Fluorescent dye compound as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fluorescent dyes, and particularly relates to a fluorescent dye compound as well as a preparation method and application thereof. The fluorescent dye compound provided by the invention has a Cy-R structure, wherein R is a group obtained by reacting with an aldehyde group; an R group is introduced on the basis of a Cy structure, so that the photophysical properties of the compound are greatly improved compared with those of a compound without R, the saturation loss laser power of a dye can be reduced, the saturation loss power in stimulated radiation loss super-resolution imaging is further reduced, the permeability and compatibility of the compound to living cells are enhanced, and the photochemical properties of the compound are improved. The dye can be well used for living cell stimulated radiation loss super-resolution imaging, and the biological imaging application range of the dye is widened; and the compound with Cy-R has better photobleaching resistance and light stability, so that the compound can be applied to super-resolution imaging of living cells and can characterize more internal details of the cells in an ultrahigh-resolution manner.

Description

Technical Field

[0001] The invention belongs to the field of fluorescent dye technology and biological imaging, and specifically relates to a fluorescent dye compound and a preparation method and application thereof. Background Art

[0002] With the progress of cell biology, molecular imaging diagnostic technology has gradually emerged, including confocal fluorescence imaging microscopes, fluorescence spectroscopy / lifetime meters and other technologies. In recent years, super-resolution imaging methods have continued to develop, breaking through the optical diffraction limit, achieving high-resolution imaging, and revealing microstructures less than 200nm. Fluorescent dyes, as key components of fluorescence imaging and super-resolution fluorescence imaging, play an important role in the development of imaging diagnostic analysis technology. Most of the existing super-resolution imaging dyes can only emit fluorescence of a single color. However, dyes that can simultaneously label and detect multiple molecules, organisms or multiple organelles are more practical. With the advancement of scientific research and technology, more and more dyes with multi-color luminescence and low energy loss characteristics have been developed, but there is still room for improvement and innovation in the development of fluorescent dyes that have both characteristics and can achieve super-resolution imaging of living cells. The multi-color luminescence performance of fluorescent dyes depends on the design of their molecular structure and emission energy levels. By adjusting the structure and chemical functional groups of the dyes, multiple emission energy levels and adjustable emission wavelengths can be achieved, thereby producing luminescence of different colors, which is of great significance for multi-color labeling and imaging in analysis and detection applications. At the same time, low energy loss is another key factor in dye design. High-power loss lasers can aggravate the photobleaching of fluorescent materials and cause significant damage to biological tissues, which leads to certain technical bottlenecks in stimulated emission depletion microscopy (STED) super-resolution imaging of living cells. The development of dyes that can emit strong fluorescence under low-energy excitation to achieve long-term fluorescence imaging and the study of super-resolution imaging of living cells are of great significance for exploring real biological structures and processes. Therefore, it is of great significance to study and develop more effective fluorescent materials and their preparation technologies for super-resolution imaging of living cells.

[0003] Commonly used fluorescent imaging materials include organic single-molecule fluorescent dyes, organic polymer fluorescent nanospheres, and inorganic nanoluminescent materials. Compared with organic polymer nanospheres and inorganic nanoluminescent materials, organic single-molecule fluorescent dyes have obvious advantages and have attracted much attention. They are a large class of materials with a wide range of applications. There are various types of dyes. The emission wavelength range of commonly used organic fluorescent dyes can be divided into blue (coumarins, naphthylimides), green (BODIPY, fluorescein), red (rhodamine, cyanine fluorescent dyes), etc. These dyes have suitable absorption and emission properties, low cytotoxicity and other advantages and are widely used in life science analysis. Among these fluorescent dyes, fluorescent dyes with red emission wavelengths, such as cyanine fluorescent dyes, have absorption and emission wavelengths, and large molar absorption coefficients (×10 5 ), suitable fluorescence quantum yield and low cytotoxicity, etc., are widely welcomed in many applications such as single molecule level in cells and small animals, especially in super-resolution imaging. However, due to the needs of biological imaging research, fluorescent dyes with different luminescence wavelengths can be used for multi-color labeling of cells and high-contrast imaging research of different biological functional areas. Therefore, it is necessary to continue to develop luminescent fluorescent dyes suitable for living cell imaging to meet application needs.

[0004] In summary, there are few types of super-resolution imaging dyes in related technologies, and it is difficult to target and label related dye biomolecules. The spatial resolution of imaging substructures of living cells is low, making it difficult to observe the dynamic changes of living cells. Summary of the invention

[0005] The purpose of the present invention is to overcome the problems in the related art that there are few types of super-resolution imaging dyes, the related dye biological molecule targeting labeling is difficult, the imaging spatial resolution of living cell substructures is low, and the dynamic change process of living cells is difficult to observe, and further provide a fluorescent dye compound, derivatives and their preparation method and application.

[0006] A fluorescent dye compound having a structure shown in the following formula B:

[0007] Cy-R formula B;

[0008] Wherein, R is a group obtained by reacting with an aldehyde group; Cy- is selected from one of the following structures:

[0009]

[0010] Wherein, R1, R2, and R3 are selected from any one of hydrogen, halogen, benzyl, ester group, amide group, carbonyl group, aldehyde group, carbonyl group, nitro group, substituted or unsubstituted C1-C30 alkyl group, substituted or unsubstituted C1-20 alkoxy group, substituted or unsubstituted C1-20 alkylamino group, substituted or unsubstituted C3-C60 cycloalkyl group, substituted or unsubstituted C1-C60 alkylthio group, substituted or unsubstituted C6-C60 aryl group, substituted or unsubstituted C3-C60 heteroaryl group;

[0011] Z - It is a negative ion;

[0012] n is an integer from 1 to 2;

[0013] Among them, the substituents in the substituted C1-C30 alkyl, substituted C1-20 alkoxy, substituted C1-20 alkylamino, substituted C3-C60 cycloalkyl, substituted C1-C60 alkylthio, substituted C6-C60 aryl, and substituted C3-C60 heteroaryl are selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C60 alkyl, C2-C60 alkenyl, C3-C60 cycloalkyl, C2-C60 alkynyl, C3-C60 cycloalkyl, C1-C60 heterocycloalkyl, C3-C60 cycloalkenyl, C1-C60 heterocycloalkenyl, and C6-C60 aryl.

[0014] In this application, the term "substituent" has its ordinary meaning as known in the art and refers to a chemical moiety that is covalently attached to or, where appropriate, fused to a parent core group.

[0015] In the present application, the term "substituted or unsubstituted" means that the functional group recorded after the term may or may not have a substituent (hereinafter, for ease of description, the substituent is collectively referred to as Rc). For example, "substituted or unsubstituted aryl" refers to an aryl or non-substituted aryl having a substituent Rc. Wherein the above-mentioned substituent, i.e., Rc, can be, for example, deuterium, halogen, cyano, C1-C60 alkyl, C3-C60 cycloalkyl, C6-C60 aryl, C1-C60 heteroaryl, and optionally, for example, deuterium, halogen group, cyano, alkyl, haloalkyl, trialkylsilyl, deuterated alkyl, aryl, heteroaryl, etc. Of course, the number of substituents Rc can be one or more. When two substituents Rc are connected to the same atom, the two substituents Rc can exist independently or be connected to each other to form a ring with the atom; when two adjacent substituents Rc exist on a functional group, the adjacent substituents Rc can exist independently or be fused to form a ring with the functional group to which they are connected.

[0016] In the present application, the term "alkyl" refers to a saturated hydrocarbon group, whether used as part of other terms or alone, which can be straight or branched. The term "C1-C60 alkyl" is derived from a monovalent substituent of a straight or branched saturated hydrocarbon having 1 to 60 carbon atoms, preferably 1 to 40 carbon atoms, and more preferably 1 to 20 carbon atoms, of course, examples thereof include but are not limited to methyl, ethyl, propyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl and hexyl.

[0017] In the present application, the terms "aryl" and "arylene" include monocyclic, polycyclic or condensed-ring aromatic groups, the rings may be interrupted by short non-aromatic units, and may contain spiro structures. Aryl groups include but are not limited to phenyl, biphenyl, terphenyl, naphthyl, phenanthryl, anthracenyl, fluorenyl, spirobifluorenyl, etc., and arylene groups include but are not limited to phenylene, biphenylene, terphenylene, naphthyl, phenanthryl, anthracenyl, fluorenyl, spirobifluorenyl, etc.

[0018] As used in the present invention, the term "substituted" refers to a hydrogen atom in a compound being replaced by another substituent. The position is not limited to a specific position, as long as the hydrogen at the position can be replaced by a substituent. When two or more substituents are present, the two or more substituents may be the same or different.

[0019] In the present application, the term "halogen" refers to an atom selected from fluorine, chlorine, bromine and iodine.

[0020] In the present application, unless otherwise specified, a hydrogen atom includes protium, deuterium and tritium.

[0021] In the present application, the range of carbon atom number is defined in the definition of the group, and the carbon atom number is any integer within the defined range. For example, C6-C60 aromatic group, the carbon atom number representing the aromatic group can be any integer within the range of 6-60, such as 6, 8, 10, 13, 15, 17, 20, 22, 25 or 30, etc.

[0022] In the present application, if a group is not limited to being substituted or unsubstituted, it means being unsubstituted.

[0023] It is understandable that for this application and similar structures include the following structures or similar structures:

[0024]

[0025] Preferably, the fluorescent dye compound has the structure shown below:

[0026]

[0027] Among them, R1-R3, Z -The definitions of , n are the same as described above.

[0028] Preferably, Z － Selected from Cl - Br - ,I - ,OTs － 、ClO4 - PF6 - ;

[0029] Preferably, R1, R2, and R3 are selected from any one of hydrogen, halogen, benzyl, ester, amide, carbonyl, aldehyde, carbonyl, nitro, substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C1-10 alkoxy, substituted or unsubstituted C1-10 alkylamino, substituted or unsubstituted C3-C30 cycloalkyl, substituted or unsubstituted C1-C30 alkylthio, substituted or unsubstituted C6-C30 aryl, and substituted or unsubstituted C3-C30 heteroaryl;

[0030] Wherein, the substituents in the substituted C1-C15 alkyl, substituted C1-10 alkoxy, substituted C1-10 alkylamino, substituted C3-C30 cycloalkyl, substituted C1-C30 alkylthio, substituted C6-C30 aryl, and substituted C3-C30 heteroaryl are selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C60 alkyl, C2-C60 alkenyl, C3-C60 cycloalkyl, C2-C60 alkynyl, C3-C60 cycloalkyl, C1-C60 heterocycloalkyl, C3-C60 cycloalkenyl, C1-C60 heterocycloalkenyl, and C6-C60 aryl;

[0031] Preferably, R1, R2, and R3 are selected from any one of hydrogen, halogen, benzyl, ester, amide, carbonyl, aldehyde, carbonyl, nitro, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-5 alkoxy, substituted or unsubstituted C1-5 alkylamino, substituted or unsubstituted C3-C15 cycloalkyl, substituted or unsubstituted C1-C150 alkylthio, substituted or unsubstituted C6-C15 aryl, and substituted or unsubstituted C3-C15 heteroaryl;

[0032] Wherein, the substituents in the substituted C1-C6 alkyl, substituted C1-5 alkoxy, substituted C1-5 alkylamino, substituted C3-C15 cycloalkyl, substituted C1-C150 alkylthio, substituted C6-C15 aryl, and substituted C3-C15 heteroaryl are selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C60 alkyl, C2-C60 alkenyl, C3-C60 cycloalkyl, C2-C60 alkynyl, C3-C60 cycloalkyl, C1-C60 heterocycloalkyl, C3-C60 cycloalkenyl, C1-C60 heterocycloalkenyl, and C6-C60 aryl;

[0033] Preferably, R1 is selected from H, D, F, Cl, Br, I, benzyl, ester group, amide group, carbonyl group, aldehyde group, carbonyl group, nitro group, substituted or unsubstituted A group, wherein the A group is selected from: phenyl, naphthyl, biphenyl group, terphenyl group, phenanthryl group, fluoranthene group, triphenylene group, dimethylfluorenyl group, diphenylfluorenyl group, spirobifluorenyl group, benzodimethylfluorenyl group, benzodiphenylfluorenyl group, benzospirobifluorenyl group, benzofuranyl group, dibenzofuranyl group, benzothiophenyl group, dibenzothiophenyl group, carbazolyl group, benzocarbazolyl group, dibenzocarbazolyl group;

[0034] wherein the substituent of the substituted A group is selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C50 alkyl, C2-C5 alkenyl, C3-C10 cycloalkyl, C2-C10 alkynyl, C3-C5 cycloalkyl, C1-C10 heterocycloalkyl, C3-C10 cycloalkenyl, C1-C10 heterocycloalkenyl, C6-C10 aryl;

[0035] Preferably, R2 and R3 are each independently selected from hydrogen, hydroxyl, a substituted or unsubstituted B group, wherein the B group is selected from methyl, phenyl, naphthyl, biphenyl, terphenyl, phenanthryl, fluoranthene, triphenylene, dimethylfluorenyl, diphenylfluorenyl, spirobifluorenyl, benzodimethylfluorenyl, benzodiphenylfluorenyl, benzospirobifluorenyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, carbazolyl, benzocarbazolyl, dibenzocarbazolyl or a group having the following structure:

[0036]

[0037] Among them, the substituents in the substituted B group are deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C50 alkyl, C2-C5 alkenyl, C3-C10 cycloalkyl, C2-C10 alkynyl, C3-C5 cycloalkyl, C1-C10 heterocycloalkyl, C3-C10 cycloalkenyl, C1-C10 heterocycloalkenyl, and C6-C10 aryl.

[0038] Preferably, R is selected from one of the following groups: -CN-S1, -C=C-S2;

[0039] Wherein, S1 is selected from hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C5-C12 heteroaryl, substituted or unsubstituted amide;

[0040] Wherein, the substituents in the substituted C1-C8 alkyl, substituted C3-C6 cycloalkyl, substituted C6-C12 aryl, substituted C5-C12 heteroaryl, and substituted amide are each independently selected from halogen, nitro, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C2-C6 alkynyl, C3-C10 cycloalkenyl, C6-C10 aryl, C3-C10 heteroaryl, and amide;

[0041] S2 is each independently selected from a substituted or unsubstituted onium group, wherein the substituents in the substituted group are each independently selected from a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C6-C12 aryl group, a substituted or unsubstituted C5-C12 heteroaryl group, a substituted or unsubstituted amide group;

[0042] Wherein, the substituents in the substituted C1-C8 alkyl, substituted C3-C6 cycloalkyl, substituted C6-C12 aryl, substituted C5-C12 heteroaryl, and substituted amide are each independently selected from halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C2-C6 alkynyl, C3-C10 cycloalkenyl, C6-C10 aryl, and C3-C10 heteroaryl;

[0043] Preferably, S1 is selected from substituted or unsubstituted D groups, wherein D groups include: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclopentyl, cyclohexyl, phenyl, tolyl, ethylphenyl, naphthyl, pyridyl, quinolyl, amide;

[0044] wherein the substituent in the substituted D group is selected from halogen, nitro, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, sulfhydryl, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C2-C6 alkynyl, C3-C10 cycloalkenyl, C6-C10 aryl, C3-C10 heteroaryl;

[0045] Preferably, the substituent in the substituted D group is selected from halogen, nitro, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, methyl, dimethylamino, tetrahydrothienyl, tetrahydrofuranyl, phalloidin, taxol, amide, or a group having the following structure:

[0046]

[0047] Preferably, each of the S2 groups is independently selected from a substituted or unsubstituted pyridinium group, a substituted or unsubstituted quinolinium group, a substituted or unsubstituted indole quaternary ammonium group, a substituted or unsubstituted benzindolium group, or a substituted or unsubstituted benzothiazolium group;

[0048] wherein the substituents in the substituted pyridinium group, substituted quinolinium group, substituted indole quaternary ammonium group, substituted benzindolium group, and substituted benzothiazolium group are each independently selected from halogen, cyano, hydroxyl, sulfonic acid, arsonyl, boric acid, carboxyl, amino, thiol, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C2-C6 alkynyl, C3-C10 cycloalkenyl, C6-C10 aryl, and C3-C10 heteroaryl;

[0049] Further preferably, the substituents in the substituted pyridinium group, substituted quinolinium group, substituted indole quaternary ammonium group, substituted benzindolium group, substituted benzothiazolium group are each independently selected from halogen, cyano group, hydroxyl group, sulfonic acid group, arsenic acid group, boric acid group, carboxyl group, amino group, thiol group or a group having the following structure:

[0050]

[0051] Preferably, S1 is selected from one of the following groups:

[0052]

[0053] Wherein, X is a halogen;

[0054] In the present application, phalloidin is a phalloidin group; taxol is a paclitaxel group;

[0055] Preferably, S2 is selected from one of the following groups:

[0056]

[0057] Preferably, the fluorescent dye compound has one of the following structures:

[0058] Preferably, the structure of the fluorescent dye compound includes the following structure:

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065] Among them, R1-R3, Z - The definitions of n are the same as those described above.

[0066] Preferably, R1, R2, and R3 are selected from any one of hydrogen, halogen, benzyl, ester, amide, carbonyl, aldehyde, carbonyl, nitro, substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C1-10 alkoxy, substituted or unsubstituted C1-10 alkylamino, substituted or unsubstituted C3-C30 cycloalkyl, substituted or unsubstituted C1-C30 alkylthio, substituted or unsubstituted C6-C30 aryl, and substituted or unsubstituted C3-C30 heteroaryl;

[0067] Wherein, the substituents in the substituted C1-C15 alkyl, substituted C1-10 alkoxy, substituted C1-10 alkylamino, substituted C3-C30 cycloalkyl, substituted C1-C30 alkylthio, substituted C6-C30 aryl, and substituted C3-C30 heteroaryl are selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C60 alkyl, C2-C60 alkenyl, C3-C60 cycloalkyl, C2-C60 alkynyl, C3-C60 cycloalkyl, C1-C60 heterocycloalkyl, C3-C60 cycloalkenyl, C1-C60 heterocycloalkenyl, and C6-C60 aryl;

[0068] Preferably, R1, R2, and R3 are selected from any one of hydrogen, halogen, benzyl, ester, amide, carbonyl, aldehyde, carbonyl, nitro, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-5 alkoxy, substituted or unsubstituted C1-5 alkylamino, substituted or unsubstituted C3-C15 cycloalkyl, substituted or unsubstituted C1-C150 alkylthio, substituted or unsubstituted C6-C15 aryl, substituted or unsubstituted C3-C15 heteroaryl; and / or,

[0069] R2 and R3 are each independently selected from any one of hydrogen-substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-5 alkoxy, substituted or unsubstituted C1-5 alkylamino, substituted or unsubstituted C3-C15 cycloalkyl, substituted or unsubstituted C1-C150 alkylthio, substituted or unsubstituted C6-C15 aryl, substituted or unsubstituted C3-C15 heteroaryl;

[0070] Wherein, the substituents in the substituted C1-C6 alkyl, substituted C1-5 alkoxy, substituted C1-5 alkylamino, substituted C3-C15 cycloalkyl, substituted C1-C150 alkylthio, substituted C6-C15 aryl, and substituted C3-C15 heteroaryl are selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C60 alkyl, C2-C60 alkenyl, C3-C60 cycloalkyl, C2-C60 alkynyl, C3-C60 cycloalkyl, C1-C60 heterocycloalkyl, C3-C60 cycloalkenyl, C1-C60 heterocycloalkenyl, and C6-C60 aryl;

[0071] Preferably, R1 is selected from H, D, F, Cl, Br, I, benzyl, ester group, amide group, carbonyl group, aldehyde group, carbonyl group, nitro group, substituted or unsubstituted A group, wherein the A group is selected from: methyl, methoxy, methoxyphenyl, phenyl, naphthyl, biphenyl, terphenyl, phenanthryl, fluoranthene, triphenylene, dimethylfluorenyl, diphenylfluorenyl, spirobifluorenyl, benzodimethylfluorenyl, benzodiphenylfluorenyl, benzospirobifluorenyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, carbazolyl, benzocarbazolyl, dibenzocarbazolyl;

[0072] wherein the substituent of the substituted A group is selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C50 alkyl, C2-C5 alkenyl, C3-C10 cycloalkyl, C2-C10 alkynyl, C3-C5 cycloalkyl, C1-C10 heterocycloalkyl, C3-C10 cycloalkenyl, C1-C10 heterocycloalkenyl, C6-C10 aryl;

[0073] Preferably, R2 and R3 are each independently selected from hydrogen, hydroxyl, a substituted or unsubstituted B group, wherein the B group is selected from methyl, phenyl, naphthyl, biphenyl, terphenyl, phenanthryl, fluoranthene, triphenylene, dimethylfluorenyl, diphenylfluorenyl, spirobifluorenyl, benzodimethylfluorenyl, benzodiphenylfluorenyl, benzospirobifluorenyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, carbazolyl, benzocarbazolyl, dibenzocarbazolyl or a group having the following structure:

[0074]

[0075] Among them, the substituents in the substituted B group are deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C50 alkyl, C2-C5 alkenyl, C3-C10 cycloalkyl, C2-C10 alkynyl, C3-C5 cycloalkyl, C1-C10 heterocycloalkyl, C3-C10 cycloalkenyl, C1-C10 heterocycloalkenyl, and C6-C10 aryl.

[0076] The present invention also provides a method for preparing the fluorescent dye compound described above, comprising the following steps:

[0077] Performing an aldehyde modification reaction on a compound having a structure as shown in the following formula A to obtain the fluorescent dye compound;

[0078] Cy-CHO formula A;

[0079] Wherein Cy- is selected from one of the following structures:

[0080]

[0081] Among them, R1-R3, Z - , n is defined as above;

[0082] Preferably, the aldehyde modification reaction is selected from an aldehyde-amino Schiff base reaction and an aldehyde-methyl dehydration condensation reaction.

[0083] Preferably, the preparation method of the structural compound shown in formula A comprises the following steps:

[0084] Sodium borohydride is added to the Cy compound solution for reaction, and then the solvent is evaporated, washed, and dried to obtain a first reaction product, and then the first reaction product is added to a phosphorus oxychloride solution for reaction, and then extracted to obtain a second reaction product, and the second product is reacted with 2,3-dichloro-5,6-dicyanobenzoquinone and purified to obtain Cy-CHO;

[0085] Wherein, the Cy compound is selected from one of the compounds having the following structure:

[0086]

[0087] Preferably, the solvent in the Cy compound solution is selected from one or more of acetonitrile, ethyl acetate, methanol, ethanol, dichloromethane, chloroform, toluene, and DMF;

[0088] Preferably, the molar ratio of the Cy compound and sodium borohydride added is (2-4):1;

[0089] Preferably, the reaction temperature of adding sodium borohydride to the Cy compound solution is 20-40° C. and the reaction time is 5-10 min.

[0090] Preferably, the solvent in the phosphorus oxychloride solution is selected from DMF;

[0091] Preferably, the molar ratio of the first reaction product to phosphorus oxychloride is 1:(30-50);

[0092] Preferably, the first reaction product is added to the phosphorus oxychloride solution and the reaction temperature is 60-80°C; the reaction time is 4-6h;

[0093] Preferably, the second product and 2,3-dichloro-5,6-dicyanobenzoquinone are added in a molar ratio of (4-6):1;

[0094] Preferably, the temperature for reacting the second product with 2,3-dichloro-5,6-dicyanobenzoquinone is 20-40° C., and the reaction time is 10-30 min.

[0095] The present invention also provides the use of the fluorescent dye compound described above and the fluorescent dye compound prepared by the preparation method described above in living cell fluorescence imaging and super-resolution imaging.

[0096] The present invention also provides a method for preparing a Cy compound, comprising the following steps:

[0097] E01: heating the compound of formula VII and the nucleophilic reagent 2-bromoethyl-1,3-dioxolane to obtain a compound of formula V;

[0098] E02: heating the compound of formula VIII and the nucleophilic reagent 2-bromoethyl-1,3-dioxolane to obtain a compound of formula VI;

[0099] E03: The compounds represented by formula V and VI are prepared in acetic anhydride under the catalysis of sodium acetate to form a compound represented by formula IV;

[0100]

[0101] E04: dissolving the compound of IV (n=1) in chloroform, and causing a condensation ring-closing reaction under concentrated sulfuric acid to obtain a compound of formula IX;

[0102]

[0103] Optionally, when R1 is hydrogen, the reaction process of step E04 is as follows:

[0104]

[0105] Optionally, when R1 is a methyl group, the reaction process of step E04 is as follows:

[0106]

[0107] Optionally, when R1 is a methoxy group, the reaction process of step E04 is as follows:

[0108]

[0109] Optionally, when R1 is phenyl, the reaction process of step E04 is as follows:

[0110]

[0111] Optionally, when R1 is a substituted phenyl group, the reaction process of step E04 is as follows:

[0112]

[0113] wherein X is selected from F, Cl, Br, I or -OCH3.

[0114] The present invention also provides a method for preparing other Cy compounds, comprising the following steps:

[0115]

[0116] S1: reacting the compound of formula VII with the compound having the structure of R3-Ar-Z at 80-120° C. for 6-36 hours to obtain a compound having the structure of formula V′; wherein the reaction solvent is selected from toluene or acetonitrile; and the added molar ratio of the compound of formula VII to the compound having the structure of R3-Ar-Z is 1:1-1:4;

[0117] S2: reacting the compound of formula VIII with the compound having the structure of R2-Ar-Z at 80-120° C. for 6-36 hours to obtain a compound having the structure of formula VI′; wherein the reaction solvent is selected from toluene or acetonitrile; and the added molar ratio of the compound of formula VII to the compound having the structure of R2-Ar-Z is 1:1-1:4;

[0118] S3 reacting the compound having the structure of formula V' with the compound having the structure of formula VI' to generate a compound having the structure of formula IV';

[0119] Wherein, R1, R2, R3, and Z are the same as defined above; Ar is selected from C1-C6 alkylene and C6-C15 arylene;

[0120] Preferably, Ar is selected from ethylene, methylene, phenylene, naphthylene;

[0121] When Z is Cl - Br - When KI or KBr is used as a catalyst, the reactions in steps S1 and S2 are carried out.

[0122] Beneficial effects of the present invention:

[0123] The fluorescent dye compound provided by the present invention has a Cy-R structure, wherein R is a group obtained by reacting with an aldehyde group; by introducing an R group on the basis of the Cy structure, the photophysical properties of the compound are greatly improved compared with the compound without R, thereby reducing the saturation loss laser power of the dye, thereby reducing the saturation loss power in stimulated emission depletion super-resolution imaging, enhancing the permeability and compatibility of the compound to living cells, and can be well used in stimulated emission depletion super-resolution imaging of living cells, thereby increasing the application range of the dye for biological imaging; and the compound having Cy-R has good photobleaching resistance and photostability, and can be applied to super-resolution imaging of living cells to characterize more cell internal details with ultra-high resolution;

[0124] Furthermore, the compounds provided by the present invention can provide nanoscale living cell imaging, provide more detailed structural information that cannot be obtained by conventional microscopic imaging, and can observe nanoscale microstructures and dynamic changes for a longer time and more clearly, which is of great significance for the in-depth understanding of life phenomena and the research of related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0125] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0126] Figure 1 This is the STED super-resolution microscopic image of local mitochondria in COS-7 cells corresponding to compound Cy3B-1;

[0128] Figure 2 This is the STED super-resolution microscopic image of local mitochondria in COS-7 cells corresponding to the fluorescent dye compound Ia6-1 in Example 4;

[0129] Figure 3 The STED super-resolution microscopic image of local mitochondria in COS-7 cells corresponding to the fluorescent dye compound Ia9-1 in Example 5.

[0130] Figure 4 This is the STED super-resolution microscopic image of local microfilaments and microtubules in Hala cells corresponding to the fluorescent dye compound Ia29-1 in Example 7.

[0131] Figure 5This is the STED super-resolution microscopic image of local mitochondria in COS-7 cells corresponding to the fluorescent dye compound Ia37-1 in Example 8.

[0132] Figure 6 This is the STED super-resolution microscopic image of COS-7 cell mitochondria corresponding to the fluorescent dye compound IIa5-1 in Example 11.

[0133] Figure 7 This is the STED super-resolution microscopic image of local mitochondria in COS-7 cells corresponding to the fluorescent dye compound IIa46-1 in Example 13.

[0134] Figure 8 This is the STED super-resolution microscopic image of local mitochondria in COS-7 cells corresponding to the fluorescent dye compound IIa53-1 in Example 14. DETAILED DESCRIPTION

[0135] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased.

[0136] Those skilled in the art will recognize that the chemical reactions described herein can be used to appropriately prepare many of the heterocyclic compounds of the present invention, and that other methods for preparing the compounds of the present invention are considered to be within the scope of the present invention. For example, the synthesis of the compounds not exemplified herein can be successfully accomplished by those skilled in the art by modification methods, such as appropriate protection of interfering groups, by utilizing other known reagents in addition to those described herein, or by making some conventional modifications to the reaction conditions. The compounds for which the synthesis methods are not mentioned in the present invention are all raw materials obtained from commercial sources.

[0137] Synthesis of some intermediates:

[0138] 1. Synthesis of compounds with the following structure:

[0139]

[0140] The synthesis of compound VI-1 comprises the following steps:

[0141] To a suspension of KI (3.2 g, 20 mol) in distilled MeCN (50 mL), 2-bromoethyl-1,3-dioxolane (5.2 mL, 44 mmol) was added under a nitrogen atmosphere and stirred at 100 ° C for 1 h under light-shielding conditions. 2,3,3-trimethyl-3H-indole (6.4 mL, 40 mmol) was added and the reaction was stirred under reflux for 48 h. The reaction was cooled to room temperature and filtered to remove the KBr salt. The solvent was removed in vacuo and purified by column chromatography (purified in MeOH / DCM and 0.5% Et3N) to obtain a dark red oil, which is compound VI-1. The structural characterization data of compound VI-1 are as follows: 1 H-NMR(400MHz,DMSO-d6):7.34(t,1H),7.06(t,1H),6.72(t,1H),6.36(t,1H),5.46(s,1H), 4.40(d,2H),4.00(m,1H),3.97(d,1H),3.87(t,2H),3.77(t,2H),1.82(d,2H),1.79(m,6H). 13 C-NMR(100MHz,DMSO-d6):26.4,29.1,37.2,57.2,64.7,73.4,106.9,108.6,16.8,120.8,126.7,140.9,146.6,160.4.HRMS-ESI:m / zcalcd M + for C 16 H 22 NO2 + ,259.1572;found,259.1576.

[0142] The synthesis method of compound VI-2 is similar to that of the present invention, except that 5-methyl-2,3,3-trimethyl-3H-indole is added to replace 2,3,3-trimethyl-3H-indole. The structural characterization data of compound VI-2 are as follows: HRMS-ESI: m / z calcd M + forC 16 H 22 NO2 + ,274.3835;found,274.3830.

[0143] The synthesis method of compound VI-3 is similar to that of the present invention, except that 5-methoxy-2,3,3-trimethyl-3H-indole is added to replace 2,3,3-trimethyl-3H-indole. The structural characterization data of compound VI-3 are as follows: HRMS-ESI: m / z calcd M + for C 17 H 24 NO3 +,290.3825,found,290.3829.

[0144] The synthesis method of compound VI-4 is similar to that of the present invention, except that 2,3,3-trimethyl-5-phenyl-3H-indole is added to replace 2,3,3-trimethyl-3H-indole. The structural characterization data of compound VI-4 are as follows: HRMS-ESI: m / z calcd M + forC 22 H 26 NO2 + ,336.4545,found,336.4550.

[0145] When X in formula VI-5 is F: The synthesis method of compound VI-5-1 is similar to that of compound VI-5-1, except that 5-fluorophenyl-2,3,3-trimethyl-3H-indole is added to replace 2,3,3-trimethyl-3H-indole. The structural characterization data of compound VI-5-1 are as follows: HRMS-ESI: m / z calcd M + for C 22 H 25 FNO2 + ,354.4449,found,354.4450.

[0146] When X in formula VI-5 is Cl: The synthesis method of compound VI-5-2 is similar to that of compound VI-5-2, except that 5-chlorophenyl-2,3,3-trimethyl-3H-indole is added to replace 2,3,3-trimethyl-3H-indole. The structural characterization data of compound VI-5-2 are as follows: HRMS-ESI: m / zcalcd M + for C 22 H 25 ClNO2 + ,370.8965,found,370.8962.

[0147] When X in formula VI-5 is Br: The synthesis method of compound VI-5-3 is similar to that of compound VI-5-5, except that 5-bromophenyl-2,3,3-trimethyl-3H-indole is added to replace 2,3,3-trimethyl-3H-indole. The structural characterization data of compound VI-5-3 are as follows: HRMS-ESI: m / zcalcd M + for C 22 H 25 BrNO2 + ,415.3505,found,415.3510.

[0148] When X in formula VI-5 is iodine: The synthesis method of compound VI-5-4 is similar to that of compound VI-5-5, except that 5-iodophenyl-2,3,3-trimethyl-3H-indole is added to replace 2,3,3-trimethyl-3H-indole. The structural characterization data of compound VI-5-4 are as follows: HRMS-ESI: m / z calcd M + for C 22 H 26 NO2 + ,336.4545,found,336.4550.

[0149] When X in formula VI-5 is methoxyphenyl: The synthesis method of compound VI-5-5 is similar to that of compound VI-5-5, except that 5-methoxyphenyl-2,3,3-trimethyl-3H-indole is added to replace 2,3,3-trimethyl-3H-indole. The structural characterization data of compound VI-5-5 are as follows: HRMS-ESI: m / z calcd M + for C 23 H 28 NO3 + ,366.4805,found,366.4809.

[0150] The synthesis of Cy3 compounds is shown in the following reaction formula:

[0151]

[0152] The synthesis of compound IV-3-1 (wherein Y is H) comprises the following steps: 1-ethyldioxolane-2,3,3-trimethylindole (compound VI-1 (Y is H)) (5.18 g, 20 mol), N, N'-diphenylformamidine (1.96 g, 10 mmol) and sodium acetate (0.41 g, 5 mmol) are placed in 5-8 mL of acetic anhydride, stirred at 80°C for 15 h under a nitrogen atmosphere and dark conditions, washed with DCM and saturated brine, dried over anhydrous sodium sulfate, the solvent is removed in vacuo, and purified by column chromatography (purified in MeOH / DCM=1:20) to obtain a dark green metallic luster solid. The structural characterization data of compound IV-3-1 (n=1) are as follows: 1H-NMR(400MHz,DMSO-d6):8.95(d,1H),8.12(d,2H),7.44(d,1H),7.34(t,1H),7.30(d,1H),7.06(t,1H),6.72(t,1H),6.55(d,1H),6.36(t,1H) ,5.64(t,1H),5.46(m,2H),5.33(d,1H),4.01(d,2H),4.0(s,4H),3.87( m,4H),3.77(t,2H),1.82(d,1H),1.79(m,6H),1.6(s,2H),1.44(m,6H). 13 C-NMR(100MHz,DMSO-d6):173.1,172.8,149.5,146.6,141.4,141.2,140.9,128.3,126.7,125.0,120.8,120.1,11 6.8,111.0,108.6,107.3,106.9,104.8,104.5,64.7,55.0,51.1,43.1,37.7,31.7,29.1,26.8,24.7.HRMS-ESI:m / z calcdM + for C 33 H 41 N2O4 + ,529.3061;found,529.3066.

[0153] The synthesis of compound IV-3-2 (Y is CH3) comprises the following steps: Compound VI-2 (Y is CH3) (2.74 g, 10 mol) and compound V (2.59 g, 10 mol), N, N'-diphenylformamidine (1.96 g, 10 mmol) and sodium acetate (0.41 g, 5 mmol) are placed in 5-8 mL of acetic anhydride, stirred at 80°C for 15 h under a nitrogen atmosphere and dark conditions, washed with DCM and saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed in vacuo, and purified by column chromatography (purified in MeOH / DCM=1:20) to obtain a dark green metallic solid compound IV-3-2 (n=1), HRMS-ESI: m / z calcd M + for C 34 H 43 N2O4 + 543.7275; found, 543.7278.

[0154] The synthesis of compound IV-3-3 (Y is OCH3) comprises the following steps which are similar to the synthesis of compound IV-3-1 except that compound VI-3 (Y is OCH3) is used to replace compound VI-1 (Y is H) to obtain compound IV-3-3 (n=1), HRMS-ESI: m / z calcd M + for C 34 H 43 N2O5 + 559.7265; found, 559.7260.

[0155] The synthesis of compound IV-3-4 (Y is ph) comprises the following steps, which are similar to the synthesis of compound IV-3-1 except that compound VI-4 (Y is ph) is used to replace compound VI-1 (Y is H) to obtain compound IV-3-4 (n=1), HRMS-ESI: m / z calcd M + for C 39 H 45 N2O4 + 605.7985; found, 605.7983.

[0156] The synthesis of compound IV-3-5 (Y is phOCH3) comprises the following steps, which are similar to the synthesis of compound IV-3-1 except that compound VI-5 (Y is phOCH3) is used to replace compound VI-1 (Y is H) to obtain compound IV-3-5 (n=1), HRMS-ESI: m / z calcd M + for C 40 H 47 N2O5 + 635.8245; found, 635.8249.

[0157] The synthesis of Cy5 compounds is shown in the following reaction formula:

[0158]

[0159] The synthesis method is similar to that of the Cy3 compounds, except that N,N'-diphenylformamidine is replaced with N-(3-phenylamino-2-propyleneylidene)aniline hydrochloride to obtain the corresponding Cy5 compounds.

[0160] The synthesis of compound IV-5-1 (wherein Y is H) comprises the following steps: 1-ethyldioxolane-2,3,3-trimethylindole (compound VI-1 (Y is H)) (5.18 g, 20 mol), hydrochloric acid-N-(3-phenylamino-2-propyleneylidene)aniline (2.58 g, 10 mmol) and sodium acetate (0.41 g, 5 mmol) are added to 5-8 mL of acetic anhydride, stirred at 80°C for 15 h under nitrogen atmosphere and dark conditions, washed with DCM and saturated brine, dried over anhydrous sodium sulfate, the solvent is removed in vacuo, and purified by column chromatography (purified in MeOH / DCM=1:20) to obtain a dark green metallic luster solid. Compound IV-5-1 is obtained, and the structural characterization data are as follows: HRMS-ESI: m / z calcd M + for C 35 H 43 N2O4 + ,555.7385;found,555.7381.

[0161] The synthesis of compound IV-5-2 (Y is CH3) comprises the following steps: Compound VI-2 (Y is CH3) (2.74 g, 10 mol) and compound V (2.59 g, 10 mol), N-(3-phenylamino-2-propyleneylidene)aniline hydrochloride (2.58 g, 10 mmol) and sodium acetate (0.41 g, 5 mmol) are added to 5-8 mL of acetic anhydride, stirred at 80°C for 15 h under a nitrogen atmosphere and dark conditions, washed with DCM and saturated brine, dried over anhydrous sodium sulfate, and the solvent is removed in vacuo, and purified by column chromatography (purified in MeOH / DCM=1:20) to obtain a dark green metallic solid compound IV-5-2, HRMS-ESI: m / z calcd M + for C 34 H 43 N2O4 + 569.7655; found, 569.7659.

[0162] The synthesis of compound IV-5-3 (Y is OCH3) comprises the following steps which are similar to the synthesis of compound IV-5-1 except that compound VI-3 (Y is OCH3) is used to replace compound VI-1 (Y is H) to obtain compound IV-5-3 (n=2), HRMS-ESI: m / z calcd M + for C 36 H 45 N2O5 + 585.7645; found, 585.7648.

[0163] The synthesis of compound IV-5-4 (Y is ph) comprises the following steps which are similar to the synthesis of compound IV-5-1 except that compound VI-4 (Y is ph) is used to replace compound VI-1 (Y is H) to obtain compound IV-5-4 (n=2), HRMS-ESI: m / z calcd M + for C 41 H 47 N2O4 + 631.8365; found, 631.8361.

[0164] The synthesis of compound IV-5-5 (Y is phOCH3) comprises the following steps which are similar to the synthesis of compound IV-5-1 except that compound VI-5 (Y is phOCH3) is used to replace compound VI-1 (Y is H) to obtain compound IV-5-5 (n=2), HRMS-ESI: m / z calcd M + for C 42 H 49 N2O5 + 661.8625; found, 661.8627.

[0165] The synthesis of Cy3B compounds is shown in the following reaction formula:

[0166]

[0167] The synthesis of compound Cy3B-1 comprises the following steps:

[0168]

[0169] Compound IV-3-1 was placed in a conical flask, anhydrous dichloromethane was added to completely dissolve it, 70-80% concentrated sulfuric acid was added dropwise while stirring vigorously, and the reaction was carried out at room temperature for 15-60 minutes. By TLC monitoring, obvious changes in the fluorescence color and intensity of the solution were observed, and the dichloromethane / chloroform layer was collected. The solvent was evaporated under vacuum to obtain the corresponding metallic luster solid, which was compound Cy3B-1. The structural characterization data of compound Cy3B-1 are as follows: 1H-NMR(400MHz,DMSO-d6):8.95(d,1H),8.12(d,1H),7.47(m,1H),7.44(d,1H),7.34(t,1H),7.30(d,1H),7.06(t,1H),6.72(t,1H),6.36(t,1H) ,3.88(t,2H),3.09(d,1H),2.99(d,1H),1.81(t,1H),1.79(m,6H),1.62 (d,1H),1.56(d,1H),1.44(m,6H),1.4(d,1H),1.36(s,1H),1.3(s,1H). 13 C-NMR(100MHz,DMSO-d6):183.5,164.4,146.6,141.2,141.4,140.9,135.2,128.3,127.9,126.7,125.0,120.8 ,120.1,116.8,111.0,109.6,108.6,78.2,76.8,52.8,50.9,49.0,43.3,31.3,27.1,26.7,25.0.HRMS-ESI:m / z calcdM + for C 29 H 31 N2O + ,423.2431,found,423.2435.

[0170] Example 1

[0171] This synthesis example provides a method for preparing the compound Cy3-CHO-1, comprising the following steps:

[0172]

[0173] Compound Cy3-H and MeOH were mixed, sodium borohydride was added for full reduction, and the mixture was reacted at room temperature for 15-30 minutes until the color of the solution changed significantly to light yellow, the solvent was evaporated, and the mixture was washed with DCM / H2O, dried over anhydrous sodium sulfate, and the solvent was evaporated to obtain the first reaction product; phosphorus oxychloride and DMF were stirred under ice bath conditions for 30 minutes, the first reaction product was gradually added dropwise, stirred at room temperature for 25 minutes, and the mixture was heated to 70°C for reaction for 5 hours. After the reaction, the mixture was poured into 40 mL of ice water, NaHCO3 was added to quench the reaction, DCM was extracted, dried over anhydrous sodium sulfate, the solvent was evaporated, DDQ was added for oxidation, and purified by column chromatography (purified in MeOH / DCM=1:20) to obtain a green metallic solid, which is compound Cy3-CHO-1. The structural characterization data of compound Cy3-CHO-1 are as follows: HRMS-ESI: m / z calcd M +for C 34 H 41 N2O5 + C 28 H 33 N2O + ,413.5845found,413.5840.

[0174] The preparation methods of other substances similar to compound Cy3-CHO-1 (the following reaction products) are similar to the preparation method of compound Cy3-CHO-1, except that different starting materials are used:

[0175]

[0176] wherein Y is selected from CH3, OCH3, Ph, PhOCH3, F, Cl, Br or I.

[0177] Example 2

[0178] This synthesis example provides a method for preparing the compound Cy5-CHO-1.

[0179]

[0180] The preparation process is similar to that of Synthesis Example 1, except that compound Cy3-H is replaced by compound Cy5-H. The structural characterization data of compound Cy5-CHO-1 are as follows: HRMS-ESI: m / z calcd M + for C 36 H 43 N2O5 + C 36 H 38 N2O4S,594.7700found,594.7720.

[0181] The preparation methods of other substances similar to compound Cy5-CHO-1 (the following reaction products) are similar to the preparation method of compound Cy5-CHO-1, except that different starting materials are used:

[0182]

[0183] wherein Y is selected from CH3, OCH3, Ph, PhOCH3, F, Cl, Br or I.

[0184] Example 3

[0185] This synthesis example provides a method for preparing the compound Cy3B-CHO-1, comprising the following steps:

[0186]

[0187] The preparation process is similar to that of Synthesis Example 1, except that compound Cy3-H is replaced by compound Cy3B-H. The structural characterization data of compound Cy3B-CHO-1 are as follows: 1 H-NMR(400MHz,DMSO-d6):9.88(d,1H),8.95(t,1H),8.12(t,1H),7.85(m,1H),7.74(m,1H),7.44(t,1H),7.30(t,1H), 7.22(m,1H),6.86(m,1H),3.88(m,2H),3.09(d,2H),1.81(t,2H),1.79(m,6H),1.63(d,2H),1.44(m,6H),1.36(d,2H). 13 C-NMR(100MHz,DMSO-d6):183.5,164.4,146.6,141.6,141.2,140.9,135.2,128.3,127.9,126.7,125.0,120.8 ,120.1,116.8,111.0,109.6,108.6,78.2,76.8,52.8,50.9,49.0,43.3,26.7,31.3,27.0,25.0.HRMS-ESI:m / z calcdM + for C 30 H 31 N2O2 + ,451.2380found,451.2386.

[0188] The preparation methods of other substances similar to compound Cy3B-CHO-1 (the following reaction products) are similar to the preparation method of compound Cy3B-CHO-1, except that different starting materials are used:

[0189]

[0190] Wherein, Y is selected from CH3, OCH3, Ph, PhOCH3.

[0191] Example 4

[0192] This example provides compound I a6 -1 preparation method, compound I a6 The structure of -1 is as follows:

[0193]

[0194] The preparation method of the raw material compound Cy5-CHO-CH3-ph-OH having the following structure is similar to the preparation method of Cy5-CHO-1, except that different starting materials are used;

[0195]

[0196] Compound I a6 -1, specifically comprising the following steps:

[0197] Compound Cy5-CHO-CH3-ph-OH (0.265 g, 0.5 mmol) and butylamine (0.073 g, 1 mmol) were dissolved in MeOH (6 mL), and 2 drops of DIPEA were added dropwise. The mixture was stirred at 45-55° C. overnight under a nitrogen atmosphere. After the reaction, KBH4 (0.54 g, 0.5 mmol) was added to the mixture for reduction reaction. The solvent was evaporated, and the mixture was extracted with DCM / H2O. DDQ (0.036 g, 0.25 mmol) was added to a CH2Cl2 solution for oxidation, and the mixture was purified by a chromatographic column (MeOH / DCM) to obtain compound I a6 -1. Compound I a6 -1 structure is characterized as follows: 1 H-NMR(400MHz,DMSO-d6):8.90(d,1H),7.73(t,1H),7.25(d,2H),7.22(d,1H), 7.18(m,1H),7.16(d,2H),6.70(m,1H),6.52(d,2H),6.55(s,1H),5.56(s,1H), 5.33(s,1H),4.81(s,1H),3.64(t,2H),3.59(t,2H),3.07(d,2H),2.60(d,2H), 2.53(d,2H),2.31(t,3H),1.79(m,6H),1.44(t,6H),1.38(t,2H),0.89(d,3H). 13 C-NMR(100MHz,DMSO-d6):173.1,172.8,148.0,144.9,140.7,138.4,138.1,137.4,134.5,130.4,128.0,125.0,127.7,125.9,126.2,127 .7,128.6,125.7,124.5,113.0,104.8,104.5,63.4,58.2,55.3,52.9,51.4,49.5,49.2,32.7,26.8,24.7,21.6,20.1,13.8.HRMS-ESI:m / z calcdM + for C 40 H50 N3O + ,558.8595found,558.8591.

[0198] Example 5

[0199] This example provides compound I a9 -1 preparation method, compound I a9 The structure of -1 is as follows:

[0200]

[0201] The preparation method of the raw material compound Cy3-CHO-OCH3-CH3-CH2SO3 having the following structure is similar to the preparation method of Cy3-CHO-1, except that different starting materials are used;

[0202]

[0203] Compound I a9 -1, specifically comprising the following steps:

[0204] Compound Cy3-CHO-OCH3-CH3-CH2SO3 (0.275 g, 0.5 mmol) and cyclohexylamine (0.099 g, 1 mmol) were dissolved in MeOH (6 mL), and 2 drops of DIPEA were added dropwise. The mixture was stirred overnight at 45-55°C under a nitrogen atmosphere. After the reaction was completed, KBH4 (0.54 g, 0.5 mmol) was added to the mixture for reduction reaction. The solvent was evaporated, and the mixture was extracted with DCM / H2O. DDQ (0.036 g, 0.25 mmol) was added to a CH2Cl2 solution for oxidation, and the mixture was purified by a chromatographic column (MeOH / DCM) to obtain compound I a9 -1. Compound I a9 -1 structure characterization is as follows: HRMS-ESI: m / z calcd M + for C 37 H 51 SN3O4 + ,633.8920found,633.8923.

[0205] Example 6

[0206] This example provides compound I a17 -1 preparation method, compound I a17 The structure of -1 is as follows:

[0207]

[0208] The preparation method of the raw material compound Cy3-CHO--ph-H-COOH having the following structure is similar to the preparation method of Cy3-CHO-1, except that different starting materials are used;

[0209]

[0210] Compound I a17 -1, specifically comprising the following steps:

[0211] Compound Cy3-CHO--ph-H-COOH (0.266 g, 0.5 mmol) and p-aminophenylarsonic acid (0.2 g, 0.8 mmol) were dissolved in MeOH (6 mL), and 2 drops of DIPEA were added dropwise. The mixture was stirred at 45-55°C overnight. After the reaction was completed, KBH4 (0.54 g, 0.5 mmol) was added to the mixture for reduction reaction. The solvent was evaporated, and the mixture was extracted with DCM / H2O. DDQ (0.036 g, 0.25 mmol) was added to CH2Cl2 solution for oxidation, and the mixture was purified by chromatography (MeOH / DCM) to obtain compound I a17 -1. Compound I a17 -1 structure characterization is as follows: HRMS-ESI: m / z calcd M + for C 43 H 47 AsS2N3O2 + ,776.9090;found776.9096.

[0212] Example 7

[0213] This example provides compound I a29 -1 preparation method, compound I a17 The structure of -1 is as follows:

[0214]

[0215] The preparation method of the raw material compound Cy5-CHO-H-phB(OH)2-OH having the following structure is similar to the preparation method of Cy5-CHO-1, except that different starting materials are used;

[0216]

[0217] Compound I a29 -1 is prepared in the same manner as compound I in Example 4 a6 -1 was prepared in a similar manner, except that the compound Cy5-CHO-CH3-ph-OH was replaced by the compound Cy5-CHO-H-phB(OH)2--OH, and the butylamine was replaced by the amidated phalloidin product.

[0218] Example 8

[0219] This example provides compound I a37 -1 preparation method, compound I a37 The structure of -1 is as follows:

[0220]

[0221] The preparation method of the raw material compound Cy3-CHO-IHH having the following structure is similar to the preparation method of Cy3-CHO-1, except that different starting materials are used;

[0222]

[0223] Compound I a37 -1 is prepared in the same manner as compound I in Example 5 a9 -1 is similar in preparation method, except that the compound Cy3-CHO-OCH3-CH3-CH2SO3 is replaced by the compound Cy3-CHO-IHH, and the cyclohexylamine is replaced by 2-dimethylamino-5-aminopyridine.

[0224] Example 9

[0225] This example provides compound I b41 -1 preparation method, compound I b41 The structure of -1 is as follows:

[0226]

[0227] The preparation method of the raw material compound Cy5-CHO-Cl-phNO2-H having the following structure is similar to the preparation method of Cy5-CHO-1, except that different starting materials are used;

[0228]

[0229] Compound I b41 -1, specifically comprising the following steps:

[0230] Compound Cy5-CHO-Cl-phNO2-H (0.259 g, 0.5 mmol) and 4-methylquinolate (0.158 g, 1 mmol) were dissolved in MeOH (6 mL), 5 drops of piperidine were added, and the mixture was refluxed at 80°C for 24 h. After the reaction, the solvent was evaporated and purified by column chromatography (MeOH / DCM) to obtain compound I b41 -1. Compound I b41 The structural characterization of -1 is as follows: 1H-NMR(400MHz,DMSO-d6):9.01(t,1H),8.95(d,1H),8.60(s,1H),8.42(s,1H),8. 23(s,1H),8.12(d,1H),8.10(s,1H),7.94(t,1H),7.71(m,1H),7.44(d,1H),7.34( m,1H),7.30(d,1H),6.62(t,1H),4.39(d,3H),7.47(t,1H),6.95(t,2H),3.88(t,2 H),3.09(d,2H),1.81(t,2H),1.79(m,6H),1.63(d,2H),1.44(d,6H),1.36(s,2H). 13 C-NMR(100MHz,DMSO-d6):183.5,164.4,157.0,146.3,145.8,141.4,141.2 ,140.8,139.8,136.0,135.2,133.4,128.3,127.9,126.9,126.8,125.0,124 .8,124.5,123.9,123.4,122.1,120.1,118.6,111.0,109.6,107.7,78.2,76 .8,52.8,51.3,49.0,45.3,43.3,31.3,27.1,26.7,25.0.RMS-ESI:m / zcalcd M + for C 42 H 43 ClN4O2 2+ ,671.2809;found,671.2806.

[0231] Example 10

[0232] This example provides compound II b51 -1 preparation method, compound II b51 The structure of -1 is as follows:

[0233]

[0234] The specific steps are as follows:

[0235] Compound Cy3B-CHO-1 (0.225 g, 0.5 mmol) and 1,3,3, trimethylbenzidindol salt (0.223 g, 1 mmol) were dissolved in MeOH (6 mL), 5 drops of piperidine were added, and the mixture was reacted at 55-65°C for 24 h. After the reaction, the solvent was evaporated and the mixture was purified by column chromatography (MeOH / DCM) to obtain compound II. b51-1. Compound II b51 The structural characterization of -1 is as follows: 1 H-NMR(400 MHz,DMSO-d6):8.95(d,1H),8.12(d,1H),8.08(s,1H),8.04(s,1H),7.79(m,1H),7 .79(m,1H),7.71(m,1H),7.65(d,1H),7.62(d,1H),7.44(d,1H),7.34(m,1H),7.32 (d,1H),6.79(d,1H),6.62(m,1H),5.67(d,1H),4.07(d,2H),3.88(t,2H),3.09(d, 2H),1.81(t,2H),1.79(m,6H),1.63(d,2H),1.44(d,6H),1.57(d,6H),1.41(m,3H). 13 C-NMR(100 MHz, DMSO-d6):167.5,164.6,164.4,152.8,150.1,145.8,141.0,140.8,140.1,135.2,129.5,127.9,127.2,126.9,126.8,124.5,123.3,120 .0,115.1,112.2,109.6,107.7,77.9,75.3,58.0,57.1,56.0,53.6,51.3,43.3,34.2,30.7,30.0,29.0,26.7,24.8,27.1.HRMS-ESI:m / zcalcd M + forC 47 H 49 N3O + C 43 H 47 N3O + ,671.9297 found,671.9293.

[0236] Embodiment 11

[0237] This example provides compound II a5 -1 preparation method, compound II a5 The structure of -1 is as follows:

[0238]

[0239] The preparation method of the raw material compound Cy3B-CHO-ph having the following structure is similar to the preparation method of Cy3B-CHO-1, except that different starting materials are used;

[0240]

[0241] Compound II a5 -1, specifically comprising the following steps:

[0242] Cy3B-CHO-ph (0.263 g, 0.5 mmol) and pentylamine (0.087 g, 1 mmol) were dissolved in MeOH (6 mL), and 2 drops of DIPEA were added. The mixture was stirred overnight at 45-55°C under a nitrogen atmosphere. After the reaction, KBH4 (0.54 g, 0.5 mmol) was added to the mixture for reduction reaction. The solvent was evaporated, and the mixture was extracted with DCM / H2O. DDQ (0.036 g, 0.25 mmol) was added to CH2Cl2 solution for oxidation, and the mixture was purified by chromatography (MeOH / DCM) to obtain compound II a5 -1. Compound II a5 -1 structure is characterized as follows: 1 H-NMR(400MHz,DMSO-d6):9.14(m,1H),8.06(m,1H),7.77(m,1H),7.75(d,2H),7.75( d,2H),7.41(m,1H),7.41(d,1H),7.37(m,1H),7.18(m,1H),6.70(m,1H),4.16(t,1H), 3.88(t,2H),3.64(t,2H),3.09(d,2H),2.53(d,2H),1.81(t,2H),1.79(m,6H),1.63( d,2H),1.44(t,6H),1.38(t,2H),1.36(s,2H),1.29(t,2H),1.28(t,2H),0.88(d,3H). 13 C-NMR(100MHz,DMSO-d6):183.5,164.4,148.6,144.9,141.4,141.2,140.7,140.3,135.2,129.2,127.9,127.7,125.9,125.0 ,124.5,109.6,78.2,76.8,53.1,52.9,51.2,49.5,49.0,43.3,31.3,30.2,29.2,26.7,27.1,25.0,22.4,14.1.HRMS-ESI:m / z calcdM + for C 41 H 48 N3O + ,598.8545found,598.8540.

[0243] Example 12

[0244] This example provides compound II a15 -1 preparation method, compound II a15 The structure of -1 is as follows:

[0245]

[0246] The preparation method of the raw material compound Cy3B-CHO-Cl having the following structure is similar to the preparation method of Cy3B-CHO-1, except that different starting materials are used;

[0247]

[0248] Compound II a5 -1 preparation, specifically comprising the following steps:

[0249] Cy3B-CHO-Cl (0.240 g, 0.5 mmol) and 4-aminophenol (0.109 g, 1 mmol) were dissolved in MeOH (6 mL), and 2 drops of DIPEA were added. The mixture was stirred at 45-55°C overnight under a nitrogen atmosphere. After the reaction, KBH4 (0.54 g, 0.5 mmol) was added to the mixture for reduction reaction. The solvent was evaporated, and the mixture was extracted with DCM / H2O. DDQ (0.036 g, 0.25 mmol) was added to a CH2Cl2 solution for oxidation, and the mixture was purified by a chromatographic column (MeOH / DCM) to obtain compound II a15 -1. Compound II a15 -1 structure is characterized as follows: 1 H-NMR(400MHz,DMSO-d6):9.44(s,1H),8.95(d,1H),8.12(d,1H),7.47(m,1 H),7.44(d,1H),7.37(m,1H),7.35(t,1H),7.30(d,1H),7.18(m,2H),7.08( d,1H),6.83(d,3H),6.70(m,1H),6.67(d,1H),4.20(t,2H),3.88(t,1H),3. 09(d,2H),1.81(t,2H),1.79(m,6H),1.63(d,2H),1.44(t,6H),1.36(s,2H). 13C-NMR(100MHz,DMSO-d6):183.5,164.4,149.3,144.6,141.6,141.2,140.7,135.2,129.7,129.5,128.3,127.9,126.7,125.0,1 24.9,120.8,120.1,113.5,113.0,111.0,109.6,78.2,76.8,52.8,51.2,49.0,48.3,43.3,31.3,27.1,26.7,25.0.HRMS-ESI:m / z calcdM + for C 36 H 37 N3ClO2 + ,579.1605found,579.1608.

[0250] Example 13

[0251] This example provides compound IIb 46 -1 preparation method, compound IIb 46 The structure of -1 is as follows:

[0252]

[0253] The preparation method of the raw material compound Cy3B-CHO-CH3 having the following structure is similar to the preparation method of Cy3B-CHO-1, except that different starting materials are used;

[0254]

[0255] Compound IIb 46 -1, specifically comprising the following steps:

[0256] Compound Cy3B-CHO-CH3 (0.233 g, 0.5 mmol) and 4-methylquinolate (0.158 g, 1 mmol) were dissolved in MeOH (6 mL), 5 drops of piperidine were added, and the mixture was refluxed at 80°C for 24 h. After the reaction, the solvent was evaporated and purified by column chromatography (MeOH / DCM) to obtain compound IIb 46 -1. Compound IIb 46 The structural characterization of -1 is as follows: 1H-NMR(400MHz,DMSO-d6):9.01(t,1H),8.95(d,1H),8.60(s,1H),8.42(s,1H),8. 23(s,1H),8.12(d,1H),8.10(s,1H),7.94(t,1H),7.71(m,1H),7.44(d,1H),7.34( m,1H),7.30(d,1H),6.62(t,1H),4.39(d,3H),7.47(t,1H),6.95(t,2H),3.88(t,2 H),3.09(d,2H),1.81(t,2H),1.79(m,6H),1.63(d,2H),1.44(d,6H),1.36(s,2H). 13 C-NMR(100MHz,DMSO-d6):183.5,164.4,157.0,146.3,145.8,141.4,141.2 ,140.8,139.8,136.0,135.2,133.4,128.3,127.9,126.9,126.8,125.0,124 .8,124.5,123.9,123.4,122.1,120.1,118.6,111.0,109.6,107.7,78.2,76 .8,52.8,51.3,49.0,45.3,43.3,31.3,27.1,26.7,25.0.RMS-ESI:m / zcalcd M + for C 42 H 43 N3O 2+ ,605.8249found,606.8244.

[0257] Embodiment 14

[0258] This example provides compound I b53 -1 preparation method, compound I b53 -1. The structure is as follows:

[0259]

[0260] The preparation method of the raw material compound Cy3-CHO-phOCH3-O-CH2CCH having the following structure is similar to the preparation method of Cy3-CHO-1, except that different starting materials are used;

[0261]

[0262] Compound I b53 -1 is prepared in the same manner as compound II in Example 10 b51-1 is similar in preparation method, except that the compound Cy3B-CHO-1 is replaced by the compound Cy3-CHO-phOCH3-O-CH2CCH, and the 1,3,3, trimethylbenzindole salt is replaced by 5-carboxy-2,2,3 trimethylindole salt.

[0263] Embodiment 15

[0264] This example provides compound II a26 -1 preparation method, compound II a26 The structure of -1 is as follows:

[0265]

[0266] The preparation method of the raw material compound Cy3B-CHO-OCH3, having the following structure, is similar to the preparation method of Cy3B-CHO-1, except that different starting materials are used;

[0267]

[0268] Compound II a26 -1 is prepared in the same manner as compound II in Example 14 b51 -1 is similar in preparation method, except that the compound Cy3B-CHO-1 is replaced by the compound Cy3B-CHO-OCH3, and the 1,3,3, trimethylbenzindole salt is replaced by the naphthylamine modified with the morpholine group. Compound II a26 -1.HRMS-ESI: m / z calcd M + for C 47 H 53 N4O4 + ,737.9645found,737.9641.

[0269] Example 16

[0270] This example provides compound II a29 -1 preparation method, compound II a29 The structure of -1 is as follows:

[0271]

[0272] The preparation method of the raw material compound Cy3B-CHO-I having the following structure is similar to the preparation method of Cy3B-CHO-1, except that different starting materials are used;

[0273]

[0274] Compound II a29 -1 is prepared in the same manner as compound II in Example 14b51 -1 was prepared in a similar manner, except that the compound Cy3B-CHO-1 was replaced by the compound Cy3B-CHO-I, and the 1,3,3, trimethylbenzindole salt was replaced by the amidated phalloidin product.

[0275] Application Examples

[0276] The preparation of biological samples is to achieve specific labeling of target structures and obtain high-resolution and high signal-to-noise ratio imaging results. The invention uses living cells (macrophages) as biological samples and adopts cell staining and labeling to conduct imaging experiments; among them, the macrophages used in the test include Hela cells, COS-7 cells, and 4T1 cells; Hela cells are a cell line derived from Henrietta Lacks' cervical cancer cells; COS-7 cells are a cell line derived from African green monkey kidney fibroblasts and transformed with SV40 virus genes; 4T1 cells are derived from mouse breast cancer cells.

[0277] Live cell imaging test, the test process is as follows:

[0278] Resuscitate and passage macrophages according to standard experimental methods, including:

[0279] Cell recovery: Before cell recovery, place frozen cells and culture medium in a water bath and heat to 37°C to dissolve. Operate on a clean bench, transfer the thawed cell suspension to a centrifuge tube, add culture medium, and centrifuge at 1500rpm for 5 minutes; discard the supernatant, add culture medium to suspend the cells at the bottom and blow gently; transfer the mixed cell solution to a cell culture bottle, place it in a cell culture incubator (37°C, 5% CO2), and culture for 24 hours before changing the medium or performing subculture according to the cell density.

[0280] Cell passaging: When the cell density reaches more than 80% under the microscope, cell passaging can be performed. First, wash the cells with PBS solution 1-2 times, add trypsin to digest, blow off the adherent cells, centrifuge the cell solution, blow it evenly, and transfer it to the culture bottle.

[0281] Cell cryopreservation: The operation of cell cryopreservation is basically the same as that of cell passage, except that the supernatant is removed after centrifugation, and the cryopreservation solution is added, blown evenly, transferred to cryopreservation tubes, and stored in liquid nitrogen tanks. The cell cryopreservation solution is composed of DMSO, FBS and cell culture medium in a ratio of 1:2:7.

[0282] After the macrophages were revived and passaged according to the standard experimental method, multiple cell culture media containing the revived and passaged macrophages were prepared, and DMSO solutions (concentration of 0.5 μM) of compound Cy3B-1, compound Ia6-1, Ia9-1, Ia29-1, Ia37-1, IIa5-1, IIa46-1, and IIa53-1 were added to the culture media respectively. After culturing at 37° C. and 5% carbon dioxide concentration for 30 minutes, the culture media were observed under a laser confocal microscope, and an excitation wavelength of 561 nm was selected to collect fluorescence above 575 nm. When STED super-resolution imaging was performed, the excitation wavelength was 561 nm, the loss light was 775 nm, and the fluorescence above 575 nm was collected to observe the confocal and STED imaging of living cells in the culture media; imaging results of the same target structure were obtained under the two acquisition modes of CLSM and STED. Figure 1 This is the STED super-resolution microscopic image corresponding to compound CyB (the image corresponds to COS-7 cells) Figure 2 This is the STED super-resolution microscopic image of COS-7 cells corresponding to compound Ia6-1; Figure 3 This is the STED super-resolution microscopic image of COS-7 cells corresponding to compound Ia9-1; Figure 4 This is the STED super-resolution microscopic image of local microfilaments and microtubules in Hala cells corresponding to compound Ia29-1. Figure 5 This is the STED super-resolution microscopic image of local mitochondria in COS-7 cells corresponding to compound Ia37-1. Figure 6 This is the STED super-resolution microscopic image of COS-7 cell mitochondria corresponding to compound IIa5-1. Figure 7 This is the STED super-resolution microscopic image of local mitochondria in the cell corresponding to compound IIa46-1. Figure 8 This is the STED super-resolution microscopic image of local mitochondria in the cell corresponding to compound IIa53-1.

[0283] The probe can achieve live cell staining within 5-10 minutes, fully proving its good biocompatibility and excellent cell permeability. Based on the use of Cy3B-1 dye for live cell imaging, the effects of Cy3B-1 confocal imaging and STED super-resolution imaging are not good. The dye can only be vaguely seen entering the mitochondria, and no specific fine structure can be observed; the image structures of the example compounds Ia6-1, Ia9-1, Ia29-1, Ia37-1, IIa5-1, IIa46-1, and IIa53-1 show that mitochondria can show more detailed information under STED super-resolution imaging conditions than CLSM, and the structure of mitochondrial cristae and microfilaments and microtubules on the cytoskeleton can be seen, and the contrast is significantly improved. The structure of the cristae is very clear, and STED super-resolution imaging of mitochondria can be achieved. It has the performance of super-resolution imaging, realizes spectral performance adjustment, improves the photostability of the dye, and exhibits excellent performance such as low phototoxicity.

[0284] The STED super-resolution imaging effect of the cells corresponding to compounds Ia17-1, Ib41-1, IIb51-1, Ib53-1IIa26-1, and IIa29-1 can also observe the structure of mitochondrial cristae, which also has a good super-resolution effect.

[0285] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A fluorescent dye compound, characterized in that: It has the structure shown in the following formula B: Cy-R formula B; Wherein, R is a group obtained by reacting with an aldehyde group; Cy- is selected from one of the following structures: Wherein, R1, R2, and R3 are selected from any one of hydrogen, halogen, benzyl, ester group, amide group, carbonyl group, aldehyde group, carbonyl group, nitro group, substituted or unsubstituted C1-C30 alkyl group, substituted or unsubstituted C1-20 alkoxy group, substituted or unsubstituted C1-20 alkylamino group, substituted or unsubstituted C3-C60 cycloalkyl group, substituted or unsubstituted C1-C60 alkylthio group, substituted or unsubstituted C6-C60 aryl group, substituted or unsubstituted C3-C60 heteroaryl group; Z - It is a negative ion; n is an integer from 1 to 2; Among them, the substituents in the substituted C1-C30 alkyl, substituted C1-20 alkoxy, substituted C1-20 alkylamino, substituted C3-C60 cycloalkyl, substituted C1-C60 alkylthio, substituted C6-C60 aryl, and substituted C3-C60 heteroaryl are selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C60 alkyl, C2-C60 alkenyl, C3-C60 cycloalkyl, C2-C60 alkynyl, C3-C60 cycloalkyl, C1-C60 heterocycloalkyl, C3-C60 cycloalkenyl, C1-C60 heterocycloalkenyl, and C6-C60 aryl.

2. The fluorescent dye compound according to claim 1, characterized in that The fluorescent dye compound has the structure shown below: Among them, R1-R3, Z - The definition of n is the same as that in claim 1.

3. The fluorescent dye compound according to claim 1 or 2, characterized in that: Z － Selected from Cl - Br - ,I - ,OTs － 、ClO4 - PF6 - ; Preferably, R1, R2, and R3 are selected from any one of hydrogen, halogen, benzyl, ester, amide, carbonyl, aldehyde, carbonyl, nitro, substituted or unsubstituted C1-C15 alkyl, substituted or unsubstituted C1-10 alkoxy, substituted or unsubstituted C1-10 alkylamino, substituted or unsubstituted C3-C30 cycloalkyl, substituted or unsubstituted C1-C30 alkylthio, substituted or unsubstituted C6-C30 aryl, and substituted or unsubstituted C3-C30 heteroaryl; Wherein, the substituents in the substituted C1-C15 alkyl, substituted C1-10 alkoxy, substituted C1-10 alkylamino, substituted C3-C30 cycloalkyl, substituted C1-C30 alkylthio, substituted C6-C30 aryl, and substituted C3-C30 heteroaryl are selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C60 alkyl, C2-C60 alkenyl, C3-C60 cycloalkyl, C2-C60 alkynyl, C3-C60 cycloalkyl, C1-C60 heterocycloalkyl, C3-C60 cycloalkenyl, C1-C60 heterocycloalkenyl, and C6-C60 aryl; Preferably, R1, R2, and R3 are selected from any one of hydrogen, halogen, benzyl, ester, amide, carbonyl, aldehyde, carbonyl, nitro, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-5 alkoxy, substituted or unsubstituted C1-5 alkylamino, substituted or unsubstituted C3-C15 cycloalkyl, substituted or unsubstituted C1-C150 alkylthio, substituted or unsubstituted C6-C15 aryl, substituted or unsubstituted C3-C15 heteroaryl; and / or, R2 and R3 are each independently selected from any one of hydrogen-substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-5 alkoxy, substituted or unsubstituted C1-5 alkylamino, substituted or unsubstituted C3-C15 cycloalkyl, substituted or unsubstituted C1-C150 alkylthio, substituted or unsubstituted C6-C15 aryl, substituted or unsubstituted C3-C15 heteroaryl; Wherein, the substituents in the substituted C1-C6 alkyl, substituted C1-5 alkoxy, substituted C1-5 alkylamino, substituted C3-C15 cycloalkyl, substituted C1-C150 alkylthio, substituted C6-C15 aryl, and substituted C3-C15 heteroaryl are selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C60 alkyl, C2-C60 alkenyl, C3-C60 cycloalkyl, C2-C60 alkynyl, C3-C60 cycloalkyl, C1-C60 heterocycloalkyl, C3-C60 cycloalkenyl, C1-C60 heterocycloalkenyl, and C6-C60 aryl; Preferably, R1 is selected from H, D, F, Cl, Br, I, benzyl, ester group, amide group, carbonyl group, aldehyde group, carbonyl group, nitro group, substituted or unsubstituted A group, wherein the A group is selected from: methyl, methoxy, methoxyphenyl, phenyl, naphthyl, biphenyl, terphenyl, phenanthryl, fluoranthene, triphenylene, dimethylfluorenyl, diphenylfluorenyl, spirobifluorenyl, benzodimethylfluorenyl, benzodiphenylfluorenyl, benzospirobifluorenyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, carbazolyl, benzocarbazolyl, dibenzocarbazolyl; wherein the substituent of the substituted A group is selected from deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C50 alkyl, C2-C5 alkenyl, C3-C10 cycloalkyl, C2-C10 alkynyl, C3-C5 cycloalkyl, C1-C10 heterocycloalkyl, C3-C10 cycloalkenyl, C1-C10 heterocycloalkenyl, C6-C10 aryl; Preferably, R2 and R3 are each independently selected from hydrogen, hydroxyl, a substituted or unsubstituted B group, wherein the B group is selected from methyl, phenyl, naphthyl, biphenyl, terphenyl, phenanthryl, fluoranthene, triphenylene, dimethylfluorenyl, diphenylfluorenyl, spirobifluorenyl, benzodimethylfluorenyl, benzodiphenylfluorenyl, benzospirobifluorenyl, benzofuranyl, dibenzofuranyl, benzothiophenyl, dibenzothiophenyl, carbazolyl, benzocarbazolyl, dibenzocarbazolyl or a group having the following structure: Among them, the substituents in the substituted B group are deuterium, halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C50 alkyl, C2-C5 alkenyl, C3-C10 cycloalkyl, C2-C10 alkynyl, C3-C5 cycloalkyl, C1-C10 heterocycloalkyl, C3-C10 cycloalkenyl, C1-C10 heterocycloalkenyl, and C6-C10 aryl.

4. The fluorescent dye compound according to any one of claims 1 to 3, characterized in that: R is selected from one of the following groups: -CN-S1, -C=C-S2; Wherein, S1 is selected from hydrogen, substituted or unsubstituted C1-C8 alkyl, substituted or unsubstituted C3-C6 cycloalkyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C5-C12 heteroaryl, substituted or unsubstituted amide; Wherein, the substituents in the substituted C1-C8 alkyl, substituted C3-C6 cycloalkyl, substituted C6-C12 aryl, substituted C5-C12 heteroaryl, and substituted amide are each independently selected from halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C2-C6 alkynyl, C3-C10 cycloalkenyl, C6-C10 aryl, and C3-C10 heteroaryl; S2 is each independently selected from a substituted or unsubstituted onium group, wherein the substituents in the substituted group are each independently selected from a substituted or unsubstituted C1-C8 alkyl group, a substituted or unsubstituted C3-C6 cycloalkyl group, a substituted or unsubstituted C6-C12 aryl group, a substituted or unsubstituted C5-C12 heteroaryl group, a substituted or unsubstituted amide group; Wherein, the substituents in the substituted C1-C8 alkyl, substituted C3-C6 cycloalkyl, substituted C6-C12 aryl, substituted C5-C12 heteroaryl, and substituted amide are each independently selected from halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, thiol, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C2-C6 alkynyl, C3-C10 cycloalkenyl, C6-C10 aryl, and C3-C10 heteroaryl; Preferably, S1 is selected from substituted or unsubstituted D groups, wherein D groups include: methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclopentyl, cyclohexyl, phenyl, tolyl, ethylphenyl, naphthyl, pyridyl, quinolyl, amide; wherein the substituent in the substituted D group is selected from halogen, cyano, hydroxyl, sulfonic acid, arsenic acid, boric acid, carboxyl, amino, sulfhydryl, C1-C6 alkyl, C2-C6 alkenyl, C3-C6 cycloalkyl, C3-C6 heterocycloalkyl, C2-C6 alkynyl, C3-C10 cycloalkenyl, C6-C10 aryl, C3-C10 heteroaryl; Preferably, each of the S2 groups is independently selected from a substituted or unsubstituted pyridinium group, a substituted or unsubstituted quinolinium group, a substituted or unsubstituted indole quaternary ammonium group, a substituted or unsubstituted benzindolium group, or a substituted or unsubstituted benzothiazolium group; Wherein, the substituents in the substituted pyridinium group, substituted quinolinium group, substituted indole quaternary ammonium group, substituted benzindolium group and substituted benzothiazolium group are each independently selected from halogen, cyano group, hydroxyl group, sulfonic acid group, arsonyl group, boric acid group, carboxyl group, amino group, thiol group, C1-C6 alkyl group, C2-C6 alkenyl group, C3-C6 cycloalkyl group, C3-C6 heterocycloalkyl group, C2-C6 alkynyl group, C3-C10 cycloalkenyl group, C6-C10 aryl group and C3-C10 heteroaryl group.

5. The fluorescent dye compound according to any one of claims 1 to 4, characterized in that: The fluorescent dye compound has one of the following structures: Preferably, S1 is selected from one of the following groups: Preferably, S2 is selected from one of the following groups:

6. The fluorescent dye compound according to any one of claims 1 to 5, characterized in that: The structure of the fluorescent dye compound includes the following structure: Among them, R1-R3, Z - , n is as defined in claim 1 or 2.

7. A method for preparing the fluorescent dye compound according to any one of claims 1 to 6, characterized in that: The steps include: Performing an aldehyde modification reaction on a compound having a structure as shown in the following formula A to obtain the fluorescent dye compound; Cy-CHO formula A; Wherein Cy- is selected from one of the following structures: Among them, R1-R3, Z - , n is as defined in claim 1; Preferably, the aldehyde modification reaction is selected from an aldehyde-amino Schiff base reaction and an aldehyde-methyl dehydration condensation reaction.

8. The preparation method according to claim 7, characterized in that: The preparation method of the structural compound shown in formula A comprises the following steps: Sodium borohydride is added to the Cy compound solution for reaction, and then the solvent is evaporated, washed, and dried to obtain a first reaction product, and then the first reaction product is added to a phosphorus oxychloride solution for reaction, and then extracted to obtain a second reaction product, and the second product is reacted with 2,3-dichloro-5,6-dicyanobenzoquinone and purified to obtain Cy-CHO; Wherein, the Cy compound is selected from one of the compounds having the following structure:

9. The preparation method according to claim 7 or 8, characterized in that: The solvent in the Cy compound solution is selected from one or more of acetonitrile, ethyl acetate, methanol, ethanol, dichloromethane, chloroform, toluene, and DMF; Preferably, the molar ratio of the Cy compound and sodium borohydride added is (2-4):1; Preferably, the reaction temperature of adding sodium borohydride to the Cy compound solution is 20-40° C. and the reaction time is 5-10 min. Preferably, the solvent in the phosphorus oxychloride solution is selected from DMF; Preferably, the molar ratio of the first reaction product to phosphorus oxychloride is 1:(30-50); Preferably, the first reaction product is added to the phosphorus oxychloride solution and the reaction temperature is 60-80°C; the reaction time is 4-6h; Preferably, the second product and 2,3-dichloro-5,6-dicyanobenzoquinone are added in a molar ratio of (4-6):1; Preferably, the temperature for reacting the second product with 2,3-dichloro-5,6-dicyanobenzoquinone is 20-40° C., and the reaction time is 10-30 min.

10. Use of the fluorescent dye compound according to any one of claims 1 to 6 or the fluorescent dye compound prepared by the preparation method according to any one of claims 7 to 9 in living cell fluorescence imaging and super-resolution imaging.

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