CN110483573B - Mitochondrial targeting hypochlorous acid ratio type two-photon fluorescent probe and preparation method and application thereof - Google Patents
Mitochondrial targeting hypochlorous acid ratio type two-photon fluorescent probe and preparation method and application thereof Download PDFInfo
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
- C07F9/02—Phosphorus compounds
- C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
- C07F9/6558—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system
- C07F9/65586—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing at least two different or differently substituted hetero rings neither condensed among themselves nor condensed with a common carbocyclic ring or ring system at least one of the hetero rings does not contain nitrogen as ring hetero atom
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- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention discloses a mitochondria-targeted hypochlorous acid ratio type two-photon fluorescent probe and a preparation method and application thereof, wherein the mitochondria-targeted hypochlorous acid ratio type two-photon fluorescent probe takes carbazole as a matrix and has the following structural formula:when the fluorescent probe is applied to HClO detection in aqueous solution, the selectivity is high, the response speed is high, and the fluorescence ratio (I) of the probe is high525nm/I465nm) Has good linear relation with the concentration of HClO, and the detection limit is as low as 35 nM. In addition, the fluorescent probe molecule also has excellent two-photon absorption performance. The cytotoxicity test shows that the probe has little toxic and side effect on cells. Two-photon confocal fluorescence microscopic imaging experiments show that the probe has good mitochondrial positioning capacity and cell transmembrane property, and is suitable for two-photon fluorescence imaging and quantitative detection of HClO in cell mitochondria.
Description
Technical Field
The invention relates to a mitochondrion targeting hypochlorous acid ratio type two-photon fluorescent probe, a preparation method and application thereof, which are used for realizing the quantitative detection of HClO in cell mitochondria by two-photon imaging and have the advantages of excellent two-photon absorption performance, low cytotoxicity, good membrane permeability and biocompatibility, high selectivity and light stability and the like.
Background
Recently, reactive oxygen species have attracted increasing attention from medical researchers due to their association with various biological processes in the body. Endogenous HClO, a highly reactive oxygen species, is produced primarily by the reaction between hydrogen peroxide and chloride ions catalyzed by Myeloperoxidase (MPO) in vivo. Endogenous HClO becomes a double-edged sword in the organism due to its strong oxidizing power. Normal physiological levels of HClO are effective in inhibiting microorganisms and pathogens in vivo and in regulating apoptosis. However, excessive production of HClO leads to cell and tissue damage and may be associated with certain diseases such as inflammatory diseases or cancer. In body fluids, hypochlorous acid and hypochlorite anions are in equilibrium at the micromolar level, which may increase to millimolar concentrations when the body is in a diseased state. Therefore, real-time monitoring of HClO levels in vivo is of great importance to the study of health sciences.
Mitochondria not only provide energy for human life activities, but also participate in a variety of complex physiological processes. Its abnormality is related to cancer, diabetes, Alzheimer's disease, etc. Mitochondria, the primary consumer of intracellular oxygen, are the primary source of intracellular Reactive Oxygen Species (ROS). HClO, a reactive oxygen species in the cell, is important to maintain proper concentrations in the mitochondria to ensure proper functioning in the cell. Monitoring hypochlorous acid at a sub-cellular level, especially in the mitochondria, is therefore of particular interest and value. The targeted mitochondria fluorescent probe is mainly based on that mitochondria have negative membrane potential, and then the mitochondria are positioned by utilizing fluorophore with positive charge or introducing groups with positive charge such as triphenylphosphine or quaternary ammonium salt in the fluorophore.
The fluorescence probe method is often used as an effective bioanalytical tool because of its advantages such as high selectivity, high sensitivity, real-time monitoring, etc. Fluorescent probes using single fluorescence intensity as the response signal are sensitive to environmental factors and instrument parameters, and ratiometric probes using a ratio of two fluorescence intensities are more suitable for quantitative analysis due to their internal calibration capabilities. Currently, many ratiometric fluorescent probes for in vivo imaging of HClO have been successfully developed, but most of them achieve in vivo imaging under single photon excitation. For biological imaging, short wavelength excitation causes problems of photodamage, shallow penetration and autofluorescence from intrinsic biomolecules in the sample, and the application of two-photon imaging greatly compensates for these defects. Two-photon fluorescent probes have thus been an important topic for researchers to study.
Carbazole compounds are often used as precursors of fluorescent probes because of their strong electron donating ability, large conjugated systems, good rigid planes, stable optical properties, and easy introduction of functional groups by chemical modification. As a classical fluorophore, carbazole-based single-photon fluorescent probes have been reported in many documents, but two-photon fluorescent probes designed by carbazole-based single-photon fluorescent probes are relatively rare.
Disclosure of Invention
The invention aims to provide a mitochondria-targeted hypochlorous acid ratio type two-photon fluorescence probe and a preparation method and application thereof, and aims to solve the technical problem that a proper fluorescence probe structure is obtained through molecular design, and the fluorescence probe has the advantages of excellent two-photon absorption performance, low cytotoxicity, good membrane permeability and biocompatibility, high selectivity and light stability and the like so as to realize quantitative detection of HClO in aqueous solution and cells mitochondria and two-photon fluorescence imaging in the mitochondria.
The invention relates to a ratio type two-photon fluorescent probe for mitochondrial targeting hypochlorous acid, which takes carbazole as a matrix, is abbreviated as MCL, and has the following structural formula:
the preparation method of the mitochondrial targeting hypochlorous acid ratio type two-photon fluorescent probe comprises the following steps:
step 1: synthesis of Compound 1
To a solution of acetone (200mL) was added potassium hydroxide (2.0g, 35.8mmol), potassium iodide (0.4g, 2.39mmol) and 1, 4-dibromobutane (7.73g, 35.8mmol) and heated at 60 ℃ for 1 hour, followed by slow addition of 3, 6-diiodocarbazole (10g, 23.9mmol) and continued heating at reflux for 12 hours; after the reaction was complete, it was cooled and spin dried, washed with water to give the crude product, which was purified by column chromatography (petroleum ether: dichloromethane ═ 10: 1 as eluent) to give intermediate 1, 6.8g, in 51.4% yield.
Step 2: synthesis of Compound 2
Compound 1(2g, 3.6mmol), 4-ethynylbenzaldehyde (1.4g, 10.8mmol), bis-triphenylphosphine palladium dichloride (0.0102g, 0.014mmol), cuprous iodide (0.0054g, 0.028mmol) and triethylamine (7mL) were dissolved in tetrahydrofuran (10mL) under nitrogen protection and reacted at 30 ℃ for 12 hours; after the reaction was complete, it was cooled and spin dried to give the crude product which was purified by column chromatography (petroleum ether: dichloromethane ═ 4: 1 as eluent) to give intermediate 2, 1.3g, in 64.5% yield.
And step 3: synthesis of Compound 3
Adding compound 2(1g, 1.8mmol) into acetonitrile (10mL) under the protection of nitrogen, reacting at 80 ℃ for 1 hour, adding triphenylphosphine (2.8g, 10.8mmol), and continuing to heat for reaction for 36 hours; after the reaction was complete, it was cooled and spin dried to give the crude product which was purified by column chromatography (dichloromethane: methanol 40: 1 as eluent) to give intermediate 3, 0.83g, 56% yield.
And 4, step 4: synthesis of target product MCL
Dissolving compound 3(0.83g, 1.0112mmol), methanesulfonic acid (20 μ L) and 2-mercaptoethanol (0.237g, 3.0334mmol) in dichloromethane (20mL) under nitrogen protection, and reacting at 25 deg.C for 12 h; after the reaction was complete, it was cooled and spin dried to give the crude product which was purified by column chromatography (dichloromethane: methanol 40: 1 as eluent) to give MCL as a white solid, 0.34g, 36% yield.
The synthesis process of the two-photon fluorescent probe MCL comprises the following steps:
the application of the two-photon fluorescent probe is used as a detection reagent when the HClO in mitochondria in cells is quantitatively detected, and the detection method comprises the following steps:
the fluorescent probe MCL of the present invention was dissolved in DMSO to prepare a 2mM stock solution, which was prepared in a volume of 5mL, and 15. mu.L of the stock solution was placed in a sample tube containing 3mL of PBS buffer solution having a pH of 7.4 to prepare a 10. mu.M assay solution.The ultraviolet absorption spectrum data after adding different equivalents of HClO are tested. The detection reagent has absorption peaks at 308nm and 340nm respectively, the absorption peaks of MCL at 308nm and 340nm gradually decrease with the increase of HClO equivalent, the absorption at 380nm gradually increases, and the absorption curve does not change after HClO reaches 5 times of equivalent. With the continuous addition of HClO (0-60. mu.M), the fluorescence maximum emission peak was observed to gradually red-shift from 465nm to 525 nm. When HClO reaches 5 times of equivalent, the fluorescence curve does not change any more, indicating that the saturation equivalent is reached. Adding HClO with different equivalent weight into 10 mu M detection reagent respectively to obtain the fluorescence ratio (I)525nm/I465nm) The maximum value can be reached within seconds. Fluorescence ratio (I) when HClO was added to 10. mu.M of the detection reagent in the range of 0 to 10. mu.M, respectively525nm/I465nm) There is a good linear relationship with the concentration of HClO (R ═ 3 δ/k), with detection limits as low as 35 nM. After 10 times of equivalent of other assay substrates are respectively added into 10 mu M detection reagent for 20min, the fluorescence spectrum change in the range of 370-650nm is detected, and the probe MCL only shows obvious fluorescence change to HClO, which shows that the probe MCL has specific response. The results of the investigation of 10. mu.M detection reagent and its fluorescence spectra in response to HClO in buffer solutions of different pH values indicate the fluorescence ratio (I) of probe MCL and its response to HClO525nm/I465nm) The kit is insensitive to the pH value within the pH range of 6-9, and is suitable for detecting weakly alkaline intramitochondrial HClO. In addition, the probe molecule and the red mitochondria are subjected to co-localization imaging of mitochondria and two-photon confocal imaging of HClO in the mitochondria.
The two-photon fluorescent probe has a simple structure and is easy to synthesize. The probe responds to HClO quickly and sensitively and shows specific response. After HClO is added, two acetal response sites in the probe structure are oxidized into two aldehyde groups (figure 1), the electron cloud density distribution is changed before and after the reaction, and the fluorescence and ultraviolet absorption properties are changed accordingly. Two-photon confocal fluorescence microscopic imaging experiments show that the probe has good permeability to HeLa cells, can effectively position mitochondria in the cells (the positioning coefficient is 0.93), and is suitable for two-photon fluorescence imaging of HClO in the mitochondria of the cells.
Drawings
FIG. 1 is a diagram showing the reaction mechanism of fluorescent probe molecules MCL and HClO of the present invention.
FIG. 2 shows (a) a UV absorption spectrum of 10. mu.M probe with HClO (0-60. mu.M); (b) fluorescence emission spectrum.
FIG. 3 shows the ratio of fluorescence emission peak intensities (I) of 10. mu.M probe to 10. mu.M probe with HClO (10. mu.M, 20. mu.M, 50. mu.M) added thereto525nm/I465nm) Graph against time.
FIG. 4 shows the ratio of fluorescence emission peak intensities (I) after HClO (0-10. mu.M) was added to 10. mu.M probe525nm/I465nm) Linear dependence on concentration.
FIG. 5 is a fluorescence selectivity plot of 10-fold equivalents of additional assay substrate added to a 10. mu.M probe.
FIG. 6 shows the ratio of fluorescence emission peak intensities (I) before and after adding HClO (50. mu.M) to 10. mu.M probe525nm/I465nm) Graph relating to pH.
FIG. 7 is a cross-sectional view showing the effective two-photon absorption before and after adding HClO to the probe MCL, with the relative two-photon fluorescence intensity (I) shown in the insetout) And input power (I)in) Graph of logarithmic relationship of (c).
FIG. 8 is a graph of HeLa cell viability at different concentrations (0. mu.M, 10. mu.M, 20. mu.M, 30. mu.M) of probe molecules.
FIG. 9 is a photograph of confocal fluorescence imaging for location verification of mitochondria of HeLa cells co-stained simultaneously with 10. mu.M probe and 1. mu.M MitoTracker red.
FIG. 10 is an image of two-photon confocal cell with different concentrations of exogenous HClO (0-40 μ M) added to HeLa cells, with a probe concentration of 10 μ M, a blue channel fluorescence emission collection range of 420-470nm, a green channel fluorescence emission collection range of 500-540nm, and an excitation wavelength of 740 nm.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: synthesis of Compound 1
To an acetone solution (200mL) were added potassium hydroxide (2.0g, 35.8mmol), potassium iodide (0.4g, 2.39mmol) and 1, 4-dibromobutane (7.73 g)35.8mmol) and heated at 60 ℃ for 1 hour. 3, 6-diiodocarbazole (10g, 23.9mmol) was slowly added and heating and refluxing continued for 12 hours. Cooling, spin-drying and washing to obtain a crude product. Purification by column chromatography (petroleum ether: dichloromethane ═ 10: 1 as eluent) gave intermediate 1, 6.8g in 51.4% yield.1H NMR(400MHz,CDCl3,ppm)δ8.34(d,J=1.5Hz,2H),7.74(d,J=1.6Hz,1H),7.71(d,J=1.6Hz,1H),7.19(s,1H),7.17(s,1H),4.29(t,J=7.0Hz,2H),3.37(t,J=6.4Hz,2H),2.06–1.98(m,2H),1.86(m,2H).13C NMR(100MHz,CDCl3,ppm)δ139.38,134.69,129.48,124.08,110.76,81.93,42.37,32.82,30.02,27.44.
Example 2: synthesis of Compound 2
Compound 1(2g, 3.6mmol), 4-ethynylbenzaldehyde (1.4g, 10.8mmol), bis-triphenylphosphine palladium dichloride (0.0102g, 0.014mmol), cuprous iodide (0.0054g, 0.028mmol) and triethylamine (7mL) were dissolved in tetrahydrofuran (10mL) under a nitrogen blanket and reacted at 30 ℃ for 12 hours. Cooling and spin-drying to obtain a crude product. Purification by column chromatography (petroleum ether: dichloromethane ═ 4: 1 as eluent) afforded intermediate 2, 1.3g, in 64.5% yield.1H NMR(400MHz,CDCl3,ppm)δ10.04(s,2H),8.33(s,2H),7.89(t,J=7.4Hz,4H),7.71(m,6H),7.42(d,J=8.5Hz,2H),4.38(m,2H),3.42(t,J=6.3Hz,1H),3.19(t,J=6.6Hz,1H),2.15–2.01(m,2H),1.92(m,2H).13C NMR(100MHz,CDCl3,ppm)δ191.47,140.67,135.13,133.12,131.92,130.18,130.14,129.67,129.59,124.63,122.62,113.56,109.14,94.83,87.58,32.84,30.74,27.59,5.54.
Example 3: synthesis of Compound 3
Compound 2(1g, 1.8mmol) was added to acetonitrile (10mL) under nitrogen, reacted at 80 ℃ for 1 hour, triphenylphosphine (2.8g, 10.8mmol) was added, and heating was continued for 36 hours. Cooling and spin-drying to obtain a crude product. Purification by column chromatography (dichloromethane: methanol ═ 40: 1 as eluent) afforded intermediate 3, 0.83g, in 56% yield.1H NMR(400MHz,DMSO-d6,ppm)δ10.05(s,2H),8.53(s,2H),7.98(d,J=8.0Hz,4H),7.87(m,3H),7.79(d,J=8.0Hz,4H),7.76–7.68(m,16H),4.51(t,J=6.9Hz,2H),3.62(t,J=15.1Hz,2H),2.00–1.93(m,2H),1.62(m,2H).13C NMR(100MHz,CDCl3,ppm)δ191.43,140.73,135.13,135.08,135.05,133.60,133.50,132.15,132.05,131.88,130.50,130.37,130.20,130.08,129.67,128.57,128.45,124.24,122.31,118.10,117.24,113.34,110.05,94.88,87.59,42.55,29.19,22.66,20.15.
Example 4: synthesis of target product MCL
Compound 3(0.83g, 1.0112mmol), methanesulfonic acid (20. mu.L), 2-mercaptoethanol (0.237g, 3.0334mmol) was dissolved in dichloromethane (20mL) under nitrogen and reacted at 25 ℃ for 12 hours. Cooling and spin-drying to obtain a crude product. Purification by column chromatography (dichloromethane: methanol 40: 1 as eluent) gave MCL as a white solid, 0.34g, 36% yield.1H NMR(400MHz,DMSO-d6,ppm)δ8.43(s,2H),7.84(m,3H),7.67(m,14H),7.62–7.59(m,2H),7.54(d,J=8.2Hz,4H),7.46(d,J=8.2Hz,4H),6.09(s,2H),4.51–4.43(m,4H),3.87(m,2H),3.58(t,J=14.6Hz,2H),3.20(m,4H),1.97–1.88(m,2H),1.58(m,2H).13C NMR(100MHz,DMSO-d6,ppm)δ140.13,139.74,134.86,133.48,131.13,130.17,129.59,126.91,124.27,122.85,121.80,118.69,117.84,112.85,110.21,91.14,87.52,85.55,71.76,41.69,33.53,20.10,19.57.
Example 5: spectroscopic testing of fluorescent probe molecules
The fluorescent probe MCL of the present invention was dissolved in DMSO to prepare a 2mM stock solution, which was prepared in a volume of 5mL, and 15. mu.L of the stock solution was placed in a sample tube containing 3mL of PBS buffer solution having a pH of 7.4 to prepare a 10. mu.M assay solution. The ultraviolet absorption spectrum data of the material containing different equivalent weight of HClO are tested. The detection reagent has absorption peaks at 308nm and 340nm respectively, the absorption peaks of MCL at 308nm and 340nm gradually decrease with the increase of HClO equivalent, the absorption at 380nm gradually increases, and the absorption curve does not change when HClO reaches 5 times of equivalent (FIG. 2 a). With the continuous addition of HClO (0-60. mu.M), the fluorescence maximum emission peak was observed to gradually red-shift from 465nm to 525 nm. When HClO reached 5-fold equivalent, the fluorescence curve did not change, indicating that saturation equivalent was reached (FIG. 2 b). Will be different fromThe amount of HClO added to each 10. mu.M of detection reagent was measured, and the fluorescence ratio (I)525nm/I465nm) The maximum value can be reached within seconds (fig. 3). Fluorescence ratio (I) when HClO was added to 10. mu.M of the detection reagent in the range of 0 to 10. mu.M, respectively525nm/I465nm) There was a good linear relationship with the concentration of HClO (R ═ 3 δ/k), with detection limits as low as 35nM (fig. 4). After 10 times of equivalent of other assay substrates were added to 10. mu.M of detection reagent respectively and acted for 20min, the fluorescence spectrum change in the range of 370-650nm was detected, and it can be seen that the probe MCL showed only a significant fluorescence change to HClO, indicating that it has a specific response (FIG. 5). The results of the investigation of 10. mu.M detection reagent and its fluorescence spectra in response to HClO in buffer solutions of different pH values indicate the fluorescence ratio (I) of probe MCL and its response to HClO525nm/I465nm) The kit is insensitive to pH value within the pH range of 6-9 (figure 6), and is suitable for detecting weakly alkaline intramitochondrial HClO.
Example 6: two-photon performance testing of fluorescent probe molecules
By using a two-photon induced fluorescence measurement technology, the probe MCL and the two-photon absorption performance of the probe MCL after responding HClO are measured. The probe MCL showed a maximum effective two-photon absorption cross-sectional value at 720nm of 50 GM. After 5 equivalents of HClO were added, the reaction product exhibited a maximum effective two-photon absorption cross-sectional value of 60GM at 760nm (FIG. 7). By varying the energy, input power (I), of the incident excitation lightin0.3-0.8 mW and relative fluorescence output energy (I)out) The logarithmic relationship, with slopes of 1.97 and 1.99, respectively, follows the law of two-photon properties (fig. 7 inset). The experiment proves that the probe MCL has two-photon absorption property and can be applied to two-photon fluorescence imaging for detecting HClO in cells.
Example 7: cell culture and cytotoxicity assays
Cell culture: HeLa cells were cultured in 90% DMEM (sugar and amino acids) and 10% FCS (fetal calf serum), mixed with 1% streptomycin to prevent bacterial contamination, and incubated at 37 deg.C with 5% CO2And (5) culturing in an incubator.
Cytotoxicity: by MTT (3- (4, 5-dimethylthia) as reported in the literatureOxazole-2) -2, 5-diphenyltetrazolium bromide salt) assay to test cytotoxicity. HeLa cells were cultured in a 96-well plate for 24 hours before the test, fresh DMEM was added before the addition of the probe, probe MCL (0,10,20 and 30. mu.M) was added at different concentrations, and the treated cells were cultured at 37 ℃ for 24 hours at 5% carbon dioxide content. Subsequently, 5mg/mL MTT (40. mu.L/well) was added to the cells and the culture was continued for 4 hours (37 ℃ C., 5% CO)2). The culture broth was pipetted into DMSO (150. mu.L/well) and the absorbance at 570nm was recorded. According to the formula for cell viability: percent cell survival ═ OD570(sample)/OD570(control) x 100, cell viability was calculated (figure 8). The test result shows that the MCL has less biological toxicity and is suitable for cell imaging.
Example 8: mitochondrial localization test
HeLa cells were cultured in DEME (invitrogen) medium, and the day before imaging, HeLa cells were placed in a laser confocal dish, 10. mu.M MCL was added to HeLa cells, and the mixture was incubated at 37 ℃ with 5% CO2The cells were incubated in the cell culture chamber for 0.5 hour, washed with neutral PBS buffer solution for 3 times, then 1 μ M commercial mitochondrial stain MitoTracker red (MTR) solution was added to the petri dish and incubation was continued for 0.5 hour, and then washed with neutral PBS buffer solution for 3 times and two-photon fluorescence confocal imaging was performed. Setting probe molecule MCL as blue channel (lambda)em=420-470nm,λex740 nm); setting commercial mitochondrial stain MitoTracker red (MTR) to red channel (λ)em=580-600nm,λex579 nm). The results showed that the fluorescence images of both overlapped well and the Pearson co-localization coefficient of MCL and MTR was calculated to be 0.93, indicating that MCL could target mitochondria in living cells well (fig. 9).
Example 9: intracellular HClO two-photon fluorescence imaging
Groups of HeLa cells were incubated with the probe for 30 minutes, and then with different concentrations of HClO (0,5,10,20 and 40 μ M) for 30 minutes, respectively, before two-photon imaging. Setting blue channel (lambda)em420-em500 and 540nm) were imaged and observed (fig. 10). In the case of the blank control group,it was observed that both the blue channel and the green channel showed significant fluorescence, whereas the blue fluorescence was stronger than the green fluorescence, both of which were attributable to the probe MCL itself. At the same time, bright fluorescence of intracellular filamentous structures, which is typical of mitochondria, can also be clearly observed. As the intensity of blue fluorescence and green fluorescence gradually decreased with increasing concentration of HClO, and blue fluorescence decreased faster, it was observed that increasing concentration of HClO resulted in a change in pseudo-color image. These results clearly indicate that probe MCL is able to quantitatively monitor the levels of HClO and its fluctuations within the mitochondria of living cells by two-photon imaging techniques.
Claims (3)
2. a method for preparing the two-photon fluorescent probe according to claim 1, which comprises the steps of:
step 1: synthesis of Compound 1
Adding potassium hydroxide, potassium iodide and 1, 4-dibromobutane into an acetone solution, heating for 1 hour at 60 ℃, then slowly adding 3, 6-diiodocarbazole, and continuing heating reflux reaction; cooling and spin-drying after the reaction is finished, washing with water to obtain a crude product, and purifying by column chromatography to obtain an intermediate 1;
step 2: synthesis of Compound 2
Under the protection of nitrogen, dissolving the compounds 1, 4-ethynylbenzaldehyde, bis-triphenylphosphine palladium dichloride, cuprous iodide and triethylamine in tetrahydrofuran, and reacting at 30 ℃; after the reaction is finished, cooling and spin-drying to obtain a crude product, and purifying by column chromatography to obtain an intermediate 2;
and step 3: synthesis of Compound 3
Under the protection of nitrogen, adding a compound 2 into acetonitrile, reacting for 1 hour at 80 ℃, adding triphenylphosphine, and continuing to heat for reaction; after the reaction is finished, cooling and spin-drying to obtain a crude product, and purifying by column chromatography to obtain an intermediate 3;
and 4, step 4: synthesis of target product MCL
Under the protection of nitrogen, dissolving a compound 3, methanesulfonic acid and 2-mercaptoethanol in dichloromethane, and reacting at 25 ℃; after the reaction was complete, it was cooled and spin-dried to give the crude product, which was purified by column chromatography to give MCL as a white solid.
3. Use of the two-photon fluorescent probe according to claim 1, wherein:
the application of the reagent in preparing the reagent for quantitatively detecting the HClO in mitochondria in cells.
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