A bioluminescent probe for detecting carboxylesterase 2 in a living organism, and a preparation method and use thereof

Abstract

The application provides a bioluminescent probe for detecting carboxylesterase 2 in vivo, and a preparation method and application thereof, and belongs to the technical field of biological detection.The bioluminescent probe is a compound shown in formula CBP, or a salt thereof, or a stereoisomer thereof.The bioluminescent probe for detecting carboxylesterase 2 in vivo provided by the application has good biocompatibility, a fast reaction speed with carboxylesterase 2, strong selectivity, almost no response to other interfering ions, high detection sensitivity, good responsiveness, low detection limit, a linear relationship between the bioluminescent intensity and carboxylesterase 2 in a certain concentration range, and stable bioluminescent signals, and can be used for qualitative and quantitative detection of carboxylesterase 2, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a bioluminescent probe for detecting carboxylesterase 2 in a living organism, and a preparation method and use thereof. BACKGROUND

[0002] Bioluminescence is a chemical luminescence that is ubiquitous in nature and depends on the normal life activities of organisms. Its essence is the interaction between enzymes and substrates, and chemical energy is generated through a series of biological reactions and released in the form of light energy. Bioluminescence imaging is carried out by using the principle that light photons are generated by chemical reactions catalyzed by enzymes in the living body. The most common bioluminescence system is the luciferase-luciferin system of fireflies, the essence of which is that firefly luciferase catalyzes the substrate luciferin in the presence of energy (ATP) and oxygen, and light photons are generated when the molecule returns to the ground state from the excited state, and oxidized luciferin (Oxyluciferin) is released. Bioluminescence imaging technology has become an important detection means due to its high sensitivity, high biocompatibility and visualization, and is widely used in various fields.

[0003] Carboxylesterases (CE) as an important phase I drug metabolizing enzyme in vivo, belong to the esterase family, and exist widely in mammalian cells. They mainly catalyze the hydrolysis of ester bonds, amide bonds and thioester bonds, and are involved in the detoxification and metabolism of various ester drugs, environmental toxins and carcinogens. In the human body, carboxylesterases mainly include two subtypes of carboxylesterase 1 (CE1) and carboxylesterase 2 (CE2), and there are significant differences between the two in tissue distribution and substrate specificity. CE2 is highly expressed in many cancer cells, and CE2 plays a role in mediating the activation of many prodrugs such as gemcitabine, irinotecan (CPT11) and capecitabine (CAPE). In addition, CE2 is considered to be an important determinant of the first metabolic pathway in the intestine, especially for oral anticancer prodrugs. In view of the key role of CE2 in the metabolism of various anticancer prodrugs, it is quite necessary to develop a suitable technology to accurately and sensitively detect CE2 under complex physiological conditions. SUMMARY

[0004] In order to solve the problems of poor selectivity, low sensitivity and harsh conditions of the existing carboxylesterase 2 optical probe, the present application provides a bioluminescent probe for detecting carboxylesterase 2 in a living organism, and a preparation method and use thereof. The bioluminescent probe provided by the present application has high selectivity and ultra-high sensitivity for carboxylesterase 2, and can realize qualitative and quantitative analysis and detection of carboxylesterase 2 with high selectivity and high sensitivity.

[0005] The present application provides a compound, or a salt thereof, or a stereoisomer thereof, the structure of the compound being shown in formula CBP:

[0006]

[0007] The present application also provides a preparation method of the aforementioned compound, which comprises the following steps:

[0008]

[0009] (1) reacting compound 1 and compound 2 in a solvent under the action of a base to obtain compound 3;

[0010] (2) reacting compound 3 and D-cysteine hydrochloride in a solvent under the action of a base to obtain compound CBP.

[0011] Further,

[0012] In step (1), the solvent is anhydrous dichloromethane;

[0013] And / or, in step (1), the base is triethylamine;

[0014] And / or, in step (2), the solvent is a mixed solution of dichloromethane, anhydrous methanol and water;

[0015] And / or, in step (2), the base is potassium carbonate.

[0016] Further,

[0017] In step (1), compound 1 is dissolved in anhydrous dichloromethane, and then a solution of compound 2 and a base in anhydrous dichloromethane is added, and then reacted to obtain compound 3;

[0018] And / or, in step (2), compound 3 is dissolved in a mixed solution of dichloromethane and anhydrous methanol, and then a mixed solution of anhydrous methanol and water containing a base and D-cysteine hydrochloride is added, and then reacted to obtain compound CBP.

[0019] Further,

[0020] In step (2), the volume ratio of dichloromethane to anhydrous methanol in the mixed solution of dichloromethane and anhydrous methanol is 1:1;

[0021] And / or, in step (2), the volume ratio of anhydrous methanol to water in the mixed solution of anhydrous methanol and water is 1:1.

[0022] Further,

[0023] In step (1), the molar ratio of compound 1, compound 2 and the base is 1:1-5:1-5;

[0024] And / or, in step (1), the reaction temperature is 0-40℃, and the reaction time is 4-10h.

[0025] And / or, in step (2), the molar ratio of compound 3 and D-cysteine hydrochloride is 1:1-5;

[0026] And / or, in step (2), the mass ratio of compound 3 and base is 1:1-2;

[0027] And / or, in step (2), the temperature of the reaction is 20-30 DEG C, and the time is 1-2 h.

[0028] Further,

[0029] In step (1), the molar ratio of compound 1, compound 2 and base is 1:2.5:2.5;

[0030] And / or, in step (1), the reaction is carried out at 0-4 DEG C for 1-3 h, and then at 25-40 DEG C for 5-6 h;

[0031] And / or, in step (2), the molar ratio of compound 3 and D-cysteine hydrochloride is 1:2;

[0032] And / or, in step (2), the mass ratio of compound 3 and base is 1:1.2-1.3.

[0033] Further,

[0034] In step (1), after the reaction, the reaction solution is purified to obtain compound 3, and the purification step is spin-drying, extraction, drying, filtration, spin-drying, column chromatography separation, and the eluent used in the column chromatography separation is a mixed solution of petroleum ether and ethyl acetate in a volume ratio of 9:1;

[0035] And / or, in step (2), after the reaction, the reaction solution is purified to obtain CBP, and the purification step is spin-drying, adding water, adjusting the pH to 1 with hydrochloric acid, precipitating a solid, and filtering.

[0036] The application also provides a use of the aforementioned compound, or a salt thereof, or a stereoisomer thereof in the preparation of a bioluminescent probe.

[0037] Further, the bioluminescent probe is a bioluminescent probe for detecting carboxylesterase 2;

[0038] Preferably, the bioluminescent probe is a bioluminescent probe for detecting carboxylesterase 2 in a living organism.

[0039] The present application synthesizes a novel bioluminescent probe CBP for detecting carboxylesterase 2 by taking 2-thiophenecarbonyl as a recognition group and hydroxyl fluorescein as a bioluminescent group. 2+ The carboxylesterase 2 can react with the ester group in the probe, so that the ester group is broken, and the hydroxyl fluorescein is released, and strong bioluminescence is generated under the action of ATP, luciferase, Mg

[0040] Compared with the prior art, the advantages of the technical scheme of the present application are as follows:

[0041] The bioluminescent probe for detecting carboxylesterase 2 in the present application has good biocompatibility, fast reaction speed with carboxylesterase 2, strong selectivity, almost no response to other interfering ions, high detection sensitivity, good responsiveness, low detection limit, linear relationship between bioluminescent intensity and carboxylesterase 2 in a certain concentration range, and stable bioluminescent signal, and can be used for qualitative and quantitative detection of carboxylesterase 2, and has wide application prospect.

[0042] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and common means in the art, other various forms of modification, replacement or change can be made without departing from the above technical idea of the present application.

[0043] The above content of the present application will be further described in detail through the specific embodiments below. However, it should not be understood that the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the probe CBP prepared in Example 1 is shown.

[0045] Figure 2 The nuclear magnetic resonance carbon spectrum of the probe CBP prepared in Example 1 is shown.

[0046] Figure 3 The bioluminescent linear response result graph of the probe CBP to carboxylesterase 2 in Test Example 1 is shown.

[0047] Figure 4 The selectivity result graph of the probe to carboxylesterase 2 in Test Example 2 is shown.

[0048] Figure 5 The bioluminescent comparison graph of the control group and the experimental group of mice (FVB-Luc + transgenic mice) in Test Example 3 is shown. Detailed Implementation

[0049] The raw materials and equipment used in the specific embodiments of the present invention are all known products, obtained by purchasing commercially available products.

[0050] 1. Materials and Instruments

[0051] Laboratory animal: Healthy male FVB-Luc + Transgenic mice (luciferase-expressing mice).

[0052] Instruments: ECZ-400S nuclear magnetic resonance spectrometer (JEOL Corporation, Japan); LB983NightOWL II small animal in vivo imaging system (BERTHOLD Corporation, Germany); YRT-3 melting point apparatus (Tianjin Tianda Tianfa Technology Co., Ltd.); ABIQ-starElite high-resolution mass spectrometer (Applied Biosystems, USA); automatic double pure water distiller (Millipore Corporation, USA); PB-21 pH meter (Sartorius Corporation, Germany); RTC basic magnetic stirrer (IKA Corporation, Germany).

[0053] 2. Solution preparation

[0054] (1) Preparation of Tris-HCl buffer solution containing MgCl2 (10 mM): First, dissolve 1.5143 g of Tris-base (tris(hydroxymethyl)aminomethane, 99.5%, MW = 121.14) in 250 mL of water, and adjust the pH to 7.2-7.4 with HCl to obtain a 50 mM Tris-HCl buffer solution; then, dissolve 0.508 g of MgCl2 (MgCl2·6H2O, MW = 203.30) in 250 mL of Tris-HCl buffer solution to obtain a MgCl2-containing buffer solution. 2+ Tris-HCl buffer solution.

[0055] (2) Preparation of luciferase solution containing ATP: Dispense luciferase into 60 μg / tubes. Add 3 mL of Tris-HCl buffer solution (50 mM, containing MgCl2 = 10 mM, pH = 7.4) containing MgCl2 (10 mM) to obtain a luciferase solution with a concentration of 20 μg / mL. Then add 3.63 mg of ATP (adenosine-5'-triphosphate disodium hydrate, 98%, MW = 605.2) to obtain a luciferase solution containing ATP (2 mM) (20 μg / mL). Prepare and use immediately.

[0056] (3) Preparation of PBS solution: Prepare a solvent system with a concentration of 25mM (pH 7.2) using purchased PBS mixed salt.

[0057] (4) Preparation of probe CBP stock solution: Dissolve probe CBP (0.39 mg, 0.001 mmol) in DMSO (333 μL) to obtain a 3 mM probe solution, and dilute the probe solution with PBS to obtain a 100 μM solution for use. Prepare fresh each time, and store at low temperature in the dark.

[0058] (5) Preparation of carboxylesterase 2 solution: Dissolve carboxylesterase 2 (50 μg) in sterile water (100 μL) to obtain a 500 μg / mL solution, and dilute the solution with sterile water to obtain (0.00, 1.50, 1.75, 2.00, 2.25, 2.50, 5.00, 10.00, 20.00 μg / mL) solutions for use.

[0059] (6) Preparation of loperamide solution: Dissolve loperamide (1.3 mg, 0.0025 mmol) in 1.265 μL of DMSO, and then add 2.53 μL of PEG 200 to obtain a 0.659 mM loperamide solution, and dilute the solution with PBS to obtain a 10 mM solution for use.

[0060] 3. Animal feeding

[0061] FVB-Luc + Transgenic mice, body weight 20-30 g. FVB-Luc + Transgenic mice can express luciferase systemically, and are ideal animal models for real-time imaging of carboxylesterase 2 in vivo by probes. + Transgenic mice are fed in a specific environment, and the animals are acclimated to the experimental environment for one week before the experiment, are housed in groups under natural photoperiod lighting conditions, the temperature is (22±2) °C, and the humidity is 50±10%, and the animals are allowed to eat and drink freely.

[0062] 4. In vivo horizontal bioluminescence imaging

[0063] Two hours before imaging, healthy male FVB-Luc + transgenic mice were divided into two groups, with 5 mice in each group. The control group was injected with 100 μL of CBP (100 μM) solution via the tail vein, and the experimental group was injected with 100 μL of loperamide solution (10 mM), a specific inhibitor of carboxylesterase 2, via the tail vein to inhibit the activity of carboxylesterase 2. One hour later, 100 μL of CBP (100 μM) solution was injected via the tail vein. Immediately, imaging was performed using a small animal in vivo imaging instrument. Select the bioluminescence mode, and the exposure time is 1 s.

[0064] Example 1, Preparation of a bioluminescence probe for detecting carboxylesterase 2 in a living organism according to the present application

[0065] The synthetic route of the bioluminescent probe for detecting carboxylesterase 2 in a living organism is as follows:

[0066]

[0067] (1) 528 mg (3.0 mmol) of 6-hydroxybenzothiazole-2-carbonitrile was dissolved in 10 mL of anhydrous dichloromethane, the temperature was brought to 0°C in an ice water bath under nitrogen protection, 5 mL of 148 mg (7.5 mmol) of triethylamine dissolved in anhydrous dichloromethane, 1.094 mg (7.5 mmol) of 2-thiophene formyl chloride was added dropwise, the reaction was continued at 0°C for 3 h, and then the temperature was increased to room temperature (26±2) °C, and the reaction was continued for 5-6 h, and then the remaining residue was dissolved in ethyl acetate, and then the organic phase was extracted with water, and then anhydrous sodium sulfate was added to the organic phase for drying, and then filtered, and then dried, and then separated by column chromatography, and then the eluent was petroleum ether / ethyl acetate (9:1, v / v), and then the intermediate 3 was obtained in the form of yellow oil, and the yield was 80%.

[0068] (2) 286 mg (1.0 mmol) of the intermediate 3 was dissolved in 10 mL of dichloromethane and 10 mL of anhydrous methanol, and then 370 mg of potassium carbonate dissolved in 2 mL of anhydrous methanol and 2 mL of distilled water, and then 450 mg (2.0 mmol) of D-cysteine hydrochloride was added dropwise under nitrogen protection, and then the reaction was continued at 20-30°C for 1-2 h, and then dried, and then 2 mL of distilled water was added, and then the pH was adjusted to 1 with 1 mM hydrochloric acid, and then a solid was precipitated, and then filtered to obtain the target product, the white solid powder probe CBP, and the structure of the probe CBP is as follows:

[0069]

[0070] 1 H NMR (400 MHz, Methanol-d4) δ 8.14 (d, J = 8.9 Hz, 1H), 8.05 (dd, J = 3.8, 1.2 Hz, 1H), 7.99 (d, J = 2.2 Hz, 1H), 7.92 (dd, J = 5.0, 1.2 Hz, 1H), 7.46 (dd, J = 8.9, 2.3 Hz, 1H), 7.27 (dd, J = 5.0, 3.8 Hz, 1H), 5.31 (t, J = 9.3 Hz, 1H), 3.77 (dd, J = 9.3, 1.7 Hz, 2H). 13 C NMR (101 MHz, Methanol-d4) δ 173.80, 164.86, 161.96, 160.48, 150.94, 149.54, 136.65, 134.88, 134.28, 132.04, 128.03, 124.32, 121.41, 114.99, 80.09, 35.32. Yield 65%, purity 90%.Figure 1 NMR hydrogen spectrum of CBP; Figure 2 NMR carbon spectrum of CBP.

[0071] The beneficial effects of the present application are demonstrated below by specific test examples.

[0072] Test Example 1, linear test of probe CBP response to carboxylesterase 2

[0073] The concentration of the probe CBP (100 μM) was fixed and different concentrations of carboxylesterase 2 (0.00, 1.50, 1.75, 2.00, 2.25, 2.50, 5.00, 10.00, 20.00 μg / mL) were added in a black 96-well plate, 50 μL of the probe solution (concentration of 100 μM) and 50 μL of different concentrations of carboxylesterase 2 solution were added to each well respectively, incubated at 37°C, and the incubation time was 30 min, then 50 μL of Luciferase (20 μg / mL) solution containing ATP (2 mM) was added, three replicates were made for each concentration, and then imaged under a live imager.

[0074] The probe (100 μM) and carboxylesterase 2 (0.00, 1.50, 1.75, 2.00, 2.25, 2.50, 5.00, 10.00, 20.00 μg / mL) were incubated in Tris-HCl buffer (50 mM, containing MgCl2=10 mM, pH=7.4) at 37°C for 30 min, then ATP (2 mM, 50 μL) and Luciferase (20 μg / mL) were added for reaction, and the relative bioluminescence intensity was measured, and the results are shown in Figure 3 , wherein the inserted graph is the linear relationship between bioluminescence intensity and carboxylesterase 2 concentration (0-20 μg / mL). According to the experiment, when the concentration of the probe is 0, the standard deviation of the measured 3 groups of values is 2967208.138, and Figure 3 , the slope is 2868266666, and the standard deviation divided by the slope gives the detection limit (LOD) of 0.001034495 mg / mL.

[0075] As can be seen from Figure 3 , with the increase of the concentration of carboxylesterase 2, the bioluminescence intensity gradually increased. In Tris-HCl buffer, the bioluminescence intensity showed a good linear relationship with the concentration of carboxylesterase 2 in the range of (0-20 μg / mL). The detection limit (LOD) of the probe CBP for carboxylesterase 2 was 0.001034495 mg / mL. The above results show that the probe CBP has good detection sensitivity and can quantitatively detect millimolar levels of carboxylesterase 2 in biological samples.

[0076] Test Example 2, selectivity study of the probe and carboxylesterase 2

[0077] The concentration of the fixed probe CBP (10 μM, 50 μL) was taken with different analytes: Blank; NaCl (1 mM); Na2CO3 (1 mM); MgCl2 (1 mM); NaNO3 (1 mM); ZnCl2 (1 mM); CaCl2 (1 mM); CuCl2 (1 mM); KCl (1 mM); Ser (1 mM); Trp (1 mM); Ala (1 mM); Thr (1 mM); Lys (1 mM); Cys (1 mM); Asp (1 mM); Phe (1 mM); ALT (5 μg / mL); AchE (5 μg / mL); Trypsin (5 μg / mL); BchE (5 μg / mL); chymotrypsin (5 μg / mL); HSA (5 μg / mL); CES1 (5 μg / mL); CES2 (5 μg / mL) were dissolved in the buffer solution, and after the addition of the probe, incubation was carried out at 37°C, the incubation time was 30 min, 50 μL of luciferase (20 μg / mL) solution containing ATP (2 mM) was added per well, and imaging was immediately carried out under the live imaging instrument. CES1 is carboxylesterase 1, and CES2 is carboxylesterase 2.

[0078] After the incubation of the probe CBP with various bioactive substances (Blank; NaCl; Na2CO3; MgCl2; NaNO3; ZnCl2; CaCl2; CuCl2; KCl; Ser; Trp; Ala; Thr; Lys; Cys; Asp; Phe; ALT; AchE; Trypsin; BchE; chymotrypsin; HSA; CES1; CES2), the bioluminescence intensity was observed, and the results are shown in Table 1. Figure 4 It can be seen that among various in vivo enzymes and molecular compounds, only carboxylesterase 2 causes relatively strong bioluminescence, and has relatively strong selectivity relative to carboxylesterase 1, and other in vivo enzymes and molecular compounds hardly produce obvious bioluminescence signals. It can be seen that the probe CBP can selectively detect carboxylesterase 2 in a living organism without interference from other substances, and the specificity of the probe CBP for the detection of carboxylesterase 2 is good.

[0079] Test Example 3, FVB-Luc + Imaging of Transgenic Mice

[0080] Animals used are luciferase transgenic mice (FVB-Luc + transgenic mice), 10 adult transgenic male mice were taken, 2 h before imaging, healthy male FVB-Luc +The transgenic mice are divided into two groups, each group of 5, and are anesthetized with isoflurane, and the control group is injected with 100 μL of a CBP (100 μM) solution via the tail vein; the experimental group is injected with 100 μL of a loperamide (10 mM) solution via the tail vein to inhibit the activity of carboxylesterase 2, and 1 h later, 100 μL of a CBP (100 μM) solution is injected via the tail vein. Then, imaging is recorded immediately under a live imaging instrument, and the time is recorded as 0 min, and then every minute thereafter until the bioluminescence intensity decreases, and the total intensity of all the light-emitting parts of the mouse except for the tail is plotted.

[0081] Two hours before imaging, healthy male FVB-Luc + The transgenic mice are divided into two groups, each group of 5, and the control group is injected with 100 μL of a CBP (100 μM) solution via the tail vein, and the experimental group is injected with 100 μL of a loperamide (10 mM) solution via the tail vein to inhibit the activity of carboxylesterase 2, and 1 h later, 100 μL of a CBP (100 μM) solution is injected via the tail vein, and imaging is immediately performed using a small animal live imaging instrument. The bioluminescence mode is selected, and the exposure time is 1 s. The bioluminescence intensity is recorded by Figure 5 It can be known that in the animal body, the probe can also detect the presence of carboxylesterase 2, and the bioluminescence intensity of the mouse injected with CBP is significantly higher than that before the probe is injected, and there is a significant statistical difference. Compared with the control group, the mouse injected with loperamide first and then injected with CBP 1 h later has a bioluminescence intensity that is significantly lower than that of the control group, and there is a significant statistical difference.

[0082] Moreover, since the experiment is performed in an anesthetized state of the mouse, the changes of the probe in the control group and the experimental group in the body can also be observed, and the normal life state of the mouse in the experiment and after the experiment is not affected. The small animal live imaging is performed, and a relatively strong bioluminescence signal can be observed, and the bioluminescence intensity reaches a maximum 1 min after the probe is injected via the tail vein, and then decreases. The above results show that the probe reacts quickly with carboxylesterase 2, and the generated bioluminescence signal is relatively strong and stable, and therefore, the probe can realize the bioluminescence imaging of endogenous carboxylesterase 2 in the living body.

[0083] In summary, the bioluminescence probe for detecting carboxylesterase 2 in a living body provided by the present application has good biocompatibility, a fast reaction speed with carboxylesterase 2, strong selectivity, almost no response to other interfering ions, high detection sensitivity, good responsiveness, a low detection limit, a linear relationship between the bioluminescence intensity and carboxylesterase 2 in a certain concentration range, and a stable bioluminescence signal, and can be used for qualitative and quantitative detection of carboxylesterase 2, and has a wide application prospect, such as being used for screening carboxylesterase 2 inhibitors.

Claims

1. A compound, or a salt thereof, or a stereoisomer thereof, characterized in that: The structure of the compound is shown as formula CBP:

2. Process for the preparation of a compound according to claim 1, characterized in that: It comprises the following steps: (1) Compound 1 and compound 2 react in a solvent under the action of a base to obtain compound 3; (2) Compound 3 and D-cysteine hydrochloride react in a solvent under the action of a base to obtain compound CBP.

3. The preparation method according to claim 2, wherein: In step (1), the solvent is anhydrous dichloromethane; And / or, in step (1), the base is triethylamine; And / or, in step (2), the solvent is a mixed solution of dichloromethane, anhydrous methanol and water; And / or, in step (2), the base is potassium carbonate.

4. The preparation method according to claim 3, wherein: In step (1), compound 1 is dissolved in anhydrous dichloromethane, and then a solution of compound 2 and a base in anhydrous dichloromethane is added, and then reacted to obtain compound 3; And / or, in step (2), compound 3 is dissolved in a mixed solution of dichloromethane and anhydrous methanol, and then a mixed solution of a base and D-cysteine hydrochloride in anhydrous methanol and water is added, and then reacted to obtain compound CBP.

5. The preparation method according to claim 4, wherein: In step (2), the volume ratio of dichloromethane to anhydrous methanol in the mixed solution of dichloromethane and anhydrous methanol is 1:1; And / or, in step (2), the volume ratio of anhydrous methanol to water in the mixed solution of anhydrous methanol and water is 1:

1.

6. The preparation method according to claim 4, wherein: In step (1), the molar ratio of compound 1, compound 2 and the base is 1:1-5:1-5; And / or, in step (1), the reaction temperature is 0-40℃, and the reaction time is 4-10h; And / or, in step (2), the molar ratio of compound 3 to D-cysteine hydrochloride is 1:1-5; And / or, in step (2), the mass ratio of compound 3 to the base is 1:1-2; And / or, in step (2), the reaction temperature is 20-30℃, and the reaction time is 1-2h.

7. The preparation method according to claim 6, wherein: In step (1), the molar ratio of compound 1, compound 2 and the base is 1:2.5:2.5; And / or, in step (1), the reaction is first carried out at 0-4℃ for 1-3h, and then carried out at 25-40℃ for 5-6h; And / or, in step (2), the molar ratio of compound 3 to D-cysteine hydrochloride is 1:2; And / or, in step (2), the mass ratio of compound 3 to the base is 1:1.2-1.

3.

8. The preparation method according to claim 2, wherein: In step (1), after the reaction, the reaction solution is purified to obtain compound 3, and the purification step is spin-drying, extraction, drying, filtration, spin-drying and column chromatography separation, and the eluent used in the column chromatography separation is a mixed solution of petroleum ether and ethyl acetate with a volume ratio of 9:

1. And / or, in step (2), the reaction solution is purified after the reaction to obtain the CBP, and the purification step is as follows: the reaction solution is spin-dried, water is added, hydrochloric acid is added to adjust the pH to 1, solid is precipitated, and filtration is performed.

9. Use of the compound of claim 1, or a salt thereof, or a stereoisomer thereof in the preparation of a bioluminescent probe, which is a bioluminescent probe for detecting carboxylesterase 2.

10. Use according to claim 9, characterized in that: The bioluminescent probe is a bioluminescent probe for detecting carboxylesterase 2 in a living organism. The bioluminescent probe is a bioluminescent probe for detecting carboxylesterase 2 in a living organism.

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