Compound, kit and method for detecting alkalization degree of ammonia death marker lysosome

Abstract

The invention provides a compound, a kit and a method for detecting the alkalization degree of an ammonia death marker lysosome, and belongs to the technical field of biological detection.The compound is EKNOH and can specifically react with ammonia under the action of an auxiliary agent hypobromous acid to generate a fluorescent substance, and the fluorescent substance is used for detecting the alkalization degree of the lysosome. The kit comprises a sample extracting solution A, a sample extracting solution B, a sample buffer solution and a protein precipitant which can effectively extract lysosomal ammonia from cells, and a substrate stock solution and a color developing auxiliary reagent which are used for color developing detection. The scheme provided by the invention is particularly suitable for cell samples, has the advantages of simplicity and convenience in operation, higher detection sensitivity, small sample dosage and the like, and provides an effective detection means for related mechanism research of ammonia death, drug development and the like.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a compound, a kit and a method for detecting the alkalinity of lysosomes, a marker of ammonia death. Background Art

[0002] Amino acid metabolism produces ammonia as a byproduct, which is toxic to cells. To detoxify ammonia, the urea cycle has evolved and occurs in hepatocytes. Therefore, in liver failure, hyperammonemia inevitably develops, which damages neural cells such as astrocytes through unknown mechanisms, leading to hepatic encephalopathy. Amino acid amino groups in extrahepatic tissues are thought to be incorporated into glutamine through transamination. This glutamine is transported to the liver through the cycle, where it releases ammonia and allows it to enter the urea cycle of hepatocytes. However, some extrahepatic cells may also produce ammonia through glutaminolysis. Although extrahepatic cells can use purine and polyamine synthesis pathways to process ammonia, these pathways cannot achieve complete clearance and residual ammonia may accumulate in cells. Rapidly proliferating cells require acetyl-CoA as a component of lipogenesis, and they must use glutaminolysis to salvage the tricarboxylic acid cycle by supplementing α-ketoglutarate, thereby producing ammonia as a byproduct.

[0003] T cells scavenge ammonia for their survival, while effector cells allow ammonia accumulation to mediate their death. Glutaminolysis-derived ammonia is deposited in lysosomes, and excess ammonia accumulation increases lysosomal pH and triggers the death of CD8+ Teff cells by disassembling mitochondria. This form of cell death is characterized by lysosomal alkalinization and mitochondrial swelling, which is different from other known forms of cell death mechanisms.

[0004] However, the deleterious effects of ammonia on cells in other tissues have not been explored. In particular, whether ammonia is physiologically produced by extrahepatic cells and exerts its effects remains largely unknown. Therefore, the development of reagents and methods related to lysosomal alkalinization based on ammonia death is of great significance for related research. Summary of the invention

[0005] In view of the technical problems existing in the background technology, the present invention provides a compound, a kit and a method for detecting the alkalinity of lysosomes, a marker of ammonia death, aiming to solve the problem that there are no detection kits and methods related to lysosome alkalinity on the market.

[0006] In the first aspect, the present invention provides a compound, specifically a new compound of 5-((4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)(ethyl)amino)-8-methoxynaphthalene-1-sulfonate sodium salt, referred to as EKNOH, with a molecular formula of C 31 H 27BrNNaO9S, molecular weight is 691.05, and the structural formula is as follows: .

[0007] In a second aspect, the present invention provides a method for preparing a compound EKNOH, comprising the following steps: S1. 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine is reacted with ethyl bromide under base catalysis to obtain compound A, whose structural formula is shown below: ; S2. Compound A is reacted with sodium 8-methoxy-5-bromonaphthalene-1-sulfonate under base catalysis to obtain EKNOH.

[0008] Preferably, in the above preparation method, the reaction temperature of step S1 is 60-80° C. In some embodiments of the present invention, the reaction solvent used in step S1 is N,N-dimethylformamide (DMF), the base used is Na2CO3, and the reaction product can be extracted with ethyl acetate first, and then column chromatography is performed using petroleum ether and ethyl acetate as eluents to purify compound A.

[0009] Preferably, in the above preparation method, the reaction temperature of step S2 is 60-80° C. In some embodiments of the present invention, the reaction solvent used in step S2 is dimethyl sulfoxide (DMSO), the base used is potassium carbonate, and the reaction product can be directly subjected to column chromatography using petroleum ether and ethyl acetate as eluents to obtain purified EKNOH.

[0010] In a third aspect, the present invention provides an application of compound EKNOH in ammonia detection, and the application is not for the purpose of disease diagnosis and treatment. Experimental data show that compound EKNOH can generate fluorescent substances with ammonia (the fluorescence intensity can be measured by a fluorescence microplate reader with an excitation wavelength of 486 nm and an emission wavelength of 535 nm), and has specificity for ammonia, and the fluorescence intensity is proportional to the ammonia content, so it can be used for specific quantitative detection of ammonia.

[0011] In some embodiments of the present invention, the compound EKNOH is used to detect the ammonia content in the cell lysosome, and the alkalinity of the lysosome in the test sample is measured according to the ratio of the difference between the ammonia content in the test sample and the ammonia content in the control sample to the ammonia content in the control sample.

[0012] In a fourth aspect, the present invention provides a kit for detecting the degree of lysosomal alkalinity of an ammonia death marker, comprising the compound EKNOH.

[0013] Preferably, the above kit includes at least the following reagents: Sample extract A, containing potassium dihydrogen phosphate, disodium hydrogen phosphate, trehalose, Triton X-100, polyvinylpyrrolidone K30 (PVP30), and bovine serum albumin (BSA); sample extract B, containing 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), sodium chloride, sucrose, BSA, and polyvinylpyrrolidone K40 (PVP40); Sample buffer, including potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Triton X-100, EDTA, sucrose, trehalose, β-cyclodextrin, mannitol, hexose, methylcellulose, sodium sulfate, dextran, and PEG6000; protein precipitants, including trichloroacetic acid and sodium dodecyl sulfate (SDS); Substrate stock solution, containing EKNOH and α-cyclodextrin; Auxiliary reagent for color development, including hypobromous acid.

[0014] More preferably, in the sample extract A, the pH is 7.1-7.6, the content of potassium dihydrogen phosphate is 12-16 g / L, the content of disodium hydrogen phosphate is 2.65-3.85 g / L, the content of trehalose is 1.5-2.2 g / L, the content of Triton X-100 is 0.6-0.8 mL / L, the content of PVP30 is 1.35-1.65 g / L, and the content of BSA is 2.8-5.6 g / L; In sample extract B, the pH is 7.2-8.0, the HEPES content is 9.5-16 g / L, the sodium chloride content is 10-15 g / L, the sucrose content is 0.8-3.6 g / L, the BSA content is 3.5-5.7 g / L, and the PVP40 content is 0.8-1.8 g / L; In the sample buffer, the pH is 7.2-7.6, the content of potassium dihydrogen phosphate is 9.5-16 g / L, the content of disodium hydrogen phosphate is 2.65-3.85 g / L, the content of sodium chloride is 7-12 g / L, the content of Triton X-100 is 0.5-0.8 mL / L, the content of EDTA is 0.1-0.5 g / L, the content of sucrose is 0.8-1.6 g / L, the content of trehalose is 3.5-5.7 g / L, the content of β-cyclodextrin is 0.8-1.8 g / L, the content of mannitol is 2-5.8 g / L, the content of hexose is 2.4-3.8 g / L, the content of methylcellulose is 0.2-0.8 g / L, the content of sodium sulfate is 0.1-0.6 g / L, the content of dextran is 0.1-0.6 g / L, and the content of PEG6000 is 0.1-0.5 g / L; In the protein precipitant, the content of trichloroacetic acid is 100-200 g / L, and the content of SDS is 50-100 g / L; In the substrate stock solution, potassium dihydrogen phosphate-sodium dihydrogen phosphate buffer was used, the pH was 7.2-7.6, the content of EKNOH was 346.3-460.2 mg / L, and the content of α-cyclodextrin was 50-100 mg / L; In the color development auxiliary reagent, potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer is used, the pH value is 7.6-8.0, and the content of hypobromous acid is 8.8-12.2 mg / mL.

[0015] More preferably, the above kit further comprises a standard solution. In some embodiments of the present invention, the standard solution is a 100 μmol / L ammonium chloride aqueous solution.

[0016] In a fifth aspect, the present invention provides a method for detecting lysosomal alkalinity, an ammonia death marker, comprising the following steps: S1, extracting ammonia in lysosomes in the cell sample to be tested to obtain a sample solution to be tested; S2. Prepare a blank group, a standard group and a test group, and detect the fluorescence intensity after incubation at 25-37°C for 10-30 min; wherein the reaction system of the blank group contains EKNOH and hypobromous acid, the reaction system of the standard group contains EKNOH, hypobromous acid and ammonium chloride (standard), and the reaction system of the test group contains EKNOH, hypobromous acid and the sample solution to be tested; S3. Calculate the lysosome alkalinity based on the test results.

[0017] In the above detection method, the excitation and emission wavelengths of the fluorescent substance obtained by the reaction of compound EKNOH with ammonia are 486 nm and 535 nm respectively, so step S2 can detect the fluorescence intensity under corresponding conditions. Preferably, in the above detection method, the reaction conditions of step S2 are: incubation at 37°C for 10 min.

[0018] Preferably, in the above detection method, step S1 uses the kit provided by the fourth aspect of the present invention to obtain the sample solution to be tested, which specifically includes the following operations: S11, adding sample extract A to the collected cells, homogenizing and centrifuging to obtain the supernatant, and then centrifuging the supernatant to obtain the precipitate; S12, adding sample extract B to the precipitate obtained in S11, mixing and centrifuging to collect the precipitate; S13, adding sample buffer and protein precipitant to the precipitate obtained in S12, and centrifuging to obtain the supernatant which is the sample solution to be tested.

[0019] In some embodiments of the present invention, the centrifugation operation of step S11 is: first, collect the supernatant by centrifugation at 500×g, then discard the precipitate of the supernatant at 5000×g, and finally collect the precipitate of the supernatant by centrifugation at 30000×g; the centrifugation operation of step S12 is: collect the precipitate by centrifugation at 30000×g; the centrifugation operation of step S12 is: centrifugation at 5000×g.

[0020] By utilizing the extraction reagent and extraction operation provided by the present invention, lysosomal subcellular organelles can be obtained in a targeted and effective manner, and then the corresponding sample to be tested can be obtained, so that the result is accurate and reliable.

[0021] Preferably, in the above detection method, step S3 includes the following: S31, calculating the ammonia content in the sample to be tested according to formula (1); (1); In the formula, = sample OF (fluorescence intensity) value - blank OF value, = OF value of standard - OF value of blank, c is the concentration of standard added to the standard group, and f is the dilution multiple before the sample test; S32, calculating the lysosomal alkalinity in the cell sample to be tested according to formula (2); (2); The control sample is a normal cell sample that has not undergone ammonia death, and the extraction and ammonia content detection of the control sample are consistent with those of the sample to be tested.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention has successfully developed a chromogenic substrate, namely compound EKNOH, which can generate fluorescent substances when reacting with ammonia and is specific to ammonia, so it has a promising future in ammonia-specific detection. Based on compound EKNOH, the present invention further developed a kit and method for detecting lysosomal alkalinization in cell samples, filling the market gap, and having the advantages of simple operation, higher detection sensitivity, and less sample usage, etc., providing detection tools and means for scientific researchers in studying the mechanism of ammonia death and the development of ammonia death-related drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.

[0024] Figure 1This is a schematic diagram of the reaction of the compound EKNOH of the present invention with ammonia to generate a fluorescent substance; Figure 2 The synthetic route of the compound EKNOH in the embodiment of the present invention; Figure 3 This is a graph showing the specificity detection results of the compound EKNOH in the embodiment of the present invention; Figure 4 It is a standard curve diagram of the compound EKNOH in the examples of the present invention. DETAILED DESCRIPTION

[0025] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention; the terms "including" and "having" and any variations thereof in this document are intended to cover non-exclusive inclusions.

[0027] In order to solve the technical problem of lack of detection kits and methods for lysosomal alkalinity, the present invention provides a compound, a kit and a method for detecting the ammonia death marker lysosomal alkalinity. Based on the reaction of the newly developed compound EKNOH with ammonia to generate a fluorescent substance, combined with the matching extraction reagents and detection methods, the quantitative detection of lysosomal alkalinity in cell samples is achieved.

[0028] Please refer to Figure 1 The embodiment of the present invention first provides a substrate that can react specifically with ammonia to generate a fluorescent substance, namely, the compound EKNOH.

[0029] Please refer to Figure 2 The embodiment of the present invention also provides a method for synthesizing the compound EKNOH, comprising the following steps: S1, reacting 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine with ethyl bromide under base catalysis to obtain compound A; S2. Compound A is reacted with sodium 8-methoxy-5-bromonaphthalene-1-sulfonate under base catalysis to obtain EKNOH.

[0030] The embodiment of the present invention also provides a kit for detecting the degree of lysosomal alkalinity of an ammonia death marker, which comprises at least the following reagents: ① Sample extract A, containing potassium dihydrogen phosphate, disodium hydrogen phosphate, trehalose, Triton X-100, PVP30 and BSA; ② Sample extraction solution B, containing HEPES, sodium chloride, sucrose, BSA and PVP40; ③ Sample buffer, including potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Triton X-100, EDTA, sucrose, trehalose, β-cyclodextrin, mannitol, hexose, methylcellulose, sodium sulfate, dextran and PEG6000; ④ Protein precipitant, including trichloroacetic acid and SDS; ⑤ Substrate stock solution, including compound EKNOH and α-cyclodextrin; ⑥ Color development auxiliary reagent, including hypobromous acid; ⑦Ammonium chloride standard solution.

[0031] It is understandable that the kit may also include other commonly used consumables, such as ELISA plates, sampling tubes, etc.

[0032] Based on the ammonia death marker lysosomal alkalinity detection kit, the embodiment of the present invention also provides an ammonia death marker lysosomal alkalinity detection method, comprising the following steps: S1, extracting ammonia in lysosomes in the cell sample to be tested to obtain a sample solution to be tested; S2. Prepare a blank group, a standard group and a test group, and detect the fluorescence intensity after incubation at 25-37°C for 10-30 min; wherein the reaction system of the blank group contains EKNOH and hypobromous acid, the reaction system of the standard group contains EKNOH, hypobromous acid and ammonium chloride, and the reaction system of the test group contains EKNOH, hypobromous acid and the sample solution to be tested; S3. First calculate the ammonia content in the test sample solution according to the test data, and then use the control sample to calculate the lysosomal alkalinity of the test sample; wherein the control sample is a cell sample that has not undergone ammonia death, and the extraction and ammonia content detection of the control sample are consistent with those of the test sample.

[0033] Some specific examples are listed below. It should be noted that the examples described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

[0034] Example 1 This example provides a method for preparing the compound EKNOH, comprising the following steps: (1) Preparation of 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine.

[0035] Dissolve 100.2 mmol of rhein in 250 mL of CCl4 and stir, add 50.5 mmol of AlCl3, drop 100.2 mmol of liquid bromine, stir and react at 70-80°C for 6 h (TLC detection), after the reaction is complete, cool to 30°C, add 200 mL of water to terminate the reaction, extract with 3×250 mL of DCM, and concentrate the extract to obtain the crude product. Then, column chromatography is performed with petroleum ether and ethyl acetate as eluents to obtain the pure product of 4-bromo-rhein with a yield of 75%.

[0036] Dissolve 50.3 mmol 4-bromo-rhubarb in 150 mL THF and stir, add 100.6 mmol potassium carbonate, add 45.6 mmol iodomethane dropwise, stir and react at 50-80°C for 12 h (TLC detection), after the reaction is complete, cool to 30°C, add 100 mL water to terminate the reaction, extract with 3×150 mL DCM, and concentrate the extract to obtain the crude product. Then, column chromatography is performed with petroleum ether and ethyl acetate as eluents to obtain the pure product of 4-bromo-6-methoxy-rhubarb, with a yield of 55%.

[0037] Dissolve 25.3 mmol 4-bromo-6-methoxy-rhubarb in 100 mL THF and stir, add 50.6 mmol potassium carbonate, add 22.8 mmol iodoethane dropwise, stir and react at 50-80°C for 12 h (TLC detection), after the reaction is complete, cool to 30°C, add 100 mL water to terminate the reaction, extract with 3×100 mL DCM, and concentrate the extract to obtain the crude product. Then, column chromatography is performed using petroleum ether and ethyl acetate as eluents to obtain the pure product of 4-bromo-1-ethoxy-6-methoxy-rhubarb, with a yield of 58%.

[0038] Add 20 mL of concentrated sulfuric acid under ice bath conditions, slowly add 10 mL of concentrated nitric acid while stirring, keep the temperature below 10°C, slowly add 12.3 mmol of 4-bromo-1-ethoxy-6-methoxy-rhubarb, stir and react at 10°C for 2 h (TLC detection), after the reaction is complete, pour the reactant into 100 mL of ice water, terminate the reaction, extract with 3×100 mL of DCM, and use 3×100 mL of 10% sodium bicarbonate aqueous solution to concentrate the extract to obtain the crude product. Then column chromatography is performed using petroleum ether and ethyl acetate as eluents to obtain the pure product of 2-nitro-4-bromo-1-ethoxy-6-methoxy-rhubarb, with a yield of 58%.

[0039] 10.2 mmol 2-nitro-4-bromo-1-ethoxy-6-methoxy-rhodamine was dissolved in 100 mL methanol, 25 mg Pd / C was added, the reaction system was placed in a hydrogen atmosphere, stirred at room temperature for 8 h (LC-MS was used to detect the reaction progress), Pd / C was removed by filtration, washed with 2×30 mL of methanol, and the filtrate was concentrated to obtain a crude product. Then, column chromatography was performed using DCM and methanol as eluents to obtain a pure product of 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine with a yield of 92%.

[0040] The prepared 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine was detected and characterized, wherein the NMR and mass spectrometry data are as follows: mp 243.5-249.2 °C; 1 H NMR (400 MHz, DMSO- d 6): 10.01 (s, 1H, OH), 6.91 (s, 1H, ArH), 6.73 (s, 1H, ArH), 5.33 (br, 2H, NH), 4.09 (m, 2H,CH2), 3.77 (s, 3H, CH3), 2.25 (s, 3H, CH3),1.34 (d, J = 2.4 Hz, 3H, CH3), ppm. HRMS (ESI): calcd for C 18 H 16 BrNO5[M] + ,405.0235; found, 405.0248.

[0041] (2) Preparation of compound A.

[0042] 8.2 mmol 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine was dissolved in 100 mL of DMF, 16.2 mmol bromoethylamine and 24.4 mmol sodium carbonate were added, and the mixture was stirred at 70°C for 12 h (LC-MS was used to detect the reaction progress). After the reaction was complete, 40 mL of water was added and the mixture was stirred at room temperature for 3 h. The reaction was terminated, and the mixture was extracted with 3×60 mL of ethyl acetate. The extract was concentrated to obtain a crude product. Column chromatography was then performed using petroleum ether and ethyl acetate as eluents to obtain a pure product of compound A with a yield of 85%.

[0043] The prepared compound A was tested and characterized, wherein the NMR and mass spectrometry data are as follows: mp 254.3-258.7 °C; 1 H NMR (400 MHz, DMSO- d 6): 10.04 (s, 1H, OH), 6.90 (s, 1H, ArH), 6.74(s, 1H, ArH), 6.30 (br, 1H, NH), 4.07 (m, 2H,CH2), 3.77 (s, 3H, CH3), 3.52 (m,2H,CH2), 2.25 (s, 3H, CH3), 1.34 (d, J = 2.4 Hz, 3H, CH3), 1.26 (d, J = 2.4 Hz,3H, CH3), ppm. HRMS (ESI): calcd for C 20 H 20 BrNO5[M] + , 433.0545; found,433.0558.

[0044] (3) Preparation of sodium 8-methoxy-5-bromonaphthalene-1-sulfonate.

[0045] 100 mmol 1-methoxynaphthalene was added to the reaction flask, 150 mL DCM was added to dissolve and stir, then 150 mmol sulfonic acid chloride was added, and the reaction was stirred at room temperature for 12 h (TLC detection). After the reaction was complete, 80 mL water was added to terminate the reaction. Column chromatography was performed using DCM and methanol as eluents to obtain 8-methoxynaphthalene-1-sulfonic acid with a yield of 71%.

[0046] Add 50 mmol 8-methoxynaphthalene-1-sulfonic acid into the reaction flask, add 150 mL CCl4 to dissolve and stir, heat to 45°C, add 300 mmol liquid bromine dropwise, stir and react at 70-80°C for 6 h (TLC detection), after the reaction is complete, cool to 30°C, add 80 mL water to terminate the reaction, add 50 mL 8M sodium hydroxide, spin dry to obtain crude 8-methoxy-5-bromonaphthalene-1-sulfonate sodium, and recrystallize from ethanol water system to obtain a pure product with a yield of 52%.

[0047] The prepared sodium 8-methoxy-5-bromonaphthalene-1-sulfonate was characterized, wherein the NMR and mass spectrometry data are as follows: mp 323.3-326.7 °C; 1 H NMR (400 MHz, DMSO- d 6): 8.54 (d, J= 2.4 Hz, 1H, ArH),8.04 ( t, J = 2.4 Hz, 1H, ArH), 7.93 (d, J = 2.4 Hz, 1H, ArH), 7.75 (d, J = 2.4 Hz,1H, ArH), 6.24 (d, J = 2.4 Hz, 1H, ArH), 3.77 (s, 3H, CH3),ppm. HRMS (ESI):calcd for C 11 H8BrNaO4S [M] + , 337.9235; found, 337.9254.

[0048] (4) Preparation of the target compound EKNOH.

[0049] Add 10 mmol of compound A to the reaction bottle, add 25 mL of DMSO to dissolve and stir, then add 12 mmol of sodium 8-methoxy-5-bromonaphthalene-1-sulfonate and 50.4 mmol of potassium carbonate, stir and react at 70°C for 12 h (TLC detection), and directly column chromatography after the reaction is complete. Column chromatography is performed using petroleum ether and ethyl acetate as eluents to obtain pure EKNOH product with a yield of 78%.

[0050] The prepared EKNOH was tested and characterized, and the NMR and mass spectrometry data are shown below: EKNOH mp 347.3-352.6 °C; 1 H NMR (400 MHz, DMSO- d 6): 8.02 (s, 1H, ArH), 7.72-7.63 (m, 2H, ArH), 7.45 (s, 1H, ArH), 6.53 (s, 1H, ArH), 6.47-6.46 (d, J =2.4 Hz,2H, ArH), 5.02 (br, 1H, OH), 3.94-3.90 (m, 2H,CH2), 3.73 (t, J = 2.4Hz, 6H, CH3),3.14-3.12 (m, 2H,CH2),2.23 (s, 3H, CH3),1.33 (d, J = 2.4 Hz, 3H,CH3), 1.12 (t, J= 2.4 Hz, 3H, CH3), ppm. HRMS (ESI): calcd for C 31 H 27 BrNNaO9S[M] + , 691.0535; found, 691.0548.

[0051] Example 2 This example demonstrates the specificity of compound EKNOH for ammonia, that is, compound EKNOH has the potential to specifically detect ammonia. The specific experimental process is as follows: Sample buffer solution was prepared by potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Triton X-100, EDTA, sucrose, trehalose, β-cyclodextrin, mannitol, hexose, methylcellulose, sodium sulfate, dextran and PEG6000, with a pH of 7.4; wherein the content of potassium dihydrogen phosphate was 10.5 g / L, the content of disodium hydrogen phosphate was 3.25 g / L, the content of sodium chloride was 10 g / L, the content of Triton X-100 was 0.8 mL / L, the content of EDTA was 0.4 g / L, the content of sucrose was 1.2 g / L, the content of trehalose was 4.3 g / L, the content of β-cyclodextrin was 1.2 g / L, the content of mannitol was 3.6 g / L, the content of hexose was 3.4 g / L, the content of methylcellulose was 0.6 g / L, the content of sodium sulfate was 0.3 g / L, the content of dextran was 0.25 g / L, and the content of PEG6000 was 0.16 g / L; Prepare substrate stock solution: dissolve EKNOH and α-cyclodextrin in potassium dihydrogen phosphate and disodium hydrogen phosphate buffer; wherein the content of EKNOH is 352.2 mg / L, the content of α-cyclodextrin is 100 mg / L, the concentration of potassium dihydrogen phosphate and disodium hydrogen phosphate buffer is 150 mM, and the pH is 7.4.

[0052] Prepare color development auxiliary reagent: dissolve hypobromous acid in potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer; wherein the content of hypobromous acid is 12.2 mg / mL, the concentration of potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer is 50 mM, and the pH is 7.8.

[0053] Specificity verification samples: Take standard ammonium chloride, phenylethylamine, aniline, ethylenediaminetetraacetic acid, phenylmethylamine cetyltrimethylammonium bromide (CTAB), dissolve them in 1.0 mL sample buffer respectively as specificity verification samples.

[0054] Using the above reagents, perform the following experimental operations: ① Blank wells and assay wells are set on the ELISA plate, wherein sample buffer, substrate stock solution and color development auxiliary reagent are added to the blank wells, and verification samples, substrate stock solution and color development auxiliary reagent are added to the assay wells; ②Incubate at 37℃ for 15 min, and measure the fluorescence intensity using a fluorescence microplate reader with an excitation wavelength of 486 nm and an emission wavelength of 535 nm.

[0055] The test results of each sample are shown in Table 1 and Figure 3 As shown in the above results, only the sample dissolved with ammonium chloride has fluorescence intensity, and the test wells corresponding to phenylethylamine, aniline, ethylenediaminetetraacetic acid, CTAB and the blank wells have no fluorescence intensity, indicating that EKNOH has no reaction, that is, the compound is specific to ammonia when used as a substrate and can be used for the specific detection of ammonia.

[0056] Table 1

[0057] According to the above test conditions, samples of ammonium chloride with different concentrations were tested, the absolute OF value was calculated, and curves were drawn with the ammonium chloride concentration and the absolute OF value as the horizontal and vertical coordinates. Figure 4 As shown, this compound can be used for the quantitative detection of ammonia.

[0058] Example 3 This example provides a lysosomal alkalinization detection kit for ammonia death markers, which includes sample extract A, sample extract B, sample buffer, protein precipitant, substrate stock solution, color development auxiliary reagent and standard solution. Specifically, the composition and preparation of each reagent are as follows: Preparation of sample extract A: weigh 14 g potassium dihydrogen phosphate, 3.85 g disodium hydrogen phosphate, 2.2 g trehalose, 0.8 mL Triton X-100, 1.65 g PVP30, and 2.8 g BSA, add 700 mL of water, stir to dissolve, adjust the pH to 7.4 with 2 M sodium hydroxide, and then make up to 1000 mL with water.

[0059] Preparation of sample extract B: Weigh 14.2 g HEPES, 15 g sodium chloride, 3.2 g sucrose, 3.5 g BSA, and 0.8 g PVP40, add 700 mL of water, stir to dissolve, adjust the pH to 7.8 with 2 M sodium hydroxide, and make up to 1000 mL with water.

[0060] Preparation of sample buffer: weigh 12 g potassium dihydrogen phosphate, 2.65 g disodium hydrogen phosphate, 12 g sodium chloride, 0.8 mL Triton X-100, 1.3 g sucrose, 3.6 g trehalose, 0.8 g β-cyclodextrin, 3.6 g mannitol, 2.6 g hexose, 0.2 g methylcellulose, 0.2 g sodium sulfate, 0.3 g dextran, 0.15 g PEG6000, and 0.4 g EDTA, add 700 mL of water, stir to dissolve, adjust the pH to 7.4 with 2 M sodium hydroxide, and then make up to 1000 mL with water.

[0061] Preparation of protein precipitant: Weigh 100 g trichloroacetic acid and 50 g SDS, dissolve them in water, and make up to 1000 mL.

[0062] Preparation of substrate stock solution: Weigh 36 mg EKNOH and 8 mg α-cyclodextrin, dissolve them in 150 mM, pH 7.4 phosphate buffer (potassium dihydrogen phosphate + sodium dihydrogen phosphate), and make up to 100 mL.

[0063] Preparation of color development auxiliary reagent: Weigh 122 mg of hypobromous acid and mix it with 10 mL of 50 mM, pH 7.6 phosphate buffer (potassium dihydrogen phosphate, potassium hydrogen phosphate).

[0064] Preparation of standard: Weigh 5.4 mg of ammonium chloride and dissolve it in water, then make up to 1000 mL.

[0065] In the actual preparation process, each reagent can be packaged according to the actual demand (such as sample extract A is packaged into 50mL vials and 2 vials are configured in each kit). It is also recommended to store the kit at 4℃ away from light.

[0066] Example 4 Based on the kit provided in Example 3, this example provides a method for detecting lysosomal alkalinization of an ammonia death marker, comprising the following steps: (1) Preparation of sample solution to be tested.

[0067] Specifically, the cell sample to be tested is prepared into a sample solution to be tested by the following operations: ① Take 1-2×10 7 The cells were centrifuged at 4°C and 500 × g for 5 minutes, and the culture medium was carefully aspirated to dryness as much as possible to collect the cells; ② Wash twice with 4℃ PBS and absorb the supernatant as much as possible after washing; ③ Add 500 μL of sample extract A at 4°C, place on ice for 10 minutes, and homogenize with a homogenizer 50-70 times; ④ Centrifuge the homogenate at 4°C, 500×g for 5 minutes, discard the precipitate, and collect the supernatant; ⑤ Centrifuge the supernatant at 4°C, 5000×g for 10 minutes, discard the precipitate, and collect the supernatant; ⑥ Centrifuge the supernatant at 4°C, 30,000×g for 20 minutes, discard the supernatant, and collect the precipitate; ⑦ Add 500 μL of 4°C sample extract B to the precipitate and mix well; ⑧ Centrifuge at 4°C, 30,000×g for 20 minutes, discard the supernatant and collect the precipitate; ⑨Add 500 μL of 4°C sample buffer to the precipitate and mix well; ⑩Take 100 μL of the mixed sample, add 100 μL of protein precipitant, mix well, and place at 4°C for 10 minutes; ⑪ Centrifuge at 4°C and 5000×g for 10 minutes, discard the precipitate, collect the supernatant, and obtain the sample solution to be tested.

[0068] (2) Testing of the sample solution to be tested.

[0069] Blank wells, standard wells, and assay wells were set on the ELISA plate. 10 μL sample buffer, 30 μL substrate stock solution, and 80 μL color development auxiliary reagent were added to the blank wells; 10 μL standard, 30 μL substrate stock solution, and color development auxiliary reagent were added to the standard wells; 10 μL sample solution to be tested (which can be diluted with sample buffer according to actual conditions before detection), 30 μL substrate stock solution, and 80 μL color development auxiliary reagent were added to the assay wells.

[0070] Incubate at 37°C for 10 min, and measure the fluorescence intensity using a fluorescence microplate reader (excitation wavelength 486 nm, emission wavelength 535 nm).

[0071] (3) Calculation of ammonia content and alkalinity in lysosomes.

[0072] First, calculate the ammonia content in the sample solution to be tested according to the following formula; ; In the formula, = sample OF value - blank OF value, = OF value of standard - OF value of blank, c is the concentration of ammonium chloride added to the standard group, and f is the dilution multiple of the sample solution before testing; Then calculate the lysosomal alkalinity in the cell sample to be tested according to the following formula; ; Among them, the control sample is a cell sample that has not undergone ammonia death, and the extraction and detection of the control sample are consistent with those of the sample to be tested.

[0073] Example 5 Based on the detection method in Example 4, this example detected the lysosomal alkalinity of ammonia-dead cells.

[0074] Referring to the existing literature, cell samples of the Hela cell-ammonia death model group (i.e., the sample to be tested) and the Hela cell-normal group (i.e., the control sample) were first prepared, and then the two cell samples were tested simultaneously according to the detection method in Example 4. The results are shown in Table 2: Table 2

[0075] Based on the detected ammonia content in Table 2, the ammonia content of the sample to be tested is calculated according to formula (2): , specifically: .

[0076] From the above data, we can see that the ammonia concentration detected in the normal group samples is lower than that in the ammonia death model group samples, and the lysosomes have become alkaline with an alkalinity of 48.6%.

[0077] In summary, the present invention provides a feasible solution and tool for the study and determination of cell ammonia death.

[0078] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are merely examples, and embodiments having substantially the same structure as the technical idea and exerting the same effects within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, within the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments, and other methods of combining some of the constituent elements in the embodiments to construct are also included in the scope of the present invention.

Claims

1. A compound, characterized in that The compound is EKNOH, and its structural formula is as follows: 。 2. Use of the compound as claimed in claim 1 in ammonia detection.

3. The use according to claim 2, characterized in that: The ammonia is the ammonia in the cell lysosome.

4. Use of the compound as claimed in claim 1 in the preparation of a product for detecting the lysosomal alkalinity of ammonia death markers.

5. A kit for detecting the lysosomal alkalinity of an ammonia death marker, characterized in that: Comprising the EKNOH described in claim 1.

6. The ammonia death marker lysosomal alkalinity detection kit according to claim 5, characterized in that: It includes sample extracting solution A, sample extracting solution B, sample buffer, protein precipitant, substrate stock solution and color development auxiliary reagent; The sample extract A contains potassium dihydrogen phosphate, disodium hydrogen phosphate, trehalose, Triton X-100, PVP30 and BSA; The sample extract B contains HEPES, sodium chloride, sucrose, BSA and PVP40; The sample buffer contains potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Triton X-100, EDTA, sucrose, trehalose, β-cyclodextrin, mannitol, hexose, methylcellulose, sodium sulfate, dextran and PEG6000; The protein precipitant comprises trichloroacetic acid and SDS; The substrate stock solution comprises EKNOH and α-cyclodextrin; The color development auxiliary reagent comprises hypobromous acid.

7. The ammonia death marker lysosomal alkalinity detection kit according to claim 6, characterized in that: In the sample extract A, the pH is 7.1-7.6, the content of potassium dihydrogen phosphate is 12-16 g / L, the content of disodium hydrogen phosphate is 2.65-3.85 g / L, the content of trehalose is 1.5-2.2 g / L, the content of Triton X-100 is 0.6-0.8 mL / L, the content of PVP30 is 1.35-1.65 g / L, and the content of BSA is 2.8-5.6 g / L; In the sample extract B, the pH is 7.2-8.0, the HEPES content is 9.5-16 g / L, the sodium chloride content is 10-15 g / L, the sucrose content is 0.8-3.6 g / L, the BSA content is 3.5-5.7 g / L, and the PVP40 content is 0.8-1.8 g / L; In the sample buffer, the pH is 7.2-7.6, the content of potassium dihydrogen phosphate is 9.5-16 g / L, the content of disodium hydrogen phosphate is 2.65-3.85 g / L, the content of sodium chloride is 7-12 g / L, the content of Triton X-100 is 0.5-0.8 mL / L, the content of EDTA is 0.1-0.5 g / L, the content of sucrose is 0.8-1.6 g / L, the content of trehalose is 3.5-5.7 g / L, the content of β-cyclodextrin is 0.8-1.8 g / L, the content of mannitol is 2-5.8 g / L, the content of hexose is 2.4-3.8 g / L, the content of methylcellulose is 0.2-0.8 g / L, the content of sodium sulfate is 0.1-0.6 g / L, the content of dextran is 0.1-0.6 g / L, and the content of PEG6000 is 0.1-0.5 g / L; In the protein precipitant, the content of trichloroacetic acid is 100-200 g / L, and the content of SDS is 50-100 g / L; In the substrate stock solution, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer is used, the pH is 7.2-7.6, the content of EKNOH is 346.3-460.2 mg / L, and the content of α-cyclodextrin is 50-100 mg / L; In the color development auxiliary reagent, potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer is used, the pH value is 7.6-8.0, and the content of hypobromous acid is 8.8-12.2 mg / mL.

8. A method for detecting lysosomal alkalinity, a marker of ammonia death, characterized in that: The following steps are involved: S1, extracting ammonia in lysosomes in the cell sample to be tested to obtain a sample solution to be tested; S2. Prepare a blank group, a standard group and a measurement group, and detect the fluorescence intensity after incubation at 25-37°C for 10-30 min; the reaction system of the blank group contains EKNOH and hypobromous acid, the reaction system of the standard group contains EKNOH, hypobromous acid and ammonium chloride, and the reaction system of the measurement group contains EKNOH, hypobromous acid and a sample solution to be tested; S3. Calculate the degree of lysosomal alkalinity.

9. The method for detecting the lysosomal alkalinity of the ammonia death marker according to claim 8, characterized in that: Step S1 includes the following steps: S11, adding sample extract A to the collected cells, homogenizing and centrifuging to obtain the supernatant, and then centrifuging the supernatant to obtain the precipitate; S12, adding sample extract B to the precipitate obtained in S11, mixing and centrifuging to collect the precipitate; S13, adding sample buffer and protein precipitant to the precipitate obtained in S12, and the supernatant obtained by centrifugation is the sample solution to be tested; The sample extract A contains potassium dihydrogen phosphate, disodium hydrogen phosphate, trehalose, Triton X-100, PVP30 and BSA, the sample extract B contains HEPES, sodium chloride, sucrose, BSA and PVP40, the sample buffer contains potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Triton X-100, EDTA, sucrose, trehalose, β-cyclodextrin, mannitol, hexose, methylcellulose, sodium sulfate, dextran and PEG6000, and the protein precipitant contains trichloroacetic acid and SDS.

10. The method for detecting lysosomal alkalinity of ammonia death marker according to claim 8, characterized in that: Step S3 includes the following: S31, calculating the ammonia content in the sample to be tested according to formula (1); （1）； In the formula, = sample OF value - blank OF value, = OF value of standard - OF value of blank, c is the concentration of ammonium chloride added to the standard group, and f is the dilution multiple before the sample test; S32, calculating the lysosomal alkalinity in the cell sample to be tested according to formula (2); （2）； The control sample is a cell sample that has not undergone ammonia death, and the extraction and ammonia content detection of the control sample are consistent with those of the sample to be tested.

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