A carboxylesterase 2 fluorescent probe and its synthesis method and application

Abstract

The present invention relates to a fluorescent probe of carboxylesterase 2 and its synthesis method and application. The fluorescent probe uses 1,8-naphthalimide as a fluorescent group, introduces 4-morpholineacetic acid to locate in lysosomes, and uses carboxylate as an enzyme cleavage site to construct a carboxylesterase 2 fluorescent probe. The probe induces the breakage and departure of its carboxylester group through an enzymatic reaction with carboxylesterase 2, and detects the presence and activity of carboxylesterase 2 by changes in the fluorescence intensity of the probe and its reaction product at 560nm. It can be used for qualitative and quantitative detection of carboxylesterase 2 activity in various living cells and subcellular levels.

Description

Technical Field

[0001] The invention provides a fluorescent probe that can be used for selectively detecting carboxylesterase 2, and relates to a fluorescent probe for carboxylesterase 2, a synthesis method and an application thereof. Background Art

[0002] Carboxylesterase is a member of the serine esterase family, and its main function is to hydrolyze esters. It is widely present in mammals, bacteria and other organisms. Human carboxylesterase 1 and human carboxylesterase 2 are mainly present in the human body, which can regulate the ester levels of many biological molecules in the human body and bacteria. This regulatory process is closely related to hormone regulation, cell component degradation, signal pathway regulation, bacterial infection and inflammation in cells, and is also related to many diseases. Therefore, the development of new methods for determining carboxylesterase activity is of great significance in medical diagnosis and drug development.

[0003] In recent years, traditional detection methods are cumbersome, time-consuming, labor-intensive, and expensive, and they cannot detect enzyme activity. Due to its advantages such as high spatiotemporal resolution, high sensitivity, high selectivity, and non-invasiveness, fluorescence analysis detection technology has been widely used in various fields such as biomolecule labeling, enzyme analysis, environmental monitoring, cell staining, and clinical laboratory diagnosis. It has been favored by researchers in particular in the detection of enzyme activity. In recent years, fluorescent probe technology has become a practical, sensitive, highly selective, and highly accurate detection method for measuring the true activity of human carboxylesterase 2 in biological samples. Current fluorescent probes are mostly concentrated on carboxylesterase 1, and some carboxylesterase 2 probes have appeared, but they lack subcellular organelle targeting, making them difficult to use for the quantitative detection of carboxylesterase 2 (such as lysosomal carboxylesterase 2) in living cells or subcellular organelles. Therefore, it is necessary to develop a fluorescent probe that uses carboxylates as metabolic substrates and is located in lysosomes. Summary of the invention

[0004] The present invention aims at the above problems and provides a fluorescent probe that can be used to selectively detect intracellular lysosomal carboxylesterase 2. The probe can selectively react with carboxylesterase 2 under physiological conditions, and the fluorescence is significantly enhanced after the reaction. Small molecule 4-bromo-1,8-naphthalenedicarboxylic anhydride is used as a fluorescent matrix, and N-(2-aminoethyl)morpholine is introduced, which has the dual functions of active site and positioning group, and the fluorescence difference between reactants and products is used to realize the selective detection of carboxylesterase 2 in living cells.

[0005] The present invention provides a method for synthesizing the fluorescent probe of carboxylesterase 2, comprising the following steps:

[0006] (1) 4-bromo-1,8-naphthalene dicarboxylic anhydride (compound 1) and n-butylamine were mixed and dissolved in ethanol, heated to reflux at 78-80°C for 3-5 hours, poured into ice water 1-5 times the volume of the solvent (ethanol), and allowed to stand for 12-15 hours, filtered, and recrystallized from chlorobenzene to obtain compound 2 (light yellow solid). The molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride (compound 1) and n-butylamine was 1:(2.5-3); the molar volume ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride to ethanol was 1:(2-3)mmol / mL.

[0007] (2) Compound 2 and sodium methoxide were mixed and dissolved in methanol, and heated under reflux for 6-7 hours under nitrogen protection. Ice water of 1-5 times the volume of the solvent (methanol) was added and allowed to stand for 12-15 hours. The mixture was filtered and recrystallized from ethanol to obtain compound 3 (yellow-green solid). The molar ratio of compound 2 and sodium methoxide was 1:(3-3.5); the molar volume ratio of compound 2 to methanol was 1:(2-3)mmol / mL.

[0008] (3) Compound 3 and 57% hydroiodic acid were mixed, and the mixture was heated under reflux for 48-72 hours under nitrogen protection. The mixture was cooled and concentrated at room temperature, and separated by column chromatography to obtain compound 4 (light yellow solid). The molar volume ratio of compound 3 and 57% hydroiodic acid was 1: (2-3) mmol / mL.

[0009] (4) Compound 4, 4-chloroacetylmorpholine and triethylamine were dissolved in dichloromethane, stirred at room temperature for 18-20 hours, the product was concentrated, purified by silica gel column, and recrystallized to obtain the final product (light yellow solid), i.e., the fluorescent probe of the new substrate metabolism, named NBM. The molar ratio of compound 4, 4-chloroacetylmorpholine and triethylamine was 1:(2-2.5):(3-4); the molar volume ratio of compound 4 to dichloromethane was 1:(8-15)mmol / mL.

[0010] The synthesis process of the probe is as follows:

[0011]

[0012] In addition, characterization was performed by means of nuclear magnetic resonance hydrogen spectrum, carbon spectrum, mass spectrum, ultraviolet spectrum and the like, indicating that the fluorescent probe NBM was successfully synthesized.

[0013] The present invention provides a fluorescent probe for carboxylesterase 2, the structural formula of which is shown in I:

[0014]

[0015] The above structural formula I can qualitatively detect carboxylesterase 2. The NBM probe of the present invention has a maximum absorption wavelength of 340 nm and a fluorescence emission wavelength of 416 nm when carboxylesterase 2 is not added. When carboxylesterase 2 is added, the maximum absorption and maximum emission are red-shifted to 450 nm and 560 nm, and the reaction generates a compound having structural formula II, thereby causing fluorescence changes.

[0016]

[0017] The above structural formula I can quantitatively detect carboxylesterase 2. Carboxylesterase 2 aqueous solutions with gradient concentrations are added to aqueous solutions containing structural formula I, and the content of carboxylesterase 2 in the test solution can be read out according to the fluorescence intensity within the linear reaction time of enzyme kinetics.

[0018] Structural formula I is used as a specific fluorescent probe for carboxylesterase 2 for rapid screening of carboxylesterase 2 inhibitors and quantitative evaluation of inhibitory ability; it is also used to detect the activity of carboxylesterase 2 in various living cells. With the help of its lysosomal targeting, it can be quickly located in the lysosome and used to detect the activity of lysosomal carboxylesterase 2.

[0019] Normal pancreatic cells and tumor cells can be distinguished based on the activity of endogenous carboxylesterase 2 in different types of cells.

[0020] The fluorescent probe of the present invention uses 1,8-naphthaleneimide as a fluorescent group, introduces 4-morpholineacetic acid to locate in lysosomes, uses carboxylate as an enzyme cleavage site, and constructs a carboxylesterase 2 fluorescent probe. The probe induces the cleavage and departure of the carboxylester group of carboxylesterase 2 through an enzymatic reaction with carboxylesterase 2, and detects the presence and activity of carboxylesterase 2 through changes in the fluorescence intensity of the probe and its reaction product at 560 nm. It can be used for qualitative and quantitative detection of carboxylesterase 2 activity in various living cells and subcellular levels.

[0021] The beneficial effects of the present invention are as follows: the fluorescence of the compound changes significantly in the presence of carboxylesterase 2, and the compound can be used to detect carboxylesterase 2 with high selectivity and high sensitivity, and can be used as a fluorescent probe of carboxylesterase 2 with high specificity.

[0022] 1) Cheap and easy to obtain: It can be obtained through simple chemical synthesis, the synthetic raw materials are cheap and easy to obtain, the synthesis process is simple and easy, the cost is low, and it is easy to promote;

[0023] 2) High specificity and high sensitivity: The probe can be specifically hydrolyzed by carboxylesterase 2 to form hydroxynaphthylamide fluorophore. The detection limit of carboxylesterase 2 was 1.986 μg / ml by the standard curve.

[0024] 3) Live cell detection: The compound can be quickly located in the lysosome by virtue of its lysosomal targeting, and can be used to detect the activity of lysosomal carboxylesterase 2. In particular, the compound can be used to detect carboxylesterase 2 in lysosomes in human pancreatic cells, and to distinguish normal pancreatic cells from cancer cells based on the different carboxylesterase 2 contents in normal pancreatic cells and cancer cells. This is of great significance for in-depth study of the dynamic mechanism of carboxylesterase 2 in physiological and pathological processes in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the principle of detecting carboxylesterase 2 using the fluorescent probe NBM provided by the present invention;

[0026] Figure 2 The probe NBM synthesized in Example 1 1 H NMR (a), 13 C NMR (b) spectrum;

[0027] Figure 3 UV-visible absorption spectrum (a) and emission spectrum (b) of the fluorescent probe NBM in response to carboxylesterase 2 in Example 2;

[0028] Figure 4 Schematic diagrams of the selectivity of the fluorescent probe NBM for carboxylesterase 2 in Example 3 (a) and (b);

[0029] Figure 5 The kinetic diagram of the fluorescent probe NBM responding to different concentrations of carboxylesterase 2 in Example 4 and the linear relationship between the reaction rate and the enzyme concentration;

[0030] Figure 6 Kinetic diagram of the reaction rate of the fluorescent probe NBM catalyzed by carboxylesterase 2 (a) and a complex system (human liver cell plasma) (b) in Example 5;

[0031] Figure 7 Schematic diagram of (a) and (b) the fluorescent probe NBM acting as an inhibitor of carboxylesterase 2 in Example 6;

[0032] Figure 8 Schematic diagram of imaging of the fluorescent probe NBM in HPDE6-C7 (a) and PANC-1 (b) cells in Example 7;

[0033] Fig. 9 Schematic diagram of the co-localization imaging of the fluorescent probe NBM in lysosomes of HPDE6-C7 (a) and PANC-1 (b) cells in Example 8. DETAILED DESCRIPTION

[0034] The examples are used to further illustrate the present invention, but the present invention is not limited to the examples.

[0035] Example 1 (Synthesis of probe NBM):

[0036] The synthetic route of the fluorescent probe NBM is as follows, which specifically includes the following steps:

[0037] (1) 0.1805 mol of 4-bromo-1,8-naphthalene dicarboxylic anhydride (Compound 1) was dissolved in 500 ml of ethanol, 0.5013 mol of n-butylamine was added, and the mixture was refluxed at 78° C. for 3 hours. The mixture was poured into 1000 ml of ice-water mixture, allowed to stand overnight, filtered, and recrystallized from chlorobenzene to obtain Compound 2 (yellow solid) with a yield of 70%.

[0038] (2) 0.1264 mol of compound 2 and 0.3875 mol of sodium methoxide were dissolved in 350 ml of methanol. The mixture was heated under reflux for 6 hours under nitrogen protection. 1000 ml of ice-water mixture was poured into the mixture and allowed to stand overnight. The mixture was filtered and recrystallized from ethanol. The mixture was dried in vacuo to obtain compound 3 (yellow solid) with a yield of 61%.

[0039] (3) 0.2118 mol of compound 3 and 500 ml of 57% wt potassium iodate were mixed, heated under reflux for 48 hours under nitrogen protection, cooled to room temperature, concentrated under reduced pressure to a solid, and separated by column chromatography to obtain compound 4 (yellow solid) with a yield of 91%.

[0040] (4) 1 mmol of compound 4 was dissolved in 10 ml of dichloromethane, and 2 mmol of 4-chloroacetylmorpholine and 3 mmol of triethylamine were added. The mixture was stirred at room temperature for 18 hours and concentrated under reduced pressure to obtain a solid. The solid was purified by column chromatography and recrystallized from dichloromethane to obtain the final product NBM (light yellow solid) with a yield of 33%.

[0041] The probe NBM prepared in this example was subjected to nuclear magnetic resonance analysis ( Figure 2 (ab)), the results are as follows: 1H NMR (400

[0042] MHz,Chloroform-d)δ8.61(t,J=8.4Hz,2H),8.23(d,J=8.4Hz,1H),7.78(t,J=7.9Hz,1H),7.58(d,J=8.1Hz,1H),4.18(t,J=7.4Hz,2 H),3.82(t,J=4.5Hz,4H),3.71(s,2H),2.77(t,J=4.4Hz,4H),1.71(p,J=7.5Hz,2H),1.45h,J=7.3Hz,2H),0.98(t,J=7.3Hz,3H).13C NMR (101MHz, CDCl3) δ167.87,163.97,163.42,151.00,131.69,131.67,129.37,127.41,127.32,125.09 ,123.07,120.67,119.32,77.33,77.22,77.02,76.70,66.81,59.46,53.32,40.32,30.20,20.38,13.84.

[0043] Example 2 (UV-visible absorption spectra (a) and emission spectra (bc) of fluorescent probe NBM and aqueous solution after metabolism):

[0044] The probe NBM was dissolved in boric acid borax buffer (50mM, PH=5) to prepare a 10uM solution. 200ul of the solution was added to a 96-well plate, and the UV-visible absorption spectrum (a) and emission spectrum (b: excitation wavelength 360nm, c: excitation wavelength 450nm) of the working solution were measured; reaction system (solvent boric acid borax buffer (50mM, PH=5)): 200ul, the final concentrations of the probe NBM and carboxylesterase 2 were 10uM and 50ug / ml respectively, incubated at 37℃ for 30min, and the absorption and emission spectra were measured. The results showed that the absorption and maximum emission wavelengths of the probe NBM were 360nm / 416nm. The absorption and maximum emission wavelengths after reaction metabolism were 450nm / 560nm.

[0045] Example 3 (Selectivity of NBM for Carboxylesterase 2):

[0046] Preliminary screening: Dispose the aqueous solutions of sarcosine oxidase, bilirubin oxidase, choline oxidase, cholesterol oxidase, lactate oxidase, L-amino acid oxidase, D-amino acid oxidase, alcohol oxidase, acetylcholinesterase, alkaline phosphatase, lipase, amine oxidase, ascorbic acid oxidase, lipoxygenase, lipase A, carboxylesterase 1, carboxylesterase 2 and aromatic acetamide deacetylase, respectively, with a concentration of 1 mg / ml, and store frozen at -80°C. Slowly dissolve on ice before use, use each portion only once, and prepare the aqueous solution of NBM (0.5mM), which is prepared immediately before use. The total volume of each reaction system is 200ul, and 4ul NBM (0.5mM) is added to 192ul 50mM PH=5 boric acid borax buffer, and 4ul of the above-mentioned different enzymes are added respectively, incubated at 37°C for 30min, and measured using an ELISA reader and a 96-well ELISA plate, and the final concentrations of the probe and enzyme are 10uM and 20ug / ml, respectively. The fluorescence emission spectrum of the working solution was measured, λex = 450nm, λem = 560nm. The results of the NBM selectivity experiment for carboxylesterase 2 are shown in Figure 4 As shown in (a), the fluorescence intensity at 560 nm is significantly enhanced. Figure 4 (a) It shows that NBM has good selectivity for carboxylesterase 2, and the fluorescence of the system is significantly enhanced.

[0047] Carboxylesterase 2 (1 mg / ml), liver microsomes (10 mg / ml) and liver cell fluid (10 mg / ml) were prepared with borate borax buffer (50 mM, pH = 5) respectively, and the reaction conditions were the same as above. Figure 4 As shown in (b), under the assay conditions, there is a response in the complex enzyme system.

[0048] Example 4 (Quantitative detection of carboxylesterase 2 by NBM):

[0049] Prepare 0.1mg / ml carboxylesterase 2, the total volume of the reaction system is 200ul, add 4ul NBM (0.5mM) to different volumes of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100ul of 0.1mg / ml carboxylesterase 2 in boric acid borax buffer (50mM, pH=5) to start the reaction, incubate at 37℃ for 30min, and measure using an ELISA reader and a 96-well ELISA plate. Measure the fluorescence emission spectrum of the working solution, λex=450nm, λem=560nm. The results show: Figure 5 As the enzyme concentration increases from 0 to 50ug / ml, the fluorescence intensity of the reaction system gradually increases, and the product generation rate increases proportionally.

[0050] Example 5 (Determination of Enzymatic Parameters of NBM):

[0051] Prepare a gradient dilution of NBM aqueous solution, the total volume of the reaction system is 50ul, add 1ul NBM (concentrations are 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2 and 2.5mM) and 1ul carboxylesterase 2 (1mg / ml) to 48ul boric acid borax buffer (50mM pH = 5) for each reaction, incubate at 37℃ for 25 minutes, measure with a microplate reader and a 96-well microplate, and measure the fluorescence emission spectrum of the working solution, λex = 450nm, λem = 560nm. The results show that: Figure 6 (a) The catalysis of probe NBM by carboxylesterase 2 is consistent with the model of enzyme kinetics, where K m =22.01μM, V max =14.40nmol / min / mg;

[0052] The total volume of the reaction system was 50ul. In each reaction, 1ul of NBM (concentrations were 0.025, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2 and 2.5mM) and 1ul of human liver cell plasma (10mg / ml) were added to 48ul of boric acid borax buffer (50mM pH=5). The reaction was incubated at 37°C for 10 minutes. The fluorescence emission spectrum of the working solution was measured using an ELISA reader and a 96-well ELISA plate. λex=450nm, λem=560nm. The results showed that: Figure 6 (b), The catalysis of probe NBM by carboxylesterase 2 conforms to the model in enzyme kinetics, Km = 135 μM, Vmax = 283.4 nmol / min / mg.

[0053] Example 6 (NBM inhibition experiment):

[0054] The screened inhibitors were prepared into a series of concentration gradient working solutions: parecoxib (concentrations were 0.1, 0.5, 1, 2.5, 5, 7.5 and 10 mM), erdosteine ​​(concentrations were 0.025, 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 7.5 and 10 mM) and nitisinone (concentrations were 0.25, 0.5, 1, 2.5, 5, 7.5, 10, 30, 60 and 90 mM). The total volume of the reaction system was 200ul. For each reaction, 2ul of the above inhibitor solution and 4ul of carboxylesterase 2 (1mg / ml) were added to 192ul of boric acid borax buffer (50mM, pH=5). 2ul of probe NBM (0.5mM) was used to initiate the reaction. The reaction was incubated at 37°C for 30min. The fluorescence emission spectrum of the working solution was measured using an ELISA reader and a 96-well ELISA plate. λex=450nm, λem=560nm. The results showed that: Figure 7, the catalytic effect of NBM on carboxylesterase 2 could be inhibited by carboxylesterase 2 inhibitors, with the calculated IC50 being 30.73, 31.55 and 34.41uM, respectively.

[0055] Example 7 (Probe NBM is used for the detection of carboxylesterase 2 in HPDE6-C7 and PANC-1 cells):

[0056] HPDE6-C7 and PANC-1 cells were cultured according to the American type Tissue Culture Collection regulations. They were inoculated in a 35 mm × 12 mm (diameter * height) culture dish and cultured for 24 hours to allow them to adhere to the wall. The culture medium was discarded before use and washed three times with 50 mM PH = 5 boric acid borax buffer. Control group: add 1ml boric acid borax buffer and incubate at 37℃ for 30min for observation; Experimental group: add 1ml NBM (final concentration 10uM) in boric acid borax buffer (50mM PH=5), incubate at 37℃ for 30min for observation; Inhibition group (parecoxib, erdosteine, nitisinone): first incubate with 1ml parecoxib (final concentration 100uM), erdosteine ​​(final concentration 100uM) and nitisinone (final concentration 100uM) (carboxylesterase 2 inhibitor) in boric acid borax buffer (50mM PH=5) at 37℃ for 30min, then add probe NBM (final concentration 10uM)) and incubate for 30min. Use confocal microscope Olympus FV1000 (Ex.405nm, Em.500-560nm) lens (×40) for observation and image acquisition. The first row is the bright field image, the second row is the fluorescence channel image, and the third row is the superimposed image. The results show that: Figure 8 (a), Fluorescence image of HPDE6-C7 cells. The control group has almost no fluorescence. After adding the probe, the fluorescence intensity increases. When the inhibitor is used, the fluorescence of the inhibition group is significantly weakened. Figure 8 (b) The response of the probe to PANC-1 cells is consistent with that to HPDE60C7 cells. Figure 8 In the second column of (a) and (b), the fluorescence intensity in pancreatic cancer cells (Panc-1 cells) is higher than that in normal pancreatic cells (HPDE6-C7 cells). This experiment proves that the probe NBM can be used to detect carboxylesterase 2 in cells and can distinguish normal pancreatic cells from cancer cells.

[0057] Example 8 (Lysosome co-localization imaging of probe NBM in HPDE6-C7 and PANC-1 cells):

[0058] HPDE6-C7 and PANC-1 cells were cultured according to the American type Tissue Culture Collection regulations, inoculated in 35 mm × 12 mm (diameter * height) culture dishes and cultured for 24 h to allow them to adhere to the wall. Before use, the culture medium was discarded, and the cells were washed three times with boric acid borax buffer (50 mM pH = 5), and stained with NBM probe and Lyso-Tracker Red. Fig. 9 As shown in (a), HPDE6-C7 cells were placed under a confocal fluorescence microscope and photographed. The first row is the bright field image, and the second row is the fluorescence channel image. Fig. 9 (a) As shown in the first column, the probe NBM (final concentration 10uM) was stained, incubated at 37℃ for 30min, and the excitation channel was 405nm. Fig. 9 (a) As shown in the second column, cells were counterstained with the commercial subcellular organelle localization reagent Lyso-Tracker Red (final concentration 50 nM), incubated at 37°C for 30 min, and excited at 543 nm. The third column is an overlay of the two channels, verifying that the probe can be localized to the cell lysosome. Fig. 9 As shown in (b), PANC-1 cells were photographed under a confocal fluorescence microscope. Fig. 9 (b) As shown in the first column, the probe NBM (final concentration 10uM) was stained, incubated at 37℃ for 30min, and the excitation channel was 405nm. Fig. 9 (b) As shown in the second column, cells were counterstained with the commercial subcellular organelle localization reagent Lyso-Tracker Red (final concentration 50 nM), incubated at 37°C for 30 min, and the excitation channel was 543 nm. The third column is an overlay of the two channels to verify that the probe can be localized to the cell lysosome. The results show that the Pearson coefficient of counterstaining with Lyso-Tracker Red is very high, and the probe can be localized to the lysosome.

Claims

1. A fluorescent probe for carboxylesterase 2, characterized in that: The structural formula of the fluorescent probe is shown in Structure I: 。 2. A method for synthesizing a fluorescent probe of carboxylesterase 2, characterized in that: The following steps are involved: (1) Dissolve 4-bromo-1, 8-naphthalene dicarboxylic anhydride (compound 1) and n-butylamine in ethanol, heat and reflux at 78-80°C for 3-5 hours, pour in a mixture of ice water 1-5 times the volume of ethanol, let stand for 12-15 hours, filter, and recrystallize from chlorobenzene to obtain compound 2; (2) Compound 2 and sodium methoxide were dissolved in methanol, heated under reflux for 6-7 hours under nitrogen protection, and an ice-water mixture of 1-5 times the volume of methanol was added and allowed to stand for 12-15 hours, then filtered and recrystallized from ethanol to obtain compound 3; (3) Compound 3 was mixed with 57% hydroiodic acid, and the mixture was heated under reflux for 48-72 hours under nitrogen protection. The mixture was cooled at room temperature and concentrated into a solid, and then separated by column chromatography to obtain compound 4; (4) Compound 4, 4-chloroacetylmorpholine and triethylamine were dissolved in dichloromethane and stirred at room temperature for 18-20 hours. The product was concentrated, purified by silica gel column and recrystallized to obtain the final product. The product was a light yellow solid, i.e., the fluorescent probe of claim 1, named NBM.

3. The method for synthesizing the fluorescent probe of carboxylesterase 2 according to claim 2, characterized in that: Place In the step (1), 4-bromo-1, 8-naphthalene dicarboxylic anhydride (Compound 1) and n-butylamine are in a molar ratio of 1: (2.5-3); the molar volume ratio of 4-bromo-1, 8-naphthalene dicarboxylic anhydride to ethanol is 1: (2-3) mmol / mL; in the step (2), Compound 2 and sodium methoxide are in a molar ratio of 1: (3-3.5); the molar volume ratio of Compound 2 to methanol is 1: (2-3) mmol / mL; in the step (3), Compound 3 and 57% hydroiodic acid are in a molar volume ratio of 1: (2-3) mmol / mL; in the step (4), Compound 4, 4-chloroacetylmorpholine and triethylamine are in a molar ratio of 1: (2-2.5): (3-4); the molar volume ratio of Compound 4 to dichloromethane is 1: (8-15) mmol / mL.

4. Use of the fluorescent probe for carboxylesterase 2 according to claim 1 in detecting carboxylesterase 2, wherein the use is not suitable for diagnosis and treatment of diseases.

5. The use of the fluorescent probe for carboxylesterase 2 according to claim 4 in detecting carboxylesterase 2, characterized in that: The compounds represented by structure I can be used for the qualitative or quantitative detection of carboxylesterase 2.

6. Use of the fluorescent probe for carboxylesterase 2 according to claim 4 or 5 in detecting carboxylesterase, characterized in that: The compound represented by structure I can be used for rapid detection of carboxylesterase 2, and its action model is consistent with kinetics.

7. Use of the fluorescent probe for carboxylesterase 2 according to claim 4 or 5 in detecting carboxylesterase 2, characterized in that: When the compound represented by structure I is applied to the detection of carboxylesterase 2, it generates a compound having structure II, thereby causing a fluorescence change; 。 8. Use of the fluorescent probe for carboxylesterase 2 according to claim 4 or 5 in detecting carboxylesterase 2, after the compound represented by structure I acts on carboxylesterase 2, the fluorescence intensity detected at 560 nm is proportional to the activity of carboxylesterase 2, and an increase in the fluorescence intensity means an increase in activity.

9. Use of the fluorescent probe for carboxylesterase 2 according to claim 4 or 5 in detecting carboxylesterase 2, characterized in that: The fluorescent probe can be used to achieve fluorescent imaging of intracellular carboxylesterase 2.

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