CN105295007B - One kind conjugation water-soluble polymer tracer material and preparation method thereof - Google Patents

Abstract

The invention relates to a water-soluble conjugated polymer tracer molecule, which has the following molecular structural formula: This material has the property of absorbing the fluorescence of the main chain of polyfluorene, and when the azo molecule of the side chain is broken, the blue light will be restored, which can be used for tumor cell tracking and drug delivery.

Description

A kind of conjugated water-soluble polymer tracer material and preparation method thereof

technical field

The invention belongs to the field of biosensing of water-soluble conjugated polymers, and in particular relates to the synthesis of water-soluble polymers whose main chain is polyfluorene structure and whose side chains have azo molecules and quaternary ammonium groups.

Background technique

Due to their excellent optical and electrical properties, conjugated conductive polymers have attracted the attention of researchers in the fields of organic electronics and bioelectronics. Water-soluble conjugated polymers can be used as highly sensitive fluorescent probes and sensing materials in chemical/biological sensing, biomedical imaging, disease diagnosis and treatment, and other fields. As a special class of conjugated polymers, water-soluble conjugated polymers mainly refer to conjugated polymers whose side chains contain ionic functional groups or non-ionic water-soluble functional groups. The combination of the water solubility of the electrolyte makes it suitable for the detection of biomolecules such as DNA and protein.

Contents of the invention

Technical problem: The object of the present invention is a water-soluble conjugated polymer tracer material and its preparation method. The material prepared in the present invention has the characteristic of absorbing the blue light of the main chain polyfluorene structure. When encountering the specific azo reductase in colon cancer tumor cells, the azo molecule breaks and the blue light recovers, so as to achieve the purpose of detecting cancer cells .

Technical solution: The water-soluble conjugated polymer tracer material of the present invention has the following molecular structure:

Wherein, n is the polymerization degree, and n is 8-12;

The 5 of the side chain is the degree of polymerization of the azo molecule, which means that one side chain is connected with 5 azo molecules.

The main chain of this material is composed of polyfluorene.

The side chain of the material is an azo molecular structure.

The preparation method of water-soluble conjugated polymer tracer material of the present invention is:

The specific steps of the method are as follows:

Compound 1: Add 2,7-dibromofluorene, n-tetrabutylammonium bromide and 1,6-dibromohexane into a two-necked round-bottomed flask with a reflux condenser and magnet; seal the entire reaction system after adding Vacuumize, then fill with nitrogen, keep the whole reaction process in nitrogen atmosphere; add 50% KOH aqueous solution, react at 90°C for 4-5h, cool the reaction mixture to room temperature; neutralize the reaction mixture with hydrochloric acid solution After reaching neutrality, extract with dichloromethane, and dry the extract with anhydrous sodium sulfate; filter, use a rotary evaporator for precipitation under reduced pressure, and remove excess 1,6-dibromohexane by distillation under reduced pressure; The product was mixed with silica gel and petroleum ether was used as the washing liquid, and the residue was recrystallized with methanol/dichloromethane after desolvation under reduced pressure to obtain white crystals;

Compound 2: Add 2,7-dibromofluorene and n-tetrabutylammonium bromide to a two-necked round-bottomed flask with a reflux condenser and a magneton. After the addition, the entire reaction system is sealed and evacuated, and then filled with nitrogen to keep The whole reaction process is carried out in a nitrogen atmosphere; inject the solvent N,N-dimethylformamide with a syringe, and after fully dissolving, inject the sodium hydroxide solution with a syringe, and stir for 30 minutes; put the device at 90°C and slowly add 3-Bromo-1-propanol, reacted for 24 hours; after the reaction was completed, extracted three times with saturated saline and dichloromethane, combined the organic layer, then dried with anhydrous sodium sulfate, and filtered the solid crude product with silica gel Using petroleum ether and ethyl acetate 4:1 as the washing solution, the silica gel chromatography was used for purification, and then the organic layer was dried and concentrated to obtain a white solid product;

Compound 3: Add compound 1, pinacol diborate and potassium acetate in a two-neck round bottom flask with a magnet, and add catalyst 1,1'-bis(diphenylphosphino)ferrocene] dichloride Palladium dichloromethane complex, after the addition, seal the entire reaction system away from light and vacuumize it, and then fill it with nitrogen to keep the entire reaction process in a nitrogen atmosphere; put the device in an environment of 90°C, and inject the solvent with a syringe N, N-dimethylformamide, reacted for 24 hours; after the reaction was completed, the crude product was dissolved with dichloromethane, passed through an alumina column, and the catalyst was removed; extracted three times with saturated saline and dichloromethane, and the organic layers were combined, and then Dry with anhydrous sodium sulfate, and after suction filtration, mix the solid crude product with silica gel, use petroleum ether and ethyl acetate 10:1 as the washing liquid, purify by silica gel chromatography, then dry and concentrate the organic layer to obtain a white solid product ;

Compound 4: Add compound 2 and compound 3, n-tetrabutylammonium bromide, and catalyst tetrakis(triphenylphosphine)palladium to a polymerization tube with a magneton, and after the addition, seal the entire reaction system away from light and evacuate it , and then filled with nitrogen to keep the whole reaction process in a nitrogen atmosphere; deoxygenate the toluene solution and potassium carbonate solution, and then use a syringe to draw the two solutions into the polymerization tube, put the device in an environment of 90 ° C, and react 4 days; after the reaction was completed, the crude product was dissolved in tetrahydrofuran, passed through an alumina column, and the catalyst was removed; after being spin-dried, it was settled in petroleum ether;

Compound 5: In a single-necked round-bottomed flask with a magnet, add Compound 4. After the addition, the entire reaction system is sealed and evacuated, and then filled with nitrogen to keep the entire reaction process in a nitrogen atmosphere; add anhydrous dichloro Methane and triethylamine, and then add 2-bromo-2-methylpropionyl bromide dropwise; put the device in an environment of 0°C and react for 2 hours; after the reaction is completed, extract the crude product with dichloromethane and water; combine The organic layer is then dried with anhydrous sodium sulfate, spin-dried after suction filtration, settled with petroleum ether in a beaker, and dried after the sedimentation;

Compound 6: Add compound 5, anisole, CuBr to a polymer tube with a magnet, freeze on dry ice, vacuumize, blow nitrogen, and thaw with ethanol, repeat the cycle three times; add trace amounts of N, N, N, N, N-pentamethyldiethylenetriamine, reacted at 100°C for 24 hours, the crude product after the reaction was passed through neutral aluminum oxide to remove the catalyst, concentrated, and mixed in methanol/water 4/1V/V Settled three times and dried under vacuum at 40°C to obtain a light yellow solid;

Compound 7: In a single-necked round-bottom flask with a magnet, add compound 6, add tetrahydrofuran, and slowly drop trimethylamine at -78°C. After 30 minutes, the solution temperature rises to room temperature. After 24 hours, distill off the solvent , to obtain a pale yellow solid.

Beneficial effects: the invention synthesizes a water-soluble conjugated polymer tracer molecule through an efficient method. Dissolve the material in dichloromethane solution. Before adding azoreductase, there is no blue light under 365nm laser irradiation. After adding azoreductase, there will be blue light under 365nm laser irradiation. This property can effectively detect Nitrogen reductase in colon cancer cells has a good application effect in the early detection of cancer cells.

detailed description

A kind of water-soluble conjugated polymer tracer material structure of the present invention is as follows:

Wherein, n is the polymerization degree, and n is 8-12;

The 5 of the side chain is the degree of polymerization of the azo molecule, which means that one side chain is connected with 5 azo molecules.

The preparation method of the water-soluble conjugated polymer tracer material of the present invention is as follows:

Compound 1: Add 2,7-dibromofluorene, n-tetrabutylammonium bromide and 1,6-dibromohexane into a two-necked round-bottomed flask with a reflux condenser and magnet; seal the entire reaction system after adding Vacuumize, then fill with nitrogen, keep the whole reaction process in nitrogen atmosphere; add 50% KOH aqueous solution, react at 90°C for 4-5h, cool the reaction mixture to room temperature; neutralize the reaction mixture with hydrochloric acid solution After reaching neutrality, extract with dichloromethane, and dry the extract with anhydrous sodium sulfate; filter, use a rotary evaporator for precipitation under reduced pressure, and remove excess 1,6-dibromohexane by distillation under reduced pressure; The product was mixed with silica gel and petroleum ether was used as washing liquid, and the residue was recrystallized with methanol/dichloromethane after desolvation under reduced pressure to obtain white crystals;

Compound 2: Add 2,7-dibromofluorene and n-tetrabutylammonium bromide to a two-necked round-bottomed flask with a reflux condenser and a magneton. After the addition, the entire reaction system is sealed and evacuated, and then filled with nitrogen to keep The whole reaction process is carried out in a nitrogen atmosphere; inject the solvent N,N-dimethylformamide with a syringe, and after fully dissolving, inject the sodium hydroxide solution with a syringe, and stir for 30 minutes; put the device at 90°C and slowly add 3-Bromo-1-propanol, reacted for 24 hours; after the reaction was completed, extracted three times with saturated saline and dichloromethane, combined the organic layer, then dried with anhydrous sodium sulfate, and filtered the solid crude product with silica gel Using petroleum ether and ethyl acetate 4:1 as the washing solution, the silica gel chromatography was used for purification, and then the organic layer was dried and concentrated to obtain a white solid product;

Compound 3: Add compound 1, pinacol diborate and potassium acetate in a two-neck round bottom flask with a magnet, and add catalyst 1,1'-bis(diphenylphosphino)ferrocene] dichloride Palladium dichloromethane complex, after the addition, seal the entire reaction system away from light and vacuumize it, and then fill it with nitrogen to keep the entire reaction process in a nitrogen atmosphere; put the device in an environment of 90°C, and inject the solvent with a syringe N, N-dimethylformamide, reacted for 24 hours; after the reaction was completed, the crude product was dissolved with dichloromethane, passed through an alumina column, and the catalyst was removed; extracted three times with saturated saline and dichloromethane, and the organic layers were combined, and then Dry with anhydrous sodium sulfate, and after suction filtration, mix the solid crude product with silica gel, use petroleum ether and ethyl acetate 10:1 as the washing liquid, purify by silica gel chromatography, then dry and concentrate the organic layer to obtain a white solid product ;

Compound 4: Add compound 2 and compound 3, n-tetrabutylammonium bromide, and catalyst tetrakis(triphenylphosphine)palladium to a polymerization tube with a magneton, and after the addition, seal the entire reaction system away from light and evacuate it , and then filled with nitrogen to keep the whole reaction process in a nitrogen atmosphere; deoxygenate the toluene solution and potassium carbonate solution, and then use a syringe to draw the two solutions into the polymerization tube, put the device in an environment of 90 ° C, and react 4 days; after the reaction was completed, the crude product was dissolved in tetrahydrofuran, passed through an alumina column, and the catalyst was removed; after being spin-dried, it was settled in petroleum ether;

Compound 5: In a single-necked round-bottomed flask with a magnet, add Compound 4. After the addition, the entire reaction system is sealed and evacuated, and then filled with nitrogen to keep the entire reaction process in a nitrogen atmosphere; add anhydrous dichloro Methane and triethylamine, and then add 2-bromo-2-methylpropionyl bromide dropwise; put the device in an environment of 0°C and react for 2 hours; after the reaction is completed, extract the crude product with dichloromethane and water; combine The organic layer is then dried with anhydrous sodium sulfate, spin-dried after suction filtration, settled with petroleum ether in a beaker, and dried after the sedimentation;

Compound 6: Add compound 5, anisole, CuBr to a polymer tube with a magnet, freeze on dry ice, vacuumize, blow nitrogen, and thaw with ethanol, repeat the cycle three times; add trace amounts of N,N,N,N, N-pentamethyldiethylenetriamine, reacted at 100°C for 24 hours, the crude product after the reaction was passed through neutral aluminum oxide to remove the catalyst, concentrated, and settled three times in a 4:1 mixture of methanol and water , dried at 40°C under vacuum to obtain a light yellow solid;

Compound 7: In a single-necked round-bottom flask with a magnet, add compound 6, add tetrahydrofuran, and slowly drop trimethylamine at -78°C. After 30 minutes, the solution temperature rises to room temperature. After 24 hours, distill off the solvent , to obtain a pale yellow solid.

In order to better understand the present invention, the technical scheme of the present invention will be further described below through specific embodiments:

Compound 1: Add 9.72g of 2,7-dibromofluorene, 2g of n-tetrabutylammonium bromide and 12ml of 1,6-dibromohexane in a two-necked round-bottomed flask with a reflux condenser and a magnet; The entire reaction system was sealed and vacuumed, and then filled with nitrogen to keep the entire reaction process in a nitrogen atmosphere; add 100ml of 50% KOH aqueous solution, react at 90°C for 4-5h, and cool the reaction mixture to room temperature; After the reaction mixture was neutralized to neutral, it was extracted with dichloromethane, and the extract was dried with anhydrous sodium sulfate; filtered, and desolvated by a rotary evaporator under reduced pressure, and the excess 1,6-dibromohexane was distilled under reduced pressure Removed; the solid crude product was mixed with silica gel and petroleum ether as the washing liquid, and the residue was recrystallized with methanol and dichloromethane after desolvation under reduced pressure to obtain white crystals, that is, compound 1, a total of 16.5 g (yield 85%);

Compound 2: Add 9.72 g of 2,7-dibromofluorene and 3 g of n-tetrabutylammonium bromide into a two-necked round-bottomed flask with a reflux condenser and a magnet. After the addition, the entire reaction system is sealed and evacuated, and then filled with Nitrogen, keep the whole reaction process in a nitrogen atmosphere; inject the solvent N,N-dimethylformamide with a syringe, after fully dissolving, inject 100ml of 50% KOH aqueous solution with a syringe, and stir for 30min; put the device into 90 Slowly add 8ml of 3-bromo-1-propanol at ℃ and react for 24 hours; after the reaction is completed, extract three times with saturated saline and dichloromethane, combine the organic layers, then dry with anhydrous sodium sulfate, and filter with suction The solid crude product was mixed with silica gel and petroleum ether and ethyl acetate 4:1 were used as washing liquid, purified by silica gel chromatography, and then the organic layer was dried and concentrated to obtain a white solid product, that is, compound 2, a total of 9.5g (yield 72 %);

Compound 3: Add a total of 6.5g of compound 1, 10g of pinacol ester of diboronic acid and 3g of potassium acetate in a two-necked round bottom flask with a magnet, and add the catalyst 1,1'-bis(diphenylphosphine)dicene Iron] palladium dichloride dichloromethane complex 150mg, after adding, the whole reaction system is sealed away from light and vacuumized, and then filled with nitrogen to keep the whole reaction process carried out in a nitrogen atmosphere; the device is placed in a 90°C environment , use a syringe to inject 60ml of solvent N,N-dimethylformamide, and react for 24 hours; after the reaction is completed, the crude product is dissolved in dichloromethane, passed through an alumina column, and the catalyst is removed; extracted with saturated saline and dichloromethane Three times, the organic layer was combined, then dried with anhydrous sodium sulfate, and the solid crude product was mixed with silica gel after suction filtration, and petroleum ether and ethyl acetate 10:1 were used as washing liquid, and purified by silica gel chromatography, and then the organic layer was Drying and concentration gave a white solid product, compound 3, a total of 4.68 g (yield 63%);

Compound 4: In the polymerization tube with magnet, add a total of 0.88g of compound 2, a total of 1.48g of compound 3, 2g of n-tetrabutylammonium bromide, and 150mg of catalyst tetrakis (triphenylphosphine) palladium, and the whole The reaction system was sealed away from light and vacuumized, and then filled with nitrogen to keep the whole reaction process in a nitrogen atmosphere; the toluene solution and the potassium carbonate solution were deoxygenated, and then 10ml of each of the two solutions was extracted with a syringe, and 3ml was added to the polymerization tube Put the device in a 90°C environment and react for 4 days; after the reaction, dissolve the crude product in tetrahydrofuran and pass it through an alumina column to remove the catalyst; spin dry and settle in petroleum ether, and the polymer after settling in the beaker Filter and dry to obtain a total of 1g of compound 4 (62% yield);

Compound 5: In a single-necked round-bottomed flask with a magnet, add a total of 0.8 g of Compound 4. After the addition, the entire reaction system is sealed and vacuumized, and then filled with nitrogen to keep the entire reaction process carried out in a nitrogen atmosphere; 10ml of dichloromethane in water, 5ml of triethylamine, and then 3ml of 2-bromo-2-methylpropionyl bromide was added dropwise; the device was placed in an environment of 0°C and reacted for 2 hours; after the reaction was completed, the crude product was washed with dichloro Extract with methane and water; combine the organic layers, then dry with anhydrous sodium sulfate, spin dry after suction filtration, settle with petroleum ether in a beaker, and dry the polymer after sedimentation to obtain a total of 0.6 g of compound 5 (yield 54 %);

Compound 6: Add 0.3 g of compound 5, 3 ml of anisole, and 30 mg of CuBr into a polymerization tube with a magnet, freeze on dry ice, vacuumize, blow nitrogen, and thaw with ethanol, repeat the cycle three times; add trace amounts of N, N, N,N,N-Pentamethyldiethylenetriamine, reacted at 100°C for 24 hours, the crude product after the reaction was passed through neutral aluminum oxide to remove the catalyst, concentrated, and mixed in methanol and water at a ratio of 4:1 Settled in the liquid three times, and dried at 40°C under vacuum to obtain a light yellow solid with a yield of 67%;

Compound 7: In a single-necked round-bottom flask with a magnet, add compound 6, add tetrahydrofuran, and slowly drop trimethylamine at -78°C. After 30 minutes, the solution temperature rises to room temperature. After 24 hours, distill off the solvent , a pale yellow solid was obtained with a yield of 71%.

Claims (4)

1. A conjugated water-soluble polymer tracer material is characterized in that the material has the following molecular structural formula: Wherein, n is the polymerization degree, and n is 8-12; The 5 of the side chain is the degree of polymerization of the azo molecule, which means that one side chain is connected with 5 azo molecules. 2. The conjugated water-soluble polymer tracer material according to claim 1, characterized in that the main chain of the material is composed of polyfluorene. 3. The conjugated water-soluble polymer tracer material according to claim 1, characterized in that the molecular side chain of the material is an azo molecular structure. 4. a preparation method of conjugated water-soluble polymer tracer material as claimed in claim 1, is characterized in that the method comprises the following steps: Compound 1: Add 2,7-dibromofluorene, n-tetrabutylammonium bromide and 1,6-dibromohexane into a two-necked round-bottomed flask with a reflux condenser and magnet; seal the entire reaction system after adding Vacuumize, then fill with nitrogen, keep the whole reaction process in nitrogen atmosphere; add 50% KOH aqueous solution, react at 90°C for 4-5h, cool the reaction mixture to room temperature; neutralize the reaction mixture with hydrochloric acid solution After reaching neutrality, extract with dichloromethane, and dry the extract with anhydrous sodium sulfate; filter, use a rotary evaporator for precipitation under reduced pressure, and remove excess 1,6-dibromohexane by distillation under reduced pressure; The product was mixed with silica gel and petroleum ether was used as the washing liquid, and the residue was recrystallized with methanol/dichloromethane after desolvation under reduced pressure to obtain white crystals; Compound 2: Add compound 1, pinacol diborate and potassium acetate in a two-necked round bottom flask with a magnet, and add the catalyst [1,1'-bis(diphenylphosphino)ferrocene]dichloro Palladium dichloromethane complex, after the addition, seal the entire reaction system away from light and vacuumize it, and then fill it with nitrogen to keep the entire reaction process in a nitrogen atmosphere; put the device in an environment of 90°C, and inject it with a syringe Solvent N,N-dimethylformamide, reacted for 24 hours; after the reaction was completed, the crude product was dissolved in dichloromethane, passed through an alumina column, and the catalyst was removed; extracted three times with saturated saline and dichloromethane, and the organic layers were combined, Then dry with anhydrous sodium sulfate, and after suction filtration, mix the solid crude product with silica gel, use petroleum ether and ethyl acetate 10:1 as washing liquid, and use silica gel chromatography to purify, then dry and concentrate the organic layer to obtain a white solid product; Compound 3: Add 2,7-dibromofluorene and n-tetrabutylammonium bromide in a two-necked round-bottomed flask with a reflux condenser and a magneton. After the addition, the entire reaction system is sealed and evacuated, and then filled with nitrogen to keep The whole reaction process is carried out in a nitrogen atmosphere; inject the solvent N,N-dimethylformamide with a syringe, and after fully dissolving, inject the sodium hydroxide solution with a syringe, and stir for 30 minutes; put the device at 90°C and slowly add 3-Bromo-1-propanol, reacted for 24 hours; after the reaction was completed, extracted three times with saturated saline and dichloromethane, combined the organic layer, then dried with anhydrous sodium sulfate, and filtered the solid crude product with silica gel Using petroleum ether and ethyl acetate 4:1 as the washing solution, the silica gel chromatography was used for purification, and then the organic layer was dried and concentrated to obtain a white solid product; Compound 4: Add compound 2 and compound 3, n-tetrabutylammonium bromide, and catalyst tetrakis(triphenylphosphine)palladium to a polymerization tube with a magneton, and after the addition, seal the entire reaction system away from light and evacuate it , and then filled with nitrogen to keep the whole reaction process in a nitrogen atmosphere; deoxygenate the toluene solution and potassium carbonate solution, and then use a syringe to draw the two solutions into the polymerization tube, put the device in an environment of 90 ° C, and react 4 days; after the reaction was completed, the crude product was dissolved in tetrahydrofuran, passed through an alumina column, and the catalyst was removed; after being spin-dried, it was settled in petroleum ether; Compound 5: In a single-necked round-bottomed flask with a magnet, add Compound 4. After the addition, the entire reaction system is sealed and evacuated, and then filled with nitrogen to keep the entire reaction process in a nitrogen atmosphere; add anhydrous dichloro Methane and triethylamine, and then add 2-bromo-2-methylpropionyl bromide dropwise; put the device in an environment of 0°C and react for 2 hours; after the reaction is completed, extract the crude product with dichloromethane and water; combine The organic layer is then dried with anhydrous sodium sulfate, spin-dried after suction filtration, settled with petroleum ether in a beaker, and dried after the sedimentation; Compound 6: Add compound 5, anisole, 4-methacrylamido-2', 3-dimethylazobenzene, CuBr, freeze on dry ice, vacuum, pass Nitrogen, ethanol thawing, repeat the cycle three times; add a small amount of N,N,N,N,N-pentamethyldiethylenetriamine, react at 100°C for 24 hours, and the crude product after the reaction is subjected to neutral dioxide Remove the catalyst from aluminum, concentrate, settle three times in methanol/water 4/1V/V mixture, and dry at 40°C under vacuum to obtain a light yellow solid; Compound 7: In a single-necked round-bottom flask with a magnet, add compound 6, add tetrahydrofuran, and slowly drop trimethylamine at -78°C. After 30 minutes, the solution temperature rises to room temperature. After 24 hours, distill off the solvent , to obtain a pale yellow solid.