CN113004310A

CN113004310A - Preparation method and application of hydrogen peroxide ratio type fluorescent molecular probe based on DCPO parent nucleus

Info

Publication number: CN113004310A
Application number: CN201911322421.5A
Authority: CN
Inventors: 曾文彬; 李石; 陈小洪
Original assignee: Hunan Chaoji Chemical Technology Co ltd
Current assignee: Hunan Chaoji Testing Technology Co ltd
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2021-06-22
Anticipated expiration: 2039-12-20
Also published as: CN113004310B

Abstract

The invention discloses a preparation method and application of a hydrogen peroxide ratio type fluorescent molecular probe based on DCPO parent nucleus, wherein the chemical structural formula is as follows:

Description

Preparation method and application of hydrogen peroxide ratio type fluorescent molecular probe based on DCPO parent nucleus

Technical Field

The invention relates to a preparation method and application of a hydrogen peroxide ratio type fluorescent molecular probe based on a DCPO parent nucleus, belonging to the technical field of analytical chemistry.

Background

Reactive Oxygen Species (ROS) is a generic name for molecules or ions with active chemical properties and high oxidation activity, and plays an important role in regulating physiological functions and pathological processes. Hydrogen peroxide (hydrogen peroxide) is an important component of the active oxygen family and has been of interest and research because of its relatively mild reactivity in biological systems. Studies have shown that hydrogen peroxide is a product of most oxidase processes and is involved in regulatory processes in the body of organisms. Therefore, it has various physiological and pathological effects within living cells according to the degree, time and location of its production. Under normal physiological concentration, hydrogen peroxide is involved in reversible oxidation of intracellular proteins, and regulates cellular processes from phosphorylation of proteins to gene expression. Excess hydrogen peroxide is an important mediator of the pathological processes of various diseases. Such as an increase in its rate of angiogenesis, contributes to the development of inflammation. Meanwhile, abnormal hydrogen peroxide concentrations in vivo have been shown to be associated with alzheimer's disease, parkinson's disease and cancer. Therefore, the development of sensitive and selective methods for detecting and quantifying hydrogen peroxide is very important for further understanding and elucidating its biological functions, thereby enhancing the research on the prevention, diagnosis and pathology of related diseases.

At present, methods such as an electrochemical method and an element analysis mass spectrum can be used for detecting hydrogen peroxide. However, these methods require complicated sample preparation and manipulation procedures and can damage cellular and tissue structures, limiting their further use in vivo. In contrast, fluorescence analysis is receiving attention because of its advantages such as high sensitivity, high selectivity, rapid response, and the ability to perform real-time visual monitoring.

CN 106928263B reports a reactive hydrogen peroxide fluorescent probe compound, which has almost no fluorescence in a mixed solvent of DMSO and water with pH =7.4, and the fluorescence of the probe solution rapidly increases at 495 nm after the hydrogen peroxide is added, while the fluorescence of the solution is not significantly changed after other active oxygen species are added, thereby realizing the specific detection of hydrogen peroxide. However, the emission wavelength of the probe is short, so that the biocompatibility of the probe is poor, the interference of background fluorescence in an organism is large, and the function of hydrogen peroxide in a cellular process and a biological growth and development process cannot be well revealed. Meanwhile, compared with an enhanced fluorescent probe, the ratiometric fluorescent probe has the characteristic of dual-wavelength emission (or excitation), and the change of the wavelength ratio value is independent of the concentration of the probe and the intensity of a light source, so that the interference of other detection conditions can be greatly reduced. Therefore, it is very necessary to develop a ratiometric near-infrared fluorescent molecular probe capable of specifically recognizing hydrogen peroxide.

Disclosure of Invention

Aiming at the defects of the existing hydrogen peroxide fluorescent molecular probe, the invention aims to provide a ratio type near-infrared fluorescent molecular probe with good water solubility, high specificity and strong sensitivity.

The second purpose of the invention is to provide a high-efficiency preparation method of the fluorescent molecular probe.

The third purpose of the invention is to provide the application of the fluorescent molecular probe in detecting hydrogen peroxide in aqueous solution and organisms.

In order to achieve the purpose, the invention adopts the technical scheme that:

the invention provides a hydrogen peroxide ratio type fluorescent molecular probe based on a DCPO parent nucleus, wherein the structural formula of the fluorescent molecular probe is shown as a formula I:

formula I

The preparation method of the hydrogen peroxide fluorescent molecular probe comprises the following steps:

(1) adding DCPO and hexamethyl-hydroxylamine into a trifluoroacetic acid solution, heating and refluxing until the reaction is finished, cooling, pouring into ice water, adjusting to be nearly neutral by using a 1 mol/L NaOH solution, carrying out suction filtration, collecting and washing a filter cake, carrying out vacuum drying, and carrying out column chromatography purification to obtain a compound 1;

(2) dissolving the purified product in absolute ethyl alcohol, adding 2-aminothiophenol, 30% hydrogen peroxide and 37% HCl, after the reaction at room temperature, pouring into ice water, carrying out suction filtration, collecting and washing a filter cake, and drying to obtain a compound 2;

(3) the purified product, the p-bromomethyl phenylboronic acid pinacol ester and K₂CO₃Dissolving in anhydrous DMF solution, heating and refluxing to complete reaction, pouring into water, and respectively usingWater, saturated NaHCO₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain the target molecular probe.

The synthesis route of the hydrogen peroxide fluorescent probe is as follows:

the invention also provides application of the hydrogen peroxide molecular fluorescent probe, and the fluorescent probe can be applied to sensing and detecting the content of hydrogen peroxide in a water environment and a biological cell system. The detection principle of the probe is as follows: by utilizing an excited-state intramolecular proton transfer (ESIPT) mechanism and utilizing the ESIPT function of the probe hindered by benzoxy pinacol borate, the probe only generates enol emission, and the ESIPT function of the probe is recovered after hydrogen peroxide is added, so that the probe recovers the keto emission, and the ratiometric detection of the hydrogen peroxide is finally realized. The detection mechanism is as follows:

further, the specific method for measuring hydrogen peroxide in the water environment comprises the following steps: the fluorescent probe was dissolved in a PBS buffer solution (10 mM, pH =7.4) at room temperature, and was configured to have a concentration of 10 μ M. Adding hydrogen peroxide water solutions with different concentrations into the system, respectively measuring the fluorescence intensity, and realizing the quantitative detection of the hydrogen peroxide through the linear relation between the fluorescence intensity and the hydrogen peroxide concentration.

Further, the specific method for measuring hydrogen peroxide in the biological cell system comprises the following steps: adding 10 μ M fluorescent probe solution into HeLa medium, and placing at 37^oC, 5% CO₂After incubation in the incubator for 30 minutes, the cells were washed three times with 0.1M PBS buffer (10 mM, pH =7.4) to remove probe molecules that did not enter the cells, and then the medium was replaced and buffered with hydrogen peroxideThe solution (30. mu.M) was incubated for 30 minutes, and the fluorescence was measured, and the presence or absence of hydrogen peroxide was determined from the change in fluorescence.

Compared with the prior art, the technical scheme of the invention has the beneficial technical effects that:

the fluorescent molecular probe for detecting hydrogen peroxide has the following advantages:

(1) the fluorescent probe has the advantages of stable and high chemical property and optical stability, and has no autoxidation and no photobleaching phenomenon;

(2) the fluorescent probe can realize the ratiometric identification of hydrogen peroxide, has the characteristic of dual-wavelength emission (or excitation), and the change of the wavelength ratio value is independent of the concentration of the probe and the intensity of a light source, so that the interference of other detection conditions can be greatly reduced, and the detection precision is improved;

(3) the fluorescent probe has the advantages of good water solubility, quick response and high specificity, can avoid the interference of other objects to be detected, is favorable for quickly detecting hydrogen peroxide in the environment, and has stronger practical application value in the field of environmental science.

(4) The probe has stronger red light emission, can effectively avoid the interference of biological autofluorescence, has good cell membrane permeability and small cytotoxicity, can be used for biological imaging of hydrogen peroxide, and has stronger practical application value in the field of life science.

Drawings

FIG. 1 is a graph showing the emission spectrum of the fluorescence intensity of the fluorescent probe varying with the concentration of hydrogen peroxide in the practice of the present invention;

FIG. 2 shows the ratio of fluorescence intensity of the fluorescent probe in the practice of the present invention (I)_{695 nm}/I_{510 nm}) And hydrogen peroxide concentration, the abscissa of the graph is the hydrogen peroxide concentration, and the ordinate is the fluorescence intensity ratio (I)_{695 nm}/I_{510 nm})；

FIG. 3 is a graph showing the selectivity of a fluorescent probe for hydrogen peroxide in the practice of the present invention;

FIG. 4 is a confocal image of fluorescence of a fluorescent probe in HeLa cells in the practice of the present invention.

Detailed Description

The following embodiments are intended to further illustrate the present invention and are not intended to limit the present invention.

Example 1

Synthesis of Compound 1

DCPO (936.9 mg, 3 mmol) and hexamethyl-hydroxylamine (1400 mg, 10 mmol) were added to a 25 mL round-bottom flask at room temperature, 25 mL trifluoroacetic acid was added to the reaction system, and the temperature was raised to 90%^oAnd C, heating and refluxing, after the reaction is finished, taking down the reaction, cooling, pouring the cooled reaction product into 500 mL of ice water, adjusting the pH value to be about 6 by using a 1 mol/L NaOH solution, separating out a large amount of precipitate, carrying out suction filtration, collecting and washing a filter cake, carrying out vacuum drying, and purifying by column chromatography to obtain 810.6 mg of a product, wherein the yield is 79.4%.¹H NMR(400 MHz, DMSO-d₆, ppm):δ 10.36 (s, 1H), 7.89-7.91 (m, 1H), 7.79 (d, 1H, J =7.6 Hz), 7.72(d, 1H, J=16.0 Hz), 7.89-7.62 (m, 3H), 7.28 (d, 1H, J=16.0 Hz,), 6.98 (s, 1H), 6.85 (d, 1H, J=8.8 Hz), 5.56 (s, 1H), 5.35(s, 1H)。

Synthesis of Compound 2

Dissolving the compound 1 (680.6 mg, 2 mmol) in 20 mL of absolute ethanol, adding 2-aminothiophenol (300 mg, 2.4 mmol), 30% hydrogen peroxide (1 mmol) and 37% HCl (1 mmol) into the system, after the reaction is finished, pouring the reaction system into ice water, performing suction filtration, collecting and washing a filter cake, and drying to obtain 646.0 mg of solid with the yield of 72.5%.¹H NMR(400 MHz, DMSO-d₆, ppm):δ=8.18(d, 1H, J=8.6 Hz), 8.01 (d, 1H, J=8.4 Hz), 7.78 (s, 1H), 7.56(t, 2H, J=8.9 Hz), 7.52(d,1H, J=7.8 Hz ), 7.26-7.24 (m, 2H), 7.16 (t, 1H, J=8.4 Hz), 7.08 (d, 1H, J=16.0 Hz), 6.85 (d, 1H, J=8.8 Hz), 5.56 (s, 1H), 5.35(s, 1H)。

Synthesis of target molecular probes

Compound 2 (445.5 mg, 1 mmol) and p-bromomethylbenzeneboronic acid pinacol ester (297.0 mg, 1 mmol) were dissolved in 5mL DMF, and K was added₂CO₃(150 mg, 1.12 mmol) and heated to reflux until the reaction was complete. GetReacting, pouring into water, and respectively using water and saturated NaHCO₃Extracting the solution with saturated saline solution, combining organic layers, drying with anhydrous magnesium sulfate, concentrating, and purifying by column chromatography to obtain target molecular probe 451.9 mg with a yield of 68.3%.¹H NMR(400MHz, DMSO-d₆, ppm):δ=8.17(d, 1H, J=8.6 Hz), 8.02 (d, 1H, J=8.4 Hz), 7.84 (s, 1H), 7.68 (d, 2H, J=8.2 Hz), 7.58(d,1H, J=7.8 Hz ), 7.53(t, 2H, J=8.8 Hz), 7.36-7.32 (m, 3H), 7.21(d, 1H, J=8.0 Hz), 7.16 (t, 1H, J=8.4 Hz), 7.00-6.96(m,2H), 6.93 (d, 1H, J=16.0 Hz), 6.82 (d, 1H, J=8.8 Hz), 5.66 (s, 1H), 5.16 (s, 2H), 1.24(s, 12H)。HR-MS (ESI), m/z: calculated [M+H]⁺: 662.22066, found:662.23196.

Example 2

Experiment of relationship between fluorescence intensity of fluorescent probe and concentration change of hydrogen peroxide

661.5 mg of the target fluorescent molecular probe having a purity of 99% or more prepared in example 1 was accurately weighed, transferred into a 50 mL volumetric flask, and dissolved in an acetonitrile solution to prepare a 1 mM probe mother solution. mu.L of the resulting solution was taken out of the mother liquor, added to a 5mL centrifuge tube, and different concentrations of a standard solution of hydrogen peroxide were added, diluted with a PBS buffer solution (10 mM, pH =7.4), adjusted to a concentration of 10. mu.M containing the probe compound in the solution, and the concentrations of hydrogen peroxide were 1. mu.M, 3. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M, 30. mu.M, and 40. mu.M, respectively, and after incubation at room temperature for 20 min, the fluorescence spectra of the different systems were measured in 10 mM cuvettes, respectively. The fluorescence spectrum is shown in FIG. 1. The result shows that the fluorescence emission intensity of the system at 510 nm gradually decreases with the increase of the concentration of the added hydrogen peroxide, a new fluorescence emission peak is generated at 695 nm, and the fluorescence intensity gradually increases with the increase of the concentration of the hydrogen peroxide. Meanwhile, a linear fit graph (FIG. 2) for detecting hydrogen peroxide was prepared, and the results showed that I was measured when the concentration of hydrogen peroxide was in the range of 0 to 15. mu.M_{695 nm}/I_{510 nm}The value of (a) has a good linear relation with the concentration of the hydrogen peroxide, the regression linear equation is y =0.1402x +0.1515, and the linear correlation coefficient is: 0.9927, and calculatesThe limit of detection (LOD) was 0.13 μ M (S/N ═ 3), indicating that the fluorescent probe had good sensitivity.

Example 3

Selectivity of fluorescent probes for hydrogen peroxide

The fluorescent probe prepared in example 1 was mixed with six active oxygen species (O)₂-, NO, ClO-, tert-butyl peroxy-alcohol, HO-, NO₃-), two mercaptoamino acids (L-cysteine, L-glutathione), a metal ion (Fe)³⁺) And respectively responding and testing the response condition. In order to ensure comparability of experimental results, the whole reaction system is set at 25^oC (10 mM, pH =7.4), the concentration of the probe molecules in the whole detection system is 10 mu M, the concentrations of other compounds to be detected are respectively 30 mu M of hydrogen peroxide, the concentrations of the other compounds are all 100 mu M, after incubation for 20 min at room temperature, the fluorescence spectra of different systems are respectively detected, the fluorescence intensity at 695 nm and 510 nm is recorded, and I is calculated_{695 nm}/I_{510 nm}The value of (c). As shown in FIG. 3, it was found that the fluorescence of the fluorescent probe was significantly changed only by the addition of hydrogen peroxide, while the probe was slightly changed or not changed by the addition of other test substances. The fluorescent probe is shown to have good selectivity and can effectively avoid the interference of other active molecules.

Example 8

Response of fluorescent probes to hydrogen peroxide in cells

First, a probe solution was added to HeLa medium and the mixture was placed in a bath of 37^oC, 5% CO₂Incubating in an incubator for 30 min under the atmosphere, washing with PBS buffer solution for three times to remove probe molecules which do not enter cells,

the cells were transferred to a fluorescence microscope for imaging, the medium was changed, the cells were further incubated with hydrogen peroxide buffer (100. mu.M) for 30 min, washed three times with PBS buffer, and the fluorescence change was observed under a fluorescence microscope, as shown in FIG. 4. Experiments show that the probe molecules entering the cell body react with the hydrogen peroxide, so that the fluorescent probe has a good imaging effect on the hydrogen peroxide in the cell and can be used for detecting the hydrogen peroxide in the organism.

Although the present invention has been described with reference to the specific embodiments shown in the drawings, it is not intended to limit the scope of the present invention, and various modifications or variations can be made by those skilled in the art from the disclosure of the present invention without inventive efforts.

Claims

1. A hydrogen peroxide ratio type fluorescent molecular probe based on DCPO parent nucleus is characterized by having a structure shown in a formula (I):

formula I.

2. The method for preparing a novel fluorescent probe for hydrogen peroxide detection according to claim 1, comprising the following steps:

adding DCPO and hexamethyl-nitril into trifluoroacetic acid solution, 90%^oC, heating and refluxing until the reaction is finished, cooling, pouring into ice water, adjusting to be nearly neutral by using 1 mol/L NaOH solution, performing suction filtration, collecting and washing a filter cake, performing vacuum drying, and performing column chromatography purification to obtain a compound 1, wherein the structural formula of the compound 1 is as follows:

dissolving the purified product in absolute ethyl alcohol, adding 2-aminothiophenol, 30% hydrogen peroxide and 37% HCl, after the reaction is finished at room temperature, pouring into ice water, performing suction filtration, collecting, washing a filter cake, and drying to obtain a compound 2, wherein the structural formula of the compound 2 is shown as follows:

the purified product, the p-bromomethyl phenylboronic acid pinacol ester and K₂CO₃Dissolving in anhydrous DMF solution, heating and refluxing to complete reaction, pouring into water, and adding water and saturated NaHCO respectively₃Extracting the solution with saturated saline solution, combining organic layers, drying, concentrating, and purifying by column chromatography to obtain the target molecular probe.

3. The hydrogen peroxide fluorescent molecular probe as claimed in claims 1 and 2, which is O-tolerant₂-, NO, ClO-, tert-butyl peroxy-alcohol, HO-, NO₃-, L-cysteine, L-glutathione, Fe³⁺The interference of (2).

4. Use of the fluorescent probe according to claims 1-3 for the detection of hydrogen peroxide in environmental systems and biological sample systems.