SCOTfluors: Small, Conjugatable, Orthogonal, and Tunable Fluorophores for In Vivo Imaging of Cell Metabolism

Abstract The transport and trafficking of metabolites are critical for the correct functioning of live cells. However, in situ metabolic imaging studies are hampered by the lack of fluorescent chemical structures that allow direct monitoring of small metabolites under physiological conditions with high spatial and temporal resolution. Herein, we describe SCOTfluors as novel small‐sized multi‐colored fluorophores for real‐time tracking of essential metabolites in live cells and in vivo and for the acquisition of metabolic profiles from human cancer cells of variable origin.

Metabolites are essential biochemical components,w ith their transport and localization regulating most biological functions.D espite advances in fluorescence imaging to label biomolecules, [1] there are few approaches to image small metabolites in live cells and intact organisms.M ost metabolites do not contain groups that allow direct visualization and need to be modified with exogenous chromophores.H owever,f luorescent labels,i np articular red and near-infrared (NIR) fluorophores,a re bulky structures that can impair metabolite traffic within cells.O ur group has recently developed fluorogenic amino acids to label peptides without affecting their properties. [2] Herein, we describe an ew strategy for direct imaging of essential metabolites in live cells and in vivo using small-sized multi-color fluorophores.
Since the report by Ghosh and Whitehouse, [3] nitrobenzodioxazole (NBD) has been widely used because of its small size and neutral character.T hese properties have facilitated labeling biomolecules with retention of their native properties. [4] However, NBD (l em % 540 nm) is incompatible with other green fluorescent reporters (e.g., GFP) and has limited application for in vivo use.T oa ddress these shortcomings,h erein we report ac ollection of fluorophores, named SCOTfluors,with tunable emission covering the entire visible spectrum. SCOTfluors include the smallest fluorophores emitting in the NIR window (650-900 nm) reported to date ( Figure 1). [*] S. Benson, [+]  Several strategies have been described to optimize the optical properties of fluorophores for live-cell imaging. [5] For instance,t he replacement of oxygen atoms with geminal dimethyl groups in rhodamine and fluorescein produced redshifted fluorophores with enhanced properties for bioimaging. [6] We envisioned that the synthesis of nitrobenzodiazoles with different groups bridging the nitroaminoaniline core would render multi-color fluorophores with tunable emission and enhanced capabilities for metabolite imaging in live cells. Thepreparation of SCOTfluors was achieved in two synthetic steps from the common intermediate 1 (Figure 1). First, the aminoaniline core was cyclized with different bridging groups. All these reactions proceeded similarly for fluoride and chloride derivatives (full list of analogues in the Supporting Information). Second, halogenated compounds (2,F igure 1) underwent substitution with primary and secondary amines to render the final fluorophores (3-7,F igure 1). Tr iazole derivatives (4)were synthesized by reaction with sodium nitrite in acidic media at r.t.,t hioderivatives (5)w ere obtained by condensation with N-thionylaniline under heating, and selenium analogues (6)w ere prepared by reaction with SeO 2 under reflux in EtOH. Finally,c arbon derivatives (3 and 7) were synthesized by Cu-catalyzed coupling using linear and cyclic ketones,respectively.
We examined the optical properties of SCOTfluors and compared them to the original NBD (Figure 1and Figure S1). With the exception of triazoles (4), all compounds showed longer emission wavelengths than NBD,l ong Stokes shifts (around 80-100 nm), solvatochromic properties ( Figure S2), and good photostability ( Figure S3). Among SCOTfluors,Seand C-bridged derivatives display red and NIR emission, respectively,l ikely owing to reduced HOMO-LUMO gaps that result in bathochromic shifts in fluorescence emission, as with heteroatom-bridged rhodamine and rhodol fluorophores. [6b,c, 7] To the best of our knowledge,this is the first example of C-bridged nitrobenzodiazoles as fluorophores with NIR emission. Furthermore,C -bridged derivatives are readily accessible through one-step coupling of aminoaniline 1 with different ketones,r epresenting an ew platform for the direct synthesis of small NIR fluorophores.S COTfluors proved compatible for experiments in live cells,s howing no significant cytotoxicityinHeLa cells ( Figure S4).
Then, we examined the properties of SCOTfluors for imaging the trafficking of essential metabolites under physiological conditions.Sphingolipids are critical components of membranes in the regulation of cellular metabolism. The dysregulation of sphingolipid metabolism is associated with several diseases (e.g.,G aucher and Niemann-Pick [8] )a nd its intracellular localization is crucial to understand metabolic disruption. We used the C-bridged nitrobenzodiazole core to generate the NIR ceramide 8 (Figure 2A)a nd monitor its intracellular localization over time by co-staining with endoplasmic reticulum (ER) and lysosome markers.Spectral analysis confirmed that the optical properties of 8 were independent of the sphingoid base and therefore could be applicable to several types of biolipids ( Figure 2C and Figure S5). Compound 8 showed insignificant aggregation in water ( Figure S6), and the incubation with liposomes highlighted its fluorogenic behavior,with around 15-fold increase in emission ( Figure 2B and Figure S7). We exploited this property to visualize the recyclingofc eramide 8 in real time in human A549 cells using fluorescence confocal microscopy.
At short times (i.e., 15 min), the ceramide 8 was mainly found at the Golgi apparatus around the ER, as shown by high co-localization with ER Tr acker Green (R = 91 %) but not LysoTracker Blue (R = 26 %). Time-lapse imaging demonstrated that the ceramide 8 translocated to the recycling lysosomes after 3h ( Figure 2C)a sh ighlighted by the increased co-staining with LysoTracker Blue (R = 75 %). Notably,t hese observations agree with prior reports of lipid mobilization, [9] and could not be obtained with af luorescein ceramide analog ( Figure S8). These results confirm the suitability of our approach to prepare neutral NIR-fluorescent probes to image biolipid function in cells.
We also examined whether SCOTfluors could be used to image in vivo tissues with high metabolic activity.Fluorescent deoxyglucose tracers can monitor glucose uptake in metabolically active cells and tissues, [10] although few have been reported for in vivo use.W es ynthesized compound 9 (Figure 3I)a sa ni nvivo-compatible glucose analog by conjugation of the nitrobenzoselenadiazole 6 with 2-deoxyglucosamine.Notably,weperformed the reaction with chloride and fluoride derivatives of 6 and observed increased reactivity and recovery for the latter ( Figure S9). Compound 9 showed emission around 605 nm with ar emarkable Stokes shift of 115 nm ( Figure S10), enabling multiplexed imaging with blue and green fluorescent proteins (i.e., BFP and GFP,F igure 3A-F). We examined the transport of 9 in HeLa cells  (7:1) liposomes (red). C) Confocal microscopy images of A549 cells treated with 8 (50 mm, red), LysoTracker Blue (magenta) and ER Tracker Green (green) after 15 min (co-localization = white arrows) and 3h(co-localization = yellow arrows). Total co-localization coefficients (R) were determined using ImageJ. Scale bar = 15 mm.
transfected with EGFP-tagged GLUT4, the main glucose transporter in mammalian cells.F luorescence microscopy showed the uptake of 9 in GLUT4-EGFP cells and colocalization with the transporters (Figure 3A-C). Notably, the uptake of 9 was blocked by competition with excess glucose (Figure 3D-F) and was increased by pre-treating HeLa cells with insulin [11] (Figure 3H and Figure S11). These results confirm that compound 9 is af unctional substrate of GLUT4 and that enables dual tracking of glucose uptake and its transporters under physiological conditions.F inally,w e tested compound 9 in vivo in zebrafish embryos to visualize regions of high glucose uptake.I nvivo administration and imaging of compound 9 in wildtype zebrafish embryos indicated bright red fluorescence staining in regions of the developing brain (e.g., midbrain and hindbrain;F igure 3J), which express GLUT2 transporters to supply glucose from circulation. [12] We confirmed that the staining was dependent on the active transport of 9 through GLUT2 by examining glut2 morpholino-injected zebrafish, which have reduced levels of GLUT2. In vivo images of 9-treated glut2 morpholino-injected zebrafish showed much weaker fluorescence in the same regions ( Figure 3J). We also tested compound 6-NEt 2 as ac ontrol and observed no tissue-specific staining in wildtype or in glut2 morpholino-injected zebrafish (Figure 3J), highlighting the role of deoxyglucose to recognize GLUT2 transporters.Altogether,compound 9 can be used to image glucose uptake in vivo and to perform non-invasive studies of glucose transport in whole organisms.
Next, we used SCOTfluors to prepare the first redfluorescent analogue of lactic acid, an essential metabolite in muscle,b lood, and cancer cells.L actic acid is known as acarbon source in cancer cells and its uptake in tumours has been recently linked to aggressive oncological behaviour, [13] yet little is known about its traffic and diffusion inside cancer cells.W ec onjugated the nitrobenzoselenadiazole 6 with l-isoserine to produce compound 10 ( Figure 4A, l em % 605 nm) as ap robe to study the transport of lactic acid in live cells. First, we confirmed increased uptake in hypoxic (1 %O 2 ) versus normoxic (20 %O 2 )c ells,s ince lactic acid can accumulate in environments with low concentrations of oxygen ( Figure 4B). [14] Figure 3. Fluorescenceimages of GLUT4-EGFPHeLa cells treated with 9.A -F) Green (GLUT4-EGFP), red (9,100 mm)a nd merged (Hoechst 33 342) images of HeLa cells without additionalg lucose (A-C) and in media containing 5mmd-glucose (D-F). White arrows identify colocalizationo fGLUT4-EGFP and 9.Scale bar = 10 mm. G) Fluorescenceemission spectra of BFP (blue), GFP (green), and 9 (red). H) Insulindependent (100 nm,1h) uptake of 9 (red, 100 mm)inGLUT4-EGFPHeLa cells. I) Chemical structures of compounds 6-NEt 2 and 9.J )Invivo images of the head in zebrafish embryos (28 hpost fertilization, hpf)a fter injection of 6-NEt 2 or 9 (both 50 pmol) to the yolk sac (blue arrowheads). Fluorescence images were taken of wildtype zebrafish or zebrafish that had been injected at one cell stage with 4.2 ng anti-sense glut2 morpholino. Yellow arrows point at midbrain and hindbrain regions within the zebrafish embryo heads. Scale bar = 100 mm. We performed flow cytometry analysis to observe that hypoxic cells were significantly brighter than normoxic cells after incubation with the same concentration of compound 10. We also performed competition assays between 10 and excess of lactic acid in normoxic cells,w hich markedly reduced the fluorescence staining,s uggesting ac ommon transporter for compound 10 and lactic acid in live cells ( Figure 4B). Encouraged by these results,weused total internal reflection fluorescence (TIRF) microscopy to image the real-time diffusion of lactic acid in normoxic and hypoxic cancer cells with super-resolution. Fort hese studies,w eu sed HeLa cells that had been treated or not with dimethyloxalylglycine (DMOG), ap ermeable prolyl 4-hydroxylase inhibitor that upregulates hypoxia-inducible factors.
We tracked the paths of over 1000 individual particles in both untreated (i.e., normoxic) and DMOG-treated (i.e., hypoxic) cells after incubation with compound 10 and measured their respective intracellular diffusion coefficients (Figures 4D-E and Figure S12, . Remarkably, particles in hypoxic cells showed higher mean diffusion coefficients than in normoxic cells,a sw ell as ar eduction of the slow diffusion species.A ltogether,t hese results suggest that hypoxic tumors might display faster recycling rates for intracellular lactic acid than normoxic tumors and demonstrate the utility of compound 10 as anew probe for imaging lactic acid metabolism in live cells with high spatiotemporal resolution. Finally,g iven the multi-color capabilities and high stability of SCOTfluors under physiological and oxidative environments ( Figure S13), we employed them to analyze the metabolic profiles of human cells from different origin. The groups of Chang and Rotello previously reported the discrimination of cancer cells using fluorescent dyes [15] or host-guest arrays. [16] In this study,weincubated human cancer cell lines with compounds 8, 10,a nd 11 ( Figure 5) as respective analogues of ceramide,l actic acid, and glucose in order to obtain metabolic uptake signatures.F irst, we plated the cells at similar densities and incubated them with the probes under the same conditions.N ext, we measured their fluorescence emission in the NIR, red, and green regions to determine their intracellular levels of ceramide,l actic acid, and glucose,r espectively.N otably,d ifferent cancer cells presented variability in their metabolic uptake,asrepresented by their intracellular glucose/lactate and ceramide/lactate ratios ( Figure 5). Whereas the biological implications of these results remain to be defined, these results demonstrate that SCOTfluors can generate multiplexed metabolic readouts from live cells,w hich is not possible in other imaging modalities.
In conclusion, we developed SCOTfluors as small-sized fluorophores covering the entire visible spectrum. SCOTfluors are readily obtained by bridging aminoanilines with different groups and include the smallest NIR-emitting fluorophores to date.W ev alidated SCOTfluors for realtime and in situ imaging of different small metabolites (e.g., lipids and sugars) in live cells and in vivo,a swell as their combination to generate multi-color fingerprints in cells.The tunability and versatility of SCOTfluors will enable noninvasive bioimaging studies of essential metabolites that cannot be performed with conventional fluorophores.