Development of a ratiometric fluorescent probe for sulfite based on a coumarin–benzopyrylium platform

Accepted Manuscript Title: Development of a ratiometric fluorescent probe for sulfite based on a coumarin-benzopyrylium platform Author: Yanhong Chen Xin Wang Xiao-Feng Yang Yaogang Zhong Zheng Li Hua Li PII: DOI: Reference:

S0925-4005(14)01121-6 http://dx.doi.org/doi:10.1016/j.snb.2014.09.052 SNB 17428

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

31-7-2014 2-9-2014 13-9-2014

Please cite this article as: Y. Chen, X. Wang, X.-F. Yang, Y. Zhong, Z. Li, H. Li, Development of a ratiometric fluorescent probe for sulfite based on a coumarin-benzopyrylium platform, Sensors and Actuators B: Chemical (2014), http://dx.doi.org/10.1016/j.snb.2014.09.052 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.

Development of a ratiometric fluorescent probe for sulfite based on a coumarin-benzopyrylium platform

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Yanhong Chen,a Xin Wang,a Xiao-Feng Yang,*,a Yaogang Zhong,b Zheng Li,b and

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Hua Lia a

Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of

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Education, College of Chemistry & Materials Science, Northwest University, Xi’an 710069, P. R .China

College of Life Sciences, Northwest University, Xi'an 710069, P. R. China

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b

Author to whom correspondence should be addressed:

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Xiao-Feng Yang, PhD,

College of Chemistry & Materials Science, Northwest University

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Fax: 86-29-88303798

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Tel: 86-29-88302604

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E-mail: [email protected]

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Abstract A ratiometric fluorescent probe, 2-(7-diethylamino-2-oxo-2H-1-benzopyran-3-yl)7-hydroxyl-1-benzopyrylium (1), has been developed for sulfite sensing. The method

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employs the nucleophilic addition of sulfite to the electrically positive benzopyrylium moiety of 1 to block the π-conjugated system of the whole molecule, which in turn

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results in significant blue shifts in the absorption and emission spectra of the sensing

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system. The fluorescence intensity ratio at 485 and 640 nm (I485 /I640) increases linearly with sulfite concentration in the range of 0.05 - 10 µM. The proposed probe

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shows excellent selectivity toward sulfite over other common anions and biothiols. The bioimaging experiment demonstrates the potential of probe 1 for the ratiometric

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fluorescent imaging of sulfite in living cells.

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Keywords: Sulfite; Fluorescent probe; Ratiometric; Benzopyrylium; Coumarin

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1. Introduction Sulfur dioxide (SO2) is an environmental pollutant and is toxic at elevated concentrations. Inhaled SO2 is easily hydrated to produce sulfite (SO32-) and bisulfite

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(HSO3-) (3:1 M / M, in neutral fluid) [1], and the toxicity of SO2 is mainly affected by

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the two derivatives. SO2 at elevated concentrations is known to induce oxidative

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damage to biomacromolecules such as proteins, lipids, and DNA. Moreover, epidemiological studies indicate that extended exposure to SO2 and/or its derivatives

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is associated with lung cancer, cardiovascular diseases, neurological disorders [2], and a change in the characteristics of voltage-gated sodium and potassium channels [3].

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On the other hand, sulfite is widely used as a preservative for food and beverages to prevent oxidation and bacterial growth and inhibit the development of both enzymatic

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and nonenzymatic browning during production and storage [4]. Since high doses of sulfite may cause adverse reactions and acute symptoms, the threshold levels of

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sulfite in foodstuffs have been rigorously controlled [5]. Therefore, the development of a sensitive and selective method for sulfite and bisulfite assays is of great importance, especially the successful application in bioimaging of living cells. Several methods such as spectrophotometry [6-8], chromatography [9, 10],

electrochemistry [11, 12], and chemiluminescence [13, 14] have been developed for sulfite detection. Fluorescent probes are valuable molecular tools for sensing and imaging trace amounts of samples due to their high sensitivity, exacting specificity, simplicity of implementation, and an ability to allow for real-time monitoring of target molecules in live cells or tissues. Accordingly, several fluorescent probes for

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sulfite (or bisulfite) have been exploited in recent years, based on the reaction of sulfite with aldehydes [15-19], levulinate esters [20-24], Michael-type additions [25-30], and coordinative interactions [31, 32]. However, aldehyde-based probes can

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only be operated in acidic conditions [15], and may suffer from the interference of

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cysteine (Cys) and homocysteine (Hcy) [33]. Probes based on the levulinate group

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show certain drawbacks such as low sensitivity, or long response time [22]. Other reported probes perform poorly in pure water solution [29]. Therefore, there remains a

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need for developing simple and effective fluorescent probes for sulfite based on alternative detection principles.

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2-(7-Diethylamino-2-oxo-2H- 1-benzopyran-3-yl)- 7-hydroxyl-1-benzopyrylium (1) constructed by hybridizing coumarin and benzopyrylium moieties. Recently, these

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kinds of dyes have received great attention due to their good photochemical properties such as high molar extinction coefficients, large fluorescence quantum yields, and

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long excitation and emission wavelengths [34, 35]. More significantly, the benzopyrylium unit can not only extend the absorption and fluorescence spectra of the fluorophore but also acts as a guest receptor, as its 4-position is an effective reaction site for nucleophiles. The nucleophilic addition to its benzopyrylium C-4 atom alters the large π-conjugated system of 1 and thereby affords remarkable blue shifts in the optical spectra of the sensing system. Since 1 contains a coumarin fluorophore, it can

still afford coumarin emission even after nucleophilic addition to its benzopyrylium unit. The coumarin-benzopyrylium platform can therefore serve as a broadly applicable platform to construct ratiometric probes based on altering the

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π-conjugation system of the fluorophore, and ratiometric fluorescent probes for Cys/Hcy [36], H2S [37] and Hg2+ [38] have been reported. On the other hand, it was reported that sulfite can react with α,β-unsaturated

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compounds efficiently in aqueous solution [39]. Considering that this reaction would

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block the π-conjugation of the system, it is possible to design a chemosensing system

hybrid

coumarin-benzopyrylium

dye,

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for sulfite based on this process. Thus, according to this mechanism, we propose a 2-(7-diethylamino-2-oxo-2H-

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1-benzopyran-3-yl)- 7-hydroxyl-1-benzopyrylium (1), as a ratiometric fluorescent probe for sulfite. The ratiometric sensing is realized via the nucleophilic attack of

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sulfite to the electrically positive benzopyrylium moiety of 1 to interrupt the large π-conjugated system of the original fluorophore. As a result, two well-resolved

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emission bands before and after adding sulfite are observed due to the distinct

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emission between 1 and the corresponding 1-SO3- adduct (Scheme 1). The

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fluorescence intensity ratio at 485 and 640 nm (I485/I640) increases linearly with sulfite

concentration in the range of 0.05 - 10 μM. The proposed probe shows excellent selectivity toward sulfite over other common anions and biothiols. Probe 1 has been

successfully applied to the ratiometric imaging of sulfite in living HepG2 cells.

2. Experimental 2.1. Materials and instrumentation All the chemical reagents and solvents were purchased from commercial suppliers and used without further purification unless for special needs. Doubly distilled water was used in the experiments. Flash chromatography was performed using Qingdao 5

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Haiyang silica gel (200 - 300 mesh). The fluorescence spectra and relative fluorescence intensity were measured with a Shimadzu RF-5301 spectrofluorimeter with a 10 mm quartz cuvette. UV/Vis spectra were made with a Shimadzu UV-2550

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spectrophotometer. IR spectra were recorded on a Bruker Vertex 70 FT-IR spectrometer using a diamond ATR attachment. High-resolution mass spectra (HRMS)

Corp., USA) in electrospray ionization (ESI) mode. 1H and

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were collected using a Bruker micrOTOF-Q II mass spectrometer (Bruker Daltonics 13

C NMR spectra were

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obtained on either a Varian INOVA-400 or Gemini 2000 (600 MHz) spectrometer

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with reference to solvent signals. The pH was measured with a Sartorius PB-10 pH meter. The fluorescence images were acquired with an Olympus BX 61 fluorescence

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scanning microscope (Tokyo, Japan). 2.2. Synthesis of compounds 1, 2 and 3

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Compound 1

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Compound 4 was synthesized according to the reported procedure [40].

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2,4-Dihydroxybenzaldehyde (0.24 g, 1.738 mmol) and 4 (0.38 g, 1.448 mmol) were dissolved in methanesulfonic acid (5 mL) and stirred at 90 oC for 6 h (Scheme 2). After cooling to room temperature, the solution was added dropwise to ice-cold brine, and the precipitate was filtered and washed with water. The crude product was then purified by flash chromatography on silica gel using CH2Cl2/CH3OH (20:1, v/v) as eluent to afford 1 as a dark blue solid (0.40 g, 76.0% yield). IR (ATR, cm-1), 2980,

2933, 1723, 1588, 1459, 1130, 842, 768, 696. 1H NMR (CDCl3, 400 MHz): δ ppm 8.45 (s, 1H), 7.86 (d, J = 7.6 Hz,1H), 7.59 (d, J = 7.6 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H), 7.31 (d, J = 9.2 Hz, 2H), 6.78(d, J = 8.8 Hz, 1H), 6.68(d, J = 7.6 Hz, 1H), 6.50 (d, J = 14 Hz, 2H), 3.48 (q, J = 6.8 Hz, 4H), 1.26 (t, J = 6.8 Hz, 6H).13C NMR (100 6

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MHz, CDCl3/CD3OD = 1:1, v/v): 184.72, 161.14, 159.93, 159.47, 158.40, 154.61, 144.67, 143.95, 132.73, 132.32, 129.44, 120.41, 111.71, 109.88, 108.70, 107.89, 104.30, 97.13, 45.96, 12.77. HRMS (ESI): [M-Cl]+ m/z 362.1399, calcd for

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C22H20NO4 362.1392.

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2'-carboxy- 2,4-dihydroxybenzophenon (5) [41]

Fluorescein was dissolved in 12 mL of 50% NaOH solution (w/v) and heated at 160

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o

C in an oil bath for 1 h. After cooling to room temperature, the mixture was poured

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into 80 mL of ice water, acidified with concentrated HCl, and allowed to stand at room temperature for 2 h. The precipitate was filtered and dried to afford the desired

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product as a beige powder (1.06 g, 80.3% yield). IR (ATR, cm-1), 3384, 2931, 2634, 1700, 1625, 1592, 1225, 1078, 843, 773.1H NMR (400 MHz, DMSO-d6): δ ppm

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13.22 (s, 1H), 12.24 (s, 1H), 10.95 (s, 1H), 7.98 (d, J=7.7 Hz, 1H), 7.71 (t, J=7.4 Hz, 1H), 7.63 (t, J=7.6 Hz, 1H), 7.41 (d, J=7.4 Hz, 1H), 6.90 (d, J=8.7 Hz, 1H), 6.39 (d,

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J=2.1 Hz, 1H), 6.31 (dd, J1=8.8 Hz, J2=1.6 Hz, 1H). HRMS (ESI) [M + Na] + m/z 281.0425, calcd for C14H10O5Na 281.0426. Compound 3 was prepared by employing the same procedure as for the synthesis of

compound 1 except that benzophenone 5 was used. Yield, 63.6%. 1H NMR (400 MHz,

CDCl3/CD3OD = 1:1, v/v): δ ppm 8.45 (s, 1H), 7.97 (d, J = 7.5 Hz, 1H), 7.68 (t, J =

7.2 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.59 (s, 1H), 7.50 (d, J = 9.2 Hz, 1H), 7.29 (d, J = 7.5 Hz, 1H), 6.91 (s, 1H), 6.76 - 6.71 (m, 3H), , 6.58 (d, J = 7.0 Hz, 1H), 6.46 (s, 1H), 3.47 (q, J = 6.8 Hz, 4H), 1.22 (t, J = 7.2 Hz, 6H).13C NMR (150 MHz, CDCl3/CD3OD = 1:1): 171.19, 160.38, 157.37, 153.10, 142.67, 134.64, 131.44,

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130.33, 129.53, 126.94, 126.00, 110.82, 110.25, 109.11, 103.48, 97.02, 45.65, 12.82. HRMS (ESI) [M-Cl]+ m/z 482.1588, calcd for C29H24NO6 482.1604.

2.3. General procedure for the spectra measurement

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Compound 2 was prepared by the procedure reported previously [37].

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The stock solutions of 1 and 2 (2.0 mM) were prepared in ethanol. Test solutions were prepared by adding 25 μL of probe 1 (2.0 mM), 1.0 mL of phosphate buffer (0.2

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M, pH 7.4), and appropriate aliquots of each analyte stock solution in a 10 mL volumetric flask, and diluting the solution to 10 mL with water. The resulting solution

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was kept at room temperature (25 oC) for 5 min, and then the absorption or

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fluorescence spectra were recorded. The fluorescence ratio of I485/I640 was measured

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respectively.

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with the excitation and emission wavelengths at 430/605 nm and 485/640 nm,

2.4. Cell culture and fluorescence imaging HepG2 cells were seeded in 6-well plates at a density of 2×104 per well and

cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS), penicillin (100 U mL-1) and streptomycin (100 μg mL-1) at 37 oC in humidified 5% CO2 atmosphere for 24 h. The cells were rinsed three times with phosphate-buffered saline (PBS) and incubated with 1 (10 μM) in PBS (containing 0.5% ethanol as a cosolvent) for 30 min, and then washed three times with PBS to remove the remaining probe. Experiments to assess sulfite uptake were performed in the same medium supplemented with sulfite (200 μM) for 20 min at 37 oC. The 8

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fluorescence images were acquired after the cells had been rinsed with PBS. 2.5. Cytotoxicity Assay HepG2 cells were seeded in 96-well plates at a density of 1 × 105 cells mL-1 and

5%

CO2

and

95%

air

at

37

°C

for

12

h.

Then,

20

μL

of

cr

of

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incubated with varied concentrations of probe 1 (0 − 30 μM) (n = 5) in an atmosphere

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] tetrazolium MTT

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solution (5.0 mg mL-1) was added to each well, followed by incubation at 37 °C for 4

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h. After that, 100 μL of the supernatant was removed, and 150 μL of DMSO was added to each well to dissolve the formed formazan. The plate was shaken for 10 min

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and then the absorbance of the solution in the well was measured at 490 nm with a

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microplate reader (ELX 800 UV, BIO-TEK Instruments Inc.).

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3. Results and discussion

3.1. Effect of substituent on the reactivity of the probe

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Initially, the influence of substituent on the reactivity of the probe was investigated

by comparing two dyes (1 and 2) with different substituents in the 7-position of the

benzopyrylium unit. The reactivity of probes 1 and 2 toward sulfite was studied in

ethanol / phosphate buffer (30:70 v/v, pH 7.4, 20 mM), respectively. As shown in Fig. S1 (supplementary data), probe 2 afforded a large hypochromatic shift when compared with probe 1. However, the emission ratio (I485/I640) of probe 1 increased

dramatically upon mixing with sulfite, whereas the intensity ratio of 2 (I485/I690) exhibited only a small increase under identical conditions (Fig. 1). This can be explained by the fact that the substituents on the benzopyrylium cations affect their 9

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reactivity toward nucleophiles. The above observation is in accordance with previous results that a benzopyrylium species with a 7-OH group shows the highest tendency toward nucleophilic addition among other substituents, such as –H and –NEt2 [42]. As

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compound 1 exhibited a much stronger fluorescence response than probe 2, it was

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selected for further studies.

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3.2. Optical response of 1 toward sulfite

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The sensing behavior of probe 1 toward sulfite was investigated with absorption and fluorescence spectroscopy in phosphate buffer (pH 7.4, 20 mM, containing 0.25%

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ethanol as a cosolvent). The free probe 1 displays a strong absorption at around 605 nm. However, the introduction of increasing concentrations of sulfite (0 - 8 equiv.) to

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the solution of 1 (5 μM) resulted in a gradual decrease of the absorption peak at 605

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nm and a progressive increase of a new absorption band around 420 nm (Fig. 2a).

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Meanwhile, a well-defined isobestic point was observed at 456 nm, indicating the formation of a new species upon the mixture of probe 1 with sulfite. Next, the fluorescence sensing behavior of probe 1 toward sulfite was examined

under the same conditions. Probe 1 itself exhibited an emission peak centered at around 640 nm, and its quantum yield was determined to be 0.056. Upon addition of sulfite solution, the fluorescence intensity at 640 nm showed a distinct decrease with the concomitant progressive increase of a new emission band at 485 nm (Fig. 2b), indicating the interruption of the π-conjugation of 1 at its benzopyrylium moiety. This large hypsochromic shift (~ 155 nm) in the emission spectra results in the emission

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peaks being well resolved before and after reacting with sulfite, and thus ratiometric sensing can be carried out, which provides a built-in correction for environmental effects. The intensity ratio of the two emission bands, I485/I640, increased from 0.034 to

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40.23, and saturated after 3 equiv of NaHS was added (φF = 0.074 using fluorescein

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as a fluorescence standard). The intensity ratios (I485/I640) were plotted as a function of

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sulfite concentration and a typical calibration graph was obtained (Fig. 3). The I485/I640 value is proportional to sulfite concentration in the range of 0.05 to 10 μM with a

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detection limit of 34 nM (3δ). This indicates that probe 1 is highly sensitive and may be potentially useful in the detection of sulfite in a variety of real samples.

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3.3. Mechanism study

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Some experiments were carried out to gain insight into the sensing mechanism

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proposed above. It has been reported that the C-2 and C-4 atoms of the benzopyrylium unit are both liable to attack by nucleophiles [43]. Therefore, to test which site is responsible for the sensing event, the carboxyphenyl was introduced at the 4-position of the benzopyrylium unit of 1 (to form compound 3) and its sensing

behavior was compared with 1 under identical conditions. As shown in Fig. S3 (supplementary data), compound 1 afforded drastic fluorescence spectra changes upon

being treated with sulfite. In the case of 3, however, no significant spectra changes were observed. The above experiments prove that the reactivity of the probe is dependent on the steric factors of the reaction site. The inherent reactivity of 2 is significantly reduced as a result of a steric hindrance effect, which also provides 11

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strong evidence that the benzopyrylium C-4 atom is the sulfite addition site. The reaction mechanism between 1 and sulfite was further examined by 1H NMR spectroscopy. Upon addition of sulfite to the solution of 1, the resonance signal at

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around δ = 8.19 ppm, corresponding to the double-bond proton Ha, disappeared;

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concurrently, a new peak, assigned to the corresponding saturated –CH- proton (Hb)

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adjacent to the SO3- group of the proposed adduct, appeared at δ = 4.71 ppm (Fig. 4), demonstrating that sulfite addition to the benzopyrylium C-4 atom indeed occurs.

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Finally, the mass spectrometry analysis of probe 1 treated with Na2SO3 in methanol-H2O (1:1, v/v) solution confirmed the formation of the 1-SO3- adduct. A

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prominent peak at m/z 442.0955 corresponding to the 1-SO3- adduct (calcd. 442.0960 for C22H20NO7S) is clearly observed in the HRMS data (Fig. S4, supplementary data).

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Additional proof of the above mechanism is provided by comparing the absorption

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and emission spectra of 1-SO3- and coumarin 4. As shown in Fig. S5 (supplementary

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data), the absorption and emission spectra of 1-sulfite are similar to those of 4. This

can be explained by the fact that the 1-SO3- adduct and compound 4 have almost the

same coumarin fluorophore. These data are in good agreement with the proposed sensing mechanism shown in Scheme 2.

3.4. Effect of pH The pH value of the solution was found to be essential to the present sensing system. Initially, the effect of pH on the stability of probe 1 was investigated in the pH range of 1.0 – 12.0. As shown in Fig. S6 (supplementary data), compound 1 displayed

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a strong fluorescence emission at 640 nm in the pH range of 6 – 10. The fluorescence intensity at 640 nm decreased significantly when pH ≥ 10. This is due to the nucleophilic addition of OH- in 2-position of the benzopyrylium unit to form a

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2-hydroxyflavene (6), which subsequently transforms to the cis-chalcone (7) by a

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tautomeric process, and the trans-chalcone (8) forms as the ultimate product via the

isomerization of 7 (Scheme 3) [44]. The above reaction may change the π-conjugated

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system of 1. Alternatively, OH- might add to the 4-position of the benzopyrylium

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cation of 1 to form a 4-hydroxyflavene. When this species occurs, however, it is unstable and only present as an intermediate product [45]. Interestingly, unlike the

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addition of sulfite to the 4-position of the benzopyrylium unit, it was observed that the absorption maximum of 8 is centered at 556 nm, which is much longer than the parent

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coumarin (Fig. S7). This redshift behavior can seemingly be ascribed to the extension of the π-conjugation of coumarin by the α,β-unsaturated ketone unit. Further, 1

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showed no significant fluorescence emission even in more basic solution (pH = 13), which is indicative of efficient photo-induced electron transfer (PET) quenching of the fluorophore by the intramolecular carbon–carbon double bond [46]. Next, the effect of pH on the present sensing system was studied. It was observed that the probe displayed a good response toward sulfite in the pH range of 4.0 – 7.4 (Fig. 5). When the pH value exceeded 7.4, the fluorescence emission at 485 nm decreased, while emission at 640 nm increased significantly, which might be due to the decomposition of the 1-SO3- adduct under basic conditions. The above results indicate that the good response of 1 toward sulfite can be obtained in physiological pH solutions.

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3.5. Kinetic studies

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The time course of the fluorescence spectra of probe 1 in the presence of sulfite was studied. Upon addition of sulfite to the solution of 1, the emission ratio (I485/I640)

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of the sensing system increased significantly, and leveled off as the reaction time was

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prolonged, while the fluorescence background in the absence of sulfite remained unchanged under identical conditions (Fig. 6). The emission ratio of the sensing

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system essentially reached a maximum after 5 min, and thereafter remained stable.

sensitivity of probe 1 toward sulfite.

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Therefore, an assay time of 5 min was chosen in the evaluation of the selectivity and

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3.6. Selective studies

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To test the selectivity of the probe for sulfite, probe 1 was treated with a series of anions (S2O32-, SO42-, CO32-, HCO3-, CH3COO-, NO2-, NO3-, Br-, Cl-, F-, I-, SCN-, CN-,

S2-) and biothiols (Cys, GSH) in phosphate buffer solution (pH 7.4) and monitored by absorption and fluorescence spectroscopy, respectively. As displayed in Fig. S8, only sulfite and bisulfite resulted in a decrease of the absorption band centered at around 605 nm and the formation of a new blueshifted absorption peak with the maximum at 420 nm. The other anions and biothiols induced no significant changes in the absorption spectra under identical conditions. In good agreement with the variations in the absorption spectra, these competitive species afforded no significant

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fluorescence changes of 1, and only sulfite and bisulfite gave dramatic ratiometric fluorescence changechanges, as shown in Fig. 7 and Fig. S8-b. In addition, the selective response of 1 toward sulfite is observable by the naked eye. When probe 1

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was treated with various species, only sulfite caused an obvious color change under

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visible or UV light (Fig. 7 and Fig. S9, supplementary data). The excellent selectivity of 1 toward sulfite could be explained as follows: although the thiol group of Cys and

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GSH is nucleophilic, it contains a cationic –NH3+ group which will prevent effective

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collisions with probe 1 because the addition site of 1 is a cationic benzopyrylium unit [37]. Thus, probe 1 shows almost no response toward these biothiols.

3.7. Intracellular sulfite imaging

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To investigate whether 1 can selectively detect sulfite in a cellular environment,

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cell-based experiments were performed. The HepG2 cells were incubated with probe

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1 for 30 min in PBS at 37 oC, and showed strong fluorescence in the red channel but weak fluorescence in the blue channel. In a control experiment, the cells were pre-treated with probe 1 (10 μM) and further incubated with Na2SO3 (200 μM). An

obvious fluorescence decrease in the red channel and an increased fluorescence in the blue channel were observed simultaneously (Fig. 8). Meanwhile, the potential toxicity of

1

was

investigated

by

the

standard

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Fig. S10). The results showed that 1 has no marked cytotoxicity at concentrations below 30 μM (Fig. S10, supplementary data). All these results demonstrate the capacity of

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probe 1 for the ratiometric imaging of sulfite in living cells.

4. Conclusion

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In summary, we have developed a coumarin-benzopyrylium platform (1) as a

cr

ratiometric fluorescent probe for the selective detection of sulfite. The ratiometric

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sensing is conducted by altering the π-conjugation of 1 by a nucleophilic addition of sulfite to the electrically positive benzopyrylium moiety of the probe. The probe

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exhibits excellent selectivity toward sulfite over other common anions and biothiols. Preliminary studies show that this probe is cell-permeable and can be used for the

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fluorescence imaging of cellular sulfite with low cytotoxicity. Thus, we believe that

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Acknowledgements

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variety of real samples.

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this design concept will be further developed for the detection of SO2 derivatives in a

This research was supported by the National Natural Science Foundation of China

(No. 21475105, 21275117, 21375105), the Science & Technology Department (No. 2012JM2004) and the Education Department (No. 12JK0518) of Shaanxi Province of China.

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(1994) 155–165. [2] N. Sang, Y. Yun, H. Li, L. Hou, M. Han, G. Li, SO2 inhalation contributes to the

cr

evelopment and progression of ischemic stroke in the brain, Toxicol. Sci. 114 (2010)

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[4] R.F. McFeeters, Use and removal of sulfite by conversion to sulfate in the preservation of salt-free cucumbers, J. Food Prot. 61 (1998) 885–890.

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[6] G.J. Mohr, A chromoreactant for the selective detection of HSO3- based on the

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[7] P.W. West, G.C. Gaeke, Fixation of sulfur dioxide as disulfitomercurate and subsequent colorimetric estimation, Anal. Chem. 28 (1956) 1816–1819. [8] M.A. Segundo, A.O.S.S. Rangel, A. Cladera, V. Cerda, Multisyringe flow system: determination of sulfur dioxide in wines, Analyst 125 (2000) 1501–1505. [9] J. Rethmeier, A. Rabenstein, M. Langer, U. Fischer, Detection of traces of oxidized and reduced sulfur compounds in small samples by combination of different high-performance liquid chromatography methods, J. Chromatogr. A 760 (1997) 17

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cr

[11] D. Huang, B. Xu, J. Tang, J. Luo, L. Chen, L. Yang, Z. Yang, S. Bi, Indirect determination of sulfide ions in water samples at trace level by anodic stripping

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″turn-on″ fluorescent sensing and bioimaging application, J. Mater. Chem. B 1 (2013)

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(1995) 31–36. [36] H. Lv, X-F. Yang, Y. Zhong, Y. Guo, Z. Li, H. Li, Native chemical ligation combined with spirocyclization of benzopyrylium dyes for the ratiometric and

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compounds, J. Am. Chem. Soc. 74 (1952) 3523–3526.

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[40] V.C. Ezeh, T.C. Harrop, A sensitive and selective fluorescence sensor for the detection of arsenic(III) in organic media, Inorg. Chem. 51 (2012) 1213–1215.

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[41] X. Xiong, F. Song, G. Chen, W. Sun, J. Wang, P. Gao, Y. Zhang, B. Qiao, W. Li, S. Sun, J. Fan, X. Peng, Construction of long-wavelength fluorescein analogues and their application as fluorescent probes, Chem. Eur. J. 19 (2013) 6538–6545. [42] H. Lietz, G. Haucke, P. Czerney, B. John, Hydrolysis of benzopyrylium dyes - an application of the concept of chemical hardness, J. prakt. Chem. 338 (1996) 725–730. [43] R. Gavara, C.A.T. Laia, A.J. Parola, F. Pina, Formation of a leuco spirolactone from

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applications of flavylium compounds: a handful of colours, Chem. Soc. Rev. 41 (2012) 869–908. [45] R.A. McClelland, S. Gedge, Hydration of the flavylium ion, J. Am. Chem. Soc.

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102 (1980) 5838–5848. [46] L. Yi, H. Li, L. Sun, L. Liu, C. Zhang, Z. Xi, A highly sensitive fluorescence

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probe for fast thiol-quantification assay of glutathione reductase, Angew. Chem. Int.

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te

d

M

an

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Ed. 48 (2009) 4034–4037.

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Figure Captions Scheme 1. Proposed mechanism for the ratiometric sensing of sulfite when using 1. Scheme 2. Synthesis of probe 1, 3 and the structure of 2.

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Scheme 3. Chemical reactions of 1 in basic medium. Fig. 1. The emission ratios of probes 1 and 2 in the absence and presence of sulfite,

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respectively. The reaction was carried out by mixing the probe (5 μM) with sulfite (2 equiv.) in ethanol / phosphate buffer (30:70 v/v, 20 mM, pH 7.4) for 5 min.

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Fig. 2. Absorption (a) and fluorescence spectra (b) of probe 1 (5 μM) upon addition of

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increasing concentrations of sulfite (0 - 8 equiv.) in phosphate buffer (pH 7.4, 20 mM, containing 0.25% ethanol as a cosolvent). The reactions were carried out for 5 min at

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room temperature.

Fig. 3. Fluorescence intensity ratio (I485/I640) of probe 1 (5 μM) as a function of sulfite

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cosolvent).

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concentration in phosphate buffer (pH 7.4, 20 mM, containing 0.25% ethanol as a

Fig. 4. Partial 1H NMR spectra of 1 in the absence (bottom) and presence (top) of

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sulfite (2 equiv.) in CD3OD/D2O (2:1, v/v). Fig. 5. The fluorescence intensities at 485 and 640 nm for probe 1 (5.0 μM) in the

presence of sulfite (10 μM) at various pH values. Fig. 6. Time-dependent emission ratio (I485/I640) changes of probe 1 (5 μM) upon

addition of sulfite (2 equiv.) in phosphate buffer (pH 7.4, 20 mM, containing 0.25% ethanol as a cosolvent).

Fig. 7. Fluorescent ratio (I485/I640) of probe 1 (5 μM) to various anion species and biothiols in phosphate buffer (20 mM, pH 7.4, containing 0.25% ethanol as a cosolvent). 1, probe 1 only; 2, HSO3-, 3, SO32-; 4: S2- (2 equiv. of each); 5, S2O32-, 6, SO42- ,7, CO32-, 8, HCO3-, 9, AcO-, 10, NO2-, 11, NO3-, 12, Br-, 13, Cl-, 14, F-, 15, I- ,

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16, SCN-, 17, CN- (10 equiv. of each); 18, Cys (1 mM), 19, GSH (1 mM). Data were recorded after 5 min. Inset: images of 1 (10 μM) in the presence of various anion species and biothiols under a UV lamp at 365 nm.

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Fig. 8. Fluorescence and bright-field images of HepG2 cells. (A) Bright-field image of HepG2 cells incubated with probe 1 (10 μM) for 30 min. (B) fluorescence image of

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(A) from the blue channel; (C) fluorescence image of (A) from the red channel; (D) Bright-field image of HepG2 cells incubated with probe 1 for 30 min, and further

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incubated with sulfite (200 μM) for 30 min. (E) fluorescence image of (D) from the

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blue channel; (F) fluorescence image of (D) from the red channel.

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SO3SO32HO

O O

O

O O

N

O

1-SO3-

1

N

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HO

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Scheme 1. Proposed mechanism for the ratiometric sensing of sulfite when using 1.

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O

CHO OH

CH3SO3H

+ N

O

HO

O

90 oC

O

O

O

N

OH 4

1

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COOH

COOH O 50% aq. NaOH

4 OH

o

160 C HO

O

CH3SO3H 90 oC

O

HO

O

OH

N

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O

N

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O O

O

3

5

N

O

cr

COOH

2

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Scheme 2. Synthesis of probe 1, 3 and the structure of 2.

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OHHO

HO

O O

O

O

N

OH O O

N

6 1

O

isomerization

OH O

O

N

OH O O

O

N

7 cis-chalcone

8 trans-chalcone

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Scheme 3. Chemical reactions of 1 in basic medium.

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HO

cr

HO light

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tautomerization

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10

8

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7 6

cr

1.5 1.0

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I485/I640 (or I485/I690)

9

0.5 0.0

2-

2 only

2-

2 + SO3

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1 + SO3

1 only

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Fig. 1

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Fig. 2

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7.5 y=0.761x-0.181 r=0.9922

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6.0

3.0

cr

I485/I640

4.5

0.0 2

4

6

8

an

0

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1.5

10

Sulfite concentration / M

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d

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Fig. 3

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Fig. 4

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600 450

cr

300 150

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Fluorescence intensity (a.u.)

485 nm 640 nm

750

0 6

8

10

an

4

pH

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Fig. 5

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12 1 only 21 + SO3

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6

cr

I485/I640

9

0 5

10

15

20

an

0

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3

Reaction time / min

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Fig. 6

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7.7

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6.3 0.8

cr

I485/I640

7.0

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0.4

an

0.0

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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Fig. 7

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Fig. 8

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Biographies Yanhong Chen is currently a master candidate in College of Chemistry and Chemical Engineering, Northwest University. Her research interests focus on developing

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fluorescent probes and chemosensors.

Xin Wang is a graduate student in College of Chemistry and Chemical Engineering,

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Northwest University. Her research is concerning on synthesis and characterization of

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new chemosensors.

Xiao-Feng Yang received his Ph.D. degree from Xiamen University in 2002. He is

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currently a professor in College of Chemistry and Chemical Engineering, Northwest University. His current interests include fluorescent molecular devices, chemical and

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biological sensors, molecular recognition and chemiluminescence. Yaogang Zhong is a graduate student in College of Life Sciences, Northwest

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Zheng Li.

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University. He is currently pursuing his doctor’s degree under the supervision of Prof.

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Zheng Li is a professor in College of Life Sciences, Northwest University. His research interests focus on functional glycomics and biochips. Hua Li received his Ph.D. degree from Changchun Institute of Applied Chemistry

Chinese Academy of Sciences in 1996. He is currently a professor in College of Chemistry and Chemical Engineering, Northwest University. His research interests focus on chemometrics and host-guest chemistry.

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Highlights

A coumarin-benzopyrylium platform has been developed for the ratiometric

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fluorescent sensing of sulfite. The method employs the nucleophilic addition of sulfite to 4-position of the

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benzopyrylium unit of the probe.

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The ratiometric sensing is carried out by interrupting the large π-conjugation system of the probe.

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The proposed probe displays high selectivity for sulfite over other anions and

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d

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biothiols.

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Ac

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pt

ed

M

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Graphical Abstract (for review)

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