[go: up one dir, main page]

WO2018130158A1 - Sonde fluorescente pour bactéries pathogènes tolérantes aux antibiotiques carbapénèmes et procédé de synthèse et utilisation associés - Google Patents

Sonde fluorescente pour bactéries pathogènes tolérantes aux antibiotiques carbapénèmes et procédé de synthèse et utilisation associés Download PDF

Info

Publication number
WO2018130158A1
WO2018130158A1 PCT/CN2018/072128 CN2018072128W WO2018130158A1 WO 2018130158 A1 WO2018130158 A1 WO 2018130158A1 CN 2018072128 W CN2018072128 W CN 2018072128W WO 2018130158 A1 WO2018130158 A1 WO 2018130158A1
Authority
WO
WIPO (PCT)
Prior art keywords
fluorescent probe
carbapenem
compound
resistant
antibiotic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2018/072128
Other languages
English (en)
Chinese (zh)
Other versions
WO2018130158A9 (fr
Inventor
谢贺新
毛梧宇
夏令英
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
East China University of Science and Technology
Original Assignee
East China University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by East China University of Science and Technology filed Critical East China University of Science and Technology
Publication of WO2018130158A1 publication Critical patent/WO2018130158A1/fr
Publication of WO2018130158A9 publication Critical patent/WO2018130158A9/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D477/00—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring
    • C07D477/10—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2
    • C07D477/12—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6
    • C07D477/14—Heterocyclic compounds containing 1-azabicyclo [3.2.0] heptane ring systems, i.e. compounds containing a ring system of the formula:, e.g. carbapenicillins, thienamycins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulphur-containing hetero ring with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 4, and with a carbon atom having three bonds to hetero atoms with at the most one bond to halogen, e.g. an ester or nitrile radical, directly attached in position 2 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 6 with hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, attached in position 3
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F5/00—Compounds containing elements of Groups 3 or 13 of the Periodic Table
    • C07F5/02—Boron compounds
    • C07F5/022—Boron compounds without C-boron linkages
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • 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
    • G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • G—PHYSICS
    • 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)
    • G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G01N2333/986—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in cyclic amides (3.5.2), e.g. beta-lactamase (penicillinase, 3.5.2.6), creatinine amidohydrolase (creatininase, EC 3.5.2.10), N-methylhydantoinase (3.5.2.6)

Definitions

  • the present invention relates to the field of compound preparation technology, and in particular to a fluorescent probe for detecting a carbapenem-resistant antibiotic pathogen and a method for synthesizing the same, and the fluorescent probe detecting carbapenemase And applications in carbapenem-resistant bacteria.
  • Carbapenem antibiotics are a class of ⁇ -lactam antibiotics with a special structure. It consists of a ⁇ -lactam ring and a five-membered ring to form an antibiotic core. The substituent on the four-membered ring is a trans structure. Carbapenem antibiotics are in contrast to other types of beta-lactam antibiotics. Carbapenem antibiotics mainly include imipenem, meropenem, doripenem and ertapenem. Carbapenem antibiotics have broad antibacterial activity and have the ability to strongly inhibit or kill different types of bacteria (J. Antimicrob. Agents. 1999, 11, 93; Antimicrob. Agents Chemother.
  • Carbapenem antibiotics have a good effect on infections caused by Gram-negative and positive bacteria. Carbapenem antibiotics also have a good effect on resistant bacteria containing penicillinase and cephalosporin. However, with the widespread use of such antibiotics, in recent years, bacteria resistant to them have gradually appeared worldwide. Studies have found that there are many reasons for the resistance of pathogenic bacteria. For example, one of the main reasons is that the target penicillin-binding protein of the ⁇ -lactam antibiotic is mutated. Another major cause is the production of an antibiotic inactivating enzyme called beta-lactamase in bacteria.
  • This enzyme rapidly hydrolyzes the ⁇ -lactam bond of the commonly used ⁇ -lactam antibiotics, making the antibiotics less effective.
  • This type of ⁇ -lactamase which disables penicillin antibiotics, is called carbapenemase.
  • the carbapenemase generally hydrolyzes carbapenem antibiotics and even hydrolyzes almost all beta-lactam antibiotics.
  • the sensitivity to carbapenem antibiotics (imipenem, meropenem, etc.) is lowered, and the therapeutic effect is deteriorated.
  • the ⁇ -lactamase (carbameptanase) can be classified into four types: A, B, C, and D (Philos Trans R Soc Lond B Biol Sci 1980; 289: 321-31). Among them, the three types A, C and D are ⁇ -lactamase containing serine.
  • Type B is a ⁇ -lactamase containing a metal zinc ion.
  • the type A ⁇ -lactamase includes KPC, a GES type ⁇ -lactamase, and the like. It can be detected in many pathogenic bacteria, such as Enterobacter, Serratia marcescens, Klebsiella, and the like.
  • phenotypic detection is typically detected by measuring the sensitivity of the bacteria to carbapenem antibiotics. It also includes agar diffusion test method, minimum inhibitory concentration measurement (MIC) method, double-paper synergy test method (DDST), modified Hodge detection method (MHT) and the like. Based on these methods, the American Institute of Clinical and Laboratory Standards has developed standard antibiotic susceptibility tests to detect the susceptibility of pathogenic bacteria. It reflects the resistance of bacteria to a certain extent, and also greatly helps the treatment of patients with serious bacterial infections. However, these methods lack good specificity and sensitivity.
  • the gene detection method is mainly carried out by polymerase chain reaction (PCR). mRNA expression of the carbapenemase-encoding gene can be detected by real-time quantitative reverse transcriptase-PCR (rt qRT-PCR).
  • rt qRT-PCR real-time quantitative reverse transcriptase-PCR
  • the gene detection method has high accuracy and sensitivity.
  • the instrument it uses is expensive, costly, and cannot detect a new carbapenemase gene.
  • the carbapenem-based assay which provides important information on bacterial antibiotic endurance by detecting carbapenemase activity.
  • the carbapenemase was successfully detected by the colorimetric principle using Nitrocefin.
  • the detection principle is as follows: the ⁇ -lactam ring of cefanitrothiophene can be rapidly hydrolyzed by ⁇ -lactamase to open the ring. The color changes from yellow to red due to changes in the conjugated structure of the substrate molecule.
  • the detection of the drug-resistant bacteria by the change of color is simple and easy to use. However, due to the slow color change and low sensitivity, the practical application of the detection method is greatly restricted.
  • fluorescent probes have received widespread attention.
  • the fluorescent probe has many advantages such as low background signal, high sensitivity, low instrument cost and the like. It has attracted much attention in the field of biomedicine.
  • more fluorescent probe compounds have been applied to the detection of ⁇ -lactamase in high sensitivity and in real time.
  • Nobel Prize winner Professor Qian Yongjian wrote in the journal Science (Science 1998, 279, 84-88).
  • a molecular fluorescent probe based on a ⁇ -lactam structure was first reported. Fluorescence energy transfer (FRET) mechanism was used to monitor the activity of ⁇ -lactamase. Subsequently, some fluorescent probe compounds for detecting ⁇ -lactamase activity have been reported.
  • FRET Fluorescence energy transfer
  • coumarin is introduced as a fluorescent reporter group, and the conformation of the 6,7-position is converted from cis to trans conformation to synthesize a series of fluorescent probe compounds.
  • selective detection of ⁇ -carbapyrenease and bacteria expressing carbapenemase is achieved.
  • the different fluorescent probes have very different selectivity and detection sensitivity for different carbapenemases.
  • a patent application by East China University of Science and Technology in 2016 also proposed a fluorescent probe resistant to carbapenem antibiotics.
  • the fluorescent probe uses an enzyme-specific recognition group of the parent nucleus structure of the carbapenem antibiotic.
  • a dye having a leaving function is introduced at the 3'-position of the carbapenem antibiotic core.
  • the ⁇ -lactam ring is opened by the action of carbapenemase to generate a fluorescent signal, and selective detection of the carbapenemase is achieved.
  • this fluorescent probe is mainly based on coumarin as a fluorophore.
  • the excitation light is located in the ultraviolet region, and the chromophoric light is located in the blue light region, which is easily interfered by the autofluorescence signal that may exist in the pathological sample, thereby reducing the sensitivity of the fluorescent probe detection.
  • a second object of the present invention is to provide a method of synthesizing the fluorescent probe.
  • a third object of the present invention is to provide a specific application method of the fluorescent probe in detecting carbapenemase and carbapenem-resistant bacteria.
  • the present invention adopts the following technical solutions.
  • a class of fluorescent probes for carbapenem-resistant antibiotic pathogens for detection wherein the structural formula of the fluorescent probe is represented by the general formula I:
  • n 1, 2.
  • M is H or a metal ion.
  • X is CH, R 1 is methyl or H; or X is S;
  • the dye is fluoroboron dipyrrole, naphthalimide, coumarin, fluorescein or rhodamine.
  • the fluorescent probe when the dye is fluoroboron dipyrrole, the fluorescent probe has the structural formula represented by Formula II:
  • M is H or a metal ion
  • R 2 , R 3 , R 4 , R 5 , R 6 are independently selected from hydrogen, methyl or ethyl;
  • R 7 is selected from an aromatic ring, hydrogen or an alkane.
  • the fluorescent probe has the structural formula represented by Formula III:
  • R 8 , R 9 , R 10 , R 11 and R 12 are independently selected from the group consisting of hydrogen, methyl, ethyl, halogen, alkoxy, hydroxy, carboxy, sulfonate, cyano, aldehyde, ester Or polyethylene glycol base.
  • R 8 , R 9 , R 10 , R 11 and R 12 are also independently selected from a lithium, sodium, potassium, magnesium or calcium salt derivative of a carboxyl or sulfonic acid group.
  • a specific structure of the fluorescent probe is represented by the structural formula i:
  • the fluorescent probe is capable of selectively recognizing the biomarker carbapenemase to function as a carbapenem-resistant antibiotic.
  • the structural formula of the fluorescent probe is represented by the general formula IV:
  • M is H or a metal ion
  • R is a 1-6 carbon alkyl group, a saturated carboxyl group, a saturated ester group or a polyethylene glycol group.
  • the ⁇ -lactam ring is opened by a carbapenemase to cause a structural change of the entire fluorescent probe, thereby producing a change in optical properties, so that the fluorescent probe has a function for detection.
  • the detection principle of penicillinase and its resistant bacteria is expressed as:
  • the present invention adopts the following technical solutions.
  • a method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen comprising the following steps:
  • reaction system was reacted at 80 ° C for 16 hours under a nitrogen atmosphere. After cooling, the reaction system was diluted with ethyl acetate. The ethyl acetate phase was washed with water and then brine, dried over anhydrous sodium sulfate
  • the obtained mixture is purified by silica gel column chromatography using petroleum ether and ethyl acetate as a mobile phase to obtain compound 3;
  • the compound 3 obtained in the step (1) is dissolved in a mixture of N-methylpyrrolidone (NMP) and N,N-dimethylformamide, N-methylpyrrolidone and N,N-dimethylformamide.
  • NMP N-methylpyrrolidone
  • the volume ratio is 1:3;
  • reaction mixture was diluted with ethyl acetate.
  • the ethyl acetate phase was washed with water and then brine
  • the obtained mixture is purified by silica gel column chromatography using petroleum ether and ethyl acetate as a mobile phase to obtain compound 4;
  • the compound 4 obtained in the step (2) is dissolved in tetrahydrofuran, and a 0.35 M phosphate buffer solution having a pH of 6.0 and activated zinc powder are added, and the reaction is carried out at 20 ° C for 1 hour;
  • the synthetic route of the fluorescent probe is as follows:
  • Compound 1 is a key intermediate and is a derivative capable of synthesizing other fluorescent probe compounds.
  • the present invention adopts the following technical solutions.
  • a fluorescent probe resistant to a carbapenem antibiotic pathogen is used as a test paper, a kit, or a test chip for detecting a carbapenemase and a carbapenem-resistant bacteria.
  • the application comprises the following steps:
  • the invention also provides a fluorescent probe of a carbapenem-resistant antibiotic pathogen, wherein the fluorescent probe has the structural formula:
  • X is a carbon atom or a sulfur atom; when X is CH, R 1 is a methyl group, which can be in the R or S configuration; or X is CH 2 or S; and the dye (dye) is fluoroboron dipyrrole (BODIPY) Any one of naphthalimide, coumarin, fluorescein or rhodamine.
  • BODIPY fluoroboron dipyrrole
  • R 2 , R 3 , R 4 , R 5 , R 6 are independently selected from the group consisting of hydrogen, methyl, and ethyl;
  • R 8 , R 9 , R 10 R 11 and R 12 are independently selected from hydrogen, methyl, ethyl, halogen, alkoxy, hydroxy, carboxy, sulfonate, cyano, aldehyde, ester or polyethyl b. a diol group, wherein the acid group (such as a carboxyl group, a sulfonic acid group) includes lithium, sodium, potassium, magnesium, and calcium salts thereof.
  • the preferential structure (CVB-1) of the fluorescent probe containing the general structure of fluoroboron dipyrrole (BODIPY) is:
  • the fluorescent probe having the preferential structure can selectively recognize the biomarker carbapenemase, thereby functioning as a carbapenemase.
  • R is selected from an alkyl group of 1 to 6 carbons, a saturated carboxyl group, a saturated ester group or a polyethylene glycol group.
  • a type of fluorescent probe resistant to carbapenem antibiotics is obtained by introducing a double bond at the 3-position of the penem antibiotic nucleus and then connecting to the 2-position of BODIPY (fluoroboron). Opening the ⁇ -lactam ring under the action of carbapenenemase, causing a certain structural change of the entire fluorescent probe, thereby producing a change in optical properties, so that the fluorescent probe has a function for detecting blue.
  • BODIPY fluoroboron
  • the present invention adopts the following technical solutions.
  • a method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen comprises the following steps:
  • reaction system was reacted for 16 hours at 80 ° C under a nitrogen atmosphere. After cooling, the reaction mixture was diluted with ethyl acetate. The ethyl acetate phase was washed with water and then brine
  • the obtained mixture was purified by silica gel column chromatography using petroleum ether and ethyl acetate.
  • the compound 3 obtained in the step (1) is dissolved in a mixture of N-methylpyrrolidone (NMP) and N,N-dimethylformamide, N-methylpyrrolidone and N,N-dimethylformamide.
  • NMP N-methylpyrrolidone
  • the volume ratio is 1:3.
  • Ammonium hydrogen difluoride (NH 4 ⁇ HF 2 ) was added and reacted at room temperature.
  • reaction mixture was diluted with ethyl acetate. EtOAc was evaporated.
  • the obtained mixture was purified by silica gel column chromatography using petroleum ether and ethyl acetate as mobile.
  • the compound 4 obtained in the step (2) was dissolved in tetrahydrofuran (THF), and 0.35 M phosphate buffer (PB) having a pH of 6.0 and activated zinc powder (Zn) were added thereto, and the mixture was reacted at 20 ° C for 1 hour.
  • THF tetrahydrofuran
  • PB phosphate buffer
  • Zn activated zinc powder
  • the synthetic route of the fluorescent probe (CVB-1) is:
  • the main feature of the synthetic route is that the compound 1 is passed through a Heck coupling reaction with a dye substituted with iodine, bromine or trifluoromethanesulfonate (the fluoroboron dipyrrole 2 substituted with iodine as an example) to realize the mother nucleus and fluorescence. Conjugative coupling of the reporter group; followed by two steps of deprotection to give the preferred fluorescent probe CVB-1.
  • the compound 1 is a key intermediate, and as a derivative, it is possible to synthesize other fluorescent probe compounds.
  • the present invention adopts the following technical solutions.
  • a test paper, a kit, or a test chip made of the fluorescent probe of the carbapenem-resistant antibiotic pathogen is used for detecting carbapenemase and carbapenem-resistant bacteria.
  • the present invention redesigned the original carbapenemase fluorescent probe. Using the fluorescence enhancement mechanism studied by myself, it overcomes the shortcomings of the originally studied fluorescent probes, and develops new fluorescent probes with higher detection sensitivity and wider detection range.
  • the fluorescent probe (CVB-1) of the present invention introduces a fluoroborodipyrrole conjugated to a mother core at the 3-position as a fluorescent reporter group, and undergoes an automatic structural change after being triggered by carbapenemase hydrolysis. A fluorescent response is produced afterwards.
  • Fluoroborodipyrrole was used as a fluorescent reporter group, and its excitation wavelength was 503 nm, and the maximum emission wavelength was 512 nm. Exciting the wavelength of the emitted light and in the green light range can greatly improve the sensitivity of detection of carbapenemase, resistant pathogenic bacteria or blood samples and urine samples containing the former two.
  • Fluorescent probe (CVB-1) can not only produce fluorescence changes but also change color under the action of carbapenemase. Therefore, samples can be detected by color change without application of instruments. It is simpler and more convenient.
  • the fluorescent probe provided by the present invention can be applied to the detection of clinical resistant bacteria. It can quickly detect bacteria with carbapenemase expression and its resistant bacteria, and has high selectivity, high precision and high sensitivity, low cost, easy and convenient, and no or less antibiotics in medical treatment. Very important.
  • 1 is a flow chart showing a method for synthesizing a fluorescent probe of a carbapenem-resistant antibiotic pathogen of the present invention.
  • Figure 3 is a graph showing the change in color of the fluorescent probe (100 ⁇ M) of Application Example 1 in the presence of carbapenemase IMP-1 (100 nM) and carbapenemase IMP-1 (0 nM).
  • Fig. 5 is a graph showing the change in fluorescence of a different ⁇ -lactamase mixed with a fluorescent probe for 1 hour in Application Example 2.
  • Fig. 6 is a graph showing the change in fluorescence of the mixed recombinant ⁇ -lactamase-containing Escherichia coli mixed with a fluorescent probe for 2 hours in Application Example 3.
  • Fig. 7 is a graph showing the relative fluorescence intensity of the application of Example 3 in which different clinical drug-resistant bacteria and wild E. coli enzymes were mixed with a fluorescent probe for 2 hours.
  • the fluorescent probe of the carbapenem-resistant antibiotic pathogen of the present invention is obtained by introducing a double bond at the 3-position of the parent core of the penem antibiotic and then connecting to the 2-position of the fluoroboron dipyrrole. Opening the ⁇ -lactam ring under the action of carbapenenemase, causing a certain structural change of the entire fluorescent probe, thereby producing a change in optical properties, so that the fluorescent probe has a function for detecting blue
  • the properties of myco-enzymes and their resistant bacteria is:
  • X is a carbon atom or a sulfur atom; when X is CH, R 1 is a methyl group, which can be in the R or S configuration; or X is CH 2 or S;
  • R 2 , R 3 , R 4 , R 5 , R 6 are independently selected from hydrogen, methyl or ethyl, wherein R 2-5 is preferably methyl and R 6 is preferably hydrogen;
  • R 7 , R 8 , R 9 , R 10 and R 11 are independently selected from hydrogen, methyl, ethyl, halogen, alkoxy, hydroxy, carboxy, sulfonate, cyano, aldehyde, ester or poly
  • a method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen wherein the preparation method of the compound 3 is:
  • reaction system was reacted at 80 ° C for 16 h under a nitrogen atmosphere. After cooling, the reaction system was diluted with ethyl acetate. The ethyl acetate phase was washed with water. It was washed with saturated brine, dried over anhydrous sodium sulfate and evaporated.
  • a method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen wherein the preparation method of the compound 4 is:
  • reaction system was diluted with ethyl acetate.
  • the ethyl acetate phase was washed with water and brine, dried over anhydrous sodium sulfate and evaporated.
  • a method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen wherein the preparation method of the fluorescent probe (CVB-1) is:
  • Example 2 A total of 13.9 mg (0.02 mmol) of the compound 4 obtained in Example 2 was dissolved in 0.5 ml of tetrahydrofuran. 0.3 mL of 0.35 M phosphate buffer (PB) having a pH of 6.0 and 26.2 mg (0.02 mmol) of activated zinc powder were added. The reaction was carried out at 20 ° C for 1 h.
  • PB phosphate buffer
  • reaction solution was filtered. Wash with chromatographic acetonitrile. Column purification was carried out using reverse phase C18. Freeze-dried. A dark purple compound, the fluorescent probe (CVB-1), was obtained.
  • test paper, the kit or the test chip made of the fluorescent probe prepared by the synthetic method of the present invention can be used for detecting carbapenemase and carbapenem-resistant bacteria in various fields of biology, preventive health care, and clinical diagnosis. Three application examples are provided below to illustrate the application of the present invention.
  • the use of the fluorescent probe prepared by the invention for detecting carbapenemase and carbapenem-resistant bacteria comprises the following steps:
  • the fluorescent probe of the carbapenem-resistant antibiotic pathogen of the present invention is mixed with a sample to be tested under certain conditions to form a compound having optical properties or color change (see Figs. 2, 3, and 4).
  • the fluorescent probe CVB-1 significantly enhanced the absorption fluorescence under the action of carbapenemase.
  • the carbapenemase was tested as a class A recombinantly expressed carbapenemase KPC-3; a zinc-containing class B recombinantly expressed carbapenemase VIM-27, IMP-1, NDM-1; D-type carbon Penemase OXA-48 and non-carbapenease TEM-1, TEM-3, CTX-M-9.
  • test sample ⁇ -lactamase or drug-resistant bacteria
  • the change in fluorescence intensity was measured by a microplate reader at room temperature (25 ° C) with an excitation wavelength of 500 nm and an emission wavelength of 535 nm, which was monitored for about 1 h.
  • Figure 5 shows different ⁇ -lactamases (Carbapenemase VIM-27, IMP-1, KPC-3, NDM-1, OXA-48, and non-carbapenease TEM-1, TEM-3, CTX -M-9 and no enzyme) mixed with a fluorescent probe for 1 hour of fluorescence change.
  • the fluorescent probe CVB-1 can be obviously formed by the action of low concentrations of carbapenemases (IM-27, IMP-1, KPC-3, NDM-1, OXA-48).
  • the fluorescence intensity was changed, and the fluorescence intensity of the fluorescent probe CVB-1 did not change significantly without the action of enzyme or high concentration of non-carbapenease. This phenomenon of whether the fluorescence intensity changes or not indicates that the fluorescent probe CVB-1 prepared by the present invention can be used for detecting or distinguishing carbapenemase.
  • Application Example 2 is only a general condition for detecting a sample. It should be noted that the test sample (enzyme sample, bacterial sample, blood sample, urine sample, etc.) used in Example 2, various reagents (buffer system, enzyme stabilizer, etc.), detection conditions (pH, temperature, etc.) It is not limited to the above-mentioned samples to be tested and general conditions.
  • test sample enzyme sample, bacterial sample, blood sample, urine sample, etc.
  • detection conditions pH, temperature, etc.
  • the fluorescent probe prepared by the present invention is used for detecting Escherichia coli and clinical bacteria expressing recombinant carbapenemase.
  • Clinical E. coli expressing different ⁇ -lactamases were cultured overnight in LB medium at 37 °C. The corresponding bacterial count was obtained by measuring the absorbance at 600 nm and expressed in CFU/mL (colony forming unit per ml). A series of dilutions were performed for each of the strains used for the study according to the four gradients of 1..10. Bacterial assays were performed on black 384-well plates (total volume 15 ⁇ L). 5 ⁇ L of the gradient-diluted bacteria were simultaneously added to each well of the 384-well plate. Then 10 ⁇ L of 7.5 ⁇ M fluorescent probe CVB-1 was added. The obtained sample to be tested was monitored for changes in fluorescence intensity by a microplate reader at room temperature of 25 °C.
  • NDM-1-Kp NDM-1 Klebsiella pneumoniae (ATCC BAA2146).
  • VIM-1-Kp is VIM-1 Klebsiella pneumoniae (NCTC 13440).
  • MDR-Ab is a multi-drug resistant Acinetobacter baumannii (ATCC BAA1605).
  • OXA-48-Kp is OXA-48 Klebsiella pneumoniae (NCTC 13442).
  • TEM-3-E.coli is TEM-3 E. coli (NCTC 13351).
  • CTX-M-9-Ec is CTXM-9 Enterobacter cloacae (NCTC 13464).
  • SHV-18-Kp is SHV-18 Klebsiella pneumoniae (ATCC 700603).
  • TEM-1-E.coli is EM-1 E. coli (ATCC 35218).
  • E. coli is a lactamase-free E. coli (LMG194).
  • the fluorescent probe of the carbapenem-resistant antibiotic pathogen of the present invention can be applied to the detection of carbapenemase and carbapenem-resistant bacteria.
  • the carbapenemase can be detected or distinguished by the phenomenon that the fluorescent probe changes in fluorescence intensity or color.
  • the pathogenic drug-resistant bacteria with carbapenemase expression can be quickly detected to guide the rational use of antibiotic drugs in medical clinics.
  • the fluorescent probe of the carbapenem-resistant antibiotic pathogen of the present invention has potential and positive application value in various fields of biology, preventive health care, and clinical diagnosis.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Pathology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Materials Engineering (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne une sonde fluorescente pour bactéries pathogènes tolérantes aux antibiotiques de type carbapénème ayant une formule générale structurale telle que représentée par la formule générale I. Le procédé de synthèse de la sonde fluorescente comprend les étapes de : (1) préparation d'un composé 3; (2) préparation d'un composé 4; et (3) préparation d'une sonde fluorescente CVB-1. La sonde fluorescente peut prendre la forme d'un papier réactif, de kits ou de puces de détection à appliquer à la détection de carbapénémases et de bactéries tolérantes aux médicaments carbapénèmes. Les carbapénémases sont détectées ou distinguées en déterminant si l'intensité de fluorescence ou la couleur de la sonde fluorescente change ou non, et en conséquence, les bactéries pathogènes tolérantes aux médicaments exprimant les carbapénémases peuvent être détectées rapidement. La sonde fluorescente guide l'utilisation raisonnable d'antibiotiques en termes de traitement ou d'application clinique, et présente une importance considérable en termes de diminution de l'utilisation d'antibiotiques ou de non recours à l'utilisation d'antibiotiques.
PCT/CN2018/072128 2017-01-13 2018-01-10 Sonde fluorescente pour bactéries pathogènes tolérantes aux antibiotiques carbapénèmes et procédé de synthèse et utilisation associés Ceased WO2018130158A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710027441.4A CN106811192B (zh) 2017-01-13 2017-01-13 耐碳青霉烯类抗生素病菌的荧光探针及其合成方法与应用
CN201710027441.4 2017-01-13

Publications (2)

Publication Number Publication Date
WO2018130158A1 true WO2018130158A1 (fr) 2018-07-19
WO2018130158A9 WO2018130158A9 (fr) 2018-11-22

Family

ID=59110883

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2018/072128 Ceased WO2018130158A1 (fr) 2017-01-13 2018-01-10 Sonde fluorescente pour bactéries pathogènes tolérantes aux antibiotiques carbapénèmes et procédé de synthèse et utilisation associés

Country Status (2)

Country Link
CN (1) CN106811192B (fr)
WO (1) WO2018130158A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113218928A (zh) * 2021-05-21 2021-08-06 宁德师范学院 一种基于荧光探针的快速测定抑菌活性的比色方法
CN115615983A (zh) * 2022-10-18 2023-01-17 山西大学 一种钼掺杂碳点纳米酶及其制备方法和应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106811192B (zh) * 2017-01-13 2019-04-23 华东理工大学 耐碳青霉烯类抗生素病菌的荧光探针及其合成方法与应用
CN111638197B (zh) * 2020-05-25 2022-10-21 南京工业大学 一种检测β-内酰胺酶的探针及其在荧光检测耐药菌上的应用
CN113045573B (zh) * 2021-03-09 2022-04-01 南开大学 一种耐碳青霉烯类抗生素病菌的探针化合物及应用
CN114487404B (zh) * 2021-12-28 2023-06-23 中国农业大学 用于检测细菌内碳青霉烯酶的免疫层析试纸条及检测方法
CN115521324B (zh) * 2022-10-13 2023-08-29 山西医科大学 一种检测耐药菌中β-内酰胺酶的近红外荧光探针的制备
CN117069794B (zh) * 2023-06-20 2024-02-13 广州中医药大学(广州中医药研究院) 一种糖肽类抗生素荧光探针化合物及其制备方法与应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012003955A1 (fr) * 2010-07-08 2012-01-12 Hans Rudolf Pfaendler Carbapénemes fluorescents
CN106279178A (zh) * 2016-07-18 2017-01-04 华东理工大学 耐碳青霉烯类抗生素病菌的荧光探针及合成方法与应用
CN106811192A (zh) * 2017-01-13 2017-06-09 华东理工大学 耐碳青霉烯类抗生素病菌的荧光探针及其合成方法与应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102443018B (zh) * 2011-10-06 2014-09-10 浙江大学 荧光标记的o6-苄基鸟嘌呤及其制备和应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012003955A1 (fr) * 2010-07-08 2012-01-12 Hans Rudolf Pfaendler Carbapénemes fluorescents
CN106279178A (zh) * 2016-07-18 2017-01-04 华东理工大学 耐碳青霉烯类抗生素病菌的荧光探针及合成方法与应用
CN106811192A (zh) * 2017-01-13 2017-06-09 华东理工大学 耐碳青霉烯类抗生素病菌的荧光探针及其合成方法与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JEESOOK PARK ET AL.: "A Ratiometric Fluorescent Probe Based on a BODIPY-DCDHF Conjugate for the Detection of Hypochlorous Acid in Living Cells", ANALYST, vol. 138, no. 12, 31 December 2013 (2013-12-31), Cambridge, United Kingdom, pages 3368 - 3371, XP055507742 *
WUYU MAO ET AL.: "Detection of Carbapenemase-Producing Organisms with a Carbapenem-Based", ANGEWANDTE CHEMIE, vol. 56, no. 16, 23 March 2017 (2017-03-23), pages 4469 - 4472, XP055507740 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113218928A (zh) * 2021-05-21 2021-08-06 宁德师范学院 一种基于荧光探针的快速测定抑菌活性的比色方法
CN115615983A (zh) * 2022-10-18 2023-01-17 山西大学 一种钼掺杂碳点纳米酶及其制备方法和应用
CN115615983B (zh) * 2022-10-18 2024-07-05 山西大学 一种钼掺杂碳点纳米酶及其制备方法和应用

Also Published As

Publication number Publication date
CN106811192B (zh) 2019-04-23
CN106811192A (zh) 2017-06-09
WO2018130158A9 (fr) 2018-11-22

Similar Documents

Publication Publication Date Title
WO2018130158A1 (fr) Sonde fluorescente pour bactéries pathogènes tolérantes aux antibiotiques carbapénèmes et procédé de synthèse et utilisation associés
An et al. Multichannel ratiometric fluorescence sensor arrays for rapid visual monitoring of epinephrine, norepinephrine, and levodopa
Kim et al. Citrate-based fluorescent materials for low-cost chloride sensing in the diagnosis of cystic fibrosis
Huang et al. A multicolor time-resolved fluorescence aptasensor for the simultaneous detection of multiplex Staphylococcus aureus enterotoxins in the milk
An et al. Ratiometric fluorescence detection of ciprofloxacin using the terbium-based coordination polymers
Peng et al. A simple design of fluorescent probes for indirect detection of β-lactamase based on AIE and ESIPT processes
CN106279178A (zh) 耐碳青霉烯类抗生素病菌的荧光探针及合成方法与应用
Shen et al. A novel ratiometric pH probe for extreme acidity based on FRET and PET
Zhu et al. Spiroboronate Si-rhodamine as a near-infrared probe for imaging lysosomes based on the reversible ring-opening process
Li et al. Two rhodamine lactam modulated lysosome-targetable fluorescence probes for sensitively and selectively monitoring subcellular organelle pH change
Zhang et al. A label-free fluorescence turn-on assay for glutathione detection by using MnO2 nanosheets assisted aggregation-induced emission-silica nanospheres
Liu et al. A naked-eye visible and turn-on fluorescence probe for Fe3+ and its bioimaging application in living cells
CN113912626B (zh) 耐超广谱β-内酰胺类和头孢菌素类抗生素病菌探针及其合成方法与应用
Mu et al. Dual-emission fluorescence biosensing of vancomycin based on AIEgen–peptide conjugates and aptamer-modified Au nanoclusters
Tong et al. A ratiometric fluorescent pH probe based on keto–enol tautomerization for imaging of living cells in extreme acidity
EP3102583B1 (fr) Dérivés de céphalosporine disubstitués en 2 et 7 utilisés comme substrats de bêta-lactamase et procédés d'utilisation de ces dérivés pour le diagnostic de la tuberculose
CN109593078B (zh) N-丁基-4-羟基-1,8-萘二甲酰亚胺-3-甲醛-(2-吡啶)腙及应用
Zhang et al. A post-synthetically modified Eu@ Zn-MOF for ratiometric fluorescence detection of tetracycline in tap water
Li et al. A water-soluble fluorescent chemosensor having a high affinity and sensitivity for Zn2+ and its biological application
CN115521324A (zh) 一种检测耐药菌中β-内酰胺酶的近红外荧光探针的制备
Wang et al. A reversible fluorescent probe for directly monitoring protein-small molecules interaction utilizing vibration-induced emission
Fu et al. A novel ratiometric fluorescent probe for the detection of mitochondrial pH dynamics during cell damage
CN114249760B (zh) 三通道同时区分次氯酸和过氧化氢的荧光探针的合成与应用
Yao et al. An innovative near-infrared fluorescent probe with FRET effect for the continuous detection of Zn2+ and PPi with high sensitivity and selectivity, and its application in bioimaging
Zhang et al. Tuning dual-channel fluorescence-enhanced chemosensor for imaging of living cells in extreme acidity

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18739435

Country of ref document: EP

Kind code of ref document: A1

DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18739435

Country of ref document: EP

Kind code of ref document: A1