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.

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.

Preparation of a Water-Soluble Glycopolymer Bearing Porphyrin Skeletons and Its Biological Properties.

A known tetraphenyl porphyrin (TPP) having an amino functional group [5-(4-aminophenyl)-10,15,20-(triphenyl)porphyrin] was converted into the corresponding monomer by means of condensation with acryloyl chloride. Simple radical polymerization of the porphyrin monomer and a glycosyl monomer in the presence of acrylamide as a regulator monomer in order to avoid steric interference gave a water-soluble glycopolymer bearing porphyrin moieties. Spectroscopic analyses suggested incorporation of porphyrin moieties in the glycopolymer. The physical properties of the water-soluble glycopolymer bearing porphyrin moieties were examined in aqueous media, and the results also indicated the incorporation of TPP moieties in the polymer. Uptake of the polymer into HeLa cells was observed, and the cytotoxicity of the polymer was confirmed by microscopic analyses. The glycopolymer bearing porphyrin moieties is promising not only for photodynamic therapy but also as an anti-cancer reagent.

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.

Synthesis of 4-O-(4-Amino-4-deoxy-ß-D-xylopyranosyl)paromomycin and 4-S-(ß-D-Xylopyranosyl)-4-deoxy-4'-thio-paromomycin and Evaluation of their Antiribosomal and Antibacterial Activity.

The design, synthesis and antiribosomal and antibacterial activity of two novel glycosides of the aminoglycoside antibiotic paromomycin are described. The first carries of 4-amino-4-deoxy-ß-D-xylopyranosyl moiety at the paromomycin 4'-position and is approximately two-fold more active than the corresponding ß-D-xylopyranosyl derivative. The second is a 4'-(ß-D-xylopyranosylthio) derivative of 4'-deoxyparomomycin that is unexpectedly less active than the simple ß-D-xylopyranosyl derivative, perhaps because of the insertion of the conformationally more mobile thioglycosidic linkage.

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.

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.

Modification of Fab Fragments by Dibromopyridazinediones Carrying Mono- and Double-Biotin Functionalities.

To verify the potencies of dibromopyridazinediones with mono- and double-biotin groups, the functions as cysteine-selective biotinylation reagents were evaluated through conjugation with a goat anti-mouse IgG Fab fragment as a functional protein model. The starting Fab was reduced with tris(2-carboxyethyl)phosphine to cleave the disulfide bond and then treated with the reagents. These reagents simultaneously introduced biotin groups into the reduced Fab and re-bridged the disulfide moiety. Furthermore, we demonstrated that the biotin-labeled Fabs were reactive to an antigen and streptavidin.

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.

Synthesis and Antibacterial Activity of Propylamycin Derivatives Functionalized at the 5''- and Other Positions with a View to Overcoming Resistance Due to Aminoglycoside Modifying Enzymes.

Propylamycin (4'-deoxy-4'-propylparomomycin) is a next generation aminoglycoside antibiotic that displays increased antibacterial potency over the parent, coupled with reduced susceptibility to resistance determinants and reduced ototoxicity in the guinea pig model. Propylamycin nevertheless is inactivated by APH(3')-Ia, a specific aminoglycoside phosphotransferase isozyme that acts on the primary hydroxy group of the ribofuranosyl moiety (at the 5''-position). To overcome this problem, we have prepared and studied the antibacterial and antiribosomal activity of various propylamycin derivatives carrying amino or substituted amino groups at the 5''-position in place of the vulnerable hydroxy group. We find that the introduction of an additional basic amino group at this position, while overcoming the action of the aminoglycoside phosphoryltransferase isozymes acting at the 5''-position as anticipated, results in a significant drop in selectivity for the bacterial over the eukaryotic ribosomes that is predictive of increased ototoxicity. In contrast, 5''-deoxy-5''-formamidopropylamycin retains the excellent across-the-board levels of antibacterial activity of propylamycin itself, while circumventing the action of the offending aminoglycoside phosphotransferase isozymes and affording even greater selectivity for the bacterial over the eukaryotic ribosomes. Other modifications to address the susceptibility of propylamycin to the APH(3')-Ia isozyme including deoxygenation at the 3'-position and incorporation of a 6',5''-bis(hydroxyethylamino) modification offer no particular advantage.

Impaired O-Glycosylation at Consecutive Threonine TTX Motifs in Mucins Generates Conformationally Restricted Cancer Neoepitopes.

Autoantibody signatures of circulating mucin fragments stem from cancer tissues, and microenvironments are promising biomarkers for cancer diagnosis and therapy. This study highlights dynamic epitopes generated by aberrantly truncated immature O-glycosylation at consecutive threonine motifs (TTX) found in mucins and intrinsically disordered proteins (IDPs). NMR analysis of synthetic mucin models having glycosylated TTX motifs and colonic MUC2 tandem repeats (TRs) containing TTP and TTL moieties unveils a general principle that O-glycosylation at TTX motifs generates a highly extended and rigid conformation in IDPs. We demonstrate that the specific conformation of glycosylated TTX motifs in MUC2 TRs is rationally rearranged by concerted motions of multiple dihedral angles and noncovalent interactions between the carbohydrate and peptide region. Importantly, this canonical conformation of glycosylated TTX motifs minimizes steric crowding of glycans attached to threonine residues, in which O-glycans possess restricted orientations permitting further sugar extension. An antiadhesive microarray displaying synthetic MUC2 derivatives elicited the presence of natural autoantibodies to MUC2 with impaired O-glycosylation at TTX motifs in sera of healthy volunteers and patients diagnosed with early stage colorectal cancer (CRC). Interestingly, autoantibody levels in sera of the late stage CRC patients were distinctly lower than those of early stage CRC and normal individuals, indicating that the anti-MUC2 humoral response to MUC2 neoepitopes correlates inversely with the CRC stage of patients. Our results uncovered the structural basis of the creation of dynamic epitopes by immature O-glycosylation at TTX motifs in mucins that facilitates the identification of high-potential targets for cancer diagnosis and therapy.

Stereospecific synthesis of methyl 2-amino-2,4-dideoxy-6S-deuterio-α-D-xylo-hexopyranoside and methyl 2-amino-2,4-dideoxy-6S-deuterio-4-propyl-α-d-glucopyranoside: Side chain conformation of the novel aminoglycoside antibiotic propylamycin.

The stereospecific syntheses of methyl 2-amino-2,4-dideoxy-4-C-propyl-α-d-glucopyranoside and of methyl 2-amino-2,4-dideoxy-α-D-xylo-hexopyranoside and of their 6S-deuterioisotopomers are described as models for ring I of the aminoglycoside antibiotics propylamycin and 4'-deoxyparomomycin, respectively. Analysis of the 1H NMR spectra of these compounds and of methyl 2-amino-2-deoxy-α-d-glucopyranoside, a model for paromomycin itself, reveals that neither deoxygenation at the 4-position, nor substitution of the C-O bond at the 4-postion by a C-C bond significantly changes the distribution of the side chain population between the three possible staggered conformations. From this it is concluded that the beneficial effect on antiribosomal and antibacterial activity of the propyl group in propylamycin does not derive from a change in side chain conformation. Rather, enhanced basicity of the ring oxygen and increased hydrophobicity and/or solvation effects are implicated.

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.

Design, Multigram Synthesis, and in Vitro and in Vivo Evaluation of Propylamycin: A Semisynthetic 4,5-Deoxystreptamine Class Aminoglycoside for the Treatment of Drug-Resistant Enterobacteriaceae and Other Gram-Negative Pathogens.

Infectious diseases due to multidrug-resistant pathogens, particularly carbapenem-resistant Enterobacteriaceae (CREs), present a major and growing threat to human health and society, providing an urgent need for the development of improved potent antibiotics for their treatment. We describe the design and development of a new class of aminoglycoside antibiotics culminating in the discovery of propylamycin. Propylamycin is a 4'-deoxy-4'-alkyl paromomycin whose alkyl substituent conveys excellent activity against a broad spectrum of ESKAPE pathogens and other Gram-negative infections, including CREs, in the presence of numerous common resistance determinants, be they aminoglycoside modifying enzymes or rRNA methyl transferases. Importantly, propylamycin is demonstrated not to be susceptible to the action of the ArmA resistance determinant whose presence severely compromises the action of plazomicin and all other 4,6-disubstituted 2-deoxystreptamine aminoglycosides. The lack of susceptibility to ArmA, which is frequently encoded on the same plasmid as carbapenemase genes, ensures that propylamycin will not suffer from problems of cross-resistance when used in combination with carbapenems. Cell-free translation assays, quantitative ribosome footprinting, and X-ray crystallography support a model in which propylamycin functions by interference with bacterial protein synthesis. Cell-free translation assays with humanized bacterial ribosomes were used to optimize the selectivity of propylamycin, resulting in reduced ototoxicity in guinea pigs. In mouse thigh and septicemia models of Escherichia coli, propylamycin shows excellent efficacy, which is better than paromomycin. Overall, a simple novel deoxy alkyl modification of a readily available aminoglycoside antibiotic increases the inherent antibacterial activity, effectively combats multiple mechanisms of aminoglycoside resistance, and minimizes one of the major side effects of aminoglycoside therapy.

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.

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.

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.

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.