Novel phosphonoglycosphingolipids containing pyruvylated galactose from the nervous system of Aplysia kurodai

We have reported the existence of a phosphonoglycosphingolipid containing a pyruvylated galactose, FGL-IIb, in nerve fibers of Aplysia kurodai (Araki, S., Abe, S., Ando, S., Kon, K., Fujiwara, N. & Satake, M. (1989) J. Biol. Chem. 264, 19922-19927). We have now isolated two other pyruvylated galactose-containing phosphonoglycosphingolipids, named FGL-V and FGL-IIa, from the nervous tissue of Aplysia, and characterized them as [3,4-O-(S-1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3[6'-O-(2- aminoethylphosphonyl)Gal alpha 1----2] (2-aminoethylphosphonyl----6) Gal beta 1----4Glc beta 1----1 ceramide and [3,4,O-(S-1-carboxyethylidene)] Gal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1----2]Gal beta 1----4Glc beta 11----ceramide, respectively. Their major aliphatic components are palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, the nervous system of Aplysia contains several pyruvylated phosphonoglycolipids.     

Highly sulphated galactan from Halymenia durvillei (Halymeniales,Rhodophyta), a red seaweed of Madagascar marine coasts

Halymenia durvillei is a red seaweed with a great potential as sulphated galactan producer collected in the coastal waters of small island of Madagascar (Nosy-be in Indian Ocean). To elucidate the structure of its polysaccharide, NMR (¹H and ¹³C), FTIR, HPAEC and different colorimetric methods were carried out. It has been shown that this polysaccharide, consisted mainly of galactose, was branched by xylose and galactose in minor amounts. Arabinose and fucose were also detected. This galactan was found highly sulphated (42%, w/w) and pyruvylated (1.8%, w/w). Analysis of glycosidic linkages by CPG-MS and ¹³C NMR indicated that the polysaccharide has the defining linear backbone of alternating 3-β-d-galactopyranosyl units and 4-linked α-l/d-galactopyranosyl residues. 3,6-Anhydrogalactose units have been also detected in minor quantity. This λ-carrageenan like polysaccharide has shown original sulphatation patterns with 2-O (26%) or 2/6-O (58%) sulphated 3-linked β-d-galactopyranosyl units and 6-O (19%) or 2/6-O (47%) 4-linked α-l/d-galactopyranosyl residues.     

Galloyl, caffeoyl and hexahydroxydiphenoyl esters of dihydrochalcone glucosides from Balanophora tobiracola

Seven galloyl, caffeoyl and (S)-hexahydroxydiphenoyl (HHDP) esters of dihydrochalcone glucosides were isolated from Balanophora tobiracola; based on spectroscopic and chemical evidence, their structures were determined to be 6'-O-galloyl-, 3',4'-di-O-galloyl-, 4',6'-di-O-galloyl-, 4',6'-O-(S)-HHDP-, 3'-O-galloyl-4',6'-O-(S)-HHDP-, 3'-O-caffeoyl-4',6'-O-(S)-HHDP-3-hydroxyphloretin 4'-O-beta-D-glucosides and 3'-O-galloyl-4',6'-O-(S)-HHDP-phloretin 4'-O-beta-D-glucoside, respectively. By contrast, these compounds were not found in the taxonomically related B. japonica. The 3'-galloyl-4',6'-HHDP esters of the dihydrochalcone glucosides showed strong inhibitory activities against alpha-glucosidase. Four known compounds were also isolated namely, (+/-)-eriodictyol 7-O-beta-D-glucoside, 1-O-caffeoyl-3-O-galloyl-beta-D-glucose, phloretin 4'-O-beta-D-glucoside, and 3-hydroxyphloretin 4'-O-beta-D-glucoside.     

Dextran-linked purine nucleosides as substrates and inhibitors of adenosine deaminase

Dextran-bound adenosine, inosine, and nebularine have been prepared by carbodiimide coupling of their 2',3'-O-(4-carboxyethyl-1-methylbutylidene) cyclic acetal derivatives to 6-aminohexyldextran or 12-aminododecanyldextran. The latter polymers were prepared by cyanogen-bromide activation of dextran T80 followed by reaction with 1,6-diaminohexane or 1,12-diaminododecane. A high CNBr concentration leads to high-molecular-weight material, probably due to cross-linking, accompanied by a decrease in the digestion velocity using endo-dextranase from Penicillium species (EC 3.2.1.11). The dextran-bound nucleosides, as well as the nucleoside 2',3'-O-(4-ethoxycarbonyl-1-methylbutylidene) acetal derivatives, were tested as substrates and inhibitors for adenosine deaminase. The Km of the adenosine acetal ester is identical to that of adenosine which shows that acetalation does not hinder complex formation. Since the maximum velocity of deamination is decreased fourfold, the modified substrate does not fit as well as the nucleoside. The polymer-bound acetals show a 3-8-fold increase of Km or Ki and unchanged V compared to the corresponding acetals while dextranase digestion of the support does not alter the kinetic data. This indicates that the length of the polysaccharide chain does not interfere either with the complex formation or with the catalytic activity of the modified substrate. Since the activation energies of the deamination reactions of adenosine, its acetal ester, and dextran-linked adenosine are all similar (29.8-32.3 kJ mol-1) it is concluded that no diffusion control of the enzymatic reaction results from the binding of the nucleoside acetals to dextran T80.     

Characterization of two novel pyruvylated glycosphingolipids containing 2'-aminoethylphosphoryl(-->6)-galactose from the nervous system of Aplysia kurodai.

Two novel acidic glycosphingolipids containing pyruvylated galactose were purified from the nervous tissue of Aplysia kurodai by successive Iatrobeads column chromatographies. By component analysis, sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structures of these acidic glycosphingolipids, named F-9 and FGL-I, were determined to be: [3,4-O-(S-1-carboxyethylidene)]Gal beta 1-->3 GalNAc alpha 1-->3[6'-O-(2-aminoethylphosphonyl)Gal alpha 1-->2] (2-aminoethylphosphoryl 1-->6)Gal beta 1-->4Glc beta 1-->1ceramide and [3,4-O-(S-1-carboxyethylidene)] Gal beta 1-->3GalNAc alpha 1-->3(Fuc alpha 1-->2)(2-aminoethylphosphonyl-->6 Gal beta 1-->4Glc beta 1-->1ceramide, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. Thus, pyruvylated glycosphingolipids containing phosphoethanolamine in addition to or in place of 2-aminoethylphosphonate are present in the nervous system of Aplysia.     

Purification and structure elucidation of the N-acetylbacillosamine-containing polysaccharide from Bacillus licheniformis ATCC 9945.

The exopolysaccharide of Bacillus licheniformis ATCC 9945 (formerly B. subtilis ATCC 9945) contains among other glycoses 4-acetamido-2-amino-2,4,6-trideoxy-D-glucose, termed N-acetylbacillosamine (Bac2N4NAc). A similar diamino glycose, 2-acetamido-4-amino-2,4,6-trideoxy-D-glucose, was found in a surface layer (S-layer) glycoprotein preparation of Clostridium symbiosum HB25. Electron microscopic studies, however, showed that B. licheniformis ATCC 9945 is not covered with an S-layer lattice, indicating that the N-acetylbacillosamine present in that organism might be a constituent of a cell wall-associated polymer. For elucidation of the structure of the N-acetylbacillosamine-containing polysaccharide, it was purified from a trichloroacetic acid extract of B. licheniformis ATCC 9945 cells. Using different hydrolysis protocols and a hydrolysate of the S-layer glycoprotein preparation from C. symbiosum HB25 as reference, the purified polysaccharide was found to contain 2,4-diamino-2,4,6-trideoxy-glucose, 2-acetamido-2-deoxy-glucose, 2-acetamido-2-deoxy-galactose and galactose in a molar ratio of 1 : 1 : 1 : 2. One- and two-dimensional NMR spectroscopy, including 800 MHz proton magnetic resonance measurements, in combination with chemical modification and degradation experiments, revealed that the polysaccharide consists of identical pyruvylated pentasaccharide repeating units with the structure: [-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-alpha-D-GlcpNAc-(1-->3)-beta-D-Bacp2N4NAc-(1-->3)-[(S)Py-(3,4)-beta-D-Galp-(1-->6)]-beta-D-GalpNAc-(1-->](n)     

Iridoid glucosides and flavones from the aerial parts of Avicennia marina

Two new iridoid glucosides, namely, 2'-O-[(2E,4E)-5-phenylpenta-2,4-dienoyl]mussaenosidic acid (1; mussaenosidic acid = [1S-(1alpha,4aalpha,7alpha,7aalpha)]-1-(beta-D-glucopyranosyloxy)-1,4a,5,6,7,7a-hexahydro-7-hydroxy-7-methylcyclopenta[c]pyran-4-carboxylic acid) and 2'-O-(4-methoxycinnamoyl)mussaenosidic acid (2), were isolated from the aerial parts of the mangrove plant Avicennia marina. Beside that, one known iridoid glucoside, 2'-O-coumaroylmussaenosidic acid (3) and four known flavones (flavone = 2-phenyl-4H-1-benzopyran-4-one) including 4',5-dihydroxy-3',7-dimethoxyflavone (4), 4',5-dihydroxy-3',5',7-trimethoxyflavone (5), 4',5,7-trihydroxyflavone (6), and 3',4',5-trihydroxy-7-methoxyflavone (7) were also isolated and identified. The structures of these compounds were elucidated by NMR spectroscopy and by low- and high-resolution mass spectrometry. The chemotaxonomic significance of these findings was discussed. In addition, each isolated compound was evaluated for the ability of alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH) radical-scavenging activity.     

A simple procedure for synthesis of diastereoisomers of thymidine cyclic 3',5'-phosphate derivatives

Diastereoisomeric thymidine cyclic (3',5')-methanephosphonates (3a), cyclic (3',5')-phosphoranilidates (3b) and cyclic (3',5')-phosphoranilidothioates (3c) were prepared by treatment of diastereoisomerically pure thymidine 3'-O-[O-(4-nitrophenyl)methanephosphonates] (2a), 3'-O-[O-(4-nitrophenyl)phosphoranilidates] (2b) or 3'-O-[O-(4-nitrophenyl)phosphoranilidothioates] (2c), respectively, with sodium hydroxide in dioxane-water solution.     

Chemical structure of a novel aminophospholipid from Hydrogenobacter thermophilus strain TK-6

The phospholipid composition of Hydrogenobacter thermophilus strain TK-6, an obligately chemolithoautotrophic, extremely thermophilic hydrogen bacterium, was analyzed. Two of four phospholipids detected from the strain were assumed to be phosphatidylinositol and phosphatidylglycerol. An aminophospholipid named PX, whose content among the phospholipids was 65%, was found to have a novel chemical structure by analysis of the dilyso form with nuclear magnetic resonance and fast atom bombardment-mass spectrometry (FAB-MS) and by analysis of the intact PX with FAB-MS as 1,2-diacyl-3-O-(phospho-2'-O-(1'-amino)-2',3',4',5'-pentanetetrol)-sn-glycerol. Structurally similar phospholipids have been identified in Methanospirillum hungatei, Methanolacinia paynteri, and Methanogenium cariaci, which all belong to the Archaea.     

Characterization of the exopolysaccharide produced by Streptococcus thermophilus 8S containing an open chain nononic acid.

The exopolysaccharide produced by Streptococcus thermophilus 8S in reconstituted skimmed milk is a heteropolysaccharide containing d-galactose, d-glucose, d-ribose, and N-acetyl-d-galactosamine in a molar ratio of 2 : 1 : 1 : 1. Furthermore, the polysaccharide contains one equivalent of a novel open chain nononic acid constituent, 3,9-dideoxy-d-threo-d-altro-nononic acid, ether-linked via C-2 to C-6 of an additional d-glucose per repeating unit. Methylation analysis and 1D/2D NMR studies (1H and 13C) performed on the native polysaccharide, and mass spectrometric and NMR analyses of the oligosaccharide obtained from the polysaccharide by de-N-acetylation followed by deamination and reduction demonstrated the 'hepta'saccharide repeating unit to be: -->4)-alpha-D-Galp-(1-->2)-beta-D-Ribf-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1--7')-Sub-(1-->4)-beta-D-GalpNAc-(1--> in which Sug is 6-O-(3',9'-dideoxy-d-threo-d-altro-nononic acid-2'-yl)-alpha-d-glucopyranose.     

Coumarin glucosides from Cruciata taurica

Two new coumarin glycosides (1 and 2) along with two known coumarin glucosides, daphnin (3) and daphnetin glucoside (4) were isolated from the aerial parts of Cruciata taurica. The structures of the new compounds were elucidated by spectral methods and chemical means as 7-O-(6' -acetoxy-beta-D-glucopyranosyl)-8-hydroxycoumarin (1) and 7-O-[6 '-O-(3',4'-dihydroxycinnamoyl)-beta-D-glucopyranosyl]-8-hydroxycoumarin (2). The phylogenetic significance of coumarins in C. taurica was discussed.     

Two aurone glycosides from heartwood of Pterocarpus santalinus

Two new aurone glycosides, 6 hydroxy 5 methyl 3',4',5' trimethoxy aurone 4-O-alpha-L-rhamnopyranoside and 6,4' dihydroxy aurone 4-O-rutinoside have been isolated from the ethanolic extract of the wood of Pterocarpus santalinus. Their structures were determined on the basis of chemical and spectroscopic analysis (UV, IR, EIMS, (1)H and (13)C NMR).     

Archaea contain a novel diether phosphoglycolipid with a polar head group identical to the conserved core of eucaryal glycosyl phosphatidylinositol.

The structure of a major ether polar lipid of the methanogenic archaeon Methanosarcina barkeri was identified as glucosaminyl archaetidylinositol. This lipid had archaeol (2,3-di-O-phytanyl-sn-glycerol) as a core lipid portion, and the polar head group consisted of 1 mol each of phosphate, myo-inositol and D-GlcN. The polar head group was identified by means of chemical degradations, phosphatidylinositol-specific phospholipase C treatment, permethylation analysis, and fast atom bombardment-mass spectrometry as glucosaminylinositol phosphate, which was linked to the glycerol backbone via a phosphodiester bond. The stereochemical configuration of the phospho-myo-inositol residue of glucosaminyl archaetidylinositol was determined to be 1-D-myo-inositol 1-phosphate by measuring optical rotation of phospho-myo-inositol prepared by nitrous acid deamination and alkaline hydrolysis from the lipid. 1H NMR of the intact lipid showed that GlcN was linked to C-6 position of myo-inositol as an alpha-anomer. It is, finally, concluded that the complete structure of this lipid is 2,3-di-O-phytanyl-sn-glycero-1-phospho- 1'[6'-O-(2"-amino-2"-deoxy-alpha-D-glucopyranosyl)]-1'-D-myo-inositol. This lipid has a hybrid nature of an archaeal feature in alkyl glycerol diether core portion and an eucaryal feature in the polar head group identical to the conserved core structure (GlcNp(alpha 1-6)-myo-inositol 1-phosphate) of glycosylated phosphatidylinositol which serves as a membrane protein anchor in eucaryal cells.     

Anti-inflammatory and antinociceptive activity of flavonoids isolated from Viscum album ssp. album

Viscum album L. has been used in the indigenous systems of medicine for treatment of headache and some inflammatory diseases. In order to evaluate this information, antinociceptive and anti-inflammatory activities of the five flavonoids (5,7-dimethoxy naringenin or 4',6'-dimethoxy chalcononaringenin) derivatives, isolated from the ethyl acetate fraction of the extract from V. album ssp. album, were investigated, namely 5,7-dimethoxy-flavanone-4'-O-beta-D-glucopyranoside (1), 2'-hydroxy-4',6'-dimethoxy-chalcone-4-O-beta-D-glucopyranoside (2), 5,7-dimethoxy-flavanone-4'-O-[2"-O-(5"'-O-trans-cinnamoyl)-beta-D-apiofuranosyl]-beta-D-glucopyranoside (3), 2'-hydroxy-4',6'-dimethoxy-chalcone-4-O-[2"-O-(5"'-O-trans-cinnamoyl)-beta-Dapiofuranosyl]-beta-D-glucopyranoside (4), 5,7-dimethoxy-flavanone-4'-O-[beta-D-apiofuranosyl-(1 --> 2)]-beta-D-glucopyranoside (5). For the antinociceptive activity assessment the p-benzoquinone-induced writhing test and for the anti-inflammatory activity the carrageenan-induced hind paw edema model in mice were used. The ethyl acetate fraction in a dose of 250 mg/kg as well as compounds 2 and 5 in a 30 mg/kg dose were shown to possess remarkable antinociceptive and anti-inflammatory activities per os without inducing any apparent acute toxicity as well as gastric damage.     

Structure of a highly pyruvylated galactan sulfate from the Pacific green alga Codium yezoense (Bryopsidales, Chlorophyta)

A polysaccharide fraction consisting of d-galactose, sulfate, and pyruvate in a molar proportion of 4:2:1 was isolated from the green seaweed Codium yezoense by water extraction followed by ion-exchange chromatography. To elucidate its structure, modified polysaccharides were prepared by desulfation, depyruvylation, and by total removal of non-carbohydrate substituents. Structures of the native polysaccharide and of the products of its chemical modifications were investigated by methylation analysis as well as by 1D and 2D (1)H and (13)C NMR spectroscopy. The polysaccharide devoid of sulfate and pyruvate was subjected to two subsequent Smith degradations to afford a rather low-molecular and essentially linear (1-->3)-beta-d-galactan. A highly ramified structure was suggested for the native polysaccharide, which contains linear backbone segments of 3-linked beta-d-galactopyranose residues connected by (1-->6) linkages, about 40% of 3-linked residues being additionally substituted at C-6, probably by short oligosaccharide residues also containing (1-->3) and (1-->6) linkages. Sulfate groups were found mainly at C-4 and in minor amounts at C-6. Pyruvate was found to form mainly five-membered cyclic ketals with O-3 and O-4 of the non-reducing terminal galactose residues. The minor part of pyruvate forms six-membered cyclic ketals with O-4 and O-6. The absolute configurations of ketals (R for six-membered ketals and S for five-membered ones) were established using NMR spectral data.     

7,5'-O-dibenzylinosines: synthesis and studies on their conformational properties

Reaction of 2',3'-O-isopropylidene inosine with benzyl bromide (1 h, rt) led to the 1,5'-O-dibenzylderivative 4, but by increasing the reaction time or the temperature, compound 4 is further transformed into the 1,7,5'-O-tribenzylinosine derivative 5. Similarly, the 7-methyl-1,5'-O-dibenzylderivative 6 has been synthesized from 4. The 1H-NMR spectra of 5 and 6 showed peculiar chemical shifts for geminal protons (H5' and H5' of the ribose, and the CH2 of the benzyl groups). Preliminary NMR studies have been performed, including NOESY experiments that point toward the predominant existence of conformers that are stabilized by an electrostatic interaction between the positively charged imidazole of the base moiety and the high electron density of the 5'-benzyl substituent.     

Synthesis and enzymatic deprotection of fully protected 2'-5' oligoadenylates (2-5A): towards a prodrug strategy for short 2-5A

Fully protected pA2'p5'A2'p5'A trimers 1a and 1b have been prepared as prodrug candidates for a short 2'-5' oligoadenylate, 2-5A, and its 3'-O-Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)-triggered deprotection in HEPES buffer (pH?7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2-5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2-5A trimer, although partial removal of the 3'-O-[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2',5'→3',5' phosphate migration and release of adenosine as side reactions.     

Structural investigation of the O-specific polysaccharides of Morganella morganii consisting of two higher sugars

The lipopolysaccharide of the bacterium Morganella morganii (strain KF 1676, RK 4222) yielded two polysaccharides, PS1 and PS2, when subjected to mild acid degradation followed by GPC. The polysaccharides were studied by 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, 1H,(13)C HMQC, and HMBC experiments. Each polysaccharide was found to contain a disaccharide repeating unit consisting of two higher sugars, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid (a derivative of 8-epilegionaminic acid, 8eLeg5Am7Ac) and 2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-2,6-dideoxy-D-galactose (shewanellose, She). The two polysaccharides differ only in the ring size of shewanellose and have the following structures:Shewanellose has been previously identified in a phenol-soluble polysaccharide from Shewanella putrefaciens A6, which shows a close structural similarity to PS2.     

Structural elucidation of a capsular polysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn's disease.

The structure of a capsular polysaccharide (CPS) from a clinical isolate of Bacteroides vulgatus was elucidated. B. vulgatus IMCJ 1204 was isolated from feces of a patient with Crohn's disease. CPS was prepared by phenol/water extraction of the bacterial cells followed by hydrophobic interaction chromatography and then gel filtration chromatography of the extract. The structure of CPS was determined by chemical analysis and NMR spectroscopy including DQF-COSY, TOCSY, ROESY, HSQC-TOCSY, HMQC and HMBC to be a polysaccharide composed of the following repeating unit: -->3)beta-D-Glcp(1-->6)[alpha-D-GalpNAc(1-->2)beta-D-Galp(1-->4)]beta-D-GlcpNAc(1-->3)alpha-D-Galp(1-->4)beta-D-Manp(1-->.