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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 intrinsic tenase complex (iXase) is an attractive antithrombotic target to treat or prevent pathological thrombosis with negligible bleeding risk. Fucosylated glycosaminoglycan (FG) is a promising anticoagulant by inhibiting iXase. A depolymerized FG (dHG-5) as an anticoagulant has been approved for clinical trials. Given that dHG-5 is a multi-component drug candidate consisting of a homologous series of oligosaccharides, it is difficult to predict a clear pharmacokinetics. Here, as a major oligosaccharide component, the tetradecasaccharide (oHG-14) was purified from dHG-5 and its structure was defined as L-Fuc 3S4S -α(1,3)-L-Δ4,5GlcA-α(1,3)-{D-GalNAc 4S6S -β(1,4)-[L-Fuc 3S4S -α(1,]3)-D-GlcA-β(1,3)-} 3 -D-GalNAc 4S6S -β(1,4)-[L-Fuc 3S4S -α(1,]3)-D-GlcA-ol. oHG-14 showed potent iXase inhibitory activity in vitro and antithrombotic effect in vivo comparable to dHG-5. After single subcutaneous administration of oHG-14 at 8, 14.4 and 32.4 mg/kg to rats, the absolute bioavailability was 71.6 %–80.9 % determined by the validated bioanalytical methods. The maximum concentration (C max) was 3.73, 8.07, and 11.95 μg/mL, respectively, and the time reaching C max (T max) was about 1 h. oHG-14 was mainly excreted by kidney as the parent compound with the elimination kinetics of first-order linear model. Anticoagulant activity of oHG-14 was positively correlated with its concentration in rat plasma. The pharmacokinetics/pharmacodynamics (PK/PD) of oHG-14 is similar to that of dHG-5. This study could provide supportive data for the clinical trial of dHG-5 and further development of pure oligosaccharide as an antithrombotic drug candidate. [Display omitted] • dHG-5 is a multi-component anticoagulant by inhibiting iXase in clinical trials. • A tetradecasaccharide oHG-14 with equal anti-iXase activity to dHG-5 is identified. • oHG-14 has predictable pharmacokinetics and is mainly excreted by kidney. • The PK/PD of oHG-14 supports pure oligosaccharide as an anticoagulant candidate. [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]

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]

Abstract: 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) n m) by binding to factor IXa with high affinity, thus offering promise for the development of new anticoagulant agents targeting the intrinsic coagulation pathway. [ABSTRACT FROM AUTHOR]

The slug Vaginulus alte is used as folk medicine in China, but the structure and activities of its galactan components remain to be clarified. Here, the galactan from V. alte (VAG) was purified. The Mw of VAG was determined as ~28.8 kDa. Chemical composition analysis showed that VAG was composed of d -galactose (75 %) and l -galactose (25 %). To elucidate its precise structure, a series of disaccharides and trisaccharides were purified from mild acid hydrolyzed VAG and their structures were characterized by 1D/2D NMR spectroscopy. Based on methylation analysis and structural analysis of oligosaccharides, VAG was elucidated as a highly branched polysaccharide and mainly composed of (1 → 6)- or (1 → 3)-linked β- d -galactose, and distinct (1 → 2)-linked α- l -galactose. The investigation of probiotic effects in vitro revealed that VAG could promote the growth of B. thetaiotaomicron and B. ovatus , while had no effect on the growth of L. acidophilus , L. rhamnosus , B. longum subsp. infantis and B. animalis subsp. lactis , but dVAG-3 with Mw ~1.0 kDa could promote the growth of L. acidophilus. These results will provide insights into specific structures and functions of polysaccharides from the V. alte. • A highly branched galactan VAG was obtained from the slug Vaginulus alte. • Structures of oligosaccharides released from VAG were identified. • VAG was consisted of (1 → 3)-, (1 → 6)-linked β- d -galactose and (1 → 2)-linked α- l -galactose. • VAG could promote the growth of B. thetaiotaomicron and B. ovatus. • dVAG-3 with Mw of 1.0 kDa could promote the growth of L. acidophilus. [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]

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]

Mild acid hydrolysis is a common method to study the chemical structure of fucosylated glycosaminoglycan (FG). It was generally considered that the fucose branches α-L-FucS-(1, of FG could be hydrolyzed selectively in mild acid. This report focused on the selectivity of glycosidic bond cleavage and extensive desulfation characteristics of the backbone during mild acid hydrolysis. The hydrolyzed product of native SvFG (dfSvFG) was prepared by mild acid hydrolysis in 0.1 M H 2 SO 4 at 100 °C for 2 h. A series of oligosaccharides were purified by GPC and SAX-HPLC from dfSvFG, then they were analyzed by HPGPC, 1D/2D NMR and ESI-Q-TOF-MS. The precise structure of these oligosaccharides was elucidated to be trisaccharides, tetrasaccharides and pentasaccharides, indicating SvFG branches hydrolyzed basically and its' backbone composed of repeating β-D-GlcA-(1,3)-D-GalNAc and β-D-GalNAc-(1,4)-D-GlcA unit. The prevalent presence of the GlcA residues at the non-reducing terminal of these oligosaccharides, suggesting the glycosidic bond of β-D-GalNAc-(1,4)-D-GlcA was more susceptible to acid than that of β-D-GlcA-(1,3)-D-GalNAc during mild acid hydrolysis. Moreover, the sulfate ester groups in GalNAc 4S6S unit could also be hydrolyzed by acid, and it at position C-4 was more susceptible to hydrolysis than that at C-6. This extensive degradation and desulfation of the backbone should be taken into consideration when mild acid hydrolysis was used in elucidating the exact structure or structure-activity relationship of native FG. [Display omitted] • Structures of oligosaccharides from mild acid hydrolysis of SvFG were analyzed. • The β-D-GalNAc-(1,4)-D-GlcA glycosidic bond was preferentially cleaved. • Sulfate ester at C-4 in GalNAc 4S6S was more susceptible to acid than that at C-6. • Desulfation should be accounted when using mild acid hydrolysis for FG structure study. [ABSTRACT FROM AUTHOR]

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]

The invasion and metastasis of tumor cells are the hallmarks of malignant diseases and the greatest obstacle to overcome. Heparanase-mediated degradation of heparan sulfate (HS) is the critical process for tumor angiogenesis and metastasis, therefore, heparanase become an attractive target for cancer research. Herein, we reported a native fucosylated glycosaminoglycan (nHG) extracted from sea cucumber Holothuria fuscopunctata and a depolymerized nHG (dHG) and its contained oligosaccharides (hs17, hs14, hs11, hs8 and hs5), acting as heparanase inhibitors. nHG and its derivatives have the ability to bind with heparanase directly, leading to significant inhibition of heparanase activity. Moreover, their apparent binding affinity to heparanase was comparable to their inhibitory effect, which was elevated along with the increase of chain length, similar to the effect of heparins. In addition, oligosaccharides inhibited the migration and invasion of 4T1 mammary carcinoma cells and human umbilical vein endothelial cells (HUVECs) and also suppressed tube formation in Matrigel matrix and angiogenesis in the chick chorioallantoic membrane (CAM) assay. In the metastatic mouse model, oligosaccharides exhibited practical antimetastatic effects on 4T1 mammary carcinoma cells. According to the reported anticoagulant activity and the low bleeding tendency of dHG and its oligosaccharides, the use of the oligosaccharides may lead to better effects on tumor patients with thrombosis tendency. [Display omitted] • Fucosylated GAG and its derivatives are discovered as heparanase inhibitors. • Oligosaccharides originated from FG inhibit cell migration and invasion in vitro. • The oligosaccharides inhibit tube formation and angiogenesis. • The oligosaccharides suppress tumor cell metastasis in mice. [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]

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]