New sphingolipid probes for metabolism and trafficking studies

Abstract

To study biological systems through the specific labeling of biomolecules by chemical reactions has grown interest in the last few years. In order to perform chemical reactions in living systems, it is essential that these processes fulfill certain criteria: 1) to proceed under physiological conditions; 2) to be high yielding and show fast reaction rates; 3) to require simple reaction conditions, and 4) to be biocompatible with the myriad of functionalities within biological systems. Along this line, to incorporate an azide functionality (chemical reporter) in the biomolecule of interest allows its selective labeling through 1,3-cycloadditions with terminal (copper-catalyzed) or cyclic alkynes. Such reactions are termed as ‘click’ reactions and are very used to label a wide number of biomolecules. Sphingolipids (SLs), a large and diverse class of lipids, are structural components of eukaryotic cell membranes. Besides its structural roles, it has been discovered that some sphingolipids are bioactive molecules which regulate cellular responses such as proliferation, differentiation, apoptosis and cell death. Moreover, sphingolipids dynamically assemble with sterols to form lipid rafts which are intimately associated with cell signaling. All these discoveries have grown interest in the development of molecular and chemical tools to study the metabolism and localization of these molecules. In the present Doctoral Thesis, we designed and synthesized various sphingolipid probes, bearing an azide functionality at omega and C1 positions of sphingoid base. The sphingolipid probes possessing an azide at omega position also comprised variations at C1 and as well as different acyl chains in the amide linkage. This family of omega-azidosphingolipids turned out to be non-cytotoxic and to metabolize as natural sphingolipids in different cell lines. Likewise, we developed new methodologies to study sphingolipid metabolism and intracellular localization based on azide-alkyne cycloaddition reactions. First of all, we developed a new approach for the quantitative analysis of sphingolipids, based on the labeling of different populations of sphingolipids with various azadibenzocyclooctyne-derived tags through click reactions. We optimized the click reaction in cell extracts containing omega-azidosphingolipids, and next we labeled cell extracts with the various cyclooctyne tags. We were able to detect a large number of sphingolipid triazoles, arising from the click reactions, by UPLC-TOF with high sensitivity. Moreover, we developed a method for the labeling of omega-azidosphingolipids in live cells through the click reaction with a fluorescent dye. We first designed and prepared the fluorescent dye, based on an azadibencyclooctyne moiety linked to a fluorescein unit. We next evaluated the internalization of this dye in cell membranes, analyzing cellular fluorescence by flow cytometry. Finally, we labeled cells with the fluorescent dye, and we observed an increase of fluorescence in cells which had been pre-treated with omega-azidosphingolipids. Moreover, we nicely visualized the resulting fluorescent click adducts by confocal microscopy. In the last application of these sphingolipid probes, we developed a method for the visualization of fluorescent ceramides in artificial lipid membranes. We incorporated omega-azidoceramide and C1-azidoceramide probes into artificial membranes and next labeled them using a fluorogenic click reaction with a non-fluorescent naphtalimide. The resulting click adducts, which became fluorescent, were nicely visualized by confocal microscopy. This methodology might be an important approach for the localization of azidoceramides in membranes of live cells by fluorescence microscopy.