Fluorescent probe for detecting proline aminopeptidase as well as preparation method and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting proline aminopeptidase and a preparation method and application thereof, and belongs to the technical field of biological medicine, the structural formula of the fluorescent probe is # imgabs0 #, and the specific fluorescent probe provided by the invention has high specificity in the application process and can be detected by a fluorescence detector. Meanwhile, the fluorescent probe DDAP provided by the invention has longer fluorescence emission wavelength, can better weaken the interference of biological background fluorescence, and has high sensitivity. In addition, the DDAP provided by the invention can be obtained through simple chemical synthesis, the synthesis process is simple and feasible, and the detection cost of a fluorescence method is low.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to a fluorescent probe for detecting proline aminopeptidase and a preparation method and application thereof. Background Art

[0002] Enterococci have long been considered harmless commensal bacteria, especially compared with other more toxic pathogens. However, there is increasing evidence that these bacteria have the ability to survive and colonize in patients under specific circumstances, causing infections such as bacteremia, peritonitis, endocarditis, and urinary tract, wound, and device-related infections. Enterococci have become an important pathogen in immunocompromised and critically ill patients, especially after the emergence of multidrug-resistant strains. At the same time, studies have found that the formation of enterococcal biofilms is closely related to enterococcal infections, which not only increases the toxicity of bacteria, but also increases the difficulty of treatment. Therefore, destroying the integrity of enterococcal cells and inhibiting the formation of bacterial biofilms have a good effect on the treatment of enterococcal infections.

[0003] L-proline (L-Pro) is the only imino acid among the 20 basic amino acids that make up proteins. It plays an important role in a variety of biological processes, such as cell growth, oxidative and osmotic stress responses, protein folding and stability, and redox signaling. Proline forms glutamate through catabolism, allowing organisms to use it as carbon, nitrogen, and energy. Existing research results show that proline also plays an important role in bacteria. First, it can participate in metabolism as a nutrient. For some bacteria, proline is the only nitrogen or carbon source. Secondly, proline is an effective compatible solute that can resist the adverse effects of high osmotic pressure on cell physiology. Third, proline has the function of resisting cold, heat, and oxidative stress. In microorganisms, proline can not only act as a heat protectant to reduce protein aggregation during heat stress, but also has almost the same cryoprotective activity as glycerol and trehalose. It is a natural non-toxic cryoprotectant and is also related to the regulation of reactive oxygen balance. Fourth, proline has the ability to improve the disease-curing ability of pathogens. Studies have found that proline biosynthesis metabolism generates a variety of genes that affect the virulence of pathogens. When proline metabolism and transport capacity are disrupted, the virulence of some pathogens can be significantly reduced. Therefore, reducing the L-Pro required for bacterial biofilm formation and pathogen virulence gene transport is helpful for enterococcal infection.

[0004] Proline aminopeptidase (PAP) is the only enzyme that can effectively release proline from the N-terminus of peptides. It exists in various organisms, including bacteria, fungi, plants and insects, and plays a biological regulatory role in a variety of cellular physiological functions and metabolic activities. The enzyme has a very narrow substrate specificity, and other N-terminal amino acids cannot be released or removed by the enzyme. In the study of the catalytic activity and structure of bacterial PAP, it was shown that it also has high specificity for N-terminal proline and hydroxyproline. It was also found that the accumulation of free proline in bacteria is positively correlated with PAP activity. Therefore, a bacterial PAP activity detection system was established, an inhibitor screening method was constructed, and the L-Pro content was reduced, providing new ideas for the treatment of enterococcal infections.

[0005] As an enzyme with low popularity, most of the existing methods for detecting PAP activity are not suitable for screening inhibitors and subsequent research. Fluorescence visualization detection and analysis methods are simple to operate and have intuitive results. As a new PAP activity detection technology, it is more suitable for experimental needs. Therefore, how to provide a specific fluorescent probe substrate with high sensitivity and strong resistance to environmental interference for in vivo and in vitro detection of proline aminopeptidase, and establish a fluorescent detection method for high-throughput screening of PAP inhibitors is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0006] In order to solve the above technical problems, the present invention proposes a fluorescent probe for detecting proline aminopeptidase and a preparation method and application thereof.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] A fluorescent probe for detecting proline aminopeptidase, named N-(4-(((6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl)oxy)methyl)phenyl)pyrrolidine-2-carboxamide (DDAP), the structural formula of the fluorescent probe for detecting proline aminopeptidase is shown in formula (1):

[0009]

[0010] The present invention also provides a method for preparing the fluorescent probe for detecting proline aminopeptidase, wherein BOC-L-proline and 4-aminobenzyl alcohol are used as raw materials to perform an addition reaction to obtain compound 1; PBr is added to compound 1; 3 , replacing the hydroxyl group in compound 1 with Br to obtain compound 2; subjecting compound 2 and 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone (DDAO) to an addition reaction to synthesize the fluorescent probe for detecting proline aminopeptidase;

[0011] Wherein, the structural formula of the compound 1 is The structural formula of compound 2 is

[0012]

[0013] The fluorescent probe for detecting proline aminopeptidase provided by the present invention is a specific fluorescent probe, which has high specificity during application and can be detected by a fluorescence detector. At the same time, the DDAP provided by the present invention has a long-wavelength fluorescence emission wavelength, which can better reduce the interference of biological background fluorescence and has high sensitivity. In addition, the DDAP provided by the present invention can be obtained by simple chemical synthesis, the synthesis process is simple and easy, and the fluorescence method detection cost is low.

[0014] Preferably, in the method for preparing the fluorescent probe for detecting proline aminopeptidase, the molar ratio of BOC-L-proline (Boc-L-Proline), 4-aminobenzyl alcohol and 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone is 1:1:0.32.

[0015] More preferably, the synthetic route of the fluorescent probe (DDAP) for detecting proline aminopeptidase is as follows:

[0016]

[0017] The specific preparation method comprises the following steps:

[0018] First, Boc-L-Proline (215 mg, 1.0 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 380 mg, 1.0 mmol) and N,N-diisopropylethylamine (DIPEA, 129 mg, 1.0 mmol) were dissolved in 10 mL of dimethylformamide (DMF), and stirred at 0°C under argon for 1 h to obtain a first mixed solution; then 4-aminobenzyl alcohol (123 mg, 1.0 mmol) was added dropwise to DMF (5 mL), and stirred at room temperature for 12 h to obtain a second mixed solution; the first mixed solution and the second mixed solution were mixed to obtain a mixture, and the mixture was treated with CH 2 Cl 2 (5 mL) and diluted with H 2 O was washed three times, and the organic phase was washed with MgSO 4 Dry on ice, evaporate under reduced pressure, and purify the crude product by silica gel chromatography (CH 2 Cl 2 :MeOH=20:1, v:v), to obtain compound 1 (yellow solid);

[0019] PBr 3(260 mg, 0.96 mmol) was added to the CH 2 Cl 2 (30 mL) solution, stirred at 0 °C for 1 h, then poured into ice water and washed with CH 2 Cl 2 Extraction, organic phase in MgSO 4 The mixture was dried on ice and evaporated under reduced pressure to give a crude product 2 (200 mg), which was used directly in the next reaction without further purification. Compound 2 (200 mg), 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone (DDAO, 100 mg, 0.32 mmol) and K 2 CO 3 (88.3 mg, 0.64 mmol) was dissolved in DMF (5 mL), and the mixed solution was stirred at room temperature for 24 h, evaporated under reduced pressure, and the crude product was redissolved in CH 2 Cl 2 (5 mL), and then CF was added dropwise 3 COOH (0.5 mL), stirred at 0 ° C for 30 minutes, evaporated under reduced pressure, and the obtained crude product was purified by silica gel chromatography (CH 2 Cl 2 :MeOH=10:1, v:v) to give the product DDAP as an orange solid.

[0020] The present invention also provides a method for detecting proline aminopeptidase, using the fluorescent probe for detecting proline aminopeptidase as a specific substrate of proline aminopeptidase to carry out a hydrolysis reaction, and then using the fluorescence intensity to quantitatively detect the generation rate of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone per unit time, thereby quantitatively determining the activity of proline aminopeptidase.

[0021] The specific fluorescent probe hydrolysis product of the present invention is a long emission wavelength fluorescent probe, which is not easily interfered by biological system matrix and impurities during the proline aminopeptidase activity detection process and can be used for quantitative determination of various recombinant proline aminopeptidases or proline aminopeptidase activities in intestinal microorganisms.

[0022] Preferably, in the method for detecting proline aminopeptidase, the reaction system of proline aminopeptidase is pre-incubated before the hydrolysis reaction;

[0023] The reaction system comprises a 0.9wt% NaCl aqueous solution and proline aminopeptidase, wherein the concentration of the proline aminopeptidase is 0-30 μg / mL, and the concentration of the proline aminopeptidase is not 0;

[0024] The pre-incubation temperature is 37° C., the pre-incubation time is 5 min, and the pre-incubation pH is 7-8.

[0025] Preferably, in the method for detecting proline aminopeptidase, in the hydrolysis reaction, the concentration of the fluorescent probe for detecting proline aminopeptidase in the reaction system is 10 μmol / L;

[0026] The temperature of the hydrolysis reaction is 37° C. and the time is 90 min.

[0027] Preferably, in the method for detecting proline aminopeptidase, the excitation wavelength (Ex) is 600 nm and the maximum emission wavelength (Em) is 658 nm.

[0028] Preferably, the proline aminopeptidase is recombinantly expressed proline aminopeptidase or proline aminopeptidase in intestinal microorganisms.

[0029] The fluorescent probe for detecting proline aminopeptidase and its hydrolysis product provided by the present invention have different optical properties, and a fluorescent detector can be used to realize rapid and sensitive detection of the product.

[0030] The present invention also provides the use of the fluorescent probe for detecting proline aminopeptidase in detecting the activity of recombinantly expressed proline aminopeptidase or endogenous proline aminopeptidase of intestinal microorganisms for non-therapeutic purposes.

[0031] The present invention also provides the application of the fluorescent probe for detecting proline aminopeptidase in the rapid screening of proline aminopeptidase inhibitors and the quantitative evaluation of the inhibitory ability.

[0032] The present invention also provides the use of the fluorescent probe for detecting proline aminopeptidase in the evaluation of proline aminopeptidase activity in intestinal microorganisms for non-therapeutic purposes.

[0033] The present invention also provides application of the fluorescent probe for detecting proline aminopeptidase in high-throughput screening of proline aminopeptidase inhibitors.

[0034] Compared with the prior art, the present invention has the following advantages and technical effects:

[0035] The specific fluorescent probe substrate provided by the present invention can be selectively hydrolyzed by proline aminopeptidase to generate a hydrolyzate with significantly changed fluorescence properties, which has high specificity and can be detected by a fluorescence detector. In addition, the activity and function of proline aminopeptidase in intestinal microorganisms can be quantitatively evaluated by using the probe reaction, and can be used for the screening of proline aminopeptidase inhibitors. At the same time, the fluorescent probe DDAP provided by the present invention has a long fluorescence emission wavelength, can better reduce the interference of biological background fluorescence, and has high sensitivity. In addition, the DDAP provided by the present invention can be obtained by simple chemical synthesis, the synthesis process is simple and easy, and the detection cost of the fluorescence method is low. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0037] Figure 1 The H NMR spectrum of the intermediate compound 1 synthesized from DDAP in Example 1 ( 1 H-NMR);

[0038] Figure 2 is the DDAP H NMR spectrum in Example 1 ( 1 H-NMR);

[0039] Figure 3 is the DDAP carbon NMR spectrum in Example 1 ( 13 C-NMR);

[0040] Figure 4 The DDAP high resolution mass spectrum in Example 1;

[0041] Figure 5 are the ultraviolet absorption and fluorescence emission spectra of DDAP and its hydrolysis product DDAO, where a is the ultraviolet absorption spectrum and b is the fluorescence emission spectrum;

[0042] Figure 6 The results of the screening experiment of different hydrolases on DDAP in Example 2;

[0043] Figure 7 The results of the screening experiment of different ions and amino acids on DDAP in Example 3;

[0044] Figure 8 is the linear change of the enzyme in the reaction of DDAP probe catalyzed by proline aminopeptidase in Example 4, wherein a is the fluorescence spectrum of DDAP to different PAP concentrations, and b is the linear relationship between fluorescence intensity and PAP concentration. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0047] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0048] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0049] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0050] Unless otherwise specified, the room temperature in the present invention is 25±2°C.

[0051] All raw materials used in the embodiments of the present invention are commercially available.

[0052] The technical solution of the present invention is further illustrated by the following embodiments.

[0053] Example 1

[0054] A fluorescent probe for detecting proline aminopeptidase, named N-(4-(((6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl)oxy)methyl)phenyl)pyrrolidine-2-carboxamide (DDAP), has a structural formula as shown in formula (1):

[0055]

[0056] The preparation method comprises the following steps:

[0057]

[0058] First, Boc-L-Proline (215 mg, 1.0 mmol), HATU (380 mg, 1.0 mmol) and DIPEA (129 mg, 1.0 mmol) were dissolved in DMF (10 mL), and stirred at 0°C under argon for 1 h to obtain a first mixed solution; then 4-aminobenzyl alcohol (123 mg, 1.0 mmol) was added dropwise to DMF (5 mL), and stirred at room temperature for 12 h to obtain a second mixed solution; the first mixed solution and the second mixed solution were mixed to obtain a mixture, and the mixture was treated with CH 2 Cl 2 (5 mL) and diluted with H 2 O was washed three times, and the organic phase was washed with MgSO 4 Dry on ice, evaporate under reduced pressure, and purify the crude product by silica gel chromatography (CH 2 Cl 2 :MeOH=20:1, v:v0 to give compound 1 (yellow solid, 200 mg, yield: 62.3%).

[0059] PBr 3 (260 mg, 0.96 mmol) was added to the CH 2 Cl 2 (30 mL) solution, stirred at 0 °C for 1 h, then poured into ice water and washed with CH 2 Cl 2 Extraction, organic phase in MgSO 4 The mixture was dried on ice and evaporated under reduced pressure to give a crude product 2 (200 mg), which was used directly in the next reaction without further purification. Compound 2 (200 mg), 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone (DDAO, 100 mg, 0.32 mmol) and K 2 CO 3 (88.3 mg, 0.64 mmol) was dissolved in DMF (5 mL), and the mixed solution was stirred at room temperature for 24 h, evaporated under reduced pressure, and the crude product was redissolved in CH 2 Cl 2 (5 mL), and then CF was added dropwise 3 COOH (0.5 mL), stirred at 0 ° C for 30 minutes, evaporated under reduced pressure, and the obtained crude product was purified by silica gel chromatography (CH 2 Cl 2:MeOH=10:1, v:v) to give the product DDAP as an orange solid (40 mg, yield: 24.2%).

[0060] The structural identification spectral data of compound 1 are as follows:

[0061] H NMR spectroscopy (eg Figure 1 ): 1 H-NMR (400 MHz, DMSO-d 6 )δ9.90(s,1H),7.54(d,J=8.4Hz,2H),7.24(d,J=8.3Hz,2H),5.16-4.97(m,1H),4.43(d,J=5.7Hz,2H),4.29-4. 14(m,1H),3.39(ddd,J=22.5,11.3,6.6Hz,2H),2.27-2.09(m,1H),1.96-1.73(m,3H),1.40(s,3H),1.27(s,6H).

[0062] The structural identification spectral data of DDAP are as follows:

[0063] H NMR spectroscopy (eg Figure 2 ): 1 H-NMR (500 MHz, DMSO-d 6 )δ10.10(s,1H),7.82(s,1H),7.68(d,J=8.1Hz,2H),7.61(d,J=8.7Hz,1H),7.45(d,J=8.1Hz,2H),7.39(s,1H),7.11(d,J=8.1 Hz,1H),5.21(s,2H),3.83-3.75(m,1H),2.95(t,J=6.6Hz,2H),2.10(dt,J=15.3,7.9Hz,1H),1.83(s,6H),1.75-1.65(m,2H).

[0064] C NMR spectroscopy (e.g. Figure 3 ): 13 C-NMR (125 MHz, DMSO-d 6 )δ172.39,162.13,147.08,140.98,140.62,139.36,138.35,135.26,133.60,133.13,13 1.11,128.83,119.16,114.79,113.75,69.77,60.62,46.58,40.11,30.27,26.03,25.46.

[0065] High-resolution mass spectrometry (eg Figure 4 ): HRMS(+)m / z 532.1165[M+Na] + , calculated value m / z 532.1273.

[0066] Figure 5 is the UV absorption and fluorescence emission spectra of DDAP and its hydrolysis product DDAO, where a is the UV absorption spectrum and b is the fluorescence emission spectrum. Figure 5 It can be seen that the best excitation of DDAO is at 600 nm. Under the excitation of 600 nm, DDAP has almost no fluorescence. Therefore, DDAP is an "OFF-ON type" fluorescent probe.

[0067] Example 2

[0068] In vitro determination of the selectivity of different hydrolases:

[0069] (1) preparing 199 μL of in vitro metabolic reaction systems of 9 different types of hydrolases, the reaction systems comprising a 0.9 wt% NaCl aqueous solution at pH 7.4 and a hydrolase (30 μg / mL), and pre-incubating at 37° C. for 5 minutes with shaking;

[0070] Among them, the 9 different types of hydrolases were human serum albumin, bovine serum albumin, β-glucuronidase, carboxylesterase 1c, arginase, human lipase, porcine lipase, neutrophil elastase, and proline aminopeptidase;

[0071] (2) adding 1 μL of 2 mM DDAP (final concentration 10 μM) to each of the nine in vitro metabolic reaction systems pre-incubated in step (1), reacting at 37° C. for 90 min, then adding 100 μL of glacial acetonitrile, shaking vigorously, and terminating the reaction;

[0072] (3) Centrifuge at 4°C and 20,000×g for 20 min in a high-speed refrigerated centrifuge, collect the supernatant, and determine the formation rate of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone by fluorescence detection (fluorescence detection conditions: Ex=600nm, Em=658nm).

[0073] like Figure 6 The fluorescence detection results show that the reaction rate of the reaction catalyzed by proline aminopeptidase (PAP) is much higher than that of other hydrolases, indicating that the reaction of DDAP catalyzed by proline aminopeptidase has good selectivity. The DDAP provided by the present invention can be used for the activity determination of proline aminopeptidase.

[0074] Example 3

[0075] In vitro determination of the effects of different ions and amino acids on DDAP selectivity:

[0076] (1) preparing 199 μL of an in vitro metabolic reaction system including different ions and amino acids, the reaction system comprising a 0.9 wt % NaCl aqueous solution at pH 7.4, proline aminopeptidase, a metal ion (final concentration 200 μM) and an amino acid (final concentration 10 μM), and pre-incubating the reaction system at 37° C. with shaking for 5 minutes;

[0077] Among them, ions include sodium ions, zinc ions, sulfate ions, chromate ions, manganese ions, calcium ions, barium ions, nickel ions, potassium ions, magnesium ions, copper ions, divalent iron ions, and trivalent iron ions;

[0078] Amino acids include lysine, serine, histidine, arginine, glutamine, glycine, tryptophan, glutathione, and proline;

[0079] (2) adding 1 μL of 2 mM DDAP (final concentration 10 μM) to the pre-incubated in vitro metabolic reaction system obtained in step (1), reacting at 37° C. for 90 min, then adding 100 μL of glacial acetonitrile, shaking vigorously, and terminating the reaction;

[0080] (3) Centrifuge at 4°C and 20,000×g for 20 min in a high-speed refrigerated centrifuge, collect the supernatant, and determine the production rate of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone by fluorescence detection (fluorescence detection conditions: Ex=600nm, Em=658nm).

[0081] like Figure 7 The fluorescence test results show that the fluorescence intensity of the probe itself is not affected by other ions and amino acids in the reaction catalyzed by proline aminopeptidase (PAP), indicating that the reaction of DDAP catalyzed by proline aminopeptidase has good anti-interference ability, and the DDAP provided by the present invention can be used for the activity determination of proline aminopeptidase.

[0082] Example 4

[0083] Linearity study of the DDAP probe reaction catalyzed by proline aminopeptidase:

[0084] (1) preparing 199 μL of an in vitro metabolic reaction system, which includes a 0.9 wt% NaCl aqueous solution at pH 7.4 and proline aminopeptidase (0-30 μg / mL), and pre-incubating at 37°C with shaking for 5 min;

[0085] (2) adding 1 μL of 2 mM DDAP (final concentration 10 μM) to the pre-incubated reaction system obtained in step (1), reacting at 37° C. for 90 min, then adding 100 μL of glacial acetonitrile, shaking vigorously, and terminating the reaction;

[0086] (3) Centrifuge at 4°C and 20,000×g for 20 min in a high-speed refrigerated centrifuge, collect the supernatant, and determine the formation rate of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone by fluorescence detection (fluorescence detection conditions: Ex=600nm, Em=658nm).

[0087] like Figure 8 The fluorescence detection results showed that the probe reaction of DDAP catalyzed by proline aminopeptidase (PAP) showed a good enzyme linear relationship in the range of 0-30 μg / mL, r 2 The value is 0.997, indicating that DDAP in the present invention can be applied to the determination of the activity and expression level of proline aminopeptidase in complex samples.

[0088] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A fluorescent probe for detecting proline aminopeptidase, characterized in that: The name is N-(4-(((6,8-dichloro-9,9-dimethyl-7-oxo-7,9-dihydroacridin-2-yl)oxy)methyl)phenyl)pyrrolidine-2-carboxamide. The structural formula of the fluorescent probe for detecting proline aminopeptidase is shown in formula (1):

2. A method for preparing a fluorescent probe for detecting proline aminopeptidase according to claim 1, characterized in that: BOC-L-proline and 4-aminobenzyl alcohol are used as raw materials to first undergo an addition reaction to obtain compound 1; PBr3 is added to compound 1 to replace the hydroxyl group in compound 1 with Br to obtain compound 2; compound 2 and 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone are subjected to an addition reaction to synthesize the fluorescent probe for detecting proline aminopeptidase; Wherein, the structural formula of the compound 1 is The structural formula of compound 2 is 3. The method for preparing a fluorescent probe for detecting proline aminopeptidase according to claim 2, characterized in that: The molar ratio of BOC-L-proline, 4-aminobenzyl alcohol and 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone is 1:1:0.

32.

4. A method for detecting proline aminopeptidase, characterized in that: The fluorescent probe for detecting proline aminopeptidase according to claim 1 is used as a specific substrate of proline aminopeptidase to carry out a hydrolysis reaction, and then the fluorescence intensity is used to quantitatively detect the generation rate of 1,3-dichloro-7-hydroxy-9,9-dimethyl-2(9H)-acridone per unit time, thereby quantitatively determining the activity of proline aminopeptidase.

5. The method for detecting proline aminopeptidase according to claim 4, characterized in that: Before the hydrolysis reaction, the reaction system of proline aminopeptidase is pre-incubated; The reaction system comprises a 0.9wt% NaCl aqueous solution and proline aminopeptidase, wherein the concentration of the proline aminopeptidase is 0-30 μg / mL, and the concentration of the proline aminopeptidase is not 0; The pre-incubation temperature is 37° C., the pre-incubation time is 5 min, and the pre-incubation pH is 7-8.

6. The method for detecting proline aminopeptidase according to claim 4, characterized in that: In the hydrolysis reaction, the concentration of the fluorescent probe for detecting proline aminopeptidase in the reaction system is 10 μmol / L; The temperature of the hydrolysis reaction is 37° C. and the time is 90 min.

7. Use of the fluorescent probe for detecting proline aminopeptidase according to claim 1 in detecting the activity of recombinantly expressed proline aminopeptidase or endogenous proline aminopeptidase of intestinal microorganisms for non-therapeutic purposes.

8. Use of the fluorescent probe for detecting proline aminopeptidase according to claim 1 in the rapid screening of proline aminopeptidase inhibitors and the quantitative evaluation of their inhibitory ability.

9. Use of the fluorescent probe for detecting proline aminopeptidase according to claim 1 in evaluating the activity of proline aminopeptidase in intestinal microorganisms of non-therapeutic purpose.

10. Use of the fluorescent probe for detecting proline aminopeptidase according to claim 1 in high-throughput screening of proline aminopeptidase inhibitors.

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