Imidazolyl BODIPY derivative viscosity fluorescent probe and application thereof

Abstract

The invention discloses an imidazolyl BODIPY derivative viscosity fluorescent probe with an aggregation-induced fluorescence enhancement (AIEE) effect and application thereof, the imidazolyl BODIPY derivative viscosity fluorescent probe is realized by the following method: taking a 2-formaldehyde BODIPY derivative (I), diphenyl ethanedione and aniline as raw materials, and carrying out Debus-Radziszewski imidazole synthesis reaction under the catalytic action of ammonium acetate, and the chemical structural formula of the imidazolyl BODIPY derivative viscosity fluorescent probe is as shown in formula (II). The preparation method is simple in reaction step, mild in reaction condition, simple and convenient to separate and purify and high in yield; the fluorescent probe has an aggregation-induced fluorescence enhancement effect, has high responsiveness to viscosity, can be used for detecting viscosity change in a solution and cells, and can provide an effective detection method for researching physiological action of viscosity in cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of organic synthesis and fluorescent probes, and particularly relates to an imidazole-based BODIPY derivative viscosity fluorescent probe with AIEE effect and application thereof. BACKGROUND

[0002] Cell viscosity, as an important physiological environment parameter, affects the physiological activities such as material transport, metabolism, signal transmission, and cell differentiation and apoptosis. Changes in cell viscosity are closely related to various diseases and dysfunctions, such as diabetes, hypertension, arteriosclerosis, Alzheimer's disease, and even cancer. Abnormal viscosity behavior of cells is always accompanied by dramatic changes in the level of related active analytes. Therefore, it is of great significance to monitor the local micro-viscosity of living cells and analyze the changes in analytes.

[0003] In recent years, fluorescence imaging has attracted extensive attention due to its high sensitivity, easy operation, high spatiotemporal resolution, and good biocompatibility. In addition, fluorescence imaging can detect cells and organisms at the cellular or subcellular level and shows great potential in disease imaging and diagnosis. It has been proven to be an effective method for monitoring viscosity levels in biological processes.

[0004] BODIPY has very excellent photochemical physical properties, has a very high molar extinction coefficient, and is beneficial to the improvement of the light sensitivity of the dye. The fluorescence quantum yield is high, generally more than 0.6, and the quantum yield of many dyes can reach 1. It still maintains a very high quantum yield in solution and does not undergo quenching phenomenon, and can be applied in the field of biological analysis. The dye has good photo-thermal stability and chemical stability, avoids rapid photodegradation caused by external interference, and ensures the stability of the optical signal. These excellent properties make the application of this type of dye develop rapidly, and it has become one of the most concerned fluorescent dyes. In addition to the above properties, its parent structure can be derivatized, substituted or functioned in a direct way, thereby generating new spectral properties. Many functionalized BODIPYs have been synthesized. At present, highly diversified BODIPY derivatives have been widely used in many fields. However, BODIPY derivatives have ACQ effect, and exhibit weak fluorescence in the aggregated state, which is in sharp contrast to their excellent luminescence in solution. These problems greatly limit the application of BODIPY derivatives as aggregates in the biological field. Therefore, it is of important scientific significance and application value to design and synthesize new BODIPY derivatives with aggregation-induced fluorescence enhancement (AIEE) properties.

[0005] Based on the excellent optical physical properties of BODIPY fluorescent dyes, the present application synthesizes an imidazole-based BODIPY derivative viscosity fluorescent probe with an aggregation-induced fluorescence enhancement effect through a Debus-Radziszewski imidazole synthesis reaction. The fluorescent probe has superior sensitivity to viscosity and can respond to changes in intracellular viscosity, and is expected to give a signal when abnormal conditions occur in vivo, more sensitive and accurate sensing of disease occurrence, and has potential application prospects in preventing, diagnosing and treating diseases. SUMMARY

[0006] The present application aims at providing an imidazole-based BODIPY derivative viscosity fluorescent probe with AIEE effect and application thereof.

[0007] Technical scheme: In order to achieve the above-mentioned application purpose, the technical scheme adopted by the present application is:

[0008] The imidazole-based BODIPY derivative viscosity fluorescent probe with AIEE effect and application thereof of the present application has the following structural formula shown in formula (II):

[0009]

[0010] The imidazole-based BODIPY derivative viscosity fluorescent probe with AIEE effect and application thereof has the following preparation steps:

[0011] At room temperature, compound (I) and diphenyl ketone are dissolved in acetic acid, aniline is added dropwise into the mixture, then ammonium acetate (CH3COONH4) is added, heated to 110 DEG C and refluxed for 2 hours, the reaction is stopped, cooled to room temperature, stirred in ice water, sodium bicarbonate (NaHCO3) is added to adjust the pH value to neutral, extracted with dichloromethane, the organic phase is separated, the organic phase is combined and dried with anhydrous sodium sulfate, the organic solvent is removed by reduced pressure distillation, and the residue is purified by silica gel column chromatography to obtain the imidazole-based BODIPY derivative viscosity fluorescent probe (II). The specific chemical reaction formula is as follows:

[0012]

[0013] In the above synthesis step, the mass ratio of 2-aldehyde BODIPY derivative (I), diphenyl ketone, aniline and CH3COONH4 is 1:1:1.5:5, and the reaction solvent is acetic acid.

[0014] Advantages of the present application

[0015] Compared with the prior art, the imidazole-based BODIPY derivative viscosity fluorescent probe (II) with AIEE effect and the application thereof have the following advantages: (1) the synthesis steps are less, the preparation method is simple and easy to operate, and the yield is higher; (2) the viscosity probe has AIEE effect, and the fluorescence intensity gradually increases with the increase of viscosity; within a certain viscosity range, the fluorescence intensity and the logarithm of the system viscosity show a good linear relationship, different viscosities in the solution can be qualitatively and quantitatively detected, and the viscosity probe has good stability in the physiological pH value range; (3) the cell toxicity is low, and the cell membrane permeability is good; (3) the viscosity probe has good selectivity, strong anti-interference property and high sensitivity, and can be used in many fields such as biological fluorescence imaging, fluorescence labeling, environment, and biomedicine. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a mass spectrum of the imidazole-based BODIPY derivative viscosity fluorescent probe (II);

[0017] Figure 2 is a fluorescence spectrum of the imidazole-based BODIPY derivative viscosity fluorescent probe (II) in a DMSO / toluene solution with different proportions;

[0018] Figure 3 is a fluorescence spectrum of the imidazole-based BODIPY derivative viscosity fluorescent probe (II) in a water-glycerol solution with different proportions;

[0019] Figure 4 is a linear relationship diagram of the fluorescence intensity and the viscosity of the imidazole-based BODIPY derivative viscosity fluorescent probe (II) in a water-glycerol mixture;

[0020] Figure 5 is a fluorescence emission spectrum of the imidazole-based BODIPY derivative viscosity fluorescent probe (II) under the coexistence of different metal ions;

[0021] Figure 6 is a confocal fluorescence imaging diagram of HeLa cells treated by the imidazole-based BODIPY derivative viscosity fluorescent probe (II). DETAILED DESCRIPTION

[0022] The application will be further described below in combination with specific drawings.

[0023] With 1 H-NMR, 13C-NMR and ESI-HRMS were used to characterize and confirm the structure of the BODIPY derivative viscosity fluorescent probe (II). The spectral properties were determined by UV-Vis spectra and fluorescence emission spectra; the instruments used were: Bruker ARX 400 type nuclear magnetic resonance instrument (deuterated chloroform as solvent), Leica SP8 laser confocal microscope, NeXion-300X type inductively coupled plasma mass spectrometer, Shimadzu UV-3100 type ultraviolet-visible spectrophotometer, and LS-55 type fluorescence spectrophotometer (the slit was 2.5 mm) of the United States.

[0024] Example 1 Preparation of imidazole-based BODIPY compound (II)

[0025] Compound (I) (140 mg, 0.21 mmol) and diphenylglyoxal (44.1 mg, 0.21 mmol) were dissolved in 5 mL of acetic acid at room temperature, and aniline (0.028 mL, 0.315 mmol) was added dropwise to the mixture; then ammonium acetate (CH3COONH4) (81 mg, 1.05 mmol) was added, and the reaction was heated to reflux at 110°C for 2 h, then cooled to room temperature. The reaction was poured into ice water and stirred, and a sodium bicarbonate solution was added to adjust the pH to neutral. The organic phase was extracted with dichloromethane, and the organic phase was separated and dried over anhydrous sodium sulfate. After removing the organic solvent, the product was purified by column chromatography on silica gel to obtain the imidazole-based BODIPY derivative viscosity fluorescent probe (II). Yield: 70%. 1 H NMR (600 MHz, CDCl3, ppm): δ 6.00 (s, 1H), 1.25 (s, 3H), 1.45 (s, 3H), 2.53 (s, 3H), 3.36 (d, J = 11.40 Hz, 6H), 3.56-3.51 (m, 4H), 3.67 (m, J = 9.6, 5.91 Hz, 4H), 3.65-3.62 (m, 4H), 3.76-3.73 (m, 4H), 3.85 (m, J = 4.80 Hz, 2H), 3.89 (m, J = 4.8 Hz, 2H), 7.57 (d, J = 7.20 Hz, 2H), 7.49 (d, J = 8.40 Hz, 2H), 7.32 (m, J = 7.80 Hz, 2H), 7.23 (m, J = 7.80, 3.40 Hz, 3H), 7.19-7.17 (m, 3H), 2.38 (s, 3H), 4.07 (d, J = 4.80 Hz, 2H), 6.95 (d, J = 8.40 Hz, 1H), 6.86-6.82 (m, 2H), 7.13-7.09 (m, 3H), 4.18 (m, J = 4.80 Hz, 2H). 13C NMR (100 MHz, CDC13 ppm): 177.1, 172.2, 159.7, 154.2, 152.8, 149.7, 146.1, 142.4, 140.0, 136.5, 133.7, 133.4, 132.5, 131.5, 130.0, 129.0, 128.3, 128.0, 127.9, 127.8, 127.3, 126.9, 123.0, 120.7, 114.5, 113.8, 71.9, 70.9, 70.8, 70.7, 70.6, 70.5, 69.7, 69.6, 69.1, 68.6, 59.1, 59.0, 14.8, 13.6, 13.0. ESI-HRMS: calculated for C 54 H 62 BF2N4O8[M+H] + : 943.4629; found: 943.4596 Figure 1 ).

[0026] Preparation of viscosity fluorescent probe (II) of imidazolyl BODIPY derivative

[0027] Similar to Example 1, the difference is that the mass ratio of 2-aldehyde BODIPY derivative (I), diphenyl ketone, aniline, CH3COONH4 in this example is 1:1:1.2:5; the reaction temperature is controlled at 100°C, and the reaction time is 3 hours. Yield: 65%.

[0028] Preparation of viscosity fluorescent probe (II) of imidazolyl BODIPY derivative

[0029] Similar to Example 1, the difference is that the mass ratio of 2-aldehyde BODIPY derivative (I), diphenyl ketone, aniline, CH3COONH4 in this example is 1:1:1.2:5; the reaction temperature is controlled at 100°C, and the reaction time is 3 hours. Yield: 65%.

[0030] AIEE effect of viscosity fluorescent probe (II) of imidazolyl BODIPY derivative

[0031] The fluorescent probe (II) was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 1 x 10 -3 mol / L. Ten portions of the stock solution were taken and different volumes of DMSO or toluene were added to prepare 3 mL solutions with different proportions of DMSO / toluene, so that the toluene content was 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, respectively. Then the fluorescence emission spectrum intensity change was measured.Figure 2 It can be seen that in DMSO / toluene mixed solution, the fluorescence emission intensity gradually increases with the increase of toluene content, and when the toluene content increases to 90%, the fluorescence intensity reaches the maximum.

[0032] Example 5 Fluorescence emission spectrum changes of viscosity fluorescence probe (II) of imidazole-based BODIPY derivative in glycerol / water solution with different proportions

[0033] The imidazole-based BODIPY derivative viscosity fluorescence probe (II) prepared in Example 1 was prepared into a 4 mM stock solution, and then the stock solution (3 mM) of the probe (II) was added into glycerol / water solutions (1 mL) with different volume proportions to obtain 40 μM probe (II) solutions with different viscosities. The fluorescence emission spectrum was measured at an excitation wavelength of 470 nm; with the increase of viscosity, the fluorescence intensity at 520 nm gradually increased. Figure 3 is the fluorescence emission spectrum diagram of the imidazole-based BODIPY derivative viscosity fluorescence probe (II) solution (40 μM) in glycerol / water solution with different proportions. By analyzing the relationship between the fluorescence intensity of the imidazole-based BODIPY derivative viscosity fluorescence probe (II) at 520 nm and the logarithm of the viscosity of the system (λ ex = 470 nm), the response ability of the probe to viscosity was evaluated, and the results showed that the logarithm of the fluorescence intensity of the probe (II) and the logarithm of the viscosity within a certain viscosity range showed a good linear relationship, and the linear relationship equation between them was log(F 520 ) = 1.09119*log(η) + 0.6165, where F 520 represents the fluorescence intensity of the probe (II) at 520 nm, and η represents the viscosity of the probe (II) solution system. The linear correlation coefficient R 2 = 0.99069. Figure 4 is the linear relationship diagram between the fluorescence intensity of the imidazole-based BODIPY derivative viscosity fluorescence probe (II) solution (40 μM) at 520 nm and the logarithm of the viscosity of the system.

[0034] Example 6 Study on the selective recognition of different ions by the imidazole-based BODIPY derivative viscosity fluorescence probe (II) with AIEE effect.

[0035] A 10 μM fluorescence probe (II) glycerol:DMSO (v / v = 1:4) solution was accurately prepared. Different ions such as Na + , Ba 2+ , Ca 2+ , Cu 2+ , Fe 2+ , Fe3+ , K + , La 3+ , Mg + , Co 2+ , Pb 2+ , Ni 2+ , Br - , Cl - , CO3 2- , F - , HPO4 - , HSO4 - , MnO 4- , PO4 3- , SO4 2- The fluorescence emission spectra of the solution were measured after shaking, and the results are shown in Figure 5 . It was found that the fluorescence spectra after adding different ions had almost no obvious change, and these results indicated that the viscosity probe (ii) had good selectivity. In addition, the probe had almost no response to different ions in addition to the obvious response to different viscosities.

[0036] Confocal fluorescence imaging of HeLa cells incubated with BODIPY derivative viscosity fluorescence probe (II) under different viscosity conditions

[0037] To investigate the response of viscosity probe (II) to cell viscosity, Hela cells were incubated with different exogenous drugs, and the cells were divided into four groups: negative control group and Hela cells, Hela cells treated with LPS (10 μM), Hela cells treated with monensin (10 μM), and Hela cells treated with nystatin (10 μM). After treatment, all cells in the groups were incubated with probe (II) at a concentration of 10 mM for 30 minutes, and then washed with water for 3 times. Finally, high-resolution fluorescence confocal microscopy was used to observe the cells, and Leica application software was used for image analysis of confocal fluorescence imaging, and the results are shown in Figure 6 . When HeLa cells were treated with viscosity probe (II) only for 30 minutes, only weak fluorescence was observed. In contrast, after HeLa cells were treated with LPS, Mon and Nys respectively, and then incubated with viscosity probe (II), the sharp increase in cell viscosity induced by LPS, Mon and Nys caused the fluorescence signal to significantly enhance, and these results indicated that viscosity probe (II) could be used to detect changes in cell viscosity.

Claims

1. An imidazolyl BODIPY derivative viscosity fluorescent probe (II) characterized by, The structural formula is shown as formula (II) below:

2. A process for the preparation of the imidazolyl BODIPY derivative viscosity fluorescent probe (II) of claim 1, characterized by, The preparation process is realized by the following steps: 2-formaldehyde BODIPY derivative (I) and benzalaniline undergo Debus-Radziszewski imidazole synthesis reaction under the catalysis of ammonium acetate to obtain an imidazole BODIPY derivative viscosity fluorescent probe (II) with an aggregation-induced fluorescence enhancement (AIEE) effect, and the chemical reaction formula of the preparation process is as follows:

3. The method for preparing a viscosity fluorescent probe of imidazole-based BODIPY derivative with AIEE effect according to claim 2, characterized in that, The method comprises the following steps: At room temperature, compound (I) and benzalaniline are dissolved in acetic acid, aniline is added dropwise into the mixture, and ammonium acetate (CH3COONH4) is added, and then heated to 110 DEG C for 2 hours, cooled to room temperature, poured into ice water, stirred, added NaHCO3 to adjust pH, extracted with dichloromethane, separated the organic phase, combined the organic phase and dried with anhydrous sodium sulfate, removed the organic solvent under reduced pressure, and purified by silica gel column chromatography to obtain the imidazole BODIPY derivative viscosity fluorescent probe (II).

4. The method for preparing the imidazole-based BODIPY derivative viscosity fluorescent probe with AIEE effect according to claim 3, characterized in that In the synthesis step, the mass ratio of compound (I), benzalaniline, aniline and CH3COONH4 is 1:1:1.5:

5.

5. The imidazole BODIPY derivative viscosity fluorescent probe (II) with AIEE effect of claim 1 is applied in viscosity detection.

6. The viscosity fluorescent probe (II) with AIEE effect of claim 1 can be applied in fluorescence imaging, fluorescence labeling, biomedicine and other fields.

Images