CN104610960B - A kind of fluorescent probe of detection cysteine and preparation method thereof and using method - Google Patents

Abstract

The invention discloses a kind of fluorescent probe of detection cysteine and preparation method thereof and using method.Benzo BODIPY of the present invention using launch wavelength near infrared region is fluorogen, and in 3 introducing benzene sulfoxide moieties for being easy to modify.Under conditions of it there is cysteine, benzene sulfoxide moiety is replaced by cysteine leaves away, and then obtains the benzo BODIPY of cysteine replacement." ratiometer type " the cysteine probe and its dedicated test test kit of two pyrroles's methine class dyestuff of fluorine boron that the present invention is provided has good response to cysteine solution, the detection to intracellular cysteine can be realized, with easy to operate, it is with low cost, response is sensitive, it is easy to the advantages of promotion and application.

Description

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

technical field

The invention belongs to the technical field of biological detection, and specifically relates to a fluoroboron dipyrromethene-phenylsulfoxide derivative used as a cysteine fluorescent probe material and a preparation method and use method thereof.

Background technique

Cysteine (Cys), glutathione (GSH) and homocysteine (Hcy) belong to intracellular reactive sulfur species (RSS), and participate in intracellular redox reactions, etc. A variety of signal transduction processes play an important role in the physiological and pathological processes of living organisms. When intracellular cysteine is at an abnormal level, it will cause a series of physiological problems such as liver damage, cardiovascular disease, and nervous system degenerative diseases (see N.J.Pace and E.Weerapana, Diverse Functional Roles of Reactive Cysteines, ACS Chem . Biol., 2013, 8: 283-296). Therefore, effective detection or monitoring of cysteine in biological samples or environmental samples has become a research hotspot in related fields in recent years.

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. Fluorescent boron dipyrromethene (BODIPY) fluorescent molecules have become one of the most important fluorescent precursors for this method because of their unique advantages such as good photostability, narrow absorption and emission wavelengths, high molar extinction coefficient and quantum yield. , has been widely used in the fluorescence detection of various molecules to be detected.

The currently developed small-molecule fluorescent probes for the detection of cysteine 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 cysteine, the 2,4-dinitrobenzenesulfonylamino group or 2,4-dinitrobenzenesulfonyl ester group in the probe molecule can be replaced by the sulfhydryl group in cysteine , 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 cysteine Gender.

However, most of the reported cysteine probes are interfered by amino acids that also contain sulfhydryl groups in organisms, such as homocysteine (Hcy) and glutathione (GSH) (see J.Bouffard, Y. Kim, T.M. Swager et al., A Highly Selective Fluorescent Probe for Thiol Bioimaging, Org. Lett., 2008, 10:37-40). In addition, most of these reported cysteine probes are fluorescent "off-on" or "on-off" type (see Y.Kim, M.Choi, S.Seo et al., A Selective Fluorescent Probe for Cysteine and Its Imaging in LiveCells, RSC Adv., 2014, 4: 64183-64186), susceptible to the detection environment, such as detection temperature, probe concentration and other conditions, it is difficult to achieve the specificity of cysteine in complex organisms Sex detection. Therefore, there is an urgent need for a novel cysteine fluorescent probe that has good biological stability and can realize "ratiometer" detection.

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 cysteine derived from fluoroborate dipyrromethene and phenylsulfoxide, as well as its preparation method and use method.

The core of the invention is to use benzo BODIPY whose emission wavelength is in the near-infrared region as a fluorophore, and introduce a phenylsulfoxide moiety at the easily modified 3-position. When there is cysteine, the phenylsulfoxide part is replaced by cysteine, and then cysteine-substituted benzo BODIPY is obtained (the sulfhydryl group on cysteine is connected to the benzo BODIPY at this time). 3-bit). Since the fluorescence intensities of the two compounds before and after the reaction are not much different but the fluorescence emission wavelengths are different, an ideal "ratiometer" fluorescence response is obtained through the above scheme, which greatly improves the detection sensitivity.

First, in order to achieve the above purpose, the present invention provides a fluoroboron dipyrromethene-phenylsulfoxide derivative represented by formula (I).

In formula (I), R ₁ is hydrogen, or any one of methyl; R ₂ is hydrogen, or methyl, or ethyl, or isopropyl, or any one of fluorine.

The compound shown in formula (I) is specifically the compound (BOD-C) shown in formula (II),

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

(1) Under the catalysis of phosphorus oxychloride, 3-chloro-isoindole-1-aldehyde shown in formula (III) reacts with pyrroles in an organic solvent to obtain substituted 3-chlorobenzene shown in formula (IV) And BODIPY.

In formula (IV), R ₁ is hydrogen, or any one of methyl.

(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 an organic solvent to obtain substituted 3-(4-R ₂ - phenylthio)-benzo BODIPY.

In formula (V), R ₂ is hydrogen, or methyl, or ethyl, or isopropyl, or any one of fluorine; In formula (VI), R ₁ is hydrogen, or any one of methyl Species; R ₂ is any one of hydrogen, or methyl, or ethyl, or isopropyl, or fluorine.

(3) Under an inert atmosphere, under an inert atmosphere, the substituted 3-(4-R ₂ -phenylthio)-benzo BODIPY shown in formula (VI) reacts with m-chloroperoxybenzoic acid in an organic solvent. The compound represented by formula (I) is obtained.

In the above preparation method, the organic solvent described in step (1) is dichloromethane, acetonitrile, 1,2-dichloroethane or tetrahydrofuran;

The pyrrole compound is pyrrole, 2-methylpyrrole or 2,4-dimethylpyrrole;

The molar ratio of 3-chloro-isoindole-1-aldehyde represented by formula (III) to the pyrrole compound is 1 to 0.1:1;

The molar ratio of 3-chloro-isoindole-1-aldehyde represented by formula (III) to phosphorus oxychloride is 0.5～5:1;

The reaction temperature is 0-80 degrees; the reaction time is 1-48 hours;

As a priority: the organic solvent described in step (1) is dichloromethane; the pyrrole compound is 2,4-dimethylpyrrole; 3-chloro-isoindole-1-aldehyde shown in formula (III) The molar ratio with the pyrrole compound is 0.5:1; the molar ratio of 3-chloro-isoindole-1-aldehyde shown in formula (III) to phosphorus oxychloride is 1:1; the reaction temperature is 25 Degree; reaction time is 24 hours.

In the above preparation method, the molar ratio of the base to the compound represented by formula (V) in step (2) is 1 to 5:1;

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 molar ratio of the substituted thiophenol represented by formula (V) to the compound represented by formula (IV) is 1 to 20:1;

The reaction temperature is 0-40 degrees, and the reaction time is 0.1-2 hours;

The reaction solvent in step 2 is an organic solvent; the organic solvent is dichloromethane, acetonitrile, 1,2-dichloroethane, tetrahydrofuran or DMF;

As preferably: the molar ratio of base described in step (2) to the compound shown in formula (V) is 2.5:1; The molar ratio of substituted thiophenol shown in formula (V) and the compound shown in formula (IV) is 10 : 1; the reaction temperature is 25 degrees; the reaction time is 1 hour; the organic solvent is dichloromethane.

In the above preparation method, the molar ratio of m-chloroperoxybenzoic acid described in step (3) to substituted 3-(4-R ₂ -thiophenolyl)-benzo BODIPY represented by formula (VI) is 1 to 5 :1;

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

The reaction temperature is 0 to 50 degrees, and the reaction time is 1 to 24 hours;

As a preference: the molar ratio of m-chloroperoxybenzoic acid described in step (3) to substituted 3-(4-R ₂ -thiophenolyl)-benzo BODIPY represented by formula (VI) is 1.5:1; The organic solvent is dichloromethane; the reaction temperature is 25 degrees; the reaction time is 4 hours.

The present invention further provides a kit for detecting cysteine, comprising the compound represented by formula (I) and a solvent.

The concentration of the compound represented by formula (I) is 0.001 mM to 100 mM, specifically 1 mM.

The solvent is any one of water, ethanol, and dimethyl sulfoxide.

The compound represented by formula (I) or the above kit is applied to the detection of cysteine in aqueous solution.

The content of cysteine in the aqueous solution is specifically detected through the following steps:

(1) Add the same concentration of the compound represented by the formula (I) to buffer salts with different concentrations of cysteine, and configure at least three standard solutions containing the compound represented by the formula (I) with different cysteine 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 5-12, specifically 7.2;

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

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

(2) Measure the fluorescence emission spectrum of the standard solution respectively, the excitation wavelength is 530nm, take the cysteine concentration as the abscissa, and take I ₅₈₄ /I ₅₅₂ or I ₅₅₂ /I ₅₈₄ as the ordinate to establish a standard curve.

I ₅₈₄ represents the fluorescent emission peak intensity value of the standard solution at a wavelength of 584nm;

I ₅₅₂ represents the fluorescent emission peak intensity value of the standard solution at a wavelength of 552nm;

(3) Add the compound shown in formula (I) in the sample to be measured, control its concentration and the concentration of the compound shown in formula (I) in described standard solution to be equal; Measure its fluorescence emission under the excitation light of 530nm at excitation wavelength Spectrum, that is, calculate the cysteine content of the sample to be tested according to the standard curve.

The fluorescence intensity in the above 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 red solid with good structure and optical stability.

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

3) The fluorescent probe provided by the present invention has emission wavelengths of 552nm and 584nm, and has a dual-wavelength response, which can greatly eliminate the influence of differences in detection conditions during detection and improve detection sensitivity.

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

The "ratiometer type" cysteine probe of BODIPY dyes provided by the present invention and its kit have good response to cysteine solution, can realize the detection of intracellular cysteine, and are easy to operate, It has the advantages of low cost, sensitive response, and easy promotion and application.

Description of drawings

Fig. 1 is the synthesis route of the fluorescent probe BOD-C prepared in Example 1.

Fig. 2 is the color response graph of the BOD-C kit prepared in Example 6 to cysteine aqueous solution.

Fig. 3 is the fluorescence response diagram of the BOD-C kit prepared in Example 6 to different cysteine aqueous solutions.

Fig. 4 is the relationship curve between the ratio I ₅₅₂ /I ₅₈₄ of the fluorescence emission intensity of the BOD-C kit prepared in Example 6 and the concentration of cysteine at wavelengths of 552nm and 584nm.

Fig. 5 is a diagram of the fluorescence response of the BOD-C kit prepared in Example 6 to common coexisting ions or small biomolecules.

Fig. 6 is the fluorescence imaging picture of cysteine in the cell to the BOD-C kit prepared in embodiment 6; Wherein, (a) is the cell fluorescence imaging picture before adding BOD-C; (b) is adding BOD- Cell fluorescence imaging image after C; (c) is cell fluorescence imaging image after adding BOD-C and cysteine.

detailed description

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 BOD-C

Step a): Under an inert atmosphere, add 0.5 g of 3-chloro-isoindole-1-al and 0.23 mL of 2,4-dimethylpyrrole into 10 mL of anhydrous dichloromethane, then add 0.25 mL of trichloro Oxyphosphorus, stirred in an ice bath for 30 minutes, then added 3.9 mL of anhydrous triethylamine and 3.9 mL of boron trifluoride ether, and stirred at room temperature for 24 hours. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.74 g of intermediate 3-chlorobenzo BODIPY (87% yield), a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.69(d, J=8.2Hz, 1H), 7.65(d, J=8.2Hz, 1H), 7.43(t, J=7.6Hz, 1H), 7.30-7.23 (m,2H), 7.19(s,1H), 5.95(s,1H), 2.48(s,3H), 2.20(s,3H).

Step b): Under an inert atmosphere, 0.5 g of 3-chlorobenzo BODIPY, 0.25 g of p-methylthiophenol and 1.0 mL of anhydrous triethylamine were dissolved in 10 mL of anhydrous dichloromethane, and stirred at room temperature for 10 minutes. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.59 g (87% yield) of 3-(4-methylphenylthio)benzo-BODIPY as a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.65(d, J=7.9Hz, 1H), 7.44(d, J=7.6Hz, 2H), 7.27(t, J=7.3Hz, 1H), 7.18(s ,1H),7.10(d,J=7.5Hz,2H),6.91(t,J=7.5Hz,1H),6.74(d,J=8.2Hz,1H),5.93(s,1H),2.48(s ,3H),2.30(s,3H),2.20(s,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,124.240,12 , 122.57, 118.01, 116.65, 113.93, 20.28, 13.48, 10.25.

Step c): Under an inert atmosphere, dissolve 0.15g of 3-(4-methylphenylthio)benzo BODIPY in 10mL of dichloromethane, add 0.10g of m-chloroperoxybenzoic acid to the system in three batches, React at 0°C for 4 hours. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 0.08 g of the final product BOD-C (yield 52%), a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.19(d, J=8.4Hz, 1H), 7.80(d, J=8.2Hz, 2H), 7.68(d, J=8.2Hz, 1H), 7.38(s ,1H),7.31(t,J=7.6Hz,1H),7.20(d,J=6.0Hz,3H),7.15(t,J=7.7Hz,1H),6.08(s,1H),2.55(s ,3H), 2.27(s,3H), 2.25(s,3H); HRMS (ESI-TOF): m/z 409.1360[M+H] ⁺ , calc'd.409.1357.

Embodiment 2, the preparation of fluorescent probe BOD-C

Step a): Under an inert atmosphere, add 0.5g of 3-chloro-isoindole-1-al and 0.54mL of 2-methylpyrrole into 10mL of anhydrous acetonitrile, then add 1.25mL of phosphorus oxychloride, ice-bath After stirring at low temperature for 30 minutes, 3.9 mL of anhydrous triethylamine and 3.9 mL of boron trifluoride ether were added, and stirred at 0°C for 48 hours. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.65 g of intermediate 3-chlorobenzo BODIPY (76% yield) as a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.69(d, J=8.2Hz, 1H), 7.65(d, J=8.2Hz, 1H), 7.43(t, J=7.6Hz, 1H), 7.30-7.23 (m,2H), 7.19(s,1H), 5.95(s,1H), 2.48(s,3H), 2.20(s,3H).

Step b): Under an inert atmosphere, 0.5g of 3-chlorobenzo BODIPY, 1.25g of p-methylthiophenol and 1.3mL of anhydrous pyridine were dissolved in 10mL of anhydrous acetonitrile, and stirred at 40°C for 6 minutes. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.50 g (yield: 74%) of 3-(4-methylphenylthio)benzo-BODIPY as a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.65(d, J=7.9Hz, 1H), 7.44(d, J=7.6Hz, 2H), 7.27(t, J=7.3Hz, 1H), 7.18(s ,1H),7.10(d,J=7.5Hz,2H),6.91(t,J=7.5Hz,1H),6.74(d,J=8.2Hz,1H),5.93(s,1H),2.48(s ,3H),2.30(s,3H),2.20(s,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,124.240,12 , 122.57, 118.01, 116.65, 113.93, 20.28, 13.48, 10.25.

Step c): Under an inert atmosphere, dissolve 0.15g of 3-(4-methylphenylthio)benzo BODIPY in 10mL of chloroform, add 0.10g of m-chloroperoxybenzoic acid to the system in three batches, The reaction was carried out for 1 hour. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 35 mg of the final product BOD-C (yield 23%), a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.19(d, J=8.4Hz, 1H), 7.80(d, J=8.2Hz, 2H), 7.68(d, J=8.2Hz, 1H), 7.38(s ,1H),7.31(t,J=7.6Hz,1H),7.20(d,J=6.0Hz,3H),7.15(t,J=7.7Hz,1H),6.08(s,1H),2.55(s ,3H), 2.27(s,3H), 2.25(s,3H); HRMS (ESI-TOF): m/z 409.1360[M+H] ⁺ , calc'd.409.1357.

Embodiment 3, the preparation of fluorescent probe BOD-C

Step a): Under an inert atmosphere, add 0.5g of 3-chloro-isoindole-1-al and 2.3mL of pyrrole to 10mL of anhydrous tetrahydrofuran, then add 0.13mL of phosphorus oxychloride, and stir for 30 minutes in an ice bath Then add 3.9 mL of anhydrous triethylamine and 3.9 mL of boron trifluoride ether, and stir at 80 degrees for 1 hour. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.36 g (42% yield) of the intermediate 3-chlorobenzo BODIPY as a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.69(d, J=8.2Hz, 1H), 7.65(d, J=8.2Hz, 1H), 7.43(t, J=7.6Hz, 1H), 7.30-7.23 (m,2H), 7.19(s,1H), 5.95(s,1H), 2.48(s,3H), 2.20(s,3H).

Step b): Under an inert atmosphere, 0.5g of 3-chlorobenzo BODIPY, 0.13g of p-methylthiophenol and 2.8g of anhydrous potassium carbonate were dissolved in 10mL of anhydrous tetrahydrofuran, and stirred at 0°C for 60 minutes. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.42 g (61% yield) of 3-(4-methylphenylthio)benzo-BODIPY as a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.65(d, J=7.9Hz, 1H), 7.44(d, J=7.6Hz, 2H), 7.27(t, J=7.3Hz, 1H), 7.18(s ,1H),7.10(d,J=7.5Hz,2H),6.91(t,J=7.5Hz,1H),6.74(d,J=8.2Hz,1H),5.93(s,1H),2.48(s ,3H),2.30(s,3H),2.20(s,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,124.240,12 , 122.57, 118.01, 116.65, 113.93, 20.28, 13.48, 10.25.

Step c): Under an inert atmosphere, dissolve 0.15g of 3-(4-methylphenylthio)benzo BODIPY in 10mL of acetonitrile, add 0.35g of m-chloroperoxybenzoic acid to the system in three batches, Under reaction for 24 hours. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 55 mg of the final product BOD-C (yield 36%), a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.19(d, J=8.4Hz, 1H), 7.80(d, J=8.2Hz, 2H), 7.68(d, J=8.2Hz, 1H), 7.38(s ,1H),7.31(t,J=7.6Hz,1H),7.20(d,J=6.0Hz,3H),7.15(t,J=7.7Hz,1H),6.08(s,1H),2.55(s ,3H), 2.27(s,3H), 2.25(s,3H); HRMS (ESI-TOF): m/z 409.1360[M+H] ⁺ , calc'd.409.1357.

Embodiment 4, the preparation of fluorescent probe BOD-C

Step a): Under an inert atmosphere, add 0.5 g of 3-chloro-isoindole-1-aldehyde and 0.07 mL of 2,4-dimethylpyrrole into 10 mL of anhydrous 1,2-dichloroethane, and then Add 0.50 mL of phosphorus oxychloride, stir in an ice bath for 30 minutes, then add 3.9 mL of anhydrous triethylamine and 3.9 mL of boron trifluoride ether, and stir at room temperature for 36 hours. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.42 g of intermediate 3-chlorobenzo BODIPY (yield 49%), a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.69(d, J=8.2Hz, 1H), 7.65(d, J=8.2Hz, 1H), 7.43(t, J=7.6Hz, 1H), 7.30-7.23 (m,2H), 7.19(s,1H), 5.95(s,1H), 2.48(s,3H), 2.20(s,3H).

Step b): Under an inert atmosphere, 0.5 g of 3-chlorobenzo BODIPY, 0.20 g of p-methylthiophenol and 0.5 g of anhydrous sodium hydroxide were dissolved in 10 mL of anhydrous 1,2-dichloroethane, Stir at room temperature for 30 minutes. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.33 g (48% yield) of 3-(4-methylphenylthio)benzo BODIPY as a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.65(d, J=7.9Hz, 1H), 7.44(d, J=7.6Hz, 2H), 7.27(t, J=7.3Hz, 1H), 7.18(s ,1H),7.10(d,J=7.5Hz,2H),6.91(t,J=7.5Hz,1H),6.74(d,J=8.2Hz,1H),5.93(s,1H),2.48(s ,3H),2.30(s,3H),2.20(s,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,124.240,12 , 122.57, 118.01, 116.65, 113.93, 20.28, 13.48, 10.25.

Step c): Under an inert atmosphere, dissolve 0.15g of 3-(4-methylphenylsulfanyl)benzo-BODIPY in 10mL of 1,2-dichloroethane, and add 0.15g of m-chlorine to the system in three batches Peroxybenzoic acid, reacted at 25 degrees for 2 hours. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 68 mg of the final product BOD-C (yield 44%), a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.19(d, J=8.4Hz, 1H), 7.80(d, J=8.2Hz, 2H), 7.68(d, J=8.2Hz, 1H), 7.38(s ,1H),7.31(t,J=7.6Hz,1H),7.20(d,J=6.0Hz,3H),7.15(t,J=7.7Hz,1H),6.08(s,1H),2.55(s ,3H), 2.27(s,3H), 2.25(s,3H); HRMS (ESI-TOF): m/z 409.1360[M+H] ⁺ , calc'd.409.1357.

Embodiment 5, the preparation of fluorescent probe BOD-C

Step a): Under an inert atmosphere, add 0.5g of 3-chloro-isoindole-1-al and 0.46mL of pyrrole to 10mL of anhydrous dichloromethane, then add 0.29mL of phosphorus oxychloride, and stir in an ice bath After 30 minutes, 3.9 mL of anhydrous triethylamine and 3.9 mL of boron trifluoride ether were added, and stirred at room temperature for 12 hours. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.60 g of intermediate 3-chlorobenzo BODIPY (70% yield), a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.69(d, J=8.2Hz, 1H), 7.65(d, J=8.2Hz, 1H), 7.43(t, J=7.6Hz, 1H), 7.30-7.23 (m,2H), 7.19(s,1H), 5.95(s,1H), 2.48(s,3H), 2.20(s,3H).

Step b): Under an inert atmosphere, 0.5 g of 3-chlorobenzo BODIPY, 0.50 g of p-methylthiophenol and 1.0 g of anhydrous sodium bicarbonate were dissolved in 10 mL of anhydrous DMF, and stirred at room temperature for 20 minutes. After the reaction was complete, the solvent was spin-dried and purified by column chromatography to obtain 0.55 g (81% yield) of 3-(4-methylphenylthio)benzo BODIPY as a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ7.65(d, J=7.9Hz, 1H), 7.44(d, J=7.6Hz, 2H), 7.27(t, J=7.3Hz, 1H), 7.18(s ,1H),7.10(d,J=7.5Hz,2H),6.91(t,J=7.5Hz,1H),6.74(d,J=8.2Hz,1H),5.93(s,1H),2.48(s ,3H),2.30(s,3H),2.20(s,3H); ¹³ C NMR(100MHz,CDCl ₃ )δ151.96,150.77,138.43,136.59,134.41,132.22,131.03,130.74,129.38,128.39,124.240,12 , 122.57, 118.01, 116.65, 113.93, 20.28, 13.48, 10.25.

Step c): Under an inert atmosphere, dissolve 0.15g 3-(4-methylphenylthio)benzo BODIPY in 10mL DMF, add 0.08g m-chloroperoxybenzoic acid to the system in three batches, The reaction was carried out for 12 hours. After the reaction was complete, the reaction solution was spin-dried, separated and purified by column chromatography to obtain 54 mg of the final product BOD-C (yield 35%), a red solid.

¹ H NMR (400MHz, CDCl ₃ ) δ8.19(d, J=8.4Hz, 1H), 7.80(d, J=8.2Hz, 2H), 7.68(d, J=8.2Hz, 1H), 7.38(s ,1H),7.31(t,J=7.6Hz,1H),7.20(d,J=6.0Hz,3H),7.15(t,J=7.7Hz,1H),6.08(s,1H),2.55(s ,3H), 2.27(s,3H), 2.25(s,3H); HRMS (ESI-TOF): m/z 409.1360[M+H] ⁺ , calc'd.409.1357.

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

Weigh 4.1mg of BOD-C, dissolve it in 10mL DMSO, and prepare the mother solution (1mM) to obtain the BOD-C kit. Add 100 μL of this mother solution dropwise to phosphate buffer saline with different concentrations of cysteine, and dilute to 10 mL with the corresponding phosphate buffer. Measure its fluorescence emission spectrum. The fluorescence emission spectrum is excited at 530nm, and the intensity ratio of the emission peak is I ₅₈₄ /I ₅₅₂ or I ₅₅₂ /I ₅₈₄ ; the slit widths of excitation and emission are 1.5/1.5, respectively.

Fig. 2 is the color response diagram of BOD-C kit to cysteine aqueous solution. As can be seen from Figure 2, when the cysteine aqueous solution was added, the color of the solution was observed to change from red to yellow, and the fluorescence of the solution also changed from orange-yellow fluorescence to bright green fluorescence. It is proved that the kit of the present invention has an intuitive color response to cysteine.

Fig. 3 is a graph showing the fluorescence response of the BOD-C kit to different cysteine aqueous solutions. Known from Fig. 3, along with the increase of cysteine concentration, the wavelength is that the fluorescence intensity of the emission peak at the 584nm place decreases gradually, and the fluorescence intensity of the emission peak at the 552nm place increases gradually, which proves that the kit of the present invention is Cysteine has a sensitive ratiometric response.

Fig. 4 is the relationship curve between the ratio I ₅₅₂ /I ₅₈₄ of the fluorescence emission intensity of the BOD-C kit at wavelengths of 552nm and 584nm and the concentration of cysteine. It can be known from Fig. 4 that the fluorescence emission ratio I ₅₅₂ /I ₅₈₄ increases gradually with the increase of the concentration of cysteine in the aqueous solution. In the range of cysteine concentration of 0-1mM, the fluorescence intensity ratio I ₅₅₂ /I ₅₈₄ of the emission peak has a good linear relationship with the concentration of cysteine in the aqueous solution (R ² =0.996). The kit of the invention is proven to perform accurate measurement of cysteine.

Figure 5 is the fluorescence response diagram of the BOD-C kit to common coexisting ions or small biological molecules. It can be seen from Figure 5 that the addition of common coexisting cations, anions, and small biomolecules cannot change the fluorescence emission ratio I ₅₅₂ /I ₅₈₄ of the solution. It is proved that the kit of the present invention has excellent selectivity to cysteine.

Embodiment 7, the mensuration of intracellular cysteine 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 BOD-C (5 μM) and incubate for 30 minutes, wash with PBS three times, and perform confocal fluorescence imaging, where the excitation wavelength is 510nm and the collection wavelength is 530-650nm. Then, add Cys (10 μM) phosphate-buffered saline solution to the above HeLa cells, continue to incubate for 10 minutes, and then perform imaging on a laser confocal microscope, wherein the excitation wavelength is 510 nm, and the collection wave band is 530-650 nm.

From Figure 6, the cells loaded with BOD-C showed orange-yellow fluorescence before adding cysteine, indicating that BOD-C can permeate the cell membrane well. When cysteine was added, the cells showed green fluorescence emission, indicating that BOD-C specifically responded to cysteine in the cells. The kit of the invention is demonstrated to detect cysteine in cells.

Finally, it should be noted that the above examples only use the BOD-C 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. However, it is not used to limit this 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. it is a kind of detection cysteine fluorescent probe, it is characterised in that：The structural formula of the probe is compound shown in formula (I),

In formula (I), R₁Any one of for hydrogen, or methyl；R₂Appointing in for hydrogen, or methyl, or ethyl, or isopropyl, or fluorine What is a kind of.

2. it is according to claim 1 it is a kind of detection cysteine fluorescent probe, it is characterised in that：The structural formula of the probe Such as formula (II),

3. a kind of preparation method of the fluorescent probe of detection cysteine, comprises the steps：

Step one：Under phosphorus oxychloride catalysis, the chloro- iso-indoles -1- aldehyde of 3- shown in formula (III) is with azoles organic molten Reaction in agent obtains 3- chlorobenzenes the BODIPY replaced shown in formula (IV)；

In formula (IV), R₁Any one of for hydrogen, or methyl；

Step 2：Under an inert atmosphere, in the presence of a base, compound shown in formula (IV) in organic solvent with formula (V) shownization Compound reaction obtains the 3- (4-R replaced shown in formula (VI)₂- thiophenyl)-benzo BODIPY；

In formula (V), R₂Any one of for hydrogen, or methyl, or ethyl, or isopropyl, or fluorine；In formula (VI), R₁For hydrogen, or Any one of methyl；R₂Any one of for hydrogen, or methyl, or ethyl, or isopropyl, or fluorine；

Step 3：Under an inert atmosphere, the 3- (4-R for replacing shown in formula (VI)₂- thiophenyl)-benzo BODIPY and m-chloro peroxide benzene Formic acid reacts compound shown in the formula of obtaining final product (I) in organic solvent.

4. it is according to claim 3 it is a kind of detection cysteine fluorescent probe preparation method, it is characterised in that：

Organic solvent described in step one is dichloromethane, acetonitrile, 1,2- dichloroethanes or tetrahydrofuran；The pyroles chemical combination Thing is pyrroles, 2- methylpyrroles or 2,4- dimethyl pyrroles；Chloro- iso-indoles -1- the aldehyde of 3- shown in formula (III) and the pyroles The mol ratio of compound is 1～0.1:1；Chloro- iso-indoles -1- the aldehyde of 3- shown in formula (III) is 0.5～5 with the mol ratio of phosphorus oxychloride: 1；The reaction temperature is 0～80 degree；Response time is 1～48 hour；

Alkali described in step 2 is 1～5 with the mol ratio of compound shown in formula (V):1；The alkali be organic base in triethylamine, Pyridine or diisopropyl ethyl amine；Or for the potassium carbonate in inorganic base, sodium carbonate, sodium hydroxide, potassium hydroxide, sodium bicarbonate or Potassium bicarbonate；The mol ratio of replacement phenylmercaptan. and compound shown in formula (IV) shown in formula (V) is 1～20:1；The reaction temperature For 0～40 degree, the response time is 0.1～2 hour；The reaction dissolvent of step 2 be organic solvent in dichloromethane, acetonitrile, 1, 2- dichloroethanes, tetrahydrofuran or DMF；

Metachloroperbenzoic acid described in step 3 and the 3- (4-R replaced shown in formula (VI)₂- phenylmercaptan. base)-benzo BODIPY Mol ratio is 1～5:1；The organic solvent is chloroform, dichloromethane, acetonitrile, DMF, DMSO or 1,2- dichloroethanes；It is described anti- Temperature is answered for 0～50 degree, the response time is 1～24 hour.

5. the preparation method of the fluorescent probe of a kind of detection cysteine according to claim 3 or 4, it is characterised in that：

Organic solvent described in step one is dichloromethane；The azoles are 2,4- dimethyl pyrroles；Formula (III) institute Show that the chloro- iso-indoles -1- aldehyde of 3- is 0.5 with the mol ratio of the azoles：1；Chloro- iso-indoles-the 1- of 3- shown in formula (III) Aldehyde is 1 with the mol ratio of phosphorus oxychloride：1；The reaction temperature is 25 degree；Response time is 24 hours；

Alkali described in step 2 is 2.5 with the mol ratio of compound shown in formula (V)：1；Replace phenylmercaptan. and formula shown in formula (V) (IV) mol ratio of compound shown in is 10：1；Reaction temperature is 25 degree；Response time is 1 hour；Described organic solvent is Dichloromethane；

Metachloroperbenzoic acid described in step 3 and the 3- (4-R replaced shown in formula (VI)₂- phenylmercaptan. base)-benzo BODIPY Mol ratio is 1.5：1；The organic solvent is dichloromethane；The reaction temperature is 25 degree；Response time is 4 hours.

6. it is a kind of detection cysteine fluorescent probe using method, it is characterised in that the method is comprised the following steps：

Step (1)：Compound shown in the formula (I) of same concentrations, configuration are added in the buffer solution of variable concentrations cysteine The standard solution containing compound shown in formula (I) of at least 3 kinds different cysteine contents；

Shown buffer solution is being phosphate buffered solution, Tris-HCl buffer solution, HEPES buffer solution, boric acid-sodium borate Any one of buffer solution；

The pH value of shown standard solution is 5～12；

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

In shown standard solution, the content of cysteine is 0.1nM～1mM；

Step (2)：The fluorescence emission spectrum of the standard solution is determined respectively, and excitation wavelength is 530nm, with semicystinol concentration For abscissa, with I₅₈₄/I₅₅₂Or I₅₅₂/I₅₈₄For vertical coordinate, standard curve is set up；

I₅₈₄Represent the standard solution in the fluorescence emission peak intensity level that wavelength is at 584nm；

I₅₅₂Represent the standard solution in the fluorescence emission peak intensity level that wavelength is at 552nm；

Step (3)：Compound shown in formula (I) is added in testing sample, its concentration is controlled with formula (I) institute in the standard solution Show that the concentration of compound is equal；Determine its fluorescence emission spectrum under exciting light of the excitation wavelength for 530nm, i.e., it is bent according to standard Line computation draws the cysteine content of testing sample；

In above-mentioned steps (2) or step (3), fluorescence intensity is detected on luminoscope.

7. it is according to claim 6 it is a kind of detection cysteine fluorescent probe using method, it is characterised in that：Step (1) in, buffer solution is phosphate buffered solution；The pH value 7.2 of standard solution.