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.
