Application of a tetrazine-based fluorescence-enhanced probe in the detection of superoxide anion

Abstract

The present invention provides an application of a tetrazine-based fluorescence-enhanced probe in the detection of superoxide anions, belonging to the field of biomedicine. The structure of the tetrazine-based fluorescence-enhanced probe includes a fluorophore, a linker chain, and a tetrazine nucleus, and the superoxide anion is the superoxide anion in solution, cells, tissues, or living animals; the detection of the superoxide anion includes the following steps: dissolving the tetrazine compound in dimethyl sulfoxide to prepare a stock solution, and then diluting it with PBS or cell culture medium to a final concentration of 1-10 μM of the tetrazine compound, and finally adding it to the system containing superoxide anions. After 25-35 minutes, the superoxide anion is detected. The tetrazine-based fluorescence-enhanced probe is used for the detection of superoxide anions, with high sensitivity, strong selectivity, and fast detection speed.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to the application of a tetrazine-based fluorescence-enhanced probe in the detection of superoxide anions. Background Art

[0002] Superoxide anion (O 2 ·- ) is an important endogenous reactive oxygen species in biological systems, the source molecule of other reactive oxygen species (ROS), and also an important signaling molecule. Intracellular superoxide anion (O 2 ·- ) is mainly produced by NADPH oxidase (NOXs) or the mitochondrial respiratory chain. Superoxide anion is rapidly converted to hydrogen peroxide (H 2 O 2 ), and H 2 O 2 may also be converted to H 2 O by cellular antioxidant proteins (such as peroxiredoxins). In addition, hydrogen peroxide reacts with metal cations (Fe 2+ ) to form hydroxyl radicals (OH . ). At the same time, O 2 ·- reacts with free nitric oxide (NO) to generate peroxynitrite anion. These oxidizing small molecules can all oxidize proteins, lipids, nucleic acids, etc., and then irreversibly damage cellular macromolecules and develop into various diseases. Since O 2 ·- is an upstream reactive oxygen species and participates in various physiological processes, but the relationship between O 2 ·- and the occurrence of diseases has not been clarified, such as aging, neurodegenerative diseases, cardiovascular diseases, cancer, etc. On the other hand, O 2 ·- has a low concentration in vivo (10 -10 M) and a short half-life (10 -6 Sec), and is easily converted into other oxidizing species, so it is challenging to accurately achieve real-time detection of O 2 ·- . Developing new specific targeting O 2 ·- detection tools is of great significance for studying the pathological development mechanism mediated by it.

[0003] Currently, the detection methods of superoxide anion mainly include electrochemical detection, spectrophotometric detection, vibrational spectroscopy detection, bioluminescence detection, and fluorescence detection. Electrochemical and other spectroscopic detections are easy to detect O 2 ·- in solution and are not applicable to O 2 in living cells or tissues.·- Detection. Fluorescence imaging is gradually becoming a new and efficient biological detection method because it can detect biomolecules in the in-situ biological environment. Given the important significance of O 2 ·- in various pathophysiological processes, there are currently various O 2 ·- probes reported, and most probes can detect O 2 ·- in living cells. For example, there is a commercially available probe, dihydroethidium, but its non-specificity and cytotoxicity limit its application. Currently, high specificity and high sensitivity for intracellular imaging O 2 ·- remain a challenge for O 2 ·- probes. Therefore, developing new fluorescent probes with high sensitivity and high selectivity to achieve the dynamic detection of O 2 ·- in the in-situ biological environment is the first technical problem to be solved in studying the relationship between O 2 ·- and the disease development mechanism.

[0004] Tetrazine compounds and their derivatives have many physiological activities, such as anti-inflammatory, analgesic, antiviral, antibacterial and anticancer. Among them, tetrazine derivatives containing fluorescent groups can be used as biological fluorescent probes. Tetrazine derivatives and the corresponding bioorthogonal chemistry have the advantages of good biocompatibility, in vivo stability, rapid reaction, etc., and have been widely used in important research fields such as chemical biology and pharmacy to explore the life processes of cells and tissues from the molecular and subcellular structure levels. More importantly, due to the inherent physical and chemical properties of tetrazine compounds, they can be designed as biological fluorescent probes. The fluorescence signal of this type of probe is only activated through bioorthogonal reactions, thereby greatly reducing the background signal of unreacted probes in the body and achieving improved image resolution and detection sensitivity. Summary of the Invention

[0005] The present invention addresses the problems existing in the prior art and provides an application of a tetrazine-based fluorescence-enhanced probe in the detection of superoxide anion with good stability, high sensitivity and high selectivity.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] An application of a tetrazine-based fluorescence-enhanced probe in the detection of superoxide anion, characterized in that the tetrazine-based fluorescence-enhanced probe has the structure of formula I:

[0008]

[0009] Wherein,

[0010] The emission wavelength of the fluorophore is 300 nm - 800 nm;

[0011] The linking chain is a single bond or a divalent group;

[0012] R is selected from H, straight-chain or branched-chain alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; said R may be substituted by one or more substituents.

[0013] Further, said R is selected from H, C 1-6 straight-chain or branched-chain alkyl, C 3-6 cycloalkyl, heterocycloalkyl, phenyl or heteroaryl, and said R may be substituted by one or more substituents.

[0014] Further, said R is selected from C 1-4 straight-chain or C 1-4 branched-chain alkyl, C 4-6 cycloalkyl, C 2-5 heterocycloalkyl, phenyl, five - six-membered heteroaryl; said R may be substituted by one or more substituents.

[0015] Further, in the heterocycloalkyl or heteroaryl, the heteroatoms are selected from N, O or S, and the number of heteroatoms is 1, 2 or 3.

[0016] Preferably, said R is selected from one of the following structures:

[0017]

[0018] Further, the emission wavelength of the fluorophore is 400 nm - 600 nm, more preferably 400 nm - 500 nm.

[0019] Further, the fluorophore fluorophore nucleus is selected from naphthylamine, quinoline, sulfonaphthylamine, 1,8-naphthalimide, xanthene, 4-nitrobenzoxadiazole, boron dipyrromethene, coumarin, carbazole, silafluorene or cyanine.

[0020] Preferably, the fluorophore is selected from one of the following structures:

[0021]

[0022] Further, the linking chain is a divalent group, and the divalent group is selected from alkylene, arylene, heteroarylene, divalent groups containing O, divalent groups containing N, divalent groups containing amide bonds or divalent groups containing ester bonds.

[0023] Further, the divalent group is selected from C 1-6 alkylene, arylene, aryloxy, arylamino, aryl-C 1-3 alkyl-oxy, aryl-C 1-3Alkyl - amino, arylene - C 1-3 Alkyl - amino - C(O) group, sub - C 1-2 -C(O)-C 1-2 -amino or sub - C 1-2 -C(O)-C 1-2 -oxy group, and the divalent group is substituted by 1 - 2 C 1-3 alkyl groups.

[0024] Furthermore, the aryl group is selected from benzene or five - to six - membered heteroarylene, and in the heteroaryl group, the heteroatoms are selected from N, O or S, and the number of heteroatoms is 1, 2 or 3.

[0025] Furthermore, the linking chain is selected from a single bond or one of the following structures:

[0026]

[0027] Furthermore, the tetrazine - type fluorescence - enhanced probe is selected from one of the following structures:

[0028]

[0029]

[0030] Preferably, the tetrazine - type fluorescence - enhanced probe is selected from one of the following structures:

[0031]

[0032] Furthermore, the superoxide anion is the superoxide anion in solution, cells, tissues or live animals.

[0033] Secondly, a method for detecting superoxide anion is provided, that is, contacting the tetrazine - type fluorescence - enhanced probe shown in formula I with superoxide radicals.

[0034] Furthermore, the tetrazine - type fluorescence - enhanced probe exists in the form of a reagent, a probe solution or a kit.

[0035] Furthermore, the method for detecting superoxide anion includes the following steps: dissolving the tetrazine compound in dimethyl sulfoxide to prepare a stock solution, then diluting it with PBS or cell culture medium to a final concentration of 1 - 10 μM of the tetrazine compound, and finally adding it to the system containing superoxide anion. After 25 - 35 min, the superoxide anion is detected. The concentration of the tetrazine compound in the stock solution is 1 - 10 mM, and the cell culture medium is DMEM.

[0036] Furthermore, the concentration of superoxide ions in the system of superoxide anion is 1 nM - 100 μM.

[0037] Finally, a method for preparing a tetrazine-based fluorescence-enhanced probe is provided, including the following steps:

[0038] (1) React R1CN with RCN under the catalysis of divalent zinc ions to obtain product A;

[0039] (2) Oxidize product A, then elute and purify to obtain the tetrazine-based fluorescence-enhanced probe;

[0040] Among them, R1CN is a cyanide-containing fluorescent compound, and RCN is a cyanide compound containing an R group;

[0041] The chemical reaction equation for preparing the tetrazine-based fluorescence-enhanced probe is as follows:

[0042]

[0043] Further, in step (1), R1 in R1CN is selected from: naphthylamine, quinoline, xanthene, boron dipyrromethene, coumarin, sila-xanthene or cyanine dye.

[0044] Further, the molar ratio of the fluorescent compound to the cyanide-substituted RCN in step (1) is 1:10 - 50.

[0045] Further, the oxidant used for oxidation in step (2) is air or oxygen.

[0046] Further, the tetrazine-based fluorescence-enhanced probe can also be applied to detect different degrees of oxidative damage in cells, where the detection is to identify different concentrations of superoxide anions in cells to distinguish different degrees of oxidative stress damage in cells.

[0047] The detection mechanism of superoxide radicals in the present invention is as follows: The tetrazine group in the probe structure is a good fluorescence quenching group, so the background fluorescence of the probe is in a quenched state with weak intensity; however, this tetrazine group can undergo a specific and efficient conversion reaction with superoxide. As the tetrazine structure is destroyed by superoxide radicals, the fluorescence intensity of the probe can be restored. The restored fluorescence intensity has a dose-dependent relationship with superoxide, so it can be used to qualitatively and quantitatively indicate superoxide.

[0048] After further research, it was found that after the tetrazine structure contacts with superoxide radicals, through mass spectrometry analysis, the molecular weight of the probe in the fluorescence recovery state is 12 less than that of the probe in the fluorescence quenching state. It is judged that the tetrazine structure is converted into an oxadiazole structure. Since the oxadiazole does not have the ability to quench fluorescence, the fluorescence recovery of the fluorescence probe can be achieved. The specific process is as follows:

[0049]

[0050] The fluorescence probe for detecting superoxide anion of the present invention has no fluorescence itself, but the tetrazine structure it contains can react specifically and rapidly with superoxide anion to generate a product with strong fluorescence, and there is a positive correlation between the fluorescence intensity and the concentration of superoxide anion; as the concentration of superoxide anion increases, the fluorescence intensity gradually increases, thus realizing the dynamic detection of superoxide anion. The fluorescence probe for detecting superoxide anion of the present invention is applicable to the real-time dynamic observation of superoxide anion in solutions, cells, tissues or living animals.

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

[0052] (1) As a fluorescence probe for detecting superoxide anion, it has good molecular stability and can be stored and used for a long time;

[0053] (2) It has high fluorescence signal brightness, high detection signal-to-noise ratio and good sensitivity;

[0054] (3) It has excellent selectivity and can specifically detect superoxide anion in complex biological samples;

[0055] (4) It has good biofilm permeability and can thus be used for the detection of superoxide anion in living cells;

[0056] (5) It has a fast detection speed;

[0057] (6) It has a sensitive detection response, and the signal intensity will increase with the increase of the content of superoxide anion in the species to be detected, and is suitable for the real-time tracing of endogenous superoxide anion in organisms. Description of the Drawings

[0058] Figure 1 It is the fluorescence intensity of Compound 4 and its responses to 1 - blank, 2 - superoxide anion, 3 - hydrogen peroxide, 4 - singlet oxygen, 5 - sodium hypochlorite, 6 - tert-butyl hydroperoxide, 7 - peroxynitrite anion, 8 - nitric oxide, 9 - hydroxyl radical at the maximum emission wavelength;

[0059] Figure 2 It is the fluorescence intensity of Compound 21 and its responses to 1 - blank, 2 - superoxide anion, 3 - hydrogen peroxide, 4 - singlet oxygen, 5 - sodium hypochlorite, 6 - tert-butyl hydroperoxide, 7 - peroxynitrite anion, 8 - nitric oxide, 9 - hydroxyl radical at the maximum emission wavelength;

[0060] Figure 3 It is the fluorescence intensity of Compound 22 and its responses to 1 - blank, 2 - superoxide anion, 3 - hydrogen peroxide, 4 - singlet oxygen, 5 - sodium hypochlorite, 6 - tert-butyl hydroperoxide, 7 - peroxynitrite anion, 8 - nitric oxide, 9 - hydroxyl radical at the maximum emission wavelength;

[0061] Figure 4 is the fluorescence intensity of Compound 30 at the maximum emission wavelength after its response to 1 - blank, 2 - superoxide anion, 3 - hydrogen peroxide, 4 - singlet oxygen, 5 - sodium hypochlorite, 6 - tert - butyl hydroperoxide, 7 - peroxynitrite anion, 8 - nitric oxide, 9 - hydroxyl radical;

[0062] Figure 5 is the fluorescence intensity of Compounds 22 and 30 after their response to different concentrations of superoxide anion (0 - 0.10 equivalent KO 2 );

[0063] Figure 6 is the fluorescence intensity of Compounds 22 and 30 after their response to different concentrations of superoxide anion (0 - 20 equivalent KO 2 );

[0064] Figure 7 is the fluorescence intensity of Compound 30 after its response to different concentrations of superoxide anion;

[0065] Figure 8 : A) are fluorescence microscopy images of Compounds 22 and 30 in HepG 2 cells in response to superoxide anion generated by induction with different concentrations of hydrogen peroxide, and B) are the quantitative results of the fluorescence intensity;

[0066] Figure 9 : A) are fluorescence microscopy images of Compounds 22 and 30 in HepG 2 cells in response to superoxide anion generated by induction with 2 mM hydrogen peroxide for different times, and B) are the quantitative results of the fluorescence intensity;

[0067] Figure 10 : A) are fluorescence microscopy images of Compounds 22 and 30, DCFHDA, and DHE in HepG 2 cells in response to superoxide anion generated under different set conditions (Groups 1 - 4), and B) are the quantitative results of the fluorescence intensity;

[0068] Figure 11 is Compound 30 imaging superoxide anion generated by cardiac ischemia - reperfusion in myocardial tissue sections;

[0069] Figure 12 is an anatomical cardiac imaging of Compound 30 detecting superoxide anion generated by cardiac ischemia - reperfusion in live animals. Detailed implementation mode

[0070] It should be noted that the raw materials used in the present invention are all ordinary commercially available products, and their sources are not specifically limited.

[0071] Example 1 Synthesis of unknown compound

[0072] Synthetic methods of compounds 22, 23, 30, and 31

[0073] The cyanide group-containing fluorophore (1 eq), acetonitrile (10 eq), zinc trifluoromethanesulfonate (0.5 eq), and 80% hydrazine hydrate (50 eq) were placed in a pressure-sealed flask and reacted at 70 °C in an oil bath for 12 h. After the reaction, ethyl acetate was added to extract the organic phase, and the aqueous phase was washed three times with water and saturated brine in sequence. The organic phase was dried over anhydrous sodium sulfate, concentrated by evaporation, and purified by silica gel column chromatography using a petroleum ether solution containing ethyl acetate (50%) as the eluent to obtain the intermediate dihydrotetrazine derivative. The intermediate dihydrotetrazine derivative was dissolved in 5 mL of methanol solution, and air was continuously bubbled in for oxidation. After the reaction, it was concentrated by evaporation and purified by silica gel column chromatography using a petroleum ether solution containing ethyl acetate (20%) or a dichloromethane solution containing methanol (10%) as the eluent to obtain compound 22 (ESI-MS (m / z): [M+H] + calc’d.for C 18 H 20 N 3 : 306.1719; found: 306.25). Compound 23 (ESI-MS (m / z): [M+H] + calc’d.for C 24 H 31 N 6 O 2 : 435.2508; found: 435.10). Compound 30 (ESI-MS (m / z): [M+H] + calc’d.for C 23 H 30 N 7 O 2 : 436.2461; found: 436.25). Compound 31 (ESI-MS (m / z): [M+H] + calc’d.for C 17 H 19 N 6 : 307.1617; found: 307.20.

[0074] Synthetic methods of compounds 5, 7, 8, 10, and 12

[0075] Dissolve fluorescein / rhodamine / silicon rhodamine / carbon rhodamine with carboxyl group (1 eq), 6-methyl-3-(4-methylbenzylamino)-1,2,4,5-tetrazine amino (1.2 eq), EDCI (2.5 eq), and HOBT (2.5 eq) in 10 mL of anhydrous dichloromethane solution. Add DIPEA (3.0 eq) under an ice bath, and stir the reaction solution for 12 h under a nitrogen atmosphere. After the reaction is complete, add water, saturated brine, and anhydrous sodium sulfate to the reaction solution in sequence to extract and dry the organic phase. Rotate to dry the solvent, and perform silica gel column chromatography purification using a dichloromethane solution containing methanol (3%) as the eluent to obtain Compound 5 (ESI-MS (m / z): [M+H] + calc’d.for C 31 H 24 F 2 N 5 O 4 : 568.1796; found: 568.25). Compound 7 (ESI-MS (m / z): [M+H] + calc’d.for C 36 H 34 N 7 O 4 + : 628.2667; found: 628.20). Compound 8 (ESI-MS (m / z): [M+H] + calc’d.for C 39 H 42 N 7 O 2 + : 640.3395; found: 640.10). Compound 10 (ESI-MS (m / z): [M+H] + calc’d.for C 40 H 40 N 7 O 3 Si + : 694.2956; found: 694.50). Compound 12 (ESI-MS (m / z): [M+H] + calc’d.for C 38 H 40 N 7 O + : 610.3289; found: 610.50).

[0076] Synthesis methods of Compounds 32 and 33

[0077] Dissolve 4-bromo-substituted naphthalimide derivative (1 eq), 6-methyl-3-benzylamino-1,2,4,5-tetrazine (1.2 eq), Pd(OAc) 2 (0.1 eq), BINAP (0.2 eq) and cesium carbonate (2.5 eq) in 10 mL of anhydrous toluene solution, heat in an 80 °C oil bath, and stir the reaction solution under a nitrogen atmosphere for 12 h. After the reaction is complete, evaporate the solvent, and purify by silica gel column chromatography using a dichloromethane solution containing methanol (1%) as the eluent to obtain compound 32 (ESI-MS (m / z): [M+H] + calc’d.for C 28 H 28 N 7 O 3 : 510.2254; found: 510.25). Compound 33 (ESI-MS (m / z): [M+H] + calc’d.for C 29 H 30 N 7 O 4 : 540.2359; found: 540.50).

[0078] Synthesis method of compound 2

[0079] Dissolve Cy7-Cl (1 eq) and 6-methyl-3-(4-methylbenzylamino)-1,2,4,5-tetrazine (5 eq) in 10 mL of anhydrous dichloromethane, add DIPEA (5 eq), stir at room temperature for 24 h. After the fluorophore is completely consumed, evaporate the solvent, and purify by silica gel column chromatography using a dichloromethane solution containing methanol (10%) as the eluent to obtain compound 2 (ESI-MS (m / z): [M+H] + calc’d.for C 43 H 48 IN 7 : 789.3016; found: 789.10).

[0080] Synthesis method of compound 14

[0081] Dissolve ethyl 9-butyryloxy-6,8,8-trimethyl-2-oxo-6,7,8,9-tetrahydro-2H-pyrano[3,2-g]quinoline-3-carboxylate (1 eq), 6-methyl-3-benzylamino-1,2,4,5-tetrazine (1.2 eq), EDCI (2.5 eq), and HOBT (2.5 eq) in 10 mL of anhydrous dichloromethane solution. Add DIPEA (3.0 eq) under an ice bath, and stir the reaction solution under a nitrogen atmosphere for 12 h. After the reaction is complete, add water, saturated brine, and anhydrous sodium sulfate to the reaction solution in sequence to extract and dry the organic phase. Rotate to dry the solvent, and purify by silica gel column chromatography using a petroleum ether solution containing ethyl acetate (33%) as the eluent to obtain compound 14 (ESI-MS (m / z): [M+H] + calc’d.for C 32 H 37 N 6 O 5 : 585.2825; found: 585.10).

[0082] Synthetic method of compound 18

[0083] Dissolve 8-(3-hydroxyphenyl)bODIPY (1 eq) and 6-methyl-3-chloro-1,2,4,5-tetrazine (5 eq) in 10 mL of anhydrous dichloromethane, add DIPEA (5 eq), and stir at room temperature for 24 h. After the fluorophore is completely consumed, rotate to dry the solvent, and purify by silica gel column chromatography using a dichloromethane solution containing methanol (10%) as the eluent to obtain compound 18 (ESI-MS (m / z): [M+H] + calc’d.for C 22 H 21 BF 2 N 6 O: 434.1838; found: 434.20).

[0084] Responsiveness of compounds 1-33 in Example 2 to superoxide radicals

[0085] Experimental method

[0086] Use compounds 1-33 (1 mg) as probe molecules respectively, and dissolve them in a small amount of dimethyl sulfoxide (DMSO) to make a 5 mM stock solution. Control group: Dilute the probe stock solution with PBS to a final concentration of 5 μM, and record the fluorescence intensity at the maximum emission wavelength. Experimental group: Take another probe stock solution and react it with 20 equivalents of superoxide anion (KO 2Dissolve it in acetonitrile containing 1% 18-crown-6). After reacting for 30 min, dilute it with PBS to a final probe concentration of 5 μM, and record the fluorescence intensity at the maximum emission wavelength. Compare the fluorescence intensities at the maximum wavelength of the control group and the experimental group. Compounds 1-33 showed a significant increase in fluorescence towards superoxide anions. The experimental results are shown in Table 1.

[0087] Table 1

[0088]

[0089]

[0090]

[0091]

[0092]

[0093] Example 3 Study on Reaction Mechanism

[0094] Dissolve Compounds 1-33 separately in DMSO to prepare 10 mM stock solutions. Take a small amount of the stock solution and dilute it to a concentration of 40 μM with chromatographically pure acetonitrile; take solid potassium superoxide and dissolve it in dry chromatographically pure acetonitrile, add a small amount of 18-crown-6 to assist dissolution, prepare a saturated solution, and measure its absorbance at 255 nm using a UV-visible spectrophotometer, and determine its concentration using the molar extinction coefficient (ε = 1460 M -1 cm -1 ). Take 20 equivalents of the superoxide solution and add it to the probe solution, and dilute the final probe concentration to 20 μM by making up the acetonitrile. Use LC-MS to characterize the reaction between superoxide and the probe. LC-MS conditions: Mobile phase A: pure water containing 0.1% formic acid, mobile phase B: methanol. Flow rate: 0.3 mL / min. Mobile phase ratio: 0 - 3 min 20% mobile phase B → 95% mobile phase B; 3 - 8 min 95% mobile phase B → 20% mobile phase B; 8 - 10 min 20% mobile phase B. Detection wavelength: 254 nm. The detection results are shown in Table 2.

[0095] Table 2

[0096]

[0097]

[0098] Example 4 Specific Fluorescent Response of Compounds 4, 21, 22, and 30 to Superoxide Anions

[0099] Compounds 4, 21, 22, and 30 (1 mg) were used as probe molecules respectively, and dissolved in a small amount of dimethyl sulfoxide (DMSO) (0.31, 0.63, 0.65, 0.46 mL) respectively to prepare mother solutions. The probe mother solutions were diluted with PBS to a final concentration of 5 μM, and the fluorescence intensities at their maximum emission wavelengths were recorded. Separately, the probe mother solutions were reacted with 20 equivalents (the equivalents in this paragraph were based on the concentrations of compounds 4, 21, 22, and 30 being 1 equivalent respectively) of superoxide anion (KO 2 dissolved in acetonitrile containing 1% 18-crown-6) for 30 min, and then diluted with PBS to a final probe concentration of 5 μM, and the fluorescence intensities at their maximum emission wavelengths were recorded. Separately, the probe mother solutions were diluted with PBS to a final probe concentration of 5 μM, and then reacted with 20 equivalents of hydrogen peroxide, 20 equivalents of singlet oxygen, 4 equivalents of sodium hypochlorite, 20 equivalents of tert-butyl hydroperoxide, 2 equivalents of peroxynitrite anion, 4 equivalents of nitric oxide, and 20 equivalents of hydroxyl radical for 30 min, and then the fluorescence intensities at their maximum emission wavelengths were recorded. The above fluorescence results were plotted as shown in Figure 1-4 Figure [Figure number not provided in the original, so it remains Figure 1-4 in translation]. Compounds 4, 21, 22, and 30 showed a significant increase in fluorescence with superoxide anion, and other oxidizing species could not cause this change, indicating that such probes have a specific fluorescence response to superoxide anion.

[0100] Example 5 Specific Fluorescence Response of Compounds 22 and 30 to Different Concentrations of Superoxide Anion

[0101] Compounds 22 (0.70 mg) and 30 (1.00 mg) were used as probe molecules respectively, and dissolved in a small amount of dimethyl sulfoxide (DMSO) (0.46 mL) to prepare mother solutions. The probe mother solutions were diluted with PBS to a final concentration of 5 μM, and their fluorescence emission spectra were recorded. Separately, the probe mother solutions were reacted with 0 - 20 equivalents of superoxide anion (KO 2 dissolved in acetonitrile containing 1% 18-crown-6) for 30 min, and then diluted with PBS to a final probe concentration of 5 μM, and their fluorescence intensities were recorded. The above fluorescence results were plotted as shown in Figure 5-6 Figure [Figure number not provided in the original, so it remains Figure 5-6 in translation]. The fluorescence intensity of the probe molecules increased with the increase in the concentration of superoxide anion. Among them, the probe of compound 30 responded to 0 - 0.1 equivalents of superoxide anion, while the probe of compound 22 only responded when the concentration of superoxide anion was greater than 0.1 equivalent, indicating that compound 30 has a more sensitive response to superoxide anion and can detect lower concentrations of superoxide anion.

[0102] Experimental Example 6 Specific Fluorescence Response of Compound 30 to Different Concentrations of Superoxide Anion Generated by Xanthine Oxidase

[0103] Compound 30 was used as a probe molecule and dissolved in a small amount of dimethyl sulfoxide (DMSO) to prepare a stock solution. The probe stock solution was diluted with PBS to a final concentration of 5 μM, and its fluorescence intensity at the maximum emission wavelength was recorded as the control group. In addition, a PBS solution of 5 μM probe was prepared, different concentrations of xanthine oxidase and xanthine were added, and after incubation for 30 min, its fluorescence intensity at the maximum emission wavelength was measured. The above fluorescence results were plotted as shown in Figure 7 As shown, as the concentration of xanthine oxidase / xanthine increased (X×10 mU / X μM) (under physiological conditions, 1 μM xanthine can be converted to generate 1 / 3 μM superoxide anion), the concentration of the generated superoxide anion (0 - 10 μM) increased, and the fluorescence intensity of the probe gradually increased.

[0104] Example 7 Fluorescence changes of superoxide anion generated by different concentrations of modeling agents in HepG2 cells detected by compounds 22 and 30

[0105] Compounds 22 and 30 were used as probe molecules. The probe molecules were dissolved in a small amount of DMSO to prepare a probe solution. The cells were pre-incubated with different concentrations of the modeling agent hydrogen peroxide (0 - 2 mM) for 2 h, then the probe was diluted to 5 μM with the medium, added to HepG2 cells and incubated for 30 min, and then imaged using a fluorescence microscope. The imaging results are shown in Figure 8 A), and as shown in Figure 8 B), compound 30 can detect superoxide anion induced by low-concentration modeling agent in living cells, and compound 22 can only detect superoxide anion induced by high-concentration modeling agent in living cells. It shows that compound 30 is more sensitive to low-concentration superoxide anion than compound 22.

[0106] Example 8 Fluorescence changes of superoxide anion generated by the same concentration of modeling agent in HepG2 cells for different modeling times detected by compounds 22 and 30

[0107] Compounds 22 and 30 were used as probe molecules. The probe molecules were dissolved in a small amount of DMSO to prepare a probe solution. The cells were pre-incubated with 2 mM modeling agent hydrogen peroxide for different times (0 - 4 h), then the probe was diluted to 5 μM with the medium, added to HepG2 cells and incubated for 30 min, and then imaged using a fluorescence microscope. The imaging results are shown in Figure 9 A), and as shown in Figure 9 B), as the modeling time extended, compounds 22 and 30 could detect the concentration change of superoxide anion in living cells.

[0108] Example 9 Fluorescence changes of superoxide anion induced by modeling agent in HepG2 cells detected by compounds 22 and 30 and fluorescence changes of superoxide anion induced by modeling agent after pre-adding superoxide anion scavenger

[0109] Compounds 22 and 30 were used as probe molecules, and the commercially available probes DCFHDA and DHE were used as control molecules. The probe molecules were dissolved in a small amount of DMSO to prepare a probe solution. The cells were divided into 4 groups. The first group (Control) was not pretreated and then the probe was diluted to 5 μM with the culture medium and added to HepG2 cells for incubation for 30 min; the second group (H 2 O 2 ) was incubated with 2 mM hydrogen peroxide as the modeling agent for 2 h and then the probe was diluted to 5 μM with the culture medium and added to HepG2 cells for incubation for 30 min; the third group (Tiron + H 2 O 2 ) was pre-incubated with 100 μM Tiron for 1 h, then incubated with 2 mM hydrogen peroxide as the modeling agent for 2 h, and then the probe was diluted to 5 μM with the culture medium and added to HepG2 cells for incubation for 30 min; the fourth group (TEMPO + H 2 O 2 ) was pre-incubated with 300 μM TEMPO for 1 h, then incubated with 2 mM hydrogen peroxide as the modeling agent for 2 h, the probe was diluted to 5 μM with the culture medium, and added to HepG2 cells for incubation for 30 min. Imaging was performed using a fluorescence microscope, and the imaging results are shown in Figure 10 A), and as shown in Figure 10 B), the fluorescence intensity of the second group of cells, i.e., the modeling group, was stronger than that of the other three groups. However, after pre-incubating with the superoxide anion scavengers Tiron and TEMPO, the fluorescence intensity of Compounds 4 and 5 could be significantly reduced, while the fluorescence of the commercially available probe DHE was not significantly reduced, indicating that Compounds 22 and 30 have higher specificity for superoxide anions. The response multiple of DCFHDA to intracellular superoxide anions is lower, indicating that Compounds 22 and 30 are more sensitive to superoxide anions.

[0110] Example 10 Detection of Superoxide Anions in Myocardial Tissue of Ischemia-Reperfusion Mice by Compound 30

[0111] Compound 30 was used as a probe molecule and dissolved in physiological saline containing a small amount of DMSO to prepare a probe solution. A mouse model of cardiac ischemia-reperfusion (IR) was established with ischemia for 45 min and reperfusion for 15 min. Then, 20 μL of the probe solution was injected into the heart. After the mice were sacrificed, heart tissues were taken for sectioning and then confocal imaging was performed. The results are shown in Figure 11 As shown, the control group was not treated and there was almost no fluorescence in the myocardial tissue, while the fluorescence of the tissue sections in the ischemia-reperfusion modeling group was significantly enhanced.

[0112] Example 11 Detection of Superoxide Anions in Living Cardiac Tissue of Ischemia-Reperfusion Mice by Compound 30

[0113] Compound 30 was used as a probe molecule and dissolved in physiological saline containing a small amount of DMSO to prepare a probe solution. A mouse model of cardiac ischemia-reperfusion (IR) was established, with ischemia for 45 min and reperfusion for 15 min. Then, 20 μL of the probe solution was injected into the heart. After the mice were sacrificed, the heart tissues were imaged using a small animal imager. The results are as Figure 12 shown. In the control group without treatment, there was almost no fluorescence in the heart tissue, while the fluorescence in the heart tissue of the ischemia-reperfusion model group was significantly enhanced.

[0114] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Application of a tetrazine-based fluorescence-enhanced probe in the detection of superoxide anion, characterized in that, the tetrazine-based fluorescence-enhanced probe is selected from one of the following structures:

2. The application according to claim 1, characterized in that, the tetrazine-based fluorescence-enhanced probe is selected from one of the following structures:

3. The application according to any one of claims 1-2, characterized in that, the superoxide anion is the superoxide anion in solution, cells, tissues or living animals.

4. A method for detecting superoxide anion, characterized in that, the tetrazine-based fluorescence-enhanced probe according to any one of claims 1-2 is contacted with superoxide radicals.

5. The detection method according to claim 4, characterized in that, the tetrazine-based fluorescence-enhanced probe exists in the form of a reagent, a probe solution or a kit.

6. A preparation method of a tetrazine-based fluorescence-enhanced probe, characterized in that, it includes the following steps: (1) React R1CN with RCN and hydrazine hydrate under the catalysis of divalent zinc ions to obtain product A; (2) Oxidize product A, then elute and purify to obtain the tetrazine-based fluorescence-enhanced probe; The chemical reaction equation for the preparation of the tetrazine-based fluorescence-enhanced probe is as follows: wherein, R1CN is a cyanide-containing fluorescent compound, and RCN is a cyanide compound containing an R group; the R1 is selected from: naphthylamine, quinoline, xanthene, boron dipyrromethene difluoride, coumarin, sila-xanthene or cyanine dye; the R is selected from one of the following structures:

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