Highly Selective Fluorescent Probe for Imaging ... - ACS Publications

0 downloads 0 Views 2MB Size Report
Dec 5, 2016 - Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of. Molecular .... Fluorescent Analytical Performance of Hcy-H2Se. The .... Hcy-H2Se, and other materials (PDF) ... J.; de Jonge, M. D.; Howard, D. L.; Musgrave, I. F.; Harris, H. H..
Article pubs.acs.org/ac

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, Shandong 250014, P. R. China S Supporting Information *

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 (hemicyanine (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.

S

benzoselenadiazole (BS), for imaging Sec in living cells and in vivo.26 Moreover, on the basis of 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

elenium (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 technique 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 and co-workers developed a fluorescent probe (Sel-green) to detect Sec with high selectivity under physiological conditions.22 Lin and co-workers reported a NIR probe to detect selenols on the basis of 2,4-dinitrobenzene.23 In addition, by utilizing a 1,2-dithiolane reporter group, Fang and co-workers 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© 2016 American Chemical Society

Received: August 11, 2016 Accepted: December 5, 2016 Published: December 5, 2016 688

DOI: 10.1021/acs.analchem.6b03136 Anal. Chem. 2017, 89, 688−693

Article

Analytical Chemistry

EtOAc, 1:6, v/v) to yield Hcy-NH2 (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 of pyridine in CH2Cl2 (4 mL) was slowly added 4-nitrophenyl chloroformate (100 mg in 1 mL of THF). The mixture was stirred at 0 °C for 0.5 h and 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 dissolved 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): δ 14.4, 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.

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, hemicyanine (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 H2Sespecific 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 4aminobenzaldehyde were synthesized according to the reported literature.36,37 H2Se was prepared 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), Lhomocysteine (Hcy), ascorbic acid (Vc), N-acetyl-L-cysteine (NAC), 3-(4,5-dimethylthiazol-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) was treated with guanidine in the presence of Cys (as a reducing agent)26 before use. 4Nitrophenyl-chloroformate and DL-dithiothreitol were obtained from Aladdin Chemical Company (Shanghai, China). N,NDimethylformamide (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 ultrahigh resolution-TOF MS system. 1H NMR and 13C NMR spectra were obtained on Bruker Advance 300 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/



RESULTS AND DISCUSSION Design and Synthesis of Hcy-H2Se. To distinguish H2Se from RSH, H2S, and Sec, a novel recognition group, 1,2dithiane-4,5-diol, was designed. Compared to the S−S bond in the chain structure, the S−S bond in 1,2-dithiane-4,5-diol is more stable and 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 Scheme 1. Synthesis of Hcy-H2Sea

a Conditions: (a) EtOH; (b) 4-nitrophenyl chloroformate, CH2Cl2, and THF.

synthesized by integrating the 2-dithiane-4,5-diol 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). 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 689

DOI: 10.1021/acs.analchem.6b03136 Anal. Chem. 2017, 89, 688−693

Article

Analytical Chemistry

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). 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, HcyH2Se could be used as a viable probe for detecting H2Se in biological samples. Selective Recognition of H2Se by Hcy-H2Se. 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 interfere with the detection of H2Se (Figure S3). 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. These results demonstrated that HcyH2Se could be employed for specific recognition of H2Se. Bioimaging of H2Se in Living Cells. 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 to our previous work, we suspect that, in the hypoxic tumor cells, Na2SeO3 is metabolized to H2Se, resulting in cell apoptosis via nonoxidative stress.49,50 To confirm our hypothesis, first, we confirmed that H2Se can be generated from Na2SeO3 in the presence of GSH in living HepG2 cells under hypoxic conditions (Figure S6). Then, we observed the real-time content levels of H2Se in the HepG2 cell

Scheme 2. Proposed Mechanism of Fluorescence Turn On of Hcy-H2Se Switched by H2Se

(469.16) of Hcy-H2Se disappeared and a new peak at 291.19 (corresponding to the Hcy-NH2 dye) was observed upon interaction with 10 equiv of H2Se with the probe (Figure S1). These results are consistent with our proposed recognition mechanism. Fluorescent Analytical Performance of Hcy-H2Se. 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). 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 HcyH2Se 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. 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

Figure 1. (a) Fluorescence spectra obtained during the titration of Hcy-H2Se (10 μM) with H2Se (up to 100 μM) for 5 min after being 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. 690

DOI: 10.1021/acs.analchem.6b03136 Anal. Chem. 2017, 89, 688−693

Article

Analytical Chemistry

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.

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 interfering agents to a 10 μM Hcy-H2Se solution. The red bars represent the addition of both H2Se (10 μM) and one interfering agent to the probe solution. (b) Fluorescence intensity changes for Hcy-H2Se (10 μM) after adding 20 equiv of ROS.

response to different concentrations of sodium selenite under hypoxic environments and normoxic conditions. As shown in Figure 4a, higher H2Se contents were observed in hypoxic environments than those 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 dose-dependent and incubation time-dependent manner under hypoxic environments. Imaging of H2Se in Vivo. 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 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.



CONCLUSIONS In summary, on the basis of the 1,2-dithiane-4,5-diol receptor unit, we designed and synthesized a novel fluorescent probe (Hcy-H2Se) 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

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 pretreated 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 imaged using a confocal microscope with 476 nm excitation. Scale bar = 100 μm. 691

DOI: 10.1021/acs.analchem.6b03136 Anal. Chem. 2017, 89, 688−693

Article

Analytical Chemistry

(7) Weekley, C. M.; Aitken, J. B.; Finney, L. A.; Vogt, S.; Witting, P.; Harris, H. H. Nutrients 2013, 5, 1734−1756. (8) Malinouski, M.; Kehr, S.; Finney, L. A.; Vogt, S.; Carlson, B. A.; Seravalli, J.; Jin, R.; Handy, D. E.; Park, T. J.; Loscalzo, J.; Hatfield, D. L.; Gladyshev, V. N. Antioxid. Redox Signaling 2012, 16, 185−192. (9) Rayman, M. P.; Infante, H. G.; Sargent, M. Br. J. Nutr. 2008, 100, 238−253. (10) Ip, C. J. Nutr. 1998, 128, 1845−1854. (11) Weekley, C. M.; Aitken, J. B.; Witting, P. K.; Harris, H. H. Metallomics 2014, 6, 2193−2203. (12) Weekley, C. M.; Shanu, A.; Aitken, J. B.; Vogt, S.; Witting, P. K.; Harris, H. H. Metallomics 2014, 6, 1602−1615. (13) Areti, S.; Verma, S. K.; Bellare, J.; Rao, C. P. Anal. Chem. 2016, 88, 7259−7267. (14) Yang, L.; Sturgeon, R. E.; McSheehy, S.; Mester, Z. J. Chromatogr. A 2004, 1055, 177−184. (15) Encinar, J. R.; Schaumloffel, D.; Ogra, Y.; Lobinski, R. Anal. Chem. 2004, 76, 6635−6642. (16) Yuan, L.; Lin, W. Y.; Zheng, K. B.; He, L. W.; Huang, W. M. Chem. Soc. Rev. 2013, 42, 622−661. (17) Guo, Z. Q.; Park, S.; Yoon, J. Y.; Shin, I. Chem. Soc. Rev. 2014, 43, 16−29. (18) Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123−128. (19) Chen, X.; Wang, F.; Hyun, J. Y.; Wei, T.; Qiang, J.; Ren, X.; Shin, I.; Yoon, J. Chem. Soc. Rev. 2016, 45, 2976−3016. (20) Yang, Z.; Cao, J.; He, Y.; Yang, J. H.; Kim, T.; Peng, X.; Kim, J. S. Chem. Soc. Rev. 2014, 43, 4563−4601. (21) Maeda, H.; Katayama, K.; Matsuno, H.; Uno, T. Angew. Chem., Int. Ed. 2006, 45, 1810−1813. (22) Zhang, B.; Ge, C.; Yao, J.; Liu, Y.; Xie, H.; Fang, J. J. Am. Chem. Soc. 2015, 137, 757−769. (23) Chen, H.; Dong, B.; Tang, Y.; Lin, W. Chem. - Eur. J. 2015, 21, 11696−11700. (24) Zhang, L.; Duan, D.; Liu, Y.; Ge, C.; Cui, X.; Sun, J.; Fang, J. J. Am. Chem. Soc. 2014, 136, 226−233. (25) Liu, Y.; Ma, H.; Zhang, L.; Cui, Y.; Liu, X.; Fang, J. Chem. Commun. 2016, 52, 2296−2299. (26) Kong, F.; Hu, B.; Gao, Y.; Xu, K.; Pan, X.; Huang, F.; Zheng, Q.; Chen, H.; Tang, B. Chem. Commun. 2015, 51, 3102−3105. (27) Hu, B.; Cheng, R.; Liu, X.; Pan, X.; Kong, F.; Gao, W.; Xu, K.; Tang, B. Biomaterials 2016, 92, 81−89. (28) Liu, X.; Hu, B.; Cheng, R.; Kong, F.; Pan, X.; Xu, K.; Tang, B. Chem. Commun. 2016, 52, 6693−6696. (29) Wallenberg, M.; Olm, E.; Hebert, C.; Björnstedt, M.; Fernandes, A. P. Biochem. J. 2010, 429, 85−93. (30) Veres, Z.; Tsai, L.; Scholz, T. D.; Politino, M.; Balaban, R. S.; Stadtman, T. C. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 2975−2979. (31) Kong, F.; Ge, L.; Pan, X.; Xu, K.; Liu, X.; Tang, B. Chem. Sci. 2016, 7, 1051−1056. (32) Letavayová, L.; Vlcková, V.; Brozmanová, J. Toxicology 2006, 227, 1−14. (33) Kobayashi, Y.; Ogra, Y.; Ishiwata, K.; Takayama, H.; Aimi, N.; Suzuki, K. T. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 15932−15936. (34) (a) Lee, M. H.; Yang, Z.; Lim, C. W.; Lee, Y. H.; Dongbang, S.; Kang, C.; Kim, J. S. Chem. Rev. 2013, 113, 5071−5109. (35) Kong, F.; Liang, Z.; Luan, D.; Liu, X.; Xu, K.; Tang, B. Anal. Chem. 2016, 88, 6450−6456. (36) Cleland, W. W. Biochemistry 1964, 3, 480−482. (37) Campaigne, E.; Budde, W. M.; Schaefer, G. F. Org. Synth. 1951, 31, 6. (38) Mealli, C.; Midollini, S.; Sacconi, L. Inorg. Chem. 1978, 17, 632− 637. (39) Matylitsky, V. V.; Shavel, A.; Gaponik, N.; Eychmüller, A.; Wachtveitl, J. J. Phys. Chem. C 2008, 112, 2703−2710. (40) Santra, S.; Kaittanis, C.; Santiesteban, O. J.; Perez, J. M. J. Am. Chem. Soc. 2011, 133, 16680−16688. (41) Lee, M. H.; Sessler, J. L.; Kim, J. S. Acc. Chem. Res. 2015, 48, 2935−2946. (42) Cao, X.; Lin, W.; Yu, Q. J. Org. Chem. 2011, 76, 7423−7430.

Figure 5. Fluorescence imaging of 10 μM Na2SeO3-treated (top) and 10 μM saline-treated (bottom) tumor-bearing mice.

showed that H2Se accumulated gradually in a hypoxic environment, which indicates that the anticancer mechanism of Se for hypoxic solid tumors occurs via nonoxidative 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

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b03136. Verification of the recognition mechanism by MS, quantum yield calculations, pH stability, spectra of Hcy-H2Se, and other materials (PDF)



AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Tel.: +86 0531-86180010. Fax: +86 0531-86180017. *E-mail: [email protected]. ORCID

Bo Tang: 0000-0002-8712-7025 Notes

The authors declare no competing financial interest.



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



REFERENCES

(1) Weekley, C. M.; Harris, H. H. Chem. Soc. Rev. 2013, 42, 8870− 8894. (2) Rayman, M. P. Lancet 2012, 379, 1256−1268. (3) Weekley, C. M.; Aitken, J. B.; Vogt, S.; Finney, L. A.; Paterson, D. J.; de Jonge, M. D.; Howard, D. L.; Musgrave, I. F.; Harris, H. H. Biochemistry 2011, 50, 1641−1650. (4) Duffield-Lillico, A. J.; Reid, M. E.; Turnbull, B. W.; Combs, G. F., Jr.; Slate, E. H.; Fischbach, L. A.; Marshall, J. R.; Clark, L. C. Cancer Epidemiol. Biomarkers Prev. 2002, 11, 630−639. (5) Weekley, C. M.; Aitken, J. B.; Vogt, S.; Finney, L. A.; Paterson, D. J.; de Jonge, M. D.; Howard, D. L.; Witting, P. K.; Musgrave, I. F.; Harris, H. H. J. Am. Chem. Soc. 2011, 133, 18272−18279. (6) Weekley, C. M.; Aitken, J. B.; Musgrave, I. F.; Harris, H. H. Biochemistry 2012, 51, 736−738. 692

DOI: 10.1021/acs.analchem.6b03136 Anal. Chem. 2017, 89, 688−693

Article

Analytical Chemistry (43) Kryukov, G. V.; Castellano, S.; Novoselov, S. V.; Lobanov, A. V.; Zehtab, O.; Guigo, R.; Gladyshev, V. N. Science 2003, 300, 1439. (44) Ganther, H. E. Biochemistry 1971, 10, 4089−4098. (45) Wallenberg, M.; Olm, E.; Hebert, C.; Björnstedt, M.; Fernandes, A. P. Biochem. J. 2010, 429, 85−93. (46) Fairweather-Tait, S. J.; Bao, Y.; Broadley, M. R.; Collings, R.; Ford, D.; Hesketh, J. E.; Hurst, R. Antioxid. Redox Signaling 2011, 14, 1337−1383. (47) Ip, C.; Hayes, C.; Budnick, R. M.; Ganther, H. E. Cancer Res. 1991, 51, 595−600. (48) Chen, T.; Wong, Y. S. Int. J. Biochem. Cell Biol. 2009, 41, 666− 676. (49) Rajasekaran, N. S.; Connell, P.; Christians, E. S.; Yan, L.-J.; Taylor, R. P.; Orosz, A.; Zhang, X.-Q.; Stevenson, T. J.; Peshock, R. M.; Leopold, J. A.; Barry, W. H.; Loscalzo, J.; Odelberg, S. J.; Benjamin, I. J. Cell 2007, 130, 427−439. (50) Labunskyy, V. M.; Lee, B. C.; Handy, D. E.; Loscalzo, J.; Hatfield, D. L.; Gladyshev, V. N. Antioxid. Redox Signaling 2011, 14, 2327−2336.

693

DOI: 10.1021/acs.analchem.6b03136 Anal. Chem. 2017, 89, 688−693