A turn-on fluorescent probe for detection of hydrogen sulfide in aqueous solution and living cells

Abstract

We report the design and synthesis of a turn-on fluorescence probe FD-NO₂ for H₂S, utilizing H₂S selective reduction of the nitro group. FD-NO₂ displays good water solubility, negligible background fluorescence, high sensitivity, and high selectivity for H₂S over other anions, reactive sulfur, oxygen, and nitrogen species. Further confocal imaging experiments demonstrate that FD-NO₂ is capable of detecting H₂S in living cells.

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Introduction

Hydrogen sulfide (H₂S) has been considered as a poisonous gas for many years.¹ However, recent studies have demonstrated that H₂S is the third endogenous gasotransmitter, in addition to nitric oxide (NO) and carbon monoxide (CO).² At physiological concentrations, it is believed that H₂S plays important roles in a number of physiological and pathological processes, including neurotransmission,³ vasorelaxation,⁴ cardioprotection,⁵ angiogenesis,⁶ anti-inflammation,⁷ and insulin signaling.⁸ On the other hand, H₂S levels beyond the physiological range can lead to serious diseases such as Alzheimer's disease,⁹ Down's syndrome,¹⁰ diabetes,¹¹ and liver cirrhosis.¹² Although the important roles of H₂S have been recognized, the complex contributions of H₂S to both healthy and diseased states still remain unknown. Therefore, development of sensitive and selective methods for detection of H₂S in aqueous solution and biological systems is strongly desired.

Although traditional methods including colorimetry,¹³ electrochemical analysis,¹⁴ gas chromatography,¹⁵ and sulfide precipitation¹⁶ have been used for detection of H₂S, these methods often require sample preprocessing and destruction of cells or tissues,¹⁷ which limit their application in biological studies. In contrast, fluorescence imaging provides a powerful method to study biomolecules in living systems because of its simplicity, high sensitivity, real-time detection, and non-destructive detection of biological samples.¹⁸ To date, various fluorescent probes for H₂S detection have been reported. These probes were designed by taking advantage of several characteristic properties of H₂S, including the binding affinity towards copper ions,¹⁹ thiolysis of dinitrophenyl ether,²⁰ nucleophilicity,²¹ efficient reducing property towards selenoxide,²² and reduction of azide, hydroxy-amine and nitro to amine.²³ It is superior to design probes with the nitro group based on the reduction feature of H₂S, because the nitro group is a strong quencher for fluorophores²⁴ and can significantly reduce the background signal in biological assays. On the other hand, rhodol has excellent photophysical properties such as high fluorescence quantum yield, photostability, good water solubility and relatively long emission wavelength.²⁵ Hence, we present a novel rhodol-based fluorescent probe FD-NO₂ for detection of H₂S in aqueous solution and living cells (Scheme 1).

Scheme 1 Synthetic strategy for FD-NO₂ and proposed reaction mechanism for H₂S.

Results and discussion

The synthetic strategy for preparing FD-NO₂ is outlined in Scheme 1. Briefly, the synthesis of FD-NO₂ was started from fluorescein, which is first conjugated with triethylene glycol monomethyl ether to yield FD-Tg. The incorporating ethylene glycolic moiety to fluorescein is in favor of the solubility of the probe in water and penetration into the cellular membrane. FD-Tg was triflated to provide FD-Tf, which was coupled with bis(pinacolato)diboron in the presence of the Pd catalyst to obtain the pinacolatoboron derivative FD-B. FD-B was then hydrolyzed to boronic acid compound, and further reacted with tert-butyl nitrite to afford FD-NO₂. FD-NO₂ was characterized by ¹H and ¹³C NMR spectroscopy, and high resolution mass spectrometry.

Since the nitro group is a strong fluorescence quencher, we anticipate that the closed lactone form FD-NO₂ should be almost no fluorescence. In the presence of H₂S, the nitro group of FD-NO₂ was reduced to generate amine group, consequently leading to concomitant ring opening of the lactone so as to form the fluorescent product FA. We believe that this mechanism can be used to design a fluorescence turn-on probe for detecting H₂S. To corroborate this design strategy, FD-NO₂ was incubated with sodium sulphide (a H₂S precursor)²⁶ in methanol. The reaction solution was analyzed by fluorimetry and MALDI-TOF, respectively. The fluorescence experiment showed that a strong emission at 518 nm was detected (Fig. S1†), indicating the occurrence of reduction reaction and the generation of FA. On the other hand, a major mass peak m/z 478.1 detected by MALDI-TOF is assigned to the mass (M + H⁺) of the fluorescent conjugated product FA (Fig. S2†). The observations demonstrate that FD-NO₂ can detect H₂S by fluorescence enhancement. On the basis of the above results, the proposed reaction mechanism is shown in Scheme 1.

Next, we assessed the photophysical properties of FD-NO₂ under simulated physiological conditions (10 mM PBS, pH 7.4, 25 °C). As expected, FD-NO₂ (2 μM) has almost no fluorescence upon excitation at 480 nm. However, upon addition of H₂S (100 μM) for 50 min, the FD-NO₂ solution showed significant fluorescent emission, and the fluorescence intensity at 518 nm (Φ = 0.55) reached a plateau in 50 min with a 25-fold enhancement (Fig. 1).

Fig. 1 (a) Fluorescence response of 2 μM FD-NO₂ to 100 μM H₂S. Spectra were acquired every 5 min after H₂S was added. (b) Fluorescence changes as a function of time for the reaction of 2 μM FD-NO₂ to 100 μM H₂S. Data were acquired at 25 °C in PBS buffer (pH 7.4), λ_ex = 480 nm.

To evaluate the capability of the probe FD-NO₂ in the measurement of H₂S concentration, FD-NO₂ was treated with H₂S under various concentrations to obtain a standard curve of fluorescence intensity at 518 nm versus H₂S concentration. The concentration of the compound FD-NO₂ was maintained at 2 μM, while the concentrations of H₂S varied from 0 to 100 μM. As shown in Fig. 2, the fluorescent signal was indeed linearly related to the concentration of H₂S at this concentration range. The limit of detection (LOD) was determined to be as low as 1 μM (S/N = 3), indicating that our probe can potentially detect H₂S with high sensitivity. Moreover, the effect of FD-NO₂ concentration on the reaction was also studied, and the results showed very similar fluorescence response when the probe concentration is in the range of 2–10 μM (Fig. S6†).

Fig. 2 (a) Fluorescence spectra of FD-NO₂ (2 μM) upon addition of H₂S (0–100 μM). (b) Fluorescence intensities of FD-NO₂ at 518 nm as a function of the concentrations of H₂S in the range of 0–100 μM. Data were acquired at 25 °C in PBS buffer (pH 7.4), λ_ex = 480 nm.

To study the selectivity profile of FD-NO₂ for H₂S, various potential interferences including anions, reactive sulfur species (RSS), reactive oxygen species (ROS), and reactive nitrogen species (RNS), were tested in parallel under identical conditions. As shown in Fig. 3, the fluorescence intensity was increased by over 25-fold in the presence of H₂S (100 μM), while other anions (N₃⁻, AcO⁻, 1 mM), RSS (SCN⁻, SO₃²⁻, S₂O₃²⁻, 1 mM), ROS (H₂O₂, HClO, ^tBuOOH, O₂⁻, 1 mM), RNS (NO, NO₂⁻, NO₃⁻, 1 mM), and in particular different thiol compounds (lipoic acid, Cys, Hcy, GSH, 1 mM) only trigger minor changes. Thus, FD-NO₂ has an ideal selectivity towards H₂S over other anions, RSS, ROS, and RNS.

Fig. 3 Fluorescence responses of FD-NO₂ (2 μM) to H₂S (100 μM) and other anions, RSS, ROS, and RNS (1 mM). Bars represent fluorescence intensity ratios after (F) and before (F₀) addition of each anion, RSS, ROS, and RNS. Data were acquired at 25 °C in PBS buffer (pH 7.4), λ_ex = 480 nm.

To further verify whether the probe is suitable for biological applications, we examined the influence of pH in the range of 3–11. The results showed that the fluorescent intensity of FD-NO₂ in the absence of H₂S is hardly affected by the pH varying from 3 to 11. However, in the presence of H₂S, FA develops a turn-on fluorescence in the pH range between 6.5 and 10.5, with a strongest and constant response between pH 7.5 and 9.5 (Fig. S4†). These data confirm that FD-NO₂ is a very good candidate for detecting H₂S in living cells.

We next tested the ability of the probe FD-NO₂ to be used to visualize H₂S in living cells (Fig. 4). After the incubation of NIH 3T3 cells with 10 μM probe FD-NO₂ in serum-free DMEM for 30 min at 37 °C, the emission channel of 500–600 nm only exhibited faint fluorescence upon excitation at 488 nm. When we treated the FD-NO₂-loaded NIH 3T3 cells with H₂S (250 μM) in serum-free DMEM for another 30 min at 37 °C, the cells displayed obvious fluorescence enhancement. To further prove whether the fluorescence enhancement in the cells arises from H₂S, ZnCl₂ (an efficient H₂S scavenger)²⁶ was used to pretreat the NIH 3T3 cells. As expected, on addition of H₂S to the Zn²⁺-pretreated NIH 3T3 cells, no obvious fluorescence enhancement was observed. Therefore, we can conclude that fluorescence enhancement originated from the H₂S reduction of the nitro group and FD-NO₂ can be used to detect H₂S variations in living cells. Considering its good membrane permeability, high sensitivity and selectivity, we also anticipate that FD-NO₂ can contribute as a useful molecular probe for the investigations on H₂S-relevant biological processes.

Fig. 4 Fluorescent imaging of H₂S in FD-NO₂-labeled NIH 3T3 cells. (a) Cells were stained with 10 μM FD-NO₂ at 37 °C for 30 min in serum-free DMEM. (b) Cells were pretreated with FD-NO₂ (10 μM) at 37 °C for 30 min and then incubated with 250 μM H₂S at 37 °C for 30 min. (c) Cells were pretreated with 500 μM ZnCl₂ at 37 °C for 30 min, incubated with FD-NO₂ (10 μM) at 37 °C for 30 min and then incubated with 250 μM H₂S at 37 °C for 30 min. (d), (e) and (f) are the bright-field images of (a), (b) and (c), respectively. Emission intensities were collected in an optical window 500–600 nm, λ_ex = 488 nm, Scale bar: 20 μm.

Conclusions

In summary, we have developed a novel turn-on fluorescent probe, FD-NO₂, for the detection of H₂S based on H₂S-selective reduction of the nitro group. The probe exhibits significant turn-on fluorescence for H₂S and high selectivity for H₂S over other anions, reactive sulfur, oxygen, and nitrogen species in aqueous solutions. Moreover, we have also demonstrated that FD-NO₂ is suitable for imaging H₂S in living cells, providing a potentially efficient tool for detection of H₂S.

Experimental

General methods

All the chemicals and solvents were purchased from Alfa Aesar, J&K Chemical Ltd, or Beijing Chemical Reagents, and used as received with the following exceptions. Tetrahydrofuran (THF) and 1,4-dioxane were distilled from sodium. ¹H and ¹³C NMR spectra were measured on a Bruker Avance-400 400 MHz NMR spectrometer and referenced to solvent signals. MALDI-TOF and HRMS-ESI were measured on Autotlex III and Bruker Apex IV Fourier transform mass spectrometers respectively. 2 was prepared according to the literature.²⁷

Synthesis of compound FD-Tg. To a solution of NaOH (1.44 g, 36.0 mmol) in methanol (150 mL) was added fluorescein (6.00 g, 18.0 mmol). The mixture was stirred until all of the fluorescein dissolved at room temperature. The resulting solution was evaporated and the residue was dried under vacuum to give the disodium salt of fluorescein 1 (6.76 g, quantitative). The disodium salt 1 (6.7 g, 18.0 mmol) was suspended in DMF (150 mL) containing 2 (90 mmol). The reaction mixture was stirred at 90 °C for 36 h, the mixture was evaporated and diluted with 5% NaHCO₃ (60 mL) and extracted with ethyl acetate (3 × 150 mL). The combined extracts were washed with brine (50 mL), dried over Na₂SO₄, and evaporated to give the corresponding ester–ether 3 as a brown oil. This substance was dissolved in methanol (100 mL) and mixed with a 2 M aqueous solution of NaOH (30 mL). The resulting mixture was stirred for 2 h at room temperature, concentrated under vacuum and diluted with water (40 mL). The aqueous phase was acidified with concentrated HCl to pH about 2. The mixture was extracted with ethyl acetate (3 × 150 mL), and the combined extracts were washed with brine (50 mL), dried over Na₂SO₄, and evaporated to give the crude product FD-Tg, which was finally purified by silica gel column chromatography using ether–ethyl acetate as the eluent to afford the desired product (2.15 g, 25%). ¹H NMR (400 MHz, d₆-acetone, ppm) δ 8.98 (s, 1H), 8.00–7.98 (d, J = 7.6 Hz, 1H), 7.83–7.79 (t, J = 7.2 Hz, 1H), 7.76–7.72 (t, J = 7.6 Hz, 1H), 7.29–7.27 (d, J = 7.6 Hz, 1H), 6.89 (s, 1H), 6.77–6.76 (d, J = 2.4 Hz, 1H), 6.73–6.72 (d, J = 1.2 Hz, 2H), 6.68–6.66 (d, J = 8.8 Hz, 1H), 6.65–6.62 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 4.24–4.21 (t, J = 4.8 Hz, 2H), 3.85–3.83 (t, J = 4.8 Hz, 2H), 3.67–3.64 (m, 2H), 3.61–3.56 (m, 4H), 3.47–3.45 (m, 2H), 3.27 (s, 3H). ¹³C NMR (400 MHz, d₆-acetone, ppm) δ 169.47, 161.67, 160.31, 153.95, 153.27, 136.06, 130.76, 130.12, 129.91, 127.76, 125.38, 124.88, 113.40, 113.02, 112.58, 111.57, 103.36, 102.22, 83.48, 72.62, 71.42, 71.24, 71.08, 70.13, 68.83, 58.76. HRMS (ESI) for C₂₇H₂₇O₈⁺ ([M + H]⁺): calcd: 479.17004, found: 479.17003.

Synthesis of compound FD-Tf. FD-Tg (957 mg, 2.0 mmol) and N-phenylbis(trifluoromethanesulfonamide) (PhN(Tf)₂) (786 mg, 2.2 mmol) were dissolved in 10 mL of anhydrous THF. N,N-Diisopropylethylamine (DIEA) (0.4 mL, 2.4 mmol) was added via a syringe, and the resulting solution was stirred at room temperature overnight under an argon atmosphere. The reaction mixture was dried by rotary evaporation, and purified by silica gel column chromatography using ether–ethyl acetate as the eluent to afford the desired product (793.8 mg, 65%). ¹H NMR (400 MHz, d₆-acetone, ppm) δ 8.05–8.03 (d, J = 7.6 Hz, 1H), 7.86–7.83 (m, 1H), 7.80–7.77 (t, J = 7.6 Hz, 1H), 7.54–7.53 (d, J = 2.4 Hz, 1H), 7.39–7.38 (d, J = 7.6 Hz, 1H), 7.26–7.23 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H), 7.14–7.12 (d, J = 8.8 Hz, 1H), 6.97 (s, 1H), 6.81 (s, 2H), 4.26–4.24 (t, J = 4.4 Hz, 2H), 3.86–3.84 (t, J = 4.8 Hz, 2H), 3.67–3.65 (m, 2H), 3.61–3.56 (m, 4H), 3.47–3.45 (m, 2H), 3.27 (s, 3H). ¹³C NMR (400 MHz, d₆-acetone, ppm) δ 169.10, 162.05, 153.49, 152.86, 152.66, 150.99, 136.50, 131.44, 131.26, 129.96, 127.19, 125.77, 124.93, 121.40, 117.97, 113.92, 111.87, 111.37, 102.41, 81.87, 72.65, 71.45, 71.27, 71.10, 70.12, 69.00, 58.77. MALDI-TOF for C₂₈H₂₆F₃O₁₀S⁺ ([M + H]⁺): calcd: 611.1, found: 611.0.

Synthesis of compound FD-B. FD-Tf (610.5 mg, 1.0 mmol), bis(pinacolato)diboron (380.8 mg, 1.5 mmol), potassium acetate (294.4 mg, 3.0 mmol), and PdCl₂(dppf)CH₂Cl₂ (81.6 mg, 0.1 mmol) were dissolved in 10 mL of anhydrous 1,4-dioxane. The resulting solution was stirred at 80 °C overnight under an argon atmosphere. The reaction mixture was dried by rotary evaporation, and purified by silica gel column chromatography using ether–ethyl acetate as the eluent to afford the desired product (352.9 mg, 60%). ¹H NMR (400 MHz, d₆-acetone, ppm) δ 8.03–8.01 (d, J = 7.6 Hz, 1H), 7.83–7.79 (m, 1H), 7.77–7.74 (m, 1H), 7.66 (s, 1H), 7.47–7.46 (m, 1H), 7.30–7.28 (d, J = 7.2 Hz, 1H), 6.94–6.93 (d, J = 2.0 Hz, 1H), 6.91–6.89 (d, J = 8.0 Hz, 1H), 6.80–6.78 (d, J = 8.8 Hz, 1H), 6.77–6.74 (m, 1H), 4.25–4.22 (t, J = 4.8 Hz, 2H), 3.86–3.84 (t, J = 4.8 Hz, 2H), 3.67–3.65 (m, 2H), 3.61–3.56 (m, 4H), 3.47–3.45 (m, 2H), 3.27 (s, 3H), 1.35 (s, 12H). ¹³C NMR (400 MHz, d₆-acetone, ppm) δ 169.41, 161.92, 154.14, 153.09, 151.56, 136.27, 130.99, 130.31, 129.92, 128.29, 127.21, 125.61, 124.84, 123.77, 123.07, 113.27, 112.15, 102.39, 85.05, 82.64, 72.67, 71.47, 71.28, 71.12, 70.16, 68.92, 58.79, 25.16. MALDI-TOF for C₃₃H₃₈BO₉⁺ ([M + H]⁺): calcd: 589.3, found: 589.1.

Synthesis of compound FD-NO₂. FD-B (294.2 mg, 0.5 mmol) was dissolved in 10 mL THF–H₂O (v/v = 1 : 1). NaIO₄ (534.7 mg, 2.5 mmol) was added, and the resulting solution was stirred at room temperature for 2 h. Then 1 M HCl (20 mL) was added, and stirred at room temperature for 5 h. The mixture was extracted with ethyl acetate (3 × 20 mL), and the combined extracts were washed with brine (20 mL), dried over Na₂SO₄, and evaporated to give the crude product 4 which was used without further purification. The crude product 4 was dried in vacuo, then 5 mL anhydrous 1,4-dioxane and tert-butyl nitrite (0.6 mL, 5.0 mmol) were added, and the resulting solution was heated at 80 °C overnight. The reaction mixture was dried by rotary evaporation, and purified by silica gel column chromatography using ether–ethyl acetate as the eluent to afford the desired product (50.7 mg, 20%). ¹H NMR (400 MHz, d₆-acetone, ppm) δ 8.18–8.17 (d, J = 2.0 Hz, 1H), 8.07–8.05 (d, J = 7.6 Hz, 1H), 7.98–7.95 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H), 7.87–7.83 (m, 1H), 7.81–7.77 (m, 1H), 7.39–7.37 (d, J = 7.6 Hz, 1H), 7.24–7.22 (d, J = 8.8 Hz, 1H), 7.01–7.00 (d, J = 2.0 Hz, 1H), 6.86–6.83 (m, 2H), 4.27–4.25 (t, J = 4.4 Hz, 2H), 3.87–3.85 (m, 2H), 3.67–3.65 (m, 2H), 3.61–3.56 (m, 4H), 3.47–3.45 (m, 2H), 3.27 (s, 3H). ¹³C NMR (400 MHz, d₆-acetone, ppm) δ 169.12, 162.20, 153.57, 152.72, 152.18, 150.00, 136.62, 131.42, 130.72, 129.95, 126.93, 126.89, 125.92, 124.92, 119.10, 114.14, 113.42, 111.63, 102.50, 81.64, 72.68, 71.49, 71.29, 71.13, 70.15, 69.08, 58.80. HRMS (ESI) for C₂₇H₂₆NO₉⁺ ([M + H]⁺): calcd: 508.16021, found: 508.15946.

Acknowledgements

We thank the National Natural Science Foundation of China (21102148 and 21125205), National Basic Research Program of China (2011CB935800), and the State Key Laboratory of Fine Chemicals, Department of Chemical Engineering, Dalian University of Technology for financial supports.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Characterization of compounds and additional spectroscopic data. See DOI: 10.1039/c4qo00048j

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