Fluorescently Labeled Ceramides and 1‑Deoxyceramides: Synthesis, Characterization, and Cellular Distribution Studies

• Published in: Journal of organic chemistry 2022-12, Vol.87 (24), p.16351-16367
• Main Authors: Izquierdo, Eduardo, López-Corrales, Marta, Abad-Montero, Diego, Rovira, Anna, Fabriàs, Gemma, Bosch, Manel, Abad, José Luís, Marchán, Vicente
• Format: Article
• Language: English
• Subjects: Ceramides - chemistry; Ceramides - metabolism; Diabetes Mellitus, Type 2; Fluorescent Dyes - chemistry; HeLa Cells; Humans; Ionophores; Sphingolipids - chemistry; Sphingolipids - metabolism
• ISSN: 0022-3263; 1520-6904
• DOI: 10.1021/acs.joc.2c02019

Ceramides (Cer) are bioactive sphingolipids that have been proposed as potential disease biomarkers since they are involved in several cellular stress responses, including apoptosis and senescence. 1-Deoxyceramides (1-deoxyCer), a particular subtype of noncanonical sphingolipids, have been linked to the pathogenesis of type II diabetes. To investigate the metabolism of these bioactive lipids, as well as to have a better understanding of the signaling processes where they participate, it is essential to expand the toolbox of fluorescent sphingolipid probes exhibiting complementary subcellular localization. Herein, we describe a series of new sphingolipid probes tagged with two different organic fluorophores, a far-red/NIR-emitting coumarin derivative (COUPY) and a green-emitting BODIPY. The assembly of the probes involved a combination of olefin cross metathesis and click chemistry reactions as key steps, and these fluorescent ceramide analogues exhibited excellent emission quantum yields, being the Stokes’ shifts of the COUPY derivatives much higher than those of the BODIPY counterparts. Confocal microscopy studies in HeLa cells confirmed an excellent cellular permeability for these sphingolipid probes and revealed that most of the vesicles stained by COUPY probes were either lysosomes or endosomes, whereas BODIPY probes accumulated either in Golgi apparatus or in nonlysosomal intracellular vesicles. The fact that the two sets of fluorescent Cer probes have such different staining patterns indicates that their subcellular distribution is not entirely defined by the sphingolipid moiety but rather influenced by the fluorophore.