Reaction-based ratiometric fluorescent probe for selective recognition of sulfide anions with a large Stokes shift through switching on ESIPT

Abstract

An ESIPT-based, highly selective, ratiometric fluorescent probe BNPT has been synthesized and characterized, which shows turn-on fluorescence response in the presence of S²⁻, attributed to the removal of a protective 2,4-dinitrobenzene (DNB) moiety, and the resulting fluorescent product has been used for the selective detection of Zn²⁺. UV-vis and fluorescence spectroscopy analysis, and DFT and TDDFT calculations were carried out to understand the sensing mechanism. The probe BNPT displays a rapid response time and good sensitivity. The probe can detect quantitatively in a concentration range of 0–6.0 μM. The detection limit is 31.3 nM. The sensing ability of BNPT has been successfully applied in real water samples. Applicability as an in-field test kit has been demonstrated by sensing with a BNPT-coated TLC plate. The probe BNPT has been successfully used for visualization of intracellular environment in living cells. The uniqueness lies in the fact that starting with the probe BNPT, sensing can be achieved by two different mechanisms – first, by selective de-protection of the probe BNPT and second, by reacting with the Zn²⁺ ensemble with S²⁻.

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Introduction

Developing fluorescent chemosensors for chemical species that can be used in detecting biologically and environmentally important anions and cations has received tremendous attention due to their crucial roles in a variety of biological and chemical processes.¹ In particular, Zn²⁺ detection has aroused immense interest, because it plays critical roles in the fundamental physiological processes of organisms. In contrast, sulfide (S²⁻), as a toxic, hazardous, and traditional pollutant, is widely spread in the environment.² It is produced naturally and as a result of human activity. Natural sources include non-specific and anaerobic bacteria reduction of sulfates and sulfur-containing amino acids in meat proteins. S²⁻ is found naturally in volcanic eruptions, crude petroleum and hot springs. It is released from stagnant or polluted waters and manure or coal pits. S²⁻ may be produced by a variety of commercial methods as a byproduct in industrial processes, for example, conversion into sulfur and sulfuric acid, dyes and cosmetic manufacturing, production of an agricultural disinfectant, paper and wood pulp, etc.³ Therefore, it is possible to bio-concentrate and bio-magnify in the food-chain. So continuous and high concentration exposure of S²⁻ would lead to various physiological and biochemical problems such as irritation in mucous membranes, unconsciousness, and respiratory paralysis.⁴ Once a sulfide anion is protonated, it becomes even more toxic. Therefore, the detection of S²⁻ has become very important from an industrial, environmental and biological point of view through newer and quicker analytical methodologies.⁵ To date, a variety of detection strategies have been developed for S²⁻, such as spectroscopy,⁶ electrochemical methods,⁷ titration,⁸ ion chromatography⁹ and chemiluminescence methods.¹⁰ Among the techniques employed, S²⁻ sensing by fluorescence spectrometry has received much attention due to its easy operability and high sensitivity.¹¹ On the other hand, Zn²⁺ is one of the most abundant transition-metal ions present in living cells, owing to its rich coordination chemistry and it plays a variety of fundamental roles in the physiological processes in organisms ranging from bacteria to mammals.¹² Pools of labile Zn²⁺ are found in the cells of mammalian organs, including the brain,¹³ pancreas and prostate.¹⁴ In contrast, overloading of Zn²⁺ in the neuronal cytoplasm and the metabolic process are associated with some serious diseases, such as Wilson's Menke's disease and various infectious diseases.¹⁵ Zn²⁺ is also associated with various anions, especially with sulfides and phosphates, by showing strong affinity.¹⁶ Recently, metal ion complexes have been used as receptors for sulfides and phosphates, and this emerged as one of the most successful strategies, since it provides specific metal ion–anion interactions.¹⁷ Among the metal complex-based receptors, the ones that exhibit fluorescence change in the presence of Zn²⁺ have attracted considerable attention because of the strong affinity of Zn²⁺ toward sulfides and phosphates.¹⁸ Thus, considerable attention has been devoted to the development of fluorescent probes for highly selective recognition of Zn²⁺. ESIPT-based fluorophores such as benzimidazole,¹⁹ benzoxazole,²⁰ benzothiazole²¹ and thiazole²² have been explored for the detection of Zn²⁺.

Although a great number of selective fluorescent probes for S²⁻ have been developed,²³ fluorescent sensing of S²⁻ in water solution still remains a challenging task because of the strong hydration nature of anions, which weakens the interaction of probes with the target anions.²⁴ A number of sensing mechanisms have been employed to overcome this issue for S²⁻ detection, such as metal displacement (ensemble),²⁵ Michael addition reaction,²⁶ cleavage reactions of 2,4-dinitrobenzenesulfonyl (DNBS) with S²⁻,²⁷ nucleophilic substitution reactions,²⁸ and disulfide exchange reactions.²⁹ These studies greatly advanced the research on the fluorescent detection of S²⁻; however, many of these probes suffer from relatively low sensitivity due to the inefficiency of the quenching group or inadequate selectivity due to the cross reactivity of the reactive unit utilized. A ratiometric method measuring the ratio of fluorescence intensities at two wavelengths provides an alternative approach, which can overcome the drawbacks of intensity-based measurements by built-in correction of two emission bands and seems to be more favorable for sensing target ions in comparison with fluorescence intensity-based probes. Therefore, there still has been a large demand to develop a new fluorescent probe that features high reactivity and specificity, excellent sensitivity and fast response toward S²⁻.

In this paper, we identify the 2,4-dinitrobenzene (DNB) moiety as a novel reactive group and reaction switch specific for S²⁻. Integration of this unique moiety into the phenylthiazole and benzothiazole fluorophores afforded a ratiometric fluorescence “off–on” probe 2-[3-benzothiazol-2-yl-4-(2,4-dinitrophenoxy)phenyl]-4-methylthiazole-5-carboxylic ethyl ester, BNPT, for highly specific and sensitive detection of sulfide. In BNPT, the DNB group is especially preferable as an efficient reactive site for S²⁻ and the benzothiazole moiety used as recognition unit for signal transducer through excited state intramolecular proton transfer (ESIPT) resulted in a ratiometric fluorescent probe. We envisioned that the probe itself gave almost no fluorescence emission in the absence of S²⁻ due to the dual-quenching effect of DNB via the intramolecular charge transfer (ICT) and the thiazole can quench the fluorescence via a photoinduced electron transfer (PET) effect. We predicted that fluorescence will be heavily quenched through the combined usage of PET–ICT dual-quenching effects.

Upon reaction with S²⁻, the fluorophore BNPT-OH was released and showed significant fluorescence enhancement attributing to the ESIPT process (Scheme 1).

Scheme 1 Structure and sensing mechanism of BNPT against S²⁻.

Results and discussion

The precursor hydroxyphenylbenzothiazole derivative 5 has been synthesized in four steps on the basis of our previously reported literature method³⁰ starting from p-hydroxybenzonitrile. All the precursor and probe molecules were fully characterized by ¹H NMR, ¹³C NMR, and ESI-MS spectroscopy. The S²⁻-promoted aromatic nucleophilic substitution reaction cleaves the specific O–C(DNB) bond of probe BNPT, which liberates precursor 5 and an intramolecular proton transfer gives ratiometric emission from the excited state.

The reactivity of probe BNPT toward different anions and their preferential selectivity toward S²⁻ over the other ions have been studied by fluorescence titrations. We found that the addition of trace amounts of S²⁻ causes the absorption and fluorescence signal to change rapidly, which is very important for real-time detection. To find the suitability of the solvent system of BNPT to sense S²⁻ in environmental and biological samples, we first investigated the emission properties of probe BNPT in pure CH₃CN and pure aqueous solution; disappointingly, in pure aqueous solutions, the probe showed no response to S²⁻. We speculate that this phenomenon was probably due to the hydrogen bond between S²⁻ and water in an aqueous solution that weakened the nucleophilicity of S²⁻ to probes and then decreased the reaction speed. Finally, the fluorescence signaling behavior of the probe BNPT was investigated in an optimized aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4) solution at room temperature.

Spectral response and detection of S²⁻ in solutions

We examined the absorption spectra of probe BNPT in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4) at room temperature. The absorption spectra of free probe BNPT displayed a maximum absorbance at 312 nm. Upon the addition of S²⁻ (Na₂S has been used as a source of S²⁻ in all the experiments), the color of the solution turned from colorless to slight greenish yellow, which was clearly recognizable by the naked eye.

At the same time, a concomitant increase in a new absorption band at 426 nm was observed with two isosbestic points at 275 nm and 347 nm (Fig. 1). The isosbestic points observed at 275 nm and 347 nm were indicative of the transition between the reactant and the product through the formation of aryl-σ-complex species.

Fig. 1 (a) UV-Vis spectral change of BNPT (1 × 10⁻⁵ M) in the presence of increasing amounts of S²⁻ (1 × 10⁻⁴ M) in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4) at room temperature. Inset: Photograph of visible color under ambient light in the absence and presence of S²⁻. (b) The linear relationship between the absorbance ratio (I₄₂₆/I₃₁₂) and the concentration of S²⁻.

Due to the specific O–C (DNB) cleavage, a distinct 114 nm red shift in absorbance was observed. Since the phenolate group is a much stronger electron donor than the ether group in BNPT, the ICT efficiency should be significantly enhanced by the interaction of probe BNPT with S²⁻ and thus shifting the absorption to a longer wavelength.

Subsequently, the fluorescence spectra of probe BNPT in the absence and presence of S²⁻ were recorded (Fig. 2). The probe BNPT exhibits very weak emission at ∼426 nm when excited at 347 nm in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4). This negligible emission of BNPT has been explained in terms of photoinduced electrontransfer (PET) as well as prohibitions of the ESIPT process produced by the 2,4-dinitrobenzene moiety. Besides these two effects, the electron-withdrawing nature of the nitro group may also be contributing toward the non-fluorescent nature of BNPT.

Fig. 2 (a) Fluorescence emission spectra (λ_ex = 347 nm) of BNPT (1 × 10⁻⁵ M) in the presence of increasing amounts of S²⁻ (1 × 10⁻⁴ M) in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4) at room temperature. Inset: Enlarged view of the isobestic point (b) concentration-dependent fluorescence intensity changes of BNPT (1 × 10⁻⁵ M) in the presence of increasing amounts of S²⁻ (1 × 10⁻⁴ M). Inset: Visual fluorescence color of BNPT before and after the addition of 5 equiv. of S²⁻.

However, upon the progressive additions of S²⁻ (0–5 equiv.) to the solution of BNPT, distinct enhancement of the fluorescent intensity at 503 nm was observed and a simultaneous decrease in emission at 426 nm, which indicated that the chemical reaction between S²⁻ and the DNB group could be attributed to the regeneration of a hydroxyl group by effective cleavage of the 2,4-dinitrobenzene moiety through the ArS_N reaction and restoration of the ESIPT process.

The observation is in good agreement with the aforementioned design concept. Accordingly, the emission changes also resulted in obvious fluorescence color changes from colorless to green as well as a well-defined isoemissive point at 437 nm. The emission intensity ratio, I₅₀₃/I₄₂₆, increases with the increase in the S²⁻ concentrations, which allows the detection of S²⁻ ratiometrically. It is noteworthy that the difference in the two emission wavelengths is very large (emission shift = 77 nm), which not only contributes to the accurate measurement of the intensities of the two emission peaks, but also results in a huge ratiometric value (I₅₀₃/I₄₂₆ = 450 fold). Under the same conditions, the ratio changes of the two fluorescence peaks (I₅₀₃/I₄₂₆) produced a good linear relationship with the concentration of S²⁻ between 0 and 0.6 μM, and the detection limit for S²⁻ was determined as 31.3 nM based on S/N = 2, which was sufficiently low for the detection of S²⁻ in environmental samples (Fig. S13, ESI†).

Based on the spectral changes, we propose that a cleavage of ether group by S²⁻ with BNPT enables it to turn-on fluorescence. Furthermore, the ratiometric emission by BNPT with S²⁻ could be explained as a consequence of ICT and ESIPT. The ratiometric emissions in the case of BNPT may be attributed to the photo tautomer of excited normal isomer (N* emission) and tautomer (T* emission) forms, which result from an ESIPT (N* ↔ T*) process (Scheme 3).

Molecules having both acidic and basic moieties in close proximity often undergo ESIPT in the excited state. In the present case, transfer of a phenolic-OH proton to the benzothiazole nitrogen through keto–enol tautomerization leads to significant differences in structure and electronic configuration from its corresponding normal species.³¹ If the phenolic-OH of the probe is protected by a suitable group, the ESIPT process is inhibited resulting in fluorescence switch off. The probe BNPT shows only local fluorescence emissions at 426 nm. The emission of BNPT at lower wavelength (426 nm) is assigned to the enol form, i.e., normal isomer (N* emission), while the keto form emits (T* emission) at the higher wavelength (503 nm). Thus, the ESIPT process is much more favorable in the probe BNPT after the S²⁻ reaction and it is highly dependent on the chemical structure.

Sensing of S²⁻ in the presence of other anions

In order to check whether the probe BNPT is sensitive to only S²⁻ or even to the other competitive anions, similar fluorescence titrations were carried out in the same medium with 16 other different anions, viz. N₃⁻, AcO⁻, SCN⁻, PO₄³⁻, H₂PO₄⁻, HCO₃⁻, CO₃²⁻, F⁻, Cl⁻, Br⁻, I⁻, NO₃⁻, SO₄²⁻, HSO₄⁻, SO₃²⁻, and S₂O₃²⁻ as well as a thiol species (cysteine, homocysteine, and glutathione), and no significant fluorescence enhancement was found in the presence of any of these ions (Fig. 3). Visual fluorescent color change experiments have been carried out to look at the behavior of probe BNPT in the presence of various anions. Under UV light, the solution of BNPT is weakly fluorescent, whereas in the presence of S²⁻, it shows an intense green fluorescent color that is otherwise not present in the case of the other ions studied (Fig. S9, ESI†). Therefore, S²⁻ can easily be differentiated by visual color change among the other anions.

Fig. 3 Fluorescence spectra of probe BNPT (10 μM) in the presence of various anions (50 μM) in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4).

In order to show the practical utility of the probe BNPT to detect S²⁻ selectively even in the presence of other anions, competitive allied anion titrations were carried out. As can be seen, these competitive species, including N₃⁻, AcO⁻, SCN⁻, PO₄³⁻, H₂PO₄⁻, HCO₃⁻, CO₃²⁻, NO₃⁻, SO₄²⁻, F⁻, Cl⁻, Br⁻, I⁻, HSO₄⁻, SO₃²⁻, S₂O₃²⁻, Cys, Hcy, and GSH, which often show strong interference to S²⁻ detection, did not lead to any significant ratiometric fluorescence changes at all, and the fluorescence emission spectrum of BNPT remained almost undisturbed (Fig. S8, ESI†). Therefore, it can be concluded that probe BNPT selectively reacts with S²⁻ even in the presence of other analytes.

Time courses of the BNPT and S²⁻ system

We further moved forward to study the reaction kinetics of the probe BNPT in the presence of S²⁻. Probe BNPT (10 μM) was incubated with 50 μM S²⁻ in HEPES buffer at 37 °C. Upon addition of increasing concentrations of S²⁻, a dramatic increase of emission intensity at 503 nm was observed within 1 min, and the emission signal essentially reached a plateau within 5 min and showed a fairly fast reaction rate (Fig. 4).

Fig. 4 (a) Time course experiment of the probe BNPT in the presence of 5 equiv. S²⁻ in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4) at room temperature. (b) Pseudo-first-order kinetic plot.

Through monitoring the fluorescence emission at 503 nm, the S²⁻ mediated reaction was found to obey a pseudo-first-order law of rate constant K′ = 0.58 min⁻¹ under S²⁻ excess conditions. The absence of S²⁻ in the tested solutions of probe BNPT meant that no changes in fluorescence response were detected; this result suggests that probe BNPT is a “fast-response” probe for S²⁻ and may be suitable for real-time sensing of S²⁻.

Secondary recognition of Zn²⁺

The release of phenolic compound 5 from BNPT by S²⁻ has been used for the secondary sensing property toward metal ions. To the solution (BNPT + S²⁻), an aqueous solution of Zinc perchlorate (5 mM) was added – a complete quenching was observed at 503 nm with concomitant increase of fluorescence at 426 nm (Fig. S5, ESI†). Fluorescence color changed from intense green to non-fluorescent. Interestingly, subsequent addition of S²⁻ (5 mM) to the above solution retrieves the fluorescence (Fig. S6, ESI†). The color of the solution gradually changed from non-fluorescent to intense green. The secondary recognition capability of the compound 5 has been carried out by titrating it with different cations, viz., Na⁺, K⁺, Mg²⁺, Ca²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, Cu²⁺, Cd²⁺, Zn²⁺, and Hg²⁺.

Compound 5 does not show any significant fluorescence change toward alkali and alkaline earth metal cations; however, it shows quenching of fluorescence intensity in the rest of the cations due to inhibition of ESIPT on chelation. Thus, in the case of zinc ions, it shows maximum quenching and the sigmoidal plot exhibits saturation at ∼3.5 equiv., as compared with all other transition-metal ions studied, which show saturation at much higher equivalents, suggesting the strong chelating affinity of Zn²⁺ toward 5 (Fig. 5). The fast and selective response of compound 5 toward Zn²⁺ as compared with many cations, including other transition-metal ions, allows it to be a selective sensing molecular system for Zn²⁺.

Fig. 5 Fluorescence spectra of BNPT (10 μM) as a function of cations/compound 5 mole ratio obtained during the titration of BNPT with different cations in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4).

Reversibility and reusability. The reusability of the phenolic compound 5 has been demonstrated by carrying out nine alternative cycles of the titration of 5 with Zn²⁺ followed by S²⁻ (Fig. 6). Zn²⁺ shows notable switch-off fluorescence changes through the formation of [Zn-5] complex, and further the titration of this non-fluorescent complex with S²⁻ enhances the same by removing the Zn²⁺ from [Zn-5] due to the recovery of ESIPT.

Fig. 6 (a) Reversible switching cycles of fluorescence intensity (λ_ex = 347 nm) by alternate addition of S²⁻ and Zn²⁺ in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4). (b) Schematic representation of reversible nature of compound 5.

The chemical reversibility behavior of the compound 5 with Zn²⁺, followed by the removal of Zn²⁺ by S²⁻, was studied in aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4). Hence, compound 5 is a reversible and reusable sensor for Zn²⁺ and its zinc complex [Zn-5] as a secondary sensor for S²⁻.

The 1 : 1 stoichiometry of [Zn-5] was established from the Job's plot (Fig. S12, ESI†). The formation of a 1 : 1 complex was further confirmed by HR-MS studies. The molecular ion peak appears at m/z 558.8810, and corresponds to the mass of [BNPT + Zn²⁺ + ClO₄]⁺ (Fig. S4, ESI†).

The binding constant for the formation of [Zn-5] complex was calculated on the basis of change of emission at 503 nm by considering 1 : 1 binding stoichiometry. The binding constant (K) determined by the Benesi–Hildbrand plot experiments was found to be 4.0 × 10⁵ M⁻¹ (Fig. S14, ESI†).

Theoretical calculations

Computational calculations were carried out using the Gaussian 09 program to explain the electronic structural properties of probe BNPT and its corresponding phenolic product 5, as well as to establish the complexation features of product 5 by Zn²⁺. To do that, initially the structures of BNPT and its corresponding phenolic product 5 were optimized in DFT. The optimized geometries and spatial distributions and orbital energies of HOMO and LUMO of BNPT and resultant product 5 are given in Fig. 7. The optimized 5 was subjected to the interaction with Zn²⁺ that was further optimized by Lanl2dz with the same ECP to obtain the complex [Zn-5]. The optimized geometries and the calculated electron distributions in the frontier molecular orbitals HOMO and LUMO of the phenolic product 5 – Zn-complex are shown in ESI† (Fig. S16 and Table S1). The major chromophore exists in a nearly planar conformation, extending from the phenoxide moieties to the acceptors thiazole and benzthiazole moieties of BNPT. Such a planar conformation enables an efficient π-conjugation and favors the efficient ICT transition and ESIPT process between the donor and the acceptor groups.

Fig. 7 The optimized configuration of (a) BNPT and (b) compound 5. HOMO–LUMO energy gaps for respective compounds and interfacial plots of the orbitals: (c) BNPT and (d) compound 5.

In particular, for the probe BNPT, the HOMOs are majorly localized on the benzothiazole and thiazole parts whereas LUMOs are widely spread out over the dinitrobenzene (DNB) part that results in PET process ON and it effects the initial weak fluorescent of the probe. The charge separation in the excited state is much more pronounced in the phenolic species that corresponds to the n → π* transition of the phenolic group to the thiazole moiety. Furthermore, the energy gap between the HOMO and LUMO of the probe BNPT was 2.87 eV higher than that of their corresponding phenolic compound (1.86 eV) (Table S2, ESI†), in good agreement with the red shift in the absorption observed upon the treatment of probes with S²⁻.

The TDDFT calculations were performed at the B3LYP/6-31+G(d,p) level to explain the optical properties of the ground and excited states behavior of BNPT and compound 5. The vertical transitions i.e., the calculated λ_max, main orbital transition, and oscillator strength (f) are listed in Table S1, (ESI†). The vertical transitions calculated by TDDFT of BNPT and compound 5 were found to have good agreement with the experimental data. The studies suggest that the vertical transitions observed at ∼319 and ∼366 nm are comparable to those of the experimentally observed spectra at ∼312 and ∼347 nm for BNPT and compound 5, respectively. The main contributing transitions for BNPT and compound 5 are S₀ → S₁₄ (∼56.60%) and S₀ → S₁ (∼95.60%), respectively, which correspond to energy states arising from HOMO−1 → LUMO and HOMO → LUMO/HOMO → LUMO+1, respectively. The calculated red shift is in good agreement with experimental results due to the n → π* transition. The TDDFT calculations have been carried out for zinc complex also and the corresponding electronic transition details are included in the ESI† (Fig. S16 and Tables S1, S2).

Detection of S²⁻ in environmental water samples

The potential utilities of BNPT were also checked by proof-of concept experiments through the analysis of S²⁻ in aqueous solution for practical applications. Because S²⁻ has carcinogenic properties and has been widely used in a variety of industrial processes, the detection of S²⁻ in aqueous samples is of interest.

Tap and river paper water were collected from the laboratory and Ganges river, respectively, and were spiked with S²⁻ (0–15 μM) into these samples. The concentration of S²⁻ in these samples was determined by fluorescence titration using BNPT. The fluorescence intensities were proportional to the concentrations of S²⁻ in the range of 0–15 μM in the two collected water samples (Fig. 8). Therefore, the presented probe BNPT could be used for trace S²⁻ analysis in these environmental samples.

Fig. 8 Fluorescence detection of S²⁻ in tap water and river water with BNPT.

To find the practical application of BNPT, the test strips were prepared by soaking the TLC plates into an aqueous acetonitrile (CH₃CN : H₂O = 3 : 2 v/v, 10 mM HEPES buffer, pH = 7.4) solution of BNPT (10 μM) and then drying in air. The test strips were utilized to sense S²⁻ (0.1 mM) in aqueous solution. A significant change in color (non-fluorescent to intense green fluorescence) was observed with S²⁻ under a 365 nm UV lamp. Therefore, the test strips based on BNPT can be used to detect the presence of trace amounts of S²⁻ in an aqueous medium quickly and conveniently (Fig. 9).

Fig. 9 Sensing of S²⁻ using a BNPT-coated TLC plate under a UV lamp.

Application in intracellular S²⁻ imaging

To demonstrate the applicability of BNPT in living cells, the suitability of the probe for visualization of S²⁻ was investigated. First, cytotoxicity of BNPT in living cells was evaluated by MTT assay (Fig. S15, ESI†). The application of BNPT in cell biology showed excellent uptake of the receptor by HADF cells. The untreated HADF cells emitted much less green auto-fluorescence. The HADF cells treated with BNPT gave green fluorescence in lower intensity. This indicated that BNPT was well taken up by the cells. Therefore, a strong green fluorescence was observed in those cells treated with BNPT and followed by 1 h sulfide treatment (Fig. 10). These results revealed that non-fluorescent BNPT is cell compatible and gives strong fluorescence. The exact spectral behavior of the BNPT was also found in fluorescence titration.

Fig. 10 Fluorescence microscopic observation of HADF cells under green filter. Fluorescence with low intensity was observed in BNPT-treated cells compared to control. After addition of S²⁻, a strong green fluorescence was emitted by BNPT, scale bar 100 μm.

Conclusion

In conclusion, we have designed and synthesized a new fluorescent chemoreactant probe BNPT based on the benzothiazole–thiazole arms, and explored its sulfide ion recognition property extensively using spectroscopic techniques. The proposed chemosensors exhibit good selectivity and sensitivity toward sulfide over other tested allied anions with ratiometric emission. The fluorescence enhancement mechanism is based on the cleavage of DNB from the fluorophore by S²⁻, which switches the weak emissive state to a strong emissive excited state. The ratiometric emission by the probe with S²⁻ was well justified in terms of ICT and ESIPT due to deprotection of the DNB group. Probe BNPT displays a color change from colorless to greenish yellow upon addition of S²⁻, and thus can serve as a “naked-eye” probe for S²⁻. Therefore, BNPT can be used as a sensitive and selective ratiometric switch on fluorescence chemoreactant for S²⁻on the basis of fluorescence, absorption, and ¹H NMR spectroscopy, ESI mass spectrometry, and visual fluorescent color changes. The in situ generated [Zn-5] complex was further explored for its secondary sensing properties for the detection of cations resulting in a selective turn-off fluorescence response toward zinc among the allied cations. The structural, electronic, and emission properties of the probe BNPT and their reaction product have been demonstrated using the DFT and TDDFT computational calculations. Furthermore, we have also demonstrated the proof-of-concept experiments with some natural water samples, wherein the probe can be used for an evaluation of sulfide ion in real water samples. Cell imaging studies were carried out using BNPT for visualization of S²⁻ in living cells. Low cytotoxicity and excellent uptake by the cells make it a good chemoreactant for visualization of intracellular S²⁻ in living cells.

Experimental section

Materials and methods

Unless otherwise mentioned, materials were obtained from commercial suppliers and were used without further purification. ¹H and ¹³C NMR spectra were recorded on a Brucker 400 MHz instrument. For NMR spectra, DMSO-d₆ and CDCl₃ were used as solvent using TMS as an internal standard. Chemical shifts are expressed in δ ppm units and ¹H–¹H and ¹H–C coupling constants in Hz. Mass spectra were carried out using a Waters QTOF Micro YA 263 mass spectrometer. Fluorescence spectra were recorded on a Perkin Elmer Model LS 55 spectrophotometer. UV spectra were recorded on a JASCO V-530 spectrophotometer.

General method of UV-vis and fluorescence titration

For UV-vis and fluorescence titrations, the stock solution of probe BNPT was prepared (c = 10.0 μM) in aqueous solution HEPES buffer (pH 7.4, 10 mM) in CH₃CN–H₂O (3 : 2, v/v). The solution of the guest anions, cations and amines in the order of 1.0 × 10⁻⁴ ML⁻¹ was also prepared in aqueous solution HEPES buffer (pH 7.4, 10 mM) in CH₃CN–H₂O (3 : 2, v/v). The spectra of these solutions were recorded by means of UV-vis and the fluorescence methods. All the solvents were purchased from local suppliers and were distilled by the standard procedure before use.

Kinetic study

In kinetic studies of the probe for S²⁻ detection, the apparent rate constant k_obs for the reaction of BNPT with S²⁻ was determined by fitting the fluorescence intensities to the pseudo-first-order equation:

Ln[(F_max − F_t)/F_max] = −k_obst

where F_t and F_max are respectively the fluorescence intensities at 503 nm at a time t and the maximum value obtained after the reaction completed.

Cell imaging study

Human adult dermal fibroblast (HADF, Himedia Laboratories, India) cells were cultured in Dulbecco's modified eagle medium (DMEM, Himedia Laboratories, India) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% antibiotic–antimycotic solution (Himedia Laboratories, India) and seeded on a clean sterile cover slip placed in 24-well plates (Thermo Scientific, USA) with a serum-free medium and incubated for 24 h at 37 °C in a humidified CO₂ incubator. Two sets of cells were treated with 10⁻⁴ M receptor (BNPT) for 1 h. After washing with phosphate buffered saline (PBS), one set of treated cells were fixed with 4% paraformaldehyde in PBS (pH 7.4) for 15 minutes at room temperature, and another set of cells were treated with 10⁻² M sulfide in serum-free DMEM followed by 1 h incubation. The sulfide-treated cells were washed with PBS and fixed using 4% paraformaldehyde. Fluorescence microscopy was carried out under a Nikon Inverted microscope (Nikon eclipse TíU, Japan) equipped with a 20× (S Plan Fluor) objective. 465–495 nm excitation filters were used and images were captured through a 515–555 nm emission filter.

Synthesis of BNPT

Probe BNPT was synthesized by stirring 2-(3-benzothiazol-2-yl-4-hydroxy-phenyl)-4-methyl-thiazole-5-carboxylic acid ethyl ester (5) and 2,4-dinitrofluorobenzene in the presence of K₂CO₃, in dry DMF at 80 °C, to give probe BNPT in 72% yield (Scheme 2).

Scheme 2 Synthesis of BNPT.

Scheme 3 Schematic representation of ESIPT process of BNPT.

Experimental procedure

To a stirred solution of compound 5 [2-(3-benzothiazol-2-yl-4 hydroxyphenyl)-4-methylthiazole-5-carboxylic acid ethyl ester] (200 mg, 0.50 mmol) in DMF (5 mL) was added K₂CO₃ (698 mg, 5.05 mmol) followed by the addition of 1-fluoro-2,4-dinitrobenzene (282 mg, 1.51 mmol) at rt. Then the reaction mixture was heated at 80 °C for 5 h. TLC showed SM was consumed and formed a polar spot. The reaction mixture was cooled at rt and then diluted with ethyl acetate (50 mL). The combined organic solution was washed with cold water (10 mL × 3), dried over sodium sulfate and concentrated. The crude residue was purified by column chromatography over silica to afford desired compound BNPT (120 mg, 42%) [2-[3-benzothiazol-2-yl-4-(2,4-dinitrophenoxy)phenyl]-4-methylthiazole-5-carboxylic ethyl ester] as a yellow solid. ¹H NMR (400 MHz, d₆-DMSO) δ (ppm): 9.14 (s, 1H), 9.01 (s, 1H), 8.49 (d, J = 9.48 Hz, 1H), 8.26 (d, J = 8.76 Hz, 1H), 8.19–8.14 (m, 2H), 7.6–7.49 (m, 4H), 4.36 (q, J = 7.08 Hz, 2H), 2.71 (s, 3H), 1.33 (t, J = 6.96 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): 166.92, 161.90, 161.15, 159.62, 154.66, 152.41, 152.27, 142.21, 139.74, 135.51, 131.97, 130.04, 129.34, 128.97, 126.98, 126.57, 125.83, 123.52, 122.85, 122.20, 122.13, 121.41, 118.39, 61.37, 17.41, 14.23. MS (ESI): m/z calc. for C₂₆H₁₈N₄O₇S₂: 562.58; found: 563.3 [M + H]⁺.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

We gratefully acknowledge DST-SERB [File No. EMR/2016/006640] for financial support. SSA, SM, AKP, and RS thank the UGC-MANF, UGC, SERB-NPDF, and UGC-RGNF, New Delhi, for the financial and research support. Special thanks are given to C. Bodhak and R. Bag from Calcutta University for characterization support.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7nj03207b

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