Dual-Site and Dual-Excitation Fluorescent Probe That Can Be Turned

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Dual-Site and Dual-Excitation Fluorescent Probe That Can Be Turned for Discriminative Detection of Cysteine, Homocystein, and Thiophenols Yang Yang, Yan Feng, Fangzhou Qiu, Kanwal Iqbal, Yingzhe Wang, Xuerui Song, Ying Wang, Guo-Lin Zhang, and Weisheng Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04163 • Publication Date (Web): 06 Nov 2018 Downloaded from http://pubs.acs.org on November 6, 2018

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Analytical Chemistry

Dual-Site and Dual-Excitation Fluorescent Probe That Can Be Turned for Discriminative Detection of Cysteine, Homocystein, and Thiophenols Yang Yang, Yan Feng, Fangzhou Qiu, Kanwal Iqbal, Yingzhe Wang, Xuerui Song, Ying Wang, Guolin Zhang*, and Weisheng Liu* Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, P. R. China

ABSTRACT: Thiols play a vital role in both the physiological process and organic synthesis field, including aliphatic thiols (e.g., Cys, Hcy, and GSH) and thiophenols. Due to the similarity of thiols in terms of molecular structure and chemical properties, it is difficult for conventional fluorescent probes to distinguish them, which hinders the progress of biological and pathological research. Keeping this in mind, a dual-site and dual-excite fluorescent probe (YY) was designed to distinguish Cys, Hcy, and thiophenols by three different reaction paths. When excited at 470 nm, YY only exhibits a fluorescence

* Corresponding author: Fax: +86-9318912582 E-mail address: [email protected] (G. Zhang); [email protected] (W. Liu)

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OFF-ON response towards thiophenols. However, when excited at 453 nm, YY not only displays a fluorescence OFF-ON response towards Hcy and thiophenols (λem = 499 and 561 nm) but also presents a two-stage fluorescence response towards Cys, which possesses a fluorescence OFF-ON response in the first stage (λem = 501 nm) and then a fluorescence ON-OFF response in the second stage (λem = 556 nm). This specific fluorescence response indicates that YY has ability to overcome the above-mentioned challenge to achieve discriminative detection of Cys, Hcy, and thiophenols qualitatively, which promotes the study of thiols in the field of physiology and pathology. Furthermore, cell-imaging studies show that YY can be applied to the imaging of exogenous Cys, Hcy, and thiophenols through two different emission channels.

Introduction Thiols, including biothiols (eg., Cys, Hcy, and GSH) and thiophenols, are vital compounds in industrial production and biological systems.1-3 Intracellular biothiols are important signal molecular and play significant role in various biological process, including protein synthesis, cellular metabolism and antioxidant defense.4 Aberrant concentration of biothiols are closely connected to a series of pathological illness. The normal levels of intracellular Cys is 30-200 μM and the abnormity of Cys can give rise to neurotoxicity, poor growth, dropsy, weakness, skin lesions, hepatic injury, decreased hematopoiesis and AIDS.5 The normal intraserous concentration of Hcy is approximately 5-12 μM and elevated concentration of Hcy may lead to cardiovascular disease, coronary


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heart disease, neural tube defect, osteoporosis and Alzheimer’s disease.6 For GSH, the normal intracellular concentration is 1-10 mM.7 Because of their importance, researchers have done a lot of work for the detection of biothiols.8-11 Due to the similarity of molecular structure and chemical properties, it is difficult for conventional fluorescent probes to distinguish Cys, Hcy and GSH, which greatly barrier their research progress in physiology and pathology. Up to now, probes having the ability to distinguish these three biothiols are few in number.12-14 In 2016, Yin’s group reported a pH-dependent fluorescent probe, which was directly synthesized by modified coumarin and acryloyl chloride. This probe can intelligently distinguish Cys and Hcy by different sensing mechanisms and the detection mechanism towards Cys is based on a two-stage process (pH = 7.8), which includes a Michael

addition

process

in

the

first

stage

and

then

an

intramolecular

cyclization-cleavage process in the second stage. But when the test occurs in an environment with slightly lower pH value (pH = 7.4), the probe no longer has the ability to distinguish Cys and Hcy. Due to the pH value of the intracellular environment is about 7.4, which is serious hinder the simultaneous determination of Cys and Hcy in vivo. Subsequently, Gong et al. reported a BODIPY-functionalized bio-probe to specifically detect Cys through an intramolecular charge transfer (ICT) quenching process. Wang and co-workers utilized fluorescein and coumarin fluorophores to synthesize a dual mode fluorescent probe to distinguish Cys and the others. The bio-probe exhibits a fast detection process towards Cys (∼5 min), showing a ratiometric fluorescence change upon



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excitation at 332 nm. Interestingly, it also shows a turn-on fluorescence enhancement when excited at a higher wavelength (450 nm). Unfortunately, the above-mentioned two probes can only be used to detect Cys, and a fluorescent probe capable of detecting Cys, Hcy and GSH is still needed. Thiophenols, also named benzenethiols, are extensively used in medicine, pesticides, polymer materials and additives for organic synthesis.15,

16

However, as a toxicant to

aquatic ecological enviroment, the median lethal dose concentrations (LC50) were reported in a range of ∼0.01-0.4 mM in fish and 46.2 mg/kg in mouse.17 Prolonged contact can lead to serious consequences such as bronchospasm, edema, respiration disorder, muscle atrophy, chemical pneumonia and pneumonedema.18 To protect the environment from the pollution caused by thiophenols, a simple, rapid, sensitive, and efficient fluorescent probe which qualitatively determines the thiophenols is urgently needed for the chemical, biological and environmental science. Both thiophenols and biothiols (Cys, Hcy and GSH) have a mercapto functional group (-SH), so they have similar chemical property, which leads to a great challenge to identify them. As far as we know, there is no open literature has been published to date which distinguishes the detection for Cys, Hcy, and thiophenols through three completely different reaction mechanisms. Keep this in mind, we reasonably designed a dual-site and dual-excite fluorescent probe (YY) that can be applied to discriminate Cys, Hcy, and thiophenols. As we have seen, YY is the first reported fluorescent probe which distinguishes Cys, Hcy, and


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thiophenols based on different reaction mechanisms (Scheme 1). Furthermore, cell-imaging studies show that YY can be used for imaging of exogenous Cys, Hcy, and thiophenols through two different emission channels.

Scheme 1. The Discriminative Detection of YY towards Cys, Hcy, and thiophenols.

Experimental section Synthesis. Compounds 2, 3, and YY were synthesized as described in Supporting Information (Scheme S1). 1H NMR (400 MHz, CDCl3) δ 8.87 (d, J= 2.7 Hz, 1H), 8.58 (s, 1H), 8.35 (dd, J= 10, 2.8 Hz, 1H), 8.17 (d, J= 16 Hz, 1H), 7.80 (d, J= 16 Hz, 1H), 7.77 (d, J= 12 Hz, 2H), 7.45 (d, J= 12 Hz, 1H), 7.16 (d, J= 8.0 Hz, 2H), 7.10 (d, J= 8.0 Hz, 1H), 6.65 (dd, J= 10, 2.5 Hz, 1H), 6.50 (d, J= 4.0 Hz, 1H), 3.48 (q, J= 8.0 Hz, 4H), 1.26 (t, J= 8.0 Hz, 6H). HRMS: calcd for C28H23O8N3 [M+H]+, 530.1563; found, 530.1553 (Figure S1-S9).



Fluorescence Imaging. HeLa cells were placed in a 96-well culture dish and incubated for 24 h in an adapt circumstance. Firstly the cells were pre-treated with N-ethylmaleimide (NEM) to get rid of the cellular biothiols and SH-containing proteins. Then, YY (10 μM) solution was injected and the cells were incubated for 30 minutes. Cys/Hcy/thiophenols solutions were subsequently added, and the cells were further incubated for 30 minutes. After washing the cells, Olympus FV1000-IX81 laser confocal microscope was used for the cells imaging of Cys/Hcy/thiophenols. Meanwhile, the other groups were performed as a control group. Determination of Thiophenols in Real Sample. As descried previously,19 the standard addition method was employed to verify the ability of YY to detect thiophenols in crude water samples (Yuxiu Lake in Lanzhou University and Yellow River in Lanzhou city of China), which were pretreated by a filtration step through a microfiltration membrane before the test. Four concentrations of thiophenols (0, 5, 10, 20 μM) were prepared, and then the recovery of each water sample was measured.

Results and discussion Design Strategy of YY. Probe YY was synthesized through a three-step method, which consists of the α,β-unsaturated ethanoylcoumarin fluorophore and a strong electron-withdrawing 2,4-dinitrobenzene group. The α,β-unsaturated ethanoylcoumarin moiety functions as an excellent biothiols recognizing site through a nucleophilic addition reaction process.20 The 2,4-dinitrobenzene group not only functions as a


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thiophenols recognizing site but also severs an outstanding fluorescence quenching group.21 Based on the above considerations, we designed a dual-site and dual-excite fluorescent probe (YY) that could effectively distinguish Cys, Hcy, and thiophenols. To our knowledge, YY is the first fluorescent probe that can effectively distinguish Cys, Hcy, and thiophenols based on different reaction mechanisms, which promotes the study of thiols in the field of physiology and pathology. Sensing Properties of YY with the Excitation at 470 nm. In order to demonstrate the selectivity of YY, analytes including N3−, F−, Cl−, Br−, I−, HSO3−, CH3COO−, SCN−, CN−, NO2−, NO3−, SO42−, CO32−, H2PO4−, ClO−, ClO4−, HCO3−, HPO4−, H2O2, HOCH2CH2SH, C6H5NH2, Na2S, and various amino acids were tested in DMSO-HEPES buffer solution (1:1, v/v, 10 mM, pH = 7.4) at 37 °C. As pictured in Figure S10, only thiophenols can causes a significant emission enhancement at 561 nm, not even other analytes result in an obvious optical change. To further prove the selectivity, competition experiments were also measured in the exist of various interfering materials. As we expected, other interfering materials don’t lead to any obvious interference for the detection process of thiophenols (Figure S11). Subsequently, the absorption and emission titration spectra of YY towards thiophenols were measured. As displayed in Figure S12, free YY exhibits weak emission intensity at 561 nm. The emission intensity dramatically enhances with the addition of thiophenols, and then shows no change with a 7.2-fold fluorescence enhancement (Figure S13). The emission intensity and the concentration of thiophenols present a good linear



relationship, the detection limit based on the IUPAC definition is calculated to be 0.84 µM (Figure S14), which is much lower than the median lethal dose concentrations (LC50). The time-dependent emission spectra of YY shows that the detection process completed in 10 min (Figure S15), which is faster than most of the probes for thiophenols already reported (Table S1). Thus, YY can be employed to fast and sensitively detect thiopheols.

Sensing Properties of YY with the Excitation at 453 nm. Encouraged by previous work,12 we further measured the absorption and emission spectra with a shorter excitation wavelength (453 nm). In the subsequent measurements some interesting phenomena was observed. Probe YY exhibits three completely different fluorescence responses after the individual addition of Cys, Hcy, and thiophenols. The fluorescence spectra of YY towards thiophenols were measured. As displayed in Figure S16, free YY displays weak emission intensity at 561 nm. Upon the continuous addition of thiophenols, a significantly enhanced emission was recorded. The emission intensity and the concentration of thiophenols present a good linear relationship (Figure S17). The detection limit is calculated to be 1.3 μM, indicating that YY has ability to sensitively detect thiophenols. In addition, no distinct spectral change was observed in presence of various interfering substances (Figure S18). The time-dependent fluorescence response shows that the detection process completed in 10 min (Figure S19). The sensing properties of YY towards biothiols were systematically studied by


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time-dependent absorption and emission spectra. The emission intensity of YY enhances intensively and shows no change in 7 min upon the addition of Cys, accompanying with a conspicuous blue shift from 561 nm to 501 nm (Figure 1a). Then the emission intensity decreases slowly and shows a red shift response, accompanying with the appearance of a second fluorescence emission centered at 556 nm (Figure 1b). The time-dependent absorption spectra of YY for Cys shows a significant blue shift response from 470 nm to 458 nm in the initial half hour and then reverses with a red shift response from 458 nm to 463 nm in the succedent 90 min, which is consistent with the emission spectra (Figure 1c,d). Interestingly, the addition of Hcy and GSH does not lead to any appearance of the second fluorescence emission centered. The fluorescence intensity of YY increases gradually and shows no change in 30 min upon the addition of Hcy, accompanying with an obvious blue shift from 561 nm to 499 nm (Figure 2a,c). The time-dependent absorption spectra of YY for Hcy display a significant blue shift response from 470 nm to 464 nm, which is also consistent with the emission spectra (Figure 2b). Moreover, the addition of GSH also induces an apparent blue shift response from 561 nm to 503 nm and balances in 60 min (Figure S20). However, compared with Cys and Hcy, the emission intensity of YY induced by GSH (at ∼500 nm) is weak and negligible. The different reactivity of Cys/Hcy/GSH leads to three different fluorescence responses, which provides a superexcellent opportunity to distinguish these three biothiols.



Figure 1. Time-dependent emission (a, b) and absorption spectra (c, d) of 10 μM YY upon addition of 30 equiv of Cys.

The spectroscopic tests indicate that YY not only reveals a good selectivity towards thiophenols but also has an outstanding selectivity towards biothiols (Cys and Hcy) over other interfering substances (Figure 2d). The varying fluorescence changes towards Cys, Hcy, and thiophenols indicate that YY can simultaneously distinguish Cys, Hcy, and thiophenols by three different fluorescence emission channels. In addition, competition experiments further demonstrate that there is no obvious interference phenomenon of other analytes in the identification process (Figure S21 and S22). Thus, YY can be used as the first fluorescent probe to effectively distinguish Cys, Hcy, and thiophenols.


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Figure 2. (a, b) Time-dependent emission and absorption change of YY (10 μM) upon addition of 30 equiv of Hcy; (c) Time-dependent emission intensity changes of YY towards 30 equiv of Hcy; (d) Fluorescence response of YY upon addition of 30 equiv of Cys, Hcy, GSH, and thiophenols.

Proposed Mechanism. According to the spectral analysis and reported literature,13, 22

the proposed mechanism of YY towards Cys, Hcy, and thiophenols is presented in

Scheme 1. Subsequently, the 1H NMR titration and HRMS spectra were carried out to prove the mechanism. As shown in Figure S23a, the signals at 8.15 and 7.79 ppm were disappeared, which pertain to the α,β-unsaturated ketone protons of YY. At the same time, a new signal was appeared at ~9.75 ppm, which belongs to the phenolic hydroxyl



proton. The signal changes indicate that the detection process of YY towards Cys consists of nucleophilic addition and intramolecular cleavage. Furthermore, we could clearly see m/z = 485.1747 and m/z = 651.1766 in the HRMS spectra, which belong to the addition product (m/z = 485.1741) and cleavage product (m/z = 651.1755), respectively (Figure 3a). For Hcy, however, we only found the disappearance of the signals of α,β-unsaturated ketone protons, indicating that a nucleophilic addition reaction was occurred. Moreover, we only found the m/z = 665.1920 in the HRMS spectra, which belongs to the addition product (m/z = 665.1912) (Figure 3b). For thiophenols, the isolated products of sensing reaction were obtained. As we expected, the compound 3 and isolated product 1 are the same compound, which can be proved by the same 1H NMR spectra. In addition, we can clearly see from the 1H NMR spectra that isolated product 2 is the compound 4 (Figure S23b).


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Figure 3. (a) HRMS spectra of probe YY-Cys system and (b) YY-Hcy system.

Fluorescence Imaging in HeLa Cells. Encouraged by the favorable spectroscopic properties, the practicability of YY to detect biothiols in living cells was investigated.



Before cell-imaging experiments, the MTT assay was employed to study the cytotoxicity of YY. The cell viability is as high as 94% even when YY is at a high concentration (100 μM), indicating that YY has slight toxicity to living cells (Figure S24). As displayed in Figure 4, the cells treated with 10 μM YY and 1 mM NEM show weak fluorescence emission in red and green channels. The cells further treated with exogenous 100 μM Cys and incubated for 10 min display bright fluorescence emission in red and green channels, and the fluorescence emission in red channel is stronger than the green channel. However, the cells further treated with Cys and incubated for 30 min show a relatively weaker fluorescence emission compared to the upper group of cells. The above phenomenon shows that two reaction processes are occurred upon the addition of Cys. For Hcy, the cells also display significant fluorescence emission in red and green channels, and the fluorescence emission in red channel is stronger than the green channel. As for GSH, however, the cells display dim fluorescence emission in red and green channels. To know the process deeply, we monitored the imaging of different incubation time (2, 5, 10, 20, and 30 min) of YY in living cells after the addition of biothiols. Upon the addition of Cys, the intracellular fluorescence emission increases both in red and green channels (0-10 min), and then the fluorescence emission gradually decreases (10-30 min), which is consistent with the time-dependent emission spectra of YY and Cys (Figure 5a). As expected, the cells incubated with Hcy or GSH display increasing fluorescence emission both in red and green channels (0-30 min), which is also consistent with the time-dependent emission spectra (Figure 5b and S25). The distinct fluorescence


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signal in red and green channels makes distinguishable detection of Cys/Hcy in vivo to become reality, which accelerates the clarification of their effects in biological and pathological progress. Furthermore, we also studied the cell imaging of thiophenols in HeLa cells. As is shown in Figure S26, it can be see clearly that the cells show increasing red fluorescence with the addition of the concentration of thiophenols. Thus, all cell-imaging studies show that YY can be applied to the real-time imaging of exogenous Cys, Hcy, and thiophenols through two different emission channels.

Figure 4. Fluorescence images of Cys, Hcy, and GSH in HeLa cells. (A1-A4) Cells incubated with NEM (1 mM) and YY (10 μM) for 30 min; (B1-B4) Cells incubated with NEM (1 mM), YY (10 μM), and Cys (100 μM) for 10 min; (C1-C4) (D1-D4) (E1-E4)



Cells incubated with NEM (1 mM), YY (10 μM), and Cys/Hcy/GSH (100 μM) for 30 min at 37 °C, respectively. λex = 458 nm, green channel is at 500 ± 20 nm; red channel is at 600 ± 30 nm.


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Figure 5. Time-dependent fluorescence images of biothiols in HeLa cells. Cells incubated with NEM (1 mM), YY (10 μM), and Cys (100 μM) (a) and Hcy (100 μM) (b)



at 37 °C. λex = 458 nm, green channel is at 500 ± 20 nm; red channel is at 600 ± 30 nm.

Real Sample Detection of Thiophenols. In order to determine the feasibility of YY to detection thiophenols in natural water sample, we measured tests in crude water samples (Yuxiu Lake in Lanzhou University and Yellow River in Lanzhou city of China). Four concentrations of thiophenols (0, 5, 10, 20 μM) were spiked in these two samples, and then treated with YY. The emission intensity of water samples at 561 nm was measured. As recorded in Table S2, thiophenols recoveries were varied from 91% to 102%, revealing that YY has potential application to detect thiophenols in real water samples.

Conclusions In conclusion, we have successfully synthesized a dual-site and dual-excite fluorescent probe (YY) based on a hybrid of α,β-unsaturated ethanoylcoumarin fluorophore and 2, 4-dinitrobenzene group, which can simultaneously detect Cys, Hcy, and thiophenols by two different emission channels. When excited at 470 nm, YY exhibits a fluorescence OFF-ON response towards thiophenols. When excited at 453 nm, YY not only shows a fluorescence OFF-ON response towards Hcy and thiophenols (λem = 499 and 561 nm) but also exhibits a two-stage fluorescence response towards Cys, which possesses a fluorescence OFF-ON response in the first stage (λem = 501 nm) and then a fluorescence ON-OFF response in the second stage (λem = 556 nm). Furthermore,


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cell-imaging studies show that YY can intelligently distinguish exogenous Cys, Hcy, and thiophenols using red and green channels. The study provides a new way to explore the function of thiols, which promotes the research process of thiols in physiology and pathology.

Acknowledgments This study was supported by the National Natural Science Foundation of China (Grants No. 21431002).

Supporting Information Materials and instruments, experimental procedure, synthesis, characteristic (1H NMR,

13C

NMR, MS, and HRMS spectra), additional spectroscopic data, cell

cytotoxicity, and supplemental fluorescence images of cells.



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Dual-Site and Dual-Excitation Fluorescent Probe That Can Be Turned

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