Ester hydrolase detection chemiluminescence probe, preparation method and application thereof

Abstract

The invention discloses an ester hydrolase detection chemiluminescence probe and a preparation method and application thereof, the ester hydrolase detection chemiluminescence probe based on adamantane-dioxetane derivatives can selectively react with chymotrypsin and generate 540nm chemiluminescence, and meanwhile, the ester hydrolase detection chemiluminescence probe has high selectivity and high sensitivity. The chemiluminescent probe for ester hydrolase detection has a structure as shown in a formula I, which is called CL-CHT-O or adamantane-dioxetane derivative for short; # imgabs0 #

Description

Technical Field

[0001] The present invention relates to a chemiluminescent probe, a preparation method and an application thereof, and more specifically to a chemiluminescent probe for detecting ester hydrolase, a preparation method and an application thereof. Background Art

[0002] A serine protease called chymotrypsin (CHT) is often found in the digestive system of mammals. It mainly hydrolyzes carboxy-aromatic amino acids and acts as a peptide bond endonuclease. To date, chymotrypsin has been shown to be involved in controlling the digestion, necrosis, and apoptosis of dietary proteins. Chymotrypsin is often used as an active ingredient in drugs to treat inflammation, edema, wound healing, or surgical infection. It can be used as an adjunct treatment for pneumonia, dermatitis, and ophthalmology. If chymotrypsin is overdosed, the body releases histamine, which can cause allergic reactions. Patients with severe liver and kidney diseases as well as abnormal coagulation function are more likely to develop anaphylactic shock. However, the toxicity of chymotrypsin has not been thoroughly studied.

[0003] In the prior art, chymotrypsin has been discovered using a variety of techniques, including traditional mass spectrometry (MS), high performance liquid chromatography (HPLC), and UV / visible spectrophotometer analysis; chymotrypsin is then used to catalyze peptide substrates to increase fluorescence, thereby improving the selectivity of the enzyme. Studies have also shown that chymotrypsin activity can be detected using protein blotting and enzyme-linked immunosorbent assay (ELISA) methods. Fluorescent probes are a new type of detection technology with advantages such as strong selectivity, fast response speed, low detection limit, biocompatibility, and visibility. It is currently impossible to create a chymotrypsin fluorescent probe with a fast reaction time, simple synthesis method, and high fluorescence quantum yield. Therefore, the development of a unique chymotrypsin activity detection technology is crucial to determine the amount of chymotrypsin used and monitor drug pathology. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a chemiluminescent probe for detecting esterase. The chemiluminescent probe for detecting esterase based on adamantane-dioxetane derivative can selectively react with chymotrypsin and produce chemiluminescence of 540 nm, and has high selectivity and high sensitivity.

[0005] The invention also provides a method for preparing the esterase detection chemiluminescent probe and the practical application of the probe in esterase detection.

[0006] The technical concept and principle of the present invention are as follows: the ester hydrolase detection chemiluminescent probe based on adamantane-dioxetane derivative can selectively react with chymotrypsin and generate 540nm chemiluminescence. The present invention is the first to prepare a chymotrypsin detection chemiluminescent probe, i.e., adamantane-dioxetane derivative, and use it for the selective detection of chymotrypsin for the first time, thereby solving the problems existing in the prior art.

[0007] The technical solution of the present invention to solve the technical problem is as follows:

[0008] The ester hydrolase detection chemiluminescent probe of the present invention has a structure as shown in Formula I, referred to as: CL-CHT-O or adamantane-dioxetane derivative;

[0009]

[0010] The preparation method of the ester hydrolase detection chemiluminescent probe of the present invention comprises the following steps: dissolving 4-((3-(((1r, 3r, 5R, 7S)-adamantane-2-ylidene)(methoxy)methyl)-2-chloro-6-((E)-3-methoxy-3-oxoprop-1-en-1-yl)phenoxy)methyl)phenyl 4-bromobutyrate and methylene blue in dichloromethane, and introducing oxygen under yellow light irradiation to generate CL-CHT-O, i.e., the ester hydrolase detection chemiluminescent probe.

[0011] The preparation method of the esterase detection chemiluminescent probe of the present invention has a further technical solution that the reaction is carried out for more than 1 hour under ice bath, yellow light irradiation and oxygen bubbling conditions. A further technical solution is to purify after the reaction is completed, and the purification steps are as follows: the crude product is filtered under reduced pressure, and the crude product is further purified on a silica gel column with n-hexane / ethyl acetate to obtain CL-CHT-O.

[0012] The application of the esterase detection chemiluminescent probe of the present invention in the detection of esterase is particularly used for the detection of chymotrypsin content in biological systems.

[0013] The present invention has the following beneficial effects:

[0014] 1) The chemiluminescent probe of the present invention has almost no fluorescence in a buffer solution of pH=7.4, but releases green chemiluminescence at 5640 nm after reacting with chymotrypsin.

[0015] 2) After using the fluorescent probe of the present invention, the detection sensitivity is high.

[0016] 3) The fluorescent probe of the present invention reacts only with chymotrypsin and has no reaction with other common ions, amino acids and enzymes, and has good selectivity and specificity and has a suitable fluorescence emission wavelength (540nm).

[0017] 4) The preparation process of the fluorescent probe of the present invention is simple and easy to carry out and can be easily mass-produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The chemiluminescent probe of Example 1 of the present invention 1 H-NMR spectrum.

[0019] Figure 2 This is the chemiluminescence time curve of the reaction between the chemiluminescent probe of Example 2 of the present invention and chymotrypsin.

[0020] Figure 3 The chemiluminescent emission spectra of the chemiluminescent probe in Example 3 of the present invention reacting with various ions.

[0021] Figure 4 This is a bar graph of the chemiluminescent intensity of various ion reactions of the chemiluminescent probe in Example 3 of the present invention.

[0022] Figure 5 This is a graph of the chemiluminescent increment of the reaction between the chemiluminescent probe of Example 4 of the present invention and chymotrypsin. DETAILED DESCRIPTION

[0023] The present invention will be described in further detail below with reference to the accompanying drawings and in combination with embodiments, but the present invention is not limited to the examples given.

[0024] The reaction equation for preparing the probe of the present invention is as follows:

[0025]

[0026] Example 1 Preparation of chemiluminescent probe for chymotrypsin detection

[0027] 80 mg of CL-CHT and a small amount of methylene blue were dissolved in 20 ml of dichloromethane solution; the mixture was stirred for 1 hour under ice bath conditions, oxygen bubbling and irradiation with a yellow deuterium lamp; after the reaction, the reaction solution was first concentrated under reduced pressure, and then the crude product was purified by silica gel column chromatography (n-hexane / ethyl acetate), and finally vacuum dried to obtain 53 mg of an oily substance, which was the pure chemiluminescent optical probe for chymotrypsin detection ( 1 H-NMR diagram Figure 1 ).

[0028] Example 2 Time response of chymotrypsin chemiluminescent probe to chymotrypsin

[0029] Weigh 6.7 mg of CL-CHT-O and prepare it into 10 mL of DMSO solution with a concentration of 1 mM as the mother solution. Weigh 6 mg of chymotrypsin with an activity of 800 U / mL and prepare it into 16 mL of PBS solution with an activity of 300 U / mL as the mother solution of the test substance.

[0030] Chemiluminescence test: 30 μL of the above mother solution was added to a certain amount of 10 mM PBS buffer solution (pH 7.4), and then 500 μL of the test substance mother solution was added to make the final concentration 50 U / mL and the final concentration of the chemiluminescent probe 10 μM. The chemiluminescence time curve was tested. The emission slit width was 10 nm and the emission wavelength was set to 540 nm. The results are as follows: Figure 2 shown.

[0031] The above results show that:

[0032] The chemiluminescence of the chemiluminescent probe reached a maximum after 15 minutes of incubation with chymotrypsin, and the chemiluminescence was basically reduced to the baseline after 80 minutes.

[0033] Example 3 Spectral properties of the reaction of chymotrypsin chemiluminescent probe with various ions

[0034] 30 μL of the mother solution in Example 2 was added to a certain amount of 10 mM PBS buffer solution (pH 7.4), and then various ions were added: (1) CHT, (2) K + ,(3)Na + ,(4)CO3 2- ,(5)SO3 2- ,(6)Cl - ,(7)ClO - ,(8)S 2- , amino acids and peptides: (9) Cys, (10) Hcy, (11) GSH, enzymes: (12) peroxidase, (13) xanthine oxidase in milk, (14) inorganic pyrophosphorylase, (15) acetylcholinesterase, (16) blank, the final concentration of the analyte is 10 μM or 20 U / mL, the final concentration of the chemiluminescent probe is 10 μM, and the chemiluminescence spectrum is tested after incubation for 15 minutes. The emission slit width is 10 nm. The results are shown in Figure 3 , 4 shown.

[0035] The above results show that:

[0036] (1) The chemiluminescent probe prepared in Example 1 is colorless in solution and emits almost no light. However, with the addition of chymotrypsin, the green chemiluminescence of the probe CL-CHT-O at 540 nm gradually increases.

[0037] (2) The chemiluminescent probe prepared in Example 1 has high selectivity and specificity for chymotrypsin and can be used to detect K + ,Na + ,CO3 2- ,SO3 2- ,Cl - ,ClO - ,S 2- Chymotrypsin can be distinguished from common ions such as Cys, Hcy, GSH and other common amino acids and peptides as well as peroxidase, xanthine oxidase in milk, inorganic pyrophosphorylase, acetylcholinesterase and other common enzymes.

[0038] Example 4 Spectral properties of the reaction product of chymotrypsin and chemiluminescent probe

[0039] 10 μL of the probe mother solution in Example 2 was added to a certain amount of 10 mM PBS buffer solution (pH 7.4), and then different equivalents of chymotrypsin were added to make the final concentration of the chemiluminescent probe 10 μM. The final concentrations of chymotrypsin were 0 U / mL, 0.2 U / mL, 0.4 U / mL, 0.8 U / mL, 1.5 U / mL, 3 U / mL, 6 U / mL, 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, and 50 U / mL, respectively. After the chymotrypsin solution was added, its chemiluminescent emission spectrum was measured immediately. The slit width of the emission was 10 nm. The incremental graph of the obtained chemiluminescent intensity is shown in Figure 5 .

[0040] The experimental results showed that the chemiluminescence intensity increased with the increase of chymotrypsin concentration after the reaction.

Claims

1. A chemiluminescent probe for detecting esterase, characterized in that: The probe has a structure as shown in Formula I, referred to as: CL-CHT-O or adamantane-dioxetane derivative; 2. A method for preparing a chemiluminescent probe for detecting esterase according to claim 1, characterized in that The following steps are involved: 4-((3-(((1r, 3r, 5R, 7S)-adamantan-2-ylidene)(methoxy)methyl)-2-chloro-6-((E)-3-methoxy-3-oxoprop-1-en-1-yl)phenoxy)methyl)phenyl 4-bromobutyrate and methylene blue were dissolved in dichloromethane, and oxygen was introduced under yellow light to generate CL-CHT-O, which is a chemiluminescent probe for ester hydrolase detection.

3. The method for preparing a chemiluminescent probe for detecting esterase according to claim 2, characterized in that: The reaction is carried out for more than 1 hour under ice bath, yellow light irradiation and oxygen bubbling conditions.

4. The method for preparing a chemiluminescent probe for detecting esterase according to claim 3, characterized in that: After the reaction is completed, purification is performed, and the purification steps are as follows: the crude product is filtered under reduced pressure, and the crude product is further purified on a silica gel column using n-hexane / ethyl acetate to obtain CL-CHT-O.

5. Use of the esterase detection chemiluminescent probe according to claim 1 in detecting esterase.

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