CN114249760A - Synthesis and application of fluorescent probe capable of simultaneously distinguishing hypochlorous acid and hydrogen peroxide in three channels - Google Patents
Synthesis and application of fluorescent probe capable of simultaneously distinguishing hypochlorous acid and hydrogen peroxide in three channels Download PDFInfo
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- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 title claims abstract description 70
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 title claims abstract description 54
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 25
- 230000015572 biosynthetic process Effects 0.000 title claims description 10
- 238000003786 synthesis reaction Methods 0.000 title claims description 10
- 239000003068 molecular probe Substances 0.000 claims abstract description 17
- 238000006243 chemical reaction Methods 0.000 claims abstract description 9
- 239000000126 substance Substances 0.000 claims abstract description 4
- -1 11-ethyl-3-(pyridin-4-yl)pyrano[2,3-b]phenothiazin-2(11H)-one Chemical compound 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
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- QVLJLWHOILVHJJ-UHFFFAOYSA-N ethyl 2-pyridin-4-ylacetate Chemical compound CCOC(=O)CC1=CC=NC=C1 QVLJLWHOILVHJJ-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention discloses a method for simultaneously distinguishing and detecting hypochlorous acid (HOCl) and hydrogen peroxide (H) through three different fluorescence emission signals of green, red and yellow2O2) The fluorescent molecular probe of (1), wherein the chemical structural formula of the molecular probe is as follows:
. The fluorescent probe can distinguish HOCl and H under the same detection condition2O2And both coexist. The probe can emit 504 nm green light and H at 350 nm excitation wavelength after reacting with HOCl2O2After reaction, the red light with 640 nm is emitted under the excitation wavelength of 430 nm, and the red light and the HOCl and H react together2O2) Then emits yellow light of 550 nm at an excitation wavelength of 440 nm. The probeSimultaneous sequential detection of HOCl and H through three channels2O2And HOCl and H in the coexistence2O2The method has the advantages of high sensitivity, good selectivity, large Stokes shift and the like, and has great application prospect in the technical fields of analytical chemistry, life science, environmental science and the like.
Description
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to synthesis of a fluorescent probe for detecting hypochlorous acid and hydrogen peroxide, and application of the fluorescent probe in quantitative analysis of hypochlorous acid and hydrogen peroxide in the environment and simultaneous differentiation of imaging hypochlorous acid and hydrogen peroxide in biological systems such as cells, tissues, living bodies and the like.
Background
Reactive Oxygen Species (ROS) are multiple species of organismsMessenger molecules essential for processing (J. Amer. Chem. Soc.2017, 139, 6911-6918), including senescence, signal transduction, cellular redox homeostasis and protection against pathogen invasion: (Spectrochim. Acta. A Mol. Biomol. Spectrosc., 2021, 246, 118927). Hydrogen peroxide (H)2O2) And hypochlorous acid (HClO) are two important reactive oxygen species that play important roles in many physiological and pathological processes. There is evidence that excess HClO is associated with various human diseases, such as cardiovascular disease, neurodegeneration, arthritis, and cancer (b) ((R))Anal. Chem.2017, 89, 10384-); in addition, H is abnormally produced in cells2O2Is involved in various pathological processes such as diabetes, cancer, cardiovascular diseases and neurodegeneration: (Anal. Chem., 2016, 88, 8019-8025). Since the change of the content of one of the two active oxygen species may cause the change of the other active oxygen species, many diseases occur in close relation to the change of the content thereof. Thus, efficient detection methods/techniques were developed for simultaneous differential detection of HOCl and H2O2The two active oxygen species have very important significance in understanding the biological functions of the two active oxygen species. The fluorescent imaging technology based on the small molecular fluorescent probe becomes an effective method for visualizing the time-space distribution of the biological molecules in the biological system due to the characteristics of real time, sensitivity, no wound and the like (Anal. Chem., 2016, 88, 8019-8025). Despite small molecules of active oxygen hypochlorous acid (HOCl) and hydrogen peroxide (H)2O2) With similar chemistry, single/dual fluorescence channel fluorescent probes have also been developed for detecting HOCl or H2O2 (e.g. usingJ. Am. Chem. Soc., 2017, 139, 6911–6918、Chem. Sci., 2018, 9, 8207–8212、Anal. Chem., 2020, 92, 3262–3269、Chem. Commun.2018, 54, 9238-9241, etc.). Only a very small number of fluorescent probes can simultaneously detect HOCl and H2O2The two active oxygen species do not realize the simultaneous separation of HOCl and H in three channels2O2And HOCl and H in the coexistence thereof2O2。
Disclosure of Invention
In view of the above, the present invention overcomes some deficiencies of the prior art, and an object of the present invention is to provide a fluorescent molecular probe for simultaneously detecting hypochlorous acid and hydrogen peroxide by using three-channel fluorescent signals and their coexistence, which can distinguish hypochlorous acid and hydrogen peroxide in biological systems such as imaging cells by using different fluorescent signals, thereby providing some analytical detection methods and ideas for the fields of analytical detection, etc. The invention also aims to provide a synthesis and application method of the fluorescent molecular probe for detecting multiple active oxygen, which has a simple preparation method.
The invention solves the problem by adopting the specific technical scheme that three channels simultaneously distinguish the synthesis and the application of the fluorescent probe of hypochlorous acid and hydrogen peroxide, and the chemical structural formula of the probe is as follows:
the fluorescent probe capable of distinguishing hypochlorous acid from hydrogen peroxide simultaneously is characterized in that the preparation method of the fluorescent molecular probe comprises the following steps:
step 1, synthesizing 11-ethyl-3- (pyridine-4-yl) pyran [2,3-b ] phenothiazin-2 (11H) -ketone
a. Under the protection of nitrogen, adding 11-ethyl-3- (pyridine-4-yl) pyran [2,3-b ] phenothiazin-2 (11H) -ketone and 4-bromomethyl phenylboronic acid pinacol ester into anhydrous acetonitrile, refluxing and stirring at 100 ℃ for 12 hours to obtain a reddish brown solution,
b. after the reaction was cooled to room temperature, it was filtered to give 4- (11-ethyl-2-oxo-2, 11-dihydropyrano [2,3-b ] phenothiazin-3-yl) -1- (4- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxan-2-yl) benzyl) pyridine-1-ammonium bromide as a reddish brown solid.
The fluorescent molecular probe of the invention simultaneously distinguishes and detects the use methods of hypochlorous acid and hydrogen peroxide: without specific reference, the probe molecules are typically dissolved at room temperature in an environment with a volume ratio of organic phase to aqueous phase of 5:5, the organic phase being dimethyl sulfoxide (DMSO) and the aqueous phase being Phosphate Buffered Saline (PBS) at pH = 7.4 and an aqueous solution of the analyte for analytical detection.
The fluorescent molecular probe disclosed by the invention has the following specific characteristics of simultaneously distinguishing hypochlorous acid and hydrogen peroxide: the molecular fluorescent probe is dissolved by dimethyl sulfoxide (DMSO), probe molecules are dissolved in an organic phase solution and a water phase solution (5:5, v/v), the probe emits green light with the wavelength of 504 nm under the excitation wavelength of 350 nm after reacting with hypochlorous acid, emits red light with the wavelength of 640 nm under the excitation wavelength of 430 nm after reacting with hydrogen peroxide, and emits yellow light with the wavelength of 550 nm under the excitation wavelength of 440 nm after reacting with the probe and hydrogen peroxide together. Thus enabling specific excitation and fluorescence emission signals for detection of specific analytes, and also enabling sequential and concurrent detection of both with a third fluorescence channel. The fluorescent molecular probe realizes the differential detection of hypochlorous acid and hydrogen peroxide under the same detection condition, and has no obvious response to other active oxygen, active sulfur, common amino acid, metal ions, active nitrogen and the like. Probe detection for HOCl and H2O2The detection limit of (A) was as low as 51.11 nM and 39.45 nM, respectively. Therefore, the fluorescent molecular probe disclosed by the invention can realize three channels to simultaneously distinguish hypochlorous acid from hydrogen peroxide.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of the fluorescent probe of the present invention.
FIG. 2 is a fluorescence spectrum diagram of the fluorescence probe of the present invention for detecting hypochlorous acid and hydrogen peroxide.
FIG. 3 shows the fluorescent probe of the present invention simultaneously imaging the intracellular hypochlorous acid (HOCl) and hydrogen peroxide (H) in RAW 264.7 (macrophage) cells by green, red and yellow fluorescence2O2) Fluorescence imaging.
Detailed Description
The invention is further described below with reference to the accompanying drawings.
The synthetic route of the fluorescent molecular probe is shown as the following formula:
EXAMPLE 1 Synthesis of 11-Ethyl-3- (pyridin-4-yl) pyran [2,3-b ] phenothiazin-2 (11H) -one
I. 1.0 g (3.69 mmol) of 10-ethyl-2-hydroxy-10H-phenothiazine-3-carbaldehyde and 608.81 mg (3.69 mmol) of ethyl 4-pyridylacetate are added into 10 mL of absolute ethyl alcohol, stirred at 95 ℃ for 8-12 hours to obtain a red solution,
after the reaction was cooled to room temperature, it was filtered to obtain 1.0 g of 11-ethyl-3- (pyridin-4-yl) pyran [2,3-b ] phenothiazin-2 (11H-one) as a red solid in a yield of 73.0%.
EXAMPLE 2 Synthesis of fluorescent Probe 4- (11-Ethyl-2-oxo-2, 11-dihydropyrano [2,3-b ] phenothiazin-3-yl) -1- (4- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxan-2-yl) benzyl) pyridine-1-ammonium bromide
A. Under the protection of nitrogen, 1.0 g (2.68 mmol) of 11-ethyl-3- (pyridin-4-yl) pyran [2,3-b ] phenothiazin-2 (11H) -one and 797.4 mg (2.68 mmol) of 4-bromomethylbenzeneboronic acid pinacol ester are added into 15 mL of anhydrous acetonitrile, and the mixture is refluxed and stirred for 12 hours at 100 ℃ to obtain a reddish brown solution,
B. after the reaction was cooled to room temperature, it was filtered to give 1.5 g of 4- (11-ethyl-2-oxo-2, 11-dihydropyrano [2,3-b ] phenothiazin-3-yl) -1- (4- (4, 4,5, 5-tetramethyl-1, 3, 2-dioxan-2-yl) benzyl) pyridine-1-ammonium bromide as a reddish brown solid in a yield of 83.3%.
Example 3 use of fluorescent molecular probes to differentially detect hypochlorous acid and hydrogen peroxide in an in vitro Environment
The experiment for detecting the spectral properties of hypochlorous acid and hydrogen peroxide by using the fluorescent molecular probe disclosed by the invention comprises the following steps: the probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution having a concentration of 1 mM, and hypochlorous acid and an aqueous hydrogen peroxide solution having a concentration of 1 mM were prepared, respectively. The specific test mode is as follows: mu.L of 1 mM probe solution, 980. mu.L of analytically pure DMSO and the required amount of PBS buffer in water, the required amount of hypochlorous acid/hydrogen peroxide in water in a 2 mL sample tube, all tests maintained a volume ratio of organic to aqueous phase of 5:5 (total volume of 2 mL per test sample). For example, when the fluorescence intensity of the probe after reaction with hypochlorous acid is required to be measured at a hypochlorous acid concentration of 20. mu.M, the samples are prepared as follows: after 20. mu.L of 1 mM probe solution, 980. mu.L of analytical DMSO, 960. mu.L of PBS buffer solution, and 40. mu.L of 1 mM hypochlorous acid aqueous solution were put in a 2 mL sample tube and shaken at room temperature for 15 minutes, the fluorescence emission intensity was measured at an excitation wavelength of 350 nm, and other test procedures were similar to the above-mentioned procedures. The detection limit of the probe on hypochlorous acid and hydrogen peroxide is respectively as low as 51.11 nM and 39.45 nM. Therefore, the fluorescent molecular probe disclosed by the invention can realize high-sensitivity distinguishing detection on the fluorescent molecular probe and the fluorescent molecular probe.
Example 4 three-channel fluorescence imaging analysis of endogenous hypochlorous acid and hydrogen peroxide in RAW 264.7 (macrophage) cells
The RAW 264.7 cells are subcultured in a confocal dish cell culture medium for 24 hours under the standard growth condition, a proper amount of probe (5 mu M) is added to continue to be cultured for 30 minutes under the standard growth condition, then photography is carried out under a confocal fluorescence microscope, fluorescence imaging is carried out on endogenous hypochlorous acid and hydrogen peroxide of the RAW 264.7 cells by using green, red and yellow fluorescence channels respectively, as can be seen from figure 3, the fluorescent probe can emit green, red and yellow fluorescence in the cells, which indicates that the probe can detect the hypochlorous acid and the hydrogen peroxide in the cells and the joint detection of the hypochlorous acid and the hydrogen peroxide, and the endogenous HOCl and H in the cells are successfully realized2O2Three-channel fluorescence imaging analysis.
The fluorescent probe capable of distinguishing hypochlorous acid from hydrogen peroxide simultaneously provided by the invention realizes different chemical reactions between the probe and hypochlorous acid and hydrogen peroxide under the same detection condition based on the same probe to generate different fluorescent substances, so that the fluorescent probes emit green, red and yellow fluorescence under specific excitation wavelength, the purpose of distinguishing and detecting hypochlorous acid and hydrogen peroxide simultaneously is achieved, and endogenous hypochlorous acid and hydrogen peroxide in cells are successfully realized through three-channel simultaneous fluorescence imaging. It is expected to provide some ideas for the development of active oxygen fluorescent probes in the future. While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Therefore, fluorescent probes that distinguish hypochlorous acid from hydrogen peroxide while having the technical features described herein fall within the scope of the present patent.
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| CN113292582A (en) * | 2021-05-11 | 2021-08-24 | 湖南师范大学 | Synthesis and application of bifunctional fluorescent probe capable of distinguishing hydroxyl free radicals and hydrogen peroxide simultaneously |
| CN114163456A (en) * | 2021-12-23 | 2022-03-11 | 西南医科大学附属医院 | A kind of phenothiazine coumarin-based pyridine salt compound and its preparation and application |
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| CN113292582A (en) * | 2021-05-11 | 2021-08-24 | 湖南师范大学 | Synthesis and application of bifunctional fluorescent probe capable of distinguishing hydroxyl free radicals and hydrogen peroxide simultaneously |
| CN114163456A (en) * | 2021-12-23 | 2022-03-11 | 西南医科大学附属医院 | A kind of phenothiazine coumarin-based pyridine salt compound and its preparation and application |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114957286A (en) * | 2022-04-25 | 2022-08-30 | 湖南省肿瘤医院 | Multi-signal fluorescent probe for early diagnosis of tumor and preparation method and application thereof |
| CN116217589A (en) * | 2022-04-25 | 2023-06-06 | 湖南省肿瘤医院 | Application of ONOO- and/or Na2S2 fluorescent probe reagent for preparing tumor early diagnosis |
| US11857644B2 (en) | 2022-04-25 | 2024-01-02 | Hunan Cancer Hospital | Multi-signal fluorescent probe for early diagnosis of tumors, and preparation and use thereof |
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