Fluorescent probe for detecting biological mercaptan as well as preparation method and application of fluorescent probe

Abstract

The invention belongs to the technical field of fluorescence detection, and particularly discloses a fluorescent probe for detecting biological mercaptan (cysteine, homocysteine, N-acetylcysteine and glutathione) as well as a preparation method and application of the fluorescent probe. The fluorescent probe disclosed by the invention is shown as a formula IV, the fluorescent probe and biological mercaptan (cysteine, homocysteine, N-acetylcysteine and glutathione) are subjected to a specific chemical reaction, and green fluorescence of 540nm is emitted under the excitation wavelength of 450nm. The fluorescent probe disclosed by the invention has no obvious response to other active oxygen, common amino acids, metal ions and active nitrogen species, and is high in detection sensitivity, good in selectivity and high in reaction rate (lt; the fluorescent probe can be used for fluorescence imaging analysis of quantitative biological mercaptan in an in-vitro environment and biological mercaptan flux in biological systems such as cells and the like, and has a relatively great application prospect in the technical fields of analytical chemistry, life science, chemical biology and the like.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescence detection, and in particular to a fluorescent probe for detecting biological thiol, and a preparation method and application thereof. Background Art

[0002] Biothiols are a class of amino acids containing active sulfhydryl groups, which are commonly found in organisms and have important biological activities and physiological functions. Biothiols mainly include cysteine ​​(Cys), homocysteine ​​(Hcy), N-acetylcysteine ​​(NAC) and glutathione (GSH). These compounds play a vital role in maintaining intracellular redox homeostasis. Cys plays an important role in protein folding and stability, and is also involved in redox reactions and the formation of thiol / disulfide bonds in the body. Hcy is an important intermediate in methionine metabolism and an independent risk factor for cardiovascular disease. NAC is a thiol-containing antioxidant and a precursor for the biosynthesis of GSH, which can regulate the redox state in cells. GSH is the most abundant antioxidant in cells, which can scavenge reactive oxygen species such as free radicals and protect cells from oxidative damage.

[0003] The detection of biothiols has important research significance in the fields of life science and environmental science. They are involved in a variety of life activities, such as metabolism, energy conversion, signal transduction, etc. The detection of biothiols can help us understand the mechanisms and processes of these activities. In the field of ecological environment, many microorganisms produce a large amount of biothiols and release them into the environment. These microorganisms play an extremely important role on the earth and help regulate the sulfur and nitrogen cycle and other geochemical cycles. Therefore, detecting the content of biothiols in the environment can help us better understand the role of microorganisms on the earth and environmental changes.

[0004] Currently, a variety of methods have been developed to detect biothiols, such as liquid chromatography-mass spectrometry, high performance liquid chromatography, gas chromatography-mass spectrometry, and fluorescence spectroscopy. Among these detection methods, fluorescent probe analysis is generally favored due to its ability of rapid response and high sensitivity, as well as spatial resolution and satisfactory biocompatibility. Therefore, it is particularly important to develop fluorescent probes for the selective detection of biothiols to achieve their quantitative analysis. Summary of the invention

[0005] In view of the above-mentioned shortcomings, the present invention provides a fluorescent probe for detecting biothiols, a preparation method and an application thereof. The fluorescent probe of the present invention can rapidly and selectively detect biothiols (cysteine, homocysteine, N-acetylcysteine, glutathione) from various bioactive substances under specific detection conditions, and has the advantages of high sensitivity, low detection limit, rapid reaction, etc. The preparation method of the fluorescent probe of the present invention is simple and has the advantages of low cost.

[0006] In order to achieve the above object, the present invention provides a fluorescent probe for detecting biological thiol, the chemical structure of the fluorescent probe for detecting biological thiol is shown in Formula IV:

[0007]

[0008] The present invention also provides a method for preparing the above-mentioned fluorescent probe for detecting biological thiol, comprising the following steps: reacting the compound of formula I, the compound of formula II, bistriphenylphosphine palladium dichloride, cuprous iodide and the compound of formula III to prepare the compound of formula IV, and the synthesis route thereof is as follows:

[0009]

[0010] Further, the following steps are included:

[0011] S1. Under the protection of an inert atmosphere, 4-chloro-7-(diethylamino)-2H-chromene-2-one, trimethylsilyl acetylene, bistriphenylphosphine palladium dichloride, cuprous iodide and diisopropylethylamine are added to anhydrous tetrahydrofuran, and the mixture is stirred at room temperature to react to obtain a reaction solution;

[0012] S2. The reaction solution is concentrated and purified by column chromatography to obtain pure 4,4'-(ethyne-1,2-diyl)bis(7-(diethylamino)-2H-chromen-2-one).

[0013] It should be noted that the inert atmosphere is preferably an inert gas, more preferably argon.

[0014] Furthermore, the molar ratio of the 4-chloro-7-(diethylamino)-2H-chromen-2-one to trimethylsilyl acetylene is 1:0.5-2; the molar amount of the bistriphenylphosphine palladium dichloride and cuprous iodide is 5%-20% of the molar amount of 4-chloro-7-(diethylamino)-2H-chromen-2-one; and the molar ratio of the diisopropylethylamine to 4-chloro-7-(diethylamino)-2H-chromen-2-one is 1-3:1.

[0015] Furthermore, in step S1, the reaction time is 4 to 24 hours.

[0016] The present invention also provides a fluorescent probe composition for detecting biothiol, comprising the fluorescent probe mentioned above.

[0017] Furthermore, the fluorescent probe composition also includes at least one of a solvent, an acid, a base, and a buffer solution.

[0018] The present invention also provides a method for detecting the presence of biothiol or determining the content of biothiol in a sample for non-disease diagnosis and treatment purposes, comprising the following steps:

[0019] S1. Dissolving the fluorescent probe according to claim 1 in an organic solvent to form a probe solution; dissolving the analyte in water to form an analyte aqueous solution;

[0020] S2. After mixing the probe solution, the organic solvent, the PBS buffer solution and the aqueous solution of the object to be tested at room temperature and shaking evenly, the fluorescence intensity at 540 nm is observed under a laser wave of 450 nm in a fluorescence spectrometer; wherein the volume ratio of the organic phase to the aqueous phase in the mixed system of the probe solution, the organic solvent, the PBS buffer solution and the aqueous solution of the object to be tested is 1:1.

[0021] For example, the organic solvent may be dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile (ACN) or ethanol (EtOH), but is not limited thereto.

[0022] In some specific embodiments of the present invention, the sample is a chemical sample or a biological sample.

[0023] In some specific embodiments of the present invention, the sample is a biological sample including water, blood, microorganisms, or cells or tissues.

[0024] The present invention also provides a kit for detecting the presence of biothiol or determining the content of biothiol in a sample, which protects the fluorescent probe of formula I.

[0025] The present invention also provides application of the fluorescent probe in quantitatively detecting biothiols in the environment.

[0026] The present invention also provides application of the fluorescent probe in cell fluorescence imaging, which is used for non-disease diagnosis and treatment purposes.

[0027] Beneficial effects of the present invention:

[0028] The fluorescent probe of the present invention is dissolved in DMSO, and the probe is dissolved in an organic phase and an aqueous phase (1:1, v / v) solution, and after reacting with cysteine, homocysteine, N-acetylcysteine ​​or glutathione for 10 minutes, emits 540nm green fluorescence at an excitation wavelength of 450nm. The fluorescent probe of the present invention has no obvious response to other active oxygen, common amino acids, metal ions and active nitrogen species, has high detection sensitivity for biothiols, good selectivity and fast reaction rate (<10min), and can be used for quantitative biothiols in an in vitro environment, and fluorescent imaging analysis of biothiols flux in biological systems such as cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The fluorescent probe synthesized in Example 1 of the present invention detects tyrosine, tryptophan, glutamic acid, leucine, threonine, taurine, arginine, valine, cystine, isoleucine, methionine, histidine, lysine, phenylalanine, urea, ethylenediamine hydrochloride, zinc sulfate, aluminum nitrate, ferric chloride, ferrous sulfate, Hcy, Cys, NaHS, NaHSO 3 , GSH, NAC, homocysteine ​​thiolactone, H 2 O 2 and fluorescence emission spectra of HClO;

[0030] Figure 2 This is a fluorescence imaging diagram of endogenous biothiols in zebrafish imaged by the fluorescent probe synthesized in Example 1 of the present invention; wherein, (a) is bright field; and (b) is fluorescence channel. DETAILED DESCRIPTION

[0031] To make the present invention easier to understand, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by professional and technical personnel in the field; unless otherwise specified, the raw materials and reagents involved in this article can be purchased from the market or prepared by known methods.

[0032] A method for preparing a fluorescent probe for detecting biological thiol comprises the following steps:

[0033] The compound of formula Ⅰ, the compound of formula Ⅱ, bistriphenylphosphine palladium dichloride, cuprous iodide and the compound of formula Ⅲ are reacted to prepare the compound of formula Ⅳ, and the synthesis route thereof is as follows:

[0034]

[0035] Specifically:

[0036] (1) Under the protection of an inert atmosphere, 4-chloro-7-(diethylamino)-2H-chromen-2-one, trimethylsilyl acetylene, bistriphenylphosphine palladium dichloride, cuprous iodide and diisopropylethylamine are added to anhydrous tetrahydrofuran, and stirred at room temperature for reaction to obtain a reaction solution;

[0037] (2) The reaction solution was concentrated and purified by column chromatography to obtain pure 4,4'-(ethynyl-1,2-diyl)bis(7-(diethylamino)-2H-chromen-2-one)

[0038] Preferably, the molar ratio of the 4-chloro-7-(diethylamino)-2H-chromen-2-one to trimethylsilyl acetylene is 1:0.5-2; the molar amount of the bistriphenylphosphine palladium dichloride and cuprous iodide is 5%-20% of the molar amount of 4-chloro-7-(diethylamino)-2H-chromen-2-one; and the molar ratio of the diisopropylethylamine to 4-chloro-7-(diethylamino)-2H-chromen-2-one is 1-3:1.

[0039] Preferably, in step S1, the reaction time is 4 to 24 hours.

[0040] The following is further described in conjunction with specific embodiments.

[0041] Example 1

[0042] A method for preparing a fluorescent probe (4,4'-(ethyne-1,2-diyl)bis(7-(diethylamino)-2H-chromene-2-one)) for detecting biological thiol comprises the following steps:

[0043] Under argon protection, 4-chloro-7-(diethylamino)-2H-chromen-2-one (200.0 mg), trimethylsilyl acetylene (85.9 mg), bistriphenylphosphine palladium dichloride (27.9 mg), cuprous iodide (15.1 mg) and diisopropylethylamine (205.4 mg) were added to 10 mL of anhydrous tetrahydrofuran and stirred at room temperature for 12 hours. After the reaction was completed, the reaction solution was concentrated and purified by column chromatography to obtain 180.0 mg of an orange-red solid product with a yield of 49.6%.

[0044] The orange-red solid product was characterized by H NMR spectrum, and its specific data are as follows: 1 H NMR (500 MHz, CDCl 3 )δ7.60(d,J=8.9Hz,2H),6.63(dd,J=8.9,2.5Hz,2H),6.49(d,J=2.5Hz,2H),6.30(s,2H),3.43(q,J=7.1Hz,8H),1.23(t,J=7.1Hz,12H). It can be seen from the H NMR spectrum that Example 1 successfully prepared 4,4'-(ethyne-1,2-diyl)bis(7-(diethylamino)-2H-chromen-2-one).

[0045] Example 2

[0046] Application of fluorescent probes for detecting biothiols in vitro:

[0047] The fluorescence probe spectral property experiment for detecting biothiols of the present invention: the probe prepared in Example 1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1 mM, and analytes such as Cys, Hcy, NAC and GSH (tyrosine, tryptophan, glutamic acid, leucine, threonine, taurine, arginine, valine, cystine, isoleucine, methionine, histidine, lysine, phenylalanine, urea, ethylenediamine hydrochloride, zinc sulfate, aluminum nitrate, ferric chloride, ferrous sulfate, Hcy, Cys, NaHS, NaHSO 3 , GSH, NAC, homocysteine ​​thiolactone, H 2 O 2 and HClO) aqueous solution. The specific test method is: take 20μL of 1mM probe solution, 980μL of analytical grade DMSO, the required amount of PBS buffered aqueous solution and the required amount of 1mM analyte aqueous solution in a 2mL sample tube. For all tests, the volume ratio of organic phase and aqueous phase is maintained at 1:1 (the total volume of each test sample is 2mL). For example, when it is required to test the spectral changes after the probe reacts with Cys when the Cys concentration is 10μM, the sample preparation is: take 20μL of 1mM probe solution, 980μL of analytical grade DMSO, 980μL of PBS buffer solution and 20μL of 1mM Cys aqueous solution in a 2mL sample tube, shake at room temperature for 10 minutes and then measure its spectral changes. After reacting with biological thiol, the probe emits 540nm green fluorescence at an excitation wavelength of 450nm ( Figure 1 ). Other test operations are similar to the above steps. Figure 1 It can be seen that the probe has high selectivity in detecting biothiols and has no obvious response to other common amino acids, metal ions, reactive oxygen and reactive nitrogen species.

[0048] Example 3

[0049] Imaging and analysis of biothiols in zebrafish:

[0050] After zebrafish embryos were cultured under standard growth conditions for 48 hours, an appropriate amount of the fluorescent probe synthesized in Example 1 (final concentration of 5 μM) was added and cultured under standard growth conditions for another 30 minutes. Then, imaging was performed under a confocal fluorescence microscope, and obvious green fluorescence ( Figure 2 ), which enables fluorescence imaging analysis of endogenous biothiols in zebrafish.

[0051] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any technician familiar with the art within the technical scope disclosed in the present invention should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

Claims

1. A fluorescent probe for detecting biological thiols, characterized in that: The chemical structure of the fluorescent probe for detecting biothiol is shown in Formula IV:

2. The method for preparing a fluorescent probe for detecting biological thiols according to claim 1, characterized in that: The following steps are involved: The compound of formula Ⅰ, the compound of formula Ⅱ, bistriphenylphosphine palladium dichloride, cuprous iodide and the compound of formula Ⅲ are reacted to prepare the compound of formula Ⅳ, and the synthesis route thereof is as follows:

3. The method for preparing a fluorescent probe for detecting biological thiols according to claim 2, characterized in that: The following steps are involved: S1. Under the protection of an inert atmosphere, 4-chloro-7-(diethylamino)-2H-chromene-2-one, trimethylsilyl acetylene, bistriphenylphosphine palladium dichloride, cuprous iodide and diisopropylethylamine are added to anhydrous tetrahydrofuran, and the mixture is stirred at room temperature to react to obtain a reaction solution; S2. The reaction solution is concentrated and purified by column chromatography to obtain pure 4,4'-(ethyne-1,2-diyl)bis(7-(diethylamino)-2H-chromen-2-one).

4. The method for preparing a fluorescent probe for detecting biological thiol according to claim 3, characterized in that: The molar ratio of the 4-chloro-7-(diethylamino)-2H-chromen-2-one to trimethylsilyl acetylene is 1:0.5-2; the molar amounts of the bistriphenylphosphine palladium dichloride and cuprous iodide are 5%-20% of the molar amount of the 4-chloro-7-(diethylamino)-2H-chromen-2-one; and the molar ratio of the diisopropylethylamine to 4-chloro-7-(diethylamino)-2H-chromen-2-one is 1-3:

1.

5. The method for preparing a fluorescent probe for detecting biological thiol according to claim 3, characterized in that: In step S1, the reaction time is 4 to 24 hours.

6. A fluorescent probe composition for detecting biothiols, characterized in that: Comprising the fluorescent probe according to claim 1.

7. The fluorescent probe composition for detecting biothiols according to claim 6, characterized in that: The fluorescent probe composition further comprises at least one of a solvent, an acid, a base and a buffer solution.

8. A method for detecting the presence of biothiols or determining the content of biothiols in a sample for non-disease diagnosis and treatment purposes, characterized in that: The following steps are involved: S1. Dissolving the fluorescent probe according to claim 1 in an organic solvent to form a probe solution; dissolving the analyte in water to form an analyte aqueous solution; S2. Mix the probe solution, the organic solvent, the PBS buffer solution and the aqueous solution of the object to be tested at room temperature and shake them evenly, and then observe the fluorescence intensity at 540 nm under the laser wave of 450 nm of the fluorescence spectrometer; wherein the volume ratio of the organic phase to the aqueous phase in the mixed system of the probe solution, the organic solvent, the PBS buffer solution and the aqueous solution of the object to be tested is 1:1; wherein the organic solvent comprises at least one of dimethyl sulfoxide, N,N-dimethylformamide, acetonitrile and ethanol.

9. Use of the fluorescent probe according to claim 1 in quantitative detection of biothiols in the environment.

10. Use of the fluorescent probe according to claim 1 in cell fluorescence imaging, wherein the use is for non-disease diagnosis and treatment purposes.

Images