Are multivalent cluster glycosides a means of controlling ligand density of glycoarrays?
Graphical abstract
Highlights
► Three mannose conjugates were prepared and tested as inhibitors of bacterial adhesion. ► Glycoarrays were fabricated from the new glycoclusters. ► Testing showed different trends of bacterial adhesion, suggesting that ligand density is varied with cluster valency.
Introduction
Every eukaryotic cell is covered with a layer of complex glycoconjugates, the so-called glycocalyx, which plays a crucial role in processes like cell-cell communication, cellular adhesion, inflammation and signalling.1, 2, 3 Due to the structural complexity of the glycoconjugates embedded into the cell membrane, elucidation of the molecular details of carbohydrate recognition is a demanding task. A specific scenario which is suited to test the details of carbohydrate recognition on a surface is bacterial adhesion to fabricated glycosylated surfaces and glycoarrays, respectively.4, 5, 6, 7, 8, 9, 10, 11 Bacteria utilise lectins, expressed as part of long protein appendages, called fimbriae, to attach to the glycosylated surface of their host cells.12, 13 This process facilitates bacterial colonisation of cell surfaces and biofilm formation and is connected to, i.a., severe inflammatory diseases of the host.
A key question in the investigation of carbohydrate recognition processes occurring on the cell surface is the composition of the glycocalyx. It is determined by the chemical nature of the carbohydrate constituents of the various glycoconjugates. But, moreover, the nature of the glycocalyx is regulated by additional parameters. These comprise (i) multivalency of sugar epitopes,14, 15, 16 (ii) their orientation and conformational flexibility of their presentation17, 18, 19 as well as (iii) ligand density and spacing of interaction partners.20, 21, 22, 23 Such parameters are difficult to adjust and to analyse and related research typically requires complex and demanding approaches. For example, carbohydrate spacing has been accomplished on DNA arrays,24 and carbohydrate density on a surface has been varied using neoglycoproteins.25 Indeed, glycoarrays offer an opportunity to address many important features of glycosylated surfaces but the effort to systematically vary and characterise their properties has remained high.26, 27, 28, 29
Here, we report on a study where cluster glycosides of different valencies were used in an attempt to vary carbohydrate ligand density of a glycoarray in a facile way. It is known from surface chemistry, that the size of molecules used for surface decoration influences the density of the formed molecular layer as a consequence of steric hindrance during the immobilisation process.30, 31 Thus, we have made mono-, di- and trivalent (cluster) mannosides to functionalise polystyrene microtiter plates. Then, mannose-specific adhesion of bacterial cells to the respective glycoarrays was assayed in three parallel test series employing systematic concentration variation (Fig. 1). We were interested to investigate, which different influences concentration and valency of sugar ligands have on the ‘stickiness’ of a glycosylated surface. This was systematically studied with three different types of mannose-coated surfaces which were employed in a lectin-mediated bacterial adhesion assay.
Section snippets
Synthesis of mannosides
A series of mono-, di- and trivalent cluster mannosides were synthesised in two variations. One set of mannosides was equipped with a primary amino function to allow covalent immobilisation of pre-functionalised microtiter plates. A second set was prepared accordingly, but with the amino group in N-acetylated form. The latter mannosides were used as soluble inhibitors of bacterial adhesion to a mannan-coated surface to determine their IC50 values.
The synthetic routes started with azidoethyl
Discussion
Our findings give a first hint that the hypothesis behind this study is promising. It has been suggested that cluster glycosides might be used to vary ligand density of glycoarrays, which can be rudimentally seen in our experiments. Apparently, concentration-dependent fabrication of glycoarrays using glycoclusters leads to different trends in adhesiveness depending on the cluster valency. Based on earlier experiments, we have no reason to assume, that the effectiveness of the immobilisation
General experimental methods
Commercially available starting materials and reagents were used without further purification unless otherwise noted. Anhydrous DMF was purchased, other solvents were dried for reactions or distilled for chromatography. Black Immobilizer Amino™ F96 MicroWell™ plates were purchased from Nunc (Thermo Fisher Scientific). Air- and/or moisture-sensitive reactions were carried out under an atmosphere of nitrogen. Thin layer chromatography was performed on silica gel plates (GF 254, Merck). Detection
Acknowledgements
Funding of this work by the DFG (collaborative network SFB677) and by the Evonik Foundation (stipend for J.W.W.) is gratefully acknowledged.
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