Synthetic construction of sugar-amino acid hybrid polymers involving globotriaose or lactose and evaluation of their biological activities against Shiga toxins produced by Escherichia coli O157:H7.

Synthetic assembly of sugar moieties and amino acids in order to create "sugar-amino acid hybrid polymers" was accomplished by means of simple radical polymerization of carbohydrate monomers having an amino acid-modified polymerizable aglycon. Amines derived from globotriaoside and lactoside as glycoepitopes were condensed with known carbobenzyloxy derivatives, including Z-Gly, Z-l-Ala and Z-ß-Ala, which had appropriate spacer ability and a chiral center to afford fully protected sugar-amino acid hybrid compounds in good yields. After deprotection followed by acryloylation, the water-soluble glycomonomers were polymerized with or without acrylamide in the presence of a radical initiator in water to give corresponding copolymers and homopolymers, which were shown by SEC analysis to have high molecular weights. Evaluation of the biological activities of the glycopolymers against Shiga toxins (Stxs) was carried out, and the results suggested that glycopolymers having highly clustered globotriaosyl residues had high affinity against Stx2 (KD = 2.7∼4.0 µM) even though other glycopolymers did not show any affinity or showed very weak binding affinity. When Stx1 was used for the same assay, all of the glycopolymers having globotriaosyl residues showed high affinity (KD = 0.30∼1.74 µM). Interestingly, couple of glycopolymers having lactosyl moieties had weaker binding affinity against Stx1. In addition, when cytotoxicity assays were carried out for both Stxs, glycopolymers having highly clustered globotriaosyl residues showed higher affinity than that of the copolymers, and only highly clustered-type glycopolymers displayed neutralization potency against Stx2.

Carbosilane glycodendrimers.

Glycodendrimers fascinate both carbohydrate chemists and biologists because of their ability to recognize lectins and enhance carbohydrate-protein interactions. These characteristics make glycodendrimers a valuable tool in glycoscience and chemical biology. Many glycodendrimers have been described to date; this tutorial review focuses specifically on carbosilane glycodendrimers. We present methodologies for synthesizing parent carbosilane dendrimers and describe their use in biological assays. We also describe representative functionalizations of parent carbosilane dendrimers at terminal positions which are necessary for chemical ligation with carbohydrate ligands. This is followed by a description of all coupling reactions between carbohydrate and carbosilane dendrimer functionalities used in the synthesis of carbosilane glycodendrimers. The major emphasis of this review is the use of carbosilane glycodendrimers as medical agents against Shiga toxins, dengue viruses, relapsing fever Borrelia, and hemagglutinin and neuraminidase of influenza viruses, as well as on the relationship between dendrimer structure and these biological activities. The last two sections introduce recent attempts to use carbosilane glycodendrimers as new versatile and widely-applicable lectin sensors, and the use of carbosilane glycodendrimers as a novel drug carrier in an active targeting drug delivery system. This review article will be of interest to scientists in the areas of organic chemistry, chemical biology, carbohydrate chemistry, heteroatom chemistry, and organosilicon chemistry.

A carbosilane dendrimer and a silacyclopentadiene analog carrying peripheral lactoses as drug-delivery systems.

A carbosilane dendrimer (4a) and its silacyclopentadiene analog (4b), both functionalized with lactoses, were tested for their abilities to act as drug-delivery systems. The critical micelle concentrations of 4a and 4b were measured using the drop-volume method in water and were 1.7 and 2.9 µM, respectively, suggesting that they could act as aggregates of glycoclusters. The amounts of the hydrophobic dye Orange OT loaded onto aqueous micelles of 4a and 4b and the stabilities of the dye/micelle complexes were determined by extracting the dyes from the complexes into chloroform. The particle sizes were measured for the loaded micelles by dynamic light scattering. Transfer of the dye from the micelles to peanut agglutinin was observed by fluorescence microscopy. Given the abilities of micelles of 4a and 4b to bind and release Orange OT, these glycocluster micelles may find use as drug-delivery systems.

Synthesis of sialyllactosamine clusters using carbosilane as core scaffolds by means of chemical and enzymatic approaches.

An efficient synthesis of sialyllactosamine (SiaLacNAc) clusters using carbosilanes as core scaffolds has been accomplished by means of chemical and enzymatic approaches. N-Acetyl-D-glucosamine (GlcNAc) clusters having O-glycosidic linkage or S-glycosidic linkage were chemically synthesized from known intermediates in high yields. The GlcNAc clusters were first used as substrates for beta1,4 galactosyl transferase using UDP-galactose (UDP-Gal) as a sugar source to provide corresponding N-acetyllactosamine clusters. Further sugar elongation of the LacNAc clusters was demonstrated using alpha2,3 sialyl transferase and CMP-neuraminic acid (CMP-NANA) to yield the corresponding SiaLacNAc clusters.

Simple and conveniently accessible bi-fluorescence-labeled substrates for amylases.

Synthesis of bi-fluorescence-labeled maltooligosaccharides for amylase assay was accomplished. Preliminary biological evaluation of both bi-fluorescence-labeled maltohexasaccharide and maltose using alpha-amylase was carried out, and the hexaosyl derivative showed unique variation on the basis of fluorescence resonance energy transfer (FRET).

Relapsing fever Borrelia binds to neolacto glycans and mediates rosetting of human erythrocytes.

A hallmark of acute relapsing fever borreliosis is severe bacteremia. Some Borrelia species, such as B. duttonii and B. crocidurae, associate with erythrocytes and induce aggregation recognized as erythrocyte rosetting. Erythrocyte rosettes contribute to disease severity by increased tissue invasiveness (such as invasion of CNS and encephalitis), hemorrhaging, and reduced blood flow in affected microcapillaries. Here we report that relapsing fever Borrelia binds to neolacto (Galbeta4GlcNAcbeta3Galbeta4Glcbeta1)-carrying glycoconjugates that are present on human erythrocytes. This interaction is of low affinity but is compensated for by the multivalency of neo-lacto-oligosaccharides on the erythrocyte cell surface. Hence, the protein-carbohydrate interaction is dependent on multivalent neolacto-glycans to mediate binding.

Synthesis of sialic acid derivatives having a C=C double bond substituted at the C-5 position and their glycopolymers.

Glycomonomers of sialic acid in which the acetamide group at C-5 was converted into two kinds of C=C double bond substituents were prepared and the fully protected glycomonomers were directly polymerized before deprotection steps. Radical polymerization with acrylamide in DMF in the presence of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine proceeded smoothly and gave corresponding sialopolymers. Interestingly glycomonomers had hemagglutination inhibitory activities not only for H1N1 but also for H3N2 of human influenza virus strains.

Systematic syntheses of influenza neuraminidase inhibitors: a series of carbosilane dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties.

In order to develop novel influenza sialidase inhibitors, we constructed a library of glycoclusters composed of twelve types of sialylated dendrimers with thioglycosidic linkage that are resistant to hydrolysis by the sialidases. These sialodendrimers were synthesized by condensation reaction between a thiosialoside modified on the aglycon terminal end by a thioacetyl group and twelve types of carbosilane dendrimers having brominated terminal ends under deacetylation conditions, and temporal re-protection was performed for purification. Removal of all protection of the glycodendrimers was accomplished by transesterification and subsequent saponification to provide corresponding water-soluble glycodendrimers in good yields. For investigation of the structure-activity relationship, dendrimer scaffolds having differences in number of the sugar moieties, such as 3-, 4-, 6- and 12-functionalized dendrimers, and in linkage patterns, such as normal aliphatic linkage, ether- and amide-linkages. Biological evaluations of these glycodendrimers showed that all of the ether- and amide-elongated compounds had inhibitory potencies for the influenza sialidases in the mM range, while compounds having normal aliphatic linkage did not have any activities except for a 12-functionalized compound.

Syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl alpha(2-->3) lactose moieties as inhibitors for human influenza viruses.

A series of carbosilane dendrimers uniformly functionalized with sialyl lactose moieties (Neu5Ac alpha2-->3Gal beta1-->4Glc) was systematically synthesized, and biological evaluations for anti-influenza virus activity using the glycodendrimers were performed. The results suggested that the glycodendrimers had unique biological activities depending on the form of their core frame, and Dumbbell(1)6-amide type glycodendrimer 7 showed particularly strong inhibitory activities against human influenza viruses [A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2)]. The results suggested that the structure-activity relationship (SAR) on the glycolibrary against various influenza viruses was observed, and dumbbell-shaped dendrimers as supporting carbohydrate moieties were found to be the most suitable core scaffolds in this study.

Synthesis and characterization of photo-responsive carbosilane dendrimers.

Preparation of photo-responsive carbosilane dendrimers bearing 4-phenylazo-benzonitrile units on their molecular surface has been accomplished, and their both photo and thermal behaviors have also been characterized. These functional dendrimers suggest that the apparent molecular sizes of the cis-isomers are smaller than those of the corresponding trans-isomers, since the molecular diameter of these dendrimers would be shorter on the basis of trans-->cis photo-isomerization of azobenzene.

Sialyl alpha(2-->3) lactose clusters using carbosilane dendrimer core scaffolds as influenza hemagglutinin blockers.

An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with sialyl alpha(2-->3) lactose (Neu5Acalpha(2-->3)Galbeta(1-->4)Glcbeta1-->) moieties was accomplished. The results of a preliminary study on biological responses against influenza virus hemagglutinin, using the sialyl lactose clusters showed unique biological activities on the basis of the structure-activity relationship according to the carbosilane scaffolds.

Use of a recycle-type SEC method as a powerful tool for purification of thiosialoside derivatives.

An efficient separation between fully acetylated thiosialoside methyl esters and fully acetylated Neu5Ac2en methyl esters was accomplished by means of a size-exclusion chromatography (SEC) method. Purity determinations and structural elucidation of the isolated compounds were performed by a combination of elemental analyses and spectroscopic analyses, including IR, (1)H, and (13)C NMR, and mass spectroscopic analyses.

Novel linear polymers bearing thiosialosides as pendant-type epitopes for influenza neuraminidase inhibitors.

A conventional synthesis of alpha-thioglycoside of sialic acid as a glycomonomer was accomplished. Radical copolymerization of the glycomonomer with vinyl acetate proceeded smoothly to afford a new class of glycopolymers having thiosialoside residues, in which all protection was removed by a combination of transesterification and saponification to provide a water-soluble thiosialoside cluster. The results of a preliminary study on biological responses against influenza virus neuraminidases using the thiosialoside polymer as a candidate for a neuraminidase inhibitor showed that the glycopolymer has potent inhibitory activity against the neuraminidases.

Lactotriaose-containing carbosilane dendrimers: syntheses and lectin-binding activities.

Carbosilane dendrimers periphery-functionalized with lactotriaose (GlcNAcbeta1-3Galbeta1-4Glc) with valencies of three, four, six, and twelve were prepared for use in a lectin-binding assay. A lactotriaose derivative was prepared from D-glucosamine and D-lactose derivatives. The N-Troc-protected glucosamine glycosyl donor and 3'-O-unprotected lactose glycosyl acceptor were condensed in the presence of silver trifluoromethanesulfonate and methylsulfenyl bromide to provide corresponding trisaccharide with new beta-1-3 linkages in 92% yield. The protection group of the trisaccharide was transformed into an acetyl group. The 4-pentenyl glycoside was prepared from the acetate via glycosyl bromide. The alkene was converted into acetyl sulfide by addition of thioacetic acid under radical conditions. The lactotriaose unit was linked with carbosilane dendrimers to afford acetyl-protected glycodendrimers. De-O-acetylation of the dendrimers was carried out in the presence of sodium methoxide and then aq NaOH to give the desired lactotriaose clusters using a carbosilane dendrimer backbone. Their biological activities toward WGA were evaluated by fluorescence methods. The binding constants of free lactotriaose and trivalent, tetravalent, hexavalent, and dodecavalent glycodendrimers to WGA were determined to be 1.1x10(3), 4.4x10(4), 5.1x10(4), 2.8x10(6), and 1.3x10(6) M-1, respectively. The hexavalent glycodendrimer showed a 2500-fold larger binding effect than that of free lactotriaose.

Thiosialoside clusters using carbosilane dendrimer core scaffolds as a new class of influenza neuraminidase inhibitors.

An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with alpha-thioglycoside of sialic acid was accomplished. The results of a preliminary study on biological responses against influenza virus sialidases using thiosialoside clusters showed that some of the glycodendrimers have inhibitory potencies against the sialidases.

Carbosilane dendrimers bearing globotriaoses: syntheses of globotrioasyl derivative and introduction into carbosilane dendrimers.

As an application of a one-pot reaction involving Birch reduction and subsequent S(N)2 reaction in liquid ammonia, synthetic assembly of trisaccharidic moieties of globotriaosyl ceramide onto carbosilane dendrimers was accomplished using tris(3-bromopropyl)phenylsilane and tris[tris(3-bromopropyl)silylpropyl]phenylsilane as the core scaffolds. The common globotriaosyl derivative having benzylsulfide functionality at the terminal of the aglycon was efficiently prepared from d-galactose and d-lactose as starting materials. The glycosyl donor derived from galactose and the glycosyl acceptor derived from lactose were condensed in the presence of silver triflate as the best promoter to provide corresponding trisaccharide with newly formed alpha-1-4 linkages in 90% yield. Fully benzylated protection of the trisaccharide was deprotected under the Birch reduction condition followed by acetylation to give an acetate in which alkene was converted into benzyl sulfide by radical addition of alpha-toluenethiol in high yields. On the other hand, carbosilane dendrimers were prepared from appropriate chlorosilanes as starting materials by a combination of hydrosylation followed by alkenylation. The terminal C=C double bonds of the carbosilanes were converted into corresponding alcohols by means of the usual hydroboration reaction, and the alcohols underwent further chemical manipulation to give carbosilane dendrimers with peripheral bromine atoms.

Carbosilane dendrimers bearing globotriaoses: construction of a series of carbosilane dendrimers bearing globotriaoses.

To enhance biological activities on the basis of the sugar cluster effect, a series of carbosilane dendrimers as core scaffolds for the construction of glycodendrimers was systematically synthesized from appropriate chlorosilanes by a combination of alkenylation and hydrosylation reactions. Those carbosilane dendrimers having terminal C=C double bonds underwent general hydroboration reactions to give corresponding primary polyols. Further transformations of the alcohols were then performed by mesylation followed by a displacement with NaBr to provide corresponding dendrimers with 4 to 36 bromine atoms at each terminal end. Assembly of trisaccharide moieties of globotriaosyl ceramide using alkyl halide-type carbosilane dendrimers as the core frame was conducted in liquid ammonia by a one-pot reaction involving selective removal of a benzyl group under the Birch reduction condition and subsequent S(N)2 reaction to yield a series of carbosilane dendrimers having appropriate numbers of trisaccharide moieties. These dendrimers have unique shapes and adequate numbers of terminal trisaccharide moieties. Some of the dendrimers showed unique biological activity against Stxs, which were produced by pathogenic Escherichia coli O157:H7.

Identification and characterization of carbohydrate molecules in mammalian cells recognized by dengue virus type 2.

The interaction between cell surface receptors and the envelope glycoprotein (EGP) on the viral membrane surface is the initial step of Dengue virus infection. To understand the host range, tissue tropism, and virulence of this pathogen, it is critical to elucidate the molecular mechanisms of the interaction of EGP with receptor molecules. Here, using a TLC/virus-binding assay, we isolated and characterized a carbohydrate molecule on mammalian cell surfaces that is recognized by dengue virus type 2 (DEN2). Structural determination by immunochemical methods showed that the carbohydrate structure of the purified glycosphingolipid was neolactotetraosylceramide (nLc4Cer). This glycosphingolipid was expressed on the cell surface of susceptible cells, such as human erythroleukemia K562 and baby hamster kidney BHK-21. All serotypes of DEN viruses, DEN1 to DEN4, reacted with nLc4Cer, and the non-reducing terminal disaccharide residue Galbeta1-4GlcNAcbeta1- was found to be a critical determinant for the binding of DEN2. Chemically synthesized derivatives carrying multiple carbohydrate residues of nLc4, but not nLc4 oligosaccharide, inhibited DEN2 infection of BHK-21 cells. These findings strongly suggested that multivalent nLc4 oligosaccharide could act as a competitive inhibitor against the binding of DEN2 to the host cells.

Structural analysis of the interaction between Shiga toxin B subunits and linear polymers bearing clustered globotriose residues.

We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.

Syntheses of a series of lacto-N-neotetraose clusters using a carbosilane dendrimer scaffold.

4-Pentenyl (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-(3,6-di-O-acetyl-2-deoxy-2-phthalimido-beta-d-glucopyranosyl)-(1-->3)-(2,6-di-O-benzoyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzoyl-beta-d-glucopyranoside (4) was synthesized by regioselective glycosylation of 4-pentenyl (2,6,-di-O-benzoyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzoyl-beta-d-glucopyranoside and (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-3,6-di-O-acetyl-2-deoxy-2-phthalimido-beta-d-glucopyranosyl chloride. By conversion of the protecting groups followed by thioacetylation, 4 was transformed into the corresponding lacto-N-neotetraose derivative, 5-(acetylthio)pentenyl (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-O-(3,6-di-O-acetyl-2-acetamido-2-deoxy-beta-d-glucopyranosyl)-(1-->3)-(2,4,6-di-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-acetyl-beta-d-glucopyranoside (6). The lacto-N-neotetraose derivative 6 was introduced into carbosilane dendrimer cores of three shapes, and three kinds of new carbosilane dendrimers peripherally functionalized by lacto-N-neotetraose were obtained.