Mitochondria-targeted coumarin mercury ion ratio fluorescent probe as well as preparation and application thereof

Abstract

The invention relates to the technical field of fluorescent probes for metal ion detection, in particular to a coumarin mercury ion ratio fluorescent probe for targeting mitochondria as well as preparation and application of the coumarin mercury ion ratio fluorescent probe. The ratiometric fluorescent probe is applied to rapid detection of mercury ions and targeted imaging of mercury ions and mitochondria in living cells. At present, reported mercury ion fluorescent probes still have some limitations, such as complex synthesis, separation and purification, single-wavelength detection, no targeted positioning function and the like. The preparation method of the ratiometric fluorescent probe comprises the following steps: carrying out a reaction on O-(4-(bromomethyl) phenyl) diphenyl selenophosphonate and 7-diethylamino-3-(4-pyridyl) group coumarin in acetonitrile to generate 4-(7-(diethylamino)-2-oxo-2H-chromene-3-yl)-1-(4-((diphenyl selenophosphoryl) oxy) benzyl) pyridine-1-ammonium bromide. The fluorescent probe disclosed by the invention is simple to prepare, separate and purify, and fluorescent probe molecules have the advantages of good selectivity, high sensitivity, high response speed and the like in a coexistence system of mercury ions and other metal ions.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescent probes for metal ion detection, and particularly relates to a mitochondrial-targeted coumarin mercury ion ratio fluorescent probe and its preparation and application. Background Art

[0002] Heavy metal pollution has attracted widespread attention due to its adverse effects on human health and environmental safety. Mercury is a highly toxic metal pollutant that comes from natural factors such as volcanic eruptions and human activities such as coal mining, chemical production, fossil fuel combustion, and urban and industrial wastewater discharge. As a result, it is inevitably present in the air, water, and soil of the human living environment and can enter the human body through the skin or mouth. Mercury exists in three forms: inorganic mercury, organic mercury, and elemental mercury. Mercury ions (Hg 2+ ) is the most common form in nature. Due to its bioaccumulation and strong binding affinity to proteins and enzymes containing sulfur and selenium groups, once Hg 2+ Chelating with proteins and enzymes will inactivate their antioxidant properties, induce oxidative stress and disrupt the redox balance. 2+ The toxicity of Hg can cause damage to organs, especially the kidneys, and lead to various diseases such as Minamata disease, movement disorders, cognitive impairment, central nervous system damage, cancer, and even death. Therefore, the development of effective Hg 2+ Detection methods are of great significance to human health and environmental protection.

[0003] So far, many methods for detecting Hg have been developed. 2+ There are a number of analytical methods, such as atomic absorption spectroscopy, high performance liquid chromatography, atomic fluorescence spectroscopy, electrochemical methods, inductively coupled plasma optical emission spectroscopy, inductively coupled plasma mass spectrometry, inductively coupled plasma atomic emission spectroscopy, surface plasmon resonance, surface enhanced Raman scattering spectroscopy, X-ray absorption spectroscopy, and colorimetry. However, these methods have some disadvantages, such as high cost, expensive and complex instruments, tedious and time-consuming sample preparation, time-consuming detection process, complicated operation steps, and the need for professional technicians, which are not suitable for Hg in biological systems. 2+ To overcome these limitations, fluorescent probes have the advantages of low cost, diverse structures, easy modification, rapid response, good selectivity, high sensitivity, simple operation, high temporal and spatial resolution, and non-invasiveness. Moreover, combined with fluorescence imaging, they can be used to detect Hg in biological systems. 2+ Therefore, many methods for detecting Hg have been developed in recent years. 2+ The reported Hg 2+ Fluorescent probes can be divided into coordination type and reaction type. Compared with the former, fluorescent probes based on chemical reaction are more sensitive to Hg2+ has higher sensitivity due to its specific reaction. However, most Hg-based 2+ fluorescent probes achieve detection by changing the fluorescence emission intensity at a single wavelength, such as fluorescence quenching or fluorescence enhancement, and are vulnerable to instrument or environmental factors. Ratio fluorescent probes can perform self-calibration through the ratio of the emission intensities at two different wavelengths, eliminating most interferences, and thus have higher sensitivity and accuracy. However, there are few Hg 2+ ratio fluorescent probes. In addition, research shows that Hg 2+ tends to accumulate in cell mitochondria, reducing mitochondrial membrane potential, generating oxidative stress, and inducing apoptosis. However, there are few mitochondrial-targeted Hg 2+ ratio fluorescent probes. Therefore, developing mitochondrial-targeted Hg 2+ ratio fluorescent probes is a challenging task.

[0004] Based on the fact that coumarin not only has excellent optical properties and a typical "donor-π-acceptor" structure, but also its structure is easy to modify. Therefore, the mitochondrial-targeted coumarin-based mercury ion ratio fluorescent probe of the present invention is a ratio fluorescent probe using 7-diethylamino-3-(4-pyridyl)coumarin as the fluorophore and diphenyl selenophosphonate as the recognition unit. After retrieval, no literature and patent applications similar to the present application were found. Summary of the Invention

[0005] In view of the above problems, the present invention provides a novel ratio fluorescent probe for identifying mercury ions and targeting mitochondria. The fluorescent probe has a short response time, can achieve quantitative detection of mercury ions, has high selectivity and sensitivity; in addition, it can also target mitochondria, thereby realizing imaging analysis at specific positions.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a mitochondrial-targeted coumarin-based mercury ion ratio fluorescent probe, which is 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-(4-((diphenylselenophosphoryl)oxy)benzyl)pyridin-1-ium bromide, and its structural formula is:

[0008]

[0009] The present invention provides a preparation method of a mitochondrial-targeted coumarin-based mercury ion ratio fluorescent probe, including the following steps:

[0010] Step 1, Synthesis of diphenylselenophosphoryl chloride: Under argon protection, diphenylphosphinous chloride and selenium powder were added to a two-necked round-bottom flask, and heated to 130 - 140 °C for stirring reaction for 3 - 6 h under solvent-free conditions. After the reaction was completed, the reaction mixture was cooled to room temperature, and the obtained crude product, namely diphenylselenophosphoryl chloride, was not purified.

[0011] Step 2, Synthesis of O-(4-(hydroxymethyl)phenyl)diphenylselenophosphonate: Under argon protection, p-hydroxymethylbenzyl alcohol and triethylamine were dissolved in anhydrous dichloromethane, and the reaction solution was cooled to 0 °C with an ice bath. Then, diphenylselenophosphoryl chloride diluted with anhydrous dichloromethane was slowly added thereto, and then the temperature was raised to room temperature for reaction overnight. After the reaction was completed, the reaction solution was washed successively with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a crude solid, which was purified by silica gel chromatography to obtain the product, namely O-(4-(hydroxymethyl)phenyl)diphenylselenophosphonate.

[0012] Step 3, Synthesis of (4-(bromomethyl)phenyl)diphenylselenophosphonate: Under argon protection, O-(4-(hydroxymethyl)phenyl)diphenylselenophosphonate was added to a two-necked round-bottom flask and dissolved in anhydrous dichloromethane. The reaction solution was cooled to 0 °C, and phosphorus tribromide was added thereto, and then the temperature was raised to room temperature for reaction overnight. After the reaction was completed, the reaction mixture was poured into saturated sodium bicarbonate solution and extracted with dichloromethane. The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a crude solid, which was purified by silica gel chromatography to obtain the solid product, namely (4-(bromomethyl)phenyl)diphenylselenophosphonate.

[0013] Step 4, Synthesis of 7-diethylamino-3-(4-pyridyl)coumarin: 4-Diethylaminosalicylaldehyde and ethyl 4-pyridineacetate were dissolved in anhydrous ethanol, and piperidine was added thereto. Then, the reaction mixture was heated to 106 - 110 °C and stirred for reaction overnight under argon. After the reaction was completed, the reaction mixture was cooled to room temperature, and the solvent was evaporated to obtain a crude solid. Ethyl acetate and petroleum ether mixed solvent was added thereto and sonicated evenly, then filtered, and washed with a small amount of ethyl acetate and petroleum ether mixed solvent. The solid was collected, namely 7-diethylamino-3-(4-pyridyl)coumarin.

[0014] Step 5, Synthesis of the Fluorescent Probe: O-(4-(Bromomethyl)phenyl)diphenylphosphonoselenate and 7-diethylamino-3-(4-pyridyl)coumarin were added to a two-necked round-bottom flask and dissolved with acetonitrile. The reaction mixture was then heated to 95-102°C and stirred under argon overnight. After completion of the reaction, the reaction solution was cooled to room temperature, and a solid was precipitated with ethyl acetate. The solid was filtered and washed with ethyl acetate. The crude solid product was collected and purified by silica gel chromatography to obtain a solid product, 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-(4-((diphenylphosphonoselenyl)oxy)benzyl)pyridinium-1-ammonium bromide.

[0015] Furthermore, the molar ratio of diphenylphosphine chloride to selenium powder in step 1 is 1:1, and the product can be used in the next reaction without purification.

[0016] In step 2, the molar ratio of p-hydroxybenzyl alcohol, triethylamine and diphenyl selenophosphonyl chloride is 1:2-4:1-1.2, and the eluents for silica gel chromatography separation are petroleum ether and ethyl acetate, with the volume ratio of petroleum ether to ethyl acetate being 4:1-2.

[0017] In step 3, the molar ratio of O-(4-(hydroxymethyl)phenyl)diphenylphosphonoselenate to phosphorus tribromide is 1:2-4, the silica gel chromatography eluent is petroleum ether and dichloromethane, and the volume ratio of petroleum ether to dichloromethane is 5:1-5.

[0018] In the step 4, the molar ratio of 4-diethylaminosalicylaldehyde to 4-pyridine ethyl acetate is 1:1.2-2. The product is homogenized by ultrasonication with a mixed solvent of ethyl acetate and petroleum ether, filtered, and washed with a mixed solvent of ethyl acetate and petroleum ether without silica gel chromatography separation and purification.

[0019] The present invention provides a ratiometric fluorescent probe for rapid detection of mercury ions and imaging of mercury ions in cells and targeting mitochondria.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. The preparation method of the fluorescent probe provided by the present invention is simple, the raw materials are easily available, and the cost is low.

[0022] 2. The fluorescent probe provided by the present invention can be used for rapid fluorescence detection of mercury ions with good selectivity and sensitivity.

[0023] 3. The fluorescent probe provided by the present invention has good biocompatibility and can be used for intracellular mercury ion imaging.

[0024] 4. The present invention utilizes the negative membrane potential characteristics of mitochondria to design a positively charged fluorescent probe, which is positioned in the mitochondria through electrostatic attraction to achieve targeted imaging of mitochondria.

[0025] 5. The fluorescence probe provided by the present invention realizes the detection of mercury ions through the fluorescence intensity ratio F at two wavelengths of 498 nm and 570 nm, overcoming the characteristic of low accuracy in single-wavelength detection in the prior art. 498nm / F 570nm Variation to achieve the detection of mercury ions, overcoming the low accuracy of single-wavelength detection in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the 1H NMR spectrum of the probe of the present invention.

[0027] Figure 2 is the 13C NMR spectrum of the probe of the present invention.

[0028] Figure 3 is the 31P NMR spectrum of the probe of the present invention.

[0029] Figure 4 is the high-resolution mass spectrum of the probe of the present invention.

[0030] Figure 5 is the UV spectrum of the fluorescence probe of the present invention for detecting 150 μM Hg 2+ changing with time.

[0031] Figure 6 is the fluorescence spectrum of the fluorescence probe of the present invention for detecting 150 μM Hg 2+ changing with time.

[0032] Figure 7 is the fluorescence spectrum and fluorescence ratio F of the fluorescence probe of the present invention for titrating different Hg 2+ concentrations and the variation diagram of 498nm / F 570nm changing.

[0033] Figure 8 is the fluorescence ratio F of the fluorescence probe of the present invention after the addition of mercury ions and the action of different metal ions 498nm / F 570nm changing diagram.

[0034] Figure 9 is the imaging of mercury ions in living cells by the fluorescence probe of the present invention.

[0035] Figure 10 is the imaging of the fluorescence probe of the present invention targeting and localizing in mitochondria.

[0036] Figure 11 is the synthesis schematic diagram of the mercury ion ratio fluorescence probe compound 1. DETAILED DESCRIPTION OF THE INVENTION

[0037] Example 1: Specific synthesis process of the fluorescence probe of the present invention

[0038] (1) The specific synthesis steps of compound 3 are as follows:

[0039]

[0040] Under argon protection, diphenylphosphinous chloride (7.94 g, 36 mmol) and selenium powder (2.86 g, 36 mmol) were added to a 50 mL two-necked round-bottom flask, and the mixture was heated to 130 °C and stirred under solvent-free conditions for 4 h. After the reaction was completed, the reaction mixture was cooled to room temperature, and the crude product compound 5 could be used for the next reaction without purification.

[0041]

[0042] Under argon protection, p-hydroxybenzyl alcohol (7.94 g, 36 mmol) and triethylamine (15 mL, 108 mmol) were dissolved in anhydrous dichloromethane (30 mL), and the reaction solution was cooled to 0 °C with an ice bath. Then, diphenylselenophosphinyl chloride (compound 5) (10.79 g, 36 mmol) diluted with anhydrous dichloromethane (20 mL) was slowly added thereto, and the mixture was then allowed to react at room temperature overnight. After the reaction was completed, the reaction solution was washed successively with water and saturated sodium chloride solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a crude solid. The crude solid was purified by silica gel chromatography (petroleum ether / ethyl acetate = 4:1 - 4:2) to obtain product compound 4 (yield 73%), namely O-(4-(hydroxymethyl)phenyl)diphenylselenophosphonate.

[0043]

[0044] Under argon protection, O-(4-(hydroxymethyl)phenyl)diphenylselenophosphonate (compound 4) (5 g, 12.9 mmol) was added to a 100 mL two-necked round-bottom flask and dissolved in anhydrous dichloromethane (30 mL). The reaction solution was cooled to 0 °C, and phosphorus tribromide (3.68 mL, 38.7 mmol) was added thereto. Then, the mixture was allowed to react at room temperature overnight. After the reaction was completed, the reaction mixture was poured into saturated sodium bicarbonate solution, and extracted with dichloromethane (3 × 30 mL). The organic phases were combined, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated to obtain a crude solid. The crude solid was purified by silica gel chromatography (petroleum ether / dichloromethane = 5:1 - 5:5) to obtain the solid product compound 3 (yield 72%), namely (4-(bromomethyl)phenyl)diphenylselenophosphonate.

[0045] (2) The specific synthesis steps of compound 2 are as follows:

[0046]

[0047] Dissolve 4 - diethylaminosalicylaldehyde (4.59 g, 23.7 mmol) and ethyl 4 - pyridineacetate (4.61 g, 27.9 mmol) in anhydrous ethanol (20 mL), and then add piperidine (0.2 mL, 2 mmol). Then heat the reaction mixture to 106 °C and stir the reaction overnight under argon. After the reaction is completed, cool the reaction mixture to room temperature, evaporate the solvent to obtain a crude solid. Add a mixed solvent of ethyl acetate and petroleum ether (1:5, 20 mL) to it, ultrasonicate to uniformity, filter, and wash with a small amount of mixed solvent of ethyl acetate and petroleum ether (1:5 v / v) to obtain the solid product Compound 2 (yield 77%), namely 7 - diethylamino - 3 - (4 - pyridyl)coumarin.

[0048] (3) The specific synthesis steps of the fluorescent probe are as follows:

[0049]

[0050] Add O - (4 - (bromomethyl)phenyl)diphenylselenophosphonate (Compound 3) (337.7 mg, 0.75 mmol) and 7 - diethylamino - 3 - (4 - pyridyl)coumarin (Compound 2) (147.2 mg, 0.5 mmol) into a 50 mL two - necked round - bottom flask, dissolve with acetonitrile (8 mL), and then heat the reaction mixture to 98 °C and stir the reaction overnight under argon protection. After the reaction is completed, cool the reaction solution to room temperature, precipitate the solid with ethyl acetate, filter and wash with ethyl acetate, collect the crude solid product, and purify it by silica gel chromatography (dichloromethane / methanol = 50:1 - 20:1) to obtain the solid product (yield 85%), namely 4 - (7 - (diethylamino)-2 - oxo - 2H - chromen - 3 - yl)-1 - (4 - ((diphenylselenophosphoryl)oxy)benzyl)pyridin - 1 - ammonium bromide. 1 HNMR(400MHz,DMSO - d6,ppm)δ9.09(d,J = 6.4Hz,2H),8.88(s,1H),8.57(d,J = 7.2Hz,2H),7.98 - 7.93(m,4H),7.66 - 7.56(m,7H),7.52(d,J = 8.4Hz,2H),7.16 - 7.14(m,2H),6.86 - 6.84(m,1H),6.62(s,1H),5.74(s,2H),3.50(q,J = 6.8Hz,4H),1.14(t,J = 6.8Hz,6H). 1313C NMR (100 MHz, DMSO-d6, ppm) δ 159.26, 157.42, 153.08, 151.47, 150.93, 146.82, 143.57, 134.02, 133.05, 132.79, 132.76, 131.84, 131.39, 131.18, 131.06, 130.01, 128.97, 128.84, 123.96, 122.02, 121.97, 110.54, 109.75, 108.45, 95.97, 61.20, 44.53, 12.36. 31 31P NMR (162 MHz, DMSO-d6, ppm) δ 86.40. HRMS for C 37 H 34 N2O3PSe + ([M - Br] + ): calcd: 665.14668, found: 665.14720.

[0051] Example 2: Detection of Hg by the fluorescent probe of the present invention 2+ UV and fluorescence spectra as a function of time.

[0052] 150 μM of Hg was added to the fluorescent probe solution (5 μM, PBS:DMSO = 1:1, pH 7.4) of the present invention 2+ and then the changes of UV and fluorescence spectra with time were measured. The results are shown in Figure 5 and Figure 6 .

[0053] As Figure 5 shown, after adding 150 μM of Hg to the fluorescent probe solution of the present invention 2+ , the absorbance at 492 nm decreased rapidly, and the absorbance at 426 nm increased gradually. The color of the solution changed from orange to light yellow visibly to the naked eye. At the same time, the fluorescence spectrum also changed significantly (excitation at 458 nm, slit: 5 nm / 5 nm). As Figure 6 (a) shown, after adding 150 μM of Hg to the fluorescent probe solution of the present invention 2+ , the fluorescence intensity at 570 nm decreased gradually, and the fluorescence intensity at 498 nm increased rapidly. Under the 365 nm UV lamp, the fluorescence color of the solution could be observed to change from orange - red to blue; it can be found from Figure 6 (b) that the fluorescence ratio F 498nm / F 570nm changed significantly and reached the maximum value within 30 min. It shows that the fluorescent probe of the present invention can detect mercury ions through the fluorescence ratio F 498nm / F 570nm and has a relatively fast response speed.

[0054] Example 3: Fluorescence spectra and fluorescence ratio F 498nm / F 570nm variation graph of the present invention's fluorescent probe for titration with different mercury ion concentrations.

[0055] After adding different concentrations of Hg 2+ to the fluorescent probe solution (5 μM, PBS:DMSO = 1:1, pH 7.4) of the present invention, its fluorescence spectrum and fluorescence ratio F 498nm / F 570nm variation with time were measured. The results are shown in Figure 7 .

[0056] As Figure 7 shown, the fluorescence spectra and fluorescence ratio F 2+ of the fluorescent probe solution of the present invention upon addition of different concentrations of Hg 498nm / F 570nm variation (excitation at 458 nm, slit: 5 nm / 5 nm). As can be seen from Figure 7 (a), as the concentration of Hg 2+ increases, the fluorescence intensity at 570 nm weakens, and the fluorescence intensity at 498 nm increases; at the same time, the fluorescence ratio F 498nm / F 570nm increases, and reaches the maximum when the concentration of Hg 2+ is 150 μM, as shown in Figure 7 (b). As can be found from Figure 7 (c), the fluorescence ratio F 498nm / F 570nm has a good linearity with Hg 2+ in the low concentration range, indicating that this fluorescent probe has high sensitivity.

[0057] Example 4: Fluorescence ratio F 498nm / F 570nm variation graph of the present invention's fluorescent probe after the addition of mercury ions and different metal ions.

[0058] 150 μM of mercury ions (Hg 2+ ), silver ions (Ag + ), potassium ions (K + ), sodium ions (Na + ), calcium ions (Ca 2+ ), zinc ions (Zn 2+ ), barium ions (Ba 2+ ), magnesium ions (Mg 2+ ), cobalt ions (Co 2+ ), manganese ions (Mn2+ ) lead ion (Pb 2+ ) iron ion (Fe 3+ ) aluminum ion (Al 3+ ) cadmium ion (Cd 2+ ) and copper ion (Cu 2+ ). After 30 min, their fluorescence spectra were measured. The results are shown in Figure 8 .

[0059] As Figure 8 shown, regardless of the presence of other metal ions, mercury ions caused a significant change in the fluorescence ratio F 498nm / F 570n m, while other metal ions showed little change. This indicates that the fluorescence probe of the present invention has good selectivity and anti-interference ability for mercury ions, and can specifically recognize mercury ions to a certain extent, providing a good basis for bioimaging.

[0060] Example 5: Imaging of mercury ions in living cells by the fluorescence probe of the present invention.

[0061] HeLa cells were cultured adherently in a low-glucose culture medium containing 10% fetal bovine serum, cultured at 37 °C in an incubator with 5% CO2 saturated humidity, the culture medium was changed every 2 - 3 days, and subculture was carried out and the cells were transferred into a confocal dish for culture. Two confocal dishes were taken out. One confocal dish was directly added with 5 μM of the fluorescence probe of the present invention and incubated for 30 min and then imaged; the other confocal dish was incubated with 5 μM of the fluorescence probe of the present invention and 150 μM of mercury ions in sequence for 30 min and then imaged. The excitation wavelength was 458 nm, the fluorescence collection wavelength in the green window was 470 - 530 nm, and the fluorescence collection wavelength in the red window was 550 - 610 nm. The results are shown in Figure 9 .

[0062] From Figure 9 it can be found that for the cells treated only with the fluorescence probe of the present invention, the fluorescence in the red window was stronger than that in the green window, and the fluorescence intensity ratio (green window / red window) was smaller; while for the cells treated with the fluorescence probe of the present invention and mercury ions in sequence, although the fluorescence in the green window did not change much, the fluorescence in the red window was significantly weakened, and the fluorescence intensity ratio (green window / red window) was larger. This indicates that this fluorescence probe can detect the change in the concentration of mercury ions in cells by ratio.

[0063] Example 6: Imaging of the fluorescence probe of the present invention targeting mitochondria.

[0064] HeLa cells were cultured adherently in a low-glucose culture medium containing 10% fetal bovine serum, cultured at 37 °C in an incubator with 5% CO2 and saturated humidity, the culture medium was changed every 2-3 days, and subculture was carried out and the cells were transferred into a confocal dish for culture. One confocal dish was taken, the culture medium in it was replaced with 1 mL of fresh culture medium, and it was incubated with 5 μM of the fluorescent probe of the present invention and 500 nM of the commercial mitochondrial-targeted fluorescent probe Mito-Tracker Deep Red FM for 30 min and then imaged. The excitation wavelength of the green window of the fluorescent probe of the present invention is 458 nm, and the fluorescence collection wavelength is 550-610 nm; the excitation wavelength of the red window of Mito-Tracker Deep Red FM is 633 nm, and the fluorescence collection wavelength is 660-710 nm. The results are shown in Figure 10 .

[0065] From Figure 10 it can be found that the overlapping of the fluorescent probe of the present invention and the commercial mitochondrial-targeted fluorescent probe is good, indicating that the fluorescent probe of the present invention is localized in cell mitochondria.

[0066] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. For those of ordinary skill in the art, without departing from the principle of the present invention, several variations and improvements can still be made, which are all included in the protection scope of the present invention.

[0067] In addition, it should be understood that although this specification is described according to embodiments, not every embodiment only contains an independent technical solution. This narrative way of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A coumarin-based mercury ion ratio fluorescence probe targeting mitochondria, characterized in that, The coumarin-based mercury ion ratio fluorescent probe targeting mitochondria is 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-(4-((diphenylselenophosphoryl)oxy)benzyl)pyridin-1-ium bromide, and its structural formula is:

2. A preparation method of the coumarin-based mercury ion ratio fluorescent probe targeting mitochondria as described in claim 1, comprising the following steps: Step 1, synthesis of diphenylselenophosphoryl chloride: Under argon protection, diphenylphosphinous chloride and selenium powder are added to a two-necked round-bottom flask, and heated to 128-135 °C for stirring reaction for 4-6 h under solvent-free conditions. After the reaction is completed, the reaction mixture is cooled to room temperature, and the obtained crude product, namely diphenylselenophosphoryl chloride, does not need to be purified. Step 2, synthesis of O-(4-(hydroxymethyl)phenyl)diphenylselenophosphonate: Under argon protection, p-hydroxymethylbenzyl alcohol and triethylamine are dissolved in anhydrous dichloromethane, and the reaction solution is cooled to 0 °C with an ice bath. Then, diphenylselenophosphoryl chloride diluted with anhydrous dichloromethane is slowly added thereto, and then the temperature is raised to room temperature for reaction overnight. After the reaction is completed, the reaction solution is washed successively with water and saturated sodium chloride solution. The organic phase is dried with anhydrous sodium sulfate, filtered, the solvent is evaporated, and a crude solid is obtained. The product is purified by silica gel chromatography to obtain the product, namely O-(4-(hydroxymethyl)phenyl)diphenylselenophosphonate. Step 3, synthesis of (4-(bromomethyl)phenyl)diphenylselenophosphonate: Under argon protection, O-(4-(hydroxymethyl)phenyl)diphenylselenophosphonate is added to a two-necked round-bottom flask, dissolved in anhydrous dichloromethane, the reaction solution is cooled to 0 °C, phosphorus tribromide is added thereto, and then the temperature is raised to room temperature for reaction overnight. After the reaction is completed, the reaction mixture is poured into saturated sodium bicarbonate solution and extracted with dichloromethane. The organic phases are combined, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered, the solvent is evaporated, and a crude solid is obtained. The solid product is purified by silica gel chromatography to obtain the solid product, namely (4-(bromomethyl)phenyl)diphenylselenophosphonate. Step 4, synthesis of the fluorescent probe: O-(4-(bromomethyl)phenyl)diphenylselenophosphonate and 7-diethylamino-3-(4-pyridyl)coumarin are added to a two-necked round-bottom flask, dissolved in acetonitrile, and then the reaction mixture is heated to 95-102 °C and stirred for reaction overnight under argon protection. After the reaction is completed, the reaction solution is cooled to room temperature, and the solid is precipitated with ethyl acetate, filtered and washed with ethyl acetate. The solid crude product is collected and purified by silica gel chromatography to obtain the solid product, namely 4-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1-(4-((diphenylselenophosphoryl)oxy)benzyl)pyridin-1-ium bromide.

3. The preparation method of a coumarin-based mercury ion ratio fluorescent probe targeting mitochondria according to claim 2, characterized in that, In step 1, the molar ratio of diphenylphosphinous chloride to selenium powder is 1:1, and it can be used for the next reaction without purification.

4. The preparation method of a coumarin-based mercury ion ratio fluorescent probe targeting mitochondria according to claim 2, characterized in that, In step 2, the molar ratio of p-hydroxymethylbenzyl alcohol, triethylamine and diphenylselenophosphoryl chloride is 1:2-4:1-1.

2. The eluent for silica gel chromatography separation is petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 4:1-2.

5. The preparation method of a coumarin-based mercury ion ratio fluorescence probe targeting mitochondria according to claim 2, characterized in that, In step 3, the molar ratio of O-(4-(hydroxymethyl)phenyl)diphenylphosphine selenate to phosphorus tribromide is 1:2 to 4, and the eluent for silica gel column chromatography is petroleum ether and dichloromethane, and the volume ratio of the petroleum ether to the dichloromethane is 5:1 to 5.

6. The preparation method of a coumarin-based mercury ion ratio fluorescence probe targeting mitochondria according to claim 2, characterized in that, In step 4, the molar ratio of 4-diethylaminosalicylaldehyde to ethyl 4-pyridylacetate is 1:1.2 to 2. The product is sonicated uniformly with a mixed solvent of ethyl acetate and petroleum ether and then filtered, and washed with a mixed solvent of ethyl acetate and petroleum ether, without silica gel column chromatography separation and purification.

7. A method for preparing a coumarin-based mercury ion ratio fluorescence probe targeting mitochondria, characterized in that, In step 5, the molar ratio of 7-diethylamino-3-(4-pyridyl)coumarin to O-(4-(bromomethyl)phenyl)diphenylphosphine selenate is 1:1.2 to 2, and the eluent for silica gel column chromatography is dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 100:2 to 5.

8. Use of a coumarin-based mercury ion ratio fluorescence probe targeting mitochondria according to claim 1, characterized in that, The ratio fluorescent probe is used for preparing a reagent for rapidly detecting mercury ions, and is applied to preparing a reagent for intracellular mercury ion imaging and targeting mitochondria.

9. Use of a coumarin-based mercury ion ratio fluorescent probe targeting mitochondria according to claim 1, characterized in that: The maximum fluorescence emission wavelength of the ratio fluorescence probe solution is located at 570 nm. When mercury ions are added, the maximum fluorescence emission wavelength of the probe solution blue-shifts to 498 nm. The presence or absence of mercury ions can be detected by the change in the ratio F 498nm / F 570nm . The detection limit for detecting mercury ions is 16.7 nM.

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