MACA Fast and Efficient Method for Detecting H2O2 by a Dual-Locked Model Chemosensor.

A pentafluorobenzene-containing fluorescent probe GW-1 was designed and synthesized for monitoring hydrogen peroxide. The probe's fluorescence was activated by a dual-locked model system that consists of a spiro location and a target analyte, which avoids the "alkalizing effect." The smart GW-1 exhibited high selectivity toward hydrogen peroxide over other reactive oxygen species (ROS) by a dual-controlled molecular switch. These features are favorable for H2O2 sensing and pH changes in bioanalytical and biomedical applications.

Introduction

Hydrogen peroxide is one of the crucial eactive oxygen species (ROS) byproducts in living systems and has attracted intense interest recently in regulating important cellular biological processes, including cytotoxicity, cell growth, apoptosis, and proliferation.[1−6] Indeed, H2O2 is involved in signal transduction and intracellular oxidant levels in living organisms, which has been increasingly supported by new data.[7−11] Thus, determining H2O2 generation is of great importance for understanding the roles and biological function of H2O2 in biological systems.[12−17]

H2O2-mediated transformation of the boronic ester moiety to phenols, which has been established by the Chang’s group, is a general strategy for the optical detection of H2O2 over other biologically relevant ROS.[18−21] A series of fluorescent probes have been proposed for imaging of H2O2.[22−29] Unfortunately, the boronate moiety can still be patchy and unreliable in complex environments.

In this paper, we developed pentafluorobenzenesulfonyl indicator (GW-1) for selective detection of H2O2 with improved sensitivity and enhanced stability. The pentafluorobenzene ring enhances the reactivity of the sulfonates toward H2O2, which are more stable to hydrolysis than esters. The morpholine moiety is introduced into the molecular backbone by a dual-controlled molecular switch, which can induce fluorescence enhancement activated by H+ and H2O2. Moreover, our results highlight a strategy that the rate constants of the reactions were constructed faster than the boronate moiety in biological systems.

Experimental Section

Materials and Instruments

All chemicals were of analytical grade and were used as received without further purification. Water used in all experiments was doubly distilled. All chemicals were purchased from commercial suppliers such as Alfa Aesar (Tianjin), Sigma-Aldrich (Beijing), J&K (Guangzhou), Sangon (Shanghai) and used as received. All fluorescence spectra were recorded using an F-7000 fluorescence spectrophotometer (Hitachi), and the UV–vis absorption spectra were recorded using a U-3900H spectrophotometer (Hitachi). Electrospray ionization mass spectroscopy (ESI-MS) was performed on a Bruker Esquire 3000 plus mass spectrometer. The 1H NMR and 13C NMR spectra were recorded at 400 and 100 MHz, respectively, on a Bruker Advance-400 spectrometer using tetramethylsilane (TMS) as the internal standard. All of the measurements were operated at room temperature (25 °C) (Scheme ).

Scheme 1

Traditional Strategy and Dual-Controlled Molecular Switch Strategy for the Sensing of H2O2

Synthesis of GW-1

Conjugate GW-1 is composed of an oxyxanthene derivative, a 4-(2-aminoethyl) morpholine unit, a pentafluorobenzene ring as the H2O2 reactive site, an oxyxanthene moiety as a fluorophore whose fluorescence intensity changes upon H2O2 release, and rhodamine spirolactam functional groups cleavable by H2O2. Details for characterization of GW-1 is provided in the Supporting Information.

The synthesis routes of GW-1 are shown in Scheme . Compounds A was synthesized according to the literature methods.[25]

Scheme 2

Synthesis of GW-1

Synthesis of B

A suspension of A (240 mg, 0.38 mmol) and m-resorcinol (84 mg, 0.38 mmol) in trifluoroacetyl (TFA) (3 mL) was stirred at 90 °C for 16 h. The reaction mixture was cooled to room temperature and then poured in ice-cold water (15 mL). The precipitate was filtered and washed with brine (10 mL) and then dried under vacuum. The target compound was isolated by flash column chromatography on silica gel using CH2Cl2/EtOAc/MeOH (10:10:3, v/v/v) for elution. Yield 62 mg, 44%. 1H NMR (500 MHz, DMSO) δ 9.83 (s, 1H), 7.97 (d, J = 7.7 Hz, 2H), 7.61 (t, J = 7.4 Hz, 2H), 7.39 (d, J = 7.5 Hz, 2H), 6.80 (d, J = 9.1 Hz, 2H), 6.39 (d, J = 6.9 Hz, 2H), 1.37 (d, J = 7.0 Hz, 4H), 1.10 (t, J = 7.0 Hz, 6H). ESI-MS: m/z calcd for [C24H21NO4]+, 387.15; found, 388.2 (M)+.

Synthesis of C

Compound B (101 mg, 0.26 mmol) and 4-(2-aminoethyl)-morpholine (100 mg, 0.78 mmol) were dissolved in ethanol (10 mL), and the solution was refluxed under a N2 atmosphere for 10 h. After the solvent was removed under reduced pressure, the residue was purified by silica gel chromatography using CH2Cl2/MeOH (20:1, v/v) as the eluent to afford compound C as a faint yellow solid (96 mg, 59.2%). 1H NMR (500 MHz, DMSO) δ 12.70 (s, 1H), 7.99 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 6.0 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 9.1 Hz, 2H), 6.14 (d, J = 6.8 Hz, 2H), 3.36 (s, 2H), 3.17 (s, 2H), 3.13 (d, J = 6.7 Hz, 2H), 2.47 (s, 2H), 2.41 (d, J = 5.5 Hz, 2H), 2.34 (s, 2H), 2.32 (d, J = 6.8 Hz, 4H), 1.09 (s, 6H). ESI-MS: m/z calcd for [C30H33N3O4]+, 499.25; found, 500.3 (M)+.

Synthesis of GW-1

A mixture of C (100 mg, 0.2 mmol), pentafluorobenzenesulfonyl chloride (67 mg, 0.25 mmol), and Cs2CO3 (98 mg, 0.3 mmol) in N,N-dimethylformamide (DMF; 10 mL) was heated at 80°C for 8 h. After cooling, 30 mL of water was added into the mixture and extracted with CH2Cl2 (10 mL × 3). The organic solutions were combined, washed with water and brine, and dried with Na2SO4. The solvents were evaporated to give the crude product, which was purified by flash chromatography (silica gel, dichloromethane/0–10% methanol) to obtain the desired products as a pale solid (97 mg). Yield: 46%. 1H NMR (500 MHz, DMSO) δ 7.99 (d, J = 9.0 Hz, 2H), 7.78 (d, J = 6.0 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 6.78 (d, J = 9.1 Hz, 2H), 6.14 (d, J = 6.8 Hz, 2H), 3.36 (s, 2H), 3.17 (s, 2H), 3.13 (d, J = 6.7 Hz, 2H), 2.47 (s, 2H), 2.41 (d, J = 5.5 Hz, 2H), 2.34 (s, 2H), 2.32 (d, J = 6.8 Hz, 4H), 1.09 (s, 6H). ESI-MS: m/z calcd for [C36H32F5N3O6S]+, 729.19; found, 730.2 (M)+.

Results and Discussion

Spectroscopic Properties and Optical Responses to H2O2

To identify the performance of GW-1, the fluorescence response of the probe by the addition of a range of concentrations of H2O2 (0–200 μM) was investigated using 5.0 mM phosphate-buffered saline (PBS) buffer at pH 7.4. As shown in Figure , a remarkable fluorescence enhancement in the fluorescence intensity was observed at about 549 nm upon gradual addition of H2O2. This is attributed to the formation of a spirolactam structure of the probe in the solution. The results suggested that GW-1 can rapidly respond to H2O2, revealing that it sufficiently sensitive for the application in biological systems.

Figure 1

Fluorescence titration spectra of GW-1 (2 μM) with H2O2 (0–200 μM) in PBS buffer solution (λex = 475 nm).

Effect of pH

The pH response of GW-1 was also investigated in buffer solution at different pH values ranging from 2 to 10. As shown in Figure , the fluorescence of GW-1 was very weak in the pH range of 2.0–10.0, and with the addition of 200 μM H2O2, the fluorescence significantly enhanced at 549 nm under each pH due to the cleavage of the quenching group. On the one hand, under acidic conditions, the nucleophilic reaction ability was weak, and the quenching group would be difficult to leave. On the other hand, with the increase in alkalinity, the probe mainly exists in a stable spirolactam form. The results showed that the optimum pH of hydrogen peroxide is 6.0. Meanwhile, a pKa value of 3.9 was obtained using a sigmoidal curve by fluorimetric titration as a function of pH at 549 nm (Figure ). A good linear relationship was obtained between the fluorescence intensity and the pH values in the range of 3.0–5.0, and the linear equation was y = 189x – 206 (R2 = 0.97), indicating that GW-1 could detect the pH value quantitatively.

Figure 2

Fluorescence spectra of 2 μM of GW-1 to pH in the absence and the presence of 200 μM of H2O2 in the buffer solution.

Selectivity Studies of GW-1 to H2O2

To demonstrate whether GW-1 could specifically monitor H2O2 under biological conditions, we next investigated the selectivity of the probe toward other biologically related species using fluorescence spectroscopy, as shown in Figure . As expected, the addition of the other representative reactive oxygen species (HOCl, KO2, ONOO–, •OH, TBHP) induced very weak or almost no fluorescence response, while the addition of H2O2 triggered marked fluorescence enhancement. These data clearly indicate that GW-1 has a high selectivity toward H2O2 over the other species examined.

Figure 3

Fluorescence responses of 2 μM GW-1 to various analytes (200 μM) in phosphate buffer (100 mM, pH 6.0). λex = 475 nm.

Time Dependence in the Detection Process of H2O2

We further investigated the influence of the time-dependent modulations of the probe GW-1 when reacted with H2O2 under physiological conditions by monitoring the change of the fluorescent intensity (Figure ). Upon addition of H2O2, the emission intensity of the probe GW-1 at 549 nm increased dramatically until it reached a plateau in about 30 min at 37 °C in buffer. These results showed that the probe offered a 24-fold turn-on emission, indicating that the sensor can achieve real-time detection of H2O2 by the fluorescence signals under physiological conditions.

Figure 4

Kinetics of GW-1 (2 μM) and hydrogen peroxide (200 μM) in aqueous solution phosphate buffer (100 mM, pH 7.4).

Reaction Mechanism

The principle of sensing H2O2 by GW-1 for the optical changes is depicted in Scheme . As expected, with the addition of H2O2, the free GW-1 undergoes a nucleophilic addition reaction through the process described in Scheme , which generated a strong conjugate system after the cleavage of the quenching group and induced the fluorescence enhancement. To further investigate the sensing mechanism of GW-1, the mass spectrometry analysis indicated corroborative evidence for the formation of the GW-1 conjugate at m/z obsd 730.2 ([GW-1 + H]+, calcd 729.19 for C36H32F5N3O6S) implying the addition product (Figure S5).

Scheme 3

Schematic Illustration of the Reaction of GW-1 with H2O2

Conclusions

In summary, we have presented a platform to effectively measure H2O2 using a pentafluorobenzene-containing fluorescent probe GW-1. The pentafluorobenzene ring enhances the reactivity of the sulfonates for monitoring hydrogen peroxide, which are more stable to hydrolysis than are esters. The morpholine moiety is introduced into the molecular backbone by a dual-locked model system that consists of a spiro location and a target analyte, which can induce fluorescence enhancement activated by H+ and H2O2. These features are favorable for H2O2 sensing and pH changes in bioanalytical and biomedical applications.