Inhibition of Alzheimer amyloid aggregation with sulfated glycopolymers.

Glycopolymers carrying sulfated saccharides with modest sugar contents (11% and 28%) were found to suppress the formation of amyloid fibrils by amyloid beta peptides (Abeta(1-42), Abeta(1-40), and Abeta(25-35)), as evaluated by thioflavin T assays and atomic force microscopy observation. Circular dichroism spectra showed that the conformation of amyloid beta peptides depended on the glycopolymer additives, and that the glycopolymer additives reduced the beta-sheet contents. Neutralization activity was confirmed by in vitro assay with HeLa cells. The sulfate group and the appropriate sugar contents were essential for the inhibitory effect.

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 Stxneutralizing 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 galactotrehalose 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.

Sulfatide and its synthetic analogues recognition by Moraxella catarrhalis.

Moraxella catarrhalis is one of the major pathogens of respiratory and middle ear infections. Attachment of this bacterium to the surface of human pharyngeal epithelial cells is the first step in the pathogenesis of infections. This study revealed that sulfatide might act as a binding molecule for the attachment of M. catarrhalis to human pharyngeal epithelial cells. Furthermore, six different synthetic sulfatides were found to inhibit the attachment of M. catarrhalis significantly at an optimum concentration of 10 microg/ml. Synthetic sulfatides may have the potential to be used as a therapy to prevent M. catarrhalis infections.

A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins.

A Gb3-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb3 antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb3 antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb3 antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb3 mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb3Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb3 mimic peptide potentially has great promise for use against Stxs.

Design of multifunctional peptides expressing both antimicrobial activity and shiga toxin neutralization activity.

We have designed novel short peptides expressing both antimicrobial and Shiga-toxin (Stx) neutralization activities by combining nuclear localization signal (NLS) peptides (RIRKKLR, PKKKRKV, and PRRRK) tandemly with globotriaoside (Gb3) mimic peptide (WHWTWL). These fusion peptides exhibited excellent antimicrobial activity against both gram-positive and gram-negative bacteria. A peptide WHWTWLRIRKKLR (Trp-His-Trp-Thr-Trp-Leu-Arg-Ile-Arg-Lys-Lys-Leu-Arg), especially, exhibited about 100 times higher activity than the original NLS peptide. SPR analysis demonstrated that the binding of this peptide to both Stxs was strong: K(d) = 6.6 x 10(-6) to Stx-1 and 6.8 x 10(-6) to Stx-2. The in vitro assay against Stx-1 using HeLa cells showed that this peptide increased the survival rate of HeLa cells against the infection of Stx-1. The peptide has been found to maintain high antimicrobial activity, Stx neutralization activity, and no cytotoxicity at its concentration of 7.8-31.3 microg/mL (4.2-16.7 microM). The present peptide design has a prospect of developing potent multifunctional drugs to destroy proteinaceous toxin-producing bacteria and to simultaneously neutralize the toxins released by bacteriolysis.

On-off switching of gene expression regulated with carbohydrate-lectin interaction.

A novel strategy for artificial regulation system of gene expression applying the specific molecular recognition between carbohydrate and lectin is proposed. Plasmid-lactose conjugates (pActin-lactose and pGFP-lactose) prepared via diazocoupling maintained the transcription activity with T7 RNA polymerase. Gel-shift assay showed that the pActin-lactose conjugates were specifically complexed with galactose-specific lectin RCA(120) with a strong binding affinity (K(a) = 7.6 x 10(5) M(-1) per Lac-unit). The complexes were observed to form aggregates of sub-several micrometer size by means of transmission electron microscopy (TEM) and atomic force microscopy (AFM). The activities of transcription and expression of the conjugates were evaluated, respectively, on the basis of the amount of transcript of pActin and the fluorescent intensity of the expressed GFP. These activities were repressed in the presence of an increasing concentration of RCA120, and then recovered by adding lactose, lactosylceramide-containing liposomes, and lactose-carrying polymers to the conjugate-RCA120 complex. Gel-shift assay and TEM observation revealed that the aggregation form of the complex was relaxed partially in the presence of the lactose derivatives, which increased the accessibility of T7 RNA polymerase to result in the recovery of transcription activity.

One-pot alpha-glycosylation pathway via the generation in situ of alpha-glycopyranosyl imidates in N,N-dimethylformamide.

Divergent pathways are disclosed in the activation of 2-O-benzyl-1-hydroxy sugars by a reagent combination of CBr4 and Ph3P, all of which afford one-pot alpha-glycosylation methods. When this reagent is used in CH2Cl2, the 1-hydroxy sugar is converted to the alpha-glycosyl bromide in a conventional way and leads to the one-pot alpha-glycosylation method based on a halide ion-catalytic mechanism. In either DMF or a mixture of DMF and CHCl3, however, alternative alpha-glycosyl species are generated. From the 1H and 13C NMR study of the products, as well as the reactions using Vilsmeier reagents [(CH3)2N+=CHX]X- (X=Br and Cl), these were identified as cationic alpha-glycopyranosyl imidates having either Br- or Cl- counter ion. The cationic alpha-glycosyl imidate (Br-), derived specifically in the presence of DMF, is more reactive than the alpha-glycosyl bromide and thus is responsible for the accelerated one-pot alpha-glycosylation. The one-pot alpha-glycosylation methodology performed in DMF was assessed also with different types of acceptor substrates including tertiary alcohols and an anomeric mixture of 1-OH sugars.

Effect of the carbohydrate side-chain on the conformation of a glycoconjugate polystyrene in aqueous solution.

An oligomaltose-carrying polystyrene "glycoconjugate polystyrene" was synthesized by the homopolymerization of 4-vinylbenzylamine oligomaltonic amides, derived from maltose, maltotriose, maltopentaose, and maltoheptaose. The resultant amphiphilic glycoconjugate polystyrenes were dissolved in 0.1 M aqueous urea, and their structures characterized by small-angle X-ray scattering and molecular modeling. "Glycoconjugate polystyrene" was found to behave as a "molecular bottle brush", composed of a large pseudo-helical polystyrene backbone and carbohydrate brushes. A large pseudo-helical polystyrene backbone is formed by a random sequence of TT, TG, and/or TTGG. The results indicate that the cross-section of a backbone chain with smaller oligosaccharide side-chains is obliged to expand more than that with longer side-chains. Even with rigid hydrophilic pendant oligosaccharide chains, the larger pseudo-helix of the main chain could orient the side-chains so as to envelop the hydrophobic backbone in aqueous solution. Thus the conformation of the main chain is determined not only by the chemical nature of an oligosaccharide chain but also by its length.

Cooperative lectin recognition of periodical glycoclusters along DNA duplexes: alternate hybridization and full hybridization.

We describe herein the construction of periodically, spatially controlled glycoclusters along DNA duplexes and their cooperative lectin recognition. Site-specifically alpha-mannosylated oligodeoxynucleotide 20-mer (Man-ODN20) was synthesized via the phosphoramidite solid-phase synthesis. Alternate hybridization of the Man-ODN20 with the half-sliding complementary ODN 20-mer (hscODN20) gave an alternately prolonged Man-cluster Man-ODN20/hscODN20. The binding of the Man-cluster to FITC-labeled ConA lectin showed sigmoidal fluorescence dependency on the concentration of Man-ODN, indicating that some mannose residues along the repeating DNA duplex were cooperatively bound to ConA (apparent affinity constant: K(af)=2.4 x 10(4)M(-1) and Hill coefficient: n=3.5). The duplex of Man-ODN20 with full complementary ODN 20-mer (fcODN20) was little bound to ConA. The binding behavior of Man-ODN20/hscODN20 is compared with that of the alternately prolonged Gal-cluster Gal-ODN20/hscODN20 previously reported. Duplexes 20-mer, 40-mer, and 60-mer presenting one, two, and three periodic galactoses were also prepared by full hybridization of 20-mer beta-galactosylated oligodeoxynucleotide (Gal-ODN20) with the periodically repeating full complementary 20-mer, 40-mer, and 60-mer ODNs. RCA(120) lectin was found to little bind the 20-mer and 40-mer duplexes and to bind weakly and non-cooperatively the 60-mer duplex (K(af)=1.1 x 10(4)M(-1)). The cooperative lectin recognition of these glycoclusters in relation with the degree of association (DA) of ODN and the numbers of glycosides along the DNA duplex is discussed.

Preparation and supramolecular properties of unadulterated glycosyl liposomes from a bis(alpha-D-mannopyranosyl)-[60]fullerene conjugate.

The bis(alpha-D-mannopyranosyl)-[60]fullerene conjugate 3 was prepared by thermal coupling of C60 and either 2-azidoethyl 2,3,4,6-tetra-O-acetyl- or 2,3;4,6-di-O-isopropylidene-alpha-D-mannopyranoside (Scheme). Compound 3 was found to readily self-assemble. Dynamic-light-scattering (DLS) and atomic-force microscopy (AFM) experiments supported that the amphiphilic compound gives rise to nano-sized supramolecular structures during sugar deprotection (Ac-group removal) performed in MeOH/CH2Cl2 solution. Encapsulation studies with an aqueous suspension of 3 showed that the self-assembling structure envelopes Ba2+ and the fluorescent dye Acridine Red during its formation, which indicates that it resembles a bilayer vesicle or an unadulterated liposome with an inner hollow space. In addition to this notable property, the unique molecular geometry of the spatially arranged mannosyl surface residues of 3 gives rise to strong binding of the carbohydrate-recognizing lectin Con A. Hence, the polar amphiphilic end of 3 mimics the structure of 3,6-branched tri-alpha-D-mannoside (6; Fig. 3), a natural ligand of the Con A protein.

Analysis of GM3-Gg3 interaction using clustered gycoconjugate models constructed from glycolipid monolayers and artificial glycoconjugate polymers.

Carbohydrate-carbohydrate interactions between clustered GM3 on the Langmuir monolayer and clustered Gg3 trisaccharide along a polystyrene chain were investigated using surface pressure-area (pi-A) isotherms and surface plasmon resonance (SPR). The pi-A isotherm of the GM3 monolayer was expanded substantially and specifically by the Gg3-trisaccharide-bearing glycoconjugate polystyrene [PN(Gg3)] even at 10(-12) M. The PN(Gg3)-induced expansion of the GM3 monolayer required no calcium ion, and the expansion was strongly inhibited in the presence of urea and acetamido sugars. SPR studies of the GM3-Gg3 interaction were carried out to estimate the affinity constant and specificity of the interaction quantitatively. PN(Gg3) was adsorbed onto the GM3 monolayer strongly and specifically with an apparent affinity constant of Ka = 2.5 x 10(6) M(-1). The mechanism of the GM3-Gg3 interaction was discussed on the basis of the relationship between affinity and structure. We found that the NHAc groups of N -acetylneuraminic acid in GM3 and of GalNAc in Gg3 play an important role in the GM3-Gg3 interaction and that PN(Gg3) recognizes not only some specified portions of GM3 but also the trisaccharide as a whole. Published in 2003.

Micropatterned carbohydrate displays by self-assembly of glycoconjugate polymers on hydrophobic templates on silicon.

We report a novel strategy for micropatterned carbohydrate displays on Si substrates. This method exploited the hydrophobic-hydrophilic microfabrication by photolithography of ODS-SAM on Si substrates and the subsequent selective self-assembly of glycoconjugate polymers onto the hydrophobic regions. Protein micropatterning by molecular recognition on the carbohydrate substrates was also successful.

Peptides binding to a Gb3 mimic selected from a phage library.

Peptides binding to a Gb3 mimic were selected from 12-mer peptide library. The self-assembled monolayer (SAM) of a Gb3 mimic was formed on the gold surface, and biopanning was carried out with the phage display peptide library. After three rounds of biopanning, four individual sequences were obtained from 10 phage clones, and the selected peptides having the specific 7-mer sequence (FHENWPS) showed affinities to the Gb3 mimic as strong as to RCA120. Molecular dynamics calculations suggested that the peptides bound to the Gb3 mimic by hydrophobic interaction and hydrogen bonding formation, and the cooperative interactions played an important role in the recognition. The Stx-1 binding was inhibited by the peptides.

Sulfatase-catalyzed assembly of regioselectively O-sulfonated p-nitrophenyl alpha-D-gluco- and alpha-D-mannopyranosides.

A chemoenzymic methodology is extended to the library synthesis of regioselectively O-sulfonated pNP D-gluco and D-mannopyranosides. The method involves the sequential reactions of chemical O-sulfonation and sulfatase-catalyzed O-desulfonation. pNP 2,6-di-O-sulfo-alpha-D-glucopyranoside and pNP 3,6-di-O-sulfo-alpha-D-mannopyranoside were obtained as sodium salts using chemical methods by way of dibutylstannylene acetals or tributylstannyl ethers. They were then applied to enzyme reactions using three molluscan enzymes (snail, limpet, and abalone). The sulfatase reactions cleaved a sulfate group at the secondary O-2 or O-3 position to yield the corresponding pNP 6-O-sulfo sugars. Neither pNP 6-O-sulfo-alpha-D-glucopyranoside nor 6-O-sulfo-alpha-D-mannopyranoside became the enzyme substrate. Evidently, the molluscan sulfatases have a tendency to cleave the secondary O-sulfo group with assistance from the 6-O-sulfo group.

Surface plasmon resonance study of carbohydrate-carbohydrate interaction between various gangliosides and Gg3-carrying polystyrene.

Carbohydrate-carbohydrate interactions between Gg3 trisaccharide-carrying polystyrene (PN(Gg3)) and monolayers of several glycosphingolipids (GSLs) were quantitatively investigated by surface plasmon resonance techniques. PN(Gg3) was adsorbed onto a GM3 monolayer strongly and specifically with an apparent affinity constant of K(a) = 2.5 x 10(6) M(-1), and the apparent affinity constants onto GSLs decreased in the following order: GM3 > LacCer > (KDN)GM3 approximately GlcCer > GM2 approximately GD3 approximately GM4 > GM1 approximately 2,6-isoGM3 > ceramide. These results suggest that PN(Gg3) recognizes not only some specified portions of GM3 but also the trisaccharide as a whole. On the other hand, PN(Lac) and PN(Cel) were bound to GSLs less strongly (K(a) approximately 10(4) M(-1)) and less selectively. The kinetic analysis revealed that the selectivity in the adsorption of PN(Gg3) onto the GM3 monolayer is dominated by the faster adsorption rate.

Design of N-acetyl-6-sulfo-beta-d-glucosaminide-based inhibitors of influenza virus sialidase.

Biological activity of N-acetyl-6-sulfo-beta-d-glucosaminides (6-sulfo-GlcNAc 1) having a structural homology to N-acetylneuraminic acid (Neu5Ac 2) and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en 3) was examined in terms of inhibitory activity against influenza virus sialidase (influenza, A/Memphis/1/71 H3N2). pNP 6-Sulfo-GlcNAc 1a was proved to show substantial activity to inhibit the virus sialidase (IC(50)=2.8 mM), though p-nitrophenyl (pNP) GlcNAc without 6-sulfo group and pNP 6-sulfo-GlcNH(3)(+) 1b without 2-NHAc showed little activity (IC(50) >50 mM). The activity was enhanced nearly 100-fold when the pNP group of 1a was converted to p-acetamidophenyl one 5 (IC(50)=30 microM) or replaced with 1-naphthyl 6 (IC(50)=10 microM) or n-propyl one 8 (IC(50)=11 microM).

Self-organized glycoclusters along DNA: effect of the spatial arrangement of galactoside residues on cooperative lectin recognition.

We describe herein the relationship between the spatial arrangement of self-organized galactose clusters and lectin recognition. beta-Galactose-modified deoxyuridine phosphoramidite was synthesized and applied to solid-phase synthesis to provide 18-, 20-, and 22-mers of site-specifically galactosylated oligodeoxynucleotides (Gal-ODNs). These Gal-ODNs were self-organized through hybridization with the corresponding 18-, 20-, and 22-mers of half-sliding complementary ODNs (hsc-ODNs) to give periodic galactoside clusters. The self-organization of ODNs was confirmed by size exclusion chromatography and gel electrophoresis. The binding of the Gal-clusters to the FITC-labeled RCA(120) lectin was analyzed by monitoring the change in fluorescence intensity. The assembly of 20-mer Gal-ODN with the 20-mer hsc-ODN was strongly and cooperatively recognized by the lectin. The 18-mer assembly was bound more weakly and less cooperatively, and the 22-mer assembly was minimally bound to the lectin. RCA(120) lectin recognized not only the density of galactoside residues, but also the spatial arrangement. The size of the Gal cluster was estimated from the association constant of Gal-ODN with hsc-ODN. The relationship between lectin-recognition and Gal-cluster size is also discussed.

Stereo- and biochemical profiles of the 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerenes.

The 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerene were studied by means of circular dichroism (CD), deuterium labeling, 1H-NMR, molecular-dynamics (MD) calculations, and a lectin-binding assay. The CD spectra of the O-acetylated derivatives allowed clear discrimination of the isomers, while the 1H-NMR spectra, with assistance from deuterium labeling and MD calculations, served to disclose the unique conformation and molecular geometry of each acetylated isomer in chloroform solution. The deprotected 5-6- and 6-6-isomers, which gave colloidal suspensions in aqueous mixtures, displayed marked activity in blocking lectin-induced hemagglutination by concanavalin A.

N-Acetyl-6-sulfo-D-glucosamine as a promising mimic of N-Acetyl neuraminic acid.

6-Sulfo-D-GlcNAc with a molecular geometry close to that of N-acetylneuraminic acid (Neu5Ac) was hypothesized to serve as a simple Neu5Ac mimic possessing high potential in biochemical and medicinal applications. The hypothesis was evidenced with a neuraminidase inhibition assay using p-nitrophenyl (pNP) 3-, 4-, and 6-sulfo-beta-D-GlcNAc (4, 5 and 2a) and 6-sulfo-beta-D-Glc 6, in which only pNP 6-sulfo-beta-D-GlcNAc 2a was found to show substantial activity.