Aminopeptidase fluorescent probe, preparation method and application thereof

Abstract

The application belongs to the technical field of fluorescent probe, and particularly relates to an aminopeptidase fluorescent probe and a preparation method and application thereof. In order to develop an APN near-infrared fluorescent probe suitable for in vivo imaging, a center-position ester group substituted pentamethine cyanine dye platform (Cy5-COOM) is used, and based on an "ester group to carboxyl group" conversion strategy, an aminopeptidase (APN) fluorescent probe Cy5-APN is developed. The probe is firstly hydrolyzed by APN, then a 1,6-elimination reaction occurs, and finally a strong fluorescent center-position carboxyl group substituted pentamethine cyanine dye Cy5-COO is released. In addition, in view of the characteristics that cancer cell surfaces overexpress APN, the probe can distinguish cancer cells / tissues and normal cells / tissues with high contrast, and therefore has great application potential in fluorescence-guided tumor surgery.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to an aminopeptidase fluorescent probe, its preparation method, and its application. Background Technology

[0002] Aminopeptidase N (APN / CD13, EC 3.4.11.2) is a zinc-dependent, membrane-bound type II metallotransmembrane glycoprotein widely distributed in mammals and present as a homodimer on the plasma membrane. It participates in various important physiological functions through both enzyme-dependent and non-enzyme-dependent pathways, including signal transduction, neuropeptide degradation, immune responses, and antigen processing. Furthermore, APN is widely considered a marker of myeloid-derived hematopoietic cells and promotes the classification of human leukemia cells through its antigenicity. Drug-induced liver injury (DILI) is considered a major cause of acute liver injury, accompanied by abnormal APN expression. APN plays a crucial role in tumor invasion, angiogenesis, and metastasis, and exhibits enhanced enzymatic activity in cancer cells; therefore, APN can serve as a cancer biomarker for cancer assessment and diagnosis. Given the important physiological and pathological functions of APN, developing highly selective and sensitive methods for real-time detection of APN will advance the diagnosis and pathological research of APN-related diseases.

[0003] Due to their high selectivity, sensitivity, spatiotemporal resolution, and non-destructive nature, fluorescent probe technology is widely used for real-time detection of various physiological and pathological processes, playing a crucial role in disease diagnosis and surgery. Furthermore, due to the strong tissue penetration of near-infrared light and the low autofluorescence in the near-infrared region, near-infrared fluorescent dyes exhibit higher spatiotemporal resolution in muscle and in vivo fluorescence imaging compared to visible-region fluorescent dyes. However, only a few APN near-infrared fluorescent probes suitable for in vivo imaging have been reported, thus highlighting the urgent need to develop novel APN near-infrared fluorescent probes. Summary of the Invention

[0004] This invention utilizes a centromeric pentamethine cyanine dye platform (Cy5-COOM) with a centrally ester-substituted group. Based on an "ester → carboxyl" conversion strategy, an aminopeptidase (APN) fluorescent probe, Cy5-APN, is developed. This probe is first hydrolyzed by APN overexpressed on the surface of cancer cells, followed by a 1,6-elimination reaction, ultimately releasing the strongly fluorescent centromeric carboxyl-substituted pentamethine cyanine dye Cy5-COO, thus illuminating the cancer cells. Cellular and in vivo experiments have confirmed that this probe can distinguish cancer cells / tissues from normal cells / tissues with high contrast, thus possessing great application potential in fluorescence-guided tumor surgery.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] An aminopeptidase fluorescent probe has the following structural formula:

[0007]

[0008] A method for preparing an aminopeptidase fluorescent probe includes the following steps:

[0009]

[0010] Step 1: Under N2 environment, DMF is dissolved in anhydrous dichloromethane, and then oxalyl chloride is gradually added. The reaction is stirred at room temperature. After the reaction is completed, the solvent is completely evaporated under reduced pressure to obtain compound 1, which is a white solid. It is used directly in the next reaction without purification.

[0011] Step 2: Compound 1 and monomethyl malonate were dissolved in anhydrous dichloromethane and refluxed. After evaporating the solvent under reduced pressure, compound 2 was obtained. Acetic anhydride, 1,2,3,3-tetramethyl-3H-indole iodide, and anhydrous sodium acetate were added to the mixture in sequence and stirred. The mixture was then diluted with water and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, then purified by vacuum distillation and column chromatography to obtain compound Cy5-COOM, which was a dark blue solid.

[0012] Step 3: Dissolve compound Cy5-COOM in a mixed solution of MeOH and NaOH, stir and react, cool, dilute with water and extract with dichloromethane, combine the organic layers and dry with anhydrous sodium sulfate, then purify by vacuum distillation and column chromatography to obtain compound Cy5-COO, which is a dark blue solid.

[0013] Step 4: Cy5-COO, compound 3, and potassium carbonate were dissolved in anhydrous N,N-dimethylformamide and stirred. After the reaction was completed, the mixture was cooled, diluted with water, and extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was redissolved in a mixed solution of trifluoroacetic acid and dichloromethane. After further stirring, the mixture was purified by reduced pressure distillation and column chromatography to obtain compound Cy5-APN, which is a dark blue solid.

[0014] Furthermore, in step 1, the molar ratio of DMF to oxaloyl chloride is 30:35, and the stirring reaction time is 2 hours.

[0015] Furthermore, in step 2, the molar ratio of compound 1 to monomethyl malonate is 2:1, the reflux reaction temperature is 40°C, and the reaction time is overnight; the molar ratio of compound 2, 1,2,3,3-tetramethyl-3H-indole iodide, and anhydrous sodium acetate is 1:2:3; the stirring reaction temperature is 90°C, and the reaction time is 4 hours; the developing solvent for column chromatography separation and purification is dichloromethane / methanol = 10 / 1 (v / v).

[0016] Furthermore, in step 3, the stirring reaction temperature is 43°C, the time is 3 hours, and the developing solvent for column chromatography separation and purification is 10-50% methanol / dichloromethane (v / v).

[0017] Furthermore, in step 4, the molar ratio of Cy5-COO, compound 3, and potassium carbonate is 1:3:2, the stirring temperature is 48°C, the time is 3 hours, the volume ratio of trifluoroacetic acid to dichloromethane is 1:1, the further stirring temperature is room temperature, the time is 20 minutes, and the developing solvent for column chromatography separation and purification is CH2Cl2 / MeOH = 8:1 (v / v).

[0018] An aminopeptidase fluorescent probe can be used to distinguish between normal cells / tissues and cancer cells / tissues, serving as a fluorescent imaging reagent in fluorescence-guided surgery.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] Most currently developed APN fluorescent probes are constructed based on the protection and deprotection of the amino group in fluorescent dyes. That is, the interaction between APN and the probe alters the dye's ICT (Inductively Coupled Transform) behavior, achieving a fluorescence on / off response and thus sensing the APN. However, the APN fluorescent probe Cy5-APN in this invention is constructed based on an excited-state π-conjugation mechanism involving a central ester group. Specifically, APN can induce an ester-to-carboxyl group transition in the probe, resulting in a large fluorescence on / off change. Furthermore, utilizing the characteristic of cancer cells overexpressing APN, this probe can distinguish normal cells / tissues from cancer cells / tissues with high contrast, thus possessing potential application value in surgical navigation. Attached Figure Description

[0021] Figure 1 The NMR and HRMS spectra of compound Cy5-COO are shown.

[0022] Figure 2 The NMR and HRMS spectra of compound Cy5-COOM are shown.

[0023] Figure 3 The NMR and HRMS spectra of compound Cy5-APN are shown.

[0024] Figure 4In the figure, (A) shows the UV-Vis absorption spectra of Cy5-APN (5μM) and APN (40ng / mL) before and after reaction under PBS (10mM, pH=7.4) conditions at 37℃; (B) shows the fluorescence spectrum changes and fluorescence intensity changes at 662nm over time after reaction under PBS (10mM, pH=7.4) conditions at 37℃.

[0025] Figure 5 HPLC and HRMS chromatograms of Cy5-APN before and after the reaction with APN; HPLC conditions: C18 column (2.1×100mm); mobile phase: MeCN / H2O (3:7 to 9:1, v / v, containing 0.1% formic acid); flow rate: 0.2mL / min;

[0026] Figure 6 In the figure, (A) shows the fluorescence spectra of Cy5-APN (5 μM) before and after reaction with increased concentrations of APN (0-40 ng / mL) under the conditions of 37℃, PBS (10 mM, pH=7.4); (B) shows the linear correlation between fluorescence intensity at 660 nm and APN concentration.

[0027] Figure 7 To determine the concentrations of Cy5-APN (5 μM) in DMEM at 37°C and in PBS (10 mM, pH 7.4), or in combination with (B) NADPH (500 μM), (C) GSH (1 mM), (D) Cys (200 μM), (E) H2O2 (100 μM), and (F) O2, respectively. ·- UV-Vis absorption spectrum changes (30 minutes) after reaction with (100 μM), (G)GGT (50 U / L), and (H)NTR (2 μg / mL, containing 0.5 mM NADPH);

[0028] Figure 8 Cell viability of A549 cells after treatment with different concentrations (0 μM, 2.0 μM, 4.0 μM, 6.0 μM, 8.0 μM and 10.0 μM) of Cy5-APN for 24 hours;

[0029] Figure 9 Cell images of cancer cells (HepG2 and A549 cells) and normal cells (BEAS-2B and LO2 cells) loaded with Cy5-APN (2.0 μM), respectively; for cancer cells, cells were incubated with the probe for 60 minutes, or pre-incubated with the inhibitor (Ube, 100 μM, 60 minutes) before incubating with the probe for 60 minutes; for normal cells, cells were incubated with the probe only for 60 minutes; images were collected at wavelengths of 640-750 nm (λ). ex=633nm), scale bar: 20μm;

[0030] Figure 10 In the image, (A) shows confocal images of HepG2 tumor tissue and right leg muscle tissue loaded with Cy5-APN (2.0 μM), respectively. For the former, the tissue was incubated with the probe for 60 minutes, or pre-incubated with the inhibitor (Ube, 100 μM, 60 minutes) before incubating with the probe for 60 minutes. For the latter, the tissue was incubated with the probe only for 60 minutes. The collection wavelength was 640-750 nm (λ). ex =633nm), scale bar: 20μm; (B) is the fluorescence quantitative chromatogram of (A);

[0031] Figure 11 In vivo imaging of HepG2 tumor-bearing mice after intratumoral injection of Cy5-APN (20 μM, 50 μL); using a 610 nm excitation filter and a 700 nm emission filter. Detailed Implementation

[0032] To further illustrate the technical solution of the present invention, the present invention will be further described below through embodiments.

[0033] Example 1

[0034] The structural formula of an aminopeptidase (APN) fluorescent probe is as follows:

[0035]

[0036] The preparation method of this fluorescent probe includes the following steps:

[0037] Step 1: Under N2 environment, DMF (2.3 mL, 30 mmol) and anhydrous dichloromethane (15 mL) were added to a dry flask, followed by the gradual addition of oxaloyl chloride (3.0 mL, 35 mmol). The mixture was stirred at room temperature for 2 hours. After the reaction was completed, the solvent was evaporated completely under reduced pressure to obtain compound 1 (3.6 g, 94.7%), a white solid, which was used directly in the next reaction without purification.

[0038] Step 2: Compound 1 (1.2 g, 10 mmol) and monomethyl malonate (0.59 g, 5 mmol) were dissolved in anhydrous dichloromethane (20 mL) and refluxed overnight at 40 °C. After evaporating the solvent under reduced pressure, compound 2 was obtained. Acetic anhydride (10 mL), 1,2,3,3-tetramethyl-3H-indole iodide (3.01 g, 10 mmol) and anhydrous sodium acetate (1.23 g, 15 mmol) were added sequentially to the solution. The mixture was stirred at 90 °C for 4 hours. The solution was then diluted with water and extracted with dichloromethane. The organic layers were combined and purified by drying with anhydrous sodium sulfate, vacuum distillation, and column chromatography (dichloromethane / methanol = 10 / 1) to obtain compound Cy5-COOM (1.1 g, 38.7%), which was a dark blue solid. 1 H NMR(600Hz,CD3Cl)δ8.52(d,J=14.4Hz,2H),7.45(t,J=7.2Hz,4H),7.33(t,J =8.4Hz,4H),7.03(d,J=15.0Hz,4H),3.99(s,6H),3.93(s,3H),1.79(s,12H); 13 C NMR (150MHz, CD3Cl) δ177.7,167.1,142.5,141.2,128.9,126.4,122.3,111.9,102.1,52.0,50.2,34.3,28.2; ESI-MS[M] + :calcd for441.2537,Found 441.2526.

[0039] Step 3: The compound Cy5-COOM (852 mg, 1.5 mmol) was dissolved in a mixed solution of MeOH (40 mL) and NaOH (2 mM, 60 mL), and the mixture was stirred at 43 °C for 3 hours. After cooling, the mixture was diluted with water and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, then purified by vacuum distillation and column chromatography (10-50% methanol / dichloromethane, v / v) to obtain the compound Cy5-COO (318 mg, 38.3%), which was a dark blue solid. 1 H NMR (600Hz, CD3OD) δ8.29(s,2H),7.45(m,8H),6.90(s,2H),3.72(s,6H),1.76(s,12H); 13 C NMR (150MHz, CD3OD) δ142.7,141.4,128.4,125.5,121.9,111.0,101.7,60.1,49.4,30.6,26.7,19.5,13.1; ESI-MS[M] +:calcdfor427.2380,Found 427.2389.

[0040] Step 4: Cy5-COO (554 mg, 1.0 mmol), compound 3 (1074 mg, 3.0 mmol), and potassium carbonate (276 mg, 2.0 mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL). The mixture was stirred at 48 °C for 3 hours. After the reaction was completed, the mixture was cooled, diluted with water, and extracted with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and redissolved in a mixed solution of trifluoroacetic acid (10 mL) and dichloromethane (10 mL). The mixture was stirred at room temperature for 20 minutes. After purification by reduced pressure distillation and column chromatography (dichloromethane / methanol = 8 / 1, v / v), compound Cy5-APN was obtained as a dark blue solid (244 mg, yield 33.4%). 1 H NMR (600Hz, CD3OD) δ8.44(s,2H),7.72(d,J=8.4Hz,2H),7.59(d,J=8.4Hz,2H),7.57(d,J=9.0Hz,2H),7.49(t,J=8.4Hz,2H),7.45(d,J= 7.8Hz,2H),7.38(t,J=7.8Hz,2H),7.04(d,J=14.4Hz,2H),5.37(s,2H),4.13(m,1H),3.67(s,6H),1.76(s,12H),1.65(d,J=7.2Hz,3H); 13 C NMR (150MHz, CD3OD) δ177.5,168.0,166.3,142.4,141.5,138.1,132.6,129.4,128.5,126.1, 122.0,120.1,111.5,101.6,65.9,60.1,49.8,49.5,30.8,26.7,19.5,16.2,13.1; ESI-MS[M] + :calcd for 603.3330,Found603.3327.

[0041] Example 2

[0042] 1. Study of photophysical properties

[0043] Previous studies have found that the conversion from "ester group to carboxyl group" in the Cy5-COOM dye platform causes a significant fluorescence off-on response. The absorption and emission peaks of Cy5-COOM and Cy5-COO in PBS are 610nm / 645nm (weak fluorescence) and 636nm / 662nm, respectively.

[0044] For the aminopeptidase fluorescent probe Cy5-APN, we investigated the absorption and emission spectra of Cy5-APN before and after the reaction with APN in PBS (10 mM, pH = 7.4). The results are as follows: Figure 4 As shown. Figure 4 As shown in (A), similar to Cy5-COOM, the maximum absorption peak of Cy5-APN is located at around 610 nm. When it reacts with APN, the absorption peak of the probe at 610 nm is red-shifted to 635 nm. This absorption peak is basically consistent with the absorption peak of Cy5-COO dye in PBS, indicating that Cy5-APN generates Cy5-COO after reacting with APN. Figure 4 As shown in Figure (B), when excited at 633 nm, the probe Cy5-APN exhibits only weak fluorescence. However, after interaction with APN, the fluorescence intensity at 662 nm gradually increases and reaches saturation at 60 minutes. This emission peak can also be attributed to the Cy5-COO dye.

[0045] Figure 5 The figures show HPLC and HRMS chromatograms of Cy5-APN before and after the reaction with APN; HPLC conditions: C18 column (2.1 × 100 mm); mobile phase: MeCN / H2O (3:7 to 9:1, v / v, containing 0.1% formic acid); flow rate: 0.2 mL / min. HPLC-MS analysis also confirmed that the reaction of Cy5-APN with APN produced Cy5-COO dye.

[0046] Figure 6 (A) shows the fluorescence spectra of Cy5-APN (5 μM) before and after reaction with increased concentrations of APN (0-40 ng / mL) under PBS (10 mM, pH = 7.4) conditions at 37℃; (B) shows the linear correlation between fluorescence intensity at 660 nm and APN concentration. The fluorescence titration experiment showed that the fluorescence intensity of the probe Cy5-APN at 662 nm was linearly correlated with the APN concentration. The calculated detection limit for APN was 0.393 ng / mL.

[0047] Figure 7 To determine the concentrations of Cy5-APN (5 μM) in DMEM at 37°C and in PBS (10 mM, pH 7.4), or in combination with (B) NADPH (500 μM), (C) GSH (1 mM), (D) Cys (200 μM), (E) H2O2 (100 μM), and (F) O2, respectively. ·-UV-Vis absorption spectrum changes (30 minutes) after reaction with (100 μM), (G)GGT (50 U / L), and (H)NTR (2 μg / mL, containing 0.5 mM NADPH). Selectivity experiments showed that the probe Cy5-APN was effective in DMEM medium or in media containing NADPH, GSH, Cys, H2O2, and O2, respectively. ·- All three proteins, including GGT and NTR, are stably present in PBS. These results demonstrate that the Cy5-APN probe is a highly selective APN probe, laying the foundation for further applications in bioimaging.

[0048] 2. Cell imaging research

[0049] Before conducting cell experiments, we first analyzed the cytotoxicity of the probe Cy5-APN using the Cell Counting Kit-8 (CCK8 reagent), and the results are as follows: Figure 8 As shown, when the probe concentration is less than 10 μM, the cell survival rate is greater than 80%, which proves the low toxicity of the probe.

[0050] Furthermore, considering that Cy5-APN is overexpressed in a variety of cancer cells, we then evaluated the probe's ability to distinguish between cancer cells and normal cells using laser confocal microscopy (CLSM). Figure 9 Fluorescence images of cancer cells (including HepG2 and A549 cells) loaded with Cy5-APN and normal cells (including LO2 and BEAS-2B cells), respectively, were generated by [the authors / organizations]. Figure 9 It was observed that under 633nm laser excitation, cancer cells exhibited a significant red fluorescence signal, while the fluorescence signal in normal cells was negligible. Furthermore, when the cancer cells were pre-incubated with the Ube inhibitor before incubating the probe, almost no fluorescence signal was observed in the cancer cells, indicating that the red fluorescence signal in cancer cells was indeed caused by APN. These results demonstrate that Cy5-APN can utilize the overexpression of APN on the surface of cancer cells to distinguish cancer cells from normal cells with high contrast.

[0051] Next, we evaluated the probe's ability to distinguish between cancerous and normal tissue. First, tumor-bearing mice were obtained by subcutaneously injecting HepG2 cells into nude mice. After dissection, the tumors and part of the leg muscle tissue were removed and prepared into 20 μm sections. Imaging was then performed on HepG2 tumor tissue loaded with Cy5-APN (2.0 μM) and right leg muscle tissue, respectively. For the former, the tissue was incubated with the probe for 60 minutes, or pre-incubated with an inhibitor (Ube, 100 M, 60 minutes) before incubating with the probe for 60 minutes; for the latter, the tissue was incubated with the probe only for 60 minutes. The results are as follows: Figure 10 As shown. From Figure 10As shown in (A), tumor tissue sections loaded with Cy5-APN exhibit a bright fluorescence signal in the red channel, which can be suppressed by Ube; normal tissue sections loaded with Cy5-APN show a negligible fluorescence signal in the red channel. Figure 10 The quantitative fluorescence data in (B) show that the average fluorescence intensity of normal tissue is 9.9 times that of normal tissue, which is much greater than the clinically acceptable threshold of 2, suggesting the potential of the probe Cy5-APN in identifying tumor tissue in patients.

[0052] Finally, Cy5-APN was used for in situ real-time imaging of tumors in tumor-bearing mice, such as... Figure 11 As shown, Cy5-APN was injected orally into the tumor and left leg of HepG2 tumor-bearing mice, respectively. The fluorescence intensity in the tumor area gradually increased and reached its maximum at 100 minutes, while the fluorescence in the normal tissue area was negligible throughout the detection time, with a maximum signal-to-noise ratio (T / N) of 19. These results indicate that Cy5-APN can distinguish between cancerous and normal tissues with high contrast, thus possessing great application potential in fluorescence-guided tumor surgery.

[0053] In summary, this invention utilizes the Cy5-COOM dye platform and employs an "ester group → carboxyl group" conversion strategy to construct an APN fluorescent probe. This probe exhibits high selectivity and sensitivity for APN, with a detection limit of 0.393 ng / mL. Taking advantage of the overexpression of APN by cancer cells, this probe can achieve high-contrast differentiation between cancer cells / tissues and normal cells / tissues, thus showing great promise for surgical navigation.

[0054] 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 within the protection scope of the present invention.

Claims

1. A fluorescent probe for aminopeptidase, characterized in that, Its structural formula is as follows: 。 2. A method for preparing the aminopeptidase fluorescent probe according to claim 1, characterized in that, Includes the following steps: Step 1: Under N2 environment, DMF is dissolved in anhydrous dichloromethane, and then oxalyl chloride is gradually added. The reaction is stirred at room temperature. After the reaction is completed, the solvent is completely evaporated under reduced pressure to obtain compound 1, which is a white solid. It is used directly in the next reaction without purification. Step 2: Compound 1 and monomethyl malonate were dissolved in anhydrous dichloromethane and refluxed. After evaporating the solvent under reduced pressure, compound 2 was obtained. Acetic anhydride, 1,2,3,3-tetramethyl-3H-indole iodide, and anhydrous sodium acetate were added to the mixture in sequence and stirred. The mixture was then diluted with water and extracted with dichloromethane. The organic layers were combined and dried over anhydrous sodium sulfate, then purified by vacuum distillation and column chromatography to obtain compound Cy5-COOM, which was a dark blue solid. Compound 1 is: Compound 2 is: The compound Cy5-COOM is: ; Step 3: Dissolve compound Cy5-COOM in a mixed solution of MeOH and NaOH, stir and react, cool, dilute with water and extract with dichloromethane, combine the organic layers and dry with anhydrous sodium sulfate, then purify by vacuum distillation and column chromatography to obtain compound Cy5-COO, which is a dark blue solid. The compound Cy5-COO is: ; Step 4: Cy5-COO, compound 3, and potassium carbonate were dissolved in anhydrous N,N-dimethylformamide and stirred. After the reaction was completed, the mixture was cooled, diluted with water, and extracted with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After concentration under reduced pressure, the mixture was redissolved in a mixed solution of trifluoroacetic acid and dichloromethane. After further stirring, the mixture was purified by vacuum distillation and column chromatography to obtain compound Cy5-APN, which is a dark blue solid. The compound 3 is: .

3. The method for preparing an aminopeptidase fluorescent probe according to claim 2, characterized in that, In step 1, the molar ratio of DMF to oxaloyl chloride is 30:35, and the stirring reaction time is 2 hours.

4. The method for preparing an aminopeptidase fluorescent probe according to claim 2, characterized in that, In step 2, the molar ratio of compound 1 to monomethyl malonate is 2:1, the reflux reaction temperature is 40°C, and the reaction time is overnight. The molar ratio of compound 2, 1,2,3,3-tetramethyl-3H-indole iodide, and anhydrous sodium acetate is 1:2:

3. The stirring reaction temperature is 90°C, and the reaction time is 4 hours. The developing solvent for column chromatography separation and purification is dichloromethane / methanol = 10 / 1, v / v.

5. The method for preparing an aminopeptidase fluorescent probe according to claim 2, characterized in that, In step 3, the stirring reaction is carried out at a temperature of 43°C for 3 hours, and the developing solvent for column chromatography separation and purification is 10-50% methanol / dichloromethane, v / v.

6. The method for preparing an aminopeptidase fluorescent probe according to claim 2, characterized in that, In step 4, the molar ratio of Cy5-COO, compound 3, and potassium carbonate is 1:3:

2. The stirring temperature is 48°C, the reaction time is 3 h, the volume ratio of trifluoroacetic acid to dichloromethane is 1:1, the further stirring temperature is room temperature, the reaction time is 20 minutes, and the developing solvent for column chromatography separation and purification is CH2Cl2 / MeOH = 8:1, v / v.

7. An application of the aminopeptidase fluorescent probe according to claim 1, characterized in that, Applications of fluorescent imaging reagents in fluorescence-guided surgery.

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