Synthetic Glycosphingolipids for Live-Cell Labeling

Abstract

Glycosphingolipids are an important component of cell membranes that are involved in many biological processes. Fluorescently labeled glycosphingolipids are frequently used to gain insight into their localization. However, the attachment of a fluorophore to the glycan part or-more commonly-to the lipid part of glycosphingolipids is known to alter the biophysical properties and can perturb the biological function of the probe. Presented here is the synthesis of novel glycosphingolipid probes with mono- and disaccharide head groups and ceramide moieties containing fatty acids of varying chain length (C4 to C20). These glycosphingolipids bear an azide or an alkyne group as chemical reporter to which a fluorophore can be attached through a bioorthogonal ligation reaction. The fluorescent tag and any linker connected to it can be chosen in a flexible manner. We demonstrate the suitability of the probes by selective visualization of the plasma membrane of living cells by confocal microscopy techniques. Whereas the derivatives with the shorter fatty acids can be directly applied to HEK 293T cells, the hydrophobic glycosphingolipids with longer fatty acids can be delivered to cells using fusogenic liposomes.

Chemical Biology Tools to Study Lipids and their Metabolism with Increased Spatial and Temporal Resolution

“…This two-step strategy allowed the visualization of the subcellular distribution of phospholipids, [22][23][24] sphingolipids [25] or cholesterol [26] in mammalian cell membranes. The most used bioorthogonal reactions for fluorophore tagging were the coppercatalysed azide-alkyne cycloaddition (CuAAC) and the strainpromoted azide-alkyne cycloaddition (SPAAC), on fixed and live cells, respectively.…”

Metabolic labeling of glycerophospholipids via clickable analogs derivatized at the lipid headgroup

“…Penno, Thiele and co-workers developed alkynyl-1deoxysphingolipid analogs to detect restrictions in metabolism and links to mitochondrial dysfunction (Alecu et al, 2017). Wittman and co-workers have reported clickable analogs of glycosphingolipids containing mono-and disaccharides at the headgroup (Dauner et al, 2016). Finally, incorporation of azido-sugars into liposomes, either through lipidation or encapsulation, has also been shown to enhance metabolic labeling of carbohydrates (Shen et al, 2019).…”

Greasing the Wheels of Lipid Biology with Chemical Tools

“…However, these probes tag the totality of a particular type of glycosylation, and the tagged lipids can then be separated from glycoproteins for in vitro analysis. Glycolipids can also be synthesized ex vivo with bioorthogonal functionality, applied to cells and then be tagged for fluorescence imaging [33]. This approach is less perturbative than one involving direct attachment of the fluorophore during the initial synthesis, affording a better chance to observe proper lipid localization relative to previous methods while ensuring adequate specificity for glycolipids.…”