CN111349091A - Novel fluorescent dye and preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of fluorescent dyes, and provides a novel fluorescent dye which has the following structural general formula I:
the preparation method comprises the following steps: s1, reacting 2-methylthiobenzothiazole with 6-bromohexanoic acid to obtain 2-methylthio-3- (5-carboxypentyl) benzothiazole; s2, mixing 2-hydroxy-4-methyl quinolone with copper powder and carbonMixing potassium and iodobenzene, and performing reflux reaction to obtain 1-phenyl-4-methyl quinolone; s3, dissolving 1-phenyl-4-methyl quinolone in dichloromethane, adding phosphorus oxychloride and dimethylformamide, and performing reflux reaction to obtain 1-phenyl-2-chloro-4-methylbenzoquinoline; s4, dissolving 1-phenyl-2-chloro-4-methylbenzoquinoline in dichloromethane, adding 2-methylthio-3- (5-carboxypentyl) benzothiazole and triethylamine, and mixing and reacting to obtain the fluorescent dye with the structural general formula I. Through the technical scheme, the problem that the effect of detecting single-strand and double-strand DNA molecules by using the electrostatic interaction force by the fluorescent dye is not good in the prior art is solved.
Description
Technical Field
The invention belongs to the technical field of fluorescent dyes, and relates to a novel fluorescent dye and a preparation method and application thereof.
Background
In recent years, scientists have designed a number of biosensing systems to perform DNA and enzyme detection. In 2002, water-soluble polythiophene derivatives are synthesized by Laclerc research and used for detecting DNA; bazan research group designed to synthesize a new type of benzothiazole-doped cationic polyfluorene, which was used to determine the concentration of single-and double-stranded DNA molecules. In recent years, this company has also synthesized a series of cationic polyfluorene derivatives and polythiophene derivatives, and used them to detect DNA, proteins, enzymes, metal ions, and the like. The common feature of these biosystem sensing systems is that a complex is formed with the polymer almost primarily by electrostatic interaction between the substrate and the polymer. However, the use of electrostatic interaction forces for DNA detection has the disadvantage of being susceptible to interference from other charged species in solution. Therefore, it is necessary to synthesize a fluorescent dye that can overcome the disadvantages of detection using electrostatic interaction and improve the ability of the probe to discriminate single-double stranded DNA molecules.
Disclosure of Invention
The invention provides a novel fluorescent dye and a preparation method and application thereof, and solves the problem that the fluorescent dye in the prior art is easily interfered by other charged substances in a solution when being used for detecting DNA by electrostatic interaction force, so that the effect of detecting single-double-stranded DNA molecules is poor.
The technical scheme of the invention is realized as follows:
a novel fluorescent dye has the following structural general formula I:
wherein R is1、R2Is a straight chain alkyl group of 1 to 6 carbons; r3Is a straight chain carboxyl group of 1 to 6 carbons.
As a further technical solution, R1、R2Is a linear alkyl group of 5 carbons, R3Is a linear carboxyl group of 5 carbons.
A preparation method of a novel fluorescent dye comprises the following steps:
s1, reacting 2-methylthiobenzothiazole with 6-bromohexanoic acid at 110 ℃ for 16 hours to obtain 2-methylthio-3- (5-carboxypentyl) benzothiazole for later use;
s2, mixing 2-hydroxy-4-methyl quinolone with copper powder, potassium carbonate and iodobenzene, and then adding the mixture into N2Carrying out reflux reaction for 48 hours under the protection of gas to obtain 1-phenyl-4-methyl quinolone;
s3, dissolving the 1-phenyl-4-methyl quinolone obtained in the step S2 in dichloromethane, adding phosphorus oxychloride, adding dimethylformamide, and carrying out reflux reaction for 24 hours to obtain 1-phenyl-2-chloro-4-methylbenzoquinoline;
s4, dissolving the 1-phenyl-2-chloro-4-methylbenzoquinoline obtained in the step S3 in dichloromethane, adding the 2-methylthio-3- (5-carboxypentyl) benzothiazole obtained in the step S1, adding triethylamine, mixing and stirring for reacting for 6 hours to obtain the fluorescent dye with the structural general formula I.
As a further technical scheme, the molar ratio of the 2-methylthiobenzothiazole to the 6-bromohexanoic acid in the step S1 is 1:1, the molar ratio of 2-hydroxy-4-methyl-quinoline to copper powder, potassium carbonate and iodobenzene in step S2 is 1: 5: 1.5: 1, the molar ratio of 1-phenyl-2-chloro-4-methylbenzoquinoline to 2-methylsulfanyl-3- (5-carboxypentyl) benzothiazole in step S4 is 1: 1.
as a further technical scheme, the method is used for DNA detection, fabric printing and dyeing, protein and biomacromolecule dyeing.
A novel fluorescent dye has the following structural general formula II:
wherein R is1、R2Is a straight chain alkyl group of 1 to 6 carbons; r3Is a straight chain alkyl group of 1 to 6 carbons.
As a further technical solution, R3Is a straight chain alkyl group of 5 carbons.
A preparation method of a novel fluorescent dye comprises the following steps:
s1, reacting 2-methylthiobenzothiazole with 6-bromohexanoic acid at 110 ℃ for 16 hours to obtain 2-methylthio-3- (5-carboxypentyl) benzothiazole for later use;
s2, mixing 2-hydroxy-4-methyl quinolone with copper powder, potassium carbonate and iodobenzene, and then adding the mixture into N2Carrying out reflux reaction for 48 hours under the protection of gas to obtain 1-phenyl-4-methyl quinolone;
s3, dissolving the 1-phenyl-4-methyl quinolone obtained in the step S2 in dichloromethane, adding phosphorus oxychloride, adding dimethylformamide, and carrying out reflux reaction for 24 hours to obtain 1-phenyl-2-chloro-4-methylbenzoquinoline;
s4, dissolving the 1-phenyl-2-chloro-4-methylbenzoquinoline obtained in the step S3 in dichloromethane, adding the 2-methylthio-3- (5-carboxypentyl) benzothiazole obtained in the step S1, adding triethylamine, mixing and stirring for reacting for 6 hours to obtain a fluorescent dye (Z) -3- (5-carboxypentyl) -2- ((2-chloro-1-phenylquinol-4 (1H) alkenyl) methyl) benzothiazole bromide compound with the structural general formula I;
s5, dissolving the (Z) -3- (5-carboxypentyl) -2- ((2-chloro-1-phenylquinol-4 (1H) alkenyl) methyl) benzothiazole bromide compound obtained in the step S4 in anhydrous N, N-dimethylformamide, adding N, N' -dicyclohexylcarbodiimide and N-hydroxysuccinamide, and stirring at room temperature for reacting for 24 hours to obtain the fluorescent dye with the structural general formula II.
As a further technical scheme, the molar ratio of the 2-methylthiobenzothiazole to the 6-bromohexanoic acid in the step S1 is 1:1, the molar ratio of 2-hydroxy-4-methyl-quinoline to copper powder, potassium carbonate and iodobenzene in step S2 is 1: 5: 1.5: 1, the molar ratio of 1-phenyl-2-chloro-4-methylbenzoquinoline to 2-methylsulfanyl-3- (5-carboxypentyl) benzothiazole in step S4 is 1:1, step S5 molar ratio of (Z) -3- (5-carboxypentyl) -2- ((2-chloro-1-phenylquinol-4 (1H) enyl) methyl) benzothiazole bromide compound to N, N' -dicyclohexylcarbodiimide, N-hydroxysuccinamide is 1: 1.17: 1.06.
as a further technical scheme, the method is used for DNA detection, fabric printing and dyeing, protein and biomacromolecule dyeing.
The working principle and the beneficial effects of the invention are as follows:
1. in the invention, a benzothiazole compound and a quinoline compound are used as raw materials, and the reaction is carried out to prepare the novel fluorescent dye in conjugated connection with the quinoline and the benzothiazole.
2. The prepared novel fluorescent dye can be used for DNA detection, protein and biomacromolecule dyeing, and can also be used for printing and dyeing fabrics such as fur, leather, viscose fiber, yarn and the like. The fluorescent dye disclosed by the invention is firm in dyeing, excellent in performance, clear in color of dyed protein and biomacromolecule, sharp in spectral band, and suitable for popularization and use.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of compound I of the present invention;
FIG. 2 is a NMR spectrum of a compound II of the present invention;
FIG. 3 is a NMR chart of Compound III of the present invention;
FIG. 4 is a NMR chart of a compound IV of the present invention;
FIG. 5 shows a NMR spectrum of Compound V of the present invention;
FIG. 6 is a fluorescence spectrum of compound I and compound II of the present invention for DNA detection;
in the figure: 1-ethidium bromide, 1-compound I and 1-compound II.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A preparation method of a fluorescent dye comprises the following steps:
S1.8mL of 2-methylthiobenzothiazole was reacted with 11.3g of 6-bromohexanoic acid at 110 ℃ for 16 hours. The crude product was cooled to room temperature to give a solid which was suspended in 20mL of methanol, then 40mL of diethyl ether was added, the precipitate filtered, washed with dioxane until no odor of 2-methylthiobenzothiazole, then with diethyl ether and dried under reduced pressure to give 11.2g of a white powder product which was 2-methylthio-3- (5-carboxypentyl) benzothiazole as compound III having the following formula III:
s2, mixing 10g of 2-hydroxy-4-methyl-quinoline with 24g of copper powder, 8.68g of potassium carbonate and 19.2g of iodobenzene, and then adding the mixture into N2The reaction was refluxed for 48 hours under a gas blanket, cooled to room temperature, separated with water and ethyl acetate, filtered, and the organic solution layer was dried over magnesium sulfate and the crude product was purified by silica gel chromatography using ethyl acetate: n-hexane ═ 1:1 to yield 8.1g of a product, designated compound iv, having the following formula iv:
s3, dissolving 2.8g of the compound IV in 20mL of dichloromethane, adding 1.85g of phosphorus oxychloride, adding dimethylformamide, and carrying out reflux stirring reaction for 24 hours to obtain 1-phenyl-2-chloro-4-methylbenzoquinoline, which is marked as a compound V and has the following structural formula V:
s4, dissolving 3.45g of the compound in 20mL of dichloromethane, adding the compound III, adding 1.3mL of triethylamine, mixing and stirring for reacting for 6 hours, and purifying a crude product by using a silica gel chromatographic column, wherein the weight ratio of ethyl acetate: chloroform: methanol 1: 1:1, recrystallizing and purifying the obtained product by using methanol/acetic acid/ethyl acetate to obtain the fluorescent dye (Z) -3- (5-carboxyl amyl) -2- ((2-chloro-1-phenyl quinolyl-4 (1H) alkenyl) methyl) benzothiazole bromide compound with the structural formula I, wherein the structural formula is as follows:
example 2
A preparation method of a fluorescent dye comprises the following steps:
S1.8mL of 2-methylthiobenzothiazole was reacted with 11.3g of 6-bromohexanoic acid at 110 ℃ for 16 hours. The crude product was cooled to room temperature to give a solid which was suspended in 20mL of methanol, then 40mL of diethyl ether was added, the precipitate filtered, washed with dioxane until no odor of 2-methylthiobenzothiazole, then with diethyl ether and dried under reduced pressure to give 11.2g of a white powder product which was 2-methylthio-3- (5-carboxypentyl) benzothiazole as compound III having the following formula III:
s2, mixing 10g of 2-hydroxy-4-methyl-quinoline with 24g of copper powder, 8.68g of potassium carbonate and 19.2g of iodobenzene, and then adding the mixture into N2The reaction was refluxed for 48 hours under a gas blanket, cooled to room temperature, separated with water and ethyl acetate, filtered, and the organic solution layer was dried over magnesium sulfate and the crude product was purified by silica gel chromatography using ethyl acetate: n-hexane ═ 1:1 to yield 8.1g of a product, designated compound iv, having the following formula iv:
s3, dissolving 2.8g of the compound IV in 20mL of dichloromethane, adding 1.85g of phosphorus oxychloride, adding dimethylformamide, and carrying out reflux stirring reaction for 24 hours to obtain 1-phenyl-2-chloro-4-methylbenzoquinoline, which is marked as a compound V and has the following structural formula V:
s4, dissolving 3.45g of the compound V in 20mL of dichloromethane, adding the compound III, adding 1.3mL of triethylamine, mixing and stirring for reacting for 6 hours, purifying a crude product by using a silica gel chromatographic column, and adding ethyl acetate: chloroform: methanol 1: 1: the obtained product is recrystallized and purified by methanol/acetic acid/ethyl acetate to obtain the fluorescent dye (Z) -3- (5-carboxyl amyl) -2- ((2-chloro-1-phenyl quinol-4 (1H) alkenyl) methyl) benzothiazole bromide compound with the general structural formula I, which is marked as a compound I and has the following structural formula:
s5, dissolving 500mg of compound I in 10mL of anhydrous N, N-dimethylformamide, adding 0.225g of N, N' -dicyclohexylcarbodiimide and 0.88g of N-hydroxysuccinamide, stirring at room temperature for 24 hours for reaction, stopping reaction, and detecting CH by TLC (thin layer chromatography)2Cl2：CH3OH ═ 20: and 3, filtering the reaction solution, adding diethyl ether into the filtrate to generate white precipitate, combining the solid obtained after filtering with the solid obtained by filtering, and drying under reduced pressure to obtain white solid. 22mg of ethylenediamine was dissolved in 2mL of anhydrous N, N-dimethylformamide, and 0.05mL of diisopropylethylamine was added thereto. The white solid obtained above was dissolved in 6mL of anhydrous N, N-dimethylformamide and slowly dropped into the reaction system. After the addition was complete, stirring was continued for 12 hours and the reaction was monitored by TLC. Stopping the reaction, precipitating the product by adding diethyl ether, filtering and drying, and purifying the product by reverse phase silica gel column chromatography, H2O：CH3OH ═ 1:1, giving a fluorescent dye of general structural formula ii, having the following structural formula ii:
fig. 1 is a nuclear magnetic resonance hydrogen spectrum of a compound i, fig. 2 is a nuclear magnetic resonance hydrogen spectrum of a compound ii, fig. 3 is a nuclear magnetic resonance hydrogen spectrum of a compound iii, fig. 4 is a nuclear magnetic resonance hydrogen spectrum of a compound iv, fig. 5 is a nuclear magnetic resonance hydrogen spectrum of a compound v, and absorption peaks of hydrogen protons can be observed from fig. 1 to 5.
The application of the compound I and the compound II in DNA detection is as follows:
respectively prepared at a concentration of 1 × 10-6M, namely a compound I, a compound II and ethidium bromide solution, 3mL of the compound I, the compound II and the ethidium bromide solution are respectively taken as a solvent by using 10mM PBS buffer solution with the pH value of 7.4 and placed in a cuvette to obtain each dye solution, an aqueous solution of Bovine Serum Albumin (BSA) with a certain concentration of 50ug/uL (750uM) is prepared, the BSA solution is dropwise added into the dye solution, the fluorescence intensity of the BSA solution is measured after the fluorescence is stabilized after 5 minutes of addition, and the used instrument is a fluorescence spectrophotometer with the model: FL0903M018, a fluorescence spectrum is shown in FIG. 6.
As can be seen from FIG. 6, under the same conditions, namely the same substrate concentration and DNA concentration, the fluorescence intensity of the compound I and the compound II after being respectively combined with calf thymus DNA is obviously enhanced compared with that of the commercial dye ethidium bromide, and the compound I and the compound II prepared by the embodiment of the invention are suitable for being used as fluorescent dyes for DNA detection.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A novel fluorescent dye is characterized by having the following structural general formula I:
wherein R is1、R2Is a straight chain alkyl group of 1 to 6 carbons; r3Is a straight chain carboxyl group of 1 to 6 carbons.
2. The novel fluorescent dye according to claim 1, wherein R is1、R2Is a linear alkyl group of 5 carbons, R3Is a linear carboxyl group of 5 carbons.
3. A preparation method of a novel fluorescent dye is characterized by comprising the following steps:
s1, reacting 2-methylthiobenzothiazole with 6-bromohexanoic acid at 110 ℃ for 16 hours to obtain 2-methylthio-3- (5-carboxypentyl) benzothiazole for later use;
s2, mixing 2-hydroxy-4-methyl quinolone with copper powder, potassium carbonate and iodobenzene, and then adding the mixture into N2Carrying out reflux reaction for 48 hours under the protection of gas to obtain 1-phenyl-4-methyl quinolone;
s3, dissolving the 1-phenyl-4-methyl quinolone obtained in the step S2 in dichloromethane, adding phosphorus oxychloride, adding dimethylformamide, and carrying out reflux reaction for 24 hours to obtain 1-phenyl-2-chloro-4-methylbenzoquinoline;
s4, dissolving the 1-phenyl-2-chloro-4-methylbenzoquinoline obtained in the step S3 in dichloromethane, adding the 2-methylthio-3- (5-carboxypentyl) benzothiazole obtained in the step S1, adding triethylamine, mixing and stirring for reacting for 6 hours to obtain the fluorescent dye with the structural general formula I.
4. The method of claim 3, wherein the molar ratio of 2-methylthiobenzothiazole to 6-bromohexanoic acid in step S1 is 1:1, the molar ratio of 2-hydroxy-4-methyl-quinoline to copper powder, potassium carbonate and iodobenzene in step S2 is 1: 5: 1.5: 1, the molar ratio of 1-phenyl-2-chloro-4-methylbenzoquinoline to 2-methylsulfanyl-3- (5-carboxypentyl) benzothiazole in step S4 is 1: 1.
5. the use of the novel fluorescent dye according to claim 1 for DNA detection, textile printing, protein and biomacromolecule staining.
6. A novel fluorescent dye is characterized by having the following structural general formula II:
wherein R is1、R2Is a straight chain alkyl group of 1 to 6 carbons; r3Is a straight chain alkyl group of 1 to 6 carbons.
7. The novel fluorescent dye according to claim 6, wherein R is1、R2Is a linear alkyl group of 5 carbons, R3Is a straight chain alkyl group of 5 carbons.
8. A preparation method of a novel fluorescent dye is characterized by comprising the following steps:
s1, reacting 2-methylthiobenzothiazole with 6-bromohexanoic acid at 110 ℃ for 16 hours to obtain 2-methylthio-3- (5-carboxypentyl) benzothiazole for later use;
s2, mixing 2-hydroxy-4-methyl quinolone with copper powder, potassium carbonate and iodobenzene, and then adding the mixture into N2Carrying out reflux reaction for 48 hours under the protection of gas to obtain 1-phenyl-4-methyl quinolone;
s3, dissolving the 1-phenyl-4-methyl quinolone obtained in the step S2 in dichloromethane, adding phosphorus oxychloride, adding dimethylformamide, and carrying out reflux reaction for 24 hours to obtain 1-phenyl-2-chloro-4-methylbenzoquinoline;
s4, dissolving the 1-phenyl-2-chloro-4-methylbenzoquinoline obtained in the step S3 in dichloromethane, adding the 2-methylthio-3- (5-carboxypentyl) benzothiazole obtained in the step S1, adding triethylamine, mixing and stirring for reacting for 6 hours to obtain a fluorescent dye (Z) -3- (5-carboxypentyl) -2- ((2-chloro-1-phenylquinol-4 (1H) alkenyl) methyl) benzothiazole bromide compound with the structural general formula I;
s5, dissolving the (Z) -3- (5-carboxypentyl) -2- ((2-chloro-1-phenylquinol-4 (1H) alkenyl) methyl) benzothiazole bromide compound obtained in the step S4 in anhydrous N, N-dimethylformamide, adding N, N' -dicyclohexylcarbodiimide and N-hydroxysuccinamide, and stirring at room temperature for reacting for 24 hours to obtain the fluorescent dye with the structural general formula II.
9. The method of claim 8, wherein the molar ratio of 2-methylthiobenzothiazole to 6-bromohexanoic acid in step S1 is 1:1, the molar ratio of 2-hydroxy-4-methyl-quinoline to copper powder, potassium carbonate and iodobenzene in step S2 is 1: 5: 1.5: 1, the molar ratio of 1-phenyl-2-chloro-4-methylbenzoquinoline to 2-methylsulfanyl-3- (5-carboxypentyl) benzothiazole in step S4 is 1:1, step S5 molar ratio of (Z) -3- (5-carboxypentyl) -2- ((2-chloro-1-phenylquinol-4 (1H) enyl) methyl) benzothiazole bromide compound to N, N' -dicyclohexylcarbodiimide, N-hydroxysuccinamide is 1: 1.17: 1.06.
10. the use of a fluorescent dye according to claim 6 for DNA detection, textile printing, protein and biomacromolecule staining.
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Cited By (3)
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| CN112635827A (en) * | 2020-12-04 | 2021-04-09 | 上海应用技术大学 | Electrolyte additive, electrolyte containing additive and lithium ion battery |
| WO2022099659A1 (en) * | 2020-11-13 | 2022-05-19 | 大连理工大学 | Leukocyte classification reagent, erythrocyte analysis reagent, reagent kit, and analysis method |
| US11952518B2 (en) | 2022-02-17 | 2024-04-09 | Beijing Fluorescence Biotechnology Co. Ltd | Synthesis and application of fluorescent dye with phenanthridine and benzothiazole conjugated |
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| WO2014089421A1 (en) * | 2012-12-07 | 2014-06-12 | Life Technologies Corporation | Detection of rna molecules using substituted unsymmetrical cyanine dyes |
| WO2019070930A1 (en) * | 2017-10-06 | 2019-04-11 | Life Technologies Corporation | Rna quality assay |
| WO2019177541A1 (en) * | 2018-03-15 | 2019-09-19 | National University Of Singapore | A dye suitable for use in the differentiation of viable and non-viable bacteria |
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| WO2022099659A1 (en) * | 2020-11-13 | 2022-05-19 | 大连理工大学 | Leukocyte classification reagent, erythrocyte analysis reagent, reagent kit, and analysis method |
| WO2022100717A1 (en) * | 2020-11-13 | 2022-05-19 | 大连理工大学 | Leukocyte classification reagent, erythrocyte analysis reagent, kits, and analysis method |
| CN112635827A (en) * | 2020-12-04 | 2021-04-09 | 上海应用技术大学 | Electrolyte additive, electrolyte containing additive and lithium ion battery |
| US11952518B2 (en) | 2022-02-17 | 2024-04-09 | Beijing Fluorescence Biotechnology Co. Ltd | Synthesis and application of fluorescent dye with phenanthridine and benzothiazole conjugated |
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