Phosphate Ion Targeted Colorimetric and Fluorescent Probe and Its

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A Phosphate Ion Targeted Colorimetric and Fluorescent Probe and Its Use to Monitor Endogeneous Phosphate Ion in a Hemichannel-Closed Cell Lin E Guo, Jun Feng Zhang, Xin Yi Liu, Li Mei Zhang, Hong Li Zhang, Jian hua Chen, Xiao Guang Xie, Ying Zhou, Kaijun Luo, and Juyoung Yoon Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503818p • Publication Date (Web): 16 Dec 2014 Downloaded from http://pubs.acs.org on December 22, 2014

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Analytical Chemistry

A Phosphate Ion Targeted Colorimetric and Fluorescent Probe and Its Use to Monitor Endogeneous Phosphate Ion in a Hemichannel-Closed Cell Lin E Guo,†, # Jun Feng Zhang,‡, # Xin Yi Liu,§ Li Mei Zhang,† Hong Li Zhang,‡ Jian Hua Chen,‡ Xiao Guang Xie,† Ying Zhou*, † Kaijun Luo*, § and Juyoung Yoon *,¶ † College of Chemical Science and Technology, Yunnan University, Kunming 650091, China, fax: +86-871-5033679, e-mail: [email protected] ‡ College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, China § College of Life Science, Yunnan University, Kunming 6500 91, China, fax: +86-871-65031412, e-mail: [email protected] ¶ Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea, fax: + 82-2-3277-3419, e-mail: [email protected]

ABSTRACT: Fluorescent probe 1, the first inorganic phosphate (Pi) targeted colorimetric and fluorescent probe to detect endogenous Pi in hemichannel-closed cells, has been developed. Probe 1 undergoes a unique Pi induced hydrolytic reaction in DMSO–HEPES (V/V = 9:1) buffered (0.02 M, pH 7.4) solutions that produces a colorimetric change associated with a 62-nm red-shift in the UV-vis absorption maximum and up to a 780-fold enhancement in the fluorescence intensity. The mechanistic proposal that these spectroscopic changes are associated with reaction Pi with 1 to form coumarin gains support from the results of theoretical calculations and mass spectrometry studies. Observations made in fluorescence imaging studies with HeLa cells and C. elegans show that 1 can be employed to monitor Pi production in-vivo caused by apyrase-catalyzed ATP hydrolysis. Moreover, probe 1 was utilized to show that apoptosis of hemichannel-closed Sf9 cells is caused by Inx3 promoted dephosphorylation of Akt (RAC serine/threonine-protein kinase), leading to an elevation of the concentration of Pi. Overall, the study has produced the first fluorescent sensor 1 for endogenous inorganic phosphate. Moreover, the utility of 1 for measuring Pi release in vitro has been demonstrated and utilized to elucidate the mechanism of Inx3 action in hemichannel-closed Sf9 cells.

INTRODUCTION As the importance of anionic species has risen in biology, medicine, catalysis, and the environment so has the interest in developing molecules that are capable of selectively recognizing and sensing these negatively charged ions 1-8. Fluorescence chemosensors have received extensive study owing to their high sensitivities and, in particular, their potential applications to monitoring in-vitro and/or in-vivo analytes. The three main approaches used thus far to design chemosensors for anion recognition rely on a “binding site-signaling subunit” protocol, a displacement approach, and the “chemodosimeter” paradigm 9. Compared with the first two, the chemodosimeter strategy takes advantage of target specific anion induced chemical reactions that generate unique fluorescence or colorimetric changes. Adenosine triphosphate (ATP) and pyrophosphate (PPi), the chemical energy carriers in living cells, both generate two molecules of inorganic phosphate (Pi) by hydrolysis under cellular conditions. However, unlike those devised for ATP 10-19 and PPi 20-35, chemosensors for selective recognition of inorganic phosphate, especially the Pi anion, have been explored to a much lesser extent 36-38. One reason for this is that the selectivity for sensing Pi is hindered by the presence of structurally similar anions in cells. Owing to the significant advantages held by chemodosimeters, we envisioned that a

strategy utilizing a predesigned Pi induced reaction, which is not promoted by ATP, AMP or PPi, would serve as the basis for a selective sensor for this important anionic species. To explore this approach, we designed chemodosimeter 1 (Scheme 1) as an endogenous Pi targeted colorimetric and fluorescent probe. Chemodosimeter 1 contains an oxalate moiety linked via an ester bond to the hydroxyl group of coumarin fluorophore. We anticipated that selective reaction of 1 with Pi would lead to cleavage of the ester bond and liberation of the coumarin fluorophores, which would bring about observable changes in both the absorption and fluorescence spectra. In the study described, below we have demonstrated that 1 is a selective colorimetric and fluorescent chemosensor for Pi and that it can be employed to detect this important anion in vivo in HeLa cells and C. elegans and used to elucidate the mechanism of Inx3 promoted apoptosis of hemichannel-closed Sf9 cells.

EXPERIMENTAL SECTION General methods. All reagents were purchased from commercial sources and were used without further purification. Flash chromatography was carried out on silica gel (230-400 mesh). 1H NMR spectra (CDCl3) were recorded using DRX 500 spectrometer; 13C NMR spectra (CDCl3) were recorded

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using AVANCEⅢ400 spectrometer; mass spectrometry was recorded with Xevo TQ-S mass spectrometer and Q-TOF B.05.01 mass spectrometer. The UV-Vis spectra were obtained using UV-240 IPC spectrophotometer. The fluorescence spectra were obtained with F-4500 FL spectrometer with a 1 cm standard quartz cell. Synthesis. To a solution of 7-hydroxy-4-methylcoumarin (176 mg, 1.0 mmol) and triethylamine (0.2 mL) in 15 mL of anhydrous dichloromethane at 0 oC, methyl chlorooxoacetate (0.2 mL, mixed with 5 mL of CH2Cl2) was added dropwise and kept stirring at this temperature for 30 min. Then the mixture was warmed to room temperature and stirred overnight. The solution was diluted with CH2Cl2 (30 mL) and washed with brine (30 mL × 2), then dried with anhydrous Na2SO4. The solvent was removed in vacuo to obtain a crude mixture solid. Finally, the target compound 1 was isolated by silica chromatography eluting with (88 % yield) CH2Cl2. 1H NMR (500 MHz, CDCl3) δ: 2.46 (S, 3H), 4.03 (s, 3H), 6.32 (s, 1H), 7.18-7.20 (m, 1H), 7.23-7.24 (d, 1H), 7.67-7.68 (d, 1H); 13C NMR (125 MHz, CDCl3) δ: 160.01 (s), 157.03 (s), 155.03 (s), 154.05 (s), 151.83 (s), 151.58 (s), 125.70 (s), 118.63 (s), 117.15 (s), 115.03 (s), 109.96 (s), 54.04 (s), 18.61 (s). HRMS (ESI): calcd for C13H10O6 [M + H]+ =263.0555, found m/z 263.0552. Scheme 1. The synthesis route of compound 1.

The tests of 2 and 3. To a mixture of 1 (10 mg, 0.038 mmol) and sodium phosphate (1.14 g, 3 mmol) in DMSO-HEPES (V/V=9:1, 4 mL), the reaction mixture was stirred at room temperature for 24 h. After evaporation of under reduced pressure, chromatography of the crude product on silica gel using CH2Cl2 as eluent to give 2 as a white solid powder in 86% isolated yield (8.6 mg). TOF MS: calcd for C10H8O3 [M + H]+ =177.0473, found m/z 176.9923. To a mixture of 1 (10 mg, 0.038 mmol) and sodium phosphate (1.14 g, 3 mmol) in DMSO-HEPES (V/V=9:1, 4 mL), the reaction mixture was stirred at room temperature for 24 h. The mess of 3 was tested by using the crude reaction mixtures, MS (EI): calcd for C2O6P [M-H]- =150.94, found m/z 151.08. (Figure S5) The linear range and detection limit. The detection limit was calculated based on the method reported in the previous literature [1]. The fluorescence emission spectrum of 1 was measured by twenty times and the standard deviation of blank measurement was achieved. The fluorescence intensity at 455 nm was plotted as a concentration of Pi. The detection limit was calculated by using detection limit 3σ/k: Where σ is the standard deviation of blank measurement, k is the slope between the fluorescence intensity versus Pi concentration. Culture of Hela cells and Fluorescent Imaging. Hela was cultured in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) supplemented with 10 % FBS (fetal bovine serum) in an atmosphere of 5 % CO2 and 95 % air at 37 ℃. The cells

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were seeded in 24-well flat-bottomed plates and then incubated for 24 h at 37 ℃ under 5 % CO2. Then the cells were incubated with 80.0 µM Pi and ATP in an atmosphere of 5 % CO2 and 95 % air for 2 h at 37 ℃, respectively. Then the cells treated with ATP were incubation with 0.5 U and 0.75 U and 1 U of apyrase for 0.5 h, respectively. Then the cells were incubation with 20.0 µM 1 for 1 h .Wash cells twice with 1 mL HEPES at room temperature, Cells were imaged using an Olympus 71 inverted fluorescence microscopy. Culture of SF9 cells and Fluorescent Imaging. All cell lines were cultured in TNM-FH insect culture medium containing 10 % fetal bovine serum (FBS, Hyclone). Cells were maintained and passaged in 25 cm2 tissue culture flasks (Corning).The infection procedure was performed: 1.0×104 cells were seeded in a 12-well culture plate (Corning) 40 min prior to infection with recombinant viruses. The infected cells were incubated at 27 °C; after 24 hrs, cells were washed in PBS, and fixed for 15 min in 3.7 % formaldehyde. Then, the cells were add 3 U of apyrase for 2 h and then the cells were incubation with 100 µM 1 for 1.5 h. Wash cells twice with 1 mL deionized water at room temperature, Cells were imaged using an Olympus 71 inverted fluorescence microscopy. Culture of C. elegans and Fluorescent Imaging. The C. elegans wild type strain N2 was acquired from the Key Biological Laboratory Center (Yunnan University). The larval stage 4 (L4) C. elegans was used. The L4 stage nematodes were washed three times with deionized water by centrifugation at 2500 r/min for 2 minutes. To expose the L4 stage nematodes to ATP, a centrifuge tube was first filled with 2 mL of deionized water supplemented with 80 µM of ATP. These L4 stage worms were then incubated in the tubes at room temperature for 7 h. After the incubation, the exposed nematodes were washed three times again with deionized water by centrifugation at 2500 r/min for 2 minutes. Then the L4 stage nematodes were exposed at the ATP hydrolysis enzyme aqueous solution, a centrifuge tube was filled with 1.5 mL of succinic acid buffer solution supplemented with 80 µM of ATP hydrolysis enzyme which was incubated at 37 °C for 0.5 h. For imaging of accumulations of Pi in the nematode, the previously exposed worms were incubated in centrifuge tube filled with 2 mL of deionized water, containing 20 µM of 1 at 20 °C for 1 h. The nematodes were washed three times again with deionized water by centrifuging at 2500 r/min for 2 minutes before being mounted onto a slide glass. The images of the mounted nematodes were acquired by using Nikon ECLIPSE 90i fluorescence microscope using UV-2A Ex 330−380 (DM400, BA420) channel.

RESULTS AND DISCUSSION Synthesis and UV-Vis and emission properties of 1 in the presence of Pi. Chemodosimeter 1 was prepared by reaction of 7-hydroxy-4-methylcoumarin with methyl chlorooxoacetate in the presence of triethylamine (Scheme 1). Reactions of 1 with the anions Pi, P2O74-, ATP, ADP, AMP, GTP, GDP, GMP, TMP, UTP, UDP, UMP, CH3COO-, Cl-, Br-, I-, NO3-, NO2-, H2S, Cys, GSH and Glu in DMSO–HEPES buffered (0.02 M, pH 7.4) (V/V=9:1) were probed using UV–vis absorption and fluorescence spectroscopy. The results show that only Pi promotes a distinct color change of the probe from


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colorless to light blue (Figure 1a) associated with a shift of the maximum of the major absorption band at 325 nm to 387 nm. Importantly, the other anions do not cause significant spectral changes under the test condition, except for UMP which produces a weak enhancement of the absorbance at 380 nm. Chemodosimeter 1 in DMSO–HEPES buffered (0.02 M, pH 7.4) (V/V=9:1) solution only weakly fluoresces. However, upon addition of Pi only, the emission band at 460 nm undergoes a 780-fold increase in its intensity. While addition of UMP and GMP to solutions of 1 results in minor fluorescence enhancement (5.3-fold for UMP and 3.6-fold for GMP) (Figure 1b), none of the other anions promote a change in emission.

Figure 1. (a) Absorption spectra of 1 (2.0×10-5 M) in DMSO-HEPES buffer (0.02 M, pH 7.4) (V/V = 9:1) with 100 equiv of Pi, P2O74-, ATP, ADP, AMP, GTP, GDP, GMP, TMP, UTP, UDP , UMP, CH3COO-, Cl-, Br-, I-, NO3-, NO2-, H2S, Cys, GSH and Glu. (b) Absorbances of 1（2.0 × 10-5 M）at 388 nm after addition of 100 equiv of selected anions. (c) Fluorescence emission spectra of 1 (2.0 × 10-5 M) in DMSO–HEPES buffer (0.02 M, pH 7.4) (V/V = 9:1) with 100 equiv of Pi, P2O74-, ATP, ADP, AMP, GTP, GDP, GMP, TMP, UTP, UDP , UMP, CH3COO-, Cl-, Br-, I-, NO3-, NO2-, H2S, Cys, GSH and Glu. (d) Fluorescence intensities of 1 (2.0×10-5 M) at 455 nm after addition of 100 equiv of selected ions. (a : UTP, b : P2O74-, c : ATP, d : ADP, e : GMP, f : GTP, g : GDP, h : AMP, i : TMP, j : UMP, k : Pi, l : UDP, m: CH3COO-, n: Cl-, o: Br-, p: I-, q: NO3-, r: NO2-, s: H2S, t: Cys, u: GSH, v: Glu).

Figure 2. Fluorescence titration spectra of 1 (2.0 × 10-5 M) in the presence of varying concentrations of Pi in DMSO–HEPES buffer (0.02 M, pH = 7.4) (V/V = 9:1).

Excitation wavelength was 385 nm. Inset: fluorescence intensity of 1 (2.0 × 10-5 M) at 455 nm as a function of varying concentrations of Pi. To obtain insight into the reaction of Pi with 1, fluorescence spectra and UV–Vis spectra of 1 in DMSO–HEPES buffer upon addition of different equivalents of Pi were recorded. The results show that 1 exhibits very weak fluorescence and that Pi addition causes a significant increase of the intensity of fluorescence at 455 nm, levelling at a 780-fold increase and associated with an observable bright blue emission. (Figure 2) The fluorescence intensity increase caused by addition of Pi to 1 is linearly proportional to Pi concentration in the 0-0.2 mM range. The detection limit is calculated to be 8.11 × 10−7 M from 3σ/k, where σ is the standard deviation of a blank measurement, and k is the slope between the fluorescence intensity vs. Pi concentration (Figure S6). In addition, upon addition of Pi to a buffered solution of 1 the absorption maximum at 326 nm decreases while a new absorbance band at 380 nm simultaneously forms with a ratio of intensities (I380/I326) ranging from 0.5 to 1.6 increase (Figure 3). Following addition of 100 equivalents of Pi, the color of the solution becomes blue. Compound 2 shows typical coumarin absorbance at 325 nm and emission at 455 nm in present test conditions. (Figure S8) It shows that the final spectral data of this sensing process by 1 are exactly same with the optical properties of 2, which give the inspiration and supporting of the sensing mechanism in Figure 4.

Figure 3. Absorption titration spectra of 1 (2.0 × 10-5 M) in the presence of varying concentrations of PO43- in DMSO-HEPES buffer (0.02 M, pH = 7.4) (V/V = 9:1). Inset: ratiometric calibration curve I380/I326 as a function PO43concentration. Mechanistic study. The design of the new chemodosimeter was based on the ester bond cleavage mechanism for reaction of 1 with Pi shown in Figure 4. To gain support for this mechanism, theoretical calculations were carried out. In the study, density functional theory (B3LYP/6-31G** level) 39 was used to describe the energy changes occurring in the reaction. It showed that the coumarin fluorophores is generated by Pi promoted ester bond cleavage, the energy change is calculated to be -19.3 kJ. A transition state was located at the sensing process, involving conversion of the mixed anhydride to the cyclic diphosphate 3, which has one imaginary frequency of -267.4 cm-1 (see OUT files in Supporting Information II) associated with a vibration depicted in Figure S9.



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Additional support for this mechanistic proposal comes from the results of a mass spectrometry (MS) study. Specifically, MS analysis of a mixture of 1 and 100 equivalents of Pi (in DMSO-HEPES buffer (0.02 M, pH = 7.4), V/V = 9:1) after stirring for 12 h contains a peak at m/z 151.08 (Figure S5) corresponding to the cyclic diphosphate 3.

Figure 4. The proposed sensing mechanism of 1 with Pi. In-Vivo imaging for exogenous and endogenous Pi.

Figure 5. (a) Time plots of ATP hydrolysis of apyrase monitored by the emission at 455 nm. (1 (2.0 × 10-6 M), ATP (2.0 × 10-4 M), DMSO–succinic acid buffer (20 mM, pH = 6.4), excitation at 385 nm)). (b) Time dependent fluorescence spectra of 1 (2.0 × 10-6 M) in the presence of 2U apyrase (0-40 min) in DMSO-succinic acid buffer (20 mM, pH = 6.4) (V/V = 9:1). Apyrase is a hydrolytic enzyme that converts both ATP and ADP into AMP and Pi 40, 41. This enzymatic process was utilized to evaluate the ability of 1 to be employed to in vivo image Pi production. The time-dependent fluorescence response of 1 in the presence of different concentrations of apyrase and ATP in DMSO-succinic acid buffer (20 mM, pH 6.4) (V/V = 9:1) were first recorded in vitro. Upon the addition of increasing amounts of apyrase to solutions of ATP and 1, an enhancement in the fluorescence intensity at 455 nm takes place, reaching a maximum after 2U apyrase are added (Figure 5). According to the response time, all the fluorescent increase stopped within 30 min, allowing 1 to sense Pi in a real-time in-vivo imaging study.

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(80 µM) and apyrase (0.5 U). (d) Probe 1 (20µM), ATP (80 µM) and apyrase (0.75 U). (e) Probe 1 (20 µM), ATP (80 µM) and apyrase (1 U). To demonstrate the feasibility of chemodosimeter 1 for use in locating endogenous and exogenous Pi in-vivo, fluorescence imaging studies were carried out using HeLa cells and C. elegans. Fluorescent imaging was first investigated using HeLa cells that are incubated with 20 µM chemodosimeter 1 (Figure 6a) and 80 µM Pi (Figure 6b), or with 80 µM ATP and apyrase (Figure 6c-6e). No obvious fluorescence is observed when the HeLa cells were incubated with 1 for 1 h. When external Pi (80 µM) is added to the cells, the fluorescence intensity increases. In order to monitor the in-vivo generation of Pi formed by the apyrase catalyzed ATP hydrolysis, HeLa cells are preincubated with ATP (80 µM) for 2 h, treated with 0.5 U, 0.75 U, or 1 U apyrase for 0.5 h, and then stained using probe 1 for 1 h. With increasing amounts of apyrase, the fluorescence intensities increase proportionally, indicating that 1 penetrates live cell membranes and where it reacts leading to strong fluorescence emission throughout the cytoplasm where apyrase-catalyzed ATP hydrolysis and inorganic phophate production take place.

Figure 7. Fluorescent imaging (top) and phase contrast (bottom) for Pi in C. elegans. (a) Probe 1 (20 µM) only. (b) Probe 1 (20 µM), ATP (80 µM) (c) Probe 1 (20 µM), ATP (80 µM) and apyrase (0.5 U). (d) Probe 1 (20 µM), ATP (80 µM) and apyrase (1 U). (e) Probe 1 (20 µM), ATP (80 µM) and apyrase (2 U). Additional studies were carried out using C. elegans larvae at developmental stage 3 (L3). The larvae were incubated in Petri dishes filled with M9 buffer containing 20 µM 1 at 20 oC for 1 h (Figure 7a). In the second test group, apyrase was added to convert ATP into AMP and Pi. In this case, to C. elegans, preincubated with ATP (80 µM) for 7 h at 37 oC, was added 0U, 0.5 U, 1 U, and 2U apyrase followed by staining with probe 1 for 1 h. No fluorescence was observed in the pretreated nematodes until the concentration of apyrase reaches 0.5 U and after addition of 2U apyrase a clear bright blue fluorescence is emitted from the intestinal region and gonads.

Figure 6. Fluorescent imaging (top) and phase contrast (bottom) for Pi in Hela cells. (a) Probe 1 (20 µM) only. (b) Probe 1 (20 µM) and Pi (80 µM). (c) Probe 1 (20 µM), ATP


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Figure 8. Fluorescence imaging (top), phase contrast (middle), and merge (bottom) for Pi in Sf9 cells treated with virus and apyrase. (a) Probe 1 (100 µM) only and uninfected Sf9 cells. (b) Probe 1 (100 µM) and apyrase (3 U) and uninfected Sf9 cells. (c) Probe 1 (100 µM) and apyrase (3 U) and infected Sf9 cells. (d) Probe 1 (100 µM) and apyrase (3 U) and Sf9 cells treated with Inx2. (e) Probe 1 (100 µM) and apyrase (3 U) and Sf9 cells treated with Inx3; (k-o) Merged images.

Figure 9. Fluorescent imaging (top) and phase contrast (bottom) for Pi in Sf9 cells treated by virus only. (a) Probe 1 (100 µM) only; (b) Probe 1 (100 µM) and Sf9 cells treated by virus vector; (c) Probe 1 (100 µM) and Sf9 cells treated by Inx2. (d) Probe 1 (100 µM) and Sf9 cells treated by Inx3. Monitoring Phosphate Ion in a Hemichannel-Closed Cell. Gap junctions serve to coordinate cell-to-cell communication within tissues by allowing the transfer of small molecules, ions, amino acids, and nucleotides, as well as second messengers such as Ca2+, cAMP, cGMP, and IP3 between cells 42, 43. Gap junction channels span two plasma membranes, resulting from the association of a hemichannels in each membrane. A hemichannel is are composed of six-innexin proteins, each containing four transmembrane domains (TM), which are linked with two extracellular loops and a cytoplasmic loop 44. Open hemichannels allow the bidirectional passage of ions and small molecules of up to 1-2 kDa between the extracellular and intracellular space, which affects cellular behaviors through the activation of paracrine signaling pathways via signaling molecules, including ATP, ADP, AMP, and adenosine, the latter three which may be degraded by ecto-ATPase 45, 46. Under pathological conditions, opening of hemichannels causes the intracellular accumulation of toxic metabolites and ATP depletion, which can lead to apoptosis and/or necrosis 47-49. Recently, innexin3 (Inx3) was found to dephosphorylate Akt to promote cell apoptosis. Using a Bac-to-Bac system (DH10Bac was used as a control named Bac), Bac-Inx3 was

found to promote movement of living cells into an apoptotic early stage and accelerated apoptotic late stages (unpublished results in a manuscript in preparation). Interestingly, hemichannels of living cells infected by bacuovirus are closed. Importantly, no direct evidence exists for the changing process in hemichannel-closed cells and that the precise roles played by Bac-Inx3 are not well defined. Considering the potential involvement of ATP hydrolysis in these events, we anticipated that fluorescent monitoring of endogenous inorganic phosphate production using chemodosimeter 1 might aid an understanding the mechanism of baculovirus induced survival via closing hemichannels. For this purpose, Sf9 (IPLB-Sf21-AE) adherent cells were derived from Spodoptera frugiperda pupal ovarian tissue 50. Inx2 and Inx3 were amplified by using PCR (polymerase chain reaction), with fourth instar S. lituralarvae hemocytes’ cDNA as a template and the primers Bac-Inx2_F (5’- GAA TTC ATG TTT GAC GTT TTC GGC T - 3’), Bac-inx2_R (5’- GC GGC CGC AAC ACA CTG TCC TT - 3’), Bac-inx3_F (5’ - GAA TTC ATG GCG GTA TTT GGT TTG G - 3’) and Bac-Inx3_R (5’ - GC GGC CGC AAC GTT TCG GTT TC - 3’) containing EcoR I and Not I sites (underline). The genes were then directionally cloned into pMD19 and sequenced. Transfer-vector plasmids were constructed to generate recombinant AcMNPV expressing Inx2, Inx3 protein. Inx2 and Inx3 were respectively cloned into the EcoR I and Not I sites of the pFastBacTMHTA vector (Invitrogen). The pFastBac-Inx2 and pFastBac-Inx3 vectors were transformed into E. coli DH10Bac cells (Gibco BRL) and positive colonies were selected in the manner described by the manufacturer. Volumes of each high-titer virus were determined empirically as those minimally required to infect all cells determined by cell cycle as suggested by Boukarabila 51. The infection procedure was performed by seeding 1.0×104 cells in a 12-well culture plate (Corning) 40 min prior to infection with recombinant viruses. The infected cells were incubated at 27 °C for 24 h, washed in PBS, and then fixed for 15 min in 3.7 % formaldehyde. The cells were then incubated with apyrase and 1. No fluorescence is observed from Sf9 cells treated with only 1 (100 µM), and not virus and apyrase (Figure 8a). Upon addition of apyrase (3U), Sf9 cells display weak fluorescence owing to the increase of Pi generated by apyrase catalyzed ATP hydrolysis. No obvious fluorescence arises from cells that are treated with the virus vector in the presence of 1 (100 µM) and apyrase (3U) (Figure 8c). Importantly, when Sf9 cells are treated with Inx2 or Inx3 under the same conditions (1 (100 µM) and apyrase (3U)), strong blue fluorescence arises. In contrast, in the presence of Bac-Inx2 and Bac-Inx3 even stronger fluorescence emission takes place, indicating that more Pi is generated in these cases. The proposal that dephosphorylation of Akt is promoted by Inx3 in Sf9 cells was probed next. Treatment of Sf9 cells with 1 and Inx3 in the absence of apyrase, leads to a clear fluorescent image (Figure 9d). This observation demonstrates that Inx3 causes dephosphorylation of Akt in hemichannel-closed cells that leads to apoptosis. Thus, by using chemodosimeter 1 we have been able for the first time to gain direct evidence for the change-inducing process taking place in hemichannel-closed cells and for the precise role of Inx3 in apotosis.



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CONCLUSIONS In the effort described above we designed chemodosimeter 1 as the first inorganic phosphate targeted colorimetric and fluorescent probe for detecting endogenous Pi. The results of the studies with this sensor show that, as predicted, treatment of 1 with Pi promotes ester bond cleavage and the liberation of the coumarin in association with a red shift in the absorption maximum from 325 nm to 387 nm and up to a 780-fold enhancement in the fluorescence intensity at 460 nm. The proposed sensing mechanism is supported by the results of theoretical calculations and mass spectrometry study. Observations made in fluorescence imaging studies, performed using HeLa cells and C. elegans, demonstrate the feasibility of using chemodosimeter 1 for localizing exogenous and endogenous Pi in vivo. These assays demonstrated that 1 serves as an excellent in-vivo Pi monitor for apyrase catalyzed ATP hydrolysis. Finally, 1 was utilized to probe Pi generation in hemichannel-closed Sf9 adherent cells. Importantly, the results of this study have provided direct evidence for the proposal that Inx3 causes dephosphorylation of Akt in hemichannel-closed cells to promote apoptosis.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected]; Fax: +86-871-65033679. * E-mail: [email protected]; Fax: +86-871-65031412. * E-mail: [email protected]; Fax: + 82-2-3277-3419.

Author Contributions #

L. E. Guo and J. F. Zhang contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS Y. Z and J. F. Zhang are grateful for financial support from the Natural Science Foundation of China (21102127, 21262045, 21262050, 21302165, 21462050), the Foundation of the Department of Science and Technology of Yunnan Province of China (2011FB013, 2011FB047, 2013HB062). K. J. Luo thanks to the support from National Basic Research Program of China (973 Program: 2013CB127600) from Major State Basic Research Development Program and a grant (2013FA003) from Yunnan Department of Science and Technology, National Natural Science Foundation of China (31260448, 31060251). J. Yoon thanks to the support from the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (No. 2012R1A3A2048814).

Supporting Information Experimental details, synthesis, characteristic data (1H NMR, 13C NMR, mass spectra) and theoretical calculations (OUT files).This material is available free of charge via the Internet at http://pubs.acs.org.

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“for TOC only”


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Phosphate Ion Targeted Colorimetric and Fluorescent Probe and Its

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