Synthesis of the Non Reducing End Oligosaccharides of Glycosphingolipids from Ascaris suum.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the non reducing end oligosaccharides of glycosphingolipids from Ascaris suum have been accomplished. Galα1→3GalNAcß1→OR (1), Galß1→3Galα1→3GalNAcß1→OR (2), Galß1→6Galα1→3GalNAcß1→OR (3), Galß1→6(Galß1→3)Galα1→3GalNAcß1→OR (4) and GlcNAcß1→6Galß1→6(Galß1→3)Galα1→3GalNAcß1→OR (5) (R = biotinylated probe) were synthesized by stepwise condensation (1-4) and block synthesis (5) using 5-(methoxycarbonylpentyl) 2-O-benzoyl-3-O-2-napthylmethyl-4,6-O-di-tert-butylsilylene-α-D-galactopyranosyl-(1→3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-ß-D-galactopyranoside (12) as a common precursor. Compound 12 was converted into two kinds of glycosyl acceptors and was condensed with suitable galactosyl donors, respectively.

Construction of Vicinal Quaternary Stereogenic Centers by Enantioselective Direct Mannich-Type Reaction Using a Chiral Bis(guanidino)iminophosphorane Catalyst.

A novel asymmetric direct Mannich-type reaction of α-iminophenylacetate esters with thionolactones, bearing a substituent at the α-position, as a less acidic pronucleophile was developed. Using bis(guanidino)iminophosphorane as the chiral organosuperbase catalyst, the reaction afforded densely functionalized amino-acid derivatives having vicinal quaternary stereogenic centers, one of which is an all-carbon quaternary stereogenic center, in good yield with high diastereo- and enantioselectivities.

Enantioselective Addition of a 2-Alkoxycarbonyl-1,3-dithiane to Imines Catalyzed by a Bis(guanidino)iminophosphorane Organosuperbase.

A chiral bis(guanidino)iminophosphorane catalyzes enantioselective addition reactions of a 1,3-dithiane derivative as a pronucleophile. The chiral uncharged organosuperbase facilitates the addition of benzyloxycarbonyl-1,3-dithiane to aromatic N-Boc-protected imines to provide optically active α-amino-1,3-dithiane derivatives, which are valuable versatile building blocks in organic synthesis.

Structural elucidation of the neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides from the marine crab Erimacrus isenbeckii.

The neutral glycosphingolipids, mono-, di-, tri- and tetraglycosylceramides (GL-1, GL-2, GL-3, GL-4a and GL-4b), were identified from whole tissues of the marine crab Erimacrus isenbeckii by successive column chromatography with ion exchange Sephadex (QAE-Sephadex), magnesium silicate (Florisil) and silicic acid (Iatrobeads) resins. Through component analysis, sugar analysis, methylation studies, exoglycosidase cleavage, and various chromatographic and spectrometric techniques, their structures were proposed to be as follows: GL-1, Glcß1-1Cer; GL-2, Manß1-4Glcß1-1Cer; GL-3, Galß1-3Manß1-4Glcß1-1Cer; and GL-4a and GL-4b, Gal3Meα1-4Galß1-3Manß1-4Glcß1-1Cer. The main molecular species of the aliphatic moiety in each purified glycolipid were 18:0, 22:0, 22:1-d14:1 (fatty acid-sphingoid) and 18:0-d16:1 for GL-1; 18:0-d16:1 and 22:1-d14:1, d16:1 for GL-2; 22:1, 24:1-d16:1 for GL-3; 22:1, 24:1-d16:1 for GL-4a; and h22:1, h24:1-d16:1 for GL-4b, respectively. By immunological detection, an arthro-series glycosphingolipid (At3Cer; GlcNAcß1-3Manß1-4Glcß1-1Cer) was also detected as a minor component. The characteristic arthro-series glycosphingolipid has been observed in most animals belonging to the phylum Arthropoda.

Development of a chiral bis(guanidino)iminophosphorane as an uncharged organosuperbase for the enantioselective amination of ketones.

Chiral bis(guanidino)iminophosphoranes were designed and synthesized as chiral uncharged organosuperbase catalysts that facilitate activation of less-acidic pro-nucleophiles. The newly developed bis(guanidino)iminophosphoranes, which possess the highest basicity among chiral organocatalysts reported to date, were proven to be a superb class of chiral organosuperbases by reaction of azodicarboxylates with 2-alkyltetralones and their analogues as the less acidic pro-nucleophiles.

Collagenase inhibitors from Viola yedoensis.

Fractionation of acetone and methanol extracts of Viola yedoensis, under the guidance of inhibition against Clostridium histolyticum collagenase (ChC), resulted in the isolation of esculetin (1) (IC(50) 12 µM) and scopoletin (2) (IC(50) 1.8 µM) as the active constituents, together with trans-p-coumaric acid (3), cis-p-coumaric acid (4), 3-O-ß-D-glucosyl-7-O-α-L-rhamnosylkaempferol (5), rutin (6), isovitexin (7), isoorientin (8), vicenin-2 (9), isoscoparin (10), vanillic acid (11) and adenosine (12). Modification of phenolic hydroxy groups of 1 showed that small O-alkyl groups largely increased the activity, whereas larger O-alkyl groups decreased the activity, and 6,7-dimethoxycoumarin (scoparone 13) potently inhibited ChC (IC(50) 24 nM).

Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of glycosphingolipids and related carbohydrate moieties from the parasite Schistosoma mansoni.

Stereocontrolled syntheses of three neutral glycosphingolipids and six oligosaccharide derivatives found from the parasite Schistosoma mansoni have been accomplished. A pentasaccharide glycosphingolipid ß-D-Galp-(1→4)-[α-L-Fucp-(1→3)]-ß-D-GlcpNAc-(1→3)-ß-D-GalpNAc-(1→4)-ß-D-Glcp-(1↔1)-Cer (1), two hexasaccharide glycosphingolipids α-L-Fucp-(1→3)-ß-D-Galp-(1→4)-[α-L-Fucp-(1→3)]-ß-D-GlcpNAc-(1→3)-ß-D-GalpNAc-(1→4)-ß-D-Glcp-(1↔1)-Cer (2) and ß-D-Galp-(1→4)-[α-L-Fucp-(1→3)]-ß-D-GlcpNAc-(1→3)-ß-D-GlcpNAc-(1→3)-ß-D-GalpNAc-(1→4)-ß-D-Glcp-(1↔1)-Cer (3), together with their non-reducing end tri- and tetrasaccharides, ß-D-Galp-(1→4)-[α-L-Fucp-(1→3)]-ß-D-GlcpNAcOR (4) and α-L-Fucp-(1→3)-ß-D-Galp-(1→4)-[α-L-Fucp-(1→3)]-ß-D-GlcpNAcOR (5), were synthesized by block synthesis. Moreover, non-reducing end oligosaccharides of schistosomal glycosphingolipids, ß-D-GalpNAc-(1→4)-[α-L-Fucp-(1→3)]-ß-D-GlcpNAcOR (6), α-L-Fucp-(1→3)-ß-D-GalpNAc-(1→4)-[α-L-Fucp-(1→3)]-ß-D-GlcpNAcOR (7), α-L-Fucp-(1→3)-ß-D-GalpNAc-(1→4)-[α-L-Fucp-(1→2)-α-L-Fucp-(1→3)]-ß-D-GlcpNAcOR (8) and α-L-Fucp-(1→3)-ß-D-GalpNAc-(1→4)-[α-L-Fucp-(1→2)-α-L-Fucp-(1→2)-α-L-Fucp-(1→3)]-ß-D-GlcpNAcOR (9) [R=2-(trimethylsilyl)ethyl], were synthesized as probes to explore their diagnostic potential to detect schistosomiasis from patients' sera.

Galα1-4Galß1-3GalNAc is the dominant epitope of Em2 antigen, the mucin-type glycoprotein from Echinococcus multilocularis.

The larval stage of Echinococcus multilocularis causes alveolar echinococcosis in human. In serodiagnosis of alveolar echinococcosis, specific reactions have been noted not only against protein antigens but also carbohydrates. With regard to protein antigens, the recent development of recombinant antigens has contributed to an improvement in serodiagnostic examination. On the contrary, the preparation of carbohydrate antigen still depends on extraction from crude antigens, and isolation is usually accompanied with difficulty; consequently, it is rare to examine individual antigenicity of carbohydrates. However, parasitic helminths express various antigenic carbohydrates. In the case of Echinococcus granulosus, antigenic glycoproteins of the laminated layer have been reported. Furthermore, the laminated layer of E. multilocularis contains Em2 antigen which is a famous mucin-type glycoprotein and which seems to play an important role in metacestode survival mechanisms within the immunologically reacting host; nevertheless, the anomeric configurations and the individual antigenicity of Em2 O-glycans have not been confirmed so far. Under these circumstances, we introduced a chemical synthesis to get pure oligosaccharides in order to assess diagnostic performance. In our previous study, 11 oligosaccharides have already been prepared by stereocontrolled syntheses. Among them, three synthetic oligosaccharides showed antigenicity. Our aim is to investigate correct sequence and serodiagnostic potential of the dominant epitope of Em2. This study provided important diagnostic information: (1) the trisaccharide Galα1-4Galß1-3GalNAc sequence is the dominant epitope of Em2 (sensitivity 95.0 %), (2) Trematoda expresses carbohydrates with the similar trisaccharide sequence, and (3) the terminal Galα1-4Gal sequence is a candidate for the widely common epitope that accounts for the cross-reaction.

Synthesis of the carbohydrate moiety from the parasite Echinococcus multilocularis and their antigenicity against human sera.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions with a Galß1-3GalNAc core of the Em2 glycoprotein antigen obtained from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Galß1-3(GlcNAcß1-6)GalNAcα1-R (G), tetrasaccharide Galß1-3(Galß1-4GlcNAcß1-6)GalNAcα1-R (J) and pentasaccharide Galß1-3(Galα1-4Galß1-4GlcNAcß1-6)GalNAcα1-R (K) (R=biotinylated probe) were synthesized by block synthesis by the use of 5-(methoxycarbonyl)pentyl 2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1→3)-2-azido-4-O-benzyl-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. Moreover, linear trisaccharide Galα1-4Galß1-3GalNAcα1-R (H) and branched tetrasaccharide Galα1-4Galß1-3(GlcNAcß1-6)GalNAcα1-R (I) were synthesized by stepwise condensation. We examined the antigenicity of these five oligosaccharides by an enzyme linked immunosorbent assay (ELISA). Our results demonstrate that biotinylated oligosaccharides H, I and K show good serodiagnostic potential to detect infections caused by the parasite E. multilocularis. Among them the linear sequence Galα1-4Galß1-3GalNAcα1-R in oligosaccharide (H) appears to show the highest sensitivity (95%). Moreover, our study clarified the dominant carbohydrate epitope of Em2 antigen.

Synthesis, inhibitory effects on nitric oxide and structure-activity relationships of a glycosphingolipid from the marine sponge Aplysinella rhax and its analogues.

The novel glycosphingolipid, ß-D-GalNAcp(1-->4)[α-D-Fucp(1-->3)]-ß-D-GlcNAcp(1-->)Cer (A), isolated from the marine sponge Aplysinella rhax has a unique structure, with D-fucose and N-acetyl-D-galactosamine moieties attached to a reducing-end N-acetyl-D-glucosamine through an α1-->3 and ß1-->4 linkage, respectively. We synthesized glycolipid 1 and some non-natural di- and trisaccharide analogues 2-6 containing a D-fucose residue. Among these compounds, the natural type showed the most potent nitric oxide (NO) production inhibitory activity against LPS-induced J774.1 cells. Our results indicate that both the presence of a D-Fucα1-3GlcNAc-linkage and the ceramide aglycon portion are crucial for optimal NO inhibition.

Synthetic studies on glycosphingolipids from protostomia phyla: synthesis of glycosphingolipids from the parasite Schistosoma mansoni.

Synthetic access to three neutral glycosphingolipids from the parasite Schistosoma mansoni adult worm has been achieved. These structures differ significantly from those of other parasites and exhibit a unique structural motif termed "schisto-core" consisting of GalNAcbeta1-->4Glcbeta1-->sequence. We have synthesized glycosphingolipids, beta-D-GalNAcp-(1-->4)-beta-D-Glcp-(1-->1)Cer (1), beta-D-GlcNAcp-(1-->3)-beta-D-GalNAcp-(1-->4)-beta-D-Glcp-(1-->1)Cer (2) and beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-GalNAcp-(1-->4)-beta-D-Glcp-(1-->1)Cer (3).

Genetic and chemical comparison of Boi (Sinomeni Caulis et Rhizoma) and Seifuto (Caulis Sinomenii).

Boi and its original plant Sinomenium acutum from Japan were compared with Seifuto and its botanical origins from China in terms of their internal transcribed spacer (ITS) sequences and major chemical components. Boi, Seifuto, and their botanical origins overall showed seven variable sites in the ITS sequence and six genotypes. Japanese S. acutum and Boi had one nucleotide variation at position 593 to show two genotypes (J1 and J2) and their heterozygote (J3). Seifuto samples and their botanical origins, S. acutum and S. acutum var. cinereum from China, showed three genotypes (C1, C2, and C3), which did not agree with the botanical classification, indicating that they cannot be distinguished according to their ITS sequences. All Seifuto samples from Henan market showed the same ITS genotype (C1). The Japanese and Chinese genotypes differed in the nucleotide position 424, which can be used to distinguish the country of origin of these materials. In the HPLC analysis of six major components, sinomenine (1), magnoflorine (2), menisperine (3), 6-O-methyllaudanosoline glucoside (4), liriodendrin (5), and menisdaurin (6), all were detected in Boi, whereas five (all except for menisdaurin) were detected in Seifuto. The main component in the rhizome of Seifuto was sinomenine, whereas magnoflorine was the main component in the rhizome and the climbing stem of Boi. The content of sinomenine in Seifuto was almost twice that in Boi. Although the individual content of alkaloids 1-4 differed between Boi and Seifuto, the total contents of these alkaloids were comparable between them both in the climbing stem and rhizome.

Different contributions of side-chains in beta-D-(1-->3,6)-galactans on intestinal Peyer's patch-immunomodulation by polysaccharides from Astragalus mongholics Bunge.

Thirteen polysaccharides isolated from an extract of the aerial portions of Astragalus mongholics Bunge demonstrated immunomodulating activity against Peyer's patch immunocompetent cells. Nine of the active polysaccharide fractions were composed of either arabinogalactans, pectic arabinogalactans or pectins. The activities of the arabinogalactans and pectic arabinogalactans were associated with beta-D-(1-->3)-galactan moieties branched with beta-D-(1-->6)-galactooligosaccharide side-chains having degrees of polymerization of 8 or less. Degradation of the beta-D-(1-->3)-galactan or beta-D-(1-->6)-galactosyl side-chains in the arabinogalactans significantly decreased immunomodulating activity. Rhamnogalacturonan I (RG-I) with beta-D-(1-->3,6)-galactosyl side-chains having terminal beta-D-GlcA showed activity in the pectin-enriched fractions. Interestingly, the terminal GlcA was not required for activity of the arabinogalactan-enriched fractions, suggesting at least two different immunomodulating structures.

Syntheses of glycoclusters containing a phosphocholine residue related to a glycosphingolipid from the earthworm Pheretima hilgendorfi.

Three types of glycoclusters related to an amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi were synthesized. The glycoclusters were prepared from a common precursor and a simple approach for the rational design of a glycocluster was developed.

[Carbohydrate study for 40 years].

This review describes the carbohydrate study and the natural product related to the glycoside chemistry. What shall the people in the field of pharmacognosy and natural products chemistry search in scene in future? Forty years before while isolating dimeric compound having naphthoquinonepyrone skeleton from the coloring material produced by the pathogen that hosted in wheat and caused rotten root disease, silica gel has to be treated with oxalic acid to reduce the absorbency before separation. However now a days, availability of reversed phase adsorbents for liquid chromatography has made the separation and isolation of complex compounds possible, easy and rapid. With the advancement of mechanical/physicochemical analytic methods, it has even been possible to isolate traces of compounds present in complex. This advancement has made it possible to determine structure of saponins and complex polysaccharides without decomposition and carry out in vitro bioassay at the same time using various cells on-line. Further, this review describes the oligosaccharide syntheses and biological activities of glycosphingolipids, focusing especially on those found in invertebrates.

Inhibitory effect of Lysichiton camtschatcense extracts on Fe2+/ascorbate-induced lipid peroxidation in rat kidney and brain homogenates.

Lysichiton camtschatcense is a well-known plant in Japan where it has been used as a traditional medicine by the "Ainu" people for the treatment of acute nephritis. It is presumed that L. camtschatcense has an inhibitory effect against nephritis caused by reactive oxygen species (ROS) owing to its antioxidant activities. Consequently, the antioxidant effect of L. camtschatcense extracts was assessed against Fe(2+)/ascorbic acid (AsA)-induced lipid peroxidation in rat kidney and brain homogenates. The antioxidant effect of the chloroform extract (CE) was more potent than that of the methanol extract (ME) for both homogenates. The antioxidant effect of both extracts was similar to those of alpha-tocopherol, a lipid-soluble antioxidant, and glutathione (GSH), a water-soluble antioxidant, which were used as reference compounds. Although CE showed a low radical-scavenging effect for superoxide anion radicals (O(2)(*-)) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, assessed by using an electron spin resonance (ESR) method, hydroxyl radicals (*OH) were markedly scavenged by more than 80%. On the other hand, ME showed more significant scavenging effect for DPPH radicals and O(2)(*-) than CE. These results suggest that the inhibitory effects of the L. camtschatcense extract on lipid peroxidation in rat kidney and brain are based on its high radical-scavenging effect against *OH, O(2)(*-) , and lipid-derived radicals generated from the cell membrane.

Synthetic studies on the carbohydrate moiety of the antigen from the parasite Echinococcus multilocularis.

Stereocontrolled syntheses of branched tri-, tetra-, and pentasaccharides displaying a Gal beta 1-->3GalNAc core in the glycan portion of the glycoprotein antigen from the parasite Echinococcus multilocularis have been accomplished. Trisaccharide Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc alpha 1-OR (A), tetrasaccharide Gal beta 1-->3(Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (D), and pentasaccharides Gal beta 1-->3(Gal beta 1-->4Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (E) and Gal beta1-->3(Gal alpha 1-->4Gal beta 1-->4GlcNAc beta 1-->6)GalNAc alpha 1-OR (F) (R = 2-(trimethylsilyl)ethyl) were synthesized by block synthesis. The disaccharide 2-(trimethylsilyl)ethyl 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-2-azido-4-O-benzyl-2-deoxy-alpha-d-galactopyranoside served as a common glycosyl acceptor in the synthesis of the branched oligosaccharides. Moreover, linear trisaccharide Gal beta 1-->4Gal beta 1-->3GalNAc alpha 1-OR (B) and branched tetrasaccharide Gal beta 1-->4Gal beta 1-->3(GlcNAc beta 1-->6)GalNAc alpha 1-OR (C) were synthesized by stepwise condensation.

Pomegranate juice inhibits sulfoconjugation in Caco-2 human colon carcinoma cells.

Several fruit juices have been reported to cause food-drug interactions, mainly affecting cytochrome P450 activity; however, little is known about the effects of fruit juices on conjugation reactions. Among several fruit juices tested (apple, peach, orange, pineapple, grapefruit, and pomegranate), pomegranate juice potently inhibited the sulfoconjugation of 1-naphthol in Caco-2 cells. This inhibition was both dose- and culture time-dependent, with a 50% inhibitory concentration (IC(50)) value calculated at 2.7% (vol/vol). In contrast, no obvious inhibition of glucuronidation of 1-naphthol in Caco-2 cells was observed by any of the juices examined. Punicalagin, the most abundant antioxidant polyphenol in pomegranate juice, was also found to strongly inhibit sulfoconjugation in Caco-2 cells with an IC(50) of 45 microM, which is consistent with that of pomegranate juice. These data suggest that punicalagin is mainly responsible for the inhibition of sulfoconjugation by pomegranate juice. We additionally demonstrated that pomegranate juice and punicalagin both inhibit phenol sulfotransferase activity in Caco-2 cells in vitro, at concentrations that are almost equivalent to those used in the Caco-2 cells. Pomegranate juice, however, shows no effects on the expression of the sulfotransferase SULT1A family of genes (SULT1A1 and SULT1A3) in Caco-2 cells. These results indicate that the inhibition of sulfotransferase activity by punicalagin in Caco-2 cells is responsible for the reductions seen in 1-naphthyl sulfate accumulation. Our data also suggest that constituents of pomegranate juice, most probably punicalagin, impair the enteric functions of sulfoconjugation and that this might have effects upon the bioavailability of drugs and other compounds present in food and in the environment. These effects might be related to the anticarcinogenic properties of pomegranate juice.

Synthesis of neutral glycosphingolipids from Zygomycetes.

Novel neutral glycosphingolipids (NGSLs) containing Gal-alpha1-->6Gal, previously found in the Zygomycetes species Mucor hiemalis, were synthesized. The structures of these compounds are different from those of other fungal GSLs, and they are expected to be involved in host-parasite interactions. A key step in their synthesis is direct 1,2-cis alpha-selective galactosylation of 4,6-diol tri- and tetrasaccharide acceptors with a galactosyl donor in the presence of N-iodosuccinimide (NIS)/trifluoromethanesulfonic acid (TfOH). The fully protected glycosides were deprotected to give the two target glycosphingolipids.

A synthetic glycosphingolipid-induced antiproliferative effect in melanoma cells is associated with suppression of FAK, Akt, and Erk activation.

Numerous studies have demonstrated the participation of glicolipids in signal transduction and the regulation of melanoma cell growth and apoptosis. Hoping to discover new anticancer drugs, we have synthesized ten glycolipids found in various invertebrates that do not have sialic acids. These compounds were tested for antiproliferative effects on a melanoma cell line, B16F10. A synthetic compound, Manbeta(1-4)[Fucalpha(1-3)]Glcbeta1-Cer, (glycosphingolipid 7), which was identified in the millipede Parafontaria laminata armigera, had an antiproliferative effect on the melanoma cells. This compound suppressed the activation of the focal adhesion kinase (FAK)-Akt pathway as well as the activation of extracellular signal-regulated kinase (Erk)1/2 pathway involved in cell proliferation. Expression of the cell cycle proteins, cyclin D1 and CDK4, was suppressed by glycosphingolipid 7. From these results, glycosphingolipid 7 suppressed the activation of the FAK-Akt pathway and of Erk1/2, which resulted in a decrease in the expression of cyclin D1 and CDK4. Glycosphingolipid 7 might be a candidate for an inhibitor of cell proliferation in melanomas.