CN101844983B - Start fluorescent polymer, initiator thereof and preparation methods for start fluorescent polymer and initiator - Google Patents

Abstract

The invention discloses a star-shaped fluorescent polymer, an initiator thereof and a preparation method thereof. The general structural formula of the fluorescent polymer is shown in formula VII. The initiator used to prepare the fluorescent polymer is shown in formula I. Using the fluorescent polymer provided by the invention to separate DNA or protein, the separation speed is fast, the separation effect is good, the capillary does not need to be modified, and the one-time glue filling can be used repeatedly and has good repeatability; moreover, the present invention can convert comparable When different topological structures are introduced into the separation medium, the influence of linear polymers on the separation results of biomacromolecules can be systematically compared; in addition, since the fluorescent polymer has a fluorescent nucleus, the fluorescent nucleus can be introduced into the separation medium to achieve simultaneous The purpose of the separation medium and biomacromolecules can be observed, so as to obtain a more intuitive image of biomacromolecules in the separation process, and provide a more powerful method for revealing the separation mechanism. (Formula VII); (Formula I).

Description

Star-shaped fluorescent polymer and its initiator and their preparation method

technical field

The invention relates to the field of biomacromolecule separation, in particular to a star-shaped fluorescent polymer used in a capillary electrophoresis separation medium, an initiator thereof and a preparation method thereof.

Background technique

Capillary electrophoresis is currently one of the most effective means to separate and analyze biomacromolecules. Taking the separation of DNA as an example, different DNA fragments have nearly the same charge-to-mass ratio, and in free solution (free solution), they have similar migration rates under the action of an external electric field. Therefore, in order to achieve the separation of DNA fragments of different lengths or structures, a separation medium must be added to the capillary. The currently widely used separation media are mainly non-crosslinked polymer solutions, and the common ones are: linear homopolymers (including linear polyacrylamide, poly(N,N-dimethylacrylamide), polyethylene oxide, polyvinyl pyrrolidone, cellulose and its derivatives, etc.), copolymers and mixtures, etc. Due to the high viscosity of linear polymers at the same weight average molecular weight, there are some attempts to use nonlinear polymers as separation media for capillary electrophoresis, see CONTROLLED-ARCHITECTUREPOLYMERS AND USE THEREOF AS SEPARATION MEDIA Klaerner G., et al. WO: 2001009204, 20020417. However, no systematic study has been conducted on the comparison of linear polymers and nonlinear polymers for the separation of biomacromolecules.

The concentration of the polymer solution often determines the separation mechanism of biomacromolecules. Taking the separation of DNA as an example, the separation mechanism of DNA in entangled polymer solution includes Ogston model, various modified snaking models, entropy barrier model and so on. The separation mechanism of DNA in dilute polymer solution includes instantaneous entanglement coupling mechanism, collision model, "collective motion" model and so on. Although there are many mechanisms for DNA separation, each model has a limited scope of application and cannot explain all the experimental results. It is very important to further reveal the mechanism of DNA separation by capillary electrophoresis: on the one hand, it can reduce the blindness in choosing the separation medium, and on the other hand, it can better explain the experimental results. For this reason, people use EB to stain DNA, and then observe the movement behavior of DNA molecules during the separation process under a fluorescent microscope, so as to infer the interaction between the separation medium and DNA molecules. At the same time, there are also attempts to observe the movement behavior of the separation medium under a fluorescent microscope after dyeing the separation medium, see Method and apparatus for screening flowable separation media for electrophoresis and related applications McWaid, T.H., etal.USA: 2003196896, 20031023. Of course, fluorescent chromophores can also be introduced into polymers by chemical methods, see Synthesis of poly(N-isopropylacrylamide) by ATRP using a fluorescein-based initiator Lu Xiaoju, et al. Polymer Bulletin 2007, 59, 195-206. However, no attempt has been made to simultaneously observe the movement of DNA molecules and separation media under a fluorescence microscope.

Contents of the invention

The object of the present invention is to provide a kind of fluorescent polymer and its initiator and their preparation method.

Initiator provided by the invention has a general structural formula as shown in formula I,

(Formula I)

In the general structural formula of formula I, R _is selected from any one of straight chain or branched chain alkyl groups with a total number of carbon atoms of 1-20, preferably a straight chain or branched chain alkyl group with a total number of carbon atoms of 6-12 Any one of, more preferably hexyl; R ₂ is a π-conjugated group, and the π-conjugated group is selected from aryl, alkenyl, alkynyl, aryl oligomer, alkenyl oligomer and at least one of alkynyl oligomers, the aryl group is selected from at least one of phenyl, thienyl, furyl and pyridyl, preferably an aryl oligomer with 1-5 repeating structural units , most preferably phenyl; m is 0 or 1, and p is 1, 2 or 3.

The compound shown in formula I is preferably a compound shown in formula II or formula III,

(Formula II)

(Formula III)

The method for preparing the compound described in m=1 and p=1 in the general structural formula of the above-mentioned formula I provided by the present invention comprises the following steps:

1) In the presence of a palladium catalyst, the compound shown in formula IV, monomethoxy substituted aryl boronic acid, and an aqueous solution of a basic compound are subjected to a Suzuki coupling reaction in an organic solvent to obtain a compound shown in formula V;

(Formula IV)

In the general structural formula of formula IV, R _is selected from any one of straight-chain or branched-chain alkyl groups with a total of 1-20 carbon atoms;

(Formula V)

In the general structural formula of formula V, R ₁ is selected from any one of linear or branched chain alkyl groups with a total carbon number of 1-20, R ₂ is a π-conjugated group, m=1, p= 1;

2) In the presence of boron tribromide, the compound shown in the formula V is demethylated in an organic solvent to obtain the compound shown in the formula VI;

(Formula VI)

In the general structural formula of the formula VI, R ₁ is selected from any one of linear or branched chain alkyl groups with a total carbon number of 1-20, R ₂ is a π-conjugated group, m=1, p= 1;

3) In the presence of triethylamine, the compound shown in the formula VI is reacted with 2-bromoisobutyryl bromide in an organic solvent to obtain the structure of the formula I. In the general formula, m=1, p=1 said compound.

In the step 1) of the method, in the general structural formula of the formula IV, R is _any one of straight chain or branched chain alkyl groups whose total carbon atoms are 6-12, preferably hexyl; the general structural formula of the formula V In the formula, R ₁ is selected from any one of linear or branched alkyl groups with a total carbon number of 6-12, preferably hexyl, and in the R ₂ , the π-conjugated group is selected from aryl, At least one of alkenyl, alkynyl, aryl oligomer, alkenyl oligomer and alkynyl oligomer, the aryl is selected from at least one of phenyl, thienyl, furyl and pyridyl species, preferably aryl oligomers with repeating structural units of 1-5, most preferably phenyl; the compound of formula IV is 5,5,10,10,15,15-hexahexyl-2,7,12- Tribromotripyrane, the palladium catalyst is selected from at least one of tetrakis(triphenylphosphine)palladium, bis(triphenylphosphine)palladium chloride and palladium acetate, and the monomethoxy substituted arylboronic acid It is p-methoxyphenylboronic acid, methoxythiophene boronic acid or methoxypyridine boronic acid, preferably p-methoxyphenylboronic acid, and the basic compound is sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide; The concentration of the aqueous solution of the basic compound is 0.5-5 mol/L, preferably 2 mol/L; The ratio is 1:3-6:10-20:0.05-0.2, preferably 1:4:15:0.1; in the Suzuki coupling reaction, the temperature is 0-30°C, preferably 20°C, and the time is 6-24 hours , preferably 12 hours;

In the step 2) in the general structural formula of the formula VI, R ₁ is selected from any one of linear or branched alkyl groups with a total carbon number of 6-12, preferably hexyl; in the R ₂ , the The π-conjugated group is selected from at least one of aryl, alkenyl, alkynyl, aryl oligomer, alkenyl oligomer and alkynyl oligomer, and the aryl is selected from phenyl, thiophene At least one of base, furyl and pyridyl, preferably an aryl oligomer with 1-5 repeating structural units, most preferably phenyl; the molar ratio of boron tribromide to the compound shown in the formula V is 5-20:1, preferably 10:1; the reaction temperature is 0-30°C, preferably 20°C, and the time is 6-24 hours, preferably 12 hours;

In the step 3), the organic solvent is at least one of dichloromethane, chloroform, ether, toluene and tetrahydrofuran; the mole of triethylamine, the compound shown in the formula VI and 2-bromoisobutyryl bromide The ratio is 10-200:1:3-15, preferably 120:1:10; in the reaction, the temperature is 0-30°C, preferably 20°C, and the time is 6-24 hours, preferably 12 hours.

In the above preparation method, when R ₁ of the compound described in formula IV in step 1) is hexyl, and the monomethoxy substituted aryl boronic acid is p-methoxyphenylboronic acid, the compound described in formula II can be obtained.

The compound described in m=1 and p=2 in the preparation formula I provided by the invention comprises the following steps:

1) In the presence of a palladium catalyst, the aqueous solution of the compound shown in formula IV, dimethoxy substituted aryl boronic acid and basic compound is subjected to Suzuki coupling reaction in an organic solvent to obtain the compound shown in formula V;

(Formula IV)

In the general structural formula of formula IV, R _is selected from any one of straight-chain or branched-chain alkyl groups with a total of 1-20 carbon atoms;

(Formula V)

In the general structural formula of formula V, R ₁ is selected from any one of linear or branched chain alkyl groups with a total carbon number of 1-20, R ₂ is a π-conjugated group, m=1, p= 2;

2) In the presence of boron tribromide, the compound shown in the formula V is demethylated in an organic solvent to obtain the compound shown in the formula VI;

(Formula VI)

In the general structural formula of the formula VI, R ₁ is selected from any one of linear or branched chain alkyl groups with a total carbon number of 1-20, R ₂ is a π-conjugated group, m=1, p= 2;

3) In the presence of triethylamine, the compound represented by the formula VI is reacted with 2-bromoisobutyryl bromide in an organic solvent to obtain the compound represented by the formula I in which m=1 and p=2.

In the step 1) of the method, in the general structural formula of the formula IV, R is _any one of straight chain or branched chain alkyl groups whose total carbon atoms are 6-12, preferably hexyl; the general structural formula of the formula V In the formula, R ₁ is selected from any one of linear or branched alkyl groups with a total carbon number of 6-12, preferably hexyl, and in the R ₂ , the π-conjugated group is selected from aryl, At least one of alkenyl, alkynyl, aryl oligomer, alkenyl oligomer and alkynyl oligomer, the aryl is selected from at least one of phenyl, thienyl, furyl and pyridyl species, preferably aryl oligomers with repeating structural units of 1-5, most preferably phenyl; the compound of formula IV is 5,5,10,10,15,15-hexahexyl-2,7,12- Tribromotripyrene, the palladium catalyst is selected from at least one of tetrakis(triphenylphosphine)palladium, bis(triphenylphosphine)palladium chloride and palladium acetate, and the dimethoxy substituted arylboronic acid It is one of 3,4-dimethoxyphenylboronic acid, dimethoxythiopheneboronic acid, and dimethoxypyridineboronic acid, preferably 3,4-dimethoxyphenylboronic acid, and the basic compound is sodium carbonate , potassium carbonate, sodium hydroxide or potassium hydroxide; the concentration of the aqueous solution of the basic compound is 0.5-5mol/L, preferably 2mol/L; the compound shown in the formula IV, the dimethoxy substituted arylboronic acid , The molar ratio of the basic compound to the palladium catalyst is 1:3-6:10-20:0.05-0.2, preferably 1:4:15:0.1; in the Suzuki coupling reaction, the temperature is 0- 30°C, preferably 20°C, for 6-24 hours, preferably 12 hours;

In the step 2) in the general structural formula of the formula VI, R ₁ is selected from any one of linear or branched alkyl groups with a total carbon number of 6-12, preferably hexyl; in the R ₂ , the The π-conjugated group is selected from at least one of aryl, alkenyl, alkynyl, aryl oligomer, alkenyl oligomer and alkynyl oligomer, and the aryl is selected from phenyl, thiophene At least one of base, furyl and pyridyl, preferably an aryl oligomer with 1-5 repeating structural units, most preferably phenyl; the molar ratio of boron tribromide to the compound shown in the formula V is 10-30:1, preferably 20:1; the reaction temperature is 0-30°C, preferably 20°C, and the time is 6-24 hours, preferably 12 hours;

In the step 3), the organic solvent is at least one of dichloromethane, chloroform, ether, toluene and tetrahydrofuran; the mole of triethylamine, the compound shown in the formula VI and 2-bromoisobutyryl bromide The ratio is 20-300:1:6-30, preferably 240:1:20; in the reaction, the temperature is 0-30°C, preferably 20°C, and the time is 6-24 hours, preferably 12 hours.

In the above preparation method, when the _R of the compound described in the formula IV in step 1) is a hexyl group, and the dimethoxy substituted arylboronic acid is 3,4-dimethoxyphenylboronic acid, the compound described in the formula III can be obtained. compound.

The fluorescent polymer provided by the present invention has a general structural formula as shown in formula VII,

(Formula VII)

In the general structural formula of the formula VII, R _is selected from any one of straight-chain or branched-chain alkyl groups with a total number of carbon atoms of 1-20, preferably a straight-chain or branched-chain alkyl group with a total number of carbon atoms of 6-12 Any one of, more preferably hexyl; R ₂ is a π-conjugated group, and the π-conjugated group is selected from aryl, alkenyl, alkynyl, aryl oligomer, alkenyl oligomer and at least one of alkynyl oligomers, the aryl group is selected from at least one of phenyl, thienyl, furyl and pyridyl, preferably an aryl oligomer with 1-5 repeating structural units , most preferably phenyl; _R3 is a water-soluble polymer chain selected from polyacrylamide, polyN, N-dimethylacrylamide, polyethylene oxide, polyvinylpyrrolidone, cellulose , at least one of cellulose nitrate, cellulose acetate, methyl cellulose and carboxymethyl cellulose, preferably poly N, N-dimethylacrylamide; m is 0 or 1; p is 1, 2 or 3; the number average molecular weight of the fluorescent polymer represented by the formula VII is 10,000-10,000,000, preferably 500,000-2,000,000, and the molecular weight dispersion is 1.5-5, preferably 2.5.

The fluorescent polymer shown in the formula VII is preferably a polymer shown in the formula VIII,

(Formula VIII)

In the polymer represented by formula VIII, m is 0 or 1, n is an integer of 100-10000, and p is 1, 2 or 3;

The fluorescent polymer shown in the formula VII is more preferably the fluorescent polymer shown in the formula IX and the formula X,

(Formula IX)

In the general structural formula of the formula IX, n is an integer of 100-10000;

(Formula X)

In the general structural formula of formula X, n is an integer of 100-10000.

The method for the polymer described in m=1 and p=1 in the general structural formula of the preparation formula VII provided by the present invention comprises the following steps: using cuprous bromide as a catalyst, and using N, N, N', N,' N″-pentamethyldiethylenetriamine is used as a ligand, and the compound described in m=1 and p=1 in the general structure of formula I is used as an initiator to initiate N,N-dimethylacrylamide to carry out atom transfer Free radical polymerization reaction, the reaction is completed to obtain the polymer described in the general structural formula of formula VII where m=1 and p=1.

In the method, the cuprous bromide, N, N, N', N, 'N"-pentamethyldiethylenetriamine, the compound of m=1, p=2 in the general structural formula of formula I The molar ratio to N,N-dimethylacrylamide is 3-4:3-4:1:100-10000, preferably 3:3:1:3000; in the atom transfer radical polymerization reaction, the temperature is 100 -150°C, preferably 120°C, for 1-7 days, preferably 4 days.

The method for the polymer described in m=1 and p=2 in the general structural formula of the preparation formula VII provided by the present invention comprises the following steps: using cuprous bromide as a catalyst, and using N, N, N', N,' N″-pentamethyldiethylenetriamine is used as a ligand, and the compound described in m=1 and p=1 in the general structure of formula I is used as an initiator to initiate N,N-dimethylacrylamide to carry out atom transfer Free radical polymerization reaction, the reaction is completed to obtain the polymer described in the general structural formula of formula VII where m=1 and p=2.

In the method, the cuprous bromide, N, N, N, N, N "-pentamethyldiethylenetriamine, m=1 in the formula I structural general formula, the compound described in p=2 and N , the molar ratio of N-dimethylacrylamide is 6-8:6-8:1:100-10000, preferably 6:6:1:3000; in the atom transfer radical polymerization reaction, the temperature is 100-150 °C, preferably 120 °C, for 1-7 days, preferably 4 days.

The application of the fluorescent polymer represented by the general structure of formula VII provided by the present invention as a separation medium in the separation of DNA or protein by capillary electrophoresis also belongs to the protection scope of the present invention.

Using the fluorescent polymer provided by the invention to separate DNA or protein, the separation speed is fast, the separation effect is good, the capillary does not need to be modified, and the one-time glue filling can be used repeatedly and has good repeatability; moreover, the present invention can convert comparable When different topological structures are introduced into the separation medium, the influence of linear polymers on the separation results of biomacromolecules can be systematically compared; in addition, since the fluorescent polymer has a fluorescent nucleus, the fluorescent nucleus can be introduced into the separation medium to achieve simultaneous The purpose of the separation medium and biomacromolecules can be observed, so as to obtain a more intuitive image of biomacromolecules in the separation process, and provide a more powerful method for revealing the separation mechanism.

Description of drawings

Fig. 1 is the NMR spectrum of the initiator S-I (ie formula II) prepared in Example 1.

Fig. 2 is the NMR spectrum of polymer S-3-PDMA prepared in Example 2.

Fig. 3 is the static light scattering Zimm diagram of polymer S-3-PDMA prepared in Example 2.

Fig. 4 is the NMR spectrum of the initiator S-II (ie formula III) prepared in Example 3.

Figure 5 is the capillary electrophoresis separation diagram of the polymer solution on ΦX174/Hae III DNA digestion solution, from top to bottom are the solution pairs of polymer S-3-PDMA with a mass percentage concentration of 1%, 3%, and 5%. Capillary electrophoresis separation diagram of ΦX174/Hae III DNA digest.

Figure 6 is a repeatability experiment graph of capillary electrophoresis separation of ΦX174/Hae III DNA digestion solution with a 5% solution of S-3-PDMA, and the three curves are the capillary electrophoresis separation graphs of three injections under this condition.

Fig. 7 is the capillary electrophoresis separation figure of polymer solution to ΦX174/Hae III DNA digestion liquid, is respectively the solution of polymer L-PDMA, S-3-PDMA and S-6-PDMA from top to bottom (mass percentage concentration 3%) for capillary electrophoresis separation of ΦX174/Hae III DNA digest.

Detailed ways

The present invention will be further described below in conjunction with specific examples, but the present invention is not limited to the following examples.

The preparation of initiator S-I shown in embodiment 1, formula II

Using 5,5,10,10,15,15-hexahexyl-2,7,12-tribromotrisindene as the starting material, it is obtained by Suzuki coupling reaction with p-methoxyphenylboronic acid under the catalysis of palladium catalyst 5,5,10,10,15,15-hexahexyl-2,7,12-tris(4-methoxyphenyl)triindene. Then demethylation under the action of boron tribromide gives 5,5,10,10,15,15-hexahexyl-2,7,12-tris(4-hydroxyphenyl)triindene. Then react with 2-bromoisobutyryl bromide under the action of triethylamine to obtain the initiator S-I shown in formula II. Concrete reaction process is shown in the following formula:

1) Preparation of compound 5,5,10,10,15,15-hexahexyl-2,7,12-tris(4-methoxyphenyl)tripolyindene:

5,5,10,10,15,15-hexahexyl-2,7,12-tribromotripolyindene (0.5g, 0.46mmol), p-methoxyphenylboronic acid (0.28g, 1.84mmol), concentration 2M aqueous sodium carbonate solution (3.7mL) and tetrahydrofuran (20mL) were added to the reaction flask, tetrakis(triphenylphosphine)palladium (0.05g, 0.04mmol) was added under nitrogen protection, and the reaction was refluxed for 20h. After the reaction, the organic phase was washed with saturated ammonium chloride aqueous solution and saturated brine respectively. The organic phase was dried over anhydrous magnesium sulfate, spinned to remove the solvent, and separated on a silica gel column with ethyl acetate/petroleum ether=1/10 as the eluent to obtain a white solid product. Yield; 78%.

The structural detection data of this compound are as follows: ¹ H NMR (CDCl ₃ , 300MHz, ppm): 8.45-8.43 (d, J=8.1Hz, 3H, Ar-H), 7.73-7.70 (d, J=8.7Hz, 6H, Ar-H), 7.68(s, 3H, Ar-H), 7.65-7.63(d, J=8.1Hz, 3H, Ar-H), 7.07-7.04(d, J=8.7Hz, 6H, Ar-H ), 3.81 (s, 9H, OCH ₃ ), 3.07-3.00 (m, 6H, CH ₂ ), 2.23-2.13 (m, 6H, CH ₂ ), 0.98-0.90 (m, 36H, CH ₂ ), 0.63- 0.59 (m _, 30H, _CH2CH3 ).

From the above structure detection data, it can be seen that the structure of the compound is correct.

2) Preparation of compound 5,5,10,10,15,15-hexahexyl-2,7,12-tris(4-hydroxyphenyl)tripolyindene:

Dissolve 5,5,10,10,15,15-hexahexyl-2,7,12-tris(4-methoxyphenyl)indene (0.26 g, 0.22 mmol) in dichloromethane (10 mL) In, boron tribromide (0.56 g, 2.23 mmol) was added dropwise under ice-cooling, and the reaction system was stirred overnight at room temperature. After the reaction, a saturated aqueous solution of sodium bicarbonate was added to the reaction system, and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, and the solvent was spun off, and then separated on a silica gel column with ethyl acetate/petroleum ether=1/5 as the eluent to obtain a white solid product. Yield: 99%.

The structural detection data of this compound are as follows: ¹ H NMR (CDCl ₃ , 300MHz, ppm): 8.41-8.37 (d, J=12Hz, 3H, Ar-H), 7.68-7.58 (m, 12H, Ar-H), 7.00 -7.96 (d, J=12Hz, 6H, Ar-H), 4.84 (s, 3H, OH), 3.04-2.97 (m, 6H, CH ₂ ), 2.20-2.06 (m, 6H, CH ₂ ), 0.88 (m, 36H, CH ₂ ), 0.62-0.56 (m, 30H, CH ₂ CH ₃ ). ¹³ C NMR (CDCl ₃ , 75MHz, ppm): δ155.0, 154.3, 144.8, 139.2, 138.5, 138.0, 134.2 , 128.3, 124.8, 124.6, 120.1, 115.7, 55.7, 37.1, 31.5, 29.7, 29.5, 23.9, 22.3, 13.9. MS (MALDI-TOF): Calcd for C ₈₄ H ₁₀₈ O ₃ : 1123. Found: 1123.

From the above structure detection data, it can be seen that the structure of the compound is correct.

(3) Preparation of compound S-I:

5,5,10,10,15,15-hexahexyl-2,7,12-tri(4-hydroxyphenyl)tripolyindene (0.26g, 0.23mmol), triethylamine (2mL, 28mmol) and Tetrahydrofuran (10 mL) was added into the reaction flask, 2-bromoisobutyryl bromide (0.53 g, 2.3 mmol) was added dropwise under ice-cooling, and the reaction system was stirred at room temperature for 12 hours. After the reaction was finished, the ammonium salt was removed by filtration, and the solvent was spun off. Separation on a silica gel column with ethyl acetate/petroleum ether=1/10 as the eluent gave a white solid product, namely compound S-I. Yield: 89%.

The proton NMR spectrum of this compound is shown in Figure 1, and the specific detection data of the proton and carbon spectra are as follows: ¹ H NMR (CDCl ₃ , 300MHz, ppm): δ8.45-8.42 (d, J=8.4Hz, 3H, Ar-H), 7.80-7.77(dd, J=8.4Hz, J=2.1Hz, 6H, Ar-H), 7.67(s, 3H, Ar-H), 7.67-7.64(d, J=8.4Hz, 3H, Ar-H), 7.29-7.27(dd, J=6.8Hz, J=1.8Hz, 6H, Ar-H), 3.04-2.99(m, 6H, CH ₂ ), 2.19-2.09(m, 6H, CH ₂ ), 2.12(s, 18H, CH ₃ ), 0.96-0.89(m, 36H, CH ₂ ), 0.63-0.56(m, 30H, CH ₂ CH ₃ ). ¹³ C NMR (CDCl ₃ , 75MHz, ppm ): δ170.4, 154.3, 150.1, 154.3, 139.7, 139.5, 138.2, 138.0, 128.1, 125.1, 124.9, 121.3, 120.6, 55.8, 55.4, 37.1, 31.5, 30.7, 29.7, 29.5, 23.9, 29.2, 1.0. MS (MALDI-TOF): Calcd _{for C93H117Br3O6} : _1570. Found: ₁₅₇₁ .

From the above structure detection data, it can be seen that the structure of the compound is correct.

The preparation of polymer (S3-PDMA) shown in embodiment 2, formula IX

The compound represented by formula II prepared in Example 1 was used as the initiator in an amount of 0.01 mmol, N, N-dimethylacrylamide was used as a monomer in an amount of 30 mmol, and CuBr was used as a catalyst in an amount of 0.03 mmol. PMDETA is used as a ligand, and the dosage is 0.03 mmol, and it is reacted at 120°C for four days to obtain the polymer represented by the structural formula IX. The molecular weight of the polymer was measured by static light scattering to be 9×10 ⁵ g/mol, and the molecular weight dispersion was 2.5.

The preparation of initiator (S-II) shown in embodiment 3, formula III

According to the same experimental method and conditions as in Example 1, only the p-methoxyphenylboronic acid used in step 1) is replaced with 3,4-dimethoxyphenylboronic acid, and the 3,4-dimethoxyphenylboronic acid The consumption of phenylboronic acid is 1.84mmol, and the initiator shown in preparation formula III. That is to use 5,5,10,10,15,15-hexahexyl-2,7,12-tribromotripolyindene as the starting material, and 3,4-dimethoxyphenylboronic acid under the catalysis of palladium catalyst Suzuki coupling reaction gives 5,5,10,10,15,15-hexahexyl-2,7,12-tris(3,4-dimethoxyphenyl)triindene. Then demethylation under the action of boron tribromide to obtain 5,5,10,10,15,15-hexahexyl-2,7,12-tris(3,4-dihydroxyphenyl)triindene. Then react with 2-bromoisobutyryl bromide under the action of triethylamine to obtain initiator S-II. Concrete reaction process is shown in the following formula:

The proton NMR spectrum of the compound S-II is shown in Figure 4, and the specific detection data of the proton and carbon spectra are as follows: ¹ HNMR (CDCl ₃ , 300MHz, ppm): δ8.46-8.43 (d, J=8.7Hz, 3H, Ar-H), 7.70-7.64(m, 9H, Ar-H), 7.59-7.58(d, J=1.8Hz, 3H, Ar-H), 7.40-7.37(d, J=8.4Hz, 3H , Ar-H), 3.02-2.98 (m, 6H, CH ₂ ), 2.23-2.10 (m, 6H, CH ₂ ), 2.13 (s, 18H, CH ₃ ), 2.11 (s, 18H, CH ₃ ), 0.95-0.90 (m, 36H, CH ₂ ), 0.64-0.55 (m, 30H, CH ₂ CH ₃ ). ¹³ C NMR (CDCl ₃ , 75 MHz, ppm): δ169.2, 154.4, 145.6, 142.2, 141.2, 140.8, 140.1, 137.9, 137.3, 125.6, 125.3, 124.9, 123.1, 121.5, 120.6, 55.9, 55.1, 55.0, 37.0, 32.8, 31.5, 30.8, 30.82, 30.80, 29.7, 29.4, 03.0, 22.2

From the above structure detection data, it can be seen that the structure of the compound is correct.

Embodiment 4, the preparation of polymer S-6-PDMA (formula X)

The compound shown in formula III prepared in Example 3 is used as an initiator, and the dosage is 0.01mmol; N, N-dimethylacrylamide is used as a monomer, and the dosage is 30mmol; cuprous bromide CuBr is used as a catalyst, and the dosage is 0.06mmol, using PMDETA as a ligand, the dosage is 0.06mmol, reacting at 120°C for four days to obtain the polymer S-6-PDMA. The molecular weight of the polymer measured by the static light scattering method was 9×10 ⁵ g/mol, and the molecular weight dispersion was 2.5.

The polymer (S-3-PDMA) shown in the resulting formula IX structural formula prepared in Example 2 was used as a separation medium, and the concentration was 100 μg/mL ΦX174/Hae III DNA digestion solution (this digestion solution was purchased from sigmaaldrich company ) for capillary electrophoresis separation: first prepare 100 μg/mL ΦX174/Hae III DNA digestion solution, and stain with fluorescent dye GeneFinder ^TM (purchased from Xiamen Baiweixin Biotechnology Co., Ltd.). Then, the prepared polymer shown in the structural formula IX is dissolved in a TBE (Tris-boric acid buffer) solution with a mass percent concentration of 1% to prepare a series of polymer solutions with different concentrations. The adopted capillary has an inner diameter of 75 microns, a total tube length of 31 cm, and an effective tube length of 21 cm. During operation, first rinse the capillary with 1M hydrochloric acid solution for 10 minutes, then rinse with deionized water for 2 minutes, then press the prepared polymer solution into the capillary at 30kpa for 8 minutes, and the baseline basically reaches balance. Then, the negative electrode was electrically injected, the voltage was -5kV/cm, and the injection time was 5s. After the sample injection is completed, use 1% TBE solution as the buffer solution, and carry out the DNA separation experiment under the conditions of 25° C. and a separation voltage of -8 kV/cm. Detection is performed with a fluorescence detector, the excitation wavelength is 488nm, and the detection wavelength is 520nm.

The separation results are shown in Figure 5. It can be seen from the figure that the ΦX174/Hae III DNA digestion solution contains a total of 11 DNA fragments, and each fragment is represented as a peak in the capillary electrophoresis diagram. First, taking the polymer S-3-PDMA with a molecular weight of 9×10 ⁵ g/mol as an example, it was formulated into polymer solutions with a mass percent concentration of 1%, 3% and 5% respectively, and the solvent in the solution was It is TBE (Tris-boric acid buffer), and its capillary electrophoresis separation result is shown in Figure 5. It can be seen that as the concentration increases, the separation effect increases, indicating that within a certain concentration range, high concentration is conducive to the improvement of separation effect. The capillary can be reused after being filled with gel once. Taking 5% S-3-PDMA as an example, the electrophoresis three times has good repeatability. The repeatability experiment results are shown in Figure 6. Select polymers S-3-PDMA, S-6-PDMA, and L-PDMA with a molecular weight of 9×10 ⁵ g/mol to prepare polymer solutions with a concentration of 3% by mass, and the solvent used is TBE (Tris-boric acid buffer) for DNA isolation. The capillary electrophoresis separation results are shown in Figure 7. It can be seen that the separation effect of S-3-PDMA is the best, and the separation time of S-6-PDMA is the shortest when the separation effect is similar to that of the linear L-PDMA polymer. The above experimental facts show that: under the molecular weight, star-shaped PDMA has better separation properties when separating DNA.

(Formula XI)

Wherein, the structural formula of the L-PDMA polymer is shown in formula X, and its preparation method is as follows:

1) Preparation of compound 9,9-dihexyl-2,7-bis(p-methoxyphenyl)fluorene:

9,9-dihexyl-2,7-dibromofluorene (1.5g, 3mmol), 4-methoxyphenylboronic acid (1.4g, 9mmol), sodium carbonate (2M, 13mL) and tetrahydrofuran (30mL) were added to In the reaction flask, tetrakis(triphenylphosphine)palladium (0.2 g, 0.018 mmol) was added under the protection of nitrogen, and the reaction was refluxed for 20 h. After the reaction, the organic phase was washed with saturated ammonium chloride aqueous solution and saturated brine respectively. The organic phase was dried over anhydrous magnesium sulfate, and the solvent was removed by spinning, separated on a silica gel column, and eluted with petroleum ether as an eluent to obtain a white solid product, which was compound 9,9-dihexyl-2,7-di(p- Methoxyphenyl)fluorene. Yield; 96%.

The structure detection data of this compound are as follows: ¹ H NMR (CDCl ₃ , 200MHz, ppm): 7.78-7.74 (dJ=8.2Hz, 2H, Ar-H), 7.66-7.62 (d, J=8.6Hz, 4H, Ar- H), 7.57-7.54(m, 4H, Ar-H), 7.06-7.02(d, J=8.6Hz, 4H, Ar-H), 3.89(s, 6H, OCH ₃ ), 2.09-2.01(m, 4H, CH ₂ ), 1.09 (m, 12H, CH ₂ ), 0.81-0.75 (m, 10H, CH ₂ CH ₃ ). ¹³ C NMR (CDCl ₃ , 50MHz, ppm): δ159.1, 151.6, 139.6, 139.5, 134.3, 128.2, 125.5, 121.0, 119.8, 114.2, 55.3, 55.2, 40.5, 31.4, 29.7, 23.8, 22.6, 14.0. MS (EI): Calcd for C ₃₉ H ₄₆ O ₂ : 546. Found: 546. (M ⁺ , 100%). Anal. Calcd for C ₃₉ H ₄₆ O ₂ : C, 85.67; H, 8.48. Found: C, 85.50; H, 8.39.

From the above structure detection data, it can be seen that the structure of the compound is correct.

2) Preparation of compound 9,9-dihexyl-2,7-bis(4-hydroxyphenyl)fluorene:

Dissolve 9,9-dihexyl-2,7-bis(4-methoxyphenyl)fluorene (0.1 g, 0.18 mmol) in dichloromethane (10 mL), and add boron tribromide (0.18 g, 0.74mmol), the reaction system was stirred overnight at room temperature. After the reaction, a saturated aqueous solution of sodium bicarbonate was added to the reaction system, and the aqueous phase was extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, spinned to remove the solvent, separated on a silica gel column, and eluted with an eluent mixed with a volume ratio of ethyl acetate:petroleum ether=1:8 to obtain a white solid product, which is the compound 9,9-Dihexyl-2,7-bis(4-hydroxyphenyl)fluorene. Yield: 99%.

The structural detection data of this compound are as follows: ¹ H NMR (CDCl ₃ , 300MHz, ppm): δ7.74-7.71 (d, J=8.1Hz, 2H, Ar-H), 7.57-7.54 (d, J=8.7Hz, 4H, Ar-H), 7.53-7.50 (m, 4H, Ar-H), 6.96-6.94 (d, J=8.7Hz, 4H, Ar-H), 4.84 (s, 2H, OH), 2.04-1.99 (m, 4H, CH ₂ ), 1.14-1.04 (m, 12H, CH ₂ ), 0.77-0.73 (m, 10H, CH ₂ CH ₃ ). ¹³ C NMR (CDCl ₃ , 75MHz, ppm): δ154.9 , 151.6, 139.6, 139.5, 134.5, 128.4, 125.5, 121.0, 119.8, 115.7, 55.1, 40.5, 31.4, 29.7, 23.8, 22.5, 14.0. HRMS (EI): Calcd for C ₃₇ H ₄₂ O ₂ : 518.3185.Found : 518.3192. (M ⁺ , 100%).

From the above structure detection data, it can be seen that the structure of the compound is correct.

(3) Preparation of linear initiator:

Add 9,9-dihexyl-2,7-bis(4-hydroxyphenyl)fluorene (0.14g, 0.27mmol), triethylamine (1.5mL, 21mmol) and tetrahydrofuran (10mL) into the reaction flask, ice-bath 2-Bromoisobutyryl bromide (0.24 g, 1 mmol) was added dropwise, and the reaction system was stirred overnight at room temperature. After the reaction was finished, the ammonium salt was removed by filtration, and the solvent was spun off. Silica gel column separation, eluting with a mixed eluent with a volume ratio of ethyl acetate:petroleum ether=1:10, a white solid product was obtained, and the linear initiator was obtained. Yield: 95%.

The structural detection data of this compound are as follows: ¹ H NMR (CDCl ₃ , 200MHz, ppm): δ7.79-7.76 (d, J=7.8Hz, 2H, Ar-H), 7.71-7.67 (d, J=8Hz, 4H , Ar-H), 7.58-7.54 (m, 4H, Ar-H), 7.26-7.22 (d, J=8Hz, 4H, Ar-H), 2.11 (s, 12H, CH ₃ ), 2.11-1.94 ( m, 4H, CH ₂ ), 1.07 (m, 12H, CH ₂ ), 0.76-0.72 (m, 10H, CH ₂ CH ₃ ). ¹³ C NMR (CDCl ₃ , 50MHz, ppm): δ170.3, 151.7, 150.1, 140.1, 139.8, 139.2, 128.2, 126.1, 121.5, 121.3, 120.1, 55.4, 55.3, 40.4, 31.4, 30.7, 29.6, 23.8, 22.5, 14.0. MS (MALDI-TOF): Calcd for C ₄₅ H ₅₂ Br _2O4 : _816. Found: 815. Anal _. Calcd for _C45H52Br2O4 : C, 66.18 _; H, 6.42. _Found : C, 66.05; H, 6.35.

From the above structure detection data, it can be seen that the structure of the compound is correct.

Take 0.01mmol of the obtained linear initiator prepared by the above method, use N,N-dimethylacrylamide as the monomer, the dosage is 30mmol, use CuBr as the catalyst, the dosage is 0.02mmol, and use PMDETA as the ligand, the dosage is 0.02mmol , and reacted at 120° C. for four days to obtain the linear L-PDMA polymer. The molecular weight of the polymer was measured by static light scattering method to be 9×10 ⁵ g/mol, and the molecular weight dispersion was 2.5.

Claims (14)

1. The compound shown in formula II or formula III, 2. A method for preparing the compound shown in formula II described in claim 1, comprising the steps of: 1) In the presence of a palladium catalyst, carry out a Suzuki coupling reaction of the compound represented by formula IV, p-methoxyphenylboronic acid and an aqueous solution of a basic compound in an organic solvent to obtain a compound represented by formula V; In the formula IV, R ₁ is hexyl; In the formula V, R ₁ is hexyl, R ₂ is phenyl, m=1, p=1; 2) In the presence of boron tribromide, the compound represented by the formula V is demethylated in an organic solvent to obtain the compound represented by the formula VI; In the formula VI, R ₁ is hexyl, R ₂ is phenyl, m=1, p=1; 3) In the presence of triethylamine, react the compound represented by the formula VI with 2-bromoisobutyryl bromide in an organic solvent to obtain the compound represented by the formula II described in claim 1. 3. The method according to claim 2, characterized in that: in the step 1), the palladium catalyst is selected from tetrakis(triphenylphosphine)palladium, bis(triphenylphosphine)palladium chloride and palladium acetate At least one of, the basic compound is sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide; In the step 3), the organic solvent is at least one of dichloromethane, chloroform, ether, toluene and tetrahydrofuran. 4. The method according to claim 2 or 3, characterized in that: in the step 1), the concentration of the aqueous solution of the basic compound is 0.5-5mol/L; the compound shown in the formula IV, p-formazine The molar ratio of oxyphenylboronic acid, the basic compound and the palladium catalyst is 1:3-6:10-20:0.05-0.2; in the Suzuki coupling reaction, the temperature is 0-30°C and the time is 6-24 hours; In the step 2), the molar ratio of boron tribromide to the compound represented by the formula V is 5-20:1; the reaction temperature is 0-30°C, and the time is 6-24 hours; In the step 3), the molar ratio of triethylamine, the compound represented by the formula VI to 2-bromoisobutyryl bromide is 10-200:1:3-15; in the reaction, the temperature is 0-30 ℃, the time is 6-24 hours. 5. The method according to claim 4, characterized in that: in the step 1), the concentration of the aqueous solution of the basic compound is 2mol/L; the compound shown in the formula IV, p-methoxyphenylboronic acid . The molar ratio of the basic compound to the palladium catalyst is 1:4:15:0.1; in the Suzuki coupling reaction, the temperature is 20°C and the time is 12 hours; In the step 2), the molar ratio of boron tribromide to the compound represented by the formula V is 10:1; the reaction temperature is 20°C, and the reaction time is 12 hours; In the step 3), the molar ratio of triethylamine, the compound represented by the formula VI to 2-bromoisobutyryl bromide is 120:1:10; in the reaction, the temperature is 0°C and the time is 12 hours . 6. A method for preparing the compound shown in formula III described in claim 1, comprising the steps of: 1) In the presence of a palladium catalyst, carry out a Suzuki coupling reaction with an aqueous solution of the compound represented by formula IV, 3,4-dimethoxyphenylboronic acid and a basic compound in an organic solvent to obtain the compound represented by formula V; In the formula IV, R ₁ is hexyl; In the formula V, R ₁ is hexyl, R ₂ is phenyl, m=1, p=2; 2) In the presence of boron tribromide, the compound represented by the formula V is demethylated in an organic solvent to obtain the compound represented by the formula VI; In the formula VI, R ₁ is hexyl, R ₂ is phenyl, m=1, p=2; 3) In the presence of triethylamine, react the compound represented by the formula VI with 2-bromoisobutyryl bromide in an organic solvent to obtain the compound represented by the formula III in claim 1. 7. The method according to claim 6, characterized in that: in the step 1), the palladium catalyst is selected from tetrakis(triphenylphosphine)palladium, bis(triphenylphosphine)palladium chloride and palladium acetate At least one of, the basic compound is sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide; In the step 3), the organic solvent is at least one of dichloromethane, chloroform, ether, toluene and tetrahydrofuran. 8. The method according to claim 6 or 7, characterized in that: in the step 1), the concentration of the aqueous solution of the basic compound is 0.5-5mol/L; the compound shown in formula IV, 3, The molar ratio of 4-dimethoxyphenylboronic acid, the basic compound and the palladium catalyst is 1:3-6:10-20:0.05-0.2; in the Suzuki coupling reaction, the temperature is 0-30 °C, the time is 6-24 hours; In the step 2), the molar ratio of boron tribromide to the compound represented by the formula V is 10-30:1; the reaction temperature is 0-30°C, and the reaction time is 6-24 hours; In the step 3), the molar ratio of triethylamine, the compound represented by the formula VI to 2-bromoisobutyryl bromide is 20-300:1:6-30; in the reaction, the temperature is 0-30 ℃, the time is 6-24 hours. 9. The method according to claim 8, characterized in that: in the step 1), the concentration of the aqueous solution of the basic compound is 2mol/L; the compound shown in the formula IV, 3,4-dimethyl The molar ratio of oxyphenylboronic acid, the basic compound and the palladium catalyst is 1:4:15:0.1; in the Suzuki coupling reaction, the temperature is 20°C and the time is 12 hours; In the step 2), the molar ratio of boron tribromide to the compound represented by the formula V is 20:1; the reaction temperature is 20°C, and the reaction time is 12 hours; In the step 3), the molar ratio of triethylamine, the compound represented by the formula VI to 2-bromoisobutyryl bromide is 240:1:20; in the reaction, the temperature is 20°C and the time is 12 hours . 10. The fluorescent polymer shown in formula IX or formula X, In the general structural formula of the formula IX, n is an integer of 100-10000; In the general structural formula of formula X, n is an integer of 100-10000. 11. A method for preparing the polymer as claimed in claim 10, comprising the steps of: using cuprous bromide as a catalyst, N, N, N', N', N "-pentamethyldiethylenetriamine As a ligand, the compound described in claim 1 is used as an initiator to initiate N,N-dimethylacrylamide to carry out atom transfer radical polymerization, and the polymer is obtained after the reaction is completed. 12. The method according to claim 11, characterized in that: said cuprous bromide, N,N,N',N',N"-pentamethyldiethylenetriamine, as claimed in claim 1 The molar ratio of the compound to N,N-dimethylacrylamide is 3-4:3-4:1:100-10000; in the atom transfer radical polymerization reaction, the temperature is 100-150°C and the time is 1- 7 days. 13. The method according to claim 12, characterized in that: the cuprous bromide, N,N,N',N',N"-pentamethyldiethylenetriamine, as claimed in claim 1 The molar ratio of compound to N,N-dimethylacrylamide is 3:3:1:3000; in the atom transfer radical polymerization reaction, the temperature is 120° C. and the time is 4 days. 14. The application of the fluorescent polymer according to claim 10 as a separation medium in capillary electrophoresis separation of DNA or protein.