CN111718319A

CN111718319A - Fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism and preparation method thereof

Info

Publication number: CN111718319A
Application number: CN202010731175.5A
Authority: CN
Inventors: 黄永飞; 阴彩霞; 霍方俊; 张永斌
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2020-07-27
Filing date: 2020-07-27
Publication date: 2020-09-29

Abstract

The invention provides a fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism, and a preparation method and application thereof. The Chinese name of the fluorescent probe is 10- (diethylamino) -3- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromen-4-yl) oxy) -5,6-dihydrobenzo [ c]Xanthene-12-onium perchlorate, having the english name 10- (diethyleneimine) -3- ((7- (diethyleneimine) -3-formyl-2-oxo-2H-chromen-4-yl) oxy) -5, 6-dihydrobenzol [ c]xanthen-12-ium perchlorate, named CM-O-AC. The invention provides a red fluorescent probe which has multiple binding sites, good water solubility and large Stokes shift. The probe shows three different emission channels for distinguishing and detecting Cys/Hcy and GSH, and is successfully applied to HeLa cells and living bodiesAnd (6) imaging. In addition, the probe can realize the effect on Cys/GSH metabolite SO₂At the cellular level, and at H₂O₂The fluorescence signal is reversible under the action. Probes can be used in physiological and pathological processes as a tool for visualizing biological metabolism. The detection process is simple, sensitive and quick, and the detection result is accurate.

Description

Fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism and preparation method thereof

Technical Field

The invention relates to a fluorescent probe, and particularly belongs to a fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism, and a preparation method and application thereof.

Background

Thiol-containing small molecules, thiol cysteine (Cys), homocysteine (Hcy), and Glutathione (GSH), play critical roles in many physiological and pathological processes. Hcy is an intermediate product in the metabolism of methionine to Cys. Hcy is converted to cysteine in cells via cystathionine mainly by transsulfuration. Cys is catalyzed by cysteine peroxidase (CDO) under aerobic conditions to produce cysteine sulfinic acid, and further catalyzed by cysteine sulfinic acid decarboxylase (CSD) and Aspartate Aminotransferase (AAT) to produce the neurotransmitters taurine and gaseous signal molecule sulfur dioxide, respectively. GSH is one of the major antioxidant substances in cells, synthesized from Cys through a two-step enzymatic reaction by gamma-glutamylcysteine synthetase (GCS) and GSH synthase (GCS). Abnormal cysteine concentrations in the body can cause slow growth in children, cardiovascular diseases and liver damage. However, the effect of Hcy has not been studied yet, and concentration abnormality may cause senile dementia. Glutathione at abnormal concentrations will directly cause cancer. Therefore, real-time differentiation detection of intracellular Cys/Hcy and GSH and visual monitoring of metabolism of Cys/GSH endogenous homeostasis are of great importance.

The fluorescent probe is one of indispensable tools in biomedicine and material science due to the characteristics of simple operation, short reaction time and the like. Many fluorescent probes for the detection of biological thiols have been reported in recent years. Since the three thiols have similar structures, only a few of these fluorescent probes can simultaneously distinguish Cys, Hcy and GSH. Whereas the metabolites of Cys/GSH have been studied almost rarely. H₂O₂As a common active oxygen, it reacts with SO₂In vivo redox cycling processes exist. However, it is well known that only a few fluorescent probes can achieve H₂O₂And SO₂Reversible sensing of (2). Development of differential fluorescenceMethod for detecting H by optical signal₂O₂And SO₂Is of great importance in clinical diagnosis.

In view of the above problems, designing a red fluorescent probe with multiple binding sites, good water solubility and large stokes shift for distinguishing and detecting thiol and monitoring Cys/GSH metabolism has become one of the leading challenges in the current biomedical development.

Disclosure of Invention

The invention aims to provide a fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism and a preparation method thereof.

Another objective of the invention is to use the fluorescent probe for differential detection of thiols and monitoring Cys/GSH metabolism, and the probe can be used for differential detection of Cys/Hcy and GSH in animals besides differential detection of intracellular thiols. In addition, the probe can realize the effect on Cys/GSH metabolite SO₂At the cellular level, and at H₂O₂The fluorescence signal is reversible under the action.

The invention provides a fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism, which is named as 10- (diethylamino) -3- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromen-4-yl) oxy) -5,6-dihydrobenzo [ c ] xanthene-12-perchloric acid onium in the Chinese, named as 10- (diazepamino) -3- ((7- (diazepamino) -3-formamyl-2-oxo-2H-chromen-4-yl) oxy) -5,6-dihydrobenzo [ c ] xanthene-12-iumchloride in the English, named as CM-O-AC, and has the structural formula:

the invention provides a synthetic method of a fluorescent probe for distinguishing and detecting mercaptan and monitoring Cys/GSH metabolism, which comprises the following steps:

(1) equimolar amounts of 4-diethylamino salicylaldehyde and 6-hydroxy-1-tetralone were added to a round-bottom flask, followed by glacial acetic acid and perchloric acid, heating to reflux for 1.5 hours, cooling, and ethyl acetate was poured: standing and filtering the solution of petroleum ether at a ratio of 1:1 to obtain 10- (diethylamino) -3-hydroxy-5, 6-dihydrobenzo [ c ] xanthene 12-perchloric acid onium (compound 1);

(2) dissolving the compound 1 prepared in the step (1) in dichloromethane, adding 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-formaldehyde in an equal molar amount with stirring, then adding triethylamine in a catalytic amount, stirring at room temperature overnight, removing the solvent under reduced pressure, and performing column chromatography on the residue to obtain the target product 10- (diethylamino) -3- ((7- (diethylamino) -3-formyl-2-oxo-2H-chromene-4-yl) oxy) -5,6-dihydrobenzo [ c ] xanthene-12-perchloric acid onium.

And the eluent of the column chromatography is 20: 1 of dichloromethane and methanol.

The invention provides a method for distinguishing and detecting mercaptan, which comprises the following steps:

(1) preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); preparing 2mM Cys, Hcy and GSH solutions by using distilled water respectively;

(2) adding 2mL of PBS buffer solution (pH 7.4) and 10 mu L of fluorescent probe stock solution into a fluorescence cuvette, measuring the fluorescence spectrum of the probe on a fluorescence spectrophotometer, then gradually adding Cys, Hcy and GSH solutions with different volumes, measuring the fluorescence spectrum on the fluorescence spectrophotometer, wherein after Cys and Hcy are added, the probe has two new fluorescence emission peaks (excitation is 380nm) at 480nm and 625nm, and the fluorescence intensity is gradually increased along with the addition of Cys/Hcy until the fluorescence intensity is basically unchanged; while the probe shows a new fluorescence emission peak only at 625nm (excitation at 380nm) after GSH addition. When the excitation is 450nm, Cys/Hcy added by the probe is basically unchanged, and two new fluorescence emission peaks appear at 545nm and 625nm of GSH, so that the differential detection can be realized;

(3) for Cys/Hcy, taking the concentration of Cys/Hcy as an abscissa and the fluorescence intensity of the probe at 480nm as an ordinate, drawing a graph and performing linear fitting to obtain a regression equation of the probe as follows: 11.659x +41.367(y 11.008x +68.748), coefficient of linear dependence R²＝0.9950(R²0.9969), the detection limit was 0.10 μ M (0.02 μ M). And GSH takes the fluorescence intensity of the probe at 545nm as the ordinate, draws a graph and performs linear fitting to obtain the GSHThe regression equation to the probe is: 5.785x +19.507, coefficient of linear correlation R²With 0.9964, the detection limit was 0.04 μ M.

The method for monitoring Cys/GSH metabolism provided by the invention comprises the following steps:

(1) preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); preparing 2mM Cys and GSH solutions with distilled water respectively; 2mM Na was prepared in distilled water₂SO₃Solution (SO)₂The donor of (a);

(2) adding 2mL of PBS buffer (pH 7.4) and 10 μ L of stock solution of fluorescent probe to a fluorescence cuvette, and measuring the fluorescence spectrum of the probe on a fluorescence spectrophotometer; adding a Cys solution with the maximum volume, measuring the fluorescence spectrum of the Cys solution on a fluorescence spectrophotometer, and allowing the probe to have two new fluorescence emission peaks at 480nm and 625nm after adding the Cys; followed by the addition of SO₂The fluorescence quenching is detected at 625nm and the fluorescence intensity is increased at 480nm by the detection of a fluorescence spectrophotometer; finally, H is continuously added on the basis of the test system₂O₂The fluorescence signal at 625nm was recovered;

(3) adding 2mL of PBS buffer (pH 7.4) and 10 μ L of stock solution of fluorescent probe to a fluorescence cuvette, and measuring the fluorescence spectrum of the probe on a fluorescence spectrophotometer; then adding GSH solution with the maximum volume, measuring the fluorescence spectrum of the GSH solution on a fluorescence spectrophotometer, and enabling the probe to generate two new fluorescence emission peaks at 545nm and 625nm after GSH is added; followed by the addition of SO₂The fluorescence at 545nm and 625nm is detected and quenched on a fluorescence spectrophotometer; finally, H is continuously added on the basis of the test system₂O₂The fluorescence signals at 545nm and 625nm were recovered.

Compared with the prior art, the invention has the following advantages and effects:

1. the synthetic method of the fluorescent probe for distinguishing and detecting Cys/Hcy and GSH is simple and convenient to operate;

2. the detection method can realize the distinguishing detection of Cys/Hcy and GSH, and is not interfered by common amino acid; meanwhile, the kit can also be used for distinguishing and detecting Cys/Hcy and GSH under cells and animal skins;

3. the invention can also realize the monitoring of the metabolism of Cys/GSH, has simple detection means and can be realized only by means of a fluorescence spectrometer;

4. the detection signal of the invention is obvious and is the change of multi-channel fluorescence.

Drawings

FIG. 1 nuclear magnetic hydrogen spectrum of fluorescent probe CM-O-AC prepared in example 1

FIG. 2 nuclear magnetic carbon spectrum of fluorescent probe CM-O-AC prepared in example 1

FIG. 3 Mass Spectroscopy of fluorescent probe CM-O-AC prepared in example 1

FIG. 4 fluorescent titration plot of the effect of the fluorescent probe CM-O-AC with Cys/Hcy and GSH

FIG. 5 working curves of the effect of the fluorescent probe CM-O-AC with Cys/Hcy and GSH

FIG. 6 fluorescent histogram of fluorescent probe CM-O-AC with various analytes

FIG. 7 fluorescent probes CM-O-AC with Cys and SO₂-H₂O₂Fluorescence titration map of action

FIG. 8 fluorescent probes CM-O-AC with GSH and SO₂-H₂O₂Fluorescence titration map of action

FIG. 9 cytographic image of fluorescent probe CM-O-AC detecting endogenous Cys/Hcy and GSH

FIG. 10 is a cytographic image of the fluorescent probe CM-O-AC for detecting exogenous Cys/Hcy and GSH

FIG. 11 in vivo imaging of Cys/Hcy and GSH detected by fluorescent probe CM-O-AC

FIG. 12 cytographic imaging of Cys metabolism monitored by fluorescent probe CM-O-AC

FIG. 13 cytographic imaging of fluorescent probe CM-O-AC monitoring of GSH metabolism

Detailed Description

The present invention will be described in further detail with reference to the following examples and drawings, but the scope of the present invention is not limited by the following examples.

Example 1

Preparation and characterization of CM-O-AC:

(1) preparation of compound 1: at 100In a mL round-bottom flask, 4-diethylamino salicylaldehyde (1.93g, 10mmol), 6-hydroxy-1-tetralone (1.62g, 10mmol) and perchloric acid (3mL) were dissolved in 20mL of acetic acid, and the mixture was refluxed for 1.5 hours. After cooling to room temperature, the solution was poured into a mixture of ethyl acetate (15ml) and petroleum ether (15 ml). The precipitate was filtered and washed with ethanol, followed by vacuum drying to give pure compound 1 as a dark purple solid (2.88g, yield: 90%).¹HNMR(600MHz,DMSO-d₆)11.11(s,1H),8.63(s,1H),8.16(d,J＝8.6Hz,1H),7.91(d,J＝9.3Hz,1H),7.41(d,J＝9.3Hz,1H),7.27(s,1H),6.95(d,J＝8.6Hz,1H),6.87(s,1H),3.67(q,J＝6.9Hz,4H),3.01(s,4H),1.24(t,J＝7.0Hz,6H).¹³C NMR(150MHz,DMSO-d₆)164.73,164.31,158.24,155.23,148.36,146.21,132.03,129.40,120.70,117.99,117.67,117.66,116.16,96.14,45.71,40.52,26.98,25.11,12.89.ESI-MS m/z:[M]⁺calcd for 320.1645；Found 320.1645.

(2) Preparation of CM-O-AC: compound 1(0.42g, 1mmol), 4-chloro-7- (diethylamino) -2-oxo-2H-chromene-3-carbaldehyde (0.28g, 1mmol) and triethylamine (208. mu.L, 1.5mmol) were dissolved in dichloromethane (10ml), and the mixture was stirred at room temperature overnight. The solution was concentrated and purified by column chromatography to give CM-O-AC as a dark purple solid (0.42g, yield: 63%).¹H NMR(600MHz,DMSO-d₆)9.87(s,1H),8.70(s,1H),8.24(d, J ═ 8.7Hz,1H),7.96(d, J ═ 9.4Hz,1H),7.49(d, J ═ 9.4Hz,1H),7.43(d, J ═ 9.2Hz,1H),7.39-7.32(m,2H),7.28-7.23(m,1H),6.81(d, J ═ 10.8Hz,1H),6.72(s,1H),3.74-3.66(m,4H),3.52(q, J ═ 6.8Hz,4H),3.03(s,4H),1.25(t, J ═ 7.0Hz,6H),1.15(t, J ═ 7.0, 6H) (fig. 1H)¹³C NMR(150MHz,DMSO-d₆)185.79,164.47,162.51,162.48,162.27,158.74,158.52,155.86,154.17,148.99,145.56,132.39,128.65,127.38,121.19,119.00,118.65,116.12,115.75,111.31,105.61,103.71,97.27,96.12,55.40,45.92,45.07,40.49,26.63,24.82,12.83. (FIG. 2) ESI-MS M/z: [ M]⁺calcd for 563.2540; found 563.2546 (fig. 3)

Example 2

Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, and preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); 2mM Cys/Hcy and GSH solutions were prepared separately with distilled water. Adding 2mL of PBS buffer (pH 7.4) and 10 μ L of fluorescent probe stock solution into a fluorescence cuvette, measuring the fluorescence spectrum of the probe on a fluorescence spectrophotometer, then gradually adding Cys/Hcy and GSH solutions with different volumes, measuring the fluorescence spectrum on the fluorescence spectrophotometer, wherein after Cys and Hcy are added, the probe shows two new fluorescence emission peaks (excitation is 380nm) at 480nm and 625nm, and the fluorescence intensity is gradually increased along with the addition of Cys/Hcy until the fluorescence intensity is basically unchanged (FIGS. 4a and b); whereas the probe showed a new fluorescence emission peak (excitation 380nm) only at 625nm after addition of GSH (fig. 4 c). When the excitation is 450nm, there is essentially no change in Cys/Hcy upon probe addition (FIG. 4d, e), while GSH shows two new fluorescence emission peaks at 545nm and 625nm (FIG. 4f), thus allowing for differential detection.

Example 3

For Cys/Hcy, taking the concentration of Cys/Hcy as an abscissa and the fluorescence intensity of the probe at 480nm as an ordinate, drawing a graph and performing linear fitting to obtain a regression equation of the probe as follows: 11.659x +41.367(y 11.008x +68.748), coefficient of linear dependence R²＝0.9950(R²0.9969), the detection limit was 0.10 μ M (0.02 μ M). And the GSH takes the fluorescence intensity of the probe at 545nm as a vertical coordinate, a graph is drawn and linear fitting is carried out, and the regression equation of the probe is obtained as follows: 5.785x +19.507, coefficient of linear correlation R²With 0.9964, the detection limit was 0.04 μ M. (see FIG. 5)

Example 4

Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, and preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); 2mM Cys/Hcy and GSH solutions were prepared separately with distilled water. 2mL of PBS buffer (pH 7.4) and 10. mu.L of stock solutions of the fluorescent probe were added to a fluorescence cuvette, followed by addition of Cys/Hcy and GSH (1-probe, 21-Cys,22-Hcy,23-GSH) solutions, respectively, and 10 equivalents of other aqueous amino acid solutions (2-Ala,3-Asp,4-Asn,5-Arg,6-Gly,7-Glu,8-Gln,9-His,10-IIe,11-Leu,12-Lys,13-Met,14-Phe,15-Pro,16-Ser,17-Tyr,18-Thr,19-Trp and 20-Val), followed by measurement of fluorescence spectra on a fluorescence spectrometer, as shown in FIG. 6. Experiments prove that the amino acids do not interfere with the detection of Cys/Hcy and GSH by the probe.

Example 5

Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, and preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); 2mM Cys and GSH solutions were prepared separately with distilled water. 2mM Na was prepared in distilled water₂SO₃Solution (SO)₂The donor of (a). Adding 2mL of PBS buffer (pH 7.4) and 10 μ L of stock solution of fluorescent probe to a fluorescence cuvette, and measuring the fluorescence spectrum of the probe on a fluorescence spectrophotometer; adding a Cys solution with the maximum volume, measuring the fluorescence spectrum of the Cys solution on a fluorescence spectrophotometer, and allowing the probe to have two new fluorescence emission peaks at 480nm and 625nm after adding the Cys; followed by the addition of SO₂The fluorescence was quenched at 625nm and increased at 480nm as measured on a fluorescence spectrophotometer (FIG. 7 a); finally, H is continuously added on the basis of the test system₂O₂The fluorescence signal at 625nm was recovered (FIG. 7 b).

Example 6

Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, and preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); 2mM Cys and GSH solutions were prepared separately with distilled water. 2mM Na was prepared in distilled water₂SO₃Solution (SO)₂The donor of (a). Adding 2mL of PBS buffer (pH 7.4) and 10 μ L of stock solution of fluorescent probe to a fluorescence cuvette, and measuring the fluorescence spectrum of the probe on a fluorescence spectrophotometer; then adding GSH solution with the maximum volume, measuring the fluorescence spectrum of the GSH solution on a fluorescence spectrophotometer, and enabling the probe to generate two new fluorescence emission peaks at 545nm and 625nm after GSH is added; followed by the addition of SO₂Fluorescence quenching was found at 545nm and 625nm as measured on a fluorescence spectrophotometer (FIG. 8 a); finally, H is continuously added on the basis of the test system₂O₂The fluorescence signals at 545nm and 625nm were recovered (FIG. 8 b).

Example 7

Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, and preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); 2mM Cys/Hcy and GSH solutions were prepared separately with distilled water. And the probe CM-O-AC distinguishes and detects Cys/Hcy and GSH in HeLa cells. First, cells were incubated with probe CM-O-AC (10 μ M) for 15 minutes, and blue, green and red channels were observed to show weak fluorescent responses by cell imaging (fig. 9B, C, D). Next, cells were incubated with NEM (N-ethylmaleimide, 0.5mM) for 30 min, then with CM-O-AC (10 μ M) and Cys/Hcy/GSH (20 μ M) for 15 min. From the imaging experiments, a significant increase in fluorescence was observed for Cys/Hcy, the blue channel (FIG. 10B, F) and the red channel (FIG. 10C, G). However, there was a fluorescent response for the green and red channels of GSH (fig. 10J, K). Thus, the probe can distinguish the exogenous Cys/Hcy and GSH in the cell level.

Example 8

Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, and preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); 2mM Cys/Hcy and GSH solutions were prepared separately with distilled water. Add 10. mu.L of CM-O-AC in DMSO to 2mL of PBS; we injected the probe solution subcutaneously into mice and observed the fluorescent signal on a live imager; subsequently, Cys/Hcy and GSH solutions were injected at the same site and a significant increase in fluorescence signal was observed over time (0-40min), see FIG. 11(1-Hcy, 2-Cys, 3-GSH).

Example 9

Preparing a PBS buffer solution with the pH value of 7.4 and the concentration of 10mM, and preparing a fluorescent probe stock solution of 2mM CM-O-AC by using dimethyl sulfoxide (DMSO); 2mM Cys and Na were prepared separately from distilled water₂S₂O₃And (3) solution. Probe CM-O-AC detects Cys/GSH metabolic process in HeLa cells. First, cells were incubated with Cys (100. mu.M) and probe CM-O-AC (10. mu.M), and a gradual increase in fluorescence emission was observed in the blue and red channels by cell imaging. The blue fluorescence continued to increase over 60-120 minutes, reflecting the depletion of Cys in the cells. Accordingly, the fluorescence of the red channel is gradually quenched, which means that SO is generated₂(FIG. 12). Second, previous literature reports Na in animals₂S₂O₃Endogenous dioxygen can be produced by TST (thiosulfate-thiotransferase) in combination with GSHAnd (4) vulcanizing. Contacting the cells with Na₂S₂O₃(500. mu.M) was incubated with probe CM-O-AC (10. mu.M), and a gradual increase in fluorescence emission was observed in the green and red channels by cell imaging. This reflects the depletion of GSH in the cells. Subsequently, over 24 minutes, the green fluorescence gradually decreased and the red channel fluorescence gradually quenched, suggesting that endogenous SO was produced₂(FIG. 13). Thus, the probe can realize the detection of Cys/GSH metabolic process at a cellular level.

Claims

1. A fluorescent probe for differentially detecting thiols and monitoring Cys/GSH metabolism, having the structural formula:

2. the method of claim 1, wherein the steps of synthesizing a fluorescent probe for differential detection of thiols and monitoring Cys/GSH metabolism comprise:

3. The method of claim 2, wherein the eluent for the column chromatography is a mixture of 20: 1 of dichloromethane and methanol.

4. A method for differentially detecting thiols, comprising the steps of:

(1) preparing 2mM of the stock solution of the fluorescent probe according to claim 1 with dimethyl sulfoxide (DMSO); 2mM Cys, Hcy and GSH solutions were prepared separately with distilled water.

(2) Adding 2mL of PBS buffer solution (pH 7.4) and 10 mu L of fluorescent probe stock solution into a fluorescence cuvette, measuring the fluorescence spectrum of the probe on a fluorescence spectrophotometer, then gradually adding Cys, Hcy and GSH solutions with different volumes, measuring the fluorescence spectrum on the fluorescence spectrophotometer, wherein after Cys and Hcy are added, the probe has two new fluorescence emission peaks (excitation is 380nm) at 480nm and 625nm, and the fluorescence intensity is gradually increased along with the addition of Cys/Hcy until the fluorescence intensity is basically unchanged; after GSH is added, a new fluorescence emission peak (excitation is 380nm) appears only at 625nm in the probe; when the excitation is 450nm, Cys/Hcy added by the probe is basically unchanged, and two new fluorescence emission peaks appear at 545nm and 625nm of GSH, so that the differential detection can be realized;

(3) for Cys/Hcy, taking the concentration of Cys/Hcy as an abscissa and the fluorescence intensity of the probe at 480nm as an ordinate, drawing a graph and performing linear fitting to obtain a regression equation of the probe as follows: 11.659x +41.367(y 11.008x +68.748), coefficient of linear dependence R²＝0.9950(R²0.9969), detection limit 0.10 μ M (0.02 μ M); and the GSH takes the fluorescence intensity of the probe at 545nm as a vertical coordinate, a graph is drawn and linear fitting is carried out, and the regression equation of the probe is obtained as follows: 5.785x +19.507, coefficient of linear correlation R²With 0.9964, the detection limit was 0.04 μ M.

5. A method of monitoring Cys/GSH metabolism comprising the steps of:

(1) preparing 2mM of the stock solution of the fluorescent probe according to claim 1 with dimethyl sulfoxide (DMSO); preparing 2mM Cys and GSH solutions with distilled water respectively; 2mM Na was prepared in distilled water₂SO₃A solution;

(3) adding 2ml of a buffer solution (pH 7.4) of LPBS and 10. mu.l of a stock solution of a fluorescent probe to a fluorescence cuvette, and measuring the fluorescence spectrum of the probe on a fluorescence spectrophotometer; then adding GSH solution with the maximum volume, measuring the fluorescence spectrum of the GSH solution on a fluorescence spectrophotometer, and enabling the probe to generate two new fluorescence emission peaks at 545nm and 625nm after GSH is added; followed by the addition of SO₂The fluorescence at 545nm and 625nm is detected and quenched on a fluorescence spectrophotometer; finally, H is continuously added on the basis of the test system₂O₂The fluorescence signals at 545nm and 625nm were recovered.

6. Use of the fluorescent probe of claim 1 in the preparation of a reagent for the differential detection of Cys/Hcy and GSH in cells and animals.

7. Use of a fluorescent probe according to claim 1 in the preparation of a metabolic reagent for monitoring Cys/GSH in a cell.

8. Use of the fluorescent probe according to claim 1 for the preparation of reagents for imaging living animals and cells.