Chemiluminescent probe, preparation method thereof and application thereof in biological thiol detection

Abstract

The application discloses a chemiluminescent probe, a preparation method thereof and application thereof in biological thiol detection, and belongs to the technical field of biosensing. The structure of the chemiluminescent probe is as follows: wherein R' is a nitrogen heterocyclic group; R is a conventional polar modification group; the probe shows obvious selectivity to biological thiol, can be widely applied to biological thiol monitoring in vivo and high-sensitivity and high-selectivity imaging, and provides a new chemiluminescent molecular probe for early detection and accurate diagnosis of biological thiol related diseases.

Description

Technical Field

[0001] This invention relates to a biological probe, specifically to a type of peroxycyclobutane chemiluminescent probe with allyl acetyl as the recognition group, and also to its preparation method, as well as the application of the chemiluminescent probe in the detection of biothiols, belonging to the field of biosensing technology. Background Technology

[0002] Biothiols, such as cysteine ​​(Cys), glutathione (GSH), and homocysteine ​​(Hcy), are important small amino acids containing sulfhydryl groups. They play crucial roles in many physiological and pathological processes, such as reversible redox homeostasis, tissue growth, and human metabolism. The quantity of biothiols is associated with many diseases. Therefore, non-invasive and highly sensitive detection methods for biothiols are essential for elucidating their various biological functions.

[0003] To achieve real-time monitoring of the biological environment, optical imaging methods such as fluorescence and bioluminescence have attracted considerable attention as non-invasive monitoring tools. Numerous fluorescent biothiol probes have been reported and successfully applied to monitor biothiol levels in living cells and within the body. However, fluorescence imaging requires an excitation light source, and the potential for fluorescent probes is limited by potential phototoxicity and reduced detection sensitivity due to autofluorescence and light scattering. In the last decade, chemiluminescence systems have increasingly been considered as an alternative to fluorescence systems.

[0004] Chemiluminescent probes have been widely used for imaging detection of target molecules in living biological tissues. The mechanism is that after the probe is activated, the high-energy chemical bonds within the molecule break and release energy, which further stimulates the dye molecules to emit light. The biggest advantage of chemiluminescent probes is that they do not require an additional excitation light source, effectively avoiding problems such as light absorption, autofluorescence, and light scattering in biological tissues. Existing chemiluminescent probes for the detection of small molecule biological thiols have greatly improved the detection sensitivity and signal-to-noise ratio, but the wavelengths are all limited to the visible light region of about 550 nm (e.g., ACS Sensors 2019, 4(1), 87-92; Anal. Chem. 2023, 95(35), 13191-13200; ACS Appl. Mater. Interfaces 2023, 15(29), 34505-34512). Therefore, in order to increase the length of the emission wavelength, there is a great need for a chemiluminescent probe for detecting biothiols with near-infrared (NIR; 650-900nm) emission, which has the advantages of deep tissue penetration, low tissue light damage and small autofluorescence interference. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the first objective of this invention is to provide a chemiluminescent probe that uses an acetyl nitrogen heterocycle as an electron-withdrawing group, peroxybutane as a luminescent group, and allyl acetyl as a biothiol recognition group. Furthermore, by introducing different types of electron-withdrawing groups at the olefin terminus of the allyl acetyl group, the probe's reactivity with biothiols is altered. This probe exhibits characteristics such as low light absorption or intrinsic light intensity in biological tissues, strong penetration into biological tissues, high imaging signal-to-noise ratio, high resolution, and high selective recognition of biothiols. It can be used for highly selective and sensitive imaging of biothiols in vivo.

[0006] The second objective of this invention is to provide a method for preparing a chemiluminescent probe, which is simple in steps, mild in conditions, high in yield, uses readily available raw materials, is low in cost, and is conducive to large-scale production.

[0007] The third objective of this invention is to provide an application of a chemiluminescent probe as a molecular probe in the detection of biothiols. Based on the characteristics of chemiluminescent probes, such as low light absorption or light intensity in biological tissues, strong penetration ability in biological tissues, high imaging signal-to-noise ratio, high resolution, and strong selectivity, its application in the detection of biothiols in vivo has the advantages of high selectivity, high sensitivity, and low biotoxicity.

[0008] To achieve the above technical objectives, the present invention provides a chemiluminescent probe having the structure of Formula 1:

[0009]

[0010] in,

[0011] R' is a nitrogen heterocyclic group;

[0012] R is hydrogen, C1-C5 alkyl, C1-C5 alkoxy, C2-C6 ester, C1-C6 aliphatic amine, C1-C5 aliphatic alcohol, halogen substituent, C1-C5 haloalkyl, cyano, sulfonic acid, aromatic heterocyclic, phenyl or substituted phenyl;

[0013] The substituted phenyl group has at least one of the following substituents on its benzene ring: C1-C5 alkyl, C1-C5 alkoxy, C2-C6 fatty acyl, C1-C6 fatty amine, amino, halogen substituent, C1-C5 haloalkyl, cyano, and sulfonic acid group.

[0014] The chemiluminescent probe of this invention has a unique molecular structure, comprising a biothiol recognition group, a peroxybutyl group, and a polar conjugated group. The acryloyl derivative, with its olefin terminus replaced by various electron-withdrawing groups, serves as the biothiol recognition group, endowing it with high selectivity for biothiols. The peroxybutyl group acts as the core framework for chemiluminescence, and the introduction of groups with larger conjugation coefficients extends its emission wavelength, resulting in low light absorption or intensity in biological tissues. The polar nitrogen heterocyclic group enhances its penetration into biological tissues. In summary, the entire chemiluminescent probe exhibits characteristics such as low light absorption or intensity in biological tissues, strong penetration into biological tissues, high imaging signal-to-noise ratio and resolution, and high selectivity for biothiols. It can be used for highly selective and sensitive imaging of biothiols in cells or other living organisms.

[0015] As a preferred embodiment, in Formula 1, R' is a nitrogen heterocyclic group, which can be a five-membered or six-membered nitrogen heterocyclic group, and more preferably a pyridine group, quinoline group, phenanthridine group or o-phenanthroline group containing a pyridine ring with strong polarity.

[0016] As a preferred embodiment, R represents hydrogen, C1-C5 alkyl (alkyl groups with 3 or more carbon atoms can be branched or straight-chain alkyl groups), C1-C5 alkoxy (alkoxy groups with 3 or more carbon atoms can be branched or straight-chain alkyl groups), C2-C6 ester group (specifically, methyl formate, methyl acetate, etc.), C1-C6 aliphatic amine group (amine groups can be primary or secondary amine groups, and the aliphatic hydrocarbon substituent on the amine group is an alkyl substituent, specifically, methyl, ethyl, etc.), C1-C5 aliphatic alcohol group (aliphatic alcohol group is an alkyl group containing a hydroxyl substituent, specifically, a methanol group), and halogen substituent (specifically, a fluorinated substituent). Substituents include: alkyl groups, chlorinated substituents, etc.), C1-C5 haloalkyl groups (haloalkyl groups are alkyl groups containing halogen substituents, such as fluorinated substituents, chlorinated substituents, etc., specifically perfluoroalkyl groups), cyano groups, sulfonic acid groups, aromatic heterocyclic groups (aromatic heterocycles are five- or six-membered aromatic heterocycles containing heteroatoms such as nitrogen, oxygen, and sulfur, specifically thiophene, pyridine, etc.), phenyl or substituted phenyl groups (substituted phenyl groups contain small molecule substituents on the benzene ring, and the benzene ring of the substituted phenyl group contains at least one of the following substituents: C1-C5 alkyl, C1-C5 alkoxy, C2-C6 fatty acyl, C1-C6 fatty amine, amino, halogen substituent, C1-C5 haloalkyl, cyano, sulfonic acid).

[0017] In a further preferred embodiment, R is hydrogen, methyl, ethyl, butyl, methyl formate, N,N'-dimethyl, methyl alcohol, formamido, methoxy, fluorinated, chlorinated, bromine, difluoromethyl, trifluoromethyl, cyano, sulfonic acid, pyridyl, thiophene, morpholino, phenyl, p-tolyl, p-aminophenyl, p-(N,N'-dimethyl)phenyl, p-tert-butylphenyl, p-methoxyphenyl, p-fluorophenyl, p-trifluorotolyl, p-cyanophenyl, or p-acetylphenyl. Most preferably, R is an electron-withdrawing group.

[0018] As an optimal solution, in Equation 1, R is trifluoromethyl, and R' is...

[0019] This invention also provides a method for preparing a chemiluminescent probe, which includes the following steps:

[0020] 1) The compound with structure 2 and the compound with structure 3 are esterified to obtain the compound with structure 4;

[0021] 2) The compound with structure 4 is subjected to a peroxidation reaction with oxygen to obtain the product;

[0022]

[0023] in,

[0024] R' is a nitrogen heterocyclic group;

[0025] R is hydrogen, C1-C5 alkyl, C1-C5 alkoxy, C2-C6 ester, C1-C6 aliphatic amine, C1-C5 aliphatic alcohol, halogen substituent, C1-C5 haloalkyl, cyano, sulfonic acid, aromatic heterocyclic, phenyl, or substituted phenyl; the benzene ring of the substituted phenyl contains at least one of the following substituents: C1-C5 alkyl, C1-C5 alkoxy, C2-C6 aliphatic acyl, C1-C6 aliphatic amine, amino, halogen substituent, C1-C5 haloalkyl, cyano, or sulfonic acid; X is a halogen substituent (specifically, for example, a chlorine substituent).

[0026] As a preferred embodiment, in Formulas 2 to 4, R' is a pyridine group, quinoline group, phenanthridine group, or o-phenanthroline group; R is hydrogen, methyl, ethyl, butyl, methyl formate, N,N'-dimethyl, methyl alcohol, formamido, methoxy, fluorinated, chlorinated, bromine, difluoromethyl, trifluoromethyl, cyano, sulfonic acid, pyridyl, thiophene, morpholino, phenyl, p-tolyl, p-aminophenyl, p-(N,N'-dimethyl)phenyl, p-tert-butylphenyl, p-methoxyphenyl, p-fluorophenyl, p-trifluorotolyl, p-cyanophenyl, or p-acetylphenyl; X is chlorine.

[0027] As a preferred embodiment, the esterification reaction is carried out under the catalysis of triethylamine at a temperature of -5 to 5°C for 0.5 to 1 hour. The esterification reaction is preferably carried out in CH2Cl2 solvent. The reaction temperature is further preferably 0°C, and the reaction time is further preferably 1.0 hour.

[0028] As a preferred embodiment, the peroxidation reaction is carried out under the following conditions: catalysis by tetraphenylporphyrin, in an ice bath environment, and under light irradiation for 0.5–1.5 h. The peroxidation reaction is preferably carried out in CH₂Cl₂ solvent. The reaction time is further preferably 1.0 h.

[0029] The specific synthesis steps and route of the chemiluminescent probe of the present invention are as follows:

[0030] Step 1: The phenolic raw material of Formula 2 and triethylamine (4.0 eq) were dissolved in dry dichloromethane; under ice bath conditions, the acyl chloride compound of Formula 3 (4.0 eq) was added dropwise to the above mixed solution, and the mixture was gradually heated to room temperature. The reaction was then carried out at room temperature for 1.0 h. After the reaction was completed, the solvent was evaporated and purified by silica gel column chromatography with petroleum ether:ethyl acetate = 20:1 as the eluent to obtain the intermediate of Formula 4.

[0031] Step 2: Add the intermediate of Formula 4 and TPP to a 25mL reaction flask. After sealing the reaction flask, oxygen is evacuated three times. CH2Cl2 is added under an oxygen atmosphere. The reaction is carried out under ice bath conditions and irradiated with a fluorescent lamp for 1 hour. After the reaction is completed, the target compound is obtained by direct separation and purification by chromatographic column. The eluent is petroleum ether:ethyl acetate = 20:1, v:v.

[0032] In the following, R in the chemiluminescent probe represents trifluoromethyl, For example, the specific synthetic route of the chemiluminescent probe is explained as follows:

[0033]

[0034] This invention also provides an application of a chemiluminescent probe, which serves as a molecular probe for the detection of biothiols.

[0035] As a preferred embodiment, the chemiluminescent probe is used for selective imaging of biothiols in living organisms.

[0036] The selective recognition mechanism of the chemiluminescent probe of this invention for biothiols is as follows (where R represents trifluoromethyl, ...). (Taking a chemiluminescent probe as an example): First, the thiol group of bio-thiols such as cysteine ​​undergoes nucleophilic addition to the terminal carbon atom of the olefin (III). Then, the amino group of cysteine ​​attacks the carbonyl carbon to undergo intramolecular cyclization. The cyclization process forms a lactam (IV) containing a sulfur atom. Finally, the recognition group leaves, thereby activating the probe decomposition and causing chemiluminescence.

[0037]

[0038] Compared with existing technologies, the beneficial technical effects of the present invention are as follows:

[0039] 1. The chemiluminescent probe provided by this invention can specifically interact with biothiols to produce chemiluminescence.

[0040] 2. The chemiluminescent probe provided by this invention has a simple synthesis procedure, readily available raw materials, low cost, mild reaction conditions, and high yield, which is conducive to industrial production.

[0041] 3. The chemiluminescent probe of the present invention has the characteristics of low light absorption or light intensity of biological tissues, strong penetration ability of biological tissues, high imaging signal-to-noise ratio, high resolution, and strong selectivity. It can be used for highly selective and sensitive imaging of biological thiols in living organisms. Attached Figure Description

[0042] Figure 1 This is the 1H NMR spectrum of compound 2.

[0043] Figure 2 This is the carbon NMR spectrum of compound 2.

[0044] Figure 3 This is the 1H NMR spectrum of compound CH.

[0045] Figure 4 This is the carbon NMR spectrum of compound CH.

[0046] Figure 5 This is the 1H NMR spectrum of compound 3.

[0047] Figure 6 The image shows the NMR fluorine spectrum of compound 3.

[0048] Figure 7 This is the carbon NMR spectrum of compound 3.

[0049] Figure 8 This is the 1H NMR spectrum of compound C-CF3.

[0050] Figure 9 This is the NMR fluorine spectrum of compound C-CF3.

[0051] Figure 10 This is the carbon NMR spectrum of compound C-CF3.

[0052] Figure 11 This is a comparison of the responses of seven chemiluminescent probes to Cys, GSH, and Hcy.

[0053] Figure 12 This study investigated the selectivity of C-CF3 for different analyzed species.

[0054] Figure 13 C-CF3 was subjected to different concentrations of ONOO - A quantitative graph of chemiluminescence intensity after 18 min of incubation.

[0055] Figure 14 This is an imaging experiment of CH and C-CF3 probes under the skin of mice.

[0056] Figure 15 This is an imaging experiment of two probes, CH and C-CF3, in the peritoneal cavity of mice. Detailed Implementation

[0057] The following specific embodiments are intended to further illustrate the content of the present invention, rather than to limit the scope of protection of the claims of the present invention.

[0058] Unless otherwise specified, the raw materials, solvents, etc. used in the following specific examples are all conventional commercially available chemical reagents.

[0059] The following are 44 chemiluminescent probes synthesized in this invention, with two selected as typical examples for detailed explanation.

[0060]

[0061] The bicyclic structure of the Matsumoto-type 1,2-dioxane contained in this chemiluminescent probe restricts the diffusion of the solvent cage, increases the probability of electron back-transfer to generate excited states, and thus has better thermal stability. At the same time, the D-π-A structure is conducive to accepting electrons and generating photocurrent, and has a high molar absorptivity. The pyridine group is introduced into the molecule as an electron-withdrawing group, which enables its emission wavelength to reach the near-infrared level of 650 nm.

[0062] Example 1

[0063] Synthesis of compound CH:

[0064]

[0065] 0.3 mmol of compound 1 and triethylamine (4.0 eq) were dissolved in dry dichloromethane; acryloyl chloride (4.0 eq) was added dropwise to the above mixed solution under ice bath conditions, and the mixture was gradually heated to room temperature, and then reacted at room temperature for 30 min; the mixture was filtered through a column with PE / EA = 10 / 1 to give 109.9 mg of a pale yellow solid, with a separation yield of 85%. 1 H NMR(400MHz,Chloroform-d)δ8.74–8.69(m,1H),8.33(d,J=16.1Hz,1H),8.16(dt ,J=7.9,1.1Hz,1H),8.01(d,J=16.1Hz,1H),7.91–7.80(m,2H),7.47(ddd,J=7.6,4 .7,1.2Hz,1H),7.28–7.14(m,2H),6.68(dd,J=17.3,1.2Hz,1H),6.41(dd,J=17.3, 10.4Hz, 1H), 6.08 (dd, J=10.4, 1.2Hz, 1H), 3.87 (s, 2H), 1.32 (s, 6H), 1.08 (s, 9H). 13 C NMR(101MHz,Chloroform-d)δ189.2,164.0,154.0,149.2,148.8,148.1,139.4,137.3,137.0, 133.4,127.7,127.5,127.4,127.1,126.9,124.7,122.9,122.9,83.2,47.3,32.5,32.5,27.3.

[0066]

[0067] 0.25 mmol of compound 2 and TPP (10%) were added to a flask, and dichloromethane was added under an oxygen atmosphere. The reaction was carried out in an ice bath under light and oxygen conditions for 1 h. After the reaction was stopped, some of the solvent was removed by vortexing, and the sample was loaded by wet method. TPP was eluted by PE / EA = 20 / 1. Then the solvent polarity was gradually increased to obtain 92.6 mg of pale yellow solid, with a separation yield of 80%. 1H NMR(400MHz,Chloroform-d)δ8.74–8.69(m,1H),8.36(d,J=16.1Hz,1H),8.16(d,J=7.9Hz ,1H),8.00(d,J=16.2Hz,1H),7.94(d,J=8.2Hz,1H),7.87(td,J=7.7,1.7Hz,1H),7.58(dd ,J=8.3,1.8Hz,1H),7.51–7.44(m,2H),6.72–6.65(m,1H),6.41(dd,J=17.3,10.5Hz,1H), 6.12–6.05(m,1H),4.58(d,J=8.3Hz,1H),3.83(d,J=8.2Hz,1H),1.25(s,6H),1.02(s,9H). 13 C NMR(101MHz,Chloroform-d)δ189.1,164.0,153.8,149.2,148.8,139.1,137.0,136.7,133.5,128.9,127.4,1 27.2,127.0,126.0,123.7,123.5,122.9,115.9,105.3,80.3,45.6,36.7,31.4,30.1,29.6,26.7,25.1,18.4.

[0068] Example 2

[0069] Synthesis of compound C-CF3:

[0070]

[0071] 0.3 mmol of compound 1 and triethylamine (4.0 eq) were dissolved in dry dichloromethane; 4,4,4-trifluorobutenoyl chloride (4.0 eq) was added dropwise to the above mixed solution under ice bath conditions, and the mixture was gradually heated to room temperature, and then reacted at room temperature for 30 min; the mixture was filtered through a column with PE / EA = 20 / 1 to give 121.3 mg of a pale yellow solid, with a separation yield of 81%. 1H NMR(400MHz,Chloroform-d)δ8.71(ddd,J=4.7,1.7,0.9Hz,1H),8.36(d,J=16.1Hz,1H ),8.17(dt,J=7.8,1.1Hz,1H),7.93(d,J=16.2Hz,1H),7.89–7.82(m,2H),7.48(ddd,J =7.6,4.7,1.3Hz,1H),7.28(dd,J=8.0,1.6Hz,1H),7.18(d,J=1.6Hz,1H),7.04(dq,J= 15.8,6.4Hz,1H),6.82(dq,J=15.8,1.9Hz,1H),3.87(s,2H),1.33(s,6H),1.08(s,9H). 19 F NMR(377MHz,Chloroform-d)δ-65.5. 13 C NMR(101MHz,Chloroform-d)δ189.1,161.9,153.8,148.8,148.5,147.9,139.5,137.0,136.9,133.5(q,J=35.8Hz),128.4, 128.3,127.8(q,J=6.1Hz),127.3,127.3,127.1,124.4,123.5,122.9,121.8(q,J=270.5Hz),83.3,47.4,32.5,29.7,27.3.

[0072]

[0073] 0.25 mmol of compound 3 and TPP (10%) were added to a flask, and dichloromethane was added under an oxygen atmosphere. The reaction was carried out in an ice bath under light and oxygen conditions for 1 h. After the reaction was stopped, some of the solvent was removed by vortexing, and the sample was loaded by wet method. TPP was eluted by PE / EA = 20 / 1. Then the solvent polarity was gradually increased to obtain 83.6 mg of pale yellow solid, with a separation yield of 63%. 1H NMR(400MHz,Chloroform-d)δ8.74–8.68(m,1H),8.39(d,J=16.2Hz,1H),8.17(d,J=7.8Hz,1H),7.97–7.83(m,3H),7.61(dd,J=8.2,1.7Hz,1H),7.49(td, J=5.0,2.1Hz,2H),7.12–6.96(m,1H),6.82(dt,J=15.8,2.0Hz,1H),4.58(d, J=8.3Hz,1H),3.83(d,J=8.2Hz,1H),1.37(s,3H),1.15(s,3H),1.01(s,9H). 19 F NMR(377MHz,Chloroform-d)δ-65.6. 13 C NMR(101MHz,Chloroform-d)δ189.0,162.0,153.7,148.9,148.6,139.3,137.1,136.4,133.7(q,J=35.8Hz),128.7,128.3,12 7.7(q,J=6.2Hz),127.2,126.6,124.4,123.2,123.0,121.7(q,J=270.6Hz),115.8,105.4,80.4,45.6,36.7,26.8,25.1,18.4.

[0074] Example 3

[0075] This example demonstrates the response of a chemiluminescent molecular probe to biothiols:

[0076] Seven chemiluminescent probes were used in response experiments with three different biothiols: cysteine, glutathione, and homocysteine. The results showed that probes substituted with electron-withdrawing groups such as trifluoromethyl, methyl formate, and fluorine groups generally exhibited higher luminescence intensity than probes substituted with electron-neutral hydrogen atoms, while probes substituted with electron-donating groups such as methyl or phenyl groups had the lowest overall luminescence intensity. Furthermore, the strongly electron-withdrawing trifluoromethyl-substituted chemiluminescent probe C-CF3 showed the strongest chemiluminescence intensity among all chemiluminescent probes in their response to the three biothiols. Specific comparative data are as follows (…). Figure 11In the cysteine ​​response experiment, the intensity of C-CF3 was 2.4 times, 2.1 times, 47.4 times, 130.0 times, 223.4 times, and 240.6 times that of CH, C-COOMe, C-Me, C-Ph-CF3, C-Ph-F, and C-Ph, respectively. In the glutathione response experiment, the intensity of C-CF3 was 11.6 times, 6.9 times, 75.1 times, 168.7 times, 310.1 times, and 372.1 times that of CH, C-COOMe, C-Me, C-Ph-CF3, C-Ph-F, and C-Ph, respectively. In the homocysteine ​​response experiment, the intensity of C-CF3 was 5.0 times, 5.5 times, 38.9 times, 121.2 times, 152.0 times, and 334.2 times that of CH, C-COOMe, C-Me, C-Ph-CF3, C-Ph-F, and C-Ph, respectively.

[0077] Example 4

[0078] This example demonstrates a selective experiment using a chemiluminescent molecular probe for biothiols.

[0079] To investigate the selectivity of chemiluminescent molecular probes for different active species, several well-known active species were selected: 1-Blank, 2-Ac - 3-β-Gal, 4-ALP, 5-S 2- ,6-TBHP,7-NO,8-OH,9-ClO - 10-H₂O₂, 11-Hcy, 12-GSH, and 13-Cys. The results are as follows... Figure 12 As shown, biothiols Cys, GSH, and Hcy exhibited significant response effects, with the luminescence intensity of the three biothiols in the order of Cys > GSH > Hcy. The luminescence intensity of other active species did not show a significant increase compared to the control group.

[0080] Example 5

[0081] This example demonstrates the determination of the detection limit of biothiols using a chemiluminescent molecular probe:

[0082] The sensitivity of C-CF3 to Cys was investigated by incubating C-CF3 (20 μM) with different concentrations of Cys ranging from 1.25 μM to 100 μM at 37 °C. Figure 13 The results showed that the chemiluminescence intensity of C-CF3 was positively correlated with the Cys concentration, and a significant linear relationship was established in the range of 1.25-50 μM (y = 40584.79x - 1547.48, R² = 0.992), with a detection limit (LOD) of 37 nM.

[0083] Example 6

[0084] This example demonstrates a bioimaging experiment using chemiluminescent molecular probes to detect subcutaneous biothiols in mice.

[0085] We selected two chemiluminescent probes, CH and C-CF3, and conducted a comparative imaging experiment in the subcutaneous region of mice. The results are as follows: Figure 14 As shown, the C-CF3 probe has a higher chemiluminescence intensity than the CH probe. Both probes reached their peak at 10 min. At 10 min, the chemiluminescence intensity of the C-CF3 group was 1.63 times that of the CH group. The chemiluminescence intensities of the C-CF3 group and the CH group were 126.5 times and 205.7 times that of the control group, respectively.

[0086] Example 7

[0087] N-acetylmaleimide (NEM) is a common biothiol inhibitor that effectively inhibits the production of biothiols in animals. Figure 15 In our experiment, we measured the response of C-CF3 and CH to biothiols in the peritoneal cavity of mice. The results showed that both C-CF3 and CH probes were significantly enhanced in vivo compared to the control group (with NEM inhibitor). The strength of C-CF3 and CH was 4.69 times and 2.77 times that of the control group, respectively. Furthermore, the strength of the C-CF3 group was 1.69 times that of the CH group.

[0088] The above embodiments are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make various changes or modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions should also fall within the protection scope of the invention, which should be defined by the claims.

Claims

1. A chemiluminescent probe, characterized in that: It has the structure of Formula 1: Formula 1: ; in R' is a pyridine group, quinoline group, phenanthridine group, or o-phenanthroline group; R is hydrogen, C2-C6 ester group, halogen substituent, C1-C5 haloalkyl, cyano or sulfonic acid group.

2. The chemiluminescent probe according to claim 1, characterized in that: R can be hydrogen, methyl formate, fluorine substituent, chlorine substituent, bromine substituent, difluoromethyl, trifluoromethyl, cyano, or sulfonic acid group.

3. A method for preparing a chemiluminescent probe according to claim 1 or 2, characterized in that: Includes the following steps: 1) The compound with structure 2 and the compound with structure 3 are esterified to obtain the compound with structure 4; 2) The compound with structure 4 is subjected to a peroxidation reaction with oxygen to obtain the product; Formula 2: ; Formula 3: ; Formula 4: ; in, R' is a pyridine group, quinoline group, phenanthridine group, or o-phenanthroline group; R is hydrogen, C2-C6 ester group, halogen substituent, C1-C5 haloalkyl group, cyano group or sulfonic acid group; X is a halogen substituent.

4. The method for preparing a chemiluminescent probe according to claim 3, characterized in that: R is hydrogen, methyl formate, fluorine substituent, chlorine substituent, bromine substituent, difluoromethyl, trifluoromethyl, cyano, or sulfonic acid group; X represents chlorine.

5. A method for preparing a chemiluminescent probe according to claim 3 or 4, characterized in that: The esterification reaction conditions are as follows: under the catalysis of triethylamine, at a temperature of -5℃ to 5℃, the reaction is carried out for 0.5 to 1 h.

6. A method for preparing a chemiluminescent probe according to claim 3, characterized in that: The conditions for the peroxidation reaction are: under the catalysis of tetraphenylporphyrin, in an ice bath environment, and under light irradiation for 0.5 to 1.5 h.

7. The application of the chemiluminescent probe according to claim 1 or 2, characterized in that: Prepare molecular probes for the detection of biothiols.

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