Bioorthogonal mass spectrometry derivatization probe molecules for abasic sites, preparation and use thereof

Abstract

The application relates to a biological ortho-mass spectrometry derivative probe molecule for a dealkylation site, preparation and application, and belongs to the technical field of analytical chemistry. The structural formula of the probe molecule is as follows: wherein n1 is an integer of 1-3, and n2 is an integer of 1-2. The probe molecule has low toxicity characteristics, has selectivity to AP sites contained on DNA, has a high-efficiency reaction with aldehyde groups released from the AP sites to form stable oxime target compounds, and has a terminal alkyne component with a biological ortho-chemical function, which can be subjected to a cycloaddition click reaction with biotin-azide, is enriched by a magnetic bead, and realizes targeted detection of AP site adducts in a complex sample. In addition, the pyridine heterocyclic structure increases the mass spectrometry response and reaction efficiency, and significantly improves the detection sensitivity and mass spectrometry response.

Description

Technical Field

[0001] The present invention relates to a bioorthogonal mass spectrometry-derived probe molecule targeting an abasic site, its preparation and application, and belongs to the technical field of analytical chemistry. Background Art

[0002] Apurinic pyrimidine sites (AP sites) are the most common type of DNA damage, arising from the breakage of glycosidic bonds in DNA. Studies have shown that each mammalian cell generates an average of approximately 10,000 endogenous AP sites per day. If these lesions are not repaired promptly, they can lead to DNA replication disorders, DNA chain breaks, and ultimately mutations and cell death. Under physiological conditions, AP sites can display highly reactive aldehyde groups, making them a potential important target for detection in DNA damage research. Through bioconjugation labeling reactions, AP sites can be covalently labeled with highly selective and specific hydroxylamine and hydrazine molecules, allowing for more in-depth research.

[0003] The article "Detection Methods of DNA Abasic Sites and Their Biological Research Progress" reported that AP sites can be used as a potential test method for DNA damage. Currently, the detection methods of AP sites mainly include chemifluorescence, liquid chromatography-mass spectrometry (LC-MS / MS), 14 C or 32 P isotope labeling method, etc.

[0004] Fluorescence analysis has enabled the monitoring and tracking of bioactive substances or targets in cells and even in vivo, thanks to its "visualization" capabilities. Compared to chemical derivatization-assisted LC-MS / MS techniques, the development of "turn-on" fluorescent probe technology offers significant advantages. By fluorescently labeling DNA, the content of the target analyte can be directly read, avoiding complex enzymatic processing steps. However, the major drawback is that while fluorescent labeling methods can be used for dynamic monitoring, they can suffer from photobleaching, which reduces signal intensity, or quenching over extended periods, compromising the stability and reliability of the fluorescent signal. The paper "Selective Labeling Aldehydes in DNA" reports the design and synthesis of a naphthalene dicarboxylic acid imide hydroxylamine probe that selectively labels all naturally occurring aldehydes present in DNA, including 5-formylcytosine, 5-formyluracil, and abasic sites. The fluorescent properties of the resulting nucleosides are studied in detail, and the reactivity of hydroxylamine and amine groups toward aldehydes in DNA is discussed, providing important insights for the design of selective DNA labeling chemicals. The AP sites labeled with fluorophore-ARP were detected by capillary electrophoresis with a detection limit of 1.2 AP sites / 10 6 However, due to the limitation of using appropriate antibodies, the universality of this method is low.

[0005] In addition, LC-MS / MS has high sensitivity and good selectivity, and has obvious advantages in the qualitative and quantitative detection of nucleotide derivatives, but there are some challenges in actual operation. The operation process of extracting DNA from cell lysis and chemical derivatization labeling is relatively complex, and due to the problem of background interference, it is impossible to accurately quantitatively analyze the AP site of low trace sample. In "Quantitation of Apurinic / Apyrimidinic Sites in Isolated DNA and in Mammalian Tissue with a Reduced Level of Artifacts", a LC-MS / MS quantitative method for derivatizing and labeling AP sites using PMOA reagent is developed, which shows that the method of directly processing AP sites in the cell lysis stage can significantly reduce false positive results compared to processing after extracting DNA. The method successfully detected about 0.9 AP sites / 10 7 Nucleotides. By extracting DNA from cell lysis and chemical derivatization labeling. However, this method relies on professional technicians and large equipment, and the sample operation is complex, which may generate additional AP sites, and is affected by sample complexity and matrix effect, resulting in inhibition of ionization efficiency of target compounds, affecting the accuracy of quantitative results, and there is an urgent need to develop a new method for qualitative and quantitative analysis of specific recognition and capture of AP sites in complex matrix. SUMMARY

[0006] In view of the poor selectivity of the existing technology fluorescent or mass spectrometry probe molecules in detecting DNA aldehyde group sites, and the problem that after cell samples are derivatized and enzymatically degraded, the matrix effect causes the AP site to be accurately quantitatively analyzed, the present application provides a biological orthogonal mass spectrometry derivatization probe molecule for abasic sites and a preparation method and application thereof. The design of the probe molecule aims to improve the selectivity and sensitivity of the abasic site, and promote the accurate analysis of DNA damage and its application in biomedical research.

[0007] To achieve the above object, the technical scheme of the present application is as follows.

[0008] A biological orthogonal mass spectrometry derivatization probe molecule for abasic sites, the structural formula of the probe molecule is: Wherein, n1 is an integer from 1 to 3, and n2 is an integer from 1 to 2.

[0009] A preparation method of the biological orthogonal mass spectrometry derivatization probe molecule for abasic sites according to the present application, the method steps comprising:

[0010] (1) Compound 1 and compound 2 are coupled via the Mizoroki-Heck reaction to obtain compound 3;

[0011] (2) Compound 3 was deprotected by K2CO3 and then halogenated to obtain compound 4;

[0012] (3) In the presence of an acid-binding agent, compound 4 reacts with N-hydroxyphthalimide to remove one molecule of hydrogen halide to obtain compound 5;

[0013] (4) Compound 5 is deprotected under the action of a reducing agent to obtain a bioorthogonal mass spectrometry-derived probe molecule targeting the abasic site;

[0014] Among them, the structural formula of compound 1 is n2 is an integer from 1 to 2; the structural formula of compound 2 is n1 is an integer from 1 to 3; the structural formula of compound 3 is The structural formula of compound 4 is X is a halogen; the structural formula of compound 5 is

[0015] Furthermore, during the halogenation reaction in step (2), SOCl2 or PBr3 is added.

[0016] Furthermore, in step (3), the acid binding agent is K2CO3 or triethylamine.

[0017] Furthermore, in step (4), the reducing agent is hydrazine or hydrazine hydrate.

[0018] An application of the bioorthogonal mass spectrometry-derived probe molecule for abasic sites according to the present invention, wherein the probe molecule is used to label AP sites.

[0019] Furthermore, after the probe molecule labels the AP site, the AP site is enriched and detected through a click reaction.

[0020] A bioorthogonal analysis method for AP sites, comprising the following steps:

[0021] (1) adding the probe molecule to DNA extracted from cells, incubating, precipitating, and enzymatically hydrolyzing to obtain a sample to be tested;

[0022] (2) mixing the sample to be tested with biotin-azide to perform a click reaction to obtain an adduct mixed solution;

[0023] (3) mixing the adduct mixed solution with avidin magnetic beads, incubating the mixture, and magnetically separating the mixture to obtain a sample to be analyzed;

[0024] (4) The samples to be analyzed are analyzed by liquid chromatography-mass spectrometry (LC-MS / MS).

[0025] Furthermore, in step (1), the probe molecule is added to a DNA hydrolyzate extracted from cells and incubated at 37° C. for 1 to 2 hours to obtain a derivatized sample solution; the derivatized sample solution is precipitated, hydrolyzed, de-enzymed, dried, and redissolved to obtain a sample to be tested.

[0026] Furthermore, in step (2), the sample to be tested is mixed evenly with biotin-azide to obtain a mixed solution; aminoguanidine and sodium ascorbate are added to the mixed solution, and the mixture is reacted at 37° C. for 1 to 2 hours under nitrogen protection to obtain an adduct mixed solution.

[0027] Furthermore, in step (4), the liquid chromatography conditions are as follows: the chromatographic column is Waters ACQUITY UPLC HSS T3, 100 mm × 2.1 mm, 1.8 μm; the mobile phase A is a 0.1% by mass formic acid aqueous solution; the mobile phase B is an acetonitrile solution; the flow rate is 0.25 mL / min, and the injection volume is 5 μL; the gradient elution is as follows: 0-1 min, phase A is 95%, phase B is 5%; 1-3 min, phase A decreases from 95% to 80%, and phase B increases from 5% to 20%; 3-7 min, phase A decreases from 80% to 50%, and phase B increases from 20% to 50%; 7-8 min, phase A increases from 50% to 95%, and phase B decreases from 50% to 5%.

[0028] Furthermore, in step (4), the mass spectrometry conditions are: positive ion scanning, multiple reaction monitoring data acquisition mode, capillary voltage of 2500 V; ion source temperature of 150°C; desolvation gas nitrogen temperature of 500°C; desolvation gas nitrogen flow rate of 800 L / h; collision gas argon flow rate of 0.15 mL / min.

[0029] Beneficial effects

[0030] In the present invention, the probe molecule has low cytotoxicity and can achieve efficient and selective derivatization of AP sites in DNA. It reacts with the aldehyde group released from the AP site to form a stable oxime target compound. The alkyne component at its end gives it bioorthogonal chemical functionality, enabling it to react with biotin-azide through a cycloaddition click reaction, and using magnetic bead enrichment technology, achieve targeted detection of AP sites in complex samples. In addition, the introduction of a pyridine heterocyclic structure significantly improves its mass spectrometry response and reaction efficiency. The present invention provides a solid foundation for the screening of genotoxic substances, the development of toxicity testing methods, and its application in the field of food and environmental toxicity screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1This is the synthetic route of the bioorthogonal probe molecule in Example 1 of the present invention.

[0032] Figure 2 The gas chromatography and mass spectrometry of the probes EOA, EMOA and EFOA in the examples and comparative examples are shown.

[0033] Figure 3 The hydrogen and carbon nuclear magnetic resonance spectra of the probes EOA, EMOA, and EFOA in the examples and comparative examples are shown.

[0034] Figure 4 These are the cytotoxicity test results of the probes EOA, EMOA, and EFOA in the examples and comparative examples.

[0035] Figure 5 These are the selectivity experimental results of different probe molecules for aldehyde sites on cellular DNA in Examples and Comparative Examples.

[0036] Figure 6 The difference in mass spectrometric response ability of probes MOA, EMOA and EFOA after addition of AP in the examples and comparative examples.

[0037] Figure 7 These are the experimental results of the in vitro AP site derivatization efficiency of the probe EMOA in the examples.

[0038] Figure 8 This is a flow chart for the enrichment detection of cell AP sites of the present invention.

[0039] Figure 9 Schematic diagram of the bioorthogonal analysis method for AP sites constructed in the present invention and comparison of peak area values ​​and peak response values ​​before and after AP site enrichment in cells.

[0040] Figure 10 is the mass spectrometric response of the blank matrix AP-EMOA in the examples.

[0041] Figure 11 This is the mass spectrum of the blank matrix AP-EMOA adduct in the example.

[0042] Figure 12 This is the background response of the AP site in HepG2 cells in the example and the response after artificially increasing the AP site by heating before enzymatic hydrolysis. DETAILED DESCRIPTION

[0043] The present invention will be further described in detail below with reference to specific embodiments.

[0044] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0045] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods.

[0046] Unless otherwise specified, all materials, reagents, etc. in the following examples and comparative examples can be obtained from commercial sources.

[0047] 1. Reagents and instruments

[0048] (1) Experimental materials: 1-(Chloromethyl)-4-ethynylbenzene, 3-bromopyridine-5-methanol, 4-bromo-2-fluorobenzyl alcohol, trimethylsilylene, copper sulfate, tris(3-hydroxypropyltriazolylmethyl)amine, ethylenediaminetetraacetic acid, aminoguanidine, sodium ascorbate, formamide (Beijing Inokai Technology Co., Ltd.); N-hydroxyphthalimide, dichlorothionyl, bis(triphenylphosphine)palladium chloride, hydrazine hydrate, cuprous chloride (Shanghai Bid Pharmaceutical Technology Co., Ltd.); potassium carbonate, dichloromethane, tetrahydrofuran, triethylamine, ethanol (Beijing Sinopharm Chemical Reagent Co., Ltd.); acetonitrile (chromatographic grade, Beijing Bailingwei Technology Co., Ltd.); alkaline phosphatase (derived from Escherichia coli), phosphodiesterase I, 6-mdA (Shanghai Sangon Biotechnology Co., Ltd.); nuclease P1 (NEB, USA); Biotin-azide (MedChemexpress Biotechnology Co., Ltd., USA); oligonucleotide chain (Shanghai Sangon Biotechnology Co., Ltd.), sequence information is shown in Table 1.

[0049] Table 1 Detailed information of oligonucleotide sequences

[0050]

[0051] (2) Instruments

[0052] GC-MS-QP2020 NX gas chromatography-mass spectrometer (Shimadzu, Japan); Bruker 600M nuclear magnetic resonance spectrometer (Bruker, USA); Mili-Q ultrapure water system (Millipore, USA); N-1210BV-W rotary evaporator (Shanghai Airo Instrument Co., Ltd.); MS105DU electronic balance (Shanghai Mettler Toledo Instrument Co., Ltd.); 3111 CO2 incubator (Thermo, USA); ACQUITY UPLC high-performance liquid chromatograph (Waters, USA); TQ-XS triple quadrupole mass spectrometer (Waters, USA); inverted microscope (Olympus, Japan); low-temperature centrifuge (Sigma, USA).

[0053] (3) Commonly used buffer solutions and reagent configurations

[0054] (1) Tris lysis buffer: 10 mM Tris-base, 0.32 M sucrose, 5 mM MgCl2, 0.1 mM deferoxamine mesylate, pH 7.5, with 1% Triton X-100.

[0055] (2) Tris-HCl solution: (100 mM, pH 8.9): Weigh 1.21 g of Tris-base into a 100 mL volumetric flask, add sterilized ultrapure water to 100 mL, prepare a Tris-HCl solution with a concentration of 100 mM and a pH of 8.9.

[0056] (3) 1 × PBS buffer solution: 20 × PBS buffer dry powder, add ultrapure water to 2 L, sterilize under high pressure, and store at 4 ℃ for later use.

[0057] (4) Proteinase K solution: (20 mg / mL): Weigh 20 mg of proteinase K into 1 mL of sterilized ultrapure water, and store at -20 ℃.

[0058] (5) Nuclease P1 solution: 10 × buffer solution and sterilized ultrapure water are used to prepare 10 U / μL.

[0059] (6) Alkaline phosphatase solution: centrifuge at 14,000 rpm for 5 min at 4 ℃, discard the supernatant (ammonium sulfate solution), add Tris-HCl solution (pH 8.9) to resuspend the precipitate, and prepare an enzyme solution with a concentration of 3 U / 20 μL;

[0060] (7) 5 mM Biotin-azide.

[0061] (8) Premixed solution of 5 mM copper sulfate (CuSO4) and 10 mM tris(3-hydroxypropyl triazole methyl) amine (THPTA).

[0062] (9) 20 mM Aminoquanidine.

[0063] (10) 20 mM Sodium ascorbate.

[0064] (11) Wash Buffer I: 1 × PBS buffer solution, 1 mM ethylenediaminetetraacetic acid (EDTA), 1 M NaCl, 0.01%-0.1% Tween-20, pH 7.5.

[0065] (12) Elution Buffer I: 95% formamide, 10 mM EDTA, pH 8.2.

[0066] Comparative Example 1

[0067] Synthesis of probe O-(4-ethynylbenzyl)hydroxylamine (EOA):

[0068] Dissolve 1-(chloromethyl)-4-ethynylbenzene (600 mg, 5 mmol) in 20 ml acetonitrile, add N-hydroxyphthalimide (980 mg, 6 mmol) and potassium carbonate (140 mg 1 mmol), and react at 60 °C for 10 h. After the reaction, cool to room temperature, filter to obtain a brown solution, add cold water under ice bath and stir, white solid precipitates, filter, wash with cold water, and dry to obtain white solid compound. Then add hydrazine hydrate dropwise in methanol, white precipitate is produced after half an hour of reaction, filter, concentrate the filtrate, and purify by silica gel column chromatography (petroleum ether: ethyl acetate = 7: 1) to obtain brown compound EOA (yield 72%, 396 mg). Gas chromatography and mass spectrometry, nuclear magnetic resonance results are shown in Figure 2-3

[0069] Nuclear magnetic resonance: 1 H NMR (600 MHz, CD2Cl2) δ 7.48 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.0 Hz, 2H), 4.65 (s, 2H), 3.13 (s, 1H), 1.26 (s, 2H); 13 C NMR (151 MHz, CD2Cl2) δ 138.95, 132.09, 132.05, 128.14, 128.12, 121.39, 83.29, 77.23, 77.12.

[0070] Comparative Example 2

[0071] Synthesis of probe O-(4-ethynyl-2-fluorobenzyl)hydroxylamine (EFOA):

[0072] ​Compound, 4-bromo-2-fluorobenzyl alcohol (2050 mg, 10 mmol), dichlorobis(triphenylphosphine)palladium(II) (731 mg, 1 mmol), copper(I) chloride (99 mg, 1 mmol) were dissolved in triethylamine, and nitrogen was bubbled for 10 minutes, then trimethylsilyl acetylene (1470 μL, 15 mmol) was added dropwise, and stirred at 70°C for 6 hours. The reaction mixture was diluted with DCM, filtered, washed with ultrapure water, dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain crude product 3. It was dissolved in a methanol solution, and potassium carbonate was added, and stirred at room temperature for 2 hours. The filtrate was collected by filtration, concentrated, and dissolved in DCM, and dichlorosulfoxide (500 μL, 4 mmol) was added under ice bath, and stirred for 10 minutes, and then reacted at room temperature for 3 hours to obtain intermediate 4. Purification by silica gel column chromatography (petroleum ether: ethyl acetate = 7:1) to obtain brown compound 4 (1475 mg, 8 mmol). It was dissolved in acetonitrile, and N-hydroxyphthalimide (1304 mg, 8 mmol) and potassium carbonate (140 mg, 1 mmol) were added, and reacted at 60°C for 10 hours. After filtration, ice water was added, and a white solid was precipitated, which was filtered and washed with cold water to obtain white solid compound 5. It was dissolved in methanol, and hydrazine hydrate (400 μL, 8 mmol) was added, and the filtrate was collected by filtration after reacting for half an hour, and concentrated to obtain yellow product, which was further purified by silica gel column chromatography to obtain compound EFOA (1020 mg, yield 61%). The results of gas chromatography and mass spectrometry, nuclear magnetic resonance are shown in Figure 2-3

[0073] Nuclear magnetic resonance: 1 H NMR (600 MHz, CD2Cl2) δ 7.39 (d, J = 8.2 Hz, 2H), 7.23 (d, J = 7.9 Hz, 1H), 4.57 (s, 2H), 4.00 (dd, 1H), 1.18 (s, 2H); 13 C NMR (151 MHz, CD2Cl2) δ 148.84, 139.35, 132.42, 128.53, 128.52, 121.79, 83.70, 77.66, 77.53.

[0074] Example 1:

[0075] Synthesis of probe O-((5-ethynylpyridin-3-yl)methyl)hydroxylamine (EMOA):

[0076] As Figure 1 ​As shown, 3-bromopyridine-5-methanol (1106 mg, 10 mmol), dichlorobis(triphenylphosphine)palladium(II) (731 mg, 1 mmol) and cuprous chloride (99 mg, 1 mmol) were dissolved in an appropriate amount of triethylamine, and trimethylsilyl acetylene (1470 μL, 15 mmol) was added dropwise after nitrogen was bubbled in. The reaction was carried out at 70 °C for 6 h. Subsequently, the reaction mixture was filtered, and the filtrate was extracted with saturated brine (50 mL x 3). The organic phase was dried and the solvent was removed under reduced pressure to obtain the crude product 3. Without purification, the product was dissolved in methanol, and potassium carbonate (140 mg, 1 mmol) was added. The reaction was carried out at 60 °C for 10 h. After cooling to room temperature, filtration was performed, and a large amount of ice water was added and stirred. Subsequently, the mixture was filtered while cold, and the white solid was dried to obtain compound 5. The compound was dissolved in 10 mL of methanol, and hydrazine hydrate (400 μL, 8 mmol) was added dropwise. After 30 min, a white precipitate was formed, and the solution was filtered and concentrated to obtain the yellow product. The product was purified by flash column chromatography (petroleum ether: ethyl acetate = 6: 1) to obtain brown compound EMOA (660 mg, yield 62%).

[0077] Compound 3 was dissolved in 50 mL of acetonitrile, and N-hydroxyphthalimide (1304 mg, 8 mmol) and potassium carbonate (140 mg, 1 mmol) were added. The reaction was carried out at 60 °C for 10 h. After cooling to room temperature, filtration was performed, and a large amount of ice water was added and stirred. Subsequently, the mixture was filtered while cold, and the white solid was dried to obtain compound 5. The compound was dissolved in 10 mL of methanol, and hydrazine hydrate (400 μL, 8 mmol) was added dropwise. After 30 min, a white precipitate was formed, and the solution was filtered and concentrated to obtain the yellow product. The product was purified by flash column chromatography (petroleum ether: ethyl acetate = 6: 1) to obtain brown compound EMOA (660 mg, yield 62%). Figure 2-3

[0078] Nuclear magnetic resonance: 1 H NMR (600 MHz, CD2Cl2) δ 8.54 (dd, J = 16.6, 2.2 Hz, 1H), 8.44 (dd, J = 23.9, 2.0 Hz, 1H), 7.72 (t, J = 2.2 Hz, 1H), 4.58 (d, J = 4.7 Hz, 2H), 3.22 (s, 1H), 1.18 (s, 2H); 13 C NMR (151 MHz, CD2Cl2) δ 151.97, 149.25, 138.90, 133.04, 118.81, 80.38, 74.54, 61.90.

[0079] Cytotoxicity experiments of the probes described in Comparative Example 1-2 and Example 1:

[0080] HepG2 cells were seeded at 1 x 10 4 ​The density of 1 cell / hole was inoculated in a 96-well plate and cultured in a 37℃, 5% CO2 incubator for a certain period of time. The probe was set at 3-1000 μmol / L, and blank and control groups were set up, each with 5 duplicate wells. After 24 h of culture, 110 μL of mixed solution of culture medium and CCK-8 reagent at a volume ratio of 10:1 was added to each well, and after 1.5 h of incubation, the OD value of each well was measured at 450 nm wavelength by an enzyme-labeled instrument. The cell survival rate was calculated as follows: cell survival rate = (measured value-blank value) / (control group-blank group) x 100%, and the prism software was used for curve fitting IC 50 value.

[0081] Results: Since the probe is to be derivatized with the AP site in the cell, there is no false positive result at the time of administration. The CCK-8 cytotoxicity experiment verified that the EMOA probe has less cytotoxicity. As shown in Figure 4 , the IC 50 value of EOA is 855.3 μM, the IC 50 value of EMOA is 758.9 μM, and the IC 50 value of EFOA is greater than 1000 μM.

[0082] Example 2

[0083] Selective experiment of different probe molecules (BOA, EOA, EMOA, EFOA, PMOA) on aldehyde groups (AP site, 5fu, 5fc) contained in cell DNA.

[0084] Standard: Under the same conditions, each probe concentration of 10 μmol and dR, 5fu, 5fc concentration of 1 μmol solution were prepared with PBS buffer, and then 200 μL of each was incubated in an EP tube at 37℃. At time points 1 h, 2 h, 3 h, 10 μL of reaction solution was taken and butyl aldehyde (10 μL, 1 mmol) was added to terminate the reaction. After dilution, the target adduct was detected by high performance liquid chromatography.

[0085] Oligonucleotide: Under the same conditions, each probe concentration of 10 μmol and AP-oligo, 5fu-oligo, 5fc-oligo concentration of 1 μmol solution were prepared with PBS buffer, and then 200 μL of each was incubated in an EP tube at 37℃. At time points 1 h, 2 h, 3 h, 10 μL of reaction solution was taken and butyl aldehyde (10 μL, 1 mmol) was added to terminate the reaction. After dilution, the target adduct was detected by high performance liquid chromatography.

[0086] Results: As shown in Figure 5 , the probe molecule has a certain selectivity for the AP site in a short time. According to the experimental data, the reaction activity is as follows: AP site > 5fu > 5fc.

[0087] Example 3

[0088] Experiment on the difference in mass spectrometry response ability after derivatization of different probe molecules with AP sites:

[0089] Under the same conditions, PBS buffer was used to prepare solutions of 10 μmol of each probe BOA, EFOA, and EMOA, and 1 μmol of dR. 200 μL of each solution was then taken and incubated in an EP tube at 37°C for 90 min. 10 μL of the reaction solution was taken and butyraldehyde (10 μL, 1 mmol) was added to terminate the reaction. After dilution, the internal standard 6-mdA was added and the mass spectrometry response of the AP adduct was detected by liquid chromatography-mass spectrometry.

[0090] Results: As Figure 6 As shown in the figure, after the EMOA probe is conjugated to the AP site, its mass spectrometry response is one to two times higher than that of other molecules.

[0091] Example 4

[0092] Experiment on the derivatization efficiency of probe molecule EMOA and AP site:

[0093] Under the same conditions, PBS buffer was used to prepare solutions of 10 μmol EMOA and 1 μmol dR. 200 μL of each solution was then placed in an EP tube and incubated at 37°C for 15, 30, 45, 60, 90, 120, 150, and 180 minutes. 10 μL of the reaction solution was then added to butyraldehyde (10 μL, 1 mmol) to terminate the reaction. After dilution, the internal standard 6-mdA was added and the reaction status was monitored by liquid chromatography-mass spectrometry.

[0094] Results: As Figure 7 As shown, the derivatization efficiency of the EMOA probe was good, reaching equilibrium in one and a half hours, and the derivatization time in cell experiments was determined.

[0095] Example 5

[0096] like Figure 8 As shown, the probe molecule EMOA is used in the AP site derivatization experiment in cells:

[0097] (1) Sample processing

[0098] 1.1 DNA extraction from HepG2 cells:

[0099] Cell digestion: When the cells adhere to the wall and grow to the logarithmic growth phase, the experiment was carried out. Take a dish of HepG2 cells in the logarithmic growth phase, rinse the cells with pre-cooled phosphate buffer, digest the cells with trypsin, and resuspend them to adjust the cell concentration to about 2-3×10 5 EP tube;

[0100] Precipitate cell nuclei: Centrifuge the cell suspension at 1000 rpm for 3 min at 4°C, remove the supernatant, and precipitate the cell nuclei in pre-chilled Tris lysis buffer. Centrifuge at 5000 rpm for 10 min at 4°C, remove the supernatant, and the precipitate is the cell nuclei.

[0101] DNA extraction: 500 μL PBS buffer, 30 μL 10% sodium dodecyl sulfate, and 20 μL proteinase K were added to the obtained cell nuclear pellet, and the solution was incubated at 37°C for 1.5 h to obtain an enzymatic solution containing HepG2 cell DNA.

[0102] 1.2 Derivatization: 29 μL of 100 mM probe (EMOA) was added to 550 μL of the above enzymatic hydrolyzate containing HepG2 cell DNA, and the mixture was incubated at 37° C. for 1.5 h to perform a derivatization reaction to obtain a derivatized sample solution.

[0103] 1.3 DNA precipitation: Add 1.5 mL of pre-cooled isopropanol at -20°C to the derivatized sample solution to precipitate DNA for 1 hour. Wash the precipitate (DNA) three times with 70% ethanol, evaporate to dryness, and collect them together.

[0104] 1.4 Enzymatic hydrolysis: Redissolve the DNA sample in 90 μL of ultrapure water, incubate with nuclease P1 (1 U) at 37°C for 2 h, and then incubate with phosphodiesterase I (0.5 U) and alkaline phosphatase (3 U) for 4 h to obtain the enzymatic hydrolysis solution.

[0105] 1.5 Preparation of the test sample solution: The enzymatic hydrolyzate was added with 1 volume of methanol and 5 volumes of acetonitrile to remove the enzyme, dried by vacuum centrifugation (1500 rpm, 40°C), and reconstituted with 50 μL of ultrapure water to obtain the test sample solution. The test sample solution was transferred to an injection vial for LC-MS / MS analysis to detect the response and peak area of ​​AP-EMOA.

[0106] like Figure 8 As shown, the probe molecule EMOA is applied to the Click reaction and enrichment experiment of AP sites in cell samples:

[0107] (1) Click reaction:

[0108] 1.1 The sample to be tested obtained in Example 5 was transferred to a new EP tube, 10 μl of biotin-azide was added, and the mixture was vortexed for 20 seconds;

[0109] A premixed solution of 2.5 μl CuSO4 and 5.0 μl THPTA was prepared and added to the mixture, and vortexed for 20 seconds.

[0110] 1.2 Add 25 μl each of aminoguanidine and sodium ascorbate to the above mixture, fill with nitrogen, vortex the mixture for 20 seconds, and place it in a metal bath at 37°C for 1 hour to obtain an adduct mixture;

[0111] (2) Magnetic bead enrichment

[0112] 2.1 Immobilization of nucleic acid: fully suspend the magnetic beads, vortex and oscillate for 20 seconds, take 75 μL of magnetic beads into a new EP tube, place it on a magnetic stand for magnetic separation, and discard the supernatant.

[0113] 2.2 Washing the magnetic beads: Add 0.5 mL of Wash Buffer I to thoroughly wash the beads. Vortex the beads for 15 seconds, perform magnetic separation, and discard the supernatant. Repeat the above steps once.

[0114] 2.3 Sample adsorption: Dilute the adduct mixture obtained in 1.2 above with 100 μL Wash Buffer I, add it to the magnetic bead sample, vortex thoroughly to suspend, and incubate on a rotary mixer for 60 minutes.

[0115] 2.4 Magnetic separation: Place the sample on a magnetic stand for 1 min and transfer the supernatant to a new EP tube for subsequent use.

[0116] 2.5 Wash: Add 1 mL of Wash Buffer I to the sample to thoroughly wash the magnetic beads. Vortex the beads for 15 seconds, perform magnetic separation, and discard the supernatant. Repeat the above steps once.

[0117] 2.6 Elution: Add 50 μL Elution Buffer I to the above sample, incubate at 65°C for 10 min, place on a magnetic stand for magnetic separation, collect the supernatant and transfer it to a new EP tube.

[0118] 2.7 Preparation of test sample solution: Centrifuge the above sample at 14,000 rpm for 10 min. Pipette the supernatant and transfer it to an injection vial for LC-MS / MS analysis to detect the response and peak area of ​​the AP-EMOA adduct.

[0119] Example 6

[0120] LC-MS / MS analysis of the sample solution

[0121] 1. Chromatographic conditions: The chromatographic column was a Waters ACQUITY UPLC HSS T3, 100 mm × 2.1 mm, 1.8 μm; the mobile phase A was 0.1% formic acid in water; the mobile phase B was acetonitrile; the flow rate was 0.25 mL / min; the injection volume for AP-EMOA and AP-EMOA adduct compounds was 5 μL.

[0122] Gradient elution was as follows:

[0123] 0-1min, phase A is 95%, phase B is 5%;

[0124] 1-3 min, phase A decreased from 95% to 80%, and phase B increased from 5% to 20%;

[0125] 3-7 min, phase A decreases from 80% to 50%, and phase B increases from 20% to 50%;

[0126] 7-8 min, phase A increased from 50% to 95%, and phase B decreased from 50% to 5%;

[0127] 2. Mass spectrometry conditions: positive ion scan, multiple reaction monitoring data acquisition mode, capillary voltage: 2500 V; ion source temperature: 150°C; desolvation gas nitrogen temperature: 500°C; desolvation gas nitrogen flow rate: 800 L / h; collision gas argon flow rate: 0.15 mL / min;

[0128] 3. Detection parameters:

[0129]

[0130] 4. Perform LC-MS / MS analysis on AP-EMOA and AP-EMOA adduct in the test sample: AP-EMOA and AP-EMOA adduct are used as the horizontal coordinates, and the chromatographic peak area and peak response of AP-EMOA and AP-EMOA adduct in the test sample solution are used as the vertical coordinates.

[0131] Results: As Figure 9 As shown, the specific capture of the target molecule is achieved through techniques such as the Click reaction and magnetic bead separation. Compared to direct derivatization detection, this enrichment effectively eliminates matrix interference, significantly improving sample purity and detection sensitivity. The mass spectrometer response is increased by approximately one order of magnitude. This method not only improves the recovery of the target molecule but also reduces background noise, making subsequent analysis more accurate and reliable.

[0132] Example 7

[0133] Validation of specificity and detection limit of AP-EMOA and AP-EMOAadduct

[0134] (1) Specificity:

[0135] 1.1 Preparation of blank matrix: HepG2 cells containing 10 μg of DNA were processed according to the HepG2 cell DNA extraction, DNA precipitation, enzymatic hydrolysis, and sample solution preparation in Example 5 to obtain a blank matrix;

[0136] 1.2 AP-EMOA and AP-EMOA adduct reference substances were added to the blank matrix to prepare AP-PMOA and AP-EMOA adduct quality control samples at a concentration of 1 ng / mL. The AP-EMOA and AP-EMOA adduct quality control samples were then analyzed by LC-MS / MS.

[0137] Figure 10 The blank matrix was added with the mass spectrometric response of AP-EMOA, which did not interfere with the determination results of AP-EMOA; Figure 11 The blank matrix was added with the mass spectrometric response of AP-EMOA adduct, which did not interfere with the determination results of AP-EMOA adduct.

[0138] (2) Detection limit:

[0139] AP-EMOA adduct control was added to the blank matrix to prepare AP-EMOA and AP-EMOA adduct solutions at concentrations of 0.02, 0.1, 1, 2, 5, 10, 20, 50, and 100 ng / mL, respectively. LC-MS / MS analysis was performed, and a signal-to-noise ratio (S / N) greater than 3 was used as the detection limit for the compound;

[0140] Results: The lowest detection limit of AP-EMOA adduct was 0.05 ppb.

[0141] Example 8

[0142] Verification experiment after artificially adding AP sites:

[0143] The other steps were identical to those in Example 5, with the exception of the enzymatic digestion step, which was performed to artificially increase AP sites by heating before digestion. The extracted DNA sample was reconstituted with 90 μL of ultrapure water, and the EMOA probe was added and incubated at 50°C for 1 h. The sample was then incubated with nuclease P1 (1 U) at 37°C for 2 h.

[0144] Results: See Figure 12 The background content of AP sites in HepG2 cells was low. After artificially increasing the AP sites by heating treatment before enzymatic hydrolysis, the AP site content increased by 5 times, indicating that the molecule has good reproducibility.

[0145] In summary, the invention includes but is not limited to the above embodiments. Any equivalent replacement or partial improvement made under the spirit and principle of the present invention shall be deemed to be within the scope of protection of the present invention.

Claims

1. Use of a compound as a bioorthogonal mass spectrometry-derived probe molecule targeting an abasic site, characterized in that: The structural formula of the compound is: ; The application is for non-disease diagnosis or treatment purposes.

2. A method for preparing a bioorthogonal mass spectrometry-derived probe molecule targeting an abasic site, characterized by: The method steps include: (1) Compound 1 and compound 2 are coupled via Heck reaction to obtain compound 3; (2) Compound 3 was deprotected with K2CO3 and then halogenated to obtain compound 4; (3) Under the action of an acid-binding agent, compound 4 reacts with N-hydroxyphthalimide to remove one molecule of hydrogen halide to obtain compound 5; (4) Compound 5 is deprotected under the action of a reducing agent to obtain a bioorthogonal mass spectrometry-derived probe molecule targeting the abasic site; the structural formula of the probe molecule is: ; Among them, the structural formula of compound 1 is , n2 is 1; the structural formula of compound 2 is , n1 is 1; the structural formula of compound 3 is ; The structural formula of compound 4 is , X is a halogen; the structural formula of compound 5 is ; During the halogenation reaction in step (2), SOCl2 or PBr3 is added; in step (3), the acid binding agent is K2CO3 or triethylamine; in step (4), the reducing agent is hydrazine or hydrazine hydrate.

3. An application of a bioorthogonal mass spectrometry-derived probe molecule targeting an abasic site, characterized in that: The structural formula of the probe molecule is: The probe molecule is used to mark the AP site, and the application is for non-disease diagnosis or treatment purposes.

4. The use of a bioorthogonal mass spectrometry-derived probe molecule targeting an abasic site according to claim 3, wherein: After the probe molecule labels the AP site, the AP site is enriched and detected through a click reaction.

5. A bioorthogonal analysis method for AP sites, characterized by: The method steps include: (1) Adding the probe molecule described in claim 1 to DNA extracted from cells, incubating, precipitating, and enzymatically hydrolyzing to obtain a sample to be tested; (2) Mixing the sample to be tested with biotin-azide to perform a click reaction to obtain an adduct mixed solution; (3) The adduct mixture solution is mixed with avidin magnetic beads, incubated, and magnetically separated to obtain the sample to be analyzed; (4) Perform LC-MS / MS analysis on the samples to be analyzed; In step (1), the probe molecule is added to the DNA enzymatic hydrolysis solution extracted from the cells, and incubated at 37°C for 1 to 2 hours to obtain a derivatized sample solution; the derivatized sample solution is precipitated, enzymatically hydrolyzed, deenzymed, dried, and redissolved to obtain a sample to be tested; In step (2), the sample to be tested and biotin-azide are mixed uniformly to obtain a mixed solution; aminoguanidine and sodium ascorbate are added to the mixed solution, and the mixture is reacted at 37° C. for 1 to 2 hours under nitrogen protection to obtain an adduct mixed solution; In step (4), the liquid chromatography conditions are as follows: the chromatographic column is Waters ACQUITY UPLC HSS T3, 100 mm×2.1 mm, 1.8 μm; the mobile phase A is a 0.1% mass fraction of formic acid in water; the mobile phase B is an acetonitrile solution; the flow rate is 0.25 mL / min, and the injection volume is 5 μL; the gradient elution is as follows: 0-1 min, phase A is 95%, phase B is 5%; 1-3 min, phase A decreases from 95% to 80%, and phase B increases from 5% to 20%; 3-7 min, phase A decreases from 80% to 50%, and phase B increases from 20% to 50%; 7-8 min, phase A increases from 50% to 95%, and phase B decreases from 50% to 5%; In step (4), the mass spectrometry conditions are: positive ion scan, multiple reaction monitoring data acquisition mode, capillary voltage of 2500 V; ion source temperature of 150°C; desolvation gas nitrogen temperature of 500°C; desolvation gas nitrogen flow rate of 800 L / h; collision gas argon flow rate of 0.15 mL / min.

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