Our group has developed several chemical probes and biochemical tools to monitor the activity of some of the enzymes of the sphingolipid metabolism.
1) Fluorogenic substrates to determine enzyme activity
Chemical probes for sphingosine-1-phosphate lyase (S1PL) activity
Sphingosine-1-phosphate lyase (SPL) is the last enzyme in the catabolism of sphingolipids. It catalyzes the retroaldol cleavage of long chain base phosphates into phosphoethanolamine and a fatty aldehyde. We have developed a new fluorescent substrate for this enzyme by incorporating a fluorescent reporter as part of the sphingoid base chain.
Sanllehí, P.; Casasampere, M.; Abad, J.-L.; Fabriàs, G.; López, O.; Bujons, J.; Casas, J.; Delgado, A. The First Fluorogenic Sensor for Sphingosine-1-Phosphate Lyase Activity in Intact Cells. Chem. Commun. 2017, 53 (39), 5441–5444.
Chemical probes for ceramidases
Ceramidases (CDases) are lipolytic amidohydrolases that catalyze the cleavage of ceramides into sphingosine and fatty acids. According to their optima pH, ceramidases are classified into acidic, neutral and alkaline, with different tissue distribution, subcellular localization and substrate specificity. We have developed a probe for the determination of acid CDase activity with applicability for the diagnosis of Farber disease, a rare disease resulting for the deficiency of this enzyme
(1) Bedia, C.; Camacho, L.; Abad, J. L.; Fabrias, G.; Levade, T. A Simple Fluorogenic Method for Determination of Acid Ceramidase Activity and Diagnosis of Farber Disease. J Lipid Res 2010, 51 (12), 3542–3547.
(2) Bedia, C.; Casas, J.; Garcia, V.; Levade, T.; Fabrias, G. Synthesis of a Novel Ceramide Analogue and Its Use in a High-Throughput Fluorogenic Assay for Ceramidases. ChemBioChem 2007, 8 (6), 642–648.
2) Azide-Tagged Sphingolipids for cellular trafficking studies
Investigation of the biological functions, localization and traffi cking of SLs requires their detection in their natural compartments, either the cell membranes or the inner organelle. In addition to the current available probes that incorporate a fluorescent reporter as part of the sphingoid backbone, we have developed minimally modified azidosphingolipids that allow a biorthogonal reaction with a suitable reporter at a later stage of the experiment.
(1) Garrido, M.; Abad, J. L.; Alonso, A.; Goni, F. M.; Delgado, A.; Montes, L.-R. In Situ Synthesis of Fluorescent Membrane Lipids (Ceramides) Using Click Chemistry. J. Chem. Biol. 2012, 5 (3), 119–123.
Azido-tagged sphingolipids can also be used for the simultaneous, quantitative sphingolipidomics from different cell populations, as illustrated in this reference:
(1) Garrido, M.; Abad, J. L.; Fabrias, G.; Casas, J.; Delgado, A. Azide-Tagged Sphingolipids: New Tools for Metabolic Flux Analysis. ChemBioChem 2015, 16 (4), 641–650.
3) Biochemical tools
HPA-12 is a CerT inhibitor. We can provide mg quantities of this useful biochemical tool upon request. Please, contact us for a quotation.
We can also provide deuterium-labelled and other tagged sphingosines and ceramides upon request. The synthesis and characterization of other small molecules can also be undertaken under official agreement.