阅读说明：本技术 一种具有高亮度、高光稳定性的深红荧光染料及其合成方法 (Deep red fluorescent dye with high brightness and high light stability and synthesis method thereof ) 是由 袁林 任天兵 蒋钢威 张晓兵 于 2021-10-08 设计创作，主要内容包括：本发明公开了一种具有高亮度、高光稳定性的深红荧光染料及其合成方法,其结构式为Ⅰ～Ⅲ中的一种：本发明通过亲核取代及硼氢化钠或氢化铝锂还原,我们可以得到中间体,之后将之与苯酮酸反应得到相应的罗丹明类荧光染料。本发明的染料表现出红移的发射光谱、更好的光稳定性及大的Stokes位移,并且能够在一定程度上抵御微环境的变化(如pH、粘度、蛋白及极性环境),在水溶液中的亮度明显增强。该染料可以被设计生成各种标记试剂或探针用于蛋白质标记及生物标志物的检测。并得益于其较高的量子产率及光稳定性,这些探针或标记试剂能够被用于长时间的先进的成像技术,如STED显微成像等。(The invention discloses a deep red fluorescent dye with high brightness and high light stability and a synthesis method thereof, wherein the structural formula of the deep red fluorescent dye is one of I to III: the invention can obtain an intermediate through nucleophilic substitution and reduction of sodium borohydride or lithium aluminum hydride, and then the intermediate is reacted with phenylketonic acid to obtain corresponding rhodamineA bright fluorescent dye. The dye disclosed by the invention shows a red-shifted emission spectrum, better light stability and large Stokes shift, can resist the change of a microenvironment (such as pH, viscosity, protein and polar environment) to a certain extent, and obviously enhances the brightness in an aqueous solution. The dye can be designed to produce various labeling reagents or probes for protein labeling and biomarker detection. And due to the high quantum yield and light stability of the probe or the labeling reagent, the probe or the labeling reagent can be used for long-term advanced imaging technology, such as STED microscopic imaging and the like.)

1. A deep red fluorescent dye with high brightness and high light stability is characterized in that the structural formula is one of I to III:

wherein: r₁Is one of H, C1-C20 alkyl and substituted alkyl; r₂Is one of H, alkyl, substituted alkyl, aryl and substituted aryl; r₃Is R_3-1、R_3-2And R_3-3One of (1):

R_3-1is one of structural formulas S1-S3:

R_3-2is one of structural formulas S4-S5:

n＝1～10；

R_3-3is one of structural formulas S6-S9:

n1＝1～10，n2＝1～10。

2. the method for preparing a deep red fluorescent dye with high brightness and high light stability according to claim 1, wherein the method for preparing the type I deep red fluorescent dye comprises the following steps:

dissolving the intermediate 1 in a solvent, stirring for dissolving, adding the compound 1, reacting at a set temperature, pouring a reaction solution into ice water after the reaction is finished, adding inorganic acid, precipitating a large amount of solids, sequentially performing suction filtration, washing the solids, drying, separating by a silica gel column, and removing the solvent under reduced pressure to obtain a purple I-type dark red fluorescent dye;

the synthetic technical route is as follows:

3. the method for preparing a deep red fluorescent dye with high brightness and high light stability according to claim 2, wherein the molar ratio of the intermediate 1 to the compound 1 is 1 (1-1.5); the solvent is methanesulfonic acid, and the mass-to-volume ratio of the intermediate 1 to the solvent is 1mmol (5-10) mL; the inorganic acid is one of sulfuric acid, hydrochloric acid, perchloric acid and p-toluenesulfonic acid; setting the temperature to be 85-95 ℃, and the reaction time to be 1-4 h; the silica gel column separation uses dichloroethane and ethanol as 100 (1-10) as eluent.

4. The method for preparing a deep red fluorescent dye with high brightness and high light stability according to claim 1, wherein the method for preparing the type II deep red fluorescent dye comprises the following steps:

dissolving the intermediate 1 in a solvent, stirring for dissolving, adding the compound 2, reacting at a set temperature, pouring a reaction solution into ice water after the reaction is finished, adding inorganic acid, precipitating a large amount of solids, sequentially performing suction filtration, washing the solids, drying, separating by a silica gel column, and removing the solvent under reduced pressure to obtain a purple II-type deep red fluorescent dye;

the synthetic route is as follows:

5. the method for preparing a deep red fluorescent dye with high brightness and high light stability according to claim 4, wherein the molar ratio of the intermediate 1 to the compound 2 is 1 (1-1.5); the solvent is methanesulfonic acid, and the mass-to-volume ratio of the intermediate 1 to the solvent is 1mmol (5-10) mL; the inorganic acid is one of sulfuric acid, hydrochloric acid, perchloric acid and p-toluenesulfonic acid; setting the temperature to be 85-95 ℃, and the reaction time to be 1-4 h; the silica gel column separation uses dichloroethane and ethanol as 100 (1-10) as eluent.

6. The method for preparing a deep red fluorescent dye with high brightness and high light stability according to claim 1, wherein the method for preparing the type III deep red fluorescent dye comprises the following steps:

(1) intermediate 2, compound 3 and BF₃·OEt₂Dissolving in dichloromethane, stirring at room temperature, adding water to the reaction solution to quench the reaction, and mixing the mixture with waterExtracting with dichloromethane, separating organic phase, drying, concentrating, and separating by column chromatography to obtain compound 4;

(2) dissolving the compound 4 in the step (1) in anhydrous tetrahydrofuran, cooling to-78 ℃, adding a tetrahydrofuran solution dissolved with n-butyllithium into the anhydrous tetrahydrofuran solution under the protection of nitrogen, and stirring for reaction at the temperature; then, dissolving dichlorodimethylsilane in a tetrahydrofuran solution, adding the tetrahydrofuran solution into the reaction solution, slowly returning to room temperature, continuously stirring for reaction, adding an HCl solution after the reaction is finished, quenching the reaction, then adding saturated sodium bicarbonate into the reaction solution to neutralize the pH of the solution, and then extracting with dichloromethane; separating the organic phase, drying with anhydrous sodium sulfate, and spin-drying to obtain solid primary product; dissolving the solid primary product in acetone, cooling to 0 ℃, stirring, and adding potassium permanganate into the solution in batches in the next 3 hours; filtering the mixture with diatomite, spin-drying the solution, and performing column chromatography separation to obtain a compound 5;

(3) dissolving the compound 5 in the step (2) in anhydrous tetrahydrofuran, and under the protection of nitrogen, dissolving the compound containing R₂Adding a group format reagent into the mixture, carrying out reflux reaction on the mixture at a set temperature, cooling to room temperature after the reaction is finished, adding an HCl solution into the mixture to quench the reaction, adding saturated sodium bicarbonate into the mixture to neutralize the pH of the solution, extracting with dichloromethane, separating an organic phase, drying with anhydrous sodium sulfate, carrying out spin drying, and separating a solid product by using column chromatography to obtain the III type deep red fluorescent dye;

the synthetic route is as follows:

7. the method for preparing a deep red fluorescent dye with high brightness and high light stability according to claim 6, wherein in the step (1), the intermediate 2, the compound 3 and BF are used₃·OEt₂The molar ratio of (1) (0.8-1.2) to (1.8-2.2), and the reaction time of 20E to E28 h; in the step (2), the molar ratio of the compound 4, n-butyllithium and dichlorodimethylsilane is 1 (2.0-2.4) to 1-3, and the molar ratio of the compound 4 to potassium permanganate is 1 (3-5); stirring for 15-25 min, and continuously stirring for 0.5-1.5 h; the concentration of the HCl solution is 1.5-2.5M; in the step (3), the compound 5 and the compound containing R₂The molar ratio of the group format reagent is 1 (9-11); the set temperature is 75-85 ℃, the reflux reaction time is 1.5-2.5 h, and the concentration of the HCl solution is 1.5-2.5M.

8. The method for preparing a deep red fluorescent dye with high brightness and high light stability according to any one of claims 2 to 7, wherein the method for preparing the intermediate 1 or 2 comprises the following steps:

when R is₃Is R_3-1The synthetic route is as follows:

the method specifically comprises the following steps:

dissolving Compound 6 or Compound 7 in acetonitrile, and adding the corresponding R thereto_3-1Performing reflux reaction on the bromine substituent and potassium carbonate for 1-4 h, removing the solvent under reduced pressure after the reaction is finished, separating by using a silica gel column, removing the solvent under reduced pressure by using petroleum ether and ethyl acetate which are 10:1 as eluent, and correspondingly obtaining a colorless oily liquid R_3-1Intermediate 1 or R_3-1Intermediate 2;

when R is₃Is R_3-2The synthetic route is as follows:

the method specifically comprises the following steps:

dissolving a compound 6 or a compound 7 in tetrahydrofuran, stirring, dropwise adding trifluoroacetic anhydride, reacting at room temperature for 5-10 min, and performing spin drying after the reaction is finished; in thatAdding tetrahydrofuran for dissolving, adding sodium borohydride and boron trifluoride diethyl etherate, refluxing for 1-3 h, cooling to room temperature after the reaction is finished, adding water for quenching reaction, extracting with ethyl acetate, drying the organic phase, removing the solvent under reduced pressure, and separating by a silica gel column; removing the solvent under reduced pressure by using petroleum ether and ethyl acetate as eluent to obtain a colorless oily liquid R_3-2Intermediate 1 or R_3-2Intermediate 2;

when R is₃Is R_3-3The synthetic route is as follows:

the method specifically comprises the following steps:

dissolving a compound 6 or a compound 7 in acetonitrile, stirring, adding potassium carbonate and bromoacetonitrile or 2-bromo-N-alkyl acetamide or N, N-dialkyl-bromoacetamide, refluxing for 1-4 h, after the reaction is finished, removing the solvent under reduced pressure, separating by a silica gel column, dissolving the product obtained by separation in tetrahydrofuran, adding sodium borohydride and boron trifluoride diethyl etherate, refluxing for 1-3 h, after the reaction is finished, cooling to room temperature, adding water to quench the reaction, extracting by ethyl acetate, drying the organic phase, removing the solvent under reduced pressure, separating by the silica gel column, removing the solvent under reduced pressure by using petroleum ether and ethyl acetate 1:1 as an eluent, and obtaining a colorless or light yellow oily liquid R correspondingly_3-3Intermediate 1 or R_3-3Intermediate 1.

9. The method for preparing a deep red fluorescent dye with high brightness and high light stability according to claim 8, wherein R is the number of moles of the compound₃Is R_3-1When compounds 6 or 7 are reacted with R_3-1The molar ratio of the bromine substituent is 1 (1.5-2); the molar ratio of the compound 6 or 7 to the potassium carbonate is 1: 3; the mass-to-volume ratio of the compound 6 or 7 to acetonitrile is 1g (10-30) mL;

when R is₃Is R_3-2When the molar ratio of the compound 6 or 7 to the trifluoroacetic anhydride is 1 (1.5-3); the molar weight of sodium borohydride and boron trifluoride diethyl etherate added later3-10 times of the compound 6 or 7; the mass-to-volume ratio of the compound 6 or 7 to tetrahydrofuran is 1g (10-30) mL;

when R is₃Is R_3-3When the molar ratio of the compound 6 or 7 to bromoacetonitrile or 2-bromo-N-alkyl acetamide or N, N-dialkyl-bromoacetamide is 1 (1.5-3), the molar amount of sodium borohydride and boron trifluoride ethyl ether is 8-bromo-1, 2,3,3a,4, 5-hexahydropyrrole [1,2-a ]]3-10 times of quinoxaline; the mass-to-volume ratio of the compound 6 or 7 to acetonitrile or tetrahydrofuran is 1g (10-30) mL.

10. The use of the high brightness, high light stability deep red fluorescent dye according to claim 1 in a variety of bioluminescent imaging applications, luminescent materials, fluorescent probes and multi-color imaging.

Technical Field

The invention belongs to the field of fluorescent dyes, and particularly relates to a deep red fluorescent dye with high brightness and high light stability and a synthesis method thereof.

Background

Fluorescence imaging techniques play an important role in many fields of research of life processes, and the development of high-resolution imaging techniques enables one to visualize molecular structures at the nano-and even single-molecule level in cells, which is crucial for understanding the progress of life processes and cell biology. The implementation of this technique relies on a sensitive and stable fluorescence signal output. The difference in fluorescence properties of chromophores seriously affects the imaging quality, wherein fluorescence intensity and light stability are two important indicators for determining whether a chromophore can be used in advanced imaging technology.

As a chromophore with excellent properties, small-molecule fluorescent dyes have been widely used in the fields of cell staining, biomolecular labeling, analysis, clinical diagnosis, and the like. However, the development of bright and light-stable fluorescent dyes has still been seriously delayed compared to the increasing innovation of imaging technology, which hinders the acquisition of high-quality imaging information. Although various methods for increasing the brightness of dyes have been developed and dyes developed using these methods can be used for super-resolution imaging, their photostability in long-term super-resolution imaging remains to be examined. The development of high-brightness and high-light-stability small-molecule fluorescent dyes which can be used for long-time super-resolution imaging has important value in the aspects of tracing the change of molecular structures in cells and obtaining the visualization of 3D structures of intracellular organelles.

Disclosure of Invention

The invention aims to provide a deep red fluorescent dye with high brightness and high light stability and a synthesis method thereof, wherein the fluorescent dye has higher fluorescence quantum yield in aqueous solution, so that the change of a molecular structure in a cell can be traced.

The deep red fluorescent dye with high brightness and high light stability has one of structural formulas I to III:

wherein: r₁Is one of H, C1-C20 alkyl and substituted alkyl; r₂Is one of H, alkyl, substituted alkyl, aryl and substituted aryl; r₃Is R_3-1、R_3-2And R_3-3One of (1):

R_3-1is one of structural formulas S1-S3:

R_3-2is one of structural formulas S4-S5:

R_3-3is one of structural formulas S6-S9:

n₁＝1～10，n₂＝1～10。

the preparation method of the I-type deep red fluorescent dye comprises the following steps:

intermediate 1 (8-methoxy-5-substituent R)₃-1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxaline) solutionDissolving in solvent under stirring, adding compound 1 (2-substituent R)₂Keto-4-disubstituted R₁Aminophenol) is reacted at a set temperature, after the reaction is finished, the reaction liquid is poured into ice water, inorganic acid is added, a large amount of solid is separated out, suction filtration, solid washing, drying, silica gel column separation and solvent removal under reduced pressure are sequentially carried out, and purple I-type dark red fluorescent dye is obtained;

the synthetic technical route is as follows:

the molar ratio of the intermediate 1 to the compound 1 is 1 (1-1.5); the solvent is methanesulfonic acid, and the mass-to-volume ratio of the intermediate 1 to the solvent is 1mmol (5-10) mL; the inorganic acid is one of sulfuric acid, hydrochloric acid, perchloric acid and p-toluenesulfonic acid; setting the temperature to be 85-95 ℃, and the reaction time to be 1-4 h; the silica gel column separation uses dichloroethane, ethanol 100 (1-5) as an eluent.

The preparation method of the II type deep red fluorescent dye comprises the following steps:

intermediate 1 (8-methoxy-5-substituent R)₃-1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxaline) in a solvent, dissolving with stirring, adding thereto the compound 1 (2-substituent R)₂Resorcinol), reacting at a set temperature, pouring the reaction liquid into ice water after the reaction is finished, adding inorganic acid to separate out a large amount of solids, sequentially performing suction filtration, washing the solids, drying, separating by a silica gel column, and removing the solvent under reduced pressure to obtain purple II-type deep red fluorescent dye;

the synthetic route is as follows:

the molar ratio of the intermediate 1 to the compound 2 is 1 (1-1.5); the solvent is methanesulfonic acid, and the mass-to-volume ratio of the intermediate 1 to the solvent is 1mmol (5-10) mL; the inorganic acid is one of sulfuric acid, hydrochloric acid, perchloric acid and p-toluenesulfonic acid; setting the temperature to be 85-95 ℃, and the reaction time to be 1-4 h; the silica gel column separation uses dichloroethane, ethanol 100 (1-5) as an eluent.

The preparation method of the III type deep red fluorescent dye comprises the following steps:

(1) intermediate 2 (8-bromo-5-substituent R)_3-3-1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxaline), compound 3 (2-bromo-4- (disubstituted alkyl R)₁Amino) benzyl alcohol) and BF₃·OEt₂Dissolving in dichloromethane, stirring at room temperature for reaction, adding water into the reaction solution to quench the reaction after the reaction is finished, extracting the mixture with dichloromethane, separating an organic phase, drying, concentrating, and performing column chromatography to obtain a compound 4 (3-bromo-4- ((8-bromo-5-substituent R)_3-3-1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxalin-7-yl) methyl) -N, N-disubstituted alkyl radicals R₁Amines);

(2) dissolving the compound 4 in the step (1) in anhydrous tetrahydrofuran, cooling to-78 ℃, adding a tetrahydrofuran solution dissolved with n-butyllithium into the anhydrous tetrahydrofuran solution under the protection of nitrogen, and stirring for reaction at the temperature; then, dissolving dichlorodimethylsilane in a tetrahydrofuran solution, adding the tetrahydrofuran solution into the reaction solution, slowly returning to room temperature, continuously stirring for reaction, adding an HCl solution after the reaction is finished, quenching the reaction, then adding saturated sodium bicarbonate into the reaction solution to neutralize the pH of the solution, and then extracting with dichloromethane; separating the organic phase, drying with anhydrous sodium sulfate, and spin-drying to obtain solid primary product; dissolving the solid primary product in acetone, cooling to 0 ℃, stirring, and adding potassium permanganate into the solution in batches in the next 3 hours; filtering the mixture with diatomite, spin-drying the solution, and separating by column chromatography to obtain compound 5(10- (disubstituted alkyl R)₁Amino) -5-substituent R_3-3-12, 12-dialkyl-1, 2,3,3a,4, 5-hexahydrobenzo [5,6 ]]Silicon group [3,2-g ]]Pyrrole [1,3-a ]]Quinoxalin-7 (12H) -one);

(3) dissolving the compound 5 in the step (2) in anhydrous tetrahydrofuran, and under the protection of nitrogen, dissolving the compound containing R₂Adding a radical form reagent, refluxing the mixture at a set temperature, and reactingAfter completion, the reaction mixture was cooled to room temperature, HCl solution was added thereto to quench the reaction, then saturated sodium bicarbonate was added to the mixture to neutralize the pH of the solution, extraction was performed with methylene chloride, the organic phase was separated, dried over anhydrous sodium sulfate, spin-dried, and the solid product was separated by column chromatography to obtain a type III deep red fluorescent dye (5-substituent R)_3-3-N, N-dialkyl R₁-12, 12-dimethyl-7-R₂-1,2,3,3a,4,5,10, 12-octahydrobenzo [5,6 ]]Silicon [3,2-g]Pyrrole [1,2-a ]]Quinoxaline-10-amine).

The synthetic route is as follows:

in the step (1), the intermediate 2, the compound 3 and BF₃·OEt₂The molar ratio of (1) (0.8-1.2) to (1.8-2.2), and the reaction time is 20-28 h;

in the step (2), the molar ratio of the compound 4, n-butyllithium and dichlorodimethylsilane is 1 (2.0-2.4) to 1-3, and the molar ratio of the compound 4 to potassium permanganate is 1 (3-5); stirring for 15-25 min, and continuously stirring for 0.5-1.5 h; the concentration of the HCl solution is 1.5-2.5M;

in the step (3), the compound 5 and the compound containing R₂The molar ratio of the group format reagent is 1 (9-11); the set temperature is 75-85 ℃, the reflux reaction time is 1.5-2.5 h, and the concentration of the HCl solution is 1.5-2.5M.

The preparation method of the intermediate 1 or 2 comprises the following steps:

when R is₃Is R_3-1The synthetic route is as follows:

the method specifically comprises the following steps:

the compound 6 (8-methoxy-1, 2,3,3a,4, 5-hexahydropyrrole [1, 2-a)]Quinoxaline) or compound 7 (8-bromo-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxaline) in acetonitrileTo which the corresponding R is added_3-1Performing reflux reaction on the bromine substituent and potassium carbonate for 1-4 h, decompressing to remove the solvent after the reaction is finished, separating by using a silica gel column, removing the solvent under reduced pressure by using petroleum ether and ethyl acetate which are 10:1 as eluent, and obtaining a colorless oily liquid R correspondingly_3-1Intermediate 1 (8-methoxy-5-substituent R)_3-1-1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxaline) or R_3-1Intermediate 2 (8-bromo-5-substituent R)_3-1-1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxaline);

when R is₃Is R_3-2The synthetic route is as follows:

the method specifically comprises the following steps:

dissolving a compound 6 or a compound 7 in tetrahydrofuran, stirring, dropwise adding trifluoroacetic anhydride, reacting at room temperature for 5-10 min, and performing spin drying after the reaction is finished; adding tetrahydrofuran for dissolving, adding sodium borohydride and boron trifluoride diethyl etherate, refluxing for 1-3 h, cooling to room temperature after the reaction is finished, adding water for quenching reaction, extracting with ethyl acetate, drying the organic phase, removing the solvent under reduced pressure, and separating by a silica gel column; the method comprises the following steps of (1) mixing petroleum ether: ethyl acetate 10:1 as eluent, and removing the solvent under reduced pressure to obtain colorless oily liquid R_3-2Intermediate 1 (8-methoxy-5-R)_3-21,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] yl]Quinoxaline) or R_3-2Intermediate 2 (8-bromo-5-R)_3-21,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] yl]Quinoxaline); wherein the content of the first and second substances,

when R is₃Is R_3-3The synthetic route is as follows:

the method specifically comprises the following steps:

dissolving compound 6 or 7 in acetonitrile, stirring, adding potassium carbonate and bromoacetonitrile or 2-bromo-N-alkylacetoacetateRefluxing amine or N, N-dialkyl-bromoacetamide for 1-4 h, decompressing to remove a solvent after the reaction is finished, separating by using a silica gel column, dissolving a product obtained by separation in tetrahydrofuran, adding sodium borohydride and boron trifluoride diethyl etherate, refluxing for 1-3 h, cooling to room temperature after the reaction is finished, adding water to quench the reaction, extracting by using ethyl acetate, drying an organic phase, decompressing to remove the solvent, separating by using the silica gel column, removing the solvent by using petroleum ether and ethyl acetate which are 1:1 as an eluent under reduced pressure to obtain a colorless or light yellow oily liquid R_3-3Intermediate 1 (8-methoxy-5-R)_3-3-1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxaline) or R_3-3Intermediate 1 (8-bromo-5-R)_3-3-1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ]]Quinoxaline);

when R is₃Is R_3-1When compounds 6 or 7 are reacted with R_3-1The molar ratio of the bromine substituent is 1 (1.5-2); the molar ratio of the compound 6 or 7 to the potassium carbonate is 1: 3; the mass-to-volume ratio of the compound 6 or 7 to the acetonitrile is 1g (10-30) mL;

when R is₃Is R_3-2When the mole of the compound 6 or 7 and the trifluoroacetic anhydride is 1 (1.5-3); the molar weight of the added sodium borohydride and boron trifluoride diethyl etherate is 3-10 times that of the compound 6 or 7; the mass-to-volume ratio of the compound 6 or 7 to tetrahydrofuran is 1g (10-30) mL;

when R is₃Is R_3-3When the molar ratio of the compound 6 or 7 to bromoacetonitrile or 2-bromo-N-alkyl acetamide or N, N-dialkyl-bromoacetamide is 1 (1.5-3), the molar amount of sodium borohydride and boron trifluoride ethyl ether is 8-bromo-1, 2,3,3a,4, 5-hexahydropyrrole [1,2-a ]]3-10 times of quinoxaline; the mass-to-volume ratio of the compound 6 or 7 to acetonitrile or tetrahydrofuran is 1g (10-30) mL.

The deep red fluorescent dye with high brightness and high light stability is applied to the fields of various biological fluorescence imaging, luminescent materials, fluorescent probes and multicolor imaging, wherein: the biological fluorescence imaging field is focusing imaging, super-resolution imaging, living body imaging and the like.

The invention has the beneficial effects that:

1) the fluorescent dye has the advantages of cheap synthetic raw materials, simple method, easy modification and functionalization and the like.

2) The fluorine-containing substituted fluorescent dye has an absorption spectrum in an aqueous solution of 550-720nm and an emission spectrum of 600-830nm, and belongs to a far-red-near-infrared spectral region.

3) The fluorine-containing substituted fluorescent dye has high fluorescence quantum yield (more than 0.6) in aqueous solution and proper Stokes shift (more than 60nm), and can effectively reduce the intensity of used laser, reduce the interference of self-quenching and improve the signal-to-noise ratio of imaging when applied to the field of biological imaging, thereby improving the sensitivity of imaging.

4) The fluorine-containing substituted fluorescent dye provided by the invention shows strong anti-solvent effect on pH, polarity, viscosity and protein environments, and can keep high fluorescence brightness in different environments, so that a fluorescence signal obtained when the fluorine-containing substituted fluorescent dye is applied is more accurate.

5) The fluorine-containing substituted fluorescent dye has good light stability, can provide more accurate fluorescent signals during imaging, and can be used in advanced imaging technical fields such as long-time super-resolution imaging.

6) The present invention is based on the DQF dyes of the previous design invention by introducing different electron withdrawing groups (R) on the N atom in the 3-position of the phenazine fused structure₃) An intermediate can be obtained through nucleophilic substitution and reduction of sodium borohydride or lithium aluminum hydride, and then the intermediate is reacted with phenylketo acid to obtain the corresponding rhodamine fluorescent dye. Compared with the classical fluorescent dye (Rhodamine, Rhodol, coumarin, Boranil), the dye of the invention shows a red-shifted emission spectrum, better light stability and large Stokes shift, can resist the change of microenvironment (such as pH, viscosity, protein and polar environment) to a certain extent, and obviously enhances the brightness in aqueous solution. The dye can be designed to produce various labeling reagents or probes for protein labeling and biomarker detection. And due to the high quantum yield and light stability of the probe or the labeling reagent, the probe or the labeling reagent can be used for long-term advanced imaging technology, such as STED microscopic imaging and the like.

Drawings

FIG. 1 nuclear magnetic spectrum hydrogen spectrum of BDQ-3 prepared in example 1.

FIG. 2 nuclear magnetic spectrum carbon spectrum of BDQ-3 prepared in example 1.

FIG. 3 nuclear magnetic spectrum hydrogen spectrum of BDQ-6 prepared in example 2.

FIG. 4 nuclear magnetic spectrum carbon spectrum of BDQ-6 prepared in example 2.

FIG. 5 nuclear magnetic hydrogen spectrum of BDQ-8 prepared in example 3.

FIG. 6 nuclear magnetic spectrum carbon spectrum of BDQ-8 prepared in example 3.

FIG. 7 nuclear magnetic spectrum hydrogen spectrum of BDQF-3 prepared in example 4.

FIG. 8 nuclear magnetic spectrum carbon spectrum of BDQF-3 prepared in example 4.

FIG. 9 nuclear magnetic spectrum hydrogen spectrum of BDQF-6 prepared in example 4.

FIG. 10 nuclear magnetic spectrum carbon spectrum of BDQF-6 prepared in example 4.

FIG. 11 nuclear magnetic hydrogen spectrum of BDQF-8 prepared in example 4.

FIG. 12 nuclear magnetic spectrum carbon spectrum of BDQF-8 prepared in example 4.

FIG. 13 nuclear magnetic hydrogen spectrum of BDQF-10 prepared in example 5.

FIG. 14 nuclear magnetic spectrum carbon spectrum of BDQF-10 prepared in example 5.

FIG. 15 nuclear magnetic hydrogen spectrum of BDQF-12 prepared in example 6.

FIG. 16 nuclear magnetic spectrum carbon spectrum of BDQF-12 prepared in example 6.

FIG. 17 UV absorption and fluorescence spectra of BDQF-6 tested in example 7 in four solvents and its optical properties.

FIG. 18 UV and fluorescence spectra of BDQF-6 and RhB tested in example 8 normalized in PBS.

FIG. 19 comparison of fluorescence properties of DQF and BDQF dyes with different substituents in PBS in example 9.

FIG. 20 change of fluorescence intensity of BDQF-6 tested in example 10 with time under 1W 530nm laser irradiation.

FIG. 21 contrast the imaging brightness of BDQF-6-Halo and RhB-Halo tested in example 11 when fully labeled in cells.

FIG. 22 comparison of the photostability of BDQF-6-Halo tested in example 12 with that of RhB-Halo when fully labeled in cells.

Detailed Description

Example 1

R₃The substituent is R_3-1Wherein R is N, N-dimethylacetoamino group of (1)_3-1The preparation steps of the intermediate 1(BDQ-3) are as follows:

synthesis of 8-methoxy-5- (N, N-dimethylacetylamino) -1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline (BDQ-3): 1.5mmol of 8-methoxy-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline is dissolved in 10ml of acetonitrile, 1.8mmol of N, N-dimethyl bromoacetamide and 2.2mmol of potassium carbonate are then added thereto, reflux is carried out for 2h, and after the reaction is completed, the solvent is removed under reduced pressure and separation is carried out by means of a silica gel column. The method comprises the following steps of (1) mixing petroleum ether: ethyl acetate 10:1 as eluent, and removing the solvent under reduced pressure to obtain BDQ-3 (8-methoxy-5- (N, N-dimethylacetylamino) -1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline) as a colorless oily liquid, which has the following structural formula:

the results of nuclear magnetic testing of the prepared BDQ-3 are shown in FIG. 1 and FIG. 2, and are as follows:

¹H NMR(400MHz,Chloroform-d)δ6.43(d,J＝8.2Hz,1H),6.09(s,2H),3.99(s,2H),3.77(s,3H),3.35(s,3H),3.05(d,J＝37.9Hz,8H),2.10(dd,J＝11.3,6.1Hz,2H),2.03–1.94(m,1H),1.47(d,J＝7.6Hz,1H).¹³C NMR(100MHz,CDCl3)δ169.90,154.02,137.05,127.91,111.55,99.36,98.43,56.79,55.64,53.61,47.88,36.97,35.87,30.26,23.74.MALDI-TOF/MS,m/z:calc 289.18,found 289.22.

as can be seen from the nuclear magnetic spectrum, the result is completely consistent with the structural formula of BDQ-3.

Example 2

R₃The substituent is R_3-2Wherein R is trifluoroethyl in (1)_3-2The preparation of intermediate 1(BDQ-6) was as follows:

synthesis of 8-methoxy-5-trifluoroethyl-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline (BDQ-6): dissolving 1.5mmol of 8-methoxy-1, 2,3,3a,4, 5-hexahydropyrrole [1,2-a ] quinoxaline in 10ml of tetrahydrofuran, stirring, dropwise adding 1.8mmol of trifluoroacetic anhydride, reacting at room temperature for 10min, and after the reaction is finished, spin-drying; adding 15ml of tetrahydrofuran for dissolving, adding 7.5mmol of sodium borohydride and 7.5mmol of boron trifluoride diethyl etherate, refluxing for 1.5h, cooling to room temperature after the reaction is finished, adding water for quenching reaction, extracting with ethyl acetate, drying an organic phase, removing the solvent under reduced pressure, separating by a silica gel column, and removing the solvent under reduced pressure by using petroleum ether and ethyl acetate which are 10:1 as an eluent to obtain BDQ-6 (8-methoxy-1, 2,3,3a,4, 5-hexahydropyrrole [1,2-a ] quinoxaline) of colorless oily liquid, wherein the structural formula is as follows:

the results of nuclear magnetic testing of the prepared BDQ-6 are shown in FIGS. 3 and 4, and are as follows:

¹H NMR(400MHz,Chloroform-d)δ6.66(d,J＝8.6Hz,1H),6.20(dd,J＝8.6,2.4Hz,1H),6.13–6.07(m,1H),3.76(d,J＝7.5Hz,4H),3.64(dd,J＝16.1,8.9Hz,1H),3.49–3.36(m,2H),3.30(t,J＝8.0Hz,2H),2.86(t,J＝10.2Hz,1H),2.09(dt,J＝10.9,6.3Hz,2H),1.97(q,J＝8.2,7.8Hz,1H),1.49–1.38(m,1H).¹³C NMR(100MHz,Chloroform-d)δ154.38,136.74,126.93,124.23,114.10,100.08,98.57,55.51,54.56,54.02,47.29,29.81,23.19.ESI/MS,m/z:calc 286.13,found 286.13.

as can be seen from the nuclear magnetic spectrum, the result is completely consistent with the structural formula of BDQ-6.

Example 3

R₃The substituent is R_3-32-aminoethyl of (2), wherein R_3-3The preparation of intermediate 1(BDQ-8) was as follows:

1.5mmol of 8-methoxy-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline is dissolved in 10ml of acetonitrile, stirred, 4.5mmol of potassium carbonate and 1.7mmol of bromoacetonitrile are added thereto, refluxed for 4 hours, after the reaction is completed, the solvent is removed under reduced pressure, silica gel column separation is carried out, the product obtained by the separation is dissolved in tetrahydrofuran, sodium borohydride and boron trifluoride diethyl etherate are added thereto, and refluxed for 3 hours. Then cooling the reaction system to room temperature, adding 10mL of water to quench the reaction, adjusting the solution to be strongly alkaline, extracting the solution for three times by using ethyl acetate, drying an organic phase, spinning, and separating by using a silica gel column. The solvent was removed under reduced pressure using petroleum ether and ethyl acetate 1:1 as eluent to give BDQ-8(2- (8-methoxy-2, 3,3a, 4-tetrahydropyrrole [1,2-a ] quinoxalin-5 (1H) -yl) ethyl-1-amine) as a colorless or pale yellow oil.

The results of nuclear magnetic testing of the prepared BDQ-8 are shown in FIGS. 5 and 6, which are as follows:

¹H NMR(400MHz,Chloroform-d)δ6.54(d,J＝8.6Hz,1H),6.14(dd,J＝8.5,

2.3Hz,1H),6.06(s,1H),4.00(s,2H),3.75(s,3H),3.58(dt,J＝13.4,6.9Hz,1H),3.35(dt,J＝20.3,10.2Hz,3H),3.27–3.15(m,2H),3.05(s,1H),2.85(dd,J＝12.7,6.6Hz,1H),2.53(t,J＝10.4Hz,1H),2.07(dt,J＝16.8,8.3Hz,2H),2.00–1.91(m,1H),1.43–1.36(m,1H).¹³C NMR(100MHz,Chloroform-d)δ153.71,137.21,128.65,112.60,99.60,98.48,77.23,56.22,56.15,55.73,53.29,47.77,39.42,30.25,23.65.

as can be seen from the nuclear magnetic spectrum, the result is completely consistent with the structural formula of BDQ-6.

Example 4

Synthesis of fluorescent dye of phenazine fused structure type i: 0.35mmol of 8-methoxy-5- (N, N-dimethylacetoamino) -1,2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline (BDQ-3 prepared in example 1) or 8-methoxy-5-trifluoroethyl-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline (BDQ-6 prepared in example 2) or 2- (8-methoxy-2, 3,3a, 4-tetrahydropyrrole [1,2-a ] quinoxalin-5 (1H) -yl) ethyl-1-amine (BDQ-8 prepared in example 3) is dissolved in 2ml of methanesulfonic acid, dissolved with stirring, to which 0.38mmol of 4-diethylaminoketo acid is added, the reaction was carried out at 90 ℃ for 2 h. Then pouring the reaction solution into ice water, adding 0.5ml of perchloric acid, separating out a large amount of solid, carrying out suction filtration, washing the solid, drying, and carrying out silica gel column separation; and (3) taking dichloroethane and ethanol as 100 (1-5) as an eluent, and removing the solvent under reduced pressure to obtain BDQF-3, BDQF-6 or BDQF-8 correspondingly, wherein the structural formula is shown as follows:

the results of nuclear magnetic tests on the prepared BDQF- (3, 6, 8) are shown in FIGS. 7-12, which are as follows:

BDQF-3：¹H NMR(400MHz,Methanol-d₄)δ8.47(t,J＝7.3Hz,1H),7.98–7.87(m,2H),7.43(t,J＝7.1Hz,1H),7.25(t,J＝9.0Hz,1H),7.02(d,J＝9.2Hz,1H),6.98(s,1H),6.81(s,1H),5.70(d,J＝7.6Hz,1H),4.26(t,J＝9.9Hz,1H),4.05–3.99(m,1H),3.97–3.87(m,2H),3.72(d,J＝7.2Hz,6H),3.53–3.46(m,1H),2.98–2.87(m,6H),2.47–2.35(m,2H),2.35–2.20(m,1H),1.79–1.70(m,1H),1.42(t,J＝6.9Hz,6H).¹³C NMR(100MHz,Methanol-d₄)δ169.23,167.80,167.23,157.28,156.19,155.62,154.43,147.48,135.50,134.89,133.66,133.20,132.11,131.68,131.23,130.65,115.95,114.29,104.10,96.68,95.72,58.97,53.27,52.81,46.26,36.74,36.04,30.57,23.67,12.90.HRMS(ESI):m/z calc.for C33H37N4O4[M]553.2809；found 553.2814.

BDQF-6：¹H NMR(400MHz,Methanol-d₄)δ8.43–8.36(m,1H),7.85(dt,J＝21.6,7.5Hz,2H),7.38(t,J＝8.4Hz,1H),7.21(t,J＝9.4Hz,1H),7.02–6.91(m,2H),6.80(s,1H),6.27(d,J＝7.5Hz,1H),3.97–3.86(m,2H),3.85–3.77(m,3H),3.68(q,J＝6.8Hz,5H),3.17(t,J＝10.8Hz,1H),2.37(dd,J＝11.9,6.2Hz,2H),2.29–2.14(m,1H),1.69(p,J＝11.7Hz,1H),1.37(t,J＝7.0Hz,6H).¹³C NMR(100MHz,Methanol-d₄)δ167.00,156.73,156.59,154.47,154.03,146.26,134.12,132.72,132.50,131.27,130.98,130.12,130.03,129.96,114.59,113.64,113.41,104.95,95.75,95.29,57.45,57.42,53.39,51.81,45.40,29.55,22.66,11.94.MALDI-TOF/MS,m/z:calc 550.23,found 550.17.

BDQF-8:¹H NMR(400MHz,Methanol-d₄)δ8.11(t,J＝8.7Hz,1H),7.68(dq,J＝13.5,6.8Hz,2H),7.44(dd,J＝19.3,9.5Hz,1H),7.29(dd,J＝20.3,7.2Hz,1H),7.03(t,J＝8.5Hz,1H),6.92(s,1H),6.75(s,1H),6.41(d,J＝13.6Hz,1H),3.82(t,J＝10.3Hz,1H),3.74–3.65(m,7H),3.28(p,J＝7.1,6.4Hz,1H),3.13–2.98(m,2H),2.37(dd,J＝9.9,5.6Hz,2H),2.25(dd,J＝22.3,8.1Hz,1H),2.01(s,2H),1.68(d,J＝8.4Hz,1H),1.50(d,J＝23.1Hz,2H),1.39(d,J＝6.5Hz,6H).¹³C NMR(100MHz,Methanol-d₄)δ157.46,156.61,156.36,155.91,154.79,147.98,134.56,134.46,133.55,132.18,132.00,131.25,131.05,130.96,115.55,114.46,104.99,96.71,96.09,58.93,52.24,49.15,47.77,46.29,30.38,23.64,14.45,12.90,9.28.

as can be seen from the nuclear magnetic spectrum, the result is completely consistent with the BDQF- (3, 6, 8) structural formula.

Example 5

Synthesis of fluorescent dye of type ii phenazine fused structure: 0.35mmol of 8-methoxy-5-trifluoroethyl-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline (BDQ-6 prepared in example 2) was dissolved in 2ml of methanesulfonic acid with stirring, and 0.38mmol of resorcinol was added thereto and reacted at 90 ℃ for 2 hours. Then pouring the reaction solution into ice water, adding 0.5ml of perchloric acid, separating out a large amount of solid, carrying out suction filtration, washing the solid, drying, and carrying out silica gel column separation; with ethylene dichloride: and (1-5) as an eluent, and removing the solvent under reduced pressure to obtain BDQF-10, wherein the structural formula is shown as follows:

the results of nuclear magnetic testing of the prepared BDQF-10 are shown in FIGS. 13 and 14, which are as follows:

¹H NMR(400MHz,Methanol-d₄)δ8.32(s,1H),7.77(dt,J＝14.6,7.1Hz,2H),7.32(t,J＝7.4Hz,1H),7.16(t,J＝8.6Hz,1H),7.07(s,1H),6.92(d,J＝8.4Hz,1H),6.80(s,1H),6.21(d,J＝7.0Hz,1H),3.83(dt,J＝39.2,16.2Hz,5H),3.66(s,1H),3.21–3.06(m,1H),2.26(s,2H),2.11(s,1H),1.72–1.51(m,1H).¹³C NMR(100MHz,Methanol-d₄)δ166.96,165.26,156.67,156.05,155.26,148.38,134.15,134.03,133.93,132.58,132.47,131.26,130.12,129.99,129.91,129.84,116.88,115.36,103.93,101.91,95.41,58.00,51.73,51.51,48.97,29.38,22.46.MALDI-TOF/MS,m/z:calc 494.15,found 495.10.[M+H]^–

as can be seen from the nuclear magnetic spectrum, the result is completely consistent with the BDQF-10 structural formula.

Example 6

The starting material from example 2 was exchanged for 8-bromo-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline to give intermediate 2 (BDQ-6') from 8-methoxy-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline.

Synthesis of fluorescent dyes of type III phenazine fused Structure:

(1) 0.9mmol of 8-bromo-5-trifluoroethyl-1, 2,3,3a,4, 5-hexahydropyrrolo [1,2-a ] quinoxaline (BDQ-6'), 1.0mmol of 2-bromo-4- (N, N-dimethylamino) benzyl alcohol and 1.5mmol of BF 3. OEt2 are dissolved in 10ml of dichloromethane and stirred at room temperature for 24 h. Water was added thereto to quench the reaction, the mixture was extracted with dichloromethane, the organic phase was separated, dried, concentrated, and separated by column chromatography to give intermediate 1.

(2)1mmol of intermediate 1 was dissolved in 5ml of anhydrous tetrahydrofuran and cooled to-78 ℃. A solution of 1M n-butyllithium (3mmol) in tetrahydrofuran was added thereto under nitrogen protection, and stirred at that temperature for 20 min. Thereafter, 3mmol of dichlorodimethylsilane was dissolved in anhydrous tetrahydrofuran and added to the above solution, slowly returned to room temperature, and stirred for 1 h. 5ml of 2M HCl solution was added to the above solution to quench the reaction. Saturated sodium bicarbonate was added to the mixture to neutralize the solution pH and extracted with dichloromethane. The organic phase was separated, dried over anhydrous sodium sulfate and spin dried. The residue was dissolved in acetone and cooled to 0 ℃ and stirred, 3mmol of potassium permanganate were added to it in portions over the next 3 h. Filtering the mixture by using kieselguhr, spin-drying the solution, and separating by column chromatography to obtain an intermediate product 2.

(3) 0.5mmol of intermediate 2 was dissolved in 5ml of dry tetrahydrofuran, 1.5mmol of o-tolyl magnesium chloride were added thereto under nitrogen protection, and the mixture was refluxed at 80 ℃ for 2 h. Cooled to room temperature and 2mmol of 2M HCl solution was added to the above solution to quench the reaction. Saturated sodium bicarbonate was added to the mixture to neutralize the solution pH and extracted with dichloromethane. The organic phase is separated, dried by anhydrous sodium sulfate, dried by spinning and separated by column chromatography to obtain BDQF-12, the structural formula of which is shown as follows:

the results of the prepared BDQF-12 line nuclear magnetic tests are shown in FIG. 15 and FIG. 16, which are specific as follows:

¹H NMR(400MHz,Methanol-d₄)δ7.49–7.33(m,3H),7.28(s,1H),7.15–7.05(m,3H),6.70(d,J＝9.0Hz,1H),6.32(d,J＝3.9Hz,1H),3.97(t,J＝10.9Hz,1H),3.82(dd,J＝23.5,13.8Hz,2H),3.72(d,J＝11.8Hz,1H),3.58(dd,J＝24.0,7.9Hz,2H),3.06(t,J＝10.6Hz,1H),2.34–2.22(m,2H),2.04(d,J＝11.4Hz,3H),1.68–1.58(m,1H),1.49–1.22(m,6H),0.98(t,J＝7.4Hz,1H),0.60(dd,J＝16.1,6.1Hz,6H).¹³C NMR(101MHz,MeOD)δ169.39,154.81,148.20,145.05,144.68,141.34,140.46,136.95,136.78,134.29,134.07,132.33,131.23,130.05,129.86,129.13,126.69,121.55,118.54,114.69,66.69,59.60,53.19,52.80,40.93,30.42,23.94,19.65,14.18.MALDI-TOF/MS,m/z:calc 534.25,found 534.13。

as can be seen from the nuclear magnetic spectrum, the result is completely consistent with the BDQF-12 structural formula.

And (3) performance testing:

the dyes are respectively dissolved in DMSO solution to prepare 1mM mother liquor of different dyes, and test solutions with different concentrations are prepared according to requirements so as to detect fluorescence spectra of the dyes and be used for fluorescence imaging in cells.

Example 7

BDQF-6 was tested for uv absorption and fluorescence emission spectra in four solvents, dichloromethane, acetonitrile, ethanol and 20mM PBS buffer (pH 7.4). Taking 5 mu L of BDQF-6 mother liquor, respectively adding 1mL of the four solvents,and (3) uniformly mixing by using a uniformly mixing instrument to obtain 5 mu M of fluorescent dye test solution, and then carrying out ultraviolet absorption and fluorescence spectrum test. And calculating the spectral properties including molar extinction coefficient and fluorescence quantum yield of BDQF-6 through corresponding formulas. And (4) measuring the fluorescence quantum yield. Quantum yield of cresyl violet in ethanol solution was used as reference (. PHI.) (phi.)_f0.58). The fluorescence quantum yield can be calculated according to the following formula:

absref and Abssam are used as reference and the absorption intensity of the liquid to be detected at the reference excitation wavelength; f_refAnd F_samIs the integral value of the fluorescence intensity at the corresponding excitation; eta_samAnd eta_refThe refractive indices of the solutions used for the samples and for the reference. The absorption of the solution to be detected and the reference solution is not higher than 0.05.

As shown in FIG. 17, the maximum absorption wavelength in the four solvents is 575-585nm, and the maximum emission wavelength is 620-640 nm. And the absorption and emission intensities of the fluorescent probe in the four solvents are relatively close, which shows that the fluorescent probe is less influenced by the polarity of the solvents and can provide relatively accurate fluorescent signals in complex physiological environments.

Example 8

And testing the ultraviolet absorption and fluorescence spectrum of BDQF-6 and RhB in PBS. And (3) adding 5 mu L of BDQF-6 and RhB mother liquor into 1mL of 20mM PBS respectively, uniformly mixing by using a mixing instrument to obtain 5 mu M of fluorescent dye test solution, then carrying out ultraviolet absorption and fluorescence spectrum tests, and normalizing the result.

As shown in FIG. 18, compared with RhB, the excitation spectrum and the emission spectrum of BDQF-6 have smaller overlap and larger Stokes shift, so that self-quenching caused by background scattering of a biological sample can be effectively avoided, and the imaging signal-to-noise ratio of the dye is increased.

Example 9

Photophysical properties of BDQF dyes of different substituents in 20mM PBS buffer (pH 7.4). Taking 5 mu L of BDQF-6 mother liquor, respectively adding 1mL of the four solutionsAnd uniformly mixing the reagent by using a mixing instrument to obtain 5 mu M fluorescent dye test solution, and then carrying out ultraviolet absorption and fluorescence spectrum test. And calculating the spectral properties including molar extinction coefficient and fluorescence quantum yield of BDQF-6 through corresponding formulas. And (4) measuring the fluorescence quantum yield. Quantum yield of cresyl violet in ethanol solution was used as reference (. PHI.) (phi.)_f0.58). The specific calculation method is described in example 6.

The results are shown in fig. 19, and the fluorescence quantum yield of BDQF dye in PBS is greatly improved compared to the previous DQF dye. The quantum yield of the trifluoroethyl substituted BDQF-6 dye is 12 times that of DQF-584 and 2 times that of a traditional symmetric dye RhB, and BDQF-6 still maintains the Stokes shift (56nm) twice that of RhB, so that the potential of the dye for acquiring high-quality images in an advanced imaging technology is further demonstrated.

Example 10

BDQF-6 was tested for the change of fluorescence intensity with time under 1W 530nm laser irradiation. 5 μ L of BDQF-6 mother liquor and 5 μ L of 1mM RhB mother liquor were added to 1mL of ethanol solution containing 0.1% trifluoroacetic acid, respectively, and mixed well. And then adding the mixture into a threaded cuvette, continuously irradiating by using laser, performing fluorescence spectrum test by respectively adopting 0min, 20min, 40 min, 60 min and 80min as time nodes, selecting a dye fluorescence emission peak value to map the time, and performing normalization treatment.

As shown in FIG. 20, the fluorescence of BDQF-6 is reduced by about 17% under 80min continuous irradiation, and at the same time, the fluorescence of RhB is reduced by about 60%, which indicates that BDQF-6 has better light stability and potential to be used for long-time imaging.

Example 11

Luminance test of BDQF-6 in cells. The stock solution of 1. mu.L BDQF-22-Halo was dissolved in 1mL of cell culture solution at 37 ℃ with 5% CO₂HeLa cells transfected with H2B-Halo protein were incubated for 4H before direct confocal imaging.

As shown in FIG. 21, the fluorescence intensity of BDQF-6-Halo is about 1.5 times that of RhB-Halo under the condition that cellular Halo protein is completely labeled and imaging conditions are completely consistent, which indicates that BDQF-6-Halo has higher imaging brightness in protein labeling, and further proves that BDQF-6 has higher brightness, can be used for low-power imaging and is beneficial to reducing damage to cells or tissues.

Example 12

Photostability test of BDQF-6 in stimulated emission depletion microscopy (STED). BDQF-6 framework-based one-Halo protein probe (BDQF-22-Halo), BDQF-22-Halo was dissolved in DMSO to form 200. mu.M stock solution. mu.L of the mother liquor was dissolved in 1mL of cell culture medium, and Halo protein-transfected U2OS cells were incubated at 37 ℃ under 5% CO2 for 4h, followed by cell fixation and imaging using STED.

As shown in FIG. 22, transfected Halo-vimentin proteins in U2OS cells were fully labeled using BDQF-6-Halo, CPY-Halo, and JF608-Halo as labeling agents, respectively. Under the same imaging conditions, BDQF-6 can obtain images of 15 frames of vimentin and still maintain relatively clear imaging effect. However, the CPY-Halo or JF608 can only image 5 frames, and the obtained image has lost much detail and the imaging effect is poor. This illustrates the potential of BDQF-6 to be used in super-resolution imaging requiring long acquisition times, such as super-resolution 3D imaging.