Gamma-glutamyltranspeptidase detection substrate as well as preparation method and application thereof

Abstract

The invention discloses a gamma-glutamyltranspeptidase detection substrate and a preparation method and application thereof, the gamma-glutamyltranspeptidase detection substrate is L-gamma-glutamyl-3-sulfonic acid-4-(N, N-diethyl) aniline, the structural formula of the gamma-glutamyltranspeptidase detection substrate is # imgabs0, the gamma-glutamyltranspeptidase detection substrate is stable and easy to dissolve in water at normal temperature, so that the gamma-glutamyltranspeptidase detection substrate can be used for dry biochemical detection of GGT activity, a chromogenic reaction product is green, the detection wavelength range is 650-670nm, and the gamma-glutamyltranspeptidase detection substrate can be used for detecting GGT activity. The influence of other active substances in a substrate and a sample on a detection result can be avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis, and specifically relates to a gamma-glutamyl transpeptidase detection substrate and a preparation method and application thereof. Background Art

[0002] γ-Glutamyltranspeptidase (GGT) is an enzyme that can cleave the C-terminal glutamic acid of proteins and peptides. Abnormally elevated activity of GGT is often associated with a variety of liver diseases, nervous system diseases, and myocardial infarction. In the diagnosis of hepatobiliary diseases, changes in GGT activity are a more sensitive indicator than alkaline phosphatase and leucine aminopeptidase. The determination of serum GGT activity has become an important auxiliary means in clinical diagnosis.

[0003] The most commonly used substrate for GGT activity determination is L-glutamyl-4-nitroaniline or its related derivatives, which have sufficient specificity for the enzyme and are easy to use and sensitive. At present, the IFCC method and Szasz method are mainly used in China for GGT activity determination. Both methods are based on the principle of enzyme kinetics and use γ-glutamyl-3-carboxy-4-p-nitroaniline and diGly peptide as substrates. GGT acts on the donor substrate γ-glutamyl-3-carboxy-4-p-nitroaniline and the acceptor substrate diGly peptide to produce γ-glutamyl diGly peptide and 3-carboxy-4-nitroaniline, and the concentration of GGT in serum is calculated by detecting the absorption peak at 405nm. However, the solubility and stability of γ-glutamyl-4-nitroaniline are poor, which is an obvious disadvantage of this method. This substrate has low solubility and the solution will fail in a very short time. Although surfactants can be added to improve its solubility, γ-glutamyl-4-nitroaniline still needs to be dissolved at a temperature of 50 to 60°C, and it will spontaneously hydrolyze above this temperature. In addition, since the wavelengths of other components in the serum sample are close to the product measurement wavelength, strict blank tests are required for the samples, making the process complicated and cumbersome.

[0004] In order to solve these problems, several γ-glutamyl-4-nitroaniline derivatives with higher solubility have been synthesized. For example, by introducing a sulfonic acid group or a carboxyl group at the 3-position of p-nitroaniline, the solubility of the substrate can be increased. Among them, γ-glutamyl-3-carboxyl-4-nitroaniline has been commercialized as a donor substrate, but its released product is strongly interfered by hemolysis, icteric or turbid samples in the absorbance range of 350-450nm. At the same time, the absorbance of γ-glutamyl-3-carboxyl-4-nitroaniline is high in the measurement wavelength range of the reaction product (400-420nm), which also affects its application in GGT detection.

[0005] Another determination method uses γ-glutamylanilide as a substrate, diazotizing the aniline cleavage product by a modified Bratton-Marshall method, and coupling it with N-(1-naphthyl)ethylenediamine. However, this substrate has poor stability in solution and requires heating or surfactants to promote dissolution, which severely limits its determination range and linear upper limit.

[0006] Another substrate used is γ-L-glutamyl-α-naphthylamide, the cleavage product of which is α-naphthylamine, which can be coupled with the diazo compound Fast Blue B salt to form a strong color dye. However, this reaction process is slow, and α-naphthylamine has been found to be carcinogenic. Summary of the invention

[0007] The present invention aims to overcome the defects of the prior art and provide a substrate for detecting γ-glutamyl transpeptidase.

[0008] Another object of the present invention is to provide a method for preparing the above-mentioned γ-glutamyl transpeptidase detection substrate.

[0009] Another object of the present invention is to provide an application of the above-mentioned γ-glutamyl transpeptidase detection substrate.

[0010] The technical solution of the present invention is as follows:

[0011] A γ-glutamyl transpeptidase detection substrate is L-γ-glutamyl-3-sulfonic acid-4-(N,N-diethyl)aniline, and its structural formula is

[0012] The preparation method of the above-mentioned γ-glutamyl transpeptidase detection substrate has a synthetic route as follows:

[0013]

[0014] In a preferred embodiment of the present invention, the steps are as follows:

[0015] (1) dissolving equal moles of N-phthaloyl-L-glutamic anhydride and 3-sulfonic acid-4-(N,N-diethyl)aniline in a reaction solvent and reacting at 0-60° C. for 1-2 h;

[0016] (2) removing the reaction solvent from the material obtained in step (1), cooling to room temperature, adding icy ethanol to precipitate crystals, and then performing solid-liquid separation to obtain a solid;

[0017] (3) dissolving the solid obtained in step (2) in a reaction solvent, then adding a hydrazine solution and stirring evenly, and reacting at room temperature for 40-50 hours;

[0018] (4) The material obtained in step (3) is filtered to obtain the γ-glutamyl transpeptidase detection substrate.

[0019] More preferably, the reaction solvent is methanol, ethanol, propanol, isopropanol, ethylene glycol, chloroform, dichloromethane, acetic acid or ethyl acetate.

[0020] Further preferably, the solute in the hydrazine solution is at least one of hydrazine, hydrazine hydrate, hydrazine sulfate and hydrazine hydrochloride.

[0021] More preferably, the reaction temperature of step (1) is 10-30°C.

[0022] Further preferably, the reaction solvent is methanol, ethanol, propanol, isopropanol, ethylene glycol, chloroform, dichloromethane, acetic acid or ethyl acetate; the solute in the hydrazine solution is at least one of hydrazine, hydrazine hydrate, hydrazine sulfate and hydrazine hydrochloride; and the reaction temperature of step (1) is 10-30°C.

[0023] A method for detecting the activity of γ-glutamyl transpeptidase, using the above-mentioned γ-glutamyl transpeptidase detection substrate for detection.

[0024] The beneficial effects of the present invention are as follows: the present invention is stable at room temperature and easily soluble in water, and therefore can be used for dry biochemical detection of GGT activity, the color development product is green, the detection wavelength range is 650-670nm, and the influence of substrates and other active substances in samples on the detection results can be avoided. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 A graph showing the correlation results in Example 2 of the present invention.

[0026] Figure 2 A graph showing the correlation results in Example 3 of the present invention. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further illustrated and described below through specific implementation modes in combination with the accompanying drawings.

[0028] Example 1 Synthesis of L-γ-glutamyl-3-sulfonic acid-4-(N,N-diethyl)aniline

[0029] Dissolve 51.8g of N-phthaloyl-L-glutamic anhydride and 48.7g of 3-sulfonic acid-4-(N,N-diethyl)aniline in a mixed solution of 250mL of chloroform and 50mL of glacial acetic acid, and heat to reflux at 60℃ in an oil bath for 1h. Remove the solvent by rotary evaporation, and add an appropriate amount of cold ethanol to precipitate crystals. Filter and separate the crystals by suction and dry to obtain 60.2g of the intermediate product N-phthaloyl-γ-glutamyl-3-sulfonic acid-4-(N,N-diethyl)aniline. Dissolve the intermediate product in 70mL of methanol, add 15mL of hydrazine hydrate solution, and stir the mixture at room temperature at 25℃ for 2d. Filter and separate the product by suction and dry to obtain 44.7g of L-γ-glutamyl-3-sulfonic acid-4-(N,N-diethyl)aniline as white crystals.

[0030] The NMR characterization data of the obtained product are as follows:

[0031] 1 H NMR (400MHz, DMSO-d6) δ1.16(6H,t,J=7.0Hz),1.85-1.99(2H,1.92(dt,J=7.6,7.4Hz) ),1.92(dt,J=7.6,7.4Hz)),2.36-2.48(2H,2.42(t,J=7.4Hz),2.42(t,J=7.4Hz)),3. 46(1H,t,J=7.6Hz),3.58-3.70(4H,3.64(q,J=7.0Hz),3.64(q,J=7.0Hz)),6.53(1H, dd,J=8.1,0.5Hz),7.40-7.63(2H,7.46(dd,J=8.1,2.0Hz),7.58(dd,J=2.0,0.5Hz)).

[0032] 13 C NMR (125MHz, DMSO-d6) δ175.3,171.6,144.3,136.1,134.3,116.6,116.3,115.4,55.4,44.6,32.9,27.4,12.9.

[0033] Example 2 Application of L-γ-glutamyl-3-sulfonic acid-4-(N,N-diethyl)aniline

[0034] The color developing layer solution shown in Table 1 below was coated on a transparent 125 μm PET film support and dried at 70°C to a thickness of 30 μm after drying. The reagent layer solution shown in Table 2 below was then coated on the above coating layer and dried at 70°C to a total thickness of 100 μm after drying. After drying, the film was cut into 1.0 cm 2A small piece of dry sheet reagent is assembled, covered with mesh as a diffusion layer, and then a black PET plastic sheet with holes is used as a substrate. It can be used for determination after being assembled into a dry sheet reagent.

[0035] Table 1 Chromogenic layer solution

[0036] Reagents concentration Borate buffer pH 8.0 50mM N-Methylaminobenzoic acid 1mM Potassium periodate 0.2％ Triton X-100 1％ gelatin 10％

[0037] Table 2 Reagent layer solution

[0038] Reagents concentration Borate buffer pH 8.0 50mM Glycine 20mM L-γ-Glutamyl-3-sulfonic acid-4-(N,N-diethyl)aniline 10mM Triton X-100 1％ gelatin 10％

[0039] Fifteen venous serum samples with gradients were randomly selected for testing. 10 μL of the sample to be tested was dripped onto the mesh layer and incubated at 37°C for 5 minutes. The serum sample quickly diffused in four directions through the diffusion layer and diffused longitudinally to the reagent layer, forming a uniform concentration distribution. The color change reaction of the dry analytical reagent was detected from the support side by the rate method at a wavelength of 670 nm, and the concentration of GGT in the sample to be tested was calculated using a pre-made calibration curve. Then compared with the hospital value, the correlation is shown in Figure 1 , the R-square value is 0.99, which shows a good correlation.

[0040] Example 3 Jaundice Sample Test

[0041] According to the operating steps of Example 2, L-γ-glutamyl-3-sulfonic acid-4-(N,N-diethyl) aniline was replaced with γ-glutamyl-3-carboxyl-4-nitroaniline to prepare a control dry sheet, and the jaundice sample detection effect was compared with the dry sheet reagent in Example 2. 10 jaundice venous blood serum samples were randomly selected for testing, 10 μL of the sample to be tested was dripped on the mesh layer, and incubated at 37 ° C for 5 minutes. The serum sample quickly diffused uniformly in four directions through the diffusion layer while diffusing longitudinally to the reagent layer to form a uniform concentration distribution. The color change reaction produced by the dry analysis reagent was detected by the rate method from the support side of the control dry sheet and the dry sheet reagent of Example 2 at wavelengths of 405nm and 670nm, and the concentration of GGT in the sample to be tested was calculated using the pre-made calibration curve.

[0042] The measured values ​​of the control dry sheet and the measured values ​​of the dry sheet reagent of Example 2 were compared with the hospital values, and the correlation was shown as follows: Figure 2 , the k value in the dry film reagent measurement result of Example 2 is closer to 1, that is, the dry film reagent measurement result of Example 2 is closer to the hospital value, indicating that compared with the control dry film, the dry film reagent of Example 2 can more effectively resist the sample background interference.

[0043] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A γ-glutamyl transpeptidase detection substrate, characterized in that: It is L-γ-glutamyl-3-sulfonic acid-4-(N,N-diethyl)aniline, and its structural formula is 2. The method for preparing a γ-glutamyl transpeptidase detection substrate according to claim 1, characterized in that: Its synthetic route is 3. The preparation method according to claim 2, characterized in that: The steps include: (1) dissolving equal moles of N-phthaloyl-L-glutamic anhydride and 3-sulfonic acid-4-(N,N-diethyl)aniline in a reaction solvent and reacting at 0-60° C. for 1-2 h; (2) removing the reaction solvent from the material obtained in step (1), cooling to room temperature, adding icy ethanol to precipitate crystals, and then performing solid-liquid separation to obtain a solid; (3) dissolving the solid obtained in step (2) in a reaction solvent, then adding a hydrazine solution and stirring evenly, and reacting at room temperature for 40-50 hours; (4) The material obtained in step (3) is filtered to obtain the γ-glutamyl transpeptidase detection substrate.

4. The preparation method according to claim 3, characterized in that: The reaction solvent is methanol, ethanol, propanol, isopropanol, ethylene glycol, chloroform, dichloromethane, acetic acid or ethyl acetate.

5. The preparation method according to claim 3, characterized in that: The solute in the hydrazine solution is at least one of hydrazine, hydrazine hydrate, hydrazine sulfate and hydrazine hydrochloride.

6. The preparation method according to claim 3, characterized in that: The reaction temperature of step (1) is 10-30°C.

7. The preparation method according to claim 3, characterized in that: The reaction solvent is methanol, ethanol, propanol, isopropanol, ethylene glycol, chloroform, dichloromethane, acetic acid or ethyl acetate; the solute in the hydrazine solution is at least one of hydrazine, hydrazine hydrate, hydrazine sulfate and hydrazine hydrochloride; the reaction temperature of step (1) is 10-30°C.

8. A method for detecting γ-glutamyl transpeptidase activity, characterized in that: The detection is performed using the γ-glutamyl transpeptidase detection substrate described in claim 1.

Images