Thisbe K. Lindhorst

Artificial Multivalent Sugar Ligands to Understand and Manipulate Carbohydrate-Protein Interactions

Multivalency plays an important functional role in carbohydrate-protein interactions. Understanding of the molecular principles underlying multivalency effects is an important goal of biological chemistry. Consequently, a large number of different synthetic mulitvalent glycoligands have been designed to interfere effectively with carbohydrate-protein interactions and to facilitate the investigation of the multiple interactions occurring during these molecular recognition events. Control of inflammation processes and of microbial adhesion are important examples where multivalent carbohydrate ligands might be developed into useful therapeutics such as in the context of an anti-adhesion therapy. Furthermore, cell-cell interactions can also be manipulated by a chemical bioengineering approach which utilizes synthetic substrates in the biosynthetic pathways leading to the assembly of the complex carbohydrate environment on cell surfaces.

Inhibition of the type 1 fimbriae-mediated adhesion of Escherichia coli to erythrocytes by multiantennary α-mannosyl clusters: The effect of multivalency

Alpha-mannosyl glycoclusters and glycodendrimers were tested as multivalent inhibitors of the type 1 (mannose-specific) fimbriae of a recombinant E. coli HB101 strain. Inhibition of haemagglutination of guinea pig erythrocytes was determined on microtiter plates. The effect of multivalency is pronounced for up to three mannosyl residues in the molecule, whereas larger derivatives do not have an appreciable effect on binding to the fimbrial carbohydrate binding domain. The best glycoclusters tested reach the binding potency of the known potent inhibitor pNPMan (3). The results support the idea of a monovalent recognition site at the adhesive protein FimH, which might best accommodate molecules with the size of a trisaccharide or those which expose up to three alpha-mannosyl residues, such as the glycocluster 8. The results obtained with the thiourea-bridged alpha-mannosyl clusters, possessing defined sugar valencies, facilitate the development of high affinity inhibitors of the fimbrial lectin on type 1 fimbriae.

Evaluation of the carbohydrate recognition domain of the bacterial adhesin FimH: design, synthesis and binding properties of mannoside ligands

Fimbriae are proteinogeneous appendages on the surface of bacteria, which mediate bacterial adhesion to the host cell glycocalyx. The so-called type 1 fimbriae exhibit specificity for alpha-d-mannosides and, therefore, they are assumed to mediate bacterial adhesion via the interaction of a fimbrial lectin and alpha-d-mannosyl residues exposed on the host cell surface. This carbohydrate-specific adhesive protein subunit of type 1 fimbriae has been identified as a protein called FimH. The crystal structure of this lectin is known and, based on this information, the molecular details of the interaction of mannoside ligands and FimH are addressed in this paper. Computer-based docking methods were used to evaluate known ligands as well as to design new ones. Then, a series of new mannosides with extended aglycon was synthesized and tested as inhibitors of type 1 fimbriae-mediated bacterial adhesion in an ELISA. The results obtained were compared to the predictions and findings as delivered by molecular modeling. This study led to an improved understanding of the ligand-receptor interactions under investigation.

Functional evaluation of carbohydrate-centred glycoclusters by enzyme-linked lectin assay: ligands for concanavalin A.

The affinities of the mannose‐specific lectin concanavalin A (Con A) towards D‐glucose‐centred mannosyl clusters differing in the anomeric configuration of the monosaccharide core, nature of the bridging functional groups and valency, have been measured by a competitive enzyme‐linked lectin assay. Pentavalent thioether‐linked ligands (5 and 7) were prepared by radical addition of 2,3,4,6‐tetra‐O‐acetyl‐1‐thio‐α‐D‐mannopyranose to the corresponding penta‐O‐allyl‐α‐ or ‐β‐D‐glucopyranose, followed by deacetylation. The distinct reactivity of the anomeric position in the D‐glucose scaffold was exploited in the preparation of a tetravalent cluster (10) that keeps a reactive aglyconic group for further manipulation, including incorporation of a reporter group or attachment to a solid support. Hydroboration of the double bonds in the penta‐O‐allyl‐α‐D‐glucopyranose derivative and replacement of the hydroxy groups with amine moieties gave a suitable precursor for the preparation of pentavalent and 15‐valent mannosides through the thiourea‐bridging reaction (17 and 20, respectively). The diastereomeric 1‐thiomannose‐coated clusters 5 and 7 were demonstrated to be potent ligands for Con A, with IC50 values for the inhibition of the Con A–yeast mannan association indicative of 6.4‐ and 5.5‐fold increases in binding affinity (valency‐corrected values), respectively, relative to the value for methyl α‐D‐mannopyranoside. The tetravalent cluster 10 exhibited a valency‐corrected relative lectin‐binding potency virtually identical to that of the homologous pentavalent mannoside 7. In sharp contrast, replacement of the 1‐thiomannose wedges of 5 with α‐D‐mannopyranosylthioureido units (17) virtually abolished any multivalent or statistic effects, with a dramatic decrease of binding affinity. The 15‐valent ligand 20, possessing classical O‐glycosidic linkages, exhibited a twofold increase in lectin affinity relative to the penta‐O‐(thioglycoside) 5; it is less efficient based on the number of mannose units. The results illustrate the potential of carbohydrates as polyfunctional platforms for glycocluster construction and underline the importance of careful design of the overall architecture in optimising glycocluster recognition by specific lectins.

Saccharide-Modified Nanodiamond Conjugates for the Efficient Detection and Removal of Pathogenic Bacteria

The detection and removal of bacteria, such as E. coli in aqueous environments by using safe and readily available means is of high importance. Here we report on the synthesis of nanodiamonds (ND) covalently modified with specific carbohydrates (glyco-ND) for the precipitation of type 1 fimbriated uropathogenic E. coli in solution by mechanically stable agglutination. The surface of the diamond nanoparticles was modified by using a Diels-Alder reaction followed by the covalent grafting of the respective glycosides. The resulting glyco-ND samples are fully dispersible in aqueous media and show a surface loading of typically 0.1 mmol g(-1). To probe the adhesive properties of various ND samples we have developed a new sandwich assay employing layers of two bacterial strains in an array format. Agglutination experiments in solution were used to distinguish unspecific interactions of glyco-ND with bacteria from specific ones. Two types of precipitates in solution were observed and characterized in detail by light and electron microscopy. Only by specific interactions mechanically stable agglutinates were formed. Bacteria could be removed from water by filtration of these stable agglutinates through 10 μm pore-size filters and the ND conjugate could eventually be recovered by addition of the appropriate carbohydrate. The application of glycosylated ND allows versatile and facile detection of bacteria and their efficient removal by using an environmentally and biomedically benign material.

Synthesis of octopus glycosides: core molecules for the construction of glycoclusters and carbohydrate-centered dendrimers

Abstract Allyl α- d -glucopyranoside was perallylated to allyl 2,3,4,6-tetra-O-allyl-α- d -glucopyranoside and this was converted into an array of uniformly functionalized spacer glycosides of an “octopus” type, taking advantage of the rich chemistry of the allyl ether function. Thus, carbohydrate-derived pentaaldehydes, pentaalcohols and pentaamines with different spacer lengths were obtained by ozonolysis, reductive amination, hydroboration or photoaddition of cysteamine hydrochloride, respectively. The new octopus glycosides are useful core molecules for the synthesis of glycoclusters and for the construction of carbohydrate-centered dendrimers.

Cluster Mannosides as Inhibitors of Type 1 Fimbriae-Mediated Adhesion ofEscherichia coli: Pentaerythritol Derivatives as Scaffolds

Pentaerythritol derivatives were used as core molecules for the synthesis of two cluster α-D-mannosides, which were designed as oligomannoside mimetics. The problem of glycosyl orthoester formation, which frequently occurs in oligo-mannosylations, was solved. The clusters were tested for their capacity to block binding of Escherichia coli to yeast mannan in vitro and were found to be more than 200 times more potent in inhibiting mannose-specific adhesion than methyl α-D-mannoside.

Squaric acid monoamide mannosides as ligands for the bacterial lectin FimH: covalent inhibition or not?

Bacteria use long proteinaceous appendages, called fimbriae or pili, to adhere to the surfaces of their host cells. Widely distributed among the Enterobacteriacae are type 1 fimbriae that mediate mannose‐specific bacterial adhesion through the lectin FimH, located at the fimbrial tips. It is possible to design synthetic mannosides such that they show high affinity for FimH and can thus inhibit mannose‐specific bacterial adhesion in a competitive manner. It has been found that mannosidic squaric acid monoamides serve especially well as inhibitors of type 1 fimbriae‐mediated bacterial adhesion, but it has remained unclear whether this effect is due to specific inhibition of the bacterial lectin FimH or to unspecific bioconjugation between the lectins carbohydrate binding site and a squaric acid monoamide. A bioconjugation reaction would result in a covalently crosslinked squaric acid diamide. Here it is shown that covalent inhibition of FimH by mannosidic squaric acid derivatives is very unlikely and that compounds of this type serve rather as excellent specific candidates for low‐molecular‐weight inhibitors of bacterial adhesion. This has been verified by testing the properties of glycosidic squaric acid monoamides in diamide formation, by two different adhesion assays with a series of selected control compounds, and by molecular docking studies that further support the results obtained in the bioassays.

Oligomannoside mimetics by glycosylation of ‘octopus glycosides’ and their investigation as inhibitors of type 1 fimbriae-mediated adhesion of Escherichia coli

The glycocalyx of eukaryotic cells is composed of glycoconjugates, which carry highly complex oligosaccharide portions. To elucidate the biological role and function of the glycocalyx in cell-cell communication and cellular adhesion processes, glycomimetics have become targets of glycosciences, which resemble the composition and structural complexity of the glycocalyx constituents. Here, we report about the synthesis of a class of oligosaccharide mimetics of a high-mannose type, which were obtained by mannosylation of spacered mono- and oligosaccharide cores. These carbohydrate-centered cluster mannosides have been targeted as inhibitors of mannose-specific bacterial adhesion, which is mediated by so-called type 1 fimbriae. Their inhibitory potencies were measured by ELISA and compared to methyl mannoside as well as to a series of mannobiosides, and finally to the polysaccharide mannan. The obtained results suggest a new interpretation of the mechanisms of bacterial adhesion according to a macromolecular rather than a multivalency effect.

Trivalent α-D-mannoside clusters as inhibitors of type-1 fimbriae-mediated adhesion of Escherichia coli: structural variation and biotinylation

Structural modifications of trivalent cluster mannosides are presented to further elucidate the ligand preferences of the type-1 fimbrial lectin of Escherichia coli. Two types of variations are performed, either regarding the aglycone part of cluster mannosides of type 2, leading to 27, or altering the spacer lengths of mannosyl clusters of type 1, leading to clusters 20–22. Biotinylation of the cluster mannoside with the highest affinity to the type-1 fimbrial lectin is also shown (33). Testing of the inhibitory potencies of the synthesised cluster glycosides as inhibitors of mannose-specific (type-1 fimbriae-mediated) binding of E. coli to mannan in an enzyme-linked immunosorbent assay (ELISA) suggests that a structural preorganisation as given in cluster 2a can be favourably combined with greater spacer flexibility as in cluster 22.