Organic fluorescent dye as well as preparation method and application thereof

Abstract

The invention relates to an organic fluorescent dye as well as a preparation method and application thereof, the structural formula of the organic fluorescent dye is as follows: # imgabs0 # organic fluorescent dye is used for a super-resolution imaging process of living cells, disulfide bonds in molecules of the organic fluorescent dye are triggered by mitochondria with reduction performance of the living cells to generate sulfydryl, and the sulfydryl is used as a fluorescent dye. Sulfydryl attacks nearby carbon atoms to form a six-membered ring, and reversible opening and closing of the six-membered ring generate a spontaneous light flickering behavior; the organic fluorescent dye provided by the invention can be used as a fluorescent molecule in a super-resolution optical imaging process of a super-resolution optical imaging microscope, can prolong the light switching time and improve the time resolution, does not need to use an imaging buffer solution, avoids the influence of the imaging buffer solution on super-resolution imaging, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the field of super-resolution dyes, and in particular to an organic fluorescent dye and a preparation method and application thereof. Background Art

[0002] In recent years, the emergence of super-resolution optical imaging microscopy has made it possible to image the ultrafine structures of biological organelles, cellular proteins and DNA. Several super-resolution microscopy techniques have been developed and applied to the study of 2D and 3D mitochondrial structures. These methods include structured illumination microscopy, stimulated emission depletion microscopy and single-molecule localization microscopy, such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM).

[0003] STORM technology is based on the optical switching of individual fluorescent molecules, achieving an imaging resolution of up to 20 nanometers in fixed samples. In principle, a small number of fluorescent molecules are randomly switched to the "on" state, localized at a single time point, and then switched to the "off" state, a process called photoblinking. Thousands of frames of photoblinking data are collected and used to reconstruct super-resolution images.

[0004] Commonly used organic fluorescent molecules, such as Alexa 647, are ideal candidates for STORM imaging, but such organic fluorescent molecules need to be used in high concentration imaging buffers to exert their advantages. The imaging buffer contains thiols, glucose, glucose oxidase, and catalase for enzymatic oxygen removal. The adducts formed by the thiols in these buffers will turn the fluorescence "off". In direct stochastic optical reconstruction microscopy (dSTORM), β-mercaptoethanol and intense laser beams are required to effectively induce photoblinking, which is very useful for super-resolution imaging in fixed cells. However, STORM imaging in living cells remains a daunting challenge. Poor temporal resolution, the need for imaging buffers, and short-term photo-switching limit the applicability of STORM in living cell imaging. Summary of the invention

[0005] The embodiments of the present invention provide an organic fluorescent dye and a preparation method and application thereof, which solve the problems that the existing super-resolution optical imaging microscope has poor time resolution, requires imaging buffer and short-term light switching when performing super-resolution optical imaging of living cells, which limits the application of super-resolution optical imaging microscopes in living cell imaging.

[0006] To achieve the above objectives, in a first aspect, an embodiment of the present invention provides an organic fluorescent dye, the structural formula of the organic fluorescent dye is:

[0007]

[0008] When the organic fluorescent dye is used in the super-resolution imaging process of living cells, the disulfide bonds in the molecules of the organic fluorescent dye are triggered by the mitochondria of the living cells with reducing properties to generate thiol groups, which attack nearby carbon atoms to form a six-membered ring, and the reversible opening and closing of the six-membered ring produces spontaneous light flashing behavior.

[0009] In a second aspect, an embodiment of the present invention provides a method for preparing the organic fluorescent dye described in the first aspect, the method comprising:

[0010] Step S1, reacting 2,3,3-trimethyl-3H-indole with ethylsulfate, S-(3-iodopropyl) ester to obtain a first compound; the structural formula of the first compound is

[0011] Step S2, heating the first compound and malondialdehyde diphenylamine hydrochloride under acid catalysis to generate a second compound; the structural formula of the second compound is

[0012] Step S3, reacting 2,3,3-trimethyl-3H-indole-5-carboxylic acid with 1-iodomethane to obtain a third compound; the structural formula of the third compound is

[0013] Step S4, heating the second compound and the third compound to generate an asymmetric cyanine dye; the asymmetric cyanine dye has a structural formula of

[0014] Step S5, reacting the asymmetric cyanine dye with 1-boc-piperazine under base catalysis to obtain a fourth compound; the structural formula of the fourth compound is

[0015] Step S6, removing the acyl group from the fourth compound under alkaline conditions to generate a fifth compound having a six-membered heterocyclic ring in the molecule; the structural formula of the fifth compound is

[0016] Step S7, washing the fifth compound with hydrochloric acid to open the six-membered heterocyclic ring of the fifth compound to obtain a Flou-SH dye containing a thiol group; the structural formula of the Flou-SH dye is

[0017] Step S8, reacting the Flou-SH dye with disulfide-containing cystamine to generate a sixth compound containing a disulfide bond; the structural formula of the sixth compound is

[0018] Step S9, reacting the sixth compound with 7-(diethylamino)-2-oxo-2-benzopyran-3-carboxylic acid to generate a seventh compound; the structural formula of the seventh compound is

[0019]

[0020] Step S10, removing the Boc group from the seventh compound in dichloromethane and trifluoroacetic acid to obtain an eighth compound; the Boc group is tert-butyloxycarbonyl; the structural formula of the eighth compound is

[0021]

[0022] Step S11, reacting the eighth compound with chloroacetic anhydride to finally obtain an organic fluorescent dye;

[0023] The structural formula of the organic fluorescent dye is:

[0024]

[0025] Preferably, the step S1 specifically includes:

[0026] A mixture of 1-bromo-3-chloropropane and potassium thioacetate is refluxed in tetrahydrofuran solvent for 1 hour, then stirred at room temperature for 24 hours, and then dissolved in acetone after removing tetrahydrofuran by rotary evaporation. Sodium iodide is added, stirred at room temperature for 6 days, and then the organic phase is removed. Then, the mixture is extracted with dichloromethane and water, and then washed with sodium thiosulfate. After removing tetrahydrofuran by rotary evaporation, the ethylsulfate, S-(3-iodopropyl) ester is obtained;

[0027] The ethylsulfate, S-(3-iodopropyl) ester and the 2,3,3-trimethyl-3H-indole are dissolved in a solvent acetonitrile and refluxed for 24 hours. After cooling to room temperature, the acetonitrile is evaporated, and diethyl ether is added to generate a viscous first purple oily substance. The upper liquid is poured out, and the viscous oil is dissolved with dichloromethane. Diethyl ether is added again to generate a second purple oily substance. The upper liquid is poured out, and the oily substance is dried to obtain the first compound.

[0028] Preferably, the step S2 specifically includes:

[0029] The first compound and the malondialdehyde diphenylamine hydrochloride are dissolved in a mixed solvent of acetic acid and acetic anhydride, heated under reflux for 1 hour and then cooled to room temperature, the mixed solvent is evaporated and ether is added, the solid residue is collected and analyzed by column chromatography to obtain the second compound;

[0030] The second compound is a dark yellow solid; the volume ratio of acetic acid to acetic anhydride is 1:1;

[0031] Preferably, the step S3 specifically includes:

[0032] Dissolving the 2,3,3-trimethyl-3H-indole-5-carboxylic acid and the 1-iodomethane in acetonitrile as a solvent to obtain a mixture;

[0033] Under an inert atmosphere, the mixture was heated at 80° C. for 4 hours, cooled to room temperature, and then subjected to rotary evaporation to remove the solvent acetonitrile to obtain a precipitate;

[0034] The precipitate was washed with diethyl ether to obtain the third compound.

[0035] Preferably, the step S4 specifically includes:

[0036] The second compound and the third compound are dissolved in ethanol solvent, triethylamine catalyst is added, and after reflux for 2 hours to complete the reaction, the ethanol is removed by rotary evaporation to obtain a solid residue, and the solid residue is purified by column chromatography to obtain the asymmetric cyanine dye;

[0037] The asymmetric cyanine dye is a dark blue solid.

[0038] Preferably, the step S5 specifically includes:

[0039] The asymmetric cyanine dye, 1-boc-piperazine and triethylamine are dissolved in dichloromethane, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is added to obtain a mixed solution;

[0040] The mixed solution was stirred at room temperature for 3 hours, then washed with 0.1 mol / L hydrochloric acid solution and brine, the organic phase was collected, the solvent was removed by rotary evaporation, and the organic matter was purified by column chromatography to obtain the fourth compound.

[0041] Preferably, the step S6 specifically includes:

[0042] The fourth compound is dissolved in methanol, potassium carbonate is added, and the mixture is stirred at room temperature for 30 minutes until the reaction is complete, thereby obtaining and generating the fifth compound having a six-membered heterocyclic ring in the molecule;

[0043] The step S7 specifically includes:

[0044] diluting the fifth compound with dichloromethane, and washing with a 0.1 mol / L aqueous hydrochloric acid solution to obtain a mixture;

[0045] The entire mixture is shaken vigorously until the organic phase in the mixture completely turns blue to obtain a precursor;

[0046] The precursor is dried and the solvent is evaporated to obtain the Flou-SH dye; the Flou-SH dye is a blue solid.

[0047] Preferably, the step S8 specifically includes:

[0048] Dissolving the Flou-SH dye in a solvent, and then adding cystamine and triethylamine to obtain a mixed liquid;

[0049] The mixed liquid was stirred at room temperature for 24 hours, and after the reaction was completed, it was washed with water, extracted with dichloromethane, and the organic phase was dried and concentrated to obtain a solid substance;

[0050] The solid material was dissolved with 1 mL of dichloromethane, 0.1 mL of triethylamine and 1 mL of ethyl acetate were added, and excess ether was added to remove the unreacted Flou-SH dye, and this process was performed twice to obtain a residue;

[0051] Purifying the residue by column chromatography to obtain the sixth compound;

[0052] The solvent is a mixture of dichloromethane and methanol in a volume ratio of 1:1.

[0053] Preferably, the step S9 specifically includes:

[0054] The sixth compound and 7-(diethylamino)-2-oxo-2-benzopyran-3-carboxylic acid are stirred at room temperature for 24 hours to obtain a mixture;

[0055] The mixture was diluted with dichloromethane and washed with 5 wt% aqueous hydrochloric acid solution, saturated aqueous sodium bicarbonate solution and brine, after which the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the organic matter was purified by column chromatography to obtain the seventh compound.

[0056] Preferably, the step S10 specifically includes:

[0057] The seventh compound is dissolved in a solvent, and stirred at room temperature for 1 hour, and the solvent is removed by rotary evaporation to remove the Boc group to obtain a residue;

[0058] The residue is diluted with dichloromethane, washed with water and an aqueous solution of sodium bicarbonate, and the organic phase is dried and concentrated to obtain the blue eighth compound;

[0059] The solvent is a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 3:1.

[0060] Preferably, the step S11 specifically includes:

[0061] dissolving the eighth compound in dichloromethane, adding chloroacetic anhydride, and then adding triethylamine as a catalyst to obtain a mixed solution;

[0062] The mixed solution was stirred at room temperature for 1 hour and washed with deionized water. The organic phase was dried and concentrated, and purified by column chromatography to obtain the organic fluorescent dye.

[0063] In a third aspect, an embodiment of the present invention provides an application of the organic fluorescent dye described in the first aspect above, wherein the organic fluorescent dye is used as a fluorescent molecule in a super-resolution optical imaging process of a super-resolution optical imaging microscope.

[0064] The embodiment of the present invention provides an organic fluorescent dye and a preparation method and application thereof. By introducing a disulfide bond (-SS-) into the molecule of the organic fluorescent dye of the present invention, the disulfide bond is triggered by the reducing mitochondria in living cells to generate a thiol group (-SH). The obtained thiol group attacks the carbon atom nearby to form a six-membered ring. The reversible opening and closing of the six-membered ring causes the organic fluorescent dye to produce spontaneous light flashing behavior. The organic fluorescent dye of the present invention is used as a fluorescent molecule in the imaging technology of a super-resolution optical imaging microscope, which can prolong the light switching time and improve the time resolution, and does not need to use an imaging buffer, thereby avoiding the influence of the imaging buffer on super-resolution imaging. The organic fluorescent dye provided by the embodiment of the present invention has broad application prospects in the field of super-resolution optical imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 A flow chart of a method for preparing an organic fluorescent dye provided in an embodiment of the present invention.

[0066] Figure 2 The specific chemical formula for preparing the organic fluorescent dye provided in Example 1 of the present invention.

[0067] Figure 3 This is a picture of the fluorescence lifetime test of the organic fluorescent dye prepared in Example 1 according to Example 2 of the present invention.

[0068] Figure 4 This is a picture of fluorescence imaging observation of the organic fluorescent dye prepared in Example 1 according to Example 2 of the present invention.

[0069] Figure 5 This is a picture of the biocompatibility test of the organic fluorescent dye prepared in Example 1 using Example 3 of the present invention.

[0070] Figure 6 This is a picture of living cell imaging using the organic fluorescent dye prepared in Example 1 according to Example 4 of the present invention. DETAILED DESCRIPTION

[0071] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0072] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments.

[0073] The embodiment of the present invention provides an organic fluorescent dye, the structural formula of which is as follows:

[0074]

[0075] When the organic fluorescent dye provided by the embodiment of the present invention is used in the super-resolution imaging process of living cells, the disulfide bonds in the molecules of the organic fluorescent dye are triggered by the mitochondria with reducing properties of the living cells to generate thiol groups, and the thiol groups attack nearby carbon atoms to form a six-membered ring. The reversible opening and closing of the six-membered ring produces spontaneous light flashing behavior.

[0076] After the protecting group of the organic fluorescent dye provided by the embodiment of the present invention is cut off by the free thiol group on the protein, the thiol group on the dye molecule is exposed and can spontaneously attack the adjacent carbon atoms to generate a six-membered ring within the molecule, switching the molecule to a dark state. The molecule can be reopened through reversible thermal equilibrium, thereby achieving reversible opening and closing of the six-membered ring.

[0077] The present invention provides a method for preparing the organic fluorescent dye. Figure 1 As shown, the specific steps include:

[0078] Step S1, reacting 2,3,3-trimethyl-3H-indole with ethyl sulfate, S-(3-iodopropyl) ester to obtain a first compound;

[0079] Specifically, a mixture of 1-bromo-3-chloropropane and potassium thioacetate is refluxed in a solvent of tetrahydrofuran for 1 hour, then stirred at room temperature for 24 hours, the tetrahydrofuran is removed by rotary evaporation, and then dissolved in acetone, sodium iodide is added, stirred at room temperature for 6 days, and the organic phase is removed, extracted with dichloromethane and water, and then washed with sodium thiosulfate, and tetrahydrofuran is removed by rotary evaporation to obtain ethylsulfate, S-(3-iodopropyl) ester;

[0080] Ethylsulfate, S-(3-iodopropyl) ester and 2,3,3-trimethyl-3H-indole were dissolved in acetonitrile and refluxed for 24 hours. After cooling to room temperature, the acetonitrile was evaporated and ether was added to generate a first viscous purple oily substance. The upper layer of liquid was poured out and the viscous oil was dissolved with dichloromethane. Ether was added again to generate a second purple oily substance. The upper layer of liquid was poured out and the oily substance was dried to obtain a first compound having the structural formula:

[0081] The first purple oily substance and the second purple oily substance are both adhered to the bottom of the container; and the drying is specifically performed by rotary evaporation at 40°C.

[0082] Step S2, heating the first compound and malondialdehyde diphenylamine hydrochloride under acid catalysis for condensation reaction to generate a second compound;

[0083] Specifically, the first compound and malondialdehyde diphenylamine hydrochloride are dissolved in a mixed solvent of acetic acid and acetic anhydride, heated under reflux for 1 hour and then cooled to room temperature, the mixed solvent is evaporated and ether is added, the solid residue is collected and analyzed by column chromatography to obtain the second compound;

[0084] Among them, the second compound is a dark yellow solid with the structural formula

[0085] The amount of ether added is 10 to 20 times the amount of dichloromethane used for dissolution in step S1;

[0086] The volume ratio of acetic acid to acetic anhydride is 1:1.

[0087] Step S3, reacting 2,3,3-trimethyl-3H-indole-5-carboxylic acid with 1-iodomethane to obtain a third compound;

[0088] Specifically, 2,3,3-trimethyl-3H-indole-5-carboxylic acid and 1-iodomethane are dissolved in acetonitrile as a solvent to obtain a mixture;

[0089] Under an inert atmosphere, the mixture was heated at 80°C for 4 hours, cooled to room temperature, and then subjected to rotary evaporation to remove the solvent acetonitrile to obtain a precipitate;

[0090] The precipitate was washed with ether to obtain the third compound;

[0091] Among them, the structural formula of the third compound is

[0092] The inert atmosphere includes any of a nitrogen atmosphere and an argon atmosphere.

[0093] Step S4, heating the second compound and the third compound to generate an asymmetric cyanine dye;

[0094] Specifically, the second compound and the third compound are dissolved in ethanol solvent, triethylamine catalyst is added, and after reflux for 2 hours to complete the reaction, the ethanol is removed by rotary evaporation to obtain a solid residue, and the solid residue is purified by column chromatography to obtain an asymmetric cyanine dye;

[0095] Asymmetric cyanine dye is a dark blue solid with the structural formula

[0096] Step S5, reacting the asymmetric cyanine dye with 1-boc-piperazine under base catalysis to obtain a fourth compound;

[0097] Specifically, an asymmetric cyanine dye, 1-boc-piperazine and triethylamine are dissolved in dichloromethane, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is added to obtain a mixed solution;

[0098] The mixed solution was stirred at room temperature for 3 hours, then washed with 0.1 mol / L hydrochloric acid solution and brine, the organic phase was collected, the solvent was removed by rotary evaporation, and the organic matter was purified by column chromatography to obtain the fourth compound with the structural formula

[0099] Step S6, removing the acyl group from the fourth compound under alkaline conditions to generate a fifth compound having a six-membered heterocyclic ring in the molecule;

[0100] Specifically, the fourth compound is dissolved in methanol, potassium carbonate is added, and the mixture is stirred at room temperature for 30 minutes until the reaction is complete, thereby obtaining and generating a fifth compound having a six-membered heterocyclic ring in the molecule, and having the structural formula:

[0101] Step S7, washing the fifth compound with hydrochloric acid to open the six-membered heterocyclic ring of the fifth compound to obtain a Flou-SH dye containing a thiol group;

[0102] Specifically, the fifth compound is diluted with dichloromethane, and washed with a hydrochloric acid aqueous solution having a molar mass of 0.1 mol / L to obtain a mixture;

[0103] The entire mixture was shaken vigorously until the organic phase in the mixture turned completely blue to obtain the precursor;

[0104] The precursor is dried and the solvent is evaporated to obtain Flou-SH dye; Flou-SH dye is a blue solid. The structural formula of Flou-SH dye is

[0105] Step S8, reacting the Flou-SH dye with disulfide-containing cystamine to generate a sixth compound containing a disulfide bond;

[0106] Specifically, the Flou-SH dye is dissolved in a solvent, and cystamine and triethylamine are added to obtain a mixed liquid;

[0107] The mixed liquid was stirred at room temperature for 24 hours. After the reaction was completed, it was washed with water and then extracted with dichloromethane. The organic phase was dried and concentrated to obtain a solid substance.

[0108] The solid material was dissolved with 1 mL of dichloromethane, 0.1 mL of triethylamine and 1 mL of ethyl acetate were added, and excess ether was added to remove the unreacted Flou-SH dye. This process was performed twice to obtain a residue;

[0109] The residue is purified by column chromatography to obtain a sixth compound;

[0110] Among them, the structural formula of the sixth compound is

[0111] The solvent is a mixture of dichloromethane and methanol in a volume ratio of 1:1.

[0112] Step S9, reacting the sixth compound with 7-(diethylamino)-2-oxo-2-benzopyran-3-carboxylic acid to generate a seventh compound;

[0113] Specifically, the sixth compound and 7-(diethylamino)-2-oxo-2-benzopyran-3-carboxylic acid are stirred at room temperature for 24 hours to obtain a mixture;

[0114] The mixture was diluted with dichloromethane and washed with 5 wt% aqueous hydrochloric acid, saturated aqueous sodium bicarbonate and brine, then the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the organic matter was purified by column chromatography to obtain the seventh compound. The structural formula of the seventh compound is

[0115]

[0116] Step S10, removing the Boc group from the seventh compound in dichloromethane and trifluoroacetic acid to obtain an eighth compound; the Boc group is tert-butyloxycarbonyl;

[0117] Specifically, the seventh compound is dissolved in a solvent, and stirred at room temperature for 1 hour, and the solvent is removed by rotary evaporation to remove the Boc group to obtain a residue;

[0118] The residue was diluted with dichloromethane, washed with water and sodium bicarbonate aqueous solution, and the organic phase was dried and concentrated to obtain a blue eighth compound having the structural formula

[0119] The solvent is a mixture of dichloromethane and trifluoroacetic acid in a volume ratio of 3:1.

[0120] Step S11, reacting the eighth compound with chloroacetic anhydride to finally obtain an organic fluorescent dye;

[0121] Specifically, the eighth compound is dissolved in dichloromethane, chloroacetic anhydride is added, and then triethylamine is added to obtain a mixed solution;

[0122] The mixed solution was stirred at room temperature for 1 hour, washed with deionized water, and the organic phase was dried and concentrated. After purification by column chromatography, an organic fluorescent dye was obtained with the structural formula:

[0123]

[0124] An embodiment of the present invention provides an application of the above-mentioned organic fluorescent dye. The organic fluorescent dye is used as a fluorescent molecule in the super-resolution optical imaging process of a super-resolution optical imaging microscope, which can prolong the light switching time and improve the time resolution. There is no need to use an imaging buffer, thereby avoiding the influence of the imaging buffer on the super-resolution imaging.

[0125] In order to better understand the technical solution provided by the present invention, the preparation process and characteristics of the organic fluorescent dye of the present invention are respectively described below with multiple specific examples.

[0126] Example 1

[0127] This embodiment provides a preparation process of an organic fluorescent dye. The specific chemical equation of the preparation process is as follows: Figure 2 shown.

[0128] Step 1, first prepare ethyl sulfate, S-(3-iodopropyl) ester, the specific process is: reflux a mixture of 27.6 mmol of 1-bromo-3-chloropropane and 23 mmol of potassium thioacetate in tetrahydrofuran for 1 hour, stir at room temperature for 24 hours, remove the solvent by rotary evaporation, dissolve in 15 ml of acetone, add 25 mmol of sodium iodide and stir at room temperature for 6 days, remove the organic phase, extract with dichloromethane and water, add sodium thiosulfate for washing, and remove the solvent by rotary evaporation to obtain ethyl sulfate, S-(3-iodopropyl) ester.

[0129] 2,3,3-trimethyl-3H-indole is reacted with ethyl sulfate, S-(3-iodopropyl) ester to obtain compound (1). The specific process is as follows: 15 mmol of 2,3,3-trimethyl-3H-indole and 15 mmol of ethyl sulfate, S-(3-iodopropyl) ester are dissolved in acetonitrile and refluxed for 24 hours. After cooling to room temperature, most of the solvent acetonitrile is evaporated, 50 ml of ether is added to generate a viscous first purple oily substance, the upper liquid is carefully poured out, and the viscous oil is dissolved with 5 ml of dichloromethane. 50 ml of ether is added to generate a second purple oily substance again, the liquid is carefully poured out, and the second purple oily substance at the bottom of the container is thoroughly dried by rotary evaporation at 40°C to obtain compound (1).

[0130] Step 2, heating compound (1) and malondialdehyde diphenylamine hydrochloride under acid catalysis for condensation reaction to generate compound (2), the specific process is: 15 mmol of compound (1) and 15 mmol of malondialdehyde diphenylamine hydrochloride are dissolved in 15 ml of a mixture of acetic acid and acetic anhydride in a volume ratio of 1:1, heated under reflux for 1 hour and then cooled to room temperature. Most of the solvent is evaporated and 30 ml of ether is added, the solid residue is collected and analyzed by column chromatography to obtain a dark yellow solid, namely compound (2).

[0131] Step 3, reacting 2,3,3-trimethyl-3H-indole-5-carboxylic acid with 1-iodomethane to obtain compound (3), the specific process is: dissolving 9.84 mmol of 2,3,3-trimethyl-3H-indole-5-carboxylic acid and 29.5 mmol of iodomethane in 10 ml of acetonitrile, heating the mixture at 80° C. for 4 hours under an argon atmosphere, cooling to room temperature, removing the solvent by rotary evaporation, and washing the precipitate with diethyl ether to obtain compound (3).

[0132] Step 4, heating compound (2) and compound (3) to generate asymmetric cyanine dye (4), the specific process is: dissolving 10 mmol of compound (2) and 10 mmol of compound (3) in 100 ml of ethanol as a solvent, adding a few drops of catalyst triethylamine, reflux for 2 hours, monitoring the complete consumption of the starting material, removing the ethanol by rotary evaporation to obtain a solid residue, and purifying the solid residue by column chromatography to obtain a dark blue solid, which is the asymmetric cyanine dye (4).

[0133] Step 5, reacting the asymmetric cyanine dye (4) with 1-boc-piperazine under base catalysis to obtain compound (5), the specific process is: dissolving 5 mmol of the asymmetric cyanine dye (4), 10 mmol of 1-boc-piperazine and a few drops of catalyst triethylamine in 200 ml of dichloromethane, then adding 10 ml of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate to obtain a mixed solution; stirring the mixed solution at room temperature for 3 hours, then washing with a hydrochloric acid solution with a molar mass of 0.1 mol / L and brine, collecting the organic phase, removing the solvent by rotary evaporation, and purifying the organic matter by column chromatography to obtain compound (5).

[0134] Step 6, removing the acyl group from compound (5) under alkaline conditions to generate compound (6) having a six-membered heterocyclic ring in the molecule. The specific process is as follows: dissolving 12 mmol of compound (5) in 500 ml of methanol, adding 40 mmol of potassium carbonate, and stirring at room temperature for 30 minutes to complete the reaction to obtain compound (6) having a six-membered heterocyclic ring in the molecule.

[0135] Step 7, washing compound (6) with hydrochloric acid to open the six-membered heterocyclic ring of compound (6) to obtain Flou-SH dye (7) containing a thiol group, wherein the specific process is as follows: diluting compound (6) with 20 ml of dichloromethane, and washing with a hydrochloric acid aqueous solution with a molar mass of 0.1 mol / L to obtain a mixture; vigorously shaking the entire mixture until the organic phase in the mixture completely turns blue to obtain a precursor; drying the precursor and evaporating the solvent to obtain a blue solid, namely Flou-SH dye (7).

[0136] Step 8, reacting Flou-SH dye (7) with disulfide-containing cystamine to generate a disulfide-containing compound (8), the specific process is: dissolving 0.5 mmol of Flou-SH dye (7) in 10 ml of a mixed solvent of dichloromethane and methanol in a volume ratio of 1:1, and then adding cystamine and triethylamine to obtain a mixed liquid; stirring the mixed liquid at room temperature for 24 hours, washing with water after the reaction is completed, extracting with dichloromethane, drying and concentrating the organic phase to obtain a solid substance; dissolving the solid substance with 1 mL of dichloromethane, adding 0.1 mL of triethylamine and 1 mL of ethyl acetate, adding excess ether, removing unreacted Flou-SH dye, this process is performed twice to obtain a residue; the residue is purified by column chromatography to obtain a compound (8). Wherein, the amount of cystamine added is twice the mole of Flou-SH dye (7), and the amount of triethylamine added as a catalyst is a few drops.

[0137] Step 9, reacting compound (8) with 7-(diethylamino)-2-oxo-2-benzopyran-3-carboxylic acid to generate compound (9), wherein the specific process is as follows: stirring 10 mmol of compound (8) and 10 mmol of 7-(diethylamino)-2-oxo-2-benzopyran-3-carboxylic acid at room temperature for 24 hours to obtain a mixture; diluting the mixture with 20 ml of dichloromethane, and washing with 5 wt% aqueous hydrochloric acid solution, saturated aqueous sodium bicarbonate solution and brine, then drying the organic phase with anhydrous sodium sulfate, filtering and concentrating, and purifying the organic matter by column chromatography to obtain compound (9).

[0138] Step 10, removing the Boc group from compound (9) in dichloromethane and trifluoroacetic acid to obtain compound (10), the specific process is: dissolving 0.2 mmol of compound (9) in a 10 ml mixed solvent of dichloromethane and trifluoroacetic acid in a volume ratio of 3:1, and stirring at room temperature for 1 hour, removing the solvent by rotary evaporation, removing the Boc group, and obtaining a residue; diluting the residue with dichloromethane, washing with water and sodium bicarbonate aqueous solution, and drying and concentrating the organic phase to obtain a blue compound (10).

[0139] Step 11, reacting compound (10) with chloroacetic anhydride to obtain an organic fluorescent dye, the specific process is: dissolving compound (10) in dichloromethane, adding 0.5 mmol chloroacetic anhydride, and then adding 0.5 mmol triethylamine to obtain a mixed solution; stirring the mixed solution at room temperature for 1 hour, and washing with deionized water, drying and concentrating the organic phase, and purifying by column chromatography to obtain an organic fluorescent dye.

[0140] Example 2

[0141] The solid powder of the organic fluorescent dye prepared in Example 1 was dissolved in dimethyl sulfoxide to obtain a dye mother solution with a molar concentration of 1 millimole per liter (mM).

[0142] The dye stock solution was diluted to 10 μM with dimethyl sulfoxide, and the fluorescence lifetime of the dye was measured using a transient and steady-state fluorescence spectrometer, such as Figure 3 As shown, the horizontal axis is time (Time, unit: ns), and the vertical axis is fluorescence intensity (Intensity, unit: au). It can be seen that the fluorescence lifetime of the organic fluorescent dye prepared in Example 1 is 1.48 ns.

[0143] The dye stock solution was dropped onto a clean glass cover, and the sample was spun to make a thin layer. Then the fluorescence phenomenon of the dye was observed using a homemade STORM imaging system. Figure 4 As shown, the fluorescence phenomenon of the dye can be clearly observed under a microscope, and the white dots in the red circle in the figure are the fluorescence phenomenon.

[0144] Example 3

[0145] The organic fluorescent dye prepared in Example 1 was dissolved in dimethyl sulfoxide at a stock concentration of 1 mM to obtain a dye mother solution.

[0146] The cell counting box CCK-8 was used to determine cell viability, and the cytotoxicity of HeLa cells was determined. First, the cells were inoculated in a 96-well plate, the cell density was controlled to 10,000 cells per well, and incubated for adhesion. After 24 hours of culture, the culture medium in each well was replaced with 100 μL of fresh culture medium (RPMI-1640) containing probes with different concentrations of organic fluorescent dyes (Mito-SS, 1~8μM, different concentrations of probes were obtained by diluting the dye mother solution with culture medium). After incubation for 3 hours, the detection reagent (CCK-8 was diluted with RPMI-1640 culture medium (10%)) was added to the corresponding wells and incubated for another 4 hours. After the above steps, the absorbance was measured at 450nm. Each group of experiments was performed in parallel 6 times. Calculate the cell survival rate. As Figure 5As shown, the horizontal axis represents the concentration value (Concentration, unit μM), and the vertical axis represents the cell viability (Cell Viability, %). The results after co-culture show that the dye has no cytotoxicity.

[0147] Example 4

[0148] The solid powder of the organic fluorescent dye prepared in Example 1 was dissolved in dimethyl sulfoxide to obtain a dye mother solution with a molar concentration of 1 millimole per liter (mM). HeLa cells were planted in a living cell culture dish. After 48 hours, the culture medium was replaced and the prepared dye mother solution was added to the culture dish to make the final concentration of the dye 1uM. The culture dish was placed at 37 degrees for 10 minutes, the culture medium was discarded, and the dish was washed 3 times with PBS buffer. The dish was placed on the stage of a laser confocal microscope, and a long-pass filter with an excitation wavelength of 633nm and an emission wavelength of 645nm was selected for observation. Figure 6 As shown, it can be seen that the organic fluorescent dye prepared in Example 1 can enter cells and has different selectivity in cells, and can be applied to the application of living cell fluorescent dyes.

[0149] The embodiment of the present invention provides an organic fluorescent dye and a preparation method and application thereof. By introducing a disulfide bond (-SS-) into the molecule of the organic fluorescent dye of the present invention, the disulfide bond is triggered by the reducing mitochondria in living cells to generate a thiol group (-SH). The obtained thiol group attacks the carbon atom nearby to form a six-membered ring. The reversible opening and closing of the six-membered ring causes the organic fluorescent dye to produce spontaneous light flashing behavior. The organic fluorescent dye of the present invention is used as a fluorescent molecule in the imaging technology of a super-resolution optical imaging microscope, which can prolong the light switching time and improve the time resolution, and does not need to use an imaging buffer, thereby avoiding the influence of the imaging buffer on super-resolution imaging. The organic fluorescent dye provided by the embodiment of the present invention has broad application prospects in the field of super-resolution optical imaging.

[0150] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An organic fluorescent dye, characterized in that: The structural formula of the organic fluorescent dye is: When the organic fluorescent dye is used in the super-resolution imaging process of living cells, the disulfide bonds in the molecules of the organic fluorescent dye are triggered by the mitochondria of the living cells with reducing properties to generate thiol groups, which attack nearby carbon atoms to form a six-membered ring, and the reversible opening and closing of the six-membered ring produces spontaneous light flashing behavior.

2. A method for preparing the organic fluorescent dye according to claim 1, characterized in that: The preparation method comprises: Step S1, reacting 2,3,3-trimethyl-3H-indole with ethyl sulfate, S-(3-iodopropyl) ester to obtain a first compound; the structural formula of the first compound is Step S2, heating the first compound and malondialdehyde diphenylamine hydrochloride under acid catalysis to generate a second compound; the structural formula of the second compound is Step S3, reacting 2,3,3-trimethyl-3H-indole-5-carboxylic acid with 1-iodomethane to obtain a third compound; the structural formula of the third compound is Step S4, heating the second compound and the third compound to generate an asymmetric cyanine dye; the asymmetric cyanine dye has a structural formula of Step S5, reacting the asymmetric cyanine dye with 1-boc-piperazine under base catalysis to obtain a fourth compound; the structural formula of the fourth compound is Step S6, removing the acyl group from the fourth compound under alkaline conditions to generate a fifth compound having a six-membered heterocyclic ring in the molecule; the structural formula of the fifth compound is Step S7, washing the fifth compound with hydrochloric acid to open the six-membered heterocyclic ring of the fifth compound to obtain a Flou-SH dye containing a thiol group; the structural formula of the Flou-SH dye is Step S8, reacting the Flou-SH dye with disulfide-containing cystamine to generate a sixth compound containing a disulfide bond; the structural formula of the sixth compound is Step S9, reacting the sixth compound with 7-(diethylamino)-2-oxo-2-benzopyran-3-carboxylic acid to generate a seventh compound; the structural formula of the seventh compound is Step S10, removing the Boc group from the seventh compound in dichloromethane and trifluoroacetic acid to obtain an eighth compound; the Boc group is tert-butyloxycarbonyl; the structural formula of the eighth compound is Step S11, reacting the eighth compound with chloroacetic anhydride to finally obtain an organic fluorescent dye; The structural formula of the organic fluorescent dye is:

3. The preparation method according to claim 2, characterized in that: The step S1 specifically includes: A mixture of 1-bromo-3-chloropropane and potassium thioacetate is refluxed in tetrahydrofuran solvent for 1 hour, then stirred at room temperature for 24 hours, and then dissolved in acetone after removing tetrahydrofuran by rotary evaporation. Sodium iodide is added, stirred at room temperature for 6 days, and then the organic phase is removed. Then, the mixture is extracted with dichloromethane and water, and then washed with sodium thiosulfate. After removing tetrahydrofuran by rotary evaporation, the ethylsulfate, S-(3-iodopropyl) ester is obtained; The ethylsulfate, S-(3-iodopropyl) ester and the 2,3,3-trimethyl-3H-indole are dissolved in a solvent acetonitrile and refluxed for 24 hours. After cooling to room temperature, the acetonitrile is evaporated, and diethyl ether is added to generate a viscous first purple oily substance. The upper liquid is poured out, and the viscous oil is dissolved with dichloromethane. Diethyl ether is added again to generate a second purple oily substance. The upper liquid is poured out, and the oily substance is dried to obtain the first compound.

4. The preparation method according to claim 2, characterized in that: The step S2 specifically includes: The first compound and the malondialdehyde diphenylamine hydrochloride are dissolved in a mixed solvent of acetic acid and acetic anhydride, heated under reflux for 1 hour and then cooled to room temperature, the mixed solvent is evaporated and ether is added, the solid residue is collected and analyzed by column chromatography to obtain the second compound; The second compound is a dark yellow solid; the volume ratio of the acetic acid to the acetic anhydride is 1:

1.

5. The preparation method according to claim 2, characterized in that: The step S3 specifically includes: Dissolving the 2,3,3-trimethyl-3H-indole-5-carboxylic acid and the 1-iodomethane in acetonitrile as a solvent to obtain a mixture; Under an inert atmosphere, the mixture was heated at 80° C. for 4 hours, cooled to room temperature, and then subjected to rotary evaporation to remove the solvent acetonitrile to obtain a precipitate; The precipitate was washed with diethyl ether to obtain the third compound.

6. The preparation method according to claim 2, characterized in that: The step S4 specifically includes: The second compound and the third compound are dissolved in ethanol solvent, triethylamine catalyst is added, and after reflux for 2 hours to complete the reaction, the ethanol is removed by rotary evaporation to obtain a solid residue, and the solid residue is purified by column chromatography to obtain the asymmetric cyanine dye; The asymmetric cyanine dye is a dark blue solid.

7. The preparation method according to claim 2, characterized in that: The step S5 specifically includes: The asymmetric cyanine dye, 1-boc-piperazine and triethylamine are dissolved in dichloromethane, and then 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate is added to obtain a mixed solution; The mixed solution was stirred at room temperature for 3 hours, then washed with 0.1 mol / L hydrochloric acid solution and brine, the organic phase was collected, the solvent was removed by rotary evaporation, and the organic matter was purified by column chromatography to obtain the fourth compound.

8. The preparation method according to claim 2, characterized in that: The step S6 specifically includes: The fourth compound is dissolved in methanol, potassium carbonate is added, and the mixture is stirred at room temperature for 30 minutes until the reaction is complete, thereby obtaining and generating the fifth compound having a six-membered heterocyclic ring in the molecule; The step S7 specifically includes: diluting the fifth compound with dichloromethane, and washing with a 0.1 mol / L aqueous hydrochloric acid solution to obtain a mixture; The entire mixture is shaken vigorously until the organic phase in the mixture completely turns blue to obtain a precursor; The precursor is dried and the solvent is evaporated to obtain the Flou-SH dye; the Flou-SH dye is a blue solid.

9. The preparation method according to claim 2, characterized in that: The step S8 specifically includes: Dissolving the Flou-SH dye in a solvent, and then adding cystamine and triethylamine to obtain a mixed liquid; The mixed liquid was stirred at room temperature for 24 hours, and after the reaction was completed, it was washed with water and then extracted with dichloromethane, and the organic phase was dried and concentrated to obtain a solid substance; The solid material was dissolved with 1 mL of dichloromethane, 0.1 mL of triethylamine and 1 mL of ethyl acetate were added, and excess ether was added to remove the unreacted Flou-SH dye, and this process was performed twice to obtain a residue; Purifying the residue by column chromatography to obtain the sixth compound; The solvent is a mixture of dichloromethane and methanol in a volume ratio of 1:

1.

10. The preparation method according to claim 2, characterized in that: The step S9 specifically includes: The sixth compound and 7-(diethylamino)-2-oxo-2-benzopyran-3-carboxylic acid are stirred at room temperature for 24 hours to obtain a mixture; The mixture was diluted with dichloromethane and washed with 5 wt% aqueous hydrochloric acid solution, saturated aqueous sodium bicarbonate solution and brine, after which the organic phase was dried over anhydrous sodium sulfate, filtered and concentrated, and the organic matter was purified by column chromatography to obtain the seventh compound.

11. The preparation method according to claim 2, characterized in that: The step S10 specifically includes: The seventh compound is dissolved in a solvent, and stirred at room temperature for 1 hour, and the solvent is removed by rotary evaporation to remove the Boc group to obtain a residue; The residue is diluted with dichloromethane, washed with water and an aqueous sodium bicarbonate solution, and the organic phase is dried and concentrated to obtain the blue eighth compound; The solvent is a mixed solution of dichloromethane and trifluoroacetic acid in a volume ratio of 3:

1.

12. The preparation method according to claim 2, characterized in that: The step S11 specifically includes: dissolving the eighth compound in dichloromethane, adding chloroacetic anhydride, and then adding triethylamine to obtain a mixed solution; The mixed solution was stirred at room temperature for 1 hour and washed with deionized water. The organic phase was dried and concentrated, and purified by column chromatography to obtain the organic fluorescent dye.

13. An application of the organic fluorescent dye according to claim 1, characterized in that: The organic fluorescent dye is used as a fluorescent molecule in the super-resolution optical imaging process of a super-resolution optical imaging microscope.

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