Visualizing Hydrogen Sulfide in Mitochondria and Lysosome of Living

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Visualizing Hydrogen Sulfide in Mitochondria and Lysosome of Living Cells and in Tumor of Living Mice with Positively Charged Fluorescent Chemosensors Zhisheng Wu, Duanwei Liang, and XinJing Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02459 • Publication Date (Web): 18 Aug 2016 Downloaded from http://pubs.acs.org on August 19, 2016

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Visualizing Hydrogen Sulfide in Mitochondria and Lysosome of Living Cells and in Tumor of Living Mice with Positively Charged Fluorescent Chemosensors Zhisheng Wu, Duanwei Liang and Xinjing Tang * State Key Laboratory of Natural and Biomimetic Drugs, the School of Pharmaceutical Sciences, Peking University, Beijing 100191, China. Email: [email protected]; Tel/Fax: 8610-82805635. ABSTRACT: Two fluorescent chemosenors, Mito-HS and Lyso-HS, were rationally designed and synthesized with positive charge at physiological conditions. The positive charge showed triple functions as target moieties for subcellular mitochondria and lysosome of living cells, soluble moieties for chemosenors, as well as effective sequesters of HS− (H2S at physiological conditions) These two probes showed faster and more efficient fluorescence H2S detection than the similar literature-reported probe without positive charge. In addition, visualizing of hydrogen sulfide in tumor of living mice was successfully achieved for the first time using probe Mito-HS.

Hydrogen sulfide (H2S), together with carbon monoxide (CO) and nitric oxide (NO) has recently viewed as the three gaseous signaling molecules and plays crucial roles in chemical and biological process.1 H2S is associated with a variety of physiological and pathological processes, such as cell growth,2 cardiovascular protection,3 angiogenesis stimulation.4 It also acts as an antioxidant or scavenger for reactive oxygen species (ROS).5,6 Accordingly, efficient methods for detecting and tracking H2S inside living cells, esp. in subcellular structures are essential for understanding its biological processes and functions. Although several analytical technologies have been developed for detection of H2S such as colorimetric7 and electrochemical methods,8,9 chromatography,10 and sulfide precipitation,11 these methods are destructive and not suitable for detection in biological systems. In contrast, fluorescence probes, together with fluorescence microscopy techniques provide attractive methods to study H2S in biological signaling pathways. Under physiological condition, H2S actually contains three forms: H2S (aq), HS− and S2− and can be transformed each other. Considering their specific reducibility, nucleophilicity and strong complexation ability with Cu2+, many examples based on different H2S specific reactions were developed for detection and imaging of exogenous or endogenous H2S.12-44 Among these reactions, selective reduction of azido moiety by H2S was widely used for rational design of H2S fluorescent chemosensors due to the simple synthesis, good selectivity, and suitable reaction time kinetics. Our previous work12,35,36 showed the sensitive detect for H2S in cells, however, H2S probes with short emission wavelength and no subcellular localization effect was developed. To explore the functions of H2S in subcellular biology, there is a need to design fluorescent probes to track H2S distribution in organelles, such as mitochondria and lysosome which are associated with a variety of biological activities. Here, we chose 1,8-naphthalimide moiety as the fluorophore that has been used in developing H2S in previous work.18,23,45 However, low aqueous solubility

or no subcellular targeting effect was not solved. On the basis of previous works on target moieties for mitochondria and lysosome, we rational designed and synthesized two positively charged fluorescence probes (Mito-HS and Lyso-HS) for H2S detection in mitochondria and lysosome. (Scheme 1) Three factors were considered: 1) Based on our previous studies in sequestration effect of charged moieties on fluoride ion detection46,47, positively charged moieties in physiological conditions could sequenster HS- and S2- (two main form of H2S in physiological pH). 2) Probes can be localized in specific subcellular organelles. 3) Elevated aqueous solubility of probes. Among many functional moieties, triphenyl phosphonium and dimethylamino moieties satisfied all three requirements and could be used for guiding probes to mitochondria and lysosome, respectively.

Scheme 1. Syntheses of Mito-HS, Lyso-HS and Con-HS.

MATERIAL AND METHODS

General experiments and instruments All solvents and chemicals were purchased from commercial suppliers and were used without further purification. Column chromatography was performed on silica gel (200~300 mesh). The probe stock solutions were prepared in DMSO. PBS buffers were prepared using distilled water, and degassed

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with N2 for 30 min. NMR spectra were measured on Bruker instrument (400 MHz for 1H NMR and 100 MHz for 13C NMR). Mass spectra (ESI-MS) were acquired on a Waters SQD. pH values of the buffers were obtained on METTLER TOLEDO FiveEasy pH FE20. UV-Visible spectra were obtained on a DU800 ultraviolet spectrometer and fluorescence measurement were performed on a Cary Eclipse fluorometer. Cell imaging was performed with a Nikon A1R confocal microscope. In vivo imaging was performed with a CRI Maestro small animal in vivo imaging system. Detection of hydrogen sulphide of tissue homogenate was performed with a TBR 4100 Free Radical Analyzer. Synthesis of Chemosensors Synthesis of (3-ammoniopropyl) triphenyl phosphonium bromide. A solution of triphenylphosphane (1.00 g, 3.8 mmol) and 3-bromopropan-1-aminium bromide (834 mg, 3.8 mmol) in 50 mL CH3CN was heated to reflux overnight. After cooling to room temperature, the precipitate was collected by filtration and washed with CH2Cl2. The solid residue was dried to afford pruduct as a white solid (800 mg, 53% yield). 1H NMR (400 MHz, DMSO-d6,) δ/ppm 8.05 (s, 3H), 7.80 (m, 15H), 3.90 (d, J= 15 Hz, 2H), 3.05 (s, 2H), 1,90 (m, 2H), 13CNMR (100 MHz, DMSO-d6) δ/ppm 135.58, 135.56, 134.15, 134.05, 130.91, 130.78, 118.10, 20.56, 19.16, 18.63; MS (ESI): C21H23NP+ (M+) calculated: 320.16, found 320.40. Synthesis of probe Mito-HS. Compound 1 was synthesized according to the literature.48 (3-ammoniopropyl)triphenyl phosphonium bromide (479 mg, 1mmol) and compound 1 (239 mg, 1mmol) was dissolved in CH3OH and 3 mL triethylamine was added. The solution was heated to reflux for 5 h. After cooling to room temperature, the solvent was removed under reduced pressure. The solid residue was purified by flash column chromatograph to obtain Mito-HS (412 mg, 66% yield). 1H NMR (400 MHz, CD3OD) δ/ppm 8.19 (d, J = 7.16 Hz, 1H), 8.13 (t, J = 7.78 Hz, 2H), 7.89 -7.75 ( m, 15H),7.55 (t, J = 7.82 Hz, 1H,) 7.34 (d, J = 7.96 Hz, 2H), 4.23 (t, J = 6.92 Hz , 1H), 3.69-3.62 (m, 2H), 13C NMR (100 MHz, CD3OD) δ/ppm 163.64, 163.16, 143.57, 135.02, 134.99, 133.57, 133.47, 131.45, 131.35, 130.29, 130.16, 128.46, 128.26, 126.49, 123.60, 121.59, 118.70, 117.83,117.62, 114.73, 40.04, 20.94,19.77 MS (ESI) m/z, C23H18N3O7+ (M + H)+ calculated: 541.18, found 541.43. Synthesis of probe Lyso-HS. A mixture of compound 1 (239 mg, 1 mmol) and N, N-dimethylpropane-1,3-diamine (153 mg ,1.8 mmol) in MeOH was heated to reflux for 5 hours. After cooling to room temperature, the solvent was removed. The solid residue was by flash column chromatography to obtain Lyso-HS as a yellow solid (200 mg, 71% yield.) 1H NMR (400 MHz, CDCl3) δ/ppm 8.63 (dd, J1 = 1.08 Hz, J2 = 7.28 Hz, 1H), 8.58 (d, J = 7.96 Hz, 1H), 8.45-8.42 (m, 1H), 7.77-7.73 (m, 1H), 7.47 (d, J = 8.00 Hz, 1H), 4.24 (t, 2H, J = 7.54 Hz), 2.47 (t, J = 7.28 Hz, 2H), 2.29 (s, 6H), 1.97-1.90 (m, 2H); 13C NMR (100 MHz, CDCl3) δ/ppm 163.92, 163.49, 143.37, 132.14, 131.63, 129.10, 128.70, 126.82, 124.30, 122.60, 118.89, 114.63, 57.23, 45.32, 38.79, 26.02. ESI-MS: m/z C23H18N3O7+ (M+H+) calculated: 324.15, found: 324.25. Synthesis of probe Con-HS. n-Butylamine (0.5 mL) and compound 1 (239 mg, 1 mmol) was dissolved in CH3OH. The solution was heated to reflux for 5 h. After cooling to room temperature, the solution was filtered and the solid residue was washed with CH3OH to obtain Con-HS (206 mg, 70% yield). 1 H NMR (400 MHz, CDCl3) δ/ppm 8.61 (d, J = 7.24 Hz, 1H), 8.56 (d, J = 7.96 Hz, 1H), 8.41 (d, J = 8.36 Hz, 1H), 7.73 (t, J

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= 7.84 Hz, 1H), 7.45 (d, J = 7.96 Hz, 1H), 4.16 (t, J = 7.54 Hz, 2H) ,1.75-1.67 (m, 2H), 1.48-1.42 (m, 2H), 0.98 (t, J = 7.34 Hz, 3H); 13C-NMR (100 MHz, CDCl3) δ/ppm 163.97, 163.55, 143.33, 132.15, 131.64, 129.11, 128.67, 126.83, 124.31, 122.66, 118.96, 114.65, 40.27, 20.40, 13.86. ESI-MS (ESI): (M + Na)+ calculated: 317.10, found 317.14. RESULTS AND DISCUSSION Rational design and characterization of fluorescent sensors for H2S. 4-amino-1,8-naphthalimide was chosen as fluorophore due to its commercial availability, readily synthesis and high fluorescent quantum yield. Compound 1 can be easily prepared from 4-bromo-1,8-naphthalic anhydride. The probes, Mito-HS and Lyso-HS were then obtained from compound 1 with (3-ammoniopropyl) triphenyl phosphonium bromide and N, N-dimethylpropane-1,3-diamine by one-step amidation. The syntheses were very straightforward without laborious procedures (Scheme 1). In addition, we also synthesized a literature-reported H2S fluorescence probe (Con-HS) for comparison.49 (See supporting information for details) Due to the positive charge (dimethylamine moiety will be partially protonized at physiological pH 7.4), the probes, Mito-HS and Lyso-HS were much higher solubility than Con-HS. Both Mito-HS and Lyso-HS were soluble in PBS buffer (containing 1% DMSO) at a concentration up to 1 mM. However, Con-HS was precipitated at the same concentration. Evaluation of Sensitivity and Selectivity. All three chemosensors, Mito-HS, Lyso-HS and Con-HS were then investigated for the evaluation of fluorescent detection of H2S in 20 mM degassed PBS buffer (pH 7.4, containing 1% DMSO) (Figure 2 and Figure S2). Under these conditions, all the probes are essentially non-fluorescent due to azide functionality. Upon addition of NaHS (a standard source for hydrogen sulfide), Mito-HS solution (10 µM) quickly turn to yellow-green fluorescence emission under hand-hold UV lamp and its fluorescence intensity at 540 nm reached its plateau in 45 min and 21-fold turn-on fluorescence was observed. For Lyso-HS, similar experimental phenomenon was observed, but with a little bit lower sensitivity. We only observed 15fold increase in fluorescence intensity upon the addition of NaHS in 60 min. However, under current conditions, literature-reported Con-HS showed much less sensitivity, and the addition of NaHS only triggered 4-fold enhancement of fluorescence emission. By fitting the data of time-dependent fluorescence responses (Figure S3), fluorescence

Figure 1. Fluorescence spectra of Mito-HS, Lyso-HS and Con-HS (10 µM) upon addition of NaHS (25 eq.). Inset is photo images of turn-on fluorescence of three chemosensors.

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Figure 2. Kinetics of fluorescent intensity of probes (10 µM) at 540 nm against time in the presence of NaHS (25 eq.). The experiments were performed in PBS buffer (20 mM, 1% DMSO, pH 7.4), λem=435 nm, slit width (ex/em): 5 nm/10 nm. turn-on rates of all three chemosensors were obtained. Pseudofirst-order kinetic rate constant (0.034 min-1) of Mito-HS was also a little bit larger than that of Lyso-HS (0.028 min-1), however both of them have much larger pseudo-first-order kinetic rate (17- and 14- fold) than that of Con-HS (0.002 min-1). Due to the same reactive functional azido moiety and the same chromophore, the sensitivity and kinetic deference in fluorescence response to H2S was caused by different attachment of imides. According to our previous observations on fluoride fluorescence probes,46,47 the positively charged triphenyl phosphonium with a flexible linker could sequester HS-1 or S2- and bring them to the surrounding of azido moiety. The azido group was then quickly reduced and triggered the fluorescence emission of 4-amino-1, 8-naphthalimide chromophore. Tertiary amine of Lyso-HS could also be partially protonized under physiological pH and the similar sequestration effect was also observed, but with less effective than that of triphenylphosphonium. However, for Con-HS with only alkyl moiety, much less efficient and slower response was observed.

Figure 3. Fluorescence intensity changes at 540 nm of the probes (10 µM) with different concentrations of NaHS (0~100 µM). λex = 435 nm, λem = 540 nm. Data was collected in 60 min after the addition of NaHS.

Next, we evaluated the concentration dependence of the triggered fluorescence intensity on H2S. Various concentrations of NaHS (0~100 µM) was added to the probe solutions (10 µM) and the fluorescence spectra were measured. As shown in Figure 3, standard curves for both probes (Mito-HS and Lyso-HS) in degassed PBS buffer were obtained between fluorescence signals at 540 nm and the concentration (µM) of NaHS. The regression equations were y = 0.91x+14.01 (R2 = 0.988) for Mito-HS and y = 0.67x+11.16 (R2 = 0.993) for Lyso-HS, respectively. The data suggested a good linear relationship between turn-on fluorescence intensity of Mito-HS and Lyso-HS and H2S concentrations in degassed PBS buffer. To evaluate the fluorescence H2S detection of Mito-HS and Lyso-HS, we investigated their selectivity in the presence of common anions, cations, reactive oxygen species, and biothiols. As shown in Figure 4, addition of commons anions (F−, Cl−, Br−, NO2−, AcO−, SCN−, CO32−, HCO3−, SO42−, HSO4−), metal ions (Na+, Mg2+, Ca2+, Zn2+), and ROS (H2O2, ClO−, tBuOOH, 1O2, O2−) did not trigger turn-on fluorescence responses even at 1 mM for both chemosensors. The addition of biothiols (such as cysteine, glutathione and homocysteine at 5 mM) slight turn on the fluorescence emission with only 2-3 fold increase for Mito-HS and Lyso-HS. However, a robust increase in fluorescence intensity was observed upon addition of 100 µM NaHS with 13- and 9- fold enhancement under current experimental conditions. These results demonstrated that both chemosensors had a high selectivity for fluorescence detection of H2S over various biologically relevant analytes.

Figure 4. Fluorescence responses of probes (A, Mito-HS; B, Lyso-HS, 10 µM) towards various species. control; F−; Cl−; Br−; NO2−; AcO−; SCN−; CO32−; HCO3−; SO42−; HSO4−; Na+; Mg2+; Ca2+; Zn2+; ClO−; H2O2 ; t-BuOOH; O2−; 1O2; GSH; Cys; Hcy; NaHS. The experiment was performed in degassed PBS buffer (20 mM, pH 7.4), λex = 435 nm. The incubation time was 60 min.

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Cell Studies. Encouraged by good sensing performance for Mito-HS and Lyso-HS towards H2S, we next investigated whether both probes can be applied for imaging H2S in mitochondria and lysosome of living cells. Firstly, standard cell viability protocols (SRB assays) for probes were conducted and the results showed that Mito-HS had higher toxicity than Lyso-HS at their high concentrations. However, the concentrations (10 µM) of both probes used for cell imaging studies were only slightly toxic even after a long incubation time (24 h). As shown in Figure 5, cells incubated with only Mito-HS or Lyso-HS also afforded an obvious green fluorescence signal (Figure 5A and D). The fluorescence signal should be induced by endogenous H2S instead of background fluorescence of probes in cells. To confirm that, HeLa cells were pre-treated with PMA (1 µg/mL) for 30 min which could decrease endogenous H2S,50,51 and then further incubated with Mito-HS or Lyso-HS (10 µM) for 30 min. Almost no fluorescence emission was observed under the same imaging conditions (Figure 5 B, E). In addition, after PMA pre-treated cells were incubated with the probes, followed by the addition of NaHS, the strong turn-on fluorescence of both probes was observed again. These results indicated that both Mito-HS and Lyso-HS are also applied for imaging exogenous and endogenous H2S. In order to further demonstrate targeting specificity of Mito-HS and Lyso-HS for mitochondria and lysosome in cells, colocalization experiments were also performed (Figure 6). The overlapped fluorescence image (Pearson’s correlation 0.94, see supporting Figure S9) of MitoTracker Red and H2Striggered Mito-HS indicated that triphenyl phosphonium moiety did bring Mito-HS to the mitochondria of living cells. The same result was obtained for lysosomal colocalization of LysoTracker Red and H2S triggered Lyso-HS with overlapped green and red fluorescence (Pearson’s correlation 0.79, see supporting Figure S9). It is reported that H2S can be produced in mitochondria52 and lysosome53. To make sure that the triggered fluorescence of Mito-HS and Lyso-HS actually happened in mitochondria and lysosome instead of cytoplasma, HeLa cells was first treated with PMA to consume H2S, and then incubated

Figure 5. Fluorescence images of H2S in HeLa cells. (A, D) Cells were incubated with 10 µM probes; (B, E) Cells were incubated with PMA (1 µg/mL) for 30 min, and was then treated with Mito-HS or Lyso-HS for another 30 min; (C, F) Cells were pre-stimulated by PMA (1 µg/ mL) for 30 min, then were treated with Mito-HS or Lyso-HS (10 µM, 30 min), followed by incubation with NaHS (200 µM, 60 min). Scale bar=50 µm.

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Figure 6. Confocal images of chemosensors colocalized in mitochondria and lysosome of HeLa cells. (A) Mito-HS (10 µM, λex=488 nm, λem=500−550 nm). (B) MitoTracker Red (1 µM, λex=561 nm, λem=570−620 nm). (C) Overlay of A and B. (D) Lyso-HS (10 µM, λex=488 nm, λem=500−550 nm). (E) LysoTracker Red (0.5 µM, λex=561 nm, λem=570−620 nm). (F) Overlay of D and E. Scale bar= 10 µm. with Mito-HS or Lyso-HS. After localization of Mito-HS or Lyso-HS in 30 min, cell washing, the cells were then incubated with NaHS. Only strong fluorescence in mitochondria or lysosome could be observed after colocalization with MitoTracker or LysoTracker. The data indicated that targeting moieties (triphenyl phosphonium and dimethylamino) could specifically bring the probes to mitochondria or lysosome and H2S in mitochondria or lysosome reduced the azido moiety and turned on the fluorescence of Mito-HS or Lyso-HS. The results showed promising applications to study the H2S functions in subcellular structures, such as mitochondria or lysosome. Finally, we examined the suitability of the sensor for detecting and visualizing H2S in living animals. BALB/C nude mice with xenograft breast cancer tumor were selected and given a skin-pop injection of probe Mito-HS in region A (breast tumor tissue) and nearby region B (no-cancerous tissue). After incubation for 2 h, the nude mice were imaged using a CRI Maestro small animal in vivo imaging system. A shown in Figure 7, no-cancerous tissue showed almost no fluorescence, while tumor tissue showed strong fluorescence of the probe according to the pseudocolor, proving that probe Mito-HS can detect endogenous H2S in breast tumor. In addition, tumor and no-cancerous tissues were cut and made to homogenate, and H2S levels of

Figure 7. Representative fluorescence images of nude mice (pseudocolor) given a skin-pop injection of probe (Mito-HS, 50 µM, 150 µL) and the signal to noise ration. Region A represent breast tumor tissue and region B represent no cancerous tissue. Images was taken in 2 hours after injection.

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these tissues were analyzed by a sulfide-specific electrode. As shown in Figure S10, electronic current for tumor tissue is obviously higher than that for no-cancerous tissue, indicating the higher concentration of H2S in tumor tissue compared with no-cancerous tissue. This is due to the factor that breast cancer cells exhibit higher expression levels of cystathionine βsynthase (CBS, a H2S producing enzyme) than adjacent cells from no-cancerous tissue or non-transformed cells.54,55 This represents the first example of imaging of H2S in breast tumor of living mice. CONCLUSIONS In summary, two fluorescence naphthalimide chemosensors (Mito-HS and Lyso-HS) with attachment of triphenylphosphonium and dimethylamino moieties were developed to specifically detect H2S in mitochondria and lysosome. These two attached moieties were positively charged under physiological pH, which could also enhance probe solubility. And the positive charge also showed sequestration effect on sulfur anions and promoted quicker and more sensitive response to H2S than literature reported Con-HS did under the same conditions. These two probes showed good selectivity over common anions, cations, reactive oxygen species, as well as biothiols. In addition to the solubility and sequestration effect of positive charges, attachment of triphenylphosphonium and dimethylamino moieties helped transport probes to mitochondria or lysosome specifically and detected H2S in situ. These results indicated that these probes could show promising applications to study H2S biology in subcellular structures, such as mitochondria and lysosome. Furthermore, Imaging of tumor H2S in living mice was successfully achieved for the first time.

ASSOCIATED CONTENT Supporting Information Experimental procedures of in vitro and in vivo measurements, Figure S1-S11 and NMR and MS of synthetic probes and their intermediates. This material is available free of charge via the Internet at http://pubs.acs.org/

AUTHOR INFORMATION Corresponding Author * Email: [email protected]; Tel/Fax: 8610-82805635.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Basic Research Program of China (‘973’ Program; grant no. 2013CB933800), the National Natural Science Foundation of China (grant nos. 21422201 and 21372018).

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