CN115521324B - Preparation of near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria - Google Patents

Preparation of near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria Download PDF

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CN115521324B
CN115521324B CN202211255743.4A CN202211255743A CN115521324B CN 115521324 B CN115521324 B CN 115521324B CN 202211255743 A CN202211255743 A CN 202211255743A CN 115521324 B CN115521324 B CN 115521324B
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drug
lactamase
near infrared
fluorescent probe
carboxyethyl
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CN115521324A (en
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郭丽霞
刘�文
田亚飞
李锦瑶
任国栋
马素芳
张承武
李丽红
刁海鹏
王浩江
王斌
闫丽丽
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Shanxi Medical University
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    • C07D501/16—Compounds having a nitrogen atom directly attached in position 7 with a double bond between positions 2 and 3
    • C07D501/20—7-Acylaminocephalosporanic or substituted 7-acylaminocephalosporanic acids in which the acyl radicals are derived from carboxylic acids
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Abstract

The invention relates to the technical field of biosensing, in particular to preparation of a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria. Specifically, 2, 3-trimethyl-3H-indole is taken as a raw material, nucleophilic substitution is carried out to obtain 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole, and further condensation and substitution reaction are carried out to obtain 2- ((E) -2- (((7S) -2-carboxyl-8-oxo-7- (2-phenylacetamide) -5-sulfur-1-azacyclo [4.2.0] oct-2-en-3-yl) methoxy) -2, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -1- (2-carboxyethyl) -3, 3-dimethyl-3H-indole-1-ammonium (CS-PA). The fluorescent probe obtained by the invention is a near infrared fluorescent probe which specifically responds to enzyme generated by drug-resistant bacteria; the adopted preparation method is simple and the condition is mild; the probe emits near infrared fluorescence through the specific combination of the response part and beta-lactamase generated by the drug-resistant bacteria, and has higher application value in the aspect of drug-resistant bacteria detection.

Description

Preparation of near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria
Technical Field
The invention relates to the technical field of biosensing, in particular to preparation of a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria.
Background
Hundreds of millions of pathogenic bacteria have been saved from the advent of antibiotics to infect patients. However, with the development of technology and the progress of society, antibiotics are abused and abused in a large amount, which causes the appearance of drug-resistant bacteria, so that the existing antibiotics are disabled, and finally, the antibiotics are developed into multi-drug-resistant bacteria. The production of antibiotic-degrading enzymes is an important cause of bacterial resistance, with beta-lactamases being the most common. Beta-lactam antibiotics are often used as the first choice for treating pathogenic bacterial infections due to their broad spectrum and high antibacterial properties. Therefore, the rapid and efficient screening of the drug-resistant bacteria has important significance for the selection of drugs.
Early diagnosis is of great importance for guiding the use of drugs and for reducing the risk of multi-drug resistant bacterial infections. Detection of enzymes produced by bacteria is an effective method for the current diagnosis of drug-resistant bacteria. However, in the existing methods, the phenotypic analysis is mostly low in specificity, low in sensitivity and long in time consumption, aggravates the risk of multi-drug resistant bacterial infection of patients, and the Polymerase Chain Reaction (PCR) is high in cost and cannot detect unknown genes. The fluorescence analysis is a novel detection method developed in recent years, has the advantages of simple operation, high sensitivity, high detection speed, low cost, noninvasive in-situ imaging capability and the like, and is becoming a powerful method for early diagnosing whether the in-vivo and in-vitro bacterial infection is drug-resistant bacterial infection or not. However, the probes reported so far have long response times, short emission wavelengths, and relatively large background interference in organisms. Therefore, it is the object of the present invention to obtain a near infrared fluorescent probe with high sensitivity and specificity to detect drug-resistant bacteria.
Disclosure of Invention
The invention aims to provide a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria and preparation thereof.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria has a structural formula as follows:
the preparation of the near infrared fluorescent probe for detecting the beta-lactamase in the drug-resistant bacteria comprises the following steps:
1) Stirring 2, 3-trimethyl-3H-indole and 3-bromopropionic acid under the condition of no solvent for reaction, cooling after the reaction is finished, ultrasonically dissolving reactants by using methylene dichloride, and then precipitating by using a solvent for three times to obtain white precipitate 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole; the reaction formula is as follows:
2) Uniformly mixing 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole, (E) -2-chloro-3- (hydroxymethyl) cyclohexene-1-formaldehyde, anhydrous sodium acetate and acetic anhydride, stirring for reaction, concentrating under reduced pressure, pouring into distilled water, extracting with dichloromethane, collecting a lower organic phase, concentrating under reduced pressure, purifying a crude product by silica gel column chromatography to obtain green solid 1- (2-carboxyethyl) -2- ((E) -2- (E) -3- (2- (E) -1- (2-carboxyethyl) -3, 3-dimethyl-2-methylene) ethylene) -2-chlorocyclohexyl-1-en-1-yl) vinyl) -3, 3-dimethyl-3H-indole; the reaction formula is as follows:
3) In N 2 Stirring 3-hydroxy thiophenol, potassium carbonate and organic solvent at 50deg.C for 30min under protection, and adding a solvent containing 1- (2-carboxyethyl) -2- ((E) -2- (E) -3- (2- (E) -1- (2-carboxyethyl) -3, 3-dimethyl)-2-methylene) ethenyl) -2-chlorocyclohexyl-1-en-1-yl-vinyl) -3, 3-dimethyl-3H-indole acetonitrile, the obtained mixed solution is stirred for 3H at 55 ℃, the mixture is concentrated under reduced pressure, and the crude product is purified by silica gel column chromatography to obtain blue solid (E) -1- (2-carboxyethyl) -2- (2- (6-hydroxy-2, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -3, 3-dimethyl-3H-indole; the reaction formula is as follows:
4) Dissolving (E) -1- (2-carboxyethyl) -2- (2- (6-hydroxy-2, 3-dihydro-1H-thioxanthene-4-yl) vinyl) -3, 3-dimethyl-3H-indole and condensing agent in DMF, stirring at room temperature for 20min, then dropwise adding DMF solution containing 7-phenylacetylamino-3-chloromethyl-4-cephalosporanic acid p-methoxybenzyl ester, stirring for 30min, concentrating under reduced pressure, purifying the crude product by silica gel column chromatography to obtain blue solid compound, the blue solid compound was then dissolved in a mixed solution of anisole, trifluoroacetic acid and dichloromethane, stirred for 1H at 0 ℃, concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel to give blue solid 2- ((E) -2- (((7S) -2-carboxy-8-oxo-7- (2-phenylacetamide) -5-thio-1-aza [4.2.0] oct-2-en-3-yl) methoxy) -2, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -1- (2-carboxyethyl) -3, 3-dimethyl-3H-indol-1-ammonium, i.e., fluorescent probe (CS-PA). The reaction formula is as follows:
further, the temperature of the reaction in the step 1) is 90 ℃, and the reaction time is 60min; the reaction conditions in step 2) were 6h at room temperature.
Further, the molar ratio of 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole, (E) -2-chloro-3- (hydroxymethylene) cyclohexene-1-carbaldehyde to anhydrous sodium acetate described in step 2) was 2:1:3.
Further, the silica gel column chromatography in the steps 2) and 3) is a mixture of dichloromethane and methanol in a volume ratio of 20:1; the silica gel column chromatography in the step 4) is a mixture of dichloromethane and methanol in a volume ratio of 25:1 or 2:1.
Further, the solvent in the step 1) is ethyl acetate; the organic solvent in the step 3) is acetonitrile or DMF; the condensing agent in the step 4) is Cs 2 CO 3 。
Still further, the volume ratio of anisole, trifluoroacetic acid and methylene chloride described in step 4) is 1:4:20.
The application of the near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria can be used for detecting the drug-resistant bacteria.
The application of near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria, wherein the probe can be specifically combined with enzyme in the drug-resistant bacteria to emit fluorescent signals.
The application of near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria, the probe can be prepared into a kit for screening drug-resistant bacteria.
Compared with the prior art, the invention has the following advantages:
the near infrared fluorescent probe CS-PA for detecting the beta-lactamase in the drug-resistant bacteria has long emission wavelength and low cytotoxicity, can be specifically combined with the beta-lactamase overexpressed in the drug-resistant bacteria, has quick response time, can be prepared into a kit for screening the drug-resistant bacteria, and has simple reaction operation and mild conditions by adopting a preparation method.
Drawings
FIG. 1 shows the ultraviolet absorption and fluorescence emission spectra of the near infrared fluorescent probe CS-PA of the present invention before and after the reaction with beta-lactamase;
FIG. 2 is a graph showing the time and temperature of the action of the near infrared fluorescent probe CS-PA and the beta-lactamase;
FIG. 3 is a diagram showing a research on the specificity of the near infrared fluorescent probe CS-PA detection of beta-lactamase;
FIG. 4 is a cytotoxicity map of the near infrared fluorescent probe CS-PA of the present invention;
FIG. 5 is a diagram showing the performance research of detecting drug-resistant bacteria by using the near infrared fluorescent probe CS-PA of the invention;
FIG. 6 is a diagram showing the time of detection of drug-resistant bacteria by using the near infrared fluorescent probe CS-PA of the invention;
FIG. 7 is a graph showing the performance of the near infrared fluorescent probe CS-PA of the invention in detecting different amounts of drug-resistant bacteria;
FIG. 8 is a graph showing the comparison of UV absorption before and after the action of the near infrared fluorescent probe CS-PA of the present invention with drug-resistant bacteria and beta-lactamase.
Detailed Description
Example 1
1) 2, 3-trimethyl-3H-indole (0.48 g,3.0 mmol) and 3-bromopropionic acid (0.46 g,3.0 mmol) were placed in a 25mL single neck round bottom flask equipped with magnetic stirring and the mixture was stirred at 90℃without solvent for 60min. After completion of the reaction, it was cooled, and the reaction mass was dissolved with methylene chloride (5 mL) by ultrasonic wave, followed by precipitation with 50mL of ethyl acetate and repeating three times to give 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole as a white precipitate (0.35 g, yield 73%).
1 H NMR(400MHz,CDCl 3 ,ppm)δ:1.62(s,6H),3.13(s,3H),3.38(t,J＝12.0Hz,2H),5.00(t,J＝12.0Hz,2H),7.54～7.64(m,3H),7.81(d,J＝4.0Hz,1H)；MALDI-TOF for C 14 H 18 NO 2 + :Calculated for 232.133[M]；Found for 232.139[M]；
2) 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole) (0.13 g,0.5 mmol), (E) -2-chloro-3- (hydroxymethylene) cyclohexene-1-carbaldehyde (0.07 g,0.250 mmol) and anhydrous sodium acetate (0.065 g,0.75 mmol) were placed in a 25mL single neck round bottom flask equipped with magnetic stirring, dissolved by adding 12mL acetic anhydride, and reacted at room temperature for 6H. After the completion of the reaction, the mixture was concentrated under reduced pressure, and then poured into 20mL of distilled water, followed by extraction with 40mL of methylene chloride. The lower organic phase was collected, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol=20:1) to give 1- (2-carboxyethyl) -2- ((E) -2- (E) -3- (2- (E) -1- (2-carboxyethyl) -3, 3-dimethyl-2-methylene) ethenylidene) -2-chlorocyclohexyl-1-en-1-yl) vinyl) -3, 3-dimethyl-3H-indole (0.06 g, yield 33%) as a green solid.
1 H NMR(400MHz,CD 3 OD,ppm)δ:1.72(s,12H),1.95(t,J＝8.0Hz,2H),2.63(t,J＝12.0Hz,4H),2.77(t,J＝12.0Hz,4H),4.40(t,J＝16.0Hz,4H),6.47(d,J＝12.0Hz,2H),7.25～7.29(m,2H),7.38～7.44(m,4H),7.51(d,J＝8.0Hz,2H),8.44(d,J＝16.0Hz,2H)。MALDI-TOF for C 36 H 40 ClN 2 O 4 + :Calculated for 599.267[M]；Found for 599.296[M]；
3) At N 2 After placing 3-hydroxybenzylthiophenol (0.25 g,2.0 mmol), potassium carbonate (0.28 g,2.0 mol) and 20mL of acetonitrile or DMF under protection in a 50mL three-necked round bottom flask equipped with magnetic stirring, the mixed solution was stirred at 50℃for 30min, then 10mL of acetonitrile containing 1- (2-carboxyethyl) -2- ((E) -2- (E) -3- (2- (E) -1- (2-carboxyethyl) -3, 3-dimethyl-2-methylene) ethylene) -2-chlorocyclohexyl-1-en-1-yl) vinyl) -3, 3-dimethyl-3H-indole (0.60 g,1.0 mmol) was added, the resulting mixed solution was stirred at 55℃for 3H, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol=20: 1) Purification gave (E) -1- (2-carboxyethyl) -2- (2- (6-hydroxy-2, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -3, 3-dimethyl-3H-indole (0.23 g, 50% yield) as a blue solid.
1 H NMR(400MHz,CD 3 OD,ppm)δ:1.84(s,6H),1.97(s,2H),2.76(s,2H),2.86(t,J＝8.0Hz,4H),4.67(s,2H),6.80～6.98(m,2H),7.10(s,2H),7.33(d,J＝8.0Hz,2H),7.49～7.74(broad,5H)；8.44(t,J＝12.0Hz,1H)；MALDI-TOF for C 28 H 28 NO 3 S + :Calculated for458.1784[M]；Found for458.1793[M]；
4) (E) -1- (2-carboxyethyl) -2- (2- (6-hydroxy-2, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -3, 3-dimethyl-3H-indole (230 mg,0.5 mmol) and Cs 2 CO 3 (424 mg,1.3 mmol) was placed in a 25mL single neck round bottom flask equipped with magnetic stirring, and 5mL of DMF was added to dissolve, stirring was performed at room temperature for 20min, then a solution of p-methoxybenzyl 7-phenylacetamido-3-chloromethyl-4-cephalosporanic acid (633 mg,1.3mmo 1) in DMF (3 mL) was added dropwise, stirring was continued at room temperature for 30min, concentrating under reduced pressure, and the crude product was purified by silica gel column chromatography (dichloromethane: methanol=25:1) to give a blue solid compound (91 mg, 20%); the blue solid compound obtained is then treated36.3mg,0.04mmo 1) was dissolved in a mixed solution of anisole (50. Mu.L), trifluoroacetic acid (200. Mu.L) and methylene chloride (1 mL), stirred at 0℃for 1h, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography (methylene chloride: methanol=2: 1) Purification gave 2- ((E) -2- (((7S) -2-carboxy-8-oxo-7- (2-phenylacetamide) -5-thio-1-azacyclo [ 4.2.0) as a blue solid]Oct-2-en-3-yl) methoxy) -2, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -1- (2-carboxyethyl) -3, 3-dimethyl-3H-indol-1-ammonium, i.e., fluorescent probe (CS-PA). (10 mg, 31.7%).
1 H NMR(400MHz,CD 3 OD,ppm)δ:1.80(s,6H),1.94(m,2H),2.55(s,1H),2.74(t,J＝6.0Hz,2H),2.81(t,J＝6.0Hz,2H),2.98(t,J＝6.0Hz,2H),3.59(d,J＝4.0Hz,2H),4.64(m,3H),4.78(s,1H),5.31(d,J＝4.0Hz,1H),5.38(d,J＝4.0Hz,1H),6.33(s,1H),6.72(d,J＝12.0Hz,1H),6.97(dd,J＝4.0,4.0Hz,1H),7.10(d,J＝4.0Hz,1H),7.30(m,8H),7.44(t,J＝6.0Hz,1H),7.56(m,3H),7.65(d,J＝8.0Hz,1H),8.44(d,J＝12.0Hz,1H)；MALDI-TOF for C 44 H 42 N 3 O 7 S 2 + :Calculated for 788.2459[M]；Found for 788.2467[M]；
Example 2
Ultraviolet and fluorescence spectroscopy test of probe CS-PA:
a certain amount of CS-PA is accurately weighed and dissolved in methanol to prepare a mother solution with the concentration of 1.0mM, beta-lactamase is prepared into a mother solution with the concentration of 365 mu M, and the mother solution is stored at the temperature of minus 20 ℃ for standby. 20. Mu.L of probe (10. Mu.M) and a certain concentration of beta-lactamase were added to PBS, and the mixture was mixed uniformly and reacted at 37℃for 7 minutes. Then, the reaction solution was transferred to a fluorescence cell, and the absorption peak was 700nm as measured on a HITACHI UH5300 ultraviolet absorber. The excitation and emission slits were 5nm and 10nm, respectively, and the excitation wavelength was 725nm, as measured on a HITACHI F-7100 fluorometer. The test was performed at room temperature and at ambient atmospheric pressure. As shown in FIG. 1, the maximum absorption wavelength of the probe CS-PA is 700nm, and the maximum absorption wavelength of the probe CS-PA after the probe CS-PA reacts with beta-lactamase is 710nm and is obviously enhanced. The fluorescence emission spectrum can show that the emission wavelength of the probe is positioned at 763nm and in the near infrared region, and the fluorescence is obviously enhanced after the probe reacts with beta-lactamase. The above results demonstrate that the probe can selectively detect beta-lactamase.
Example 3
Test of probe CS-PA response time and temperature to beta-lactamase:
to the PBS buffer, 20. Mu.L of probe CS-PA (10. Mu.M) and 1.6. Mu.L (200 nM) of beta-lactamase were added, respectively, and after mixing well, the mixture was reacted at 37℃and the fluorescence spectrum was scanned every one minute. Then, 4 sets of mixed solutions of probe CS-PA (10. Mu.M) and beta-lactamase (200 nM) were incubated at 20℃and 30℃and 37℃and 40℃for 7min, respectively, and fluorescence spectra were measured after the incubation was completed. The conditions for the fluorescence test were the same as in example 2, and the fluorescence intensity at 763nm was plotted against time. The test result is shown in FIG. 2, the probe CS-PA and beta-lactamase can reach the platform after about 5min, and the optimal test temperature is 37 ℃.
Example 4
Probe CS-PA response selectivity test for beta-lactamase:
20. Mu.L of probe CS-PA (10. Mu.M) and different ions, amino acids and enzymes, ca, were added to PBS buffer, respectively 2+ (1mM)、Mg 2+ (1mM)、ClO - (100μM)、S 2 O 3 2- (100. Mu.M), proline (1 mM), cysteine (1 mM), phenylalanine (1 mM), lysine (1 mM), glutamic acid (1 mM), glucose (10 mM), H 2 O 2 (hydrogen peroxide, 100. Mu.M), nitroreductase (2. Mu.g/mL), beta-lactamase (200 nM). After mixing uniformly, incubating for 7min at 37 ℃, and measuring fluorescence spectra of the materials respectively after incubation. The conditions for the fluorescence test were the same as in example 2, and the fluorescence intensity at 763nm was plotted against time. As shown in FIG. 3, the fluorescence intensity of the probe CS-PA is enhanced by about 3.5 times by adding the beta-lactamase, which shows that the probe CS-PA has better selectivity for detecting the beta-lactamase.
Example 5
Cytotoxicity test of probe CS-PA:
cytotoxicity of the probe CS-PA on human endometrial epithelial cells (hEC cells) was measured by MTT method. Spreading the uniformly mixed cells in 96-well plate, wherein each well contains about 8000 cells, CO 2 Cell attachment after 24h culture in incubatorThe old medium was discarded and medium of different concentrations of probe CS-PA was added. After further incubation for 24h, 10. Mu.L of 5mg.mL was added to each well -1 The MTT solution of (C) was further cultured for 2 to 4 hours. Finally, taking out the 96-well plate, putting the 96-well plate into an enzyme labeling instrument, vibrating for 1min, and measuring the absorbance value of each well at 490 nm. The calculation method of the cell viability CR comprises the following steps:
CR＝A/A 0 ×100％
wherein A is the absorbance value of the cells of the experimental group treated by the probe CS-PA, A 0 Absorbance values for cells of the control group without probe CS-PA. As shown in FIG. 4, when the concentration of the probe CS-PA reaches 50. Mu.M, the bacterial survival rate can still reach more than 85%, which indicates that the probe has lower cytotoxicity.
Example 6
Detection of bacterial liquid by the probe CS-PA:
1) Culturing bacteria:
the ultra-clean bench is sterilized by turning on an ultraviolet lamp for 20-30 min, the surface of the ultra-clean bench is wiped by 75% alcohol, and a sterilized 50mL centrifuge tube, LB culture medium, NB culture medium, 1 XPBS and bacterial liquid are taken into the ultra-clean bench. Taking out a 50mL centrifuge tube, sucking 10mL LB or NB liquid culture into a 50mL sterile centrifuge tube, respectively taking 10 mu L of different strains into corresponding culture mediums, wherein the strains are enterococcus faecalis, staphylococcus aureus, methicillin-resistant staphylococcus aureus, klebsiella pneumoniae, pseudomonas aeruginosa, escherichia coli, multiple drug-resistant pseudomonas aeruginosa and drug-resistant escherichia coli. Shake culturing at 37 deg.C and 180rpm for 8-10 hr.
2) Detection of bacterial liquid:
centrifuging (7100 rpm,2 min) liquid culture medium of different strains in a super clean bench, precipitating bacteria, washing the precipitated bacteria with 1 XPBS, centrifuging again, repeating twice, discarding supernatant, re-suspending the bacterial solution in 1 XPBS, and adjusting OD 600 1.5. Preparing lysozyme mother solution with concentration of 50mg/mL for later use, taking 1mL of different bacterial suspensions, adding 200 mu L of the lysozyme solution, uniformly mixing, incubating for 1 hour at 37 ℃, then crushing for 3min in an ultrasonic crusher, and carrying out ice bath.20 mu L (10 mu M) of the probe and 20 mu L of the bacterial liquid of the different treated bacteria are added into PBS buffer solution respectively, and the mixture is evenly mixed and incubated for 7min at 37 ℃. The influence of enzyme inhibitor PCA on the detection of enzymes by probes was studied for drug-resistant strains, and the bacterial solution of the drug-resistant strain was incubated with enzyme inhibitor PCA (100. Mu.M) for 2min, followed by addition of probes and incubation at 37℃for 7min. The resulting solution was transferred to a fluorescence cell for measurement of fluorescence spectrum, and fluorescence test conditions were as in example 2. As shown in FIG. 5, the probe CS-PA was responsive only to the resistant strain producing beta-lactamase and not to methicillin-resistant Staphylococcus aureus and normal strains.
Example 7
Probe CS-PA and bacterial liquid (OD) 600 =1.0) test of response time:
the pretreatment of the bacterial solution was the same as in example 6, and 20. Mu.L of probe CS-PA (10. Mu.M) and 200. Mu.L of drug-resistant bacterial solution were added to PBS buffer, mixed well, incubated at 37℃and fluorescence spectrum scanned every one minute. Fluorescence intensity at 763nm was plotted against time. As shown in FIG. 6, the fluorescence intensity was the strongest when the probe CS-PA was allowed to act on the drug-resistant bacterial liquid for about 7 minutes, indicating that the optimal test time was 7 minutes.
Example 8
Probe CS-PA and different volumes of bacterial liquid (OD 600 =1.0) response test
The pretreatment of the bacterial liquid was the same as in example 6, 20. Mu.L of the probe (10. Mu.M) and different volumes of the bacterial liquid were added to the PBS buffer, and after the mixture was homogenized, the incubation was carried out at 37℃for 7 minutes, and after the incubation was completed, the fluorescence spectra were measured. As shown in FIG. 7, the fluorescence intensity of the probe CS-PA was gradually increased with the increase of the bacterial liquid volume, indicating that the probe had a concentration dependence on the enzyme produced in the drug-resistant bacteria.
Example 9
Absorption spectrum test before and after probe CS-PA responds to bacterial liquid:
the pretreatment of the bacterial solution was the same as in example 6, and 20. Mu.L of the probe (10. Mu.M) and 200. Mu.L of the bacterial solution were added to the PBS buffer, and the mixture was mixed uniformly and incubated at 37℃for 7 minutes. After the incubation, the sample was transferred to a quartz cell to measure the ultraviolet-visible absorption spectrum. As shown in FIG. 8, after the probe CS-PA acts on the bacterial liquid of the drug-resistant bacteria, the absorption peak at 710nm is obviously enhanced, and the absorption spectrum is close to that of the beta-lactamase after the probe CS-PA acts on the beta-lactamase, which indicates that the beta-lactamase is secreted in the drug-resistant bacteria, and the probe can monitor the beta-lactamase in the drug-resistant bacteria.
What is not described in detail in the present specification belongs to the prior art known to those skilled in the art. While the foregoing describes illustrative embodiments of the present invention to facilitate an understanding of the present invention by those skilled in the art, it should be understood that the present invention is not limited to the scope of the embodiments, but is to be construed as protected by the accompanying claims insofar as various changes are within the spirit and scope of the present invention as defined and defined by the appended claims.

Claims (9)

1. A near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria is characterized by comprising the following structural formula:
2. the method for preparing a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria according to claim 1, comprising the following steps:
1) Stirring 2, 3-trimethyl-3H-indole and 3-bromopropionic acid under the condition of no solvent for reaction, cooling after the reaction is finished, ultrasonically dissolving reactants by using methylene dichloride, and then precipitating by using a solvent for three times to obtain white precipitate 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole;
2) Adding 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole, (E) -2-chloro-3- (hydroxymethyl) cyclohexene-1-formaldehyde and anhydrous sodium acetate into acetic anhydride, stirring for reaction, concentrating under reduced pressure, pouring into distilled water, extracting with dichloromethane, collecting a lower organic phase, concentrating under reduced pressure, purifying a crude product by silica gel column chromatography to obtain green solid 1- (2-carboxyethyl) -2- ((E) -2- (E) -3- (2- (E) -1- (2-carboxyethyl) -3, 3-dimethyl-2-methylene) ethylene) -2-chlorocyclohexyl-1-en-1-yl) vinyl) -3, 3-dimethyl-3H-indole;
3) In N 2 Stirring 3-hydroxybenzylthiophenol, potassium carbonate and an organic solvent at 50 ℃ for 30min under protection, then adding acetonitrile containing 1- (2-carboxyethyl) -2- ((E) -2- (E) -3- (2- (E) -1- (2-carboxyethyl) -3, 3-dimethyl-2-methylene) ethylene) -2-chlorocyclohexyl-1-en-1-yl) vinyl) -3, 3-dimethyl-3H-indole, stirring the obtained mixed solution at 55 ℃ for 3H, concentrating under reduced pressure, and purifying the crude product by silica gel column chromatography to obtain blue solid (E) -1- (2-carboxyethyl) -2- (2- (6-hydroxy-2, 3-dihydro-1H-thianthrene-4-yl) vinyl) -3, 3-dimethyl-3H-indole;
4) Dissolving (E) -1- (2-carboxyethyl) -2- (2- (6-hydroxy-2, 3-dihydro-1H-thioxanthene-4-yl) vinyl) -3, 3-dimethyl-3H-indole and condensing agent in DMF, stirring at room temperature for 20min, then dropwise adding DMF solution containing 7-phenylacetylamino-3-chloromethyl-4-cephalosporanic acid p-methoxybenzyl ester, stirring for 30min, concentrating under reduced pressure, purifying the crude product by silica gel column chromatography to obtain blue solid compound, the blue solid compound was then dissolved in a mixed solution of anisole, trifluoroacetic acid and dichloromethane, stirred for 1H at 0 ℃, concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel to give blue solid 2- ((E) -2- (((7S) -2-carboxy-8-oxo-7- (2-phenylacetamide) -5-thio-1-azacyclo [4.2.0] oct-2-en-3-yl) methoxy) -2, 3-dihydro-1H-thioxanthen-4-yl) vinyl) -1- (2-carboxyethyl) -3, 3-dimethyl-3H-indol-1-ammonium, i.e., fluorescent probe.
3. The method for preparing a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria according to claim 2, wherein the temperature of the reaction in the step 1) is 90 ℃, and the reaction time is 60min; the reaction conditions in step 2) were 6h at room temperature.
4. The method for preparing a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria according to claim 2, wherein the molar ratio of 1- (2-carboxyethyl) -2, 3-trimethyl-3H-indole, (E) -2-chloro-3- (hydroxymethylene) cyclohexene-1-carbaldehyde to anhydrous sodium acetate in step 2) is 2:1:3.
5. The method for preparing a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria according to claim 2, wherein the silica gel column chromatography in the steps 2) and 3) is a mixture of dichloromethane and methanol in a volume ratio of 20:1; the silica gel column chromatography in the step 4) is a mixture of dichloromethane and methanol in a volume ratio of 25:1 or 2:1.
6. The method for preparing a near infrared fluorescent probe for detecting beta-lactamase in a drug-resistant bacterium according to claim 2, wherein the solvent in the step 1) is ethyl acetate; the organic solvent in the step 3) is acetonitrile or DMF; the condensing agent in the step 4) is Cs 2 CO 3 。
7. The method for preparing a near infrared fluorescent probe for detecting beta-lactamase in drug-resistant bacteria according to claim 2, wherein the volume ratio of anisole, trifluoroacetic acid and dichloromethane in the step 4) is 1:4:20.
8. The use of a near infrared fluorescent probe for detecting beta-lactamase in a drug-resistant bacterium as claimed in claim 1, wherein the probe is used for preparing a detection reagent for the drug-resistant bacterium.
9. The use of a near infrared fluorescent probe for detecting beta-lactamase in a drug-resistant bacterium according to claim 1 for preparing a kit, wherein the kit is used for screening the drug-resistant bacterium.
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Publication number Priority date Publication date Assignee Title
CN106279178A (en) * 2016-07-18 2017-01-04 华东理工大学 The fluorescent probe of Carbapenem-resistant class antibiotic pathogenic bacteria and synthetic method and application
CN106811192A (en) * 2017-01-13 2017-06-09 华东理工大学 The fluorescence probe of Carbapenem-resistant class antibiotic germ and its synthetic method and application
CN110950893A (en) * 2019-12-03 2020-04-03 华南理工大学 Multifunctional fluorescent probe and preparation method and application thereof
CN113912626A (en) * 2021-08-24 2022-01-11 南开大学 Broad spectrum resistant beta-lactam and cephalosporin antibiotics pathogen probe and synthetic method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106279178A (en) * 2016-07-18 2017-01-04 华东理工大学 The fluorescent probe of Carbapenem-resistant class antibiotic pathogenic bacteria and synthetic method and application
CN106811192A (en) * 2017-01-13 2017-06-09 华东理工大学 The fluorescence probe of Carbapenem-resistant class antibiotic germ and its synthetic method and application
CN110950893A (en) * 2019-12-03 2020-04-03 华南理工大学 Multifunctional fluorescent probe and preparation method and application thereof
CN113912626A (en) * 2021-08-24 2022-01-11 南开大学 Broad spectrum resistant beta-lactam and cephalosporin antibiotics pathogen probe and synthetic method and application thereof

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