Photosensitive mass spectrum probe as well as preparation method and application thereof in mass spectrum detection

Abstract

The invention discloses a photosensitive mass spectrum probe as well as a preparation method and application thereof in mass spectrum detection, and belongs to the technical field of organic chemistry and analytical chemistry. According to the invention, a thioxanthone oxime benzenesulfonate structure is used as a basis and is bonded with an active reaction group to construct a high-efficiency photosensitive mass spectrum probe, so that rapid labeling of an analyte and online high-efficiency photolysis of a labeled product can be realized, and the labeled product with a sulfonic acid group is released. The invention also provides a preparation method of the photosensitive mass spectrum probe, the photosensitive mass spectrum probe with a target structure is synthesized in a simple and feasible manner, and the preparation method has good guidance and feasibility. When the photosensitive mass spectrum probe is applied to metabolite labeling and mass spectrum detection, the detection sensitivity is improved, the ion inhibition effect caused by excessive labeling reagents is reduced, high-flux and high-sensitivity detection analysis of low-ionization-efficiency analytes is realized, and the probe has a wide prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of organic chemistry and analytical chemistry, and in particular to a photosensitive mass spectrometry probe and a preparation method thereof and an application thereof in mass spectrometry detection. Background Art

[0002] Flow injection-high resolution mass spectrometry (FI-HRMS) is a commonly used method for high-throughput metabolite analysis due to its advantages such as fast analysis speed, low cost and simple operation steps (no need to undergo column equilibration, chromatographic gradient condition optimization, etc.). However, in practical applications, this type of technology still faces two important challenges. First, since some metabolites lack easily ionized groups, they are difficult to be effectively ionized in the electrospray ionization (ESI) source, making mass spectrometry (MS) detection difficult; second, since there is no chromatographic separation process, complex biological sample matrices will lead to more serious ion suppression and matrix effects, affecting the sensitivity of metabolite detection and the coverage of metabolite analysis.

[0003] In order to solve the problem that metabolites lack easily ionized groups, chemical derivatization technology can enhance the ionization ability of analytes in ESI source by modifying their structures, thereby improving the detection sensitivity. Trends in Analytical Chemistry 2019, 119 , 115608.) introduced the LC-MS technique combined with chemical derivatization for mass spectrometry detection of various low-abundance, low-ionization-efficiency functional group small molecule metabolites, thereby achieving highly sensitive detection of metabolites. However, the derivatization process usually uses a large amount of derivatization reagents. If not separated by chromatography, the excess derivatization reagents will continue to enter the mass spectrometer with the sample, causing serious matrix effects, thereby affecting the analytical sensitivity.

[0004] In summary, how to improve the ionization efficiency of metabolites while effectively controlling the influence of matrix effects is the key to achieving high-throughput FI-HRMS metabolomics analysis. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, in the first aspect of the present invention, a type of photosensitive mass spectrometry probe that can realize rapid labeling of analytes and online efficient photolysis of labeled products is provided. The photosensitive mass spectrometry probe includes a photosensitive portion (iTASO) and an active reactive group (RG), wherein the active reactive group includes ONH2, NH2, NHS, and MAL. The structural formula of the photosensitive mass spectrometry probe is as follows: .

[0006] In the second aspect of the present invention, a method for preparing a type of photosensitive mass spectrometry probe according to the first aspect of the present invention is provided, comprising the following steps: (1) 2-isopropylthioxanthone and hydroxylamine hydrochloride are dissolved in a solvent, and then sodium hydroxide is added and reacted to obtain compound A, whose structural formula is as follows: ; (2) Compound A is dissolved in a solvent, and triethylamine (TEA) and p-bromomethylphenylsulfonyl chloride are then added and reacted to obtain compound B, whose structural formula is as follows: ; (3) An active reactive group is introduced through a substitution reaction to replace the chlorine atom in compound B to obtain a photosensitive mass spectrometry probe with the corresponding structure.

[0007] Preferably, in step (1), the amount of 2-isopropylthioxanthone used is 1.0 equiv., the amount of hydroxylamine hydrochloride used is 6-10 equiv., and the amount of sodium hydroxide used is 10-15 equiv.

[0008] Preferably, in step (1), the reaction temperature is 100-120°C and the reaction time is 8-20 h.

[0009] Preferably, in the step (2), the amount of compound A used is 1.0 equiv., the amount of triethylamine used is 2.0-3.0 equiv., and the amount of p-bromomethylphenylsulfonyl chloride used is 1.0-3.0 equiv.

[0010] Preferably, in step (2), the reaction is carried out at room temperature and the reaction time is 30-60 min.

[0011] In the process, the synthesis of compound A and compound B is carried out in a solution environment. Those skilled in the art can select the appropriate solvent type and amount according to the substrates, product physical and chemical properties and actual conditions of each reaction. For example, in the synthesis of compound A, a mixture of pyridine / methanol (1:1, v / v) can be used to create a solution environment for the reaction; in the synthesis of compound B, dichloromethane (DCM) can be used. Other suitable types of solvents and their mixtures can also achieve the purpose of synthesis. In addition, at the specific operational level, sodium hydroxide is preferably added under ice bath conditions; and p-bromomethylphenylsulfonyl chloride is preferably added in batches under ice bath and protective atmosphere. The above operations are all helpful to ensure the safety and smooth progress of the reaction.

[0012] Preferably, when the active reaction group is ONH2, the process of step (3) is as follows: N-hydroxyphthalimide is dissolved in a solvent, potassium carbonate is added to carry out a first-stage reaction, and compound B is added after solid appears in the reaction solution to carry out a second-stage reaction; after the reaction is completed, the product is collected and dissolved in a solvent, and then hydrazine hydrate is added and refluxed to obtain a photosensitive mass spectrometry probe (iTASO-ONH2).

[0013] The structural formula of iTASO-ONH2 is as follows: .

[0014] Further preferably, the amount of N-hydroxyphthalimide used is 1.0-1.5 equiv., the amount of potassium carbonate used is 2.0-3.0 equiv., the amount of compound B used is 1.0 equiv., and the amount of hydrazine hydrate used is 20-30 equiv.

[0015] Further preferably, the first stage reaction is carried out at room temperature; the second stage reaction is carried out at room temperature for 12-16 h; the temperature of the reflux reaction is 80-90 °C and the reaction time is 20-30 min.

[0016] Based on the general selection principle of solvents in this field, technicians can also select the appropriate solvent type and amount according to the substrate type, etc. In this step, the first and second stage reactions can be carried out in a solution environment created by N,N-dimethylformamide (DMF). After the reaction is completed, the corresponding product can be simply obtained by adding water and filtering, and then the product is dissolved in ethanol for subsequent reflux reaction. Other suitable solvents or separation methods can also achieve the above purpose.

[0017] Preferably, when the active reaction group is NH2, the process of step (3) is as follows: compound B and potassium phthalimide are dissolved in a solvent and reacted; after the reaction is completed, the product is collected and dissolved in a solvent, and then hydrazine hydrate is added and refluxed to obtain a photosensitive mass spectrometry probe (iTASO-NH2).

[0018] The structural formula of iTASO-NH2 is as follows: .

[0019] Further preferably, the amount of compound B used is 1.0 equiv., the amount of potassium phthalimide used is 1.5-2.0 equiv., and the amount of hydrazine hydrate used is 20-30 equiv.

[0020] Further preferably, the reaction is carried out at room temperature for 12-16 h; the temperature of the reflux reaction is 80-90° C. for 20-30 min.

[0021] Similarly, this step can also use DMF as the reaction solvent. After the reaction is completed, water is added and filtered to obtain the corresponding product, and then the product is dissolved in ethanol for reflux reaction. Other suitable solvents or separation methods can also achieve the above purpose.

[0022] Preferably, when the active reaction group is NHS, the process of step (3) is as follows: iTASO-NH2 and triethylamine are dissolved in a solvent, and then succinic anhydride is added to carry out a first-stage reaction to obtain an intermediate; the intermediate and N-hydroxysuccinimide are dissolved in a solvent, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) is added to carry out a second-stage reaction to obtain a photosensitive mass spectrometry probe (iTASO-NHS).

[0023] The structural formula of iTASO-NHS is as follows: .

[0024] In iTASO-NHS, the process of introducing NHS can be regarded as first preparing iTASO-NH2, and then further reacting on the basis of iTASO-NH2 to obtain the target iTASO-NHS. The iTASO-NH2 used can be synthesized by the method provided by the present invention.

[0025] Further preferably, the amount of iTASO-NH2 is 1.0 equiv., the amount of triethylamine is 2.0-3.0 equiv., the amount of succinic anhydride is 1.0-1.5 equiv., the amount of N-hydroxysuccinimide is 1.0-1.5 equiv., and the amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1.5-2.0 equiv.

[0026] Further preferably, the first stage reaction is carried out at room temperature, and the reaction time is 30-60 min; the second stage reaction is carried out at room temperature, and the reaction time is 30-60 min.

[0027] Similarly, this step can use DMF as the solvent for the first stage reaction and DCM as the solvent for the second stage reaction. During the operation, succinic anhydride is preferably added in batches, and EDCI is preferably added under ice bath conditions.

[0028] Preferably, when the active reaction group is MAL, the process of step (3) is as follows: iTASO-NH2 and 4-maleimidobutyric acid are dissolved in a solvent, and then triethylamine and 2-(7-azobenzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HATU) are added and reacted to obtain a photosensitive mass spectrometry probe (iTASO-MAL).

[0029] The structural formula of iTASO-MAL is as follows: .

[0030] Similarly, in iTASO-MAL, the process of introducing MAL can be regarded as first preparing iTASO-NH2, and then further reacting on its basis to obtain the target iTASO-MAL.

[0031] Further preferably, the amount of iTASO-NH2 used is 1.0 equiv., the amount of 4-maleimidobutyric acid used is 1.0-2.0 equiv., the amount of triethylamine used is 2.0-3.0 equiv., and the amount of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate used is 1.3-2.0 equiv.

[0032] Further preferably, the reaction is carried out at room temperature and the reaction time is 30-60 min.

[0033] Similarly, DMF can be used as the solvent for the reaction in this step. Other suitable solvents can also achieve the above purpose.

[0034] In the above synthesis, technicians can use common methods in the art to monitor the progress of the reaction and appropriately purify the products of each reaction.

[0035] In the third aspect of the present invention, provided is the application of a type of photosensitive mass spectrometry probe according to the first aspect of the present invention or a photosensitive mass spectrometry probe prepared by the method of the second aspect of the present invention, specifically the application of the photosensitive mass spectrometry probe in metabolite labeling and mass spectrometry detection.

[0036] Preferably, the application includes labeling and mass spectrometry detection of carbonyl analytes, carboxylic acid analytes, amino analytes, and thiol analytes.

[0037] The online photolysis FI-ESI(-)-HRMS analysis method established based on this probe can improve the sensitivity of analyte detection while being less susceptible to the ion suppression caused by excessive labeling reagents, thereby achieving high-throughput and high-sensitivity detection of analytes with low ionization efficiency.

[0038] Preferably, the application includes sample detection by FI-ESI(-)-HRMS using the labeled product of the photosensitive mass spectrometry probe.

[0039] Based on the above technical scheme, the design idea and principle of the present invention lies in that the inventor team noticed that photoacid generators (PAGs) are a type of photosensitive substances that can generate acid under light conditions and are widely used in photoresists, 3D printing, dental materials, and biochemical research. As a type of PAG, thioxanthone oxime benzene sulfonate can efficiently release sulfonic acid under 365 nm wavelength light radiation. As a strong electron-withdrawing group, sulfonic acid is easily ionized in the ESI negative ion mode and has a good mass spectrometry response. In addition, compared with the ESI positive ionization mode, the ESI negative ionization mode generally has lower background noise, so to a certain extent, the ESI negative ion mode shows a weaker matrix effect.

[0040] Therefore, the present invention uses the thioxanthone oxime benzene sulfonate structure as the basis to bond the active reaction group to construct a set of efficient photosensitive mass spectrometry probes. The product labeled with this type of probe can efficiently release the labeled product with sulfonic acid group under ultraviolet light, thereby achieving high ionization efficiency and low background noise mass spectrometry detection of the analyte in ESI negative ion mode, thereby improving the detection sensitivity and coverage of FI-HRMS analysis.

[0041] Compared with the prior art, the present invention has the following advantages and beneficial effects: The present invention provides a type of photosensitive mass spectrometry probe, which can realize rapid labeling of analytes and online efficient photolysis of labeled products to release labeled products with sulfonic acid groups.

[0042] The present invention provides a method for preparing a type of photosensitive mass spectrometry probe, which can synthesize a photosensitive mass spectrometry probe of a target structure in a simple and feasible manner, and has good guidance and feasibility.

[0043] The present invention provides an application of a type of photosensitive mass spectrometry probe, which realizes high-throughput and high-sensitivity detection of analytes with low ionization efficiency, and has broad prospects in metabolite labeling and mass spectrometry detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 This is the nuclear magnetic resonance spectroscopy (NMR) image of iTASO-ONH2; Figure 2 is the NMR spectrum of iTASO-NH2; Figure 3 is the NMR spectrum of iTASO-NHS; Figure 4 is the NMR spectrum of iTASO-MAL; Figure 5 Mass spectrometric characteristics of iTASO-ONH2 labeled products and photolysis products of carbonyl analytes.

[0045] Figure 6 Mass spectrometric characteristics of the iTASO-NH2 labeled products and photolysis products of carboxylic acid analytes.

[0046] Figure 7 Mass spectrometric characteristics of iTASO-NHS labeling products and photolysis products of amino analytes.

[0047] Figure 8 Mass spectrometric characteristics of iTASO-MAL labeled products and photolysis products of thiol analytes.

[0048] Fig. 9 ac are the combination of photosensitive mass spectrometry probe and FI-HRMS analysis: a) Schematic diagram of online photolysis and FI-HRMS apparatus; b) signal comparison of analyte before and after probe labeling; c) effect of excess probe reagent on FI-MS analysis. DETAILED DESCRIPTION

[0049] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0050] In the following examples, the preparation process of the present invention and the structural formula of the related products are as follows: ; ; ; .

[0051] Example 1 Synthesis of photosensitized mass spectrometry probe iTASO-ONH2: (E)-2-isopropyl-9H-thioxanthen-9-one O-((4-((aminooxy) methyl)phenyl)sulfonyl) oxime: ; The reactants 2-isopropylthioxanthone 1a (20.0 g, 78.6 mmol) and hydroxylamine hydrochloride (32.3 g, 471.8 mmol) were dissolved in pyridine / methanol (1:1, v / v, 400 mL), and NaOH (31.5 g, 786.3 mmol) was slowly added in batches under ice bath conditions, and then the temperature was raised to 100 °C for 12 h. After TLC detection, the reaction mixture was concentrated under reduced pressure to remove part of the solvent, and then water (300 mL) was added and extracted with ethyl acetate (3 × 100 mL). The combined organic layer was washed with 1M HCl (4 × 100 mL) and saturated brine (2 × 200 mL) in turn, dried over anhydrous sodium sulfate and filtered, and concentrated under reduced pressure to obtain a crude product; the crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 - 10 / 1) to obtain compound 2a (light yellow solid, 14.0 g, yield 66%); Compound 2a (13.0 g, 48.3 mmol, 1.0 equiv.), solvent dichloromethane (150 mL) and TEA (13.0 mL, 96.5 mmol, 2.0 equiv.) were added to a dry three-necked round-bottom flask, and then p-bromomethylphenylsulfonyl chloride 3a (19.5 g, 72.4 mmol) was added in batches under ice bath conditions and nitrogen protection, and the temperature was raised to room temperature for reaction for 30 min. After TLC detection showed that the reaction of the raw material was complete, a saturated NH4Cl aqueous solution (300 mL) was added to quench the reaction, the solution was separated and the organic phase was collected, and the aqueous phase was extracted with dichloromethane (2 × 100 mL). The combined organic layer was washed with saturated brine (2 × 200 mL) in turn, dried over anhydrous sodium sulfate and filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1:0-10:1) to obtain compound 4a (white solid, 19.0 g, yield 86%). N-hydroxyphthalimide 5a (1.1 g, 6.6 mmol) was dissolved in DMF (20 mL), and K2CO3 (1.2 g, 8.7 mmol) was added. After the reaction at room temperature, a solid appeared. Compound 4a (2.0 g, 4.4 mmol) was added, and the reaction was continued at room temperature for 12 h. After TLC detected that the reaction of the raw material was complete, water (60 mL) was slowly added, and a solid precipitated. The solid was filtered and the filter cake was dissolved in EtOH (20 mL). Hydrazine hydrate (85% aqueous solution, 7.8 mL, 131.0 mmol) was added and refluxed at 80 °C for 20 min. After TLC monitored that the reaction was complete, water (60 mL) was added to the reaction solution, and ethyl acetate (3 × 40 mL) was used for extraction. The combined organic layer was washed with saturated brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 10:1 - 2:1) to obtain compound iTASO-ONH2 (light yellow solid, 1.5 g, yield 76%).

[0052] 1 H NMR (400 MHz, DMSO- d 6) δ 8.14 – 8.11 (m, 0.5H), 8.06 – 7.98 (m,2H), 7.96 (d, J = 2.0 Hz, 0.5H), 7.78 – 7.70 (m, 1H), 7.69 – 7.45 (m, 7H), 6.23(s, 2H), 4.71 (d, J = 4.8 Hz, 2H), 3.04 – 2.93 (m, 1H), 1.22 (dd,J = 8.0, 6.8Hz, 6H). 13 C NMR (101 MHz, DMSO- d 6) δ 155.79, 155.06, 148.13, 147.01, 146.97,134.50, 133.45, 133.42, 133.08, 131.75, 131.49, 131.39, 131.17, 130.49,130.00, 129.92, 129.20, 129.08, 128.97, 128.81, 128.68, 127.75, 127.60,127.54, 127.46, 127.42, 127.30, 126.82, 126.45, 124.78, 123.13, 76.01, 33.47,33.36, 24.00. (NMR spectrum as shown Figure 1 as shown).

[0053] Example 2 Synthesis of photosensitive mass spectrometry probe iTASO-NH2: (E)-2-isopropyl-9H-thioxanthen-9-one O-((4- (aminomethyl)phenyl)sulfonyl) oxime: ; The synthesis steps of compound 4a are shown in Example 1; Compound 4a (12.0 g, 26.2 mmol, 1.0 equiv.) and potassium phthalimide salt 6a (7.3 g, 39.3 mmol, 1.5 equiv.) were dissolved in DMF (120 mL) and reacted at room temperature for 12 h. After TLC showed that the reaction of the raw material was complete, water (300 mL) was slowly added, and the solid precipitated. The solid was filtered and the filter cake was dissolved in EtOH (100 mL). Hydrazine hydrate (85% aqueous solution, 47.0 mL, 786.0 mmol) was added and refluxed at 80 °C for 20 min. After TLC showed that the reaction was complete, the solvent was removed by concentration under reduced pressure, and then water (100 mL) was added to the residue and extracted with ethyl acetate (3 × 50 mL). The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate and filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (eluent: dichloromethane / methanol = 30:1 - 5:1) to obtain compound iTASO-NH2 (light yellow solid, 8.5 g, yield 74%).

[0054] 1 H NMR (400 MHz, DMSO- d 6) δ 8.14 – 8.11 (m, 0.5H), 8.06 – 7.98 (m,2H), 7.96 (d, J = 2.0 Hz, 0.5H), 7.78 – 7.70 (m, 1H), 7.69 – 7.45 (m, 7H), 6.23(s, 2H), 4.71 (d, J = 4.8 Hz, 2H), 3.04 – 2.93 (m, 1H), 1.22 (dd, J = 8.0, 6.8Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 149.66, 147.67, 134.99, 133.76, 131.58,130.40, 129.81, 129.50, 129.34, 129.22, 128.64, 127.52, 127.46, 126.60,125.86, 125.49, 125.32, 45.86, 33.77, 23.85. (NMR spectrum as shown in Figure 2 as shown).

[0055] Example 3 Synthesis of photosensitive mass spectrometry probe iTASO-NHS: 2,5-dioxopyrrolidin-1-yl(E)-4-((4-((((2-isopropyl-9H-thioxanthen-9-ylidene)amino)oxy)sulfonyl)benzyl)amino)-4-oxobutanoate: ; iTASO-NH2 was synthesized using the steps of Example 2; Compound iTASO-NH2 (2.3 g, 5.2 mmol) and TEA (1.5 mL, 10.5 mmol) were dissolved in DMF (20 mL), and then succinic anhydride 7a (680 mg, 6.8 mmol) was added in batches and reacted at room temperature for 30 min. After TLC detected that the reaction of the raw material was complete, water (60 mL) was added to the mixture, and extracted with ethyl acetate (3 × 30 mL). The combined organic layer was washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 8a. The crude product 8a and N-hydroxysuccinimide 9a (904 mg, 7.9 mmol) were dissolved in DCM (30 mL), and then EDCI (1.5 g, 7.9 mmol) was added in an ice bath and reacted at room temperature for 30 min. After TLC detected that the reaction of the raw material was complete, water (90 mL) was added to the mixture, and extracted with dichloromethane (3 × 30 mL). The combined organic layer was saturated brine (50 The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 - 1:1.5) to obtain the product iTASO-NHS (light yellow solid, 3.0 g, yield 90%).

[0056] 1 H NMR (400 MHz, DMSO- d 6) δ 8.62 (q, J = 5.8 Hz, 1H), 8.11 (dd, J = 7.8,1.6 Hz, 0.5H), 8.04 – 7.92 (m, 2.5H), 7.75 – 7.70 (m, 1H), 7.69 – 7.61 (m,1H), 7.62 – 7.42 (m, 6H), 4.42 (dd, J = 6.0, 3.6 Hz, 2H), 3.00 – 2.90 (m, 1H), 2.94 (t, J = 6.8 Hz, 2H), 2.85 – 2.80 (m, 4H), 2.62 – 2.55 (m, 2H), 1.21 (dd, J =6.8, 5.6 Hz, 6H). 13 C NMR (101 MHz, DMSO- d6) δ 170.14, 170.09, 168.67, 168.65, 155.31,154.59, 147.67, 147.24, 147.19, 146.54, 134.06, 132.64, 132.59, 131.25,131.02, 130.94, 130.68, 129.99, 129.54, 129.43, 128.89, 128.65, 128.63,128.12, 128.01, 127.26, 127.16, 127.08, 126.98, 126.95, 126.82, 126.33,125.97, 125.94, 124.32, 122.68, 41.84, 40.15, 39.94, 39.73, 39.52, 39.31,39.10, 38.89, 33.02, 32.91, 29.28, 25.98, 25.46, 25.30, 25.24, 23.53. (NMR spectrum as shown Figure 3 as shown).

[0057] Example 4 Synthesis of the photosensitive mass spectrometry probe iTASO-MAL: (E)-4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(4-((((2-isopropyl-9H-thioxanthen-9-ylidene)amino)oxy)sulfonyl)benzyl)butanamide: ; iTASO-NH2 was synthesized using the steps of Example 2; Compound iTASO-NH2 (1.8 g, 4.1 mmol) and 4-maleimidobutyric acid 10a (980 mg, 5.3 mmol) were dissolved in DMF (20 mL), and then TEA (1.2 mL, 8.2 mmol) and HATU (2.0 g, 5.3 mmol) were added and reacted at room temperature for 30 min. After TLC detected that the reaction of the raw materials was complete, water (60 mL) was added to the mixture, and ethyl acetate (3 × 30 mL) was used for extraction. The combined organic layer was washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 - 1:1.5) to obtain the product iTASO-MAL (light yellow solid, 1.2 g, yield 48%).

[0058] 1 H NMR (400 MHz, DMSO- d 6) δ 8.45 (q, J = 5.6 Hz, 1H), 8.10 (d, J = 7.9 Hz,0.5H), 8.04 – 7.91 (m, 2.5H), 7.73 (dd, J = 8.4, 2.8 Hz, 1H), 7.65 (d, J = 8.0Hz, 0.5H), 7.61 – 7.41 (m, 6.5H), 6.98 (d, J = 2.2 Hz, 2H), 4.36 (t, J = 5.4 Hz,2H), 3.45 – 3.39 (m, 2H), 3.03 – 2.89 (m, 1H), 2.16 (t, J = 7.6 Hz, 2H), 1.83 –1.70 (m, 2H), 1.20 (t, J = 7.4 Hz, 6H). 13 C NMR (101 MHz, DMSO- d 6) δ 171.58, 171.56, 171.06, 155.31, 154.56,147.65, 147.46, 147.39, 146.54, 134.45, 134.04, 132.63, 132.52, 131.27,131.02, 130.93, 130.69, 130.00, 129.53, 129.45, 128.90, 128.65, 128.62,128.13, 128.00, 127.25, 127.15, 127.07, 126.99, 126.83, 126.34, 125.98,125.95, 124.29, 122.66, 41.72, 36.83, 33.01, 32.90, 32.46, 24.12, 24.09,23.53. (NMR spectrum as shown Figure 4 as shown).

[0059] Example 5 Labeling and mass spectrometric characteristics of aldehyde and ketone (carbonyl) analytes using the photosensitive mass spectrometry probe iTASO-ONH2: The labeling reaction is as follows: ; Labeling conditions: 10 μL of aldehyde and ketone mixed standard solution (5 μM), iTASO-ONH2 (10 μL, 10 mM) and acetonitrile solution containing 1% acetic acid (80 μL) were added to a brown centrifuge tube (1.5 mL) in sequence. The mixture was shaken at 40 °C for 20 min and then dried with a stream of nitrogen. The residue can be reconstituted with 100 μL of acetonitrile for mass spectrometry analysis. In the absence of ultraviolet radiation, the probe-labeled product can be detected by ESI positive ion mode; under ultraviolet radiation, the labeled photolysis product can be detected by ESI negative ion mode (the labeling and photolysis mass spectra are shown in Figure 2). Figure 5 as shown).

[0060] Example 6 Labeling and mass spectrometric characteristics of carboxylic acid analytes using the photosensitive mass spectrometry probe iTASO-NH2: The labeling reaction is shown below: ; Labeling conditions: 10 μL of carboxylic acid mixed standard solution (5 μM), acetonitrile (60 μL), TBTU (10 μL, 2 mM), TEA (10 μL, 2 mM) and iTASO-NH2 (10 μL, 10 mM) were added to a brown centrifuge tube (1.5 mL) in sequence. The mixture was shaken at 40°C for 20 minutes. After that, 200 μL of saline was added to the reaction solution, and then extracted with HPLC grade ethyl acetate (3 × 100 mL). The organic layers were combined and dried with a nitrogen stream. The residue was reconstituted in 100 μL of acetonitrile for mass spectrometry analysis (labeling and photolysis mass spectra are shown in Figure 2). Figure 6 as shown).

[0061] Example 7 Labeling and mass spectrometric characteristics of amino analytes using the photosensitive mass spectrometry probe iTASO-NHS: The labeling reaction is shown below: ; Labeling conditions: 10 μL of amino standard solution (5 μM), acetonitrile (70 μL), TEA (10 μL, 2 mM) and iTASO-NHS (10 μL, 10 mM) were added to a brown centrifuge tube (1.5 mL) in sequence. The mixture was shaken at 40°C for 20 minutes and then dried under nitrogen flow. The residue was reconstituted in 100 μL of acetonitrile and used for mass spectrometry analysis (labeling and photolysis mass spectra are shown in Figure 7 as shown).

[0062] Example 8 Labeling and mass spectrometric characteristics of thiol analytes using the photosensitive mass spectrometry probe iTASO-MAL: The labeling reaction is shown below: ; Labeling conditions: 10 μL of thiol standard mixed solution (5 μM), β-ME (10 mM, 10 μL), and EDTA (1 mM, 0.05% formic acid, 10 μL) were added to a centrifuge tube (1.5 mL) in sequence. The mixture was shaken at 40°C for 1 hour and then dried with a nitrogen stream. The residue was mixed with acetonitrile (70 μL), NH4OAc buffer solution (1 mM, pH 5.8, 20 μL), and iTASO-MAL (10 μL, 10 mM). The mixture was shaken at 40°C for 20 minutes and then dried with a nitrogen stream. The residue was reconstituted in 100 μL of acetonitrile for mass spectrometry analysis (labeling and photolysis mass spectra are shown in Figure 2). Figure 8 as shown).

[0063] Example 9 Online photolysis FI-ESI(-)-HRMS high-throughput analysis method based on iTASO-ONH2 probe: After the carbonyl analyte is labeled with the iTASO-ONH2 probe, the sample is detected by FI-ESI(-)-HRMS. In addition to the commercial liquid phase mass spectrometry instrument, the device also includes: 1. Use a UV-transparent fluorinated ethylene propylene (FEP) tube (inner diameter 0.25 mm, outer diameter 1 / 16 inch, length 500 mm) to connect the autosampler outlet and the ion source of the mass spectrometer; 2. Use an LED point light source (~365 nm, 2 W / cm²) for online light radiation. The UV light source is aimed at any position of the FEP tube. After the labeled product is injected, photolysis occurs at the irradiation of the UV point light source to produce a labeled product with a sulfonic acid group, which is subjected to mass spectrometry analysis by the ESI negative ion mode of the mass spectrometer (the schematic diagram of the device is shown in the figure). Fig. 9 a).

[0064] Online photolysis FI-ESI(-)-HRMS analysis method of probe-labeled products: (samples were directly injected into the mass spectrometer through the HPLC autosampler without chromatographic separation) Carrier liquid: acetonitrile / ultrapure water = 1 / 1; gradient flow rate: first, the flow rate was linearly decreased from 0.6 mL / min to 0.15 mL / min from 0 to 0.11 minutes, then the flow rate was kept constant at 0.15 mL / min for 0.51 minutes, and finally the flow rate was linearly increased from 0.15 mL / min to 0.6 mL / min from 0.62 to 1 min. The injection volume was 5 µL. Mass spectrometry: Full-scan MS mode, ESI negative ion mode detection.

[0065] The signal response of the analyte before and after iTASO-ONH2 probe labeling was analyzed by using a mixed standard solution of four carbonyl standards. The results showed that the unlabeled analyte was not detected in the mass spectrometry, while the mass spectrometry response signal after probe labeling was significantly improved. The signal response of the labeled product with sulfonic acid group after further photolysis was about one order of magnitude higher than that of the unphotolyzed probe labeled product (results shown in Fig. 9 b).

[0066] The effect of the amount of probe on FI-HRMS detection was analyzed by changing the concentration of iTASO-ONH2 probe. The results showed that the amount of probe had little effect on the mass spectrometry detection of the labeled product with sulfonic acid group after photolysis, and the highest detection inhibition was 34%. However, when the labeled product was directly analyzed without photolysis, the excess amount of probe greatly inhibited the mass spectrometry detection of the labeled product, with the inhibition reaching up to 94% (results shown in Fig. 9 c).

[0067] The sensitivity of the method was investigated by measuring the detection limit of the labeled product in the standard solution, where the detection limit (LOD) was selected when the signal-to-noise ratio was 3. Table 1 shows the sensitivity of the carbonyl labeled product.

[0068] Table 1: Sensitivity of carbonyl labeled products

[0069] The results in Table 1 show that the LOD of the four carbonyl compounds can reach 5-31.5 fmol after being labeled with iTASO-ONH2 probe and online photolysis FI-ESI(-)-HRMS analysis method.

[0070] In summary, the present invention uses the thioxanthone oxime benzene sulfonate structure as the basis to bond the active reaction group, and constructs and synthesizes a class of efficient photosensitive mass spectrometry probes. The product labeled with this type of probe can efficiently release the labeled product with sulfonic acid group under ultraviolet light, thereby achieving high ionization efficiency and low background noise mass spectrometry detection of the analyte in ESI negative ion mode, thereby improving the detection sensitivity and coverage of FI-HRMS analysis, and achieving high-throughput and high-sensitivity detection of low ionization efficiency analytes, which has broad prospects in metabolite labeling and mass spectrometry detection.

[0071] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. A photosensitive mass spectrometry probe, characterized in that: This type of photosensitive mass spectrometry probe includes a photosensitive portion and an active reaction group. The active reaction group includes ONH2, NH2, NHS, and MAL. The structural formula of the photosensitive mass spectrometry probe is as follows: 。 2. A method for preparing a photosensitive mass spectrometry probe as claimed in claim 1, characterized in that: (1) 2-isopropylthioxanthone and hydroxylamine hydrochloride are dissolved in a solvent, and then sodium hydroxide is added and reacted to obtain a compound A, whose structural formula is as follows: ； (2) Compound A is dissolved in a solvent, and then triethylamine and p-bromomethylphenylsulfonyl chloride are added and reacted to obtain compound B, whose structural formula is as follows: ； (3) An active reactive group is introduced through a substitution reaction to replace the chlorine atom in compound B to obtain a photosensitive mass spectrometry probe with the corresponding structure.

3. The method for preparing a photosensitive mass spectrometry probe according to claim 2, characterized in that: In the step (1), the amount of 2-isopropylthioxanthone used is 1.0 equiv., the amount of hydroxylamine hydrochloride used is 6-10 equiv., and the amount of sodium hydroxide used is 10-15 equiv.; the reaction temperature is 100-120° C., and the reaction time is 8-20 h.

4. The method for preparing a photosensitive mass spectrometry probe according to claim 2, characterized in that: In the step (2), the amount of compound A used is 1.0 equiv., the amount of triethylamine used is 2.0-3.0 equiv., and the amount of p-bromomethylphenylsulfonyl chloride used is 1.0-3.0 equiv.; the reaction is carried out at room temperature and the reaction time is 30-60 min.

5. The method for preparing a photosensitive mass spectrometry probe according to claim 2, characterized in that: When the active reaction group is ONH2, the process of step (3) is as follows: N-hydroxyphthalimide is dissolved in a solvent, potassium carbonate is added to carry out a first-stage reaction, and compound B is added after solid appears in the reaction solution to carry out a second-stage reaction; after the reaction is completed, the product is collected and dissolved in a solvent, and then hydrazine hydrate is added and refluxed to obtain a photosensitive mass spectrometry probe; The dosage of N-hydroxyphthalimide is 1.0-1.5 equiv., the dosage of potassium carbonate is 2.0-3.0 equiv., the dosage of compound B is 1.0 equiv., and the dosage of hydrazine hydrate is 20-30 equiv.; the first stage reaction is carried out at room temperature; the second stage reaction is carried out at room temperature, and the reaction time is 12-16 h; the temperature of the reflux reaction is 80-90 ° C, and the reaction time is 20-30 min.

6. The method for preparing a photosensitive mass spectrometry probe according to claim 2, characterized in that: When the active reaction group is NH2, the process of step (3) is as follows: dissolving compound B and potassium phthalimide in a solvent and reacting; after the reaction is completed, collecting the product and dissolving it in a solvent, then adding hydrazine hydrate and performing a reflux reaction to obtain a photosensitive mass spectrometer probe; The dosage of compound B is 1.0 equiv., the dosage of potassium phthalimide is 1.5-2.0 equiv., and the dosage of hydrazine hydrate is 20-30 equiv.; the reaction is carried out at room temperature, and the reaction time is 12-16 h; the temperature of the reflux reaction is 80-90° C., and the reaction time is 20-30 min.

7. The method for preparing a photosensitive mass spectrometry probe according to claim 2, characterized in that: When the active reactive group is NHS, the process of step (3) is as follows: iTASO-NH2 and triethylamine are dissolved in a solvent, and then succinic anhydride is added to carry out a first-stage reaction to obtain an intermediate; the intermediate and N-hydroxysuccinimide are dissolved in a solvent, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added to carry out a second-stage reaction to obtain a photosensitive mass spectrometry probe; The dosage of iTASO-NH2 is 1.0 equiv., the dosage of triethylamine is 2.0-3.0 equiv., the dosage of succinic anhydride is 1.0-1.5 equiv., the dosage of N-hydroxysuccinimide is 1.0-1.5 equiv., and the dosage of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 1.5-2.0 equiv.; the first stage reaction is carried out at room temperature, and the reaction time is 30-60 min; the second stage reaction is carried out at room temperature, and the reaction time is 30-60 min.

8. The method for preparing a photosensitive mass spectrometry probe according to claim 2, characterized in that: When the active reaction group is MAL, the process of step (3) is as follows: iTASO-NH2 and 4-maleimidobutyric acid are dissolved in a solvent, and then triethylamine and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate are added and reacted to obtain a photosensitive mass spectrometry probe; The amount of iTASO-NH2 used is 1.0 equiv., the amount of 4-maleimidobutyric acid used is 1.0-2.0 equiv., the amount of triethylamine used is 2.0-3.0 equiv., and the amount of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate used is 1.3-2.0 equiv.; the reaction is carried out at room temperature and the reaction time is 30-60 min.

9. An application of a photosensitive mass spectrometer probe according to claim 1 or a photosensitive mass spectrometer probe prepared by the preparation method according to any one of claims 2 to 8, characterized in that: Application of photosensitive mass spectrometry probes in metabolite labeling and mass spectrometry detection.

10. The use according to claim 9, characterized in that: Applications include labeling and mass spectrometric detection of carbonyl analytes, carboxylic acid analytes, amino analytes, and sulfhydryl analytes; and sample detection of labeled products of photosensitive mass spectrometric probes by FI-ESI(-)-HRMS.

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