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A NKT cell repertoire is characterized by the expression of the Valpha19-Jalpha26 invariant TCR alpha chain (Valpha19 NKT cell). This repertoire, as well as a well-established Valpha14-Jalpha281 invariant TCR alpha(+) NKT cell subset (Valpha14 NKT cell), has been suggested to have important roles in the regulation of the immune system and, thus, is a major therapeutic target. Here, we attempted to find specific antigens for Valpha19 NKT cells. Valpha19 as well as Valpha14 NKT cells exhibited reactivity to alpha-galactosyl ceramide (alpha-GalCer). Thus, a series of monoglycosyl ceramides with an axially oriented glycosidic linkage between the sugar and ceramide moiety were synthesized and their antigenicity to Valpha19 NKT cells was determined by measuring their immune responses in culture with glycolipids. Comprehensive examinations revealed substantial antigenic activity for Valpha19 NKT cells by alpha-mannosyl ceramide.

We previously reported the generation of Valpha14 invariant TCR+ (Valpha14i) NK1.1+ natural killer T (NKT) cells in the cytokine-activated suspension culture of murine fetal liver cells. In this study, we attempted to apply this finding to the induction of Valpha14i NKT cell differentiation in the culture of hematopoietic precursors residing in bone marrow or peripheral blood. Preferential generation of NKT cells was found in the culture of Thy-1(+)-depleted bone marrow cells in the presence of culture supernatant from Con A-stimulated spleen T cells and a combination of recombinant IL-3, IL-4, IL-7 and GM-CSF. NKT cell development from peripheral blood hematopoietic precursors was induced when they were cultured on stromal cell monolayers prepared from Thy-1(+)-depleted bone marrow or fetal liver cells, suggesting that certain environments derived from hematopoietic organs are required for the induction of NKT cells from precursors in vitro. A significant fraction of NKT cells generated in the culture were positive for staining with CD1-alpha-galactosylceramide tetramer, indicating that Valpha14i NKT cells were the major subset among the NKT cells. The present methods for obtaining NKT cells in the culture of bone marrow or peripheral blood cells are applicable to the treatment of patients suffering from diseases with numerical and functional disorders of NKT cells.

We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of a native glyco-enzyme. As a representative of this class of enzyme, beta-glucosidase was selected, and the reaction was analyzed. Our EMMA was based on the plug-plug interaction of enzyme and substrate plugs, which is essential to reduce quantities of materials. Furthermore, we have addressed the problem of incompatibility of the enzymatic reaction and separation of the reactants. As a result, EMMA of native glycosidase was achieved with a reaction volume of approximately 20 nL and the Michaelis constant was estimated according to the Lineweaver-Burk plot. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme (ng order) and substrate (pmol order) required for a reaction.

Oligosaccharides, commonly found on the cell surfaces, are deeply involved in a variety of important biological functions, yet demanding difficulties synthesizing such structures limit the investigation of their functions. Technologies to chemically synthesize these oligosaccharides have dramatically advanced during the last two decades mainly due to the introduction of good anomeric leaving groups. In addition, tactical analyses have been addressed to enhance the overall efficiency of oligosaccharide synthesis. Based on the advancement of solution-phase chemistry, solid-phase technologies are being investigated in connection with the current trend of combinatorial chemistry and high throughput screening. This review summarizes the necessary solution-phase methodologies, the status of solid-phase synthesis of oligosaccharides, and combinatorial synthesis of oligosaccharide libraries.

The compound Neu5Ac3alphaF-DSPE (4), in which the C-3 position was modified with an axial fluorine atom, inhibited the catalytic hydrolysis of influenza virus sialidase and the binding activity of hemagglutinin. The inhibitory activities to sialidases were independent of virus isolates examined. With the positive results obtained for inhibition of hemagglutination and hemolysis induced by A/Aichi/2/68 virus, the inhibitory effect of Neu5Ac3alphaF-DSPE (4) against MDCK cells was examined, and it was found that 4 inhibits the viral infection with IC50 value of 5.6 microM based on the cytopathic effects. The experimental results indicate that compound 4 not only inhibits the attachment of virus to the cell surface receptor but also disturbs the release of the progeny viruses from infected cells by inhibiting both hemagglutinin and sialidase of the influenza viruses. The study suggested that the compound is a new class of bifunctional drug candidates for the future chemotherapy of influenza.

Background: Oligosaccharide processing enzymes are important classes of catalysts involved in synthesizing specific oligosaccharide structures on proteins and sphingolipids. Development of specific inhibitors of such enzymes is of current interest as these inhibitors may be used to control cellular functions. Five-membered iminocyclitols have been shown to be potent inhibitors of such enzymes. Since a rational design and synthesis of inhibitors is often extremely difficult due to the limited information regarding the structure of the active site, we carried out a combinatorial library approach. Results: To create diversity, we decided to use an aldehyde group of a protected iminocyclitol for reductive amination and the Strecker reaction. After transformation of the nitrile group introduced by the Strecker reaction into an amine and amide and complete deprotection, a small library of five-membered iminocyclitols consisting of 27 compounds was synthesized. A series of compounds obtained by reductive amination was first screened as potential inhibitors of glycosidases and glycosyltransferases. Among them, compounds carrying a C 10 -alkyl group showed marked enhancement of inhibitory activity against α-mannosidase at 10 μM concentration when compared with its parent compound and deoxymannojirimycin. Furthermore, compounds having the phenylethyl group showed an extremely strong inhibitory effect against α-galactosaminidase at a K i value of 29.4 nM. Compounds with an aminomethyl and amide group at the C -1′ position of these two molecules showed a decrease in inhibitory activities. Conclusions: A combinatorial approach based on five-membered iminocyclitols with a galacto -configuration was exploited. The potential usefulness of the library as a source of inhibitors of glycoenzymes is clearly shown in this study.

In an effort to develop new structures as inhibitors of both influenza virus proteins hemagglutinin and neuraminidase, a series of sialic acid derivatives, including those with one of the hydrogen atoms at the C-3 position replaced by either OH or F, were synthesized. The sialic acid derivative with a 3-eq-OH group was first synthesized by means of a new process and used as the key intermediate for further derivatization at the C-3 position. The stability of these compounds under acid- and sialidase-catalyzed hydrolysis conditions was studied, and the results showed that these compounds exhibit stronger resistance towards both conditions than their parent p-nitrophenyl α-sialoside. Further inhibition assay indicated that the 3-ax-OH or F derivatives 4, 5, and 24, the 4-epimer of 4, are effective specific inhibitors of the sialidases from Clostridium perfringens, among other bacterial sialidases tested. The 3-eq-OH derivative 3, however, showed little inhibition. The same tendency was observed for the inhibition of human influenza sialidases N1 and N2. Compounds 3−5 and sialic acid were then converted into the distealoylphosphatidylethanolamine conjugates. Of these liposome-like compounds, the ones from 4 and 5 showed potent and selective inhibitory activities against the hemagglutinin H3 subtype, but displayed resistance to the influenza virus neuraminidases N1 and N2.

This paper describes the synthesis of 1,1-linked galactosyl mannosides as sialyl Lewis X mimetics that contain a spiro-ring to position the carboxylate group in a well-defined orientation. It was found that compound 4 is more active as a P-selectin inhibitor (IC50 = 19 microM) than the parent disaccharide 2, which contains a flexible carboxyl group (IC50 = 193 microM). This result is consistent with that observed in the previous NMR study of sialyl Lewis X bound to P-selectin. The chemistry described here should be useful for the development of selective inhibitors of E-, P-, and L-selectins.

Just tagging along. For the nondestructive quantitative monitoring of solid-phase oligosaccharide synthesis a

Based on the principle of a multivalent interaction, the amphiphilic polymer 1, present in solution as an aggregate (see below right), is able to inhibit infection with the influenza virus. After recognition of a specific sialyllactose epitope through hemaglutinin (HA) on the virus surface, the sphingosine residues and the fluorescent tag form a stable complex with HA through hydrophobic interactions. Polymer 1 shows in vitro inhibitory activity 106 -fold greater than that of sialyllactose. PGA=polyglutamic acid.

A single reaction of an unprotected β- d -GlcNAc glycoside with tetra -O- acetyl -α- d -galactopyranosyl trichloroacetimidate in dioxane, catalyzed by BF 3 -etherate, was shown to yield all six possible Gal-GlcNAc disaccharides. This result is surprising not only because significant amounts of α-linked disaccharides were formed, despite the presence of a participating group at O-2 of the glycosyl donor, but also because glycosylation of the primary OH-6 is not the dominant reaction. These results suggest ‘random-glycosylation’ to be a valid strategy for the rapid production of oligosaccharide libraries.

Two of the most difficult problems in Glycobiology are how to discover new biologically-active oligosaccharide ligands and how to enhance the binding affinity of such ligands once they are identified. These are important problems since many examples of physiologically important recognition phenomena are being discovered (Varki, 1993) where the ability to intervene with synthetic carbohydrate-based inhibitors may lead to new modes of therapy for disease. Key targets for drug development include inhibitors of glycosyltrans-ferases which could alter cell surface glycosylation (Hindsgaul et al., 1991) and inhibitors of selectin binding to sialylated and sulfated oligosaccharides which would prevent the inflammatory response (Lasky, 1992).

Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. For example, α1,3-fucosylation, the addition of a fucose moiety abundant in Drosophila neural cells, is essential for neural development, function, and behavior. However, it remains largely unknown how neural-specific α1,3-fucosylation is regulated. In the present study, we searched for genes involved in the glycosylation of a neural-specific protein using a Drosophila RNAi library. We obtained 109 genes affecting glycosylation that clustered into nine functional groups. Among them, members of the RNA regulation group were enriched by a secondary screen that identified genes specifically regulating α1,3-fucosylation. Further analyses revealed that an RNA–binding protein, second mitotic wave missing (Swm), upregulates expression of the neural-specific glycosyltransferase FucTA and facilitates its mRNA export from the nucleus. This first large-scale genetic screen for glycosylation-related genes has revealed novel regulation of fucTA mRNA in neural cells., Author Summary Glycosylation plays crucial regulatory roles in various biological processes such as development, immunity, and neural functions. Accordingly, some glycans are generated in a stage- and tissue-specific manner. To address how such distinct glycosylation is regulated in different tissues, we performed a large-scale screen for genes involved in glycosylation of a neural-specific protein. We identified 109 genes, 95 of which are assigned for the first time as directly or indirectly involved in glycosylation. We further found that neural-specific glycosylation is regulated at the RNA level, which is a novel regulatory mechanism of tissue-specific glycosylation.

The potential applications of N-hexyl-4-aminobutyl glycosides in the mass spectrometric investigation of glycan structure and in the investigation of glycan functions were studied. Under collision-induced dissociation (CID) conditions, sodiated glycosides carrying N-hexyl-4-aminobutyl groups effectively produced a hemiacetal species (C-ions), which is important in mass-spectrometry-based structural investigation. The usefulness of N-hexyl-4-aminobutyl glycosides in biological analysis was also confirmed by obtaining a binding constant for the binding of dipyrrometheneboron difluoride C3-labeled N-hexyl-4-aminobutyl beta-lactoside with an Erythrina cristagalli lectin, and by visualizing cellular organelles using a more hydrophobic BODIPY-labeled compound.

A microfluidic chip carrying three reaction chambers was designed and constructed to examine sequential multiple enzymatic reactions. The synthesis of oligosaccharides in living cells is carried out in the Golgi apparatus where multiple enzymes such as glycosidase and glycosyltransferases act on a variety of substrates to generate glycoconjugates that include glycolipids and glycoproteins. The regulatory mechanism of the process however remains unknown. A microchip-based analysis platform may provide a valuable tool with which to address the issue by mimicking the Golgi function. We thus examined 3 sequential glycosyltransfer reactions on a chip, and succeeded in the synthesis of a tetrasaccharide using immobilized enzymes. Also, the kinetic parameters for a recently identified glycosyltransferase, proteoglycan GalT-I, were obtained for the first time.

Several new strategies have been introduced into the realm of synthetic carbohydrate chemistry during the period covered by this review. Regioselective glycosylation of lightly protected glycosyl acceptors has been applied successfully to the synthesis of molecules which are related to cell adhesion. Intramolecular reactions are becoming promising methods of obtaining the β-mannosides found in the core region of N-linked glycoproteins.

The glycosphingolipids isolated from spermatozoa of a fresh-water bivalve, Hyriopsis schlegelii , have a unique structure containing one or two mannosyl residues, novel linkages including an internal fucopyranosyl residue, as well as terminal xylosyl and 4- O -methyl- d -glucopyranosyluronic acid groups. The trisaccharide derivatives that constitute the partial structure of lipid IV were synthesized as follows. Condensation of 4,6-di- O -acetyl-2-azido-2-deoxy-3- O -methyl-α- d -galactopyranosyl bromide with 2-(trimethylsilyl)ethyl 4- O -acetyl-2- O -benzyl-β- l -fucopyranoside, in the presence of mercuric cyanide and mercuric bromide, gave the corresponding disaccharide in 87% yield. Condensation of methyl (2,3-di- O -acetyl-4- O -methyl-α- d -glucopyranosyl bromide)uronate with the appropriate OH-4-free disaccharide derivative afforded the corresponding precursor of 4- O -Me-β- d -Glc p A-(1→4)-[3- O -Me-α- d -Gal p NAc-(1→3)]- l -Fuc, namely 2-(trimethylsilyl)ethyl 2- O -benzyl-3- O -(4,6-di- O -acetyl-2-azido-2-deoxy-3- O -methyl-α- d -galactopyranosyl]-4- O -[methyl (2,3-di- O -acetyl-4- O -methyl-β- d -glucopyranosyl)uronate]-β- l -fucopyranoside.

Methyl 5-Acetamido-4,7,8,9-Tetra-O-Acetyl-2-Chloro-2,3,5-Trideoxy-D-Glycero-β-D-Galacto-2-Nonulopyranosonate (1) Underwent Displacement With Fluorine Ion, To Yield, After 2-S-Acetylation, Methyl 5-Acetamido-4,7,8, 9-Tetra-O-Acetyl-2-S-Acetyl-3,5-Dideoxy-D-Glycero-β-D-Galacto-2-Nonulopyranosonate (4), Which Was Converted, By Selective S-Deacetylation, Into The Corresponding Sodium Salt (6). Condensation Of The α- And β-Sodium Salts (5 And 6) With 2′,3′-Di-O-Acetyl-N 4-Benzoyl-5′-Bromo-5′-Deoxycytidine (9), Derived From N 4-Benzoylcytidine (7) In Two Steps, Gave Their Respective Coupled Products. These Were Converted By O-Deacetylation, N 4-Debenzoylation, And Hydrolysis Of The Methyl Ester Group, Into The Title Compounds.

3-S-α-D-Neuraminyl-(2→3)-D-galactose derivatives were synthesized. As the glycosyl acceptors, 4, 6-O-ethylidene-1,2-O-isopropylidene-3-O-trifluoromethanesulfonyl-α-D-gulopyranose (6) and 1, 2-di-O-acetyl-4,6-O)-isopropylidene-3-O-trifluoromethanesulf onyl-β-D-gulopyranose (13) were prepared from 4,6-O-ethylidene-1,2-O-isopropylidene-a-o-galactopyranose (3) in several steps. Condensation of 6 or 13 with the sodium salt of methyl 5-acetamido-4,7,8, 9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-α-D-galacto-2-nonulopyranosonate (2) gave the corresponding 3-S-(N-acetyl-α-D-neuraminyl)-3-thio-D-galactose derivatives (14 and 15). Compound 15 was converted, via O-deisopropylidenation and subsequent acetylation, into the desired product (17).

Recently, great interest has been focussed on the synthesis of oligosaccharides containing N-acetylneuraminic acid (Neu5Ac) because of its important roles in a variety of biological recognitions.2 However, many difficulties in the synthesis of naturally occurring α-glycosides still remained.3 We have recently reported the stereo-selective synthesis of a series of α- and β-2-thio-neuraminyl glycosides.1-4 In the meantime, the utility of thioglycosides in oligosaccharide synthesis has been widely developed.5 Particularly noteworthy is the dimethyl(methylthio)sulfonium triflate (DMTST) promoted glyco-sylation method5k.1 because excellent yields are achieved due to the high thiophilicity of this reagent.

Glycans, a family of compounds often attached to proteins and ceramides, are diverse molecules involved in a wide range of biological functions. Their structural analysis is necessary and is often carried out at the microscale level. Methods based on mass spectrometry are therefore used, although they do not provide information regarding isomeric structures often found in glycan structures. If one finds "factors" characteristic of a certain isomer, this information can be used to elucidate an unknown oligosaccharide sequence. One potential technique is to use energy-resolved mass spectrometry (ERMS) that has been used to distinguish a pair of isomeric compounds. Thus, compounds in a combinatorial library might be effectively used for this purpose. We analyzed a set of 16 isomeric disaccharides, the structures of which consisted of all possible combinations of anomeric configurations and interglycosidic linkage positions. All of the compounds were distinguished based on ERMS where normal collision-induced dissociation could distinguish only seven compounds. Furthermore, it was shown that alpha-glycosidic linkages of fucose were more reactive than the beta-isomers and the secondary glycosides were more reactive than the primary glycosides.

Glycosylation of proteins can modulate their function in a striking variety of systems, including immune responses, neuronal activities and development. The Drosophila protein, Chaoptin (Chp), is essential for the development and maintenance of photoreceptor cells. This protein is heavily glycosylated, but the possible role of this glycosylation is not well-understood. Here we show that mutations introduced into about 1/3 of 16 potential N-linked glycosylation sites within Chp impaired its cell adhesive activities when expressed in Drosophila S2 cells. Mutation of 2/3 of the glycosylation sites resulted in a marked decrease in Chp protein abundance. These results suggest that N-linked glycosylation of Chp is essential for its stability and activity.

細胞表面に存在する糖タンパク、糖脂質や細胞間マトリックスであるプロテオグリカン等の複合糖質は、複数の糖加水分解酵素、糖転移酵素の連続的な作用により合成される。したがって、各々の糖鎖関連酵素の選択的な阻害物質は糖鎖の機能解析のために重要な役割を担うと考えられる。従来、天然物質として種々のアルカロイドやヌクレオチド類似物質が阻害能を有することが知られている。近年、糖鎖関連酵素の3次元構造の解析、特に糖加水分解酵素の結晶構造に基づき酵素阻害剤のデザイン合成が行われるようになってきた。また、コンビナトリアル合成の方法論を取り込んだ半デザイン合成によるアプローチも注目される。