A pair of isotopic probes for the detection and quantification of active electrophilic small molecules, their preparation methods and applications

Abstract

This invention discloses a pair of isotope probes, including probe-126 and probe-127, wherein the probes consist of a thiophenol group as the warhead portion, a dimethylpiperidine group as the reporter group, and a diamide group as the linking group, and an element is introduced on the reporter group or the linking group. 13 Composed of C isotopes. This pair of probes can not only screen electrophilic small molecules in a single sample and discover novel target compounds, but also simultaneously perform relative quantitative analysis of electrophilic small molecules in a pair of samples in complex environments. This elucidates their concentration differences in different tissues and cells, and under different physiological and pathological states, thereby identifying key novel electrophilic small molecules and providing important research data for in-depth study of their mechanisms of action. The probes of this invention have high sensitivity, low false positive rate, can react with a variety of electrophilic small molecules for screening, and can also discover novel target compounds from complex matrices, while simultaneously conducting qualitative and quantitative studies.

Description

Technical Field

[0001] This invention relates to the field of probe compound technology, and more specifically, to a pair of isotopic probes for detecting and quantifying active electrophilic small molecules, their preparation methods, and applications. Background Technology

[0002] Multistage mass spectrometry (LC-MS) based methods have been widely applied in metabolomics research and the study of natural products (plant secondary metabolites). Characteristic daughter ions generated from second- or multistage mass spectrometry data are often used to identify parent ions detected by first-stage mass spectrometry. Currently, in plant metabolite research, small-molecule probes labeled with target metabolites are employed to detect and guide the separation of these metabolites. The principles include using the UV absorption of aromatic groups as a reporter signal, but UV absorption spectroscopy has low sensitivity and poor spectral specificity, making it difficult to discover novel metabolites with low abundance. Another approach utilizes halogen-specific isotope peaks as reporter signals to discover novel metabolites via LC-MS, but it is difficult to avoid false positives caused by co-eluents due to complex matrices, posing significant challenges to subsequent discovery and separation. The raw UV or mass spectrometry data generated by existing small-molecule probes require manual analysis, making it difficult to efficiently process large amounts of spectral data. This limits their application to targeted studies of metabolites with specific parent nuclei, hindering the large-scale discovery of novel metabolites using non-targeted methods and preventing in-depth exploration of the unknown chemical structures of novel metabolites.

[0003] Existing research indicates that the formation, distribution, and elimination of electrophilic products exhibit high tissue and organ specificity and are closely related to physiological and pathological states. Active electrophilic molecules exert a range of effects, including anti-inflammatory, antioxidant, and antitumor activity, through cross-linking with cysteine ​​residues in proteins, demonstrating significant drug-like properties and research value. However, active electrophilic molecules are diverse, low in abundance, and exist in complex chemical environments. They are also prone to degradation during conventional extraction and purification processes. Furthermore, existing probe technologies suffer from limitations such as high false-positive rates, low detection sensitivity, labor-intensive data analysis, and difficulties in quantification. Therefore, given the numerous and extremely low abundance of electrophilic products, using small molecule probes with quantitative functions to elucidate their concentration differences in different tissues and cells under various physiological and pathological states can more accurately identify key small molecules and provide crucial research data for in-depth studies of their mechanisms of action. Patent CN112321489A discloses an electrophilic molecular probe based on an active thiol group and a reporter ion. This probe can only be used to detect target molecules in a single sample and cannot simultaneously analyze a pair of samples to determine the abundance ratio of the target molecule in the pair. It lacks relative quantitative functionality, and its sensitivity needs further improvement. Therefore, there is an urgent need for an electrophilic molecular probe that can simultaneously analyze a pair of samples, determine 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 this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide a pair of isotope probes.

[0005] A second objective of this invention is to provide a method for preparing the aforementioned pair of isotope probes.

[0006] A third objective of this invention is to provide the application of the above-mentioned probe in the relative quantitative analysis of active electrophilic small molecules in a pair of samples.

[0007] A fourth objective of this invention is to provide a method for the relative quantitative analysis of active electrophilic molecules in a pair of samples using the aforementioned probe.

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

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

[0010]

[0011] In equations (I) and (II), * represents isotopes. 13 C.

[0012] Furthermore, the probe comprises a thiophenol group as the warhead portion, a dimethylpiperidine group as the reporter group, and a diamide group as the linking group, and an element is introduced onto the reporter group or the linking group. 13 Composition of C isotopes.

[0013] The isotope probe designed and synthesized in this invention uses a thiophenol group as the warhead to simulate a cysteine ​​thiol group, a dimethylpiperidine group as the reporter group, and a diamide group as the linking group to improve polarity. Simultaneously, a [missing information - likely a specific component or element] is introduced into either the reporter group or the linking group. 13The C isotope is used to achieve relative quantitative analysis. Thiophene, acting as a nucleophilic reactive group, undergoes a Michael addition reaction with electrophilic products (including electrophilic metabolites, electrophilic natural products, electrophilic extracts, electrophilic reaction products, electrophilic synthetics, etc.) to form conjugates (i.e., the reaction mixture formed by the small molecule probe of this invention covalently linking to the aforementioned electrophilic products). The reaction mixture is analyzed using a liquid chromatography-mass spectrometry (LC-MS) system. After the conjugates are detected by mass spectrometry (MS1), the reporter group dimethylpiperidine generates a reporter ion in a high-energy collision cell (HCD) and is detected by mass spectrometry (MS2). The presence or absence of the reporter ion locks onto the target conjugate precursor ion. The m / z of the target conjugate precursor ion is analyzed, and the precise molecular weight of the target electrophilic small molecule is calculated accordingly. The precise molecular weight is then matched with the chemical formula, and possible structural formulas are further screened using methods such as database searches. Finally, standards are purchased or synthesized for verification.

[0014] This invention uses dimethylpiperidine as a reporter group covalently attached to electrophilic products. This reporter group has extremely high fragmentation efficiency in the HCD fragmentation cell, providing the high sensitivity necessary for the detection of low-abundance electrophilic products. At the same time, the reporter ion formed by the fragmentation of this reporter group has a lower mass-to-charge ratio than most daughter ions, making it less likely to be confused with other daughter ions in secondary mass spectrometry, and has an extremely low false positive rate.

[0015] The probe of this invention mimics the cysteine ​​residue in its tip portion, targeting potential cysteine ​​regulators. Numerous studies have shown that key sites on cysteine ​​proteins can be covalently modified with many electrophiles, including metabolites, synthetic compounds, and natural products. Some of these compounds have been shown to effectively regulate the function of target proteins. The probe of this invention mimics the cysteine ​​residue, screening for novel electrophiles that can bind to it. These electrophiles are potential cysteine ​​regulators and have significant value in bioactivity research.

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

[0017] S1. Bromoacetic acid with and without isotopic labeling was subjected to a substitution reaction with 2,6-dimethylpiperidine to obtain compound 1 with and without isotopic labeling;

[0018] The structures of compound 1 with and without isotopic labeling are shown below:

[0019]

[0020] S2. The isotopically labeled compound 1 is condensed with glycine to obtain compound 2; the unlabeled compound 1 is condensed with the isotopically labeled glycine to obtain compound 4.

[0021]

[0022] S3. Compounds 2 and 4 are condensed with 4,4'-dithiodiphenylamine to obtain compounds 3 and 5;

[0023]

[0024] S4. Compound 3 and compound 5 were reduced in the presence of tris(2-carboxyethyl)phosphohydrochloride to obtain the above isotopes Probe-127 and Probe-126 (i.e., Probe-127 and Probe-126).

[0025]

[0026] Further, 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 involves dissolving bromoacetic acid, with or without isotopic labeling, in NaOH solution, stirring at 0°C, then slowly adding 2,6-dimethylpiperidine dropwise, stirring for 1 hour after the addition is complete, and then transferring to room temperature for 3 days of reaction.

[0029] Furthermore, in step S2, the equivalent ratio of compound 1 to glycine is 1:1.5 to 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 involves dissolving compound 1 (with or without isotopic labeling), NHS (N-hydroxysuccinimide), and EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) in DMSO, stirring at room temperature, adding triethylamine and glycine, and continuing the reaction.

[0032] Further, in step S3, the equivalent ratio of compound 2 or compound 4 to 4,4'-dithiodiphenylamine is 1:0.5 to 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 involves dissolving compound 2 or 4, 4,4'-dithiodiphenylamine, HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate) in THF (tetrahydrofuran), and then adding triethylamine and reacting overnight at room temperature.

[0035] Further, in step S4, the reduction reaction involves dissolving compound 3 or 5 in THF, then adding TCEP·HCl and H2O. The air in the reaction flask is replaced with argon, and the reaction is carried out overnight at room temperature under argon protection. TCEP is a disulfide bond reducing agent; its presence inhibits the formation of dimers in the probe, thus facilitating probe preservation. Simultaneously, a small amount of TCEP also promotes the reaction of the probe with electrophilic small molecules.

[0036] This invention utilizes isotopes to label the reporter group and linker portion of a probe molecule. When the same active electrophile in a pair of samples reacts with excess probe-126 and probe-127 respectively, the electrophile captured by the probe, i.e., the probe-electrophile conjugate, produces precursor ions with identical mass-to-charge ratios in primary mass spectrometry, exhibiting the same elution time (the same peak). These two precursor ions are indiscriminately sent to a fragmentation cell (HCD or other types of fragmentation cell) for fragmentation. The resulting ordinary reporter ion and isotope-labeled reporter ion are detected by secondary mass spectrometry (i.e., characteristic fragment ion peaks of probe-126 and probe-127, 126.1277 and 127.1311, respectively, can be observed in the secondary mass spectrometer). The ratio of their ion intensities represents the concentration ratio (i.e., abundance ratio) of the electrophile in the pair of samples, thus achieving relative quantification of the same electrophile in two samples. This invention solves the problem of the lack of standards for unknown electrophilic small molecules, making absolute quantification impossible. This invention can perform relative quantification of multiple unknown or known electrophilic products in samples from different groups, thereby discovering electrophilic products with significant biological activity or biological significance.

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

[0038] Furthermore, the samples are natural products; extracts of animal or plant cells, tissues or organs; extracts or samples of bacteria or viruses; soil, mineral samples, atmospheric samples, water samples and their extracts.

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

[0040] S1. Add equal amounts of stoichiometric amounts of probe-126 and probe-127 to the two groups of samples, respectively. After the reaction is complete, mix the two groups of samples in equal volumes, dilute, and then perform LC-MS.n On-machine testing;

[0041] S2.LC-MS n Data processing and analysis: First, valid spectral data are determined by the presence of reporter ions in MS2. Then, the m / z of the conjugate precursor ion is obtained from MS1 corresponding to the valid MS2 spectrum. The spectrum number, retention time, m / z of the conjugate precursor ion, and absolute intensities of reporter ions 126 and 127 are recorded. The difference between the m / z of the conjugate precursor ion and the m / z of the probe precursor ion is calculated to obtain the molecular weight of the electrophilic small molecule. The ratio of reporter ions 126 and 127 is calculated to obtain the abundance ratio of the electrophilic small molecule in this pair of samples.

[0042] Furthermore, the preferred signals with large abundance differences and molecular weights between 100 and 1000, or other signals suitable for the molecular weight range of the research object, are further studied.

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

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

[0045] Preferably, step S1 involves taking a pair of biological samples (from different sources; if the samples are cell samples, they are lysed first), removing proteins and nucleic acids from the samples using a suitable method (generally chloroform-methanol precipitation + microporous membrane filtration), and retaining the methanol-soluble extract (if it is a natural extract, start from here). The extract is dried, weighed, and reconstituted to prepare two sets of extract methanol solutions with the same concentration. Equal and excess amounts of Probe-127 and Probe-126 are then added to the two sets of extract solutions. Both solutions are sealed and stirred at room temperature for 24 hours. After the reaction is complete, equal volumes of the reaction solution are accurately transferred, mixed, diluted, filtered through a microporous membrane, and the supernatant is collected by centrifugation to prepare a solution with a total mass concentration of 30 μg / mL (30 ppm). LC-MS is then used for further analysis. n On-machine testing.

[0046] Preferably, step S2 involves analyzing liquid chromatography-mass spectrometry (LC-MS) data using the commercial software Xcalibur (Thermo Fisher Scientific). Target precursor ions are screened based on the presence or absence of reporter ions. The intensities of reporter ions 126 and 127 represent the abundance ratio of the target compound in the two sample groups. Signals with significantly different abundance ratios in the two sample groups are selected. Combined with information such as the molecular weight, origin, and structural characteristics (α-β-unsaturated aldehyde / ketone fragments, epoxides, β-lactams, etc.) of the small molecule compounds, the possible corresponding chemical structures are searched in various databases (such as the metabolite database HMDB: https: / / hmdb.ca / ). Potential small molecule compounds are purchased or synthesized, reasonable experiments are designed, activities are tested, and small molecules with high potential are selected for further investigation of mechanisms and target identification.

[0047] This invention utilizes this method to analyze the differences in small molecule metabolites between exhausted T cells and polar T cells in mice, screening out more than seventy valid data points. Among them, more than thirty compounds showed a difference in content of more than two times, and five showed a difference of more than five times. One signal was selected, and structural analysis and activity testing were performed, successfully screening out three active electrophilic small molecule compounds that can significantly inhibit the exhaustion of mouse T cells.

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

[0049] This invention provides a pair of isotope probes, including probe-126 and probe-127; the probes consist of a thiophenol group as the warhead portion, a dimethylpiperidine group as the reporter group, and a diamide group as the linking group, and an element is introduced on the reporter group or the linking group. 13 Composed of C isotopes. This pair of probes can not only screen electrophilic small molecules in a single sample and discover novel target compounds, but also simultaneously perform relative quantitative analysis of electrophilic small molecules in a pair of samples in complex environments, elucidating their concentration differences in different tissues and cells and under different physiological and pathological states. This allows for the identification of key novel electrophilic small molecules, providing important research data for in-depth studies of their mechanisms of action. The probes of this invention have high sensitivity, low false positive rate (the simultaneous appearance of probe-126 and probe-127 reporter ions is equivalent to secondary confirmation of the conjugate), can react with electrophilic small molecules with multiple parent nuclei, and have a high degree of automated data analysis, enabling qualitative and quantitative studies without the need for enrichment and purification of target compounds. Attached Figure Description

[0050] Figure 1 The NMR spectrum for Probe-127 detection.

[0051] Figure 2 Mass spectrometry for the detection of Probe-127.

[0052] Figure 3 The NMR spectrum for Probe-126 detection.

[0053] Figure 4 Mass spectrometry for the detection of Probe-126.

[0054] Figure 5 This describes the structure and working principle of an isotope quantitative probe. Figure 5 In this paper, A represents the design and structure of the small molecule probe of the present invention; B represents the usage process and principle of the small molecule probe of the present invention, taking natural products (NPs) as an example; and C represents the principle of the small molecule probe of the present invention to complete relative quantification by using the isotope-labeled reporter ion part and the linking part.

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

[0056] Figure 7 This is a magnified secondary mass spectrometry image of the parent ion of the Sample AF conjugate.

[0057] Figure 8 The correlation curves between the test values ​​and theoretical values ​​of 2-nonenal for each sample are shown.

[0058] Figure 9 A full secondary mass spectrometry plot serving as a valid example of small molecule metabolite differences between exhausted T cells and polar T cells in mice.

[0059] Figure 10 A magnified secondary mass spectrometry image serving as a valid example of small molecule metabolite differences between exhausted T cells and polar T cells in mice. Detailed Implementation

[0060] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

[0062] Example 1: Synthesis of Isotope Probes

[0063] Synthetic route of isotope quantitative probes:

[0064]

[0065] 1. Synthesis of Probe-127

[0066] (1) Synthesis of compound 1:

[0067] Take a 25 mL round-bottom flask, add 593 mg (1.2 eq) of isotopically-containing bromoacetic acid, and dissolve it in 4 mL of 3.3 M NaOH solution. Stir at 0 °C. After 5 min, slowly add 400 mg (482 μL, 1 eq) of 2,6-dimethylpiperidine to the above solution. After the addition is complete, stir for 1 h, then transfer to room temperature and react for 3 days. Monitor the reaction using TLC with dichloromethane:methanol:acetic acid = 5:1:0.2 as the developing solvent. After the reaction is complete, adjust the pH of the reaction solution to ≈2 with concentrated hydrochloric acid and concentrate under reduced pressure at 50 °C. Add a small amount of methanol to the residue, and take the soluble residue for column chromatography purification. The elution conditions are: methanol / dichloromethane 0-7% 12 mL / min 100 min. Collect and combine the target components, concentrate and dry.

[0068] 250 mg of white solid was obtained. Yield: 41.8%, confirmed by NMR mass spectrometry.

[0069] (2) Synthesis of compound 2:

[0070] Take a 25 mL narrow-mouth bottle, add 330 mg (1.0 eq) of compound 1, 266.3 mg (1.2 eq) of NHS (N-hydroxysuccinimide), and 441.4 g (1.2 eq) of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), dissolve in 5 mL of DMSO, and stir at room temperature for 18 h. After 18 h, add 975 mg (5 eq) of triethylamine and 289.4 mg (2 eq) of glycine, and continue the reaction for 6 h. Monitor the reaction using TLC with dichloromethane:methanol = 3:1 as the developing solvent. After the reaction is complete, concentrate the reaction solution under reduced pressure at 45 °C, then evacuate to minimize the solvent, and immediately purify by column chromatography. Elution conditions are: methanol / dichloromethane 0% 30 min; 15-35% 90 min; 35-50% 30 min. Collect the product fraction, concentrate under reduced pressure, and dry under vacuum.

[0071] 530 mg of a yellow oily substance was obtained, with a yield of 89.9%, which was confirmed by nuclear magnetic resonance mass spectrometry.

[0072] (3) Synthesis of compound 3:

[0073] Take a 10 mL round-bottom flask, add 50 mg (1 eq) of compound 2, 43.4 mg (0.8 eq) of 4,4'-dithiodiphenylamine, and 67 mg (0.8 eq) of HATU (2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate), dissolve in 1 mL of THF, and then add 45 mg of triethylamine. React overnight at room temperature. Monitor the reaction using TLC with dichloromethane:methanol = 5:1 as the developing solvent. After the reaction is complete, concentrate the reaction solution under reduced pressure at 45 °C, purify by column chromatography, and elute under the following conditions: methanol / dichloromethane 0-5% 12 mL / min 150 min. Collect the product fraction, concentrate, and dry.

[0074] 42 mg of product was obtained, a yellow oil. The yield was 42%, confirmed by NMR mass spectrometry.

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

[0076] Take a 25 mL round-bottom flask, add 148 mg (1 eq) of compound 3, dissolve in 3 mL of THF, then add 110.6 mg (1.2 eq) of TCEP·HCl (51805-45-9) and 11.6 mg (2 eq) of H2O. Replace the air in the reaction flask with argon, and then allow the reaction to proceed overnight at room temperature under argon protection. Monitor the reaction using acetone:cyclohexane = 1:1 as the TLC developing solvent. After the reaction is complete, evaporate the solvent, purify by column chromatography, using the following elution conditions: acetone / cyclohexane 5-20% 60 min; D / M 30-30% 40 min. Collect the product fraction, concentrate, and dry.

[0077] 124 mg of product (containing TCEP) was obtained, a grayish-white solid. The NMR mass spectrometry results are as follows: Figure 1 and Figure 2 As shown, the successful synthesis of Probe-127 is confirmed. The probe contains a small amount of TCEP, a disulfide bond reducing agent. Its presence inhibits the formation of dimers, thus facilitating probe preservation. Simultaneously, a small amount of TCEP also promotes the reaction of the probe with electrophilic small molecules.

[0078] 2. Synthesis of Probe-126

[0079] (1) Synthesis of compound 4:

[0080] The procedure is the same as in "Synthesis of Compound 2". Confirmed by NMR mass spectrometry.

[0081] (2) Synthesis of compound 5:

[0082] The procedure is the same as in "Synthesis of Compound 3". Confirmed by NMR mass spectrometry.

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

[0084] The procedure is the same as for "Synthesis of Probe-127".

[0085] The detection results of nuclear magnetic resonance mass spectrometry are as follows: Figure 3 and Figure 4 As shown, the successful synthesis of Probe-126 is confirmed.

[0086] Example 2: Quantitative testing using 2-nonenal as a standard

[0087] I. Experimental Methods

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

[0089] Take six small reaction flasks and number them AF sequentially.

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

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

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

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

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

[0095] Add 200 μL of solution ② and 100 μL of solution ③ precisely transferred to bottle F.

[0096] Six groups of mixed solutions were each sealed and stirred at room temperature for 24 hours. After the reaction, equal volumes of the reaction solution were mixed, diluted, filtered through a microporous membrane, and centrifuged according to the formulas “A+F”, “A+E”, “B+F”, “C+F”, “C+E”, “B+D”, and “C+D”, respectively. The supernatant was collected to prepare seven samples with a total mass concentration of 30 μg / mL (30 ppm), named “Sample AF”, “Sample AE”, ..., “Sample CD”, respectively. LC-MS was then used to prepare these samples. n On-machine testing.

[0097] (2) Liquid chromatography-mass spectrometry

[0098] LC-MS analysis was performed using a Q Exactive mass spectrometer (Thermofisher Scientific, USA), equipped with an electrospray ionization source and a Dionex UltiMate 3000U HPLC (Thermofisher Scientific, USA). Mobile phase (A) was 0.1% formic acid aqueous solution, and mobile phase (B) was acetonitrile. Samples (2 μL) were analyzed using a Hypersil GOLD-C18 high-performance liquid chromatography column (2.1 × 100 mm, 1.9 μm, Thermofisher Scientific, USA) at a flow rate of 0.3 mL / min. Detailed specifications of the HPLC gradient and mass spectrometry settings are as follows:

[0099] Table 1. Chromatographic method parameters (Dionex UltiMate 3000, Hypersil GOLD-C 18)

[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] II. Experimental Results

[0104] According to probe design principles (such as...) Figure 5 As shown in the figure, Sample AE-CD should give the same precursor ion (477.2976±5 ppm) in LC-MS1, and different reporter ion ratios (126.1277:127.1311) in LC-MS2, with theoretical values ​​of 10:1, 5:1, 2:1, 1:1, 1:2, 1:5, and 1:10, respectively. Taking Sample AF as an example, its LC-MS2 spectrum is shown below. Figure 6 The report shows peaks 126 and 127 of ions as follows: Figure 7 All sample data were compiled and processed to create charts showing the difference between theoretical and test values ​​(Table 3). Figure 8 ), r 2 =0.9992, which meets the requirements.

[0105] In summary, this invention demonstrates that by labeling the reporter group and linker portion of a probe molecule with isotopes, the electrophilic product captured by the probe (i.e., the probe-electrophilic conjugate) produces precursor ions with identical mass-to-charge ratios in primary mass spectrometry. These two precursor ions are indiscriminately fed into a fragmentation cell (HCD or other types of fragmentation cell) for fragmentation. The resulting ordinary reporter ion and isotopically labeled reporter ion are detected by secondary mass spectrometry. The intensity ratio of these two ions represents the concentration ratio of the two electrophilic products in the sample, thus achieving relative quantification of the same electrophilic product in two samples. This invention solves the problem of the lack of standards for unknown electrophilic small molecules, making absolute quantification impossible. This invention can perform relative quantification of multiple unknown or known electrophilic products in different groups of samples, thereby discovering electrophilic products with significant biological activity or biological significance.

[0106] Table 3 shows the measured and theoretical values ​​of the reported ion in the secondary spectra of the parent ion in each sample.

[0107]

[0108] Example 3: Quantitative analysis of small molecule metabolite differences between exhausted T cells and polar T cells in mice using isotope probes.

[0109] I. Experimental Methods

[0110] (1) Sample pretreatment

[0111] EP tubes containing mouse polar T cells (labeled "AR") and mouse exhausted T cells (labeled "CL") were repeatedly freeze-thawed at -20°C to ensure complete cell lysis. The cell lysate was transferred to two new EP tubes in small amounts several times with methanol and then dried. A suitable amount of methanol-chloroform (1:1) solution was added to the dried residue to precipitate proteins, followed by centrifugation at 12000 rpm, 4°C, for 20 min. The supernatant was filtered through a 0.22 μm microporous membrane into two sample vials. The filtrates were dried and weighed to obtain 1.34 mg of "AR" extract and 1.49 mg of "CL" extract, which were then used for further processing.

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

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

[0114] Take a 4 mL sample vial, label it "G", add a stir bar, then add 50 μL ① and 200 μL ④ sequentially, and react at room temperature in a sealed environment for 24 h. Take another sample vial, label it "H", add a stir bar, then add 50 μL ② and 200 μL ⑤ sequentially, and react at room temperature in a sealed environment for 24 h. After the reactions in "G" and "H" are complete, accurately transfer 50 μL of each to an EP tube, dilute to 1 mL with HPLC-grade methanol, filter the diluent through a 0.22 μm microporous membrane, and centrifuge the filtrate at 8000 rpm, room temperature, and 5 min. After centrifugation, take 150 μL of the supernatant into a 2 mL LC-MS vial, add 450 μL of HPLC-grade methanol, and obtain 600 μL of 50 ppm LC-MS sample. n On-machine testing.

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

[0116] Valid data should be selected from the raw data according to the following rules: Rule 1: The secondary spectrum must contain at least one of 126.1277 and 127.1311 (in most cases, both are present, and the greater the difference in their relative intensities, the higher the spectrum priority), and the relative intensity of the higher peak should not be less than 70% (the closer to 100%, the higher the spectrum priority); Rule 2: The parent ion peak must be present and be 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 should not exceed 2 ppm (the smaller the error, the higher the spectrum priority); Rule 4: The m / z of the parent ion should be greater than 350 and dimer peaks (m / z = 671.3320) should be excluded.

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

[0118] II. Experimental Results

[0119] Based on the above rules, this method was used to analyze the differences in small molecule metabolites between exhausted T cells and polar T cells in mice, identifying over seventy valid data points. Among these, over thirty compounds showed a difference in content of more than two times, and five showed a difference of more than five times. The inventors selected one of these signals for structural analysis and activity testing, successfully identifying three active compounds that significantly inhibited mouse T cell exhaustion. The mass spectrum of one of the valid data points is shown below. Figure 9 and Figure 10As shown in the figure, and the data obtained from the processed mass spectrum, are presented in Table 4. Combining information such as the molecular weight, origin (mouse T cells), and structural characteristics (α-β unsaturated aldehyde / ketone fragments, epoxides, β-lactams, etc.) of the small molecule compounds, the possible chemical structures corresponding to the small molecules were searched on the metabolite website (HMDB: https: / / hmdb.ca / ). Potential small molecule compounds were purchased or synthesized, reasonable experiments were designed, activities were tested, and small molecules with high potential were selected for further investigation of mechanisms and target identification.

[0120] Table 4 shows the processed mass spectra of the above valid data.

[0121]

Claims

1. A pair of isotope probes, characterized in that, Including probe-126 and probe-127; the structural formula of probe-126 is shown in formula (I), and the structural formula of probe-127 is shown in formula (II): ， In equations (I) and (II), * represents isotopes. 13 C.

2. The method for preparing the isotope probe according to claim 1, characterized in that, Includes the following steps: S1. Bromoacetic acid with and without isotope labeling was subjected to substitution reaction with 2,6-dimethylpiperidine to obtain compound 1 with and without isotope labeling, respectively. S2. The isotopically labeled compound 1 is condensed with glycine to obtain compound 2; the unlabeled compound 1 is condensed with the isotopically labeled glycine to obtain compound 4. S3. Compounds 2 and 4 were condensed with 4,4'-dithiodiphenylamine to obtain compounds 3 and 5, respectively. S4. Compound 3 and compound 5 are respectively reduced in the presence of tris(2-carboxyethyl)phosphohydrochloride to obtain the isotope probe-127 and probe-126 as described in claim 1; The structures of compound 1 with and without isotopic labeling are shown below: and ; The structures of compounds 2 and 4 are shown below: and ; The structures of compounds 3 and 5 are shown below: and .

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

5.

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

6. The application of the isotope probe of claim 1 in the relative quantitative analysis of active electrophilic small molecules in a pair of samples, characterized in that, The application is for purposes other than disease diagnosis and / or treatment.

7. The application according to claim 6, wherein the sample is a natural product; an extract of animal or plant cells, tissues or organs; an extract or sample of bacteria or viruses; a soil, mineral sample, atmospheric sample, water sample or extract thereof.

8. A method for relative quantitative analysis of a pair of active electrophilic molecules in a sample using the isotope probe of claim 1, characterized in that, Includes the following steps: S1. Add equal amounts of stoichiometric amounts of probe-126 and probe-127 to the two groups of samples, respectively. After the reaction is complete, mix the two groups of samples in equal volumes, dilute, and then perform LC-MS. n On-machine testing; S2.LC-MS n Data processing and analysis: First, valid spectral data are determined by the presence of reporter ions in MS2. Then, the m / z of the conjugate precursor ion is obtained from the MS1 corresponding to the valid MS2 spectrum. The spectrum number, retention time, m / z of the conjugate precursor ion, and absolute intensities of reporter ions 126 and 127 are recorded. The difference between the m / z of the conjugate precursor ion and the m / z of the probe precursor ion is calculated to obtain the molecular weight of the electrophilic small molecule. The ratio of reporter ions 126 and 127 is calculated to obtain the abundance ratio of the electrophilic small molecule in this pair of samples. The method described is not for disease diagnosis and / or treatment purposes.

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, In step S2, the valid spectral data is the secondary spectrum containing one or both of the characteristic peaks 126.1277 and 127.1311.

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