Anatoly I. Usov

13C-n.m.r. spectra of xylo-oligosaccharides and their application to the elucidation of xylan structures

Abstract 13 C-N.m.r. spectra of thirteen xylo-oligosaccharides [a complete series of α- and β- d -xylopyranosyl derivatives of methyl α- d -xylopyranoside, β- d -xylopyranosyl derivatives of methyl 4- O -β- d -xylopyranosyl- d -xylopyranoside, methyl O -α- d -xylopyranosyl-(1→3)- O -β- d -xylopyranosyl-(1→4)- d -xylopyranoside, and a branched methyl β-xylotetraoside] have been interpreted. The data obtained have been used for the carbon signal assignment in the spectra of a number of red-algal xylans. 13 C-N.m.r. spectroscopy is shown to be a rapid and convenient method for the structural analysis of xylose-rich polysaccharides.

STRUCTURE OF A SULFATED XYLOGALACTAN FROM THE CALCAREOUS RED ALGA CORALLINA PILULIFERA P. ET R. (RHODOPHYTA, CORALLINACEAE)

The structure of a sulfated polysaccharide isolated from the calcareous red alga Corallina pilulifera was studied by methylation analysis before and after desulfation or Smith degradation, as well as by 1D and 2D 1H and 13C NMR spectroscopy. The polysaccharide was shown to consist of D-galactose, L-galactose, 2-O-methyl-L-galactose, 3-O-methyl-L-galactose, 6-O-methyl-D-galactose, D-xylose, and sulfate in a molar ratio of 29:20:5:2:1:20:23. Its agaran-like backbone built up of alternating 3-linked beta-D-galactopyranose and 4-linked alpha-L-galactopyranose residues bears single beta-D-xylopyranosyl substituents at position 6 of beta-D-galactose residues, whereas sulfate and O-methyl groups occupy positions 2 and 3 of alpha-L-galactose and position 6 of beta-D-galactose residues.

Synthesis, NMR and Conformational Studies of Fucoidan Fragments 1:1 Desulfated 2,3- and 3,4-Branched Trisaccharide Fragments and Constituting Disaccharides

ABSTRACT Two fucotriosides with vicinal disubstitution α-L-Fuc-(1→2)[α-L-Fuc-(1→3)]α-L-Fuc-OPr (1) and α-L-Fuc-(1→3)[α-L-Fuc-(1→4)]α-L-Fuc-OPr (2), which are related to fragments of natural polysaccharides fucoidans, have been synthesized together with constituent disaccharides 3-5. Spectral and conformational properties of tri- and disaccharides have been investigated by 1H, 13C and NOE NMR spectroscopy.

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.

Synthesis, NMR and Conformational Studies of Fucoidan Fragments. V.[1] Linear 4,4′,4″‐Tri‐O‐Sulfated and Parent Non‐sulfated (1→3)‐Fucotrioside Fragments

Propyl 4,4′,4″‐tri‐O‐sulfated and non‐sulfated (1→3)‐α‐l‐fucotriosides which are related to fragments of natural fucoidans have been synthesized. Their spectral and conformational properties have been investigated by 1H and 13C NMR, NOE and molecular modeling. Molecular mechanics calculations of the tri‐O‐sulfated compound as a trianion did not give agreement with the experimental NOE values, while the model with the non‐dissociated sulfo group on the non‐reducing end worked successfully. (1→3)‐Fucobioside fragments in both trisaccharides investigated were shown to have the same range of conformations as in previously described propyl (1→3)‐α‐l‐fucobiosides, but with the increase of the relative population of the conformer with the spatial proximity of H‐1′ and H‐4 in the case of non‐sulfated fucotrioside.

Anticoagulant activity of fucoidans from brown algae

The anticoagulant activity of polysaccharide fucoidans from 11 species of brown algae was studied. The anticoagulant activity was measured by the activated partial thromboplastin time (APTT), prothrombin time, and thrombin time. Inhibitory action of these fucoidans significantly varied from one species to another. Fucoidans from Laminaria saccharina and Fucus distichus exhibited high anticoagulant activity, while fucoidans from Cladosiphon okamuranus and Analipus japonicus were almost inactive. Other fucoidans exhibited intermediate inhibitory activity. The inhibitory effect of fucoidans on thrombin and factor Xa was investigated in the presence or in the absence of natural thrombin inhibitor, antithrombin III (AT III). In contrast to the best-studied anticoagulant, heparin, most of these fucoidans inhibited thrombin in the absence of AT III. In the presence of AT III the inhibitory effect of fucoidans considerably increased. In contrast to heparin, fucoidans weakly influenced factor Xa activity in the presence of AT III and their inhibitory effect was not observed in the absence of AT III. There was no correlation between the anticoagulant activities of this series of fucoidans and their anti-inflammatory action, studied earlier. It is suggested that these two types of fucoidan activities depend on different structural features of fucoidans. Results of this study demonstrate a possibility of preparation of fucoidans with high anti-inflammatory activity but low anticoagulant activity. Anticoagulant activity of the fucoidans did not exhibit direct dependence on the content of fucose, the other neutral sugars and sulfates; no dependence was also found between the anticoagulant activity and the structure of the backbone of their molecules.

Effect of Enzyme Preparation from the Marine Mollusk Littorina kurila on Fucoidan from the Brown Alga Fucus distichus

A fucoidanase preparation from the marine mollusk Littorina kurila cleaved some glycosidic bonds in fucoidan from the brown alga Fucus distichus, but neither fucose nor lower oligosaccharides were produced. The main product isolated from the incubation mixture was a polysaccharide built up of disaccharide repeating units →3)-α-L-Fucp-(2,4-di-SO3−)-(1→4)-α-L-Fucp-(2SO3−)-(1→, the structure coinciding with the idealized formula proposed for the initial substance. A polymer fraction with the same carbohydrate chain but sulfated only at positions 2 and nonstoichiometrically acetylated at positions 3 and 4 of fucose residues was isolated as a minor component. It is suggested that the native polysaccharide should contain small amounts of non-sulfated and non-acetylated fucose residues, and only their glycosidic bonds are cleaved by the enzyme. The enzymatic hydrolysis showed that irregular regions of the native polysaccharide containing acetylated and partially sulfated repeating units were assembled in blocks.

Synthesis, NMR, and Conformational Studies of Fucoidan Fragments. VII.1 Influence of Length and 2,3‐Branching on the Conformational Behavior of Linear (1→3)‐Linked Oligofucoside Chains

The conformational behavior of linear (1→3)‐linked propyl di‐, tri‐, tetrafucosides and 2,3‐branched tetrafucosides with linear (1→3)‐linked trisaccharide backbone related to fragments of natural fucoidans were studied by theoretical molecular modeling and experimental determination of transglycosidic vicinal coupling constants 3JC,H. The application of NOE NMR‐spectroscopy, which is traditionally used in conformational analysis of oligosaccharides, was accompanied by experimental difficulties in the case of tetrafucosides, due to the overlap of cross‐peaks and their trend to be close to zero. It was shown that conformations of difucoside units in the studied compounds depend on their position within the oligosaccharide backbone, on the chain length, and on the presence or absence of 2,3‐branch point. The comparison of experimental and calculated values of transglycosidic constants 3JC,H showed good coincidence for the middle disaccharide units of tetrafucosides, indicating that these units are more rigid than terminal ones.

The Synthesis and NMR and Conformational Studies of Fucoidan Fragments: VI.1 Fragments with an α-(1 → 2)-Linked Fucobioside Unit

A series of selectively sulfated di- and trisaccharide derivatives corresponding to the potential fragments of fucoidans with a α-(1 → 2)-linked fucobioside unit were synthesized and studied by 1Н and 13C NMR spectroscopy. NOE experiments and molecular modeling were used for a conformational analysis of the compounds synthesized. In the case of disaccharides, the experimental NOE values were found to agree with those obtained using modeling with the use of density functional theory (DFT) and differ from those resulting from modeling by the molecular mechanics MM3 force field. Trisaccharide fragments partially or completely sulfated in position 4 turned out to be correctly described by both MM3 force field and DFT computation.