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.

TEMPO-Substituted Poly(ethylene sulfide) for Solid-State Electro-Chemical Charge Storage.

A poly(ethylene sulfide) backbone is introduced as the main chain of a radical polymer. Anionic ring-opening polymerization of an episulfide monomer substituted with 2,2,6,6tetramethylpiperidin1oxyl (TEMPO), a robust nitroxide radical, yields the corresponding polythioether. Compared to the traditional poly(ethylene oxide) backbone, the new polymer shows a lower glass transition temperature (-10 °C), and about threefold higher solid-state ionic conductivity. The polythioether is also shown to improve the charge/discharge properties of a cathode in solid-state lithium-ion batteries.

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.

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.

2-Benzoylpyridine Ligand Complexation with Gold Critical for Propargyl Ester-Based Protein Labeling.

In previously reported work, AuIII complexes coordinated with 2-benzoylpyridine ligand, BPy-Au, were prebound to a protein and used to discover a novel protein-directed labeling approach with propargyl ester functional groups. In this work, further examination discovered that gold catalysts devoid of the 2-benzoylpyridine ligand (e.g., NaAuCl4) had significantly reduced levels of protein labeling. Mechanistic investigations then revealed that BPy-Au and propargyl esters undergo a rare example of C(sp2 )-C(sp) aryl-alkynyl cross-coupling, likely through spontaneous reductive elimination. Overall, these observations appear to suggest that BPy-Au-mediated, propargyl ester-based protein labeling acts via an activated ester intermediate, which contributes to our understanding of this process and will aid the expansion/optimization of gold-catalyst usage in future bioconjugation applications, especially in vivo.

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.

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.

Total Synthesis of Kehokorins A-E, Cytotoxic p-Terphenyls.

This paper describes a general method for the synthesis of kehokorins A-E, novel cytotoxic p-terphenyls. 2,4,6-Trihydroxybenzaldehyde served as a common building block for preparation of the central aromatic ring. Construction of their p-terphenyl skeletons was achieved by a stepwise Suzuki-Miyaura coupling, whereas the phenyldibenzofuran moiety was built up by an intramolecular Ullmann reaction. Introduction of an l-rhamnose residue into partly protected kehokorin B was performed by the trichloroacetimidate method.

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.

Synthesis of Fluorinated Polymers and Evaluation of Wettability.

Two kinds of fluorinated polymers were synthesized: an acrylate polymer having a fluorinated triethylene glycol as a pendant group (2a) and a fluoroalkyl acrylate polymer (2b). The contact angle of these fluorinated polymers against water, non-fluorinated alcohols and fluorinated alcohols were evaluated. As compared with the fluoroalkyl polymer (2b), fluoroethylene glycol polymer (2a) showed smaller contact angle against water and non-fluorinated alcohols. This supports the proposition that changing the alkyl chain into the ethylene glycol-type chain gave some interaction between etheric oxygen and water or non-fluorinated alcohols. In addition, fluoroalkyl acrylate polymer (2b) showed remarkably low values of critical surface tension.

Effect of aglycon structure on saccharide elongation by cells.

Alkyl N-acetyl-ß-D-glucosaminide (GlcNAc primers) with different aglycon moieties were synthesized and used to determine the effect of the aglycon structure on cellular saccharide elongation. Dodecyl N-acetyl-ß-D-glucosaminide (GlcNAc-C12), tridecan-7-yl N-acetyl-ß-D-glucosaminide (GlcNAc-2C6), and pentacosan-13-yl N-acetyl-ß-D-glucosaminide (GlcNAc-2C12) primers were synthesized by glycosylation of dodecan-1-ol, tridecan-7-ol, and pentacosan-13-ol, respectively, with peracetylglucosamine. These primers were introduced to mouse B16 melanoma cells to prepare glycolipids. After 48 h incubation, results showed that GlcNAc-C12 was elongated to give NeuAc-Gal-GlcNAc-C12. GlcNAc-2C6 was also elongated to afford Gal-GlcNAc-2C6 and NeuAc-Gal-GlcNAc-2C6. On the other hand, GlcNAc-2C12 primer was not elongated. Significantly, the results demonstrated that the amount of glycosylated product increased 1.5-times by modifying the aglycon structure of GlcNAc from C12 to 2 C6 despite having almost the same number of C-units.

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.