A pyrazoline derivative-based lysosome-targeted biothiol fluorescent probe and its preparation method and application

Abstract

The present invention belongs to the field of chemical analysis and detection technology, and provides a pyrazoline derivative lysosome-targeted biological thiol fluorescent probe and its preparation method and application. The preparation method of the pyrazoline derivative lysosome-targeted biological thiol fluorescent probe of the present invention comprises the following steps: mixing compound 1, 2-chloro-3,5-dinitropyridine, a base and an organic solvent and reacting to obtain a pyrazoline derivative lysosome-targeted biological thiol fluorescent probe. The fluorescent probe of the present invention can monitor the concentration fluctuations of Cys, Hcy and GSH in living cell lysosomes in real time by fluorescence imaging, and has the advantages of high selectivity, high sensitivity and rapid response; good selectivity and low cytotoxicity make it an ideal tool for simultaneous detection of Cys, Hcy and GSH.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis and detection, and in particular to a lysosome-targeted biological thiol fluorescent probe of a pyrazoline derivative type, and a preparation method and application thereof. Background Art

[0002] Biothiols are essential reactive sulfur species in the human body and often play key roles in physiological and pathological processes, such as undergoing reversible oxidation, regulating oxidative stress, inactivating reactive free radicals, signal transduction, and chelating harmful metal ions. Reduced glutathione (GSH), the most potent non-protein biothiol in cells, maintains the balance between cysteine ​​(Cys) and homocysteine ​​(Hcy) on proteins. Abnormal GSH levels can cause hypertension, leukopenia, psoriasis, liver damage, and cancer. Cys, a precursor of reduced glutathione, plays an important role in many physiological processes, including detoxification, protein metabolism, and synthesis. Cys has been reported to be a risk factor for obesity, hair loss, slow growth in children, skin lesions, edema, lethargy, muscle loss, and liver damage. Furthermore, Hcy, a precursor of Cys, is associated with numerous diseases, including cardiovascular disease, birth defects, folate and cobalamin deficiencies, and cognitive impairment in the elderly. Therefore, selective detection of these three biothiols is of great significance in biological systems.

[0003] Eukaryotic cells contain numerous organelles, and most biological events occur within specific organelles, such as mitochondria, lysosomes, endoplasmic reticulum, lipid droplets, and the Golgi apparatus. These well-organized organelles play key roles in fundamental metabolic processes, including cell growth, division, fusion, signal transduction, and apoptosis. Lysosomes have a slightly acidic pH (4.5–5.5) and contain over 60 hydrolases, which primarily break down and metabolize intracellular biomacromolecules such as proteins, nucleic acids, and polysaccharides. Thiols are closely associated with intralysosomal proteolysis by reducing disulfide bonds. For example, GSH is a potent stimulator of albumin hydrolysis in renal lysosomes, and Cys can effectively stimulate albumin degradation in hepatic lysosomes. To better understand the role of lysosomal thiols, effective monitoring and detection of lysosomal thiols is crucial.

[0004] Because biothiols are closely associated with various diseases, much research has focused on developing diverse methods to monitor their presence and concentration in vitro and in vivo. Conventional methods for detecting biothiols include UV-visible absorption spectroscopy, capillary electrophoresis, electrochemical detection, high-performance liquid chromatography, and mass spectrometry. Compared to these detection techniques, fluorescent probes offer several advantages, including low cost, rapid response, high sensitivity, relative ease of operation, and real-time, non-destructive imaging. Currently reported fluorescent probes for biothiols primarily rely on fluorophores such as BODIPY, rhodamine, tetraphenylethylene, coumarin, and naphthalimide, and achieve biothiols detection through sensing mechanisms such as Michael addition, cleavage reaction, cyclization with aldehydes, nucleophilic substitution, conjugate addition and cyclization, and metal complex substitution and coordination. Pyrazoline derivatives, as an important class of nitrogen-containing five-membered heterocyclic compounds, possess significant advantages, including prominent blue emission, high fluorescence quantum yield, narrow emission wavelength, and exceptional color purity.

[0005] Therefore, the development of a lysosome-targeted biothiol fluorescent probe based on pyrazoline derivatives for the detection of cysteine, homocysteine ​​and glutathione has important application value. Summary of the Invention

[0006] The purpose of the present invention is to provide a lysosome-targeted biological thiol fluorescent probe of a pyrazoline derivative type in order to overcome the deficiencies of the prior art, as well as a preparation method and application thereof.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a lysosome-targeted biothiol fluorescent probe based on a pyrazoline derivative. The structural formula of the biothiol fluorescent probe is:

[0009]

[0010] The present invention also provides a method for preparing the lysosome-targeted biological thiol fluorescent probe based on pyrazoline derivatives, comprising the following steps:

[0011] Compound 1, 2-chloro-3,5-dinitropyridine, a base and an organic solvent are mixed and reacted to obtain a pyrazoline derivative-type lysosome-targeted biothiol fluorescent probe;

[0012] The structural formula of the compound 1 is:

[0013]

[0014] Preferably, the base is sodium acetate, sodium carbonate, potassium carbonate, sodium hydroxide, triethylamine or ammonium acetate.

[0015] Preferably, the organic solvent is methanol, ethanol, acetonitrile, tetrahydrofuran or N,N-dimethylformamide.

[0016] Preferably, the reaction temperature is 25 to 100° C., and the reaction time is 2 to 12 hours.

[0017] Preferably, the molar ratio of compound 1, 2-chloro-3,5-dinitropyridine and base is 1:1-2:0.6-1.2; the molar volume ratio of compound 1 and organic solvent is 1 mol:20-40 mL.

[0018] Preferably, the product obtained after the reaction is completed is sequentially extracted, washed, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain a lysosome-targeted biological thiol fluorescent probe of a pyrazoline derivative type.

[0019] The present invention also provides the use of the pyrazoline derivative-based lysosome-targeted biothiol fluorescent probe in the preparation of a product for detecting biothiol.

[0020] The beneficial effects of the present invention include:

[0021] 1) The present invention is based on the derivatization of pyrazoline as the structural parent nucleus, introducing a lysosome-targeted morpholine group to synthesize a novel lysosome-targeted blue fluorescent dye. At the same time, a recognition group with a specific reaction to biothiol is introduced, and a lysosome-targeted biothiol fluorescent probe of a pyrazoline derivative is constructed by means of a light-induced electron transfer (PET) mechanism. The fluorescent probe of the present invention has a strong electron-withdrawing property due to 2-chloro-3,5-dinitropyridine, which causes the light-induced electron transfer (PET) effect to be turned on. The fluorescent probe itself does not have fluorescent properties. When the probe is treated with biothiol, the biothiol has a strong nucleophilicity and can immediately undergo a nucleophilic substitution reaction with the probe molecule, resulting in the removal of the fluorescence quencher accompanied by the release of the blue fluorophore, thereby causing a change in the fluorescence signal. Strong fluorescence in the blue channel is observed at 470nm, achieving qualitative detection and quantitative analysis of biothiol.

[0022] 2) Through molecular design, the present invention has developed an organic small molecule structure that can specifically detect biothiols and target lysosomes. This allows for real-time monitoring of Cys, Hcy, and GSH concentration fluctuations within the lysosomes of living cells via fluorescence imaging, demonstrating advantages such as high selectivity, high sensitivity, and rapid response. The fluorescent probe of the present invention has a detection range of 1 to 200 μmol / L for Cys, Hcy, and GSH, with detection limits of 16.5 nmol / L (Cys), 16.8 nmol / L (Hcy), and 21.3 nmol / L (GSH), respectively. Its excellent selectivity and low cytotoxicity make it an ideal tool for the simultaneous detection of Cys, Hcy, and GSH.

[0023] 3) The fluorescent probe of the present invention has good cell membrane permeability and is capable of imaging endogenous and exogenous biothiols in living HeLa cells. The probe has been successfully applied to confocal imaging of biothiols in HeLa cell lysosomes. Therefore, the fluorescent probe of the present invention can be used to detect biothiols in aqueous solutions and complex biological systems, and is a promising molecular tool for studying the functions and synergistic effects of Cys, Hcy, and GSH in physiological processes. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a hydrogen nuclear magnetic resonance spectrum of the lysosome-targeted biological thiol fluorescent probe based on pyrazoline derivatives of Example 1;

[0025] Figure 2 This is the C NMR spectrum of the lysosome-targeted pyrazoline derivative biothiol fluorescent probe of Example 1;

[0026] Figure 3 The fluorescence spectra of the fluorescent probe of Example 1 after reacting with different concentrations of cysteine; wherein the concentrations of cysteine ​​are, from small to large, 0 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmol / L;

[0027] Figure 4 Graph showing the fluorescence intensity of the fluorescent probe of Example 1 at 470 nm versus cysteine ​​concentration;

[0028] Figure 5 : The fluorescence spectra of the fluorescent probe of Example 1 after reacting with different concentrations of homocysteine; wherein, the concentration of homocysteine ​​is 0 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmol / L from small to large;

[0029] Figure 64 is a graph showing the fluorescence intensity of the fluorescent probe of Example 1 at 470 nm and the concentration of homocysteine;

[0030] Figure 7 The fluorescence spectra of the fluorescent probe of Example 1 after reacting with different concentrations of glutathione; wherein the concentrations of glutathione are, from small to large, 0 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmol / L;

[0031] Figure 8 Graph showing the fluorescence intensity of the fluorescent probe at 470 nm and glutathione concentration in Example 1;

[0032] Figure 9 Schematic diagram of the selectivity and competitiveness of the fluorescent probe of Example 1 for biothiols at 470 nm; wherein a to z are sequentially: a is a blank group, b is Ca 2+ 、c is Mg 2+ d is Pb 2+ 、e is Cu 2+ 、f is Hg 2+ , g is Asp, h is Gly, i is Thr, j is Ser, k is Met, l is His, m is Phe, n is Gln, o is Glu, p is Val, q is Lys, r is Pro, s is Arg, t is NO3 - 、u is SO3 2- 、v is CO3 2- 、w is ClO4 - , x is GSH, y is Hcy, z is Cys;

[0033] Figure 10 This is a colocalization imaging image of HeLa cells cultured with the fluorescent probe of Example 1 and Lyso Tracker Red (a commercial lysosomal localization dye), where a is the red channel, b is the blue channel, c is the superimposed merge of the red channel, blue channel, and bright field, d is the bright field, e is the colocalization coefficient, and f is the corresponding intensity profile; Figure 10 The scale bars are all 10 μm;

[0034] Figure 11These are fluorescence imaging images of the fluorescent probe of Example 1 detecting endogenous biothiols and exogenous biothiols in HeLa cells; wherein A1 to A3 are bright field images, 405 nm excited fluorescence images, and overlay images of HeLa cells cultured with the fluorescent probe, respectively; B1 to B3 are bright field images, 405 nm excited fluorescence images, and overlay images of HeLa cells pre-treated with the thiol blocker NEM and then cultured with the fluorescent probe, respectively; C1 to C3 are bright field images, 405 nm excited fluorescence images, and overlay images of HeLa cells pre-treated with the thiol blocker NEM and then cultured with cysteine ​​and the fluorescent probe, respectively; D1 to D3 are bright field images, 405 nm excited fluorescence images, and overlay images of HeLa cells pre-treated with the thiol blocker NEM and then cultured with homocysteine ​​and the fluorescent probe, respectively; E1 to E3 are bright field images, 405 nm excited fluorescence images, and overlay images of HeLa cells pre-treated with the thiol blocker NEM and then cultured with glutathione and the fluorescent probe, respectively. DETAILED DESCRIPTION

[0035] The present invention provides a lysosome-targeted biothiol fluorescent probe based on a pyrazoline derivative. The structural formula of the biothiol fluorescent probe is:

[0036]

[0037] The present invention also provides a method for preparing the lysosome-targeted biological thiol fluorescent probe based on pyrazoline derivatives, comprising the following steps:

[0038] Compound 1, 2-chloro-3,5-dinitropyridine, a base and an organic solvent are mixed and reacted to obtain a pyrazoline derivative-type lysosome-targeted biothiol fluorescent probe;

[0039] The structural formula of the compound 1 is:

[0040]

[0041] In the present invention, the structural formula of 2-chloro-3,5-dinitropyridine is:

[0042]

[0043] In the present invention, the base is preferably sodium acetate, sodium carbonate, potassium carbonate, sodium hydroxide, triethylamine or ammonium acetate.

[0044] In the present invention, the organic solvent is preferably methanol, ethanol, acetonitrile, tetrahydrofuran or N,N-dimethylformamide.

[0045] In the present invention, the reaction temperature is preferably 25-100° C., more preferably 55-95° C., more preferably 80-90° C.; the reaction time is preferably 2-12 h, more preferably 4-10 h, more preferably 6-8 h.

[0046] In the present invention, the molar ratio of compound 1, 2-chloro-3,5-dinitropyridine and base is preferably 1:1-2:0.6-1.2, more preferably 1:1.2-1.8:0.7-1.0, and more preferably 1:1.4-1.5:0.8-0.9; the molar volume ratio of compound 1 and organic solvent is preferably 1 mol:20-40 mL, more preferably 1 mol:25-35 mL, and more preferably 1 mol:30 mL.

[0047] In the present invention, the product obtained after the reaction is completed is preferably separated and purified in sequence to obtain a lysosome-targeted biological thiol fluorescent probe of a pyrazoline derivative type; separation is preferably performed in sequence by extraction, washing, drying, and reduced pressure concentration, and purification is preferably performed by silica gel column chromatography.

[0048] In the present invention, the extraction reagent is preferably ethyl acetate, and the number of extractions is preferably 2 to 4 times, more preferably 3 times; the washing reagent is preferably saturated brine, and the number of washings is preferably 2 to 4 times, more preferably 3 times; the drying reagent is preferably anhydrous sodium sulfate; in the silica gel column chromatography purification, the volume ratio of dichloromethane to ethyl acetate is preferably 75 to 85:1, more preferably 78 to 82:1, and more preferably 80:1.

[0049] The present invention also provides the use of the pyrazoline derivative-based lysosome-targeted biothiol fluorescent probe in the preparation of a product for detecting biothiol.

[0050] In the present invention, a fluorescent probe is used to prepare a product for qualitatively and quantitatively detecting biothiols in an ethanol / PBS mixed solution (the volume ratio of ethanol to PBS is 3:7) or a biological system, and biothiols in cell lysosomes are successfully detected.

[0051] In the present invention, the biothiol fluorescent probe is dissolved in an ethanol PBS buffer solution, and cysteine, homocysteine ​​or glutathione in the PBS solution is quantitatively detected. In the PBS solution, the volume ratio of water to anhydrous ethanol is preferably 6-8:2-4, and more preferably 7:3.

[0052] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0053] In the embodiment, the structural formula of compound 1 is:

[0054]

[0055] Example 1

[0056] 0.5 mmol of compound 1, 0.75 mmol of 2-chloro-3,5-dinitropyridine, and 0.45 mmol of potassium carbonate were placed in 15 mL of acetonitrile and refluxed at 90°C for 6 h. After completion of the reaction, the reaction solution was added to 15 mL of water, extracted three times with ethyl acetate, and washed twice with saturated brine. The combined organic layers were dried over anhydrous sodium sulfate for 20 min and concentrated under reduced pressure at -0.1 MPa for 10 min to remove the solvent. The residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate, volume ratio 80:1) to obtain an orange solid product—a pyrazoline derivative-based lysosomal-targeted biothiol fluorescent probe.

[0057] The mass of the biothiol fluorescent probe of this embodiment is 150.1 mg, and the yield is 53.0%. The structural formula of the lysosome-targeted biothiol fluorescent probe of the pyrazoline derivative type is:

[0058]

[0059] The nuclear magnetic resonance hydrogen spectrum of the pyrazoline derivative lysosome-targeted biological thiol fluorescent probe of this embodiment is as follows: Figure 1 Shown: 1H NMR (600MHz, DMSO) δ9.33(d,J=2.3Hz,1H),9.27(d,J=2.3Hz,1H),7.71(d,J=7.3Hz,1H),7 .50(t,J=7.3Hz,1H),7.46–7.36(m,2H),7.02(t,J=7.8Hz,2H),6.91(d,J=8.5Hz,2H),6.7 8(d,J=8.6Hz,2H),6.65(t,J=7.2Hz,1H),6.59(d,J=8.1Hz,2H),5.28(dd,J=12.0,5.5Hz, 1H), 3.84 (dd, J=17.2, 12.1Hz, 1H), 3.71–3.67 (m, 4H), 3.05 (d, J=5.6Hz, 1H), 3.02 (s, 4H).

[0060] The nuclear magnetic resonance carbon spectrum of the pyrazoline derivative lysosome-targeted biological thiol fluorescent probe of this embodiment is as follows: Figure 2 Shown: 13C NMR (151 MHz, DMSO) δ 158.54, 148.82, 148.59, 143.99, 139.72, 133.07, 129.09, 126.91, 123.96, 119.26, 115.67, 113.23, 66.51, 62.07, 48.63, 44.68.

[0061] Example 2

[0062] 0.5 mmol of compound 1, 0.6 mmol of 2-chloro-3,5-dinitropyridine, and 0.4 mmol of sodium hydroxide were placed in 12.5 mL of methanol and refluxed at 55°C for 10 hours. After completion of the reaction, the reaction solution was added to 15 mL of water, extracted twice with ethyl acetate, and washed twice with saturated brine. The combined organic layers were dried over anhydrous sodium sulfate for 20 minutes and concentrated under reduced pressure at -0.1 MPa for 10 minutes to remove the solvent. The residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate, volume ratio 78:1) to obtain an orange solid product—a pyrazoline derivative-based lysosomal-targeted biothiol fluorescent probe.

[0063] The mass of the biothiol fluorescent probe of this embodiment is 132.3 mg, and the yield is 46.7%. The structural formula of the lysosome-targeted biothiol fluorescent probe of the pyrazoline derivative type is:

[0064]

[0065] Example 3

[0066] 0.5 mmol of compound 1, 0.9 mmol of 2-chloro-3,5-dinitropyridine, and 0.5 mmol of triethylamine were placed in 17.5 mL of N,N-dimethylformamide and refluxed at 80°C for 8 h. After completion of the reaction, the reaction solution was added to 18 mL of water, extracted four times with ethyl acetate, and washed three times with saturated brine. The combined organic layers were dried over anhydrous sodium sulfate for 20 min and concentrated under reduced pressure at -0.1 MPa for 10 min to remove the solvent. The residue was purified by silica gel column chromatography (dichloromethane:ethyl acetate, volume ratio 82:1) to obtain an orange solid product—a pyrazoline derivative-based lysosomal-targeted biothiol fluorescent probe.

[0067] The mass of the biothiol fluorescent probe of this embodiment is 110.7 mg, and the yield is 39.1%. The structural formula of the lysosome-targeted biothiol fluorescent probe of the pyrazoline derivative type is:

[0068]

[0069] Application Example 1 Spectral Determination of Biothiols by Fluorescent Probes

[0070] Fluorescence spectroscopy was used to compare the probe's reactivity with Cys, Hcy, and GSH, as well as its ability to quantitatively sense biothiols, to investigate the applicability of the fluorescent probe. The fluorescent probe prepared in Example 1 was dissolved in dimethyl sulfoxide to obtain a stock solution with a fluorescent probe concentration of 1 mmol / L. This stock solution was then added to a 10 mmol / L, pH 7.4 phosphate buffer solution (containing 30% ethanol by volume) to obtain a probe solution with a fluorescent probe concentration of 10 μmol / L. In a 3 mL quartz test tube, different concentrations of Cys, Hcy, and GSH (the concentrations of Cys, Hcy, and GSH were 0 μmol / L, 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L, 150 μmol / L, and 200 μmol / L, respectively) were placed in the above-mentioned probe solution, mixed evenly, and then tested.

[0071] The fluorescence spectra of the fluorescent probe of Example 1 after reacting with different concentrations of cysteine ​​are shown in FIG. Figure 3 As shown, the fluorescence spectra of the fluorescent probe of Example 1 after reacting with different concentrations of homocysteine ​​are shown in FIG. Figure 5 As shown, the fluorescence spectra of the fluorescent probe of Example 1 after reacting with different concentrations of glutathione are shown in FIG. Figure 7 As shown by Figure 3 、 Figure 5 、 Figure 7 It can be seen that when different concentrations of Cys, Hcy, and GSH are added to the probe solution, the fluorescence emission at 470 nm is gradually enhanced, and the fluorescence intensity increases by 16 times, 15 times, and 13 times, respectively. The fluorescence intensity of the fluorescent probe at 470 nm of Example 1 and the cysteine ​​concentration are shown in FIG. Figure 4 As shown in FIG. 1 , the fluorescence intensity of the fluorescent probe of Example 1 at 470 nm and the homocysteine ​​concentration are shown in FIG. Figure 6 As shown in FIG. 1 , the fluorescence intensity of the fluorescent probe of Example 1 at 470 nm and the glutathione concentration are shown in FIG. Figure 8 As shown. Figure 4 、 Figure 6 、 Figure 8 It can be seen that at 470 nm, the fluorescence intensity is linearly correlated with the concentration of Cys, Hcy, and GSH in the range of 1 to 10 μmol / L (Cys: y = 24.435x + 47.018, R 2 =0.9969;Hcy:y=24.735x+50.79,R 2=0.9936; GSH: y=18.506x+55.87, R 2 =0.9962), and the detection limits (LODs) of the fluorescent probe for Cys, Hcy, and GSH were calculated to be 16.5 nmol / L, 16.8 nmol / L, and 21.3 nmol / L, respectively, based on 3σ / k. Figures 3 to 8 It can be seen that pyrazoline derivatives-based lysosome-targeted biothiol fluorescent probes can be used as a promising molecular quantitative reagent to track Cys, Hcy and GSH.

[0072] Application Example 2: Selective and competitive testing of fluorescent probes for biothiols

[0073] Selectivity is one of the most important characteristics of fluorescent probes. In order to study the selectivity of fluorescent probes for Cys, Hcy, and GSH, the 10 μmol / L probe solution of Application Example 1 (the fluorescent probe of Example 1 was dissolved in dimethyl sulfoxide) was used to detect the selectivity of Ca2+, which was 200 μmol / L. 2+ Mg 2+ , Pb 2+ 、Cu 2+ 、Hg 2+ 、Aspartic acid (Asp), glycine (Gly), threonine (Thr), serine (Ser), methionine (Met), histidine (His), phenylalanine (Phe), glutamine (Gln), glutamic acid (Glu), valine (Val), lysine (Lys), proline (Pro), arginine (Arg), NO3 - 、SO3 2- 、CO3 2- 、ClO4 - , GSH, Hcy, and Cys were used for fluorescence response, and the results were as follows: Figure 9 As shown. Figure 9 It can be seen that the fluorescent probe of Example 1 only showed obvious fluorescence signal enhancement at 470 nm for Cys, Hcy, and GSH, while other analytes (Ca 2+ Mg 2+ , Pb 2+ 、Cu 2+ 、Hg 2+ ,Asp,Gly,Thr,Ser,Met,His,Phe,Gln,Glu,Val,Lys,Pro,Arg,NO3 - 、SO3 2- 、CO3 2- 、ClO4 - ) will not cause significant fluctuations in the fluorescence intensity of the fluorescent probe. Figure 9The competitive experiments showed that the fluorescent probe can detect Cys, Hcy, and GSH even in the presence of coexisting species (analyte + GSH, analyte + Hcy, analyte + Cys, and the analyte is Ca 2+ Mg 2+ , Pb 2+ 、Cu 2+ 、Hg 2+ ,Asp,Gly,Thr,Ser,Met,His,Phe,Gln,Glu,Val,Lys,Pro,Arg,NO3 - 、SO3 2- 、CO3 2- 、ClO4 - ), it also has excellent anti-interference ability, indicating that the fluorescent probe of the present invention has the ability to detect Cys, Hcy, and GSH in complex biological systems.

[0074] Application Example 3: Cellular Localization Test of Fluorescent Probes

[0075] In order to study the lysosomal targeting performance of the fluorescent probe, a commercial lysosomal dye (LysoTracker Red, 1 μmol / L) was used in combination with the fluorescent probe of Example 1 (10 μmol / L) at a density of 1×10 5 Colocalization test was performed in HeLa cells, as shown in Figure 10 As shown. Figure 10 It can be seen that the blue channel of the fluorescent probe and the fluorescence image of Lyso Tracker Red overlap well, and the colocalization coefficient is calculated to be 0.95. These results show that the fluorescent probe of the present invention can be well localized in the lysosomes of living cells.

[0076] Application Example 4: Cellular Imaging Test of Exogenous and Endogenous Biothiols Using Fluorescent Probes

[0077] The density is 1×10 5 After the HeLa cells were incubated with the 10 μmol / L probe solution of Application Example 1, the bright field image, 405 nm excitation fluorescence image (blue channel) and overlay image were as follows: Figure 11 As shown in A1, A2 and A3 in Figures A1 to A3, fluorescent signals are emitted in the blue channel, indicating that the fluorescent probe prepared in Example 1 can enter HeLa cells and interact with endogenous biological thiols. After incubating HeLa cells with 1 mmol / L thiol blocker N-ethylmaleimide (NEM) for 30 minutes, they were co-incubated with 10 μmol / L probe solution of Application Example 1. The bright field image, 405 nm excitation fluorescence image (blue channel) and superimposed image are shown as follows: Figure 11As shown in Figure B1, Figure B2 and Figure B3. As can be seen from Figures B1 to B3, no fluorescence signal was found in the blue channel. After HeLa cells were pretreated with 1mmol / L thiol blocker NEM, cysteine, homocysteine ​​and glutathione were added respectively, and then the probe solution of Example 1 was added and incubated continuously. The bright field image, 405nm excitation fluorescence image (blue channel) and superimposed image of HeLa cells incubated with 100μmol / L cysteine ​​and the fluorescent probe of Example 1 are shown as follows: Figure 11 As shown in C1, C2 and C3 in the figure; the bright field image, 405nm excited fluorescence image (blue channel) and superimposed image of HeLa cells incubated with 100μmol / L homocysteine ​​and the fluorescent probe of Example 1 are shown respectively. Figure 11 As shown in D1, D2 and D3 in the figure; the bright field image, 405nm excited fluorescence image (blue channel) and superimposed image of HeLa cells incubated with 100μmol / L glutathione and the fluorescent probe of Example 1 are shown in FIG. Figure 11 As shown in E1, E2 and E3. Figure 11 As shown in Figures C1-C3, D1-D3, and E1-E3, bright fluorescence signals were observed in the blue channel of HeLa cells incubated with cysteine, homocysteine, and glutathione. These results indicate that the fluorescent probe of the present invention can detect endogenous and exogenous biothiols in living HeLa cells.

[0078] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A pyrazoline derivative-based lysosomal-targeted biothiol fluorescent probe, characterized in that: The structural formula of the biothiol fluorescent probe is: 。 2. The method for preparing the lysosomal-targeted biothiol fluorescent probe of pyrazoline derivatives according to claim 1, characterized in that: The following steps are included: Compound 1, 2-chloro-3,5-dinitropyridine, a base and an organic solvent are mixed and reacted to obtain a pyrazoline derivative-type lysosome-targeted biothiol fluorescent probe; The structural formula of the compound 1 is: 。 3. The preparation method according to claim 2, characterized in that The base is sodium acetate, sodium carbonate, potassium carbonate, sodium hydroxide, triethylamine or ammonium acetate.

4. The preparation method according to claim 2 or 3, characterized in that The organic solvent is methanol, ethanol, acetonitrile, tetrahydrofuran or N,N-dimethylformamide.

5. The preparation method according to claim 4, characterized in that The reaction temperature is 25-100°C, and the reaction time is 2-12 hours.

6. The preparation method according to claim 2 or 5, characterized in that The molar ratio of compound 1, 2-chloro-3,5-dinitropyridine and base is 1:1~2:0.6~1.2; the molar volume ratio of compound 1 and organic solvent is 1 mol:20~40 mL.

7. The preparation method according to claim 6, characterized in that After the reaction is completed, the product obtained is sequentially extracted, washed, dried, concentrated under reduced pressure, and purified by silica gel column chromatography to obtain a lysosome-targeted biological thiol fluorescent probe of a pyrazoline derivative.

8. Use of the pyrazoline derivative-based lysosome-targeted biothiol fluorescent probe according to claim 1 in the preparation of a product for detecting biothiols, wherein the purpose of the use is not to diagnose or treat a disease.

Images