Design and Synthesis of a Fluorescent Probe with a Large Stokes Shift for Detecting Thiophenols and Its Application in Water Samples and Living Cells

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Article Design and Synthesis of a Fluorescent Probe with a Large Stokes Shift for Detecting Thiophenols and Its Application in Water Samples and Living Cells

Hua Liu 1,2,3,4,†, Chuanlong Guo 1,5,†, Shuju Guo 1,2,3, Lijun Wang 1,2,3,* and Dayong Shi 1,2,3,4,* 1 CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China; [email protected] (H.L.); [email protected] (C.G.); [email protected] (S.G.) 2 Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China 3 Center for Ocean Mega-Science, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao 266071, China 4 University of Chinese Academy of Sciences, Beijing 100049, China 5 Department of Pharmacy, College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao 266042, China * Correspondence: [email protected] (L.W.); [email protected] (D.S.); Tel.: +86-532-8289-8741 (L.W.); +86-532-8289-8719 (D.L.) † These authors contributed equally to this work.

Received: 3 January 2019; Accepted: 21 January 2019; Published: 21 January 2019

Abstract: A turn-on florescent probe (probe-KCP) was developed for highly selective detection of thiophenols based on a donor-excited photo-induced electron transfer mechanism. Herein, the synthesis of the probe, a chalcone derivative, through a simple straightforward combination of a carbazole-chalcone fluorophore with a 2,4-dinitrophenyl functional group. In a kinetic study of the probe-KCP for thiophenols, the probe displayed a short response time (~30 min) and significant fluorescence enhancement. In selection and competition experiments, the probe-KCP exhibited excellent selectivity for thiophenols over glutathione (GSH), cysteine (Cys), sodium hydrosulfide (NaSH), and ethanethiol (C2H5SH) in addition to common anions and metal ions. Using the designed probe, we successfully monitored and quantified thiophenols, which are highly toxic. This turn-on fluorescence probe features a remarkably large Stokes shift (130 nm) and a short response time (30 min), and it is highly selective and sensitive (~160-fold) in the detection of thiophenols, with marked fluorescence in the presence of thiophenols. probe-KCP responds to thiophenols with a good range of linearity (0–15 µM) and a detection limit of 28 nM (R2 = 0.9946) over other tested species mentioned including aliphatic thiols, thiophenol analogues, common anions, and metal ions. The potential applications of this carbazole-chalcone fluorescent probe was successfully used to determine of thiophenols in real water samples and living cells with good performance and low cytotoxicity. Therefore, this probe has great potential application in environment and biological samples.

Keywords: ﬂuorescent probe; thiophenol; water samples; chalcone; living cells

1. Introduction Thiophenols are small molecules that serve as essential raw materials in the synthetic chemical industry. For example: they are used in the preparation of pesticides, polymers, and pharmaceuticals [1,2]. However, thiophenols are also pollutants which are highly toxic to

Molecules 2019, 24, 375; doi:10.3390/molecules24020375 www.mdpi.com/journal/molecules Molecules 2019, 24, 375 2 of 14 organisms, including humans, where thiophenol toxicity can have adverse physiological and psychological effects. Prolonged exposure to thiophenols can cause damage to the central nervous system, breathing difficulties, muscle weakness, paralysis of the limbs, a coma, and even death [3,4]. Previous research reported that the median lethal dose of thiophenols to fish and mice was 0.01–0.4 mmol/L and 46.2 mg/kg3, respectively. Thiophenols are considered a priority pollutant by the U.S. Environmental Protection Agency (EPA waste code: P014) [5,6]. There is an urgent need for the development of a rapid, selective, and sensitive method for monitoring of thiophenols in environmental samples and biological samples. There are various traditional methods such as high-performance liquid chromatography, gas chromatography, and nonlinear spectroscopy, as well as nanomaterial-based sensors, available for the detection of thiophenols [7–11]. However, most of these can detect thiophenols only in vitro. Fluorescent probe-based methods for the detection of thiophenols have many advantages [12–20]. These include high sensitivity and high selectivity, real-time bio-imaging, operational simplicity, and short response times, all of which are important factors in the detection of thiophenols. The study of small molecule fluorescent probes has attracted more and more attention [21]., However, many probes are affected by interference from glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) which have similar chemical structures with thiophenols, or probes have a narrow Stokes shift (<60 nm) which is detrimental for biological applications due to excitation backscattering effects [22–27]. Designing a fluorescent probe that can distinguish between aliphatic thiols and aromatic thiophenols in living cells is a huge challenge. The biological activities of chalcone derivatives are well known [28–33]. These derivatives also exhibit some excellent optical properties. Dyes based on chalcone derivatives are characterized by large diversity and flexible modes of synthesis, intramolecular charge transfer, large Stoke shifts, bright fluorescence, and a high quantum yield [34,35]. Therefore, chalcone derivatives have attracted intense attention in recent years. In the present study, a turn-on fluorescent probe-KCP, based on a donor-excited photo-induced electron transfer mechanism was designed and applied to detect thiophenols. The probe-KCP was synthesized through a simple straightforward combination of a chalcone unit, which served as a fluorescent chromophore, and a 2,4-dinitrophenyl moiety, which acted as a fluorescent quenching unit. As expected, the designed probe shown pronounced selectivity for thiophenols over common metal ions, anions, amino acids and aliphatic thiols. The probe-KCP was substantially nonfluorescent due to the strong quenching effect of the 2,4-dinitrophenyl group. Thus, the probe-KCP has excellent detection abilities for thiophenols and possesses marked fluorescence intensity enhancement (~160-fold). It also has a conspicuously large Stokes shift (~130 nm). Therefore, the probe has been successfully applied for the quantitative detection of thiophenols in real water samples and fluorescent imaging of thiophenols in living cells.

2. Results and Discussion

2.1. Synthesis of the Probe-KCP The synthesis procedure of the target chalcone fluorescent probe-KCP for the detection of thiophenols is outlined in Scheme1. The probe-KCP was synthesized with chalcones as a strong fluorophore linked to a 2,4-dinitrophenyl group acted simultaneously as recognition unit and a fluorophore quencher. KCP-OCH3 was reacted with boron tribromide in dry dichloromethane and cooled to −78 ◦C. The crude product was conveniently converted to the probe-KCP in dichloromethane at room temperature through a straightforward reaction with 2,4-dinitro-fluorobenzene. The structure of the probe was characterized by 1H-NMR, 13C-NMR, and MS spectra. The experimental characterization data are given in the Electronic Supplementary Information. Molecules 2019, 24, 375 3 of 14

2.2. The Absorption and Emission Spectroscopic Properties of the Probe-KCP To determine the fluorescence properties of the probe-KCP for thiophenols, we first examined the absorption and fluorescent spectra of probe. The reaction response time of the probe-KCP for thiophenols was investigated in DMSO/PBS (1:1, v/v, 20 mM, pH = 7.4). As shown Molecules 2019, 24, x FOR PEER REVIEW 3 of 14 in Figure1, the absorption spectra of the probe (10 µM) solution displayed one band at 406of 4‐ nmmethoxythiophenol only after the addition(100 μM). of The 4-methoxythiophenol absorption maximum (100 at 403µM). nm The (Φ= absorption 0.027, ε = 6.2 maximum × 103 M−1 3 −1 −1 atcm 403−1) displayed nm (Φ = a 0.027 slight, redε = 6.2shift× 310 nmM to 406cm nm) (Φ displayed= 0.19; ε = a2.12 slight × 104 redM−1 cm shift−1). 3In nmthe fluorescence to 406 nm 4 −1 −1 (spectra,Φ = 0.19 ;significantε = 2.12 × fluorescence10 M cm enhancement). In the fluorescence (~160‐fold) spectra, was observed significant at fluorescence 540 nm after enhancement 4‐methoxy‐ (~160-fold)thiophenol was added observed to the at DMSO/PBS 540 nm after solution 4-methoxy-thiophenol containing the probe was‐KCP added (10 to μM) the and DMSO/PBS incubated ◦ solutionfor 30 min containing at 37 °C. The the probe-KCPsolution of the (10 probeµM) and‐KCP incubated alone exhibited for 30 min weak at fluorescence 37 C. The solution intensity, of with the probe-KCPexcitation at alone 410 nm. exhibited After the weak addition fluorescence of 4‐methoxythiophenol intensity, with excitation to the probe at 410‐KCP nm. solution, After the it addition emitted ofrelatively 4-methoxythiophenol a strong fluorescent to the signal probe-KCP at 540 solution,nm and an it absorption emitted relatively maximum a strong at 410 fluorescent nm. The addition signal atof 540 4‐methoxythiophenol nm and an absorption resulted maximum in obvious at 410 green nm. fluorescence The addition from of 4-methoxythiophenol the probe‐KCP solution resulted when init obviouswas exposed green to fluorescence daylight or fromto a hand the probe-KCP‐held UV lamp. solution (Figure when S8). it These was exposed results todemonstrated daylight or tothat a hand-heldthe probe‐KCP UV lamp. is a promising (Figure S8). fast These (30 min) results turn demonstrated‐on fluorescence that probe, the probe-KCP with a large is a Stokes promising shift fast(130 (30nm) min) for turn-onthe detection fluorescence of thiophenols. probe, with a large Stokes shift (130 nm) for the detection of thiophenols.

Figure 1. (A) Absorption spectra and (B) fluorescence spectra of probe-KCP (10 µM) in the presence Figure 1. (A) Absorption spectra and (B) fluorescence spectra of probe‐KCP (10 μM) in the presence of 100 µM 4-methoxythiophenol (red) in DMSO/PBS (1:1, v/v, 20 mM, pH = 7.4). λex = 410 nm and of 100 μM 4‐methoxythiophenol (red) in DMSO/PBS (1:1, v/v, 20 mM, pH = 7.4). λex = 410 nm and λem λem = 540 nm. = 540 nm. To further investigate the sensitivity of the probe-KCP for thiophenols, the fluorescence To further investigate the sensitivity of the probe‐KCP for thiophenols, the fluorescence properties of the probe in the presence of different concentrations of thiophenols were investigated. properties of the probe in the presence of different concentrations of thiophenols were investigated. The probe-KCP detected thiophenols in DMSO/PBS (v/v =1:1, 20 mM, pH = 7.4) solvent at 37 ◦C. The probe‐KCP detected thiophenols in DMSO/PBS (v/v =1:1, 20 mM, pH = 7.4) solvent at 37 °C. The The probe-KCP (10 µM) on its own showed a maximum absorption peak at 403 nm and almost no probe‐KCP (10 μM) on its own showed a maximum absorption peak at 403 nm and almost no fluorescence. However, when 4-methoxythiophenol was added to the probe solution, the maximum fluorescence. However, when 4‐methoxythiophenol was added to the probe solution, the maximum absorption peak red-shifted to 406 nm. As shown in Figure2, the fluorescence intensity was absorption peak red‐shifted to 406 nm. As shown in Figure 2, the fluorescence intensity was proportional to the concentration of thiophenol and showed a rapid growth trend. When the proportional to the concentration of thiophenol and showed a rapid growth trend. When the concentration of 4-methoxythiophenol reached 60 µM, the fluorescence intensity was enhanced by concentration of 4‐methoxythiophenol reached 60 μM, the fluorescence intensity was enhanced by about 160-fold and no change in the fluorescence at subsequent concentrations. An excellent linearly about 160‐fold and no change in the fluorescence at subsequent concentrations. An excellent linearly dependent (R2 = 0.9946) between the enhancement of fluorescence intensity and the concentration of dependent (R2 = 0.9946) between the enhancement of fluorescence intensity and the concentration of 4-methoxythiophenol (0 µM to 15 µM), thereby indicating that the probe-KCP had the potential to be 4‐methoxythiophenol (0 μM to 15 μM), thereby indicating that the probe‐KCP had the potential to be used for quantitatively monitoring thiophenols. The detection limit of the samples was calculated used for quantitatively monitoring thiophenols. The detection limit of the samples was calculated using the following equation: using the following equation: LD = 3σ/k (1) LD = 3σ/k (1) where σ is the standard deviation of a blank measurement, and k is the slope. where σ is the standard deviation of a blank measurement, and k is the slope. The kinetic study showed that the fluorescence intensity of the probe‐KCP solution increased significantly 30 min after adding 4‐methoxythiophenol at 37 °C and that the fluorescence intensity did not change substantially after 30 min (Figure S6, Figure S7). The results pointed to the potential use of the probe for real‐time monitoring of thiophenols.

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The kinetic study showed that the fluorescence intensity of the probe-KCP solution increased significantly 30 min after adding 4-methoxythiophenol at 37 ◦C and that the fluorescence intensity did not change substantially after 30 min (Figures S6 and S7). The results pointed to the potential use of theMolecules probe 2019 for, 24 real-time, x FOR PEER monitoring REVIEW of thiophenols. 4 of 14

Figure 2. (A) Fluorescence spectra of 10 µM probe-KCP upon the addition of 4-methoxy thiophenol Figure 2. (A) Fluorescence spectra of 10 μM probe‐KCP upon the addition of 4‐methoxy thiophenol (0–100 µM); (B) The linearity of the probe-KCP fluorescence intensity versus thiophenol concentration (0–100 μM); (B) The linearity of the probe‐KCP fluorescence intensity versus thiophenol concentration (0–15 µM). λex = 410 nm and λem = 540 nm. Relative standard deviation (RSD), n = 3. (0–15 μM). λex = 410 nm and λem = 540 nm. Relative standard deviation (RSD), n = 3. 2.3. The Effect of pH on the Probe 2.3. The Effect of pH on the Probe The pH of a system can sometimes exert a marked effect on the fluorescence intensity. We tested the responseThe pH of of a system the probe-KCP can sometimes to 4-methoxythiophenol exert a marked effect over on the a fluorescence wide pH range intensity. from We 1 totested 10. Thethe response influence of of the pH probe on the‐KCP fluorescence to 4‐methoxythiophenol spectra of the probe-KCPover a wide to pH 4-methoxythiophenol range from 1 to 10. wasThe tested.influence At of a pH pH of on <6, the no fluorescence significant changesspectra of in the the probe fluorescence‐KCP to intensity4‐methoxythiophenol of the probe-KCP was tested. solution At werea pH observed of <6, no at significant 540 nm, and changes similar resultsin the werefluorescence observed intensity when the of pH the exceeded probe‐KCP 10.0. However,solution were at a pHobserved of >6, the at 540 fluorescence nm, and intensitysimilar results of the probe-KCPwere observed (10 µ whenM) decreased the pH significantlyexceeded 10.0. in the However, presence at of a 4-methoxythiophenolpH of >6, the fluorescence at a concentration intensity of the of probe 100 µM.‐KCP As (10 shown μM) in decreased Figure S2, significantly the probe-KCP in the functioned presence atof 540 4‐methoxythiophenol nm in a pH range from at 7.0a concentration to 10.0. The fluorescent of 100 μM. intensity As shown of the probe-KCPin Figure S2, (10 µtheM) probe was stable‐KCP overfunctioned a wide at pH 540 range, nm in which a pH indicatedrange from that 7.0 the to 10.0. probe The was fluorescent stable in theintensity pH range of the of probe physiological‐KCP (10 conditions.μM) was stable Thus, over in subsequenta wide pH range, live cell which imaging indicated studies, that a the pH probe of 7.4 was was stable selected in the as pH optimal range for of DMSO/PBSphysiological buffer conditions. (1:1, v/ Thus,v, 20 mM). in subsequent live cell imaging studies, a pH of 7.4 was selected as optimal for DMSO/PBS buffer (1:1, v/v, 20 mM). 2.4. Sensitivity and Selectivity of the Probe for Thiophenols 2.4. Sensitivity and Selectivity of the Probe for Thiophenols Next, the spectral responses of the probe-KCP toward various thiophenols, thiols, amino acids,Next, other the potentially spectral responses interfering of substances, the probe‐KCP common toward anions various and metalthiophenols, ions were thiols, investigated amino acids, by fluorescenceother potentially spectroscopy. interfering As shown substances, in Figures common S3 and anions S4, only and the introductionmetal ions ofwere thiophenols investigated such asby pfluorescence-CH3−C6H4 spectroscopy.SH, p-NH2−C As6H shown4SH, p-F in− FigureC6H4SH S3 and Figurep-CH3 OS4,− Conly6H4 theSH introduction to the probe-KCP of thiophenols solution 3 6 4 2 6 4 6 4 3 6 4 inducedsuch as p a‐CH significantly−C H SH, enhanced p‐NH −C theH fluorescenceSH, p‐F−C H intensity.SH and p Reactivity‐CH O−C H comparison:SH to the probe p-CH‐3KCPO−C solution6H4SH 3 6 4 >inducedp-F−C6 aH significantly4SH > p-NH enhanced2−C6H4SH the > fluorescencep-CH3−C6H intensity.4SH. However, Reactivity the comparison: addition of other p‐CH biologicallyO−C H SH > relevantp‐F−C6H analytes4SH > p‐NH resulted2−C6H in4SH no change> p‐CH3 in−C the6H fluorescence4SH. However, intensity the addition of the compounds of other biologically in the probe-KCP relevant solution.analytes Theseresulted results in no clearly change suggested in the fluorescence that the probe-KCP intensity was of particularly the compounds selective in forthe thiophenols probe‐KCP oversolution. other interferingThese results species. clearly In other suggested words, thethat probe-KCP the probe displayed‐KCP was high particularly selectivity for selective thiophenols for (4-fluorothiophenol,thiophenols over other 4-toluenethiol, interfering species. 4-methoxythiophenol, In other words, the and probe 4-aminothiophenol).‐KCP displayed high As shownselectivity in Figurefor thiophenols3, it is worth (4‐fluorothiophenol, noting that there 4 was‐toluenethiol, no significant 4‐methoxythiophenol, fluorescence signal and change 4‐aminothiophenol). after the probe reactedAs shown with in theFigure other 3, it potentially is worth noting interfering that there substances was no significant interferences fluorescence (10 eq.), such signal as change mercaptan, after nucleophilicthe probe reacted species, with and the metal other ions potentially in the DMSO/PBS interfering buffer substances (1:1, v /interferencesv, 20 mM, pH (10 = 7.4)eq.), at such 37 ◦ Cas mercaptan, nucleophilic species, and metal ions in the DMSO/PBS buffer (1:1, v/v, 20 mM, pH = 7.4) at 37 °C for 30 min. These results showed that the probe‐KCP could be used to monitor thiophenols in the presence of competitive interference from other substances.

Molecules 2019, 24, 375 5 of 14 for 30 min. These results showed that the probe-KCP could be used to monitor thiophenols in the presenceMolecules 2019 of, competitive 24, x FOR PEER interference REVIEW from other substances. 5 of 14

µ Figure 3. The fluorescence ﬂuorescence intensity atat 540540 nmnm ((λλexex = 410 410 nm) nm) of of probe probe-KCP‐KCP (10 μM) in the presence of ◦ 100 μµMM ofof variousvarious analytesanalytes in in DMSO/PBS DMSO/PBS buffer (1:1, vv//vv,, 20 20 mM, mM, pH pH 7.4) 7.4) at 37 37 °CC prior prior to to (black (black bars) bars) and after (red bars) addition of 100 μµM 4 4-methoxythiophenol‐methoxythiophenol to the individual probe/analyte solution. solution.

(1) blank, (2) (2) GSH,(3) GSH,(3) Cys, Cys, (4) (4) Hcy, Hcy, (5) (5) Ala, Ala, (6) (6) Ser, Ser, (7) (7) NaSH, NaSH, (8) (8) C2H C25SH,H5SH, (9) Ph (9)‐OH, Ph-OH, (10) (10)Ph‐NH Ph-NH2, (11)2, (11) NaCl, (12) FeCl , (13) KCl, (14) CaCl , (15) FeCl , (16) MgSO , (17) NaNO , (18) KF, (19) NaHCO , NaCl, (12) FeCl2, (13)2 KCl, (14) CaCl2, (15)2 FeCl3, (16)3 MgSO4, (17)4 NaNO3, (18)3 KF, (19) NaHCO3, (20)3 (20)4‐toluenethiol, 4-toluenethiol, (21) 4 (21)‐methoxythiophenol, 4-methoxythiophenol, (22) 4 (22)‐aminothiophenol, 4-aminothiophenol, (23) 4 (23)‐fluorothiophenol. 4-fluorothiophenol. Data were Data wererecorded recorded 30 min 30 after min afteraddition addition of analytes/ of analytes/ thiophenols thiophenols with withexcitation excitation at 410 at nm 410 and nm andemission emission at 540 at 540nm. nm.Error Error bars bars are relative are relative standard standard deviations deviations (RSD), (RSD), n=3.n =3. 2.5. Plausible Mechanism of Detection of Thiophenols 2.5. Plausible Mechanism of Detection of Thiophenols To determine a plausible mechanism for the detection of thiophenols, 1H-NMR, 13C-NMR, To determine a plausible mechanism for the detection of thiophenols, 1H‐NMR, 13C‐NMR, and and mass spectrometry (Figures S1 and S9) were employed to monitor the reaction of the probe-KCP mass spectrometry (Figure S1, Figure S9) were employed to monitor the reaction of the probe‐KCP with 4-methoxythiophenol. Two products (probe-OH and S-NO2) were isolated and their structural with 4‐methoxythiophenol. Two products (probe‐OH and S‐NO2) were isolated and their structural characterizations are shown in Figure S1. characterizations are shown in Figure S1. To better understand the reaction mechanism of the probe-KCP with thiophenols, density To better understand the reaction mechanism of the probe‐KCP with thiophenols, density functional theory was applied using the Gaussian 09 program (Figure4 and Figure S10). As shown in functional theory was applied using the Gaussian 09 program (Figure 4 and Figure S10). As shown Figure4A, for the probe-OH moiety, electron clouds on both HOMO and LUMO essentially resided in Figure 4A, for the probe‐OH moiety, electron clouds on both HOMO and LUMO essentially on its entire skeleton. By contrast, in the case of the probe-KCP (Figure4B), the electron clouds on resided on its entire skeleton. By contrast, in the case of the probe‐KCP (Figure 4B), the electron clouds the HOMO and LUMO+2 were mainly distributed in the electron-donating moiety of the probe-OH. on the HOMO and LUMO+2 were mainly distributed in the electron‐donating moiety of the probe‐ However, those on the LUMO and LUMO+1 were primarily located on the electron-withdrawing OH. However, those on the LUMO and LUMO+1 were primarily located on the electron‐ 2,4-dinitrophenyl group. These findings indicated that the probe-KCP could bear efficient electron withdrawing 2,4‐dinitrophenyl group. These findings indicated that the probe‐KCP could bear transfer from the moiety of the probe-OH to the 2,4-dinitrophenyl moiety, resulting in weak or no efficient electron transfer from the moiety of the probe‐OH to the 2,4‐dinitrophenyl moiety, resulting fluorescence of the probe. After the addition of 4-methoxythiophenol, the electron transfer from in weak or no fluorescence of the probe. After the addition of 4‐methoxythiophenol, the electron the moiety of the probe-OH to the 2,4-dinitrophenyl was blocked following the elimination of the transfer from the moiety of the probe‐OH to the 2,4‐dinitrophenyl was blocked following the 2,4-dinitrophenyl group. The resulting almost complete overlap of electrons on the transition orbitals elimination of the 2,4‐dinitrophenyl group. The resulting almost complete overlap of electrons on the may induce strong fluorescence emission from the moiety of the probe-OH. transition orbitals may induce strong fluorescence emission from the moiety of the probe‐OH.

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Figure 4. (A) Frontier molecular orbital plots of dye probe‐OH (CPCM model); (B) Frontier molecular FigureFigure 4.4. (A) Frontier molecular orbital plots of dye probe-OHprobe‐OH (CPCM(CPCM model); ((B)) Frontier molecular orbital plots of probe‐KCP (CPCM model). The fluorescence emission of probe‐KCP moieties is orbitalorbital plotsplots ofof probe-KCPprobe‐KCP (CPCM(CPCM model).model). The fluorescencefluorescence emission of probe-KCPprobe‐KCP moietiesmoieties isis quenched by d‐PET. quenchedquenched byby d-PET.d‐PET. 2.6. Measurements of Thiophenols in Water Samples 2.6. Measurements of Thiophenols in Water Samples To evaluate whether the probe could directly detect thiophenols in a a real real environmental environmental sample, sample, To evaluate whether the probe could directly detect thiophenols in a real environmental sample, the probe was tested in experiments involving deionized water, artificial artificial seawater, and tap water the probe was tested in experiments involving deionized water, artificial seawater, and tap water samples. The water samples were spiked with different concentrations of 4-methoxythiophenol4‐methoxythiophenol and samples. The water samples were spiked with different concentrations of 4‐methoxythiophenol and then incubated with the probe forfor 30 min at 3737 ◦°C.C. then incubated with the probe for 30 min at 37 °C. As shownshown in in Figure Figure5, 5, the the experimental experimental results results showed showed that thethat fluorescence the fluorescence intensity intensity of the probeof the As shown in Figure 5, the experimental results showed that the fluorescence intensity of the increasedprobe increased in proportion in proportion to the concentration to the concentration of 4-methoxythiophenol of 4‐methoxythiophenol added and added that the and recovery that the of probe increased in proportion to the concentration of 4‐methoxythiophenol added and that the 4-methoxythiophenolrecovery of 4‐methoxythiophenol was 92–107%. was This 92–107%. finding This indicated finding that indicated the probe that wasthe probe capable was of capable detecting of recovery of 4‐methoxythiophenol was 92–107%. This finding indicated that the probe was capable of thiophenolsdetecting thiophenols in real water in real samples. water The samples. results The shown results in Table shown1 was in Table reported 1 was as thereported mean as± thestandard mean detecting thiophenols in real water samples. The results shown in Table 1 was reported as the mean deviation± standard of deviation triplicate of experiments triplicate experiments for thiophenol for thiophenol spiked from spiked 0 to 10 fromµM. 0 to 10 μM. ± standard deviation of triplicate experiments for thiophenol spiked from 0 to 10 μM.

Figure 5. A linear calibration curve between the fluorescence intensities of probe-KCP and Figure 5. A linear calibration curve between the fluorescenceµ intensities of probe‐KCP and concentrationFigure 5. A oflinear 4-methoxythiophenol calibration curve in between the range the of 0–10 fluorescenceM in real intensities water samples. of probe (A) Fluorescent‐KCP and concentration of 4‐methoxythiophenol in the range of 0–10 μM in real water samples. (A) Fluorescent detectionconcentration of different of 4‐methoxythiophenol concentrations of in 4-methoxythiophenolthe range of 0–10 μM in in real “tap water water”, samples. “deionized (A) Fluorescent water”, detection of different concentrations of 4µ‐methoxythiophenol in “tap water”, “deionizedB water”,D and anddetection “artificial of different water” byconcentrations probe-KCP (10 of 4‐M)methoxythiophenol in PBS buffer (20 in mM) “tap with water”, 50% “deionized DMSO; ( – water”,Linear and of fluorescence“artificial water” intensity by probe changes‐KCP of probe-KCP(10 μM) in against PBS buffer the of (20 4-methoxythiophenol mM) with 50% DMSO; for real (B– waterD) Linear sample. of “artificial water” by probe‐KCP (10 μM) in PBS buffer (20 mM) with 50% DMSO; (B–D) Linear of

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Table 1. Determination of thiophenol concentrations in water samples.

Thiophenol Spiked Thiophenol Recovered Sample Recovery (%) (µM) (µM) 0 not detected 2 1.99 ± 0.334 100% 4 3.79 ± 0.5484 95% Artiﬁcial seawater 6 5.63 ± 0.560 94% 8 8.41 ± 0.617 105% 10 10.12 ± 0.777 101% 0 not detected 2 1.84 ± 0.105 92% 4 4.19 ± 0.411 105% Tap water 6 6.12 ± 0.397 102% 8 8.57 ± 0.464 107% 10 9.99 ± 0.405 100% 0 not detected 2 2.11 ± 0.304 106% 4 3.80 ± 0.453 95% Deionized water 6 6.21 ± 0.3555 104% 8 8.11 ± 0.605 101% 10 9.62 ± 0.592 96%

2.7. Determination of the Potential Cytotoxicity of the Probe To evaluate the potential cytotoxicity of the probe before applying it in live cell imaging, we performed an MTT assay in A549 cells. Different concentrations of the probe-KCP (0, 2.5, 5, 10, 20, and 50 µM) were added to the A549 cells and incubated for 24 h. As shown in Figure S5, the probe-KCP was nontoxic to the cultured cells under the experimental conditions, which demonstrated that the probe was safe and suitable for cell imaging.

2.8. Cell Imaging Given the favorable properties of the probe-KCP (e.g., high selectivity, excellent sensitivity, a short response time, and lack of cytotoxicity) in terms of the detection of thiophenols in vitro in DMSO/PBS buffer (1:1, v/v, 20 mM, pH = 7.4), we tested the potential applications of the probe-KCP in live cell imaging assays. The A549 cells were treated with the probe-KCP and then observed under a confocal fluorescence microscope. As shown in Figure6, the probe-KCP (10 µM) was incubated with the A549 cells for 30 min at 37 ◦C. Almost no intracellular fluorescence signals were observed in the green channel by CLSM. However, following the addition of 300 µM 4-methoxythiophenol to the probe-KCP solution and incubation with the A549 cells for 30 min, green fluorescence signals were observed in the green channel. These findings indicated that the probe-KCP could be employed to detect thiophenols in A549 cells via CLSM. Thus, the designed probe-KCP is cell membrane permeable and can detect thiophenols with obvious fluorescence signals in living cells. Molecules 2019,, 24,, x 375 FOR PEER REVIEW 88 of of 14

Figure 6. Imaging of 4-methoxythiophenol in living A549 cells using the probe-KCP. The A549 cells Figure 6. Imaging of 4‐methoxythiophenol in living A549 cells using the probe‐KCP. The A549 cells were pretreated with 4-methoxythiophenol (300 µM) for 30 min at 37 ◦C and then incubated with the were pretreated with 4‐methoxythiophenol (300 μM) for 30 min at 37 °C and then incubated with the probe-KCP (10 µM) for 30 min at 37 ◦C. probe‐KCP (10 μM) for 30 min at 37 °C. 3. Experimental 3. Experimental 3.1. Starting Materials and Instruments 3.1. StartingCarbazole Materials (CAS and No. Instruments 86-74-8, 98%), bromoethane (CAS No. 74-96-4, 98%), 40-methoxy-acetophenoneCarbazole (CAS No. (CAS 86‐74 No.‐8, 100-06-1,98%), bromoethane 99%), 2,4-dinitrofluorobenzene (CAS No. 74‐96 (CAS‐4, 98%), No. 70-34-8,4′‐methoxy 99%),‐ acetophenoneboron tribromide (CAS (CAS No. No. 100 10294-33-4),‐06‐1, 99%), and 2,4 cesium‐dinitrofluorobenzene carbonate (CAS No.(CAS 534-17-8, No. 70 99%)‐34‐8, were 99%), obtained boron tribromidecommercially (CAS and No. used 10294 without‐33‐4), furtherand cesium purification. carbonate Other (CAS reagentsNo. 534‐17 (phosphorus‐8, 99%) were oxychloride, obtained commerciallyethanol, ethyl acetate,and used petroleum without ether, further dichloromethane, purification. sodiumOther reagents bicarbonate, (phosphorus and anhydrous oxychloride, sodium ethanol,sulfate) wereethyl of acetate, commercial petroleum analytical ether, grade dichloromethane, and were used withoutsodium furtherbicarbonate, purification. and anhydrous Silica gel sodium(200−300 sulfate) mesh, were Qingdao of commercial Makall Group analytical Co., Ltd., grade Qingdao, and were China) used was without used further in silica purification. gel column Silicachromatography. gel (200−300 Themesh, fins Qingdao salt was Makall obtained Group commercially Co., Ltd., Qingdao, (Qingdao China) Haike was General used Sea in silica Salt Co.,gel columnLtd., Qingdao, chromatography. China). The The pH fins was salt determined was obtained by commercially a pH meter (LEICI, (Qingdao Shanghai, Haike General China). Sea UV-vis Salt Co.,spectra Ltd., were Qingdao, measured China). on a The UV-Vis pH was spectrophotometer determined by (Agilenta pH meter Technologies, (LEICI, Shanghai, Waldbronn, China). Germany). UV‐vis spectraFluorescence were spectrameasured were on recorded a UV‐Vis on spectrophotometer a Tecan Infinite M1000 (Agilent Pro Technologies, Microplate Reader Waldbronn, (Tecan, Germany).Männedorf, Fluorescence Switzerland). spectra1H-NMR were and recorded13C-NMR on spectraa Tecan were Infinite recorded M1000 on Pro a Bruker Microplate Avance Reader IIITM (Tecan,500 MHz Männedorf, NB Digital Switzerland). NMR spectrometer 1H‐NMR (Bruker-Biospin, and 13C‐NMR spectra Billerica, were MA, recorded USA) in on CDCl a Bruker3 or DMSO- Avanced6 IIIwithTM 500 TMS MHz as an NB internal Digital standard. NMR spectrometer High-resolution (Bruker mass‐Biospin, spectra Billerica, (HRMS) MA, were USA) obtained in CDCl with3 or a DMSOMALDI‐d SYNAPT6 with TMS G2 as HDMS an internal system standard. (Waters, Milford, High‐resolution MA, USA). mass In general, spectra the(HRMS) designed were probe-KCP obtained withcompounds a MALDI were SYNAPT synthesized G2 HDMS as depicted system in (Waters, Scheme 1Milford,, and the MA, experimental USA). In general, details arethe shown designed in probeScheme‐KCP1. compounds were synthesized as depicted in Scheme 1, and the experimental details are shown in Scheme 1.

Molecules 2019, 24, 375 9 of 14 Molecules 2019, 24, x FOR PEER REVIEW 9 of 14

Scheme 1. The synthetic routes and the chemical structures of the probe-KCP. Reagents and Scheme 1. The synthetic routes and the chemical structures of the probe‐KCP. Reagents and conditions: ◦ conditions:(a) NaH, DMF, bromoethane; (b) POCl3, DMF, 90 C; (c)C2H5OH, C2H5ONa, (a)0 NaH, DMF, bromoethane; (b) POCl3, DMF,◦ 90 °C; (c) C2H5OH, C2H5ONa, 4′‐methoxy◦ ‐ 4 -methoxy-acetophenone; (d) BBr3, DCM, −78 C; (e) 2,4-dinitroﬂuorobenzene, Cs2CO3, DCM, 30 C. acetophenone; (d) BBr3, DCM, −78 °C; (e) 2,4‐dinitrofluorobenzene, Cs2CO3, DCM, 30 °C.

3.2. Synthesis of KCP-OCH3 3.2. Synthesis of KCP‐OCH3 The synthesis of carbazole compound 2 was performed in accordance with methods described in The synthesis of carbazole compound 2 was performed in accordance with methods described the literature [36,37]. The synthesis of the compound KCP-OCH3 was also based on the literature [38]. inCompound the literature2 (1 [36,37]. mmol) The was synthesis added to of a the solution compound of 40-methoxyacetophenone KCP‐OCH3 was also based (1 mmol) on the in literature ethanol (10[38]. mL), Compound and fresh 2 sodium(1 mmol) ethoxide was added (4 mmol) to a solution solution of (5 4 mL)′‐methoxyacetophenone was then added slowly. (1 mmol) The mixture in ethanol was (10stirred mL), at and room fresh temperature sodium ethoxide overnight. (4 mmol) The resulting solution precipitate (5 mL) was was then filtered added and slowly. recrystallized The mixture from was stirred at room temperature overnight. The resulting precipitate was filtered and recrystallized ethanol to give (E)-3-(9-ethyl-9H-carbazol-3-yl)-1-(4-methoxyphenyl)-prop-2-en-1-one (KCP-OCH3) from ethanol to give (E)‐3‐(9‐ethyl‐91H‐carbazol‐3‐yl)‐1‐(4‐methoxyphenyl)‐prop‐2‐en‐1‐one (KCP‐ as a faint yellow solid in 85% yield: H-NMR (500 MHz, CDCl3) δH 8.36 (s, 1H), 8.14 (d, J = 7.7 Hz, 1 1H),OCH 8.103) as (d, a faintJ = 8.5 yellow Hz, 2H), solid 8.06 in 85% (d, J yield:= 15.5 H Hz,‐NMR 1H), (500 7.77 MHz, (d, J = CDCl 8.4 Hz,3) δ 1H),H 8.36 7.60 (s, (d,1H),J = 8.14 15.5 (d, Hz, J = 1H), 7.7 Hz,7.50 1H), (t, J =8.10 7.6 (d, Hz ,J 1H),= 8.5 7.40Hz, (dd,2H), J8.06= 13.6, (d, J 8.3 = 15.5 Hz, Hz, 2H), 1H), 7.29 7.77 (t, J (d,= 7.4 J = Hz,8.4 Hz, 1H), 1H), 7.00 7.60 (d, J(d,= 8.5J = Hz,15.5 2H),Hz, 1H), 7.50 (t, J = 7.6 Hz, 1H), 7.40 (dd, J = 13.6, 8.3 Hz, 2H), 7.2913 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 8.5 Hz, 4.34 (q, J = 7.1 Hz, 2H), 3.89 (s, 3H), 1.44 (t, J = 7.2 Hz, 3H); C-NMR (125 MHz, CDCl3) δC 188.73, 13 163.08,2H), 4.34 145.52, (q, J = 141.25, 7.1 Hz, 140.36, 2H), 3.89 131.53, (s, 3H), 130.62, 1.44 126.20, (t, J = 7.2 126.00, Hz, 3H); 123.35, C 122.78,‐NMR 121.40,(125 MHz, 120.56, CDCl 119.57,3) δC 188.73, 118.69, 163.08, 145.52, 141.25, 140.36, 131.53, 130.62, 126.20, 126.00, 123.35, 122.78, 121.40, 120.56, 119.57,+ 113.68, 108.79, 77.00, 55.40, 37.67, and 13.78. The HRMS (ESI) m/z was calculated for C24H22NO2 118.69,[M + H] 113.68,+: 356.1651, 108.79, Found 77.00, 356.1641. 55.40, 37.67, and 13.78. The HRMS (ESI) m/z was calculated for C24H22NO2+ [M + H]+: 356.1651, Found 356.1641. 3.3. Synthesis of the probe-KCP 3.3. Synthesis of the probe‐KCP Boron tribromide (as a 1.0 M solution in dichloromethane; 2 mL, 2 mmol) was added dropwise overBoron a 10 min tribromide period (as to aa suspension1.0 M solution of KCP-OCHin dichloromethane;3 (0.5 mmol) 2 mL, in 2 dry mmol) dichloromethane was added dropwise (15 mL overcooled a 10 to min 0 ◦ periodC). The to resultinga suspension mixture of KCP was‐OCH stirred3 (0.5 mmol) at 0 ◦C in todry room dichloromethane temperature. (15 WatermL cooled and todichloromethane 0 °C). The resulting were mixture added. was The stirred organic at layer 0 °C wasto room separated temperature. and dried Water with and anhydrous dichloromethane Na2SO4. Afterwere added. concentrate The organic under reducedlayer was pressure, separated the and crude dried product with anhydrous was used forNa2 theSO4. next After step, concentrate without underseparation reduced or purification. pressure, the Then, crude Cs2 COproduct3 (Equation was used (2)) was for addedthe next and step, stirred without at room separation temperature or purification.for 15 min. Subsequently, Then, Cs2CO 2,4-dinitrofluorobenzene3 (2 eq.) was added wasand addedstirred and at stirredroom attemperature room temperature for 15 for min. 2 h. Subsequently,The resulting precipitate 2,4‐dinitrofluorobenzene was filtered and was recrystallized added and from stirred ethyl at acetate: room petroleumtemperature ether for (2v :vh.= The 2:8) resultingto (E)-1-(4-(2,4-dinitrophenoxy)phenyl)-3-(9-ethyl-9 precipitate was filtered and recrystallized Hfrom-carbazol-3-yl)prop-2-en-1-one ethyl acetate: petroleum ether (probe-KCP) (v:v = 2:8) asto 1 (aE give)‐1‐(4 yellow‐(2,4‐dinitrophenoxy)phenyl) solid (yield 76%): H-NMR‐3‐(9‐ethyl (500‐9H MHz,‐carbazol CDCl‐3‐3yl)prop) δH 8.84‐2‐en (s,‐1 1H),‐one 8.43–8.28(probe‐KCP) (m, as 2H), a give8.22–8.02 yellow (m, solid 4H), (yield 7.78 (d, 76%):J = 8.31H‐ Hz,NMR 1H), (500 7.59–7.47 MHz, CDCl (m, 2H),3) δH 7.42 8.84 (t, (s,J 1H),= 8.5 8.43–8.28 Hz, 2H), (m, 7.32–7.18 2H), 8.22–8.02 (m, 3H), 13 (m,7.12 4H), (d, J 7.78= 9.0 (d, Hz J ,= 1H), 8.3 Hz, 4.37 1H), (d, J 7.59–7.47= 6.9 Hz, (m, 2H), 2H), 1.45 7.42 (t, J =(t, 6.8 J = Hz,8.5 Hz, 3H). 2H),C-NMR 7.32–7.18 (125 (m, MHz, 3H), CDCl 7.123 (d,) δ CJ 188.63,= 9.0 Hz, 156.93, 1H), 154.91,4.37 (d, 147.21, J = 6.9 142.02,Hz, 2H), 141.55, 1.45 (t, 140.41, J = 6.8 136.66, Hz, 3H). 131.10, 13C‐ 128.85,NMR (125 126.41, MHz, 125.54, CDCl 123.43,3) δC 188.63, 122.70, 122.09,156.93, 121.75,154.91, 120.56, 147.21, 119.83 142.02, (d, 141.55,J = 11.5 140.41, Hz), 119.55, 136.66, 118.10, 131.10, 108.92, 128.85, 77.00, 126.41, 37.74, 125.54, and 13.80. 123.43, The 122.70, HRMS + + (ESI)122.09,m /121.75,z was calculated120.56, 119.83 for (d, C29 JH =22 11.5NO Hz),2 [M 119.55, + H] :118.10, 508.1509, 108.92, Found 77.00, 508.1503. 37.74, and 13.80. The HRMS (ESI) m/z was calculated for C29H22NO2+ [M + H]+: 508.1509, Found 508.1503.

3.4. Preparation of the Test Solutions The fluorescence properties of the probe‐KCP for thiophenols were evaluated in DMSO/PBS buffer (1:1, v/v, 20 mM, pH = 7.4). A stock solution of the probe‐KCP was prepared at a concentration

Molecules 2019, 24, 375 10 of 14

3.4. Preparation of the Test Solutions The fluorescence properties of the probe-KCP for thiophenols were evaluated in DMSO/PBS buffer (1:1, v/v, 20 mM, pH = 7.4). A stock solution of the probe-KCP was prepared at a concentration of 2.5 mM in 5 mL of DMSO and then diluted to the desired concentration with PBS (20 mM, pH = 7.4, containing 50% DMSO). Stock solutions of thiophenols and other analytes, in addition to those of common anions and metal ions, were prepared at a concentration of 5 mM in distilled water and then diluted to the desired concentrations with PBS (20 mM, pH = 7.4, containing 50% DMSO). Absorbance and fluorescence spectral data were recorded 30 min after the addition of the analytes. For fluorescence measurements, the excitation wavelength was 410 nm, and emissions were acquired from 450 nm to 800 nm.

3.5. General Procedure for Analysis Stock solutions (100 µM) of thiophenols (4-toluenethiol, 4-fluorothiophenol, 4-methoxythiophenol, and 4-aminothiophenol) and other analytes, including glutathione (GSH), cysteine (Cys), and homocysteine (Hcy); alanine (Ala), serine (Ser), aniline (Ph-NH2), phenol (Ph-OH), sodium hydrosulfide (NaSH) and ethanethiol (C2H5SH), sodium chloride (NaCl), Ferrous chloride (FeCl2), Potassium chloride (KCl), calcium chloride (CaCl2), ferric chloride (FeCl3), magnesium sulfate (MgSO4), Sodium nitrate (NaNO3), Potassium fluoride (KF) and Sodium bicarbonate (NaHCO3) were prepared with PBS buffer (20 mM, pH = 7.4, containing 50% DMSO). The resulting solution was kept at 37 ◦C for 30 min before recording its absorption and fluorescence spectra.

3.6. Kinetic Study To determine the reaction time of the probe-KCP, the increase in fluorescence intensity upon the addition of thiophenols (100 µM) to the probe solution (10 µM) in DMSO/PBS buffer (1:1, v/v, 20 mM, pH = 7.4) was investigated using a microplate reader. As shown in Figure S1, the addition of 4-methoxythiophenol immediately led to significant fluorescence enhancement at 540 nm for up to 30 min, followed by steady-state fluorescence. Taken together, these data indicated that the reaction time for the detection of thiophenols was 30 min. Thus, 30 min was considered an appropriate reaction time in subsequent experiments. The kinetic study indicated that the probe-KCP had a rapid response time, with high sensitivity for the detection of thiophenols.

3.7. Fluorescence Quantum Yield The ﬂuorescence quantum yield of the probe-KCP was determined according to methods described in the literature [39] using rhodamine 6G (Φ = 0.95, λex = 530 nm, in ethanol solutions) as a ﬂuorescence standard. The u values of the samples were calculated using the following equation:

F A n2 = u × s × u × φu 2 φs (2) Fs Au ns where the subscripts u and s denote the test and standard respectively, Φ is the ﬂuorescence quantum yield, A is the absorbance at the excitation wavelength, F is the area under the corrected emission curve, and n is the refractive index of the solvent used. The subscripts s and u refer to the standard and the sample.

3.8. Reaction Mechanism A possible reaction mechanism of the probe-KCP with thiophenols was proposed based upon studies of the UV-vis spectra, the ﬂuorescence intensity and other experiments. The reaction products were investigated by 1H-NMR, 13C-NMR, and mass spectrometry. The likely interaction mechanism of the probe-KCP with thiophenols is shown in Scheme2. The probe-KCP (Equation (1)) and 4-methoxythiophenol (Equation (1)) was dissolved in the DMSO/PBS (20 mM, pH = 7.4) solution, Molecules 2019, 24, 375 11 of 14 followed by stirring at room temperature for 2 h. The mixture was diluted with 10 mL of water and extracted by ethyl acetate (3 × 10 mL). The organic layer was evaporated under reduced pressure and puriﬁed by silica gel column chromatography to give probe-OH and S-NO2. The characterization data Molecules 2019, 24, x FOR PEER REVIEW 11 of 14 for the probe-OH and S-NO2 are given in the Electronic Supplementary Information (Figure S1).

Scheme 2. Proposed thiophenol detection mechanism of the probe. Scheme 2. Proposed thiophenol detection mechanism of the probe. 3.9. Detection of Thiophenols in Water Samples 3.9. Detection of Thiophenols in Water Samples Deionized water, artificial sea water, and tap water were filtered using a microfiltration membrane beforeDeionized use. The studiedwater, artificial water samples sea water, were keptand attap 4 ◦Cwater before were use. filtered The water using samples a microfiltration were spiked withmembrane different before concentrations use. The studied of thiophenol water samples (0, 2.0, 4.0, were 6.0, kept 8.0, 10.0at 4 µ°CM). before The solutions use. The werewater incubated samples forwere 30 spiked min before with measurementdifferent concentrations and the fluorescence of thiophenol intensity (0, 2.0, was 4.0, observed 6.0, 8.0, at10.0 540 μM). nm. The solutions were incubated for 30 min before measurement and the fluorescence intensity was observed at 540 3.10.nm. Cell Culture A human lung cancer cell line, A549, was purchased from the Cell Bank, Chinese Academy 3.10. Cell Culture of Sciences (Shanghai, China). The cells were maintained in F-12K, supplemented with 10% FBS, 100 U/mLA human of penicillin, lung cancer and cell 100 line,µg/mL A549, of streptomycin was purchased at 37from◦C. the The Cell cells Bank, were Chinese cultured Academy at 37 ◦C in of a humidifiedSciences (Shanghai, CO2 (5%) China). atmosphere. The cells were maintained in F‐12K, supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C. The cells were cultured at 37 °C in a 3.11.humidified Determination CO2 (5%) of Cellatmosphere. Viability Cell viability was determined using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium 3.11. Determination of Cell Viability bromide (MTT) assay. Cells in growth phase were plated into a 96-well culture plate at a density of 3 × 10Cell3 cells viability per well was and determined incubated forusing 24 h. a The3‐(4,5 cells‐dimethyl were then‐2‐thiazolyl) exposed to‐2,5 2.5,‐diphenyl 5, 10, 20, tetrazolium and 50 µM ofbromide the probe-KCP (MTT) assay. for 24 Cells h. Following in growth this phase treatment, were plated the cells into were a 96 further‐well culture incubated plate with at a MTTdensity for of 4 h.3 The× 103 medium cells per inwell each and well incubated was carefully for 24 h. removed, The cells and were DMSO then (150exposedµL) wasto 2.5, then 5, 10, added 20, and to each 50 μM well. of Thethe probe samples‐KCP were for thoroughly 24 h. Following agitated this for treatment, 10 min on the a shaker.cells were Finally, further the incubated absorbance with of the MTT samples for 4 h. at 490The nmmedium was measured in each well using was a microplatecarefully removed, reader. and DMSO (150 μL) was then added to each well. The samples were thoroughly agitated for 10 min on a shaker. Finally, the absorbance of the samples 3.12.at 490 Fluorescence nm was measured Microscopic using Cell a Imagingmicroplate reader. The A549 cells were seeded on a cover glass and incubated overnight. The probe-KCP dissolved in3.12. DMSO Fluorescence was added Microscopic to the cell Cell medium Imaging at a final concentration of 10 µM for 30 min. The medium was thenThe removed, A549 cells and were the cells seeded were on washed a cover with glass PBS and three incubated times. overnight. For comparison The probe purposes,‐KCP dissolved the cells ◦ werein DMSO pretreated was added with 4-methoxythiophenolto the cell medium at (300a finalµM) concentration at 37 C for 30of min,10 μM followed for 30 min. by washing The medium three times.was then Subsequently, removed, and the cellsthe cells were were treated washed with thewith probe-KCP PBS three for times. 30 min. For Thecomparison cells were purposes, imaged on the a FV1000cells were confocal pretreated laser with scanning 4‐methoxythiophenol microscope (CLSM, (300 Olympus, μM) at 37 Tokyo, °C for Japan). 30 min, followed by washing three times. Subsequently, the cells were treated with the probe‐KCP for 30 min. The cells were 4. Conclusions imaged on a FV1000 confocal laser scanning microscope (CLSM, Olympus, Tokyo, Japan). In summary, we described the synthesis of a safe and efficient chalcone fluorescent probe-KCP for the4. Conclusions detection of thiophenols. The probe-KCP responded selectively to thiophenols over other amino acidsIn and summary, common we metal described ions andthe synthesis to potential of a interference safe and efficient from otherchalcone substances fluorescent in DMSO/PBS probe‐KCP 2 (1:1,for thev/v detection, 20 mM, pH of =thiophenols. 7.4). The detection The probe limit‐KCP of the responded probe was 28selectively nM (R = to 0.9946). thiophenols Thus, we over conclude other thatamino the acids probe-KCP and common exhibits highmetal selectivity ions and toward to potential thiophenols. interference We used from the probeother tosubstances successfully in monitorDMSO/PBS and (1:1, quantify v/v, 20 highly mM, pH toxic = thiophenols.7.4). The detection This turn-on limit of fluorescence the probe was probe 28 nM features (R2 = 0.9946). a remarkably Thus, largewe conclude Stokes shift that (130 the probe nm) and‐KCP a fast exhibits response high time selectivity (30 min). toward It is highly thiophenols. selective We and used sensitive the probe in terms to successfully monitor and quantify highly toxic thiophenols. This turn‐on fluorescence probe features a remarkably large Stokes shift (130 nm) and a fast response time (30 min). It is highly selective and sensitive in terms of the detection of thiophenols, with significant fluorescence enhancement. In addition, this probe can be successfully applied to detect thiophenol in real water samples and in living cells. Therefore, this work provides a chalcone fluorescent probe (probe‐KCP) for the detection of the highly toxic thiophenols found in environmental and biological samples.

Molecules 2019, 24, 375 12 of 14 of the detection of thiophenols, with significant fluorescence enhancement. In addition, this probe can be successfully applied to detect thiophenol in real water samples and in living cells. Therefore, this work provides a chalcone fluorescent probe (probe-KCP) for the detection of the highly toxic thiophenols found in environmental and biological samples.

Supplementary Materials: The following are available online, Figure S1. date for investigation of the sensing mechanism. Figure S2. The effect of pH on the fluorescence intensity (λem = 540 nm) of probe-KCP (10 µM, λex = 410 nm) in DMSO/PBS buffer (1:1, v/v, 20 mM) upon addition of 100 µM 4-methoxy thiophenol after incubation at 37 ◦C for 20 min. Figure S3. Fluorescence responses of probe-KCP (10 µM) to thiophenol and other various analytes (100 µM) in PBS buffer solution (20 mM, pH = 7.4) containing 50 % DMSO. Figure S4. Enhanced fluorescence response at 540 nm of the probe-KCP (10 µM) to thiophenol and other various analytes (100 µM) in PBS buffer solution (20 mM, pH = 7.4) containing 50 % DMSO. Figure S5. Percentage of viable A549 cells after treatment with different concentrations of the probe-KCP after 24 h using an MTT assay. Figure S6. (A)Time-dependent the fluorescence response of probe-KCP (10 µM) in the absence (blank) and presence of 4-methoxythiophenol, GSH, Cys, Hcy, NaSH or C2H5SH (10 equiv) in PBS buffer solution (20 mM, pH = 7.4) containing 50 % DMSO. (B) Time-dependent the fluorescence response of probe-KCP (10 µM) in the absence (blank) and presence of 4-methoxythiophenol, GSH, Cys, Hcy, NaSH or C2H5SH (10 equiv) at 0 min, 10 min, 20 min, 30 min. Figure S7. Time-dependent the fluorescence response of probe-KCP (10 µM) in the presence of 4-methoxythiophenol (10 equiv) in PBS/ DMSO solution (v:v = 1:1, 20 mM, pH = 7.4). Figure S8. Photograph of probe-KCP solutions (10 µM) in the presence of 4-methoxythiophenol (10 equiv) under natrual light and UV irradiation (365 nm). Author Contributions: H.L. performed probe-KCP synthesis and characterization and data analysis, drafted the manuscript and finalized the manuscript; C.G. participated cell culture, determination of cell viability, fluorescence microscopic cell imaging; S.G. helped on probe-KCP synthesis and data analysis; L.W. helped on data analysis; D.S. guided the experiments. Funding: This work was supported by the National Natural Science Foundation of China (No. 81773586, 81703354, 81600782), and Shandong Provincial Natural Science Foundation for Distinguished Young Scholars (JQ201722), and Key Research Program of Frontier Sciences, CAS (QYZDB-SSW-DQC014), and the Project of Discovery, Evaluation and Transformation of Active Natural Com-pounds, Strategic Biological Resources Service Network Program of Chinese Academy of Sciences (ZSTH-026) and National Program for Support of Top-notch Young Professionals, and Taishan scholar Youth Project of Shandong province and Key research and development project of Shandong province (2018GSF118223) and Natural Science Foundation of Shandong Province (ZR2016DQ07). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds probe-KCP is available from the authors.

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