Dual-responsive fluorescent probe for ferrous ions and viscosity in Parkinson's disease models, as well as preparation method and application thereof

Abstract

The present invention discloses a dual-responsive fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model. The probe is an "off-on" fluorescent probe that can respond well to both ferrous ions and viscosity. It has strong specificity, low background interference, high detection sensitivity, and an emission wavelength with good biocompatibility and low biological background fluorescence. It has been successfully applied to fluorescence imaging in living cells and in the brain tissue of nematodes and fruit flies. The present invention also discloses a preparation method and application. The preparation method is simple to operate and the conditions are mild. The fluorescent probe of the present invention has been successfully applied to biological systems including living cells, Caenorhabditis elegans, and fruit flies, and ferrous ion and viscosity levels have been successfully detected using a confocal microscope.

Description

Technical Field

[0001] The present invention relates to the technical field of organic functional molecules and fluorescent probes, and in particular to a fluorescent probe that responds 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. To date, there is a lack of effective diagnostic methods, which prevents early diagnosis of the disease and there is no clinical treatment plan that accurately targets 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. Therefore, monitoring Fe 2+ The dynamic changes of viscosity and 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 relatively 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 for ferrous ions and viscosity in Parkinson's disease models, which has the advantages of strong specificity, low background interference, high detection sensitivity, emission wavelength, good biocompatibility, and low biological background fluorescence, and has been 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-responsive fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model for use in the preparation of a dual-responsive 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 dual-responsive fluorescent probe to ferrous ions and viscosity in a Parkinson's disease model is provided, which has the structure of the following formula (I):

[0009]

[0010] (I).

[0011] 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:

[0012] Step 1: 5-diethylamino-2-nitrosophenol hydrochloride and tert-butyl-2-hydroxyphenylethylcarbamate are mixed as raw materials and subjected to multi-step reactions to prepare an intermediate compound of formula (II);

[0013] 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 performing column chromatography to prepare the compound of formula (I);

[0014]

[0015] (II).

[0016] Preferably, in step 2, the molar ratio of the compound of formula (II), m-chloroperbenzoic acid, and sodium bicarbonate is 1:2:1.

[0017] Preferably, in step 2, the organic solvent is dichloromethane.

[0018] Preferably, in step 2, the reaction conditions are: reaction at room temperature and reaction time is 3 h.

[0019] Preferably, in step 2, the eluent used for column chromatography is dichloromethane and methanol in a volume ratio of 5:1.

[0020] Preferably, in step 1, the mixing molar ratio of 5-diethylamino-2-nitrosophenol hydrochloride to tert-butyl-2-hydroxyphenethylcarbamate is 1:1.

[0021] The purpose of the present invention can be further achieved by using a dual-responsive fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model in the preparation of a ferrous ion and viscosity detection probe.

[0022] Preferably, the fluorescent probe specifically detects ferrous ions and viscosity at the cellular level.

[0023] Preferably, the fluorescent probe is used to detect a nematode model of Parkinson's disease or a fruit fly model of Parkinson's disease in nematodes or fruit flies.

[0024] The present invention has at least the following beneficial effects:

[0025] 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, and low biological background fluorescence. It has been successfully applied to fluorescence imaging in living cells and in the brain tissues of nematodes and fruit flies.

[0026] 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 simple reaction operation and mild conditions.

[0027] Third, the dual-responsive 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 at the PC-12 cell level, nematode and Drosophila models.

[0028] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of compound 5 prepared in Inventive Example 1;

[0030] Figure 2 is the mass spectrum of compound 5 prepared in Inventive Example 1;

[0031] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe P5 prepared in Example 1 of the present invention;

[0032] Figure 4 is the carbon NMR spectrum of the fluorescent probe P5 prepared in Example 1 of the present invention;

[0033] Figure 5 is the mass spectrum of the fluorescent probe P5 prepared in Example 1 of the present invention;

[0034] 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 ;

[0035] 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;

[0036] 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 spectrum in B is the fluorescence spectrum 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 ;

[0037] Figure 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;

[0038] Figure 10 This is a graph showing the effect of fluorescent probe P5 on PC12 cell viability in Inventive Example 6;

[0039] Figure 11 The fluorescence probe P5 in Example 7 of the present invention is used to detect Fe 2+ and viscosity-specific bioimaging;

[0040] Figure 12 The fluorescent probe P5 in Example 8 of the present invention is Fe 2+ and fluorescence imaging of viscosity detection;

[0041] Figure 13 Fe ions of the fluorescent probe P5 in the Parkinson's disease fruit fly brain model in Example 9 of the present invention 2+ Fluorescence imaging of viscosity detection. DETAILED DESCRIPTION

[0042] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0043] It should be understood that terms such as “having,” “including,” and “comprising” used herein do not prescribe the existence or addition of one or more other elements or combinations thereof.

[0044] 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.

[0045] Main equipment: UV-visible spectrophotometer (UH-5300, Hitachi, Japan), fluorescence spectrophotometer (F-7100, Hitachi, Japan), rotary evaporator (RE-2000B, Hangzhou Ruijia Precision Scientific Instrument), microplate reader (SpectraMax190, Meigu Molecular Instrument), laser confocal microscope (FV-1000, Olympus, Japan).

[0046] Main chemical reagents: methanol (AR, Thermo Scientific (China)), dichloromethane (CH2Cl2) (AR, Tianjin Damao Chemical Reagent Factory), DMEM high glucose basal medium (Wuhan Punosai Life Science Technology Co., Ltd.), fetal bovine serum (FBS) (Wuhan Punosai Life Science Technology Co., Ltd.), penicillin-streptomycin solution (Wuhan Punosai Life Science Technology Co., Ltd.), 0.25% trypsin solution (Wuhan Punosai Life Science Technology Co., Ltd.), anhydrous ethanol (AR, Tianjin Zhiyuan Chemical Reagent), nystatin (Biyuntian), Erastin (Aladdin), Fer-1 (Aladdin), ferrous chloride (B&K).

[0047] Example 1

[0048] A dual-responsive fluorescent probe for ferrous ions and viscosity in a Parkinson's disease model has the following structure:

[0049]

[0050] The specific synthetic route is as follows:

[0051]

[0052]

[0053] The specific synthesis steps are as follows:

[0054] 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 reaction 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 shown as follows Figure 1 As shown, the mass spectrum Figure 2 As shown;

[0055] 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 at room temperature for 3 h. After the TLC plate is monitored and the reaction is confirmed to be complete, the reaction solution is dried under reduced pressure and purified by silica gel column chromatography (dichloromethane: methanol = 5:1, v / v) to obtain fluorescent probe P5 as a yellow solid (263 mg, 50%). The hydrogen spectrum, carbon spectrum and mass spectrum of the fluorescent probe P5 are as follows: Figure 3 、 Figure 4 and Figure 5 shown.

[0056] Example 2

[0057] The fluorescent probe P5 prepared in Example 1 was detected at different concentrations of Fe 2+ Mid-UV absorption and fluorescence spectroscopy test:

[0058] 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 to prepare a P5 stock solution with a final concentration of 1 mM. The solution was stored at -20°C for later use. Different concentrations of Fe 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 as follows: 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.

[0059] Example 3

[0060] Ultraviolet absorption and fluorescence spectrum tests of the fluorescent probe P5 prepared in Example 1 in different proportions of glycerol:

[0061] 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. The solution was stored at -20°C for later use. HEPES buffer solutions with different proportions of glycerol (volume percentages of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, and 90%) were prepared and stored at 4°C in the dark for later use. 20 μL of P5 stock solution was added to the HEPES buffer solutions with different proportions of glycerol to make the final concentration of P5 10 μM. After mixing evenly, the solution was transferred to a fluorescence cell to test the absorption spectrum and fluorescence emission spectrum of the solution system. The results are shown in the figure below. Figure 7 Middle A, Figure 7 Middle B, by Figure 7 It can be seen that with the increase of the glycerol ratio, the absorption intensity and fluorescence intensity of P5 will increase slightly.

[0062] Example 4

[0063] 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:

[0064] 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 to prepare a P5 stock solution with a final concentration of 1 mM. The solution was stored at -20°C until 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 in the dark at 4°C until use. 2+ 20 μL of P5 stock solution was added to HEPES buffer containing 10% glycerol by volume to make the final concentration of P5 10 μM. After mixing evenly, the solution was transferred to a fluorescence cell to test the absorption spectrum and fluorescence emission spectrum of the solution system. The results are shown in the figure. 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.

[0065] Example 5

[0066] The fluorescent probe P5 prepared in Example 1 was 2+ UV absorption and fluorescence spectrum tests of glycerol with different proportions:

[0067] 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 to prepare a P5 stock solution with a final concentration of 1 mM. The solution was stored at -20°C until 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 of 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 a fluorescence cell to measure the absorption spectrum and fluorescence emission spectrum of the solution system. The results are shown in the figure below. Figure 9 Middle A, Figure 9 Middle B, by Figure 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.

[0068] Example 6

[0069] Effect of the fluorescent probe P5 prepared in Example 1 on the survival rate of PC12 cells:

[0070] 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 cultured in a 37°C incubator for 4 h. The absorbance at λmax = 490 nm was measured with a microplate reader, and the cell survival rate was calculated. The test results are shown in Figure 2. Figure 10 As shown by Figure 10 It can be seen that P5 has low cytotoxicity and high biosafety.

[0071] Example 7

[0072] The fluorescent probe P5 prepared in Example 1 was used to detect Fe 2+ Cell imaging with glycerol:

[0073] 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 a clean bench, PC12 cells were seeded in a culture dish. When the cells grew to about 70%, nystatin and exogenous Fe 2+PC12 cells were treated with ferroptosis activators and ferroptosis inhibitors for 4 hours. The cells were then washed three times with PBS and incubated in a 10 mM P5 medium. The treated PC12 cells were placed in an incubator for 30 minutes. The cells were then washed three times with PBS and quickly imaged under a confocal microscope. The test results are shown in Figure 2. Figure 11 As shown, after the ferroptosis activator Erastin induced PC-12 cells, the intracellular Fe 2+ and viscosity will increase. Figure 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 have a good response.

[0074] Example 8

[0075] The fluorescent probe P5 prepared in Example 1 is effective for Fe 2+ and fluorescence imaging of glycerol:

[0076] First synchronize the nematodes so that the growth cycles of wild-type and Parkinson's model nematodes are consistent. Pick out 5 nematodes in the egg-laying period from the culture dish of wild-type nematodes and put them into the corresponding culture dish, pick out 5 nematodes in the egg-laying period from the culture dish of Parkinson's nematodes and put them into the corresponding culture dish, pick out 5 nematodes in the egg-laying period from the culture dish of Parkinson's nematodes and put them into the culture dish containing iron death inhibitor, and then put them into a constant temperature and humidity incubator at 20°C for culture. 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 these nematodes in a PBS solution with a concentration of 100μM probe for 5 minutes. Then pick the nematodes onto the culture medium without OP50. After the food and probes on the surface of the nematodes' body are removed, pick the nematodes onto a 2% agar slide for fixation and perform confocal fluorescence imaging. The test results are as follows: Figure 12 As shown by Figure 12 It can be seen that the fluorescent probe P5 of the present invention can distinguish Parkinson's disease nematodes from wild-type nematodes.

[0077] Example 9

[0078] The fluorescent probe P5 prepared in Example 1 is effective for Fe 2+ and fluorescence imaging of glycerol:

[0079] First, synchronize the fruit flies so that the growth cycles of wild-type and Parkinson's model fruit flies are consistent. Among them, a part 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: Figure 13 As shown by Figure 13 It can be seen that the fluorescent probe P5 of the present invention can distinguish Parkinson's disease nematodes from wild-type nematodes.

[0080] 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 Parkinson's disease progression and can be extended to fluorescence imaging of other disease-related biomarkers.

[0081] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and exemplary embodiments. They can be applied to a variety of fields suitable for the present invention. Further modifications will be readily apparent to those skilled in the art. Therefore, the present invention is not limited to the specific details and illustrations shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims

1. A dual-responsive 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 to ferrous ions and viscosity in a Parkinson's disease model as claimed in claim 1, characterized in that: The steps include: Step 1: 5-diethylamino-2-nitrosophenol hydrochloride and tert-butyl-2-hydroxyphenylethylcarbamate are mixed as raw materials and subjected 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 performing column chromatography to prepare the compound of formula (I); (II).

3. The method according to claim 2, wherein 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, wherein In step 2, the organic solvent is dichloromethane.

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

6. The method according to claim 2, wherein 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, wherein In step 1, the mixing molar ratio of 5-diethylamino-2-nitrosophenol hydrochloride and tert-butyl-2-hydroxyphenethylcarbamate 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 nematodes or fruit flies.

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