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CN113321638A - High-performance fluorescent dye suitable for various fluorescence detection scenes and preparation method thereof - Google Patents

High-performance fluorescent dye suitable for various fluorescence detection scenes and preparation method thereof Download PDF

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CN113321638A
CN113321638A CN202110640477.6A CN202110640477A CN113321638A CN 113321638 A CN113321638 A CN 113321638A CN 202110640477 A CN202110640477 A CN 202110640477A CN 113321638 A CN113321638 A CN 113321638A
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fluorescence detection
fluorescent dye
membered ring
compound
dye suitable
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向国兵
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Nanjing Superyears Gene Technology Co ltd
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Nanjing Superyears Gene Technology Co ltd
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    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/78—Ring systems having three or more relevant rings
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    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems
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    • C07F7/0803—Compounds with Si-C or Si-Si linkages
    • C07F7/081—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te
    • C07F7/0812—Compounds with Si-C or Si-Si linkages comprising at least one atom selected from the elements N, O, halogen, S, Se or Te comprising a heterocyclic ring
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
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    • C09K2211/1018—Heterocyclic compounds
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    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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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
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
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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
    • C09K2211/1044—Heterocyclic compounds characterised by ligands containing two nitrogen atoms as heteroatoms
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
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    • C09K2211/10—Non-macromolecular compounds
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  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The invention relates to the technical field of fluorescent dyes, and discloses a high-performance fluorescent dye suitable for various fluorescence detection scenes and a preparation method thereof, wherein corresponding raw materials are obtained based on preparation requirements and are prepared and dried to obtain a first compound; adding phosphorus oxychloride into the first compound under the protection of inert gas, stirring for 3 hours at 70 ℃, and dissolving in dry dichloromethane after vacuum drying; and then adding 3-methyl aminobutyric acid and triethylamine under the ice bath condition, stirring the solution in acetonitrile for 30 minutes, washing with slightly acidic water, extracting the water phase with dichloromethane, combining dichloromethane solutions, drying with sodium sulfate, and purifying by using a silica gel column to obtain a second compound. The dyes have high light and high thermal stability, high light absorption efficiency and high emission efficiency, the quantum yield is more than 50%, and the design and other application scenes of test kits with 8 colors, 9 colors, 10 colors and more colors are facilitated.

Description

High-performance fluorescent dye suitable for various fluorescence detection scenes and preparation method thereof
Technical Field
The invention relates to the technical field of fluorescent dyes, in particular to a high-performance fluorescent dye suitable for various fluorescence detection scenes and a preparation method thereof.
Background
Some applications require two 6-color kits to cover. Development of 8 or more color locus kits can cover all locus loci in one box, greatly improving efficiency. The key to designing an 8-color or more kit is the selection of 8 fluorescent dyes, and the existing dyes cannot meet the perfect 8-color or more kit design.
Although many rhodamine-like fluorescent dyes exist, dozens of rhodamine-like fluorescent dyes exist, rhodamine-like fluorescent dyes with specific wavelengths are not easy to find, the rhodamine-like fluorescent dyes are relatively few, and the rhodamine-like fluorescent dyes containing silicon are fewer. In particular, fluorescent dyes that are thermostable, high photostable, highly absorbing, high quantum yield, and suitably water soluble are less readily found and do not facilitate the design and other application scenarios of 8-color, 9-color, 10-color and higher color test kits.
Disclosure of Invention
The invention aims to provide a high-performance fluorescent dye suitable for various fluorescence detection scenes and a preparation method thereof, and provides convenience for the design of test kits with 8 colors, 9 colors, 10 colors and more and other application scenes.
In order to achieve the above object, in a first aspect, the present invention provides a high performance fluorescent dye suitable for various fluorescence detection scenarios, where the high performance fluorescent dye suitable for various fluorescence detection scenarios includes three rhodamine-like fluorescent dyes with different structures, and a structural general formula of the rhodamine-like fluorescent dye with three different structures is:
Figure RE-GDA0003185667690000021
wherein X is any one of O, -C (CH3)2 or-Si (CH3) 2;
three rhodamine-like fluorescent dyes with different structures respectively comprise R1, R2, R3, R4, X0, X1, X2, X3, X4 and L.
Wherein R1, R2, R3 and R4 are any one of H, CH3, CH2CH3, six-membered ring (CH2)5-, five-membered ring (CH2)4-, substituted six-membered ring or substituted five-membered ring respectively.
Wherein, X0, X1, X2, X3 and X4 are respectively one of H, CH3, CH2CH3, Cl, Br, OH, NH2, SO3H, CH2SO3H, -OPO3H2 or-PO 3H 2.
Wherein L is any one of-benzene ring-, substituted benzene ring, five-membered ring, six-membered ring, 3-6 carbon alkane, substituted five-membered ring, substituted six-membered ring or substituted 3-6 carbon alkane.
Wherein the difference of the emission wavelengths of two adjacent rhodamine-like fluorescent dyes is less than 5 nm.
In a second aspect, the present invention provides a method for preparing a high performance fluorescent dye suitable for use in various fluorescence detection scenarios as described in the first aspect, comprising the following steps:
obtaining corresponding raw materials based on preparation requirements, and performing preparation drying to obtain a first compound;
adding phosphorus oxychloride into the first compound under the protection of inert gas, stirring for 3 hours at 70 ℃, and dissolving in dry dichloromethane after vacuum drying; and then adding 3-methyl aminobutyric acid and triethylamine under the ice bath condition, stirring the solution in acetonitrile for 30 minutes, washing with slightly acidic water, extracting the water phase with dichloromethane, combining dichloromethane solutions, drying with sodium sulfate, and purifying by using a silica gel column to obtain a second compound.
The invention relates to a high-performance fluorescent dye suitable for various fluorescence detection scenes and a preparation method thereof, wherein corresponding raw materials are obtained based on preparation requirements and are prepared and dried to obtain a first compound; adding phosphorus oxychloride into the first compound under the protection of inert gas, stirring for 3 hours at 70 ℃, and dissolving in dry dichloromethane after vacuum drying; then under the ice bath condition, 3-methyl aminobutyric acid and triethylamine are added, the solution in acetonitrile is stirred for 30 minutes, then the solution is washed by slightly acidic water, the water phase is extracted by dichloromethane, the dichloromethane solution is combined and dried by sodium sulfate, a silica gel column is utilized for purification to obtain a second compound, the maximum absorption is 500nm to 800nm or higher, and the emitted light is green light to near infrared light. The dyes have high light and high thermal stability, high light absorption efficiency and high emission efficiency, and the quantum yield is more than 50%. The difference of the emission wavelengths of two adjacent high-performance fluorescent dyes is less than 5nm, such as 1-4nm and 2-3nm, and the kit provides convenience for the design of test kits with 8 colors, 9 colors, 10 colors and more and other application scenes. Fluorescent dyes with different water solubility, from hydrophilic to hydrophobic, can be synthesized to adapt to different biological application scenes.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a schematic structural diagram of a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a first embodiment of the present invention.
Fig. 2 is a schematic structural diagram of a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a second embodiment of the present invention.
Fig. 3 is a schematic structural diagram of a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a third embodiment of the present invention.
Fig. 4 is a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a fourth embodiment of the present invention.
Fig. 5 is a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a fifth embodiment of the present invention.
Fig. 6 is a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a sixth embodiment of the present invention.
Fig. 7 is a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a seventh embodiment of the present invention.
Fig. 8 is a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to an eighth embodiment of the present invention.
Fig. 9 is a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a ninth embodiment of the present invention.
Fig. 10 is a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a tenth embodiment of the present invention.
FIG. 11 is a schematic diagram of a basic structure of a high-performance fluorescent dye suitable for various fluorescence detection scenarios provided by the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
In the description of the present invention, it is to be understood that the terms "length", "width", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on the orientations or positional relationships illustrated in the drawings, and are used merely for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the devices or elements referred to must have a particular orientation, be constructed in a particular orientation, and be operated, and thus, are not to be construed as limiting the present invention. Further, in the description of the present invention, "a plurality" means two or more unless specifically defined otherwise.
Referring to fig. 1, a general structural formula of a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a first embodiment of the present invention is shown in fig. 1, where X is any one of O, -C (CH3)2 or-Si (CH3) 2; the three rhodamine-like fluorescent dyes with different structures all comprise R1, R2, R3, R4, X0, X1, X2, X3, X4 and L, and R1, R2, R3 and R4 are respectively one of H, CH3, CH2CH3, six-membered ring (CH2)5-, five-membered ring (CH2)4-, substituted six-membered ring or substituted five-membered ring; x0, X1, X2, X3 and X4 are each any one of H, CH3, CH2CH3, Cl, Br, OH, NH2, SO3H, CH2SO3H, -OPO3H2 or-PO 3H 2; l is any one of-benzene ring-, substituted benzene ring, five-membered ring, six-membered ring, 3-6 carbon alkane, substituted five-membered ring, substituted six-membered ring or substituted 3-6 carbon alkane.
Referring to fig. 2, a general structural formula of a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a second embodiment of the present invention is shown in fig. 2, wherein X is any one of O, -C (CH3)2 or-Si (CH3) 2; the three rhodamine-like fluorescent dyes with different structures all comprise R1, R2, R3, R4, X0, X1, X2, X3, X4 and L, and R1, R2, R3 and R4 are respectively one of H, CH3, CH2CH3, six-membered ring (CH2)5-, five-membered ring (CH2)4-, substituted six-membered ring or substituted five-membered ring; x0, X1, X2, X3 and X4 are each any one of H, CH3, CH2CH3, Cl, Br, OH, NH2, SO3H, CH2SO3H, -OPO3H2 or-PO 3H 2; l is any one of-benzene ring-, substituted benzene ring, five-membered ring, six-membered ring, 3-6 carbon alkane, substituted five-membered ring, substituted six-membered ring or substituted 3-6 carbon alkane.
Referring to fig. 3, a general structural formula of a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a third embodiment of the present invention is shown in fig. 3, wherein X is any one of O, -C (CH3)2 or-Si (CH3) 2; the three rhodamine-like fluorescent dyes with different structures all comprise R1, R2, R3, R4, X0, X1, X2, X3, X4 and L, and R1, R2, R3 and R4 are respectively one of H, CH3, CH2CH3, six-membered ring (CH2)5-, five-membered ring (CH2)4-, substituted six-membered ring or substituted five-membered ring; x0, X1, X2, X3 and X4 are each any one of H, CH3, CH2CH3, Cl, Br, OH, NH2, SO3H, CH2SO3H, -OPO3H2 or-PO 3H 2; l is any one of-benzene ring-, substituted benzene ring, five-membered ring, six-membered ring, 3-6 carbon alkane, substituted five-membered ring, substituted six-membered ring or substituted 3-6 carbon alkane.
In the above three embodiments, the general formula of the basic structure is shown in fig. 11. COOH is converted to the corresponding activated ester COONHS to react with an amino group (-NH2) in the biomolecule to form a dye-labeled biomolecule. COOH may also be converted to other reactive groups, - (CH2) nNH2(n ═ 1 to 10), -NHNH2, maleimido, - (CH2) nN3(n ═ 1 to 10), - (CH2) nC ═ CH (n ═ 0 to 10); the dye has a maximum absorption of 500nm to 800nm or more and emits light from green to near-infrared light; the difference of the emission wavelengths of two adjacent high-performance fluorescent dyes is less than 5nm, such as 1-4nm and 2-3 nm; one or more water-soluble groups, such as-SO 3H, -PO3H2, -OPO3H2, -OH, -NH2, -OCH2CH2O-, are introduced to increase the water solubility of the fluorescent dye. The design and other application scenes of the test kit with 8 colors, 9 colors, 10 colors and more are facilitated. Fluorescent dyes with different water solubility, from hydrophilic to hydrophobic, can be synthesized to adapt to different biological application scenes.
Referring to fig. 4, a fourth embodiment of the present invention provides a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios, including the following steps:
a100 mL three-necked flask was charged with 2.75g (FW:137.18, 20mmol) of 3-N, N' -dimethylaminophenol, 1.48g (FW:148.12,10mmol) of phthalic anhydride and 10mL of concentrated sulfuric acid. Stirring under the protection of N2, slowly heating to 180 ℃, and keeping the temperature for reaction for 6 hours. After cooling to room temperature, the reaction mixture was poured in portions into 40mL of an ice-water mixture and stirred vigorously. Suction filtration, washing with water for several times and drying to obtain 1, 3.5g of compound.
1.94g of dried compound 1(FW:387.45,5mmol) in 200ml of 1, 2-dichloroethane are added 16.7ml of phosphorus oxychloride (POCl3, 167mmol) under argon. The reaction was stirred at 70 ℃ for 3 hours, the solvent was removed, and the reaction mixture was dried in vacuo and dissolved in 100ml of dry dichloromethane. The reaction was stirred in an ice bath and a solution of 1.76g of 3-methylaminobutyric acid (FW:117.15, 15mmol), triethylamine (3.25ml, 22.5mmol) in 30ml of dry acetonitrile was added in one portion under argon. After stirring for 30 minutes, the mixture is washed with slightly acidic water, the aqueous phase is extracted again with dichloromethane, and the dichloromethane solutions are combined and dried over sodium sulfate. Purification on a silica gel column afforded 2g of Compound 2.
Referring to fig. 5, a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a fifth embodiment of the present invention is provided.
Specifically, the process steps adopted in this embodiment are the same as those adopted in the fourth embodiment of the present invention, and therefore, the description thereof is omitted here.
Referring to fig. 6, a sixth embodiment of the present invention provides a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios, including the following steps:
approximately 5mmol of lithium reagent was formed in 25ml of dry THF at-78 deg.C (cf. Eur. J. org. chem. 2010, 3593-3610). 309mg of Compound 5(1mmol, Eur. J. org. chem. 2010, 3593-3610) are added to the lithium reagent at-78 ℃ with stirring in 12ml of dry THF. After stirring at-78 ℃ for 6 hours and then at 0 ℃ overnight, the mixture was poured into acetic acid-containing ethanol (30ml of ethanol, 2ml of acetic acid) with stirring at 0 ℃. After removing all the solvent, the extract was separated and purified by a silica gel column to obtain 350mg of a solid. All 350mg of solid were placed in 20ml of concentrated hydrochloric acid and 10ml of water and heated overnight at 80 ℃ with stirring, with reflux (approximately 18 hours). 30ml of water, 40ml of dichloromethane and 60ml of ethyl acetate are added and neutralized by adding 22g of sodium hydrogencarbonate with vigorous stirring. The aqueous phase was separated and extracted 5 times with dichloromethane until the aqueous phase was colourless, 40ml each time. The combined organic phases were dried over sodium sulfate, drained and purified over a silica gel column to give 210mg of Compound 6.
Dried 207mg of Compound 6(FW:413.53,0.5mmol) in 20ml of 1, 2-dichloroethane are added under argon protection with 1.67ml of phosphorus oxychloride (POCl3, 16.7 mmol). The reaction was stirred at 70 ℃ for 3 hours, the solvent was removed, and the reaction mixture was dried in vacuo and dissolved in 10ml of dry dichloromethane. The reaction was stirred in an ice bath and a solution of 0.18g of 3-methylaminobutyric acid (FW:117.15, 1.5mmol), triethylamine (0.33ml, 2.25mmol) in 3ml of dry acetonitrile was added in one portion under argon. After stirring for 30 minutes, the mixture is washed with slightly acidic water, the aqueous phase is extracted again with dichloromethane, and the dichloromethane solutions are combined and dried over sodium sulfate. Purification on a silica gel column afforded 180mg of Compound 7.
Referring to fig. 7, a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to a seventh embodiment of the present invention is provided.
Specifically, the process steps adopted in this embodiment are the same as those adopted in the sixth embodiment of the present invention, and therefore, the description thereof is omitted here.
Referring to fig. 8, a flowchart of a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios according to an eighth embodiment of the present invention is provided.
Specifically, the process steps adopted in this embodiment are the same as those adopted in the sixth embodiment of the present invention, and therefore, the description thereof is omitted here.
Referring to fig. 9, a ninth embodiment of the present invention provides a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios, including the following steps:
compound 14 (Natmethods.2015Mar; 12(3): 244-250) (FW 652.61, 653mg, 1mmol, 1 equiv.), Pd2dba3(92mg, 0.1mmol, 0.1 equiv.), XPhos (144mg, 0.3mmol, 0.3 equiv.) and Cs2CO3(909mg, 2.8mmol, 2.8 equiv.) were added to the vial. The vial was sealed and purged with nitrogen 3 times, dimethylamine (2MTHF, 10mL, 20mmol, 20 equiv.) was added and the reaction vial was stirred at 100 ℃ for 2 hours, then cooled to room temperature, diluted with CH2Cl2, deposited on celite, and concentrated to dryness. Purification by silica gel chromatography (0-40% linear gradient of ethyl acetate/hexanes, reaction adsorbed onto celite) afforded 460mg of compound 15 (92.0%).
Compound 15(FW499.74, 250mg, 0.5mml, 1 single dose).
Dried 250mg of compound 15(FW499.74, 0.5mml, 1% strength) are introduced into 20ml of 1, 2-dichloroethane, under argon protection, with 1.67ml of phosphorus oxychloride (POCl3, 16.7 mmol). The reaction was stirred at 70 ℃ for 3 hours, the solvent was removed, and the reaction mixture was dried in vacuo and dissolved in 10ml of dry dichloromethane. The reaction was stirred in an ice bath and a solution of 0.18g of 3-methylaminobutyric acid (FW:117.15, 1.5mmol), triethylamine (0.33ml, 2.25mmol) in 3ml of dry acetonitrile was added in one portion under argon. After stirring for 30 minutes, the mixture is washed with slightly acidic water, the aqueous phase is extracted again with dichloromethane, and the dichloromethane solutions are combined and dried over sodium sulfate. Purification on a silica gel column afforded 220mg of compound 16.
Referring to fig. 10, a tenth embodiment of the present invention provides a method for preparing a high-performance fluorescent dye suitable for various fluorescence detection scenarios, including the following steps:
a vial was charged with Compound 17 (Natmethods.2015Mar; 12(3): 244-250) (FW 694.69, 695mg, 1mmol, 1 equiv.), Pd2dba3(92mg, 0.1mmol, 0.1 equiv.), XPhos (144mg, 0.3mmol, 0.3 equiv.) and Cs2CO3(909mg, 2.8mmol, 2.8 equiv.). The vial was sealed and purged with nitrogen 3 times, dimethylamine (2MTHF, 10mL, 20mmol, 20 equiv.) was added and the reaction vial was stirred at 100 ℃ for 2 hours, then cooled to room temperature, diluted with CH2Cl2, deposited on celite, and concentrated to dryness. Purification by silica gel chromatography (0-40% linear gradient of ethyl acetate/hexanes, reaction adsorbed onto celite) afforded 450mg of compound 18 (92.6%).
Compound 18(FW485.71, 243mg, 0.5mml, 1 single dose)
Dried 243mg of compound 18(FW485.71, 0.5mml, 1% w) in 20ml of 1, 2-dichloroethane are added, under argon protection, 1.67ml of phosphorus oxychloride (POCl3, 16.7 mmol). The reaction was stirred at 70 ℃ for 3 hours, the solvent was removed, and the reaction mixture was dried in vacuo and dissolved in 10ml of dry dichloromethane. The reaction was stirred in an ice bath and a solution of 0.18g of 3-methylaminobutyric acid (FW:117.15, 1.5mmol), triethylamine (0.33ml, 2.25mmol) in 3ml of dry acetonitrile was added in one portion under argon. After stirring for 30 minutes, the mixture is washed with slightly acidic water, the aqueous phase is extracted again with dichloromethane, and the dichloromethane solutions are combined and dried over sodium sulfate. Purification on a silica gel column afforded 200mg of compound 19.
Wherein, the first compound can be compound 1, compound 3, compound 6, compound 9, compound 12, compound 15, compound 18; the second compound may be compound 2, compound 4, compound 7, compound 10, compound 13, compound 16, compound 19. Wherein, the compounds 2 and 4 belong to rhodamine fluorescent dye when X is O, and have high quantum yield and high stability. The synthesis of compound 4 is similar to compound 2. And more rhodamine fluorescent dyes with X being O can be synthesized by changing the substituent.
The compounds 7, 10 and 13 belong to the rhodamine-like fluorescent dye when X is-C (CH3)2, and have high quantum yield and high stability.
The synthesis of compounds 10, 13 is similar to that of compound 7. The substituent can be changed to synthesize more rhodamine-like fluorescent dyes with X being-C (CH3) 2.
The compounds 16 and 19 belong to the same type of the silicon rhodamine fluorescent dye with X being-Si (CH3)2, and have high quantum yield and high stability. The substituent can be changed to synthesize more than one silicorhodamine fluorescent dye with X being-Si (CH3) 2.
All compounds 2, 4, 7, 10, 13, 16, 19 belong to different classes of rhodamine series fluorescent dyes.
The invention relates to a high-performance fluorescent dye suitable for various fluorescence detection scenes and a preparation method thereof, wherein corresponding raw materials are obtained based on preparation requirements and are prepared and dried to obtain a first compound; adding phosphorus oxychloride into the first compound under the protection of inert gas, stirring for 3 hours at 70 ℃, and dissolving in dry dichloromethane after vacuum drying; then under the ice bath condition, 3-methyl aminobutyric acid and triethylamine are added, the solution in acetonitrile is stirred for 30 minutes, then the solution is washed by slightly acidic water, the water phase is extracted by dichloromethane, the dichloromethane solution is combined and dried by sodium sulfate, a silica gel column is utilized for purification to obtain a second compound, the maximum absorption is 500nm to 800nm or higher, and the emitted light is green light to near infrared light. The dyes have high light and high thermal stability, high light absorption efficiency and high emission efficiency, and the quantum yield is more than 50%. The difference of the emission wavelengths of two adjacent high-performance fluorescent dyes is less than 5nm, such as 1-4nm and 2-3nm, and the kit provides convenience for the design of test kits with 8 colors, 9 colors, 10 colors and more and other application scenes. Fluorescent dyes with different water solubility, from hydrophilic to hydrophobic, can be synthesized to adapt to different biological application scenes.
While the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (6)

1. A high-performance fluorescent dye suitable for various fluorescence detection scenes is characterized in that,
the high-performance fluorescent dye suitable for various fluorescence detection scenes comprises three rhodamine-like fluorescent dyes with different structures, and the rhodamine-like fluorescent dyes with the three different structures have the structural general formula:
Figure FDA0003107411150000011
wherein X is any one of O, -C (CH3)2 or-Si (CH3) 2;
three rhodamine-like fluorescent dyes with different structures respectively comprise R1, R2, R3, R4, X0, X1, X2, X3, X4 and L.
2. The high performance fluorescent dye suitable for use in various fluorescence detection scenarios as claimed in claim 1,
r1, R2, R3 and R4 are any one of H, CH3, CH2CH3, six-membered ring (CH2)5-, five-membered ring (CH2)4-, substituted six-membered ring or substituted five-membered ring respectively.
3. The high performance fluorescent dye suitable for use in various fluorescence detection scenarios as claimed in claim 1,
x0, X1, X2, X3 and X4 are respectively H, CH3, CH2CH3, Cl, Br, OH, NH2, SO3H, CH2SO3H, -OPO3H2 or-PO 3H 2.
4. The high performance fluorescent dye suitable for use in various fluorescence detection scenarios as claimed in claim 2,
l is any one of-benzene ring-, substituted benzene ring, five-membered ring, six-membered ring, 3-6 carbon alkane, substituted five-membered ring, substituted six-membered ring or substituted 3-6 carbon alkane.
5. The high performance fluorescent dye suitable for use in various fluorescence detection scenarios as claimed in claim 1,
the difference of the emission wavelengths of two adjacent rhodamine-like fluorescent dyes is less than 5 nm.
6. A method for preparing a high performance fluorescent dye suitable for use in various fluorescence detection scenarios as claimed in any of claims 1 to 5, comprising the steps of:
obtaining corresponding raw materials based on preparation requirements, and performing preparation drying to obtain a first compound;
adding phosphorus oxychloride into the first compound under the protection of inert gas, stirring for 3 hours at 70 ℃, and dissolving in dry dichloromethane after vacuum drying; and then adding 3-methyl aminobutyric acid and triethylamine under the ice bath condition, stirring the solution in acetonitrile for 30 minutes, washing with slightly acidic water, extracting the water phase with dichloromethane, combining dichloromethane solutions, drying with sodium sulfate, and purifying by using a silica gel column to obtain a second compound.
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