Scutellaria Root extract-induced hepatocytotoxicity can be controlled by regulating its baicalin content.

Scuellaria Root (SR, root of Scutellaria baicalensis), which has potent anti-inflammatory effects, is a component of useful Kampo formulae. Albeit a low frequency, SR induces serious interstitial pneumonia and liver dysfunction. In this study, to control the adverse effects of SR, we investigated the causal constituent responsible for its hepatocytotoxicity and aimed to develop a method to control it. As a result, we revealed that the hepatocytotoxicity of SR was correlated with its baicalin content, a major constituent in SR. It was confirmed by preparing a baicalin-free SR extract, which exhibited reduced hepatocytotoxicity. The addition of baicalin to the baicalin-free SR extract restored the hepatocytotoxicity, indicating that the hepatocytotoxicity of SR is dependent on its baicalin content. Thus, SR extract-induced hepatocytotoxicity can be controlled by regulating its baicalin content.

Repeated psychological stress, chronic vicarious social defeat stress, evokes irritable bowel syndrome-like symptoms in mice.

Increasing evidence has demonstrated that emotional states and intestinal conditions are inter-connected in so-called "brain-gut interactions." Indeed, many psychiatric disorders are accompanied by gastrointestinal symptoms, such as the irritable bowel syndrome (IBS). However, the functional connection remains elusive, partly because there are few useful experimental animal models. Here, we focused on a highly validated animal model of stress-induced psychiatric disorders, such as depression, known as the chronic vicarious social defeat stress (cVSDS) model mice, which we prepared using exposure to repeated psychological stress, thereafter examining their intestinal conditions. In the charcoal meal test and the capsaicin-induced hyperalgesia test, cVSDS model mice showed a significantly higher intestinal transit ratio and increased visceral pain-related behaviors, respectively. These changes persisted over one month after the stress session. On the other hand, the pathological evaluations of the histological and inflammatory scores of naive and cVSDS model mice did not differ. Furthermore, keishikashakuyakuto-a kampo medicine clinically used for the treatment of IBS-normalized the intestinal motility change in cVSDS model mice. Our results indicate that cVSDS model mice present IBS-like symptoms such as chronic intestinal peristaltic changes and abdominal hyperalgesia without organic lesion. We therefore propose the cVSDS paradigm as a novel animal model of IBS with wide validity, elucidating the correlation between depressive states and intestinal abnormalities.

Elucidation of Chemical Interactions between Crude Drugs Using Quantitative Thin-Layer Chromatography Analysis.

To elucidate the interactions between crude drugs in Kampo medicines (traditional Japanese medicines), it is important to determine the content of the constituents in a cost-effective and simple manner. In this study, we quantified the constituents in crude drug extracts using thin-layer chromatography (TLC), an inexpensive and simple analytical method, to elucidate the chemical interactions between crude drugs. We focused on five crude drugs, for which quantitative high-performance liquid chromatography (HPLC) methods are stipulated in the Japanese Pharmacopoeia XVIII (JP XVIII) and compared the analytical data of HPLC and TLC, confirming that the TLC results corresponded with the HPLC data and satisfied the criteria of JP XVIII. (Z)-ligustilide, a major constituent in Japanese Angelica Root, for which a method of quantification has not been stipulated in JP XVIII, was also quantitatively analyzed using HPLC and TLC. Furthermore, Japanese Angelica Root was combined with 26 crude drugs to observe the variation in the (Z)-ligustilide content from each combination by TLC. The results revealed that combinations with Phellodendron Bark, Citrus Unshiu Peel, Scutellaria Root, Coptis Rhizome, Gardenia Fruit, and Peony Root increased the (Z)-ligustilide content. Quantifying the constituents in crude drug extracts using the inexpensive and simple TLC method can contribute to elucidating interactions between crude drugs in Kampo medicines, as proposed by the herbal-pair theory.

Synthesis of the Carbohydrate Moiety of Glycoproteins from the Parasite Echinococcus granulosus and Their Antigenicity against Human Sera.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the carbohydrate moiety of glycoprotein from Echinococcus granulosus have been accomplished. Trisaccharide Galß1-3Galß1-3GalNAcα1-R (A), tetrasaccharide Galα1-4Galß1-3Galß1-3GalNAcα1-R (B), and pentasaccharide Galα1-4Galß1-3Galß1-3Galß1-3GalNAcα1-R (C), (R = biotinylated probe) were synthesized by stepwise condensation and/or block synthesis by the use of 5-(methoxycarbonyl)pentyl 2-azido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside as a common glycosyl acceptor. The synthesis of the tetrasaccharide and the pentasaccharide was improved from the viewpoint of reducing the number of synthetic steps and increasing the total yield by changing from stepwise condensation to block synthesis. Moreover, hexasaccharide E, which contains the oligosaccharide sequence which occurs in E. granulosus, was synthesized from trisaccharide D. We examined the antigenicity of these five oligosaccharides by an enzyme-linked immunosorbent assay (ELISA). Although compounds of C-E did not exhibit antigenicity against cystic echinococcosis (CE) patient sera, compounds B, D, and E showed good serodiagnostic potential for alveolar echinococcosis (AE).

Changes in the extracted amounts and seasonally variable constituents of Diospyros kaki at different growth stages.

Persimmon Calyx is a crude drug derived from the persistent calyx of mature fruit of Diospyros kaki Thunberg (Ebenaceae) and is used for the treatment of intractable hiccups. Although there are several reports on the isolation of constituents from Persimmon Calyx, its active constituents have not been elucidated. In this study, by focusing on the medicinal part of Persimmon Calyx, calyx on mature fruit of D. kaki, we examined the changes in the extraction amounts of 3 cultivars of D. kaki ('Hiratanenashi', 'Jiro', and 'Tonewase') to identify and quantify seasonally variable constituents during the maturation process by analysing their chemical compositions. We found that the extraction weight of the calyx, fruit of persimmons, and total tannin content in calyxes were significantly increased during maturation. Lupeol (1), betulinic acid (2), pomolic acid (3), ursolic acid (4), ß-sitosterol (5), rotungenic acid (6), barbinervic acid (7), catechin (8), gallocatechin (9), and sucrose (10) were identified in the calyx of D. kaki. Compounds 1, 6, and 7 were isolated from Persimmon Calyx for the first time. Moreover, the isolated compounds (1-7) and their analogue (oleanolic acid) were quantitatively analysed, and the results showed that the amounts of 4 and oleanolic acid were reduced during maturation, whereas that of 2, 3, 6, and 7 were increased.

Structures and Inhibitory Activities for Interleukin-2 Production of Seasonally Variable Constituents in Flower Parts of Magnolia kobus at Different Growth Stages.

Magnolia Flower is a crude drug used for the treatment of headaches, toothaches, and nasal congestion. Here, we focused on Magnolia kobus, one of the botanical origins of Magnolia Flower, and collected the flower parts at different growth stages to compare chemical compositions and investigate potential inhibitory activities against interleukin-2 (IL-2) production in murine splenic T cells. After determining the structures, we examined the inhibitory effects of the constituents of the bud, the medicinal part of the crude drug, against IL-2 production. We first extracted the flower parts of M. kobus from the bud to fallen bloom stages and analysed the chemical compositions to identify the constituents characteristic to the buds. We found that the inhibitory activity of the buds against IL-2 production was more potent than that of the blooms. We isolated two known compounds, tiliroside (1) and syringin (2), characteristic to the buds from the methanol (MeOH) extract of Magnolia Flower. Moreover, we examined the inhibitory activities of both compounds against IL-2 production and found that tiliroside (1) but not syringin (2), showed strong inhibitory activity against IL-2 production and inhibited its mRNA expression. Thus, our strategy to examine the relationship between chemical compositions and biological activities during plant maturation could not only contribute to the scientific evaluation of medicinal parts of crude drugs but also assist in identifying biologically active constituents that have not yet been reported.

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.

Quantitative analysis of the anti-inflammatory activity of orengedokuto II: berberine is responsible for the inhibition of NO production.

Orengedokuto is a Kampo formula that has been used for removing "heat" and "poison" to treat inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. We report here our analysis of the anti-inflammatory effect of the component crude drugs of orengedokuto and their constituents using the inhibition of nitric oxide (NO) production in the murine macrophage-like cell line J774.1. An initial comparison of NO production inhibitory activities of the extracts of the component crude drugs and their combinations revealed that the activity could be attributed to Phellodendron Bark and Coptis Rhizome. Berberine (1), the major constituent of these crude drugs, showed potent activity (IC50 4.73 ± 1.46 µM). Quantitative analysis of 1 in the extracts of all combinations of component crude drugs revealed that the amount of 1 in each extract of the combination of Scutellaria Root with either Phellodendron Bark and/or Coptis Rhizome was lower than that in the corresponding mixtures of the extracts of the individual crude drugs and that 1 was present in the precipitates formed during the decoction process. To the contrary, the differences in the amounts of 1 were smaller in the extracts containing Gardenia Fruit. These results indicated that the constituents of Scutellaria Root precipitated with 1 and that the constituents of Gardenia Fruit dissolved the precipitates. To identify the constituents affecting the solubility of 1, we fractionated the hot-water extracts of Scutellaria Root based on solubility tests of 1 to give baicalin (2), wogonin (3) and oroxyloside (4), which formed precipitates with 1.

Synergistic effect of baicalein, wogonin and oroxylin A mixture: multistep inhibition of the NF-κB signalling pathway contributes to an anti-inflammatory effect of Scutellaria root flavonoids.

Scutellaria root, the root of Scutellaria baicalensis Georgi, is a crude drug used for inflammatory diseases. In our previous report, the combination of flavonoids contained in Scutellaria root have been found to inhibit PGE2 production more strongly than individual flavonoids. Here, to investigate the mechanism of the synergistic effect, we examined the effects of an equimolar mixture (F-mix) of baicalein (1), wogonin (2) and oroxylin A (3) on the production of PGE2 in LPS-treated J774.1 cells. Although 1 and 3 inhibited COX-2 activity, the F-mix showed no synergistic effect on COX-2 inhibition. Therefore, we investigated the steps leading to the activation of COX-2 protein. Compounds 1-3 and F-mix inhibited the expression of COX-2 protein. However, only 2 inhibited the expression of COX-2 mRNA among the flavonoids, and the F-mix showed no synergistic effect. Only 1 inhibited NF-κB translocation into the nucleus, and the F-mix showed no synergistic effect. Although 2 did not affect NF-κB translocation, it strongly inhibited NF-κB-dependent transcriptional activity, and the F-mix inhibited the activity slightly more than 2. Compounds 1-3 also inhibited NO production, and the F-mix showed a synergistic effect. However, the effects of each flavonoid on the expression of iNOS mRNA were not consistent with those on COX-2 mRNA. Because the flavonoids inhibit different steps in the production of PGE2 and NO, and their mixture did not show apparent synergistic effects in each step, we conclude that the synergistic effect of the flavonoid mixture reflects the total effect of the flavonoids inhibiting different steps in the NF-κB signalling pathway.

Synthesis and Antigenicity against Human Sera of a Biotin-Labeled Oligosaccharide Portion of a Glycosphingolipid from the Parasite Echinococcus multilocularis.

Synthesis of a biotinylated analog of the carbohydrate portion of a glycosphingolipid from the parasite Echinococcus multilocularis has been achieved. We synthesized ß-D-Galp-(1→6)-ß-D-Galp-(1→6)-[α-L-Fucp-(1→3)]-ß-D-Galp-(1→R: biotin probe) (1) and compared the antigenicity by an enzyme linked immunosorbent assay (ELISA) with biotinylated trisaccharide α-D-Galp-(1→4)-ß-D-Galp-(1→3)-α-D-Galp-(1→R: biotin probe) (F), which has been shown to have significant antigenicity. Both of the oligosaccharides reacted with sera of alveolar echinococcosis (AE) patients, but showed different reactivity. Among the 60 sera of AE patients, more sera reacted with the linear sequence Galα1→4Galß1→3GalNAcα1→R of oligosaccharide (F) than for branched compound 1. Some sera showed high specificity to one of the compound, indicating that the antibodies in the sera of AE patients differ in their specificity to recognize carbohydrate sequences of glycosphingolipids. Our results demonstrate that both of the biotinylated oligosaccharides 1 and F have good serodiagnostic potential and are complementary to detect infections caused by the parasite Echinococcus multilocularis.

Application of a new method, orthogonal projection to latent structure (OPLS) combined with principal component analysis (PCA), to screening of prostaglandin E2 production inhibitory flavonoids in Scutellaria Root.

A new method consisting of orthogonal projection to latent structure (OPLS) and modified principal component analysis (PCA) was applied to a screening of bioactive compounds from natural products. In this report, extracts of 52 Scutellaria Root (the root of Scutellaria baicalensis Georgi) samples were analyzed by high-performance liquid chromatography (HPLC), and their inhibitory activities towards prostaglandin E2 (PGE2) production in a murine macrophage-like cell line J774.1 were examined. Wogonin and oroxylin A were predicted to be strong inhibitors of PGE2 production by OPLS analysis of the data. However, 6-methoxywogonin, which has been reported to have inhibitory activity, was omitted. Modified PCA was then applied to these data as a filter to exclude compounds less relevant to the activity, and OPLS analysis was applied to the modified data. As a result, this method predicted wogonin, oroxylin A and 6-methoxywogonin to be strong inhibitors of PGE2 production without any prior knowledge. The predictions by the OPLS combined with PCA method of PGE2 production inhibitory activities of 52 samples showed good agreement with the actual data. This method is simple and effective and can be used in screening of bioactive natural compounds without any prior knowledge.

Synthesis of a new glycosphingolipid, neurosporaside, from Neurospora crassa.

The glycosphingolipid neurosporaside (α-D-Glcp-(1 → 2)-ß-D-Galp-(1 → 6)-ß-D-Galp-(1 → 6)-ß-D-Galp-(1 →)-Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A) and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluoromethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds (ß-D-Galp-(1 → 6)-ß-D-Galp-(1 → 6)-ß-D-Galp) were formed stereoselectively, leading to the synthesis of glycosphingolipid 2.

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: carbohydrate-protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar.

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate-protein interactions. We investigated whether carbohydrate-lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp-GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

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.

The surface carbohydrates of the Echinococcus granulosus larva interact selectively with the rodent Kupffer cell receptor.

The larvae of the cestodes belonging to the genus Echinococcus dwell primarily in mammalian liver. They are protected by the laminated layer (LL), an acellular mucin-based structure. The glycans decorating these mucins constitute the overwhelming majority of molecules exposed by these larvae to their hosts. However, their decoding by host innate immunity has not been studied. Out of 36 mammalian innate receptors with carbohydrate-binding domains, expressed as Fc fusions, only the mouse Kupffer cell receptor (KCR; CLEC4F) bound significantly to the Echinococcus granulosus LL mucins. The receptor also bound the Echinococcus multilocularis LL. Out of several synthetic glycans representing Echinococcus LL structures, the KCR bound strongly in particular to those ending in Galα1-4Galß1-3 or Galα1-4Galß1-4GlcNAc, both characteristic LL carbohydrate motifs. LL carbohydrates may be optimized to interact with the KCR, expressed only in liver macrophages, cells known to contribute to the tolerogenic antigen presentation that is characteristic of this organ.

Further structural characterization of the Echinococcus granulosus laminated layer carbohydrates: the blood-antigen P1-motif gives rise to branches at different points of the O-glycan chains.

The glycobiology of the cestodes, a class of parasitic flatworms, is still largely unexplored. An important cestode species is Echinococcus granulosus, the tissue-dwelling larval stage of which causes hydatid disease. The E. granulosus larva is protected from the host by a massive mucin-based extracellular matrix termed laminated layer (LL). We previously reported ( Díaz et al. 2009. Biochemistry 48:11678-11691) the molecular structure of the most abundant LL O-glycans, comprising up to six monosaccharide residues. These are based on Cores 1 and 2, in cases elongated by a chain of Galpß1-3 residues, which can be capped by Galpα1-4. In addition, the Core 2 GlcNAcp residue can be decorated with the Galpα1-4Galpß1-4 disaccharide. Larger glycans also detected contained additional HexNAc residues that could not be explained by the structural repertoire described above. In this work, we elucidate, by mass spectrometry (MS) and nuclear magnetic resonance (NMR), six additional glycans from the E. granulosus LL between six and eight residues in size. Their structures are related to those already described but in cases bear GlcNAcpß1-6 or Galpα1-4Galpß1-4GlcNAcpß1-6 as ramifications on the core Galpß1-3 residue. We also obtained evidence that noncore Galpß1-3 residues can be similarly ramified. Thus, the new motif together with the previous information may explain all the glycan compositions detected in the LL by MS. In addition, we show that the anti-Echinococcus monoclonal antibody E492 (Parasite Immunol 21:141, 1999) recognizes Galpα1-4Galpß1-4GlcNAcp (the blood P(1)-antigen motif). This explains the antibody's reactivity with a range of Echinococcus tissues, as the P(1)-motif is also carried on non-LL N-glycans and glycolipids from this genus.

Quantitative analysis of anti-inflammatory activity of orengedokuto: importance of combination of flavonoids in inhibition of PGE2 production in mouse macrophage-like cell line J774.1.

Orengedokuto is a Kampo formula which has been used for removing "heat" and "poison" to treat inflammation, hypertension, gastrointestinal disorders, and liver and cerebrovascular diseases. In this report, we quantitatively analyzed the anti-inflammatory effect of the component crude drugs of orengedokuto and their constituents, using inhibition of prostaglandin E2 (PGE2) production in the murine macrophage-like cell line J774.1. First, we compared PGE2 production inhibitory activities of extracts of combinations of the component crude drugs, which showed that the activity could be ascribed to Scutellaria Root. Next, as baicalin (1), one of the major constituents of Scutellaria Root, did not show any activity, and baicalein (2), the aglycon of 1, showed only weak activity (IC50 92 µM), a hot-water extract of Scutellaria Root was fractionated under the guidance of the activity to give wogonin (3) (IC50 28 µM), 6-methoxywogonin (4) (IC50 7.2 µM) and oroxylin A (5) (IC50 45 µM) from the most active fraction. However, the activities of these compounds at concentrations equivalent to those in the extract were weaker than that of the extract, and none of these compounds alone could explain the activity of the extract. Therefore, we examined the activity of combinations of compounds 2-5. Comparison of all combinations of the four compounds in a ratio which is the same as in the extract revealed that wogonin (3) had an essential role in the activity, and a combination of baicalein (2) and wogonin (3), together with 6-methoxywogonin (4) or oroxylin A (5), was necessary to show activity equivalent to that of the extract.

Sulfatides inhibit adhesion, migration, and invasion of murine melanoma B16F10 cell line in vitro.

Endogenous sulfatide, such as 3-sulfated galactosylceramide (3-sulfatide) has been reported to be involved in neuronal development and regulation of tumor cell metastasis. Recently, a new 6-sulfated glucosylceramide (6-sulfatide) has been isolated from the ascidian, Ciona intestinalis. To determine the antitumor function of the new sulfatide, we examined the effects of synthetic 6-sulfatide and 3-sulfatide on the metastatic features of a murine melanoma cell line, B16F10. Both sulfatides significantly inhibited the adhesion of melanoma cells onto fibronectin-coated tissue plates and, the motility and invasion of the cells, with 6-sulfatide showing stronger inhibitory activities. In addition, both sulfatides inhibited α(5)-, and ß(1)- but not α(v)- or ß(3)-integrin expression. Furthermore, these sulfatides inhibited the activation of focal adhesion kinase, Akt, and extracellular signal-regulated kinase signaling pathways, which are thought to be important for cell migration and invasion. Therefore, these sulfatides may serve as promising drug candidates for the treatment of cancer metastasis.

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.

Synthesis, antigenicity against human sera and structure-activity relationships of carbohydrate moieties from Toxocara larvae and their analogues.

Stereocontrolled syntheses of biotin-labeled oligosaccharide portions containing the Galß1-3GalNAc core of the TES-glycoprotein antigen obtained from larvae of the parasite Toxocara and their analogues have been accomplished. Trisaccharides Fuc2Meα1-2Gal4Meß1-3GalNAcα1-OR (A), Fucα1-2Gal4Meß1-3GalNAcα1-OR (B), Fuc2Meα1-2Galß1-3GalNAcα1-OR (C), Fucα1-2Galß1-3GalNAcα1-OR (D) and a disaccharide Fuc2Meα1-2Gal4Meß1-OR (E) (R = biotinylated probe) were synthesized by block synthesis using 5-(methoxycarbonyl)pentyl-2,3,4,6-tetra-O-acetyl-ß-D-galactopyranosyl-(1-->3)-2-azide-4-O-benzyl-2-deoxy-α-D-galactopyranoside as a common glycosyl acceptor. We examined the antigenicity of these five oligosaccharides by enzyme linked immunosorbent assay (ELISA). Our results demonstrate that the O-methyl groups in these oligosaccharides are important for their antigenicity and the biotinylated oligosaccharides A, B, C and E have high serodiagnostic potential to detect infections caused by Toxocara larvae.