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CN-121991264-A - Double-end fluorescent labeling polymer and preparation method and application thereof

CN121991264ACN 121991264 ACN121991264 ACN 121991264ACN-121991264-A

Abstract

The invention discloses a double-end fluorescent labeling polymer and a preparation method and application thereof. The structural formula of the double-end fluorescent marking polymer is shown as a formula V, wherein n is 30-300, and x is 1-10. The preparation method comprises the following steps of taking 2-bromo-2-methylpropanoic acid-2-hydroxyethyl ester with hydroxyl at the tail end as an initiator, carrying out ATRP active polymerization reaction to obtain a compound with the tail end blocked by hydroxyl and bromo, reacting the hydroxyl end in the compound with perylene fluorescent dye shown in a formula II, carrying out azide reaction on the bromo end, and then carrying out click chemistry reaction with ATTO647N dye to obtain the double-end fluorescent marked polymer shown in a formula V. The polymer with the double fluorescent molecular ends has FRET effect, can realize single-molecule fluorescent microscopic imaging, and has important significance for basic research such as representing micro-nano structure evolution of analytical materials.

Inventors

  • Ju Jinzhou
  • ZHAO JIANG

Assignees

  • 中国科学院化学研究所

Dates

Publication Date
20260508
Application Date
20241107

Claims (9)

  1. 1. A double-end fluorescent labeling polymer has a structural formula shown in a formula V: in the formula V, n is 30-300, and x is an integer of 1-10.
  2. 2. A process for preparing a double-ended fluorescent-labeled polymer of formula V according to claim 1, comprising the steps of 1) subjecting a monomer to ATRP living polymerization reaction using 2-bromo-2-methylpropanoic acid-2-hydroxyethyl ester having hydroxyl groups at the terminal as an initiator to obtain a compound of formula I having hydroxyl groups and bromo groups at the terminal; 2) The hydroxyl end of the compound shown in the formula I reacts with perylene fluorescent dye shown in the formula II to obtain a compound shown in the formula III; 3) The bromine end in the compound shown in the formula III is subjected to an azide reaction to obtain a compound shown in the formula IV, and then the compound is subjected to a click chemical reaction with ATTO647N dye to obtain the double-end fluorescent marked polymer shown in the formula V; Wherein n is 30 to 300 and x is an integer of 1 to 10 in the formula I, II, III, IV.
  3. 3. The method according to claim 2, wherein in the step 1), the polymerization reaction is carried out under the conditions that 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 in a volume ratio of 92.5:7.5; The molar ratio of the initiator to the ligand to the CuBr to the monomer is 1:1:1:20-100, the reaction temperature is 40 o C ~70 o ℃, the reaction time is 4-8 hours, and oxygen is introduced to finish the polymerization reaction; the molecular weight distribution PDI of the compound shown in the formula I is below 1.2.
  4. 4. A method according to claim 2 or 3, characterized in that in step 1) the purification treatment of the system after the polymerization is as follows: Adding methanol containing HBr into the system as a precipitator, stirring, separating out a product, dissolving with acetone, precipitating again, eluting, repeating the above steps for three times to obtain the poly (n-butyl methacrylate) shown in the formula I, and then drying; the methanol containing HBr is prepared by mixing aqueous HBr solution with the volume percentage concentration of 40% with methanol, the volume ratio of the aqueous HBr solution to the methanol is 1:15-25, and the drying temperature is 25 o C~45 o ℃ and the drying is carried out under reduced pressure.
  5. 5. The process according to any one of claims 2 to 4, wherein in step 2) the reaction is carried out in the presence of a catalyst, said catalyst being diisopropyl azodicarboxylate and triphenylphosphine; the reaction is carried out in the presence of a solvent, wherein the solvent is THF; the reaction temperature is 20 o C ~25 o ℃ and the reaction time is 4-8 hours.
  6. 6. The method according to any of claims 2 to 5, characterized in that in step 2) the post-treatment of the reaction is the removal of the perylene fluorescent dye of formula II by gel column chromatography and the detection of the free dye removal by fluorescence correlation spectroscopy.
  7. 7. The method according to any one of claims 2 to 6, wherein in step 3) the azide reaction is performed in the presence of a solvent, the solvent being DMF; The reaction temperature is 20 o C ~25 o ℃ and the reaction time is 8-16 hours.
  8. 8. The method according to any one of claims 2-8, wherein in step 3) the click chemistry reaction is performed 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 chemistry reaction is 20 o C ~25 o ℃ and the time is 24-48 hours.
  9. 9. Use of a double-ended fluorescent tagged polymer of formula V according to claim 1, for characterizing the micro-nano structure of analytical materials.

Description

Double-end fluorescent labeling polymer and preparation method and application thereof Technical Field The invention belongs to the field of basic research of polymer physics and polymer chemistry, and relates to a double-end fluorescent marked polymer and a preparation method and application thereof. Background The microstructure change of the material often has a great influence on the end-use performance of the material, and the Fluorescence Resonance Energy Transfer (FRET) effect is used as a technology capable of quantitatively measuring the distance between two different luminous groups, the measurement accuracy can reach within 10 nm, and the technology is often used for representing the physical and chemical processes such as the change of microstructure conformation in the detection material, the evolution of interaction force and the like. Compared with the traditional ensemble detection method, the single-molecule detection method has the unique advantages of high spatial resolution, for example, compared with the ensemble detection method, only one average value can be obtained, local dynamic information fluctuation along with time in a microstructure can be further seen, microscopic tracking of specific sites and the like can be realized, and therefore research on physical and chemical mechanisms in a plurality of micro-nano fields is realized on the scale of single molecules. Conventional FRET techniques can only calculate FRET efficiency by detecting changes in fluorescence intensity or fluorescence lifetime between fluorescent acceptor and donor dye, and obtain ensemble information such as intermolecular distance changes or interaction forces by further formula fitting. However, with the development of technology, the requirements on the material performance are more and more 'severe', and the material structure is dissected from a more microscopic view, so that the material with higher performance is of great importance. Based on the method, a FRET detection method for a single molecular level is developed, and the FRET technology is combined with a single molecular fluorescence microscopy imaging technology, so that the development of a nano-microstructure of a single molecular scale analysis material is facilitated, a more accurate physicochemical process mechanism is obtained, and a material with better performance is prepared. Disclosure of Invention The invention aims to provide a double-end fluorescent marked polymer and a preparation method and application thereof. According to the invention, two fluorescent dyes with high quantum yield and good photo-bleaching resistance, namely perylene dyes and ATTO647N, are selected as end-capped fluorescent marking groups, are respectively used as a donor and a receptor in the FRET effect, and are combined with a carefully designed chemical synthesis path, so that the tracking of the FRET effect on a single molecular level is realized, the application range of the FRET effect is expanded, and the fluorescent dye has wider application in the basic research fields of surface interfaces, ultrathin films and the like. The structural formula of the double-end fluorescent labeling polymer provided by the invention is shown as formula V: in the formula V, n is 30-300, and x is 1-10. The invention also provides a preparation method of the double-end fluorescent marked polymer shown in the formula V, which comprises the following steps of 1) taking 2-bromo-2-methylpropanoic acid-2-hydroxyethyl ester with hydroxyl at the end of a monomer as an initiator, and carrying out ATRP active polymerization reaction to obtain a compound shown in the formula I with the end blocked by hydroxyl and bromo; In the formula I, n is 30-300, and x is an integer of 1-10; 2) The hydroxyl end of the compound shown in the formula I reacts with perylene fluorescent dye shown in the formula II to obtain a compound shown in the formula III; 3) The bromine end in the compound shown in the formula III is subjected to an azide reaction to obtain a compound shown in the formula IV, and then the compound is subjected to a click chemical reaction with ATTO647N dye to obtain a double-end fluorescent marked polymer shown in the formula V; Wherein n is 30 to 300 and x is an integer of 1 to 10 in the formula I, II, III, IV. In the method, in the step 1), the polymerization reaction is carried out under the conditions that the catalyst is CuBr, the ligand is N, N, N ', N, ' ' N ' ' -pentamethyldiethylenetriamine (PMEDTA for short in English), and the reaction solvent system is a mixed solution of isopropanol and water in a volume ratio of 92.5:7.5; The molar ratio of the initiator to the ligand to the CuBr to the monomer can be 1:1:20-100, the reaction temperature can be 40 oC ~70o C, specifically 40 o C or 70 o C, the reaction time can be 4-8 hours, and oxygen is introduced to finish the polymerization reaction; the molecular weight distribution PDI of the compound shown in the formula I is