Azide‐Tagged Sphingolipids: New Tools for Metabolic Flux Analysis

Abstract

Several diseases involve alterations in sphingolipid metabolism, so the development of tools for the analysis of sphingolipid metabolic fluxes is of interest. In this work, ω-azidosphingolipids 1-3 have been synthesized and tested as tracers in live cells. The synthesis starts from (S)-Garner's aldehyde and uses bromide or tosyloxy precursors for the introduction of the azido group into the sphingoid base. Studies in HGC-27 cells showed that probes 1-3 compete with the natural metabolites and are incorporated into sphingolipid pathways without affecting cell viability. The reactivity and bioorthogonality of the terminal azido group have been exploited by means of click reactions with different azadibenzocyclooctyne tags. This allows the mass spectrometric characterization of azidosphingolipidomes in pooled samples from different cell populations after independent treatments, providing proof of concept of the applicability of this technology in sphingolipid metabolic flux analysis.

Tools for the analysis of metabolic flux through the sphingolipid pathway

“…Alkyne lipids, containing a triple bond, are not intrinsically fluorescent but can be visualized by microscopy after a chemical reaction. Such click chemistry-based probes have recently been used for tracing fatty acid, cholesterol and sphingolipid flux in mammalian cells [51][52][53][54].…”

Greasing the Wheels of Lipid Biology with Chemical Tools

“…To overcome the issue of general metabolic precursors, such as fatty acids, being incorporated into undesired pathways, researchers have also prepared bioorthogonally functionalized analogs of several lipid species, including sterols [16,17] and sphingolipids [18,19] (Table 1). Given that these lipids are bound for a narrower realm of metabolic fates, they are more useful for revealing precise lipid localizations within cells.…”

3-Ketosphinganine provokes the accumulation of dihydroshingolipids and induces autophagy in cancer cells

“…The metabolic fate of several free bases has been studied over the years. Dragusin et al 13 investigated the metabolism of have also been examined, 15,16 as well as those of o-azidosphingosine 17 and o-alkynylsphingosine. 18 Although enzymatic conversions of KSa into Sa, 19 ketosphingosine 20 and sphingosine (So) 21 were reported in cell free systems in the early 70s, only the reduction step has been later extensively confirmed in cell culture.…”