A coumarin-based biothiol ratiometric fluorescent probe targeting mitochondria and preparation and application thereof
By preparing a coumarin-based biothiol ratio fluorescent probe targeting mitochondria, the complexity and accuracy issues of detecting biothiols in existing technologies have been resolved, achieving rapid, quantitative, and targeted imaging.
Patent Information
- Application Number
- CN202410616946.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-05-17
- Publication Date
- 2025-10-24
- Estimated Expiration
- 2044-05-17
AI Technical Summary
Existing fluorescent probes for detecting biothiols have problems such as complex synthesis routes, long response time, lack of targeted positioning function, and low single-wavelength detection accuracy.
A mitochondrial-targeted coumarin-based biothiol ratiometric fluorescent probe was designed. By simplifying the preparation method and introducing the targeting function, the negative membrane potential of mitochondria was utilized to achieve targeted positioning, and the probe was detected by the ratio change of fluorescence intensity at two wavelengths, 498nm and 567nm.
It enables rapid and quantitative detection of biothiols with high selectivity and sensitivity, and can target mitochondria for specific location imaging, simplifying the preparation process and reducing costs.
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Figure CN118515651B_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of optical imaging, in particular to a coumarin-based biothiol ratiometric fluorescent probe targeting mitochondria and a preparation and application thereof. BACKGROUND
[0002] Intracellular biothiols, such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), play a crucial role in many physiological and pathological processes. Abnormal levels of Cys / Hcy / GSH have been shown to be closely related to a variety of diseases. For example, growth retardation, skin damage, liver damage, edema, lethargy and hair discoloration are associated with lower than normal levels of Cys. Diseases such as Parkinson's disease and Alzheimer's disease are associated with higher than normal levels of Hcy. Neurodegenerative diseases, cancer and AIDS are associated with abnormal levels of GSH. Therefore, given the important physiological and physiological functions of these biothiols, it is of great significance to develop effective detection methods.
[0003] In recent years, fluorescent probes have attracted much attention due to their high sensitivity, good selectivity, simple operation, low cost, non-destructive detection, and fast analysis speed. According to the relevant literature reports, chromene derivatives are good detection sites for biothiols, but the reported fluorescent probes still have some limitations, such as complex synthesis route, long response time, single-wavelength detection, and no targeting localization function. Therefore, it is of great significance and value to develop new biothiol fluorescent probes with chromene derivatives as recognition groups. SUMMARY
[0004] To solve the above problems, the present application provides a novel ratiometric fluorescent probe for recognizing biothiols and targeting mitochondria. The fluorescent probe has a short response time, can realize quantitative detection of biothiols, has high selectivity and sensitivity, and can also target mitochondria to realize imaging analysis at a specific location.
[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:
[0006] The present application provides a coumarin-based biothiol ratiometric fluorescent probe targeting mitochondria, which is 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chroman-7-yl)methyl)pyridine-1- bromide, and its structural formula is:
[0007]
[0008] The present application provides a preparation method of a coumarin-based biothiol ratiometric fluorescent probe targeting mitochondria, comprising the following steps:
[0009] Step 1, synthesis of 5-hydroxymethyl salicylaldehyde: under argon protection, salicylaldehyde was dissolved in concentrated hydrochloric acid, then mass fraction 37% formaldehyde aqueous solution was added. The mixture was heated to 25-70°C and stirred for 24h, after the reaction was completed, it was cooled to room temperature, filtered, the solid was washed with ultrapure water, the solid was collected and dissolved in dimethyl sulfoxide and ultrapure water, then copper sulfate pentahydrate was added, the mixture was heated to 100-120°C and stirred for 3-14h, after the reaction was completed, it was cooled to room temperature, extracted with dichloromethane, the organic phase was combined and washed with brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography to obtain a solid product, i.e. 5-hydroxymethyl salicylaldehyde.
[0010] Step 2, synthesis of 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b] chromen-1(2H)-one: under argon protection, 2-hydroxy-5-hydroxymethylbenzaldehyde, 2-cyclopenten-1-one and imidazole were dissolved in tetrahydrofuran and ultrapure water, and the mixture was stirred at room temperature for 48-96h. After the reaction was completed, the mixture was extracted with dichloromethane, the organic phase was combined and washed with ultrapure water and brine in turn, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography to obtain a solid product, i.e. 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one.
[0011] Step 3, synthesis of 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one: under argon protection, 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one and triphenylphosphine were dissolved in dichloromethane, then N-bromosuccinimide was added. The mixture was stirred at room temperature for 1-14h, then washed with water and brine in turn, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography to obtain a solid product, i.e. 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one.
[0012] Step 4, synthesis of 7-diethylamino-3-(4-pyridyl)coumarin: under argon protection, 4-(diethylamino)salicylaldehyde and 4-pyridine acetic acid hydrochloride were dissolved in dichloromethane, which was cooled to 0°C in an ice bath, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine were added under ice bath. Then, the reaction mixture was slowly warmed to room temperature and stirred for 14-20h. After the reaction was completed, it was washed with water and brine in turn, and dried over anhydrous sodium sulfate. The solvent was evaporated to obtain a crude solid, the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography to obtain a solid product, i.e. 7-diethylamino-3-(4-pyridyl)coumarin.
[0013] Step 5, synthesis of fluorescent probe: under argon protection, 7-diethylamino-3-(4-pyridyl) coumarin and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b] chromen-1(2H)-one are dissolved in acetonitrile, the reaction mixture is heated to 90-100℃ and stirred for 3-20h, after the reaction is completed, it is cooled to room temperature, solid is precipitated with ethyl acetate, filtered and washed with ethyl acetate, and the collected solid crude product is separated by silica gel column chromatography to obtain solid product, i.e. 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide.
[0014] Further, in step 1, the molar ratio of salicylaldehyde, formaldehyde and copper sulfate is 1:1:1, the eluent for column chromatography separation is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 3:1-3.
[0015] In step 2, the molar ratio of 2-hydroxy-5-hydroxymethylbenzaldehyde, 2-cyclopenten-1-one and imidazole is 1:1.5-3:1-2, the eluent for column chromatography separation is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 3:1-3.
[0016] In step 3, the molar ratio of 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one, triphenylphosphine and N-bromosuccinimide is 1:1.5-3:1-2, the eluent for column chromatography separation is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 20:4-5.
[0017] In step 4, the molar ratio of 4-(diethylamino) salicylaldehyde, 4-pyridine acetate hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine is 1:1.2-2:1.2-3:0.1-0.2:3-5, the eluent for column chromatography separation is dichloromethane and ethyl acetate, and the volume ratio of dichloromethane to ethyl acetate is 1:1.
[0018] In step 5, the molar ratio of 7-diethylamino-3-(4-pyridyl) coumarin and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one is 1:1.2-2, the eluent for column chromatography separation is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100:2-5.
[0019] The present application provides a ratio fluorescent probe for rapid detection of biological thiols and imaging of intracellular biological thiols and targeting mitochondria.
[0020] Compared with the prior art, the present application has the following advantages:
[0021] 1. The preparation method of the fluorescent probe is simple, raw materials are easy to obtain, and the cost is low.
[0022] 2. The fluorescent probe has good selectivity and sensitivity in rapid fluorescence detection of biological thiols.
[0023] 3. The fluorescent probe has good biocompatibility and can be applied to intracellular biological thiol imaging.
[0024] 4. The present application utilizes the characteristics of mitochondrial negative membrane potential to design a positively charged fluorescent probe, which is positioned in mitochondria through electrostatic attraction, realizing the targeted imaging of mitochondria.
[0025] 5. The fluorescent probe provided by the present application realizes the detection of biological thiols through the change of the fluorescence intensity ratio (F 498nm / F 567nm ) at two wavelengths of 498 nm and 567 nm, overcoming the low accuracy of single-wavelength detection in the prior art. BRIEF DESCRIPTION OF DRAWINGS
[0026] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of the probe of the present application.
[0027] Figure 2 is the nuclear magnetic resonance carbon spectrum of the probe of the present application.
[0028] Figure 3 is the ultraviolet and fluorescence spectrum of the fluorescent probe of the present application before and after reaction with biological thiols.
[0029] Figure 4 is the ultraviolet spectrum of the fluorescent probe of the present application for detecting 50 μM Cys with time change.
[0030] Figure 5 is the fluorescence spectrum of the fluorescent probe of the present application for detecting 50 μM Cys with time change.
[0031] Figure 6 is the fluorescence ratio (F 498nm / F 567nm ) change graph of the fluorescent probe of the present application for different Cys concentration titration.
[0032] Figure 7 is the fluorescence ratio (F 498nm / F 567nm ) change graph of the fluorescent probe of the present application after adding biological thiols and different amino acids.
[0033] Figure 8 is the imaging of the fluorescent probe of the present application for biological thiols in living cells.
[0034] Figure 9 is the imaging of the present application fluorescent probe targeted to the mitochondria. DETAILED DESCRIPTION
[0035] Example 1: The specific synthesis process of the fluorescent probe of the present application
[0036] (1) The specific synthesis steps of compound 3 are as follows:
[0037]
[0038] Under the protection of argon, salicylaldehyde (compound 6) (5.0 mL, 50 mmol) was dissolved in concentrated hydrochloric acid (50 mL), and then a 37% mass fraction aqueous formaldehyde solution (4 mL) was added. The mixture was stirred at room temperature for 24 h, and after the reaction was completed, it was filtered, the solid was washed with ultrapure water, the solid was collected, and then it was dissolved in dimethyl sulfoxide (DMSO, 30 mL) and ultrapure water (15 mL), and then copper sulfate pentahydrate (12.48 g, 50 mmol) was added. The mixture was heated to 110°C and stirred for 3 h, and after the reaction was completed, it was cooled to room temperature, extracted with dichloromethane, and the combined organic phase was washed with brine, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1:3~1:1) to obtain solid product compound 5 (yield 40%), i.e. 5-hydroxymethyl salicylaldehyde.
[0039]
[0040] Under the protection of argon, 5-hydroxymethyl salicylaldehyde (compound 5) (1.52 g, 10 mmol), 2-cyclopenten-1-one (1.23 g, 15 mmol) and imidazole (1.02 g, 15 mmol) were dissolved in tetrahydrofuran (THF, 5 mL) and ultrapure water (5 mL), and the mixture was stirred at room temperature for 72 h. After the reaction was completed, the mixture was extracted with dichloromethane, and the combined organic phase was washed with ultrapure water and brine in sequence, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1:3~1:1) to obtain solid product compound 4 (yield 26%), i.e. 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b] chroman-1(2H)-one.
[0041]
[0042] Under argon protection, 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one (compound 4) (432.4 mg, 2 mmol) and triphenylphosphine (1.05 g, 4 mmol) were dissolved in dichloromethane (DCM, 20 mL), and then N-bromosuccinimide (NBS, 711.9 mg, 4 mmol) was added. The mixture was stirred at room temperature for 2 h, and then washed with water and brine in turn, dried over anhydrous sodium sulfate, and the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography (ethyl acetate / petroleum ether = 1:5~1:4) to obtain a solid product compound 3 (yield 33%), i.e., 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one.
[0043] (2) The specific synthesis steps of compound 2 are as follows:
[0044]
[0045] Under argon protection, 4-(diethylamino)salicylaldehyde (compound 7) (0.97 g, 5 mmol) and 4-pyridine acetic acid hydrochloride (compound 8) (1.04 g, 6 mmol) were dissolved in dichloromethane (30 mL), which was cooled to 0°C in an ice bath, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.38 g, 7.2 mmol), 4-dimethylaminopyridine (DMAP, 61.1 mg, 0.5 mmol) and triethylamine (TEA, 2.1 mL, 15 mmol) were added thereto under ice bath. Then, the reaction mixture was slowly raised to room temperature and stirred for 14 h. After the reaction was completed, it was washed with water and brine in turn, and dried over anhydrous sodium sulfate. The filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography (dichloromethane / ethyl acetate = 1:1) to obtain a solid product compound 2 (yield 45%), i.e., 7-diethylamino-3-(4-pyridyl)coumarin.
[0046] (3) The specific synthesis steps of the fluorescent probe are as follows:
[0047]
[0048] Under argon, 7-diethylamino-3-(4-pyridyl)coumarin (compound 2) (88.2 mg, 0.3 mmol) and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one (compound 3) (111.6 mg, 0.4 mmol) were dissolved in acetonitrile (3 mL), the reaction mixture was heated to 90°C and stirred for 5 h, after the reaction was completed, it was cooled to room temperature, and the solid was precipitated with ethyl acetate, filtered and washed with ethyl acetate, and the solid crude product was collected and separated by silica gel column chromatography (dichloromethane / methanol = 100:2 ~ 5) to obtain the solid product, 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide. 1 HNMR (400 MHz, d6-DMSO, ppm) δ 9.08 (d, J = 6.0 Hz, 2H), 8.88 (s, 1H), 8.59 (d, J = 6.4 Hz, 2H), 7.63-7.60 (m, 2H), 7.53 (d, J = 8.4 Hz, 1H), 7.21 (s, 1H), 7.03 (d, J = 8.4 Hz, 1H), 6.88 (d, J = 8.8 Hz, 1H), 6.64 (s, 1H), 5.71 (s, 2H), 5.34 (t, J = 7.6 Hz, 1H), 3.52-3.50 (m, 4H), 2.65-2.58 (m, 1H), 2.46-2.34 (m, 2H), 2.08-1.97 (m, 1H), 1.17 (t, J = 6.4 Hz, 6H). 13 CNMR (100 MHz, d6-DMSO, ppm) δ 200.97, 159.32, 157.45, 155.29, 153.10, 151.47, 146.79, 143.51, 132.96, 132.79, 131.86, 130.96, 128.36, 125.34, 123.98, 122.25, 116.93, 110.56, 109.83, 108.47, 96.00, 75.57, 61.31, 44.56, 36.66, 27.60, 12.38.
[0049] Example 2: UV and fluorescence spectra of the fluorescent probe of the present application before and after reaction with biological thiols.
[0050] After 50 μM of Cys, GSH or Hcy was added to the solution of the fluorescent probe of the present application (10 μM, PBS:DMSO = 1:1) for 15 min, the UV and fluorescence spectrum changes were measured. The results are shown in Figure 3 .
[0051] AsFigure 3 As shown in (a) of FIG. 14, the maximum ultraviolet absorption peak of the fluorescent probe solution of the present application is located at 490 nm before and 426 nm after the addition of Cys, GSH or Hcy. From (b) of FIG. 14, it can be found that the change of the fluorescence emission spectrum (458 nm excitation, slit: 2.5 nm / 2.5 nm) is from 567 nm to 498 nm. It is illustrated that the fluorescent probe of the present application has reacted with biological thiols (Cys, GSH and Hcy), and can be used for tracing biological thiols. Figure 3
[0052] Example 3: Ultraviolet and fluorescence spectra of the fluorescent probe of the present application for detecting Cys over time.
[0053] After 50 μM of Cys is added to the fluorescent probe solution (10 μM, PBS:DMSO = 1:1) of the present application, the ultraviolet and fluorescence spectra over time are determined. The results are shown in FIG. 15. Figure 4 Figure 5
[0054] As shown in (a) of FIG. 16, after 50 μM of Cys is added to the fluorescent probe solution of the present application, the absorbance at 490 nm rapidly decreases, the absorbance at 426 nm gradually increases, and the color of the solution can be visually observed to change from orange to light yellow. Meanwhile, the fluorescence spectrum also obviously changes, as shown in (b) of FIG. 16. Figure 5 Figure 5 As shown in (a) of FIG. 17, after 50 μM of Cys is added to the fluorescent probe solution of the present application, the fluorescence intensity at 567 nm gradually decreases, and the fluorescence intensity at 498 nm rapidly increases. The fluorescence color of the solution can be observed to change from orange to blue under a 365 nm ultraviolet lamp. From (b) of FIG. 17, it can be found that within 15 min, the fluorescence ratio (F 498nm / F 567nm ) obviously changes and reaches a maximum value. It is illustrated that the fluorescent probe of the present application can realize the detection of biological thiols through the fluorescence ratio (F 498nm / F 567nm ), and has a relatively fast response speed.
[0055] Example 4: Change diagram of the fluorescence ratio (F 498nm / F 567nm ) of the fluorescent probe of the present application for different Cys concentration titrations.
[0056] After different concentrations of Cys are added to the fluorescent probe solution (10 μM, PBS:DMSO = 1:1) of the present application, the fluorescence ratio (F 498nm / F 567nm ) over time is determined. The results are shown in FIG. 18. Figure 6
[0057] As shown in (a) of FIG. 19, after different concentrations of Cys are added to the fluorescent probe solution of the present application, the fluorescence ratio (F 498nm / F 567nm ) over time is determined. The results are shown in (b) of FIG. 19.As shown in the figure, the fluorescence ratio (F 498nm / F 567nm ) changes (458nm excitation, gap: 2.5nm / 2.5nm). Figure 6 (a) and Figure 6 As shown in (b), the fluorescence ratio (F 498nm / F 567nm ) increased more and faster, and reached the maximum when the Cys concentration was 50 μM; Figure 6 In (c), it can be found that the fluorescence ratio (F 498nm / F 567nm ) has good linearity with Cys in the low concentration range, indicating that the fluorescent probe has high sensitivity.
[0058] Example 5: Fluorescence ratio (F) of the fluorescent probe of the present invention after adding biothiol and different amino acids 498nm / F 567nm )Change graph.
[0059] 50 μM of cysteine (Cys), glutathione (GSH), homocysteine (Hcy), alanine (Ala), arginine (Arg), aspartic acid (Asp), glutamine (Gln), glutamic acid (Glu), histidine (His), leucine (Leu), lysine (Lys), proline (Pro) and serine (Ser) were added to the fluorescent probe solution of the present invention (10 μM, PBS:DMSO=1:1), and the fluorescence spectrum was measured after 15 minutes. The results are shown in Figure 7 .
[0060] like Figure 7 As shown, only biothiols (Cys, GSH and Hcy) cause the fluorescence ratio (F 498nm / F 567nm ) significantly increased, while other amino acids remained almost unchanged. This indicates that the fluorescent probe of the present invention has good selectivity for biothiols (Cys, GSH, and Hcy) and can specifically recognize biothiols to a certain extent, providing a good foundation for biological imaging.
[0061] Example 6: Imaging of biothiols in living cells using the fluorescent probe of the present invention.
[0062] HeLa cells were cultured in a low-glucose culture medium containing 10% fetal bovine serum, and cultured in a saturated humidity incubator with 5% CO2 at 37°C. The culture medium was replaced every 2-3 days, and the cells were subcultured and moved into confocal dishes for culture. Two confocal dishes were taken out, and 5uM of the fluorescent probe of the present invention was directly added to one of the confocal dishes and incubated for 30 minutes before imaging; the other confocal dish was first incubated with 1mM thiol scavenger N-ethylmaleimide (NEM) for 30 minutes, washed with phosphate buffered saline (PBS), and then 5uM of the fluorescent probe of the present invention was added and incubated for 30 minutes before imaging. The excitation wavelength was 458nm, the green window fluorescence collection wavelength was 475-525nm, and the red window fluorescence collection wavelength was 550-600nm. See the results. Figure 8 .
[0063] from Figure 8 It was found that in cells treated only with the fluorescent probe of the present invention, the green window fluorescence was stronger than the red window fluorescence, and the fluorescence intensity ratio (green window / red window) was larger. In contrast, in cells treated first with NEM and then with the fluorescent probe of the present invention, the green window fluorescence was weaker than the red window fluorescence, and the fluorescence intensity ratio (green window / red window) was smaller. This demonstrates that the fluorescent probe can ratiometrically detect changes in intracellular biothiol concentrations.
[0064] Example 7: Imaging of the fluorescent probe of the present invention targeted to mitochondria.
[0065] HeLa cells were cultured in a low-glucose culture medium containing 10% fetal bovine serum, and cultured in a saturated humidity incubator with 5% CO2 at 37°C. The culture medium was replaced every 2-3 days, and the cells were subcultured and moved into a confocal dish for culture. Take 1 confocal dish, replace the culture medium with 1 mL of fresh culture medium, first incubate with 1 mM thiol scavenger N-ethylmaleimide (NEM) for 30 minutes, wash with phosphate buffered saline (PBS), and then add 5 uM of the fluorescent probe of the present invention and 500 nM of the commercial mitochondrial targeted fluorescent probe Mito-Tracker Deep Red FM and incubate for 30 minutes before imaging. The green window excitation wavelength of the fluorescent probe of the present invention is 458 nm, and the fluorescence collection wavelength is 550-600 nm; the red window excitation wavelength of Mito-Tracker Deep Red FM is 633 nm, and the fluorescence collection wavelength is 660-710 nm. See the results. Figure 9 .
[0066] from Figure 9 It can be found that the fluorescent probe of the present invention has good overlap with the commercial mitochondrial-targeted fluorescent probe, indicating that the fluorescent probe of the present invention is localized in the cell mitochondria.
[0067] The above embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above embodiments, and several modifications and improvements can be made by those skilled in the art without departing from the principles of the present application, and all of them are included in the protection scope of the present application.
[0068] In addition, it should be understood that, although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that those skilled in the art can understand.
Claims
1. A coumarin-based biothiol ratiometric fluorescent probe targeting mitochondria, characterized in that, The targeting mitochondria coumarin biological sulfydryl ratio fluorescent probe is 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide, and the structural formula is: 。 2. A preparation method of the targeting mitochondria coumarin biological sulfydryl ratio fluorescent probe according to claim 1, comprising the following steps: Step 1, synthesis of 5-hydroxymethylsalicylaldehyde: under argon protection, salicylaldehyde is dissolved in concentrated hydrochloric acid, and then a 37% mass fraction formaldehyde aqueous solution is added; the mixture is heated to 25-70 DEG C and stirred for reaction for 24 h, after the reaction is completed, it is cooled to room temperature, filtered, the solid is washed with ultrapure water, the solid is collected, and then the solid is dissolved in dimethyl sulfoxide and ultrapure water, and then copper sulfate pentahydrate is added; the mixture is heated to 100-120 DEG C and stirred for reaction for 3-14 h, after the reaction is completed, it is cooled to room temperature, extracted with dichloromethane, the combined organic phase is washed with brine, dried with anhydrous sodium sulfate, and the filtrate is concentrated to obtain a crude product, which is separated by silica gel column chromatography to obtain a solid product, i.e. 5-hydroxymethylsalicylaldehyde; Step 2, synthesis of 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one: under argon protection, 5-hydroxymethylsalicylaldehyde, 2-cyclopenten-1-one and imidazole are dissolved in tetrahydrofuran and ultrapure water, and the mixture is stirred at room temperature for reaction for 48-96 h; after the reaction is completed, the mixture is extracted with dichloromethane, the combined organic phase is washed with ultrapure water and brine in sequence, dried with anhydrous sodium sulfate, and the filtrate is concentrated to obtain a crude product, which is separated by silica gel column chromatography to obtain a solid product, i.e. 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one; Step 3, synthesis of 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one: under argon protection, 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one and triphenylphosphine are dissolved in dichloromethane, and then N-bromosuccinimide is added; the mixture is stirred at room temperature for reaction for 1-14 h, and then washed with water and brine in sequence, dried with anhydrous sodium sulfate, and the filtrate is concentrated to obtain a crude product, which is separated by silica gel column chromatography to obtain a solid product, i.e. 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one; Step 4, synthesis of 7-diethylamino-3-(4-pyridyl)coumarin: under argon protection, 4-(diethylamino)salicylaldehyde and 4-pyridine acetate hydrochloride were dissolved in dichloromethane, and the reaction mixture was cooled to 0°C in an ice bath, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine were added under ice bath; then, the reaction mixture was slowly warmed to room temperature and stirred for 14-20 h; after the reaction was completed, the reaction mixture was washed with water and brine, and dried over anhydrous sodium sulfate; the solvent was evaporated to obtain a crude solid, and the filtrate was concentrated to obtain a crude product, which was separated by silica gel column chromatography to obtain a solid product, i.e. 7-diethylamino-3-(4-pyridyl)coumarin; Step 5, synthesis of the fluorescent probe: under argon protection, 7-diethylamino-3-(4-pyridyl)coumarin and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one were dissolved in acetonitrile, and the reaction mixture was heated to 90-100°C and stirred for 3-20 h; after the reaction was completed, the reaction mixture was cooled to room temperature, and solid was precipitated with ethyl acetate, filtered and washed with ethyl acetate, and the solid crude product was collected and separated by silica gel column chromatography to obtain a solid product, i.e. 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-((1-oxo-1,2,3,3a-tetrahydrocyclopenta[b]chromen-7-yl)methyl)pyridin-1-ium bromide.
3. The method for preparing a coumarin-based biothiol ratio fluorescent probe targeting mitochondria according to claim 2, characterized in that, In step 1, the molar ratio of salicylaldehyde, formaldehyde and copper sulfate is 1:1:1, and the eluent for column chromatography is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 3:1-3.
4. The method for preparing a mitochondrial-targeting coumarin-based biothiol ratiometric fluorescent probe according to claim 2, wherein: In step 2, the molar ratio of 2-hydroxy-5-hydroxymethylbenzaldehyde, 2-cyclopenten-1-one and imidazole is 1:1.5-3:1-2, and the eluent for column chromatography is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 3:1-3.
5. The method for preparing a mitochondrial-targeting coumarin-based biothiol ratiometric fluorescent probe according to claim 2, wherein: In step 3, the molar ratio of 7-(hydroxymethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one, triphenylphosphine and N-bromosuccinimide is 1:1.5-3:1-2, and the eluent for column chromatography is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 20:4-5.
6. The method for preparing a mitochondrial-targeting coumarin-based biothiol ratiometric fluorescent probe according to claim 2, wherein: In step 4, the molar ratio of 4-(diethylamino)salicylaldehyde, 4-pyridine acetate hydrochloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and triethylamine is 1:1.2-2:1.2-3:0.1-0.2:3-5, and the eluent for column chromatography is dichloromethane and ethyl acetate, and the volume ratio of dichloromethane to ethyl acetate is 1:
1.
7. The method of claim 2, wherein the method is characterized by: In step 5, the molar ratio of 7-diethylamino-3-(4-pyridyl)coumarin and 7-(bromomethyl)-3,3a-dihydrocyclopenta[b]chromen-1(2H)-one is 1:1.2-2, and the eluent for column chromatography is dichloromethane and methanol, and the volume ratio of dichloromethane to methanol is 100:2-5.
8. Use of a coumarin-based biothiol ratio fluorescent probe targeting mitochondria according to claim 1, characterized in that, The ratio fluorescent probe is used for preparing a reagent for rapidly detecting biological thiols, and is applied to preparation of a reagent for imaging biological thiols in cells and targeting mitochondria.