Good-water-solubility near-infrared fluorescent probe TM-N2 for detecting nitroreductase as well as preparation method and application thereof

Abstract

The invention relates to the technical field of fluorescent probes, in particular to a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase as well as a preparation method and application of the near-infrared fluorescent probe TM-N2. The good-water-solubility fluorescent probe TM-N2 for near-infrared red light detection of nitroreductase has good water solubility, the wavelength can reach the near-infrared band, and the preparation method is simple; the probe has high sensitivity and good selectivity. The detection of nitroreductase under the hypoxia condition in the zebra fish body and the detection of nitroreductase in tumor cells are realized; the method has a wide application prospect in the fields of biological living body imaging analysis and tumor detection.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescent probes, and in particular to a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, and its preparation method and application. Background Art

[0002] Under the current medical technology, cancer has become one of the important causes of human death. The detection of cancer relies on tissue section biopsy in the late stage of cancer, with a late discovery time and a long detection time. Therefore, it is of great significance to develop methods for early diagnosis and identification of cancer. If parenchyma in human tissues undergoes neoplastic changes, the expression level of nitroreductase in tumor cells is directly associated with the hypoxic state of cells. Therefore, it is necessary to monitor tumor cells because they produce nitroreductase under hypoxic conditions. By detecting nitroreductase, its hypoxic condition can be clarified, and then the occurrence of parenchymal neoplastic changes in the body can be detected.

[0003] In recent years, the detection of the tumor marker nitroreductase has received extensive attention. Fluorescent probes have important applications in the detection of nitroreductase due to their high specificity, high sensitivity, and excellent biocompatibility. Currently, most of the probes for detecting nitroreductase have wavelengths around 600 nm; in the detection of short-wavelength probes, the biological penetrability is poor, while the biological penetrability of red light detection is good and the harm is small; secondly, other types of probes have poor water solubility and require more co-solvents for solubilization, and more co-solvents cause greater harm to organisms. Probes with good water solubility can enter the body through water transport, cause less harm to organisms, and do not require a large amount of chemical reagents. Therefore, it is very important to design a near-infrared fluorescent probe with good water solubility for detecting nitroreductase to achieve sensitive detection of nitroreductase. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, and its preparation method and application, aiming at the deficiencies of the above-mentioned prior art. The fluorescent probe has the characteristics of high sensitivity, good stability, and less harm to organisms, with a high yield and simple synthesis. It can be used to prepare reagents for detecting hypoxic lesion sites in organisms, and can also be used to prepare tumor cell detection reagents.

[0005] The present invention provides a near-infrared fluorescent probe for detecting nitroreductase with good water solubility and TM-N2. The chemical structural formula of the fluorescent probe TM-N2 is: .

[0006] The present invention provides a preparation method of the above-mentioned near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, which includes the following steps: S1. Dissolve 4 - diethylamino ketonic acid and 4 - aminoacetophenone in concentrated sulfuric acid, heat under reflux. After cooling to room temperature, dropwise add it to an ice - water mixture, then add perchloric acid and let it stand for precipitation. Filter by suction to obtain the intermediate product; S2. Take the intermediate product prepared in S1 and mix it with K2CO3, dissolve it in acetonitrile, then add 4 - nitrobenzyl bromide and react at room temperature. Separate by column chromatography to obtain the desired product TM - N2.

[0007] According to the preparation method provided by the present invention, the molar volume ratio of 4 - diethylamino ketonic acid, 4 - aminoacetophenone, concentrated sulfuric acid, ice - water mixture and perchloric acid in S1 is (1 - 2) mmol:(1 - 2) mmol:(5 - 10) mL:(20 - 40) g:(1 - 2) mL. The concentration of the concentrated sulfuric acid is 98%, and the mass fraction of perchloric acid is 70%.

[0008] According to the preparation method provided by the present invention, the temperature of the heating reflux reaction in S1 is 90 °C, and the time of the heating reflux reaction is 0.5 - 5 h.

[0009] According to the preparation method provided by the present invention, the molar volume ratio of the intermediate product, K2CO3, acetonitrile and 4 - nitrobenzyl bromide in S2 is (0.334 - 0.835) mmol:(0.334 - 0.835) mmol:(1.3 - 2) mL:(0.334 - 0.835) mmol.

[0010] According to the preparation method provided by the present invention, the conditions of the column chromatography are as follows: in a silica gel column, using methanol / dichloromethane as the eluent, and the elution ratio is 1 / (2 - 15).

[0011] The present invention also provides an application of the near - infrared fluorescent probe TM - N2 for detecting nitroreductase with good water solubility. The fluorescent probe TM - N2 can be used to prepare reagents for detecting hypoxic lesion sites in organisms, and can also be used to prepare tumor cell detection reagents.

[0012] The present invention has the following advantages compared with the prior art: The near - infrared fluorescent probe TM - N2 for detecting nitroreductase with good water solubility provided by the present invention has good water solubility, its wavelength can reach the near - infrared stage, and the preparation method is simple; the probe has high sensitivity and good selectivity. It realizes the detection of nitroreductase under hypoxic conditions in zebrafish and the detection of nitroreductase in tumor cells; it has broad application prospects in the fields of in vivo bio - imaging analysis and tumor detection. Brief Description of the Drawings

[0013] To more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the attached drawings required for the description of the embodiments or the prior art. Obviously, the attached drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other attached drawings can also be obtained based on these attached drawings.

[0014] Figure 1 is the 1H NMR spectrum of probe TM-N2; Figure 2 is the 13C NMR spectrum of probe TM-N2; Figure 3 is the mass spectrum of probe TM-N2; Figure 4 is the fluorescence spectrum of probe TM-N2 for nitroreductase at different times; Figure 5 is the fluorescence spectrum of probe TM-N2 for nitroreductase with different concentrations; Figure 6 is the selectivity schematic diagram of probe TM-N2, where the vertical coordinate is the fluorescence intensity of probe TM-N2 at 650 nm; Figure 7 is the change diagram of probe TM-N2 for nitroreductase in zebrafish; Figure 8 is the confocal image after co-culturing HeLa cells with TM-N2 for 30 minutes under different oxygen content conditions. Detailed implementation manners

[0015] Example 1

[0016] This example provides a preparation method for a near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase. The specific steps are as follows: S1. Place 1 mmol of 4-diethylamino ketone acid and 1 mmol of 4-aminobenzophenone in a 25 mL round-bottom flask, add 5 mL of 98% concentrated sulfuric acid (the concentration of concentrated sulfuric acid is 98%), react at a temperature of 90 °C for 2 h. After cooling to room temperature, drop it into 20 g of ice-water mixture, then add 1 mL of 70% perchloric acid, and let it stand for 12 h to precipitate. Filter by suction to obtain the required compound TM, which is a purple solid;

[0017] S2. Take 0.334 mmol of TM prepared in S1 and 0.334 mmol of K2CO3, dissolve them in 1.3 mL of acetonitrile. After dissolution, add 0.334 mmol of 4-nitrobenzyl bromide and react at room temperature for 12 h. After the reaction, separate by column chromatography to obtain the required product TM-N2, which is a purple solid; The conditions for column chromatography are as follows: in a silica gel column, methanol / dichloromethane is used as the eluent, and the elution ratio is 1 / (2 - 15).

[0018]

[0019] Example 2 This example provides a preparation method of a near-infrared fluorescent probe TM-N for detecting nitroreductase with good water solubility, and the specific steps are as follows: 2 : S1. Place 2 mmol of 4-diethylaminoketone acid and 2 mmol of 4-aminophenylethanone in a 25 mL round-bottom flask, add 10 mL of concentrated sulfuric acid, react at 90 °C for 2 h. After cooling to room temperature, add dropwise to 40 g of ice-water mixture, then add 2 mL of 70% perchloric acid, let stand for 12 h to precipitate, and filter to obtain the required compound TM, which is a purple solid; S2. Take 0.334 mmol of TM prepared in S1 and 0.835 mmol of K 2 CO 3 Dissolve in 2 mL of acetonitrile. After dissolution, add 0.835 mmol of 4-nitrobenzyl bromide and react at room temperature for 12 h. After the reaction, separate by column chromatography to obtain the required product TM-N 2 , which is a purple solid; The conditions for column chromatography are as follows: in a silica gel column, methanol / dichloromethane is used as the eluent, and the elution ratio is 1 / (2 - 15).

[0020] Example 3 This example provides a preparation method of a near-infrared fluorescent probe TM-N for detecting nitroreductase with good water solubility, and the specific steps are as follows: 2 : S1. Place 1.5 mmol of 4-diethylaminoketone acid and 1.5 mmol of 4-aminophenylethanone in a 25 mL round-bottom flask, add 7.5 mL of concentrated sulfuric acid, react at 90 °C for 2 h. After cooling to room temperature, add dropwise to 30 g of ice-water mixture, then add 1.5 mL of 70% perchloric acid, let stand for 12 h to precipitate, and filter to obtain the required compound TM, which is a purple solid; S2. Take 0.334 mmol of TM prepared in S1 and 0.58 mmol of K 2 CO 3 Dissolve in 1.7 mL of acetonitrile. After dissolution, add 0.58 mmol of 4-nitrobenzyl bromide and react at room temperature for 12 h. After the reaction, separate by column chromatography to obtain the required product TM-N 2 , which is a purple solid; The conditions for column chromatography are as follows: in a silica gel column, methanol / dichloromethane is used as the eluent, and the elution ratio is 1 / (2 - 15).

[0021] Example 4 This example provides a probe TM-N prepared in Example 1 2 Response to nitroreductase; Probe TM-N 2 was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution, a 500 μg / mL nitroreductase solution was prepared with a PBS stock solution at pH 7.4, and a 1 mM NADH stock solution was prepared with reduced coenzyme I (NADH); In a PBS solution of probe TM-N at a concentration of 10 μM 2 , the fluorescence signal of the probe itself was low. After adding 250 μL of the NADH stock solution and 2 μg / mL of nitroreductase, a strong fluorescence signal was emitted at 650 nm.

[0022] As shown in the figure, Figure 4 are the fluorescence spectra before and after adding nitroreductase, Figure 5 are the fluorescence images after adding different concentrations of nitroreductase; The results of the comparative experiment show that the probe TM-N 2 can achieve a highly sensitive response to nitroreductase, and it was found that the fluorescence signal of compound TM-N 2 increased significantly after adding nitroreductase.

[0023] Example 5 This example provides a probe TM-N prepared in Example 1 2 selectivity; Common interfering substances were added to a PBS solution of probe TM-N at a concentration of 10 μM 2 , such as: inorganic ions (K + , Mg 2+ , Ca 2+ , HSO 3 – , SO 3 2– , HS – ), glucose, Vc, VB 6 , HSA, Glu, Arg, Ser, Asp, Pro, reactive oxygen species (HClO, 1 O 2 , H 2 O 2 , ·OH) and biothiols (GSH, Cys, Hcy and DTT), to investigate the selectivity of the probe for nitroreductase.

[0024] As Figure 6 shown, after adding the above-mentioned interfering substances such as inorganic ions (K + , Mg 2+ , Ca 2+ , HSO 3 – , SO 3 2– , HS – ), glucose, Vc, VB 6 , HSA, Glu, Arg, Ser, Asp, Pro, reactive oxygen species (HClO, 1 O 2 , H 2 O 2 , ·OH) and biothiols (GSH, Cys, Hcy and DTT), the fluorescence of the probe at 650 nm is basically not enhanced, indicating that the probe has high selectivity for nitroreductase.

[0025] Example 6 This example provides an application of the probe TM-N prepared in Example 1 2 for zebrafish imaging.

[0026] As Figure 7 shown, the first group is the control group; the second group is added with the probe TM-N 2 (10 μM); the third group is added with the probe TM-N 2 and after placing for 30 min, the zebrafish changes from normoxia to hypoxia; the fourth group is added with the probe TM-N 2 (10 μM) and the nitroreductase inhibitor dicoumarol (50 μM), and the hatching time of the zebrafish is 30 min for all groups; As can be seen from the figure, the zebrafish in the third group are hatched in the probe TM-N at a concentration of 10 μM 2 and PBS solution for 20 min, and the probe TM-N 2 has strong fluorescence at specific parts in the zebrafish; during the 30 min of observing the fish out of water, the fish changes from the normoxic state to the hypoxic state, and the content of nitroreductase in the fish body changes, and the fluorescence intensity gradually increases.

[0027] In the fourth group of zebrafish, 50 μM dicoumarol, a nitroreductase inhibitor, is added. After the nitroreductase in the fish body is removed, there is no strong fluorescence, and its probe TM-N 2 only has a strong fluorescence signal at specific parts; indicating that the probe TM-N 2 has high selectivity and sensitivity in vivo and also has the potential to detect hypoxia in organisms.

[0028] Example 7 This example provides a probe TM-N prepared in Example 1 2 for application in Hela cell imaging.

[0029] Inoculate Hela cells into a 15 mm culture dish and place it in an incubator at a temperature of 37 °C and containing 5% CO 2 for 12 h. After the cells adhere to the wall, divide them into three groups for culture; The three conditions are: oxygen content of 21%, oxygen content of 1%, oxygen content of 1% and adding the inhibitor dicoumarol; After culturing for 6 h, add 10 μM of the probe TM-N to the cells under the three culture conditions respectively 2 , continue to culture for 30 min, discard the cell culture medium, wash 3 times with PBS buffer, and use a confocal microscope to take pictures of the cells under the three culture conditions respectively. The pictures taken are as follows Figure 8 shown.

[0030] It is found from Figure 8 that the fluorescence intensity of the cells cultured under the condition of 1% oxygen content in the second group is significantly stronger after the action of the probe TM-N 2 ; The fluorescence intensity of the cells cultured under the condition of 1% oxygen content in the third group and adding the inhibitor dicoumarol is significantly reduced.

[0031] It is proved that under hypoxic conditions, the content of nitroreductase in cells has changed, and the probe TM-N 2 can effectively detect the change of nitroreductase in Hela cells.

[0032] The above are only the preferred embodiments of the present invention, and do not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiments according to the technical essence of the invention still fall within the protection scope of the technical solution of the present invention.

Claims

1. A near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase, characterized in that: The chemical structural formula of the fluorescent probe TM-N2 is: 。 2. A method for preparing the near-infrared fluorescent probe TM-N2 with good water solubility for detecting nitroreductase as claimed in claim 1, characterized in that: The following steps are involved: S1, dissolving 4-diethylamino keto acid and 4-aminoacetophenone in concentrated sulfuric acid, heating under reflux for reaction, and after cooling to room temperature, adding dropwise to a mixture of ice and water, adding perchloric acid, standing and precipitating, and filtering to obtain an intermediate product; S2. Mix the intermediate product prepared in S1 with K2CO3, dissolve it in acetonitrile, add 4-nitrobenzyl bromide, react at room temperature, and separate by column chromatography to obtain the desired product TM-N2.

3. The preparation method according to claim 2, characterized in that: The molar volume ratio of 4-diethylaminoketo acid, 4-aminoacetophenone, concentrated sulfuric acid, ice-water mixture and perchloric acid in S1 is (1-2) mmol: (1-2) mmol: (5-10) mL: (20-40) g: (1-2) mL, the concentration of the concentrated sulfuric acid is 98%, and the mass fraction of the perchloric acid is 70%.

4. The preparation method according to claim 2, characterized in that: The temperature of the heating reflux reaction in S1 is 90° C., and the time of the heating reflux reaction is 0.5 to 5 h.

5. The preparation method according to claim 2, characterized in that: The molar volume ratio of the intermediate product, K2CO3, acetonitrile and 4-nitrobenzyl bromide in S2 is 0.334 mmol: (0.334-0.835) mmol: (1.3-2) mL: (0.334-0.835) mmol.

6. The preparation method according to claim 2, characterized in that: The column chromatography conditions are as follows: in a silica gel column, methanol / dichloromethane is used as the eluent, and the elution ratio is 1 / (2-15).

7. A use of the near-infrared fluorescent probe TM-N2 for detecting nitroreductase with good water solubility as claimed in claim 1, characterized in that: The fluorescent probe TM-N2 can be used to prepare a reagent for detecting hypoxic lesions in an organism, and can also be used to prepare a tumor cell detection reagent.

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