Article pubs.acs.org/ac
Targetable Fluorescent Probe for Monitoring Exogenous and Endogenous NO in Mitochondria of Living Cells Haibo Yu,† Xinfu Zhang,† Yi Xiao,*,† Wei Zou,‡ Liping Wang,‡ and Liji Jin*,‡ †
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China School of Life Science and Technology, Dalian University of Technology, Dalian 116024, China
‡
S Supporting Information *
ABSTRACT: Nitric oxide (NO) is a ubiquitous cellular messenger molecule in the cardiovascular, nervous, and immune systems. Mitochondrion is the main area where endogenous NO is synthesized by inducible NOS enzymes in mammalian cells. Thus, real-time monitoring NO in mitochondria is very meaningful for NO chemical biology. Although a variety of fluorescent probes for NO have been successfully developed, they are not suited for detecting mitochondrial NO because none of them can specifically localize in mitochondria. Herein, Mito-Rh-NO, the first mitochondria-targetable “turn-on” fluorescent probe for NO, has been developed through attaching a triphenylphosphonium to a rhodamine spirolactam. The characteristics of this probe are as following: (1) Mito-Rh-NO exhibits high sensitivity toward NO. In solution, Mito-Rh-NO responds to NO by significant fluorescence enhancement up to 60-fold, and its NO detection limit is as low as 4.0 nM. (2) The NO sensing of Mito-Rh-NO is highly selective, which will not interfere with the other reactive oxygen and nitrogen species. (3) Mito-Rh-NO has a low cytotoxic effect: after being treated with 10 μM Mito-Rh-NO for 24 h, the survival rate is higher than 90%. (4) Mito-Rh-NO specifically localizing in mitochondria: colocalization experiment of Mito-Rh-NO and Rh 123, a typical mitotracker, shows the merged fluorescent microcopy image with a high Pearson’s colocalization coefficient 0.92 and overlap coefficient 0.99. (5) Mito-Rh-NO demonstrates high applicability for realtime monitoring of mitochondrial NO in live cells. Both the exogenous NO released by the donor NOC13 and endogenous NO generated in cells under stimulation have been visualized under confocal microscopy.
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mitochondrial NO can modulate the respiratory chain by binding to the heme group of cytochrome c oxidase or by controlling mitochondrial pH.21−23 Second, NO endogenously generated in mitochondria can be further oxidized into ONOO−, N2O3, NO2−, and NO3− by excess reactive oxygen species and induces nitrosative damage to proteins, ultimately resulting in apoptosis of cells.24,25 Finally, NO and oxidation product ONOO− in mitochondria can easily diffuse into other subcellular structures such as lysosomes and lipid membrane and be a well-known inflammatory mediator in regulating of autophagy.26−28 Although studies of mitochondrial NO have helped people to understand many NO-relative physiological processes such as aging of the cardiovascular system,29 fatty livers,30 and lipid peroxidation of the brain,31 unknown functions of mitochondrial NO and the dependency relationship of NO with calcium ions and copper(I) in mitochondria is still not well established.32−34 Since there remain many other aspects awaiting discovery, it is very meaningful for NO chemical biology to search for methods and tools to monitor mitochondrial NO.
itric oxide (NO), a messenger molecule, plays key roles in various physiological as well as pathological processes. Endogenous NO has mediated multiple processes in the various physiological systems such as the cardiovascular system, immune system, and the central and peripheral nervous system.1−3 Exogenous NO released from NO donors such as sodium nitroprusside (SNP), nitroglycerin, S-nitroso-N-acetylpenicillamine (SNAP), and spermine NONOate was widely used as a modulator in preclinical treatments of cancers and other diseases.4−7 Moreover, excessive and unregulated NO synthesis has been implicated as a causal or contributing factor to various pathophysiological conditions including cancer,8,9 endothelial dysfunction,10,11 and neurodegenerative diseases.12−14 It has been reported that upregulation of nitric oxide synthase (NOS) in head and neck squamous cell carcinoma (HNSCC) is linked with oral carcinogenesis.15,16 The free radical nature of NO makes it undergo a variety of reactions in tissues with other radicals, leading to the generation of highly reactive nitrosating species. These toxic species would cause nitrosation or oxidation of zinc finger-containing proteins involved in DNA repair.17 Hence, nitric oxide has been a hot area of research in recent years. Mitochondrion is the main area where endogenous NO is synthesized by inducible NOS enzymes in mammalian cells and plays a critical role in regulating of cell functions.18−20 First, © XXXX American Chemical Society
Received: January 16, 2013 Accepted: July 5, 2013
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dx.doi.org/10.1021/ac401916z | Anal. Chem. XXXX, XXX, XXX−XXX
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for 24 h. After the materials were consumed completely (monitored via thin-layer chromatography), the reaction mixture was washed with water and extracted with dichloromethane. The organic phase was dried over MgSO4. The solvent was evaporated and the residue was purified by silica gel column chromatography using eluent (CH2Cl2/CH3OH 50:1). Compound 2 was obtained as a white solid (300 mg; yield, 67%) mp 259−260 °C. 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS): δ 7.89 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 7.09 (s, 1H), 6.84 (t, J = 8.0 Hz, 1H), 6.69 (br. s, 2H), 6.54 (d, J = 8.0 Hz, 1H), 6.44 (br. s, 2H), 6.28 (s, 2H), 6.18 (t, J = 8.0 Hz, 1H), 5.90 (d, J = 8.0 Hz, 1H), 4.46 (s, 2H), 4.38 (s, 1H), 2.90 (s, 12H). 13C NMR (100 MHz, DMSO-d6, 25 °C, TMS): δ 164.6, 153.3, 152.6, 151.1, 145.6, 132.0, 131.2, 128.9, 128.3, 127.5, 127.0, 125.9, 123.3, 120.6, 116.0, 115.5, 108.6, 106.3, 98.1, 83.3, 82.6, 66.7, 30.6, 22.1, 14.0. ES(+)TOF-MS calcd for C32H29N4O2 [M + H]+ 501.2291, found 501.2287. Synthesis of Mito-Rh-NO. The compound 2 (100 mg, 0.20 mmol), (3-azidopropyl)triphenylphosphonium bromide (85 mg, 0.20 mmol), sodium ascorbate (4.0 mg, 0.02 mmol), and CuSO4·5H2O (2.25 mg, 0.01 mmol) were suspended in a mixture of THF and water (v/v 5 mL/2 mL) in a 50 mL roundbottom flask equipped with a small magnetic stirring bar. Under a nitrogen atmosphere, the mixture was stirred at room temperature for 24 h. After the materials were consumed completely (monitored via thin-layer chromatography), the reaction mixture was washed three times with water and extracted with dichloromethane. The organic phase was dried over MgSO4. The solvent was evaporated, and the residue was purified by silica gel column chromatography using eluent (CH2Cl2/CH3OH 20:1). An offwhite solid (100 mg, yield 54%) was obtained, mp 286−287 °C. 1H NMR (400 MHz, DMSO-d6, 25 °C, TMS) δ 8.74 (s, 1H), 8.09 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 7.9 Hz, 1H), 7.92−7.61 (m, 21H), 7.45 (s, 1H), 6.94−6.10 (m, 11H), 5.93 (d, J = 7.5 Hz, 1H), 4.54 (s, 2H), 4.48 (s, 2H), 3.65 (br. s, 2H), 2.89 (s, 12H), 2.12 (br. s, 2H). 13C NMR (100 MHz, DMSO-d6, 25 °C, TMS) δ 165.1, 154.1, 152.6, 151.1, 145.7, 145.6, 135.1, 135.0, 133.7, 133.6, 130.3, 130.2, 129.0, 128.2, 127.5, 125.3, 123.7, 122.9, 120.9, 119.9, 118.5, 117.7, 116.1, 115.6, 108.6, 98.1, 66.9, 49.5, 49.3, 22.8, 18.3, 17.7. ES(+)-TOF-MS calcd for C53H49N7O2P 846.3685, found 846.3621; atmospheric pressure ionization (API)-ES(−) Br 79.0 Preparation and Calibration of Reactive Oxygen and Nitrogen Species. NOC13 (l-Hydroxy-2-oxo-3-(3-aminopropyl)-3-methyl-l-triazene) was synthesized according to the method reported in the literature.52 NOC13 is the NO releaser, and it’s half-life period is 13.7 min at 22 °C in pH 7.4 phosphate buffer. Hydrogen peroxide (H2O2) was diluted immediately from a stabilized 30% solution, and its concentration was determined by using its molar absorption coefficient of 43.6 M−1 cm−1 at 240 nm.53 Freshly prepared aqueous solutions of NaNO2 and NaNO3 were used as nitrite (NO2−) and nitrate (NO3−) sources, respectively. Singlet oxygen was chemically generated from the −OCl/H2O2 system in buffer.54 Hydroxyl radicals (•OH) were generated in the Fenton system from ferrous ammonium sulfate and hydrogen peroxide.55 ONOO− was synthesized from sodium nitrite (0.6 M) and H2O2 (0.65 M) (excess H2O2 was used to minimize nitrite contamination). After the reaction, the solution was treated with MnO2 to eliminate the excess H2O2. The concentration of the ONOO− stock solution was assayed by
So far, the strong desire for a reliable method to monitor mitochondrial NO has hardly been achieved. Among various cellular biology tools, fluorescent probes take the advantages of high resolution, high sensitivity, and noninvasive damage to biological specimens. For this reason, a variety of NO sensitive fluorescent molecular probes have been developed by researchers all over the world.35−50 Among these works, those of Nagano’s37,46−48 group and Lippard’s38 group are pioneering. The extensive applications of these fluorescent probes have provided good opportunities to assess cellular NO and have greatly promoted NO-related biological investigations. Nevertheless, although these probes are claimed to be able to map NO’s distributions in cells, none of them is well suited for mitochondrial applications, because they are not mitochondriatargeting. Actually, in a previous investigation, DAF-FM,48−50 a commercially available NO probe mainly localizing in cytoplasm but not in mitochondria, was adopted to detect NO generated in mitochondria,50 which would lead to inexact information as fluorescence response to NO synthesized by NOS in cytoplasm could not be ruled out. Thus, development of mitochondrion-specific fluorescent probes will benefit NO chemical biology very much.
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EXPERIMENTAL SECTION Synthesis of Compound 1. 6-Bromo-tetramethylrhodmaine was obtained according to the method we previously reported.51 A solution of 6-bromotetramethylrhodamine (6-BrTMR) (1 g, 2.15 mmol) in dry 1,2-dichloroethane (80 mL) was stirred until the solid dissolved completely, and phosphorus oxychloride (0.36 mL) was added with vigorous stirring at room temperature for 10 min. Then the solution was refluxed for 4 h. The reaction mixture was cooled and evaporated in vacuo to give 6-bromotetramethylrhodamine acid chloride, which was used without further purification. The crude acid chloride was dissolved in dry acetonitrile (50 mL) and added dropwise to a solution of o-phenylenediamine (227 mg, 2.10 mmol) in CH3CN (30 mL) and NEt3 (8 mL). After stirring overnight, the crude product was purified through silica gel column chromatography with a mixture of dichloromethane and methanol (v/v 50:1) as the eluent. Compound 1 was obtained as a colorless powder (0.86 g; yield, 72%) mp, 188− 189 °C. 1H NMR (400 MHz, CDCl3, 25 °C, TMS) δ 7.89 (d, J = 8.0 Hz, 1H), 7.67 (dd, J = 8.0, 1.6 Hz, 1H), 7.34 (d, J = 1.5 Hz, 1H), 6.94 (t, J = 8.0, 1H), 6.70 (d, J = 8.8 Hz, 2H), 6.54 (dd, J = 8.0, 1.2 Hz, 1H), 6.46− 6.36 (m, 3H), 6.31 (d, J = 2.0 Hz, 2H), 6.06 (dd, J = 8.0, 1.3 Hz, 1H), 2.95 (s, 12H). 13C NMR (100 MHz, CDCl3, 25 °C, TMS) δ 165.5, 154.4, 153.5, 151.5, 144.3, 132.0, 130.6, 128.8, 128.7, 128.3, 127.5, 127.4, 125.0, 121.9, 118.4, 117.3, 108.6, 106.9, 98.8, 77.4, 77.1, 76.7, 67.7, 53.5, 40.3. Electrospray(+)-time-of-flight mass spectrometry (ES(+)-TOF-MS) calcd for C30H27N4O2Br 554.1317, found 554.1309 Synthesis of Compound 2. To a 10 mL microwave tube equipped with magnetic stirrer, compound 1 (500 mg, 0.90 mmol), trimethylsilylacetylene (143 mg, 1.46 mmol), triethylamine (1.5 mL), PdCl2(PPh3)2 (0.2%), and toluene (4 mL) was added under a nitrogen atmosphere. The sealed tube was placed in a microwave reactor. Then its contents were irradiated, with vigorous stirring, for 15 min at 150 °C (constant temperature mode). After completion of the reaction, the solvent was evaporated in vacuo, and the residue was dissolved in 40 mL of methanol containing KOH (200 mg, 3.6 mmol). The offwhite solvent was stirred at room temperature B
dx.doi.org/10.1021/ac401916z | Anal. Chem. XXXX, XXX, XXX−XXX
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Scheme 1. Synthesis of Mito-Rh-NO
Figure 1. Changes in the absorption (a) and emission spectra (b) of Mito-Rh-NO (1.0 μM) in CH3CN/H2O (20:80, v/v) at pH 7.4 (0.1 mM sodium phosphate buffer) upon the gradual addition of NO solution from 0 to 25 μM. Inset: the linear response of fluorescent intensity at 585 nm in the concentration of NO from 1 to 10 μM (Ex slit 1 nm, Em slit 1 nm). (c) Emission spectra of Mito-Rh-NO (0.1 μM) in the presence of various amounts NO from 0 to 0.07 μM (Ex slit 1 nm, Em slit 3 nm). (d) Fluorescent intensity at 585 nm upon the gradual addition of NO solution (from 0 to 0.07 μM).
Preparation of NO Stock Solutions. The preparation and concentration of NO was determined by the Griess method
measuring the absorbance at 302 nm with a molar extinction coefficient of 1670 M−1 cm−1.56 C
dx.doi.org/10.1021/ac401916z | Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 2. Fluorescence response (a), intensity change at 585 nm of Mito-Rh-NO (1.0 μM) for NO over (b) other reactive nitrogen, oxygen species, DAA, and AA (0.1 mM, 100 equiv) in CH3CN/H2O (20:80, v/v) at pH 7.4 (0.1 mM sodium phosphate buffer) at 25 °C. Fluorescence is collected 30 min after the addition of reactive nitrogen, oxygen species, and other molecules.
reported previously.57 Since O2 can rapidly oxidize NO to form NO2, all apparatus were carefully degassed with argon for 30 min to remove O2. The NO gas was bubbled through a saturated NaOH solution to eliminate NO2 generated from the reaction of NO with O2. To produce a NO solution as a stock solution, this gas was bubbled through 10 mL of deoxygenated deionized water for 30 min and kept under an NO atmosphere. The concentration of this stock solution was determined by the Griess method. Aliquots (50 μL) of this solution were added to 1 mL of potassium phosphate buffer (0.1 mM, pH 7.4) containing sulfanilamide solution (17 mM) and N-(1naphthyl)ethylenediamine (0.4 mM). The solution was immediately mixed by inversion and incubated at room temperature for 5 min. The colorimetric product was measured in each well at 490 nm by use of a UV−vis spectrophotometer HP-8453. Sodium nitrite standards were used to normalize the assay reactivity and associated absorbance. Effects on Cell Growth/Viability. The cytotoxic effects of probe Mito-Rh-NO were assessed using the MTT assay. Briefly, 1.0 × 104 cells/well were seeded onto 96-well plates and allowed to grow for 24 h prior to treatment with different MitoRh-NO concentrations. The final concentrations of probe Mito-Rh-NO were kept from 0 to 30 μM (n = 3). After 24 h, the Mito-Rh-NO-containing medium was replaced with PBS. At the end of this time, MTT was then added to each well (final concentration 0.5 mg/mL) for 4 h at 37 °C and formazan crystals formed through MTT metabolism by viable cells were dissolved in DMSO. Optical densities were measured at 570 nm using a microplate reader AC100-120. For analysis of statistical differences between the treatment groups and the control group, the t test was performed. Statistical significance was reached when the p-value was
