A near-infrared fluorescent probe dual-responsive to tyrosinase and hydrogen peroxide and its preparation and application

Abstract

The present invention discloses a near-infrared fluorescent probe with dual response to tyrosinase and hydrogen peroxide, which is prepared by connecting a semi-cyanine compound with a borate compound. Under the joint action of tyrosinase and hydrogen peroxide, the probe can light up near-infrared fluorescent signals with high sensitivity and selectivity, and simultaneously respond to the two characteristic factors of tyrosinase and H2O2 in melanoma, thereby avoiding the interference of "false positive" signals, and realizing highly selective fluorescent imaging analysis of melanoma. A near-infrared fluorescent probe with dual response to tyrosinase and hydrogen peroxide of the present invention has the advantages of novel structure, simple and fast synthesis process, high targeting and good imaging performance, and is of great significance for promoting accurate diagnosis of tumors.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probe detection, and specifically relates to a tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe and a preparation method thereof, and also relates to the application of the fluorescent probe in fluorescent imaging. Background Art

[0002] Melanoma is a highly invasive skin malignancy characterized by rapid growth and early metastasis. Statistics show that the 5-year relative survival rate is 23% in the late stage of melanoma. Currently, the clinical treatment of melanoma is still mainly surgical resection, and accurate diagnosis of melanoma is the key to clinical melanoma treatment. Biomarker-activated fluorescent probes, especially near-infrared (NIR) fluorescent probes, provide a powerful means for the diagnosis of melanoma. In recent years, researchers at home and abroad have developed a variety of melanoma characteristic factor-activated fluorescent probes for melanoma-specific fluorescence imaging detection (Wang Chenyu et al., Chinese Journal of Dermatology, 2021, 54, 60-63; Li, Z. et al., Anal. Chem. 2018, 90, 3666–3669; Park,SY et al., Sensors and Actuators B: Chemical , 2020, 319, 128306). However, the fluorescent probes currently developed usually only respond to a single characteristic factor in melanoma, which is prone to interference from "false positive" signals (Wu, L. et al., Nat. Rev. Chem. 2021, 5, 406-421). Therefore, the development of near-infrared fluorescent probes that respond to multiple characteristic factors in melanoma is of great significance for the precise imaging detection of melanoma.

[0003] Tyrosinase is a multifunctional, glycosylated, melanocyte-specific copper-containing oxidase that is mainly present in the organelle melanosomes. It mediates the oxidation of tyrosine into some quinones, which triggers the formation of melanin. Studies have shown that tyrosinase plays an important role in physiological processes related to tyrosine, and the tyrosinase content in melanoma is much higher than that in normal cells and tissues. Due to the overexpression of tyrosinase in melanoma cells, tyrosinase is used as an independent biomarker for the diagnosis and prognosis of melanoma. In addition, hydrogen peroxide (H2O2) is the most stable reactive oxygen species (ROS) in the body. As a signaling molecule and maintainer of redox homeostasis, low levels of H2O2 regulate various physiological processes such as cell growth, proliferation, and migration. However, high concentrations of H2O2 promote oxidative stress and lead to the development of various diseases such as cancer. The content of H2O2 in melanoma is 10 times that of normal cells. Therefore, constructing a fluorescent probe co-activated by tyrosinase and H2O2 in melanoma can effectively distinguish melanoma from normal tissues, which is of great significance for the imaging detection of melanoma. Summary of the invention

[0004] The object of the present invention is to provide a near-infrared fluorescent probe that is dual-responsive to tyrosinase and hydrogen peroxide; another object of the present invention is to provide a preparation method of the fluorescent probe and its application.

[0005] 1. Tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe and its preparation

[0006] The near-infrared fluorescent probe of the present invention that is dual-responsive to tyrosinase and hydrogen peroxide has the structural formula:

[0007]

[0008] Wherein: the substituents R1, R2, and R3 are H, methyl, ethyl, propyl, methoxy, hydroxyl, nitro, amino, halogen, aldehyde, carboxyl, sulfonic acid, thiol, ester, cyano, amide, or alkynyl;

[0009] The substituent R4 is H, methoxy, hydroxy, nitro, amino, halogen, aldehyde, carboxyl, sulfhydryl, sulfonic acid, mercapto, ester, cyano, amide or alkynyl;

[0010] X - is iodide ion, bromide ion, chloride ion, nitrate ion, sulfate ion, carbonate ion or sulfite ion; n is an integer of 1 to 20; Y is an oxyether group, a thiol group or an imino group.

[0011] The preparation method of the tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe of the present invention comprises the following steps: adding a hemicyanine compound, a base and triphosgene to an organic solvent, reacting at 0-25°C for 0.01-1 hour, adding a borate compound, reacting at 0-80°C for 0.1-20 hours, concentrating under reduced pressure, and separating by column chromatography to obtain the probe.

[0012] The structural formula of the hemicyanine compound is as follows:

[0013]

[0014] wherein the substituent R4 is H, methoxy, hydroxyl, nitro, amino, halogen, aldehyde, carboxyl, sulfhydryl, sulfonic acid, sulfhydryl, ester, cyano, amide or alkynyl;

[0015] X - is iodide ion, bromide ion, chloride ion, nitrate ion, sulfate ion, carbonate ion or sulfite ion; n is an integer from 1 to 20; Y is hydroxyl, thiol or amino. The preparation method of hemicyanine compounds is shown in the literature ( Anal. Chem., 2021, 93, 15080−15087).

[0016] The structural formula of the borate ester compound is as follows:

[0017]

[0018] Wherein, the substituents R1, R2, and R3 are H, methyl, ethyl, propyl, methoxy, hydroxyl, nitro, amino, halogen, aldehyde, carboxyl, sulfonic acid, mercapto, ester, cyano, amide, or alkynyl.

[0019] The molar ratio of the hemicyanine compound to the borate compound is 1:0.2-1:5; the molar ratio of the hemicyanine compound to triphosgene is 1:0.1-1:0.5; and the molar ratio of the hemicyanine compound to the base is 1:1-1:5.

[0020] The organic solvent is at least one of tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide, dichloromethane, benzene, toluene, xylene, chlorobenzene, methanol, ethanol, and chloroform, preferably dichloromethane.

[0021] The base is an organic base or an inorganic base; the organic base is triethylamine, pyridine or 4-dimethylaminopyridine; the inorganic base is sodium carbonate, potassium carbonate, sodium hydroxide, cesium carbonate, sodium bicarbonate, potassium bicarbonate or barium carbonate; preferably 4-dimethylaminopyridine.

[0022] 2. Application of fluorescent probes in fluorescence imaging

[0023] The near-infrared fluorescent probe of the present invention can simultaneously respond to two melanoma characteristic factors, tyrosinase and H2O2, and is beneficial to the imaging analysis of melanoma in cells or in vivo by utilizing the advantages of strong penetrability of near-infrared fluorescent signals and low background interference. The application of the fluorescent probe of the present invention in fluorescence imaging is described below using the prepared fluorescent probe 7 as an example.

[0024] 1. Application of fluorescent probes in tumor cell imaging analysis

[0025] First, A549 cells were cultured in a cell culture incubator at 37°C, 21% oxygen, and 5% carbon dioxide for 24 hours, and then the cells were digested with trypsin and transferred to a cell imaging dish, which was divided into an experimental group and a control group. The experimental group was added with the near-infrared fluorescent probe 7 solution prepared in Example 1 of the present invention, tyrosinase, and H2O2; the control group a was only added with the near-infrared fluorescent probe 7 solution prepared in Example 1 of the present invention; the control group b was added with the near-infrared fluorescent probe 7 solution prepared in Example 1 of the present invention and H2O2; the control group c was added with the near-infrared fluorescent probe 7 solution prepared in Example 1 of the present invention and tyrosinase. The above four groups of cells were further cultured in a cell culture incubator at 37°C and 5% carbon dioxide for 4 hours, the culture medium in the imaging dish was washed away with PBS buffer, and fluorescence imaging was performed using a laser scanning confocal microscope. Figure 5It can be seen that the cells in the experimental group showed strong fluorescence, indicating that the probe can enter the cells and be activated by tyrosinase and H2O2 to produce significant fluorescence signals. In contrast, no obvious fluorescence signal was observed in the cells in the control group, indicating that the mechanism interference was small.

[0026] 2. Application of near-infrared fluorescent probes in in vivo fluorescence imaging analysis of tumors

[0027] After two tumor-bearing mice were injected with 0.1 mL of 100 μM near-infrared fluorescent probe 7, the control group mice were injected with 0.1 mL of normal saline, and the drug group mice were injected with 0.1 mL of a mixed solution of tyrosinase and H2O2. After 8 hours of drug administration, the above mice were imaged using a small animal in vivo fluorescence imager. The results are shown in Figure 2. Figure 6 shown. Figure 6 Mouse No. 1 is the control group mouse, and mouse No. 2 is the drug-treated group mouse. As can be seen from the figure, the fluorescent signal at the tumor site of the drug-treated group mouse is more significant, compared with no fluorescent signal at the tumor site of the control group mouse. This is because the probe in the drug-treated group mouse interacts with tyrosinase and H2O2, causing the near-infrared fluorescent signal to light up, realizing the imaging analysis of the tumor in the animal body.

[0028] Semi-cyanine compounds have near-infrared fluorescence emission properties. The present invention connects the fluorescent group semi-cyanine compounds with the quenching group borate compounds. Since the borate compounds quench the fluorescence of the fluorescent group semi-cyanine compounds, the semi-cyanine compounds have no fluorescence emission properties. After the borate group is activated by the high concentration of H2O2 in the tumor, the intermediate product is further activated by tyrosinase to release the fluorescent group semi-cyanine compounds. Through this cascade activation, the fluorescent group semi-cyanine compounds light up the near-infrared fluorescence signal, realizing the simultaneous detection of two melanoma characteristic factors, tyrosinase and H2O2. Compared with the currently developed fluorescent probes that only activate a single characteristic factor in the tumor (tyrosinase, H2O2, microacidity and hypoxia, etc.), the dual-response near-infrared fluorescent probe provided by the present invention can simultaneously respond to the two characteristic factors of tyrosinase and H2O2 in the tumor, with high selectivity and strong sensitivity to tumors, which is of great significance for the accurate detection of melanoma.

[0029] The beneficial effects of the present invention are:

[0030] 1. The present invention provides a near-infrared fluorescent probe that is dual-responsive to tyrosinase and hydrogen peroxide, which has the advantages of novel structure, high targeting, strong sensitivity and good imaging performance. It can simultaneously respond to the two characteristic factors of tyrosinase and H2O2 in melanoma, avoid the interference of "false positive" signals, and realize highly selective fluorescence imaging analysis of melanoma.

[0031] 2. The present invention provides a method for preparing a near-infrared fluorescent probe that is dual-responsive to tyrosinase and hydrogen peroxide. The synthetic process is simple and effective, and the fluorescent probe can be obtained with high yield and high purity.

[0032] 3. In view of the good imaging performance, the tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe provided in this application is of great significance for promoting the imaging detection of melanoma and has potential application value in the field of visualization detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of compound 5 prepared by the present invention;

[0034] Figure 2 is the carbon nuclear magnetic resonance spectrum of compound 5 prepared by the present invention;

[0035] Figure 3 is the hydrogen nuclear magnetic resonance spectrum of the fluorescent probe 7 prepared by the present invention;

[0036] Figure 4 is the carbon nuclear magnetic resonance spectrum of the fluorescent probe 7 prepared by the present invention;

[0037] Figure 5 This is an imaging diagram of the near-infrared fluorescent probe 7 of the present invention in A549 cells;

[0038] Figure 6 This is an in vivo imaging diagram of the near-infrared fluorescent probe 7 of the present invention in tumor-bearing mice. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0040] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies. Example

[0041] (1) Preparation of compound 5:

[0042]

[0043] In a round-bottom flask, add compound 3 (1.0 mmol) and compound 4 (1.1 mmol), add dry acetonitrile (10 mL), and then add potassium carbonate (3 mmol). After stirring at room temperature for 30 minutes under nitrogen protection, continue to react at 60°C for 5 hours, filter, dry, and separate by column chromatography to obtain compound 5; the reaction formula is as follows:

[0044]

[0045] The H NMR spectrum of the prepared compound 5 is shown in Figure 1 As shown, the carbon NMR spectrum is as follows Figure 2 As shown, 1 H NMR and 13 C NMR data are as follows: 1 H NMR (400 MHz, CDCl3): δ 7.82 (d, J =7.6 Hz, 2H), 7.43 (d, J =8.0 Hz, 2H), 7.24-7.28 (m, 1H), 7.00 (s, 1H), 6.94 (d, J =7.6 Hz, 1H), 6.88-6.90 (m, 1H), 5.10 (s, 2H), 4.66 (d, J =5.2 Hz, 2H), 1.35 (s, 12H). 13 C NMR (101 MHz, CDCl3): δ : 159.0, 142.6, 140.2, 135.1, 129.6, 126.5, 119.4, 114.1,113.3, 83.9, 69.8, 65.2, 24.9.

[0046] (2) Preparation of fluorescent probe 7:

[0047]

[0048] In a round-bottom flask, compound 6 (1 mmol) and dry dichloromethane (50 mL) were added with p-dimethylaminopyridine (4 mmol), and triphosgene (0.4 mmol) was added at 0°C. After stirring at 0°C for 30 minutes, compound 5 (2 mmol) was added. After continuing the reaction at room temperature for 8 hours, the mixture was concentrated under reduced pressure and separated by column chromatography to obtain fluorescent probe 7. The preparation method of compound 6 is described in the literature ( Anal. Chem., 2021, 93, 15080−15087). The reaction formula is as follows:

[0049]

[0050] The nuclear magnetic resonance hydrogen spectrum of the fluorescent probe 7 prepared in this example is as follows Figure 3 As shown, the carbon NMR spectrum is as follows Figure 4 As shown, 1 H NMR and13 C NMR data are as follows: 1 H NMR (400 MHz, DMSO- d 6): δ 10.44 (s, 1H), 8.54 (d, J =15.2 Hz, 1H), 7.79-7.80 (m, 2H), 7.67-7.69 (m, 2H), 7.44-7.57 (m, 6H), 7.31-7.33 (d, J =8.0 Hz, 2H), 7.11 (s, 1H), 6.99-7.03 (m, 2H), 6.55 (d, J =15.2 Hz,1H), 5.17 (d, J =5.6 Hz, 4H), 3.88 (s, 3H), 2.67-2.72 (m, 4H), 1.83 (m, 2H), 1.75 (s, 6H), 1.258 (s, 12H). 13 C NMR (101 MHz, DMSO- d 6): δ 178.2, 160.5,158.8, 153.6, 142.8, 142.4, 140.8, 138.3, 135.0, 130.2, 128.1, 127.6, 127.3,120.7, 117.0, 114.9, 114.7, 114.2, 104.3, 84.1, 69.4, 50.8, 33.1, 30.1, 27.6,25.1. The fluorescence imaging results are shown in Figure 2. Figure 5 , Figure 6 .

Claims

1. A near-infrared fluorescent probe that is dual-responsive to tyrosinase and hydrogen peroxide, having the structural formula: 。 2. The method for preparing a near-infrared fluorescent probe of dual response type of tyrosinase and hydrogen peroxide as claimed in claim 1, comprising adding a hemicyanine compound, a base and triphosgene to an organic solvent, reacting at 0°C for 0.5 hours, then adding a borate compound, reacting at room temperature for 8 hours, concentrating under reduced pressure, and separating by column chromatography to obtain the hemicyanine compound; the structural formula of the hemicyanine compound is as follows: ； The structural formula of the borate ester compound is as follows: 。 3. The method for preparing a tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe as claimed in claim 2, characterized in that: The molar ratio of the hemicyanine compound to the borate ester compound is 1:

2.

4. The method for preparing a tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe as claimed in claim 2, characterized in that: The organic solvent is at least one of tetrahydrofuran, acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide, dichloromethane, benzene, toluene, xylene, chlorobenzene, methanol, ethanol and chloroform.

5. The method for preparing a tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe as claimed in claim 2, characterized in that: The base is an organic base or an inorganic base; the organic base is triethylamine, pyridine or 4-dimethylaminopyridine; the inorganic base is sodium carbonate, potassium carbonate, sodium hydroxide, cesium carbonate, sodium bicarbonate or potassium bicarbonate.

6. The method for preparing a tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe as claimed in claim 2, characterized in that: The molar ratio of the hemicyanine compound to triphosgene is 1:0.

4.

7. The method for preparing a tyrosinase and hydrogen peroxide dual-responsive near-infrared fluorescent probe as claimed in claim 2, characterized in that: The molar ratio of the hemicyanine compound to the base is 1:1 to 1:

5.

8. Use of the near-infrared fluorescent probe as claimed in claim 1 in the preparation of a tyrosinase and hydrogen peroxide dual-responsive fluorescent imaging reagent.

Images