English

• 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) .

  图 1

  

  图 1  Synthesis of probe 1 for detection of H₂S.

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

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  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) .

  图 1

  

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

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

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  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) .

  图 2

  

  图 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) . λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

  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) . λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

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  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].

  图 3

  

  图 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. λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

  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. λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

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  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.

  图 4

  

  图 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) . λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

  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) . λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

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  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.

  图 5

  

  图 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.

  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.

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  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.

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• 

  Figure 1  Synthesis of probe 1 for detection of H₂S.

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

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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) . λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

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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. λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

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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) . λ_ex = 310 nm, λ_em = 484 nm. Slits: 5/5 nm.

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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.

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•