Leucine aminopeptidase fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a leucine aminopeptidase fluorescent probe as well as a preparation method and application thereof, and belongs to the technical field of fine chemical engineering, the probe takes a fluorescent dye methylene blue MB as a structural basis, and the final fluorescent probe for detecting leucine aminopeptidase LAP is constructed by introducing an LAP recognition group L-leucine to destroy a conjugated structure of the fluorescent dye methylene blue MB. After the fluorescent probe with the structural formula of # imgabs0 # responds to LAP, the recognition group leaves, and the probe recovers the conjugated structure of MB, so that strong near-infrared fluorescence is shown at 690nm, and the detection limit of 1.65 mU / mL is realized; the LAP can specifically recognize N-terminal residues of L-leucine in a probe structure, so that the probe shows high selectivity on LAP detection; meanwhile, MB is used as a dye with photodynamic performance, and the photodynamic performance of MB is activated after the probe and LAP act; finally, the probe is successfully used for fluorescence imaging and photodynamic therapy of cancer cells.

Description

Technical Field

[0001] The present invention relates to the technical field of fine chemical industry, and in particular to a leucine aminopeptidase fluorescent probe and a preparation method and application thereof. Background Art

[0002] Leucine aminopeptidase (LAP) is an important metallopeptidase in lysosomes that selectively catalyzes the hydrolysis of N-terminal leucine residues in protein or peptide substrates. Abnormal expression or catalytic function of LAP is associated with a variety of diseases, including hepatitis, liver cancer, ovarian cancer, breast cancer, and kidney damage. In the liver, LAP is primarily distributed in hepatocytes and bile duct epithelial cells, participating in protein metabolism and maintaining cellular homeostasis. As an important biomarker for hepatobiliary diseases, changes in LAP levels reflect the pathological state of hepatocellular injury or biliary obstruction. The mechanisms involved include cell damage release, changes in bile duct pressure, and cholestatic reactions. Furthermore, high expression levels of LAP are positively correlated with clinical characteristics of patients with related cancers, such as tumor size, grade, depth of invasion, and metastasis. Therefore, in situ monitoring and analysis of intracellular LAP activity has important clinical significance for the early diagnosis, treatment, and management of tumors.

[0003] To date, many methods for detecting LAP in vitro have been developed both domestically and internationally. Common methods for detecting LAP include enzyme cycling assays, electrochemical analysis, high-performance liquid chromatography, and capillary electrophoresis. However, these methods are unable to detect changes in LAP levels in living cells and tissues in situ and in real time. Fluorescent probes have attracted widespread attention in the scientific community due to their advantages such as high sensitivity, high selectivity, fast reaction speed, low detection limit, and simple operation. They have great application potential in the field of LAP visualization research. Therefore, the use of fluorescent probes to achieve precise imaging of LAP is an important means to achieve timely diagnosis of cancer and study the pathogenesis of cancer. Since cells contain a large number of substances that spontaneously fluoresce in the visible light region, the development of activated fluorescent probes is an effective strategy to reduce probe background fluorescence, improve the signal-to-noise ratio, and thus achieve precise detection of LAP and achieve cancer treatment. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, such as the inability to detect changes in LAP levels in living cells and tissues in situ and in real time, in order to overcome the shortcomings of the existing technology, the purpose of the present invention is to provide a leucine aminopeptidase fluorescent probe and its preparation method and application, and introduce the LAP specific recognition group L-leucine group into the methylene blue MB conjugated structure to form a non-π conjugated non-fluorescent form. In the presence of LAP, a specific hydrolysis reaction occurs to form methylene blue MB, thereby restoring the intramolecular charge transfer ICT effect of the probe and forming a fluorescent form with a conjugated structure. The maximum emission wavelength of the probe reaches 690nm, which effectively avoids the interference of biological autoluminescence and improves the accuracy of LAP detection. Subsequently, through cancer cell imaging experiments, it was confirmed that the probe has good imaging capabilities of LAP in cancer cells, thereby accurately diagnosing cancer cells and implementing photodynamic therapy.

[0005] In order to solve the problems of the prior art, the technical solution adopted by the present invention is:

[0006] A leucine aminopeptidase fluorescent probe is provided. The leucine aminopeptidase fluorescent probe is based on the fluorescent dye methylene blue MB and is connected to the leucine aminopeptidase LAP recognition group L-leucine. The conjugated structure of the fluorescent dye is destroyed, and its fluorescent signal can be activated by LAP. The structural formula of the leucine aminopeptidase fluorescent probe is shown in formula (I):

[0007]

[0008] (I), denoted as probe MB-Leu.

[0009] As an improvement, the MB fluorescent dye structure is based on the introduction of the LAP-specific recognition group L-leucine to destroy the conjugated structure of MB. The L-leucine group of the probe can be specifically recognized by LAP and hydrolyzed and removed, the MB conjugated structure is restored, and the intramolecular charge transfer ICT effect is restored, so that the near-infrared fluorescence at 690 nm is restored.

[0010] A method for preparing a leucine aminopeptidase fluorescent probe comprises the following steps:

[0011] Step 1: Add 3.8-3.9 g of potassium carbonate, 1.9-2.1 g of methylene blue MB, and 4.9-5.0 g of sodium dithionite to a 500 mL two-necked flask in a mass-volume ratio, and continue to add 80-90 mL of H2O and 30-35 mL of dichloromethane, dehydrate and deoxygenate the reaction system, then heat to 40-45°C and stir for 1-2 hours. After cooling to room temperature, add 50-55 mg of trifluoroacetic acid and stir for 5 minutes. Cool to 0°C, add 1.24 g of triphosgene, and add 1.4 mL of ultra-dry DCM with a syringe under an ice bath and N2 atmosphere, stir at 40°C for 1 hour; add 15-25 mL of DCM and 5-15 mL of H2O, terminate the reaction, extract the reaction solution, combine the organic phases, dry over anhydrous sodium sulfate, remove the solvent under reduced pressure, purify on a silica gel column using PE / DCM (1:1, v / v) as the eluent, and finally concentrate under reduced pressure to obtain a blue product (IV);

[0012]

[0013] Step 2: Add 300-320 mg of Boc-Leu and 720-740 mg of HATU to a 50 mL Shrek tube in a mass-to-volume ratio, heat the reaction system under vacuum to remove water and oxygen, and inject 3-6 mL of ultra-dry DCM and 240-250 mg of DIEA with a syringe under a N2 atmosphere, and finally inject 170-180 mg of PABA. Stir at room temperature for 10-12 hours, cool to room temperature, wash the reaction solution with water three times, remove the product solvent under reduced pressure, and purify by silica gel column chromatography using DCM / MeOH (50:1, v / v) as eluent. Finally, concentrate under reduced pressure to obtain a yellow product (III).

[0014]

[0015] Step 3: Add 80-90 mg of compound (III), 90-100 mg of K2CO3, and 30-40 mg of DMAP to a 50 mL Shrek tube in a mass-to-volume ratio; heat the reaction system under vacuum to remove water and oxygen, inject 90-100 mg of compound (IV) and 2-3 mL of ultra-dry DCM into the reaction solution using a syringe under a N2 atmosphere, and stir at room temperature for 1-2 h. After the reaction, filter the reaction solution to remove K2CO3, remove the product solvent under reduced pressure, and purify the product by column chromatography using DCM / MeOH (200:1, v / v) as the eluent on a silica gel column. Finally, concentrate under reduced pressure to obtain a bluish-white product (II);

[0016]

[0017] Step 4: Add 100-120 mg of compound (II) to a 50 mL Shrek tube reaction vessel in a mass-to-volume ratio, heat the reaction system under vacuum to remove water and oxygen, add 3-5 mL of ultra-dry dichloromethane (DCM) and 3-5 mL of trifluoroacetic acid by syringe in an ice bath and N2 atmosphere, and stir at room temperature for 1-2 hours; terminate the reaction, remove the product solvent under reduced pressure, and purify by silica gel column chromatography using DCM / MeOH (20:1, v / v) as the eluent. Finally, concentrate under reduced pressure to obtain the blue product compound leucine aminopeptidase fluorescent probe (I).

[0018]

[0019] As an improvement, in the third step, the post-treatment operation should be to directly remove K2CO3 by filtration, then remove the product solvent under reduced pressure, and finally purify by column chromatography.

[0020] As an improvement, the specific process parameters for removing water and oxygen in the first, second, third and fourth steps are: vacuuming with a vacuum pump for 10-20 minutes, maintaining at room temperature for 5-10 minutes, and then replacing nitrogen into the system.

[0021] The present application also discloses the use of any of the above-mentioned leucine aminopeptidase fluorescent probes or the leucine aminopeptidase fluorescent probes prepared by any of the above-mentioned preparation methods in cell fluorescence labeling.

[0022] As an improvement, the application is specifically the use of a leucine aminopeptidase fluorescent probe for the detection of LAP in cancer cells.

[0023] Beneficial effects:

[0024] Compared with the prior art, the leucine aminopeptidase fluorescent probe of the present invention and its preparation method and application have the following beneficial effects:

[0025] 1. This application uses the fluorescent dye methylene blue MB as the probe backbone and introduces the LAP-specific recognition group L-leucine to destroy the conjugated structure of the dye, forming a non-fluorescent form, thereby achieving accurate detection of LAP in cancer cells;

[0026] 2. In the presence of LAP, the L-leucine group of the fluorescent probe prepared in this application is hydrolyzed and removed, restoring the conjugated structure of the fluorescent dye, thereby restoring the ICT effect of the probe and significantly enhancing the fluorescence at 690 nm;

[0027] 3. The fluorescent probe prepared in this application showed a 52-fold fluorescence enhancement after 45 minutes of reaction with LAP, achieving a low detection limit of 1.65 mU / mL. At the same time, the photodynamic performance was restored, and singlet oxygen was generated under 660 nm light and completely degraded the added ABDA singlet oxygen detection reagent within 1 minute;

[0028] 4. The fluorescent probe prepared by this invention has successfully achieved in situ real-time imaging of LAP in cancer cells, providing new tools and design insights for the development of fluorescent probes for precise identification of tumor locations;

[0029] 5. After incubating the probe with cancer cells for 4 hours and then irradiating with 660nm light for 5 minutes, the fluorescence intensity of the reactive oxygen species detection reagent in cancer cells was significantly enhanced, nearly tenfold, while that of normal cells was only enhanced by less than twofold. At the same time, live-dead cell imaging experiments using calcein and propidium iodide showed that the probe had a significant therapeutic effect, with a cell killing rate of nearly 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a spectral change diagram of the leucine aminopeptidase fluorescent probe 10 μM in 10 mM PBS 7.4 solution and LAP (3.6 U / mL) for 45 minutes, wherein A is the absorption spectrum change diagram, the horizontal axis is wavelength (nm), and the vertical axis is absorbance (Abs); B is the fluorescence spectrum change diagram, the excitation wavelength is 650 nm, the horizontal axis is wavelength (nm), and the vertical axis is fluorescence intensity (au);

[0031] Figure 2 This is a graph showing the change in fluorescence intensity of the leucine aminopeptidase fluorescent probe 10 μM in this application after it reacts with different concentrations of LAP and other competing species in a 10 mM PBS 7.4 solution, wherein A is the fluorescence intensity I of the leucine aminopeptidase fluorescent probe 10 μM in a 10 mM PBS 7.4 solution after it reacts with different concentrations of LAP (0.6, 1.2, 1.8, 2.4, 3.0, 3.6 U / mL) for 45 minutes. 690nm The curve of the change with LAP concentration. B is the change of fluorescence intensity at 690 nm after 10 μM leucine aminopeptidase fluorescent probe was reacted with LAP 3.6 U / mL and other competing species in 10 mM PBS 7.4 solution for 30 min. (1: Blank; 2: LAP; 3: β-Gal; 4: ALP; 5: Lysozyme; 6: Cellulase; 7: ATP; 8: GSH; 9: HCy; 10: Cys; 11: Tyr; 12: Trp; 13: H2O2; 14: ClO - ;15:ONOO - ; 16: 1 O2;17:·OH;18:O2 ·- );

[0032] Figure 3A is the fluorescent probe of leucine aminopeptidase in this application. 10 μM was reacted with 3.6 U / mL LAP in 10 mM PBS 7.4 solution for 70 min, and then 30 μM ABDA was added. The fluorescence was detected by laser (660 nm, 15 mW / cm 2 ) is the absorption spectrum change graph after irradiation for 60 s, B is the fluorescence intensity change curve at 690 nm after 10 μM of the leucine aminopeptidase fluorescent probe of the present application was treated with 3.6 U / mL LAP in 10 mM PBS 7.4 solution for 30 min (as well as the control group of the probe MB-Leu alone), and with PBS 7.4 (10 mM) solutions of different pH values (4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0) for 30 min;

[0033] Figure 4 Confocal fluorescence imaging of 10 μM leucine aminopeptidase fluorescent probe MB-Leu after incubation with different cells (4T1, 4T1 with LAP enzyme inhibitor, Hela, Lx2) for 4 hours. Blue channel: Ex / Em = 360-390 / 410-420 nm, red channel: Ex / Em = 530-550 / 575-625 nm, green channel: Ex / Em = 460-495 / 510-550 nm;

[0034] Figure 5 The 10 μM leucine aminopeptidase fluorescent probe MB-Leu was co-incubated with different cells (4T1, Lx2) for 4 h. The probe alone was not lasered and lasered (660 nm, 15 mW / cm 2 ) irradiated for 5 minutes 1 Fluorescence imaging of O2 generation. DCFH was used as a fluorescent probe for ROS imaging. Green channel: Ex / Em = 460-495 / 510-550nm, blue channel: Ex / Em = 360-390 / 410-420nm;

[0035] Figure 6 In this application, 10 μM leucine aminopeptidase fluorescent probe MB-Leu was co-incubated with 4T1 cells for 4 h, and laser (660 nm, 15 mW / cm 2 ) Calcein AM (green) and PI (red) double-stained images before and after 5 minutes of irradiation. Green channel: Ex / Em = 460-495 / 510-550 nm, red channel: Ex / Em = 530-550 / 575-625 nm. DETAILED DESCRIPTION

[0036] The present invention will be further described below by way of examples, the purpose of which is only to provide a better understanding of the present invention. Therefore, the examples given do not limit the scope of protection of the present invention.

[0037] Example 1

[0038] A leucine aminopeptidase fluorescent probe, which is based on the fluorescent dye methylene blue MB and is connected to the leucine aminopeptidase LAP recognition group L-leucine. The conjugated structure of the fluorescent dye is destroyed, and its fluorescent signal can be activated by LAP. The structural formula of the leucine aminopeptidase fluorescent probe is shown in formula (I);

[0039]

[0040] (I), denoted as probe MB-Leu.

[0041] Based on the MB fluorescent dye structure, the LAP-specific recognition group L-leucine is introduced to destroy the conjugated structure of MB. The L-leucine group of the probe can be specifically recognized by LAP and hydrolyzed and removed, thereby restoring the MB conjugated structure and the intramolecular charge transfer (ICT) effect, thereby restoring the near-infrared fluorescence at 690 nm.

[0042] The preparation method of the leucine aminopeptidase fluorescent probe comprises the following steps:

[0043] Step 1: Add potassium carbonate 3.88g, 4.02mmol, methylene blue MB2.0g, 1.11mmol, sodium dithionite 4.9g, 4.0mmol in a 500mL two-necked flask according to the mass volume ratio, and continue to add H2O 80mL and dichloromethane 32mL to dehydrate and deoxygenate the reaction system, then heat to 40℃ and stir for 1h. After cooling to room temperature, add trifluoroacetic acid 50mg, 1.4mmol and stir for 5min. Cool to 0℃, add triphosgene 1.24g, 0.6mmol, add ultra-dry DCM 1.4mL with a syringe under ice bath and N2 atmosphere, stir at 40℃ for 1h; add 20mL DCM and 10mL After the reaction was terminated, the reaction solution was extracted and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was purified on a silica gel column using PE / DCM (1:1, v / v) as the eluent and finally concentrated under reduced pressure to obtain 163.4 mg of the blue product (IV) with a yield of 78.5%.

[0044]

[0045] 1H NMR (400MHz, DMSO-d6) δ7.41(d,J=8.9Hz,2H),6.78(d,J=2.8Hz,2H),6.70(dd,J=9.0,2.8Hz,2H),2.92(s,12H). 13 C NMR(101MHz,DMSO-d6)δ149.26,148.51,127.05,110.80,109.84.HRMS-ESI(m / z):[M+H] + Calcd for C 17 H 19 ClN3OS + 348.0932,found348.0942.

[0046] Step 2: Boc-Leu 300 mg, 0.98 mmol, HATU 740 mg, 1.5 mmol were added to a 50 mL Shrek tube in a mass-to-volume ratio. The reaction system was heated under vacuum to remove water and oxygen. Under a nitrogen atmosphere, ultra-dry DCM 3 mL, DIEA 243 mg, 1.5 mmol, and finally PABA 175 mg, 1.1 mmol were injected using a syringe. The mixture was stirred at room temperature for 12 h, cooled to room temperature, and the reaction solution was washed with water three times. The product solvent was removed under reduced pressure and purified by column chromatography using DCM / MeOH (50:1, v / v) as the eluent. Finally, it was concentrated under reduced pressure to obtain 220 mg of the yellow product (III) in a yield of 66.9%.

[0047]

[0048] 1 H NMR (400MHz, DMSO-d6) δ9.88(s,1H),7.54(d,J=8.5Hz,2H),7.23(d,J=8.3Hz,2H),7.00(d,J=8.1Hz,1H),5.09(s,1H),4.43( s,2H),4.11(td,J=9.1,5.4Hz,1H),1.70–1.57(m,1H),1.56–1.48(m,1H),1.46–1.40(m,1H),1.38(s,9H),0.92–0.86(m,6H). 13 C NMR(101MHz,DMSO-d6)δ171.69,155.49,137.70,137.38,126.93,119.00,78. 01,62.63,53.51,40.72,28.24,24.38,23.01,21.60.HRMS-ESI(m / z):[M+Na] +Calcd for C 18 H 28 N2NaO4 + 359.1942, found 359.1949.

[0049] Step 3: Compound (III) 80 mg, 0.24 mmol, K2CO3 98 mg, 3.0 mmol, and DMAP 35 mg, 0.95 mmol were added to a 50 mL Shrek tube in a mass-to-volume ratio; the reaction system was heated under vacuum to remove water and oxygen, and compound (IV) MB-CI 100 mg, 1.2 mmol and ultra-dry DCM 2.5 mL were injected into the reaction solution under N2 atmosphere by syringe, and stirred at room temperature for 2 h; after the reaction, the reaction solution was filtered to remove K2CO3, and the product solvent was removed under reduced pressure. The product was purified by column chromatography using DCM / MeOH (200:1, v / v) as the eluent and silica gel column. Finally, it was concentrated under reduced pressure to obtain 89 mg of bluish-white product (II) in a yield of 57.4%;

[0050]

[0051] 1 H NMR(400MHz, DMSO-d6)δ10.02(s,1H),7.60(d,J=8.2Hz,2H),7.29(t,J=9.0Hz,4H),7.05(d,J=8.0Hz,1H),6.69–6.65(m,3H),6.65–6.62(m,1H), 5.09(s,2H),4.12(td,J=9.2,5.5Hz,1H),2.87(s,12H),1.70–1.59(m,1H ),1.57–1.48(m,1H),1.47–1.41(m,1H),1.38(s,9H),0.92–0.86(m,6H). 13 C NMR(101MHz,DMSO-d6)δ171.93,155.50,153.62,148.62,138.88,131.99,130.98,128.50,127.56,127.0 5,119.09,110.89,109.74,78.03,66.94,54.96,53.5428.23,24.37,22.98,21.60.HRMS-ESI(m / z):[M+H] + Calcd for C 35 H 46 N5O5S + 648.3215, found 648.3224.

[0052] Step 4: Compound (II) 100 mg, 0.11 mmol were added to a 50 mL Shrek tube reaction vessel in a mass-to-volume ratio. The reaction system was heated to remove water and oxygen under vacuum pump evacuation. 4 mL of ultra-dry dichloromethane (DCM) and 4 mL of trifluoroacetic acid were added by syringe in an ice bath and N2 atmosphere, and stirred at room temperature for 1.5 h. The reaction was terminated and the product solvent was removed under reduced pressure. The product was purified by column chromatography using a volume ratio of DCM / MeOH (20:1, v / v) as the eluent and silica gel column purification. Finally, 32 mg of the blue product compound leucine aminopeptidase fluorescent probe (I) was obtained by concentration under reduced pressure in a yield of 53.2%.

[0053]

[0054] 1 H NMR(400MHz, DMSO-d6)δ7.62(d,J=7.8Hz,2H),7.29(dd,J=12.8,8.3Hz,4H),6.70–6.65(m,3H),6.65–6.62(m,1 H),5.09(s,2H),2.88(s,12H),1.81–1.68(m,1H),1.47(s,1H),1.35–1.32(m,1H),0.89(dd,J=10.2,6.7Hz,6H). 13 C NMR(101MHz,DMSO-d6)δ174.75,153.63,148.62,138.77,131.99,130.93,128.46,127.56,127 .05,119.03,110.88,109.74,66.96,44.01,29.04,24.24,23.20,21.91.HRMS-ESI(m / z):[M+H] + Calcd for C 30 H 38 N5O3S + 548.2690,found 548.2698.

[0055] Example 2

[0056] Time-dependent absorption and fluorescence spectral analysis of the response of the leucine aminopeptidase fluorescent probe MB-Leu to LAP in Example 1:

[0057] The leucine aminopeptidase fluorescent probe MB-Leu prepared in Example 1 was dissolved in PBS buffer (10 mM, pH 7.4) and the sensing performance of MB-Leu to LAP was tested at different times. In the presence of LAP (3.6 U / mL), the UV-Vis absorption of the probe at 665 nm gradually increased with time at the same concentration. Figure 1 This change in UV-vis absorption is accompanied by a gradual increase in near-infrared fluorescence emission at 690 nm, as shown in Figure 2. Figure 1 As shown in B. Figure 1 This is the spectral change of the leucine aminopeptidase fluorescent probe 10 μM in 10 mM PBS 7.4 solution and LAP 3.6 U / mL for 45 minutes, where A is the absorption spectrum change, the horizontal axis is wavelength (nm), and the vertical axis is absorbance (Abs); B is the fluorescence spectrum change, the excitation wavelength is 650 nm, the horizontal axis is wavelength (nm), and the vertical axis is fluorescence intensity (au).

[0058] Example 3

[0059] Detection limit and selectivity of the leucine aminopeptidase fluorescent probe MB-Leu in response to LAP in Example 1:

[0060] The fluorescence value of the probe MB-Leu at 690 nm has a good linear relationship with the concentration of LAP. Figure 2 As shown in A. The detection limit (LOD) of the probe for LAP can be calculated as 1.65mU / mL through the standard curve. When the probe is combined with common molecules in biological systems (including β-Gal, ALP, Lysozyme, Cellulase, ATP, GSH, HCy, Cys, Tyr, Trp, H2O2, ClO - , ONOO - , 1 O2, OH, O2 ·_ ), its fluorescence signal was significantly weaker than that of LAP, indicating that the probe has good selectivity for LAP. Figure 2 As shown in B. Figure 2 A in the middle is the fluorescence intensity I of the leucine aminopeptidase fluorescent probe 10 μM in 10 mM PBS 7.4 solution after acting with different concentrations of LAP (0.6, 1.2, 1.8, 2.4, 3.0, 3.6 U / mL) for 45 minutes. 690nm The graph shows the change in fluorescence intensity at 690 nm after 10 μM of the leucine aminopeptidase fluorescent probe of the present application reacted with 3.6 U / mL of LAP and other competing species in a 10 mM PBS 7.4 solution for 30 min (1: Blank; 2: LAP; 3: β-Gal; 4: ALP; 5: Lysozyme; 6: Cellulase; 7: ATP; 8: GSH; 9: HCy; 10: Cys; 11: Tyr; 12: Trp; 13: H2O2; 14: ClO - ;15:ONOO - ; 16: 1O2;17:·OH;18:O2 ·- ).

[0061] Example 4

[0062] Verification of the photodynamic performance of the leucine aminopeptidase fluorescent probe MB-Leu after its response to LAP in Example 1 and the fluorescence spectrum test of the responsiveness of the probe to LAP under different pH conditions:

[0063] like Figure 3 As shown in A, the probe MB-Leu (10 μM) was stabilized with LAP (3.6 U / mL) in PBS 7.4 (10 mM) solution for 70 min, and then 30 μM ABDA was added and the laser (660 nm, 15 mW / cm 2 ) irradiated for 60s, the absorption peak of ABDA decreased significantly. These results indicate that MB-Leu generates MB after LAP response, and its photodynamic properties are activated, verifying the activation-type photodynamic mechanism of the probe MB-Leu. Figure 3 Figure B shows the change curve of the fluorescence intensity at 690 nm after the probe (10 μM) was reacted with LAP (3.6 U / mL) in PBS (10 mM) buffer solutions of different pH values for 30 min. 690nm The fluorescence intensity is maximized in the acidic and alkaline environments, while LAP shows weaker activity, indicating that under physiological conditions, LAP has the best response effect and good biocompatibility, providing an experimental basis for subsequent cell imaging and treatment.

[0064] Example 5

[0065] Imaging images of the leucine aminopeptidase fluorescent probe MB-Leu incubated with different cells in Example 1:

[0066] The leucine aminopeptidase fluorescent probe MB-Leu 10 μM prepared in Example 1 was incubated with 4T1, Hela, and Lx2 cells for 4 hours to test the ability of the probe to enter the cells. Under the same concentration and exposure time conditions, 4T1 and Hela cells incubated with the probe MB-Leu had stronger imaging signals, indicating that the L-leucine-modified probe has better cancer cell targeting ability and can quickly enter cancer cells. Figure 4 Compared with the 4T1 control group (with LAP inhibitor), the red fluorescence of the probe MB-Leu is weaker, indicating that the cancer cell imaging signal comes from the interaction between MB-Leu and LAP.

[0067] Example 6

[0068] Cell imaging image of the reactive oxygen species generation capability verified after the interaction of the leucine aminopeptidase fluorescent probe MB-Leu with LAP in Example 1:

[0069] After incubating 4T1 and Lx2 cells with the leucine aminopeptidase fluorescent probe MB-Leu prepared in Example 1 for 4 h, the generation of reactive oxygen species (ROS) was detected using 2,7-dichlorofluorescein dichlorodiacetate (DCFH-DA). When irradiated with 660 nm light, significant green fluorescence was generated in 4T1 cells. Figure 5 As shown, the fluorescence of 4T1 cells without illumination was weak, indicating that the ability of the probe MB-Leu to generate reactive oxygen species can be activated in cancer cells. In Lx2 cells, the green fluorescence was weak both before and after illumination, indicating that normal cells cannot activate the photodynamic properties of the probe.

[0070] Example 7

[0071] Cell imaging images of the photodynamic therapy effect verification of the leucine aminopeptidase fluorescent probe MB-Leu in Example 1:

[0072] The leucine aminopeptidase fluorescent probe MB-Leu (10 μM) prepared in Example 1 was co-incubated with 4T1 cells for 4 h, and the live or dead state of the cells was confirmed using Calcein AM and propidium iodide (PI) double staining. Figure 6 As shown in the figure, 4T1 cells died less in the presence of MB-Leu probe alone, indicating that MB-Leu probe has excellent biocompatibility. 2 ) After 5 minutes, a large amount of reactive oxygen species was produced to induce cell apoptosis. These results prove that the probe MB-Leu has a significant photodynamic therapy effect on cancer cells, providing an important theoretical basis for in vivo imaging and treatment.

[0073] The above is a schematic description of the present invention and its embodiments, which is not restrictive. The drawings show only one embodiment of the present invention, and the actual structure is not limited thereto. Therefore, if a person skilled in the art is inspired by this and, without departing from the purpose of the present invention, designs a structure and embodiment similar to this technical solution without inventiveness, they shall fall within the scope of protection of the present invention.

Claims

1. A leucine aminopeptidase fluorescent probe, characterized in that: The leucine aminopeptidase fluorescent probe is based on the fluorescent dye methylene blue MB and is connected to the leucine aminopeptidase LAP recognition group L-leucine. The conjugated structure of the fluorescent dye is destroyed and its fluorescent signal can be activated by LAP. The structural formula of the leucine aminopeptidase fluorescent probe is shown in formula (I): (I), denoted as probe MB-Leu.

2. A leucine aminopeptidase fluorescent probe according to claim 1, characterized in that: Based on the MB fluorescent dye structure, the LAP-specific recognition group L-leucine is introduced to destroy the conjugated structure of MB. The L-leucine group of the probe can be specifically recognized by LAP and hydrolyzed and removed, thereby restoring the MB conjugated structure and the intramolecular charge transfer (ICT) effect, thereby restoring the near-infrared fluorescence at 690 nm.

3. A method for preparing the leucine aminopeptidase fluorescent probe according to claim 1, characterized in that: The specific steps include: Step 1: Add 3.8-3.9 g of potassium carbonate, 1.9-2.1 g of methylene blue MB, and 4.9-5.0 g of sodium dithionite to a 500 mL two-necked flask in a mass-volume ratio, and continue to add 80-90 mL of H2O and 30-35 mL of dichloromethane to dehydrate and deoxygenate the reaction system, then heat to 40-45°C and stir for 1-2 hours. After cooling to room temperature, add 50-55 mg of trifluoroacetic acid and stir for 5 minutes. Cool to 0°C, add 1.24 g of triphosgene, and add 1.4 mL of ultra-dry DCM with a syringe under an ice bath and N2 atmosphere, and stir at 40°C for 1 hour; add 15-25 mL of DCM and 5-15 mL of H2O to terminate the reaction, extract the reaction solution, combine the organic phases, dry over anhydrous sodium sulfate, and remove the solvent under reduced pressure. Purify on a silica gel column using PE / DCM (1:1, v / v) as the eluent, and finally concentrate under reduced pressure to obtain a blue product (IV); Step 2: Add 300-320 mg of Boc-Leu and 720-740 mg of HATU to a 50 mL Shrek tube in a mass-to-volume ratio, heat the reaction system under vacuum to remove water and oxygen, and inject 3-6 mL of ultra-dry DCM and 240-250 mg of DIEA with a syringe under N2 atmosphere, and finally inject 170-180 mg of PABA. Stir at room temperature for 10-12 hours, cool to room temperature, wash the reaction solution with water three times, remove the product solvent under reduced pressure, and purify by silica gel column chromatography using DCM / MeOH (50:1, v / v) as eluent. Finally, concentrate under reduced pressure to obtain a yellow product (III). Step 3: Add 80-90 mg of compound (III), 90-100 mg of K2CO3, and 30-40 mg of DMAP to a 50 mL Shrek tube in a mass-to-volume ratio; heat the reaction system under vacuum to remove water and oxygen, inject 90-100 mg of compound (IV) and 2-3 mL of ultra-dry DCM into the reaction solution using a syringe under a N2 atmosphere, and stir at room temperature for 1-2 h. After the reaction, filter the reaction solution to remove K2CO3, remove the product solvent under reduced pressure, and purify the product by column chromatography using DCM / MeOH (200:1, v / v) as the eluent on a silica gel column. Finally, concentrate under reduced pressure to obtain a bluish-white product (II); Step 4: Add 100-120 mg of compound (II) to a 50 mL Shrek tube reaction vessel in a mass-to-volume ratio, heat the reaction system under vacuum to remove water and oxygen, add 3-5 mL of ultra-dry dichloromethane (DCM) and 3-5 mL of trifluoroacetic acid using a syringe in an ice bath and N2 atmosphere, and stir at room temperature for 1-2 hours; terminate the reaction, remove the product solvent under reduced pressure, and purify by silica gel column chromatography using DCM / MeOH (20:1, v / v) as the eluent. Finally, concentrate under reduced pressure to obtain the blue product compound leucine aminopeptidase fluorescent probe (I).

4. The method for preparing the leucine aminopeptidase fluorescent probe according to claim 3, wherein: In the third step, the post-treatment operation should be to directly remove K2CO3 by filtration, remove the product solvent under reduced pressure, and finally purify by column chromatography.

5. The method for preparing the leucine aminopeptidase fluorescent probe according to claim 3, wherein: The specific process parameters for removing water and oxygen in the first, second, third and fourth steps are: vacuuming with a vacuum pump for 10-20 minutes, maintaining at room temperature for 5-10 minutes, and then replacing nitrogen into the system.

6. Use of the leucine aminopeptidase fluorescent probe according to claim 1 or 2 or the leucine aminopeptidase fluorescent probe prepared by the preparation method according to any one of claims 3 to 5 in cell fluorescent labeling.

7. The use according to claim 6, characterized in that: The application is specifically the use of a leucine aminopeptidase fluorescent probe for the detection of LAP in cancer cells.

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