Design and synthesis of novel glycopolythiophene assemblies for colorimetric detection of influenza virus and E. coli

A novel family of glycopolythiophenes containing sialic acid or mannose ligands were prepared and evaluated for their ability to bind lectins, virus, and bacteria. For the set of glycopolythiophenes studied, the spacer-length between the polymer backbone and the ligand was varied to optimize binding interactions. The glycopolymers were blue-shifted (absorbance of ca. 400 nm) relative to the corresponding homo-polythiophenes (absorbance ca. 440 nm), suggesting a twisted conformation for the glycopolymers. The altered conformation is likely due to electrostatic or H-bonding interactions between the polymer chains, arising from the carbohydrate ligand. Further conformational changes in the polythiophene backbone were detected by the binding of specific receptors; lectins (wheat germ agglutinin, concanavalin A), Influenza virus, and Escherichia coli. The binding interactions result in an unusual red-shift in the visible absorption of the polymer backbone, suggesting a lengthening of the effective conjugated length upon interaction of the ligand with its congnate receptor. These conjugated glycopolymeric systems offer a potentially new platform for the detection of molecular binding interactions.     

Sequence-defined glycopolymer segments presenting mannose: synthesis and lectin binding affinity

We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Functional building blocks displaying alkyne moieties and hydrophilic ethylenedioxy units were assembled stepwise on solid phase. The resulting polymer segments were conjugated with mannose sugars via 1,3-dipolar cycloaddition. The obtained mono-, di-, and trivalent mannose structures were then subject to Con A lectin binding. Surface plasmon resonance studies showed a nonlinear increase in binding regarding the number and spacing of sugar ligands. The results of Con A lectin binding assays indicate that the chemical composition of the polymeric scaffold strongly contributes to the binding activities as well as the spacing between the ligands and the number of presented mannose units. Our approach now allows for the synthesis of highly defined glycooligomers and glycopolymers with a diversity of properties to investigate systematically multivalent effects of polymeric ligands.     

Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions

A series of glycopolymers composed of 2-deoxy-2-methacrylamido glucopyranose (MAG) and the primary amine-containing N-(2-aminoethyl) methacrylamide (AEMA) were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The colloidal stability of the polyplexes formed with three diblock glycopolymers and pDNA was assessed using dynamic light scattering, and the polyplexes were found to be stable against aggregation in the presence of salt and serum over the 4 h time period studied. Delivery experiments were performed in vitro to examine the cellular uptake, transfection efficiency, and cytotoxicity of the glycopolymer/pDNA polyplexes in cultured HeLa cells and the diblock copolymer with the shortest AEMA block was found to be the most effective. Additionally, the ability of the diblock glycopolymers to deliver siRNA to U-87 (glioblastoma) cells was screened, and the diblock copolymer with the longest AEMA block was found to have gene knockdown efficacy similar to Lipofectamine 2000.     

RAFT-based tri-component fluorescent glycopolymers: synthesis,characterization and application in lectin-mediated bacterial binding study

A group of fluorescent statistical glycopolymers, prepared via reversible addition–fragmentation chain-transfer (RAFT)-based polymerizations, were successfully employed in lectin-mediated bacterial binding studies. The resultant glycopolymers contained three different monomers: N-(2-hydroxyethyl) acrylamide (HEAA), N-(2-aminoethyl) methacrylamide (AEMA) and N-(2-glyconamidoethyl)-methacrylamides possessing different pendant sugars. Low dispersities (≤1.32) and predictable degrees of polymerization were observed among the products. After the polymerization, the glycopolymers were further modified by different succinimidyl ester fluorophores targeting the primary amine groups on AEMA. With their binding specificities being confirmed by testing with lectin coated agarose beads, the glycopolymers were employed in bacterial binding studies, where polymers containing α-galactose or β-galactose as the pendant sugar were specifically bound by two clinically important pathogens Pseudomonas aeruginosa and Staphylococcus aureus, respectively. This is the first report of using RAFT-based glycopolymers in bacterial binding studies, and the ready access to tri-component statistical glycopolymers also warrants further exploration of their utility in other glycobiological applications.     

Chemoenzymatic synthesis and application of glycopolymers containing multivalent sialyloligosaccharides with a poly(L-glutamic acid) backbone for inhibition of infection by influenza viruses

Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers.     

Effects of polymer structure on the inhibition of cholera toxin by linear polypeptide-based glycopolymers

A variety of important biological events are mediated by the multivalent interaction between relevant oligosaccharides and multiple saccharide receptors on lectins, toxins, and cell surfaces; a variety of glycopolymeric materials have therefore been investigated in studies aimed at manipulating these events. The synthesis of protein- and polypeptide-based glycopolymers via protein engineering and other methods offers opportunities to control both the number and the spacing of saccharides on a scaffold, as well as the conformation of the polymer backbone, and will therefore facilitate the structure-based design of polymers for inhibition of multivalent binding events. In initial studies, we have synthesized a family of galactose-functionalized glycopolymers with a poly(L-glutamic acid) backbone, in which the density and linker length of the pendant carbohydrate moiety were varied. The composition of the glycopolymers was determined via (1)H NMR spectroscopy, and the impact of saccharide density and linker length, as well as the potential for these polypeptide-based glycopolymers to act as high-affinity inhibitors of the cholera toxin, has been indicated via competitive enzyme-linked immunosorbent assay and fluorescence titration experiments. The results of these studies suggest strategies for optimizing the binding of linear glycopolymers to bacterial toxins and will aid in the design of additional protein-based materials for studying the impact of multivalency, spacing, and backbone rigidity in a variety of biologically relevant binding events.     

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-β-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 (K_D?=?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 (K_D?=?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.     

Synthesis and characterization of glucose-grafted biodegradable amphiphilic glycopolymers P(AGE-glucose)-b-PLA

Novel glucose-grafted biodegradable amphiphilic glycopolymers P(AGE-glucose)-b-PLA were synthesized through a facile and efficient way. First, the block copolymer intermediates PAGE-b-PLA bearing double bonds in the side chains were synthesized by ring-opening polymerization of LA using PAGE as macroinitiator and Sn(Oct)₂ as catalyst; Then, 2-mercaptoethyl-β-glucoside (MEGlu) was conjugated to the side chains of PAGE-b-PLA via free-radical coupling reaction to give the glycopolymer P(AGE-glucose)-b-PLA. The micellization behavior of the glycopolymers P(AGE-glucose)-b-PLA in aqueous media was investigated by fluorescence (FL), ¹H nuclear magnetic resonance spectroscopy (¹H NMR), dynamic light scattering (DLS), and transmission electron microscope (TEM). The results showed that these glycopolymers P(AGE-glucose)-b-PLA formed spherical micelles with diameters about 200 nm.     

Syntheses of sulfated glycopolymers and analyses of their BACE-1 inhibitory activity

The glycopolymers for glycosaminoglycan mimic were synthesized, and the inhibitory effects of Alzheimer’s β-secretase (BACE-1) were examined. The regio-selective sulfation was conducted on N-acetyl glucosamine (GlcNAc), and the acrylamide derivatives were synthesized with the consequent sulfated GlcNAc. The glycopolymers were synthesized with acrylamide using radical initiator. The glycopolymer with sulfated GlcNAc showed the strong inhibitory effect on BACE-1, and the inhibitory effects were dependent on the sulfation positions. Especially, glycopolymers carrying 3,4,6-O-sulfo-GlcNAc showed the strong inhibitory effect. The docking simulation suggested that glycopolymers bind to the active site of BACE-1.     

Immobilized sialyloligo-macroligand and its protein binding specificity

We report a chemoenzymatic synthesis of chain-end functionalized sialyllactose-containing glycopolymers with different linkages and their oriented immobilization for glycoarray and SPR-based glyco-biosensor applications. Specifically, O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were synthesized by enzymatic α2,3- and α2,6-sialylation of a lactose-containing glycopolymer that was synthesized by cyanoxyl-mediated free radical polymerization. (1)H NMR showed almost quantitative α2,3- and α2,6-sialylation. The O-cyanate chain-end functionalized sialyllactose-containing glycopolymers were printed onto amine-functionalized glass slides via isourea bond formation for glycoarray formation. Specific protein binding activity of the arrays was confirmed with α2,3- and α2,6-sialyl specific binding lectins together with inhibition assays. Further, immobilizing O-cyanate chain-end functionalized sialyllactose-containing glycopolymers onto amine-modified SPR chip via isourea bond formation afforded SPR-based glyco-biosensor, which showed specific binding activity for lectins and influenza viral hemagglutinins (HA). These sialyloligo-macroligand derived glycoarray and SPR-based glyco-biosensor are closely to mimic 3D nature presentation of sialyloligosaccharides and will provide important high-throughput tools for virus diagnosis and potential antiviral drug candidates screening applications.     

Glycosaminoglycan-mimetic biomaterials. 3. Glycopolymers prepared from alkene-derivatized mono- and disaccharide-based glycomonomers

Mono- and disaccharide-containing glycopolymers were synthesized by two different free-radical processes, and their ability to act as heparan sulfate glycomimetics in promoting the binding of Fibroblast Growth Factor-2 (FGF-2) to its receptor (FGFR-1) was evaluated using an in vitro cell-based assay. Cyanoxyl (*OC triple bond N)-mediated polymerization of acrylamide with alkene-derivatized mono- and disaccharides including sulfated or nonsulfated N-acetyl-D-glucosamine is described. The results of this approach are compared to those obtained via the classical ammonium peroxodisulfate (APS)/N,N,N',N'-tetramethylethylenediamine (TMEDA) initiating system and confirm the capacity of cyanoxyl-mediated polymerization to generate a variety of glycopolymers with high saccharide contents and low polydispersity indexes. In vitro assays demonstrate that specific glycopolymers can potentiate FGF-2/FGFR-1 binding interactions.     

Emulsifying properties of biodegradable polylactide-grafted dextran copolymers

Amphiphilic glycopolymers, polylactide-grafted dextran copolymers (Dex-g-PLA), were synthesized with a well-controlled architecture obtained through a three-step procedure: partial silylation of the dextran hydroxyl groups, ring-opening polymerization of D,L-lactide initiated from remaining hydroxyl groups, silylether deprotection under very mild conditions. Depending on their proportion in polylactide (PLA), these copolymers exhibited solubility either in water or in organic solvents. The emulsifying properties of these glycopolymers were studied: depending on their PLA-to-dextran ratio, they were able to stabilize either direct or inverse emulsions. Droplet size was related to the amount of amphiphilic copolymer in the continuous phase. The aging mechanism of both direct and inverse emulsions was shown to be Ostwald ripening in the first weeks following preparation. Finally inverse miniemulsion copolymerization of acrylamide and N, N'-methylenebisacrylamide was performed in the presence of an amphiphilic Dex-g-PLA stabilizer. Polyacrylamide hydrogel nanoparticles were prepared in that way.     

Chemoenzymatic synthesis, characterization, and application of glycopolymers carrying lactosamine repeats as entry inhibitors against influenza virus infection

To control interspecies transmission of influenza viruses, it is essential to elucidate the molecular mechanisms of the interaction of influenza viruses with sialo-glycoconjugate receptors expressed on different host cells. Competitive inhibitors containing mimetic receptor carbohydrates that prevent virus entry may be useful tools to address such issues. We chemoenzymatically synthesized and characterized the glycopolymers that were carrying terminal 2,6-sialic acid on lactosamine repeats as influenza virus inhibitors. In vitro and in vivo infection experiments using these glycopolymers demonstrated marked differences in inhibitory activity against different species of viruses. Human viruses, including clinically isolated strains, were consistently inhibited by glycopolymers carrying lactosamine repeats with higher activity than those containing a single lactosamine. A swine virus also showed the same recognition properties as those from human hosts. In contrast, avian and equine viruses were not inhibited by any of the glycopolymers examined carrying single, tandem, or triplet lactosamine repeats. Hemagglutination inhibition and solid-phase binding analyses indicated that binding affinity of glycopolymers with influenza viruses contributes dominantly to the inhibitory activity against viral infection. Sequence analysis and molecular modeling of human viruses indicated that specific amino acid substitutions on hemagglutinin may affect binding affinity of glycopolymers carrying lactosamine repeats with viruses. In conclusion, glycopolymers carrying lactosamine repeats of different lengths are useful to define molecular mechanisms of virus recognition. The core carbohydrate portion as well as sialyl linkages on the receptor glycoconjugate may affect host cell recognition of human and swine viruses.     

Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

Synthetic glycopolymers are instrumental and versatile tools used in various biochemical and biomedical research fields. An example of a facile and efficient synthesis of well-controlled fluorescent statistical glycopolymers using reversible addition-fragmentation chain-transfer (RAFT)-based polymerization is demonstrated. The synthesis starts with the preparation of β-galactose-containing glycomonomer 2-lactobionamidoethyl methacrylamide obtained by reaction of lactobionolactone and N-(2-aminoethyl) methacrylamide (AEMA). 2-Gluconamidoethyl methacrylamide (GAEMA) is used as a structural analog lacking a terminal β-galactoside. The following RAFT-mediated copolymerization reaction involves three different monomers: N-(2-hydroxyethyl) acrylamide as spacer, AEMA as target for further fluorescence labeling, and the glycomonomers. Tolerant of aqueous systems, the RAFT agent used in the reaction is (4-cyanopentanoic acid)-4-dithiobenzoate. Low dispersities (≤1.32), predictable copolymer compositions, and high reproducibility of the polymerizations were observed among the products. Fluorescent polymers are obtained by modifying the glycopolymers with carboxyfluorescein succinimidyl ester targeting the primary amine functional groups on AEMA. Lectin-binding specificities of the resulting glycopolymers are verified by testing with corresponding agarose beads coated with specific glycoepitope recognizing lectins. Because of the ease of the synthesis, the tight control of the product compositions and the good reproducibility of the reaction, this protocol can be translated towards preparation of other RAFT-based glycopolymers with specific structures and compositions, as desired.     

Redefining the facilitated transport of mannose in human cells: absence of a glucose-insensitive, high-affinity facilitated mannose transport system

Current evidence suggests that extracellular mannose can be transported intracellularly and utilized for glycoprotein synthesis; however, the identity and the functional characteristics of the transporters of mannose are controversial. Although the glucose transporters are capable of transporting mannose, it has been postulated that the entry of mannose in mammalian cells is mediated by a transporter that is insensitive to glucose [Panneerselvam, K., and Freeze, H. (1996) J. Biol. Chem. 271, 9417-9421] or by a transporter induced by cell treatment with metformin [Shang, J., and Lehrman, M. A. (2004) J. Biol. Chem. 279, 9703-9712]. We performed a detailed analysis of the uptake of mannose in normal human erythrocytes and in leukemia cell line HL-60. Short uptake assays allowed the identification of a single functional activity involved in mannose uptake in both cell types, with a K(m) for transport of 6 mM. Transport was inhibited in a competitive manner by classical glucose transporter substrates. Similarly, the glucose transporter inhibitors cytochalasin B, genistein, and myricetin inhibited mannose transport by 100%. Using long uptake experiments, we identified a second, high-affinity component associated with the intracellular trapping of mannose in the HL-60 cells that is not directly involved in the transport of mannose via the glucose transporters. Thus, the transport of mannose via glucose transporters is a process which is kinetically and biologically separable from its intracellular trapping. A general survey of human cells revealed that mannose uptake was entirely blocked by concentrations of cytochalasin B that obliterates the activity of the glucose transporters. The transport and inhibition data demonstrate that extracellular mannose, whose physiological concentration is in the micromolar range, enters cells in the presence of physiological concentrations of glucose. Overall, our data indicate that transport through the glucose transporter is the main mechanism by which human cells acquire mannose.     

Biotinylated glycopolymers synthesized by atom transfer radical polymerization

Biotinylated glycopolymers that bind to the protein streptavidin were synthesized by atom transfer radical polymerization (ATRP). Poly(methacrylate)s with pendent N-acetyl-d-glucosamines were prepared by polymerizing the protected monomer, followed by deprotection. Alternatively, the unprotected monomer was directly polymerized. Both paths provided well-defined glycopolymers with narrow molecular weight distributions (PDI = 1.07-1.23). The number-average molecular weights determined by gel permeation chromatography increased with increasing initial monomer-to-initiator ratios. The polymers were synthesized using a biotin-functionalized initiator for ATRP. Confirmation of the end group and binding to the protein streptavidin was achieved by (1)H NMR and surface plamon resonance.     

Facile synthesis of chain-end functionalized glycopolymers for site-specific bioconjugation

A series of derivatized arylamine initiators were used to generate chain-end functionalized glycopolymers by cyanoxyl-mediated free-radical polymerization. Significant features of this strategy include the capacity to produce polymers of low polydispersity (PDI < 1.5) under aqueous conditions using unprotected monomers bearing a wide range of functional groups. In addition, the presence of a phenyl ring simplifies calculation of polymer saccharide content and molar mass by (1)H NMR. It is particularly noteworthy, however, that derivatized arylamine initiators in conjunction with the presence of a terminal cyanate group provide a convenient approach for synthesizing polymers with a variety of distinct functional groups at alpha and omega chain ends. In the process, the capacity to label glycopolymers or otherwise conjugate them to proteins or other molecules is greatly enhanced.     

Structure of a major oligosaccharide of Taka-amylase A.

The structures of a major oligosaccharide of Taka-amylase A, shown below, is proposed based on the results of chemical (methylation and acetolysis) and enzymatic (digestions with exo and endo-glycosidases) analyses. This structure is an amendment of that proposed by Yamaguchi et al. (1971) (J. Biochem. 70, 587-594), in which one more mannose residue is attached (Formula: see text) through an alpha 1,2 linkage to the mannose residue which is alpha 1,3-linked to the intermost mannose residue.     

Impedance analysis of valinomycin activity in nano-BLMs

Nano-black lipid membranes (nano-BLMs) were obtained by functionalization of highly ordered porous alumina substrates with an average pore diameter of 60 nm based on a self-assembled alkanethiol submonolayer followed by spreading of 1,2-diphytanoyl-sn-glycero-3-phosphocholine dissolved in n-decane on the hydrophobic substrate. By means of impedance spectroscopy, we analyzed the influence of the self-assembled alkanethiol submonolayer on the electrical properties of the nano-BLMs as well as their long-term stability. We were able to stably integrate nano-BLMs into a flow through system, which allowed us to readily exchange buffer solutions several times and accounts for mass transport phenomena. The ionophore valinomycin was successfully inserted into nano-BLMs and its transport activity monitored as a function of different potassium and sodium ion concentrations reflecting the specificity of valinomycin for potassium ions.     

Mannosylation of Virus-Like Particles Enhances Internalization by Antigen Presenting Cells

Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.