A small-molecule fluorescent probe based on coumarin-quinoline structure

Abstract

The application discloses a kind of design, synthesis and application of small molecule fluorescent probe based on coumarin-quinoline structure. Probe is named SDS-Bio, by coupling coumarin unit with quinolinium cation through α, β-unsaturated carbonyl group (as reaction site), the detection of biological thiol is realized. In the molecular structure, coumarin group is as luminophore, and quinolinium cation enhances water solubility, and improves the electrophilicity of β-carbon, so as to enhance the sensitivity of probe to biological thiol. When thiol group is attacked by 1,4-michael addition, the extended π-conjugated system is destroyed, and the intramolecular charge transfer process is blocked, resulting in the maximum absorption peak from 480 nm blue shift to 450 nm, while presenting significant fluorescence opening response at 495 nm. The probe exhibits short response time, high sensitivity, and has superior selectivity to cysteine, homocysteine and glutathione than other detection materials. In addition, experimental results prove that the probe has important application value in the study of ferroptosis, a regulatory cell death pathway.

Description

[0001] This invention belongs to the field of synthesis and detection technology, specifically relating to the design, synthesis and application of a small molecule fluorescent probe based on a coumarin-quinoline structure. Background Technology

[0002] Biothiols, including cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH), are essential low-molecular-weight thiol compounds in biological systems. They maintain intracellular redox balance, scavenge reactive oxygen species, protect cells from oxidative damage, and play a key role in disease pathogenesis. Abnormal biothiol metabolism is closely related to a variety of diseases. For example, cysteine ​​deficiency easily leads to oxidative stress and immune dysfunction; elevated homocysteine ​​levels are closely related to cardiovascular and cerebrovascular diseases; and glutathione metabolism disorders are closely related to various cancers (including lung cancer and prostate cancer).

[0003] Currently, methods for detecting biothiols include high-performance liquid chromatography (HPLC), mass spectrometry (MS), colorimetry, and electrochemical methods. Among these, fluorescence-based methods have become a research hotspot due to their superior sensitivity, real-time monitoring capabilities, and non-invasive operation. However, existing biothiol fluorescent probes often suffer from poor water solubility, slow response kinetics, insufficient sensitivity, and susceptibility to interference from other sulfur compounds, making them unsuitable for detection in complex biological systems. Therefore, developing a novel biothiol fluorescent probe with fast response, high sensitivity, good selectivity, and excellent biocompatibility has significant application value. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying a fluorescent probe for the detection of biothiols. The probe is named SDS-Bio, which solves the problems of poor water solubility, slow response, and poor selectivity of existing probes. At the same time, it has good biocompatibility and can realize the detection and imaging of biothiols in living cells.

[0005] The technical solution of the present invention is as follows: A biothiol detection probe, wherein the probe uses a coumarin group as the luminescent group, a quinolineonium cation as the water-soluble enhancing group, and an α,β-unsaturated carbonyl group as the biothiol-specific response site, and the chemical structure is shown below. .

[0006] The preparation method of the above-mentioned bio-thiol detection probe includes the following steps: (1) 4-(diethylamino)-2-hydroxybenzaldehyde and ethyl acetoacetate were reacted with pyridine catalysis and nitrogen protection by heating and reflux in anhydrous ethanol. After cooling, the mixture was filtered under reduced pressure to obtain a golden yellow solid compound 1. (2) Compound 1 and 3-quinoline carbaldehyde were reacted under piperidine catalysis and nitrogen protection in anhydrous ethanol under reflux. The solvent was removed by rotary evaporation and purified by column chromatography to obtain red solid compound 2. (3) Add methyl trifluoromethanesulfonate dropwise to anhydrous dichloromethane of compound 2 at room temperature, stir the reaction, filter, wash and dry to obtain yellow solid target probe SDS-Bio.

[0007] Preferably, in step (1), 4-(diethylamino)-2-hydroxybenzaldehyde, anhydrous ethanol, and ethyl acetoacetate are added sequentially into a round-bottom flask, and anhydrous piperidine is added dropwise. The mixture is refluxed at 80°C and stirred for 16 hours under nitrogen protection. After cooling, the reaction solution is filtered under reduced pressure to obtain a golden yellow solid compound 1.

[0008] Preferably, in step (2), compound 1 and 3-quinoline carbaldehyde are dissolved in anhydrous ethanol, piperidine is added dropwise, and the mixture is refluxed at 80°C for 16 hours under nitrogen protection. The solvent is then removed by rotary evaporation under reduced pressure. The resulting residue is purified by column chromatography using a mixture of dichloromethane and ethyl acetate as the eluent at a volume ratio of 15:1 to obtain a red solid compound 2. The molar ratio of compound 1 to 3-quinoline carbaldehyde is 1.5:1.

[0009] Preferably, according to the preparation method of claim 2, in step (3), compound 2 is dissolved in anhydrous dichloromethane, methyl trifluoromethanesulfonate is slowly added dropwise at room temperature, the mixture is stirred and reacted for 6 hours, filtered under reduced pressure, the resulting solid is washed with cold dichloromethane, and dried to obtain a yellow solid target probe SDS-Bio. The molar ratio of compound 2 to methyl trifluoromethanesulfonate is 1:7.08.

[0010] Application of the above-mentioned fluorescent probe or the fluorescent probe prepared by the above-mentioned preparation method in the detection of biothiols.

[0011] Preferably, the solvent environment of the biothiol includes cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH).

[0012] Furthermore, the solvent environment for detecting biothiols was a mixture of dimethyl sulfoxide (DMSO) and PBS buffer at a volume ratio of 3:7, a pH of 7.4, and a concentration of 10 mM.

[0013] Furthermore, the final test concentration of the probe in the solution was 10 μM, and it was incubated at 37°C for 30 min before detection. The excitation wavelength for fluorescence detection was 450 nm, and the emission wavelength was 495 nm.

[0014] Furthermore, the probe is used for imaging endogenous and exogenous biothiols in A549 cells and for monitoring the dynamic fluctuations of intracellular biothiols during Erastin-induced ferroptosis, which has application value in studying this regulatory cell death pathway.

[0015] Beneficial effects of the present invention (1) The structural design is reasonable and solves the problem of poor water solubility of traditional probes; (2) Fast response speed and high sensitivity; (3) Excellent selectivity; (4) The preparation method is simple and the reaction conditions are mild; (5) The probe has excellent biocompatibility and low cytotoxicity. Attached Figure Description

[0016] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments of this application and their descriptions are used to explain this application and do not constitute an undue limitation of this application.

[0017] Figure 1 These are preliminary spectral results of the response of SDS-Bio (10 μM) to biothiols (300 μM). (a) is the UV-Vis spectrum, and (b) is the fluorescence spectrum. Figure 2 These are fluorescence titration experiments of SDS-Bio (10 μM) in response to biothiols (0-500 μM). Among them, (a) is a fluorescence titration experiment in response to cysteine, (b) is a fluorescence titration experiment in response to homocysteine, and (c) is a fluorescence titration experiment in response to glutathione. Figure 3 The linear relationship between fluorescence intensity and concentration of SDS-Bio (10 μM) in response to biothiols (0-10 μM) is shown. Among them, (a) is the linear relationship between fluorescence intensity and concentration in response to cysteine, (b) is the linear relationship between fluorescence intensity and concentration in response to homocysteine, and (c) is the linear relationship between fluorescence intensity and concentration in response to glutathione. Figure 4 This is a time-dependent assay of SDS-Bio (10 μM) response to biothiols (50 / 500 μM). (a) shows the change in fluorescence intensity over time in response to cysteine, (b) shows the change in fluorescence intensity over time in response to homocysteine, and (c) shows the change in fluorescence intensity over time in response to glutathione. Figure 5 The fluorescence spectra of SDS-Bio (10 μM) in response to other analytes (500 μM) and biothiols are shown. Figure 6The fluorescence spectra of SDS-Bio (10 μM) in response to biothiols (500 μM) under different pH conditions; Figure 7 The cytotoxicity assays were performed on SDS-Bio at different concentrations (0, 10, 20, 30, 40 μM). Figure 8 This is a fluorescence image of biothiols in A549 cells; Figure 9 This is a fluorescence image of biothiol fluctuations during Erastin-induced ferroptosis in A549 cells; Figure 10 The fluorescence intensity for cell imaging is (±SD, n=3). Detailed Implementation

[0018] The present invention will be further described below with reference to the embodiments. However, the scope of protection of the present invention is not limited to the following embodiments.

[0019] Example 1: Synthesis of probe SDS-Bio (1) Synthesis of Compound 1 4-(diethylamino)-2-hydroxybenzaldehyde (3.86 g, 20 mmol) was added to a 150 mL round-bottom flask, followed by 50 mL of anhydrous ethanol, ethyl acetoacetate (3.12 g, 24 mmol), and 5 drops of anhydrous piperidine. The mixture was refluxed and stirred at 80 °C for 16 hours under nitrogen protection. After cooling, the reaction solution was filtered under reduced pressure to give a golden solid, compound 1 (3.96 g, 76.4% yield). ¹H NMR (600 MHz, DMSO) δ 8.47 (s, 1H), 7.64 (d, J = 9.0Hz, 1H), 6.78 (dd, J = 9.0, 2.4 Hz, 1H), 6.56 (d, J = 2.4 Hz, 1H), 3.48 (q, J= 7.2 Hz, 4H), 2.49 (s, 3H), 1.13 (t, J = 7.2 Hz, 6H). ¹³C NMR (151 MHz, DMSO-d6) δ 194.69, 160.34, 158.74, 153.42, 148.12, 132.90, 115.45, 110.59, 107.94,96.25, 44.89, 30.60, 12.80. HRMS m / z: calculated for C 15 H 17 NO3 + [M+H] +:260.1287, found: 260.1285; (2) Synthesis of Compound 2 Compound 1 (1.94 g, 7.5 mmol) and 3-quinoline carbaldehyde (785 mg, 5 mmol) were placed in a 50 mL round-bottom flask and dissolved in 20 mL of anhydrous ethanol. Three drops of piperidine were added dropwise to the reaction mixture, and the mixture was refluxed at 80 °C for 16 h under nitrogen protection. After cooling, the solvent was removed by rotary evaporation under reduced pressure. The residue was purified by column chromatography (dichloromethane / ethyl acetate = 15 / 1, v / v) to give compound 2 (796 mg, 40% yield) as a red solid. 1 H NMR (600 MHz, CDCl3) δ 9.19(d, J = 2.1 Hz, 1H), 8.59 (s, 1H), 8.42 (d, J = 2.1 Hz, 1H), 8.38 (d, J =15.9 Hz, 1H), 8.11 (dd, J = 8.4, 1.2 Hz, 1H), 7.96 (d, J = 15.9 Hz, 1H), 7.91– 7.86 (m, 1H), 7.74 (ddd, J = 8.4, 6.9, 1.5 Hz, 1H), 7.58 (ddd, J = 8.0,6.9, 1.2 Hz, 1H), 7.45 (d, J = 9.0 Hz, 1H), 6.65 (dd, J = 9.0, 2.4 Hz, 1H), 6.51 (d, J = 2.4 Hz, 1H), 3.48 (q, J = 7.2 Hz, 4H), 1.26 (t, J = 7.2 Hz, 6H). 13 C NMR (151 MHz, CDCl3) δ186.26, 161.19, 158.99, 153.35, 150.62, 149.21,148.75, 139.62, 135.51, 132.14, 130.56, 129.55, 128.64, 128.60, 127.95,127.38, 126.93, 116.41, 110.17, 108.87, 96.82, 45.38, 12.63. HRMS m / z:calculated for C 25 H 22 N2O3 + [M+H] + : 399.1703, found: 399.1705; (3) Synthesis of probe SDS-Bio Compound 2 (500 mg, 1.25 mmol) was dissolved in 15 mL of anhydrous dichloromethane and placed in a 25 mL round-bottom flask. Methyl trifluoromethanesulfonate (1 mL, 8.85 mmol) was slowly added dropwise at room temperature, and the mixture was stirred for 6 h. After the reaction was complete, the mixture was filtered under reduced pressure, and the resulting solid was washed with cold dichloromethane and dried to obtain a yellow solid target probe, SDS-Bio (326 mg, 46% yield). 1 H NMR (600 MHz, DMSO- d 6) δ 9.95 (d, J = 1.8 Hz, 1H), 9.59 – 9.56 (m,1H), 8.66 (s, 1H), 8.50 (ddd, J = 18.6, 8.4, 1.2 Hz, 2H), 8.33 – 8.26 (m,2H), 8.08 (ddd, J = 8.0, 6.9, 0.9 Hz, 1H), 7.86 (d, J = 15.9 Hz, 1H), 7.72(d, J = 9.0 Hz, 1H), 6.85 (dd, J = 9.0, 2.4 Hz, 1H), 6.64 (d, J = 2.4 Hz,1H), 4.67 (s, 3H), 3.52 (q, J = 7.2 Hz, 4H), 1.16 (t, J = 7.2 Hz, 6H). 13C NMR (151 MHz, DMSO-) d 6) δ 185.59, 160.43, 158.98, 153.90, 150.75, 149.49, 145.00,138.42, 136.41, 135.37, 133.18, 131.35, 130.93, 130.38, 129.58, 129.43,119.74, 115.12, 111.03, 108.53, 96.49, 46.05, 45.05, 12.86. HRMS m / z:calculated for C 26 H 25 N2O3 + [M] + : 413.1860, found: 413.1858.

[0020] Example 2: Solution preparation and spectral testing conditions (1) Preparation of stock solution Probe SDS-Bio stock solution: Weigh 2.83 mg of probe solid powder and dissolve it completely in 503 μL of DMSO to prepare a 10 mM stock solution; PBS buffer: Dissolve 2L of phosphate buffer powder in ultrapure water, and after adjusting the volume, prepare a series of buffer solutions with pH values ​​of 4.0-10.0 for later use; Selective analyte stock solutions: Cys, Hcy, GSH, methionine (Met), glutamic acid (Glu), leucine (Leu), histidine (His), arginine (Arg), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), KCl, NaF, KBr, NaCl, FeCl2, ZnCl2, CuSO4, and NaHS were all prepared to 10 mM using ultrapure water; (2) Spectral testing conditions The final test concentration of the probe SDS-Bio was 10 μM, and all spectroscopic experiments were performed in DMSO / PBS buffer (v:v = 3:7, 10 mM, pH 7.4) at 37 °C for 30 minutes. The excitation wavelength was fixed at 450 nm, and the emission wavelength was recorded in the range of 465–550 nm. The excitation slit width was 5 nm; the emission slit width was 10 nm; and the excitation voltage was 800 V.

[0021] Example 3: Cell Culture and Cytotoxicity Experiments A549 cells were cultured and incubated in RPMI-1640 medium containing 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin mixture. Cells were cultured in a constant temperature incubator at 37 °C and 5% CO2. The cytotoxic effect of SDS-Bio on A549 cells was tested using the MTT assay: A549 cells treated with trypsin-EDTA digestion solution containing phenol red were divided into groups of 6 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 hours. SDS-Bio solution with final concentrations of 0, 10, 20, 30, and 40 µM was then added, and incubation continued for another 24 hours. The culture medium was removed, and MTT chromogenic reagent (5 mg / L) was added to each well. After reacting for 4 hours, the supernatant was aspirated, and 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the generated formazan crystals. Finally, the absorbance of each well was measured at 570 nm using a microplate reader, and cell viability was calculated. The results showed that cell viability exceeded 80% in the concentration range of 10 to 40 μM, demonstrating that SDS-Bio has low cytotoxicity, good biocompatibility, and can be applied to live cell imaging experiments.

[0022] Example 4: Cell Imaging Experiment (1) Cell imaging of endogenous and exogenous biothiols A549 cells were seeded in 6-well plates (1.2 × 10⁶ cells per well). 5 (100 cells) were cultured for 24 hours, and the experiment was divided into five groups: Group 1: Cells were incubated with SDS-Bio (10 μM) for 30 minutes before imaging; Group 2: Cells were incubated sequentially with medium containing NEM (thiol scavenger) (200 μM) and then with medium containing probe SDS-Bio (10 μM) for 30 minutes. Groups 3-5: Cells were incubated sequentially with medium containing NEM (200 μM), medium containing Cys / GSH / Hcy (500 μM), and medium containing SDS-Bio (10 μM), for 30 minutes each. After washing three times with PBS, each group was imaged using an inverted fluorescence microscope with an excitation wavelength of 450 nm and an emission wavelength of 495 nm. The results showed that group 1 exhibited a significant fluorescence signal, indicating that the probe responded to endogenous biothiols. However, the fluorescence signal in group 2 was significantly reduced, indicating that NEM effectively removed endogenous biothiols. The fluorescence intensity in groups 3-5 recovered significantly. These results demonstrate that the SDS-Bio probe can specifically detect endogenous biothiols in living cells and maintain its responsiveness to exogenous thiols. (2) Cellular imaging of ferroptosis induced by Erastin A549 cells were seeded in 6-well plates (1.2 × 10⁶ cells per well). 5 (100 cells) were cultured for 24 h, and the experiment was divided into three groups: Group 1: Cells were incubated in a medium containing SDS-Bio (10 µM) for 30 min; Group 2: First, treat with medium containing Erastin (5 µM) for 4 h, then treat with medium containing SDS-Bio (10 µM) for 30 min; Group 3: First, treat with medium containing Erastin (5 µM) for 4 h, then treat with medium containing Ferrostatin-1 (5 µM) for 4 h, and finally treat with medium containing SDS-Bio (10 µM) for 30 min. After washing, fluorescence imaging and quantitative analysis showed that the fluorescence signal in group 2 was significantly weaker than that in group 1, suggesting the depletion of intracellular biothiols during Erastin-induced ferroptosis. The fluorescence signal in group 3 recovered compared to group 2, indicating that Fer-1 can effectively promote the recovery of intracellular biothiols. These results confirm that SDS-Bio can serve as an effective tool for monitoring fluctuations in biothiol levels during ferroptosis.

[0023] Example 5: Fluorescence spectral changes of the reaction between probe SDS-Bio and biothiols over time. Take 20 μL of the probe stock solution from Example 2 and add it to a 5 mL centrifuge tube. Add 580 μL of DMSO and 10 / 100 μL of a 10 mmol / L Cys / Hcy / GSH solution. Then, dilute to 2 mL with 10 mmol / L PBS buffer (pH 7.4) to prepare a test solution with a probe concentration of 10 μmol / L, a Cys / Hcy / GSH concentration of 50 / 500 μM, and a DMSO:PBS volume ratio of 3:7. Measure the fluorescence spectrum over time using an excitation wavelength of 450 nm. Figure 4 As shown, the fluorescence at 495 nm gradually increased and then remained stable with increasing time, reaching its maximum value at 30 s, 200 s and 600 s, respectively.

[0024] Example 6: Selectivity study of probe SDS-Bio against different interfering substances Take 20 μL of the probe stock solution from Example 2 and add it to a 5 mL centrifuge tube. Add 580 μL of DMSO and then add less than 100 μL of each of the following analytes (interferant stock solution concentration: 10 mmol / L; cysteine, glutathione, and homocysteine ​​concentrations: 10 mmol / L): Cl- HS - , Br - , F - Zn 2+ , K + Fe 2+ Cu 2+ Met, Glu, Leu, His, Arg, Phe, Pro, Thr, Ser, Cys, GSH, Hcy were then added and diluted to 2 ml with PBS buffer (10 mmol / L, pH 7.4) to prepare a probe concentration of 10 μmol / L and an analyte concentration of 500 μmol / L. The fluorescence spectrum of the test solution was detected after 30 min of reaction. Figure 5 It was observed that the fluorescence intensity of the test solutions did not change significantly upon the addition of various ions and amino acids. However, the fluorescence intensity of the test solutions containing cysteine, glutathione, and homocysteine ​​was significantly enhanced. The experimental results indicate that the probe SDS-Bio has good selectivity for biothiols. Subsequently, PBS buffer solution (concentration 10 mmol / L, pH=7.4) was used to make up to 2 ml to prepare a probe concentration of 10 μmol / L and an analyte concentration of 500 μmol / L. After reacting for 30 min, the fluorescence spectrum changes of the test solutions were detected. Figure 5 It was observed that the fluorescence intensity of the test solution did not change significantly upon the addition of various ions and amino acids. However, the fluorescence intensity of the test solution significantly increased upon the addition of cysteine, glutathione, and homocysteine. These experimental results demonstrate that the probe SDS-Bio exhibits excellent selectivity for biothiols.

[0025] Example 7: Fluorescence spectral changes of probe SDS-Bio before and after reaction with biothiols at different pH values Take 20 μL of the probe stock solution from Example 2 and add it to a 5 mL centrifuge tube. Add 580 μL of DMSO, 100 μL of a 10 mmol / L Cys / Hcy / GSH solution, and then add PBS buffer solutions of different pH values ​​to bring the volume to 2 mL to prepare a probe concentration of 10 μmol / L and a Cys / Hcy / GSH concentration of 500 μmol / L. After reacting for 30 min, test the fluorescence spectrum change of the probe using a fluorescence spectrometer (excitation wavelength 450 nm). Figure 6 As shown, without the addition of Cys / Hcy / GSH, the fluorescence intensity of the probe SDS-Bio did not change significantly in the pH range of 4.0-10.0; after the addition of Cys / Hcy / GSH, the fluorescence intensity changed significantly in the pH range of 7.0-10.0, and reached its peak at around pH 7.4.

[0026] Example 8: Cell Culture and Cytotoxicity Experiments A549 cells were cultured in RPMI-1640 medium containing 10% (v / v) fetal bovine serum and 1% penicillin-streptomycin mixture. Cells were cultured in a constant temperature incubator at 37 °C and 5% CO2. The cytotoxicity of SDS-Bio to A549 cells was tested using the MTT assay: A549 cells treated with trypsin-EDTA digestion solution containing phenol red were cultured at a density of 6 × 10⁶ cells / well. 4 Cells were seeded at a density of [number] cells / well in 96-well plates and cultured for 24 hours. SDS-Bio solution was then added to final concentrations of 0, 10, 20, 30, and 40 μmol / L, and incubated for another 24 hours. The culture medium was removed, and MTT chromogenic reagent (5 mg / L) was added to each well. After reacting for 4 hours, the supernatant was aspirated, and 100 µL of dimethyl sulfoxide (DMSO) was added to dissolve the generated formazan crystals. Finally, the absorbance of each well was measured at 570 nm using a microplate reader, and cell viability was calculated. The results showed that the cell viability exceeded 80% in the concentration range of 10 to 40 μmol / L, which proves that SDS-Bio has low cytotoxicity, good biocompatibility, and can be applied to live cell imaging experiments.

[0027] Example 9: Cellular Imaging of Endogenous and Exogenous Biothiols A549 cells were seeded in 6-well plates (1.2 × 10⁶ cells per well). 5 (100 cells) were cultured for 24 h, and the experiment was divided into five groups: Group 1: Cells were incubated with SDS-Bio (10 μmol / L) for 30 min before imaging; Group 2: Cells were incubated sequentially with medium containing NEM (thiol scavenger) (200 μmol / L) and then with medium containing probe SDS-Bio (10 μmol / L) for 30 min. Groups 3-5: Cells were incubated sequentially with medium containing NEM (200 μmol / L), medium containing Cys / GSH / Hcy (500 μmol / L), and medium containing SDS-Bio (10 μmol / L), for 30 min each time. After washing three times with PBS, each group was imaged using an inverted fluorescence microscope. The results showed that group 1 exhibited a significant fluorescence signal, indicating that the probe responded to endogenous biothiols. However, group 2 showed a significantly reduced fluorescence signal, indicating that NEM effectively removed endogenous biothiols. The fluorescence intensity of groups 3-5 recovered significantly. These results demonstrate that the SDS-Bio probe can specifically detect endogenous biothiols in living cells and maintain its responsiveness to exogenous thiols.

[0028] The coumarin-quinoline-based small molecule fluorescent probe SDS-Bio of this invention features a simple preparation method and a rationally designed structure, overcoming the problem of poor water solubility in traditional probes. This probe exhibits short response time, high sensitivity, and superior selectivity for cysteine, homocysteine, and glutathione compared to other detectable substances. Furthermore, in vitro experiments have confirmed its successful application in imaging endogenous and exogenous biothiols in A549 cells. More importantly, it can monitor the dynamic fluctuations of intracellular biothiols during Erastin-induced ferroptosis, highlighting its potential application in studying this regulatory cell death pathway.

[0029] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A small molecule fluorescent probe SDS-Bio based on a coumarin-quinoline structure, characterized in that, The structure of the fluorescent probe is shown below:

2. The method for preparing the small molecule fluorescent probe based on the coumarin-quinoline structure according to claim 1, characterized in that, Includes the following steps: ； (1) Synthesis of Compound 1 4-(diethylamino)-2-hydroxybenzaldehyde was reacted with ethyl acetoacetate under pyridine catalysis and nitrogen protection in anhydrous ethanol under reflux. After cooling, the mixture was filtered under reduced pressure to obtain a golden yellow solid compound 1. (2) Synthesis of Compound 2 Compound 1 was reacted with 3-quinoline carbaldehyde under piperidine catalysis and nitrogen protection by heating and reflux in anhydrous ethanol. The solvent was removed by rotary evaporation and purified by column chromatography to obtain compound 2 as a red solid. (3) Synthesis of probe SDS-Bio Compound 2 was added dropwise to anhydrous dichloromethane at room temperature with methyl trifluoromethanesulfonate. After stirring and reacting, the mixture was filtered, washed, and dried to obtain a yellow solid target probe, SDS-Bio.

3. The preparation method according to claim 2, characterized in that, In step (1), 4-(diethylamino)-2-hydroxybenzaldehyde, anhydrous ethanol, and ethyl acetoacetate were added sequentially to a round-bottom flask, and anhydrous piperidine was added dropwise. The mixture was refluxed at 80°C and stirred for 16 hours under nitrogen protection. After cooling, the reaction solution was filtered under reduced pressure to obtain a golden-yellow solid compound 1. The molar ratio of 4-(diethylamino)-2-hydroxybenzaldehyde to ethyl acetoacetate was 1:1.

2.

4. The preparation method according to claim 2, characterized in that, Step (2) Compound 1 and 3-quinoline carbaldehyde were dissolved in anhydrous ethanol, piperidine was added dropwise, and the mixture was refluxed at 80°C for 16 hours under nitrogen protection. The solvent was removed by rotary evaporation under reduced pressure. The residue was purified by column chromatography using a mixture of dichloromethane and ethyl acetate as the eluent at a volume ratio of 15:1 to obtain a red solid compound 2. The molar ratio of compound 1 to 3-quinoline carbaldehyde was 1.5:

1.

5. The preparation method according to claim 2, characterized in that, In step (3), compound 2 was dissolved in anhydrous dichloromethane, and methyl trifluoromethanesulfonate was slowly added dropwise at room temperature. The mixture was stirred for 6 hours, filtered under reduced pressure, and the resulting solid was washed with cold dichloromethane and dried to obtain the yellow solid target probe SDS-Bio. The molar ratio of compound 2 to methyl trifluoromethanesulfonate was 1:7.

08.

6. The application of the fluorescent probe according to claim 1 or the fluorescent probe prepared by the preparation method according to claims 2-5 in the detection of biothiols.

7. The application according to claim 6, characterized in that, The solvent environment of the biothiols includes cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH).

8. The application according to claim 6, characterized in that, The solvent environment for detecting biothiols is a mixture of dimethyl sulfoxide (DMSO) and PBS buffer at a volume ratio of 3:7, pH 7.4, and concentration of 10 mM.

9. The application according to claim 8, characterized in that, The final test concentration of the probe in the solution was 10 μM. Before detection, it was incubated at 37°C for 30 minutes. The excitation wavelength for fluorescence detection was 450 nm, and the emission wavelength was 495 nm.

10. The application according to claim 6, characterized in that, The probe is used for imaging endogenous and exogenous biothiols in A549 cells and for monitoring the dynamic fluctuations of intracellular biothiols during Erastin-induced ferroptosis, and has application value in studying this regulatory cell death pathway.

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