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CN114437057A - Fluorescent dye and preparation method and application thereof - Google Patents

Fluorescent dye and preparation method and application thereof Download PDF

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CN114437057A
CN114437057A CN202011196170.3A CN202011196170A CN114437057A CN 114437057 A CN114437057 A CN 114437057A CN 202011196170 A CN202011196170 A CN 202011196170A CN 114437057 A CN114437057 A CN 114437057A
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compound
alkyl
fluorescent dye
carboxyl
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冯剑军
王译责
宋莲
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Shenzhen Reetoo Biotechnology Co Ltd
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    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
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    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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    • C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/104—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with other heteroatoms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/302—Stain compositions

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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

The application discloses a fluorescent dye and a preparation method and application thereof. The fluorescent dye has an asymmetric structure shown as a formula I, wherein R is used for increasing the fluorescence intensity5The substituent can enhance the stability of the overall structure of the fluorescent dye. The fluorescent dye has good dyeing effect on nucleic acid, leucocyte, nucleated erythrocyte and reticulocyte, can emit stable fluorescence in a near infrared region, can avoid the fluorescence background interference generated by organisms, and improves the accuracy of measurementThe kit has high sensitivity and sensitivity, and can be widely applied to flow cytometry classification and blood cell analysis. The fluorescent dye provides a novel dyeing scheme and a novel dyeing way with small fluorescent background interference and good light stability for dyeing a biological sample.

Description

Fluorescent dye and preparation method and application thereof
Technical Field
The application relates to the technical field of organism dyeing, in particular to a fluorescent dye and a preparation method and application thereof.
Background
Staining of organisms mainly refers to staining cells or nucleic acids in cells with fluorescent dyes. Currently, commonly used fluorescent dyes include new methylene blue, brilliant toluene blue, ethidium bromide, and the like; the fluorescent dyes can form a complex by combining with nucleic acid molecules, and obvious fluorescence can be observed under a microscope; however, the fluorescent dye itself can also form a fluorescent complex, i.e., the fluorescent dye itself has background fluorescence, which causes strong fluorescence interference and reduces the detection accuracy.
At present, a kind of red excitation fluorescent dye exists, and the fluorescent dye with the structure can mark and dye RNA and/or DNA in cells. However, in the specific application process, the fluorescent dye has poor light stability and is difficult to meet the requirements of large-scale production and application.
In conclusion, the existing fluorescent dyes generally have the problems of background fluorescence interference, poor light stability and the like. How to reduce the background fluorescence of the fluorescent dye and improve the light stability of the fluorescent dye still remains the research focus and difficulty of the organism dyeing technology.
Disclosure of Invention
The application aims to provide a novel fluorescent dye and a preparation method and application thereof.
In order to achieve the purpose, the following technical scheme is adopted in the application:
the first aspect of the application discloses a fluorescent dye, which has a structure shown as a formula I; is like
Figure BDA0002754075770000011
Wherein X is C (CH)3)2、C(C2H5)2、CH3CC2H5O, S or Se;
R1and R4Each independently selected from H, OH, OR7Mercapto group, carboxyl group, carbonyl group, C1-C18Alkenyl radical, C1-C18Alkynyl, cyano, nitro, amino, trifluoromethyl, difluoroethyl, trifluoromethoxy, sulfonic acid, phosphoramidite, phosphonic acid, C1-C18Alkyl radical, C1-C8Alkyl OR6、C1-C18Alkylsulfonic acid group, benzyl group, phenyl group, furyl group, thienyl group, thiazolyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, oxazolyl group, isoxazolyl group, triazolyl group, tetrazolyl group, oxadiazolyl group, pyrazinyl group, pyrimidinyl group, pyridazinyl group, pyridyl group, quinolyl group, indolyl group, acridinyl group, C1-C4Alkyl pyridyl radical, C1-C18Haloalkyl, C1-C18Alkyl phosphate group or C1-C18Alkyl SR6Wherein benzyl is substituted benzyl optionally substituted with the following substituents: a boronic acid group, a hydroxyl group, a halogen, a mercapto group, a cyano group, a nitro group, an alkyl group, an aryl group, an alkoxy group, an amide group, a carboxyl group, a sulfonic acid group, an amino group, a heterocyclic group, and an alkylamino group;
R2and R3Each independently selected from H, C1-C18Alkyl group COOR7、C1-C18Alkyl OR7、C1-C18Alkyl NHR6、C1-C18Alkyl N (R)7)(R7)、C1-C18Alkylsulfonic acid group, C1-C18Alkyl phosphoric acid group, C1-C18Alkylamide group, C1-C18Alkenyl radical, C1-C18Alkynyl, C1-C18Alkyl, sulfonic acid group, carbonyl group, carboxyl group, amino group, phenyl group, phenylboronic acid group, pyridyl group, cyano group, or benzyl group; wherein benzyl is a substituted benzyl group optionally substituted with the following substituents: hydroxyl, halogen, mercapto, cyano, nitro, alkyl, aryl, alkoxy, amide, carboxyl, sulfonic, amino, heterocyclic, and alkylamino;
R5selected from OH, OR7Mercapto, carboxyl, carbonyl, C1-C18Alkenyl radical, C1-C18Alkynyl, cyano, nitro, amino, trifluoromethyl, difluoroethyl, trifluoromethoxy, sulfonic acid, phosphoramidite, phosphonic acid, C1-C18Alkyl radical, C1-C8Alkyl OR6、C1-C18Alkylsulfonic acid group, benzyl group, phenyl group, furyl group, thienyl group, thiazole groupA group selected from the group consisting of an alkyl group, an aryl group, a cycloalkyl group, an aryl group, a cycloalkyl group, an arylalkyl group, a cycloalkyl group, a heterocycloalkyl group, a tetrazolyl group, an oxadiazolyl group, a tetrazolyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a pyridyl group, a quinolyl group, an indolyl group, an acridinyl group, a tetrazolyl group, a pyrazinyl group, a pyrimidinyl group, a pyridazinyl group, a pyridyl group, a quinolyl group, an indolyl group, an acridinyl group, a pyridyl group, a tetrazolyl group, a pyridyl group, a salt thereof, and a salt thereof1-C4Alkyl pyridyl radical, C1-C18Haloalkyl, C1-C18Alkyl phosphate group or C1-C18Alkyl SR6Wherein benzyl is substituted benzyl optionally substituted with the following substituents: a boronic acid group, a hydroxyl group, a halogen, a mercapto group, a cyano group, a nitro group, an alkyl group, an aryl group, an alkoxy group, an amide group, a carboxyl group, a sulfonic acid group, an amino group, a heterocyclic group, and an alkylamino group;
R6is C1-C18Alkyl, H, carboxyl, C1-C4Carboxyl group, amino group, hydroxyl group, ether group derivative, C1-C6Ether group or C1-C6An ether group derivative;
R7is C1-C18Alkyl, H, carboxyl, C1-C18An alkylcarboxyl, amino, hydroxyl, ether or phenyl group; wherein the phenyl is a substituted phenyl optionally substituted with the following substituents: hydroxyl, halogen, mercapto, cyano, nitro, alkyl, aryl, alkoxy, heterocyclyl, haloalkyl, amino, alkylamino, amidoamino, carbonyl, carboxyl, phosphate, and phosphite;
Y-is the single existence of negative ions or the negative ions carried by the functional groups.
The fluorescent dye of the present application has an asymmetric structure and is represented by R5The substituent can enhance the stability of the whole structure of the fluorescent dye, so that the light stability of the fluorescent dye is improved; the fluorescent dye has good dyeing effect on nucleic acid, leucocyte, nucleated erythrocyte, reticulocyte, lymphocyte, monocyte, neutrophil, eosinophil and mature erythrocyte; can emit stable fluorescence in a near infrared region, can avoid fluorescence background interference generated by organisms, improves the accuracy and sensitivity of measurement, and can be widely applied to flow cell classification and blood cell analysis. And, atIn one implementation of the present application, R1、R2、R3、R4When the groups have hydroxyl or other water-soluble groups, the water solubility of the dye can be increased, and the dye is convenient to use in an aqueous system.
Preferably, R1、R4And R5Each independently selected from H, C1-C18Alkenyl radical, C1-C18Alkynyl, C1-C18Alkyl radical, C1-C8Alkyl OR6、C1-C18Alkyl sulfonic acid group and benzyl group, wherein the benzyl group is a substituted benzyl group which is optionally substituted by the following substituents: hydroxyl, halogen, alkyl, aryl, alkoxy, carboxyl, sulfonic acid, and amino.
In addition, R is as defined above1、R4And R5The preferred group can improve the light stability of the fluorescent dye and simultaneously obtain better water solubility, thereby facilitating the fluorescent dye to be used in an aqueous system.
Preferably, R2And R3Each independently selected from H, C1-C18Alkyl group COOR7、C1-C18Alkyl OR7、C1-C18Alkenyl radical, C1-C18Alkynyl, C1-C18Alkyl or benzyl, wherein benzyl is substituted benzyl optionally substituted with the following substituents: hydroxyl, halogen, alkyl, aryl, alkoxy, carboxyl, sulfonic acid, and amino.
In addition, R is as defined above2And R3Can further enhance the water solubility of the fluorescent dye, thereby facilitating the use of the fluorescent dye in aqueous systems.
Preferably, R6Is C1-C18An alkyl group.
R7Is C1-C18Alkyl radical H, C1-C18Alkyl carboxyl or phenyl, wherein phenyl is substituted phenyl optionally substituted with the following substituents: hydroxyl, halogen, alkyl, aryl, alkoxy, carbonyl, and carboxyl.
Note that, the above isR6And R7Can further enhance the water solubility of the fluorescent dye, thereby facilitating the use of the fluorescent dye in an aqueous system.
In one implementation of the present application, Y-Is halogen anion, BF4 -、ClO4 -、IO4 -At least one of sulfonic acid group anions and alkylsulfonic acid group anions.
In one implementation of the present application, X is C (CH)3)2S or Se; and/or, R1And R2Each independently selected from C1-C6Alkyl radical, C1-C3Alkylsulfonic acid groups, sulfonic acid groups, carbonyl groups, carboxyl groups, amino groups, phenyl groups; and/or, R3And R4Each independently selected from C1-C6Alkyl radical, C1-C4Alkyl OR6、C1-C4Alkyl group COOR7Phenylboronic acid group, C1-C4Alkylsulfonic acid group, sulfonic acid group, benzyl group, phenyl group, C1-C3Alkenyl radical, C1-C3Alkynyl, pyridyl or cyano, and R6Is C1-C6Alkyl or C1-C4Carboxyl or H or C1-C6Ether group or C1-C6An ether group derivative; and/or, R5Is selected from C1-C6Alkyl, benzyl, phenyl, C1-C3Alkenyl radical, C1-C4Ether group, furyl group, C1-C4Alkyl pyridyl, triazolyl, C1-C4Alkyl OR6And R is6Is C1-C6Alkyl or H or C1-C6Ether group or C1-C6An ether group derivative; and/or, Y-Selected from Cl-、Br-、I-、BF4 -、ClO4 -At least one of sulfonic acid group anion and alkyl sulfonic acid group anion.
In one implementation of the present application, R1And R2Same, and/or R3And R4Different.
In one implementation of the present application, the fluorescent dye is any one of compound a to compound Z, and compound XA, compound XB.
A second aspect of the present application discloses a conjugate having a fluorescent dye of the present application attached thereto.
It should be noted that the conjugate of the present application refers to a compound formed by linking a fluorescent dye to other molecules through covalent bonds; in general, molecules capable of linking to the fluorochromes of the present application include proteins or peptide fragments of various functions, or nucleic acid molecules, such as DNA, RNA, etc. The conjugates of the present application may have different functions and effects depending on the molecule to which they are attached; for example, conjugates formed by linking proteins that specifically bind to cells or cellular components can be used for cell staining, and thus for fluorescence activated cell sorters or solid phase immunoassays; among them, proteins include, but are not limited to, antibodies. Antigens, receptors, ligands, enzymes, coenzymes, and the like. For another example, a conjugate formed by connecting DNA or RNA can achieve target localization or detection of nucleic acid, etc., depending on the specificity of DNA or RNA.
A third aspect of the present application discloses a kit for staining a biological sample, comprising a fluorescent dye according to the present application or a conjugate according to the present application.
It should be noted that the fluorescent dye of the present application has good permeability to cell membranes and good and stable staining effect to both DNA and RNA of organisms, and thus can be used for staining biological samples. Likewise, the conjugates of the present application may also be used for cell staining or nucleic acid staining and thus also for staining of biological samples. It will be appreciated that the kit of the present application, which is critical to staining a biological sample with the fluorochrome or conjugate of the present application, may also contain other reagents necessary for staining, and is not particularly limited herein.
A fourth aspect of the present application discloses a cell mimetic particle for use in a fluorescent staining assay, which is a cell, RNA or DNA stained with a fluorescent dye of the present application or a conjugate of the present application; wherein the cell comprises at least one of lymphocyte, monocyte, neutrophil, eosinophil, basophil, erythrocyte, reticulocyte, nucleated erythrocyte and platelet.
In one embodiment of the present invention, the cell-stimulating particle can stimulate the fluorescence and volume properties of other blood cells by staining red blood cells of a specific volume size, and can be used as a control or calibrator of a blood analyzer.
The fifth aspect of the application discloses a method for classifying and detecting cells in a blood sample, which comprises the steps of dyeing the blood sample by adopting the fluorescent dye, obtaining a scatter diagram according to a dyeing signal, and classifying and/or counting the cells in the blood sample according to the distribution of the scatter diagram; the cell comprises at least one of lymphocyte, monocyte, neutrophil, eosinophil, basophil, erythrocyte, reticulocyte, nucleated erythrocyte and platelet.
The fluorescent dye of the present application can be used as a nucleic acid staining agent in flow cytometry, and can improve the detection accuracy, and is particularly suitable for staining leukocytes, nucleated erythrocytes, and reticulocytes. Therefore, the fluorescent dye based on the present application can perform cell classification and detection on blood samples.
The sixth aspect of the application discloses a method for synthesizing the fluorescent dye, which comprises the steps of mixing a compound with a structure shown in a formula II and a compound with a structure shown in a formula III, and heating to react to obtain the fluorescent dye with a structure shown in a formula I;
formula II
Figure BDA0002754075770000051
Formula III
Figure BDA0002754075770000052
X, R in formula II1、R3And R5And Y in formula III-、R2And R4Are all the same as the formula one.
In one implementation mode of the application, the heating is carried out for 2-5 hours by adopting oil bath; and after the heating in the oil bath, further cooling the product of the heating reaction to room temperature, then adding the reaction product into diethyl ether or methyl tert-butyl ether to separate out the solid, filtering to obtain the solid, and then continuously washing and drying to obtain the required fluorescent dye. In another implementation manner of the present application, the method further comprises performing column chromatography purification or recrystallization on the solid obtained by filtration to obtain the fluorescent dye with higher purity.
In the synthesis method of the present application, both the compound having the structure represented by formula two and the compound having the structure represented by formula three can be obtained by commercial purchase; wherein, the compound of the formula II can also be prepared by the following synthetic route:
Figure BDA0002754075770000053
due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
the fluorescent dye can well dye biological samples such as nucleic acid, cells and the like, has less background interference, high sensitivity and good light stability; when the method is applied to an instrument with a flow cytometry principle, parameters required by measurement can be met. The fluorescent dye provides a novel dyeing scheme and a novel dyeing way with small fluorescent background interference and good light stability for dyeing a biological sample.
Drawings
FIG. 1 is a graph showing the results of an absorbance test of a fluorescent dye prepared in example one of the present application;
FIG. 2 is a graph showing the results of photostability test of a fluorescent dye prepared in example one of the present application;
FIG. 3 is a graph showing the results of a cell-staining classification test using a fluorescent dye of Compound A in example one of the present application;
FIG. 4 is a graph showing the results of a classification test of cell staining using a fluorescent dye of Compound B in example one of the present application;
FIG. 5 is a graph showing the results of a classification test of cell staining using a fluorescent dye of Compound C in example one of the present application;
FIG. 6 is a graph showing the results of a cell-staining classification test using a fluorescent dye of Compound D in example one of the present application;
FIG. 7 is a graph showing the results of a differential cell-staining assay using a fluorescent dye of Compound E in example one of the present applications;
FIG. 8 is a graph showing the results of a differential cell-staining assay using fluorescent dye of Compound F in example I of the present application;
FIG. 9 is a graph showing the results of a classification test of cell staining using a fluorescent dye of Compound G in example one of the present application;
FIG. 10 is a graph showing the results of a differential detection of cell staining using the fluorescent dye of control 1 in the first example of the present application;
FIG. 11 is a graph showing the results of a differential detection of cell staining using fluorescent dye of control 2 in example one of the present application;
FIG. 12 is a graph showing the results of the classification and detection of cell staining using the fluorescent dye of control 3 in the first example of the present application.
Detailed Description
Conventional fluorescent dyes, for example, the fluorescent dyes described in patent nos. CN101231243B, CN101349644B, and CN101743469B, generally have a problem of poor light stability. According to the fluorescent dye, a series of researches are carried out on cyanine dyes, and a novel fluorescent dye which is good in light stability and has a good and stable dyeing effect on nucleic acid, namely, the fluorescent dye with the structure shown in the formula I is finally developed.
Compared with other existing fluorescent dyes, the fluorescent dye has the following advantages:
(1) has good permeability to cell membrane, and has good and stable dyeing effect on DNA of organism.
(2) The product has high light stability and has good and stable dyeing effect on DNA or RNA.
(3) The self background interference of organisms can be avoided, and the accuracy and precision of detection are improved, for example, experiment 3 in the first embodiment of the application proves that the fluorescent dye can accurately distinguish and detect various white blood cells, the boundaries among the various white blood cells are clear and obvious, and the accuracy and precision of detection are improved.
(4) The dye can be used as a nucleic acid dye in the flow cytometry analysis technology, improves the detection precision, and is particularly suitable for dyeing white blood cells, nucleated red blood cells and reticulocytes.
The present application is described in further detail below with reference to specific embodiments and the attached drawings. The following examples are intended to be illustrative of the present application only and should not be construed as limiting the present application. In the following examples, all reagents, materials and equipment were commercially available unless otherwise specified.
Example one
In this example, compound a, compound B, compound C, compound D, compound E, compound F, and compound G were synthesized, and seven fluorescent dyes were used to perform an absorbance test and a light stability test, and seven fluorescent dyes were used to dye cells, respectively, to perform a cell classification test. Meanwhile, in this example, the same cells were stained by using fluorescent dyes of patents CN101743469B, CN101231243B and CN101349644B as a contrast, and a cell classification test was performed; the cell classification detection effect of the fluorescent dye synthesized in the example and the existing fluorescent dye is compared and analyzed. The method comprises the following specific steps:
preparation of Compound A
Figure BDA0002754075770000071
Adding 20mL of acetic anhydride into a clean three-neck flask, sequentially adding 10mmol of benzothiazole quaternary ammonium salt bromide and 10.1mmol of quinoline quaternary ammonium salt bromide, heating in an oil bath for 2h, and cooling to room temperature after TLC (thin layer chromatography) detection raw materials completely disappear; pouring the reaction solution into 250mL of diethyl ether, stirring for 30min, and separating out a solid; the solid obtained by filtration is washed by ether and dried to obtain a bluish violet solid product.
The structure was confirmed by nuclear magnetic and mass spectrometry, confirming that the synthesized product was compound a in 67.4% yield.
The nuclear magnetic data are as follows:
1H NMR:(400MHZ，CD3OD,TMS):0.9(m,6H),1.30(m,4H),1.52(m,4H)1.9-2.5(m,6H),3.6(m，2H),3.8(m,2H),5.12(t,2H),5.5(d,1H),6.4(s,1H),6.75(m,3H),7.48(d,3H),8.06(m,1H),8.21(m,1H),8.7(d,2H)
EI data are: MS (EI) C29H35BrN2OS m/z：461.3[M-Br]+
Preparation of Compound B
Figure BDA0002754075770000081
Adding 20mL of acetic anhydride into a clean three-neck flask, sequentially adding 10mmol of benzothiazole quaternary ammonium salt bromide and 10.1mmol of N-hydroxyethyl quinoline quaternary ammonium salt bromide, heating in an oil bath for 3.5h, and cooling to room temperature after TLC detection raw materials completely disappear; pouring the reaction solution into 250mL of diethyl ether, stirring for 30min, and separating out a solid; the solid obtained by filtration is washed with ethyl ether-ethyl acetate and dried to obtain a dark purple solid product.
The structure was confirmed by nuclear magnetic and mass spectrometry, confirming that the synthesized product was compound B in 56.8% yield.
The nuclear magnetic data are as follows:
1H NMR:(400MHZ,CD3OD,TMS):3.11(m.2H),3.74(m,2H),5.8(d,1H),6.04(s,1H),6.5-6.7(m,4H),7.2(m,2H),7.3-7.7(m,11H),8.0(m,1H),8.31(m,1H),8.6-8.8(m,2H)
EI data are: MS (EI) C34H29BrN2OS m/z：515.6[M-Br]+
Preparation of Compound C
Figure BDA0002754075770000082
Adding 20mL of acetic anhydride into a clean three-neck flask, sequentially adding 10mmol of benzothiazole quaternary ammonium salt bromide and 10.1mmol of quinoline quaternary ammonium salt bromide, heating in an oil bath for 3.5h, and cooling to room temperature after TLC (thin layer chromatography) detection raw materials completely disappear; pouring the reaction solution into 300mL of diethyl ether, stirring for 30min, and separating out a solid; filtering to obtain solid, drying, and purifying by column chromatography to obtain a purple solid product.
The structure was confirmed by nuclear magnetic and mass spectrometry, confirming that the synthesized product was compound C in 61.1% yield.
The nuclear magnetic data are as follows:
1H NMR:(400MHZ,CD3OD,TMS):1.4-1.7(m,6H),2.0-2.2(m,4H),2.6-3.1(m,4H),3.82(m,2H),5.4(m,2H),5.7(d,1H),6.62(s,1H),6.72(m,3H),7.4(m,3H),8.11(m,1H),8.34(m,1H),8.5-8.8(m,2H)
EI data are: MS (EI) C28H34N2O6S3 m/z：590.1[M-H]-
Preparation of Compound D
Figure BDA0002754075770000091
Adding 20mL of acetic anhydride into a clean three-neck flask, sequentially adding 10mmol of 3, 3-dimethyl indole quaternary ammonium salt bromide and 10.1mmol of quinoline quaternary ammonium salt bromide, heating in an oil bath for 3.5h, detecting by TLC that the raw materials completely disappear, and cooling to room temperature; pouring 300mL of ether into the reaction solution, stirring for 30min, and separating out a solid; filtering to obtain solid, drying, and purifying by column chromatography to obtain a purple solid product.
The structure was confirmed by nuclear magnetic resonance and mass spectrometry, confirming that the synthesized product is compound D in 53.0% yield.
The nuclear magnetic data are as follows:
1H NMR:(400MHZ,CD3OD,TMS):1.2-1.5(m,10H),2.1-2.3(m,5H),3.2(m,2H),3.73(m,2H),4.25(m,2H),5.3(s,1H),5.92(d,1H),6.84(m,3H),7.45(m,2H),7.67(m,1H),8.42(m,1H),8.54(m,1H),8.93(m,1H)
EI data are: MS (EI) C30H37BrN2O3 m/z：473.5[M-Br]+
Preparation of Compound E
Figure BDA0002754075770000092
Adding 20mL of acetic anhydride into a clean three-neck flask, sequentially adding 10mmol of 3, 3-dimethyl indole quaternary ammonium salt bromide and 10.1mmol of quinoline quaternary ammonium salt bromide, heating in an oil bath for 3.5h, detecting by TLC that the raw materials completely disappear, and cooling to room temperature; pouring the reaction solution into 300mL of diethyl ether, stirring for 30min, and separating out a solid; filtering to obtain solid, recrystallizing with ethanol, and drying to obtain purple solid product.
The structure was confirmed by nuclear magnetic and mass spectrometry, confirming that the synthesized product was compound E in 48.0% yield.
The nuclear magnetic data are as follows:
1H NMR:(400MHZ,CD3OD,TMS):0.91(m,3H),1.35(m,6H),1.46(s,6H),1.54(m,2H),2.2(s,1H),3.93(m,2H),4.15(m,2H),5.14(s,1H),5.7(d,1H),6.93(s,1H),7.13(d,1H),7.34(d,1H),7.42(d,1H),7.76(d,1H),7.92(d,1H),8.12(d,1H),9.21(m,1H)
EI data is: MS (EI) C31H40N2O6S2 m/z：600.1[M-H]-
Preparation of Compound F
Figure BDA0002754075770000101
Adding 20mL of acetic anhydride into a clean three-neck flask, sequentially adding 10mmol of 3, 3-dimethyl indole quaternary ammonium salt and 10.1mmol of quinoline quaternary ammonium bromide, heating in an oil bath for 5 hours, and cooling to room temperature after TLC detection raw materials completely disappear; pouring the reaction solution into 500mL of methyl tert-butyl ether, stirring for 1h, placing in a refrigerator overnight, and separating out solids; the solid obtained by filtration is recrystallized by methanol and dried to obtain a dark purple solid product.
The structure was confirmed by nuclear magnetic and mass spectrometry, confirming that the synthesized product was compound F in 32.0% yield.
The nuclear magnetic data are as follows:
1H NMR:(400MHZ,CD3OD,TMS):0.91(m,6H),1.34(m,6H),1.45(s,6H),1.51(m,2H),3.89(s,2H),3.96(m,2H),4.06(m,2H),5.1(d,1H),5.74(d,1H),6.84(m,3H),7.2-7.6(m,7H),7.81(d,1H),7.98(s,1H),8.16(d,1H),9.22(d,1H)
EI data are: MS (EI) C37H42N2O3S m/z：595.1[M+H]-
Preparation of Compound G
Figure BDA0002754075770000102
Adding 20mL of acetic anhydride into a clean three-neck flask, sequentially adding 5mmol of selenoindole quaternary ammonium salt and 5.1mmol of quinoline quaternary ammonium salt bromide, heating in an oil bath for 3h, detecting that the raw materials completely disappear by TLC, and cooling to room temperature; pouring the reaction solution into 120mL of methyl tert-butyl ether, stirring for 30min, and separating out solids; the solid was obtained by filtration, recrystallized from methanol and dried to give a tan solid product.
The structure was confirmed by nuclear magnetic and mass spectrometry, confirming that the synthesized product was compound G, with a yield of 47.0%.
The nuclear magnetic data are as follows:
1H NMR:(400MHZ,CD3OD,TMS):2.23(m,2H),3.63(d,2H),3.86(m,2H),4.35(d,2H),5.13(s,1H),5.87(m,3H),6.73(m,3H),7.3-7.5(m,6H),7.94(d,2H),8.32(m,1H),8.66(d,1H),9.12(d,1H)
EI data are: MS (EI) C31H30BN2O6Se+m/z：617.1[M-H]-
The fluorescent dye of patent CN101743469B used in this example is of the following formula, control 1
Figure BDA0002754075770000111
The fluorescent dye of patent CN101231243B used in this example is of the following formula, control 2
Figure BDA0002754075770000112
The fluorescent dye of patent CN101349644B used in this example is of the following formula, control 3
Figure BDA0002754075770000113
Test 1 Absorbance test
In the test, a compound A, a compound B, a compound C, a compound D, a compound E and a compound F, a compound G are respectively prepared into 20ppm solutions by using ethylene glycol, and the absorbance of each fluorescent dye is analyzed by measuring the spectrum of 400-850nm by using an ultraviolet visible spectrophotometer UV-2450 (Shimadzu). The test results are shown in fig. 1.
The results in FIG. 1 show that the maximum absorption peak of the seven fluorescent dyes synthesized in this example is between 630 and 670nm, and if an excitation light source of 633nm is used, the generated fluorescent signals are not interfered by stray light and noise, and the staining and measuring accuracy of organism DNA or RNA can be effectively improved.
The comparative analysis shows that:
(1) compared with the compound B, the compound A and the compound B contain hydroxyl groups, so that the compound B is favorable for penetrating cell membranes and quickly combining with DNA or RNA, and the reaction speed and the fluorescence intensity are improved.
(2) Compared with the compound F, the compound F contains a sulfonic acid group, has better water solubility and is particularly suitable for being used in an aqueous environment.
(3) The compound G is particularly suitable for fluorescent labeling of protein due to the introduction of phenylboronic acid groups, and is helpful for molecular recognition of cells.
(4) Compared with the compound C and the compound E, although both contain two sulfonic acid groups, the compound E introduces a long hydrocarbon chain group, so that the hydrophilic and hydrophobic functions of the compound are improved, and the fluorescent labeling of the protein is facilitated through hydrophobic interaction.
Test 2 light stability test
The test is carried out by adopting the solution with the concentration of 20ppm of the compound A, the compound B, the compound C, the compound D, the compound E, the compound F and the compound G prepared in the test 1, seven fluorescent dyes are respectively arranged in a cuvette, a 600W iodine-tungsten lamp is used for irradiating at the distance of 20cm, the absorbance is tested every 1h, and the stability of the fluorescent dyes is analyzed. The test results are shown in fig. 2.
The results in fig. 2 show that the light stability of the seven fluorescent dyes is slightly different, which may be related to the molecular structure itself, but the overall light stability is very good, and is very suitable for the fluorescence recognition of organisms.
Test 3 use of dyes in cell sorting
Fresh blood of healthy humans was stained with compound a, compound B, compound C, compound D, compound E, compound F and compound G, and control 1, control 2 and control 3, respectively, for this test. Wherein, fresh blood of a healthy human body is used for a dyeing test after being subjected to EDTA-2K anticoagulation treatment. The method comprises the following specific steps:
(1) dyeing liquid
Ten kinds of fluorescent dyes, i.e., a compound A, a compound B, a compound C, a compound D, a compound E, a compound F, a control 1, a control 2, and a control 3, were dissolved in ethylene glycol, respectively, to prepare fluorescent dye solutions having a final concentration of 7ppm, which were used as staining solutions.
(2) Hemolytic agent
Figure BDA0002754075770000121
PH＝6.1
(3) Cell staining and differential detection
Taking 0.6mL of hemolytic agent, heating to 40 ℃, adding 50 muL of prepared staining solution and 5 muL of EDTA-2K anticoagulated healthy human fresh blood, incubating for 20 seconds at 40 ℃, preparing a test sample, measuring the sample on a flow cytometry instrument with a laser light source of 633nm, and recording cell classification signals. The test results are shown in fig. 3 to 12, and fig. 3 to 12 are the test results after staining compound a, compound B, compound C, compound D, compound E, compound F, compound G, control 1, control 2 and control 3 in this order.
The results in FIGS. 3 to 12 show that Compound A, Compound B, Compound C, Compound D, Compound E, Compound F, and Compound G of this example are well-classified into lymphocytes, monocytes, neutrophils, and eosinophils, as shown in FIGS. 3 to 9. In contrast, the leukocyte classification effect of the control 1, the control 2 and the control 3 is general, and the classification limit of partial cells is not obvious; for example, the fluorescent dye of control 1 did not have distinct classification limits for lymphocytes, monocytes and neutrophils, as shown in fig. 10; the fluorescent dye of control 2, with no apparent classification boundary for lymphocytes and monocytes, is shown in fig. 11; the fluorescent dye of control 3, also did not clearly demarcate lymphocytes and monocytes, as shown in FIG. 12.
The comparative analysis shows that:
(1) in leukocyte classification, compound a was classified better than compound B, and in scatter plots, the stray point names were significantly less, probably due to the difference in the R5 substituents.
(2) In leukocyte classification, compound F has stronger fluorescence intensity than compound A after the compound F acts, so that lymphocyte and monocyte groups are separated.
(3) Boric acid groups are introduced into the structure of the compound G, and after the compound G reacts with leukocytes, the leukocyte subgroup classification is very obvious, and the colony is relatively gathered.
(4) In leukocyte classification, compared with compound E, compound C has stronger fluorescence after being acted on under the same experimental conditions, which may have a certain relation with the difference of hydrophilicity and hydrophobicity of the compound.
Example two
In this example, based on example one, compounds H to Z, compound XA, and compound XB were synthesized using the same synthesis strategy, and a total of 21 fluorescent dyes were synthesized. The synthetic raw materials of the 21 fluorescent dyes can be directly obtained from commercial purchase. The details of the 21 fluorescent dyes are shown in Table 2.
TABLE 2 structural formula of the synthetic fluorescent dye of this example
Figure BDA0002754075770000131
Figure BDA0002754075770000141
Figure BDA0002754075770000151
The 21 fluorescent dyes of this example were subjected to an absorbance test, a photostability test and a cell sorting test in the same manner as in example one. The result shows that the maximum absorption peak of 21 fluorescent dyes is also between 630 and 670nm, and the fluorescence signal generated by using an excitation light source with 633nm is not interfered by stray light and noise, so that the DNA or RNA can be accurately measured. Furthermore, the 21 fluorescent dyes of the present example are excellent in stability and suitable for fluorescence recognition of living bodies. The cell classification result shows that the 21 fluorescent dyes of the embodiment can also perform classification tests on lymphocytes, monocytes, neutrophils and eosinophils, and the classification limit of each cell is obvious and the detection effect is good.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. It will be apparent to those skilled in the art from this disclosure that many more simple derivations or substitutions can be made without departing from the spirit of the disclosure.

Claims (10)

1. A fluorescent dye, characterized by: the fluorescent dye has a structure shown as a formula I;
is like
Figure FDA0002754075760000011
In the first formula, the first reaction solution is,x is C (CH)3)2、C(C2H5)2、CH3CC2H5O, S or Se;
R1and R4Each independently selected from H, OH, OR7Mercapto group, carboxyl group, carbonyl group, C1-C18Alkenyl radical, C1-C18Alkynyl, cyano, nitro, amino, trifluoromethyl, difluoroethyl, trifluoromethoxy, sulfonic acid, phosphoramidite, phosphonic acid, C1-C18Alkyl radical, C1-C8Alkyl OR6、C1-C18Alkylsulfonic acid group, benzyl group, phenyl group, furyl group, thienyl group, thiazolyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, oxazolyl group, isoxazolyl group, triazolyl group, tetrazolyl group, oxadiazolyl group, pyrazinyl group, pyrimidinyl group, pyridazinyl group, pyridyl group, quinolyl group, indolyl group, acridinyl group, C1-C4Alkyl pyridyl radical, C1-C18Haloalkyl, C1-C18Alkyl phosphate group or C1-C18Alkyl SR6Wherein benzyl is substituted benzyl optionally substituted with the following substituents: a boronic acid group, a hydroxyl group, a halogen, a mercapto group, a cyano group, a nitro group, an alkyl group, an aryl group, an alkoxy group, an amide group, a carboxyl group, a sulfonic acid group, an amino group, a heterocyclic group, and an alkylamino group;
R2and R3Each independently selected from H, C1-C18Alkyl group COOR7、C1-C18Alkyl OR7、C1-C18Alkyl NHR6、C1-C18Alkyl N (R)7)(R7)、C1-C18Alkylsulfonic acid group, C1-C18Alkyl phosphoric acid group, C1-C18Alkylamide group, C1-C18Alkenyl radical, C1-C18Alkynyl, C1-C18Alkyl, sulfonic acid group, carbonyl group, carboxyl group, amino group, phenyl group, phenylboronic acid group, pyridyl group, cyano group, or benzyl group; wherein benzyl is a substituted benzyl group optionally substituted with the following substituents: hydroxy, halogen, mercapto, cyano, nitro, alkyl, aryl, alkoxy, amido, carboxyl, and the like,Sulfonic acid groups, amino groups, heterocyclic groups, and alkylamino groups;
R5selected from OH, OR7Mercapto group, carboxyl group, carbonyl group, C1-C18Alkenyl radical, C1-C18Alkynyl, cyano, nitro, amino, trifluoromethyl, difluoroethyl, trifluoromethoxy, sulfonic acid, phosphoramidite, phosphonic acid, C1-C18Alkyl radical, C1-C8Alkyl OR6、C1-C18Alkylsulfonic acid group, benzyl group, phenyl group, furyl group, thienyl group, thiazolyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, oxazolyl group, isoxazolyl group, triazolyl group, tetrazolyl group, oxadiazolyl group, pyrazinyl group, pyrimidinyl group, pyridazinyl group, pyridyl group, quinolyl group, indolyl group, acridinyl group, C1-C4Alkyl pyridyl radical, C1-C18Haloalkyl, C1-C18Alkyl phosphate group or C1-C18Alkyl SR6Wherein benzyl is substituted benzyl optionally substituted with the following substituents: a boronic acid group, a hydroxyl group, a halogen, a mercapto group, a cyano group, a nitro group, an alkyl group, an aryl group, an alkoxy group, an amide group, a carboxyl group, a sulfonic acid group, an amino group, a heterocyclic group, and an alkylamino group;
R6is C1-C18Alkyl, H, carboxyl, C1-C4Carboxyl group, amino group, hydroxyl group, ether group derivative, C1-C6Ether group or C1-C6An ether group derivative;
R7is C1-C18Alkyl, H, carboxyl, C1-C18An alkylcarboxyl, amino, hydroxyl, ether or phenyl group; wherein the phenyl is a substituted phenyl optionally substituted with the following substituents: hydroxyl, halogen, mercapto, cyano, nitro, alkyl, aryl, alkoxy, heterocyclyl, haloalkyl, amino, alkylamino, amidoamino, carbonyl, carboxyl, phosphate, and phosphite;
y-is a single anion or a anion of the functional group itself.
2. Fluorescent dye according to claim 1, characterized in thatThe method comprises the following steps: said Y is-Is halogen anion, BF4 -、ClO4 -、IO4 -At least one of sulfonic acid group anions and alkylsulfonic acid group anions.
3. The fluorescent dye according to claim 1, wherein:
x is C (CH)3)2S or Se;
and/or
The R is1And R2Each independently selected from C1-C6Alkyl radical, C1-C3Alkylsulfonic acid groups, sulfonic acid groups, carbonyl groups, carboxyl groups, amino groups, phenyl groups;
and/or
The R is3And R4Each independently selected from C1-C6Alkyl radical, C1-C4Alkyl OR6、C1-C4Alkyl group COOR7Phenylboronic acid group, C1-C4Alkylsulfonic acid group, sulfonic acid group, benzyl group, phenyl group, C1-C3Alkenyl radical, C1-C3Alkynyl, pyridyl or cyano, and R6Is C1-C6Alkyl or H or C1-C6Ether group or C1-C6An ether group derivative;
and/or
Said R is5Is selected from C1-C6Alkyl, benzyl, phenyl, C1-C3Alkenyl radical, C1-C4Ether group, furyl group, C1-C4Alkyl pyridyl, triazolyl, C1-C4Alkyl OR6And R is6Is C1-C6Alkyl or H or C1-C6Ether group or C1-C6An ether group derivative;
and/or
Said Y is-Selected from Cl-、Br-、I-、BF4 -、ClO4 -At least one of sulfonic acid group anions and alkylsulfonic acid group anions.
4. The fluorescent dye according to claim 1, wherein: r1And R2Are the same, and/or R3And R4Different.
5. The fluorescent dye according to claim 1, wherein: the fluorescent dye is any one of a compound A to a compound Z and a compound XA and a compound XB;
compound A
Figure FDA0002754075760000031
Compound B
Figure FDA0002754075760000032
Compound C
Figure FDA0002754075760000033
Compound D
Figure FDA0002754075760000034
Compound E
Figure FDA0002754075760000035
Compound F
Figure FDA0002754075760000036
Compound G
Figure FDA0002754075760000041
Compound H
Figure FDA0002754075760000042
Compound I
Figure FDA0002754075760000043
Compound J
Figure FDA0002754075760000044
Compound K
Figure FDA0002754075760000045
Compound L
Figure FDA0002754075760000051
Compound M
Figure FDA0002754075760000052
Compound N
Figure FDA0002754075760000053
Compound O
Figure FDA0002754075760000054
Compound P
Figure FDA0002754075760000055
Compound Q
Figure FDA0002754075760000056
Compound R
Figure FDA0002754075760000061
Compound S
Figure FDA0002754075760000062
Compound T
Figure FDA0002754075760000063
Compound U
Figure FDA0002754075760000064
Compound V
Figure FDA0002754075760000065
Compound W
Figure FDA0002754075760000066
Compound X
Figure FDA0002754075760000071
Compound Y
Figure FDA0002754075760000072
Compound Z
Figure FDA0002754075760000073
Compound XA
Figure FDA0002754075760000074
Compound XB
Figure FDA0002754075760000075
6. A conjugate, characterized by: the conjugate having attached thereto the fluorescent dye of any one of claims 1-5.
7. A kit for staining a biological sample, characterized by: the kit contains the fluorescent dye according to any one of claims 1 to 5 or the conjugate according to claim 6.
8. A cell-mimic particle for use in fluorescence staining analysis, comprising: the cell mimetic particle is a cell, RNA or DNA stained with the fluorescent dye of any one of claims 1 to 5 or the conjugate of claim 6; wherein the cells comprise at least one of lymphocytes, monocytes, neutrophils, eosinophils, basophils, erythrocytes, reticulocytes, nucleated erythrocytes, and platelets.
9. A method for cell classification detection of a blood sample, which is characterized by comprising the following steps: comprising staining a blood sample with a fluorescent dye according to any one of claims 1 to 5, obtaining a scatter plot from the staining signals, classifying and/or counting cells in the blood sample according to the distribution of the scatter plot; the cell comprises at least one of lymphocyte, monocyte, neutrophil, eosinophil, basophil, erythrocyte, reticulocyte, nucleated erythrocyte and platelet.
10. A method of synthesizing a fluorescent dye according to any one of claims 1 to 5, wherein: mixing a compound with a structure shown in a formula II with a compound with a structure shown in a formula III, and heating for reaction to obtain the fluorescent dye with the structure shown in the formula I;
formula II
Figure FDA0002754075760000081
Formula III
Figure FDA0002754075760000082
X, R in formula II1、R3And R5And Y in formula III-、R2And R4Are all the same as the formula one.
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