In Situ Lysosomal Cysteine-Specific Targeting and Imaging During

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In Situ Lysosomal Cysteine-Specific Targeting and Imaging During Dexamethasone Induced Apoptosis Yongkang Yue, Fangjun Huo, Ping Yue, Xiangming Meng, James C. Salamanca, Jorge O. Escobedo, Robert M. Strongin, and Caixia Yin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01406 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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Analytical Chemistry

In Situ Lysosomal Cysteine-Specific Targeting and Imaging During Dexamethasone Induced Apoptosis Yongkang Yue,†,§ Fangjun Huo,‡,§ Ping Yue,┴ Xiangming Meng,┴ James C. Salamanca,¶ Jorge O. Escobedo,¶ Robert M. Strongin,¶ and Caixia Yin†,* †

Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Key Laboratory of Materials for Energy Conversion and Storage of Shanxi Province, Institute of Molecular Science and ‡Research Institute of Applied Chemistry, Shanxi University, Taiyuan 030006, China. *E-mail: [email protected]. Tel/Fax: +86-351-7011022. ┴ Department of Chemistry, Anhui University, Hefei 230601, China ¶

Department of Chemistry, Portland State University, Portland, Oregon 97201, United States

ABSTRACT: Herein we utilize the similar though divergent nucleophilic properties of cysteine, homocysteine, and glutathione to achieve the selective detection of cysteine under mildly acidic conditions. This enables the specific in situ detection of lysosomal cysteine. Employing time dependent fluorescent imaging of probe-labeled A549 cells, we demonstrate that dexamethasone-induced apoptosis is not dependent on lysosomal cysteine. This methodology can thus produce useful information about pathogenesis associated with cysteine and lysosomes.

In mammalian cells, cysteine (Cys) is generated enzymatically from methionine, which is converted to homocysteine (Hcy) via the intermediate Sadenosylmethionine.1-3 The reaction of Hcy and serine, catalyzed by cystathionine beta-synthase, produces cystathionine which is a substrate for cystathionine gamma-lyase and is converted to cysteine and alpha-ketobutyrate. Cys plays a central role in sulfur metabolism as a key redox-active protein residue and as a precursor to glutathione (GSH), inorganic sulfur, and taurine. Elevated and reduced Cys has been reported to be associated with various diseases like rheumatoid arthritis, Parkinson’s disease, Alzheimer’s disease, slowed growth, edema, and liver damage.4-6 In recent years, Cys fluorescent probes have been featured as a less-invasive diagnostic tool that is applicable for living cell imaging.7 However, the similar structures and properties of the three small molecular thiols, especially for Hcy, create several challenges for selective and discriminative detection for each individual thiol.8-10 In 2004, the Strongin group used a xanthene dye possessing aldehyde groups that upon reaction with Cys and Hcy produce thiazolidines and thiazinanes respectively, resulting in fluorescence quenching and color change.11-13 This result prompted the Peng group to investigate the selective detection of Cys over Hcy, GSH and other amino acids utilizing a fluorogenic aldehyde-containing dye via the formation and hydrolysis of a thiazolidine derivative. Later, Strongin and coworkers reported the acrylate-based fluorescent probe for discriminative detection of Cys and Hcy through a successively nucleophilic addition reaction and intramolecular cyclization process.14 Based on these multistage reaction processes between probes and Cys with specific fluorescent responses, significant progress has been made for selective detection of Cys.15-22 However, these probes can also produce reactions with Hcy or GSH while exhibiting

interfering fluorescent responses with that of Cys processes. This would not only cause the consumption of the probes, but also affect the integrity of the fluorescent signals. Particularly due to intracellular GSH concentration being tenfold than Cys.23-25 Recently, we reported a coumarin derivative that displayed selective, pH-adjustable fluorescent responses toward Cys, Hcy, and GSH in the order of reactivity Cys > Hcy > GSH.26 We envisaged that selective Cys imaging could be achieved in subcellular organelles as a function of their compartmentalized internal pH values. Lysosomes display a unique acidic pH range (pH 3.8~5.0), and contain many different types of hydrolytic enzymes to regulate cellular homeostasis, biomolecular degradation, apoptosis and cell death.27-29 These enzymes require a highly reductive environment for optimal functionality and has been speculated that this environment is regulated in part by free intracellular biothiols. Introduction of reactive oxygen species due to oxidative stress or metabolic dysfunction affects biothiol concentration within the lysosome and could be a possible indicator of diseased states.29,30 Imaging lysosomal Cys during apoptosis would provide insight into its specific role in the process. Guided by our previous studies, we selected an α,βunsaturated ketone as the Cys-reactive probe.6,26,31 Importantly, the reactions between α,β-unsaturated ketones and Cys are generally reversible.6,32,33 The probe structure was modified with a lysosome targeting moiety28,34,35 via a click reaction to afford Ly-1 (Figure 1) for the selective detection of Cys in lysosomes. In addition, the introduction of triazole and morpholine moieties improved the water solubility of Ly-1. As expected, Ly-1 exhibited a turn-on fluorescent response toward Cys in pH 5.0 buffer. Through time dependent confocal fluorescent imaging of LysoTracker Red (a commercially available lysosome-targeting dye) and Ly-1 co-

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loaded A549 cells, we achieved in situ lysosomal Cys detection and found that dexamethasone-induced A549 cell apoptosis was lysosomal Cys independent. Furthermore, the lysosomal membrane was not ruptured during the apoptosis process.

Figure 1. Probe design and the detection mechanism of Ly-1 toward Cys.

EXPERIMENTAL SECTION Materials and Methods. All chemicals were purchased from commercial suppliers and used without further purification. All solvents were purified prior to use. Distilled water was used after passing through a water ultra-purification system. TLC analysis was performed using precoated silica plates. Hitachi F−7000 fluorescence spectrophotometer was employed to measure fluorescence spectra. Hitachi U-3900 UV-vis spectrophotometer was employed to measure UV-vis spectra. Shanhai Huamei Experiment Instrument Plants, China provided a PO-120 quartz cuvette (10 mm). 1H NMR and 13C NMR experiments were performed with a BRUKER AVANCE III HD 600 MHz and 151 MHz NMR spectrometer, respectively (Bruker, Billerica, MA). Coupling constants (J values) are reported in hertz. HR-MS determinations were carried out on an AB SCIEX TripleTOF 5600 Instruments. The cell imaging experiments were measured by Zeiss LSM880 Airyscan confocal laser scanning microscope. The zebrafish imaging experiments were measured by an Olympus FV1200 confocal laser scanning microscope. Synthesis. Compounds were synthesized according to the general procedure in Scheme S1 in the Supporting Information. Synthesis of Compound 9. Compound 826 (2 mmol, 0.52 g) and compound 336 (2.4 mmol, 0.35 g) was dissolved in 20 mL EtOH. 200 µL piperidine, 100 µL glacial acetic acid was added and the mixture was refluxed for 70 hours. After cooling to room temperature, the solvent was separated under reduce pressure. The residue was purified by column chromatography using ethyl acetate/ dichloromethane (12:500) to give 0.31 g (0.8 mmol) compound 9 as a red solid. 1H NMR (600 MHz, DMSO-d6) δ 8.60 (s, 1H), 7.93 (d, J = 15.8 Hz, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.68 (t, J = 12.9 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 6.82 (dd, J = 9.0, 1.7 Hz, 1H), 6.61 (s, 1H), 3.51 (dd, J = 13.8, 6.8 Hz, 4H), 1.16 (t, J = 7.0 Hz, 6H). 13C NMR (151 MHz, DMSO-d6) δ 185.8, 160.4, 158.7, 153.5, 148.9, 141.7, 141.4, 132.8, 132.3, 130.6, 125.0, 120.2, 115.9, 110.7, 108.4, 96.4, 44.9, 12.8. Synthesis of Probe Ly-1. Compound 9 (0.5 mmol, 0.19 g) and compound 537 (0.5 mmol, 0.7 g) was dissolved in THF, then Et3N (0.25 mmol) and CuI (0.1 mmol) was added under argon atmosphere successively. The mixture was stirred at room temperature for 6 hours. After the solvent was evaporated under reduce pressure, the residue was dissolved in dichloromethane and washed with brine. The organic layer was concentrated and separated by column chromatography using methanol/ dichloromethane (1:20) to give 0.07 g (0.14 mmol)

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probe Ly-1 as an orange solid. 1H NMR (600 MHz, CDCl3) δ 8.57 (s, 1H), 8.21 (d, J = 15.7 Hz, 1H), 8.00 (s, 1H), 7.86 – 7.75 (m, 5H), 7.44 (d, J = 8.9 Hz, 1H), 6.64 (d, J = 9.5 Hz, 1H), 6.50 (s, 1H), 3.78 (s, 2H), 3.77 – 3.72 (m, 4H), 3.48 (q, J = 7.1 Hz, 4H), 2.60 (s, 4H), 1.26 (t, J = 7.1 Hz, 6H). 13C NMR (151 MHz, CDCl3) δ 186.2, 161.0, 158.8, 153.2, 149.0, 141.1, 137.7, 135.9, 131.9, 130.0, 126.3, 120.4, 116.3, 110.0, 108.7, 96.6, 66.8, 53.6, 53.4, 45.2, 12.5. Preparation of Solutions of Probe Ly-1 and Analytes. Stock solution of probe Ly-1 (2 mM) and Nethylmaleimide (200 mM) were prepared in DMSO. Stock solutions of 200 mM GSH, 20 mM Cys, Hcy, and other amino acids were prepared by direct dissolution in deionized water. All chemicals used were of analytical grade. General Fluorescence Spectra Measurements. All the detection experiments were measured in PB/DMSO (9/1 v/v, 200 mM, pH 5.0) or PBS/DMSO (9/1 v/v, 10 mM, pH 7.2~7.4). The procedure was as follows: into a PB/DMSO (9/1 v/v, 200 mM, pH 5.0) or PBS/DMSO (9/1 v/v, 10 mM, pH 7.2~7.4) solution, containing 20 µM probe Ly-1, an analyte sample was added. The process was monitored by fluorescence spectrometer. Cell Culture and Imaging. The A549 cells were grown in 1640 medium supplemented with 12% Fetal Bovine Serum and 1% antibiotics at 37 °C in humidified environment of 5% CO2. Cells were plated on 14 mm glass coverslips and allowed to adhere for 24 h. Before the experiments, cells were washed with PBS 3 times. For the colocalization experiments, cells were incubated with Ly-1 (5 µM in PBS) for 10 min and LysoTracker Red (0.5 µM in PBS) for another 15 min before imaging. For the time dependent experiments, confocal fluorescent imaging was performed immediately after analyte added.

RESULTS AND DISCUSSION Spectroscopic responses of Ly-1 to Cys in the Solution. We initially evaluated the sensing properties of Ly-1 toward Cys systematically via the addition of analytes to Ly-1 in PB/DMSO (9/1 v/v, 200 mM, pH 5.0). As shown in Figure 2a, Cys addition induced distinctive turn-on fluorescent emission at 498 nm. The signal saturated in 40 min, with a 90fold fluorescent enhancement (Figure S1). Correspondingly, the UV-vis absorption peak changed from 469 nm to 452 nm with a distinct isosbestic point at 459 nm, indicating the formation of a single species (Figure 2b). Other amino acids including Hcy and GSH induced negligible fluorescent responses in the detection system (Figure 2c, 2d and S2). Moreover, the existence of Hcy (1 equiv.) or GSH (10 equiv.) did not interfere with Cys detection (Figure 2e). In contrast, time dependent UV-vis spectral changes of Ly-1 upon addition of Hcy, GSH, and H2O demonstrated that Ly-1 was inert toward Hcy and GSH (Figure S3). The addition of NEM (N-ethylmaleimide, a thiol scavenging reagent) led to recovery of the Ly-1 signal, thereby showing that the process was reversible (Figure S4). The corresponding detection limit based on the IUPAC definition (CDL = 3 Sb/m) was 1.2 µM from 10 blank solutions (Figure 2f). These results showed that Ly-1 could detect Cys with high selectivity and sensitivity at pH 5.0.


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Analytical Chemistry experiments of Ly-1 and LysoTracker Red with A549 cells revealed (Figure 3), that both the green and red channels displayed strong emission, and their intensity profiles merged well. This result demonstrated that Ly-1 was membrane permeable and was lysosome specific. Furthermore, the Ly-1 incubation exhibited fluorescent emission in the green channel might be caused by the reaction of Ly-1 with endogenous Cys.

Figure 2. Time dependent fluorescence a) and absorption b) responses of Ly-1 (20 µM) toward Cys (1.2 mM) in PB/DMSO (9/1 v/v, 200 mM, pH 5.0); c) Time dependent fluorescent changes of Ly-1 (20 µM) toward Cys (1.2 mM), Hcy (1.2 mM), and GSH (12 mM) at 498 nm; d) Fluorescent spectra obtained 60 min after addition of various amino acids (1.2 mM) to Ly-1 (20 µM); e) Cys (1.2 mM) detection in the presence of Hcy (1.2 mM) or GSH (12 mM); f) Working curve of Ly-1 to detect Cys obtained 60 min after addition of various concentrations of Cys (0−500 µM) to 20 µM Ly-1; Error bars represent standard deviations, n = 3; λex = 430 nm, slit: 5 nm/5 nm.

The selectivity of Ly-1 toward Cys at higher pH (PBS/DMSO 9/1 v/v, 10 mM, pH 7.2~7.4) was not maintained. Hcy addition to the solution of Ly-1 also induced turn-on fluorescent response (Figure S5). GSH, however, did not induce an interfering response. These results are in agreement with our prior findings, and further supported the design strategy involving pH-regulated Cys-selective detection. Detection mechanism of Ly-1 to Cys. To verify the detection mechanism of Ly-1 toward Cys, we performed a titration, monitored by 1H NMR, via adding Cys (in D2O) to a Ly-1 DMSO-d6 solution (Figure S6). The signals at 7.72 and 7.74 ppm (C=C protons of the α,β-unsaturated ketone) diminished as two new doublets appeared at 4.56 ppm. The HR-MS (Figure S7) exhibited a peak at m/z = 635.2641 corresponding to [Ly-1-Cys + H]+. Along with a blue-shift in the UV-vis absorption spectrum, this evidence confirmed that the detection process was based on the conjugate addition of the thiol to the α,β-unsaturated ketone in a single-stage process. In situ lysosomal Cys specific imaging. To evaluate the applicability of Ly-1 to live cell imaging, its cytotoxicity was measured via a CCK-8 assay with A549 cells. After incubation with 10 µM Ly-1 for 5 h, A549 cells displayed 82.9% viability, thereby demonstrating relatively low cytotoxicity associated with Ly-1 (Figure S8). As Ly-1 was designed with a morpholine moiety, we investigated whether Ly-1 could stain lysosomes selectively in live cells. Co-staining

Figure 3. Confocal fluorescent image of A549 cells incubated with Ly-1 (5 µM) for 10 min and LysoTracker Red (0.5 µM) for 15 min; a) green channel; b) red channel; c) bright field; d) merged image of a), b), and c); e) ROI of d); f) intensity profiles within the ROI (red line in Figure 3e) of Ly-1 and LysoTracker Red across A549 cells; Green channel: λex = 405 nm, λem = 470520 nm; Red channel: λex = 461 nm, λem = 580-600 nm; Scale bars = 20 µm.

In order to confirm its endogenous Cys sensing capacity, Ly-1 was further applied for cell imaging in Cys presence or absence A549 cells (Figure 4). Consistently, cells stained directly with Ly-1 displayed strong fluorescent emission. However, cells treated with NEM prior to staining Ly-1 exhibited no fluorescent emission. Cys addition of the NEMtreated cells containing Ly-1 resulted in strong fluorescent emission (Figure S9). Control experiments with cells successively treated with NEM, then Hcy or GSH and Ly-1 also exhibited no fluorescence emission. The emission of Ly1-incubated A549 cells was effectively quenched via further incubation with NEM. These results demonstrated that Ly-1 could image Cys specifically and reversibly in living A549 cells.

Figure 4. a) Confocal fluorescent image of A549 cells incubated with Ly-1 (5 µM) for 10 min; b) confocal fluorescent image of A549 cells pretreated with NEM (1 mM) for 30 min and then incubated with Ly-1 (5 µM) for 10 min; c) and d) confocal fluorescent image of A549 cells successively treated with NEM (1 mM) for 30 min, Hcy (15 µM) or GSH (1 mM) for 30 min, and then incubated with Ly-1 (5 µM) for 10 min, respectively; e), f),



g), and h) The corresponding merge images of the green channel and bright field; λex = 405 nm, λem = 470-520 nm; Scale bars = 20 µm.

Since Ly-1 could react with Cys both in pH 5.0 and pH 7.4 systems with similar fluorescent signals, we pondered whether the cellular fluorescent signal was caused by in situ reaction of Ly-1 and lysosomal Cys or by the lysosome accumulation of the reaction product of Ly-1 and cytoplasmic Cys. To clarify this, LysoTracker Red-labeled A549 cells were cultured with Ly-1 and time-dependent imaging was initiated. As shown in Figure 5a and 5c (detailed in Figure S10), a fluorescent signal in the green channel emerged within 1 min and increased gradually over the following 10 min, consistent with the reaction of Ly-1 with Cys. Interestingly, the signals in the red channel and green channels could be superimposed during the imaging process with a lowest Pearson coefficient of 0.86 (Figure 5b). These results showed that the fluorescent signal in the green channel originated in lysosomes directly, which further demonstrated the specificity of Ly-1 toward lysosomal Cys.

Figure 5. a) Time dependent confocal fluorescent images of LysoTracker Red (0.5 µM) loaded A549 cells incubated with Ly1 (5 µM) while imaging; b) corresponding Pearson coefficients calculated from the intensity correlation plots; c) corresponding normalized fluorescent intensity changes of green channel and red channel; green channel: λex = 405 nm, λem = 470-520 nm; red channel: λex = 461 nm, λem = 580-600 nm.

Lysosomal Cys fluctuation during dexamethasone induced apoptosis. Cys is a natural cellular reductant that counteracts oxidative stress. During apoptosis, oxidative stress is involved in cell death.38-40 Thus, we investigated the utility of Ly-1 to image lysosomal Cys dynamics during lysosomeinitiated apoptosis. Dexamethasone, a synthetic glucocorticoid, was reported to induce a variety of different cells to undergo apoptosis through stimulation of sphingosine which accumulates in the lysosome and induces membrane permeabilization.27,39,41 When cells loaded with Ly-1 and LysoTracker Red were incubated with dexamethasone, the cells contracted significantly after 40 min incubation (Figure 6a) indicating gradual apoptosis (detailed in Figure S11).42,43

During this process, the corresponding fluorescent intensities of the dexamethasone-treated and the control group in the green channel quenched synchronously after 18 times exposure (Figure 6b). This phenomenon was caused by the photobleaching of Ly-1-Cys and displayed that dexamethasone induced A549 cells apoptosis was lysosomal Cys independent. Further, the Pearson coefficients of green and red channels were stable during the apoptosis process (Figure 6c). Combined with the impervious fluorescent emission of Ly-1-Cys at both pH 5.0 and pH 7.4, the lysosomal membrane was not ruptured (LMNR) in this process.

Figure 6. Time dependent confocal fluorescent images of LysoTracker Red (0.5 µM) and Ly-1 (5 µM) loaded A549 cells incubated with dexamethasone (4 µM, dexamethasone group) or PBS (control group) while imaging; b) corresponding normalized fluorescent intensity changes of dexamethasone group and control group in the green channel; c) corresponding Pearson coefficients calculated from the intensity correlation plots; Error bars represent standard deviations, n = 2; green channel: λex = 405 nm, λem = 470-520 nm; red channel: λex = 461 nm, λem = 580-600 nm.

In vivo Cys imaging. Next a Zebrafish study was undertaken to validate the feasibility of Ly-1 for imaging Cys in vivo. As shown in Figure 7a, a 5-day-old zebrafish incubated with Ly-1 displayed strong green fluorescent emission. For comparison, zebrafish was pretreated with NEM followed by incubation with Ly-1 displayed only minimal fluorescence (Figure 7b). Further, zebrafish successively incubated with NEM, Ly-1 and Cys displayed distinct


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Analytical Chemistry fluorescent emission (Figure 7c). These results implied that Ly-1 was tissue-permeable and could image endogenous and exogenous Cys specifically in live zebrafish.

Figure 7. Confocal fluorescent images of Ly-1 responding to endogenous and exogenous Cys in a 5-day-old zebrafish. a) Zebrafish was incubated with Ly-1 (5 µM) for 20 min; b) zebrafish was pre-treated with NEM (200 µM) for 20 min and then incubated with Ly-1 (5 µM) for 20 min; c) zebrafish was treated with NEM (200 µM) for 20 min, Ly-1 (5 µM) for 20 min, and then Cys (200 µM) for 20 min; d), e) and f) The corresponding merge images of the green channel and bright field; λex = 405 nm, λem = 470-520 nm.

CONCLUSIONS In conclusion, we have developed a coumarin probe Ly-1 to detect Cys selectively at pH 5.0 via pH-regulated thiol nucleophilic reactivity. The specific one-step Michael addition of sulfhydryl to the α,β-unsaturated ketone in Ly-1 and turnon fluorescent response enables the probe as a potential tool to label Cys in biological systems. The limit of detection of Ly-1 toward Cys was 1.2 µM. Cellular experiments demonstrated lysosomal Cys specific imaging capacity. Co-labeling of LysoTracker Red and Ly-1 enabled us to image lysosomal Cys dynamics during a dexamethasone-induced A549 cell apoptosis process, along with corresponding lysosomal membrane changes. These results are strong evidence that Ly1 type probes facilitate the study of lysosomal Cys dynamics under biological conditions such as oxidative stress and autophagy, and can enhance our understanding of the roles of Cys in lysosome functionality.

ASSOCIATED CONTENT Supporting Information Synthesis of probe and the structure characterizations, additional UV-vis and fluorescence spectra and additional fluorescence images (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

Author Contributions §

Y.Y. and F.H. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the National Natural Science Foundation of China (No. 21672131, 21775096, 21705102), Talents Support Program of Shanxi Province (2014401), Shanxi Province Foundation for Re-

turness (2017-026) and Scientific Instrument Center of Shanxi University.

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