Fluorescent probe for detecting cysteine as well as preparation method and use method of fluorescent probe

Abstract

The invention discloses a fluorescent probe for detecting cysteine as well as a preparation method and a use method of the fluorescent probe. The invention designs a ratio type fluorescent probe BDP-SO-PEG7 (Benzo Bodipy) based on benzo Bodipy and PEG (Polyethylene Glycol) sulfoxide. A PEG sulfoxide part of the probe is a good leaving group and can be subjected to nucleophilic substitution reaction with sulfydryl of Cys to generate an intermediate product BDP-S-Cys, and then a final product BDP-N-Cys is generated through intramolecular rearrangement, so that the fluorescence change of the probe is caused. The ratio type fluorescent probe based on the benzo-Bodipy dye provided by the invention has good response performance to cysteine, and can realize sensitive, rapid and quantitative detection of trace cysteine in a sample. The probe has the advantages of simplicity and convenience in operation, low cost, sensitive response and the like, and is easy to popularize and apply.

Description

Technical Field

[0001] The present invention belongs to the field of organic small molecule fluorescent probes, and particularly relates to a preparation method and a usage method of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole for use as a cysteine fluorescent probe. Background Art

[0002] Cysteine (Cys) is a small molecule thiol widely distributed in organisms and has various biological functions. Cys participates in the synthesis of proteins in organisms and is the core component for the formation of disulfide bonds in proteins; at the same time, it is also a key cell signal transduction molecule and plays an important role in maintaining the redox homeostasis in organisms. The abnormal metabolism of Cys and its level changes are closely related to various diseases, such as neurodegenerative diseases, cardiovascular diseases, and cancers. Therefore, Cys can be used as an effective biomarker to label the oxidative stress state caused by diseases. Achieving specific detection of Cys levels and clarifying its role in physiological and pathological processes are of profound significance for the diagnosis and treatment of diseases.

[0003] Compared with traditional detection methods such as high performance liquid chromatography (HPLC), mass spectrometry (MS), and surface enhanced Raman scattering (SERS), the fluorescent probe technology has the advantages of simple operation, rapid response, high sensitivity, high signal-to-noise ratio, and good biocompatibility, and can truly reflect the spatio-temporal distribution of cysteine (Cys) in organisms. Therefore, the fluorescent probe technology has become a widely used and effective tool in clinical and on-site detections.

[0004] At present, fluorescent probes of various different reaction types have been used to identify cysteine (Cys). These fluorescent probes can be roughly divided into the following categories: The first category is fluorescent probes based on aromatic nucleophilic substitution (SNAr) reaction and Smiles rearrangement, in which Cys reacts with aromatic electrophiles (such as 7-nitrobenzofuran (NBD), nitrophenol or nitrothiophenol) to form a C–S bond, and then a fluorescent product is generated through Smiles rearrangement; The second category is fluorescent probes based on the Michael addition reaction of acrylate analogs, in which the unsaturated double bond undergoes an addition reaction with the thiol group of Cys, and then the fluorescence signal is changed through intramolecular cyclization and elimination reactions; The third category is fluorescent probes based on the addition cyclization reaction of Cys to aldehyde groups. After the probe adds to the thiol group of Cys, it then reacts with the amino group to form a cyclic acetal structure; The fourth category is fluorescent probes with dual reaction sites. After undergoing aromatic nucleophilic substitution and Smiles rearrangement, based on the difference in the chain lengths of Cys and homocysteine (Hcy), the distinction between Cys / Hcy is achieved by using the different rates of addition reactions with double bonds.

[0005] However, most of the fluorescent probes reported so far are fluorescence switch types, and the fluorescence signal intensity of their single emission band is affected by other factors unrelated to the analyte, such as probe concentration, non-uniform staining, light source intensity, and instrument sensitivity (see review: Rizwana Asghar; Yong-chuang Li; Fang-jun Huo; Cai-xia Yin. Chemi. & Biomed. Imaging, 2024, 2, 250–269.). Ratiometric fluorescent probes are based on the intensity changes of multiple emission bands caused by target biomolecules, and the content of the target biomolecule is determined by the ratio of fluorescence intensities at different wavelengths, thus overcoming the influence of light source intensity and instrument sensitivity. Summary of the Invention

[0006] In order to overcome the above-mentioned defects in the prior art, the present invention proposes a ratiometric fluorescent probe (BDP-SO-PEG7) for quantitative detection of cysteine. Using the present invention, cysteine in a sample can be quantitatively detected.

[0007] BODIPY has excellent properties such as a high fluorescence quantum yield, outstanding chemical stability and photostability, and low sensitivity to solvent polarity and pH. Based on these characteristics, we synthesized a ratiometric fluorescence probe BDP-SO-PEG7 based on a benzobenzodipyrromethene fluorophore and PEG sulfoxide. In this probe, the PEG sulfoxide moiety linked to BODIPY is a good leaving group. When the probe reacts with cysteine (Cys), the PEG sulfoxide is first nucleophilically substituted by the thiol group of Cys to generate an intermediate product BDP-S-Cys, and then an intramolecular rearrangement occurs to form the final product BDP-N-Cys. In contrast, homocysteine (Hcy) has a larger steric hindrance and weaker nucleophilic ability, so the rates of both nucleophilic substitution and intramolecular rearrangement are slower when it reacts with the probe. And glutathione (GSH) cannot undergo intramolecular rearrangement after thiol nucleophilic substitution due to its large steric hindrance, so its fluorescence signal does not change. Based on the above mechanism, BDP-SO-PEG7 can achieve specific detection of Cys.

[0008] The cysteine fluorescence probe described in the present invention is named 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole, and its structural formula is shown in formula (I):

[0009]

[0010] The preparation method of the above-mentioned fluorescent probe is as follows: A certain amount of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (1), mercaptoheptaethylene glycol monomethyl ether (2) and triethylamine (3) are dissolved in anhydrous dichloromethane. After reaction at room temperature, the solvent is evaporated to dryness to obtain 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (4). A certain amount of (4) is dissolved in anhydrous dichloromethane, and a solution of m-chloroperbenzoic acid (5) in anhydrous dichloromethane is added dropwise. After the addition is complete, the mixture is transferred to room temperature for reaction, and then the solvent is evaporated to dryness. The purple oily compound 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (6), namely BDP-SO-PEG7, is obtained through column chromatography.

[0011] Preferably, in the above (i), the molar ratio of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (1), mercaptoheptaethylene glycol monomethyl ether (2) and triethylamine (3) is 0.1 - 1:1:2 - 5;; the reaction temperature is 10 - 35 °C, and the reaction time is 0.5 - 3 hours; 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo

[0012] [1', 2':1,6][1,3,2]Diazabenzophenanthro[4,3-a]isoindole and anhydrous dichloromethane have a mass-volume ratio of 10:1 to 8; in the said ii), the molar ratio of 7-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1', 2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (4) to m-chloroperbenzoic acid (5) is 0.5 to 1:1; the anhydrous dichloromethane is 1 to 20 mL; the reaction temperature is 0 to 25 °C, and the reaction time is 1 to 3 hours; the mass-volume ratio of 7-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1', 2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (4) to anhydrous dichloromethane is 10:1 to 8.

[0013] Preferably, in the said i), the molar ratio of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1', 2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (1), mercaptoheptaethylene glycol monomethyl ether (2) and triethylamine (3) is 1:0.95:3; the mass-volume ratio of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1', 2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole to anhydrous dichloromethane is 5:1; the reaction temperature is 25 °C, and the reaction time is 2 hours;

[0014] In the said ii), the molar ratio of 7-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1', 2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (4) to m-chloroperbenzoic acid (5) is 1:1.1; 7-

[0015] ((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1', 2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole (4) and anhydrous dichloromethane have a mass-volume ratio of 17:5; the reaction temperature is 10 °C, and the reaction time is 2 hours.

[0016] The preparation reaction formula of the above probe is as follows:

[0017]

[0018] The usage method of the above-mentioned cysteine fluorescent probe is as follows:

[0019] Step 1: Add cysteine with different concentrations to the phosphate buffer solution (PBS) of the compound shown in formula (I) with the same concentration to prepare at least 5 standard solutions containing the compound shown in formula (I) with different cysteine contents;

[0020] The concentration of the compound shown in formula (I) in the standard solution is 1 nM to 10 μM;

[0021] The content of cysteine in the standard solution is 0.1 nM to 1 mM;

[0022] Step 2: Measure the fluorescence emission spectra of the standard solutions respectively. The excitation wavelength is 515 nm. Taking the cysteine concentration as the abscissa and I 551 / I 585 as the ordinate, establish a standard curve;

[0023] I 551 represents the fluorescence emission peak intensity value of the standard solution at a wavelength of 551 nm, I 585 represents the fluorescence emission peak intensity value of the standard solution at a wavelength of 585 nm, I 551 / I 585 is the ratio of the fluorescence emission peak intensity value at a wavelength of 551 nm to the fluorescence emission peak intensity value at a wavelength of 585 nm;

[0024] Step 3: Add the compound shown in formula (I) to the sample to be measured, and control its concentration to be equal to the concentration of the compound shown in formula (I) in the standard solution; measure its fluorescence emission spectrum under the excitation light with an excitation wavelength of 515 nm, and then calculate the cysteine content of the sample to be measured according to the standard curve.

[0025] The present invention has the following characteristics:

[0026] 1) The fluorescent probe provided by the present invention is a purple oily compound with good optical stability.

[0027] 2) The solution of the fluorescent probe provided by the present invention is sensitive to the concentration of cysteine. As the cysteine concentration increases, the fluorescence of its aqueous solution changes from orange to yellow when observed under ultraviolet light.

[0028] 3) The emission wavelength of the fluorescent probe provided by the present invention is 585 nm. After reacting with cysteine, the emission wavelength shifts to 551 nm, which is a ratio-type fluorescence response under two wavelengths. It can effectively overcome the influence of interference factors such as probe concentration, non-uniform staining, light source intensity and instrument sensitivity during detection, and improve the detection sensitivity.

[0029] 4) The fluorescence probe provided by the present invention has a linear relationship with the cysteine concentration and can be used for accurate measurement of the cysteine concentration.

[0030] The ratio-type cysteine fluorescence probe based on benzoxaborole dye provided by the present invention has a specific response to cysteine solution, can realize sensitive quantitative detection of cysteine in samples, and has the advantages of simple operation, low cost, sensitive response, easy promotion and application, etc. Brief Description of the Drawings

[0031] Figure 1 : 1H NMR spectrum of the fluorescence probe BDP-SO-PEG7.

[0032] Figure 2 : Color response diagram of the fluorescence probe BDP-SO-PEG7 to cysteine phosphate buffer solution.

[0033] Figure 3 : Fluorescence response diagram of the fluorescence probe BDP-SO-PEG7 to cysteine phosphate buffer solution.

[0034] Figure 4 : UV titration curve of the fluorescence probe BDP-SO-PEG7 to cysteine, where the probe concentration is 10.0 μM.

[0035] Figure 5 : Fluorescence titration curve of the fluorescence probe BDP-SO-PEG7 to cysteine, where the excitation wavelength is 515 nm and the probe concentration is 10.0 μM.

[0036] Figure 6 : Fluorescence response diagram of the fluorescence probe BDP-SO-PEG7 to common small molecule reactive sulfur species and amino acids in vivo, where the excitation wavelength is 515 nm, the probe concentration is 10.0 μM, and the concentration of the analyte is 40.0 μM. Detailed Description of the Embodiments

[0037] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0038] The materials, reagents, etc. used in the following examples are all obtained from commercial channels unless otherwise specified.

[0039] The compound numbers in the examples correspond to the numbers in the above compounds.

[0040] Example 1. Synthesis of the compound BDP-SO-PEG7.

[0041] Synthesis of compound 7-((2,5,8,11,14,17,20-hexaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4).

[0042] Dissolve 35 mg of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (1) (0.11 mmol) in 3 mL of anhydrous dichloromethane, add 0.05 mL of triethylamine (3) (0.36 mmol), and then slowly drip a 1 mL anhydrous dichloromethane solution of 40 mg of mercaptoheptaethylene glycol monomethyl ether (2) (0.11 mmol) into the reaction solution. After reacting at room temperature for 2 hours, evaporate to dryness to obtain 67 mg of the product 7-((2,5,8,11,14,17,20-hexaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) with a yield of 93%.

[0043] 1 H NMR (400 MHz, Chloroform-d) δ 7.89 (d, J = 8.2 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.29 (s, 1H), 6.02 (s, 1H), 3.71 (t, J = 6.6 Hz, 2H), 3.67–3.51 (m, 24H), 3.47 (t, J = 6.7 Hz, 2H), 3.36 (s, 3H), 2.54 (s, 3H), 2.27 (s, 3H).

[0044] Synthesis of compound 7-((2,5,8,11,14,17,20-hexaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (6).

[0045] Dissolve 34 mg of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) (0.05 mmol) in 3 mL of anhydrous dichloromethane. Add a 1 mL dichloromethane solution of 9 mg of m-chloroperbenzoic acid (5) (0.05 mmol) to it in an ice bath at 0 °C. Then transfer the reaction to room temperature and continue the reaction for 2 hours. After evaporation to dryness, separate the product by column chromatography to obtain 32 mg of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (6) with a yield of 91%.

[0046] 1 H NMR (400 MHz, Chloroform-d) δ 8.52 (d, J = 8.4 Hz, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.46 (d, J = 6.5 Hz, 2H), 7.29 (t, J = 7.6 Hz, 1H), 6.10 (d, J = 3.1 Hz, 1H), 4.05 (qt, J = 10.9, 5.8 Hz, 2H), 3.67–3.52 (m, 24H), 3.49 (q, J = 6.1, 5.1 Hz, 2H), 3.36 (s, 3H), 2.53 (s, 3H), 2.31 (s, 3H).

[0047] Example 2. Synthesis of compound BDP-SO-PEG7.

[0048] Synthesis of compound 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4).

[0049] Dissolve 3.5 mg of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (1) in 0.35 mL of anhydrous dichloromethane, add 0.034 mL of triethylamine (3), and then dropwise add a 0.23 mL dichloromethane solution of 40 mg of mercaptoheptaethylene glycol monomethyl ether (2) to the reaction solution. After reacting at 10 °C for 0.6 h, rotary evaporate to obtain 62 mg of the product 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) with a yield of 90%.

[0050] Synthesis of compound 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (6).

[0051] Dissolve 17 mg of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) in 1 mL of anhydrous dichloromethane. At 15 °C, add a 0.5 mL dichloromethane solution of 9 mg of m-chloroperbenzoic acid (5). Then transfer the reaction to room temperature and continue to react for 1 h. Rotary evaporate, and then separate by column chromatography to obtain 31 mg of the product 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (6) with a yield of 89%.

[0052] Example 3. Synthesis of compound BDP-SO-PEG7.

[0053] Synthesis of compound 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4).

[0054] Dissolve 18 mg of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (1) in 18 mL of anhydrous dichloromethane, add 0.08 mL of triethylamine (3), and then dropwise add a 2 mL dichloromethane solution of 40 mg of mercaptoheptaethylene glycol monomethyl ether (2) to the reaction solution. Then, react at 35 °C for 3 hours and then evaporate to dryness to obtain 67 mg of the product 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) with a yield of 91%.

[0055] Synthesis of compound 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (6).

[0056] Dissolve 48 mg of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) in 18 mL of anhydrous dichloromethane, add a 2 mL dichloromethane solution of 9 mg of m-chloroperbenzoic acid (5) at 25 °C, then transfer the reaction to room temperature and continue to react for 3 hours and then evaporate to dryness. After column chromatography separation, 32 mg of the product 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (6) is obtained with a yield of 91%.

[0057] Example 4. Color response of compound BDP-SO-PEG7 to cysteine.

[0058] Prepare a test stock solution of the fluorescence probe BDP-SO-PEG7 for detecting cysteine of the present invention with a concentration of 1 mM in ethanol (EtOH) for later use. Measure 100 μL of this stock solution and add it to a phosphate buffer solution with a certain concentration, and make up the volume to 10 mL with the corresponding phosphate buffer solution so that the concentration of the probe in the test solution is 10.0 μM and the concentration of cysteine is 10.0 μM for color response testing. As Figure 2 and 3As shown, after adding the cysteine solution, the color of the solution changed from purplish red to light yellow visually, and at the same time, the fluorescence of the solution also changed from orange fluorescence to yellowish green fluorescence, indicating that the probe BDP-SO-PEG7 has an intuitive colorimetric response to cysteine.

[0059] Example 5. UV titration detection of different concentrations of cysteine on compound BDP-SO-PEG7.

[0060] Prepare a test stock solution of the fluorescence probe BDP-SO-PEG7 for detecting cysteine of the present invention with a concentration of 1 mM in ethanol (EtOH) for later use. Measure 100 μL of this stock solution and add it to a phosphate buffer solution with a certain concentration, and make up the volume to 10 mL with the corresponding phosphate buffer solution, so that in the test solution, the concentration of the probe is 10.0 μM and the cysteine concentration is 0 - 50.0 μM for absorption spectrum testing. Obtain the UV absorption curves of each system and establish a standard curve of absorbance vs. cysteine concentration. As Figure 4 shown, as the cysteine concentration increases, the absorbance at 568 nm gradually decreases, and the absorbance at 491 nm gradually increases, and the ratio of A 491 / A 568 has a good linear relationship with the cysteine concentration (0 - 40.0 μM).

[0061] Example 6. Fluorescence titration detection of different concentrations of cysteine on compound BDP-SO-PEG7.

[0062] Prepare a test stock solution of the fluorescence probe BDP-SO-PEG7 for detecting cysteine of the present invention with a concentration of 1 mM in ethanol (EtOH) for later use. Measure 100 μL of this stock solution and add it to a phosphate buffer solution with a certain concentration, and make up the volume to 10 mL with the corresponding phosphate buffer solution, so that in the test solution, the concentration of the probe is 10.0 μM and the cysteine concentration is 0 - 50.0 μM for fluorescence detection (λex = 515 nm, λem = 551 nm / 585 nm). Obtain the fluorescence intensity of each system and calculate the fluorescence intensity ratio I 551 / I 585 , and establish a standard curve of the fluorescence intensity ratio vs. cysteine concentration. As Figure 5 shown, as the cysteine concentration increases, the fluorescence intensity ratio of the system (I 551 / I 585 ) gradually increases. When the cysteine concentration reaches 40.0 μM, the fluorescence intensity ratio of the reaction system (I 551 / I 585 ) reaches the maximum value, that is, the equilibrium state.

[0063] Example 7, Selectivity of compound BDP-SO-PEG7 towards common small reactive sulfur species and amino acids in vivo.

[0064] Prepare a test stock solution of the fluorescence probe BDP-SO-PEG7 for detecting cysteine according to the present invention with a concentration of 1 mM in ethanol (EtOH) for later use. Measure 100 μL of this stock solution and add it to the phosphate buffer solutions of different small molecules to be measured, and then make up the volume to 10 mL with the corresponding phosphate buffer solution, so that the concentration of the probe in the test solution is 10.0 μM for fluorescence detection (λex = 515 nm, λem = 551 nm / 585 nm). Obtain the fluorescence intensities in each system and establish a bar chart showing the relationship between the fluorescence intensity ratio (I 551 / I 585 ) and each analyte to be measured. As Figure 6 shown, other common small reactive sulfur molecules and amino acid molecules to be measured have almost no effect on the fluorescence of the probe BDP-SO-PEG7.

Claims

1. A fluorescent probe for detecting cysteine, characterized in that: Its molecular formula is C30H43BF2N2O8S, abbreviated as BDP-SO-PEG7, and its structural formula is formula (I); 2. Preparation of a fluorescence probe for detecting cysteine according to claim 1 A method, characterized in that The synthesis steps are as follows: Under nitrogen protection and low temperature conditions, compound 7-chloro-5,5-difluoro-1,3-dimethyl- 5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazabenzophenanthro[4,3-a] isoindole, mercaptoheptaethylene glycol monomethyl ether and triethylamine are dissolved in anhydrous dichloromethane, and after reacting for a certain time, the product 7-((2,5,8,11,14,17,20-heptaoxadocosane-22-yl) thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1', 2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole is obtained; then the product is dissolved in anhydrous di chloromethane and reacted with m-chloroperbenzoic acid for a certain time to obtain 7- ((2,5,8,11,14,17,20-heptaoxadocosane-22-yl)sulfinyl)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazabenzophenanthro[4,3-a]isoindole, namely BDP-SO-PEG7.

3. A method for using a fluorescence probe for detecting cysteine; characterized in that: 1) Different concentrations of cysteine are added to the phosphate buffer solution of the compound shown in formula (I) with the same concentration to prepare at least 5 standard solutions containing the compound shown in formula (I) with different cysteine contents; The concentration of the compound shown in formula (I) in the standard solution is 1 nM to 10 μM; The content of cysteine in the standard solution is 0.1 nM to 1 mM; 3) The compound shown in formula (I) is added to the sample to be tested, and its concentration is controlled to be equal to the concentration of the compound shown in formula (I) in the standard solution; The fluorescence emission spectrum is measured under the excitation light with an excitation wavelength of 515 nm, and the cysteine content of the sample to be tested is calculated according to the standard curve. 2) Measure the fluorescence emission spectra of the standard solutions separately, with an excitation wavelength of 515 nm. Using the cysteine concentration as the abscissa and I 551 / I 585 as the ordinate, establish a standard curve; I 551 represents the fluorescence emission peak intensity value of the standard solution at a wavelength of 551 nm, I 585 represents the fluorescence emission peak intensity value of the standard solution at a wavelength of 585 nm, I 551 / I 585 is the ratio of the fluorescence emission peak intensity value at a wavelength of 551 nm to the fluorescence emission peak intensity value at a wavelength of 585 nm; 4. The preparation method of a fluorescence probe for detecting cysteine according to claim 2, characterized in that: ​ ​ In the step (i), the molar ratio of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (1), mercaptoheptaethylene glycol monomethyl ether (2) and triethylamine (3) is 0.1-1:1:2-5; the reaction temperature is 10-35 °C, and the reaction time is 0.5-3 hours; the mass-volume ratio of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole to anhydrous dichloromethane is 10:1-8; in the step (ii), the molar ratio of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) and m-chloroperbenzoic acid (5) is 0.5-1:1; the amount of anhydrous dichloromethane is 1-20 mL; the reaction temperature is 0-25 °C, and the reaction time is 1-3 hours; the mass-volume ratio of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) to anhydrous dichloromethane is 10:1-8.

5. The preparation method of a fluorescence probe for detecting cysteine according to claim 1, wherein: In the step (i), the molar ratio of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (1), mercaptoheptaethylene glycol monomethyl ether (2) and triethylamine (3) is 1:0.95:3; the mass-volume ratio of 7-chloro-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole to anhydrous dichloromethane is 5:1; the reaction temperature is 25 °C, and the reaction time is 2 hours; In the above (ii), the molar ratio of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) to meta-chloroperbenzoic acid (5) is 1:1.1; the mass-volume ratio of 7-((2,5,8,11,14,17,20-heptaoxadocosan-22-yl)thio)-5,5-difluoro-1,3-dimethyl-5H-4λ4,5λ4-pyrrolo[1',2':1,6][1,3,2]diazaborinin[4,3-a]isoindole (4) to anhydrous dichloromethane is 17:5; the reaction temperature is 10 °C, and the reaction time is 2 hours.

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