Sugar‐Decorated Sugar Vesicles: Lectin–Carbohydrate Recognition at the Surface of Cyclodextrin Vesicles

An artificial glycocalix self‐assembles when unilamellar bilayer vesicles of amphiphilic β‐cyclodextrins are decorated with maltose and lactose by host–guest interactions. To this end, maltose and lactose were conjugated with adamantane through a tetra(ethyleneglycol) spacer. Both carbohydrate–adamantane conjugates strongly bind to β‐cyclodextrin (K_a≈4×10⁴ M ^?1). The maltose‐decorated vesicles readily agglutinate (aggregate) in the presence of the lectin concanavalin A, whereas the lactose‐decorated vesicles agglutinate in the presence of peanut agglutinin. The orthogonal multivalent interaction in the ternary system of host vesicles, guest carbohydrates, and lectins was investigated by using isothermal titration calorimetry, dynamic light scattering, UV/Vis spectroscopy, and cryogenic transmission electron microscopy. It was shown that agglutination is reversible, and the noncovalent interaction can be suppressed and eliminated by the addition of competitive inhibitors, such as D ‐glucose or β‐cyclodextrin. Also, it was shown that agglutination depends on the surface coverage of carbohydrates on the vesicles.     

Fullerene Sugar Balls: A New Class of Biologically Active Fullerene Derivatives

Among the large variety of bioactive C₆₀ derivatives, fullerene derivatives substituted with sugar residues, that is, glycofullerenes, are of particular interest. The sugar residues are not only solubilizing groups; their intrinsic biological properties also provide additional appealing features to the conjugates. The most recent advances in the synthesis and the biological applications of glycofullerenes are summarized in the present review article with special emphasis on globular glycofullerenes, that is, fullerene sugar balls, constructed on a hexa‐substituted fullerene scaffold. The high local concentration of carbohydrates around the C₆₀ core in fullerene sugar balls is perfectly suited to the binding of lectins through the “glycoside cluster effect”, and these compounds are potential anti‐adhesive agents against bacterial infection. Moreover, mannosylated fullerene sugar balls have shown antiviral activity in an Ebola pseudotyped infection model. Finally, when substituted with peripheral iminosugars, dramatic multivalent effects have been observed for glycosidase inhibition. These unexpected observations have been rationalized by the interplay of interactions involving the catalytic site of the enzyme and non‐glycone binding sites with lectin‐like abilities.     

Unique aggregation of anthrax (Bacillus anthracis) spores by sugar-coated single-walled carbon nanotubes

There has been significant interest in the binding of anthrax spores by molecular species, but with only limited success. Proteins and more recently peptides were used. However, despite the known presence of carbohydrates on the spore surface, carbohydrate-carbohydrate interactions have hardly been explored likely because of the lack of required specific platform for synthetic carbohydrates. We report the successful use of single-walled carbon nanotubes as a truly unique scaffold for displaying multivalent monosaccharide ligands that bind effectively to anthrax spores with divalent cation mediation to cause significant spore aggregation. The work should have far-reaching implications in development of countermeasure technologies.     

Site-selective modification of proteins for the synthesis of structurally defined multivalent scaffolds

A combination of classical site-directed mutagenesis, genetic code engineering and bioorthogonal reactions delivered a chemically modified barstar protein with one or four carbohydrates installed at specific residues. These protein conjugates were employed in multivalent binding studies, which support the use of proteins as structurally defined scaffolds for the presentation of multivalent ligands.     

Cation-mediated interaction of dextran sulfate with phospholipid vesicles: binding, vesicle surface polarity, leakage and fusion

 The interaction of dextran sulfate (DS) with dimyristoylphosphatidylcholine (DMPC) large unilamellar vesicles was investigated. DS of different molecular weights (1, 8, 40 and 500 kDa) and divalent cations (Ca²⁺, Mg²⁺ and Mn²⁺) and the trivalent cation La³⁺ were used in the experiments. Binding of DS was studied by use of the microelectrophoresis and monolayer technique. Binding depends strongly on cation and NaCl concentrations in the medium and does not occur in the absence of multivalent cations. Binding is modulated by the molecular weight of the polymers; DS with lower molecular weights lead to less negative zeta potentials at identical concentrations. A comparable monomer of DS, glucose-6-sulfate, does not change the zeta potential of DMPC vesicles. Monolayer experiments revealed a decrease in surface pressure after addition of multivalent cations and DS, indicating a stronger interaction of the cation–polymer complex with the phosphatidylcholine headgroups than its penetration into the phospholipid (PL) bilayer. The cation-mediated binding of DS to the vesicles leads to aggregation of the vesicles. The tendency to promote aggregation of DMPC vesicles is La³⁺>Ca²⁺>Mn²⁺≥ Mg²⁺. The aggregated vesicles show a stacklike arrangement of the bilayers as shown by freeze-fracture electron microscopy. The strong aggregation is accompanied by lipid mixing measured by the 1,4-nitrobenzo-2-oxa-1,3-diazole–phosphatidylethanolamine (PE)/lissamine rhodamine B sulfonyl-PE assay. At low ionic strength substantial lipid mixing can be observed in the previously mentioned order of the cations. This lipid mixing is accompanied by an increase in the permeability of the vesicles as revealed by the 1-aminonaphthalene-3,6,8-trisulfonic acid/p-xylenebis (pyridiium bromide) assay. The extent of leakage is determined by the cation used and the DS molecular weight. These interaction processes between the opposing bilayers are connected with a decrease in the water content in the gap between the opposing PL bilayers. As a measure for the change of the polar properties of the vesicle surface the shift of the emission wavelength of the fluorescent probe dansylphosphatidylethanolamine was measured. The effectiveness of divalent/trivalent cations to decrease the surface dielectric constant of DMPC vesicles also followed the sequence of ions as found for binding, PL mixing and leakage. The results are discussed in terms of the changed hydration at the bilayer surface induced by DS in the presence of multivalent ions. Received: 16 December 1998/Accepted: 17 December 1999     

Functionalized self-assembled monolayers for measuring single molecule lectin carbohydrate interactions

The specific interactions between sugar-binding proteins (lectins) and their complementary carbohydrates mediate several complex biological functions. There is a great deal of interest in uncovering the molecular basis of these interactions. In this study, we demonstrate the use of an efficient one-step amination reaction strategy to fabricate carbohydrate arrays based on mixed self-assembled monolayers. These allow specific lectin carbohydrate interactions to be interrogated at the single molecule level via AFM. The force required to directly rupture the multivalent bonds between Concanavalin A (Con A) and mannose were subsequently determined by chemical force microscopy. The mixed self-assembled monolayer provides a versatile platform with active groups to attach a 1-amino-1-deoxy sugar or a protein (Con A) while minimizing non-specific adhesion enabling quick and reliable detection of rupture forces. By altering the pH of the environment, the aggregation state of Con A was regulated, resulting in different dominant rupture forces, corresponding to di-, tri- and multiple unbinding events. We estimate the value of the rupture force for a single Con A-mannose bond to be 95 ± 10 pN. The rupture force is consistent even when the positions of the binding molecules are switched. We show that this synthesis strategy in conjunction with a mixed SAM allows determination of single molecules bond with high specificity, and may be used to investigate lectin carbohydrate interactions in the form of carbohydrate arrays as well as lectin arrays.     

Vesicle formed by amphiphilc cucurbit[6]uril: versatile, noncovalent modification of the vesicle surface, and multivalent binding of sugar-decorated vesicles to lectin

We report a novel vesicle formed by an amphiphilic CB[6] derivative, the surface of which can be easily modified via host-guest interactions by taking advantage of molecular cavities, readily accessible at the vesicle surface, and their strong affinity toward polyamines. Amphiphilic CB[6] derivative 1 synthesized by reaction between (allyloxy)12CB[6] and 2-[2-(2-methoxyethoxy)ethoxy]ethanethiol affords a vesicle that has been characterized by TEM, light scattering, and fluorescent dye entrapment experiments. Treatment of vesicle 1 with FITC (fluorescein isothiocyanate)-spermine conjugate ligand 2, in which spermine serves as a binding motif to CB[6] and FITC as a fluorescent tag, produced a surface-modified vesicle, which can be easily visualized by a confocal microscope. This result provides us with a new noncovalent, modular approach to the modification of vesicle surfaces. By treating the vesicle derived from the amphiphilic CB[6] with a tag-attached polyamine, we can easily decorate the surface of the vesicle with the tag. Sugar-decorated vesicles were prepared by this noncovalent method, and their interactions with concanavalin A (ConA) were studied. The binding constant of the vesicle decorated with mannose-spermidine conjugate 3 to ConA was measured to be approximately 3 x 104 M-1, which is almost 3 orders of magnitude higher than that of free ligand 3 to ConA (K = approximately 50 M-1). On the other hand, the binding constant of the vesicle coated with galactose-spermidine conjugate 4 to ConA was too small to be measured. These results illustrate the specific and multivalent interactions between the mannose-decorated vesicle and ConA. The ability for facile surface modification suggests many practical applications, including its use in targeted drug delivery and immunization.     

Fluorescence Turn‐On Sensing of Lectins with Mannose‐Substituted Tetraphenylethenes Based on Aggregation‐Induced Emission

The synthesis of mannose‐substituted tetraphenylethenes (TPEs) and their aggregation‐induced emission (AIE) behavior, induced by interactions with concanavalin A (Con A), are reported. A mixture of the mannose‐TPE conjugates and Con A in a buffer solution displays an intense blue emission on agglutination within a few seconds, which serves as a “turn‐on” fluorescent sensor for lectins. The sensing is also selective: the conjugates act as a sensor for Con A, but do not sense a galactose‐binding lectin, PNA. Con A‐recognition is not affected even in the presence of other proteins in a mixture. The conjugates also exhibit high sensitivity to detect Con A. An increased sensitivity of the conjugates results if mannopyranoside substituents are linked to the TPE‐core unit with a flexible chain and/or when the number of mannose residues increases.     

RNA selectively interacts with vesicles depending on their size

RNA and vesicles are two important molecular classes in the origin of life and early evolution, but they are not generally considered as interacting partners. The present paper reports about the interaction between tRNA (Esherichia coli) and vesicles made of the zwitterionic surfactant POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), partially positively charged with small molar fractions (max 10%) of the single-chained CTAB (cetyltrimethylammonium bromide). CTAB is capable to insert efficiently in POPC vesicles (as determined by zeta-potential measurements), and the binding of tRNA to such charged vesicles operates a strong selection being critically dependent upon the vesicle size. The binding of tRNA to the vesicles is size-selective as it induces a strongly pronounced process of aggregation of large vesicles (ca. 160-nm diameter) but not of small ones (ca. 80-nm diameter) that are stable against vesicle aggregation (as followed by dynamic light-scattering and optical density measurements). The aggregation of the large vesicles is fully reversible upon the addition of RNase A. The selective behavior of tRNA with respect to differently sized vesicles is observable in separated samples as well as in a mixture of both populations. In the latter case, the fraction of large vesicles readily aggregates in the presence of the small ones that remain unaltered in the mixture. This kind of discrimination capability of RNA might have been of importance in the early phases of the formation of the protocells.     

Vesicle aggregation by multivalent ligands: relating crosslinking ability to surface affinity

In an effort to improve the stability of our tissue-mimetic vesicle aggregates, we have investigated how increasing the valency of our multivalent crosslinking ligand, poly-l-histidine, affected both the extent of vesicle aggregation and the affinity of the multivalent ligand for the synthetic receptor Cu(1) embedded in the vesicle membranes. Although increasing ligand valency gave the anticipated increase in the size of the vesicle aggregates, isothermal calorimetric studies did not show the expected increase in the valence-corrected binding constant for the embedded receptors. To explain both observations, we have developed a simple new binding model that encompasses both multivalent binding to receptors on a single vesicle surface (intramembrane binding) and vesicle crosslinking (intermembrane binding).     

Naphthalene Imide Conjugates: Formation of Supramolecular Assemblies,and the Encapsulation and Release of Dyes through DNA‐Mediated Disassembly

We report the synthesis of two new amphiphilic conjugates 1 and 2 based on naphthalene di‐ and monoimide chromophores and the investigation of their photophysical, self‐assembly and DNA‐binding properties. These conjugates showed aqueous good solubility and exhibited strong interactions with DNA and polynucleotides such as poly(dG?dC)–poly(dG?dC) and poly(dA?dT)–poly(dA?dT). The interaction of these conjugates with DNA was evaluated by photo‐ and biophysical techniques. These studies revealed that the conjugates interact with DNA through intercalation with association constants in the order of 5–8×10⁴ M ^?1. Of these two conjugates, bolaamphiphile 1 exhibited a supramolecular assembly that formed vesicles with an approximate diameter of 220 nm in the aqueous medium at a critical aggregation concentration of 0.4 mM , which was confirmed by SEM and TEM. These vesicular structures showed a strong affinity for hydrophobic molecules such as Nile red through encapsulation. Uniquely, when exposed to DNA the vesicles disassembled, and therefore this transformation could be utilised for the encapsulation and release of hydrophobic molecules by employing DNA as a stimulus.     

Dissecting Molecular Aspects of Cell Interactions Using Glycodendrimersomes with Programmable Glycan Presentation and Engineered Human Lectins

Glycodendrimersomes with programmable surface display of glycan, together with artificially engineered galectins, were used to understand the physiological significance of human lectins with homodimeric and tandem‐repeat‐type displays. The mode of topological surface presentation and the density of glycan affected vesicle aggregation mediated by multivalent carbohydrate–protein interactions. The cross‐linking capacity of homodimeric lectins was enhanced by covalent connection of the two carbohydrate‐binding sites. These findings highlight the value of glycodendrimersomes as versatile cell membrane mimetics, and assays provide diagnostic tools for protein functionality. This work also provides guidelines for the design of cell separators, bioactive matrices, bioeffectors, and other biomedical applications.     

Potent Glycosidase Inhibition with Heterovalent Fullerenes: Unveiling the Binding Modes Triggering Multivalent Inhibition

Glycosidases are key enzymes in metabolism, pathogenic/antipathogenic mechanisms and normal cellular functions. Recently, a novel approach for glycosidase inhibition that conveys multivalent glycomimetic conjugates has emerged. Many questions regarding the mechanism(s) of multivalent enzyme inhibition remain unanswered. Herein we report the synthesis of a collection of novel homo‐ and heterovalent glyco(mimetic)‐fullerenes purposely conceived for probing the contribution of non‐catalytic pockets in glysosidases to the multivalent inhibitory effect. Their affinities towards selected glycosidases were compared with data from homovalent fullerene conjugates. An original competitive glycosidase–lectin binding assay demonstrated that the multivalent derivatives and the substrate compete for low affinity non‐glycone binding sites of the enzyme, leading to inhibition by a “recognition and blockage” mechanism. Most notably, this work provides evidence for enzyme inhibition by multivalent glycosystems, which will likely have a strong impact in the glycosciences given the utmost relevance of multivalency in Nature.     

Cell aggregation by scaffolded receptor clusters

The aggregation of cells by lectins or antibodies is important for biotechnological and therapeutic applications. One strategy to augment the avidity and aggregating properties of these mediators is to maximize the number of their ligand binding sites. The valency of lectins and antibodies, however, is limited by their quaternary structures. To overcome this limitation, we explored the use of polymers generated by ring-opening metathesis polymerization (ROMP) as scaffolds to noncovalently assemble multiple copies of a lectin, the tetravalent protein concanavalin A (Con A). We demonstrate that complexes between Con A and multivalent scaffolds aggregate cells of a T cell leukemia line (Jurkat) more effectively than Con A alone. We anticipate that synthetic scaffolds will offer a new means of facilitating processes that rely on cell aggregation, such as pathogen clearance and immune recognition.     

Multivalent binding of galactosylated cyclodextrin vesicles to lectin

Amphiphilic beta-cyclodextrins with alkylthio chains at the primary-hydroxyl side and galactosylthio-oligo-(ethylene glycol) units at the secondary-hydroxyl side, which form nanoparticles and vesicles, show multivalent effects in their binding to lectin.     

A Neoglycoprotein-Immobilized Fluorescent Magnetic Bead Suspension Multiplex Array for Galectin-Binding Studies

Carbohydrate-protein conjugates have diverse applications. They have been used clinically as vaccines against bacterial infection and have been developed for high-throughput assays to elucidate the ligand specificities of glycan-binding proteins (GBPs) and antibodies. Here, we report an effective process that combines highly efficient chemoenzymatic synthesis of carbohydrates, production of carbohydrate-bovine serum albumin (glycan-BSA) conjugates using a squarate linker, and convenient immobilization of the resulting neoglycoproteins on carboxylate-coated fluorescent magnetic beads for the development of a suspension multiplex array platform. A glycan-BSA-bead array containing BSA and 50 glycan-BSA conjugates with tuned glycan valency was generated. The binding profiles of six plant lectins with binding preference towards Gal and/or GalNAc, as well as human galectin-3 and galectin-8, were readily obtained. Our results provide useful information to understand the multivalent glycan-binding properties of human galectins. The neoglycoprotein-immobilized fluorescent magnetic bead suspension multiplex array is a robust and flexible platform for rapid analysis of glycan and GBP interactions and will find broad applications.     

Photoresponsive molecular recognition and adhesion of vesicles in a competitive ternary supramolecular system

A competitive photoresponsive supramolecular system is formed in a dilute aqueous solution of three components: vesicles of amphiphilic α-cyclodextrin host 1a, divalent p-methylphenyl guest 2 or divalent p-methylbenzamide guest 3, and photoresponsive azobenzene monovalent guest 5. Guests 2 and 3 form weak inclusion complexes with 1a (K(a)≈10(2) M(-1)), whereas azobenzene guest 5 forms a strong inclusion complex (K(a)≈10(4) M(-1)), provided it is in the trans state. The aggregation and adhesion of vesicles of host 1a is mediated by guest 2 (or 3) due to the formation of multiple intervesicular noncovalent links, as confirmed by using isothermal titration calorimetry (ITC), optical density measurements at 600 nm (OD600), dynamic light scattering (DLS), and cryogenic transmission electron microscopy (cryo-TEM). The addition of excess monovalent guest trans-5 to vesicles of 1a aggregated by divalent guest 2 (or 3) causes the dispersion of vesicles of 1a because trans-5 displaces 2 (as well as 3) from the vesicle surface. Upon UV irradiation of a dilute ternary mixture of vesicles of 1a, guest 2 (or 3), and competitor trans-5, compound trans-5 isomerizes to cis-5, and renewed aggregation of vesicles of 1a by guest 2 (or 3) occurs because 2 (as well as 3) displaces cis-5 from the vesicle surface. Subsequent visible irradiation causes the redispersion of vesicles of 1a because cis-5 reisomerizes into trans-5, which again displaces guest 2 (or 3) from the vesicle surface. In this way, the competitive photoresponsive aggregation and dispersion of vesicles can be repeated for several cycles.     

Electrochemical, microscopic, and spectroscopic characterization of prevesicle nanostructures and vesicles on mixed cationic surfactant systems

Several experimental techniques (conductivity, zeta potential, transmission electronic microscopy, and steady-state fluorescence spectroscopy) have been used to study the formation of mixed colloidal aggregates consisting of a cationic double-chain surfactant, di-dodecyldimethylammonium bromide (di-C12DMAB), and a single-chain alkyltrimethylammonium bromide with 10 and/or 14 carbon atoms (decyltrimethylammonium bromide, C10TAB, and/or tetradecyltrimethylammonium bromide, C14TAB). Special interest has been devoted to the prevesicle domain, within which the formation of aggregated nanostructures was first reported in our laboratory. For that purpose, studies have been carried out on the very dilute region by means of conductivity experiments, confirming the existence of two critical aggregation concentrations in that concentration domain: the so-called mixed critical aggregate concentration, CAC, and the mixed critical vesicle concentration, CVC. By carrying out TEM experiments on negatively stained samples, we were surprised to find a number of aggregates without a clear aggregation pattern and with a variety of sizes and shapes at concentrations below CAC, where only monomers were expected. However, the nanoaggregates found at concentrations between CAC and CVC, also by TEM microscopy, show a clear and ordered "fingerprint"-like aggregation pattern similar to the liquid-crystalline phases reported for DNA-liposome complexes and/or DNA packed with viral capsids. Finally, at total surfactant concentrations above CVC, the aggregates were confirmed, by means of cryo-TEM micrographs and zeta potential measurements, to be essentially unilamellar spherical vesicles with a medium polydispersity and a net-averaged surface density charge of around 12 x 10(-3) C m(-2). The fluorescence emission of two probes, TNS (anionic) and PRODAN (nonionic), allows for the analysis of the micropolarity and microviscosity of the different microenvironments present in aqueous surfactant solutions where the above-mentioned vesicle and prevesicle aggregates are present.     

Interactions between antimicrobial polynorbornenes and phospholipid vesicles monitored by light scattering and microcalorimetry

Antimicrobial polynorbornenes composed of facially amphiphilic monomers have been previously reported to accurately emulate the antimicrobial activity of natural host-defense peptides (HDPs). The lethal mechanism of most HDPs involves binding to the membrane surface of bacteria leading to compromised phospholipid bilayers. In this paper, the interactions between biomimetic vesicle membranes and these cationic antimicrobial polynorbornenes are reported. Vesicle dye-leakage experiments were consistent with previous biological assays and corroborated a mode of action involving membrane disruption. Dynamic light scattering (DLS) showed that these antimicrobial polymers cause extensive aggregation of vesicles without complete bilayer disintegration as observed with surfactants that efficiently solubilize the membrane. Fluorescence microscopy on vesicles and bacterial cells also showed polymer-induced aggregation of both synthetic vesicles and bacterial cells. Isothermal titration calorimetry (ITC) afforded free energy of binding values (Delta G) and polymer to lipid binding ratios, plus revealed that the interaction is entropically favorable (Delta S>0, Delta H>0). It was observed that the strength of vesicle binding was similar between the active polymers while the binding stoichiometries were dramatically different.     

On-bead synthesis and binding assay of chemoselectively template-assembled multivalent neoglycopeptides

The investigation of recognition events between carbohydrates and proteins, especially the control of how spatial factors and binding avidity are correlated in, remains a great interest for glycomics. Therefore, the development of efficient methods for the rapid evaluation of new ligands such as multivalent glycoconjugates is essential for diverse diagnostic or therapeutic applications. In this paper we describe the synthesis of chemoselectively-assembled multivalent neoglycopeptides and the subsequent recognition assay on a solid support. Aminooxylated carbohydrates (betaLac-ONH(2) 4, alphaGalNAc-ONH(2) 9 and alphaMan-ONH(2) 13) have been prepared as carbohydrate-based recognition elements and assembled as clusters onto a cyclopeptidic scaffold by an oxime-based strategy in solid phase. Further binding tests between lectins and beads of resin derivatized with neoglycopeptides displaying clustered lactoses, N-acetylgalactoses and mannoses (18-20) have shown specific recognition and enhanced affinity through multivalent interactions, suggesting that the local density of carbohydrate-based ligands at the bead surface is crucial to improve the interaction of proteins of weak binding affinity. This solid phase strategy involving both molecular assembly and biological screening provides a rapid and efficient tool for various applications in glycomics.