WO2020124688A1 - Full-spectrum high-brightness and high-stability fluorescent dyes, and synthesis and application thereof - Google Patents

Abstract

Provided in the present invention are full-spectrum high-brightness and high-stability fluorescent dyes, the dyes being one or several of the following mixed according to any ratio: 4-amido substituted naphthalimide dye, bisalkoxy substituted naphthalimide fluorescent dye, bisamino substituted naphthalimide fluorescent dye, 9, 10-bisamino substituted perylene imide, six-membered ring rhodamine dye, five-membered ring rhodamine dye, and silicon-based rhodamine dye. Compared to current commercial dyes, the fluorescent dyes of the present invention have higher photostability and narrower full width at half maximum (25 nm), and are not sensitive to various external environments including pH, polarity, and temperature. By means of the introduction of active groups such as click groups, protein tags, and drug molecules, the obtained functionalised fluorescent molecules have high biocompatibility, and can quickly and specifically dye living cells and bodies. Due to the increased photostability and fluorescent brightness, the present series of dyes can implement super-resolution fluorescence imaging in Storm, STED, and SIM modes.

Description

Full-spectrum high-brightness, high-stability fluorescent dye and its synthesis and application Technical field

The invention belongs to the field of fluorescent dyes, and in particular relates to the synthesis and application of a full-spectrum high-brightness, high-stability fluorescent dye.

Background technique

Organic fluorescent dyes can be divided into Resonant dyes and Charge (Transfer, CT) dyes from the fluorescence structure-activity relationship, and the fluorescent properties are determined by the electron transfer between the ground state and the excited state, so that the study of the structure-activity relationship can be Achieve structural modification with different light performance requirements, or accurately predict the luminous performance of known structures. Fluorescein, rhodamine, Bodipy, and most cyanine dyes are all classified as resonance dyes, which have narrow absorption and emission peaks, solvent insensitivity, small Stokes shift, high molar extinction coefficient, and high quantum yield. Performance; while charge transfer dyes (such as 1,8-naphthalimide and coumarin etc.) have a clear electron donor and electron acceptor, which has a wide absorption and emission peak, solvent sensitivity, large Stokes Displacement, relatively low molar absorption coefficient and other luminous properties. In addition, the spectral properties of charge transfer dyes are greatly affected by the strong polar environment, and the fluorescence quantum yield in the near infrared region is extremely low. Researchers should choose appropriate fluorescent dyes according to different biological application needs. For example: fluorescent dyes usually choose resonance dyes with high brightness and environmental insensitivity; while fluorescent probes often choose environment-sensitive charge transfer dyes.

At present, the small organic fluorescent dyes used for fluorescent labeling mainly focus on fluorescein, rhodamine, Bodipy and cyanine dyes with high quantum yield and narrow half-width. In 1953, fluorescein was first used for immunofluorescence labeling, and then a variety of organic small molecule fluorescent dyes such as rhodamine dyes were gradually developed and should be used for fluorescent labeling. Despite the wide variety of organic fluorescent dyes and different properties, each dye has obvious disadvantages. The luminescent form of fluorescein is a negatively charged fluorophore. This form is extremely sensitive to pH and has poor stability. And this negatively charged form has poor cell permeability, which seriously affects its labeling of target molecules in living cells. On the contrary, rhodamine dye is a positive ion fluorescent dye with good membrane permeability and stability. Due to the pain in the mitochondria, this positive ion form can easily enter the mitochondria for non-specific labeling. Cyanine dyes are often used for imaging of living bodies and tissues with their near-infrared fluorescence emission wavelength, which has strong penetration ability in tissues and shields autofluorescence. However, the light stability and low quantum yield of cyanine dyes have always been a barrier limiting their applications.

With the development of super-resolution fluorescence technology, organic fluorescent dyes have been widely used due to their high quantum yield, small molecular structure, and easy functionalization. However, they also put forward higher requirements for the performance of fluorescent dyes. Rhodamine and cyanine dyes have been successfully applied to dSTORM and PALM fluorescence imaging due to their higher meso-position activity (literature). In addition, the extremely high stability allows Rhodamine to be used in STED. In contrast, fluorescein and Bodipy are rarely used in this field because of their poor stability. In 2015, the Yamaguchi research group developed a new phosphorous-doped fluorescent dye C-Naphox and successfully applied it to STED super-resolution fluorescence imaging (Figure). PB430 exhibits higher light stability than Alexa Fluor 488 in STED super-resolution fluorescence imaging. However, its low molar extinction coefficient results in a brightness of only 6900M ^-1 cm ^-1 (ε×Φ).

Although a large number of commercial dyes have been developed and widely used, they still need to overcome the limitations of existing dyes to meet their needs in different fields. The development of super-resolution technology further highlights the urgency of this demand. Organic fluorescent dyes with small molecular weight, high light absorption capacity, high stability, high cell permeability and narrow emission peak still need to be further developed, derived and even applied.

Summary of the invention

One of the objectives of the present invention is to provide a full-spectrum, high-brightness, high-stability fluorescent dye. The fluorescent dye of this system has extremely high biocompatibility, and can stain living cells within 20 seconds to 5 minutes. Functionalized fluorescent molecules with such dyes can be able to specifically label target molecules or organelles.

Another object of the present invention is to provide a method for synthesizing fluorescent dyes with high brightness and high stability, which has the advantages of simple operation, easy derivation, and easy purification.

The invention provides a high-brightness and high-stability fluorescent dye, which takes naphthalimide and perylene imide as fluorescent groups, and introduces two amino substituents at one end of the power supply group to greatly improve the stability and fluorescent brightness. In addition, these dyes are insensitive to external microenvironments such as viscosity, pH, and temperature.

The invention provides a class of functionalized fluorescent molecules based on novel fluorescent dyes, which have high cell permeability, can quickly stain a variety of living cells, and are successfully used in the fields of fluorescent labeling, fluorescent imaging, and the like.

The invention provides a full-spectrum, high-brightness, high-stability fluorescent dye whose excitation wavelength covers the full wavelength band. The dye suppresses intramolecular twisting at the end of the electron donor through the adjustment of the rigid cyclic amine structure, achieves an increase in fluorescence quantum efficiency, and stabilizes light The dye is improved by 4-amide substituted naphthalimide dyes, dialkoxy substituted naphthalimide fluorescent dyes, diamino substituted naphthalimide fluorescent dyes, 9,10-bisamino One or more of the substituted perylene imides, six-membered ring-rhodamine dyes, five-membered ring-rhodamine dyes, and silicon-based rhodamine-based dyes are mixed in any ratio.

The bisalkoxy substituted naphthalimide fluorescent dye has an absorption wavelength at 390 nm and a fluorescence emission wavelength at 405 nm excitation, and its structural formula is as follows:

若R ₃，R ₄不独立则为

以整体结构存在

p为0-2整数。 R ₃ and R ₄ are respectively independent

If R ₃ and R _{4 are} not independent, it is

Exist as a whole structure

p is an integer from 0-2.

The synthesis steps of the dialkoxy substituted naphthalimide fluorescent dyes are as follows:

Specific steps are as follows:

(1) Synthesis of dye N-butyl-4,5-dialkoxy-1,8-naphthalimide:

Dissolve the polyhydric alcohol in dry tetrahydrofuran, add Na block under nitrogen blowing, and add N-butyl-4-bromo-5-nitro-1,8-naphthalimide to the reaction solution after 0.5-1h and heat To 60-90 ℃, 2-10h. After removing the solvent under reduced pressure, the silica gel column was separated, using dichloromethane and petroleum ether = 1:1 to 4 by volume as the eluent, and the solvent was removed under reduced pressure to obtain a white solid N-butyl-4,5-dioxane Oxy-1,8-naphthalimide.

In step (1), the mass ratio of polyol and sodium block is 2-1:1; the mass ratio of polyol and N-butyl-4-bromo-5-nitro-1,8-naphthalimide is 2:1-12; the volume ratio of polyol to tetrahydrofuran is 1-10:1mg/mL;

The diamino-substituted naphthalimide fluorescent dye has an absorption wavelength of 440-490nm, can be excited by 450nm and 488nm lasers, and can be used as the following dyes by changing the targeting group: mitochondrial fluorescent dye, SNAP- Tag fluorescent dyes, Halo-tag fluorescent dyes, reactive ester fluorescent dyes, drug targeting fluorescent dyes, etc., the structural formula is as follows:

Where R ₂ is H, C1-16 alkyl, aryl, substituted aryl, (CH ₂ CH ₂ O) _n H, (CH ₂ ) _m COOMe and (CH ₂ ) _m SO ₃ H, heteroaryl or substituted Heteroaryl, biological targeting groups such as N-ethylmorpholine, benzylguanine, n-hexane-triphenylphosphine, folic acid, colchicine, paclitaxel, 6-chlorohexane and so on.

中的一种，若R ₅，R ₆不独立则为

以整体结构存在，

R ₅ and R ₆ are respectively independent

One of them, if R ₅ and R _{6 are} not independent, it is

Exist as a whole structure,

R ₇ and R ₈ are each independently H, C1-4 alkyl, (CH ₂ CH ₂ O) _n H; if R _{7 is} not H, R ₈ must be a non-H substituent;

Y is sulfone group, sulfoxide group, dimethylsilyl group, boryl group;

m and n are integers from 0-4.

The synthesis steps of the diamino substituted naphthalimide fluorescent dyes are as follows:

Specific steps are as follows:

(1) Synthesis of intermediate N-alkyl-4-bromo-5-nitro-1,8-naphthalimide:

Dissolve 4-bromo-5-nitro-1,8-naphthalic anhydride and benzylamine in absolute ethanol; heat the reaction solution to 40-90°C and stir for 1-10h; after cooling the reaction solution to room temperature, After removing the solvent under reduced pressure, the silica gel column was separated, using petroleum ether and dichloromethane or dichloromethane and methanol with a volume ratio of 800-100:1 as the eluent, and the solvent was removed under reduced pressure to obtain an off-white solid N-alkyl- 4-bromo-5-nitro-1,8-naphthalimide;

(2) Synthesis of dye N-alkyl-4,5-difatty amino-1,8-naphthalimide:

Dissolve N-alkyl-4-bromo-5-nitro-1,8-naphthalimide in ethylene glycol methyl ether, and add alicyclic amine to it; slowly warm the reaction solution to 50-140°C , And react under nitrogen protection for 10-24h; remove the solvent under reduced pressure, and separate it with a silica gel column, using dichloromethane and methanol in a volume ratio of 400-30:1 as the eluent, and remove the solvent to obtain a brownish yellow solid N-alkane Group-4,5-difatty amino-1,8-naphthalimide;

In step (1): the mass ratio of 4-bromo-5-nitro-1,8-naphthalic anhydride: primary fatty amine is 1:0.5-2; 4-bromo-5-nitro-1,8-naphthalic anhydride The volume ratio of the mass to absolute ethanol is 1:20-80g/mL.

Fatty primary amines include linear alkyl amines such as methylamine, ethylamine, butylamine, n-dodecylamine, n-hexadecylamine, benzylamine analogs, amino-substituted alkyl sulfonic acids, amino alcohols, and the like.

In step (2): the mass ratio of N-alkyl-4-bromo-5-nitro-1,8-naphthalimide to alicyclic amine is 1:1-3; N-alkyl-4-bromo The volume ratio of -5-nitro-1,8-naphthalimide to ethylene glycol methyl ether is 1:50-200g/mL;

Alicyclic amines are aziridine, azetidine, tetrahydropyrrole, piperidine, cycloheximide, ethylenediamine derivatives and cyclohexanediamine derivatives.

A full-spectrum, high-brightness, high-stability fluorescent dye that is disubstituted with 9,10 bisamino-substituted peryleneimide dyes. It is characterized by being used for 680nm and 710nm lasers. Its structure is as follows:

Among them: R ₁₁ is

中的一种，若R ₉，R ₄不独立则为

以整体结构存在，

R ₉ and R ₁₀ are respectively independent

One of them, if R ₉ and R _{4 are} not independent, it is

Exist as a whole structure,

R ₇ and R ₈ are each independently H, C1-4 alkyl, (CH ₂ CH ₂ O) _n H; if R _{7 is} not H, R ₈ must be a non-H substituent; n is an integer of 0-4.

The synthesis route of a double-substituted perylene imide dye in a full-spectrum high-brightness, high-stability fluorescent dye is as follows:

Specific steps are as follows:

(1) Synthesis of intermediate N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide:

Dissolve 9,10-dibromo-1,6,7,12-tetrachloroperyleneimide and primary alcohol amine or fatty primary amine in a mixture of N-methylpyrrolidone and glacial acetic acid; heat the reaction solution to 100 -140°C, stirring for 1-10h; cool the reaction solution to room temperature, pour it into ice water and filter to obtain a black solid, vacuum dry, separate with a 200-300 mesh silica gel column, dichloromethane with a volume ratio of 1:0.25～6 Methane: petroleum ether is used as the eluent, and the solvent is removed under reduced pressure to obtain dark red solid N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide;

(2) Synthesis of probe N-alkyl-9,10-difatty amino-1,6,7,12-tetrachloroperyleneimide:

Dissolve N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide in ethylene glycol methyl ether, and add fatty amine to it; then warm the reaction solution slowly To 90-130 ℃, and react under nitrogen protection for 10-24h; remove the solvent under reduced pressure, 200-300 mesh separation, using dichloromethane: petroleum ether with a volume ratio of 1:0-1 as eluent, under reduced pressure The solvent was removed to obtain a blue solid probe N-alkyl-9,10-difatty amino-1,6,7,12-tetrachloroperyleneimide.

In step (1), the mass ratio of the 9,10-dibromo-1,6,7,12-tetrachloroperyleneimide to the primary alcohol amine or aliphatic primary amine is 1-10:1; the 9 , The mass-volume ratio of 10-dibromo-1,6,7,12-tetrachloroperyleneimide and N-methylpyrrolidone is 1:20-120g/mL; the N-methylpyrrolidone and glacial acetic acid The volume ratio is 1-3:3-4;

In step (2), the mass ratio of the N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide to fatty amine is 1:6-8; the fat The mass to volume ratio of amine to ethylene glycol methyl ether is 5-120:1mg/mL; the fatty amines include ammonia, aziridine, azetidine, tetrahydropyrrole, piperidine or cyclohexanediamine derivatives Wait.

A full-spectrum, high-brightness, high-stability fluorescent dye used in the excitation of 532nm six-membered ring rhodamine dyes, its structure is as follows:

R ₁₂ is a 5-position parallel six-membered ring or a 7-position parallel six-membered ring;

R ₁₃ is H or C1-4 alkane.

The synthesis route of the six-membered ring-rhodamine dyes is as follows:

Specific steps are as follows:

(1) Synthesis of intermediate N-alkyl-5-hydroxytetrahydroquinolinyl benzophenone acid

Dissolve the intermediates N-alkyl-5-hydroxytetrahydroquinoline and phthalic anhydride in toluene. After heating and refluxing for 4-8h, stop the reaction. After cooling to room temperature, let stand in an ice water bath for 30min-60min. , Filter, the filter cake was washed with a small amount of petroleum ether, and the filter cake was dried to obtain the crude intermediate product N-alkyl-5-hydroxytetrahydroquinolinyl benzophenone acid;

(2) Synthesis of target dyes

Intermediate N-alkyl-5-hydroxytetrahydroquinoline (or its analogue N-substituted-7-hydroxytetrahydroquinoline) and intermediate N-alkyl-5-hydroxytetrahydroquinolinobenzoic acid Dissolved in a mixed acidic solvent of methanesulfonic acid and trifluoroacetic acid, heated to 140°C under the protection of nitrogen. After two days of reaction, most of the solvent was removed under reduced pressure, and the pH was adjusted to 9-10 with a weak alkaline aqueous solution. Dichloro Methane extraction, organic phase collection and drying, after removing organic solvent, silica gel column separation, eluent is dichloromethane and methanol in a volume ratio of 20-5:1, the solvent is removed under reduced pressure to obtain the final product;

In step (1), the mass ratio of the intermediate N-alkyl-5-hydroxytetrahydroquinolinyl keto acid and phthalic anhydride is 1:1-2, and the intermediate N-alkyl-5-hydroxyl The mass ratio of tetrahydroquinolinyl benzophenone acid to toluene is 1:40-80g/mL;

In step (2), the intermediate N-alkyl-5-hydroxytetrahydroquinoline (or its analog N-substituted-7-hydroxytetrahydroquinoline) and the intermediate N-alkyl-5-hydroxytetrahydroquinoline The mass ratio of quinolinyl benzophenic acid is 1:2-4; the volume ratio of trifluoroacetic acid and methanesulfonic acid is 1:1-5; the intermediate N-alkyl-5-hydroxytetrahydroquinoline (or its The volume ratio of the analog N-substituted-7-hydroxytetrahydroquinoline) to trifluoroacetic acid is 1:30-80 g/mL.

A full-spectrum, high-brightness, high-stability fluorescent dye with five-membered ring rhodamine dyes, its structure is as follows:

Among them, R ₁₃ is H or C1-4 alkane.

The synthetic route of the five-membered ring-rhodamine dyes is as follows:

Specific steps are as follows:

(1) Synthesis of intermediate N-alkyl-4-hydroxyindolinyl benzophenone acid

Intermediate N-alkyl-4 hydroxyindoline and phthalic anhydride are dissolved in toluene. After heating and refluxing for 4-8h, the reaction is stopped. After cooling to room temperature, after standing in an ice water bath for 30min-60min, filtering and filtering The cake was washed with a small amount of petroleum ether, and the filter cake was dried to obtain the intermediate N-alkyl-4-hydroxyindolinyl benzophenone acid;

(2) Synthesis of target dyes

Intermediate N-alkyl-4 hydroxyindoline and intermediate N-alkyl-4-hydroxyindoline benzophenone acid are dissolved in a mixed acidic solvent of methanesulfonic acid and trifluoroacetic acid, heated under nitrogen protection After reacting at 140°C for two days, most of the solvent was removed under reduced pressure. The pH was adjusted to 9-10 with a weakly alkaline aqueous solution, extracted with dichloromethane, the organic phase was collected and dried, after removing the organic solvent, the silica gel column was separated and eluted The agent is dichloromethane and methanol in a volume ratio of 20-5:1, and the final product is obtained after removing the solvent under reduced pressure;

In step (1), the mass ratio of intermediate N-alkyl-4hydroxyindoline to phthalic anhydride is 1:1-2, and the mass of intermediate N-alkyl-4hydroxyindoline and toluene The volume ratio is 1:20-40g/mL;

In step (2), the mass ratio of the intermediate N-alkyl-4hydroxyindoline to the intermediate N-alkyl-4-hydroxyindoline benzophenone acid is 1:2-3, trifluoroacetic acid and The volume ratio of methanesulfonic acid is 1:1-5, and the volume ratio of the mass of intermediate N-alkyl-4hydroxyindoline to trifluoroacetic acid is 1:10-30g/mL.

A full-spectrum, high-brightness, high-stability fluorescent dye of silicon-based rhodamine dyes, its structure is as follows:

Among them: q=0 or 1;

R ₁₃ is H, C1-4 alkyl.

The synthetic route of silicon-based rhodamine dyes is as follows:

Specific steps are as follows:

(1) Synthesis of target fluorescent molecule symmetrical silyl rhodamine

Add 2-bromobenzoic acid tert-butyl ester to Shrek bottle, add tetrahydrofuran solution under the protection of nitrogen, add butyllithium solution under -78 ℃ stirring condition, stir for 20-40min; add intermediate Si-keto tetrahydrofuran solution, l Stir at room temperature and avoid light overnight; after quenching with saturated aqueous ammonium chloride solution, dilute with water, extract with ethyl acetate, collect the organic phase, wash and dry, remove the organic solvent under reduced pressure to obtain the crude product, and separate it through a 200-300 mesh silica gel column, The eluent is petroleum ether and ethyl acetate in a volume ratio of 50-20:1 to obtain the target fluorescent dye molecule;

Specific steps are as follows:

(2) Synthesis of target fluorescent dye asymmetric silyl rhodamine

Add 2-bromobenzoic acid tert-butyl ester to Shrek bottle, add tetrahydrofuran solution under the protection of nitrogen, add butyllithium solution under -78℃ stirring condition, stir for 20-40min; add intermediate Si-ketos tetrahydrofuran solution, l Stir to room temperature and avoid light overnight; after quenched with saturated aqueous ammonium chloride solution, dilute with water, extract with ethyl acetate, collect the organic phase, wash and dry, and remove the organic solvent under reduced pressure to obtain the crude product; the crude product is separated by column chromatography and washed The removal agent is petroleum ether and ethyl acetate with a volume ratio of 50-20:1, and the target fluorescent dye molecule is obtained after removing the organic solvent under reduced pressure;

In step (1), the mass ratio of intermediate Si-keto to 2-bromobenzoic acid tert-butyl ester is 1:4-8, and the mass ratio of intermediate Si-keto to butyllithium solution is 10-20: 1mg/mL;

In step (2), the mass ratio of intermediate Si-ketos to tert-butyl 2-bromobenzoate is 1:4-7, the volume ratio of butyllithium solution to tetrahydrofuran is 1:30-50, intermediate Si- The volume ratio of ketos to butyllithium is 1:10-20mg/μL.

The above-mentioned fluorescent dyes used in a class of high brightness and high stability have high biocompatibility. After functionalization, they can perform real-time fluorescence imaging of different organelles and different protein targets of living cells, and super-resolution in STED and SIM. It can be used in fluorescence microscopy.

A full-spectrum high-brightness, high-stability fluorescent dye is used in the field of fluorescent imaging of living cells, tissues, and living bodies.

A full-spectrum high-brightness, high-stability fluorescent dye is used in the field of SNAP-tag and Halo-tag identification tags.

A full-spectrum high-brightness, high-stability fluorescent dye is used in the field of fluorescent imaging of living cells, tissues, and living bodies. The invention has the following characteristics:

The dyes involved in the present invention have the advantages of simple synthetic methods, cheap raw materials and easy functionalization.

Some dyes involved in the present invention have a fluorescence emission half-peak width of less than 40nm in different organic solvents, and the narrowest can reach 25nm; the fluorescence quantum yield is significantly improved, and can reach 0.80 in water; the light stability is significantly higher than fluorescein, rhodamine, Fluoroboron pyrrole dyes.

The functionalized molecules based on this kind of dye fluorescent precursor have high biocompatibility, and can complete the fluorescent labeling of cells within 20s-5min. Among them, the mitochondrial series of fluorescent probes can completely label mitochondria of various living cells within 2 minutes. After the protein-targeted fluorescent probe is combined with the protein, it realizes a ten-fold increase in fluorescence, enabling no-wash fluorescence imaging in living cells. The lipid droplet dye can accurately locate lipid droplets in various cell lines such as HT29 (colon cancer cells), MCF (breast cancer cells), and fat cells; at the same time, it can mark the zebrafish living lipid metabolism center (liver) And fluorescence imaging.

The improvement of the light stability of the dye involved in the present invention enables the dye to achieve super-resolution fluorescence imaging, which is more light stable than the traditional fluorescent dye Alexa 488.

BRIEF DESCRIPTION

FIG. 1 is the hydrogen spectrum of the NMR spectrum of Lyso-DAze prepared in Example 23. FIG.

2 is a nuclear magnetic spectrum hydrogen spectrum of the Nu-DAC prepared in Example 24.

3 is a hydrogen spectrum of the NMR spectrum of the CM-DAze prepared in Example 30.

4 is a hydrogen spectrum of the NMR spectrum of the NHSB-DAC prepared in Example 40. FIG.

5 is a hydrogen spectrum of the nuclear magnetic spectrum of BuLD-DAze prepared in Example 49.

FIG. 6 is a nuclear magnetic spectrum hydrogen spectrum of the dye SiR-1 prepared in Example 60. FIG.

7 is a high-resolution mass spectrum of the DTX-DAC prepared in Example 46.

FIG. 8 is the normalized fluorescence excitation spectrum and fluorescence emission spectrum of the dye material BuAN-DAze prepared in Example 5 in ethanol. The abscissa is the wavelength, the ordinate is the fluorescence intensity, and the concentration of the fluorescent dye is 10 μM.

FIG. 9 is the normalized fluorescence excitation spectrum and fluorescence emission spectrum of the dye material OLD-710 prepared in Example 52 in ethanol. The abscissa is the wavelength, the ordinate is the fluorescence intensity, and the concentration of the fluorescent dye is 10 μM.

10 is a normalized fluorescence excitation spectrum and fluorescence emission spectrum of the dye material SiR-1 prepared in Example 60 in ethanol, the abscissa is the wavelength, the ordinate is the fluorescence intensity, and the concentration of the fluorescent dye is 10 μM.

FIG. 11 is a graph of the fluorescence intensity of the dye material BuAN-DAze prepared in Example 5 at 495 nm as a function of time under the irradiation of a 500 W tungsten lamp. Commercial green mitochondrial dye, rhodamine 123, fluorescein, and Bodipy were selected as reference dyes.

FIG. 12 is a fluorescence confocal imaging of live cells of RWPE cells prepared with the dye Mito-DAze prepared in Example 20. FIG.

13 is a light-illuminated micro-imaging image of the living cell structure of the RWPE cell of the dye Mito-DAze prepared in Example 20. FIG.

14 is a fluorescence confocal imaging diagram of HeLa cell live cell fluorescent dye Mito-DAC prepared in Example 19. FIG.

FIG. 15 is a fluorescent confocal imaging image of adipose cell live cell fluorescence of the lipid droplet dye OLD-DAze prepared in Example 50. FIG.

16 is a fluorescent confocal imaging of HeLa cells transfected with HALO-H2B dye prepared in Example 14 by the dye Halo-DAze, and the concentration of the fluorescent probe is 1 μM.

17 is a fluorescent confocal imaging of HeSN cells of pSNAP _f -H2B transfected with the dye SNAP-DAC prepared in Example 17, and the concentration of the fluorescent probe is 1 μM.

18 is a microscopic image of stimulated radiation loss of HeLa cells of pSNAP _f -H2B transfected with the dye SNAP-DAC prepared in Example 17, and the concentration of the fluorescent probe is 1 μM.

FIG. 19 is a structured light illumination microcolor imaging of RWPE cells with Rho-4 prepared in Example 58 and Nu-DAC prepared in Example 33

FIG. 20 is a structured light illumination microscope multicolor imaging diagram of OLD-DAze prepared in Example 50 and Nu-DAC prepared in Example 33 on HT29 cells.

detailed description

Example 1

Synthesis of DOAN:

Ethylene glycol (20 mg, 0.33 mmol) was dissolved in 10 mL of dry tetrahydrofuran, Na block (20 mg, 0.89 mmol) was added under nitrogen, and N-butyl-4-bromo-5-nitrate was added to the reaction solution after 0.5 h -1,8-naphthalimide (120mg, 0.31mmol) and heated to 80°C for 10h. After the solvent was removed under reduced pressure, the silica gel column was separated, using dichloromethane: petroleum ether = 1:2 as the eluent, and the solvent was removed under reduced pressure to obtain 12 mg of white solid DOAN with a yield of 12%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.44 (d, J=8.3 Hz, 2H), 7.23 (d, J=8.4 Hz, 2H), 4.31 (s, 4H), 4.06-3.96 (m, 2H), 1.60 (td, J = 13.2, 6.2Hz, 2H), 1.34 (td, J = 14.2, 7.3Hz, 2H), 0.95 (t, J = 7.2Hz, 3H).

After examination, the structure is as shown in the above formula BAm. The dye has strong absorption at 405 nm and can be used for excitation at 405 nm.

Example 2

Synthesis of OEOAN:

Ethylene glycol (100 mg, 1.67 mmol) was dissolved in 10 mL of dry tetrahydrofuran, Na block (77 mg, 3.34 mmol) was added under nitrogen, and N-butyl-4-bromo-5-nitro was added to the reaction solution after 1 h -1,8-naphthalimide (50mg, 0.13mmol) and heated to 60 °C to continue the reaction for 3h. After removing the solvent under reduced pressure, the silica gel column was separated, using dichloromethane: methanol = 100:1 as the eluent, and the solvent was removed under reduced pressure to obtain 40 mg of white solid OEOAN with a yield of 81%. The specific data of the nuclear magnetic spectrum hydrogen spectrum and carbon spectrum of the OEOAN prepared in Example 4 are:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.43 (d, J=8.4 Hz, 2H), 7.22 (d, J=8.5 Hz, 2H), 5.21 (t, J=5.7 Hz, 2H), 4.23 (s, 4H), 4.06-3.96 (m, 2H), 3.91 (d, J = 4.0 Hz, 4H), 1.60 (td, J = 13.5, 6.3 Hz, 2H), 1.34 (td, J = 14.4, 7.2 Hz, 2H), 0.93 (t, J=7.1Hz, 3H). ¹³ C NMR (101MHz, DMSO-d6) δ163.69, 162.86, 134.26, 131.88, 114.04, 114.01, 107.88, 71.42, 60.20, 30.21, 20.31 , 14.21.

After testing, the structure is as shown in the above formula OEOAN. The fluorescence emission wavelength of OEOAN in acetonitrile, chloroform, ethanol, dimethyl sulfoxide, and water is 420-450nm. The fluorescence wavelength does not basically change with the polarity of the solvent, and the half-width is less than 50nm. It avoids the interference of different polar environments on the fluorescence signal in fluorescence imaging and detection. OEOAN's ultraviolet absorption wavelength in acetonitrile, chloroform, ethanol, dimethyl sulfoxide, and water has strong absorption at 405nm, which is convenient for excitation to obtain high-brightness fluorescence. In addition, the dye is less affected by changes in polarity.

Example 3

Synthesis of DOEOAN:

Diethylene glycol (50 mg, 0.47 mmol) was dissolved in 5 mL of dry tetrahydrofuran, Na block (50 mg, 2.18 mmol) was added under nitrogen, and N-butyl-4-bromo-5-nitrate was added to the reaction solution after 0.5 h -1,8-naphthalimide (50mg, 0.13mmol) and heated to 60 ° C to continue the reaction for 2h. After the solvent was removed under reduced pressure, the silica gel column was separated, using dichloromethane: methanol = 50:1 as the eluent, and the solvent was removed under reduced pressure to obtain light yellow semi-solid DOEOAN 14 mg, with a yield of 23%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.45 (d, J=8.2 Hz, 2H), 7.21 (d, J=8.6 Hz, 2H), 5.46 (t, J=5.8 Hz, 2H), 4.25 (s, 4H), 4.06-3.94 (m, 2H), 3.92-3.65 (m, 12H), 1.61 (td, J = 13.0, 6.2 Hz, 2H), 1.36 (td, J = 142, 7.1 Hz, 2H ), 0.95 (t, J = 7.2Hz, 3H).

After examination, the structure is as shown in the above formula DOEOAN. The dye has strong absorption at 405 nm and can be used for excitation at 405 nm.

Example 4

Synthesis of DMOAN:

Dissolve 1,2-dimethylethylene glycol (30mg, 0.33mmol) in 30mL of dry tetrahydrofuran, add Na block (15mg, 0.87mmol) under nitrogen, and add N-butyl to the reaction solution after 0.5h 4-Bromo-5-nitro-1,8-naphthalimide (126mg, 0.33mmol) and heated to 90°C for 8h. After removing the solvent under reduced pressure, the silica gel column was separated, and dichloromethane: petroleum ether = 1:2 was used as the eluent. The solvent was removed under reduced pressure to obtain 9 mg of white solid DMOAN with a yield of 8%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.41 (d, J=8.2 Hz, 2H), 7.25 (d, J=8.4 Hz, 2H), 4.47 (m, 2H), 4.01-3.92 (m, 2H), 2.47 (d, J = 8.7 Hz, 6H), 1.61 (td, J = 13.1, 6.4 Hz, 2H), 1.33 (td, J = 14.2, 7.8 Hz, 2H), 0.96 (t, J = 7.2 Hz, 3H).

After examination, the structure is as shown in the above formula DMOAN. The dye has strong absorption at 405nm and can be used for excitation at 405nm.

Example 5

Synthesis of N-butyl-4,5-diazacyclobutane-1,8 naphthalimide (BuAN-DAze)

N-butyl-4-bromo-5-nitro-1,8-naphthalimide (100 mg, 0.26 mmol) was dissolved in 20 mL of ethylene glycol methyl ether, and azetidine (300 mg) was added thereto , 5.26mmol). The reaction solution was slowly heated to 120°C and reacted for 24h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 150:1, V/V) to obtain 80 mg of a yellow solid with a yield of 51%. The nuclear magnetic spectrum hydrogen spectrum and carbon spectrum of BuAN-DAze prepared in Example 8 are shown in Figures 3 and 4, respectively. The specific data are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.38 (d, J=8.5 Hz, 2H), 6.38 (d, J=8.5 Hz, 2H), 4.21-4.12 (m, 2H), 4.05 (s, 2H) , 2.42 (s, 8H), 1.69 (dt, J = 15.2, 7.6 Hz, 2H), 1.43 (dq, J = 14.8, 7.4 Hz, 2H), 0.95 (t, J = 7.3 Hz, 3H). ¹³ C NMR (101MHz, CDCl ₃ ) δ164.44, 155.52, 133.05, 132.82, 110.29, 108.05, 106.30, 54.79, 39.68, 30.41, 20.49, 16.90, 13.93.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value calcd for C ₂₂ H ₂₆ N ₃ O ₂ [M+H] ⁺ 364.2025, measured value 364.2035.

After testing, its structure is shown in the above formula BuAN-DAze, and its fluorescent properties are as follows:

Dissolve BuAN-DAze in DMSO solution to prepare 2mM mother liquor, and prepare test solutions with different concentrations as needed to detect its fluorescence spectrum and excitation spectrum.

Each time, 20 μL of the mother liquor of dye was added to 4 mL of ethanol to prepare a 10 μM fluorescent probe test solution, and fluorescence excitation and emission spectrum tests were performed.

The fluorescence excitation and emission spectrum of BuAN-DAze is shown in Fig. 34: The excitation wavelength of BuAN-DAze in ethanol is 480nm, the fluorescence emission wavelength is 488nm, and the half-width of the fluorescence emission is only 32nm. This shows that BuAN-Daze can be applied to multi-color fluorescence imaging.

Example 6

Synthesis of N-butyl-4,5-bis(azacyclopentyl)-1,8 naphthalimide (BuAN-DAzo)

N-butyl-4-bromo-5-nitro-1,8-naphthalimide (50 mg, 0.13 mmol) was dissolved in 5 mL of ethylene glycol methyl ether, and 200 mg of tetrahydropyrrole was added thereto. The reaction solution was slowly heated to 120°C and reacted for 10h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V/V) to obtain 38 mg of a yellow solid with a yield of 75%. The nuclear magnetic spectrum hydrogen spectrum of BuAN-DAzo prepared in Example 9 is shown in FIG. 5, and the specific data of hydrogen spectrum and carbon spectrum are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.32 (d, J=8.7 Hz, 2H), 6.65 (d, J=8.7 Hz, 2H), 4.18 (t, J=7.0 Hz, 2H), 3.60 (s , 2H), 3.37 (d, J = 4.9 Hz, 1H), 3.28 (d, J = 4.2 Hz, 2H), 2.67 (d, J = 8.0 Hz, 2H), 2.19 (s, 2H), 1.97 (s , 4H), 1.70 (dt, J=15.2, 7.6Hz, 2H), 1.57 (s, 2H), 1.45 (dt, J=15.1, 7.4Hz, 2H), 0.96 (t, J=7.3Hz, 3H) . ¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.33, 154.20, 133.79, 132.69, 109.52, 108.97, 106.15, 52.27, 49.49, 39.53, 30.45, 25.89, 25.54, 20.51, 13.97.

The high-resolution mass spectrometry data is as follows: the theoretical value of the resolution mass spectrum C ₂₄ H ₃₀ N ₃ O ₂ [M+H1 ⁺ 392.2338, the actual value is 392.2343.

After testing, its structure is as shown in the above formula BuAN-DAzo, and its fluorescent properties are as follows:

Dissolve BuAN-DAzo in DMSO solution to prepare 2mM mother liquor, and formulate test solutions with different concentrations as needed to detect its fluorescence spectrum and excitation spectrum.

Each time, 20 μL of the mother liquor of dye was added to 4 mL of ethanol to prepare a 10 μM fluorescent probe test solution, and fluorescence excitation and emission spectrum tests were performed.

The fluorescence excitation and emission spectrum of BuAN-DAzo is shown in Figure 35: The excitation wavelength of BuAN-DAzo in ethanol is 485nm, the fluorescence emission wavelength is 495nm, and the fluorescence emission half-width is only 40nm, which is suitable for 488nm excitation.

Example 7

Synthesis of N-butyl-4,5-diazacycloheptyl-1,8 naphthalimide (BuAN-DHMI)

N-butyl-4-bromo-5-nitro-1,8-naphthalimide (80 mg, 0.21 mmol) was dissolved in 15 mL of ethylene glycol methyl ether, and 400 mg of hexamethyleneimine was added thereto . The reaction solution was slowly heated to 120°C and reacted for 20h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=200:1, V/V) to obtain 80 mg of a yellow solid with a yield of 85%. The nuclear magnetic spectrum hydrogen spectrum of the BuAN-DHMI prepared in Example 11 is shown in FIG. 7, and the specific data are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.32 (d, J=8.6 Hz, 2H), 6.88 (d, J=8.7 Hz, 2H), 4.23-3.97 (m, 2H), 3.47 (s, 8H) , 1.82-1.31 (m, 20H), 0.96 (t, J = 7.3 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.42, 156.62, 134.48, 132.31, 110.55, 109.52, 109.48, 52.71, 39.60 , 30.46, 28.33, 27.80, 20.52, 13.95.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₈ H ₃₈ N ₃ O ₂ [MH ⁺ ] 448.2964, found value 448.2973.

After testing, its structure is as shown in the above formula BuAN-DHMI, and its fluorescent properties are as follows: in the aqueous solution, its fluorescence emission wavelength is 520 nm, and its absorption wavelength reaches 485 nm.

Example 8

Synthesis of BuAN-450

BuAN-DBu (100 mg, 0.25 mmol) was dissolved in 10 mL of toluene, and then the reaction solution was slowly heated to 100° C. and reacted for 24 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=200:1, V/V) to obtain 39.6 mg of a yellow solid with a yield of 40%. The hydrogen spectrum of the nuclear magnetic spectrum of BuAN-450 prepared in Example 12 is shown in FIG. 8, and the specific data of hydrogen spectrum and carbon spectrum are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.55 (d, J=8.2 Hz, 1H), 8.50 (d, J=8.4 Hz, 1H), 7.23 (d, J=8.2 Hz, 1H), 6.75 (d , J=8.5Hz, 1H), 4.26-4.12(m, 2H), 4.04-3.90(m, 2H), 2.84-2.72(m, 2H), 1.70(dt, J=7.7, 6.5Hz, 2H), 1.61-1.50 (m, 2H), 1.45 (td, J = 14.9, 7.4 Hz, 2H), 1.13 (t, J = 7.4 Hz, 3H), 1.07 (t, J = 7.4 Hz, 3H), 0.97 (t , J=7.4Hz, 3H).

The high-resolution mass spectrometry data is as follows: high-resolution mass spectrometry theoretical value C ₂₄ H ₃₀ N ₃ O ₂ [M+H] ⁺ 392.2338, the actual value is 392.2352.

After testing, its structure is shown in the above formula BuAN-450, and its fluorescent properties are as follows: the fluorescence emission wavelength in methylene chloride is 475nm, and the absorption wavelength reaches 458nm, which is suitable for excitation by a 450nm laser.

Example 9

Synthesis of N-butyl-4-azacyclobutyl-5-cycloethylamino-1,8 naphthalimide (BuAN-AzeAzi)

N-butyl-4-bromo-5-nitro-1,8-naphthalimide (100 mg, 0.26 mmol) was dissolved in 10 ml of ethylene glycol methyl ether, and aziridine (30 μL) was added thereto . After the reaction solution was stirred at 50°C for 2h, ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 200: 1, V/V) to obtain a brown solid 78mg, yield 80%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.52 (d, J=8.1 Hz, 1H), 8.36 (d, J=7.9 Hz, 1H), 8.06 (d, J=7.9 Hz, 1H), 7.40-7.08 (m, 1H), 4.28-4.02 (m, 2H), 2.54 (s, 4H), 1.84-1.58 (m, 2H), 1.43 (dd, J = 14.9, 7.4 Hz, 2H), 0.97 (t, J = 7.3 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ163.89,163.44,157.69,133.68,132.98,131.48,131.17,125.58,125.35,122.47,118.61,117.16,40.23,32.63,30.14,20.38, 13.85.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₁₈ H ₁₈ BrN ₂ O ₂ [M+H] ⁺ 373.0554, the actual value is 373.0561.

Synthesis of N-butyl-4-azacyclobutyl-5-cycloethylamino-1,8 naphthalimide (BuAN-AzeAzi)

BuAN-BrAzi (50 mg, 0.13 mmol) was dissolved in 10 mL of ethylene glycol methyl ether and 200 mg of azetidine was added to the reaction solution, and then the reaction solution was slowly heated to 120° C. and reacted for 12 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V/V) to obtain 22 mg of a dark yellow solid with a yield of 47%. The hydrogen spectrum of the NMR spectrum of BuAN-AzeAzi prepared in Example 13 is shown in FIG. 9, and the specific data are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.43 (d, J=5.9 Hz, 1H), 8.41 (d, J=6.3 Hz, 1H), 7.02 (d, J=8.1 Hz, 1H), 6.53 (d , J=8.5Hz, 1H), 4.28-4.08 (m, 6H), 2.38 (dt, J=15.0, 7.5Hz, 2H), 2.26 (s, 4H), 1.68 (dt, J=15.2, 7.6Hz, 2H), 1.43 (dq, J = 14.7, 7.3H [z, 2H), 0.95 (t, J = 7.3 Hz, 3H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.41, 164.31, 159.01, 155.45, 132.87, 132.52, 132.33, 115.99, 115.20, 115.15, 111.06, 107.80, 55.77, 39.80, 30.32, 30.26, 20.45, 16.97, 13.90.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₁ H ₂₄ N ₃ O ₂ [M+H] ⁺ 350.1869, actual value 350.1872.

After testing, its structure is as shown in the above formula BuAN-AzeAzi, and its fluorescence performance is as follows: the fluorescence emission wavelength in water is 530nm, and the absorption wavelength reaches 468nm, which is suitable for excitation by a 450nm laser.

Example 10

Synthesis of N-butyl-4-azacyclopentyl-5-azacyclobutyl-1,8 naphthalimide (BuAN-AzeAzo)

Synthesis of N-butyl-4-bromo-5-azacyclobutyl-1,8 naphthalimide (BuAN-BrAze)

N-butyl-4-bromo-5-nitro-1,8-naphthalimide (100 mg, 0.26 mmol) was dissolved in 8 ml of ethylene glycol methyl ether, and 40 mg of azetidine was added thereto . After the reaction solution was stirred at 50°C for 1 h, ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=200:1, V/V) to obtain a brown solid 75 mg, yield 72%.

Synthesis of N-butyl-4-azacyclopentyl-5-azacyclobutyl-1,8 naphthalimide (BuAN-AzeAzo)

BuAN-BrAze (80 mg, 0.21 mmol) was dissolved in 10 mL of ethylene glycol methyl ether and 200 mg of tetrahydropyrrole was added to the reaction solution, and then the reaction solution was slowly heated to 120° C. and reacted for 12 h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V/V) to obtain a deep yellow solid 52mg, yield 67%. The hydrogen spectrum of the NMR spectrum of BuAN-AzeAzo prepared in Example 15 is shown in FIG. 10, and the specific data are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.28 (dd, J=10.1, 8.7 Hz, 2H), 6.61 (d, J=8.7 Hz, 1H), 6.27 (d, J=8.5 Hz, 1H), 4.16 -3.90 (m, 4H), 3.68-3.49 (m, 4H), 2.95 (s, 2H), 2.45-2.23 (m, 2H), 2.09-1.89 (m, 2H), 1.87 (s, 2H), 1.62 (dt, J = 15.2, 7.6 Hz, 2H), 1.36 (dq, J = 14.7, 7.4 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H0. ¹³ C NMR (101 MHz, CDCl ₃ ) δ 163.50 ,163.32,155.24,152.31,132.38,131.85,131.67,109.05,108.50,107.97,105.48,104.86,54.66,52.05,50.07,38.62,29.41,28.68,24.70,19.49,15.78,12.92.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₃ H ₂₈ N ₃ O ₂ [M+H] ⁺ 378.2182, actual value 378.2093.

After testing, its structure is as shown in the above formula BuAN-AzeAzo, and its fluorescence performance is as follows: the fluorescence emission wavelength in water is 493nm, and the absorption wavelength reaches 481nm, which is suitable for 488nm laser excitation.

Example 11

Synthesis of N-butyl-4,5-ethylenediamine-1,8 naphthalimide (BuAN-EDA)

N-butyl-4-bromo-5-nitro-1,8-naphthalimide (100 mg, 0.27 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and 150 mg of ethylenediamine was added thereto. The reaction solution was slowly heated to 70°C and reacted for 24h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 70:1, V/V) to obtain 71 mg of a yellow solid with a yield of 87%. The hydrogen spectrum of the nuclear magnetic spectrum of BuAN-EDA prepared in Example 17 is shown in FIG. 12, and the specific data of the hydrogen spectrum and the carbon spectrum are:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.29 (s, 2H), 8.03 (d, J = 8.6 Hz, 2H), 6.67 (d, J = 8.7 Hz, 2H), 4.01-3.92 (m, 2H), 3.51 (s, 4H), 1.54 (dt, J = 14.9, 7.5Hz, 2H), 1.31 (dt, J = 14.8, 7.4Hz, 2H), 0.90 (t, J = 7.3Hz, 3H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 163.36, 155.59, 135.28, 133.35, 110.27, 107.36, 105.79, 46.73, 38.97, 30.40, 20.35.

After testing, its structure is as shown in the above formula BuAN-EDA, and its fluorescence performance is as follows; the fluorescence emission wavelength in water is 487 nm, and the absorption wavelength reaches 481 nm, which is suitable for excitation by a 488 nm laser.

Example 12

Synthesis of N-butyl-4,5-(1,2-cyclohexanediamine)yl-1,8 naphthalimide (BuAN-DAC)

N-butyl-4-bromo-5-nitro-1,8-naphthalimide (100 mg, 0.27 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 350 mg of cyclohexanediamine was added thereto. The reaction solution was slowly heated to 120°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 70:1, V/V) to obtain a yellow solid 31 mg with a yield of 32%. The nuclear magnetic spectrum hydrogen spectrum of the BuAN-DAC prepared in Example 18 is shown in FIG. 13, and the specific data are:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.04 (d, J=8.6 Hz, 2H), 7.50 (s, 2H), 6.83 (d, J=8.7 Hz, 2H), 4.04-3.83 (m, 2H), 3.16 (t, J = 7.0 Hz, 2H), 2.19 (d, J = 11.2 Hz, 2H), 1.73 (d, J = 8.1 Hz, 2H), 1.54 (dt, J = 14.9, 7.6 Hz, 2H), 1.30 (dq, J = 14.3, 7.2 Hz, 6H), 0.90 (t, J = 7.3 Hz, 3H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 163.43, 154.52, 134.70, 133.32, 110.56, 107.86, 106.52, 59.52, 55.38, 32.09, 30.40, 23.64, 20.35, 14.28.

Theoretical value of high-resolution mass spectrometry C ₂₂ H ₂₆ N ₃ O ₂ [M+H] ⁺ 364.2025, found value 364.2029.

After testing, its structure is as shown in the above-mentioned BuAN-DAC, and its fluorescence performance is as follows: the fluorescence emission wavelength in water is 488nm, and the absorption wavelength reaches 481nm, which is suitable for excitation by a 488nm laser.

Example 13

Synthesis of N-butyl-4,5-(1,2-cyclohexanediamine)yl-1,8 naphthalimide (BuAN-DMC)

N-butyl-4-bromo-5-nitro-1,8-naphthalimide (100 mg, 0.27 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and N,N'-dimethyl was added thereto Cyclohexanediamine 350mg. The reaction solution was slowly heated to 120°C and reacted for 12h. The ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V/V) to obtain a yellow solid 31 mg with a yield of 30%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.40 (d, J=8.2 Hz, 1H), 6.81 (d, J=8.4 Hz, 1H), 4.35-3.94 (m, 1H), 3.10 (s, 2H) , 2.22 (s, 1H), 1.81 (d, J = 8.3 Hz, 1H), 1.70 (dt, J = 15.2, 7.5 Hz, 1H), 1.44 (dq, J = 14.8, 7.4 Hz, 1H), 1.20 ( s, 1H), 0.96 (t, J=7.3Hz, 2H).

After testing, its structure is as shown in the above formula BuAN-DMC, and its fluorescence performance is as follows: its fluorescence emission wavelength in water is 515nm, and its absorption wavelength reaches 460nm, which is suitable for 450nm laser excitation.

Example 14

Synthesis of Halo-DAze

Synthesis of Intermediate N-(2-(2-Hydroxy)-ethoxy)ethyl-4,5-diazacyclobutane-1,8 naphthalimide (OAN-DAze)

OAN-Br (50 mg, 0.12 mmol) was dissolved in 5 mL of ethylene glycol methyl ether, and 150 mg of azetidine was added thereto. The reaction solution was slowly heated to 120°C and reacted for 10h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=80:1, V/V) to obtain a yellow solid 25 mg with a yield of 52%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.37 (d, J = 8.5 Hz, 2H), 6.38 (d, J = 8.5 Hz, 2H), 4.42 (d, J = 5.3 Hz, 2H), 4.09 (s , 8H), 3.83 (t, J = 5.4 Hz, 2H), 3.68 (s, 4H), 2.42 (s, 4H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.74, 155.77, 133.31, 133.16, 109.85 , 107.84, 106.39, 72.18, 68.91, 61.94, 55.25, 38.99, 16.89.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₂ H ₂₆ N ₃ O ₄ [M+H] ⁺ 396.1923, found value 396.1919.

Synthesis of Halo-DAze

Halo-OH (30 mg, 0.08 mmol) and NaH (6 mg, 0.25 mmol) were placed in a 10 mL Shrek bottle and replaced with nitrogen three times. After dissolving 15 μL of 1-iodo-6-chlorohexane in 6 mL of dried DMF, the reaction solution was added. After stirring at room temperature for 5 hours, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 200:1, V/V) to obtain a brown solid 20 mg with a yield of 50%. The hydrogen spectrum of the Halo-DAze NMR spectrum prepared in Example 20 is shown in FIG. 14, and the specific data of the hydrogen spectrum and the carbon spectrum are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.37 (d, J = 8.5 Hz, 2H), 6.38 (d, J = 8.5 Hz, 2H), 4.41 (t, J = 6.5 Hz, 2H), 4.07 (s , 8H), 3.78(t, J=6.5Hz, 2H), 3.71-3.65(m, 2H), 3.60-3.54(m, 2H), 3.43(t, J=6.6Hz, 2H), 2.43(s, 4H), 2.02 (dd, J = 14.1, 7.1 Hz, 2H), 1.80-1.70 (m, 2H), 1.54 (dd, J = 13.8, 6.9 Hz, 2H), 1.41 (dd, J = 15.2, 7.8 Hz , 2H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.41, 155.61, 133.22, 132.44, 110.11, 108.02, 107.86, 106.32, 77.22, 71.21, 70.13, 68.21, 54.55, 38.61, 33.56, 29.70, 26.74, 25.42 , 25.38.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₈ H ₃₇ ClN ₃ O ₄ [M+H] ⁺ 514.2473, the actual value is 514.2477.

After testing, the structure is as shown in the above formula Halo-DAze, the ultraviolet absorption wavelength in water is 484nm, and the fluorescence emission wavelength is 493nm, which can be used for Halo-tag fluorescent labeling.

Example 15

Synthesis of Halo-DAC

Synthesis of N-dodecyl-4,5-(1,2-cyclohexanediamine)yl-1,8 naphthalimide (DDAN-DAC)

OAN-NBr (100 mg, 0.24 mmol) was dissolved in 5 mL of ethylene glycol methyl ether, and 100 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 100°C and reacted for 24h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated on a silica gel column (dichloromethane: methanol = 70:1, V/V) to obtain 34 mg of a yellow solid with a yield of 35%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d6) δ 8.04 (d, J=8.6 Hz, 21H), 7.54 (s, 2H), 6.83 (d, J=8.7 Hz, 2H), 4.59 (t, J=4.7 Hz, 1H), 4.15 (t, J = 6.8 Hz, 2H), 3.56 (t, J = 6.8 Hz, 2H), 3.46 (s, 4H), 3.15 (d, J = 9.4 Hz, 2H), 2.20 ( d, J = 12.0 Hz, 2H), 1.73 (d, J = 7.2 Hz, 2H), 1.43-1.22 (m, 4H). ¹³ C NMR (101 MHz, DMSO d6) δ 163.43, 154.65, 134.84, 133.40, 110.63, 107.62, 106.40, 72.53, 67.69, 60.66, 59.48, 46.17, 32.07, 23.63.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₂ H ₂₆ N ₃ O ₄ [M+H] ⁺ 396.1923, found value 396.1919.

Synthesis of Halo-DAC

OAN-DAC (50 mg, 0.13 mmol) and NaH (10 mg, 0.42 mmol) were placed in a 10 mL Shrek bottle and replaced with nitrogen three times. After dissolving 50 μL of 1-iodo-6-chlorohexane in 5 mL of dried DMF, the reaction solution was added. After stirring at room temperature for 1 h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V/V) to obtain a brown solid 36 mg with a yield of 56%. The hydrogen spectrum of the nuclear magnetic batch diagram of Halo-DAC prepared in Example 2 is shown in FIG. 2, and the specific data of hydrogen spectrum and carbon spectrum are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.25 (d, J=8.3 Hz, 2H), 6.52 (d, J=8.3 Hz, 2H), 5.00 (s, 2H), 4.39 (t, J=6.2 Hz , 2H), 3.81 (t, J = 6.2 Hz, 2H), 3.70 (s, 2H), 3.58 (d, J = 4.4 Hz, 2H), 3.41 (dd, J = 11.1, 6.3 Hz, 2H), 3.21 (d, J = 7.7 Hz, 2H), 2.13 (d, J = 11.4 Hz, 2H), 1.86 (d, J = 7.5 Hz, 2H), 1.79-1.64 (m, 2H), 1.56-1.27 (m, 10H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.33, 152.36, 133.81, 114.40, 110.73, 110.57, 107.77, 71.20, 70.18, 70.12, 68.15, 59.46, 45.18, 38.69, 33.58, 32.67, 32.56, 29.51, 26.75, 25.41, 23.61.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₈ H ₃₇ ClN ₃ O ₄ [M+H] ⁺ 514.2473, the actual value is 514.2477.

After testing, the structure is as shown in the above formula Halo-DAC, and its fluorescence properties are as follows: Halo-DAC has a water light emission wavelength of about 490nm, an excitation wavelength of 480nm, and a fluorescence half-width of only 40nm.

Example 16

Synthesis of SNAP-DAze:

Synthesis of BA-DAze

BA-NBr (300 mg, 0.68 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and 300 mg of azetidine was added thereto. The reaction solution was slowly heated to 120°C and reacted for 10h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 60:1, V/V) to obtain 52 mg of a yellow solid with a yield of 18%. The NMR spectrum hydrogen spectrum is shown in Figure 1, the specific data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.38 (d, J=8.5 Hz, 2H), 7.50 (d, J=7.8 Hz, 2H), 7.25 (d, J=5.9 Hz, 2H), 6.38 (d , J = 8.5Hz, 2H), 5.36 (s, 2H), 4.61 (s, 2H), 4.11 (s, 8H), 2.44 (s, 4H).

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₆ H ₂₆ N ₃ O ₃ [M+H] ⁺ 428.1974, actual value 428.1997.

Synthesis of SNAP-DAze

BA-DAze (40 mg, 0.09 mmol), BG ⁺ (40 mg, 0.16 mmol), potassium tert-butoxide (40 mg, 0.36 mmol) were placed in a 10 mL Shrek bottle, replaced with nitrogen three times and 5 mL of dry DMF was added. After stirring at room temperature for 6 hours, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 20:1, V/V) to obtain a brown solid 24 mg with a yield of 45%. The hydrogen spectrum of the nuclear magnetic spectrum prepared in Example 22 is shown in FIG. 15, and the specific data of the hydrogen spectrum and carbon spectrum of the nuclear magnetic spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.40 (s, 1H), 8.17 (d, J=8.4 Hz, 2H), 7.79 (s, 1H), 7.41 (d, J=7.4 Hz, 2H) , 7.28 (d, J = 7.6 Hz, 2H), 6.48 (d, J = 8.5 Hz, 2H), 6.26 (s, 2H), 5.42 (s, 2H), 5.22 (s, 2H), 4.14 (s, 8H), 2.38 (s, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ163.47, 160.31, 160.09, 156.01, 155.65, 138.90, 138.22, 135.60, 133.37, 132.87, 128.87, 127.74, 113.97, 108.21 , 106.94, 106.73, 56.50, 54.63, 42.37, 19.02.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₃₁ H ₂₉ N ₈ O ₃ [M+H] ⁺ 561.2363, actual value 561.2380.

After testing, its structure is as shown in the above formula SNAP-DAze, and its fluorescence properties are as follows: SNAP-DAze has a light emission wavelength of about 490nm in acetonitrile, chloroform, dimethyl sulfoxide, ethanol, and water, and fluoresces with the change of polarity There is no obvious change in emission wavelength and fluorescence peak shape.

Example 17

Synthesis of SNAP-DAC

Synthesis of BA-DAC

BA-NBr (200 mg, 0.45 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and 400 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 140°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 80:1, V/V) to obtain 93 mg of a yellow solid with a yield of 48%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.05 (d, J=8.6 Hz, 21H), 7.56 (s, 2H), 7.24 (d, J=8.2 Hz, 2H), 7.20 (d, J= 8.3 Hz, 2H), 6.83 (d, J = 8.7 Hz, 2H), 5.14 (s, 2H), 5.10 (t, J = 5.7 Hz, 2H), 4.42 (d, J = 5.7 Hz, 2H), 3.16 (d, J = 9.2 Hz, 2H), 2.19 (d, J = 12.0 Hz, 2H), 1.72 (d, J = 7.3 Hz, 2H), 1.49-1.18 (m, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ163.39, 154.73, 141.39, 137.45, 134.91, 133.49, 127.79, 126.77, 110.69, 107.60, 106.41, 63.16, 59.47, 42.36, 32.06, 23.62

Synthesis of SNAP-DAC

BA-DAC (40 mg, 0.09 mmol), BG ⁺ (95 mg, 0.37 mmol), potassium tert-butoxide (84 mg, 0.75 mmol) were placed in a 10 mL Shrek bottle, replaced with nitrogen four times and 3 mL of dry DMF was added. After stirring at room temperature for 10 h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 25:1, V/V) to obtain a brown solid 28 mg, with a yield of 53%. The hydrogen spectrum of the nuclear magnetic spectrum is shown in Figure 16, and the specific data of the hydrogen spectrum and the carbon spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.39 (s, 1H), 8.05 (d, J=8.6 Hz, 2H), 7.79 (s, 1H), 7.56 (s, 2H), 7.40 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 8.0 Hz, 2H), 6.84 (d, J = 8.7 Hz, 2H), 6.27 (s, 2H), 5.41 (s, 2H), 5.17 (s, 2H), 3.16 (d, J = 8.5 Hz, 2H), 2.19 (d, J = 11.3 Hz, 2H), 1.73 (d, J = 6.6 Hz, 2H), 1.40-1.25 (m, 4H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ163.39, 160.30, 160.09, 155.65, 154.76, 138.94, 138.22, 135.61, 134.96, 133.53, 128.86, 127.99, 113.94, 110.71, 107.56, 106.40, 99.99, 66.98, 59.47, 42.38 , 32.06, 23.62.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₃₁ H ₂₉ N ₈ O ₃ [M+H] ⁺ 561.2363, actual value 561.2380.

After testing, its structure is as shown in the above formula SNAP-DAC, its fluorescence emission wavelength in water is 485nm, absorption wavelength is about 479nm, and it can carry out no-wash mark on SNAP-tag.

Example 18

Synthesis of SNAP-AzeAzo

Synthesis of BA-AzeAzo

BA-NBr (300 mg, 0.68 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and 39 mg of azetidine was added thereto. The reaction solution was slowly heated to 50°C and reacted for 1 h. 200 mg of tetrahydropyrrole was added dropwise to the reaction, which was gradually heated to 130° C., and the reaction was continued at this temperature for 12 hours to remove ethylene glycol methyl ether under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol=70:1) , V/V), to obtain a yellow solid 30mg, yield 10%.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₇ H ₂₈ N ₃ O ₃ [M+H] ⁺ 442.2131, the actual value is 442.2142.

Synthesis of SNAP-AzeAzo

BA-AzeAzo (20 mg, 0.05 mmol), BG ⁺ (36 mg, 0.14 mmol), potassium tert-butoxide (51 mg, 0.45 mmol) were placed in a 10 mL Shrek bottle, replaced with nitrogen three times and 4 mL of dry DMF was added. After stirring at room temperature for 8h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 20:1, V/V) to obtain a brown solid 14mg, yield 52%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.45 (s, 1H), 8.13 (dd, J=15.7, 8.0 Hz, 2H), 7.80 (s, 1H), 7.41 (d, J=5.9 Hz, 2H), 7.29 (d, J = 7.2 Hz, 2H), 6.76 (dd, J = 18.6, 9.6 Hz, 1H), 6.42 (t, J = 8.4 Hz, 1H), 6.25 (s, 2H), 5.43 ( s, 2H), 5.23 (s, 2H), 3.63 (s, 2H), 3.19 (s, 2H), 2.90 (s, 2H), 2.07 (m, 6H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ163.51,163.36,160.09,156.69,155.65,154.68,153.83,138.19,133.73,132.76,132.66,132.59,128.82,127.77,113.99,107.87,107.39,107.15,106.84,106.24,67.01,52.46,49.75,42.31 , 25.93, 25.63, 25.30, 16.69.

After testing, its structure is as shown in the above formula SNAP-AzeAzo, its fluorescence emission wavelength in water is 494nm, absorption wavelength is about 485nm, and it can carry out no-wash mark on SNAP-tag.

Example 19

Synthesis of fluorescent probe Mito-DAC

Dissolve N-(6-triphenylphosphinohexyl)-4-bromo-5-nitro-1,8-naphthalic anhydride (100 mg, 0.13 mmol) in 10 ml of ethylene glycol methyl ether, and add 1 , 2-diaminocyclohexanediamine (60 mg, 0.52 mmol). The reaction solution was slowly heated to 120°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 200:1, V/V) to obtain a yellow solid 40 mg, yield 89%. The nuclear magnetic spectrum hydrogen spectrum and carbon Spectral data is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (d, J=8.5 Hz, 2H), 7.83 (t, J=6.8 Hz, 3H), 7.68 (dd, J=13.9, 6.4 Hz, 12H), 6.83 (d, J = 8.5 Hz, 2H), 5.86 (s, 2H), 4.02 (t, J = 6.5 Hz, 2H), 3.42-3.31 (m, 2H), 3.18 (d, J = 9.7 Hz, 2H) , 2.33 (d, J = 12.5 Hz, 2H), 1.80 (d, J = 8.2 Hz, 2H), 1.63 (s, 4H), 1.48 (d, J = 9.7 Hz, 2H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ164.31, 153.34, 135.46, 134.31, 133.53, 133.43, 130.75, 130.63, 118.30, 117.44, 111.04, 109.26, 107.18, 59.65, 38.94, 32.67, 29.71, 27.28, 25.53, 23.65.

High-resolution mass spectrometry data are as follows: C ₄₂ H ₄₃ N ₁₀ O ₂₁ P ⁺ [M] ⁺ calculated value: 652.3087, experimental value: 652.3128.

After testing, the structure of the compound is shown as Mito-DAC, suitable for imaging of live cell mitochondria in a variety of physiological states and the light performance is not affected by the microenvironment, high brightness and strong stability can meet the long-term dynamics of mitochondria by super-resolution imaging Tracking, the fluorescence emission wavelength is around 481nm.

Example 20

Synthesis of fluorescent probe Mito-DAze.

The compound N-(6-triphenylphosphinohexyl)-4-bromo-5-nitro-1,8-naphthalic anhydride (100 mg, 0.13 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and nitrogen was added thereto Heterocyclobutane (30 mg, 0.52 mmol). The reaction solution was slowly heated to 120°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=200:1, V/V) to obtain 40 mg of a yellow solid with a yield of 89%. The hydrogen spectrum of the Mito-DAze NMR spectrum prepared in Example 28 is shown in FIG. 17, and the specific data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.31 (d, J=8.4 Hz, 2H), 7.76 (dd, J=21.9, 9.4 Hz, 15H), 6.38 (d, J=8.4 Hz, 2H), 4.22 -3.83 (m, 10H), 3.50 (s, 2H), 2.43 (s, 4H), 1.66 (s, 4H), 1.38 (s, 4H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 155.69, 135.22 , 133.65, 133.55, 132.86, 130.68, 130.56, 118.51, 109.73, 107.73, 106.34, 55.05, 39.31, 29.67, 27.53, 26.15, 22.51, 16.92.

High-resolution mass spectrometry data are as follows: C ₄₂ H ₄₃ N ₃ O ₂ P ⁺ [M] ⁺ calculated value: 652.3088, experimental value: 652.3109.

After detection, the structure of the above product is Mito-DAze, the compound can be quickly and accurately located in the mitochondria in live cell imaging experiments, with high brightness and strong stability.

Example 21

Synthesis of HAN-DAze

BA-DAze (50 mg, 0.12 mmol) was dissolved in 10 mL of dimethyl sulfoxide, and 200 mg of triethylamine was added thereto. The reaction solution was slowly heated to 140°C and reacted for 10h. 100 mL of dichloromethane was added to the reaction solution and washed with water to obtain an organic phase, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 50: 1, V/V), 24 mg of yellow solid was obtained with a yield of 67%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 9.2 (s, 1H), 8.36 (d, J=8.7 Hz, 2H), 7.52 (d, J=7.7 Hz, 2H), 4.21 (s, 8H), 2.46 (s, 4H).

After testing, its structure is shown in the above formula HAN-DAze, and its fluorescent properties are as follows: its fluorescence emission wavelength in water is 491 nm, and its absorption is 482 nm.

Example 22

Synthesis of HAN-DMEDA

BA-DMEDA (50 mg, 0.12 mmol) was dissolved in 15 mL of dimethyl sulfoxide, and 350 mg of triethylamine was added thereto. The reaction solution was slowly heated to 140°C and reacted for 12h. 150mL of dichloromethane was added to the reaction solution and washed with water (50mL*4) to obtain an organic phase, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 50: 1, V/V) to obtain 14 mg of a yellow solid with a yield of 40%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.05 (s, 1H), 8.18 (d, J=8.5 Hz, 2H), 6.84 (d, J=8.6 Hz, 2H), 3.60 (s, 4H) , 3.11 (s, 6H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 164.43, 155.77, 134.18, 131.97, 116.90, 111.50, 110.18, 57.80, 41.65.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₁₉ H ₂₀ N ₃ O ₂ [M+H] ⁺ 336.1712, actual value 336.1733.

After testing, its structure is shown in the above formula HAN-DMEDA, and its fluorescent properties are as follows: its fluorescence emission wavelength in water is 520 nm and absorption is 475 nm.

Example 23

Synthesis of Lyso-DAze

Lyso-NBr (50 mg, 0.12 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 100 mg of azetidine was added thereto. The reaction solution was slowly heated to 120°C and reacted for 15h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 50:1, V/V) to obtain 6 mg of a yellow solid with a yield of 12%. The nuclear magnetic spectrum hydrogen spectrum of Lyso-DAze prepared in Example 32 is shown in FIG. 18, and the specific data are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.37 (d, J=8.5 Hz, 2H), 6.39 (d, J=8.5 Hz, 2H), 4.37-4.30 (m, 2H), 4.07 (s, 8H) , 3.75-3.66 (m, 4H), 2.72-2.66 (m, 2H), 2.62 (s, H), 2.43 (s, 4H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ164.37, 155.62, 133.17, 132.88, 110.06, 107.98, 106.34, 67.13, 56.42, 54.62, 53.80, 36.67, 16.90.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₄ H ₂₉ N ₄ O ₃ [M+H] ⁺ 421.2240, the actual value is 421.2251.

After testing, its structure is as shown in the above formula Lyso-DAze, and its fluorescence performance is as follows: its fluorescence emission wavelength in water is 493 nm and absorption is 483 nm, and it can be used for intracellular lysosomal labeling.

Example 24

Synthesis of Nu-DAC

Lyso-NBr (100 mg, 0.23 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 300 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 90°C and reacted for 18h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 50:1, V/V) to obtain 48 mg of a yellow solid with a yield of 50%. The nuclear magnetic spectrum hydrogen spectrum of the Nu-DAC prepared in Example 33 is shown in FIG. 19, and the specific data of the hydrogen spectrum and the carbon spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.04 (d, J=8.6 Hz, 2H), 7.52 (s, 2H), 6.83 (d, J=8.7 Hz, 2H), 4.10 (t, J= 7.0Hz, 2H), 3.62-3.51(m, 4H), 3.15(d, J=9.2Hz, 2H), 2.45(s, 4H), 2.19(d, J=11.9Hz, 2H), 1.73(d, J = 7.1 Hz, 2H), 1.44-1.20 (m, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ163.39, 154.58, 134.78, 133.35, 110.59, 107.75, 106.45, 66.66, 59.50, 56.42, 53.89, 36.50, 32.08, 23.64.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₄ H ₂₉ N ₄ O ₃ [M+H] ⁺ 421.2240, the actual value is 421.2248.

After testing, its structure is as shown in the above formula Nu-DAC, its fluorescence performance is as follows: its fluorescence emission wavelength in water is 487nm, absorption is 480nm, it can be used for intracellular nuclear labeling.

Example 25

The synthesis route and product structure of carbonic anhydrase probe SML-DAze are as follows:

Take SML-NBr (0.1g, 0.2mmol) and azetidine (0.13g, 2mmol) in 5mL ethylene glycol monomethyl ether, warm to 140°C for 12h, cool the reaction solution to room temperature and remove under reduced pressure The solvent was evaporated, and the residue was separated by silica gel column chromatography (dichloromethane/methanol=50/1, V/V) to obtain 0.06 g of SML-Daze as an orange solid with a yield of 60%. The SML-DAze NMR spectrum prepared in Example 34 is shown in FIG. 20, and the specific data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.38 (d, J=8.5 Hz, 2H), 7.80 (d, J=8.2 Hz, 2H), 7.61 (d, J=8.2 Hz, 2H), 6.40 (d , J = 8.5Hz, 2H), 5.41 (s, 2H), 4.78 (s, 2H), 4.14 (s, 8H), 2.43 (s, 4H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ 159. 52,151.17,139.17,135.63,128.66,128.54,124.32,121.70,104.75,102.98,101.71,53.72,37.96,12.12.

After testing, its structure is shown in the above formula SML-DAze, its emission wavelength in ethanol is 485nm, it has high brightness and light stability, and can quickly label intracellular carbonic anhydrase.

Example 26

The synthesis route and product structure of carbonic anhydrase probe SML-AzeAzo are as follows:

Take SML-NBr (0.1g, 0.2mmol) and azetidine (0.01g, 0.2mmol) in 5mL ethylene glycol methyl ether, warm to 60°C for 5h, then cool the reaction solution to room temperature, remove the steam under reduced pressure No post-treatment process is needed after the solvent. 5mL of ethylene glycol methyl ether and tetrahydropyrrole (0.15g, 2mmol) are added. After heating to 140°C for 12h, the reaction solution is cooled to room temperature and the solvent is removed under reduced pressure. The residue is passed through a silica gel column Chromatographic separation (dichloromethane/methanol=40/1, V/V) gave 0.04 g of SML-AzeAzo as an orange solid with a yield of 40%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.13 (dd, J = 15.6, 8.0 Hz, 2H), 7.74 (d, J = 7.8 Hz, 2H), 7.41 (d, J = 7.9 Hz, 2H) , 7.26 (s, 1H), 6.88-6.70 (m, 1H), 6.46 (d, J=8.6Hz, 1H), 5.27 (s, 2H), 4.16 (s, 2H), 3.66 (s, 4H), 3.19(s, 2H), 2.34(s, 2H), 2.02(s, 2H), 1.86(s, 2H).

After testing, its structure is as shown in the above formula SML-DAzeAzo, its emission wavelength in ethanol is 485nm, it has high brightness and light stability, and can quickly label intracellular carbonic anhydrase.

Example 27

The synthesis route and product structure of carbonic anhydrase probe SML-DAzo are as follows:

Take SML-NBr (0.1g, 0.2mmol) and tetrahydropyrrole (0.15g, 2mmol) in 5mL ethylene glycol methyl ether, warm to 140°C for 12h, cool the reaction solution to room temperature, and remove the evaporated solvent under reduced pressure. The residue was separated by silica gel column chromatography (dichloromethane/methanol=50/1, V/V) to obtain 0.06 g of SML-DAzo as an orange solid with a yield of 60%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.11 (d, J=8.8 Hz, 2H), 7.73 (d, J=8.3 Hz, 2H), 7.41 (d, J=8.3 Hz, 2H), 7.26 (s, 2H), 6.78 (d, J = 8.9 Hz, 2H), 5.26 (s, 2H), 3.82-3.61 (m, 2H), 3.38 (dd, J = 13.5, 7.3 Hz, 2H), 3.27- 3.16 (m, 2H), 2.23-1.86 (m, 8H), 1.66-1.47 (m, 2H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 163.31, 154.79, 143.36, 142.89, 134.19, 132.66, 127.92, 126.11, 108.56, 106.94, 52.51, 49.80, 42.32, 25.96, 25.42.

After testing, its structure is as shown in the above formula SML-DAzo, its emission wavelength in ethanol is 495nm, it has high brightness and light stability, and can quickly label intracellular carbonic anhydrase.

Example 28

Synthesis of SO3-DAze

SO3-NBr (50 mg, 0.12 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 100 mg of azetidine was added thereto. The reaction solution was slowly heated to 120°C and reacted for 10h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=50:1, V/V) to obtain 7 mg of a yellow solid with a yield of 14%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.34 (d, J=8.5 Hz, 2H), 8.28 (s, 1H), 6.38 (d, J=8.5 Hz, 2H), 4.18 (s, 8H), 2.45 (s, 4H).

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₁₉ H ₂₀ N ₃ O ₅ S[M+H] ⁺ 402.1124, actual value 402.1140.

After testing, its structure is shown in the above formula SO3-DAze, its emission wavelength in ethanol is 490nm, and the fluorescence quantum yield reaches 0.85.

Example 29

Tro-DAC synthesis

Tro-NBr (150 mg, 0.30 mmol) was dissolved in 20 mL of ethylene glycol methyl ether, and 400 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 100°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 50:1, V/V) to obtain 102 mg of a yellow solid with a yield of 70%. The nuclear magnetic spectrum hydrogen spectrum of the Tro-DAC prepared in Example 38 is shown in FIG. 21, and the specific data are:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.11 (d, J=7.1 Hz, 2H), 7.57 (s, 2H), 6.87 (d, J=7.3 Hz, 2H), 6.67 (s, 2H) , 5.13 (s, 2H), 3.72 (s, 6H), 3.64 (s, 3H), 3.18 (s, 2H), 2.21 (s, 2H), 1.75 (s, 2H), 1.33 (s, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 163.52, 154.78, 153.11, 136.99, 134.96, 134.88, 133.58, 110.76, 107.61, 106.44, 105.83, 60.45, 59.51, 56.31, 42.85, 32.09, 23.64.

After examination, its structure is shown in the above formula SO3-DAze, its emission wavelength in ethanol is 487nm, and its absorption wavelength is 480nm.

Example 30

Synthesis of CM-DAze

Synthesis of Intermediate PhAN-DAze

PhAN-NBr (150 mg, 0.26 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and 400 mg of azetidine was added thereto. The reaction solution was slowly heated to 120°C and reacted for 10h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V/V) to obtain 51 mg of a dark yellow solid with a yield of 35%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.35 (d, J=8.5 Hz, 2H), 7.87-7.80 (m, 2H), 7.69 (s, 2H), 6.37 (d, J=8.6 Hz, 2H) , 4.42-4.30 (m, 2H), 4.08 (s, 8H), 3.87-3.80 (m, 2H), 3.71 (dd, J = 14.1, 6.4 Hz, 4H), 3.66 (d, J = 18.8 Hz, 2H ), 2.44 (s, 4H).

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₃₂ H ₃₂ N ₄ O ₆ Na[M+Na] ⁺ 591.2220, actual value 591.2284.

Synthesis of CM-DAze

PhAN-DAze (20 mg, 0.03 mmol) was dissolved in 20 mL of ethanol, and 200 μL of an aqueous hydrazine hydrate solution (85%) was added thereto. After the reaction solution was refluxed for 3 h, ethanol was removed under reduced pressure, and the residue was dissolved in 20 mL of dichloromethane. The organic phase was washed with 3×50 mL of saturated brine, and the organic phase was dried over anhydrous sodium sulfate overnight. The brown-yellow solid obtained by removing the solvent under reduced pressure was dissolved in 10 mL of acetonitrile, and Chol-ANBr (71 mg, 0.14 mmol) and K ₂ CO ₃ (15 mg, 0.14 mmol) were added. The reaction solution was heated to reflux for 3 h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 50:1, V/V) to obtain a dark yellow solid 26 mg, with a total yield of 58% in two steps. The NMR spectrum of the CM-DAze prepared in Example 39 is shown in FIG. 22, and the specific data are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.38 (d, J=8.6 Hz, 2H), 6.51-6.20 (m, 2H), 5.35 (s, 2H), 4.69 (d, J=8.1 Hz, 2H) , 4.47 (d, J = 4.7Hz, 2HD, 4.23-3.46 (m, 20H), 2.90 (s, 2H), 2.39 (dd, J = 28.2, 12.6Hz, 8H), 2.06-1.32 (m, 40H) , 1.21-0.82 (m, 42H). ¹³ C NMR (101MHz, CDCl ₃ ) δ164.89, 155.94, 139.47, 133.42, 122.82, 121.66, 109.31, 107.51, 106.48, 69.78, 56.79, 56.21, 50.10, 42.32, 39.76 , 39.51, 38.04, 36.98, 36.58, 36.19, 35.78, 31.93, 31.81, 29.70, 28.22, 28.01, 27.76, 24.28, 23.84, 22.82, 22.57, 21.04, 19.29, 18.73, 16.92, 14.13, 11.86.

After examination, its structure is shown in the above formula CM-DAze, its ultraviolet absorption wavelength in ethanol is 482nm, and its fluorescence emission wavelength is 497nm.

Example 31

Synthesis of cell membrane probe DDAN-DAC.

DDAN-NBr (0.25g, 0.51mmol) was dissolved in 20mL ethylene glycol methyl ether, and 1,2-cyclohexanediamine (0.35g, 3.1mmol) was added thereto, and the reaction solution was slowly heated to 130°C, And react for 18h. The ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane/methanol=150/1, V/V) to obtain 0.06 g of a yellow solid with a yield of 25%. The specific data of the nuclear magnetic spectrum hydrogen spectrum and carbon spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.04 (d, J=8.6 Hz, 2H), 7.46 (s, 2H), 6.82 (d, J=8.6 Hz, 2H), 3.94 (t, J= 7.2Hz, 2H), 3.15(s, 2H), 2.19(d, J=11.6Hz, 2H), 1.73(d, J=6.3Hz, 2H), 1.54(s, 2H), 1.43-1.12(m, 22H), 0.84 (t, J = 6.5 Hz, 3H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 163.42, 154.48, 134.69, 133.28, 110.54, 107.93, 106.56, 59.54, 32.09, 31.76, 29.48, 29.46, 29.43, 29.39,

29.27, 29.17, 28.16, 27.06, 23.65, 22.55, 14.41.

After testing, its structure is as shown in the above formula DDAN-DAC, its ultraviolet absorption wavelength in ethanol is 475nm, the fluorescence emission wavelength is 485nm, has a high brightness and light stability, is not sensitive to the environment and can accurately locate the cell membrane of living cells .

Example 32

Synthesis of cell membrane probe HexAN-DAC.

N-hexadecyl-4-bromo-5-nitro-1,8-naphthalimide (0.3g, 0.55mmol) was dissolved in 20mL of ethylene glycol methyl ether, and 1,2- was added thereto Cyclohexanediamine (0.45g, 3.99mmol), the reaction solution was slowly heated to 130°C, and reacted for 18h, ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane/methanol=150) /1, V/V) to obtain 0.1 g of yellow solid HexAN-DAC with a yield of 35%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.06 (d, J=8.7 Hz, 2H), 7.51 (s, 2H), 6.85 (d, J=8.5 Hz, 2H), 3.96 (t, J= 7.2 Hz, 2H), 3.25 (s, 2H), 2.19 (d, J = 11.3 Hz, 2H), 1.73 (d, J = 6.3 Hz, 2H), 1.54 (s, 2H), 1.47-1.02 (m, 30H), 0.85 (t, J = 6.5 Hz, 3H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 163.55, 154.62, 134.77, 133.20, 110.54, 108.05, 106.70, 59.55, 32.12, 31.76, 29.48, 29.46, 29.33, 29.32, 29.19, 28.20, 27.07, 23.68, 22.57, 14.43.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₃₄ H ₅₀ N ₃ O ₂ [M+H] ⁺ 532.3903, actual value 532.3930.

After testing, its structure is as shown in the above formula HexAN-DAC, its ultraviolet absorption wavelength in ethanol is 475nm, the fluorescence emission wavelength is 485nm, has a high brightness and light stability, is not sensitive to the environment and can accurately locate the cell membrane of living cells .

Example 33

Synthesis of cell membrane probe MBSO3-DAC.

Synthesis of intermediate MBAN-DAC:

MBAN-NBr (50 mg, 0.09 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 1,2-cyclohexanediamine (200 mg, 1.77 mmol) was added thereto, and the reaction solution was slowly heated to 130° C. and reacted At 18h, ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane/methanol=50/1, V/V) to obtain a yellow solid MBAN-DAC 25 mg, with a yield of 52%. The hydrogen spectrum of the nuclear magnetic spectrum is shown in FIG. 23, and the specific data of the hydrogen spectrum and the carbon spectrum are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.04 (d, J=8.2 Hz, 2H), 6.63 (d, J=8.4 Hz, 2H), 5.70 (s, 2H), 4.17 (s, 2H), 3.16 (s, 4H), 3.07-2.92 (m, 2H), 2.78 (s, 3H), 2.26 (d, J = 11.0 Hz, 4H), 1.83 (d, J = 6.6 Hz, 3H), 1.53-1.44 ( m, 6H), 1.10 (d, J = 5.4 Hz, 4H), 1.01-0.76 (m, 12H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 164.06, 153.39, 134.31, 133.59, 111.06, 108.74, 106.81 , 59.35, 54.89, 54.09, 40.04, 39.06, 36.85, 32.57, 31.12, 29.71, 27.89, 24.57, 23.62, 22.71, 22.58, 19.30, 14.14.

After testing, its structure is shown in the above MBAN-DAC.

Synthesis of cell membrane probe MBSO3-DAC:

MBAN-DAC (20 mg, 0.04 mmol) and 1,3-propane sultone (5 mg, 0.04 mmol) were dissolved in 5 mL of acetonitrile, and the reaction solution was slowly heated to 60° C. for 18 h. The solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane/methanol=20/1, V/V) to obtain 12 mg of a dark yellow solid MBSO3-DAC with a yield of 48%.

The high-resolution mass spectrum of MBSO3-DAC prepared in Example 42 is shown in FIG. 31, and the mass spectrum data is: theoretical value of high-resolution mass spectrum C ₃₅ H ₅₃ N ₄ O ₅ S [M+H] ⁺ 641.3737, measured value 641.3762.

After testing, its structure is as shown in the above formula MBSO3-DAC. Its ultraviolet absorption wavelength in ethanol is 475nm and its fluorescence emission wavelength is 485nm. It has high brightness and light stability, is not sensitive to the environment and can accurately locate the cell membrane of living cells. .

Example 34

Synthesis of CMN-DAC.

Synthesis of intermediate CFAN-DAC:

CFAN-NBr (150 mg, 0.33 mmol) was dissolved in 20 mL of ethylene glycol methyl ether, and 1,2-cyclohexanediamine (400 mg, 3.54 mmol) was added thereto, and the reaction solution was slowly heated to 130° C. for 18 h. . The ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 100:1, V/V) to obtain a dark yellow solid CFAN-DAC 73 mg, with a yield of 50%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.46 (s, 1H), 8.05 (d, J=8.4 Hz, 2H), 7.52 (s, 2H), 6.83 (d, J=8.6 Hz, 2H) , 4.16 (s, 2H), 3.46 (d, J = 4.8 Hz, 2H), 3.16 (s, 2H), 2.20 (d, J = 10.7 Hz, 2H), 1.73 (s, 2H), 1.30 (dd, J = 28.5, 18.2 Hz, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 163.68, 156.68, 154.59, 134.95, 133.35, 110.56, 107.83, 106.50, 59.52, 38.24, 38.19, 32.08, 23.64.

After testing, its structure is shown in the above formula CFAN-DAC.

Synthesis of intermediate EDA-DAC:

CFAN-DAC (50 mg, 0.11 mmol) was dissolved in 20 mL of methanol, and potassium carbonate (200 mg, 1.4 mmol) was added thereto, the reaction solution was slowly heated to 70° C., and reacted for 6 h. The methanol was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane/methanol=40/1, V/V) to obtain 33 mg of a dark yellow solid EDA-DAC with a yield of 85%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.05 (d, J=8.6 Hz, 2H), 7.57 (s, 2H), 6.84 (d, J=8.7 Hz, 2H), 4.12 (t, J= 6.4Hz, 2H), 3.15 (d, J = 8.8Hz, 2H), 2.91 (t, J = 6.4Hz, 2H), 2.20 (d, J = 11.5Hz, 2H), 1.73 (d, J = 6.7Hz , 2H), 1.49-1.16 (m, 4H). ¹³ C NMR (101MHz, DMSO-d ₆ ) δ163.74, 154.68, 134.97, 133.40, 110.64, 107.69, 106.35, 59.48, 55.39, 32.06, 23.62.

After testing, its structure is shown in the above formula EDA-DAC.

Synthesis of CMN-DAC:

EDA-DAC (20mg, 0.06mmol), potassium iodide (10mg, 0.06), potassium carbonate (30mg, 0.22mmol), Chol-ANBr (29mg, 0.06mmol) was dissolved in 10mL acetonitrile, and the reaction solution was slowly heated to 85 At ℃, and react for 6h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane/methanol=80/1, V/V) to obtain a dark yellow solid CMN-DAC 29mg, yield 65%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.38 (d, J=8.6 Hz, 2H), 7.62 (s, 2H), 6.51-6.20 (m, 2H), 5.35 (s, 2H), 4.71 (d, J = 8.1 Hz, 2H), 4.57 (d, J = 4.7 Hz, 2H), 4.23-3.46 (m, 10H), 2.39 (dd, J = 28.2, 12.6 Hz, 8H), 2.16-1.28 (m, 16H ), 1.21-0.75 (m, 22H).

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₄₉ H ₆₉ N ₄ O ₄ [M+H] ⁺ 777.5319, actual value 777.5365.

After testing, its structure is as shown in the above formula CMN-DAC. Its ultraviolet absorption wavelength in ethanol is 475nm, and its fluorescence emission wavelength is 485nm. It has high brightness and light stability, is not sensitive to the environment, and can accurately locate the cell membrane of living cells. .

Example 35

Synthesis of DDAN-DAze.

DDAN-NBr (150 mg, 0.26 mmol) was dissolved in 30 mL of ethylene glycol methyl ether, and azetidine (400 mg, 7 mmol) was added thereto, the reaction solution was slowly heated to 120° C., and reacted for 10 h under reduced pressure Ethylene glycol methyl ether was removed, and the residue was separated through a silica gel column (dichloromethane/methanol=100/1, V/V) to obtain 51 mg of a dark yellow solid with a yield of 35%.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₂ H ₂₆ N ₃ O ₃ [M+H] ⁺ 364.2025, actual value 364.2082.

After testing, its structure is as shown in the above formula DDAN-DAze, its fluorescence emission wavelength in ethanol is 490nm, it has high brightness and light stability, is not sensitive to the environment and can accurately locate the cell membrane of living cells.

Example 36

Synthesis of COOH-DAze

(1) Synthesis of COMe-DAze

COMe-NBr (200 mg, 0.49 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 400 mg of azetidine was added thereto. The reaction solution was slowly heated to 120°C and reacted for 10h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V/V) to obtain 60 mg of a dark yellow solid with a yield of 31%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.74 (d, J=7.9 Hz, 1H), 8.54 (d, J=7.7 Hz, 1H), 8.24 (d, J=7.9 Hz, 1H), 7.94 (d , J = 7.8Hz, 1H), 4.93 (s, 2H), 4.23 (q, J = 7.2Hz, 2H), 4.19-3.90 (m, 8H), 2.43 (s, 4H), 1.32 (t, J = 7.2Hz, 3H).

(2) Synthesis of COOH-DAze

COMe-DAze (40 mg, 0.10 mmol) was dissolved in 4 mL of methanol, and 4 mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition was completed, the reaction liquid was reacted at room temperature for 1 h, and methanol was distilled off under reduced pressure. The turbid liquid was filtered and the filter cake was washed with 4 mL of water and dried to obtain COOH-DAze 32 mg, with a yield of 86%. The hydrogen spectrum of the COOH-DAze NMR spectrum prepared in Example 45 is shown in FIG. 24, and the specific data are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.15 (d, J=8.3 Hz, 2H), 6.48 (d, J=8.3 Hz, 2H), 4.49 (s, 2H), 4.06 (s, 8H) , 2.39 (s, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ163.49, 155.72, 133.21, 132.50, 109.02, 107.32, 106.59, 54.80, 43.24, 16.81.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₀ H ₂₀ N ₃ O ₄ [M+H] ⁺ 366.1454, found value 366.1440.

After testing, its structure is shown in the above formula COOH-DAze, and its fluorescent properties are as follows:

The emission wavelength of COOH-DAze in different solvents is 480-495nm, the half-width of fluorescence emission is less than 35nm, and the fluorescence wavelength does not change with the change of polarity.

The ultraviolet absorption wavelength of COOH-DAze in different solvents is 470-485nm, and the absorption wavelength does not change with the change of polarity, which can keep the fluorescence signal stability as much as possible.

Example 37

Synthesis of COOH-DAC

(1) Synthesis of COMe-DAC

COMe-NBr (200 mg, 0.49 mmol) was dissolved in 20 mL of ethylene glycol methyl ether, and 400 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 100°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 80:1, V/V) to obtain 124 mg of a dark yellow solid with a yield of 64%.

The high-resolution mass spectrometry data is as follows: high-resolution mass spectrometry theoretical value C ₂₂ H ₂₄ N ₃ O ₄ [M+H] ⁺ 394.1767, the actual value is 394.1788.

(2) Synthesis of COOH-DAC

COMe-DAC (60 mg, 0.10 mmol) was dissolved in 3 mL of methanol, and 3 mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition was completed, the reaction solution was reacted at room temperature for 3 hours, and methanol was distilled off under reduced pressure. The turbid liquid was filtered and the filter cake was washed with 3 mL of water and dried to obtain COOH-DAC 46 mg, with a yield of 83%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.72 (s, 1H), 8.03 (d, J=8.6 Hz, 2H), 7.59 (s, 2H), 6.84 (d, J=8.7 Hz, 2H) , 4.62 (s, 2H), 3.16 (d, J = 5.9 Hz, 2H), 2.20 (d, J = 11.7 Hz, 2H), 1.73 (d, J = 6.9 Hz, 2H), 1.31 (dt, J = 31.3, 16.1 Hz, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 170.62, 163.06, 154.85, 135.08, 133.45, 110.71, 107.28, 106.37, 59.46, 41.02, 32.06, 23.62.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₀ H ₂₀ N ₃ O ₄ [M+H] ⁺ 366.1454, found value 652.3109.

After testing, its structure is as shown in the above formula COOH-DAC, its ultraviolet absorption wavelength in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield is as high as 0.80.

Example 38

Synthesis of BCOOH-DAC

(1) Synthesis of BCOMe-DAC

BCOMe-NBr (200 mg, 0.46 mmol) was dissolved in 10 mL of ethylene glycol methyl ether, and 600 mg of 1,2-cyclohexanediamine was added thereto. The reaction solution was slowly heated to 100°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 80:1, V/V) to obtain 103 mg of a dark yellow solid with a yield of 53%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.04 (d, J=8.6 Hz, 2H), 7.51 (s, 2H), 6.82 (d, J=8.7 Hz, 2H), 4.00 (dt, J= 14.1, 5.3 Hz, 4H), 3.14 (d, J = 8.8 Hz, 2H), 2.30 (t, J = 7.5 Hz, 2H), 2.19 (d, J = 11.7 Hz, 2H), 1.89-1.80 (m, 2H), 1.73 (d, J = 6.8 Hz, 2H), 1.31 (dt, J = 30.1, 15.8 Hz, 4H), 1.14 (t, J = 7.1 Hz, 3H). ¹³ C NMR (101 MHz, DMSO-d6 ) δ172.88, 163.49, 154.56, 134.79, 133.35, 110.58, 107.74, 106.44, 60.18, 59.48, 38.55, 32.07, 31.80, 23.75, 23.63, 14.53.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₄ H ₂₈ N ₃ O ₄ [M+H] ⁺ 422.2080, the actual value is 422.2108.

(2) Synthesis of BCOOH-DAC

BCOMe-DAC (80 mg, 0.19 mmol) was dissolved in 5 mL of methanol, and 8 mL of 2M sodium hydroxide solution was slowly added dropwise to the reaction solution. After the dropwise addition was completed, the reaction liquid was reacted at room temperature for 1 h, and methanol was distilled off under reduced pressure. The turbid liquid was filtered and the filter cake was washed with 5 mL of water and dried to obtain 65 mg of BCOOH-DAC, with a yield of 87%. The data of hydrogen spectrum and carbon spectrum of NMR spectrum are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.01 (s, 1H), 8.04 (d, J=8.6 Hz, 2H), 7.51 (s, 2H), 6.82 (d, J=8.7 Hz, 2H) , 3.99 (dd, J = 9.2, 4.6 Hz, 2H), 3.15 (d, J = 9.1 Hz, 2H), 2.21 (dd, J = 16.7, 9.3 Hz, 4H), 1.88-1.76 (m, 2H), 1.72 (d, J = 8.0 Hz, 2H), 1.42-1.19 (m, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 174.48, 163.50, 154.57, 134.79, 133.36, 110.58, 107.76, 106.47, 59.50, 47.97, 33.82, 32.08, 31.90, 25.79, 24.93, 23.86, 23.63.

The high-resolution mass spectrometry data is as follows: high-resolution mass spectrometry theoretical value C ₂₂ H ₂₄ N ₃ O ₄ [M+H] ⁺ 394.1767, the actual value is 394.1824.

After testing, the structure is as shown in the above formula BOOH-DAC, its ultraviolet absorption wavelength in water is 481nm, the fluorescence emission wavelength is 489nm, and the fluorescence quantum yield is as high as 0.80.

Example 39

Synthesis of NHSM-DAC

COOH-DAC (20 mg, 0.05 mmol) and dicyclohexylcarbylene (DCC) (100 mg, 0.50 mmol) were dissolved in 1 mL of N,N-dimethylformamide and stirred at room temperature for 30 min. After dissolving N-hydroxysuccinimide (200 mg, 1.74 mmol) in 2 mL of N,N-dimethylformamide, it was added dropwise to the reaction solution. After 5h, the solvent was removed under reduced pressure, and the silica gel column was separated. Dichloromethane: ethyl acetate = 6: 1 was used as the eluent, and the solvent was removed to obtain 22 mg of yellowish-yellow solid with a yield of 87%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.10-7.83 (m, 2H), 7.56 (s, 2H), 6.84 (d, J=8.7 Hz, 2H), 4.25 (s, 2H), 3.18 ( d, J = 9.1 Hz, 2H), 2.82 (s, 4H), 2.19 (d, J = 11.4 Hz, 2H), 1.73 (d, J = 7.2 Hz, 2H), 1.33 (dt, J = 27.8, 15.1 Hz, 4H).

After testing, its structure is as shown in the above formula NHSM-DAC, its fluorescence emission wavelength in water is 487nm, and it can be condensed with active amino groups at room temperature.

Example 40

Synthesis of NHSB-DAC

BCOOH-DAC (50 mg, 0.12 mmol) and dicyclohexylcarbylene (DCC) (112 mg, 0.54 mmol) were dissolved in 2 mL of N,N-dimethylformamide and stirred at room temperature for 20 min. After dissolving N-hydroxysuccinimide (200 mg, 1.74 mmol) in 2 mL of N,N-dimethylformamide, it was added dropwise to the reaction solution. After 3h, the solvent was removed under reduced pressure, and the silica gel column was separated. Dichloromethane: ethyl acetate = 5: 1 was used as the eluent, and the solvent was removed to obtain an earthy yellow solid 55mg, yield 89%. The hydrogen spectrum of the NHSB-DAC nuclear magnetic spectrum prepared in Example 50 is shown in FIG. 25, and the specific data are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.19-7.93 (m, 2H), 7.53 (s, 2H), 6.83 (d, J=8.7 Hz, 2H), 4.05 (t, J=6.5 Hz, 2H), 3.15 (d, J = 9.2 Hz, 2H), 2.80 (s, 4H), 2.72 (t, J = 7.7 Hz, 2H), 2.19 (d, J = 11.4 Hz, 2H), 1.97-1.88 ( m, 2H), 1.73 (d, J = 7.2 Hz, 2H), 1.31 (dt, J = 28.8, 15.2 Hz, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 170.66, 169.11, 163.47, 154.65, 134.87, 133.42, 110.63, 107.66, 106.43, 59.48, 38.35, 32.07, 28.69, 25.90, 23.73, 23.63.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₆ H ₂₇ N ₄ O ₆ [M+H] ⁺ 491.1931, the actual value is 491.1981.

After testing, its structure is as shown in the above formula NHSB-DAC, its fluorescence emission wavelength in water is 487nm, and it can be condensed with active amino groups at room temperature.

Example 41

Synthesis of NEAN-DAC

NEAN-NBr (50 mg, 0.12 mmol) was dissolved in 5 mL of ethylene glycol methyl ether, and 100 mg of cyclohexanediamine was added thereto. The reaction solution was slowly heated to 120°C and reacted for 12h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane:methanol=100:1, V/V) to obtain a yellow solid 13 mg with a yield of 27%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.06 (d, J=8.7 Hz, 2H), 7.48 (s, 2H), 6.85 (d, J=8.6 Hz, 2H), 4.02-3.79 (m, 2H), 2.36 (t, J = 7.0 Hz, 2H), 2.21 (d, J = 11.2 Hz, 2H), 1.73-1.65 (m, 4H), 1.54 (dt, J = 14.9, 7.6 Hz, 2H), 1.30 (dq, J=14.3, 7.2Hz, 6H).

After testing, its structure is as shown in the above formula NEAN-DAC, and its fluorescence emission wavelength in water is 487nm, which can be used for biological orthogonal reaction with alkynyl groups.

Example 42

Synthesis of NEBAN-DAzi

N3BAN-NBr (80 mg, 0.19 mmol) was dissolved in 5 mL of ethylene glycol methyl ether, and aziridine 40 mg was added thereto. The reaction solution was slowly heated to 100°C and reacted for 24h. Ethylene glycol methyl ether was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 150:1, V/V) to obtain 5 mg of a yellow solid with a yield of 7%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.13 (d, J=8.7 Hz, 2H), 6.11 (d, J=8.8 Hz, 2H), 3.71 (t, J=6.5 Hz, 2H), 2.52 (s , 8H), 2.31 (t, J = 6.6Hz, 2H), 1.43 (m, 2H), 1.40 (m, 2H).

After testing, its structure is as shown in the above formula NEBAN-DAzi. Its fluorescence emission wavelength in water is 475Hm and absorption wavelength is 468nm, which can be used for orthogonal reaction with alkynyl bio.

Example 43

Synthesis of Pyne-DAze

OAN-DAze (30 mg, 0.08 mmol) and NaH (15 mg, 0.63 mmol) were placed in a 10 mL Shrek bottle and replaced with nitrogen three times. After dissolving 30 μL of bromopropyne in 1 mL of dried DMF, the reaction solution was added. After stirring at room temperature for 5h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 100: 1, V/V) to obtain a brown solid 5mg, yield 15%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.36 (d, J=8.6 Hz, 2H), 6.36 (d, J=8.5 Hz, 2H), 4.42 (t, J=6.7 Hz, 2H), 4.27 (s , 2H), 4.05 (s, 8H), 3.80 (t, J = 6.6Hz, 2H), 3.70-3.63 (m, 2H), 3.44 (t, J = 6.6Hz, 2H), 3.11 (s, 1H) , 2.44 (s, 4H).

After testing, its structure is as shown in the above formula OAN-DAze, its fluorescence emission wavelength in water is 492nm, absorption wavelength is 484nm, which can be used for biological positive reaction with azide.

Example 44

Synthesis of Pyne-DAC

OAN-DAC (30 mg, 0.08 mmol) and NaH (3 mg, 0.13 mmol) were placed in a 10 mL Shrek bottle and replaced with nitrogen three times. After dissolving 15 μL of bromopropyne in 2 mL of dried DMF, the reaction solution was added. After stirring at room temperature for 5 hours, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (dichloromethane: methanol = 100:1, V/V) to obtain a brown solid 7 mg with a yield of 21%.

The high-resolution mass spectrometry data are as follows: high-resolution mass spectrometry theoretical value C ₂₅ H ₂₈ N ₃ O ₄ [M+H] ⁺ 434.2080, the measured value is 434.2108.

After testing, its structure is as shown in the above formula Pyne-DAC, its fluorescence emission wavelength in water is 487nm, absorption wavelength is 480nm, which can be used for orthogonal reaction with azide biological.

Example 45

Synthesis of Col-DAC

NHSB-DAC (20 mg, 0.04 mmol) and aminocolchicine (15 mg, 0.04 mmol) were placed in a 5 mL Shrek bottle and replaced with nitrogen three times. 5 μL of diisopropylethylamine (DIPEA) was dissolved in 2 mL of dimethyl sulfoxide (DMSO), and then the mixture was added to the reaction bottle. After stirring at room temperature for 3 h, the reaction solution was poured into 10 mL of water, and extracted with 50 mL of dichloromethane to obtain an organic phase, which was dried over anhydrous sodium sulfate and separated by a silica gel column (dichloromethane: methanol = 80: 1, V/V) 23mg brown solid, yield 77%.

The high-resolution mass spectrum of Col-DAC prepared in Example 55 is shown in FIG. 32, and the mass spectrum data is: high-resolution mass spectrometry theoretical value C ₄₂ H ₄₅ N ₄ O ₈ [M+H] ⁺ 733.3237, the measured value 733.3220.

After testing, its structure is as shown in the above formula Col-DAC, its fluorescence emission wavelength in water is 489nm, absorption is 481nm.

Example 46

Synthesis of DTX-DAC

NHSB-DAC (10 mg, 0.02 mmol) and paclitaxel (14 mg, 0.02 mmol) were placed in a 5 mL Shrek bottle and replaced with nitrogen three times. 3 μL of diisopropylethylamine (DIPEA) was dissolved in 1 mL of dimethyl sulfoxide (DMSO), and then the mixture was added to the reaction bottle. After stirring at room temperature for 2 h, the reaction solution was poured into 10 mL of water, and extracted with 50 mL of dichloromethane to obtain an organic phase, which was dried over anhydrous sodium sulfate and separated by a silica gel column (dichloromethane:methanol=20:1, V/V). Brownish yellow solid 16mg, yield 72%.

The high-resolution mass spectrum of DTX-DAC prepared in Example 56 is shown in FIG. 33, and the mass spectrum data are: high-resolution mass spectrometry theoretical value C ₆₀ H ₆₇ N ₄ O ₁₅ [M+H] ⁺ 1083.4603, measured value 1083.4603.

After testing, its structure is as shown in the above formula DTX-DAC, its fluorescence emission wavelength in water is 488nm, absorption is 481nm.

Example 47

Synthesis of UNAA-DAC

NHSB-DAC (30 mg, 0.06 mmol) and 4-(4-methyl-2,3,5,6-azaphenyl)benzylamine hydrochloride (19 mg, 0.06 mmol) were placed in a 5 mL Shrek bottle, Replace with nitrogen 4 times. 20 L of diisopropylethylamine (DIPEA) was dissolved in 2 mL of dimethyl sulfoxide (DMSO), and then the mixture was added to the reaction bottle. After stirring at room temperature for 2 h, the reaction solution was poured into 10 mL of water and extracted with 100 mL of dichloromethane to obtain an organic phase, which was dried over anhydrous sodium sulfate and separated by a silica gel column (dichloromethane:methanol=40:1, V/V) Brownish yellow solid 21mg, yield 60%. The nuclear magnetic spectrum hydrogen spectrum of UNAA-DAC prepared in Example 57 is shown in FIG. 26, and the specific data of hydrogen spectrum and carbon spectrum are:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.54 (t, J=5.9 Hz, 1H), 8.46 (d, J=8.3 Hz, 2H), 8.11 (d, J=8.6 Hz, 2H), 7.58 (d, J = 8.4 Hz, 4H), 6.88 (d, J = 8.7 Hz, 2H), 4.43 (d, J = 5.9 Hz, 2H), 4.07 (t, J = 7.1 Hz, 2H), 3.21 (d , J = 9.1 Hz, 2H), 3.04 (s, 3H), 2.27 (dd, J = 16.9, 8.9 Hz, 4H), 1.93 (dd, J = 14.8, 7.5 Hz, 2H), 1.78 (d, J = 7.9 Hz, 2H), 1.40 (dd, J = 21.3, 9.5 Hz, 4H). ¹³ C NMR (101 MHz, DMSO-d ₆ ) δ 172.39, 167.51, 163.65, 163.51, 154.57, 144.96, 134.79, 133.38, 130.74 , 128.47, 127.87, 110.59, 107.79, 106.45, 59.49, 42.30, 39.02, 33.79, 32.07, 24.80, 23.63, 21.27.

After testing, its structure is as shown in the above formula UNAA-DAC, its fluorescence emission wavelength in water is 487nm, absorption is 481nm; the fluorescence is effectively quenched due to the introduction of tetrazole.

Example 48

Synthesis of MLD-DAze fluorescent dye

Synthesis of dye N-methyl-9,10-di-aziridinyl-1,6,7,12-tetrachloroperyleneimide:

Combine N-butyl-1,6,7,12-tetrachloro-9,10-dibromo-3,4-peryleneimide MLD-DBr (200 mg, 0.32 mmol) and aziridine (25 mg, 0.46 mmol ) Dissolve in 20 mL of ethylene glycol methyl ether and heat it to 120°C. After 12h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane = 1:3, V/V) to obtain a blue-green solid 18mg, a yield of 11%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.55 (s, 2H), 6.57 (s, 2H), 4.31 (s, 3H), 2.76 (s, 8H).

After testing, its structure is shown as MLD-DAzi, and its fluorescence emission wavelength reaches 670-700nm in different solvents, achieving near infrared fluorescence emission.

Example 49

Synthesis of fluorescent dye BuLD-DAze

Synthesis of the dye N-butyl-9,10-diazacyclobutyl-1,6,7,12-tetrachloroperyleneimide:

Combine N-butyl-1,6,7,12-tetrachloro-9,10-dibromo-3,4-peryleneimide (200mg, 0.30mmol) with azetidine (86mg, 1.50mmol) Dissolve in 10 mL of ethylene glycol methyl ether and heat it to 120°C. After 12h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane = 1:4, V/V) to obtain a blue-green solid 46mg, a yield of 25%. The nuclear magnetic spectrum of N-butyl-9,10-diazetidine-1,6,7,12-tetrachloroperyleneimide (BuLD-DAze) prepared in Example 59 is shown in FIG. 27 The specific data are as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.52 (s, 2H), 6.55 (s, 2H), 4.25-4.19 (m, 2H), 4.09 (s, 8H), 2.48 (s, 4H), 1.79- 1.67 (m, 2H), 1.48 (dd, J = 14.9, 7.4Hz, 2H), 0.99 (t, J = 7.4Hz, 3H).

After testing, the structure is shown in the above formula BuLD-DAze, and the fluorescence emission wavelength in ethanol is around 720nm, which has reached the near infrared region.

Example 50

Synthetic method of fluorescent dye OLD-DAze.

Dye N-(2-(2-hydroxy)-ethoxy)ethyl-9,10-di-azetidin-1,6,7,12-tetrachloroperyleneimide (OLD-DAze) Synthesis:

LD-DBr (200 mg, 0.28 mmol) and azetidine (104 mg, 1.42 mmol) were dissolved in 10 mL of ethylene glycol methyl ether and heated to 120°C. After 24h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (developing agent: dichloromethane) to obtain a blue-green solid 60mg, with a yield of 32%.

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.53 (s, 2H), 6.57 (s, 2H), 4.57-4.37 (m, 2H), 4.10 (s, 8H), 3.88 (d, J=4.9 Hz, 2H), 3.71 (s, 4H), 2.50 (s, 4H).

After testing, the structure is shown in the above formula OLD-DAze, and the fluorescence emission wavelength in ethanol is about 750nm, which has reached the near infrared region.

Example 51

Synthesis method of near infrared dye BuLD-710.

Synthesis of dye N-butyl-9,10-cyclohexanediamine-1,6,7,12-tetrachloroperyleneimide:

Combine N-butyl-1,6,7,12-tetrachloro-9,10-dibromo-3,4-peryleneimide (200mg, 0.30mmol) with transcyclohexanediamine (228mg, 2.00mmol) ) Dissolve in 10 mL of ethylene glycol methyl ether and heat it to 130°C. After 14h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane = 1:4, V/V) to obtain a blue-green solid 56mg, a yield of 30%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.24 (d, J = 3.7 Hz, 2H), 8.11 (s, 1H), 7.96 (s, 1H), 7.17 (d, J = 10.8 Hz, 2H) ,, 4.28 (t, J = 6.5 Hz, 2H), 3.21 (d, J = 10.0 Hz, 2H), 2.26 (d, J = 10.9 Hz, 2H), 1.94-1.68 (m, 2H), 1.77 (s , 2H), 1.56-1.38 (m, 2H), 1.41 (s, 2H), 1.25 (s, 2H), 0.99 (t, J = 7.1Hz, 3H).

After testing, the structure is shown in the above formula BuLD-710, and the fluorescence emission wavelength in ethanol is about 750nm, which has reached the near infrared region.

Example 52

Synthesis method of fluorescent dye OLD-710.

Combine N-(2-(2-hydroxy)-ethoxy)ethyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide (200mg, 0.28mmol) with trans Cyclohexanediamine (1200 mg, 10.5 mmol) was dissolved in 10 mL of ethylene glycol methyl ether and heated to 120°C. After 24h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (developing agent: dichloromethane) to obtain 80 mg of a blue-green solid with a yield of 43%. N-(2-(2-hydroxy)-ethoxy)ethyl-9,10-cyclohexanediamine-1,6,7,12-tetrachloroperyleneimide (OLD- 710) The nuclear magnetic spectrum hydrogen spectrum is shown in Figure 28, the specific data are as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.24 (d, J=3.7 Hz, 2H), 8.10 (s, 1H), 7.94 (s, 1H), 7.17 (d, J=10.8 Hz, 2H) , 4.60 (s, 1H), 4.26 (t, J = 6.5 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H), 3.48 (s, 4H), 3.21 (d, J = 10.0 Hz, 2H) , 2.26 (d, J = 10.9 Hz, 2H), 1.76 (s, 2H), 1.40 (s, 2H), 1.22 (s, 2H).

After testing, its structure is shown in the above formula OLD-710, and its fluorescent properties are as follows:

Dissolve the dye in DMSO solution, prepare 2mM mother liquor, prepare test solutions with different concentrations according to the needs, detect the change of fluorescence spectrum and fluorescence imaging of cells and in vivo lipid droplets.

Fluorescence excitation and emission spectrum test in ethanol. Each time, 20 μL of the mother liquor of dye was added to 4 mL of ethanol to prepare a 10 μM fluorescent probe test solution, and fluorescence excitation and emission spectrum tests were performed.

The excitation and emission spectra of OLD-710 in ethanol are shown in Figure 36: Normalized fluorescence spectra of OLD-710 in ethanol. The fluorescence emission wavelength in OLD-710 ethanol reached 750nm, the excitation wavelength was 712nm, and the excitation and emission wavelength reached the near infrared emission wavelength.

Example 53

Synthesis of dye ELD-DAC

ELD-DBr (200 mg, 0.27 mmol) and 1,2-cyclohexanediamine (228 mg, 2.00 mmol) were dissolved in 20 mL of ethylene glycol methyl ether and heated to 120°C. After 16h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane = 1:4, V/V) to obtain a blue-green solid 75mg, a yield of 40%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.25 (s, 2H), 7.93 (s, 2H), 7.15 (s, 2H), 4.11 (t, J=6.6 Hz, 2H), 3.97 (dt, J = 13.3, 6.5 Hz, 2H), 3.21 (d, J = 9.6 Hz, 2H), 2.38 (t, J = 7.3 Hz, 2H), 2.26 (d, J = 11.8 Hz, 2H), 1.97-1.87 ( m, 2H), 1.77 (d, J = 6.4Hz, 2H), 1.35 (dd, J = 21.4, 8.0Hz, 4H), 1.13 (t, J = 7.1Hz, 3H).

After testing, its structure is shown as the above-mentioned ELD-DAC, its fluorescence emission wavelength in ethanol is about 752nm, and the emission wavelength has reached the near infrared region.

Example 54

Synthesis of BuLD-DAzi

N-butyl-1,6,7,12-tetrachloro-9,10-dibromo-3,4-peryleneimide BuL]D-DBr (200mg, 0.30mmol) and aziridine (100mg, 2.32 mmol) was dissolved in 20 mL of ethylene glycol methyl ether and heated to 120°C. After 12h, the solvent was removed under reduced pressure, and the residue was separated through a silica gel column (petroleum ether: dichloromethane = 1:3, V/V) to obtain a blue-green solid 14mg, a yield of 8%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 8.54 (s, 2H), 6.56 (s, 2H), 4.25-4.19 (m, 2H), 2.78 (s, 8H), 1.79-1.66 (m, 2H), 1.47 (dd, J = 14.9, 7.4 Hz, 2H), 0.99 (t, J = 7.4 Hz, 3H).

After testing, the structure is as shown in the above formula BuLD-DAzi, the fluorescence emission wavelength in ethanol is about 730nm, and the emission wavelength has reached the near infrared region.

Example 55

Synthesis of target dye Rho-1

Weigh 500mg of 5-hydroxytetrahydroquinoline and 595mg of phthalic anhydride in a flask, add 30mL of toluene under the protection of nitrogen, stir under heating at reflux for 5h, stop the reaction, cool to room temperature and ice bath for 30min, filter with suction, The filter cake was washed with petroleum ether and dried to obtain 458 mg of crude product with a yield of 46%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 13.15 (s, 1H), 9.07 (d, J = 111.2 Hz, 1H), 7.87 (d, J = 56.8 Hz, 1H), 7.44 (s, 2H) , 6.91 (s, 1H), 6.42 (d, J = 64.0Hz, 1H), 3.34 (s, 4H), 2.67 (t, J = 6.6Hz, 2H), 1.91 (s, 2H).

Weigh 135 mg of 5-hydroxytetrahydroquinoline and 290 mg of intermediate 5-hydroxytetrahydroquinoline benzophenic acid respectively, and under the protection of nitrogen, add 4 mL of methanesulfonic acid and 4 mL of trifluoroacetic acid. The temperature was first raised to 120°C, then to 150°C, and stirred for two days. Most of the acid was removed under reduced pressure, and an aqueous solution of sodium carbonate was added to adjust the pH to 9-10. Dichloromethane was extracted, the organic solvent was removed under reduced pressure, and the silica gel column was separated. Using dichloromethane: methanol 20:1-5:1 as the eluent, the organic solvent was removed under reduced pressure to obtain 178 mg of purple-red solid with a yield of 48%. The nuclear magnetic spectrum hydrogen spectrum of Rho-1 prepared in Example 64 is shown in FIG. 29, and the specific data are:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.93 (d, J=7.6 Hz, 1H), 7.74 (t, 1H), 7.66 (t, J=7.4 Hz, 1H), 7.22 (d, J= 7.6Hz, 1H), 6.29(s, 2H), 6.22(q, J=8.7Hz, 4H), 3.20(s, 4H), 2.84(t, J=6.3Hz, 4H), 1.95-1.82(m, 4H).

After testing, its structure is shown in the above formula Rho-1, which can be used to image mitochondria in cells, and its optical properties are as follows:

The absorption and emission spectrum of Rho-1 dye molecule Rho-1 in ethanol. The rhodamine dye molecule Rho-1 obtained in Example 64 was dissolved in DMSO to prepare a 2 mM stock solution. 20 μL of the mother liquor was dissolved in 4 mL of ethanol and configured as a test solution with a final concentration of 10 μM, and its absorption and emission spectra were measured.

The absorption and emission spectra of Rho-1 in ethanol are shown in Figure 37: The absorption of Rho-1 in ethanol is 533nm and the emission wavelength is 558nm. The fluorescence quantum yield in ethanol is calculated to be 0.91.

Example 56

Synthesis of target dye Rho-2

Weigh 100 mg of 5-hydroxytetrahydroquinoline and 215 mg of intermediate 5-hydroxytetrahydroquinoline benzophenic acid, respectively, and add 4 mL of methanesulfonic acid and 4 mL of trifluoroacetic acid under nitrogen protection. The temperature was first raised to 120°C, then to 150°C, and stirred for two days. Most of the acid was removed under reduced pressure, and an aqueous solution of sodium carbonate was added to adjust the pH to 9-10. Dichloromethane was extracted, the organic solvent was removed under reduced pressure, and the silica gel column was separated. Using dichloromethane: methanol 20:1-5:1 as the eluent, the organic solvent was removed under reduced pressure to obtain 144 mg of purple-red solid with a yield of 52%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.93 (d, J=7.4 Hz, 1H), 7.75 (t, J=6.0 Hz, 1H), 7.66 (t, J=7.1 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 6.33 (t, J = 14.3 Hz, 2H), 6.26-6.19 (m, 2H), 6.17 (dd, J = 9.0, 2.1 Hz, 1H), 6.09 (d, J = 11.5 Hz, 1H), 3.18 (dd, J = 10.9, 4.9 Hz, 4H), 2.81 (dt, J = 20.3, 6.4 Hz, 2H), 2.44 (dd, J = 16.3, 6.4 Hz, 2H), 1.72-1.65 (m, 2H), 1.23 (s, 2H).

After examination, the structure is as shown in the above formula Rho-2, and its optical properties are as follows: the dye Rho-2 absorbs in ethanol at 534 nm and the emission wavelength at 559 nm, and the fluorescence quantum yield in ethanol is calculated to be 0.85.

Example 57

Synthesis of target dye Rho-3

Weigh 500mg of N-ethyl-5-hydroxytetrahydroquinoline and 654mg of phthalic anhydride in a flask, add 30mL of toluene under the protection of nitrogen, stir under heating and reflux for 5h, stop the reaction, cool to room temperature and ice bath 30min, suction filtration, washing the filter cake with petroleum ether and drying to obtain 514mg of crude product with a yield of 56%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.94 (s, 1H), 7.94 (dd, 1H, J=7.8, 1.1 Hz), 7.66 (td, 1H, J=7.5, 1.3 Hz), 7.59 ( td, 1H, J = 7.6, 1.3 Hz), 7.33 (dd, 1H, J = 7.6, 1.1 Hz), 6.40 (s, 1H), 3.35 (s, 1H), 3.25 (t, 2H, J = 5.7 Hz ), 3.23 (t, 2H, J = 6.2Hz), 2.59 (t, 2H, J = 6.4Hz), 1.85 (m, 2H), 1.67 (t, 2H).

Weigh 200 mg of N-ethyl-5-hydroxytetrahydroquinoline and 404 mg of intermediate N-ethyl-5-hydroxybenzophenone acid respectively, under nitrogen protection, add 5 mL of methanesulfonic acid and 5 mL of trifluoroacetic acid. The temperature was first raised to 120°C, then to 150°C, and stirred for two days. Most of the acid was removed under reduced pressure, and an aqueous solution of sodium carbonate was added to adjust the pH to 9-10. Dichloromethane was extracted, the organic solvent was removed under reduced pressure, and the silica gel column was separated. Using dichloromethane: methanol 20:1-5:1 as the eluent, the organic solvent was removed under reduced pressure to obtain 173 mg of purple solid, with a yield of 36%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.91 (d, J=7.6 Hz, 1H), 7.64 (t, 1H), 7.56 (t, J=7.4 Hz, 1H), 7.22 (d, J= 7.6Hz, 1H), 6.26(s, 2H), 6.12(q, J=8.7Hz, 4H), 3.40(s, 4H), 3.37(q, J=7.1Hz 4H), 2.84(t, J=6.3Hz , 4H), 1.95-1.82 (m, 4H), 1.10-1.14 (t, 6H)

After testing, its structure is as shown in the above formula Rho-3, and its optical properties are as follows: The absorption and fluorescence emission wavelengths of Rho-3 in ethanol are: absorption wavelength is 538nm, emission wavelength is 561nm, and the fluorescence quantum in ethanol is calculated The yield was 0.81.

Example 58

Synthesis of target dye Rho-4

Weigh 1.0g of 4-hydroxyindoline and 1.3g of phthalic anhydride in a flask, add 30mL of toluene under the protection of nitrogen, stir under heating and reflux for 5h, stop the reaction, cool to room temperature and ice bath for 30min, filter with suction , The filter cake was washed with petroleum ether and dried to obtain the crude product white solid 1.0g, yield 48%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 9.55 (s, 1H), 7.95 (d, J=7.6 Hz, 1H), 7.63 (d, J=7.9 Hz, 1H), 7.55 (s, 1H) , 7.46 (t, J=7.4 Hz, 1H), 7.31 (d, J=7.3 Hz, 1H), 7.00 (t, J=8.0 Hz, 1H), 6.51 (d, J=8.1 Hz, 1H), 3.64 (t, 2H), 2.87 (t, J=8.4Hz, 2H).

Weigh 135 mg of 4-hydroxyindoline and 290 mg of intermediate 4-hydroxyindoline benzophenic acid, add 8 mL of methanesulfonic acid and 2 mL of trifluoroacetic acid, under nitrogen protection. The temperature was first raised to 120°C, then to 150°C, and stirred for two days. Remove most of the acid under reduced pressure, add sodium carbonate aqueous solution to adjust the pH to 9-10. Extract with dichloromethane, remove the organic solvent under reduced pressure, and separate the silica gel column, wash with dichloromethane: methanol 20:1-5:1 The agent was removed and the organic solvent was removed under reduced pressure to obtain 106 mg of a white solid with a yield of 27.6%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CD ₃ OD) δ 8.21-8.16 (m, 1H), 7.68-7.62 (m, 2H), 7.29-7.23 (m, 1H), 7.08 (d, J=8.9 Hz, 2H) , 6.68 (d, J = 8.9 Hz, 2H), 4.03 (t, J = 8.8 Hz, 4H), 3.42-3.37 (m, 4H).

After testing, the structure is as shown in the above formula Rho-5, which can be used to image mitochondria in cells. The optical properties are as follows: Rho-5 absorption in ethanol is 551nm, emission wavelength is 574nm, and Stokes shift is 23nm. The half-width of the emission spectrum is 26nm, and the fluorescence quantum yield in ethanol is calculated to be 0.93.

Example 59

Synthesis of target dye Rho-5

Weigh 500mg of N-ethyl-4-hydroxyindoline and 454mg of phthalic anhydride in a flask, add 20mL of toluene under the protection of nitrogen, stir under heating at reflux for 5h, stop the reaction, cool to room temperature and ice bath for 30min , Suction filtration, washing the filter cake with petroleum ether and drying to obtain 391 mg of crude product as a white solid with a yield of 41%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.04 (s, 1H), 8.44 (t, J=7.5 Hz, 1H), 8.18 (t, J=7.9 Hz, 1H), 7.96 (d, 1H) , 7.03 (d, J = 7.4 Hz, 1H), 6.39 (d, J = 7.3 Hz, 1H), 3.61 (t, J = 8.0 Hz, 2H), 3.40 (m, J = 8.1 Hz, 2H), 3.12 (t, J = 8.4Hz, 2H), 1.12 (s, 3H).

Weigh 100 mg of N-ethyl-4-hydroxyindoline and 190 mg of N-7yl-4-hydroxyindoline benzophenone acid respectively, add 4 mL of methanesulfonic acid and 1 mL of trifluoroacetic acid, under nitrogen protection. The temperature was first raised to 120°C, then to 150°C, and stirred for two days. Remove most of the acid under reduced pressure, add sodium carbonate aqueous solution to adjust the pH to 9-10, extract with dichloromethane, remove the organic solvent under reduced pressure, and separate on a silica gel column (200-300 mesh), using dichloromethane: methanol 20: 1-5:1 is the eluent, and the organic solvent is removed under reduced pressure to obtain 70 mg of white solid with a yield of 26%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CD ₃ OD) δ 8.19-8.15 (m, 1H), 7.67-7.63 (m, 2H), 7.20-7.15 (m, 1H), 7.07 (d, J = 8.9 Hz, 2H) , 6.68 (d, J = 8.9 Hz, 2H), 3.99 (t, J = 8.8 Hz, 4H), 3.42-3.37 (m, 4H), 3.33 (m, J = 8.8 Hz, 4H), 1.12 (t, J=8.8Hz, 6H).

After testing, its structure is shown in the above formula Rho-5, and its optical properties are as follows:

The absorption and emission wavelengths of Rho-5 in ethanol are: absorption wavelength is 553nm, emission wavelength is 577nm, and the fluorescence quantum yield in ethanol is calculated to be 0.90.

Example 60

Synthesis of target dye SiR-1

96 mg of tert-butyl 2-bromobenzoate was added to a Shrek bottle, and a solution of tetrahydrofuran was added under the protection of nitrogen, and 220 μL of a 1.7 M isobutyl lithium heptane solution was added under stirring at -78°C, and the mixture was stirred for 30 min. Add 20 mg of the intermediate Si-ket6 in tetrahydrofuran, warm to room temperature and stir to avoid light overnight. After quenched with saturated aqueous ammonium chloride solution, diluted with water, extracted with ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure to obtain a crude product. The crude product was separated through a silica gel column. The eluent was petroleum ether: ethyl acetate = 30:1. After removing the organic solvent under reduced pressure, 14 mg of a colorless solid was obtained with a yield of 55%. The nuclear magnetic spectrum hydrogen spectrum of SiR-1 prepared in Example 69 is shown in FIG. 30, and the specific data are:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.87 (d, J=7.6 Hz, 1H), 7.48 (td, J=7.5, 1.0 Hz, 1H), 7.39 (td, J=7.4 Hz, 1H), 7.08 (d, J=7.7 Hz, 1H), 6.78 (d, J=8.8 Hz, 2H), 6.49 (d, J=8.9 Hz, 2H), 3.26 (t, J=6.1 Hz, 4H), 2.97-2.89 (m, J = 11.1, 5.5 Hz, 4H), 2.01 (t, 4H), 0.76 (s, 3H), 0.67 (s, 3H).

After testing, the structure is as shown in the above formula SiR-1, which can be used to image mitochondria in cells, and its optical properties are as follows:

Absorption and emission spectrum test of silicon-based substituted rhodamine dye molecule SiR-1 in ethanol. The dye molecule SiR-1 obtained in Example 1 was dissolved in a DMSO solution to prepare a 2 mM stock solution of different dyes. 20 μL of the stock solution was taken in 4 mL of methanol to prepare a test solution with a final concentration of 10 μM to test its absorption and fluorescence spectrum.

The normalized absorption and emission spectra of SiR-1 in ethanol are shown in Figure 38: the absorption of dye molecule SiR-1 in methanol is 648 nm, the emission wavelength is 663 nm, and the calculated quantum yield is 0.34.

Example 61

Synthesis of target dye SiR-2

93 mg of 2-bromobenzoic acid tert-butyl ester was added to a Shrek bottle. A solution of tetrahydrofuran was added under the protection of nitrogen, and 0.21 mL of a 1.7M isobutyl lithium heptane solution was added under -78°C stirring conditions. After stirring for 30 min. Intermediate Si-ket5 (18 mg) was added to dissolve in tetrahydrofuran solution and injected into the reaction system, and the temperature was raised to room temperature and stirred and protected from light overnight. After quenched with saturated aqueous ammonium chloride solution, diluted with water, extracted with ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure to obtain a crude product. The crude product was separated through a silica gel column. The eluent was petroleum ether: ethyl acetate = 30:1. After removing the organic solvent under reduced pressure, a colorless solid 11 mg was obtained with a yield of 47%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.92 (d, J=7.6 Hz, 1H), 7.60 (td, J=7.5, 1.1 Hz, 1H), 7.49 (td, 1H), 7.24 (d, 1H) , 6.71 (d, J = 8.5 Hz, 2H), 6.31 (d, J = 8.5 Hz, 2H), 3.42 (dd, J = 15.5, 8.3 Hz, 2H), 3.32 (dd, J = 17.0, 8.6 Hz, 2H), 3.12 (dd, 4H), 2.71 (s, 6H), 0.65 (s, J = 16.3Hz, 6H).

After testing, its structure is as shown in the above formula SiR-2, and its optical properties are as follows:

Normalized graph of absorption and fluorescence of the dye molecule SiR-2 in methanol. The absorption is 649 nm, the emission wavelength is 659 nm, and the calculated quantum yield is as high as 0.4.

Example 62

Synthesis of target dye SiR-3

In a Shrek bottle, 36 mg of tert-butyl 2-bromobenzoate was added with tetrahydrofuran solution under the protection of nitrogen, and 80 μL of a 1.7 M isobutyl lithium heptane solution was added under stirring at -78°C. After stirring for 30 min. Add intermediate Si-kets5 silicone 7mg dissolved in tetrahydrofuran solution and inject into the reaction system, warm to room temperature, stir and avoid light overnight. After quenched with saturated aqueous ammonium chloride solution, diluted with water, extracted with ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure to obtain a crude product. The crude product was separated through a silica gel column. The eluent was petroleum ether: ethyl acetate = 30:1. After removing the organic solvent under reduced pressure, 2 mg of a colorless solid was obtained with a yield of 24%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.87 (d, J=7.7 Hz, 1H), 7.52 (t, J=7.5 Hz, 1H), 7.43 (t, 2H), 7.16 (d, J=7.7 Hz , 1H), 6.64 (d, J = 8.5Hz, 1H), 6.59 (s, 1H), 6.57 (s, 2H), 6.23 (d, J = 8.5Hz, 1H), 3.65 (t, J = 7.0Hz , 5H), 3.20 (t, J = 8.2 Hz, 2H), 3.04 (t, J = 8.2 Hz, 2H), 2.73 (s, 4H), 2.64 (s, 3H), 0.57 (s, J = 3.1 Hz , 3H), 0.53 (s, 3H).

After testing, its structure is shown in the above formula SiR-3, and its optical properties are as follows: the absorption of the dye molecule SiR-3 in methanol is 662 nm, the emission wavelength is 684 nm, and the calculated quantum yield is 0.36.

Example 63

Synthesis of target dye SiR-4

115 mg of 2-bromobenzoic acid tert-butyl ester was added to a Shrek bottle, a solution of tetrahydrofuran was added under nitrogen protection, and 0.26 mL of a 1.7M isobutyl lithium heptane solution was added under stirring at -78°C, after stirring for 30 min. Add intermediate Si-ketBu (22mg) dissolved in tetrahydrofuran solution and inject into the reaction system, warm to room temperature, stir and avoid light overnight. After quenched with saturated aqueous ammonium chloride solution, diluted with water, extracted with ethyl acetate, the organic phase was collected, dried over anhydrous sodium sulfate, and the organic solvent was removed under reduced pressure to obtain a crude product. The crude product was separated through a silica gel column. The eluent was petroleum ether: ethyl acetate = 30:1. After removing the organic solvent under reduced pressure, a colorless solid 13 mg was obtained with a yield of 49%. The hydrogen spectrum data of the nuclear magnetic spectrum is as follows:

¹ H NMR (400 MHz, CDCl ₃ ) δ 7.92 (d, J=7.6 Hz, 1H), 7.60 (td, J=7.5, 1.1 Hz, 1H), 7.49 (td, 1H), 7.24 (d, 1H) , 6.71 (d, J = 8.5 Hz, 2H), 6.31 (d, J = 8.5 Hz, 2H), 3.42 (dd, J = 15.5, 8.3 Hz, 2H), 3.32 (dd, J = 17.0, 8.6 Hz, 2H), 3.12 (dd, 4H), 2.71 (t, J = 8.5Hz, 4H), 1.2-1.6c (m, J = 8.2Hz, 8H), 1.06 (s, 6H). 0.65 (s, J = 16.3Hz, 6H).

After testing, the structure is as shown in the above formula SiR-4, and its optical properties are as follows: The absorption and fluorescence wavelengths of SiR-4 in methanol are: absorption wavelength is 660nm, emission wavelength is 681nm, and the calculated quantum yield is 0.31 .

The present invention involves dissolving the dyes in DMSO solution and preparing 2mM mother liquors of different dyes, and preparing different concentrations of test solutions as needed to detect changes in their fluorescence spectra and intracellular fluorescence imaging.

Example 64

BuAN-DAze tested the fluorescence intensity with time under the irradiation of 500W tungsten lamp. Take 20μL of BuAN-DAze and commercial dye stock solution into 4mL PBS (Phosphate buffer, pH 7.4), and then add it to the threaded cuvette. Place it on the front at 50cm of the tungsten lamp. Take 0, 0.5, 1, 1.5, 2, respectively. 3,4,6,8,10h are the time nodes to carry out the fluorescence spectrum test, and the fluorescence emission peak of each dye is selected and the time is plotted.

The photostability of different dyes is shown in Figure 11: BuAN-DAze fluorescence intensity can still maintain a higher intensity (97%) after 10 hours of illumination, while commercial green mitochondrial dyes, rhodamine 123, fluorescein, Bodipy and other fluorescence intensity are all Significantly reduced, which shows that BuAN-DAze light stability is extremely high.

Example 65

Some dyes in this project are fluorescence confocal imaging and structured light illumination microscopic imaging in living cells (RWPE, HeLa, fat cells, etc.). Take more than 0.5 μL of the probe mother solution and dissolve it in 1 mL of cell culture solution, and then incubate the cells at 37° C. for 10-30 minutes for fluorescence imaging.

The fluorescence confocal imaging of live cell RWPE cells of the dye Mito-DAze is shown in Fig. 12: accurate dye positioning, clear mitochondrial contours, and a good co-localization effect with the commercial dye MitoTracker Deep.

The mitochondrial dye Mito-DAze's RWPE cell live cell structure light illumination microscopic image is shown in Figure 13: RWPE cell mitochondria are clearly line-shaped, and can clearly see the mitochondrial ridge.

Mito-DAC imaging of live cell mitochondria is shown in Figure 14: the dye Mito-DAC can specifically label HeLa cell mitochondria and has a high signal-to-noise ratio.

The imaging diagram of OLD-DAze on live cell lipid droplets is shown in Figure 15: The lipid droplet dye OLD-DAze can specifically mark lipid droplets in adipocytes, and it can monitor lipid droplet particles of different sizes.

Example 66

Confocal fluorescence imaging of SNAP-tag or Halo-tag in transfected HeLa cells fused with tag protein. Dissolve 0.5 μL of the dye stock solution in 1 mL of cell culture solution and incubate at 37° C. and 5% CO ₂ for 30 minutes before using it for fluorescence confocal imaging.

The confocal imaging of Halo-DAze on the nucleus of living cells is shown in Figure 16. The dye Halo-DAze can accurately locate the nucleus and specifically react with the histone protein fused with Halo-tag, and the nuclear contour is clear.

SNAP-DAze's confocal imaging of the nucleus in living cells is shown in Figure 17: The dye SNAP-DAC can accurately locate the nucleus and react specifically with histones fused with SNAP-tag. The nuclear contour is clear and the signal is noisy Relatively high.

Example 67

SNAP-DAC was STED super-resolution fluorescence imaging in transfected pSNAP _f -H2B HeLa cells. Take 0.5μL of SNAP-DAC mother liquor dissolved in 1mL cell culture solution, incubate at 37°C, 5% CO ₂ for 30 minutes, fix the cells with 4% formaldehyde solution and place in 1mL PBS buffer for STED super resolution Fluorescence imaging.

The super-resolution image of SNAP-DAC on the nucleus is shown in Figure 18: SNAP-DAC can specifically label the nucleus in HeLa cells. Due to the improvement of light stability, SNAP-DAC can perform multiple imaging and reconstruction under GW/cm ² high-intensity laser to obtain higher resolution images.

Example 68

Rho-4 and Nu-DAC experiments on structured light illumination microcolor imaging of RWPE cells. Take more than 0.5 μL of the probe mother solution and dissolve it in 1 mL of cell culture solution at the same time, and then incubate the cells at 37°C for 10-30 minutes for fluorescence imaging.

Rho-4 and Nu-DAC's structured light illumination microcolor imaging of RWPE cells is shown in Figure 19: (a) Rho-4 channel imaging, which can specifically label the cell mitochondria; (b) It is a Nu-DAC channel imaging map, which can specifically stain the cell nucleus; (c) is an overlay of the above two. This shows that Rho-4 and Nu-DAC can be used simultaneously for multi-color fluorescence imaging of living cells.

Example 69

OLD-DAze and Nu-DAC experiments on structured light illumination microcolor imaging of HT29 cells. Take more than 0.5 μL of the probe mother solution and dissolve it in 1 mL of cell culture solution at the same time, and then incubate the cells at 37° C. for 10-30 minutes for fluorescence imaging.

OLD-DAze and Nu-DAC's structured light illumination microcolor imaging of HT29 cells is shown in Figure 20: (a) is the OLD-DAze channel imaging map, which can specifically mark cell lipid droplets; (b ) Is the Nu-DAC channel imaging map, which can specifically stain the cell nucleus. This shows that OLD-DAze and Nu-DAC can be used simultaneously for multi-color fluorescence imaging of living cells.

Claims (16)

A full-spectrum, high-brightness, high-stability fluorescent dye, characterized in that the dye suppresses intramolecular torsion at the end of the electron donor through the adjustment of the rigid cyclic amine structure, and realizes the improvement of fluorescence quantum efficiency and light stability The dye consists of 4-amide substituted naphthalimide dyes, bisalkoxy substituted naphthalimide fluorescent dyes, bisamino substituted naphthalimide fluorescent dyes, 9,10-bisamino substituted perylene acyl One or more of amine, six-membered cyclic rhodamine dyes, five-membered cyclic rhodamine dyes, and silicon-based rhodamine dyes are mixed in any ratio. The full-spectrum high-brightness, high-stability fluorescent dye according to claim 1, wherein the bisalkoxy substituted naphthalimide fluorescent dye has an absorption wavelength at 390 nm and a fluorescence emission wavelength is in use Excited at 405nm, its structural formula is as follows:

R ₃ and R ₄ are respectively independent

If R ₃ and R _{4 are} not independent, it is

Exist as a whole structure

p is an integer from 0-2. The full-spectrum high-brightness, high-stability fluorescent dye according to claim 1, wherein the diamino-substituted naphthalimide fluorescent dye has an absorption wavelength of 440-490nm and can pass 450nm and 488nm lasers Upon excitation, it can be used as the following dyes by changing the targeting group: mitochondrial fluorescent dye, SNAP-tag fluorescent dye, Halo-tag fluorescent dye, active ester fluorescent dye, drug targeting fluorescent dye, and its structural formula is as follows:

Where R ₂ is H, C1-16 alkyl, aryl, substituted aryl, (CH ₂ CH ₂ O) _n H, (CH ₂ ) _m COOMe and (CH ₂ ) _m SO ₃ H, heteroaryl or substituted Heteroaryl, biological targeting groups such as N-ethylmorpholine, benzylguanine, n-hexane-triphenylphosphine, folic acid, colchicine, paclitaxel, 6-chlorohexane and so on. R ₅ and R ₆ are respectively independent

One of them, if R ₅ and R _{6 are} not independent, it is

Exist as a whole structure,

R ₇ and R ₈ are each independently H, C1-4 alkyl, (CH ₂ CH ₂ O) _n H; if R _{7 is} not H, R ₈ must be a non-H substituent; Y is sulfone group, sulfoxide group, dimethylsilyl group, boryl group; m and n are integers from 0-4. The full-spectrum high-brightness, high-stability fluorescent dye according to claim 1, characterized in that the 9,10-position diamino substituted peryleneimide dye can be used for 680nm and 710nm lasers, and It can realize the imaging of lipid droplets in cells and in vivo, and its structural formula is as follows:

Among them: R ₁₁ is

R ₉ and R ₁₀ are respectively independent

One of them, if R ₉ and R _{4 are} not independent, it is

Exist as a whole structure,

R ₇ and R ₈ are each independently H, C1-4 alkyl, (CH ₂ CH ₂ O) _n H; if R _{7 is} not H, R ₈ must be a non-H substituent; n is an integer of 0-4. The full-spectrum high-brightness, high-stability fluorescent dye according to claim 1, wherein the six-membered ring-rhodamine dye can be used for 532nm excitation, and its structural formula is as follows:

R ₁₂ is a 5-position parallel six-membered ring or a 7-position parallel six-membered ring; R ₁₃ is H or C1-4 alkane. The full-spectrum high-brightness, high-stability fluorescent dye according to claim 1, wherein the five-membered ring-rhodamine dye can be used for 550nm excitation, and its structural formula is as follows:

Among them, R ₁₃ is H or C1-4 alkane. The full-spectrum high-brightness and high-stability fluorescent dye according to claim 1, wherein the silicon-based rhodamine dye can be used for excitation at 640 nm for fluorescent labeling and imaging, and its structural formula is as follows:

Among them: q=0 or 1; R ₁₃ is H, C1-4 alkyl. A method for synthesizing a full-spectrum, high-brightness, high-stability fluorescent dye according to claim 2, characterized in that the steps include: (1) Synthesis of dye N-butyl-4,5-dialkoxy-1,8-naphthalimide: Dissolve the polyhydric alcohol in dry tetrahydrofuran, add Na block under nitrogen blowing, and add N-butyl-4-bromo-5-nitro-1,8-naphthalimide to the reaction solution after 0.5-1h and heat To 60-90 ℃, 2-10h. After removing the solvent under reduced pressure, the silica gel column was separated, using dichloromethane and petroleum ether = 1:1 to 4 by volume as the eluent, and the solvent was removed under reduced pressure to obtain a white solid N-butyl-4,5-dioxane Oxy-1,8-naphthalimide; Among them, the mass ratio of polyol and sodium block is 2-1:1; the mass ratio of polyol and N-butyl-4-bromo-5-nitro-1,8-naphthalimide is 2:1- 12; The volume ratio of polyol to tetrahydrofuran is 1-10:1mg/mL. A method for synthesizing a full-spectrum high-brightness, high-stability fluorescent dye according to claim 3, characterized in that the steps include: (1) Synthesis of intermediate N-alkyl-4-bromo-5-nitro-1,8-naphthalimide: Dissolve 4-bromo-5-nitro-1,8-naphthalic anhydride and primary fatty amine in absolute ethanol; heat the reaction solution to 40-90°C and stir for 1-10 h; cool the reaction solution to room temperature After removing the solvent under reduced pressure, the silica gel column was separated. Using petroleum ether and dichloromethane or dichloromethane and methanol with a volume ratio of 800-100:1 as eluents, the solvent was removed under reduced pressure to obtain an off-white solid N-( 4-hydroxymethyl)benzyl-4-bromo-5-nitro-1,8-naphthalimide; Among them, the mass ratio of 4-bromo-5-nitro-1,8-naphthalic anhydride: primary fatty amine is 1:0.5-2; the mass of 4-bromo-5-nitro-1,8-naphthalic anhydride and no The volume ratio of water ethanol is 1:20-80g/nL. Fatty primary amines include linear alkyl amines such as methylamine, ethylamine, butylamine, n-dodecylamine, n-hexadecylamine, benzylamine analogs, amino-substituted alkyl sulfonic acids, and amino alcohols; (2) Synthesis of dye N-alkyl-4,5-difatty amino-1,8-naphthalimide: Dissolve N-alkyl-4-bromo-5-nitro-1,8-naphthalimide in ethylene glycol methyl ether, and add alicyclic amine to it; warm the reaction solution slowly to 100-140℃ , And react under nitrogen protection for 10-24h; remove the solvent under reduced pressure and separate it with a silica gel column, using dichloromethane and methanol in a volume ratio of 400-30:1 as the eluent, remove the solvent to obtain a brownish yellow solid N-alkane Group-4,5-difatty amino-1,8-naphthalimide; Among them, the mass ratio of N-alkyl-4-bromo-5-nitro-1,8-naphthalimide to aliphatic cyclic amine is 1:1-3; N-alkyl-4-bromo-5-nitro The volume ratio of the base-1,8-naphthalimide to ethylene glycol methyl ether is 1:50-200g/mL; the alicyclic amine is aziridine, azetidine, tetrahydropyrrole, Piperidine, cyclohexaneimine, ethylenediamine derivatives and cyclohexanediamine derivatives. A method for synthesizing a full-spectrum, high-brightness, high-stability fluorescent dye according to claim 4, characterized in that the steps include: (1) Synthesis of intermediate N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide: Dissolve 9,10-dibromo-1,6,7,12-tetrachloroperyleneimide and primary alcohol amine or fatty primary amine in a mixture of N-methylpyrrolidone and glacial acetic acid; heat the reaction solution to 100 -140°C, stirring for 1-10h; cool the reaction solution to room temperature, pour it into ice water and filter to obtain a black solid, vacuum dry, separate with a 200-300 mesh silica gel column, dichloromethane with a volume ratio of 1:0.25～6 Methane: petroleum ether is used as the eluent, and the solvent is removed under reduced pressure to obtain dark red solid N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide; Among them, the mass ratio of 9,10-dibromo-1,6,7,12-tetrachloroperyleneimide to primary alcohol amine or fatty primary amine is 1-10:1; 9,10-dibromo-1, The mass-volume ratio of 6,7,12-tetrachloroperyleneimide and N-methylpyrrolidone is 1:20-120g/mL; the volume ratio of N-methylpyrrolidone and glacial acetic acid is 1-3:3- 4; (2) Synthesis of probe N-alkyl-9,10-difatty amino-1,6,7,12-tetrachloroperyleneimide: Dissolve N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide in ethylene glycol methyl ether, and add fatty amine to it; then warm the reaction solution slowly To 90-130 ℃, and react under nitrogen protection for 10-24h; remove the solvent under reduced pressure, 200-300 mesh separation, using dichloromethane: petroleum ether with a volume ratio of 1:0-1 as eluent, under reduced pressure Remove the solvent to obtain the blue solid probe N-alkyl-9,10-difatty amino-1,6,7,12-tetrachloroperyleneimide; Among them, the mass ratio of N-alkyl-9,10-dibromo-1,6,7,12-tetrachloroperyleneimide to fatty amine is 1:6-8; the mass of fatty amine and ethylene glycol methyl ether The volume ratio is 5-120:1mg/mL; alicyclic amines are aziridine, azetidine, tetrahydropyrrole, piperidine, cycloheximide, ethylenediamine derivatives and cyclohexanediamine derivatives . A method for synthesizing a full-spectrum, high-brightness, high-stability fluorescent dye according to claim 5, characterized in that the steps include: (1) Synthesis of intermediate N-alkyl-5-hydroxytetrahydroquinolinyl benzophenone acid Dissolve the intermediates N-alkyl-5-hydroxytetrahydroquinoline and phthalic anhydride in toluene. After heating and refluxing for 4-8h, stop the reaction. After cooling to room temperature, let stand in an ice water bath for 30min-60min. , Filter, the filter cake was washed with a small amount of petroleum ether, and the filter cake was dried to obtain the crude intermediate product N-alkyl-5-hydroxytetrahydroquinolinyl benzophenone acid; Among them, the mass ratio of intermediate N-alkyl-5-hydroxytetrahydroquinolinyl keto acid and phthalic anhydride is 1:1-2, intermediate N-alkyl-5-hydroxytetrahydroquinolinyl The volume ratio of benzophenic acid to toluene is 1:40-80g/mL; (2) Synthesis of target dyes Intermediate N-alkyl-5-hydroxytetrahydroquinoline (or its analogue N-substituted-7-hydroxytetrahydroquinoline) and intermediate N-alkyl-5-hydroxytetrahydroquinolinobenzoic acid Dissolved in a mixed acidic solvent of methanesulfonic acid and trifluoroacetic acid, heated to 140°C under the protection of nitrogen. After two days of reaction, most of the solvent was removed under reduced pressure, and the pH was adjusted to 9-10 with a weak alkaline aqueous solution. Dichloro Methane extraction, organic phase collection and drying, after removing organic solvent, silica gel column separation, eluent is dichloromethane and methanol in a volume ratio of 20-5:1, the solvent is removed under reduced pressure to obtain the final product; Among them, the intermediate N-alkyl-5-hydroxytetrahydroquinoline (or its analogue N-substituted-7-hydroxytetrahydroquinoline) and the intermediate N-alkyl-5-hydroxytetrahydroquinolinebenzene The mass ratio of keto acid is 1:2-4; the volume ratio of trifluoroacetic acid and methanesulfonic acid is 1:1-5; the intermediate N-alkyl-5-hydroxytetrahydroquinoline (or its analog N- The volume ratio of substituted-7-hydroxytetrahydroquinoline) to trifluoroacetic acid is 1:30-80g/mL. A method for synthesizing a full-spectrum high-brightness, high-stability fluorescent dye according to claim 6, characterized in that the steps include: (1) Synthesis of intermediate N-alkyl-4-hydroxyindolinyl benzophenone acid Intermediate N-alkyl-4 hydroxyindoline and phthalic anhydride are dissolved in toluene. After heating and refluxing for 4-8h, the reaction is stopped. After cooling to room temperature, after standing in an ice water bath for 30min-60min, filtering and filtering The cake was washed with a small amount of petroleum ether, and the filter cake was dried to obtain the intermediate N-alkyl-4-hydroxyindolinyl benzophenone acid; Among them, the mass ratio of intermediate N-alkyl-4hydroxyindoline to phthalic anhydride is 1:1-2; the mass ratio of intermediate N-alkyl-4hydroxyindoline to toluene is: 1:20-40g/mL; (2) Synthesis of target dyes Intermediate N-alkyl-4 hydroxyindoline and intermediate N-alkyl-4-hydroxyindoline benzophenone acid are dissolved in a mixed acidic solvent of methanesulfonic acid and trifluoroacetic acid, heated under nitrogen protection After reacting at 140°C for two days, most of the solvent was removed under reduced pressure. The pH was adjusted to 9-10 with a weakly alkaline aqueous solution, extracted with dichloromethane, the organic phase was collected and dried, after removing the organic solvent, the silica gel column was separated and eluted The agent is dichloromethane and methanol in a volume ratio of 20-5:1, and the final product is obtained after removing the solvent under reduced pressure; Among them, the mass ratio of intermediate N-alkyl-4 hydroxyindoline and intermediate N-alkyl-4-hydroxyindoline benzophenone acid is 1:2-3; the ratio of trifluoroacetic acid and methanesulfonic acid The volume ratio is 1:1-5; the volume ratio of the mass of intermediate N-alkyl-4hydroxyindoline to trifluoroacetic acid is 1:10-30g/mL. A method for synthesizing a full-spectrum high-brightness, high-stability fluorescent dye according to claim 7, characterized in that the steps include the following steps: (1) Synthesis of target fluorescent molecule symmetrical silyl rhodamine Add 2-bromobenzoic acid tert-butyl ester to Shrek bottle, add tetrahydrofuran solution under the protection of nitrogen, add butyllithium solution under -78 ℃ stirring condition, after stirring for 20-40min; add intermediate Si-keto tetrahydrofuran solution, l Stir at room temperature and avoid light overnight; after quenching with saturated aqueous ammonium chloride solution, dilute with water, extract with ethyl acetate, collect the organic phase, wash and dry, remove the organic solvent under reduced pressure to obtain the crude product, and separate it through a 200-300 mesh silica gel column. The eluent is petroleum ether and ethyl acetate in a volume ratio of 50-20:1 to obtain the target fluorescent dye molecule; Among them, the mass ratio of the intermediate Si-keto to tert-butyl 2-bromobenzoate is 1:4-8; the volume ratio of the mass of the intermediate Si-keto to the volume of butyllithium solution is 10-20:1 mg/mL; (2) Synthesis of target fluorescent dye asymmetric silyl rhodamine Add 2-bromobenzoic acid tert-butyl ester to Shrek bottle, add tetrahydrofuran solution under the protection of nitrogen, add butyllithium solution under -78℃ stirring condition, stir for 20-40min; add intermediate Si-ketos tetrahydrofuran solution, l Stir to room temperature and avoid light overnight; after quenched with saturated aqueous ammonium chloride solution, dilute with water, extract with ethyl acetate, collect the organic phase, wash and dry, and remove the organic solvent under reduced pressure to obtain the crude product; the crude product is separated by column chromatography and washed The removal agent is petroleum ether and ethyl acetate with a volume ratio of 50-20:1, and the target fluorescent dye molecule is obtained after removing the organic solvent under reduced pressure; Among them, the mass ratio of intermediate Si-ketos to tert-butyl 2-bromobenzoate is 1:4-7; the volume ratio of butyllithium solution to tetrahydrofuran is 1:30-50; the mass of intermediate Si-ketos and The volume ratio of butyl lithium is 1:10-20 mg/μL. The application of a full-spectrum high-brightness, high-stability fluorescent dye according to claim 1 in the field of fluorescence imaging of living cells, tissues and living bodies. The application of a full-spectrum high-brightness, high-stability fluorescent dye according to claim 1 in the field of identification marks of SNAP-tag and Halo-tag. The application of a full-spectrum high-brightness, high-stability fluorescent dye according to claim 1 in the field of fluorescent imaging of living cells, tissues and living bodies.

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