CN110950893B - Multifunctional fluorescent probe and preparation method and application thereof - Google Patents

Multifunctional fluorescent probe and preparation method and application thereof Download PDF

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CN110950893B
CN110950893B CN201911226167.9A CN201911226167A CN110950893B CN 110950893 B CN110950893 B CN 110950893B CN 201911226167 A CN201911226167 A CN 201911226167A CN 110950893 B CN110950893 B CN 110950893B
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fluorescent probe
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multifunctional fluorescent
lactamase
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张雷
余攀
李杉
颜金武
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South China University of Technology SCUT
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Abstract

The invention relates to the technical field of specific molecular recognition materials, in particular to a multifunctional fluorescent probe which can detect AmpC beta-lactamase, can screen AmpC beta-lactamase inhibitors and can screen drug resistance of strains, and a preparation method and application thereof. The compound 1 is synthesized into the multifunctional fluorescent probe capable of detecting AmpC beta-lactamase, screening AmpC beta-lactamase inhibitor and screening the drug resistance of bacterial strains through three steps, and the invention has the advantages of few synthesis steps, simple post-treatment process, easy operation and easy obtaining of products. The multifunctional fluorescent probe is designed and modified based on the cephalosporin nucleus, and has high sensitivity, good specificity and better bacteriostatic effect; can be simultaneously used for detecting AmpC beta-lactamase and screening AmpC beta-lactamase inhibitors, can detect the drug resistance of bacteria at the bacterial level, and can identify the drug resistance mechanism of the bacteria.

Description

Multifunctional fluorescent probe and preparation method and application thereof
Technical Field
The invention relates to the technical field of specific molecular recognition materials, in particular to a multifunctional fluorescent probe which can detect AmpC beta-lactamase, can screen AmpC beta-lactamase inhibitors and can screen drug resistance of strains, and a preparation method and application of the multifunctional fluorescent probe.
Background
In 1940, β -lactam antibiotics were first used for the treatment of bacterial infections, and since they have a very good therapeutic effect in the treatment of infectious diseases, they were widely used. In recent 20 years, due to abuse and misuse of antibiotics, part of bacteria generate drug resistance to the antibiotics, so that the treatment effect is poor and the health of people is endangered. It has been found that a major cause of drug resistance of pathogenic bacteria is caused by the generation of various beta-lactamase enzymes in the bacteria, which can hydrolyze and inactivate beta-lactam antibiotics with high efficiency. If the species of β -lactamase produced by the bacteria can be specifically detected, antibiotics that can be hydrolyzed by such enzymes can be avoided, other suitable antibiotics can be selected, or more precise treatment can be achieved by using a combination of enzyme inhibitor-antibiotic therapy. The method can detect the condition of enzyme in time in clinical treatment, and has important significance for doctors to judge the state of illness, determine bacteria and put forward a corresponding treatment scheme.
Beta-lactamases can be classified into A, B, C, D four classes according to the Ambler classification, with class A and class C being the two most important classes. There have been many reports of the identification and detection of class a beta-lactamases, but there have been few studies on the specific detection of class C beta-lactamases, and the AmpC beta-lactamases, which are the most common of class C beta-lactamases, are the most active beta-lactamases that hydrolyze beta-lactam antibiotics.
On the other hand, as the phenomenon of drug resistance of bacteria is more and more common, and a plurality of antibiotics cannot exert good treatment effect when being used alone, the treatment of patients by adopting the mode of combining the antibiotics and the inhibitor can well ensure the treatment effect. Therefore, the search for inhibitors having a good inhibitory effect on β -lactamases and screening methods thereof is a key direction for the development of new drugs. The most currently used screening method is to use nitrocefin as a substrate and screen a suitable inhibitor by the ultraviolet and color change of the substrate after the substrate is acted by an enzyme. Although the detection only by the change of color is simple in operation and convenient in use, the sensitivity is not high.
Fluorescent probes have attracted extensive attention in the field of analytical testing due to their advantages of low background signal, high sensitivity, low cost, and the like. Therefore, a probe is expected to have two functions of detecting AmpC beta-lactamase and screening AmpC beta-lactamase inhibitor, and can well guide the development and clinical medication of new drugs.
Disclosure of Invention
In order to solve the defects and shortcomings of the prior art, the invention provides the multifunctional fluorescent probe which can detect AmpC beta-lactamase and screen AmpC beta-lactamase inhibitors, can be used for screening the drug resistance of strains and can identify the drug resistance mechanism of bacteria.
In another aspect of the present invention, a method for preparing the multifunctional fluorescent probe is provided.
In another aspect of the invention, the multifunctional fluorescent probe is applied to detection of AmpC beta-lactamase, screening of AmpC beta-lactamase inhibitors, preparation of antibacterial drugs and identification of bacterial drug resistance mechanisms.
In order to achieve the purpose, the invention adopts the following technical scheme.
In a first aspect of the present invention, there is provided a multifunctional fluorescent probe, having a structural formula:
Figure BDA0002300914270000021
in another aspect of the present invention, there is provided a method for preparing the multifunctional fluorescent probe, comprising the steps of:
s1, reacting the compound 1 with NaI in a hydrophilic organic solvent, removing the hydrophilic organic solvent after the reaction is finished, adding a lipophilic organic solvent for dissolving, washing and drying an organic phase, adding triphenylphosphine for reacting, and separating a product after the reaction is finished to obtain an intermediate 2.
Preferably, the molar ratio of the compound 1 to the NaI to the triphenylphosphine is 1: 4-5: 1-1.5.
More preferably, the molar ratio of the compound 1, NaI and triphenylphosphine is 1: 4.4: 1.2.
Preferably, the reaction temperature in step S1 is 10-35 ℃.
S2, dissolving the intermediate 2 in a lipophilic organic solvent, adding an aqueous solution of NaOH to react, removing a water phase after the reaction is finished, drying an organic phase, adding the compound 3 to react, and separating a product after the reaction is finished to obtain an intermediate 4.
Preferably, the feeding molar ratio of the intermediate 2, NaOH and the compound 3 is 1 to (8-10) to (0.6-0.9).
More preferably, the molar ratio of the intermediate 2, NaOH and compound 3 is 1: 9: 0.7.
Preferably, the concentration of the aqueous NaOH solution is 1 mol/L.
Preferably, the reaction temperature in step S2 is 10-35 ℃.
S3, adding the intermediate 4, anisole and trifluoroacetic acid into a lipophilic organic solvent, and separating a product after the reaction is finished to obtain the multifunctional fluorescent probe.
Preferably, the feeding molar ratio of the intermediate 4, the anisole and the trifluoroacetic acid is 1: (10-15): (80-100).
More preferably, the molar ratio of intermediate 4, anisole and trifluoroacetic acid is 1: 12.3: 89.7.
Preferably, the reaction temperature in step S3 is 0 ℃.
The structural formulas of the compound 1, the intermediate 2, the compound 3 and the intermediate 4 are as follows.
The structural formula of compound 1 is:
Figure BDA0002300914270000041
the structural formula of the intermediate 2 is as follows:
Figure BDA0002300914270000042
the structural formula of compound 3 is:
Figure BDA0002300914270000043
the structural formula of the intermediate 4 is as follows:
Figure BDA0002300914270000044
in still another aspect of the present invention, there is provided a use of the multifunctional fluorescent probe described above.
The multifunctional fluorescent probe is used for detecting AmpC beta-lactamase.
The multifunctional fluorescent probe is used for screening AmpC beta-lactamase inhibitor.
The multifunctional fluorescent probe is applied to preparing antibacterial drugs.
Preferably, the bacteriostatic drug is a drug for inhibiting staphylococcus aureus.
The multifunctional fluorescent probe is applied to identifying a bacterial drug resistance mechanism.
Compared with the prior art, the invention has the beneficial effects that:
the compound 1 is synthesized into the multifunctional fluorescent probe for detecting AmpC beta-lactamase and screening AmpC beta-lactamase inhibitor and for screening the drug resistance of bacterial strains through three steps, and the multifunctional fluorescent probe has the advantages of few synthesis steps, simple post-treatment process, easy operation and easy obtaining of products. The multifunctional fluorescent probe is designed and modified based on the cephalosporin nucleus, and has high sensitivity, good specificity and better bacteriostatic effect; can be simultaneously used for detecting AmpC beta-lactamase and screening AmpC beta-lactamase inhibitors, and can detect the drug resistance of bacteria and identify the drug resistance mechanism of the bacteria at the bacterial level.
Drawings
FIG. 1 is a synthetic scheme of the multifunctional fluorescent probe of example 1;
FIG. 2 shows a multi-functional fluorescent probe according to the present invention1H-NMR spectrum;
FIG. 3 shows a multi-functional fluorescent probe according to the present invention13A C-NMR spectrum;
FIG. 4a is a graph of the UV absorbance intensity as a function of time (10min) for a multifunctional fluorescent probe of the present invention after mixing with 100U/mL AmpC beta-lactamase;
FIG. 4b is a graph of fluorescence intensity over time (10min) for a multifunctional fluorescent probe of the present invention when mixed with 100U/mL AmpC beta-lactamase;
FIG. 5 is a graph showing the inhibitory effect of two inhibitors, sulbactam sodium and tazobactam acid, on AmpC beta-lactamase when the multifunctional fluorescent probe of the present invention is used as a substrate;
FIG. 6a is a bacteriostatic circle diagram obtained by the interaction of the multifunctional fluorescent probe (2), oxacillin sodium (1), compound 1(3), cefazolin sodium (4), solvent (5, blank group) and Staphylococcus aureus ATCC 25923;
FIG. 6b is a bacteriostatic circle diagram obtained by the interaction of the multifunctional fluorescent probe (2), oxacillin sodium (1), compound 1(3), cefazolin sodium (4), solvent (5, blank group) and Staphylococcus aureus ATCC 29213;
FIG. 6c is a bacteriostatic circle diagram obtained by the interaction of the multifunctional fluorescent probe (2), oxacillin sodium (1), compound 1(3), cefazolin sodium (4), solvent (5, blank group) and Staphylococcus aureus ATCC 43300.
FIG. 6d is a bacteriostatic circle diagram obtained by the interaction of the multifunctional fluorescent probe (2), oxacillin sodium (1), compound 1(3), cefazolin sodium (4), solvent (5, blank group) and Enterobacter cloacae ATCC 13047.
FIG. 7 is a graph showing the change of fluorescence intensity with time (10min) after mixing the multifunctional fluorescent probe of the present invention with 4 kinds of bacteria (Staphylococcus aureus ATCC 25923, ATCC 29213, ATCC 43300, and Enterobacter cloacae ATCC 13047).
Detailed Description
In order to more fully understand the technical contents of the present invention, the technical solutions of the present invention will be further described and illustrated with reference to the following specific embodiments.
In addition to the reaction conditions selected for the synthesis process shown in the examples (e.g., charge ratios between reactants, reaction temperatures, etc.), in other embodiments, the molar ratio of compound 1, NaI, triphenylphosphine in the synthesis of intermediate 2 may be in the range of 1: (4-5): (1-1.5), and the reaction temperature may be in the range of 10-35 ℃; when the intermediate 4 is synthesized, the feeding molar ratio of the intermediate 2, NaOH and the compound 3 can be in the range of 1 to (8-10) to (0.6-0.9), and the reaction temperature can be in the range of 10-35 ℃; when the multifunctional fluorescent probe CDC-559 is synthesized, the feeding molar ratio of the intermediate 4, the anisole and the trifluoroacetic acid can be in the range of 1: 10-15: 80-100. Examples are provided to further illustrate the technical content of the present invention so that the reader can easily understand that the embodiments of the present invention are not limited to the following examples.
For parameters not particularly noted, it can be carried out with reference to conventional techniques. The nuclear magnetic spectrum is measured by AVANCE II 400M/600M nuclear magnetic resonance instrument of Bruker company, Germany, and deuterated chloroform and deuterated DMSO are used as solvents. The ultraviolet spectrum was measured by using a UV-Vis 2450 ultraviolet spectrometer manufactured by SHIMADZU, Japan. Fluorescence Spectroscopy and semi-Inhibitory Concentration (IC)50) The measurement was carried out using EnSpire-2300 multi-functional microplate reader Perkinelmer, USA.
Examples
This example provides a multifunctional fluorescent probe and a method for synthesizing the multifunctional fluorescent probe, wherein the synthetic route is shown in FIG. 1, and CDC-559 is a target fluorescent compound, i.e., a multifunctional fluorescent probe. The specific synthesis steps are as follows:
1. synthesis of intermediate 2
Compound 1(3.0g, 6.1mmol) was dissolved in acetone (40mL), NaI (4.0g, 26.7mmol) was added, stirred at room temperature for 1h and then spun dry, dissolved in 100mL ethyl acetate, the organic phase was taken up with 10% Na in turn2S2O3(50 mL. times.1), water (50 mL. times.1), and saturated brine (50 mL. times.1), anhydrous MgSO4After drying, the organic phase was concentrated to 40mL and triphenylphosphine (1.9g, 7.2mmol) was added rapidly under nitrogen and reacted overnight at room temperature (25 ℃ C.) in the absence of light. The reaction solution was suction-filtered, and the obtained solid was washed with n-hexane to give 4.0g of a yellow solid, with a yield of 77%.
2. Synthesis of intermediate 4
Intermediate 2(4.0g) was dissolved in dichloromethane, 43mL of 1mol/L NaOH solution was added, the mixture was stirred vigorously at room temperature (25 ℃) for 30min, the organic phase was separated, and anhydrous MgSO4Drying, filtration and then addition of compound 3(0.8g, 3.3mmol) stirring at room temperature for 6 h. After spin-drying, column chromatography was performed to obtain 620mg of a red solid, the yield was 28%.
The data of nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum of the intermediate 4 are as follows:
1H NMR(400MHz，CDCl3)δ7.74(d，J＝16.5Hz，1H)，7.58(s，1H)， 7.40-7.21(m，8H)，6.91-6.83(m，3H)，6.57(d，J＝8.6Hz，1H)，6.43-6.38(m，2H)， 5.81(dd，J＝9.1，4.8Hz，1H)，5.25(q，J＝12.1Hz，2H)，4.96(d，J＝4.7Hz，1H)， 3.78(s，3H)，3.66(m，2H)，3.65(d，J＝2.8Hz，2H)，3.42(q，J＝6.9Hz，4H)，1.22 (t，J＝6.9Hz，6H).
13C NMR(101MHz，CDCl3)δ171.20，164.67，162.05，161.56，159.85， 155.96，150.89，137.77，133.86，130.69，129.45，129.38，129.13，128.57，127.66， 127.08，126.61，123.70，123.18，116.69，114.01，109.29，108.92，97.12，67.87， 59.17，57.90，55.26，44.92，43.35，24.52，12.53.
HRMS(ESI，m/z)Calcd.for C38H37N3O7S 679.2352；found：702.2254[M +Na]+(Calcd.702.2352).
3. synthesis of multifunctional fluorescent probe CDC-559
Intermediate 4(100mg, 0.15mmol) was dissolved in dichloromethane (5mL), cooled to 0 deg.C, and then anisole (200. mu.L) and trifluoroacetic acid (1mL) were added, and the reaction was continued at this temperature for 1 h. After completion of the reaction, the solvent was dried by spinning, 1mL of diethyl ether was added, and the precipitated solid was filtered and washed with diethyl ether (1 mL. times.3) to obtain 24mg of a red solid with a yield of 30%. The nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum of the obtained product are respectively shown in FIG. 2 and FIG. 3.
The nuclear magnetic hydrogen spectrum, nuclear magnetic carbon spectrum and mass spectrum data of the product are as follows:
1H NMR(600MHz，DMSO-d6)δ9.09(d，J＝8.2Hz，1H)，7.76(s，1H)， 7.68(d，J＝16.6Hz，1H)，7.47(d，J＝8.7Hz，1H)，7.27(m，6H)，6.70(d，J＝8.3 Hz，1H)，6.53(s，1H)，6.43(d，J＝16.6Hz，1H)，5.48(dd，J＝7.6，4.8Hz，1H)， 5.00(d，J＝4.3Hz，1H)，3.65-3.49(m，4H)，3.43(q，J＝6.9Hz，4H)，1.13(t，J＝ 6.6Hz，6H).
13C NMR(151MHz，DMSO)δ171.44，164.85，163.85，160.36，155.84， 151.15，141.18，136.31，130.18，129.50，128.70，126.97，125.64，125.47，125.39， 116.20，109.92，108.81，96.69，65.39，59.72，58.38，44.64，42.07，31.16，23.93， 15.64，12.84.
HRMS(ESI，m/z)calcd.for C30H29N3O6S+559.1777，found 582.1669 (M+Na+)(Calcd.582.1777).
the multifunctional fluorescent compound CDC-559 prepared in the embodiment is used for performing an in vitro response experiment on AmpC beta-lactamase, a functional verification experiment which can be used for screening AmpC beta-lactamase, a bacteriostasis experiment and a test for identifying a bacterial drug resistance mechanism.
EXPERIMENTAL 1 in vitro RESPONSE EXPERIMENTAL OF COMPOUND CDC-559 TO AMpC beta-lactamase
The experimental method comprises the following steps: dissolving a compound CDC-559 in dimethyl sulfoxide to prepare 10mmol/L stock solution, diluting the stock solution into 10 mu mol/L test solution by using HEPES buffer solution, mixing the test solution with 100U/mL AmpC beta-lactamase, and rapidly scanning by using a fluorescence spectrophotometer (scanning for 10 times, and each time interval is 1 min).
The change of the ultraviolet absorbance and the relative fluorescence intensity of the compound CDC-559 and 100U/mL AmpC beta-lactamase within 10min is shown in FIG. 4a and FIG. 4 b.
As can be seen from FIGS. 4a and 4b, after the compound CDC-559 was combined with AmpC beta-lactamase, the maximum UV absorption was slightly reduced in intensity at 440nm, while the fluorescence intensity was rapidly reduced by 93% at 539 nm.
Experiment 2 Compound CDC-559 can be used in functional verification test for screening AmpC beta-lactamase
The experimental method comprises the following steps: dissolving a compound CDC-559 in dimethyl sulfoxide to prepare 10mmol/L stock solution, and diluting the stock solution into 15 mu mol/L solution to be detected by using HEPES buffer solution; sulbactam sodium and tazobactam acid are dissolved in distilled water to prepare 10mmol/L stock solution, and the stock solution is diluted into different concentrations by HEPES. Firstly, 62.5U/mL AmpC beta-lactamase is mixed with sulbactam sodium or tazobactam acid with different concentrations, the mixture is stood for 10min at room temperature, and then a compound CDC-559 is added, and then the detection is rapidly carried out by using a microplate reader.
The inhibitory effects of two inhibitors (sulbactam sodium and tazobactam acid) on AmpC-lactamase when compound CDC-559 was used as a substrate are shown in fig. 5.
As can be seen from FIG. 5, both sulbactam sodium and tazobactam acid had a very good inhibitory effect on AmpC beta-lactamase, and IC of both inhibitors was tested using the compound CDC-559 as a substrate50Values are within the reported range in the literature, indicating that compound CDC-559 can be used to screen AmpC beta-lactamase inhibitors.
Experiment 3 bacteriostatic experiment of Compound CDC-559
The experimental method comprises the following steps: after 1 mu g/mu L of solution is prepared by oxacillin sodium (1), compound CDC-559(2), compound 1(3), cefazolin sodium (4) and solvent (5, blank group), 1 mu L of solution is dripped on a paper sheet, then the paper sheet is respectively placed on culture dishes which are paved with staphylococcus aureus ATCC 25923, ATCC 29213 (sensitive bacteria), ATCC 43300 (drug-resistant bacteria, the drug-resistant mechanism is that the bacteria have genes for expressing PBP2a protein) and Enterobacter cloacae ATCC 13047 (drug-resistant bacteria, the drug-resistant mechanism is that the bacteria can express AmpC beta-lactamase in large quantity), and the paper sheet is placed in an incubator at 37 ℃ for 18 hours and then observed and photographed.
The inhibition zones obtained by culturing the 5 samples on 3 staphylococcus aureus are shown in fig. 6a, fig. 6b and fig. 6c, and the inhibition zone obtained by culturing on enterobacter cloacae is shown in fig. 6 d.
As can be seen from FIGS. 6a to 6d, Staphylococcus aureus ATCC 25923 and ATCC 29213 are sensitive to oxacillin sodium and cefazolin sodium, while Staphylococcus aureus ATCC 43300 and Enterobacter cloacae ATCC 13047 are resistant to oxacillin sodium and cefazolin sodium, which indicates that the test system meets the requirements. When the compound CDC-559 acts on sensitive bacteria ATCC 25923 and ATCC 29213, compared with the cephalosporin nucleus (compound 1) before modification, the compound CDC-559 has a much larger antibacterial ring which is only a little smaller than the first generation antibiotic cefazolin sodium, which shows that the compound CDC-559 has better antibacterial activity; when the antibacterial activity of the compound CDC-559 is acted with drug-resistant bacteria ATCC 43300 and ATCC 13047, no inhibition zone is generated, which is consistent with an expected result, and the compound CDC-559 is proved to be capable of detecting the drug resistance of bacteria on a bacterial level, and meanwhile, a thought is provided for developing a novel antibiotic later, and the compound CDC-559 can be applied to the preparation of antibacterial drugs.
Experiment 4 experiment for identifying bacterial drug resistance mechanism by CDC-559 compound
The experimental method comprises the following steps: dissolving the compound CDC-559 in dimethyl sulfoxide to prepare a 10mmol/L stock solution. Will OD600 Staphylococcus aureus ATCC 25923, ATCC 29213 (sensitive bacteria), ATCC 43300 (drug-resistant bacteria, the drug-resistant mechanism is that the bacteria carry genes expressing PBP2a protein) and Enterobacter cloacae ATCC 13047 (drug-resistant bacteria, the drug-resistant mechanism is that the bacteria can express AmpC beta-lactamase in a large amount) of 0.5. Mixing 15 μ L of supernatant with the above stock solution, diluting with HEPES buffer solution to make CDC-559 initial concentration 10 μmol/L, mixing, and rapidly detecting with microplate reader.
The change in fluorescence intensity within 10min after mixing of the compound CDC-559 with 4 bacteria (Staphylococcus aureus ATCC 25923, ATCC 29213, ATCC 43300 and Enterobacter cloacae ATCC 13047) is shown in FIG. 7.
As can be seen from FIG. 7, Staphylococcus aureus ATCC 25923 and ATCC 29213 have almost no influence on the fluorescence intensity of the compound CDC-559, Staphylococcus aureus ATCC 43300 slightly reduces the fluorescence intensity of the compound CDC-559, while the fluorescence intensity of the compound CDC-559 is reduced by 76% within 10 minutes under the action of Enterobacter cloacae ATCC 13047, which indicates that the compound CDC-559 can distinguish not only bacterial sensitivity or drug resistance, but also even bacterial drug resistance mechanism.
The technical contents of the present invention are further illustrated by the examples, so as to facilitate the understanding of the reader, but the embodiments of the present invention are not limited thereto, and any technical extension or re-creation based on the present invention is protected by the present invention.

Claims (11)

1. A multifunctional fluorescent probe is characterized in that the structural formula of the multifunctional fluorescent probe is as follows:
Figure FDA0002300914260000011
2. the method for preparing the multifunctional fluorescent probe as claimed in claim 1, which comprises the following steps:
s1, reacting the compound 1 with NaI in a hydrophilic organic solvent, removing the hydrophilic organic solvent after the reaction is finished, adding a lipophilic organic solvent for dissolving, washing and drying an organic phase, adding triphenylphosphine for reacting, and separating a product after the reaction is finished to obtain an intermediate 2;
s2, dissolving the intermediate 2 in a lipophilic organic solvent, adding an aqueous solution of NaOH to react, removing a water phase after the reaction is finished, drying an organic phase, adding a compound 3 to react, and separating a product after the reaction is finished to obtain an intermediate 4;
s3, adding the intermediate 4, anisole and trifluoroacetic acid into a lipophilic organic solvent, and separating a product after the reaction to obtain the multifunctional fluorescent probe;
the structural formula of the compound 1 is as follows:
Figure FDA0002300914260000012
the structural formula of the intermediate 2 is as follows:
Figure FDA0002300914260000013
the structural formula of the compound 3 is as follows:
Figure FDA0002300914260000021
the structural formula of the intermediate 4 is as follows:
Figure FDA0002300914260000022
3. the method for preparing a multifunctional fluorescent probe according to claim 2, wherein in step S1, the molar ratio of the compound 1, NaI and triphenylphosphine is 1: 4-5: 1-1.5.
4. The method of claim 2, wherein the feeding molar ratio of the intermediate 2, NaOH and the compound 3 is 1: 8-10: 0.6-0.9 in step S2.
5. The method of claim 4, wherein the concentration of the aqueous solution of NaOH is 1mol/L in step S2.
6. The method for preparing a multifunctional fluorescent probe according to claim 2, wherein in step S3, the molar ratio of the intermediate 4, anisole and trifluoroacetic acid is 1: (10-15): (80-100).
7. The multifunctional fluorescent probe of claim 1 for detecting AmpC β -lactamase.
8. The multifunctional fluorescent probe of claim 1 for screening AmpC beta-lactamase inhibitor.
9. The use of the multifunctional fluorescent probe of claim 1 in the preparation of a bacteriostatic drug.
10. The use of the multifunctional fluorescent probe of claim 9 in the preparation of a bacteriostatic drug, wherein the bacteriostatic drug is a drug that inhibits staphylococcus aureus.
11. Use of the multifunctional fluorescent probe of claim 1 for identifying a bacterial resistance mechanism.
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