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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]

Fucosylated chondroitin sulfate (FCS) extracted from Phyllophorella kohkutiensis (PkFCS) is composed of d -GalNAc, d -GlcA, l -Fuc and -SO 4 2−. According to the defined structures revealed by NMR spectra of the branches released by mild acid hydrolysis and oligosaccharides generated by β-eliminative depolymerization, the backbone of PkFCS is CS-E, and the branch types attached to C-3 of d -GlcA include l -Fuc 2S4S , l -Fuc 3S4S , l -Fuc 4S, and the disaccharide α- d -GalNAc-1,2-α- l -Fuc 3S4S with the ratio of 43:13:22:22. Notably, novel heptasaccharide and hendecasaccharide were identified that are branched with continuous distribution of the disaccharide. The structural sequences of the oligosaccharides indicate that three unique structural motifs are present in the entire PkFCS polymer, including a motif branched with randomly distributed different sulfated l -Fuc units, a motif containing regular l -Fuc 2S4S branches and a motif enriched in α- d -GalNAc-1,2-α- l -Fuc 3S4S. This is the first report about the distribution pattern of diverse branches in natural FCS. Natural PkFCS exhibited potent anticoagulant activity on APTT prolonging and anti-iXase activity. Regarding the structurally defined oligosaccharides with sulfated fucosyl side chains, octasaccharide (Pk4b) is the minimum fragment responsible for its anticoagulant activity correlated with anti-iXase. However, further glycosyl modification with a non-sulfated d -GalNAc at the C-2 position of l -Fuc 3S4S could significantly decrease the anticoagulant and anti-iXase activity. [Display omitted] [ABSTRACT FROM AUTHOR]

• A new glycosaminoglycan (LF-GAG) was obtained from the slug Limacus flavus. • Physicochemical properties of LF-GAG were analyzed. • Four oligosaccharides were obtained by deacetylation-deamination and purification. • The precise structure of LF-GAG was determined. • Structure and heparinase inhibitory activity relationships were discussed. A new glycosaminoglycan (LF-GAG) was purified from the slug Limacus flavus. Its unique chemical structure and heparanase inhibitory activity were studied in this work. The native LF-GAG was composed of L-iduronic acid (L-IdoA) and N -acetyl-D-glucosamine (D-GlcNAc), with a Mw of 22,700 Da. To elucidate the precise structure and structure-activity relationship, its deacetylation-deaminative depolymerized product (dLF-GAG) was prepared, and from which four oligosaccharides were purified. Combining the NMR spectral analysis of LF-GAG and its derived oligosaccharides, the structure of LF-GAG was deduced to be -4)-L-IdoA 2R -(α1,4)-D-GlcNAc-(α1-, in which R was −OH (˜80%) or –OSO 3 − (˜20%). Bioactivity assays showed that LF-GAG could potently inhibit human heparanase (IC 50 , 0.10 μM). dLF-GAG and LF-3 were less potent but also active for heparanase inhibition. Structure-activity relationship analysis indicated that the chain length and sulfate substitution of LF-GAG are essential for its heparanase inhibitory activity. [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]

Fucoidan or fucan sulfate, a sulfated polysaccharide from algae or echinoderm mainly containing fucoses, possesses complex chemical structure and various biological activities. Herein, three fucan sulfates consisted of distinctive simplest repeating units were isolated from Holothuria fuscopunctata, Thelenota ananas and Stichopus horrens . The structural sequences of these fucan sulfates from H. fuscopunctata, T. ananas and S. horrens are →4-α- l -Fuc p -(3 S O 3 − )-1→, →4-α- l -Fuc p -(2 S O 3 − )-1→ and →3-α- l -Fuc p -(2 S O 3 − )-1→, respectively, revealing the existence of the highly regular homogeneous fucan sulfates and their structural diversity in the sea cucumber species for the first time. Pharmacological assays indicated their specific sulfation pattern and position of the glycosidic linkage may contribute to the anticoagulant action. Further mechanism analysis suggested that these fucan sulfates may exhibit strong inhibition of the intrinsic coagulation pathway by targeting the intrinsic coagulation factor Xase. Our results provide novel information to enrich knowledge on structural types of fucan sulfate and to illustrate its functionality. [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]

Edible snails have been widely used as a health food and medicine in many countries. A unique glycosaminoglycan (AF-GAG) was purified from Achatina fulica . Its structure was analyzed and characterized by chemical and instrumental methods, such as Fourier transform infrared spectroscopy, analysis of monosaccharide composition, and 1D/2D nuclear magnetic resonance spectroscopy. Chemical composition analysis indicated that AF-GAG is composed of iduronic acid (IdoA) and N -acetyl-glucosamine (GlcNAc) and its average molecular weight is 118 kDa. Structural analysis clarified that the uronic acid unit in glycosaminoglycan (GAG) is the fully epimerized and the sequence of AF-GAG is →4)-α-GlcNAc (1 → 4)-α-IdoA2S (1→. Although its structure with a uniform repeating disaccharide is similar to those of heparin and heparan sulfate, this GAG is structurally highly regular and homogeneous. Anticoagulant activity assays indicated that AF-GAG exhibits no anticoagulant activities, but considering its structural characteristic, other bioactivities such as heparanase inhibition may be worthy of further study. [ABSTRACT FROM AUTHOR]

Peroxidative depolymerization is often used to elucidate the structure and structure-activity relationship of fucosylated glycosaminoglycan (FG), while the selectivity of bond cleavage and structural characteristics of the resulting fragments remain to be confirmed. Here, the FG from Stichopus variegatus (SvFG) was depolymerized by H 2 O 2 , and a series of yielded mono- and oligo-saccharides were purified. Almost all the non-reducing ends of oligosaccharides were d -GalNAc 4S6S , suggesting that GlcA-β1,3-GalNAc 4S6S linkage was preferentially cleaved. The model reactions showed the glycosidic bond of uronate was more susceptible than those of N -acetyl hexosamine and fucose, which should be due to bond energy of the anomeric C H. The reducing ends of oligosaccharides include C4–C6 saccharic acid and GalNAc or GalNAcA, which should be derived from the oxidation of the reducing end. A hexasaccharide with tartaric acid exhibited increased anti-iXase activity, suggesting the oxidation of reducing end did not impair the anti-iXase activity of FG-derived oligosaccharides. [Display omitted] [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]

Highlights: [•] DFG with the anhydro-d-talose units at new reducing ends was obtained by degradation. [•] The reaction conditions of the deacetylation and deaminative cleavage were investigated. [•] The chemical structures of DFGs such as sulfated fucose chains may remain stable. [•] Decreasing of molecular weight (M w) may weaken the anticoagulant activity in vitro. [•] M w might not affect inhibition of factor Xase and HCII-mediated inhibition of thrombin. [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]

Free radical depolymerization is a common method in structural analysis of polysaccharides, the major challenge is the analysis of the cleavage site and characterization of newly formed ends in this reaction. Here, a fucosylated glycosaminoglycan from H. fuscopunctata (HfFG) was depolymerized by H 2 O 2 and a series of oligosaccharides were purified and their structures were elucidated. For non-reducing ends of the trisaccharides were intact GalNAc 4S6S , the cleavage site should mainly be the β(1,3) linkages between GlcA and GalNAc in the backbone of FG. Meanwhile, the reducing ends of the disaccharides and trisaccharides were almost dicarboxylic acid derivatives of GlcA, possibly arising from oxidative breaking of the C C bond of GlcA at the reducing ends. In addition, glycosidic linkages in D-GalNAc-β(1,4)-D-GlcA and L-FucS-α(1,3)-D-GlcA located at the reducing end could be cleaved, and the released GalNAc 4S6S were oxidized to N -acetylgalactosaminic acid. • Structures of oligosaccharides from free radical depolymerized HfFG were determined. • The preferential cleavage site in FG during depolymerization was identified. • Oligosaccharides with dicarboxylic acid as the reducing end were found. • The possible mechanism of free radical depolymerization of FG was proposed. [ABSTRACT FROM AUTHOR]

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]

• A fucan sulfate with a regular structure was obtained from Stichopus herrmanni. • Structure of the fucan sulfate was clarified. • A possible process of peroxide depolymerization of this fucan sulfate was proposed. • Molecular weight and anti-thrombin activity relationships were discussed. Marine sulfated polysaccharides have aroused widespread concern for their various structures and bioactivities. Peroxide depolymerization is a common strategy in analysis of structures and structure-activity relationships of polysaccharides. However, confirming the depolymerization process and exact structures of the degradation products is still a considerable challenge. This study reported the structures of a fucan sulfate (FS) from sea cucumber Stichopus herrmanni and its depolymerized products (dFS) prepared by peroxide degradation. The FS was elucidated with a highly regular structure, {-3)- L -Fuc 2S -(α1-} n. Structure analysis of oligosaccharides purified from dFS suggested that peroxide degradation involved in cleavage of glycosidic bonds and oxidative modification of reducing end of sugar residue, while no break in sugar ring was observed. Both FS and series of dFSs exhibited significant anticoagulant activities due to their anti-thrombin effects in presence of heparin cofactor II and their potencies were related to their molecular sizes, dFS with ∼ 20 kDa showed the strongest 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]