Polypeptide fluorescent probe capable of in-situ specific detection of fibroblast activation protein and preparation method and application thereof

Abstract

The application provides a polypeptide fluorescent probe capable of specifically detecting fibroblast activation protein in situ and a preparation method thereof, the polypeptide fluorescent probe comprising biotin, a specific dipeptide substrate (GP) and a dye molecule ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile (FL)), wherein the biotin and the FL are connected through the GP. The polypeptide fluorescent probe has high specificity and selective response capacity for FAP, high biological safety, good tissue penetration, long imaging time and stability, and can distinguish cancer and normal tissue by detecting the content change of FAP, thereby realizing in-situ fluorescence imaging of FAP.

Description

Technical Field

[0001] This invention relates to the field of organic synthesis and analytical detection technology, and more specifically to a polypeptide fluorescent probe capable of in situ specific detection of fibroblast activation proteins, its preparation method, and its application. Background Technology

[0002] Fibroblast activation protein (FAP) is a type II transmembrane serine protease with dipeptidyl peptidase and endopeptidase activities. FAP is expressed at low levels in normal adult tissues but is upregulated in various cancer tissues, particularly in cancer-associated fibroblasts (CAFs), which are widely present in the stroma of epithelial cancers including breast cancer, lung cancer, colorectal cancer, and ovarian cancer. FAP plays a crucial role in tumorigenesis, progression, and invasion; therefore, its overexpression is often associated with poor prognosis and is frequently considered an important target for cancer therapy. Furthermore, studies suggest a positive correlation between FAP expression levels and clinical stage of breast cancer, and a weak positive correlation between pathological grade and FAP expression; that is, higher FAP expression levels indicate later clinical stage and higher pathological grade of breast cancer. Therefore, developing a highly sensitive, specific, and in-situ real-time visualized method for detecting FAP activity is of great significance for tumor diagnosis, staging, and prognosis assessment.

[0003] Fluorescence detection technology offers numerous advantages, including ease of operation, high specificity and sensitivity, non-invasiveness, and real-time imaging capabilities. It is commonly used for real-time monitoring of many physiological and pathological processes at the cellular and even molecular levels. The detection principle of common fluorescent probes is that after a specific response occurs between the target substrate and the probe, the probe generates a fluorescence signal change under excitation light. This signal is detected by the instrument, allowing the determination of the content and location of the target analyte based on the signal change.

[0004] Currently, there are many probes being developed for in situ real-time visualization detection of FAP activity. For example, a near-infrared dual-modal optical probe with a lipophilic fluorophore and a hydrophilic quaternary ammonium salt structure can achieve fluorescence / photoacoustic dual-modal imaging of FAP in a mouse liver fibrosis model. However, the fluorescence intensity of this probe decays significantly within 60 minutes, resulting in poor long-term stable imaging. Another example is a bioluminescent nanoprobe (PABC) using an amphiphilic block polymer and its loaded luciferin with bioluminescent function. This probe can quantitatively detect FAP and provide cell bioluminescence imaging for up to 6 hours. However, the signal intensity of this bioluminescent probe only reaches its maximum value at 120 minutes, after which the luminescence intensity gradually decays, and its long-term stable imaging capability is still poor. Furthermore, existing probes for FAP detection are mainly limited to the detection of FAP activity levels in cell lines and tumor-bearing mice. There are no reports on the detection of FAP activity in human cancer tissue samples, thus failing to provide a reference for subsequent clinical applications.

[0005] Existing technical documents:

[0006] Chinese Patent: CN117820417A

[0007] Chinese Patent: CN112370533A. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of existing technologies by providing a polypeptide fluorescent probe for in situ specific detection of fibroblast activation protein (FAP) and its preparation method. This probe exhibits high specificity and selectivity for FAP, high biosafety, good tissue penetration, and long and stable imaging time. By detecting changes in FAP content, it can distinguish between cancerous and normal tissues, thereby achieving in situ fluorescence imaging of FAP.

[0009] According to a first aspect of the present invention, a polypeptide fluorescent probe is provided for in situ specific detection of fibroblast activation proteins, the polypeptide fluorescent probe comprising biotin, a specific dipeptide substrate (GP), and a dye molecule ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile (FL), wherein the biotin and FL are linked by GP;

[0010] The molecular structural formulas of Biotin, GP and FL are shown in Formula I, Formula II and Formula III, respectively.

[0011] Formula I;

[0012] Formula II;

[0013] Formula III.

[0014] As an optional implementation, the molecular structure of the polypeptide fluorescent probe is shown in Formula IV, denoted as Biotin-GP-FL;

[0015] Formula IV.

[0016] According to a second aspect of the present invention, a method for preparing the aforementioned polypeptide fluorescent probe capable of in situ specific detection of fibroblast activation proteins is provided, comprising the following steps:

[0017] 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile was prepared by isophorone (denoted as F1), and 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile was denoted as F2;

[0018] ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile was prepared by F2, and ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile was denoted as FL;

[0019] Biotin-GP-OH was synthesized from Fmoc-Pro-OH, Fmoc-Gly-OH and D-Biotin via a solid-state reaction.

[0020] Biotin-GP-OH was reacted with FL, and the reactants were purified and separated to obtain a polypeptide fluorescent probe, denoted as Biotin-GP-FL.

[0021] As an optional embodiment, the preparation of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile via isophorone includes the following preparation process:

[0022] F1 and malononitrile were dissolved in ethanol, glacial acetic acid and piperidine were added, and the mixture was heated to reflux under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature and the ethanol was removed under vacuum. The mixture was purified by rapid column chromatography and dried by rotary evaporation to obtain F2.

[0023] As an optional implementation, the molar ratio of F1, malononitrile, glacial acetic acid and piperidine is 10:(10-15):0.1:1.

[0024] As an optional implementation, the preparation of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile via F2 includes the following preparation process:

[0025] F2 and 4-aminobenzaldehyde were dissolved in ethanol, piperidine was added, and the mixture was heated to reflux under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered to collect the precipitate, and dried to obtain FL.

[0026] As an optional implementation, the molar ratio of F2, 4-aminobenzaldehyde and piperidine is (2-3):2.4:1.

[0027] As an optional implementation, the synthesis of Biotin-GP-OH from Fmoc-Pro-OH, Fmoc-Gly-OH, and D-Biotin via a solid-state reaction includes the following preparation process:

[0028] Fmoc-Pro-OH was first inoculated into CTC resin, deprotected with 20% piperidine solution, washed with N,N-dimethylformamide (DMF), and deprotection was verified to be complete by ninhydrin detection. Then, Fmoc-Gly-OH and D-Biotin were inoculated sequentially until Biotin-GP-CTC resin was obtained. After that, Biotin-GP-OH was cleaved off the resin with trifluoroethanol and freeze-dried to obtain Biotin-GP-OH.

[0029] As an optional implementation, the ratio of CTC resin, Fmoc-Pro-OH, Fmoc-Gly-OH and D-Biotin is 2 g:1.6 mmol:(1.6-3) mmol:(1-2) mmol.

[0030] As an optional implementation, the reaction of Biotin-GP-OH with FL, followed by purification and separation of the reactants to obtain the polypeptide fluorescent probe, includes the following preparation process:

[0031] Biotin-GP-OH and FL were dissolved in DMF, and O-benzotriazole-tetramethylurea hexafluorophosphate (HATU), 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylethylamine (DIEA) were added to activate Biotin-GP-OH. The activated Biotin-GP-OH was then added dropwise to the FL solution to react. After purification and lyophilization, Biotin-GP-FL was obtained.

[0032] As an optional implementation, the molar ratio of Biotin-GP-OH, HATU, HOBT, DIEA and FL is (1:1:1:2:1.2)-(1:3:3:3:1.2).

[0033] In a third aspect of the present invention, an application is provided of the aforementioned polypeptide fluorescent probe capable of in situ specific detection of fibroblast activation proteins or the polypeptide fluorescent probe prepared by the aforementioned method in the detection of fibroblast activation proteins.

[0034] The advantages of the polypeptide fluorescent probe of the present invention, which can specifically detect fibroblast activation proteins in situ, include the following:

[0035] 1. High specificity: Based on inhibition experiment analysis, FAP can specifically recognize and cleave the FAP-specific dipeptide substrate GP in the Biotin-GP-FL polypeptide fluorescent probe and release free fluorescent dye molecules FL, generating a strong fluorescent signal, thereby achieving specific detection of FAP.

[0036] 2. High selectivity: Fluorescence intensity tests after mixing the probe with different anions, cations, amino acids, proteins, and various enzymes showed that Biotin-GP-FL only produces a significant fluorescence signal enhancement in the presence of FAP, indicating that the polypeptide fluorescent probe of the present invention has high selectivity.

[0037] 3. High biocompatibility: Based on the results of cytotoxicity experiments, the polypeptide fluorescent probe Biotin-GP-FL of the present invention showed low biocompatibility against different breast cancer cell lines (MDA-MB-468, MCF-7, MDA-MB-231 and 4T1), and had high biocompatibility.

[0038] 4. Strong targeting: Based on biotin targeting experiments, the polypeptide fluorescent probe Biotin-GP-FL of the present invention has biotin targeting, that is, the polypeptide fluorescent probe not only has FAP specificity but can also specifically target the biotin receptor, thereby further enhancing the targeting of tumor tissue and realizing specific imaging of FAP in tumor tissue.

[0039] 5. Excellent tissue penetration: The polypeptide fluorescent probe Biotin-GP-FL of the present invention has fluorescence imaging capability in different cell lines. By measuring the tissue penetration effect of the polypeptide fluorescent probe Biotin-GP-FL of the present invention on human breast cancer tissue sections and corresponding adjacent normal tissue (300 μm) after soaking for 60 min, it can be seen that the polypeptide fluorescent probe Biotin-GP-FL of the present invention also has in situ imaging capability for human breast cancer tissue.

[0040] 6. Long-term stable in-situ imaging capability: By measuring the fluorescence intensity changes of human breast cancer tissue sections after different immersion times using the Biotin-GP-FL polypeptide fluorescent probe of the present invention, it was found that the fluorescence signal reached its maximum value at 100 min and remained stable within 180 min. Furthermore, the fluorescent dye dicyanoisophorone used in the polypeptide fluorescent probe of the present invention has high photostability and is not prone to photobleaching, thus possessing the characteristic of maintaining a stable fluorescence signal during long-term imaging, making it suitable for dynamic monitoring and long-term observation. Therefore, the polypeptide fluorescent probe of the present invention has the potential for long-term stable in-situ imaging. Attached Figure Description

[0041] Figure 1 This is a synthetic route diagram of the polypeptide fluorescent probe Biotin-GP-FL of the present invention.

[0042] Figure 2 This is a diagram illustrating the mechanism of action of the polypeptide fluorescent probe of the present invention.

[0043] Figure 3 shows the 1H NMR spectrum (A), 1C NMR spectrum (B), and mass spectrum (C) of compound F2.

[0044] Figure 4 shows the 1H NMR spectrum (A), 1C NMR spectrum (B), and mass spectrum (C) of compound FL.

[0045] Figure 5 shows the high performance liquid chromatography retention time (A) and mass spectrum (B) of the compound Biotin-GP-FL.

[0046] Figure 6 This is an inhibition experiment of the polypeptide fluorescent probe of the present invention.

[0047] Figure 7 This is the response fluorescence spectrum of the polypeptide fluorescent probe Biotin-GP-FL and rhFAP of the present invention.

[0048] Figure 8 shows the reaction kinetics performance test of the polypeptide fluorescent probe Biotin-GP-FL with rhFAP (A) and MCF-7 cell suspension (B) of the present invention.

[0049] Figure 9 This invention relates to the concentration gradient test of the polypeptide fluorescent probe Biotin-GP-FL and rhFAP.

[0050] Figure 10 This invention relates to the selective assay of the polypeptide fluorescent probe Biotin-GP-FL.

[0051] Figure 11This invention relates to a cytotoxicity test of different concentrations of the polypeptide fluorescent probe Biotin-GP-FL; wherein, A is MDA-MB-468, B is MCF-7, C is MDA-MB-231, and D is 4T1.

[0052] Figure 12 This invention relates to the FAP specificity and biotin targeting assay of the polypeptide fluorescent probe Biotin-GP-FL; wherein, A is MDA-MB-468 and B is MDA-MB-231.

[0053] Figure 13 shows the expression levels of FAP protein in different breast cancer cell lines (A) and fluorescence imaging of the present invention's polypeptide fluorescent probe Biotin-GP-FL in different breast cancer cell lines (B).

[0054] Figure 14 This is an image of a human breast cancer tissue sample slice incubated with the polypeptide fluorescent probe Biotin-GP-FL and excited at a wavelength of 488 nm.

[0055] Figure 15 The images (A) and quantization curves (B) show human breast cancer tissue samples incubated with the same concentration of Biotin-GP-FL probe at different times and excited at 488 nm.

[0056] Figure 16 This is an image of a human breast cancer tissue sample section stained with Biotin-GP-FL and excited at 488 nm, along with the corresponding HE staining image. Detailed Implementation

[0057] To better understand the technical content of the present invention, specific embodiments are described below in conjunction with the accompanying drawings.

[0058] Various aspects of the invention are described in this disclosure with reference to the accompanying drawings, in which numerous illustrative embodiments are shown. The embodiments of this disclosure are not necessarily intended to encompass all aspects of the invention. It should be understood that the various concepts and embodiments described above, as well as those described below in more detail, can be implemented in any of a number of ways.

[0059] Peptide fluorescent probe

[0060] In an exemplary embodiment of the present invention, a polypeptide fluorescent probe is provided for in situ specific detection of fibroblast activation proteins. The polypeptide fluorescent probe includes biotin, a specific dipeptide substrate (GP), and a dye molecule ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile (FL), wherein biotin and FL are linked by GP.

[0061] Biotin receptors are highly expressed in various cancers such as breast cancer, ovarian cancer, and lung cancer. Therefore, modifying the peptide fluorescent probe with biotin molecules can further enhance the probe's specific targeting effect.

[0062] GP is a FAP-specific dipeptide substrate. The middle part of the polypeptide fluorescent probe Biotin-GP-FL acts as a link between FL and the Biotin targeting group, and can be specifically recognized and cleaved by FAP.

[0063] The molecular structural formulas of Biotin, GP and FL are shown in Formula I, Formula II and Formula III, respectively.

[0064] Formula I;

[0065] Formula II;

[0066] Formula III.

[0067] The molecular structure of the polypeptide fluorescent probe is shown in Formula IV, denoted as Biotin-GP-FL;

[0068] Formula IV.

[0069] Preparation method

[0070] Combination Figure 1 As shown, in another exemplary embodiment of the present invention, a method for preparing the aforementioned polypeptide fluorescent probe capable of in-situ specific detection of fibroblast activation proteins is provided, characterized by comprising the following steps:

[0071] 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile was prepared by isophorone (denoted as F1), and 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile was denoted as F2;

[0072] ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile was prepared by F2, and ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile was denoted as FL;

[0073] Biotin-GP-OH was synthesized from Fmoc-Pro-OH, Fmoc-Gly-OH and D-Biotin via a solid-state reaction.

[0074] Biotin-GP-OH was reacted with FL, and the reactants were purified and separated to obtain a polypeptide fluorescent probe, denoted as Biotin-GP-FL.

[0075] As an optional example, the preparation of 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile via isophorone includes the following preparation process:

[0076] F1 and malononitrile were dissolved in ethanol, glacial acetic acid and piperidine were added, and the mixture was heated to reflux under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature and the ethanol was removed under vacuum. The mixture was purified by rapid column chromatography and dried by rotary evaporation to obtain F2.

[0077] As an optional example, the molar ratio of F1, malononitrile, glacial acetic acid and piperidine is 10:(10-15):0.1:1, and is particularly preferred to be 10:12:0.1:1.

[0078] As an optional example, the preparation of ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile via F2 includes the following preparation process:

[0079] F2 and 4-aminobenzaldehyde were dissolved in ethanol, piperidine was added, and the mixture was heated to reflux under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered to collect the precipitate, and dried to obtain FL.

[0080] As an optional example, the molar ratio of F2, 4-aminobenzaldehyde and piperidine is (2-3):2.4:1, and is particularly preferred to be 2.16:2.4:1.

[0081] As an alternative example, Biotin-GP-OH is synthesized from Fmoc-Pro-OH, Fmoc-Gly-OH, and D-Biotin via a solid-state reaction, including the following preparation process:

[0082] Fmoc-Pro-OH was first inoculated into CTC resin, deprotected with 20% piperidine solution, washed with DMF, and deprotection was verified to be complete by ninhydrin detection. Then, Fmoc-Gly-OH and D-Biotin were inoculated sequentially until Biotin-GP-CTC resin was obtained. After that, Biotin-GP-OH was cleaved off the resin with trifluoroethanol and freeze-dried to obtain Biotin-GP-OH.

[0083] As an optional example, the ratio of CTC resin, Fmoc-Pro-OH, Fmoc-Gly-OH and D-Biotin is 2 g: 1.6 mmol: (1.6-3) mmol: (1-2) mmol, and is particularly preferred to be 2 g: 1.6 mmol: 3 mmol: 2 mmol.

[0084] In one specific example, Fmoc-Pro-OH was mixed with CTC resin, and dichloromethane (DCM) and DIEA were added to the reactor and reacted at 30 °C. Then, 20% piperidine was added to remove the Fmoc protecting group. The mixture was washed with DMF, and the color of the resin was detected by the ninhydrin method to verify whether the deprotection was successful.

[0085] Fmoc-Gly-OH was added, along with an appropriate amount of HOBT, N,N-diisopropylcarbodiimide (DIC), and the mixture was reacted in a reactor at 30 °C to obtain Fmoc-GP-CTC resin. 20% piperidine solution was then added to remove the protective layer.

[0086] D-Biotin was weighed and added to a reactor, along with an appropriate amount of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (HBTU), HOBT, and DIEA. Biotin-GP-CTC resin was obtained in the reactor and reacted at 30 °C. Then, Biotin-GP-OH was cleaved off the resin with 20% trifluoroethanol and lyophilized.

[0087] Understandably, in the above reaction process, there is no strictly defined proportional relationship between Fmoc-Pro-OH, DCM, and DIEA; between Fmoc-Gly-OH, HOBT, and DIC; and between D-Biotin, HBTU, HOBT, and DIEA, which can be adjusted according to specific circumstances.

[0088] In one example, the ratio of CTC resin, Fmoc-Pro-OH, DCM and DIEA was 2 g:1.6 mmol: 20 mL:3.2 mmol; the molar ratio of Fmoc-Gly-OH, HOBT and DIC was 3:4.5:4.5; and the molar ratio of D-Biotin, HBTU, HOBT and DIEA was 2:3:3:4.

[0089] As an optional example, Biotin-GP-OH is reacted with FL, and the reactants are purified and separated to obtain a polypeptide fluorescent probe, including the following preparation process:

[0090] Biotin-GP-OH and FL were dissolved in DMF, and HATU, HOBT and DIEA were added to activate Biotin-GP-OH. The activated Biotin-GP-OH was then added dropwise to the FL solution to react. After purification and lyophilization, Biotin-GP-FL was obtained.

[0091] As an optional example, the molar ratio of Biotin-GP-OH, HATU, HOBT, DIEA and FL is (1:1:1:2:1.2)-(1:3:3:3:1.2), and is particularly preferred to be 1:3:3:3:1.2.

[0092] Below, as a specific example, the preparation process of a polypeptide fluorescent probe capable of in situ specific detection of fibroblast activation proteins includes the following steps:

[0093] Step 1: Dissolve F1 and malononitrile in ethanol, add glacial acetic acid and piperidine, and heat under reflux for 12 h under nitrogen protection; after the reaction is completed, cool to room temperature and remove ethanol under vacuum, purify by rapid column chromatography, and evaporate to dryness to obtain 2-(3,5,5-trimethylcyclohex-2-en-1-yl)malononitrile; which is F2; ​​wherein, the molar ratio of F1, malononitrile, glacial acetic acid and piperidine is 10:(10-15):0.1:1.

[0094] Step 2: Dissolve F2 and 4-aminobenzaldehyde obtained in Step 1 in ethanol, add piperidine, and heat under reflux for 12 h under nitrogen protection. After the reaction is complete, cool to room temperature, filter to collect the precipitate, and dry to obtain ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile; which is FL; wherein the molar ratio of F2, 4-aminobenzaldehyde and piperidine is (2-3):2.4:1.

[0095] Step 3: Mix Fmoc-Pro-OH with CTC resin, add DCM and DIEA to the reactor and react at 30 °C for 2 h. Then add 20% piperidine and react for 20 min to remove the Fmoc protecting group. Wash with DMF and use the ninhydrin method to detect the color of the resin to verify whether the deprotection was successful.

[0096] Fmoc-Gly-OH and an appropriate amount of HOBT were added. DIC was reacted in the reactor at 30 °C for 1 h to obtain Fmoc-GP-CTC resin. 20% piperidine solution was added to remove the protection.

[0097] D-Biotin was weighed and added to the reactor, along with appropriate amounts of HBTU, HOBT, and DIEA, to obtain Biotin-GP-CTC resin. The resin was reacted at 30 °C for 30 min, and then Biotin-GP-OH was cleaved off the resin using 20% ​​trifluoroethanol and freeze-dried.

[0098] The ratio of CTC resin, Fmoc-Pro-OH, Fmoc-Gly-OH and D-Biotin is 2 g: 1.6 mmol: (1.6-3) mmol: (1-2) mmol.

[0099] Weigh out Biotin-GP-OH and dissolve it in DMF. Add 3 times the amount of HATU+HOBT+DIEA and activate it for 10 min. Then weigh out 1.2 times the amount of FL and dissolve it in DMF. Add the activated Biotin-GP-OH dropwise to the FL solution and react at 30 °C for 1 h. Purify and separate to obtain Biotin-GP-FL.

[0100] In other examples of the present invention, the application of the aforementioned polypeptide fluorescent probe capable of in situ specific detection of fibroblast activation proteins or the polypeptide fluorescent probe prepared by the aforementioned method in the detection of fibroblast activation proteins is also provided.

[0101] Combination Figure 2 As shown, when FAP is present, GP is specifically recognized and cleaved, thereby releasing free fluorescent dye molecules FL, generating a strong fluorescent signal, thus achieving specific detection of FAP.

[0102] To facilitate better understanding, the present invention will be further illustrated below with several specific examples, but the preparation process is not limited to these examples, and the content of the present invention is not limited to these examples.

[0103] Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0104] Example 1

[0105] [Reference Figure 1 [Preparation of Biotin-GP-FL fluorescent probe via the shown synthetic route]

[0106] Step 1: Dissolve F1 (1.38 g, 10 mmol) and malononitrile (0.8 g, 12 mmol) in 40 mL of ethanol, add glacial acetic acid (5 μL, 0.1 mmol) and piperidine (1 μL, 1 mmol), and heat under reflux for 12 h under nitrogen protection. After the reaction is complete, cool to room temperature and remove ethanol under vacuum. Purify by rapid column chromatography and evaporate to dryness to obtain 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malononitrile, which is F2.

[0107] Step 2: Dissolve F2 (0.4 g, 2.16 mmol) and 4-aminobenzaldehyde (0.4 g, 2.4 mmol) obtained in Step 1 in 30 mL of ethanol, add piperidine (1 μL, 1 mmol), and heat under reflux for 12 h under nitrogen protection. After the reaction is complete, cool to room temperature, filter to collect the precipitate, and dry to obtain ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile, which is FL.

[0108] Step 3: Mix Fmoc-Pro-OH (0.43 g, 1.6 mmol) with CTC (2 g) resin, add 20 mL of DCM and DIEA (0.41 g, 3.2 mmol) to the reactor and react at 30 °C for 2 h. Then add 30 mL of 20% piperidine and react for 20 min to remove the Fmoc protecting group. Wash with DMF and verify that the resin is reddish-brown by the ninhydrin method, indicating successful deprotection.

[0109] Weigh out Fmoc-Gly-OH (0.74 g, 3 mmol), and add HOBT (0.62 g, 4.5 mmol) and DIC (0.62 g, 4.5 mmol) to the reactor. React at 30 °C for 1 h to obtain Fmoc-GP-CTC resin. Add 30 mL of 20% piperidine solution for deprotection, wash with DMF, and verify that the resin is reddish-brown using the ninhydrin method, indicating successful deprotection.

[0110] D-Biotin (0.49 g, 2 mmol) was weighed and added to the reactor, along with HBTU (1.18 g, 3 mmol), HOBT (0.41 g, 3 mmol), and DIEA (1.17 g, 4 mmol). The mixture was reacted at 30 °C for 30 min to obtain Biotin-GP-CTC resin. Subsequently, Biotin-GP-OH was cleaved from the CTC resin using 30 mL of 20% trifluoroethanol and then lyophilized to obtain Biotin-GP-OH.

[0111] Biotin-GP-OH (0.40 g, 1 mmol) was weighed and dissolved in 10 mL of DMF. HATU (1.18 g, 3 mmol) + HOBT (0.41 g, 3 mmol) + DIEA (0.44 g, 3 mmol) were added and activated at 30 °C for 10 min. FL (0.350 g, 1.2 mmol) was weighed and dissolved in 10 mL of DMF. The activated Biotin-GP-OH was added dropwise to FL and reacted at 30 °C for 1 h. The final product Biotin-GP-FL was obtained by purification and separation by high performance liquid chromatography.

[0112] Example 2

[0113] [Characteristics and Mechanism of Biotin-GP-FL]

[0114] The intermediate products F2 and FL generated during the preparation of the polypeptide fluorescent probe Biotin-GP-FL, as well as the final structure of the Biotin-GP-FL polypeptide fluorescent probe, can be completely confirmed by the 1H NMR spectrum, 1C NMR spectrum, HPLC retention time, and mass spectrometry shown in Figures 3-5. This proves that the present invention has successfully synthesized the polypeptide fluorescent probe Biotin-GP-FL, with the structural formula shown in Formula IV.

[0115] like Figure 2 As shown, the Biotin-GP-FL polypeptide fluorescent probe involved in this invention can first selectively target tumor sites based on Biotin and Biotin receptor. After reaching the tumor site, under the action of FAP, the intermediate linker of the Biotin-GP-FL polypeptide fluorescent probe, namely the specific dipeptide substrate GP of FAP, is cleaved, releasing free fluorescent molecules FL, generating fluorescence, and thus realizing the quantitative detection of FAP.

[0116] Example 3

[0117] [In vitro testing of Biotin-GP-FL]

[0118] The reaction system for in vitro testing was as follows: each well had a total volume of 100 μL, including 1 μL of 1 mM Biotin-GP-FL stock solution, 0.125 μL of 400 μg / mL recombinant human fibroblast activator protein (rhFAP) stock solution, and 98.875 μL of 10 mM Triton-PBS (pH 7.4). The Biotin-GP-FL stock solution was prepared from DMSO, with a final test concentration of 10 μM, and the final test concentration of rhFAP was 0.5 μg / mL. The reaction system was incubated on a constant temperature shaker at 37°C and 220 rpm. This system was used for all rhFAP tests and is referred to as reaction system 1.

[0119] (1) Inhibition experiment, specifically: MCF-7 cells were resuspended in 0.05% Triton-PBS (10 mM, pH 7.4) solution to a cell density of 4 × 10⁻⁶. 6 / mL; Take 500 μL of the above cell suspension, add 4.975 μL of FAP-specific inhibitor SP-13786 (10 mM, DMSO) to a final concentration of 50 μM, and 490 μL of 0.05% Triton-PBS (10 mM, pH 7.4), and pre-incubate at 37 ℃ and 220 rpm for 2 h. The control group is added with the same volume of DMSO. After incubation, add 1 μL of Biotin-GP-FL (10 mM, DMSO) and 4.025 μL of DMSO, and continue incubating at 37 ℃ for 2 h. The control group is added with the same volume of DMSO. Then, detect the fluorescence intensity of the control group and the inhibitor group at an excitation wavelength of 480 nm.

[0120] like Figure 6 As shown, compared with the control group, the fluorescence intensity of the inhibitor group decreased significantly, indicating that the polypeptide fluorescent probe of the present invention exhibits a specific response to FAP (λex = 480 nm).

[0121] (2) The responsiveness of Biotin-GP-FL to rhFAP was tested using reaction system 1, and the results are as follows: Figure 7 As shown, when the reaction system is only Biotin-GP-FL, i.e. without rhFAP and the same volume of 0.05% Triton-PBS (10 mM, pH 7.4) is added, weak fluorescence can be observed at 660 nm wavelength when a 480 nm laser is used as the excitation wavelength; while the fluorescence intensity is significantly enhanced after incubation with rhFAP (0.5 μg / mL) for 6 h.

[0122] The above results demonstrate that the Biotin-GP-FL of the present invention can detect changes in FAP activity (λex = 480 nm).

[0123] (3) The reaction system 1 was used for testing, and samples were taken at incubation times of 0, 30, 60, 120, 180, 240, 300, 360, 420, and 480 min. The results are as follows: Figure 8A As shown, when the concentrations of Biotin-GP-FL and rhFAP are 10 μM and 0.5 μg / mL, respectively, the fluorescence intensity of the reaction system at an incubation temperature of 37 °C gradually increases with the extension of incubation time.

[0124] MCF-7 cells were resuspended in 0.05% Triton-PBS (10 mM, pH 7.4) to a cell density of 4 × 10⁻⁶ cells / year. 6 / mL; Take 500 μL of the above cell suspension, add 1 μL of Biotin-GP-FL (10 mM, DMSO) and 499 μL of 0.05% Triton-PBS (10 mM, pH 7.4) solution, and incubate at 37 ℃. Samples are taken for testing at incubation times of 0, 15, 30, 45, 60, 90, 120, 150, and 180 min. The results are as follows. Figure 8B As shown, the fluorescence intensity reached a plateau (λex = 480 nm) after incubation for about 120 min.

[0125] The above results indicate that the Biotin-GP-FL of the present invention has a relatively fast response rate to FAP in MCF-7 cells.

[0126] (4) The responsiveness of Biotin-GP-FL to different concentrations of rhFAP was tested using reaction system 1. Specifically, in a 0.05% Triton-PBS (10 mM, pH 7.4) reaction system, when the concentration of Biotin-GP-FL was 10 μM, it was mixed with different concentrations of rhFAP (0, 0.2, 0.4, 0.6, 0.8, and 1.0 μg / mL) and incubated at 37 ℃ for 2 h to obtain the fluorescence spectra (λex = 480 nm) corresponding to different concentrations of rhFAP. The results are as follows: Figure 9 As shown.

[0127] As can be seen from the figure, the fluorescence intensity of Biotin-GP-FL after reacting with rhFAP gradually increases with the increase of rhFAP concentration, indicating that Biotin-GP-FL of the present invention can quantitatively detect changes in FAP activity.

[0128] (5) The selectivity performance of Biotin-GP-FL was tested using reaction system 1, specifically: 35 common inorganic anions and cations, amino acids, proteins and biological enzymes were selected, where 1: rhFAP (0.5 μg / mL); 2-31: K + Na + Cu 2+ Fe 2+ Fe 3+ Al 3+ Zn 2+ Mg 2+ , Cr 2+ , Cl - NO 2- ClO4 - HS - P3O4 2- SO32- SO4 2- CO3 2- HSO3 - , Vit C, Vit E, ATP, GSH, Trp, Gly, Tyr, Ala, Lys, Arg, Cys, Put (100 μM); 32-33: HSA, BSA (10 μg / mL); 34-36: Enterokinase, Carboxylesterase, Acetylcholinesterase (1 U / mL); 37: Blank; In a 0.05% Triton-PBS (10 mM, pH 7.4) reaction system, the above interfering substances were added sequentially to a final concentration of 10 μM for Biotin-GP-FL. The mixture was incubated at 37 ℃ for 2 h, and the fluorescence intensity at 660 nm (λex = 480 nm) was measured. The results are as follows: Figure 10 As shown.

[0129] As can be seen from the figure, there is a significant enhancement of fluorescence signal only when rhFAP is added, while there is no significant enhancement of fluorescence signal when other interfering substances are added. This result indicates that the Biotin-GP-FL of the present invention has a high selectivity for the response to rhFAP.

[0130] Example 4

[0131] [Intracellular assay of Biotin-GP-FL]

[0132] (1) Cytotoxicity analysis of Biotin-GP-FL, specifically as follows: different breast cancer cell lines (MDA-MB-468, MCF-7, MDA-MB-231 and 4T1) after trypsin digestion were divided into groups of 1 × 10⁻⁶ cells. 4 Cells were seeded in 96-well plates and incubated at 37 °C for 24 h until adherence. The supernatant was discarded, and different concentrations of Biotin-GP-FL (0, 5, 10, 20, 50, and 100 μM) were added to the wells. Incubation continued for 24 h, the supernatant was discarded, and the cells were washed with PBS. 50 μL of 1× MTT was added, and incubation continued at 37 °C for 4 h. The supernatant was discarded, and 150 μL of DMSO was added and shaken on a shaker to dissolve the cells. The absorbance of each well was then measured at 490 nm. The results are shown below. Figure 11 As shown.

[0133] As can be seen from the figure, after incubating different concentrations of Biotin-GP-FL with MDA-MB-468, MCF-7, MDA-MB-231 and 4T1 cells for 24 h, the cell survival rate can reach about 80%, which shows that the Biotin-GP-FL of the present invention has high biosafety.

[0134] (2) To verify the FAP specificity and biotin targeting of Biotin-GP-FL, specifically: MDA-MB-468 and MCF-7 breast cancer cells digested with trypsin were subjected to a reaction at a concentration of 1 × 10⁻⁶. 5 Cells were seeded in 6-well plates and incubated at 37 °C for 24 h until adherence. After washing with PBS, 1 mL / well of SP-13786 (10 μM, 1% DMSO) and free Biotin (2 mM, 1% DMSO) were added, and the plates were pre-incubated at 37 °C for 2 h, with 1% DMSO as a control. After incubation, cells were washed with PBS, and 1 mL / well of Biotin-GP-FL solution (10 μM, 1% DMSO) was added, and the plates were incubated at 37 °C for 60 min. After washing with PBS, cells were digested with trypsin, centrifuged, and resuspended in 1% BSA-PBS buffer for analysis. Results are shown below. Figure 12 As shown.

[0135] As can be seen from the figure, compared with the control group, the fluorescence intensity of SP-13786 and the free Biotin pre-incubation group decreased, which indicates that the Biotin-GP-FL of the present invention has FAP specific response capability and biotin targeting.

[0136] (3) Imaging effect test of Biotin-GP-FL on different breast cancer cells (MDA-MB-468, MCF-7 and MDA-MB-231): Biotin-GP-FL (10 μM, 1% DMSO) was incubated with MDA-MB-468, MCF-7 and MDA-MB-231 breast cancer cells at 37 ℃ for 60 min. After washing with PBS, live cell imaging was performed under a confocal fluorescence microscope. The results are shown in Figure 13.

[0137] As can be seen from the figure, according to the Western blot results (A), the highest FAP expression level was found in MDA-MB-468 among the three breast cancer cell lines; the fluorescence imaging results in Figure B show that Biotin-GP-FL has the strongest fluorescence intensity in MDA-MB-468 cell imaging, indicating that the Biotin-GP-FL of the present invention can quantitatively detect FAP activity in living cells.

[0138] Example 5

[0139] [In-tissue testing of Biotin-GP-FL]

[0140] To verify the penetration effect of Biotin-GP-FL on human breast cancer tissue, imaging tests were performed on human breast cancer tissue sections using Biotin-GP-FL. Imaging tests were conducted on human breast cancer tissue and adjacent tissue sections under 488 nm laser light.

[0141] Cancerous and adjacent normal tissue sections with a thickness of 300 μm were placed in 1.5 mL centrifuge tubes, and 100 μL of 60 μM Biotin-GP-FL (5% DMSO) was added. The tubes were incubated at 37 ℃ for 60 min. Cancerous tissue + PBS and adjacent normal tissue + Biotin-GP-FL served as controls. The tissue sections were then placed on glass slides, covered with coverslips, and imaged under a confocal fluorescence microscope.

[0142] The results are as follows Figure 14 As shown, the fluorescence intensity of cancer tissue sections is much higher than that of adjacent normal tissue sections, and almost complete penetration is achieved. This result proves that the Biotin-GP-FL of the present invention has excellent penetration effect on tissue sections.

[0143] Meanwhile, to demonstrate the time stability of Biotin-GP-FL for in situ imaging of human breast cancer tissue, human breast cancer tissue was immersed in 60 μM Biotin-GP-FL (5% DMSO) and incubated at 37 ℃. The changes in fluorescence imaging intensity at different incubation times were observed (A) and the corresponding quantitative fluorescence results (B).

[0144] The results are as follows Figure 15 As shown, the fluorescence intensity of the polypeptide fluorescent probe reaches a plateau after 100 min of incubation and remains stable within 180 min, thus demonstrating that the Biotin-GP-FL of the present invention has good long-term stable imaging capability.

[0145] Furthermore, to verify the imaging capability of Biotin-GP-FL for human breast cancer tissue, human breast cancer tissue was stained with DAPI + Biotin-GP-FL, and another corresponding tissue sample was stained with HE. The in situ imaging capability of Biotin-GP-FL for human breast cancer tissue was analyzed by comparing the results of the two studies. The results are as follows: Figure 16 As shown, the imaging results of Biotin-GP-FL of the present invention correspond to the HE staining results, and have the potential to distinguish between normal and cancerous tissues.

[0146] As shown above, this invention successfully synthesized the polypeptide fluorescent probe Biotin-GP-FL, with an emission wavelength of approximately 660 nm. It features low background fluorescence interference, high specificity, good selectivity, strong targeting, good biosafety, excellent tissue penetration depth, and stable imaging over long periods. Furthermore, the polypeptide fluorescent probe Biotin-GP-FL exhibits stable imaging capability for human breast cancer tissue samples over long periods and possesses excellent tissue penetration, exhibiting strong contrast with adjacent normal tissue. This enables in-situ real-time quantitative detection of FAP activity levels within cancer tissue, providing a reference for the subsequent clinical application of this polypeptide fluorescent probe.

[0147] While the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the invention. Those skilled in the art can make various modifications and refinements without departing from the spirit and scope of the invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A polypeptide fluorescent probe for in situ specific detection of fibroblast activation proteins, characterized in that, This polypeptide fluorescent probe consists of biotin, a specific dipeptide substrate, and a dye molecule. The specific dipeptide substrate is glycine-proline (GP), and the dye molecule is (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonitrile (FL). The biotin and FL are linked by GP. The molecular structure of the polypeptide fluorescent probe is shown in Formula IV, denoted as Biotin-GP-FL; Formula IV; The molecular structural formulas of Biotin, GP and FL are shown in Formula I, Formula II and Formula III, respectively. Formula I; Formula II; Formula III.

2. A method for preparing a polypeptide fluorescent probe for in situ specific detection of fibroblast activation proteins as described in claim 1, characterized in that, Includes the following steps: 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile was prepared by isophorone, and 2-(3,5,5-trimethylcyclohexyl-2-en-1-yl)malonitrile was designated as F2; ((E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malonadionitrile (FL) was prepared by F2. Biotin-GP-OH was synthesized from Fmoc-Pro-OH, Fmoc-Gly-OH and D-Biotin via a solid-state reaction. Biotin-GP-OH was reacted with FL, and the reactants were purified and separated to obtain a polypeptide fluorescent probe, denoted as Biotin-GP-FL.

3. The method for preparing a polypeptide fluorescent probe capable of in-situ specific detection of fibroblast activation proteins according to claim 2, characterized in that, The preparation of 2-(3,5,5-trimethylcyclohex-2-en-1-yl)malononitrile via isophorone includes the following preparation process: Isophorone and malononitrile were dissolved in ethanol, and glacial acetic acid and piperidine were added. The mixture was heated to reflux under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature and the ethanol was removed under vacuum. The mixture was purified by rapid column chromatography and dried by rotary evaporation to obtain F2. The molar ratio of isophorone, malononitrile, glacial acetic acid and piperidine was 10:(10-15):0.1:

1.

4. The method for preparing a polypeptide fluorescent probe capable of in-situ specific detection of fibroblast activation proteins according to claim 2, characterized in that, The preparation of (E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-yl)malononitrile via F2 includes the following preparation process: F2 and 4-aminobenzaldehyde were dissolved in ethanol, piperidine was added, and the mixture was heated to reflux under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature, filtered to collect the precipitate, and dried to obtain FL. The molar ratio of F2, 4-aminobenzaldehyde and piperidine was (2-3):2.4:

1.

5. The method for preparing a polypeptide fluorescent probe capable of in-situ specific detection of fibroblast activation proteins according to claim 2, characterized in that, The synthesis of Biotin-GP-OH via solid-phase reaction of Fmoc-Pro-OH, Fmoc-Gly-OH, and D-Biotin includes the following preparation process: Fmoc-Pro-OH was first inoculated into CTC resin, deprotected with 20% piperidine solution, washed with N,N-dimethylformamide (DMF), and deprotection was verified to be complete by ninhydrin detection. Then, Fmoc-Gly-OH and D-Biotin were inoculated sequentially until Biotin-GP-CTC resin was obtained. After that, Biotin-GP-OH was cleaved off the resin with trifluoroethanol and freeze-dried to obtain Biotin-GP-OH.

6. The method for preparing a polypeptide fluorescent probe capable of in-situ specific detection of fibroblast activation proteins according to claim 5, characterized in that, The ratio of CTC resin, Fmoc-Pro-OH, Fmoc-Gly-OH and D-Biotin is 2 g: 1.6 mmol: (1.6-3) mmol: (1-2) mmol.

7. The method for preparing a polypeptide fluorescent probe capable of in-situ specific detection of fibroblast activation proteins according to claim 2, characterized in that, The process of reacting Biotin-GP-OH with FL, purifying and separating the reactants to obtain a polypeptide fluorescent probe includes the following preparation steps: Biotin-GP-OH and FL were dissolved in DMF, and O-benzotriazole-tetramethylurea hexafluorophosphate (HATU), 1-hydroxybenzotriazole (HOBT) and N,N-diisopropylethylamine (DIEA) were added to activate Biotin-GP-OH. The activated Biotin-GP-OH was then added dropwise to the FL solution to react. After purification and lyophilization, Biotin-GP-FL was obtained.

8. The method for preparing a polypeptide fluorescent probe capable of in situ specific detection of fibroblast activation proteins according to claim 7, characterized in that, The molar ratio of Biotin-GP-OH, HATU, HOBT, DIEA and FL is (1:1:1:2:1.2)-(1:3:3:3:1.2).

9. The application of a polypeptide fluorescent probe according to claim 1 for in situ specific detection of fibroblast activation proteins, or a polypeptide fluorescent probe prepared by the method described in any one of claims 2-8, in the preparation of fibroblast activation protein detection products.

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