The central hub of sphingolipid metabolism is occupied by ceramides. 1 They play important roles in signal transduction, cell cycle regulation, and programmed cell death (e.g., apoptosis) and serve as biosynthetic precursors for many highly bioactive lipids. 2 The delivery of ceramides and functionalized analogs to cells has been a long-standing challenge due to the very limited cell permeability and bioactivity of exogenously added long-chain ceramides. To overcome this limitation, shortchain ceramide analogs (e.g. C2, C6, C8) have been employed. These exhibit improved cellular uptake and have markedly increased bioactivity compared to long-chain variants, which are abundant in cells. 3,4 Photoswitchable lipids containing an azobenzene tail modification allow for the optical modulation of various aspects of lipid biology, 5 including the modulation of ion channels, [6][7][8] G protein-coupled receptors, [9][10][11] nuclear hormone receptors, [12][13][14] immune receptors, 15,16 quorum sensing receptors, 17 and enzymes. 18,19 They include photoswitchable derivatives of sphingosine and sphingosine-1-phosphate 10,20 and photoswitchable ceramides, which have afforded optical control over lipid rafts 21 and sphingolipid metabolism. 19 So far, the photoswitchable ceramides could not be used successfully to control ceramide-mediated signaling pathways in cells, including apoptosis. We reasoned that this limitation could potentially be overcome through the design of short-chain photoswitchable ceramide analogs.

We here describe the synthesis and application of two novel short-chain, clickable, and photoswitchable ceramides (scaCer1 and 2, Figure 1). These show improved cellular uptake, bioactivity, and cellular imaging compared to their long-chain congeners (caCer3 and 4, Figure 1). These new molecular tools enable optical control of apoptosis in HeLa cells, while the longchain analogs are completely inactive under comparable conditions. Similar to caCer3 and 4, they exhibit optical control of their metabolic conversion, as shown through light-dependent metabolism by sphingomyelin synthase 2 (SMS2).

Design, Synthesis, and Photophysical Characterization of Short-Chain caCer Analogs. The clickable and azobenzene-containing ceramide analogs caCer3 and caCer4 contain an azobenzene moiety in their sphingoid base and an alkyne handle for click-derivatization in their N-acyl chain. 19 We envisioned designing short clickable and azobenzenecontaining ceramide analogues scaCer1 and scaCer2, which contain identical sphingoid bases and a short N-acyl chain (Figure 1). This design strategy allows for direct comparison to the established caCers and assessment of chain length variations on cellular uptake and bioactivity. scaCer1 and scaCer2 were synthesized through an amide coupling reaction from aSph1 and aSph2 in good yields (Figure 1B). The synthesis of aSph1 and aSph2 has been previously described. 19 Using UV-vis spectroscopy, we then evaluated the photophysical properties of scaCer1 and scaCer2, in direct comparison with caCer3 and caCer4 (SI Figure 1). The photolipids were either kept in the dark (trans) or treated with blue (λ = 460 nm) or with UV-A (λ = 365 nm) light. The resulting UV-vis spectra demonstrated wavelength-dependent switching as expected for dialkyl-substituted azobenzenes (SI Figure 1A-D). Photoswitching could be repeated over multiple cycles, and all photolipids exhibit considerable thermal stability in the cis state in the absence of light (SI Figure 1E-H).

scaCers Exhibit Enhanced Cell Permeability. To quantify the relative cellular uptake of caCer 3, caCer4, scaCer1, and scaCer2, we incubated HeLa cells with the respective functionalized ceramide analog (dark-adapted), conducted lipid extraction, and used mass spectrometry (Figure 2B). We found that both scaCer variants show markedly increased cellular uptake (>10-fold) compared to the previously reported caCers. We further envisioned that scaCers could exhibit enhanced utility for the visualization using a clickable fluorophore (diSulfo-Cy5 azide). After labeling and fixation, the scaCers were clicked onto diSulfo-Cy5 azide via a copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC). scaCer1 and scaCer2 showed higher fluorescence compared to their long-chain structural counterparts caCer3 and caCer4 indicative of higher cell loading after incubation (Figure 2C,D). We observed that an increased number of washing steps led to a decrease of the fluorescence especially for scaCer2. This result and the more diffuse subcellular localization of labeled scaCer1 and scaCer2 suggest that the click product exhibits decreased cellular retention compared to the click product of long-chain analogs.

scaCers Enable Optical Control of Apoptosis. Ceramides are pro-apoptotic lipids, and increased ceramide levels can induce apoptotic cell death (Figure 3A). 22 It has been shown that ceramide translocation to the outer mitochondrial membrane is sufficient to induce apoptosis in cells. 23 Increased ceramide production at the outer mitochondrial membrane and exogenous addition of ceramides also induce cell death. 24,25 However, due to the limited cellular uptake of long-chain ceramides, short-chain analogs remain important tools for investigating the biological function of these lipids. 26 In order to test if scaCers exhibit potency for apoptosis induction and potentially allow for optical control, we treated HeLa cells with trans or cis isomers of scaCers and conducted a cell viability assay comparing cells in the presence or absence of pulsed irradiation (10 ms every 10 s using a Cell DISCO system). 27 While this irradiation protocol did not change the viability in the presence of DMSO or C8-Ceramide, it had significant effects on cells treated with scaCer1 and scaCer2 (Figure 3B). In each case, the trans isomer (dark-adapted condition) was found be more potent than the cis isomer (pulsed irrdatiation). To show that the biological effects observed are due to the induction of apoptosis, we immunoblotted the cell lysates of HeLa cells treated with isomers of caCers and scaCers for cleaved Poly-ADP-Ribose Polymerase (PARP), which is a well-known downstream marker of apoptotic activity (Figure 3C,D). We found that scaCers can induce PARP cleavage in a light-dependent manner (Figure 3D), whereas caCers were inactive (Figure 3C). Both assays are consistent and demonstrate that scaCer1 and scaCer2 show enhanced apoptogenic activity in their trans form. These results show that scaCers have increased bioactivity as compared to long-chain analogs and suggest that they could be useful tools for the optical control of ceramide-dependent biological processes in cell culture.

scaCers Are Light-Sensitive Substrates of SM Synthase. We have previously shown that caCers are lightdependent substrates for sphingolipid-metabolizing enzymes and were therefore interested to investigate if scaCers also act as photoswitchable substrates ("photosubstrates"). 19 To this end, we determined their conversion into sphingomyelin by the enzyme sphingomyelin synthase 2 (SMS2) heterologously expressed in Saccharomyces cerevisiae (Figure 4). Crude membrane preparations containing SMS2-V5, but not empty vector control membranes, were capable of SM synthesis (Figure 4C). Moreover, SM production was isomer-dependent: the trans isomer (kept in the dark) of scaCer1 and scaCer2 was converted faster than the corresponding cis isomer (pretreated with UV-A light, Figure 4D). Interestingly, for the long-chain analogues caCer3 and caCer4, the opposite trend was observed: under the identical experimental conditions, both were preferably converted as cis isomers (Figure 4D). Thus, scaCer1 and scaCer2, like their long-chain analogues, may be amenable for light-controlled manipulation of ceramide biology.

Concluding Remarks. Herein, we report new short-chain bifunctional ceramides (scaCers) that incorporate an azobenzene photoswitch to attain optical control of ceramide biology and an alkyne for click chemistry to visualize and quantify these designer lipids or the metabolites thereof. Both chemical modifications are made to the lipid tails, which allows the retention of the headgroup, leading to a functionalized analog with similar bioactivity. We demonstrated that scaCers exhibit markedly improved cellular uptake compared to previously reported long-chain analogs. We further showed induction and optical control of ceramide-dependent apoptosis, which notably could not be induced or controlled with the long-chain analogs caCer3 and caCer4. We further demonstrated that scaCer1 and scaCer2 are photoswitchable substrates for ceramide metabolism with light-dependent conversion with SMS2. Taken together, these results suggest that scaCers could be useful tools for the spatiotemporal control of ceramide biology. Recently, the Wells laboratory published an alternative strategy to the optical control of apoptosis using a photoreceptor engineered caspase. 28 In contrast to this genetically engineered system, scaCers do not require genetic manipulations and could therefore be more widely applicable. Optical control of apoptosis could be particularly interesting in the context of complex cell networks, e.g., for the ablation of cells in development or nervous systems.