1, 2, 4, 5-tetrazine compound excitation type chemiluminescent probe as well as preparation method and application thereof

Abstract

The invention relates to the field of molecular recognition, biosensing, optical analysis and small-molecule chemiluminescence probes, in particular to a 1, 2, 4, 5-tetrazine compound excitation type chemiluminescence probe as well as a preparation method and application of the 1, 2, 4, 5-tetrazine compound excitation type chemiluminescence probe. Based on the fact that chemiluminescence can be initiated by excitation of a 1, 2, 4, 5-tetrazine compound, target non-zymoprotein is marked by the 1, 2, 4, 5-tetrazine compound, chemiluminescence is activated through biological orthogonal reaction of the 1, 2, 4, 5-tetrazine compound, and detection of non-zymoprotein in living cells and living bodies is achieved. Specifically, the ifenprodil can be specifically combined with an N-methyl-D-aspartic acid receptor NR2B subunit, the ifenprodil modified by the 1, 2, 4, 5-tetrazine compound can specifically target living cells, the N-methyl-D-aspartic acid receptor NR2B subunit and a chemiluminescence probe in a living body can be activated by the 1, 2, 4, 5-tetrazine compound, chemiluminescence imaging is carried out, and the ifenprodil modified by the 1, 2, 4, 5-tetrazine compound can be used for detecting the NR2B subunit of the N-methyl-D-aspartic acid receptor. Therefore, non-zymoprotein in living cells and living bodies can be detected.

Description

Technical Field

[0001] This invention relates to the fields of molecular recognition, biosensing, optical analysis and small molecule chemiluminescent probes, specifically to a 1,2,4,5-tetraazine compound-excited chemiluminescent probe and its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Compared to fluorescence technology, chemiluminescence originates from a chemical reaction, requires no external excitation light source, and possesses the unique advantages of being unaffected by light scattering and autofluorescence. Compared to bioluminescence, chemiluminescence also does not rely on the specific reactions of various insect luciferase-substrate pairs. Compared to luminol, a traditional chemiluminescent substrate, the 1,2-dioxane chemiluminescence system has been widely used in biochemical detection and optical imaging in recent years due to its ease of structural modification and spectral tunability across the visible-near-infrared region. Taking chemiluminescent immunoassay (CLIA) as an example, it is a diagnostic technique that combines chemiluminescence systems with immunological methods. By amplifying and converting immune signals into chemiluminescent signals, the intensity of the light is detected to determine the content of the corresponding substances. Chemiluminescent immunoassay (CLIA) can be used to detect various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs.

[0004] However, current chemiluminescence detection systems are subject to two key limitations: first, the short chemiluminescence half-life (<2 h) of these systems results in a short window for operation and processing by testing personnel; second, the reliance on reactive biomolecules (such as reactive oxygen species or enzymes) to trigger the decomposition of the 1,2-dioxane chemiluminescence system means that current chemiluminescence analysis techniques cannot directly detect non-enzymatic proteins and can only perform indirect detection through enzyme-labeled immunoreactions as described above. Furthermore, the requirement to simultaneously contain enzymes, antigens / antibodies, and chemiluminescent substrates leads to high production, transportation, and storage costs for chemiluminescence-related analytical reagents, limiting their further application in clinical testing, bioimaging, and other fields. Summary of the Invention

[0005] To overcome the above problems, the present invention provides a 1,2,4,5-tetraazine compound-excited chemiluminescent probe, its preparation method and application.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a 1,2,4,5-tetraazine compound-excited chemiluminescent probe, the structural formula of which is shown in formula (I), formula (II) or formula (III).

[0007] Equation (I);

[0008] Formula (II);

[0009] Formula (Ⅲ); The structure of R in equations (I), (II), and (III) is shown below: .

[0010] In a second aspect, the present invention provides an intermediate for a 1,2,4,5-tetraazine compound-excited chemiluminescent probe as described in the first aspect, wherein the intermediate for the 1,2,4,5-tetraazine compound-excited chemiluminescent probe shown in Formula (I) is shown in Formula (IV), the intermediate for the 1,2,4,5-tetraazine compound-excited chemiluminescent probe shown in Formula (II) is shown in Formula (V), and the intermediate for the 1,2,4,5-tetraazine compound-excited chemiluminescent probe shown in Formula (III) is shown in Formula (VI).

[0011] Formula (Ⅳ);

[0012] Formula (V);

[0013] Formula (VI); The structure of R in equations (Ⅳ), (Ⅴ), and (Ⅵ) is shown below: .

[0014] A third aspect of the present invention provides a method for preparing the 1,2,4,5-tetraazine compound-excited chemiluminescent probe described in the first aspect, comprising the following steps: (1) The compound shown in formula (Ⅳ) is added to the first organic solvent as a reaction substrate and the first photosensitizer is added. Photocatalytic oxidation is carried out under light in an atmosphere containing oxygen to obtain the 1,2,4,5-tetraazine compound excited chemiluminescent probe shown in formula (Ⅰ). (2) The compound shown in formula (V) was added to the second organic solvent as a reaction substrate, and a second photosensitizer was added. Photocatalytic oxidation was carried out under light in an atmosphere containing oxygen to obtain the 1,2,4,5-tetraazine compound excited chemiluminescent probe shown in formula (II). (3) The compound shown in formula (VI) is added to the third organic solvent as a reaction substrate, and a third photosensitizer is added. Photocatalytic oxidation is carried out under light in an atmosphere containing oxygen to obtain the 1,2,4,5-tetraazine compound excited chemiluminescent probe shown in formula (III).

[0015] Formula (Ⅳ);

[0016] Formula (V);

[0017] Formula (VI); The structure of R in equations (Ⅳ), (Ⅴ), and (Ⅵ) is shown below: .

[0018] In one or more embodiments, in steps (1), (2) and (3), the first organic solvent, the second organic solvent and the third organic solvent are all dichloromethane.

[0019] In one or more embodiments, in steps (1), (2) and (3), the first photosensitizer, the second photosensitizer and the third photosensitizer are all methylene blue.

[0020] In one or more embodiments, in steps (1), (2) and (3), the light used for illumination is blue light with a wavelength of 380~500 nm.

[0021] In one or more embodiments, in step (1), the concentration of the compound represented by formula (Ⅳ) in the first organic solvent is 4~6 mM.

[0022] In one or more embodiments, in step (2), the concentration of the compound represented by formula (V) in the second organic solvent is 4 to 6 mM.

[0023] In one or more embodiments, in step (3), the concentration of the compound represented by formula (VI) in the third organic solvent is 4 to 6 mM.

[0024] In one or more embodiments, the method for preparing the compound shown in formula (Ⅳ) includes: Compound 3 was dissolved in a fourth organic solvent containing triphosgene and pyridine, and the reaction was stirred. After the reaction was completed, the triphosgene was removed. Then (R,E)-cyclooct-2-en-1-ol and pyridine were added, and the reaction was stirred again to obtain the compound shown in formula (Ⅳ). The structural formula of compound 3 is shown below: .

[0025] Preferably, the fourth organic solvent is dichloromethane.

[0026] Preferably, the molar ratio of compound 3, triphosgene, and (R,E)-cyclooct-2-en-1-ol is 1:(1.8~2.2):(1.8~2.2), more preferably 1:2:2.

[0027] Preferably, the stirring reaction time is 1-4 h; the second stirring reaction time is 3-5 h.

[0028] In one or more embodiments, the method for preparing the compound represented by formula (V) includes: Compound 3 and compound 4 were reacted under alkaline conditions to obtain the compound shown in formula (V); The structural formula of compound 3 is shown below: ; The structural formula of compound 4 is shown below: .

[0029] The molar ratio of compound 3 to compound 4 is 1:(2~2.5), preferably 1:2.35.

[0030] In one or more embodiments, the method for preparing the compound represented by formula (VI) includes: In an oxygen atmosphere, compound 3 reacts with tetravinyltin in the presence of copper acetate to obtain the compound shown in formula (VI); The structural formula of compound 3 is shown below: .

[0031] Preferably, the molar ratio of compound 3 to tetravinyltin is 3:(3.5~5), more preferably 3:4.

[0032] A fourth aspect of the present invention provides the application of the 1,2,4,5-tetraazine compound-excited chemiluminescent probe described in the first aspect in the detection and imaging of non-enzymatic proteins in living cells and organisms.

[0033] A fifth aspect of the present invention provides a method for detecting and imaging non-enzymatic proteins in living cells or living organisms, comprising the following steps: Aifendil modified with a 1,2,4,5-tetraazine compound was incubated with live cells to be tested, followed by the addition of a 1,2,4,5-tetraazine compound-excited chemiluminescent probe, and after incubation again, chemiluminescent imaging was performed. The structure of 1,2,4,5-tetraazine-modified ivendil is shown below: .

[0034] In one or more embodiments, the incubation temperature is 35~40 ℃, preferably 37 ℃; the incubation time is 20~40 min, preferably 30 min.

[0035] In one or more embodiments, the temperature for re-incubation is 35~40 ℃, preferably 37 ℃; the incubation time is 2.5~4 h, preferably 3 h.

[0036] The beneficial effects of this invention are as follows: (1) The 1,2,4,5-tetraazine compound-excited chemiluminescent probe provided by the present invention can be excited by 1,2,4,5-tetraazine compounds to induce chemiluminescence in 1,2-dioxane. Specifically, the 1,2,4,5-tetraazine compound-excited chemiluminescent probe includes two parts: one is a bioorthogonal reaction module, which consists of trans-cyclooctene (TCO), isonitrile (-NC), and vinyl ether (VE); all of which can undergo bioorthogonal reactions with tetraazine; the other is a 1,2-dioxane chemiluminescent module, in which the phenolic oxygen is not chemiluminescent when protected by the bioorthogonal module, but after undergoing a bioorthogonal reaction with tetraazine, the phenolic oxygen is deprotected, inducing chemiluminescence in 1,2-dioxane.

[0037] (2) Based on the ability to be excited by 1,2,4,5-tetraazine compounds and thus trigger chemiluminescence, this invention uses 1,2,4,5-tetraazine compounds to label target non-enzymatic proteins, and activates chemiluminescence through the bioorthogonal reaction of 1,2,4,5-tetraazine compounds to realize the detection of non-enzymatic proteins in living cells and living organisms; specifically, fenfendil can specifically bind to the N-methyl-D-aspartate receptor NR2B subunit. Using 1,2,4,5-tetraazine compounds to modify fenfendil can specifically target the N-methyl-D-aspartate receptor NR2B subunit in living cells and living organisms. The chemiluminescent probe can be activated by 1,2,4,5-tetraazine compounds to perform chemiluminescent imaging and realize the detection of non-enzymatic proteins in living cells and living organisms.

[0038] (3) The 1,2,4,5-tetraazine compound-excited chemiluminescent probe provided by the present invention has a long luminescence half-life (5~18h), which extends the time window available for detection personnel to operate and process.

[0039] (4) The 1,2,4,5-tetraazine compound-excited chemiluminescent probe provided by this invention does not require active small molecules or enzymes to trigger chemiluminescence. Utilizing the high specificity and high biocompatibility of bioorthogonal chemistry, it can detect and image non-enzymatic proteins without an enzyme immunoassay process. Compared with traditional chemiluminescent immunoassay techniques, small molecule labeling greatly improves the labeling efficiency of single target proteins compared with large molecule enzyme labeling, and also greatly simplifies the labeling process, reduces the operational difficulty, and can be used for the detection and imaging of target proteins in living cells. Attached Figure Description

[0040] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0041] Figure 1 This is the preparation route for the 1,2,4,5-tetraazine compound-excited chemiluminescent probe in this invention; Figure 2 The chemiluminescence spectra of CL-TCO, CL-NC, and CL-VE after reaction with 1,2,4,5-tetraazine compounds are shown. Figure 3 The graph shows the change in chemiluminescence intensity over time after CL-TCO (A), CL-NC (B), and CL-VE (C) react with 1,2,4,5-tetraazine compounds. Figure 4 The effects of different intracellular bioactive substances on the chemiluminescence of CL-TCO, CL-NC, and CL-VE; Figure 5 For CL-NC bioorthogonal-chemiluminescence imaging of N-methyl-D-aspartate receptors in different living cells; Figure 6 To study the relationship between the bioorthogonal-chemiluminescence intensity of CL-NC and the concentration of 1,2,4,5-tetraazine compounds, where A represents the chemiluminescence imaging of mixed solutions of Tz-IFDL and CL-NC at different concentrations, and B represents the quantification of the chemiluminescence intensity in A. Figure 7 The tissue penetration depth of bioorthogonal-chemiluminescence. Detailed Implementation

[0042] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0043] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0044] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0045] Example 1 Figure 1 The preparation route of the 1,2,4,5-tetraazine compound-excited chemiluminescent probe in this invention is described in reference [reference]. Figure 1 Synthesize chemiluminescent probes excited by 1,2,4,5-tetraazine compounds as shown in formula (I), (II), or (III).

[0046] (1) Synthesis of compound 3: Compound 1 (50 mg, 0.17 mmol), piperidine (34 µL, 0.34 mmol), and compound 2 (35 mg, 0.17 mmol) were dissolved in 3 mL of acetonitrile; the mixture was stirred at 90 °C for 18 h; after the reaction was complete, the reaction mixture was diluted with ethyl acetate (100 mL) and washed with 0.5 M HCl (100 mL); the mixture was allowed to stand, the organic layer was separated, washed with saturated brine, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The product was purified by rapid column chromatography (DCM:MeOH = 50:1, v / v, Rf = 0.5); an orange solid, compound 3 (43 mg, 52% yield), was obtained.

[0047] Nuclear magnetic resonance and mass spectrometry characterization: 1H NMR (400 MHz, CDCl3): δ 8.91 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 16.0Hz, 1H), 7.75-7.68 (m, 2H), 7.45 (d, J = 8.0 Hz, 2H), 6.93 (dd, J1 = 8.8 Hz,J2 = 2.8 Hz, 1H), 6.83 (d, J = 2.8 Hz, 1H), 6.82 (s, 2H), 6.69 (d, J = 16.0Hz, 1H), 6.09 (s, 1H), 3.38 (s, 1H), 3.31 (s, 1H), 2.14 (t, J = 3.2 Hz, 1H), 2.02-1.72 (m, 13H). 13 C NMR (101 MHz, CDCl3) δ 158.3, 157.8, 153.1, 152.4, 139.5, 138.6,137.3, 134.6, 132.7, 127.6, 126.9, 125.9, 125.8, 118.4, 117.9, 117.7, 117.0,116.5, 116.2, 115.9, 106.4, 61.6, 57.0, 39.0, 36.9, 32.6, 29.9, 28.2. HRMS (ESI) m / z: calcd for C 32 H 28 N2O3[MH ]- 487.2027, found 487.1951. (2) Synthesize the compound shown in formula (Ⅳ): At 0 °C, pyridine (20 µL, 0.24 mmol) was added dropwise to a DCM (2 mL) solution containing triphosgene (120 mg, 0.4 mmol); then, compound 3 (100 mg, 0.2 mmol) was added dropwise at 0 °C; the reaction was stirred at room temperature for 3 h; nitrogen was bubbled through the solution to remove as much excess triphosgene as possible; (R,E)-cyclooct-2-en-1-ol (50 mg, 0.4 mmol) and pyridine (42 µL, 0.5 mmol) were added to the reaction mixture; after stirring for 4 h, the reaction mixture was washed with DCM and brine, dried over Na2SO4, and evaporated under reduced pressure; the product was purified by silica gel chromatography (DCM, Rf=0.5); an orange solid, namely the compound of formula (Ⅳ) (35 mg, 28% yield), was obtained.

[0048] Nuclear magnetic resonance and mass spectrometry characterization: 1 H NMR (400 MHz, CDCl3): δ 8.92 (d, J = 9.6 Hz, 1H), 7.93 (d, J = 16.4Hz, 1H), 7.84-7.71 (m, 2H), 7.47 (d, J = 9.6 Hz, 3H), 7.31-7.26 (m, 2H), 7.18(s, 1H), 6.87 (s, 1H), 6.80 (d, J = 16.4 Hz, 1H), 5.60-5.44 (m, 1H), 3.39 (s,1H), 3.29 (s, 4H), 2.24 (d, J = 16.0 Hz, 2H), 2.15 (s, 2H), 2.04-1.98 (m,1H), 1.84 (s, 1H), 1.80 (s, 1H), 1.72 (s, 10H). HRMS (ESI) m / z: calcd. for C 41 H 40 N₂O₅[M+Na] + 663.2829, found 663.2807. (3) Preparation of the chemiluminescent probe excited by the 1,2,4,5-tetraazine compound shown in formula (Ⅰ): The compound shown in formula (Ⅳ) (30 mg, 0.046 mmol) and a catalytic amount of methylene blue were dissolved in 10 mL of DCM; oxygen was bubbled through the solution while irradiating it with blue light (wavelength 380~500 nm); after the reaction was completed, the solvent was concentrated under reduced pressure and the product was purified by column chromatography; an orange solid product (6 mg, 20% yield) was obtained, which is the 1,2,4,5-tetraazine compound excited chemiluminescent probe shown in formula (Ⅰ), abbreviated as CL-TCO.

[0049] Mass spectrometry characterization: HRMS (ESI) m / z: calcd for C 41 H 40 N₂O₇[M+Na] + 695.2728; found 695.2631. (4) Synthesize the compound shown in formula (Ⅳ): Anhydrous Cs₂CO₃ (236 mg, 0.72 mmol) was added to an anhydrous DMF (1.6 mL) solution containing compound 3 (250 mg, 0.48 mmol), and the mixture was stirred for 15 min. Anhydrous DMF (0.8 mL) solution containing compound 4 (344 mg, 1.13 mmol) was added dropwise. The mixture was stirred at 60 °C for 4 h. The solution was cooled to room temperature, and the solid residue was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was dissolved in DCM (3 × 50 mL), and the organic layer was washed with water (2 × 50 mL) and brine (1 × 100 mL). The residue was dried with Na₂SO₄, filtered, and evaporated. The residue was purified by silica gel chromatography (DCM:PE = 1:1, v / v, Rf = 0.4) to obtain the desired orange solid compound, which is the compound shown in formula (Ⅳ), with a yield of 85 mg (32%).

[0050] Nuclear magnetic resonance and mass spectrometry characterization: 1 H NMR (400 MHz, CDCl3): δ 8.12 (d, J = 15.6 Hz, 1H), 7.94 (d, J =8.0 Hz, 1H), 7.78 (d, J = 8.8 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.29 (q, J =4.0 Hz, 2H), 7.20 (s, 1H), 7.04 (d, J = 15.6 Hz, 1H), 6.93 (d, J1 = 8.8 Hz,J2 = 2.8 Hz, 1H), 6.82 (d, J = 2.8 Hz, 1H), 4.15 (t, J = 5.6 Hz, 2H), 3.67(t, J = 6.4 Hz, 2H), 3.59 (d, J = 6.4 Hz, 3), 3.37 (s, 1H), 3.31 (s, 3H), 2.21-2.15 (m, 2H), 1.96-1.68 (m, 13H). 13C NMR (101 MHz, CDCl3) δ 161.1, 158.5, 158.3, 157.6, 152.4, 146.0,140.3, 137.5, 133.6, 131.4, 131.3, 129.1, 126.7, 125.8, 123.9, 123.5, 117.2,117.1, 116.0, 114.7, 104.9, 99.2, 63.4, 56.8, 50.7, 39.0, 38.8, 38.4, 38.4,38.3, 37.0, 32.5, 29.6, 28.9, 28.2, 25.8, 24.5. HRMS (ESI) m / z: calcd for C 36 H 33 N3O3[M+Na] + 578.2414, found 578.2420. (5) Synthetic chemiluminescent probe excited by the 1,2,4,5-tetraazine compound shown in formula (II): The compound shown in formula (Ⅳ) (30 mg, 0.054 mmol) and a catalytic amount of methylene blue were dissolved in 10 mL of DCM. While irradiating the solution with blue light (wavelength 380–500 nm), oxygen was bubbled through the solution. After completion, the solvent was concentrated under reduced pressure, and the product was purified by column chromatography. An orange solid product (16 mg, 52% yield) was obtained, which is the 1,2,4,5-tetraazine compound excited-type chemiluminescent probe shown in formula (Ⅱ), abbreviated as CL-NC.

[0051] Nuclear magnetic resonance and mass spectrometry characterization: 1H NMR (400 MHz, CDCl3): δ 8.91 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 8.51 (d, J = 16.0 Hz, 1H), 7.74 (td, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 7.69 (d,J = 8.8 Hz, 1H), 7.59 (dd, J1 = 8.8 Hz, J2 = 1.6 Hz, 1H), 7.53 (d, J = 2.8Hz, 1H), 7.46 (td, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 7.13 (dd, J1 = 8.8 Hz, J2 =2.8 Hz, 1H), 6.87 (s, 1H), 6.66 (d, J = 16.0 Hz, 1H), 4.22 (t, J = 6.0 Hz,2H), 3.98 (s, 3H), 3.68 (t, J = 6.4 Hz, 2H), 3.40-3.18 (s, 2H), 2.22-1.26 (s,14H). 13 C NMR (101 MHz, CDCl3) δ 166.8, 159.4, 157.6, 152.9, 152.4, 137.2,134.6, 134.6, 131.0, 129.4, 129.0, 126.0, 125.8, 125.7, 119.8, 119.1, 118.8,117.8, 116.8, 116.3, 115.7, 106.9, 106.5, 64.0, 62.7, 57.0, 52.6, 40.1, 37.0,32.7, 29.8, 28.9, 28.8, 28.3, 28.2. HRMS (ESI) m / z: calcd for C 36 H 33 N3O5[M+Na] + 610.2312; found 610.2245. (6) Synthesize the compound shown in formula (VI): Anhydrous Cu(OAc)₂ (73 mg, 0.4 mmol) was added to an acetonitrile (3 mL) solution containing compound 3 (150 mg, 0.3 mmol); the mixture was purged under vacuum, O₂ was introduced, tetravinyltin (73 µL, 0.4 mmol) was added, and the mixture was stirred overnight; after the reaction was complete, the mixture was poured into a 25% NH₄OAc aqueous solution (10 mL) and stirred for 10 min; then, the aqueous phase was extracted three times with ethyl acetate, the combined organic layers were washed with brine, dried over anhydrous Na₂SO₄ and concentrated; finally, the residue was purified by silica gel chromatography (DCM:PE = 1:2, v / v, Rf = 0.2) to give the desired orange solid compound (92 mg, 60% yield), which is the compound with yield shown in formula (VI).

[0052] Nuclear magnetic resonance and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO-d6): δ 7.74 (dd, J1 = 8.4 Hz, J2 = 1.2 Hz, 1H), 8.05 (d, J = 8.8 Hz, 1H), 7.94 (td, J1 = 8.4 Hz, J2 = 1.2 Hz, 1H), 7.84 (d, J= 8.0 Hz ,1H), 7.63 (td, J1 = 8.4 Hz, J2 = 1.2 Hz, 1H), 7.54-7.46 (m, 2H),7.22 (dd, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 7.05-7.00 (m, 2H), 6.96 (d, J = 2.8Hz,1H), 4.88 (dd, J1 = 13.6 Hz, J2 = 1.6 Hz, 1H), 4.62 (dd, J1 = 6.0 Hz, J2= 1.6 Hz, 1H), 3.24 (s, 3H), 2.00-1.67 (m, 13H). 13 C NMR (101 MHz, DMSO) δ 159.4, 157.8, 153.3, 152.4, 148.1, 139.9, 139.0, 137.0, 135.9, 132.5, 129.6, 128.5, 126.7, 125.3, 119.2, 118.8, 118.3,117.6, 117.2, 115.9, 107.8, 98.9, 60.9, 57.0, 55.4, 36.9, 33.2, 29.7, 28.1. HRMS (ESI) m / z: calcd for C 34 H 30 N₂O₃[M+Na] + 537.2149, found 537.2085. (7) Synthetic chemiluminescent probe of the 1,2,4,5-tetraazine compound shown in formula (III): The compound shown in formula (VI) (28 mg, 0.054 mmol) and a catalytic amount of methylene blue were dissolved in 10 mL of DCM; oxygen was bubbled through the solution while irradiating it with blue light (wavelength 380~500 nm); after the reaction was completed, the solvent was concentrated under reduced pressure and the product was purified by column chromatography; an orange solid product (18 mg, 62% yield) was obtained; this is the 1,2,4,5-tetraazine compound excited chemiluminescent probe shown in formula (III), abbreviated as CL-VE.

[0053] Nuclear magnetic resonance and mass spectrometry characterization: 1 H NMR (400 MHz, DMSO-d6): δ 8.73 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 8.03 (dd, J1 = 8.8 Hz, J2 = 3.2 Hz, 1H), 7.93 (td, J1 = 8.8 Hz, J2 = 1.6 Hz, 1H), 7.83 (d, J = 16.0 Hz, 1H), 7.62 (td, J1 = 8.4 Hz, J2 = 1.2 Hz, 1H), 7.52 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 7.46 (d, J = 16.0 Hz, 1H), 7.20 (dd, J1 =8.8 Hz, J2 = 2.8 Hz, 1H), 7.04-6.99 (m, 2H), 6.95 (d, J = 2.8 Hz, 1H), 4.87 (dd, J1 = 13.2 Hz, J2 = 1.6 Hz, 1H), 4.61 (dd, J1 = 6.0 Hz, J2 = 1.6 Hz, 1H),3.23 (s, 3H), 2.02-1.66 (m, 13H). 13C NMR (100 MHz, CDCl3): δ 166.1, 158.1, 157.3, 152.8, 151.8, 147.2,139.4, 137.9, 135.6, 135.4, 131.5, 131.1, 128.8, 128.0, 126.2, 124.8, 118.7,118.3, 117.8, 117.1, 117.1, 116.6, 115.7, 106.7, 96.9, 60.4, 56.5, 54.8,52.7, 36.4, 32.2, 29.2, 27.6. HRMS (ESI) m / z: calcd for C 34 H 30 N₂O₅[M+H] + 547.2228; found 547.2190. Example 2 CL-TCO, CL-NC, CL-VE, and the 1,2,4,5-tetraazine compound were all diluted with DMSO to prepare 10 mM stock solutions. 100 μL of each stock solution was diluted with DMSO to 1 mL to obtain 1 mM working solutions. 250 μL of each of the CL-TCO, CL-NC, and CL-VE working solutions were added to 500 μL of PBS solution, followed by 250 μL of the 1,2,4,5-tetraazine compound working solution, to obtain 1 mL of each of the CL-TCO, CL-NC, and CL-VE bioorthogonal-chemiluminescence assay solutions (the final concentrations of the three chemiluminescent probes and the 1,2,4,5-tetraazine compound were all 0.25 mM, and the volume ratio of PBS to DMSO was 1:1). The three mixed solutions were incubated at 37 °C, and the chemiluminescence spectra were measured at 1.5 h, 3 h, and 5 h of incubation.

[0054] The results are as follows Figure 2 As shown, from Figure 2 As can be seen, the maximum emission peak of chemiluminescence produced by CL-TCO, CL-NC and CL-VE after reacting with 1,2,4,5-tetraazine compounds is around 675 nm.

[0055] Example 3 Chemiluminescence kinetics of CL-TCO, CL-NC and CL-VE: CL-TCO, CL-NC, CL-VE, and the 1,2,4,5-tetraazine compound were all diluted with DMSO to prepare 10 mM stock solutions. 100 μL of each of the CL-TCO, CL-NC, and CL-VE stock solutions were added, followed by 300 μL of DMSO and 500 μL of PBS solution, and then 100 μL of the 1,2,4,5-tetraazine compound stock solution was added to each, yielding 1 mL of each of the bioorthogonal-chemiluminescence assay solutions for CL-TCO, CL-NC, and CL-VE (the final concentrations of the three chemiluminescent probes and the 1,2,4,5-tetraazine compound were all 1 mM, and the volume ratio of PBS to DMSO was 1:1). Immediately after adding the 1,2,4,5-tetraazine compound, the change in chemiluminescence intensity of the three mixed solutions over time was measured. The experimental results are as follows: Figure 3 As shown, the chemiluminescent half-lives of CL-TCO, CL-NC, and CL-VE are 5.2 h, 13.4 h, and 18 h, respectively.

[0056] Example 4 Investigating the effects of different intracellular bioactive substances on the chemiluminescence of CL-TCO, CL-NC, and CL-VE: CL-TCO, CL-NC, CL-VE, and 1,2,4,5-tetraazine compounds were diluted with DMSO to prepare 10 mM stock solutions. 100 μL of each stock solution was diluted with DMSO to 1 mL to obtain 1 mM working solutions. 100 μL of each of the CL-TCO, CL-NC, and CL-VE working solutions were added to 400 μL of DMSO and 400 μL of PBS solution, respectively. Then, 100 μL of 1 mM aqueous solutions of different reactive oxygen, nitrogen, and sulfur species and metal ions were added to each solution to obtain mixed solutions of three chemiluminescent probes and different intracellular active substances (final concentration 0.1 mM, aqueous phase to DMSO volume ratio 1:1). Experimental results are as follows: Figure 4 As shown in the figure, the three probes exhibit good bioorthogonal selectivity; none of them are triggered to emit chemiluminescence by common intracellular bioactive substances (reactive oxygen, nitrogen, and sulfur species and metal ions). Only the 1,2,4,5-tetraazine compound can trigger the chemiluminescence of all three probes. These experimental results demonstrate the feasibility of probes in cellular applications.

[0057] Example 5 N-methyl-D-aspartate receptor in live cells using CL-NC bioorthogonal-chemiluminescence imaging: Further investigation was conducted using CL-NC as an example to study its ability to image non-enzymatic proteins in live cells. Using the N-methyl-D-aspartate receptor (NMDA receptor) as the target protein and fenfenadil, a specific, non-competitive ligand for the NMDA receptor NR2B subunit, as the targeting group, different live cell lines (HT22 mouse hippocampal neurons, Bend.3 mouse brain microvascular endothelial cells, HEK293 human embryonic kidney cells, AML-12 mouse normal hepatocytes, H9C2 rat cardiomyocytes, and HeLa cells) were incubated with 1,2,4,5-tetraazine-modified fenfenadil (Tz-IFDL) (50 μM) at 37 °C for 30 min to target the NMDA receptor. After washing with PBS buffer, these cells were incubated with CL-NC (50 µM) at 37 °C for 3 h, followed by chemiluminescence imaging. The results are as follows: Figure 5 As shown, cells that hardly express NR2B (HEK293, AML-12, H9C2, and HeLa) show almost no chemiluminescence, while cell lines that highly express NR2B (HT22 and Bend.3) exhibit significant chemiluminescence. Furthermore, NR2B expression was knocked down by siRNA in HT22 and Bend.3 cells (siRNA sequence: sense strand: 5'-AGCUCGUUCCCAAAAGAGCUU-3', antisense strand: 3'-UUUCGAGCAAGGGUUUUCUCG-5'). Figure 5 The chemiluminescent intensity of “NR2B KD” (NR2B knock down) in these cells was lower than that in normal HT22 and Bend.3 cells. Therefore, by modifying the target protein with 1,2,4,5-tetraazine compounds, chemiluminescent probes can be used to detect and image the expression of the target protein.

[0058] Example 6 The relationship between chemiluminescence intensity and the concentration of 1,2,4,5-tetraazine compounds was investigated using CL-NC: Both CL-NC and 1,2,4,5-tetraazine-modified efenidil were diluted with DMSO to prepare 10 mM stock solutions. 100 μL of each stock solution was diluted with DMSO to 1 mL to obtain 1 mM working solutions. 50 μL of the CL-NC working solution was added to six wells of a 96-well plate, followed by 100 μL of PBS solution, and then 50 μL, 40 μL, 30 μL, 20 μL, 10 μL, and 0 μL of DMSO, respectively (the final concentration of CL-NC was 0.25 mM, the final concentration of the 1,2,4,5-tetraazine compound was 0–0.25 mM, and the PBS to DMSO volume ratio was 1:1). These mixed solutions were incubated at 37 ℃ for 3 h, and then chemiluminescence imaging was performed to quantify the chemiluminescence intensity. The results are as follows: Figure 6 As shown, the chemiluminescence intensity is positively correlated with the concentration of 1,2,4,5-tetraazine-modified iverm (Rf). 2 =0.9922).

[0059] Example 7 Tissue penetration depth of bioorthogonal-chemiluminescence: like Figure 7 As shown, the tissue penetration depth of bioorthogonal-chemiluminescence was further investigated. CL-NC and the 1,2,4,5-tetraazine compound were diluted with DMSO to prepare 10 mM stock solutions. 100 μL of each stock solution was diluted with DMSO to 1 mL to obtain a 1 mM working solution. 250 μL of the CL-NC working solution was added to 500 μL of PBS solution, followed by 250 μL of the 1,2,4,5-tetraazine compound working solution, to obtain 1 mL of the CL-NC bioorthogonal-chemiluminescence test solution (the final concentrations of both CL-NC and the 1,2,4,5-tetraazine compound were 0.25 mM, and the PBS to DMSO volume ratio was 1:1). Chicken breast tissue of different thicknesses was then placed on top of this mixed solution, and imaging was performed using both chemiluminescence and fluorescence modes to study the tissue penetration depth of chemiluminescence generated by bioorthogonalization. At all tissue depths (0–6 cm), the signal-to-background ratio of chemiluminescence was higher than that of fluorescence. Chemiluminescence penetrated to a tissue depth of up to 6 cm, while solution fluorescence was barely detectable in tissues 2 cm or thicker. The SBR of chemiluminescence was 209 times that of fluorescence without tissue coverage, and 2.74 times that of fluorescence at a tissue thickness of 6 cm. These results demonstrate the potential of bioorthogonal-chemiluminescence for deep tissue imaging.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A chemiluminescent probe excited by a 1,2,4,5-tetraazine compound, characterized in that, Its structural formula is shown in formula (I), formula (II) or formula (III). Equation (I); Formula (II); Formula (Ⅲ); The structure of R in equations (I), (II), and (III) is shown below: 。 2. The intermediate of the 1,2,4,5-tetraazine compound-excited chemiluminescent probe according to claim 1, characterized in that, The intermediate of the 1,2,4,5-tetraazine compound-excited chemiluminescent probe shown in Formula (I) is shown in Formula (IV), the intermediate of the 1,2,4,5-tetraazine compound-excited chemiluminescent probe shown in Formula (II) is shown in Formula (V), and the intermediate of the 1,2,4,5-tetraazine compound-excited chemiluminescent probe shown in Formula (III) is shown in Formula (VI). Formula (Ⅳ); Formula (V); Formula (VI); The structure of R in equations (Ⅳ), (Ⅴ), and (Ⅵ) is shown below: 。 3. The method for preparing the 1,2,4,5-tetraazine compound-excited chemiluminescent probe according to claim 1, characterized in that, Includes the following steps: (1) The compound shown in formula (Ⅳ) is added to the first organic solvent as a reaction substrate and the first photosensitizer is added. Photocatalytic oxidation is carried out under light in an atmosphere containing oxygen to obtain the 1,2,4,5-tetraazine compound excited chemiluminescent probe shown in formula (Ⅰ). (2) The compound shown in formula (V) was added to the second organic solvent as a reaction substrate, and a second photosensitizer was added. Photocatalytic oxidation was carried out under light in an atmosphere containing oxygen to obtain the 1,2,4,5-tetraazine compound excited chemiluminescent probe shown in formula (II). (3) The compound shown in formula (VI) is added to the third organic solvent as a reaction substrate, and a third photosensitizer is added. Photocatalytic oxidation is carried out under light in an atmosphere containing oxygen to obtain the 1,2,4,5-tetraazine compound excited chemiluminescent probe shown in formula (III). Formula (Ⅳ); Formula (V); Formula (VI); The structure of R in equations (Ⅳ), (Ⅴ), and (Ⅵ) is shown below: 。 4. The preparation method according to claim 3, characterized in that, In steps (1), (2) and (3), the first organic solvent, the second organic solvent and the third organic solvent are all dichloromethane; Alternatively, in steps (1), (2), and (3), the first photosensitizer, the second photosensitizer, and the third photosensitizer are all methylene blue; Alternatively, in steps (1), (2) and (3), the light used for illumination is blue light (wavelength 380~500 nm).

5. The preparation method according to claim 3, characterized in that, The preparation method of the compound shown in formula (Ⅳ) includes: Compound 3 was dissolved in a fourth organic solvent containing triphosgene and pyridine, and the reaction was stirred. After the reaction was completed, the triphosgene was removed. Then (R,E)-cyclooct-2-en-1-ol and pyridine were added, and the reaction was stirred again to obtain the compound shown in formula (Ⅳ). The structural formula of compound 3 is shown below: 。 6. The preparation method according to claim 3, characterized in that, The preparation method of the compound shown in formula (V) includes: Compound 3 and compound 4 were reacted under alkaline conditions to obtain the compound shown in formula (V); The structural formula of compound 3 is shown below: ； The structural formula of compound 4 is shown below: 。 7. The preparation method according to claim 3, characterized in that, The preparation method of the compound shown in formula (VI) includes: In an oxygen atmosphere, compound 3 reacts with tetravinyltin in the presence of copper acetate to obtain the compound shown in formula (VI); The structural formula of compound 3 is shown below: 。 8. The application of the 1,2,4,5-tetraazine compound-excited chemiluminescent probe of claim 1 in the detection and imaging of non-enzymatic proteins in living cells and organisms.

9. A method for detecting and imaging non-enzymatic proteins in living cells or living organisms, characterized in that, Includes the following steps: Aifendil modified with a 1,2,4,5-tetraazine compound was incubated with live cells to be tested, and then the 1,2,4,5-tetraazine compound-excited chemiluminescent probe of claim 1 was added. After incubation again, chemiluminescent imaging was performed. The structure of tetrazine-modified ivendil is shown below: 。 10. The method as described in claim 9, characterized in that, The incubation temperature is 35~40 ℃, preferably 37 ℃; the incubation time is 20~40 min, preferably 30 min; Alternatively, the temperature for the second incubation is 35~40 ℃, preferably 37 ℃; the incubation time is 2.5~4 h, preferably 3 h.

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