CN113698413B - Coumarin derivative SS-590 as well as preparation method and application thereof - Google Patents
Coumarin derivative SS-590 as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a coumarin derivative SS-590, a preparation method and an application thereof, wherein the coumarin derivative SS-590 is named as (E) -1,4-diethyl-8- (3- (4-hydroxy-3-methoxyphenyl) acryloyl) -1,2,3,4-tetrahydro-7H-pyran [2,3-g ] quinoxaline-7-one in Chinese, and is named as (E) -1,4-diethyl-8- (3- (4-hydroxy-3-methoxy) acryl) -1,2,3,4-tetrahydro-7H-pyrano [2,3-g ] quinoxalin-7-one in English, and is abbreviated as SS-590. The invention provides a method for specifically detecting human serum albumin, which is based on coumarin derivative SS-590 to detect human serum albumin in PBS. The human serum albumin labeled by the coumarin derivative SS-590 can be used for SDS-PAGE Gel analysis. Has wide application prospect in the field of biomolecule detection.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, and particularly belongs to a synthetic method of coumarin derivatives SS-590 and SS-590, and application of the SS-590 in specific detection and covalent labeling of human serum albumin.
Background
Human serum albumin is a main protein in a human blood system, is synthesized by the liver and enters a blood circulation system, and plays a key role in maintaining the osmotic pressure of blood and carrying various endogenous and exogenous substances. In clinic, the reduction of the concentration of human serum albumin has indication significance for various diseases such as gastrointestinal inflammation, liver cirrhosis and the like. As an endogenous substance of a human body, the circulation time of albumin modified drug molecules in the body is obviously prolonged, the corresponding drug anaphylactic reaction is obviously weakened, and the albumin modified drug molecules are an effective way for drug optimization. Therefore, it is clinically significant to accurately analyze the concentration of human serum albumin.
In recent years, fluorescent probe-based methods have received much attention for the quantitative detection of human serum albumin. However, these probes are generally based on the non-covalent binding of a fluorescent dye to albumin to trigger a change in the fluorescent signal of the test system. Therefore, these fluorescent probes cannot be used for quantitative analysis and labeling of albumin by SDS-PAGE Gel. The development of fluorescent probes that are capable of covalently binding to human serum albumin and which are accompanied by a change in specific fluorescent signal is extremely deficient.
The invention provides a coumarin derivative SS-590 and a preparation method thereof, and realizes specific covalent labeling and detection of human serum albumin based on the derivative SS-590.
Disclosure of Invention
The invention aims to provide a coumarin derivative and a preparation method thereof, and the coumarin derivative can be used as a detection reagent for specific covalent labeling and detection of human serum albumin; the selectivity and the sensitivity are high when the human serum albumin is detected; the derivative labeled human serum albumin can be used for SDS-PAGE Gel analysis.
The invention provides a coumarin derivative, wherein the name of the coumarin derivative is (E) -1,4-diethyl-8- (3- (4-hydroxy-3-methoxyphenyl) acryloyl) -1,2,3,4-tetrahydro-7H-pyran [2,3-g ] quinoxaline-7-one, the name of the coumarin derivative is (E) -1,4-diethyl-8- (3- (4-hydroxy-3-methoxy) acryloyl) -1,2,3,4-tetrahydro-7H-pyrano [2,3-g ] quinoxalin-7-one, and the abbreviation is SS-590. The structural formula is as follows:
the coumarin derivative shows excellent sensitivity and selectivity to human serum albumin in PBS, and the detection process is simple, convenient and sensitive.
The preparation method of SS-590 comprises the following steps:
reacting 8-acetyl-1, 4-diethyl-1, 2,3,4-tetrahydro-7H-pyrano [2,3-g]Quinoxalin-7-ones
And vanillin according to a molar ratio of 1: 1.2 dissolving in ethanol, adding a catalytic amount of piperidine, and refluxing for 70h until the reaction is complete; the solvent is evaporated to dryness under reduced pressure to obtain a crude product, and then the crude product is separated by column chromatography (eluent is ethyl acetate: petroleum ether 1: 1 according to the volume ratio) to obtain a fluorescent probe SS-590
The application of the coumarin derivative SS-590 is as follows: the derivative can be used for specifically detecting human serum albumin, and the human serum albumin marked by the dye can be used for SDS-PAGE Gel analysis; the detection comprises fluorescence detection and SDS-PAGE Gel imaging analysis.
Compared with the prior art, the invention has the following beneficial effects:
1. the coumarin derivative is simple to synthesize, low in cost and easy for large-scale production;
2. the detection method can realize the specific detection of the human serum albumin, and other amino acids, inorganic ions, bovine serum albumin and mouse serum albumin do not interfere the determination of the human serum albumin;
3. the detection method can be used for SDS-PAGE Gel analysis of human serum albumin.
Description of the drawings:
FIG. 1SS-590 hydrogen spectrum characterization.
FIG. 2SS-590 carbon spectrum characterization.
FIG. 3SS-590 Mass Spectrometry characterization.
FIG. 4 fluorescence spectrum of the interaction of SS-590 with human serum albumin in example 2.
FIG. 5 is a graph comparing the fluorescence intensity at 590nm for example 3SS-590 with that of various analytes.
FIG. 6 example 4 SDS-PAGE Gel analysis after interaction of SS-590 with human serum albumin.
The specific implementation mode is as follows:
the present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1
Preparation and characterization of SS-590:
dissolving 8-acetyl-1, 4-diethyl-1, 2,3,4-tetrahydro-7H-pyrano [2,3-g ] quinoxaline-7-one (0.5mmol,0.15g) and vanillin (0.6mmol,0.09g) in 20mL of ethanol, adding 3-5 drops of piperidine, and refluxing for 70H until the reaction is complete; the system is decompressed and the solvent is evaporated to dryness to obtain a crude product, and then the crude product is separated by column chromatography (eluent is ethyl acetate according to the volume ratio: petroleum ether-1: 1) to obtain the fluorescent probe SS-590.
1H NMR (600MHz, Chloroform-d) δ 8.52(s,1H),8.07(d, J ═ 15.6Hz,1H),7.78(d, J ═ 15.6Hz,1H), 7.23-7.18 (m,2H),6.92(d, J ═ 8.6Hz,1H),6.54(s,1H),6.43(s,1H),5.89(s,1H),3.96(s,3H), 3.59-3.55 (m,2H),3.45(q, J ═ 7.1Hz,2H),3.36(q, J ═ 7.1Hz,2H), 3.27-3.23 (m,2H),1.25(t, J ═ 7.1Hz,3H),1.21(t, J ═ 7.1H) (fig. 1H).13C NMR(151MHz,CDCl3) δ 186.73,161.52,153.39,148.12,147.96,146.70,143.38,132.50,128.26,124.20,122.83,116.37,114.59,109.71,109.35,107.79,95.49,56.06,47.77,46.36,45.46,44.51,10.62,9.70 (fig. 2). HR-MS [ SS-1+ H]+M/z theoretical 435.1914, experimental 435.1740 (FIG. 3).
Example 2
Preparing a PBS solution, preparing a DMSO solution of 2mM SS-590, and preparing an aqueous solution of 20mg/mL human serum albumin; 1950. mu.L PBS and 5. mu.L SS-590 in DMSO were added to a fluorescence cuvette, 50. mu.L human serum albumin in water was added and mixed, and the mixture was detected on a fluorescence spectrophotometer (505nm excitation) after 10 min. The fluorescence intensity at 590nm gradually increased, and the fluorescence emission pattern is shown in FIG. 4.
Example 3
Preparing PBS solution, preparing DMSO solution of 2mM SS-590, preparing aqueous solution of 20mg/mL human serum albumin, preparing aqueous solution of 20mg/mL bovine serum albumin, preparing aqueous solution of 10mg/mL mouse serum albumin, and respectively preparing 8mM Glu, Ile, Lys, Leu, Met, Phe, Ser, Hyp, Gln, Pro, Tyr, Cystine, Arg, Thr, Asp and Na2SO3，Na2S，H2O2NaClO, 4mM Hcy, 40mM Cys and 80mM GSH in water; in 27 fluorescence cuvettes, 1950. mu.L of PBS, 5. mu.L of SS-590 in DMSO were added to the fluorescence cuvettes, and 50. mu.L of PBS, Glu, Ile, Lys, Leu, Met, Phe, Ser, Hyp, Gln, Pro, Tyr, Cystine, Arg, Thr, Asp, Na, respectively2SO3，Na2S，Cys，Hcy，GSH，H2O2NaClO, Gly, mouse serum albumin, bovine serum albumin and human serum albumin solution are mixed uniformly, and the fluorescence intensity (505nm excitation) is detected on a fluorescence spectrophotometer after 10 min. Human serum onlyThe protein added group showed a significant increase in fluorescence intensity. The fluorescence emission intensity is shown in FIG. 5, where 1 blank; glu; 3. Ile; lys; 5, Leu; met; phe; ser; hyp; gln; pro; tyr; cystine; arg; thr; asp; 17.Na2SO3；18.Na2S；19.Cys；20.Hcy；21.GSH；22.H2O2(ii) a NaClO; gly; 25. mouse serum albumin; 26. bovine serum albumin; 27. human serum albumin.
Example 4
Preparing a DMSO solution of 2mM SS-590, and preparing an SDS (2%) aqueous solution of 2mg/mL human serum albumin; mu.L of human serum albumin in SDS (2%) aqueous solution was placed in 2 centrifuge tubes, and 1. mu.L of PBS and SS-590 solution were added to each tube, and 10. mu.L of loading buffer was added to each tube. Mixing for 10min, separating with 4-12% SDS-PAGE Precast Gel, performing 80V electrophoresis for 20min and 120V electrophoresis for 40min, taking out the Gel strip, washing with PBS, and imaging with Gel imager. Only the fluorescence signal is present in the stained band. The image of the fluorescence is shown in FIG. 6.
Claims (5)
1. A coumarin derivative SS-590 is characterized in that the structural formula is as follows:
2. the process for preparing the coumarin derivative SS-590 as claimed in claim 1, wherein the process comprises the steps of dissolving 8-acetyl-1, 4-diethyl-1, 2,3,4-tetrahydro-7H-pyrano [2,3-g ] quinoxalin-7-one and vanillin in ethanol, adding a catalytic amount of piperidine, and refluxing to completion of the reaction; the system is decompressed and the solvent is evaporated to dryness to obtain a crude product, and then the crude product is separated by column chromatography to obtain a target compound SS-590;
wherein the molar ratio of 8-acetyl-1, 4-diethyl-1, 2,3,4-tetrahydro-7H-pyrano [2,3-g ] quinoxalin-7-one and vanillin is 1: 1-1.5.
3. Process for the preparation of coumarin derivative SS-590 according to claim 2, wherein the molar ratio between 8-acetyl-1, 4-diethyl-1, 2,3,4-tetrahydro-7H-pyrano [2,3-g ] quinoxalin-7-one and vanillin is 1: 1.2.
4. the method for preparing the coumarin derivative SS-590 as claimed in claim 2, wherein the column chromatography eluent comprises ethyl acetate: petroleum ether is 1: 1.
5. use of the coumarin derivative SS-590 as claimed in claim 1 in the preparation of a reagent for the detection of human serum albumin.
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