Synthesis and characterization of bichromophoric 1-deoxyceramides as FRET probes

The suitability as FRET probes of two bichromophoric 1-deoxydihydroceramides containing a labelled spisulosine derivative as a sphingoid base and two differently omega-labelled fluorescent palmitic acids has been evaluated. The ceramide synthase (CerS) catalyzed metabolic incorporation of omega-azido palmitic acid into the above labeled spisulosine to render the corresponding omega-azido 1-deoxyceramide has been studied in several cell lines. In addition, the strain-promoted click reaction between this omega-azido 1-deoxyceramide and suitable fluorophores has been optimized to render the target bichromophoric 1-deoxydihydroceramides. These results pave the way for the development of FRET-based assays as a new tool to study sphingolipid metabolism.

Automated summary

The suitability of two bichromophoric 1-deoxydihydroceramides as FRET probes, containing a labelled spisulosine derivative and two differently omega-labelled fluorescent palmitic acids, has been evaluated. The metabolic incorporation of omega-azido palmitic acid into the labeled spisulosine to produce omega-azido 1-deoxyceramide via ceramide synthase (CerS) has been studied. Optimization of the strain-promoted click reaction between the omega-azido 1-deoxyceramide and suitable fluorophores has enabled the development of the target bichromophoric 1-deoxydihydroceramides, paving the way for FRET-based assays for studying sphingolipid metabolism.