Fluorescent probe with dual response to ferrous ions and viscosity in Parkinson's disease model as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a dual-response fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model, which is an off-on type fluorescent probe, can well realize dual response to ferrous ions and viscosity, and has the advantages of strong specificity, low background interference, high detection sensitivity, emission wavelength, better biocompatibility, low biological background fluorescence and the like. The fluorescent probe is successfully applied to fluorescence imaging in living cells and brain tissues of nematodes and drosophila melanogaster. The invention further discloses a preparation method and application. The preparation method is easy to operate and mild in condition. The fluorescent probe is successfully applied to biological systems including living cells, caenorhabditis elegans and drosophila melanogaster, and ferrous ion and viscosity level detection is successfully carried out by using a confocal microscope.

Description

Technical Field

[0001] The invention relates to the technical field of organic functional molecules and fluorescent probes, and in particular to a fluorescent probe with dual response to ferrous ions and viscosity in a Parkinson's disease model, and a preparation method and application thereof. Background Art

[0002] Parkinson's Disease (PD) is the second most common neurodegenerative disease. Statistics show that half of the world's patients are in China. Parkinson's disease has become the third largest killer of middle-aged and elderly people in my country. So far, there is a lack of effective diagnostic methods, which cannot achieve early diagnosis of the disease, and there is no clinically targeted treatment plan for the PD phenotype. Ferroptosis is an iron-dependent programmed cell death. In recent years, many studies have shown that ferroptosis is involved in the pathological process and pathogenesis of PD. The cell death form of ferroptosis is closely related to abnormal iron metabolism and lipid peroxidation. Ferrous ions and viscosity changes play an important role in inducing cell death. It can be seen that monitoring Fe 2+ The dynamic changes of viscosity are of great significance for the early diagnosis of Parkinson's disease.

[0003] Existing Fe 2+ Fluorescent probes still have the disadvantages of low specificity and sensitivity, and are subject to large background interference when used as fluorescent probes for biological analysis. Summary of the invention

[0004] An object of the present invention is to solve at least the above problems and / or disadvantages and to provide at least the advantages which will be described hereinafter.

[0005] Another object of the present invention is to provide a dual-responsive fluorescent probe to ferrous ions and viscosity in a Parkinson's disease model, which has the advantages of strong specificity, low background interference, high detection sensitivity, emission wavelength, good biocompatibility, low biological background fluorescence, etc., and is successfully applied to fluorescence imaging in living cells and in the brain tissues of nematodes and fruit flies.

[0006] Another object of the present invention is to provide a method for preparing a fluorescent probe that responds dually to ferrous ions and viscosity in a Parkinson's disease model, wherein the reaction operation is simple and the conditions are mild.

[0007] Another object of the present invention is to provide a dual-response fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model for use in the preparation of a dual-response probe for ferrous ions and viscosity in a Parkinson's disease model, which can achieve the purpose of ferrous ion and viscosity detection at the PC-12 cell level, nematode and fruit fly models.

[0008] In order to achieve these purposes and other advantages according to the present invention, a fluorescent probe with dual response to ferrous ions and viscosity in a Parkinson's disease model is provided, which has the structure of the following formula (I): (I).

[0009] The purpose of the present invention can be further achieved by a method for preparing a dual-responsive fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model, the preparation method comprising the following steps: Step 1, using 5-diethylamino-2-nitrosophenol hydrochloride and tert-butyl-2-hydroxyphenylethyl carbamate as raw materials, mixing and subjecting to multi-step reactions to prepare an intermediate compound of formula (II); Step 2: reacting the compound of formula (II) with m-chloroperbenzoic acid and sodium bicarbonate in an organic solvent, removing the solvent by vortexing, and preparing the compound of formula (I) by column chromatography; (II).

[0010] Preferably, in step 2, the molar ratio of the compound of formula (II), m-chloroperbenzoic acid and sodium bicarbonate is 1:2:1.

[0011] Preferably, in step 2, the organic solvent is dichloromethane.

[0012] Preferably, in step 2, the reaction conditions are: reaction at room temperature and reaction time is 3 h.

[0013] Preferably, in step 2, the eluent used for column chromatography is dichloromethane and methanol in a volume ratio of 5:1.

[0014] Preferably, in step 1, the mixing molar ratio of 5-diethylamino-2-nitrosophenol hydrochloride to tert-butyl-2-hydroxyphenethylcarbamate is 1:1.

[0015] The purpose of the present invention can be further achieved by using a dual-response fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model in the preparation of a ferrous ion and viscosity detection probe.

[0016] Preferably, the fluorescent probe specifically detects ferrous ions and viscosity at the cellular level.

[0017] Preferably, the fluorescent probe is used to detect a nematode model of Parkinson's disease or a fruit fly model of Parkinson's disease on a nematode or a fruit fly.

[0018] The present invention has at least the following beneficial effects: First, the dual-responsive fluorescent probe for ferrous ions and viscosity in the Parkinson's disease model of the present invention has the advantages of strong specificity, low background interference, high detection sensitivity, emission wavelength, good biocompatibility, low biological background fluorescence, etc., and has been successfully applied to fluorescence imaging in living cells and in the brain tissues of nematodes and fruit flies.

[0019] Second, the method of the present invention for preparing a dual-responsive fluorescent probe to ferrous ions and viscosity in a Parkinson's disease model has a simple reaction operation and mild conditions.

[0020] Third, the dual-response fluorescent probe for ferrous ions and viscosity in the Parkinson's disease model of the present invention can achieve the purpose of ferrous ion and viscosity detection in PC-12 cell level, nematode and fruit fly models.

[0021] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of compound 5 prepared in Inventive Example 1; Figure 2 is the mass spectrum of compound 5 prepared in Inventive Example 1; Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe P5 prepared in Example 1 of the present invention; Figure 4 is the carbon nuclear magnetic resonance spectrum of the fluorescent probe P5 prepared in Example 1 of the present invention; Figure 5 is the mass spectrum of the fluorescent probe P5 prepared in Example 1 of the present invention; Figure 6 A is the fluorescence probe P5 in Example 2 of the present invention at different concentrations of Fe 2+ The UV normalized spectrum in B is the fluorescence spectrum of the fluorescent probe P5 in Example 2 of the present invention at different concentrations of Fe 2+ Fluorescence emission spectra in ; Figure 7 A is the UV normalized spectrum of the fluorescent probe P5 in Example 3 of the present invention in different proportions of glycerol, and B is the fluorescence emission spectrum of the fluorescent probe P5 in Example 3 of the present invention in different proportions of glycerol; Figure 8 A is the fluorescence probe P5 in Example 4 of the present invention in 10% glycerol with different concentrations of Fe 2+ The UV normalized spectra in B are the fluorescence spectra of the fluorescent probe P5 in Example 4 of the present invention at different concentrations of Fe in 10% glycerol. 2+ Fluorescence emission spectra in ; Fig. 9 The fluorescence probe P5 in Example 5 of the present invention is 100 μM Fe 2+ UV normalized spectra in different proportions of glycerol, B is the UV normalized spectra of the fluorescent probe P5 in Example 5 of the present invention in 100 μM Fe 2+ Fluorescence emission spectra in different ratios of glycerol; Fig.10 This is a graph showing the effect of the fluorescent probe P5 on the survival rate of PC12 cells in Invention Example 6; Fig.11 The fluorescence probe P5 in Example 7 of the present invention is used to detect Fe 2+ and viscosity-specific bioimaging; Fig.12 The fluorescent probe P5 in Example 8 of the present invention is used to detect Fe in the Parkinson's disease nematode model. 2+ and fluorescence imaging of viscosity detection; Fig.13 The Fe of the fluorescent probe P5 in Example 9 of the present invention in the Parkinson's disease fruit fly brain model 2+ Fluorescence imaging of the viscosity detection. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0026] It should be understood that terms such as “having”, “including” and “comprising” used herein do not specify the existence or addition of one or more other elements or combinations thereof.

[0027] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0028] Main equipment: UV-visible spectrophotometer (UH-5300, Hitachi, Japan), fluorescence spectrophotometer (F-7100, Hitachi, Japan), rotary evaporator (RE-2000B, Hangzhou Ruijia Precision Scientific Instruments), microplate reader (SpectraMax190, Meigu Molecular Instruments), laser confocal microscope (FV-1000, Olympus, Japan).

[0029] Main chemical reagents: methanol (AR, Thermo Scientific (China)), dichloromethane (CH 2 Cl 2) (AR, Tianjin Damao Chemical Reagent Factory), DMEM high glucose basal medium (Wuhan Pronose Life Science Technology Co., Ltd.), fetal bovine serum (FBS) (Wuhan Pronose Life Science Technology Co., Ltd.), penicillin-streptomycin solution (Wuhan Pronose Life Science Technology Co., Ltd.), 0.25% trypsin solution (Wuhan Pronose Life Science Technology Co., Ltd.), anhydrous ethanol (AR, Tianjin Zhiyuan Chemical Reagent), nystatin (Biyuntian), Erastin (Aladdin), Fer-1 (Aladdin), ferrous chloride (B&K).

[0030] Example 1 A fluorescent probe that responds to ferrous ions and viscosity in a Parkinson's disease model has the following structure (I): The specific synthetic route is as follows: The specific synthesis steps are as follows: Among them, the synthetic steps from compound 1 to compound 4 are referenced as follows: ACS Chem. Biol . 2020, 15,1913-1920, the synthesis steps of compound 4 to compound 5 are as follows: compound 4 (1 mmol, 283 mg) was dissolved in 10 mL of dichloromethane to form a suspension, 0.2 mL of triethylamine was added thereto, and stirred until a homogeneous solution was obtained, cooled to -20°C, acetyl chloride (1 mmol, 71 μL) was slowly added, and after reacting for 15 min, silica gel column chromatography was performed for separation and purification (dichloromethane: methanol = 8:1, v / v) to obtain compound 5 (283 mg, yield 87%). The H NMR spectrum of compound 5 is as follows Figure 1 As shown, the mass spectrum Figure 2 As shown; The synthesis steps from compound 5 to compound P5 are as follows: weigh compound 5 (500 mg, 1.54 mmol), m-chloroperbenzoic acid (532 mg, 3.08 mmol) and sodium bicarbonate (129 mg, 1.54 mmol) in a 100 mL round-bottom flask with magnetic stirring, add 30 mL of dichloromethane, and stir the reaction at room temperature for 3 h. Monitor with a TLC plate. After confirming that the reaction is complete, the reaction solution is decompressed and dried, and separated and purified by silica gel chromatography (dichloromethane: methanol = 5:1, v / v) to obtain fluorescent probe P5 as a yellow solid (263 mg, 50%). Among them, the hydrogen spectrum, carbon spectrum and mass spectrum of the fluorescent probe P5 are as follows: Figure 3 , Figure 4 and Figure 5 shown.

[0031] Example 2 The fluorescent probe P5 prepared in Example 1 was detected at different concentrations of Fe 2+ Mid-UV absorption and fluorescence spectroscopy test: 6.0 mg of the pure fluorescent probe P5 prepared in Example 1 was accurately weighed on an analytical balance and dissolved in 18 mL of ethanol solvent to prepare a P5 stock solution with a final concentration of 1 mM, which was stored at -20°C for later use. 2+ (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 μM) HEPES buffer, stored at 4 °C in the dark until use, at different concentrations of Fe 2+ 20 μL P5 stock solution was added to the HEPES buffer to make the final concentration of P5 10 μM. After mixing evenly, the solution was transferred to the fluorescence cell to test the absorption spectrum and fluorescence emission spectrum of the solution system. The results are shown in Figure 6 Middle A, Figure 6 Middle B, by Figure 6 It can be seen that with the increase of Fe 2+ With the increase of concentration, the absorption intensity and fluorescence intensity of P5 will increase significantly.

[0032] Example 3 Ultraviolet absorption and fluorescence spectrum tests of the fluorescent probe P5 prepared in Example 1 in different proportions of glycerol: An analytical balance was used to accurately weigh 6.0 mg of the pure fluorescent probe P5 prepared in Example 1, and the solution was dissolved in 18 mL of ethanol solvent to prepare a P5 stock solution with a final concentration of 1 mM, which was stored at -20°C for later use. HEPES buffer solutions with different proportions of glycerol (volume percentages were 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%, respectively) were prepared and stored at 4°C in the dark for later use. 20 μL P5 stock solution was added to the HEPES buffer solutions with different proportions of glycerol so that the final concentration of P5 was 10 μM. After mixing evenly, the solution was transferred to a fluorescence cell, and the absorption spectrum and fluorescence emission spectrum in the solution system were tested. The results are shown in the figure. Figure 7 Middle A, Figure 7 Middle B, by Figure 7 It can be seen that with the increase of glycerol ratio, the absorption intensity and fluorescence intensity of P5 will increase slightly.

[0033] Example 4 The fluorescent probe P5 prepared in Example 1 was exposed to different concentrations of Fe in 10% glycerol. 2+ UV absorption and fluorescence spectrum test: 6.0 mg of the pure fluorescent probe P5 prepared in Example 1 was accurately weighed on an analytical balance and dissolved in 18 mL of ethanol solvent to prepare a P5 stock solution with a final concentration of 1 mM, which was stored at -20°C for later use. 2+ (0, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 μM) and 10% glycerol in HEPES buffer and store at 4°C in the dark until use. 2+ 20 μL P5 stock solution was added to HEPES buffer containing 10% glycerol by volume, respectively, to make the final concentration of P5 10 μM. After mixing evenly, the solution was transferred to the fluorescence cell to test the absorption spectrum and fluorescence emission spectrum of the solution system. The results are shown in Figure 8 Middle A, Figure 8 Middle B. By Figure 8 It can be seen that with the increase of Fe 2+ With the increase of concentration, the absorption intensity and fluorescence intensity of P5 will increase significantly.

[0034] Example 5 The fluorescent probe P5 prepared in Example 1 was 2+ UV absorption and fluorescence spectra test of glycerol with different proportions: 6.0 mg of the pure fluorescent probe P5 prepared in Example 1 was accurately weighed on an analytical balance and dissolved in 18 mL of ethanol solvent to prepare a P5 stock solution with a final concentration of 1 mM, which was stored at -20°C for later use. Different proportions of glycerol (volume percentages of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%) and 100 μM Fe 2+ 20 μL P5 stock solution was added to HEPES buffer containing different proportions of glycerol to make the final concentration of P5 10 μM. After mixing evenly, the solution was transferred to the fluorescence cell to test the absorption spectrum and fluorescence emission spectrum of the solution system. The results are shown in the figure. Fig. 9 Middle A, Fig. 9 Middle B, by Fig. 9 It can be seen that with the increase of the glycerol ratio, the absorption intensity and fluorescence intensity of P5 are the highest in the environment with a volume percentage of 10% glycerol.

[0035] Example 6 Effect of the fluorescent probe P5 prepared in Example 1 on the survival rate of PC12 cells: PC12 cells were seeded in 96-well plates. When the cells grew to about 70%, different concentrations of P5 (0, 2.5, 5, 10, 15, and 20 μM) were used for 24 h. Then MTT was added and the cells were placed in a 37°C incubator for 4 h. The expression of P5 in the plates was measured using an enzyme-labeled instrument. = absorbance value at 490 nm, and then calculate the cell survival rate. The test results are as follows Fig.10 As shown by Fig.10 It can be seen that P5 has low cytotoxicity and high biosafety.

[0036] Example 7 Example 1: The fluorescence probe P5 prepared in PC12 cells 2+ Cell Imaging with Glycerol: PC12 cells were used as the research object and nystatin, exogenous Fe 2+ , ferroptosis activators, and ferroptosis inhibitors to induce PC12 cells to increase the intracellular Fe 2+ The performance of the fluorescent probe P5 was studied. In the clean bench, PC12 cells were inoculated in a culture dish. When the cells grew to about 70%, nystatin, exogenous Fe 2+ PC12 cells were treated with ferroptosis activators and ferroptosis inhibitors for 4 h. The cells were then washed three times with PBS, and the treated PC12 cells were incubated in a culture medium containing 10 μM P5 for 30 min. The cells were then washed three times with PBS and quickly imaged under a confocal microscope. The test results are shown in the figure. Fig.11 As shown, after the ferroptosis activator Erastin induced PC-12 cells, intracellular Fe 2+ and viscosity will increase. Fig.11 The imaging results show that the fluorescence of probe P5 is the strongest after being induced by the ferroptosis activator Erastin, which proves that probe P5 can activate Fe 2+ And viscosity has a good response.

[0037] Example 8 Example 1 Fluorescent probe P5 prepared for Fe 2+ Fluorescence imaging of glycerol: First synchronize the nematodes to make the growth cycles of wild-type and Parkinson's model nematodes consistent. Pick out 5 nematodes in the egg-laying stage from the culture dish of wild-type nematodes and put them in the corresponding culture dish, pick out 5 nematodes in the egg-laying stage from the culture dish of Parkinson's nematodes and put them in the corresponding culture dish, pick out 5 nematodes in the egg-laying stage from the culture dish of Parkinson's nematodes and put them in the culture dish containing iron death inhibitor, and then culture them in a 20°C constant temperature and humidity incubator. After 4 days, it was observed that the nematodes on the surface of the culture medium had grown into adults. Pick out 5 nematodes from each group of culture medium, and soak them in a PBS solution with a concentration of 100μM probe for 5 minutes. Then pick out the nematodes to the culture medium without OP50. After the food and probes on the surface of the nematodes' body are removed, pick the nematodes to a 2% agar slide for fixation and perform confocal fluorescence imaging. The test results are as follows: Fig.12 As shown by Fig.12 It can be seen that the fluorescent probe P5 of the present invention can distinguish Parkinson's disease type nematodes from wild type nematodes.

[0038] Example 9 Example 1 Fluorescent probe P5 prepared in the Parkinson's disease fruit fly brain model Fe 2+ Fluorescence imaging of glycerol: First, synchronize the fruit flies to make the growth cycles of wild-type and Parkinson's model fruit flies consistent. A portion of the Parkinson's model fruit flies were transferred to a culture bottle containing an iron death inhibitor. They were divided into normal type (WT), Parkinson's disease type (PD), and Parkinson's disease type group treated with an iron death inhibitor. After culturing at 23 degrees Celsius for 20 days, the brain tissue was peeled off and placed in PBS in a 96-well plate. A PBS solution containing 100 μM probe was prepared, and the peeled fruit fly brain was transferred into the prepared probe solution. The probe was incubated for 30 minutes and confocal fluorescence imaging was performed. The test results are as follows: Fig.13 As shown by Fig.13 It can be seen that the fluorescent probe P5 of the present invention can distinguish Parkinson's disease-type nematodes from wild-type nematodes.

[0039] The dual-response fluorescent probe for ferrous ions and viscosity in the Parkinson's disease model of the present invention is an "off-on" fluorescent probe that can well respond to Fe 2+ The probe has excellent properties such as near-infrared fluorescence emission at 650 nm and can simultaneously monitor Fe 2+ It has good selectivity, low background interference, high detection sensitivity and good biocompatibility. It has been successfully applied to biological systems including living cells, Caenorhabditis elegans and Drosophila. Fe 2+This study provides a promising near-infrared fluorescence method for monitoring the progression of Parkinson's disease, which can be extended to the fluorescence imaging of other disease-related biomarkers.

[0040] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily realized. Therefore, without departing from the general concept defined by the claims and equivalent scope, the present invention is not limited to the specific details and the illustrations shown and described here.

Claims

1. A dual-response fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model, characterized in that: It has the structure of formula (I): （I）。 2. A method for preparing the dual-responsive fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model as claimed in claim 1, characterized in that: The steps include: Step 1, using 5-diethylamino-2-nitrosophenol hydrochloride and tert-butyl-2-hydroxyphenylethyl carbamate as raw materials, mixing and subjecting to multi-step reactions to prepare an intermediate compound of formula (II); Step 2: reacting the compound of formula (II) with m-chloroperbenzoic acid and sodium bicarbonate in an organic solvent, removing the solvent by vortexing, and preparing the compound of formula (I) by column chromatography; (II).

3. The method according to claim 2, characterized in that In step 2, the molar ratio of the compound of formula (II), m-chloroperbenzoic acid and sodium bicarbonate is 1:2:

1.

4. The method according to claim 2, characterized in that In step 2, the organic solvent is dichloromethane.

5. The method according to claim 2, characterized in that In step 2, the reaction conditions are: room temperature reaction, and the reaction time is 3 h.

6. The method according to claim 2, characterized in that In step 2, the eluent used for column chromatography is dichloromethane and methanol in a volume ratio of 5:

1.

7. The method according to claim 2, characterized in that In step 1, the mixing molar ratio of 5-diethylamino-2-nitrosophenol hydrochloride and tert-butyl-2-hydroxyphenylethylcarbamate is 1:

1.

8. Use of the dual-responsive fluorescent probe to ferrous ions and viscosity in a Parkinson's disease model as claimed in claim 1 in preparing a probe for detecting ferrous ions and viscosity.

9. The use according to claim 8, characterized in that The fluorescent probe specifically detects ferrous ions and viscosity at the cellular level.

10. The use according to claim 8, characterized in that The fluorescent probe is used to detect a Parkinson's disease nematode model or a Parkinson's disease fruit fly model on a nematode or a fruit fly.

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