Highly Selective Fluorescent Probe for Imaging H2Se in Living Cells

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A Highly Selective Fluorescent Probe for Imaging H2Se in Living Cells and in vivo Based on the Disulfide Bond Fanpeng Kong, Yuehui Zhao, Ziye Liang, Xiaojun Liu, Xiaohong Pan, Dongrui Luan, Kehua Xu, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b03136 • Publication Date (Web): 05 Dec 2016 Downloaded from http://pubs.acs.org on December 5, 2016

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Analytical Chemistry

A Highly Selective Fluorescent Probe for Imaging H2Se in Living cells and in vivo Based on the Disulfide Bond Fanpeng Kong, Yuehui Zhao, Ziye Liang, Xiaojun Liu, Xiaohong Pan, Dongrui Luan, Kehua Xu* and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of Molecular and Nano Science, Shandong Normal University, Jinan 250014, P. R. China . E-mail: [email protected], Tel.: +86 0531-86180010; Fax: +86 0531-86180017; [email protected]

ABSTRACT: Hydrogen selenide (H2Se) is an important metabolite of dietary Se compounds and has been implicated in various pathological and physiological processes. The development of highly sensitive and selective methods for the sensing of H2Se is therefore very important. Herein, we developed a fluorescent probe (Hcy-H2Se) for detecting H2Se based on a new H2Se-specific receptor unit, 1,2-dithiane-4,5-diol. Hcy-H2Se showed high selectivity toward H2Se over thiols (RSH), hydrogen sulfide (H2S), and selenocysteine (Sec) and was further exploited for the fluorescence imaging of H2Se both in living cells and in vivo. Furthermore, with the aid of Hcy-H2Se, we demonstrated that H2Se can be generated and gradually accumulated in HepG2 cells under hypoxic conditions and in the solid tumor after treatment with Na2SeO3.

INTRODUCTION Selenium (Se) is an essential trace element for various physiological functions in the human body and is associated with a number of diseases.1-2Importantly, selenium is of potential use in the prevention and treatment of cancer.3-7 The biological activity of selenium is dependent upon its chemical form.8-10 Se exists as different forms in vivo, such as selenocysteine (Sec), selenophosphate, thioredoxin reductase (TrxR), and selenodiglutathione. To clarify the biological function of Se, numerous detection methods for detecting Se have been developed.11-15Among these methods, fluorescence imaging is a powerful techniques for real-time, noninvasive monitoring of biomolecules with high spatial and temporal resolution.16-20 To date, few fluorescent probes for detecting the metabolite of Se have been developed. Maeda et al. reported the first fluorescent probe, BESThio, to discriminate Sec from its counterpart, Cys, based on the difference in their pKa values. 21 Fang et al. developed a fluorescent probe (Sel-green) to detect Sec with high selectivity under physiological conditions.22 Lin et al. reported a NIR probe to detect selenols based on 2,4dinitrobenzene. 23 In addition, by utilizing a 1, 2-dithiolane reporter group, Fang et al. developed the fluorescent probes TRFS-green and Mito-TRFS to selectively image TrxR in living cells. 24-25 Our group has exploited a novel recognition group, 2, 1, 3-benzoselenadiazole (BS), for imaging Sec in living cells and in vivo.26 Moreover, based on the rapid substitution reaction of the Au-S bond by selenol, we have developed fluorescent nanosensors for detecting selenol in vivo. 27-28 Hydrogen selenide (H2Se), which has a structure similar to that of H2S, is an important metabolite of dietary Se compounds that are generated by reducing selenite via GSH and

other reduction systems29 and is involved in many physiological and pathological processes. 30 To date, only one fluorescent probe for imaging H2Se in living cells has been developed, which was reported by our group. 31 However, the introduction of additional Se (released from the BS unit) in the recognition process would have an impact on the cellular homeostasis. 32-33 Therefore, it remains a challenge to design H2Se-specific fluorescent probes with good biocompatibility. Previously, the disulfide bond in the chain structure could be cleaved by a sulfhydryl group via nucleophilic substitution, which was used to establish probes for thiol detection and to selectively deliver drugs. 34-35 Inspired by this recognition mechanism, we suggested the hypothesis that the more stable disulfide bond in the six-membered ring may only react with H2Se because of its higher reaction activity compared to thiols. To confirm our point of view, we designed and synthesized a novel fluorescent probe, Hcy-H2Se, containing 1, 2-dithiane-4, 5-diol group for H2Se. As expected, the results showed that Hcy-H2Se rapidly responds to H2Se with high selectivity over H2S, Sec, biological thiols, and reactive oxygen species (ROS). The probe was also successfully applied to image H2Se generated from Na2SeO3 in living cells under normoxic and hypoxic conditions. We hope that this new H2Se-specific receptor unit will pave the way for design and development of fluorescent probes with biocompatibility to understand the physiological function of H2Se and the anticancer mechanism of Se. EXPERIMENTAL SECTION Materials and Instruments 1, 2-dithiane-4, 5-diol and 4-aminobenzaldehyde were synthesized according to the reported literature. 36-37 H2Se was pre-

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pared by the reaction of Al2Se3 with H2O in an N2 atmosphere for 30 min at room temperature before each use. 38-39 Glutathione(GSH), L-cysteine (L-cys), L-homocysteine (Hcy), ascorbic acid (Vc), N-acetyl-L-cysteine (NAC), 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), L-buthionine sulphoximine (BSO), Thioredoxin Reductase (TrxR) and sodium nitroferricyanide(III) dehydrate (SNP) were all purchased from Sigma-Aldrich Co. Ltd. Thioredoxin Reductase (TrxR). TrxR was treated by guanidine in the present of Cys (as a reducing agent) 26 before use. 4Nitrophenyl-chloroformate and DL-dithiothreitol were obtained from Aladdin Chemical Company (Shanghai, China). N, N-Dimethyl formamide (DMF), tetrahydrofuran (THF), dichloromethane, methanol, sodium hydroxide, sodium sulfate, pyridine, ether, and aluminum oxide were purchased from Sinopharm Chemical Reagent. Co. Ltd. (Shanghai, China). The silica gel for the flash chromatography was purchased from Qingdao Haiyang Chemical Co. (China). Sartorius ultrapure water (18.2 MΩ cm) was purified with a Sartorius Arium 611 VF system (Sartorius AG, Germany). The solvents were used after appropriate distillation or purification. High-resolution mass spectral analyses were performed on a Bruker maXis ultra-high resolution-TOF MS system. 1H NMR and 13C NMR spectra were obtained on Bruker Advance 300 MHz and 400 MHz spectrometers (Bruker, Germany). The fluorescence spectra measurements were performed using an FLS-920 Edinburgh fluorescence spectrometer. Absorption spectra were recorded on a UV-1700 UV-vis spectrophotometer (Shimadzu, Japan). All pH measurements were performed using a pH-3c digital pH meter (Shanghai Lei Ci Device Works, Shanghai, China) with a combined glass calomel electrode. The MTT assay was measured with a microplate reader (RT 6000, Rayto, United States). The fluorescence imaging studies were performed with a Leica DMI6000 fluorescence microscope (Leica Co., Ltd., Germany). The in vivo fluorescence imaging was performed using an IVIS Lumina III in vivo imaging system. Synthesis of Hcy-NH2 1, 2, 3, 3-Tetramethyl-3H-indolium iodide (0.94 g, 7.78 mmol) and 4-aminobenzaldehyde (2.45 g, 7.78 mmol) were dissolved in ethanol (27 mL). The reaction mixture was stirred for 12 h under an Ar atmosphere and monitored by TLC. After completion, the mixture was added to ether (300 mL), filtered through a fritted glass funnel, and rinsed with ether (50 mL). The resulting crude product was purified by flash column chromatography (silica gel, MeOH : EtOAc, 1:6, v/v) to yield HcyNH2 (1.92 g, 46 %). 1H NMR (400 MHz, DMSO-d6 ): δ 1.38 (s, 3H), 1.75 (s, 6H), 4.55 (s, 2H), 7.23 (d, J = 16 Hz, 2H), 7.47-7.55 (m, 2H), 7.72-7.78 (m, 2H), 8.04 (s, 2H), 8.30 (d, J = 16 Hz, 2H). 13C NMR (100 MHz, DMSO-d6 ): δ 13.6, 27.0, 51.2, 104.0, 113.7, 114.7, 122.9, 123.3, 127.8, 129.3, 135.6, 141.2, 143.2, 155.5, 157.1, 179.2. HR MS [M-I] +: m/z calcd. 291.1855; found 291.1872. Synthesis of Hcy-H2Se To a solution of Hcy-NH2 (48.1 mg, 0.1 mmol) and 20 μL pyridine in CH2Cl2 (4 mL) was slowly added 4-nitrophenyl chloroformate (100 mg in1 mL THF). The mixture was stirred at 0 ºC for 0.5 h, then stirred for 3 h at room temperature and monitored by TLC. After completion, the mixture was added to a solution of 100 mg of 1, 2-dithiane-4, 5-diol that was dis-

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solved in 3 mL of THF and 1 mL of pyridine. The resulting mixture was stirred for 12 h under an Ar atmosphere. After completion, the mixture was added to ether (100 mL), filtered through a fritted glass funnel, and rinsed with ether (50 mL). The resulting crude product was purified by flash column chromatography (neutral alumina, CH2Cl2 : MeOH, 100:0 20:0, v/v) to yield Hcy-H2Se (19.1 mg, 32 %).1H NMR (400 MHz, CDCl3 ): δ 1.53 (s, 3H), 1.71 (s, 6H), 2.29-3.14 (m, 3H), 3.31-3.34 (m, 1H),4.01 (s, 1H), 4.52 (s, 1H), 5.11 (s, 1H), 5.30 (s, 1H), 7.46-7.55 (m, 2H), 7.65-7.69 (m, 2H), 7.77-7.79 (m, 2H), 7.96-8.05 (m, 3H), 8.19 (s, 2H), 10.54(s, 1H).13C NMR (100 MHz,CDCl3): δ14.4, 27.4, 29.7, 39.0, 41.0, 43.2, 51.8, 71.6, 79.0, 110.0, 115.4, 118.3, 118.7, 122.2, 127.9, 129.4, 130.0, 131.0, 133.3, 140.3, 142.8, 145.9, 153.2, 154.7, 180.4, 191.2. HR MS [M-I] +: m/z calcd. 469.1614; found 469.1620. RESULTS AND DISCUSSION Design and Synthesis of Hcy-H2Se Scheme1. Synthesis of Hcy-H2Se

Conditions: (a) EtOH; (b) 4-nitrophenyl chloroformate, CH2Cl2, THF To distinguish H2Se from RSH, H2S and Sec, a novel recognition group, 1, 2-dithiane-4, 5-diol, was designed. Compared to the S-S bond in the chain structure, the S-S bond in 1, 2dithiane-4, 5-diol is more stableand can be specifically cleaved by H2Se because of its superior nucleophilic character. The synthetic methodology for Hcy-H2Se is outlined in scheme 1. Hcy-H2Se was synthesized by integrating the 2-dithiane-4, 5diol moiety into a hemicyanine dye via an ester linker, the structure of which was confirmed by 1H NMR, 13C NMR and HRMS (see Supporting Information). Scheme 2. Proposed Mechanism of Fluorescence Turn On of Hcy-H2Se Switched by H2Se

The proposed recognition mechanism of Hcy-H2Se toward H2Se is shown in scheme 2. In the presence of H2Se, the stable disulfide bond of the probe is cleaved. Then, the intermediates participate in the intramolecular cyclization, 40-42 releasing the strong fluorescent dye. In the mass spectra, the intrinsic peak (469.16) of Hcy-H2Se disappeared and a new peak at 291.19 (corresponding to the Hcy-NH2 dye) was observed upon inter-

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action with 10 equiv of H2Se with the probe (Figure. S1 in the Supporting Information). These results are consistent with our proposed recognition mechanism. Fluorescent Analytical Performance of Hcy-H2Se

In addition, the cytotoxicity of the probe in HepG2 cells was determined via a conventional MTT assay (Figure 2b), which indicated that the Hcy-H2Se probe exhibited low biotoxicity. Therefore, Hcy-H2Se could be used as a viable probe for detecting H2Se in biological samples. Selective Recognition of H2Se by Hcy-H2Se

Figure 1. (a) Fluorescence spectra obtained during the titration of Hcy-H2Se (10 μM) with H2Se (up to 100 μM) for 5 min after in PBS buffer (pH = 7.4, 10 mM) at λex = 470 nm. (b) Linear correlation between the emission intensity at 535 nm and H2Se concentration. The fluorescence spectra of Hcy-H2Se in the absence and presence of H2Se were first recorded in PBS (10 mM, pH = 7.4) aqueous solution (Figure S2 in the Supporting Information). The results showed that Hcy-H2Se has very weak fluorescence intensity. After treatment of Hcy-H2Se (10 μM) with 10 equiv of H2Se, a marked enhancement in ﬂuorescence quantum yield (from 0.023 to 0.081) was observed, indicating the electron-donor amino group was released in Hcy-H2Se via a cyclization reaction triggered by H2Se. Next, we performed fluorescence titration studies of Hcy-H2Se for H2Se. The spectra of the solution of Hcy-H2Se treated with a series of H2Se (0 to 100 μM) were recorded. As shown in Figure 1a, upon treatment with H2Se, the fluorescence intensities at 535 nm gradually increased with increasing concentration of H2Se. The emission intensity of 535 nm showed a good linear relationship with H2Se concentrations (0 to 100 µM). The regression equation was F = 1043.59 + 101.83 × [H2Se] (10 -6 M) with a linear coefficient of 0.9941 and a detection limit of 6.8 × 10 -7 M, respectively. Kinetics and MTT experiment

Figure 3. (a) Fluorescence intensity changes for Hcy-H2Se (10 μM) after adding 100 equiv. of Cys, GSH, NAC, Sec, Hcy, Na2S, DTT, and Vc. The black bars show the addition of one of these interferents to a 10 μM Hcy-H2Se solution. The red bars represent the addition of both H2Se (10 μM) and one interferent to the probe solution. (b) Fluorescence intensity changes for Hcy-H2Se (10 μM) after adding 20 equiv. of ROS. To illustrate the good selectivity of this newly developed system, a series of thiols, the reactive oxygen species (ROS), and amino acids were evaluated. Under identical conditions, addition of 100 equiv of thiols, Na2S, Sec, NAC, DTT, and Vc produced negligible fluorescence compared to that of H2Se (Figure 3a). The GSH in high concentration (10 mM), bovine serum albumin (BSA, 200 μM) and thioredoxin reductase (TrxR, 2.5 µg / mL) also did not interfer the detection of H2Se Figure S3 in the Supporting Information). The reactivity of Hcy-H2Se toward ROS was also tested. Figure 3b shows that biologically relevant ROS, including H2O2, NO, -O2 and 1O2, did not trigger any fluorescence changes in the probe solution. Furthermore, the interference from amino acids was tested, the results of which are shown in Figure S4 in the supporting information. These results demonstrated that Hcy-H2Se could be employed for specific recognition of H2Se. Bioimaging of H2Se in Living Cells

Figure 2. (a) Time course for the fluorescence intensity of 10 μM Hcy-H2Se with 100 μM H2Se (red line) and without H2Se (black line) in 10 mM PBS, pH = 7.4, at room temperature. (b) MTT assay of HepG2 cells in the presence of different concentrations of Hcy-H2Se. Considering the variable nature and quick metabolism of endogenous H2Se in biological systems, a fast-responding method for H2Se detection is necessary. The response of Hcy-H2Se to H2Se was evaluated via a kinetics experiment. The results indicated that the fluorescence intensity immediately increased to its maximum after adding H2Se to the probe solution, which indicated that the probe instantly responds to H2Se (Figure 2a).

Figure 4.(a) Fluorescence images of living HepG2 cells pretreated with different concentrations of Na2SeO3 for 12 h, followed by incubation with 10 μM Hcy-H2Se for 15 min under normoxic (20 % pO2) and hypoxic (1 % pO2) conditions. (b) Fluorescence images of living HepG2 cells pre-treated with 10 μM Na2SeO3 for different durations, followed by incubation with 10 μM Hcy-H2Se for 15 min under normoxic (20 % pO2) and hypoxic (1 % pO2) conditions. The fluorescence was im-

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aged using a confocal microscope with 476 nm excitation. Scale bar = 100 µm. The above results indicate that the Hcy-H2Se probe can respond to H2Se instantaneously with high sensitivity and selectivity as well as low biotoxicity. These features of Hcy-H2Se make it favorable for imaging H2Se in biological samples. To show the practical utility of the probe in the detection of cellular H2Se, fluorescence microscopy studies were performed in living HepG2 cells. Sodium selenite (Na2SeO3), a precursor of selenols, is often used as an anticancer reagent in cancer treatment. 43-48 According our previous work, we suspect that in the hypoxic tumor cells, Na2SeO3 is metabolized to H2Se, resulting in cell apoptosis via non-oxidative stress.49-50 To confirm our hypothesis, firstly, we confirmed that H2Se can be generated from Na2SeO3 in the presence of GSH in living HepG2 cells under hypoxic conditions (Figure S6 in the Supporting Information). Then, we observed the real-time content levels of H2Se in HepG2 cell response to difference concentrations of sodium selenite under hypoxic environments and normoxic conditions. As shown in Figure 4a, higher H2Se contents were observed in hypoxic environments than under normoxic conditions. Similar results were obtained in parallel experiments, in which the HepG2 cells were incubated with 10 μM Na2SeO3 for different lengths of time. In summary, the H2Se contents in the cancer cells increased in a Na2SeO3 dosedependent and incubation time-dependent manner under hypoxic environments. Imaging of H2Se in vivo

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CONCLUSIONS In summary, based on the 1, 2-dithiane-4, 5-diol receptor unit, we designed and synthesized a novel fluorescent probe (HcyH2Se) for the detection of H2Se. Hcy-H2Se displayed remarkable ﬂuorescence enhancement and quick response time, as well as excellent selectivity toward H2Se over other biological thiols, H2S, and Sec. The probe was also successfully applied to image H2Se generated from Na2SeO3 in living cells and in vivo. Furthermore, the imaging results in living HepG2 cells under hypoxic condition and in solid tumors treated by Na2SeO3 showed that H2Se accumulated gradually in a hypoxic environment, which indicates that the anticancer mechanism of Se for hypoxic solid tumors occurs via non-oxidative stress. We anticipate that the current probe will provide an ideal tool for further studies into the biological functions of H2Se and Se anticancer mechanisms.

ASSOCIATED CONTENT AUTHOR INFORMATION Corresponding Author Notes * E-mail: [email protected], [email protected] Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by 973 Program (2013CB933800), National Natural Science Foundation of China (21390411, 21535004, 21275092, 21575081 and 21405098).

Supporting Information Verified the recognition mechanism by MS, quantum yield calculations, pH stability, spectra of Hcy-H2Se and other materials. The Supporting Information is available free of charge on the ACS Publications website.

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Figure 5. Fluorescence imaging of 10 μM Na2SeO3-treated (top) and 10 μM saline-treated (bottom) tumour-bearing mice. To evaluate its potential for detecting endogenous H2Se in vivo, the buffer solutions containing Hcy-H2Se (10 μM) and sodium selenite (10 μM) were orthotopically injected into the tumor region of the mice bearing subcutaneously implanted tumors grown from murine hepatoma cell line H22, and fluorescence images were then obtained at different times using an in vivo imaging system (IVIS). As shown in Figure 5, the fluorescence signal of the probe was exclusively observed in the tumor region, where the fluorescence intensity increased from 3 h to 12 h post injection. In contrast experiments, no fluorescence was observed after orthotopic injection of the saline water. The results indicated that H2Se can be generated from sodium selenite in the hypoxic solid tumor and gradually accumulated.

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