CN114907302B - Fluorescent probe for rapidly detecting elastase and preparation method and application thereof - Google Patents
Fluorescent probe for rapidly detecting elastase and preparation method and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07D—HETEROCYCLIC COMPOUNDS
- 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
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/966—Elastase
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Abstract
The invention discloses a fluorescent probe for rapidly detecting elastase, a preparation method and application thereof, which is characterized in that the molecular formula is C 20 H 11 F 8 NO 4 The structural formula is shown as formula (I),
the fluorescent probe has strong water solubility, strong affinity to elastase, high sensitivity and quick response.
Description
Technical Field
The invention relates to the field of chemical analysis and detection, in particular to a fluorescent probe for rapidly detecting elastase, and a preparation method and application thereof.
Background
Neutrophil elastase is a hematopoietic serine protease consisting of 267 amino acid residues, has high proteolytic activity, is an important regulator of immune response, and is used for preventing invasion of microorganisms and eliminating extracellular pathogens. Studies have shown that neutrophil elastase is considered a biomarker for human disease progression and a therapeutic target for drug discovery. Overexpression of elastase can lead to the development of a number of diseases such as tissue injury, acute lung injury, chronic obstructive pulmonary disease, and acute respiratory distress syndrome. Finding suitable inhibitors is a major approach to the treatment of elastase-related diseases, and commercially available elastase inhibitors are mainly Sivelestat, elastatinal, etc., but these inhibitors are generally expensive. Therefore, it is important to develop a method for detecting elastase and for use in inhibitor screening.
At present, methods for detecting elastase mainly comprise a colorimetry method, an electrochemical method, an electrophoresis method, a high performance liquid chromatography method and a fluorescent probe method, wherein the fluorescent probe method has the advantages of simplicity in operation, high response time, high sensitivity and the like.
Although the fluorescent probe method is widely used for detection of metal ions, small molecules, biological enzymes, and the like, few fluorescent probes for elastase have been reported so far. It is therefore interesting to develop a fluorescent probe for sensitive detection of elastase for in vitro and in vivo detection of elastase.
Disclosure of Invention
In view of the above, the present application provides a fluorescent probe for rapidly detecting elastase, and a preparation method and application thereof, where the fluorescent probe has strong water solubility, strong affinity to elastase, high sensitivity, and rapid response.
In order to achieve the technical purpose, the application adopts the following technical scheme:
in a first aspect, the present application provides a fluorescent probe for rapid detection of elastase having a formula C 20 H 11 F 8 NO 4 The structural formula is shown as formula (I),
in a second aspect, the present application provides a method for preparing a fluorescent probe for rapid detection of elastase, comprising the steps of:
s1, taking 4-amino benzyl alcohol and pentafluoropropionic anhydride as raw materials, reacting in a solvent in the presence of pyridine, and separating and purifying to obtain an intermediate a;
s2, dispersing the intermediate a in a solvent, adding phosphorus tribromide under the ice bath condition, then reacting at room temperature, adding alkali for neutralization, and separating and purifying to obtain an intermediate b;
s3, dissolving 7-hydroxy-4- (trifluoromethyl) coumarin and alkali in a solvent, adding an intermediate b, reacting at room temperature, and separating and purifying to obtain a fluorescent probe, namely HFC-NE;
the synthetic route is as follows:
preferably, the molar ratio of 4-aminobenzyl alcohol to pentafluoropropionic anhydride is 1:1-3.
Preferably, the molar ratio of 4-aminobenzyl alcohol to pyridine is 1:2-5.
Preferably, the molar ratio of compound a to phosphorus tribromide is 1:1-3.
Preferably, the molar ratio of 7-hydroxy-4- (trifluoromethyl) coumarin to compound b is 1:1-3.
In a third aspect, the present application provides the use of a fluorescent probe for rapid detection of elastase for screening for elastin inhibitors or for in vitro or in vivo detection of elastase.
Preferably, the fluorescent probe is used for qualitative and quantitative detection of the elastase in the aqueous solution.
Preferably, the fluorescent probes are used for detection and imaging of endogenous elastase in living cells and in vivo zebra fish models, the cells being used for non-therapeutic purposes.
Preferably, the elastase inhibitor is screened using a fluorescent probe as a substrate.
Preferably, fluorescent probes are used to distinguish HepG2 cells from non-HepG 2 cells, the cells being used for non-therapeutic purposes.
The beneficial effects of this application are as follows: the fluorescent probe has the advantages of good water solubility, rapid response, low fluorescent background and high sensitivity (LOD=0.011U mL) -1 ) High fluorescence enhancement factor (176 times); the near infrared fluorescent probe containing the self-breaking group has high selectivity, low cytotoxicity, high sensitivity and strong affinity to elastase, can detect and image living cells and endogenous elastase of living zebra fish, and can be used for distinguishing HepG2 cells from other cells; the preparation method has the advantages of easily available raw materials, simple reaction conditions and few synthesis steps.
Drawings
FIG. 1 is a schematic diagram showing the detection mechanism of elastase by the fluorescent probe of the present invention.
FIG. 2 shows the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe of the present invention.
FIG. 3 is a nuclear magnetic resonance carbon spectrum of a fluorescent probe of the present invention.
FIG. 4 is a HRMS spectrum of a fluorescent probe of the invention.
FIG. 5 is a graph showing fluorescence spectra of response time dependence of the fluorescent probe of the present invention and elastase.
FIG. 6 is a graph showing fluorescence spectra of the response of the fluorescent probe of the present invention to elastase at different concentrations.
FIG. 7 shows the fluorescence probe of the present invention and the concentration range (1-10U mL) -1 ) Linear relationship diagram of elastase.
FIG. 8 is a graph showing the fit of the Michaelis equation of the enzyme kinetics of the fluorescent probe of the present invention to elastase.
FIG. 9 is a graph showing the selectivity of the fluorescent probe of the present invention for elastase.
FIG. 10 is a graph showing detection of imaged elastase in living cells by fluorescent probes of the invention.
FIG. 11 is a graph showing detection of imaging elastin in living zebra fish by fluorescent probes of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The application provides a fluorescent probe for rapidly detecting elastase, which has a molecular formula of C 20 H 11 F 8 NO 4 The structural formula is shown as formula (I),
in the prior art, the problems to be solved by the fluorescent probe for detecting elastase are as follows: 1) The water solubility of the probe needs to be enhanced so as to improve the detection capability of the probe in an aqueous solution; 2) The sensitivity of the probe and the affinity of the probe to enzyme are required to be improved so as to improve the practicability of the probe; 3) Probes are needed for detection and imaging of endogenous elastase in living cells and in vivo models. Whereas the fluorescent probe in the present application has the following advantages: the fluorescent probe has good water solubility and is beneficial to the application in biological imaging; the fluorescent probe has better fluorescent response effect on elastase, and the fluorescence enhancement multiple of the fluorescent probe is up to 167 times; the fluorescent probe has larger Stokes displacement, so that the fluorescent probe has lower background interference and larger penetrating power on biological tissues; finally, the fluorescent probe provided by the invention can be used for detecting and imaging living cells and living zebra fish, can be used for distinguishing HepG2 cells from other cells, and has an application prospect in diagnosing liver cancer and inflammatory diseases.
In addition, the application provides a preparation method of a fluorescent probe for rapidly detecting elastase, which comprises the following steps:
s1, dissolving 1 molar equivalent of 4-amino benzyl alcohol in anhydrous dichloromethane, adding 2-5 molar equivalents of pyridine, dropwise adding 1-3 molar equivalents of pentafluoropropionic anhydride under ice bath conditions, reacting for 6-12 hours, and separating and purifying to obtain a pale yellow solid compound a;
s2, dissolving 1 molar equivalent of the compound a in anhydrous dichloromethane, adding 1-3 molar equivalents of phosphorus tribromide under ice bath conditions, reacting for about 3-9 hours at room temperature, stopping the reaction after the raw materials are reacted, adding sodium bicarbonate solution for neutralization, and extracting with ethyl acetate and saturated saline water to obtain a white solid compound b;
s3, adding 1 molar equivalent of 7-hydroxy-4- (trifluoromethyl) coumarin and 1-3 molar equivalents of anhydrous potassium carbonate into a 25mL round bottom flask, adding anhydrous acetonitrile for dissolution, adding 1-3 molar equivalents of compound b, reacting for 12-24 hours, stopping the reaction after the reaction of the raw material 7-hydroxy-4- (trifluoromethyl) coumarin is completed, and separating and purifying to obtain a white solid compound, namely a fluorescent probe (HFC-NE);
the synthetic route is as follows:
meanwhile, the application provides an application of the fluorescent probe for rapidly detecting elastase, wherein the fluorescent probe is used for screening elastin inhibitors or in-vitro or in-vivo detection of elastase, and the screening of the fluorescent probe for the elastin inhibitors is carried out by taking the fluorescent probe as a substrate; fluorescent probes for in vitro detection of elastase include, but are not limited to, using fluorescent probes for qualitative and quantitative detection of elastase in aqueous solution; fluorescent probes for in vivo detection of elastase include, but are not limited to, fluorescent probes for detection and imaging of endogenous elastase in living cells and in living zebra fish models, cells for non-therapeutic purposes, including, but not limited to, fluorescent probes for distinguishing HepG2 cells from non-HepG 2 cells, cells for non-therapeutic purposes. In the application, the action mechanism of the fluorescent probe and the elastase is shown in a figure 1, the fluorescent probe (I) is in a quenching state under the action of a self-breaking group and a recognition group, an amide bond is broken under the catalysis of the elastase, then intramolecular charge transfer occurs in the self-breaking group, a fluorescent group (II) is released, strong fluorescence is shown, and the activity of the elastase is detected by detecting the fluorescence intensity of a product fluorescent group.
The present embodiment will be described below by way of specific examples.
Example 1
The preparation method of the fluorescent probe for rapidly detecting elastase specifically comprises the following steps:
synthesis of compound a: 4-amino benzyl alcohol (1 g,8.0938 mmol) was weighed, 15mL of anhydrous dichloromethane was added for dissolution, pyridine (1.281g, 16.1876 mmol) was added, then pentafluoropropionic anhydride (3.764 g,12.1407 mmol) was added dropwise under ice bath condition, the reaction was completed for about 12 hours, after the raw materials were reacted, the reaction was completed, extracted with ethyl acetate, after three times of washing with saturated salt water, ethyl acetate layer was collected, dried with anhydrous sodium sulfate, the solvent was concentrated to obtain a crude product, which was chromatographed on silica gel column with eluent PE: EA=20:1, to obtain pale yellow solid compound a 1.616g with a yield of 34%. The resulting product was validated: 1 H NMR(400MHz,DMSO-d 6 )δ11.27(s,1H),7.61(d,J＝8.4Hz,2H),7.35(d,J＝8.2Hz,2H),5.22(t,J＝5.6Hz,1H),4.48(d,J＝5.6Hz,2H).
synthesis of compound b: compound a (1 g, 3.015 mmol) was weighed, 15mL of anhydrous dichloromethane was added, phosphorus tribromide (1.5085 g,5.573 mmol) was added under ice bath conditions, the reaction was stopped after the reaction of the raw materials was completed at room temperature, the reaction was neutralized by adding sodium bicarbonate solution, extraction was performed with ethyl acetate and saturated saline, the ethyl acetate layer liquid was collected, dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation to give compound b (0.85 g) as a white solid in 68.90% yield. Without further purification, it was used directly in the next step. The resulting product was validated: 1 H NMR(400MHz,Chloroform-d)δ8.10(s,1H),7.56(d,J＝8.0Hz,2H),7.42(d,J＝8.2Hz,2H),4.48(s,2H).
synthesis of probe HFC-NE: 7-hydroxy-4- (trifluoromethyl) coumarin (200 mg,0.869 mmol) and anhydrous potassium carbonate (194.66 g,1.74 mmol) were weighed into a 25mL round bottom flask, 10mL of anhydrous acetonitrile was added, and after stirring for 30min, compound b (432.85 mg,1.30 mmol) was added, the reaction was stopped after the reaction of the starting material 7-hydroxy-4- (trifluoromethyl) coumarin was completed, ethyl acetate and water were used for extraction, and ethyl acetate layer liquid was collected, and thenAfter drying over anhydrous sodium sulfate, the liquid was removed by rotary evaporation, and the obtained crude product was subjected to silica gel column chromatography (PE: ea=50:1-5:1) to obtain 151.44mg of a white solid with a yield of 36%. The obtained product is verified, the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe HFC-NE is shown in figure 2, 1 H NMR(400MHz,DMSO-d 6 ) δ11.37 (s, 1H), 7.78-7.49 (m, 5H), 7.24 (s, 1H), 7.14 (d, j=11.6 hz, 1H), 6.86 (s, 1H), 5.27 (s, 2H). The nuclear magnetic resonance carbon spectrum of the fluorescent probe HFC-NE is shown in figure 3, 13 c NMR (101 MHz, DMSO-d 6) delta 162.43,159.17,156.26,155.75,139.68,136.53,134.01,129.11,126.34,121.91,114.45,113.93,107.09,103.09,70.01 the HRMS spectrum of fluorescent probe HFC-NE is shown in FIG. 4, HR-MS (ESI) calculated for C 20 H 11 F 8 NO 4 + [M+H] + 482.0639；Found:482.06329
Test example 1
Determination of the change over time in the fluorescence intensity of the reaction of the fluorescent probe obtained in example 1 with elastase: to the cuvette were added a solution of the above compound HFC-NE (working concentration 10 μm), PBS (ph=7.4) buffer, elastase (working concentration 100U/mL), and the above system was subjected to primary fluorescence spectra at reaction times 0, 4, 7, 10, 15, 20, 25, 30, 45, 60, 75, 90, 120, 150, 180min, as shown in fig. 5.
As can be seen from FIG. 5, the fluorescence was significantly enhanced at 4 minutes of reaction, and the probe responded very rapidly to elastase. With the increase of the reaction time, the fluorescence intensity of the reaction system at 505nm is gradually enhanced, and when the reaction time reaches 1.5h, the fluorescence intensity is kept unchanged and reaches the platform.
Test example 2
Determination of the fluorescence spectra of the fluorescent probes obtained in example 1 reacted with elastase at different concentrations: a solution of HFC-NE (working concentration: 10. Mu.M), PBS (pH=7.4) buffer and elastase (working concentrations: 0, 1, 2, 4, 6, 8, 10, 15, 20, 40, 60, 80, 100U mL-1) were added to the cuvette, and the fluorescence spectrum after 1.5 hours of the reaction system was examined, as shown in FIG. 6.
As can be seen from FIG. 6, in a certain concentration range, the fluorescence intensity of the reaction system at 505nm increases with the increase of the elastase concentration, which indicates that the probe can detect elastase at different concentrations.
Test example 3
Determination of the minimum limit of detection of elastase by the fluorescent probe obtained in example 1: a linear plot of fluorescence intensity at 505nm and elastase after 1.5h of reaction of a solution of HFC-NE (working concentration 10. Mu.M), PBS (pH=7.4) buffer and (0.5, 1, 2, 4, 6, 8, 10U/mL) elastase was added to the cuvette, as shown in FIG. 7.
As can be seen from FIG. 7, the fluorescence intensity at 505nm and the elastase concentration have a good linear relationship, indicating that the fluorescence probe can quantitatively detect the activity of elastase in the range of (0-10U/mL). Meanwhile, according to the linear relation, the detection limit of the probe for detecting the elastase can be calculated to be 0.011U mL -1 The detection limit of the probe on the elastase is lower, which indicates that the probe has higher sensitivity on the elastase.
Test example 4
Determination of the fitted map of the enzymatic kinetic Mies equation of the fluorescent probe obtained in example 1 for elastase: 100U/mL elastase was reacted with fluorescent probes at different concentrations (1, 2, 5, 10, 20, 30, 40. Mu.M) and the change in fluorescence intensity with time was measured. The slope of the fluorescence intensity versus reaction time curve is the reaction rate. The reaction rate is plotted on the ordinate and the fluorescent probe concentration is plotted on the abscissa, and the elastase is tested for K for this probe m And V max As shown in fig. 8.
K of elastase on the probe was calculated by Mi equation fitting m And V max 2.85. Mu.M and 1.68. Mu.M min, respectively -1 Its K m Smaller, indicating that elastase has good affinity for the probe.
Test example 5
The fluorescent probe obtained in example 1 was assayed for elastase selectivity: 1. Mu.L of the fluorescent probe obtained in example 1 (working concentration: 10. Mu.M, dissolved in PBS buffer solution having pH of 7.4 at 37 ℃) and different analytes were added to the cuvette, respectivelyMixed solution (a: ca) 2+ ；b:Mg 2+ ；c:K + ；d:Cu 2+ ；e:Li + ；f:Fe 3+ ；g:Mn 2+ ；h:Zn 2+ ；I:Cl - ；j:SO4 2- ；l:HCO 3 - The method comprises the steps of carrying out a first treatment on the surface of the m is L-Ala; glu; o is L-Val; p is D-Val; q is D-Arg; L-Arg; s is L-Ser; t is D-Gln; u is D-Ser; v: GSH; hcy; trypsin; chymotrypsin; z elastase), the fluorescence intensity of each mixture was measured at 505nm after 1.5 hours of reaction. As shown in FIG. 9, the fluorescence signal of the fluorescent probe is basically unchanged in the presence of various anions, metal cations, amino acids and various biological enzymes, and the fluorescence of the fluorescent probe is obviously enhanced after elastase is added, so that the fluorescent probe has high selectivity to elastase.
Test example 6
Assay of the fluorescent probes obtained in example 1 elastase in living cells was detected: the fluorescent probe obtained in example 1 (10. Mu.M) was incubated with living cells (HeLa, SKOV3 and HepG2 cells) for 30 minutes, and the cells were imaged after washing with PBS, as shown in FIG. 10.
From the figure, the fluorescence of the probe in HeLa cells and SKOV3 cells is very weak, which indicates that the content of endogenous elastase in HeLa cells and SKOV3 cells is low, but the fluorescence in HepG2 cells is obvious, which indicates that elastase is over-expressed in HepG2 cells. The experiment shows that the probe can be used for distinguishing HepG2 cells from other two cells.
Test example 7
Measurement of the imaging detection of elastase by the fluorescent probe obtained in example 1: zebra fish seedlings grown for 0-7 days are cultured in a 6-well plate and divided into three groups, wherein the first group is shown in a control group in fig. 11 (a-c), namely, after the probe with the concentration of 10 mu M is added to incubate with the zebra fish for 2 hours, the zebra fish seedlings are washed three times by PBS and imaged; a second group (d-f), wherein after pretreatment of zebra fish by adding LPS (elastase inducer, which can induce the generation of elastase in zebra fish), probe is added for incubation, and imaging is performed after washing with PBS three times; and the third group (g-i), after the zebra fish is pretreated by adding the inducer LPS and Sivelestat (elastase inhibitor, which can inhibit the activity of elastase), the probe is added, and the mixture is washed three times with PBS and imaged, and the experimental result is shown in FIG. 11.
As can be seen from the figure, when the probe is added to incubate with the zebra fish, green fluorescence is generated in the zebra fish body, the green fluorescence is enhanced after the probe is pretreated by an inducer, and the fluorescence is obviously inhibited when the probe is treated by the inducer and the inhibitor, so that the experiments can fully prove that the HFC-NE can be used for specific detection and imaging of elastase in living organisms.
In addition, compared with the detection of a fluorescent probe recognizing elastase in the related art, the differences between the present application are shown in Table 1:
TABLE 1 characterization of different fluorescent probes
In the prior art, the problems to be solved by the fluorescent probe for detecting elastase are as follows: 1) The water solubility of the probe is enhanced, and the detection capability of the probe in aqueous solution is improved; 2) The sensitivity of the probe and the affinity of the probe to enzyme are improved, and the practicability of the probe is improved; 3) The probe is applied to detection and imaging of endogenous elastase of living cells and living models. Whereas the fluorescent probe in the present application has the following advantages: the fluorescent probe has good water solubility and is beneficial to the application in biological imaging; the fluorescent probe has better fluorescent response effect on elastase, and the fluorescence enhancement multiple of the fluorescent probe is up to 167 times; the fluorescent probe has larger Stokes displacement, so that the fluorescent probe has lower background interference and larger penetrating power on biological tissues; finally, the fluorescent probe provided by the invention can be used for detecting and imaging living cells and living zebra fish, can be used for distinguishing HepG2 cells from other cells, and has an application prospect in diagnosing liver cancer and inflammatory diseases.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention.
Claims (9)
1. A fluorescent probe for rapidly detecting elastase is characterized in that the molecular formula is C 20 H 11 F 8 NO 4 The structural formula is shown as formula (I),
2. A method for preparing a fluorescent probe for rapid detection of elastase according to claim 1, comprising the steps of:
s1, taking 4-amino benzyl alcohol and pentafluoropropionic anhydride as raw materials, reacting in a solvent in the presence of pyridine, and separating and purifying to obtain an intermediate a;
s2, dispersing the intermediate a in a solvent, adding phosphorus tribromide under the ice bath condition, then reacting at room temperature, adding alkali for neutralization, and separating and purifying to obtain an intermediate b;
s3, dissolving 7-hydroxy-4- (trifluoromethyl) coumarin and alkali in a solvent, adding an intermediate b, reacting at room temperature, and separating and purifying to obtain the fluorescent probe;
the synthetic route is as follows:
3. the method for preparing a fluorescent probe for rapid detection of elastase according to claim 2, wherein the molar ratio of 4-aminobenzyl alcohol to pentafluoropropionic anhydride is 1:1-3.
4. The method for preparing a fluorescent probe for rapid detection of elastase according to claim 2, wherein the molar ratio of 4-aminobenzyl alcohol to pyridine is 1:2-5.
5. The method for preparing a fluorescent probe for rapid detection of elastase according to claim 2, wherein the molar ratio of the compound a to the phosphorus tribromide is 1:1-3.
6. The method for preparing a fluorescent probe for rapid detection of elastase according to claim 2, wherein the molar ratio of the 7-hydroxy-4- (trifluoromethyl) coumarin to the compound b is 1:1-3.
7. Use of a fluorescent probe for rapid detection of elastase according to claim 1 for the preparation of an in vitro or in vivo detection reagent for elastase.
8. The use of a fluorescent probe for rapid detection of elastase according to claim 7, wherein the fluorescent probe is used for preparing a qualitative and quantitative detection reagent for elastase in aqueous solution.
9. The use of a fluorescent probe for rapid elastase detection according to claim 7, wherein the fluorescent probe is used for preparing reagents for detection and imaging of endogenous elastase in living cells and living zebra fish models.
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