Preparation method and application of hypochlorous acid near-infrared fluorescent probe

Abstract

The invention relates to the technical field of organic small-molecule fluorescent probes, in particular to a preparation method and application of a hypochlorous acid near-infrared fluorescent probe. According to the invention, dicyanoisophorone derivatives with excellent optical properties and more modification sites are selected as a fluorescent matrix; the fluorescence probe with the emission wavelength in a near-infrared band is designed and synthesized by selecting dimethylaminothioformate as a recognition site of hypochlorite, the optical performance of the probe on hypochlorite response is evaluated by using an ultraviolet spectrophotometer and a fluorescence spectrophotometer, the cytotoxicity of the probe is tested by using a CCK8 method, and the detection result is accurate. And finally, the probe is applied to detection of the level change of hypochlorite in the cells.

Description

Technical Field

[0001] The present invention relates to the technical field of organic small molecule fluorescent probes, and specifically relates to a preparation method and use of a near-infrared fluorescent probe for hypochlorous acid. Background Art

[0002] Hypochlorite (ClO - ) is an important reactive oxygen species produced during human metabolism and plays a key role in physiological activities such as immune defense and cell signaling. As an important part of the human immune system, hypochlorite is produced by myeloperoxidase in neutrophils and can effectively kill invading pathogenic microorganisms. However, abnormal levels of hypochlorite are closely related to the occurrence and development of various diseases, including inflammatory diseases, cardiovascular diseases, neurodegenerative diseases, and cancers. Therefore, real-time monitoring of the concentration changes of hypochlorite in biological systems is of great significance for understanding its physiological and pathological mechanisms of action.

[0003] In recent years, fluorescent probe technology has been regarded as a powerful tool for detecting hypochlorite in biological systems due to its excellent sensitivity, high selectivity, and real-time monitoring ability. However, most of the currently reported hypochlorite fluorescent probes have obvious limitations: First, the emission wavelengths of these probes are usually in the visible light region and are easily interfered by the autofluorescence of biological samples; Second, the tissue penetration ability of short-wavelength light is poor, which limits its application in in vivo imaging; In addition, some probes may have problems such as poor photostability and slow response speed. These defects may lead to large errors in detection results and are difficult to meet the actual needs of biomedical research.

[0004] To solve the above problems, the development of new near-infrared fluorescent probes for hypochlorite detection has become a hot research direction. Near-infrared fluorescent probes have many advantages: their emission wavelengths are within the "optical window" range of biological tissues, which can significantly reduce background fluorescence interference and improve the signal-to-noise ratio; At the same time, near-infrared light has stronger tissue penetration ability, which is conducive to realizing deep tissue imaging and in vivo real-time monitoring. Therefore, designing and synthesizing near-infrared hypochlorite fluorescent probes with high sensitivity, high selectivity, and good biocompatibility has important scientific significance and application value for in-depth study of the mechanism of action of hypochlorite in physiological and pathological processes, as well as the early diagnosis and treatment of related diseases. Summary of the Invention

[0005] The technical objective to be achieved by the present invention is: The present invention designs a preparation method and use of a near-infrared fluorescent probe for hypochlorous acid, which is applied to detect the level changes of hypochlorite in cells.

[0006] The method for preparing a near-infrared fluorescent probe for detecting hypochlorite adopted by the present invention is specifically implemented according to the following steps:

[0007] Step 1, Dissolve isophorone and malononitrile in absolute ethanol, heat to dissolve, add piperidine dropwise, under a nitrogen atmosphere, heat to reflux at 85 °C for 4 - 6 hours. After the reaction is completed, cool to room temperature, extract three times with dichloromethane and saturated brine, combine the organic phases, dry with anhydrous sodium sulfate, concentrate in vacuo, and separate and purify by column chromatography (V PE / V EA = 60:1) to obtain a white solid powder, which is Compound 1, with a yield of 75%;

[0008] Step 2, Dissolve Compound 1 and p-hydroxybenzaldehyde in an acetonitrile solution, add piperidine dropwise, raise the temperature to 85 °C and reflux for 6 hours. After the reaction is completed, cool to room temperature, extract three times with dichloromethane and saturated brine, combine the organic phases, dry with anhydrous sodium sulfate, concentrate in vacuo, and separate and purify by column chromatography (V PE / V EA = 10:1) to obtain a light yellow solid powder, which is Compound 2, with a yield of 65%;

[0009] Step 3, Dissolve Compound 2 and hexamethylenetetramine (HMTA) in trifluoroacetic acid, heat to reflux at 75 °C for 3 hours. After the reaction is completed, cool to room temperature, extract three times with dichloromethane and ice water, combine the organic phases, dry with anhydrous sodium sulfate, concentrate in vacuo, and separate and purify by column chromatography (V PE / V EA = 15:1) to obtain a yellow solid powder, which is Compound 3, with a yield of 40%;

[0010] Step 4, Dissolve Compound 3 and ethoxycarbonylmethylenetriphenylphosphine in toluene, stir at 50 °C for 12 hours under nitrogen protection and in the dark, then concentrate in vacuo after completion, and separate and purify by column chromatography (V PE / V EA = 5:1) to obtain an orange-red solid powder, which is Compound 4, with a yield of 85%;

[0011] Step 5, Dissolve Compound 4 in dichloromethane, add DIPEA at 0 °C and under a nitrogen atmosphere, then add dimethylaminothiocarbonyl chloride and stir for 5 minutes, transfer to room temperature and stir for 2 - 3 days. After the reaction is completed, concentrate in vacuo, and separate and purify by column chromatography (V PE / V EA = 10:1) to obtain a light yellow solid powder DCI-OEt-HClO.

[0012] Preferably, in Step 1, the molar ratio of isophorone to malononitrile is 1:3, and the piperidine is 200 μL;

[0013] Preferably, in Step 2, the molar ratio of Compound 1 to p-hydroxybenzaldehyde is 1:1, and the piperidine is 200 μL;

[0014] Preferably, in step 3, the molar ratio of compound 2 to hexamethylenetetramine is 1:2;

[0015] Preferably, in step 4, the molar ratio of compound 3 to ethoxycarbonylmethylene triphenylphosphine is 1:1.5;

[0016] Preferably, in step 5, the molar ratio of compound 4, DIPEA, and dimethylaminothiocarbonyl chloride is 1:2:4;

[0017] Preferably, the near-infrared fluorescent probe can be used for detecting hypochlorite in solutions and cells.

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) In the present invention, a dicyanoisophorone derivative with excellent optical properties and many modification sites is selected as the fluorescent matrix, and dimethylaminothiocarbonate is selected as the recognition site for hypochlorite, and a fluorescent probe with an emission wavelength in the near-infrared band is synthesized and designed. The optical properties of the probe's response to hypochlorite were evaluated using a UV spectrophotometer and a fluorescence spectrophotometer, the cytotoxicity of the probe was tested by the CCK-8 method, and finally the probe was applied to detect the level change of hypochlorite in cells.

[0020] (2) The present invention shows extremely high sensitivity and selectivity to hypochlorite, can accurately identify and detect hypochlorite, and is not interfered by other reactive oxygen species, ensuring the reliability of the detection results. The detection method is simple and easy to perform, and only a common fluorescence spectrophotometer is needed to complete it, without complex instruments or operation steps, reducing the use threshold and cost. This reagent can be used to quickly detect hypochlorite in living cells, can monitor the dynamic changes of hypochlorite in cells in real time, and provides a powerful tool for studying its physiological and pathological effects. This reagent has little damage to cells and has good biocompatibility, suitable for long-term living cell experiments, and provides a safe and reliable means for biological research at the cell level. Due to its excellent performance and biocompatibility, this reagent has broad application prospects in the fields of biomedical research, disease diagnosis and treatment, etc., especially has great potential in the research of diseases related to hypochlorite such as inflammation and cancer. Description of the Drawings

[0021] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] The above and other aspects of the present invention will now be described with reference to the accompanying drawings, by way of example only, wherein:

[0023] Figure 1 is a flow chart of the preparation method of the near-infrared fluorescence probe of the present invention;

[0024] Figure 2 is the synthetic route for preparing the near-infrared fluorescence probe of the present invention;

[0025] Figure 3 is the ultraviolet spectrum of the near-infrared fluorescence probe in response to hypochlorite;

[0026] Figure 4 is the fluorescence spectrum of the near-infrared fluorescence probe in response to hypochlorite;

[0027] Figure 5 is according to Figure 3 made working curve graph of the near-infrared fluorescence probe;

[0028] Figure 6 is the fluorescence spectrum of the near-infrared fluorescence probe in response to hypochlorite under PBS conditions with different pH values (3, 4, 5, 6, 7, 7.4, 8, 9, 10, 11);

[0029] Figure 7 is the reaction kinetic fluorescence spectrum of the near-infrared fluorescence probe;

[0030] Figure 8 is the fluorescence spectrum of the anti-interference ability of the near-infrared fluorescence probe;

[0031] Figure 9 is the cytotoxicity histogram of the near-infrared fluorescence probe;

[0032] Figure 10 is the fluorescence imaging diagram of detecting hypochlorite in HT22 cells with different probe concentrations;

[0033] Figure 11 is the chemical structure diagram of the near-infrared fluorescence probe DCI-OEt-HClO.

[0034] Note: The test system is a mixed solution of 1200 μL PBS (pH = 7.4) and 800 μL DMF Detailed implementation manners

[0035] In order to better understand the above technical solutions, the above technical solutions will be described in detail below in conjunction with the accompanying drawings of the specification and specific implementation manners.

[0036] As Figures 1-11 shown, Example 1: The method for preparing a near-infrared fluorescence probe for detecting hypochlorite adopted by the present invention is specifically implemented according to the following steps:

[0037] Step 1: Isophorone and malononitrile were dissolved in absolute ethanol, heated for dissolution, and piperidine was added dropwise. Under a nitrogen atmosphere, it was heated under reflux at 85 °C for 4 - 6 hours. After the reaction ended, it was cooled to room temperature, extracted three times with dichloromethane and saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under vacuum, and separated and purified by column chromatography (V PE / V EA = 60:1) to obtain a white solid powder, which is Compound 1, with a yield of 75%;

[0038] Step 2: Compound 1 and p-hydroxybenzaldehyde were dissolved in an acetonitrile solution, piperidine was added dropwise, and the temperature was raised to 85 °C and refluxed for 6 hours. After the reaction ended, it was cooled to room temperature, extracted three times with dichloromethane and saturated brine, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under vacuum, and separated and purified by column chromatography (V PE / V EA = 10:1) to obtain a light yellow solid powder, which is Compound 2, with a yield of 65%;

[0039] Step 3: Compound 2 and hexamethylenetetramine (HMTA) were dissolved in trifluoroacetic acid and heated under reflux at 75 °C for 3 hours. After the reaction ended, it was cooled to room temperature, extracted three times with dichloromethane and ice water, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under vacuum, and separated and purified by column chromatography (V PE / V EA = 15:1) to obtain a yellow solid powder, which is Compound 3, with a yield of 40%;

[0040] Step 4: Compound 3 and ethoxycarbonylmethylenetriphenylphosphine were dissolved in toluene, stirred at 50 °C for 12 hours under nitrogen protection and in the dark, then concentrated under vacuum after completion, and separated and purified by column chromatography (V PE / V EA = 5:1) to obtain an orange-red solid powder, which is Compound 4, with a yield of 85%;

[0041] Step 5: Compound 4 was dissolved in dichloromethane, DIPEA was added at 0 °C and under a nitrogen atmosphere, then dimethylaminothiocarbonyl chloride was added and stirred for 5 minutes, and it was transferred to room temperature and stirred for 2 - 3 days. After the reaction ended, it was concentrated under vacuum, and separated and purified by column chromatography (V PE / V EA = 10:1) to obtain a light yellow solid powder DCI-OEt-HClO.

[0042] In Step 1, the molar ratio of isophorone to malononitrile was 1:3, and the amount of piperidine was 200 μL;

[0043] In Step 2, the molar ratio of Compound 1 to p-hydroxybenzaldehyde was 1:1, and the amount of piperidine was 200 μL;

[0044] In step 3, the molar ratio of compound 2 to hexamethylenetetramine is 1:2;

[0045] In step 4, the molar ratio of compound 3 to ethoxycarbonylmethylenetriphenylphosphine is 1:1.5;

[0046] In step 5, the molar ratio of compound 4, DIPEA, and dimethylaminothiocarbonyl chloride is 1:2:4.

[0047] Example 2: The spectral properties of the fluorescent probe prepared in this example were tested as follows:

[0048] 2.1. Preparation of the probe: The probe concentration was 2 mmol / L. 0.0019 g of DCI-OEt-HClO was weighed and dissolved in 2 mL of DMSO;

[0049] 2.2. UV titration experiment of the fluorescent probe: The wavelength scanning range was 300 - 700 nm. The test system was a solution with a volume ratio of DMF to PBS with a pH of 7.4 of 4:6. 15 μmol / L of the probe (2 mmol / L) was added. As the hypochlorite was added, the absorption peak at 400 nm decreased, and a new absorption peak appeared at 500 nm and gradually increased. When the concentration of hypochlorite finally reached 70 μmol / L, the absorbance no longer changed;

[0050] 2.3. Fluorescence titration experiment of the fluorescent probe: The test system was a solution with a volume ratio of DMF to PBS with a pH of 7.4 of 4:6. 15 μmol / L of the probe (2 mmol / L) was added. The excitation wavelength of the fluorescence spectrophotometer was set to 610 nm, and the wavelength scanning range was 620 - 800 nm. When the concentration of hypochlorite increased, the fluorescence intensity of the probe DCI-OEt-HClO at 708 nm increased accordingly. When the concentration of hypochlorite reached 70 μmol / L, the fluorescence intensity increased by approximately 121 times and reached the maximum value;

[0051] 2.4. Determination of the detection limit (LOD) of the fluorescent probe: The test system was a solution with a volume ratio of DMF to PBS with a pH of 7.4 of 4:6. 15 μmol / L of the probe (2 mmol / L) was added. When the concentration of hypochlorite was in the range of 0 - 70 μmol / L, as the concentration of hypochlorite increased, the fluorescence intensity value of DCI-OEt-HClO increased linearly. The regression equation of the probe DCI-OEt-HClO was y = 2776.55128 + 1655.84141 (R2 = 0.999). According to the calculation formula for the detection limit 3δ / k (σ: standard deviation of the fluorescence intensity of the probe 10 times, k: slope of the linear equation), the LOD was calculated, and the detection limit of the probe DCI-OEt-HClO for hypochlorite was 85.5 nmol / L;

[0052] 2.5. The stability of the fluorescent probe DCI-OEt-HClO was tested within the pH range of 3 to 11. The fluorescence intensity of the pure probe was very weak within the pH range of 3 to 11, indicating that the probe was very stable. When hypochlorite was added, the fluorescence intensity of the probe changed significantly within the pH range of 7 to 11;

[0053] 2.6. The change in fluorescence intensity of the probe DCI-OEt-HClO with time after adding 70 μmol / L hypochlorite was tested. After adding hypochlorite, the fluorescence intensity increased rapidly within 15 seconds, reached a plateau at 16 seconds, and remained stable within 10 minutes;

[0054] 2.7. The specificity of the probe DCI-OEt-HClO was tested. The concentration of the probe was 15 μmol / L and the concentration of the analyte to be tested was 200 μM. We selected Na + , NO 2 - , Ca 2+ , Cl - , Mg 2+ , SO 4 2- , Cu 2+ , Zn 2+ , Fe 3+ , Fe 2+ , HCO 3 2- , CO 3 2- , Hcy, GSH, Cys, H 2 , S, TBHP, NO, ONOO - ; 1 O 2 , H 2 O 2 , . OH, NaClO; as analytes to be tested, whether the probe could recognize and respond to them was tested. At the emission wavelength of 708 nm, only the addition of sodium hypochlorite led to a significant increase in the fluorescence signal of the probe DCI-OEt-HClO, and the addition of other analytes to be tested did not cause an obvious change in the fluorescence signal of the probe. The results indicated that the probe DCI-OEt-HClO had a specific recognition response to hypochlorite;

[0055] Example 3: Cytotoxicity of the probe DCI-OEt-HClO:

[0056] 3.1. Cell culture: HT22 cells were cultured using DMEM medium containing 10% FBS. The cell culture dish was placed in a CO 2 incubator (37 °C, 5% CO2 ) Cultured. The cells grow in a monolayer in an adherent state. When the cells grow to about 80-90% of the bottom surface of the culture dish, 0.25% trypsin is used to digest the adherent cells for subculture or subsequent cell experiments;

[0057] 3.2, Cytotoxicity test: HT22 cells were seeded in 96-well plates at a cell density of about 10 5 cells / mL, shaken on a microplate shaker, and left standing overnight in an incubator. After removing the original culture medium, 90 μL of fresh culture medium containing different concentrations of the probe (0, 1, 5, 10, 15, and 20 μmol / L) was added, and then the 96-well plate was placed in a CO 2 incubator and incubated for 24 hours. After 24 hours, CCK-8 reagent (10 μL per well) was added, and the 96-well plate was incubated in the incubator for 2 hours. After completion, the absorbance (OD) of each well was measured at 450 nm using a microplate reader. The cell viability was calculated according to the formula. Six parallel wells were set for each concentration, and the test was repeated 3 times. Through the analysis of the experimental results, it was found that when the probe concentration was 20 μmol / L, the cell survival rate was still above 82%, the cytotoxicity was very small, and it had good biocompatibility;

[0058] Example 4: Cell fluorescence imaging experiment of probe DCI-OEt-HClO:

[0059] 4.1. In the cell fluorescence imaging experiment, a probe concentration-dependent experiment was carried out. The probe concentrations were set to 1 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, and 20 μmol / L. After 10 minutes, confocal imaging was performed. As the probe concentration increased, the fluorescence intensity increased;

[0060] For those of ordinary skill in the art, various modifications to the present disclosure will be obvious, and the general principles defined herein can be applied to other variations without departing from the scope of the present disclosure. Therefore, the present disclosure is not limited to the examples and designs described herein, but should be accorded the widest scope consistent with the principles and novel features disclosed herein. Although one or more exemplary embodiments of the present disclosure have been described with reference to the accompanying drawings, those of ordinary skill in the art will understand that various changes in form and detail can be made therein without departing from the spirit and scope of the present disclosure as defined by the appended claims.

Claims

1. A method for preparing a hypochlorous acid near-infrared fluorescent probe, characterized in that: The method is specifically implemented according to the following steps: Step 1, dissolving isophorone and malononitrile in anhydrous ethanol, heating to dissolve, adding piperidine dropwise, heating and reflux at 85°C for 4-6 hours under a nitrogen atmosphere, cooling to room temperature after the reaction, extracting three times with dichloromethane and saturated brine, combining the organic phases, drying over anhydrous sodium sulfate, concentrating in vacuo, and performing column chromatography (V PE / V EA =60:1) to obtain a white solid powder, which is compound 1, with a yield of 75%; Step 2, compound 1 and p-hydroxybenzaldehyde are dissolved in acetonitrile solution, piperidine is added dropwise, the temperature is raised to 85°C and refluxed for 6 hours, after the reaction is completed, the mixture is cooled to room temperature, extracted three times with dichloromethane and saturated brine, the organic phases are combined, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography (V PE / V EA =10:1) separation and purification to obtain a light yellow solid powder, compound 2, with a yield of 65%; Step 3, compound 2 and hexamethylenetetramine (HMTA) were dissolved in trifluoroacetic acid, heated under reflux at 75°C for 3 hours, and after the reaction was completed, the mixture was cooled to room temperature and extracted three times with dichloromethane and ice water. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated in vacuo, and purified by column chromatography (V PE / V EA =15:1) separation and purification to obtain a yellow solid powder, compound 3, with a yield of 40%; Step 4, compound 3 and ethoxycarbonyl methylene triphenylphosphine were dissolved in toluene, stirred at 50°C for 12 hours under nitrogen protection and light protection, and then concentrated in vacuo. PE / V EA =5:1) separation and purification to obtain an orange-red solid powder, compound 4, with a yield of 85%; Step 5, compound 4 was dissolved in dichloromethane, DIPEA was added at 0°C under nitrogen atmosphere, and then dimethylaminothiocarbonyl chloride was added and stirred for 5 minutes, and then the mixture was transferred to room temperature and stirred for 2 to 3 days. After the reaction was completed, the mixture was concentrated in vacuo and purified by column chromatography (V PE / V EA =10:1) to obtain a pale yellow solid powder DCI-OEt-HClO by separation and purification.

2. A method for preparing a near-infrared fluorescent probe according to claim 1, characterized in that: In the step 1, the molar ratio of isophorone to malononitrile is 1:3, and the amount of piperidine is 200 μL.

3. A method for preparing a near-infrared fluorescent probe according to claim 1, characterized in that: In the step 2, the molar ratio of compound 1 to p-hydroxybenzaldehyde is 1:1, and piperidine is 200 μL.

4. The method for preparing a near-infrared fluorescent probe according to claim 1, characterized in that: In the step 3, the molar ratio of compound 2 to hexamethylenetetramine is 1:

2.

5. The method for preparing a near-infrared fluorescent probe according to claim 1, characterized in that: In the step 4, the molar ratio of compound 3 to ethoxyformylmethylenetriphenylphosphine is 1:1.

5.

6. The method for preparing a near-infrared fluorescent probe according to claim 1, characterized in that: In the step 5, the molar ratio of compound 4, DIPEA and dimethylaminothiocarbonyl chloride is 1:2:

4.

7. The method for preparing a near-infrared fluorescent probe according to claim 1, characterized in that: The near-infrared fluorescent probe is used for detecting hypochlorite in solutions and cells.

Images