Andrey S. Dmitrenok

Low-molecular-weight, biologically active compounds from marine Pseudoalteromonas species

We have examined the ability of marine Proteobacteria from the Pseudoalteromonas genus and Alteromonas macleodii to produce low-molecular-weight, biologically active compounds with antimicrobial and surface-active properties. A new marine bacterium, Pseudoalteromonas issachenkonii, exhibited a high level of biological activity and produced antifungal and hemolytic compounds. A detailed spectroscopic investigation based on UV, IR, high-resolution mass spectrometry, and 2D 1H and 13C nuclear magnetic resonance revealed that the former was indole-2,3-dione (isatin). The chemical structure of red-brown pigment (C9H7N3OS3) responsible for hemolytic activity remained unclear. Four of the 15 strains studied (P. luteoviolacea, P. rubra, P. undina, and P. issachenkonii) produced cell-bound, two (P. elaykovii and P. carrageenovora) produced extracellular, and one strain (P. citrea) produced cell-bound and extracellular fatty acids and phospholipids with surface activity. Neither peptides nor glycolipids with surface activity were detected.

Pyranoside‐into‐Furanoside Rearrangement: New Reaction in Carbohydrate Chemistry and Its Application in Oligosaccharide Synthesis

Great interest in natural furanoside-containing compounds has challenged the development of preparative methods for their synthesis. Herein a novel reaction in carbohydrate chemistry, namely a pyranoside-into-furanoside (PIF) rearrangement permitting the transformation of selectively O-substituted pyranosides into the corresponding furanosides is reported. The discovered process includes acid-promoted sulfation accompanied by rearrangement of the pyranoside ring into a furanoside ring followed by solvolytic O-desulfation. This process, which has no analogy in organic chemistry, was shown to be a very useful tool for the synthesis of furanoside-containing complex oligosaccharides, which was demonstrated by synthesizing disaccharide derivatives α-D-Galp-(1→3)-β-D-Galf-OPr, 3-O-s-lactyl-β-D-Galf-(1→3)-β-D-Glcp-OPr, and α-L-Fucf-(1→4)-β-D-GlcpA-OPr related to polysaccharides from the bacteria Klebsiella pneumoniae and Enterococcus faecalis and the brown seaweed Chordaria flagelliformis.

Structural characterization of fucosylated chondroitin sulfates from sea cucumbers Apostichopus japonicus and Actinopyga mauritiana

Two samples of fucosylated chondroitin sulfate (FCS), AJ and AM, were isolated from holothurian species Apostichopus japonicus and Actinopyga mauritiana, respectively. Purification of FCS was performed by ion exchange chromatography followed by gel filtration. Structure of the biopolymers was elucidated using chemical and NMR spectroscopic methods. Both polysaccharides were shown to contain a typical chondroitin core built up of repeating disaccharide units →3)-β-d-GalNAc-(1→4)-β-d-GlcA-(1→ and decorated by sulfate groups and α-l-Fuc branches. Two polysaccharides were different in pattern of sulfation of GalNAc and fucosyl branches connected to O-3 of GlcA. The ratio of GalNAc4S6S:GalNAc4S for AJ was about 2:1, whereas for AM this value was approximately 1:1. AJ contained Fucp2S4S and Fucp3S4S residues linked to O-3 of GlcA in a ratio of 3:1, while for AM this ratio was 1:4. Small portions of Fucp4S units attached to O-3 of GlcA were also found in both polysaccharides. Moreover, in a structure of AM the presence of Fucp3S residues linked to O-6 of GalNAc were determined using the data of NMR spectra.

Sterols from six marine sponges

The free sterol fractions from marine sponges Darwinella australiensis, Haliclona sp., Agelas mauritiana, Clathria major, Didiscus aceratusand Teichaxinella labirinticafrom Western Australia were isolated and studied by HPLC, GLC, GLC-MS, and NMR methods. D. australiensis contained 7 -, 5 -, 5,7 -, 5,7,9(11) -sterols, and cholest-7-en-3β-ol was shown to be a main sterol. The free sterols from A. mauritiana proved to be stanols and 7 -series compounds, chondrillasterol was identified as a predominant constituent. Haliclona sp. contained 5 -sterols with cholesterol as a main constituent. C. major and D. aceratus contained 5 -sterols, and clionasterol was shown to be a main sterol. T. labirintica was shown to contain 3β-hydroxymethyl-A-nor-sterols. Absolute configurations at C-24 of major sterols from C. major, D. aceratusand A. mauritiana were established by NMR method. Distribution of different sterols in the studied species was discussed to provide additional viewpoint on the probable application of these natural products as chemotaxonomic markers and to understand biological roles of unusual sterols in sponges using an idea of so-called biochemical coordination.  2003 Elsevier Ltd. All rights reserved.

Structure and biological activity of a fucosylated chondroitin sulfate from the sea cucumber Cucumaria japonica.

A fucosylated chondroitin sulfate (FCS) was isolated from the body wall of Pacific sea cucumber Cucumaria japonicaby extraction in the presence of papain followed by Cetavlon precipitation and anion-exchange chromatography. FCS was shown to contain D-GalNAc, D-GlcA, L-Fuc and sulfate in molar proportions of about 1:1:1:4.5. Structure of FCS was elucidated using NMR spectroscopy and methylation analysis of the native polysaccharide and products of its desulfation and carboxyl reduction. The polysaccharide was shown to contain a typical chondroitin core → 3)-β-D-GalNAc-(1 → 4)-β-D-GlcA-(1 →. Sulfate groups in this core occupy O-4 and the majority of O-6 of GalNAc. Fucosyl branches are represented by 3,4- and 2,4-disulfated units in a ratio of 4:1 and are linked to O-3 of GlcA. In addition, ∼ 33% of GlcA are 3-O-sulfated, and hence, the presence of short fucooligosaccharide chains side by side with monofucosyl branches cannot be excluded. FCS was shown to inhibit platelets aggregation in vitro mediated by collagen and ristocetin, but not adenosine diphosphate, and demonstrated significant anticoagulant activity, which is connected with its ability to enhance inhibition of thrombin and factor Xa by antithrombin III, as well as to influence von Willebrand factor activity. The latest property significantly distinguished FCS from low-molecular-weight heparin.

Anticoagulant and antithrombotic activities of modified xylofucan sulfate from the brown alga Punctaria plantaginea.

Selectively and totally sulfated (1 → 3)-linked linear homofucans bearing ∼ 20 monosaccharide residues on average have been prepared from the branched xylofucan sulfate isolated from the brown alga Punctaria plantaginea. Anticoagulant and antithrombotic properties of the parent biopolymer and its derivatives were assessed in vitro. Highly sulfated linear fucan derivatives were shown to inhibit clot formation in APTT assay and ristocetin induced platelets aggregation, while the partially sulfated analogs were inactive. In the experiments with purified proteins, fucan derivatives with degree of sulfation of ∼ 2.0 were found to enhance thrombin and factor Xa inhibition by antithrombin III. The effect of sulfated fucans on thrombin inhibition, which was similar to those of heparinoid Clexane(®) (enoxaparin) and of a fucoidan from the brown alga Saccharina latissima studied previously, can be explained by the multicenter interaction and formation of a ternary complex thrombin-antithrombin III-polysaccharide. The possibility of such complexation was confirmed by computer docking study.

Synthesis of the Oligosaccharides Related to Branching Sites of Fucosylated Chondroitin Sulfates from Sea Cucumbers

Natural anionic polysaccharides fucosylated chondroitin sulfates (FCS) from sea cucumbers attract great attention nowadays due to their ability to influence various biological processes, such as blood coagulation, thrombosis, angiogenesis, inflammation, bacterial and viral adhesion. To determine pharmacophore fragments in FCS we have started systematic synthesis of oligosaccharides with well-defined structure related to various fragments of these polysaccharides. In this communication, the synthesis of non-sulfated and selectively O-sulfated di- and trisaccharides structurally related to branching sites of FCS is described. The target compounds are built up of propyl β-d-glucuronic acid residue bearing at O-3 α-l-fucosyl or α-l-fucosyl-(1→3)-α-l-fucosyl substituents. O-Sulfation pattern in the fucose units of the synthetic targets was selected according to the known to date holothurian FCS structures. Stereospecific α-glycoside bond formation was achieved using 2-O-benzyl-3,4-di-O-chloroacetyl-α-l-fucosyl trichloroacetimidate as a donor. Stereochemical outcome of the glycosylation was explained by the remote participation of the chloroacetyl groups with the formation of the stabilized glycosyl cations, which could be attacked by the glycosyl acceptor only from the α-side. The experimental results were in good agreement with the SCF/MP2 calculated energies of such participation. The synthesized oligosaccharides are regarded as model compounds for the determination of a structure-activity relationship in FCS.

A highly regular fucosylated chondroitin sulfate from the sea cucumber Massinium magnum: Structure and effects on coagulation

A fucosylated chondroitin sulfate MM was isolated from the sea cucumber Massinium magnum. Structure of this polysaccharide was determined using chemical and NMR spectroscopic methods. The backbone of MM was shown to consist mainly of chondroitin sulfate E units with a small portion (about 10%) of chondroitin sulfate A fragments. Practically one type of branches Fuc3S4S attached to O-3 of GlcA residues was found in the polysaccharide molecules. The main repeating units of MM are →4)-[α-l-Fuc3S4S-(1→3)]-β-d-GlcA-(1→3)-β-d-GalNAc4S6S-(1→, whereas the minor repeating units are →4)-[α-l-Fuc3S4S-(1→3)]-β-d-GlcA-(1→3)-β-d-GalNAc4S-(1→. Anticoagulant activity of MM determined in APTT and TT tests was shown to be lower than that of heparin, but higher than that of enoxaparin. In the experiments with purified proteins MM effectively potentiated inhibition of thrombin and factor Xa by ATIII. Besides, MM did not induce platelets aggregation in platelets rich plasma.

Definitive Structural Assessment of Enterococcal Diheteroglycan

Enterococcus faecalis is one of most important nosocomial and often multi-antibiotic resistant pathogens responsible for infections that are difficult to treat. Previously, a cell-wall polysaccharide termed diheteroglycan (DHG) was isolated and characterized as a promising vaccine candidate. However, the configuration of its lactic acid (LA) residue attached to the galactofuranoside unit was not assessed, although it influences conformation of DHG chain in terms of biological recognition and immune evasion. This study proves the R configuration of the LA residue by means of chemical analysis, investigation of intramolecular NMR nuclear Overhauser effects and molecular dynamics simulations of native DHG and corresponding R and S models, which were obtained by using pyranoside-into-furanoside rearrangement. As alternative treatment and prevention strategies for E. faecalis are desperately needed, this discovery may offer the prospect of a synthetic vaccine to actively immunize patients at risk.

Two fucosylated chondroitin sulfates from the sea cucumber Eupentacta fraudatrix

Two fucosylated chondroitin sulfates EF1 and EF2 were isolated from the sea cucumber Eupentacta fraudatrix. Separation of the polysaccharides was performed using anion-exchange chromatography on DEAE-Sephacel by elution of 0.75M and 1.0M NaCl solutions. The structures of biopolymers were determined by chemical and NMR spectroscopic methods. The backbone of EF1 was found to be composed of chondroitin sulfate A and E units in a ratio of about 1:1. The core of EF2 along with chondroitin sulfate A and E fragments contained unusual disaccharide repeating units →4)-β-d-GlcpA2S3S-(1→3)-β-d-GalpNAc6S-(1→. The main type of branches in both polysaccharides was α-l-Fucp3S4S unit attached to O-3 of GlcA residues. Another type of branches was found to be the disaccharide fragment α-l-Fucp-(1→2)-α-l-Fucp3S4S-(1→ linked to O-3 of GlcA. The presence of structurally different fucosylated chondroitin sulfates in one species of sea cucumber is rather unusual and has not been described previously.