A fluorescent probe for detecting glycine cholate hydrolase, and a preparation method and application thereof

Abstract

The application relates to a fluorescent probe for detecting glycine cholic acid hydrolase, a preparation method and application thereof, and belongs to the technical field of biological medicines. The structural formula of the fluorescent probe is shown in formula (1). The preparation method comprises the following steps: taking anhydrous cholic acid and aminobenzyl alcohol as raw materials, preparing an intermediate 1 through an acylation reaction, preparing an intermediate 2 through oxidation, and then preparing the fluorescent probe for detecting glycine cholic acid hydrolase through a Brain Coupling reaction of the intermediate 2 and 6-(2,3,3-trimethylindole-1-oxonium-1-yl) hexanoic acid bromide. The application further discloses application of the fluorescent probe in detection of glycine cholic acid hydrolase activity. The fluorescent probe provided by the application can be selectively hydrolyzed by glycine cholic acid hydrolase, has high specificity, can be detected by a fluorescent detector, and has high sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a fluorescent probe for detecting glycocholic acid hydrolase, its preparation method and application. Background Technology

[0002] Glycylcholine hydrolase (GCH), also known as choylglycine hydrolase, is a type of bile salt hydrolase (BSH). It is primarily expressed in intestinal microorganisms and catalyzes the hydrolysis of conjugated bile acids in the intestine into free bile acids, participating in bile acid metabolism. In recent years, with the continuous improvement of people's living standards, high-fat and high-sugar diets have led to a significant increase in patients with abnormal glucose and lipid metabolism. Abnormal glucose and lipid metabolism often causes metabolic-related diseases such as obesity, hyperglycemia, hyperlipidemia, and hypercholesterolemia.

[0003] Studies have shown that inhibiting BSH activity in gut microbiota reduces its hydrolytic capacity, leading to an increase in conjugated bile acids. BSH is a natural antagonist of Fxr (ferrous oxalate), and can improve hyperglycemia and non-alcoholic fatty liver disease by inhibiting the gut Fxr-ceramide axis. Therefore, the development of GCH inhibitors is of great significance for the treatment of metabolic diseases.

[0004] Therefore, how to provide a specific fluorescent probe substrate with high sensitivity and strong anti-interference ability suitable for in vivo and in vitro detection of glycocholic acid hydrolase activity, and provide an efficient method for the study of BSH activity regulators in intestinal bacteria, is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a highly specific, inexpensive, readily available, and highly sensitive fluorescent probe for detecting glycocholic acid hydrolase, along with its preparation method and applications.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A fluorescent probe for detecting glycocholic acid hydrolase, the structure of which is shown in formula (1):

[0008] (1).

[0009] A method for preparing a fluorescent probe for detecting glycocholic acid hydrolase includes the following steps:

[0010] Anhydrous cholic acid and aminobenzyl alcohol were used as raw materials to prepare intermediate 1 through acylation reaction, and then intermediate 2 was obtained by oxidation. Then, intermediate 2 was obtained by reacting intermediate 2 with 6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide through the Brainwell reaction to obtain the fluorescent probe for detecting glycocholic acid hydrolase.

[0011] Beneficial Effects: The probe molecule structure provided by this invention mainly comprises two groups. The cholic acid group is the recognition group for glycocholic acid hydrolase, facilitating the recognition of the probe molecule and its binding to the target hydrolase. 6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide is used as the fluorescent group; its free molecules after hydrolysis can produce a significant fluorescent signal. Secondly, the hexanoic acid fragment in the 6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide group, as an acidic group, facilitates the metabolic recognition by glycocholic acid hydrolase. Furthermore, the cholic acid group and the fluorescent group are linked by an amide bond of aminobenzyl alcohol. This amide bond is the metabolic hydrolysis site of glycocholic acid hydrolase, facilitating its metabolic hydrolysis and the release of fluorescent product molecules.

[0012] Preferably, DMF is used as a solvent and HATU and DIPEA are used as catalysts in the acylation reaction.

[0013] More preferably, the acylation reaction includes the following steps:

[0014] Anhydrous cholic acid, HATU, and DIPEA were dissolved in DMF and stirred at room temperature for 30 minutes. Then, aminobenzyl alcohol was added, and the mixture was stirred and reacted before extraction, drying, and evaporation under reduced pressure to obtain intermediate 1.

[0015] The ratio of the added anhydrous choline, HATU, DIPEA, DMF and aminobenzyl alcohol is 1 mmol: 1 mmol: 1 mmol: 2 ml: 1 mmol.

[0016] The extraction is performed using water and dichloromethane as the extraction solution, wherein the volume ratio of DMF, water and dichloromethane is 2:20:10.

[0017] The stirring reaction was carried out at room temperature for 8-12 hours.

[0018] The desiccant used in the drying process is anhydrous sodium sulfate.

[0019] Beneficial effects: This invention uses bile acid molecules as starting material, effectively reducing process costs, and the reaction process is mostly carried out at room temperature and atmospheric pressure, making the process operation relatively convenient. Furthermore, the yield of intermediate 1 in this invention is high, reaching 91.8%.

[0020] Preferably, the oxidation includes the following steps: mixing intermediate 1 with dichloromethane and manganese dioxide, stirring and reacting at room temperature, filtering, and evaporating under reduced pressure to obtain intermediate 2.

[0021] More preferably, the ratio of intermediate 1, dichloromethane and manganese dioxide added is 1 mmol: 30 mL: 10 mmol.

[0022] The stirring reaction time is 2 hours.

[0023] Beneficial effects: Intermediate 2 requires no further purification and has a high yield of 82.4%.

[0024] Preferably, the brain Wenger reaction includes the following steps: mixing intermediate 2, 6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide and acetonitrile, adding piperidine under a protective atmosphere, stirring the reaction, evaporating the solvent and purifying to obtain the fluorescent probe for detecting glycocholic acid hydrolase.

[0025] More preferably, the ratio of the amount of intermediate 2, 6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide, acetonitrile and piperidine added is 0.1 mmol: 0.2 mmol: 20 mL: 0.5 mL.

[0026] The stirring reaction temperature was 90℃, and the reaction time was 2 hours.

[0027] The purification was carried out by silica gel column chromatography, and the eluent used in the purification process was a mixture of dichloromethane and methanol at a volume ratio of 10:1.

[0028] Beneficial effects: In the final stage of the synthesis process of this invention, 2,6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide is used as the later-stage raw material, which reduces the loss of this raw material and lowers the process cost.

[0029] Application of a fluorescent probe for detecting glycocholic acid hydrolase in the detection of glycocholic acid hydrolase activity.

[0030] Beneficial effects: The hydrolysis product of the specific fluorescent probe in this invention is a long-emission wavelength fluorescent probe, which is not easily affected by the biological matrix and impurities in the detection process of glycocholic acid hydrolase activity. It can be used for the quantitative determination of endogenous glycocholic acid hydrolase activity in complex biological samples.

[0031] A method for detecting glycocholic acid hydrolase includes the following steps: using the above-mentioned fluorescent probe as a specific substrate for glycocholic acid hydrolase to carry out a hydrolysis reaction, and then using fluorescence detection to quantitatively detect the change pattern of fluorescence signal per unit time to determine the formation rate of 2-[(1E)-2-(4-aminophenyl)vinyl]-1-(5-carboxypentyl)-3,3-dimethyl-3H-indole, thereby quantitatively determining the activity of glycocholic acid hydrolase.

[0032] Beneficial effects: The above detection process relies on the detection of fluorescence signals (λ562 nm) and is applicable to various types of fluorescence detection instruments, especially fluorescence microplate readers.

[0033] Preferably, the reaction system of glycocholic acid hydrolase is pre-incubated before the hydrolysis reaction;

[0034] The reaction system includes a buffer solution and glycocholic acid hydrolase, and the concentration of glycocholic acid hydrolase is 0-40 U / mL;

[0035] The pre-incubation temperature was 37°C, the pre-incubation time was 3 minutes, and the pre-incubation pH was 5-6.

[0036] Beneficial effects: The present invention enables the detection of enzyme activity in a near-neutral system, which is beneficial for the application of this probe in the visual detection of enzyme activity in biological systems.

[0037] Preferably, in the hydrolysis reaction, the concentration of the fluorescent probe in the reaction system is 10 μmol / L; the hydrolysis reaction temperature is 37℃ and the time is 30 min.

[0038] Beneficial effects: This invention requires only 30 minutes of catalytic reaction time, demonstrating the rapid reaction rate of the probe.

[0039] Preferably, the excitation wavelength in the fluorescence detection is 500 nm, and the maximum emission wavelength is 562 nm.

[0040] Beneficial effects: The fluorescent probe and its hydrolysis products provided by this invention have different optical properties, and the products can be detected rapidly and sensitively using a fluorescent detector.

[0041] Preferably, the enzyme activity detection range is 0-40 U / mL.

[0042] Beneficial effects: In the reaction system of the fluorescent probe and glycocholate hydrolase provided by this invention, the fluorescence intensity and enzyme activity showed a good linear relationship at 0, 2.5, 5, 10, 15, 20, 25, 30, 35, 37.5, and 40 U / mL. 2 The value is 0.9813, indicating that the probe GCA-DP can be used to determine the activity of glycocholic acid hydrolase.

[0043] This invention discloses a fluorescent probe for detecting glycocholic acid hydrolase, its preparation method, and its application. The specific fluorescent probe substrate provided by this invention can be selectively hydrolyzed by glycocholic acid hydrolase to generate hydrolysis products with significantly altered fluorescence properties, exhibiting high specificity and detectable by a fluorescence detector. The activity of glycocholic acid hydrolase can be quantitatively determined based on fluorescence intensity. Furthermore, the fluorescent probe GCA-DP provided by this invention has a fluorescence emission wavelength greater than 500 nm, which can effectively reduce interference from biological background fluorescence and provides high sensitivity. In addition, the GCA-DP provided by this invention can be obtained through simple chemical synthesis, the synthesis process is simple and easy to implement, and the fluorescence detection method is low-cost. Attached Figure Description

[0044] The accompanying drawings, which form part of this application, are used to provide a further understanding of this application. The illustrative embodiments and descriptions of this application are used to explain this application and do not constitute an undue limitation of this application. In the drawings:

[0045] Figure 1 The hydrogen nuclear magnetic resonance spectrum of GCA-DP in Example 1 ( 1 H-NMR);

[0046] Figure 2 The carbon NMR spectrum of GCA-DP in Example 1 ( 13 C-NMR);

[0047] Figure 3 The high-resolution mass spectrometry of GCA-DP in Example 1;

[0048] Figure 4 The UV absorption spectrum and fluorescence emission spectrum of GCA-DP and its hydrolysis products obtained in Example 1 are shown.

[0049] Among them, (a) is the ultraviolet absorption spectrum of GCA-DP and its hydrolysis products, and (b) is the fluorescence emission spectrum of GCA-DP and its hydrolysis products.

[0050] Figure 5 The results of the screening experiment for GCA-DP using different hydrolases in Example 1 are shown.

[0051] Figure 6 The results of the screening experiment for GCA-DP using different ions and amino acids in Example 2 are shown.

[0052] Figure 7 The linear change of enzyme in the reaction of GCA-DP probe catalyzed by glycocholate hydrolase in Example 3 is shown.

[0053] Among them, (a) is the fluorescence spectrum under the catalysis of different enzyme activities, and (b) is the correlation analysis between enzyme activity and fluorescence intensity. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0055] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0056] All raw materials used in the embodiments of this invention were purchased through commercial channels.

[0057] Example 1

[0058] A fluorescent probe for detecting glycocholate hydrolase has the structural formula shown in formula (1):

[0059] (1).

[0060] The fluorescent probe is named 1-(5-carboxypentyl)-2-[(1E)-2-(4-{[(4R)-1-oxo-1-yl-4-[(1R,3bR,7R,11S)-4,7,11-trihydroxy-9a,11a-dimethylhexadecyl-1H-cyclopenta[1,2-a]phenanthrene-1-yl]pentyl]amino}phenyl)vinyl]-3,3-dimethyl-3H-indole (GCA-DP).

[0061] A method for preparing a fluorescent probe for detecting glycocholic acid hydrolase, the synthetic route of which is as follows:

[0062] .

[0063] The preparation method specifically includes the following steps:

[0064] (1) Synthesis of intermediate 1: Anhydrous cholic acid (4080 mg, 10 mmol), HATU (3800 mg, 10 mmol) and DIPEA (1290 mg, 10 mmol) were added to a flask, followed by 20 mL of DMF. After stirring at room temperature for 30 minutes, p-aminobenzyl alcohol (1231 mg, 10 mmol) was added, and the mixture was stirred at room temperature for 8-12 hours. The next day, the reaction mixture was added to 200 mL of water and extracted three times with 100 mL of dichloromethane (DCM). The organic phase was dried with anhydrous sodium sulfate (Na2SO4) and then evaporated under reduced pressure to obtain intermediate 1 (white powder product) 4720 mg, with a yield of 91.8%.

[0065] (2) Synthesis of intermediate 2: Intermediate 1 (518 mg, 1 mmol) was added to a flask, followed by 30 mL of dichloromethane (DCM) and manganese dioxide (870 mg, 10 mmol). The mixture was stirred at room temperature for 2 h. The manganese dioxide was removed by filtration, and the product was evaporated under reduced pressure to obtain 421 mg of intermediate 2 (white solid product), with a yield of 82.4%.

[0066] (3) Synthesis of GCA-DP: Intermediate 2 (51 mg, 0.1 mmol) and 6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide (71 mg, 0.2 mmol) were added to a flask, followed by the addition of 20 mL of acetonitrile. Under nitrogen protection, 0.5 mL of piperidine was added to the reaction mixture, and the mixture was heated to 90 °C and stirred for 2 hours. The solvent was evaporated to dryness, and the product was purified by silica gel column chromatography (eluent: dichloromethane:methanol = 10:1, v:v) to obtain 17 mg of GCA-DP (yellow solid product), with a yield of 20.4%.

[0067] The obtained structural identification spectral data of GCA-DP are as follows:

[0068] Nuclear magnetic resonance hydrogen spectrum (e.g.) Figure 1 ): 1 H-NMR (600 MHz, CD3OD) δ 8.43 (d, J = 16.2 Hz,1H), 8.05 (d, J = 9.0 Hz, 1H), 7.84 (d, J = 9.0 Hz, 1H), 7.81 (m, 1H), 7.77(m, 1H), 7.64 (m, 2H), 7.55 (d, J = 16.2 Hz, 1H), 4.66 (d, J = 7.8 Hz, 1H), 2.51 (m, 1H), 2.38 (m, 1H), 2.32 (m, 3H), 2.23 (m, 2H), 1.97 (m, 9H), 1.86(s, 6H), 2.38(m, 1H), 1.73 (m, 7H), 1.57 (m, 8H), 1.47 (m, 4H), 1.33 (m,4H), 1.07 (d, J = 6.0 Hz, 3H), 0.99 (m, 2H), 0.92 (s, 3H), 0.89 (m, 1H), 0.73 (s, 3H).

[0069] Carbon nuclear magnetic resonance spectroscopy (such as) Figure 2 ): 13C-NMR (150 MHz, CD3OD) δ 182.18, 175.76, 174.33,154.64, 144.54, 143.67, 140.81, 131.72, 129.45, 129.21, 122.72, 119.57,114.58, 109.91, 72.67, 71.46, 67.65, 52.44, 46.60, 46.29, 46.10, 41.78,41.65, 39.60, 39.07, 35.54, 35.07, 34.50, 33.73, 33.00, 31.50, 29.78, 28.22, 27.97, 27.32, 26.50, 25.68, 25.33, 24.09, 22.84, 21.75, 16.38, 11.58.

[0070] High-resolution mass spectrometry (e.g.) Figure 3 ):(+) HR-ESI-MS theoretical value [M] + m / z 767.4994, measured value m / z 767.4988.

[0071] Application Example 1

[0072] In vitro determination of the selectivity of different hydrolases:

[0073] (1) Dissolve the GCA-DP obtained in Example 1 in DMSO to prepare a GCA-DP solution with a concentration of 1 mM;

[0074] (2) Prepare 200 µL of in vitro metabolic reaction system containing 8 different types of hydrolytic enzymes. The reaction system consists of a buffer buffer (KH2PO4, K2HPO4, final concentration 100 mM) with pH 5-6 and hydrolytic enzyme (final concentration 30 U / mL). The system is pre-incubated at 37°C with shaking for 3 minutes to obtain the pre-incubated in vitro metabolic reaction system.

[0075] Among them, the eight different types of hydrolases are carbon glycosyltransferase, fatty acid amidotransferase, lipase, bovine serum albumin, leucine aminopeptidase, penicillin G acylase, human serum albumin, and glycocholic acid hydrolase.

[0076] (3) Add 2µL of 1 mM (final concentration 10 μM) GCA-DP solution to each of the eight pre-incubated in vitro metabolic reaction systems in step (2), react at 37 °C for 30 min, then add 100µL of ice-cold acetonitrile, shake vigorously, and terminate the reaction.

[0077] (4) Centrifuge at 4℃ and 20000×g for 20 min using a high-speed refrigerated centrifuge. Take the supernatant and determine the generation rate of 2-[(1E)-2-(4-aminophenyl)vinyl]-1-(5-carboxypentyl)-3,3-dimethyl-3H-indole-1-cation by fluorescence detection (fluorescence detection conditions: Ex=500 nm, Em=562 nm).

[0078] like Figure 4 As shown, the absorption spectrum of GCA-DP undergoes a red shift after being treated with glycocholic acid hydrolase; and its fluorescence signal is significantly enhanced under 500 nm excitation light after treatment with glycocholic acid hydrolase.

[0079] like Figure 5 As shown in the fluorescence detection results, the reaction rate of the glycocholate hydrolase (GCH) catalyzed reaction is much higher than that of other hydrolases, indicating that the glycocholate hydrolase catalyzes the reaction of GCA-DP with good selectivity. The GCA-DP provided by this invention can be applied to the activity determination of glycocholate hydrolase.

[0080] Application Example 2

[0081] In vitro determination of the effects of different metal ions and amino acids on the fluorescence intensity of GCA-DP:

[0082] (1) Dissolve the GCA-DP obtained in Example 1 in DMSO to prepare a GCA-DP solution with a concentration of 1 mM;

[0083] (2) Add different types of ion solutions (final concentration 200 μM) or amino acids (final concentration 10 μM) to the pH 5-6 buffer (KH2PO4, K2HPO4, 100 mM) respectively, and incubate them at 37°C with shaking for 3 minutes.

[0084] The ions in the above ionic solutions are carbonate ions, sulfate ions, nickel ions, calcium ions, manganese ions, potassium ions, sodium ions, ferrous ions, zinc ions, barium ions, copper ions, and magnesium ions, respectively.

[0085] The amino acids are lysine (Lys), glutamine (Gln), tryptophan (Try), L-cysteine ​​(Cys), glycine (Gly), serine (Ser), arginine (Arg), glutathione (Gsh), and glutamic acid (Glu);

[0086] (3) Add 2µL of 1 mM (final concentration 10μM) GCA-DP solution to the in vitro metabolic reaction system obtained in step (2), react at 37℃ for 30 min, then add 100µL of ice acetonitrile, shake vigorously, and terminate the reaction.

[0087] (4) Centrifuge at 4℃ and 20000×g for 20 min using a high-speed refrigerated centrifuge, take the supernatant, and determine the formation rate of 2-[(1E)-2-(4-aminophenyl)vinyl]-1-(5-carboxypentyl)-3,3-dimethyl-3H-indole by fluorescence detection (fluorescence detection conditions: Ex=500 nm, Em=562 nm).

[0088] like Figure 6 Fluorescence test results showed that various metal ions or amino acids had no effect on the fluorescence intensity of GCA-DP obtained in Example 1, indicating that GCA-DP has high anti-interference ability.

[0089] Application Example 3

[0090] Linearity study of the GCA-DP probe reaction catalyzed by glycocholate hydrolase:

[0091] (1) Dissolve the GCA-DP obtained in Example 1 in DMSO to prepare a GCA-DP solution with a concentration of 1 mM;

[0092] (2) Prepare an in vitro metabolic reaction system of 200 µL. The reaction system consists of a buffer solution (KH2PO4, K2HPO4, final concentration 100 mM) at pH 7.4 and glycocholate hydrolase 1 (final concentrations of 0, 2.5, 5, 10, 15, 20, 25, 30, 35, 37.5, 40 U / mL), and pre-incubate with shaking at 37 °C for 3 min.

[0093] (3) Add 2 µL of 1 mM (final concentration 10 μM) GCA-DP solution to the pre-incubated reaction system obtained in step (2), react at 37 °C for 30 min, then add 100 µL of ice acetonitrile, shake vigorously, and terminate the reaction.

[0094] (4) Centrifuge at 4℃ and 20000×g for 20 min using a high-speed refrigerated centrifuge, take the supernatant, and determine the formation rate of 2-[(1E)-2-(4-aminophenyl)vinyl]-1-(5-carboxypentyl)-3,3-dimethyl-3H-indole by fluorescence detection (fluorescence detection conditions: Ex=500 nm, Em=562 nm).

[0095] like Figure 7As shown, fluorescence detection results indicate that the reaction of GCA-DP obtained in Example 1 catalyzed by glycocholate hydrolase (GCH) exhibits good enzyme linearity in the range of 0-40 U / mL. 2 The value was 0.9813, indicating that GCA-DP can be applied to the determination of the activity and expression level of glycocholic acid hydrolase in complex samples.

[0096] The above are merely preferred embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A fluorescent probe for detecting glycocholic acid hydrolase, characterized in that, The structure of the fluorescent probe is shown in formula (1): （1）。 2. The method for preparing a fluorescent probe for detecting glycocholic acid hydrolase as described in claim 1, characterized in that, Includes the following steps: Anhydrous cholic acid and aminobenzyl alcohol were used as raw materials to prepare intermediate 1 by acylation reaction, and then intermediate 2 was obtained by oxidation. Then, intermediate 2 was obtained by reacting intermediate 2 with 6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide through the Brainwell reaction to obtain the fluorescent probe for detecting glycocholic acid hydrolase. The structural formula of intermediate 1 is as follows: ; The structural formula of intermediate 2 is as follows: .

3. The method for preparing a fluorescent probe for detecting glycocholic acid hydrolase according to claim 2, characterized in that, The acylation reaction is carried out using DMF as a solvent and HATU and DIPEA as catalysts.

4. The method for preparing a fluorescent probe for detecting glycocholic acid hydrolase according to claim 2, characterized in that, The oxidation process includes the following steps: intermediate 1 is mixed with dichloromethane and manganese dioxide, stirred and reacted at room temperature, then filtered and evaporated under reduced pressure to obtain intermediate 2.

5. The method for preparing a fluorescent probe for detecting glycocholic acid hydrolase according to claim 2, characterized in that, The brain wenger response The process includes the following steps: intermediate 2, 6-(2,3,3-trimethylindol-1-onth-1-yl)hexanoic acid bromide and acetonitrile are mixed, piperidine is added under a protective atmosphere, the reaction is stirred, the solvent is evaporated and purified to obtain the fluorescent probe for detecting glycocholic acid hydrolase.

6. The use of the fluorescent probe for detecting glycocholic acid hydrolase as described in claim 1 in the preparation of reagents for detecting glycocholic acid hydrolase activity.

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