A kind of thiophene polar fluorescent probe and its preparation method and application

Abstract

The present invention relates to the field of fluorescent probe technology, and in particular to a thiophene-based polar fluorescent probe and its preparation method and application. The basic skeleton of the probe molecule is thiophene, and electron-donating groups and electron-withdrawing groups are introduced at both ends of thiophene to form a strong intramolecular charge transfer system, so that the molecule responds to polarity. At the same time, because the electron-withdrawing group carries a positive charge, the probe molecule has good mitochondrial localization ability.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probes, and in particular to a thiophene-based polar fluorescent probe and a preparation method and application thereof. Background Art

[0002] Polarity is an extremely important microenvironment parameter. Polarity changes can affect various life activities in the biological microenvironment, such as cell membrane permeability, biological signal conduction, etc. Mitochondria, as a very important organelle, not only have important functions such as providing energy for cells and participating in metabolism, but also play an important role in important biological processes such as cell signal transduction. Abnormal changes in mitochondrial structure and function are an important cause of neurodegenerative diseases, cardiovascular diseases, cancer and other diseases. Changes in mitochondrial polarity can affect many processes in cell life activities, such as cell membrane permeability, enzyme activity and stability, cell proliferation, metabolic regulation and cell homeostasis. Therefore, for a variety of diseases caused by abnormal changes in mitochondrial polarity, real-time and accurate monitoring of polarity can be used to achieve early prevention and diagnosis and play a positive role in clinical treatment.

[0003] Fluorescent probes have become important detection tools in chemistry, environment and life sciences due to their high sensitivity, good selectivity, easy synthesis, low cost and good biological applications. So far, polar fluorescent probes for detecting solvents and cells have been reported one after another. The parent structures of polar fluorescent probes targeting mitochondria mainly involve fluorophores such as coumarin, naphthylimide and cyanine. As a classic fluorophore, thiophene not only has a large conjugated system and coplanar structure, but also has good photostability and low toxicity. So far, there are very few reports on the use of polar fluorescent probes with thiophene as a fluorophore for monitoring mitochondrial polarity during cell apoptosis. Summary of the invention

[0004] The present invention aims to provide a thiophene-based polar fluorescent probe and a preparation method and use thereof. The technical problem to be solved is to obtain a suitable fluorescent probe structure through molecular design so that it has a larger conjugated system and a coplanar structure, excellent mitochondrial targeting performance, high selectivity and membrane permeability, low cytotoxicity, high photostability and biocompatibility, so as to achieve quantitative detection of polarity changes in solutions and confocal imaging detection of polarity changes in mitochondria during cell apoptosis.

[0005] The present invention is achieved through the following technical solutions:

[0006] A thiophene-based polar fluorescent probe, abbreviated as TpIn, is based on thiophene and has the following structural formula:

[0007] .

[0008] The present invention also provides a method for preparing the thiophene-based polar fluorescent probe, comprising the following steps:

[0009]

[0010] Step 1: Preparation of compound 1

[0011] Take appropriate amounts of 5-bromothieno[3,2-b]thiophene-2-carboxaldehyde and N,N-dimethyl-4-vinylaniline, palladium acetate, tri(o-methylphenyl)phosphine (P-(o-tol)3), and triethylamine in a two-necked bottle, add N,N-dimethylformamide and ultrasonically dissolve, react at 90°C for 24 hours under nitrogen protection, extract with dichloromethane, wash the organic phase with saturated brine, spin dry, and purify with dichloromethane and methanol as eluents to obtain compound 1;

[0012] Step 2: Preparation of target probe TpIn

[0013] Compound 1, 1-ethyl-2,3,3-trimethylindolyl iodonium and piperidine were respectively placed in a two-necked bottle, an appropriate amount of ethanol was added for ultrasonic dissolution, the mixture was reacted at 80°C for 6 hours, the solvent was dried after cooling to room temperature, and purified with eluents such as dichloromethane and methanol to finally obtain the target probe TpIn.

[0014] Further, the molar ratio of 5-bromothieno[3,2-b]thiophene-2-carboxaldehyde to N,N-dimethyl-4-vinylaniline is 1:1.25;

[0015] The molar ratio of 5-bromothieno[3,2-b]thiophene-2-carboxaldehyde to palladium acetate is 1.05:0.04;

[0016] The molar ratio of 5-bromothieno[3,2-b]thiophene-2-carboxaldehyde to tri(o-methylphenyl)phosphine is 1.05:0.19;

[0017] Furthermore, the volume ratio of dichloromethane to methanol is 50:1.

[0018] Furthermore, the molar ratio of the compound 1 to 1-ethyl-2,3,3-trimethylindolyl iodonium is 1:2.

[0019] The present invention also provides the use of the thiophene polar fluorescent probe in solvent polarity detection. Under 530nm wavelength excitation, the fluorescence intensity of the thiophene polar fluorescent probe gradually weakens as the polarity of the test solvent system increases, and is not affected by the pH environment.

[0020] The invention also provides application of the thiophene polar fluorescent probe in mitochondrial localization.

[0021] The present invention also provides the use of the thiophene polar fluorescent probe in detecting the change of mitochondrial polarity during cell apoptosis.

[0022] The beneficial technical effects of the present invention are as follows:

[0023] The fluorescent probe of the present invention has a simple structure and is easy to synthesize. The basic skeleton of the molecule is thiophene, and electron-donating groups and electron-withdrawing groups are introduced at both ends of thiophene to form a strong intramolecular charge transfer system, so that the molecule responds to polarity. At the same time, because the electron-withdrawing group carries a positive charge, the probe molecule has good mitochondrial localization ability.

[0024] Under 530nm wavelength excitation, the fluorescence intensity of the fluorescent probe gradually weakens with the increase of the polarity of the test solvent system (dioxane < dichloromethane < ethanol < methanol < water), and the change is obvious. The fluorescent probe has a good response to polarity changes and is not affected by the pH environment. In addition, the probe has good mitochondrial localization ability and can be used to detect polarity changes in the process of cisplatin-induced HK-2 cell apoptosis. As the cells apoptosis, the polarity of the mitochondria decreases and the fluorescence intensity increases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a high-resolution mass spectrum of the probe TpIn prepared in Example 1;

[0026] Figure 2 is the hydrogen nuclear magnetic resonance spectrum of the probe TpIn prepared in Example 1;

[0027] Figure 3 This is the UV absorption spectrum of 10 μM probe TpIn in different polar organic solvents;

[0028] Figure 4 This is the fluorescence spectra of 10 μM probe TpIn in different polar organic solvents;

[0029] Figure 5 This is the fluorescence emission spectra of 10 μM probe TpIn in water / dioxane mixed solvents with different volume ratios;

[0030] Figure 6 This is the linear relationship between solvent polarity and fluorescence intensity of 10 μM probe TpIn in water / dioxane mixed solvents with different volume ratios;

[0031] Figure 7 This is the pH stability graph of 10 μM probe TpIn in pure water;

[0032] Figure 8 It is the cell survival rate under the action of different concentrations (0μM, 2μM, 5μM, 10μM, 15μM) of the probe molecule TpIn;

[0033] Fig. 9 This is a confocal fluorescence imaging image of HK-2 cells mitochondrial localization verification using 5 μM probe TpIn and 100 nM commercial mitochondrial red fluorescent dye co-stained simultaneously;

[0034] Fig.10 These are confocal images of HK-2 cell apoptosis induced by 5 μM probe TpIn in the presence of different concentrations of cisplatin. DETAILED DESCRIPTION

[0035] The present invention will be further described below by way of examples.

[0036] Example 1

[0037] Synthesis of compound 1

[0038] Weigh dried 5-bromothieno[3,2-b]thiophene-2-carbaldehyde (4.25mmol, 1.05g), tri(o-methylphenyl)phosphine (0.19mmol, 0.06g), and palladium acetate (0.04mmol, 0.01g) into a 100mL two-necked bottle. After sealing with a glass stopper, use a double-row tube to charge and discharge nitrogen three times to remove the air and a small amount of water in the bottle. While filling with nitrogen, use a glass syringe to inject an appropriate amount of N,N-dimethylformamide (DMF) and N,N-dimethyl-4-vinylaniline (5.10mmol, 0.75g) dissolved in DMF. Stir at room temperature for 30 minutes. Finally, inject triethylamine (13.0mL) and react at 90°C for 24h under nitrogen protection. After the reaction, a vacuum distillation apparatus was used to remove the high-boiling DMF and a small amount of triethylamine, and then the inorganic salts were removed by extraction. The organic phase was collected, dried over anhydrous Na2SO4 and filtered, and the organic solvent was removed by a rotary evaporator. The remaining solid was dissolved with an appropriate amount of dichloromethane, and the sample was prepared by silica gel. Column chromatography was used for separation, and dichloromethane: methanol (DCM: MeOH, v / v, 50:1) was used as the eluent to purify and obtain compound 1, 1.10 g of orange solid powder, and the yield was 83%. 1 HNMR (400MHz, CDCl3) δ9.91 (s, 1H), 7.84 (s, 1H), 7.76 (s, 1H), 7.41 (s, 2H), 7.15 (s, 1H), 7.02 (d, J=19.3Hz, 1H), 6.71 (s, 2H), 3.01 (s, 6H).

[0039] Synthesis of target probe TpIn

[0040] Weigh compound 1 (0.2mmol, 0.06g) and 1-ethyl-2,3,3-trimethyl-3H-indol-1-ium iodide (0.4mmol, 0.13g) in a 100mL round-bottom flask, add an appropriate amount of ethanol (30mL) to dissolve, stir evenly at room temperature, add 5 drops of piperidine, and heat to reflux at 80°C for 4h. After the reaction is completely cooled to room temperature, the solvent is dried, the product is dissolved in an appropriate amount of dichloromethane, silica gel sample is prepared, column chromatography is used for separation, and dichloromethane: methanol (DCM: MeOH, v / v, 30:1) is used as the eluent to purify and obtain the target probe TpIn, 0.02g of blue-purple solid powder, and the yield is: 21%. The hydrogen nuclear magnetic resonance spectrum is shown in Figure 2 As shown. 1 HNMR (400MHz, CDCl3) δ8.89 (s, 2H), 7.52 (s, 4H), 7.45 (s, 3H), 7.16 (s, 1H), 7.06 (d, J=8.8Hz, 1H), 6. 84(s, 1H), 5.34(s, 1H), 4.72(s, 2H), 3.05(s, 6H), 2.22(s, 1H), 1.99(d, J=16.8Hz, 3H), 1.88(s, 6H).

[0041] The reaction equation is as follows:

[0042] .

[0043] Example 2 Spectral test of fluorescent probe molecules

[0044] The fluorescent probe TpIn prepared in Example 1 of the present invention was dissolved in DMSO to prepare a 1mM mother solution, and 5mL was prepared. 30μL of the mother solution was taken into a 3mL sample tube, and the concentration of the test solution was prepared to be 10μM. The spectral data in different polar solvents were tested. The UV spectrum showed that the UV absorption peak of the probe in different polar solutions was between 600-700nm. Only the absorption peak in dichloromethane was red-shifted by about 80nm. The absorption peak shift in other solvents was not obvious. Figure 3 .

[0045] It can be seen from the fluorescence spectrum that under the excitation wavelength of 530nm, with the increase of solvent polarity, the fluorescence intensity of the probe at 600nm decreases significantly. Figure 4 The above data preliminarily indicate that the probe molecule TpIn prepared in Example 1 responds to polarity. In order to further prove the polarity response characteristics of the probe TpIn, the fluorescence spectrum data of the probe molecule in mixed solvents of water / 1,4-dioxane with different volume ratios were tested. When the volume of dioxane in the mixed solvent increased from 0% to 100%, the polarity of the mixed system decreased, and the fluorescence intensity of the probe at 560nm increased with a certain regularity. The results are shown in Table 1. Figure 5 , and there is a certain linear relationship between the fluorescence intensity and the polarity value, see Figure 6 .

[0046] In order to eliminate the influence of pH, the fluorescence spectra of the probe molecule TpIn in solutions with different pH values ​​were tested. It can be seen that the fluorescence intensity of the probe TpIn does not change much in phosphate buffers of different pH values. Figure 7 ,The experimental results show that the pH value has little effect on the probe TpIn, and the probe TpIn is not sensitive to changes in pH.

[0047] Example 3 Cell culture and cytotoxicity test

[0048] Cell culture: HK-2 cells were cultured in 90% DMEM (sugar and amino acids) and 10% FCS (fetal calf serum), and 1% penicillin-streptomycin mixture was added to prevent bacterial contamination. The cells were cultured in a constant temperature incubator containing 5% CO2 at 37°C.

[0049] Cytotoxicity: CCK-8 was used to determine the cytotoxicity of the probe molecule TpIn prepared in Example 1 to HK-2. HK-2 was subcultured into a 96-well cell culture plate and the cell density was controlled to be 70%. HK-2 cells were incubated in an incubator for one day with the probe molecule TpIn at concentrations of 0, 2, 5, 10 and 15 μM, respectively; the cells were then treated with CCK-8 and incubated for another 2 hours. The 96-well plate was then placed in an ELISA reader, the absorbance at 450 nm was recorded, and the cell survival rate was calculated. The results are shown in FIG. Figure 8 shown.

[0050] The cell survival rate (%) was calculated according to the following formula:

[0051] Cell survival rate % = OD450 (experimental group) / OD450 (control group) × 100%.

[0052] from Figure 8 It can be seen that when the probe concentration reaches 15 μM, the cell survival rate is still about 88%, indicating that the fluorescent probe molecule TpIn of the present invention is non-toxic to cells.

[0053] Example 4 Cell localization test

[0054] The probe molecule TpIn (5 μM) prepared in Example 1 and 100 nM commercial mitochondrial green fluorescent probe (MitoGreen) were added to the adherent HK-2 cells and incubated for 30 minutes. After washing with PBS three times, confocal imaging was performed. The excitation wavelengths were 488 nm (MitoGreen) and 520 nm (TpIn), respectively; the corresponding collection bands were 500-550 nm and 550-650 nm, respectively. Fig. 9It can be seen that the overlap coefficient between TpIn and MitoGreen is 0.85, indicating that TpIn is mostly localized in mitochondria, indicating that it can be used to detect cell mitochondrial polarity.

[0055] Example 5

[0056] In order to study the changes in mitochondrial polarity during apoptosis and verify the effectiveness of TpIn for mitochondrial polarity imaging, we used cisplatin to induce apoptosis. Adherent HK-2 cells were stimulated with different concentrations of cisplatin (0.4, 0.8, 1.2 mM) for 12 hours, then rinsed three times with PBS, incubated with 5 μM probe TpIn for 15 minutes, and then rinsed three times with PBS before imaging. The excitation wavelength was 520 nm, and the corresponding collection band was 550-650 nm. Fig.10 It can be seen that the fluorescence intensity of the cells themselves is weak. With the increase of the incubation cisplatin concentration (0.4, 0.8 and 1.2 mM), the red fluorescence signal gradually increases, indicating that during the apoptosis of HK-2 cells, the mitochondrial polarity gradually decreases, verifying that TpIn can monitor the changes in mitochondrial polarity in real time.

Claims

1. A thiophene-based polar fluorescent probe, characterized in that: The fluorescent probe is based on thiophene, and its structural formula is as follows: , abbreviated as TpIn.

2. A method for preparing a thiophene-based polar fluorescent probe as claimed in claim 1, characterized in that: The steps include: Step 1: Preparation of compound 1 Take appropriate amounts of 5-bromothieno[3,2-b]thiophene-2-carboxaldehyde and N,N-dimethyl-4-vinylaniline, palladium acetate, tri(o-methylphenyl)phosphine and triethylamine in a two-necked bottle, add N,N-dimethylformamide and ultrasonically dissolve, react at 90°C for 24 hours under nitrogen protection, extract with dichloromethane and wash the organic phase with saturated brine, spin dry and purify with dichloromethane and methanol as eluents to obtain compound 1; Step 2: Preparation of target probe TpIn Compound 1, 1-ethyl-2,3,3-trimethylindolyl iodonium and piperidine were respectively placed in a two-necked bottle, an appropriate amount of ethanol was added for ultrasonic dissolution, the mixture was reacted at 80°C for 6 hours, the solvent was dried after cooling to room temperature, and purified with eluents such as dichloromethane and methanol to finally obtain the target probe TpIn.

3. The method for preparing the thiophene-based polar fluorescent probe according to claim 2, characterized in that: The molar ratio of 5-bromothieno[3,2-b]thiophene-2-carboxaldehyde to N,N-dimethyl-4-vinylaniline is 1:1.25; The molar ratio of 5-bromothieno[3,2-b]thiophene-2-carboxaldehyde to palladium acetate is 1.05:0.04; The molar ratio of 5-bromothieno[3,2-b]thiophene-2-carboxaldehyde to tri(o-methylphenyl)phosphine is 1.05:0.

19.

4. The method for preparing the thiophene-based polar fluorescent probe according to claim 2, characterized in that: The volume ratio of dichloromethane to methanol is 50:

1.

5. The method for preparing the thiophene-based polar fluorescent probe according to claim 2, characterized in that: The molar ratio of the compound 1 to 1-ethyl-2,3,3-trimethylindolyl iodonium is 1:

2.

6. Use of the thiophene-based polar fluorescent probe as claimed in claim 1 in detecting solvent polarity.

7. The use of the thiophene polar fluorescent probe according to claim 1 in solvent polarity detection, characterized in that: Under excitation at a wavelength of 530 nm, the fluorescence intensity of the thiophene-based polar fluorescent probe gradually weakens as the polarity of the test solvent system increases, and is not affected by the pH environment.

8. Use of the thiophene polar fluorescent probe as claimed in claim 1 in detecting mitochondrial polarity for purposes other than disease diagnosis and treatment.

9. A use of the thiophene polar fluorescent probe as claimed in claim 1 for detecting changes in mitochondrial polarity during cell apoptosis for purposes other than disease diagnosis and treatment.

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