Synthetic ganglioside analogues for sensitive biosensing : improved probes for antibodies and bacterial toxins

This thesis describes the synthesis of analogues of human gangliosides and applications thereof for the detection and inhibition of bacterial toxins and antibodies. An efficient glycosylation method was developed for the synthesis of ω-functionalized alkyl lactosides (Chapter 2). These lactosides were further used as starting compounds in chemo-enzymatic syntheses of analogues of human gangliosides GM3, GM2, GM1, GD1a and GalNAc-GD1a (Chapters 3 and 4). In addition, divalent, tetravalent and octavalent GM2 and GM1 gangliosides were obtained (Chapter 5). A complete NMR characterization of the synthesized compounds was performed (Chapter 3). The potential of these ganglioside mimics to detect toxins and antibodies was shown in a variety of diagnostic tests: - in inhibition studies of the synthesized oligosaccharide-linked dendrimers with the cholera toxin B-subunit (Chapter 5) unprecedentedly large multivalency effects were observed: the tetra- and octa- GM1-substituted dendrimers are, respectively, 80 000 and 380 000 times stronger than a monovalent GM1 derivative as binding ligands for the toxin; - in various modified ELISAs the synthetic ganglioside analogues were used to modify the plates (Chapters 3, 4 and 6) and thus tested for their performance in the detection of the cholera toxin B-subunit and the B subunit of E. coli heat-labile enterotoxin; - for the detection of IgG and IgM antibodies in serum samples from neuropathy patients by the covalently attached ganglioside analogues a proof of principle was demonstrated (Chapter 6). <br/

Efficient Stereoselective Glycosylations of Alcohols by Sugar Perpivalates: The First Use of 1-O-Pivaloylated Glycosyl Donors

1-O-Pivaloyl glycosides were shown to be efficient glycosyl donors by using the perpivaloylated derivatives of lactose, galactose and glucose in the direct ZnCl2-promoted glycosylations of various alcohols. The corresponding glycosides were isolated in good yields and ß-selectivit

Syntheses of alkenylated carbohydrate derivatives toward the preparation of monolayers on silicon surfaces

This note describes the synthesis of different alkenylated carbohydrate derivatives suitable for direct attachment to hydrogen-terminated silicon surfaces. The derivatives were alkenylated at the C-1 position, while the remaining hydroxyl groups were protected. The development of such new carbohydrate-based sensing elements opens the access to new classes of biosensor

Visible-light attachment of Si-C linked functionalized organic monolayers on silicon surfaces

Organic monolayers on hydrogen-terminated silicon surfaces were prepared under extremely mild conditions using visible light and analyzed by a variety of surface-sensitive techniques: (angle-resolved) X-ray photoelectron spectroscopy (ARXPS), scanning tunneling microscopy (STM), high-resolution electron energy loss spectroscopy (HREELS), and attenuated total reflection infrared spectroscopy (ATR-IR). Detailed XPS and STM analysis of non-functionalized monolayers displays detailed mechanistic and structural information. Additionally, we present the first attachment of a disaccharide to the silicon surface, and a characterization thereof by ATR-IR and HREELS

The Influence of Ligand Valency on Aggregation Mechanisms for Inhibiting Bacterial Toxins

Divalent and tetravalent analogues of ganglioside GM1 are potent inhibitors of cholera toxin and Escherichia coli heat-labile toxin. However, they show little increase in inherent affinity when compared to the corresponding monovalent carbohydrate ligand. Analytical ultracentrifugation and dynamic light scattering have been used to demonstrate that the multivalent inhibitors induce protein aggregation and the formation of space-filling networks. This aggregation process appears to arise when using ligands that do not match the valency of the protein receptor. While it is generally accepted that multivalency is an effective strategy for increasing the activity of inhibitors, here we show that the valency of the inhibitor also has a dramatic effect on the kinetics of aggregation and the stability of intermediate protein complexes. Structural studies employing atomic force microscopy have revealed that a divalent inhibitor induces head-to-head dimerization of the protein toxin en route to higher aggregate

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol–water media, which for the first time were shown to be compatible with the C. jejuni α-(2→3)-sialyltransferase (CST-06) and β-(1→4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain–Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with 13C–1H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all 1H and 13C chemical shifts is presented