CN112608466B - Monomer compound, preparation method thereof, water-soluble fluorescent conjugated molecule and preparation method thereof - Google Patents

Abstract

The invention provides a monomer compound, a preparation method thereof, a water-soluble fluorescent conjugated molecule and a preparation method thereof, and belongs to the field of water-soluble fluorescent organic small molecules. Its preparation method is that polyethylene glycol monomethyl ether and p-toluenesulfonyl chloride synthesize the compound shown in formula (1), the compound shown in (1) and 2-bromofluorene are synthesized the compound shown in formula (2), and formula (2 ) is coupled with 2,5-bis(2-ethylhexyl)-3,6-bis(5-trimethyltin)-diketopyrrolopyrrole to obtain a water-soluble fluorescent conjugated molecule. The water-soluble fluorescent conjugated molecule has excellent biological tissue penetration depth and is easily soluble in water.

Description

A monomer compound, its preparation method, water-soluble fluorescent conjugated molecule and its preparation method

technical field

The present invention relates to the field of a water-soluble fluorescent organic small molecule, in particular to a monomer compound used to prepare a water-soluble fluorescent conjugated molecule. The invention also relates to a preparation method of the monomer compound. Further, the invention also relates to A water-soluble fluorescent conjugated molecule prepared by using the monomer compound and a preparation method thereof.

Background technique

In the past few decades, the rapid development of nanotechnology has promoted the emergence and development of nanodiagnostic technology, making the integration of nanodiagnostic technology and nanomedicine possible. Medical imaging has become a clinically important diagnostic technique including computed tomography (CT), among others. However, traditional imaging techniques still have disadvantages such as long time to acquire images and high cost. In fluorescence (FL) imaging, the camera can quickly collect the fluorescence of the target, even imaging within milliseconds.

Fluorescence imaging technology has attracted the attention of the biomedical field due to its short image acquisition time, high detection sensitivity, environmental protection and economy. In recent years, fluorescent materials have developed rapidly and produced many types, such as organic dyes, quantum dots, rare earth complexes, etc. Compared with other fluorescent materials, organic materials have good biocompatibility and eliminate the biological toxicity caused by heavy metals from the root, which makes organic materials have great potential in life sciences. The design and synthesis of new conjugated molecules have almost unlimited variability. The optical properties of conjugated molecules can be adjusted by adjusting the type and quantity of fluorophores and changing the length of the conjugated structure. It has the advantages of easy preparation and low processing temperature (0- 120°C) has attracted much attention. In the near-infrared (NIR) region, especially the second near-infrared window (1000-1700nm, NIR-II), the absorption, scattering and autofluorescence of biological tissues are relatively low. Near-infrared light can achieve greater penetration depth and deep tissue imaging in biological tissues. However, since fluorophores are mainly hydrophobic groups, hydrophobic fluorescent molecules often cannot be directly applied to bioimaging and will reduce biocompatibility. This presents great difficulties in the biological application of fluorescent materials.

Contents of the invention

In order to solve the defects of poor water solubility and short fluorescence emission wavelength of organic fluorescent dyes in the prior art, the present invention provides a monomer compound that can be used to prepare water-soluble fluorescent conjugated molecules and water-soluble fluorescent conjugated molecules. Water-soluble fluorescent conjugated molecules have excellent penetration depth into biological tissues and are easily soluble in water.

Further, the present invention provides a preparation method of 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene shown in formula (2).

Furthermore, the present invention also provides a near-infrared two-region conjugated fluorescent molecule prepared as one of the raw materials of 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene represented by formula (2), namely the formula (3) The water-soluble fluorescent conjugated molecule shown in (3) and its preparation method.

To achieve the above object, the present invention adopts the following technical solutions:

A monomeric compound has a structure shown in formula (2):

n is an integer of 10-50, preferably 20.

A preparation method of the monomer compound, comprising the steps of:

S1, polyethylene glycol monomethyl ether and p-toluenesulfonyl chloride synthetic formula (1) shown in polyethylene glycol monomethyl ether sulfonyl benzene methane:

n is an integer of 10-50, preferably 20;

S2. The compound shown in formula (1) and 2-bromofluorene are synthesized into 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene shown in formula (2).

Preferably, in the step S1, polyethylene glycol monomethyl ether and p-toluenesulfonyl chloride are dissolved in dichloromethane, the ice-water bath is kept at 0°C, sodium hydroxide is added in batches under vigorous stirring, and the reaction is carried out for 5-7 hours Stop, after the reaction finishes, remove sodium hydroxide by filtration, the solution obtained carries out vacuum distillation, obtains the compound shown in formula (1).

Further preferably, the molar ratio of polyethylene glycol monomethyl ether, p-toluenesulfonyl chloride and sodium hydroxide in the step S1 is 1: (1-1.2): (8-12), and the sodium hydroxide is divided into 3 - 4 additions with an interval of 2-5 minutes, reacting at 0-5°C for 5-7 hours.

Preferably, the step S2 is: dissolving 2-bromofluorene and the phase transfer catalyst in dimethyl sulfoxide, stirring and mixing evenly, adding potassium hydroxide aqueous solution, and finally adding the compound shown in formula (1) for reaction; the reaction ends Finally, wash the extraction reaction solution with dichloromethane for 3 times, take the organic layer and carry out vacuum distillation to remove dichloromethane, and use a small amount of distilled water to dissolve the remaining solution and perform dialysis. After the dialysis, the resulting solution is freeze-dried to obtain Compound shown, the phase transfer catalyst is tetrabutylammonium bromide or 18 crown ether 6.

Further preferably, the step S2 is: the 2-bromofluorene, the molar ratio of the compound represented by formula (1) to tetrabutylammonium bromide is 1: (2-2.2): (0.01-0.03), hydrogen Potassium oxide solution concentration is 40-50wt%, the volume ratio of dimethyl sulfoxide and potassium hydroxide solution is (3-5): 1, and reaction temperature is 60-80 ℃, and reaction time is 6-10 hour; Dialysis used for dialysis The passing molecular weight of the bag is 3000.

A water-soluble fluorescent conjugated molecule having a structure shown in formula (3):

Wherein, n is an integer of 10-50, preferably 20.

A method for preparing the water-soluble fluorescent conjugated molecule, comprising the steps of:

Step S3, dissolving 2,5-bis(2-ethylhexyl)-3,6-bis(5-trimethyltin)-diketopyrrolopyrrole, the compound represented by formula (2) and the coupling agent in Toluene, anhydrous and oxygen-free reaction, that is.

Preferably, 2,5-bis(2-ethylhexyl)-3,6-bis(5-trimethyltin)-diketopyrrolopyrrole, the compound represented by formula (2) and the coupling agent are dissolved in In toluene, the reaction is carried out under anhydrous and oxygen-free conditions; after the reaction is completed, the reaction solution is cooled to room temperature, and the crude product is obtained by distillation under reduced pressure, which is further separated and purified to obtain the compound shown in formula (3), and the coupling agent is four Triphenylphosphopalladium and/or bistriphenylphosphopalladium dichloride.

Further preferably, in step S3, the compound represented by formula (2) The molar ratio to the coupling agent is 1: (1-2.4): (0.01-0.05), the reaction temperature is 110-120°C, and the reaction time is 30-50 hours; the coupling agent is tetrakistriphenylphosphine Palladium and/or bistriphenylphosphine palladium dichloride.

The further separation and purification method is as follows: the reaction solution is distilled under reduced pressure to obtain the crude product, poured into 100-200mL of anhydrous n-hexane for sedimentation, and then suction filtered to obtain the crude product; the crude product is dissolved in chloroform and placed on a silica gel column (silica gel 80-100 mesh) purification; vacuum distillation collected chloroform solution, and then vacuum distillation and vacuum drying, that is.

Compared with the prior art, the present invention has the following beneficial effects:

1. The water-soluble fluorescent conjugated molecule shown in the formula (3) provided by the invention comprises two parts: a part is the structure conjugated main chain shown in the formula (4) that contains the π-π conjugated system of fluorenes. The structure ensures the optical properties of fluorescent molecules; the other part is water-soluble side group polyethylene glycol monomethyl ether 2000, so as to meet the solubility of fluorescent molecules in the water phase.

2. The present invention contains a water-soluble group, and the water-soluble fluorescent conjugated molecule can emit fluorescence in the second near-infrared region under the excitation of 808nm laser, and the emission wavelength is 900nm-1300nm, and the fluorescence at this wavelength is relative to the near-infrared one Area fluorescence has better penetration performance on biological tissues.

Description of drawings

In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings that need to be used in the description of the specific embodiments or the prior art. Obviously, the accompanying drawings in the following description The drawings show some implementations of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative work.

Fig. 1 is the absorption spectra of fluorescent molecules obtained in Examples 1, 2, and 3 in water and chloroform.

Fig. 2 is the fluorescence spectrum of the fluorescent molecules obtained in Examples 1, 2, and 3 in water and chloroform.

Fig. 3 is the NMR spectra of the fluorescent molecules obtained in Examples 1, 2, and 3.

Figure 4 is a comparison of the imaging effects of the second near-infrared region and the first near-infrared region in application example 1.

Detailed ways

The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Apparently, the described embodiments are part of the embodiments of the present invention, but not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

In addition, the technical features involved in different embodiments of the present invention described below can be combined with each other as long as they do not constitute conflicts with each other.

The invention provides a water-soluble fluorescent conjugated molecule, which is composed of monomer compound 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene represented by formula (2), and 2,5-di (2-Ethylhexyl)-3,6-bis(5-trimethyltin)-diketopyrrolopyrrole coupled. Its reaction formula is as follows:

The preparation method of the water-soluble fluorescent conjugated molecule of the present invention has the following examples:

Example 1:

S1, polyethylene glycol monomethyl ether sulfonyl benzene methane shown in the preparation formula (1):

Dissolve polyethylene glycol monomethyl ether 2000 (4g, 2mmol) and p-toluenesulfonyl chloride (0.3812g, 2mmol) in dichloromethane (50ml), keep the ice-water bath at 0°C, and add hydroxide in batches under vigorous stirring. Sodium (0.96g, 24mmol), the sodium hydroxide was added in 4 times with an interval of 3 minutes, and the reaction was stopped after 6 hours. After the reaction was finished, the sodium hydroxide was removed by filtration, and the obtained solution was distilled under reduced pressure to obtain polyethylene glycol monomethyl ether sulfonyl benzene methane represented by the white powder formula (1), with a yield of about 90%.

S2. Preparation of 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene shown in formula (2):

Dissolve 2-bromofluorene (0.324g, 1mmol) and tetrabutylammonium bromide (7.8mg, 0.024mmol) in dimethyl sulfoxide, stir, add potassium hydroxide (5ml, 45wt%) aqueous solution, and finally add the formula The compound (4.8192 g, 2.2 mmol) shown in (1) was reacted at a reaction temperature of 75° C. and a reaction time of 8 hours. After the reaction was finished, the extraction reaction solution was washed 3 times with dichloromethane, and the organic layer was removed by distillation under reduced pressure to remove the dichloromethane, and the remaining solution was dissolved in a small amount of distilled water and dialyzed. After the dialysis, the resulting solution was freeze-dried to obtain 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene represented by formula (2) in the form of yellow powder, with a yield of about 30%. The dialysis bag used for dialysis has a molecular weight of 3000.

S3. Synthesis of water-soluble fluorescent conjugated molecules

2,5-bis(2-ethylhexyl)-3,6-bis(5-trimethyltin)-diketopyrrolopyrrole (50mg, 0.042mmol), compound represented by formula (2) (420mg, 0.1mmol), tetrakistriphenylphosphopalladium (2.4mg, 0.002mmol) was dissolved in toluene, and the reaction was carried out under anhydrous and oxygen-free conditions. The reaction temperature was 120°C, and the reaction time was 36 hours. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was distilled under reduced pressure to obtain the crude product, which was poured into 100-200mL of anhydrous n-hexane for sedimentation, and then suction-filtered to obtain the crude product; the crude product was dissolved in chloroform and placed on a silica gel column (silica gel 80-100 mesh) Purification; the collected chloroform solution was distilled under reduced pressure, then distilled under reduced pressure and dried in vacuo to obtain 260 mg of the water-soluble fluorescent conjugated molecule represented by the dark green solid formula (3), with a yield of about 55%.

Example 2:

S1, polyethylene glycol monomethyl ether sulfonyl benzene methane shown in the preparation formula (1):

Dissolve polyethylene glycol monomethyl ether 2000 (8g, 4mmol) and p-toluenesulfonyl chloride (0.762g, 4.8mmol) in dichloromethane (50ml), keep the ice-water bath at 0°C, and add hydrogen in batches under vigorous stirring Sodium oxide (0.64g, 16mmol), the sodium hydroxide was added in 3 times with an interval of 2 minutes, and the reaction was stopped after 6 hours. After the reaction was finished, sodium hydroxide was removed by filtration, and the obtained solution was distilled under reduced pressure to obtain polyethylene glycol monomethyl ether sulfonyl benzene methane shown in white powdery formula (1), with a yield of about 86%.

S2. Preparation of 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene shown in formula (2):

2-bromofluorene (0.648g, 2mmol) and 18 crown ether 6 (10.7mg, 0.02mmol) were dissolved in dimethyl sulfoxide, stirred, potassium hydroxide (10ml, 40wt%) aqueous solution was added, and finally the formula (1 ) The compound (8.758g, 4.0mmol) shown in ) was reacted, the reaction temperature was 70°C, and the reaction time was 7 hours. After the reaction finishes, wash the extraction reaction solution 3 times with methylene chloride, get the organic layer and carry out distillation under reduced pressure to remove methylene chloride, and the remaining solution is dissolved and dialyzed with a small amount of distilled water. After the dialysis, the resulting solution was freeze-dried to obtain 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene represented by the yellow powder formula (2). The yield is about 25%. The molecular weight of the dialysis bag used for dialysis was 3000.

S3. Synthesis of water-soluble fluorescent conjugated molecules

2,5-bis(2-ethylhexyl)-3,6-bis(5-trimethyltin)-diketopyrrolopyrrole (50 mg, 0.042 mmol), compound represented by formula (2) (336 mg, 0.1mmol), tetrakistriphenylphosphopalladium (1.2mg, 0.001mmol) was dissolved in toluene, and the reaction was carried out under anhydrous and oxygen-free conditions. The reaction temperature was 120°C, and the reaction time was 36 hours. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was distilled under reduced pressure to obtain the crude product, which was poured into 100-200mL of anhydrous n-hexane for sedimentation, and then suction-filtered to obtain the crude product; the crude product was dissolved in chloroform and placed on a silica gel column (silica gel 80-100 mesh) Purification; the collected chloroform solution was distilled under reduced pressure, then distilled under reduced pressure and dried in vacuo to obtain 213 mg of a dark green solid water-soluble fluorescent conjugated molecule represented by formula (3), with a yield of about 45%.

Example 3:

S1, polyethylene glycol monomethyl ether sulfonyl benzene methane shown in the preparation formula (1):

Dissolve polyethylene glycol monomethyl ether 2000 (4g, 2mmol) and p-toluenesulfonyl chloride (0.3812g, 2mmol) in dichloromethane (50ml), keep the ice-water bath at 0°C, and add hydroxide in batches under vigorous stirring. Sodium (0.64g, 16mmol), the sodium hydroxide was added in 4 times with an interval of 3 minutes, and the reaction was stopped after 6 hours. After the reaction was finished, the sodium hydroxide was removed by filtration, and the obtained solution was distilled under reduced pressure to obtain polyethylene glycol monomethyl ether sulfonyl benzene methane represented by the white powder formula (1), with a yield of about 88%.

S2. Preparation of 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene shown in formula (2):

Dissolve 2-bromofluorene (0.324g, 1mmol) and tetrabutylammonium bromide (6.5mg, 0.02mmol) in dimethyl sulfoxide, stir, add potassium hydroxide (5ml, 40wt%) aqueous solution, and finally add the formula The compound shown in (1) (4.8192 g, 2.2 mmol) was reacted at a reaction temperature of 75° C. and a reaction time of 8 hours. After the reaction was finished, the extraction reaction solution was washed 3 times with dichloromethane, and the organic layer was removed by distillation under reduced pressure to remove the dichloromethane, and the remaining solution was dissolved in a small amount of distilled water and dialyzed. After the dialysis, the resulting solution was freeze-dried to obtain 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene represented by formula (2) in the form of yellow powder, with a yield of about 33%. The molecular weight of the dialysis bag used for dialysis was 3000.

S3. Synthesis of water-soluble fluorescent conjugated molecules

2,5-bis(2-ethylhexyl)-3,6-bis(5-trimethyltin)-diketopyrrolopyrrole (60 mg, 0.05 mmol), compound represented by formula (2) (420 mg, 0.1 mmol), bistriphenylphosphine palladium dichloride (2.2 mg, 0.001 mmol) was dissolved in toluene, and the reaction was carried out under anhydrous and oxygen-free conditions. The reaction temperature was 120°C, and the reaction time was 36 hours. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was distilled under reduced pressure to obtain the crude product, which was poured into 100-200mL of anhydrous n-hexane to settle, and then filtered with suction to obtain the crude product; the crude product was dissolved in chloroform and placed on a silica gel column (silica gel 80-100 mesh) Purification; The collected chloroform solution was distilled under reduced pressure, then distilled under reduced pressure and dried under vacuum to obtain 235 mg of a water-soluble fluorescent conjugated molecule represented by formula (3) as a dark green solid. The yield is about 50%.

Example 4:

S1, polyethylene glycol monomethyl ether sulfonyl benzene methane shown in the preparation formula (1):

Dissolve polyethylene glycol monomethyl ether 2000 (4g, 4mmol) and p-toluenesulfonyl chloride (0.831g, 4.8mmol g) in dichloromethane (50ml), keep the ice-water bath at 0°C, and add them in batches under vigorous stirring Sodium hydroxide (1.92 g, 48 mmol), the sodium hydroxide was added in 4 times with an interval of 5 minutes, and the reaction was stopped after 6 hours. After the reaction was finished, sodium hydroxide was removed by filtration, and the obtained solution was distilled under reduced pressure to obtain polyethylene glycol monomethyl ether sulfonyl benzene methane shown in white powdery formula (1), and the yield was about 93%.

S2. Preparation of 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene shown in formula (2):

Dissolve 2-bromofluorene (0.324g, 1mmol) and tetrabutylammonium bromide (9.7mg, 0.03mmol) in dimethyl sulfoxide, stir, add potassium hydroxide (5ml, 50wt%) aqueous solution, and finally add the formula The compound shown in (1) (4.381 g, 2.2 mmol) was reacted at a reaction temperature of 75° C. and a reaction time of 8 hours. After the reaction finishes, wash the extraction reaction liquid 3 times with dichloromethane, get the organic layer and carry out vacuum distillation to remove dichloromethane, and the remaining solution is dissolved and dialyzed with a small amount of distilled water. After the dialysis, the resulting solution was freeze-dried to obtain 2-bromo-9,9-dipolyethylene glycol monomethyl ether fluorene represented by formula (2) in the form of yellow powder, with a yield of about 37%. The molecular weight of the dialysis bag used for dialysis was 3000.

S3. Synthesis of water-soluble fluorescent conjugated molecules

2,5-bis(2-ethylhexyl)-3,6-bis(5-trimethyltin)-diketopyrrolopyrrole (120 mg, 0.1 mmol), compound represented by formula (2) (1 g, 0.24mmol), tetrakistriphenylphosphopalladium (5.8mg, 0.005mmol) was dissolved in toluene, and reacted under anhydrous and oxygen-free conditions. The reaction temperature was 120°C, and the reaction time was 36 hours. After the reaction was completed, the reaction solution was cooled to room temperature. The reaction solution was poured into 150mL of anhydrous methanol, then suction filtered to obtain the crude product. The crude product was dissolved in chloroform and purified on a silica gel column (silica gel 80-100 mesh). The chloroform solution collected by distillation under reduced pressure, the obtained solid was dissolved in chloroform as little as possible, then added to 120mL of anhydrous methanol, then suction filtered and vacuum-dried to obtain 283mg of dark green solid represented by the formula (3). Sexual fluorescent conjugated molecules, the yield is about 60%.

Fig. 1 is the absorption spectrum of fluorescent molecules obtained in water and chloroform in embodiment 1,2,3,4, the fluorescence spectrum of fluorescent molecules obtained in Fig. 2 embodiment 1,2,3,4 in water and chloroform, Fig. 3 is the nuclear magnetic spectrum of fluorescent molecules obtained in Examples 1, 2, 3, and 4. Figures 1 and 2 show the optical properties of fluorescent molecules, and Figure 3 demonstrates the successful synthesis of fluorescent molecules.

Application example 1

Pork slices were used to cover capillary tubes filled with aqueous solutions of fluorescent molecules, and then imaging was performed in the first near-infrared region and the second near-infrared region. The two imaging modalities were compared by increasing the number of pork slices to increase the thickness of the biological tissue. The results show that the tissue penetration depth using the near-infrared zone 2 imaging mode is more than 5mm, while the tissue tissue penetration depth using the near-infrared zone 1 imaging mode is only 3mm. The thickness of pork slices is the average value of the total thickness of ten slices, with an average of 1 mm per slice. Figure 4 is a comparison of the imaging effects of the second near-infrared region and the first near-infrared region in Application Example 1, illustrating the superiority of the fluorescent molecules of the present application in imaging in the second near-infrared region, that is, having deeper tissue penetration.

Apparently, the above-mentioned embodiments are only examples for clearly illustrating, rather than limiting the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in different forms can also be made. It is not necessary and impossible to exhaustively enumerate all implementation modes here. And the obvious changes or variations derived therefrom are still within the scope of protection of the present invention.

Claims (8)

1. A water-soluble fluorescent conjugated molecule having a structure represented by formula (3):

wherein n is an integer of 10 to 50.

2. A method of preparing the water-soluble fluorescent conjugated molecule of claim 1, comprising the steps of:

step S1, synthesizing polyethylene glycol monomethyl ether sulfonyl phenylmethane shown in formula (1) by polyethylene glycol monomethyl ether and p-toluenesulfonyl chloride:

n is an integer of 10 to 50;

step S2, synthesizing 2-bromo-9, 9-dipolyethylene glycol monomethyl ether fluorene shown in formula (2) by using the compound shown in formula (1) and 2-bromofluorene:

n is an integer of 10 to 50;

and S3, dissolving the 2, 5-di (2-ethylhexyl) -3, 6-di (5-trimethyltin) -pyrrolopyrrole diketone, the compound shown in the formula (2) and a coupling agent in toluene, and reacting under anhydrous and oxygen-free conditions to obtain the compound.

3. The method of claim 2, wherein the step of preparing the water-soluble fluorescent conjugated molecule,

dissolving polyethylene glycol monomethyl ether and p-toluenesulfonyl chloride in dichloromethane in the step S1, keeping the temperature of an ice water bath at 0 ℃, adding sodium hydroxide in batches under the condition of vigorous stirring, stopping the reaction for 5-7 hours, filtering to remove the sodium hydroxide after the reaction is finished, and distilling the obtained solution under reduced pressure to obtain the compound shown in the formula (1).

4. The method for preparing a water-soluble fluorescent conjugated molecule according to claim 3,

the molar ratio of the polyethylene glycol monomethyl ether, the p-toluenesulfonyl chloride and the sodium hydroxide in the step S1 is 1: (1-1.2): (8-12), adding the sodium hydroxide for 3-4 times at an interval of 2-5 minutes each time, and reacting for 5-7 hours at the temperature of 0-5 ℃.

5. The method for preparing a water-soluble fluorescent conjugated molecule according to claim 4,

the step S2 is as follows: dissolving 2-bromofluorene and a phase transfer catalyst in dimethyl sulfoxide, stirring and mixing uniformly, adding a potassium hydroxide aqueous solution, and finally adding a compound shown in the formula (1) for reaction; after the reaction, the extracted reaction solution was washed with dichloromethane 3 times, the organic layer was distilled under reduced pressure to remove dichloromethane, the remaining solution was dissolved with a small amount of distilled water and dialyzed, and after the dialysis was completed, the resulting solution was freeze-dried to obtain a compound represented by formula (2).

6. The method for preparing a water-soluble fluorescent conjugated molecule according to claim 5,

the step S2 is as follows: the molar ratio of the 2-bromofluorene, the compound shown in the formula (1) and the phase transfer catalyst is 1: (2-2.2): (0.01-0.03), the concentration of the potassium hydroxide solution is 40-50wt%, the volume ratio of the dimethyl sulfoxide to the potassium hydroxide solution is (3-5) to 1, the reaction temperature is 60-80 ℃, and the reaction time is 6-10 hours; the passage molecular weight of the dialysis bag used for dialysis was 3000.

7. The method for preparing a water-soluble fluorescent conjugated molecule according to claim 2, wherein the step S3 is:

s3, dissolving 2, 5-di (2-ethylhexyl) -3, 6-di (5-trimethyltin) -pyrrolopyrrole dione, a compound shown in a formula (2) and a coupling agent in toluene, and reacting in the absence of water and oxygen; after the reaction is finished, cooling the reaction solution to room temperature, carrying out reduced pressure distillation to obtain a crude product, and further separating and purifying to obtain the compound shown in the formula (3).

8. The method for preparing a water-soluble fluorescent conjugated molecule according to claim 7, wherein the step S3 is:

step S3, adding 2, 5-di (2-ethylhexyl) -3, 6-di (5-trimethyltin) -pyrrolopyrrole-dione, a compound shown as a formula (2), and tetrakistriphenylphosphine palladium in a molar ratio of 1: (1-2.4): (0.01-0.05), the reaction temperature is 110-120 ℃, and the reaction time is 30-50 hours;

the further separation and purification method comprises the following steps: distilling the reaction solution under reduced pressure to obtain a crude product, pouring the crude product into 100-200mL of anhydrous n-hexane for sedimentation, and then carrying out suction filtration to obtain a crude product; dissolving the crude product in chloroform, and purifying on 80-100 mesh silica gel column; distilling the collected chloroform solution under reduced pressure, and then distilling under reduced pressure and drying in vacuum to obtain the final product.

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