Lionel Chevolot

Further data on the structure of brown seaweed fucans: relationships with anticoagulant activity.

The composition, molecular weight (MW), anticoagulant activity and nuclear magnetic resonance spectra of various low-molecular-weight fucans (LMWFs) obtained by partial hydrolysis or radical depolymerization of a crude fucoidan extracted from the brown seaweed Ascophyllum nodosum are compared. Fucose units were found mainly sulfated at O-2, to a lesser extent at O-3, and only slightly at O-4, contrary to previously published results for fucoidans from other brown seaweeds, and fucose 2, 3-O-disulfate residues were observed for the first time. As the sulfation pattern excluded an alpha-(1-->2)-linked fucose backbone and a high proportion of alpha-(1-->4) linkages was found, it would appear that the concept of fucoidan structure needs to be revised. Anticoagulant activity is apparently related not only to MW and sulfation content, as previously determined, but also (and more precisely) to 2-O-sulfation and 2,3-O-disulfation levels.

A disaccharide repeat unit is the major structure in fucoidans from two species of brown algae

The predominant repeating structure of a fraction of the fucoidan from Ascophyllum nodosum prepared by acid hydrolysis and centrifugal partition chromatography (LMWF) was established as: [-->3)-alpha-L-Fuc(2SO3-)-(1-->4)-alpha-L-Fuc(2,3diSO3-)-(1]n by NMR spectroscopy and methylation analysis. The proton and carbon NMR spectra of this unit have been assigned and found to correspond with features in the spectra of the whole purified fucan from A. nodosum which account for most of the integrated intensity. The same structure has also been recognised in the fucoidan of Fucus vesiculosus. The fraction LMWF has in vitro anticoagulant activity, indicating that the above structure may be partly responsible for biological activity in the native fucoidan.

Interaction of fucoidan with the proteins of the complement classical pathway.

Fucoidan inhibits complement by mechanisms that so far remain to be unraveled, and the objective of this work was to delineate the mode of inhibition by this sulfated polysaccharide. For that purpose, low molecular weight fractions of algal (Ascophyllum nodosum) fucoidan containing the disaccharide unit [-->3)-alpha-L-Fuc(2SO3(-))-(1-->4)-alpha-L-Fuc(2,3diSO3(-))-(1-->](n) have been studied. Gel co-affinity electrophoresis and a new affinity capillary electrophoresis (ACE) method have been implemented to characterize fucoidan-complement protein complexes. Fucoidan binds C1q, likely to its collagen-like region through interactions involving lysine residues, and then prevents the association of the C1r(2)-C1s(2) subunit, required to form the fully active C1. In addition to C1q, fucoidan forms a complex with the protein C4 as observed by ACE. The fucoidan inhibits the first steps of the classical pathway activation that is of relevance in view of the proinflammatory effects of the subsequent products of the cascade. This study shows that a high level of inhibitory activity can be achieved with low molecular weight carbohydrate molecules and that the potential applicability of fucoidan oligosaccharides for therapeutic complement inhibition is worthy of consideration.

NMR characterization and molecular modeling of fucoidan showing the importance of oligosaccharide branching in its anticomplementary activity.

Fucoidan is a potent inhibitor of the human complement system whose activity is mediated through interactions with certain proteins belonging to the classical pathway, particularly the protein C4. Branched fucoidan oligosaccharides displayed a higher anticomplementary activity as compared to linear structures. Nuclear magnetic resonance (NMR) characterization of the branched oligosaccharides and saturation transfer difference-NMR experiment of the interaction with the protein C4 allowed the identification of the glycan residues in close contact with the target protein. Transferred nuclear Overhauser effect spectroscopy experiment and molecular modeling of fucoidan oligosaccharides indicated that the presence of side chains reduces the flexibility of the oligosaccharide backbone, which thus adopts a conformation which is very close to the one recognized by the protein C4. Together, these results suggest that branching of fucoidan oligosaccharides, determining their conformational state, has a major impact on their anticomplementary activity.

Improvement purification of sulfated oligofucan by ion-exchange displacement centrifugal partition chromatography

Centrifugal partition chromatography in ion-exchange displacement mode was used to fractionate mixtures of sulfated oligofucans obtained by partial depolymerization of brown seaweed fucoidans. Diluted (10%, v/v) protonated LA2 (a lipophilic secondary amine) is used as a weak exchanger. In an attempt to improve this method, several solvents (methyl isobutyl ketone, methyl tert.-butyl ether, BuOH) were tested to dissolve LA2H+. MtBE produced less bleeding than MiBK, whereas BuOH proved unsuitable. The sample injected needs to be highly diluted in water to ensure participation in the chromatographic process. A comparison of data (NMR, composition, molecular mass) indicated the homogeneity of the fractions obtained as well as the differences between them.

Preliminary report on fractionation of fucans by ion-exchange displacement centrifugal partition chromatography.

A new method combining ion-exchange displacement chromatography with centrifugal partition chromatography (CPC) was used for the fractionation of partially depolymerized fucans (polysulphated polysaccharides). The ion-exchanger was Amberlite LA2, a high-molecular-mass liquid secondary amine miscible with most common organic solvents and immiscible with aqueous solutions. Ion-exchange displacement centrifugal partition chromatography was performed with LA2 in methyl isobutyl ketone (MiBK) as the stationary phase, water as the mobile phase, Cl- as the carrier and OH- as the displacer. A complex mixture of partially depolymerized fucans was resolved into adjacent families characterized by their peak molecular mass and polydispersity. The Dubois test (sugar) and the azur A test (SO3-) confirmed the displacement mode of the process, and size-exclusion chromatographic controls confirmed its efficiency.

Polysaccharide labelling: impact on structural and biological properties

Three sulphated polysaccharides labelled at the reducing end with three different chemical treatments (2-aminopyridine (2-AP), 8-amino-1,3,6-trisulphonic acid (ANTS) and 6-(biotinyl)-aminocaproyl-hydrazide (BACH)) were compared. Molecular weight, chemical composition, nuclear magnetic resonance features and anticoagulant activity expressed as activated partial thromboplastin time were studied to determine whether structural and biological properties were retained or not after labelling. Partial depolymerisation and reduced biological activity were observed when reductive amination was performed with 2-AP. The initial properties were better retained with ANTS, and BACH did not modify the anticoagulant and structural properties.