Sugar Ligands Research Articles

Quantitative assessment of the multivalent protein-carbohydrate interactions on silicon.

A key challenge in the development of glycan arrays is that the sensing interface be fabricated reliably so as to ensure the sensitive and accurate analysis of the protein-carbohydrate interaction of interest, reproducibly. These goals are complicated in the case of glycan arrays as surface sugar density can influence dramatically the strength and mode of interaction of the sugar ligand at any interface with lectin partners. In this Article, we describe the preparation of carboxydecyl-terminated crystalline silicon (111) surfaces onto which are grafted either mannosyl moieties or a mixture of mannose and spacer alcohol molecules to provide "diluted" surfaces. The fabrication of the silicon surfaces was achieved efficiently through a strategy implicating a "click" coupling step. The interactions of these newly fabricated glycan interfaces with the lectin, Lens culinaris, have been characterized using quantitative infrared (IR) spectroscopy in the attenuated total geometry (ATR). The density of mannose probes and lectin targets was precisely determined for the first time by the aid of special IR calibration experiments, thus allowing for the interpretation of the distribution of mannose and its multivalent binding with lectins. These experimental findings were accounted for by numerical simulations of lectin adsorption.

Separation of Rebaudiana A from Steviol glycoside using a polymeric adsorbent with multi-hydrogen bonding in a non-aqueous system.

Rebaudioside A (RA) and stevioside (SS) are the primary effective glycoside components in Stevia Rebaudiana. The RA glycoside is sweeter, and it tastes similarly to sucrose. Because extracts with a high RA content can be used as natural sweeteners for food additives approved by the FAO and FDA, RA should generate high market demand. In this study, an efficient method for separating RA was established based on the synergistic multi-hydrogen bonding interaction between a polymeric adsorbent and the RA glycoside. To overcome the destruction of the hydrophobic affinity required for the selective adsorption of RA, an innovative non-aqueous environment was established for adsorption and separation. To this end, an initial polymeric adsorbent composed of a glycidyl methacrylate and trimethylolpropane trimethacrylate (GMA-co-TMPTMA) copolymer matrix was synthesized, and polyethylene polyamine was employed as a functional reagent designed to react with the epoxy group on GME-co-TMPTMA to form a highly selective macroporous adsorbent. The effects of the different functional reagents and the solvent polarity on the adsorption selectivity for RA and SS, respectively, were investigated. Matching the structure of the polyethylene polyamine and sugar ligand on the glycoside molecule was essential in ensuring that the maximum synergistic interaction between adsorbent and adsorbate would be achieved. Moreover, the hydrogen-bonding force was observed to increase when the polarity of the adsorption solvent decreased. Therefore, among the synthesized macroporous polymeric adsorbents, the GTN4 adsorbent-bonding tetraethylenepentamine functional group provided the best separation in an n-butyl alcohol solution. Under the optimized gradient elution conditions, RA and SS can be effectively separated, and the contents of RA and SS increased from 33.5% and 51.5% in the initial crude extract to 95.4% and 78.2% after separation, respectively. Compared to conventional methods, the adsorption-desorption process is more advanced due to its procedural simplicity, low cost and adaptability for industrial production.

Scavenger receptor CL-P1 mainly utilizes a collagen-like domain to uptake microbes and modified LDL

BackgroundCollectins are considered to play a role in host defense via complement activation and opsonization, and are composed of a collagen-like domain and a carbohydrate recognition domain (CRD). Collectin placenta 1 (CL-P1) showed scavenger receptor activity as functions in vitro, and has three candidate domains: a coiled-coil domain, a collagen-like domain and CRD. MethodsWe constructed seven types of CL-P1 deletion mutants to determine the site of each ligand binding domain, and observed whether the specific binding to sugar ligand, microbes, or oxidized LDL decreases or not in cells with CL-P1 deletion mutants and CL-P1 containing mutations of amino acid, respectively. ResultsCL-P1 mainly interacted with ligands of microbes through the collagen-like domain and it binds a sugar ligand through the CRD. Additionally it could bind oxidized low density lipoprotein (OxLDL) due to the coiled-coil domain as well as the collagen-like domain. This binding study using mutants at three positively charged sites in the collagen-like domain reveals that the site of R496 K499 K502 plays the most important role in ligand binding functions for microbes and OxLDL. ConclusionsCL-P1 has three unique functional domains: the collagen-like domain mainly acts against most negatively charged ligands, and the CRD specifically does against sugar substances, while the coiled-coil domain additionally acts on modified LDL. General significanceWe considered that the binding activity for various ligands due to the association of a coiled-coil domain, a collagen-like domain and/or a CRD in CL-P1, might play a role in physiological functions in the animal body.

Photoswitchable precision glycooligomers and their lectin binding.

The synthesis of photoswitchable glycooligomers is presented by applying solid-phase polymer synthesis and functional building blocks. The obtained glycoligands are monodisperse and present azobenzene moieties as well as sugar ligands at defined positions within the oligomeric backbone and side chains, respectively. We show that the combination of molecular precision together with the photoswitchable properties of the azobenzene unit allows for the photosensitive control of glycoligand binding to protein receptors. These stimuli-sensitive glycoligands promote the understanding of multivalent binding and will be further developed as novel biosensors.

Synthesis and surface-spectroscopic characterization of photoisomerizable glyco-SAMs on Au(111).

Photoisomerizable glyco-SAMs (self-assembled monolayers), utilizing synthetic azobenzene glycoside derivatives were fabricated. The ultimate goal of this project is to assay the influence of the 3D arrangement of sugar ligands on cell adhesion, and eventually make cell adhesion photoswitchable. However, it is a prerequisite for any biological study on the spatial conditions of carbohydrate recognition, that photoisomerization of the surface molecules can be verified. Here, we employed IRRAS and XPS to spectroscopically characterize glyco-SAMs. In particular and unprecedented to date, we prove reversible E→Z→E isomerization of azobenzene glycoside-terminated SAMs.

Carbohydrate-Lectin Recognition of Sequence-Defined Heteromultivalent Glycooligomers

Multivalency as a key principle in nature has been successfully adopted for the design and synthesis of artificial glycoligands by attaching multiple copies of monosaccharides to a synthetic scaffold. Besides their potential in various applied areas, e.g. as antiviral drugs, for the vaccine development and as novel biosensors, such glycomimetics also allow for a deeper understanding of the fundamental aspects of multivalent binding of both artificial and natural ligands. However, most glycomimetics so far neglect the purposeful arranged heterogeneity of their natural counterparts, thus limiting more detailed insights into the design and synthesis of novel glycomimetics. Therefore, this work presents the synthesis of monodisperse glycooligomers carrying different sugar ligands at well-defined positions along the backbone using for the first time sequential click chemistry and stepwise assembly of functional building blocks on solid support. This approach allows for straightforward access to sequence-defined, multivalent glycooligomers with full control over number, spacing, position, and type of sugar ligand. We demonstrate the synthesis of a set of heteromultivalent oligomers presenting mannose, galactose, and glucose residues. All heteromultivalent structures show surprisingly high affinities toward Concanavalin A lectin receptor in comparison to their homomultivalent analogues presenting the same number of binding ligands. Detailed studies of the ligand/receptor interaction using STD-NMR and 2fFCS indeed indicate a change in binding mechanism for trivalent glycooligomers presenting mannose or combinations of mannose and galactose residues. We find that galactose residues do not participate in the binding to the receptor, but they promote steric shielding of the heteromultivalent glycoligands and thus result in an overall increase in affinity. Furthermore, the introduction of nonbinding ligands seems to suppress receptor clustering of multivalent ligands. Overall these results support the importance of heteromultivalency specifically for the design of novel glycoligands and help to promote a fundamental understanding of multivalent binding modes.

Assessment of weak sugar-binding ability using lectin tetramer and membrane-based glycans.

To consider biological significance of glycosylation of proteins, it is necessary to evaluate the importance of sugar-recognition processes mediated by lectins. Though the interaction between sugars and proteins, especially animal lectins, is quite weak with K d approximately 10(-4) M, cellular and molecular recognitions mediated via sugar-protein interaction increase their avidity by 1-3 orders of magnitude by the self-association of both receptors and their ligands on cell surfaces. To assess the weak interaction between lectins and their sugar ligands, we established lectin tetramer binding to cell surface glycans using flow cytometry. This strategy is highly sensitive, and useful to determine whether or not a putative lectin domain may have sugar-binding ability.

Modifications of Chitosan for Use in Nanoparticulate Drug Delivery Systems

Nanoparticles with naturally occurring biodegradable polymers have emerged as potential carriers of various therapeutic agents. Chitosan, a cationic polysaccharide, is one of such biodegradable polymers, which has been extensively exploited for the preparation of nanoparticles with several therapeutic agents. In recent years, the area of focus has shifted from chitosan to chitosan derivatized polymers due to its vastly improved properties such as better drug retention capability, improved permeation, enhanced mucoadhesion, and sustained release of therapeutic agents. Chitosan derivatized polymers are primarily the quaternized chitosan derivatives, chitosan cyclodextrin complexes, thiolated chitosan, pegylated chitosan, chitosan combined with sugar ligands, chitosan with cholanic acid, chitosan-linolenic acid and chitosan-DTPA. This review article covers the various modifications of chitosan, their preparation procedures and various works done with them. 

Hybrid Material for Protein Sensing Based on Electrosynthesized MIP on a Mannose Terminated Self‐Assembled Monolayer

A novel strategy to prepare a surface confined molecularly imprinted polymer (MIP) film directly on a transducer surface for protein sensing is achieved by combining interaction with a natural binding receptor and binding to a fully synthetic MIP. A thiolated oligoethyleneglycol (OEG)/mannose conjugate is first self‐assembled on the transducer surface. Then the carbohydrate binding protein, concanavalin A (ConA), is “vectorially” immobilized as a submonolayer on the underlying mannose modified surface. Afterwards, an ultrathin polyscopoletin film with the thickness comparable to that of the protein is electrodeposited on the top. This architecture ensures that the target is confined to the film surface. The resulting functional material shows an approximately 20‐fold higher affinity than that obtained from the mannose self‐assembled monolayer. This result shows a synergism between multivalent binding of the natural sugar ligand and the non‐covalent interactions of the target within the MIP cavities. Recognition capability of the film is characterized by a real‐time measurement using quartz crystal microbalance. In comparison to the non‐imprinted film, the imprinted film reveals 8.6 times higher binding capacity towards ConA. High discrimination towards the target protein's homologues shows size and shape specificity of the imprint.

Synthesis of homo- and heteromultivalent carbohydrate-functionalized oligo(amidoamines) using novel glyco-building blocks

We present the solid phase synthesis of carbohydrate-functionalized oligo(amidoamines) with different functionalization patterns utilizing a novel alphabet of six differently glycosylated building blocks. Highly efficient in flow conjugation of thioglycosides to a double-bond presenting diethylentriamine precursor is the key step to prepare these building blocks suitable for fully automated solid-phase synthesis. Introduction of the sugar ligands via functionalized building blocks rather than postfunctionalization of the oligomeric backbone allows for the straightforward synthesis of multivalent glycoligands with full control over monomer sequence and functionalization pattern. We demonstrate the potential of this building-block approach by synthesizing oligomers with different numbers and spacing of carbohydrates and also show the feasibility of heteromultivalent glycosylation patterns by combining building blocks presenting different mono- and disaccharides.

Stepwise Orthogonal Click Chemistry toward Fabrication of Paclitaxel/Galactose Functionalized Fluorescent Nanoparticles for HepG2 Cell Targeting and Delivery

In this report, we used stepwise orthogonal click chemistry (SOCC) involving strain-promoted azide-alkyne cycloaddition (SPAAC) and microwave-assisted Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to assemble an anticancer drug (paclitaxel, PTX) and a targeting ligand (trivalent galactoside, TGal) on a fluorescent silicon oxide nanoparticle (NP) by using dialkyne linker 8 as a bridge. The fluorescent NH2@Cy3SiO2NP was fabricated using a competition method to incorporate Cy3 without loss of the original surface amine density on the NPs. The concept of SOCC was first investigated in a solution-phase model study that showed quantitative reaction yield. In the fabrication of TGal-PTX@Cy3SiO2NP, the expensive compound azido-functionalized PTX 12 used in SPAAC can be easily recovered due to the absence of other reagents in the reaction mixture. High loading of the sugar ligand on the NP surface serves a targeting function and also overcomes the low water solubility of PTX. Confocal fluorescence microscopy and cytotoxicity assay showed that TGal-PTX@Cy3SiO2NP was taken up by HepG2 cells and was affected by the microtubule skeleton in these cells and inhibited the proliferation of these cells in a dose-dependent manner. The presence of a fluorescent probe, a targeting ligand, and an anticancer drug on the multifunctional TGal-PTX@Cy3SiO2NP allows for real-time imaging, specific cancer-cell targeting, and the cell-killing effect which is better than free PTX.

Sensors: Fluorogenic Probing of Specific Recognitions between Sugar Ligands and Glycoprotein Receptors on Cancer Cells by an Economic Graphene Nanocomposite (Adv. Mater. 30/2013)

Ingenious detection of the universal sugar-protein interactions at a cellular level has long remained a formidable task, hampering the advancement of Glycomics. Guo-Rong Chen, Jia Li, Xiao-Peng He and co-workers devise an economic system in work described on page 4097. The work involves simply stacking glycosyl rhodamines to graphene oxide for the generation of a fluorogenic sugary nanomaterial suited for probing transmembrane glycoprotein receptors that are uniquely pervaded on cancer cells in situ.

ChemInform Abstract: Cyclodextrin‐Based Multivalent Glycodisplays: Covalent and Supramolecular Conjugates to Assess Carbohydrate—Protein Interactions

AbstractReview: 176 refs.

Magnetic Porous Sugar-Functionalized PEG Microgels for Efficient Isolation and Removal of Bacteria from Solution

Here, we present a new microparticle system for the selective detection and magnetic removal of bacteria from contaminated solutions. The novelty of this system lies in the combination of a biocompatible scaffold reducing unspecific interactions with high capacity for bacteria binding. We apply highly porous poly(ethylene glycol) (PEG) microparticles and functionalize them, introducing both sugar ligands for specific bacteria targeting and cationic moieties for electrostatic loading of superparamagnetic iron oxide nanoparticles. The resulting magnetic, porous, sugar-functionalized (MaPoS) PEG microgels are able to selectively bind and discriminate between different strains of bacteria Escherichia coli . Furthermore, they allow for a highly efficient removal of bacteria from solution as their increased surface area can bind three times more bacteria than nonporous particles. All in all, MaPoS particles represent a novel generation of magnetic beads introducing for the first time a porous, biocompatible and easy to functionalize scaffold and show great potential for various biotechnological applications.

Fluorogenic Probing of Specific Recognitions between Sugar Ligands and Glycoprotein Receptors on Cancer Cells by an Economic Graphene Nanocomposite

Economical nanocomposites based on π-stacking of N-acetyl glycosyl rhodamine B to graphene oxide (GO) are simply prepared. These "sweet" GO-materials are proven to be admirable for the fluorogenic recognition of specific intercellular sugar-based ligand-glycoprotein receptor interactions of interest.

Targeting Hepatic Cancer Cells with PEGylated Dendrimers Displaying N‐Acetylgalactosamine and SP94 Peptide Ligands

Poly(amidoamine) (PAMAM) dendrimers are branched water-soluble polymers defined by consecutive generation numbers (Gn) indicating a parallel increase in size, molecular weight, and number of surface groups available for conjugation of bioactive agents. In this article, we compare the biodistribution of N-acetylgalactosamine (NAcGal)-targeted [(14) C]1 -G5-(NH2 )5 -(Ac)108 -(NAcGal)14 particles to non-targeted [(14) C]1 -G5-(NH2 )127 and PEGylated [(14) C]1 -G5-(NH2 )44 -(Ac)73 -(PEG)10 particles in a mouse hepatic cancer model. Results show that both NAcGal-targeted and non-targeted particles are rapidly cleared from the systemic circulation with high distribution to the liver. However, NAcGal-targeted particles exhibited 2.5-fold higher accumulation in tumor tissue compared to non-targeted ones. In comparison, PEGylated particles showed a 16-fold increase in plasma residence time and a 5-fold reduction in liver accumulation. These results motivated us to engineer new PEGylated G5 particles with PEG chains anchored to the G5 surface via acid-labile cis-aconityl linkages where the free PEG tips are functionalized with NAcGal or SP94 peptide to investigate their potential as targeting ligands for hepatic cancer cells as a function of sugar conformation (α versus β), ligand concentration (100-4000 nM), and incubation time (2 and 24 hours) compared to fluorescently (Fl)-labeled and non-targeted G5-(Fl)6 -(NH2 )122 and G5-(Fl)6 -(Ac)107 -(cPEG)15 particles. Results show G5-(Fl)6 -(Ac)107 -(cPEG[NAcGalβ ])14 particles achieve faster uptake and higher intracellular concentrations in HepG2 cancer cells compared to other G5 particles while escaping the non-specific adsorption of serum protein and phagocytosis by Kupffer cells, which make these particles the ideal carrier for selective drug delivery into hepatic cancer cells.

Are multivalent cluster glycosides a means of controlling ligand density of glycoarrays?

Bacterial adhesion to the glycocalyx of human host cells is of biological and medicinal importance. This process is often initiated by the interaction of bacterial lectins and specific carbohydrate ligands. Thus, adhesion of bacterial cells to glycosylated surfaces is a suitable model system to study various parameters of lectin-mediated carbohydrate recognition. Glycoarrays have become important tools to study such lectin-mediated carbohydrate recognition. However, it is difficult to adjust the characteristics of a specific glycoarray regarding its carbohydrate density or the clustering of sugar ligands, respectively. Thus, we have made an attempt to use synthetic cluster glycosides of different valencies to vary carbohydrate density on a polystyrene surface. A series of mono-, di- and trivalent mannosides were synthesised for immobilisation on pre-functionalised polystyrene microtiter plates and the resulting glycoarrays were tested as adhesive surfaces in mannose-specific adhesion of Escherichia coli. Our measurements give first promising hints about the potential of this approach to alter ligand density of glycoarrays in a systematic way.

Cyclodextrin-based multivalent glycodisplays: covalent and supramolecular conjugates to assess carbohydrate–protein interactions

Covalent attachment of biorecognizable sugar ligands in several copies at precise positions of cyclomaltooligosaccharide (cyclodextrin, CD) macrocycles has proven to be an extremely flexible strategy to build multivalent conjugates. The commercial availability of the native CDs in three different sizes, their axial symmetry and the possibility of position- and face-selective functionalization allow a strict control of the valency and spatial orientation of the recognition motifs (glycotopes) in low, medium, high and hyperbranched glycoclusters, including glycodendrimer-CD hybrids. "Click-type" ligation chemistries, including copper(i)-catalyzed azide-alkyne cycloaddition (CuAAC), thiol-ene coupling or thiourea-forming reactions, have been implemented to warrant full homogeneity of the adducts. The incorporation of different glycotopes to investigate multivalent interactions in heterogeneous environments has also been accomplished. Not surprisingly, multivalent CD conjugates have been, and continue to be, major actors in studies directed at deciphering the structural features ruling carbohydrate recognition events. Nanometric glycoassemblies endowed with the capability of adapting the inter-saccharide distances and orientations in the presence of a receptor partner or capable of mimicking the fluidity of biological membranes have been conceived by multitopic inclusion complex formation, rotaxanation or self-assembling. Applications in the fields of sensors, site-specific drug and gene delivery or protein stabilization attest for the maturity of the field.

10 SPERM BINDING TO PORCINE UTERINE EPITHELIAL CELLS MIGHT BE LECTIN MEDIATED

Evidence suggests that porcine spermatozoa bind to the endometrium lining after entering the uterus. In previous studies, it was verified that this binding is specific to porcine uterine epithelial cells (UEC). Here, we present trials aimed at identifying the engaged binding mechanism. Several interactions of spermatozoa with the female reproductive tract are known to be lectin mediated. The respective sugar ligands are species specific for the oviductal as well as zona pellucida surface membrane. Therefore, we hypothesised that sperm-endometrium interactions are mediated by lectins, too. To block possibly existing sugar moieties on the sperm surface, we pre-incubated spermatozoa with lectins selected for their strong binding to viable spermatozoa [wheat germ agglutinin WGA, succinylated WGA (sWGA), concanavalin A (ConA)] and subsequently undertook co-incubation studies using an established cell culture model from primary porcine uterine epithelial cells. All trials were carried out in modified Dulbecco’s Modified Eagle’s Medium containing 20% serum (D20) because the UEC monolayer proved to react sensitive to PBS as well as to semen extender. Pre-trials confirmed that in D20 sperm vitality remained unaffected and lectin binding efficiency did not differ compared with PBS. The sperm-rich fraction of the ejaculate from 3 German Landrace boars was collected in D20 and washed by centrifugation. The pellet was resuspended in D20 and the concentration adjusted to 100 × 106 spermatozoa mL–1. Then, the samples were incubated with 1 of the 3 lectins (10 µg mL–1) for 15 min at 37°C. As a control, 1 aliquot of the sperm suspension was incubated without lectins. After another washing step, the pellet was resuspended in D20. For co-incubation with UEC, 500 µL of lectin-incubated sperm was released onto a UEC monolayer and the binding activity observed under a phase contrast microscope. The binding density was quantified by area under view and compared to results from the control incubation with untreated sperm. Images (2 repeats/boar and lectin) were divided into fields of 61.6 µm2 and the fields with and without sperm were counted. For statistical analysis, a Tukey Test was carried out. Sperm treated with WGA and sWGA, both binding to glucosamine molecules, showed significantly reduced binding activity to the UEC (P < 0.05), whereas ConA-treated and untreated sperm bound in the dense pattern as shown in previous trials. The results indicate that the sperm-UEC binding mechanism is mediated by glucosamine moieties on the sperm surface, which interact with matching lectins placed on the UEC membrane surface. Investigations for further confirmation of the suggested binding mechanism between porcine sperm and the uterine epithelial layer are ongoing. Supported by IMV Technologies, L’Aigle, France.

Synthesis of Biotinylated Multipodal Glycoclusters on a Solid Support

AbstractTrivalent glycoconjugates, each bearing a biotin side arm in addition to three different sugar ligands, have been synthesized on a solid support. The conjugates were assembled on an orthogonally protected pentaerythrityl tetramine core that was anchored to the support through a backbone amide linker. Peptide coupling chemistry was applied to elongate three of the branches with β‐alanine and a fully acylated glycosylacetic acid. The fourth branch was levulinoylated and oximated with aminooxy‐derivatized D‐biotin, followed by acidolytic release into solution. The acyl protecting groups were removed by methoxide‐catalyzed transesterification in methanol.