An ESIPT based fluorescent probe for imaging hydrogen sulfide with a large turn-on fluorescence signal

Abstract

Through an intramolecular nucleophilic substitution reaction and ESIPT mechanism, we designed and synthesized a fluorescent hydrogen sulfide probe with a large turn on fluorescence signal (400-fold). The new probe exhibits high selectivity, good membrane-permeability and is suitable for the visualization of exogenous and endogenous hydrogen sulfide in living cells.

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Hydrogen sulfide (H₂S), well known for its pungent smell and noxious nature, is an important endogenous gasotransmitter, which is associated with diverse physiological processes.¹ It is clear that hydrogen sulfide now sits with nitric oxide and carbon monoxide as one of three gaseous mediators in biology. H₂S is endogenously produced by enzymes such as cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), cysteine aminotransferase (CAT) and 3-mercaptopyruvate sulphurtransferase (MST).² H₂S has both beneficial and detrimental sides for organisms. On the one hand, accumulating evidence has confirmed that hydrogen sulfide is involved in a vast number of physiological and pathological processes, such as ischemia reperfusion injury, vasodilation, apoptosis, insulin signaling, and oxygen sensing.³ On the other hand, overexposure to the corrosive and flammable gas can cause eye irritation, and inflammation in the respiratory tract.⁴ In the human body, abnormal H₂S levels have also been associated with various diseases including Alzheimer's disease,⁵ Down's syndrome,⁶ diabetes,⁷ and liver cirrhosis.⁸ Therefore, it is very necessary to develop an efficient method for sensitively and selectively probing H₂S in living systems.

Several analytical techniques including colorimetric,⁹ electrochemical assay,¹⁰ polarographic analysis,¹¹ and sulfide precipitation¹² have been devoted in detection of H₂S. Compared with those detection methods, fluorescence imaging has been widely used as a powerful tool for monitoring biomolecules within the context of living systems with high spatial and temporal resolution.¹³ Recently, some groups have made great progresses in the detection of H₂S in blood and in cells.¹⁴ Among them, various photomechanisms were used in the design of fluorescent H₂S probe, including internal charge transfer (ICT),^14e–h Förster resonance energy transfer (FRET),^14i–l and rhodamine close–open ring process.^14m,n Excited state intramolecular proton transfer (ESIPT) is also a significantly important fluorescent phenomenon, which generally involves a hydroxyl proton transfer to an acceptor in the excited state. In comparison with the other fluorescent processes, such as electron transfer and energy transfer, ESIPT process can occur at a much faster rate.¹⁵ Fluorescence sensing based on ESIPT is quite promising by virtue of low background signal in aqueous media, good photo-stability, intramolecular hydrogen-bonded property, and spectral sensitivity to the surrounding medium. Moreover, ESIPT dyes generally have large Stokes shift, which can reduce the interference from the excitation light.¹⁶

Among ESIPT dyes, the molecule 2-(2′-hydroxyphenyl)-benzothiazole (HBT) and its derivatives are a potentially interesting moiety as an fluorophore being widely used.¹⁷ In addition, azido group is well-known for sensitively and selectively responding to H₂S, based on the specific reduction of azide by H₂S to generate the corresponding amine.¹⁸

Herein, in this work, we employed the HBT chromophore as the signal reporter and azido group as the responding site for H₂S to construct a fluorescent H₂S probe (ESIPT-HS) based on ESIPT process. Compared with the previous reports,^17b,19 the probe ESIPT-HS possesses a large turn-on fluorescence signal enhancement (an approximately 400-fold) and could be used to imaging endogenous H₂S in living cells (as shown in Table S1†). This is mainly attributed to the probe with relatively low background fluorescence and high sensitivity to H₂S. We investigated its optical properties, response rate and selectivity toward various reactive sulphur, ROSs and reactive nitrogen species. The result suggests that it possesses high selectivity. Besides, the cell imaging confirmed that the probe ESIPT-HS can be used to monitor the level of H₂S in living cells.

Compound HBT and compound 1 were prepared according to a literature procedure.²⁰ The target compound ESIPT-HS was readily synthesized in one simple step. Treatment of compound 1 with DCC and DMAP in DCM and added HBT in room temperature afforded sensor ESIPT-HS in good yield (Scheme 1). The structure of the target compound synthesized was fully characterized by standard ¹H-NMR, ¹³C-NMR and mass spectrometry (see ESI†).

Scheme 1 Synthesis and response mechanism of the fluorescent probe ESIPT-HS.

With the probe ESIPT-HS in hand, we firstly evaluated the capability of ESIPT-HS to detect H₂S in aqueous buffer. The titration of Na₂S was carried out and employed to replace H₂S in this work. H₂S is a weak acid in aqueous solutions (pK_a1 = 7.04, pK_a2 = 11.96), equilibrating mainly with HS⁻ at physiological pH. For example, at pH 7.4 and temperature of 37 °C, 18.5% of free hydrogen sulfide exists as H₂S molecule and the remainder is almost all hydrosulfide anion (HS⁻) with a negligible contribution of S₂⁻.²¹ So, as the ion distribution of Na₂S is the same with that of H₂S in a neutral buffer solution. As shown in Fig. 1, ESIPT-HS has nearly no absorption in the visible region. However, with the addition of Na₂S to the solution, the absorption at 381 nm was gradually increased, as well as the absorption peak at 300 nm was also increased slightly. In good agreement with the absorption spectra, upon excitation at 410 nm, the free probe ESIPT-HS displays a weak fluorescent emission, owing to the inhabited of ESIPT process, and the fluorescence quantum yield of compound ESIPT-HS is only 0.00281. By contrast, addition of Na₂S renders the fluorescence intensity a drastic large (up to 400-fold) fluorescence increase in the emission of 462 nm was observed, owing to the cleavage reaction of ESIPT-HS would trigger by H₂S and release the fluorescent of HBT with a stronger ESITP emission. In this case the fluorescence quantum efficiency is as high as 0.265, after the probe response with Na₂S. Consistently, the visual emission color of the probe ESIPT-HS solution turned from no color to pale yellow and the fluorescent emission under a UV lamp with excitation of 365 nm could be observed by naked eyes and turned from no color to blue color (Fig. S1†). A linear calibration curve was obtained between the fluorescence signal at 462 nm and the concentration of Na₂S up to 60 μM, (Fig. S2†), which indicated that the chemodosimeter is suitable for quantitative determination of H₂S. In addition, the probe showed relatively high sensitivity to H₂S with a detection limit of 2.21 × 10⁻⁶ M under the experimental conditions, which is much lower than the physiological H₂S concentration in mammalian serum (30–100 μM) and in the brain (160 μM),²² (Fig. S3†), indicating that the probe ESIPT-HS has enough sensitive to determine the H₂S in physiological condition.

Fig. 1 The absorption (a) and fluorescence (b) spectral changes of probe ESIPT-HS (10 μM) upon addition of increasing concentrations of Na₂S (0–20 equiv.) (Ex = 410 nm) in 20 mM PBS buffer, pH 7.4, containing 30% DMSO as a cosolvent.

To shed light on the H₂S-triggered fluorescence turn-on response, we decided to characterize the reduced product. Incubation of ESIPT-HS with Na₂S afforded the product, which was isolated and characterized by standard ¹H-NMR (Fig. 2). Compared with the ¹H-NMR of HBT, we found that the ¹H-NMR spectra of the isolated compound is the same as that of HBT. From the above experimental results, we can deduce the recognition mechanism of the probe to H₂S. ESIPT-HS can react with H₂S to form the intermediate ESIPT-NH. However, ESIPT-NH is a NH₂ containing compound, which can spontaneously cyclize with ester groups to release the fluorophore HBT and the other by-product BP-N.

Fig. 2 (a) ¹H NMR spectrum of the compound HBT. (b) ¹H NMR spectrum of the isolated product of the probe ESIPT-HS reacted with Na₂S.

We then decided to examine the effect of pH on the fluorescence properties of ESIPT-HS and its response to H₂S. As shown in Fig. 3, the emission intensities of ESIPT-HS are quite low and do not change significantly over a wide ranges of pH from 1.5–10.5, indicating that the free probe was stable in the wide pH range. Upon addition of Na₂S, ESIPT-HS emits weak fluorescent emission in the acid environment range of pH from 1.5 to 6.4. However, with the increase of pH from 6.4 to 10.5, an enhancement trend is observed in ESIPT-HS fluorescence intensity of response to H₂S. We speculate that such results are mainly due to the pH effect on the fluorescence spectra of the reaction product HBT. As shown in Fig. S4,† the trend of fluorescence intensity of HBT at 462 nm with the change of pH value was consistent with the response of the probe to H₂S at in different pH test solution. ESIPT-HS displays a relatively strong fluorescence response at the pH range of 7.4–8.5, implying that the probe can be suitable to detect H₂S at a physiological pH conditions.

Fig. 3 Fluorescence intensity changes of the probe ESIPT-HS (10 μM) at different pH values in the absence () or presence () of Na₂S (50 μM) in PBS buffer (containing 30% DMSO as a cosolvent).

The time course of the fluorescence intensities of ESIPT-HS (10 μM) in the presence of H₂S in PBS buffer pH 7.4 is displayed in Fig. S5.† Notably, a drastic increase in fluorescence intensity was observed in 40 min, and a plateau was reached within 70 minutes in the presence of H₂S (50 μM). In spite of the speed of the probe response to H₂S is not very fast, but within few minutes the fluorescence enhancement ratio is enough to apply to cell imaging. Therefore, the results of timescale may allow ESIPT-HS to sense H₂S in real-time intracellular imaging.

Generally, realizing higher selectivity toward a specific analyte over other potential competing species is necessary for a fluorescence chemosensor. To examine the selectivity, the probe ESIPT-HS was treated with various biologically relevant analytes to establish a selectivity profile, such as anions, reactive oxygen species, small molecule thiols and Na₂S in PBS buffer pH 7.4 (containing 30% DMSO as a cosolvent). As exhibited in Fig. 4 and S6,† the representative species Br⁻; F⁻; H₂PO₄⁻; HPO₄²⁻; S₂O₃²⁻; SO₃²⁻; HSO₃⁻; ClO⁻; H₂O₂ were all prepared at 50 μM. Remarkably, all these representative interfering species induced no marked fluorescence response. However, upon addition of Na₂S a large fluorescence spectrum was observed, around 462 nm. Biological thiols (Cys, GSH, Hcy) exerted a negligible change on the fluorescence response for ESIPT-HS. These results suggest that the probe ESIPT-HS is highly selective for H₂S over other tested species. In this regard, ESIPT-HS can be considered as a good off–on chemosensor for specific recognition of H₂S.

Fig. 4 Fluorescence intensities of ESIPT-HS (10 μM) treated with various species (50 μM) in PBS buffer (pH 7.4, containing 30% DMSO as a cosolvent).

Encouraged by the above mentioned desirable properties of the spectroscopic data establishing that the probe can selectively respond to H₂S in aqueous solution with a large turn on fluorescence signal, we evaluated ESIPT-HS imaging assays of H₂S in live cells, and fluorescence imaging experiments were carried out in living cells (HeLa cells) on confocal laser scanning microscopy. The cytotoxicity of ESIPT-HS was examined toward HeLa cells by a MTT assay (Fig. S7†). The result indicates that the probe ESIPT-HS at the low micromolar concentrations has no marked cytotoxicity to the cells after a long period (>90% HeLa cells survived after 24 h with 30.0 μM ESIPT-HS incubation). Thus, the favorable properties of the probe include low cytotoxicity, which may render the probe ESIPT-HS suitable for imaging H₂S in living cells.

The utility of probe ESIPT-HS for fluorescence imaging of exogenous H₂S in living cells was investigated. As shown in Fig. 5, ESIPT-HS (5 μM) was initially incubated with HeLa cells for 20 min, and then rinsed three times by PBS buffer to remove excess of ESIPT-HS. The HeLa cells incubated with only the probe ESIPT-HS exhibited very weak fluorescence signal seen from the confocal imaging (Fig. 5b; λ_ex = 405 nm; λ_em = 425–475 nm). In contract, when incubated with 5.0 μM probe ESIPT-HS for 20 min and then treated with 50 μM Na₂S for another 30 min, HeLa cells displayed enhanced fluorescence in the blue channel at the same test conditions (Fig. 5e). These data established that the probe ESIPT-HS with suitable amphipathicity is cell membrane permeable and able to display a fluorescence turn-on response to H₂S in the living cells.

Fig. 5 Imaging of exogenous H₂S in HeLa cells stained with the probe ESIPT-HS (a) brightfield image of HeLa cells costained only with ESIPT-HS; (b) fluorescence images of (a) from blue channel; (c) overlay of (a) and (b); (d) brightfield image of HeLa cells costained with ESIPT-HS and treated with Na₂S; (e) fluorescence images of (d) from blue channel; (f) overlay of the brightfield image (d) and blue channel (e).

To obtain insight into the efficacy of the probe in the detection of endogenous H₂S, we determine to detect intrinsically biosynthesized H₂S inside the cells. Cysteine could be catalyzed by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) in living cells for H₂S production.²³ HeLa cells were treated with 10.0 μM ESIPT-HS and then stimulated with 200 μM cysteine for 2 hour. As shown in Fig. 6, the cells without stimulated with cysteine and loaded with only the probe ESIPT-HS (10.0 μM) displayed a weak fluorescence in the blue channel (Fig. 6b). However, the cells stimulated with cysteine and then treated with probe ESIPT-HS exhibit a dramatic enhancement in the blue channel (Fig. 6e). These results demonstrate that ESIPT-HS is capable of detecting not only exogenous H₂S in living cells, but also H₂S biologically produced by the cells.

Fig. 6 Imaging of endogenous H₂S in living cells. (a) Brightfield image of HeLa cells costained with ESIPT-HS; (b) fluorescence images of (a) from blue channel; (c) overlay of (a) and (b); (d) brightfield image of HeLa cells stimulated with cysteine (100 μM mL⁻¹) and costained with ESIPT-HS, (e) fluorescence images of (d) from blue channel; (f) overlay of the brightfield image (d) and blue channel (e).

In summary, the probe ESIPT-HS was developed fluorescent H₂S probe based on ESIPT process with a large turn-on fluorescence signal. The addition of H₂S to ESIPT-HS results in a large fluorescence enhancement at 462 nm because of the fluorescent of the probe triggered by H₂S and released the blue fluorescent of HBT with a stronger ESIPT-state emission. The probe exhibited favorable properties such as large turn on fluorescence signal, large Stokes shift, good selectivity and low cytotoxicity. Fluorescence imaging shows that ESIPT-HS was membrane-permeable and suitable for visualization of exogenous and endogenous H₂S in living cells.

Acknowledgements

This work was financially supported by NSFC (21172063, 21472067, 21502067), the Natural Science Foundation of Shandong Province, China (ZR2014BP001), Taishan Scholar Foundation (TS 201511041), and the startup fund of University of Jinan (160082101).

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Footnote

† Electronic supplementary information (ESI) available: Characterization data, and additional spectra. See DOI: 10.1039/c6ra12127f

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