Synthesis and Biological Applications of Glycoconjugates

Infection by bacteria is often initiated by the specific recognition of host epithelial surfaces by adhesins and lectins. These glycan binding proteins (GBP) are therefore virulence factors that play a role in the first step of adhesion and invasion. The adhesins are part of organelles, they are generally located at the tip of pili or fimbriae. On the opposite, soluble lectins occur either as individual proteins, or in association with toxins or enzymes. The human targets for bacterial adhesins and lectins are mostly fucosylated human histo-blood groups and/or sialylated epitopes. The binding of lectins and adhesins to host epithelial glycoconjugates is amplified by the multivalent presentation of the binding sites. The analysis of the available crystal structures of bacterial lectins and adhesins helps deciphering the structure/function relationship for this important class of proteins. It is a prerequisite for the development of multivalent high affinity ligands in antibacterial strategies.

Adhesion of bacteria to glycosylated cells and surfaces is largely facilitated through adhesive organells projecting from the bacterial surface, which are called fimbriae. The most important fimbriae in Escherichia coli and in most of the other enterobacteria are the type 1 fimbriae which mediate adhesion in an α-D-mannoside-specific manner. The lectin that is responsible for binding terminal α-D-mannosyl residues is called FimH and is located at the tips of type 1 fimbriae. FimH is known to mediate shear-enhanced adhesion of bacteria to mannosylated cells or surfaces. Like many adhesins, FimH is a two-domain protein consisting of a pilin domain, that anchors FimH to the fimbrial shaft, and a distal, N-terminal lectin domain, harboring the carbohydrate binding pocket. A variety of natural as well as synthetic ligands for type 1 fimbriated bacteria and FimH, respectively, have been tested in order to improve our understanding of this protein-carbohydrate interaction in cellular adhesion and in an attempt to develop effective low-molecular weight compounds for an anti-adhesion therapy. Besides tailor-made ligands that have been designed based on the computer docking, various multivalent cluster mannosides have revealed interesting activities. In this review, carbohydrate binding by FimH will be discussed and modern means for its investigation including photoaffinity labelling will be described. In addition, very recent crystallographic work will be documented that provides an explanation for shear-enhanced binding of type 1 fimbriated E. coli.

Calixarenes, the cyclic oligomers obtained by condensation of phenols or resorcinols with aldehydes, are ideal scaffolds for the construction of multivalent glycosylated ligands with unique properties. Thanks to the possibility that we can vary their size, valency and conformation and finely tune the topology of the saccharide units in the space, a wide variety of glycocalixarenes could have been prepared in the last 15 years. In this account, after a brief review on the basic chemistry of calixarenes, we will focus the attention on the most convenient methodologies used so far for the synthesis of glycocalixarenes and on their aggregation and biological properties. Glycocalixarenes often show, in fact, an impressive ability in the recognition and inhibition of carbohydrate binding proteins (lectins) disclosing remarkable multivalent effects. The amphiphilic character, due to the simultaneous presence of highly hydrophilic saccharide units and of a lipophilic aromatic backbone, confers to glycocalixarenes a marked tendency to self-aggregate in water solution. A quite unique peculiarity of these glycoclusters is related to their combined ability to firmly bind small organic molecules and to strongly and selectively interact with lectins, which makes them ideal candidates for tomorrow's site-specific drug-delivery systems. Other important biological functions have been envisaged to be influenced by the multivalency of glycocalixarenes which span gene-delivery, inhibition of tumor cell migration and proliferation to stimulation of the immunogenic system.

Studies on multivalent carbohydrate protein interactions critically depend on the nature of protein's binding sites, their number and also their relative three dimensional orientations. Although a wide range of neoglycoconjugates including glycodendrimers has been synthesized to address this issue, no systematic rules exist yet that can predict the optimal shapes, size, and number of required exposed carbohydrate ligands. This chapter will illustrate a few examples of bacterial and human lectins together with bacterial toxins having varied number of carbohydrate recognition domains that necessitated multivalent glycoconjugates. In particular, fullerenes, because of their particular physical properties, have been used to describe novel synthetic strategies and possible fit with concomitant lectins.

Cholera and travellers' diarrhoea are caused by AB5 protein toxins that bind to ganglioside GM1 at the surface of the cells lining the intestine. Inhibition of this protein-carbohydrate interaction would prevent the toxin from entering the cells, and thus prevents toxin-induced diarrhoea. In this review we will describe the structures of the cholera and E. coli heat-labile toxins, and summarize the main strategies that have led to the development of monovalent and multivalent inhibitors of these toxins. A number of key design concepts emerge from these studies including the importance of pre-organization of the sugar residues within the monovalent ligands, and also the pre-organization of monovalent ligand groups within larger multivalent ligands. The importance of chelation and protein aggregation as mechanisms of multivalent inhibition is also discussed.

The biological implications of lectins have prompted a large number of research projects at the interface between biology and chemistry for a better understanding of their roles. Several synthetic high affinity ligands have been designed in order to inhibit their negative effects such as bacterial or viral infections and cancer. Among these receptor proteins, galectins are galactose-binding lectins implicated in inflammation or cancer and are important biological targets for the design of treatment against cancer. The careful design of high affinity ligands for galectins has been investigated through several studies using either (1) a “medicinal chemistry” approach in which the native ligand (i.e. galactose) is modified on one or several positions or (2) based on a multivalent approach in which galactose is repeated n times at the periphery of a core scaffold. Both strategies yielded essential information about the fundamental aspects of galectin-ligand interactions and provided a better knowledge of the implications of galectins in biology. The present review with 130 references will focus particularly on the past decade and present the most recent results obtained in this field for monovalent and multivalent ligands of galectins.

Dendrimers, displaying a regularly branched tree-like structure, offer an optimal synthetic platform to explore multivalency effects, in particular to construct bioactive glycoclusters as ligands for lectins. We have used combinatorial peptide chemistry to prepare glycopeptide dendrimer libraries as glycoclusters of the general structure (sugar-XXX)4(LysXXX)2LysXXXNH2 (X = alpha-amino acid, Lys = branching lysine residue). Binding assay was performed on such combinatorial libraries bearing an alpha-C-fucoside endgroup, and led to the discovery of strong ligands for the fucose specific lectin LecB from Pseudomonas aeruginosa. These dendrimers are potent inhibitors of biofilms, and require a tetravalent ligand for activity. In a related approach, involving binding assay to cancer cells using galactosylated glycopeptide dendrimers led to drug-delivery dendrimers. In both cases the amino acid composition of the dendrimer branches strongly influences the biological activity of the glycoclusters in addition to the simple multivalency effects, demonstrating the utility of peptide chemistry for systematically optimizing physicochemical and biological properties of the dendrimers.

The emergence of glycomics has deeply stimulated the design of new bioactive glycoclusters over the past decade. Among the increasing number of multivalent synthetic structures reported so far, cyclopeptide-based glycoclusters (CBGs) have recently shown promising interest for diverse biological applications. This chapter aims at describing the major advances in this field, with a special focus on the inhibition of carbohydrate-protein interactions and the synthetic vaccines.

Nucleic acids have been widely investigated as biological tools for various applications including gene silencing, DNA chips and drug targets. Interactions involving nucleic acids are generally well known making their rationalization straightforward. However, they generally suffer from a lack of bioavailability. One of the main strategies to overcome these limitations is the conjugation of oligonucleotides with an appropriate reporter. As carbohydrates are involved in a huge variety of biological processes including molecular and cell surface recognitions, oligonucleotide glycoconjugates are perhaps one of the most promising approaches to improving cellular delivery of oligonucleotides. Other applications of these conjugates have emerged like the use of DNAdirected immobilization to prepare carbohydrate biochips or the construction of glycoclusters by self organization of oligonucleotides. This chapter summarizes the applications of these conjugates and approaches to prepare them through some recent examples.

Multivalent sugar-based materials have attracted attention since the functional role of carbohydrates in biology has been disclosed. The design of artificial systems that mimics the polyvalent carbohydrate organization at cell surface has been envisaged as a strategy to study and intervene in carbohydrate-mediated interactions. One of the first synthetic glycomaterials which appeared in the literature were glycoliposomes, dynamic systems that resemble the glycocalix in the phospholipidic bilayer of cell membranes. Glycoliposomes are non-covalent systems which have been used since the seventies as multivalent tools in carbohydrate-based interactions against pathogens, for enhancing immunity and as molecular carriers in drug delivery. In former years, the advent of nanotechnology has allowed the design and construction of new materials similar in size to biologically relevant molecules (proteins, nucleic acids, etc) and displaying unique physical properties. The bio-functionalization of metallic nanomaterials with carbohydrates generated a new class of glycomaterials, named glyconanoparticles, which present carbohydrates in a highly multivalent way and in high local concentrations. At the same time, the quantum size properties of metallic nanoclusters can be used for biosensing, diagnostics, and (in perspective) therapy. This review focuses on glycoliposomes and covalently-functionalized glyconanoparticles which make use of the “glyco-code” to address specifically pathogens or pathological-related problems.

Chemoselective glycosylation (CG) methods have rapidly developed and diffused in glycochemist and glycobiologist's community in the last fifteen years, and an increasing number of applications confirms the enormous potential of these techniques. CG has been successfully used for the synthesis of glycoproteins, glycopeptides, glycosylated natural compounds, carbohydrate-functionalized surfaces and nanoparticles. CG techniques can be considered as subset of click reactions that allow the efficient, high-yield conjugation of sugars with unprotected aglycons in aqueous media. In this review the CG methods have been divided into two main groups: “direct” CG that uses unmodified glycans, and “indirect” CG, in which the sugar has to be chemically modified before the glycosylation reaction. Recent applications of both CG techniques are reviewed, with special focus on the chemistry of CG reactions: mechanisms, stereochemistry issues, structure and stability of the glycoconjugates.

In recent years, carbohydrate-processing enzymes have become the enzymes of choice in many applications thanks to their stereoselectivity and efficiency. This chapter presents recent development in glycosidase-catalyzed synthesis of unnatural semisynthetic carbohydrate structures via two complementary approaches: the use of wild-type enzymes with engineered substrates and mutant glycosidases. Genetic engineering has recently produced glucuronyl synthases, an inverting xylosynthase and the first mutant endo-β-Nacetylglucosaminidase. A careful selection of enzyme producing strains and aptly modified substrates has resulted in rare glycostructures, such as N-acetyl-β-galactosaminuronates, β1,4-linked mannosides and β1,4-linked galactosides. The efficient selection of mutant enzymes is facilitated by high-throughput screening assays involving the co-expression of coupled enzymes or chemical complementation. Selective glycosidase inhibitors and highly specific glycosidases are finding attractive applications in biomedicine, biology and proteomics.

[4+2] Cycloadditions between “in situ” generated α,α-dioxothiones and unprotected or variously protected glycals provide a powerful synthetic way to obtain glycomimetics chemo-, regio- and stereoselectively. Examples of glycopeptidomimetics and mimetic of tumor antigens are reported.

A wide range of assays has been used to characterize protein-carbohydrates interactions allowing access to various quantitative information including thermodynamic ones such as stoichiometry of binding, binding constant, enthalpy of binding as well as kinetics and mechanistic ones. An understanding of each technique is essential in order to propose and validate appropriate interaction models that rationalize experimental data. Due to the complexity of carbohydrate-lectins interactions as an inherent consequence of multivalent interactions, a careful control of experimental conditions is necessary in order to avoid misinterpretation. This article will briefly present the various techniques that are commonly used to characterize sugar-lectin interaction: inhibition of hemagglutination assay (HIA), enzyme-linked lectin assays (ELLA), isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). Recently introduced, force spectroscopy by atomic force microscopy (AFM) is a new approach for measuring carbohydrate-protein interaction at the level of a single protein.