A pair of isotope probes used for detecting and quantifying active electrophilic small molecule substances, and preparing method and applications thereof

Abstract

The invention discloses a pair of isotope probes, which comprises a probe 126 and a probe 127, and the probes are formed by taking a thiophenol group as a warhead part, taking dimethyl piperidine as a reporter group, taking a bisamide group as a linking group, and introducing a 13C isotope on the reporter group or the linking group. The pair of probes not only can screen electrophilic small molecules in a single sample and discover novel target compounds, but also can perform relative quantitative analysis on the electrophilic small molecules in a pair of samples in a complex environment at the same time, and clarify the concentration difference of the electrophilic small molecules in different tissue cells and different physiological and pathological states; therefore, key novel electrophilic micromolecules are determined, and important research data are provided for deeply researching the action mechanism of the micromolecules. The probe disclosed by the invention is high in sensitivity and low in false positive rate, can react with various electrophilic small molecules for screening, can discover a novel target compound from a complex matrix, and simultaneously can perform qualitative and quantitative research.

Description

Technical Field

[0001] The present invention relates to the technical field of probe compounds, and more specifically, to a pair of isotope probes for detecting and quantifying active electrophilic small molecule substances, and a preparation method and application thereof. Background Art

[0002] Liquid chromatography-mass spectrometry based on multi-stage mass spectrometry has been widely used in metabolomics research and natural product (plant secondary metabolites) research, and the characteristic daughter ions generated by secondary or multi-stage mass spectrometry data are often used to identify the parent ions detected by the primary mass spectrometry. At present, in the study of plant metabolites, small molecule probes that can label target metabolites are used to detect and guide the separation of target metabolites. Its principle includes using the ultraviolet absorption of aromatic groups as a reporter signal, but the ultraviolet absorption spectrum has low sensitivity and poor spectroscopic specificity, making it difficult to discover low-abundance new metabolites; there are also methods that use the specific isotope peaks of halogen atoms as reporter signals to discover new metabolites through liquid chromatography-mass spectrometry, but it is difficult to avoid false positive results caused by co-eluting substances caused by complex matrices, which causes great difficulties for subsequent discovery and separation work. The raw ultraviolet or mass spectrometry data generated by existing small molecule probes all need to be manually analyzed, and it is difficult to efficiently process a large amount of spectral data, which limits its application scope to targeted studies of metabolites targeting specific parent nuclei, and it is difficult to use non-targeted methods to discover new metabolites on a large scale, and it is impossible to deeply reveal the unknown chemical structure space of new metabolites.

[0003] Existing studies have shown that the generation, distribution and elimination of electrophilic products have a high degree of tissue and organ specificity, and are also closely related to physiological and pathological conditions. Active electrophilic molecules play a series of roles such as anti-inflammatory, anti-oxidative and anti-tumor through cross-linking protein cysteine ​​residues, and have good drug properties and research value. However, there are many types of active electrophilic molecules, low abundance, and complex chemical environments. They are also easy to deteriorate during conventional extraction and purification, and existing probe technologies have limitations such as high false positive rate, low detection sensitivity, labor-intensive data analysis, and quantitative difficulties. Therefore, in the face of many types of electrophilic products with extremely low abundance, the concentration differences under different tissue cells and different physiological and pathological conditions can be clarified by small molecule probes with quantitative functions, which can more accurately determine key small molecules and provide important research data for in-depth study of their mechanism of action. Patent CN112321489A discloses an electrophilic molecular probe based on active thiol and reporter ion, which can only be used to find the target molecule in a sample, and cannot simultaneously analyze a pair of samples, and obtain the abundance ratio of the target molecule in a pair of samples, and does not have a relative quantitative function, and the sensitivity needs to be further improved. Therefore, there is an urgent need for an electrophilic molecular probe that can analyze a pair of samples simultaneously to obtain the abundance ratio of the target molecule in the pair of samples and has quantitative function and further improved sensitivity. Summary of the invention

[0004] The purpose of the present invention is to overcome the above defects and shortcomings in the prior art and provide a pair of isotope probes.

[0005] The second object of the present invention is to provide a method for preparing the above pair of isotope probes.

[0006] The third object of the present invention is to provide the use of the above probe in relative quantitative analysis of active electrophilic small molecules in a pair of samples.

[0007] The fourth object of the present invention is to provide a method for relative quantitative analysis of active electrophilic molecules in a pair of samples using the above-mentioned probe.

[0008] The above-mentioned object of the present invention is achieved through the following technical solutions:

[0009] The present invention provides a pair of isotope probes, including probe-126 and probe-127; the structural formula of the probe-126 (Probe-126) is shown in formula (I), and the structural formula of the probe-127 (Probe-127) is shown in formula (II):

[0010]

[0011] Wherein, the * in formula (I) and formula (II) represents an isotope 13 C.

[0012] Furthermore, the probe comprises a thiophenol group as a warhead, dimethyl piperidine as a reporter group, a bisamide group as a linker group, and a 13 C isotopic composition.

[0013] The isotope probe designed and synthesized in the present invention uses a thiophenol group as the warhead to simulate the cysteine ​​sulfhydryl group, uses dimethyl piperidine as the reporter group, and uses a bisamide group as the linker group to improve the polarity. 13C isotope is used to realize the relative quantitative function. Among them, thiophenol, as a nucleophilic reaction group, undergoes Michael addition reaction with electrophilic products (including electrophilic metabolites, electrophilic natural products, electrophilic extracts, electrophilic reaction products, electrophilic syntheses, etc.) to form a conjugate (i.e., the small molecule probe of the present invention is connected with the above-mentioned electrophilic product by covalent bonds to form a reaction mixture product). The reaction mixture is analyzed by a liquid chromatography-mass spectrometry system, and after the conjugate is detected by a primary mass spectrometer (MS1), the reporter group dimethyl piperidine generates a reporter ion in a high energy collision cell (HCD) and is detected by a secondary mass spectrometer (MS2). The target conjugate parent ion is locked according to the presence or absence of the reporter ion, the m / z of the target conjugate parent ion is analyzed, and the precise molecular weight of the target electrophilic small molecule is calculated accordingly, and then the chemical formula is matched according to the precise molecular weight, and the possible structural formula is further screened by database retrieval and other methods, and finally the standard is purchased or synthesized and verified.

[0014] The present invention uses dimethyl piperidine as a reporter group covalently linked to the electrophilic product. The fragmentation efficiency of the reporter group in the HCD fragmentation pool is extremely high, providing the high sensitivity necessary for discovering low-abundance electrophilic products. At the same time, the mass-to-charge ratio of the reporter ion formed by the fragmentation of the reporter group is lower than that of most daughter ions, and it is not easy to be confused with other daughter ions in the secondary mass spectrometry, and has an extremely low false positive rate.

[0015] The warhead part of the probe of the present invention simulates cysteine ​​and targets potential cysteine ​​regulators. Currently, many literatures show that key sites of cysteine ​​proteins can be covalently modified with many electrophilic products, including metabolites, synthetic compounds and natural products. Some of these compounds have been shown to effectively regulate the function of target proteins. The warhead part of the present invention simulates cysteine ​​and screens new electrophilic products that can bind to it. These electrophilic products are all potential cysteine ​​regulators and have great value in biological activity research.

[0016] The present invention provides a method for preparing the above pair of isotope probes, comprising the following steps:

[0017] S1. performing a substitution reaction between isotope-labeled and non-isotope-labeled bromoacetic acid and 2,6-dimethylpiperidine to obtain isotope-labeled and non-isotope-labeled compound 1;

[0018] The structures of the isotope-labeled and non-isotope-labeled compound 1 are shown below:

[0019]

[0020] S2. condensing the isotope-labeled compound 1 with glycine to obtain compound 2; condensing the non-isotope-labeled compound 1 with isotope-labeled glycine to obtain compound 4;

[0021]

[0022] S3. Compound 2 and Compound 4 are subjected to a condensation reaction with 4,4'-dithiodiphenylamine to obtain Compound 3 and Compound 5;

[0023]

[0024] S4. Compound 3 and Compound 5 are subjected to a reduction reaction in the presence of tris(2-carboxyethyl)phosphine hydrochloride to obtain the above-mentioned isotope probes-127 and -126 (ie, Probe-127 and Probe-126);

[0025]

[0026] Furthermore, in step S1, the equivalent ratio of bromoacetic acid to 2,6-dimethylpiperidine is 1 to 1.5:1.

[0027] Preferably, in step S1, the equivalent ratio of bromoacetic acid to 2,6-dimethylpiperidine is 1.2:1.

[0028] Preferably, the substitution reaction in step S1 is to dissolve isotopically or non-isotopically labeled bromoacetic acid in a NaOH solution, stir at 0°C, then slowly dropwise add 2,6-dimethylpiperidine, stir for 1 hour after the dropwise addition is completed, and then transfer to room temperature for reaction for 3 days.

[0029] Furthermore, in step S2, the equivalent ratio of compound 1 to glycine is 1:1.5-2.5.

[0030] Preferably, in step S2, the equivalent ratio of compound 1 to glycine is 1:2.

[0031] Preferably, the condensation reaction in step S2 is to dissolve the isotopically labeled or unlabeled compound 1, NHS (N-hydroxysuccinimide), and EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) in DMSO, stir at room temperature, add triethylamine and glycine, and continue the reaction.

[0032] Furthermore, in step S3, the equivalent ratio of compound 2 or compound 4 to 4,4'-dithiodiphenylamine is 1:0.5-1.

[0033] Preferably, in step S3, the equivalent ratio of compound 2 or compound 4 to 4,4'-dithiodiphenylamine is 1:0.8.

[0034] Preferably, the condensation reaction in step S3 is to dissolve compound 2 or 4, 4,4'-dithiodiphenylamine, and HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate) in THF (tetrahydrofuran), and then add triethylamine to react at room temperature overnight.

[0035] Furthermore, the reduction reaction in step S4 is to dissolve compound 3 or 5 in THF, and then add TCEP·HCl and H2O. The air in the reaction bottle is replaced with argon, and then the reaction is carried out at room temperature overnight under argon protection. Among them, TCEP is a disulfide bond reducing agent, and its presence can inhibit the formation of dimers of the probe, thereby facilitating the preservation of the probe. At the same time, a small amount of TCEP is also conducive to the reaction of the probe with electrophilic small molecules.

[0036] The present invention uses isotopes to respectively label the reporter group part and the linker part of the probe molecule. When the same active electrophilic product in a pair of samples reacts with excess probe-126 and probe-127 respectively, the electrophilic product captured by the probe, i.e., the probe-electrophilic product conjugate, generates a parent ion with exactly the same mass-to-charge ratio in the primary mass spectrometer, which is manifested as the same peak at the same time. The two parent ions are indiscriminately sent to a fragmentation pool (HCD or other types of fragmentation pools) for fragmentation, and the generated common reporter ions and isotope-labeled reporter ions are detected by the secondary mass spectrometer (i.e., the characteristic fragment ion peaks 126.1277 and 127.1311 of probe-126 and probe-127 can be observed in the secondary mass spectrometer, respectively). The ratio of the ion intensities of the two can represent the concentration ratio of the electrophilic product in the pair of samples (i.e., the abundance ratio), thereby completing the relative quantification of the same electrophilic product in the two samples. The present invention solves the problem that there is no standard product for unknown electrophilic small molecules and that the standard product cannot be used for absolute quantification. The present invention can perform relative quantification of a variety of unknown or known electrophilic products in different groups of samples, thereby discovering electrophilic products with significant biological activity or biological significance.

[0037] Therefore, the present invention also provides the use of the probe in relative quantitative analysis of active electrophilic small molecules in a pair of samples.

[0038] Furthermore, the sample is a natural product; an extract of an animal or plant cell, tissue or organ; an extract or sample of bacteria or virus; a soil, mineral sample, atmospheric sample, water sample and an extract thereof.

[0039] The present invention also provides a method for relative quantitative analysis of active electrophilic molecules in a pair of samples using the probe, comprising the following steps:

[0040] S1. Add equal amounts of probe-126 and probe-127 to the two groups of samples. After the reaction is completed, mix the two groups of samples in equal amounts, dilute them, and perform LC-MSn Computer test;

[0041] S2.LC-MS n Data processing and analysis: First, determine the valid spectrum data by the appearance of the reporter ion in MS2, and then obtain the conjugate parent ion m / z from the MS1 corresponding to the valid MS2 spectrum; record the spectrum number, retention time, conjugate parent ion m / z, and the absolute intensity of reporter ions 126 and 127, calculate the difference between the conjugate parent ion m / z and the probe parent ion m / z, and obtain the molecular weight of the electrophilic small molecule; calculate the ratio of reporter ions 126 and 127 to obtain the abundance ratio of the electrophilic small molecule in the pair of samples.

[0042] Furthermore, the preferred signals with large abundance difference and molecular weight between 100-1000 or other molecular weight ranges suitable for the research object are further studied.

[0043] Furthermore, the reaction time in step S1 is 22 to 26 hours.

[0044] Furthermore, the valid spectrum data in step S2 is one or two of the characteristic peaks 126.1277 and 127.1311 in the secondary spectrum.

[0045] Preferably, step S1 is to take a pair of biological samples (samples from different sources; if they are cell samples, they are lysed first), use a suitable method (generally chloroform methanol precipitation + microporous membrane filtration) to remove proteins and nucleic acids in the samples, and retain the extracts that can be dissolved in methanol (if they are natural extracts, start here). The extracts are dried, weighed, and redissolved to prepare two groups of extract methanol solutions with the same concentration, and then equal and excess Probe-127 and Probe-126 are added to the two groups of extract solutions. The two groups of solutions are sealed and stirred at room temperature for 24 hours. After the reaction is completed, an equal amount of reaction solution is accurately transferred, mixed, diluted, passed through a microporous filter membrane, and centrifuged to obtain the supernatant to prepare a solution with a total mass concentration of 30ug / mL (30ppm), and LC-MS n Test on the computer.

[0046] Preferably, step S2 is to use commercial software Xcalibur (Thermo Fisher Scientific) to analyze the LC-MS data, and screen out the target parent ion based on the presence or absence of the reporter ion. The intensity of the reporter ions 126 and 127 represents the abundance ratio of the target compound in the two groups of samples. Select the signal with a significant difference in abundance ratio between the two groups of samples, and combine the molecular weight, source, structural characteristics (α-β unsaturated aldehyde ketone fragments, epoxide, β-lactam, etc.) and other information of the small molecule compound to search for the chemical structure that the small molecule may correspond to in various databases (such as the metabolite database HMDB: https: / / hmdb.ca / ). Purchase or synthesize possible small molecule compounds, design reasonable experiments, test the activity, and select small molecules with great potential to further study the mechanism and find targets.

[0047] The present invention uses this method to analyze the differences in small molecule metabolites between mouse exhausted T cells and polarized T cells, and screens out more than 70 valid data, of which more than 30 compounds have a content difference of more than two times, and five compounds have a content difference of more than five times. One of the signals was selected for structural analysis and activity testing, and three active electrophilic small molecule compounds that can significantly inhibit mouse T cell exhaustion were successfully screened out.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] The present invention provides a pair of isotope probes, including probe-126 and probe-127; the probes are composed of a thiophenol group as a warhead, dimethyl piperidine as a reporter group, a bisamide group as a linker group, and a 13 C isotope composition. This pair of probes can not only screen electrophilic small molecules in a single sample and discover new target compounds, but also can simultaneously perform relative quantitative analysis of electrophilic small molecules in a pair of samples in a complex environment, clarifying the concentration differences in different tissue cells and different physiological and pathological states, thereby determining key new electrophilic small molecules, and providing important research data for in-depth research on their mechanism of action. The probe of the present invention has high sensitivity, low false positive rate (probe-126 and probe-127 report ions appear at the same time, which is equivalent to the conjugate being confirmed twice), can react with electrophilic small molecules of various parent nuclei, and has a high degree of data automation analysis, and qualitative and quantitative research can be performed without enriching and purifying the target compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 This is the detection NMR spectrum of Probe-127.

[0051] Figure 2 This is the mass spectrum of Probe-127 detection.

[0052] Figure 3 This is the detection NMR spectrum of Probe-126.

[0053] Figure 4 This is the detection mass spectrum of Probe-126.

[0054] Figure 5 The structure and working principle of the isotope quantitative probe. Figure 5 A is the design and structure of the small molecule probe of the present invention; B is the use process and principle of the small molecule probe of the present invention, taking natural products (NPs) as an example; C is the principle of the small molecule probe of the present invention using isotope-labeled reporter ion part and linker part to achieve relative quantification.

[0055] Figure 6 This is the full image of the secondary mass spectrum of the parent ion of Sample AF conjugate.

[0056] Figure 7 This is a partial enlargement of the secondary mass spectrum of the parent ion of Sample AF conjugate.

[0057] Figure 8 It is the correlation curve between the test value and theoretical value of 2-nonenal of each sample.

[0058] Fig. 9 Full image of the secondary mass spectrum showing an example of valid data showing the differences in small molecule metabolites between mouse exhausted T cells and polarized T cells.

[0059] Fig.10 A zoomed-in image of the secondary mass spectrometry showing an example of valid data showing the differences in small molecule metabolites between mouse exhausted T cells and polarized T cells. DETAILED DESCRIPTION

[0060] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0061] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0062] Example 1 Synthesis of isotope probe

[0063] Synthesis route of isotope quantitative probe:

[0064]

[0065] 1. Synthesis of Probe-127

[0066] (1) Synthesis of Compound 1:

[0067] Take a 25mL eggplant-shaped bottle, add 593mg (1.2eq) of bromoacetic acid with isotope, dissolve in 4mL 3.3M NaOH solution, and stir at 0℃. After 5min, slowly add 400mg (482μL, 1eq) of 2,6-dimethylpiperidine to the above solution, stir for 1h after the addition, and then transfer to room temperature for reaction for 3 days. Use dichloromethane: methanol: acetic acid = 5: 1: 0.2 as TLC developer to monitor the reaction. After the reaction is completed, adjust the pH of the reaction solution to 2 with concentrated hydrochloric acid, and concentrate at 50℃ under reduced pressure. Add a small amount of methanol to the residue, take the soluble matter for column chromatography purification, and the elution conditions are: methanol / dichloromethane 0-7% 12mL / min 100min. Collect and combine the target components, concentrate and dry.

[0068] 250 mg of white solid was obtained, with a yield of 41.8%, confirmed by NMR-MS.

[0069] (2) Synthesis of Compound 2:

[0070] Take a 25mL narrow-necked bottle, add 330mg (1.0eq) of compound 1, 266.3mg (1.2eq) of NHS (N-hydroxysuccinimide), 441.4g (1.2eq) of EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride), 5mL of DMSO to dissolve, and stir at room temperature for 18h. After 18h, add 975mg (5eq) of triethylamine and 289.4mg (2eq) of glycine, and continue to react for 6h. Monitor the reaction with dichloromethane:methanol=3:1 as TLC developing agent. After the reaction is completed, the reaction solution is concentrated under reduced pressure at 45℃, and then vacuumed to reduce the solvent as much as possible, followed by column chromatography purification, and the elution conditions are: methanol / dichloromethane 0% 30min; 15-35% 90min; 35-50% 30min. Collect the product components, concentrate under reduced pressure, and dry in vacuum.

[0071] 530 mg of yellow oil was obtained with a yield of 89.9%, confirmed by NMR-MS.

[0072] (3) Synthesis of Compound 3:

[0073] Take a 10mL eggplant-shaped bottle, add 50mg (1eq) of compound 2, 43.4mg (0.8eq) of 4,4'-dithiodiphenylamine, 67mg (0.8eq) of HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate), dissolve in 1mL THF, and then add 45mg of triethylamine. React at room temperature overnight. Monitor the reaction with dichloromethane:methanol=5:1 as TLC developing agent. After the reaction is completed, the reaction solution is concentrated under reduced pressure at 45°C and purified by column chromatography. The elution conditions are: methanol / dichloromethane 0-5% 12mL / min 150min. Collect the product components and concentrate to dryness.

[0074] The product was obtained in the form of yellow oil (42 mg), with a yield of 42%, confirmed by NMR-MS.

[0075] (4) Synthesis of Probe-127:

[0076] Take a 25mL eggplant-shaped bottle, add 148mg (1eq) of compound 3, dissolve it in 3mL THF, and then add 110.6mg (1.2eq) of TCEP·HCl (51805-45-9) and 11.6mg (2eq) of H2O. Replace the air in the reaction bottle with argon, and then react at room temperature overnight under argon protection. Monitor the reaction with acetone: cyclohexane = 1:1 as TLC developing agent. After the reaction is completed, spin dry the solvent and purify it by column chromatography. The elution conditions are: acetone / cyclohexane 5-20% 60min; D / M 30-30% 40min. Collect the product components and concentrate to dryness.

[0077] The product was 124 mg (containing TCEP) as an off-white solid. Figure 1 and Figure 2 As shown, it is confirmed that Probe-127 was successfully synthesized. The probe contains a small amount of TCEP, which is a disulfide bond reducing agent. Its presence can inhibit the formation of dimers of the probe and is beneficial to the preservation of the probe. At the same time, a small amount of TCEP is also beneficial to the reaction of the probe with electrophilic small molecules.

[0078] 2. Synthesis of Probe-126

[0079] (1) Synthesis of compound 4:

[0080] The operation was the same as that of “Synthesis of Compound 2”. Confirmed by NMR-MS.

[0081] (2) Synthesis of Compound 5:

[0082] The operation was the same as that of “Synthesis of Compound 3”. Confirmed by NMR-MS.

[0083] (3) Synthesis of Probe-126:

[0084] The operation is the same as “Synthesis of Probe-127”.

[0085] The results of NMR mass spectrometry are as follows Figure 3 and Figure 4 As shown, it was confirmed that Probe-126 was successfully synthesized.

[0086] Example 2 Quantitative test using 2-nonenal as standard

[0087] 1. Experimental Methods

[0088] (1) Accurately weigh 3 mg of each of Probe-127 and Probe-126 prepared in Example 1, and dissolve them in 1 mL of anhydrous methanol, respectively, to obtain 3 mg / mL of Probe-127 working solution ① and 3 mg / mL of Probe-126 working solution ② (① and ② are also applicable to Examples 3 and 4). Accurately weigh 4.30 mg of 2-nonenal, and dissolve it in 3 mL of methanol, to obtain 1.33 mg / mL of 2-nonenal working solution ③.

[0089] Take six small reaction bottles and number them AF in sequence.

[0090] Add 200 μL of solution ①, 10 μL of solution ③, and 90 μL of methanol into bottle A;

[0091] Add 200 μL of solution ①, 50 μL of solution ③, and 50 μL of methanol into bottle B;

[0092] Add 200 μL of solution ① and 100 μL of solution ③ into bottle C;

[0093] Add 200 μL of solution ②, 10 μL of solution ③, and 90 μL of methanol into bottle D;

[0094] Add 200 μL of solution ②, 50 μL of solution ③, and 50 μL of methanol into bottle E;

[0095] Add 200 μL of solution ② and 100 μL of solution ③ that have been accurately pipetted into bottle F.

[0096] Each of the six mixed solutions was sealed and stirred at room temperature for 24 hours. After the reaction was completed, the reaction solutions were mixed, diluted, filtered through a microporous filter membrane, and centrifuged according to "A+F", "A+E", "B+F", "C+F", "C+E", "B+D", and "C+D". The supernatant was taken to prepare seven samples with a total mass concentration of 30ug / mL (30ppm), named "Sample AF", "Sample AE", ... "Sample CD" in sequence, and LC-MS n Test on the computer.

[0097] (2) Liquid quality test

[0098] LC-MS analysis was performed using a Q Exactive mass spectrometer (Thermofisher Scientific, USA) equipped with an electrospray source and a Dionex UltiMate 3000UHPLC (Thermofisher Scientific, USA). The mobile phase (A) was 0.1% formic acid in water and the mobile phase (B) was acetonitrile. The sample (2 μL) was analyzed using a Hypersil GOLD-C18 HPLC column (2.1×100 mm, 1.9 μm, Thermofisher Scientific, USA) with a mobile phase flow rate of 0.3 mL / min. The detailed description of the HPLC gradient and mass spectrometry settings are as follows:

[0099] Table 1 Chromatographic method parameters (Dionex UltiMate 3000, Hypersil GOLD-C 1 8)

[0100] Time(min) A％ B％ 0 90 1 0 5 70 30 1 6 5 95 1 7 5 95 1 7.1 90 1 0 20 90 1 0

[0101] Table 2 Mass spectrometry method parameters (Thermofisher)

[0102]

[0103] 2. Experimental Results

[0104] According to the probe design principle (such as Figure 5 Sample AE-CD should give the same parent ion (477.2976±5ppm) in LC-MS1 and different reporter ion ratios (126.1277:127.1311) in LC-MS2. The theoretical values ​​are 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10. Taking Sample AF as an example, its LC-MS2 spectrum is as follows: Figure 6 , the reporter ions 126 and 127 peaks are as follows Figure 7 All sample data were summarized and processed to make a chart showing the gap between theoretical values ​​and test values ​​(Table 3, Figure 8 ), r 2 =0.9992, which meets the requirements.

[0105] In summary, the present invention proves that the reporter group part and the linker part of the probe molecule are respectively labeled with isotopes, so that the electrophilic product captured by the probe, i.e., the probe-electrophilic product conjugate, produces a parent ion with exactly the same mass-to-charge ratio in the primary mass spectrometer, and these two parent ions are indiscriminately sent to the fragmentation pool (HCD or other types of fragmentation pools) for fragmentation, and the generated common reporter ions and isotope-labeled reporter ions are detected by the secondary mass spectrometer. The ratio of the intensities of the two can represent the ratio of the concentrations of the two electrophilic products in the sample, thus completing the relative quantification of the same electrophilic products in the two samples. The present invention solves the problem that there is no standard product for unknown electrophilic small molecules and that the standard product cannot be used for absolute quantification. The present invention can perform relative quantification of a variety of unknown or known electrophilic products in samples of different groups, thereby discovering electrophilic products with significant biological activity or biological significance.

[0106] Table 3 Test values ​​and theoretical values ​​of reporter ions in the secondary spectra of parent ions in each sample

[0107]

[0108] Example 3 Quantitative analysis of small molecule metabolites between exhausted T cells and polarized T cells in mice using isotope probes

[0109] 1. Experimental Methods

[0110] (1) Sample pretreatment

[0111] Place the EP tubes containing mouse polar T cells (marked "AR") and mouse exhausted T cells (marked "CL") at -20°C and freeze and thaw repeatedly to fully lyse the cells. Use methanol to transfer the cell lysate to two new EP tubes in small amounts and multiple times and dry them. Add appropriate amounts of methanol-chloroform (1:1) solution to the dried residue to precipitate the protein, and then centrifuge at 12000rpm, 4°C, and 20min. The supernatant after centrifugation is filtered through a 0.22μm microporous filter membrane into two sample bottles. Dry the filtrate and weigh it to obtain 1.34mg of "AR" extract and 1.49mg of "CL" extract for later use.

[0112] (2) Reaction of extract with isotope probe

[0113] Add 0.67 mL of HPLC grade methanol to the above "AR" extract and fully dissolve to obtain a 2 mg / mL working solution ④. Add 0.745 mL of HPLC grade methanol to the above "CL" extract and fully dissolve to obtain a 2 mg / mL working solution ⑤.

[0114] Take a 4mL sample bottle, number "G", add a stirrer, then add 50μL① and 200μL④ in sequence, and react in a sealed state at room temperature for 24 hours. Take another sample bottle, number "H", add a stirrer, then add 50μL② and 200μL⑤ in sequence, and react in a sealed state at room temperature for 24 hours. After the reaction of "G" and "H", accurately transfer 50μL of each into an EP tube, add HPLC grade methanol to dilute to 1mL, pass the dilution through a 0.22μm microporous filter membrane, and centrifuge the filtrate at 8000rpm, room temperature, and 5min. After centrifugation, take 150μL of the supernatant into a 2mL liquid mass spectrometer bottle, add 450μL of HPLC grade methanol, and obtain 600μL of 50ppm liquid mass sample for LC-MS n Test on the computer.

[0115] (3) LC-MS n Data processing and analysis

[0116] Valid data were screened from the raw data according to the following rules: Rule 1, at least one of 126.1277 and 127.1311 must be present in the secondary spectrum (in most cases both are present, and the greater the relative intensity difference between the two, the higher the spectrum priority), and the relative intensity of the peak with higher peak height must not be less than 70% (the closer to 100%, the higher the spectrum priority); Rule 2, the parent ion peak exists and is the peak with the largest m / z; Rule 3, the error between the m / z of the parent ion peak and the m / z of the parent ion selected by the software must not be higher than 2ppm (the smaller the error, the higher the spectrum priority); Rule 4, the m / z of the parent ion should be greater than 350 and the dimer peak (m / z=671.3320) should be excluded.

[0117] Valid data were screened and the absolute intensities of the conjugate parent ion m / z and reporter ions 126 and 127 were recorded. The difference between the m / z of the conjugate parent ion and the m / z of the probe parent ion is the molecular weight of the electrophilic small molecule, and the absolute intensity ratio of 126 and 127 represents the abundance ratio of the small molecule in mouse polarized T cells and exhausted T cells.

[0118] 2. Experimental Results

[0119] According to the above rules, this method was used to analyze the differences in small molecule metabolites between mouse exhausted T cells and polarized T cells, and more than 70 valid data were screened out, of which more than 30 compounds had a content difference of more than two times, and five had a content difference of more than five times. The inventor selected one of the signals, performed structural analysis and activity testing, and successfully screened out three active compounds that can significantly inhibit mouse T cell exhaustion. The mass spectrum of a valid data obtained by screening is shown in the figure below: Fig. 9 and Fig.10As shown in Table 4, the data obtained from the mass spectrum are processed. Combined with the molecular weight, source (mouse T cells), structural characteristics (α-β unsaturated aldehyde ketone fragments, epoxide, β-lactam, etc.) of the small molecule compound, the chemical structure that may correspond to the small molecule is retrieved on the metabolite website (HMDB: https: / / hmdb.ca / ). Purchase or synthesize possible small molecule compounds, design reasonable experiments, test activity, and select small molecules with great potential to further study the mechanism and find targets.

[0120] Table 4 The above effective data mass spectrum after processing data

[0121]

Claims

1. A pair of isotope probes, characterized in that: It includes probe-126 and probe-127; the structural formula of the probe-126 is shown in formula (I), and the structural formula of the probe-127 is shown in formula (II): Wherein, the * in formula (I) and formula (II) represents an isotope 13 C.

2. The method for preparing the isotope probe according to claim 1, characterized in that: The steps include: S1. performing substitution reaction of isotope-labeled and non-isotope-labeled bromoacetic acid with 2,6-dimethylpiperidine to obtain isotope-labeled and non-isotope-labeled compound 1, respectively; S2. condensing the isotope-labeled compound 1 with glycine to obtain compound 2; condensing the non-isotope-labeled compound 1 with isotope-labeled glycine to obtain compound 4; S3. Compound 2 and Compound 4 are subjected to condensation reaction with 4,4'-dithiodiphenylamine to obtain Compound 3 and Compound 5; S4. Compound 3 and compound 5 are respectively subjected to reduction reaction in the presence of tris(2-carboxyethyl)phosphine hydrochloride to obtain the isotope probe-127 and probe-126 according to claim 1; The structures of the isotope-labeled and non-isotope-labeled compound 1 are shown below: The structures of compounds 2 and 4 are shown below: The structures of compounds 3 and 5 are shown below:

3. The preparation method according to claim 2, characterized in that: In the step S1, the equivalent ratio of bromoacetic acid to 2,6-dimethylpiperidine is 1 to 1.5:

1.

4. The preparation method according to claim 2, characterized in that: In the step S2, the equivalent ratio of compound 1 to glycine is 1:1.5-2.

5.

5. The preparation method according to claim 2, characterized in that: In the step S3, the equivalent ratio of compound 2 or compound 4 to 4,4'-dithiodiphenylamine is 1:0.5-1.

6. Use of the isotope probe according to claim 1 in relative quantitative analysis of active electrophilic small molecules in a pair of samples.

7. The use according to claim 6, characterized in that the sample is a natural product; an extract of an animal or plant cell, tissue or organ; an extract or sample of bacteria or virus; a soil, mineral sample, air sample, water sample and an extract thereof.

8. A method for relative quantitative analysis of active electrophilic molecules in a pair of samples using the isotope probe according to claim 1, characterized in that: The following steps are involved: S1. Add equal amounts of probe-126 and probe-127 to the two groups of samples. After the reaction is completed, mix the two groups of samples in equal amounts, dilute them, and perform LC-MS n Computer test; S2.LC-MS n Data processing and analysis: First, determine the valid spectrum data by the appearance of the reporter ion in MS2, and then obtain the conjugate parent ion m / z from the MS1 corresponding to the valid MS2 spectrum; record the spectrum number, retention time, conjugate parent ion m / z, and the absolute intensity of reporter ions 126 and 127, calculate the difference between the conjugate parent ion m / z and the probe parent ion m / z, and obtain the molecular weight of the electrophilic small molecule; calculate the ratio of reporter ions 126 and 127 to obtain the abundance ratio of the electrophilic small molecule in the pair of samples.

9. The method according to claim 8, characterized in that: The reaction time in step S1 is 22 to 26 hours.

10. The method according to claim 8, characterized in that: The valid spectrum data in step S2 is one or two of the characteristic peaks 126.1277 and 127.1311 in the secondary spectrum.

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