Mass spectrum imaging probe for detecting peroxynitrite as well as preparation method and application of mass spectrum imaging probe

Abstract

The invention discloses a mass spectrum imaging probe for detecting peroxynitrite as well as a preparation method and application of the mass spectrum imaging probe. The invention provides a compound with a novel structure, the hemicyanine structure of the compound has a positive electric center, and the compound can be used as a mass spectrum detection label to capture a signal; the tetrazine structure of the compound reacts with peroxynitrite to generate an oxadiazole structure, the oxadiazole structure can also be detected by mass spectrometry, the response abundance of the oxadiazole structure is obviously higher than that of the oxadiazole structure after the oxadiazole structure reacts with other common active oxygen and active nitrogen, and the oxadiazole structure shows excellent peroxynitrite selectivity and can be used as a mass spectrometry probe for detecting the peroxynitrite level in a biological sample. The probe can detect the signal intensity of a corresponding product through a mass spectrum channel to evaluate the generation of peroxynitrite in an in-vivo sample, and the spatial distribution of peroxynitrite in a biological sample (such as ischemia-reperfusion injury liver tissue) is rapidly, simply and accurately analyzed by virtue of a mass spectrum imaging technology. The compound as well as the preparation method and the application thereof are not disclosed in the prior art.

Description

Technical Field

[0001] The invention belongs to the field of chemistry, relates to a novel probe and its application in mass spectrometry imaging, and specifically relates to a mass spectrometry imaging probe for detecting peroxynitrite, and a preparation method and application thereof. Background Art

[0002] Mass spectrometry has become a powerful tool in analytical chemistry that can accurately and efficiently reveal the complexity of molecular structures, elucidate chemical composition, and gain insight into biological processes, such as liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption / ionization (MALDI) mass spectrometry. MALDI mass spectrometry has the advantages of high detection sensitivity, limited sample preparation, strong salt tolerance, and fast speed. It has shown great potential in in situ analysis and can achieve high-throughput in situ imaging of complex biological tissues at the molecular level.

[0003] Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have the characteristics of high reactivity and short-lived existence in physiological and pathological metabolic activities, making their detection in vivo difficult. In addition, their similar structures and reactivity make the specific detection of a single ROS or RNS extremely challenging. In order to capture short-lived and variable redox signals, analytical tools for the specific detection of ROS or RNS must be developed. Peroxynitrite is a type of RNS produced by the coupling reaction of nitric oxide and superoxide radicals, which is easily oxidized and nucleophilic with various molecules. It can interact with biological macromolecules in various physiological and pathological conditions, leading to microenvironmental disorders. During liver ischemia, a series of redox reactions occur, producing a large amount of peroxynitrite. At present, fluorescent probes are mostly developed for the in vivo detection of peroxynitrite. The change in fluorescence signal caused by energy resonance or electron transfer represents the change of a class of compounds with the same structure. Mass spectrometry has the accuracy and efficiency of detecting specific molecules, so the development of mass spectrometry probes is of great significance for the detection of ROS and RNS in vivo. Summary of the invention

[0004] The present invention aims to provide a mass spectrometry imaging probe for detecting peroxynitrite and a preparation method and application thereof.

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0006] A compound consisting of a cation represented by formula I and a pharmaceutically acceptable anion:

[0007]

[0008] Preferably, the anion is a chloride ion, and the structure is shown in Formula II:

[0009]

[0010] A method for preparing the above compound, characterized in that it comprises the following steps:

[0011] Step S1: Place appropriate amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride in a reaction vessel, add appropriate amounts of organic solvent to dissolve, and stir at room temperature for a certain period of time;

[0012] Step S2: adding an appropriate amount of (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methylamine hydrochloride dissolved in an organic solvent to the solution obtained in step S1, and continuing to stir and react at room temperature for a certain period of time;

[0013] Step S3: After the reaction in step S2 is completed, the reaction solution is poured into an appropriate amount of ice water, and an appropriate amount of organic solvent is added for extraction. The organic phase is collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to remove the solvent to obtain a crude product;

[0014] Step S4: Load the crude product obtained in step S3 onto normal phase silica gel column chromatography, elute with an eluent, and separate and purify to obtain the product.

[0015] Preferably, in step S1, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride is 2:1:1, and the organic solvent is dichloromethane.

[0016] More preferably, the reaction is stirred for 2 hours in step S1.

[0017] Preferably, the molar ratio of the (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methylamine hydrochloride solution in step S2 to the 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride in step S1 is 4:5, and the organic solvent is dichloromethane.

[0018] More preferably, the stirring reaction is continued for 24 hours in step S2.

[0019] Preferably, the organic solvent in step S3 is dichloromethane.

[0020] Preferably, the eluent in step S4 is a mixed solvent of toluene and methanol in a volume ratio of 10:1.

[0021] The compound is used as a mass spectrometry probe to detect the level of peroxynitrite.

[0022] The compound is used as a mass spectrometry probe to detect the spatial distribution of peroxynitrite in biological samples.

[0023] Beneficial effects:

[0024] The present invention provides a novel compound HCY-TzN, wherein the semicyanine structure of the compound has a positively charged center and can be used as a mass spectrometry detection label to capture signals; the tetrazine structure of HCY-TzN reacts with peroxynitrite to generate an oxadiazole structure, which can also be detected by mass spectrometry, and the response abundance is significantly higher than that after reacting with other common ROS and RNS, showing excellent peroxynitrite selectivity, and can be used as a mass spectrometry probe to detect the level of peroxynitrite in biological samples.

[0025] The probe molecules provided by the present invention can evaluate the production of peroxynitrite in vivo samples by detecting the signal intensity of the corresponding products from the mass spectrometry channel, and can quickly, simply and accurately analyze the spatial distribution of peroxynitrite in biological samples (such as liver slices with liver ischemia-reperfusion injury) with the help of mass spectrometry imaging technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the target compound HCY-TzN obtained in Example 1.

[0027] Figure 2 This is the high-resolution mass spectrum of the target compound HCY-TzN obtained in Example 1.

[0028] Figure 3 This is the UV-visible absorption spectrum of HCY-TzN in Example 2 before and after the addition of peroxynitrite.

[0029] Figure 4 This is the high-resolution mass spectrum of HCY-TzN in Example 2 before and after the addition of peroxynitrite.

[0030] Figure 5 The bar graph is a mass spectrometry abundance ratio of the products of HCY-TzN in Example 3 after adding common ROS and RNS.

[0031] Figure 6 This is a comparison of mass spectrometry images of HCY-TzN detecting peroxynitrite in liver sections of sham-operated mice and model mice in Example 4. DETAILED DESCRIPTION

[0032] The essential contents of the present invention are described in detail below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto.

[0033] Example 1: Synthesis of mass spectrometry imaging probe HCY-TzN

[0034]

[0035] In a 100 ml round-bottom flask, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (38 mg, 0.2 mmol), 4-dimethylaminopyridine (24 mg, 0.2 mmol), 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride (32 mg, 0.1 mmol) were added in sequence, and then 45 ml of dichloromethane was added, and the mixture was stirred and reacted at room temperature for 2 hours. Subsequently, a dichloromethane solution of (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methylamine hydrochloride (20 mg, 0.08 mmol) was added, and the mixture was stirred and reacted at room temperature for 24 hours. After the reaction, the reaction solution was poured into 15 ml of ice water, and dichloromethane was added for extraction (10 ml × 3 times). The dichloromethane extracts were collected and combined, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to remove the organic solvent to obtain a crude product; the crude product was separated and purified by a normal phase silica gel column with an eluent (toluene: methanol = 10: 1, volume ratio) to obtain a mass spectrometry imaging probe HCY-TzN (21.2 mg, 37.8%) with a purity of 93.2%.

[0036] The structure of the probe was characterized by hydrogen nuclear magnetic resonance spectroscopy. 1 H-NMR (400MHz, CDCl3) δ1.45(m,2H),1.52(s,6H),1.62(m,2H),1.87(m,2H) ,2.24(t,J=7.2Hz,2H),2.84(s,3H),2.99(s,3H),4.36(d,J=5.6Hz,2H),4.4 6(t,J=7.6Hz,2H),7.52(d,J=8.4Hz,2H),7.61(m,3H),7.82(dd,J=4.8,3.6 Hz, 1H), 7.98 (t, J = 4.4Hz, 1H), 8.41 (d, J = 8.4Hz, 2H), 8.61 (t, J = 5.6Hz, 1H). Among them, the chemical shift of 1.52ppm is the proton peak of the semi-cyanine structure, the chemical shift of 2.99ppm is the methyl proton peak of the tetrazine, and the chemical shift of 8.61ppm is the proton peak of the amide NH. The nuclear magnetic resonance hydrogen spectrum is as follows Figure 1 shown.

[0037] In addition, its molecular mass was verified by high-resolution mass spectrometry, and HRMS (ESI) m / z was obtained: calcd.for C 27 H 33 ON6 + [M] + 457.2710, observed 457.2702, as Figure 2 shown.

[0038] Example 2: Test of the responsiveness of mass spectrometry imaging probe HCY-TzN to peroxynitrite

[0039] The mass spectrometry imaging probe HCY-TzN prepared in Example 1 was dissolved in ethanol to form a 5 mM test mother solution, and the test mother solution was diluted with ethanol to form a 100 μM sample solution, and the UV-visible absorption spectrum and high-resolution mass spectrum were measured.

[0040] Peroxynitrite was added to the sample solution to a final concentration of 50 μM. After reacting for 10 minutes, the UV-visible absorption spectrum and high-resolution mass spectrum were measured.

[0041] The UV-visible absorption spectrum shows that the maximum absorption wavelength of HCY-TzN is at 268nm. With the addition of peroxynitrite, an absorption peak appears in the range of 352nm, indicating that peroxynitrite can react with HCY-TzN to generate new substances. The results are shown in Figure 3 The high-resolution mass spectrometry results showed that the molecular formula of the cation in the mass spectrometry imaging probe HCY-TzN is C 27 H 33 ON6 + [M] + 457.2710, observed 457.2702, the molecular formula of the cation in the oxidation product HCYO is C 27 H 33 O2N4 + [M] + 445.2598, observed 445.2591, as Figure 4 shown.

[0042] Example 3: Response of mass spectrometry imaging probe HCY-TzN to different types of interferents

[0043] The mass spectrometry imaging probe HCY-TzN prepared in Example 1 was dissolved in ethanol to form a 5 mM test mother solution, and the test mother solution was diluted with ethanol to form a 100 μM sample solution. Different types of interfering substances (hydrogen peroxide H2O2, hypochlorite ClO - 、Nitrite NO2 - 、Nitrate NO3 - 、alkoxy anion ROO - 、Superoxide anion O2 - , nitric oxide (NO), to a final concentration of 100 μM, and the mass spectrometry response after adding different interfering substances was tested, and compared with the addition of blank samples and the addition of peroxynitrite (ONOO) -There were no oxadiazole molecules with significant responses in all the interference groups. The matrix peak correction method was used to compare the mass spectrometry responses of all groups. The response of the product ion peak of peroxynitrite after correction was significantly higher than that of other groups, indicating that the mass spectrometry imaging probe HCY-TzN has a significant effect on peroxynitrite ONOO - With good selectivity, the results are shown in Figure 5 .

[0044] Example 4: Detection of peroxynitrite in liver sections of model mice using mass spectrometry imaging probe HCY-TzN

[0045] Take the mass spectrometry imaging probe HCY-TzN prepared in Example 1 and dissolve it into a certain concentration of liquid medicine with a PBS solution of 10% ethanol and 10% polyoxyethylene castor oil. Take ICR male mice (8 weeks old) and anesthetize them to create a liver ischemia model. The specific method is as follows: take ICR male mice that have been fasting for 14-16 hours and anesthetize them. After the mice are completely anesthetized, open the abdomen and use vascular clamps to block the arterial and portal vein blood flow of the left and middle lobes of the liver for 40 minutes. The liver tissue with blocked blood flow changes from reddish brown to pale. Carefully remove the vascular clamp after 40 minutes of ischemia, and the ischemic liver lobe returns to reddish brown after blood flow is restored. Cover the mouse with sterile gauze, place it on an electric blanket to keep warm, and replenish sterile saline in time through the peritoneal cavity.

[0046] In this experiment, after blocking the arterial and portal vein blood flow of the left and middle lobes of the liver for 40 minutes, the vascular clamp was removed, and the HCY-TzN probe solution (30 mg / kg) was given to the portal vein at the same time. The mice were killed 10 minutes later, and the livers were frozen and sectioned and stored at -80°C. The sections were pre-treated before testing. The specific method was as follows: the complete liver tissues of the sham and model mice were taken out from the -80°C refrigerator and immediately transferred to the freezing microtome, and 8 μm thick mouse liver sections were prepared at -25°C. The prepared liver sections were placed in a vacuum drying oven to dry, and the DHB matrix solution was evenly sprayed on the dried mouse liver sections, followed by MALDI mass spectrometry imaging analysis. The results are shown in Figure 6 .

[0047] from Figure 6 It can be seen that since a large amount of peroxynitrite is produced in the model group (model), the probe molecules react with peroxynitrite to generate HCYO molecules and are detected by mass spectrometry, while the response level of HCYO molecules in the sham group (sham) is significantly lower than that in the model group. Therefore, the probe can be used to detect the level of peroxynitrite in biological samples, and then to detect the spatial distribution of peroxynitrite in biological samples.

[0048] In summary:

[0049] The present invention provides a novel compound HCY-TzN, wherein the semicyanine structure of the compound has a positively charged center and can be used as a mass spectrometry detection label to capture signals; the tetrazine structure of HCY-TzN reacts with peroxynitrite to generate an oxadiazole structure, which can also be detected by mass spectrometry, and the response abundance is significantly higher than that after reacting with other common ROS and RNS, showing excellent peroxynitrite selectivity, and can be used as a mass spectrometry probe to detect the level of peroxynitrite in biological samples.

[0050] The probe molecules provided by the present invention can evaluate the production of peroxynitrite in vivo samples by detecting the signal intensity of the corresponding products from the mass spectrometry channel, and can quickly, simply and accurately analyze the spatial distribution of peroxynitrite in biological samples (such as liver slices with liver ischemia-reperfusion injury) with the help of mass spectrometry imaging technology.

[0051] It should also be noted that those skilled in the art should know through the above experiments that the above uses of HCY-TzN depend on its cationic structure, and the compounds formed by the cation and other pharmaceutically acceptable anions (such as bromide ions, iodide ions, nitrate ions) must also have the same uses.

[0052] The purpose of the above-mentioned embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims

1. A compound consisting of a cation represented by formula I and a pharmaceutically acceptable anion:

2. The compound according to claim 1, wherein the anion is a chloride ion, and the structure is shown in Formula II:

3. A method for preparing the compound according to claim 2, characterized in that: The steps include: Step S1: Place appropriate amounts of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride in a reaction vessel, add appropriate amounts of organic solvent to dissolve, and stir at room temperature for a certain period of time; Step S2: adding an appropriate amount of (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methylamine hydrochloride dissolved in an organic solvent to the solution obtained in step S1, and continuing to stir and react at room temperature for a certain period of time; Step S3: After the reaction in step S2 is completed, the reaction solution is poured into an appropriate amount of ice water, and an appropriate amount of organic solvent is added for extraction. The organic phase is collected, dried over anhydrous sodium sulfate, and then distilled under reduced pressure to remove the solvent to obtain a crude product; Step S4: Load the crude product obtained in step S3 onto normal phase silica gel column chromatography, elute with an eluent, and separate and purify to obtain the product.

4. The preparation method according to claim 3, characterized in that: In step S1, the molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride is 2:1:1, and the organic solvent is dichloromethane.

5. The preparation method according to claim 4, characterized in that: In step S1, the reaction was stirred for 2 hours.

6. The preparation method according to claim 3, characterized in that: In step S2, the molar ratio of the (4-(6-methyl-1,2,4,5-tetrazine-3-yl)phenyl)methylamine hydrochloride solution to the 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride in step S1 is 4:5, and the organic solvent is dichloromethane.

7. The preparation method according to claim 6, characterized in that: In step S2, the reaction was continued under stirring for 24 hours.

8. The preparation method according to claim 3, characterized in that: In step S3, the organic solvent is dichloromethane.

9. Use of the compound according to claim 1 or 2 as a mass spectrometry probe for detecting peroxynitrite levels.

10. Use of the compound according to claim 1 or 2 as a mass spectrometry probe for detecting the spatial distribution of peroxynitrite in a biological sample.

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