A fluorescent probe based on silicon-substituted rhodamine derivatives and its preparation method and application

Abstract

The present invention discloses a fluorescent probe based on a silicon-substituted rhodamine derivative, its preparation method, and its application, relating to the field of biochemistry. The present invention provides the structural formula of the fluorescent probe based on the silicon-substituted rhodamine derivative and also provides a corresponding preparation method. The present invention designs a leucine aminopeptidase-responsive near-infrared fluorescent small molecule probe that can effectively identify head and neck squamous cell carcinoma, effectively addressing the low sensitivity, specificity, and response speed issues of existing technologies.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to a fluorescent probe based on a silicon atom-substituted rhodamine derivative, and a preparation method and application thereof. Background Art

[0002] Head and neck squamous cell carcinoma (HNSCC) ranks among the top malignant tumors in terms of global morbidity and mortality, with the prevalence projected to reach approximately 1 million cases by 2030. Therefore, improving the diagnosis and treatment of HNSCC is a critical and urgent task. Currently, a comprehensive treatment approach, primarily consisting of surgery, chemotherapy, radiotherapy, and targeted therapy, is used for patients with advanced HNSCC, with surgery being a key component. Negative surgical margins are crucial for reducing tumor recurrence and improving patient survival. It is generally accepted that in the treatment of HNSCC, a surgical margin greater than 5 mm from the tumor is considered adequate, while a margin less than 5 mm is considered inadequate. However, despite the introduction of numerous new technologies over the past 30 years, the overall rate of inadequate margins for head and neck tumors remains the highest among all surgical procedures, at approximately 15-30%. Currently, the most commonly used method for intraoperative resection margin control is frozen section analysis (FSA). However, this technology not only has the subjectivity of sampling, but also requires a long operation time, usually 20-30 minutes. Its application has certain limitations. Therefore, there is an urgent need to develop a rapid and accurate method to diagnose and identify intraoperative resection margin samples for head and neck tumors to avoid incomplete resection of tumor lesions and unnecessary resection of normal tissue.

[0003] With the rapid development of molecular imaging technologies, fluorescent probes, as a powerful molecular detection technique, have attracted extensive research interest in the field of biological detection. Compared with other detection methods, fluorescent probes offer unique advantages in detecting substances in vivo, such as high sensitivity and excellent temporal and spatial resolution. Fluorescent probe technology can monitor physiological and pathological processes at the molecular and cellular levels, making it an ideal technology for tumor cell monitoring and imaging. Although a variety of responsive fluorescent probes for tumor detection have been reported, and their good sensitivity and specificity have been demonstrated in vitro, few fluorescent probes have been reported that can achieve rapid and high-precision imaging of tumors in vivo. Furthermore, rapid probe responsiveness is crucial for physicians to quickly determine tumor boundaries during surgery, which helps reduce the incidence of positive resection margins and improve surgical success rates. Therefore, the development of probes that can rapidly and accurately detect specific substances in head and neck squamous cell carcinoma is highly desirable, but remains a significant challenge.

[0004] Leucine aminopeptidase (LAP) is an important proteolytic enzyme in the human body, involved in a variety of pathological processes, including tumor cell invasion, proliferation, and angiogenesis. It has been used as a tumor-associated marker for liver cancer, ovarian cancer, and epithelial cancers. Head and neck squamous cell carcinoma (HNSCC) is also an epithelial tumor, and currently, there are few reports of fluorescent probes for HNSCC. Therefore, by designing and developing a fluorescent probe activated by leucine aminopeptidase, we are developing a responsive fluorescent probe for rapid and accurate imaging of HNSCC. This probe can be used for rapid verification of pathological sections during clinical surgery, which undoubtedly has important research value and practical significance. Summary of the Invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide a fluorescent probe based on silicon atom-substituted rhodamine derivatives, as well as its preparation method and application. A near-infrared fluorescent small molecule probe responsive to leucine aminopeptidase is designed, which can effectively identify head and neck squamous cell carcinoma, effectively solving the problems of low recognition sensitivity, specificity and response speed in the existing technology.

[0006] The present invention solves the above-mentioned technical problem with the following technical solution: providing a fluorescent probe based on a silicon atom-substituted rhodamine derivative, the structural formula of which is:

[0007]

[0008]

[0009] The present invention also provides a method for preparing the fluorescent probe based on the silicon atom-substituted rhodamine derivative, comprising the following steps:

[0010] (1) 2-Bromo-4-fluorobenzaldehyde and potassium carbonate were dissolved in N,N-dimethylformamide, and then diallylamine was added. The mixture was heated to 100°C and refluxed for 8 hours. The mixture was then filtered, extracted, dried, and distilled under reduced pressure to obtain compound 1.

[0011] (2) Compound 1 obtained in step (1) was dissolved in methanol, sodium borohydride was added to 0°C, and the mixture was stirred at room temperature for 1 hour. The solvent was then evaporated under reduced pressure, water was added to the residue, and the mixture was extracted with dichloromethane. The organic layer was collected, dried, and evaporated under reduced pressure to obtain Compound 2.

[0012] (3) adding boron trifluoride etherate to a dichloromethane solution of 1-(3-bromophenyl)tetrahydropyrrole or 7-bromo-1,2,2,4-tetramethyl-1,2-dihydroquinoline and the compound 2 obtained in step (2) at 0°C, stirring at room temperature for 8 hours, and then extracting, drying, evaporating to dryness and purifying in sequence to obtain compound 3;

[0013] (4) Compound 3 obtained in step (3) was dissolved in anhydrous tetrahydrofuran, and n-butyl lithium was added under a nitrogen atmosphere at -78°C and stirred for 1 hour. Then SiMe2Cl2 was added, and the temperature was raised to room temperature and stirred overnight. Water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic layer was collected, washed, dried, and evaporated to dryness to obtain Compound 4.

[0014] (5) Compound 4 obtained in step (4) was dissolved in acetone at -5°C, potassium permanganate was added and stirred for 3 hours, and then filtered, evaporated to dryness and purified to obtain compound 5;

[0015] (6) 1-Bromo-2,6-dimethoxybenzene was dissolved in anhydrous tetrahydrofuran, n-butyl lithium was added under a nitrogen atmosphere at -78°C and stirred for 1 hour, then compound 5 obtained in step (5) was added, the temperature was raised to room temperature and stirred overnight, hydrochloric acid was added and extracted with dichloromethane, the organic layer was collected, and then washed, dried and evaporated to dryness in sequence to obtain compound 6;

[0016] (7) Compound 6 obtained in step (6) was added with 1,3-dimethylbarbituric acid and tetrakistriphenylphosphine palladium in dichloromethane, and then reacted under an argon atmosphere for 24 hours. The reaction was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic layer was collected, dried, concentrated, and purified to obtain compound 7;

[0017] (8) Compound VII obtained in step (7) was dissolved in N,N-dimethylformamide, HATU and DIPEA were added, and the mixture was stirred at 0°C for 10 min. Then, BOC-L-leucine dissolved in N,N-dimethylformamide was added dropwise at room temperature, and the mixture was stirred at room temperature for 6 h. After concentration under reduced pressure, water was added, and the mixture was extracted with dichloromethane. The organic layer was collected, dried, evaporated under reduced pressure, and purified to obtain Compound VIII.

[0018] (9) Compound 8 obtained in step (8) was dissolved in dichloromethane, and trifluoroacetic acid was added, stirred at 0°C for 1 min, and then distilled under reduced pressure, washed, dried and evaporated under reduced pressure to obtain a fluorescent probe based on a silicon atom-substituted rhodamine derivative.

[0019] Furthermore, in step (1), the molar volume ratio of 2-bromo-4-fluorobenzaldehyde, potassium carbonate, N,N-dimethylformamide and diallylamine is 50 mmol:50 mmol:100 mL:20 mL.

[0020] Furthermore, in step (2), the molar ratio of compound 1 to sodium borohydride is 50 mmol:60 mmol.

[0021] Furthermore, in step (3), the molar ratio of 1-(3-bromophenyl)tetrahydropyrrole or 7-bromo-1,2,2,4-tetramethyl-1,2-dihydroquinoline, compound II and boron trifluoride ether is 50 mmol:50 mmol:50 mmol or 71.2 mmol:71.2 mmol:142.4 mmol.

[0022] Furthermore, in step (4), the molar ratio of the compound tributyl lithium, n-butyl lithium and SiMe2Cl2 is 10mmol:22mmol:10mmol or 12.3mmol:25.8mmol:12.9mmol.

[0023] Furthermore, in step (6), the molar ratio of 1-bromo-2,6-dimethoxybenzene, n-butyl lithium and compound V is 10 mmol:10 mmol:1 mmol or 7.1 mmol:7.1 mmol:0.5 mmol.

[0024] Furthermore, in step (7), the molar ratio of 1,3-dimethylbarbituric acid to tetrakistriphenylphosphine palladium is 2 mmol:0.22 mmol or 0.7 mmol:0.04 mmol.

[0025] Furthermore, in step (8), the molar ratio of compound VII, HATU, DIPEA and BOC-L-leucine is 0.2 mmol: 0.4 mmol: 0.4 mmol: 0.2 mmol.

[0026] The present invention also provides the use of the fluorescent probe based on the silicon atom-substituted rhodamine derivative in a head and neck squamous cell carcinoma detection instrument.

[0027] The present invention has the following beneficial effects:

[0028] 1. The present invention provides a near-infrared fluorescent small molecule probe that responds to leucine aminopeptidase (LAP). The probe can effectively identify head and neck squamous cell carcinoma and improve the sensitivity, specificity and response speed of identification, thereby achieving the goal of assisting in the rapid determination of resection margins during surgery for head and neck squamous cell carcinoma.

[0029] 2. This invention verifies the various properties of fluorescent probes based on silicon-atom-substituted rhodamine derivatives, providing new ideas and methods for rapidly determining tumor boundaries during surgery. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the hydrogen spectrum of the product obtained in Example 1;

[0031] Figure 2 is the carbon spectrum of the product obtained in Example 1;

[0032] Figure 3This is the high-resolution mass spectrum of the product obtained in Example 1;

[0033] Figure 4 is the hydrogen spectrum of the product obtained in Example 2;

[0034] Figure 5 is the carbon spectrum of the product obtained in Example 2;

[0035] Figure 6 This is the high-resolution mass spectrum of the product obtained in Example 2;

[0036] Figure 7 is the UV absorption spectrum and fluorescence emission curve;

[0037] Figure 8 is the fluorescence intensity test result;

[0038] Figure 9 This is the in vivo imaging result;

[0039] Figure 10 Schematic diagram of frozen section during clinical operation. DETAILED DESCRIPTION

[0040] The principles and features of the present invention are described below. The examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, conventional conditions or manufacturer-recommended conditions were used. Reagents or instruments used where the manufacturer is not specified are conventional products that can be purchased commercially.

[0041] Example 1

[0042] A fluorescent probe based on a silicon-substituted rhodamine derivative, the preparation method of which comprises the following steps (the roadmap is shown below):

[0043]

[0044] (1) 2-Bromo-4-fluorobenzaldehyde (10.6 g, 50 mmol) and potassium carbonate (6.9 g, 50 mmol) were dissolved in 100 mL of N,N-dimethylformamide, and then 20 mL of diallylamine was added. The mixture was heated to reflux at 100°C for 8 h. Solid impurities were removed by filtration, and water was added followed by extraction with dichloromethane. The mixture was repeated three times, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain compound A1.

[0045] (2) Compound 1 (14.0 g, 50 mmol) obtained in step (1) was dissolved in methanol, and sodium borohydride (2.3 g, 60 mmol) was added to the mixture and the temperature was raised to 0°C. The mixture was stirred at room temperature for 1 h, and then the solvent was evaporated under reduced pressure. Water was added to the residue, and the mixture was extracted with dichloromethane. The organic layer was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain compound 2 A2.

[0046] (3) Boron trifluoride etherate (12 mL, 50 mmol) was added to a dichloromethane solution of 1-(3-bromophenyl)tetrahydropyrrole (11.3 g, 50 mmol) and compound II obtained in step (2) (14.1 g, 50 mmol) at 0°C, and the mixture was stirred at room temperature for 8 h. After adding water, the mixture was extracted with dichloromethane. The organic layer was collected, dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography to obtain compound III A3;

[0047] (4) Compound 3 obtained in step (3) (4.9 g, 10 mmol) was dissolved in anhydrous tetrahydrofuran, and n-butyl lithium (8.8 mL, 22 mmol) was added under a nitrogen atmosphere at -78°C and stirred for 1 h. Then, SiMe2Cl2 (0.97 mL, 10 mmol) was added, and the mixture was warmed to room temperature and stirred overnight. Water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic layer was collected, washed with saturated brine, dried over sodium sulfate, and evaporated to dryness to obtain compound 4A4;

[0048] (5) Compound 4 obtained in step (4) was dissolved in acetone at -5°C, and then potassium permanganate (4.7 g, 30 mmol) was added and stirred for 3 h. The mixture was filtered, evaporated to dryness, and purified by column chromatography to obtain compound 5 A5.

[0049] (6) 1-Bromo-2,6-dimethoxybenzene (2.17 g, 10 mmol) was dissolved in anhydrous tetrahydrofuran, and n-butyl lithium (2.5 mL, 10 mmol) was added under a nitrogen atmosphere at -78°C and stirred for 1 h. Then, compound 5 (402 mg, 1 mmol) obtained in step (5) was added, and the mixture was warmed to room temperature and stirred overnight. 2N hydrochloric acid was added and extracted with dichloromethane. The organic layer was collected, washed with brine, dried over sodium sulfate, and evaporated to dryness to obtain compound 6A6;

[0050] (7) Compound 6 obtained in step (6) was added with 1,3-dimethylbarbituric acid (312 mg, 2 mmol) and tetrakistriphenylphosphine palladium (254 mg, 0.22 mmol) in dichloromethane, and then reacted under argon atmosphere for 24 h. The reaction was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic layer was collected, dried with saturated sodium bicarbonate solution, and dried over sodium persulfate, concentrated to dryness, and purified by column chromatography to obtain compound 7 A7;

[0051] (8) Compound VII (89 mg, 0.2 mmol) obtained in step (7) was dissolved in N,N-dimethylformamide, HATU (150 mg, 0.4 mmol) and DIPEA (69 μL, 0.4 mmol) were added, and the mixture was stirred at 0°C for 10 min. Then, BOC-L-leucine (47 mg, 0.2 mmol) dissolved in N,N-dimethylformamide was added dropwise at room temperature, and the mixture was stirred at room temperature for 6 h. After concentration under reduced pressure, water was added, and the mixture was extracted with dichloromethane. The organic layer was collected, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and purified by column chromatography to obtain compound VIII A8.

[0052] (9) Compound A8 obtained in step (8) was dissolved in dichloromethane, and then 3 mL of trifluoroacetic acid was added. The mixture was stirred at 0°C for 1 min, distilled under reduced pressure, washed with saturated brine, dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure to obtain a fluorescent probe A9 based on a silicon atom-substituted rhodamine derivative.

[0053] The hydrogen spectrum, carbon spectrum and high-resolution mass spectrum of the fluorescent probe A9 based on silicon-atom-substituted rhodamine derivatives obtained in this example are as follows: Figure 1-3 shown.

[0054] Example 2

[0055] A fluorescent probe based on a silicon-substituted rhodamine derivative, the preparation method of which comprises the following steps (the roadmap is shown below):

[0056]

[0057] (1) 2-Bromo-4-fluorobenzaldehyde (10.6 g, 50 mmol) and potassium carbonate (6.9 g, 50 mmol) were dissolved in 100 mL of N,N-dimethylformamide, and then 20 mL of diallylamine was added. The mixture was heated to reflux at 100°C for 8 h. Solid impurities were removed by filtration, and water was added followed by extraction with dichloromethane. The mixture was repeated three times, dried over anhydrous sodium sulfate, and distilled under reduced pressure to obtain compound A1.

[0058] (2) Compound 1 (14.0 g, 50 mmol) obtained in step (1) was dissolved in methanol, and sodium borohydride (2.3 g, 60 mmol) was added to the mixture and the temperature was raised to 0°C. The mixture was stirred at room temperature for 1 h, and then the solvent was evaporated under reduced pressure. Water was added to the residue, and the mixture was extracted with dichloromethane. The organic layer was collected, dried over anhydrous sodium sulfate, and evaporated under reduced pressure to obtain compound 2 A2.

[0059] (3) Boron trifluoride etherate (17 mL, 142.4 mmol) was added to a dichloromethane solution of 7-bromo-1,2,2,4-tetramethyl-1,2-dihydroquinoline (19 g, 71.2 mmol) and compound II obtained in step (2) (20 g, 71.2 mmol) at 0°C, and the mixture was stirred at room temperature for 8 h. After adding water, the mixture was extracted with dichloromethane. The organic layer was collected, dried over anhydrous sodium sulfate, evaporated to dryness, and purified by column chromatography to obtain compound III B3;

[0060] (4) Compound 3 (6.5 g, 12.3 mmol) obtained in step (3) was dissolved in anhydrous tetrahydrofuran, and n-butyl lithium (10.3 mL, 25.8 mmol) was added under a nitrogen atmosphere at -78°C and stirred for 1 h. Then, SiMe2Cl2 (1.3 mL, 12.9 mmol) was added, and the mixture was warmed to room temperature and stirred overnight. Water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic layer was collected, washed with saturated brine, dried over sodium sulfate, and evaporated to dryness to obtain compound 4 B4;

[0061] (5) Compound 4 obtained in step (4) was dissolved in acetone at -5°C, and then potassium permanganate (5.8 g, 36.9 mmol) was added and stirred for 3 h. The mixture was filtered, evaporated to dryness, and purified by column chromatography to obtain compound 5 B5;

[0062] (6) 1-Bromo-2,6-dimethoxybenzene (1.5 g, 7.1 mmol) was dissolved in anhydrous tetrahydrofuran, and n-butyl lithium (2.9 mL, 7.1 mmol) was added under a nitrogen atmosphere at -78°C and stirred for 1 h. Then, compound 5 (210 mg, 0.5 mmol) obtained in step (5) was added, and the mixture was warmed to room temperature and stirred overnight. 2N hydrochloric acid was added and extracted with dichloromethane. The organic layer was collected, washed with brine, dried over sodium sulfate, and evaporated to dryness to obtain compound 6 B6;

[0063] (7) Compound 6 obtained in step (6) was added with 1,3-dimethylbarbituric acid (130 mg, 0.7 mmol) and tetrakistriphenylphosphine palladium (42 mg, 0.04 mmol) in dichloromethane, and then reacted under argon atmosphere for 24 h. The reaction was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic layer was collected, dried with saturated sodium bicarbonate solution, and dried over sodium persulfate, concentrated to dryness, and purified by column chromatography to obtain compound 7 B7;

[0064] (8) Compound VII (88 mg, 0.2 mmol) obtained in step (7) was dissolved in N,N-dimethylformamide, HATU (139 mg, 0.4 mmol) and DIPEA (64 μL, 0.4 mmol) were added, and the mixture was stirred at 0°C for 10 min. Then, BOC-L-leucine (42 mg, 0.2 mmol) dissolved in N,N-dimethylformamide was added dropwise at room temperature, and the mixture was stirred at room temperature for 6 h. After concentration under reduced pressure, water was added, and the mixture was extracted with dichloromethane. The organic layer was collected, dried over anhydrous sodium sulfate, evaporated under reduced pressure, and purified by column chromatography to obtain compound VIII B8;

[0065] (9) Compound B8 obtained in step (8) was dissolved in dichloromethane, and then 3 mL of trifluoroacetic acid was added. The mixture was stirred at 0°C for 1 min, distilled under reduced pressure, washed with saturated brine, dried over anhydrous Na2SO4, and evaporated to dryness under reduced pressure to obtain a fluorescent probe B9 based on a silicon atom-substituted rhodamine derivative.

[0066] The hydrogen spectrum, carbon spectrum and high-resolution mass spectrum of the fluorescent probe B9 based on silicon-atom-substituted rhodamine derivatives obtained in this example are as follows: Figure 4-6 shown.

[0067] Test example

[0068] 1. The fluorescent probe based on the silicon-atom-substituted rhodamine derivative obtained in Example 1 was prepared into a 10 mM DMSO mother solution, and a PBS solution was added to measure its ultraviolet absorption spectrum and fluorescence spectrum to obtain the ultraviolet absorption spectrum and fluorescence emission curve, as shown in FIG. Figure 7 shown.

[0069] Depend on Figure 7 It can be seen that the probe has a maximum absorption at 546 nm and almost no fluorescence emission.

[0070] 2. Different amounts (0-8 U / L) of leucine aminopeptidase were added to a 0.01 mol / L phosphate buffer solution containing 10 mmol of the probe obtained in Example 1, and the fluorescence intensity was tested. The results are shown in FIG. Figure 8 shown.

[0071] Depend on Figure 8 It can be seen that with the addition of leucine aminopeptidase, the fluorescence intensity gradually increased, indicating that the LAP can well recognize the probe and has good fluorescence activation ability.

[0072] 3. The probe obtained in Example 1 was prepared into 100 μM, and the volume was 100 μL of physiological saline solution, which was injected into the body of mice with in situ head and neck squamous cell carcinoma. The imaging was performed using a living imaging device. The results are shown in FIG. Figure 9 shown.

[0073] Depend on Figure 9It can be seen that the fluorescence signal at the tumor site was significantly enhanced, indicating that the probe can clearly identify tumor tissue.

[0074] 4. Imaging experiment of probe in human intraoperative frozen sections: 50 μM of the probe obtained in Example 1 was sprayed on clinical intraoperative frozen sections, and the results were as follows: Figure 10 shown.

[0075] Depend on Figure 10 It can be seen that obvious fluorescence activation was observed, and the probe clearly displayed the intertissue structure and cell morphology with high resolution, and had a significant effect on distinguishing the boundaries between tumor tissue, inflammatory tissue and normal tissue. More importantly, the fluorescence imaging results were highly consistent with pathological HE (hematoxylin-eosin staining). Clinical specimens are an important bridge connecting preclinical research and clinical research. The good results achieved in clinical specimens demonstrate its reliability and translational potential. The probe's imaging experiment in human intraoperative clinical frozen sections showed good tumor recognition ability, and compared with the gold standard method for clinical intraoperative frozen section detection - HE staining, the time saved was about half.

[0076] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A fluorescent probe based on a silicon atom-substituted rhodamine derivative, characterized in that: Its structural formula is: or .

2. The method for preparing a fluorescent probe based on a silicon atom-substituted rhodamine derivative according to claim 1, characterized in that: The following steps are involved: (1) 2-Bromo-4-fluorobenzaldehyde and potassium carbonate were dissolved in N,N-dimethylformamide, and then diallylamine was added. The mixture was heated to reflux at 100 °C for 8 h, and then filtered, extracted, dried, and distilled under reduced pressure to obtain compound 1. (2) Compound 1 obtained in step (1) was dissolved in methanol, sodium borohydride was added to 0°C, and the mixture was stirred at room temperature for 1 h. The solvent was then evaporated under reduced pressure, water was added to the residue, and the mixture was extracted with dichloromethane. The organic layer was collected, and the mixture was dried and evaporated under reduced pressure to obtain Compound 2. (3) Add boron trifluoride ether to a dichloromethane solution of 1-(3-bromophenyl)tetrahydropyrrole or 7-bromo-1,2,2,4-tetramethyl-1,2-dihydroquinoline and compound 2 obtained in step (2) at 0°C, stir at room temperature for 8 h, and then extract, dry, evaporate to dryness and purify in sequence to obtain compound 3; (4) Compound 3 obtained in step (3) was dissolved in anhydrous tetrahydrofuran, and n-butyl lithium was added under a nitrogen atmosphere at -78 °C and stirred for 1 h, followed by the addition of SiMe2Cl2. The mixture was warmed to room temperature and stirred overnight. Water was added to quench the reaction, and the mixture was extracted with dichloromethane. The organic layer was collected and then washed, dried, and evaporated to dryness to obtain compound 4. (5) Compound 4 obtained in step (4) was dissolved in acetone at -5°C, and then potassium permanganate was added and stirred for 3 h. The mixture was then filtered, evaporated to dryness, and purified to obtain compound 5. (6) 1-Bromo-2,6-dimethoxybenzene was dissolved in anhydrous tetrahydrofuran, n-butyl lithium was added under a nitrogen atmosphere at -78 °C and stirred for 1 h, then compound 5 obtained in step (5) was added, the temperature was raised to room temperature and stirred overnight, hydrochloric acid was added and extracted with dichloromethane, the organic layer was collected, and then washed, dried and evaporated to dryness in sequence to obtain compound 6; (7) Compound 6 obtained in step (6) was added with 1,3-dimethylbarbituric acid and tetrakistriphenylphosphine palladium in dichloromethane, and then reacted under an argon atmosphere for 24 h. The reaction was quenched with saturated sodium bicarbonate solution, extracted with dichloromethane, and the organic layer was collected and then dried, concentrated, and purified to obtain compound 7; (8) Compound VII obtained in step (7) was dissolved in N,N-dimethylformamide, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine were added, and the mixture was stirred at 0°C for 10 min. Then, BOC-L-leucine dissolved in N,N-dimethylformamide was added dropwise at room temperature, and the mixture was stirred at room temperature for 6 h. After concentration under reduced pressure, water was added, and the mixture was extracted with dichloromethane. The organic layer was collected, dried, evaporated under reduced pressure, and purified to obtain compound VIII. (9) Compound VIII obtained in step (8) was dissolved in dichloromethane, and trifluoroacetic acid was added, stirred at 0°C for 1 min, and then subjected to vacuum distillation, washing, drying and vacuum evaporation in sequence to obtain a fluorescent probe based on a silicon atom-substituted rhodamine derivative.

3. The method for preparing a fluorescent probe based on a silicon atom-substituted rhodamine derivative according to claim 2, wherein: In step (1), the molar volume ratio of 2-bromo-4-fluorobenzaldehyde, potassium carbonate, N,N-dimethylformamide and diallylamine is 50 mmol:50 mmol:100 mL:20 mL.

4. The method for preparing a fluorescent probe based on a silicon atom-substituted rhodamine derivative according to claim 2, wherein: In step (2), the molar ratio of compound 1 to sodium borohydride is 50 mmol:60 mmol.

5. The method for preparing a fluorescent probe based on a silicon atom-substituted rhodamine derivative according to claim 2, wherein: In step (3), the molar ratio of 1-(3-bromophenyl)tetrahydropyrrole or 7-bromo-1,2,2,4-tetramethyl-1,2-dihydroquinoline, compound II and boron trifluoride ether is 50 mmol:50 mmol:50 mmol or 71.2 mmol:71.2 mmol:142.4 mmol.

6. The method for preparing a fluorescent probe based on a silicon atom-substituted rhodamine derivative according to claim 2, wherein: In step (4), the molar ratio of the compound tributyl lithium, n-butyl lithium and SiMe2Cl2 is 10 mmol:22 mmol:10 mmol or 12.3 mmol:25.8 mmol:12.9 mmol.

7. The method for preparing a fluorescent probe based on a silicon atom-substituted rhodamine derivative according to claim 2, wherein: In step (6), the molar ratio of 1-bromo-2,6-dimethoxybenzene, n-butyl lithium and compound V is 10 mmol:10 mmol:1 mmol or 7.1 mmol:7.1 mmol:0.5 mmol.

8. The method for preparing a fluorescent probe based on a silicon atom-substituted rhodamine derivative according to claim 2, wherein: In step (7), the molar ratio of 1,3-dimethylbarbituric acid to tetrakistriphenylphosphine palladium is 2 mmol:0.22 mmol or 0.7 mmol:0.04 mmol.

9. The method for preparing a fluorescent probe based on a silicon atom-substituted rhodamine derivative according to claim 2, wherein: In step (8), the molar ratio of compound VII, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and BOC-L-leucine is 0.2 mmol:0.4 mmol:0.4 mmol:0.2 mmol.

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