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.

中文翻译：

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荧光标记的神经酰胺和 1-脱氧神经酰胺：合成、表征和细胞分布研究

神经酰胺 (Cer) 是具有生物活性的鞘脂，已被提议作为潜在的疾病生物标志物，因为它们参与多种细胞应激反应，包括细胞凋亡和衰老。1-脱氧神经酰胺 (1-deoxyCer) 是非经典鞘脂的一种特殊亚型，与 II 型糖尿病的发病机制有关。为了研究这些生物活性脂质的代谢，以及更好地了解它们参与的信号传导过程，必须扩展显示互补亚细胞定位的荧光鞘脂探针的工具箱。在此，我们描述了一系列标记有两种不同有机荧光团的新型鞘脂探针，一种是发射远红光/NIR 的香豆素衍生物 (COUPY)，另一种是发射绿光的 BODIPY。探针的组装涉及烯烃交叉复分解和点击化学反应的组合作为关键步骤，这些荧光神经酰胺类似物表现出优异的发射量子产率，COUPY 衍生物的斯托克斯位移远高于 BODIPY 对应物。在 HeLa 细胞中进行的共聚焦显微镜研究证实了这些鞘脂探针具有出色的细胞渗透性，并揭示了大多数被 COUPY 探针染色的囊泡是溶酶体或核内体，而 BODIPY 探针则在高尔基体或非溶酶体细胞内囊泡中积累。两组荧光 Cer 探针具有如此不同的染色模式这一事实表明它们的亚细胞分布并不完全由鞘脂部分决定，而是受荧光团的影响。