Improved Aromatic SubstitutionâRearrangement-Based Ratiometric

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An Improved Aromatic Substitution-rearrangement-based Ratiometric Fluorescent Cysteine-specific Probe and Its Application of Real-time Imaging under Oxidative Stress in Living Zebrafish Longwei He, Xueling Yang, Kaixin Xu, and Weiying Lin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02649 • Publication Date (Web): 09 Aug 2017 Downloaded from http://pubs.acs.org on August 9, 2017

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Analytical Chemistry

An

Improved

Aromatic

Substitution-rearrangement-based

Ratiometric Fluorescent Cysteine-specific Probe and Its Application of Real-time Imaging under Oxidative Stress in Living Zebrafish

Longwei He, Xueling Yang, Kaixin Xu, Weiying Lin*

Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of Materials Science and Engineering, University of Jinan, Shandong 250022, P.R. China * Corresponding author. Tel: +86–531–82769031; Fax: +86–531–82769031 E-mail address: [email protected]

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Abstract: Biothiols, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a crucial role in many physiological processes. Cys production and metabolism is closely connected with Hcy and GSH, meanwhile, the dynamic antioxidant defenses network by Cys is independent on GSH system, and Cys can serve as a more effective biomarker of oxidative stress. Hence, it is significantly and urgently to develop an efficient method for specific detection of Cys over other biothiols (Hcy/GSH). However, most of present Cys-specific fluorescent probes distinguished Cys from Hcy through response time, which would be suffered to an unavoidable interference from Hcy in long time detection. In this work, in order to improve the selectivity, we employed an improved aromatic substitution-rearrangement strategy to develop a ratiometric Cys-specific fluorescent probe (Cou-SBD-Cl) based on a new FRET coumarin-sulfonyl benzoxadiazole (Cou-SBD) platform for discrimination of Hcy and GSH. Response of Cou-SBD-Cl to Cys would switch FRET on and generate a new yellow fluorescence emission with a 56.1-fold enhancement of ratio signal and a 99 nm emission shift. The desirable dual-color ratiometric imaging was achieved in living cells and normal zebrafish. In addition, probe Cou-SBD-Cl was also applied to real-time monitor Cys fluctuation in lipopolysaccharide-mediated oxidative stress in zebrafish.

Key

Words:

Cysteine-specific;

Ratiometric

fluorescent

probe;

Aromatic

substitution-rearrangement; Coumarin-sulfonyl benzoxadiazole dyad; Oxidative stress


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INTRODUCTION

Biothiol, including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a crucial role in many physiological processes, such as biological redox homeostasis, signal transduction, detoxify-cation of xenobiotics, and post-translational modifications.1-3 On the one hand, Cys production and metabolism is closely connected with Hcy and GSH. For example, Hcy is a precursor of Cys, which endogenously produced by cystathionine β-synthase (CBS) and cysathionine γ-lyase (CSE),4 meanwhile, GSH is synthesized from Cys by the consecutive actions of γ-glutamylcysteine synthetase (GSH I) and glutamine synthetase (GSH II).5 On the other hand, the dynamic antioxidant defenses network by Cys is independent on GSH system, and Cys is more effective to be oxidized than GSH, hence Cys can serve as a more effective biomarker for oxidative stress status.6 The concentration level of Cys would be abnormal in oxidative stress and further leads to various diseases including Alzheimer's

disease,

Parkinson's

disease,

liver

damage,

slowed

growth,

and

neurodegeneration.7,8 Therefore, it is significantly and urgently to develop an efficient method for specific detection of Cys over other biothiols (Hcy/GSH). Dual-color fluorescence imaging has attracted great attentions because of its favorable multi-channel fluorescence signal output and the independent ratiometric signal from probe concentration, probe environment, and equipment efficiency.9,10 Accordingly, ratiometric imaging has emerged as one important non-invasive technique to real-time monitor biomolecules and biological processes in the context of living systems with high temporal and spatial resolution.11-22 However, it is still very challenging to develop selective fluorescent probes for discriminating GSH/Cys/Hcy from each other, especially Cys from 3 ACS Paragon Plus Environment


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Hcy, owing to the similar structures and chemical properties of them. After years of efforts, there has been development of four main strategies for constructing Cys-specific probes, such as cyclization with aldehydes, conjugate addition-cyclization with acrylates, native chemical ligation, and aromatic substitution-rearrangement.23,24 Most of fluorescent probes based on those strategies distinguish Cys from Hcy through response time, because a bigger reaction rate constant exists between probes and Cys under the same condition.25-32 From this, specific detection of Cys will be subject to an unavoidable interference from Hcy in long time detection. In order to improve the selectivity of fluorescent probe for Cys, we utilized a reformation strategy of aromatic substitution-rearrangement to enhance the response selectivity and constructed a highly selective and sensitive fluorescent probe (Cou-SBD-Cl) for specificly detecting Cys over Hcy and GSH. The coumarin and sulfonyl benzoxadiazole (SBD) chromophore compose a new fluorescence resonance energy transfer (FRET) dyad linked with rigid piperazine group as the dual-channel fluorescence signal reporter, and the chlorine unit at 4-position on SBD chromophore was served as the Cys-specific recognition site. Free probe Cou-SBD-Cl exhibits inherent blue fluorescence of coumarin chromophore as the FRET process remains off. However, up addition of Cys, probe will undergo sequential substitution and rearrangement reactions and yielding a 4-amino SBD derivative, which results in triggering FRET on and generation of a new yellow fluorescence emission accompanied with blue fluorescence weakness (Emission shift: 99 nm). In contrast, owing to incapable occurrence of rearrangement, encounter of probe with Hcy or GSH produces a 4-sulfydryl SBD derivative and induces almost negligible fluorescence intensity changes. We 4 ACS Paragon Plus Environment

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demonstrated the versatile utility of Cou-SBD-Cl by monitoring Cys in biological contexts using confocal fluorescence imaging. Moreover, we demonstrated that Cou-SBD-Cl can be employed to visualize Cys fluctuation in LPS-induced oxidative stress in living zebrafish. Therefore, development of probe Cou-SBD-Cl may offer a useful tool for elucidation of the intricate roles of Cys in living systems.

MATERIAL AND METHODS Materials and instruments. Unless otherwise stated, all reagents were purchased from commercial suppliers and used without further purification. Twice distilled water was used throughout all experiments. The instruments used in this work were listed in supporting information.

Cell culture. HeLa cells were cultured in DMEM (Dulbecco’s modified Eagle's medium) supplemented with 10% FBS (fetal bovine serum) and grown in the constant-temperature incubator with an atmosphere of 5% CO2 and 95% air at 37 °C. Fluorescence imaging of Cys in living cells. HeLa cells were incubated with Cou-SBD-Cl (5 µM) for 20 min, then imaged after washing by PBS buffer, as the control group. HeLa cells were pretreated with Cou-SBD-Cl (5 µM) for 20 min, subsequently incubated with Cys (50, 100 and 250 µM) or Hcy (500 µM) for another 60 min, then imaged after washing by PBS buffer, as the experimental group of imaging exogenous Cys. The confocal microscopic imaging uses Nikon A1MP confocal microscope with excitation filter of 405 nm and the collection wavelength range is from 425-475 nm (blue channel) and 550-600 nm (green 5 ACS Paragon Plus Environment


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channel). The cellular imaging was repeated three times, and the mean value of fluorescence intensity in blue/green channel was calculated. The fluorescence intensity ratio values (Igreen/Iblue) were calculated by division of the intensity of the green channel to that of the blue channel. The ratiometric images and related statistics of ratio value were processed and obtained on Nikon A1MP analysis software.

Fluorescence imaging of Cys in living zebrafish. Wild type Zebrafish were obtained from the Nanjing Eze-Rinka Biotechnology Co., Ltd.. 5-day-old zebrafish were incubated with Cou-SBD-Cl (10 µM) for 60 min, then imaged after washing by PBS buffer, as the control group. Zebrafish were pretreated with NEM (0.5 mM) for 30 min, subsequently incubated with Cou-SBD-Cl for 60 min, then imaged after washing by PBS buffer, as the negative control group. Zebrafish were pretreated with Cys (200 and 500 µM) for 30 min, subsequently incubated with Cou-SBD-Cl for 60 min, then imaged after washing by PBS buffer, as the experimental group. To monitor the fluctuation of Cys in zebrafish under oxidative stress status, zebrafish were was mediated by lipopolysaccharide (LPS, 100 µg/mL) for 30, 60, or 120 min, subsequently incubated with Cou-SBD-Cl for 60 min, and then imaged the zebrafish after washing by PBS buffer. The confocal microscopic imaging uses Nikon A1MP confocal microscope with excitation filter of 405 nm and the collection wavelength range is from 425-475 nm (blue channel) and 550-600 nm (green channel). The cellular imaging was repeated three times, and the mean value of fluorescence intensity in blue/green channel was calculated. The ratiometric images and related statistics of ratio value were processed and obtained on Nikon A1MP analysis software.


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Synthesis of compound Cou-SBD-Cl. The piperazyl-coumarin 3 was prepared by previous methods

in

laboratory.33

our

The

raw

materials

4-chloro-7-chlorosulfonyl-2,1,3-benzoxadiazole (4, 25 mg, 0.1 mmol) and compound 3

(33

mg, 0.1 mmol) were dissolved in 3 mL of dichloromethane, followed by addition of a drop of trimethylamine, and then stirred for 1 hour at room temperature. After complete reaction, the solvent was removed under reduced pressure and the solid residue was purified by flash chromatography column using methanol/dichloromethane (1/70, v/v) to afford a yellow powder as compound Cou-SBD-Cl (40 mg, yield 73.2%). 1H NMR (400 MHz, CDCl3), δ (ppm): 1.21-1.24 (t, J = 6.8 Hz, 6H), 3.41-3.49 (m, 10H), 3.84 (2H), 6.47 (s, 1), 6.61-6.63 (d, J = 7.2 Hz, 1H), 7.28-7.31 (d, J = 8.8 Hz, 1H), 7.56-7.58 (d, J = 7.2 Hz, 1H), 7.86 (s, 1H), 7.95-7.97 (d, J = 7.2 Hz, 1H);

13

C NMR (100 MHz, CDCl3): δ (ppm):42.16, 45.11, 45.84,

46.15, 47.30, 96.99, 107.82, 109.68, 115.02, 125.88, 128.33, 129.14, 130.08, 134.54, 145.53, 146.17, 148.87, 151.89, 157.37, 159.24, 165.22. HRMS (ESI) m/z calcd for C24H24ClN5O6S ([M+1]+): 546.1209. Found 546.1209.

Synthesis of compound SBD-Cl. The product SBD-Cl was prepared by the similar synthetic method of Cou-SBD-Cl with a yield of 81.6 %. 1H NMR (400 MHz, CD3OD): δ 1.07-1.11 (t, J = 7.0 Hz, 6H), 3.31-3.36 (q, J = 7.0 Hz, 4H), 7.89-7.91 (d, J = 7.2 Hz, 1H), 8.06-8.08 (d, J = 7.6 Hz, 1H).

13

C NMR (100 MHz, CD3OD): δ 14.35, 42.15, 125.18, 126.89, 130.65,

134.88, 145.28, 148.78. HRMS (ESI) m/z calcd for C10H13ClN3O3S ([M+1]+): 290.0361. Found 290.0365.



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RESULTS AND DISCUSSION Design and synthesis of compound dyad Cou-SBD-Cl. Yang et. al confirmed that 4-chloro-7-nitrobenzofurazan (NBD-Cl) can detect Cys and Hcy with turn-on fluorescence based on aromatic substitution-rearrangement, and the reaction rate of NBD-Cl with Cys is around seven times larger than that of Hcy under the same conditions.34 Based on this research result, we predicted the 4-chloro-N,N-diethylsulfonylamino benzoxadiazole (SBD-Cl) is able to trigger substitution and rearrangement reactions with Cys, but the rearrangement process can hardly occur in the reaction between SBD-Cl and Hcy (Scheme 1A), resulting in an improvement in selectivity for Cys over Hcy. The results of response spectra demonstrated our supposition. An apparent fluorescence enhancement was generated in the mixture of SBD-Cl and Cys, on the contrary, addition of Hcy induced no fluctuation of emission intensity within 1 hour (Figure 1A), which can be ascribed to the higher chemical reactivity of 4-position on SBD platform with relative mild electron withdrawing sulfonylamino group than that on NBD platform with strong electron withdrawing nitro group. Hence the chlorine group at 4-position on SBD chromophore is an appropriate Cys-specific recognition site without interference from Hcy. To achieve well-defined dual-channel fluorescence signals, energy donor coumarine and energy acceptor SBD chromophore were chosen as two signal reporting groups and constructed an innovative FRET platform (Cou-SBD) with large shift emission. As shown in Figure 1B, the emission spectrum of coumarin has almost no overlap with absorption spectrum of SBD-Cl, while efficiently overlaps with the red-shifted absorption spectrum of SBD-Cl in the presence of Cys, indicating Cys is a feasible trigger for FRET process in fluorescent dyad Cou-SBD-Cl. 8 ACS Paragon Plus Environment

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Therefore, we employed an improved aromatic substitution-rearrangement strategy to rationally design a ratiometric Cys-specific fluorescent probe (Cou-SBD-Cl) based on new Cou-SBD FRET platform (Scheme 1B). Upon reaction with Cys, the FRET process might switch on and the emission color of Cou-SBD-Cl might be changed from blue (coumarin) to yellow (SBD). The targeted fluorescent probe and control compound were synthesized through simple synthetic steps (for synthetic and characterization details, see the Supporting Information).

Scheme 1. Design of an improved aromatic substitution-rearrangement-based ratiometric fluorescent Cys-specific probe (Cou-SBD-Cl) based on coumarin-sulfonyl benzoxadiazole FRET dyad.



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Figure 1. (a) The fluorescence spectra of SBD-Cl in the absence (black) and presence of Cys (Red), Hcy (Blue), or GSH (Pink). (b) The normalized absorption spectra of energy acceptor SBD-Cl before (green) and after (red) addition of Cys and the normalized fluorescence spectrum of energy donor coumarin 3 (blue).

Optical response of probe Cou-SBD-Cl to Cys. With the desired probe in hands, we first test the dynamic range of probe (10 µM) when responding to Cys in aqueous solution (pH 7.4, 25 mM PBS buffer solution containing 20% DMF). In titrimetric spectra analysis, as shown in Figure 2, free probe Cou-SBD-Cl only fluoresces at the maxima of 481 nm. However, upon addition of incremental Cys (0-100 equiv.), the initial blue fluorescence faded away gradually, and accompanied with generation of a new yellow emission band centered at 580 nm (Figure 2A). According to a general calculation method ([(fluorescence of donor -fluorescence of donor in cassette)/fluorescence of donor] × 100%),35 the energy transfer efficiency of the coumarin-sulfonyl benzoxadiazole dyad was calculated to be 97.05% (Figure S1). The ratio value of fluorescence intensity (I580/I481) has measured a 56.1-fold increase and has a good linear relationship with Cys concentration ranging from 30 to 200 µM (Figure 2B), accordingly the detection limit is determined to be 1.4×10-6 M (S/N = 3). In 10 ACS Paragon Plus Environment

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absorptive response spectra, after encounter of probe with Cys, the absorption band centered at 330 nm was disappeared and simultaneously enhanced absorption at 420 nm (Figure S2), indicating the reaction between Cou-SBD-Cl and Cys yields the Cys-induced substitution-rearrangement product Cou-SBD-Cys, which was verified by the high resolution mass spectrometry (HRMS) of probe in the presence of Cys (Figure S3). The results of spectra study probe Cou-SBD-Cl possesses significant sensitivity and selectivity for Cys with ratiometric signal and large emission shift (99 nm).

Figure 2. (a) Fluorescence spectra of Cou-SBD-Cl (10 μM) with 0–100 equiv. of Cys in aqueous solution (pH 7.4, 25 mM PBS buffer solution containing 20% DMF). Incubation time: 60 min. Excitation: 450 nm. The inset shows the visual fluorescence color of Cou-SBD-Cl before (left) and after (right) addition of Cys (UV lamp, 365 nm) and the scattergram profile of ratio values of fluorescence intensity at 580 and 481 nm. (b) The linear relationship between the fluorescence intensity ratio (I580/I481) and the concentration of Cys (30-200 µM).



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Responding time and effect of pH assay. We then evaluated the response time of probe Cou-SBD-Cl to Cys. As shown in Figure 3A, the ratio values of fluorescence intensity at 580 and 481 nm (I580/I481) reached a plateau within about 1 hour in the presence of 30 or 60 equiv. of Cys. However, even treatment of probe with 120 equiv. of Hcy induced negligible changes of fluorescence intensity during the same time. In addition, the pH effect on probe was also evaluated. As shown in Fig. 3B, there was almost no change of ratio values (I580/I481) for free probe at pH 4-10, suggesting Cou-SBD-Cl is stable under acidic and alkaline conditions. However, an obvious enhancement of ratiometric signals was observed within the normal physiological ranges when being treated with Cys. These results indicate that probe Cou-SBD-Cl has the potential of discrimination of Cys from Hcy in physiological conditions.

Figure 3. (a) Reaction-time profile of probe Cou-SBD-Cl (10 µM) in the presence of Cys (300 or 600 µM) or Hcy (1200 µM) in aqueous solution (pH 7.4, 25 mM PBS buffer solution containing 20% DMF). (b) pH-dependence of probe Cou-SBD-Cl (10 µM) in the absence or presence of Cys (600 µM) in different pH (range from 4.0 to 10.0) PBS solution (25 mM, containing 20% DMF). Incubation time: 60 min. Excitation: 450 nm.


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Selectivity of probe Cou-SBD-Cl. Selectivity, a vital feature for fluorescent probes, of Cou-SBD-Cl was evaluated in the presence of various biological analyte species, including essential amino acid (Ala, Arg, Asp, Glu, His, Ser, Thr, Val, Ile, Phe, Tyr), biothiols (Cys, Hcy, GSH), anions (Br-, AcO-, NO3-, PO43-, S2-, ascorbate), cations (Na+, Ca2+, Cu2+, Fe3+), and reactive oxygen/nitrogen/sulfur species (H2O2, HClO, 1O2, BuO•, O2•-, NO). As shown in Figure 4, Cou-SBD-Cl exhibits negligible fluorescence intensity ratio (I580/I481) in the presence of these interferents (1.0 mM). It was worth noting that the homogeneous biothiols, such as Hcy and GHS, induced no obvious fluorescence changes (Figure S4). However, a remarkable ratio enhancement was observed after treated with the same level of Cys, which suggested probe Cou-SBD-Cl has superior selectivity for Cys over other biological species, especially Hcy and GSH. The MS results indicate that Hcy/GSH can indeed react with the probe in the aqueous solution (Figure S5 and S6) as mentioned above. We hypothesized that the reaction between the probe and Cys may produce the fluorescent 4-amino SBD group and the reaction with Hcy/GSH may afford the non-fluorescent 4-sulfydryl SBD moiety. That is to mean that Cou-SBD-Cl has great potential in specific distinguishment of Cys from Hcy and GSH in complex and changeable living systems.



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Figure 4. Fluorescence responses of the probe Cou-SBD-Cl (10 µM) to various relevant species (1.0 mM) in aqueous solution (pH 7.4, 25 mM PBS buffer solution containing 20% DMF). Incubation time: 60 min. Excitation: 450 nm. 1, blank; 2, Ala; 3, Arg; 4, Asp; 5, Glu; 6, His; 7, Ser; 8, Thr; 9, Val; 10, Ile; 11, Phe; 12, Try; 13, Hcy; 14, GSH; 15, Cys; 16, Br-; 17, AcO-; 18, NO3-; 19, PO43-; 20, S2-; 21, ascorbate; 22, Na+; 23, Ca2+; 24, Cu2+; 25, Fe3+; 26, H2O2; 27, HClO; 28, 1O2; 29, BuO•; 30, O2•-; 31, NO.

Ratiometric fluorescence imaging of Cys in living cells. Encouraged by the favorable spectral response of probe Cou-SBD-Cl to Cys in vitro, we further assessed the capability of probe for monitoring Cys in living cells by dual-color and ratiometric fluorescence imaging. Before that, the cell cytotoxity of Cou-SBD-Cl was evaluated by standard MTT assays, which suggested that Cou-SBD-Cl possesses low cytotoxicity to living HeLa cells (Figure S7). HeLa cells incubated with only Cou-SBD-Cl (5 µM) showed bright emission in the blue channel while almost no fluorescence in the green channel (Figure 5A). However, pretreatment with incremental Cys (50, 100 and 250 µM), though amount of co-existence of endogenous GSH, the blue fluorescence intensity gradually decreased accompanied by enhancement of emission in the green channel, which is consistent with results of responding 14 ACS Paragon Plus Environment

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spectra in aqueous solution. The signal ratios of fluorescence intensity at two channel (Fgreen/Fblue) were also calculated, inducing a maximum of around 50 folds enhancement (Figure 5F). Delightedly, even treatment with 500 µM Hcy, a most potential interferent, leaded to very small ratio increase (Figure 5E). Thus, Cou-SBD-Cl is capable to detect Cys over Hcy and GSH by the dual-color fluorescence imaging in the living cells.

Figure 5. Fluorescence images of probe Cou-SBD-Cl responding to Cys and Hcy in living HeLa cells by confocal fluorescence imaging. (A) Cells were incubated with probe Cou-SBD-Cl (5 µM, 20 min), then imaged; (B-E) Cells were treated with probe Cou-SBD-Cl (5 µM, 20 min), subsequently incubated with Cys (50 µM (B), 100 µM (C), or 250 µM (D)) or Hcy (250 µM (E)) for 60 min, then imaged. The fluorescence images were captured from the blue channel of 425-475 nm (second column) and green channel of 550-600 nm (third column) with excitation at 405 nm. Fourth column: ratiometric images of



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green channel to blue channel. Scale bar: 20 µm. (F) Fluorescence intensity ratio (Igreen/Iblue) in Fig. A-E. Data are expressed as mean ± SD of three parallel experiments.

Ratiometric fluorescence imaging of Cys in living zebrafish. To evaluate efficacy of Cou-SBD-Cl on monitoring Cys in living animal model, we chose 5-day-old vertebrate zebrafish as the testing biological samples. The zebrafish incubated with only Cou-SBD-Cl (10 µM) showed bright blue emission and very dim fluorescence in the green channel (Figure 6A). In negative control experiment, the zebrafish pretreated with NEM (N-ethylmaleimide, a scavenger for biothiols) performed strong blue fluorescence with slight decrease of green fluorescence (Figure 6B), indicating that endogenous GSH has little interference on outputting of the fluorescent signal of Cou-SBD-Cl. However, upon addition of increasing dosages of Cys (200-500 µM), the fluorescence was significantly enhanced in the green channel and the blue emission gradually weakened at the same time (Figure 6C-D). The ratios of fluorescence intensity (Fgreen/Fblue) were determined to be 3.9 and 8.7 folds enhancement (Figure 6E), respectively. The results suggested that probe Cou-SBD-Cl has the capacity to detect Cys without interference from GSH in living small animals.


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Figure 6. Fluorescence images of probe Cou-SBD-Cl responding to Cys in living 5-day-old zebrafish by confocal fluorescence imaging. (A) Zebrafish were incubated with Cou-SBD-Cl (10 µM, 60 min), then imaged; (B) Zebrafish were pretreated with NEM (0.5 mM, 30 min), subsequently incubated with probe Cou-SBD-Cl (10 µM, 60 min), then imaged; (C-D) Zebrafish were pretreated with NEM (0.5 mM, 30 min), subsequently incubated with Cys (200 or 500 µM, 20 min) and probe Cou-SBD-Cl (10 µM, 60 min), then imaged. The fluorescence images were captured from the blue channel of 425-475 nm (first row) and green channel of 550-600 nm (second row) with excitation at 405nm. Third row: ratiometric images of green channel to blue channel. Scale bar: 500 µm. (F) Fluorescence intensity ratio (Igreen/Iblue) in Fig. A-D. Data are expressed as mean ± SD of three parallel experiments.

Ratiometric fluorescence imaging of Cys in living zebrafish under oxidative stress status. Oxidative stress refers to the cellular status with enhanced production of intracellular reactive oxygen species and decrease of Cys concentration level, which impairs the function of the 17 ACS Paragon Plus Environment


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cellular anti-oxidant defense system.36,37 According to the preliminary imaging studies in living cells and normal zebrafish, we further evaluated the applicability of the probe Cou-SBD-Cl for monitoring Cys fluctuation in zebrafish under oxidative stress status. The oxidative stress of zebrafish was mediated by lipopolysaccharide (LPS),38,39 and imaged the zebrafish at a set of times from 30 min to 120 min. As shown in Figure 7, comparing with the control zebrafish incubated with free probe Cou-SBD-Cl, a slight fluorescence increase in the blue channel and small decrease of fluorescence in the green channel was generated in zebrafish pretreated with LPS (100 µg/mL), which was in line with the reduced concentration of Cys in oxidative stress status. The decrease of ratio intensity (Fgreen/Fblue) was also observed in LPS-mediated zebrafish along with the time (Figure 7E). Therefore, our probe Cou-SBD-Cl was demonstrated to be able to image fluctuation of endogenous Cys level in living animals and be a potential powerful tool for elucidation of the intricate roles of Cys in oxidative stress responses.


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Figure 7. Fluorescence images of probe Cou-SBD-Cl responding to Cys in living 5-day-old zebrafish under oxidative stress status by confocal fluorescence imaging. (A) Zebrafish were incubated with Cou-SBD-Cl (10 µM, 60 min), then imaged; (B-D) Zebrafish were was mediated by lipopolysaccharide (LPS, 100 µg/mL) for 30 (B), 60 (C), or 120 (D) min, subsequently incubated with Cou-SBD-Cl (10 µM) for 60 min, then imaged. The fluorescence images were captured from the blue channel of 425-475 nm (first row) and green channel of 550-600 nm (second row) with excitation at 405 nm. Third row: ratiometric images of green channel to blue channel. Scale bar: 500 µm. (E) Fluorescence intensity ratio (Igreen/Iblue) in Fig. A-D. Data are expressed as mean ± SD of three parallel experiments.

CONCLUSION In summary, we have developed a ratiometric Cys-specific fluorescent probe (Cou-SBD-Cl) based on new FRET coumarin-sulfonyl benzoxadiazole platform with large emission shift (99 nm) through an improved aromatic substitution-rearrangement strategy. The chlorine group on SBD moiety with moderate reactivity was subtly regarded as the recognition site for specific discrimination of Cys from the most potential interference of Hcy. Reaction of Cou-SBD-Cl with Cys will produce sequential substitution and rearrangement reactions at 4-position on benzoxadiazole chromophore, resulting in triggering FRET on and generation of a new yellow fluorescence emission accompanied with blue fluorescence weakness with a 56.1-fold ratio signal enhancement and a 99 nm emission shift. The desirable dual-color ratiometric imaging was achieved in living cells and normal zebrafish. In addition, probe Cou-SBD-Cl was also applied to real-time monitor Cys fluctuation in LPS-mediated oxidative stress in zebrafish. Thus, development of Cou-SBD-Cl provides a new strategy to 19 ACS Paragon Plus Environment


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improve the selectivity of fluorescent probes for Cys and a new FRET platform to construct ratiometic fluorescent probes, and offers a potential powerful tool for elucidation of the intricate roles of Cys in oxidative stress responses.

ASSOCIATED CONTENT Supporting Information Instruments used in this work, synthetic route, absorption spectra, cytotoxicity of probe, and characterizations of new compounds. The material is available and free of charge on the ACS Publications website. ACKNOWLEDGMENTS This work was financially supported by NSFC (21472067, 21672083, and 21605059), SDNSF (ZR2016BB26), Taishan Scholar Foundation (TS 201511041), and the startup fund of the University of Jinan (309-10004 and XBS1629).

REFERENCES (1) Shahrokhian, S. Anal. Chem. 2001, 73, 5972–5978. (2) Zhang, S. Y.; Ong, C.-N.; Shen, H.-M. Cancer Lett. 2004, 208, 143–153. (3) Dalton, T. P.; Shertzer, H. G.; Puga, A. Annu. Rev. Pharmacol. Toxicol. 1999, 39, 67–101. (4) Jakubowski, H. J. Biol. Chem. 1997, 272, 1935–1942. (5) Anderson, M. E. Chem.-Biol. Interact. 1998, 111, 1–14. (6) Stobiecka, M., Deeb, J.; Hepel, M. Biophys. Chem. 2010, 146, 98–107. (7) Reddie, K. G.; Carroll, K. S. Curr. Opin. Chem. Biol. 2008, 12, 746−754.


Page 21 of 23

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


(8) Weerapana, E.; Wang, C.; Simon, G. M.; Richter, F.; Khare, S.; Dillon, M. B. D.; Bachovchin, D. A.; Mowen, K.; Baker, D.; Cravatt, B. F. Nature 2010, 468, 790−795. (9) Demchenko, A. P. J. Fluoresc. 2010, 20, 1099−1128. (10)

Kikuchi, K.; Takakusa, H.; Nagano, T. TrAC, Trends Anal. Chem. 2004, 23,

407–415. (11)

Yuan, L.; Lin, W.; Zheng, K.; Zhu, S. Acc. Chem. Res. 2013, 46, 1462–1473.

(12)

Lee, M. H.; Kim, J. S.; Sessler, J. L. Chem. Soc. Rev. 2015, 44, 4185–4191.

(13)

Zhang, W.; Liu, T.; Huo, F.; Ning, P.; Meng, X.; Yin, C. Anal. Chem. 2017,

DOI: 10.1021/acs.analchem.7b01580. (14)

Zhou, Y.; Zhang, X.; Yang, S.; Li, Y.; Qing, Z.; Zheng, J.; Li, J.; Yang, R.

Anal. Chem. 2017, 89, 4587−4594. (15)

He, X.; Xu, Y.; Shi, W.; Ma, H. Anal. Chem. 2017, 89, 3217−3221.

(16)

Fu, Z.-H.; Han, X.; Shao, Y.; Fang, J.; Zhang, Z.-H.; Wang, Y.-W.; Peng, Y.

Anal. Chem. 2017, 89, 1937−1944. (17)

Jia, X.; Chen, Q.; Yang, Y.; Tang, Y.; Wang, R.; Xu, Y.; Zhu, W.; Qian, X. J.

Am. Chem. Soc. 2016, 138, 10778−10781. (18)

Han, X.; Song, X.; Yu, F.; Chen, L. Adv. Funct. Mater. 2017, 1700769.

(19)

Dong, B.; Song, X.; Kong, X.; Wang, C.; Tang, Y.; Liu, Y.; Lin, W. Adv.

Mater. 2016, 28, 8755–8759. (20)

Li,

Z.;

Xu,

Y.;

Zhu

H.;

Qian,

Y.

Chem.

Sci.

2017,

DOI:

10.1039/C7SC00373K. (21)

Liu, C.; Wang, Y.; Tang, C.; Liu, F.; Ma, Z.; Zhao, Q.; Wang, Z.; Zhu, B.;

Zhang, X. J. Mater. Chem. B 2017, 5, 3557–3564. 21 ACS Paragon Plus Environment


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

(22)

Page 22 of 23

Shen, S.-L.; Zhao, X.; Zhang, X.-F.; Liu, X.-L.; Wang, H.; Dai, Y.-Y.; Miao,

J.-Y.; Zhao, B.-X. J. Mater. Chem. B 2017, 5, 289–295. (23)

Niu, L.-Y.; Chen, Y.-Z.; Zheng, H.-R.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z.

Chem. Soc. Rev. 2015, 44, 6143–6160. (24)

Chen, H.; Tang, Y.; Lin, W.; TrAC, Trends Anal. Chem. 2016, 76, 166–181.

(25)

Lee, H. Y.; Choi, Y. P.; Kim, S.; Yoon, T.; Guo, Z.; Lee, S.; Swamy,

K. M.

K.; Kim, G.; Lee, J. Y.; Shin, I.; Yoon, J. Chem. Commun. 2014, 50, 6967–6969. (26)

Li, H.; Fan, J.; Wang, J.; Tian, M.; Du, J.; Sun, S.; Sun, P.; Peng, X. Chem.

Commun. 2009, 5904–5906. (27)

Yang, X.; Guo, Y.; Strongin, R. M. Angew. Chem. Int. Ed. 2011, 50,

10690–10693. (28)

Niu, L.-Y.; Guan, Y.-S.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang, Q.-Z. J.

Am. Chem. Soc. 2012, 134, 18928–18931. (29)

Liu, J.; Sun, Y.-Q.; Huo, Y.; Zhang, H.; Wang, L.; Zhang, P.; Song, D.; Shi,

Y.; Guo, W. J. Am. Chem. Soc. 2014, 136, 574–577. (30)

Yang, X.-F.; Huang, Q.; Zhong, Y.; Li, Z.; Li, H.; Lowry, M.; Escobedo, J.

O.; Strongin, R. M. Chem. Sci. 2014, 5, 2177–2183. (31)

Jung, H. S.; Pradhan, T.; Han, J. H.; Heo, K. J.; Lee, J. H.; Kang, C.; Kim, J.

S. Biomaterials 2012, 33, 8495–8502. (32)

Jung, H. S.; Han, J. H.; Pradhan, T.; Kim, S.; Lee, S. W.; Sessler, J. L.; Kim,

T. W.; Kang, C., Kim, J. S. Biomaterials 2012, 33, 945–953. (33)

Yuan, L.; Lin, W.; Xie, Y.; Chen, B.; Song, J. Chem. Commun. 2011, 47,

9372–9374. (34)

Niu, L.-Y.; Zheng, H.-R.; Chen, Y.-Z.; Wu, L.-Z.; Tung, C.-H.; Yang,

Q.-Z.

Analyst 2014, 139, 1389–1395. 22 ACS Paragon Plus Environment

Page 23 of 23

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


(35)

Bhalla, V. ; Kumar, M. ; Sharma, P. R.; Kaur, T. Inorg. Chem. 2012, 51,

2150–2156. (36)

Buttke, T. M.; Sandstrom, P. A. Immunol. Today 1994, 15, 7–10.

(37)

Iyer, S. S.; Jones, D. P.; Brigham, K. L.; Rojas, M. Am. J. Respir. Cell Mol.

Biol. 2009, 40, 90–98. (38)

Ki-Hoon Parka, Kyung-Hyun Cho, Fish Shellfish Immun. 2011, 31, 904–910.

(39)

Lee, S.-H.; Ko, C.-I.; Jee, Y.; Jeong, Y.; Kim, M.; Kim, J.-S.; Jeon, Y.-J.

Carbohyd. Polym. 2013, 92, 84–89.

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