A mass spectrometry imaging probe for detecting peroxynitrite and its preparation method and application

Abstract

The present invention discloses a mass spectrometry imaging probe for detecting peroxynitrite, and its preparation method and application. The present invention provides a novel structural compound, the semicyanine structure of which has a positive center and can be used as a mass spectrometry detection label to capture signals; the tetrazine structure of the compound reacts with peroxynitrite to generate an oxadiazole structure, which can also be detected by mass spectrometry, and the response abundance is significantly higher than the response after reaction with other common reactive oxygen and reactive nitrogen species, showing excellent peroxynitrite selectivity, and can be used as a mass spectrometry probe for detecting peroxynitrite levels in biological samples. The probe can evaluate the production of peroxynitrite in in vivo samples by detecting the signal intensity of the corresponding product from the mass spectrometry channel, and quickly, simply and accurately analyze the spatial distribution of peroxynitrite in biological samples (such as liver tissue with ischemia-reperfusion injury) with the help of mass spectrometry imaging technology. The prior art has not disclosed the compound of the present invention, its preparation method and application.

Description

Technical Field

[0001] The present invention belongs to the field of chemistry and relates to a novel probe and its application in mass spectrometry imaging, and in particular 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, accurately and efficiently revealing the complexity of molecular structures, elucidating chemical composition, and providing insights into biological processes. Methods such as liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption / ionization (MALDI) mass spectrometry have demonstrated significant potential for in situ analysis, enabling high-throughput in situ imaging of complex biological tissues at the molecular level. MALDI mass spectrometry offers advantages such as high sensitivity, limited sample preparation, strong salt tolerance, and rapid speed.

[0003] Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are highly reactive and short-lived in physiological and pathological metabolic processes, making their detection in vivo challenging. Furthermore, their similar structures and reactivities make specific detection of individual ROS or RNS extremely challenging. To capture these transient and variable redox signals, analytical tools for their specific detection are essential. Peroxynitrite, a type of RNS produced by the coupled reaction of nitric oxide and superoxide radicals, readily undergoes oxidative and nucleophilic reactions with a variety of molecules. In various physiological and pathological conditions, it can interact with biomacromolecules, leading to microenvironmental disturbances. During hepatic ischemia, a series of redox reactions occur, generating large amounts of peroxynitrite. Currently, fluorescent probes are primarily developed for the in vivo detection of peroxynitrite. Fluorescent signal changes caused by energy resonance or electron transfer characterize changes in a class of compounds with the same structure. Mass spectrometry offers the accuracy and efficiency to detect specific molecules, making the development of mass spectrometry probes crucial for the in vivo detection of ROS and RNS. 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 an appropriate amount of an organic solvent to dissolve them, and stir the mixture at room temperature for a certain period of time;

[0012] Step S2: adding an appropriate amount of (4-(6-methyl-1,2,4,5-tetrazin-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: The crude product obtained in step S3 is loaded onto a normal phase silica gel column for chromatography, eluted with an eluent, and separated and purified 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-tetrazin-3-yl)phenyl)methanamine 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. The hemicyanine 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. The response abundance is significantly higher than that after reacting with other common ROS and RNS, showing excellent peroxynitrite selectivity. It 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 in vivo samples by detecting the signal intensity of the corresponding product through the mass spectrometry channel, and use mass spectrometry imaging technology to quickly, simply and accurately analyze the spatial distribution of peroxynitrite in biological samples (such as liver slices with liver ischemia-reperfusion injury). 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 This is a bar graph of the mass spectrometry abundance ratio of HCY-TzN products after adding common ROS and RNS in Example 3.

[0031] Figure 6 This is a comparison of mass spectrometry imaging 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 with reference to 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] To a 100 ml round-bottom flask, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (38 mg, 0.2 mmol), 4-dimethylaminopyridine (24 mg, 0.2 mmol), and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride (32 mg, 0.1 mmol) were added in sequence, followed by 45 ml of dichloromethane. The mixture was stirred at room temperature for 2 hours. Subsequently, a dichloromethane solution of (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride (20 mg, 0.08 mmol) was added, and the mixture was stirred at room temperature for 24 hours. After the reaction, the reaction solution was poured into 15 ml of ice water, and extracted with dichloromethane (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: 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 the 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 solution, which was then diluted with ethanol to form a 100 μM sample solution. 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 results of high-resolution mass spectrometry 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 the mass spectrometry imaging probe HCY-TzN to different types of interfering substances

[0043] The mass spectrometry imaging probe HCY-TzN prepared in Example 1 was dissolved in ethanol to form a 5 mM test solution, which was then diluted with ethanol to form a 100 μM sample solution. Different types of interfering substances (hydrogen peroxide H2O2, hypochlorite ClO2, and chlorine ions) were added to the sample solution. - , nitrite NO2 - 、nitrate NO3 - , alkoxy anion ROO - 、Superoxide anion O2 - , nitric oxide (NO), to a final concentration of 100 μM, and the mass spectrometric response after adding different interfering substances was tested, and compared with the addition of blank samples and the addition of peroxynitrite (ONOO) -There was no significant response to the oxadiazole structure molecules 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 is sensitive to 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 perform surgery to create a liver ischemia model after anesthesia. The specific method is: take ICR male mice that have been fasting for 14-16 hours and anesthetize them. After the mice are fully 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 the blood flow is restored. Cover the mouse with sterile gauze, place it on an electric blanket to keep warm, and replenish sterile saline through the abdominal cavity in time.

[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 HCY-TzN probe solution (30 mg / kg) was administered through the portal vein. The mice were killed 10 minutes later and the livers were frozen and sectioned and stored at -80°C. The sections were pre-processed before testing. The specific method was as follows: the complete liver tissues of the sham and model mice were taken out of the -80°C refrigerator and immediately transferred to a freezing microtome. 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. MALDI mass spectrometry imaging analysis was then performed. The results are shown in the figure. 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. The hemicyanine 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. The response abundance is significantly higher than that after reacting with other common ROS and RNS, showing excellent peroxynitrite selectivity. It 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 in vivo samples by detecting the signal intensity of the corresponding product through the mass spectrometry channel, and use mass spectrometry imaging technology to quickly, simply and accurately analyze the spatial distribution of peroxynitrite in biological samples (such as liver slices with liver ischemia-reperfusion injury).

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

[0052] The purpose of the above 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 this specific embodiment.

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: 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, 4-dimethylaminopyridine, and 1-(5-carboxypentyl)-2,3,3-trimethyl-3H-indol-1-ium chloride are placed in a reaction vessel, an organic solvent is added to dissolve them, and the mixture is stirred at room temperature for a certain period of time; Step S2: adding (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine 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 ice water, and an 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: The crude product obtained in step S3 is loaded onto a normal phase silica gel column for chromatography, eluted with an eluent, and separated and purified to obtain the product.

4. The preparation method according to claim 3, wherein: 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, wherein: In step S2, the molar ratio of (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine hydrochloride to 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 with stirring for 24 hours.

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

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

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

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