阅读说明：本技术 多信号荧光探针的合成及其同时区分Cys、SO2、GSH和Hcy的应用 (Synthesis of multi-signal fluorescent probe and simultaneous Cys and SO distinguishing thereof2GSH and Hcy ) 是由 尹鹏 贺梦雪 尹国兴 甘亚兵 李海涛 于 2021-03-17 设计创作，主要内容包括：本发明公开了一种通过不同的激发与荧光发射信号同时区分检测半胱氨酸(Cys)、二氧化硫(SO-(2))、谷胱甘肽(GSH)和同型半胱氨酸(Hcy)的多信号荧光分子探针,该多信号分子探针的化学结构式如下：。该多信号荧光探针能在同一检测条件下利用探针与Cys、NaHSO-(3)、GSH和Hcy发生不同的化学反应,生成不同的荧光物质,达到同时区分检测Cys、SO-(2)、GSH和Hcy的目的。探针与Cys反应后在376 nm激发波长下发射464 nm的蓝光,与NaHSO-(3)反应后在425 nm激发波长下发射520 nm的绿光,与GSH作用后在450 nm激发波长下发射536 nm的黄绿光,与Hcy作用后在490 nm激发波长下发射564 nm的橘光。该多信号荧光分子探针能够用于在体外环境中同时定量,在细胞中同时荧光成像Hcy、Cys、SO-(2)和GSH。(The invention discloses a method for simultaneously distinguishing and detecting cysteine (Cys) and sulfur dioxide (SO) through different excitation and fluorescence emission signals 2 ) Glutathione (GSH) and homocysteine (Hcy), wherein the chemical structural formula of the multi-signal molecular probe is as follows: . The multi-signal fluorescent probe can utilize the probe and Cys and NaHSO under the same detection condition 3 GSH and Hcy react to generate different fluorescent substances, SO as to detect Cys and SO simultaneously 2 GSH and Hcy. The probe emits 464 nm blue light at 376 nm excitation wavelength after reacting with Cys, and NaHSO 3 After the reaction, 520 nm green light is emitted at 425 nm excitation wavelength, 536 nm yellow green light is emitted at 450 nm excitation wavelength after the reaction with GSH, and 564 nm orange light is emitted at 490 nm excitation wavelength after the reaction with Hcy. The multi-signal fluorescent molecular probe can be used for simultaneous quantification in an in vitro environment and simultaneous fluorescence imaging of Hcy, Cys and SO in cells 2 And GSH.)

1. Simultaneously distinguish Cys and SO₂The multiple-signal fluorescent probe for GSH and Hcy is characterized in that the chemical structural formula of the fluorescent molecular probe is as follows:

。

2. the synthesis of the multi-signal fluorescent probe of claim 1, wherein the preparation method of the fluorescent molecular probe comprises the following steps:

step 1. Synthesis of 4-hydroxy-7- (pyrrolidin-1-yl) -2H-chromen-2-one

a. Proper amount of diphenyl malonate and 3- (pyrrolidine-1-yl) phenol are added into anhydrous toluene to react for 8 to 12 hours at 100 ℃,

b. cooling the reaction to room temperature, filtering, washing the solid with diethyl ether, and drying in vacuum to obtain a gray-green solid;

step 2. Synthesis of 4-chloro-2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde

I. Under the protection of nitrogen, adding a proper amount of dry redistilled N, N-Dimethylformamide (DMF) slowly into equal volume of phosphorus oxychloride (POCl)₃) Stirring at 20-50 deg.C for 30 min to obtain red solution,

II, dissolving 4-hydroxy-7- (pyrrolidine-1-yl) -2H-chromen-2-one in a proper amount of N, N-dimethylformamide, dropwise adding the mixture into the mixed solution in the step I, continuously stirring the mixture at 60 ℃ under the protection of nitrogen for reaction for 12 hours,

III, pouring the reaction liquid obtained in the step II into a proper amount of ice water, adjusting the pH value to 5-6 by using a 20% NaOH solution, generating a large amount of precipitates, filtering, washing the solids for 3 times by using a proper amount of deionized water, and drying in vacuum to obtain 4-chloro-2-oxo-7- (pyrrolidine-1-yl) -2H-methylene-3-formaldehyde;

step 3. Synthesis of 4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde

i. Dissolving 4-chloro-2-oxo-7- (pyrrolidine-1-yl) -2H-methylene-3-formaldehyde and n-butylmercaptan in dichloromethane, adding appropriate amount of triethylamine dropwise, stirring at room temperature for reaction,

ii. step (iii)Purifying the reaction liquid column to obtain 4- (butylthio) -2-oxo-7- (pyrrolidine-1-yl) -2H-methylene-3-formaldehyde;

step 4. Synthesis of multiple Signal fluorescent probes (E) -2- (benzo [ 2 ], ]d]Thiazol-2-yl) -3- (4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-pyran-3-yl) acrylonitrile

A. 4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde and corresponding 2- (benzo [ b ], ]d]Thiazole-2-yl) acetonitrile is added into a proper amount of absolute ethyl alcohol and a proper amount of p-toluenesulfonic acid, stirred at room temperature for reaction,

B. after the reaction is finished, filtering, and drying the obtained solid in vacuum to obtain the multi-signal fluorescent molecular probe of claim 1.

3. The synthesis of the multi-signal fluorescent probe of claim 2, wherein the 4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde prepared in the step A and 2- (benzo [ 2 ], [ solution ]d]Thiazol-2-yl) acetonitrile in a molar ratio of 1: 1.1-1.2.

4. The multi-signal fluorescent probe of claim 1 simultaneously distinguishing Cys and SO₂And GSH and Hcy characterized in thatThe fluorescent molecular probe can be prepared to quantitatively detect Cys and SO in the environment₂GSH and Hcy, and imaging Cys and SO simultaneously and differentially in cells₂GSH and Hcy.

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a multi-signalSynthesis of fluorescent probe and simultaneous quantitative detection of Cys and SO in environment by using fluorescent probe₂GSH and Hcy, and imaging Cys and SO simultaneously and differentially in cells₂GSH and Hcy.

Background

Bioactive molecules such as active sulfur, active nitrogen and active oxygen play an extremely important role in maintaining the redox homeostasis in the cell. Wherein, the active Sulfur components (RSS) with the highest content in active molecules in cells mainly comprise biological thiol (cysteine (Cys), homocysteine (Hcy), Glutathione (GSH)), Sulfur dioxide (SO)₂) Hydrogen sulfide (H)₂S) and N-acetylcysteine (NAC) as active moleculesAnal. Chem. 2018, 90, 533-555.). Hcy can be synthesized from methionine in mammals with the aid of various enzymes, and has a normal serum concentration of 5-12 μ M, a normal intracellular concentration of 30-200 μ M Cys, GSH which is the most abundant low molecular weight biological thiol in cells, a normal concentration of 0.5-10 mM, and sulfur dioxide (SO)₂) As a metabolite of sulfur-containing amino acids, sulfite (HSO) is used under physiological conditions₃ ^-And SO₃ ^2-) Is present in the organism, the serum sulphite content is generally less than 10. mu.M. Biological mercaptan plays an important role in cell metabolism, the content of the biological mercaptan in cells is higher than the normal level in patients with cancer, Alzheimer disease, Parkinson disease and the like, and the content of the biological mercaptan in blood directly reflects the health condition of organisms. Because these four active sulfur species have similar structures and reactivity, there is a biological metabolic process in cells, i.e., the conversion of Hcy to Cys and the conversion of Cys to GSH, wherein the change of one content may cause the associated change of the other two, and many diseases occur in close relation to the change of their content. Therefore, efficient detection methods/techniques were developed for simultaneous differential detection of Cys and SO₂The four sulfur-containing compounds GSH and Hcy have important significance. In recent years, multi-signal fluorescent probes have received much attention because of their ability to simultaneously monitor and visualize two or more analytes in living cells but haveThe reported thiol fluorescent probe cannot realize simultaneous differential detection of Cys and SO by using four channels₂Four sulfur-containing compounds, GSH and Hcy, develop and simultaneously distinguish Cys and SO₂The multiple-signal fluorescent probe of GSH and Hcy has great application value in the fields of analysis and detection, environmental science, biochemistry and the like.

Disclosure of Invention

In view of the above, the present invention overcomes some of the deficiencies of the prior art and provides a method for simultaneously detecting Cys and SO by using four-channel fluorescence signals₂Multiple signal fluorescent molecular probe of GSH and Hcy for quantitative detection of Cys and SO in environment and food₂GSH and Hcy, and imaging Cys and SO simultaneously and differentially in cells₂And GSH and Hcy, and provides some analysis and detection methods and ideas for the fields of analysis and detection, environmental science and the like.

The invention also aims to provide a synthesis and application method of the multi-signal fluorescent molecular probe with simple preparation method.

The specific technical scheme adopted by the invention for solving the problems is that the synthesis of a multi-signal fluorescent probe and the simultaneous differential detection of Cys and SO are adopted₂GSH and Hcy, the chemical structural formula of the probe is as follows:

。

simultaneously distinguishing and detecting Cys and SO₂And synthesis of GSH and Hcy multi-signal fluorescent probes, wherein the preparation method of the fluorescent molecular probe comprises the following steps:

step 1. Synthesis of 4-hydroxy-7- (pyrrolidin-1-yl) -2H-chromen-2-one

a. Proper amount of diphenyl malonate and 3- (pyrrolidine-1-yl) phenol are added into anhydrous toluene to react for 8 to 12 hours at 100 ℃,

b. cooling the reaction product to room temperature, filtering, washing the solid with diethyl ether, and drying in vacuum to obtain a grey-green solid 4-hydroxy-7- (pyrrolidine-1-yl) -2H-chromen-2-one;

step 2. Synthesis of 4-chloro-2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde

I. Under the protection of nitrogen, adding a proper amount of dry redistilled N, N-Dimethylformamide (DMF) slowly into equal volume of phosphorus oxychloride (POCl)₃) Stirring at 20-50 deg.C for 30 min to obtain red solution,

II, dissolving 4-hydroxy-7- (pyrrolidine-1-yl) -2H-chromen-2-one in a proper amount of N, N-dimethylformamide, dropwise adding the mixture into the mixed solution in the step I, continuously stirring the mixture at 60 ℃ under the protection of nitrogen for reaction for 12 hours,

III, pouring the reaction liquid obtained in the step II into a proper amount of ice water, adjusting the pH value to 5-6 by using a 20% NaOH solution, generating a large amount of precipitates, filtering, washing the solids for 3 times by using a proper amount of deionized water, and drying in vacuum to obtain 4-chloro-2-oxo-7- (pyrrolidine-1-yl) -2H-methylene-3-formaldehyde;

step 3. Synthesis of 4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde

i. Dissolving 4-chloro-2-oxo-7- (pyrrolidine-1-yl) -2H-methylene-3-formaldehyde and n-butylmercaptan in dichloromethane, adding appropriate amount of triethylamine dropwise, stirring at room temperature for reaction,

ii. step (iii)Purifying the reaction liquid column to obtain 4- (butylthio) -2-oxo-7- (pyrrolidine-1-yl) -2H-methylene-3-formaldehyde;

step 4. Synthesis of multiple Signal fluorescent probes (E) -2- (benzo [ 2 ], ]d]Thiazol-2-yl) -3- (4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-pyran-3-yl) acrylonitrile

A. 4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde and corresponding 2- (benzo [ b ], ]d]Thiazole-2-yl) acetonitrile is added into a proper amount of absolute ethyl alcohol, a proper amount of p-toluenesulfonic acid is added, stirring reaction is carried out at room temperature,

B. after the reaction is finished, filtering, and drying the obtained solid in vacuum to obtain the multi-signal fluorescent molecular probe of claim 1.

The multi-signal fluorescent molecular probe of the invention can be used for differential detection at the same timeMeasuring Cys and SO₂GSH and Hcy methods of use: 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 method is characterized in that: the molecular fluorescent probe is dissolved by dimethyl sulfoxide (DMSO), probe molecules are dissolved in an organic phase solution and a water phase (5:5, v/v) solution, and after the probe molecules react with Cys at room temperature for 15 minutes, strong blue fluorescence of 464 nm is emitted under the excitation wavelength of 376 nm; with NaHSO₃After reacting for 15 minutes at room temperature, emitting strong green fluorescence at 520 nm under the excitation wavelength of 425 nm; after reacting with GSH for 15 minutes, emitting yellow green fluorescence of 536 nm under the excitation wavelength of 450 nm; after 15 minutes of interaction with Hcy, an orange fluorescence of 564 nm was emitted at an excitation wavelength of 490 nm. The probe has no obvious fluorescence, strong blue fluorescence can be generated only by excitation at 376 nm when Cys is detected, and NaHSO is used for detection₃The excitation wavelength of/GSH/Hcy (425/450/490 nm) is non-fluorescent or weak, and the other three are also the same. Thus, detection of a particular analyte using a particular excitation and fluorescence emission signal is achieved, and when both are present, the four can be well distinguished using different excitation and fluorescence emission signals. The fluorescent molecular probe realizes the simultaneous differential detection of Cys and NaHSO under the same detection condition₃GSH and Hcy, has no obvious response to other common amino acids, active oxygen and active nitrogen species, and has no obvious response to Cys and NaHSO₃The detection limits for GSH and Hcy were 0.0132, 0.0757, 0.0231 and 0.0218 μ M, respectively. Therefore, the multi-signal fluorescent molecular probe disclosed by the invention can realize high-sensitivity distinguishing detection of the four.

Drawings

FIG. 1 shows that the multi-signal fluorescent probe of the invention detects Cys and NaHSO₃Fluorescence spectra of GSH and Hcy.

FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the multi-signal fluorescent probe of the present invention.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

The synthetic route of the multi-signal fluorescent molecular probe is as follows:

EXAMPLE 1 Synthesis of 4-hydroxy-7- (pyrrolidin-1-yl) -2H-chromen-2-one

a. Diphenyl malonate (6.28 g, 24.51 mmol) and 3- (pyrrolidin-1-yl) phenol (2 g, 12.25 mmol) were added to dry toluene (20 mL), the reaction mixture was heated to 110 ℃ to react for 8 hours,

b. after completion of the reaction, filtration was carried out, and the filter cake was washed 3 times with n-hexane and dried under vacuum to give 4-hydroxy-7- (pyrrolidin-1-yl) -2H-chromen-2-one (2.0 g, 68% yield) as a pale yellow solid.

EXAMPLE 2 Synthesis of 4-chloro-2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde

Under nitrogen protection, 1.8 mL of dry, redistilled N, N-Dimethylformamide (DMF) was slowly added to an equal volume of phosphorus oxychloride (POCl)₃) Stirring at 20-50 deg.C for 30 min to obtain red solution,

4-hydroxy-7- (pyrrolidin-1-yl) -2H-chromen-2-one (1.0 g, 4.32 mmol) was dissolved in 5 mL of N, N-dimethylformamide and added dropwise to stepThe mixture is continuously stirred and reacted for 12 hours at the temperature of 60 ℃ under the protection of nitrogen,

after the reaction is completed, the stepSlowly pouring the reaction solution into 500 mL of ice water, adjusting the pH value to 6 by using a 20% NaOH solution to generate a large amount of precipitate, filtering, washing a filter cake for 3 times by using a proper amount of deionized water, and drying the obtained solid in vacuum to obtain the 4-chloro-2-oxo-7- (pyrrolidine-1-yl) -2H-methylene-3-formaldehyde

0.74 g, yield 62%.

EXAMPLE 3 Synthesis of 4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde

0.20 g (720.19 mmol) of 4-chloro-2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde and 64.95 mg (720.19 mmol) of n-butylmercaptan were added to 20 mL of anhydrous dichloromethane, 70. mu.L of triethylamine was added dropwise thereto, the reaction was stirred at room temperature,

after the reaction is completed, the stepPerforming column chromatography on the reaction solution to obtain 4- (butylthio) -2-oxo-7- (pyrrolidine-1-yl) -2H-methylene-3-formaldehyde.

Example 4 Synthesis of multiple Signal fluorescent probes: (E) -2- (benzo [ 2 ], ]d]Thiazol-2-yl) -3- (4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-pyran-3-yl) acrylonitrile

4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-methylene-3-carbaldehyde (0.20 g, 0.603 mmol) and 2- (benzo [ d ] o]Thiazol-2-yl) acetonitrile (137 mg, 0.664 mmol) was added to 20 mL of anhydrous ethanol, an appropriate amount of p-toluenesulfonic acid was added, and the reaction mixture was stirred at room temperature overnight. After the reaction was complete, the filter cake was filtered and washed with cold ethanol and the solid was dried under vacuum to give a red product (CE) -2- (benzo [ 2 ], ]d]Thiazol-2-yl) -3- (4- (butylsulfanyl) -2-oxo-7- (pyrrolidin-1-yl) -2H-pyran-3-yl) acrylonitrile.

Example 5 Multi-Signal fluorescent molecular Probe for differential detection of Cys and SO in an in vitro Environment₂GSH and Hcy

The spectrum property experiment of the multi-signal fluorescent molecular probe comprises the following steps: the probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1mM probe solution, and Cys and NaHSO were prepared at 1mM concentrations, respectively₃（SO₂Mainly in the form of sulfite under physiological conditions), GSH and Hcy in aqueous solution. The specific test mode is as follows: 20 μ L of 1mM probe solution, 980 μ L of assay pure DMSO, the required amount of 1mM Cys/NaHSO₃The aqueous GSH/Hcy solution and the required amount of aqueous PBS buffer in a 2 mL sample tube, all with a volume ratio of organic phase to aqueous phase of 5:5 (total volume of 2 mL each), such as the fluorescence intensity after reaction of probe with GSH when a GSH concentration of 20 μ M is required, was prepared as follows: 20 μ L of 1mM probe solution, 980 μ L of analytically pure DMSO, 40 μ L of 1mM GSH aqueous solution and 960 μ L of PBS buffer solution are placed in a 2 mL sample tube, shaken and shaken for 15 minutes at room temperature, and then the fluorescence emission intensity can be measured by using the excitation wavelength of 450 nm, and other testing operations are similar to the above steps. The multi-signal probe molecule realizes the distinguishing detection of Cys and NaHSO by using different excitation wavelengths and fluorescence emission signals₃The four active sulfur species of GSH and Hcy have high sensitivity, the detection limit is respectively as low as 0.0132, 0.0757, 0.0231 and 0.0218 mu M, and the method is very suitable for the endogenous Cys and NaHSO of living cells₃Imaging analysis of GSH and Hcy.

The synthesis of the multi-signal fluorescent probe and the simultaneous differential detection of Cys and NaHSO thereof₃GSH and Hcy, develops a high-efficiency and simple probe capable of distinguishing multiple signals of 4 sulfur-containing compounds at the same time, and utilizes the probe and Cys and NaHSO under the same detection condition based on the same probe₃GSH and Hcy have different chemical reactions to generate different fluorescent substances, so that the fluorescent substances emit blue, green, yellow and orange fluorescence under specific excitation wavelength, the aim of distinguishing and detecting simultaneously is fulfilled, and the method has huge application prospect in simultaneously quantitatively analyzing the four sulfur-containing compounds. Hopefully for in the futureThe development of multi-signal fluorescent probes provides several ideas. 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. Thus, synthesis of a multi-signal fluorescent probe having the features described herein and its simultaneous Cys, SO discrimination₂GSH and Hcy, all fall within the scope of this patent.