Camphor-based fluorescent probe capable of specifically distinguishing different biological mercaptans as well as preparation method and application of camphor-based fluorescent probe

Abstract

The invention discloses an acryloyl-based fluorescent probe with a camphor derivative structure, which takes a camphor derivative as a fluorophore and acryloyl as a recognition group, and has a structural formula as shown in (I): # imgabs0. The probe molecule emits orange fluorescence in a solution, and after the probe molecule responds to cysteine (Cys), the solution fluorescence is quenched; after response with homocysteine (Hcy) / glutathione (GSH), solution fluorescence is enhanced, and yellow fluorescence is emitted. The fluorescent probe disclosed by the invention not only can be used for detecting biological mercaptan in a water sample and a biological sample, but also can be used for distinguishing cysteine from homocysteine / glutathione. The fluorescent probe provided by the invention has very high sensitivity, good selectivity and anti-interference performance, can rapidly distinguish biological mercaptan, shortens response time, and realizes real-time effective detection of biological mercaptan. The fluorescent probe disclosed by the invention can realize biological mercaptan imaging in cells and organisms, is expected to play a role in industrial production, food detection and clinical medicine detection, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescent probes, and specifically relates to a fluorescent probe with a camphor derivative as a fluorescent group and an acryloyl group as a recognition group and its application in distinguishing and detecting biothiols. The present invention also provides a preparation method of the fluorescent probe. Background Art

[0002] Cysteine ​​(Cys), homocysteine ​​(Hcy) and glutathione (GSH) are common biothiols in organisms, playing important roles in physiological and pathological processes such as oxidative stress regulation, active free radical scavenging, signal transduction, and metal ion chelation. Abnormal levels of biothiols are signals of many diseases. For example, abnormally elevated cysteine ​​levels are associated with diseases such as hair depigmentation, edema, liver damage, slow growth in children, skin lesions, and muscle loss. Abnormally elevated homocysteine ​​levels are a risk factor for Alzheimer's disease, neural tube defects, cardiovascular disease, inflammatory bowel disease, and ischemic stroke. Glutathione is the most abundant non-protein thiol in cells and an important component of intracellular redox activity, signal transduction, and gene regulation. Some diseases such as cancer, heart disease, and liver damage are associated with abnormal glutathione levels. Therefore, identifying different biothiols is of great significance for the early diagnosis of certain diseases and the assessment of disease progression.

[0003] So far, there are many methods for detecting biothiols, including spectrophotometry, capillary electrophoresis, electrochemical detection, mass spectrometry, high performance liquid chromatography, etc. These methods can meet the needs of biothiols detection, but there are still some problems, such as complex sample processing, expensive experimental instruments, and the need for professional analysts. In recent years, fluorescence detection technology based on small molecule fluorescent probes has been recognized as one of the most attractive molecular detection technologies. It has the advantages of simplicity, convenience, and high sensitivity, and is widely used in the visual detection of target analytes in chemistry, biology, and the environment.

[0004] At present, the main design strategy of biothiol probes is to use the strong nucleophilicity of thiol to react with probe molecules, thereby changing the fluorescence signal of the probe. Based on this strategy, a series of fluorescent probes for biothiol detection have been developed, and the detection mechanism includes cyclization reaction with aldehyde, Michael addition reaction, disulfide bond cleavage reaction, sulfonate and sulfonamide cleavage reaction, etc. However, due to the similarity of the structures of the three biothiols, a large part of the fluorescent probes respond to all three biothiols and cannot distinguish the three. For example, the recently published document "An ortho-activation strategy to develop NIR fluorescent probe for rapid imaging of biothiols in vivo" discloses a fluorescent probe with fluorescein as the fluorescent group and acryloyl as the recognition group (Talanta, 2024, 266: 125110.). When the probe is applied to biothiol detection, the three biothiols can cause the fluorescence signal of the probe to be enhanced, but the three cannot be effectively distinguished. In addition, some fluorescent probes can only respond to one biothiol, but not to the other two biothiols. For example, the recent paper "Sequential activation strategy of triazinyl resorufin for high selectivity fluorescence GSH detection" reports a fluorescent probe with resorufin as the fluorescent group and triazine as the recognition group. This probe can only be used for glutathione detection, but not for cysteine ​​and homocysteine ​​detection (Talanta, 2024, 269: 125477.). At present, only a small number of fluorescent probes can respond to three biothiols at the same time and distinguish the three biothiols. These probes often use dual fluorescent group strategies or complex molecular structures. For example, the document "A triple-emission fluorescent probe for discriminatory detection of cysteine / homocysteine, glutathione / hydrogen sulfide, and thiophenol in living cells" discloses a fluorescent probe with a salicylaldehyde-coumarin structure as the core, 2,4-dinitrophenyl and 7-nitrobenz[c][1,2,5]oxadiazole (NBD) as recognition groups, in which NBD also plays the role of a fluorescent group (ACSSensors, 2018, 3: 1863-1869.).The probe emits green fluorescence in the solution after responding to cysteine ​​and homocysteine, and emits blue fluorescence in the solution after responding to glutathione, thereby achieving the purpose of simultaneously responding to and distinguishing the three biothiols. However, to achieve this goal, the design strategy of the probe is very complex.

[0005] At the same time, many fluorescent probes used for biothiol detection use fluorescent groups that can be divided into rhodamine, BODIPY, hemicyanine, fluorescein, coumarin, etc., according to their structures. However, there are very few reports on the construction of fluorescent groups by structural modification of renewable natural camphor. Camphor, as an important source of natural terpenoid compounds, is widely distributed in Lauraceae plants and can be used as an ideal starting material for the preparation of various compounds, such as antitumor drugs, anesthetics, antibacterial agents and catalysts. In addition, camphor derivatives have good biocompatibility and low cytotoxicity. Chinese patent document CN117263958A discloses a fluorescent probe based on the structure of camphor derivatives, which uses camphor derivatives as fluorescent groups and 2,4-dinitrophenol as recognition groups. It can respond to thiophenol compounds with high selectivity and sensitivity. In the presence of thiophenols, the fluorescence intensity decreases significantly. It can be used for qualitative and quantitative detection of thiophenol compounds without interference from aliphatic thiols such as glutathione (GSH), cysteine ​​(Cys), and homocysteine ​​(Hcy). At present, there is no report on a fluorescent probe based on the natural camphor structure that can respond to three biological thiols at the same time and can distinguish the three biological thiols. Summary of the invention

[0006] In order to solve the deficiencies of the prior art, the technical problem to be solved by the present invention is to provide a fluorescent probe based on camphor derivatives, which can specifically react with three biothiols (cysteine, homocysteine, and glutathione), and can distinguish the three biothiols, and is not interfered by other amino acids, ions and other substances in the sample. Another technical problem to be solved by the present invention is to provide a method for preparing the fluorescent probe. Another technical problem further solved by the present invention is to provide an application of the fluorescent probe to image biothiols in living cells and in vivo, and can distinguish the three biothiols in imaging.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] A fluorescent probe based on camphor derivatives with acryloyl as the recognition group.

[0009] In a specific example of the present invention, the fluorescent probe structure is shown in formula (I):

[0010]

[0011] The present invention also discloses the use of the fluorescent probe in preparing an indicator for detecting thiol compounds. Preferably, the thiol compound is a biothiol. More preferably, the biothiol includes cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH).

[0012] The fluorescent probe aqueous solution of the present invention has a maximum ultraviolet absorption wavelength of 470nm, and the color of the solution changes from light yellow to strong fluorescence with a maximum emission wavelength of 568nm under visible light. Under a 365nm ultraviolet lamp, the color of the solution changes to orange.

[0013] When cysteine ​​is detected in the sample, the maximum absorption wavelength of the solution's ultraviolet absorption red-shifts to 480nm. Under visible light, the color of the solution changes from slightly yellow to slightly red. The fluorescence intensity of the solution at 568nm is weakened by 150 times. Under 365nm ultraviolet light, the color of the solution changes from orange to purple.

[0014] When homocysteine ​​is detected in the sample, the maximum absorption wavelength of the solution's ultraviolet absorption blue-shifts to 454nm. Under visible light, the color of the solution remains unchanged. The fluorescence emission wavelength of the solution blue-shifts to 556nm, and the intensity is enhanced to twice the original. Under 365nm ultraviolet light, the color of the solution changes from orange to yellow.

[0015] When glutathione is present in the test sample, the maximum absorption wavelength of the solution's ultraviolet absorption blue-shifts to 445nm. Under visible light, the color of the solution remains unchanged. The fluorescence emission wavelength of the solution blue-shifts to 556nm, and the intensity increases to 2.75 times of the original. Under 365nm ultraviolet light, the color of the solution changes from orange to yellow.

[0016] The fluorescent probe of the present invention can be used for qualitative or quantitative detection of biothiols.

[0017] A specific example of the above application is fluorescence imaging of liquids, cells or organisms (in vivo).

[0018] The fluorescent probe of the present invention is used as a diagnostic reagent for the diagnosis of diseases related to abnormal biothiol content, wherein the diseases related to abnormal biothiol content are selected from liver damage, skin damage, central nervous system damage, muscle weakness, vomiting, and coma. The fluorescent probe of the present invention is also used as a detection reagent to further study the kinetic mechanism of the transport and accumulation of biothiols in the environment and in vivo.

[0019] In a specific example, the application of the fluorescent probe (I) described in the present invention comprises the following steps:

[0020] (1) preparing a fluorescent probe system;

[0021] (2) preparing solutions of biothiols (Cys, Hcy, GSH);

[0022] (3) adding the biothiol solution to the fluorescent probe system to prepare mixed solutions with different biothiol concentrations;

[0023] (4) testing the fluorescence intensity of the mixed solution at 568 nm, and drawing a standard curve based on the relationship between the fluorescence intensity and the biothiol concentration;

[0024] (5) Quantitatively detecting the content of biothiol in the test solution according to the standard curve.

[0025] The steps of preparing the fluorescent probe system include:

[0026] (1) dissolving the fluorescent probe (I) in an organic solvent to obtain a mother solution, wherein the concentration of the probe in the mother solution is 5×10 -7 ~5×10 -3 mol / L.

[0027] (2) diluting the concentration of the mother solution with a buffer solution to obtain a fluorescent probe system.

[0028] (3) The organic solvent for preparing the fluorescent probe mother solution is one or more of methanol, ethanol, acetonitrile, tetrahydrofuran, acetone, acetic acid, N,N-dimethylformamide, N,N-dimethylacetamide, and dimethyl sulfoxide.

[0029] (4) The buffer used to dilute the mother solution is one or more of PBS buffer, HEPES buffer, and Tris buffer.

[0030] Another object of the present invention is to provide a method for preparing the fluorescent probe (I) of the present invention, comprising the following steps:

[0031] S1: reacting camphorquinone with 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine in an organic solvent to prepare compound (II),

[0032]

[0033] The organic solvent is selected from one or more of formic acid, acetic acid, propionic acid, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, toluene, xylene, 1,4-dioxane, and 2-methylpyrrolidone.

[0034] The reaction temperature is preferably 0°C to 200°C.

[0035] S2: Compound (II) and 4-hydroxyphenylboronic acid pinacol ester are dissolved in an organic solvent, and reacted with a catalyst and a base under nitrogen protection to obtain compound (III):

[0036]

[0037] The organic solvent is selected from one or more of acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, ethylene glycol dimethyl ether and water.

[0038] The selected catalyst is selected from one or more of Pd(PPh3)4, Pd(PPh3)2Cl2, Pd(dppf)Cl2, Pd(dba)2-PCy3, PdCl2, Pd(OAc)2, Pd / C, and NiCl2(dppf).

[0039] The selected base is selected from one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t-BuONa, t-BuOK, Ba(OH)2, NaOH, NaHCO3, triethylamine, N,N-diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0040] The reaction temperature is preferably 0°C to 200°C.

[0041] S3: Compound (III) and acryloyl chloride are dissolved in an organic solvent, and a base is added to react to obtain a fluorescent probe (I):

[0042]

[0043] The organic solvent is selected from one or more of dichloromethane, chloroform, acetone, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, and ethylene glycol dimethyl ether.

[0044] The selected base is selected from one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t-BuONa, t-BuOK, Ba(OH)2, NaOH, NaHCO3, sodium acetate, potassium acetate, triethylamine, N,N-diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU).

[0045] The reaction temperature is preferably 0°C to 200°C.

[0046] A specific preparation method of the present invention is as follows:

[0047] S1: Dissolve camphorquinone and 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine in a reaction solvent, heat and stir. After the reaction is completed, cool the reaction mixture to room temperature and filter to obtain a crude product. Column chromatography is used to separate the product (II).

[0048] S2: Compound (II), 4-hydroxyphenylboronic acid pinacol ester, base and catalyst are dissolved in a reaction solvent, and heated and stirred in a nitrogen atmosphere. After the reaction is completed, the reaction is cooled to room temperature, most of the solvent is removed by rotary evaporation, and then extracted with ethyl acetate. The combined organic layer is washed with saturated brine, and dried by adding anhydrous Na2SO4. The product (III) is separated by column chromatography.

[0049] S3: Dissolve compound (III), acryloyl chloride and alkali in a reaction solvent, heat and stir. After the reaction is completed, cool the reaction to room temperature, remove most of the reaction solvent by rotary evaporation, then add water to the reaction system and extract with ethyl acetate. Wash the combined organic layer with saturated brine, add anhydrous Na2SO4 and dry. Separate by column chromatography to obtain the fluorescent probe (I).

[0050] The above specific reaction conditions can be found in the examples.

[0051] The present invention is beneficial in that:

[0052] (1) The fluorescent probe provided by the present invention introduces natural camphor molecules into the dye as a fluorescent matrix, uses the simplest acryloyl group as the recognition group, and can respond to three biological thiols (cysteine, homocysteine, and glutathione) with high selectivity and high sensitivity, and will not be interfered by other amino acids, ions and other substances in the sample. At the same time, the probe can effectively distinguish cysteine ​​from homocysteine / glutathione by virtue of the different pKa values ​​of the three biological thiols (the pKa values ​​of Cys, Hcy and GSH are 8.53, 10.0 and 9.20 respectively) and the large difference in steric hindrance (Cys has the smallest steric hindrance, followed by Hcy, and GSH has the largest steric hindrance), and has a good application value.

[0053] (2) The fluorescent probe provided by the present invention has low toxicity to cells and good biocompatibility. It can not only detect and distinguish biothiols in solution, but also detect and distinguish biothiols in cells and zebrafish. It can be used to deeply study the kinetic mechanism of biothiols' transport and accumulation in the environment and in organisms, and also help to clarify the biological role and mechanism of abnormal biothiols content in the pathogenic process. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0055] Figure 1 The UV spectrum (a), fluorescence spectrum (b) and solution color change (inset) of fluorescent probe (Ⅰ) (10μM) after adding cysteine ​​(1000μM) for 0.5h. The detection system is DMSO-PBS (pH=7.4, v / v=20 / 80), and the temperature during detection is 37℃. The excitation wavelength of the fluorescence spectrum is 440nm, and the slit is 5 / 5nm.

[0056] Figure 2 The UV spectrum (a), fluorescence spectrum (b) and solution color change (inset) of fluorescent probe (Ⅰ) (10μM) after adding homocysteine ​​(1000μM) for 0.5h. The detection system is DMSO-PBS (pH=7.4, v / v=20 / 80), and the temperature during detection is 37℃. The excitation wavelength of the fluorescence spectrum is 440nm, and the slit is 5 / 5nm.

[0057] Figure 3 The UV spectrum (a), fluorescence spectrum (b) and solution color change (inset) of fluorescent probe (Ⅰ) (10μM) after adding glutathione (1000μM) for 0.5h. The detection system is DMSO-PBS (pH=7.4, v / v=20 / 80), and the temperature during detection is 37℃. The excitation wavelength of the fluorescence spectrum is 440nm, and the slit is 5 / 5nm.

[0058] Figure 4 The fluorescence spectrum (a) of the fluorescent probe (Ⅰ) (10μM) after adding different concentrations of cysteine ​​(0-1000μM) and the fluorescence intensity at 568nm (b) change with the cysteine ​​concentration. Inset: Linear fitting diagram of fluorescence intensity and concentration in the range of 0-50μM. The detection system is DMSO-PBS (pH=7.4, v / v=20 / 80), the temperature during detection is 37℃, the incubation time is 0.5h, the excitation wavelength is 440nm, and the slit is 5 / 5nm.

[0059] Figure 5The fluorescence spectrum (a) and fluorescence intensity (b) of fluorescent probe (Ⅰ) (10μM) after adding cysteine ​​(1000μM) over time. Inset: Changes in fluorescence spectrum from 0 to 4min. The detection system was DMSO-PBS (pH=7.4, v / v=20 / 80), the temperature during detection was 37℃, the excitation wavelength was 440nm, and the slit was 5 / 5nm.

[0060] Figure 6 The fluorescence intensity (a) of the solution after adding cysteine ​​(1000 μM) to the fluorescent probe (Ⅰ) (10 μM) under different pH conditions and the fluorescence spectrum (b) at pH = 6. The detection system is DMSO-PBS (pH = 7.4, v / v = 20 / 80), the temperature during detection is 37 ° C, the incubation time is 0.5 h, the excitation wavelength is 440 nm, and the slit is 5 / 5 nm.

[0061] Figure 7 It is a fluorescent probe (Ⅰ) (10μM) added with 1000μM of biothiols (Cys, Hcy, GSH) or 1000μM of various analytes (Ala, Arg, Asp, Gln, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, SH - 、S2O3 2- 、SO4 2- , Cl - 、NO2 - 、NO3 - 、Na + , K + Mg 2+ and Ca 2+ ) after addition of DMSO-PBS (pH=7.4, v / v=20 / 80), the detection temperature was 37°C, the incubation time was 0.5h, the excitation wavelength was 440nm, and the slit was 5 / 5nm.

[0062] Figure 8 It is the color of the solution after adding 1000μM of biological thiols (Cys, Hcy, GSH) or 1000μM of various analytes (Ala, Arg, Asp, Gln, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, NaSH, Na2S2O3, MgSO4, NaNO2, KNO3 and CaCl2) to the fluorescent probe (I) (10μM) under 365nm ultraviolet light (a) and visible light (b). The detection system is DMSO-PBS (pH=7.4, v / v=20 / 80), the temperature during detection is 37℃, and the incubation is 0.5h.

[0063] Fig. 9 It is a fluorescent probe (Ⅰ) (10μM) and 1000μM cysteine ​​and 1000μM of various analytes (Ala, Arg, Asp, Gln, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, Na + , K + Mg 2+ , Ca 2+ , Cl - SH - 、S2O3 2- 、SO4 2- 、NO2 - and NO3 - ) The fluorescence intensity change of the fluorescence spectrum at 568nm after addition of DMSO-PBS (pH=7.4, v / v=20 / 80), the temperature during detection was 37℃, the incubation time was 0.5h, the excitation wavelength was 440nm, and the slit was 5 / 5nm.

[0064] Fig.10 This is a diagram showing the mechanism of using fluorescent probe (I) to distinguish three types of biothiols.

[0065] Fig.11 This is a high-resolution mass spectrometry (pH = 7.4) of the solution after the fluorescent probe (I) detects cysteine. Before the detection, 0.6 mg of the probe (I) was dissolved in 0.3 mL of DMSO, and 6 mg of cysteine ​​was dissolved in 1.2 mL of PBS (pH 7.4, 10 mM). During the detection, the two solutions were mixed, incubated in a water bath at 37°C for 0.5 h, and then the high-resolution mass spectrometry was measured.

[0066] Fig.12 This is the high-resolution mass spectrometry (pH=6) of the solution after the fluorescent probe (I) detects cysteine. Before the detection, 0.6 mg of the probe (I) was dissolved in 0.3 mL of DMSO, and 6 mg of cysteine ​​was dissolved in 1.2 mL of PBS (pH 6.0, 10 mM). During the detection, the two solutions were mixed, incubated in a water bath at 37°C for 0.5 h, and then the high-resolution mass spectrometry was measured.

[0067] Fig.13 This is a high-resolution mass spectrometry (pH = 7.4) of the solution after the fluorescent probe (I) detects homocysteine. Before the test, 0.6 mg of the probe (I) was dissolved in 0.3 mL of DMSO, and 6.9 mg of homocysteine ​​was dissolved in 1.2 mL of PBS (pH 7.4, 10 mM). During the test, the two solutions were mixed, incubated in a water bath at 37°C for 0.5 h, and then the high-resolution mass spectrometry was measured.

[0068] Fig.14This is the high-resolution mass spectrometry (pH=7.4) of the solution after the fluorescent probe (I) detected glutathione. Before the detection, 0.6 mg of the probe (I) was dissolved in 0.3 mL of DMSO, and 15.7 mg of glutathione was dissolved in 1.2 mL of PBS (pH 7.4, 10 mM). During the detection, the two solutions were mixed, incubated in a water bath at 37°C for 0.5 h, and then the high-resolution mass spectrometry was measured.

[0069] Fig.15 These are laser confocal microscopy photos taken at different time points when the fluorescent probe (I) (10 μM) was used to detect endogenous cysteine ​​in HeLa cells.

[0070] Fig.16 This is a laser confocal microscopy photograph of the fluorescent probe (Ⅰ) (10μM) used to detect endogenous and exogenous biothiols in HeLa cells.

[0071] Fig.17 This is an inverted fluorescence microscope photograph of the fluorescent probe (Ⅰ) (10μM) used to detect endogenous and exogenous biothiols in zebrafish. DETAILED DESCRIPTION

[0072] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention and should not be used to limit the protection scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the protection scope of the present invention.

[0073] Example 1: Preparation of fluorescent probe

[0074]

[0075] S1: Preparation of 4,11-dibromo-6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methyl[1,2,5]thiadiazole[3,4-b]phenazine (II)

[0076] Camphorquinone (1.66 g, 10 mmol) and 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine (3.24 g, 10 mmol) were dissolved in a reaction solvent (120 mL) and stirred at 120°C for 12 h. After the reaction was completed (monitored by TLC), the mixture was cooled to room temperature and the solvent was removed by evaporation under reduced pressure. The residue was dissolved in dichloromethane (300 mL) and washed three times with a saturated NaHCO3 solution (100 mL). The organic layer was washed with saturated brine and dried over anhydrous Na2SO4. The product was separated by column chromatography (eluent ethyl acetate: petroleum ether = 1:100 to elute the target compound). 2.41 g of yellow solid (II) was obtained with a yield of 53%.

[0077] Structure determination:

[0078] 1 H NMR(600MHz, CDCl3)δ3.26(d,J=4.6Hz,1H),2.44–2.33(m,1H),2.15(dd,J=16 .8,5.4Hz,1H),1.59(t,J=8.5Hz,2H),1.50(s,3H),1.20(s,3H),0.73(s,3H); 13 C NMR (150MHz, CDCl3) δ169.4,167.7,151.6,151.5,139.7,139.6,114.0,113.4,54.4,53.2,52.6,31.8,24.7,20.9,18.2,9.9; HRMS (ESI + )calcd for C 16 H 14 Br2N4S[M+H] + 452.9379, found 452.9378.

[0079] S2: Preparation of 4,4'-(6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methyl[1,2,5]thiadiazole[3,4-b]phenazine-4,11-diyl)diphenol (III)

[0080] 4,11-Dibromo-6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methyl[1,2,5]thiadiazole[3,4-b]phenazine (II, 454 mg, 1.0 mmol), 4-hydroxyphenylboronic acid pinacol ester (880 mg, 4.0 mmol), and Na2CO3 (420 mg, 4.0 mmol) were dissolved in a mixed solvent of toluene / ethanol / H2O (10 / 6 / 5 mL). PdCl2(dppf) (74 mg, 0.1 mmol) was added to the reaction system. The system was heated to 70°C and stirred for 6 h under a nitrogen atmosphere. After the reaction was completed (monitored by TLC), the reaction mixture was cooled to room temperature, the solvent was removed by rotary evaporation, and then extracted with dichloromethane (3×15 ml). The combined organic layer was washed with saturated brine and dried over anhydrous Na2SO4. The product was separated by column chromatography (eluent: ethyl acetate: petroleum ether = 1:5 to elute the target compound), to obtain 306 mg of a red solid (V), with a yield of 63.6%.

[0081] Structure determination:

[0082] 1 H NMR (600MHz, DMSO) δ9.66(d,J=8.1Hz,2H),7.70(d,J=7.9Hz,2H),7.58(d,J=7.8Hz,2H),6.94(d,J=7.5Hz,4H),2 .95(s,1H),2.27(s,1H),2.04(t,J=10.1Hz,1H),1.41(t,J=10.5Hz,2H),1.23(s,3H),1.08(s,3H),0.63(s,3H); 13 C NMR (150MHz, DMSO) δ166.3,164.8,157.7,152.7,152.4,137.6,137.2,134.5,134.1,129.2,129.0,12 6.3,126.0,114.8,114.6,53.8,52.8,52.5,31.9,31.4,24.8,22.5,20.7,18.3,14.4,10.1; HRMS(ESI + )calcd for C 28 H 24 N4O2S[M+H] + 481.1693, found 481.1697.

[0083] S3: Preparation of fluorescent probe (I)

[0084] Dissolve 4,4'-(6,12,12-trimethyl-6,7,8,9-tetrahydro-6,9-methyl[1,2,5]thiadiazole[3,4-b]phenazine-4,11-diyl)diphenol (Ⅲ, 96 mg, 0.2 mmol) and triethylamine (0.3 mL) in dichloromethane (5 mL), slowly add acryloyl chloride (720 mg, 0.8 mmol), and stir at room temperature for 12 h. After the reaction is completed (monitored by TLC), remove the solvent by rotary evaporation, then add 50 mL of water, and extract with dichloromethane (3×15 ml). Wash the combined organic layer with saturated brine, add anhydrous Na2SO4 and dry. Separate the product by column chromatography (eluent ethyl acetate: petroleum ether = 1:15 to elute the target compound). 40 mg of yellow solid (Ⅰ) was obtained, with a yield of 34%.

[0085] Structure determination: 1 H NMR(600MHz, CDCl3)δ7.98(s,1H),7.96(s,1H),7.89(s,1H),7.88(s,1H),7.40–7.37(m,4H),6.69 (dd,J=4.3,1.0Hz,1H),6.66(dd,J=4.3,1.0Hz,1H),6.42(dd,J=10.4,2.8Hz,1H),6.39(dd,J=10. 5,2.8Hz,1H),6.07(dd,J=12.0,1.1Hz,1H),6.06(dd,J=12.0,1.1Hz,1H),3.03(d,J=4.4Hz,1H),2 .32–2.26(m,1H),2.07–2.01(m,1H),1.49(d,J=9.4Hz,2H),1.31(s,3H),1.12(s,3H),0.70(s,3H); 13 C NMR (150MHz, CDCl3) δ166.9,165.4,164.6,164.5,152.6,152.4,150.5,150.4,138.0,137.8,133.9,133.6,13 2.8,132.7,132.6,129.0,128.9,128.1,120.9,120.5,54.0,53.2,52.4,31.9,24.8,20.7,18.2,9.7; HRMS (ESI + )calcd for C 34 H 28 N4O4S[M+H] + 589.1904, found 589.1904.

[0086] Example 2: Basic experimental conditions for detecting biothiols using fluorescent probe (I)

[0087] The fluorescent probe (I) was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe stock solution with a concentration of 10mM, which was stored in a 4°C medicine cool cabinet. During the test, the probe stock solution was diluted with DMSO to a concentration of 1mM as a stock solution for spectral testing to explore the changes in the ultraviolet and fluorescence spectral properties of the probe after responding to biothiols. To simulate physiological experiments, the experiments were carried out at pH = 7.4 (DMSO-PBS, pH = 7.4, v / v = 20 / 80) and 37°C. The excitation wavelength of the fluorescence spectrum was 440nm, and the slit width was 5nm / 5nm.

[0088] Example 3: Spectral response of fluorescent probe (I) to cysteine ​​(Cys)

[0089] A 100 mM cysteine ​​solution was prepared with water as solvent. The cysteine ​​solution was mixed with the above-mentioned probe stock solution to make the final concentration of the probe 10 μM and the cysteine ​​concentration 1000 μM. The solution was shaken and balanced for 0.5 h, and the UV spectrum and fluorescence spectrum were measured.

[0090] The changes in the UV absorption spectrum of probe (I) before and after the addition of cysteine ​​are shown in Figure 1 As shown in a. Figure 1 a It can be seen that after adding cysteine, the ultraviolet absorption of the detection solution decreased significantly, and the maximum absorption wavelength red-shifted from 470nm to 480nm. Under visible light, the color of the solution changed from slightly yellow to slightly red ( Figure 1 aInset).

[0091] The fluorescence spectrum of probe (I) before and after adding cysteine ​​is shown in Figure 1 As shown in b. Figure 1 As can be seen in b: before adding cysteine, the fluorescence of the test solution is very strong, and the maximum emission wavelength is 568nm; after adding cysteine, the fluorescence intensity of the solution at 568nm is weakened by 150 times. Under 365nm ultraviolet light, the color of the solution changes from orange to purple ( Figure 1 b illustration).

[0092] The above research results show that the fluorescent probe (I) has a good response to cysteine ​​through changes in ultraviolet and fluorescence spectra as well as naked eye observation.

[0093] Example 4: Spectral response of fluorescent probe (I) to homocysteine ​​(Hcy)

[0094] A homocysteine ​​solution with a concentration of 100 mM was prepared using water as the solvent. The homocysteine ​​solution was mixed with the probe solution, the final concentration of the probe was 10 μM, and the homocysteine ​​concentration was 1000 μM. The solution was shaken and balanced for 0.5 h, and the UV spectrum and fluorescence spectrum were measured.

[0095] The changes in the UV absorption spectrum of probe (I) before and after the addition of homocysteine ​​are shown in Figure 2 As shown in a. Figure 2 a It can be seen that after adding homocysteine, the ultraviolet absorption of the detection solution decreased significantly, and the maximum absorption wavelength shifted from 470nm to 454nm. Under visible light, the color of the solution remained unchanged and was always slightly yellow ( Figure 2 aInset).

[0096] The fluorescence spectrum of probe (I) before and after adding homocysteine ​​is shown in Figure 2 As shown in b. Figure 2 As can be seen in Figure b: before adding homocysteine, the fluorescence of the test solution is very strong, and the maximum emission wavelength is 568nm; after adding homocysteine, the emission wavelength of the solution blue-shifts to 556nm, and the fluorescence intensity of the solution increases to twice the original. Under 365nm ultraviolet light, the color of the solution changes from orange to yellow ( Figure 2 b illustration).

[0097] The above research results show that the fluorescent probe (I) has a good response to homocysteine ​​through changes in ultraviolet and fluorescence spectra as well as naked eye observation.

[0098] Example 5: Spectral response of fluorescent probe (I) to glutathione (GSH)

[0099] A 100 mM glutathione solution was prepared with water as solvent. The glutathione solution was mixed with the probe solution, the final concentration of the probe was 10 μM, and the concentration of glutathione was 1000 μM. The solution was shaken and balanced for 0.5 h, and the UV spectrum and fluorescence spectrum were measured.

[0100] The changes of the UV absorption spectrum of probe (Ⅰ) before and after adding glutathione are shown in Figure 3 As shown in a. Figure 3 a It can be seen that after adding glutathione, the ultraviolet absorption of the test solution decreased significantly, and the maximum absorption wavelength shifted from 470nm to 445nm. Under visible light, the color of the solution remained unchanged and was always slightly yellow ( Figure 3 aInset).

[0101] The fluorescence spectrum of probe (I) before and after adding glutathione is shown in Figure 3 As shown in b. Figure 3As can be seen in b: before adding glutathione, the fluorescence of the test solution is very strong, and the maximum emission wavelength is 568nm; after adding glutathione, the emission wavelength of the solution blue-shifts to 556nm, and the fluorescence intensity of the solution increases to 2.75 times the original. Under 365nm ultraviolet light, the color of the solution changes from orange to yellow ( Figure 3 b illustration).

[0102] The above research results show that the fluorescent probe (I) has a good response to glutathione through changes in ultraviolet and fluorescence spectra as well as naked eye observation.

[0103] In addition, comprehensive Figure 1 , Figure 2 , Figure 3 The fluorescent probe (I) exhibits completely different performances in detecting cysteine, homocysteine, and glutathione, and can effectively distinguish cysteine ​​from homocysteine / glutathione, and has great application value.

[0104] Example 6: Detection limit of fluorescent probe (I) for cysteine

[0105] A mixed solution consisting of DMSO and PBS buffer solution (DMSO-PBS, pH = 7.4, v / v = 20 / 80) was used as the reaction system to prepare a reaction mixture of 10 μM probe (Ⅰ) and different concentrations of cysteine. The prepared reaction mixture was incubated at 37°C for 0.5 h and then the fluorescence spectrum was measured. The excitation wavelength of the fluorescence spectrum was 440 nm and the slit width was 5 nm / 5 nm. The detection limit of probe (Ⅰ) for cysteine ​​was calculated according to the 3σ / k method, where σ represents the standard deviation of the emission intensity values ​​of the blank sample measured ten times, and k represents the slope of the fitted straight line between the fluorescence intensity and the cysteine ​​concentration.

[0106] The fluorescence spectra (a) and the fluorescence intensity changes at 568 nm (b) of probe (I) after adding different concentrations of cysteine ​​(0-1000 μM) are shown in Figure 2. Figure 4 As shown. Before adding cysteine, the fluorescence intensity of probe (I) was very strong. As the concentration of cysteine ​​increased, the fluorescence at 568nm of the solution rapidly weakened. The fluorescence intensity was linearly related to cysteine ​​in the range of 0-50μM, and the linear regression equation was y=-29.195x+3519.1, R 2 =0.9988( Figure 4 b illustration). According to the 3σ / k method, the detection limit of probe (I) for cysteine ​​is 1.38 μM. This proves that cysteine ​​can be quantitatively analyzed with the help of this fluorescent probe.

[0107] Example 7: Response time of fluorescent probe (I) to cysteine

[0108] A mixed solution consisting of DMSO and PBS buffer solution (DMSO-PBS, pH = 7.4, v / v = 20 / 80) was used as the reaction system to prepare a reaction mixed solution of 10 μM probe (I) and 1000 μM cysteine, and the prepared reaction mixed solution was measured for fluorescence spectrum at different time points (0-40 min) at 37°C. The excitation wavelength of the fluorescence spectrum was 440 nm, and the slit width was 5 nm / 5 nm.

[0109] The fluorescence spectrum (a) and the change of fluorescence intensity at the emission wavelength (b) of the probe (I) after adding cysteine ​​are shown in Figure 2. Figure 5 After adding cysteine, the emission wavelength of the solution immediately blue-shifted to 556 nm, and the fluorescence intensity increased rapidly and reached a peak within 4 min ( Figure 5 aInset and Figure 5 b); then the fluorescence intensity of the solution at 556 nm will gradually weaken, and drop to the lowest point within 30 minutes and reach a plateau ( Figure 5 a and Figure 5 b), therefore the detection time of cysteine ​​is controlled within 30 min.

[0110] Example 8: Effect of pH on the detection of cysteine ​​by fluorescent probe (I)

[0111] A mixed solution consisting of DMSO and PBS buffer solution (DMSO-PBS, v / v=20 / 80) was used as the reaction system, and the pH of the mixed solution was adjusted using 3 mol / L hydrochloric acid and 3 mol / L NaOH solution. Subsequently, a reaction mixed solution of 10 μM probe (Ⅰ) and 1000 μM cysteine ​​was prepared using these reaction systems with different pH values, and the prepared reaction mixed solution was incubated at 37°C for 0.5 h before measuring the fluorescence spectrum. The excitation wavelength of the fluorescence spectrum was 440 nm, and the slit width was 5 nm / 5 nm.

[0112] The stability of probe (I) under different pH conditions and its ability to detect cysteine ​​are shown in Figure 2. Figure 6 As shown in a. It can be clearly seen from the figure that the fluorescent probe has good stability in a long pH range (2-10). When pH <6, the probe does not respond to cysteine; when pH ≥6, the probe shows different response performance to cysteine: when cysteine ​​is added to the detection system with pH ≥7, the fluorescence intensity of the solution drops sharply; when cysteine ​​is added to the detection system with pH = 6, the fluorescence intensity of the solution is enhanced by 1 times, and the emission wavelength of the solution is blue-shifted from 568nm to 556nm ( Figure 6 b).

[0113] Therefore, the fluorescent probe (I) can meet the needs of detecting biothiols in cells and animals under physiological conditions.

[0114] Example 9: Selectivity of fluorescent probe (I) for biothiol recognition

[0115] Generally speaking, high selectivity for target analytes is the core requirement of all detection methods. According to Example 2, various biothiols, sulfur-containing salts, amino acids, and metal salt solutions (Cys, Hcy, GSH, Ala, Arg, Asp, Gln, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, NaSH, Na2S2O3, MgSO4, NaNO2, KNO3, and CaCl2) with a concentration of 100 mM were prepared using deionized water as a solvent. In order to study the selectivity of probe (I) for biothiols, a mixed solution consisting of DMSO and PBS buffer solution (DMSO-PBS, pH = 7.4, v / v = 20 / 80) was used as a reaction system to prepare a reaction mixture solution of 10 μM probe (I) and 1000 μM biothiols or 1000 μM various sulfur-containing salts, amino acids, and metal salt solutions, and the prepared reaction mixture solution was incubated at 37°C for 0.5 h and then the fluorescence spectrum was measured. The excitation wavelength of the fluorescence spectrum is 440 nm, and the slit width is 5 nm / 5 nm.

[0116] The fluorescence spectrum (a) and fluorescence intensity (b) of probe (I) after adding 1000 μM biothiol or 1000 μM various sulfur-containing salts, amino acids, metal salts are shown in Figure 2. Figure 7 As shown. Only biothiols can cause significant changes in the fluorescence spectrum of the probe, while other analytes will not cause significant changes in the fluorescence spectrum of the probe, indicating that probe (I) has high selectivity for biothiols. In addition, the three biothiols (cysteine, homocysteine, and glutathione) cause different changes in the fluorescence spectrum: cysteine ​​quenches the fluorescence of the solution, while homocysteine / glutathione enhances the fluorescence intensity of the solution.

[0117] The selectivity of probe (I) can also be demonstrated by observing the change in solution color with the naked eye. Figure 8 As shown in the figure, under 365nm ultraviolet light, when other analytes are added, the color of the probe solution is still orange; after adding cysteine, the color of the solution quickly turns to purple and the fluorescence is quenched; after adding homocysteine / glutathione, the color of the solution quickly turns to bright yellow and the fluorescence is enhanced. Under visible light, when other analytes, homocysteine ​​and glutathione are added, the color of the probe solution is still slightly yellow; when cysteine ​​is added, the color of the solution quickly turns to light red.

[0118] The above results show that the probe is highly selective for biothiols. When applied, it can not only effectively distinguish biothiols from other analytes, but also distinguish cysteine ​​from homocysteine / glutathione, and has great application value.

[0119] Example 10: Competitive experiment of fluorescent probe (I)

[0120] In order to prove that the fluorescent probe has good application potential, it is necessary to test the anti-interference performance. Analytes that may interfere with the detection of the fluorescent probe (I) are added to the detection system to examine whether the detection ability of the fluorescent probe (I) will be interfered in the presence of these analytes.

[0121] A mixed solution consisting of DMSO and PBS buffer solution (DMSO-PBS, pH = 7.4, v / v = 20 / 80) was used as the reaction system to prepare a reaction mixed solution containing 10 μM probe (I), 1000 μM cysteine, and 1000 μM sulfur salts, amino acids, and metal salt solutions (Ala, Arg, Asp, Gln, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, NaSH, Na2S2O3, MgSO4, NaNO2, KNO3, and CaCl2). The prepared reaction mixed solution was incubated at 37°C for 0.5h and then the fluorescence spectrum was measured. The excitation wavelength of the fluorescence spectrum was 440nm, and the slit width was 5nm / 5nm. Fig. 9 As shown, in the presence of other analytes, the fluorescence of the solution is quenched after the probe (I) detects cysteine, proving that the presence of other analytes will not interfere with the detection of cysteine ​​by the probe, indicating that the anti-interference performance of the probe (I) is very good.

[0122] Example 11: Mechanism of fluorescent probe (I) to distinguish different biothiols

[0123] Fig.10This is a diagram of the mechanism of fluorescent probe (I) distinguishing three types of biothiols. In the basic structure of fluorescent probe (I), the acryloyl group as a recognition group can effectively inhibit the rotation of the phenol group, thereby inhibiting the torsional intramolecular charge transfer (TICT) effect, resulting in a strong fluorescence intensity of the solution. During the detection process, the sulfhydryl (-SH) of the three biothiols reacts with the acryloyl group to produce Michael addition products (CQXPA-Cys, CQXPA-Hcy, CQXPA-GSH) with large side chains, which can more effectively inhibit the rotation of the phenol group and the TICT effect, so the fluorescence intensity of the solution is enhanced. However, the Michael addition product CQXPA-Cys generated by the detection of cysteine ​​is very unstable and is prone to intramolecular cyclization reaction to hydrolyze its side chain to generate CQXP. The rotation of the phenol group is no longer inhibited, and the TICT process is restored, which greatly induces the non-radiative relaxation of the energy of the excited state molecules, resulting in a decrease in fluorescence intensity.

[0124] To prove the above reaction mechanism, the solutions after the fluorescent probe (I) was used to detect the three biothiols were subjected to high-resolution mass spectrometry analysis. Fig.11 This is the high-resolution mass spectrometry (pH = 7.4) of the solution after the fluorescent probe (I) detected cysteine. Before the detection, 0.6 mg of the probe (I) was dissolved in 0.3 mL of DMSO, and 6 mg of cysteine ​​was dissolved in 1.2 mL of PBS (pH 7.4, 10 mM). During the detection, the two solutions were mixed and incubated in a water bath at 37°C for 0.5 h, and then the high-resolution mass spectrometry was measured. It was found that only the molecular ion peak of CQXP could be found, proving that in the detection system at pH = 7.4, CQXPA was converted into CQXP through Michael addition-cyclization reaction.

[0125] Fig.12 This is the high-resolution mass spectrometry (pH = 6.0) of the solution after the fluorescent probe (I) detected cysteine. Before the test, 0.6 mg of the probe (I) was dissolved in 0.3 mL of DMSO, and 6 mg of cysteine ​​was dissolved in 1.2 mL of PBS (pH 6.0, 10 mM). During the test, the two solutions were mixed and incubated in a water bath at 37°C for 0.5 h, and then the high-resolution mass spectrometry was measured. It was found that only the molecular ion peak of CQXPA-Cys could be found, proving that in the detection system at pH = 6.0, CQXPA was converted into CQXPA-Cys by Michael addition reaction.

[0126] Fig.13This is the high-resolution mass spectrometry of the solution after the fluorescent probe (I) detected homocysteine. Before the detection, 0.6 mg of the probe (I) was dissolved in 0.3 mL of DMSO, and 6.9 mg of homocysteine ​​was dissolved in 1.2 mL of PBS (pH 7.4, 10 mM). During the detection, the two solutions were mixed and incubated in a water bath at 37°C for 0.5 h, and then the high-resolution mass spectrometry was measured, and the molecular ion peak of CQXPA-Hcy was found, proving that when detecting homocysteine, CQXPA was converted into CQXPA-Hcy through the Michael addition reaction.

[0127] Fig.14 This is the high-resolution mass spectrometry of the solution after the fluorescent probe (I) detected glutathione. Before the detection, 0.6 mg of the probe (I) was dissolved in 0.3 mL of DMSO, and 15.7 mg of glutathione was dissolved in 1.2 mL of PBS (pH 7.4, 10 mM). During the detection, the two solutions were mixed and incubated in a water bath at 37°C for 0.5 h, and then the high-resolution mass spectrometry was measured, and it was found that the molecular ion peak corresponding to CQXPA-GSH could be found, proving that when detecting glutathione, CQXPA was converted into CQXPA-GSH through the Michael addition reaction.

[0128] Figures 11 to 14 Well proved Fig.10 The mechanism of the proposed fluorescent probe (I) to distinguish the three biothiols.

[0129] Example 12: Changes in fluorescence signal over time when fluorescent probe (I) detects endogenous cysteine ​​in cells

[0130] HeLa cells were pre-cultured in a confocal dish for 24 hours to allow the cells to adhere to the wall, and laser confocal imaging was performed under a laser confocal microscope (488 nm excitation wavelength, 500 nm to 700 nm fluorescence collection). 0.5 mM N-ethylmaleimide (NEM) was added to the HeLa cells and incubated for 15 minutes to remove the biothiols in the cells, followed by incubation with 10 μM probe (I) for 30 minutes, and finally cysteine ​​(500 μM) was added and imaged under a laser confocal microscope at different time points.

[0131] Fig.15 This reflects the change in fluorescence signal when the fluorescent probe (I) detects endogenous cysteine ​​in cells. From the results, the fluorescence signal of the blank group is the strongest. When cysteine ​​is added and incubated for a period of time, the fluorescence signal in the cells gradually weakens with the extension of time, which is in line with the experimental expectations, proving that the fluorescent probe (I) can be used for the detection of intracellular cysteine.

[0132] Example 13: Fluorescent probe (I) for the detection of three biothiols in cells

[0133] HeLa cells were pre-cultured in a laser confocal dish for 24 h to allow the cells to adhere to the wall, and then imaged under a FV1000 laser confocal microscope (Olympus, Japan) with an excitation wavelength of 488 nm and fluorescence collected at 500-600 nm.

[0134] 10 μM probe (I) was added to the first group of HeLa cells and incubated for 30 min, and then imaged under a laser confocal microscope; 0.5 mM N-ethylmaleimide (NEM) was added to the second group of HeLa cells and incubated for 15 min to remove endogenous biothiols in the cells, followed by incubation with 10 μM probe (I) for 30 min, and then imaged under a laser confocal microscope; 0.5 mM NEM was added to the third group of HeLa cells and incubated for 15 min, followed by incubation with 10 μM probe (I) for 30 min, and finally 500 μM C ys and incubated for 30 min, and imaged under a laser confocal microscope; to the fourth group of HeLa cells, 0.5 mM NEM was added and incubated for 15 min, followed by incubation with 10 μM probe (Ⅰ) for 30 min, and finally 500 μM Hcy was added and incubated for 30 min, and imaged under a laser confocal microscope; to the fifth group of HeLa cells, 0.5 mM NEM was added and incubated for 15 min, followed by incubation with 10 μM probe (Ⅰ) for 30 min, and finally 500 μM GSH was added and incubated for 30 min, and imaged under a laser confocal microscope.

[0135] Fig.16This is the result of using fluorescent probe (I) to image three types of biothiols in cells. Glutathione is the biothiol with the highest content in cells, so the first group of laser confocal microscopy photos shows the results of fluorescent probe (I) detecting endogenous glutathione in cells, and the fluorescence intensity in the first group of cells is very strong, which is consistent with the characteristic of signal enhancement after fluorescent probe (I) detects glutathione; after NEM was added to the second group of cells, the influence of endogenous biothiols in cells was eliminated, and its fluorescence intensity reflects the fluorescence intensity of the probe itself in cells; after NEM was added to the third group of cells to eliminate the influence of endogenous biothiols in cells, its fluorescence intensity The fluorescence intensity reflects the change of the probe's fluorescence intensity after detecting exogenous cysteine ​​in the cell. The results show that the fluorescence of the third group is sharply weakened, which is consistent with the characteristic of fluorescence quenching after probe (I) detects cysteine. After adding NEM to the fourth and fifth groups of cells to eliminate the influence of endogenous biological thiols in the cell, the fluorescence intensity reflects the change of the probe's fluorescence intensity after detecting exogenous homocysteine ​​and glutathione in the cell. The results show that the fluorescence of the fourth and fifth groups is sharply enhanced, which is consistent with the characteristic of fluorescence enhancement after probe (I) detects homocysteine ​​and glutathione. Therefore, it can be proved that fluorescent probe (I) can be used for the detection of both endogenous biological thiols in the cell and exogenous biological thiols in the cell, and can distinguish Cys from Hcy / GSH.

[0136] Example 14: Fluorescent probe (I) for the detection of biothiols in zebrafish

[0137] Three-day-old zebrafish were imaged under a DMi8 inverted fluorescence microscope (Leica, Germany) using the RHOD (rhodamine) channel, an excitation wavelength range of 511–521 nm, and collection of fluorescence at 565–605 nm.

[0138] 10 μM probe (I) was added to the first group of zebrafish and incubated for 30 min, and then imaged under an inverted fluorescence microscope; 0.1 mM NEM was added to the second group of zebrafish and incubated for 30 min to remove biothiols in the zebrafish, followed by incubation with 10 μM probe (I) for 30 min, and then imaged under an inverted fluorescence microscope; 0.1 mM NEM was added to the third group of zebrafish and incubated for 30 min, followed by incubation with 10 μM probe (I) for 30 min, and finally incubated with 500 μM Cys for 3 0 min, and imaging was performed under an inverted fluorescence microscope; 0.1 mM NEM was added to the fourth group of zebrafish and incubated for 30 min, followed by incubation with 10 μM probe (Ⅰ) for 30 min, and finally incubated with 500 μM Hcy for 30 min, and imaging was performed under an inverted fluorescence microscope; 0.1 mM NEM was added to the fifth group of zebrafish and incubated for 30 min, followed by incubation with 10 μM probe (Ⅰ) for 30 min, and finally incubated with 500 μM GSH for 30 min, and imaging was performed under an inverted fluorescence microscope.

[0139] Fig.17 This is the result of using fluorescent probe (I) to image biothiols in zebrafish. Glutathione is the most abundant biothiol in zebrafish, so the first group of inverted fluorescence microscope photos shows the results of fluorescent probe (I) detecting endogenous glutathione in zebrafish. The fluorescence intensity in the first group of zebrafish is very strong, which is consistent with the characteristic of signal enhancement after fluorescent probe (I) detects glutathione. After adding NEM to the second group of zebrafish, the influence of endogenous biothiols in zebrafish is eliminated, and its fluorescence intensity reflects the fluorescence intensity of the probe itself in zebrafish. After adding NEM to the third group of zebrafish to eliminate the influence of endogenous biothiols in zebrafish, Its fluorescence intensity reflects the change in fluorescence intensity of the probe after detecting exogenous cysteine ​​in zebrafish. From the results, the fluorescence of the third group is sharply weakened, which is consistent with the characteristic of fluorescence quenching after probe (Ⅰ) detects cysteine; after adding NEM to the fourth and fifth groups of cells to eliminate the influence of endogenous biological thiols in zebrafish, its fluorescence intensity reflects the change in fluorescence intensity of the probe after detecting exogenous homocysteine ​​and glutathione in zebrafish. From the results, the fluorescence of the fourth and fifth groups is sharply enhanced, which is consistent with the characteristic of fluorescence enhancement after probe (Ⅰ) detects homocysteine ​​and glutathione. Therefore, it can be proved that fluorescent probe (Ⅰ) can be used for the detection of endogenous biological thiols in zebrafish, as well as the detection of exogenous biological thiols in zebrafish, and can distinguish Cys from Hcy / GSH.

[0140] It should be noted that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation shall fall within the protection scope of the present invention.

Claims

1. A fluorescent probe based on a camphor derivative of acryloyl group, characterized in that: The structural formula is shown in formula (I):

2. Use of the fluorescent probe according to claim 1 in preparing an indicator for detecting thiol compounds.

3. The use according to claim 2, characterized in that The thiol compound is biothiol.

4. The use according to claim 3, characterized in that The biothiol is selected from cysteine, homocysteine ​​and glutathione.

5. The use according to claim 2, characterized in that The detection is qualitative or quantitative.

6. The use according to claim 4, characterized in that The application is fluorescence imaging of solutions, cells or organisms.

7. The use according to claim 4, characterized in that The fluorescent probe is used as a diagnostic reagent in the diagnosis of diseases caused by abnormal biothiol content.

8. The use according to claim 7, characterized in that The disease caused by abnormal biothiol content is selected from liver damage, skin damage, slow growth, Alzheimer's disease, depression, cardiovascular disease, neural tube defects, inflammatory bowel disease, and osteoporosis.

9. The method for preparing a fluorescent probe according to claim 1, characterized in that The following steps are involved: S1: Compound (II) is prepared by reacting camphorquinone with 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine. S2: Compound (II) prepared in S1 is reacted with 4-hydroxyphenylboronic acid pinacol ester to prepare compound (III), S3: The compound (III) prepared in S2 is reacted with acryloyl chloride and triethylamine to obtain the fluorescent probe (I) 10. The preparation method according to claim 9, characterized in that: The following steps are involved: S1: dissolving camphorquinone and 4,7-dibromobenzo[c][1,2,5]thiadiazole-5,6-diamine in an organic solvent and heating to obtain compound (II), wherein the solvent is selected from one or more of formic acid, acetic acid, propionic acid, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, toluene, xylene, 1,4-dioxane, and 2-methylpyrrolidone, and the reaction temperature is 0°C to 200°C; S2: Compound (II) and 4-hydroxyphenylboronic acid pinacol ester are dissolved in an organic solvent, and reacted with a catalyst and a base under nitrogen protection to obtain compound (III), wherein the solvent is selected from one or more of acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, ethylene glycol dimethyl ether, and water, and the catalyst is selected from Pd(PPh3)4, Pd(PPh3)2Cl2, Pd(dppf)Cl2, Pd(PPh3)2 ... One or more of d(dba)2-PCy3, PdCl2, Pd(OAc)2, Pd / C, NiCl2(dppf), the selected base is one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t-BuONa, t-BuOK, Ba(OH)2, NaOH, NaHCO3, triethylamine, N,N-diisopropylethylamine, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), and the reaction temperature is 0°C to 200°C; S3: Compound (III) and acryloyl chloride are dissolved in an organic solvent, and a base is added to react to obtain a fluorescent probe (I). The solvent is selected from one or more of dichloromethane, chloroform, acetone, acetonitrile, tetrahydrofuran, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, toluene, xylene, 1,4-dioxane, 2-methylpyrrolidone, ethanol, methanol, and ethylene glycol dimethyl ether. The base is selected from one or more of K2CO3, K3PO4, Na2CO3, CsF, Cs2CO3, t-BuONa, t-BuOK, Ba(OH)2, NaOH, NaHCO3, sodium acetate, potassium acetate, triethylamine, N,N-diisopropylethylamine, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). The reaction temperature is 0°C to 200°C.

Images