阅读说明：本技术 一种检测***根的近红外荧光探针的制备方法及应用 (Preparation method and application of near-infrared fluorescent probe for detecting selenious acid roots ) 是由 *** 张迪 刘继红 李漫 王铁良 王红旗 徐孟生 王允 曹成 王俊艳 郑嘉 于 2019-09-26 设计创作，主要内容包括：本发明属于亚硒酸跟(SeO<Sub>3</Sub><Sup>2-</Sup>)检测领域,涉及一种检测亚硒酸根的近红外荧光探针的制备方法及应用。通过催化剂EDC和DMAP对乙酰丙酸进行活化,该活化产物与丙二腈异佛尔酮类物质中间体1(2-[3-(4-羟基苯乙烯基)-5,5-二甲基-环己-2-烯-1-亚基]丙二腈)进行缩合酰化反应,得到一种基于丙二腈异佛尔酮类的化合物,该化合物在乙醇-水溶液中可以用做亚硒酸根的高选择性荧光探针,其荧光发射波长在650 nm以上,实现了近红外荧光光谱识别。结果表明：探针SeP1对亚硒酸根具有高效专一的选择性,具有较强的抗干扰能力。该探针对亚硒酸根的最低检测限为0.068μM,具有应用于环境中亚硒酸根的检测的实用价值。(The present invention belongs to selenious acid heel (SeO) 3 2‑ ) The detection field relates to a preparation method and application of a near-infrared fluorescent probe for detecting selenious acid roots. Activating levulinic acid by catalysts EDC and DMAP, and reacting the activated product with malononitrile isophorone intermediate 1 (2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene)]Malononitrile) to obtain a compound based on malononitrile isophorone which can be used as a high choice for selenite in ethanol-water solutionThe fluorescence emission wavelength of the sex fluorescent probe is above 650 nm, and near-infrared fluorescence spectrum recognition is realized. The results show that: the probe SeP1 has high-efficiency and specific selectivity on selenite and strong anti-interference capability. The lowest detection limit of the probe to selenite is 0.068 mu M, and the probe has practical value of being applied to detection of selenite in the environment.)

1. A near-infrared fluorescent probe molecule for detecting selenite roots is characterized in that the structural formula of the fluorescent probe is as follows:

。

2. The method for preparing the near-infrared fluorescent probe molecule for detecting selenite root as claimed in claim 1, which comprises the following steps:

(1) Dissolving levulinic acid, EDC and DMAP in an anhydrous dichloromethane solution, and stirring and reacting at room temperature for a period of time to obtain a solution I;

(2) Dissolving the intermediate 1 in an anhydrous dichloromethane solution to obtain an intermediate 1 solution, then dropwise adding the intermediate 1 solution into the solution I obtained in the step (1) at room temperature, and reacting for a period of time after dropwise adding to obtain a solution II;

(3) and (3) washing the solution II obtained in the step (2) with a saturated sodium chloride solution, drying the obtained organic phase with anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, and separating by silica gel column chromatography to obtain a product SeP1, namely the fluorescent probe molecule.

3. The method of claim 2, wherein: in the step (1), the molar ratio of the levulinic acid to the EDC to the DMAP is 1 (0.5-4) to 0.1-2, and the reaction is carried out for 10-60 minutes under stirring at room temperature.

4. the method of claim 2, wherein: in the step (2), the intermediate 1 is 2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene ] malononitrile, the molar ratio of the levulinic acid in the solution I to the intermediate 1 is 1 (0.5-2), and the reaction time is 8-24 hours after the dropwise addition is finished.

5. The method of claim 2, wherein: the silica gel column chromatography separation in the step (3) adopts ethyl acetate and petroleum ether eluent with the volume ratio of 1 (3-12), and the yield is 40-80%.

6. The near-infrared fluorescent probe molecule of claim 1, applied to the field of high-sensitivity and specific detection of selenious acid root.

Technical Field

The present invention belongs to selenious acid heel (SeO)₃ ^2-) The detection field relates to a preparation method and application of a near-infrared fluorescent probe for detecting selenious acid roots.

Background

Selenium is one of essential trace elements in human body, participates in the synthesis of various selenium-containing enzymes and selenium-containing proteins in human body, and has various physiological functions of resisting oxidation, resisting tumor and the like. Due to the important function of selenium, the proper amount of selenium supplement for human bodies can play roles in preventing organ aging and pathological changes, delaying aging, enhancing immunity and resisting harmful heavy metals.

Selenium exists in nature in two forms of organic selenium and inorganic selenium. The organic selenium is formed by combining selenium with amino acid through biotransformation, mainly exists in the form of selenomethionine, participates in the synthesis of protein in a living body, and plays important functions of resisting oxidation and enhancing the immunity of the human body. The inorganic selenium mainly refers to sodium selenate and sodium selenite, and has low biological effectiveness, high toxicity and easy environmental pollution. The toxicity of inorganic selenium is obviously higher than that of organic selenium, and the toxicity of selenite is slightly higher than that of sodium selenate. Therefore, the method has important significance for detecting and evaluating selenium with different forms, particularly inorganic selenium.

At present, the inorganic selenium form analysis method mainly adopts a method of combining chromatographic separation and atomic fluorescence technology, but the method has high requirements on instruments and equipment, has strict requirements on the technical level of operators, needs expensive reagents and has high cost, and is not beneficial to wide popularization.

The organic fluorescent molecular sensing technology developed in recent years is applied to the detection of various ions due to its advantages of high sensitivity, high selectivity, no need of separation, easy observation, etc., and also becomes a popular research field. Therefore, it becomes very significant to design fluorescent probes for rapid detection of selenous acid heel. The fluorescent probe has the advantages of good selectivity, high sensitivity, simple and rapid operation, less damage to a detected object and the like, and is widely applied to the aspects of detecting metal cations, anions, active small molecules in organisms and the like in environments and biological systems. The fluorescent probe detection method not only makes up the defects of large sample reagent amount requirement, difficult real-time online analysis, complicated steps, unsuitability for biological and toxicological researches and the like of the traditional molecular and ion analysis method, but also has more attractive advantages compared with the traditional detection method. The research on fluorescent probes has grown considerably over the past decades; patent 201510178114.X discloses a malononitrile isophorone copper ion fluorescent probe and a preparation method thereof, the probe can selectively identify copper ions in a buffer solution, and the copper ions are represented by low detection limit (only 0.2 mu M), large Stokes displacement, fluorescence enhancement, long-wavelength emission and color change under visible light, and the copper ions can be detected by naked eyes by using the fluorescence enhancement and the color change under the visible light; the fluorescence enhancement response of the fluorescent probe to the copper ions can be used for carrying out qualitative or quantitative analysis on the copper ions; but the method is mainly used for researching the quantitative and qualitative analysis of cation-copper ions, the lowest detection limit of the probe is higher, and a recognition group in the chemical structure of the probe cannot be used for detecting anions, so that the probe cannot be applied to detecting the anion selenite; patent 201310125465.5 discloses a method for detecting selenate ion content and reducing to selenite ion online, which is used to reduce hexavalent selenate ion online to tetravalent selenite ion, thereby realizing liquid chromatography/ion chromatography and atomic fluorescence combined morphological analysis method of selenate ion high-sensitivity steam generation-atomic fluorescence detection, the patent detects selenite ion by chromatography and atomic fluorescence combined analysis, the detection limit of the method can not reach the requirement of detecting selenite in the environment, and the detection method is complex, not beneficial to real-time rapid detection, the fluorescence probe analysis method adopted by the invention directly detects tetravalent selenite ion, and is essentially different from the patent methods; therefore, the development prospect of developing fluorescent probes for recognizing inorganic selenium with high sensitivity and high selectivity is still a huge challenge and brings huge returns.

Disclosure of Invention

In order to solve the technical problems, the invention provides a preparation method and application of a near-infrared fluorescent probe for detecting selenite, levulinic acid is activated through catalysts EDC and DMAP, the activated product and malononitrile isophorone intermediate 1 (2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene ] malononitrile) are subjected to condensation acylation reaction to obtain a compound based on malononitrile isophorone, the compound can be used as a high-selectivity fluorescent probe for selenite in ethanol-water solution, the fluorescence emission wavelength of the compound is above 650 nm, and near-infrared fluorescence spectrum identification is realized.

The technical scheme of the invention is realized as follows:

A near-infrared fluorescent probe molecule for detecting selenite roots is disclosed, wherein the structural formula of the fluorescent probe is as follows:

。

the method for preparing the near-infrared fluorescent probe molecule for detecting selenite roots has the following synthetic route:

。

The preparation steps are as follows:

(1) dissolving levulinic acid, EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and DMAP (4-dimethylaminopyridine) in an anhydrous dichloromethane solution, and stirring at room temperature for reaction for a period of time to obtain a solution I;

(2) Dissolving the intermediate 1 (2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene ] malononitrile) in an anhydrous dichloromethane solution to obtain an intermediate 1 solution, then dropwise adding the intermediate 1 solution into the solution I obtained in the step (1) at room temperature, and reacting for a period of time after dropwise adding to obtain a solution II;

(3) And (3) washing the solution II obtained in the step (2) with a saturated sodium chloride solution, drying the obtained organic phase with anhydrous sodium sulfate, filtering, removing the solvent under reduced pressure, and separating by silica gel column chromatography to obtain a product SeP1, namely the fluorescent probe molecule.

in the step (1), the molar ratio of the levulinic acid to the EDC to the DMAP is 1 (0.5-4) to 0.1-2, and the reaction is carried out for 10-60 minutes under stirring at room temperature.

In the step (2), the intermediate 1 is 2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene ] malononitrile, the molar ratio of the levulinic acid in the solution I to the intermediate 1 is 1 (0.5-2), and the reaction time is 8-24 hours after the dropwise addition is finished.

The silica gel column separation in the step (3) adopts ethyl acetate and petroleum ether eluent with the volume ratio of 1 (3-12), and the yield is 40-80%.

The near-infrared fluorescent probe molecule is applied to the field of high-sensitivity and specific detection of selenious acid roots.

The Latin reagent company, such as chemical reagent, solvent, and metal ion, used in the process of preparing the fluorescent probe SeP1 according to the present invention. A DTX-400 nuclear magnetic resonance spectrometer of Bruke company is adopted in the process of confirming and testing the performance of the fluorescent probe SeP1, the solvent is deuterated chloroform, and a hydrogen spectrum and a carbon spectrum of nuclear magnetic resonance are recorded by taking TMS as an internal standard. High resolution mass spectral data were recorded using a Q-exact HR-MS mass spectrometer from Thermo. The fluorescence spectrum was recorded using a F-7000 fluorescence spectrometer from Hitachi, Japan.

The invention has the following beneficial effects:

(1) The fluorescent probe is a compound based on malononitrile isophorone, namely an intermediate 1 (2- [3- (4-hydroxystyryl) -5, 5-dimethyl-cyclohex-2-en-1-ylidene)]Malononitrile) has strong fluorescence when the hydroxyl group is exposed, and hardly has fluorescence when the hydroxyl group is protected. According to the invention, an acetyl propyl group is adopted to protect the hydroxyl in the structure of the intermediate 1 to obtain the fluorescent probe SeP1, and when a selenite ion and the acetyl propyl group perform a specific reaction, the intermediate 2 is generated and separated, so that the hydroxyl in the structure is deprotected to regenerate the intermediate 1 and release strong fluorescence (a specific mechanism diagram is shown in figure 8). The reaction mechanism is verified by means of high-resolution mass spectrometry (as shown in figure 9), and the result of the high-resolution mass spectrometry test of the solution after the fluorescent probe SeP1 recognizes selenite is 289.1347 ([ M-H ]⁺]^-) The theoretical calculation of anion for intermediate 1 was 289.1341. This data corroborates the mechanism of action shown in FIG. 8.

(2) The invention researches the situation that the probe SeP1 is in CH by a fluorescence spectrometer₃CH₂OH-H₂The identification performance of the O solution and related analytes is that the fluorescence intensity (657 nm) obtained by adding selenite in the presence of metal cations and common anions is basically the same as that obtained by adding selenite alone, as shown in FIG. 5, and the result shows that the probe SeP1 has stronger anti-metal cation and common anion interference performance on the detection of selenite and can overcome the interference of complex background matrix fluorescence in organisms and environments.

(3) The invention adopts a fluorescence spectrometer to detect the selenite root of the probe SeP1Minimum detection limit in CH₃CH₂OH-H₂In O (1:1, v/v) solution, the concentration of the immobilized probe SeP1 is 16.7 mu M, the response intensity of the immobilized probe to selenite with different concentrations is measured, the system fluorescence intensity is continuously enhanced along with the increase of the selenite concentration (figure 6), and the research shows that the solution fluorescence intensity value is linear (R is between 0 and 2.5 equivalent of the selenite concentration)²= 0.985), according to IUPAC rules, the detection limit of the probe molecule to the bisulfite is 0.068 mu M through calculation (3 sigma/k), and the test result shows that the probe SeP1 has practical value for detecting selenite in the environment, and has wide application prospect in the detection field of environment and biological systems.

drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows the NMR spectrum of the fluorescent probe SeP1 of the present invention.

FIG. 2 is a nuclear magnetic resonance carbon spectrum of the fluorescent probe SeP1 of the present invention.

FIG. 3 is a high resolution mass spectrum of the fluorescent probe SeP1 of the present invention.

FIG. 4 is a graph showing fluorescence selectivity of the fluorescent probe SeP1 of the present invention, with an excitation wavelength of 550 nm.

FIG. 5 shows that the fluorescent probe SeP1 of the present invention recognizes SeO₃ ^2-The excitation wavelength is 550 nm, and the emission wavelength is 657 nm.

FIG. 6 shows that the fluorescent probe SeP1 of the present invention recognizes SeO₃ ^2-Fluorescence titration graph of (1), excitation wavelength 550 nm.

FIG. 7 shows that the fluorescent probe SeP1 of the present invention recognizes SeO₃ ^2-The excitation wavelength is 550 nm, and the emission wavelength is 657 nm.

FIG. 8 shows a fluorescent probe of the present inventionSeP1 identifying SeO₃ ^2-the reaction mechanism diagram of (1).

FIG. 9 shows SeO recognition by fluorescent probe SeP1 according to the present invention₃ ^2-High resolution verification of the reaction mechanism (2).

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without inventive effort based on the embodiments of the present invention, are within the scope of the present invention.

the preparation method of the intermediate 1 comprises the following steps:

。

Commercial starting material (3,5, 5-trimethylcyclohex-2-enylidene) malononitrile (compound A, 186 mg, 1 mmol) and p-hydroxybenzaldehyde (244 mg, 2 mmol) were dissolved in 6 mL of an ethanol solution, one to two drops of piperidine and acetic acid were added dropwise to the above system, respectively, and the reaction was heated under reflux for 4 hours, and cooled to room temperature after TLC monitoring completion of the reaction. The reaction solution was poured into 15ml of an ice-water mixture, and a precipitate was formed. The precipitate was filtered off and redissolved in dichloromethane and isolated by column chromatography to give intermediate 1 (203 mg) as a yellow solid in 70% yield.