A highly sulfite-selective ratiometric fluorescent probe based on ESIPT

Abstract

The levulinate ester of 2-(benzothiazol-2-yl)phenol, 1, has been developed as a ratiometric fluorescent probe for identifying and quantitating sulfite anions. The mechanism of action is based on the sulfite-triggered intramolecular cleavage of the levulinate moiety to give 1 which, when excited with 310 nm light, decays to its ground state via an excited state intramolecular proton transfer (ESIPT) mediated pathway. We show that the intensity of the ESIPT fluorescent signal relative to that of 1 is proportional to sulfite concentration. This new probe shows good selectivity and high sensitivity for sulfite over other typically encountered anions (F⁻, Cl⁻, Br⁻, I⁻, HPO₄²⁻, SO₄²⁻, NO₃⁻, AcO⁻, ClO₄⁻, N₃⁻, HCO₃⁻) when measured in CH₃CN/H₂O (50 : 50, v/v) solution.

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Introduction

Large quantities of mono- and divalent metal salts of the sulfite anion have been used in industry since the mid-1800's. The first major use of sodium sulfite was in the manufacture of wood pulp for the paper industry in 1867. Subsequently these materials have found wide use in the food industry¹ where they are used as preservatives, anti-oxidants, bacteriostasis agents, and to control fruit and vegetable degradation caused by enzymatic or non-enzymatic reactions.^2–4 In spite of their many beneficial uses, the willful or accidental release of large quantities of these chemicals into waste water streams can constitute a major environmental hazard. For example, sulfite contaminated waste water can seriously deplete the amount of oxygen in water to levels which are unsustainable to aquatic life forms.⁵ Moreover, sulfite can constitute a life-threatening hazard to allergy prone individuals; in-take of these anions by sensitized patients can cause acute breathing difficulties, asthma attacks, skin sensitivity and gastrointestinal reactions within minutes of ingestion.^6,7 Additionally, their adverse effects can extend to general populations where they are known to be inimical to some vitamins and are suspected to be the cause of a variety of chronic health conditions.⁸ As a result, the presence of sulfite in the environment has aroused great concern and the use of sulfite in food stuffs and in waste water is strictly regulated in many countries. Recognition of these potential adverse effects has stimulated considerable interest in the development of methods to detect and quantify sulfite contaminants.

As a consequence of the concerns enumerated above, a variety of disparate schemes have been proposed for sulfite determination including electrochemistry,^9–11 chromatography,¹² chemiluminescence,^13,14 electrochemical and enzymatic techniques.^10,11 However, most of these conventional methods are either time-consuming, have poor selectivities, are expensive or can involve complicated procedures. Owing to operational simplicity and potential high sensitivity, fluorescent probes have been developed as alternative options for the detection of anions during the past decade.^15–17 An important contribution in this regard has been made by Chang et al. who reported a turn-on type fluorescent probe for sulfite by chemical deprotection of resorufin levulinate.¹⁸ However, this intensity-based fluorescent sensor suffers from errors associated with the determination of probe concentration, environmental effects and instrument factors.^19–24 Therefore, ratiometric fluorescent probes for sulfite detection are more desirable. So far, there is only one ratiometric probe for sulfite reported.²⁵ The goal of the work reported herein was to develop a new ratiometric probe for sulfite that would allow us to more easily and accurately detect and quantitate this anionic species.

2-(Benzothiazol-2-yl)phenol and its derivatives^26,27 are known to exhibit an excited state intramolecular proton transfer (ESIPT) from the phenol form to the keto form which results in an easily detectable fluorescence signal due to an accompanying large Stokes shift. To date, several derivatives of 2-(benzothiazol-2-yl)phenol have been reported as ratiometric fluorescent probes for F⁻, Hg²⁺, Zn²⁺, anionic species, pyrophosphate ions (PPi), and phosphates.^28–33

These reports, coupled with Chang's findings that sulfite is capable of cleaving levulinate-protected phenol moieties selectively under mild and neutral conditions, inspired us to develop a novel ratiometric fluorescent probe 1,^18,34 levulinate of 2-(benzothiazol-2-yl)phenol, for the detection of sulfite anions. Probe 1 was synthesized by the reaction of 2-(benzothiazol- 2-yl)phenol with levulinyl chloride in 65% yield, as shown in Scheme 1. Ester 1 shows a blue-violet fluorescence centered at 373 nm. Upon addition of sulfite, 1 is converted into phenolic dye 2 which, because of its ESIPT-mediated emission, exhibits a significant red-shifted fluorescent band centered at 468 nm which corresponds to a 95 nm red shift. The possible sulfite-selective signaling mechanism is shown in Scheme 2.

Scheme 1 Synthesis of ratiometric probe 1 for sulfite.

Scheme 2 Possible sulfite-selective signaling mechanism.

Experimental

Materials and equipments

All titrations were carried out in HEPES buffer solution (10 mM, pH = 7.4, 50% acetonitrile, v/v). Unless otherwise noted, all chemicals obtained from commercial suppliers are analytical reagent grade and used as received. The fluorescence emission spectra are measured after 60 min of mixing. THF were refluxed with sodium and distilled under an atmospheric pressure. TLC plates and silica gel (200–300 mesh) were purchased from Qingdao Ocean Chemicals, China. The UV-Vis absorption spectra and fluorescence emission spectra were measured using a Shimadzu UV-2450 spectrophotometer and a HITACHI F-4600 spectrometer, respectively. ¹H NMR spectra in CDCl₃ were recorded using a Bruker 400 M AVANCE-III spectrometer with chemical shifts reported as ppm (TMS as the internal standard). Mass spectra were measured on a Shimadzu LC-MS-IT-TOF mass spectrometer.

Synthesis

2-(Benzothiazol-2-yl)phenol 2 and levulinyl chloride were synthesized according to the literature methods.^35,36 Probe 1 was prepared as follows: to a stirred mixture of 2-(benzothiazol-2-yl)phenol 2 (227 mg, 1.0 mmol) and NaH (31 mg, 1.3 mmol) in 10 mL dry THF under an argon atmosphere, was added in a dropwise manner levulinyl chloride (174 mg, 1.3 mmol) at room temperature. The resulting reaction mixture was allowed to stir at room temperature overnight. The reaction was quenched with 1 mL of water and the solvent was evaporated under vacuum. The resulting solid product was extracted twice with 50 mL portions of dichloromethane. After drying over anhydrous Na₂SO₄, the organic solvent was filtered and removed by distillation to yield a yellow solid. Product 1 was purified by column chromatography on silica gel (3 : 1 hexane/ethyl acetate as eluent) and isolated as a pale yellow solid (210 mg, 65%). ¹H NMR (400 MHz, CDCl₃): δ (ppm) = 8.30 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 8.4 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.51 (t, J = 7.6 Hz, 2H), 7.43–7.38 (m, 2H), 7.28 (s, 1H), 3.06 (t, J = 6.4 Hz, 2H), 2.93 (t, J = 6.4 Hz, 2H), 2.23 (s, 3H). m/z (LCMS-IT-TOF) M⁺ + 1: calculated, 326.0851; found, 326.0861.

Results and discussion

The absorption and fluorescence spectra of probe 1 and dye 2 are illustrated in Fig. 1. Due to the ESIPT process, the dye 2 displayed an emission band centered at 468 nm which is attributable to its keto tautomer. Because a corresponding ESIPT mechanism is not possible for 1, only the enol-like emission at 373 nm was observed. After addition of sulfite to a solution of 1 in HEPES buffer (10 mM, pH = 7.4, 50% acetonitrile), the fluorescence undergoes a 95 nm red shift and the emission band at 373 nm steadily decreases with the appearance of the emission band at 468 nm (Fig. 2a). There is a clear isoemission point at 426 nm. The above results indicate that sulfite can easily induce cleavage of the levulinate protection group while concomitantly generating the phenol moiety for the ESIPT–mediated emission.

Fig. 1 The absorption and emission spectra (△) of probe 1 and dye 2 in H₂O/CH₃CN (50%, v/v). Dashed line (a, c): 1; solid line (b, d): 2.

Fig. 2 (a) The fluorescence emission spectra (λ_ex = 310 nm) of probe 1 (10 μM) upon addition of sulfite (0–100 equiv.) in HEPES buffered (pH 7.4, 10 mM) H₂O/CH₃CN (50%, v/v). Inset: fluorescence intensity change based on the peak heights of 373 and 468 nm. (b) The fluorescence intensity ratiometric I₄₆₈/I₃₇₃ of probe 1 (10 μM) upon addition of sulfite (0–100 equiv.) in HEPES buffered (pH 7.4, 10 mM) H₂O/CH₃CN (50%, v/v). Inset: enlarged displays of the linear correlation between ratiometric I₄₆₈/I₃₇₃ and the low concentration of sulfite.

Inspection of Fig. 2b shows that the fluorescence intensity ratio (I₄₆₈/I₃₇₃) changes from 0.37 to 9.2 (25-fold) when the concentration of sulfite increases from 0 to 1000 μM with good linearity, R² = 0.99208, occurring in the range of 0–320 μM. Specifically, the detection limit of probe 1 to sulfite was calculated to be 5 μM.

To evaluate the selectivity of probe 1, other typically encountered anions, including F⁻, Cl⁻, Br⁻, I⁻, HPO₄²⁻, SO₄²⁻, NO₃⁻, AcO⁻, ClO₄⁻, N₃⁻ and HCO₃⁻, were added to the solution of probe 1 and the fluorescence response was measured (Fig. 3). Our results show that within this family of anions, sulfite is the only one that significantly causes the transformation of Probe 1 into phenol 2. The result demonstrates that probe 1 can be used to detect sulfite with good selectivity over other anions.

Fig. 3 Anion selectivity in the fluorescence intensity ratiometric I₄₆₈/I₃₇₃ of probe 1. Inset: The fluorescence spectra (λ_ex = 310 nm) of probe 1 (10 μM) upon addition of 100 equiv. of various anions in HEPES buffered (pH 7.4, 10 mM) H₂O/CH₃CN (50%, v/v).

In order to check the practical ability of the probe 1 to act as a selective fluorescent probe for sulfite, we conducted competitive experiments in the presence of sulfite at 100 equiv. mixed with F⁻, Cl⁻, Br⁻, I⁻, HPO₄²⁻, SO₄²⁻, NO₃⁻, AcO⁻, ClO₄⁻, N₃⁻ and HCO₃⁻ (200 equiv. each). The results, shown in Fig. 4a and Fig. 4b, indicate that the addition of these potential interfering anions, even at higher concentrations, does not adversely affect the fluorescence response of 1.

Fig. 4 (a) The fluorescence spectra (λ_ex = 310 nm) of probe 1 (10 μM) upon addition of sulfite (100 equiv.) and in the presence of interfering anions (200 equiv.) in HEPES buffered (pH 7.4, 10 mM) H₂O/CH₃CN (50%, v/v). (b) The fluorescence intensity ratio (I_{1+anion+sulfite}/I_1+sulfite) of probe 1 (10 μM) at 468 nm upon addition of sulfite (100 equiv.) and in the presence of interfering anions (200 equiv.) in HEPES buffered (pH 7.4, 10 mM) H₂O/CH₃CN (50%, v/v).

We further investigated the influence of pH on the fluorescence of probe 1 and the parent dye 2, respectively. As for probe 1, the fluorescence intensity ratio (I₄₆₈/I₃₇₃) was barely affected when the pH ranged from 4.0 to 10.0, while the decomposition of probe 1 was observed from the fluorescence change when the pH was over 10 (shown in Fig. 5a). The pH has a slight effect on the fluorescence of the parent dye 2, as shown in Fig. 5b. The data suggests that probe 1 can be used for the detection of sulfite at a wide pH range.

Fig. 5 (a) The fluorescence intensity ratio (I₄₆₈/I₃₇₃) of probe 1 (10 μM) at various pH values in H₂O/CH₃CN (50%, v/v) solution. (b) Fluorescence spectra (λ_ex = 310 nm) of compound 2 (10 μM) in H₂O/CH₃CN (50%, v/v) solution at different pH conditions.

Conclusions

In summary, we have developed a novel ESIPT-based, ratiometric fluorescent probe (1) which can detect sulfite qualitatively and quantitatively. It displays a 95 nm red-shift of fluorescence emission as a result of the sulfite-selective deprotection of levulinate. Moreover, this probe shows good selectivity and high sensitivity for sulfite over other anions in a wide working pH range. This work also suggests that O-functionalized 2-(benzothiazol-2-yl)phenols can potentially be used for the design of ratiometric fluorescent probes for other ions.

Acknowledgements

The research was supported by NSF of China (No. 21072235) and the Program for New Century Excellent Talents in University (No. NCET-10-0793).

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Footnote

† Electronic Supplementary Information (ESI) available. See DOI: 10.1039/c2ra21471g

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