Synthesis and Characterization of Fluorescent Probes for the Development of a Ceramide Synthase FRET-Based Assay

Ceramides are recognized to occupy a pivotal position in sphingolipid metabolism, playing a fundamental role as membrane structural components, as well as second messengers. Ceramide synthases (CerS) are a family of enzymes that catalyze the N-acylation of sphingosine and dihydrosphingosine, thus controlling the levels of intracellular ceramides. In this work, we present the synthesis and the fluorescent properties of new probes for the development of a FRET-based assay for CerS activity. Based on our previous discovery of spisulosine as a suitable probe for CerS activity in cells, the use of a modified NBD-spisulosine with a clickable fatty acid as CerS substrates may allow the ultimate formation of a bichromophoric reporter for a FRET-based analysis of CerS activity in cells.