Bioluminescent probe (ZD-1) for detecting butyrylcholine esterase in living body as well as preparation method and application of bioluminescent probe

Abstract

The invention provides a bioluminescent probe for detecting butyrylcholine esterase in a living body as well as a preparation method and application of the bioluminescent probe, and belongs to the field of organic synthesis and detection. In the work, an open-type bioluminescent probe ZD-1 for detecting butyrylcholine esterase with high selectivity and high sensitivity is developed. The probe is manufactured by adding a recognition group of a cyclopropylformyl group to a hydroxyfluorescein skeleton. Cell and in-vivo imaging experiments verify that the probe has the capability of detecting butyrylcholine esterase in a biological system, and can provide a powerful tool for screening butyrylcholine esterase inhibitors. The probe ZD-1 has the advantages of simple preparation method, high yield, low cost, high efficiency, specificity, rapidness, sensitivity and the like, can qualitatively and quantitatively detect butyrylcholine esterase, and is easy to popularize and apply.

Description

Technical Field

[0001] The present invention provides a bioluminescent probe (ZD-1) for detecting butyrylcholinesterase in an organism, and a preparation method and application thereof. Background Art

[0002] Bioluminescence is a chemiluminescence that is ubiquitous in nature and depends on the normal life activities of organisms. Its essence is the interaction between enzymes and substrates, which produces chemical energy through a series of biological reactions and releases it in the form of light energy. Bioluminescence imaging is based on the principle that chemical reactions catalyzed by enzymes in organisms produce photons. The most common bioluminescence system is the firefly luciferase-luciferin system. Its essence is that firefly luciferase catalyzes the substrate luciferin under the conditions of energy (ATP) and oxygen, causing electronic transitions. When the molecule returns from the excited state to the steady state, photons are generated and oxidized luciferin is released. Bioluminescence imaging technology has become a very important means of detection due to its high sensitivity, high biocompatibility and visualization, and is widely used in various fields.

[0003] To evaluate the activity of butyrylcholinesterase (BChE), we designed and developed a new probe (ZD-1) that can be rapidly activated by BChE and emit light. Butyrylcholinesterase is an esterase synthesized by the human liver and widely distributed in many tissues such as liver, brain, and blood. It is a sensitive indicator for determining the protein synthesis function of hepatocytes and can be used for a variety of liver diseases such as liver cancer and cirrhosis. In addition, changes in BChE levels are also associated with many other diseases such as diabetes, cardiovascular disease, and Alzheimer's disease. Therefore, in clinical diagnosis, accurate detection of BChE activity is crucial for evaluating liver function and BChE-related diseases. Several effective methods for evaluating BChE activity have been established. As an attractive alternative, bioluminescent probes have become an indispensable tool for exploring bioactive molecules. However, these technical methods usually require time-consuming and complicated procedures and are costly, which may largely limit their application in rapid detection. In order to solve this problem, the present invention develops a bioluminescent probe with high selectivity and ultra-sensitivity, which exhibits excellent selectivity and responsiveness to butyrylcholinesterase and is also suitable for the study of butyrylcholinesterase in the field of biological imaging, providing a powerful research method for solving this problem. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a bioluminescent probe for detecting butyrylcholinesterase in organisms, as well as a preparation method and application thereof, so as to solve the problems of poor selectivity, low sensitivity and harsh conditions of existing butyrylcholinesterase optical probes.

[0005] The technical problem to be solved by the present invention is to provide a bioluminescent probe for detecting butyrylcholinesterase in organisms, as well as a preparation method and application thereof, so as to achieve qualitative and quantitative analysis and detection of butyrylcholinesterase with high selectivity and high sensitivity.

[0006] The present invention provides a bioluminescent probe for detecting butyrylcholinesterase in an organism, and its structural formula is:

[0007]

[0008] The present invention provides a method for preparing a bioluminescent probe for detecting butyrylcholinesterase in an organism, and the specific steps are as follows:

[0009] (1) Dissolve hydroxybenzothiazole in a solvent to obtain a hydroxybenzothiazole solution, add triethylamine, and then dropwise add cyclopropylcarbonyl chloride under nitrogen protection, react at room temperature, spin dry, extract, dry, filter, spin dry, and separate by column chromatography to obtain intermediate 3.

[0010] (3) Dissolve the intermediate 3 in a solvent to obtain an intermediate 3 solution, add potassium carbonate and D-cysteine ​​hydrochloride dissolved in the solvent dropwise under nitrogen protection, react for 1 to 2 hours, spin dry, add distilled water, adjust the pH with hydrochloric acid, precipitate solids, filter, and obtain a highly selective butyrylcholinesterase bioluminescent probe ZD-1 with a yield of 78%. The molar ratio of the intermediate 3, potassium carbonate and D-cysteine ​​hydrochloride is 1:1:1 to 1:2:2, and the concentration of the intermediate 3 solution is 30 to 40%.

[0011] In the step (1), the solvent is anhydrous dichloromethane, and diluted triethylamine is added dropwise at 0° C. The concentration of the triethylamine after dilution is 30-35%.

[0012] In the step (1), the reaction temperature is 25° C. to 40° C., the reaction time is 4 to 6 hours, and the column chromatography separation is performed using dichloromethane and petroleum ether in a volume ratio of 4:1.

[0013] The solvent in step (2) is dichloromethane and anhydrous methanol, and the solvents for potassium carbonate and D-cysteine ​​hydrochloride are anhydrous methanol and distilled water.

[0014] In the step (2), the reaction temperature is 20° C. to 30° C., the reaction time is 1 to 2 h, and the pH is adjusted to 1 to 2 using 1 M HCl.

[0015] The invention discloses a bioluminescent probe for detecting butyrylcholinesterase in organisms, which is used for qualitative and quantitative detection of butyrylcholinesterase. The bioluminescent probe has fast reaction speed with butyrylcholinesterase, high detection sensitivity, good responsiveness and broad application prospects.

[0016] The present invention uses cyclopropylformyl as the recognition group and hydroxyl fluorescein as the bioluminescent group to synthesize the novel bioluminescent probe for detecting butyrylcholinesterase. The preparation method of the bioluminescent probe is simple, high in yield and low in cost. Butyrylcholinesterase can react with the ester group in the optical probe, thereby breaking the ester group and releasing hydroxyl fluorescein. 2+ Strong bioluminescence is produced under the action of the above substances, and the bioluminescence intensity shows an excellent linear relationship with the butyrylcholinesterase concentration within a certain range, which can be used as a basis for the qualitative and quantitative detection of butyrylcholinesterase.

[0017] The probe ZD-1 of the present invention has high sensitivity and strong selectivity to butyrylcholinesterase, has almost no response to other interfering ions, and serves as a basis for qualitative and quantitative detection of butyrylcholinesterase, and has broad application prospects.

[0018] The technical solution of the present invention has the following advantages:

[0019] The bioluminescent probe for identifying butyrylcholinesterase provided by the present invention has the advantages of good selectivity, high sensitivity, low detection line and good biocompatibility.

[0020] In Tris-HCl buffer, the bioluminescence intensity showed a good linear relationship with the concentration of butyrylcholinesterase, indicating that the probe is suitable for quantitative detection of butyrylcholinesterase. The probe also achieved in vivo levels (FVB-Luc + The method has the advantages of high sensitivity detection of endogenous butyrylcholinesterase in transgenic mice, rapid response and stable bioluminescent signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 A nuclear magnetic resonance hydrogen spectrum of a bioluminescent probe for detecting butyrylcholinesterase in a living organism prepared in Example 1;

[0022] Figure 2 The carbon nuclear magnetic resonance spectrum of a bioluminescent probe for detecting butyrylcholinesterase in organisms prepared in Example 1;

[0023] Figure 3 A mass spectrum of a bioluminescent probe for detecting butyrylcholinesterase in an organism prepared in Example 1;

[0024] Figure 4 The linear response of the probe ZD-1 to butyrylcholinesterase in Example 2: The probe (100 μM) and butyrylcholinesterase (0.00, 0.10, 0.20, 0.40, 0.80, 1.60, 3.20, 6.40, 12.8, 25.6 U / mL) were mixed in Tris-HCl buffer (50 mM, containing MgCl 2=10 mM, pH=7.4, 1% DMSO) and incubated at 37°C for 30 min, then ATP (2 mM, 50 μL) and Luciferase (2 μg / mL) were added for reaction, and the relative bioluminescence intensity was measured;

[0025] Figure 5 The selectivity of the probe for butyrylcholinesterase in Example 1: the probe (100 μM) and butyrylcholinesterase, other different enzymes, molecular compounds (Blank; Gly (100 μM); NaHCO 3 (100μM);Sodium citrate(100μM);KCl(100μM);NaCl(100μM);KAl(SO 4 ) 2 (100 μM); MgSO 4 (100μM); Citric acid (100μM); CH 3 COONa (100 μM); CaCl 2 (100 μM); KH 2 PO 4 (100 μM); PdCl 2 (100 μM); GSH (100 μM); H 2 O 2 (100 μM); NADH (100 μM); NQO1 (5 μg / mL); Trypsin (5 μg / mL); GST (15 U / mL); BChE (15 U / mL)) in Tris-HCl buffer (50 mM, containing MgCl 2 =10 mM, pH=7.4, 1% DMSO) at 37°C for 30 min, and then ATP (2 mM, 50 μL) and Luciferase (2 μg / mL) were added for reaction, and the relative bioluminescence intensity was detected;

[0026] Figure 6 The mice in the control group and experimental group (FVB-Luc + Bioluminescence statistics of transgenic mice) The control group was intraperitoneally injected with 100 μL ZD-1 (100 μM) solution to induce male healthy mice (FVB-Luc + transgenic mice) luminescence; the experimental group was injected with LPS (10 mg / kg) solution through the tail vein to induce an inflammatory model, and 1 hour later, 100 μL ZD-1 (100 μM) solution was injected through the tail vein. DETAILED DESCRIPTION

[0027] For a better understanding of the present invention, the content of the present invention is further described below in conjunction with the examples, but the present invention is not limited to the following examples. The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercial products.

[0028] 1. Implementation Methods

[0029] 1. Materials and instruments

[0030] Experimental animals: healthy male FVB-Luc + Transgenic mice (luciferase expressing mice).

[0031] Instruments: ECZ-400S nuclear magnetic resonance instrument (JEOL, Japan); LB983NightOWL II small animal living imager (BERTHOLD, Germany); YRT-3 melting point meter (Tianjin Tianda Tianfa Technology Co., Ltd.); ABIQ-starElite high-resolution mass spectrometer (Applied Biosystems, USA); automatic double pure water distiller (Millipore, USA); PB-21 pH meter (Sartorius, Germany); RTC basic magnetic stirrer (IKA, Germany).

[0032] 2. Solution preparation

[0033] (1) Containing MgCl 2 Preparation of Tris-HCl buffer (10 mM): first dissolve 1.5143 g Tris-base (tris(hydroxymethyl)aminomethane, 99.5%, MW = 121.14) in 250 mL water, adjust the pH to 7.2-7.4 with HCl to obtain a 50 mM Tris-HCl buffer solution; then take 0.508 g MgCl 2 (MgCl 2 6H 2 O, MW = 203.30) was dissolved in 250 mL Tris-HCl buffer to obtain Mg 2+ Tris-HCl buffer solution.

[0034] (2) Preparation of luciferase solution containing ATP: luciferase was dispensed into 60 μg / tubes, 3 mL of Tris-HCl solution was added before use to obtain a luciferase solution with a concentration of 20 μg / mL, and then 3.63 mg of ATP (adenosine-5'-triphosphate disodium salt hydrate, 98%, MW = 605.2) was added to obtain a luciferase solution (20 μg / mL) containing ATP (2 mM), which was prepared and used immediately.

[0035] (3) Preparation of PBS solution: Use purchased PBS mixed salt to prepare a solvent system with a concentration of 25 mM (pH 7.2).

[0036] (4) Preparation of probe ZD-1 stock solution: Dissolve probe ZD-1 (2.0 mg, 0.00574 mmol) in DMSO (574 μL) to obtain a 10 mM probe solution, and dilute the stock solution to a 10 μM solution for later use. Prepare it before use and store it at low temperature and away from light.

[0037] 3. Animal husbandry

[0038] FVB-Luc + Transgenic mice, weight 20-30g. FVB-Luc + Transgenic mice can express luciferase throughout the body, making them an ideal animal model for real-time imaging of butyrylcholinesterase in vivo. + The transgenic mice were raised in a specific environment. The animals were adapted to the experimental environment one week before the experiment and were housed in groups under natural circadian lighting conditions. The temperature was (22±2)°C and the humidity was 50±10%. The animals had free access to food and water.

[0039] 4. In vivo bioluminescence imaging

[0040] 2 h before imaging, healthy male FVB-Luc + Transgenic mice were divided into two groups, 6 mice in each group. The control group was injected with 100 μL ZD-1 (100 μM) solution through the tail vein, and the experimental group was injected with LPS (10 mg / kg) solution through the tail vein to induce an inflammatory model. One hour later, 100 μL ZD-1 (100 μM) solution was injected through the tail vein. Immediately imaged using a small animal live imager. The bioluminescence mode was selected, and the exposure time was 1 s.

[0041] 2. Embodiment

[0042] Example 1: Synthesis of probe ZD-1

[0043] (1) 300 mg (1.70 mmol) of hydroxybenzothiazole was dissolved in 10 mL of anhydrous dichloromethane. The temperature was brought to 0°C in an ice-water bath under nitrogen protection. 861 mg (8.51 mmol) of triethylamine and 195.78 mg (1.87 mmol) of cyclopropylcarbonyl chloride dissolved in 5 mL of anhydrous dichloromethane were added dropwise. The mixture was reacted at 0°C for 3 h. The mixture was then moved to room temperature for further reaction. The reaction time was 5 to 6 h. The mixture was dried by spin drying, extracted, dried, filtered, dried by spin drying, and separated by column chromatography. The eluent was dichloromethane / petroleum ether (4:1, v / v). A white solid intermediate 3 was obtained with a yield of 88.96%.

[0044] (2) Dissolve 200 mg (0.82 mmol) of intermediate 3 in 2 mL of dichloromethane and 1 mL of anhydrous methanol. Under nitrogen protection, add dropwise 124.47 mg of potassium carbonate and 158.19 mg (0.90 mmol) of D-cysteine ​​hydrochloride monohydrate dissolved in 1 mL of anhydrous methanol and 0.5 mL of distilled water. React at 20°C to 30°C for 1 to 2 h. Dried by vortexing, add 2 mL of distilled water, adjust the pH to 1 to 2 with 1 mM hydrochloric acid, precipitate solids, and filter to obtain the target product, white solid powder probe ZD-1 (240 mg, 0.69 mmol). 1 H NMR (400 MHz, DMSO-d 6 )δ13.22(s,1H),8.19(d,J=8.9Hz,1H),8.06(d,J=2.3Hz,1H),7.41(dd,J=8.9,2.4Hz,1H),5.46(dd,J=9.8, 8.4Hz,1H),3.81(dd,J=11.3,9.9Hz,1H),3.76-3.66(m,1H),1.96(tt,J=7.8,4.7Hz,1H),1.16-1.02(m,3H); 13 C NMR (101 MHz, DMSO-d 6 )δ173.45,171.54,164.90,161.54,150.88,149.76,136.45,125.12,122.56,116.32,78.61,35.31,13.09,9.73; MS m / z calcd.for C 15 H 12 N 2 O 4 S 2 [M+H]+349.0317, found 349.0344. Yield: 84.14%.

[0045] Example 2: Linearity test of probe ZD-1 response to butyrylcholinesterase

[0046] The concentration of fixed probe ZD-1 (100 μM), the probe and butyrylcholinesterase (0.00, 0.10, 0.20, 0.40, 0.80, 1.60, 3.20, 6.40, 12.8, 25.6 U / mL) were placed in a black 96-well plate. 50 μL of probe solution and 50 μL of butyrylcholinesterase solution of different concentrations were added to each well, and the plates were incubated at 37°C for 30 min. Then, 50 μL of a mixed solution of luciferase and ATP was added. Three replicate wells were made for each concentration, and then images were taken under a living imager.

[0047] Example 3: Investigation of probe selectivity

[0048] The concentration of probe ZD-1 was fixed (100 μM), and a Tris-HCl solution with a butyrylcholinesterase concentration of (15 U / mL) was prepared. Different analytes (Blank; Gly (100 μM); NaHCO 3 (100μM);Sodium citrate(100μM);KCl(100μM);NaCl(100μM);KAl(SO 4 ) 2 (100 μM); MgSO 4 (100μM); Citric acid (100μM); CH 3 COONa (100 μM); CaCl 2 (100 μM); KH 2 PO 4 (100 μM); PdCl 2 (100 μM); GSH (100 μM); H 2 O 2 (100 μM); NADH (100 μM); NQO1 (5 μg / mL); Trypsin (5 μg / mL); GST (15 U / mL); BChE (15 U / mL)) were prepared into a solution with a buffer solution, and after adding the probe, incubated at 37°C for 30 min, 50 μL / well of luciferase solution containing ATP was added, and imaging was immediately performed under a live imaging instrument.

[0049] Example 4: FVB-Luc + Transgenic mouse imaging

[0050] The animals used were luciferase transgenic mice. Ten adult transgenic male mice were selected and healthy male FVB-Luc + Transgenic mice were divided into two groups, 5 in each group, and anesthetized with isoflurane. The control group was injected with 100 μL ZD-1 (100 μM) solution through the tail vein; the experimental group was injected with LPS (10 mg / kg) solution through the tail vein to induce an inflammatory model. One hour later, 100 μL ZD-1 (100 μM) solution was injected through the tail vein. Immediately, the images were recorded under a live imaging device, with the time being recorded as 0 min, and then recorded every one minute until the bioluminescence intensity decreased. The total intensity of all luminous parts of the mouse except the tail was plotted.

[0051] 3. Effect Verification

[0052] 1. Linear relationship between probe and butyrylcholinesterase reaction

[0053] The probe ZD-1 (100 μM) was incubated with different concentrations of butyrylcholinesterase (0.00, 0.10, 0.20, 0.40, 0.80, 1.60, 3.20, 6.40, 12.8, 25.6 U / mL) to observe the relationship between the bioluminescence intensity and the butyrylcholinesterase concentration. Figure 4 It can be seen that with the increase of butyrylcholinesterase concentration, the bioluminescence intensity gradually increases, and a linear relationship is present in the range of 0 to 0.8 U. After calculation, the linear equation is Y = 259471667X + 2510500, X is the concentration, Y is the bioluminescence intensity, the standard deviation when the concentration is 0 is 614925.47, and the calculated detection line is 0.0071 U / ml. The above results show that probe ZD-1 can detect BChE well.

[0054] 2. Study on the selectivity of probe and butyrylcholinesterase

[0055] The probe ZD-1 was combined with various bioactive substances (Blank; Gly; NaHCO 3 ; Sodium citrate; KCl; NaCl; KAl(SO 4 ) 2 MgSO 4 ;Citricacid;CH 3 COONa; CaCl 2 ; KH 2 PO 4 ; PdCl 2 ;GSH;H 2 O 2 NADH; NQO1 (5 μg / mL); Trypsin (5 μg / mL); GST (15 U / mL); BChE (15 U / mL).) and observe the bioluminescence intensity. Figure 5 It can be seen that among various enzymes and molecular compounds in the body, only butyrylcholinesterase caused strong bioluminescence and had strong selectivity, while other enzymes and molecular compounds in the body hardly produced obvious bioluminescence signals. It can be seen that probe ZD-1 can selectively detect butyrylcholinesterase in organisms without being interfered by other substances.

[0056] 3. In vivo bioluminescence imaging

[0057] 2 h before imaging, healthy male FVB-Luc +Transgenic mice were divided into two groups, 5 mice in each group. The control group was injected with 100 μL ZD-1 (100 μM) solution through the tail vein, and the experimental group was injected with LPS (10 mg / kg) solution through the tail vein to induce an inflammatory model. One hour later, 100 μL ZD-1 (100 μM) solution was injected through the tail vein and the images were immediately taken using a small animal in vivo imager. The bioluminescence mode was selected and the exposure time was 1 s. Figure 6 It can be seen that the probe can also detect the presence of butyrylcholinesterase in animals. The bioluminescence intensity of mice injected with ZD-1 was significantly higher than that before the probe was injected, with significant statistical differences. Compared with the control group, the bioluminescence intensity of mice injected with LPS first and then injected with ZD-1 1h later was significantly higher than that of the control group, with significant statistical differences. Moreover, since the experiment was conducted under anesthesia of mice, we can also observe the changes of the probe in the control group and the experimental group, and it does not affect the normal life state of mice during and after the experiment. In vivo imaging of small animals can observe strong bioluminescence signals, and the bioluminescence intensity reaches the maximum 1min after the probe is injected into the tail vein, and then tends to decrease. The above results show that the probe reacts quickly with butyrylcholinesterase, and the generated bioluminescence signal is strong and relatively stable. Therefore, the probe can realize bioluminescence imaging of endogenous butyrylcholinesterase in vivo.

[0058] The above description is only a preferred embodiment of the present invention. For ordinary technicians in this field, several improvements and changes can be made without departing from the creative concept of the present invention, all of which belong to the protection scope of the present invention.

Claims

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

2. A method for preparing the compound according to claim 1, characterized in that: The method 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 ZD-1.

3. The preparation method according to claim 2, characterized in that: In step (1), the solvent is anhydrous dichloromethane; And / or, in step (1), the base is triethylamine or potassium carbonate; 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 triethylamine or potassium carbonate.

4. The method according to claim 3, characterized in that: In step (1), the solvent is anhydrous dichloromethane, the reaction temperature is 25° C. to 40° C., and the reaction time is 4 to 6 hours.

5. The preparation method according to claim 3, characterized in that: The solvent in step (2) is anhydrous dichloromethane and anhydrous methanol; the solvents for potassium carbonate and D-cysteine ​​hydrochloride monohydrate are anhydrous methanol and distilled water. The reaction temperature is 20°C to 30°C, and the reaction time is 1 to 2 hours.

6. The preparation method according to claim 3, characterized in that: In step (1), the molar ratio of compound 1, compound 2 and base is 1:1-2:1-2; And / or, in step (1), the reaction is first carried out at 0-4°C for 1-2 hours, and then at 20-40°C for 3-4 hours; And / or, in step (2), the molar ratio of compound 3 to D-cysteine ​​hydrochloride is 1:1-2; And / or, in step (2), the mass ratio of the compound 3 to the base is 1:1-2.

7. The preparation method according to claim 5, characterized in that: In step (2), 1M HCl is used to adjust the pH to 1-2.

8. The preparation method according to claim 2, characterized in that: In step (1), after the reaction, the reaction solution is purified to obtain compound 3, and the purification step comprises distilling the reaction solution under reduced pressure, extracting, drying, filtering, distilling under reduced pressure, and separating by column chromatography, wherein the eluent used in the column chromatography separation is a mixed solution of dichloromethane and petroleum ether in a volume ratio of 4:1; And / or, in step (2), after the reaction, the reaction solution is purified to obtain compound ZD-1, and the purification step comprises the steps of drying the reaction solution, adding water, adjusting the pH to 1-2 with hydrochloric acid, precipitating solids, and filtering.

9. Use of the compound according to claim 1, or a salt thereof, or a stereoisomer thereof in the preparation of a bioluminescent probe.

10. The use according to claim 9, characterized in that: The bioluminescent probe is a bioluminescent probe for detecting butyrylcholinesterase in organisms.

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