A real-time colorimetric and ratiometric fluorescent probe for rapid detection of SO₂ derivatives in living cells based on a near-infrared benzopyrylium dye

Abstract

A benzopyrylium-based dye, Probe 1, was developed as a near-infrared fluorescent probe for the detection of SO₂ derivatives (sulfite/bisulfite) in pure aqueous solution. In the presence of sulfite/bisulfite, the solution of Probe 1 displayed significant changes in its absorption (from 650 nm to 420 nm) and emission (from 690 nm to 489 nm) spectra. Probe 1 exhibits excellent selectivity and high sensitivity toward sulfite/bisulfite and the detection limit (S/N = 3) for bisulfite is as low as 10.4 nM. Specifically, Probe 1 shows a fast response time (within 10 s) toward sulfite/bisulfite, which makes it capable for real-time sensing or imaging both in vitro and in vivo assays. Probe 2, an analogue of Probe 1, as a turn-on fluorescent probe was also developed and displayed similar properties for the detection of sulfite/bisulfite. The demonstration of Probe 1 as a ratiometric fluorescent probe and Probe 2 as a turn-on fluorescent probe for the detection of SO₂ derivatives was achieved in living HeLa cells and A431 cells, respectively.

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Introduction

Sulphur dioxide (SO₂), with a characteristic strong pungent odor, was conventionally regarded as an environmental pollutant and toxin for several decades.¹ In physiological systems, SO₂ mainly exists in two forms, sulfite and bisulfite (SO₃²⁻/HSO₃⁻). Toxicological studies have implied that SO₂ and its derivatives can alter the intracellular thiol levels, hinder the redox balance in cells and thereby cause severe neuron damage.² Epidemiological studies further suggest that SO₂ and its derivatives are also linked with various symptoms and diseases including many respiratory problems, lung cancer, cardiovascular diseases and neurological disorders.³ Despite that SO₃²⁻/HSO₃⁻ have toxicological effects at high concentrations, they may play important roles in many physiological processes at a physiological concentration. Generally, endogenous SO₃²⁻/HSO₃⁻ are generated from sulphur-containing amino acids such as cysteine or glutathione, which plays a crucial role in maintaining the biological sulphur balance in the body.⁴ Recent studies have demonstrated that SO₃²⁻/HSO₃⁻ at low concentrations (less than 450 μM) have an endothelium-dependent vasorelaxing effect and are regarded as novel messengers in cardiovascular system.⁵ Furthermore, some studies have found that SO₂ derivatives have regulatory effects on lipid metabolism and maintain blood insulin levels.⁶

In order to further understanding the physiological effects and detailed functions of SO₃²⁻/HSO₃⁻, a variety of disparate approaches have been utilized for SO₃²⁻/HSO₃⁻ determination including electrochemistry, chromatography, flow injection analysis, chemiluminescence, electrochemical and enzymatic techniques.⁷ However, most of these conventional methods are time-consuming, have poor selectivity, require expensive instruments or involve complicated procedures, which limits their applications in biological samples. Therefore, the effective methods for rapid and accurate detection of the intracellular SO₃²⁻/HSO₃⁻ are of great importance. Owing to the operational simplicity, potential high spatial and temporal resolution, fluorescent probes have become powerful tools for visualizing morphological details and detecting biomolecules in living system.⁸ In the past decades, numerous fluorescent probes have been developed for detecting intracellular ions,⁹ small molecules¹⁰ and biomacromolecules.¹¹ Up to now, some fluorescent probes for SO₃²⁻/HSO₃⁻ have been constructed based on the specific reactions with an aldehyde¹² or levulinate group.¹³ However, fluorescent probes equipped with an aldehyde or levulinate group are generally subject to poor selectivity to SO₃²⁻/HSO₃⁻ over biothiols, which may be inapplicable for intracellular SO₃²⁻/HSO₃⁻ detection or imaging. Moreover, most of these reported fluorescent probes are intensity-based, which suffer from errors associated with probe concentration, environment effects and instrumental factors.¹⁴ In contrast, ratiometric fluorescent probes allow the measurement of two different emission peaks and are less susceptible to the aforementioned shortcomings.¹⁵ Therefore, new strategies are highly in demand to construct ratiometric fluorescent probes for SO₂ derivatives. It was reported that SO₃²⁻/HSO₃⁻ could add to α,β-unsaturated compounds in aqueous environment due to their strong nucleophilicity.¹⁶ By employing this strategy, several ratiometric fluorescent probes have recently been developed and exhibit good sensitivity and selectivity for the detection of SO₃²⁻/HSO₃⁻.¹⁷ However, probes that could instantaneously response to SO₃²⁻/HSO₃⁻ in pure aqueous solution are rare.¹⁸ Thus, it has remained a challenge to explore ratiometric fluorescent probes for the rapid detection of SO₃²⁻/HSO₃⁻ in pure aqueous environments.

Benzopyrylium dyes have been used as laser dyes,¹⁹ electrophotographic sensitizers²⁰ and chemosensors²¹ because of their excellent photophysical properties, such as emission in near-infrared region, high absorption coefficients, relatively high fluorescence quantum yield, and sufficient stability. Recently, Guo et al. reported Probe 1 as a ratiometric fluorescent probe for the detection of H₂S based on the nucleophilic addition reaction with the benzopyrylium moiety in DMF-PBS buffer solution.²² In order to obtain a good sensitivity for H₂S detection, DMF is needed as a co-solvent to accelerate the addition reaction. Considering the strong nucleophilicity of SO₃²⁻/HSO₃⁻ in aqueous environment, we envisioned that Probe 1 could be a good ratiometric fluorescent probe of the detection of SO₃²⁻/HSO₃⁻ and exhibit better sensitivity toward SO₃²⁻/HSO₃⁻ than H₂S in pure aqueous solution. In this work, we reported the selective and sensitive detection of SO₃²⁻/HSO₃⁻ by using Probes 1 and 2 in pure aqueous solution (shown in Scheme 1). The positive-charged benzopyrylium moiety in these two probes provides excellent water solubility and functions as the recognition group. The application of these two probes for the detection of SO₃²⁻/HSO₃⁻ in living cells was successfully demonstrated. Importantly, owning to the fast response time (within 10 s), Probe 1 was applied to a real-time monitoring of SO₂ releasing in vitro assay.

Scheme 1 Synthesis of Probes 1 and 2.

Results and discussion

Optical properties of Probes 1 and 2

The absorption and emission spectra of Probes 1 and 2 (5.0 μM) in different solvents (CH₃Cl, Toluene, THF, CH₃CN, MeOH, DMSO) are shown in Fig. S1–S4, ESI.† Both of the absorption and emission peaks of the probes are in NIR region. In particular, the emission spectra of the probes covers most part of the biological window (650–900 nm), which is favorable for in vivo imaging. As the polarity of the solvent increases, the fluorescence efficiency of these two probes decreases. The photophysical properties of Probes 1 and 2 in pH 7.4 HEPES buffer were summarized in Table 1. Gratefully, Probes 1 and 2 exhibit good solubility in pure aqueous solution. The quantum yield of Probe 1 were determined to be 0.039. However, Probe 2 was almost non-fluorescent (Φ < 0.001). Hence, Probe 1 can be used as a ratiometric fluorescent probe and Probe 2 will be an off-on fluorescent probe. Moreover, both of Probes 1 and 2 exhibit good photostability (Fig. S5–S7, ESI†).

Table 1 Photophysical data of Probes 1 and 2 in pH 7.4 HEPES buffer

Compound	λabs/nm^a	εmax/M^−1 cm^−1	λem/nm^b	Φf^c	Stokes shift
a The maximal absorption of the probe.b The maximal emission of the probe.c The fluorescence quantum yield of the probe by using Probe 1 (Φf = 0.34 in CH3CN) as a fluorescence standard.
Probe 1	650	76 400	690	0.039	40 nm
Probe 2	663	81 200	718	<0.001	55 nm

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Sensing properties of Probes 1 and 2 to SO₃²⁻/HSO₃⁻

Probe 1 exhibits an absorption band with a maximum at 650 nm in HEPES buffer (20.0 mM, pH = 7.4). Upon treatment with increasing concentrations of NaHSO₃ (0.0–25.0 μM), the absorption peak at 650 nm decreased gradually as well as a new peak maximizing at 421 nm concomitantly emerged (Fig. 1a). A well-defined isosbestic point at 450 nm was obtained, indicating the formation of a new compound. Meanwhile, a significant color change from blue to yellow was observed, which could be easily observed by the naked eyes. Next, the fluorescence properties of Probe 1 towards HSO₃⁻ were examined in HEPES buffer (20.0 mM, pH = 7.4). Upon excited, Probe 1 shows an emission band centered at 690 nm (Fig. 1b). In contrast, the addition of NaHSO₃ to the solution of Probe 1 resulted in a new emission band centered at 489 nm. The distinct emission spectral shift (about 200 nm) is favorable for ratiometric fluorescence detection. Similar results were obtained when the solution of Probe 1 was treated with sulfite (Fig. S8, ESI†). Moreover, the emission ratio (I₄₈₉/I₆₉₀) of Probe 1 showed a dramatic variation (240-fold) before and after treatment with HSO₃⁻ (25.0 μM), as shown in Fig. S9, ESI.† The emission ratio of I₄₈₉/I₆₉₀ was proportional to the concentration of HSO₃⁻ in the range of 0.0–22.5 μM with a good linearity (R = 0.9925). The detection limit (S/N = 3) was found to be 10.4 nM for HSO₃⁻. These results indicated that Probe 1 could detect HSO₃⁻ qualitatively and quantitatively.

Fig. 1 Absorption (a) and emission (b) spectra of Probe 1 (5.0 μM) upon the addition of increasing concentrations of NaHSO₃ (0.0–25.0 μM) in HEPES buffer (20.0 mM, pH = 7.4). Insets: color variations of absorption (a) and fluorescence (b) of Probe 1 in the absence (1) and presence (2) of NaHSO₃.

In addition, time-dependent fluorescence of Probe 1 in the absence/presence of HSO₃⁻ was investigated in HEPES buffer (20.0 mM, pH = 7.4). There was no obvious change in the fluorescence spectrum of Probe 1 within a reasonable measuring time, indicating that Probe 1 had good stability (Fig. S10, ESI†). However, an instant fluorescence change (less than 10 s) was observed when the solution of Probe 1 was incubated with HSO₃⁻ (20.0 μM) (Fig. 2), suggesting that Probe 1 was an ideal candidate for real-time detection of SO₂ derivatives. The sensing behavior of Probe 2 toward HSO₃⁻ was also investigated under the same condition. A rapid (less than 15 s) and significant fluorescence enhancement was obtained upon the addition of NaHSO₃, indicating that Probe 2 could function as an efficient turn-on fluorescent probe for HSO₃⁻.

Fig. 2 (a) Time-dependent fluorescence intensity at 489 nm of Probe 1 (5.0 μM) upon the addition of different concentrations of NaHSO₃ (5.0, 15.0 and 30.0 μM) in HEPES buffer (20.0 mM, pH = 7.4). (b) Time-dependent fluorescence intensity at 690 nm of Probe 1 (5.0 μM) in the presence of different amounts of NaHSO₃ (5.0, 15.0 and 30.0 μM) in HEPES buffer (20.0 mM, pH = 7.4).

Mechanism studies

In order to provide intuitive evidence for the sensing mechanism, ¹H NMR titration experiment was conducted on Probe 1 with different amounts of NaHSO₃ in d₆-DMSO/D₂O (v/v = 4 : 1) solution (shown in Fig. 3). In the presence of NaHSO₃, the signals at 7.42 ppm (H₁) and 6.98 ppm (H₂), assigned to the protons on the benzopyrylium moiety in Probe 1, were gradually decreased and finally disappeared while new peaks at 6.51 ppm and 4.54 ppm appeared and increased with increasing the addition amounts of NaHSO₃. This result clearly indicated the formation of 1-SO₃ adduct, as shown in Scheme 2. For a further confirmation, mass spectral analysis of Probe 1 with NaHSO₃ in CH₃CN/HEPES buffer (v/v = 1 : 1) was conducted and the appearance of the peak at m/z = 497.1727, which is almost identical to the molecular weight of 1-SO₃ adduct (calcd for C₂₆H₂₉N₂O₆S = 497.1752) (Fig. S14, ESI†), provided strong support for the proposed mechanism.

Fig. 3 ¹H NMR spectra of Probe 1 (10.0 mM) in presence of different equivalents of NaHSO₃ in d₆-DMSO/D₂O (v/v = 4 : 1) solution. Peaks marked with (▼) belong to Probe 1, and peaks marked with (*) belong to the 1-SO₃ adduct.

Scheme 2 Proposed sensing mechanism of Probe 1 for SO₂ derivatives.

Selectivity studies

To evaluate the selectivity of Probe 1 toward to SO₂ derivatives, various analytes were used to investigate the fluorescence behavior of Probe 1 in HEPES buffer (20.0 mM, pH = 7.4). As shown in Fig. 4 and Fig. S15, ESI,† the addition of 25.0 μM of representative anions (F⁻, Cl⁻, Br⁻, I⁻, N₃⁻, NO₂⁻, NO₃⁻, SO₄²⁻, S₂O₃²⁻, AcO⁻ and HS⁻), biological metal ions (Mg²⁺, Zn²⁺, Ca²⁺ and K⁺) as well as 50.0 μM of biological reactive oxygen species to the solution of Probe 1 resulted in negligible fluorescence change. It is noteworthy that the fluorescence of Probe 1 showed no changes in the presence of biological thiols (0.5 mM Cys, 0.5 mM Hcy and 4 mM GSH), which might be ascribed to the effective electrostatic repulsion between the cationic benzopyrylium moiety of Probe 1 and the cationic ammonium group of biological thiols.²² To explore the practical ability of Probe 1, we conducted competitive experiments for the detection of HSO₃⁻ with the co-existence of many potential interfering analytes. As shown in Fig. S16, ESI,† all these typically encountered analytes had little impact on the detection of HSO₃⁻. Similarly, Probe 2 exhibited good selectivity toward SO₂ derivatives (Fig. S17, ESI†).

Fig. 4 The emission ratio (I₄₈₉/I₆₉₀) of Probe 1 (5.0 μM) upon the addition of corresponding analytes, HSO₃⁻ (25.0 μM), reactive oxygen species (50.0 μM), Cys and Hcy (0.5 mM), GSH (4 mM) and others (25.0 μM) in HEPES buffer (20.0 mM, pH = 7.4). Inset: Emission images of Probe 1 with different analytes under UV 365 nm lamp.

Real-time monitoring of the SO₂ releasing

Fluorescent probes are indispensable tools for real-time monitoring of analytes in biology.²³ Encouraged by the fast response time of Probe 1 to SO₂ derivatives, we expected Probe 1 could be used for the real-time detection. Thus, a SO₂ donor, sulfonamide, was prepared according to the literature method.²⁴ SO₂ can be slowly released when the SO₂ donor was in the presence of Cys in pH 7.4 buffer (Fig. S18 and S19, ESI†). Thus, we treated Probe 1 (5.0 μM) with SO₂ donor (50.0 μM) and Cys (500.0 μM) in HEPES buffer (containing 2% DMF) and investigated the absorption and emission spectral changes with time. As the reaction time increased, the absorption peak at 660 nm decreased and the new peak at 358 nm increased (Fig. 5a). Impressively, a time-dependent ratiometric fluorescence response was observed that the emission at 690 nm decreased and the new peak at 489 nm increased with increasing reaction time (Fig. 5b). These results demonstrated that Probe 1 could be a useful tool to monitor real-time SO₂ releasing.

Fig. 5 Time-dependent absorption (a) and emission (b) spectra of Probe 1 (5.0 μM) with SO₂ donor (50.0 μM) and 500.0 μM Cys in HEPES buffer (20.0 mM, pH = 7.4, containing 2% DMF). Insets: color (a) and fluorescence (b) images of Probe 1 incubated for 30 min with Cys (400.0 μM) (1), SO₂ donor (40. 0 μM) (2) and Cys (400.0 μM) + SO₂ donor (40.0 μM) (3).

pH studies

We also investigated pH effect on the fluorescence spectrum of Probe 1 in the absence/presence of HSO₃⁻. The fluorescence intensity ratio (I₄₈₉/I₆₉₀) of Probe 1was barely affected in the pH range 2.0–11.0, which suggested that Probe 1 was stable within a wide pH range (Fig. 6). In the presence of 5.0 equiv. of HSO₃⁻, the fluorescence intensity ratio (Mg²⁺, Zn²⁺, Ca²⁺ and K⁺) changed a little in pH range of 5.0–11.0, indicating that Probe 1 could be used for the detection of HSO₃⁻ under physiological condition.

Fig. 6 The fluorescence intensity ratio (I₄₈₉/I₆₉₀) of Probe 1 (5.0 μM) in the absence/presence of HSO₃⁻ (25.0 μM) at different pH values.

Intracellular imaging of SO₂ derivatives

Owing to the excellent sensing properties of Probes 1 and 2 for SO₂ derivatives in vitro assay, we further demonstrated their capability for fluorescence imaging of SO₂ derivatives in living cells (Fig. 7). HeLa cells, incubated with Probe 1 (5.0 μM) in PBS buffer (pH = 7.4) for 30 min at 37 °C, exhibited strong red fluorescence (Fig. 7c) and relatively weak green fluorescence (Fig. 7b). In contrast, when cells were pre-treated with NaHSO₃ (50.0 μM) for 30 min, washed with PBS buffer (20.0 mM, pH = 7.4) and further incubated with Probe 1 (5.0 μM) for 30 min, the red fluorescence signals significantly decreased (Fig. 7f) and the green fluorescence was strongly enhanced (Fig. 7e). These fluorescence variations are in consistent with the ratiometric fluorescence responses of Probe 1 toward SO₂ derivatives in vitro assay. The biological experiment indicated that Probe 1 was cell-permeable and could be used as a ratiometric fluorescent probe for real-time imaging of SO₂ derivatives in living cells. In addition, Probe 2 was also used for the detection of the intracellular SO₂ derivatives in living A431 cells (Fig. S22, ESI†). As expected, Probe 2 displayed the ability for the intracellular detection of SO₂ derivatives in living cells. In addition, the MTT assays for Probes 1 and 2 indicated that both of the probes had minimal cytotoxicity (Fig. S23 and S24, ESI†).

Fig. 7 Images of living HeLa cells. Top row: cells incubated with Probe 1 (5.0 μM) for 30 min. Bottom row: washed with PBS buffer (20.0 mM, pH = 7.4) and further incubated with Probe 1 (5.0 μM) for 30 min. (a) and (d) Bright field images (b) and (e) fluorescence images (excited with a blue light) and (c) and (f) fluorescence images (excited with a green light). Scale bar = 100 μm.

Experimental section

Materials and instrumentations

Unless otherwise noted, all reagents were obtained commercially and used without further purification. All solvents were dried by standard methods. Twice-distilled water was used in the experiments. NMR spectra were performed on a BRUKER 500 spectrometer, using TMS as an internal standard. Mass spectra were performed on a micrOTOF-Q II mass spectrometer (Bruker Daltonics, Germany). A Shimadzu UV-2450 spectrophotometer was used to obtain the UV-Vis absorption spectra. Fluorescence spectra were measured using a HITACHI F7000 fluorescence spectrophotometer and both of the excitation and emission slit widths were set at 5.0 nm. Unless otherwise noted, 450 nm was used as the excitation wavelength all fluorescence measurements. Cell imaging was performed on an Olympus IX83 inverted microscope. TLC silica gel plates and silica gel (mesh 200–300) for column chromatography were purchased from Qingdao Ocean Chemicals. The pH measurement was carried out on a Leici PHS-3C meter.

General procedures for the spectra measurement

The stock solutions of Probe 1 were prepared at 1.0 mM in CH₃CN. The solutions of various testing species were prepared from NaCl, Na₂SO₄, NaF, KI, NaCl, NaBr, NaN₃, NaNO₃, Na₂S₂O₃·5H₂O, NaNO₂, CH₃COONa, MgCl₂, ZnCl₂, CuCl₂, homocysteine, GSH, cysteine, H₂O₂, NaClO, Na₂SO₃ and NaHSO₃ in twice-distilled water. The test solutions of Probes 1 and 2 (5.0 μM) were prepared by placing 0.01 mL of the corresponding stock solution into 1.94 mL HEPES buffer (20.0 mM, pH = 7.4). The resulting solution was shaken well and incubated with 0.05 mL appropriate testing species for 1 min at 37 °C before recording the spectra.

Cell cultures and fluorescence imaging

HeLa and A431 cells were seeded in a 12-well plate in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin. The cells were incubated under an atmosphere of 5% CO₂ and 95% air at 37 °C for 24 h. Before the experiments, cells were washed with HEPES buffered solution three times. For the experiment of imaging of NaHSO₃, cells were pretreated with 50.0 μM of NaHSO₃ for 30 min at 37 °C for 30 min. After washing three times with HEPES buffered solution, the pretreated cells were incubated with the probes (5.0 μM) at 37 °C for additional 30 min. Fluorescence imaging was performed after washing the cells three times with HEPES buffer (20.0 mM, pH 7.4) and observed under an Olympus IX 83 fluorescence microscope.

Determination of the fluorescence quantum yield

The fluorescence quantum yields of the Probes 1 and 2 were measured in HEPES buffer (20.0 mM, pH 7.4). The fluorescent quantum yield is determined using the equation:

Φ/Φ_R = I/I_R × OD_R/OD × n²/n_R² (1)

where I is the integrated fluorescence intensity, OD is the optical density, and n is the refractive index of the solvents. The subscript R refers to the reference fluorophore with a known fluorescent quantum yield. The excitation wavelength is at 655 nm while keeping the optical density at this wavelength below 0.05. Probe 1 was used as a reference (Φ = 0.34 in CH₃CN).^19b

Synthesis

Synthesis of Probes 1 and 2. Probe 1 was synthesized according to the literature method.^19b To a round bottom flask were added 4-diethylaminosalicyladehyde (compound 3) (0.5 mmol, 96 mg) and 3-acetyl-7-diethylamino-chromen-2-one (compound 4) (0.5 mmol, 130 mg) in conc. H₂SO₄ (3.0 mL). The resulting mixture was stirred at 90 °C for 2 hours and cooled to the room temperature. Then, the reaction mixture was pouring into ice (30 g) and 70% perchloric acid (0.5 mL) was added. The resulting precipitate was collected by filtration, washed with water and dried to afford the crude product. Pure Probe 1 was obtained by further purification by silica gel flash chromatography (CH₂Cl₂/CH₃OH = 20 : 1) as a black solid (150 mg, 57% yield). ¹H NMR (500 MHz, DMSO) δ 9.07 (s, 1H), 8.64 (d, J = 8.4 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 9.4 Hz, 1H), 7.72 (d, J = 9.2 Hz, 1H), 7.42 (d, J = 9.3 Hz, 1H), 7.24 (s, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.72 (s, 1H), 3.70 (q, J = 6.9 Hz, 4H), 3.64–3.54 (m, 4H), 1.27 (t, J = 7.0 Hz, 6H), 1.19 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, DMSO) δ 163.45, 159.16, 158.37, 156.06, 154.97, 148.68, 146.26, 133.34, 132.65, 118.09, 117.83, 112.25, 111.29, 109.96, 106.15, 96.89, 96.13, 45.96, 45.47, 12.99; ESI-HRMS: m/z calcd for C₂₆H₂₉N₂O₃: 417.2173, found: 417.2172.

Probe 2 was prepared using the same procedure for Probe 1. Yield: 162 mg, 60%. ¹H NMR (500 MHz, DMSO) δ 8.81 (s, 1H), 8.53 (d, J = 8.5 Hz, 1H), 8.17 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 9.3 Hz, 1H), 7.33 (dd, J = 9.3, 2.4 Hz, 1H), 7.25 (s, 1H), 7.13 (d, J = 2.0 Hz, 1H), 3.66 (q, J = 7.0 Hz, 4H), 3.47 (dd, J = 11.8, 6.7 Hz, 4H), 2.72 (dd, J = 12.3, 6.1 Hz, 4H), 1.94–1.86 (m, 4H), 1.26 (t, J = 7.1 Hz, 6H); ¹³C NMR (126 MHz, DMSO) δ 163.95, 158.64, 158.50, 155.56, 153.08, 151.36, 148.09, 144.92, 132.32, 128.93, 121.88, 117.12, 117.04, 111.08, 110.22, 105.80, 103.96, 96.08, 50.88, 50.28, 45.80, 27.05, 20.70, 19.70, 19.65, 12.96; ESI-HRMS: m/z calcd for C₂₈H₂₉N₃O₃: 441.2178, found: 441.2174.

Conclusions

In conclusion, we have developed Probe 1 as a novel colorimetric and ratiometric fluorescent probe and Probe 2 as a turn-on fluorescent probe for the real-time detection of SO₂ derivatives in pure aqueous environment based on a specific addition reaction. Both of these two probes displayed a fast reaction time, good sensitivity and excellent selectivity. Probe 1 displayed a significant blue shift (about 200 nm) in its fluorescence spectra in the presence of SO₂ derivatives. It is noteworthy these two probes exhibited an excellent selectivity toward SO₂ derivatives over other biologically relevant species. Importantly, the application of these two fluorescent probes for the detection of SO₂ derivative was successfully demonstrated in living cells.

Acknowledgements

The research was supported by the State Key Laboratory of Fine Chemicals (KF1202), Hunan Nonferrous Metals Holding Group Co. Ltd. and The Science and Technology projects of Hunan Province (no. 2011FJ6044 and 2010FJ4019).

Notes and references

1. X. Shi, J. Inorg. Biochem., 1994, 56, 155–165.
2. (a) L. Migliore and F. Coppedè, Mutat. Res., 2009, 674, 73–84; (b) N. Sang, Y. Yun, G. Yao, H. Li, L. Guo and G. Li, Toxicol. Sci., 2011, 124, 400–413.
3. (a) S. Iwasawa, Y. Kikuchi, Y. Nishiwaki, M. Nakano, T. Michikawa, T. Tsuboi, S. Tanaka, T. Uemura, A. Ishigami and H. Nakashima, J. Occup. Health, 2009, 51, 38–47; (b) N. Sang, Y. Yun, H. Li, L. Hou, M. Han and G. Li, Toxicol. Sci., 2010, 114, 226–236.
4. (a) M. H. Stipanuk, Annu. Rev. Nutr., 1986, 6, 179–209; (b) M. H. Stipanuk and I. Ueki, J. Inherited Metab. Dis., 2011, 34, 17–32.
5. (a) J. Li, R. Li and Z. Meng, Eur. J. Pharmacol., 2010, 645, 143–150; (b) X. Wang, H. Jin, C. Tang and J. Du, Eur. J. Pharmacol., 2011, 670, 1–6.
6. (a) S. S. Haider, Ind. Health, 1984, 23, 81–87; (b) M. Lovati, C. Manzoni, M. Daldossi, S. Spolti and C. Sirtori, Arch. Toxicol., 1996, 70, 164–173.
7. C. S. Pundir and R. Rawal, Anal. Bioanal. Chem., 2013, 405, 3049–3062.
8. (a) M. Schäferling, Angew. Chem., Int. Ed., 2012, 51, 3532–3554; (b) L. Yuan, W. Lin, K. Zheng, L. He and W. Huang, Chem. Soc. Rev., 2013, 42, 622–661.
9. (a) X. Chen, T. Pradhan, F. Wang, J. S. Kim and J. Yoon, Chem. Rev., 2011, 112, 1910–1956; (b) Y. Yang, Q. Zhao, W. Feng and F. Li, Chem. Rev., 2012, 113, 192–270.
10. C. Yin, F. Huo, J. Zhang, R. Martínez-Máñez, Y. Yang, H. Lv and S. Li, Chem. Soc. Rev., 2013, 42, 6032–6059.
11. (a) M. R. Gill, J. Garcia-Lara, S. J. Foster, C. Smythe, G. Battaglia and J. A. Thomas, Nat. Chem., 2009, 1, 662–667; (b) C. Li, M. Yu, Y. Sun, Y. Wu, C. Huang and F. Li, J. Am. Chem. Soc., 2011, 133, 11231–11239; (c) X. Peng, T. Wu, J. Fan, J. Wang, S. Zhang, F. Song and S. Sun, Angew. Chem., Int. Ed., 2011, 50, 4180–4183.
12. (a) K. Chen, Y. Guo, Z. Lu, B. Yang and Z. Shi, Chin. J. Chem., 2010, 28, 55–60; (b) X. Cheng, H. Jia, J. Feng, J. Qin and Z. Li, Sens. Actuators, B, 2013, 184, 274–280; (c) Y. Q. Sun, P. Wang, J. Liu, J. Zhang and W. Guo, Analyst, 2012, 137, 3430–3433; (d) G. Wang, H. Qi and X. F. Yang, Luminescence, 2013, 28, 97–101; (e) H. Xie, F. Zeng, C. Yu and S. Wu, Polym. Chem., 2013, 4, 5416–5424; (f) X. F. Yang, M. Zhao and G. Wang, Sens. Actuators, B, 2011, 152, 8–13; (g) Y. Yang, F. Huo, J. Zhang, Z. Xie, J. Chao, C. Yin, H. Tong, D. Liu, S. Jin and F. Cheng, Sens. Actuators, B, 2012, 166, 665–670; (h) C. Yu, M. Luo, F. Zeng and S. Wu, Anal. Methods, 2012, 4, 2638–2640.
13. (a) S. Chen, P. Hou, J. Wang and X. Song, RSC Adv., 2012, 2, 10869–10873; (b) M. G. Choi, J. Hwang, S. Eor and S. K. Chang, Org. Lett., 2010, 12, 5624–5627; (c) X. Gu, C. Liu, Y. Zhu and Y. Zhu, J. Agric. Food Chem., 2011, 59, 11935–11939.
14. D. Srikun, E. W. Miller, D. W. Domaille and C. J. Chang, J. Am. Chem. Soc., 2008, 130, 4596–4597.
15. (a) S. Deo and H. A. Godwin, J. Am. Chem. Soc., 2000, 122, 174–175; (b) M. S. Tremblay, M. Halim and D. Sames, J. Am. Chem. Soc., 2007, 129, 7570–7577.
16. M. Morton and H. Landfield, J. Am. Chem. Soc., 1952, 74, 3523–3526.
17. (a) G. Li, Y. Chen, J. Wang, Q. Lin, J. Zhao, L. Ji and H. Chao, Chem. Sci., 2013, 4, 4426–4433; (b) Y. Sun, D. Zhao, S. Fan, L. Duan and R. Li, J. Agric. Food Chem., 2014, 62, 3405–3409; (c) Y. Q. Sun, J. Liu, J. Zhang, T. Yang and W. Guo, Chem. Commun., 2013, 49, 2637–2639; (d) H. Tian, J. Qian, Q. Sun, H. Bai and W. Zhang, Anal. Chim. Acta, 2013, 788, 165–170; (e) H. Tian, J. Qian, Q. Sun, C. Jiang, R. Zhang and W. Zhang, Analyst, 2014, 139, 3373–3377; (f) M. Y. Wu, K. Li, C. Y. Li, J. T. Hou and X. Q. Yu, Chem. Commun., 2014, 50, 183–185; (g) M. Y. Wu, T. He, K. Li, M. B. Wu, Z. Huang and X. Q. Yu, Analyst, 2013, 138, 3018–3025.
18. W. Chen, Q. Fang, D. Yang, H. Zhang, X. Song and J. Foley, Anal. Chem., 2015, 87, 609–616.
19. (a) E. Brecht, Anal. Chem., 1986, 58, 384–387; (b) P. Czerney, G. Graneß, E. Birckner, F. Vollmer and W. Rettig, J. Photochem. Photobiol., A, 1995, 89, 31–36.
20. H. Sato and M. Ikeda, J. Appl. Phys., 1972, 43, 4108–4113.
21. H. Lv, X. F. Yang, Y. Zhong, Y. Guo, Z. Li and H. Li, Anal. Chem., 2014, 86, 1800–1807.
22. J. Liu, Y. Q. Sun, J. Zhang, T. Yang, J. Cao, L. Zhang and W. Guo, Chem.–Eur. J., 2013, 19, 4717–4722.
23. (a) N. Boens, V. Leen and W. Dehaen, Chem. Soc. Rev., 2012, 41, 1130–1172; (b) A. P. De Silva, H. N. Gunaratne, T. Gunnlaugsson, A. J. Huxley, C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97, 1515–1566.
24. S. R. Malwal, D. Sriram, P. Yogeeswari, V. B. Konkimalla and H. Chakrapani, J. Med. Chem., 2011, 55, 553–557.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra15067h

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