CN107602643B - Naphthalimide-based β -glucuronidase fluorescent probe and application thereof - Google Patents

Abstract

Naphthalimide-basedβThe invention relates to a fluorescent probe of glucuronidase and application thereof, belonging to the technical field of biological medicine, which can be used for measuring the activity of β -glucuronidase in biological systems of different sources, HC-glu is used as a specific probe reaction substrate, the β -glucuronidase in-vitro reaction system is used, β -glucuronide hydrolysis reaction is used as a probe reaction, and the activity of β -glucuronidase in various biological samples is measured by quantitatively detecting the generation amount of aglycone metabolites in unit time.

Description

Naphthalimide-based β -glucuronidase fluorescent probe and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a fluorescent probe of β -glucuronidase based on naphthalimide and application thereof.

Background

βGlucuronidase (D)βGlucuronidase) is a naturally occurring hydrolase that catalyzes a wide variety of typesβHydrolysis of the glucuronide bond with release of the acidβGlucuronic acid and the corresponding ligands. At present, microorganisms (bacteria such as staphylococcus, lactobacillus, and ruminococcus, penicillium, aspergillus, and yeast) and plants (astragalus, arabidopsis, tobacco, rice, and corn) and animals (human, monkey, dog, and mouse), are foundβ-the presence of glucuronidase. In animals and humans, the enzyme is present in almost all tissues and body fluids, localized in lysosomes and microsomes of cells of these organs, is an eosinophilic lysosomal enzyme, and is involved in the metabolism of endogenous substances, such as steroid hormones and bilirubin. In addition, glycoside compounds widely present in natural products, such as natural products such as glycyrrhizic acid, are also required to be purified byβThe glucuronidase is hydrolyzed into aglycon form, and then can be absorbed and utilized by the body.βGlucuronidase is also present in higher plants and microorganisms, and is involved in physiological activities such as plant biosynthesis and microbial glycolysis.

In recent years, the human body is treated withβThe study of glucuronidase is becoming increasingly widespread.

βThe activity of glucuronidase is evaluated by the consumption of a substrate or the production of a product, and a chromogenic/fluorogenic substrate/product is widely used in the evaluation of the activity of the enzyme due to the convenience of detection and high sensitivity. At present, it has been reportedβThe fluorogenic substrate of glucuronidase is 5-bromo-4-chloro-3-indole-βGlucoside acid ester, hydroxyquinoline-βGlucoside, p-nitrophenol-βGlucoside, 4-methylumbelliferone-βGlucuronide and the like, and colored products such as indole, hydroxyquinoline, p-nitrophenol, 4-methylumbelliferone and the like are respectively used as enzyme activity detection indexes. However, two problems are common to these substrates. Firstly, the selectivity is not high, and the hydrolysis reaction is not only carried outβGlucuronidase hydrolysis, possibly by other enzymes such as esterases and the like; secondly, a chromogenic reaction or coumarin fluorescence is adopted, so that large substrate interference exists, and for example, a complex system such as a biological sample has color or fluorescence with similar wavelength. Therefore, the development of a high selectivity and low interferenceβA specificity fluorescent probe substrate of glucuronidase, and has important application value.

Disclosure of Invention

The invention provides a naphthalimide-based β -glucuronidase fluorescent probe and application thereof, wherein a specific fluorescent probe substrate can be usedβ-selective hydrolysis of glucuronidase to produce a hydrolysate with significantly altered fluorescence properties, which can be detected by a fluorescence detector. The probe reaction can be used in various biological systemsβQuantitative evaluation of the distribution and function of glucuronidase and can be used forβScreening of inhibitors of glucuronidase.

The invention disclosesβThe specific fluorescent probe substrate of glucuronidase is N-N-butyl-4-O-β-D-glucuronic acid-1, 8-naphthalimide (abbreviated as HN-1-glu).

HN-1-glu

The invention also discloses the compound asβApplication of a specific fluorescent probe substrate for glucuronidase: using the above HN-1-glu asβ-a substrate specific for glucuronidase, subjected to hydrolysis reactions, for quantitative determination of different organisms by detecting the amount of hydrolysis products produced per unit of timeSystems (including recombinant expression)βGlucuronidase, human or animal tissue preparation liquid, various tissue cells, microorganisms, plant and other biological systems)β-glucuronidase activity; the specific reaction conditions are as follows:

HN-1-glu is used as a specific probe substrate in the system; the substrate concentration was selected to be 10. mu.M.

-in phosphate buffer, the reaction temperature is between 20 ℃ and 60 ℃, preferably 37 ℃ for optimal reaction time; the pH value of the incubation system is between 5.5 and 10.5, and the optimal reaction pH value is preferably pH 6.0;

the reaction time is 0-60 minutes, and the reaction is terminated when the corresponding hydrolysate of the substrate reaches the quantitative limit and the substrate conversion rate does not exceed 20%;

-determining the amount of substrate reduction or the amount of hydrolysate formed per unit time asβ-an evaluation index of glucuronidase activity.

The invention providesβApplication of a specific fluorescent probe substrate for glucuronidase, the probe substrate and its hydrolysate having different optical properties, and rapid and sensitive detection of the product can be achieved by using a fluorescence detector; the fluorescent detection conditions of the hydrolysate are respectively as follows: the excitation wavelength is 450 nm, and the maximum emission wavelength is 556 nm.

The specific probe substrate is a longer emission wavelength fluorescent probeβThe detection process of the glucuronidase activity is not easily interfered by a biological system matrix and impurities, and can be used for various recombinationsβGlucuronidase, human and animal tissue preparation liquid and various tissue cellsβ-quantitative determination of glucuronidase activity; can also be used as carrier and animal whole bodyβA probe substrate for glucuronidase, evaluationβIndividual and species differences of glucuronidase. The fluorescent detection method of the probe substrate and the hydrolysis metabolite can also be used forβRapid screening of glucuronidase inhibitors and quantitative evaluation of the inhibitory capacity.

As of high specificityβSubstrates for fluorogenic probes of glucuronidase, which compounds can be used for the detectionβGlucuronidase activity, particularly suitable for the production of cloned expression systems for bacteria, insect cells, mammalian cells and yeastsβAssay of glucuronidase Activity, and measurement of the Activity in various biological samples derived from microsomes derived from mammalian tissues and organs, S-9, and the likeβCalibration of the activity of glucuronidase.

According to the inventionβSpecific probe substrate detection of glucuronidaseβThe in vitro activity of glucuronidase has the following outstanding advantages:

(1) high specificity: HN-1-glu can be replaced byβGlucuronidase is metabolized with high specificity to hydrolysis products.

(2) The method is cheap and easy to obtain: HN-1-glu can be obtained by chemical synthesis, the synthesis process is simple and easy to implement, and the detection cost of the fluorescence method is low.

(3) High sensitivity: HN-1-glu has longer fluorescence emission wavelength and can better weaken the interference of biological background fluorescence.

Drawings

FIG. 1 shows emission spectra of HN-1-glu and its enzymatic hydrolysate.

FIG. 2 shows the results of the HN-1-glu screening experiment with different hydrolases.

FIG. 3βResults of experiments for inhibiting hydrolysis of HN-1-glu by glucuronidase.

FIG. 4 different sourcesβResults of experiments on the reaction rate of HN-1-glu catalyzed by glucuronidase.

Detailed Description

Example 1 in vitro determination of the Selectivity of different hydrolases

(1) Preparing a 99 mu L in vitro metabolic reaction system which comprises a phosphate buffer solution (50 mM) with pH 6.0 and different types of hydrolytic enzymes (0.1 mg/mL) in advance, and shaking for pre-incubation for 3 minutes at 37 ℃;

(2) 1 muL of HN-1-glu with the concentration of 1 mM (the final concentration of 10 muM) is added into the reaction system to start the reaction;

(3) after 60 minutes, adding 50 mu L of glacial acetonitrile, and stopping reaction after violent shaking;

(4) by usingAfter high-speed centrifugation at 20,000 × g at 4 ℃ for 20 minutes, the supernatant was subjected to fluorescence detection (HN-1-glu: Ex =450 nm, Em =556 nm)βGlucuronidase (GLU) catalyzed reactions, with much higher rates than other hydrolases, indicatingβThe reaction of-glucuronidase catalysis HN-1-glu has good selectivity, and the invention can be applied toβAssay of the activity of glucuronidase (FIG. 2).

Example 2 in vitroβInhibition assay of glucuronidase

(1) Preparing in advance a 99 muL in vitro metabolic reaction system including a phosphate buffer (50 mM) of pH 6.0,βGlucuronidase (0.1 mg/mL), and various concentrations of inhibitors (50, 200, 600. mu.M final concentration) were pre-incubated at 37 ℃ for 3 minutes with shaking;

(2) 1 muL of HN-1-glu with the concentration of 1 mM (the final concentration of 10 muM) is added into the reaction system to start the reaction;

(3) after 60 minutes, adding 50 mu L of glacial acetonitrile, and stopping reaction after violent shaking;

(4) after centrifuging at 20,000 × g for 20 min at 4 deg.C with a high-speed refrigerated centrifuge, the supernatant was subjected to fluorescence detection (HN-1-glu: Ex =450 nm, Em =556 nm)βThe catalytic reaction of Glucuronidase (GLU), whereas BNPP and LAP (carboxylesterase) and α -galactonase (galactose) do not inhibitβThe catalytic activity of Glucuronidase (GLU), descriptionβThe reaction of-glucuronidase catalysis HN-1-glu has good selectivity, and the invention can be applied toβScreening of inhibitors of glucuronidase (FIG. 3).

Example 3 in different individuals and organsβDetermination of the Activity of glucuronidase

(1) A99 μ L in vitro metabolic reaction system including phosphate buffer (50 mM) at pH 7.4, different sources was prepared in advanceβGlucuronidase (0.1 mg/mL), pre-incubation with shaking at 37 ℃ for 3 minutes;

(2) 1 muL of HN-1-glu with the concentration of 1 mM (the final concentration of 10 muM) is added into the reaction system to start the reaction;

(3) after 60 minutes, adding 50 mu L of glacial acetonitrile, and stopping reaction after violent shaking;

(4) after centrifugation at 20,000 × g at 4 ℃ for 20 minutes in a high-speed refrigerated centrifuge, the supernatant was subjected to fluorescence detection (HN-1-glu: Ex =450 nm, Em =556 nm)βGlucuronidase, including the liver of 16 individual donors and S9 prepared from mixed liver, intestinal, brain and kidney organs, all catalyzing the HN-1-glu reaction, but from a variety of sourcesβGlucuronidase with significantly different reaction rates, indicating HN-1-glu asβSpecific probe substrates for glucuronidase sensitive to different individual, organ originsβThe activity of glucuronidase, the invention can be applied between individuals and organsβEvaluation of glucuronidase Activity (see FIG. 4).

Claims (2)

1. The application of β -glucuronidase fluorescent probe based on naphthalimide for non-diagnosis and non-treatment purposes is characterized in that a probe substrate can be specifically catalyzed by β -glucuronidase to hydrolyze glucuronide bonds in the probe substrate, and the probe is N-N-butyl-4-O-βThe structural formula of the-D-glucuronic acid-1, 8-naphthalimide is shown as a formula (1),

formula (1)

The application of the probe is as follows: adopts N-N-butyl-4-O-βusing-D-glucuronic acid-1, 8-naphthalimide as a specific substrate of β -glucuronidase to carry out hydrolysis reaction, and quantitatively determining the activity of β -glucuronidase by quantitatively detecting the generation rate of an aglycone product in unit time under the conditions that the substrate concentration is between 1/10 and 10 Km, and an incubation bodyThe pH value is 6.0; the reaction temperature is between 20 and 60 ℃, and the elimination rate of the substrate or the generation rate of the product is lower than 20 percent; the enzymolysis product of the probe substrate has fluorescence, the detection condition of the enzymolysis product is excitation wavelength 450 nm, and the maximum emission wavelength is 556 nm.

2. The application of the fluorescent probe based on β -glucuronidase of naphthalimide in non-diagnosis and non-treatment purposes as claimed in claim 1, is characterized in that the probe substrate can also be used for rapid screening of β -glucuronidase inhibitor and quantitative evaluation of inhibition ability:

(1) preparing in advance a 99 μ L in vitro metabolic reaction system comprising 50 mM phosphate buffer solution of pH 6.0, 0.1 mg/mLβGlucuronidase, and inhibitors loperamide, α -galactose, di-p-nitrophenyl phosphate, baicalin and epicatechin gallate at final concentrations of 50, 200, 600 μ M, respectively, at 37 ℃ with shaking for 3 minutes;

(2) adding 1 mu L of N-N-butyl-4-O-ion with the concentration of 1 mM into a reaction systemβ-D-glucuronic acid-1, 8-naphthalimide starting reaction;

(3) after 60 minutes, adding 50 mu L of glacial acetonitrile, and stopping reaction after violent shaking;

(4) centrifuging at 4 deg.C and 20000 × g for 20 min, collecting supernatant, performing fluorescence detection, and detecting with Ex =450 nm and Em =556 nm to show that baicalin is inhibited in dose-dependent mannerβ-catalytic reaction of glucuronidase.

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