CN102633694A - Fluorescent probe for detecting mercapto compounds as well as preparation method and using method of fluorescent probe - Google Patents

Abstract

The invention discloses a fluorescent probe for detecting mercapto compounds as well as the preparation method and the using method of the fluorescent probe. The prior art difficultly meets the requirements for biological detection and fluorescent imaging. The fluorescent probe consists of two parts, wherein 2,4-dinitrobenzol sulfonyl is an identification group, and dansyl chloride derivative is an information report functional group; and when mercapto exists in a system, the mercapto can perform specific reaction with 2,4-dinitrobenzol sulfonyl, so that the fluorescence of the information report functional group is changed, and the specific detection of the mercapto group is realized. The probe is simple and convenient in molecular synthesis and good in stability, can be stored and used for a long term, has high mercapto detection sensitivity and excellent selectivity, does not act with other common interfering molecules, has long fluorescence emission wavelength (more than 500nm), can effectively avoid the interference from background fluorescence of which biological macromolecules are smaller than 500nm, and can be applied to detection of biological systems.

Description

A fluorescent probe for detecting sulfhydryl compounds and its preparation method and use method

technical field

The invention belongs to the technical field of bioengineering, and in particular relates to a fluorescent probe for detecting sulfhydryl compounds, a preparation method and an application method thereof.

Background technique

Sulfhydryl groups are important components of many proteins and small molecules in living organisms, and they are the most reactive functional groups related to enzyme activity. A large number of biological phenomena are considered to depend on these thiol-containing thiols, such as redox reactions, methyl transfer reactions, carbon dioxide fixation reactions, and reactions involving coenzyme A. The content of certain sulfhydryl molecules in the body is directly related to various diseases, such as cancer, Parkinson's disease, cardiovascular disease, etc. Therefore, it is of great significance to develop a rapid, sensitive and simple method for detecting thiols in biochemistry and clinical chemistry. Traditional detection methods include mass spectrometry, high performance liquid chromatography, electrochemical methods, spectrophotometry, colorimetry, etc., but these methods have low sensitivity and cannot be adapted to the determination of sulfhydryl compounds in some biological samples. Due to the special photophysical and photochemical properties of the probe, the fluorescence method has the characteristics of high sensitivity and wide dynamic response range. More importantly, it can realize real-time visual tracking of living organisms and even single cells, and has become a widely used method for detecting intracellular An important means of thiols.

In view of this, sulfhydryl fluorescent probes have become the focus of research in recent years, and great progress has been made. For example, the developed aryl halides, dansylaziridines, pyrenes, etc. have good selectivity and sensitivity, and the detection limit for sulfhydryl groups is generally around nanomolar (nmol), but these probes themselves have Strong fluorescence, which will cause low signal-to-noise ratio when used in live cell imaging; benzofuransulfonyl halide probes, although their autofluorescence is weak, need to react with sulfhydryl groups under alkaline and high temperature conditions, and cannot Applied to fluorescence imaging of living cells; while acetyl halide derivatives are sulfhydryl derivatives, which can quickly react with sulfhydryl at room temperature and physiological pH, but the photostability is poor, especially when it is in solution, and it is easy to lose the fluorophore , so it is difficult to meet the requirements of biological detection and fluorescence imaging. So far, highly sensitive and high-response probes that can be truly used for the detection of sulfhydryl compounds are relatively rare.

Contents of the invention

Aiming at the deficiencies of the prior art, the present invention provides a fluorescent probe based on 2,4-dinitrobenzenesulfonyl modified dansyl chloride derivatives that can be used for the detection of mercapto compounds and its preparation and use methods.

The fluorescent probe for detecting biothiol consists of two parts: 2,4-dinitrobenzenesulfonyl is the recognition group, and dansyl chloride derivative is the information reporting functional group, and its structure is shown in formula I.

The preparation route of the fluorescent probe for detecting biothiol is as follows:

The preparation method of the fluorescent probe that detects sulfhydryl compound comprises the following steps:

Step 1, preparation of formula II: Dissolve 0.2-0.4 g of hydroquinone in 50 mL of dichloromethane, then add 0.6 mL of triethylamine, add 0.6-1.2 g of dansyl chloride at 0°C, and then The reaction was stirred at room temperature for 10-15 hours. After the reaction was completed, the solvent was evaporated to dryness and separated with a silica gel column.

Step 2, preparation of formula I: Dissolve 0.6-1.2 g of compound of formula II in 50 mL of dichloromethane, and add 1.5 mL of triethylamine, stir for 5 minutes, then dropwise add 0.9-1.8 g of 10 mL of dichloromethane solution of 2,4-dinitrobenzenesulfonyl chloride, after the dropwise addition is complete, continue to stir at room temperature for 10-15 hours, remove the solvent under reduced pressure, separate and purify the crude product by silica gel column chromatography, and use petroleum Ether: ethyl acetate = 4:1 is the eluent for elution, and the pure product of the probe molecule is obtained.

The fluorescent probe for detecting sulfhydryl compounds can be applied to the detection of sulfhydryl compounds in chemical systems, and can be developed and applied to the analysis and detection of sulfhydryl groups in biological living cells and living tissues.

The method for using the fluorescent probe for detecting biothiols comprises the following steps:

Step 1, measuring the NMR spectrum of the probe molecule

Step 2, measuring the selectivity of the probe molecule to the sulfhydryl group

Dissolve the probe molecules in a small amount of DMSO, and then make a solution with absolute ethanol; add the samples to be tested dissolved in absolute ethanol, so that the final concentration of the probe molecules is 0.1 mM, and the concentration of the sample to be tested is 5 mM ; After 2 hours of reaction, it was measured on a fluorescence spectrometer, and then the selectivity of the probe molecule to sulfhydryl was determined.

Step 3, measure the fluorescence-concentration curve of probe molecule to thioglycolic acid

Dissolve the probe molecule with a small amount of DMSO, and then configure it into a solution with absolute ethanol; add different concentrations of thioglycolic acid respectively, so that the final concentration of the probe molecule is 0.1 mM; measure it on a fluorescence spectrometer after reacting for 2 hours, Then the curve of fluorescence intensity versus concentration change was obtained.

Step 4, measure the fluorescence-time curve of probe molecule to thioglycolic acid

Dissolve the probe molecule with a small amount of DMSO, and then configure it into a solution with absolute ethanol; add thioglycolic acid to make the final concentration of the probe molecule 0.1 mM, while the concentration of thioglycolic acid is 5 mM; react for a certain period of time, The fluorescence intensity is detected, and then the curve of the fluorescence intensity versus time is obtained.

Beneficial effects of the present invention:

1. The probe molecule is easy to synthesize, has good stability, and can be stored and used for a long time;

2. High detection sensitivity of sulfhydryl group;

3. It has excellent selectivity and has no effect on other common interfering molecules;

4. It has a long fluorescence emission wavelength (>500nm), which can effectively avoid the interference from the background fluorescence of biological macromolecules less than 500nm, and can be applied to the detection of biological systems.

Description of drawings

Fig. 1 is the proton nuclear magnetic resonance spectrum of fluorescent probe molecule;

Fig. 2 is the selectivity of fluorescent probe molecule and sulfhydryl reaction;

Fig. 3 is the fluorescence intensity-wavelength curve when concentration changes;

Fig. 4 is fluorescence intensity-concentration curve;

Fig. 5 is the fluorescence intensity-wavelength curve when time changes;

Figure 6 is the fluorescence intensity-time curve.

Detailed ways

The invention discloses a fluorescent probe for detecting sulfhydryl groups, a preparation method and application thereof. The fluorescent probe is characterized in that it is composed of two parts, wherein 2,4-dinitrobenzenesulfonyl is a recognition group, and dansyl chloride derivative is an information reporting functional group. When there is a sulfhydryl group in the system, the sulfhydryl group can specifically react with 2,4-dinitrobenzenesulfonyl to change the fluorescence of the information reporting functional group, thereby realizing the specific detection of the sulfhydryl group.

The following examples are to further illustrate the present invention, but not to limit the scope of the present invention.

Example 1:

(1) Preparation of probe molecules

The reaction process is represented by the following reaction scheme:

Step 1, preparation of formula II: Dissolve 0.2 g of hydroquinone in 50 mL of dichloromethane, then add 0.6 mL of triethylamine, add 0.6 g of dansyl chloride at 0°C, and then stir the reaction at room temperature After 10 hours, after the reaction was completed, the solvent was evaporated to dryness and separated with a silica gel column.

Step 2, preparation of formula I: Dissolve 0.6 g of compound of formula II in 50 mL of dichloromethane, and add 1.5 mL of triethylamine, stir for 5 minutes, then add dropwise 0.9 g of 2,4- Dinitrobenzenesulfonyl chloride dichloromethane solution 10 mL, after the dropwise addition is complete, continue to stir at room temperature for 10 hours, remove the solvent under reduced pressure, separate and purify the crude product by silica gel column chromatography, and use petroleum ether: ethyl acetate = 4:1 is the eluent elution, and the pure product of the probe molecule is obtained.

(2) NMR spectra of probe molecules

¹ H NMR (400 MHz, CDCl ₃ ): 8.61 (d, 1H, J = 8.4 Hz), 8.48 (d, 1H, J = 8.8 Hz), 8.06 (d, 1H, J = 7.2 Hz), 7.70 (t , 1H, J = 8.0 Hz), 7.46 (t, 1H, J = 8.0 Hz), 7.27 (d, 1H, J = 8.8 Hz), 2.93 (s, 6H).

(3) Determination of the selectivity of probe molecules to sulfhydryl groups

Dissolve the probe molecules in a small amount of DMSO, and then make a solution with absolute ethanol; add the samples to be tested dissolved in absolute ethanol, so that the final concentration of the probe molecules is 0.1 mM, and the concentration of the sample to be tested is 5 mM ; After 2 hours of reaction, it was measured on a fluorescence spectrometer, and then the selectivity of the probe molecule to sulfhydryl was determined.

It can be seen from Figure 2 that the probe molecule has a high selectivity to the active sulfhydryl group and can specifically react with the sulfhydryl group, while common interfering molecules cannot react with the probe molecule. Therefore, the probe molecule can be used in Detection and imaging of sulfhydryl compounds.

(4) Determination of the fluorescence-concentration curve of the probe molecule to thioglycolic acid

Dissolve the probe molecule with a small amount of DMSO, and then configure it into a solution with absolute ethanol; add different concentrations of thioglycolic acid respectively, so that the final concentration of the probe molecule is 0.1 mM; measure it on a fluorescence spectrometer after reacting for 2 hours, Then the curve of fluorescence intensity versus concentration change was obtained.

As shown in Figure 3 and Figure 4, the probe molecule has sufficient detection sensitivity and can meet the requirements of the concentration change of thiol compounds in actual detection.

(5) Measure the fluorescence-time curve of probe molecule to thioglycolic acid

Dissolve the probe molecule with a small amount of DMSO, and then configure it into a solution with absolute ethanol; add thioglycolic acid to make the final concentration of the probe molecule 0.1 mM, while the concentration of thioglycolic acid is 5 mM; react for a certain period of time, The fluorescence intensity is detected, and then the curve of the fluorescence intensity versus time is obtained.

As shown in Figure 5 and Figure 6, the probe molecule has a change in fluorescence intensity after 5 minutes of reaction, indicating that it has sufficient response sensitivity and can fully meet the requirements for the detection of thiol compounds in practical applications.

Example 2:

(1) Preparation of probe molecules

Reaction process is with embodiment 1:

Step 1, preparation of formula II: Dissolve 0.3 g of hydroquinone in 50 mL of dichloromethane, add 0.6 mL of triethylamine, add 0.9 g of dansyl chloride at 0°C, and then stir the reaction at room temperature for 12 hour, after the reaction was finished, the solvent was evaporated to dryness and separated with a silica gel column;

Step 2, preparation of formula I: Dissolve 0.9 g of compound of formula II in 50 mL of dichloromethane, and add 1.5 mL of triethylamine, stir for 5 minutes, then add dropwise 1.4 g of 2,4- Dinitrobenzenesulfonyl chloride dichloromethane solution 10 mL, after the dropwise addition is complete, continue to stir at room temperature for 12 hours, remove the solvent under reduced pressure, separate and purify the crude product by silica gel column chromatography, and use petroleum ether: ethyl acetate = 4:1 is the eluent elution, and the pure product of the probe molecule is obtained.

(2) Determination of the NMR spectrum of the probe molecule

Same as Example 1, the result is as shown in Figure 1.

(3) Determination of the selectivity of probe molecules to sulfhydryl groups

Same as Example 1, as shown in Figure 2, the probe molecule has a high selectivity to the active sulfhydryl group, and can specifically react with the sulfhydryl group, while common interfering molecules cannot react with the probe molecule, therefore, the probe molecule can Applied to the detection and imaging of sulfhydryl compounds.

(4) Determination of the fluorescence-concentration curve of the probe molecule to thioglycolic acid

Same as Example 1, as shown in Figure 3 and Figure 4, the probe molecule has sufficient detection sensitivity and can meet the requirements of the concentration change of thiol compounds in actual detection.

(5) Measure the fluorescence-time curve of probe molecule to thioglycolic acid

Same as Example 1, as shown in Figure 5 and Figure 6, the probe molecule has a change in fluorescence intensity when reacting for 5 minutes, indicating that it has sufficient response sensitivity and can fully meet the requirements for the detection of mercapto compounds in practical applications .

Example 3:

(1) Preparation of probe molecules

Reaction process is with embodiment 1:

Preparation of Formula II: Dissolve 0.4 g of hydroquinone in 50 mL of dichloromethane, add 0.6 mL of triethylamine, add 1.2 g of dansyl chloride at 0°C, then stir and react at room temperature for 15 hours, and wait for After the reaction was completed, the solvent was evaporated to dryness and separated with a silica gel column;

Preparation of formula I: Dissolve 1.2 g of compound of formula II in 50 mL of dichloromethane, and add 1.5 mL of triethylamine, stir for 5 minutes, then add dropwise 1.8 g of 2,4-dinitro 10 mL of dichloromethane solution of benzenesulfonyl chloride, after the dropwise addition is complete, continue to stir at room temperature for 15 hours, remove the solvent under reduced pressure, separate and purify the crude product by silica gel column chromatography, and use petroleum ether: ethyl acetate = 4:1 For the eluent elution, the pure probe molecule is obtained.

(2) Determination of the NMR spectrum of the probe molecule

Same as Example 1, the result is as shown in Figure 1.

(3) Determination of the selectivity of probe molecules to sulfhydryl groups

Same as Example 1, as shown in Figure 2, the probe molecule has a high selectivity to the active sulfhydryl group, and can specifically react with the sulfhydryl group, while common interfering molecules cannot react with the probe molecule, therefore, the probe molecule can Applied to the detection and imaging of sulfhydryl compounds.

(4) Determination of the fluorescence-concentration curve of the probe molecule to thioglycolic acid

Same as Example 1, as shown in Figure 3 and Figure 4, the probe molecule has sufficient detection sensitivity and can meet the requirements of the concentration change of thiol compounds in actual detection.

(5) Measure the fluorescence-time curve of probe molecule to thioglycolic acid

Same as Example 1, as shown in Figure 5 and Figure 6, the probe molecule has a change in fluorescence intensity when reacting for 5 minutes, indicating that it has sufficient response sensitivity and can fully meet the requirements for the detection of mercapto compounds in practical applications .

Claims (2)

1. A method for preparing a fluorescent probe for detecting thiol compounds, characterized in that: the fluorescent probe for detecting biological thiols consists of two parts: 2,4-dinitrobenzenesulfonyl is the recognition group, and dansyl chloride derivatives The substance is an information reporting functional group, and its structure is as in formula I;

The preparation route of the fluorescent probe for detecting biothiol is as follows:

A method for preparing a fluorescent probe for detecting sulfhydryl compounds, comprising the following steps, Step 1, preparation of formula II: Dissolve 0.2-0.4 g of hydroquinone in 50 mL of dichloromethane, then add 0.6 mL of triethylamine, add 0.6-1.2 g of dansyl chloride at 0°C, and then Stir and react at room temperature for 10 to 15 hours. After the reaction is completed, evaporate the solvent to dryness and separate with a silica gel column; Step 2, preparation of formula I: Dissolve 0.6-1.2 g of compound of formula II in 50 mL of dichloromethane, and add 1.5 mL of triethylamine, stir for 5 minutes, then dropwise add 0.9-1.8 g of 10 mL of dichloromethane solution of 2,4-dinitrobenzenesulfonyl chloride, after the dropwise addition is complete, continue to stir at room temperature for 10-15 hours, remove the solvent under reduced pressure, separate and purify the crude product by silica gel column chromatography, and use petroleum Ether: ethyl acetate = 4:1 is the eluent for elution, and the pure product of the probe molecule is obtained. 2. A method of using a fluorescent probe for detecting biothiols may further comprise the steps: Step 1, measuring the NMR spectrum of the probe molecule Step 2, measuring the selectivity of the probe molecule to the sulfhydryl group Dissolve the probe molecules in a small amount of DMSO, and then make a solution with absolute ethanol; add the samples to be tested dissolved in absolute ethanol, so that the final concentration of the probe molecules is 0.1 mM, and the concentration of the sample to be tested is 5 mM ; After 2 hours of reaction, measure on the fluorescence spectrometer, and then determine the selectivity of the probe molecule to the sulfhydryl group; Step 3, measure the fluorescence-concentration curve of probe molecule to thioglycolic acid Dissolve the probe molecule with a small amount of DMSO, and then configure it into a solution with absolute ethanol; add different concentrations of thioglycolic acid respectively, so that the final concentration of the probe molecule is 0.1 mM; measure it on a fluorescence spectrometer after reacting for 2 hours, Then obtain the curve of fluorescence intensity versus concentration change; Step 4, measure the fluorescence-time curve of probe molecule to thioglycolic acid Dissolve the probe molecule with a small amount of DMSO, and then configure it into a solution with absolute ethanol; add thioglycolic acid to make the final concentration of the probe molecule 0.1 mM, while the concentration of thioglycolic acid is 5 mM; react for a certain period of time, The fluorescence intensity is detected, and then the curve of the fluorescence intensity versus time is obtained.

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