Structural characterization of a distinct fucan sulfate from Pattalus mollis through an oligosaccharide mapping approach.

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-Fuc2S (major) and L-Fuc3S (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-Fuc2S-α1,4-(L-Fuc2S/3S-α1,4)x-}n, where R is L-Fuc(2S)4S-α1,3/4-L-Fuc4S(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.

Structure characterization of a highly branched galactan from the slug Vaginulus alte and its utilization by human gut microbiota.

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 â†’ 6)- or (1 â†’ 3)-linked ß-d-galactose, and distinct (1 â†’ 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.

Peroxidative depolymerization of fucosylated glycosaminoglycan: Bond-cleavage pattern and activities of oligosaccharides.

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 H2O2, and a series of yielded mono- and oligo-saccharides were purified. Almost all the non-reducing ends of oligosaccharides were d-GalNAc4S6S, suggesting that GlcA-ß1,3-GalNAc4S6S 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 CH. 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.

A Fucan Sulfate with Pentasaccharide Repeating Units from the Sea Cucumber Holothuriafloridana and Its Anticoagulant Activity.

A fucan sulfate (HfFS) was isolated from the sea cucumber Holothuriafloridana after proteolysis-alkaline treatment and purified with anion-exchange chromatography. The molecular weight (Mw) of HfFS was determined to be 443.4 kDa, and the sulfate content of HfFS was 30.4%. The structural analysis of the peroxidative depolymerized product (dHfFS-1) showed that the primary structure of HfFS was mainly composed of a distinct pentasaccharide repeating unit -[l-Fuc2S4S-α(1,3)-l-Fuc-α(1,3)-Fuc-α(1,3)-l-Fuc2S-α(1,3)-l-Fuc2S-α(1,3)-]n-. Then, the "bottom-up" strategy was employed to confirm the structure of HfFS, and a series of fucooligosaccharides (disaccharides, trisaccharides, and tetrasaccharides) were purified from the mild acid-hydrolyzed HfFS. The structures identified through 1D/2D NMR spectra showed that these fucooligosaccharides could be derivates from the pentasaccharide units, while the irregular sulfate substituent also exists in the units. Anticoagulant activity assays of native HfFS and its depolymerized products (dHf-1~dHf-6) in vitro suggested that HfFS exhibits potent APTT-prolonging activity and the potencies decreased with the reduction in molecular weights, and HfFS fragments (dHf-4~dHf-6) with Mw less than 11.5 kDa showed no significant anticoagulant effect. Overall, our study enriched the knowledge about the structural diversity of FSs in different sea cucumber species and their biological activities.

Structure Elucidation of Fucan Sulfate from Sea Cucumber Holothuria fuscopunctata through a Bottom-Up Strategy and the Antioxidant Activity Analysis.

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.

The glycosidic bond cleavage and desulfation investigation of fucosylated glycosaminoglycan during mild acid hydrolysis through structural analysis of the resulting oligosaccharides.

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 H2SO4 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 ß-D-GlcA-(1,3)-D-GalNAc and ß-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 ß-D-GalNAc-(1,4)-D-GlcA was more susceptible to acid than that of ß-D-GlcA-(1,3)-D-GalNAc during mild acid hydrolysis. Moreover, the sulfate ester groups in GalNAc4S6S 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.

Five distinct fucan sulfates from sea cucumber Pattalus mollis: Purification, structural characterization and anticoagulant activities.

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-FucS-α(1→, where FucS were Fuc2S4S (10%), Fuc2S (44%), Fuc0S (10%), Fuc4S (36%), that of FSIII was →4)-l-Fuc2S-(α1 â†’ 4)-l-Fuc2S-(α1 â†’ 4)-l-Fuc0S/3S-(α1→, where Fuc0S and Fuc3S were in equal molar, and that FSIV was →4)-l-Fuc2S3S-(α1 â†’ 4)-l-Fuc2S3S-(α1 â†’ 4)-l-Fuc2S-(α1→4)-l-Fuc2S-(α1 â†’ 4)-l-Fuc2S-(α1 â†’ 4)-l-Fuc2S-(α1 â†’ . 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.

Structural Characterization and Heparanase Inhibitory Activity of Fucosylated Glycosaminoglycan from Holothuria floridana.

Unique fucosylated glycosaminoglycans (FG) have attracted increasing attention for various bioactivities. However, the precise structures of FGs usually vary in a species-specific manner. In this study, HfFG was isolated from Holothuria floridana and purified by anion exchange chromatography with the yield of ~0.9%. HfFG was composed of GlcA, GalNAc and Fuc, its molecular weight was 47.3 kDa, and the -OSO3-/-COO- molar ratio was 3.756. HfFG was depolymerized by a partial deacetylation-deaminative cleavage method to obtain the low-molecular-weight HfFG (dHfFG). Three oligosaccharide fragments (Fr-1, Fr-2, Fr-3) with different molecular weights were isolated from the dHfFG, and their structures were revealed by 1D and 2D NMR spectroscopy. HfFG should be composed of repeating trisaccharide units -{(L-FucS-α1,3-)d-GlcA-ß1,3-d-GalNAc4S6S-ß1,4-}-, in which sulfated fucose (FucS) includes Fuc2S4S, Fuc3S4S and Fuc4S residues linked to O-3 of GlcA in a ratio of 45:35:20. Furthermore, the heparanase inhibitory activities of native HfFG and oligosaccharide fragments (Fr-1, Fr-2, Fr-3) were evaluated. The native HfFG and its oligosaccharides exhibited heparanase inhibitory activities, and the activities increased with the increase of molecular weight. Additionally, structural characteristics such as sulfation patterns, the terminal structure of oligosaccharides and the presence of fucosyl branches may be important factors affecting heparanase inhibiting activity.

Characterization of the Hydrolysis Kinetics of Fucosylated Glycosaminoglycan in Mild Acid and Structures of the Resulting Oligosaccharides.

: Mild acid hydrolysis is a common method for the structure analysis of fucosylated glycosaminoglycan (FG). In this work, the effects of acid hydrolysis on the structure of FG from S. variegatus (SvFG) and the reaction characteristic were systemically studied. The degree of defucosylation (DF) and molecular weights (Mw) of partial fucosylated glycosaminoglycans (pFs) were monitored by 1H NMR and size-exclusion chromatography, respectively. The kinetic plots of DF, degree of desulfation (DS) from fucose branches, and degree of hydrolysis (DH) of the backbone are exponentially increased with time, indicating that acid hydrolysis of SvFG followed a first-order kinetics. The kinetic rate constants kDF, kDS, and kDH were determined to be 0.0223 h-1, 0.0041 h-1, and 0.0005 h-1, respectively. The structure of the released sulfated fucose branches (FucS) from SvFG and HfFG (FG from H. fuscopunctata) was characterized by 1D/2D NMR spectroscopy, suggesting the presence of six types of fucose: α/ß Fuc2S4S, Fuc3S4S, Fuc3S, Fuc4S, Fuc2S, and Fuc. The Fuc3S4S was more susceptible to acid than Fuc2S4S, and that the sulfate ester in position of O-2 and O-3 than in O-4 of fucose. The structure characteristic of pF18 indicated the cleavage of backbone glycosidic bonds. The APTT prolonged activity reduced with the decrease of the DF and Mw of the pFs, and became insignificant when its DF was 87% with Mw of 3.5 kDa.