CN109781678B

CN109781678B - Preparation and application of ratiometric fluorescent probe applied to intramitochondrial hypochlorous acid detection

Info

Publication number: CN109781678B
Application number: CN201711118691.5A
Authority: CN
Inventors: 申世立; 葛燕青; 曹晓群
Original assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Current assignee: Shandong First Medical University and Shandong Academy of Medical Sciences
Priority date: 2017-11-14
Filing date: 2017-11-14
Publication date: 2022-03-18
Anticipated expiration: 2037-11-14
Also published as: CN109781678A

Abstract

The invention relates to preparation and application of a ratiometric fluorescent probe applied to intramitochondrial hypochloric acid detection. The probe is made of imidazo [1,5-a]The pyridine fluorophore is used as an energy donor, the rhodamine fluorophore is used as an energy acceptor, and the pyridinium structure is a mitochondrion targeting group. The probe can react with hypochlorous acid with high selectivity and high sensitivity. In the absence of hypochlorous acid, the probe emits 467 nm fluorescence; when hypochlorous acid is present, the probe emits fluorescent light at 467 nm and 575 nm simultaneously. The fluorescence at 575 nm gradually increases and the fluorescence at 467 nm gradually decreases with the increase of the hypochlorous acid concentration, the ratio of the two (I)₅₇₅/I₄₆₇) Has good linear relation with the hypochlorous acid concentration within a certain range (0.5-3.5 mu M). The probe can be used for imaging analysis of hypochlorous acid in mitochondria and has wide application prospect.

Description

Preparation and application of ratiometric fluorescent probe applied to intramitochondrial hypochlorous acid detection

Technical Field

The invention relates to preparation and application of a hypochlorous acid ratiometric fluorescent probe, which can be used for detecting the concentration of the hypochlorous acid in mitochondria.

Background

Hypochlorous acid is an important signal molecule and can regulate and control various cell physiological activities. Hypochlorous acid in the organism is generated by hydrogen peroxide and chloride ions under the catalysis of myeloperoxidase. In the immune system, hypochlorous acid can play a certain role in defending against the invasion of microorganisms. However, excessive hypochlorous acid levels in the body can easily cause diseases such as arthritis, atherosclerosis, and cancer. Therefore, the real-time monitoring of intracellular hypochlorous acid concentration has important biological significance.

Various analysis methods are currently used for measuring hypochlorous acid, wherein fluorescent probes are widely concerned and developed due to the characteristics of high sensitivity, good selectivity, low detection limit, small damage to organisms and the like. Many hypochlorous acid probes in the literature measure hypochlorous acid through the change of fluorescence intensity of a single emission peak, and the detection result is difficult to be influenced by environmental factors. In order to reduce measurement errors, a ratio type hypochlorous acid fluorescent probe is designed and synthesized, and the ratio determination of hypochlorous acid is realized.

Mitochondria are the main site of intracellular generation of active oxygen, and monitoring of change in mitochondrial hypochlorous acid concentration is important because the intracellular hypochlorous acid level is closely related to the intracellular redox balance.

However, there are few reports on ratiometric hypochlorous acid fluorescent probes for mitochondrial targeting.

Based on the design, the inventors design that an imidazo [1,5-a ] pyridine structure is used as an energy donor, a rhodamine structure is used as an energy acceptor, and a mitochondria targeting group, namely a pyridinium structure, is introduced into the structure to construct a ratio type hypochlorous acid fluorescent probe. The probe has a mitochondrial targeting function and can be applied to measuring the concentration of hypochlorous acid in mitochondria.

Disclosure of Invention

1. The invention aims to provide preparation and application of a ratio-type hypochlorous acid fluorescent probe for mitochondrion targeting.

2. The invention discloses a ratio type hypochlorous acid fluorescent probe for mitochondrion targeting, which has the following structure (IRP):

3. the invention also aims to provide the application of the fluorescent probe in fluorescence imaging for detecting the concentration of hypochlorous acid in mitochondria.

The preparation method of the fluorescent probe disclosed by the invention comprises the following steps (figure 1).

A solution of the hypochlorous acid probe in ethanol and a PBS (0.01M) buffer solution (V/V = 3:7, pH 7.40) was prepared. In the absence of hypochlorous acid, the probe emits 467 nm fluorescence; when hypochlorous acid is present, the probe emits fluorescent light at 467 nm and 575 nm simultaneously. The fluorescence at 575 nm gradually increased with the increase of the hypochlorous acid concentration, while the fluorescence intensity at 467 nm decreased, the ratio of the two (I)₅₇₅/I₄₆₇) Shows a good linear relationship with the hypochlorous acid concentration within a certain range (0.5-3.5. mu.M), as shown in FIG. 2.

A certain amount of active nitrogen, active oxygen and other common species are respectively added into ethanol and PBS (0.01M) buffer solutions of the probe, and the fluorescence test result shows that the probe has good selectivity on hypochlorous acid, which is shown in figure 3.

The probe and the mitochondrial dye Mito Tracker Deep Red are used for respectively incubating RAW264.7 cells, and then imaging analysis is carried out, and the result of figure 4 shows that the probe can target mitochondria.

When RAW264.7 cells are incubated with the probe of the present invention and then imaging is performed after further treating the cells with LPS, the ratio of blue/red fluorescence intensity of the probe is significantly reduced, so that the probe can be used for detecting mitochondrial hypochlorous acid, as shown in FIG. 5.

The ratio type hypochlorous acid fluorescent probe can be applied to the detection of hypochlorous acid in a solution and the detection of hypochlorous acid in cell mitochondria, and has wide application prospect.

Drawings

FIG. 1 is a scheme for synthesizing the probe of the present invention.

FIG. 2 shows the change in fluorescence of the probe of the present invention with the change in hypochlorous acid concentration. FIG. 2 (a) is a fluorescence emission spectrum; FIG. 2 (b) is a graph showing the change in the fluorescence intensity ratio at different concentrations of hypochlorous acid.

FIG. 3 shows the selectivity of the probes of the present invention for HOCl and other common species in organisms.

FIG. 4 shows the distribution of the probes of the present invention in RAW264.7 cells. FIG. 4 (a) is a fluorescence image collected from the blue channel (410-520 nm) of the probe; FIG. 4 (b) is a fluorescence image of Mito Tracker Deep Red; FIG. 4 (c) is a superimposed view of (a) and (b); FIG. 4 (d) is a bright field image; the co-localization coefficient was 0.95.

FIG. 5 is fluorescence imaging of HOCl in mitochondria of RAW264.7 cells with the probe of the present invention. FIG. 5 (a) shows the intracellular fluorescence of the probe before and after LPS treatment; fig. 5 (b) is the ratio of blue to red light.

Detailed description of the invention

Example 1: the synthetic route of the probe is shown in figure 1:

compound 1 (1 mmol) and triethylAmine (3 mmol) dissolved in 20 mL anhydrous CH₂Cl₂Then adding chloroacetyl chloride (2 mmol) into the solution, continuing stirring for 4 hours, and removing the solvent by reduced pressure distillation to obtain a red solid, namely the compound 2 (which is not separated and purified and is directly used for the next reaction).

Compound 2 (1 mmol) and pyridine (1 mL) were dissolved in 30 mL of anhydrous acetonitrile and refluxed for 12 hours. The solvent was removed under reduced pressure and then purified by column chromatography (dichloromethane: methanol = 10: 1) to give a yellow solid, which was the hypochlorous acid fluorescent probe of the present invention.

To be provided with¹H NMR、¹³It was characterized by C NMR and HRMS with the following data:

¹H NMR (400 MHz, d ₆-DMSO), δ (ppm): 0.92 (t, 3H, J = 7.6 Hz, CH3), 1.09 (t, 6H, J = 6.8 Hz, CH3), 1.32 (q, 2H, J = 7.6 Hz, CH2), 1.68-1.73 (m, 2H, CH2), 2.92 (t, 2H, J = 7.4 Hz, CH2), 3.26 (br, 4H, CH2), 3.32 (br, 4H, CH2 ), 3.68 (br, 4H, CH2), 5.48 (s, 2H, CH2), 6.34 (d, 2H, J = 9.2 Hz, ArH), 6.49 (d, 1H, J = 8.8 Hz, ArH), 6.56-6.61 (m, 2H, ArH), 6.68 (d, 1H, J = 2.0 Hz, ArH), 6.75 (d, 1H, J = 7.2 Hz, ArH), 7.05 (d, 1H, J = 6.8 Hz, ArH), 7.52 (s, 1H, ArH), 7.54-7.62 (m, 2H, ArH), 7.85 (d, 1H, J = 6.8 Hz, ArH ), 8.14-8.17 (m, 2H, ArH), 8.29 (d, 1H, J = 7.2 Hz, ArH), 8.70 (t, 1H, J = 8.0 Hz, ArH), 8.74 (d, 2H, J = 5.6 Hz, ArH), 10.60 (s, 1H, NH)。

¹³C NMR (100 MHz, d ₆-DMSO), δ (ppm): 167.6, 164.2, 163.8, 153.4, 153.1, 151.8, 151.7, 149.0, 146.9, 146.1, 138.9, 134.1, 129.5, 129.4, 129.2, 128.5, 128.2, 126.4, 124.3, 123.7, 123.3, 122.8, 118.7, 115.9, 112.4, 111.7, 108.5, 108.4, 104.1, 101.9, 97.5, 65.5, 60.6, 47.9, 44.1, 29.4, 29.1, 25.6, 22.1, 14.1, 12.9。

HRMS m/z: calcd for C₄₇H₄₈ClN₈O₄S [M]+: 823.3487; found: 823.3552. m/z: calcd for C₄₇H₄₉ClN₈O₄S [M+H]²⁺/2: 412.1782; found: 412.1790。

example 2: the probe of the invention changes with the fluorescence when the concentration of hypochlorous acid changes.

PBS buffer solution (containing 30% ethanol, pH 7.40) for probe preparation. Hypochlorous acid solutions of different concentrations were added to the above buffer solutions, respectively, and then subjected to fluorescence analysis, λ ex = 370 nm. The results are shown in FIG. 2.

Example 3: selectivity of the probes of the invention.

PBS buffer solution (containing 30% ethanol, pH 7.40) for probe preparation. Certain amount of certain interfering substances were added separately, and then the above samples were subjected to fluorescence test analysis, the results of which are shown in FIG. 3. (1) Blank, (2) H₂O₂, (3): t-BuOOH, (4): t-BuO•, (5): ¹O₂, (6): ^-O₂, (7): ON, (8): •OH, (9): ONOO^-, (10): NO₂ ^-, (11): AcO^-, (12): Br^-, (13): Cl^-, (14): I^-, (15): CO₃ ^2-, (16): SO₄ ^2-, (17): ^-And (4) OCl. Wherein the hypochlorous acid concentration is 4 mu M, the (2) - (9) concentration is 40 mu M, and the concentration of other ions is 1 mM.

Example 4: distribution of the probes of the invention within the cell.

RAW264.7 cells were first incubated with 1. mu.M of the probe of the invention for 30 minutes, followed by addition of 0.2. mu.M Mito Tracker Deep Red for further incubation for 30 minutes. Fluorescence imaging was performed using a confocal laser scanning microscope, and a fluorescence image of the probe (410-. Overlay analysis was then performed to calculate co-localization coefficients, see fig. 4.

Example 5: the probe is used for imaging analysis of the intracellular hypochlorous acid.

Experimental groups: firstly, 1 mu g/mL LPS is used for stimulating RAW264.7 cells for 6 hours, then 1 mu g/mL PMA is added for continuing the culture for 30 minutes, and then 1 mu M of the probe is added into a serum-free culture medium for culturing for 30 minutes; performing fluorescence imaging by using a laser confocal microscope, collecting the fluorescence of 410-520 nm and 560-700 nm, and counting the ratio of the two lights;

control group: adding 1 mu M of probe into a serum-free culture medium, and culturing for 30 minutes; the cells were washed 3 times with PBS, fluorescence imaging was performed, and the fluorescence at 410-.

Claims

1. The chemical structural formula of the hypochlorous acid fluorescent probe IRP is as follows:

；

the preparation method of the hypochlorous acid fluorescent probe IRP comprises the following steps:

1 mmol of compound 1 and 3 mmol of triethylamine are dissolved in 20 mL of anhydrous CH₂Cl₂Stirring at 0 ℃ to obtain a solution, adding 2 mmol of chloroacetyl chloride into the solution, continuously stirring for 4 hours, and removing the solvent by reduced pressure distillation to obtain a red solid, namely a compound 2;

1 mmol of compound 2, 1 mL of pyridine are dissolved in 30 mL of anhydrous acetonitrile, refluxed for 12 hours, the solvent is removed under reduced pressure, and then column chromatography is carried out using V dichloromethane: v methanol = 10: 1, purifying to obtain a yellow solid, namely the hypochlorous acid fluorescent probe IRP;

the synthetic route is as follows:

。

2. use of the probe IRP according to claim 1 for the detection of a sample containing hypochlorous acid.

3. Use of the probe IRP of claim 2 for detecting a sample containing hypochlorous acid, wherein said sample containing hypochlorous acid is a solution containing hypochlorous acid and biological cells.