Synthesis of Multivalent Carbohydrate‐Centered Glycoclusters as Nanomolar Ligands of the Bacterial Lectin LecA from Pseudomonas aeruginosa

A family of fifteen glycoclusters based on a cyclic oligo‐(1→6)‐β‐D ‐glucosamine core has been designed as potential inhibitors of the bacterial lectin LecA with various valencies (from 2 to 4) and linkers. Evaluation of their binding properties towards LecA has been performed by a combination of hemagglutination inhibition assays (HIA), enzyme‐linked lectin assays (ELLA), and isothermal titration microcalorimetry (ITC). Divalent ligands displayed dissociation constants in the sub‐micromolar range and tetravalent ligands displayed low nanomolar affinities for this lectin. The influence of the linker could also be demonstrated; aromatic moieties are the best scaffolds for binding to the lectin. The affinities observed in vitro were then correlated with molecular models to rationalize the possible binding modes of these glycoclusters with the bacterial lectin.     

Probing carbohydrate-lectin recognition in heterogeneous environments with monodisperse cyclodextrin-based glycoclusters

A series of β-cyclodextrin (βCD)-scaffolded glycoclusters exposing heterogeneous yet perfectly controlled displays of α-mannosyl (α-Man) and β-lactosyl (β-Lact) antennas were synthesized to probe the mutual influence of varying densities of the saccharide motifs in the binding properties toward different plant lectins. Enzyme-linked lectin assay (ELLA) data indicated that the presence of β-Lact residues reinforced binding of α-Man to the mannose-specific lectin concanavalin A (Con A) even though homogeneous β-Lact clusters are not recognized at all by this lectin, supporting the existence of synergic recognition mechanisms (heterocluster effect). Conversely, the presence of α-Man motifs in the heteroglycoclusters also resulted in a binding-enhancing effect of β-Lact toward peanut agglutinin (PNA), a lectin strongly binding multivalent lactosides but having no detectable affinity for α-mannopyranosides, for certain architectural arrangements. Two-site, sandwich-type ELLA data corroborated the higher lectin clustering efficiency of heterogeneous glycoclusters compared with homogeneous displays of the putative sugar ligand with identical valency. A turbidity assay was also consistent with the previous observations. Most revealingly, the lectin cross-linking ability of heterogeneous glycoclusters was sensitive to the presence of high concentrations of the non-ligand sugar, strongly suggesting that "mismatching" saccharide motifs may modulate carbohydrate-lectin specific recognition in a lectin-dependent manner when present in highly dense displays together with the "matching" ligand, a situation frequently encountered in biological systems.     

First synthesis of "Majoral-Type" glycodendrimers bearing covalently bound alpha-D-mannopyranoside residues onto a hexachlocyclotriphosphazene core

A short and efficient strategy for the first synthesis of "Majoral-Type" multivalent glycodendrimers bearing covalently bound alpha-D-mannopyranosides onto a cyclotriphosphazene scaffold assembled using single-step Sonogashira and click chemistry is reported. New glycoclusters with valencies ranging from 6 to 18 and different epitope spatial arrangements were obtained. Cross-linking abilities of this series of glycodendrimers were evaluated with the model lectin from Canavalia ensiformis (Concanavalin A). The decameric mannoside 23, built around 19, was shown to be much faster in cross-linking the tetravalent lectin Concanavalin A than the positive control, which is the polysaccharide mannan from yeast. The new glycoconjugates reported may be promising tools as probes or effectors of biological processes involving multivalent carbohydrate-binding proteins.     

Rational design and synthesis of optimized glycoclusters for multivalent lectin-carbohydrate interactions: influence of the linker arm

The design of multivalent glycoclusters requires the conjugation of biologically relevant carbohydrate epitopes functionalized with linker arms to multivalent core scaffolds. The multigram-scale syntheses of three structurally modified triethyleneglycol analogues that incorporate amide moiety(ies) and/or a phenyl ring offer convenient access to a series of carbohydrate probes with different water solubilities and rigidities. Evaluation of flexibility and determination of preferred conformations were performed by conformational analysis. Conjugation of the azido-functionalized carbohydrates with tetra-propargylated core scaffolds afforded a library of 18 tetravalent glycoclusters, in high yields, by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The compounds were evaluated for their ability to bind to PA-IL (the LecA lectin from the opportunistic pathogen Pseudomonas aeruginosa). Biochemical evaluation through inhibition of hemagglutination assays (HIA), enzyme-linked lectin assays (ELLA), surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) revealed improved and unprecedented affinities for one of the monovalent probes (K(d)=5.8?μM) and also for a number of the tetravalent compounds that provide several new nanomolar ligands for this tetrameric lectin.     

Synthesis of dodecavalent fullerene-based glycoclusters and evaluation of their binding properties towards a bacterial lectin

Multivalency is playing a major role in biological processes and particularly in lectin-carbohydrate interactions. The design of high-affinity ligands of lectins should provide molecules capable of interfering with these biological processes and potentially inhibit bacterial or viral infections. Azide-alkyne "click" chemistry was applied to the synthesis of dodecavalent fullerene-based glycoclusters. The conjugation could be efficiently performed from alkyne or azide functions on either partners (i.e. hexakis-fullerene adduct or glycoside). PA-IL is a bacterial lectin from the opportunistic pathogen Pseudomonas aeruginosa and is involved in the recognition of glycoconjugates on human tissues. The glycoclusters obtained were evaluated as ligands of PA-IL and for their potential for competing with its binding to glycosylated surfaces. The affinities measured by hemagglutination inhibition assay (HIA), enzyme-linked lectin assay (ELLA), and surface plasmon resonance (SPR) displayed a significant "glycoside cluster effect" with up to a 12,000-fold increase in binding when comparing a monovalent carbohydrate reference probe with a dodecavalent fullerene-based glycocluster, albeit with some differences depending on the analytical technique.     

CuAAC synthesis of resorcin[4]arene-based glycoclusters as multivalent ligands of lectins

Synthetic multivalent glycoclusters show promise as anti-adhesives for the treatment of bacterial infections. Here we report the synthesis of a family of tetravalent galactose and lactose functionalised macrocycles based on the resorcin[4]arene core. The development of diastereoselective synthetic routes for the formation of lower-rim propargylated resorcin[4]arenes and their functionalistion via Cu-catalyzed azide-alkyne click chemistry is described. ELLA binding studies confirm that galactose sugar clusters are effective ligands for the PA-IL bacterial lectin of Pseudomonas aeruginosa while poor binding for the lactose-based monovalent probe and no binding could be measured for the multivalent glycoclusters was observed for the human galectin-1.     

Rational Design and Synthesis of Optimized Glycoclusters for Multivalent Lectin–Carbohydrate Interactions: Influence of the Linker Arm

The design of multivalent glycoclusters requires the conjugation of biologically relevant carbohydrate epitopes functionalized with linker arms to multivalent core scaffolds. The multigram‐scale syntheses of three structurally modified triethyleneglycol analogues that incorporate amide moiety(ies) and/or a phenyl ring offer convenient access to a series of carbohydrate probes with different water solubilities and rigidities. Evaluation of flexibility and determination of preferred conformations were performed by conformational analysis. Conjugation of the azido‐functionalized carbohydrates with tetra‐propargylated core scaffolds afforded a library of 18 tetravalent glycoclusters, in high yields, by Cu^I‐catalyzed azide–alkyne cycloaddition (CuAAC). The compounds were evaluated for their ability to bind to PA‐IL (the LecA lectin from the opportunistic pathogen Pseudomonas aeruginosa). Biochemical evaluation through inhibition of hemagglutination assays (HIA), enzyme‐linked lectin assays (ELLA), surface plasmon resonance (SPR), and isothermal titration microcalorimetry (ITC) revealed improved and unprecedented affinities for one of the monovalent probes (K_d=5.8 μM ) and also for a number of the tetravalent compounds that provide several new nanomolar ligands for this tetrameric lectin.     

Reactivity Tournament of Isothiocyanato-Functionalized Saccharides with 1,6-Diamino-3,6-oxaoctane

Summary.  Thiourea-bridged glycoclusters and glycodendrimers are described in the literature as mimetics of (oligoantennary) oligosaccharides to serve as high-affinity lectin ligands. In extension of this concept, the assembly of different, structurally varied isothiocyanato-functionalized sugar derivatives on an oligoamine scaffold would lead to novel mixed glycoclusters. To control this approach, the relative reactivities of the isothiocyanates used in the thiourea-bridging reaction have to be known. Therefore, competition experiments with six different sugar isothiocyanates were carried out using 1,8-diamino-3,6-dioxaoctane as a symmetrical difunctionalized core molecule. Reactivities were ranked on the basis of integration ratios in the ¹H NMR spectra. A first mixed thiourea-bridged glycocluster was successfully prepared. Received June 13, 2001. Accepted October 31, 2001     

Probing the Nature of the Cluster Effect Observed with Synthetic Multivalent Galactosides and Peanut Agglutinin Lectin

We designed a set of multi‐galactosides with valencies ranging from one to seven and different spacer‐arm lengths. The compounds display a high structural homology for a strict assessment of multivalent phenomena. The multimers were first evaluated by an enzyme‐linked lectin assay (ELLA) toward the peanut agglutinin (PNA). The binding affinity was shown to be dependent on the spacer‐arm length, and cluster effects were observed for the galactosides bearing the shortest and the longest linkers. The latter compounds were shown to be much more potent PNA cross‐linkers in a “sandwich assay”. Dynamic light scattering (DLS) experiments also revealed the formation of soluble aggregates between heptavalent derivatives with medium or long linkers and the labeled PNA. ELLA experiments performed with valency‐controlled clusters and labeled lectins are therefore not always devoid from aggregative processes. The precise nature of the multivalent interaction observed by ELLA for the compounds bearing the shortest linkers, which are unable to form PNA aggregates, was further investigated by atomic force microscopy (AFM). The galactosides were grafted onto the tip of a cantilever and the PNA lectin onto a gold surface. Similar unbinding forces were registered when the valency of the ligands was increased, thus showing that the multimers cannot interact more strongly with PNA. Multiple binding events to the PNA were also never observed, thus confirming that a chelate binding mode does not operate with the multivalent galactosides, probably because the linkers are too short. Altogether, these results suggest that the cluster effect that operates in ELLA with the multimers is not related to additional PNA stabilizations and can be ascribed to local concentration effects that favor a dynamic turnover of the tethered galactosides in the PNA binding sites.     

Optimizing saccharide-directed molecular delivery to biological receptors: design, synthesis, and biological evaluation of glycodendrimer-cyclodextrin conjugates

Dendritic beta-cyclodextrin (betaCD) derivatives bearing multivalent mannosyl ligands have been prepared and assessed for their binding efficiency toward the tetrameric plant lectin concanavalin A (Con A) and a mammalian mannose/fucose specific cell surface receptor from macrophages. The synthetic strategy exploits the reactivity between isothiocyanate and amine functionalities for the high-yielding assembly via thioureido links of the various building blocks, including host, spacer, branching, and carbohydrate ligand elements. The methodology has been applied to the preparation of a series of betaCD-polymannoside scaffolds differing in the ligand valency and geometry. This series allowed us to explore: (i) The effects of the glycodendritic architecture on the binding efficiency; (ii) the mutual influence between the cyclodextrin core and the glycodendritic moieties on the molecular inclusion and lectin-binding properties; and (iii) the consequence of inclusion complex formation, using the anticancer drug docetaxel (Taxotère) as a target guest, on biological recognition. Our results confirm the high drug solubilization capability of this new type of betaCD-dendrimer construct and indicate that subtle changes in the architecture of the conjugate may have important consequences on receptor affinity. Interestingly, the host-guest interaction can be monitored to build up supramolecular dynamic glycoclusters with increased lectin affinity. Alternatively, the information obtained from the structure-lectin-binding avidity-inclusion capability studies has been put forward in the design of very efficient molecular transporters for docetaxel based on glycodendritic CD dimers.     

Linear Precision Glycomacromolecules with Varying Interligand Spacing and Linker Functionalities Binding to Concanavalin A and the Bacterial Lectin FimH

A series of precision glycomacromolecules is prepared following previously established solid phase synthesis allowing for controlled variations of interligand spacing and the overall number of carbohydrate ligands. In addition, now also different linkers are installed between the carbohydrate ligand and the macromolecular scaffold. The lectin binding behavior of these glycomacromolecules is then evaluated in isothermal titration calorimetry (ITC) and kinITC experiments using the lectin Concanavalin A (Con A) in its dimeric and tetrameric form. The results indicate that both sterical and statistical effects impact lectin binding of precision glycomacromolecules. Moreover, ITC results show that highest affinity toward Con A can be achieved with an ethyl phenyl linker, which parallels earlier findings with the bacterial lectin FimH. In this way, a first set of glycomacromolecule structures is selected for testing in a bacterial adhesion–inhibition study. Here, the findings point to a one‐sugar binding mode mainly affected by sterical restraints of the nonbinding parts of the respective glycomacromolecule.     

Toward the Rational Design of Galactosylated Glycoclusters That Target Pseudomonas aeruginosa Lectin A (LecA): Influence of Linker Arms That Lead to Low‐Nanomolar Multivalent Ligands

Anti‐infectious strategies against pathogen infections can be achieved through antiadhesive strategies by using multivalent ligands of bacterial virulence factors. LecA and LecB are lectins of Pseudomonas aeruginosa implicated in biofilm formation. A series of 27 LecA‐targeting glycoclusters have been synthesized. Nine aromatic galactose aglycons were investigated with three different linker arms that connect the central mannopyranoside core. A low‐nanomolar (K_d=19 nm , microarray) ligand with a tyrosine‐based linker arm could be identified in a structure–activity relationship study. Molecular modeling of the glycoclusters bound to the lectin tetramer was also used to rationalize the binding properties observed.     

Synthesis and Langmuir studies of bivalent and monovalent alpha-D-mannopyranosides with lectin Con A

Highly avid interaction between carbohydrate ligands and lectin receptors nominally requires the ligand presentation in a clustered form. We present herein an approach involving Langmuir monolayer formation of the sugar ligands and the assessment of their lectin binding at the air-water interface. Bivalent alpha-D-mannopyranoside containing the glycolipid ligand was used to study its binding profiles with lectin Con A, in comparison to the corresponding monovalent glycolipid. In addition to the bivalent and monovalent nature of the glycolipid ligands at the molecular level, the ligand densities at the monolayer level were varied with the aid of a nonsugar lipid molecule so as to obtain mixed monolayers with various sugar-nonsugar ratios. Lectin binding of bivalent and monovalent ligands at different ratios was monitored by differential changes in the surface area per molecule of the mixed monolayer, with and without the lectin. The present study shows that maximal binding of the lectin to the bivalent ligand occurs at lower sugar densities at the interface ( approximately 10% sugar in the mixed monolayer) than for that of the monovalent ligand ( approximately 20% sugar in the mixed monolayer). It is observed that complete coverage of the monolayer with only the sugar ligands does not allow all of the sugars to be functionally active.     

A comparison of biological and calorimetric analyses of multivalent glycodendrimer ligands for concanavalin A

The cluster glycoside effect — the observation that multivalent glycosides bind to their polyvalent protein receptors with apparent affinities greater than those that can be rationalized solely on the basis of valency — is by now a well established phenomenon. As part of a continuing effort to provide a molecular basis for the cluster glycoside effect, we report here the synthesis of two series of mannosylated dendritic ligands and their performance in a range of competitive and non-competitive binding assays, including hemeagglutination inhibition (HIA), enzyme-linked lectin assays (ELLA) and isothermal titration microcalorimetry (ITC). The first series of ligands contained a semi-rigid glycylglycine spacer and showed no significant performance enhancement in any binding studies. The second series of ligands contained a flexible tetraethylene glycol spacer; these ligands showed marked enhancements at tetravalent and hexavalent levels in both HIA (IC₅₀=3 and<0.8 μM, respectively) and ITC (K_A=6.2×10⁴ and 1.5×10⁶ M⁻¹, respectively) studies. In all cases, the thermodynamic parameters of association are consistent with non-specific aggregation rather than enhanced lectin–ligand affinity. This conclusion is reinforced by the lack of enhancements in ligand activity observed in ELLA studies.     

Synthesis of Oligomeric Mannosides and Their Structure‐Binding Relationship with Concanavalin A

Small glycodendrimers with α‐mannosyl ligands were synthesized by using copper‐catalyzed azide–alkyne coupling chemistry and some of these molecules were used as multivalent ligands to study the induction of concanavalin A (Con A) precipitation. The results showed that the monovalent mannose ligand could induce the precipitation of Con A. This unexpected finding initiated a series of studies to characterize the molecular basis of the ligand–lectin interaction. The atypical precipitation is found to be specific to the mannose, fluorescein moiety (FITC), and Con A. Apparently the mannose ligand binds to Con A through hydrogen‐bonding interactions, whereas the binding of FITC is mediated by hydrophobic forces.     

Selectivity among two lectins: probing the effect of topology, multivalency and flexibility of "clicked" multivalent glycoclusters

The design of multivalent glycoconjugates has been developed over the past decades to obtain high-affinity ligands for lectin receptors. While multivalency frequently increases the affinity of a ligand for its lectin through the so-called "glycoside cluster effect", the binding profiles towards different lectins have been much less investigated. We have designed a series of multivalent galactosylated glycoconjugates and studied their binding properties towards two lectins, from plant and bacterial origins, to determine their potential selectivity. The synthesis was achieved through copper(I)-catalysed azide-alkyne cycloaddition (CuAAC) under microwave activation between propargylated multivalent scaffolds and an azido-functionalised carbohydrate derivative. The interactions of two galactose-binding lectins from Pseudomonas aeruginosa (PA-IL) and Erythrina cristagalli (ECA) with the synthesized glycoclusters were studied by hemagglutination inhibition assays (HIA), surface plasmon resonance (SPR) and isothermal titration microcalorimetry (ITC). The results obtained illustrate the influence of the scaffold's geometry on the affinity towards the lectin and also on the relative potency in comparison with a monovalent galactoside reference probe.     

Dendri-RAFTs: a second generation of cyclopeptide-based glycoclusters

Synthetic glycoclusters and their related biological applications have stimulated increasing interest over the last decade. As a prerequisite to discovering active and selective therapeuticals, the development of multivalent glycoconjugates with diverse topologies is faced with inherent synthetic and structural characterisation difficulties. Here we describe a new series of molecularly-defined glycoclusters that were synthesized in a controlled manner using a robust and versatile divergent protocol. Starting from a Regioselectively Addressable Functionalized Template (RAFT) carrier, either a polylysine dendritic framework or a second RAFT, then 16 copies of βGal, αMan, βLac or cancer-related Thomsen-Freidenreich (αTF) antigen were successively conjugated within the same molecule using oxime chemistry. We thus obtained a new generation of dendri-RAFTs glycoclusters with high glycosidic density and variable spatial organizations. These compounds displaying 16 endgroups were unambiguously characterized by NMR spectroscopy and mass spectrometry. Further biological assays between a model lectin from Canavalia ensiformis (ConA) and mannosylated glycoclusters revealed a higher inhibition potency than the tetravalent counterpart, in particular for the hexadecavalent polylysine skeleton. Together with the efficiency of the synthetic and characterisation processes, this preliminary biological study provided clear evidence of promising properties that make the second generation of cyclopeptide-based glycoclusters attractive for biomedical applications.     

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.     

Binding affinity properties of dendritic glycosides based on a beta-cyclodextrin core toward guest molecules and concanavalin A.

The inclusion behavior and concanavalin A binding properties of hepta-antennated and newly synthesized tetradeca-antennated C-6-branched mannopyranosyl and glucopyrannosyl cyclomaltoheptaose (beta-cyclodextrin) derivatives have been evaluated by isothermal titration microcalorimetry and enzyme-linked lectin assay (ELLA), respectively. The synthesis of three first-order dendrimers based on a beta-cyclodextrin core containing 14 1-thio-beta-D-glucose, 1-thio-beta-mannose, and 1-thio-beta-rhamnose residues was performed following a convergent approach and involving (1) preparation of a thiolated bis-branched glycoside building block and (2) attachment of the building block onto heptakis(6-deoxy-6-iodo)-beta-cyclodextrin. Calorimetric titrations performed at 25 degrees C in buffered aqueous solution (pH 7.4) gave the affinity constants and the thermodynamic parameters for the inclusion complex formation of these beta-cyclodextrin derivatives with guests sodium 8-anilino-1-naphthalensulfonate (ANS) and 2-naphthalenesulfonate. The host capability of the persubstituted beta-cyclodextrins decreased with respect to the native beta-CD when sodium 2-naphthalenesulfonate was used as a guest and improved when ANS was used as a guest molecule. Heptavalent mannoclusters based on beta-CD cores enhance the lectin binding affinity due to the cluster effect; however, the increase of the valency from 7 to 14 ligands did not contribute to the improvement of the concanavalin A binding affinity. In addition, the synthesized hyperbranched mannoCDs lost completely the capability as a host molecules.     

Influencing receptor-ligand binding mechanisms with multivalent ligand architecture

Multivalent ligands can function as inhibitors or effectors of biological processes. Potent inhibitory activity can arise from the high functional affinities of multivalent ligand-receptor interactions. Effector functions, however, are influenced not only by apparent affinities but also by alternate factors, including the ability of a ligand to cluster receptors. Little is known about the molecular features of a multivalent ligand that determine whether it will function as an inhibitor or effector. We envisioned that, by altering multivalent ligand architecture, ligands with preferences for different binding mechanisms would be generated. To this end, a series of 28 ligands possessing structural diversity was synthesized. This series provides the means to explore the effects of ligand architecture on the inhibition and clustering of a model protein, the lectin concanavalin A (Con A). The structural parameters that were varied include scaffold shape, size, valency, and density of binding elements. We found that ligands with certain architectures are effective inhibitors, but others mediate receptor clustering. Specifically, high molecular weight, polydisperse polyvalent ligands are effective inhibitors of Con A binding, whereas linear oligomeric ligands generated by the ring-opening metathesis polymerization have structural properties that favor clustering. The shape of a multivalent ligand also influences specific aspects of receptor clustering. These include the rate at which the receptor is clustered, the number of receptors in the clusters, and the average interreceptor distance. Our results indicate that the architecture of a multivalent ligand is a key parameter in determining its activity as an inhibitor or effector. Diversity-oriented syntheses of multivalent ligands coupled with effective assays that can be used to compare the contributions of different binding parameters may afford ligands that function by specific mechanisms.