阅读说明：本技术 一种检测焦谷氨酸氨肽酶的生物发光探针的制备方法及其应用 (Preparation method and application of bioluminescent probe for detecting pyroglutamic acid aminopeptidase ) 是由 柯博文 李敏勇 陈新新 胡世龙 康婷 于 2019-09-09 设计创作，主要内容包括：本发明公开了一种式I所示的化合物。本发明还公开了式I所示的化合物在制备生物发光探针,特别是检测焦谷氨酸氨肽酶的生物发光探针中的用途。本发明所提供的探针对目标产物表现出了良好的灵敏性、选择性、生物相容性。本发明提供的探针不仅可以在体外环境中以良好的线性半定量检测焦谷氨酸氨肽酶,还具备活体水平可视化检测内源性焦谷氨酸氨肽酶的能力。本发明探针的制备方法简单、收率高、成本低,在底物检测方面效率高、专一性强、快速、灵敏,并且可同时实现定性、半定量检测和分析焦谷氨酸氨肽酶,易于推广和应用。<Image he="254" wi="700" file="DDA0002196271760000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention discloses a compound shown as a formula I. The invention also discloses application of the compound shown in the formula I in preparing a bioluminescent probe, in particular to a bioluminescent probe for detecting pyroglutamic acid aminopeptidase. The probe provided by the invention has good sensitivity, selectivity and biocompatibility on a target product. The probe provided by the invention not only can be used for detecting pyroglutamic acid aminopeptidase in an in vitro environment in a good linear semi-quantitative manner, but also has the capability of detecting endogenous pyroglutamic acid aminopeptidase in a living body level visualization manner. The probe of the invention has the advantages of simple preparation method, high yield, low cost, high efficiency in substrate detection, strong specificity, rapidness and sensitivity, can realize qualitative and semi-quantitative detection and analysis of pyroglutamic acid aminopeptidase simultaneously, and is easy to popularize and apply.)

1. A compound characterized by: the structure of the compound is shown as formula I:

Wherein X is S, Se or NH; r is H or C_1-3An alkyl group; y is S, Se or NH.

2. The compound of claim 1, wherein: the structure of the compound is shown as formula CX-1:

3. A process for the preparation of a compound according to claim 2, characterized in that: the method comprises the following steps:

4. The production method according to claim 3, characterized in that: the method comprises the following specific steps:

(1) Reacting the compound 1 with the compound 2 in an organic solvent to prepare a compound 3;

(2) reacting the compound 3 with trifluoroacetic acid in an organic solvent to prepare a compound 4;

(3) Reacting the compound 4 and D-cysteine hydrochloride in an organic solvent to obtain CX-1.

5. the method of claim 4, wherein: in the step (1), the molar ratio of the compound 1 to the compound 2 is 1: (0.5-1.5), preferably 1: 1; the organic solvent is acetonitrile, an alcohol solvent or DMF, and is preferably acetonitrile; the reaction temperature is 0-25 ℃, the reaction time is 3-9 h, preferably, the reaction temperature is 25 ℃, and the reaction time is 5 h; the reaction is carried out under the protection of inert gas, the reaction is carried out under the action of a catalyst, preferably, the catalyst is N-methylmorpholine and isobutyl chloroformate, and more preferably, the molar ratio of the compound 2 to the N-methylmorpholine and the isobutyl chloroformate is 1: 1: 1.

6. The method of claim 4, wherein: in the step (2), the molar ratio of the compound 3 to trifluoroacetic acid is 1: (2-4), preferably 1: 3; the organic solvent is an alcohol solvent, dichloromethane or acetonitrile, preferably dichloromethane; the reaction temperature is 20-60 ℃, the reaction time is 2-6 h, preferably, the reaction temperature is 40 ℃, and the reaction time is 3 h.

7. The production method according to any one of claims 4 to 6, characterized in that: in the step (3), the molar ratio of the compound 4 to the D-cysteine hydrochloride is 1: (1 to 3), preferably 1: 2; the solvent is dichloromethane, an alcohol solvent or a mixed solvent of the alcohol solvent and water, and preferably a mixed solvent of methanol and water; the reaction is carried out in the presence of an inorganic base, preferably the inorganic base is potassium carbonate, cesium carbonate, sodium carbonate or sodium bicarbonate, more preferably the molar ratio of compound 4 to inorganic base is 1: (2-3); the reaction temperature is 20-50 ℃, the reaction time is 1-5 h, preferably, the reaction temperature is 25 ℃, and the reaction time is 1 h.

8. use of a compound according to claim 1 or 2 for the preparation of a bioluminescent probe.

9. use according to claim 8, characterized in that: the bioluminescent probe is used for detecting pyroglutamic acid aminopeptidase.

10. Use according to claim 8 or 9, characterized in that: the bioluminescent probe can detect pyroglutamic acid aminopeptidase in vivo and/or in vitro;

And/or, the bioluminescent probe is capable of effecting bioluminescent imaging of endogenous pyroglutamate aminopeptidase in a living body;

And/or the bioluminescent probe can be detected in an aqueous solution system with the pH of 6-10.

Technical Field

the invention particularly relates to a preparation method and application of a bioluminescent probe for detecting pyroglutamic acid aminopeptidase.

background

Bioluminescence is a type of chemiluminescence that is ubiquitous in nature and relies on the normal vital activities of an organism. The essence of the method is the interaction between enzyme and substrate, and chemical energy is generated through a series of biological reactions and is released in the form of light energy. Bioluminescence imaging (Bioluminescence imaging) is performed using the principle that an enzyme-catalyzed chemical reaction occurs in a living body to generate photons. The most common bioluminescent system is the firefly Luciferase (Luciferase) -Luciferin (Luciferase) system, which essentially catalyzes the substrate Luciferin in the presence of energy (ATP) and oxygen, generates an electronic transition, generates a photon when the molecule returns from an excited state to a steady state, and releases Oxyluciferin (Oxyluciferin). The bioluminescence imaging technology has become an important detection means by virtue of the characteristics of high sensitivity, high biocompatibility, visualization and the like, and is widely applied to various fields.

Pyroglutamic acid aminopeptidase is an enzyme which specifically releases L-pyroglutamic acid residue at the amino terminal end of a protein or peptide (including some important anti-inflammatory proteins), and is known to be widely present in the brain, lung, serum or pituitary of various animals as well as plants or microorganisms. Previous studies have shown that pyroglutamic acid aminopeptidase is involved in immune responses in cells, however, it is unclear whether pyroglutamic acid aminopeptidase is involved in inflammatory reactions in vivo and acts as a new inflammatory cytokine, and detection of change and distribution of pyroglutamic acid aminopeptidase in cells, organisms during inflammation will help to better explain this problem. However, relevant reports on solving this problem are also currently found.

Disclosure of Invention

In order to solve the above problems, the present invention provides a bioluminescent probe having high selectivity and ultrasensitiveness, which exhibits superior selectivity and responsiveness to pyroglutamic acid aminopeptidase, and which is also suitable for the study of pyroglutamic acid aminopeptidase in the field of bioimaging.

The invention aims to provide a bioluminescent probe for detecting pyroglutamic acid aminopeptidase in a living body, a preparation method and application thereof, and solves the problems of poor selectivity, low sensitivity and harsh conditions of the existing pyroglutamic acid aminopeptidase optical probe so as to realize qualitative and quantitative analysis and detection of the pyroglutamic acid aminopeptidase with high selectivity and high sensitivity.

The invention provides a compound, which has a structure shown in a formula I:

Wherein X is S, Se or NH; r is H or C_1-3An alkyl group; y is S, Se or NH.

Further, the structure of the compound is shown as formula CX-1:

The invention also provides a preparation method of the compound shown as the formula CX-1, which comprises the following steps:

further, the method comprises the following specific steps:

(1) Reacting the compound 1 with the compound 2 in an organic solvent to prepare a compound 3;

(2) Reacting the compound 3 with trifluoroacetic acid in an organic solvent to prepare a compound 4;

(3) reacting the compound 4 and D-cysteine hydrochloride in an organic solvent to obtain CX-1.

Further, in the step (1), the molar ratio of the compound 1 to the compound 2 is 1: (0.5-1.5), preferably 1: 1; the organic solvent is acetonitrile, an alcohol solvent or DMF, and is preferably acetonitrile; the reaction temperature is 0-25 ℃, the reaction time is 3-9 h, preferably, the reaction temperature is 25 ℃, and the reaction time is 5 h; the reaction is carried out under the protection of inert gas, the reaction is carried out under the action of a catalyst, preferably, the catalyst is N-methylmorpholine and isobutyl chloroformate, and more preferably, the molar ratio of the compound 2 to the N-methylmorpholine and the isobutyl chloroformate is 1: 1: 1.

Further, in the step (2), the molar ratio of the compound 3 to trifluoroacetic acid is 1: (2-4), preferably 1: 3; the organic solvent is an alcohol solvent, dichloromethane or acetonitrile, preferably dichloromethane; the reaction temperature is 20-60 ℃, the reaction time is 2-6 h, preferably, the reaction temperature is 40 ℃, and the reaction time is 3 h.

Further, in the step (3), the molar ratio of the compound 4 to the D-cysteine hydrochloride is 1: (1 to 3), preferably 1: 2; the solvent is dichloromethane, an alcohol solvent or a mixed solvent of the alcohol solvent and water, and preferably a mixed solvent of methanol and water; the reaction is carried out in the presence of an inorganic base, preferably the inorganic base is potassium carbonate, cesium carbonate, sodium carbonate or sodium bicarbonate, more preferably the molar ratio of compound 4 to inorganic base is 1: (2-3); the reaction temperature is 20-50 ℃, the reaction time is 1-5 h, preferably, the reaction temperature is 25 ℃, and the reaction time is 1 h.

the invention also provides application of the compounds shown in the formula I and the formula CX-1 in preparation of bioluminescent probes.

further, the bioluminescent probe is a bioluminescent probe for detecting pyroglutamic acid aminopeptidase.

Further, the bioluminescent probe is capable of detecting pyroglutamic acid aminopeptidase in vivo and/or in vitro;

and/or, the bioluminescent probe is capable of effecting bioluminescent imaging of endogenous pyroglutamate aminopeptidase in a living body;

And/or the bioluminescent probe can be detected in an aqueous solution system with the pH of 6-10.

Experimental results show that the Boc-L-pyroglutamine is used as a specific recognition group of pyroglutamic acid aminopeptidase to synthesize a bioluminescent probe CX-1 for detecting the pyroglutamic acid aminopeptidase, and the bioluminescent probe CX-1 has good sensitivity, selectivity and biocompatibility on target products.

The probe CX-1 provided by the invention not only can be used for detecting pyroglutamic acid aminopeptidase in an in vitro environment in a good linear semi-quantitative manner, but also has the capability of detecting endogenous pyroglutamic acid aminopeptidase in a visual manner at a living level. The probe CX-1 has the advantages of simple preparation method, high yield, low cost, high efficiency in substrate detection, strong specificity, rapidness and sensitivity, can realize qualitative and semi-quantitative detection and analysis of pyroglutamic acid aminopeptidase simultaneously, and is easy to popularize and apply.

obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

the present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a bioluminescent probe for detecting pyroglutamic acid aminopeptidase, prepared in example 1;

FIG. 2 is a nuclear magnetic resonance carbon spectrum of a bioluminescent probe for detecting pyroglutamic acid aminopeptidase, prepared in example 1;

FIG. 3 is a graph showing the bioluminescence of probe CX-1 according to the present invention in the in vitro assay as a function of the concentration of pyroglutamic acid aminopeptidase;

FIG. 4 is a graph showing the results of selective detection of the probe of the present invention;

FIG. 5 is a bioluminescence map of mice in a blank Control group (Control) and an experimental group (D-Gal induced acute liver inflammation model);

FIG. 6 is a graph showing the quantification of total photon flux (p/sec/cm) in mice (excluding the tail part)²/sr) data are expressed as mean ± SD (n ═ 3);

FIG. 7 is a graph showing the quantification of photon flux (p/sec/cm) at each time point of mice (excluding the tail portion)²/sr) data are expressed as mean ± SD (n ═ 3).

Detailed Description

The raw materials and equipment used in the invention are known products and are obtained by purchasing commercial products.