A method for preparing a probe for analyzing the regulation of bsh activity by gut microbiota

Abstract

The present application belongs to the technical field of chemical probes, and particularly relates to a preparation method of a probe for analyzing intestinal flora regulating BSH activity. The preparation method of the probe for analyzing intestinal flora regulating BSH activity provided by the present application uses 7-amino-4-methyl-3-coumarin acetic acid (AMCA) as raw material, sequentially carries out a first reaction with a chlorinating agent and an alcohol reagent, and carries out a second reaction with a CA compound in the presence of a condensing agent and a catalyst, and the required CA-AMCA probe can be obtained through a hydrolysis reaction. The preparation method of the BSH probe provided by the present application not only has easily obtained raw materials and a simple process route, but also has a total reaction yield as high as 64%, which can effectively guarantee the product yield.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chemical probes, and particularly relates to a preparation method of a probe for analyzing the regulation of intestinal microbiota on BSH activity. BACKGROUND

[0002] Human gut microbiota (HGM) is known as the "second brain" of the human body, which greatly affects the physiological immune function of the host by affecting the host's internal balance, including metabolism, neurobiology and physiological functions. Among them, bile salt hydrolase (BSH), as a core metabolic enzyme widely distributed in HGM, its activity change is closely related to the occurrence and development of many diseases such as type 2 diabetes, obesity and other diseases. It is reported that the main function of BSH is to cut the amide bond of conjugated bile acids (BAs) from glycine / taurine residues, open the BA pool for 7-dehydroxylation, and then lead to the formation of secondary BAs. Secondary BAs are considered to be a key mediator of intestinal bacterial composition and many aspects of host metabolism, such as fat digestion and utilization.

[0003] Studies have shown that base pair nuclear receptors (such as farnesoid X receptor (FXR)) and membrane receptors (including G protein-coupled receptors, TGR5) have different effects. For example, the enterohepatic circulation of secondary BAs allows different effects on nuclear receptors (such as FXR and G protein-coupled receptors). TGR5 directly affects lipid and sugar metabolism in various tissues, making the formation rate of secondary BA crucial for maintaining metabolic homeostasis. Secondary bases such as deoxycholic acid promote the development of colon cancer, and it is found that the increase in deoxycholic acid concentration in feces is in a high-risk group for colon cancer. Recently, the restoration of bile acid metabolism leads to the enrichment of secondary BA pool and is associated with the potential prolongation of mouse lifespan to prevent age-related diseases. Although bile acids play an important role in human health, due to the lack of detection tools, the potential mechanism of intestinal microbiota regulating BSH enzyme function is not clear.

[0004] In recent years, global scientific research teams are working hard to build an enzyme probe molecule engineering platform with high selectivity, in order to break through the technical barriers of traditional biochemical detection through new substrate design. In the field of bile salt hydrolase (BSH) research, the development of probes based on the structure-function relationship has made some progress, among which the BSH probe based on coumarin as the mother nucleus (CA-AMCA) has been reported in many literatures, but there is no commercial purchase way.

[0005]

[0006] At present, the synthesis route of the reported coumarin-based BSH probe CA-AMCA is as follows.

[0007] The end product was purified using a reverse-phase high-performance liquid chromatography column, with a yield of 24.3%, as reported in Brandvold KR, Weaver JM, Whidbey C, Wright AT. Sci Rep. 2019, 9(1): 1359. However, not only is the reaction yield extremely low, but the technical route is difficult to repeat (Professor Michael Gütschow reported that the target product was not obtained by the above technical route), especially the target product and the raw material have similar polarity, which is difficult to purify by ordinary silica gel column, and can only be separated and collected by liquid chromatography or ultra-high performance liquid chromatography, which is high in cost and time-consuming, and difficult to realize industrialization of kilogram accumulation.

[0008]

[0009] As reported in Keuler T, Wolf V, Lemke C, Voget R, Braune A, Gütschow M. Bioorg Chem. 2023, 138: 106574., the total yield of the end product is about 54%. However, the key reagent ethyl chloroformate is a highly toxic and carcinogenic chemical that is strictly controlled and cannot be purchased for use.

[0010]

[0011] Therefore, it is desirable to develop a simpler method for synthesizing BSH probes (CA-AMCA). SUMMARY

[0012] To solve the above technical problems, the present application provides a preparation method of a probe for analyzing the regulation of BSH activity by intestinal microbiota, which has a simpler synthesis route, readily available raw materials, controllable cost, and is suitable for industrial promotion.

[0013] The preparation method of a probe for analyzing the regulation of BSH activity by intestinal microbiota according to the present application comprises the following steps:

[0014] (1) In a first solvent, 7-amino-4-methyl-3-coumarin acetic acid (AMCA), a chlorinating agent and an alcohol reagent are used as raw materials to perform a first reaction to obtain compound 1;

[0015] ;

[0016] (2) In a second solvent, compound 1 and cholic acid (CA) are used as raw materials to perform a second reaction in the presence of a condensing agent and a first catalyst to obtain compound 2;

[0017]

[0018] ;

[0019] (3) in the third solvent, with the compound 2 as raw material, hydrolysis reaction is carried out in the presence of the second catalyst, and the required probe CA-AMCA is obtained;

[0020]

[0021] .

[0022] Specifically, in the preparation method of the probe for analyzing the regulation of intestinal flora on BSH activity, in step (1), the chlorinating agent is thionyl chloride;

[0023] The molar ratio of the chlorinating agent to the AMCA raw material is 1.2-4.0:1.

[0024] Specifically, in the preparation method of the probe for analyzing the regulation of intestinal flora on BSH activity, in step (1), the alcohol reagent is ethanol.

[0025] It should be noted that the alcohol reagent and the AMCA raw material do not have a fixed molar ratio, and a large excess is used in the reaction process, which can be used as part of the solvent.

[0026] Specifically, in the preparation method of the probe for analyzing the regulation of intestinal flora on BSH activity, in step (2), the condensing agent is 2-(7-azobenzenetriazole)-N,N,N',N'-tetramethyl urea hexafluorophosphate (HATU).

[0027] The molar ratio of the condensing agent to the CA raw material is 1.1-3:1.

[0028] Specifically, in the preparation method of the probe for analyzing the regulation of intestinal flora on BSH activity, in step (2), the first catalyst is N,N-diisopropyl ethylamine (DIPEA).

[0029] The molar ratio of the catalyst to the CA raw material is 6.0-10:1.

[0030] Specifically, in the preparation method of the probe for analyzing the regulation of intestinal flora on BSH activity, in step (3), the second catalyst is lithium hydroxide.

[0031] The molar ratio of the second catalyst to the compound 2 is 20-30:1.

[0032] Specifically, in the preparation method of the probe for analyzing the regulation of intestinal flora on BSH activity:

[0033] In the step (1), the temperature of the first reaction is 80-100℃; and / or,

[0034] In the step (2), the temperature of the second reaction is 30-60℃; and / or,

[0035] In the step (3), the temperature of the hydrolysis reaction is 25-60℃.

[0036] Specifically, in the preparation method of the probe for analyzing the BSH activity regulated by the intestinal flora, the first solvent, the second solvent and the third solvent are independently selected from N,N-dimethylformamide (DMF), tetrahydrofuran or water.

[0037] Preferably, the first solvent is N,N-dimethylformamide (DMF).

[0038] Preferably, the second solvent is N,N-dimethylformamide (DMF).

[0039] Preferably, the third solvent is tetrahydrofuran.

[0040] Specifically, in the preparation method of the probe for analyzing the BSH activity regulated by the intestinal flora, the steps (1), (2) or (3) independently comprise a step of using a silica gel column for product separation and purification.

[0041] Specifically, in the preparation method of the probe for analyzing the BSH activity regulated by the intestinal flora, the silica gel column comprises normal phase silica gel elution column chromatography or reverse phase C-18 column chromatography.

[0042] The preparation method of the probe for analyzing the BSH activity regulated by the intestinal flora provided by the application uses 7-amino-4-methyl-3-coumarin acetic acid (AMCA) as a raw material, performs a first reaction with a chlorinating agent and an alcohol reagent, and performs a second reaction with cholic acid (CA) in the presence of a condensing agent and a catalyst, and then performs a hydrolysis reaction to obtain the required CA-AMCA probe. The preparation method of the BSH probe provided by the application not only has easy-to-obtain raw materials and a simple process route, but also has a total reaction yield as high as 64%, which can effectively guarantee the product yield and realize industrial transformation of kilogram-level accumulation.

[0043] The preparation method of the probe for analyzing the BSH activity regulated by the intestinal flora provided by the application can use a silica gel column for separation throughout the reaction, has a high product yield and simple separation, and is suitable for industrial promotion. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to make the technical solutions in the embodiment or prior art of the present application more clear, the drawings needed in the description of the embodiment or prior art will be briefly introduced as follows. Obviously, the drawings described below are some embodiments of the present application, and all other embodiments obtained by those skilled in the art without creative work based on the embodiments in the present application also belong to the present application.

[0045] Figure 1 is the H NMR spectrum of compound 1 1 H NMR spectrum;

[0046] Figure 2 is the H NMR spectrum of compound 1 13 C NMR spectrum;

[0047] Figure 3 is the H NMR spectrum of compound 2 1 H NMR spectrum;

[0048] Figure 4 is the H NMR spectrum of compound 2 13 C NMR spectrum;

[0049] Figure 5 is the H NMR spectrum of the BSH probe CA-AMCA 1 H NMR spectrum;

[0050] Figure 6 is the chromatographic peak and retention time of CA-AMCA metabolized by biliary salt hydrolase

[0051] Figure 7 is the pre-experiment result of incubation time of CA-AMCA metabolized by biliary salt hydrolase EMBODIMENT

[0052] In order to make the technical solutions in the embodiment or prior art of the present application more clear, the drawings needed in the description of the embodiment or prior art will be briefly introduced as follows. Obviously, the drawings described below are some embodiments of the present application, and all other embodiments obtained by those skilled in the art without creative work based on the embodiments in the present application also belong to the present application.

[0053] The present application provides a preparation method of a probe for analyzing the activity of BSH regulated by intestinal flora in the following examples, which comprises the following steps:

[0054] (1) in a first solvent, 7-amino-4-methyl-3-coumarin acetic acid (AMCA), a chlorinating agent and an alcohol reagent are used as raw materials to perform a first reaction, and compound 1 is obtained;

[0055] (2) in the second solvent, with the compound 1 and cholic acid (CA) as raw materials, in the presence of condensing agent and first catalyst, the second reaction is carried out, and compound 2 is obtained;

[0056] (3) in the third solvent, with the compound 2 as raw material, in the presence of second catalyst, hydrolysis reaction is carried out, and the required probe CA-AMCA is obtained.

[0057] The preparation method of the probe for analyzing the regulation of intestinal flora on BSH activity provided by the application uses 7-amino-4-methyl-3-coumarin acetic acid (AMCA) as raw material, carries out the first reaction with chlorinating agent and alcohol reagent, carries out the second reaction with CA compound in the presence of condensing agent and catalyst, and then carries out hydrolysis reaction to obtain the required CA-AMCA probe.

[0058] In some specific embodiments, in the step (1), the chlorinating agent is thionyl chloride.

[0059] The molar ratio of the chlorinating agent to the AMCA raw material is 1.2-4.0:1.

[0060] In some specific embodiments, in the step (1), the alcohol reagent is ethanol; the alcohol reagent has no fixed molar ratio with the AMCA raw material, and a large excess is used in the reaction process, which can be used as part of the solvent.

[0061] In some specific embodiments, in the step (1), the temperature of the first reaction is 80-100 DEG C.

[0062] In some specific embodiments, in the step (1), the first solvent is N, N-dimethylformamide (DMF).

[0063] In some specific embodiments, in the step (2), the condensing agent is 2-(7-azobenzenetriazole)-N, N, N', N'-tetramethylurea hexafluorophosphate (HATU).

[0064] The molar ratio of the condensing agent to the CA raw material is 1.1-3:1.

[0065] In some specific embodiments, in the step (2), the first catalyst is N, N-diisopropyl ethylamine (DIPEA).

[0066] The molar ratio of the catalyst to the CA raw material is 6.0-10:1.

[0067] In some specific embodiments, in the step (1), the temperature of the second reaction is 30-60 DEG C.

[0068] In some embodiments, the second solvent in step (2) is N,N-dimethylformamide (DMF).

[0069] In some embodiments, the second catalyst in step (3) is lithium hydroxide.

[0070] The molar ratio of the second catalyst to the compound 2 is 20-30:1.

[0071] In some embodiments, the temperature of the hydrolysis reaction in step (3) is 25-60°C.

[0072] In some embodiments, the third solvent in step (3) is tetrahydrofuran.

[0073] In some embodiments, steps (1), (2) or (3) independently comprise a step of using a silica gel column for product separation and purification.

[0074] In some embodiments, the silica gel column comprises normal phase silica gel elution column chromatography, reverse phase C-18 column chromatography, etc.

[0075] Example 1

[0076] The preparation method of the BSH activity probe in this example comprises the following steps.

[0077]

[0078] Take 7-amino-4-methyl-3-coumarin acetic acid (AMCA) (100 mg, 0.43 mmol) and dissolve it in anhydrous DMF, stir until completely dissolved, and mix with anhydrous ethanol (1 mL, 17.2 mmol) under argon protection. Then place the reaction system in an ice water bath, slowly add thionyl chloride (0.124 mL, 1.72 mmol) dropwise at 0°C, remove the ice water bath after the dropwise addition is complete, and heat the mixture to reflux at 80°C for 6 hours. After the reaction is completed, add a small amount of sodium carbonate solid to neutralize the acidic reaction system, filter out the insoluble impurities, and evaporate the solvent under reduced pressure to obtain the crude product. The crude product is separated by normal phase silica gel elution column chromatography, the eluent is methanol-dichloromethane (2 / 100, v / v), and the target component is collected to obtain compound 1. The 1 H NMR spectrum and 13 C NMR spectrum are shown in Figures Figures 1-2 It can be seen that the structure of compound 1 prepared in this step is correct, and the calculated product yield is about 90%.

[0079] 1H NMR (400 MHz, DMSO) δ 7.47 (d, J = 8.7 Hz, 1H), 6.58 (dd, J = 8.7, 2.1Hz, 1H), 6.41 (d, J = 2.1 Hz, 1H), 6.07 (s, 2H), 4.07 (q, J = 7.1 Hz, 2H), 3.57(s, 2H), 2.28 (s, 3H), 1.18 (t, J = 7.1 Hz, 3H)。

[0080] 13 C NMR (101 MHz, DMSO) δ 170.47, 161.33, 154.10, 152.58, 151.97,149.94, 127.44, 126.43, 112.21, 111.42, 109.02, 98.38, 60.27, 32.38, 14.79,14.06。

[0081] Cholic acid (106 mg, 0.26 mmol) and HATU (107 mg, 0.28 mmol) were weighed and dissolved in an appropriate amount of anhydrous DMF, and N,N- diisopropylethylamine (0.44 mL, 2.5 mmol) was added to the solution, which was then reacted at room temperature for 15 min. Then, compound 1 (74 mg, 0.28 mmol) was added, and the mixture was reacted at 50°C for 7 h in the dark. After the reaction, the organic solvent was evaporated under reduced pressure to obtain a crude product, which was separated by column chromatography using normal-phase silica gel and eluted with methanol-dichloromethane (5 / 100, v / v) to obtain compound 2. The 1 H NMR spectrum and 13 C NMR spectrum are shown in FIGS. 1 and 2, respectively. As can be seen, the structure of compound 2 prepared in this step is correct, and the calculated yield of the product is about 75%. Figures 3-4

[0082] 1 H NMR (400 MHz, DMSO) δ 10.31(s, 1H), 8.67 (s, 1H), 8.46 (d, J = 8.3Hz, 1H), 7.77 (d, J ​= 8.6 Hz, 2H), 7.56 – 7.32 (m, 2H), 4.29 (s, 2H), 3.99 (s,2H), 3.79 (s, 2H), 3.59 (d, J = 16.4 Hz, 4H), 2.38 (s, 4H), 1.86 – 1.57 (m,12H), 1.40 – 1.18 (m, 14H), 0.96 (dd, J =16.1, 6.1 Hz, 4H), 0.59 (d, J = 4.4 Hz,6H)。

[0083] 13 C NMR: 13 C NMR (101 MHz, DMSO) δ 172.54, 170.02, 160.74, 152.36,149.14, 142.30, 126.09, 117.00, 115.23, 114.89, 105.21, 71.01, 70.43, 66.24,60.42, 45.75, 41.37, 35.11, 34.38, 33.58, 32.62, 31.21, 30.40, 28.56, 27.25,26.21, 22.79, 22.60, 17.15, 14.93, 14.03, 12.32.

[0084] The compound 2 (100 mg, 0.15 mmol) was weighed and dissolved in a suitable amount of anhydrous tetrahydrofuran, and lithium hydroxide hydrate (157 mg, 3.75 mmol) was dissolved in a suitable amount of a mixture of tetrahydrofuran and pure water (1 / 1, v / v). The lithium hydroxide solution was mixed with the compound 2 solution, heated to 40°C, and incubated for 5 hours. After the reaction was completed, the pH of the reaction solution was adjusted to 3-4 using dilute hydrochloric acid, and then the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was separated and purified by reverse phase C-18 column chromatography, using water and 60% methanol in water as eluents, to obtain the desired probe CA-AMCA. The 1 The H NMR spectrum is shown in FIG. 1. As can be seen, the structure of the CA-AMCA prepared in this step is correct, and the calculated yield of the product is about 95%. Figure 5

[0085] 1 ​H NMR (400 MHz, DMSO) δ 10.33 (s, 1H), 8.28 (s, 1H), 7.96 - 7.67 (m, 2H), 7.48 (d, J = 7.5 Hz, 1H), 3.80 (s, 1H), 3.58 (d, J = 30.6 Hz, 4H), 3.17 (s, 2H), 2.35 (s, 4H), 2.06 - 1.54 (m, 12H), 1.35 (m, 14H), 0.98 (d, J = 5.2 Hz, 3H), 0.83 (d, J = 14.7 Hz, 3H), 0.60 (s, 3H).

[0086] In summary, the synthesis yield of the CA-AMCA according to the present application is 90% x 75% x 95% = 64.125%, and the product synthesis yield is relatively high.

[0087] Example 2

[0088] The preparation principle and synthesis path of the BSH active probe according to the present embodiment are the same as those of Example 1.

[0089] 7-amino-4-methyl-3-coumarin acetic acid (AMCA) (100 mg, 0.43 mmol) was weighed into anhydrous DMF and stirred until completely dissolved. Anhydrous ethanol (0.5 mL, 8.6 mmol) was added under argon protection, and then the reaction system was placed in an ice water bath. Sulfurous acid chloride (0.124 mL, 1.72 mmol) was slowly added dropwise at 0°C. After the dropwise addition was completed, the ice water bath was removed, and the mixture was heated to reflux at 90°C for 6 hours. After the reaction was completed, a small amount of sodium carbonate solid was added to neutralize the reaction system. The insoluble impurities were removed by filtration, and the solvent was evaporated under reduced pressure to obtain the crude product. The crude product was separated by column chromatography using normal phase silica gel, with methanol-dichloromethane (2 / 100, v / v) as the eluent. The target component was collected to obtain compound 1. The structure of compound 1 prepared in this step was correct, and the calculated product yield was about 90%.

[0090] Take cholic acid (106 mg, 0.26 mmol) and HATU (107 mg, 0.52 mmol), dissolve in an appropriate amount of anhydrous DMF, dissolve in an appropriate amount of anhydrous DMF, and dissolve in an appropriate amount of anhydrous DMF. N,N-diisopropylethylamine (0.44 mL, 2.5 mmol) was mixed and dissolved, and the reaction was carried out at room temperature for 15 minutes. Then add compound 1 (74 mg, 0.28 mmol), and incubate at 40°C for 7 hours in the dark. After the reaction is completed, the organic solvent is evaporated under reduced pressure to obtain the crude product. The crude product is separated by column chromatography using normal phase silica gel, and the eluent is methanol-dichloromethane (5 / 100, v / v). The target component is collected to obtain compound 2. The structure of compound 2 prepared in this step is correct, and the calculated product yield is about 75%.

[0091] Take the compound 2 (100 mg, 0.15 mmol) and stir to dissolve in an appropriate amount of anhydrous tetrahydrofuran. Take lithium hydroxide hydrate (157 mg, 3.75 mmol) and dissolve in an appropriate amount of a mixture of tetrahydrofuran and pure water (1 / 1, v / v). Mix the lithium hydroxide solution with the compound 2 solution, heat to 60°C, and incubate for 3 hours. After the reaction is completed, adjust the pH of the reaction solution to 3-4 using dilute hydrochloric acid, and then evaporate the solvent under reduced pressure to obtain the crude product. The crude product is separated and purified by reverse phase C-18 column chromatography, and the eluent is pure water and methanol (gradient elution, methanol volume ratio from 5%-100%). The desired probe CA-AMCA is obtained. The structure of CA-AMCA prepared in this step is correct, and the calculated product yield is about 95%.

[0092] Example 3

[0093] The preparation principle and synthesis path of the BSH activity probe described in this example are the same as those of Example 1.

[0094] Take 7-amino-4-methyl-3-coumarin acetic acid (AMCA) (100 mg, 0.43 mmol) and dissolve in anhydrous DMF. Stir until completely dissolved, and then add anhydrous ethanol (0.2 mL, 3.44 mmol) under argon protection. Then place the reaction system in an ice water bath, and slowly add thionyl chloride (62 µL, 0.86 mmol) dropwise at 0°C. After the addition is complete, remove the ice water bath, and heat the mixture to reflux at 80°C for 6 hours. After the reaction is completed, add a small amount of sodium carbonate solid to neutralize the reaction system, filter out the insoluble impurities, and evaporate the solvent under reduced pressure to obtain the crude product. The crude product is separated by column chromatography using normal phase silica gel, and the eluent is methanol-dichloromethane (2 / 100, v / v). The target component is collected to obtain compound 1. The structure of compound 1 prepared in this step is correct, and the calculated product yield is about 90%.

[0095] Compound 2 was prepared by the following method. Compound 2 (100 mg, 0.15 mmol) was dissolved in tetrahydrofuran (THF) and stirred at room temperature. Lithium hydroxide (63 mg, 1.5 mmol) was dissolved in a mixture of THF and water (1 / 1, v / v) and added to the solution of Compound 2. The mixture was heated to 50 °C and stirred for 8 h. After the reaction was completed, the pH of the reaction solution was adjusted to 3-4 using dilute hydrochloric acid. The solvent was then removed under reduced pressure to obtain a crude product. The crude product was separated and purified by reverse phase C-18 column chromatography using water and methanol (gradient elution, methanol volume ratio from 5% to 100%) as the eluent to obtain the desired probe CA-AMCA. The structure of CA-AMCA prepared in this step was correct, and the calculated product yield was about 50%.

[0096] Compound 2 (100 mg, 0.15 mmol) was dissolved in tetrahydrofuran (THF) and stirred at room temperature. Lithium hydroxide (63 mg, 1.5 mmol) was dissolved in a mixture of THF and water (1 / 1, v / v) and added to the solution of Compound 2. The mixture was heated to 50 °C and stirred for 8 h. After the reaction was completed, the pH of the reaction solution was adjusted to 3-4 using dilute hydrochloric acid. The solvent was then removed under reduced pressure to obtain a crude product. The crude product was separated and purified by reverse phase C-18 column chromatography using water and methanol (gradient elution, methanol volume ratio from 5% to 100%) as the eluent to obtain the desired probe CA-AMCA. The structure of CA-AMCA prepared in this step was correct, and the calculated product yield was about 50%.

[0097] Experimental Example

[0098] 1. Metabolic verification

[0099] This experimental example is based on liquid chromatography to verify the metabolic effect of bile salt hydrolase on CA-AMCA.

[0100] This example is based on the fluorescent probe CA-AMCA synthesized in Example 1. Based on an in vitro incubation system, bile salt hydrolase catalyzes the hydrolysis of CA-AMCA to generate the fluorescent product AMCA. To accurately detect the content of CA-AMCA and its hydrolysis product AMCA, ultra-high performance liquid chromatography (UHPLC) combined with a fluorescence detector was used for analysis.

[0101] By optimizing the detection conditions, the characteristic fluorescence spectrum parameters of the target compound were determined as follows: excitation wavelength (Ex) 350 nm, emission wavelength (Em) 450 nm. Under these conditions, the chromatographic peaks of CA-AMCA and AMCA were well separated, the baseline was stable, and no obvious interference peaks were present.

[0102] As Figure 6As shown in the results, two completely separated peaks are presented in the chromatogram, in which the retention time of the substrate CA-AMCA is around 10.594 minutes, and the retention time of the hydrolysis product AMCA is around 7.105 minutes.

[0103] 2. Results of in vitro incubation pre-experiment of bile salt hydrolase

[0104] The present experimental example is based on the fluorescent probe CA-AMCA synthesized in Example 1 for in vitro incubation pre-experiment of bile salt hydrolase, and the results are shown in the following table. Figure 7

[0105] According to the analysis of the pre-experiment results, the catalytic reaction of the bile salt hydrolase shows a significant linear relationship in the time range of 10 to 50 minutes, that is, the product generation amount is positively correlated with the reaction time (see the following table). Figure 7

[0106] In order to ensure the accuracy and repeatability of the experimental data, the present experimental example selects the subsequent experiments in the linear reaction stage. Based on the linear kinetic characteristics, the incubation time is finally determined to be 30 minutes. The selection of this time point can fully reflect the catalytic activity of the enzyme, and has high controllability and stability in the subsequent related experimental operations.

[0107] In summary, the preparation method of the probe for analyzing the regulation of intestinal microbiota on BSH activity according to the present application uses 7-amino-4-methyl-3-coumarin acetic acid (AMCA) as raw material, performs a first reaction with chlorinating agent and alcohol reagent, and performs a second reaction with CA compound in the presence of condensing agent and catalyst, and then obtains the required CA-AMCA probe through hydrolysis reaction. The preparation method of the BSH probe according to the present application not only has readily available raw materials and simple process route, but also has a total reaction yield as high as 64%, which can effectively guarantee the product yield and realize industrial transformation of kilogram accumulation.

[0108] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present application, and not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present application.​​

Claims

1. A method for preparing a probe for analyzing the regulation of BSH activity by gut microbiota, characterized in that, Includes the following steps: (1) In the first solvent, 7-amino-4-methyl-3-coumarin acetate AMCA, chlorinating agent and alcohol reagent were used as raw materials to carry out the first reaction to obtain compound 1; ； (2) In the second solvent, using compound 1 and cholic acid CA as raw materials, a second reaction is carried out in the presence of a condensing agent and a first catalyst to obtain compound 2; ； (3) In a third solvent, the compound 2 is used as a raw material and hydrolyzed in the presence of a second catalyst to obtain the desired probe CA-AMCA; ; In step (1), the chlorinating agent is thionyl chloride; the molar ratio of the chlorinating agent to the AMCA raw material is 1.2-4.0:1; In step (2), the condensing agent is 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate; The molar ratio of the condensing agent to the CA raw material is 1.1-3:1; In step (2), the first catalyst is N,N-diisopropylethylamine; The molar ratio of the first catalyst to the CA raw material is 6.0-10:

1.

2. The method for preparing the probe for analyzing the regulation of BSH activity by gut microbiota according to claim 1, characterized in that, In step (1), the alcohol reagent is ethanol.

3. The method for preparing the probe for analyzing the regulation of BSH activity by gut microbiota according to claim 1, characterized in that, In step (3), the second catalyst is lithium hydroxide; The molar ratio of the second catalyst to compound 2 is 20-30:

1.

4. The method for preparing the probe for analyzing the regulation of BSH activity by gut microbiota according to claim 1, characterized in that: In step (1), the temperature of the first reaction is 80-100℃; and / or, In step (2), the temperature of the second reaction is 30-60℃; and / or, In step (3), the temperature of the hydrolysis reaction is 25-60℃.

5. The method for preparing the probe for analyzing the regulation of BSH activity by gut microbiota according to claim 1, characterized in that, The first solvent, the second solvent, and the third solvent are independently selected from N,N-dimethylformamide, tetrahydrofuran, or water.

6. The method for preparing a probe for analyzing the regulation of BSH activity by gut microbiota according to any one of claims 1-5, characterized in that, Each of the steps (1), (2), or (3) includes, independently of each other, the step of separating and purifying the product using a silica gel column.

7. The method for preparing the probe for analyzing the regulation of BSH activity by gut microbiota according to claim 6, characterized in that, The silica gel column includes normal-phase silica gel elution column chromatography or reverse-phase C-18 column chromatography.

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