Chinese Chemical Letters  2016, Vol. 27 Issue (12): 1793-1796   PDF    
Cascade reaction-based fluorescent probe for detection of H2S with the assistance of CTAB micelles
Hai-Rong Zhenga,c, Li-Ya Niua,b, Yu-Zhe Chena, Li-Zhu Wua, Chen-Ho Tunga, Qing-Zheng Yangb     
a Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing 100190, China ;
b College of Chemistry, Beijing Normal University, Beijing 100875, China ;
c University of the Chinese Academy of Sciences, Beijing 100049, China
Abstract: We report a turn-on fluorescent probe for H2S through a cascade reaction using a new trap group 4-(bromomethyl)benzoate, based on excited-state intramolecular proton transfer (ESIPT) sensing mechanism. The probe showed good selectivity and high sensitivity towards H2S and it was capable of detecting and imaging H2S in living HeLa cells, indicating its potential biological applications.
Key words: Hydrogen sulfide     Fluorescent probe     Cascade reaction     ESIPT     Micelles    
1. Introduction

For centuries, hydrogen sulfide has been recognized as a toxic molecular. When exposed to this colorless, flammable gas, which has a distinctive smell of rotten eggs, people may suffer from respiratory failure, loss of consciousness, sudden cardiac death, hepatic and olfactory paralysis. However, more recent studies have challenged this traditional view. H2S is now identified as the third biological signaling molecular besides nitric oxide (NO) and carbon monoxide (CO) and plays important roles in maintaining normal physiology [1]. Endogenous H2S can be produced from sulfur- containing biomolecules such as cysteine and homocysteine, which is catalyzed by cystathionine beta synthase (CBS) , cysteine aminotransferase and mercaptopyruvate sulfurtransferase (CAT/ MST) [2, 3]. These enzymes are widely spread in human tissues ranging from the heart and vasculature, brain, kidney, liver, lungs, indicating the important physiological roles of H2S in the body. Besides, H2S can also be produced from non-enzymatic processes, including release from sulfur stores and metabolism of polysulfide [4, 5]. The abnormal level of H2S can resulted in many diseases, such as Alzheimer's disease, Down's syndrome, diabetes, and liver cirrhosis [6-9]. Therefore, methods for monitoring the production, trafficking, and consumption of H2S in living systems are highly desired.

Compared with the traditional methods including colorimetric assays [10, 11] and gas chromatography[12, 13], fluorescent probes present lots of advantages, such as rapid response, high sensitivity and excellent selectivity [14-16]. Recently, several fluorescent probes have been reported forthe detection ofH2S in living systems [17-20]. Common strategies include: H2S mediated reduction of azide to amine [21, 22], H2S trapped by nucleophilic addition [23-25], copper sulfide precipitation [26, 27], and thiolysis of dinitro- phenyl ether [28, 29]. Among these, H2S trapped by nucleophilic addition strategy has been widely applied. Fluorescent probes based on this strategy usually take advantage of the dual nucleophilicity of H2S. Such probes usually contain a H2S trap group with two electrophilic reaction sites and a fluorescent reporter, which discriminates the H2S from other biothiols.

Recently, excited state intramolecular proton transfer (ESIPT) compounds have been widely used in designing sensors for anions [30, 31], cations [32, 33], amino acids [34-36] and small neutral molecules [37, 38], because of their intrinsic properties, ultra-fast reaction rate and huge bathochromic shift in the emission signal.

Herein, we reported a fluorescent probe using 2-2 (hyroxyphe- nyl) benzothiazole (HBT) , a typical ESIPT molecule, as fluorescent reporter, and 4- (bromomethyl) benzoate as trap group, which showed fast response to H2S through cascade reaction. As the hydroxyl group was protected by 2- (bromomethyl) benzoate, the HBT moiety of the probe showed enol-like fluorescence. The initial nucleophilic attack of H2S towards bromomethyl group would lead to an intermediate thiol, which was followed by a cyclization cascade reaction towards the adjacent ester carbonyl to release the HBT with keto emission. Thus, the detection of H2S was realized.

2. Experimental

Probe 1 was synthesized using a simple procedure with HBT and 2- (bromomethyl) benzoic acid as starting materials, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) as a coupling reagent, and 4-dimethylaminopyridine (DMAP) as catalyst (Scheme 1) .

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Scheme. 1. Synthesis of probe 1 for detection of H2S.

2- (2'-Hydroxy-3'-methoxyphenyl) benzothiazole (HMBT) : A solution of 2-aminothiophenol (0.3 mL, 4.2 mmol) and o-vanillin (0.48 g, 3.15 mmol) in EtOH (10 mL) , aq. H2O2 (30%, 18.9 mmol) and aq HCl (37%, 9.45 mmol) was stirred at rt for 90 min. The solution was quenched by 10 mL H2O. The precipitate was filtered, dried under vacuum and recrystallized from EtOH to afford the desired product as a light brown solid (0.64 g, 79% yield) . 1H NMR (CDCl3, 400 MHz, ppm) : d 12.75 (s, 1H) , 8.01 (d, 1H, J = 7.6 Hz) , 7.91 (d, 1H, J = 7.2 Hz) , 7.51 (t, 1H, J = 6.8 Hz) , 7.42 (t, 1H, J = 7.2 Hz) , 7.33 (dd, 1H, J1 = 1.2Hz, J2 = 8.0Hz) , 6.99 (dd, 1H, J1 = 1.2Hz, J2 = 8.0Hz) , 6.91 (t, 1H, J =8.0 Hz) , 3.96 (s, 1H) .

Probe 1: To a solution of EDCI (0.346 g, 1.5 mmol) in CH2Cl2 (20 mL) was added 2-bromodomethylbenzoic acid (0.312 g, 1.5 mmol) followed by DMAP (0.012 g, 0.1 mmol) and HMBT (0.346 g, 1 mmol) . The mixture was stirred at room temperature for 12 h and then filtered, washed with water. The filtrate was concentrated to afford the crude product, then purified by silica gel column chromatography (PE:CH2Cl2 = 1:1) to afford compound 1 as a white solid (0.24 g, 40%) . 1H NMR (400 MHz, CDCl3, ppm) : d 8.41 (d, 1H, J = 4.0 Hz) , 7.93-7.99 (m, 2H) , 7.83 (d, 1H, J = 4.0 Hz) , 7.34-7.56 (m, 6H) , 7.15 (d, 1H, J = 4.0 Hz, ) , 4.94-5.14 (d, 2H) , 3.90 (s, 3H) . 13C NMR (100 MHz, CDCl3) : 164.2, 162.3, 153.0, 152.1, 140.0, 139.8, 138.1, 135.5, 134.0, 133.3, 132.3, 132.0, 130.9, 129.0, 128.7.128.5.128.2.127.7.126.9.126.3.125.8.125.3.123.4.122.1, 121.1, 121.5, 121.4, 114.2, 69.6, 56.4, 44.1, 31.0. HRMS: calcd.: 454.010, found: 454.011.

3. Results and discussion

The proposed sensing mechanism is shown in Fig. 1. As the hydroxyl group is protected by 2- (bromomethyl) benzoate, the excited state intramolecular proton transfer (ESIPT) process was forbidden. As a result, the HBT moiety of probe 1 shows enol-like fluorescence. The initial nucleophilic attack of H2S towards bromomethyl group would lead to an intermediate thiol, which is followed by a cyclization cascade reaction towards the adjacent ester carbonyl to release the HBT. Upon irradiation, the resulting HBT generated the excited state intramolecular proton transfer (ESIPT) tautomer, which shows keto emission. To identify the proposed sensing mechanism, probe 1 was treated with excess NaHS and Et3N in CH3CN. After the reaction, the HBT was released, with the formation of cyclization product 4. All products were separated and confirmed with 1H NMR (Figs. S1 and S2 in Supporting information) .

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Figure 1. The proposed sensing mechanism of probe 1 for H2S.

We first tested the absorption spectra of probe 1 in HEPES buffer. However, the obvious variation of the absorption spectra of 1 in 12 h suggested that it was unstable in HEPES buffer, probably due to its poor solubility in pure water (Fig. S3 in Supporting information) . Then we used 50% ethanol as co-solvent, and the stability of 1 was improved (Fig. S4 in Supporting information) . As shown in Fig. S5 in Supporting information, with the addition of 200 mmol/L NaHS in ethanol/HEPES buffer (1:1, v/v, 20 mmol/L, pH 7.4) , the emission at 350 nm (which is attributed to the enol form) decreased, followed by a new peak appearing at 484 nm (which is attributed to the keto form) . However, the fluorescence at 484 nm was quite weak, as the intramolecular hydrogen bond is strongly disturbed in polar solvents [39]. We speculated that the ESIPT would be enhanced in the nonpolar core of CTAB micelles. Probe 1 was stable in HEPES buffer (20 mmol/L, pH 7.4) containing 1 mmol/L CTAB (Fig. S6 in Supporting information) . As we expected, upon addition of NaSH to probe 1, the original emission at 350 nm decreased, and a significant fluorescence enhancement at 484 nm of approximately 60-fold was observed (Fig. 2) . In contrast, an enhancement of only 5-fold was observed in ethanol/ HEPES buffer (1:1, v/v, 20 mmol/L, pH 7.4) (Fig. S5) . The results indicated that ESIPT process was greatly enhanced with the assistance of CTAB micelles. Therefore, the following studies were carried out in HEPES buffer (20 mmol/L, pH 7.4, containing 1 mmol/L CTAB) . The probe 1 (10 mmol/L) upon reaction with NaHS (2 mmol/L) in HEPES buffer (20 mmol/L, pH 7.4, containing 1 mmol/L CTAB) exhibited a pseudo first order reaction kinetics with rate constant k = 0.59 min-1 (t1/2 = 1.17 min) , indicating the fast response of 1 towards H2S (Fig. S7 in Supporting information) .

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Figure 2. Time-dependent fluorescence response of probe 1 (10 mmol/L) upon addition of NaHS (200 mmol/L) in HEPES buffer (20 mmol/L, pH 7.4, containing 1 mmol/L CTAB) . λex = 310 nm, λem = 484 nm. Slits: 5/5 nm.

Subsequently, quantitative response of probe 1 in HEPES buffer (20 mmol/L, pH 7.4, containing 1 mmol/L CTAB) towards H2S was estimated. As shown in Fig. 3a, with the increasing concentrations of NaHS, the original emission at 350 nm decreased, and a significant fluorescence enhancement at 484 nm was observed. Moreover, the fluorescence intensity of 484 nm showed linear relationship with NaHS concentrations ranging from 0 to 100 mmol/L, suggesting the potential application for quantitative determination of H2S (Fig. 3b) . The detection limit for HS- was estimated to be 0.50 mmol/L (S/N = 3) , which is much lower than the concentration required to cause physiological response [40].

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Figure 3. (a) Fluorescence spectra of probe 1 (10 mmol/L) upon addition of NaHS (0-400 mmol/L) in HEPES buffer (20 mmol/L, pH 7.4, containing 1 mmol/L CTAB) . (b) Fluorescence response of probe 1 at 484 nm to NaHS concentration (0-100 mmol/L) . Spectra were recorded after incubation with different concentrations of NaHS for 1 h. λex = 310 nm, λem = 484 nm. Slits: 5/5 nm.

To evaluate the selectivity of the probe to H2S, emission spectra changes upon addition of 20 equivalents of different interfering species, such as cysteine (Cys) , glutathione (GSH) , homocysteine (Hcy) , sodium acetate (CH3COONa) , sodium fluoride (NaF) , sodium persulfate (Na2S2O4) , sodium dihydrogen phosphate (NaH2PO4) , sodium sulfate (Na2SO4) , sodium chloride (NaCl) , sodium tartrate (C4H4Na2O6) , sodium gluconate (C6H11NaO7) , sodium carbonate (Na2CO3) , sodium bicarbonate (NaHCO3) , sodium nitrate (NaNO3) , sodium bromide (NaBr) , sodium hydrogen sulfite (NaHSO3) were studied. As shown in Fig. 4, in most cases, little change in emission intensity was observed. In contrast, a great enhancement in emission intensity was only observed when adding NaHS. Thus, these results demonstrated that probe 1 has a high selectivity towards H2S.

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Figure 4. Fluorescence response of probe 1 (10 mmol/L) upon addition of various species in HEPES buffer (20 mmol/L, pH 7.4, containing 1 mmol/L CTAB) . λex = 310 nm, λem = 484 nm. Slits: 5/5 nm.

We further examined the capability of probe 1 to H2S in living cells (Fig. 5) . After incubated with probe 1 (20 mmol/L) for 45 min in culture medium, HeLa cells showed almost no fluorescence in green channel. By contrast, when HeLa cells were pretreated with NaHS (200 mmol/L) before incubated with probe 1 (20 mmol/L) for 45 min, the obvious fluorescence was observed. It revealed that probe 1 has potential for visualizing H2S levels in living cells.

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Figure 5. Images of H2S in HeLa cells using probe 1 (20 mmol/L) at 37 ℃. (a) Fluorescence and (b) bright-field images of HeLa cells incubated with probe 1 for 45 min. (c) Fluorescence and (d) bright-field images of HeLa cells incubated with NaHS (200 mmol/L) for 30 min and further incubated with probe 1 for 45 min. Scale bar: 50 mm.

4. Conclusion

In conclusion, we designed and synthesized a novel fluorescent probe 1 for the detection of H2S based on ESIPT mechanism with the assistance of CTAB. As the hydroxyl group is protected by 2- (bromomethyl) benzoate, probe 1 showed weak enol-like fluorescence at 350 nm. The 2- (bromomethyl) benzoate group showed fast response to H2S through cascade reaction and released free HBT, which showed strong fluorescence at 484 nm in CTAB micelles. The emission at 484 nm showed linear relationship with the H2S concentration at 0-100 mmol/Lwith the detection limit of 0.50 mmol/L. The high selectivity and sensitivity of our probe to H2S may give new insights for the development of fluorescent probe to selectively detect H2S in biological systems.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in theonlineversion, at http://dx.doi.org/10.1016/j.cclet.2016.04.023.

Acknowledgments We thank Prof. Z. Niu and Dr. Y. Tian for providing HeLa cells. This work was financially supported by the 973 Program (No. 2013CB933800),National Natural Science Foundation of China (Nos. 21525206,21402216,21272243),the Fundamental Research Funds for the Central Universities and Beijing Municipal Commission of Education.
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