A highly selective hydrogen sulfide ratio fluorescence probe based on boron dipyrromethene dye and its preparation and application

Abstract

The present invention discloses a novel boron-dipyrromethene-based ratiometric fluorescent probe for detecting hydrogen sulfide, and its molecular structural formula is shown as formula (I). Based on boron-dipyrromethene dyes, the present invention constructs a ratiometric fluorescent probe for hydrogen sulfide by means of a design strategy of enhancing molecular conjugation through consecutive nucleophilic reactions. This probe selectively recognizes hydrogen sulfide. The fluorescence emission intensity of the probe gradually increases at 594 nm and gradually decreases at 542 nm. The intensity ratio of the two shows a linear relationship with the concentration of hydrogen sulfide within a certain range, and it can achieve dual-channel ratiometric fluorescence imaging of hydrogen sulfide in cells.

Description

Technical Field

[0001] The present invention relates to a ratio fluorescence sensing and its application, in particular to the preparation and application of a highly selective hydrogen sulfide ratio fluorescence probe based on fluorinated BODIPY, belonging to the technical field of organic fluorescence sensing. Background Art

[0002] Hydrogen sulfide (H2S) is the third gaseous neurotransmitter after nitric oxide (NO) and carbon monoxide (CO), and has received more attention due to its multiple characteristics in biological progress. Intracellular H2S is produced by enzymes such as cysteine γ-lyase (CSE) and cysteine β-synthase (CBS) catalyzed by sulfur-containing amino acids, and further plays an important role in regulating various physiological activities such as vascular tone, neural regulation, inflammation and apoptosis. Abnormal H2S concentration is usually associated with various diseases, including diabetes, Alzheimer's disease and cirrhosis. Therefore, monitoring the intracellular H2S level is of great significance for thoroughly understanding the functional role of H2S in living cells.

[0003] Fluorescent probes are one of the most powerful detection methods in biological systems due to their high sensitivity, high selectivity and high spatiotemporal resolution (Chem. Soc. Rev. 2015, 44, 6143–6160). Among many fluorophores, fluorinated boron dipyrrole (BODIPY) dyes have been widely used in fluorescence sensing and imaging due to their high absorption coefficient, excellent fluorescence quantum yield and high photostability. So far, many BODIPY-based fluorescent probes have been used for the monitoring and imaging of endogenous hydrogen sulfide in living cells, and have high sensitivity and low detection limits. However, most of these reported fluorescent probes detect hydrogen sulfide through the detection mode of fluorescence intensity enhancement or weakening, but there are also some detection problems. For example, the change in single fluorescence intensity is also easily affected by environmental factors, excitation light sources and probe concentrations, resulting in detection errors. Ratio fluorescence probes detect the concentration change of hydrogen sulfide through the ratio of fluorescence intensities at two channel wavelengths, thus showing better detection accuracy and avoiding systematic errors in biological imaging. In addition, the design strategies of H2S fluorescent probes are mainly the reaction of the probe with hydrogen sulfide, including nucleophilic reaction, Michael addition reaction, reduction of nitro or azide, metal ion complex type, etc. Among them, the nucleophilic addition reaction of hydrogen sulfide with the probe realizes fluorescence recovery by changing the intramolecular charge transfer of the probe or blocking the photoinduced electron transfer process. However, this nucleophilic reaction is easily interfered by cellular reactive sulfur sources (cysteine and glutathione), resulting in poor sensing selectivity. Therefore, it is necessary to develop a fluorescent probe based on fluorinated boron dipyrrole dye, which has a ratio fluorescence intensity change for hydrogen sulfide and excellent detection selectivity. Summary of the Invention

[0004] The present invention provides a fluoroboron dipyrrole-based ratiometric fluorescence probe, a preparation method thereof, and a technique for highly efficient fluorescence detection of hydrogen sulfide.

[0005] The chemical structural formula of the ratiometric fluorescence probe described in the present invention is shown in Formula (I):

[0006]

[0007] The fluoroboron dipyrrole fluorescence probe of the present invention undergoes consecutive nucleophilic substitution reactions with hydrogen sulfide, resulting in enhanced molecular conjugation and a red shift in the fluorescence emission spectrum, thereby enabling highly selective and accurate recognition and detection of hydrogen sulfide. In the test system of DMSO / PBS buffer solution (v / v, 1 / 1, pH 7.0), the probe has strong green fluorescence at 542 nm. After reacting with hydrogen sulfide, the fluorescence emission red-shifts to 594 nm, achieving dual-wavelength ratiometric fluorescence detection, and having strong detection selectivity and detection sensitivity, and can be used for naked-eye qualitative identification and fluorescence quantitative detection.

[0008] The present invention also provides a preparation method of the ratiometric fluorescence probe, comprising the following steps:

[0009] According to the method for synthesizing asymmetric fluoroboron dipyrrole, 2,4-dimethyl-3-ethylpyrrole and 5-chloro-2-formylpyrrole are synthesized under the action of trifluoroacetic acid and boron trifluoride diethyl ether / triethylamine to obtain the 3-chloro-substituted fluoroboron dipyrrole compound 2. This compound reacts in iodine monochloride to obtain compound 3. Finally, compound 3 undergoes Suzuki coupling reaction with 2-formylphenylboronic acid under a Pd catalyst. After the reaction is completed, the ratiometric fluorescence probe is obtained through post-treatment.

[0010] The chemical reaction formula for the preparation is as follows

[0011]

[0012] The present invention also provides an application of the novel ratiometric fluorescence probe in the spectral response to NaHS and cell imaging.

[0013] The ratiometric fluorescence probe described in the present invention exhibits a maximum emission peak of fluorescence at 542 nm in DMSO / PBS (v / v, 1:1, pH 7.4). After adding NaHS and undergoing consecutive nucleophilic substitution reactions, the absorption and emission spectra of the probe are significantly red-shifted. At this time, the fluorescence at 542 nm weakens, and fluorescence with a maximum emission of 594 nm is generated, thereby achieving ratiometric fluorescence detection of hydrogen sulfide, as Figure 3 and 4 shown, where the fluorescence intensity ratio of the probe (I 594nm / I 542nm) It shows a linear relationship change within the concentration range of NaHS (0 - 75 μM), indicating that the fluorescence described in the present invention can quantitatively detect the concentration of hydrogen sulfide, as Figure 5 shown.

[0014] The ratiometric fluorescence probe described in the present invention is applied to the detection and imaging of hydrogen sulfide in living cells. After the probe (5 μM) is incubated in HeLa cells for 30 minutes, there is a strong fluorescence signal in the green channel (510 - 560 nm), while there is almost no signal in the red channel (570 - 630 nm). When NaHS (300 μM) is added to the cell culture medium and incubated for 60 minutes, the fluorescence signal in the green channel weakens, while the emission signal in the red channel increases significantly, indicating that the probe can monitor hydrogen sulfide in living cells through the change of dual-channel fluorescence signals, as Figure 8 shown.

[0015] The beneficial effects of the present invention are as follows: The ratiometric fluorescence probe can highly selectively detect hydrogen sulfide. As the concentration of hydrogen sulfide increases, the fluorescence emission intensity of the probe gradually increases at 594 nm and gradually decreases at 542 nm; the fluorescence ratio of the two shows a linear relationship with the concentration of hydrogen sulfide within a certain range. The ratiometric fluorescence probe not only has excellent selectivity and sensitivity, but also can be used for dual-channel ratiometric fluorescence imaging of hydrogen sulfide in living cells. Brief Description of the Drawings

[0016] Figure 1 is the synthesis route of the ratiometric fluorescence probe of the present invention.

[0017] Figure 2 is the response sensing mechanism of the ratiometric fluorescence probe of the present invention to NaHS.

[0018] Figure 3 is the absorption change spectrum of the ratiometric fluorescence probe of the present invention after the action of different concentrations of NaHS.

[0019] Figure 4 is the fluorescence emission change spectrum of the ratiometric fluorescence probe of the present invention after the action of different concentrations of NaHS.

[0020] Figure 5 is the fluorescence ratio change spectrum (I 594nm / I 542nm ) of the ratiometric fluorescence probe of the present invention after the action of different concentrations of NaHS (0 - 75 μM).

[0021] Figure 6 is the fluorescence emission ratio change spectrum over time of the ratiometric fluorescence probe of the present invention after the action of NaHS (500 μM).

[0022] Figure 7 is the fluorescence ratio change spectrum (I 594nm / I 542nm ) Spectrogram.

[0023] Figure 8 This is the fluorescence imaging map of the ratio fluorescence probe of the present invention in Hela cells. Detailed implementation mode

[0024] Example 1

[0025] Dissolve 5-chloro-pyrrole-2-carbaldehyde (0.39 g, 3 mmol) and 2,4-dimethyl-3-ethylpyrrole (0.36 g, 3 mmol) in CH2Cl2 (80 mL) solution. Add three drops of trifluoroacetic acid under N2 and stir the reaction overnight. Add Et3N (3 mL) and BF3Et2O (3 mL) in sequence and continue to stir the reaction for 2 hours. Then, wash the mixture with brine, dry the organic layer with sodium sulfate, filter, and concentrate under vacuum. Purify the residue by column chromatography to obtain compound 2 (0.36 g, 43%). 1 1H NMR (400 MHz, CDCl3) δ: 6.98 (s, 1H), 6.79 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 3.2 Hz, 1H), 2.58 (s, 3H), 2.43 - 2.37 (m, 2H), 2.17 (s, 3H), 1.08 (t, J = 3.6 Hz, 3H); 13 13C NMR (100 MHz, CDCl3) δ: 163.9, 140.9, 136.9, 136.0, 135.3, 131.6, 125.4, 122.1, 115.1, 17.2, 14.2, 13.3, 9.4.

[0026] Example 2

[0027] Dissolve compound 2 (0.28 g, 1 mmol) in a mixed solution of N,N-dimethylformamide and methanol (5 mL / 10 mL). Slowly add iodine monochloride (0.19 g, 1.2 mmol) dropwise at room temperature and stir the reaction for 1 hour. Quench the reaction with sodium thiosulfate solution (20 mL) and further extract with dichloromethane (40 mL * 2). Dry the organic layer with sodium sulfate, filter, concentrate, and further purify by silica gel column chromatography to obtain red solid compound 3 (0.3 g, 74%). 1 1H NMR (400 MHz, CDCl3) δ: 6.92 (s, 1H), 6.88 (s, 1H), 2.58 (s, 3H), 2.40 (q, J = 7.6 Hz, 2H), 2.18 (s, 3H), 1.08 (t, J = 7.6 Hz, 3H); 1313C NMR (100 MHz, CDCl3) δ: 166.1, 142.0, 138.9, 136.3, 132.5, 130.3, 120.9, 77.0, 17.3, 14.0, 13.3, 9.4。

[0028] Example 3

[0029] A solution of compound 3 (50 mg, 0.12 mmol) and compound 4 (32 mg,, 0.13 mmol) in THF (10 mL) was bubbled with argon for 10 minutes, then Pd(PPh3)4 (7 mg, 6 μmol) and K2CO3 (2 M, 0.2 ml) were added. Under nitrogen protection, the mixture was heated at 80 °C for 8 hours. After cooling to room temperature, ethyl acetate (20 mL) and water (10 mL) were poured into the mixture. The organic layer was collected, dried over sodium sulfate, filtered and concentrated. The residue was purified by silica gel column chromatography to give the probe as an orange solid (32 mg, 68%). 1 1H NMR (400 MHz, CDCl3) δ: 10.06 (s, 1H), 8.01 (d, J = 8.0 Hz, 1H), 7.63 - 7.65 (m, 1H), 7.47 - 7.52 (m, 2H), 7.06 (s, 1H), 6.85 (s, 1H), 2.63 (s, 3H), 2.42 - 2.47 (m, 2H), 2.22 (s, 3H), 1.12 (t, J = 8 Hz, 3H); 13 13C NMR (100 MHz, CDCl3) δ: 192.0, 166.2, 141.7, 136.3, 134.0, 133.7, 131.4, 130.7, 128.2, 127.6, 125.1, 121.8, 17.3, 14.1, 13.6, 9.5; MS(ESI): calculated for C 20 H 18 BClF2N2O [M+Na] + 409.1065, found 409.1060。

[0030] Example 4

[0031] Spectral change diagrams of the probe for different concentrations of hydrogen sulfide: The probe was added to a DMSO / PBS (pH 7.4, 20 mM, v / v 1:1) test solution to prepare a solution with a concentration of 5 μmol / L, and then different concentrations of aqueous NaHS solution were added dropwise (since hydrogen sulfide is a gas, NaHS was used as a model for testing). After equilibrium, the absorption and fluorescence emission spectra were measured respectively. The results are shown in Figure 3 and Figure 4 。

[0032] From Figure 3 and 4 it can be seen that after adding NaHS, the spectrum of the probe is significantly redshifted, the maximum absorption peak is redshifted to 568 nm, and the color of the solution changes from orange to purple. At the same time, the fluorescence emission of the probe at 594 nm is significantly enhanced, while the fluorescence at 542 nm is weakened. Therefore, the probe can be used as a ratio-type fluorescence probe for hydrogen sulfide to achieve its quantitative detection.

[0033] Example 5

[0034] The probe was added to a DMSO / PBS (pH 7.4, 20 mM, v / v 1:1) test solution to prepare a solution with a concentration of 5 μmol / L, and then 500 μmol / L of NaHS was added. The fluorescence spectra at different times were recorded, as Figure 6 shown.

[0035] The results show that Figure 6 as time increases, the fluorescence intensity I 594nm of the probe increases, and I 542nm decreases until it stabilizes after 60 minutes, indicating that the probe can be used as a ratio-type fluorescence probe for detecting hydrogen sulfide.

[0036] Example 6

[0037] Test the selectivity of the ratio fluorescence probe: The probe was added to a DMSO / PBS (v / v, 1:1, pH 7.4) test solution to prepare a solution with a concentration of 5 μmol / L, and then various interfering substances were added, including Ala, Glu, GSH, Cys, Hcy, H2O2, S2O3 2- , Cl - , Br - , NO2 - , SO4 2- , ClO - , Mg 2+ and K + , and the change in the fluorescence intensity ratio (I 594nm / I 542nm ) was tested.

[0038] It can be seen from Figure 7 that the fluorescence intensity ratio of the probe changes significantly only for hydrogen sulfide, and the influence of other species is very weak, indicating that the selectivity of the probe for hydrogen sulfide is higher than that of other species.

[0039] Example 7

[0040] Intracellular fluorescence imaging test: After transferring Hela cells to a glass bottle for confocal imaging and incubating for 24 h, the experimental group was incubated with the probe (5 μM) solution for 30 minutes, and then washed three times with PBS for confocal cell imaging detection. The control group was incubated with the probe (5 μM) in cell culture for 30 minutes, then added with NaHS (300 μM) solution and incubated for 60 minutes, and then washed three times with PBS for confocal cell imaging detection. The emission of the green channel and red channel of the probe was collected in the emission ranges of 510 - 560 nm and 570 - 630 nm, respectively, and excited at 488 nm and 543 nm. As shown Figure 8 .

[0041] It can be seen from Figure 8 that the probe has a strong fluorescence signal in the green channel of HeLa cells, but almost no signal in the red channel; once the cells are further incubated with NaHS (300 μM) in the culture medium for 60 minutes, the fluorescence signal in the green channel weakens, while the emission signal in the red channel significantly increases, indicating that the probe can monitor intracellular hydrogen sulfide in living cells.

Claims

1. A ratiometric fluorescence probe based on an asymmetrical boron dipyrromethene dye, characterized in that, The structural formula of the ratio fluorescence probe is as follows:

2. A method for preparing the ratiometric fluorescence probe according to claim 1, characterized in that, It includes the following steps: (1) 2,4-Dimethyl-3-ethylpyrrole and 5-chloro-2-formylpyrrole are used to obtain compound 3-chloro-substituted fluoroboron dipyrrole through the asymmetric fluoroboron dipyrrole method; (2) The compound obtained in step (1) undergoes an iodination reaction with iodine monochloride, followed by a Suzuki coupling reaction with 2-formylphenylboronic acid. After the reaction is completed and processed, the ratio fluorescence probe is obtained.

3. Use of a ratiometric fluorescence probe as described in claim 1 in the preparation of a reagent for detecting hydrogen sulfide, characterized in that, The ratio fluorescence probe is applied to the spectral response or cell imaging of hydrogen sulfide.

4. Use of the ratiometric fluorescent probe according to claim 3 in the preparation of a reagent for detecting hydrogen sulfide, characterized in that, The fluorescence probe is for the spectral response of hydrogen sulfide.

5. Use of the ratiometric fluorescence probe according to claim 3 or 4 in the preparation of a reagent for detecting hydrogen sulfide, characterized in that, For the detection of the spectral response of the fluorescence probe to hydrogen sulfide, the specific method is as follows: The fluorescence probe is configured into a test solution, and then the sample to be tested is added. The absorption spectra before and after the test solution are measured. If the absorption spectrum shows an obvious red shift and the solution color changes from orange to purple, it indicates that hydrogen sulfide exists in the sample to be tested.

6. Use of the ratiometric fluorescence probe according to claim 3 or 4 in the preparation of a reagent for detecting hydrogen sulfide, characterized in that, For the detection of the spectral response of the fluorescence probe to hydrogen sulfide, the specific method is as follows: (1) The fluorescence probe is configured into a test solution, and then the sample to be tested is added, and its fluorescence spectrum change is observed; (2) Calculate the fluorescence intensity ratio I 594nm / I 542nm , and determine the concentration of hydrogen sulfide based on the fluorescence intensity ratio I 594nm / I 542nm . Calculate the detection limit according to the 3σ / k method.

7. Use of the ratiometric fluorescence probe according to claim 6 in the preparation of a reagent for detecting hydrogen sulfide, characterized in that, The preparation method of the test solution is as follows: DMSO / PBS is added to the fluorescence probe to configure a test solution with a concentration of 5 μmol / L.

8. Use of the ratiometric fluorescence probe according to claim 3 or 4 in the preparation of a reagent for detecting hydrogen sulfide, characterized in that, The fluorescence probe is used for intracellular fluorescence imaging tests. The specific method is as follows: The cells to be tested are incubated with fresh FBS-free medium containing the probe, then washed three times with PBS, and confocal cell imaging detection is carried out; During confocal cell imaging detection, the excitation wavelengths are 488 nm and 543 nm respectively, the green channel collection wavelength is 510 - 560 nm, and the red channel collection wavelength is 570 - 630 nm; After the probe is incubated in HeLa cells for 30 minutes, there is a strong fluorescence signal in the green channel, while there is almost no signal in the red channel; in addition, the cells are further incubated with NaHS in the medium for 60 minutes, where the concentration of NaHS is 300 μM. The fluorescence signal in the green channel weakens, while the emission signal in the red channel significantly increases.

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