- Email: [email protected]
A dual-selective fluorescent probe for GSH and Cys detection: Emission and pH dependent selectivity
Accepted Manuscript A dual-selective fluorescent probe for GSH and Cys detection: Emission and pH dependent selectivity Yunqiang Tang, Longyi Jin, Bingzhu Yin PII:
S0003-2670(17)31077-2
DOI:
10.1016/j.aca.2017.09.028
Reference:
ACA 235453
To appear in:
Analytica Chimica Acta
Received Date: 16 June 2017 Revised Date:
7 September 2017
Accepted Date: 20 September 2017
Please cite this article as: Y. Tang, L. Jin, B. Yin, A dual-selective fluorescent probe for GSH and Cys detection: Emission and pH dependent selectivity, Analytica Chimica Acta (2017), doi: 10.1016/ j.aca.2017.09.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphic abstract
ACCEPTED MANUSCRIPT
A dual-selective fluorescent probe for GSH and Cys detection: emission and pH dependent selectivity Yunqiang Tang, Longyi Jin* and Bingzhu Yin* Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules of the
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a
Ministry of Education, Department of Chemistry, Yanbian University, Yanji 133002, PR China
Abstract
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e-mail: [email protected]
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A novel fluorescent probe 1 based on acridine orange was developed for the selective detection and bioimaging of biothiols. The probe exhibits higher selectivity and turn-on fluorescence response to cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) than to other amino acids. Importantly, the probe responds to GSH and Cys/Hcy with distinct fluorescence emissions in PBS
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buffer at pH of 7.4. The Cys/Hcy-triggered tandem SNAr-rearrangement reaction and GSH-induced SNAr reaction with the probe led to the corresponding amino-acridinium and
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thio-acridinium dyes, respectively, which can discriminate GSH from Cys/Hcy through different emission channels. Interestingly, Cys finishes the tandem reaction with the probe and
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subsequently forms amino-acridinium and Hcy/GSH induces SNAr reaction with the probe to form thio-acridiniums at weakly acidic conditions (pH 6.0), enabling Cys to be discriminated from Hcy/GSH at different emissions. Finally, we demonstrated that probe 1 can selectively probe GSH over Cys and Hcy or Cys over GSH and Hcy in HeLa cells through multicolor imaging.
Key Words: Acridine orange; fluorescent probe; GSH; Cys; detection; cell imaging.
ACCEPTED MANUSCRIPT 1. Introduction Intracellular thiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play critical roles in many biological processes [1-4]. Although they exhibit similar reactions, their
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functions in vivo are entirely different. GSH has a balance with its oxidation state, glutathione disulfide (GSSG). GSH can be oxidated to GSSG by cellular oxidizing species, which limit the concentration of cellular oxides and prevent the stacking of these compounds. Changes cellular
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GSH levels are associated with many diseases, such as liver damage, leucocyte loss, psoriasis,
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cancer, and HIV infection [5-9]. Cys is a basic amino acid for protein synthesis and related to the formation of the three-dimensional protein structures. Moreover, Cys can strongly bind to metal ions and balance their intracellular concentrations, thereby protecting cells from damages caused by high concentrations of heavy ions. Several studies indicated that abnormal intracellular Cys
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levels are closely related to several diseases, such as Parkinson's disease and Alzheimer's disease, and adverse pregnancy outcomes [10,11]. Meanwhile, Hcy has a similar molecular structure with Cys, and its intracellular concentration is rather low. Moreover, it is an independent factor for
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some diseases, including cardiovascular disease and Alzheimer's disease [12-14]. Thus, Hcy is
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attracing considerable attention, and Hcy level is considered as one of the important parameters for some diseases. Meanwhile, developing methods for selective and sensitive detection of biothiols is significant in the area of clinical diagnosis. Considerable progress is already achieved in this undertaking particularly in the development of fluorescent probes for thiol detection, in which the strong nucleophilicity of the thiol group is exploited [15-19]. However, although these probes can highly selectively distinguish these biothiols from other amino acids, most of them cannot distinguish Cys, Hcy and GSH from one another because of the structures and reactivity of
ACCEPTED MANUSCRIPT these biothols are similar. Based on the two strategies of by Strongin et al. [20, 21], who first achieved ring formation of Cys with aldehydes or acrylates, numerous specific probes for Cys [22-36] or Hcy [37-43] were recently developed on the basis of various molecular recognition or
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thiol-specific reaction strategies. Similarly, inspired by the GSH-induced SNAr substitution reaction developed by Yang et al. [44], several researchers explored some specific probes for GSH [45-55]. Meanwhile, constructing two or three thiol-responsive probes is highly valuable but more
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challenging than constructing single thiol-responsive fluorescent probes. However, to the best of
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our knowledge, only one fluorescent probe has been developed for simultaneous detection and multicolor imaging of GSH and Cys at different emission channels [56]. Observations in previous studies showed that Cys is more reactive in nucleophilic reactions than Hcy and GSH owing to the differences among their pKa values, Yoon et al. [57] reported recently that an
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aryl-thioether-substituted nitrobenzothiadiazole probe is more selective to Cys over Hcy and GSH under weakly acidic conditions (pH 6.0).
Basing on the results of these studies, we designed and synthesized a thiol-selective
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fluorescence probe by incorporating a 3,4-dimethoxythiophenol moiety to the 9-position of
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acridinium dye. The strategy used for the design of present fluorescence probe exploits the reaction activity of the 9-position of acridines and acridiniums [58-61]. The probe not only can discriminate GSH from Cys or Hcy through different emission channels but also can discriminate Cys from GSH or Hcy through the adjustment of the pH of the solution contained in a PBS buffer with relatively fast kinetics and obvious fluorescence turn-on response. In addition, the probe can simultaneously and selectively detect Cys and GSH in HeLa cells through multicolor imaging.
2. Experimental
ACCEPTED MANUSCRIPT 2.1. General information and materials Commercially available compounds were used without further purification. Solvents were dried according to standard procedures [62]. All reactions were magnetically stirred and monitored
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by thin-layer chromatography (TLC) using Qingdao GF254 silica gel coated plates. Fluorescence spectra were carried out on a Shimadzu RF-5301PC fluorescence spectrophotometer. UV–vis spectra were recorded with a Shimadzu UV-2550 spectrophotometer. NMR spectra were recorded 13
C), and chemical shifts
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on a Bruker AV-300 Spectrometer (300 MHz for 1H and 75 MHz for
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were referenced relative to tetramethylsilane (δH/δC = 0). Mass data were obtained by a Shimadzu AXIMA-CFRTM plus mass spectrometry, using a 1,8,9-anthracenetriol (DITH) matrix. The high-resolution mass spectra were obtained using a Bruker micro TOF II focus spectrometer (ESI).
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2.2. Synthesis and characterization
The starting material acridine-oringe base was purchased from J&K chemical ltd. and was
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used without further purification. Synthesis of probe 1, the intermediates, and the control
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compounds are given in supplementary information (SI).
2.3. Cell culture and fluorescence imaging HeLa cells were obtained from American Type Culture collection and grown in Dulbecco
Modified Eagle Medium (Free DMEM/high: with 4500 mg/L Glucose, 4.0 mM L-Glutamine, and 110 mg/L Sodium Pyruvate). The cells were incubated in a 5% CO2 humidified incubator at 37°C and typically passaged with sub-cultivation ratio of 1:4 for two days. The HeLa cells were seeded in 6-well culture plate overnight. Stock solutions of probe 1 (3 mM) and NMM (0.03 M) in
ACCEPTED MANUSCRIPT ethanol, Cys, Hcy and GSH (0.15 M) in PBS buffer (10 mM) were prepared at the same day of experiment. Before the experiments, cells were washed with PBS buffer 3 times. Then, the cells were
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incubated with probe 1 (10 µM), or pretreated with Cys (500 µM, 30 min), Hcy (500 µM, 30 min) or GSH (500 µM, 30 min) in DMEM medium at 37°C. After each treatment, the cells were washed with PBS buffer 3 times. The cells were imaged using an inverted microscope
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(OLYMPUS IX73). The fluorescence in green channel was excited at 450-480 nm, and in red
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channel was excited at 530-550 nm.
2.4. General Procedure for Spectral Measurements
Deionized water and spectroscopic grade EtOH were used as solvents for the spectral studies. The spectral determinations were conducted in 10 mm quartz cuvettes at room temperature. Probe
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1 was dissolved in EtOH to prepare a concentration of 1 × 10−3 M stock solution, which was diluted to 1 × 10−5 M with pH 7.4 PBS buffer (10 mM, 1% EtOH) and pH 6.0 PBS buffer (10 mM,
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20% EtOH), respectively. The thiols were dissolved in pH 7.4 PBS buffer (10 mM, 1 % EtOH) and pH 6.0 PBS buffer (10 mM, 20 % EtOH), respectively, to prepare the stock solutions. The
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absorbance was measured from 220 to 800 nm in the corresponding blank PBS buffers, and 10 equiv. of thiols were added to the 10 µM host solution (3 mL). The emission was measured from 400 nm to 800 nm, and the different amounts of thiols solutions were added to the 10 µM host solution (3 mL) in portions (total volumes of 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 36, 42, 48, 54, 60, 75, 90, 120 and 150 µL of 1 × 10−3 M GSH, and 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 75, 90, 120, 150, 180, 210, 240, 270 and 300 µL of 5 × 10−4 M Cys). The resultant solution was shaken well and the absorption and emission spectra were recorded immediately.
ACCEPTED MANUSCRIPT 3. Results and discussion
3.1. Synthesis of probe 1
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The synthetic route of probe 1 is depicted in Scheme 1. Intermediate acridinium 2 was readily prepared from an acridine orange base and ethyl bromoacetate at an 89% yield. The reaction of acridinium 2 with sodium cyanide in DMSO-water (95:5) solvent system causes a color change
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from brick-red to dark blue. The reaction shows that a cyanide-adduct forms initially and rapidly reacts with air to produce acridinone 3 at a moderate yield (49%). The direct conversion of
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acridinone 3 to probe 1 through the successive treatments with trifluoroacetic anhydride and 3,4-dimethoxythiophenol, a procedure employed for the pyroninone [63], ended in failure. Fortunately, 9-chloroacridinium 4, which was obtained from the chlorination reaction of acridinone 3 with phosphorus oxychloride, was successfully converted to the 9-position
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aryl-thioether-substituted acridinium dye (probe 1) through a SNAr substitution with 3,4-dimethoxythiophenol in the presence of triethylamine in CH3CN. The chemical structures of
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HRMS spectra.
13
C-NMR, MALDI-TOF MS and
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the intermediates and probe 1 were confirmed by 1H-NMR,
Scheme 1
Furthermore, 3,4-dimethoxythiophenol moiety was introduced to acridinium dye because it
functions not only as a leaving group in the thiol-induced SNAr substitution reaction and as a fluorescence quencher via the photo-induced electron transfer (PET) process, which ensures a low fluorescence background. A Cys- or Hcy-induced SNAr-rearrangement cascade reaction with the probe leads to the corresponding amino-acridinium 5a (or 5b). However, in the case of GSH, the
ACCEPTED MANUSCRIPT initial SNAr reaction lead to thio-acridinium 6, which does not occur at the subsequent intramolecular rearrangement. The reason is that to thio-acridinium 6 has a bulky tripeptide and unstable 10-membered macrocyclic transition state (if any), as shown in Scheme 2. Given the
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distinct photophysical properties of amino- and thio-acridinium dyes, probe 1 is likely to sense Cys/Hcy and GSH from two different emission channels. In addition, assuming that the fluorescence behavior of the probe toward thiols is dependent on the pH value of the employed
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medium, that is, only Cys can induce tandem SNAr-rearrangement reaction to form corresponding
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amino-acridinium 5a under weak acidic conditions. However, the reaction trigged by Hcy and GSH stayed at SNAr reaction stage to provide thio-acridiniums 6 and 7 at same condition. A combination of these factors can serve as the basis for a selective response of probe 1 to Cys from Hcy and GSH. Overall, this design can potentially facilitate the selective discrimination of Cys
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and GSH on the basis of the differences between their chemical structures and identification of the distinct photophysical properties of the corresponding amino-acridiniums and thio-acridiniums.
Scheme 2.
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The design concepts for the probe are summarized in Scheme 2.
3.2. pH dependent studies
The major determinant of selectivity in the tandem reaction of thiols with present probe 1 is
expected to be the nature of the nucleophile, such as pKa and the pI values of the thiols. At initial SNAr reaction stage, thiolate anions are more reactive than free thiols. However, during sequential thiol–amine rearrangement reaction step, free amines are more reactive than ammonium cations. Thus, for an analogous set of substrates, the presence of an increased fraction of thiolate or free
ACCEPTED MANUSCRIPT amine at any given pH is expected to generate an amplified relative reactivity [64]. Given that the pKa values of the Cys, Hcy, and GSH are 8.53, 10.00, and 9.20, respectively [65], and their pI values are 5.02, 5.27, and 5.93, respectively [66, 67], investigating the effect of pH on the probe
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towards Cys, Hcy, and GSH and determining the optimal sensing conditions is important. In the present study, the fluorescence pH titrations of probe 1 were performed in PBS buffer solutions containing 1% and 20% EtOH to identify the conditions under which the ability of the probe to
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detect biothols can be explored. First, a PBS buffer solution containing 1% EtOH was selected.
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Upon excitation by 537 nm, no fluorescence response by probe 1 was observed after the addition of GSH at pH below 6.0. Probe 1 suddenly had a response to GSH at pH 6.0 and remained unaffected between pH 6.0 and 12. By contrast, the addition of Cys (or Hcy) barely showed any emission response in pH of 2.0–12 (Fig, 1a). The excitation wavelength of 438 nm was then
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selected to probe Cys or Hcy. A strong fluorescence response of probe 1 to Cys (or Hcy) was observed at approximately pH 7.4. Response to GSH had no significant fluorescence response at pH of 2.0–12 (Fig. S1a). Afterward, the effect of pH on the ability of probe 1 to detect thiols in a
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PBS buffer solution containing 20% EtOH was examined at pH range from 2 to 12. In contrast to
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the above-mentioned case, the reaction of probe 1 with GSH had no sensing response upon 438 nm excitation in this pH range (Fig. 1b). Interestingly, a rapid sensing response of probe 1 to Cys was observed in the pH range of 6.0–8.0. However, with regard to the response of the probe to Hcy, the pH range of sensing lagged behind, showing a pH range from 7.0 to 9.0. Second, the excitation wavelength of 537 nm was selected to probe the biothiols. As seen in Fig. S1b, after the addition of Cys to a solution containing probe 1, only a minimal sensing response in the fluorescence spectra was observed. In the case of GSH, probe 1 showed a good sensing ability in
ACCEPTED MANUSCRIPT the pH range of 6 to 12, but Hcy showed a narrow sensing range at approximately pH 6. These results indicated that fluorescence response of probe 1 towards GSH and Cys is pH dependent, that is, probe 1 can selectively probe GSH at pH 7.4 and Cys at pH 6.0, respectively, at different
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emissions. Notably, the best pH values corresponding to the highest intensity are 6.0 for Cys and 7.4 for GSH, which are within the biologically relevant pH range of 5.5–7.5 [68], This result
probe in living cells [69].
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3.3. Spectral response and sensing mechanism studies
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suggested that probe 1 could potentially be applied as a GSH and Cys dual response fluorescent
Basing on the results of pH titration experiments, pH 7.4 PBS buffer containing 1% EtOH and pH 6.0 PBS buffer containing 20% EtOH were selected to examine the reactivity of probe 1 towards
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Cys, Hcy, and GSH. As shown in Fig. 1c, free probe 1 showed a main absorption at 538 nm in pH
Figure 1.
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7.4 PBS buffer (1% EtOH) and pH 6.0 buffer (20% EtOH), thereby exhibiting characteristics of a thioacridinium dye. First, upon addition of 10 equiv. of Cys, the initial absorption peak decreased,
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along with the emergence of a blue-shifted peak at 438 nm in the PBS buffer (1%EtOH). On the basis of the aforementioned speculation and well-established tandem SNAr-rearrangement reaction mechanism [44], the new absorption peak at 438 nm can be assigned to amino-acridinium 5a. This assignment is also in accordance with the data of MALDI-TOF MS experiment (Fig. S2) and control compound AO-N-Bu. The absorption maximum of AO-N-Bu was found at approximately 428 nm (Fig. S3), which was close to that of amino-acridinium 5a. The thiol-induced tandem SNAr-rearrangement reaction mechanism can be supported further by the fact that probe 1 is inert
ACCEPTED MANUSCRIPT to the thiol-free amino acids (Fig. 3). A similar observation was obtained for Hcy under identical experimental conditions (Figs. S3). Next, we explored the sensing ability of probe 1 toward GSH at same experimental condition. In contrast to Cys, upon addition of 10 equiv. of GSH, the
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solution of probe 1 led only to the decrease of the initial absorption band at 457 nm and did not induce any shift of the initial absorption band at 538 nm (Fig. 1d). This represents the production of thio-acridinium 6 due to the bulkiness of its tripeptide and the unstable 10-membered
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macrocyclic transition state, precluding the possibility of its subsequent rearrangement to
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amino-acridinium derivative. The assignment was also supported by the data of MALDI-TOF MS experiment (Fig. S2) and control compound AO-S-Et (Fig. S3). A prominent peak at m/z 656.9 corresponding to [6]+ (calcd. 656.8 for C32H42N5O8S+), which was generated from the incubation of probe 1 with GSH in EtOH–H2O, was clearly observed in the MALDI-TOF mass spectrum. In
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addition, the absorption maximum (Absmax: 538 nm) of AO-S-Et matched with thio-acridinium 6 perfectly (Fig. S3). In the pH 7.4 PBS buffer containing 1% EtOH, the reaction of the probe with GSH produced thio-acridinium but not amino-acridinium from the subsequent rearrangement
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reaction between 6 and excess GSH, indicating that probe 1 can be used to discriminate GSH from
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Cys or Hcy. Then, the reactivity of probe 1 toward Cys, Hcy, and GSH under weakly acidic conditions was explored. As expected, the addition of Cys to pH 6.0 PBS buffer solution (20% EtOH) induced a big blue shift in the absorption maximum of probe 1 from 538 nm to 442 nm (Fig. 1d). However, Hcy and GSH caused no obvious change in the UV/vis spectrum of the probe. These results showed that probe 1 reacts with Cys in a highly selective manner. That is, probe 1 can selectively distinguish Cys from Hcy and GSH under weakly acidic conditions. Overall, the above results are in good accordance with our original design concept and the proposed sensing
ACCEPTED MANUSCRIPT mechanism (Scheme 2).
Figure 2.
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Owing to the desirable UV–Vis absorption spectra and pH effect results, two solvents (pH 7.4 PBS buffer containing 1% EtOH and pH 6.0 PBS buffer containing 20% EtOH) and two excitation wavelengths (438 and 537 nm) were used to examine the fluorescence emission
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behaviors of probe 1 in the presence of Cys, Hcy, and GSH (Figs. 2a and c). The selected
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excitation wavelengths at 438 and 537 nm were near the absorption maximums of amino-acridinium 5a (5b) and thio-acridinium 6 and 7. Under two experimental conditions, probe 1 nearly had no fluorescence because of the PET from the 3,4-dimethoxythiophenol to the acridinium. Upon excitation at 537 nm, a new emission peak appeared at 567 nm after the addition
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of 10 equiv. of GSH (Fig. 2a) and reached equilibrium within 2 min in the pH 7.4 PBS buffer (Fig. 2b). In this instance, approximately 150-fold increase in fluorescence signal was observed because of the production of thio-acridinium 6, as indicated by the UV–Vis results. By contrast, the
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addition of Cys or Hcy barely caused significant changes in the fluorescence of probe 1 at the
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same condition (Figs. 2a and b). At 438 nm excitation wavelength, the addition of 10 equiv. of Cys to a solution of probe 1 elicited a distinct fluorescence enhancement (46-fold) at 518 nm because of the production of amino-acridinium 5a. Unfortunately, the addition of 10 equiv. of Hcy produced a minor emission at approximately 518 nm possibly because of the amino-coumarin derivative 5b. Moreover, the addition of 10 equiv. of GSH resulted only in a poor emission at approximately 572 nm. Poor emission is a characteristic of thio-acridinium 6 (Fig. S4a). These results indicated that the probe can discriminate GSH from Cys or Hcy but cannot distinguish
ACCEPTED MANUSCRIPT between Cys and Hcy. Next, the fluorescence responses of the probe toward Cys, Hcy, and GSH in pH 6.0 PBS buffer (20% EtOH) was examined to achieve the selective discrimination of Cys from Hcy and GSH, Indeed, changing the medium to pH 6.0 PBS buffer (20% EtOH) did change
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the selectivity of probe 1 towards Cys. An extremely weak emission of probe 1 (λem = 515 nm, λex = 438 nm) changed significantly in presence of Cys in a “turn on” manner whereby the emission intensity increased by 52-fold, as shown in Fig. 2c. The emission was identified to be
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amino-acridinium 5a because it is nearly identical to emission obtained in pH 7.4 PBS buffer (1%
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EtOH). The kinetic studies revealed that the emission intensity reaches a maximum value within 10 mins after Cys addition (Fig. 2d). In the other hand, although Hcy and GSH present slight fluorescence increases but the increase is time consuming because of the low thiolate and the amine concentrations at this pH condition. In contrast, with excitation at 537 nm, addition of Hcy
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(or GSH) caused hardly significant fluorescence changes in probe 1 at 518 nm, but immediately elicited a significant fluorescence enhancement of 101-fold at 567 nm (or 95-folds at 570 nm) (Fig. S4b), revealing that the corresponding thio-acridiniums were produced. These results confirmed
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that probe 1 is capable of highly selective detection of Cys from Hcy and GSH under weak acidic
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condition but does not distinguish between Hcy and GSH. In addition, GSH and Cys can easily be distinguished macroscopically when illuminated by a
hand-held UV lamp at 365 nm (Fig. 2). Introducing GSH to the solution of probe 1 results in a strong orange emission color, whereas Cys/Hcy elicits a bright green emission in pH 7.4 PBS buffer (1% EtOH) (Fig. 2a inset). By contrast, upon the addition of Cys in pH 6.0 PBS buffer (20% EtOH), probe 1 induces a strong green fluorescence, but Hcy/GSH results in a significant orange emission (Fig. 2c inset).
ACCEPTED MANUSCRIPT 3.4. Selectivity and competitiveness To evaluate the specific nature of probe 1 for GSH and Cys, the probes was separately incubated with other amino acids and various biologically relevant analytes under the same
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experimental conditions. The biologically important Na+ and K+ cations were excluded because of their high contents in PBS buffers. With the excitation of 537 nm, probe 1 exhibit hardly significant fluorescence enhancement to various analytes, in order as 0) blank, 1) Ser, 2) Lys, 3)
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Asp, 4) Glu, 5) His, 6) Val, 7) Asn, 8) Gln, 9) Leu, 10) Thr, 11) Pro, 12) Trp, 13) Ile, 14) Ala, 15)
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Sar, 16) Phe, 17) Arg, 18) Tyr, 19) Gly, 20)Met, 21) ATP, 22) Gluc, 23) Zn2+, 24) Mg2+, 25) Ca2+, 26) Fe3+, 27) Cys-Cys, 28) Cys, 29) Hcy in pH 7.4 PBS buffer (1% EtOH). In fact, only GSH elicited a remarkable increase in the fluorescence intensity at 567 nm (Fig. 3a). With excitation at 438 nm, all other competitive species, including Hcy and GSH, showed nearly no fluorescence
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intensity changes in probe 1. Only Cys caused significant fluorescence turn-on at 518 nm in pH 6.0 PBS buffer (20% EtOH; Fig. 3b). Importantly, Cys and Hcy, which are difficult to distinguish
acidic condition.
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through the SNAr-rearrangement mechanism, can be distinguish among one another under weak
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Fluorescence interference experiments were performed in the presence of various competing analytes under the same experimental condition to determine the feasibility of using probe 1 as a GSH- and Cys-selective probe. Other background analytes had small or no obvious interference with the detection of GSH, except for cystine, Cys and Hcy, upon excitation at 537 nm in pH 7.4 PBS buffer (1% EtOH). Cys or Hcy might form the amino-substituted derivatives with the probe through a sequential SNAr and rearrangement reactions, which induced a blue shift in the fluorescence spectra of low intensity associated with Cys/Hcy. The disturbance of cystine
ACCEPTED MANUSCRIPT (Cys–Cys) can be attributable to the Cys, which was formed through the GSH triggered disulfide cleavage reaction of Cys–Cys (Fig. 3a) [70]. As expected, the probe was not interfered by other various competing analytes and Cys–Cys for the detection of Cys upon the excitation of 438 nm in
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pH 6.0 PBS buffer (20% EtOH). However, the coexistence of GSH or Hcy with Cys disturbed the detection of Cys at the same condition due to the formation of thio-acridiniums 6 and 7 through
fluorescence spectra associated with GSH/Hcy (Fig. 3b).
3.5. Fluorescence titration study
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Figure 3.
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the initial thio-substitution reaction, which similarly induced a red shift in the low-intensity
Basing on its excellent selectivity, the researchers subsequently investigated the ability of
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quantitative detection of probe 1 toward GSH and Cys through fluorescent titration. Titration experiments resulted in good concentration-dependent fluorescence changes. As shown in Fig. 4a. with increasing GSH concentration, the fluorescence emission intensity of probe 1 at λmax 567 nm
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increased gradually in pH 7.4 PBS buffer (1% EtOH) when undergoing excitation at 537 nm.
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When GSH concentration was increased to ca. 10 µM (1.0 equiv.), the fluorescence emission intensity became saturated. The plot of emission intensities of the probe 1 as a function of added GSH concentration was linear up to 7 µM (Fig. 4a, inset), useful for the determination of low-level GSH. A Job's plot (Fig. S6a) and MALDI-TOF mass data (Fig. S2) indicate that the reaction between probe 1 and GSH has a 1:1 stoichiometry. The detection limit [71] is determined to be 5.0 nM based on 3σ/S (Fig. 4a, inset), which is significantly below the physiological levels of GSH in live cells (1–10 mM) [72]. This result is indeed important in view of the few
ACCEPTED MANUSCRIPT fluorescent probes that can selectively and quantitatively detect GSH over Cys/Hcy in the literature so far. Similarly, the sensing ability of probe 1 to Cys was investigated by fluorescence titration in pH 6.0 PBS buffer (20% EtOH) as shown in Fig. 4b. Probe 1 can quantitatively detect
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Cys and produces a strong fluorescent signal at very low concentrations of Cys (0 µM to 25 µM). Moreover, analysis of the fluorescence intensity at 518 nm vs Cys concentration was found to be linear (R = 0.9946) with a detection limit calculated to be 0.11 µM (S/N = 3) (Fig. 4b, inset). In
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addition, a Job's plot exhibits a maximum at 0.5 molecular fraction, which indicates that the
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reaction between probe 1 and GSH has a 1:1 stoichiometry (Fig. S6b). Accurate stoichiometry of
Figure 4.
the adduct 1-Cys was further confirmed by MALDI-TOF mass spectrometry analysis (Fig. S2).
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These findings suggest that Probe 1 is indeed a sensitive detector and quantitative monitor of Cys under weak acidic condition.
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3.6. Cell imaging experiments
Finally, to demonstrate the potential biological relevance, probe 1 was used to monitor the
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levels of thiols in HeLa cells. HeLa cells incubated with probe 1 (10 µM) for 30 min showed strong fluorescence in two emission channels, green (500 nm to 550 nm) and red (570 nm to 620 nm) (Fig. 5A1 and 5B1), confirming that this probe is capable of permeating into cells and reacting with endogenous thiols to produce discernible fluorescence responses. Subsequent control experiments were performed to gain further support for this conclusion. In one control experiment, the HeLa cells were pretreated with the thiol-blocking reagent N-methylmaleimide (NMM) (100 µM) for 30 min and then incubated with probe 1 for 30 min. The inversed microscopy image of
ACCEPTED MANUSCRIPT cells treated in this manner does not display the fluorescence (Fig. 5A2 and 5B2). Moreover, incubation of the NMM-pretreated HeLa cells with Cys (500 µM) or Hcy (500 µM) causes a significant fluorescence increase in green emission (Fig. 5A3 and 5A4), while almost no
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fluorescence is observed upon incubation with GSH (Fig. 5A5). However, incubation of the cells with GSH (500 µM) caused a marked fluorescence increase in red emission (Fig. 5B5), while both Cys and Hcy barely elicited any significant fluorescence changes of probe 1 (Fig. 5B3 and 5B4),
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indicative of the high selectivity of probe 1 to GSH from Hcy and Cys in living cells.
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Subsequently, similar bioimaging under the condition of pH 6.0 was performed in HeLa cells (Fig. 5C and 5D). By contrast, the relative weak fluorescence emission of cells when incubated with probe 1 (10 µM) was found both in green emission and red emission (5C1 and 5D1). Similar to at pH 7.4, the HeLa cells pretreated with the thiol-blocking reagent N-methylmaleimide (NMM)
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(100 µM) for 30 min and then incubated with probe 1 (10 µM) for 30 min does not display the fluorescenc (5C2 and 5D2). As expected, a strong fluorescence in the cells was observed at green
Figure 5.
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channel but no emission was observed at red channel after the pre-treated cells with NMM were
incubated further with Cys (500 µM) for 30 min and then incubated with probe 1 (10 µM) for 30 min (5C3 and 5D3), which are in good accordance with the results of fluorescence spectra. Importantly, in the cases of both Hcy and GSH, almost no fluorescence is observed (5C4 and 5C5) at pH 6.0 condition. These results are in agreement with the specificity of probe 1 for Cys or GSH, and also demonstrate the potential of probe 1 to sense intracellular Cys and GSH selectively under different pH conditions and emission channels.
ACCEPTED MANUSCRIPT 4. Conclusion We
developed
an
acridinium
salt-based
fluorescent
probe
1
having
a
3,4-dimethoxythiophenol leaving group that undergoes reaction with thiols. The probe was able to
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selectively react with biothiols over other thiol-free amino acids through breaking PET process under physiological conditions. Importantly, the probe shows emission-dependent selectivity for GSH over Cys and Hcy and pH-dependent selectivity for Cys over Hcy and GSH in PBS buffer.
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Moreover, the fluorescence response is free from disturbance by other amino acids and
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biologically related analytes tested. The probe can detect as low as 5.0 × 10−9 M−1 GSH and 1.1 × 10−7 M−1 Cys, respectively. In addition, the probe has good water solubility, cell-penetration ability, and biocompatibility, and could thus be used to separately sense intracellular GSH and Cys by bioimaging by adjusting the emission channel and pH environment. Finally, the present
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strategy is expected to inspire the exploration of new systems that can respond to biothiols with multiple selectivity and high sensitivity for probing biothiols function in biological systems.
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Acknowledgments
This work is supported by the National Natural Science Foundation of China (No. 21262039
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and 2156020075).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.
References [1] G. I. Giles, Curr. Pharm. Des., 2006, 12(34), 4427-4443. [2] N. Brandes, S. Schmitt, U. Jakob, Antioxid. Redox Signaling, 2009, 11(5), 997-1014.
ACCEPTED MANUSCRIPT [3] C. K. Sen, Curr. Top. Cell. Regul., 2000, 36, 1-30. [4] T. P. Dalton, H. G. Shertzer and A. Puga, Annu. Rev. Pharmacol. Toxicol., 1999, 39, 67-101. [5] S. C. Lu, Mol. Aspects Med., 2009, 30(1-2), 42-59.
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[6] D. M. Townsend, K. D. Tew, H. Tapiero, Biomed. Pharmacother., 2003, 57(3-4), 145-155. [7] J. M. Estrela, A. Ortega, E. Obrador, Crit. Rev. Clin. Lab. Sci., 2006, 43, 143-181. [8] L. M. Hyman, K. J. Fanz, Coord. Chem. Rev., 2012, 256, 2333-2356.
M AN U
[10] S. Shahrokhian, Anal. Chem., 2001, 73(24), 5972-5978.
SC
[9] X. Chen, X. Tian, I. Shin, J. Yoon, Chem. Soc. Rev., 2011, 40, 4783-4804.
[11] M. T. Heafield, S. Fearn, G. B. Steventon, R. H. Waring, A. C. Williams, S. G. Sturman, Neurosci. Lett., 1990, 110(1-2), 216-220.
[12] P. Sachdev, R. Parslow, C. Salonikas, O. Lux, W. Wen, R. Kumar, D. Naidoo, H. Christensen,
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A. Jorm, Arch. Neurol., 2004, 61, 1369-1376.
[13] P. Sachdev, J. Neurol. Sci., 2004, 226(1-2), 25-29. [14] B. Hooshmand, T. Polvikoski, M. Kivipelto, M. Tanskanen, L. Myllykangas, T. Erkinjuntti,
EP
M. Mäkelä, M. Oinas, A. Paetau, P. Scheltens, E. C. van Straaten, R. Sulkava, A. Solomon,
AC C
Brain, 2013, 136(9), 2707-2716.
[15] X. Chen, Y. Zhou, X. Peng, J. Yoon, J. Chem. Soc. Rev. 2010, 39, 2120-2135. [16] M. E. Jun, M. B. Roy, K. H. Ahn, Chem. Commun. 2011, 47, 7583-7601. [17] Y. Zhou, J. Yoon, Chem. Soc. Rev. 2012, 41, 52-67. [18] C. Yin, F. Huo, J. Zhang, R. Martínez-Máñez, Y. Yang, H. Lv, S. Li, Chem. Soc. Rev., 2013, 42, 6032-6059 [19] L.-Y. Niu, Y.-Z. Chen, H.-R. Zheng, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, Chem. Soc. Rev.,
ACCEPTED MANUSCRIPT 2015, 44, 6143-6160. [20] O. Rusin, N. N. St. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang, I. M. Warner, F. B. Dawan, K. Lian, R. M. Strongin, J. Am. Chem. Soc., 2004, 126, 438-439.
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[21] X. Yang, Y. Guo, R. M. Strongin, Angew. Chem. Int. Ed., 2011, 50, 10690-10693. [22] Z. Guo, S. Nam, S. Park, J. Yoon, Chem. Sci., 2012, 3, 2760-2765.
[23] H. Wang, G. Zhou, H. Gai and X. Chen, Chem. Commun., 2012, 48, 8341-8343.
SC
[24] J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han and B. R. Cho, J. Am. Chem. Soc., 2010, 132(4),
M AN U
1216-1217.
[25] H. S. Jung, J. H. Han, T. Pradhan, S. Kim, S. W. Lee, J. L. Sessler, T. W. Kim, C. Kang, J. S. Kim, Biomaterials, 2012, 33(3), 945-953.
[26] H. Li, J. Fan, J. Wang, M. Tian, J. Du, S. Sun, P. Sun, X. Peng, Chem. Commun., 2009,
TE D
5904-5906.
[27] L. Yuan, W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275-6277. [28] J. Shao, H. Sun, H. Guo, S. Ji, J. Zhao, W. Wu, X. Yuan, C. Zhang, T. D. James, Chem. Sci.,
EP
2012, 3, 1049-1061.
AC C
[29] L. Long, W. Lin, B. Chen, W. Gao and L. Yuan, Chem. Commun., 2011, 47, 893-895. [30] H. Kwon, K. Lee, H.-J. Kim, Chem. Commun., 2011, 47, 1773-1775. [31] Y.-K. Yang, S. Shim, J. Tae, Chem. Commun., 2010, 46, 7766-7768. [32] H. Li, L.-Y. Jin, Y. Kan, B. Yin, Sensors and Actuators B, 2014, 196, 546-554. [33] X. Zhou, X. Jin, G. Sun, D. Li, X. Wu, Chem. Commun., 2012, 48, 8793-8795. [34] L.-Y. Niu, Y.-S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, Chem. Commun., 2013, 49, 1294-1296.
ACCEPTED MANUSCRIPT [35] L. Ma, J. Qian, H. Tian, M. Lan, W. Zhang, Analyst, 2012, 137, 5046-5050. [36] D. Lee, G. Kim, J. Yin, J. Yoon, Chem. Commun., 2015, 51, 6518-6520. [37] J. O. Escobedo, W. Wang, R. M. Strongin, Nat. Protocols, 2006, 1, 2759-2762.
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[38] T.-K. Kim, D.-N. Lee, H.-J. Kim, Tetrahedron Lett., 2008, 49(33), 4879-4881. [39] W. Wang, J. O. Escobedo, C. M. Lawrence, R. M. Strongin, J. Am. Chem. Soc., 2004, 126(11), 3400-3401.
SC
[40] H. Chen, Q. Zhao, Y. Wu, F. Li, H. Yang, T. Yi and C. Huang, Inorg. Chem., 2007, 46(26),
M AN U
11075-11081.
[41] K.-S. Lee, T.-K. Kim, J. H. Lee, H.-J. Kim and J.-I. Hong, Chem. Commun., 2008, 6173-6175.
[42] S.-K. Sun, H.-F. Wang and X.-P. Yan, Chem. Commun., 2011, 47, 3817-3819.
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[43] H. Y. Lee, Y. P. Choi, S. Kim, T. Yoon, Z. Guo, S. Lee, K. M. K. Swamy, G. Kim, J. Y. Lee, I. Shin, J. Yoon, Chem. Commun., 2014, 50, 6967-6969. [44] L.-Y. Niu, Y.-S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, J. Am. Chem. Soc.,
EP
2012, 134, 18928-18931.
AC C
[45] N. Shao, J. Jin, H. Wang, J. Zheng, R. Yang, W. Chan,Z. Abliz, J. Am. Chem. Soc., 2010, 132, 725-736.
[46] Y. Guo, X. Yang, L. Hakuna, A. Barve, J. O. Escobedo, M. Lowry, R. M. Strongin, Sensors, 2012, 12, 5940-5950.
[47] Y. Xu, B. Li, P. Han, S. Sun, Y. Pang, Analyst, 2013, 138, 1004-1007. [48] M.-Y. Jia, L.-Y. Niu, Y. Zhang, Q.-A. Yang, C.-H. Tung, Y.-F. Guan, L. Feng, ACS Appl. Mater. Interfaces, 2015, 7, 5907-5914.
ACCEPTED MANUSCRIPT [49] J. Liu, Y.-Q. Sun, H. Zhang, Y. Huo, Y. Shi, W. Guo, Chem. Sci., 2014, 5, 3183-3188. [50] Y. Liu, X. Lv, J. Liu, Y.-Q. Sun, W. Guo, Chem. Eur. J., 2015, 21, 4747-4754. [51] S.-Y. Lim, K.-H. Hong, D. I. Kim, H. Kwon, H.-J. Kim, J. Am. Chem. Soc., 2014, 136,
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7018-7025. [52] J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J.-H. Ryu, J. Yoon, J. Am. Chem. Soc., 2014, 136, 5351-5358.
SC
[53] X.-F. Yang, Q. Huang, Y. Zhong, Z. Li, H. Li, M. Lowry, J. O. Escobedoc, R. M. Strongin,
M AN U
Chem. Sci., 2014, 5, 2177-2183.
[54] F. Wang, L. Zhou, C. Zhao, R. Wang, Q. Fei, S. Luo, Z. Guo, H. Tian, W.-H. Zhu, Chem. Sci., 2015, 6, 2584-2589.
[55] C. Xu, H. Li, B. Yin, Biosens. Bioelectron., 2015, 72, 275-281.
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[56] J. Liu, Y.-Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi, W. Guo, J. Am. Chem. Soc., 2014, 136, 574-578.
[57] D. Lee, G. Kim, J. Yin, J. Yoon, Chem. Commun., 2015, 51, 6518-6520.
AC C
420-422.
EP
[58] L. M. Mironovich, L. S. Ageeva, A. Yu. Podol’nikova, Russ. J. Gen. Chem., 2016, 86(2),
[59] C. Kaufmann, A. Albertini, Ber., 1909, 42, 1999-2008. [60] J. W. Happ, E. G. Janzen, B. C. Rudy, J. Org. Chem., 1970, 35, 3382-3389. [61] Y.-K. Yang, J. Tae, Org. Lett., 2006, 8, 5721-5723. [62] D. D.Perrin, W. L. F. Armarego, Purification of Laboratory Chemicals, Pergamon Press: New York, 1988 and references cited therein. [63] L. Wu and K. Burgess, Org. Lett., 2008, 10(9), 1779-1782.
ACCEPTED MANUSCRIPT [64] R. E. Benesch, R. Benesch, J. Am. Chem. Soc., 1955, 77, 5877-5881. [65] H. S. Jung, J. H. Han, T. Pradhan, S. Kim, S. W. Lee, J. L. Sessler, T. W. Kim, C. Kang, J. S. Kim, Biomaterials, 2012, 33(3), 945-953.
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[66] Y. Ma, S. Liu, H. Yang, Y. Wu, C. Yang, X. Liu, Q. Zhao, H. Wu, J. Liang, F. Li, W. Huang, J. Mater. Chem., 2011, 21, 18974-18982.
[67] S. C. Chai, A. A. Jerkins, J. J. Banik, I. Shalev, J. L. Pinkham, P. C. Uden, M. J. Maroney, J.
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Biol. Chem., 2005, 280, 9865-9869.
M AN U
[68] Z. Shi, X. Tang, X. Zhou, J. Cheng, Q. Han, J. Zhou, B. Wang, Y. Yang, W. Liu, D. Bai, Inorg. Chem., 2014, 52, 12668-12673.
[69] X. Dong, C.H. Heo, S. Chen, H.M. Kim, Z. Liu, Anal. Chem., 2014, 86, 308-311. [70] P. K. Pullela, T. Chiku, M. J. Carvan III, D. S. Sem, Anal. Biochem., 2006, 352, 265-273.
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[71] Y. Sun, Y. Liu, M. Chen, W. Guo, Talanta, 2009, 80, 996-1000.
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[72] C. Hwang, A. L. Sinskey, H. F. Lodish, Science, 1992, 257, 1496-1502.
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Figure Captions
Scheme 1. Synthesis route of probe 1
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Scheme 2. Proposed sensing mechanisms of probe 1 for Cys, Hcy and GSH.
Figure 1. The Fluorescence intensities of probe 1 (10 µM) in the presence of 10 equiv of Cys, Hcy
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and GSH as a function of pH in PBS buffer containing 1% (a) and 20% EtOH (b) excited at 537 (a) and 438 nm (b), respectively (a: λem = 567 nm, b: λem = 515 nm). Changes in absorption spectra of probe 1 (10 µM) measured in PBS buffer (10 mM, pH = 7.4, 1 % EtOH) (c) and PBS buffer (10
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mM, pH = 6.0, 20% EtOH) (d) upon addition of Cys, Hcy and GSH.
Figure 2. Fluorescence spectra of probe 1 (10 µM) after addition of 10 equiv. of Cys, Hcy or GSH in pH 7.4 PBS buffer containing 1% EtOH (λex = 537 nm, λem = 567 nm) (a) and in pH 6.0 PBS
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buffer containing 20% EtOH (λex = 438 nm, λem = 518 nm) (c), and the corresponding
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time-dependent fluorescence intensity changes. (b and d) Slits: 1.5/3 nm for (a) and (b), 3/3 nm for (c) and (d).
Figure 3. Fluorescence responses of probe 1 (10 µM) toward various analytes in pH 7.4 PBS buffer containing 1% EtOH (a) and in pH 6.0 PBS containing 20% EtOH (b), respectively. Black bar represent the addition of a single analyte (10 µM) in order of 0) blank, 1) Ser, 2) Lys, 3) Asp, 4) Glu, 5) His, 6) Val, 7) Asn, 8) Gln, 9) Leu, 10) Thr, 11) Pro, 12) Trp, 13) Ile, 14) Ala, 15) Sar, 16) Phe, 17) Arg, 18) Tyr, 19) Gly, 20) Met, 21) ATP, 22) Gluc, 23) Zn2+, 24) Mg2+, 25) Ca2+, 26)
ACCEPTED MANUSCRIPT Fe3+, 27) Cys-Cys, 28) Cys, 29) Hcy and 30) GSH. Red bar represent the subsequent addition of GSH (a) or Cys (b) to the mixture. Excitation wavelengths: 537 nm for (a) and 438 nm for (b).
Figure 4. Fluorescence spectra of probe 1 (10 µM) after addition of increasing amounts of GSH (a,
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λex = 537 nm) and Cys (b, λex = 438 nm) in pH 7.4 PBS buffer (1% EtOH) and pH 6.0 PBS buffer (20% EtOH), respectively. Insets: fluorescence intensity at 567 nm as a function of GSH (a) and
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Cys (b) concentrations.
Figure 5. Fluorescence images of living HeLa cells. A / B and C/ D incubated in pH 7.4 and pH
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6.0 PBS, respectively. The cells were incubated with probe 1(10 µM) for 30 min (A1, B1, C1 and D1); The cells were pre-incubated with NMM (0.1 mM) for 30 min, and then treated with probe 1 (10 µM) for 30 min (A2, B2, C2 and D2); The pre-incubated cells were incubated in sequence with Cys (500 µM) and probe 1 (10 µM)(A3, B3, C3 and D3); The pre-incubated cells were
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incubated in sequence with Hcy (500 µM) and probe 1 (10 µM)(A4, B4, C4 and D4); The pre-incubated cells were incubated in sequence with GSH (500 µM) and probe 1 (10 µM)(A5, B5,
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C5 and D5). The cells were incubated with 10 µM probe 1 for 30 min in pH 7.4 PBS buffer (1 %
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EtOH) for A and B, and in pH 6.0 PBS buffer (20 % EtOH) for C and D.
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Scheme 1
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Figure 5.
ACCEPTED MANUSCRIPT Highlights： ： 1. The probe is structurally simple and easy to synthesize 2. The probe can discriminates GSH from Cys and Hcy in pH 7.4 PBS buffer upon excitation of 537 nm.
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3. The probe can distinguishs Cys from GSH and Hcy in pH 6.0 PBS buffer upon excitation of 438 nm.
4. The probe has good cell-permeability and selectively sense intracellular GSH and
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Cys by multicolor imaging in HeLa cells at different pH environment.
1
S0003-2670(17)31077-2
DOI:
10.1016/j.aca.2017.09.028
Reference:
ACA 235453
To appear in:
Analytica Chimica Acta
Received Date: 16 June 2017 Revised Date:
7 September 2017
Accepted Date: 20 September 2017
Please cite this article as: Y. Tang, L. Jin, B. Yin, A dual-selective fluorescent probe for GSH and Cys detection: Emission and pH dependent selectivity, Analytica Chimica Acta (2017), doi: 10.1016/ j.aca.2017.09.028. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Graphic abstract
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A dual-selective fluorescent probe for GSH and Cys detection: emission and pH dependent selectivity Yunqiang Tang, Longyi Jin* and Bingzhu Yin* Key Laboratory of Natural Resources of Changbai Mountain & Functional Molecules of the
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Ministry of Education, Department of Chemistry, Yanbian University, Yanji 133002, PR China
Abstract
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e-mail: [email protected]
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A novel fluorescent probe 1 based on acridine orange was developed for the selective detection and bioimaging of biothiols. The probe exhibits higher selectivity and turn-on fluorescence response to cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) than to other amino acids. Importantly, the probe responds to GSH and Cys/Hcy with distinct fluorescence emissions in PBS
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buffer at pH of 7.4. The Cys/Hcy-triggered tandem SNAr-rearrangement reaction and GSH-induced SNAr reaction with the probe led to the corresponding amino-acridinium and
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thio-acridinium dyes, respectively, which can discriminate GSH from Cys/Hcy through different emission channels. Interestingly, Cys finishes the tandem reaction with the probe and
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subsequently forms amino-acridinium and Hcy/GSH induces SNAr reaction with the probe to form thio-acridiniums at weakly acidic conditions (pH 6.0), enabling Cys to be discriminated from Hcy/GSH at different emissions. Finally, we demonstrated that probe 1 can selectively probe GSH over Cys and Hcy or Cys over GSH and Hcy in HeLa cells through multicolor imaging.
Key Words: Acridine orange; fluorescent probe; GSH; Cys; detection; cell imaging.
ACCEPTED MANUSCRIPT 1. Introduction Intracellular thiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), play critical roles in many biological processes [1-4]. Although they exhibit similar reactions, their
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functions in vivo are entirely different. GSH has a balance with its oxidation state, glutathione disulfide (GSSG). GSH can be oxidated to GSSG by cellular oxidizing species, which limit the concentration of cellular oxides and prevent the stacking of these compounds. Changes cellular
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GSH levels are associated with many diseases, such as liver damage, leucocyte loss, psoriasis,
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cancer, and HIV infection [5-9]. Cys is a basic amino acid for protein synthesis and related to the formation of the three-dimensional protein structures. Moreover, Cys can strongly bind to metal ions and balance their intracellular concentrations, thereby protecting cells from damages caused by high concentrations of heavy ions. Several studies indicated that abnormal intracellular Cys
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levels are closely related to several diseases, such as Parkinson's disease and Alzheimer's disease, and adverse pregnancy outcomes [10,11]. Meanwhile, Hcy has a similar molecular structure with Cys, and its intracellular concentration is rather low. Moreover, it is an independent factor for
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some diseases, including cardiovascular disease and Alzheimer's disease [12-14]. Thus, Hcy is
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attracing considerable attention, and Hcy level is considered as one of the important parameters for some diseases. Meanwhile, developing methods for selective and sensitive detection of biothiols is significant in the area of clinical diagnosis. Considerable progress is already achieved in this undertaking particularly in the development of fluorescent probes for thiol detection, in which the strong nucleophilicity of the thiol group is exploited [15-19]. However, although these probes can highly selectively distinguish these biothiols from other amino acids, most of them cannot distinguish Cys, Hcy and GSH from one another because of the structures and reactivity of
ACCEPTED MANUSCRIPT these biothols are similar. Based on the two strategies of by Strongin et al. [20, 21], who first achieved ring formation of Cys with aldehydes or acrylates, numerous specific probes for Cys [22-36] or Hcy [37-43] were recently developed on the basis of various molecular recognition or
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thiol-specific reaction strategies. Similarly, inspired by the GSH-induced SNAr substitution reaction developed by Yang et al. [44], several researchers explored some specific probes for GSH [45-55]. Meanwhile, constructing two or three thiol-responsive probes is highly valuable but more
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challenging than constructing single thiol-responsive fluorescent probes. However, to the best of
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our knowledge, only one fluorescent probe has been developed for simultaneous detection and multicolor imaging of GSH and Cys at different emission channels [56]. Observations in previous studies showed that Cys is more reactive in nucleophilic reactions than Hcy and GSH owing to the differences among their pKa values, Yoon et al. [57] reported recently that an
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aryl-thioether-substituted nitrobenzothiadiazole probe is more selective to Cys over Hcy and GSH under weakly acidic conditions (pH 6.0).
Basing on the results of these studies, we designed and synthesized a thiol-selective
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fluorescence probe by incorporating a 3,4-dimethoxythiophenol moiety to the 9-position of
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acridinium dye. The strategy used for the design of present fluorescence probe exploits the reaction activity of the 9-position of acridines and acridiniums [58-61]. The probe not only can discriminate GSH from Cys or Hcy through different emission channels but also can discriminate Cys from GSH or Hcy through the adjustment of the pH of the solution contained in a PBS buffer with relatively fast kinetics and obvious fluorescence turn-on response. In addition, the probe can simultaneously and selectively detect Cys and GSH in HeLa cells through multicolor imaging.
2. Experimental
ACCEPTED MANUSCRIPT 2.1. General information and materials Commercially available compounds were used without further purification. Solvents were dried according to standard procedures [62]. All reactions were magnetically stirred and monitored
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by thin-layer chromatography (TLC) using Qingdao GF254 silica gel coated plates. Fluorescence spectra were carried out on a Shimadzu RF-5301PC fluorescence spectrophotometer. UV–vis spectra were recorded with a Shimadzu UV-2550 spectrophotometer. NMR spectra were recorded 13
C), and chemical shifts
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on a Bruker AV-300 Spectrometer (300 MHz for 1H and 75 MHz for
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were referenced relative to tetramethylsilane (δH/δC = 0). Mass data were obtained by a Shimadzu AXIMA-CFRTM plus mass spectrometry, using a 1,8,9-anthracenetriol (DITH) matrix. The high-resolution mass spectra were obtained using a Bruker micro TOF II focus spectrometer (ESI).
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2.2. Synthesis and characterization
The starting material acridine-oringe base was purchased from J&K chemical ltd. and was
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used without further purification. Synthesis of probe 1, the intermediates, and the control
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compounds are given in supplementary information (SI).
2.3. Cell culture and fluorescence imaging HeLa cells were obtained from American Type Culture collection and grown in Dulbecco
Modified Eagle Medium (Free DMEM/high: with 4500 mg/L Glucose, 4.0 mM L-Glutamine, and 110 mg/L Sodium Pyruvate). The cells were incubated in a 5% CO2 humidified incubator at 37°C and typically passaged with sub-cultivation ratio of 1:4 for two days. The HeLa cells were seeded in 6-well culture plate overnight. Stock solutions of probe 1 (3 mM) and NMM (0.03 M) in
ACCEPTED MANUSCRIPT ethanol, Cys, Hcy and GSH (0.15 M) in PBS buffer (10 mM) were prepared at the same day of experiment. Before the experiments, cells were washed with PBS buffer 3 times. Then, the cells were
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incubated with probe 1 (10 µM), or pretreated with Cys (500 µM, 30 min), Hcy (500 µM, 30 min) or GSH (500 µM, 30 min) in DMEM medium at 37°C. After each treatment, the cells were washed with PBS buffer 3 times. The cells were imaged using an inverted microscope
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(OLYMPUS IX73). The fluorescence in green channel was excited at 450-480 nm, and in red
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channel was excited at 530-550 nm.
2.4. General Procedure for Spectral Measurements
Deionized water and spectroscopic grade EtOH were used as solvents for the spectral studies. The spectral determinations were conducted in 10 mm quartz cuvettes at room temperature. Probe
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1 was dissolved in EtOH to prepare a concentration of 1 × 10−3 M stock solution, which was diluted to 1 × 10−5 M with pH 7.4 PBS buffer (10 mM, 1% EtOH) and pH 6.0 PBS buffer (10 mM,
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20% EtOH), respectively. The thiols were dissolved in pH 7.4 PBS buffer (10 mM, 1 % EtOH) and pH 6.0 PBS buffer (10 mM, 20 % EtOH), respectively, to prepare the stock solutions. The
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absorbance was measured from 220 to 800 nm in the corresponding blank PBS buffers, and 10 equiv. of thiols were added to the 10 µM host solution (3 mL). The emission was measured from 400 nm to 800 nm, and the different amounts of thiols solutions were added to the 10 µM host solution (3 mL) in portions (total volumes of 0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 36, 42, 48, 54, 60, 75, 90, 120 and 150 µL of 1 × 10−3 M GSH, and 0, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 75, 90, 120, 150, 180, 210, 240, 270 and 300 µL of 5 × 10−4 M Cys). The resultant solution was shaken well and the absorption and emission spectra were recorded immediately.
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3.1. Synthesis of probe 1
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The synthetic route of probe 1 is depicted in Scheme 1. Intermediate acridinium 2 was readily prepared from an acridine orange base and ethyl bromoacetate at an 89% yield. The reaction of acridinium 2 with sodium cyanide in DMSO-water (95:5) solvent system causes a color change
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from brick-red to dark blue. The reaction shows that a cyanide-adduct forms initially and rapidly reacts with air to produce acridinone 3 at a moderate yield (49%). The direct conversion of
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acridinone 3 to probe 1 through the successive treatments with trifluoroacetic anhydride and 3,4-dimethoxythiophenol, a procedure employed for the pyroninone [63], ended in failure. Fortunately, 9-chloroacridinium 4, which was obtained from the chlorination reaction of acridinone 3 with phosphorus oxychloride, was successfully converted to the 9-position
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aryl-thioether-substituted acridinium dye (probe 1) through a SNAr substitution with 3,4-dimethoxythiophenol in the presence of triethylamine in CH3CN. The chemical structures of
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HRMS spectra.
13
C-NMR, MALDI-TOF MS and
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the intermediates and probe 1 were confirmed by 1H-NMR,
Scheme 1
Furthermore, 3,4-dimethoxythiophenol moiety was introduced to acridinium dye because it
functions not only as a leaving group in the thiol-induced SNAr substitution reaction and as a fluorescence quencher via the photo-induced electron transfer (PET) process, which ensures a low fluorescence background. A Cys- or Hcy-induced SNAr-rearrangement cascade reaction with the probe leads to the corresponding amino-acridinium 5a (or 5b). However, in the case of GSH, the
ACCEPTED MANUSCRIPT initial SNAr reaction lead to thio-acridinium 6, which does not occur at the subsequent intramolecular rearrangement. The reason is that to thio-acridinium 6 has a bulky tripeptide and unstable 10-membered macrocyclic transition state (if any), as shown in Scheme 2. Given the
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distinct photophysical properties of amino- and thio-acridinium dyes, probe 1 is likely to sense Cys/Hcy and GSH from two different emission channels. In addition, assuming that the fluorescence behavior of the probe toward thiols is dependent on the pH value of the employed
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medium, that is, only Cys can induce tandem SNAr-rearrangement reaction to form corresponding
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amino-acridinium 5a under weak acidic conditions. However, the reaction trigged by Hcy and GSH stayed at SNAr reaction stage to provide thio-acridiniums 6 and 7 at same condition. A combination of these factors can serve as the basis for a selective response of probe 1 to Cys from Hcy and GSH. Overall, this design can potentially facilitate the selective discrimination of Cys
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and GSH on the basis of the differences between their chemical structures and identification of the distinct photophysical properties of the corresponding amino-acridiniums and thio-acridiniums.
Scheme 2.
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The design concepts for the probe are summarized in Scheme 2.
3.2. pH dependent studies
The major determinant of selectivity in the tandem reaction of thiols with present probe 1 is
expected to be the nature of the nucleophile, such as pKa and the pI values of the thiols. At initial SNAr reaction stage, thiolate anions are more reactive than free thiols. However, during sequential thiol–amine rearrangement reaction step, free amines are more reactive than ammonium cations. Thus, for an analogous set of substrates, the presence of an increased fraction of thiolate or free
ACCEPTED MANUSCRIPT amine at any given pH is expected to generate an amplified relative reactivity [64]. Given that the pKa values of the Cys, Hcy, and GSH are 8.53, 10.00, and 9.20, respectively [65], and their pI values are 5.02, 5.27, and 5.93, respectively [66, 67], investigating the effect of pH on the probe
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towards Cys, Hcy, and GSH and determining the optimal sensing conditions is important. In the present study, the fluorescence pH titrations of probe 1 were performed in PBS buffer solutions containing 1% and 20% EtOH to identify the conditions under which the ability of the probe to
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detect biothols can be explored. First, a PBS buffer solution containing 1% EtOH was selected.
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Upon excitation by 537 nm, no fluorescence response by probe 1 was observed after the addition of GSH at pH below 6.0. Probe 1 suddenly had a response to GSH at pH 6.0 and remained unaffected between pH 6.0 and 12. By contrast, the addition of Cys (or Hcy) barely showed any emission response in pH of 2.0–12 (Fig, 1a). The excitation wavelength of 438 nm was then
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selected to probe Cys or Hcy. A strong fluorescence response of probe 1 to Cys (or Hcy) was observed at approximately pH 7.4. Response to GSH had no significant fluorescence response at pH of 2.0–12 (Fig. S1a). Afterward, the effect of pH on the ability of probe 1 to detect thiols in a
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PBS buffer solution containing 20% EtOH was examined at pH range from 2 to 12. In contrast to
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the above-mentioned case, the reaction of probe 1 with GSH had no sensing response upon 438 nm excitation in this pH range (Fig. 1b). Interestingly, a rapid sensing response of probe 1 to Cys was observed in the pH range of 6.0–8.0. However, with regard to the response of the probe to Hcy, the pH range of sensing lagged behind, showing a pH range from 7.0 to 9.0. Second, the excitation wavelength of 537 nm was selected to probe the biothiols. As seen in Fig. S1b, after the addition of Cys to a solution containing probe 1, only a minimal sensing response in the fluorescence spectra was observed. In the case of GSH, probe 1 showed a good sensing ability in
ACCEPTED MANUSCRIPT the pH range of 6 to 12, but Hcy showed a narrow sensing range at approximately pH 6. These results indicated that fluorescence response of probe 1 towards GSH and Cys is pH dependent, that is, probe 1 can selectively probe GSH at pH 7.4 and Cys at pH 6.0, respectively, at different
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emissions. Notably, the best pH values corresponding to the highest intensity are 6.0 for Cys and 7.4 for GSH, which are within the biologically relevant pH range of 5.5–7.5 [68], This result
probe in living cells [69].
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3.3. Spectral response and sensing mechanism studies
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suggested that probe 1 could potentially be applied as a GSH and Cys dual response fluorescent
Basing on the results of pH titration experiments, pH 7.4 PBS buffer containing 1% EtOH and pH 6.0 PBS buffer containing 20% EtOH were selected to examine the reactivity of probe 1 towards
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Cys, Hcy, and GSH. As shown in Fig. 1c, free probe 1 showed a main absorption at 538 nm in pH
Figure 1.
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7.4 PBS buffer (1% EtOH) and pH 6.0 buffer (20% EtOH), thereby exhibiting characteristics of a thioacridinium dye. First, upon addition of 10 equiv. of Cys, the initial absorption peak decreased,
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along with the emergence of a blue-shifted peak at 438 nm in the PBS buffer (1%EtOH). On the basis of the aforementioned speculation and well-established tandem SNAr-rearrangement reaction mechanism [44], the new absorption peak at 438 nm can be assigned to amino-acridinium 5a. This assignment is also in accordance with the data of MALDI-TOF MS experiment (Fig. S2) and control compound AO-N-Bu. The absorption maximum of AO-N-Bu was found at approximately 428 nm (Fig. S3), which was close to that of amino-acridinium 5a. The thiol-induced tandem SNAr-rearrangement reaction mechanism can be supported further by the fact that probe 1 is inert
ACCEPTED MANUSCRIPT to the thiol-free amino acids (Fig. 3). A similar observation was obtained for Hcy under identical experimental conditions (Figs. S3). Next, we explored the sensing ability of probe 1 toward GSH at same experimental condition. In contrast to Cys, upon addition of 10 equiv. of GSH, the
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solution of probe 1 led only to the decrease of the initial absorption band at 457 nm and did not induce any shift of the initial absorption band at 538 nm (Fig. 1d). This represents the production of thio-acridinium 6 due to the bulkiness of its tripeptide and the unstable 10-membered
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macrocyclic transition state, precluding the possibility of its subsequent rearrangement to
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amino-acridinium derivative. The assignment was also supported by the data of MALDI-TOF MS experiment (Fig. S2) and control compound AO-S-Et (Fig. S3). A prominent peak at m/z 656.9 corresponding to [6]+ (calcd. 656.8 for C32H42N5O8S+), which was generated from the incubation of probe 1 with GSH in EtOH–H2O, was clearly observed in the MALDI-TOF mass spectrum. In
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addition, the absorption maximum (Absmax: 538 nm) of AO-S-Et matched with thio-acridinium 6 perfectly (Fig. S3). In the pH 7.4 PBS buffer containing 1% EtOH, the reaction of the probe with GSH produced thio-acridinium but not amino-acridinium from the subsequent rearrangement
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reaction between 6 and excess GSH, indicating that probe 1 can be used to discriminate GSH from
AC C
Cys or Hcy. Then, the reactivity of probe 1 toward Cys, Hcy, and GSH under weakly acidic conditions was explored. As expected, the addition of Cys to pH 6.0 PBS buffer solution (20% EtOH) induced a big blue shift in the absorption maximum of probe 1 from 538 nm to 442 nm (Fig. 1d). However, Hcy and GSH caused no obvious change in the UV/vis spectrum of the probe. These results showed that probe 1 reacts with Cys in a highly selective manner. That is, probe 1 can selectively distinguish Cys from Hcy and GSH under weakly acidic conditions. Overall, the above results are in good accordance with our original design concept and the proposed sensing
ACCEPTED MANUSCRIPT mechanism (Scheme 2).
Figure 2.
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Owing to the desirable UV–Vis absorption spectra and pH effect results, two solvents (pH 7.4 PBS buffer containing 1% EtOH and pH 6.0 PBS buffer containing 20% EtOH) and two excitation wavelengths (438 and 537 nm) were used to examine the fluorescence emission
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behaviors of probe 1 in the presence of Cys, Hcy, and GSH (Figs. 2a and c). The selected
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excitation wavelengths at 438 and 537 nm were near the absorption maximums of amino-acridinium 5a (5b) and thio-acridinium 6 and 7. Under two experimental conditions, probe 1 nearly had no fluorescence because of the PET from the 3,4-dimethoxythiophenol to the acridinium. Upon excitation at 537 nm, a new emission peak appeared at 567 nm after the addition
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of 10 equiv. of GSH (Fig. 2a) and reached equilibrium within 2 min in the pH 7.4 PBS buffer (Fig. 2b). In this instance, approximately 150-fold increase in fluorescence signal was observed because of the production of thio-acridinium 6, as indicated by the UV–Vis results. By contrast, the
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addition of Cys or Hcy barely caused significant changes in the fluorescence of probe 1 at the
AC C
same condition (Figs. 2a and b). At 438 nm excitation wavelength, the addition of 10 equiv. of Cys to a solution of probe 1 elicited a distinct fluorescence enhancement (46-fold) at 518 nm because of the production of amino-acridinium 5a. Unfortunately, the addition of 10 equiv. of Hcy produced a minor emission at approximately 518 nm possibly because of the amino-coumarin derivative 5b. Moreover, the addition of 10 equiv. of GSH resulted only in a poor emission at approximately 572 nm. Poor emission is a characteristic of thio-acridinium 6 (Fig. S4a). These results indicated that the probe can discriminate GSH from Cys or Hcy but cannot distinguish
ACCEPTED MANUSCRIPT between Cys and Hcy. Next, the fluorescence responses of the probe toward Cys, Hcy, and GSH in pH 6.0 PBS buffer (20% EtOH) was examined to achieve the selective discrimination of Cys from Hcy and GSH, Indeed, changing the medium to pH 6.0 PBS buffer (20% EtOH) did change
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the selectivity of probe 1 towards Cys. An extremely weak emission of probe 1 (λem = 515 nm, λex = 438 nm) changed significantly in presence of Cys in a “turn on” manner whereby the emission intensity increased by 52-fold, as shown in Fig. 2c. The emission was identified to be
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amino-acridinium 5a because it is nearly identical to emission obtained in pH 7.4 PBS buffer (1%
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EtOH). The kinetic studies revealed that the emission intensity reaches a maximum value within 10 mins after Cys addition (Fig. 2d). In the other hand, although Hcy and GSH present slight fluorescence increases but the increase is time consuming because of the low thiolate and the amine concentrations at this pH condition. In contrast, with excitation at 537 nm, addition of Hcy
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(or GSH) caused hardly significant fluorescence changes in probe 1 at 518 nm, but immediately elicited a significant fluorescence enhancement of 101-fold at 567 nm (or 95-folds at 570 nm) (Fig. S4b), revealing that the corresponding thio-acridiniums were produced. These results confirmed
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that probe 1 is capable of highly selective detection of Cys from Hcy and GSH under weak acidic
AC C
condition but does not distinguish between Hcy and GSH. In addition, GSH and Cys can easily be distinguished macroscopically when illuminated by a
hand-held UV lamp at 365 nm (Fig. 2). Introducing GSH to the solution of probe 1 results in a strong orange emission color, whereas Cys/Hcy elicits a bright green emission in pH 7.4 PBS buffer (1% EtOH) (Fig. 2a inset). By contrast, upon the addition of Cys in pH 6.0 PBS buffer (20% EtOH), probe 1 induces a strong green fluorescence, but Hcy/GSH results in a significant orange emission (Fig. 2c inset).
ACCEPTED MANUSCRIPT 3.4. Selectivity and competitiveness To evaluate the specific nature of probe 1 for GSH and Cys, the probes was separately incubated with other amino acids and various biologically relevant analytes under the same
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experimental conditions. The biologically important Na+ and K+ cations were excluded because of their high contents in PBS buffers. With the excitation of 537 nm, probe 1 exhibit hardly significant fluorescence enhancement to various analytes, in order as 0) blank, 1) Ser, 2) Lys, 3)
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Asp, 4) Glu, 5) His, 6) Val, 7) Asn, 8) Gln, 9) Leu, 10) Thr, 11) Pro, 12) Trp, 13) Ile, 14) Ala, 15)
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Sar, 16) Phe, 17) Arg, 18) Tyr, 19) Gly, 20)Met, 21) ATP, 22) Gluc, 23) Zn2+, 24) Mg2+, 25) Ca2+, 26) Fe3+, 27) Cys-Cys, 28) Cys, 29) Hcy in pH 7.4 PBS buffer (1% EtOH). In fact, only GSH elicited a remarkable increase in the fluorescence intensity at 567 nm (Fig. 3a). With excitation at 438 nm, all other competitive species, including Hcy and GSH, showed nearly no fluorescence
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intensity changes in probe 1. Only Cys caused significant fluorescence turn-on at 518 nm in pH 6.0 PBS buffer (20% EtOH; Fig. 3b). Importantly, Cys and Hcy, which are difficult to distinguish
acidic condition.
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through the SNAr-rearrangement mechanism, can be distinguish among one another under weak
AC C
Fluorescence interference experiments were performed in the presence of various competing analytes under the same experimental condition to determine the feasibility of using probe 1 as a GSH- and Cys-selective probe. Other background analytes had small or no obvious interference with the detection of GSH, except for cystine, Cys and Hcy, upon excitation at 537 nm in pH 7.4 PBS buffer (1% EtOH). Cys or Hcy might form the amino-substituted derivatives with the probe through a sequential SNAr and rearrangement reactions, which induced a blue shift in the fluorescence spectra of low intensity associated with Cys/Hcy. The disturbance of cystine
ACCEPTED MANUSCRIPT (Cys–Cys) can be attributable to the Cys, which was formed through the GSH triggered disulfide cleavage reaction of Cys–Cys (Fig. 3a) [70]. As expected, the probe was not interfered by other various competing analytes and Cys–Cys for the detection of Cys upon the excitation of 438 nm in
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pH 6.0 PBS buffer (20% EtOH). However, the coexistence of GSH or Hcy with Cys disturbed the detection of Cys at the same condition due to the formation of thio-acridiniums 6 and 7 through
fluorescence spectra associated with GSH/Hcy (Fig. 3b).
3.5. Fluorescence titration study
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Figure 3.
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the initial thio-substitution reaction, which similarly induced a red shift in the low-intensity
Basing on its excellent selectivity, the researchers subsequently investigated the ability of
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quantitative detection of probe 1 toward GSH and Cys through fluorescent titration. Titration experiments resulted in good concentration-dependent fluorescence changes. As shown in Fig. 4a. with increasing GSH concentration, the fluorescence emission intensity of probe 1 at λmax 567 nm
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increased gradually in pH 7.4 PBS buffer (1% EtOH) when undergoing excitation at 537 nm.
AC C
When GSH concentration was increased to ca. 10 µM (1.0 equiv.), the fluorescence emission intensity became saturated. The plot of emission intensities of the probe 1 as a function of added GSH concentration was linear up to 7 µM (Fig. 4a, inset), useful for the determination of low-level GSH. A Job's plot (Fig. S6a) and MALDI-TOF mass data (Fig. S2) indicate that the reaction between probe 1 and GSH has a 1:1 stoichiometry. The detection limit [71] is determined to be 5.0 nM based on 3σ/S (Fig. 4a, inset), which is significantly below the physiological levels of GSH in live cells (1–10 mM) [72]. This result is indeed important in view of the few
ACCEPTED MANUSCRIPT fluorescent probes that can selectively and quantitatively detect GSH over Cys/Hcy in the literature so far. Similarly, the sensing ability of probe 1 to Cys was investigated by fluorescence titration in pH 6.0 PBS buffer (20% EtOH) as shown in Fig. 4b. Probe 1 can quantitatively detect
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Cys and produces a strong fluorescent signal at very low concentrations of Cys (0 µM to 25 µM). Moreover, analysis of the fluorescence intensity at 518 nm vs Cys concentration was found to be linear (R = 0.9946) with a detection limit calculated to be 0.11 µM (S/N = 3) (Fig. 4b, inset). In
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addition, a Job's plot exhibits a maximum at 0.5 molecular fraction, which indicates that the
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reaction between probe 1 and GSH has a 1:1 stoichiometry (Fig. S6b). Accurate stoichiometry of
Figure 4.
the adduct 1-Cys was further confirmed by MALDI-TOF mass spectrometry analysis (Fig. S2).
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These findings suggest that Probe 1 is indeed a sensitive detector and quantitative monitor of Cys under weak acidic condition.
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3.6. Cell imaging experiments
Finally, to demonstrate the potential biological relevance, probe 1 was used to monitor the
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levels of thiols in HeLa cells. HeLa cells incubated with probe 1 (10 µM) for 30 min showed strong fluorescence in two emission channels, green (500 nm to 550 nm) and red (570 nm to 620 nm) (Fig. 5A1 and 5B1), confirming that this probe is capable of permeating into cells and reacting with endogenous thiols to produce discernible fluorescence responses. Subsequent control experiments were performed to gain further support for this conclusion. In one control experiment, the HeLa cells were pretreated with the thiol-blocking reagent N-methylmaleimide (NMM) (100 µM) for 30 min and then incubated with probe 1 for 30 min. The inversed microscopy image of
ACCEPTED MANUSCRIPT cells treated in this manner does not display the fluorescence (Fig. 5A2 and 5B2). Moreover, incubation of the NMM-pretreated HeLa cells with Cys (500 µM) or Hcy (500 µM) causes a significant fluorescence increase in green emission (Fig. 5A3 and 5A4), while almost no
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fluorescence is observed upon incubation with GSH (Fig. 5A5). However, incubation of the cells with GSH (500 µM) caused a marked fluorescence increase in red emission (Fig. 5B5), while both Cys and Hcy barely elicited any significant fluorescence changes of probe 1 (Fig. 5B3 and 5B4),
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indicative of the high selectivity of probe 1 to GSH from Hcy and Cys in living cells.
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Subsequently, similar bioimaging under the condition of pH 6.0 was performed in HeLa cells (Fig. 5C and 5D). By contrast, the relative weak fluorescence emission of cells when incubated with probe 1 (10 µM) was found both in green emission and red emission (5C1 and 5D1). Similar to at pH 7.4, the HeLa cells pretreated with the thiol-blocking reagent N-methylmaleimide (NMM)
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(100 µM) for 30 min and then incubated with probe 1 (10 µM) for 30 min does not display the fluorescenc (5C2 and 5D2). As expected, a strong fluorescence in the cells was observed at green
Figure 5.
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channel but no emission was observed at red channel after the pre-treated cells with NMM were
incubated further with Cys (500 µM) for 30 min and then incubated with probe 1 (10 µM) for 30 min (5C3 and 5D3), which are in good accordance with the results of fluorescence spectra. Importantly, in the cases of both Hcy and GSH, almost no fluorescence is observed (5C4 and 5C5) at pH 6.0 condition. These results are in agreement with the specificity of probe 1 for Cys or GSH, and also demonstrate the potential of probe 1 to sense intracellular Cys and GSH selectively under different pH conditions and emission channels.
ACCEPTED MANUSCRIPT 4. Conclusion We
developed
an
acridinium
salt-based
fluorescent
probe
1
having
a
3,4-dimethoxythiophenol leaving group that undergoes reaction with thiols. The probe was able to
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selectively react with biothiols over other thiol-free amino acids through breaking PET process under physiological conditions. Importantly, the probe shows emission-dependent selectivity for GSH over Cys and Hcy and pH-dependent selectivity for Cys over Hcy and GSH in PBS buffer.
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Moreover, the fluorescence response is free from disturbance by other amino acids and
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biologically related analytes tested. The probe can detect as low as 5.0 × 10−9 M−1 GSH and 1.1 × 10−7 M−1 Cys, respectively. In addition, the probe has good water solubility, cell-penetration ability, and biocompatibility, and could thus be used to separately sense intracellular GSH and Cys by bioimaging by adjusting the emission channel and pH environment. Finally, the present
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strategy is expected to inspire the exploration of new systems that can respond to biothiols with multiple selectivity and high sensitivity for probing biothiols function in biological systems.
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Acknowledgments
This work is supported by the National Natural Science Foundation of China (No. 21262039
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and 2156020075).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at http://dx.
References [1] G. I. Giles, Curr. Pharm. Des., 2006, 12(34), 4427-4443. [2] N. Brandes, S. Schmitt, U. Jakob, Antioxid. Redox Signaling, 2009, 11(5), 997-1014.
ACCEPTED MANUSCRIPT [3] C. K. Sen, Curr. Top. Cell. Regul., 2000, 36, 1-30. [4] T. P. Dalton, H. G. Shertzer and A. Puga, Annu. Rev. Pharmacol. Toxicol., 1999, 39, 67-101. [5] S. C. Lu, Mol. Aspects Med., 2009, 30(1-2), 42-59.
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[6] D. M. Townsend, K. D. Tew, H. Tapiero, Biomed. Pharmacother., 2003, 57(3-4), 145-155. [7] J. M. Estrela, A. Ortega, E. Obrador, Crit. Rev. Clin. Lab. Sci., 2006, 43, 143-181. [8] L. M. Hyman, K. J. Fanz, Coord. Chem. Rev., 2012, 256, 2333-2356.
M AN U
[10] S. Shahrokhian, Anal. Chem., 2001, 73(24), 5972-5978.
SC
[9] X. Chen, X. Tian, I. Shin, J. Yoon, Chem. Soc. Rev., 2011, 40, 4783-4804.
[11] M. T. Heafield, S. Fearn, G. B. Steventon, R. H. Waring, A. C. Williams, S. G. Sturman, Neurosci. Lett., 1990, 110(1-2), 216-220.
[12] P. Sachdev, R. Parslow, C. Salonikas, O. Lux, W. Wen, R. Kumar, D. Naidoo, H. Christensen,
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A. Jorm, Arch. Neurol., 2004, 61, 1369-1376.
[13] P. Sachdev, J. Neurol. Sci., 2004, 226(1-2), 25-29. [14] B. Hooshmand, T. Polvikoski, M. Kivipelto, M. Tanskanen, L. Myllykangas, T. Erkinjuntti,
EP
M. Mäkelä, M. Oinas, A. Paetau, P. Scheltens, E. C. van Straaten, R. Sulkava, A. Solomon,
AC C
Brain, 2013, 136(9), 2707-2716.
[15] X. Chen, Y. Zhou, X. Peng, J. Yoon, J. Chem. Soc. Rev. 2010, 39, 2120-2135. [16] M. E. Jun, M. B. Roy, K. H. Ahn, Chem. Commun. 2011, 47, 7583-7601. [17] Y. Zhou, J. Yoon, Chem. Soc. Rev. 2012, 41, 52-67. [18] C. Yin, F. Huo, J. Zhang, R. Martínez-Máñez, Y. Yang, H. Lv, S. Li, Chem. Soc. Rev., 2013, 42, 6032-6059 [19] L.-Y. Niu, Y.-Z. Chen, H.-R. Zheng, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, Chem. Soc. Rev.,
ACCEPTED MANUSCRIPT 2015, 44, 6143-6160. [20] O. Rusin, N. N. St. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang, I. M. Warner, F. B. Dawan, K. Lian, R. M. Strongin, J. Am. Chem. Soc., 2004, 126, 438-439.
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[21] X. Yang, Y. Guo, R. M. Strongin, Angew. Chem. Int. Ed., 2011, 50, 10690-10693. [22] Z. Guo, S. Nam, S. Park, J. Yoon, Chem. Sci., 2012, 3, 2760-2765.
[23] H. Wang, G. Zhou, H. Gai and X. Chen, Chem. Commun., 2012, 48, 8341-8343.
SC
[24] J. H. Lee, C. S. Lim, Y. S. Tian, J. H. Han and B. R. Cho, J. Am. Chem. Soc., 2010, 132(4),
M AN U
1216-1217.
[25] H. S. Jung, J. H. Han, T. Pradhan, S. Kim, S. W. Lee, J. L. Sessler, T. W. Kim, C. Kang, J. S. Kim, Biomaterials, 2012, 33(3), 945-953.
[26] H. Li, J. Fan, J. Wang, M. Tian, J. Du, S. Sun, P. Sun, X. Peng, Chem. Commun., 2009,
TE D
5904-5906.
[27] L. Yuan, W. Lin and Y. Yang, Chem. Commun., 2011, 47, 6275-6277. [28] J. Shao, H. Sun, H. Guo, S. Ji, J. Zhao, W. Wu, X. Yuan, C. Zhang, T. D. James, Chem. Sci.,
EP
2012, 3, 1049-1061.
AC C
[29] L. Long, W. Lin, B. Chen, W. Gao and L. Yuan, Chem. Commun., 2011, 47, 893-895. [30] H. Kwon, K. Lee, H.-J. Kim, Chem. Commun., 2011, 47, 1773-1775. [31] Y.-K. Yang, S. Shim, J. Tae, Chem. Commun., 2010, 46, 7766-7768. [32] H. Li, L.-Y. Jin, Y. Kan, B. Yin, Sensors and Actuators B, 2014, 196, 546-554. [33] X. Zhou, X. Jin, G. Sun, D. Li, X. Wu, Chem. Commun., 2012, 48, 8793-8795. [34] L.-Y. Niu, Y.-S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, Chem. Commun., 2013, 49, 1294-1296.
ACCEPTED MANUSCRIPT [35] L. Ma, J. Qian, H. Tian, M. Lan, W. Zhang, Analyst, 2012, 137, 5046-5050. [36] D. Lee, G. Kim, J. Yin, J. Yoon, Chem. Commun., 2015, 51, 6518-6520. [37] J. O. Escobedo, W. Wang, R. M. Strongin, Nat. Protocols, 2006, 1, 2759-2762.
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[38] T.-K. Kim, D.-N. Lee, H.-J. Kim, Tetrahedron Lett., 2008, 49(33), 4879-4881. [39] W. Wang, J. O. Escobedo, C. M. Lawrence, R. M. Strongin, J. Am. Chem. Soc., 2004, 126(11), 3400-3401.
SC
[40] H. Chen, Q. Zhao, Y. Wu, F. Li, H. Yang, T. Yi and C. Huang, Inorg. Chem., 2007, 46(26),
M AN U
11075-11081.
[41] K.-S. Lee, T.-K. Kim, J. H. Lee, H.-J. Kim and J.-I. Hong, Chem. Commun., 2008, 6173-6175.
[42] S.-K. Sun, H.-F. Wang and X.-P. Yan, Chem. Commun., 2011, 47, 3817-3819.
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[43] H. Y. Lee, Y. P. Choi, S. Kim, T. Yoon, Z. Guo, S. Lee, K. M. K. Swamy, G. Kim, J. Y. Lee, I. Shin, J. Yoon, Chem. Commun., 2014, 50, 6967-6969. [44] L.-Y. Niu, Y.-S. Guan, Y.-Z. Chen, L.-Z. Wu, C.-H. Tung, Q.-Z. Yang, J. Am. Chem. Soc.,
EP
2012, 134, 18928-18931.
AC C
[45] N. Shao, J. Jin, H. Wang, J. Zheng, R. Yang, W. Chan,Z. Abliz, J. Am. Chem. Soc., 2010, 132, 725-736.
[46] Y. Guo, X. Yang, L. Hakuna, A. Barve, J. O. Escobedo, M. Lowry, R. M. Strongin, Sensors, 2012, 12, 5940-5950.
[47] Y. Xu, B. Li, P. Han, S. Sun, Y. Pang, Analyst, 2013, 138, 1004-1007. [48] M.-Y. Jia, L.-Y. Niu, Y. Zhang, Q.-A. Yang, C.-H. Tung, Y.-F. Guan, L. Feng, ACS Appl. Mater. Interfaces, 2015, 7, 5907-5914.
ACCEPTED MANUSCRIPT [49] J. Liu, Y.-Q. Sun, H. Zhang, Y. Huo, Y. Shi, W. Guo, Chem. Sci., 2014, 5, 3183-3188. [50] Y. Liu, X. Lv, J. Liu, Y.-Q. Sun, W. Guo, Chem. Eur. J., 2015, 21, 4747-4754. [51] S.-Y. Lim, K.-H. Hong, D. I. Kim, H. Kwon, H.-J. Kim, J. Am. Chem. Soc., 2014, 136,
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7018-7025. [52] J. Yin, Y. Kwon, D. Kim, D. Lee, G. Kim, Y. Hu, J.-H. Ryu, J. Yoon, J. Am. Chem. Soc., 2014, 136, 5351-5358.
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[53] X.-F. Yang, Q. Huang, Y. Zhong, Z. Li, H. Li, M. Lowry, J. O. Escobedoc, R. M. Strongin,
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Chem. Sci., 2014, 5, 2177-2183.
[54] F. Wang, L. Zhou, C. Zhao, R. Wang, Q. Fei, S. Luo, Z. Guo, H. Tian, W.-H. Zhu, Chem. Sci., 2015, 6, 2584-2589.
[55] C. Xu, H. Li, B. Yin, Biosens. Bioelectron., 2015, 72, 275-281.
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[56] J. Liu, Y.-Q. Sun, Y. Huo, H. Zhang, L. Wang, P. Zhang, D. Song, Y. Shi, W. Guo, J. Am. Chem. Soc., 2014, 136, 574-578.
[57] D. Lee, G. Kim, J. Yin, J. Yoon, Chem. Commun., 2015, 51, 6518-6520.
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420-422.
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[58] L. M. Mironovich, L. S. Ageeva, A. Yu. Podol’nikova, Russ. J. Gen. Chem., 2016, 86(2),
[59] C. Kaufmann, A. Albertini, Ber., 1909, 42, 1999-2008. [60] J. W. Happ, E. G. Janzen, B. C. Rudy, J. Org. Chem., 1970, 35, 3382-3389. [61] Y.-K. Yang, J. Tae, Org. Lett., 2006, 8, 5721-5723. [62] D. D.Perrin, W. L. F. Armarego, Purification of Laboratory Chemicals, Pergamon Press: New York, 1988 and references cited therein. [63] L. Wu and K. Burgess, Org. Lett., 2008, 10(9), 1779-1782.
ACCEPTED MANUSCRIPT [64] R. E. Benesch, R. Benesch, J. Am. Chem. Soc., 1955, 77, 5877-5881. [65] H. S. Jung, J. H. Han, T. Pradhan, S. Kim, S. W. Lee, J. L. Sessler, T. W. Kim, C. Kang, J. S. Kim, Biomaterials, 2012, 33(3), 945-953.
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[66] Y. Ma, S. Liu, H. Yang, Y. Wu, C. Yang, X. Liu, Q. Zhao, H. Wu, J. Liang, F. Li, W. Huang, J. Mater. Chem., 2011, 21, 18974-18982.
[67] S. C. Chai, A. A. Jerkins, J. J. Banik, I. Shalev, J. L. Pinkham, P. C. Uden, M. J. Maroney, J.
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Biol. Chem., 2005, 280, 9865-9869.
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[68] Z. Shi, X. Tang, X. Zhou, J. Cheng, Q. Han, J. Zhou, B. Wang, Y. Yang, W. Liu, D. Bai, Inorg. Chem., 2014, 52, 12668-12673.
[69] X. Dong, C.H. Heo, S. Chen, H.M. Kim, Z. Liu, Anal. Chem., 2014, 86, 308-311. [70] P. K. Pullela, T. Chiku, M. J. Carvan III, D. S. Sem, Anal. Biochem., 2006, 352, 265-273.
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[71] Y. Sun, Y. Liu, M. Chen, W. Guo, Talanta, 2009, 80, 996-1000.
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[72] C. Hwang, A. L. Sinskey, H. F. Lodish, Science, 1992, 257, 1496-1502.
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Figure Captions
Scheme 1. Synthesis route of probe 1
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Scheme 2. Proposed sensing mechanisms of probe 1 for Cys, Hcy and GSH.
Figure 1. The Fluorescence intensities of probe 1 (10 µM) in the presence of 10 equiv of Cys, Hcy
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and GSH as a function of pH in PBS buffer containing 1% (a) and 20% EtOH (b) excited at 537 (a) and 438 nm (b), respectively (a: λem = 567 nm, b: λem = 515 nm). Changes in absorption spectra of probe 1 (10 µM) measured in PBS buffer (10 mM, pH = 7.4, 1 % EtOH) (c) and PBS buffer (10
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mM, pH = 6.0, 20% EtOH) (d) upon addition of Cys, Hcy and GSH.
Figure 2. Fluorescence spectra of probe 1 (10 µM) after addition of 10 equiv. of Cys, Hcy or GSH in pH 7.4 PBS buffer containing 1% EtOH (λex = 537 nm, λem = 567 nm) (a) and in pH 6.0 PBS
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buffer containing 20% EtOH (λex = 438 nm, λem = 518 nm) (c), and the corresponding
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time-dependent fluorescence intensity changes. (b and d) Slits: 1.5/3 nm for (a) and (b), 3/3 nm for (c) and (d).
Figure 3. Fluorescence responses of probe 1 (10 µM) toward various analytes in pH 7.4 PBS buffer containing 1% EtOH (a) and in pH 6.0 PBS containing 20% EtOH (b), respectively. Black bar represent the addition of a single analyte (10 µM) in order of 0) blank, 1) Ser, 2) Lys, 3) Asp, 4) Glu, 5) His, 6) Val, 7) Asn, 8) Gln, 9) Leu, 10) Thr, 11) Pro, 12) Trp, 13) Ile, 14) Ala, 15) Sar, 16) Phe, 17) Arg, 18) Tyr, 19) Gly, 20) Met, 21) ATP, 22) Gluc, 23) Zn2+, 24) Mg2+, 25) Ca2+, 26)
ACCEPTED MANUSCRIPT Fe3+, 27) Cys-Cys, 28) Cys, 29) Hcy and 30) GSH. Red bar represent the subsequent addition of GSH (a) or Cys (b) to the mixture. Excitation wavelengths: 537 nm for (a) and 438 nm for (b).
Figure 4. Fluorescence spectra of probe 1 (10 µM) after addition of increasing amounts of GSH (a,
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λex = 537 nm) and Cys (b, λex = 438 nm) in pH 7.4 PBS buffer (1% EtOH) and pH 6.0 PBS buffer (20% EtOH), respectively. Insets: fluorescence intensity at 567 nm as a function of GSH (a) and
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Cys (b) concentrations.
Figure 5. Fluorescence images of living HeLa cells. A / B and C/ D incubated in pH 7.4 and pH
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6.0 PBS, respectively. The cells were incubated with probe 1(10 µM) for 30 min (A1, B1, C1 and D1); The cells were pre-incubated with NMM (0.1 mM) for 30 min, and then treated with probe 1 (10 µM) for 30 min (A2, B2, C2 and D2); The pre-incubated cells were incubated in sequence with Cys (500 µM) and probe 1 (10 µM)(A3, B3, C3 and D3); The pre-incubated cells were
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incubated in sequence with Hcy (500 µM) and probe 1 (10 µM)(A4, B4, C4 and D4); The pre-incubated cells were incubated in sequence with GSH (500 µM) and probe 1 (10 µM)(A5, B5,
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C5 and D5). The cells were incubated with 10 µM probe 1 for 30 min in pH 7.4 PBS buffer (1 %
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EtOH) for A and B, and in pH 6.0 PBS buffer (20 % EtOH) for C and D.
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Scheme 1
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Scheme 1
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Scheme 2
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
ACCEPTED MANUSCRIPT Highlights： ： 1. The probe is structurally simple and easy to synthesize 2. The probe can discriminates GSH from Cys and Hcy in pH 7.4 PBS buffer upon excitation of 537 nm.
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3. The probe can distinguishs Cys from GSH and Hcy in pH 6.0 PBS buffer upon excitation of 438 nm.
4. The probe has good cell-permeability and selectively sense intracellular GSH and
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Cys by multicolor imaging in HeLa cells at different pH environment.
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