Near-infrared fluorescent probe for detecting hypochlorous acid as well as preparation method and application of near-infrared fluorescent probe

Abstract

The invention discloses a near-infrared fluorescent probe for detecting hypochlorous acid as well as a preparation method and application of the near-infrared fluorescent probe, and belongs to the technical field of fluorescent probe detection and analysis of hypochlorous acid. The near-infrared fluorescent probe for detecting hypochlorous acid is (E)-2-(5, 5-dimethyl-3-(4-thiomorpholine styryl) cyclohex-2-ene-1-subunit) malononitrile (NMS), and the structural formula of the near-infrared fluorescent probe is shown in the specification. The invention also specifically discloses a preparation method of the near-infrared fluorescent probe for detecting hypochlorous acid and application of the near-infrared fluorescent probe for detecting hypochlorous acid in simultaneous rapid and high-selectivity recognition of hypochlorous acid and preparation of an imaging detection reagent for hypochlorous acid in an organism. The fluorescent probe prepared by the invention can realize colorimetric and fluorescence detection of hypochlorous acid, and has the advantages of fast response, low detection limit and high selectivity.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probe detection and analysis of hypochlorous acid (HClO), and specifically relates to a near-infrared fluorescent probe for detecting hypochlorous acid, a preparation method and an application thereof. Background Art

[0002] Hypochlorous acid (HClO) has always attracted much attention as an important member of the reactive oxygen family. Endogenous HClO is catalyzed by myeloperoxidase (MPO) to convert H2O2 and Cl - Produced, mostly in the form of HClO / ClO - The form of HClO exists in the physiological environment. HClO, which has strong oxidizing properties, is an important protective agent against the invasion of bacteria and pathogens, while abnormal endogenous HClO concentration is associated with various oxidative stress diseases, and excessive HClO concentration is also used as a biomarker for some inflammation-related diseases. Therefore, it is very important to develop a reliable tool to evaluate and observe the level of HClO in inflammatory tissues and cells. Although the diagnosis of related diseases is currently more dependent on X-ray imaging and magnetic resonance imaging, the lack of sensitivity and inability to apply on a large scale for early inflammation diagnosis are still difficult to avoid. Fluorescence imaging technology has been favored by current clinical and basic research due to its high sensitivity, high resolution and non-invasive characteristics. The present invention introduces thiomorpholine into dicyanoisophorone emission group to construct a near-infrared fluorescent probe NMS. After the fluorescent probe NMS reacts with HClO, the sulfur atom of thiomorpholine is oxidized by HClO, and the intramolecular photoinduced electron transfer process is blocked, releasing red fluorescence, which can quickly and sensitively identify HClO. There is currently no relevant report in this regard. Summary of the invention

[0003] The purpose of the present invention is to provide a near-infrared fluorescent probe for detecting hypochlorous acid and a preparation method thereof. The fluorescent probe prepared by the method can be used for rapid and highly selective identification of hypochlorous acid in organisms and for biological imaging.

[0004] The present invention adopts the following technical scheme to achieve the above-mentioned purpose: a near-infrared fluorescent probe for detecting hypochlorous acid, characterized in that: the fluorescent probe is (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS), and its structural formula is

[0005]

[0006] The preparation method of the near-infrared fluorescent probe for detecting hypochlorous acid of the present invention is characterized by the specific process of: using ethanol as a reaction medium, using 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile and 4-thiomorpholine benzaldehyde as reaction raw materials, using piperidine as a catalyst, reacting at 70-80°C under the protection of an inert gas, filtering to remove the solvent after the reaction is complete, and then washing with ethanol and ether to obtain the target product, the near-infrared fluorescent probe for detecting hypochlorous acid.

[0007] It is further defined that the molar ratio of the 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile, 4-thiomorpholinebenzaldehyde and piperidine is 1:1:0.01-0.05.

[0008] The synthetic route in the preparation method of the near-infrared fluorescent probe for detecting hypochlorous acid of the present invention is:

[0009]

[0010] The application of the near-infrared fluorescent probe for detecting hypochlorous acid in the rapid and highly selective identification of hypochlorous acid is characterized by the specific process that: the fluorescent probe appears pink in a mixed solution of phosphate buffer and acetonitrile in a volume ratio of 8:1, and has no fluorescence under a 365nm portable ultraviolet lamp; after a solution containing hypochlorous acid is added to the mixed system, the color of the mixed system changes to light yellow, and exhibits bright red fluorescence under the irradiation of a 365nm portable ultraviolet lamp.

[0011] The invention discloses an application of the near-infrared fluorescent probe for detecting hypochlorous acid in the preparation of an imaging detection reagent for hypochlorous acid in a living body.

[0012] The invention discloses an application of the near-infrared fluorescent probe for detecting hypochlorous acid in the preparation of an imaging detection reagent for hypochlorous acid in cells.

[0013] The invention discloses an application of the near-infrared fluorescent probe for detecting hypochlorous acid in preparing an imaging detection reagent for colitis in an organism.

[0014] Compared with the existing fluorescent probe technology for detecting hypochlorous acid, the fluorescent probe for detecting hypochlorous acid in an organism prepared by the present invention has a simple preparation process, can be obtained by only one-step reaction, is convenient for post-processing, and has a high yield. The fluorescent probe prepared by the present invention can detect hypochlorous acid in the near-infrared region. The fluorescent probe for detecting hypochlorous acid in an organism prepared by the present invention can realize colorimetric and fluorescent detection of hypochlorous acid, and has a fast response, a low detection limit, and a high selectivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1It is the UV-visible absorption spectrum of the fluorescent probe NMS prepared in Example 1 after adding 3 times equivalent of hypochlorous acid into the solution and the fluorescence spectrum at an excitation wavelength of 475 nm.

[0016] Figure 2 The UV (left) and fluorescence (right) spectra of the fluorescent probe NMS prepared in Example 1 after adding different equivalents of hypochlorous acid to the solution are shown in the insets, and the solution colors before and after adding hypochlorous acid under sunlight and hand-held UV 365nm irradiation.

[0017] Figure 3 It is a linear fit between the fluorescence intensity of the fluorescent probe NMS prepared in Example 1 after reacting with different equivalents of hypochlorous acid in a solution and the concentration of the added detection substance.

[0018] Figure 4 The graph is a curve showing the change of the fluorescence intensity of the solution at 675 nm over time after adding different concentrations of hypochlorous acid to the solution of the fluorescent probe NMS prepared in Example 1.

[0019] Figure 5 The fluorescence spectrum (left) and intensity bar graph (right) of the fluorescent probe NMS prepared in Example 1 reacting with different interfering ions in aqueous solution (0-blank; 1-H2O2; 2-t-BuOOH; 3-·OH; 4-O2 ·- ; 5- 1 O2;6-ONOO - ;7-NO;8-HSO3 - ;9-HS - ;10-Cys;11-Hcy;12-GSH;13-ClO - ).

[0020] Figure 6 This is a cell imaging picture of the fluorescent probe NMS prepared in Example 1 reacting with hypochlorous acid in HeLa cells.

[0021] Figure 7 This is a cell imaging picture of the fluorescent probe NMS prepared in Example 1 reacting with hypochlorous acid in RAW 264.7 cells.

[0022] Figure 8 This is the imaging picture of the fluorescent probe NMS prepared in Example 1 in colitis model mice. DETAILED DESCRIPTION

[0023] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0024] Example 1

[0025] Synthesis of Fluorescent Probe Compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS)

[0026] The fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) was reacted by the following synthetic route:

[0027]

[0028] Under nitrogen protection, 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile (267.8 mg, 1.29 mmol) and 4-thiomorpholinebenzaldehyde (240.5 mg, 1.29 mmol) were added to a single-mouth bottle, and then 10 mL of ethanol and 2-3 drops of piperidine were added. The reaction was carried out at 70-80 ° C for 12 hours. After the reaction was completed, it was cooled to room temperature and filtered to obtain a crude product. It was then washed with ethanol and ether to obtain 411 mg of a purple-red solid product with a yield of 84%.

[0029] The purple solid product prepared in this example has a hydrogen nuclear magnetic resonance spectrum: 1 H NMR (400MHz, CDCl3, 25℃, TMS): δ = 7.42 (d, J = 8.8Hz, 2H, Ar–H), 7.00 (d, J = 16.0Hz, 1H, = H), 6.77–6.86 (m, 4H, Ar– H&=H),3.72-3.74(m,4H,CH2),2.71-2.73(m,4H,CH2),2.58(s,2H,CH2),2.45(s,2H,CH2),1.07(s,6H,CH3)ppm.

[0030] Its carbon NMR spectrum is: 13 C NMR (100MHz, CDCl3, 25℃, TMS): δ = 163.9, 154.8, 151.1, 137.3, 129.4, 126.0 ,125.8,122.2,115.5,114.0,113.3,50.8,43.0,39.2,32.0,28.1,26.0ppm.

[0031] Its high resolution mass spectrum is: HRMS (ESI) m / z:calcd.for C 23 H 26 N3S + [M+H + ]:376.1842,found376.1846.

[0032] This proves that the purple product is (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS), and its structural formula is

[0033]

[0034] Example 2

[0035] UV and fluorescence responses of fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) to hypochlorous acid

[0036] The fluorescent probe NMS prepared in Example 1 was dissolved in a mixed solution of 0.1 M pH 7.4 phosphate buffer and acetonitrile, wherein the volume ratio of the phosphate buffer to the acetonitrile was 8:1, and finally a test solution with a concentration of 10 μM fluorescent probe NMS was obtained. Figure 1 The UV-visible absorption spectrum and fluorescence spectrum of the fluorescent probe NMS prepared in Example 1 at an excitation wavelength of 475 nm after adding the detection substance hypochlorous acid are given. It can be seen from the figure that the maximum absorption peak of the fluorescent probe NMS is located at 450 nm and the emission peak is located at 675 nm after adding 3.0 times the equivalent of hypochlorous acid. Therefore, the Stokes shift is determined to be 225 nm, which is a relatively large value that makes the probe NMS very suitable for biological imaging applications. Figure 2 As shown in (left), after adding different equivalents of hypochlorous acid aqueous solution, as the concentration of HClO increases, the absorption of the fluorescent probe NMS solution at 485nm decreases rapidly, while the absorption at 450nm gradually increases, thus forming an isosbestic point at a wavelength of 475nm, and the color of the reaction solution changes from light pink to light yellow. Figure 2 (Right) As shown in Figure 1, when excited at 475 nm, the solution of the fluorescent probe NMS has an obvious fluorescence opening process at 675 nm. The fluorescence intensity increases with the increase of HClO equivalents and reaches a maximum value at 3 times the equivalents. Obvious red fluorescence can be observed in the solution under a portable UV lamp. Figure 3 The fluorescence spectrum of the test solution with a concentration of 10 μM fluorescent probe NMS after adding 0-3.5 times equivalent hypochlorous acid, excited at 475 nm, after adding hypochlorous acid solutions of different concentrations, the fluorescence intensity at 675 nm increases with the increase of hypochlorous acid concentration. The fluorescence intensity at 675 nm and the concentration of added hypochlorous acid are linearly fitted, and the detection limit of the fluorescent probe NMS for hypochlorous acid is calculated to be 22 nM (S / N=3). Figure 4As shown in the figure, after adding 1, 2, and 3 times equivalent of hypochlorous acid to the solution of fluorescent probe NMS, the fluorescence intensity of the fluorescent probe solution at 675nm increased rapidly, and as the HClO equivalent increased, the fluorescence intensity increased; when the HClO equivalent increased to 3 times equivalent, the reaction reached an equilibrium within 5 seconds, which shows that the response of fluorescent probe NMS to hypochlorous acid is very sensitive and fast. Figure 1 , Figure 2 , Figure 3 and Figure 4 The results show that the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) prepared by the present invention can quickly realize the colorimetric and fluorescence detection of hypochlorous acid and is very suitable for biological imaging applications.

[0037] Example 3

[0038] Selective Detection of Fluorescent Probe Compound (E)-2-(5,5-Dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS)

[0039] To the test solution of the fluorescent probe NMS prepared in the above example with a concentration of 10 μM, interfering active oxygen (H2O2, t-BuOOH, ·OH, O2 ·- , 1 O2、ONOO - , NO), active sulfur (HSO3 - , HS - , Cys, Hcy, GSH) to a concentration of 100 μM. When the solution was excited at 475 nm, it was found that the fluorescent probe NMS only had obvious fluorescence at 675 nm after reacting with hypochlorous acid, but there was no obvious change with other active compounds (such as Figure 5 The above data show that the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) has a good selective detection effect on hypochlorous acid.

[0040] Example 4

[0041] Imaging application of fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) in HeLa cells

[0042] Hela cells were cultured in DMEM high glucose medium supplemented with 100 U / mL penicillin-streptomycin. The cell culture medium was placed in a humidified incubator at 37°C with air containing 5% CO2. 4 The cells were seeded on a 14 mm glass coverslip at a density of 10 μM. Then, the fluorescent probe NMS was added to the culture medium at a concentration of 10 μM for 1 hour, and then washed three times with phosphate buffered saline (PBS, pH = 7.4). Thereafter, hypochlorous acid (10 μM) and hypochlorous acid (20 μM) were added to the cells, respectively, and incubated at 37°C for 60 minutes. The cells were rinsed three times with PBS, and then sealed with a sealing agent and imaged on a fluorescence microscope system.

[0043] Figure 6 The results of imaging of hypochlorous acid using the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) are given. Figure 6 In the first row, it can be seen that the fluorescent probe NMS has no obvious fluorescence in the red channel; in the second row where 10 μM hypochlorous acid is added and the third row where 20 μM hypochlorous acid is added, it can be found that the fluorescent probe NMS can be observed in the red channel after being co-cultured with hypochlorous acid for 60 minutes, and the fluorescence intensity increases with the increase of hypochlorous acid equivalent. Therefore, the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinophenylvinyl)cyclohex-2-ene-1-ylidene) malononitrile (NMS) prepared by the present invention can well realize the imaging detection of hypochlorous acid in HeLa cells.

[0044] Example 5

[0045] Imaging application of fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) in macrophages

[0046] Figure 7 The results of the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) on hypochlorous acid are given. Figure 7In the first row, it can be seen that the macrophages cultured with the fluorescent probe NMS have no obvious fluorescence in the red channel; while in the second row, after the macrophages stimulated with LPS and PMA were co-cultured with the fluorescent probe NMS for 60 minutes, obvious red fluorescence can be observed in the red channel. Therefore, the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene) malononitrile (NMS) prepared by the present invention can well realize the imaging detection of hypochlorous acid in the macrophages of organisms.

[0047] Example 6

[0048] Imaging application of fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) in colitis model mice

[0049] The colitis model mice were established by oral administration of 3% dextran sodium sulfate solution for one week.

[0050] Figure 8 The results of the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) on hypochlorous acid are given. Figure 8 In (a), it can be found that by comparing the HE staining results of normal mice (top) and colitis mice (bottom), it can be found that the epithelial structure of normal mice is intact, and there is no infiltration of inflammatory cells. Compared with the control group, the model group showed partial tissue mucosal or muscular layer defects, infiltration of inflammatory cells such as lymphocytes and neutrophils, and severe pathological damage to the colon tissue of mice in the model group, indicating that the colitis mouse model was successfully constructed. Afterwards, we injected the fluorescent probe NMS (1mM, 100μL) into the mice through the tail vein injection and observed the fluorescence intensity at different times. Figure 8 As shown in (b), the mouse was photographed at 0, 30, and 60 minutes. It can be found that as time goes by, the fluorescent probe NMS shows obvious fluorescent signals in the mouse abdomen, which indicates that the fluorescent probe NMS can achieve imaging of colitis. After real-time imaging, the mouse was dissected and the main organs were imaged. Figure 8 As shown in (c), only the colon part of the mouse induced to produce inflammation showed obvious fluorescent signals, and other organs did not have obvious fluorescent signals, indicating that the fluorescent probe NMS would not have a significant effect on other organs. Therefore, the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) prepared by the present invention can well realize hypochlorous acid imaging detection of colitis model mice.

[0051] As can be seen from the above, compared with the existing fluorescent probe technology in the field of hypochlorous acid detection, the fluorescent probe compound (E)-2-(5,5-dimethyl-3-(4-thiomorpholinostyryl)cyclohex-2-ene-1-ylidene)malononitrile (NMS) prepared by the present invention can well realize the detection of hypochlorous acid, has the advantage of simple synthesis, and shows good selectivity and sensitivity for the detection of hypochlorous acid, and can realize the imaging detection of hypochlorous acid in cells and colitis model mice.

[0052] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent probe for detecting hypochlorous acid, Features: The fluorescent probe is (E)-2-(5,5-dimethyl-3-(4-thiomorpholinophenylvinyl)cyclohex-2-ene-1-ylidene)malononitrile (NMS), and its structural formula is 2. A method for preparing the near-infrared fluorescent probe for detecting hypochlorous acid according to claim 1, Features The specific process is: using ethanol as the reaction medium, 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile and 4-thiomorpholinebenzaldehyde as the reaction raw materials, and piperidine as the catalyst, the reaction is carried out at 70-80°C under the protection of inert gas. After the reaction is completed, the solvent is removed by filtration, and then the target product is washed with ethanol and ether to obtain a near-infrared fluorescent probe for detecting hypochlorous acid.

3. The method for preparing the near-infrared fluorescent probe for detecting hypochlorous acid according to claim 2, Features: The molar ratio of the 2-(3,5,5-trimethylcyclohex-2-ene-1-ylidene)malononitrile, 4-thiomorpholine benzaldehyde and piperidine is 1:1:0.01-0.

05.

4. The method for preparing the near-infrared fluorescent probe for detecting hypochlorous acid according to claim 2, Features The synthetic route is:

5. Use of the near-infrared fluorescent probe for detecting hypochlorous acid according to claim 1 in rapid and highly selective identification of hypochlorous acid.

6. The use according to claim 5, Features The specific process is as follows: the fluorescent probe appears light pink in a mixed solution of phosphate buffer and acetonitrile in a volume ratio of 8:1, and has no fluorescence under a 365nm portable ultraviolet lamp; after a solution containing hypochlorous acid is added to the mixed system, the color of the mixed system turns light yellow, and exhibits bright red fluorescence under the irradiation of a 365nm portable ultraviolet lamp.

7. Use of the near-infrared fluorescent probe for detecting hypochlorous acid according to claim 1 in preparing an imaging detection reagent for hypochlorous acid in an organism.

8. Use of the near-infrared fluorescent probe for detecting hypochlorous acid according to claim 1 in preparing an imaging detection reagent for hypochlorous acid in cells.

9. Use of the near-infrared fluorescent probe for detecting hypochlorous acid according to claim 1 in preparing an imaging detection reagent for colitis in an organism.

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