阅读说明：本技术 一种可用于亚铁离子高选择性检测的新型荧光探针制备和应用 (Preparation and application of novel fluorescent probe for high-selectivity detection of ferrous ions ) 是由 不公告发明人 于 2019-11-13 设计创作，主要内容包括：本发明涉及一种可用于亚铁离子高选择性检测的新型荧光探针制备和应用,属于分析化学技术领域。本发明的荧光探针是包含N-O功能团的香豆素衍生物,其化学结构如式(I)所示。该荧光探针经2步即可合成,与亚铁离子作用后,在PBS缓冲溶液(10 mM,pH=7.4)中生成具有红色荧光发射能力的产物,从而实现对亚铁离子高选择性检测。此外,本发明的荧光探针还可用于环境和活细胞等不同体系中的亚铁离子的快速检测,具有很好的应用前景。式(I)。(The invention relates to preparation and application of a novel fluorescent probe for high-selectivity detection of ferrous ions, and belongs to the technical field of analytical chemistry. The fluorescent probe is a coumarin derivative containing an N-O functional group, and the chemical structure of the coumarin derivative is shown as a formula (I). The fluorescent probe can be synthesized by 2 steps, and after the fluorescent probe reacts with ferrous ions, a product with red fluorescence emission capability is generated in a PBS (10 mM, pH = 7.4) buffer solution, so that the high selectivity detection of the ferrous ions is realized. In addition, the fluorescent probe can be used for rapidly detecting ferrous ions in different systems such as environment, living cells and the like, and has good application prospect.)

1. A novel fluorescent probe for highly selective detection of ferrous ions, characterized by the formula (I):

formula (I)

The method for preparing a novel fluorescent probe for high-selectivity detection of ferrous ions according to claim 1, wherein the method comprises the following steps: dissolving the coumarin derivatives in absolute ethyl alcohol, adding 2-methylpyridine salt and piperidine, and carrying out water bath reaction at 40 ℃ until the reaction is complete.

2. Evaporating the system to dryness under reduced pressure, purifying by silica gel column chromatography, and evaporating the solvent to dryness under reduced pressure to obtain mauve solid powder.

3. Dissolving the purified product in ethyl acetate 0^oAdding under CmAnd (4) reacting at room temperature to be complete, filtering, concentrating the filtrate, purifying by silica gel column chromatography, and removing the solvent by rotary evaporation to obtain the target molecular probe.

4. Use of the fluorescent probe according to claims 1 and 2 for the detection and quantitative fluorescence analysis of ferrous ions in environmental and biological samples;

use of a fluorescent probe according to any of claims 1 to 3, characterized in that the detection conditions for ferrous ions are: the excitation wavelength is 380 nm, the fluorescence emission spectrum detection is carried out in the range of 400-700 nm, the pH of the detection system is 6.0-8.6, and the solvent of the detection system is PBS buffer solution.

5. The use of claim 4, wherein the sample to be tested is added into the detection solution of the fluorescent probe, if the fluorescence of the solution is red, the sample to be tested contains ferrous ions, and if no obvious red fluorescence exists, the sample to be tested does not contain ferrous ions, and the fluorescence intensity of the determination solution can also be used as an evaluation index of the concentration of the ferrous ions.

Technical Field

The invention relates to a fluorescent probe, in particular to a preparation method of a novel fluorescent probe for high-selectivity detection of ferrous ions, and particularly relates to application of the fluorescent molecular probe in the aspects of ferrous ion detection in environment and organisms, belonging to the technical field of chemical analysis and biological analysis detection.

Background

Iron (iron, Fe) is one of the essential trace elements in the organism, and is also the most abundant transition metal element in the human body. Iron exists in two forms in human body, one is functional iron, accounts for more than 60% of total iron, and exists in erythrocytes mainly in the form of the active center of hemoglobin; the other is iron storage, which exists in the liver, reticuloendothelial cells and bone marrow in the form of ferritin and the like. Iron has redox activity and plays an important role in physiological activities in vivo, such as globulin oxygen transport, cytochrome electron transfer, cytochrome P450 oxidase and ribonucleotide reductase synthesis, and C-H functionalization of non-heme homologues. However, at the same time, the high reactivity also brings about a potential risk, for example, when the iron content is too high, high reactive oxygen species are abnormally generated by the Fenton chemical reaction. Indeed, there have been studies that indicate that excess iron in humans is associated with the development of a number of serious diseases, such as neurodegenerative diseases like alzheimer's disease and parkinson's disease, hepatitis and mesothelioma, and even cancer. When the iron content is too low, diseases such as low immunity, intelligence reduction, nerve dysfunction and the like can be caused, wherein iron deficiency anemia is the most common. Therefore, it is very important and meaningful to develop an analysis method capable of detecting the concentration of iron ions under physiological level conditions for further understanding the physiological and pathological functions thereof.

The fluorescence detection method becomes Fe due to its advantages of high sensitivity, strong specificity, little damage to biological samples, and being applicable to real-time monitoring²⁺Research hotspot of quantitative detection. At present, conventional chelator-based fluorescent probes have been developed, however, due to Fe²⁺The probes exhibit a change in "Turn-Off" fluorescence, and are less specific and may also cause fluorescence quenching due to environmental or other reasons, and thus such fluorescence-quenched probes are not suitable for Fe²⁺The analysis and detection of (2). On the contrary, the fluorescent molecular probe based on the specific chemical reaction can effectively overcome the defects, thereby having wider application prospect. Among them, fluorescent probes based on N-oxide reduction reaction have been the focus of attention of researchers due to their high selectivity. For example, CN 105985299B reports a benzothiazole ethylene fluorescent probe containing N-O functional group, which is respectively reacted with Fe³⁺Potassium ion, calcium ion, magnesium ion, sodium ion, aluminum ion and other ions in human body can not cause obvious change of fluorescence spectrum, thereby realizing Fe²⁺Has high specificity, and the needle can carry out Fe under the physiological level condition²⁺The measurement of (1). CN 109776564A also reports a fluorescent probe based on xanthene structure, and ferrous ions can reduce N-oxide to hydroxyl throughThe hydrolysis of the intracellular esterase generates a fluorescent substance with high fluorescence emission capability, the fluorescence emission at 630 nm is obviously enhanced, and the concentration of ferrous ions in cells can be measured by detecting the fluorescence intensity before and after response. However, all of the above fluorescent molecular probes have a problem of poor water solubility, and are not suitable for use in vivo Fe²⁺And (6) detecting. Therefore, fluorescent probes with long wavelength, high selectivity and good water solubility are developed, so that the fluorescent probes are more suitable for Fe in the environment and in the living body²⁺Detection becomes an important direction for the development of the probe in the future.

Disclosure of Invention

Fe against existing assay²⁺The fluorescent probe has the defects that the fluorescent probe has good water solubility and can realize Fe²⁺The fluorescent molecular probe for high specificity detection.

The second purpose of the invention is to provide a method for preparing the fluorescent molecular probe, which is simple to operate and has easily available raw materials.

The third purpose of the invention is to provide Fe in aqueous solution and organism by the fluorescent molecular probe²⁺The detection application of (1).

In order to achieve the technical purpose, the invention provides a fluorescent probe, which has a structure shown in formula I:

formula I

The preparation method of the fluorescent probe is preferably as follows:

dissolving coumarin derivatives (compound 1) in absolute ethyl alcohol, adding 2-methylpyridine salt and piperidine, and reacting in water bath at 40 ℃ until the reaction is complete. Evaporating the system to dryness under reduced pressure, purifying by silica gel column chromatography, and evaporating the solvent to dryness under reduced pressure to obtain mauve solid powder. Dissolving the purified product in ethyl acetate 0^oAdding under CmAnd (4) reacting at room temperature to be complete, filtering, concentrating the filtrate, purifying by silica gel column chromatography, and removing the solvent by rotary evaporation to obtain the target molecular probe.

The synthesis of the invention is as follows:

the invention provides application of the fluorescent probe, which can be applied to Fe²⁺Detection of (3). The detection principle of the probe is as follows: fe²⁺The N-oxide in the probe can be reduced to generate a fluorescent substance with strong fluorescence emission, which can be obtained by adding Fe²⁺Front and rear fluorescence intensity changes to achieve Fe²⁺Detection of (3). The detection mechanism is shown in the figure:

the invention provides a method for measuring Fe by using the fluorescent probe²⁺A method. The determination method comprises the following steps: under the condition of room temperature, the fluorescent probe is dissolved in PBS buffer solution, and solution prepared by acetonitrile, dichloromethane or dimethyl sulfoxide and the PBS buffer solution according to a certain proportion is prepared, and the concentration of the fluorescent probe is configured to be 10 mu M-40 mu M. Different concentrations of Fe²⁺Adding the aqueous solution into a probe system, measuring the fluorescence intensity of the solution, and determining the fluorescence intensity of the solution according to the fluorescence intensity and Fe²⁺The linear relationship of the concentrations is realized for Fe²⁺And (4) carrying out quantitative detection.

In the above detection method, preferably, the solvent system is PBS buffer solution.

Preferably, the pH of the detection method is 7.4.

In the above detection method, the concentration of the fluorescent probe is preferably 10. mu.M.

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

(1) the fluorescent probe has the advantage of good selectivity, namely NaCl, KCl and MgCl₂，CaCl₂，MnCl₂，ZnCl₂，Pb(NO₃)₂，CuCl₂，AgNO₃And FeCl₃Equi-para Fe²⁺None of the detections of (2) interfered.

(1) The probe has stronger red light emission, can effectively avoid the interference of biological autofluorescence, has good cell membrane permeability and low cytotoxicity, and can be used for Fe²⁺OfThe object imaging has stronger practical application value in the life science field.

(2) The fluorescent probe provided by the invention is simple in preparation method, low in cost, free of complex instruments, and beneficial to large-scale production, so that the fluorescent probe is suitable for popularization and application.

Drawings

FIG. 1 shows fluorescence intensity of the fluorescent probe according to Fe in the practice of the present invention²⁺Emission spectrum of concentration change;

FIG. 2 shows a fluorescent probe pair containing Fe in the practice of the present invention²⁺A selectivity profile of (a);

FIG. 3 is a photograph of fluorescent confocal images of fluorescent probes in HepG2 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 2

Compound 1 (538.60 mg, 2 mmol), 2-methylpyridine salt (470.12 mg, 2.4 mmol) was weighed out and dissolved in absolute ethanol, and 1 drop of piperidine was added and reacted in a water bath at 40 ℃ until completion. The system was evaporated to dryness under reduced pressure, purified by silica gel column chromatography, and the solvent was evaporated to dryness under reduced pressure to give 770.36 mg of a purple solid powder with a yield of 79.2%.¹H NMR (CDC1₃, 400 MHz) δ 1.97-1.99 (m, 4H), 2.76-2.79 (t, 2H, J=6.4 Hz), 2.88-2.90 (t, 2 H, J= 6.4 Hz), 3.35-3.39 (m, 4H), 4.38 (s, 3H), 6.64(d, 1H, J=8.2 Hz), 6.85(d, 1H, J=8.2 Hz), 6.98 (s, 1H), 7.29 (d, 1H, J=7.8 Hz), 7.66 (t, 1H, J=7.9 Hz), 7.83 (s, 1H), 8.10 (t, 1H, J=8.1 Hz), 8.93 (d, 1H, J=8.2 Hz)。

Synthesis of target molecular probes

Compound 2 (486.35 mg, 1 mmol) was dissolved in 10 mL ethyl acetate and then at 0^oAdding under CmCPBA (172.57 mg, 1 mmol), reaction to completion at room temperature, filtration and concentration of the filtrate. Purification by silica gel column chromatography and evaporation of the solvent under reduced pressure gave 681.17 mg of a solid powder with a yield of 67.8%.¹H NMR (CDC1₃, 400 MHz) δ 2.34-2.36 (m, 4H), 2.76-2.78 (m, 4H), 3.32-3.35 (m, 4H), 4.39 (s, 3H), 6.64(d, 1H, J=8.2 Hz), 6.85(d, 1H, J=8.2 Hz), 7.29 (d, 1H, J=7.8 Hz), 7.56 (s, 1H), 7.65 (t, 1H, J=8.0 Hz), 7.81 (s, 1H) 8.11 (t, 1H, J=8.3 Hz), 8.91 (d, 1H, J=8.3 Hz)。HRMS-ESI (C₂₃H₂₃IN₂O₃) m/z: calc. for [M-I]⁺: 375.4398; found：375.3812。

Example 2

Preparation of fluorescent probe mother liquor

The product isolated above and having a purity of 99% is accurately weighed at 5.02 mg and carefully transferred into a 50 mL volumetric flask, to which CH is added at room temperature₃CN solution is dissolved completely, and the volume is determined to the scale mark, thus obtaining the probe mother liquor with the concentration of 1 mM. During the test, 20. mu.L of the above solution was taken out by a micro-injector each time, and dissolved in the test system so that the total volume per test was 2 mL, at which time the concentration of the fluorescent probe was 10. mu.M.

Example 3

Fe²⁺Preparation of mother liquor

Fe²⁺5 mL of stock solutions were prepared in different concentration gradients (0.1 mM, 0.2 mM, 0.3 mM, 0.5 mM, 1 mM, 1.5 mM, 2.0 mM, 3.0 mM) using PBS buffer. The other tests required the use of metal ions, respectively, in PBS buffer solution to make 3 mM stock solution.

Example 4

Fluorescent intensity of fluorescent probe and Fe²⁺Relation of concentration

4.900 mL of PBS buffer solution was measured, 50. mu.L of 1 mM probe stock solution was dissolved therein, and 50. mu.L of Fe of different concentrations was transferred²⁺In the mother liquor, the concentration of the probe of the whole detection system is 10 mu M and Fe²⁺The concentrations of (A) were 1. mu.M, 2. mu.M, 3. mu.M, 5. mu.M, 10. mu.M, 15. mu.M, 20. mu.M and 30. mu.M, respectively. After incubation at room temperature for 20 min, the fluorescence spectra of the different systems were tested in 10 mm cuvettes, respectively (FIG. 1). The results show that with Fe²⁺The concentration is gradually increased, and the fluorescence emission intensity of the system at 597 nm is gradually increased.

Example 5

Fluorescent probe pair Fe²⁺Selectivity of detection

50. mu.L of 1 mM probe stock solution was dissolved in 4.900 mL PBS buffer solution, and 50. mu.L of 3 mM NaCl, KCl, MgCl were pipetted₂，CaCl₂，MnCl₂，ZnCl₂，Pb(NO₃)₂，CuCl₂，AgNO₃And FeCl₃Adding the mother liquor into the system, incubating at room temperature for 20 min, measuring the fluorescence spectra, and recording the fluorescence intensity at 597 nm, with the result shown in figure 2. The results show that only Fe was added²⁺When the fluorescent probe is added with other test metal ions, the fluorescence of the fluorescent probe is obviously enhanced, and no or only weak fluorescence change exists. The fluorescent probe is shown to have good selectivity.

Example 6

Fluorescent probes for Fe in cells²⁺Response to (2)

10 μ M of fluorescent probe solution was added to HepG2 medium and placed 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 replaced with FeCl₂The buffer solution (50. mu.M) was incubated for 30 minutes, washed three times with 0.1M PBS buffer (10 mM, pH = 7.4), and the change in fluorescence was observed under a fluorescence microscope, and the results are shown in FIG. 3. Experiments show that the probe molecules and Fe entering the cell body²⁺In response, strong red fluorescence is emitted, and the fluorescent probe thus targets Fe in the cell²⁺Has good imaging effect, and can be used for detecting Fe in organism²⁺。

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.