecular weight, and sulfate content. BaFSI was proposed to carry a unique distribution pattern of sulfate groups as a novel sequence composed of domain A and domain B that formed by different Fuc S residues, markedly differing from FS reported before, according to the analyses of 12 oligosaccharides and a representative residual saccharide chain. BaFSII possessed a highly regular structure {4-L-Fuc 3S -α1,} n according to its peroxide depolymerized product. BaFSIII was confirmed as a FS mixture bearing similar structural characteristics with BaFSI and BaFSII by means of mild acid hydrolysis and oligosaccharide analysis. Bioactivity assays showed that BaFSI and BaFSII could potently inhibit P-selectin binding to PSGL-1 and HL-60 cells. Structure-activity relationship analysis showed that molecular weight and sulfation pattern were the essential factors for the potent inhibition. Meanwhile, an acid hydrolysate of BaFSII with a molecular weight about 15 kDa exhibited a comparable inhibition with the native BaFSII. Given the potent activity and highly regular structure of BaFSII, it shows great potential for development as a P-selectin inhibitor. [Display omitted] [ABSTRACT FROM AUTHOR]

Sulfated polysaccharides such as fucosylated glycosaminoglycan and fucan sulfate from echinoderm possess complex chemical structure and various biological activities. The two sulfated polysaccharides were purified from the low-value sea cucumber Holothuria coluber . Their physicochemical properties and chemical structures were analyzed and characterized by chemical and instrumental methods. Structural analysis clarified that the sea cucumber fucosylated glycosaminoglycan contains a chondroitin sulfate-like backbone and fucosyl branches with four various sulfation patterns. The fucan sulfate with molecular weight of 64.6 kDa comprises a central core of regular α(1 → 4)-linked tetrasaccharide repeating units, each of which is linked by a 4- O -sulfated fucose residue. Anticoagulant assays indicated that these sulfated polysaccharides possessed strong APTT prolonging activities and intrinsic factor Xase inhibitory activities, both of which decreased with the reduction of their molecular weights. Our results expand knowledge on the structural types of sulfated polysaccharides from sea cucumbers and further illustrate their functionality. [ABSTRACT FROM AUTHOR]

A fucan sulfate was isolated and purified from the sea cucumber Holothuria albiventer by papain enzymolysis, alkaline hydrolysis and ion-exchange chromatography. The water-soluble polysaccharide had high molecular weight and contained fucose and sulfate in a molar ratio of about 1:0.83. Methylation analysis of the native polysaccharide indicated that its glycosidic linkages and sulfate substituents might be at O- 3 or O -3, 4 or O -2, 3, or O -2, 3, 4 positions. FT-IR and 2D NMR spectroscopies further revealed that the fucan sulfate is characteristically composed of a regular α (1 → 3) linked hexasaccharide repeating unit which is substituted with sulfate esters in a distinctive pattern. Anticoagulant properties of the fucan sulfate and its depolymerized product were assessed in vitro in comparison with a low-molecular-weight heparin. The fucan sulfate exhibits strong APTT and TT prolonging activities and intrinsic factor Xase inhibitory activity, and its molecular size seemed to be required for these activities. [ABSTRACT FROM AUTHOR]

The digestibility of fucosylated glycosaminoglycan (FG) and its effects on digestive enzymes were investigated using an in vitro digestion model. Results showed that the molecular weight and the reducing sugar content of FG were not significantly changed, and no free monosaccharides released from FG were detected after the salivary, gastric and intestinal digestion, indicating that both the backbone and the sulfated fucose branches of FG are resistant to be cleaved in the saliva and gastrointestinal tract environments. Furthermore, FG can dose-dependently inhibit digestive enzymes such as α-amylase, pepsin and pancreatic lipase in different degrees under the simulated digestion conditions due to the sulfate and carboxyl groups. These physiological effects of FG may help control the postprandial glucose concentration and have the potential in the prevention or treatment of reflux disease and obesity. The findings may provide information on the digestibility and beneficial physiological effects of FG as a potential natural product to promote human health. [ABSTRACT FROM AUTHOR]

Selective inhibition of the endogenous coagulation pathway is a promising strategy for developing new anticoagulants. Fucosylated glycosaminoglycan (FG), a structurally complex glycosaminoglycan, has distinct anticoagulant properties, especially the strong intrinsic factor Xase inhibitory activity that is recognized as a new target with potential physiological and therapeutic applications. Detailed knowledge of FG structures is necessary for developing a clinically effective intrinsic FXase inhibitor. However, challenges remain to elucidate FG structures as a basis for pharmaceutical development. Herein, using the highly selective β-elimination method, oligosaccharides with regular structures were prepared from the depolymerization products. Analysis of oligosaccharides further confirmed the precise structural sequence of the FG. Furthermore, biological activity assay suggested that these pure homogeneous oligosaccharides, particularly an octasaccharide, exhibit strong inhibition of the intrinsic coagulation pathway by inhibiting human intrinsic factor Xase. Our finding is significant for discovery of a new class of anticoagulant agents as intrinsic factor Xase inhibitors. [ABSTRACT FROM AUTHOR]

• Structural refinement of fucan sulfate from Stichopus herrmanni (Sh FS) was employed. • 21 oligosaccharides were elucidated from the mild acid hydrolysate of Sh FS. • Factors and depolymerization mechanism of mild acid hydrolysis were analyzed. • Moderate saccharide chain conferred potent anticoagulant and anti-thrombin activities. Fucan sulfate (FS) from sea cucumbers possesses linear sequences with repeating units that differ principally in the pattern of sulfation and position of glycosidic linkage. FS from Stichopus herrmanni (Sh FS) was preliminarily identified as the relatively simple structure {-3)-L-Fuc 2S -(α1-}n. Herein, mild acid hydrolysis was employed on Sh FS to yield 21 oligosaccharides. Analyses on their structures complemented the features of Sh FS to refine its spectral signal assignments. Combining with the methylation analysis, unit L-Fuc 2S4S was determined as the micro-component in its intact structure. The irregularity came from minor sulfation on O-4. Temperature and acid concentration were the critical parameters to the depolymerization and formation of oligosaccharides. Meanwhile, a three-step cleavage of the hydrolysis mechanism involving the partial O-2 de-sulfation and preferential cleavage between Fuc 0S and Fuc 2S4S was proposed. Bioactivity assays revealed that the Mw 15–16 kDa conferred the potent anticoagulation via inhibiting the thrombin activity mediated by heparin cofactor II. [ABSTRACT FROM AUTHOR]

Fucosylated chondroitin sulfate (FCS) from sea cucumber Ludwigothurea grisea (FCS Lg) is the first one that reported to bear the di-fucosyl branches. Here we deciphered it by analyzing the physicochemical properties and its derivatives. Oligosaccharides prepared by selective cleavage of glycosidic linkages presented the mono-fucose and heterodisaccharide branches in FCS Lg. The disaccharide branch was determined as d -GalNAc R1 -(α1,2)- l -Fuc R2 rather than the di-fucosyl branch, where R1 was 4-mono-O- or 4,6-di-O-sulfation, and R2 was 3-mono-O- or 3,4-di-O-sulfation, respectively. The diversity of sulfation patterns in branches complicated the structure. These results give us a new understanding of FCS Lg and provided a reliable method to decipher the FCS with complex branches. Bioanalysis of chemically modified derivatives showed that modulating the molecular mass could enhance the Xase target selectivity. Side chains conferred the Xase complex inhibition by binding to FIXa with a high affinity. Whether monosaccharide and disaccharide branches have differential effects needs to be further explored. [Display omitted] [ABSTRACT FROM AUTHOR]

Fucosylated chondroitin sulfate (FCS) from sea cucumber with complex structure has potent anticoagulant activity by inhibition of intrinsic tenase; however, it could activate factor XII and platelet. To obtain FCS’ fragments with selective inhibition on intrinsic tenase, a method for β-eliminative depolymerization of FCS was developed by treating FCS benzyl esters with alkaline in anhydrous solution. Our results demonstrated that the glycosidic linkages of GalNAc-β1, 4-GlcA were selectively cleaved and distinctive Δ 4,5 unsaturated hexuronic acid was formed at non-reducing end of resulting fragments, while the main structures were essentially stable during depolymerization. By this method, five depolymerized fragments (dFCSs) with various molecular sizes were prepared and their anticoagulant activities and activation activities of factor XII and platelet were compared. Overall, dFCSs with M w 3.2–8.8 kDa reserved potent anticoagulant activities by inhibition of intrinsic tenase, and activation activities of factor XII or platelet could be diminished or eliminated. [ABSTRACT FROM AUTHOR]

Abstract: Fucosylated chondroitin sulfate (FuCS) is a glycosaminoglycan from sea cucumber, made up of alternating β-d-glucuronic acid and N-acetyl-β-d-galactosamine units. The β-d-glucuronic acid residues have branches of sulfated fucose, while the fucose branches may have distinguishable patterns and proportions of sulfate substitution. The structure of the sulfated fucose branches of the fucosylated chondroitin sulfate from the sea cucumber Thelenata ananas has been characterized by mild acid hydrolysis and NMR technology. The results showed that the fucose residues are made up of 3-,4-mono-O-sulfated fucose and 2,4-di-O-sulfated fucose with about 25:22:53, respectively. The sulfated fucose branches are essential for the anticoagulant action of FuCS, and this potent effect is possibly related to the occurrence of 2,4-di-O-sulfated fucose units. Furthermore, these branches could constitute the structural requirement for the binding of the glycosaminoglycan to coagulant enzymes, such as thrombin by HCII, factor X by the intrinsic tenase complex. [Copyright &y& Elsevier]

Glycosaminoglycan HnFG was extracted from sea cucumber Holothuria nobilis. Its chemical structure was characterized by analyzing the physicochemical properties, oligosaccharides from its mild acid hydrolysates and depolymerized products. The disaccharide d -GalNAc 4S6S -α1,2- l -Fuc 3S -ol found in its mild acid hydrolysates provided a clue for the presence of a unique disaccharide-branch in HnFG. Furthermore, it was confirmed by a series of oligosaccharides from the low-molecular weight HnFG prepared by β-eliminative depolymerization. Combining with the analysis of its peroxide depolymerized products, the precise structure of HnFG was determined: A chondroitin sulfate E (CS-E)-like backbone branched with sulfated monofucoses (~67%) and disaccharides d -GalNAc S -α1,2- l -Fuc 3S (~33%) at O-3 position of each GlcUA. This is the first report on the novel branches in glycosaminoglycan. Biologically, the native and depolymerized HnFG showed potent activities in prolonging the activated partial thrombin time (APTT) and inhibiting intrinsic coagulation Xase (iXase), whereas the oligosaccharides (degree of polymerization ≤6) had no obvious effects. • A glycosaminoglycan HnFG were isolated from sea cucumber Holothuria nobilis. • Structures of fifteen oligomers from hydrolysates and depolymerized HnFG were clarified. • HnFG was partially branched with unique disaccharide GalNAc S -α1,2-Fuc 3S. • Anticoagulant and anti-iXase activities of (d)HnFG and oligomers were discussed. [ABSTRACT FROM AUTHOR]

Intrinsic Xase (iXase), the last and rate-limiting enzyme complex in the intrinsic coagulation pathway, may be an ideal target for antithrombotic treatment. A depolymerized fraction of fucosylated glycosaminoglycan from sea cucumber Holothuria fuscopunctata , dHG-5 (Mw 5.2 kDa), showed potent and selective inhibition of iXase (IC 50 , 14 nM). In this work, the series of oligosaccharides contained in dHG-5 were purified and their precise structures were confirmed by 2D NMR and MS spectra. The relationships between anti-iXase, f.IXa-binding, anticoagulant and antithrombotic activities ( y ) and molecular weight ( x ) could be approximately expressed as the power function ( y = a × x b), and these activity potencies of dHG-5 were approximately equivalent to the weighted average sum of that of its oligosaccharides. Given the prominent pharmacological properties, well-defined chemical composition and explicable relationships between dHG-5 and its oligosaccharides in pharmacological behaviors, dHG-5 is expected to be an ideal novel anticoagulant medicine. Image 1 • The selective iXase inhibitor dHG-5 is a potential candidate for antithrombosis. • The clear structures and contents of oligosaccharides contained in dHG-5. • Contribution of oligosaccharides components to dHG-5 in pharmacology. • Pharmacological characteristics of oligosaccharides contained in dHG-5. • oHG-17 with the minimum chain length required to achieve full activity. [ABSTRACT FROM AUTHOR]

• A novel FG (AmFG) were extracted from sea cucumber Acaudina molpadioides. • The structures of eight oligosaccharides from depolymerized AmFG were determined. • AmFG branched with unusual disaccharide GalNAc-α1,2-Fuc, besides monosaccharides. • The anticoagulant and anti-iXase activities of AmFG and oligomers were discussed. A fucosylated glycosaminoglycan (AmFG) was extracted from the sea cucumber Acaudina molpadioides. And a series of oligosaccharides were purified from the size-homogeneous fractions, which were prepared from the β-eliminative depolymerized AmFG. According to "bottom-up" strategy, the precise structure of AmFG was elucidated by analyzing the structures of these purified oligosaccharides, combining with NMR analysis of its free-radical depolymerized product. It contained a CS-E-like backbone, and each GlcUA was branched with a mono- or di-sulfated fucose (Fuc) at O -3. Intriguingly, besides two types of monosaccharide branches, Fuc 2S4S (60 %) and Fuc 4S (25 %), that were common in FG, AmFG also contained an unusual disaccharide branch GalNAc-α1,2-Fuc 3S4S (15 %); this is the first report of such a structure in a glycosaminoglycan. Biological assays indicated that native AmFG and its oligosaccharides had potent anticoagulant and intrinsic tenase (iXase) inhibitory activities in a chain length-dependent manner. For these oligosaccharides, octasaccharide was the minimum structural fragment for potent anti-iXase activity, and the disaccharide branch might enhance this activity. [ABSTRACT FROM AUTHOR]

• Oligosaccharide fractions H1 – H4 from depolymerized HcFG was purified and characterized. • HcFG was deduced as -{→4)-[ l -FucS-α(1→3)]- d -GlcA-β(1→3)- d -GalNAc 4S6S - β(1}n-. • FG branched with Fuc 3S4S was susceptible to β -eliminative depolymerization. • Anticoagulant activities of native and depolymerized HcFG were assessed. A glycosaminoglycan was isolated from the sea cucumber Holothuria coluber (HcFG). A series of oligosaccharide fragments (dp range 3–11) were prepared from its β -eliminative depolymerized product (dHcFG). Extensive NMR characterization of the oligosaccharides indicated the d -GlcA- β 1,3- d -GalNAc 4S6S repeating disaccharide backbone was substituted by monosaccharide branches comprising of Fuc 2S4S , Fuc 3S4S and Fuc 4S , linked to O-3 of d -GlcA. For the prevailing Fuc 3S4S at nonreducing end of dHcFG, the β- eliminative depolymerization process of HcFG was compared with those of the FGs from Actinopyga miliaris (AmFG, branched with Fuc 3S4S) and Stichopus variegatus (SvFG, branched with Fuc 2S4S). The result suggested that d -GlcA substituted with Fuc 3S4S was more susceptible to depolymerization than that with Fuc 2S4S. It might be due to the larger steric hindrance effects from Fuc 2S4S on the esterification of GlcA. Biological assays confirmed that the minimum chain length (dp8), regardless of the Fuc branch types, was required for the potent anti-iXase and anticoagulant activities in FG fragments. [ABSTRACT FROM AUTHOR]

• Three FGs were isolated from three species of sea cucumbers. • The structures of A1 - A4 , H1 - H4 and S1 - S4 from depolymerized FGs were determined. • Three FGs were deduced as -{→4)-[ L -FucS-α(1→3)]- D -GlcA-β(1→3)- D - GalNAc 4S6S -β(1}n-. • The effects of fucose types on anti-intrinsic tenase activity were discussed. Fucosylated glycosaminoglycan (FG), a glycosaminoglycan derivative containing distinct sulfated fucose (FucS) branches, displays potent anticoagulant activity by inhibiting the intrinsic tenase complex (iXase). Herein, AmFG, SvFG and HaFG from three species of sea cucumbers were isolated and depolymerized by β-eliminative cleavage. Three series of fragments, A1 - A4 , S1 - S4 and H1 - H4 , were purified from the depolymerized FGs. Based on structural analysis of these fragments, three FGs were deduced as -{→4)-[ L -FucS-α(1→3)]- D -GlcA-β(1→3)- D -GalNAc 4S6S -β(1} n -. The structures differed in sulfation types of FucS, namely, most of FucS in AmFG was Fuc 3S4S , but the FucS in SvFG was Fuc 2S4S , while the FucS in HaFG was Fuc 3S4S , Fuc 2S4S and Fuc 4S. However, all FucS branches attached to C -3 of GlcA as monosaccharides. Anticoagulant and anti-iXase assays showed the octasaccharide is the minimum fragment for potent anticoagulant activity via anti-iXase irrespective of FucS types. Among FG fragments with same degree of polymerization, oligosaccharides containing Fuc 2S4S had more potent anti-iXase activity. [ABSTRACT FROM AUTHOR]