Double-terminal fluorescently-labeled polymer as well as preparation method and application thereof

Abstract

The invention discloses a double-terminal fluorescence labeled polymer as well as a preparation method and application thereof. The structural formula of the double-terminal fluorescence labeled polymer is shown as a formula V. In the formula V, n is 30-300, and x is 1-10. The preparation method comprises the following steps: carrying out ATRP (Atom Transfer Radical Polymerization) active polymerization reaction on a monomer by taking 2-bromo-2-methyl propionate 2-hydroxyethyl ester with hydroxyl at the tail end as an initiator to obtain a compound of which the tail end is terminated by hydroxyl and bromo; a hydroxyl terminal in the compound reacts with a perylene fluorescent dye shown in a formula II, a bromine terminal is subjected to an azidation reaction and then is subjected to a click chemical reaction with an ATTO647N dye, and the double-terminal fluorescence labeled polymer shown in a formula V is obtained. The double-fluorescent-molecule end-capped polymer disclosed by the invention has an FRET effect; the double-fluorescent-molecule-terminated polymer can realize single-molecule fluorescence microscopic imaging, and has important significance on basic researches such as micro-nano structure evolution of characterization and analysis materials and the like.

Description

Technical Field

[0001] This invention belongs to the field of basic research in polymer physics and polymer chemistry, and relates to a dual-terminal fluorescently labeled polymer, its preparation method and application. Background Technology

[0002] Changes in the microstructure of materials often have a significant impact on their final performance. Fluorescence resonance energy transfer (FRET), a technique that can quantitatively measure the distance between two different luminescent groups with a measurement accuracy within 10 nm, is commonly used to characterize and probe changes in the conformation of the internal microstructure of materials, the evolution of interaction forces, and other physicochemical processes. Single-molecule detection has unique advantages over traditional ensemble detection methods, such as high spatial resolution. While ensemble detection only provides an average value, it allows for observation of local dynamic fluctuations in the microstructure over time, and also enables microscopic tracking of specific sites. This allows for the study of numerous micro- and nano-scale physicochemical mechanisms at the single-molecule scale.

[0003] Traditional FRET technology calculates FRET efficiency by detecting changes in fluorescence intensity or fluorescence lifetime between the fluorescent acceptor and donor dye, and then uses further formula fitting to obtain ensemble information such as intermolecular distance changes or interaction forces. However, with the advancement of technology, the requirements for material performance are becoming increasingly stringent. Dissecting material structures at a more microscopic level to obtain higher-performance materials is of great significance. Based on this, this study develops a FRET detection method applicable to the single-molecule level. By combining FRET technology with single-molecule fluorescence microscopy, it helps to further analyze the evolution of nanostructures at the single-molecule scale, obtain more precise physicochemical process mechanisms, and thus prepare materials with increasingly superior performance. Summary of the Invention

[0004] The purpose of this invention is to provide a dual-terminal fluorescently labeled polymer, its preparation method, and its application.

[0005] This invention utilizes two fluorescent dyes, perylene dyes and ATTO647N, which have high quantum yields and good photobleaching resistance, as end-capped fluorescent labeling groups, serving as the donor and acceptor in the FRET effect, respectively. Combined with a carefully designed chemical synthesis route, it achieves the tracking of the FRET effect at the single-molecule level, expanding the application scope of the FRET effect and expecting it to have wider applications in basic research fields such as interfaces and ultrathin films.

[0006] This invention provides a dual-terminal fluorescently labeled polymer, the structural formula of which is shown in Formula V:

[0007] In Equation V, n is 30~300 and x is 1~10.

[0008] The present invention also provides a method for preparing the dual-terminal fluorescently labeled polymer as shown in Formula V, comprising the following steps: 1) using 2-bromo-2-methylpropionic acid-2-hydroxyethyl ester with hydroxyl groups at the ends as an initiator to carry out ATRP living polymerization reaction to obtain the compound shown in Formula I with hydroxyl and bromine groups at the ends;

[0009] In Formula I, n is 30~300, and x is an integer from 1 to 10; 2) The hydroxyl terminus of the compound represented by Formula I reacts with the perylene fluorescent dye represented by Formula II to obtain the compound represented by Formula III;

[0010] 3) The bromine terminus of the compound shown in Formula III is azidated to obtain the compound shown in Formula IV, which is then reacted with ATTO647N dye via a click chemistry reaction to obtain the dual-terminal fluorescently labeled polymer shown in Formula V.

[0011] In Equations I, II, III, and IV, n is 30 to 300, and x is an integer from 1 to 10.

[0012] In the above method, in step 1), the polymerization reaction conditions are as follows: the catalyst is CuBr, the ligand is N,N,N',N,'N''-pentamethyldiethylenetriamine (PMEDTA), and the reaction solvent system is a mixed solution of isopropanol and water with a volume ratio of 92.5:7.5. The molar ratio of the initiator, the ligand, the CuBr, and the monomer can be 1:1:1:20~100, and the reaction temperature can be 40°C. o C ~70 o C, specifically 40 o C or 70 o C, the reaction time can be 4 to 8 hours, and oxygen is introduced to end the polymerization reaction; The molecular weight distribution (PDI) of the compound represented by Formula I is below 1.2.

[0013] In the above method, step 1) involves the purification treatment of the system after the polymerization reaction as follows: Methanol containing HBr was added to the system as a precipitant, and the mixture was stirred. The product precipitated, dissolved in acetone, and then washed off. This process was repeated three times to obtain the poly(n-butyl methacrylate) shown in Formula I, which was then dried. The methanol containing HBr is prepared by mixing an HBr aqueous solution with methanol at a volume percentage concentration of 40%, with a volume ratio of 1:15~25; the drying temperature can be 25°C. o C~45 o C-pressure drying.

[0014] In the above method, in step 2), the reaction is carried out in the presence of a catalyst, which is di-isopropyl azodicarboxylate and triphenylphosphine. The reaction is carried out in the presence of a solvent, which may be THF. The temperature of the reaction can be 20°C. o C ~25 o C, the time can be 4 to 8 hours.

[0015] In the above method, in step 2), the post-processing of the reaction is to remove the perylene fluorescent dye shown in Formula II by gel column chromatography, and to detect whether the free dye has been completely removed by fluorescence correlation spectroscopy.

[0016] In this invention, the perylene fluorescent dye represented by Formula II is a donor fluorescent dye named 3,4,9,10-perylenetetracarboxydiimide, and its chemical structure is shown in Formula II. Since it only needs to be attached to the hydroxyl end and a high end-capping rate is required, it needs to be added in excess during the reaction.

[0017] In the above method, in step 3), the azide reaction is carried out in the presence of a solvent, wherein the solvent is DMF; The temperature of the reaction can be 20°C. o C ~25 o C, the time can be 8 to 16 hours.

[0018] In the above method, in step 3), the click chemistry reaction is carried out in the presence of a catalyst, a ligand, and a solvent; The catalyst is CuBr, the ligand is N,N,N',N,'N''-pentamethyldiethylenetriamine, and the solvent is DMF; The temperature of the click chemical reaction can be 20°C. o C ~25 o C, the time can be 24~48 hours.

[0019] The dual-terminal fluorescently labeled polymer of Formula V described in this invention is used in characterizing and analyzing the micro- and nano-structures of materials.

[0020] The present invention has the following beneficial effects: 1. The dual-fluorescent molecularly capped polymer of this invention exhibits the FRET effect; 2. The dual-fluorescent molecularly capped polymer of this invention can realize single-molecule fluorescence microscopy imaging, which is of great significance for basic research such as characterizing and analyzing the evolution of micro and nano structures of materials. Attached Figure Description

[0021] Figure 1 In this embodiment of the invention, a series of GPC maps of molecular weight distribution PnBMA were synthesized using the method in step (1).

[0022] Figure 2 The above is the hydrogen NMR spectrum of Formula 2 of this invention.

[0023] Figure 3 This is the hydrogen NMR spectrum of Embodiment 3 of the present invention.

[0024] Figure 4 This is a comparison of the infrared spectra before and after the reaction in Formulas 3 to 5 in the embodiments of the present invention.

[0025] Figure 5 This is a dual-channel single-molecule fluorescence defocusing microscopy image of the dual-terminal fluorescently labeled polymer synthesized by the present invention. Detailed Implementation Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] Implementation Case 1: Preparation of a dual-fluorescent molecularly capped polymer with FRET effect.

[0028] (1) Synthesis of poly(n-butyl methacrylate) with a narrow molecular weight distribution capped by hydroxyl and bromine groups via ATRP atom transfer radical polymerization: Initiator 2-bromo-2-methylpropionic acid-2-hydroxyethyl ester (153 mg, 0.7 mmol), ligand PMEDTA (125 mg, 0.7 mmol), n-butyl methacrylate monomer (5 g, 35.2 mmol, structural formula as shown in Formula 1), 23.125 ml isopropanol / 1.875 ml pure water (V / V = 92.5 / 7.5) were added to a reaction flask. Oxygen was removed by three freeze-thaw cycles, and then catalyst CuBr (110 mg, 0.7 mmol) was added. The reaction was carried out at 40 °C. o C. Reaction time: 6 hours.

[0029]

[0030] (2) The reaction was terminated by introducing oxygen. The reaction system was then passed through a neutral alumina column to remove copper. Methanol with a concentration of 40% HBr was added as a precipitant (methanol:40% HBr = 20:1, V / V) to precipitate the resulting polymer product. This product was then dissolved in as little acetone as possible. After three cycles, a pure polymer product OH-PnBMA-Br was obtained, as shown in Formula 2. The structure was confirmed as follows: Figure 2 As shown.

[0031]

[0032] In Equation 2, n is 37, 44, 55, 67, 75, or 113, and its molecular weight is below 1.2. Figure 1 The results of GPC are shown below. The GPC plot of the molecular weight distribution PnBMA is shown in the figure. Figure 1 As shown in the image.

[0033] (3) Take 200 mg of the above polymer product OH-PnBMA-Br, perylene fluorescent dye (3,4,9,10-perylenetetracarboxydiimide, 789 mg, 4.0 mmol), triphenylphosphine (500 mg, 4.0 mmol), and ultra-dry THF (5 ml) into the reaction flask, bring it to 0°C through an ice-water bath, then add DIAD (0.38 ml, 4.0 mmol), evacuate and purge with nitrogen, and continue the reaction in an ice-water bath for half an hour, then turn to room temperature and react for 5 hours. After the reaction is complete, the reaction solution needs to be diluted first, then centrifuged at 7000 rads / min for 20 minutes. The supernatant is then concentrated by rotary evaporation. The resulting polymer product is precipitated using methanol with 40% HBr added (methanol:40% HBr = 20:1, V / V). The precipitate is then dissolved in a small amount of acetone, and this process is repeated three times. The resulting product is then dissolved in as little ultra-dry THF as possible and passed through a gel column to remove excess free dye molecules (perylene fluorescent dyes). During the gel column chromatography, the diffusion coefficient needs to be continuously monitored using FCS to ensure complete removal of free dyes, thus obtaining a pure polymer PDI-PnBMA-Br with perylene dye attachment, as shown in Equation 3. The structure is confirmed as follows. Figure 3 As shown, Figure 3 The peaks on the benzene ring of the perylene dye appear in the range of chemical shifts 8.5 to 9.0, as shown in the green circle.

[0034]

[0035] (4) Take 80 mg of the above product PDI-PnBMA-Br into a reaction flask and dissolve it with 3 ml of ultra-dry DMF. Then add 10 mg of sodium azide NaN3 under nitrogen protection and react at room temperature for 48 hours. The obtained polymer product PDI-PnBMA-N3 (as shown in Formula 4) is precipitated by adding methanol with a concentration of 40% HBr as a precipitant (methanol: 40% HBr = 20:1, V / V). Repeat three times.

[0036]

[0037] (5) Take 20 mg of the above product PDI-PnBMA-N3 and 1 mg of ATTO647N-Alkyne into a reaction flask and dissolve them with 2 ml of ultra-dry DMF. Then, under nitrogen protection, add 2 mg of cuprous bromide CuBr and 3 µl of PMEDTA. After reacting at room temperature for 36 hours, the reaction is terminated by purging oxygen. Then, add ultra-dry THF to dilute the reaction solution and pass it through a neutral alumina column to remove excess copper. Then, concentrate by rotary evaporation. The obtained polymer product PDI-PnBMA-N3 is precipitated by adding 40% HBr in methanol as a precipitant (methanol:40% HBr = 20:1, V / V). Repeat three times to obtain the dual-dye-terminated polymer PDI-PnBMA-ATTO647N with FRET effect, as shown in Formula 5.

[0038] The infrared spectra of the compounds before and after the reaction in this step are compared as follows: Figure 4 As shown, through Figure 4 The appearance and disappearance of the azide group peak in the gray region further confirms that the reaction produces the compound shown in Formula 5.

[0039]

[0040] (6) By using a dual-channel single-molecule fluorescence defocusing imaging system with both long and short wavelengths built in the laboratory, it is possible to track and detect the FRET effect at the single-molecule level, such as... Figure 5 As shown. From Figure 5 It can be seen that fluorescent dye signals appeared in both the long and short wavelength channels only under 532nm laser excitation. Using a DV2 and a 635nm beam splitter, the long and short wavelength regions were separated. Figure 5 The short-wavelength signal on the right corresponds to the pattern formed by the perylene fluorescent dye 3,4,9,10-perylenetetracarboxydiimide, which is directly excited by a 532nm laser; the long-wavelength signal on the left corresponds to ATTO647N, which is excited by the emission light of the donor dye through the FRET effect.

Claims

1. A dual-terminal fluorescently labeled polymer, the structural formula of which is shown in Formula V: In Equation V, n is 30 to 300, and x is an integer from 1 to 10.

2. A method for preparing a dual-terminal fluorescently labeled polymer as shown in Formula V according to claim 1, comprising the following steps: 1) using 2-bromo-2-methylpropionic acid-2-hydroxyethyl ester with hydroxyl groups at the ends as an initiator to carry out ATRP living polymerization reaction to obtain a compound shown in Formula I with hydroxyl and bromine groups at the ends; 2) The hydroxyl terminus of the compound represented by Formula I reacts with the perylene fluorescent dye represented by Formula II to obtain the compound represented by Formula III; 3) The bromine end of the compound shown in Formula III is azidated to obtain the compound shown in Formula IV, which is then reacted with ATTO647N dye via click chemistry to obtain the dual-terminal fluorescently labeled polymer shown in Formula V. in, In equations I, II, III, and IV, n is 30 to 300, and x is an integer from 1 to 10.

3. The method according to claim 2, characterized in that, In step 1), the polymerization reaction conditions are as follows: the catalyst is CuBr, the ligand is N,N,N',N,'N''-pentamethyldiethylenetriamine, and the reaction solvent system is a mixed solution of isopropanol and water with a volume ratio of 92.5:7.

5. The molar ratio of the initiator, the ligand, the CuBr, and the monomer is 1:1:1:20~100, and the reaction temperature is 40°C. o C ~70 o C, the reaction time is 4-8 hours, and oxygen is introduced to end the polymerization reaction; The molecular weight distribution (PDI) of the compound represented by Formula I is below 1.

2.

4. The method according to claim 2 or 3, characterized in that, In step 1), the purification process of the system after the polymerization reaction is as follows: Methanol containing HBr was added to the system as a precipitant, and the mixture was stirred. The product precipitated, dissolved in acetone, and then washed off. This process was repeated three times to obtain the poly(n-butyl methacrylate) shown in Formula I, which was then dried. The methanol containing HBr is prepared by mixing a 40% (v / v) HBr aqueous solution with methanol in a volume ratio of 1:15-25; the drying temperature is 25°C. o C~45 o C-pressure drying.

5. The method according to any one of claims 2-4, characterized in that, In step 2), the reaction is carried out in the presence of a catalyst, which is diisopropyl azodicarbonate and triphenylphosphine; The reaction is carried out in the presence of a solvent, namely THF. The reaction temperature is 20°C. o C ~25 o C, the time is 4 to 8 hours.

6. The method according to any one of claims 2-5, characterized in that, In step 2), the post-processing of the reaction is to remove the perylene fluorescent dye shown in Formula II by gel column chromatography, and to detect whether the free dye has been completely removed by fluorescence correlation spectroscopy.

7. The method according to any one of claims 2-6, characterized in that, In step 3), the azide reaction is carried out in the presence of a solvent, namely DMF; The reaction temperature is 20°C. o C ~25 o C, the time is 8~16 hours.

8. The method according to any one of claims 2-8, characterized in that, In step 3), the click chemistry reaction is carried out in the presence of a catalyst, ligand, and solvent; The catalyst is CuBr, the ligand is N,N,N',N,'N''-pentamethyldiethylenetriamine, and the solvent is DMF; The temperature of the click chemical reaction is 20°C. o C ~25 o C, the time is 24~48 hours.

9. The application of the dual-terminal fluorescently labeled polymer of Formula V as claimed in claim 1 in characterizing and resolving the micro / nanostructure of materials.

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