A compound and its use as a mass spectrometry probe

Abstract

This invention discloses a compound and its use as a mass spectrometry probe. The invention provides a compound with a novel chemical structure that can be used as a mass spectrometry probe to improve the separation of metabolite isomers and enhance their mass spectrometry response. Compared with existing compounds Dns-PP or TMT-PP, the compound provided by this invention has a superior effect on improving the separation of fatty acid, bile acid, or phosphate metabolite isomers and also a superior effect on enhancing the mass spectrometry response of these metabolite isomers. The prior art has not disclosed the structure and use of the compound of this invention, nor does it offer any technical guidance. The technical solution of this invention has outstanding substantive features and significant progress.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of biological detection, and relates to synthesis of a new compound and application in mass spectrometry. BACKGROUND

[0002] Mass spectrometry has advantages of fast analysis speed, high sensitivity, wide dynamic range, and can provide qualitative and quantitative information of compounds. At present, mass spectrometry, especially chromatography-mass spectrometry, has become a mainstream analysis technology for endogenous metabolites, and is also widely used in fields of organic chemistry, drug metabolism, toxicology, pesticide determination, environmental protection, food chemistry and the like.

[0003] There are still many problems in qualitative and quantitative analysis of endogenous metabolites by using mass spectrometry: endogenous metabolites have large polarity, poor chromatographic retention, and difficult quantification; ionization efficiency is low, and high sensitivity analysis cannot be achieved; structural and property similarities between isomers cannot be separated, identified and accurately quantified. Chemical labeling by using a mass spectrometry probe is an effective method to solve the above problems, and introduction of a hydrophobic group can reduce the polarity of a derivatization product and enhance its chromatographic retention; introduction of an easy-ionization group can improve the mass spectrometry response of the derivatization product, so that it is suitable for analysis of trace metabolites; differences in physicochemical properties between isomers are amplified, so that separation and identification of isomers on a conventional chromatographic column are achieved. Therefore, the combination of the probe and mass spectrometry can improve its sensitivity and selectivity, so as to further expand its application range.

[0004] In recent years, many new types of mass spectrometry probes have been developed, which are designed for analytes with specific functional groups such as amines, phenols, carboxylic acids, steroids, thiols and alcohols. Among them, the mass spectrometry probe for carboxylic acid analytes modifies the analytes by acid-amine condensation reaction of carboxyl with amino, piperazinyl or reaction with acyl chloride group, acyl bromide group. However, the existing mass spectrometry probe for carboxylic acid substances cannot simultaneously achieve improvement of isomer separation and improvement of mass spectrometry response, and the derivatization efficiency of different subtypes of a class of substances is uneven. In addition, the existing mass spectrometry probe has a long synthesis route, complicated steps and low yield. Therefore, exploring a new type of mass spectrometry probe with high efficiency and low cost synthesis route, which can not only enhance the mass spectrometry response, but also achieve separation and identification of multiple types of isomers, has important significance for development and wide application of mass spectrometry analysis method. SUMMARY

[0005] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a compound and use as a mass spectrometry probe.

[0006] The above purpose of the present application is achieved by the following technical scheme:

[0007] A compound, the chemical structure of which is as follows:

[0008]

[0009] Use of the above compound as a mass spectrometry probe.

[0010] Use of the above compound as a mass spectrometry probe to improve the resolution of fatty acid metabolite isomers.

[0011] Use of the above compound as a mass spectrometry probe to enhance the mass spectrometry response of fatty acid metabolites.

[0012] Use of the above compound as a mass spectrometry probe to improve the resolution of bile acid metabolite isomers.

[0013] Use of the above compound as a mass spectrometry probe to enhance the mass spectrometry response of bile acid metabolites.

[0014] Use of the above compound as a mass spectrometry probe to improve the resolution of phosphate metabolite isomers.

[0015] Use of the above compound as a mass spectrometry probe to enhance the mass spectrometry response of phosphate metabolites.

[0016] Beneficial effects:

[0017] The present application provides a compound with a novel chemical structure, which can be used as a mass spectrometry probe to improve the resolution of metabolite isomers and enhance their mass spectrometry response. Compared with the existing compounds Dns-PP or TMT-PP, the compound provided by the present application has a more optimal effect as a mass spectrometry probe for improving the resolution of fatty acid, bile acid or phosphate metabolite isomers, and also has a more optimal effect for enhancing the mass spectrometry response of fatty acid, bile acid or phosphate metabolite isomers. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a mass spectrometry detection chart of the compound of the present application;

[0019] Figure 2 is a nuclear magnetic resonance hydrogen spectrum chart of the compound of the present application;

[0020] Figure 3 is the chemical structure of the compound TMT-Pro-PP of the present application and the compounds Dns-PP and TMT-PP in the prior art;

[0021] Figure 4 is the separation effect of TMT-Pro-PP, Dns-PP and TMT-PP as mass spectrometry probes for detecting fatty acid isomers;

[0022] Figure 5 is the separation effect of TMT-Pro-PP, Dns-PP and TMT-PP as mass spectrometry probes for detecting bile acid isomers;

[0023] Figure 6 To test the separation effect of TMT-Pro-PP, Dns-PP and TMT-PP as mass spectrometry probes for detecting phosphoric acid metabolite isomers. DETAILED DESCRIPTION

[0024] The substantial content of the present application will be described in detail below in combination with examples, but the protection scope of the present application is not limited by this.

[0025] Example 1: Synthesis and structure confirmation of new compound (TMT-Pro-PP)

[0026]

[0027] The synthesis route is as follows:

[0028]

[0029] Synthesis steps:

[0030] (1) N2, N2, N4, N4-tetramethyl-2, 4-diamine-6-chloro-1, 3, 5-triazine (T1)

[0031] Into a 100 mL round flask, 6-chloro-2, 4-diamino-1, 3, 5-triazine (300 mg, 2.068 mmol) was added, sodium hydride (300 mg, 12.5 mmol) was added, a magnetic son was put in, DMF (20 mL) was added, and it was placed on a flat plate magnetic stirrer, and iodomethane (1.5 mL) was added dropwise while stirring, and the reaction was stirred at room temperature for 4 h. 0.5 ml of the reaction solution was taken into a 2 ml EP tube, 1 mL of H2O and 0.5 mL of ethyl acetate solution were added, shaken well for 1 min, and then separated into layers by standing. The upper organic phase was taken by capillary and detected by thin layer chromatography (petroleum ether: ethyl acetate = 5:1, second point). The reaction solution was transferred to a separatory funnel, H2O (50 mL) was added, and ethyl acetate was extracted (30 mL x 3 times). The extracted organic phase was concentrated by rotary evaporation, and the obtained crude product was purified by column chromatography (petroleum ether: ethyl acetate = 10:1 start) to obtain a light yellow solid, which was intermediate T1.

[0032] (2) (2, 4-bis (dimethylamino) -1, 3, 5-triazinyl) -6-L-proline (T2)

[0033] Into a 100 mL flask, add T1 (201 mg, 1 mmol), add L-proline (172 mg, 1.5 mmol), put into a magnetic stirrer, add anhydrous methanol (20 mL), add triethylamine 0.3 mL, fix to a constant temperature heating magnetic stirrer, heat under reflux at 85°C for 8 h under nitrogen protection. After the reaction is completed, stop heating, and naturally cool to room temperature. TLC detection shows that the reaction is completed (dichloromethane:methanol=25:1, third point). Concentrate the reaction solution by rotary evaporation, and purify the obtained crude product by column chromatography (dichloromethane:methanol=40:1, start). A white solid, intermediate T2, is obtained.

[0034] (3) 6-((2,4-bis(dimethylamino)-1,3,5-triazin-2-yl)-D-prolyl) piperazine-1-carboxylic acid tert-butyl ester (T3)

[0035] Into a 100 mL flask, add T2 (100 mg, 0.35 mmol), add Boc-piperazine (200 mg, 1.07 mol), add HATU (135.7 mg, 0.35 mmol), put into a magnetic stirrer, add anhydrous acetonitrile (20 mL), fix to a constant temperature heating magnetic stirrer, heat under reflux at 45°C for 5 h under nitrogen protection. After the reaction is completed, stop heating, and naturally cool to room temperature. Concentrate the reaction solution by rotary evaporation, and obtain a yellow solid product, which is the crude product of intermediate T3. Without further purification, it is directly used in the next step of the synthesis reaction.

[0036] (4) (R)-N2,N2,N4,N4-tetramethyl-2,4-diamino-6-(2-pyrrolidinylcarbonylpiperazinyl)-1,3,5-triazine (TMT-Pro-PP)

[0037] Dissolve the crude product of intermediate T3 in dichloromethane (10 mL), put into a magnetic stirrer, add trifluoroacetic acid (1 mL), put on a flat plate magnetic stirrer, and stir under ambient temperature conditions for 4 h. After the reaction is completed, wash the reaction solution with saturated NaHCO3 solution (20 mL x 3 times), and then wash with H2O (20 mL x 3 times). After washing, dry the reaction solution by adding anhydrous NaSO4, and detect the completion of the reaction by TLC (dichloromethane:methanol=20:1, third point). Concentrate the reaction solution by rotary evaporation, and purify the obtained crude product by column chromatography (dichloromethane:methanol=40:1, start). A white solid final product is obtained, and the purity is 98.87%.

[0038] Structure confirmation:

[0039] Mass spectrometry identification: TMT-Pro-PP: M=348.24; [M+H] + = 349.25; [M+Na] + = 471.19, asFigure 1 .

[0040] NMR identification: 1 HNMR (500 MHz, DMSO-d6): δ 4.87 (s, 1H), 3.02 (s, 12H), 3.34 (s, 2H), 3.56 (dd, J = 4.2 Hz, 4H), 2.70 (dd, J = 4.4 Hz, 4H), 1.87 (s, 2H), 1.73 (s, 2H), 1.71 (d, J = 39.9 Hz, 1H). As Figure 2 .

[0041] Example 2: Use of TMT-Pro-PP as a mass spectrometry probe

[0042] 1. Solution preparation

[0043] Preparation of fatty acid standard solution: accurately weigh a certain amount of each fatty acid standard, add methanol to prepare a 500 mM concentration of each fatty acid standard stock solution, store at -20°C for standby, and dilute to the appropriate concentration as needed before use.

[0044] Preparation of bile acid standard solution: accurately weigh a certain amount of each bile acid standard, add methanol to prepare a 500 mM concentration of each bile acid standard stock solution, store at -20°C for standby, and dilute to the appropriate concentration as needed before use.

[0045] Preparation of phosphoric acid metabolite standard solution: accurately weigh a certain amount of each phosphoric acid metabolite standard, add methanol-water (1 / 1, v / v) to prepare a 500 mM concentration of each phosphoric acid metabolite standard stock solution, store at -20°C for standby, and dilute to the appropriate concentration as needed before use.

[0046] Dns-PP solution: accurately weigh a certain amount of Dns-PP, dissolve in acetonitrile, and prepare a Dns-PP solution of the desired concentration, store at -20°C for standby.

[0047] TMT-PP solution: accurately weigh a certain amount of TMT-PP, dissolve in acetonitrile, and prepare a TMT-PP solution of the desired concentration, store at -20°C for standby.

[0048] TMT-Pro-PP solution: accurately weigh a certain amount of TMT-Pro-PP, dissolve in acetonitrile, and prepare a TMT-Pro-PP solution of the desired concentration, store at -20°C for standby.

[0049] HATU solution: accurately weigh a certain amount of HATU, dissolve in acetonitrile, and prepare a HATU solution of the desired concentration, store at -20°C for standby.

[0050] 2. Derivatization reaction

[0051] Fatty acid and bile acid derivatization reaction: The fatty acid and bile acid standard stock solutions were precisely pipetted, respectively, and methanol was added to prepare a fatty acid and bile acid mixed standard solution with a concentration of 1 mM for standby. 20 μL of each of the prepared fatty acid and bile acid mixed standard solution was precisely pipetted into a 1.5 mL centrifuge tube, 20 μL of HATU solution with a concentration of 6 mM was added, vortexed for 1 min, and left to stand for 10 min. 20 μL of mass spectrometry probe (Dns-PP, TMT-PP, TMT-Pro-PP) solution with a concentration of 9 mM was added, 40 μL of methanol-water (1 / 1, v / v) solution was added, vortexed for 1 min, and reacted at 45°C in a metal bath for 50 min. Vortexed for 1 min, low-temperature high-speed centrifugation (14000 rpm, 4°C, 10 min), and 60 μL of supernatant was precisely pipetted and transferred to an injection vial for LC-MS / MS analysis.

[0052] Phosphorus metabolite derivatization reaction: The phosphorus metabolite standard stock solutions were precisely pipetted, and methanol was added to prepare a phosphorus metabolite mixed standard solution with a concentration of 1 mM for standby. The mass spectrometry probe solution reaction liquid was prepared, 250 μL of each 9 mM mass spectrometry probe (Dns-PP, TMT-PP, TMT-Pro-PP) solution was added, 250 μL of EDCI solution (125 μl of methanol + 125 μL of DMSO / 6 mM) was added, 3.375 μL of N-methylmorpholine was added, vortexed for 1 min, and placed in a 1.5 mL centrifuge tube. 50 μL of the phosphorus metabolite mixed standard solution and 50 μL of the mass spectrometry probe solution reaction liquid were precisely pipetted into a 1.5 mL centrifuge tube, vortexed for 1 min, reacted at 40°C in a metal bath for 30 min, low-temperature high-speed centrifugation (14000 rpm, 4°C, 10 min), and 60 μL of supernatant was precisely pipetted and transferred to an injection vial for LC-MS / MS analysis.

[0053] 3. Analysis conditions

[0054] (1) Chromatographic conditions

[0055] Fatty acid chromatographic separation condition: Column: Agilent Zorbax Eclipse XDB-C18 (2.1 x 100 mm, 1.8 μm); injector temperature: 4 °C; column temperature: 50 °C; flow rate: 0.4 mL / min; mobile phase: 0.1% formic acid water - methanol (A-B), gradient elution program as follows: 0-9 min (30%-44% B), 9-18 min (44%-59% B), 18-25 min (69%-73% B), 18-25 min (59%-69% B), 25-30 min (69%-73% B), 30-37 min (73%-80% B), 37-42 min (80%-83% B), 42-48 min (83%-94% B), 48-53 min (94%-96% B), 53-54 min (96%-100% B), 54-56 min (100% B), 56-56.5 min (100%-40% B), 56.5-60 min (30% B); injection volume: 2 μL.

[0056] Bile acid chromatographic separation condition: Column: Agilent Zorbax Eclipse XDB-C18 (2.1 x 100 mm, 1.8 μm); injector temperature: 4 °C; column temperature: 50 °C; flow rate: 0.4 mL / min; mobile phase: 0.1% formic acid water - methanol (A-B), gradient elution program as follows: 0-2 min (40%-44% B), 2-18 min (44%-59% B), 18-25 min (59%-69% B), 25-32 min (69%-73% B), 32-37 min (73%-80% B), 37-42 min (80%-83% B), 42-48 min (83%-94% B), 48-53 min (94%-96% B), 53-54 min (96%-100% B), 54-56 min (100% B), 56-56.5 min (100%-40% B), 56.5-60 min (40% B); injection volume: 2 μL.

[0057] Phospho-metabolite chromatographic separation condition: Column: Waters ACQUITY UPLC BEH C18 (2.1 x 100 mm, 1.7 μm); injector temperature: 4 °C; column temperature: 40 °C; flow rate: 0.3 mL / min; mobile phase: 10 mM ammonium acetate / 0.1% ammonia water / water-10 nM ammonium acetate / 0.1% ammonia water / 95% methanol (A-B), gradient elution program as follows: 0-13.5 min (25%-33% B), 13.5-14 min (33%-40% B), 14-22 min (40% B), 22-25 min (40%-60% B), 25-27 min (60%-60% B), 27-30 min (60%-100% B), 30-31 min (100% B), 31-32 min (100%-25% B), 32-35 min (25% B); injection volume: 2 μL.

[0058] (2) Mass spectrometry condition

[0059] Mass spectrometry detection condition: ESI positive ion detection; interface voltage: 4.0 kV; MRM mode; heating gas flow rate: 10.0 L / min; atomizing gas: 3.0 L / min; drying gas: nitrogen, 10.0 L / min; desolvation temperature: 526 °C; heating block temperature: 400 °C; interface temperature: 300 °C; DL tube temperature: 250 °C; collision gas: argon, 270 kPa.

[0060] Different mass spectrometry probe-labeled products of fatty acid, bile acid and phospho-metabolite and mass spectrometry detection conditions are shown in Tables 1-3, respectively.

[0061] Table 1 Mass spectrometry detection condition of fatty acid isomer-labeled product

[0062]

[0063]

[0064] Table 2 Mass spectrometry detection condition of bile acid isomer-labeled product

[0065]

[0066]

[0067] Table 3 Mass spectrometry detection condition of phospho-metabolite isomer-labeled product

[0068]

[0069] 4. Experimental results

[0070] Dns-PP, TMT-PP are two compounds in prior art, used for comparison with TMT-Pro-PP to prove the advantage of TMT-Pro-PP over prior art. The chemical structure comparison of the three compounds is shown as follows. Figure 3

[0071] Figure 4 Figure 5 Figure 6 The above three compounds are used as mass spectrometry probes to compare the separation effect of fatty acid isomers, bile acid isomers and phosphoric acid isomers (the upper graph is the separation chromatogram, and the lower graph is the separation degree of adjacent chromatographic peaks).

[0072] Compared with Dns-PP, TMT-Pro-PP significantly improves the separation degree (R) of fatty acid isomers, which is increased by about 2 times in some cases; significantly improves the separation degree of bile acid isomers, which is increased by about 3 times in some cases; significantly improves the separation degree of phosphoric acid metabolite isomers, which is increased by about 2 times in some cases.

[0073] Compared with TMT-PP, TMT-Pro-PP significantly improves the separation degree (R) of fatty acid isomers, which is increased by about 3 times in some cases; significantly improves the separation degree of bile acid isomers, which is increased by about 3.5 times in some cases; significantly improves the separation degree of phosphoric acid metabolite isomers, which is increased by about 4 times in some cases.

[0074] Compared with Dns-PP, TMT-Pro-PP significantly improves the mass spectrometry response of fatty acid, bile acid and phosphoric acid metabolite isomer labeling products, which is increased by about 1000 times in some cases; compared with TMT-PP, TMT-Pro-PP significantly improves the mass spectrometry response of fatty acid, bile acid and phosphoric acid metabolite isomer labeling products, which is increased by about 10 times in some cases. The mass spectrometry response data is shown in Table 4 (the response is calculated by peak area, and is normalized based on TMT-PP); wherein: the comparison data is the sum of the isomer peak areas (C5:C5-1 / C5-2 / C5-3; C6:C6-1 / C6-2 / C6-3; C8:C8-1 / C8-2 / C8-3; C9:C9-1 / C9-2; C18:1:C18:1-1 / C18:1-2; C20:1:C20:1-1 / C20:1-2; trihydroxy bile acid: αMCA / ωMCA / CA / 5αCA; dihydroxy bile acid: HDCA / 3βHDCA / iso-UDCA / UDCA / CDCA / DCA; monohydroxy bile acid: 5α-LCA / iso-LCA / LCA; phosphoric acid sugar: G-1-P / G-6-P / F-6-P; phosphoric acid ribose: 5-P-R / 5-P-Rb; phosphoric acid glycerate: C-2-P / C-3-P; diphosphate metabolite: IPP / DPP).​​​

[0075] Table 4 Mass spectrometry relative response data (four decimal places)

[0076]

[0077]

[0078] In summary, compared with the existing compounds Dns-PP or TMT-PP, the compound provided by the present application has a more optimal effect on improving the separation degree of fatty acid, bile acid or phosphoric acid metabolite isomers as a mass spectrometry probe, and also has a more optimal effect on enhancing the mass spectrometry response of fatty acid, bile acid or phosphoric acid metabolite isomers.

[0079] The above examples serve to specifically introduce the essential content of the present application, but those skilled in the art should know that the protection scope of the present application should not be limited to the specific examples.

Claims

1. A compound, characterized in that, The chemical structure is as follows: 。 2. Use of a compound according to claim 1 as a mass spectrometry probe.

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