Alexey A. Grachev

Structure of a fucoidan from the brown seaweed Fucus evanescens C.Ag.

A fucoidan consisting of L-fucose, sulfate and acetate in a molar proportion of 1:1.23:0.36 was isolated from the Pacific brown seaweed Fucus evanescens. The structures of its desulfated and de-O-acetylated derivatives were investigated by 1D and 2D (1)H and (13)C NMR spectroscopy, and the data obtained were confirmed by methylation analysis of the native and desulfated polysaccharides. The fucoidan was shown to contain a linear backbone of alternating 3- and 4-linked alpha-L-fucopyranose 2-sulfate residues: -->3)-alpha-L-Fucp(2SO(3)(-))-(1-->4)-alpha-L-Fucp(2SO(3)(-))-(1-->. Additional sulfate occupies position 4 in a part of 3-linked fucose residues, whereas a part of the remaining hydroxyl groups is randomly acetylated.

Fucans, but Not Fucomannoglucuronans, Determine the Biological Activities of Sulfated Polysaccharides from Laminaria saccharina Brown Seaweed

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed.

Further studies on the composition and structure of a fucoidan preparation from the brown alga Saccharina latissima

The polysaccharide composition of a fucoidan preparation isolated from the brown alga Saccharina latissima (formerly Laminaria saccharina) was reinvestigated. The preparation was fractionated by anion-exchange chromatography, and the fractions obtained were analyzed by chemical methods combined with NMR spectroscopy. Several 2D procedures, including HSQC, HMQC-TOCSY, and HMQC-NOESY, were used to obtain reliable structural information from the complex spectra, and the signal assignments were additionally confirmed by comparison with the literature spectra of the related polysaccharides and synthetic oligosaccharides. In accordance with the previous data, the main polysaccharide component was shown to be a fucan sulfate containing a backbone of 3-linked alpha-l-fucopyranose residues sulfated at C-4 and/or at C-2 and branched at C-2 by single sulfated alpha-l-fucopyranose residues. In addition, three other types of sulfated polysaccharide molecules were detected in the total fucoidan preparation: (i) a fucogalactan having a backbone of 6-linked beta-d-galactopyranose residues branched mainly at C-4 and containing both terminal galactose and fucose residues; (ii) a fucoglucuronomannan having a backbone of alternating 4-linked beta-d-glucopyranosyluronic acid and 2-linked alpha-d-mannopyranose residues with alpha-l-fucopyranose residues as single branches at C-3 of alpha-d-Manp; and (iii) a fucoglucuronan having a backbone of 3-linked beta-d-glucopyranosyluronic acid residues with alpha-l-fucopyranose residues as single branches at C-4. Hence, even a single algal species may contain, at least in minor amounts, several sulfated polysaccharides differing in molecular structure. Partial resolution of these polysaccharides has been accomplished, but unambiguous evidence on their presence as separate entities was not obtained.

Synthesis and Molecular Recognition Studies of the HNK-1 Trisaccharide and Related Oligosaccharides. The Specificity of Monoclonal Anti-HNK-1 Antibodies as Assessed by Surface Plasmon Resonance and STD NMR

The human natural killer cell carbohydrate, HNK-1, plays function-conducive roles in peripheral nerve regeneration and synaptic plasticity. It is also the target of autoantibodies in polyneuropathies. It is thus important to synthesize structurally related HNK-1 carbohydrates for optimizing its function-conducive roles, and for diagnosis and neutralization of autoantibodies in the fatal Guillain-Barré syndrome. As a first step toward these goals, we have synthesized several HNK-1 carbohydrate derivatives to assess the specificity of monoclonal HNK-1 antibodies from rodents: 2-aminoethyl glycosides of selectively O-sulfated trisaccharide corresponding to the HNK-1 antigen, its nonsulfated analogue, and modified structures containing 3-O-fucosyl or 6-O-sulfo substituents in the N-acetylglucosamine residues. These were converted, together with several related oligosaccharides, into biotin-tagged probes to analyze the precise carbohydrate specificity of two anti-HNK-1 antibodies by surface plasmon resonance that revealed a crucial role of the glucuronic acid in antibody binding. The contribution of the different oligosaccharide moieties in the interaction was shown by saturation transfer difference (STD) NMR of the complex consisting of the HNK-1 pentasaccharide and the HNK-1 412 antibody.

SYNTHESIS, NMR, AND CONFORMATIONAL STUDIES OF FUCOIDAN FRAGMENTS. III.[1] EFFECT OF BENZOYL GROUP AT O-3 ON STEREOSELECTIVITY OF GLYCOSYLATION BY 3-O- AND 3,4-DI-O-BENZOYLATED 2-O-BENZYLFUCOSYL BROMIDES

The effect of a benzoyl group at O-3 on stereoselectivity of glycosylation by 3-O- and 3,4-di-O-benzoylated 2-O-benzyl-L-fucopyranosyl bromides was studied by direct chemical experiments and computational chemistry. The influence of a benzoyl group at O-3 of the fucosyl donors was shown to have a larger effect on the efficiency of α-fucosylation than a benzoyl group at O-4. It is hypothesized that this is a result of the ability of a benzoyl group at O-3 to participate in glycosyl cation stabilization.

Acid-promoted synthesis of per-O-sulfated fucooligosaccharides related to fucoidan fragments.

The synthesis of per-O-sulfated derivatives of di-, tetra-, hexa-, octa-, dodeca-, and hexadecafucosides related to natural fucoidans of different types has been performed with the use of previously reported acid-promoted protocol for per-O-sulfation of polyols by SO(3) complexes. During the treatment of (1→3)-linked oligofucosides under these conditions with the promotion by TfOH, the unusual rearrangement of the reducing pyranose residue into furanose one was observed. To avoid the formation of rearrangement by-products, the use of a series of strong acids as promoters of sulfation of large oligofucosides was studied and the improved protocol was developed based on the use of TFA instead of TfOH. The efficiency of the new method was demonstrated by the syntheses of per-O-sulfated derivatives of dodeca- and hexadecafucosides. The described method of O-sulfation opens access to the preparation of the oligosaccharides related to fucoidan fragments and their per-O-sulfated derivatives interesting for elucidation of the relationship between their structure and biological activity.

SYNTHESIS, NMR, AND CONFORMATIONAL STUDIES OF FUCOIDAN FRAGMENTS 4[1]: 4-MONO- AND 4,4′-DISULFATED (1→3)-α-l-FUCOBIOSIDE AND 4-SULFATED FUCOSIDE FRAGMENTS

Propyl 4-O-sulfonato- and 4,4′-di-O-sulfonato-3-O-α-l-fucopyranosyl-α-l-fucopyranosides, which are related to fragments of brown algal fucoidans, have been synthesized. Their spectral (1H and 13C NMR, NOE) and conformational properties have been studied in combination with molecular modeling and compared with the respective non-sulfated propyl fucobioside. Correlations between chemical shifts and conformational properties of these compounds were investigated.

[Polysaccharides of algae: 60. Fucoidan from the Pacific brown alga Analipus japonicus (Harv.) Winne (Ectocarpales, Scytosiphonaceae)].

A fucoidan containing L-fucose, sulfate, and O-acetyl groups at a molar ratio 3:2:1, as well as minor amounts of xylose, galactose, and uronic acids was isolated from the brown alga Analipus japonicus collected in the Sea of Japan. The structures of the native polysaccharide and the products of its desulfation and deacetylation were studied by the methods of methylation, periodate oxidation, and NMR spectroscopy. It was shown that a polysaccharide molecule mainly consists of a linear carbohydrate chain of (1→3)-linked α-L-fucopyranose residues, which bears numerous branches in the form of single α-L-fucopyranose residues (three branches at position 4 and one branch at position 2 per each ten residues of the main chain). Sulfate groups occupy positions 2 and (to a lesser extent) 4, most of the terminal nonreducing fucose residues being sulfated twice. The acetyl groups are located predominantly at positions 4. The structural role of minor monosaccharides was not established.

A sulfated glucuronofucan containing both fucofuranose and fucopyranose residues from the brown alga Chordaria flagelliformis

A fucoidan fraction composed of l-fucose, sulfate, and d-glucuronic acid in a molar proportion of about 1:1:0.25 and small amount of acetyl groups was isolated from the brown alga Chordaria flagelliformis. Several modified polysaccharides were prepared from the native fucoidan using solvolytic desulfation, carboxyl reduction, and partial acid hydrolysis. Polysaccharide structures were elucidated by methylation analysis and 1D and 2D NMR spectroscopy. The fucoidan was shown to contain a backbone of 3-linked alpha-l-fucopyranose residues, about one-third of which are glycosylated at C-2 by alpha-d-glucopyranosyluronic acid residues. About half of the latter residues are glycosylated at C-4 by single alpha-L-fucofuranose residues or by disaccharides alpha-L-Fucf-(1-->2)-alpha-L-Fucf-(1-->. Fucofuranose residues are mono- and disulfated at different positions, whereas some additional sulfate groups occupy C-2 and C-4 of the backbone, the latter position being also partially acetylated.

Preliminary investigation of a highly sulfated galactofucan fraction isolated from the brown alga Sargassum polycystum

A fucoidan preparation was isolated from the brown alga Sargassum polycystum (Fucales, Sargassaceae). The preparation was fractionated by anion-exchange chromatography, and two highly sulfated fractions F3 and F4 were obtained. The fractions were quite similar in composition, but different in chemical structure. F4 was analyzed by chemical methods, including desulfation, methylation, Smith degradation, and partial acid hydrolysis with mass-spectrometric monitoring, as well as by NMR spectroscopy. Several 2D NMR procedures, including HMQC-TOCSY and HMQC-NOESY, were used to obtain reliable structural information from the complex spectra. Molecules of F4 were shown to contain a backbone built up mainly of 3-linked α-L-fucopyranose 4-sulfate residues, as in many other fucoidans, but rather short sequences of these residues are interspersed by single 2-linked α-D-galactopyranose residues also sulfated at position 4. This rather unusual structural feature should have a great influence on the conformation of the polymeric molecule and may be important for biological activity of the polysaccharide. Hence, F4 is an example of a new sulfated galactofucan isolated from the brown alga. According to the data obtained, the distribution of galactose residues along the polysaccharide backbone seems to be not strictly regular, but the definitive sequence of monomers in the polymeric molecules awaits additional investigation.