Design synthesis and evaluation method based on dicyanoisophorone fluorescent probe

Abstract

The invention discloses a design synthesis and evaluation method based on a dicyanoisophorone fluorescent probe. According to the invention, based on the design principle of the fluorescent probe and a classical organic synthesis method, a series of bifunctional fluorescent probes DCX-1-20 which are based on dicyanoisophorone fluorophores and have targeting capability and related index detection capability are designed and successfully synthesized for the first time, and the bifunctional fluorescent probes DCX-1-20 can be accurately positioned to hepatocytes, lysosomes and mitochondria; the probe can be used for detecting biomarkers such as H2S, Cys, hydrogen peroxide, carboxylesterase, acetylcholin esterase, butyrylcholin esterase and the like. The synthesized representative liver-targeting fluorescent probe DCX-1 has excellent photophysical characteristics, good selectivity to Cys, no response to other analytes, excellent anti-interference capability, good stability, low cytotoxicity and excellent living cell imaging and liver-targeting capability, can be used for Cys imaging in zebra fish bodies, and can be used for detecting Cys imaging in zebra fish bodies. The visualization of the Cys in a living body, especially in hepatocytes, is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent probes, and specifically relates to a design, synthesis and evaluation method based on a dicyanoisophorone fluorescent probe. Background Art

[0002] As a practical detection tool with important prospects, fluorescent probes play an increasingly important role in specific detection, including but not limited to qualitative and quantitative detection of environmental pollutants, early detection of markers in pathological models, etc., and are particularly prominent in biological cell imaging. Compared with traditional methods such as electrochemical analysis, elemental analysis, and mass spectrometry, fluorescent probe technology has significant advantages such as low cost, strong real-time performance, high sensitivity, easy analysis of results, and high detection accuracy. As a research hotspot in fluorescent probe technology, the design and development of small molecule fluorescent probes have attracted attention from multiple disciplines such as molecular biology, biomedicine, and chemical biology. In the past few decades, traditional fluorescent groups, such as coumarin, BODIPY, rhodamine, cyanine and hemicyanine dyes, have been widely used to construct excellent probes, making significant contributions to the monitoring and tracking of important targets in living cells.

[0003] However, while the above fluorophores demonstrate their powerful capabilities, they also expose the following shortcomings:

[0004] 1. Photostability: Although the DCIP group may have good photostability, the fluorescent probe may still undergo photobleaching under continuous or strong excitation light.

[0005] 2. Biodegradability: The biodegradability of fluorescent probes is a key factor in their biomedical applications. If the probes cannot be effectively cleared by the organism, they may accumulate and become toxic.

[0006] 3. Biodistribution and clearance: The distribution and clearance mechanisms of probes in vivo need to be carefully studied to ensure that they can reach the target site and act effectively while reducing nonspecific distribution. Summary of the invention

[0007] The purpose of the present invention is to provide a design, synthesis and evaluation method of a dicyanoisophorone-based fluorescent probe in order to solve the above-mentioned problems.

[0008] The technical solution adopted by the present invention is as follows: a design and synthesis method based on a dicyanoisophorone fluorescent probe, the synthesis method comprising the following steps:

[0009] S1: First, prepare the intermediate for synthesizing dicyanoisophorone fluorescent probe; weigh 1.5g D-galactose and add it to a 150mL eggplant-shaped bottle, then add 5mL acetic anhydride to dissolve the D-galactose, then transfer the system to an ice-water bath environment to react for 30 minutes, then slowly drop 0.1mL perchloric acid, after stirring for 6 hours, monitor the reaction completion by TLC; adjust the pH of the system to neutral with saturated sodium bicarbonate, extract with ethyl acetate; wash with saturated sodium chloride solution, absorb excess water with anhydrous sodium sulfate, and finally filter the dried organic layer, and perform vacuum rotary evaporation on the collected filtrate to obtain a crude product in the form of light yellow syrup; no purification is required. The crude product of the previous step was dissolved in 10 mL of dichloromethane, and the reaction was continued in an ice-water bath for 30 minutes, and then 0.35 mL of hydrobromic acid was slowly added dropwise. After the addition was completed, the reaction system was transferred to room temperature and the reaction was continued for 6 hours. The reaction was monitored to be complete by TLC. The post-treatment of the reaction system was the same as the previous step. After multiple extractions, drying, suction filtration, and vacuum rotary evaporation, a white solid acetyl brominated galactose was obtained. The product did not need to be purified and the next step was continued. The white solid acetyl brominated galactose obtained above was dissolved in 20 mL of DMSO. At room temperature, NaN3 was added to the reaction system while stirring. After reacting for 4 hours, the reaction system was quenched with water, extracted with ethyl acetate, washed with saturated sodium chloride solution, and excess water was sucked out with anhydrous sodium sulfate. Finally, suction filtration, concentration, and purification with a silica gel column were performed to obtain a white compound 1.

[0010] S2: The white solid 1 was dissolved in 10 mL of dry methanol, and sodium methoxide was added under stirring to adjust the pH of the reaction system to 10, and the reaction was allowed to react at room temperature for 4 hours. The reaction was complete when detected by TLC; then a deionized exchange resin was added to adjust the reaction system to neutrality, and then the reaction was filtered, and the solvent was removed by vacuum rotary evaporation, and finally the white solid compound 2 was purified and separated by silica gel column;

[0011] S3: Weigh the compounds triphenylphosphine and 1,4-dibromobutane and place them in a dry 100 ml eggplant-shaped bottle, add 20 ml toluene to fully dissolve them, reflux them at 120°C under nitrogen protection for 12 hours, cool them to room temperature after the reaction, remove the solvent by vacuum filtration, and wash them three times with toluene to obtain a white powder solid compound 3;

[0012] S4: Weigh compound 3 and place it in a dry 25 ml round-bottom flask, dissolve it with 5 ml N,N-dimethylformamide, then add sodium azide, react at room temperature for 3 hours under nitrogen protection, add 10 ml deionized water to quench the reaction, extract with dichloromethane, combine the organic phases and add anhydrous sodium sulfate to absorb excess water, remove the solvent under reduced pressure, and then use an oil pump to pump out excess N,N-dimethylformamide to obtain a brown solid compound 4;

[0013] S5: Weigh N-morpholine and sodium azide and dissolve them in 20 ml of N,N-dimethylformamide. Reflux at 50°C for 3 hours under nitrogen protection. After the reaction is completed, cool to room temperature, add a large amount of water, extract with ethyl acetate, and then remove the solvent under reduced pressure to obtain compound 5;

[0014] S6: Isophorone, malononitrile, sodium acetate trihydrate and 9 ml of ethanol were placed in a flask; the mixture was stirred and heated at 50° C. overnight; the solvent was then removed under reduced pressure; the residue was eluted with petroleum ether / dichloromethane to obtain a white solid compound 6;

[0015] S7: 2,4-dihydroxybenzaldehyde, 3-bromopropyne and potassium carbonate were dissolved in acetonitrile; the reaction mixture was refluxed at 80° C. for 30 minutes under a nitrogen environment; the solvent was then removed under reduced pressure; the residue was eluted with dichloromethane / petroleum ether to obtain a white solid compound 7;

[0016] S8: Compound 7 and compound 6 were dissolved in acetonitrile, and then piperidine was added; the mixture was refluxed at 80° C. for 6 hours under a nitrogen environment, and then the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain a yellow compound 8;

[0017] S9: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen atmosphere at 0°C; the reaction mixture was stirred at 0°C for 30 minutes; acryloyl chloride was then gradually added to the solution and reacted for 30 minutes, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain yellow solid compound 9;

[0018] S10: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen environment at 0°C; the reaction mixture was stirred at 0°C for 30 minutes, and then 3-thiophenecarbonyl chloride was gradually added to the solution for reaction for 1 hour, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain yellow solid compound 10;

[0019] S11: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen atmosphere at 0°C; the reaction mixture was stirred at 0°C for 30 minutes, and then acetyl chloride was gradually added to the solution to react for 1 hour, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain an orange-yellow solid compound 11;

[0020] S12: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen environment at 0°C; the reaction mixture was stirred at 0°C for 30 minutes, and then benzoyl chloride was gradually added to the solution to react for 40 minutes, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain compound 12;

[0021] S13: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen environment at 0°C; the reaction mixture was stirred at 0°C for 30 minutes, and then cyclopropylcarbonyl chloride was gradually added to the solution to react for 30 minutes, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain compound 13;

[0022] S14: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, triethylamine was gradually added to the solution under a nitrogen atmosphere at 0°C, the reaction mixture was stirred at 0°C for 30 minutes, dimethylaminoformyl chloride was gradually added to the solution and reacted for 50 minutes, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain an orange solid compound 14;

[0023] S15: Compound 8, 4-nitrobenzyl bromide and potassium carbonate were dissolved in 15 ml of N,N-dimethylformamide, and the reaction mixture was stirred at room temperature for 4 hours. The solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain yellow solid compound 15;

[0024] S16: Compound 8, 2,4-dinitrofluorobenzene and potassium carbonate were dissolved in 20 ml of N,N-dimethylformamide, the reaction mixture was stirred at room temperature for 6 hours, and the solvent was removed under reduced pressure; the residue was purified by column chromatography, wherein petroleum ether / ethyl acetate = 15:1, to obtain orange compound 16;

[0025] S17: Synthesis of the target compound: sodium ascorbate and CuSO4.5H2O were dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 9 and compound 2, and the reaction was carried out for 12 hours; extraction was performed with dichloromethane, and water was removed with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure; dichloromethane / methanol was used as an eluent to obtain a yellow solid DCX-1, and then the entire design and synthesis method based on dicyanoisophorone fluorescent probe was completed.

[0026] In a preferred embodiment, in step S17, sodium ascorbate and CuSO4.5H2O are dissolved in H2O, and the mixture is stirred at room temperature for 30 minutes. The mixture is then added to a THF solution containing compound 10 and compound 2, and the reaction is allowed to proceed overnight. The mixture is extracted with dichloromethane, excess water is removed with anhydrous sodium sulfate, and the solvent is removed under reduced pressure. The residue is eluted with dichloromethane / methanol to obtain a yellow solid DCX-2.

[0027] In a preferred embodiment, sodium ascorbate and CuSO4.5H2O are dissolved in H2O, and the mixture is stirred at room temperature for 30 minutes. The mixture is then added to a THF solution containing compound 11 and compound 2, and the reaction is allowed to proceed overnight. The mixture is extracted with dichloromethane, excess water is dried with anhydrous sodium sulfate, and the solvent is removed under reduced pressure. The residue is eluted with dichloromethane / methanol to obtain a yellow compound DCX-3.

[0028] In a preferred embodiment, sodium ascorbate and CuSO4.5H2O are dissolved in H2O, and the mixture is stirred at room temperature for 30 minutes. The mixture is then added to a THF solution containing compound 13 and compound 2, and the reaction is allowed to proceed overnight. The mixture is extracted with dichloromethane, excess water is absorbed with anhydrous sodium sulfate, and the solvent is removed under reduced pressure. The residue is eluted with dichloromethane / methanol to obtain a yellow solid DCX-5.

[0029] In a preferred embodiment, sodium ascorbate and CuSO4.5H2O are dissolved in H2O, and the mixture is stirred at room temperature for 30 minutes. The mixture is then added to a THF solution containing compound 14 and compound 2, and the reaction is allowed to proceed overnight. The mixture is extracted with dichloromethane, the water is removed with anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure. The residue is eluted with dichloromethane / methanol to obtain the yellow target DCX-6.

[0030] In a preferred embodiment, the evaluation method comprises the following steps:

[0031] S1: To conduct spectral testing, a certain amount of DCX-1 and Cys were weighed and dissolved in DMSO and PBS respectively to prepare 10mM probe DCX-1 stock solution and 50mM Cys stock solution. Other test solutions, such as small molecule thiols, anions, cations, amino acids, etc., were dissolved in PBS to prepare a solution with a concentration of 50mM. The scanning range of the UV spectrum and fluorescence spectrum is 460-800nm. The slit width is 5nm×5nm. The test content includes UV spectroscopy experiments, fluorescence selectivity experiments, probe anti-interference experiments, concentration titration experiments, Job plot experiments, etc.

[0032] S2: Cytotoxicity assay, LO2 cells were seeded into 96-well plates, incubated at 37°C, 5% CO2 for 24 hours, and then treated with different concentrations of probes for 24 hours. The supernatant was removed and 10 μL MTT was added. The cells were incubated at 37°C for another 4 hours, and the absorbance at 488 nm was recorded using an ELX800 absorbance microplate reader.

[0033] S3 was used to detect the probe in cells. First, HepG2 cells were evenly inoculated into 24-well plates and cultured for 24 hours. Then, the cultured HepG2 cells were treated differently and divided into blank cell group, pure probe group, Cys supplement group and NEM pretreatment group. Pure probe group: 10μM probe DCX-1 was incubated for 30 minutes, then fixed and anti-fluorescence quencher was added. Cys supplement group: 10μM probe DCX-1 was incubated for 30 minutes and washed 3 times with PBS. Then the probe DCX-1 was incubated with 100μM Cys for 60 minutes, washed 3 times with PBS, and then fixed and anti-fluorescence quencher was added. NEM pretreatment group: cells were pretreated with 1mM NEM for 2 hours, washed 3 times with PBS, and then 10μM probe DCX-1 was added for incubation for 30 minutes, fixed and anti-fluorescence quencher was added.

[0034] In order to explore whether the probe DCX-1 can accurately locate hepatocytes, four cells, HepG2, A549, Hela and SGC-7901, were treated in the same way. Then the anti-fluorescence quencher was dropped onto the slide, fastened and sealed. All images were collected by a Leica sp8 laser scanning confocal microscope. The excitation wavelength of the probe was 488nm, and the emission wavelength was collected in the range of 560-650nm.

[0035] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0036] 1. In the present invention, 8 hepatocyte-targeted fluorescent probes with β-galactose as the targeting group were designed and synthesized. By connecting different recognition groups (acetyl chloride, acryloyl chloride, thiophene carboxylic acid chloride, benzoyl chloride, 2,4-dinitrofluorobenzene, etc.), they were used to detect biomarkers such as Cys, H2S, hydrogen peroxide, nitroreductase, and acetylcholinesterase. 1 H NMR, 13 The structure of each compound was determined by C NMR and HR-ESI-MS characterization. Six lysosomal-targeted fluorescent probes with morpholine as the targeting group were designed and synthesized. By connecting different recognition groups (acryloyl chloride, 4-nitrobenzyl bromide, benzoyl chloride, thiophenecarboxylic acid chloride, etc.), they were used to detect biomarkers such as Cys, H2S, hydrogen peroxide, carboxylesterase, and chymosin. 1 H NMR, 13 CNMR and HR-ESI-MS characterizations confirmed the structure of each compound.

[0037] 2. In the present invention, six mitochondrial targeting fluorescent probes with triphenylphosphine as the targeting group were designed and synthesized. By connecting different recognition groups (4-nitrobenzyl bromide, thenoyl chloride, acetyl chloride, cyclopropylcarbonyl chloride, etc.), they were used to detect biomarkers such as H2S, Cys, hydrogen peroxide, carboxylesterase, acetylcholinesterase, butyrylcholinesterase, etc. 1 H NMR, 13 CNMR and HR-ESI-MS characterizations confirmed the structure of each compound.

[0038] 3. In the present invention, the probe DCX-1 designed and synthesized contains an acrylate unit in its structure, which can selectively react with cysteine ​​to form a thiol compound and remove a molecule of a seven-membered ring, releasing a fluorescent structure, thereby emitting strong fluorescence. The probe DCX-1 has a strong selectivity for cysteine ​​and can only respond to cysteine, but not to other analytes (ClO - 、CO3 2- 、SO4 2- ,I - Br - , HS - , Cu 2+ 、Na + , Ca 2+ , Fe 3+ , H2O2, GSH, Hcy, Gln, Gly, Tyr, Arg, Lys, BSA, trypsin, lysozyme, vitamin C, urea, etc.) have no response. Probe DCX-1 has excellent anti-interference ability and is not affected by GSH, Hcy, Gln, Tyr, Arg, Ala, ILE, Lys, Phe, Met, Thr, Gly, and His. The Stokes shift of probe DCX-1 can reach 173nm, and the detection limit is as low as 0.31μM. It can respond quickly to cysteine ​​and is stable under physiological pH conditions. Probe DCX-1 has good cell and tissue penetration and can be used to detect endogenous and exogenous cysteine ​​in cells and zebrafish. Due to the introduction of galactose groups, it has excellent hepatocyte targeting and low cytotoxicity. Probe DCX-1 can be used for the precise detection of cysteine ​​in organisms, providing an effective tool for the detection of cysteine-related diseases and helping to understand the role and function of cysteine ​​in life systems.

[0039] 4. In the present invention, based on the design principle of fluorescent probes and the classic organic synthesis method, a series of dual-function fluorescent probes DCX-1 to DCX-20 based on dicyanoisophorone fluorescent groups with both targeting and related index detection capabilities are designed and successfully synthesized for the first time. They can accurately locate hepatocytes, lysosomes and mitochondria, and can be used to detect biomarkers such as H2S, Cys, hydrogen peroxide, carboxylesterase, acetylcholinesterase, butyrylcholinesterase, etc. The synthesized representative liver-targeted fluorescent probe DCX-1 has excellent photophysical properties, good selectivity for Cys, and good selectivity for other analytes (ClO - ,CO3 2- ,SO4 2- ,I - ,Br - ,HS - ,Cu 2+ ,Na + ,Ca 2+ ,Fe 3+ , H2O2, GSH, Hcy, Gln, Gly, Tyr, Arg, Lys, BSA, trypsin, lysozyme, vitamin C, urea, etc.) have no response, excellent anti-interference ability, good stability, low cytotoxicity, excellent live cell imaging and liver targeting ability, and can be used for Cys imaging in zebrafish, realizing the visualization of Cys in vivo, especially in liver cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The schematic diagram is a UV spectrum response diagram of the probe DCX-1 of the present invention to Cys;

[0041] Figure 2 Schematic diagram of the fluorescence spectrum response of the probe DCX-1 to Cys in the present invention;

[0042] Figure 3 Schematic diagram of the functional relationship between the fluorescence intensity of the probe DCX-1 and the Cys concentration in the present invention;

[0043] Figure 4 Schematic diagram of the selectivity of the probe DCX-1 for Cys in the present invention;

[0044] Among them: 1: blank; 2: glutathione; 3: homocysteine; 4: glutamine; 5: tyrosine; 6: arginine; 7: alanine; 8: isoleucine; 9: lysine; 10: phenylalanine; 11: methionine; 12: threonine; 13: glycine; 14: histidine; 15: Na + ; 16:Ca 2+ ; 17:Fe 3+ ; 18: Cu 2+ ; 19: C10- ; 20:CO3 2- ;21:SO4 2- ; 22:I - ;23:Br - ;24:HS - ; 25: bovine serum albumin; 26: trypsin; 27: lysozyme; 28: hydrogen peroxide; 29: vitamin C; 30: urea; 31: polyphenylene ether; 32: phospholipids; 33: tert-butyl peroxide; 34: chymotrypsin; 35: glyoxal oxidase; 36: carboxylesterase; 37: cysteine;

[0045] Figure 5 This is a schematic diagram of the anti-interference property of the probe DCX-1 in the present invention;

[0046] Figure 6 This is a schematic diagram of the change of the fluorescence intensity of the probe DCX-1 in the present invention over time;

[0047] Figure 7 Schematic diagram of pH stability of probe DCX-1 in the present invention. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0049] Reference Figure 1-7 ,

[0050] Example:

[0051] A design and synthesis method based on dicyanoisophorone fluorescent probe, the synthesis method comprises the following steps:

[0052] S1: First, prepare the intermediate of the synthetic dicyanoisophorone fluorescent probe; weigh 1.5g D-galactose and add it to a 150mL eggplant-shaped bottle, then add 5mL acetic anhydride to dissolve the D-galactose, then transfer the system to an ice-water bath environment for reaction for 30 minutes, then slowly drip 0.1mL perchloric acid, and after stirring for 6 hours, TLC monitors the reaction to be complete. The pH of the system is adjusted to neutral with saturated sodium bicarbonate and extracted with ethyl acetate. Wash with saturated sodium chloride solution, absorb excess water with anhydrous sodium sulfate, and finally filter the dried organic layer, and perform vacuum rotary evaporation on the collected filtrate to obtain a crude product in the form of a light yellow syrup. No purification is required, and the next step is directly performed. The crude product of the previous step is dissolved with 10mL dichloromethane, placed in an ice-water bath environment and continued to react for 30 minutes, and then 0.35mL hydrobromic acid is slowly dripped. After the addition is complete, the reaction system is transferred to room temperature and continued to react for 6 hours, and the reaction is completely monitored by TLC. The post-treatment of the reaction system is the same as the previous step. After multiple extractions, drying, suction filtration, and vacuum rotary evaporation, a white solid all-acetyl brominated galactose is obtained. The obtained product does not need to be purified and continues to the next step. The above-obtained white solid all-acetyl brominated galactose is dissolved in 20mL DMSO, and NaN3 (0.9g) is added to the reaction system while stirring at room temperature. After reacting for 4 hours, the reaction system is quenched with water, extracted with ethyl acetate, washed with saturated sodium chloride solution, and excess water is sucked out with anhydrous sodium sulfate. Finally, it is filtered, concentrated, and purified by silica gel column (petroleum ether / ethyl acetate = 5:1) to obtain white compound 1;

[0053] S2: Dissolve the white solid 1 (2.5 g, 6.70 mmol) in 10 mL of dry methanol, add sodium methoxide to adjust the pH of the reaction system to 10 while stirring, react at room temperature for 4 hours, and the reaction is complete when detected by TLC. Then add deionized exchange resin to adjust the reaction system to neutrality, filter, remove the solvent by vacuum rotary evaporation, and finally purify and separate by silica gel column (dichloromethane / methanol=10:1) to obtain a white solid compound 2;

[0054] S3: Weigh triphenylphosphine (0.15 g, 0.57 mmol) and 1,4-dibromobutane (0.10 ml, 0.86 mmol) into a dry 100 ml eggplant-shaped bottle, add 20 ml toluene to fully dissolve, reflux at 120 ° C under nitrogen protection for 12 hours, cool to room temperature after the reaction, remove the solvent by vacuum filtration, and wash with toluene three times to obtain a white powder solid compound 3;

[0055] S4: Weigh compound 3 (50 mg, 104 mmol) and place it in a dry 25 ml round-bottom flask, dissolve it with 5 ml N,N-dimethylformamide, then add sodium azide (13.54 mg, 208.38 mmol), react at room temperature for 3 hours under nitrogen protection, add 10 ml of deionized water to quench the reaction, extract with dichloromethane, combine the organic phases and add anhydrous sodium sulfate to absorb excess water, remove the solvent under reduced pressure, and then use an oil pump to pump out excess N,N-dimethylformamide to obtain a brown solid compound 4;

[0056] S5: Weigh N-(3-chloropropyl)morpholine (1 g, 6.11 mmol) and sodium azide (476.72 mg, 7.33 mmol) and dissolve in 20 ml of N,N-dimethylformamide, reflux at 50 °C for 3 hours under nitrogen protection. After the reaction is completed, cool to room temperature, add a large amount of water, extract with ethyl acetate, and then remove the solvent under reduced pressure to obtain compound 5;

[0057] S6: Isophorone (1.38 g, 10 mmol), malononitrile (0.66 g, 10 mmol), sodium acetate trihydrate (0.4 g, 5 mmol) and 9 ml of ethanol were placed in a flask. The mixture was stirred and heated at 50 °C overnight. The solvent was then removed under reduced pressure. The residue was eluted with petroleum ether / dichloromethane (50:1, v / v) to obtain a white solid compound 6;

[0058] S7: 2,4-dihydroxybenzaldehyde (800.00 mg, 5.79 mmol), 3-bromopropyne (689.02 mg, 5.79 mmol) and potassium carbonate (1.20 g, 8.69 mmol) were dissolved in acetonitrile (10 ml). The reaction mixture was refluxed at 80 ° C for 30 minutes under nitrogen. The solvent was then removed under reduced pressure. The residue was eluted with dichloromethane / petroleum ether (1:1, v / v) to obtain white solid compound 7;

[0059] S8: Compound 7 (1.00 g, 5.68 mmol) and compound 6 (1.06 g, 5.68 mmol) were dissolved in acetonitrile (8 ml), and then piperidine (96.67 mg, 1.14 mmol) was added. The mixture was refluxed at 80 ° C for 6 hours under nitrogen atmosphere, and then the solvent was removed under reduced pressure. The residue was eluted with petroleum ether / ethyl acetate (10:1, v / v) to obtain yellow compound 8;

[0060] S9: Compound 8 (1 g, 2.90 mmol) was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine (587.62 mg, 5.80 mmol) was gradually added to the solution under a nitrogen atmosphere at 0°C. The reaction mixture was stirred at 0°C for 30 minutes. Acryloyl chloride (262.78 mg, 2.90 mmol) was then gradually added to the solution and reacted for 30 minutes, and the solvent was removed under reduced pressure. The residue was eluted with petroleum ether / ethyl acetate (12:1, v / v) to obtain a yellow solid compound 9;

[0061] S10: Compound 8 (1 g, 2.90 mmol) was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine (587.62 mg, 5.80 mmol) was gradually added to the solution under a nitrogen atmosphere at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, and then 3-thiophenecarbonyl chloride (425.62 mg, 2.90 mmol) was gradually added to the solution for 1 hour, and the solvent was removed under reduced pressure. The residue was eluted with petroleum ether / ethyl acetate (15:1, v / v) to obtain yellow solid compound 10;

[0062] S11: Compound 8 (1 g, 2.90 mmol) was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine (587.62 mg, 5.80 mmol) was gradually added to the solution under a nitrogen atmosphere at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, and then acetyl chloride (227.91 mg, 2.90 mmol) was gradually added to the solution for 1 hour, and the solvent was removed under reduced pressure. The residue was eluted with petroleum ether / ethyl acetate (12:1, v / v) to obtain an orange-yellow solid compound 11;

[0063] S12: Compound 8 (1 g, 2.90 mmol) was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine (587.62 mg, 5.80 mmol) was gradually added to the solution under a nitrogen atmosphere at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, and then benzoyl chloride (408.13 mg, 2.90 mmol) was gradually added to the solution for 40 minutes, and the solvent was removed under reduced pressure. The residue was eluted with petroleum ether / ethyl acetate (15:1, v / v) to obtain compound 12;

[0064] S13: Compound 8 (1 g, 2.90 mmol) was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine (587.62 mg, 5.80 mmol) was gradually added to the solution under a nitrogen atmosphere at 0°C. The reaction mixture was stirred at 0°C for 30 minutes, and then cyclopropylcarbonyl chloride (303.51 mg, 2.90 mmol) was gradually added to the solution for 30 minutes, and the solvent was removed under reduced pressure. The residue was eluted with petroleum ether / ethyl acetate (12:1, v / v) to obtain compound 13;

[0065] S14: Compound 8 (1 g, 2.90 mmol) was dissolved in 10 ml of anhydrous dichloromethane, triethylamine (587.62 mg, 5.80 mmol) was gradually added to the solution under a nitrogen atmosphere at 0°C, the reaction mixture was stirred at 0°C for 30 minutes, then dimethylaminoformyl chloride (468.35 mg, 4.36 mmol) was gradually added to the solution and reacted for 50 minutes, and the solvent was removed under reduced pressure. The residue was eluted with petroleum ether / ethyl acetate (20:1, v / v) to obtain an orange solid compound 14;

[0066] S15: Compound 8 (1 g, 2.90 mmol), 4-nitrobenzyl bromide (940.88 mg, 4.36 mmol), and potassium carbonate (802.55 mg, 5.80 mmol) were dissolved in 15 ml of N,N-dimethylformamide. The reaction mixture was stirred at room temperature for 4 hours, and the solvent was removed under reduced pressure. The residue was eluted with petroleum ether / ethyl acetate (10:1, v / v) to obtain yellow solid compound 15;

[0067] S16: Compound 8 (1 g, 2.90 mmol), 2,4-dinitrofluorobenzene (1.08 g, 5.80 mmol), and potassium carbonate (802.55 mg, 5.80 mmol) were dissolved in 20 ml of N,N-dimethylformamide, and the reaction mixture was stirred at room temperature for 6 hours, and the solvent was removed under reduced pressure. The residue was purified by column chromatography, wherein petroleum ether / ethyl acetate = 15:1 (v / v), to obtain orange compound 16;

[0068] S17: Synthesis of the target compound: Sodium ascorbate (0.816 g, 0.412 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (4 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (6 ml) solution containing compound 9 (0.328 g, 0.824 mmol) and compound 2 (0.169 g, 0.824 mmol) and reacted for 12 hours. Extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. Using dichloromethane / methanol (15:1, v / v) as the eluent, a yellow solid DCX-1 was obtained, after which the entire design and synthesis method based on dicyanoisophorone fluorescent probe was completed.

[0069] In step S17, sodium ascorbate (0.816 g, 0.412 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (4 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (6 ml) solution containing compound 10 (0.374 g, 0.824 mmol) and compound 2 (0.169 g, 0.824 mmol), and the reaction was allowed to proceed overnight. The mixture was extracted with dichloromethane, excess water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (10:1, v / v) to obtain a yellow solid DCX-2;

[0070] Sodium ascorbate (0.816 g, 0.412 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (4 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (6 ml) solution containing compound 11 (0.318 g, 0.824 mmol) and compound 2 (0.169 g, 0.824 mmol) and reacted overnight. The mixture was extracted with dichloromethane, and the excess water was dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (20:1, v / v) to obtain a yellow compound DCX-3

[0071] In step S17, sodium ascorbate (0.816 g, 0.412 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (4 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (6 ml) solution containing compound 12 (0.369 g, 0.824 mmol) and compound 2 (0.169 g, 0.824 mmol), and the reaction was allowed to proceed overnight. The mixture was extracted with dichloromethane, excess water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (15:1, v / v) to obtain a yellow solid DCX-4;

[0072] Sodium ascorbate (0.816 g, 0.412 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (4 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a solution of compound 13 (0.340 g, 0.824 mmol) and compound 2 (0.169 g, 0.824 mmol) in THF (6 ml) and reacted overnight. The mixture was extracted with dichloromethane, excess water was absorbed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (12:1, v / v) to obtain a yellow solid DCX-5.

[0073] In step S17, sodium ascorbate (0.816 g, 0.412 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (4 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (6 ml) solution containing compound 14 (0.342 g, 0.824 mmol) and compound 2 (0.169 g, 0.824 mmol), and the reaction was allowed to proceed overnight. The mixture was extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was eluted with dichloromethane / methanol (15:1, v / v) to obtain the yellow target DCX-6;

[0074] Sodium ascorbate (0.816 g, 0.412 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (4 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a solution of compound 15 (0.395 g, 0.824 mmol) and compound 2 (0.169 g, 0.824 mmol) in THF (6 ml) and reacted overnight. The mixture was extracted with dichloromethane, dehydrated with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was eluted with dichloromethane / methanol (20:1, v / v) to obtain a yellow solid DCX-7.

[0075] In step S17, sodium ascorbate (0.816 g, 0.412 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (4 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (6 ml) solution containing compound 16 (0.421 g, 0.824 mmol) and compound 2 (0.169 g, 0.824 mmol), and the reaction was allowed to proceed overnight. The mixture was extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (15:1, v / v) to obtain a yellow solid DCX-8;

[0076] Sodium ascorbate (1.632 g, 0.824 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in 5 ml H2O, and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (15 ml) solution containing compound 9 (0.328 g, 0.824 mmol) and compound 5 (0.21 g, 1.236 mmol) and reacted for 10 hours. The mixture was extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (15:1, v / v) to obtain a yellow solid DCX-9.

[0077] In step S17, sodium ascorbate (1.632 g, 0.824 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (5 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (15 ml) solution containing compound 10 (0.374 g, 0.824 mmol) and compound 5 (0.21 g, 1.236 mmol) and reacted for 10 hours. The mixture was extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (20:1, v / v) to obtain a yellow solid DCX-10;

[0078] Sodium ascorbate (1.632 g, 0.824 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (5 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (15 ml) solution containing compound 11 (0.318 g, 0.824 mmol) and compound 5 (0.21 g, 1.236 mmol) and reacted for 10 hours. The mixture was extracted with dichloromethane, dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (14:1, v / v) to obtain a yellow solid DCX-11.

[0079] In step S17, sodium ascorbate (1.632 g, 0.824 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (5 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (15 ml) solution containing compound 12 (0.370 g, 0.824 mmol) and compound 5 (0.21 g, 1.236 mmol) and reacted for 10 hours. The mixture was extracted with dichloromethane, dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (15:1, v / v) to obtain a yellow product DCX-12;

[0080] Sodium ascorbate (1.632 g, 0.824 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (5 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (15 ml) solution containing compound 13 (0.34 g, 0.824 mmol) and compound 5 (0.21 g, 1.236 mmol) and reacted for 10 hours. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (8:1, v / v) to obtain yellow solid DCX-13;

[0081] Sodium ascorbate (1.632 g, 0.824 mmol) and CuSO4.5H2O (0.992 g, 0.412 mmol) were dissolved in H2O (5 ml), and the mixture was stirred at room temperature for 30 minutes. The mixture was then added to a THF (15 ml) solution containing compound 15 (0.395 g, 0.824 mmol) and compound 5 (0.21 g, 1.236 mmol) and reacted for 10 hours. The mixture was extracted with dichloromethane, water was absorbed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (10:1, v / v) to obtain yellow solid DCX-14.

[0082] In step S17, sodium ascorbate (3.264 g, 1.648 mmol) and CuSO4.5H2O (1.984 g, 0.824 mmol) were dissolved in H2O (10 ml) and stirred at room temperature for 30 minutes. The mixed solution was then added to a THF (30 ml) solution containing compound 9 (0.656 g, 1.648 mmol) and compound 4 (0.89 g, 2.472 mmol) and reacted for 12 hours. The mixture was extracted with dichloromethane, dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was purified by chromatography, and the chromatography fluid was dichloromethane / methanol = 20:1 (v / v) to obtain a yellow product DCX-15;

[0083] Sodium ascorbate (3.264 g, 1.648 mmol) and CuSO4.5H2O (1.984 g, 0.824 mmol) were dissolved in H2O (10 ml) and stirred at room temperature for 30 minutes. The mixed solution was then added to a THF (30 ml) solution containing compound 10 (0.75 g, 1.648 mmol) and compound 4 (0.89 g, 2.472 mmol) and reacted for 12 hours. The mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (15:1, v / v) to obtain a yellow solid DCX-16;

[0084] Sodium ascorbate (3.264 g, 1.648 mmol) and CuSO4.5H2O (1.984 g, 0.824 mmol) were dissolved in H2O (10 ml) and stirred at room temperature for 30 minutes. The mixed solution was then added to a THF (30 ml) solution containing compound 11 (0.637 g, 1.648 mmol) and compound 4 (0.89 g, 2.472 mmol) and reacted for 12 hours. The mixture was extracted with dichloromethane, dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (16:1, v / v) to obtain a yellow product DCX-17.

[0085] In step S17, sodium ascorbate (3.264 g, 1.648 mmol) and CuSO4.5H2O (1.984 g, 0.824 mmol) were dissolved in H2O (10 ml) and stirred at room temperature for 30 minutes. The mixed solution was then added to a THF (30 ml) solution containing compound 12 (0.74 g, 1.648 mmol) and compound 4 (0.89 g, 2.472 mmol) and reacted for 12 hours. The mixture was extracted with dichloromethane, and residual water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (20:1, v / v) to obtain yellow solid DCX-18;

[0086] Sodium ascorbate (3.264 g, 1.648 mmol) and CuSO4.5H2O (1.984 g, 0.824 mmol) were dissolved in H2O (10 ml) and stirred at room temperature for 30 minutes. The mixed solution was then added to a THF (30 ml) solution containing compound 13 (0.656 g, 1.648 mmol) and compound 4 (0.89 g, 2.472 mmol) and reacted for 12 hours. The mixture was extracted with dichloromethane, and the residual water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (18:1, v / v) to obtain yellow solid DCX-19;

[0087] Sodium ascorbate (3.264 g, 1.648 mmol) and CuSO4.5H2O (1.984 g, 0.824 mmol) were dissolved in H2O (10 ml) and stirred at room temperature for 30 minutes. The mixed solution was then added to a THF (30 ml) solution containing compound 15 (0.791 g, 1.648 mmol) and compound 4 (0.89 g, 2.472 mmol) and reacted for 12 hours. The mixture was extracted with dichloromethane, and the residual water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The residue was eluted with dichloromethane / methanol (10:1, v / v) to obtain a yellow product DCX-20.

[0088] List of target compounds synthesized

[0089]

[0090] Table (continued)

[0091]

[0092]

[0093] Table (continued)

[0094]

[0095]

[0096] The evaluation method includes the following steps:

[0097] S1: To test the spectrum, weigh a certain amount of DCX-1 and Cys and dissolve them in DMSO and PBS respectively to prepare 10mM probe DCX-1 stock solution and 50mM Cys stock solution. Other test solutions, such as small molecule thiols (GSH, Hcy), anions (ClO - ,CO3 2- ,SO4 2- ,I - ,Br - ,HS - ), cation (Cu 2+ ,Na + ,Ca 2+ ,Fe 3+ ), amino acids (GSH, Hcy, Gln, Tyr, Arg, Ala, ILE, Lys, Phe, Met, Thr, Gly, His), etc., were dissolved in PBS to make a solution with a concentration of 50mM. The scanning range of UV spectrum and fluorescence spectrum is 460-800nm. The slit width is 5nm×5nm. The test content includes UV spectrum experiment, fluorescence selectivity experiment, probe anti-interference experiment, concentration titration experiment, Job plot and other experiments

[0098] S2: Cytotoxicity experiment, LO2 cells were seeded into 96-well plates, incubated at 37°C, 5% CO2 for 24 hours, and then treated with different concentrations of probes for 24 hours. The supernatant was removed and 10 μL MTT (5 mg / mL) was added. The cells were incubated at 37°C for another 4 hours, and the absorbance at 488 nm was recorded using an ELX800 absorbance microplate reader.

[0099] S3 probe detection in cells, first HepG2 cells were evenly seeded into 24-well plates and cultured for 24 hours, then the cultured HepG2 cells were treated with different methods and divided into blank cell group, pure probe group, Cys supplement group and NEM (N-ethylmaleimide, a thiol blocker) group.

[110] ) pretreatment group. Pure probe group: 10μM probe DCX-1 was incubated for 30 minutes, then fixed and anti-fluorescence quencher was added. Cys supplemented group: 10μM probe DCX-1 was incubated for 30 minutes, washed 3 times with PBS (pH=7.4). Then the probe DCX-1 was incubated with 100μM Cys for 60 minutes, washed 3 times with PBS (pH=7.4), then fixed and anti-fluorescence quencher was added. NEM pretreatment group: cells were pretreated with 1mM NEM for 2 hours, washed 3 times with PBS (pH=7.4), and then 10μM probe DCX-1 was added for incubation for 30 minutes, fixed and anti-fluorescence quencher was added.

[0100] In order to explore whether the probe DCX-1 can accurately locate hepatocytes, four cells, HepG2, A549, Hela and SGC-7901, were treated in the same way. Then the anti-fluorescence quencher was dropped onto the slide, fastened and sealed. All images were collected by a Leica sp8 laser scanning confocal microscope. The excitation wavelength of the probe was 488nm, and the emission wavelength was collected in the range of 560-650nm.

[0101] Experimental results and discussion of evaluation methods

[0102] Evaluation of UV spectrum experimental results of probe DCX-1:

[0103] In order to explore the sensitivity of probe DCX-1 to Cys, the UV absorption spectrum of probe DCX-1 in PBS / DMSO (v / v=7:3, pH=7.4) solution was studied. Figure 1 As shown in the figure, in the absence of Cys, the probe DCX-1 solution showed a strong absorption peak near 405nm. After the probe was co-cultured with 50μM Cys for 10 minutes, the new absorption peak red-shifted to 438nm. With the increase of Cys concentration, the absorption intensity gradually increased. From the changes in the UV spectrum, it can be preliminarily determined that the probe DCX-1 responds to Cys.

[0104] Evaluation of fluorescence titration results:

[0105] The corresponding fluorescence titration experiment was carried out in the PBS:DMSO=7:3 (v / v, pH=7.4) system. Figure 2 As shown in the figure, in the absence of Cys, the probe DCX-1 has an emission peak at 611nm, and the corresponding fluorescence intensity is 473.784. After adding Cys, as the Cys concentration increases, the fluorescence emitted by the probe DCX-1 at 611nm is significantly enhanced. When the Cys concentration reaches 50μM, the fluorescence intensity increases to 2037.838, which is 4.3 times higher.

[0106] In addition, from Figure 3It can be seen that in the range of 0-35 μM, the fluorescence intensity of DCX-1 is linearly related to the Cys concentration, and the regression equation is y=48.71x+449.2(R 2 =0.9827), and the detection limit (LOD) was calculated to be 0.31 μM according to the formula 3σ / k. The detection limit of probe DCX-1 is very low, reaching the μM level, indicating that even a small amount of Cys can be detected by probe DCX-1.

[0107] Evaluation of the selectivity of probe DCX-1 for Cys:

[0108] The specificity of probe DCX-1 for Cys was evaluated in a PBS:DMSO=7:3 (v / v, pH=7.4) solution system. Figure 4 The fluorescence of DCX-1 solution was significantly enhanced after adding Cys, but was not significantly enhanced after adding anions (ClO - 、CO3 2- 、SO4 2- ,I - Br - , HS - ), cation (Cu 2+ 、Na + , Ca 2+ , Fe 3+ ), reactive oxygen (H2O2), biothiols (GSH, Hcy), representative amino acids (Gln, Tyr, Arg, Ala, Ile, Lys, Phe, Met, Thr, Gly, His) and some other biomarkers (BSA, trypsin, lysozyme, glyoxal oxidase, urea, vitamin C, tert-butyl peroxide, etc.), the fluorescence intensity decreased slightly, indicating that the probe DCX-1 does not respond to these biomarkers. After adding Cys, the fluorescence intensity increased significantly, increasing by about 4 times. The main reason is that in physiological media, Cys is more nucleophilic than other ions or biothiols to be measured, and it is easier to react with the acrylate structure to release the fluorophore, thereby producing strong fluorescence. The results show that the probe DCX-1 has good selectivity for Cys.

[0109] Evaluation of the competitive experiment results of probe DCX-1 on Cys:

[0110] Fluorescent probes are often affected by other substances in practical applications, so competition experiments with other thiols and amino acids were conducted on the probe DCX-1 to explore its anti-interference ability. Figure 5As shown in the figure, when the same amount of GSH, Hcy, Gln, Tyr, Arg, Ala, ILE, Lys, Phe, Met, Thr, Gly, and His were added to the DCX-1 test solution, there was no significant change in fluorescence intensity compared with the blank control group (black bar). On this basis, the same amount of Cys was added, and the fluorescence intensity was significantly enhanced (red bar), indicating that the detection of Cys by probe DCX-1 is not interfered by other substances and has good anti-interference ability.

[0111] Evaluation of the response time results of the probe DCX-1:

[0112] Response time is another important indicator to consider whether the probe can play its own advantages in practical applications. If the response time between the probe and the detected object is very long, the detection efficiency will be greatly reduced, which also goes against the original intention of the probe design. In order to evaluate the sensitivity of the probe, its response time was measured. Figure 6 As shown in the figure, after adding Cys, the fluorescence intensity of the probe DCX-1 changes immediately and increases to about 500. As time goes by, the fluorescence intensity gradually increases and reaches the maximum value at 20 minutes. After reaching the maximum value, it remains stable and no fluorescence quenching occurs. This shows that the probe DCX-1 has good stability, a short response time to Cys, and high sensitivity, and can be used to monitor Cys in life systems in real time.

[0113] Evaluation of pH stability of probe DCX-1:

[0114] In order to explore whether the probe DCX-1 can respond to Cys in a complex physiological environment and find the most suitable pH detection environment, this experiment selected a pH range of 3.0-12.0 to study the changes in the fluorescence intensity of the probe DCX-1. Figure 7 As shown in the figure, within the pH range of 3.0-9.0, the fluorescence intensity of the probe DCX-1 does not change much. After adding Cys, within the pH range of 5.0-7.0, the fluorescence intensity of the probe DCX-1 increases significantly, and the fluorescence intensity reaches a maximum at around 7.0. The pH value of the physiological environment in a healthy human body is between 7.35-7.45. It can be seen that the probe can detect Cys under the pH of the human physiological environment, and the fluorescence intensity can reach the maximum value, which has great advantages for detecting complex life systems with rapidly changing pH values.

[0115] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprise" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0116] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A design and synthesis method based on dicyanoisophorone fluorescent probe, characterized in that: The synthesis method comprises the following steps: S1: First, prepare the intermediate for synthesizing dicyanoisophorone fluorescent probe; weigh 1.5g D-galactose and add it to a 150mL eggplant-shaped bottle, then add 5mL acetic anhydride to dissolve the D-galactose, then transfer the system to an ice-water bath environment to react for 30 minutes, then slowly drop 0.1mL perchloric acid, after stirring for 6 hours, monitor the reaction completion by TLC; adjust the pH of the system to neutral with saturated sodium bicarbonate, extract with ethyl acetate; wash with saturated sodium chloride solution, absorb excess water with anhydrous sodium sulfate, and finally filter the dried organic layer, and perform vacuum rotary evaporation on the collected filtrate to obtain a crude product in the form of light yellow syrup; no purification is required. The crude product of the previous step was dissolved in 10 mL of dichloromethane, and the reaction was continued in an ice-water bath for 30 minutes, and then 0.35 mL of hydrobromic acid was slowly added dropwise. After the addition was completed, the reaction system was transferred to room temperature and the reaction was continued for 6 hours. The reaction was monitored to be complete by TLC. The post-treatment of the reaction system was the same as the previous step. After multiple extractions, drying, suction filtration, and vacuum rotary evaporation, a white solid acetyl brominated galactose was obtained. The product did not need to be purified and the next step was continued. The white solid acetyl brominated galactose obtained above was dissolved in 20 mL of DMSO. At room temperature, NaN3 was added to the reaction system while stirring. After reacting for 4 hours, the reaction system was quenched with water, extracted with ethyl acetate, washed with saturated sodium chloride solution, and excess water was sucked out with anhydrous sodium sulfate. Finally, suction filtration, concentration, and purification with a silica gel column were performed to obtain a white compound 1. S2: The white solid 1 was dissolved in 10 mL of dry methanol, and sodium methoxide was added under stirring to adjust the pH of the reaction system to 10, and the reaction was allowed to react at room temperature for 4 hours. The reaction was complete when detected by TLC; then a deionized exchange resin was added to adjust the reaction system to neutrality, and then the reaction was filtered, and the solvent was removed by vacuum rotary evaporation, and finally the white solid compound 2 was purified and separated by silica gel column; S3: Weigh the compounds triphenylphosphine and 1,4-dibromobutane and place them in a dry 100 ml eggplant-shaped bottle, add 20 ml toluene to fully dissolve them, reflux them at 120°C under nitrogen protection for 12 hours, cool them to room temperature after the reaction, remove the solvent by vacuum filtration, and wash them three times with toluene to obtain a white powder solid compound 3; S4: Weigh compound 3 and place it in a dry 25 ml round-bottom flask, dissolve it with 5 ml N,N-dimethylformamide, then add sodium azide, react at room temperature for 3 hours under nitrogen protection, add 10 ml deionized water to quench the reaction, extract with dichloromethane, combine the organic phases and add anhydrous sodium sulfate to absorb excess water, remove the solvent under reduced pressure, and then use an oil pump to pump out excess N,N-dimethylformamide to obtain a brown solid compound 4; S5: Weigh N-morpholine and sodium azide and dissolve them in 20 ml of N,N-dimethylformamide. Reflux at 50°C for 3 hours under nitrogen protection. After the reaction is completed, cool to room temperature, add a large amount of water, extract with ethyl acetate, and then remove the solvent under reduced pressure to obtain compound 5; S6: Isophorone, malononitrile, sodium acetate trihydrate and 9 ml of ethanol were placed in a flask; the mixture was stirred and heated at 50° C. overnight; the solvent was then removed under reduced pressure; the residue was eluted with petroleum ether / dichloromethane to obtain a white solid compound 6; S7: 2,4-dihydroxybenzaldehyde, 3-bromopropyne and potassium carbonate were dissolved in acetonitrile; the reaction mixture was refluxed at 80° C. for 30 minutes under a nitrogen environment; the solvent was then removed under reduced pressure; the residue was eluted with dichloromethane / petroleum ether to obtain a white solid compound 7; S8: Compound 7 and compound 6 were dissolved in acetonitrile, and then piperidine was added; the mixture was refluxed at 80° C. for 6 hours under a nitrogen environment, and then the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain a yellow compound 8; S9: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen atmosphere at 0°C; the reaction mixture was stirred at 0°C for 30 minutes; acryloyl chloride was then gradually added to the solution and reacted for 30 minutes, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain yellow solid compound 9; S10: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen environment at 0°C; the reaction mixture was stirred at 0°C for 30 minutes, and then 3-thiophenecarbonyl chloride was gradually added to the solution for reaction for 1 hour, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain yellow solid compound 10; S11: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen atmosphere at 0°C; the reaction mixture was stirred at 0°C for 30 minutes, and then acetyl chloride was gradually added to the solution to react for 1 hour, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain an orange-yellow solid compound 11; S12: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen environment at 0°C; the reaction mixture was stirred at 0°C for 30 minutes, and then benzoyl chloride was gradually added to the solution to react for 40 minutes, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain compound 12; S13: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, and triethylamine was gradually added to the solution under a nitrogen environment at 0°C; the reaction mixture was stirred at 0°C for 30 minutes, and then cyclopropylcarbonyl chloride was gradually added to the solution to react for 30 minutes, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain compound 13; S14: Compound 8 was dissolved in 10 ml of anhydrous dichloromethane, triethylamine was gradually added to the solution under a nitrogen atmosphere at 0°C, the reaction mixture was stirred at 0°C for 30 minutes, dimethylaminoformyl chloride was gradually added to the solution and reacted for 50 minutes, and the solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain an orange solid compound 14; S15: Compound 8, 4-nitrobenzyl bromide and potassium carbonate were dissolved in 15 ml of N,N-dimethylformamide, and the reaction mixture was stirred at room temperature for 4 hours. The solvent was removed under reduced pressure; the residue was eluted with petroleum ether / ethyl acetate to obtain yellow solid compound 15; S16: Compound 8, 2,4-dinitrofluorobenzene and potassium carbonate were dissolved in 20 ml of N,N-dimethylformamide, the reaction mixture was stirred at room temperature for 6 hours, and the solvent was removed under reduced pressure; the residue was purified by column chromatography, wherein petroleum ether / ethyl acetate = 15:1, to obtain orange compound 16; S17: Synthesis of target compound: Sodium ascorbate and CuSO4 . 5H2O is dissolved in H2O, and the mixture is stirred at room temperature for 30 minutes; then the mixture is added to a THF solution containing compound 9 and compound 2, and the reaction is carried out for 12 hours; it is extracted with dichloromethane, and the water is removed by anhydrous sodium sulfate, and the solvent is evaporated under reduced pressure; dichloromethane / methanol is used as an eluent to obtain a yellow solid DCX-1, and then the entire design and synthesis method based on dicyanoisophorone fluorescent probe is completed.

2. The design and synthesis method based on dicyanoisophorone fluorescent probe as claimed in claim 1, characterized in that: In step S17, sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 10 and compound 2, and the reaction was allowed to proceed overnight; the mixture was extracted with dichloromethane, excess water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-2; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 11 and compound 2, and the reaction was allowed to proceed overnight; the mixture was extracted with dichloromethane, excess water was dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow compound DCX-3.

3. The design and synthesis method based on dicyanoisophorone fluorescent probe as claimed in claim 1, characterized in that: In step S17, sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 12 and compound 2, and the reaction was allowed to proceed overnight; the mixture was extracted with dichloromethane, excess water was absorbed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-4; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 13 and compound 2, and the reaction was allowed to proceed overnight; the mixture was extracted with dichloromethane, excess water was absorbed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-5.

4. The design and synthesis method based on dicyanoisophorone fluorescent probe according to claim 1, characterized in that: In step S17, sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 14 and compound 2, and the reaction was allowed to proceed overnight; the mixture was extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain the yellow target DCX-6; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 15 and compound 2, and the reaction was allowed to proceed overnight; the mixture was extracted with dichloromethane, water was removed with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-7.

5. The design and synthesis method based on dicyanoisophorone fluorescent probe as claimed in claim 1, characterized in that: In step S17, sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 16 and compound 2, and the reaction was allowed to proceed overnight; the mixture was extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-8; Sodium ascorbate and CuSO4 . 5H2O was dissolved in 5ml H2O, and the mixture was stirred at room temperature for 30 minutes; then the mixture was added to a THF solution containing compound 9 and compound 5, and the reaction was carried out for 10 hours; it was extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-9.

6. The design and synthesis method based on dicyanoisophorone fluorescent probe according to claim 1, characterized in that: In step S17, sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 10 and compound 5, and the reaction was continued for 10 hours; the mixture was extracted with dichloromethane, the water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-10; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 11 and compound 5, and the reaction was continued for 10 hours; the mixture was extracted with dichloromethane, the water was dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-11.

7. The design and synthesis method based on dicyanoisophorone fluorescent probe according to claim 1, characterized in that: In step S17, sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 12 and compound 5, and the reaction was continued for 10 hours; the mixture was extracted with dichloromethane, dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow product DCX-12; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 13 and compound 5, and the reaction was continued for 10 hours; the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain yellow solid DCX-13; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O, and the mixture was stirred at room temperature for 30 minutes; the mixture was then added to a THF solution containing compound 15 and compound 5, and the reaction was continued for 10 hours; the mixture was extracted with dichloromethane, water was absorbed by anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-14.

8. The design and synthesis method based on dicyanoisophorone fluorescent probe according to claim 1, characterized in that: In step S17, sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O and stirred at room temperature for 30 minutes; the mixed solution was then added to a THF solution containing compound 9 and compound 4 and reacted for 12 hours; the mixture was extracted with dichloromethane, dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was purified by chromatography, and the chromatography fluid was dichloromethane / methanol = 20:1 to obtain a yellow product DCX-15; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O and stirred at room temperature for 30 minutes; the mixed solution was then added to a THF solution containing compound 10 and compound 4 and reacted for 12 hours; the mixture was extracted with dichloromethane, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-16; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O and stirred at room temperature for 30 minutes; the mixed solution was then added to a THF solution containing compound 11 and compound 4 and reacted for 12 hours; extracted with dichloromethane, dehydrated with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow product DCX-17.

9. The design and synthesis method based on dicyanoisophorone fluorescent probe according to claim 1, characterized in that: In step S17, sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O and stirred at room temperature for 30 minutes; the mixed solution was then added to a THF solution containing compound 12 and compound 4 and reacted for 12 hours; the mixture was extracted with dichloromethane, residual water was removed with anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain yellow solid DCX-18; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O and stirred at room temperature for 30 minutes; the mixed solution was then added to a THF solution containing compound 13 and compound 4 and reacted for 12 hours; the mixture was extracted with dichloromethane, and the residual water was removed by anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow solid DCX-19; Sodium ascorbate and CuSO4 . 5H2O was dissolved in H2O and stirred at room temperature for 30 minutes; the mixed solution was then added to a THF solution containing compound 15 and compound 4 and reacted for 12 hours; the mixture was extracted with dichloromethane, and the residual water was removed by anhydrous sodium sulfate, and the solvent was removed under reduced pressure; the residue was eluted with dichloromethane / methanol to obtain a yellow product DCX-20.

10. The evaluation method based on dicyanoisophorone fluorescent probe according to claim 1, characterized in that: The evaluation method comprises the following steps: S1: To conduct spectral testing, a certain amount of DCX-1 and Cys were weighed and dissolved in DMSO and PBS respectively to prepare 10mM probe DCX-1 stock solution and 50mM Cys stock solution; other test solutions, such as small molecule thiols, anions, cations, amino acids, etc., were dissolved in PBS to prepare a solution with a concentration of 50mM; the scanning range of UV spectrum and fluorescence spectrum was 460-800nm; the slit width was 5nm×5nm; the test content included UV spectrum experiment, fluorescence selectivity experiment, probe anti-interference experiment, concentration titration experiment, Job plot experiment, etc. S2: Cytotoxicity experiment, LO2 cells were seeded into 96-well plates, incubated at 37°C, 5% CO2 for 24 hours, and then treated with different concentrations of probes for 24 hours; the supernatant was removed and 10 μL MTT was added; incubated at 37°C for another 4 hours, and finally the absorbance at 488 nm was recorded using an ELX800 absorbance microplate reader. S3 was used to detect the probe in cells. First, HepG2 cells were evenly inoculated into 24-well plates and cultured for 24 hours. Then, the cultured HepG2 cells were treated differently and divided into blank cell group, pure probe group, Cys supplement group and NEM pretreatment group; pure probe group: 10 μM probe DCX-1 was incubated for 30 minutes, then fixed and anti-fluorescence quencher was added; Cys supplement group: 10 μM probe DCX-1 was incubated for 30 minutes, washed 3 times with PBS; then probe DCX-1 was incubated with 100 μM Cys for 60 minutes, washed 3 times with PBS, then fixed and anti-fluorescence quencher was added; NEM pretreatment group: cells were pretreated with 1 mM NEM for 2 hours, washed 3 times with PBS, then 10 μM probe DCX-1 was added and incubated for 30 minutes, fixed and anti-fluorescence quencher was added; In order to explore whether the probe DCX-1 can accurately locate hepatocytes, four types of cells, HepG2, A549, Hela and SGC-7901, were treated in the same way; then the anti-fluorescence quencher was dropped onto the slide, fastened and sealed; all images were collected by a Leicasp8 laser scanning confocal microscope; the excitation wavelength of the probe was 488nm, and the emission wavelength was collected in the range of 560-650nm.

Images