Bio-orthogonal mass spectrum derivative probe molecule aiming at base removal site, preparation and application thereof

Abstract

The invention relates to a biological orthogonal mass spectrum derivative probe molecule aiming at a base removal site as well as preparation and application of the biological orthogonal mass spectrum derivative probe molecule, and belongs to the technical field of analytical chemistry. The structural formula of the probe molecule is # imgabs0 #, n1 is an integer from 1 to 3, and n2 is an integer from 1 to 2. The probe molecule has the characteristic of low toxicity, has selectivity on an AP site contained on DNA, and efficiently reacts with an aldehyde group released by the AP site to form a stable oxime target compound; the method comprises the following steps: carrying out cycloaddition click reaction on a terminal alkyne component which is used for detecting the AP site adduct and has a biological orthogonal chemical function and biotin-azide, and combining magnetic bead enrichment to realize targeted detection on the AP site adduct in a complex sample; in addition, due to a pyridine heterocyclic structure, the mass spectrum response and the reaction efficiency are improved, and the detection sensitivity and the mass spectrum response are remarkably improved.

Description

Technical Field

[0001] The invention relates to a bioorthogonal mass spectrometry-derived probe molecule targeting an abasic site, preparation and application thereof, 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, which originate from the breakage of glycosidic bonds in DNA. Studies have shown that each mammalian cell generates an average of about 10,000 endogenous AP sites per day. If these damages are not repaired in time, they may lead to obstacles in DNA replication, breaks in DNA chains, and further induce mutations and cells. Under physiological conditions, AP sites can show highly active aldehyde groups, making them potential important detection targets in DNA damage research. Through bioconjugation labeling reactions, some highly selective and specific hydroxylamine and hydrazine molecules can be used to covalently label AP sites 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] Among them, the fluorescence analysis method has achieved the monitoring and tracing of biologically active substances or targets in cells and even in vivo in terms of "visualization". Compared with the chemical derivatization-assisted LC-MS / MS technology, the developed "Turn On" fluorescent probe technology has obvious advantages. By fluorescently labeling DNA, the content of the target analyte can be directly read, avoiding the complex enzymatic treatment steps. The biggest disadvantage is that although the fluorescent labeling method can be used for dynamic monitoring, photobleaching may occur during long-term observation, reducing the signal intensity, or quenching, affecting the stability and reliability of the fluorescent signal. "Selective Labeling Aldehydes in DNA" reports the design and synthesis of a naphthalene dicarboxylic acid imide hydroxylamine probe to selectively label the entire natural aldehyde present in DNA, including 5-formylcytosine, 5-formyluracil and abasic sites. The fluorescence properties of the generated nucleosides were studied in detail, and the reactivity of hydroxylamine and amine groups to aldehydes in DNA was discussed, providing an important reference for the design of DNA selective 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 Nucleotides, but the method has low universality due to the need to use appropriate antibodies.

[0005] In addition, LC-MS / MS has high sensitivity and good selectivity, and has obvious advantages in the qualitative and quantitative detection of nucleoside derivatives, but there are some challenges in actual operation. The operation process of cell lysis to extract DNA and perform chemical derivatization labeling is relatively complicated. Due to background interference problems, it is impossible to accurately quantify AP sites in low-trace samples. "Quantitation of Apurinic / Apyrimidinic Sites in Isolated DNA and in Mammalian Tissue with a Reduced Level of Artifacts" developed an LC-MS / MS quantitative method using PMOA reagent to derivatize and label AP sites, showing that directly processing AP sites in the cell lysis stage can significantly reduce false positive results compared to post-processing of DNA extraction. This method successfully detected approximately 0.9 AP sites / 10 in mouse liver tissue samples. 7 Nucleotides. DNA is extracted by cell lysis and chemically derivatized. However, this method relies on professional technicians and large equipment, and the sample operation is relatively complicated, which may produce additional AP sites. It is also affected by problems such as sample complexity and matrix effects, resulting in the ionization efficiency of the target compound being suppressed, affecting the accuracy of the quantitative results. It is urgent to develop new qualitative and quantitative analysis methods that can specifically identify and capture AP sites in complex matrices. Summary of the invention

[0006] In view of the poor selectivity of fluorescent or mass spectrometry probe molecules in the prior art when detecting DNA aldehyde sites, and the inability to accurately and quantitatively analyze AP sites in low-trace samples due to matrix effects after derivatization and enzymatic hydrolysis of cell samples, the present invention proposes a bio-orthogonal mass spectrometry derivatization probe molecule for abasic sites and its preparation method and application. The design of the probe molecule aims to improve the selectivity and sensitivity of abasic sites, promote the accurate analysis of DNA damage and its application in biomedical research.

[0007] To achieve the above objectives, the technical solution of the present invention is as follows.

[0008] A bioorthogonal mass spectrometry-derived probe molecule for an abasic site, wherein the structural formula of the probe molecule is: Here, n1 is an integer of 1 to 3, and n2 is an integer of 1 to 2.

[0009] A method for preparing a bioorthogonal mass spectrometry-derived probe molecule for an abasic site according to the present invention, 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 a molecule of hydrogen halide to obtain compound 5;

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

[0014] Among them, the structural formula of compound 1 is n2 is an integer of 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 described in 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, the method steps comprising:

[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, and magnetically separating to obtain a sample to be analyzed;

[0024] (4) Performing liquid chromatography-mass spectrometry (LC-MS / MS) analysis on the samples to be analyzed.

[0025] Furthermore, in step (1), the probe molecule is added to a DNA hydrolysate 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 is 2500V; ion source temperature is 150°C; desolvation gas nitrogen temperature is 500°C; desolvation gas nitrogen flow rate is 800L / h; collision gas argon flow rate is 0.15mL / 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 a bioorthogonal chemical function, and can react with biotin-azide through a cycloaddition click reaction, and utilizes magnetic bead enrichment technology to 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 applications in the field of food and environmental toxicity screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1This is the synthesis 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 embodiments 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 embodiments and comparative examples are shown.

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

[0035] Figure 5 The results are the selectivity experiments of different probe molecules for aldehyde sites on cellular DNA in the examples and comparative examples.

[0036] Figure 6 The difference in mass spectrometry response ability after the probes MOA, EMOA and EFOA in the examples and comparative examples are added with AP.

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

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

[0039] Fig. 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] Fig.10 It is the mass spectrometric response of the blank matrix AP-EMOA in the example.

[0041] Fig.11 The mass spectrum of the blank matrix AP-EMOA adduct in the example.

[0042] Fig.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 in conjunction with 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, cupric 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, 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 instrument (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 reagents

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

[0055] (2) Tris-HCl solution: (100 mM, pH 8.9): Accurately weigh 1.21 g of Tris-base into a 100 mL volumetric flask, add sterile ultrapure water to make up to 100 mL, and 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 powder, add ultrapure water to 2 L, sterilize by high pressure, and store at 4°C until use.

[0057] (4) Protease solution: (20 mg / mL): Weigh 20 mg of proteinase K and dissolve it in 1 mL of sterile ultrapure water. Store it in a freezer at -20°C.

[0058] (5) Nuclease P1 solution: Prepare 10U / μL using 10× buffer solution and sterile ultrapure water.

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

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

[0061] (8) A premixed solution of 5 mM copper sulfate (CuSO4) and 10 mM tris(3-hydroxypropyltriazolylmethyl)amine (THPTA).

[0062] (9) 20 mM aminoguanidine.

[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 of acetonitrile, add N-hydroxyphthalimide (980 mg, 6 mmol) and potassium carbonate (140 mg 1 mmol), and react at 60°C for 10 hours. After the reaction, cool to room temperature and filter to obtain a brown solution. Add cold water and stir under an ice bath. A white solid precipitates. After filtering, washing with cold water, and drying, a white solid compound is obtained. Then, hydrazine hydrate is added dropwise to the methanol. After half an hour of reaction, a white precipitate is produced. After filtration, concentration of the filtrate and purification by silica gel column chromatography (petroleum ether: ethyl acetate = 7:1), a brown compound EOA (yield 72%, 396 mg) is obtained. The results of gas chromatography, mass spectrometry, and nuclear magnetic resonance are as follows: Figure 2-3 shown.

[0069] NMR: 1 H NMR (600MHz, CD2Cl2) δ7.48(d,J=8.2Hz,2H),7.32(d,J=8.0Hz,2H),4.65(s,2H),3.13(s,1H),1.26(s,2H); 13 C NMR (151MHz, CD2Cl2) δ138.95,132.09,132.05,128.14,128.12,121.39,83.29,77.23,77.12.

[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), cuprous chloride (99 mg, 1 mmol) were dissolved in triethylamine, nitrogen was introduced for 10 minutes, trimethylsilyl acetylene (1470 μL, 15 mmol) was added dropwise, and magnetic stirring was carried out at 70°C for 6 hours. The reaction mixture was diluted with DCM and filtered, washed with ultrapure water, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain a crude product 3. It was dissolved in a methanol solution and potassium carbonate was added, stirred at room temperature for 2 hours, the filtrate was collected by filtration and concentrated, and re-dissolved in DCM, dichlorothionyl (500 μL, 4 mmol) was added under ice bath, magnetic stirring was carried out for 10 minutes, and then the reaction was carried out at room temperature for 3 hours to obtain intermediate 4. It was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 7:1) to obtain a brown compound 4 (1475 mg, 8 mmol). Redissolve in acetonitrile, add N-hydroxyphthalimide (1304 mg, 8 mmol) and potassium carbonate (140 mg, 1 mmol), react at 60°C for 10 h, filter and add ice water to precipitate a white solid, filter and wash with cold water to obtain a white solid compound 5. Dissolve in methanol, add hydrazine hydrate (400 μL, 8 mmol), react for half an hour, filter and collect the filtrate, concentrate to obtain a yellow product, and purify by silica gel column chromatography to obtain compound EFOA (1020 mg, yield 61%). The results of gas chromatography, mass spectrometry and nuclear magnetic resonance are as follows: Figure 2-3 shown.

[0073] NMR: 1 H NMR (600MHz, CD2Cl2) δ7.39(d,J=8.2Hz,2H),7.23(d,J=7.9Hz,1H),4.57(s,2H),4.00(dd,1H),1.18(s,2H); 13 C NMR (151MHz, CD2Cl2) δ148.84,139.35,132.42,128.53,128.52,121.79,83.70,77.66,77.53.

[0074] Embodiment 1:

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

[0076] like Figure 1As 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 first dissolved in an appropriate amount of triethylamine, and trimethylsilyl acetylene (1470 μL, 15 mmol) was added dropwise after nitrogen was introduced, and the mixture was reacted at 70°C for 6 h. The reaction mixture was then filtered, and the filtrate was extracted with saturated brine (50 ml × 3). The organic phase was dried and the solvent was removed under reduced pressure to obtain a crude product 3. Without purification, the product was dissolved in methanol and potassium carbonate was added, stirred at room temperature for 2 h, the filtrate was collected by filtration, concentrated and re-dissolved in DCM, dichlorothionyl (500 μL, 4 mmol) was added under ice bath, magnetic stirring was performed for 10 minutes, and the mixture was reacted at room temperature for 3 h. A large amount of dichloromethane (DCM) was added, and the mixture was extracted with saturated brine. The organic phase was dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the product 4. The residue was rapidly separated and purified by silica gel column (petroleum ether:ethyl acetate=5:1) to obtain brown compound 4 (1050 mg, 8 mmol).

[0077] Compound 3 was redissolved in 50 mL of acetonitrile, and N-hydroxyphthalimide (1304 mg, 8 mmol) and potassium carbonate (140 mg, 1 mmol) were added, and the mixture was reacted at 60 °C for 10 h. After cooling to room temperature, the mixture was filtered, and a large amount of ice water was added and stirred. Then, the mixture was filtered while cold and dried to obtain a white solid compound 5. The mixture was dissolved in 10 mL of methanol, and hydrazine hydrate (400 μL, 8 mmol) was added dropwise. After 30 minutes of reaction, a white precipitate was precipitated. After filtration, the solution was concentrated to obtain a yellow product, which was quickly separated and purified by a silica gel column (petroleum ether: ethyl acetate = 6:1) to obtain a brown compound EMOA (660 mg, yield 62%). The results of gas chromatography, mass spectrometry, and nuclear magnetic resonance are as follows: Figure 2-3 shown.

[0078] NMR: 1 H NMR (600MHz, CD2Cl2) δ8.54(dd,J=16.6,2.2Hz,1H),8.44(dd,J=23.9,2.0Hz,1H),7.72(t,J=2.2Hz,1H),4.58(d,J=4.7Hz,2H),3.22(s,1H),1.18(s,2H); 13 C NMR (151MHz, CD2Cl2) δ151.97,149.25,138.90,133.04,118.81,80.38,74.54,61.90.

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

[0080] HepG2 cells were collected at 1×10 4Cells were inoculated at a density of 100 / well in a 96-well plate and cultured in a 37°C, 5% CO2 incubator for a certain period of time. 3-1000 μmol / L probes, blank groups, and control groups were set up respectively, with 5 replicate wells in each group. After 24 hours of culture, 110 μL of a mixed solution of culture medium and CCK-8 reagent in a volume ratio of 10:1 was added to each well. After incubation for 1.5 hours, the OD value of each well was measured at a wavelength of 450 nm using an enzyme reader, and the cell survival rate was calculated: cell survival rate = (measured value - blank value) / (control group - blank group) × 100%, and the curve was fitted using prism software IC 50 value.

[0081] Results: Since the probe is derivatized with the AP site in the cell, there is no false positive result when the drug is administered. The CCK-8 cytotoxicity test verified that the EMOA probe has low cytotoxicity. Figure 4 As shown, the IC of EOA 50 The value is 855.3 μM, and the IC of EMOA 50 The value is 758.9 μM, and the IC 50 The value is greater than 1000 μM.

[0082] Example 2

[0083] Selectivity experiment of different probe molecules (BOA, EOA, EMOA, EFOA, PMOA) for aldehyde sites (AP site, 5fu, 5fc) contained in cellular DNA.

[0084] Standard products: Under the same conditions, PBS buffer was used to prepare solutions with a concentration of 10 μmol of each probe and a concentration of 1 μmol of dR, 5fu, and 5fc. Then 200 μL of each solution was taken and incubated at 37°C in an EP tube. 10 μL of the reaction solution at time points 1h, 2h, and 3h was taken and butyraldehyde (10 μL, 1 mmol) was added to terminate the reaction. After dilution, the target adducts were detected by HPLC.

[0085] Oligonucleotides: Under the same conditions, PBS buffer was used to prepare solutions of 10 μmol of each probe and 1 μmol of AP-oligo, 5fu-oligo, and 5fc-oligo. Then 200 μL of each solution was incubated in an EP tube at 37°C. 10 μL of the reaction solution was taken at 1h, 2h, and 3h and butyraldehyde (10 μL, 1 mmol) was added to terminate the reaction. After dilution, the target adducts were detected by HPLC.

[0086] Results: Figure 5 It is shown that the probe molecularization has a certain selectivity for the AP site in a short time, and the reaction activity is shown by the experimental data: 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. Then, 200 μL of each solution was 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 AP adducts was detected by liquid mass spectrometry.

[0090] Results: Figure 6 As shown, after the EMOA probe is coupled 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 efficiency of derivatization of probe molecule EMOA and AP site:

[0093] Under the same conditions, PBS buffer was used to prepare probe EMOA concentration of 10 μmol and dR concentration of 1 μmol solutions, and then 200 μL of each solution was taken and incubated in an EP tube at 37°C, with time points of 15min; 30min; 45min; 60min; 90min; 120min; 150min; 180min. 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 reaction status was detected by liquid mass spectrometry;

[0094] Results: Figure 7 As shown, the derivatization efficiency of EMOA probe is 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. A dish of HepG2 cells in the logarithmic growth phase was taken, and the cells were rinsed with pre-cooled phosphate buffer, and then the cells were digested with trypsin and resuspended to adjust the cell concentration to about 2-3×10 5 pcs / 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-cooled Tris lysis buffer, then centrifuge at 5000 rpm for 10 min at 4°C, remove the supernatant, and the precipitate is the cell nucleus;

[0101] Extraction of DNA: Add 500 μL of PBS buffer, 30 μL of 10% sodium dodecyl sulfate, and 20 μL of proteinase K to the obtained cell nuclear precipitate, and incubate the solution 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 the above 550 μL enzymatic hydrolyzate containing HepG2 cell DNA, and 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 naturally and collect them together.

[0104] 1.4 Enzymatic hydrolysis: The DNA sample was re-dissolved in 90 μL of ultrapure water, incubated with nuclease P1 (1 U) at 37°C for 2 h, and then incubated 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: Add 1 volume of methanol and 5 volumes of acetonitrile to the enzymatic hydrolyzate to remove the enzyme, dry it by vacuum centrifugation (1500 rpm, 40°C), and reconstitute it 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 above mixture, and vortexed for 20 seconds;

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

[0111] (2) Magnetic beads enrichment

[0112] 2.1 Immobilization of nucleic acid: fully suspend the magnetic beads, vortex 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 Wash Buffer I, wash the magnetic beads thoroughly, vortex the magnetic 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 and suspend it thoroughly, and incubate it on a rotating mixer for 60 min.

[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 Washing: Add 1 mL of Wash Buffer I to the above sample, wash the magnetic beads thoroughly, vortex the magnetic 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 sample solution: Place the above sample in a centrifuge at 14000 rpm for 10 min, transfer the supernatant to an injection vial for LC-MS / MS analysis, and detect the response and peak area of ​​AP-EMOA adduct.

[0119] Example 6

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

[0121] 1. Chromatographic conditions: 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% formic acid aqueous solution; the mobile phase B is an acetonitrile solution; the flow rate is 0.25 mL / min; the injection volume for measuring AP-EMOA and AP-EMOA adduct compounds is 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] 3-7min, 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 scanning, 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 sample to be tested: 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 sample solution to be tested are used as the vertical coordinates.

[0131] Results: Fig. 9 As shown in the figure, it is specifically captured through Click reaction, magnetic bead separation and other technologies. Compared with direct derivatization detection, the matrix interference problem is effectively removed after enrichment, and the purity and detection sensitivity of the sample are significantly improved. The mass spectrometry response is improved by about one order of magnitude. This method not only improves the recovery rate of the target molecule, but also reduces the background noise, making the 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 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 Add AP-EMOA and AP-EMOA adduct reference substances to the blank matrix to prepare AP-PMOA and AP-EMOA adduct quality control samples with a concentration of 1 ng / mL, thus obtaining AP-EMOA and AP-EMOA adduct quality control samples. Perform LC-MS / MS analysis.

[0137] Fig.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; Fig.11 The blank matrix was added with the mass spectrometry 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 reference was added to the blank matrix, and AP-EMOA and AP-EMOA adduct solutions with concentrations of 0.02, 0.1, 1, 2, 5, 10, 20, 50, and 100 ng / mL were prepared for LC-MS / MS analysis, and the signal-to-noise ratio (S / N) greater than 3 was taken as the detection limit of the compound;

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

[0141] Example 8

[0142] Verification experiment after artificially adding AP sites:

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

[0144] Results: See Fig.12 The background content of AP sites in HepG2 cells was low. After heating treatment before enzymatic hydrolysis to artificially increase AP sites, the content of AP sites 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 substitution or partial improvement made under the spirit and principle of the invention shall be deemed to be within the protection scope of the invention.

Claims

1. A bioorthogonal mass spectrometry-derived probe molecule targeting an abasic site, characterized in that: The structural formula of the probe molecule is: Here, n1 is an integer of 1 to 3, and n2 is an integer of 1 to 2.

2. A method for preparing a bioorthogonal mass spectrometry-derived probe molecule targeting an abasic site as claimed in claim 1, characterized in that: The method steps include: (1) Compound 1 and compound 2 are coupled via Heck reaction to obtain compound 3; (2) Compound 3 was deprotected by K2CO3 and then halogenated to obtain compound 4; (3) In the presence of an acid-binding agent, compound 4 reacts with N-hydroxyphthalimide to remove a molecule of hydrogen halide to obtain compound 5; (4) Compound 5 is deprotected under the action of a reducing agent to obtain a bio-orthogonal mass spectrometry-derived probe molecule targeting the abasic site; Among them, the structural formula of compound 1 is n2 is an integer of 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 3. The method for preparing a bioorthogonal mass spectrometry-derived probe molecule for an abasic site according to claim 2, characterized in that: 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.

4. A use of the bioorthogonal mass spectrometry-derived probe molecule for abasic sites as claimed in claim 1, characterized in that: The probe molecule is used to mark the AP site.

5. The use of a bioorthogonal mass spectrometry-derived probe molecule for an abasic site as claimed in claim 4, characterized in that: After the probe molecule labels the AP site, the AP site is enriched and detected through a click reaction.

6. A bioorthogonal analysis method for AP sites, characterized in that: The method steps include: (1) adding the probe molecule described in claim 1 to DNA extracted from cells, and performing incubation, precipitation, and enzymatic hydrolysis 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) mixing the adduct mixed solution with avidin magnetic beads, incubating, and magnetically separating to obtain a sample to be analyzed; (4) Perform LC-MS / MS analysis on the samples to be analyzed.

7. A bioorthogonal analysis method for AP sites as claimed in claim 1, characterized in that: In step (1), the probe molecule is added to the DNA enzymatic hydrolyzate 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 subjected to precipitation, enzymatic hydrolysis, enzyme removal, drying and re-dissolution to obtain a sample to be tested.

8. A bioorthogonal analysis method for AP sites as claimed in claim 7, characterized in that: 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.

9. A bioorthogonal analysis method for AP sites as claimed in claim 8, characterized in that: In step (4), the liquid chromatography conditions are as follows: the chromatographic column is Waters ACQUITY UPLC HSS T3, 100mm×2.1mm, 1.8μm; the mobile phase A is a formic acid aqueous solution with a mass fraction of 0.1%; the mobile phase B is an acetonitrile solution; the flow rate is 0.25mL / min, and the injection volume is 5μL; the gradient elution is as follows: 0-1min, phase A is 95%, and phase B is 5%; 1-3min, phase A decreases from 95% to 80%, and phase B increases from 5% to 20%; 3-7min, phase A decreases from 80% to 50%, and phase B increases from 20% to 50%; 7-8min, phase A increases from 50% to 95%, and phase B decreases from 50% to 5%.

10. A bioorthogonal analysis method for AP sites as claimed in claim 9, characterized in that: In step (4), the mass spectrometry conditions are: positive ion scanning, multiple reaction monitoring data acquisition mode, capillary voltage of 2500V; ion source temperature of 150°C; desolvation gas nitrogen temperature of 500°C; desolvation gas nitrogen flow rate of 800L / h; collision gas argon flow rate of 0.15mL / min.

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