CN104673278A - Fluorescence probe for detecting glutathione as well as preparation method and use method of fluorescence probe - Google Patents

Abstract

The invention discloses a fluorescent probe for detecting glutathione, a preparation method and an application method thereof. The invention utilizes 1,8-naphthalimide to construct a classic ICT system, and introduces a phenylsulfoxide part at the 4-position to regulate the ICT effect of the probe molecule. In the absence of glutathione, the probe molecule has no fluorescence emission due to the strong electron-withdrawing effect of the 4-position phenylsulfoxide; while in the presence of glutathione, the phenylsulfoxide part can be absorbed by glutathione Peptide substitution, and then intramolecular electron transfer from the 4-position sulfur atom (from glutathione) to 1,8-naphthoimide, and this ICT effect makes the probe molecule emit strong fluorescence. The invention can realize the detection of intracellular glutathione, and has the advantages of simple operation, low cost, sensitive response, easy popularization and application, and the like.

Description

A fluorescent probe for detecting glutathione and its preparation method and use method

technical field

The invention belongs to the technical field of biological detection, and in particular relates to a naphthalimide-phenylsulfoxide derivative used as a glutathione fluorescent probe material and a preparation method and use method thereof.

Background technique

Glutathione (GSH) is a tripeptide molecule composed of glutamic acid, cysteine and glycine, and is the most abundant thiol-containing amino acid in the human body. In the living body, glutathione plays an important role in maintaining intracellular redox balance, metabolism of exogenous substances, intracellular signal conversion and gene regulation. Abnormality of intracellular glutathione concentration will lead to a series of physiological diseases, such as leukocyte loss, psoriasis, liver damage, cancer and AIDS, etc. in HIV disease, Proc. Natl. Acad. Sci. USA., 1997, 94: 1967-1972). Therefore, quantitative and real-time monitoring of glutathione content in organisms has always been a research hotspot in biochemistry and chemical biology.

Due to its excellent detection sensitivity and selectivity, and the ability to realize real-time and on-line detection of biological samples, fluorescence detection has attracted extensive attention from researchers. Naphthalimide fluorescent molecules have become one of the most important fluorescent precursors for this method because of their unique advantages such as good photostability, high molar extinction coefficient, and quantum yield. a wide range of applications.

The currently developed small molecule fluorescent probes for the detection of glutathione are mainly based on the specificity between sulfhydryl and 2,4-dinitrobenzenesulfonylamino or 2,4-dinitrobenzenesulfonyl ester groups. Designed for sexual chemistry. In the presence of glutathione, the 2,4-dinitrobenzenesulfonylamino or 2,4-dinitrobenzenesulfonyl ester group in the probe molecule can be replaced by the sulfhydryl group in glutathione , the original 2,4-dinitrobenzenesulfonylamino group or 2,4-dinitrobenzenesulfonyl ester group leaves, resulting in a change in the fluorescence properties of the probe molecule, thereby achieving specificity for glutathione Gender.

However, most of the reported glutathione probes are interfered by amino acids that also contain sulfhydryl groups in organisms, such as cysteine (Cysteine, Cys) and homocysteine (Homocysteine, Hcy) (see J. Bouffard, Y.Kim, T.M.Swager, etc., A Highly Selective Fluorescent Probe for Thiol Bioimaging, Org.Lett., 2008, 10: 37-40; X.-D.Jiang, J.Zhang, X.Shao, etc., A selective fluorescent turn-on NIRprobe for cysteine, Org. Biomol. Chem., 2012, 10: 1966-1968), it is difficult to realize the specific detection of glutathione in complex organisms. Therefore, there is an urgent need for a novel fluorescent probe for the detection of glutathione in vivo that has good biological stability and can achieve excellent specific response.

Contents of the invention

In order to overcome the above-mentioned defects in the prior art, the present invention aims to provide a fluorescent probe for detecting glutathione derived from naphthalene imide and phenyl sulfoxide and its preparation method and use method.

The core of the invention is to use 1,8-naphthoimide to construct a classic ICT system, and introduce a phenylsulfoxide moiety at the 4-position to regulate the ICT effect of the probe molecule. In the absence of glutathione, the probe molecule has no fluorescence emission due to the strong electron-withdrawing effect of the 4-position phenylsulfoxide; while in the presence of glutathione, the phenylsulfoxide part can be absorbed by glutathione Peptide substitution, and then intramolecular electron transfer from the 4-position sulfur atom (from glutathione) to 1,8-naphthoimide, and this ICT effect makes the probe molecule emit strong fluorescence. Through the above scheme, a highly glutathione-specific "off-on" type fluorescence response is obtained, which greatly improves the selectivity and sensitivity of detection.

First, in order to achieve the above object, the present invention provides a naphthalimide-phenylsulfoxide derivative as shown in formula (I).

In formula (I), n is the atomic number of 0～18; R ₁ is hydrogen, or methyl, or ethyl, or isopropyl, or fluorine; R ₂ is methyl, or hydroxyl, or carboxyl, or sulfonate Acid group, or (triphenyl) phosphorus group, or any of any kind.

The compound shown in formula (I) is specifically the compound (Nap-G) shown in formula (II),

The preparation method of above-mentioned probe comprises the following steps:

Step 1: Under an inert atmosphere, react 4-bromo-1,8-naphthalic anhydride shown in formula (III) with an amino compound in alcohol to obtain substituted 4-bromo-1,8- Naphthalimide.

In formula (IV), n is the number of atoms from 0 to 18, and R is _methyl , or hydroxyl, or carboxyl, or sulfonic acid, or (triphenyl)phosphoryl, or any of the.

Step 2: Under an inert atmosphere, in the presence of a base, react the compound shown in formula (IV) with the compound shown in formula (V) in alcohol to obtain substituted 4-thiophenol-1 shown in formula (VI), 8-naphthalimide.

In formula (VI), n is the number of atoms from 0 to 18; R ₁ is hydrogen, or methyl, or ethyl, or isopropyl, or fluorine; R ₂ is methyl, or hydroxyl, or carboxyl, or sulfonate acid group, or (triphenyl)phosphoryl group, or any of the.

Step 3: Under an inert atmosphere, react the compound represented by formula (VI) with m-chloroperoxybenzoic acid in an organic solvent to obtain the compound represented by formula (I).

The amino compound described in step 1 is specifically n-butylamine; the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride represented by formula (III) to the amino compound is 1 to 50:1; the alcohol It is methanol, ethanol, n-propanol, isopropanol, n-butanol or ethylene glycol monomethyl ether; the reaction temperature is 50-120 degrees, and the reaction time is 1-20 hours;

As preferably, the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride shown in formula (III) and the amino compound is 10:1; the alcohol is ethanol; the reaction temperature is 80 degrees; the reaction time for 5 hours.

In the above preparation method, the alcohol described in step 2 is methanol, ethanol, propanol, butanol, ethylene glycol monomethyl ether or diethylene glycol monomethyl ether; 4-bromo-1,8- The molar ratio of naphthalimide and thiophenol compound shown in formula (V) is 1～10:1;

The base is an organic base or an inorganic base;

The organic base is triethylamine, pyridine or diisopropylethylamine; the inorganic base is potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or potassium bicarbonate;

The reaction temperature is 0-140 degrees; the reaction time is 1-48 hours.

As preferably, alcohol ethylene glycol monomethyl ether described in step 2; 4-bromo-1,8-naphthalimide shown in formula (IV) and the mol ratio of thiophenol compound shown in formula (V) are 5 : 1; the reaction temperature is 100 degrees; the reaction time is 24 hours.

The molar ratio of m-chloroperoxybenzoic acid described in step (3) to substituted 4-thiophenol-1,8-naphthalimide represented by formula (VI) is 0.5 to 1.5:1;

The organic solvent is chloroform, dichloromethane, acetonitrile, DMF, DMSO or 1,2-dichloroethane;

The reaction temperature is 0-100 degrees; the reaction time is 1-24 hours.

As preferably, the mol ratio of m-chloroperoxybenzoic acid and substituted 4-thiophenol-1,8-naphthalimide shown in formula (VI) is 1:1; the organic solvent is dichloromethane; the reaction The temperature is 25 degrees; the reaction time is 5 hours.

A method for using a fluorescent probe for detecting glutathione; the method specifically includes the following steps:

Step 1: adding the same concentration of the compound represented by the formula (I) to buffer salts with different concentrations of glutathione, and preparing at least three standard solutions containing the compound represented by the formula (I) with different glutathione contents.

The buffer solution shown is any one of phosphate buffer solution, Tris-HCl buffer solution, HEPES buffer solution, boric acid-sodium borate buffer solution, specifically phosphate buffer solution;

The pH value of the standard solution shown is 6-11, specifically 7.2;

The concentration of the compound shown in the formula (I) in the shown standard solution is 1nM～10μM;

The content of glutathione in the standard solution shown is 0.1nM～1mM;

Step 2: Measure the fluorescence emission spectrum of the standard solution respectively, the excitation wavelength is 366nm, the glutathione concentration is taken as the abscissa, and the _I482 is taken as the ordinate to establish a standard curve.

I ₄₈₂ represents the fluorescent emission peak intensity value of the standard solution at a wavelength of 482nm;

Step 3: Add the compound shown in formula (I) to the sample to be tested, control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission under the excitation light with excitation wavelength of 366nm Spectrum, that is, calculate the glutathione content of the sample to be tested according to the standard curve.

The above steps: the fluorescence intensity in step 2 or step 3 is detected on a fluorometer.

The present invention has following characteristics:

1) The fluorescent probe provided by the present invention is a white solid, and the molecular structure characteristics of naphthalimide and sulfoxide ensure the structure and optical stability of the probe.

2) The solution of the fluorescent probe provided by the present invention is sensitive to the concentration of glutathione. As the concentration of glutathione increases, the fluorescence of its aqueous solution changes from colorless to bright green under ultraviolet light.

3) The fluorescent probe provided by the present invention has an emission wavelength of 482nm and belongs to the "off-on" type of fluorescence response, which can greatly eliminate the influence of background differences on the results during detection and improve the sensitivity of detection.

4) The fluorescent probe provided by the present invention has excellent response sensitivity to glutathione, and commonly coexisting amino acids containing sulfhydryl groups, such as cysteine and homocysteine, do not interfere with the detection.

5) The fluorescent probe provided by the present invention has a linear relationship with the concentration of glutathione, so as to be used for accurate measurement of glutathione.

The "off-on" glutathione probe of naphthalimide dyes provided by the present invention and its kit have good response to glutathione solution, and can realize the detection of intracellular glutathione, It has the advantages of simple operation, low cost, sensitive response, easy popularization and application, and the like.

Description of drawings

Fig. 1 is the synthetic route of fluorescent probe Nap-G prepared in Example 1.

Fig. 2 is the color response diagram of the Nap-G kit prepared in Example 6 to glutathione aqueous solution.

Fig. 3 is the fluorescence response graph of the Nap-G kit prepared in Example 6 to different glutathione aqueous solutions.

Fig. 4 is the ratio I ₄₈₂ of the fluorescence emission intensity of the Nap-G kit prepared in Example 6 at a wavelength of 482nm and the glutathione concentration curve.

Fig. 5 is a graph showing the fluorescence response of the Nap-G kit prepared in Example 6 to common coexisting ions or small biomolecules.

Fig. 6 is the fluorescence imaging figure of the Nap-G kit prepared in embodiment 6 to intracellular glutathione; Wherein, (a) is the cell fluorescence imaging figure before adding Nap-G; (b) is adding Nap- Cell fluorescence imaging after G; (c) is cell fluorescence imaging after adding Nap-G and glutathione.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

As shown in Figure 1, the preparation of embodiment 1, fluorescent probe Nap-G

Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 100mL of absolute ethanol, then inject 2.5mL of n-butylamine, and reflux at a temperature of 50°C for 6 Hour. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 4.20 g of intermediate N-n-butyl-4-bromo-1,8-naphthalimide (80% yield).

Step b): Under an inert atmosphere, add 1.00g of N-n-butyl-4-bromo-1,8-naphthalimide and 1.87g of p-methylthiophenol into a 50mL three-necked bottle, inject 20mL of ethylene di Alcohol monomethyl ether and 2.1 mL of triethylamine were reacted under reflux for 5 hours at a temperature of 0°C. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 0.967g (yield 85%), yellow solid powder.

¹ H NMR (400MHz, CDCl ₃ ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).

Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalimide in 20mL of dichloromethane, and add to the system in three batches Add 0.45g m-chloroperoxybenzoic acid to it, and react at room temperature for 1 hour. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.6 g of the final product Nap-G (62% yield), a white solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H), 1.42-1.28(m, 2H), 0.88(t, J=7.4Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ163.50, 163.28, 147.97, 142.62, 140.95, 131.49, 130.63, 130.38, 128.33 ,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82; HRMS(ESI-TOF):m/z 391.1245[M+H] ⁺ ,calc'd.

Embodiment 2, the preparation of fluorescent probe Nap-G

Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalic anhydride to 100mL of anhydrous methanol, then inject 5.0mL of n-butylamine, and reflux at a temperature of 80°C for 1 Hour. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 4.40 g of intermediate N-n-butyl-4-bromo-1,8-naphthalimide (yield 84%).

Step b): Under an inert atmosphere, add 1.00g N-n-butyl-4-bromo-1,8-naphthalimide and 3.94g p-methylthiophenol into a 50mL three-necked bottle, inject 20mL butanol and 2.5 mL of diisopropylethylamine was refluxed at 100°C for 1 hour. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 1.0 g (yield 88%), yellow solid powder.

¹ H NMR (400MHz, CDCl ₃ ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).

Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylthio)-1,8-naphthalimide in 20mL of chloroform, and add to the system in three batches 0.45g m-chloroperoxybenzoic acid was reacted for 5 hours at a temperature of 0°C. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.56 g (57% yield) of the final product Nap-G, a white solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H), 1.42-1.28(m, 2H), 0.88(t, J=7.4Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ163.50, 163.28, 147.97, 142.62, 140.95, 131.49, 130.63, 130.38, 128.33 ,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82; HRMS(ESI-TOF):m/z 391.1245[M+H] ⁺ ,calc'd.

Embodiment 3, the preparation of fluorescent probe Nap-G

Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalic anhydride to 100mL of anhydrous n-propanol, then inject 7.5mL of n-butylamine, and reflux at a temperature of 120°C React for 5 hours. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 4.60 g of intermediate N-n-butyl-4-bromo-1,8-naphthoimide (yield 87%).

Step b): Under an inert atmosphere, add 1.00g of N-n-butyl-4-bromo-1,8-naphthalimide and 0.38g of p-methylthiophenol into a 50mL three-necked bottle, inject 20mL of diethyl Glycol monomethyl ether and 2.9 g of potassium carbonate were refluxed for 48 hours at a temperature of 140°C. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 0.67g (yield 59%), yellow solid powder.

¹ H NMR (400MHz, CDCl ₃ ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).

Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylthio)-1,8-naphthalimide in 20mL of 1,2-dichloroethane, Add 0.23g m-chloroperoxybenzoic acid to the system in three batches, and react at 0°C for 24 hours. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain the final product Nap-G 0.32g (yield 34%), white solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H), 1.42-1.28(m, 2H), 0.88(t, J=7.4Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ163.50, 163.28, 147.97, 142.62, 140.95, 131.49, 130.63, 130.38, 128.33 ,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82; HRMS(ESI-TOF):m/z 391.1245[M+H] ⁺ ,calc'd.

Embodiment 4, the preparation of fluorescent probe Nap-G

Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 100mL of anhydrous n-butanol, then inject 10.0mL of n-butylamine, and reflux at a temperature of 50°C React for 20 hours. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 4.84 g of intermediate N-n-butyl-4-bromo-1,8-naphthoimide (yield 88%).

Step b): Under an inert atmosphere, add 1.00g of N-n-butyl-4-bromo-1,8-naphthalimide and 2.87g of p-methylthiophenol into a 50mL three-necked bottle, inject 20mL of ethanol and 3.5g of potassium hydroxide was refluxed for 12 hours at a temperature of 100°C. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 0.84g (yield 74%), yellow solid powder.

¹ H NMR (400MHz, CDCl ₃ ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).

Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalimide in 20mL of DMSO, and add to the system in three batches 0.30g m-chloroperoxybenzoic acid was reacted at 100°C for 1 hour. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.23 g (yield 24%) of the final product Nap-G, a white solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H), 1.42-1.28(m, 2H), 0.88(t, J=7.4Hz, 3H); ¹³ C NMR (100MHz, CDCl ₃ ) δ163.50, 163.28, 147.97, 142.62, 140.95, 131.49, 130.63, 130.38, 128.33 ,127.96,127.49,125.64,124.99,123.70,123.50,40.41,30.12,21.41,20.34,13.82; HRMS(ESI-TOF):m/z 391.1245[M+H] ⁺ ,calc'd.

Embodiment 5, the preparation of fluorescent probe Nap-G

Step a): Under an inert atmosphere, add 5.00g of 4-bromo-1,8-naphthalene dicarboxylic anhydride to 100mL of anhydrous ethylene glycol monomethyl ether, then inject 0.5mL of n-butylamine, at a temperature of 80°C Down, reflux reaction for 20 hours. After the reaction was complete, needle-like crystals precipitated after standing overnight, filtered and washed three times with cold ethanol to obtain 3.2 g of intermediate N-n-butyl-4-bromo-1,8-naphthalimide (yield 60%).

Step b): Under an inert atmosphere, add 1.00g N-n-butyl-4-bromo-1,8-naphthalimide and 2.15g p-methylthiophenol into a 50mL three-necked flask, inject 20mL propanol and 2.0 mL of pyridine was refluxed for 8 hours at a temperature of 50°C. After the reaction is complete, the reaction solution is poured into 200mL of ice water, a large amount of solids precipitate out, filtered, washed, and dried in vacuo to obtain N-n-butyl-4-(p-methylphenylsulfanyl)-1,8-naphthalene Amine 0.76g (67% yield), yellow solid powder.

¹ H NMR (400MHz, CDCl ₃ ) δ8.63(d, J=7.9Hz, 2H), 8.32(d, J=7.9Hz, 1H), 7.77(t, J=7.9Hz, 1H), 7.45(d ,J=7.9Hz,2H),7.28(d,J=7.9Hz,2H),7.16(d,J=7.9Hz,1H),4.22-4.09(m,2H),2.43(s,3H),1.70 (dt, J = 15.2, 7.6 Hz, 3H), 1.44 (dq, J = 14.8, 7.3 Hz, 3H), 0.97 (t, J = 7.3 Hz, 4H).

Step c): Under an inert atmosphere, dissolve 0.9g of N-n-butyl-4-(p-methylphenylthio)-1,8-naphthalimide in 20mL of acetonitrile, and add to the system in three batches 0.45g m-chloroperoxybenzoic acid was reacted at room temperature for 2 hours. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.55 g (57% yield) of the final product Nap-G as a white solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.65 (d, J=7.7Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 8.40 (dd, J=8.1, 2.7Hz, 2H), 7.68 (t, J=7.9Hz, 1H), 7.49(d, J=8.1Hz, 2H), 7.13(d, J=8.0Hz, 2H), 4.16-3.96(m, 2H), 1.68-1.53(m, 2H),1.42-1.28(m,2H),0.88(t,J=7.4Hz,3H); ¹³ CNMR(100MHz,CDCl ₃ )δ163.50,163.28,147.97,142.62,140.95,131.49,130.63,130.38,128.33, 127.96, 127.49, 125.64, 124.99, 123.70, 123.50, 40.41, 30.12, 21.41, 20.34, 13.82; HRMS (ESI-TOF): m/z 391.1245[M+H] ⁺ , calc'd.391.1242.

Spectral properties of the compound shown in embodiment 6, formula (I)

Weigh 3.9mg of Nap-G, dissolve it in 10mL DMSO, and prepare the mother solution (1mM) to obtain the Nap-G kit. Add 100 μL of this mother solution dropwise to phosphate buffer saline with different concentrations of glutathione, and dilute to 10 mL with the corresponding phosphate buffer. Measure its fluorescence emission spectrum. When measuring the fluorescence emission spectrum, excitation was performed at 366 nm, and the intensity of the emission peak was I ₄₈₂ ; the slit widths of excitation and emission were 5/5, respectively.

Figure 2 is the color response diagram of Nap-G kit to glutathione aqueous solution. As can be seen from Figure 2, after the glutathione aqueous solution was added, the naked eye observed that the color of the solution did not change significantly, but the fluorescence of the solution changed from no fluorescence when no glutathione was added to bright green fluorescence after addition. It is proved that the kit of the present invention has an intuitive color response to glutathione.

Fig. 3 is the fluorescence response graph of Nap-G kit to different glutathione aqueous solutions. As can be seen from Figure 3, with the increase of glutathione concentration, the fluorescence intensity of the emission peak at a wavelength of 482nm gradually increases, which proves that the kit of the present invention has a sensitive "off-on" type response to glutathione.

Fig. 4 is the curve of the relationship between the fluorescence emission intensity I ₄₈₂ and the glutathione concentration of the Nap-G kit at a wavelength of 482nm. It can be seen from Figure 4 that with the increase of glutathione concentration in the aqueous solution, the fluorescence emission intensity I ₄₈₂ increases gradually. When the glutathione concentration is in the range of 0-1 mM, the fluorescence emission intensity I ₄₈₂ of the emission peak has a good linear relationship with the concentration of glutathione in the aqueous solution (R ² =0.995). The kit of the invention is proven to perform accurate measurements of glutathione.

Figure 5 is the fluorescence response graph of the Nap-G kit to common coexisting ions or small biological molecules. It can be known from Figure 5 that the addition of common coexisting cations, anions, and small biomolecules cannot change the fluorescence emission intensity I ₄₈₂ of the solution. It is proved that the kit of the present invention has excellent selectivity to glutathione.

Embodiment 7, the mensuration of intracellular glutathione content

1) Under the conditions of 37 degrees and 5% (v/v) CO ₂ , use DMEM medium containing 10% (v/v) FBS (fetal bovine serum), 100 U/mL penicillin, and 100 μg/mL streptomycin Culture HeLa cells. Cells were washed with PBS buffer before use.

2) Add PBS (pH 7.4) to HeLa cells, then add Nap-G (10 μM) and incubate for 30 minutes, wash with PBS three times, and perform confocal fluorescence imaging, where the excitation wavelength is 366nm and the collection wavelength is 400-650nm. Then, add GSH (1mM) phosphate-buffered saline solution to the above HeLa cells, continue to incubate for 30min, and perform imaging on a laser confocal microscope, wherein the excitation wavelength is 366nm, and the collection waveband is 400-650nm.

As can be seen from Figure 6, before the addition of glutathione, the cells loaded with Nap-G were in the "off" state of fluorescence, without fluorescence emission; however, after adding glutathione, the cells were in the state of "on" fluorescence, It emits green fluorescence, indicating that Nap-G can penetrate the cell membrane well and respond specifically to glutathione in the cell. The kit of the present invention is demonstrated to detect glutathione in cells.

Finally, it should be noted that the above examples only use the Nap-G compound as the fluorescent reagent, and the concentration and experimental excitation band selection of the remaining fluorescent reagents are not listed one by one due to their similar structures and properties. invention. Any person skilled in the art should make various modifications and changes without departing from the spirit and scope of the present invention.

Claims (7)

1. a fluorescent probe for detecting glutathione, characterized in that: the probe is a compound shown in formula (I), In formula (I), n is the atomic number of 0～18; R ₁ is hydrogen, or methyl, or ethyl, or isopropyl, or fluorine; R ₂ is methyl, or hydroxyl, or carboxyl, or sulfonate acid group, or (triphenyl)phosphoryl group, or any of the. 2. a kind of fluorescent probe that detects glutathione according to claim 1 is characterized in that: this probe structural formula is as formula (II), 3. the preparation method of a kind of fluorescent probe that detects glutathione described in claim 1 is characterized in that, the method comprises the following steps: Step 1: Under an inert atmosphere, react 4-bromo-1,8-naphthalic anhydride shown in formula (III) with an amino compound in alcohol to obtain substituted 4-bromo-1,8- Naphthalimide; In formula (IV), n is the number of atoms from 0 to 18, and R is _methyl , or hydroxyl, or carboxyl, or sulfonic acid, or (triphenyl)phosphoryl, or any of the Step 2: Under an inert atmosphere, in the presence of a base, react the compound shown in formula (IV) with the compound shown in formula (V) in alcohol to obtain substituted 4-thiophenol-1 shown in formula (VI), 8-naphthalimide; In formula (VI), n is the number of atoms from 0 to 18; R ₁ is hydrogen, or methyl, or ethyl, or isopropyl, or fluorine; R ₂ is methyl, or hydroxyl, or carboxyl, or sulfonate acid group, or (triphenyl)phosphoryl group, or any of the Step 3: Under an inert atmosphere, react the compound represented by formula (VI) with m-chloroperoxybenzoic acid in an organic solvent to obtain the compound represented by formula (I). 4. a kind of preparation method that detects the fluorescent probe of glutathione according to claim 3, is characterized in that: The amino compound described in step 1 is specifically n-butylamine; the molar ratio of 4-bromo-1,8-naphthalene dicarboxylic anhydride represented by formula (III) to the amino compound is 1 to 50:1; the alcohol It is methanol, ethanol, n-propanol, isopropanol, n-butanol or ethylene glycol monomethyl ether; the reaction temperature is 50-120 degrees, and the reaction time is 1-20 hours; The alcohol described in step 2 is methanol, ethanol, propanol, butanol, ethylene glycol monomethyl ether or diethylene glycol monomethyl ether; 4-bromo-1,8-naphthalimide shown in formula (IV) The molar ratio of the thiophenol compound shown in formula (V) is 1 to 10:1; the base is an organic base or an inorganic base; the organic base is triethylamine, pyridine or diisopropylethylamine ; The inorganic base is potassium carbonate, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or potassium bicarbonate; the reaction temperature is 0 to 140 degrees; the reaction time is 1 to 48 hours; The molar ratio of m-chloroperoxybenzoic acid described in step (3) to substituted 4-thiophenol-1,8-naphthalimide shown in formula (VI) is 0.5～1.5:1; the organic The solvent is chloroform, dichloromethane, acetonitrile, DMF, DMSO or 1,2-dichloroethane; the reaction temperature is 0-100 degrees; the reaction time is 1-24 hours. 5. a kind of preparation method that detects the fluorescent probe of glutathione according to claim 3 or 4, is characterized in that: In step one, the molar ratio of 4-bromo-1,8-naphthalic anhydride shown in formula (III) to the amino compound is 10:1; the alcohol is ethanol; the reaction temperature is 80 degrees; the reaction The time is 5 hours; The alcohol ethylene glycol monomethyl ether described in step 2; the molar ratio of 4-bromo-1,8-naphthalimide shown in formula (IV) and thiophenol compound shown in formula (V) is 5:1; The reaction temperature is 100 degrees; the reaction time is 24 hours; In step 3, the molar ratio of m-chloroperoxybenzoic acid to substituted 4-thiophenol-1,8-naphthalimide represented by formula (VI) is 1:1; the organic solvent is dichloromethane; The reaction temperature is 25 degrees; the reaction time is 5 hours. 6. A method for using a fluorescent probe for detecting glutathione; it is characterized in that the method specifically comprises the following steps: Step 1: Add the same concentration of the compound shown in formula (I) to the buffer solutions of different concentrations of glutathione, and configure at least 3 standard solutions containing the compound shown in formula (I) with different glutathione contents; Shown buffer solution is any one in phosphate buffer solution, Tris-HCl buffer solution, HEPES buffer solution, boric acid-sodium borate buffer solution, The pH value of the standard solution shown is 6-11, specifically 7.2; The concentration of the compound shown in the formula (I) in the shown standard solution is 1nM～10μM; The content of glutathione in the standard solution shown is 0.1nM～1mM; Step 2: Measure the fluorescence emission spectrum of the standard solution respectively, the excitation wavelength is 366nm, take the glutathione concentration as the abscissa, and take _I482 as the ordinate to establish a standard curve; I ₄₈₂ represents the fluorescent emission peak intensity value of the standard solution at a wavelength of 482nm; Step 3: Add the compound shown in formula (I) to the sample to be tested, control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission under the excitation light with excitation wavelength of 366nm Spectrum, that is, calculate the glutathione content of the sample to be tested according to the standard curve; The above steps: the fluorescence intensity in step 2 or step 3 is detected on a fluorometer. 7. The method for using a fluorescent probe for detecting glutathione according to claim 6; it is characterized in that: the buffer solution in step 1 is a phosphate buffer solution.