Yoshihiro Nishida

A novel sugar-probe biosensor for the deadly plant proteinous toxin, ricin

Because of the illegal use of highly toxic ricin from the castor-oil plant, Ricinus communis, in bioterrorism and suspected white powder cases, anti-terrorism measures for the toxin are urgently required. Here we demonstrate a facile and sensitive detection method using synthetic analogues of beta-lactosyl- and beta-d-galactosyl ceramides as the ligands based on the fact that ricin binds cell-surface oligosaccharides. Sugar-probes having lipoic acids as anchor functions were synthesized via either a chemical or chemoenzymatic way and were immobilized on the sensor chips by a self-assembled monolayer technique. Surface plasmon resonance (SPR) analysis using these carbohydrate probes allowed us to detect the toxin in a highly sensitive and facile manner (10 pg/mL, 5 min), being the best benchmark as a method for detecting the toxin. In addition, a visual monitoring method was developed, in which sugar-coated Au nanoparticles were utilized for discriminating ricin from other proteins in a facile manner, taking 10-30 min for judgment.

Glycochips from polyanionic glycopolymers as tools for detecting shiga toxins

An alternating layer‐by‐layer adsorption methodology was applied to the assembly of glycochips by using synthetic polyanionic glycopolymers. Three glycochips carrying globobioside (Gb2), β‐lactoside (β‐Lac), or α‐D‐mannoside (α‐Man) residues were prepared, and used for the detection of Shiga toxins, Stx‐1 and Stx‐2, by using surface plasmon resonance (SPR). Using this method, we could confirm that both Stx‐1 and Stx‐2 show binding specificity for the Gb2 glycochip as well as a weak affinity for the β‐Lac glycochip. The affinity constants of these toxins depended strongly on the sugar content of the Gb2 polymer used to prepare the glycochip. Greater affinity was observed for chips with a higher sugar content (up to 43 %) in the Gb2 glycopolymer. The maximal affinity constants of Stx‐1 and Stx‐2 (Ka=108–109 M−1) enabled highly sensitive and facile analysis (10 ng mL−1, 30 min). When Gb2 glycopolymers were used as competitors, Stx‐1 and Stx‐2 behaved differently from one another in terms of their SPR response; this allowed us to perform discriminative analysis between the two toxins.

Synthesis and fungicidal activity of novel 2,5-disubstituted-1,3,4-oxadiazole derivatives.

A novel series of 1,3,4-oxadiazole derivatives containing a 5-phenyl-2-furan moiety were synthesized from the intermediates diacylhydrazine 3 and acylhydrazone 5 via an efficient approach under microwave irradiation in good yields. Their structures were characterized by IR, (1)H NMR, and elemental analysis. The antifungal tests indicated that the title compounds showed in vivo fungicidal activity against Botrytis cinerea and Rhizoctonia solanii at 500 μg/mL obviously. Some tested compounds even had a superiority effect over the commercial fungicides 40% Pyrimethanil SC and 3% Validamycin AS. The activity between the title compound and their precursors diacylhydrazine 3 and acylhydrazone 5 was also compared and discussed.

Molecular design, synthesis and bioactivity of glycosyl hydrazine and hydrazone derivatives: notable effects of the sugar moiety.

Assuming that the water solubility of our previous hydrazone derivatives would improve after modification with sugars while keeping or modulating their notable biological activities, we designed and synthesized some glycosyl hydrazine and hydrazone derivatives. Bioassay results indicated that the antitumor activity of our previously prepared hydrazones reduced or disappeared after modification with sugars. On the contrary, some glycosyl derivatives displayed much better antifungal activity against selected fungi. Obviously, a small sugar can change the biological activity of hydrazones significantly.

Synthesis and absolute structures of Mycoplasma pneumoniae β-glyceroglycolipid antigens

Just recently, a pair of beta-glycolipids was isolated from the cell membrane of Mycoplasma pneumoniae as a mixture of the two compounds. They are the major immunodeterminants of this pathogenic Mycoplasma and indicate high medicinal potential. They have a beta-(1-->6)-linked disaccharide structure close to each other; one has beta-d-galactopyranoside (beta-Gal-type 1) at the non-reducing terminal, and another has beta-d-glucopyranoside (beta-Glc-type 2). In the present study, the first stereoselective synthesis was conducted for each of the two beta-glycolipids 1 and 2. (1)H NMR and TLC-immunostaining studies of the synthetic compounds enable us to establish the absolute structures having the beta-(1-->6)-linked disaccharides at the glycerol sn-3 position.

An easy α-glycosylation methodology for the synthesis and stereochemistry of mycoplasma α-glycolipid antigens

Summary Mycoplasma fermentans possesses unique α-glycolipid antigens (GGPL-I and GGPL-III) at the cytoplasm membrane, which carry a phosphocholine group at the sugar primary (6-OH) position. This paper describes a practical synthetic pathway to a GGPL-I homologue (C16:0) and its diastereomer, in which our one-pot α-glycosylation method was effectively applied. The synthetic GGPL-I isomers were characterized with 1H NMR spectroscopy to determine the equilibrium among the three conformers (gg, gt, tg) at the acyclic glycerol moiety. The natural GGPL-I isomer was found to prefer gt (54%) and gg (39%) conformers around the lipid tail, while adopting all of the three conformers with equal probability around the sugar position. This property was very close to what we have observed with respect to the conformation of phosphatidylcholine (DPPC), suggesting that the Mycoplasma glycolipids GGPLs may constitute the cytoplasm fluid membrane together with ubiquitous phospholipids, without inducing stereochemical stress.

Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin.

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase high-performance liquid chromatography. These oligosaccharides are structurally characterized using mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-L-Rha-(1-->, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rha-(1-->, which was identified in a previous study.

Design, Synthesis and Bioactivity of N-Glycosyl-N'-(5-substituted phenyl-2-furoyl) Hydrazide Derivatives

Condensation products of 5-substituted phenyl-2-furoyl hydrazide with different monosaccharides d-glucose, d-galactose,d-mannose, d-fucose and d-arabinose were prepared. The anomerization and cyclic-acyclic isomers were investigated by 1H NMR spectroscopy. The results showed that, except for the d-glucose derivatives, which were in the presence of β-anomeric forms, all derivatives were in an acyclic Schiff base form. Their antifungal and antitumor activities were studied. The bioassay results indicated that some title compounds showed superior effects over the commercial positive controls.

Interaction of 70-kDa heat shock protein with glycosaminoglycans and acidic glycopolymers

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments.

Preparation and evaluation of lactose-modified monoliths for the adsorption and decontamination of plant toxins and lectins

A series of sugar-modified porous silica monoliths with different sugar ligands (β-lactoside, β-N-acetyllactosaminide, β-d-galactoside, β-d-N-acetylgalactosaminide and β-d-glucoside) and linkers were prepared and evaluated using plant toxins and lectins including ricin and a Ricinus communis agglutinin (RCA(120)). Among these sugar monoliths, a lactose monolith carrying a triethylene glycol spacer adsorbed ricin and RCA(120) with the highest efficiency. The monolith showed no binding with albumin, globulin, and lectins from Jack beans, Osage orange, Amur maackia and wheat germ. All these data support the utility of the lactose-modified monolith as a tool for adsorption and decontamination of plant toxins.