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.

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.

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, 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).

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.

Structural characterization of glycosylinositolphospholipids with a blood group type B sugar unit from the edible mushroom, Hypsizygus marmoreus.

Edible fungi, mushrooms, are a popular food in Japan and over 15 cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Manalpha1-2Ins1-[PO(4)]-Cer and extensions of Man, Gal, and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO(4)]-Cer (AGL0), Manalpha1-2Ins1-[PO(4)]-Cer (AGL1), Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL2), Fucalpha1- 2Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL3), Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL4), Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL5), and Galalpha1-2Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (1)H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids. By immunochemical detection, the terminal structure of AGL4, Galalpha1-3(Fucalpha1-2)Galbeta-, was shown to have blood group type B activity. Galalpha1-2 and its repeating sequence in AGL5 and AGL6 are novel structures on the nonreducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.

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.

Synthetic studies on glycosphingolipids from Protostomia phyla: syntheses and biological activities of amphoteric glycolipids containing a phosphocholine residue from the earthworm Pheretima hilgendorfi.

Two types of amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi, PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (1) and PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (2), and their derivatives (4, 5) were synthesized. These were examined for their ability to enhance production of interleukin-8 (IL-8), a potent inflammatory cytokine involved in neutrophil chemotaxis, in a TNFalpha-stimulated granulocytic HL-60 cells. Compounds 1 and 2 were found to be potent enhancers of IL-8 production.

Synthesis of a novel glycosphingolipid from the millipede, Parafontaria laminata armigera, and the assembly of its carbohydrate moiety into multivalent structures.

A novel glycosphingolipid, beta-D-Manp-(1-->4)-[alpha-L-Fucp-(1-->3)]-beta-D-Glcp-(1-->1)-Cer, found in the millipede, Parafontaria laminata armigera, and multivalent derivatives of its carbohydrate moiety were synthesized. As the key step, the target glycolipid (1) was obtained through an inversion reaction at the 2-position of a beta-glucopyranoside residue yielding a beta-mannopyranoside. In addition, the synthesis of fluorescently labeled trimer and tetramer glycoconjugates (2, 3) was achieved by iterative amide bond formation using a monomer unit (24).

Syntheses of new peptidic glycoclusters derived from beta-alanine: di- and trimerized glycoclusters and glycocluster-clusters.

Fluorescence-labeled glycoclusters 1 and 2 have been synthesized to elongate glycocluster units that contain beta-alanine derivative 6 and sugar unit 7. Similarly, di- and trimerized glycoclusters 3 and 4 have been obtained by coupling glycocluster 17 with succinyl chloride and/or trimesoyl chloride, respectively. Furthermore, glycocluster-cluster 5 was also synthesized by a convergent growth approach.

Highly potent anti-HIV-1 activity isolated from fermented Polygonum tinctorium Aiton.

A water-soluble extract of fermented Polygonum tinctorium Aiton (Polygonaceae) called Sukumo, exhibited a potent inhibitory activity against HIV type 1 in vitro. The extract potently suppressed acute HIV-1 (IIIB) infection in MT-4 cells with EC50 values of 0.5 microg/ml but exhibited low cytotoxicity to MT-4 cells even at a high concentration (CC50 > 1000 microg/ml). It also inhibited giant cell formation in co-cultures of HIV-infected cells and uninfected Molt-4 cells. Sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. There was a good correlation between the extract's anti-HIV-1 activity and its inhibitory effects on HIV-1 binding. It also suppressed replication of herpes simplex virus type 1 in Vero cells with an EC 50 of 11.56 microg/ml. On the other hand, there was no appreciable activity against influenza A virus, poliovirus or SARS corona virus when tested at concentrations ranging from 3.2-400 microg/ml as shown by microscopic image analysis for cytopathic effect (CPE). Physico-chemical studies revealed that the anti-HIV activity in the extract was essentially maintained after boiling at 100 degrees C in 1N HCl or 1N NaOH, and after treatment with 100 mM NaIO4. The inhibitory activity of the extract was also not reduced after pronase digestion. The active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000-50,000.

Beta1-adrenergic receptor mediation in the lichen glucan PB-2-induced enhancement of long-term potentiation in the rat dentate gyrus in vivo.

The mechanism underlying the enhancement of long-term potentiation (LTP) induced by the systemic administration of PB-2, an alpha(1-3)(1-4)glucan-containing fraction extracted from the lichen Flavoparmelia baltimorensis and a putative LTP-enhancing agent, was investigated in the rat dentate gyrus in vivo. Particular attention was paid to the role of adrenaline beta-receptors. An intravenous (i.v.) injection of PB-2 enhanced the induction of LTP, which was in turn inhibited by an i.v. injection of the adrenaline beta1-receptor antagonist atenolol. An intracerebroventricular injection of atenolol did not affect the induction of LTP, but completely suppressed the PB-2-induced enhancement of LTP. The infusion of atenolol into the recording site attenuated the PB-2-induced facilitation of LTP. These results suggest that the adrenaline beta1-receptors contribute to the enhancement of LTP induced by the systemic administration of PB-2, and that the functional beta1-receptors are located both centrally and peripherally.

Dual effects of the lichen glucan PB-2, extracted from Flavoparmelia baltimorensis, on the induction of long-term potentiation in the dentate gyrus of the anesthetized rat: possible mediation via adrenaline beta- and interleukin-1 receptors.

We have previously found that oral or intravenous (i.v.) administration of the polysaccharide fraction PB-2, extracted from the lichen Flavoparmelia baltimorensis, facilitated the induction of long-term potentiation (LTP) in the dentate gyrus (DG) in vivo. In this study, the mechanism underlying the effect of PB-2 on the induction of LTP was investigated in the DG of anesthetized rat focusing on the contribution of the interleukin-1 (IL-1) receptor and the adrenaline beta-receptor. An i.v. injection of IL-1ra (10(-9) g/kg), an antagonist of the IL-1 receptor, had no effect on the basal response in the DG; however, this treatment augmented the enhancement of LTP induced by a single i.v. injection of PB-2 (10(-3) g/kg). This potentiating effect was also observed following intracerebroventricular (i.c.v.) injection of IL-1ra (10(-15)-10(-11) g). An i.v. injection of IL-1beta (3.5 x 10(-15)-3.5 x 10(-9) g/kg) inhibited the induction of LTP, which was diminished by the previous application of IL-1ra. These results suggest that the activation of the IL-1 receptor induces the suppression of LTP in PB-2-treated rats, and that endogenous IL-1beta contributes to the IL-1 receptor activation. An i.c.v. infusion of metoprolol (7.5 x 10(-6) g), an antagonist of the adrenaline beta(1)-receptor, attenuated the enhancement of LTP induced by an i.v. injection of PB-2. These results suggest that PB-2 has two different effects on the LTP, an enhancing effect and an inhibiting one, and that it exhibited the significant enhancing effect on the LTP as a total balance of these effects.

Structural elucidation of novel phosphocholine-containing glycosylinositol-phosphoceramides in filamentous fungi and their induction of cell death of cultured rice cells.

Novel ZGLs (zwitterionic glycosphingolipids) have been found in and extracted from the mycelia of filamentous fungi ( Acremonium sp.) isolated from soil. Five ZGLs (ZGL1-ZGL5) were structurally elucidated by sugar compositional analysis, methylation analysis, periodate oxidation, matrix-assisted laser-desorption ionization-time-of-flight MS, (1)H-NMR spectroscopy and fast-atom bombardment MS. Their chemical structures were as follows: GlcN(alpha1-2)Ins1-P-1Cer (ZGL1), Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL2), Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL3), PC-->6Man(alpha1-6)GlcN(alpha1-2)Ins1- P -1Cer (ZGL4), and PC-->6Man(alpha1-6)Man(alpha1-6)GlcN(alpha1-2)Ins1-P-1Cer (ZGL5) (where Cer is ceramide and PC is phosphocholine). In addition, one acidic glycosphingolipid, which was the precursor of ZGLs, was also characterized as inositol-phosphoceramide. The core structure of the ZGLs, GlcN(alpha1-2)Ins1- P, is rather different from those found in other fungi, such as Man(alpha1-2)Ins1- P and Man(alpha1-6)Ins1- P. Interestingly, the terminal mannose residue of ZGL4 and ZGL5 was modified further with a PC group. The presence of PC-containing glycosylinositol-phosphoceramides has not been reported previously in any organism. The ceramide constituents of both ZGLs and acidic glycosphingolipid were essentially the same, and consisted of a 4-hydroxyoctadecasphinganine (phytosphingosine) as the sole sphingoid base and 2-hydroxytetracosanoic acid (>90%) as the major fatty acid. ZGLs were found to cause cell death in suspensions of cultured rice cells. The cell death-inducing activity of ZGLs is probably due to the characteristic glycan moiety of Man(alpha1-6)GlcN, and PC-containing ZGLs had high activity. This study is the first to demonstrate that fungal glycosylinositol-phosphoceramides induce cell death in cultured rice cells.

PB-2, a polysaccharide fraction from lichen Flavoparmelia baltimorensis, peripherally promotes the induction of long-term potentiation in the rat dentate gyrus in vivo.

We have previously found that oral or intravenous administration of PC-2, a polysaccharide fraction purified from extracts of lichen Flavoparmelia caperata, facilitates the induction of long-term potentiation (LTP) in the dentate gyrus of anesthetized rats. PC-2 could be useful in the development of therapeutic drugs for senile dementia. However, it has been very difficult to obtain the material Flavoparmelia caperata because of its scarcity. In the present study, we therefore investigated whether PB-2, a polysaccharide fraction from another lichen Flavoparmelia baltimorensis, has similar biological effects. Oral administration of PB-2 (100-200 mg/kg) did not affect basal evoked potentials, but significantly promoted the induction of LTP following tetanic stimulation (30 pulses at 60 Hz) in the dentate gyrus of anesthetized rats. Intravenous injection of PB-2 (1-5 mg/kg) was also effective in promoting the induction of LTP, but intracerebroventricular injection of PB-2 (1-2 mg/brain) was ineffective. PB-2, as well as PC-2, should be valuable in identifying factors that promote synaptic plasticity in the hippocampus.

Anti-allergic activity of a Kampo (Japanese herbal) medicine "Sho-seiryu-to (Xiao-Qing-Long-Tang)" on airway inflammation in a mouse model.

Effects of a Kampo (Japanese herbal) medicine "Sho-seiryu-to (SST, Xiao-Qing-Long-Tang in Chinese)", which has been used for the treatment of allergic bronchial asthma clinically, were examined on ovalbumin (OVA)-sensitized allergic airway inflammation model (i.e., bronchial asthma) in a mouse. When SST was orally administered at 0.5 g/kg/day from day 1 to 6 days after OVA inhalation, SST reduced the OVA-specific IgE antibody titer in bronchoalveolar lavage (BAL) fluids at 7 days after the OVA inhalation. CD4(+) T cells obtained from the mouse lung produced more interleukin (IL)-4 and IL-5 but less interferon (IFN)-gamma than T cells from nonsensitized control animals. However, oral administration of SST reduced the production of IL-4 and IL-5 and the production of IFN-gamma returned to the control level. In addition, the IL-4 level was increased in the BAL fluid of the OVA-sensitized animals compared to the nonsensitized control, while the IFN-gamma levels decreased. SST reduced the IL-4 levels in the BAL fluids and returned the IFN-gamma level to control levels. Nerve growth factor (NGF) was increased in the BAL fluids of the OVA-sensitized mice over that of nonsensitized mice, but oral administration of SST augmented the NGF levels to approximately 2 times higher than in the sensitized mice. Although lung cells obtained from sensitized mice produced higher levels of NGF than nonsensitized mice, oral administration of SST augmented the production of NGF by the lung cells even higher ( approximately 2 times more than cells from sensitized mice). Administration of anti-NGF antibody to the airway blocked the effects of SST. These results suggest that SST modulates Th1/Th2 balance in the lungs and augmentation of NGF in the lungs may be related to the effects of SST. Pinellic acid (9S, 12S, 13S-trihydroxy-10E-octadecenoic acid), one component of the herbs of SST [Int. Immunopharmacol. 2 (2002) 1183], was purified from the tuber of Pinellia ternata Breitenbach. Oral administration of pinellic acid (50 microg/kg/day) also reduced the OVA-specific IgE antibody titer in BAL fluids from the sensitized mouse. This result suggests that pinellic acid is one of active ingredient(s) in SST.