WO2023208233A1 - 重氮类化合物及其制备方法和应用 - Google Patents

重氮类化合物及其制备方法和应用 Download PDF

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WO2023208233A1
WO2023208233A1 PCT/CN2023/091869 CN2023091869W WO2023208233A1 WO 2023208233 A1 WO2023208233 A1 WO 2023208233A1 CN 2023091869 W CN2023091869 W CN 2023091869W WO 2023208233 A1 WO2023208233 A1 WO 2023208233A1
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acid
small molecule
during
diazo compound
diazo
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PCT/CN2023/091869
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French (fr)
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陈知行
权力
李聪
程昆仑
赵其锦
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北京大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C245/00—Compounds containing chains of at least two nitrogen atoms with at least one nitrogen-to-nitrogen multiple bond
    • C07C245/12—Diazo compounds, i.e. compounds having the free valencies of >N2 groups attached to the same carbon atom
    • C07C245/14—Diazo compounds, i.e. compounds having the free valencies of >N2 groups attached to the same carbon atom having diazo groups bound to acyclic carbon atoms of a carbon skeleton
    • C07C245/18—Diazo compounds, i.e. compounds having the free valencies of >N2 groups attached to the same carbon atom having diazo groups bound to acyclic carbon atoms of a carbon skeleton the carbon skeleton being further substituted by carboxyl groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D215/14—Radicals substituted by oxygen atoms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/72—Mass spectrometers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8624—Detection of slopes or peaks; baseline correction
    • G01N30/8631—Peaks
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G01N2030/067—Preparation by reaction, e.g. derivatising the sample

Definitions

  • the present invention relates to the field of analytical chemistry, and specifically to a diazo compound and its preparation method and application.
  • the quantitative detection of small molecule carboxylic acid metabolites mainly includes two technical routes: one is enzymatic coupling technology, and the other is chromatography-mass spectrometry technology.
  • chromatography-mass spectrometry technology has high sensitivity and high throughput, can detect multiple metabolites simultaneously, and greatly reduces sample consumption.
  • Enzymatic coupling technology uses the enzyme's specific recognition of small molecule carboxylic acid metabolites to correlate the concentration of small molecule carboxylic acid metabolites with the enzymatic reaction rate, and ultimately through the change in light absorption of a specific wavelength (that is, the enzymatic reaction rate speed) to indirectly reflect the concentration of small molecule carboxylic acid metabolites.
  • concentration range of enzymatic coupling technology for quantitative detection of small molecule carboxylic acid metabolites in biological samples is between 10 and 100 ⁇ M.
  • Enzymatic coupling technology is characterized by easy operation, relatively few steps, low requirements for experimental conditions and personnel, easy access to instruments, and the experiment can basically be completed on a biological laboratory bench.
  • Chromatography-mass spectrometry technology combines the qualitative and quantitative characteristics of chromatographic separation and mass spectrometry, and is currently recognized as the gold standard instrument for qualitative and quantitative analysis in various industries. Chromatography can separate different substances in a sample, as each substance has a unique retention time. Collaborative mass spectrometry can provide real-time molecular weight information of the chromatographic elution components, which can be used to assist in determining the chemical information of the elution components. Among them, liquid chromatography is the most widely used chromatographic technology in biological and pharmaceutical analysis. Triple quadrupole mass spectrometry and high-resolution mass spectrometry are the current mainstream mass spectrometry technologies. The former has high sensitivity and has advantages in quantification of known compounds. The latter It has high resolution and has advantages in the characterization of unknown compounds.
  • Liquid chromatography-mass spectrometry is a common technique for quantitative detection of small molecule metabolites.
  • liquid chromatography includes reversed-phase chromatography, hydrophilic interaction chromatography, and ion pairing chromatography. Due to the strong hydrophilicity and close polarity of small molecule carboxylic acid metabolites, reversed-phase chromatography cannot effectively distinguish them, resulting in different small molecule carboxylic acid metabolites co-eluting and inhibiting ionization of each other. Both hydrophilic interaction chromatography and ion pairing chromatography can separate different small molecule carboxylic acid metabolites, but both require special mobile phases and columns.
  • Hydrophilic interaction chromatography requires a special hydrophilic interaction chromatography column and an alkaline mobile phase.
  • Ion pairing chromatography requires the addition of n-butylamine and other ion pairing reagents to the mobile phase, which limits its use with mass spectrometry technology and has been basically eliminated.
  • Output liquid mass spectrometry range There are two main ideas for detecting characteristic metabolic small molecule carboxylic acids based on liquid mass spectrometry technology:
  • Disadvantage 2 In order to improve retention, the direct detection method will use HILIC hydrophilic chromatography mode. The detection time is at least 20 minutes. Compared with the commonly used reversed-phase chromatography, the time is increased by 2 to 3 times, which is not conducive to high-throughput testing.
  • the main purpose of the present invention is to provide a diazo compound and its preparation method and application to solve the problem of low mass spectrum response and low sensitivity in the prior art when detecting small molecule carboxylic acids, especially small molecule carboxylic acid metabolites. ; Or the instrument analysis time is long, which is not conducive to high-throughput sample detection; or there is a need for special chromatographic columns, which increases the cost of analysis; or there is a long process of processing biological samples, and unstable small molecule carboxylic acid metabolites may be present in the sample. Degradation during the treatment process; or there may be problems such as the need to configure special mobile phases, increased analysis costs, etc.
  • a diazo compound has a structure shown in formula I.
  • R 1 represents H, alkyl, halogen, alkoxy or Alkylamino
  • R 2 represents an aryl group.
  • R 1 represents H, C1-C6 alkyl, halogen, C1-C6 alkoxy or dimethylamino
  • R 2 represents methylenequinolyl or ethyl-N,N-dimethylanilino .
  • a method for preparing the above-mentioned diazo compound includes: performing an esterification reaction on a first dispersion liquid containing a phenylacetic acid compound and an alcohol compound, Generate intermediate product A; perform a diazotization reaction on the second dispersion containing intermediate product A and diazo group transfer reagent to generate diazo compounds; among them, phenylacetic acid compounds have
  • alcohol compounds have a structure represented by R 2 -OH;
  • R 1 represents H, alkyl, halogen, alkoxy or alkylamino;
  • R 2 represents an aromatic group.
  • the reaction temperature is 0-30°C, and the reaction time is 0.5-24h; preferably, during the diazotization reaction, the reaction temperature is 0-30°C, and the reaction time is 1-24h; preferably, during the esterification reaction, the molar ratio of the phenylacetic acid compound and the alcohol compound is (0.5 ⁇ 2):1; preferably, during the diazotization reaction, the molar ratio of the intermediate product A and the diazo group transfer reagent is (1 ⁇ 3):1; preferably, during the diazotization reaction, the diazo group transfer reagent is 4-acetamidobenzenesulfonyl azide, p-toluenesulfonyl azide, or 4-carboxybenzenesulfonyl azide , one or more of 1H-imidazole-1-sulfonyl azide hydrochloride or 2-azido-1,3-dimethylimidazole hexaflu
  • a detection method for quantitative analysis of small molecule carboxylic acids is provided.
  • Small molecule carboxylic acids represent carboxylic acids with a molecular weight between 46 and 500.
  • the detection method includes: derivatization treatment: using derivatization reagents Derivatize the sample containing small molecule carboxylic acid to obtain a derivatized sample; liquid chromatography-mass spectrometry analysis: perform liquid chromatography-mass spectrometry analysis on the derivatization sample to obtain a liquid chromatography-mass spectrum, and according to the liquid chromatography-mass spectrometry Chromatography-mass spectrometry quantitatively analyzes the small molecule carboxylic acid components in the sample; wherein the derivatization reagent is the above-mentioned diazo compound, or the diazo compound prepared by the above preparation method.
  • the derivatization treatment includes: preparing an acetonitrile solution of 20-100mM derivatization reagent, which is marked as liquid A; preparing an aqueous solution of 20-100mM hydroxylamine compound, which is marked as liquid B; and containing a small molecule carboxylic acid.
  • preparing an acetonitrile solution of 20-100mM derivatization reagent which is marked as liquid A
  • preparing an aqueous solution of 20-100mM hydroxylamine compound which is marked as liquid B
  • containing a small molecule carboxylic acid are examples of the sample with liquid A, and centrifuge it for 5 to 8 minutes at a centrifugal speed of 10,000 to 17,000 rpm and a temperature of 4 to 30°C.
  • the sample is subjected to a derivatization reaction to obtain a derivatized sample; the preferred derivatization reaction time is 10 to 60 minutes; preferably, the sample is plasma, serum, urine, tears, tissue fluid, cells, tissue homogenate, bacterial culture fluid, and blood spots. Or feces; preferably, the small molecule carboxylic acid is myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, arachidonic acid, lactic acid, pyruvic acid, fumaric acid, oxaloacetic acid , one or more of ⁇ -ketoglutaric acid, succinic acid, malic acid, citric acid or isocitric acid.
  • the preferred derivatization reaction time is 10 to 60 minutes; preferably, the sample is plasma, serum, urine, tears, tissue fluid, cells, tissue homogenate, bacterial culture fluid, and blood spots. Or feces; preferably, the small molecule carboxy
  • the mobile phase used in liquid chromatography analysis includes phase A and phase B, wherein phase A is a mixed solution of water and formic acid, and phase B is a mixed solution of acetonitrile and formic acid, and
  • the liquid chromatography elution program used in the liquid chromatography analysis process is a gradient elution program.
  • the liquid chromatography elution program includes the first equilibrium process, the first elution process, the second elution process and the second elution process in sequence.
  • VA is 70 ⁇ 80%
  • VB is 20 ⁇ 30%
  • the time of the first equilibrium process is 0 ⁇ 1 min
  • VA is from 70 ⁇ 80%.
  • VB is in the dynamic change process of gradually switching from 20 ⁇ 30% to 70 ⁇ 80%
  • the time of the first elution process is 2 ⁇ 5 minutes
  • the second wash During the elution process, VA is in a dynamic change process of gradually switching from 20 to 30% to 0 to 5%
  • V B is in a dynamic change process of gradually switching from 70 to 80% to 95 to 100%
  • the second elution process The time is 1 to 3 minutes; during the second equilibrium process, V A is in a dynamic change process from 0 to 5% to 70 to 80%, and V B is in a dynamic change process from 95 to 100% to 20 to 30%.
  • the time of the second balancing process is 1 ⁇ 1.5min; or, during the first balancing process, VA is 45 ⁇ 55%, VB is 45 ⁇ 55%, and the time of the first balancing process is 0 ⁇ 1min;
  • VA is in a dynamic change process of gradually switching from 45 to 55% to 0 to 5%
  • V B is in a dynamic change process of gradually switching from 45 to 55% to 95 to 100%
  • the first The time of the elution process is 3 to 4 minutes
  • V A is 0 to 5%
  • V B is 95 to 100%
  • the time of the second elution process is 2 to 4 minutes
  • the second equilibrium process V A is in a dynamic change process from 0 to 5% to 50 to 55%, V B is in a dynamic change process from 95 to 100% to 45 to 55%, and the time of the second equilibrium process is 1 to 1.5min.
  • the application includes in situ detection of metabolic products of living cells by using diazo compounds; preferably, the drugs include agonists and/or inhibitors; preferably, the metabolites are carboxylic acid metabolism in the tricarboxylic acid cycle Products;
  • the carboxylic acid metabolites in the tricarboxylic acid cycle refer to small molecule carboxylic acids with a molecular weight between 46 and 500; more preferably, the small molecule carboxylic acids are myristic acid, palmitic acid, palmitoleic acid, of stearic acid, oleic acid, linoleic acid, arachidonic acid, lactic acid, pyruvic acid, fumaric acid, oxaloacetic acid, alpha-ketoglutarate, succinic acid, malic acid, citric acid or isocitric acid
  • the above-mentioned detection method for quantitative analysis of small molecule carboxylic acids is used for in-situ detection.
  • a kit which includes the above-mentioned diazo compound, or the diazo compound prepared by the above-mentioned preparation method.
  • the present invention can more effectively achieve in-situ derivatization of cell samples, thereby shortening the processing process of biological samples and reducing the amount of small-molecule carboxylic acids in biological samples (such as life activities in the body or The degradation of small molecule carboxylic acid metabolites at any time point in metabolic activity, etc., during sample processing further improves detection accuracy.
  • Figure 1 shows the flow chart of the 96-well plate living cell metabolic flux measurement experiment using U-C13-glucose as the carbon source according to the present invention
  • Figure 2 shows a schematic diagram of the principle of the 96-well plate living cell metabolic flux measurement experiment using U-C13-glucose as the carbon source according to the present invention
  • Figure 3 shows a schematic diagram of the metabolic flow of tricarboxylic acid cycle substrates in neonatal rat cardiomyocytes treated simultaneously with rotenone, an inhibitor of mitochondrial respiratory chain complex I, and antimycin A, an inhibitor of complex III, according to the present invention
  • Figure 4 shows a schematic diagram of the metabolic flow of tricarboxylic acid cycle substrates in neonatal rat cardiomyocytes treated with oligomycin, an inhibitor of mitochondrial respiratory chain complex V, according to the present invention
  • FIG. 5 shows a schematic diagram of the metabolic flow of tricarboxylic acid cycle substrates in neonatal rat cardiomyocytes treated with the uncoupling agent carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP) according to the present invention
  • Figure 6 shows a schematic diagram of the absolute content of tricarboxylic acid cycle metabolites in normal human serum measured in one embodiment of the present invention
  • Figure 7 shows a LC/MS schematic diagram of the diazo compound in Example 1 of the present invention.
  • Figure 8 shows the LC/MS schematic diagram of the diazo compound in Example 2 of the present invention.
  • Figure 9 shows the secondary mass spectrum-chromatogram of the pyruvate standard sample (1nM) after derivatization with the diazo compound in Example 1 of the present invention
  • Figure 10 shows the secondary mass spectrometry-chromatogram of the ⁇ -ketoglutarate standard sample (1 nM) derivatized with the diazo compound in Example 1 of the present invention
  • Figure 11 shows the secondary mass spectrometry-chromatogram of the ⁇ -ketovaline standard sample (2nM) derivatized with the diazo compound in Example 1 of the present invention
  • Figure 12 shows the secondary mass spectrum-chromatogram of the succinic acid standard sample (5nM) derivatized with the diazo compound in Example 1 of the present invention
  • Figure 13 shows the secondary mass spectrometry-chromatogram of a lactic acid standard sample (5 nM) derivatized with the diazo compound in Example 1 of the present invention.
  • the present invention proposes a diazo compound.
  • the diazo compound has the structure shown in formula I, wherein R 1 represents H, alkyl, halogen, alkoxy or alkylamino; R 2 Represents aromatic group.
  • the inventors of the present invention creatively discovered that the above-mentioned diazo compounds can be used as derivatization reagents to derivatize the test sample containing small molecule carboxylic acids.
  • Treatment is to perform an esterification reaction between the diazo compound and the small molecule carboxylic acid in the test sample containing small molecule carboxylic acid) to obtain a derivatized sample.
  • the derivatized sample can significantly enhance the mass spectrum response of the small molecule carboxylic acid, improve the detection sensitivity, and thereby improve the detection accuracy.
  • the derivatized small molecule carboxylic acid does not require special chromatographic columns or special mobile phases. It can achieve the best separation effect in a shorter time at a lower cost and is more conducive to high-throughput samples. detection.
  • the present invention can more effectively achieve in-situ derivatization of cell samples, thereby shortening the processing process of biological samples and reducing the amount of small-molecule carboxylic acids in biological samples (such as in vivo
  • the degradation of small molecule carboxylic acid metabolites at any time point in life activities or metabolic activities, etc.) during sample processing further improves the detection accuracy.
  • R 1 represents H, C1-C6 alkyl, halogen, C1-C6 alkoxy or dimethylamino
  • R 2 represents methylenequinolyl or ethyl-N,N- Dimethylaniline group.
  • the diazo compound can be selected from the following compounds:
  • the invention also provides a method for preparing the above-mentioned diazo compounds.
  • the preparation method includes: performing an esterification reaction on the first dispersion liquid containing the phenylacetic acid compound and the alcohol compound to generate intermediate product A; Product A and the second dispersion of the diazo group transfer reagent undergo a diazotization reaction to generate diazo compounds; wherein, the phenylacetic acid compound has the formula
  • the alcohol compound has the structure shown by the formula R 2 -OH; R 1 represents H, alkyl, halogen, alkoxy or alkylamino; R 2 represents an aromatic group.
  • the intermediate product A has the formula The structure is shown and the synthesis route is as follows:
  • the present invention uses the diazo compounds obtained based on the above preparation method to detect characteristic metabolic small molecule carboxylic acids using liquid mass spectrometry technology as a derivatization reagent to detect small molecule carboxylic acids.
  • the sample to be tested is derivatized. After derivatization, the mass spectrum response of small molecule carboxylic acids can be significantly enhanced, the detection sensitivity can be improved, and the accuracy of detection can be improved.
  • the derivatized small molecule carboxylic acid does not need to be equipped with a special chromatographic column or a special mobile phase. It can achieve the best separation effect in a shorter time at a lower cost and is more conducive to high throughput. Sample testing with better detection accuracy.
  • the beneficial effects of in-situ derivatization of cell samples can be effectively achieved, thereby shortening the processing process of biological samples and reducing the loss of small molecule carboxylic acids in biological samples during sample processing. degradation, further improving detection accuracy.
  • the preparation method has a simpler operation process, the raw materials are more readily available, and the yield and purity of the obtained product (diazo compound) are higher.
  • those skilled in the art can directly perform an esterification reaction between phenylacetic acid compounds and alcohol compounds to generate the above-mentioned intermediate product A.
  • those skilled in the art can also first prepare the phenylacetic acid compound into an acid chloride compound, and then perform an esterification reaction between the acid chloride compound and the alcohol compound to generate the above-mentioned intermediate product A. Its synthesis route is as follows:
  • the reaction temperature is 0 to 30°C, for example, it can be 0°C, 5°C, 10°C, 15°C, 20°C, 25°C or 30°C;
  • the reaction time is 0.5 ⁇ 24h, for example, it can be 0.5h, 5h, 10h, 15h, 20h or 24h.
  • the reaction temperature is 0 to 30°C, for example, it can be 0°C, 5°C, 10°C, 15°C, 20°C, 25°C or 30°C;
  • the reaction time is 1 to 24h, and the reaction time is more preferably It is 10-24h, for example, it can be 10h, 15h, 20h or 24h.
  • the molar ratio of the phenylacetic acid compound and the alcohol compound is (0.5 ⁇ 2):1 , for example, it can be 0.5:1, 1:1, 1.5:1 or 2:1.
  • the molar ratio of intermediate product A and diazo group transfer reagent is (1-3):1, for example, it can be 1:1, 2:1 or 3:1.
  • the diazo group transfer reagent is 4-acetamidobenzenesulfonyl azide Nitrogen, p-toluenesulfonyl azide, 4-carboxybenzenesulfonyl azide, 1H-imidazole-1-sulfonyl azide hydrochloride or 2-azido-1,3-dimethylimidazole hexafluorophosphate of one or more.
  • the first dispersion liquid contains a first solvent, and the first solvent is one of dichloromethane, chloroform, N,N-dimethylformamide, tetrahydrofuran or diethyl ether, or Various.
  • the first dispersion also contains a first catalyst, and the first catalyst is triethylamine, N,N-diisopropylethylamine or an alkali metal carbonate (potassium carbonate, carbonate sodium, cesium carbonate, etc.).
  • the second dispersion liquid contains a second solvent, and the second solvent is acetonitrile and/or dimethyl sulfoxide.
  • the second dispersion also contains a second catalyst, and further preferably the second catalyst is 1,8-diazabicyclo[5.4.0]undec-7-ene, triethyl One or more of amine, sodium bicarbonate, sodium carbonate, potassium carbonate, potassium hydroxide or potassium acetate.
  • the synthetic route for the diazo compounds is as follows:
  • the synthetic route of diazo compounds is as follows:
  • the synthetic route of diazo compounds is as follows:
  • the synthetic route of diazo compounds (DQhB) is as follows:
  • phenylacetic acid (1 equivalent) can be dissolved in methylene chloride, and oxalyl chloride (1.1 equivalent) can be added dropwise at 0 to 5°C. and N,N-dimethylformamide (DMF) (0.01 equivalent), the reaction mixture was stirred at 20°C for 12 hours, and the crude reaction product was rotary evaporated under reduced pressure to obtain acid chloride compounds. Then dissolve quinolinol (1 equivalent) in dichloromethane, add triethylamine (2.5 equivalents), and slowly add the above acid chloride compound (1.1 equivalents) at low temperature. The reaction mixture is stirred at 4-30°C for 8-24 hours.
  • DMF N,N-dimethylformamide
  • the reaction mixture is stirred at 4-30°C for 8-24 hours.
  • the reaction solution was concentrated under reduced pressure and spun to dryness.
  • the crude product was purified by column chromatography to obtain the above-mentioned diazo compound.
  • those skilled in the art can use raw materials with different groups (phenylacetic acid compounds and alcohol compounds), and adopt the above preparation method to obtain the corresponding diazo compounds, which will not be described in detail here.
  • the invention also provides a detection method for quantitative analysis of small molecule carboxylic acid, which represents carboxylic acid with a molecular weight between 46 and 500.
  • the detection method includes derivatization treatment and liquid chromatography-mass spectrometry analysis.
  • Derivatization treatment includes: using derivatization reagents to derivatize the sample to obtain a derivatized sample.
  • Liquid chromatography-mass spectrometry analysis includes: performing liquid chromatography-mass spectrometry analysis on the derivatized sample to obtain a liquid chromatography-mass spectrum, and quantitatively analyzing the small molecule carboxylic acid components in the sample based on the liquid chromatography-mass spectrum; among which, derivatization
  • the chemical reagent is the above-mentioned diazo compound, or the diazo compound prepared by the above-mentioned preparation method.
  • the inventors of the present invention creatively discovered that based on the above diazo compounds as derivatization reagents, small molecule carboxylic acids containing small molecules can be detected.
  • the sample of acid to be tested is derivatized. After derivatization, the mass spectrum response of small molecule carboxylic acid can be significantly enhanced, the detection sensitivity is improved, and the detection accuracy is improved.
  • the derivatized small molecule carboxylic acid does not require special chromatographic columns or configuration.
  • the special mobile phase can achieve the best separation effect in a shorter time at a lower cost, which is more conducive to high-throughput sample detection.
  • the sample processing is simple and fast, suitable for detection by various types of liquid quality instruments, and can detect a variety of small molecule carboxylic acids in a short time ( ⁇ 30min) with high throughput.
  • the derivatization treatment includes: preparing an acetonitrile solution of 20-100mM derivatization reagent, which is recorded as liquid A; preparing an aqueous solution of 20-100mM hydroxylamine compound, which is recorded as liquid B; The sample containing small molecule carboxylic acid is mixed with liquid A, and centrifuged at a centrifugal speed of 10000 to 17000 rpm and a temperature of 4 to 30°C for 5 to 8 minutes.
  • the supernatant after centrifugation is mixed with liquid B and centrifuged at 50 to 80 Under temperature conditions of °C, the sample is subjected to a derivatization reaction (specifically, an esterification reaction) to obtain a derivatized sample; the preferred derivatization reaction time is 10 to 60 minutes. Based on this, the efficiency of the derivatization reaction is higher, and after the sample is derivatized through the above process, the mass spectrum response of the small molecule carboxylic acid metabolites can be further enhanced, and the detection sensitivity can be further improved, thereby improving the detection accuracy. At the same time, the best separation effect of each component in the sample to be tested can be achieved in a shorter time at a lower cost.
  • a derivatization reaction specifically, an esterification reaction
  • the sample can be plasma, serum, urine, tears, tissue fluid, cells, tissue homogenate (crushed), bacterial culture fluid, blood spots or feces and other types of organisms that can be ground and extracted. sample.
  • test objects of the above test method of the present invention can also be non-biological samples, such as environmental samples (such as carboxylic acids in water samples and air samples) and food samples (carboxylic acids in additives and preservatives), etc.
  • the mass spectrometry analysis process can use a triple quadrupole mass spectrometer with a source temperature of 150 ⁇ 160°C, cone voltage is 30 ⁇ 35kV, capillary voltage is 2 ⁇ 5kV, desolvation temperature is 400 ⁇ 450°C, cone gas flow is 20 ⁇ 25L/Hr, desolvation gas flow is 1000 ⁇ 1100L/Hr.
  • a source temperature of 150 ⁇ 160°C cone voltage is 30 ⁇ 35kV
  • capillary voltage is 2 ⁇ 5kV
  • desolvation temperature is 400 ⁇ 450°C
  • cone gas flow is 20 ⁇ 25L/Hr
  • desolvation gas flow is 1000 ⁇ 1100L/Hr.
  • the chromatographic column used in the liquid chromatography analysis process is Cortecs HSS T3100mm; the flow used
  • the phase includes phase A and phase B, where phase A is a mixed solution of water and formic acid, phase B is a mixed solution of acetonitrile and formic acid, and the liquid chromatography elution program used in the liquid chromatography analysis process is a gradient elution program.
  • the liquid chromatography elution procedure includes the first equilibrium process, the first elution process, the second elution process and the second equilibrium process in sequence; the volume of phase A is recorded as V A and the volume of phase B is recorded as V B , the flow rate of the liquid chromatography mobile phase is recorded as V n , and V n is 0.2 to 0.6 mL/min.
  • the volume ratio of water and formic acid is 200:(0.1 ⁇ 0.3);
  • the volume ratio of acetonitrile and formic acid is 200:(0.1 ⁇ 0.3 ).
  • phase A is a mixed solution of ultrapure water or filtered distilled water suitable for UPLC and chromatographically pure formic acid
  • phase B is a mixed solution of chromatographically pure anhydrous acetonitrile and chromatographically pure formic acid.
  • the optional needle washing conditions are: 90% ACN + 10% H 2 O, once every 5 to 10 minutes or injecting each needle. Before/after, 5 to 10 seconds each time; the optional column washing conditions are: after completing all sample tests, wash with H 2 O for 5 minutes, wash with CAN for 10 to 20 minutes, and the flow rate is 0.05 to 0.4mL/min.
  • the liquid chromatography elution procedure is as follows: During the first equilibrium process, VA is 70 ⁇ 80%, V B is 20-30%, and the time of the first equilibrium process is 0-1 min; during the first elution process, V A is in a dynamic change process of gradually switching from 70-80% to 20-30%.
  • V B is in a dynamic change process that gradually switches from 20 to 30% to 70 to 80%, and the first elution process takes 2 to 5 minutes; during the second elution process, V A is in a dynamic change process from 20 to 30% to 0 ⁇ 5% of the dynamic change process of gradual switching, V B is in the dynamic change process of gradually switching from 70 ⁇ 80% to 95 ⁇ 100%, and the time of the second elution process is 1 ⁇ 3 minutes; during the second equilibrium process, V A is in a dynamic change process that gradually switches from 0 to 5% to 70 to 80%, V B is in a dynamic change process that gradually switches from 95 to 100% to 20 to 30%, and the time of the second equilibrium process is 1 to 1.5 min.
  • test objects in the samples to be tested include, but are not limited to, one or more of lactic acid, pyruvic acid, fumaric acid, oxaloacetic acid, ⁇ -ketoglutarate, succinic acid, malic acid, citric acid or isocitric acid. kind.
  • the liquid chromatography elution procedure is as follows: during the first equilibrium process, VA is 45-55%, VB is 45-55%, and the time of the first equilibrium process is 0-55%. 1min; during the first elution process, VA is in a dynamic change process of gradually switching from 45 to 55% to 0 to 5%, V B is in a dynamic change process of gradually switching from 45 to 55% to 95 to 100%, and The time of the first elution process is 3 to 4 minutes; in the second elution process, VA is 0 to 5%, V B is 95 to 100%, and the time of the second elution process is 2 to 4 minutes; the second elution process is During the balancing process, V A is in a dynamic changing process from 0 to 5% to 50 to 55%, V B is in a dynamic changing process from 95 to 100% to 45 to 55%, and the time of the second balancing process is 1 ⁇ 1.5min.
  • test objects in the sample to be tested include fatty acids, including but not limited to one or more of myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid or arachidonic acid.
  • the invention also provides the application of the above-mentioned diazo compound or the diazo compound prepared by the above-mentioned preparation method in drug screening related to the functions of mitochondrial respiratory chain complexes.
  • the carboxylic acid metabolites in the tricarboxylic acid cycle refer to small-molecule carboxylic acids with molecular weights between 46 and 500; small-molecule carboxylic acids include myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid.
  • small-molecule carboxylic acids include myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid.
  • One or more of acid, arachidonic acid, lactic acid, pyruvic acid, fumaric acid, oxaloacetic acid, ⁇ -ketoglutarate, succinic acid, malic acid, citric acid or isocitric acid is used in the present invention.
  • FIG. 1 the flow chart of the 96-well plate living cell metabolic flux measurement experiment using U-C13-glucose as the carbon source is shown in Figure 1, and the schematic diagram of the principle is shown in Figure 2.
  • cells were labeled with U-C13-glucose as the carbon source, and the newly generated acetyl-auxiliary Enzyme A is labeled with M+2, and the citric acid-isocitric acid and downstream metabolites produced by using acetyl-coenzyme A as the entrance into the tricarboxylic acid cycle are labeled with M+2.
  • the accurate determination of the metabolic flux of unstable metabolites such as ⁇ -ketoglutarate and oxaloacetate in the metabolites of living cells can be greatly improved, especially through derivatization Reaction, in situ cells fix their initial metabolic state at the time point of response through chemical reactions, and the response is better, so that more stable and credible metabolic flow data can be obtained.
  • it is used in liquid mass spectrometry technology Higher sensitivity.
  • the present invention can effectively realize in-situ derivatization of cell samples, thereby shortening the processing process of biological samples and reducing the degradation of small molecule carboxylic acid metabolites during the sample processing process, thus Significantly improve the accuracy of detection results.
  • the above application includes screening drugs related to the functions of mitochondrial respiratory chain complexes through in situ detection of living cell metabolism, and the drugs include agonists and/or inhibitors.
  • the effects of compounds on oxidative phosphorylation and glycolysis can be determined, which can be used for drug screening related to the functions of mitochondrial respiratory chain complexes.
  • the drug to be studied was added to treat primary neonatal rat cardiomyocytes.
  • the labeling ratio is used to determine whether the drug promotes or inhibits oxidative phosphorylation and glycolysis.
  • This method can quickly and high-throughput screen drugs targeting mitochondrial respiratory function, which is crucial for the screening of mitochondria-related drugs.
  • Drugs that target mitochondria, whether they promote or inhibit mitochondrial respiratory function may become drugs. This needs to be determined based on the specific pathogenesis of the disease. For example, if the pathogenic mechanism of a certain disease is that the enzyme activity of a certain enzyme during aerobic respiration is too high, then you should look for inhibitors; if the pathogenic mechanism is that the enzyme activity during aerobic respiration is reduced, you should look for Activator.
  • the present invention provides a high-throughput screening solution for searching for potential mitochondrial respiration agonists or inhibitors, which is very beneficial for subsequent drug development.
  • suckling rat cardiomyocytes are treated with rotenone, an inhibitor of mitochondrial respiratory chain complex I, and antimycin A, an inhibitor of complex III, at the same time, except for succinic acid.
  • both significantly inhibited the metabolic flux of other tricarboxylic acid cycle substrates as shown in Figure 3.
  • treatment of neonatal rat cardiomyocytes with oligomycin, an inhibitor of mitochondrial respiratory chain complex V (ATPase) significantly inhibits the metabolic flux of oxaloacetate and isocitrate. Inhibits the metabolic flux of ⁇ -ketoglutarate, fumarate and malate to a certain extent, see As shown in Figure 4.
  • treatment of neonatal rat cardiomyocytes with the uncoupling agent carbonyl cyanide-4-trifluoromethoxyphenylhydrazone significantly enhanced oxaloacetate and ⁇ -ketoglutarate.
  • FCCP carbonyl cyanide-4-trifluoromethoxyphenylhydrazone
  • 0.5 to 2 mL of normal human blood is collected through venous blood collection and placed on ice; 2) Centrifuge at a centrifugation rate of 1500 to 4500 rpm and a temperature of 4 to 25°C for 10 to 30 minutes.
  • mitochondria are the main place where the tricarboxylic acid cycle occurs.
  • Mitochondrial diseases are diseases in which mitochondria function abnormally in multiple organs or tissues in the human body. Therefore, we believe that the tricarboxylic acid cycle metabolites in plasma can reflect mitochondrial function in humans. Based on this, those skilled in the art can use the tricarboxylic acid cycle metabolites in human serum measured by the above method to reflect the mitochondrial function in the human body.
  • the invention also provides a kit, which includes the above-mentioned diazo compound, or the diazo compound prepared by the above-mentioned preparation method.
  • the instructions for use of the above-mentioned kit are as follows: Collect 3 to 5 mL of blood from the subject through venous blood collection and place it on ice; 2) 1500 to 4500 rpm centrifugation rate, 4 to 25 Centrifuge for 10 to 30 minutes under temperature conditions of °C; 3) Collect the supernatant after centrifugation as plasma to be tested, and store it at -80 °C; 4) Prepare an acetonitrile solution of 20 to 100 mM derivatization reagent (the above-mentioned diazo compound) , record it as liquid A; prepare an aqueous solution of 20-25mM hydroxylamine hydrochloride, record it as liquid B; 5) Add 20-32 ⁇ L of liquid A to 5-8 ⁇ L of plasma to be tested, mix and centrifuge, and draw 20uL of supernatant solution, then add 20 to 60 ⁇ L of solution B, mix and heat at 70°C for 20 minutes, centrifuge and take
  • the dried material was purified by column chromatography to obtain quinoline p-toluene acetate (6.8 g).
  • the above-mentioned quinoline p-toluene acetate was dissolved in acetonitrile (45 ml), and p-ABSA (9.21 g, 2 equivalents) and DBU (10.7 g, 3 equivalents) were added successively at low temperature.
  • the reaction mixture was stirred at 4-8°C for 8-10 hours.
  • the reaction solution was concentrated under reduced pressure and spun to dryness.
  • the crude product was purified by column chromatography to obtain the target compound DQmB (5.8 g, purity 95.1%).
  • Liquid chromatography analysis can be performed according to the following process:
  • the chromatographic column used is Cortecs HSST3100mm; the mobile phase used includes phase A and phase B.
  • Phase A 200mL ultrapure water plus 0.1% (v/v) formic acid
  • phase B 200mL acetonitrile plus 0.1% (v/v)Formic acid.
  • test objects in the sample to be tested include, but are not limited to, one or more of lactic acid, pyruvic acid, fumaric acid, oxaloacetic acid, alpha-ketoglutarate, succinic acid, malic acid, citric acid or isocitric acid.
  • the gradient change curve is the built-in gradient change curve of Waters brand liquid chromatography-mass spectrometry instrument, which represents the speed of phase B change.
  • test object in the sample to be tested includes but is not limited to one or more of myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid or arachidonic acid, the elution conditions See table below:
  • FIG. 9 shows the secondary mass spectrometry-chromatogram of the pyruvate standard sample (1 nM) derivatized with the diazo compound in Example 1 of the present invention
  • Figure 10 shows the ⁇ -ketoglutarate standard sample (1 nM) Secondary mass spectrometry-chromatogram after derivatization with the diazo compound in Example 1 of the present invention
  • Figure 11 shows the ⁇ -ketovaline standard sample (2nM) after derivatization with the diazo compound in Example 1 of the present invention.
  • FIG. 12 shows the secondary mass spectrometry-chromatogram of the succinic acid standard sample (5nM) derivatized with the diazo compound in Example 1 of the present invention
  • Figure 13 shows the lactic acid standard sample (5nM)
  • the secondary mass spectrometry-chromatogram after derivatization of the diazo compound in Example 1 of the present invention is used. From Figures 9 to 13, it can be found that the signal-to-noise ratio of the secondary mass spectrum is 17, 43, 3, 8, and 17, which means that the detection limit of each acid is not higher than the concentration marked in the figure (which is 1nM, 1nM, 2nM, 5nM, 5nM).

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Abstract

本发明提供了一种重氮类化合物及其制备方法和应用。该重氮类化合物具有式(I)所示结构，其中，R1表示H、烷基、卤素、烷氧基或烷基氨基；R2表示芳香基。基于上述重氮类化合物作为衍生化试剂，衍生化处理后能够明显增强小分子羧酸的质谱响应，提高检测灵敏度，从而提高检测的准确性。而且，衍生化处理后的小分子羧酸，无需配置特殊的色谱柱、无需配置特殊的流动相，在更低成本基础上即可在更短时间内实现最佳分离效果，更利于高通量样品检测，且检测准确性更佳。尤其是，基于上述重氮类化合物作为衍生化试剂，能够有效地实现原位衍生细胞样品的有益效果。

Description

重氮类化合物及其制备方法和应用
本申请是以CN申请号为202210469035.4，申请日为2022年4月29日的中国申请为基础，并主张其优先权，该CN申请的公开内容再次作为整体引入本申请中。
技术领域
本发明涉及分析化学领域，具体而言，涉及一种重氮类化合物及其制备方法和应用。
背景技术
小分子羧酸代谢物的定量检测主要包括两条技术路线：一是酶促偶联技术，二是色谱-质谱联用技术。与酶促偶联技术相比，色谱-质谱联用技术的灵敏度高、通量大，可同时检测多种代谢物，极大地减低样品消耗量。
酶促偶联技术利用酶对小分子羧酸代谢物的特异性识别，将小分子羧酸代谢物浓度与酶促反应速率关联，最终通过特定波长光吸收的变化大小(也就是酶促反应速率快慢)来间接反映小分子羧酸代谢物的浓度高低。一般而言，酶促偶联技术定量检测生物样品中小分子羧酸代谢物的浓度范围在10～100μM之间。酶促偶联技术的特点是操作简便，步骤相对较少，对实验条件和人员要求不高，仪器易获得，实验基本可以在生物实验台上完成。
色谱-质谱联用技术结合了色谱分离和质谱定性定量的特点，是目前各行业公认的定性定量金标准仪器。色谱能够将样品中不同物质分离，具体表现为每一种物质具有独特的保留时间，协同质谱能够实时给出色谱洗脱组份的分子量信息，可用于辅助确定该洗脱组份的化学信息。其中，液相色谱是在生物、医药分析中最广泛使用的色谱技术，三重四极杆质谱和高分辨质谱是目前质谱技术的主流，前者灵敏度高、在已知化合物的定量方面具有优势，后者分辨率高、在未知化合物的定性方面具有优势。
液相色谱-质谱联用技术是定量检测小分子代谢物的常用技术。针对小分子羧酸代谢物，常用的液相色谱有反相色谱、亲水相互作用色谱、离子配对色谱。由于小分子羧酸代谢物亲水性强、极性接近，反相色谱无法有效区分，导致不同小分子羧酸代谢物共同洗脱、互相抑制电离。亲水相互作用色谱和离子配对色谱均能够分离不同小分子羧酸代谢物，但两者都需要特殊的流动相和色谱柱。亲水相互作用色谱需要特殊的亲水相互作用色谱柱和碱性流动相，离子配对色谱需要在流动相中添加正丁基胺等离子配对试剂，限制了与质谱技术进行联用，已基本被淘汰出液质联用范围。目前基于液质联用技术对特征代谢小分子羧酸进行检测主要有以下两种思路：
思路1，经典方法是将样品提取后直接进样。这一方法的优点是样品前处理简单，快捷，一般是1∶3的样品和有机溶剂混合后涡旋离心，核心是萃取所需的目标化合物并除去细胞膜，蛋白质等固体杂质。其缺点1，是在质谱负离子模式下，检测灵敏度不高，仅比酶联免疫法高出10倍左右，检出限在100nM～10μM之间。这是质谱负离子模式本身灵敏度所限，无法通过 优化方法进行更多改进。缺点2，直接检测法为了改进保留，会选用HILIC亲水色谱模式，其检测时间至少为20min以上，相比于常用的反相色谱耗时增加了2～3倍，不利于高通量测试。
思路2，对样品进行衍生化前处理，再进行质谱分析。属于这种思路的衍生方法有oBHA(”Derivatization of the tricarboxylic acid intermediates with O-benzylhydroxylamine for liquid chromatography-tandem mass spectrometry detection”，Analytical Biochemistry，2014.)等。然而，oBHA衍生方法有以下缺点：乳酸的检测限较高，不适用于痕量分析；不能用于96孔培养细胞的原位衍生；生物样本的衍生过程中需要数次萃取、旋干、复溶，操作时间长，容易引起代谢物的降解。
综上，现有技术中或存在质谱响应低、灵敏度低；或存在仪器分析时间长、不利于高通量样品检测；或存在需要特殊的色谱柱、增加分析成本；或存在生物样本的处理过程长、不稳定的小分子羧酸代谢物可能会在样本处理过程中降解；或存在需要配置特殊的流动相、增加分析成本等等的问题。故而，有必要提供一种新的衍生试剂，以改善上述问题。
发明内容
本发明的主要目的在于提供一种重氮类化合物及其制备方法和应用，以解决现有技术中在检测小分子羧酸，尤其是小分子羧酸代谢物时或存在质谱响应低、灵敏度低；或存在仪器分析时间长、不利于高通量样品检测；或存在需要特殊的色谱柱、增加分析成本；或存在生物样本的处理过程长、不稳定的小分子羧酸代谢物可能会在样本处理过程中降解；或存在需要配置特殊的流动相、增加分析成本等的问题。
为了实现上述目的，根据本发明的一个方面，提供了一种重氮类化合物，重氮类化合物具有式I所示结构，式I中，R1表示H、烷基、卤素、烷氧基或烷基氨基；R2表示芳香基。
进一步地，R1表示H、C1～C6烷基、卤素、C1～C6烷氧基或二甲基氨基；R2表示亚甲基喹啉基或乙基-N，N-二甲基苯胺基。
为了实现上述目的，根据本发明的一个方面，提供了一种上述的重氮类化合物的制备方法，制备方法包括：对包含苯乙酸类化合物和醇类化合物的第一分散液进行酯化反应，生成中间产物A；对包含中间产物A和重氮基转移试剂的第二分散液进行重氮化反应，生成重氮 类化合物；其中，苯乙酸类化合物具有所示结构，醇类化合物具有R2-OH所示结构；R1表示H、烷基、卤素、烷氧基或烷基氨基；R2表示芳香基。
进一步地，酯化反应过程中，反应温度为0～30℃，反应时间为0.5～24h；优选地，重氮化反应过程中，反应温度为0～30℃，反应时间为1～24h；优选地，酯化反应过程中，苯乙酸类化合物和醇类化合物的摩尔比为(0.5～2)∶1；优选地，重氮化反应过程中，中间产物A和重氮基转移试剂的摩尔比为(1～3)∶1；优选地，重氮化反应过程中，重氮基转移试剂为4-乙酰氨基苯磺酰叠氮、对甲苯磺酰叠氮、4-羧基苯磺酰叠氮、1H-咪唑-1-磺酰叠氮盐酸盐或2-叠氮-1，3-二甲基咪唑六氟磷酸盐中的一种或多种；优选地，第一分散液中包含有第一溶剂，第一溶剂为二氯甲烷、三氯甲烷、N，N-二甲基甲酰胺、四氢呋喃或乙醚中的一种或多种；优选地，第一分散液中还包含有第一催化剂，第一催化剂为三乙胺、N，N-二异丙基乙胺或碱金属碳酸盐中的一种或多种；优选地，第二分散液中包含有第二溶剂，第二溶剂为乙腈和/或二甲基亚砜；优选地，第二分散液中还包含有第二催化剂，进一步优选第二催化剂为1，8-二氮杂双环[5.4.0]十一碳-7-烯、三乙胺、碳酸氢钠、碳酸钠、碳酸钾、氢氧化钾或醋酸钾中的一种或多种。
根据本发明的另一方面，提供了一种定量分析小分子羧酸的检测方法，小分子羧酸表示分子量在46～500之间的羧酸，检测方法包括：衍生化处理：采用衍生化试剂对包含有小分子羧酸的样品进行衍生化处理，得到衍生化样品；液相色谱-质谱分析：对衍生化样品进行液相色谱-质谱分析，得到液相色谱-质谱图，并根据液相色谱-质谱图定量分析样品中的小分子羧酸成分；其中，衍生化试剂为上述的重氮类化合物，或者为上述的制备方法制备得到的重氮类化合物。
进一步地，衍生化处理包括：配制20～100mM衍生化试剂的乙腈溶液，将其记为A液；配制20～100mM羟胺类化合物的水溶液，将其记为B液；将包含有小分子羧酸的样品和A液混合，在10000～17000rpm离心转速、4～30℃温度条件下离心处理5～8min后，将离心处理后上清液和B液混合，在50～80℃温度条件下，使样品进行衍生化反应，得到衍生化样品；优选衍生化反应的时间为10～60min；优选地，样品为血浆、血清、尿液、泪液、组织液、细胞、组织匀浆、细菌培养液、血斑或粪便；优选地，小分子羧酸为肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、花生四烯酸、乳酸、丙酮酸、富马酸、草酰乙酸、α-酮戊二酸、琥珀酸、苹果酸、柠檬酸或异柠檬酸中的一种或多种。
进一步地，液相色谱-质谱分析过程中，液相色谱分析采用的流动相包含A相和B相，其中，A相为水和甲酸的混合溶液，B相为乙腈和甲酸的混合溶液，且液相色谱分析过程中采用的液相色谱洗脱程序为梯度洗脱程序，液相色谱洗脱程序包括顺次进行的第一平衡过程、第一洗脱过程、第二洗脱过程及第二平衡过程；将A相的体积记为VA、B相的体积记为VB，将液相色谱流动相的流速记为Vn，Vn为0.2～0.6mL/min；优选地，水和甲酸的混合溶液中，水和甲酸的体积比为200∶(0.1～0.3)；优选地，乙腈和甲酸的混合溶液中，乙腈和甲酸的体积比为200∶(0.1～0.3)。
进一步地，第一平衡过程中，VA为70～80％，VB为20～30％，且第一平衡过程的时间为0～1min；第一洗脱过程中，VA处于由70～80％向20～30％逐渐切换的动态变化过程，VB处于由20～30％向70～80％逐渐切换的动态变化过程，且第一洗脱过程的时间为2～5min；第二洗脱过程中，VA处于由20～30％向0～5％逐渐切换的动态变化过程，VB处于由70～80％向95～100％逐渐切换的动态变化过程，且第二洗脱过程的时间为1～3min；第二平衡过程中，VA处于由0～5％向70～80％切换的动态变化过程，VB处于由95～100％向20～30％切换的动态变化过程，且第二平衡过程的时间为1～1.5min；或者，第一平衡过程中，VA为45～55％，VB为45～55％，且第一平衡过程的时间为0～1min；第一洗脱过程中，VA处于由45～55％向0～5％逐渐切换的动态变化过程，VB处于由45～55％向95～100％逐渐切换的动态变化过程，且第一洗脱过程的时间为3～4min；第二洗脱过程中，VA为0～5％，VB为95～100％，且第二洗脱过程的时间为2～4min；第二平衡过程中，VA处于由0～5％向50～55％切换的动态变化过程，VB处于由95～100％向45～55％切换的动态变化过程，且第二平衡过程的时间为1～1.5min。
根据本发明的另一方面，提供了一种上述的重氮类化合物，或者上述的制备方法制备得到的重氮类化合物在线粒体呼吸链复合物功能相关的药物筛选中的应用。
进一步地，应用包括通过利用重氮类化合物对活细胞代谢产物进行原位检测；优选地，药物包括激动剂和/或抑制剂；优选地，代谢产物为三羧酸循环中的羧酸类代谢产物；优选地，三羧酸循环中的羧酸类代谢产物指分子量在46～500之间的小分子羧酸；更优选地，小分子羧酸为肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、花生四烯酸、乳酸、丙酮酸、富马酸、草酰乙酸、α-酮戊二酸、琥珀酸、苹果酸、柠檬酸或异柠檬酸中的一种或多种；更优选地，采用上述的定量分析小分子羧酸的检测方法进行原位检测。
根据本发明的另一方面，提供了一种试剂盒，试剂盒包括上述的重氮类化合物，或者上述的制备方法制备得到的重氮类化合物。
基于上述重氮类化合物作为衍生化试剂，本发明能够更有效地对细胞样品实现原位衍生，从而缩短了生物样本的处理过程，减少了生物样本中小分子羧酸(诸如生物体体内生命活动或者代谢活动中任意时间点的小分子羧酸代谢产物等等)在样本处理过程中的降解，进一步提高了检测准确性。
附图说明
构成本申请的一部分的说明书附图用来提供对本发明的进一步理解，本发明的示意性实施例及其说明用于解释本发明，并不构成对本发明的不当限定。在附图中：
图1示出了本发明以U-C13-葡萄糖为碳源的96孔板活细胞代谢流测定实验的流程图；
图2示出了本发明以U-C13-葡萄糖为碳源的96孔板活细胞代谢流测定实验的原理示意图；
图3示出了本发明以线粒体呼吸链复合物I的抑制剂鱼藤酮和复合物III的抑制剂抗霉素A同时处理乳鼠心肌细胞的三羧酸循环底物的代谢流示意图；
图4示出了本发明以线粒体呼吸链复合物V的抑制剂寡霉素处理乳鼠心肌细胞的三羧酸循环底物的代谢流示意图；
图5示出了本发明以解偶联剂碳酰氰-4-三氟甲氧基苯腙(FCCP)处理乳鼠心肌细胞的三羧酸循环底物的代谢流示意图；
图6示出了本发明一种实施例测得的正常人血清中三羧酸循环代谢物的绝对含量示意图；
图7示出了本发明实施例1中重氮化合物的LC/MS示意图；
图8示出了本发明实施例2中重氮化合物的LC/MS示意图；
图9示出了丙酮酸标准样品(1nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图；
图10示出了α-酮戊二酸标准样品(1nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图；
图11示出了α-酮基缬氨酸标准样品(2nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图；
图12示出了琥珀酸标准样品(5nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图；
图13示出了乳酸标准样品(5nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
正如本发明背景技术部分所描述的，现有技术中在检测小分子羧酸，尤其是小分子羧酸代谢物时存在或质谱响应低、灵敏度低；或仪器分析时间长、不利于高通量样品检测；或需要特殊的色谱柱、增加分析成本；或生物样本的处理过程长、不稳定的小分子羧酸代谢物可能会在样本处理过程中降解；或需要配置特殊的流动相、增加分析成本等的问题。为了解决这一问题，本发明提出了一种重氮类化合物，重氮类化合物具有式I所示结构，其中，R1表示H、烷基、卤素、烷氧基或烷基氨基；R2表示芳香基。
在应用液质联用技术对小分子羧酸进行定量分析时，本发明发明人创造性地发现，基于上述重氮类化合物作为衍生化试剂，以对包含小分子羧酸的待测样品进行衍生化处理(衍生化处理即为将该重氮类化合物和包含小分子羧酸的待测样品中的小分子羧酸进行酯化反应)，得到衍生化样品。这样，在后续检测过程中，衍生化处理后的样品能够明显增强其中小分子羧酸的质谱响应，提高检测灵敏度，进而提高检测准确性。而且，衍生化处理后的小分子羧酸，无需特殊的色谱柱、无需配置特殊的流动相，在更低成本基础上即可在更短时间内实现最佳分离效果，更利于高通量样品检测。
尤其是，基于上述重氮类化合物作为衍生化试剂，本发明能够更有效地对细胞样品实现原位衍生，从而缩短了生物样本的处理过程，减少了生物样本中小分子羧酸(诸如生物体体内生命活动或者代谢活动中任意时间点的小分子羧酸代谢产物等等)在样本处理过程中的降解，进一步提高了检测准确性。
在一些优选的实施方式中，R1表示H、C1～C6烷基、卤素、C1～C6烷氧基或二甲基氨基；R2表示亚甲基喹啉基或乙基-N，N-二甲基苯胺基。基于此，衍生化处理后的小分子羧酸的质谱响应更高、检测灵敏度更高、检测精准性更佳。
在一些可选实施方式中，重氮类化合物可选自以下化合物：
本发明还提供了一种上述的重氮类化合物的制备方法，该制备方法包括：对包含苯乙酸类化合物和醇类化合物的第一分散液进行酯化反应，生成中间产物A；对包含中间产物A和重氮基转移试剂的第二分散液进行重氮化反应，生成重氮类化合物；其中，苯乙酸类化合物具有式所示结构，醇类化合物具有式R2-OH所示结构；R1表示H、烷基、卤素、烷氧基或烷基氨基；R2表示芳香基。中间产物A具有式所示结构，合成路线如下所示：
基于前文的各项原因，本发明基于上述制备方法得到的重氮类化合物在应用液质联用技术对特征代谢小分子羧酸进行检测时，以其作为衍生化试剂，对包含小分子羧酸的待测样品进行衍生化处理，衍生化处理后能够明显增强小分子羧酸的质谱响应，提高检测灵敏度，从而提高检测的准确性。而且，衍生化处理后的小分子羧酸，无需配置特殊的色谱柱、无需配置特殊的流动相，在更低成本基础上即可在更短时间内实现最佳分离效果，更利于高通量样品检测，且检测准确性更佳。尤其是，基于上述重氮类化合物作为衍生化试剂，能够有效地实现原位衍生细胞样品的有益效果，从而缩短了生物样本的处理过程，减少了生物样本中小分子羧酸在样本处理过程中的降解，进一步提高了检测准确性。另外，该制备方法操作过程更简单，原材料更易得，得到的产物(重氮化合物)的收率和纯度均更高。
在一种可选的实施方式中，本领域技术人员可直接使苯乙酸类化合物和醇类化合物进行酯化反应，即可生成上述中间产物A。本领域技术人员也可以先把苯乙酸类化合物制备成酰氯类化合物，再将酰氯类化合物和醇类化合物进行酯化反应生成上述中间产物A。其合成路线如下所示：
为了进一步提高酯化反应和重氮化反应的反应稳定性，从而进一步提高产物的收率，在一种优选的实施方式中，酯化反应过程中，反应温度为0～30℃，例如可以为0℃、5℃、10℃、15℃、20℃、25℃或30℃；反应时间为0.5～24h，例如可以为0.5h、5h、10h、15h、20h或24h。 重氮化反应过程中，反应温度为0～30℃，如可以为0℃、5℃、10℃、15℃、20℃、25℃或30℃；反应时间为1～24h，更优选反应时间为10～24h，例如可以为10h、15h、20h或24h。
为了进一步提高酯化反应和重氮化反应的产物收率，在一种优选的实施方式中，酯化反应过程中，苯乙酸类化合物和醇类化合物的摩尔比为(0.5～2)∶1，例如可以为0.5∶1、1∶1、1.5∶1或2∶1。重氮化反应过程中，中间产物A和重氮基转移试剂的摩尔比为(1～3)∶1，例如可以为1∶1、2∶1或3∶1。
为了进一步高效地提高酯化反应和重氮化反应的产物收率和纯度，在一种优选的实施方式中，重氮化反应过程中，重氮基转移试剂为4-乙酰氨基苯磺酰叠氮、对甲苯磺酰叠氮、4-羧基苯磺酰叠氮、1H-咪唑-1-磺酰叠氮盐酸盐或2-叠氮-1，3-二甲基咪唑六氟磷酸盐中的一种或多种。在一种优选的实施方式中，第一分散液中包含有第一溶剂，第一溶剂为二氯甲烷、三氯甲烷、N，N-二甲基甲酰胺、四氢呋喃或乙醚中的一种或多种。在一种优选的实施方式中，第一分散液中还包含有第一催化剂，第一催化剂为三乙胺、N，N-二异丙基乙胺或碱金属碳酸盐(碳酸钾、碳酸钠、碳酸铯等)中的一种或多种。在一种优选的实施方式中，第二分散液中包含有第二溶剂，第二溶剂为乙腈和/或二甲基亚砜。在一种优选的实施方式中，第二分散液中还包含有第二催化剂，进一步优选第二催化剂为1，8-二氮杂双环[5.4.0]十一碳-7-烯、三乙胺、碳酸氢钠、碳酸钠、碳酸钾、氢氧化钾或醋酸钾中的一种或多种。
在此可列举一些上述重氮类化合物的合成路线，诸如，在本发明一些实施方式中，重氮类化合物(DQclB)的合成路线如下所示：
重氮类化合物(DQmoB)的合成路线如下所示：
重氮类化合物(DQdmaB)的合成路线如下所示：
重氮类化合物(DQhB)的合成路线如下所示：
具体地，在本发明一种可选的实施方式中，以反应原料苯乙酸为例，可将苯乙酸(1当量)溶解于二氯甲烷，在0～5℃滴加草酰氯(1.1当量)和N，N-二甲基甲酰胺(DMF)(0.01当量)，反应混合物在20℃下搅拌12小时，将反应粗产物减压旋蒸即得到酰氯类化合物。再将喹啉醇(1当量)溶解于二氯甲烷，加入三乙胺(2.5当量)，低温下缓慢加入上述酰氯类化合物(1.1当量)。反应混合物在4～30℃下搅拌8～24小时。将反应液倒入纯水中，分离水相，用二氯甲烷依次萃取两遍。合并有机相，以无水硫酸钠干燥，过滤，减压旋干。旋干物以柱层析色谱纯化，得到对氯苯乙酸喹啉酯。将上述对氯苯乙酸喹啉酯溶解于乙腈中，在低温下依次加入4-乙酰氨基苯磺酰叠氮(p-ABSA)(2当量)和1，8-二氮杂双环[5.4.0]十一碳-7-烯(DBU)(3当量)。反应混合物在4～30℃下搅拌8～24小时。减压浓缩、旋干反应液，粗产物以柱层析色谱纯化，得到上述重氮类化合物。在实际应用中，本领域技术人员可采用具有不同基团的原料(苯乙酸类化合物和醇类化合物)，采用上述制备方法，均可以得到相应的重氮类化合物，在此不多赘述。
本发明还提供了一种定量分析小分子羧酸的检测方法，小分子羧酸表示分子量在46～500之间的羧酸。该检测方法包括衍生化处理及液相色谱-质谱分析。衍生化处理包括：采用衍生化试剂对样品进行衍生化处理，得到衍生化样品。液相色谱-质谱分析包括：对衍生化样品进行液相色谱-质谱分析，得到液相色谱-质谱图，并根据液相色谱-质谱图定量分析样品中的小分子羧酸成分；其中，衍生化试剂为上述的重氮类化合物，或者为上述的制备方法制备得到的重氮类化合物。
基于前文的各项原因，在应用液质联用技术对特征代谢小分子羧酸进行检测时，本发明发明人创造性地发现，基于上述重氮类化合物作为衍生化试剂，以对包含小分子羧酸的待测样品进行衍生化处理，衍生化处理后能够明显增强小分子羧酸的质谱响应，提高检测灵敏度，从而提高检测准确性。而且，衍生化处理后的小分子羧酸，无需特殊的色谱柱、无需配置特 殊的流动相，在更低成本基础上即可在更短时间内实现最佳分离效果，更利于高通量样品检测。样品处理简单快速，适用多种型号液质仪器检测，可短时间(＜30min)高通量检测多种小分子羧酸。
具体地，对上述衍生化处理后的小分子羧酸进行液相色谱-质谱分析，得到液相色谱-质谱图，并使用商业化软件对液相色谱-质谱图中峰进行积分，通过积分面积来定量分析样品中的小分子羧酸，这是本领域技术人员可以根据公知自行实现的，在此不多赘述。
在一种优选的实施方式中，衍生化处理包括：配制20～100mM衍生化试剂的乙腈溶液，将其记为A液；配制20～100mM羟胺类化合物的水溶液，将其记为B液；将包含有小分子羧酸的样品和A液混合，在10000～17000rpm离心转速、4～30℃温度条件下离心处理5～8min后，将离心处理后上清液和B液混合，在50～80℃温度条件下，使样品进行衍生化反应(具体为酯化反应)，得到衍生化样品；优选衍生化反应的时间为10～60min。基于此，衍生化反应的效率更高，且样品经过上述过程衍生化处理后能够进一步增强小分子羧酸代谢物的质谱响应，同时可以进一步提高检测灵敏度，从而提高检测精确性。同时，在更低成本基础上即可在更短时间内实现待测样品中各组分最佳分离效果。
在一些可选的实施方式中，样品可以为血浆、血清、尿液、泪液、组织液、细胞、组织匀浆(碎)、细菌培养液、血斑或粪便等其他可研磨浸提的各类生物样品。当然，本发明上述测试方法的测试对象也可以为非生物样品，如环境样品(如水样和空气样的羧酸)和食品样品(添加剂、防腐剂中的羧酸)等。
为了进一步提高检测过程稳定性，同时进一步提高检测精确度，在一种优选的实施方式中，液相色谱-质谱分析过程中，质谱分析过程可采用三重四级杆质谱仪、源温度为150～160℃、锥孔电压为30～35kV、毛细管电压为2～5kV、脱溶剂温度为400～450℃、锥孔气体流量为20～25L/Hr、脱溶剂气体流量为1000～1100L/Hr。额外说明的是，以上为Waters Xevo TQ-S micro质谱仪测试条件，本领域技术人员在使用其他质谱仪时可根据实际经验对相应参数调整优化。
为了进一步提高检测过程稳定性，同时进一步提高检测精确度，在一种优选的实施方式中，液相色谱-质谱分析过程中，液相色谱分析过程采用的色谱柱为Cortecs HSS T3100mm；采用的流动相包含A相和B相，其中，A相为水和甲酸的混合溶液，B相为乙腈和甲酸的混合溶液，且液相色谱分析过程中采用的液相色谱洗脱程序为梯度洗脱程序，液相色谱洗脱程序包括顺次进行的第一平衡过程、第一洗脱过程、第二洗脱过程及第二平衡过程；将A相的体积记为VA、B相的体积记为VB，将液相色谱流动相的流速记为Vn，Vn为0.2～0.6mL/min。优选地，水和甲酸的混合溶液中，水和甲酸的体积比为200∶(0.1～0.3)；优选地，乙腈和甲酸的混合溶液中，乙腈和甲酸的体积比为200∶(0.1～0.3)。更优选地，A相为超纯水或经过滤适用于UPLC的蒸馏水和色谱纯甲酸的混合溶液，B相为色谱纯无水乙腈和色谱纯甲酸的混合溶液。额外说明的是，以上为Waters ACQUITY UPLC I-Class液相色谱分析仪测试条件，本领域技术人员在使用其他液相色谱分析仪时可根据实际经验对相应参数调整优化。
考虑到衍生剂的残留问题，出于维护仪器的目的，在一种优选的实施方式中，可选洗针条件为：90％ACN+10％H2O，5～10min一次或每针进样前/后，每次5～10s；可选洗柱条件为：完成所有样品测试之后，H2O洗5min，CAN洗10～20min，流速为0.05～0.4mL/min。
在本发明一种实施方式中，在测试生物体体内生命活动或者代谢活动中任意时间点的小分子羧酸物质时，液相色谱洗脱程序如下：第一平衡过程中，VA为70～80％，VB为20～30％，且第一平衡过程的时间为0～1min；第一洗脱过程中，VA处于由70～80％向20～30％逐渐切换的动态变化过程，VB处于由20～30％向70～80％逐渐切换的动态变化过程，且第一洗脱过程的时间为2～5min；第二洗脱过程中，VA处于由20～30％向0～5％逐渐切换的动态变化过程，VB处于由70～80％向95～100％逐渐切换的动态变化过程，且第二洗脱过程的时间为1～3min；第二平衡过程中，VA处于由0～5％向70～80％逐渐切换的动态变化过程，VB处于由95～100％向20～30％逐渐切换的动态变化过程，且第二平衡过程的时间为1～1.5min。其待测样品中的测试对象包括但不限于乳酸、丙酮酸、富马酸、草酰乙酸、α-酮戊二酸、琥珀酸、苹果酸、柠檬酸或异柠檬酸中的一种或多种。
在另一种优选的实施方式中，液相色谱洗脱程序如下：第一平衡过程中，VA为45～55％，VB为45～55％，且第一平衡过程的时间为0～1min；第一洗脱过程中，VA处于由45～55％向0～5％逐渐切换的动态变化过程，VB处于由45～55％向95～100％逐渐切换的动态变化过程，且第一洗脱过程的时间为3～4min；第二洗脱过程中，VA为0～5％，VB为95～100％，且第二洗脱过程的时间为2～4min；第二平衡过程中，VA处于由0～5％向50～55％切换的动态变化过程，VB处于由95～100％向45～55％切换的动态变化过程，且第二平衡过程的时间为1～1.5min。其待测样品中的测试对象包括脂肪酸，脂肪酸包括但不限于肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸或花生四烯酸中的一种或多种。
本发明还提供了一种上述的重氮类化合物，或者上述的制备方法制备得到的重氮类化合物在线粒体呼吸链复合物功能相关的药物筛选中的应用。
三羧酸循环中的羧酸类代谢产物指分子量在46～500之间的小分子羧酸；小分子羧酸为肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、花生四烯酸、乳酸、丙酮酸、富马酸、草酰乙酸、α-酮戊二酸、琥珀酸、苹果酸、柠檬酸或异柠檬酸中的一种或多种在本发明的一种实施方式中，应用包括通过利用重氮类化合物对活细胞代谢产物进行原位检测，其检测包括以下步骤：1)在96孔细胞板中接种1×104原代乳鼠心肌细胞，细胞贴壁，培养1天后，换新鲜的培养基培养2～3天；弃去96孔板中乳鼠心肌细胞的培养基，以无底物培养基洗涤细胞2次以上；2)以含有20mM U-C13-葡萄糖为底物的DMEM培养基，分别标记原代乳鼠心肌细胞0，5，10，30，60min；3)弃掉上述含有U-C13-葡萄糖的DMEM培养基，在96孔板的每个孔加入15～30μL前述A液∶B液(v/v)＝1∶1的混合物，充分覆盖原代乳鼠心肌细胞的表面，70℃温度条件下反应20min后，取反应体系上清液，对其进行液相色谱-质谱分析，计算M+2和M+0的比率，从而得到代谢物的标记信息和流经不同分支途径的通量比例。
额外说明的是，以U-C13-葡萄糖为碳源的96孔板活细胞代谢流测定实验的流程图如图1所示、原理示意图如图2所示。具体地，以U-C13-葡萄糖为碳源标记细胞，新生成的乙酰辅 酶A带有M+2标记，以乙酰辅酶A为入口进入三羧酸循环生成的柠檬酸-异柠檬酸及下游代谢物均带有M+2标记。分别计算关键的代谢物(诸如(异)柠檬酸、丙酮酸、富马酸、草酰乙酸、α-酮戊二酸、琥珀酸、苹果酸及乳酸)的无标记形式M+0和标记形式M+2的比率，即可根据中间代谢物的标记信息计算流经不同分支途径的通量比例。其中，在没有加入U-C13-葡萄糖的情况下，所有羧酸代谢物中C13的丰度非常低，几乎都是C12，此时记为M+0；加入U-C13-葡萄糖后，葡萄糖分解后进入三羧酸循环，由于每次是以丙酮酸(含有两个C13原子)的形式进入三羧酸循环，所以下游的羧酸代谢产物的C12原子会逐渐被C13取代，此时质量会增加2，所以记为M+2。故而，根据M+2和M+0的比例随时间的变化快慢，即可推测出细胞三羧酸循环过程中每一步的速率变化(即酶活)，进而可以推测出某化合物抑制或激活了三羧酸循环过程中的哪一步。
基于本发明上述重氮类化合物作为衍生化试剂，可以极大地改善活细胞代谢产物中的α-酮戊二酸和草酰乙酸等不稳定代谢物的代谢流的精确测定，特别是通过衍生化反应，在原位细胞通过化学反应将其初始代谢状态固定在响应的时间点，且响应性更佳从而可以获得更稳定、更可信的代谢流数据，同时，其在液质联用技术中灵敏度更高。尤其是，基于本发明上述衍生化试剂，在少量细胞中即可得到较为可靠的结果，大大减少了代谢流实验中同位素标记的成本(同位素标记的葡萄糖和脂肪酸都比较昂贵)。另外，基于上述重氮类化合物作为衍生化试剂，本发明可有效实现原位衍生细胞样品，从而缩短了生物样本的处理过程，减少了小分子羧酸代谢物在样本处理过程中的降解，从而显著改善检测结果精确性。
在本发明的一种实施方式中，上述应用包括通过活细胞代谢原位检测筛选与线粒体呼吸链复合物功能相关的药物，药物包括激动剂和/或抑制剂。基于上述活细胞代谢原位检测可判断化合物对氧化磷酸化和糖酵解过程的影响，从而用于线粒体呼吸链复合物功能相关的药物筛选。具体地，在使用含有U-C13-葡萄糖为底物的DMEM培养基培养细胞前，加入待研究的药物处理原代乳鼠心肌细胞，可根据不同时间点细胞内三羧酸循环代谢产物的同位素标记比例来判断该药物对氧化磷酸化和糖酵解有无促进或抑制作用。
本方法能够快速、高通量地筛选靶向线粒体呼吸功能的药物，这对于线粒体相关药物的筛选至关重要。以线粒体为靶点的药物，无论是促进还是抑制线粒体呼吸功能，都有可能成药，这需要根据疾病的具体发病机理来决定。例如，某种疾病的致病机理是有氧呼吸过程中某种酶的酶活过高所致，那么就应该寻找抑制剂；如果致病机理是有氧呼吸中的酶活降低，就应该寻找激活剂。本发明为寻找潜在的线粒体呼吸激动剂或抑制剂提供了高通量筛选方案，对于后续药物开发是非常有益的。
例如，在本发明的一些实施方式中，以线粒体呼吸链复合物I的抑制剂鱼藤酮(Rotenone)和复合物III的抑制剂抗霉素A(Antimycin A)同时处理乳鼠心肌细胞，除琥珀酸外，二者均显著抑制其他三羧酸循环底物的代谢流，见图3所示。在本发明的一种实施方式中，以线粒体呼吸链复合物V(ATPase)的抑制剂寡霉素(oligomycin)处理乳鼠心肌细胞，则显著抑制草酰乙酸和异柠檬酸的代谢流，一定程度地抑制α-酮戊二酸，富马酸和苹果酸的代谢流，见 图4所示。在本发明的一种实施方式中，以解偶联剂碳酰氰-4-三氟甲氧基苯腙(FCCP)处理乳鼠心肌细胞，则显著增强草酰乙酸，α-酮戊二酸和富马酸的代谢流，见图5所示。
需说明的是，衍生化处理与标记速率(或标记比例变化快慢)的相关性如下：原位衍生可直接将代谢物固定于试剂加入的时刻，无需分步骤进行细胞代谢终止，也无需细胞收集和提取过程，理论上能最大程度减少不稳定代谢产物的降解，使标记速率的测定更为可靠。灵敏度的显著提升使得实施方式中只需要培养少量(如96孔板)细胞，在减少细胞用量的同时也同比例减少了培养细胞过程中昂贵同位素标记营养物质(如此处的U-C13-葡萄糖)和待研究药物的加入，极大地降低了研究成本。
在本发明一种实施方式中，通过静脉采血的方式分别采集0.5～2mL的正常人的血液，置于冰上；2)1500～4500rpm离心速率、4～25℃温度条件下离心处理10～30min；3)收集离心处理后上清液作为待检测血浆，储存于-20～-80℃；4)配制20～25mM衍生化试剂的乙腈溶液，将其记为A液；配制20～25mM盐酸羟胺的水溶液，将其记为B液；5)向5～8μL待检测血浆中加入20～32μL A液(4倍待检测血浆体积)，混匀后离心，吸取20uL上清液，再加入20～60μL B液，混匀后70℃加热20min，离心处理后取上清液进行上述液相色谱-质谱分析。使用上述方法测得的正常人血清中三羧酸循环代谢物的绝对含量见图6所示，HMDB(Human Metabolite Database)公开数据库中正常人血清中三羧酸循环代谢物的绝对含量数据汇总见下表所示。

对比图6和上述表格中绝对含量数据汇总表，可以发现通过本发明上述检测方法，测得的上述羧酸含量是合理、准确的。其中，公开数据库中虽然没有草酰乙酸的含量数据，但从测得数据的数量级来看，微摩尔每升的浓度也是相对合理的。
通常，线粒体是三羧酸循环进行的主要场所，线粒体疾病是人体内多个器官或组织中线粒体功能异常的疾病。因此，我们认为，血浆中三羧酸循环代谢产物可以反映出人体内线粒体功能。基于此，本领域技术人员可使用上述方法测得的人体血清中三羧酸循环代谢物，以反映出人体内线粒体功能。
本发明还提供了一种试剂盒，试剂盒包括上述的重氮类化合物，或者上述的制备方法制备得到的重氮类化合物。
在本发明一种实施方式中，上述试剂盒的使用说明书如下：通过静脉采血的方式分别采集3～5mL的待测者的血液，置于冰上；2)1500～4500rpm离心速率、4～25℃温度条件下离心处理10～30min；3)收集离心处理后上清液作为待检测血浆，储存于-80℃；4)配制20～100mM衍生化试剂(上述的重氮类化合物)的乙腈溶液，将其记为A液；配制20～25mM盐酸羟胺的水溶液，将其记为B液；5)向5～8μL待检测血浆中加入20～32μL A液，混匀后离心，吸取20uL上清液，再加入20～60μL B液，混匀后70℃加热20min，离心处理后取上清液进行上述液相色谱-质谱分析。基于此，本发明可对待测者进行快速初筛，为后续临床样本的检测与临床疾病的诊断、疗效评价和预后评估提供更稳定可靠、快速便捷、低成本的方法和途径。
以下结合具体实施例对本申请作进一步详细描述，这些实施例不能理解为限制本申请所要求保护的范围。
实施例1
DQmB的制备
将喹啉醇(6克，1当量)溶解于二氯甲烷(36毫升)，加入三乙胺(13.1毫升，2.5当量)，低温下缓慢加入对甲基苯乙酰氯(6.99克，1.1当量)。反应混合物在5-10℃下搅拌8-12小时。将反应液倒入纯水(30毫升)中，分离水相，用二氯甲烷(15毫升，10毫升)依次萃取两遍。合并有机相，以无水硫酸钠干燥，过滤，减压旋干。旋干物以柱层析色谱纯化，得到对甲基苯乙酸喹啉酯(6.8克)。将上述对甲基苯乙酸喹啉酯溶解于乙腈(45毫升)中，在低温下依次加入p-ABSA(9.21克，2当量)和DBU(10.7克，3当量)。反应混合物在4-8℃下搅拌8-10小时。减压浓缩、旋干反应液，粗产物以柱层析色谱纯化，得到目标化合物DQmB(5.8克，纯度95.1％)。产品核磁：
1HNMR(400MHz，CDCl3)：δ8.94(dd，J＝1.6，4.4Hz，1H)，8.16-8.12(m，2H)，7.84(d，J＝0.8Hz，1H)，7.75(dd，J＝2.0，4.0Hz，1H)，7.44-7.37(m，3H)，7.21(d，J＝8.0Hz，2H)，5.49(s，2H)，2.35(s，3H).13CNMR(101M Hz，CDCl3)：δ165.1，150.8，148.0，136.1，135.9，134.2，130.0，129.7，129.2，128.0，127.0，124.2，121.8，121.4，66.0，21.0。
LC/MS见图7所示。
实施例2
DDAB的制备
将对(N，N-二甲基)氨基苯乙醇溶解于二氯甲烷中，在低温下依次加入三乙胺(2当量)、苯乙酰氯(1.1当量)。反应混合物在5-10℃下搅拌8-12小时。将反应液倒入纯水中，分离水相，用二氯甲烷依次萃取两遍。合并有机相，以无水硫酸钠干燥，过滤，减压旋干。旋干物以柱层析色谱纯化，得到苯乙酸(N，N-二甲基)氨基苯乙酯。将上述苯乙酸酯溶解于乙腈中，在低温下依次加入TsN3(2当量)和DBU(3当量)。反应混合物在4-8℃下搅拌8-10小时。减压浓缩、旋干反应液，粗产物以柱层析色谱纯化，得到目标化合物DDAB。产品核磁：
1HNMR(400MHz，CDCl3)：δ7.48-7.46(d，J＝7.6Hz，2H)，7.40-7.37(m，2H)，7.21-7.19(m，1H)，7.13(d，J＝8.4Hz，2H)，6.73(d，J＝8.4Hz，2H)，4.43(t，J＝7.2Hz，2H)，2.95-2.92(m，8H).13CNMR(101MHz，CDCl3)：δ165.1.149.5，129.6，128.9，125.7，125.6，125.4，124.0，112.9，66.0，40.7，34.3.
LC/MS见图8所示。
液相色谱分析可按照如下过程进行：
配制20mMDQmB的乙腈溶液(A液)和20mM盐酸羟胺的水溶液(B液)。
取5uL小鼠血浆于200uL离心管，加入4倍体积的A液，振荡混匀，12000rpm4℃离心5min，取上清液于200uL离心管，加入2倍体积B液，70℃加热反应20min，上样前12000rpm、4℃、离心5min。
液相色谱分析过程中：采用的色谱柱为CortecsHSST3100mm；采用的流动相包含A相和B相，A相：200mL超纯水加0.1％(v/v)甲酸；B相：200mL乙腈加0.1％(v/v)甲酸。色谱柱：CortecsHSST3100mm。
待测样品中的测试对象包括但不限于乳酸、丙酮酸、富马酸、草酰乙酸、α-酮戊二酸、琥珀酸、苹果酸、柠檬酸或异柠檬酸中的一种或多种时，洗脱条件见下表：
注：梯度变化曲线是沃特世品牌的液相色谱-质谱仪器内置的梯度变化曲线，表示的是B相变化的快慢。一共有11种曲线，以数字1-11标记，其中6号是直线，即B相比例随时间均匀变化；1-5号曲线代表变化先快后慢，其中1号曲线几乎瞬间变化到目的比例；7-11曲线代表变化先慢后快，其中11号曲线刚开始时变化极其缓慢，后段变化极快。
质谱分析过程中：采用三重四级杆质谱仪。
质谱参数见下表：
质谱测定母离子、子离子和碰撞能量的信息见下表。

其待测样品中的测试对象包括但不限于肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸或花生四烯酸中的一种或多种时，洗脱条件见下表：
质谱测定母离子、子离子和碰撞能量的信息见下表。

灵敏度表征：
质谱信噪比S/N为3.3时的酸浓度即为检测限。其中，图9示出了丙酮酸标准样品(1nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图；图10示出了α-酮戊二酸标准样品(1nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图；图11示出了α-酮基缬氨酸标准样品(2nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图；图12示出了琥珀酸标准样品(5nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图；图13示出了乳酸标准样品(5nM)采用本发明实施例1中重氮化合物衍生后的二级质谱-色谱图。从图9至13可以发现，二级质谱的信噪比依次是17、43、3、8、17，这意味着各酸的检测限不高于图中标注浓度(其依次是1nM、1nM、2nM、5nM、5nM)。
以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (11)

  1. 一种重氮类化合物，其特征在于，所述重氮类化合物具有式I所示结构：
    所述式I中，R1表示H、烷基、卤素、烷氧基或烷基氨基；R2表示芳香基。
  2. 根据权利要求1所述的重氮类化合物，其特征在于，所述R1表示H、C1～C6烷基、卤素、C1～C6烷氧基或二甲基氨基；R2表示亚甲基喹啉基或乙基-N，N-二甲基苯胺基。
  3. 一种权利要求1或2所述的重氮类化合物的制备方法，其特征在于，所述制备方法包括：
    对包含苯乙酸类化合物和醇类化合物的第一分散液进行酯化反应，生成中间产物A；
    对包含所述中间产物A和重氮基转移试剂的第二分散液进行重氮化反应，生成所述重氮类化合物；
    其中，所述苯乙酸类化合物具有所示结构，所述醇类化合物具有R2-OH所示结构；R1表示H、烷基、卤素、烷氧基或烷基氨基；R2表示芳香基。
  4. 根据权利要求3所述的制备方法，其特征在于，所述酯化反应过程中，反应温度为0～30℃，反应时间为0.5～24h；
    优选地，所述重氮化反应过程中，反应温度为0～30℃，反应时间为1～24h；
    优选地，所述酯化反应过程中，所述苯乙酸类化合物和所述醇类化合物的摩尔比为(0.5～2)∶1；
    优选地，所述重氮化反应过程中，所述中间产物A和所述重氮基转移试剂的摩尔比为(1～3)∶1；
    优选地，所述重氮化反应过程中，所述重氮基转移试剂为4-乙酰氨基苯磺酰叠氮、对甲苯磺酰叠氮、4-羧基苯磺酰叠氮、1H-咪唑-1-磺酰叠氮盐酸盐或2-叠氮-1，3-二甲基咪唑六氟磷酸盐中的一种或多种；
    优选地，所述第一分散液中包含有第一溶剂，所述第一溶剂为二氯甲烷、三氯甲烷、N，N-二甲基甲酰胺、四氢呋喃或乙醚中的一种或多种；
    优选地，所述第一分散液中还包含有第一催化剂，所述第一催化剂为三乙胺、N，N- 二异丙基乙胺或碱金属碳酸盐中的一种或多种；
    优选地，所述第二分散液中包含有第二溶剂，所述第二溶剂为乙腈和/或二甲基亚砜；
    优选地，所述第二分散液中还包含有第二催化剂，进一步优选所述第二催化剂为1，8-二氮杂双环[5.4.0]十一碳-7-烯、三乙胺、碳酸氢钠、碳酸钠、碳酸钾、氢氧化钾或醋酸钾中的一种或多种。
  5. 一种定量分析小分子羧酸的检测方法，所述小分子羧酸表示分子量在46～500之间的羧酸，其特征在于，所述检测方法包括：
    衍生化处理：采用衍生化试剂对包含有所述小分子羧酸的样品进行衍生化处理，得到衍生化样品；
    液相色谱-质谱分析：对所述衍生化样品进行液相色谱-质谱分析，得到液相色谱-质谱图，并根据所述液相色谱-质谱图定量分析所述样品中的小分子羧酸成分；
    其中，所述衍生化试剂为权利要求1或2所述的重氮类化合物，或者由权利要求3或4所述的制备方法制备得到的重氮类化合物。
  6. 根据权利要求5所述的定量分析小分子羧酸的检测方法，其特征在于，所述衍生化处理包括：
    配制20～100mM所述衍生化试剂的乙腈溶液，将其记为A液；
    配制20～100mM羟胺类化合物的水溶液，将其记为B液；
    将包含有所述小分子羧酸的样品和所述A液混合，在10000～17000rpm离心转速、4～30℃温度条件下离心处理5～8min后，将离心处理后上清液和所述B液混合，在50～80℃温度条件下，使所述样品进行衍生化反应，得到所述衍生化样品；优选所述衍生化反应的时间为10～60min；
    优选地，所述样品为血浆、血清、尿液、泪液、组织液、细胞、组织匀浆、细菌培养液、血斑或粪便；
    优选地，所述小分子羧酸为肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、花生四烯酸、乳酸、丙酮酸、富马酸、草酰乙酸、α-酮戊二酸、琥珀酸、苹果酸、柠檬酸或异柠檬酸中的一种或多种。
  7. 根据权利要求5或6所述的定量分析小分子羧酸的检测方法，其特征在于，所述液相色谱-质谱分析过程中，液相色谱分析采用的流动相包含A相和B相，其中，
    所述A相为水和甲酸的混合溶液，所述B相为乙腈和甲酸的混合溶液，且所述液相色谱分析过程中采用的液相色谱洗脱程序为梯度洗脱程序，所述液相色谱洗脱程序包括顺次进行的第一平衡过程、第一洗脱过程、第二洗脱过程及第二平衡过程；将所述A相的 体积记为VA、所述B相的体积记为VB，将所述液相色谱流动相的流速记为Vn，Vn为0.2～0.6mL/min；
    优选地，水和甲酸的混合溶液中，水和甲酸的体积比为200∶(0.1～0.3)；
    优选地，乙腈和甲酸的混合溶液中，乙腈和甲酸的体积比为200∶(0.1～0.3)。
  8. 根据权利要求7所述的定量分析小分子羧酸的检测方法，其特征在于，
    所述第一平衡过程中，VA为70～80％，VB为20～30％，且所述第一平衡过程的时间为0～1min；所述第一洗脱过程中，VA处于由70～80％向20～30％逐渐切换的动态变化过程，VB处于由20～30％向70～80％逐渐切换的动态变化过程，且所述第一洗脱过程的时间为2～5min；所述第二洗脱过程中，VA处于由20～30％向0～5％逐渐切换的动态变化过程，VB处于由70～80％向95～100％逐渐切换的动态变化过程，且所述第二洗脱过程的时间为1～3min；所述第二平衡过程中，VA处于由0～5％向70～80％切换的动态变化过程，VB处于由95～100％向20～30％切换的动态变化过程，且所述第二平衡过程的时间为1～1.5min；或者，
    所述第一平衡过程中，VA为45～55％，VB为45～55％，且所述第一平衡过程的时间为0～1min；所述第一洗脱过程中，VA处于由45～55％向0～5％逐渐切换的动态变化过程，VB处于由45～55％向95～100％逐渐切换的动态变化过程，且所述第一洗脱过程的时间为3～4min；所述第二洗脱过程中，VA为0～5％，VB为95～100％，且所述第二洗脱过程的时间为2～4min；所述第二平衡过程中，VA处于由0～5％向50～55％切换的动态变化过程，VB处于由95～100％向45～55％切换的动态变化过程，且所述第二平衡过程的时间为1～1.5min。
  9. 一种权利要求1或2所述的重氮类化合物，或者由权利要求3或4所述的制备方法制备得到的重氮类化合物在线粒体呼吸链复合物功能相关的药物筛选中的应用。
  10. 根据权利要求9所述的应用，其特征在于，利用所述重氮类化合物对活细胞代谢产物进行原位检测，筛选与线粒体呼吸链复合物功能相关的药物；
    优选地，所述药物包括激动剂和/或抑制剂；
    优选地，所述代谢产物为三羧酸循环中的羧酸类代谢产物；
    优选地，所述三羧酸循环中的羧酸类代谢产物指分子量在46～500之间的小分子羧酸；
    更优选地，所述小分子羧酸为肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸、油酸、亚油酸、花生四烯酸、乳酸、丙酮酸、富马酸、草酰乙酸、α-酮戊二酸、琥珀酸、苹果酸、柠檬酸或异柠檬酸中的一种或多种；
    更优选地，采用权利要求5至8中任一项所述的定量分析小分子羧酸的检测方法进行所述原位检测。
  11. 一种试剂盒，其特征在于，所述试剂盒包括权利要求1或2所述的重氮类化合物，或者权利要求3或4所述的制备方法制备得到的重氮类化合物。
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