A Triple-Emission Fluorescent Probe for Discriminatory Detection of

Subscriber access provided by Kaohsiung Medical University

Article

A triple-emission fluorescent probe for discriminatory detection of cysteine/ homocysteine, glutathione/hydrogen sulfide and thiophenol in living cells Lei Yang, Yuanan Su, Yani Geng, Yun Zhang, Xiaojie Ren, Long He, and Xiangzhi Song ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00685 • Publication Date (Web): 22 Aug 2018 Downloaded from http://pubs.acs.org on August 22, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors

A triple-emission fluorescent probe for discriminatory detection of cysteine/homocysteine, glutathione/hydrogen sulfide and thiophenol in living cells Lei Yang, Yuanan Su, Yani Geng, Yun Zhang, Xiaojie Ren, Long He and Xiangzhi Song * College of Chemistry & Chemical Engineering, Central South University, Changsha 410083, China. Email: [email protected]. KEYWORDS ：triple-emission, fluorescent probe, thiols, discriminatory, imaging ABSTRACT: Thiols, such as cysteine (Cys), homocysteine (Hcy), glutathione (GSH), hydrogen sulfide (H2S) and thiophenol are metabolically correlated with each other via redox reactions. Due to the similarity of chemical properties between Cys, Hcy, GSH, H2S and thiophenol, it’s very challenging to develop an effective methodology to differentiate them. In this work, a triple-emission fluorescent probe, NCQ, was reported for the simultaneous detection Cys/Hcy, GSH/H2S and thiophenol with high sensitivity and selectivity. The solution of NCQ displayed distinct fluorescent signals toward Cys/Hcy, GSH/H2S and thiophenol: blue and green for Cys/Hcy, blue for GSH/H2S, blue and red for thiophenol. Through the blue-green-red emission color combination, Cys/Hcy, GSH/H2S and thiophenol could be discriminatively detected in solution and in living cells.

Intracellular thiols including cysteine (Cys), homocysteine (Hcy), glutathione (GSH) and hydrogen sulfide (H2S) play pivotal roles in many physiological processes. For examples: GSH, a thiol containing tripeptide, is the most important endogenous regulator of apoptosis and antioxidant;1, 2 Cys is essentially involved in the stabilization of protein structures;3 Hcy plays an essential role in cellular homeostasis through metabolic correlation with amino acid methionine;4 endogenous H2S has been proved to be as neuromodulator, cytoprotectant, neuroprotectant, apoptosis, anti-inflammation agent, antioxidant, and messenger to inhibit insulin.5-8 As a consequence, the aberrant levels of these biothiols can lead to various health problems such as preeclampsia, Parkinson’s diseases, cancer, premature delivery, HIV, liver damage and dementia.9-11 In addition, thiophenols are important for organic synthesis, and are widely used to produce pharmaceuticals and agrochemicals.12 However, thiophenol is very toxic, and the media lethal concentration (LC50) for fish is between 0.01 mM and 0.4 mM.13 And the exposure to thiophenol may cause coughing, nausea, vomiting, headache, burning sensation, and even death by damaging central nervous and other biological systems.14-17 Current research have found that GSH, Cys, Hcy, H2S are metabolically correlated with each other in the redox regulation of physiological processes through thiol-disulfide exchanges. Hcy can be catabolized in the transsulfuration pathway via cystathionine to cysteine.18 Cys is the precursor in the biosynthesis of GSH.19 Endogenous H2S is mainly produced from Cys and Hcy with the enzymatic catalysation.20 On the other hand, GSH serves as the intracellular reservoir of Cys.21 Under physiological condition, intracellular thiophenols can readily undergo autoxidation to form diphenyl disulphides

which are subsequently reduced by GSH back to thiophenols. In such process, the concentration of intracellular GSH is decreased and active oxygen species are generated .14, 16 Hence, the development of effective methodologies that can selectively distinguish these above thiols is very important. Fluorescent probes for the detection and imaging of biological species have drawn much attention due to their advantages such as high selectivity, good sensitivity, real-time sensing and easy operation.22 Many fluorescent probes have been designed for detecting thiols by utilizing the strong nucleophilicity of thiols.23-29 While being useful, most of these probes cannot differentiate Cys, Hcy, GSH, H2S and thiophenol from each other because of their similar chemical properties: all of these thiols have a nucleophilic sulfydryl group (–SH); and their pKa is in the order of H2S (6.9),30 thiophenol (7.8),31 Cys (8.3),31 Hcy (8.9),32 and GSH (9.2)33. There are some fluorescent probes reported for singly selective detection of Cys, Hcy, GSH, H2S or thiophenol (Table S1),34-43 however, few of them can simultaneously differentiate these above-mentioned thiols. Herein, we deliberately integrated two sensing groups, 2, 4-dinitrobenzene (DNB) and 7-nitro-1,2,3-benzoxadiazole (NBD), and two fluorophores into one molecule through ether bonds to design and synthesize a fluorescent probe, NCQ, to simultaneously detect Cys/Hcy, GSH/H2S, and thiophenol (Scheme 1). The synthetic route of NCQ was outlined in Scheme 2. The intermediates and their analogues were prepared according to the literature methods (Figure S1). DNB has been widely used as a recognition group for thiophenols and NBD serves as both a sensing group and a fluorophore to differentiate Cys/Hcy over GSH/H2S. The

ACS Paragon Plus Environment

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

fluorescence of NCQ would be remarkably quenched by DNB and NBD moieties. There are two reaction sites in NCQ. For site 1, the NBD ether bond can be cut off by all the five thiol substrates to release the blue-emitting CQ and the non-fluorescent sulfur-substitued NBD intermidiates (NBD-S-Cys/Hcy, NBD-S-GSH/NBD-SH and NBD-SPh). Subsequently, NBD-S-Cys/Hcy will undergo an intramolecular Smiles rearrangement to form greenfluorescent amino substituted NBD fluorophores (NBD-NCys/Hcy). However, NBD-S-GSH/NBD-SH and NBD-SPh are stable. In contrast, site 2 is only reactive to thiophenol and inert to Cys/Hcy/GSH/H2S. Thiophenol can cleave both of the DNB and NBD ether bonds to release of blueemitting coumarin 1 and to subsequently form the redemitting TQC. As a result, three sets of distinct fluorescent signals can be obtained when NCQ reacts with respective thiols: blue and green for Cys/Hcy, blue for GSH/H2S, blue and red for thiophenol. Therefore, the simultaneous differentiation of Cys/Hcy, GSH/H2S and thiophenol can be realized by a single-molecule probe.

Page 2 of 8

(200-300 mesh, Qingdao Ocean Chemicals, China). Deionized water was used in all experiments. All the optical measurements except pH study in solutions were performed in PBS buffer (10 mM, pH = 7.4, containing 30% acetonitrile). Scheme 2. Synthetic route of NCQ. (a) TsOH, toluene, reflux. (b) Piperidine, dichloromethane/EtOH (1:1), 45 °C. (c) Acetone, K2CO3, rt. HO

O

HO

O

O

O

a O OH

NC

CN

O

OH

O

1

2 NO2 NO2

HO

O

NO2

O NO2 +

O O

N

b

N

O

N

CHO

O

N

CN

O O

2

O NO2

Scheme 1. The sensing mechanism of NCQ for distinguishing Cys/Hcy, GSH/H2S and thiophenol.

N

N

N

O

PhSH

NCQ

O O

O GSH/H2S

NO2

624 nm

NO2 490 nm NO2

490 nm

N

OH

N

O

O

CN

CN TQC

CN N

O

O O

+

HO

O

490 nm O

O

O

+ CQ NO2

N

O

CQ

NO2

O Coumarin 1 +

N

544 nm O

O

N N SR SH n

n=1: NBD-N-Cys n=2: NBD-N-Hcy

OH

NO2

NO2

SPh

SPh

N

NH HOOC

O

NO2 N

+

O HOOC

H N

NH2

N H O

NO2

N

O

R=

O

O

NCQ

O

O

NO2

N

N

NO2

O O

O

OH

O

N

Group for biothiols

CN

O

O CN

O NO2

N

N

NO2 N

Site 1

Group for thiophenol

Cys/Hcy

N

l -C BD

O

Site 2

N

O

CQ

3 c

NO2

OH CN

NBD-S-GSH

COOH

R= H

NBD-SH

NBD-SPh

DNB-SPh

EXPERIMENTAL SECTION Materials and Instruments. Absorption spectra were carried out on a Shimadzu UV-2450 spectrometer. And the emission spectra were performed on an F-280 (GANGDONG, TIANJIN) spectrometer. 1H NMR and 13C NMR spectra were recorded on a Bruker 400/500 MHz spectrometer with tetramethylsilane as an internal standard. High-resolution mass spectrometry (HRMS) was performed on a Bruker Daltonics MICROTOF-Q II mass spectrometer. A Shimadzu LC-16 series instrument was used to conduct High performance liquid chromatography (HPLC) studies. pH values of the aqueous media in pH studies were measured with a Leici PHS-3C meter. Confocal laser microscopic (CLSM) images were acquired by an Olympus FV 1000-IX81 (Japan) confocal laser scanning microscope. HeLa cells were obtained from Xiangya Hospital at Central South University (Changsha, China). All commercial reagents and solvents were used as received. Column chromatography was conducted over silica gel

Synthesis of Compounds 1 and 3. The synthesis of compounds 1 and 3 was described in the literature.39, 44 Synthesis of Compound 2. Compound 1 (500 mg, 2.6 mmol), cyanoacetic acid (440 mg, 5.2 mmol) and ptoluenesulfonic acid (45 mg, 0.26 mmol) were dissolved in 15 mL toluene. The reaction mixture was refluxed for 6 hours. Then, cool the reaction mixture to room temperature and filtrate to obtain a solid, which was washed with dichloromethane to afford compound 2 as a cream-colored solid (490 mg, 73%). HRMS (ESI) m/z: [M-H] calcd for C13H8NO5, 258.0408; found, 258.0420. 1H NMR (400 MHz, DMSO-d6): δ 7.59 (d, J = 8.7 Hz, 1H), 6.81 (dd, J = 8.7, 2.1 Hz, 1H), 6.76 (d, J = 2.1 Hz, 1H), 6.31 (s, 1H), 5.44 (s, 2H), 4.23 (s, 2H), 3.86 (s, 1H). 13C NMR (100 MHz, DMSOd6): δ 166.2, 164.5, 161.9, 160.6, 155.5, 150.1, 126.5, 113.6, 109.4, 108.6, 102.9, 63.2, 25.0. Synthesis of Compound CQ. The solution of compound 3 (101 mg, 0.25 mmol) and piperidine (20 μL) in dichloromethane (7.5 mL) was added into a solution of compound 2 (97 mg, 0.37 mmol) in absolute ethanol (7.5 mL). The obtained reaction mixture was heated at 45 °C for 10 hours. Distill the solvent and the residue was purified by silica gel chromatography (eluent: dichloromethane/ethyl acetate = 15:1 and dichloromethane/methanol = 20:1, v/v) to afford pure CQ as a red solid (50 mg, 30%). HRMS (ESI) m/z: [M-H] calcd for C32H26N5O10, 640.1684; found, 640.1666. 1H NMR (400 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.83 (d, J = 2.4 Hz, 1H), 8.42 (dd, J = 9.3, 2.4 Hz, 1H), 8.14 (s, 1H), 7.62 (s, 1H), 7.52 (d, J = 9.3 Hz, 1H), 7.14 (d, J = 9.3 Hz, 1H), 6.82-6.67 (m, 2H), 6.61 (s, 1H), 6.12 (s, 1H), 5.42 (s, 2H), 3.59 (s, 2H), 3.46-3.39 (m, 2H), 1.23 (s, 4H), 1.18 (t, J = 7.0 Hz, 3H), 1.06 (t, J = 6.9 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.3, 161.9, 160.5, 156.1, 155.5, 150.5, 150.0, 145.3, 144.3, 141.6,

ACS Paragon Plus Environment

2

Page 3 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors 138.9, 133.0, 130.3, 126.4, 122.3, 118.6, 118.0, 113.5, 110.5, 109.5, 108.6, 106.6, 102.9, 101.9, 91.3, 62.9, 47.6, 45.9, 45.3, 44.3, 11.2, 9.9. Synthesis of NCQ. Compound CQ (64 mg, 0.1 mmol), NBDCl (40 mg, 0.2 mmol) and K2CO3 (60 mg, 0.4 mmol) were added into 10 mL acetone in a flask. The obtained suspension was stirred at 25℃ for 3 hours. Next, distill the solvent under vacuum and the obtained residue was purified by silica gel chromatography (eluent: dichloromethane/ethyl acetate = 20:1, v/v) to give NCQ (21 mg, 26%) as a fuchsia solid. HRMS (ESI) m/z: [M-H] calcd for C38H27N8O13, 803.1698; found, 803.4059. 1H NMR (400 MHz, DMSO-d6) δ 8.86 (d, J = 2.8 Hz, 1H), 8.67 (d, J = 8.3 Hz, 1H), 8.43 (dd, J = 9.3, 2.8 Hz, 1H), 8.17 (s, 1H), 7.93 (d, J = 8.8 Hz, 1H), 7.65-7.57 (m, 2H), 7.40 (dd, J = 8.8, 2.4 Hz, 1H), 7.16 (d, J = 9.3 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.63 (s, 1H), 6.42 (s, 1H), 5.54 (s, 2H), 3.59 (d, J = 4.9 Hz, 2H), 3.43 (dd, J = 14.1, 7.0 Hz, 2H), 1.31-1.22 (m, 4H), 1.18 (t, J = 7.0 Hz, 3H), 1.06 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, DMSO-d6) δ 163.3, 161.9, 160.5, 156.1, 155.5, 150.5, 150.0, 145.3, 144.3, 141.6, 138.9, 133.0, 130.3, 126.4, 122.3, 118.7, 118.0, 113.5, 110.5, 109.5, 108.6, 106.6, 102.9, 101.9, 91.4, 62.9, 47.6, 45.9, 45.3, 44.3, 34.1, 31.8, 29.5, 29.2, 22.6, 14.4, 11.2, 10.0. Synthesis of the Reaction Products of NCQ with Thiols. TQC, NBD-SPh, NBD-SH and DNB-SPh were synthesized according to the literature methods.45-48 The solutions of NBD-N-Cys/Hcy, NBD-S-GSH in this work were prepared from NBD-Cl with Cys, Hcy and GSH in aqueous solution (containing 30% acetonitrile).30 Cell Culture and Imaging Experiments. HeLa cells was grown at 37 °C for 24 h in DEME medium with a supplement containing 10% fetal bovine serum and 1% penicillin under an atmosphere containing 5% CO2. Discard the nutrition medium and wash cells with PBS buffer 3 times. Then, divide cells into six groups. The first five groups of cells were treated with NEM (Nethylmaleimide, 1.0 mM) for 30 min, washed 3 times with PBS buffer. Next, four groups of NEM-pretreated cells were incubated respectively with Cys, GSH, H2S, thiophenol (500 μM) for 30 min, then washed 3 times with PBS, and finally incubated with NCQ (10.0 μM) for 30 min. One group of NEM-pretreated cells were incubated with NCQ (10.0 μM) for 30 min as a control. The last group of cells was only incubated with NCQ (10 μM) for 30 min. Cells were washed with PBS buffer before imaging experiments.

linear relationships were obtained between the fluorescence intensities of NCQ (10 μM) and the concentration of these thiols within a certain range (10 μM for Cys, 10 μM for Hcy, 6 μM for GSH, 8 μM for H2S and 70 μM for thiophenol), indicating that NCQ could quantitatively detect thiols. Moreover, the fluorescence intensity reached a plateau when the solution of NCQ (10 μM) was treated with 15 μM Cys, 30 μM Hcy, 10 μM GSH, 15 μM H2S or 70 μM thiophenol. The detection limit (S/N = 3) of NCQ for Cys, Hcy, GSH, H2S and PhSH were estimated to be 0.57 μM, 0.65 μM, 0.49 μM, 0.52 μM and 0.34 μM, respectively. Moreover, the absorption spectral changes were also studied when NCQ was treated biothiols and thiophenol, respectively. (Figure S2).

RESULTS AND DISCUSSION Sensing Properties of NCQ toward Thiols. The fluorescence responses of NCQ toward H2S, biothiols and thiophenol were studied. NCQ was non-fluorescent. In the presence of Cys/Hcy, the solution of NCQ showed a strong fluorescence in blue and green channels with two maximums at 490 nm and 544 nm under an excitation at 423 nm (Figure 1). In contrast, only blue fluorescence with λmax = 490 nm was observed when the solution of NCQ was in the presence of GSH/H2S under the excitation at 423 nm. With regard to thiophenol, blue and red fluorescence with maxima at 490 nm and 624 nm was observed under a 432 nm excitation. As seen in Figure 1 (A2-E2), the fluorescence signals of NCQ increased with increasing the concentration of these thiols, and good

Figure 1. Concentration-dependent fluorescence of NCQ with Cys (A), Hcy (B), GSH (C), H2S (D), PhSH (E). First column: fluorescence spectra; second column: plot of fluorescence intensities as a function of the concentration of thiols; inset: the linear correlation between the fluorescence intensity at respective wavelength (490 nm, 544 nm and 624 nm) and the low concentration of thiols.

ACS Paragon Plus Environment

3

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Kinetic Studies. Time-dependent fluorescence experiments were conducted on NCQ (10 μM) with thiols (Figure 2). The solution of NCQ (10 μM) with 15 μM Cys displayed two emissions with maxima at 490 nm and 544 nm, whose intensities were maximized within 5 and 10 min, respectively. The longer respond time for the formation of green fluorescence also supported the fact that NBD-N-Cys was formed from NBD-S-Cys through Smiles rearrangement reaction. Similar to Cys, the maximized fluorescence signals at 490 nm and 544 nm for Hcy (30 μM) were obtained within 15 and 20 min, respectively. In the case of GSH (10 μM) and H2S (15 μM), blue fluorescence signal at 490 nm levelled off within 5 min and 15 min. The addition of thiophenol to the solution of NCQ quickly induced blue (λmax = 490 nm) and red (λmax = 624 nm) fluorescence, both of which were maximized within 20 min. Without these thiols, however, the fluorescence of NCQ was negligible during the same interval.

Figure 2. Fluorescence intensities at 490 nm, 544 nm and 624 nm for NCQ with/without of Cys (15 μM), Hcy (30 μM), GSH (10 μM), H2S (15 μM), PhSH (70 μM) as a function of time. Excitation wavelength: 423 nm for Cys, Hcy, GSH and H2S, 432 nm for PhSH.

Selectivity Studies. The selectivity of NCQ was performed by investigating its fluorescence in the presence of various relevant biological amino acids (Ala, Phe, Met, Gly, Glu, Arg, Lys, Tyr, Leu, Pro, Trp, Ser), anions (PO43-, S2O32-, SCN-, SO42-, Cl-, CO32-, NO2-, SO32-, NO3-, AcO-, N3-, I-), cations (Na+, K+, Ca2+, Mg2+) and reactive oxygen species (H2O2 and ClO-). As depicted in Figure 3, only Cys/Hcy, GSH/H2S and thiophenol induced strong distinct fluorescence signals. However, other interfering species hardly caused any fluorescence change. These results suggested that NCQ was selective for the simultaneous detection of Cys/Hcy, GSH/H2S and thiophenol. Mechanism Studies. NBD-N-Cys/Hcy, NBD-S-GSH, NBDSPh, NBD-SH, TQC and coumarin 1 were prepared and their absorption and emission spectra were measured (Figures S3-S5). In the same media, coumarin 1, NBD-NCys/Hcy and TQC exhibited emissions with maxima at 490 nm, 544 nm and 624 nm, respectively (Figure S4). Furthermore, the lifetimes (τ) of NCQ in the presence of Cys, GSH and thiophenol and the expected products were

Page 4 of 8

determined (Figure S6). The lifetimes of coumarin 1, NBDN-Cys and TQC were 5.91 ns, 2.26 ns and 1.67 ns, respectively. The solution of NCQ with Cys displayed two lifetimes (τ1 = 5.96 ns and τ2 = 2.43 ns) which were almost equal to the lifetimes of coumarin 1 (τ = 5.91 ns) and NBDN-Cys (τ = 2.26 ns). The mixture of NCQ with GSH only showed a single lifetime (τ = 5.83 ns), indicating the formation of coumarin 1. Two lifetimes, τ1 = 5.97 ns and τ2 = 1.75 ns, were observed for NCQ with PhSH, suggesting the formation of coumarin 1 (τ = 5.91 ns) and TQC (τ = 1.67 ns). The optical changes (absorption, fluorescence and lifetime) of NCQ with thiols supported our proposed sensing mechanism.

Figure 3. Fluorescence behavior of NCQ (10 μM) in the presence of (1) Ala, (2) Phe, (3) Met, (4) Gly, (5) Glu, (6) Arg, (7) Lys, (8) Tyr, (9) Leu, (10) Pro, (11) Trp, (12) Ser, (13) PO43-, (14) S2O32-, (15) SCN-, (16) SO42-, (17) Cl-, (18) CO32-, (19) NO2-, (20) SO32-, (21) NO3-, (22) AcO-, (23) N3-, (24) I-, (25) Na+, (26) K+, (27) Ca2+, (28) Mg2+, (29) H2O2, (30) ClO-, (31) PhSH, (32) Cys, (33) Hcy, (34) GSH, (35) H2S. Concentration: 1 mM for (1)-(30), 70 μM for (31), 15 μM for (32), 30 μM for (33), 10 μM for (34) and 15 μM for (35). (a): Emission spectra. (b), (c) and (d): Fluorescence intensity at 490 nm, 544 nm, and 624 nm.

To further prove the sensing mechanism of NCQ toward these thiols, HPLC analysis was carried out on NCQ, the reaction mixtures of NCQ with thiols (Cys/Hcy, GSH/H2S and thiophenol) and the expected products including CQ, TQC, coumarin 1 and other substituted compounds. HPLC chromatograms showed that NCQ, CQ and NBD-N-Cys displayed single peaks at 15.0, 9.1 and 4.3 min, respectively. When 0.5 or 1.0 equiv. of Cys was added into the solution of NCQ, the height of the peak at 15.0 min decreased and two new peaks at 9.1 min and 4.3 min occurred (Figure 4). When excessive Cys (1.5 equiv.) was added, the peak at 15.0 min disappeared and only two peaks at 9.1 min and 4.3 min were left. Similarly, HPLC chromatograms of the solution of NCQ with Hcy, GSH, H2S and thiophenol displayed expected peaks corresponding to the proposed reaction products (Figures S7-S10). Moreover, mass spectra of the mixtures of NCQ with thiols were obtained. As seen in Figure S11, the mixture of NCQ with Cys exhibited two peaks at 640.1686 and 283.0141, which were nearly identical to the exact molecular weights of the expected reaction products CQ (m/z (M-H): 640.1684) and NBD-N-Cys (m/z (M-H): 283.0143). Similarly, two peaks at 640.1671 and 297.0287 were found

ACS Paragon Plus Environment

4

Page 5 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors in the mass spectra of the reaction mixture of NCQ with Hcy, corresponding to CQ (m/z (M-H): 640.1684) and NBD-N-Hcy (m/z (M-H): 297.0294) (Figure S12). As expected, the signals assigned to NBD-S-GSH (m/z for (MH): calcd 469.0778, found 469.0783) and CQ (m/z for (MH): calcd 640.1684, found 640.1675) were also found in the reaction mixture of NCQ with GSH (Figure S13). For H2S, the peaks of NBD-SH (m/z for (M-H): calcd 195.9817, found 195.9835) and CQ (m/z for (M-H): calcd 640.1684, found 640.1683) appear in the reaction solution (Figure S14). In the reaction mixture of NCQ with thiophenol, the supposed products, TQC (m/z: calcd 283.1321, found 283.1271) and coumarin 1 (m/z for (M+H): calcd 193.0501, found 193.0474) were clearly observed (Figure S15). Therefore, HPLC and mass spectral analysis clearly supported the sensing mechanism (Scheme 1).

NCQ was used to culture cells for 24 h at 37°C (Figure S16), indicating the less cytotoxicity of NCQ. Then, we set out to conduct the fluorescence imaging experiments. Cells incubated with NCQ (10 μM), exhibited strong blue fluorescence and very weak green fluorescence (Figure 6F). When NEM-pretreated cells were incubated with NCQ, however, negligible fluorescence was observed in all three channels (Figure 6E). In contrast, strong fluorescence signals occurred from blue and green channels when NEMpretreated cells were treated with Cys and then incubated with NCQ (Figure 6A). Furthermore, only blue fluorescence was given off from inside cells when NEMpretreated cells were incubated with GSH/H2S and then NCQ (Figures 6B and 6D). As expected, strong fluorescence signals were shown in blue and red channels when NEMpretreated cells were cultured with thiophenol and then NCQ (Figure 6C). These imaging experiments clearly showed that NCQ could distinguish Cys/Hcy, GSH/H2S and thiophenol in living cells.

Figure 4. HPLC chromatograms of NCQ, NCQ with Cys (0.0, 0.5, 1.0, 1.5 equiv.) measured after 30 min incubation, CQ and NBD-N-Cys. The concentrations of NCQ, CQ and NBD-N-Cys were 50 µM. Conditions: eluent, CH3CN/H2O (7: 3, v: v); flow rate, 0.5 mL/min; temperature, 25 °C; detection wavelength, 350 nm; injection volume, 20.0 µL.

pH Effect Studies. The fluorescence performance of NCQ in aqueous media at different pH values was investigated in the presence/absence of thiols (Figure 5). In the absence of these thiols, NCQ showed negligible fluorescence within a wide pH range (4-10). Upon the addition of Cys/Hcy, GSH/H2S or thiophenol to the solution of NCQ, strong fluorescence signals were seen within pH 710: blue and green for Cys/Hcy, blue for GSH/H2S and blue and red for thiophenol. These results suggested that NCQ had the potential to detect thiols under physiological condition.

Figure 5. Fluorescence intensities of NCQ (10 µM) at 490 nm (a), 544 nm (b), 624 nm (c) in the absence/presence of Cys (15 μM), Hcy (30 μM), GSH (10 μM), H2S (15 μM) and PhSH (70 μM) at different pH values in PBS buffer (10 mM, containing 30% acetonitrile). Excitation wavelength: 323 nm for (a), 470 nm for (b), and 475 nm for (c).

Fluorescence Imaging in Living Cells. Finally, the application of NCQ in living cells was carried out. First, MTT assays were performed on NCQ to evaluate its cytotoxicity. The cell viability was 92.5% when 10 μM of

Figure 6. Images of cells. (A-D) NEM-pretreated cells, incubated with 500 μM Cys/GSH/H2S/thiophenol for 30 min, then incubated with 10 μM NCQ for 30 min. (E) NEMpretreated cells, and incubated with 10 μM NCQ for 30 min. (F) Cells were incubated with 10 μM NCQ for 30 min. First column: blue channel (470-510 nm with excitation at 405 nm); second column: green channel (525-565 nm with excitation at 488 nm); third column: red channel (605-645 nm with excitation at 488 nm); fourth column: bright field; fifth column: merged (bright field with blue, green and red channels).

CONCLUSION In conclusion, a triple-emission fluorescent probe, NCQ, to distinguish Cys/Hcy, GSH/H2S and thiophenol was developed with good selectivity and high sensitivity using different combination from three distinct fluorescence signals (blue-green-red). The sensing mechanism of this

ACS Paragon Plus Environment

5

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

probe toward to thiols was firmly proved by the optical (absorption, fluorescence and lifetime), HPLC and mass spectral analysis. This work potentially provided an approach to developing more multiple-signals fluorescent probes for the discrimination of thiols.

ASSOCIATED CONTENT Supporting Information Chemical structures of compounds, additional spectral data, 1H NMR, 13C NMR and HRMS spectra of compounds. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Fax: +86-731-88836954; Tel: +86-731-88836954; E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. U1608222), Special Fund for Agroscientific Research in the Public Interest of China (No. 201503108) and the State Key Laboratory of Chemo/Biosensing and Chemometrics (2016005).

REFERENCES (1)Meister, A.; Anderson, M. E., Glutathione. Annu. Rev. Biochem. 1983, 52 (6), 711-760. (2)Chen, X.; Tian, X.; Shin, I.; Yoon, J., Fluorescent and luminescent probes for detection of reactive oxygen and nitrogen species. Chem. Soc. Rev. 2011, 40 (9), 4783-4804. (3)Weerapana, E., Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature 2010, 468 (7325), 790795. (4)Fowler, B., Homocysteine: overview of biochemistry, molecular biology, and role in disease processes. Semin. Vasc. med. 2005, 5 (2), 77-86. (5)Yang, G.; Wu, L.; Jiang, B.; Yang, W.; Qi, J.; Cao, K.; Meng, Q.; Mustafa, A. K.; Mu, W.; Zhang, S.; Snyder, S. H.; Wang, R., H2S has a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine γ-lyase. Science 2008, 322 (5901), 587-590. (6)Abe, K.; Kimura, H., The possible role of hydrogen sulfide as an endogenous neuromodulator. J. Neurosci. 1996, 16 (3), 10661071. (7)Calvert, J. W.; Jha, S.; Gundewar, S.; Elrod, J. W.; Ramachandran, A.; Pattillo, C. B.; Kevil, C. G.; Lefer, D. J., Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ. Res. 2009, 105 (4), 365-374. (8)Flannigan, K. L.; Wallace, J. L., Hydrogen sulfide: its production, release and functions. Springer Vienna 2013. (9)Townsend, D. M.; Tew, K. D.; Tapiero, H., The importance of glutathione in human disease. Biomed. Pharmacother. 2003, 57 (3-4), 145-155. (10)Seshadri, S.; Beiser, A.; Selhub, J.; Jacques, P. F.; Rosenberg, I. H.; D'Agostino, R. B.; Wilson, P. W. F.; Wolf, P. A., Plasma homocysteine as a risk factor for dementia and Alzheimer's disease. New Engl. J. Med. 2002, 346 (7), 476-483.

Page 6 of 8

(11)Elkhairy, L.; Vollset, S. E.; Refsum, H.; Ueland, P. M., Plasma total cysteine, pregnancy complications, and adverse pregnancy outcomes: the hordaland homocysteine study. Am. J. Clin. Nutr. 2003, 77 (2), 467-472. (12)Shimada, K.; Mitamura, K., Derivatization of thiolcontaining compounds. J. Chromatogr. B Biomed. Appl. 1994, 659 (1-2), 227-241. (13)Heil, T. P.; Lindsay, R. C., Toxicological properties of thioand alkylphenols causing flavor tainting in fish from the upper Wisconsin river. J. Environ. Sci. Heal. B 1989, 24 (4), 349-360. (14)Amrolia, P.; Sullivan, S. G.; Stern, A.; Munday, R., Toxicity of aromatic thiols in the human red blood cell. J. Appl. Toxicol. 1989, 9 (2), 113-118. (15)Juneja, T. R.; Gupta, R. L.; Samanta, S., Activation of monocrotaline, fulvine and their derivatives to toxic pyrroles by some thiols. Toxicol. Lett. 1984, 21 (2), 185-189. (16)Munday, R., Toxicity of aromatic disulphides. I. Generation of superoxide radical and hydrogen peroxide by aromatic disulphides in vitro. J. Appl. Toxicol. 1985, 5 (6), 402-408. (17)Proctor, N. H.; Hughes, J. P.; Hathaway, G. J., Proctor and Hughes' chemical hazards of the workplace. Proctor & Hughes Chemical Hazards of the Workplace 2004. (18)Hultberg, B.; Andersson, A.; Isaksson, A., The cell-damaging effects of low amounts of homocysteine and copper ions in human cell line cultures are caused by oxidative stress. Toxicology 1997, 123 (1–2), 33-40. (19)Ebisch, I. M.; Peters, W. H.; Thomas, C. M.; Wetzels, A. M.; Peer, P. G.; Steegers-Theunissen, R. P., Homocysteine, glutathione and related thiols affect fertility parameters in the (sub)fertile couple. Hum. Reprod. 2006, 21 (7), 1725-1733. (20)Dominy, J. E.; Stipanuk, M. H., New roles for cysteine and transsulfuration enzymes: production of H2S, a neuromodulator and smooth muscle relaxant. Nutr. Rev. 2010, 62 (9), 348-353. (21)Hidalgo, J.; Garvey, J. S.; Armario, A., On the metallothionein, glutathione and cysteine relationship in rat liver. J. Pharmacol. Exp. Ther. 1990, 255 (2), 554-564. (22)Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y., New strategies for fluorescent probe design in medical diagnostic imaging. Chem. Rev. 2010, 110 (5), 2620-2640. (23)Jiang, W.; Fu, Q.; Fan, H.; Ho, J.; Wang, W., A highly selective fluorescent probe for thiophenols. Angew. Chem. 2007, 46 (44), 8445-8448. (24)Li, J.; Zhang, C. F.; Yang, S. H.; Yang, W. C.; Yang, G. F., A coumarin-based fluorescent probe for selective and sensitive detection of thiophenols and its application. Anal. Chem. 2014, 86 (6), 3037-3042. (25)Liu, H. W.; Zhang, X. B.; Zhang, J.; Wang, Q. Q.; Hu, X. X.; Wang, P.; Tan, W., Efficient two-photon fluorescent probe with red emission for imaging of thiophenols in living cells and tissues. Anal. Chem. 2015, 87 (17), 8896-8903. (26)Liu, T.; Huo, F.; Li, J.; Chao, J.; Zhang, Y.; Yin, C., An off-on fluorescent probe for specifically detecting cysteine and its application in bioimaging. Sensor. Actuat. B-Chem. 2016, 237, 127132. (27)Shao, X.; Kang, R.; Zhang, Y.; Huang, Z.; Peng, F.; Zhang, J.; Wang, Y.; Pan, F.; Zhang, W.; Zhao, W., Highly selective and sensitive 1-amino BODIPY-based red fluorescent probe for thiophenols with high off-to-on contrast ratio. Anal. Chem. 2015, 87 (1), 399-405. (28)Sun, Q.; Yang, S. H.; Wu, L.; Yang, W. C.; Yang, G. F., A highly sensitive and selective fluorescent probe for thiophenol designed via a twist-blockage strategy. Anal. Chem. 2016, 88 (4), 22662272. (29)Yu, D.; Huang, F.; Ding, S.; Feng, G., Near-infrared fluorescent probe for detection of thiophenols in water samples and living cells. Anal. Chem. 2014, 86 (17), 8835-8841. (30)He, L.; Yang, X.; Xu, K.; Kong, X.; Lin, W., A multi-signal fluorescent probe for simultaneously distinguishing and sequentially sensing cysteine/homocysteine, glutathione, and hydrogen sulfide in living cells. Chem. Sci. 2017, 8 (9), 6257-6265.

ACS Paragon Plus Environment

6

Page 7 of 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sensors (31)Danehy, J. P.; Noel, C. J., The relative nucleophilic character of several mercaptans toward ethylene oxide1. J. Am. Chem. Soc. 1960, 82 (10), 2511-2515. (32)Nekrassova, O.; Lawrence, N. S.; Compton, R. G., Analytical determination of homocysteine: a review. Talanta 2003, 60 (6), 1085-1095. (33)Kosower, N. S.; Kosower, E. M., The glutathione status of cells. Int. Rev. Cytol. 1978, 54 (54), 109-160. (34)Chen, C.; Liu, W.; Xu, C.; Liu, W., A colorimetric and fluorescent probe for detecting intracellular GSH. Biosens. Bioelectron. 2015, 71, 46-52. (35)Chen, W.; Luo, H.; Liu, X.; Foley, J. W.; Song, X., Broadly applicable strategy for the fluorescence based detection and differentiation of glutathione and cysteine/homocysteine: demonstration in vitro and in vivo. Anal. Chem. 2016, 88 (7), 3638-3646. (36)Chen, W.; Yue, X.; Li, W.; Hao, Y.; Zhang, L.; Zhu, L.; Sheng, J.; Song, X., A phenothiazine coumarin-based red emitting fluorescent probe for nanomolar detection of thiophenol with a large Stokes shift. Sensor. Actuat. B-Chem. 2017, 245, 702-710. (37)Dai, X.; Wang, Z. Y.; Du, Z. F.; Cui, J.; Miao, J. Y.; Zhao, B. X., A colorimetric, ratiometric and water-soluble fluorescent probe for simultaneously sensing glutathione and cysteine/homocysteine. Anal. Chim. Acta 2015, 900, 103-110. (38)Ding, S.; Feng, G., Smart probe for rapid and simultaneous detection and discrimination of hydrogen sulfide, cysteine/homocysteine, and glutathione. Sensor. Actuat. B-Chem. 2016, 235, 691-697. (39)Liu, X.; Qi, F.; Su, Y.; Chen, W.; Yang, L.; Song, X., A red emitting fluorescent probe for instantaneous sensing of thiophenol in both aqueous medium and living cells with a large Stokes shift. J. Mater. Chem.C 2016, 4 (19), 4320-4326. (40)Liu, X.; Yang, D.; Chen, W.; Yang, L.; Qi, F.; Song, X., A redemitting fluorescent probe for specific detection of cysteine over homocysteine and glutathione with a large Stokes shift. Sensor. Actuat. B-Chem. 2016, 234, 27-33. (41)Liu, X. L.; Niu, L. Y.; Chen, Y. Z.; Yang, Y.; Yang, Q. Z., A ratiometric fluorescent probe based on monochlorinated BODIPY for the discrimination of thiophenols over aliphatic thiols in water samples and in living cells. Sensor. Actuat. B-Chem. 2017, 252, 470-476. (42)Niu, L. Y.; Yang, Q. Q.; Zheng, H. R.; Chen, Y. Z.; Wu, L. Z.; Tung, C. H.; Yang, Q. Z., BODIPY-based fluorescent probe for the simultaneous detection of glutathione and cysteine/homocysteine at different excitation wavelengths. RSC Adv. 2014, 5 (6), 3959-3964. (43)Wang, P.; Wang, Y.; Li, N.; Huang, J.; Wang, Q.; Gu, Y., A novel DCM-NBD conjugate fluorescent probe for discrimination of Cys/Hcy from GSH and its bioimaging applications in living cells and animals. Sensor. Actuat. B-Chem. 2017, 245, 297-304. (44)Ji, W.; Liu, G.; Wang, F.; Zhu, Z.; Feng, C., Galactosedecorated light-responsive hydrogelator precursors for selectively killing cancer cells. Chem. Commun. 2016, 52 (85), 12574-12577. (45)Jagtap, A. R.; Satam, V. S.; Rajule, R. N.; Kanetkar, V. R., The synthesis and characterization of novel coumarin dyes derived from 1,4-diethyl-1,2,3,4-tetrahydro-7-hydroxyquinoxalin-6carboxaldehyde. Dyes Pigm. 2009, 82 (1), 84-89. (46)Rotili, D.; De Luca, A.; Tarantino, D.; Pezzola, S.; Forgione, M.; Morozzo della Rocca, B.; Falconi, M.; Mai, A.; Caccuri, A. M., Synthesis and structure–activity relationship of new cytotoxic agents targeting human glutathione-S-transferases. Eur. J. Med. Chem. 2015, 89, 156-171. (47)Montoya, L. A.; Pearce, T. F.; Hansen, R. J.; Zakharov, L. N.; Pluth, M. D., Development of selective colorimetric probes for hydrogen sulfide based on nucleophilic aromatic substitution. J. Org. Chem. 2013, 78 (13), 6550-6557. (48)Pant, P. L.; Shankarling, G. S., Deep eutectic solvent: an efficient and recyclable catalyst for synthesis of thioethers. Chemistryselect 2017, 2 (25), 7645-7650.

ACS Paragon Plus Environment

7

ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 8

Table of Contents Site 1

Site 2 NO2

Group for biothiols

Group for thiophenol

O N O

O

cy

N

NO2 N

s/ H Cy

NO2

GSH/H2S

CN

Ph S

N O O

NCQ

Cys/Hcy

H

O O

GSH/H2S

ACS Paragon Plus Environment

PhSH

8