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

Abstract

The invention discloses a chemiluminescent probe, a preparation method thereof and application of the chemiluminescent probe in biological mercaptan detection, and belongs to the technical field of biosensing. The structure of the chemiluminescent probe is as follows: # imgabs0 #, wherein R'is a nitrogen heterocyclic group; r is a conventional polar modification group; the probe shows obvious selectivity on biological mercaptan, can be widely applied to biological mercaptan monitoring and high-sensitivity and high-selectivity imaging in a living body, and provides a new chemiluminescent molecular probe for early detection and precise diagnosis of biological mercaptan related diseases.

Description

Technical Field

[0001] The present invention relates to a biological probe, in particular to a peroxycyclobutane chemiluminescent probe with allyl acetyl as a recognition group, and also to a preparation method thereof and application of the chemiluminescent probe in biological thiol detection, belonging to the field of biosensor technology. Background Art

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

[0003] To achieve real-time monitoring in biological environments, optical imaging methods such as fluorescence and bioluminescence have attracted great attention as non-invasive monitoring tools. Many fluorescent biothiol probes have been reported and successfully applied to monitor biothiol levels in living cells and in vivo. However, fluorescence imaging requires an excitation light source, which limits the capabilities of fluorescent probes due to potential phototoxicity and reduced detection sensitivity caused by autofluorescence and light scattering. In the past decade, chemiluminescence systems have been increasingly considered as an alternative to fluorescence systems.

[0004] Chemiluminescent probes have been widely used in imaging detection of target molecules in living biological tissues. The mechanism is: after the probe is activated, the high-energy chemical bonds in the molecule break and release energy, further stimulating the dye molecules to emit light. The biggest advantage of chemiluminescent probes is that they do not require additional excitation light sources, effectively avoiding problems such as light absorption, autofluorescence and light scattering in biological tissues. Existing chemiluminescent probes for the detection of small molecule biothiols have greatly improved the sensitivity and signal-to-noise ratio of detection, but the wavelength is limited to the visible light region of about 550nm (for example: 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, chemiluminescent probes for detecting biothiols with near-infrared (NIR; 650-900 nm) emission that have the advantages of deep tissue penetration, low tissue photodamage, and little autofluorescence interference are highly desired. Summary of the invention

[0005] In view of the defects of the prior art, the first object of the present invention is to provide a chemiluminescent probe, which uses an acetyl nitrogen heterocycle as an electron-withdrawing group, a peroxycyclobutane as a luminescent group, and an allyl acetyl group as a biothiol recognition group. At the same time, different types of electron-withdrawing groups are introduced at the olefin end of the allyl acetyl group to change the reactivity of the probe to biothiols. The probe has the characteristics of low light absorption by biological tissues or low self-light intensity, strong penetration ability into biological tissues, high imaging signal-to-noise ratio and resolution, and highly selective recognition of biothiols. It can be used for highly selective and highly sensitive imaging of biothiols in living bodies.

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

[0007] The third object of the present invention is to provide a chemiluminescent probe for use as a molecular probe in the detection of biological thiols. Based on the characteristics of the chemiluminescent probe, such as low light absorption in biological tissues or low light intensity, strong penetration into biological tissues, high imaging signal-to-noise ratio, high resolution, and strong selectivity, it is applied to the detection of biological thiols in vivo, and has the advantages of high selectivity, high sensitivity, and low biological toxicity.

[0008] In order to achieve the above technical objectives, the present invention provides a chemiluminescent probe having a 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 fatty amine, C1-C5 fatty alcohol, halogen substituent, C1-C5 haloalkyl, cyano, sulfonic acid, aromatic heterocyclic, phenyl or substituted phenyl;

[0013] The benzene ring of the substituted phenyl group contains at least one substituent selected from the group consisting of a C1-C5 alkyl group, a C1-C5 alkoxy group, a C2-C6 fatty acyl group, a C1-C6 fatty amine group, an amino group, a halogen substituent, a C1-C5 haloalkyl group, a cyano group, and a sulfonic acid group.

[0014] The chemiluminescent probe of the present invention has a special molecular structure, which simultaneously comprises a biothiol recognition group, a peroxycyclobutane group and a polar conjugated group, wherein an acryloyl derivative substituted by various electron-withdrawing groups at the olefin terminal is used as the biothiol recognition group, which gives it a highly selective recognition ability for biothiol, and the peroxycyclobutane group is used as the chemiluminescent core skeleton, and a group with a larger conjugation coefficient is introduced to extend its emission wavelength, so that it has the characteristics of low light absorption by biological tissue or low self-light intensity, and the polar nitrogen heterocyclic group makes it have strong penetration ability for biological tissue; in general, the entire chemiluminescent probe exhibits the following characteristics: low light absorption by biological tissue or low self-light intensity, strong penetration ability for biological tissue, high imaging signal-to-noise ratio, high resolution, high selective recognition of biothiol, and the like, and can be used for high-selectivity and high-sensitivity imaging of biothiol in cells or other living bodies.

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

[0016] As a preferred embodiment, R is hydrogen, C1-C5 alkyl (the alkyl group having 3 or more carbon atoms may be a branched alkyl group or a straight-chain alkyl group), C1-C5 alkoxy (the alkyl group having 3 or more carbon atoms in the alkoxy group may be a branched alkyl group or a straight-chain alkyl group), C2-C6 ester group (for example, methyl formate group, methyl acetate group, etc.), C1-C6 fatty amine group (the amine group may be a primary amine group or a secondary amine group, and the fatty hydrocarbon substituent on the amine group is an alkyl substituent, for example, methyl group, ethyl group, etc.), C1-C5 fatty alcohol group (the fatty alcohol group is an alkyl group containing a hydroxyl substituent, for example, a methanol group), a halogen substituent (for example, a fluorine substituent). substituted groups, chlorine substituents, etc.), C1~C5 haloalkyl (halogenated alkyl is an alkyl containing a halogen substituent, the halogen substituent is a fluorine substituent, a chlorine substituent, etc., such as a perfluoroalkyl), cyano, sulfonic acid, aromatic heterocyclic group (aromatic heterocyclic group is a five-membered or six-membered aromatic heterocyclic group containing heteroatoms such as nitrogen, oxygen, sulfur, such as thiophene, pyridine, etc.), phenyl or substituted phenyl (substituted phenyl contains a small molecule substituent on the benzene ring, and the substituted phenyl contains at least one substituent selected from C1~C5 alkyl, C1~C5 alkoxy, C2~C6 fatty acyl, C1~C6 fatty amine, amino, halogen substituent, C1~C5 haloalkyl, cyano, and sulfonic acid on the benzene ring).

[0017] In a further preferred embodiment, R is hydrogen, methyl, ethyl, butyl, methyl formate, N, N'-dimethyl, methanol, formamide, methoxy, fluorine substituent, chlorine substituent, bromine substituent, difluoromethyl, trifluoromethyl, cyano, sulfonic acid, pyridyl, thienyl, morpholinyl, phenyl, p-tolyl, p-aminophenyl, p-(N, N'-dimethyl)phenyl, p-tert-butylphenyl, p-methoxyphenyl, p-fluorophenyl, p-trifluoromethylphenyl, p-cyanophenyl or p-acetylphenyl. The most preferred R is an electron withdrawing group.

[0018] As a most preferred embodiment, in Formula 1, R is trifluoromethyl, and R' is

[0019] The present invention also provides a method for preparing a chemiluminescent probe, which comprises the following steps:

[0020] 1) esterifying the compound of formula 2 with the compound of formula 3 to obtain a compound of formula 4;

[0021] 2) subjecting the compound of formula 4 to a peroxidation reaction with oxygen to obtain;

[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 fatty amine, C1-C5 fatty alcohol, halogen substituent, C1-C5 haloalkyl, cyano, sulfonic acid, aromatic heterocyclic group, phenyl or substituted phenyl; the substituted phenyl has at least one substituent selected from C1-C5 alkyl, C1-C5 alkoxy, C2-C6 fatty acyl, C1-C6 fatty amine, amino, halogen substituent, C1-C5 haloalkyl, cyano and sulfonic acid on its benzene ring; X is a halogen substituent (for example, a chlorine substituent).

[0026] As a preferred embodiment, in Formula 2 to Formula 4, R' is a pyridine group, a quinoline group, a phenanthridine group or a phenanthroline group; R is hydrogen, methyl, ethyl, butyl, methyl formate, N,N'-dimethyl, methanol, formamide, methoxy, fluorine substituent, chlorine substituent, bromine substituent, difluoromethyl, trifluoromethyl, cyano, sulfonic acid, pyridyl, thienyl, morpholinyl, phenyl, p-tolyl, p-aminophenyl, p-(N,N'-dimethyl)phenyl, p-tert-butylphenyl, p-methoxyphenyl, p-fluorophenyl, p-trifluorotolyl, p-cyanophenyl or p-acetylphenyl; and X is chlorine.

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

[0028] As a preferred solution, the peroxidation reaction conditions are: under the catalysis of tetraphenylporphyrin, in an ice bath environment, under light for 0.5 to 1.5 hours. The peroxidation reaction is preferably carried out in a CH2Cl2 solvent. The reaction time is more preferably 1.0 hour.

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

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

[0031] Step 2: Add the intermediate of formula 4 and TPP into a 25 mL reaction bottle. After the reaction bottle is sealed, evacuate and fill it with oxygen three times. Add CH2Cl2 under an oxygen atmosphere. Under ice bath conditions, irradiate with a fluorescent lamp to react for 1 h. After the reaction is completed, directly separate and purify the target compound using a chromatographic column. The eluent is petroleum ether: ethyl acetate = 20:1, v:v.

[0032] In the following, R in the chemiluminescent probe is trifluoromethyl, Taking the chemiluminescent probe as an example, the specific synthesis route is described as follows:

[0033]

[0034] The present invention also provides an application of a chemiluminescent probe, which is used as a molecular probe for detecting biothiols.

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

[0036] The principle of the selective recognition of biothiols by the chemiluminescent probe of the present invention is as follows (where R is trifluoromethyl, Take the chemiluminescent probe of as an example): First, the thiol group of biological thiol such as cysteine ​​first nucleophilically adds to the terminal carbon atom of the olefin III, and then the amino group of cysteine ​​attacks the carbonyl carbon for intramolecular cyclization. The cyclization process forms a lactam IV containing a sulfur atom, and finally the recognition group leaves to activate the decomposition of the probe, resulting in chemiluminescence.

[0037]

[0038] Compared with the prior art, the technical solution of the present invention has the following beneficial technical effects:

[0039] 1. The chemiluminescent probe provided by the present invention can specifically react with biological thiols to produce chemiluminescence.

[0040] 2. The synthesis steps of the chemiluminescent probe provided by the present invention are simple, the raw materials are easily available, the cost is low, the reaction conditions are mild, the yield is high, and it is conducive to industrial production.

[0041] 3. The chemiluminescent probe of the present invention has the characteristics of low light absorption by biological tissues or low light intensity, strong penetration into biological tissues, high imaging signal-to-noise ratio, high resolution, and strong selectivity, and can be used for high-selectivity and high-sensitivity imaging of biological thiols in living bodies. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

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

[0047] Figure 6 This is the NMR fluorine spectrum of compound 3.

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

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

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

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

[0052] Fig.11 The figure shows the comparison of the responsiveness of seven chemiluminescent probes to Cys, GSH and Hcy.

[0053] Fig.12 This is an experiment to test the selectivity of C-CF3 for different analytical species.

[0054] Fig.13 C-CF3 is reacted with different concentrations of ONOO - Quantification of chemiluminescence intensity after 18 min of incubation.

[0055] Fig.14 This is a picture of the imaging experiment of CH and C-CF3 probes under the mouse skin.

[0056] Fig.15 This is a picture of the imaging experiment of CH and C-CF3 probes in the mouse peritoneal cavity. DETAILED DESCRIPTION

[0057] The following specific examples are intended to further illustrate 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. in the following specific examples are all conventional commercially available chemical reagents.

[0059] The following are 44 chemiluminescent probes synthesized by the present invention, two of which are selected as typical examples for specific description.

[0060]

[0061] The bicyclic structure of the Matsumoto-type 1,2-dioxetane contained in this chemiluminescent probe limits the diffusion of the solvent cage and increases the probability of electron back transfer to produce an excited state, thus having better thermal stability. At the same time, due to its D-π-A structure, it is conducive to accepting electrons and generating photocurrent, and has a higher molar absorption coefficient. The pyridine group is introduced into the molecule as an electron-withdrawing group, so that its emission wavelength reaches the near-infrared level of 650nm.

[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 mixed solution under ice bath conditions, gradually heated to room temperature, and then reacted at room temperature for 30 min; PE / EA = 10 / 1 was passed through a column to obtain 109.9 mg of a light yellow solid with an isolated 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 stopped, part of the solvent was spun off and the sample was loaded by wet method; PE / EA=20 / 1 was used to elute TPP, and then the solvent polarity was gradually increased to obtain 92.6 mg of a light yellow solid with an isolated 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 mixed solution under ice bath conditions, gradually heated to room temperature, and then reacted at room temperature for 30 min; PE / EA = 20 / 1 was passed through a column to obtain 121.3 mg of a light yellow solid with an isolated 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 stopped, part of the solvent was spun off and the sample was loaded by wet method; PE / EA=20 / 1 was used to elute TPP, and then the solvent polarity was gradually increased to obtain 83.6 mg of a light yellow solid with an isolated 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 is an experiment on the response of chemiluminescent molecular probe to biological thiols:

[0076] Seven chemiluminescent probes were tested for their responsiveness to three different biothiols: cysteine, glutathione, and homocysteine. The results showed that the luminescence intensity of probes substituted with electron-withdrawing groups such as trifluoromethyl, methyl formate, and fluorine was higher than that of probes substituted with electrically neutral hydrogen atoms, while the overall luminescence intensity of probes substituted with electron-donating groups such as methyl or phenyl was the lowest. In addition, the chemiluminescent probe C-CF3 substituted with a strong electron-withdrawing trifluoromethyl group had the strongest chemiluminescence intensity among all chemiluminescent probes in the response recognition of the three biothiols. The specific comparison data is as follows ( Fig.11): In the response experiment of cysteine, the intensity of C-CF3 is 2.4 times, 2.1 times, 47.4 times, 130.0 times, 223.4 times and 240.6 times of that of CH, C-COOMe, C-Me, C-Ph-CF3, C-Ph-F and C-Ph, respectively. In the response experiment of glutathione, the intensity of C-CF3 is 11.6 times, 6.9 times, 75.1 times, 168.7 times, 310.1 times and 372.1 times of that of CH, C-COOMe, C-Me, C-Ph-CF3, C-Ph-F and C-Ph, respectively. In the response experiment of homocysteine, 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 is a chemiluminescent molecular probe selectivity experiment for biological thiols:

[0079] In order to explore the selectivity of chemiluminescent molecular probes for different active species, a variety of 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 Fig.12 As shown, the biothiols Cys, GSH and Hcy have obvious response effects. The luminescence intensity of the three biothiols is Cys>GSH>Hcy, and the luminescence intensity of other active species is not significantly enhanced compared with the blank group.

[0080] Example 5

[0081] This example is the detection limit determination of biothiols by chemiluminescent molecular probes:

[0082] C-CF3 (20 μM) was incubated with different concentrations of Cys ranging from 1.25 μM to 100 μM at 37°C to study the sensitivity of C-CF3 to Cys. Fig.13 The results showed that the chemiluminescence intensity of C-CF3 was positively correlated with the Cys concentration, and an obvious linear relationship was established in the range of 1.25-50 μM (y=40584.79x-1547.48, R^2=0.992), with a detection limit (LOD) of 37 nM.

[0083] Example 6

[0084] This example is a bioimaging experiment of subcutaneous biothiols in mice using chemiluminescent molecular probes:

[0085] We selected two chemiluminescent probes, CH and C-CF3, to conduct imaging comparison experiments under the skin of mice. Fig.14 As shown, the C-CF3 probe has a higher chemiluminescence intensity than the CH probe. Both probes reached their peak at the 10th minute. At 10 minutes, 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-Ethylmaleimide (NEM) is a common biothiol inhibitor that can effectively inhibit the production of biothiols in animals. Fig.15 In the experiment, we measured the response of C-CF3 and CH to biothiols in the peritoneal cavity of mice and found that the two probes, C-CF3 and CH, had a significant enhancement in the animal body compared with the control group (with NEM inhibitor). C-CF3 and CH were 4.69 times and 2.77 times the intensity of the control group, respectively. In addition, the intensity of the C-CF3 group was 1.69 times that of the CH group.

[0088] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Technicians in the relevant technical field may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also fall within the protection scope of the present invention, and the protection scope of the present invention should be defined by the claims.

Claims

1. A chemiluminescent probe, characterized in that: It has the structure of formula 1: in, R' is a nitrogen heterocyclic group; R is hydrogen, C1-C5 alkyl, C1-C5 alkoxy, C2-C6 ester, C1-C6 fatty amine, C1-C5 fatty alcohol, halogen substituent, C1-C5 haloalkyl, cyano, sulfonic acid, aromatic heterocyclic, phenyl or substituted phenyl; The benzene ring of the substituted phenyl group contains at least one substituent selected from the group consisting of a C1-C5 alkyl group, a C1-C5 alkoxy group, a C2-C6 fatty acyl group, a C1-C6 fatty amine group, an amino group, a halogen substituent, a C1-C5 haloalkyl group, a cyano group, and a sulfonic acid group.

2. A chemiluminescent probe according to claim 1, characterized in that: R' is a pyridine group, a quinoline group, a phenanthridine group or an o-phenanthroline group; R is hydrogen, methyl, ethyl, butyl, methyl formate, N,N'-dimethyl, methanol, formamide, methoxy, fluorine substituent, chlorine substituent, bromine substituent, difluoromethyl, trifluoromethyl, cyano, sulfonic acid, pyridyl, thienyl, 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.

3. The method for preparing a chemiluminescent probe according to claim 1 or 2, characterized in that: The following steps are involved: 1) esterifying the compound of formula 2 with the compound of formula 3 to obtain a compound of formula 4; 2) subjecting the compound of formula 4 to a peroxidation reaction with oxygen to obtain; in, R' is a nitrogen heterocyclic group; R is hydrogen, C1-C5 alkyl, C1-C5 alkoxy, C2-C6 ester, C1-C6 fatty amine, C1-C5 fatty alcohol, halogen substituent, C1-C5 haloalkyl, cyano, sulfonic acid, aromatic heterocyclic, phenyl or substituted phenyl; The substituted phenyl group has at least one substituent selected from the group consisting of a C1-C5 alkyl group, a C1-C5 alkoxy group, a C2-C6 fatty acyl group, a C1-C6 fatty amine group, an amino group, a halogen substituent, a C1-C5 haloalkyl group, a cyano group, and a sulfonic acid group on its benzene ring; X is a halogen substituent.

4. The method for preparing a chemiluminescent probe according to claim 3, characterized in that: R' is a pyridine group, a quinoline group, a phenanthridine group or an o-phenanthroline group; R is hydrogen, methyl, ethyl, butyl, methyl formate, N,N'-dimethyl, methanol, formamide, methoxy, fluorine substituent, chlorine substituent, bromine substituent, difluoromethyl, trifluoromethyl, cyano, sulfonic acid, pyridyl, thienyl, morpholinyl, 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.

5. The method for preparing a chemiluminescent probe according to claim 3 or 4, characterized in that: The conditions of the esterification reaction are: under the catalysis of triethylamine, at a temperature of -5°C to 5°C, and reacting for 0.5 to 1 hour.

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

7. Use of a chemiluminescent probe according to claim 1 or 2, characterized in that: Used as a molecular probe to detect biothiols.

8. The use of a chemiluminescent probe according to claim 7, characterized in that: Application to selective imaging of biothiols in vivo.

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