Synthetic assembly of α-O-linked-type GlcNAc using polymer chemistry affords sugar clusters, which effectively bind to lectins.

Fischer's glycoside synthesis was applied to linker precursor alcohols of two different lengths having appropriate alkane chains to obtain the corresponding α-glycoside and it was found to be applicable with moderate yields. Water-soluble glycomonomers were systematically prepared from N-acetyl-d-glucosamine (GlcNAc) by introducing two kinds of alcohols having different methylene lengths. Typical radical polymerizations of the glycomonomers with acrylamide as a modulator for control of the distance between carbohydrate residues in water in the presence of ammonium persulfate (APS)-N,N,N',N'-tetramethylethylenediamine (TEMED) gave a series of glycopolymers with various α-glycoside-type GlcNAc residue densities. Fluorometric analysis of the interaction of wheat germ agglutinin (WGA) with the glycopolymers was performed and the results showed unique binding specificities based on structural differences.

Synthetic assembly of a series of glycopolymers having sialyl α2-3 lactose moieties connected with longer spacer arms.

Glycopolymers having sialyl α2-3 lactose moieties via longer spacer arms were systematically prepared from the corresponding glycomonomers. Radical polymerization of glycomonomers gave a series of glycopolymers displaying various sugar densities. Fluorometric analyses of wheat germ agglutinin (WGA) against the glycopolymers were conducted and the results showed unique binding specificities on the basis of sugar densities.

Proteolytic polymer: polyacrylamides functionalized with amino acids cleave bovine and human serum albumins.

Polyacrylamides with various compositions of serine, aspartic acid, and histidine, which are the amino acids involved in the catalytic triad of natural serine protease chymotrypsin, were synthesized and their protein cleavage activity was investigated. SDS-PAGE analysis showed that some of the synthesized ternary copolymers showed cleavage activity against bovine and human serum albumins. Polyacrylamides incorporating a single type of amino acid were also able to cleave the protein substrates. These homopolymers exhibited unique cleavage profiles and pH and temperature sensitivities that differed from those of α-chymotrypsin. The results indicate the potential of polymers functionalized with amino acids as proteolytic artificial enzymes.

Dendritic maleimide-thiol adducts carrying pendant glycosides as high-affinity ligands.

We synthesized N-acetylglucosamine-terminated hexavalent carbosilane dendrimers and investigated their binding to wheat germ agglutinin (WGA). The glycodendrimers were prepared by the conjugation of 3-mercaptopropyl, 4-mercaptobutyl, or 5­mercaptopentyl glycosides to maleimide-terminated hexavalent carbosilane dendrimers. Titration of WGA with the glycodendrimers yielded quenching of tryptophan fluorescence. All of the glycodendrimers exhibited high affinity with nanomolar dissociation constants (KD values). The best dendrimers were 1a and 1b with KD values of 6.5 ± 1.7 and 5.3 ± 1.7 nM, respectively. The magnitude of fluorescence quenching increased with decrease in the length of the thioalkyl spacer. Maleimide-pendant carbosilane dendrimers provide ready access to multivalent ligands with high-affinity potential.

Preparation of glycopolymers having sialyl α2 → 3 lactose moieties as the potent inhibitors for mumps virus.

A water-soluble glycomonomer having a sialyl α2 â†’ 3 lactose (SLac) moiety was prepared from a known imidate derivative of the SLac and an acrylamide alcohol by means of Schmidt's protocol followed by transesterification. Polymerization of the monomer proceeded in water as the solvent in the presence of ammonium persulfate (APS)-tetramethylethylenediamine (TEMED). Since acryl amide (AAm) was used as a regulator for the arrangement of sugar density, three kinds of glycopolymers having different sugar densities were obtained. Infection inhibition assays of mumps virus (MuV) for Vero cells using the glycopolymers were performed, and the results showed that a glycopolymer having a low sugar density has the highest inhibitory potency. In comparison to sialyl Lewis X (SLeX) as the strongest inhibitor in a previous study, SLac polymer with the low sugar density showed ten-times stronger inhibitory potency than that of SLex. This finding suggested that multivalent conversion of the monomeric SLac with appropriate spatial arrangement are able to effectively inhibit the interaction between the attachment glycoprotein of MuV and glycan receptors on Vero cells.

Fluorogenic glycopolymers available for determining the affinity of lectins by intermolecular FRET.

A convenient assembly of fluorogenic glycopolymers having various polymer compositions was accomplished from the corresponding glycomonomer and dansyl monomer by means of radical polymerization, and the water-soluble glycopolymers gave typical fluorescence spectroscopic profiles due to the dansyl moieties on the glycopolymer in aqueous media. Biological evaluation of the polymer against wheat germ agglutinin (WGA) was accomplished on the basis of fluorescence changes due to tryptophan residues on WGA, and the affinities between the glycopolymers and WGA were estimated to be 4.7 × 105 to 9.3 × 105 M-1. In order to apply the fluorogenic glycopolymers for further biological measurements, efficient resonance energy transfer from tryptophan moieties on WGA to dansyl moieties on the fluorogenic glycopolymers was examined. FRET profiles of both fluorophores were similar compared to the binding profiles on the basis of fluorescence changes of tryptophan residues. This approach is applicable for the determination of an affinity constant between a carbohydrate and a lectin in which no fluorophore exists near the binding site.

Neuraminidase-triggered activation of prodrug-type substrate of 4-nitroaniline.

Artificial substrates for probing neuraminidase activity are powerful tools for studying the physiological and pathological roles of neuraminidases. Most of the substrates are α-O-linked sialosides involving hydroxyl-containing reporters for visualization, and neuraminidase-catalyzed cleavage of the sialic acid residues directly activates the reporters. However, the use of amine-containing reporters has been avoided because α-N-linked sialosides are marginal substrates for neuraminidases. To expand the applicability of reporters to amine-containing compounds, we have focused on prodrug design. Herein we describe the synthesis and enzymatic study of a model substrate involving 4-nitroaniline as an amine-containing chromogenic reporter. The substrate can respond to neuraminidase from Clostridium perfringens. Neuraminidase-mediated hydrolysis of the sialic acid moiety of the substrate initiates self-immolative elimination of the linker moiety, leading the liberation of yellow-colored reporter 4-nitroaniline. The elimination process involves generation of quinone methide intermediate, which causes to neutralize neuraminidase. The substrate, thus, works as not only a chromogenic substrate but also a suicide inactivator.

A constraint scaffold enhances affinity of a bivalent N-acetylglucosamine ligand against wheat germ agglutinin.

Bivalent glycoconjugates have a minimal valence with avidity potential on protein-carbohydrate interactions as well as simplicity of chemical structures enabling simple synthesis with low cost. Understanding the way to maximize the affinities of bivalent glycoconjugates is important for the development of cost-effective tools for therapeutic and diagnostic research. However, there has been little discussion about the effects of constraints imposed from ligand scaffolds on the binding abilities. We synthesized three kinds of biantennary N-acetylglucosamine glycosides with different scaffolds using isobutenyl bis(propargyl)ether as a common scaffold precursor. Decoration of the scaffold branches with GlcNAc moieties through copper-catalyzed azide-alkyne cycloaddition and grafting of the alkenyl focal point to another bivalent biotin dendron through thiol-ene and nucleophilic substitution reactions were successfully carried out in an orthogonal manner. The association constants of the ligands against wheat germ agglutinin were determined by a fluorometric titration assay. A bivalent biotin counterpart provided higher affinity than an isobutyl scaffold, whereas an isobutenyl scaffold yielded more enhancement than a bivalent biotin counterpart. The present work suggested that the constraint and steric bulk of ligand scaffolds are possible factors for improving binding properties of glycoconjugates against lectins or proteins.

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.

Molasses melanoidin-like products enhance phytoextraction of lead through three Brassica species.

Previously, it has been suggested that melanoidin-like products (MLP) from sugarcane molasses may accelerate copper phytoextraction. In this study, we evaluated the facilitatory effect of MLP on phytoextraction in a medium including cadmium or lead, the concentrations of which were adjusted around the regulation values of the Soil Contamination Countermeasures Act in Japan. Three Brassica species were tested based on their fast growth, high biomass productivity, and high heavy metal absorption. Both biomass and lead uptake in the nutrient medium with 1 mM lead nitrate were significantly increased by the addition of MLP, and almost all of the lead was accumulated in the root tissue. Therefore, MLP were able both to detoxify lead ions and to improve their bioavailability in Brassica species. In contrast, only these species with MLP or citric acid survived in the nutrient medium with 1 mM cadmium sulfate. The phytoextraction of cadmium using these species was therefore impractical under the Act.

Preparation of Functional Monomers as Precursors of Bioprobes from a Common Styrene Derivative and Polymer Synthesis.

CM-Str (4-(Chloromethyl)styrene) was used as a useful starting material for the construction of a series of functional monomers. Substitution of the chlorine to the corresponding azide was performed, and the reduction of the azide proceeded smoothly to afford an aminostyrene, which was used as a common precursor for the preparation of functional monomers. Condensation of the amine with a fluorophore, biotin and carbohydrate was accomplished. Among the monomers, a carbohydrate monomer was polymerized with or without acrylamide as a model polymerization to yield the corresponding water-soluble glycopolymers, and biological evaluations of the glycopolymers for a lectin, and wheat germ agglutinin (WGA), were carried out on the basis of the fluorescence change of tryptophan in the WGA.

Iodoacetyl-functionalized pullulan: A supplemental enhancer for single-domain antibody-polyclonal antibody sandwich enzyme-linked immunosorbent assay for detection of survivin.

Survivin, an inhibitor of the apoptosis protein family, is a potent tumor marker for diagnosis and prognosis. The enzyme-linked immunosorbent assay (ELISA) is one of the methods that has been used for detection of survivin. However, ELISA has several disadvantages caused by the use of conventional antibodies, and we have therefore been trying to develop a novel ELISA system using camelid single-domain antibodies (VHHs) as advantageous replacements. Here we report a supplemental approach to improve the VHH-polyclonal antibody sandwich ELISA for survivin detection. Iodoacetyl-functionalized pullulan was synthesized, and its thiol reactivity was characterized by a model reaction with l-cysteine. The thiophilic pullulan was applied to an immunoassay asan additive upon coating of standard assay plates with an anti-survivin VHH fusion protein with C-terminal cysteine. The results showed that the mole ratio of the additive to VHH had a significant effect on the consequent response. Mole ratios of 0.07, 0.7, and 7 led to 90% lower, 15% higher, and 69% lower responses, respectively, than the response of a positive control in which no additive was used. The background levels observed in any additive conditions were as low as that of a negative control lacking both VHH and the additive. These results indicate the applicability of the thiol-reactive pullulan as a response enhancer to VHH-based ELISA.

Synthetic Assembly of Mannose Moieties Using Polymer Chemistry and the Biological Evaluation of Its Interaction towards Concanavalin A.

Protein-carbohydrate interactions exhibit myriad intracellular recognition events, so understanding and investigating their specific interaction with high selectivity and strength are of crucial importance. In order to examine the effect of multivalent binding on the specificity of protein-carbohydrate interactions, we synthesized mannose glycosides as a novel type of glycosylated monomer and glycopolymers of polyacrylamide derivatives with α-mannose (α-Man) by radical polymerization and monitored their strength of interaction with concanavalin A (Con A) by surface plasmon resonance (SPR) detection. In a quantitative test using the Con A-immobilized sensor surface, the kinetic affinity for the synthesized polymers, 8a (KD = 3.3 × 10-6 M) and 8b (KD = 5.3 × 10-5 M), were concentration-dependent, showing strong, specific molecular recognition abilities with lectin. Our study showed the enhancement in recognition specificity for multivalent saccharides, which is often mediated by cell surface carbohydrate-binding proteins that exhibit weak affinity and broad specificity for the individual ligands.

Synthetic assembly of novel avidin-biotin-GlcNAc (ABG) complex as an attractive bio-probe and its interaction with wheat germ agglutinin (WGA).

A tetravalent GlcNAc pendant glycocluster was constructed with terminal biotin through C6 linker. To acquire the multivalent carbohydrate-protein interactions, we synthesized a glycopolymer of tetrameric structure using N-acetyl-d-glucosamine (GlcNAc) as the target carbohydrate by the use of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) as coupling reagent, followed by biotin-avidin complexation leading to the formation of glycocluster of avidin-biotin-GlcNAc conjugate (ABG complex). The dynamic light scattering (DLS) system was implied for size detection and to check the binding affinity of GlcNAc conjugate with a WGA lectin we use fluorometric assay by means of specific excitation of tryptophan at λex 295nm and it was found to be very high Ka∼1.39×10(7) M(-1) in case of ABG complex as compared to GlcNAc only Ka∼1.01×10(4) M(-1) with the phenomenon proven to be due to glycocluster effect.

A secreted protein with plant-specific cysteine-rich motif functions as a mannose-binding lectin that exhibits antifungal activity.

Plants have a variety of mechanisms for defending against plant pathogens and tolerating environmental stresses such as drought and high salinity. Ginkbilobin2 (Gnk2) is a seed storage protein in gymnosperm that possesses antifungal activity and a plant-specific cysteine-rich motif (domain of unknown function26 [DUF26]). The Gnk2-homologous sequence is also observed in an extracellular region of cysteine-rich repeat receptor-like kinases that function in response to biotic and abiotic stresses. Here, we report the lectin-like molecular function of Gnk2 and the structural basis of its monosaccharide recognition. Nuclear magnetic resonance experiments showed that mannan was the only yeast (Saccharomyces cerevisiae) cell wall polysaccharide that interacted with Gnk2. Gnk2 also interacted with mannose, a building block of mannan, with a specificity that was similar to those of mannose-binding legume lectins, by strictly recognizing the configuration of the hydroxy group at the C4 position of the monosaccharide. The crystal structure of Gnk2 in complex with mannose revealed that three residues (asparagine-11, arginine-93, and glutamate-104) recognized mannose by hydrogen bonds, which defined the carbohydrate-binding specificity. These interactions were directly related to the ability of Gnk2 to inhibit the growth of fungi, including the plant pathogenic Fusarium spp., which were disrupted by mutation of arginine-93 or the presence of yeast mannan in the assay system. In addition, Gnk2 did not inhibit the growth of a yeast mutant strain lacking the α1,2-linked mannose moiety. These results provide insights into the molecular basis of the DUF26 protein family.

Synthesis and structural revision of a brominated sesquiterpenoid, aldingenin C.

This paper describes a short step synthesis of the proposed structure for aldingenin C from trans-limonene oxide. The tetrahydropyran-fused 2-oxabicyclo[3.2.2]nonane skeleton as the structural feature was constructed by an intramolecular epoxide-opening reaction and a brominative cyclization. The spectral data of the synthetic compound did not match those of the natural product reported. Re-examination of the reported NMR data using new CAST/CNMR Structure Elucidator suggests that the structure of aldingenin C should be revised to that of known caespitol.

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 thermoacidophile-specific protein family, DUF3211, functions as a fatty acid carrier with novel binding mode.

STK_08120 is a member of the thermoacidophile-specific DUF3211 protein family from Sulfolobus tokodaii strain 7. Its molecular function remains obscure, and sequence similarities for obtaining functional remarks are not available. In this study, the crystal structure of STK_08120 was determined at 1.79-Å resolution to predict its probable function using structure similarity searches. The structure adopts an α/ß structure of a helix-grip fold, which is found in the START domain proteins with cavities for hydrophobic substrates or ligands. The detailed structural features implied that fatty acids are the primary ligand candidates for STK_08120, and binding assays revealed that the protein bound long-chain saturated fatty acids (>C14) and their trans-unsaturated types with an affinity equal to that for major fatty acid binding proteins in mammals and plants. Moreover, the structure of an STK_08120-myristic acid complex revealed a unique binding mode among fatty acid binding proteins. These results suggest that the thermoacidophile-specific protein family DUF3211 functions as a fatty acid carrier with a novel binding mode.

Use of a Longer Aglycon Moiety Bearing Sialyl α(2→3) Lactoside on the Glycopolymer for Lectin Evaluation.

A polymerizable alcohol having 9 PEG repeats was prepared in order to mimic an oligosaccharide moiety. Sialyl α(2→3) lactose, which is known as a sugar moiety of GM3 ganglioside, was also prepared, and the polymerizable alcohol was condensed with the sialyl α(2→3) lactose derivative to afford the desired glycomonomer, which was further polymerized with or without acrylamide to give water-soluble glycopolymers. The glycopolymers had higher affinities than those of glycopolymers having sialyl lactose moieties with shorter aglycon moieties.

Preparation of N-Linked-Type GlcNAc Monomers for Glycopolymers and Binding Specificity for Lectin.

Glycomonomers having N-glycosidic linkages were prepared from a known glycosyl amine, N-acetyl-d-glucosamine (GlcNAc). Radical polymerization of the glycomonomers gave a series of glycopolymers displaying various sugar densities, which were models of the core structure of Asn-linked-type glycoproteins. In addition, fluorometric analyses of wheat germ agglutinin (WGA) against the glycopolymers were carried out, and the results showed unique binding specificities on the basis of flexibility of sugar moieties.