Exploiting Diels-Alder Reactions with Inverse Electron Demand for Bioorthogonal Labeling of Alkene-Substituted Fucosylated Glycoproteins in Cells

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Glycosylation is a ubiquitous form of posttranslational modification that has been estimated to occur on more than 50% of the proteins from eukaryotic cells. Among the nine monosaccharides found in the glycoproteins of vertebrates, fucose is distinguished by its occurrence in numerous epitopes associated with cell-cell interactions and regulation of protein function. Fucose is also contained in a number of tumor-associated antigens that are upregulated in cancerous tissues and, therefore, have been suggested to act as cancer biomarkers. Profiling fucosylated glycoproteins is a possible strategy for the detection of these biomarkers. The aim of this project is to develop and apply methods that allow the detection of fucosylated glycoproteins of living cells. Metabolic glycoengineering (MGE) will be used to incorporate fucose derivatives into cellular glycans. Connected to the fucose derivatives are chemical reporter groups that can react in a bioorthogonal ligation reaction, thus, enabling the attachment of a detectable probe (fluorescence dye, biotin). In the first funding period of this project we synthesized a series of fucose derivatives containing terminal alkenes and applied them to MGE. Labeling of the alkenes was achieved by an inverse-electron-demand Diels-Alder (DAinv) reaction with tetrazines which is an example of a bioorthogonal ligation reaction that proceeds in high yields without metal catalysis and that is orthogonal to click chemistry. To improve the detection efficiency of the fucose derivatives we will now explore cell lines with blocked fucose de novo synthesis pathway as well as alternative ligation reactions. Protein-specific detection of fucosylation will be achieved by a combination of bioorthogonal labeling and immunoprecipitation and will be applied to study fucosylation of alpha-1-acid glycoprotein, an established cancer biomarker. In the first funding period we proved that the DAinv reaction can be combined with click chemistry for the simultaneous detection of two different carbohydrates. We will now use fluorescence resonance energy transfer (FRET) to detect the proximity of two different carbohydrates within the same glycan as indication for the occurrence of certain tumor-associated antigens.