A biological thiol fluorescent small molecule probe and its preparation method and application

Abstract

The present invention discloses an ultra-sensitive fluorescent small molecule probe for biothiols, a preparation method thereof, and an application thereof. The present invention proposes a dual fluorescence quenching mechanism combining the PET effect and the "ester to carboxylic acid" conversion strategy to construct an MCB-NBD probe with "zero" intrinsic fluorescence. The small molecule fluorescent probe MCB-NBD of the present invention overcomes the problems of high background fluorescence, low sensitivity, and high detection limit of existing small molecule fluorescent probes. The fluorescent small molecule probe MCB-NBD of the present invention exhibits a very low detection limit for biothiols. When 200nM biothiols are added, the fluorescence response multiple exceeds 3 times, and it can significantly distinguish between liver-damaged plasma and normal plasma at 80-fold dilution. The fluorescent small molecule probe MCB-NBD of the present invention monitors the fluctuation of biothiols in dihydroartemisinin-induced ferroptosis, reflecting the application prospect of the present invention in studying new strategies for clinical tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic fluorescent probes, and relates to a biothiol fluorescent small molecule probe and a preparation method and application thereof, and specifically relates to an ultra-sensitive biothiol fluorescent small molecule probe with zero intrinsic fluorescence and high signal-to-background ratio. Background Art

[0002] Abnormal activity or concentration of biomarkers in cells, tissues, and blood can lead to a range of bodily dysfunctions. Early detection of abnormal biomarker levels is crucial for understanding their function and early disease management. Analyte-activated fluorescent small molecule probes are powerful tools for detecting and imaging biomarkers in living systems due to their unique advantages of rapid analysis, real-time, and non-invasive imaging.

[0003] However, many probes often face problems such as insufficient sensitivity and accuracy, and low response times, which are caused by the intrinsic fluorescence of the probes in the detection system. It is still of great significance to design fluorescent small molecule probes with low background fluorescence and high signal-to-noise ratio to deal with complex biological systems. At present, through strategies such as protein- or lipid-assisted fluorescence signal amplification, generation or modification of conjugated structures, the fluorescence signal after response can be significantly enhanced or the intrinsic fluorescence of the probe can be significantly reduced, thereby improving the signal-to-background ratio and detection sensitivity. These strategies are certainly effective and have made great progress, but they require complex design procedures and are not generally applicable.

[0004] Generally, intramolecular charge transfer (ICT) and photoinduced electron transfer (PET) mechanisms are widely used to construct activatable fluorescent small molecule probes. However, most ICT-based probes exhibit high background due to partial quenching of some of their own fluorescence, resulting in low signal-to-background ratio and high detection limit. PET-based fluorescent small molecule probes always show low intrinsic fluorescence when the energy gap between the fluorophore and the recognition / activation group is well matched. However, PET is particularly dependent on distance (usually less than 1 nm) and may be interfered by local environmental properties, such as ICT structure. Molecular probes that combine PET and ICT mechanisms can not only produce diverse fluorescence signal outputs, but also achieve complete quenching of the probe's intrinsic fluorescence. Therefore, developing new probes that combine multiple fluorescence quenching effects is a very effective strategy to eliminate intrinsic fluorescence. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a near-infrared emitting, dual-quenching ultra-sensitive biothiol fluorescent small molecule probe and its preparation method. The probe is used to detect biothiols in highly diluted plasma and can also perform high-fidelity imaging of biothiol fluctuations in ferroptosis-induced cells and living tumors.

[0006] The present invention provides a biothiol fluorescent small molecule probe MCB-NBD, the structural formula of the probe is as follows:

[0007]

[0008] The reaction mechanism of the biothiol fluorescent small molecule probe is as follows:

[0009]

[0010] The present invention also provides a method for preparing the biothiol fluorescent small molecule probe, comprising the following steps:

[0011] (1) 2,5,8,11-tetraoxatridecan-13-ol is dissolved in tetrahydrofuran, sodium hydroxide solution is added, and the mixture is cooled to a predetermined temperature. Then, a tetrahydrofuran solution of 4-toluenesulfonyl chloride is added dropwise, and the mixture is stirred for reaction at room temperature. The mixture is concentrated under reduced pressure, extracted and purified, and the resulting product is dissolved in tetrahydrofuran, 4-hydroxybenzaldehyde and potassium carbonate are added, and the mixture is stirred for reaction at room temperature. The mixture is concentrated under reduced pressure, and extracted and purified again to obtain compound 1;

[0012] (2) 4-(Hydroxymethyl)phenol was dissolved in dimethylformamide, 4-chloro-7-nitrobenzo[1,2,5]oxadiazole and potassium carbonate were added, and the mixture was refluxed at a predetermined temperature under inert atmosphere. The mixture was concentrated under reduced pressure. After extraction and purification, the product was dissolved in dichloromethane, phosphorus tribromide was added, and the reaction was carried out at a set temperature. The mixture was concentrated under reduced pressure, and after extraction and purification, compound 2 was obtained.

[0013] (3) 2,4-dimethylpyrrole was dissolved in dichloromethane, methyl oxalyl chloride was added dropwise at a predetermined temperature and reacted, triethylamine and boron trifluoride ether solution were added in sequence, the reaction was stirred at room temperature, the mixture was concentrated under reduced pressure, and compound 3 was obtained after extraction and purification;

[0014] (4) Compound 1 and Compound 3 were dissolved in anhydrous acetonitrile, acetic acid, piperidine, and a small amount of 3A molecular sieves were added, and the mixture was refluxed at a set temperature under inert atmosphere protection. The mixture was concentrated under reduced pressure, and after extraction and purification, Compound 4 was obtained;

[0015] (5) Compound 4 is dissolved in ethyl acetate, lithium iodide is added, and the mixture is refluxed at a set temperature under an inert atmosphere. The mixture is concentrated under reduced pressure. After extraction and purification, compound 2 and lithium carbonate are added to the obtained product, and the mixture is dissolved in dimethylformamide. The mixture is refluxed at a set temperature under an inert atmosphere. The mixture is concentrated under reduced pressure. After extraction and purification, the biothiol fluorescent small molecule probe MCB-NBD is obtained.

[0016] In a preferred embodiment, in step (1), the molar ratio of 2,5,8,11-tetraoxatridecan-13-ol, 4-toluenesulfonyl chloride and 4-hydroxybenzaldehyde is 1:(1.2-1.5):(1.5-2).

[0017] In a preferred embodiment, in step (1), extraction is performed with saturated brine and dichloromethane, and separation and purification is performed by silica gel column using a mixed solution of petroleum ether and ethyl acetate as eluent.

[0018] Furthermore, in step (1), the volume ratio of petroleum ether to ethyl acetate is 20:(1-2).

[0019] In a preferred embodiment, in step (1), the temperature is cooled to -2 to 0°C.

[0020] In a preferred embodiment, in step (2), the molar ratio of 4-(hydroxymethyl)phenol, 4-chloro-7-nitrobenzo[1,2,5]oxadiazole and phosphorus tribromide is 1:(1-1.2):(1.5-1.7).

[0021] In a preferred embodiment, in step (2), extraction is performed with saturated brine and dichloromethane, and separation and purification is performed by silica gel column using a mixed solution of petroleum ether and ethyl acetate as eluent.

[0022] Furthermore, in step (2), the volume ratio of petroleum ether to ethyl acetate is 20:(1-2).

[0023] In a preferred embodiment, in step (2), the reaction mixture is refluxed at 50-55° C. under the protection of an inert atmosphere.

[0024] In a preferred embodiment, in step (2), phosphorus tribromide is added and the reaction is carried out at -2 to 0°C.

[0025] In a preferred embodiment, in step (3), the molar ratio of 2,4-dimethylpyrrole solution, methyl oxalyl chloride, triethylamine and boron trifluoride ether solution is (2-2.2):1:(3.6-4):(6.6-7).

[0026] In a preferred embodiment, in step (3), extraction is performed with saturated brine and dichloromethane, and separation and purification is performed by silica gel column using a mixed solution of dichloromethane and methanol as eluent.

[0027] Furthermore, in step (3), the volume ratio of dichloromethane to methanol is 100:(1-5).

[0028] In a preferred embodiment, in step (3), methyl chlorothiophene is added dropwise at -78 to -75°C and reacted.

[0029] In a preferred embodiment, in step (4), the molar ratio of compound 1 to compound 3 is (3-3.2):1.

[0030] In a preferred embodiment, in step (4), separation and purification are performed by silica gel column using a mixed solution of dichloromethane and methanol as eluent.

[0031] Furthermore, in step (4), the volume ratio of dichloromethane to methanol is 100:(1-5).

[0032] In a preferred embodiment, in step (4), the reaction mixture is refluxed at 80-85° C. under the protection of an inert atmosphere.

[0033] In a preferred embodiment, in step (5), the molar ratio of compound 4, lithium iodide and compound 2 is 1:(3-5):(1.5-2).

[0034] In a preferred embodiment, in step (5), extraction is performed with saturated brine and dichloromethane, and separation and purification is performed by silica gel column using a mixed solution of dichloromethane and methanol as eluent.

[0035] Furthermore, in step (5), the volume ratio of dichloromethane to methanol is 100:(1-5).

[0036] In a preferred embodiment, in step (5), the reaction mixture is refluxed at 90-95° C. under the protection of an inert atmosphere.

[0037] The present invention also provides an application of the biothiol fluorescent small molecule probe, which is applied to a biothiol detection kit to detect biothiols in plasma, cells and tissues.

[0038] Boron dipyrromethene (BODIPY) dyes are widely used as backbones for activatable probes based on the PET effect due to their outstanding optical properties, including chemical stability against reactive oxygen species (ROS), large extinction coefficients, good photostability, and intense fluorescence. This study utilizes the PET effect and a dual quenching mechanism of an "ester-to-carboxylic acid" conversion strategy to synthesize a zero intrinsic fluorescence MCB-NBD probe.

[0039] This paper proposes a dual fluorescence quenching mechanism that combines the PET effect with an "ester to carboxylic acid" conversion strategy to synthesize an ultrasensitive biothiol fluorescent small molecule probe, MCB-NBD. Due to the dual fluorescence quenching mechanism, the probe exhibits almost zero intrinsic fluorescence. However, after reacting with biothiols, the probe releases a fluorophore, generating a strong near-infrared fluorescence signal. Therefore, the probe is capable of ultrasensitively detecting biothiols in highly diluted plasma and high-fidelity imaging of dihydroartemisinin-induced ferroptosis. The present invention can effectively overcome the interference of background fluorescence of traditional near-infrared fluorescent small molecule probes and improve the sensitivity and accuracy of biothiol imaging analysis in cells, plasma, and in vivo.

[0040] Compared with the prior art, the present invention has the following beneficial technical effects:

[0041] 1) The small molecule probe MCB-NBD has very low intrinsic fluorescence and high sensitivity, and its fluorescence response to biological thiols is as high as 180 times.

[0042] 2) The small molecule probe MCB-NBD exhibits an extremely low detection limit for biothiols. When 200 nM biothiols are added, the fluorescence response multiple exceeds 3 times, and it can significantly distinguish between liver injury plasma and normal plasma at 80-fold dilution.

[0043] 3) The small molecule probe MCB-NBD monitored the fluctuation of biothiols during dihydroartemisinin-induced ferroptosis, demonstrating the application prospect of the present invention in studying new strategies for clinical tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The process from "zero" background fluorescence to high brightness after the probe MCB-NBD reacts with biological thiols.

[0045] Figure 2 The fluorescence spectrum of the probe MCB-NBD in response to 0-100 μM GSH (A), the fluorescence spectrum of the probe MCB-NBD in response to low concentration (0-1 μM) GSH (B), the calibration curve of the probe response to low concentration (0-1 μM) GSH (C), and the UV-visible absorption spectrum of the probe MCB-NBD after responding to 0-100 μM GSH (D).

[0046] Figure 3 The fluorescence spectrum of the probe MCB-NBD in response to 0-100 μM Cys (A), the fluorescence spectrum of the probe MCB-NBD in response to low concentration (0-1 μM) Cys (B), the calibration curve of the probe response to low concentration (0-1 μM) Cys (C), and the UV-visible absorption spectrum of the probe MCB-NBD after responding to 0-100 μM Cys (D).

[0047] Figure 4 Confocal imaging of biothiols in cells using the probe MCB-NBD (A), relative fluorescence intensity in Figure A (B), flow cytometry experiment corresponding to the experiment in Figure A (C), and DILI schematic diagram (D).

[0048] Figure 5 Figure 3 shows the stability test of MCB-NBD probe on reactive oxygen species in cells (A) and the relative fluorescence intensity in Figure A (B).

[0049] Figure 6 Imaging of liver damage in living mice using the probe MCB-NBD (A) and relative fluorescence intensity in Figure A (B).

[0050] Figure 7Confocal imaging of mouse liver tissue sections using the probe MCB-NBD (A), relative fluorescence intensity in Figure A (B), and H&E staining of mouse liver tissues after different treatments (C).

[0051] Figure 8 The feasibility test of plasma sample experiment (A), the test of different degrees of liver damage by probe MCB-NBD (B), and the detection of plasma with different dilution multiples by probe MCB-NBD (C).

[0052] Figure 9 Fluorescence imaging of plasma with different dilutions of thiol inhibitors added using a small animal imager (A), fluorescence imaging of plasma with different dilutions using a small animal imager (B), and fluorescence imaging of 0-100 μM Cys using a small animal imager (C).

[0053] Figure 10 Comparison of the fluorescence intensity of the probe MCB-NBD in the human normal plasma group and the thiol inhibitor treatment group at different dilution multiples (A), the fluorescence intensity of the probe MCB-NBD in 10 groups of normal human plasma samples and 10 groups of plasma samples from patients with drug-induced liver injury (B), and the detection of biothiols in 10 groups of normal human plasma samples and 10 groups of plasma samples from patients with drug-induced liver injury by the total thiol kit (C).

[0054] Figure 11 Fluorescence imaging of ferroptosis in cells using the probe MCB-NBD (A)(B), flow cytometry experiments corresponding to the experiments in Figures A and B (C)(D), and relative fluorescence intensity of the images in Figures A and B (E)(F).

[0055] Figure 12 Fluorescence imaging of ferroptosis in tumor-bearing mice using the probe MCB-NBD (A) and the relative fluorescence intensity in Figure A (B). DETAILED DESCRIPTION

[0056] The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods described are conventional methods unless otherwise specified, and the raw materials described can be obtained from public commercial channels unless otherwise specified.

[0057] Example 1 Probe MCB-NBD

[0058] (1) 2,5,8,11-tetraoxatridecan-13-ol was dissolved in tetrahydrofuran (10 mL), and a sodium hydroxide aqueous solution (10 mL) was added. The mixture was cooled to 0°C, and then a tetrahydrofuran solution of 4-toluenesulfonyl chloride (10 mL) was added dropwise. The mixture was stirred at room temperature for 3 hours, and the mixture was concentrated under reduced pressure. The mixture was extracted with saturated brine and dichloromethane, and petroleum ether / ethyl acetate was used as the mobile phase. The crude product was purified by thin layer chromatography. After purification, the product was dissolved in tetrahydrofuran (20 mL), 4-hydroxybenzaldehyde and potassium carbonate were added, and the mixture was stirred at room temperature for 3 hours. The mixture was concentrated under reduced pressure, extracted with saturated brine and dichloromethane, and petroleum ether / ethyl acetate was used as the mobile phase. The crude product was purified by thin layer chromatography to obtain compound 1;

[0059] (2) 4-(Hydroxymethyl)phenol was dissolved in dimethylformamide (10 mL), 4-chloro-7-nitrobenzo[1,2,5]oxadiazole and potassium carbonate were added, and the mixture was refluxed at 50°C for 1 hour under nitrogen protection. The mixture was concentrated under reduced pressure, extracted with saturated brine and dichloromethane, and the crude product was purified by thin layer chromatography using petroleum ether / ethyl acetate as the mobile phase. After purification, the product was dissolved in dichloromethane (10 mL), phosphorus tribromide was added, and the reaction was carried out at 0°C for 2 hours. The mixture was concentrated under reduced pressure, extracted with saturated brine and dichloromethane, and the crude product was purified by thin layer chromatography using petroleum ether / ethyl acetate as the mobile phase to obtain compound 2;

[0060] (3) 2,4-Dimethylpyrrole was dissolved in dichloromethane (10 mL), and methyl oxalyl chloride was added dropwise at -78°C and reacted for 2 hours. Triethylamine and boron trifluoride ether solution were then added in sequence and stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure and extracted with saturated brine and dichloromethane. The crude product was purified by thin layer chromatography using dichloromethane / methanol as the mobile phase to obtain compound 3;

[0061] (4) Compound 1 and compound 3 were dissolved in anhydrous acetonitrile (10 mL), acetic acid, piperidine, and a small amount of 3A molecular sieves were added, and the mixture was refluxed at 80°C for 2 hours under nitrogen protection. The mixture was concentrated under reduced pressure and extracted with saturated brine and dichloromethane. The crude product was purified by thin layer chromatography using dichloromethane / methanol as the mobile phase to obtain compound 4;

[0062] The structure of compound 4:

[0063]

[0064] NMR and mass spectrometry characterization of dyes:

[0065] 1H NMR(400MHz,Chloroform-d)δ7.63–7.49(m,6H),6.96(d,J=8.2Hz,4H),6.72(s,2H),4.20(t,J=4.7Hz,4H),4 .00(s,3H),3.91(t,J=4.8Hz,4H),3.81–3.64(m,20H),3.57(dd,J=5.8,3.3Hz,4H),3.40(s,6H),2.19(s,6H).

[0066] 13 C NMR (101MHz, CDCl3) δ159.90,129.45,129.25,114.98,71.94,70.87,70.64,70.54,69.67,67.52,59.07,12.78.

[0067] ESI-MS: calculated for C 47 H 61 BF2N2O 12 [M+Na + ] + 917.42,found 917.77.

[0068] (5) Compound 4 was dissolved in ethyl acetate (10 mL), lithium iodide was added, and the mixture was refluxed at 90°C for 3 hours under nitrogen protection. The mixture was concentrated under reduced pressure, extracted with saturated brine and dichloromethane, and the crude product was purified by thin layer chromatography using dichloromethane / methanol as the mobile phase. Then, compound 2 and lithium carbonate were added, dissolved in dimethylformamide (10 mL), refluxed at 90°C for 3 hours under nitrogen protection, the mixture was concentrated under reduced pressure, extracted with saturated brine and dichloromethane, and the crude product was purified by thin layer chromatography using dichloromethane / methanol as the mobile phase to obtain the probe MCB-NBD as a dark green solid.

[0069] The structure of the probe MCB-NBD:

[0070]

[0071] Mass spectrometry and NMR characterization of probe MCB-NBD:

[0072] 1H NMR(400MHz,Chloroform-d)δ8.45(d,J=8.1Hz,1H),7.75–7.46(m,9H),7.32(d,J=8.1Hz,2H),6.97(d,J=8.3Hz,4H),6.71(s,2H),6.5 6(d,J=8.3Hz,1H),4.20(t,J=4.8Hz,4H),3.91(t,J=4.8Hz,4H),3.82–3.61(m,22H),3.57(t,J=4.6Hz,4H),3.40(s,6H),2.13(s,6H).

[0073] 13 C NMR (101MHz, CDCl3) δ160.04,133.09,131.68,129.30,121.37,115.03,71.96,70.89,70.65,70.63,70.54,69.67,67.57,59.05,29.68,13.00.

[0074] ESI-MS: calculated for C 47 H 61 BF2N2O 12 [M+Na + ] + 1172.45,found 1172.98.

[0075] The synthesis process of the probe MCB-NBD is as follows:

[0076]

[0077] Example 2 Detection of biothiols using the MCB-NBD probe in a buffer system

[0078] Dissolve 11.49 mg of the probe in 10 mL of DMSO to prepare a 1 mM stock solution, and store it at -20°C. The detection system is a PBS buffer solution (10 mM, pH 7.4, containing 50% DMSO). Shake the reaction system of the probe MCB-NBD and biothiol at room temperature for 40 minutes, and then measure its fluorescence emission and UV-visible absorption spectrum. Set the excitation wavelength of the fluorescence instrument to 590 nm and the emission wavelength receiving range to 600-700 nm. Set the measurement range of the absorption spectrum to 500-800 nm. The results are as follows Figure 2 As shown, from Figure 2 、 3 It can be seen that the probe MCB-NBD has a good response to biothiols, the near-infrared fluorescence spectrum shows a fluorescence enhancement of nearly 180 times, and the absorption spectrum shows a significant change.

[0079] Example 3 Detection of Biothiols in Drug-Induced Liver Injury Models Using MCB-NBD Probe in Cells and in Vivo

[0080] 11.49 mg of probe was dissolved in 10 mL of DMSO to prepare a 1 mM stock solution and stored at -20°C. 5 μL of probe was incubated in cells and the confocal microscope was set to an excitation wavelength of 640 nm and an emission wavelength range of 663-738 nm. Figure 4 、 5 As shown, from Figure 4 、 5 It can be seen that the probe MCB-NBD has a good response to the fluctuation of intracellular biothiols. The probe was prepared into 200μM and dissolved in PBS / DMSO, v / v=7:3, pH 7.4. 100μL of the probe was injected into the mouse through the tail vein, and then the small animal living fluorescence imaging was performed. The excitation wavelength of the small animal imaging instrument was set to 640nm and the emission wavelength receiving range was 660-700nm. The results are shown in Figure 2. Figure 6 As shown, from Figure 6 It can be seen that the probe MCB-NBD can well monitor the fluctuation of biothiols in mouse liver, and the imaging contrast is very high. Figure 7 It can be seen that the probe MCB-NBD can well monitor the fluctuation of biothiols in tissue sections.

[0081] Example 4 Detection of Biothiols in Plasma of Drug-Induced Liver Injury Model Using Probe MCB-NBD

[0082] Dissolve 11.49 mg of the probe in 10 mL of DMSO to prepare a 1 mM stock solution, and store it at -20°C. The detection system is a PBS buffer solution (10 mM, pH 7.4, containing 50% DMSO). Shake the reaction system of the probe MCB-NBD and plasma with different dilution multiples at room temperature for 40 minutes, and then measure its fluorescence emission spectrum. Set the excitation wavelength of the fluorescence instrument to 590 nm and the emission wavelength receiving range to 600-700 nm. Set the excitation wavelength of the small animal imaging instrument to 640 nm and the emission wavelength receiving range to 660-700 nm. The results are as follows Figure 8 As shown, from Figure 8 It can be seen that the probe MCB-NBD has a good response to biothiols in plasma and can significantly distinguish liver injury plasma from normal plasma at 80-fold dilution. Even at a dilution of up to 200-fold, the probe MCB-NBD can still show significant fluorescence enhancement, thereby accurately detecting extremely low concentrations of biothiols. Figure 9It can be seen that the probe MCB-NBD simultaneously images multiple samples in a 96-well plate using a small animal imaging instrument, indicating that the probe MCB-NBD has the potential for high-throughput detection. Figure 10 It can be seen that the detection results of the probe MCB-NBD for human plasma are similar to those of the commercial kit, indicating the clinical detection potential of the probe MCB-NBD.

[0083] Example 5 Imaging Detection of Ferroptosis-Induced Biothiol Fluctuations in Cells and Living Tumors Using the MCB-NBD Probe

[0084] 111.49 mg of probe was dissolved in 10 mL of DMSO to prepare a 1 mM stock solution and stored at -20°C. 5 μL of probe was incubated in cells and the confocal microscope was set to an excitation wavelength of 640 nm and an emission wavelength range of 663-738 nm. Figure 11 As shown, from Figure 11 It can be seen that the probe MCB-NBD has a good response to the fluctuation of biothiol induced by intracellular ferroptosis. The probe was prepared into 200μM and dissolved in DPBS / DMSO, v / v=7:3, pH 7.4. 20μL of the probe solution was injected into the tumor, and then the small animal living fluorescence imaging was performed. The excitation wavelength of the small animal imaging instrument was set to 640nm, and the emission wavelength receiving range was 660-700nm. The results are shown in Figure 2. Figure 12 As shown, from Figure 12 It can be seen that the probe MCB-NBD can well monitor the fluctuation of biothiols in mouse tumors, and the imaging contrast is very high.

[0085] This invention discloses an ultrasensitive fluorescent small-molecule biothiol probe with a dual quenching mechanism, as well as its preparation and application. The authors studied the detection of biothiols in buffer solutions using the fluorescent small-molecule probe MCB-NBD; the detection of biothiols in drug-induced liver injury models in cells and in vivo; the detection of biothiols in plasma from drug-induced liver injury models using the fluorescent small-molecule probe MCB-NBD; and the imaging detection of biothiol fluctuations induced by ferroptosis in cells and in vivo tumors using the fluorescent small-molecule probe MCB-NBD. This invention, for the first time, proposes a dual quenching mechanism using the PET effect and an "ester to carboxylic acid" conversion strategy, constructing an ultrasensitive biothiol fluorescent small-molecule probe MCB-NBD with "zero" intrinsic fluorescence. The method overcomes the problems of high background fluorescence, low sensitivity, and high detection limit of existing fluorescent small molecule probes; the near-infrared emitting, double-quenched ultra-sensitive biothiol fluorescent small molecule probe MCB-NBD responds rapidly to biothiols, has high sensitivity, and a low detection limit, and can be used for ultra-sensitive detection of biothiols in highly diluted plasma and high-fidelity imaging of dihydroartemisinin-induced ferroptosis.

[0086] The present invention proposes a dual fluorescence quenching mechanism that combines the PET effect and the "ester to carboxylic acid" conversion strategy to construct an MCB-NBD probe with "zero" intrinsic fluorescence; the small molecule fluorescent probe MCB-NBD of the present invention overcomes the problems of high background fluorescence, low sensitivity, and high detection limit of existing small molecule fluorescent probes; the fluorescent small molecule probe MCB-NBD of the present invention exhibits a very low detection limit for biothiols. When 200nM biothiols are added, the fluorescence response multiple is more than 3 times, and it can significantly distinguish between liver-damaged plasma and normal plasma at 80-fold dilution; the fluorescent small molecule probe MCB-NBD of the present invention monitors the fluctuation of biothiols in dihydroartemisinin-induced ferroptosis, reflecting the application prospects of the present invention in studying new strategies for clinical tumor treatment.

[0087] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A biothiol fluorescent small molecule probe MCB-NBD, characterized in that: The structural formula of the probe is as follows: 。 2. The method for preparing a biothiol fluorescent small molecule probe according to claim 1, characterized in that: The following steps are involved: (1) 2,5,8,11-tetraoxatridecan-13-ol is dissolved in tetrahydrofuran, sodium hydroxide solution is added, and the mixture is cooled to a predetermined temperature. Then, a tetrahydrofuran solution of 4-toluenesulfonyl chloride is added dropwise, and the mixture is stirred for reaction at room temperature. The mixture is concentrated under reduced pressure, extracted and purified, and the resulting product is dissolved in tetrahydrofuran, 4-hydroxybenzaldehyde and potassium carbonate are added, and the mixture is stirred for reaction at room temperature. The mixture is concentrated under reduced pressure, extracted and purified again to obtain compound 1; (2) 4-(Hydroxymethyl)phenol is dissolved in dimethylformamide, 4-chloro-7-nitrobenzo[1,2,5]oxadiazole and potassium carbonate are added, and the mixture is refluxed at a predetermined temperature under inert atmosphere. The mixture is concentrated under reduced pressure, extracted and purified, and the product is dissolved in dichloromethane. Phosphorus tribromide is added and the reaction is carried out at a set temperature. The mixture is concentrated under reduced pressure, extracted and purified again to obtain compound 2; (3) 2,4-Dimethylpyrrole was dissolved in dichloromethane, methyl oxalyl chloride was added dropwise at a predetermined temperature and reacted, triethylamine and boron trifluoride ether solution were added in sequence, the reaction was stirred at room temperature, and the mixture was concentrated under reduced pressure. After extraction and purification, compound 3 was obtained; (4) Compound 1 and compound 3 were dissolved in anhydrous acetonitrile, acetic acid, piperidine and a small amount of 3A molecular sieves were added, and the mixture was refluxed at a set temperature under inert atmosphere. The mixture was concentrated under reduced pressure, and after extraction and purification, compound 4 was obtained; (5) Compound 4 is dissolved in ethyl acetate, lithium iodide is added, and the mixture is refluxed at a set temperature under an inert atmosphere. The mixture is concentrated under reduced pressure. After extraction and purification, compound 2 and lithium carbonate are added to the obtained product, and the mixture is dissolved in dimethylformamide. The mixture is refluxed at a set temperature under an inert atmosphere. The mixture is concentrated under reduced pressure. After extraction and purification, the biothiol fluorescent small molecule probe MCB-NBD is obtained; The reaction formula is as follows: 。 3. The method for preparing a biothiol fluorescent small molecule probe according to claim 2, characterized in that: In step (1), the molar ratio of 2,5,8,11-tetraoxatridecan-13-ol, 4-toluenesulfonyl chloride and 4-hydroxybenzaldehyde is 1:(1.2-1.5):(1.5-2).

4. The method for preparing a biothiol fluorescent small molecule probe according to claim 2, characterized in that: In step (2), the molar ratio of 4-(hydroxymethyl)phenol, 4-chloro-7-nitrobenzo[1,2,5]oxadiazole and phosphorus tribromide is 1:(1-1.2):(1.5-1.7).

5. The method for preparing a biothiol fluorescent small molecule probe according to claim 2, characterized in that: In step (3), the molar ratio of 2,4-dimethylpyrrole solution, methyl oxalyl chloride, triethylamine and boron trifluoride ether solution is (2-2.2):1:(3.6-4):(6.6-7).

6. The method for preparing a biothiol fluorescent small molecule probe according to claim 2, characterized in that: In step (4), the molar ratio of compound 1 to compound 3 is (3-3.2):

1.

7. The method for preparing a biothiol fluorescent small molecule probe according to claim 2, characterized in that: In step (5), the molar ratio of compound 4, lithium iodide and compound 2 is 1:(3-5):(1.5-2).

8. The use of the biothiol fluorescent small molecule probe according to claim 1, characterized in that: It was applied to the biothiol detection kit.

Images