Combinatorial Strategy to Identify Fluorescent Probes for Biothiol and

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Combinatorial Strategy to Identify Fluorescent Probes for Biothiol and Thiophenol Based on Diversified Pyrimidine Moieties and Their Biological Applications Xilei Xie, Mengmeng Li, Fuyan Tang, Yong Li, Lei Lei ZHANG, Xiaoyun Jiao, Xu Wang, and Bo Tang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04608 • Publication Date (Web): 07 Feb 2017 Downloaded from http://pubs.acs.org on February 8, 2017

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Analytical Chemistry

Combinatorial Strategy to Identify Fluorescent Probes for Biothiol and Thiophenol Based on Diversified Pyrimidine Moieties and Their Biological Applications Xilei Xie,† Mengmeng Li,† Fuyan Tang,† Yong Li,† Leilei Zhang,‡ Xiaoyun Jiao,† Xu Wang,*,† 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 ‡ Beijing Key Laboratory of Active Substances Discovery and Druggability Evaluation, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 10050, China; E-mail: [email protected]; [email protected] ABSTRACT: We present a feasible paradigm of developing original fluorescent probes for target biomolecules via combinatorial chemistry. In this developmental program, pyrimidine moieties were investigated and optimized as unique recognition units for thiols for the first time through a parallel synthesis in combination with rapid screening process. This time-efficient and cost-saving process effectively facilitated the developmental progress and provided detailed structure−reactivity relationships. As a result, ResBiot and Flu-Pht were identified as optimal fluorescent probes for biothiol and thiophenol respectively. Their favorable characteristics and superior applicability have been well demonstrated in both chemical and biological contexts. In particular, Res-Biot enables the direct visualization of biothiol fluctuations during oxidative stress and cell apoptosis, indicating its suitability in elucidation of a specific pathophysiological process in both living cells and living animals. Meanwhile, Flu-Pht is competent to visualize thiophenols without the interference from endogenous biothiols in living cells.

Fluorescent probe based assay has emerged as an irreplaceable methodology to provide critical insight into function and mechanism of bioactive molecules due to its high resolution, operational simplicity, biocompatibility, and noninvasive characteristic.1−3 A typical fluorescent probe is constructed by incorporating the specific recognition unit into a classic fluorophore platform, such as cyanine,4 fluorescein,5 and resorufin,6 etc. Generally, the recognition unit mainly accounts for the reactivity and specificity of fluorescent probes.7−13 Even nowadays, confined by chemists’ limited ability to predict and simulate using theoretical calculations, rational design of original recognition units still remains an enormous challenge, especially when there is no definite structural requirements for a target analyte of interest. Combinatorial chemistry, characterized by parallel synthesis and high-throughput screening, has been demonstrated an efficacious methodology in drug discovery and development and can partially overcome the limitation of rational design strategy.14−16 Thereby, it is expected that combinatorial strategy will facilitate the discovery and optimization of original recognition units so as to construct novel fluorescent probes with improved performance. To this end, thiols were selected as the target analytes of interest to develop novel probes via combinatorial methodology. Thiols, mainly comprising biothiol and thiophenol, are both of considerable biological significance. The former refers to bioactive aliphatic thiols, mainly consists of glutathione (GSH), cysteine (Cys), and homocysteine (Hcy). Biothiols are extensively involved in redox homeostasis, signal transduction,

drug metabolism, and other pathophysiological processes.17−19 On the other hand, thiophenol serves as a pollutant for the environment and a poison for living organisms. Exposure to thiophenol may induce severe kidney and liver injuries, central nervous system damage, and even death.20−22 Fluorescent probes will be powerful promotion to comprehensively elucidate the biological function of biothiol and the toxic mechanism of thiophenol. Biothiol and thiophenol, especially their corresponding deprotonated thiolate anions, can serve as strong nucleophiles to initiate nucleophilic aromatic substitution (SNAr). Several fluorescent probes for biothiol and thiophenol have been constructed by taking advantage of this unique chemical feature.23−33 At the same time, we noticed that pyrimidine moiety has the potential to function as a novel recognition unit for biothiol/thiophenol due to its electrophilicity34−36 and biocompatibility.37,38 However, it has never been investigated to design fluorescent probes. Thus, we speculated that incorporating pyrimidine moiety onto a fluorophore platform might generate a novel fluorescent probe candidate for biothiol/thiophenol. Besides, the reactivity and specificity profiles of the probe candidate can be easily modulated by regulating the electronic characteristic of the pyrimidine moiety. Hence a probe library can be created by altering substituents on the pyrimidine ring, which provides great potential to generate ideal fluorescent probes with suitable reactivity and high specificity towards biothiol and thiophenol.

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To test the above hypothesis, we implemented a developmental program to identify novel reaction-based fluorescent probes for biothiol and thiophenol based on pyrimidine chemistry via a combinatorial strategy. To accelerate the developmental progress, probe candidates were prepared by parallel synthesis and crude products were directly screened by fluorescence measurement without elaborate purification. In consequence, Res-Biot and Flu-Pht were identified as fluorescent probes for biothiol and thiophenol respectively. Their practical utility in chemical and biological contexts was well demonstrated. Particularly, Res-Biot enabled the direct visualization of endogenous biothiol fluctuations during oxidative stress and cell apoptosis in both living cells and living animals. At the same time, Flu-Pht demonstrated its capability of visualizing thiophenol without the interference from endogenous biothiols in living cells.

EXPERIMENTAL SECTION Materials and Instruments. Unless otherwise stated, all reagents and solvents were obtained from commercial suppliers and used without further purification. Sartorius ultrapure water (18.2 MΩ cm) was used throughout the experiments. HRMS analysis was operated on a Bruker MaXis UHR-TOF system. 1H NMR and 13C NMR spectra were taken on a Bruker Advance 300 MHz or 400 MHz spectrometer using tetramethylsilane as internal standard. The pH values were measured with a Model pH-3c digital pH meter. Fluorescence spectra were recorded by a FLS-980 fluorescence spectrometer equipped with a xenon lamp and 1.0-cm quartz cells at the slits of 2.0/2.0 nm. Cell imaging was taken with a Zeiss LSM 880 confocal laser scanning microscope with an objective lens (×20). In vivo imaging was carried out on IVIS Lumina III system with a metal halide lamp (150 W). Parallel Synthesis of the Fluorescent Probe Library. Resorufin (21.3 mg, 0.1 mmol) or fluorescein (33.2 mg, 0.1 mmol) was dissolved in 1.5 mL of DMF, 1.0 mmol of pyrimidine reagent (2-chloropyrimidine, 2-chloro-4(trifluoromethyl)pyrimidine, 4-chloropyrimidine, or 2-chloro5-nitropyrimidine) and K2CO3 (27.6 mg, 0.2 mmol) were added. The reaction mixture was stirred at 70 °C. Chemical conversion was monitored by TLC analysis. After resorufin or fluorescein was completely consumed, the above mixture was added into 70 mL of ethyl acetate, and washed with 50 mL of saturated NaCl solution three times. The organic solvent was evaporated under reduced pressure. The crude product was obtained and confirmed by HRMS analysis, and then screened directly without further purification. Rapid Screening of Probe Candidates. The above crude products were dissolved in DMSO and diluted to 40 µM with 50 mM PBS buffer solution, and then incubated with GSH (500 µM) or thiophenol (500 µM) at 37 °C for 60 min. For resorufin analogs, the fluorescence intensity was measured at λex/em = 560/585 nm. For fluorescein analogs, the fluorescence intensity was measured at λex/em = 460/519 nm. Preparation and Characterization of Res-Biot and FluPht. Res-Biot was synthesized from resorufin and 2-chloro-5nitropyrimidine according to the above general procedure. The obtained crude product was further purified by column chromatography on silica gel (dichloromethane/methanol, 100:1 v/v) to afford a yellow solid (28 mg, 83% yield). 1H NMR (400 MHz, DMSO-d6): δ 9.50 (s, 2H), 7.96 (d, J = 8.7 Hz, 1H), 7.61 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 9.9 Hz, 1H), 7.42

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(dd, J = 8.7, 2.0 Hz, 1H), 6.86 (dd, J = 9.8, 1.2 Hz, 1H), 6.31 (d, J = 1.2 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): δ 185.60, 165.63, 156.78, 155.41, 154.69, 149.37, 148.24, 144.29, 139.92, 135.10, 134.69, 131.18, 119.22, 109.69, 106.09. HRMS (ESI): calculated for C16H9N4O5+ (M+H+) 337.0567, found 337.0506. Flu-Pht was synthesized from fluorescein and 4chloropyrimidine according to the above general procedure. The obtained crude product was further purified by column chromatography on silica gel (dichloromethane/methanol, 100:1 v/v) to afford a light yellow solid (22 mg, 45% yield). 1 H NMR (400 MHz, CDCl3): δ 8.80 (s, 2H), 8.63 (d, J = 5.6 Hz, 2H), 8.06 (d, J = 7.5 Hz, 1H), 7.73 (t, J = 7.3 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.16 (s, 2H), 6.98 (d, J = 5.6 Hz, 2H), 6.90 (s, 4H). 13C NMR (100 MHz, CDCl3): δ 169.13, 168.65, 158.98, 158.80, 153.84, 152.74, 152.08, 135.44, 130.30, 129.47, 126.51, 125.46, 124.28, 117.84, 116.67, 110.38, 109.00, 81.84. HRMS (ESI): calculated for C28H17N4O5+ (M+H+) 489.1193, found 489.1158.

RESULTS AND DISCUSSION Design, Synthesis, and Screening of the Fluorescent Probe Library. The pyrimidine moiety is expected to function as a novel recognition unit for biothiol/thiophenol. The πelectron density of pyrimidine ring is decreased to a large extent, which facilitates the SNAr pathway. Therefore, pyrimidine is expected to possess potential reactivity to nucleophile thiols.34−36 Besides, pyrimidine ring can be modified by diverse substituents to adjust its electronic characteristic. Accordingly, the chemical profile of the probe candidate can be easily manipulated, which provides great potential to generate ideal fluorescent probes with optimized reactivity and specificity. Additionally, cytosine, thymine, and uracil, these three pyrimidine derivatives are nucleobases of living organisms, implying the excellent biocompatibility of pyrimidine moiety.37,38 As for the fluorophore platform, commercially available resorufin and fluorescein are selected due to their outstanding photophysical characteristics and excellent biocompatibility. Besides, their fluorescence OFF/ON can be easily switched by masking or exposing the hydroxyl group of these two fluorophores.5,6 Incorporating diversely substituted pyrimidine moieties onto resorufin or fluorescein via ether linkage generates a novel probe library for thiols. We envisioned that fluorescence emission of probe candidates would be quenched by hydroxyl protection. Once thiolyzed by biothiol or thiophenol, the fluorophore would be released and fluorescence emission restored. On the basis of the above consideration, we proposed a diversity-oriented probe library consisting of eight candidates (Figure 1). Coupling of pyrimidin-2-yl, 4(trifluoromethyl)pyrimidin-2-yl, pyrimidin-4-yl, and 5nitropyrimidin-2-yl moieties with resorufin furnishes probe Res-1−Res-4 respectively. Similarly, integrating the four pyrimidine moieties onto fluorescein platform generates probe Flu-1−Flu-4.

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Figure 1. Chemical structures of designed fluorescent probe candidates.

The probe candidates were prepared via one-step parallel synthesis shown in Scheme S1. Excess pyrimidine reagents were added to ensure that the resorufin/fluorescein was completely consumed. Inside the reaction system, there were mainly the newly generated probe candidates and remained pyrimidine reagents, which was confirmed by TLC analysis. The reagents were always non-fluorescent due to its short conjugated structure. Considering there was no other substance except the probe candidates to induce fluorescence variation in the reaction system, the crude products were directly evaluated for their reactivity and specificity by fluorescence measurement without elaborate purification.39 GSH is the most abundant and comprises about 90% of non-protein biothiols inside the cell,40 thereby selected as the representative biothiol in the experiments. After incubation with GSH/thiophenol, Res-1, Res-2, Flu-1, and Flu-2 could not be lighted on by GSH or thiophenol (Figure S1a, S1b, S1e, and S1f), inferring pyrimidin-2-yl moiety and its trifluoromethyl analog were chemically stable toward GSH and thiophenol. To the contrary, pyrimidin-4-yl moiety presented moderate reactivity. When it was installed onto fluorescein, the resultant probe (Flu-3, also called Flu-Pht) specifically responded to thiophenol (Figure S1g), while the corresponding resorufin-based counterpart (Res-3) displayed no response toward thiophenol (Figure S1c), corroborating the close relationship between the reactivity of a probe and its fluorophore platform. Furthermore, fluorescence of Res-4 (also called Res-Biot) and Flu-4 was turned on by both GSH and thiophenol (Figure S1d and S1h), revealing that strong electron-withdrawing substituent on pyrimidine ring enhanced the reactivity of the probe. Thereby, structure−reactivity relationships were outlined as follows. First, despite possessing analogical structure, pyrimidin-4-yl moiety presents higher reactivity than pyrimidin-2-yl. Second, the reactivity of a probe mainly depends on its recognition unit, and is partially related to its fluorophore in certain case. Third, strong electron-withdrawing substituent (e.g. nitro group) is preferred to enhance the reactivity of a thiol probe, but probably reduces the specificity. This parallel synthesis in combination with rapid screening process, characterized by time-efficient and cost-saving, effectively facilitated the developmental progress and provided detailed structure−reactivity relationships which would be valuable for further thiol probe design. Compared to Flu-4, Res-Biot was preferred due to its red fluorescence emission. It is worth noting that Res-Biot unavoidably responds to thiophenol due to its higher nucleophilicity than that of biothiol.41 However, interference from thiophenol can be excluded in living system because it is an exogenous species. Therefore, Res-Biot and Flu-Pht were identified as fluorescent probes for biothiol and thiophenol

respectively, and selected for extensive evaluation in both chemical and biological contexts. These two probes were renewedly prepared and characterized by HRMS, 1H NMR, and 13 C NMR. Fluorescence Response and Reaction Mechanism. The fluorescence spectra variation of Res-Biot toward GSH was depicted in Figure 2a. As can be seen, Res-Biot presented nearly no fluorescence emission at 585 nm in the absence of GSH. Upon introduction of GSH, the maximum fluorescence intensity at 585 nm increased and exhibited an excellent linear correlation (R2 = 0.994) with the GSH concentrations in range of 0 to 20 µM (Figure 2a, insert). The limit of detection (LOD) value was calculated to be as low as 0.29 µM based on S/N = 3. The fluorescence spectra of Res-Biot toward GSH were identical to those of authentic resorufin, which was ascribed to the release of resorufin fluorophore by thiolysis of GSH. The reaction mechanism between Res-Biot and GSH was further confirmed by HRMS analysis. After incubation, the peaks at m/z 212.0387 and 429.0881 were both detected out in the reaction mixture (Figure S2), which agreed well with resorufin and the pyrimidine derivative respectively. The result testified the proposed reaction pathway shown in Scheme 1a. Additionally, we also investigated the reaction mechanism of Res-Biot for Cys and Hcy. HRMS spectra (Figure S3 and S4) indicated that Res-Biot was thiolyzed to resorufin (m/z 212.0368 or 212.0376) and the corresponding pyrimidine derivative (m/z 243.0208 or 257.0376), and the reaction pathway (Scheme S2) was similar to that proposed by GSH.

Figure 2. (a) Emission spectra (excited at 560 nm) of 10 µM ResBiot toward varied concentrations of GSH (0−100 µM) in HEPES buffer (20 mM, pH 7.4) at 37 °C for 30 min, insert: linear curve derived from fluorescence titration. (b) Emission spectra (excited at 460 nm) of 10 µM Flu-Pht toward varied concentrations of thiophenol (0−100 µM) in PBS buffer (50 mM, pH 7.4) at 37 °C for 2 h, insert: linear curve derived from fluorescence titration.

The same investigation was performed on the response of Flu-Pht toward thiophenol. Briefly, the emission maximum at 519 nm increased gradually with the increasing concentration of thiophenol (Figure 2b). There was excellent linearity (R2 = 0.992) between the fluorescence intensity at 519 nm and thiophenol concentrations in range of 0 to 100 µM (Figure 2b,

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insert). The LOD value was found to be 1.8 µM. Additionally, the fluorescence spectra of Flu-Pht in the presence of thiophenol were identical to those of original fluorescein, suggesting the liberation of fluorescein fluorophore by thiolysis of thiophenol. The HRMS analysis results (Figure S5) validated the proposed reaction pathway in Scheme 1b. Scheme 1. Proposed Reaction Mechanism of (a) Res-Biot and (b) Flu-Pht

The effect of pH value was estimated, revealing that ResBiot and Flu-Pht exhibited higher OFF/ON ratio toward their own target analytes in the pH 6.0−8.0 (Figure S6). This was well consistent with the physiological pH range, indicating their potential application in living systems. Specificity Investigation. Next, the specificity of Res-Biot and Flu-Pht was evaluated by screening their fluorescence response to biologically relevant species under physiological conditions. As shown in Figure 3a, Res-Biot displayed significant fluorescence increments only in the present of biothiols (GSH, Cys, and Hcy). Negligible fluorescence changes were induced by potential competing species, including other natural animo acids (Gly, Met, and Lys), reactive oxygen and nitrogen species (H2O2, ClO−, •OH, 1O2, and NO), and metal ions (K+, Fe2+, and Zn2+).

Figure 3. (a) Fluorescence responses of Res-Biot (10 µM) toward various species (100 µM): (1) blank, (2) GSH, (3) Cys, (4) Hcy, (5) Gly, (6) Met, (7) Lys, (8) H2O2, (9) ClO−, (10) •OH, (11) 1O2, (12) NO, (13) K+, (14) Fe2+, (15) Zn2+, λex/em = 560/585 nm. (b) Fluorescence responses of Flu-Pht (10 µM) toward various species (100 µM): (1) blank, (2) thiophenol, (3) GSH, (4) Cys, (5) Hcy, (6) Gly, (7) phenol, (8) aniline, (9) NaN3, (10) KI, (11) H2O2, (12) ClO−, (13) •OH, (14) 1O2, (15) NO, λex/em = 460/519 nm.

The fluorescence of Flu-Pht was exclusively triggered on by thiophenol, while the biothiols (GSH, Cys, and Hcy) induced no observable fluorescence signal enhancements, demonstrating its capability of discriminating thiophenol from biothiol. Additionally, Flu-Pht presented excellent immunity to interference from other potential interfering substances, including some nucleophilic species (Gly, phenol, aniline, NaN3, and KI), reactive oxygen and nitrogen species (H2O2, ClO−, •OH, 1O2, and NO) (Figure 3b). Above all, these results collectively indicated the high specificity of Res-Biot and Flu-Pht toward their own target analytes. Fluorescence Imaging in Living Cells. The aforementioned favorable properties of Res-Biot and Flu-Pht in chemi-

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cal system encouraged us to investigate their practical utilities in living cells. Prior to being applied in cell imaging, MTT assay was first performed on HepG2 cells to assess their biocompatibility (Figure S7). The IC50 values of Res-Biot and Flu-Pht were up to 200 µM and 256 µM respectively, inferring their low cytotoxicity and superior practical capability in biological samples. Res-Biot was employed to visualize the endogenous biothiols in Hela cells. Phorbol 12-myristate 13-acetate (PMA) is a well-established protein kinase C activator to trigger cellular oxidative stress.42 L-Buthionine sulfoximine (BSO) serves as an inhibitor of γ-glutamylcysteine synthetase to decline GSH level and finally results in cell apoptosis.43 These two widely approved reagents were applied to induce intracellular biothiol fluctuation. As expected, after incubation with Res-Biot, bright red fluorescence was observed inside the cells (Figure 4a), indicating the cell-permeability of Res-Biot and the abundant contents of intracellular biothiols. Pretreating the cells with a thiol-specific scavenger, N-ethylmaleimide (NEM),44 the fluorescence signal was obviously suppressed (Figure 4b), confirming the specific fluorescence response to cellular biothiols. Furthermore, a pronounced fluorescence decrease was observed when cells were pretreated with PMA (Figure 4c) or BSO (Figure 4d), indicating the down-regulated biothiol level during the oxidative stress and cell apoptosis process. These outcomes collectively demonstrated that Res-Biot was capable of visualizing endogenous biothiol fluctuation during a specific pathophysiological process in living cells.

Figure 4. Fluorescence images of endogenous biothiols in Hela cells. Cells were treated with (a) vehicle, (b) 1 mM NEM, (c) 1 µg/mL PMA, or (d) 100 µM BSO for 30 min followed by incubation with 10 µM Res-Biot for 20 min. (e−h) Second row shows the corresponding bright-field images for the first row. The images were acquired using a confocal microscope with 543-nm excitation and 560−620-nm collection. Scale bar: 20 µm.

Similar experiments were performed on Flu-Pht toward thiophenol in HepG2 cells. Cells treated with Flu-Pht exhibited almost no fluorescence signal (Figure 5a), revealing that endogenous biothiols could not illuminate the fluorescence of Flu-Pht and there was no endogenous thiophenol inside the cells. When pretreated with thiophenol, a significant fluorescence enhancement was observed (Figure 5b), demonstrating the capability of discriminating thiophenol from endogenous biothiols in living cells.

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Analytical Chemistry the first time that pyrimidine functionality is exploited to construct optical probes for thiols. The present work evidently exhibited the advantage of combinatorial strategy in fluorescent probe exploitation and showed that in principle, pyrimidine moiety can be installed onto alternative near-infrared or two-photon fluorophores to generate next-generation fluorescent sensors for thiols with improved performance.

ASSOCIATED CONTENT Supporting Information Figure 5. Fluorescence images of thiophenol in HepG2 cells. Cells were treated with (a) vehicle or (b) 50 µM thiophenol for 20 min followed by incubation with 5 µM Flu-Pht for 20 min. (c, d) Second row shows the corresponding bright-field images for the first row. The images were acquired using a confocal microscope with 488-nm excitation and 500−560-nm collection. Scale bar: 20 µm.

Visualizing Biothiols in Animal Models. Inspired by the red fluorescence emission of Res-Biot, we subsequently explored its feasibility of visualizing endogenous biothiols during LPS-induced inflammation response45 and BSO-induced apoptotic process in living animals. As shown in Figure 6, the mouse treated with Res-Biot emitted high fluorescence intensity (Figure 6a). By contrast, the fluorescence was apparently attenuated in the mouse pretreated with NEM (Figure 6b), which was attributed to the specific biothiol depletion by NEM. At the same time, fluorescence emission was efficiently blocked in the mouse pretreated with LPS (Figure 6c) or BSO (Figure 6d), indicating the depressed biothiol level during inflammation response and apoptotic process. Collectively, ResBiot successfully visualized LPS- and BSO-induced endogenous biothiol fluctuation, demonstrating its practical capability in studying biothiol-related pathophysiological process in living animals.

Figure 6. Fluorescence imaging (pseudocolor) of endogenous biothiols in nude mice. The mice were injected with (a) vehicle, (b) NEM (1 mM, 200 µL), (c) LPS (1 mg/mL, 200 µL), or (d) BSO (1 mM, 200 µL) followed by injection of Res-Biot (100 µM, 200 µL). The excitation filter was 540 nm, and the emission filter was 570 nm.

CONCLUSION In summary, we implemented a combinatorial developmental program to identify novel fluorescent probes for biothiol and thiophenol by exploiting the specific reaction between pyrimidine moieties and thiols. The parallel synthesis in combination with rapid screening is time-efficient and costeffective. In consequence, Res-Biot and Flu-Pht were optimized as the new fluorescent probes for biothiol and thiophenol respectively and were used to visualize biothiol and thiophenol in living systems. To the best of our knowledge, it is

The Supporting Information is available free of charge on the ACS Publications website. Experimental details, supplementary data, synthesis and characterization of compounds (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected] *Fax: +86-531-86180017

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by 973 Program (2013CB933800) and National Natural Science Foundation of China (21390411, 21535004, 21375080, and 21505088).

REFERENCES (1) Chan, J.; Dodani, S. C.; Chang, C. J. Nat. Chem. 2012, 4, 973−984. (2) Li, X.; Gao, X.; Shi, W.; Ma, H. Chem. Rev. 2014, 114, 590−659. (3) Yang, Y.; Zhao, Q.; Feng, W.; Li, F. Chem. Rev. 2013, 113, 192−270. (4) Sun, W.; Guo, S.; Hu, C.; Fan, J.; Peng, X. Chem. Rev. 2016, 116, 7768−7817. (5) Chen, X.; Pradhan, T.; Wang, F.; Kim, J. S.; Yoon, J. Chem. Rev. 2012, 112, 1910−1956. (6) Lavis, L. D.; Raines, R. T. ACS Chem. Biol. 2014, 9, 855−866. (7) Xie, X.; Yang, X.; Wu, T.; Li, Y.; Li, M.; Tan, Q.; Wang, X.; Tang, B. Anal. Chem. 2016, 88, 8019−8025. (8) Li, Y.; Wang, X.; Yang, J.; Xie, X.; Li, M.; Niu, J.; Tong, L.; Tang, B. Anal. Chem. 2016, 88, 11154−11159. (9) Wang, X.; Sun, J.; Zhang, W.; Ma, X.; Lv, J.; Tang, B. Chem. Sci. 2013, 4, 2551−2556. (10) Wang, X.; Lv, J.; Yao, X.; Li, Y.; Huang, F.; Li, M.; Yang, J.; Ruan, X.; Tang, B. Chem. Commun. 2014, 50, 15439−15442. (11) Feng, W.; Liu, D.; Feng, S.; Feng, G. Anal. Chem. 2016, 88, 10648−10653. (12) Xue, S.; Ding, S.; Zhai, Q.; Zhang, H.; Feng, G. Biosens. Bioelectron. 2015, 68, 316−321. (13) Yu, D.; Huang, F.; Ding, S.; Feng, G. Anal. Chem. 2014, 86, 8835−8841. (14) Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893−930. (15) Potyrailo, R. A.; Mirsky, V. M. Chem. Rev. 2008, 108, 770−813. (16) Vendrell, M.; Zhai, D.; Er, J. C.; Chang, Y. T. Chem. Rev. 2012, 112, 4391−4420. (17) Townsend, D. M.; Tew, K. D.; Tapiero, H. Biomed. Pharmacother. 2003, 57, 145−155. (18) Giles, N. M.; Watts, A. B.; Giles, G. I.; Fry, F. H.; Littlechild, J. A.; Jacob, C. Chem. Boil. 2003, 10, 677−693. (19) Refsum, H.; Ueland, P. M.; Nygard, O.; Vollset, S. E. Annu. Rev. Med. 1998, 49, 31−62.

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(20) Munday, R. Free Radical Biol. Med. 1989, 7, 659−673. (21) Munday, R. J. Appl. Toxicol. 1985, 5, 402−408. (22) Munday, R. J. Appl. Toxicol. 1985, 5, 409−413. (23) Jung, H. S.; Chen, X.; Kim, J. S.; Yoon, J. Chem. Soc. Rev. 2013, 42, 6019−6031. (24) Chen, H.; Tang, Y.; Lin, W. TrAC, Trends Anal. Chem. 2016, 76, 166−181. (25) Chen, H.; Tang, Y.; Ren, M.; Lin, W. Chem. Sci. 2016, 7, 1896−1903. (26) Anees, P.; Joseph, J.; Sreejith, S.; Menon, N. V.; Kang, Y.; Yu, S. W. K.; Ajayaghosh, A.; Zhao, Y. Chem. Sci. 2016, 7, 4110−4116. (27) Zhou, P.; Yao, J.; Hu, G.; Fang, J. ACS Chem. Biol. 2016, 11, 1098−1105. (28) Tang, B.; Xing, Y.; Li, P.; Zhang, N.; Yu, F.; Yang, G. J. Am. Chem. Soc. 2007, 129, 11666−11667. (29) Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W. Angew. Chem., Int. Ed. 2007, 46, 8445−8448. (30) Sun, Q.; Yang, S. H.; Wu, L.; Yang, W. C.; Yang, G. F. Anal. Chem. 2016, 88, 2266−2272. (31) Liu, H. W.; Zhang, X. B.; Zhang, J.; Wang, Q. Q.; Hu, X. X.; Wang, P.; Tan, W. Anal. Chem. 2015, 87, 8896−8903. (32) Shao, X.; Kang, R.; Zhang, Y., Huang; Z., Peng, F.; Zhang, J.; Wang, Y.; Pan, F.; Zhang, W.; Zhao, W. Anal. Chem. 2015, 87, 399−405. (33) Zhang, W.; Liu, X.; Zhang, H.; Feng, C.; Liu, C.; Yu, M.; Wei, L.; Li, Z. J. Mater. Chem. C 2015, 3, 8248−8254. (34) Smith, S. M.; Buchwald, S. L. Org. Lett. 2016, 18, 2180−2183. (35) Cui, J.; Jin, J.; Chaudhary, A. S.; Hsieh, Y. H.; Zhang, H.; Dai, C.; Damera, K.; Chen, W.; Tai, P. C.; Wang, B. ChemMedChem. 2016, 11, 43−56. (36) Delia, T. J.; Kanaar, J. B.; Knefelkamp, E. J. Heterocyclic Chem. 2002, 39, 347−350. (37) Parker, W. B. Chem. Rev. 2009, 109, 2880−2893. (38) Taylor, R. D.; MacCoss, M.; Lawson, A. D. J. Med. Chem. 2014, 57, 5845−5859. (39) Mello, J. V.; Finney, N. S. J. Am. Chem. Soc. 2005, 127, 10124−10125. (40) Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52, 711−760. (41) Liu, X. L.; Duan, X. Y.; Du, X. J.; Song, Q. H. Chem.−Asian J. 2012, 7, 2696−2702. (42) Balasubramanyam, M.; Koteswari, A. A.; Kumar, R. S.; Monickaraj, S. F.; Maheswari, J. U.; Mohan, V. J. Biosci. 2003, 28, 715−721. (43) Armstrong, J. S.; Steinauer, K. K.; Hornung, B.; Irish, J. M.; Lecane, P.; Birrell, G. W.; Peehl, D. M.; Knox, S. J. Cell Death Differ. 2002, 9, 252−263. (44) Gregory, J. D. J. Am. Chem. Soc. 1955, 77, 3922−3923. (45) Lee, D.; Khaja, S.; Velasquez-Castano, J. C.; Dasari, M.; Sun, C.; Petros, J.; Taylor, W. R.; Murthy, N. Nat. Mater. 2007, 6, 765−769.

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