Aza [3.2. 1] bridged ring substituted fluorescent dye as well as preparation method and application thereof

Abstract

The invention provides an aza [3.2. 1] bridged ring substituted fluorescent dye as well as a preparation method and application of the aza [3.2. 1] bridged ring substituted fluorescent dye. According to the fluorescent dye, an aza [3.2. 1] bridge ring is adopted as a substituent group of the fluorescent dye, so that the brightness and the light stability of the fluorescent dye are remarkably improved, the light blue shift phenomenon generated after the dye is illuminated for a long time is thoroughly avoided, meanwhile, the water solubility of the fluorescent dye can be effectively improved, and good cell membrane permeability of the fluorescent dye can be kept. The invention also provides an embodiment of the functionalized derivative of the dye for single-molecule imaging, confocal imaging and super-resolution imaging in a biological sample.

Description

Technical Field

[0001] The present invention relates to the field of fluorescent dyes in biological fluorescence analysis, and in particular to an aza[3.2.1] bridge ring substituted fluorescent dye and a preparation method and application thereof. Background Art

[0002] In recent years, fluorescent dyes have been widely used in the fields of biomacromolecule labeling, ion detection, single molecule and super-resolution imaging, and have become an irreplaceable and important tool for life science research, which also puts higher requirements on the physical and chemical properties of fluorescent dyes. Traditional fluorescent dyes such as fluorescein, tetramethylrhodamine, coumarin, etc. usually lack sufficient brightness, water solubility and photostability. In the past three decades, chemists have introduced different new substituent groups on the fluorescent dye matrix to improve the fluorescence quantum yield, photostability, resistance to photooxidative dealkylation (photoblue shift), water solubility and cell membrane permeability of the dyes, but the challenge is that the strategies for optimizing fluorescent dyes that have been reported can only optimize certain properties individually and cannot comprehensively improve all the above core properties of the dyes.

[0003] Reference Nature Methods. 2015, 12: 244-250. The brightness of fluorescent dyes was systematically improved by substitution with azetidine, but the photostability of such dyes was poor and they did not have the ability to resist photooxidative dealkylation (photoblue shift); Reference J. Am. Chem. Soc. 2008, 130, 52: 17652-17653. The brightness, photostability and resistance to photooxidative dealkylation (photoblue shift) of dyes were improved by substitution with azabicyclo[2.2.1]heptane for rhodamine, but the water solubility and cell membrane permeability of such dyes were poor, which hindered their application in living cell imaging; the introduction of sulfonic acid groups into fluorescent dye matrix can greatly improve the water solubility of dyes, increase antibody labeling efficiency and reduce nonspecific staining, but it also hinders the dyes from penetrating cell membranes, making sulfonated dyes unable to be used in living cells.

[0004] Therefore, those skilled in the art are in urgent need of developing a class of fluorescent dyes that can simultaneously improve fluorescence quantum yield, photostability, resistance to photooxidative dealkylation (photoblue shift), water solubility and cell membrane permeability. Summary of the invention

[0005] The main purpose of the present invention is to provide an aza[3.2.1] bridged ring substituted fluorescent dye and a preparation method and application thereof, so as to solve the problem in the prior art that the fluorescence quantum yield, photostability, resistance to photooxidative dealkylation (photoblue shift), water solubility and cell membrane permeability of fluorescent dyes cannot be optimized simultaneously.

[0006] In order to achieve the above object, according to one aspect of the present invention, there is provided an aza[3.2.1] bridged ring substituted fluorescent dye, wherein the aza[3.2.1] bridged ring substituted fluorescent dye has a structure shown in the following formula (I), formula (II) or formula (III):

[0007]

[0008] R a , R b , R c , R d , R e , R f , R g , R h , R i , R j , R k , R l , R m , R n , R o , R p , R q Each is independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C4-C20 cycloalkylalkyl, C4-C20 alkylcycloalkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C1-C20 ester, substituted or unsubstituted C1-C20 amide;

[0009] R A Selected from substituted or unsubstituted C6-C40 aryl, substituted or unsubstituted C6-C40 aralkyl, and substituted or unsubstituted C5-C40 heteroaryl;

[0010] X is selected from oxygen, sulfur, substituted or unsubstituted C1-C20 alkylene,

[0011] Y is selected from oxygen, sulfur, nitrogen, substituted or unsubstituted C1-C20 alkylene, substituted or unsubstituted C1-C20 alkyleneoxy, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 ester, Among them, R B is selected from a substituted or unsubstituted C1-C20 alkyl group, a substituted or unsubstituted C1-C20 alkoxy group, a substituted or unsubstituted C1-C20 acyl group, and a substituted or unsubstituted C1-C20 ester group.

[0012] Z - Selected from F - , Cl - Br- ,I - ,OAc - 、HSO4 - 、H2PO4 - 、ClO4 - CF3COO - CF3SO3 - 、CH3SO3 - 、NO3 - 、C5H7O5COO - PF6 - ; m is an integer from 1 to 3.

[0013] Furthermore, R a , R b , R c , R d , R e , R f , R g , R h , R i , R j , R k , R l , R m , R n , R o , R p , R q Each is independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C10 cycloalkylalkyl, C4-C10 alkylcycloalkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 amide; and / or, R A is selected from substituted or unsubstituted C6-C30 aryl, substituted or unsubstituted C6-C30 aralkyl, substituted or unsubstituted C5-C30 heteroaryl; and / or, X is selected from oxygen, sulfur, substituted or unsubstituted C1-C10 alkyl, and / or, Y is selected from oxygen, sulfur, nitrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 ester, Among them, R B is selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 ester; and / or, Z - Selected from F - , Cl - Br- ,I - ,OAc - 、HSO4 - 、H2PO4 - 、ClO4 - CF3COO - CF3SO3 - 、CH3SO3 - 、NO3 - 、C5H7O5COO - PF6 - ; and / or, m is an integer from 1 to 3.

[0014] Furthermore, the aza[3.2.1] bridged ring substituted fluorescent dye of formula (I) has a structure as shown in the following formula (IV):

[0015]

[0016] R1 and R5 are each independently selected from hydrogen, carboxyl, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, nitro, amino, hydroxyl,

[0017] Where R D R2, R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, and n is an integer of 1 to 6; R2, R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C10 cycloalkylalkyl, C4-C10 alkylcycloalkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 ester,

[0018] or any other functional groups for bioconjugation or subcellular organelle localization.

[0019] Furthermore, the aza[3.2.1] bridged ring substituted fluorescent dye is selected from at least one of the following formulas IA1-IA3, IIA1-IIA3, IIIA1-IIIA3, IB1-IB3, IC1-IC3, ID1-ID3, IE1-IE3, IF1-IF3, IG1-IG3:

[0020]

[0021]

[0022] Further, the aza[3.2.1] bridged ring substituted fluorescent dye is selected from the following formulas: A528, A532, A555, A586, A591, A620, A634, A640, A655, A720, A586-2Me, A586-2Me-5-COOH, A528-6-COOH, A532-6-COOH, A555-6-COOH, A586-6-COOH, A591-6-COOH, A620-6-COOH, A634-5-COOH, A640-6-COOH, A655-6-COOH, A528-HTL, A532-HTL, A555-HTL, A586-HTL, A At least one of A520-HTL, A655-HTL, A555-STL, A528-NHS, A620-STL, A655-STL, A634-NHS, A586-NHS, A591-HTL, A640-HTL, A555-TMP3, A620-TMP3, A655-TMP3, A655-4-Hoechst, A591-TMP3, A640-TMP3, A591-STL, A640-STL, A528-NCN-HTL, A555-NCN-HTL, A620-NCN-HTL, B350, B356, C622, and C654, wherein Me in the following formula represents a methyl group:

[0023]

[0024]

[0025]

[0026]

[0027] In order to achieve the above object, according to one aspect of the present invention, there is provided a method for preparing an aza[3.2.1] bridged ring substituted fluorescent dye, the method comprising: step S11, mixing compound A, an aza[3.2.1] bridged ring compound and a first catalyst in a first reaction solvent for reaction to obtain compound B; step S12, mixing compound B and an HZ aqueous solution for reaction to obtain an aza[3.2.1] bridged ring substituted fluorescent dye of formula (I); compound A has a structure as shown in the following formula (V), and the aza[3.2.1] bridged ring compound has a structure as shown in the following formula (VI):

[0028]

[0029] Among them, R A , Ra , R b , R c , R d , R e , R f , X, and Y each independently have the same meaning as in the first aspect, and Z in the HZ aqueous solution - Has the same meaning as in the first aspect, OTf is a trifluoromethanesulfonate group.

[0030] Further, the first catalyst includes a palladium catalyst, a palladium catalyst ligand and a basic catalyst.

[0031] Furthermore, the palladium catalyst includes at least one of tetrakistriphenylphosphine palladium, palladium acetate, trisdibenzylideneacetone dipalladium, diphenylphosphinothiocene palladium dichloride, ditriphenylphosphine palladium dichloride, and palladium chloride.

[0032] Further, the palladium catalyst includes at least one of tetrakistriphenylphosphine palladium, palladium acetate or trisdibenzylideneacetone dipalladium.

[0033] Further, the palladium catalyst ligand includes at least one of bis(dibenzylideneacetonepalladium), 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl, and 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl.

[0034] Further, the palladium catalyst ligand includes at least one of bis(dibenzylideneacetonepalladium) and 2,2'-bis(diphenylphosphino)-1,1'-binaphthyl.

[0035] Furthermore, the alkaline catalyst includes at least one of cesium carbonate, sodium tert-butoxide, potassium carbonate, sodium carbonate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide.

[0036] Furthermore, the alkaline catalyst includes at least one of cesium carbonate, sodium tert-butoxide, and potassium tert-butoxide.

[0037] Furthermore, the first reaction solvent includes at least one of n-hexane, tetrahydrofuran, 1,4-dioxane, diethyl ether and toluene.

[0038] In order to achieve the above object, according to one aspect of the present invention, a method for preparing an aza[3.2.1] bridged ring substituted fluorescent dye is provided, the method comprising: mixing a compound C, an aza[3.2.1] bridged ring compound and a second catalyst in a second reaction solvent to obtain an aza[3.2.1] bridged ring substituted fluorescent dye of formula (II);

[0039] Compound C has the structure shown in the following formula (VII):

[0040]

[0041] R g , R h , R i , R j , R k , X each independently have the same meaning as in the first aspect.

[0042] Further, the second catalyst and the first catalyst have the same meaning.

[0043] Further, the second reaction solvent and the first reaction solvent have the same meaning.

[0044] In order to achieve the above-mentioned purpose, according to one aspect of the present invention, there is provided a method for preparing an aza[3.2.1]bridged ring substituted fluorescent dye, the method comprising: step S31, mixing compound D, an aza[3.2.1]bridged ring compound and a third catalyst in a third reaction solvent to obtain compound E; step S32, mixing compound E and an oxidant in a fourth reaction solvent to obtain compound F; step S33, mixing compound F and an HZ aqueous solution to obtain an aza[3.2.1]bridged ring substituted fluorescent dye represented by formula (III).

[0045] Compound D has the structure shown in the following formula (VIII):

[0046]

[0047] R l , R m , R n , R o , R p , R q , X, and Y independently have the same meaning as in the first aspect, and Z in the HZ aqueous solution - Has the same meaning as the first aspect.

[0048] Further, the third catalyst and the first catalyst have the same meaning.

[0049] Further, the third reaction solvent and the first reaction solvent have the same meaning.

[0050] Furthermore, in step S32, the fourth reaction solvent is a mixed solvent of dichloromethane and water, and the volume ratio of dichloromethane to water is (10-30):1.

[0051] Further, in step S32, the oxidant includes at least one of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, potassium permanganate, hydrogen peroxide, and m-chloroperbenzoic acid.

[0052] Furthermore, the oxidant is 2,3-dichloro-5,6-dicyano-1,4-benzoquinone.

[0053] Furthermore, the molar ratio of the oxidant to the compound E is (1.0-2.0):1.

[0054] In order to achieve the above-mentioned object, according to one aspect of the present invention, there is provided an aza[3.2.1]bridge-ring substituted fluorescent dye or an aza[3.2.1]bridge-ring substituted fluorescent dye obtained by a preparation method of the aza[3.2.1]bridge-ring substituted fluorescent dye as a fluorescent marker in the fields of fluorescence analysis, fluorescence detection and fluorescence imaging.

[0055] Furthermore, the aza[3.2.1] bridge ring substituted fluorescent dye is used as a fluorescent marker in fluorescence analysis, fluorescence detection, biomacromolecule labeling, immunofluorescence imaging, living cell imaging, tissue imaging, in vivo imaging, single molecule imaging, and super-resolution fluorescence imaging.

[0056] By applying the technical solution of the present application, the aza[3.2.1] bridge ring substituted fluorescent dye provided by the present application adopts the aza[3.2.1] bridge ring as a substituent of the fluorescent dye, which not only significantly improves the brightness and photostability of the fluorescent dye, but also completely avoids the blue shift phenomenon of the dye after long-term illumination, and at the same time can effectively improve the water solubility of the fluorescent dye and maintain its good cell membrane permeability. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The drawings in the specification, which constitute a part of the present application, are used to provide further understanding of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute improper limitations on the present application.

[0058] Figure 1 shows the absorption spectra of the aza[3.2.1] bridged ring substituted fluorescent dyes according to Examples 1 to 4, 6, 7, 9, 18, 20, and 21 of the present application;

[0059] Figure 2 The emission spectra of the aza[3.2.1] bridged ring substituted fluorescent dyes according to Examples 1 to 4, 6, 7, 9, 18, 20, and 21 of the present application are shown;

[0060] Figure 3 A comparison curve of the photostability differences in the orange channel according to Examples 3 and 10 of the present application and Comparative Examples 1 to 4 is shown;

[0061] Figure 4 A comparison curve of the photostability difference in the far-red channel of Example 7 and Comparative Example 5 of the present application is shown;

[0062] Figure 5Shows a single molecule imaging photograph on fixed U2OS cells according to Example 27 of the present application;

[0063] Figure 6 A comparison of single molecule brightness on fixed U2OS cells according to Example 27 of the present application and Comparative Examples 6 and 7 is shown;

[0064] Figure 7 A comparison chart of the photostability of Example 27 and Comparative Examples 6 and 7 on fixed U2OS cells according to the present application is shown;

[0065] Figure 8 The confocal fluorescence imaging photograph of TMP-Halo-SNAP-GFP-H2B protein labeled in living Hela cells according to Example 27 of the present application is shown;

[0066] Fig. 9 Shows a confocal fluorescence imaging photograph of TMP-Halo-SNAP-GFP-H2B protein labeled in living Hela cells according to Example 31 of the present application;

[0067] Fig.10 Shows a confocal fluorescence imaging photograph of TMP-Halo-SNAP-GFP-H2B protein labeled in living Hela cells according to Example 34 of the present application;

[0068] Fig.11 A comparison of the relative fluorescence intensity of TMP-Halo-SNAP-GFP-H2B protein labeled in living Hela cells according to Example 27 of the present application and Comparative Examples 6 and 8 is shown;

[0069] Fig.12 A comparison of the relative fluorescence intensity of TMP-Halo-SNAP-GFP-H2B protein labeled in living Hela cells according to Example 31 of the present application and Comparative Example 9 is shown;

[0070] Fig.13 The confocal fluorescence imaging photograph of TMP-Halo-SNAP-GFP-H2B protein labeled in living Hela cells according to Example 28 of the present application is shown;

[0071] Fig.14 A comparison of the relative fluorescence intensity of TMP-Halo-SNAP-GFP-H2B protein labeled in living Hela cells according to Example 28 of the present application and Comparative Example 10 is shown;

[0072] Fig.15 Shows a wash-free super-resolution fluorescence imaging photograph of Lifeact-Halo protein labeled in living Hela cells on a STED microscope according to Example 34 of the present application;

[0073] Fig.16 A comparison chart showing the photostability of Lifeact-Halo protein labeled in living Hela cells under STED microscope super-resolution fluorescence imaging conditions according to Example 34 of the present application and Comparative Example 11 is shown. DETAILED DESCRIPTION

[0074] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0075] As described in the background technology of the present application, in the prior art, an azetidine group is introduced into the fluorescent dye matrix to improve the brightness of the fluorescent dye, but this type of fluorescent dye has poor photostability and does not have the ability to resist photooxidative dealkylation (photoblue shift); in addition, an azabicyclo[2.2.1]heptane is introduced into the fluorescent dye matrix to replace rhodamine to improve the fluorescence quantum yield, photostability, and resistance to photooxidative dealkylation (photoblue shift) of the dye, but this type of dye has poor water solubility and cell membrane permeability, which hinders its application in living cell imaging; the introduction of a sulfonic acid group into the fluorescent dye matrix can improve the water solubility of the dye, improve the antibody labeling efficiency and reduce nonspecific staining, but it will hinder the dye from passing through the cell membrane, making the sulfonated dye unable to be used in living cells. In order to solve this problem, the present application provides an aza[3.2.1] bridge ring substituted fluorescent dye and its preparation method and application.

[0076] In a typical embodiment of the present application, an aza[3.2.1] bridged ring substituted fluorescent dye is provided, wherein the aza[3.2.1] bridged ring substituted fluorescent dye has a structure shown in the following formula (I), formula (II) or formula (III):

[0077]

[0078]

[0079] R a , R b , R c , R d , R e , R f , R g , R h , R i , R j , R k , R l , R m , R n , R o , R p , R qEach is independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C4-C20 cycloalkylalkyl, C4-C20 alkylcycloalkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C1-C20 ester, substituted or unsubstituted C1-C20 amide.

[0080] R A It is selected from a substituted or unsubstituted C6-C40 aryl group, a substituted or unsubstituted C6-C40 aralkyl group, and a substituted or unsubstituted C5-C40 heteroaryl group.

[0081] X is selected from oxygen, sulfur, substituted or unsubstituted C1-C20 alkylene,

[0082] Y is selected from oxygen, sulfur, nitrogen, substituted or unsubstituted C1-C20 alkylene, substituted or unsubstituted C1-C20 alkyleneoxy, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 ester, Among them, R B Selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 ester;

[0083] Z - Selected from F - , Cl - Br - ,I - ,OAc - 、HSO4 - 、H2PO4 - 、ClO4 - CF3COO - CF3SO3 - 、CH3SO3 - 、NO3 - 、C5H7O5COO - PF6 - ;

[0084] In the above formula (I), Z - It can exist in ionic form and can also be bonded to R A Connection, when m ≥ 2, Z - The two forms can exist at the same time, and m is an integer from 1 to 3.

[0085] In the present application, [aryl] refers to a monocyclic or condensed polycyclic ring derived from an aromatic hydrocarbon, and includes phenyl, biphenyl, terphenyl, naphthyl, binaphthyl, phenylnaphthyl, naphthylphenyl, fluorenyl, phenylfluorenyl, benzofluorenyl, dibenzofluorenyl, phenanthrenyl, phenylphenanthrenyl, anthracenyl, indenyl, triphenylene, pyrene, condensed tetraphenyl, perylenyl, quinacyl, naphthacene, allenylfluorenyl, and the like.

[0086] "Heteroaryl" refers to a heteroaryl group, which refers to an aromatic group containing at least one heteroatom selected from the group consisting of N, O, and S as the main chain atom of the ring, which can be a monocyclic ring system such as furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, thiadiazolyl, isothiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, triazinyl, tetrazinyl, triazolyl, tetrazolyl, furazanyl, pyridinyl, pyrazinyl , pyrimidinyl, pyridazinyl, etc., or a condensed ring condensed with at least one benzene ring, such as benzofuranyl, benzothiophenyl, isobenzofuranyl, dibenzofuranyl, dibenzothiophenyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, quinolyl, isoquinolyl, cinnolinyl, quinazolinyl, quinoxalinyl, carbazolyl, phenanthroxazolyl, phenanthridinyl, benzodiacenaphthenyl, dihydroacridinyl, etc.

[0087] The term "substituted" in "substituted or unsubstituted" means that a hydrogen atom in a functional group is replaced by another atom or group (i.e., a substituent). Each of the substituents is independently selected from at least one of the following groups: deuterium, halogen, C1-C10 alkyl, C1-C10 alkoxy, C6-C10 aryl, C5-C10 heteroaryl, C5-C10 heteroaryl substituted by C6-C10 aryl, imidazolyl, benzimidazolyl, C3-C10 cycloalkyl, C5-C7 heterocycloalkyl, tri-(C1-C10)alkylsilyl, tri-(C1-C10)arylsilyl, di-(C1-C10)silyl, 10) alkyl-(C6-C10) arylsilyl, C1-C10 alkyldi-(C6-C10) arylsilyl, C2-C10 alkenyl, C2-C30 alkynyl, cyano, di-(C1-C30) alkylamino, di-(C6-C30) arylboryl, di-(C1-C30) alkylboryl, C1-C30 alkyl, C6-C10 arylC1-C10 alkyl, C1-C10 alkylC6-C10 aryl, carboxyl, nitro and hydroxyl,

[0088]

[0089] or any other functional groups for bioconjugation or subcellular organelle localization, R Dis selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C4, substituted or unsubstituted C1-C6 alkoxy, and n is a positive integer of 1 to 6. In addition, the range of carbon atoms herein can be extended from the lower limit to the upper limit, for example, C6-C10 means that the carbon atoms can be 6, 7, 8, 9 or 10.

[0090] In this application, "*" represents a connecting bond.

[0091] By applying the technical solution of the present application, the aza[3.2.1] bridge ring substituted fluorescent dye provided by the present application adopts the aza[3.2.1] bridge ring as a substituent of the fluorescent dye, which not only significantly improves the brightness and photostability of the fluorescent dye, but also completely avoids the blue shift phenomenon of the dye after long-term illumination, and at the same time can effectively improve the water solubility of the fluorescent dye and maintain its good cell membrane permeability.

[0092] In order to further improve the performance of the above-mentioned aza[3.2.1] bridged ring substituted fluorescent dye, R a , R b , R c , R d , R e , R f , R g , R h , R i , R j , R k , R l , R m , R n , R o , R p , R q Each is independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C10 cycloalkylalkyl, C4-C10 alkylcycloalkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 amide.

[0093] In some embodiments, preferably R A It is selected from a substituted or unsubstituted C6-C30 aryl group, a substituted or unsubstituted C6-C30 aralkyl group, and a substituted or unsubstituted C5-C30 heteroaryl group.

[0094] In other embodiments, X is preferably selected from oxygen, sulfur, substituted or unsubstituted C1-C10 alkyl, Preferably, Y is selected from oxygen, sulfur, nitrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 ester, Among them, R B is selected from a substituted or unsubstituted C1-C20 alkyl group, a substituted or unsubstituted C1-C20 alkoxy group, a substituted or unsubstituted C1-C20 acyl group, and a substituted or unsubstituted C1-C20 ester group.

[0095] In some other embodiments, Z - Selected from F - , Cl - Br - ,I - ,OAc - 、HSO4 - 、H2PO4 - 、ClO4 - CF3COO - CF3SO3 - 、CH3SO3 - 、NO3 - 、C5H7O5COO - PF6 - , m is a positive integer from 1 to 3, OAc - It is the acetate ion.

[0096] In order to further improve the performance of the above-mentioned aza[3.2.1] bridged ring substituted fluorescent dye, it is preferred that the aza[3.2.1] bridged ring substituted fluorescent dye has a structure as shown in the following formula (IV):

[0097]

[0098] R1 and R5 are each independently selected from hydrogen, carboxyl, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, nitro, amino, hydroxyl,

[0099] Where R D It is selected from hydrogen, halogen, cyano, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C1-C6 alkoxy, and n is an integer of 1-6.

[0100] R2, R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C10 cycloalkylalkyl, C4-C10 alkylcycloalkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 ester,

[0101] or any other functional groups for bioconjugation or subcellular organelle localization.

[0102] In some specific embodiments of the present application, the aza[3.2.1] bridged ring substituted fluorescent dye is selected from at least one of the following formulas IA1-IA3, IIA1-IIA3, IIIA1-IIIA3, IB1-IB3, IC1-IC3, ID1-ID3, IE1-IE3, IF1-IF3, IG1-IG3, wherein Ph is phenyl:

[0103]

[0104]

[0105] In other specific embodiments of the present application, the aza[3.2.1] bridged ring substituted fluorescent dye is selected from the following formulas: A528, A532, A555, A586, A591, A620, A634, A640, A655, A720, A586-2Me, A586-2Me-5-COOH, A528-6-COOH, A532-6-COOH, A555-6-COOH, A586-6-COOH, A591-6-COOH, A620-6-COOH, A634-5-COOH, A640-6-COOH, A655-6-COOH, A528-HTL, A532-HTL, A555-HTL At least one of L, A586-HTL, A620-HTL, A655-HTL, A555-STL, A528-NHS, A620-STL, A655-STL, A634-NHS, A586-NHS, A591-HTL, A640-HTL, A555-TMP3, A620-TMP3, A655-TMP3, A655-4-Hoechst, A591-TMP3, A640-TMP3, A591-STL, A640-STL, A528-NCN-HTL, A555-NCN-HTL, A620-NCN-HTL, B350, B356, C622, C654:

[0106]

[0107]

[0108]

[0109]

[0110] In a second typical embodiment of the present application, a method for preparing an aza[3.2.1] bridged ring substituted fluorescent dye of formula (I) is also provided, the preparation method comprising: step S11, mixing compound A, an aza[3.2.1] bridged ring compound and a first catalyst in a first reaction solvent to obtain compound B; step S12, mixing compound B and an HZ aqueous solution to obtain an aza[3.2.1] bridged ring substituted fluorescent dye of formula (I).

[0111] Compound A has a structure as shown in the following formula (V), and the aza[3.2.1] bridged ring compound has a structure as shown in the following formula (VI):

[0112]

[0113] R A , R a , R b , R c , R d , R e , R f , X, Y and R in the first typical embodiment A , R a , R b , R c , R d , R e , R f , X, and Y have the same meaning. Z in HZ aqueous solution - In the first exemplary embodiment, Z - They have the same meaning and are not repeated here.

[0114] In some embodiments, the reaction diagram is shown in the following formula (IX):

[0115]

[0116] In order to further improve the reaction efficiency, in the above step S11, the first catalyst includes a palladium catalyst, a palladium catalyst ligand and an alkaline catalyst, wherein the palladium catalyst includes at least one of tetrakistriphenylphosphine palladium, palladium acetate, trisdibenzylideneacetone dipalladium, diphenylphosphinothiocene palladium dichloride, ditriphenylphosphine palladium dichloride, and palladium chloride, and is preferably at least one of tetrakistriphenylphosphine palladium, palladium acetate or trisdibenzylideneacetone dipalladium.

[0117] In the present application, palladium catalysts and palladium ligands are mainly used for catalytic reactions. Different palladium catalyst ligands have different electron-rich substituents, which have a great influence on the yield of the reaction. In order to further improve the yield of the reaction, the palladium catalyst ligand is preferably at least one of bis(dibenzylideneacetonepalladium), 2,2'-bis(diphenylphosphino-1,1'-binaphthyl, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl, and 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl, and is further preferably at least one of bis(dibenzylideneacetonepalladium) and 2,2'-bis(diphenylphosphino-1,1'-binaphthyl.

[0118] The specific type of the alkaline catalyst is not limited. Common alkaline catalysts in the art include at least one of cesium carbonate, sodium tert-butoxide, potassium carbonate, sodium carbonate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide, preferably at least one of cesium carbonate, sodium tert-butoxide, and potassium tert-butoxide. The above alkaline catalysts can not only improve the ability of deprotonation, but also increase the compatibility of functional groups in the reaction compounds, thereby improving the reaction efficiency.

[0119] In the above step S11, in order to further improve the yield of the aza[3.2.1] bridge-ring substituted fluorescent dye, it is preferred that the above reaction is carried out under anhydrous and oxygen-free conditions, and the air in the reaction container is replaced with an inert gas, and the inert gas includes but is not limited to at least one of nitrogen, helium, and argon.

[0120] In the above step S11, in order to further improve the preparation efficiency of the aza[3.2.1] bridged ring substituted fluorescent dye, the molar ratio of the palladium catalyst to the compound A is preferably (0.1-1.0):1 (such as 0.1:1, 0.2:1, 0.4:1, 0.6:1, 0.8:1, 1:1 or a range consisting of any two numerical values), the molar ratio of the palladium catalyst ligand to the compound A is preferably (0.3-1.2):1 (such as 0.3:1, 0.6:1, 0.9:1, 1.2:1 or a range consisting of any two numerical values), and the molar ratio of the basic catalyst to the compound A is preferably (1.2-2.0) :1 (such as 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1 or a range value composed of any two numerical values), wherein the palladium catalyst and the palladium catalyst ligand are both precious metal catalysts, which are relatively expensive. If the feed amount is too large, the cost of the reaction will be increased; if the feed amount is too small, the effect of catalyzing the reaction cannot be achieved, resulting in a decrease in the reaction yield. Therefore, the palladium catalyst and the palladium catalyst ligand within the above range can reduce the reaction cost when the reaction is sufficient. Too little feed amount of the alkaline catalyst will lead to a decrease in the reaction yield, and too much feed amount of the alkaline catalyst will lead to complicated post-processing. Within the above range, the efficiency of the reaction can be further improved.

[0121] The specific type of the first reaction solvent is not specifically limited, and any commonly used reaction solvent in the art can be used, including but not limited to: at least one of n-hexane, tetrahydrofuran, 1,4-dioxane, ether, and toluene, which can completely dissolve the reactants, thereby improving the efficiency of the reaction.

[0122] In some embodiments, when the reactant is a benzene ring compound, toluene is often used as the first reaction solvent; when the reactant is a heterocyclic compound, 1,4-dioxane is often used as the first reaction solvent. According to the solubility of the reactant in the organic solvent, a suitable first reaction solvent is selected to improve the reaction efficiency.

[0123] In the above step S11, the reaction temperature is 60-120°C (such as 60°C, 70°C, 80°C, 90°C, 100°C, 110°C, 120°C or a range value composed of any two numerical values). If the reaction temperature is too high, a large amount of by-products will be generated, which may cause the decomposition of the intermediate. If the reaction temperature is too low, the reaction rate is low. If the reaction temperature is within the above range, the yield and reaction efficiency of the reaction can be further improved. The reaction time is 4-18h. When the reaction proceeds to a certain extent, increasing the reaction time will not only not increase the yield of the reaction, but may generate a large amount of by-products and reduce the yield of the reaction. If the reaction time is too short, the reaction is incomplete, resulting in a reduction in the reaction yield. Within the above range, the yield of the reaction can be improved while saving reaction time.

[0124] In some embodiments, in the above step S12, the reaction temperature is 20-30° C. The lower the reaction temperature, the simpler the reaction operation and the higher the reaction yield.

[0125] In the above step S12, in order to avoid violent reaction and release of a large amount of heat, in some embodiments, the HZ aqueous solution is slowly dripped into the reaction system of step S11 to neutralize the positive charge of the intermediate, so that the obtained aza[3.2.1] bridge ring substituted fluorescent dye is electrically neutral. Wherein, the molar concentration of the HZ aqueous solution is 1 to 6 mol / L, preferably 1 to 3 mol / L. If the molar concentration of the HZ aqueous solution is too high, the reaction will be violent, the reaction rate will be too fast, and the reaction will be difficult to control. If the molar concentration of the HZ aqueous solution is too low, a large amount of HZ aqueous solution needs to be added. Within the above range, the reaction rate can be effectively increased.

[0126] In some embodiments, in order to further improve the purity of the aza[3.2.1] bridge ring substituted fluorescent dye, post-treatment is performed after the reaction is completed, wherein the post-treatment process includes common post-treatment processes such as extraction, drying, column chromatography separation, etc.

[0127] In the third typical embodiment of the present application, a method for preparing an aza[3.2.1] bridge ring substituted fluorescent dye shown in formula (II) is also provided. The preparation method comprises: mixing compound C, an aza[3.2.1] bridge ring compound and a second catalyst in a second reaction solvent to obtain an aza[3.2.1] bridge ring substituted fluorescent dye shown in formula (II).

[0128] Compound C has the structure shown in the following formula (VII):

[0129]

[0130] R g , R h , R i , R j , R k , X and R in the first typical embodiment g , R h , R i , R j , R k , X have the same meaning, Z in HZ aqueous solution - In the first exemplary embodiment, Z - They have the same meaning and are not repeated here.

[0131] The second catalyst and the first catalyst have the same meaning; the second reaction solvent and the first reaction solvent have the same meaning, which will not be repeated here.

[0132] In some embodiments, the reaction diagram is shown in the following formula (X):

[0133]

[0134] The third typical embodiment of the present application is the same as the second typical embodiment except that the reaction compounds are different, the catalysts, the ratios of the catalysts to the compounds, the solvents, the reaction process parameters, and the post-treatment processes are the same, and therefore, they are not described in detail here.

[0135] In the fourth typical embodiment of the present application, a method for preparing an aza[3.2.1] bridge ring substituted fluorescent dye shown in formula (III) is also provided, and the preparation method comprises: step S31, mixing compound D, an aza[3.2.1] bridge ring compound and a third catalyst in a third reaction solvent to obtain compound E; step S32, mixing compound E and an oxidant in a fourth reaction solvent to obtain compound F; step S33, mixing compound F and an HZ aqueous solution to obtain an aza[3.2.1] bridge ring substituted fluorescent dye shown in formula (III).

[0136] Compound D has the structure shown in the following formula (VIII);

[0137]

[0138] R l , R m , R n , R o , R p , R q , X, Y and R in the first typical embodiment l , R m , R n , R o , R p , R q , X, and Y have the same meaning. Z in HZ aqueous solution - In the first exemplary embodiment, Z - They have the same meaning and are not repeated here.

[0139] The third catalyst and the first catalyst have the same meaning, and the third reaction solvent and the first reaction solvent have the same meaning, which will not be repeated here.

[0140] In some embodiments, the reaction diagram is shown in the following formula (XI):

[0141]

[0142] In the fourth typical embodiment of the present application, steps S31 and S33 are the same as steps S11 and S12 in the second typical embodiment except for the different reaction compounds. The catalysts, the ratio of the catalyst to the compound, the solvents, the reaction process parameters, and the post-treatment procedures are the same, and therefore, they are not described in detail here.

[0143] In the above step S32, the oxidant includes at least one of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, potassium permanganate, hydrogen peroxide (30% aqueous solution), and m-chloroperbenzoic acid, preferably 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, and the molar ratio of the oxidant to the compound D is (1.0-2.0):1 (such as 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1 or a range of any two values)

[0144] In order to further improve the yield of the reaction, in the above step S32, the fourth reaction solvent is preferably a mixed solvent of dichloromethane and water, wherein the volume ratio of dichloromethane to water is (10-30):1.

[0145] In the above step S32, the reaction temperature is 40 to 120° C. and the reaction time is 2 to 24 hours.

[0146] In the fifth typical embodiment of the present application, there is also provided the use of an aza[3.2.1] bridge ring substituted fluorescent dye as a fluorescent marker in the fields of fluorescence analysis, fluorescence detection, and fluorescence imaging. The aza[3.2.1] bridge ring substituted fluorescent dye is the aza[3.2.1] bridge ring substituted fluorescent dye provided in the first typical embodiment.

[0147] The invention relates to the use of aza[3.2.1] bridge ring substituted fluorescent dyes as fluorescent markers in fluorescence analysis, fluorescence detection, biomacromolecule labeling, immunofluorescence imaging, living cell imaging, tissue imaging, biological in vivo imaging, single molecule imaging and super-resolution fluorescence imaging.

[0148] The beneficial effects of the present application will be further illustrated below in combination with embodiments and comparative examples.

[0149] Example 1 Compound A528

[0150]

[0151] The compound A528 was prepared according to the following steps:

[0152]

[0153] (1) Under anhydrous and oxygen-free conditions, compound 1 (1 mol) and compound 2 (3 mol) were dissolved in toluene solvent, and palladium acetate (0.2 mol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (0.3 mol) and cesium carbonate (1.5 mol) were added in sequence. The reaction temperature was 100° C. and the reaction time was 18 h to obtain compound 3.

[0154] (2) A 2 mol / L hydrochloric acid aqueous solution (0.5 mL) was slowly dripped into the reaction system containing compound 3. The reaction time was 0.5 h and the reaction temperature was 25° C. After extraction, drying and column chromatography separation, compound A528 was obtained. The product was a red solid with a yield of 33%.

[0155] 1 H NMR (400MHz, CDCl3) δ8.03(d,J=7.6Hz,1H),7.71(td,J=7.5,1.2Hz,1H),7.63(td,J=7.4,1.2Hz,1H),7.28(d,J=7.6Hz,1H),6.72(d,J=8.7Hz,2H), 6.55(d,J=2.4Hz,2H),6.44(dd,J=8.8,2.4Hz,2H),4.71–4.54(m,4H),3. 57–3.36(m,4H),3.17–3.00(m,4H),2.64–2.48(m,4H),2.34–2.15(m,4H).

[0156] 13 C NMR (101MHz, DMSO-d6) δ168.71,152.42,152.13,145.56,135.57,130.13,129.27,126. 41,124.64,124.19,111.48,108.54,101.37,83.27,54.68,54.64,52.98,52.96,26.42.

[0157] HRMS (ESI) theoretical value C 32 H 30 N2O7S2[M+H] + 619.1562, actual value 619.1573.

[0158] Example 2 Compound A532

[0159]

[0160] The preparation method of this example is the same as that of Example 1. The product is a red solid with a yield of 19%.

[0161] 1H NMR (400MHz, Methanol-d4) δ8.37(dd,J=7.7,1.5Hz,1H),7.89(td,J=7.5,1.5Hz,1H),7.84(td,J=7.6,1.5Hz,1H),7.46(dd,J=7.4,1.4Hz,1H), 7.33–7.28(m,4H),7.25(dd,J=9.3,2.3Hz,2H),5.10–4.99(m,4H),3.91 –3.79(m,4H),2.82–2.72(m,4H),2.40–2.27(m,4H),2.14–2.05(m,4H).

[0162] 13 C NMR(101MHz,DMSO-d6)δ168.82,153.05,146.97,131.54,130.18,129.90,125.76,1 25.19,124.48,112.57,108.34,102.34,70.38,53.35,52.79,44.70,29.63,28.52.

[0163] HRMS (ESI) theoretical value C 32 H 30 N2O5S2[M+H] + 587.1663, actual value 587.1632.

[0164] Example 3 Compound A555

[0165]

[0166] The preparation method of this example is the same as that of Example 1. The product is a purple solid with a yield of 40%.

[0167] 1 H NMR (400MHz, CDCl3) δ8.03(d,J=7.6Hz,1H),7.66(td,J=7.5,1.3Hz,1H),7.60(td,J=7.4,1.1Hz,1H),7.21(d,J=7.6Hz,1H),6.64(d,J=8. 8Hz,2H),6.56(d,J=2.4Hz,2H),6.45(dd,J=8.8,2.4Hz,2H),4.12–4.03(m,4H),3.92–3.84(m,4H),3.57–3.49(m,4H),2.16–1.97(m,8H).

[0168] 13C NMR(101MHz,DMSO-d6)δ166.66,152.21,148.26,137.07,130.83,130.24,129.4 1,129.18,120.15,115.01,112.97,108.34,72.30,70.24,57.22,34.94,27.20.

[0169] HRMS (ESI) theoretical value C 32 H 30 N2O5[M+H] + 523.2222, actual value 523.2256.

[0170] Example 4 Compound A586

[0171]

[0172] The preparation method of this example is the same as that of Example 1. The product is a white solid with a yield of 12%.

[0173] 1 H NMR(400MHz, CDCl3)δ8.01(d,J=7.6Hz,1H),7.65(td,J=7.5,1.2Hz,1H),7.58(t d,J=7.4,1.2Hz,1H),7.13(d,J=7.6Hz,1H),6.91(d,J=2.5Hz,2H),6.70(d,J=8. 7Hz,2H),6.55(dd,J=8.8,2.4Hz,2H),4.77–4.63(m,4H),3.54–3.35(m,4H),3.1 6–2.98(m,4H),2.62–2.49(m,4H),2.30–2.18(m,4H),1.83(s,3H),1.75(s,3H).

[0174] 13 C NMR (101MHz, CDCl3) δ170.77,155.28,147.06,143.81,135.04,130.29,129.31,126.47,125.06,124 .23,122.10,113.98,111.66,86.47,54.70,54.63,53.46,53.44,38.41,35.31,33.49,27.06,27.04.

[0175] HRMS (ESI) theoretical value C 35 H 36 N2O6S2[M+H] +645.2082, actual value 645.2048.

[0176] Example 5 Compound A591

[0177]

[0178] The preparation method of this example is the same as that of Example 1. The product is a blue-purple solid with a yield of 10%.

[0179] MS (ESI) theoretical value C 35 H 36 N2O4S2[M+H] + 613.21,actual value: 613.21.

[0180] Example 6 Compound A620

[0181]

[0182] The preparation method of this example is the same as that of Example 1. The product is a blue solid with a yield of 18%.

[0183] 1 H NMR (400MHz, CDCl3) δ7.99 (dd, J=7.0, 1.4Hz, 1H), 7.60 (td, J=7.4, 1.4Hz, 1H ),7.55(td,J=7.4,1.2Hz,1H),7.08(d,J=7.6Hz,1H),6.97(d,J=2.3Hz,2H), 6.60(d,J=8.7Hz,2H),6.54(dd,J=8.8,2.4Hz,2H),4.11–4.06(m,4H),3.93– 3.86(m,4H),3.56–3.50(m,4H),2.12–1.96(m,8H),1.82(s,3H),1.73(s,3H).

[0184] 13 C NMR (101MHz, CDCl3) δ170.83,155.31,147.80,147.15,134.58,129.49,129.02,127.14,125. 00,124.00,121.34,114.35,112.26,87.70,69.79,69.77,57.06,38.52,35.58,32.60,26.88.

[0185] HRMS (ESI) theoretical value C 35 H 36 N2O4[M+H] + 549.2742, actual value 549.2748.

[0186] Example 7 Compound A634

[0187]

[0188] The preparation method of this example is the same as that of Example 1. The product is a blue solid with an overall yield of 8%.

[0189] HRMS (ESI) theoretical value C 35 H 41 N2O4S2Si + [M] + 645.2272, actual value 645.2305.

[0190] Example 8 Compound A640

[0191]

[0192] The preparation method of this example is the same as that of Example 1. The product is a white solid with a yield of 5%.

[0193] MS (ESI) theoretical value C 34 H 36 N2O4S2Si[M+H] + 629.10, actual value 629.19.

[0194] Example 9 Compound A655

[0195]

[0196] The preparation method of this example is the same as that of Example 1. The product is a blue solid with a yield of 14%.

[0197] 1 H NMR (400MHz, Methanol-d4) δ8.22(d,J=7.9Hz,1H),7.77(td,J=7.5,1.4Hz,1H),7.70(td,J=7.6,1.3Hz,1H),7.36(d,J=2.7Hz,2H),7.31(dd,J=7.6 ,1.3Hz,1H),6.96(d,J=9.4Hz,2H),6.77(dd,J=9.5,2.7Hz,2H),4.62(s, 4H), 3.72 (q, J = 11.0Hz, 8H), 2.26–2.01 (m, 7H), 0.63 (s, 3H), 0.58 (s, 3H).

[0198] HRMS (ESI) theoretical value C 34 H 36 N2O4Si[M+H]+ 565.2517, actual value 565.2528.

[0199] Example 10 Compound A528-6-COOH

[0200]

[0201] The preparation method of this example is the same as that of Example 1. The product is a red solid with a yield of 28%.

[0202] 1 H NMR (400MHz, DMSO-d6) δ8.27(d,J=7.6,1H),8.20(d,J=8.4Hz,1H),7.80(s,1H),6.66- 7.12(m,6H),4.93(s,4H),3.20-3.45(m,8H),2.36(d,J=7.2Hz,4H),2.07-2.22(m,4H).

[0203] 13 C NMR(101MHz,Methanol-d4)δ167.48,159.29,158.56,158.20,155.14,148.82,137.04, 131.38,130.79,120.54,117.64,114.73,113.92,100.79,82.72,56.31,53.88,28.09.

[0204] HRMS (ESI) theoretical value C 33 H 30 N2O9S2[M+H] + 663.1465, actual value 663.1467.

[0205] Example 11 Compound A555-6-COOH

[0206]

[0207] The preparation method of this example is the same as that of Example 1. The product is a purple solid with a yield of 37%.

[0208] 1 H NMR (400MHz, Methanol-d4) δ8.44–8.37(m,2H),7.99(s,1H),7.16–7.07(m,6H),4.68–4.57(m,4H),3.82–3.66(m,8H),2.26–2.07(m,8H).

[0209] 13C NMR(101MHz,Methanol-d4)δ167.67,167.38,159.74,159.65,154.68,135.99,135.98, 135.36,132.85,132.70,132.36,132.32,116.89,115.43,98.98,72.75,59.30,27.45.

[0210] HRMS (ESI) theoretical value C 37 H 30 N2O7[M+H] + 567.2126, actual value 567.2148.

[0211] Example 12 Compound A586-6-COOH

[0212]

[0213] The preparation method of this example is the same as that of Example 1. The product is a white solid with a yield of 16%.

[0214] 1 H NMR (400MHz, DMSO-d6) δ8.18(dd,J=8.0,1.3Hz,1H),8.11(d,J=8.0Hz,1H),7.49(s,1H),7.20(d,J=2.5Hz,2H),6.77(dd,J=8.9,2.4H z,2H),6.55(d,J=8.7Hz,2H),4.97–4.78(m,4H),3.32–3.18(m,8H),2.37–2.25(m,4H),2.18–2.00(m,4H),1.86(s,3H),1.76(s,3H).

[0215] 13 C NMR (101MHz, DMSO-d6) δ169.41,166.48,155.59,147.59,144.62,137.63,130.85,129.51,129. 36,125.79,124.40,120.28,114.78,112.69,55.01,53.21,53.17,38.68,34.56,33.62,26.88.

[0216] HRMS (ESI) theoretical value C 36 H 36 N2O8S2[M+H] + 689.1986, actual value 689.1990.

[0217] Example 13 Compound A591-6-COOH

[0218]

[0219] The preparation method of this example is the same as that of Example 1. The product is a blue solid with a yield of 8%.

[0220] MS (ESI) theoretical value C 36 H 36 N2O6S2[M+H] + 657.21, actual value 657.32.

[0221] Example 14 Compound A620-6-COOH

[0222]

[0223] The preparation method of this example is the same as that of Example 1. The product is a blue solid with a yield of 16%.

[0224] 1 H NMR(400MHz,DMSO-d6)δ8.28–8.13(m,2H),7.68(s,1H),7.40–7.19(m,2H), 6.91–6.61(m,4H),4.77–4.49(m,4H),3.73-3.53(m,8H),2.12–1.86(m,8H).

[0225] 13 C NMR (101MHz, DMSO-d6) δ169.99,164.41,155.36,147.78,146.98,137.76,130.05,129.93,129. 38,124.97,124.80,120.51,114.38,112.27,82.34,56.99,56.97,38.36,35.24,33.02,26.80.

[0226] HRMS (ESI) theoretical value C 36 H 36 N2O8S2[M+H] + 593.2646, actual value 593.2641.

[0227] Example 15 Compound A634-5-COOH

[0228]

[0229] The preparation method of this example is the same as that of Example 1. The product is a blue solid with an overall yield of 2%.

[0230] MS (ESI) theoretical value C 36 H 41 N2O6S2Si + [M] + 689.22, actual value 689.42.

[0231] Example 16 Compound A640-6-COOH

[0232]

[0233] The preparation method of this example is the same as that of Example 1. The product is a blue solid with a yield of 6%.

[0234] MS (ESI) theoretical value C 35 H 36 N2O6S2Si[M+H] + 673.19, actual value 673.40.

[0235] Example 17 Compound A655-6-COOH

[0236]

[0237] The preparation method of this example is the same as that of Example 1. The product is a blue solid with a yield of 14%.

[0238] 1 H NMR (400MHz, Methanol-d4) δ8.28(dd,J=8.1,1.5Hz,1H),8.22(d,J=8.1Hz,1H),7.87(d,J=1.4Hz,1H),7.32(d,J=2.6Hz,2H),6.89(d,J=9.2 Hz,2H),6.76(dd,J=9.4,2.7Hz,2H),4.53–4.48(m,4H),3.85–3.73(m,4H),3.72–3.59(m,4H),2.18–1.95(m,8H),0.65(s,3H),0.57(s,3H).

[0239] HRMS (ESI) theoretical value C 35 H 36 N2O6Si[M+H] + 609.2415, actual value 609.2420.

[0240] Example 18 Compound A720

[0241]

[0242] The preparation method of this example is the same as that of Example 1. The product is a green solid with an overall yield of 8%.

[0243] MS (ESI) theoretical value C 39 H 40 N2O3P + [M] + 615.28, actual value 615.57.

[0244] Example 19 Compound A586-2Me

[0245]

[0246] The preparation method of this example is the same as that of Example 1. The product is a purple solid with an overall yield of 18%.

[0247] MS (ESI) theoretical value C 36 H 41 N2O4S2 + [M] + 629.25, actual value 629.38.

[0248] Example 20 Compound B350

[0249]

[0250] The compound B350 was prepared according to the following steps:

[0251]

[0252] Under anhydrous and oxygen-free conditions, compound 4 (1 mol) and compound 5 (3 mol) were dissolved in toluene solvent, and palladium acetate (0.2 mol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (0.3 mol) and cesium carbonate (1.5 mol) were added in sequence. The reaction temperature was 100°C and the reaction time was 18 h. Compound B350 was obtained by extraction, drying and column chromatography. The product was a colorless solid with a yield of 40%.

[0253] 1 H NMR (400MHz, DMSO-d6) δ7.63(d,J=8.8Hz,1H),6.98(dd,J=8.8,2.5Hz,1H),6.94(d,J=2.4Hz,1H),6.09(q,J=1.3Hz,1H) ,4.93–4.87(m,2H),3.31–3.28(m,2H),3.27–3.20(m,2H),2.37(d,J=1.2Hz,3H),2.36–2.33(m,2H),2.18–2.13(m,2H).

[0254] 13 C NMR(101MHz,DMSO-d6)δ160.97,155.93,154.05,147.47,127.44,112.21,111.52,110.3 8,101.78,55.61,53.69,40.74,40.54,40.33,40.12,39.91,39.70,39.49,26.94,18.59.

[0255] HRMS (ESI) theoretical value C 16 H 17 NO4S[M+H] + 320.0957, actual value 320.0962.

[0256] Example 21 Compound B356

[0257]

[0258] The preparation method of this example is the same as that of Example 20. The product is a colorless solid with a yield of 70%.

[0259] 1 H NMR (400MHz, CDCl3) δ7.43(d,J=8.8Hz,1H),6.68(dd,J=8.8,2.4Hz,1H),6.59(d,J=2.4Hz,1H),6.02(q,J=1.2Hz,1H ),4.18–4.11(m,2H),3.86(d,J=10.9Hz,2H),3.56(dt,J=11.1,1.1Hz,2H),2.36(d,J=1.2Hz,3H),2.20–2.03(m,4H).

[0260] 13 C NMR (101MHz, CDCl3) δ162.00,156.04,152.90,149.79,126.10,111.69,110.95,110.23,101.57,77.48,77.16,76.84,69.60,56.97,27.08,18.62.

[0261] HRMS (ESI) theoretical value C 16 H 17 NO3[M+H] + 272.1287, actual value 272.1280.

[0262] Example 22 Compound C622

[0263]

[0264] The compound C622 was prepared according to the following steps:

[0265]

[0266] (1) Under anhydrous and oxygen-free conditions, compound 6 (1 mol) and compound 7 (3 mol) were dissolved in toluene solvent, and palladium acetate (0.2 mol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (0.3 mol) and cesium carbonate (1.5 mol) were added in sequence. The reaction temperature was 100° C. and the reaction time was 18 h to obtain compound 8.

[0267] (2) Compound 8 was dissolved in a mixed solvent (100 mL) of dichloromethane / water = 20 / 1, and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (1.6 mol) was added. The reaction temperature was 25° C. and the reaction time was 3 h;

[0268] (3) A 2 mol / L hypochlorous acid (5 mL) aqueous solution was slowly dripped into the reaction system containing compound 7. The reaction time was 0.5 h and the reaction temperature was 25° C. After extraction, drying and column chromatography separation, compound C622 was obtained. The product was a blue solid with an overall yield of 35%.

[0269] HRMS (ESI) theoretical value C 24 H 26 N3O5S2 + [M] + 500.13, actual value 500.14.

[0270] Example 23 Compound C654

[0271]

[0272] The preparation method of this example is the same as that of Example 22. The product is a blue solid and the total yield is 52%.

[0273] HRMS (ESI) theoretical value C 24 H 26 N3O3 + [M] + 404.20, actual value 404.19.

[0274] The compounds listed below are functionalized derivatives of the above fluorescent matrix. Their synthesis is to dissolve the corresponding dye matrix with a carboxyl group and the functional group with an amino group in DMF (dimethylformamide), add 1.2 equivalents of Carter condensation agent and 2.0 equivalents of DIPEA (diisopropylethylamine), stir at room temperature for 1 hour, and then use high performance liquid chromatography for separation and purification to obtain the final product.

[0275] Example 24 Compound A528-HTL

[0276]

[0277] Compound A528-HTL was obtained by condensation of Example 10 (A528-6-COOH) and HaloTag (O2)amines. The product was a red solid with a yield of 78%.

[0278] HRMS (ESI) theoretical value C 43 H 50 ClN3O 10 S2[M+H] + 868.2699, actual value 868.2720.

[0279] Example 25 Compound A528-NCN-HTL

[0280]

[0281] Compound A528-NCN-HTL is composed of compound (A528-NCN-6-COOH) and HaloTag(O2)amines were condensed to obtain the product as a red solid with a yield of 81%.

[0282] MS (ESI) theoretical value C 44 H 54 ClN5O9S2[M+H] + 896.31, actual value 896.70.

[0283] Example 26 Compound A532-HTL

[0284]

[0285] Compound A532-HTL is composed of compound (A532-6-COOH) and HaloTag(O2)amines were condensed to obtain the product as a red solid with a yield of 72%.

[0286] HRMS (ESI) theoretical value C 43 H 50 ClN3O8S2[M+H] + 836.2801, actual value 836.2830.

[0287] Example 27 Compound A555-HTL

[0288]

[0289] Compound A555-HTL was obtained by condensing Example 11 (A555-6-COOH) and HaloTag (O2)amines. The product was a purple solid with a yield of 82%.

[0290] HRMS (ESI) theoretical value C 43 H 50 ClN3O8[M+H] + 772.3359, actual value 772.3383.

[0291] Example 28 Compound A555-STL

[0292]

[0293] Compound A555-STL was obtained by condensation of Example 11 (A555-6-COOH) and BG-NH2. The product was a purple solid with a yield of 65%.

[0294] MS (ESI) theoretical value C 46 H 42 N8O7[M+H] + 819.32, actual value 819.68.

[0295] Example 29 Compound A555-TMP3

[0296]

[0297] Compound A555-TMP3 was obtained by condensing Example 11 (A555-6-COOH) and TMP3-NH2. The product was a purple solid with a yield of 74%.

[0298] MS (ESI) theoretical value C 59 H 60 N8O 10 [M+2H] 2+ 521.23, actual value 521.70.

[0299] Example 30 Compound A586-HTL

[0300]

[0301] Compound A586-HTL was obtained by condensation of Example 12 (A586-6-COOH) and HaloTag (O2)amines.

[0302] The product was a white solid with a yield of 52%.

[0303] HRMS (ESI) theoretical value C 46 H56 ClN3O9S2[M+H] + 894.3219, actual value 894.3216.

[0304] Example 31 Compound A620-HTL

[0305]

[0306] Compound A620-HTL was obtained by condensation of Example 14 (A620-6-COOH) and HaloTag (O2)amines. The product was a blue solid with a yield of 62%.

[0307] HRMS (ESI) theoretical value C 46 H 56 ClN3O7[M+H] + 798.3880, actual value 798.3872.

[0308] Example 32 Compound A620-STL

[0309]

[0310] Compound A620-STL was obtained by condensation of Example 14 (A620-6-COOH) and BG-NH2. The product was a blue solid with a yield of 51%.

[0311] HRMS (ESI) theoretical value C 49 H 48 N8O6[M+H] + 845.3770, actual value 845.3776.

[0312] Example 33 Compound A620-TMP3

[0313]

[0314] Compound A620-TMP3 was obtained by condensation of Example 14 (A620-6-COOH) and TMP3-NH2. The product was a blue solid with a yield of 42%.

[0315] MS (ESI) theoretical value C 62 H 66 N8O9[M+2H] 2+ 534.26, actual value 534.42.

[0316] Example 34 Compound A655-HTL

[0317]

[0318] Compound A655-HTL was obtained by condensation of Example 17 (A655-6-COOH) and HaloTag (O2)amines. The product was a blue solid with a yield of 86%.

[0319] HRMS (ESI) theoretical value C 45 H 56 ClN3O7Si[M+H] + 814.3649, actual value 814.3650.

[0320] Example 35 Compound A655-STL

[0321]

[0322] Compound A655-STL was obtained by condensation of Example 17 (A655-6-COOH) and BG-NH2. The product was a blue solid with a yield of 60%.

[0323] MS (ESI) theoretical value C 48 H 48 N8O6Si[M+H] + 861.35, actual value 861.49.

[0324] Example 36 Compound A655-TMP3

[0325]

[0326] Compound A655-TMP3 was obtained by condensation of Example 17 (A655-6-COOH) and TMP3-NH2. The product was a blue solid with a yield of 57%.

[0327] MS (ESI) theoretical value C 61 H 66 N8O9Si[M+2H] 2+ 542.24, actual value 542.68.

[0328] Example 37 Compound A655-4-Hoechst

[0329]

[0330] Compound A655-4-Hoechst was obtained by condensing Example 17 (A655-6-COOH) and Hoechst-NH2. The product was a green solid with a yield of 32%.

[0331] MS (ESI) theoretical value C 64 H 67 N9O6Si[M+H] +1086.51, actual value 1086.22.

[0332] Comparative Example 1 Compound JF 549

[0333]

[0334] Comparative Example 2 Compound TMRE

[0335]

[0336] Comparative Example 3 Compound Rhodamine B

[0337]

[0338] Comparative Example 4 Compound Cy3

[0339]

[0340] Comparative Example 5 Compound Atto647N

[0341]

[0342] Comparative Example 6 Compound TMR-HTL

[0343]

[0344] Comparative Example 7 Compound JF549-HTL

[0345]

[0346] Comparative Example 8 Compound JFX554-HTL

[0347]

[0348] Comparative Example 9 Compound CPY-HTL

[0349]

[0350] Comparative Example 10 Compound TMR-STL

[0351]

[0352] Comparative Example 11 Compound SiR-HTL

[0353]

[0354] Test Example 1

[0355] Determination of basic photophysical properties of fluorescent dyes

[0356] Example 1 (A528), Example 2 (A532), Example 3 (A555), Example 4 (A586), Example 6 (A620), Example 7 (A634), Example 9 (A655), Example 18 (A720), Example 20 (B350), and Example 21 (B356) were subjected to the determination of the basic photophysical properties of the fluorescent dyes, as shown in FIG. Figure 1 and Figure 2 shown.

[0357] The method for determining the basic photophysical properties of fluorescent dyes is as follows: the fluorescent dye is prepared into a solution with a concentration of 1 μmol / L, the solvent of which is a PBS buffer solution (phosphate buffered saline) with a pH of 7.4, and then the solution is placed in a Duetta fluorescence and absorption spectrometer for testing at 25°C, and the test is performed 3 times.

[0358] Among them, the fluorescence quantum yield test method of the fluorescent dye is as follows: the fluorescence quantum yield is measured using the reference method, the fluorescent dye to be tested or the reference fluorescent dye is prepared into a solution with a concentration of 1 μmol / L, and the solvent is a PBS buffer solution (phosphate buffered saline solution) with a pH of 7.4, and then the solution is placed in a Duetta fluorescence and absorption spectrometer to scan its absorption spectrum and fluorescence spectrum at 25°C, and its fluorescence spectrum is integrated, and the fluorescence quantum yield of the dye is obtained by the ratio of the fluorescence spectrum integral of the fluorescent dye to be tested and the reference fluorescent dye; the fluorescence quantum yield calculation formula of the fluorescent dye is:

[0359]

[0360] Among them, Φ sample , Φ ref , are the fluorescence quantum yields of the fluorescent dye to be tested and the reference fluorescent dye respectively;

[0361] OD sample , O.D. ref , are the absorption values ​​of the fluorescent dye to be tested and the reference fluorescent dye at the excitation wavelength respectively;

[0362] I sample , I ref , are the integral values ​​of the excitation spectra of the fluorescent dye to be tested and the reference fluorescent dye respectively;

[0363] d sample , d ref , are the refractive indices of the test solvents for the fluorescent dye to be tested and the reference fluorescent dye, respectively.

[0364] As can be seen from Table 1, the emission wavelength of the aza[3.2.1] bridged ring substituted fluorescent dye obtained in the present application covers the ultraviolet and visible light regions, and the brightness of the fluorescent dyes of various colors is 1.2-16 times higher than that of the traditional dimethylamino or diethylamino substituted fluorescent dyes. Among them, the ultraviolet, orange and red emitting dyes have quantum yields close to the theoretical limit.

[0365] Table 1

[0366]

[0367] Test Example 2: Fluorescent Dye Photostability Test

[0368] The fluorescent dye photostability test was performed on Example 3 (A555), Example 10 (A528-6-COOH), Example 7 (A634), Comparative Example 2 (TMRE), Comparative Example 1 (JF 549), Comparative Example 3 (Rhodamine B), Comparative Example 4 (Cy3) and Comparative Example 5 (Atto647N). Figure 3 and Figure 4 shown.

[0369] The photostability test method of fluorescent dyes is as follows: the fluorescent dye with a concentration of 10 mol / L is dissolved in a PBS buffer solution (phosphate buffered saline) with a pH of 7.4; the dye solution is irradiated with an LED lamp (light emitting diode) that matches the excitation wavelength of the fluorescent dye, and the absorption spectrum is measured every 10 minutes for a total of 30 minutes.

[0370] pass Figure 3 It can be found that in the orange channel, the stability of Example 3 and Example 10 is improved by 3.3 to 7.6 times compared with Comparative Example 2, Comparative Example 1, Comparative Example 3 and Comparative Example 4, and the fluorescent dyes of Comparative Example 2, Comparative Example 1, Comparative Example 3 and Comparative Example 4 all experience absorption spectrum blue shifts ranging from 12 to 22 nm, while Example 3 and Example 10 of the present application do not experience light blue shifts.

[0371] pass Figure 4 It can be seen that in the far-red channel, the photostability of Example 7 of the present application is improved by 1 times compared with that of Comparative Example 5, and no blue shift of light occurs.

[0372] Combination Figure 3 and Figure 4 This indicates that the photostability of the aza[3.2.1] bridge-ring substituted fluorescent dye prepared in the present application is greatly improved and the photooxidative dealkylation (light blue shift) process is completely avoided.

[0373] Test Example 3

[0374] Testing of fluorescent dye functionalized derivatives in fixed cell single molecule experiments

[0375] Example 27 (A555-HTL), Comparative Example 6 (TMR-HTL), and Comparative Example 7 (JF549-HTL) were tested for fluorescent dye functionalized derivatives in fixed cell single molecule experiments. Figure 5 , Figure 6 and Figure 7 shown.

[0376] The testing method of the fluorescent dye functionalized derivatives in the fixed cell single molecule experiment is as follows: 5pM fluorescent dye was incubated with U2OS cells for 30 minutes, and then the cells were washed three times with pH 7.4 PBS buffer, and the cells were fixed with 4% paraformaldehyde, and single molecule experiments were performed on a total internal reflection fluorescence microscope (100mW, 561nm excitation, excitation intensity of 6%).

[0377] pass Figure 6 It can be obtained that the average single molecule fluorescence intensities of Comparative Example 6, Comparative Example 7 and Example 27 are 4354, 5466 and 8176 respectively. In terms of photostability, Example 27 is improved by one time compared with Comparative Example 7.

[0378] Combination Figure 5 , Figure 6 and Figure 7 This indicates that the aza[3.2.1] bridge ring substituted fluorescent dye prepared in the present application is not only competent for single-molecule imaging experiments, but also has better performance in terms of brightness and photostability.

[0379] Test Example 4

[0380] Testing of fluorescent dye functionalized derivatives in live cell confocal microscopy experiments

[0381] Example 27 (A555-HTL), Example 31 (A620-HTL), Example 34 (A655-HTL), Example 28 (A555-STL), Comparative Example 6 (TMR-HTL), Comparative Example 8 (JFX554-HTL), Comparative Example 9 (CPY-HTL), Comparative Example 10 (TMR-STL) were tested for fluorescent dye functionalized derivatives in living cell confocal microscopy imaging experiments. Figure 8 , Fig. 9 , Fig.10 , Fig.11 , Fig.12 , Fig.13 , Fig.14 shown.

[0382] The testing method of the fluorescent dye functionalized derivatives in the living cell confocal microscopy experiment is as follows: 200nM fluorescent dye is used to perform confocal fluorescence imaging of TMP-Halo-SNAP-GFP-H2B protein in living Hela cells, and the brightness is compared with that of traditional dyes of the same color.

[0383] pass Fig.11 It can be seen that the brightness of Example 27 is 50% higher than that of the high-brightness dye comparative example 8 reported in the literature JACS Au. 2021, 1: 690-696., and 158% higher than that of comparative example 6 (confocal imaging 561nm excitation, excitation intensity of 10%; detector 566nm, no gate);

[0384] pass Fig.12 It can be seen that the brightness of Example 31 is 350% higher than that of the high-brightness dye comparative example 9 reported in the literature Angew. Chem. Int. Ed. 2016, 55: 3290-3294 (confocal imaging 640nm excitation, excitation intensity 5%; detector 664nm, nogate);

[0385] pass Fig.14 It can be seen that the brightness of Example 28 is 223% higher than that of Comparative Example 10 (confocal imaging 561nm excitation, excitation intensity 10%; detector 566nm, no gate).

[0386] Test Example 5

[0387] Testing of fluorescent dye functionalized derivatives in living cell super-resolution microscopy experiments

[0388] Example 34 (A655-HTL) and Comparative Example 11 (SiR-HTL) were tested for fluorescent dye functionalized derivatives in a living cell super-resolution microscopic imaging experiment. Fig.15 and Fig.16 shown.

[0389] The testing method of the fluorescent dye functionalized derivatives in the living cell super-resolution microscopy experiment is as follows: 1 μM fluorescent dye functionalized derivatives are used to perform wash-free super-resolution imaging of Lifeact-Halo protein in living Hela cells (STED imaging 640nm excitation, excitation intensity of 2%, loss light intensity of 30%, detector 650nm, no gate).

[0390] Combination Fig.15 and Fig.16 It can be seen that Example 34 exhibits higher photostability on STED than the far-red wash-free dye comparative example 11 reported in the literature Nature Chemistry. 2013, 5: 132-139.

[0391] From the above description, it can be seen that the above-mentioned embodiments of the present invention achieve the following technical effects: the aza[3.2.1] bridge ring substituted fluorescent dye provided in the present application adopts an aza[3.2.1] bridge ring as a substituent of the fluorescent dye, which can not only improve the brightness of the fluorescent dye, but also improve the photostability of the fluorescent dye while avoiding the optical blue shift of the dye, and at the same time can effectively improve the water solubility of the fluorescent dye and maintain its good cell membrane permeability, and can simultaneously improve the fluorescence quantum yield, photostability, resistance to photooxidative dealkylation (optical blue shift), water solubility and cell membrane permeability of the fluorescent dye.

[0392] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An aza[3.2.1] bridged ring substituted fluorescent dye, characterized in that: The aza[3.2.1] bridged ring substituted fluorescent dye has a structure as shown in the following formula (I), formula (II) or formula (III): R a , R b , R c , R d , R e , R f , R g , R h , R i , R j , R k , R l , R m , R n , R o , R p , R q Each is independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C3-C20 cycloalkyl, substituted or unsubstituted C4-C20 cycloalkylalkyl, C4-C20 alkylcycloalkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C1-C20 ester, substituted or unsubstituted C1-C20 amide; R A Selected from substituted or unsubstituted C6-C40 aryl, substituted or unsubstituted C6-C40 aralkyl, and substituted or unsubstituted C5-C40 heteroaryl; X is selected from oxygen, sulfur, substituted or unsubstituted C1-C20 alkylene, Y is selected from oxygen, sulfur, nitrogen, substituted or unsubstituted C1-C20 alkylene, substituted or unsubstituted C1-C20 alkyleneoxy, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 ester, Among them, R B Selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 ester; Z - Selected from F - , Cl - Br - ,I - ,OAc - 、HSO4 - 、H2PO4 - 、ClO4 - CF3COO - CF3SO3 - 、CH3SO3 - 、NO3 - 、C5H7O5COO - PF6 - ; m is an integer from 1 to 3.

2. The aza[3.2.1]bridged ring substituted fluorescent dye according to claim 1, characterized in that The R a , the R b , the R c , the R d , the R e , the R f , the R g , the R h , the R i , the R j , the R k , the R l , the R m , the R n , the R o , the R p , the R q Each is independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C10 cycloalkylalkyl, C4-C10 alkylcycloalkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 ester, substituted or unsubstituted C1-C10 amide; and / or, the R A Selected from substituted or unsubstituted C6-C30 aryl, substituted or unsubstituted C6-C30 aralkyl, substituted or unsubstituted C5-C30 heteroaryl; And / or, X is selected from oxygen, sulfur, substituted or unsubstituted C1-C10 alkyl, and / or, Y is selected from oxygen, sulfur, nitrogen, substituted or unsubstituted C1-C10 alkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 acyl, substituted or unsubstituted C1-C10 ester, Among them, R B Selected from substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 alkoxy, substituted or unsubstituted C1-C20 acyl, substituted or unsubstituted C1-C20 ester; and / or, the Z - Selected from F - , Cl - Br - ,I - ,OAc - 、HSO4 - 、H2PO4 - 、ClO4 - CF3COO - CF3SO3 - 、CH3SO3 - 、NO3 - 、C5H7O5COO - PF6 - ; And / or, m is an integer of 1-3.

3. The aza[3.2.1]bridged ring substituted fluorescent dye according to claim 1 or 2, characterized in that The aza[3.2.1] bridged ring substituted fluorescent dye represented by formula (I) has a structure represented by the following formula (IV): R1 and R5 are each independently selected from hydrogen, carboxyl, halogen, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, nitro, amino, hydroxyl, Where R D R2, R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-C6 alkyl, substituted or unsubstituted C1-C6 alkoxy, and n is an integer of 1 to 6; R2, R3 and R4 are each independently selected from hydrogen, halogen, carboxyl, substituted or unsubstituted C1-10 alkyl, substituted or unsubstituted C3-C10 cycloalkyl, substituted or unsubstituted C4-C10 cycloalkylalkyl, C4-C10 alkylcycloalkyl, substituted or unsubstituted C1-C10 alkoxy, substituted or unsubstituted C1-C10 ester, 4. The aza[3.2.1]bridged ring substituted fluorescent dye according to claim 3, characterized in that The aza[3.2.1] bridged ring substituted fluorescent dye is selected from at least one of the following formulas IA1-IA3, IIA1-IIA3, IIIA1-IIIA3, IB1-IB3, IC1-IC3, ID1-ID3, IE1-IE3, IF1-IF3, IG1-IG3:

5. The aza[3.2.1]bridged ring substituted fluorescent dye according to any one of claims 1 to 4, characterized in that The aza[3.2.1] bridged ring substituted fluorescent dye is selected from the following formulas: A528, A532, A555, A586, A591, A620, A634, A640, A655, A720, A586-2Me, A586-2Me-5-COOH, A528-6-COOH, A532-6-COOH, A555-6-COOH, A586-6-COOH, A591-6-COOH, A620-6-COOH, A634-5-COOH, A640-6-COOH, A655-6-COOH, A528-HTL, A532-HTL, A555-HTL, A586-HTL, A620-HTL, A655-HTL, A55 5-STL, A528-NHS, A620-STL, A655-STL, A634-NHS, A586-NHS, A591-HTL, A640-HTL, A555-TMP3, A620-TMP3, A655- TMP3, A655-4-Hoechst, A591-TMP3, A640-TMP3, A591-STL, A640-STL, A528- At least one of NCN-HTL, A555-NCN-HTL, A620-NCN-HTL, B350, B356, C622, C654:

6. A method for preparing an aza[3.2.1] bridged ring substituted fluorescent dye, characterized in that: The preparation method comprises: Step S11, mixing compound A, an aza[3.2.1] bridged ring compound and a first catalyst in a first reaction solvent for reaction to obtain compound B; Step S12, mixing the compound B and the HZ aqueous solution to react, to obtain the aza[3.2.1] bridge ring substituted fluorescent dye represented by formula (I); The compound A has a structure as shown in the following formula (V), and the aza[3.2.1] bridged ring compound has a structure as shown in the following formula (VI): Among them, R A , R a , R b , R c , R d , R e , R f , X, and Y each independently have the same meaning as in any one of claims 1 to 5, and Z in the HZ aqueous solution - has the same meaning as in any one of claims 1 to 5; Preferably, the first catalyst comprises a palladium catalyst, a palladium catalyst ligand and a basic catalyst; Preferably, the palladium catalyst comprises at least one of tetrakistriphenylphosphine palladium, palladium acetate, trisdibenzylideneacetone dipalladium, diphenylphosphinothiocene palladium dichloride, ditriphenylphosphine palladium dichloride, and palladium chloride, preferably at least one of tetrakistriphenylphosphine palladium, palladium acetate, or trisdibenzylideneacetone dipalladium; Preferably, the palladium catalyst ligand includes at least one of bis(dibenzylideneacetonepalladium), 2,2'-bis(diphenylphosphino-1,1'-binaphthyl, 2-dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl, and 2-dicyclohexylphosphino-2',6'-dimethoxybiphenyl, preferably at least one of bis(dibenzylideneacetonepalladium) and 2,2'-bis(diphenylphosphino-1,1'-binaphthyl; Preferably, the alkaline catalyst comprises at least one of cesium carbonate, sodium tert-butoxide, potassium carbonate, sodium carbonate, sodium methoxide, sodium ethoxide, and potassium tert-butoxide, preferably at least one of cesium carbonate, sodium tert-butoxide, and potassium tert-butoxide; Preferably, the first reaction solvent includes at least one of n-hexane, tetrahydrofuran, 1,4-dioxane, diethyl ether and toluene.

7. A method for preparing an aza[3.2.1] bridged ring substituted fluorescent dye, characterized in that: The preparation method comprises: Compound C, an aza[3.2.1] bridged ring compound and a second catalyst are mixed and reacted in a second reaction solvent to obtain an aza[3.2.1] bridged ring substituted fluorescent dye represented by formula (II); The compound C has a structure shown in the following formula (VII): Formula (VII); R g , R h , R i , R j , R k , X each independently has the same meaning as in any one of claims 1 to 5; Preferably, the second catalyst and the first catalyst have the same meaning; Preferably, the second reaction solvent and the first reaction solvent have the same meaning.

8. A method for preparing an aza[3.2.1] bridged ring substituted fluorescent dye, characterized in that: The preparation method comprises: Step S31, mixing compound D, an aza[3.2.1] bridged ring compound and a third catalyst in a third reaction solvent to react to obtain compound E; Step S32, mixing the compound E and the oxidant in a fourth reaction solvent to react to obtain a compound F; Step S33, mixing the compound F and the HZ aqueous solution to react, to obtain the aza[3.2.1] bridge ring substituted fluorescent dye represented by formula (III); The compound D has a structure as shown in the following formula (VIII): R l , R m , R n , R o , R p , R q , X, and Y each independently have the same meaning as in any one of claims 1 to 5, and Z in the HZ aqueous solution - has the same meaning as in any one of claims 1 to 5; Preferably, the third catalyst and the first catalyst have the same meaning; Preferably, the third reaction solvent and the first reaction solvent have the same meaning; Preferably, in step S32, the fourth reaction solvent is a mixed solvent of dichloromethane and water, and the volume ratio of the dichloromethane to the water is (10-30):

1. Preferably, in step S32, the oxidant comprises at least one of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, potassium permanganate, hydrogen peroxide, and m-chloroperbenzoic acid, preferably 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; Preferably, the molar ratio of the oxidant to the compound E is (1.0-2.0):

1.

9. Use of the aza[3.2.1] bridged ring substituted fluorescent dye according to any one of claims 1 to 5 or the aza[3.2.1] bridged ring substituted fluorescent dye obtained by the preparation method of the aza[3.2.1] bridged ring substituted fluorescent dye according to any one of claims 6 to 8 as a fluorescent marker in the fields of fluorescence analysis, fluorescence detection and fluorescence imaging.

10. The use of the aza[3.2.1]bridged ring substituted fluorescent dye according to claim 9, characterized in that: The aza[3.2.1] bridge ring substituted fluorescent dye is used as a fluorescent marker in fluorescence analysis, fluorescence detection, biomacromolecule labeling, immunofluorescence imaging, living cell imaging, tissue imaging, biological in vivo imaging, single molecule imaging, and super-resolution fluorescence imaging.

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