Photosensitive mass spectrometry probe, preparation method thereof and application thereof in mass spectrometry detection

Abstract

The application discloses a photosensitive mass spectrometry probe, a preparation method thereof and application thereof in mass spectrometry detection, and belongs to the technical field of organic chemistry and analytical chemistry. The photosensitive mass spectrometry probe is constructed by bonding an active reaction group on the basis of thioxanthone oxime phenyl sulfonate structure, and can realize rapid labeling of an analyte and on-line efficient photolysis of a labeled product, so that the labeled product with a sulfonic acid group is released. The application also provides a preparation method of the photosensitive mass spectrometry probe, which is simple and feasible to synthesize the photosensitive mass spectrometry probe with the target structure, and has good guidance and implementability. In the application of the photosensitive mass spectrometry probe in metabolite labeling and mass spectrometry detection, the detection sensitivity is improved, ion suppression effect caused by excessive labeling reagents is reduced, high-throughput and high-sensitivity detection and analysis of low ionization efficiency analytes are realized, and the photosensitive mass spectrometry probe has a wide prospect.

Description

Technical Field

[0001] This invention relates to the fields of organic chemistry and analytical chemistry, and in particular to a class of photosensitive mass spectrometry probes, their preparation methods, and their applications in mass spectrometry detection. Background Technology

[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 (no need for column equilibration, chromatographic gradient condition optimization, etc.). However, in practical applications, this technology still faces two important challenges. First, some metabolites lack easily ionizable groups, making effective ionization difficult in electrospray ionization (ESI) sources, resulting in difficulties in mass spectrometry (MS) detection. Second, the lack of chromatographic separation means that complex biological sample matrices can lead to severe ion inhibition and matrix effects, affecting the sensitivity and coverage of metabolite detection.

[0003] To address the issue of metabolites lacking readily ionizable groups, chemical derivatization techniques modify the analyte structure to enhance its ionization ability in ESI sources, thereby improving detection sensitivity. For example, the literature (T.-Y. Zhang, S.Li, Q.-F. Zhu, Q. Wang, D. Hussain, Y.-Q. Feng, ...) Trends in Analytical Chemistry 2019, 119 (115608.) This paper introduces the use of LC-MS technology combined with chemical derivatization for the mass spectrometric 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 reagent. If not separated by chromatography, the excess derivatization reagent will continuously enter the mass spectrometer with the sample, causing a serious matrix effect and thus affecting the analytical sensitivity.

[0004] In summary, the key to achieving high-throughput FI-HRMS metabolomics analysis lies in effectively controlling the matrix effect while improving the ionization efficiency of metabolites. Summary of the Invention

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

[0006] .

[0007] In a 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:

[0008] (1) Dissolve 2-isopropylthioxanone and hydroxylamine hydrochloride in a solvent, then add sodium hydroxide and react to obtain compound A, whose structural formula is as follows:

[0009] ;

[0010] (2) Compound A was dissolved in a solvent, and then triethylamine (TEA) and p-bromomethylbenzoic acid chloride were added and reacted to obtain compound B, whose structural formula is as follows:

[0011] ;

[0012] (3) By introducing an active reactive group through a substitution reaction to replace the chlorine atom in compound B, a photosensitive mass spectrometry probe with the corresponding structure is obtained.

[0013] 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.

[0014] Preferably, in step (1), the reaction temperature is 100-120 °C and the reaction time is 8-20 h.

[0015] Preferably, in step (2), the amount of compound A is 1.0 equiv., the amount of triethylamine is 2.0-3.0 equiv., and the amount of p-bromomethylbenzoic acid chloride is 1.0-3.0 equiv.

[0016] Preferably, in step (2), the reaction is carried out at room temperature for 30-60 min.

[0017] In this process, the synthesis of compounds A and B is carried out in a solution environment. Those skilled in the art can select appropriate solvent types and amounts based on the substrates, physicochemical properties of the products, and actual conditions. For example, in the synthesis of compound A, a pyridine / methanol mixture (1:1, v / v) can be used to create the solution environment; in the synthesis of compound B, dichloromethane (DCM) can be used. Other suitable types of solvents and their mixtures can also achieve the synthetic objectives. Furthermore, at the operational level, sodium hydroxide is preferably added under ice bath conditions; while p-bromomethylbenzoic acid chloride is preferably added in batches under an ice bath and protective atmosphere. All of these operations help ensure the safety and smooth progress of the reaction.

[0018] Preferably, when the active reactive group is ONH2, the process of step (3) is as follows: N-hydroxyphthalimide is dissolved in a solvent, potassium carbonate is added for the first stage reaction, and compound B is added after a solid appears in the reaction solution for the second stage reaction; after the reaction is completed, the product is collected and dissolved in a solvent, then hydrazine hydrate is added and refluxed to obtain the photosensitive mass spectrometry probe (iTASO-ONH2).

[0019] The structural formula of iTASO-ONH2 is as follows:

[0020] .

[0021] More preferably, the amount of N-hydroxyphthalimide is 1.0-1.5 equiv., the amount of potassium carbonate is 2.0-3.0 equiv., the amount of compound B is 1.0 equiv., and the amount of hydrazine hydrate is 20-30 equiv.

[0022] More 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 reflux reaction temperature is 80-90 ℃ and the reaction time is 20-30 min.

[0023] Based on the general principles of solvent selection in this field, those skilled in the art can also select appropriate solvent types and amounts according to the substrate type, etc. In this step, the first and second stages of the reaction can be carried out in a solution environment created by N,N-dimethylformamide (DMF). After the reaction is complete, adding water and filtering yields the corresponding product, which is then dissolved in ethanol for subsequent reflux reaction. Other suitable solvents or separation methods can also achieve the above objectives.

[0024] Preferably, when the active reactive group is NH2, the process of step (3) is as follows: dissolve compound B and potassium phthalimide in a solvent and react them; after the reaction is completed, collect the product and dissolve it in a solvent, then add hydrazine hydrate and reflux to obtain the photosensitive mass spectrometry probe (iTASO-NH2).

[0025] The structural formula of iTASO-NH2 is as follows:

[0026] .

[0027] More preferably, the amount of compound B is 1.0 equiv., the amount of potassium phthalimide is 1.5-2.0 equiv., and the amount of hydrazine hydrate is 20-30 equiv.

[0028] More preferably, the reaction is carried out at room temperature for 12-16 h; the reflux reaction temperature is 80-90 °C and the reaction time is 20-30 min.

[0029] Similarly, DMF can be used as the reaction solvent in this step. After the reaction is completed, water is added and the mixture is filtered to obtain the corresponding product. The product is then dissolved in ethanol and refluxed. Other suitable solvents or separation methods can also achieve the above purpose.

[0030] Preferably, 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 the 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 the second stage reaction to obtain the photosensitive mass spectrometry probe (iTASO-NHS).

[0031] The structural formula of iTASO-NHS is as follows:

[0032] .

[0033] In iTASO-NHS, the process of introducing NHS can be considered as first preparing iTASO-NH2, and then further reacting it to obtain the target iTASO-NHS. The iTASO-NH2 used can be synthesized using the method provided in this invention.

[0034] More 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.

[0035] Further preferably, the first stage reaction is carried out at room temperature for 30-60 min; the second stage reaction is carried out at room temperature for 30-60 min.

[0036] Similarly, DMF can be used as the solvent for the first stage reaction, and DCM as the solvent for the second stage reaction. During operation, succinic anhydride should be added in batches, while EDCI is best added under ice bath conditions.

[0037] Preferably, when the active reactive group is MAL, the process of step (3) is as follows: iTASO-NH2 and 4-maleimide butyric acid are dissolved in a solvent, and then triethylamine and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) are added and reacted to obtain the photosensitive mass spectrometry probe (iTASO-MAL).

[0038] The structural formula of iTASO-MAL is as follows:

[0039] .

[0040] Similarly, in iTASO-MAL, the process of introducing MAL can be regarded as first preparing iTASO-NH2, and then further reacting on it to obtain the target iTASO-MAL.

[0041] More preferably, the amount of iTASO-NH2 is 1.0 equiv., the amount of 4-maleimidebutyric acid is 1.0-2.0 equiv., the amount of triethylamine is 2.0-3.0 equiv., and the amount of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate is 1.3-2.0 equiv.

[0042] More preferably, the reaction is carried out at room temperature for 30-60 minutes.

[0043] Similarly, DMF can be used as the solvent for this step. Other suitable solvents can also achieve the same purpose.

[0044] In the above synthesis, those skilled in the art can use methods commonly used in the field to monitor the progress of the reaction and to properly purify the products of each reaction.

[0045] In a third aspect of the invention, the application of a type of photosensitive mass spectrometry probe of the first aspect of the invention or a photosensitive mass spectrometry probe prepared by the method of the second aspect of the invention is provided, specifically the application of the photosensitive mass spectrometry probe in metabolite labeling and mass spectrometry detection.

[0046] Preferably, the application includes labeling and mass spectrometry detection of carbonyl analytes, carboxylic acid analytes, amino analytes, and thiol analytes.

[0047] The online photolysis FI-ESI(-)-HRMS analysis method based on this probe can improve the detection sensitivity of analytes while being less susceptible to the ion inhibition caused by excessive labeling reagents, thus achieving high-throughput and high-sensitivity detection of analytes with low ionization efficiency.

[0048] Preferably, the application includes the detection of labeled products of photosensitive mass spectrometry probes by FI-ESI(-)-HRMS.

[0049] Based on the above technical solutions, the design concept and principle of this invention lie in the fact that the inventors noticed that photoacid generators (PAGs) are a class of photosensitive substances that can produce acid under light conditions, and are widely used in photoresists, 3D printing, dental materials, and biochemical research. Thioxanone oxime benzene sulfonate, as a type of PAG, can efficiently release sulfonic acid under 365 nm wavelength light radiation. Sulfonic acid, as a strong electron-withdrawing group, is easily ionized in ESI negative ion mode, exhibiting good mass spectrometry response. Furthermore, compared to ESI positive ionization mode, ESI negative ionization mode generally has lower background noise; therefore, to a certain extent, ESI negative ion mode exhibits a weaker matrix effect.

[0050] Therefore, this invention constructs a set of highly efficient photosensitive mass spectrometry probes by bonding reactive groups to the thioxanone oxime benzene sulfonate structure. Products labeled with this type of probe can efficiently release labeled products containing sulfonic acid groups under ultraviolet light, thereby achieving high ionization efficiency and low background noise mass spectrometry detection of analytes in ESI negative ion mode, and thus improving the detection sensitivity and coverage of FI-HRMS analysis.

[0051] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0052] This invention provides a type of photosensitive mass spectrometry probe that enables rapid labeling of analytes and efficient online photolysis of labeled products, releasing labeled products with sulfonic acid groups.

[0053] This invention provides a method for preparing a type of photosensitive mass spectrometry probe, which can synthesize photosensitive mass spectrometry probes with target structures in a simple and feasible manner, and has good guidance and feasibility.

[0054] This invention provides an application of a class of photosensitive mass spectrometry probes, enabling high-throughput and high-sensitivity detection of analytes with low ionization efficiency, and has broad prospects in metabolite labeling and mass spectrometry detection. Attached Figure Description

[0055] Figure 1 The image shows the nuclear magnetic resonance (NMR) spectrum of iTASO-ONH2.

[0056] Figure 2 The NMR spectrum of iTASO-NH2;

[0057] Figure 3 NMR spectrum of iTASO-NHS;

[0058] Figure 4 The NMR spectrum of iTASO-MAL;

[0059] Figure 5 Mass spectrometric characteristics of iTASO-ONH2 labeled products and photolysis products of carbonyl analytes.

[0060] Figure 6 Mass spectrometric characteristics of iTASO-NH2 labeled products and photolysis products of carboxylic acid analytes.

[0061] Figure 7 Mass spectrometric characteristics of iTASO-NHS labeled products and photolysis products of amino analytes.

[0062] Figure 8 Mass spectrometric characteristics of iTASO-MAL labeled products and photolysis products of thiol analytes.

[0063] Figure 9 Ac represents the combination of photosensitive mass spectrometry probes and FI-HRMS analysis: a) Schematic diagram of online photolysis and FI-HRMS setup; b) Comparison of analyte signals before and after probe labeling; c) Effect of excess probe reagent on FI-MS analysis. Detailed Implementation

[0064] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0065] In the following embodiments, the preparation process and structural formulas of the related products of the present invention are described as follows:

[0066] ;

[0067] ;

[0068] ;

[0069] .

[0070] Example 1

[0071] Synthesis of the photosensitive mass spectrometry probe iTASO-ONH2:

[0072] (E)-2-isopropyl-9H-thioxanthen-9-one O-((4-((aminooxy) methyl)phenyl)sulfonyl) oxime:

[0073] ;

[0074] 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). NaOH (31.5 g, 786.3 mmol) was slowly added in portions under ice bath conditions. The mixture was then heated to 100 °C and reacted for 12 h. After the reaction proceeded to completion as determined by TLC, the reaction mixture was concentrated under reduced pressure to remove some of the solvent. Water (300 mL) was then added, followed by extraction with ethyl acetate (3 × 100 mL). The combined organic layers were washed sequentially with 1M HCl (4 × 100 mL) and saturated brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 1 / 0 - 10 / 1) to give compound 2a (pale yellow solid, 14.0 g, yield 66%).

[0075] Compound 2a (13.0 g, 48.3 mmol, 1.0 equiv.), dichloromethane (150 mL), and TEA (13.0 mL, 96.5 mmol, 2.0 equiv.) were added to a dry three-necked round-bottom flask. Then, p-bromomethylbenzoic acid chloride 3a (19.5 g, 72.4 mmol) was added in portions under ice bath conditions and nitrogen protection. The mixture was heated to room temperature and reacted for 30 min. After the reaction was confirmed to be complete by TLC, saturated NH4Cl aqueous solution (300 mL) was added to quench the reaction. The solution was separated into layers, and the organic phase was collected. The aqueous phase was extracted with dichloromethane (2 × 100 mL). The combined organic layers were washed with saturated brine (2 × 200 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the 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%).

[0076] 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 solid appeared at room temperature, compound 4a (2.0 g, 4.4 mmol) was added, and the reaction was continued at room temperature for 12 h. After the reaction was confirmed to be complete by TLC, 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 the mixture was refluxed at 80 °C for 20 min. After the reaction was confirmed to be complete by TLC, water (60 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (3 × 40 mL). The combined organic layers were washed with saturated brine (2 × 30 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was subjected to silica gel column chromatography (petroleum ether / ethyl acetate = 10:1 - The compound iTASO-ONH2 (pale yellow solid, 1.5 g, yield 76%) was obtained by purification at a ratio of 2:1.

[0077] 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).

[0078] 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).

[0079] Example 2

[0080] Synthesis of the photosensitive mass spectrometry probe iTASO-NH2:

[0081] (E)-2-isopropyl-9H-thioxanthen-9-one O-((4- (aminomethyl)phenyl)sulfonyl) oxime:

[0082] ;

[0083] The synthesis steps of compound 4a are as shown in Example 1;

[0084] Compound 4a (12.0 g, 26.2 mmol, 1.0 equiv.) and potassium phthalimide 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 the reaction was complete as monitored by TLC, water (300 mL) was slowly added, and a 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 the mixture was refluxed at 80 °C for 20 min. After the reaction was complete as monitored by TLC, the solvent was removed by concentration under reduced pressure. The residue was then extracted with water (100 mL) and ethyl acetate (3 × 50 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was subjected to silica gel column chromatography (eluent: dichloromethane / methanol = 30:1 - The compound iTASO-NH2 (pale yellow solid, 8.5 g, yield 74%) was obtained by purification at a ratio of 5:1.

[0085] 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).

[0086] 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 spectra are shown below) Figure 2 (As shown).

[0087] Example 3

[0088] Synthesis of the photosensitive mass spectrometry probe iTASO-NHS:

[0089] 2,5-dioxopyrrolidin-1-yl(E)-4-((4-((((2-isopropyl-9H-thioxanthen-9-ylidene)amino)oxy)sulfonyl)benzyl)amino)-4-oxobutanoate:

[0090] ;

[0091] iTASO-NH2 was synthesized using the steps in Example 2;

[0092] Compound iTASO-NH2 (2.3 g, 5.2 mmol) and TEA (1.5 mL, 10.5 mmol) were dissolved in DMF (20 mL), followed by the addition of succinic anhydride 7a (680 mg, 6.8 mmol) in portions. The mixture was reacted at room temperature for 30 min. After the reaction was confirmed by TLC, water (60 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with saturated brine (3 × 50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 8a. 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. The mixture was reacted at room temperature for 30 min. After the reaction was confirmed by TLC, water (90 mL) was added to the mixture, and the mixture was extracted with dichloromethane (3 × 30 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain crude product 8a. Wash with anhydrous sodium sulfate (mL), dry and filter, concentrate under reduced pressure to obtain crude product, and purify the crude product by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 - 1:1.5) to obtain product iTASO-NHS (pale yellow solid, 3.0 g, yield 90%).

[0093] 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).

[0094] 13 C NMR (101 MHz, DMSO- d 6) 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 spectra are shown below) Figure 3 (As shown).

[0095] Example 4

[0096] Synthesis of the photosensitive mass spectrometry probe iTASO-MAL:

[0097] (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:

[0098] ;

[0099] iTASO-NH2 was synthesized using the steps in Example 2;

[0100] Compound iTASO-NH2 (1.8 g, 4.1 mmol) and 4-maleimidebutyric acid 10a (980 mg, 5.3 mmol) were dissolved in DMF (20 mL), followed by the addition of TEA (1.2 mL, 8.2 mmol) and HATU (2.0 g, 5.3 mmol), and the reaction was carried out at room temperature for 30 min. After the reaction was confirmed to be complete by TLC, water (60 mL) was added to the mixture, and the mixture was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (petroleum ether / ethyl acetate = 5:1 - 1:1.5) to obtain product iTASO-MAL (pale yellow solid, 1.2 g, yield 48%).

[0101] 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).

[0102] 13 C NMR (101 MHz, DMSO- d6) 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 spectra are shown below) Figure 4 (As shown).

[0103] Example 5

[0104] Labeling and mass spectrometric characteristics of aldehydes and ketones (carbonyl groups) analytes using the photosensitive mass spectrometry probe iTASO-ONH2:

[0105] The labeling reaction is shown below:

[0106] ;

[0107] Labeling conditions: 10 μL of aldehyde-ketone mixed standard solution (5 μM), 10 μL of iTASO-ONH2 (10 mM), and 80 μL of acetonitrile solution containing 1% acetic acid were sequentially added to a brown centrifuge tube (1.5 mL). The mixture was shaken at 40 °C for 20 min and then dried under a nitrogen stream. The residue was redissolved in 100 μL of acetonitrile for mass spectrometry analysis. Under UV-free conditions, the probe-labeled product could be detected using ESI positive ion mode; under UV-irradiation conditions, the photolysis product after labeling could be detected using ESI negative ion mode (labeling and photolysis mass spectra are shown in Figure 1). Figure 5 (As shown).

[0108] Example 6

[0109] Labeling and mass spectrometric characteristics of carboxylic acid analytes by the photosensitive mass spectrometry probe iTASO-NH2:

[0110] The labeling reaction is shown in the following formula:

[0111] ;

[0112] Labeling conditions: 10 μL of a carboxylic acid 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 sequentially added to a brown centrifuge tube (1.5 mL). The mixture was shaken at 40°C for 20 min. Then, 200 μL of saline solution was added to the reaction mixture, followed by extraction with HPLC-grade ethyl acetate (3 × 100 mL). The organic layers were combined and dried over a nitrogen stream. The residue was reconstituted with 100 μL of acetonitrile for mass spectrometry analysis (labeling and photolysis mass spectra are shown below). Figure 6 (As shown).

[0113] Example 7

[0114] Labeling and mass spectrometric characteristics of amino analytes using the photosensitive mass spectrometry probe iTASO-NHS:

[0115] The labeling reaction is shown in the following formula:

[0116] ;

[0117] 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 sequentially added to a brown centrifuge tube (1.5 mL). The mixture was shaken at 40°C for 20 min, then dried under a nitrogen stream. The residue was reconstituted in 100 μL of acetonitrile for mass spectrometry analysis (labeling and photolysis mass spectra are shown below). Figure 7 (As shown).

[0118] Example 8

[0119] Labeling and mass spectrometric characteristics of thiol analytes using the photosensitive mass spectrometry probe iTASO-MAL:

[0120] The labeling reaction is shown in the following formula:

[0121] ;

[0122] Labeling conditions: 10 μL of thiol-based mixed standard solution (5 μM), β-ME (10 mM, 10 μL), and EDTA (1 mM, 0.05% formic acid, 10 μL) were sequentially added to a centrifuge tube (1.5 mL). The mixture was shaken at 40°C for 1 hour and then dried under 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 under a nitrogen stream. The residue was reconstituted in 100 μL of acetonitrile for mass spectrometry analysis (labeling and photolysis mass spectra are shown below). Figure 8 (As shown).

[0123] Example 9

[0124] A high-throughput online photolysis FI-ESI(-)-HRMS analysis method based on the iTASO-ONH2 probe:

[0125] After labeling carbonyl analytes with the iTASO-ONH2 probe, the samples are detected by FI-ESI(-)-HRMS. In addition to commercial liquid chromatography-mass spectrometry (LC-MS) instruments, the device includes: 1. A UV-transmitting fluorinated ethylene propylene (FEP) tube (0.25 mm inner diameter, 1 / 16 inch outer diameter, 500 mm length) connecting the autosampler outlet to the ion source of the mass spectrometer; 2. An LED point light source (~365 nm, 2 W / cm²) for online illumination. This UV light source is aimed at any position on the FEP tube. After the labeled product is injected, photolysis occurs at the UV point light source, producing a labeled product with sulfonic acid groups. This product is then analyzed by mass spectrometry in ESI negative ion mode (see simplified diagram of the device). Figure 9 (as shown in a).

[0126] Online photolysis FI-ESI(-)-HRMS analysis method for probe-labeled products: (Samples are directly injected into the mass spectrometer via the HPLC autosampler without chromatographic separation) Carrier solution: Acetonitrile / ultrapure water = 1 / 1; Gradient flow rate: Initially, the flow rate linearly decreases from 0.6 mL / min to 0.15 mL / min over 0.11 min, then maintains a constant flow rate of 0.15 mL / min for 0.51 min, and finally linearly increases from 0.15 mL / min to 0.6 mL / min over 0.62–1 min. Injection volume: 5 µL. Mass spectrometry: Full-scan MS mode, ESI negative ion mode detection.

[0127] The signal responses of analytes before and after iTASO-ONH2 probe labeling were analyzed using a mixed standard solution of four carbonyl standards. The results showed that the unlabeled analytes were not detected in the mass spectrometer, while the mass spectrometric response signal was significantly improved after probe labeling. Furthermore, the signal response of the labeled product with sulfonic acid groups after photolysis was approximately one order of magnitude higher than that of the unlabeled probe-labeled product (results are shown in Figure 1). Figure 9 (as shown in b).

[0128] The effect of iTASO-ONH2 probe concentration on FI-HRMS detection was analyzed. The results showed that the probe amount had little effect on the mass spectrometry detection of labeled products with sulfonic acid groups after photolysis, with the highest detection inhibition rate being 34%. However, when the labeled products were analyzed directly without photolysis, excess probe significantly inhibited the mass spectrometry detection of the labeled products, with the highest inhibition rate reaching 94% (see results below). Figure 9 (as shown in c).

[0129] The sensitivity of this method was evaluated by determining the detection limit of the labeled product in the standard solution, where the detection limit (LOD) was selected at a signal-to-noise ratio of 3. Table 1 shows the sensitivity of the carbonyl-labeled product.

[0130] Table 1: Sensitivity of carbonyl-labeled products

[0131]

[0132] The results in Table 1 show that the LOD of the four carbonyl compounds after being labeled with the iTASO-ONH2 probe by the online photolysis FI-ESI(-)-HRMS method can reach 5-31.5 fmol.

[0133] In summary, this invention constructs and synthesizes a class of highly efficient photosensitive mass spectrometry probes by bonding reactive groups to the thioxanone oxime benzene sulfonate structure. The products labeled with these probes can efficiently release labeled products containing sulfonic acid groups under ultraviolet light, thereby achieving high ionization efficiency and low background noise mass spectrometry detection of analytes in ESI negative ion mode. This improves the detection sensitivity and coverage of FI-HRMS analysis, enabling high-throughput and high-sensitivity detection of analytes with low ionization efficiency, and shows broad prospects in metabolite labeling and mass spectrometry detection.

[0134] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A type of photosensitive mass spectrometry probe, characterized in that, The structural formula of the photosensitive mass spectrometry probe is as follows:

2. A method for preparing a photosensitive mass spectrometry probe as described in claim 1, characterized in that: (1) Dissolve 2-isopropylthioxanone and hydroxylamine hydrochloride in a solvent, then add sodium hydroxide and react to obtain compound A, whose structural formula is as follows: ； (2) Compound A was dissolved in a solvent, and then triethylamine and p-bromomethylbenzoic acid chloride were added and reacted to obtain compound B, whose structural formula is as follows: ； (3) By introducing an active reactive group through a substitution reaction to replace the chlorine atom in compound B, a photosensitive mass spectrometry probe with the corresponding structure is obtained; When the reactive group is ONH2, the procedure for this step is as follows: N-hydroxyphthalimide was dissolved in a solvent, and potassium carbonate was added to carry out the first stage reaction. After a solid appeared in the reaction solution, compound B was added to carry out the second stage reaction. After the reaction was completed, the product was collected and dissolved in a solvent. Then hydrazine hydrate was added and refluxed to obtain a photosensitive mass spectrometry probe. The amount of N-hydroxyphthalimide used was 1.0-1.5 equiv., the amount of potassium carbonate was 2.0-3.0 equiv., the amount of compound B was 1.0 equiv., and the amount of hydrazine hydrate was 20-30 equiv.; the first stage reaction was carried out at room temperature; the second stage reaction was carried out at room temperature for 12-16 h; the reflux reaction temperature was 80-90 ℃, and the reaction time was 20-30 min; When the active reactive group is NH2, the procedure is as follows: Compound B and potassium phthalimide are dissolved in a solvent and reacted; after the reaction is complete, the product is collected and dissolved in a solvent, then hydrazine hydrate is added and refluxed to obtain the photosensitive mass spectrometry probe iTASO-NH2; the structural formula of iTASO-NH2 is as follows: The amount of compound B was 1.0 equiv., the amount of potassium phthalimide was 1.5-2.0 equiv., and the amount of hydrazine hydrate was 20-30 equiv.; the reaction was carried out at room temperature for 12-16 h; the reflux reaction was carried out at 80-90℃ for 20-30 min. When the reactive group is NHS, the procedure for this step is as follows: iTASO-NH2 and triethylamine were dissolved in a solvent, and then succinic anhydride was added to carry out the first stage reaction to obtain an intermediate. The intermediate and N-hydroxysuccinimide were dissolved in a solvent, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added to carry out the second stage reaction to obtain a photosensitive mass spectrometry probe. The amounts of iTASO-NH2 used were 1.0 equiv., triethylamine 2.0-3.0 equiv., succinic anhydride 1.0-1.5 equiv., N-hydroxysuccinimide 1.0-1.5 equiv., and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride 1.5-2.0 equiv.; the first stage reaction was carried out at room temperature for 30-60 min; the second stage reaction was carried out at room temperature for 30-60 min. When the reactive group is MAL, the procedure for this step is as follows: iTASO-NH2 and 4-maleimide butyric acid were dissolved in a solvent, followed by the addition of triethylamine and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate and reaction to obtain a photosensitive mass spectrometry probe. The amount of iTASO-NH2 used was 1.0 equiv., the amount of 4-maleimidebutyric acid was 1.0-2.0 equiv., the amount of triethylamine was 2.0-3.0 equiv., and the amount of 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate was 1.3-2.0 equiv.; the reaction was carried out at room temperature for 30-60 min. The structural formula of the corresponding photosensitive mass spectrometry probe is as follows:

3. The method for preparing the photosensitive mass spectrometry probe according to claim 2, characterized in that: 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.; the reaction temperature is 100-120 ℃, and the reaction time is 8-20 h.

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

5. The application of a photosensitive mass spectrometry probe as described in claim 1 or a photosensitive mass spectrometry probe prepared by the preparation method as described in any one of claims 2-4, characterized in that: This includes labeling and mass spectrometry detection of carbonyl, carboxylic acid, amino, and thiol analytes; and sample detection of labeled products of photosensitive mass spectrometry probes via FI-ESI(-)-HRMS.

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