Compounds, kits and methods for detecting lysosomal alkalinity as an ammonia death marker

Abstract

The present invention provides compounds, kits and methods for detecting the lysosomal alkalinity as an ammonia death marker, belonging to the field of biological detection technologies. The compound is EKNOH, which can specifically react with ammonia under the action of an auxiliary agent hypobromous acid to generate a fluorescent substance. The kit includes a sample extraction solution A, a sample extraction solution B, a sample buffer solution, a protein precipitant for effectively extracting lysosomal ammonia from cells, a substrate stock solution for colorimetric detection, and a color development auxiliary reagent. The solution of the present invention is particularly applicable to cell samples, and has the advantages of simple operation, higher detection sensitivity, less sample consumption, etc., providing an effective detection means for the research on the mechanism related to ammonia death and drug development, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and particularly relates to a compound, a kit and a method for detecting the lysosomal alkalinity as an ammonia death marker. Background Art

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

[0003] T cells eliminate ammonia for their survival, while effector cells allow the accumulation of ammonia to mediate their death. Ammonia derived from glutamine catabolism deposits in lysosomes, and the accumulation of excessive ammonia increases the lysosomal pH value and triggers the death of CD8+ Teff cells by decomposing 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 harmful effects of ammonia on other tissue cells have not been explored. In particular, whether ammonia is physiologically produced by extrahepatic cells and plays its role remains largely unknown. Therefore, the development of reagents and methods related to lysosomal alkalinity 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 art, the present invention provides a compound, a kit and a method for detecting the lysosomal alkalinity as an ammonia death marker, aiming to solve the problem that there is no detection kit and detection method related to lysosomal alkalinity on the existing 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-sulfonic acid sodium, abbreviated as EKNOH, and its molecular formula is C 31 H 27BrNNaO9S, with a molecular weight of 691.05, and its structural formula is as follows:

[0007] .

[0008] In a second aspect, the present invention provides a method for preparing a compound EKNOH, comprising the following steps:

[0009] S1. React 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine with bromoethane under the catalysis of a base to obtain a compound A, and its structural formula is as follows:

[0010] ;

[0011] S2. React the compound A with sodium 8-methoxy-5-bromonaphthalene-1-sulfonate under the catalysis of a base to obtain EKNOH.

[0012] 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 first extracted with ethyl acetate and then subjected to column chromatography using petroleum ether and ethyl acetate as eluents to purify the compound A.

[0013] 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.

[0014] In a third aspect, the present invention provides the use of the compound EKNOH in ammonia detection, and this use is not for the diagnosis and treatment of diseases. Experimental data show that the compound EKNOH can form a fluorescent substance with ammonia (the fluorescence intensity can be measured by using a fluorescence microplate reader with an excitation wavelength of 486 nm and an emission wavelength of 535 nm), and it has specificity for ammonia, and the fluorescence intensity is proportional to the ammonia content, so it can be used for the specific quantitative detection of ammonia.

[0015] In some embodiments of the present invention, the ammonia content in cell lysosomes is detected using the compound EKNOH, and the alkalinization degree of lysosomes 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.

[0016] In a fourth aspect, the present invention provides a kit for detecting the alkalinization degree of ammonia death marker lysosomes, which contains the compound EKNOH.

[0017] Preferably, the above kit at least includes the following reagents:

[0018] Sample extraction solution A, containing potassium dihydrogen phosphate, disodium hydrogen phosphate, trehalose, Triton X-100, polyvinylpyrrolidone K30 (PVP30) and bovine serum albumin (BSA);

[0019] Sample extraction solution B, containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride, sucrose, BSA and polyvinylpyrrolidone K40 (PVP40);

[0020] Sample buffer, containing potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Triton X-100, EDTA, sucrose, trehalose, β-cyclodextrin, mannitol, hexose, methylcellulose, sodium sulfate, dextran and PEG6000;

[0021] Protein precipitant, containing trichloroacetic acid and sodium dodecyl sulfate (SDS);

[0022] Substrate stock solution, containing EKNOH and α-cyclodextrin;

[0023] Chromogenic auxiliary reagent, containing hypobromous acid.

[0024] More preferably, in sample extraction solution 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;

[0025] In sample extraction solution B, the pH is 7.2 - 8.0, the content of HEPES is 9.5 - 16 g / L, the content of sodium chloride is 10 - 15 g / L, the content of sucrose is 0.8 - 3.6 g / L, the content of BSA is 3.5 - 5.7 g / L, and the content of PVP40 is 0.8 - 1.8 g / L;

[0026] 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 methyl cellulose 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;

[0027] In the protein precipitant, the content of trichloroacetic acid is 100 - 200 g / L, and the content of SDS is 50 - 100 g / L;

[0028] 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;

[0029] In the color - developing auxiliary reagent, potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer is used, the pH is 7.6 - 8.0, and the content of hypobromous acid is 8.8 - 12.2 mg / mL.

[0030] More preferably, the above - mentioned kit further includes a standard solution. In some embodiments of the present invention, the standard solution is a 100 μmol / L aqueous ammonium chloride solution.

[0031] In the fifth aspect, the present invention provides a method for detecting the lysosomal alkalinity of ammonia - related death markers, including the following steps:

[0032] S1. Extract ammonia in the lysosomes of the cell sample to be tested to obtain a sample solution to be tested;

[0033] S2. Prepare a blank group, a standard group, and a determination group, and detect the fluorescence intensity after incubating 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 product), and the reaction system of the determination group contains EKNOH, hypobromous acid, and the sample solution to be tested;

[0034] S3. Calculate the lysosomal alkalinity according to the detection results.

[0035] 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. Therefore, the fluorescence intensity can be detected under corresponding conditions in step S2. Preferably, in the above detection method, the reaction conditions for step S2 are: incubation at 37 °C for 10 min.

[0036] Preferably, in the above detection method, step S1 uses the kit provided in the fourth aspect of the present invention to obtain the sample solution to be tested, which specifically includes the following operations:

[0037] S11. Add sample extraction solution A to the collected cells, homogenize and then centrifuge to obtain the supernatant, and then centrifuge the supernatant to obtain the precipitate;

[0038] S12. Add sample extraction solution B to the precipitate obtained in S11, mix well and then centrifuge to collect the precipitate;

[0039] S13. Add sample buffer and protein precipitant to the precipitate obtained in S12, and the supernatant obtained by centrifugation is the sample solution to be tested.

[0040] In some embodiments of the present invention, the centrifugation operation in step S11 is: first centrifuge at 500×g to collect the supernatant, then discard the precipitate from the supernatant at 5000×g, and finally centrifuge the obtained supernatant at 30000×g to collect the precipitate; the centrifugation operation in step S12 is: centrifuge at 30000×g to collect the precipitate; the centrifugation operation in step S12 is: centrifuge at 5000×g.

[0041] Using the extraction reagent and extraction operation provided by the present invention, the lysosomal sub-organelle can be obtained specifically and effectively, and then the corresponding sample to be tested can be obtained, making the result accurate and reliable.

[0042] Preferably, in the above detection method, step S3 includes the following:

[0043] S31. Calculate the ammonia content in the sample to be tested according to formula (1);

[0044] (1);

[0045] In the formula, = sample OF (fluorescence intensity) value - blank OF value, = standard product OF value - blank OF value, c is the concentration of the standard product added in the standard group, and f is the dilution factor before sample testing;

[0046] S32. Calculate the lysosomal alkalinity in the sample cell to be tested according to formula (2);

[0047] (2);

[0048] The control sample is a normal cell sample without ammonia-induced death, and the extraction and ammonia content detection of the control sample are the same as those of the sample to be tested.

[0049] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0050] The present invention has successfully developed a chromogenic substrate, namely compound EKNOH. This chromogenic substrate can react with ammonia to generate a fluorescent substance and is specific for ammonia, so it has prospects in the specific detection of ammonia. Based on compound EKNOH, the present invention has further developed a kit and method for detecting lysosomal alkalization in cell samples, filling the market gap, and having the advantages of simple operation, higher detection sensitivity, and less sample consumption, providing detection tools and means for scientific researchers in studying the mechanism of ammonia-induced death, developing drugs related to ammonia-induced death, and other aspects. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] In order to more clearly illustrate the technical solutions of the present invention, the drawings used in the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0052] Figure 1 It is a schematic diagram of the reaction of compound EKNOH of the present invention with ammonia to generate a fluorescent substance;

[0053] Figure 2 It is a synthetic route diagram of compound EKNOH in the embodiment of the present invention;

[0054] Figure 3 It is a diagram of the specificity detection result of compound EKNOH in the embodiment of the present invention;

[0055] Figure 4 It is a calibration curve diagram of compound EKNOH in the embodiment of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0056] The embodiments of the technical solutions of the present invention will be described in detail below with reference to the drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, so they are only examples and cannot be used to limit the protection scope of the present invention.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill 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 herein are intended to cover non-exclusive inclusion.

[0058] To solve the technical problem of the lack of detection kits and methods related to lysosomal alkalinity, the present invention provides a compound, a kit and a method for detecting the lysosomal alkalinity as an ammonia death marker. Based on the developed new compound EKNOH that reacts with ammonia to generate a fluorescent substance, in combination with the supporting extraction reagent and detection method, the quantitative detection of the lysosomal alkalinity in cell samples is achieved.

[0059] Please refer to Figure 1 , the embodiment of the present invention first provides a substrate that can specifically react with ammonia and generate a fluorescent substance, namely the compound EKNOH.

[0060] Please refer to Figure 2 , the embodiment of the present invention also provides a method for synthesizing the compound EKNOH, including the following steps:

[0061] S1. React 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine with bromoethane under the catalysis of a base to obtain compound A;

[0062] S2. React compound A with 8-methoxy-5-bromonaphthalene-1-sulfonate under the catalysis of a base to obtain EKNOH.

[0063] The embodiment of the present invention also provides a detection kit for the lysosomal alkalinity as an ammonia death marker, including at least the following reagents:

[0064] ① Sample extraction solution A, containing potassium dihydrogen phosphate, disodium hydrogen phosphate, trehalose, Triton X-100, PVP30 and BSA;

[0065] ② Sample extraction solution B, containing HEPES, sodium chloride, sucrose, BSA and PVP40;

[0066] ③ Sample buffer solution, containing potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, Triton X-100, EDTA, sucrose, trehalose, β-cyclodextrin, mannitol, hexose, methylcellulose, sodium sulfate, dextran and PEG6000;

[0067] ④ Protein precipitant, containing trichloroacetic acid and SDS;

[0068] ⑤ Substrate stock solution, containing compound EKNOH and α-cyclodextrin;

[0069] ⑥ Color development auxiliary reagent, containing hypobromous acid;

[0070] ⑦ Ammonium chloride standard solution.

[0071] It can be understood that the kit may also include other common consumables, such as enzyme-linked immunosorbent assay (ELISA) plates, sampling tubes, etc.

[0072] Based on the lysosomal alkalinity detection kit for ammonia-induced cell death markers, an embodiment of the present invention also provides a method for detecting the lysosomal alkalinity of ammonia-induced cell death markers, including the following steps:

[0073] S1. Extract ammonia in lysosomes from the cell sample to be tested to obtain a sample solution to be tested;

[0074] 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; among them, 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 the sample solution to be tested;

[0075] S3. First calculate the ammonia content in the sample solution to be tested based on the detection data, and then calculate the lysosomal alkalinity of the sample to be tested using a control sample; the control sample is a cell sample that has not undergone ammonia-induced cell death, and the extraction and ammonia content detection of the control sample are the same as those of the sample to be tested.

[0076] The following are some specific examples. 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 a limitation of the present invention. For those not specified in the examples regarding specific techniques or conditions, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0077] Example 1

[0078] This example provides a method for preparing the compound EKNOH, including the following steps:

[0079] (1) Preparation of 5-(4-bromo-1-ethoxy-8-hydroxy-6-methoxy-3-methyl-9,10-dioxo-9,10-dihydroanthracen-2-yl)amine.

[0080] Dissolve 100.2 mmol of emodin in 250 mL of CCl4 with stirring, add 50.5 mmol of AlCl3, dropwise add 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, and extract with 3 × 250 mL of DCM. The extract is concentrated to obtain a crude product. Then, column chromatography is carried out using petroleum ether and ethyl acetate as eluents to obtain the pure product of 4-bromoemodin, with a yield of 75%.

[0081] Dissolve 50.3 mmol of 4-bromoemodin in 150 mL of stirred THF, add 100.6 mmol of potassium carbonate, dropwise add 45.6 mmol of methyl iodide, stir and react at 50 - 80 °C for 12 h (monitored by TLC). After the reaction is complete, cool to 30 °C, add 100 mL of water to terminate the reaction, and extract with 3 × 150 mL of DCM. The concentrated extract gives the crude product. Then, column chromatography is carried out using petroleum ether and ethyl acetate as eluents to obtain pure 4-bromo-6-methoxymodin with a yield of 55%.

[0082] Dissolve 25.3 mmol of 4-bromo-6-methoxymodin in 100 mL of stirred THF, add 50.6 mmol of potassium carbonate, dropwise add 22.8 mmol of ethyl iodide, stir and react at 50 - 80 °C for 12 h (monitored by TLC). After the reaction is complete, cool to 30 °C, add 100 mL of water to terminate the reaction, and extract with 3 × 100 mL of DCM. The concentrated extract gives the crude product. Then, column chromatography is carried out using petroleum ether and ethyl acetate as eluents to obtain pure 4-bromo-1-ethoxy-6-methoxymodin with a yield of 58%.

[0083] Under ice bath conditions, add 20 mL of concentrated sulfuric acid, 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-methoxymodin, stir and react at 10 °C for 2 h (monitored by TLC). After the reaction is complete, pour the reactant into 100 mL of ice water to terminate the reaction, and extract with 3 × 100 mL of DCM. The extract is then extracted with 3 × 100 mL of 10% aqueous sodium bicarbonate solution. The concentrated extract gives the crude product. Then, column chromatography is carried out using petroleum ether and ethyl acetate as eluents to obtain pure 2-nitro-4-bromo-1-ethoxy-6-methoxymodin with a yield of 58%.

[0084] Dissolve 10.2 mmol of 2-nitro-4-bromo-1-ethoxy-6-methoxymodin in 100 mL of methanol, add 25 mg of Pd / C, place the reaction system in a hydrogen atmosphere, stir and react at room temperature for 8 h (monitor the reaction progress by LC-MS). Filter to remove Pd / C, wash with 2 × 30 mL of methanol, and concentrate the filtrate to obtain the crude product. Then, column chromatography is carried out using DCM and methanol as eluents to obtain pure 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%.

[0085] 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, and 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。

[0086] (2) Preparation of compound A.

[0087] 8.2 mmol of 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 of bromoethylamine and 24.4 mmol of sodium carbonate were added, and the mixture was stirred and reacted at 70 °C for 12 h (the reaction progress was detected by LC-MS). 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 the crude product. Then, column chromatography was carried out using petroleum ether and ethyl acetate as the eluent to obtain the pure product of compound A, with a yield of 85%.

[0088] The prepared compound A was detected and characterized, and 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。

[0089] (3)Preparation of sodium 8-methoxy-5-bromonaphthalene-1-sulfonate.

[0090] Add 100 mmol of 1-methoxynaphthalene into the reaction flask, add 150 mL of DCM to dissolve and stir, then add 150 mmol of sulfonyl chloride, stir and react at room temperature for 12 h (detected by TLC). After the reaction is complete, add 80 mL of water to terminate the reaction. Column chromatography is carried out with DCM and methanol as eluents to obtain 8-methoxynaphthalene-1-sulfonic acid, with a yield of 71%.

[0091] Add 50 mmol of 8-methoxynaphthalene-1-sulfonic acid into the reaction flask, add 150 mL of CCl4 to dissolve and stir, heat to 45 °C, dropwise add 300 mmol of liquid bromine, stir and react at 70 - 80 °C for 6 h (detected by TLC). After the reaction is complete, cool to 30 °C, add 80 mL of water to terminate the reaction, add 50 mL of 8M sodium hydroxide, spin dry to obtain the crude product of sodium 8-methoxy-5-bromonaphthalene-1-sulfonate, and recrystallize with an ethanol-water system to obtain the pure product, with a yield of 52%.

[0092] The prepared sodium 8-methoxy-5-bromonaphthalene-1-sulfonate was detected and characterized, and 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。

[0093] (4)Preparation of the target compound EKNOH.

[0094] Add 10 mmol of compound A into a reaction flask, add 25 mL of DMSO to dissolve and stir, then add 12 mmol of 8-methoxy-5-bromonaphthalene-1-sulfonate and 50.4 mmol of potassium carbonate, stir and react at 70 °C for 12 h (monitored by TLC). After the reaction is complete, directly perform column chromatography using petroleum ether and ethyl acetate as eluents to obtain pure EKNOH product with a yield of 78%.

[0095] Detect and characterize the prepared EKNOH. The NMR and mass spectrometry data are as follows:

[0096] 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。

[0097] Example 2

[0098] This example demonstrates the specificity of compound EKNOH for ammonia, that is, compound EKNOH has the potential for specific ammonia detection. The specific experimental procedure is as follows:

[0099] Preparation of sample buffer: An aqueous solution prepared from 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 is 10.5 g / L, the content of disodium hydrogen phosphate is 3.25 g / L, the content of sodium chloride is 10 g / L, the content of Triton X-100 is 0.8 mL / L, the content of EDTA is 0.4 g / L, the content of sucrose is 1.2 g / L, the content of trehalose is 4.3 g / L, the content of β-cyclodextrin is 1.2 g / L, the content of mannitol is 3.6 g / L, the content of hexose is 3.4 g / L, the content of methylcellulose is 0.6 g / L, the content of sodium sulfate is 0.3 g / L, the content of dextran is 0.25 g / L, and the content of PEG6000 is 0.16 g / L;

[0100] Preparation of substrate stock solution: Dissolve EKNOH and α-cyclodextrin in potassium dihydrogen phosphate and disodium hydrogen phosphate buffer solution; 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 solution is 150 mM, and the pH is 7.4.

[0101] Preparation of chromogenic auxiliary reagent: Dissolve hypobromous acid in potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer solution; wherein, the content of hypobromous acid is 12.2 mg / mL, the concentration of potassium dihydrogen phosphate and dipotassium hydrogen phosphate buffer solution is 50 mM, and the pH is 7.8.

[0102] Specificity verification sample: Take standard products ammonium chloride, phenethylamine, aniline, ethylenediaminetetraacetic acid, phenylmethylamine cetyltrimethylammonium bromide (CTAB), and dissolve them in 1.0 mL of sample buffer respectively as specificity verification samples.

[0103] Using the above reagents, conduct the following experimental operations:

[0104] ① Set blank wells and measurement wells on the enzyme-linked immunosorbent assay (ELISA) plate. Among them, add sample buffer, substrate stock solution, and chromogenic auxiliary reagent to the blank wells, and add verification samples, substrate stock solution, and chromogenic auxiliary reagent to the measurement wells;

[0105] ② Incubate at 37 °C for 15 min, use a fluorescence microplate reader with an excitation wavelength of 486 nm and an emission wavelength of 535 nm to measure its fluorescence intensity.

[0106] The detection results of each sample are shown in Table 1 and Figure 3As shown above. From the above results, it can be seen that only the sample in which ammonium chloride is dissolved has fluorescence intensity, while the test wells corresponding to phenethylamine, aniline, ethylenediaminetetraacetic acid, CTAB, and the blank well have no fluorescence intensity, indicating that EKNOH did not react, that is, this compound has specificity for ammonia when used as a substrate and can be used for the specific detection of ammonia.

[0107] Table 1

[0108]

[0109] According to the above detection conditions, samples of ammonium chloride with different concentrations were detected, the absolute OF value was calculated, and curves were plotted with the ammonium chloride concentration and the absolute OF value as the abscissa and ordinate, respectively. The results are as Figure 4 shown, indicating that this compound can be used for the quantitative detection of ammonia.

[0110] Example 3

[0111] This example provides a kit for detecting lysosomal alkalinization as an ammonia death marker. The kit includes sample extraction solution A, sample extraction solution B, sample buffer, protein precipitant, substrate stock solution, color development auxiliary reagent, and standard product solution. Specifically, the composition and preparation of each reagent are as follows:

[0112] Preparation of sample extraction solution A: Weigh 14 g of potassium dihydrogen phosphate, 3.85 g of disodium hydrogen phosphate, 2.2 g of trehalose, 0.8 mL of Triton X-100, 1.65 g of PVP30, 2.8 g of BSA, add 700 mL of water, stir to dissolve, adjust the pH to 7.4 with 2 M sodium hydroxide, and then make up the volume to 1000 mL with water.

[0113] Preparation of sample extraction solution B: Weigh 14.2 g of HEPES, 15 g of sodium chloride, 3.2 g of sucrose, 3.5 g of BSA, 0.8 g of PVP40, add 700 mL of water, stir to dissolve, adjust the pH to 7.8 with 2 M sodium hydroxide, and then make up the volume to 1000 mL with water.

[0114] Preparation of sample buffer: Weigh 12 g of potassium dihydrogen phosphate, 2.65 g of disodium hydrogen phosphate, 12 g of sodium chloride, 0.8 mL of Triton X-100, 1.3 g of sucrose, 3.6 g of trehalose, 0.8 g of β-cyclodextrin, 3.6 g of mannitol, 2.6 g of hexose, 0.2 g of methylcellulose, 0.2 g of sodium sulfate, 0.3 g of dextran, 0.15 g of PEG6000, 0.4 g of EDTA, add 700 mL of water, stir to dissolve, adjust the pH to 7.4 with 2 M sodium hydroxide, and then make up the volume to 1000 mL with water.

[0115] Preparation of protein precipitant: Weigh 100 g of trichloroacetic acid and 50 g of SDS, dissolve them in water, and make up the volume to 1000 mL.

[0116] Preparation of substrate stock solution: Weigh 36 mg of EKNOH and 8 mg of α-cyclodextrin, dissolve them in 150 mM phosphate buffer (potassium dihydrogen phosphate + disodium hydrogen phosphate) with pH 7.4, and make up the volume to 100 mL.

[0117] Preparation of chromogenic auxiliary reagent: Weigh 122 mg of hypobromous acid, and mix it evenly with 10 mL of 50 mM phosphate buffer (potassium dihydrogen phosphate, dipotassium hydrogen phosphate) with pH 7.6.

[0118] Preparation of standard product: Weigh 5.4 mg of ammonium chloride, dissolve it in water, and make up the volume to 1000 mL.

[0119] In the actual preparation process, each reagent can be aliquoted according to the actual demand (for example, sample extraction solution A is aliquoted into 50 mL vials and 2 vials are configured in each kit), and it is also recommended to store the kit in the dark at 4°C.

[0120] Example 4

[0121] Based on the kit provided in Example 3, this example provides a method for detecting lysosomal alkalization of ammonia death markers, including the following steps:

[0122] (1) Preparation of the sample solution to be measured.

[0123] Specifically, the sample solution to be measured is prepared from the cell sample to be measured through the following operations:

[0124] ① Take 1 - 2×10 7 cells, centrifuge at 4°C and 500×g for 5 minutes, carefully aspirate the culture medium, and dry it as much as possible to collect the cells;

[0125] ② Wash twice with 4°C PBS, and dry the supernatant as much as possible after washing;

[0126] ③ Add 500 μL of sample extraction solution A at 4°C, place it on ice for 10 minutes, and homogenize it 50 - 70 times with a homogenizer;

[0127] ④ Centrifuge the homogenate at 4°C and 500×g for 5 minutes, discard the precipitate, and collect the supernatant;

[0128] ⑤ Centrifuge the supernatant at 4°C and 5000×g for 10 minutes, discard the precipitate, and collect the supernatant;

[0129] ⑥ Centrifuge the supernatant at 4°C and 30000×g for 20 minutes, discard the supernatant, and collect the precipitate;

[0130] ⑦ Add 500 μL of sample extraction solution B at 4°C to the precipitate and mix well;

[0131] ⑧ Centrifuge at 4°C and 30,000×g for 20 minutes, discard the supernatant, and collect the precipitate;

[0132] ⑨ Add 500 μL of sample buffer at 4°C to the precipitate and mix well;

[0133] ⑩ Take 100 μL of the mixed sample, add 100 μL of protein precipitant, mix well, and place at 4°C for 10 minutes;

[0134] ⑪ Centrifuge at 4°C and 5,000×g for 10 minutes, discard the precipitate, and collect the supernatant to obtain the sample solution to be measured.

[0135] (2) Detection of the sample solution to be measured.

[0136] Set blank wells, standard wells, and measurement wells on the enzyme-linked immunosorbent assay (ELISA) plate. Among them, add 10 μL of sample buffer, 30 μL of substrate stock solution, and 80 μL of chromogenic auxiliary reagent to the blank well, add 10 μL of standard product, 30 μL of substrate stock solution, and chromogenic auxiliary reagent to the standard well, and add 10 μL of the sample solution to be measured (which can be diluted with sample buffer according to the actual situation before detection), 30 μL of substrate stock solution, and 80 μL of chromogenic auxiliary reagent to the measurement well.

[0137] Incubate at 37°C for 10 min, and detect the fluorescence intensity with a fluorescence microplate reader (excitation light wavelength 486 nm, emission light wavelength 535 nm).

[0138] (3) Calculation of the ammonia content and alkalinity in lysosomes.

[0139] First, calculate the ammonia content in the sample solution to be measured according to the following formula;

[0140] ;

[0141] In the formula, = sample OF value - blank OF value, = standard product OF value - blank OF value, c is the concentration of ammonium chloride added in the standard group, and f is the dilution factor of the sample solution before testing;

[0142] Then, calculate the alkalinity of lysosomes in the cell sample to be measured according to the following formula;

[0143] ;

[0144] 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 the same as those of the sample to be measured.

[0145] Example 5

[0146] Based on the detection method in Example 4, the lysosomal alkalinity of ammonia-induced dead cells was detected in this example.

[0147] Referring to the existing literature, cell samples of the Hela cell-ammonia-induced death model group (i.e., the test sample) and the Hela cell-normal group (i.e., the control sample) were first prepared and obtained, and then these two cell samples were synchronously tested according to the detection method in Example 4. The results are shown in Table 2:

[0148] Table 2

[0149]

[0150] Based on the ammonia content detected in Table 2, calculate the of the test sample according to formula (2), specifically:

[0151] .

[0152] From the above data, it can be seen that the ammonia concentration detected in the normal group sample is less than that of the ammonia-induced death model group sample, the lysosome has become alkalized, and the alkalinity is 48.6%.

[0153] In summary, the present invention provides a feasible solution and tool for the research and determination of cell ammonia-induced death.

[0154] It should be noted that the present invention is not limited to the above embodiments. The above embodiments are only examples, and embodiments having the same constitution and the same function and effect as the technical idea within the technical scope of the present invention are all included in the technical scope of the present invention. In addition, within the scope not departing from the gist of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments, and other ways constructed by combining some constituent elements of the embodiments 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 according to claim 1 in the detection of ammonia for non-diagnostic and non-therapeutic purposes.

3. The application according to claim 2, wherein The ammonia is the ammonia in cell lysosomes.

4. Use of the compound according to claim 1 in the preparation of a product for detecting the lysosomal alkalinity of an ammonia death marker.

5. A kit for detecting the lysosomal alkalinity of an ammonia death marker, characterized in that, It includes EKNOH according to claim 1.

6. The lysosomal alkalinity detection kit for ammonia death markers according to claim 5, wherein It includes sample extract A, sample extract 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 bovine serum albumin; The sample extract B contains HEPES, sodium chloride, sucrose, bovine serum albumin, 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 contains trichloroacetic acid and SDS; The substrate stock solution contains EKNOH and α-cyclodextrin; The color development auxiliary reagent contains hypobromous acid.

7. The kit for detecting the lysosomal alkalinity of an ammonia death marker according to claim 6, wherein 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 bovine serum albumin is 2.8 - 5.6 g / L; in the sample extract B, the pH is 7.2 - 8.0, the content of HEPES is 9.5 - 16 g / L, the content of sodium chloride is 10 - 15 g / L, the content of sucrose is 0.8 - 3.6 g / L, the content of bovine serum albumin is 3.5 - 5.7 g / L, and the content of PVP40 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, with a pH of 7.2 - 7.6, the content of EKNOH being 346.3 - 460.2 mg / L, and the content of α - cyclodextrin being 50 - 100 mg / L; In the color - developing auxiliary reagent, potassium dihydrogen phosphate - dipotassium hydrogen phosphate buffer is used, with a pH of 7.6 - 8.0, and the content of hypobromous acid being 8.8 - 12.2 mg / mL.

8. A method for detecting the lysosomal alkalinity of an ammonia death marker for non-diagnostic and non-therapeutic purposes, characterized in that, It includes the following steps: S1. Extract ammonia in lysosomes from the cell sample to be tested to obtain the sample solution to be tested; S2. Prepare a blank group, a standard group, and a determination group, and detect the fluorescence intensity after incubating 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 determination group contains EKNOH, hypobromous acid, and the sample solution to be tested; S3. Calculate the 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. Add sample extraction solution A to the collected cells, homogenize and then centrifuge to take the supernatant, and then centrifuge the supernatant to obtain a precipitate; S12. Add sample extraction solution B to the precipitate obtained in S11, mix well and then centrifuge to collect the precipitate; S13. Add 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 extraction solution A contains potassium dihydrogen phosphate, disodium hydrogen phosphate, trehalose, Triton X - 100, PVP30, and bovine serum albumin; the sample extraction solution B contains HEPES, sodium chloride, sucrose, bovine serum albumin, 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 contains trichloroacetic acid and SDS.

10. The method for detecting the lysosomal alkalinity of ammonia death markers according to claim 8, wherein, Step S3 includes the following: S31. Calculate the ammonia content in the sample to be tested according to formula (1); （1）； Wherein, = OF value of sample - OF value of blank, = OF value of standard - OF value of blank, c is the concentration of ammonium chloride added in the standard group, f is the dilution factor before sample testing, and the OF value is the fluorescence intensity value; S32. Calculate 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 the same as those of the sample to be tested.

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