Hirofumi Dohi

Synthesis of an artificial glycoconjugate polymer carrying Pk-antigenic trisaccharide and its potent neutralization activity against Shiga-like toxin.

Fluorescence-labeled glycoconjugate polymers carrying carbohydrate segments of a globotriaosyl ceramide (Gb3) were synthesized and subjected to biological assays using Escherichia coli O-157 strains and Shiga-like toxins (Stx-I and Stx-II). For the fluorescence labeling, a new polymerizable fluorescent monomer with a TBMB carbonyl chromophore (Ex. 325 nm, Em. 410 nm) was designed. A glycosyl monomer of the trisaccharide segment of Gb3 was prepared from p-nitrophenyl beta-lactoside and copolymerized with acrylamide and the fluorescent monomer to prepare a fluorescence-labeled glycoconjugate copolymer carrying [alpha-D-galactopyranosyl-(1-->4)-beta-D-galactopyranosyl]-(1-->4)-beta- D-glucopyranoside. The polymer showed potent neutralization activity against Stx-I and also binding activity onto E. coli O-157 strains.

An easy access to halide ion-catalytic α-glycosylation using carbon tetrabromide and triphenylphosphine as multifunctional reagents

The reaction of a 2-O-benzyl-1-hydroxy sugar with CBr4 and Ph3P generates a glycosyl bromide in situ, which is coupled with an acceptor alcohol in the presence of N,N-tetramethylurea to afford an alpha-glycosyl product virtually quantitatively. In a proposed pathway, the reagent combination plays multiple roles such as the generation of a glycosyl donor, the activation of glycosylation, and the dehydration of the reaction system. These roles allow a simple alpha-glycosylation to be performed without special attention to dehydration. Various alpha-glycosyl (D-gluco-, D-galacto- and L-fuco-) products including glycosyl glycerols and cholesterols have been prepared with this method.

Synthesis of artifical glycoconjugate polymers carrying biologically active trisaccharides with α-d-galactopyranosyl (1→3) and (1→4)-linkage

Abstract Starting from p -nitrophenyl-β-lactoside, two types of neoglycoconjugate carrying either a trisaccharide segment of globotriaosyl ceramide (Gb3) or its regioisomeric Galα1,3Galβ1,4Glc were synthesized via a common intermediate. The former polymer carrying the Gb3 trisaccharide showed strong neutralization activity against Shiga-like toxin-I in the assay using human ACHN cells.

Neutralizing Activity of Polyvalent Gb3, Gb2 and Galacto‐Trehalose Models against Shiga Toxins

Shiga toxin (Stx) is one of the most critical factors in the development of hemolytic uremic syndrome and other systemic complications following enterohemorrhagic Escherichia coli (EHEC) infection. Substances neutralizing Stx by interfering with toxin‐receptor binding have been explored as therapeutic candidates for EHEC infection. In this study, we examined globotriaosyl (Gb3), galabiosyl (Gb2) and galacto‐trehalose, each of which was synthetically conjugated with a polyacrylamide backbone, for Stx‐neutralizing activity. Galacto‐trehalose was designed as a Gb2 mimicking, unnatural Stx‐ligand that was expected to show tolerance to enzymatic degradation in vivo. Galacto‐trehalose copolymer showed neutralizing activity against Stx‐1 but not Stx‐2 in a HeLa cell cytotoxicity assay. It was thought that galacto‐trehalose copolymer could be a lead compound for the treatment of Stx‐mediated diseases, although it requires modification to show neutralizing activity to Stx‐2. The Gb3 copolymer with high sugar unit density showed stronger neutralizing activity against Stx‐2 than those with lower density. However, the density‐dependency of the neutralizing activity was less obvious against Stx‐1. Intravenous administration of the Gb3 copolymer prevented death in mice lethally infected with Stx‐1‐ and Stx‐2‐producing E. coli O157:H7. Thus, we demonstrated that the artificial Gb3 copolymer could neutralize Stx‐1 and the more clinically relevant Stx‐2 in vitro and effectively inhibit Stx toxicity in vivo.

Convenient use of non-malodorous thioglycosyl donors for the assembly of multivalent globo- and isoglobosyl trisaccharides

New thioglycosyl donors (o-methoxycarbonylphenyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside and its 6-O-acetyl analogue) were designed and used for the synthesis of glycoconjugate polymers carrying Gb(3) [Gal(alpha1-->4)Gal(beta1-->4)Glc] and isoGb(3) [Gal(alpha1-->3)Gal(beta1-->4)Glc] clusters as side chains. These donors scarcely evolved the unpleasant odor of thiophenols and showed a high alpha-anomeric selectivity in the galactosylation of p-nitrophenyl beta-lactoside derivatives, although in moderate yields. The derived trisaccharides were converted to multivalent carbohydrate ligands and were subjected to a biological assay with Shiga toxins. The multivalent Gb(3) ligand was highly active in inhibiting the toxicity, while the isoGb(3) ligand showed no activity, indicating that Stx-I discriminates between the carbohydrate structures.

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.

Enzymatic Synthesis of 4-Hydroxyphenyl β-D-Oligoxylosides and Their Notable Tyrosinase Inhibitory Activity

We have purified and characterized an oligoxylosyl transfer enzyme (OxtA) from Bacillus sp. strain KT12. In the present study, a N-terminally His-tagged recombinant form of the enzyme, OxtA(H)E, was overproduced in Escherichia coli and applied to the reaction with xylan and hydroquinone to produce 4-hydroxyphenyl β-D-oligoxylosides, β-(Xyl)n-HQ (n=1–4), by one step reaction. The obtained β-(Xyl)n-HQ inhibited mushroom tyrosinase, which catalyzes the oxidation of L-DOPA to L-DOPA quinine, and the IC50 values of β-Xyl-HQ, β-(Xyl)2-HQ, β-(Xyl)3-HQ, and β-(Xyl)4-HQ were 3.0, 0.74, 0.48, and 0.18 mM respectively. β-(Xyl)4-HQ showed 35-fold more potent inhibitory activity than β-arbutin (4-hydroxyphenyl β-D-glucopyranoside), of which the IC50 value was measured to be 6.3 mM. Kinetic analysis revealed that β-(Xyl)2-HQ, β-(Xyl)3-HQ, and β-(Xyl)4-HQ competitively inhibited the enzyme, and the corresponding K i values were calculated to be 0.20, 0.29, and 0.057 mM respectively.

CONVENIENT ACCESS TO HALIDE ION-CATALYZED α-GLYCOSYLATION FREE FROM NOXIOUS FUMES AT THE DONOR SYNTHESIS

a-Selective glycosylation is a highly challenging and meaningful objective in carbohydrate chemistry. This is mainly because many biologically active oligosaccharides and other glycoconjugates in nature carry an a-glycoside linkage at the non-reducing terminal such as a-L-fucoside in sialyl Lewis antigens and a-D-galactobioside in P antigens. However, a-glycosylation is not straightforward and requires optimization of the glycosyl donors, promoters, solvents, and other reaction conditions. In this respect, a halide ion-catalyzed a-glycosylation method proposed by Lemieux et al. in 1975 has provided one of the few definitive ways. a-Selectivity is nearly perfect for many acceptor sugars so far examined as long as 2-O-benzyl glycosyl bromides are employed as donors. – d] Moreover, the method requires no heavy metal promoters or strong Lewis acid catalysts. These properties are of high significance for large-scale production of ‘‘sugar-based’’ therapeutic agents and biochemical materials. In our study on the structure and immunogenic activity of a-D-glucopyranosyl-snglycerophospholipids (GGPLs) isolated from Mycoplasma fermentans, we applied

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.