CN114591632B - Azaindole-hemicyanine dye, and synthetic method and application thereof - Google Patents
Azaindole-hemicyanine dye, and synthetic method and application thereof Download PDFInfo
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- CN114591632B CN114591632B CN202210083686.XA CN202210083686A CN114591632B CN 114591632 B CN114591632 B CN 114591632B CN 202210083686 A CN202210083686 A CN 202210083686A CN 114591632 B CN114591632 B CN 114591632B
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- azaindole
- dye
- compound
- hemicyanine
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- C07D—HETEROCYCLIC COMPOUNDS
- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
- C07D471/04—Ortho-condensed systems
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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Abstract
The invention discloses an azaindole-hemicyanine dye, a synthesis method and application thereof. This type of dye has longer absorption and emission wavelengths than conventional hemicyanine dyes. And the related experiments prove that the novel dye molecules have better biocompatibility compared with the traditional hemicyanine dye, and can be applied to the aspects of biological identification imaging, cell imaging, protein labeling, antibody specific identification, nucleic acid labeling, fluorescent probes, DNA sequencing, tumor photodynamic therapy and the like.
Description
Technical Field
The invention relates to the technical field of organic dyes, in particular to an azaindole-hemicyanine dye, a synthesis method and application thereof.
Background
The hemicyanine dye has the advantages of large stokes shift, large molar extinction coefficient and the like, so that the hemicyanine dye is continuously used as a dye matrix to be developed into a fluorescent probe or a photosensitizer, and particularly, in recent years, researchers continuously develop a plurality of fluorescent probes with excellent performance by utilizing the unique optical properties of the hemicyanine dye, and the hemicyanine dye is used for protein marking, gene sequencing and in-vivo fluorescent imaging; or the hemicyanine dye is used as a matrix to design a photosensitizer, and active oxygen is generated under illumination for photodynamic therapy of tumors in organisms.
However, the hemicyanine dyes themselves have limitations, in particular their parent structure is relatively fixed, which results in the maximum absorption wavelength of the hemicyanine dye molecules being limited between 650nm and 720nm and the emission wavelength being limited between 680nm and 750 nm. In the aspects of deep imaging in organisms, photodynamic and the like, due to the problems of autofluorescence in organisms and tissue thickness, the absorption wavelength and the emission wavelength of a required probe and a photosensitizer are longer, and dye molecules which absorb at 720nm or more and emit at 750nm or more are more suitable for application in such scenes. However, in the prior art, no related literature exists, and how to properly modify the parent of the hemicyanine dye is disclosed, so that the hemicyanine dye structure with longer absorption and emission wavelength can be synthesized, and the novel hemicyanine dye has more application prospects, which is also a technical problem to be solved by the technicians in the field.
Disclosure of Invention
In order to solve the problems, the invention provides a novel azaindole-hemicyanine dye, a synthesis method thereof and application of the dye in the fields of biology and medicine.
The first aspect of the present application is to protect a class of azaindole-hemicyanine dyes having the structural formula I:
in the general formula I, the components are shown in the specification,
R 1 and R is 3 Each independently selected from one of hydrogen, halogen, alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, aryl, arylcarboxylic acid, alkylsulfonate, arylsulfonate, alkylsulfonate, or arylsulfonate; most preferably selected from one of hydrogen, halogen, carboxyalkyl having 1 to 8 carbons, and alkylsulfonate;
R 2 、R 4 and R is 5 Each independently selected from the group consisting of hydrogen, alkyl having 1-18 carbons, carboxyalkyl having 1-18 carbons, hydroxyalkyl having 1-18 carbons, alkyl having 1-18 carbonsOne of sulfonate, alkyl sulfonate having 1 to 18 carbons, aryl carboxylic acid, alkyl sulfonate, aryl sulfonate, alkyl sulfonate, or aryl sulfonate; more preferably one of hydrogen, alkyl having 1 to 8 carbons, carboxyalkyl having 1 to 8 carbons, aryl, alkylsulfonate having 1 to 8 carbons, arylcarboxylic acid group; most preferably one of hydrogen, aryl, alkyl having 1 to 8 carbons, alkyl sulfonate having 1 to 8 carbons;
x is selected from one of oxygen, sulfur, selenium and tellurium; most preferably one of oxygen and sulfur;
Y - selected from halogen ions, clO 4 - 、BF 4 - 、CH 3 COO - 、CF 3 COO - Or OTs - One of the following; most preferably halogen ions, clO 4 - One of them.
The second aspect of the present application is directed to a method for synthesizing a class of azaindole-hemicyanine dyes, comprising the steps of:
(1) In an organic solvent at 60-120deg.C, containing R 1 Substituted J-1 and R 2 Substituted haloalkane (N alkylating reagent) reacts for 3-24h, and N-R is obtained through recrystallization 2 Quaternary ammonium salt J-2 substituted for side chain; wherein R is contained in 1 Substituted compounds J-1 and N alkylating agent R 2 The molar ratio of substituted haloalkane is from 1:1 to 10, most preferably from 1:3 to 8;
(2) At 20-80 ℃, S-1 and R are mixed 3 、R 4 、R 5 Dissolving the substituted S-2 in a polar solvent, reacting for 10-24 hours under the catalysis of inorganic base, extracting, concentrating and purifying to obtain the catalyst containing R 3 、R 4 、R 5 Substituted intermediate S-3;
(3) In an organic solvent, R is added at 50-120 DEG C 1 And R is 2 Substituted J-2 and R 3 、R 4 、R 5 Dissolution of substituted S-3 in organic solventIn the agent, condensation reaction is carried out under the catalysis of organic base, and the aza-hemicyanine near infrared fluorescent dye I is obtained through recrystallization and purification.
For the technical scheme described above, preferably, in the step (1), the organic solvent is selected from any one of benzene, toluene, o-dichlorobenzene, DMF; the recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone and propanol;
for the technical scheme described above, preferably, in the step (2), the polar solvent is selected from mixed solvents of one or a combination of several of DMSO, DMF, methanol, acetonitrile; the inorganic base is selected from any one of sodium hydroxide, potassium carbonate, cesium carbonate, sodium acetate and sodium ethoxide;
for the technical scheme described above, preferably, in the step (3), the organic solvent is selected from any one or a combination of mixed solvents of several of ethanol, acetic acid, acetic anhydride and DMF;
for the above-described technical scheme, preferably, in the step (3), the solvent used for recrystallization is selected from a mixed solvent of any one or a combination of several of methanol, ethanol, acetonitrile, water, ethyl acetate, diethyl ether, acetone and propanol; the organic base is selected from any one of triethylamine, pyridine and DIPEA.
A third aspect of the present application consists in protecting the use of said azaindole-hemicyanine dyes in the biological and pharmaceutical fields.
For the technical solutions described above, more preferred application areas include: the azaindole-hemicyanine dye deep in vivo imaging and tumor treatment field; for example: the method can be applied to cell imaging, protein labeling, antibody specific recognition, nucleic acid labeling, fluorescent probes, DNA sequencing and tumor photodynamic therapy.
For the technical scheme, preferably, the excitation wavelength of the azaindole-hemicyanine dye is 600-950nm, and the fluorescence detection wavelength is 650-1000nm.
For the technical scheme, the working concentration of the azaindole-hemicyanine dye is preferably lower than 12 mu mol/L.
Compared with the prior art, the beneficial effect of this application:
1. the azaindole-hemicyanine dye disclosed by the invention has the advantages that the molecular charge separation degree and the electron mobility are increased by changing the position of quaternary ammonium nitrogen and the type of electron donating group, so that the azaindole-hemicyanine dye has longer absorption and emission wavelength.
Compared with the traditional hemicyanine dye, the azaindole-hemicyanine dye has a greatly red shift of the maximum absorption wavelength, and according to the embodiment of the application, the maximum absorption wavelength of the compound 1 is 728nm, the maximum absorption wavelength of the compound 2 is 754nm, and compared with the maximum absorption wavelength 671nm of the traditional hemicyanine dye compound 3, the maximum absorption wavelength of the compound 1 is 57nm and 83nm respectively.
Compared with the traditional hemicyanine dye, the azaindole-hemicyanine dye has a large red shift in maximum emission wavelength, and according to the embodiment of the application, the maximum emission wavelengths of the compound 1 and the compound 2 are respectively red-shifted by 45nm and 58nm compared with the maximum emission wavelength 719nm of the compound 3 (the traditional hemicyanine dye).
The dye is more suitable for being applied to scenes of long-wavelength absorption and emission, and can be well applied to the fields of deep in-vivo imaging and tumor treatment.
2. Compared with the traditional semi-cyanine dye with changed photophysical properties and good biocompatibility, the dye provided by the invention still shows good survival rate after MCF-7 cells are cultured for 48 hours by using the compound 1 and the compound 2 with different concentrations, and even if the concentration is increased to 12 mu mol/L, the survival rate of the cells is still high, so that the azaindole-semi-cyanine dye has good biocompatibility and can not generate toxic or side effect on the cells in a working concentration range; can be applied to aspects such as biological identification imaging, cell imaging, protein labeling, antibody specific identification, nucleic acid labeling, fluorescent probes, DNA sequencing, tumor photodynamic therapy and the like.
Drawings
FIG. 1 is a high resolution mass spectrum of Compound 1;
FIG. 2 is a high resolution mass spectrum of Compound 2;
FIG. 3 is a graph of normalized absorption spectra of Compound 1, compound 2, and Compound 3 in methanol;
FIG. 4 is a graph of normalized fluorescence spectra of Compound 1, compound 2, and Compound 3 in methanol;
FIG. 5 is a graph of MTT assay for Compound 1 and Compound 2.
Detailed Description
The present invention will be described in further detail below.
Unless otherwise indicated, the terms used herein have the following meanings.
The term "halogen" as used herein includes fluorine, chlorine, bromine and iodine.
The term "alkyl" as used herein includes both straight chain alkyl and branched alkyl groups.
The term "MTT" as used herein refers to a method of detecting cell survival and growth.
Y is used herein - Represents anions, which may be any suitable anions, including inorganic anions and organic anions, such as but not limited to halide, clO 4 - 、PF 6 - 、BF 4 - 、CH 3 COO - 、CF 3 COO - Or OTs - 。
Instruments and devices employed in the examples:
in the column chromatography process, 200-300 mesh column chromatography silica gel purchased from Qingdao Megao group Co., ltd., 100-200 mesh column chromatography silica gel and 20-40 mesh analytically pure quartz sand purchased from Tianda chemical reagent factory are adopted.
In the process of detecting the compound, a mass spectrometer adopts a Synta G2-Si HDMS high-resolution mass spectrometer of Waters company in the United states, and adopts a double-needle electrospray ion source to detect the positive and negative modes of the compound.
Dye absorption and emission spectra were measured using a Cary 60 UV visible spectrophotometer and a Cary Eclipse fluorescence spectrophotometer from Agilent corporation.
Cytotoxicity assays were measured using a Varioskan LUX Multimode Microplate Reader instrument from thermofsher, usa.
EXAMPLE 1 production of Compound 1
example 1.1
To 2-hydrazinopyridine (2.182 g,20 mmol) dissolved in 60mL of toluene was added 3-methyl-2-butanone (3.4475 g,40 mmol) at room temperature. Stirring, heating and refluxing under the protection of nitrogen, and stopping the reaction after reacting for 12 hours. Cooled to room temperature. Removing most of toluene, adding 12mL of polyphosphoric acid to the residue, heating and stirring at 140 ℃ for 45min, pouring the mixture into 200mL of ice water, adding dropwise ammonia water, adjusting pH to be slightly alkaline, extracting with ethyl acetate and extracting with anhydrous Na 2 SO 4 Drying and solvent evaporation followed by purification on a silica gel column gave a nitrogen yellow solid compound 1.1 (1.280 g,8mmol, y=40%).
Example 1.2
Compound 1.1 (0.640 g,4mmol,1.0 eq) was added to 15mL of o-dichlorobenzene, 1, 3-propanesultone (0.977 g,8mmol,2.0 eq) was added, the reaction was stopped at 65℃for 12h, cooled to room temperature, 100mL of ethyl acetate was added, and the precipitate was collected to give Compound 1.2 (0.960 g,3.4mmol, Y=85%).
Example 1.3
9mL of phosphorus tribromide was slowly added dropwise to a mixed solution of 12mL of DMF and 25mL of chloroform at 0℃and, after stirring for 1h, the temperature was raised to room temperature, 4mL of cyclohexanone was added and the reaction was stopped after stirring for 12 h. The solution was introduced into 100mL ice water and NaHCO was added 3 The solution was neutralized with CH 2 Cl 2 Extraction, washing with saturated saline solution, anhydrous Na 2 SO 4 Drying and evaporation of the solvent gave the title compound 1.3 (6.0 g,31.74mmol, y=73.8%).
Example 1.4
Compound 1.1 (2.93 g,15.5mmol,1.0 eq) and 4-diethylaminosalicylaldehyde (3.0 g,15.5mmol,1.0 eq) were added to 15mL of DMF at room temperature, cesium carbonate (5.05 g,15.5mmol,1.0 eq) was added and the reaction stopped after stirring for 16h, the solution was poured into 100mL of water with CH 2 Cl 2 Extraction, washing with saturated saline solution, anhydrous Na 2 SO 4 The organic phase was dried, concentrated and purified by silica gel column to give yellow-brown compound 1.4 (1.4 g,4.94mmol, y=31.9%).
Production of Compound 1
Compound 1.2 (0.100 g,0.35mmol,1 eq) and compound 1.4 (0.149 g,0.53mmol,1.5 eq) were dissolved in 10mL acetic anhydride at 100 ℃, 0.2mL of triethylamine was added to catalyze the reaction, the reaction was stopped after stirring for 2h, the reaction solution was added dropwise to 150mL of ethyl acetate after cooling to room temperature, and the obtained crude product was recrystallized by silica gel column to obtain compound 1 (0.068 g,0.12mmol, y=35.2%), and the high resolution mass spectrum was shown in fig. 1.
EXAMPLE 2 production of Compound 2
example 2.1
To 2-hydrazino-4-bromopyridine (1.00 g,5.32mmol,1.0 eq) dissolved in 20mL of toluene was added 3-methyl-2-butanone (0.92 g,10.64mmol,2.0 eq) at room temperature. Stirring, heating and refluxing under the protection of nitrogen, and stopping the reaction after reacting for 12 hours. Cooled to room temperature. Removing most of toluene, adding 12mL of polyphosphoric acid into the residue, heating and stirring at 140 ℃ for 45min, pouring the mixture into 200mL of ice water, dropwise adding ammonia water, adjusting pH to be slightly alkaline, extracting with ethyl acetate and extracting with Na 2 SO 4 Drying and solvent evaporation followed by purification on a silica gel column gave a nitrogen yellow solid compound 2.1 (0.760 g,3.18mmol, y=59.8%).
Example 2.2
To 15mL of acetonitrile was added compound 2.1 (0.500 g,2.09mmol,1.0 eq), benzyl bromide (0.710 g,4.18mmol,2.0 eq) was added, the reaction was stopped at 70℃for 12h, the reaction was cooled to room temperature, added to 100mL of ethyl acetate, and the precipitate was collected to give compound 2.2 (0.610 g,1.49mmol, Y=71.1%).
Production of Compound 2
Compound 1.2 (0.100 g,0.35mmol,1 eq) and compound 2.2 (0.217 g,0.53mmol,1.5 eq) were dissolved in 10mL acetic anhydride at 100 ℃, 0.2mL of triethylamine was added to catalyze the reaction, the reaction was stopped after stirring for 2h, after the reaction solution cooled to room temperature, it was added dropwise to 150mL of ethyl acetate to recrystallize, and the crude product obtained was purified by silica gel column to give compound 2 (0.095 g,0.14mmol, y=39.8%), high resolution mass spectrum was shown in fig. 2.
EXAMPLE 3 production of Compound 3
The structural formula of compound 3 is as follows: for use as a comparative example
Example 3.1
4-iodophenylhydrazine (2.00 g,8.55mmol,1 eq) and 3-methyl-2-butanone (1.47 g,17.09mmol,2 eq) were dissolved in 20mL acetic acid at room temperature, heated under reflux under nitrogen for 12h, cooled to room temperature after stopping the reaction, the majority of the solvent was removed under reduced pressure, and the remaining solution was poured into saturated NaHCO 3 In aqueous solution, extracted with ethyl acetate and extracted with Na 2 SO 4 Drying and purification by evaporation of the solvent followed by column chromatography on silica gel gave compound 3.1 as a yellowish brown color (1.85 g,6.49mmol, y=75.9%).
Example 3.2
To 15mL of acetonitrile was added compound 3.1 (0.500 g,1.75mmol,1.0 eq), methyl iodide (1.37 g,8.77mmol,5.0 eq) was added, the reaction was stopped at 70℃for 12 hours, cooled to room temperature, added to 100mL of diethyl ether, and the precipitate was collected to give compound 3.2 (0.58 g,1.31mmol, Y=75.0%).
Example 3.3
Compound 1.1 (2 g,10.58mmol,1.0 eq) and 4-methoxysalicylaldehyde (1.61 g,10.58mmol,1.0 eq) were added to 15mL of DMF at room temperature, cesium carbonate (2 g,10.58mmol,1.0 eq) was added as stirring for 16h and the reaction stopped, the solution was poured into 100mL of water using CH 2 Cl 2 Extraction, washing with saturated saline solution, anhydrous Na 2 SO 4 Drying, concentrating the organic phase, dissolving the crude product in 20mL of dichloromethane, dropwise adding 5mL of boron tribromide in an ice bath, stirring at room temperature for 12h, pouring the solution into ice water, adding NaHCO 3 The aqueous solution is adjusted to be neutral by acetic acidEthyl ester extraction and use of Na 2 SO 4 Drying and purification by evaporation of the solvent followed by column chromatography on silica gel gave compound 3.3 as a yellowish brown color (0.75 g,3.10mmol, y=29.3%).
Production of Compound 3
Compound 3.3 (0.100 g,0.438mmol,1 eq) and compound 3.2 (0.900 g,0.6570mmol,1.5 eq) were dissolved in 10mL acetic anhydride at 100deg.C, and 0.2mL triethylamine was added to catalyze the reaction, the reaction was stopped after stirring for 2h, and after cooling to room temperature, the reaction solution was added dropwise to 150mL ethyl acetate and recrystallized, the crude product obtained was purified by silica gel column to give compound 3 (0.088 g,0.135mmol, Y=30.8%).
Example 4 UV-visible absorption Spectrometry and fluorescence Spectrometry of Compounds 1, 2 and 3
Precisely weighing the dye subjected to vacuum drying by using a ten-thousandth balance, preparing 5mmol/L DMSO dye mother solution into a brown sample bottle, and storing in a refrigerator at 4 ℃ for later use.
When testing ultraviolet visible absorption spectrum and fluorescence spectrum, 1.2 mu L of dye mother liquor is measured by a micropipette, and is dissolved in a quartz cuvette containing 3mL of solvent to be tested, and the mixture is uniformly mixed to obtain the dye with the concentration of 2.0 mu mol/L for testing the absorption spectrum and the fluorescence emission spectrum. All tests were completed at 25 ℃.
As shown in fig. 3, in methanol solution, the conventional hemicyanine dye compound 3 had a maximum absorption wavelength of 671nm, and compound 1 had a maximum absorption wavelength of 728nm, and compound 2 had a maximum absorption wavelength of 754nm, which were red-shifted by 57nm and 83nm, respectively, as compared with compound 3. The maximum absorption wavelength of the novel azaindole-hemicyanine dye is greatly red-shifted compared with that of the traditional hemicyanine dye.
As shown in FIG. 4, the maximum emission wavelengths of the compound 1 and the compound 2 in methanol are 764nm and 777nm, compared with the maximum emission wavelength 719nm of the compound 3, the maximum emission wavelengths are respectively red shifted by 45nm and 58nm, which shows that the novel azaindole-hemicyanine dye is greatly red shifted compared with the maximum emission wavelength of the traditional hemicyanine dye, so that the dye is more suitable for being applied to scenes of long wavelength absorption and emission, and can be well applied to the fields of deep in-vivo imaging and tumor treatment.
Example 5 cytotoxicity experiments of Compounds 1 and 2
Toxicity of dye molecules to cells was assessed by MTT assay. The principle is as follows: succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water insoluble blue-violet crystalline Formazan (Formazan) and deposits in cells, whereas dead cells do not. Dimethyl sulfoxide (DMSO) can dissolve formazan in cells, and the light absorption value can be measured at 570nm wavelength by using an enzyme-labeled instrument, so that the number of living cells can be indirectly reflected.
MCF-7 cells are inoculated in a 96-well plate, after a period of culture, compound 1 or compound 2 with a certain concentration is respectively added into different wells, the compound concentration is respectively 0-12 mu mol/L, and after the cells are continuously incubated for 48 hours, the cell activity is detected through an MTT experiment. Experimental data as shown in fig. 5, after MCF-7 cells were cultured for 48 hours using different concentrations of compound 1 and compound 2, the cells still showed good viability, even though the concentration was increased to 12 μmol/L, the viability of the cells was still very high, indicating that the azaindole-hemicyanine dye has very good biocompatibility, and no toxic or side effect was generated to the cells in the working concentration range, thus being applicable to the fields of biology and medicine.
The foregoing is a further description of the invention in connection with specific preferred embodiments thereof, and is not intended to limit the practice of the invention to such description. It is intended that all such variations and modifications as would be included within the scope of the invention are within the scope of the following claims.
Claims (6)
1. An azaindole-hemicyanine dye, which has a structural formula as shown in a general formula I:
in the general formula I, the components are shown in the specification,
R 1 and R is 3 Each independently selected from hydrogenOne of halogen, carboxyalkyl having 1 to 8 carbons;
R 2 、R 4 and R is 5 Each independently selected from one of hydrogen, alkyl having 1-8 carbons, alkyl sulfonate having 1-8 carbons;
x is selected from oxygen or sulfur;
Y - selected from halogen ions or ClO 4 - 。
2. The method for synthesizing azaindole-hemicyanine dye as claimed in claim 1, wherein: the method comprises the following synthesis steps:
(1) In an organic solvent at 60-120deg.C, containing R 1 Substituted J-1 and R 2 Substituted halogenated alkane reacts for 3-24h, and N-R is obtained after recrystallization 2 Quaternary ammonium salt J-2 substituted for side chain; wherein R is contained in 1 Substituted J-1 and R 2 The molar ratio of the substituted halogenated alkane is 1:1-10;
(2) At 20-80 ℃, S-1 and R are mixed 3 、R 4 、R 5 Dissolving the substituted S-2 in a polar solvent, reacting for 10-24 hours under the catalysis of inorganic base, extracting, concentrating and purifying to obtain the catalyst containing R 3 、R 4 、R 5 Substituted intermediate S-3;
(3) In an organic solvent, R is added at 50-120 DEG C 1 And R is 2 Substituted J-2 and R 3 、R 4 、R 5 The substituted S-3 is dissolved in an organic solvent, and is subjected to condensation reaction under the catalysis of organic base, and is subjected to recrystallization and purification to obtain the azahemicyanine near infrared fluorescent dye I.
3. The synthesis method according to claim 2, characterized in that: in the step (1), the organic solvent is selected from benzene, toluene, o-dichlorobenzene and DMF; the recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, ethyl acetate, diethyl ether, acetone and propanol;
in the step (2), the polar solvent is selected from mixed solvents of one or more of DMSO, DMF, methanol and acetonitrile; the inorganic base is selected from any one of sodium hydroxide, potassium carbonate, cesium carbonate, sodium acetate and sodium ethoxide;
in the step (3), the organic solvent is selected from any one or a mixture of several of ethanol, acetic acid, acetic anhydride and DMF; the recrystallization solvent is selected from any one or a mixture of several of methanol, ethanol, acetonitrile, water, ethyl acetate, diethyl ether, acetone and propanol; the organic base is selected from any one of triethylamine, pyridine and DIPEA.
4. The synthesis method according to claim 2, characterized in that: in the above step (1), R is contained 1 Substituted J-1 and R 2 The molar ratio of the substituted haloalkane is 1:3-8.
5. Use of an azaindole-hemicyanine dye as claimed in claim 1, in the biological and pharmaceutical fields, characterized in that: the application comprises the application of azaindole-hemicyanine dye in cell imaging, protein labeling, specific identification of antibodies, nucleic acid labeling and DNA sequencing for the purpose of non-disease diagnosis and treatment.
6. The use according to claim 5, wherein: the excitation wavelength of the azaindole-hemicyanine is 600-950nm, and the fluorescence detection wavelength is 650-1000nm.
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