CN112710838A - Application of quinolinecarbonitrile derivative in protein detection by protein imprinting method and preparation method thereof - Google Patents

Application of quinolinecarbonitrile derivative in protein detection by protein imprinting method and preparation method thereof Download PDF

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CN112710838A
CN112710838A CN202011359196.5A CN202011359196A CN112710838A CN 112710838 A CN112710838 A CN 112710838A CN 202011359196 A CN202011359196 A CN 202011359196A CN 112710838 A CN112710838 A CN 112710838A
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朱为宏
王琪
周体健
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East China University of Science and Technology
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Abstract

The invention relates to application of quinoline nitrile derivatives in protein imprinting detection and a preparation method thereof, wherein the quinoline nitrile derivatives are shown as a compound shown in a formula I. The invention improves the problems of short luminol luminescence time, narrow detection range, poor quantitative repeatability, high commercial fluorescence detection cost and the like of the traditional reagent based on the chemiluminescence mechanism in the protein imprinting method, and the probe molecule based on the quinoline nitrile matrix for constructing the aggregation-induced emission mechanism realizes low-cost, long-time stability, wide range and quantitative detection on the protein in the protein imprinting method.
Figure DDA0002803501190000011

Description

Application of quinolinecarbonitrile derivative in protein detection by protein imprinting method and preparation method thereof
Technical Field
The invention belongs to the technical field of fine chemical engineering, and particularly relates to a method for improving the problems of short luminescence time, narrow detection range, poor quantitative repeatability, high commercial fluorescence detection cost and the like of a traditional reagent Luminol (Luminol) based on a chemiluminescence mechanism in a protein imprinting method (Western blot).
Background
In 2001, the university of hong kong science and technology, down-council academy, discovered the compound of aggregation-induced emission (AIE) (chem.commun.,2001,1740-1741) HPS, which exhibits a luminescent property completely opposite to that of conventional fluorescent molecules, and since this AIE phenomenon is widely used in various fields, especially in the field of bioimaging (angelw.chem.int.ed.2020, 59,9812). The Zhu-Macro topic group obtains a novel AIE parent quinoline nitrile (QM) (ACS appl.Mater.Interfaces 2013,5,192) by reasonably designing and changing an oxygen atom of a traditional benzopyran nitrile into a nitrogen ethyl group, has a methyl site capable of carrying out a Knoevenagel reaction, and synthesizes a plurality of quinoline nitrile derivatives with different wavelengths and adjustable morphologies for various biological imaging (Angew.chem.int.Ed.2020,59, 9812-. However, conventional AIE molecules lack water-solubilizing groups, which results in their high initial fluorescence in aqueous solutions, which results in their greatly limited application in biological assays.
The traditional protein printing method (Western blot) has complicated steps and large workload, and the main steps comprise protein extraction, gel running, membrane transfer, primary antibody incubation, secondary antibody incubation and color development of a color developing agent (anal. biochem.2009,384, 348). Among them, the existing color developing agent mostly uses Luminol (Luminol) reagent based on a chemiluminescence mechanism, and realizes qualitative detection of protein by detecting horseradish peroxidase (HRP) connected with a secondary antibody, but the reagent has the defects of short luminescence time, narrow detection range, poor quantitative repeatability and the like, so that errors and workload of experiments are greatly increased (Journal of immunological methods, 2010,353 and 148). In addition, existing fluorescence assays require the attachment of a fluorescent molecule to an antibody, which makes commercial fluorescence detection reagents expensive and not widely available in the laboratory (clin. chim. acta.2011,412, 107.). Therefore, how to realize low cost, long-time stability, wide range and quantitative detection has very important significance.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to overcome the defects in the prior art, provide the application of the water-soluble quinoline nitrile derivative without initial fluorescence in protein detection by a protein imprinting method and a preparation method thereof, and realize the long-time stable, wide-range and quantitative detection of the protein in Western blot.
One of the purposes of the invention is to provide application of quinoline nitrile derivatives in protein detection by protein imprinting method, wherein the quinoline nitrile derivatives are compounds shown as formula I
Figure BDA0002803501170000021
In the formula I, the compound has the following structure,
R1independently selected from: any one or none of monophenyl phosphate or 4- (thiophen-2-yl) phenyl phosphate;
R2is hydroxyl or halogen or dimethylamino or trimethylamine or carboxyl or none;
R3is halogen or methoxy or hydroxyl or dimethylamino or trimethylamine group or N, N-dimethylaniline or triphenylamine or pyridine or carboxyl or aldehyde group or cyano or nitro or phosphoric acid or monophenyl phosphate or none.
The technical effects are as follows:
1. according to the preparation method, the hydrophilic phosphate group is introduced to the AIE parent quinoline nitrile to construct the ALP lighting type probe I-1, and the ALP lighting type probe has the advantages of high phase response fluorescence intensity, good selectivity, high linear relation between the response fluorescence intensity and the enzyme activity and the like.
2. The invention realizes the color development of the protein in Western blot by designing fluorescent molecules of an aggregation-induced emission mechanism, thereby realizing the application of detecting the protein in a protein imprinting method.
3. The application of the invention has the same color development accuracy with the commercial ECL reagent. Compared with the commercially available ECL color developing agent, the application of the invention greatly improves the retention time after color development, and has important practical application significance. Compared with the commercially available ECL color developing agent, the probe I-1 has higher stability and higher protein quantification capability all the time on a time scale, and provides a practical new method for protein quantification.
4. The probe I-1 obtained by the application and development of the invention has low cost, and simultaneously, the application range of the secondary antibody (ALP connection) is expanded.
Drawings
FIG. 1 shows nuclear magnetic hydrogen spectrum of dye I-1.
FIG. 2 nuclear magnetic carbon spectrum of dye I-1.
FIG. 3 high resolution mass spectrum of dye I-1.
FIG. 4 is a UV absorption spectrum of dye I-1 with increasing water content in a mixed solvent of dimethyl sulfoxide and water (10)- 5mol/L)。
FIG. 5 fluorescence emission spectrum of dye I-1 with increasing water content in a mixed solvent of dimethyl sulfoxide and water (10)- 5mol/L)。
FIG. 6 is a UV absorption spectrum of dye I-1 with increasing water content in a mixed solvent of tetrahydrofuran and water (10)- 5mol/L)。
FIG. 7 shows fluorescence emission spectrum of dye I-1 with increasing water content in a mixed solvent of tetrahydrofuran and water (10)- 5mol/L)。
FIG. 8 is an ultraviolet absorption spectrum (10) of dye I-1 with increasing water content in a mixed solvent of ethanol and water-5mol/L)。
FIG. 9 fluorescence emission spectrum of dye I-1 with increasing water content in a mixed solvent of ethanol and water (10)-5mol/L)。
FIG. 10 shows the absorption spectrum of dye I-1 with time after addition of alkaline phosphatase (150U/L) at 25 ℃.
FIG. 11 shows fluorescence emission spectra of dye I-1 with time after addition of alkaline phosphatase (150U/L) at 25 ℃.
FIG. 12 is a linear graph of the fluorescence intensity (560nm) of dye I-1 at 25 ℃ in relation to the alkaline phosphatase content.
FIG. 13 shows fluorescence emission spectra of dye I-1 with time after addition of alkaline phosphatase (150U/L) at 37 ℃.
FIG. 14 shows fluorescence emission spectra of dye I-1 with time after addition of different amounts of alkaline phosphatase at 37 ℃.
FIG. 15 is a linear graph of the fluorescence intensity (560nm) of dye I-1 at 37 ℃ in relation to the alkaline phosphatase content.
FIG. 16 is a graph showing luminescence change with time and intensity analysis of commercially available Luminol after addition of horseradish peroxidase (1U/mL).
FIG. 17 is a graph showing luminescence change with time and intensity analysis after adding alkaline phosphatase (150U/L) to dye I-1.
FIG. 18 is a time-dependent analysis of the intensity of the chromoprotein of dye I-1.
FIG. 19 is a graph showing the protein band and gray level analysis after 40min of protein development by dye I-2.
FIG. 20 is a graph showing the protein band and gray level analysis after 2h of protein development by dye I-3.
FIG. 21 is a graph showing the protein band and gray level analysis after 6h of protein development by dye I-4.
FIG. 22 is a graph showing the protein band and gray level analysis after the dye I-5 develops the protein for 24 hours.
FIG. 23 protein bands after 10min of dye I-6 development on different cell-derived proteins.
FIG. 24 protein bands of dye I-7 after 40min of color development on different cell-derived proteins.
FIG. 25 is a color development and gray scale analysis of dye I-8 on concentration gradient protein.
FIG. 26 is a graph showing the color development of dye I-9 on concentration gradient protein for 60min and gray scale analysis.
Detailed Description
The invention is further illustrated by the following examples, which are intended only for a better understanding of the contents of the invention. The examples given therefore do not limit the scope of protection of the invention:
example 1
(1) Synthesis of dye I-1:
Figure BDA0002803501170000041
in a 50mL single-neck flask, 4-dicyanomethylene-2-methylquinolinecarbonitrile (470.6mg, 2.0mmol), acetonitrile (20mL), p-hydroxybenzaldehyde (244.2mg, 2.0mmol), piperidine 1.0mL, and under argon atmosphere were added and reacted at 110 ℃ for 10 hours. Cooling and spinningThe solvent was evaporated and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) afforded an orange-red solid (350mg,1.03mmol) with 51.5% yield.1H NMR(400MHz,DMSO-d6,ppm):δ＝1.46(t,J＝7.2Hz,3H,-CH2CH3),4.61(q,J＝7.2Hz,2H,-CH2CH3),6.90(d,J＝8.8Hz,2H,phenyl-H),7.06(s,1H,quinoline-H),7.38(m,J＝15.6Hz，2H,alkene-H),7.65(t,J＝7.6Hz,1H,phenyl-H),7.73(d,J＝8.4Hz,2H,phenyl-H),7.97(t,J＝8.0Hz,1H,phenyl-H),8.13(d,J＝9.2Hz,1H,phenyl-H),8.96(d,J＝8.4Hz,1H，phenyl-H),10.06(s,1H，-OH).Mass spectrometry(ESI positive ion mode for[M+Na]+):Calcd.for C22H17N3O:362.1269；found:362.1276.
Figure BDA0002803501170000042
In a 100mL single-neck flask, the product of the previous step (51mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran were added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine were added under ice bath, the mixture was stirred at room temperature for 2 hours under nitrogen protection, 20mL of water was then added to the reaction mixture, the mixture was stirred at room temperature for 30 minutes, the tetrahydrofuran was dried by spinning, dichloromethane (3 × 50mL) was added for extraction, the organic phase was washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried over anhydrous sodium sulfate, the mixture was applied to a column, and column chromatography was performed (dichloromethane: methanol ═ 5: 1). 15mg of a yellow solid are obtained, yield 24%.1H NMR(400MHz,DMSO-d6,ppm):δ＝1.40(t,J＝7.2Hz,3H,-CH2CH3),4.57(t,J＝7.2Hz,2H,-CH2CH3),7.03(s,1H,quinoline-H),7.26(d,J＝8.4Hz,2H,phenyl-H),7.46(m,J＝15.6Hz,2H,alkene-H),7.63(t,J＝7.6Hz,1H,phenyl-H),7.84(d,J＝8.8Hz,2H,phenyl-H),7.94(t,J＝7.6Hz,1H,phenyl-H),8.10(d,J＝8.8Hz,1H,phenyl-H),8.94(d,J＝8.4Hz,1H,phenyl-H).Mass spectrometry(ESI negative ion mode for[M-H]-):Calcd.for C22H18N3O:418.0957；found:418.0956.
(2) Synthesis of dye I-2:
in a 50mL single-neck flask, 5-bromothiophene-2-carbaldehyde (1.91g, 10mmol), 4-hydroxyphenylboronic acid (1.38g,10mmol), potassium carbonate (41.5g,300mmol), tetrahydrofuran (90mL), water (270mL), and tetrakis (triphenylphosphine) palladium (0.10g,0.1mmol) were added, respectively. The reaction was stirred at reflux for 12 h. Cooling, rotary evaporation of the solvent, extraction with dichloromethane (200mL), drying of the organic phase by counting anhydrous sodium sulfate, rotary drying of the organic phase and separation by silica gel column chromatography (DCM: hexane ═ 1: 1) gave a solid (1.26g,6.2mmol) in 62% yield.
Figure BDA0002803501170000051
In a 100mL single-neck flask, the product of the previous step (410mg, 2.0mmol), 4-dicyanomethylene-2-methylquinolinecarbonitrile (470mg, 2.0mmol), acetonitrile (20mL), piperidine 1.0mL, under argon, was added and reacted at 110 ℃ for 10 h. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave a red solid (280mg,0.66mmol) in 33.2% yield.
Figure BDA0002803501170000052
In a 100mL single-neck flask, the product of the previous step (64mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran are added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine are added under ice bath, the mixture is stirred at room temperature for 2h under nitrogen protection, 20mL of water is added to the reaction solution, the mixture is stirred at room temperature for 30min, the tetrahydrofuran is dried by spinning, dichloromethane (3 × 50mL) is added for extraction, the organic phase is washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried with anhydrous sodium sulfate, the mixture is applied to a column by spinning, and column chromatography is carried out (dichloromethane: methanol ═ 5: 1). 22mg of a yellow solid are obtained in 30% yield.
Figure BDA0002803501170000061
(3) Synthesis of dye I-3:
in a 50mL single-neck flask, 6-nitro-4-dicyanomethylene-2-methylquinolinecarbonitrile (560mg, 2.0mmol), acetonitrile (20mL), p-hydroxybenzaldehyde (244.2mg, 2.0mmol), and piperidine (1.0 mL) were added, respectively, and reacted at 110 ℃ for 10 hours under argon. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave an orange-red solid (404mg,1.14mmol) with 57% yield.
Figure BDA0002803501170000062
In a 100mL single-neck flask, the product of the previous step (53mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran were added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine were added under ice bath, the mixture was stirred at room temperature for 2 hours under nitrogen protection, 20mL of water was then added to the reaction mixture, the mixture was stirred at room temperature for 30 minutes, the tetrahydrofuran was dried by spinning, dichloromethane (3 × 50mL) was added for extraction, the organic phase was washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried over anhydrous sodium sulfate, the mixture was applied to a column, and column chromatography was performed (dichloromethane: methanol ═ 5: 1). 22mg of a yellow solid are obtained in 32% yield.
Figure BDA0002803501170000063
(4) Synthesis of dye I-4:
in a 50mL single-neck flask, 6-pyridine-4-dicyanomethylene-2-methylquinolinecarbonitrile (624mg, 2.0mmol), acetonitrile (20mL), p-hydroxybenzaldehyde (244.2mg, 2.0mmol), and piperidine (1.0 mL) were added, respectively, and reacted at 110 ℃ for 10 hours under argon. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave an orange-red solid (460mg,1.11mmol) with 55.3% yield.
Figure BDA0002803501170000071
In a 100mL single-neck flask, the product of the previous step (62mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran were added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine were added under ice bath, the mixture was stirred at room temperature for 2 hours under nitrogen protection, 20mL of water was then added to the reaction mixture, the mixture was stirred at room temperature for 30 minutes, the tetrahydrofuran was dried by spinning, dichloromethane (3 × 50mL) was added for extraction, the organic phase was washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried over anhydrous sodium sulfate, the mixture was applied to a column, and column chromatography was performed (dichloromethane: methanol ═ 5: 1). 22mg of a yellow solid are obtained, yield 29%.
Figure BDA0002803501170000072
(5) Synthesis of dye I-5:
in a 50mL single-neck flask, 5-bromothiophene-2-carbaldehyde (1.91g, 10mmol), 4-hydroxyphenylboronic acid (1.38g,10mmol), potassium carbonate (41.5g,300mmol), tetrahydrofuran (90mL), water (270mL), and tetrakis (triphenylphosphine) palladium (0.10g,0.1mmol) were added, respectively. The reaction was stirred at reflux for 12 h. Cooling, rotary evaporation of the solvent, extraction with dichloromethane (200mL), drying of the organic phase by counting anhydrous sodium sulfate, rotary drying of the organic phase and separation by silica gel column chromatography (DCM: hexane ═ 1: 1) gave a solid (1.26g,6.2mmol) in 62% yield.
Figure BDA0002803501170000073
In a 100mL single-neck flask, the product of the previous step (560mg, 2.0mmol), 6-nitro-4-dicyanomethylene-2-methylquinolinecarbonitrile (470mg, 2.0mmol), acetonitrile (20mL), piperidine (1.0 mL) were added, and the mixture was reacted at 110 ℃ for 10 hours under argon. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave a red solid (355mg,0.76mmol) in 38% yield.
Figure BDA0002803501170000081
In a 100mL single-neck flask, the product of the previous step (70mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran are added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine are added under ice bath, the mixture is stirred at room temperature for 2h under nitrogen protection, 20mL of water is added to the reaction solution, the mixture is stirred at room temperature for 30min, the tetrahydrofuran is dried by spinning, dichloromethane (3 × 50mL) is added for extraction, the organic phase is washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried with anhydrous sodium sulfate, the mixture is applied to a column by spinning, and column chromatography is carried out (dichloromethane: methanol ═ 5: 1). 16mg of a yellow solid are obtained in 20% yield.
Figure BDA0002803501170000082
(6) Synthesis of dye I-6:
in a 50mL single-neck flask, 5-bromothiophene-2-carbaldehyde (1.91g, 10mmol), 4-hydroxyphenylboronic acid (1.38g,10mmol), potassium carbonate (41.5g,300mmol), tetrahydrofuran (90mL), water (270mL), and tetrakis (triphenylphosphine) palladium (0.10g,0.1mmol) were added, respectively. The reaction was stirred at reflux for 12 h. Cooling, rotary evaporation of the solvent, extraction with dichloromethane (200mL), drying of the organic phase by counting anhydrous sodium sulfate, rotary drying of the organic phase and separation by silica gel column chromatography (DCM: hexane ═ 1: 1) gave a solid (1.26g,6.2mmol) in 62% yield.
Figure BDA0002803501170000083
In a 100mL single-neck flask, the product of the previous step (560mg, 2.0mmol), 6-pyridine-4-dicyanomethylene-2-methylquinolinecarbonitrile (624mg, 2.0mmol), acetonitrile (20mL), piperidine (1.0 mL) were added, and the mixture was reacted at 110 ℃ for 10 hours under argon. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave a red solid (409mg,0.82mmol) in 41% yield.
Figure BDA0002803501170000091
In a 100mL single-neck flask, the product of the previous step (75mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran are added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine are added under ice bath, the mixture is stirred at room temperature for 2h under nitrogen protection, 20mL of water is added to the reaction solution, the mixture is stirred at room temperature for 30min, the tetrahydrofuran is dried by spinning, dichloromethane (3 × 50mL) is added for extraction, the organic phase is washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried with anhydrous sodium sulfate, the mixture is applied to a column by spinning, and column chromatography is carried out (dichloromethane: methanol ═ 5: 1). 28mg of a yellow solid are obtained in 32% yield.
Figure BDA0002803501170000092
(7) Synthesis of dye I-7:
in a 50mL single-neck flask, 6-bromo-4-dicyanomethylene-2-methylquinolinecarbonitrile (628mg, 2.0mmol), acetonitrile (20mL), p-hydroxybenzaldehyde (244.2mg, 2.0mmol), and piperidine (1.0 mL) were added, respectively, and reacted at 110 ℃ for 10 hours under argon. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave an orange-red solid (502mg,1.20mmol) with 60% yield.
Figure BDA0002803501170000093
In a 100mL single-neck flask, the product of the previous step (63mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran were added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine were added under ice bath, the mixture was stirred at room temperature for 2 hours under nitrogen protection, 20mL of water was then added to the reaction mixture, the mixture was stirred at room temperature for 30 minutes, the tetrahydrofuran was dried by spinning, dichloromethane (3 × 50mL) was added for extraction, the organic phase was washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried over anhydrous sodium sulfate, the mixture was applied to a column, and column chromatography was performed (dichloromethane: methanol ═ 5: 1). 27mg of a yellow solid are obtained, yield 36%.
Figure BDA0002803501170000101
(8) Synthesis of dye I-8:
in a 50mL single-neck flask, 5-bromothiophene-2-carbaldehyde (1.91g, 10mmol), 4-hydroxyphenylboronic acid (1.38g,10mmol), potassium carbonate (41.5g,300mmol), tetrahydrofuran (90mL), water (270mL), and tetrakis (triphenylphosphine) palladium (0.10g,0.1mmol) were added, respectively. The reaction was stirred at reflux for 12 h. Cooling, rotary evaporation of the solvent, extraction with dichloromethane (200mL), drying of the organic phase by counting anhydrous sodium sulfate, rotary drying of the organic phase and separation by silica gel column chromatography (DCM: hexane ═ 1: 1) gave a solid (1.26g,6.2mmol) in 62% yield.
Figure BDA0002803501170000102
In a 100mL single-neck flask, the product of the previous step (560mg, 2.0mmol), 6-bromo-4-dicyanomethylene-2-methylquinolinecarbonitrile (628mg, 2.0mmol), acetonitrile (20mL), piperidine 1.0mL, under argon and reaction at 110 ℃ for 10h were added. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave a red solid (355mg,0.76mmol) in 38% yield.
Figure BDA0002803501170000103
In a 100mL single-neck flask, the product of the previous step (70mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran are added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine are added under ice bath, the mixture is stirred at room temperature for 2h under nitrogen protection, 20mL of water is added to the reaction solution, the mixture is stirred at room temperature for 30min, the tetrahydrofuran is dried by spinning, dichloromethane (3 × 50mL) is added for extraction, the organic phase is washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried with anhydrous sodium sulfate, the mixture is applied to a column by spinning, and column chromatography is carried out (dichloromethane: methanol ═ 5: 1). 16mg of a yellow solid are obtained in 20% yield.
Figure BDA0002803501170000111
(9) Synthesis of dye I-9:
in a 50mL single-neck flask, 6-methoxy-4-dicyanomethylene-2-methylquinolinecarbonitrile (530mg, 2.0mmol), acetonitrile (20mL), 4-N, N-dimethylbenzaldehyde (298mg, 2.0mmol), piperidine (1.0 mL), and argon were added, respectively, and reacted at 110 ℃ for 10 hours. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave a yellow solid (253mg,0.64mmol) with 32% yield.
Figure BDA0002803501170000112
In a 100mL single-neck flask, the product of the previous step (237mg, 0.60mmol), 35mL of ultra-dry tetrahydrofuran were added, 3mL of a 1mol/L boron tribromide/dichloromethane solution was added under ice-bath stirring for 30min, then stirred at room temperature for 4h, then 20mL of water was added to the reaction solution, stirred at room temperature for 30min, tetrahydrofuran was dried by spinning, dichloromethane (3 × 50mL) was added for extraction, followed by drying over anhydrous sodium sulfate, spin-drying on the column, and column chromatography (dichloromethane: methanol ═ 10: 1). 150mg of a yellow solid are obtained, yield 65%.
Figure BDA0002803501170000113
In a 100mL single-neck flask, the product of the previous step (57mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran were added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine were added under ice bath, the mixture was stirred at room temperature for 2 hours under nitrogen protection, 20mL of water was then added to the reaction mixture, the mixture was stirred at room temperature for 30 minutes, the tetrahydrofuran was dried by spinning, dichloromethane (3 × 50mL) was added for extraction, the organic phase was washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried over anhydrous sodium sulfate, the mixture was applied to a column, and column chromatography was performed (dichloromethane: methanol ═ 5: 1). 22mg of a yellow solid are obtained in 32% yield.
Figure BDA0002803501170000121
(10) Synthesis of dye I-10:
in a 50mL single-neck flask, 5-bromothiophene-2-carbaldehyde (1.91g, 10mmol), 4-N, N-dimethylbenzeneboronic acid (1.65g,10mmol), potassium carbonate (41.5g,300mmol), tetrahydrofuran (90mL), water (270mL), and tetrakis (triphenylphosphine) palladium (0.10g,0.1mmol) were added, respectively. The reaction was stirred at reflux for 12 h. Cooling, rotary evaporation of the solvent, extraction with dichloromethane (200mL), drying of the organic phase by counting anhydrous sodium sulfate, rotary drying of the organic phase and separation by silica gel column chromatography (DCM: hexane ═ 1: 1) gave a solid (1.26g,6.2mmol) in 62% yield.
Figure BDA0002803501170000122
In a 50mL single-neck flask, 6-methoxy-4-dicyanomethylene-2-methylquinolinecarbonitrile (530mg, 2.0mmol), acetonitrile (20mL), 5-phencyclyldiaminothiophene-2-carbaldehyde (462mg, 2.0mmol), piperidine 1.0mL, and under argon protection, were added and reacted at 110 ℃ for 10 hours. Cooling, rotary evaporation of the solvent and silica gel column chromatography (DCM: MeOH ═ 10:1 separation) gave an orange-red solid (354mg,0.74mmol) with 37% yield.
Figure BDA0002803501170000123
In a 100mL single-neck flask, the product of the previous step (303mg, 0.60mmol), 35mL of ultra-dry tetrahydrofuran were added, 3mL of a 1mol/L boron tribromide/dichloromethane solution was added under ice-bath stirring for 30min, then stirred at room temperature for 4h, then 20mL of water was added to the reaction solution, stirred at room temperature for 30min, tetrahydrofuran was spun dry, dichloromethane (3 × 50mL) was added for extraction, followed by drying over anhydrous sodium sulfate, spin-drying on the column, and column chromatography (dichloromethane: methanol ═ 10: 1). 224mg of a yellow solid are obtained, yield 80%.
Figure BDA0002803501170000131
In a 100mL single-neck flask, the product of the previous step (70mg, 0.15mmol), 35mL of ultra-dry tetrahydrofuran are added, 0.2mL of ultra-dry phosphorus oxychloride and 0.1mL of ultra-dry pyridine are added under ice bath, the mixture is stirred at room temperature for 2h under nitrogen protection, 20mL of water is added to the reaction solution, the mixture is stirred at room temperature for 30min, the tetrahydrofuran is dried by spinning, dichloromethane (3 × 50mL) is added for extraction, the organic phase is washed with an anhydrous copper sulfate solution (3 × 50mL), and then dried with anhydrous sodium sulfate, the mixture is applied to a column by spinning, and column chromatography is carried out (dichloromethane: methanol ═ 5: 1). 15mg of a yellow solid are obtained, yield 18%.
Figure BDA0002803501170000132
Example 2
Absorption and fluorescence spectra of dye I-1 in a dimethyl sulfoxide (DMSO)/water system
The dye I-1 prepared in example 1 was dissolved in analytically pure dimethyl sulfoxide to give a solution of 1.0X 10-3Stock solutions of M. 2970 μ L of DMSO/water mixed solvent in different ratios was then prepared. 30 mu.L of the stock solution is added into prepared DMSO/water mixed solvents with different proportions, and the mixed solution is transferred into an optical quartz cuvette (10X 10mm) to test the absorption and fluorescence spectrum of the mixed solution. As shown in FIGS. 4 and 5, dye I-1 exhibited a broad absorption peak at 300-550nm, and the maximum absorption wavelength was located at 432 nm; with 432nm as an excitation wavelength, the maximum emission peak of the dye I-1 is approximately positioned at 560nm, and the Stokes shift reaches 128 nm; and dye I-1 is non-fluorescent in aqueous phase and has potential for biological applications.
Example 3
Absorption and fluorescence spectra of dye I-1 in Tetrahydrofuran (THF)/water system
The dye I-1 prepared in example 1 was dissolved in analytically pure THF to give a solution of 1.0X 10-3Stock solutions of M. Then 2970. mu.L of THF/water mixed solvent in different ratios was prepared. 30 mu.L of the stock solution is added into THF/water mixed solvents prepared in different proportions, and the mixture is transferred into an optical quartz cuvette (10X 10mm) to test the absorption and fluorescence spectrum. As shown in FIGS. 6 and 7, dye I-1 exhibited a broad absorption peak at 300-550nm, and the maximum absorption wavelength was located at 432 nm; with 432nm as an excitation wavelength, the maximum emission peak of the dye I-1 is approximately positioned at 560nm, and the Stokes shift reaches 128 nm; and dye I-1 is non-fluorescent in aqueous phase and has potential for biological applications.
Example 4
Absorption and fluorescence spectra of dye I-1 in ethanol/water system
The dye I-1 prepared in example 1 was dissolved in analytically pure ethanol to give a solution of 1.0X 10-3Stock solutions of M. Then 2970 μ L of ethanol/water mixed solvent of different proportions was prepared. 30 mu L of the stock solution is added into the prepared ethanol/water mixed solvent with different proportions, and the mixture is transferred into an optical quartz cuvette (10 x 10mm) to test the absorption and fluorescence spectrum of the mixture. As shown in FIG. 8 and9, dye I-1 exhibits a broad absorption peak at 300-550nm, and the maximum absorption wavelength is at 432 nm; with 432nm as an excitation wavelength, the maximum emission peak of the dye I-1 is approximately positioned at 560nm, and the Stokes shift reaches 128 nm; and dye I-1 is non-fluorescent in aqueous phase and has potential for biological applications.
Example 5
Temporal fluorescence response of dye I-1 to alkaline phosphatase (ALP)
To investigate the response of dye I-1 to ALP, we first formulated the concentration to be 1X 10-52970 μ L of a solution of I-1 in mol/L in water (Tris/DMSO 95:5, v/v,50mM, pH 7.4). Then, 30. mu.L of ALP having an activity of 1500U/L was added to the solution to dilute it at a concentration of 150U/L. After mixing well, the mixture was quickly transferred to an optical cuvette and measured for absorbance and fluorescence spectra every two minutes at 25 ℃. As shown in FIGS. 10 to 15, the absorption spectrum intensity gradually decreased and the fluorescence intensity gradually increased with increasing time, and the fluorescence enhancement at 560nm had a very good linear relationship (R)2＝0.991)。
Example 6
Luminescence time variation and grayscale analysis of horseradish peroxidase (HRP) with commercial ECL reagent
Transferring 744 μ L of commercial ECL reagent A liquid B liquid to an optical quartz cuvette (10 × 10mm), transferring 12 μ L of HRP solution (260U/mL), adding into the cuvette to dilute 1U/mL, mixing well, and taking luminescence pictures in the cuvette every 20 s. The results of the grey value analysis using Photoshop software are shown in FIG. 16.
Example 7
Luminescence time variation and gradation analysis of dye I-1 for alkaline phosphatase (ALP)
Firstly, the concentration is prepared to be 1 multiplied by 10-52970 μ L of a solution of I-1 in mol/L in water (Tris/DMSO 95:5, v/v,50mM, pH 7.4). Then, 30. mu.L of ALP having an activity of 1500U/L was added to the solution to dilute it at a concentration of 150U/L. After being mixed uniformly, the mixture is quickly transferred to an optical cuvette and the luminescent pictures in the cuvette are taken every 30 s. The results of the grey value analysis using Photoshop software are shown in FIG. 17.
Example 8
Long-time protein color development and quantitative analysis by dye I-1
Six groups of 120 mu g of protein liquid extracted from Hela cells are transferred into the two fast gel holes, and then the components are cut into two PVDF membranes after gel running, membrane transferring, sealing and washing, and the corresponding Caspase 3, alpha-tubulin, Catalase and Ras rabbit primary antibody are incubated for 12 hours at 4 ℃. Three more groups were incubated with rabbit secondary antibody (HRP-linked) and rabbit secondary antibody (ALP-linked) for 1h at room temperature. Before exposure and color development on a computer, two PVDF films are respectively dripped with a commercially available ECL reagent (solution A + solution B) and 1 × 10-5mol/L of I-1 in water (incubation for 20 min). Finally, the ImageQuant LAS4000 instrument was used to expose the samples for 30s at different time points. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 18.
Example 9
Dye I-2 bands after 40min of gradient concentration protein development and quantitative analysis
To the gel wells 290,230,170,140,120,60 μ g of protein solution extracted from Hela cells were transferred, followed by gel running, membrane transfer, blocking, washing, and incubation of the corresponding rabbit primary antibodies (. beta. -Catenin, Ras and Catalase) at 4 ℃ for 12 hours. Rabbit secondary antibodies (ALP ligation) were then incubated for 1h at room temperature. Before exposure and color development on a computer, 1X 10 is dripped into PVDF film-5mol/L of I-2 in water (incubation for 20 min). After 40min, each was exposed for 30s using an ImageQuant LAS4000 instrument. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 19.
Example 10
Strip of dye I-3 after gradient concentration protein color development for 2h and quantitative analysis
To the gel wells 290,230,170,140,120,60 μ g of protein solution extracted from Hela cells were transferred, followed by gel running, membrane transfer, blocking, washing, and incubation of the corresponding rabbit primary antibodies (. beta. -Catenin, Ras, Catalase and GAPDH) at 4 ℃ for 12 h. Rabbit secondary antibodies (ALP ligation) were then incubated for 1h at room temperature. Before exposure and color development on a computer, 1X 10 is dripped into PVDF film-5mol/L of I-3 in water (incubation for 20 min). After 2h, each was exposed for 30s using an ImageQuant LAS4000 instrument. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 20.
Example 11
Strip of dye I-4 after gradient concentration protein color development for 6h and quantitative analysis
To the gel wells 290,230,170,140,120,60 μ g of protein solution extracted from Hela cells were transferred, followed by gel running, membrane transfer, blocking, washing, and incubation of the corresponding rabbit primary antibodies (. beta. -Catenin, Ras, Catalase and GAPDH) at 4 ℃ for 12 h. Rabbit secondary antibodies (ALP ligation) were then incubated for 1h at room temperature. Before exposure and color development on a computer, 1X 10 is dripped into PVDF film-5mol/L of I-4 in water (incubation for 20 min). After 6h, each was exposed for 30s using an ImageQuant LAS4000 instrument. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 21.
Example 12
Strip of dye I-5 after 24h of gradient concentration protein development and quantitative analysis
To the gel wells 290,230,170,140,120,60 μ g of protein solution extracted from Hela cells were transferred, followed by gel running, membrane transfer, blocking, washing, and incubation of the corresponding rabbit primary antibodies (. beta. -Catenin, Ras, Catalase and GAPDH) at 4 ℃ for 12 h. Rabbit secondary antibodies (ALP ligation) were then incubated for 1h at room temperature. Before exposure and color development on a computer, 1X 10 is dripped into PVDF film-5mol/L of I-5 in water (incubation for 20 min). After 24h, each was exposed for 30s using an ImageQuant LAS4000 instrument. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 22.
Example 13
Commercial ECL reagent and dye I-6 develop color for 10min experiment on protein from multiple sources
Five groups of 120 mu g of protein solution extracted from human breast cancer cells (MCF-7), human ovarian cancer cells (SKOV-3), human lung adenocarcinoma cells (H-1975), human breast cancer cells (MBA-MD-231), human gastric adenocarcinoma cells (SGC-7901) and Hela cells are respectively transplanted into the same two gel wells, and then gel running, membrane transferring and sealing are carried out, two PVDF membranes are washed, cut and respond to incubation of beta-Catenin, Catalase and Ras rabbit primary antibody for 12 hours at 4 ℃. The secondary membranes were incubated with rabbit secondary antibody (HRP-linked) and rabbit secondary antibody (ALP-linked) for 1h at room temperature. Before exposure and color development on a computer, two PVDF films are respectively dripped with a commercially available ECL reagent (solution A + solution B) and 1 × 10-5mol/L of I-1 in water (incubation for 20 min). After 10min, the samples were separated by ImageQuant LAS4000 instrumentAnd exposing for 30 s. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 23.
Example 14
Commercial ECL reagent and dye I-7 develop color for 40min experiment on protein from various sources
Five groups of 120 mu g of protein solution extracted from human breast cancer cells (MCF-7), human ovarian cancer cells (SKOV-3), human lung adenocarcinoma cells (H-1975), human breast cancer cells (MBA-MD-231), human gastric adenocarcinoma cells (SGC-7901) and Hela cells are respectively transplanted into the same two gel wells, and then gel running, membrane transferring and sealing are carried out, two PVDF membranes are washed, cut and respond to incubation of beta-Catenin, Catalase and Ras rabbit primary antibody for 12 hours at 4 ℃. The secondary membranes were incubated with rabbit secondary antibody (HRP-linked) and rabbit secondary antibody (ALP-linked) for 1h at room temperature. Before exposure and color development on a computer, two PVDF films are respectively dripped with a commercially available ECL reagent (solution A + solution B) and 1 × 10-5mol/L of I-1 in water (incubation for 20 min). After 40min, each was exposed for 30s using an ImageQuant LAS4000 instrument. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 24.
Example 15
Strip and quantitative analysis of gradient concentration protein developed by dye I-8 for 0min
To the gel wells 290,230,170,140,120,60 μ g of protein solution extracted from Hela cells were transferred, followed by gel running, membrane transfer, blocking, washing, and incubation of the corresponding rabbit primary antibodies (. beta. -Catenin, Ras, Catalase and caspase 3) at 4 ℃ for 12 h. Rabbit secondary antibodies (ALP ligation) were then incubated for 1h at room temperature. Before exposure and color development on a computer, 1X 10 is dripped into PVDF film- 5The I-5 solution in mol/L (incubation for 20min) was exposed to light for 30s using ImageQuant LAS4000 instrument. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 25.
Example 16
Strip of dye I-9 after gradient concentration protein color development for 60min and quantitative analysis
To the gel wells 290,230,170,140,120,60 μ g of protein solution extracted from Hela cells were transferred, followed by gel running, membrane transfer, blocking, washing, and incubation of the corresponding rabbit primary antibodies (. beta. -Catenin, Ras, Catalase and caspase 3) at 4 ℃ for 12 h. Then room temperatureRabbit secondary antibodies (ALP ligation) were incubated for 1 h. Before exposure and color development on a computer, 1X 10 is dripped into PVDF film- 5mol/L of I-5 in water (incubation for 20 min). After 60min, each was exposed for 30s using an ImageQuant LAS4000 instrument. Photoshop software performed the band gray value analysis, and the results are shown in FIG. 26.

Claims (12)

1. The application of the quinoline nitrile derivative in protein detection by a protein imprinting method is that the quinoline nitrile derivative is shown as a compound shown in a formula I:
Figure RE-FDA0002973981640000011
in the formula I, the compound has the following structure,
R1independently selected from: any one of monophenyl phosphate, 4- (thiophen-2-yl) benzene phosphate, N-dimethyl-4- (thiophen-2-yl) aniline, N-diphenyl-4- (thiophen-2-yl) aniline, triphenylamine or thiophen triphenylamine;
R2hydroxyl, halogen, dimethylamino, trimethylamine, carboxyl or none;
R3halogen, methoxy, hydroxyl, dimethylamino, trimethylamine group, N-dimethylaniline, triphenylamine, pyridine, carboxyl, aldehyde group, cyano, nitro, phosphoric acid, monophenyl phosphate or nothing.
2. The use of the quinolinecarbonitrile derivative of claim 1 for protein detection by western blotting, said quinolinecarbonitrile derivative being one of:
Figure RE-FDA0002973981640000021
3. the method for preparing quinolinecarbonitrile derivatives for detecting proteins by western blotting as claimed in claim 2, wherein the probe for ALP luminescent type I-1:
synthesis of dye I-1:
Figure RE-FDA0002973981640000031
4. the process for preparing quinolinecarbonitrile derivatives for the detection of proteins by western blotting according to claim 2, wherein the synthesis of dye I-2:
Figure RE-FDA0002973981640000032
5. the process for preparing quinolinecarbonitrile derivatives for detecting proteins by Western blotting as claimed in claim 2, wherein,
synthesis of dye I-3:
Figure RE-FDA0002973981640000041
6. the process for preparing quinolinecarbonitrile derivatives for the detection of proteins by western blotting according to claim 2, wherein the synthesis of dye I-4:
Figure RE-FDA0002973981640000042
7. the process for preparing quinolinecarbonitrile derivatives for the detection of proteins by western blotting according to claim 2, wherein the synthesis of dye I-5:
Figure RE-FDA0002973981640000043
Figure RE-FDA0002973981640000051
8. the process for preparing quinolinecarbonitrile derivatives for the detection of proteins by western blotting according to claim 2, wherein the synthesis of dye I-6:
Figure RE-FDA0002973981640000052
9. the process for preparing quinolinecarbonitrile derivatives for the detection of proteins by western blotting according to claim 2, wherein the synthesis of dye I-7:
Figure RE-FDA0002973981640000053
10. the process for preparing quinolinecarbonitrile derivatives for the detection of proteins by western blotting according to claim 2, wherein the synthesis of dye I-8:
Figure RE-FDA0002973981640000054
Figure RE-FDA0002973981640000061
11. the process for preparing quinolinecarbonitrile derivatives for the detection of proteins by western blotting according to claim 2, wherein the synthesis of dye I-9:
Figure RE-FDA0002973981640000062
12. the process for preparing quinolinecarbonitrile derivatives for the detection of proteins by western blotting according to claim 2, wherein the synthesis of dye I-10:
Figure RE-FDA0002973981640000063
Figure RE-FDA0002973981640000071
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