Discriminative Fluorescence Sensing of Biothiols in Vitro and in Living

Feb 17, 2015 - ... Yanmei Zhou , Wenli Gao , Junli Zhang , Haohan Song , Xiao Wang ... Near-Infrared and Naked-Eye Fluorescence Probe for Direct and ...
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Discriminative Fluorescence Sensing of Biothiols in Vitro and in Living Cells Qingqing Miao, Qing Li, Qingpan Yuan, Lingli Li, Zijuan Hai, Shuang Liu, and Gaolin Liang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac504836a • Publication Date (Web): 17 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Discriminative Fluorescence Sensing of Biothiols in Vitro and in Living Cells Qingqing Miao,† Qing Li,‡ Qingpan Yuan,† Lingli Li,† Zijuan Hai,† Shuang Liu,† and Gaolin Liang*,† CAS Key Laboratory of Soft Matter Chemistry, †Department of Chemistry and ‡Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, China. Fax no.: (+86)-551-63600730; E-mail: [email protected] ABSTRACT: Simultaneous discriminative sensing of biothiols in vitro and in living cells has remained challenging. Herein, we report a new sulfonamide-based self-quenched fluorescent probe 1 for this purpose with high sensitivity and good selectivity. Treatment of 1 with Cys, Hcy, or GSH yields Aminoluciferin, CBTHcy, or CBT, turning “on” the fluorescence at wavelengths of 522, 517, or 490 nm, respectively. Kinetic study indicated that 1 reacts with Cys faster than with Hcy or GSH. With these unique properties of 1, we applied 1 for highly sensitive sensing of Cys, Hcy, and GSH among other 19 natural AAs with good selectivity. Confocal fluorescence microscopic imaging of 1-treated HepG2 cells at two channels (522 ± 8 nm and 490 ± 8 nm), together with quantitative analysis, indicated that the “turn-on” fluorescence was induced by intracellular Cys-dominating condensation and reduction of 1 but not by intracellular GSH-dominating reduction of 1. This suggests that 1 could be applied for discriminative sensing of intracellular Cys from the abundant GSH. Further development of 1 might bring about an efficient tool for probing cellular functions that relate to biothiols.

INTRODUCTION Intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play crucial roles in maintaining redox homeostasis in biological systems through equilibrium between the reduced free thiol and oxidized disulfide forms.1 Generally, alternations in the level of cellular thiols are closely associated with certain diseased states including leucocyte loss, psoriasis, liver damage, cancer, acquired immune deficiency syndrome (AIDS), and cardiovascular diseases.2,3 Specifically, Cys deficiency is involved in many syndromes including retarded growth, hair depigmentation, edema, lethargy, liver damage, muscle and fat loss, and skin lesions.4,5 Elevated level of Hcy in plasma is a risk factor for Alzheimer's and cardiovascular diseases.6,7 And GSH, the most abundant intracellular thiol, serves many cellular functions including maintenance of intracellular redox activity, xenobiotic metabolism, intracellular signal transduction, and gene regulation.8 Consequently, assessments of the levels of these mercapto biomolecules in biological systems may aid early diagnoses of some diseases. Over the past several decades, a considerable effort has been devoted to the development of effective analytical methods for the detection of thiols in biological systems, for instances, high performance liquid chromatography (HPLC),9 capillary electrophoresis,10,11 spectrophotometry,12,13 voltammetry,14 mass spectrometry (MS),15-17 and HPLC-MS/MS.18 Among the various analytical methods available, fluorometry has its own advantages over these techniques in its operational simplicity and high sensitivity. In recent years, numerous fluorescent probes have been developed to distinguish biothiols from other amino acids (AAs) by utilizing specific reactions that can take place between thiols and the probes, including Michael addition,19 cyclization with aldehyde,20 cleavage of sulfonamide and sulfonate ester by thiols,21 cleavage of selenium–nitrogen

bond by thiols,22 cleavage of disulfide by thiols,23 metal complexes related,24 nano-material related,25-27 and others.28,29 Nevertheless, simultaneous discriminative sensing of biothiols in vitro and even in living cells has remained challenging. Guo and co-workers designed a fluorescent probe whose emission shifts from red to green or blue upon Micheal additions of GSH or Cys, respectively,30 for discriminative detection of GSH and Cys. Several sulfonamide-fluorescent probes, based on the different cleavage ability of biothiols on their sulfonamide bond to “turn-on” (or increase) the fluorescence, have been developed for the discriminative detection of biothiols. For example, Zhang and co-workers developed a coumarin-based sulfonamide fluorescent probe for simultaneous detection of GSH and Cys.21 Recently, Yoon and Ryu reported a promising cyanine-based sulfonamide-fluorescent probe, which displayed high selectivity for GSH over Cys and Hcy, for highly selective detection of GSH in living cells and in vivo.31 Inspired by these pioneering studies mentioned above, we aimed to design a fluorescent probe for simultaneously discriminative sensing of biothiols (i.e., GSH, Cys, and Hcy herein) in physiological conditions (e.g., buffers at pH 7.4) with high sensitivity. To achieve this, as shown in Figure 1, we rationally designed a sulfonamide-based self-quenched fluorescent probe 1 whose fluorescence is turned “on” at a maximum of 522, 517, or 490 nm upon that addition of Cys, Hcy, or GSH, respectively. Unlike previous sulfonamidefluorescent probes which are only subjected to biothiolreductions, our probe 1 is subjected to GSH-reduction, Cyscondensation and reduction, Hcy-condensation and reduction to yield respective products of 2-cyano-6-aminobenzothiazole (CBT), Aminoluciferin, CBTHcy which have different fluorescent emission maximums. Employing its high sensitivity and good selectivity to GSH, Cys, and Hcy, we successfully applied 1 for discriminative sensing of these biothiols among

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Figure 1. Schematic illustration of fluorescence “turn on” of probe 1 for discriminative sensing of biothiols.

the 20 natural AAs in vitro. Furthermore, probe 1 was also successfully used for discriminative sensing of Cys and GSH in living cells by ratiometric quantification. EXPERIMENTAL SECTION Materials. All the starting materials were obtained from Sigma or Sangon Biotech. Commercially available reagents were used without further purification, unless noted otherwise. All chemicals were reagent grade or better. General Methods. 1H NMR and 13C NMR spectra were obtained on a Bruker AV 300. Matrix-assisted laser desorption (MALDI) ionization-time of flight (TOF)/TOF and ESI mass spectra were obtained on a time-of-flight Ultrflex II mass spectrometer (Bruker Daltonics) and on a Finnigan LCQ Advantage ion trap mass spectrometer (ThermoFisher Corporation) equipped with a standard ESI source, respectively. High performance liquid chromatography (HPLC) purification was performed on a Shimazu UFLC system equipped with two LC-20AP pumps and an SPD-20A UV/vis detector using a Shimazu PRC-ODS column. HPLC analyses were performed on an Agilent 1200 system equipped with a G1322A pump and in-line diode array UV detector an Agilent Zorbax 300SDC18 RP column, with CH3CN (0.1% of TFA) and water (0.1% of TFA) as the eluent. Cell images were obtained on the IX71 or IX81 fluorescence microscope (Olympus, Japan) for general fluorescence images or confocal microscope images, respectively. Fluorescence spectra were recorded on an F-4600 fluorescence spectrophotometer (Hitachi High-Techonologies Corporation, Japan) with excitation wavelength set to 350 nm. Syntheses and Characterizations. Synthesis of 1. 2,4-dinitrobenzensufonyl chloride (374 mg, 1.406 mmol) was added to a solution of 2-cyano-6aminobenzothiazole (CBT, 105 mg, 0.600 mmol) in anhydrous pyridine (6 mL) at 0 °C for 1 hour, and then the solution was stirred for 8 hours at room temperature. After the reaction, the mixture was diluted with ethyl acetate and washed with water. The organic solvent was removed under reduced pressure and the reaction mixture was subjected to HPLC purification to yield pure compound 1 (168 mg, 69.1%). Synthesis of Aminoluciferin. Cysteine (121 mg, 1.00 mmol) was dissolved in water and the pH value was adjusted to 8 with sodium carbonate and then CBT (140 mg, 0.800 mmol) dissolved in CH3OH was added. CBT was consumed evidenced by thin-layer chromatography (TLC). The methanol was removed under reduced pressure and the remaining aqueous solution was acidified to pH 3 with 1 M HCl. With ad-

justment of the pH, yellow solid precipitated continuously. The precipitation was filtered and washed three times with water (2 mL × 3) to yield Aminoluciferin (170 mg, 76.2%) dried under a freeze-dryer. Synthesis of CBTHcy. Homocysteine (Hcy) (135 mg, 1.00 mmol) was dissolved in water and the pH value was adjusted to 8 with sodium carbonate and then CBT (140 mg, 0.800 mmol) dissolved in CH3OH was added. CBT was consumed evidenced by thin-layer chromatography (TLC). The methanol was removed under reduced pressure and the remaining aqueous solution was acidified to pH 3 with 1 M HCl. With adjustment of the pH, yellow solid precipitated continuously. The precipitation was filtered and washed three times with water (2 mL × 3) to yield CBTHcy (160 mg, 68.3%) dried under a freeze-dryer. Characterization of 1. MS of 1: calculated for C14H7N5O6S2, [(M+H)+]: 405.9916, obsvd. HR-MALDITOF/MS: 405.9917 (Figure S8). 1H NMR of compound 1 (300 MHz, CD3CN, Figure S9) δ (ppm): 8.40 (s, 1 H), 8.31 (dd, J = 3.0 Hz, 9.0 Hz, 1 H), 8.22 (d, J = 6.0 Hz, 1 H), 7.86 (d, J = 6.0 Hz, 1 H), 7.68 (s, 1 H), 7.23 (d, J = 8.0 Hz, 1 H); 13C NMR of compound 1 (75 MHz, CD3CN, Figure S10) δ (ppm): 150.53, 149.10, 149.01, 142.21, 140.04, 137.85, 135.69, 133.09, 127.38, 125.87, 124.55, 120.71, 114.09, 114.04. Characterization of Aminoluciferin. MS of Aminoluciferin: calculated for C11H9N3O2S2, [(M+H)+]: 280.0214, obsvd. HR-MALDI-TOF/MS: 280.0214 (Figure S11). 1H NMR of Aminoluciferin (d6-DMSO, 300 MHz, Figure S12) δ (ppm): 7.78 (d, J = 6.0 Hz, 1 H), 7.07 (s, 1 H), 6.87 (dd, J1 = 3.0 Hz, J2 = 9.0 Hz, 1 H), 5.86 (br, 2 H), 5.37 (t, J = 6.0 Hz, 1 H), 3.66 (m, 2 H). Characterization of CBTHcy. MS of CBTHcy: calculated for C12H11N3O2S2, [(M+H)+]: 294.0371, obsvd. HR-MALDITOF/MS: 294.0371 (Figure S13). 1H NMR of CBTHcy (d6DMSO, 300 MHz, Figure S14) δ (ppm): 7.72 (d, J = 9.0 Hz, 1 H), 7.04 (s, 1 H), 6.81 (dd, J1 = 3.0 Hz, J2 = 9.0 Hz,1 H), 5.73 (br, 2 H), 4.58 (m, 1 H), 3.06-3.30 (br, 2 H), 2.19 (m, 1 H), 1.91 (m, 1 H). Cell Culture. The hepatocellular carcinoma HepG2 cells were cultured in Dulbecco’s modified eagle medium (DMEM, GIBCO) supplemented with 10% fetal bovine serum (FBS, GIBCO) and streptomycin (100 µg/mL). The cells were expanded in tissue culture dishes and kept in a humid atmosphere of 5% CO2 at 37 °C. The medium was changed every other day.

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MTT Assay. The cytotoxicity was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay with HepG2 cells. Cells growing in log phase were seeded into 96-well cell-culture plate at 3 × 103/well. The cells were incubated for 12 h at 37 oC under 5% CO2. The solutions of 1 (100 µL/well) at concentrations of 20, 40, or 80 µM in 100 µL medium were added to the wells, respectively. The cells were incubated for 2, 4, or 8 h at 37 °C under 5% CO2. A solution of 5 mg/mL MTT dissolved in phosphate buffered saline (PBS) (pH 7.4) (10 µL /well) was added to each well of the 96-well plate. A solution of 10% SDS dissolved in 0.01M HCl (100 µL/well) was added to dissolve the formazan after an additional 4 h-incubation. The data were obtained using an ELISA reader (VARIOSKAN FLASH) to detect its absorption at 570/680 nm. The following formula was used to calculate the viability of cell growth: Viability (%) = (mean of Absorbance value of treatment group / mean of Absorbance value of control) ×100. Cell Imaging. The hepatocellular carcinoma HepG2 cells were plated on 3.5 cm cell culture dish at 50% cell density in the next day. Then the HepG2 cells were washed for three times with phosphate buffered saline (PBS) and incubated with 40 µM 1 in serum-free medium at 37 °C for 0.5 h in a CO2 incubator. Then, the cells were washed with PBS for another three times prior to confocal microscopic imaging. For the thiol-blocking group, the HepG2 cells were pretreated with maleimidobutyric acid (MBA, one type of thiol-blocking reagents) at 2 mM in serum-free medium at 37 °C for 0.5 h, washed with PBS for three times, then incubated with 40 µM 1 in serum-free medium at 37 °C for 0.5 h, washed with PBS for another three times prior to confocal microscopic imaging. For the Cys or GSH recovering group, after pretreated with MBA at the same condition, cells were incubated with Cys or GSH at 10 mM for 0.5 h, and then washed with PBS and incubated with 40 µM 1 in serum-free medium at 37 °C for 0.5 h. Then, the cells were washed with PBS for another three times prior to confocal microscopic imaging. The fluorescence images were obtained with an Olympus IX81 confocal microscope. The double-channel fluorescence images at 490 ± 8 and 522 ± 8 nm are shown in Figure 4a. Fluorescence intensities of the images from the two channels were analyzed with Image J (Universal Imaging Corp.), and quantification of fluorescence intensity ratio between these two channels were obtained and analyzed (Figure 4b). RESULTS AND DISCUSSION Syntheses and Rationale of the Design. We began the study with the synthesis of probe 1. The syntheses are facile and straightforward as follows, which are according to the literature method (Scheme S1)32: 2,4-dinitrobenzensufonyl chloride was coupled with CBT in anhydrous pyridine. After the reaction, the mixture was diluted with ethyl acetate and washed with water. After the organic solvent was removed under reduced pressure, the leftover syrup was dissolved in CH3OH/H2O (70/30, v/v) and pure compound 1 was obtained after HPLC purification. 1 was designed to have three components as following: (1) a bifunctional CBT moiety which can not only discriminatively condense with Cys or Hcy (not GSH) but also provide the fluorophore;33-35 (2) 2,4dinitrobenzenesulfonyl (DNBS), a strong electronwithdrawing moiety, proposed for the intermolecular charge transfer (ICT) pathway to quench the fluorescence of CBT

moiety, and (3) a thiol-responsive sulfonamide group for thiol cleavage. As outlined in Figure 1, probe 1 is nonfluorescent due to the electrons from CBT are diverted to the electronwithdrawing moiety DNBS, resulting in the quenching of CBT fluorescence. Interestingly, treatment of 1 with excessive Cys results in the condensation reaction between CBT moiety and Cys, followed by cleavage of the sulfonamide bond by Cys to yield the final product Aminoluciferin and thus turns “on” the fluorescence centered at 522 nm (λem = 522 nm). Similarly, treatment of 1 with excessive Hcy yields the condensation and cleavage product of CBTHcy and thereby its fluorescence centering at 517 nm (λem = 517 nm) is turned “on”. But treatment of 1 with excessive GSH only yields the sulfonamide-cleaved product of CBT and whose fluorescence centers at 490 nm (λem = 490 nm) is turned “on”. With the unique properties of 1 towards biothiols, we were able to apply 1 for discriminative fluorescence sensing of Cys, Hcy, and GSH in vitro and even in cells. Selectivity of 1 to Biothiols and Mechanism Study. Selectivity is one of the important parameters to evaluate the performance of a new fluorescence probe. Particularly, for a cellular imaging probe which potentially has biomedical applications, a highly selective response to the target over other potentially competing species is a necessity. Therefore, the selectivity study of 1 to biothiols over other 19 natural AAs was conducted. As shown in Figure 2a, after 10 µM of probe 1 was respectively treated with 100 µM analyte (Cys, Hcy, GSH, Glu, Leu, Ala, Lys, Gly, Phe, Tyr, Pro, Gln, Met, Asn, Thr, Arg, Val, Trp, Ser, Asp, His, Ile) at 37 °C for 2 h, remarkable fluorescent enhancements were observed from the addition of Cys, Hcy, or GSH with intensity increased at 29.3, 11.4, or 16.5 folds, respectively, while no obvious changes took place with other 19 AAs. Obviously, mixing 1 with Cys, Hcy, or GSH led to the “turn-on” of the fluorescence with emission peaks at 522, 517, or 490 nm, respectively. The fluorescent photographs of 1 in a cuvette in the presence of different analyte (100 µM) under a UV lamp, corresponding to Figure 2a, were shown in Figure 2c. Fluorescence responses of 10 µM 1 to Cys, Hcy, or GSH in the presence of other 19 AAs (each at 100 µM) were also measured (Figure 2b), and the results indicated that the sensing of Cys, Hcy, or GSH was hardly influenced by these analytes (29.3 vs. 24.5, 11.4 vs. 10.7, or 16.5 vs. 14.3 folds of fluorescence enhancement for Cys, Hcy, or GSH in the absence/presence of other 19 AAs, respectively).

Figure 2. (a) The fluorescence spectra of 1 (10 µM, λex = 350 nm) in the presence of Cys, Hcy, GSH and other 19 amino acides (100 µM) in phosphate buffer (10 mM, pH 7.4) at 37 °C for 2 h. (b) Fluorescence responses of 1 (10 µM) toward different analytes. Mix represents other 19 natural AAs except Cys (each at 100 µM). (c) The corresponding fluorescent photographs of 1 (10 µM) in a cuvette in the presence of different AAs (100 µM) under a UV lamp.

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To further confirm the abovementioned fluorescence emissions are indeed from the three products proposed in Figure 1, we synthesized Aminoluciferin and CBTHcy and studied their fluorescence properties together with CBT. Our results indicated that the fluorescence emissions of as-synthesized Aminoluciferin, CBTHcy, or CBT center at 522, 517, or 490 nm (Figure S15, Supporting Information), which are consistent with the abovementioned emissions of 1 treated with Cys, Hcy, or GSH, respectively. Besides, effects of pH values on the quantum yields of as-synthesized Aminoluciferin, CBTHcy, or CBT were also studied and the results indicated that, at physiological conditions (i.e., buffers at pH 7.4), these three compounds have the highest fluorescence intensities (Figure S16, Supporting Information). Additionally, the photobleaching behavior of CBT, CBTHcy and Aminoluciferin have been also investigated, showing that CBT undergo slower bleaching rate than that of Aminoluciferin or CBTHcy (Figure S17, Supporting Information). After confirmation of the final products, we started to monitor the chemical reaction processes of 1 with Cys, Hcy, or GSH using HPLC and mass spectrometry. Firstly, during the incubation of 10 µM 1 with 1 mM Cys at 37 °C, we injected the incubation mixture into a HPLC system for analysis. Clearly we found that the HPLC peak of 1 at rentention time of 29.9 min disappeared within 5 min and a new peak at rentention time of 26.6 min appeared, corrsponding to the condensation product of 1 with Cys (i.e., A, Figures S1&2, Supporting Information). 5 min later, time course HPLC analysis indicated that the peak of Aminoluciferin at retention time of 11.2 min increased with the peak of A decreased, demonstrating that excessive Cys starts to cleave the sulfonamide bond of A to yield the final product Aminoluciferin (Figure S1b, Supporting Information). This indicates that the condensation reaction between 1 and Cys is prior to its sulfonamide reduction by Cys (Figure S1a, Supporting Information). Similarly, we found that 1 is prone to condensing with Hcy to yield the intermediate B, followed by sulfonamide-reduction of B by Hcy to yield the final product CBTHcy, as shown in Figures S3&4. Interestingly, incubation of 1 with GSH only resulted the sulfonamide-reduction product CBT, since the CBT moiety does not condense with GSH (Figure S5, Supporting Information). Secondly, we studied the kinetic constants of the condensation reactions between Cys (or Hcy) and CBT, as well as those of reductions between 1 and Cys, Hcy, or GSH. For second-order condensation reactions, 200 µM CBT and 200 µM Cys (or Hcy) were added in phosphate buffer (0.2 M, pH = 7.4) and the reaction mixture was monitored with HPLC at different time points (Figures S6a&7a, Supporting Information). We plotted 1/[CBT] vs. reaction time (Figures S6b&7b, Supporting Information) and the linear regression analyses give the second-order rate constant value of 26.8 M-1·S-1 (formula (4) in Supporting Information) for Cys condensation or 13.6 M-1·S-1 for Hcy condensation with 1 (formula (5) in Supporting Information), which is hundred-fold larger than that of a typical copper-free [3 + 2] azide-alkyne click reaction.36 Then we studied the pseudo first order reaction constants of 10 µM 1 treated with 1 mM Cys, Hcy, or GSH. For 1 with Cys, using the time course HPLC traces in Figure S1b, we were able to construct a Ln[A] vs. time curve (Figure S1c). Linear regression analysis of Ln[A] vs. time gave the pseudo first order reaction constant 2.29 × 10-2 min-1 of 1 with Cys (formula (1) in Supporting Information). Similarly, the pseudo first order reaction constant of 1 with Hcy was calculated to be 7.94 × 10-3 min-1 (Figure S3c

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and formula (2), Supporting Information). For 1 with GSH, linear regression analysis of Ln[1] vs. time gave the pseudo first order reaction constant of 7.41 × 10-3 min-1 (Figure S5c and formula (3), Supporting Information). From these results above, we can conclude that, at this reaction condition, condensation reaction of 1 is much faster than its reduction and therefore the later should be the rate-determining step (RDS). Among the pseudo first order reduction constants, that of Cys (2.29 × 10-2 min-1) is the biggest one while those of Hcy and GSH are much smaller and close to each other (7.94 × 10-3 min-1 vs. 7.41 × 10-3 min-1), suggesting that probe 1 is more specific for sensing of Cys.

Figure 3. (a, c, e) Fluorescence spectra of 1 (10 µM, λex = 350 nm) in the presence of various concentrations of Cys, Hcy, or GSH in phosphate buffer (10 mM, pH 7.4) at 37 °C for 2 h, respectively. (b, d, f) Fitted calibration curve of the fluorescence intensities at 522 nm in Figure 3a, at 517 nm in Figure 3c, or at 490 nm in Figure 3e as a function of Cys, Hcy, or GSH concentrations, respectively. The inset fluorescent photographs show the fluorescence changes of 1 at 10 µM before and after addition of 100 µM corresponding thiols under a UV lamp.

Discriminative Sensing of Cys, Hcy, or GSH in Vitro with 1. We then evaluated the capability of 1 for qualitative and quantitative sensing of Cys, Hcy, or GSH. The fluorescence responses of 1 with different concentrations of Cys, Hcy, or GSH and their linear relationships are shown in Figure 3. Fluorescence spectra of 1 at 10 µM (λex = 350 nm) in the presence of various concentrations of Cys, Hcy, or GSH (0 - 2 mM for Cys, 0 - 4 mM for Hcy, 0 - 2 mM for GSH) in phosphate buffer (10 mM, pH 7.4) at 37 °C for 2 h are shown in Figures 3a, c, and e. With increasing concentrations of Cys, Hcy, or GSH, the spectra clearly showed gradual increase of emission intensity at 522, 517, or 490 nm and eventually reached 196-fold, 74-fold, or 47-fold of their originals, respectively. By correlating the value of the fluorescence intensities with the concentrations of the thiols, we constructed calibration curves for the determination of Cys, Hcy, or GSH

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Figure 4. (a) Confocal microscopic images of HepG2 cells. Fluorescence microscopic cell images were obtained using an excitation wavelength of 405 nm and two band-path (490 ± 8 and 522 ± 8 nm) emission filters. First row: Differential interference contrast (DIC) and fluorescence images of HepG2 cells incubated with 40 µM 1 for 30 min. Second row: DIC and fluorescence images of HepG2 cells pretreated with 2 mM maleimidobutyric acid (MBA) for 30 min and then incubated with 40 µM 1 for 30 min. Third row: DIC and fluorescence images of HepG2 cells pretreated with 2 mM MBA for 30 min, added with 10 mM cysteine, and then incubated with 40 µM 1 for 30 min. Fourth row: DIC and fluorescence images of HepG2 cells pretreated with 2 mM MBA for 30 min, added with 10 mM GSH, and incubated with 40 µM 1 for 30 min. All the cells were washed with PBS for three times at each step prior to imaging. Scale bar: 20 µm. (b) The fluorescence ratio between the intensities of the 490 ± 8 and 522 ± 8 bands (R522/490).

in phosphate buffer. As shown in Figure 3b, a linear relationship between the value of the fluorescence intensity at 522 nm with Cys concentration (Y = 9.24507 + 4.67271X, R2 = 0.989) was obtained over the range of 0 - 200 µM. The limit of detection (LOD) of Cys in this assay was 0.518 µM (S/N = 3). For Hcy detection, as shown in Figure 3d, the linear regression equation (Y = 4.61996 + 1.57933X, R2 = 0.995) was obtained over the range of 0 - 500 µM, with a limit of detection (LOD) of 0.658 µM (S/N = 3). Similarly, as shown in Figure 3f, a linear relationship between the value of the fluorescence intensity at 490 nm with GSH concentration (Y = 24.86488 +2.75745X, R2 = 0.999) was obtained over the range of 0 - 100 µM. The limit of detection (LOD) of GSH in this assay was 0.246 µM (S/N = 3). Therefore, probe 1 can be used for highly sensitive detection of biothiols in vitro, compared with those fluorescence probes recently reported (Table S2, Supporting Information). Discriminative Sensing of Cys and GSH in Living Cells. To determine the biological relevance of 1, we applied 1 for fluorescence imaging of intracellular thiols. Before that, we measured the water solubility of 1 at 20 °C to be 54 µg/mL (134 µM). Then we studied the cytotoxicity of 1. 3-(4,5dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide (MTT) assay indicated, after being incubated with 1 at 20 µM, 40 µM, or 80 µM for 2 h, 107%, 104%, or 96% of the HepG2 cells survived respectively (Figure S18), suggesting 1 is safe for HepG2 cell imaging. When the cells were incubated with 20 µM, 40 µM, or 80 µM 1 for 8 h, 99%, 100% or 88% of the cells survived respectively, suggesting 1 is not toxic to the cells until 8 h (Figure S18). As shown in Figure S19, after the healthy HepG2 cells were incubated with 10, 20, or 40 µM of

1 in serum-free culture medium at 37 °C for 0.5 h and washed three times with phosphate buffered saline (PBS, pH 7.4) prior to imaging, increased blue green fluorescence from intracellular compartment could be observed with the increase of the concentrations of 1, suggesting that 1 is cell-permeable and react with intracellular thiols to turn “on” the fluorescence. Since Hcy is hardly detectable in healthy mammalian cell, thus the above fluorescence should be ascribed to the products of 1 that reacts with Cys or GSH. To quantitatively analyze the fluorescence emission from Aminoluciferin (i.e., product of 1 reacts with intracellular Cys, λem = 522 nm) and CBT (i.e., product of 1 reacts with intracellular GSH, λem = 490 nm), we conducted the following cell imaging experiments with a confocal fluorescence microscopy equipped with two bandpath (490 ± 8 and 522 ± 8 nm) emission filters. As shown in Figure 4, after the HepG2 cells were incubated with 40 µM 1 at 37 °C for 30 min, bright fluorescence images of the cells from both of these two channels (490 ± 8 nm and 522 ± 8 nm) were clearly observed, with the fluorescence intensity from channel of 522 ± 8 nm (Cys reacts with 1) much higher than that from 490 ± 8 nm channel (GSH reacts with 1) (first row of Figure 4a). Using Image J, we calculated the and fluorescence ratio between the intensities in the bands of 522 ± 8 nm and 490 ± 8 nm (R522/490) to be 12.5 ± 3.6 (Figure 4b). When the cells were pretreated with 2 mM thiol-reactive maleimidobutyric acid (MBA) for 30 min to consume all of the free thiols within the cells and then incubated with 40 µM 1 for 30 min, neither of the two channels show detectable fluorescence images of the cells (second row of Figure 4a), suggesting the above fluorescence cell images are induced by the turned “on” fluorescence of 1 that reacted with

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intracellular thiols. To further validate whether the above fluorescence cell images were induced by Cys-dominating condensation and reduction of 1, or by GSH-dominating reduction of 1, we pretreated the HepG2 cells with MBA, then respectively incubated them with additional Cys or GSH, and quantitated the fluorescence ratios R522/490 between the two channels. In detail, as shown in the third and the forth rows of Figure 4a, after the cells were pretreated with 2 mM MBA for 30 min, the cells were added with 10 mM cysteine (or GSH), and then incubated with 40 µM 1 for 30 min prior to imaging. Quantitative analyses indicated that cells treated with Cys addition have a fluorescence ratio R522/490 of 11.8 ± 3.0 between these two channels while the cells treated with GSH addition have a R522/490 ratio of 9.1 ± 1.4, and the difference between the ratios is obvious (p = 0.005) (Figure 4b). From comparison, we know that the R522/490 ratio of HepG2 directly incubated with 1 (i.e., 12.5 ± 3.6) is close to that of cells treated with Cys addition (i.e., 11.8 ± 3.0) and the difference between the ratios is not obvious (p = 0.279), but is far away from that of cells treated with GSH addition (i.e., 9.1 ± 1.4) and difference between the ratios is obvious (p = 0.002) (Figure 4b). Therefore, we can conclude that fluorescence from 1-treated HepG2 cells (i.e., first row of Figure 4a) is induced by intracellular Cys-dominating condensation and reduction of 1, but not by intracellular GSH-dominating reduction of 1. This echoes well with above kinetic study which shows that the reaction between 1 and Cys is much faster than that of 1 with GSH. Cell imaging study suggests that our probe 1 could be applied for discriminative sensing of intracellular Cys from the more abundant intracellular GSH. CONCLUSIONS In conclusion, by rational design of sulfonamide-based selfquenched fluorescent probe 1, we report for the first time using 1 for discriminative sensing of biothiols in vitro and in cells. Condensation of 1 with Cys (or Hcy), followed by Cys (or Hcy)-cleavage of the sulfonamide bond of 1 yields Aminoluciferin (λem = 522 nm) or CBTHcy (λem = 517 nm) with different fluorescence emission peaks. And GSH-reduction of 1 yields CBT (λem = 490 nm) with another fluorescence emission. Kinetic study indicated that 1 has much higher pseudo first order reduction constant with Cys (2.29 × 10-2 min-1) than those of Hcy (7.94 × 10-3 min-1) or GSH (7.41 × 10-3 min-1). With these unique properties of 1, we were able to apply 1 for highly sensitive sensing of Cys, Hcy, and GSH among other 19 natural AAs with good selectivity. Using a confocal fluorescence microscopy equipped with two band-path (522 ± 8 nm and 490 ± 8) emission filters, we conducted fluorescence imaging of living HepG2 cells with 1 and quantitate the fluorescence ratio between these two channels (R522/490) with Image J. By comparing the R522/490 value with those of Cys (or GSH)-addition cell imaging experiments, we found that the fluorescence from 1-treated HepG2 cells was induced by intracellular Cys-dominating condensation and reduction of 1, but not by intracellular GSH-dominating reduction of 1. The cell imaging results well echo with the kinetic study, suggesting that our probe 1 could be applied for discriminative sensing intracellular Cys from the abundant intracellular GSH. Our results strongly suggest that the new probe 1 might be developed as an efficient tool for probing cellular functions that relate to biothiols.

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ASSOCIATED CONTENT Supporting Information Additional experimental details as described in text. Synthetic routes for 1, Aminoluciferin, CBTHcy; HPLC monitoring of the reactions of 1 with Cys, Hcy or GSH; Kinetic Studies of the reaction of free CBT with Cys or Hcy; Scheme S1-3; Figure S1-S19, Table S1-S2. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author E-mail: [email protected] (G.-L. L.).

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by Collaborative Innovation Center of Suzhou Nano Science and Technology, the Major Program of Development Foundation of Hefei Center for Physical Science and Technology, and the National Natural Science Foundation of China (Grants 21175122 and 21375121).

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