Carbohydrate-based chemical probes for the proteomic profiling of glucosidases and the emerging cancer marker galectin-3

Abstract

This thesis describes the synthesis of carbohydrate-based chemical probes to either profile glucosidases, or specifically label the emerging cancer marker galectin-3 in cell lysates. In general, chemical probe based methods can tag certain classes of proteins, and covalently label a protein or a protein family. This method should allow quantification within a mixture of other proteins. It could also give information about the localization of the protein in the cell, and about interactions of the target with other proteins. The method is fast, robust and easy to perform. It starts with incubation of the probe with the target protein or a cell lysate of interest, followed by photolabeling. In the next step a fluorescent reporter molecule is introduced to allow visualization of the captured proteins in a gel. An alkyne moiety on the probe allows for “click chemistry” to introduce the fluorescent reporter molecule. Instead of the fluorescent reporter, a biotin-tag or a 6xhistidine-tag was used in attempts to enrich the labeled proteins from the proteome. Competition experiments with a known ligand confirmed selective binding of the probe. One part of the project focuses on profiling glucosidases within a proteome. A glucose-based probe and iminosugar-based probes were synthesized, as well as a probe based on an acyclic mimic of the iminosugar. One iminosugar-based probe showed very high affinity towards non-lysosomal glucoceramidase, and was able to specifically label lysosomal glucocerebrosidase in the presence of a human cell lysate. This enzyme is defective in Gaucher patients. The other part of the project, which is the main part, focuses on the synthesis and evaluation of chemical probes to label galectin-3. This human protein is considered an emerging cancer marker in the literature. Because techniques used to obtain information about its function (mostly antibody based) often give conflicting results, chemical probes were designed and synthesized to selectively label galectin-3. A divalent lactose probe was synthesized, which increases the affinity for the protein over its monovalent derivative, due to a multivalency effect. Peptidic photoprobes were also synthesized, which were able to label galectin-3. The best probes in labeling galectin-3 are based on the high-affinity thiodigalactoside ligand. The probes were synthesized with benzophenone or acetophenone moieties as the photolabel for covalent attachment to the protein. Besides labeling the protein, these aromatic photolabels also greatly enhance the affinity of the probes towards galectin-3, due to the interaction of the photolabel with two arginine guanidinium groups of the protein. The linkage between the sugar and the photolabel was varied as an ester, an amide, and a triazole. For the amide and triazole derivatives, a versatile synthetic route towards a symmetrical 3-azido-3-deoxy-thiodigalactoside was developed. The results described in this thesis clearly show that creating a succesful probe is a multidimensional challenge involving issues of lipophilicity, affinity and photoreactivity. Above all, one of the probes showed such a good selectivity for its target protein in the presence of a human cancer cell lysate that this compound may have potential in future cancer research or diagnostics