WO2018130158A1 - Carbapenem antibiotic-tolerant pathogenic bacterium fluorescent probe and synthesis method and use thereof - Google Patents

Abstract

Disclosed in the present invention is a carbapenem antibiotic-tolerant pathogenic bacterium fluorescent probe with a structural general formula as represented by general formula I. The synthesis method for the fluorescent probe comprises the steps of: (1) preparation of a compound 3; (2) preparation of a compound 4; and (3) preparation of a fluorescent probe CVB-1. The fluorescent probe can be made into a test paper, kits or detection chips to be applied to the detection of carbapenemases and carbapenem drug-tolerant bacteria. Carbapenemases are detected or distinguished by determining whether the fluorescence intensity or colour of the fluorescent probe changes or not, and accordingly, pathogenic drug-tolerant bacteria expressing carbapenemases can be detected rapidly. The fluorescent probe guides the reasonable utilization of antibiotic drugs in terms of treatment or clinical application, and has important significance in terms of using no or less antibiotic drugs.

Description

Fluorescent probe for carbapenem-resistant antibiotic pathogen and synthesis method and application thereof Technical field

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.

Background technique

Carbapenem antibiotics are a class of β-lactam antibiotics with a special structure. It consists of a β-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. 2011, 55, 4943), This class of antibiotics has become the last line of defense against serious bacterial infections (Emerg. Infect. Dis. 2011, 17, 1791-1798). 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 β-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 β-lactam bond of the commonly used β-lactam antibiotics, making the antibiotics less effective. This type of β-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.

According to the Ambler classification, the β-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 β-lactamase containing serine. Type B is a β-lactamase containing a metal zinc ion. The type A β-lactamase includes KPC, a GES type β-lactamase, and the like. It can be detected in many pathogenic bacteria, such as Enterobacter, Serratia marcescens, Klebsiella, and the like. As early as 1996, the United States isolated Klebsiella containing KPC enzyme from a clinical case in North Carolina. The study found that this strain is resistant to all antibiotics. Later, New Delhi-containing metallo-β-lactamase (NDM), a class B β-lactamase, was discovered in medical institutions and the ecological environment in New Delhi. The active center zinc ions of these enzymes can bind to the amide bond, and the antibiotic hydrolysis loses its efficacy. The pathogenic bacteria containing metallo-beta-lactamase in New Delhi is also referred to as "superbug". This type of “superbug” was quickly spread to patients around the world after being discovered, causing worldwide concerns. In China, the proportion of Klebsiella pneumoniae resistant to carbapenem antibiotics increased from 2.4% to 13.4% between 2005 and 2014 (Clin Microbiol Infect 2016; 22:S9–S14).

At present, there are three main methods for detecting bacteria having carbapenemase expression: phenotypic detection, gene detection, and carbapenemase-based detection. The phenotypic assay 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. And the detection takes a long time, usually takes 24 to 48 hours. In addition, such methods do not provide the necessary information to select antibiotics in a timely manner. 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). The gene detection method has high accuracy and sensitivity. However, 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 β-lactam ring of cefanitrothiophene can be rapidly hydrolyzed by β-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.

In recent years, 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. At present, more fluorescent probe compounds have been applied to the detection of β-lactamase in high sensitivity and in real time. In 1998, Nobel Prize winner Professor Qian Yongjian wrote in the journal Science (Science 1998, 279, 84-88). A molecular fluorescent probe based on a β-lactam structure was first reported. Fluorescence energy transfer (FRET) mechanism was used to monitor the activity of β-lactamase. Subsequently, some fluorescent probe compounds for detecting β-lactamase activity have been reported. However, these fluorescent probe compounds are designed based on the cephalosporin core. They cannot distinguish between normal β-lactamases and carbapenases. International Patent Application No. WO 2012/003955 A1 discloses a "Carbapenemase fluorescent probe having a certain fluorescent property based on a carbapenem core structure". However, the fluorescent probe described in this patent application does not specify specific optical properties, and does not introduce a typical fluorescent reporter group, so its detection performance is unclear. In 2014, Rao et al. reported a fluorescent probe for detecting Enterobacteriaceae resistant to carbapenem (Angew. Chem. Int. Ed. 2014, 53, 8113-8116). The fluorescent probe is designed based on the common cephalosporin core. Through structural adjustment, 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. Thereby, selective detection of β-carbapyrenease and bacteria expressing carbapenemase is achieved. However, the different fluorescent probes have very different selectivity and detection sensitivity for different carbapenemases. In addition, 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 β-lactam ring is opened by the action of carbapenemase to generate a fluorescent signal, and selective detection of the carbapenemase is achieved. However, 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.

Summary of the invention

It is an object of the present invention to overcome the deficiencies of the prior art and to provide a fluorescent probe that is resistant to carbapenem antibiotics. It is based on penicillin antibiotic design. It is used to detect the pathogen producing carbapenemase. It has the advantages of high sensitivity, high selectivity, and low cost of use. 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.

In order to achieve the above object, 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:

Where: n = 1, 2.

In an embodiment, M is H or a metal ion.

In one embodiment, X is CH, R ¹ is methyl or H; or X is S;

In one embodiment, the dye is fluoroboron dipyrrole, naphthalimide, coumarin, fluorescein or rhodamine.

In one embodiment, when the dye is fluoroboron dipyrrole, the fluorescent probe has the structural formula represented by Formula II:

In the formula:

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.

In one embodiment, further, the fluorescent probe has the structural formula represented by Formula III:

Wherein 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.

In one embodiment, 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.

In one embodiment, a specific structure of the fluorescent probe is represented by the structural formula i:

In one embodiment, the fluorescent probe is capable of selectively recognizing the biomarker carbapenemase to function as a carbapenem-resistant antibiotic.

In one embodiment, when the dye is naphthalimide, the structural formula of the fluorescent probe is represented by the general formula IV:

In the formula:

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.

In one embodiment, the β-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:

In order to achieve the above second object, 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:

(1) Preparation of Compound 3

Compound 1 and Compound 2 were used as initial reactants in a reaction flask, and N,N-dimethylformamide (DMF) and triethylamine (TEA) were added to the reaction flask, and the resulting mixture was frozen- After degassing under vacuum-dissolving for three cycles, palladium acetate (PdOAc ₂ ) and tris(o-methylphenyl)phosphine (P(o-Tol) ₃ were added and degassed twice;

The 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;

(2) Preparation of Compound 4

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. The volume ratio is 1:3;

Add ammonium hydrogen difluoride (NH ₄ ·HF ₂ ) and react at room temperature;

After the reaction, the 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;

(3) Preparation of fluorescent probe

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 reaction solution was filtered, washed with EtOAc EtOAc (EtOAc)EtOAc.

In one embodiment, the synthetic route of the fluorescent probe is as follows:

In one embodiment, Compound 1 is a key intermediate and is a derivative capable of synthesizing other fluorescent probe compounds.

In order to achieve the above third object, 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.

In an embodiment, the application comprises the following steps:

(1) mixing a fluorescent probe of a carbapenem-resistant antibiotic pathogen with a sample to be tested under certain conditions to form a compound having optical properties;

(2) Measuring optical signal changes of a compound having optical properties, including fluorescence, ultraviolet absorption, or color change, thereby determining the content or concentration of carbapenemase or carbapenem-resistant bacteria in the sample to be tested.

The invention also provides a fluorescent probe of a carbapenem-resistant antibiotic pathogen, wherein the fluorescent probe has the structural formula:

Wherein: 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.

Further, the general structure of the fluoroboron dipyrrole (BODIPY) is:

In the formula:

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.

Further, the preferential structure (CVB-1) of the fluorescent probe containing the general structure of fluoroboron dipyrrole (BODIPY) is:

Further, the fluorescent probe having the preferential structure can selectively recognize the biomarker carbapenemase, thereby functioning as a carbapenemase.

Further, the general structure of the naphthalimide is:

In the formula:

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 β-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 are described as:

In order to achieve the above second object, 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:

(1) Preparation of Compound 3

Compound 1 and Compound 2 were placed in a reaction flask, N,N-dimethylformamide (DMF) and triethylamine (TEA) were added, and the resulting mixture was degassed by freeze-vacuum-dissolution three cycles, and then added. Palladium acetate (PdOAc ₂ ) and tris(o-methylphenyl)phosphine (P(o-Tol) ₃ were degassed twice.

The 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.

(2) Preparation of Compound 4

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. The volume ratio is 1:3.

Ammonium hydrogen difluoride (NH ₄ ·HF ₂ ) was added and reacted at room temperature.

After the completion of the reaction, the 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.

(3) Preparation of fluorescent probe (CVB-1)

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.

The reaction solution was filtered, washed with EtOAc EtOAc (EtOAc EtOAc).

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.

Further, the compound 1 is a key intermediate, and as a derivative, it is possible to synthesize other fluorescent probe compounds.

In order to achieve the above third object, 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.

Further, the application includes the following steps:

(1) mixing a fluorescent probe of a carbapenem-resistant antibiotic pathogen with a sample to be tested under certain conditions to form a compound having optical properties;

(2) Measuring optical signal changes of a compound having optical properties, including fluorescence, ultraviolet absorption, or color change, thereby determining the content or concentration of carbapenemase or carbapenem-resistant bacteria in the sample to be tested.

Positive effects of the invention:

(1) 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.

(2) Based on the carbapenem antibiotic as the mother nucleus, the specific response of the fluorescent probe to the carbapenemase is ensured, and the non-carbonicillase is not responded.

(3) 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.

(4) 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.

(5) 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.

(6) 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.

DRAWINGS

1 is a flow chart showing a method for synthesizing a fluorescent probe of a carbapenem-resistant antibiotic pathogen of the present invention.

2 is a graph showing changes in absorption spectra of a fluorescent probe in Application Example 1 under carbapenemase IMP-1.

Figure 3 is a graph showing the change in color of the fluorescent probe (100 μM) of Application Example 1 in the presence of carbapenemase IMP-1 (100 nM) and carbapenemase IMP-1 (0 nM).

4 is a graph showing changes in fluorescence spectra of a fluorescent probe of Application Example 1 under a certain concentration of carbapenemase over time.

Fig. 5 is a graph showing the change in fluorescence of a different β-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 β-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.

detailed description

The specific embodiments of the present invention and related detection effects will be specifically described below with reference to the accompanying drawings. However, it should be noted that the implementation of the present invention is not limited to the following embodiments. The experimental methods in the examples which do not specify the specific conditions are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer.

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 β-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. Its structural formula is:

In the 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;

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 The ethylene glycol group, wherein R ^7-11 is preferably hydrogen; wherein the acid group (for example, carboxyl group, sulfonic acid group) includes lithium, sodium, potassium, magnesium, calcium salts thereof.

A specific embodiment of a method for synthesizing a fluorescent probe of the carbapenem-resistant antibiotic pathogen of the present invention is described below. Description: In the practice of the present invention, ¹ H-NMR was measured by a Bruker 400Mz type instrument, and the solvent was deuterated chloroform (CDCl ₃ ), and the internal standard was tetramethylsilane (TMS); all solvents were chromatographically pure. Analytically pure or chemically pure, the anhydrous solvent used is obtained by drying according to standard methods. The purification of the product was carried out by silica gel column chromatography unless otherwise specified, and the silica gel used was 200 to 300 mesh.

Example 1 (see Figure 1)

A method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen, wherein the preparation method of the compound 3 is:

Compound 1 [0.39 mmol (mmol), 189 mg (mg)], Compound 2 (0.3 mmol, 135 mg) was placed in a reaction flask. 1.2 mL of N,N-dimethylformamide and 0.3 mL of triethylamine were added. After the resulting mixture was degassed by freeze-vacuum-dissolution three cycles, 0.03 mmol (6.7 mg) of palladium acetate and 0.045 mmol (13.7 mg) of tris(o-methylphenyl)phosphine were added. Degas again twice.

The 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.

The obtained mixture was purified by silica gel column chromatography using petroleum ether and ethyl acetate as a mobile phase to give compound 3 (57 mg).

Its synthetic route and structural formula are:

The spectral characteristics are: ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.20 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 7.49 (m, 4H), 7.30 - 7.27 (m, 2H), 6.71 (d, J = 16.7 Hz, 1H), 6.04 (s, 1H), 5.44 (d, J = 14.0 Hz, 1H), 5.25 (d, J = 14.0 Hz, 1H), 4.31 – 4.24 (m, 1H), 4.22 (dd, J=9.2, 2.5 Hz, 1H), 3.54–3.44 (m, H), 3.24 (dd, J=5.5, 2.6 Hz, 1H), 2.68 (s, 3H) ), 2.58 (s, 3H), 1.46 (s, 3H), 1.38 (s, 3H), 1.27 (d, J = 3.3 Hz, 3H), 1.26 (d, J = 3.2 Hz, 3H), 0.86 (s) , 9H), 0.09 (s, 3H), 0.08 (s, 3H). ¹³ C NMR (101MHz, CDCl ₃ ) δ 172.75, 161.20, 157.60, 154.57, 150.50, 147.64, 144.75, 143.16, 142.07, 138.71, 134.92, 132.47 , 131.08, 129.38, 129.29, 128.13, 128.11, 127.67, 127.34, 124.27, 123.79, 122.39, 121.89, 66.02, 65.14, 59.10, 56.23, 38.93, 25.80, 22.58, 18.04, 17.20, 14.86, 14.71, 13.87, 13.17,- 4.13, -4.85. HRMS (ESI) m/z calcd for C ₄₄ H ₅₁ BF ₂ N ₄ NaO ₆ Si (M+Na) ⁺ 831.3537, found 831.3547.

Example 2 (see Figure 1)

A method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen, wherein the preparation method of the compound 4 is:

A total of 57 mg (0.07 mmol) of the compound 3 prepared in Example 1 was dissolved in 0.7 ml of N-methylpyrrolidone/N,N-dimethylformamide. The volume ratio of N-methylpyrrolidone to N,N-dimethylformamide was 1:3.

Thereafter, 15.9 mg (0.28 mmol) of ammonium hydrogen dihydrogenate was added. The reaction was carried out at room temperature (25 ° C) for 36 h.

After the reaction was completed, the reaction system was diluted with ethyl acetate. The ethyl acetate phase was washed with water and brine, dried over anhydrous sodium sulfate and evaporated.

The obtained mixture was purified by silica gel column chromatography using petroleum ether and ethyl acetate as a mobile phase to give compound 4 of 39.6 mg.

Its synthetic route and structural formula are:

The spectral characteristics are: ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.22 (d, J = 8.8 Hz, 2H), 7.66 (d, J = 8.8 Hz, 2H), 7.54 - 7.48 (m, 3H), 7.45 (d, J = 16.8 Hz, 1H), 7.29 - 7.26 (m, 2H), 6.72 (d, J = 16.7 Hz, 1H), 6.05 (s, 1H), 5.49 (d, J = 13.8 Hz, 1H) , 5.23 (d, J = 13.9 Hz, 1H), 4.31 - 4.25 (m, 1H), 4.23 (dd, J = 9.0, 2.5 Hz, 1H), 3.61 - 3.51 (m, 1H), 3.28 (dd, J = 6.7, 2.5 Hz, 1H), 2.66 (s, 3H), 2.58 (s, 3H), 1.44 (s, 3H), 1.38 (s, 3H), 1.28 (s, 3H), 1.27 (s, 3H) .

Example 3 (see Figure 1)

A method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen, wherein the preparation method of the fluorescent probe (CVB-1) is:

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.

The 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.

The synthetic route and structural formula of the fluorescent probe (CVB-1) are:

The spectral characteristics are: ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.63 (d, J = 16.9 Hz, 1H), 7.50 - 7.44 (m, 2H), 7.32 - 7.26 (m, 2H), 6.46 (d, J=16.9 Hz, 1H), 6.00 (s, 1H), 4.30–4.19 (m, 1H), 4.15 (d, J=8.9 Hz, 1H), 3.47–3.37 (m, 1H), 3.20 (d, J) = 4.7 Hz, 1H), 2.70 (s, 3H), 2.56 (s, 3H), 1.42 (s, 3H), 1.37 (s, 3H), 1.33 (d, J = 6.0 Hz, 3H), 1.22 (d) , J = 7.1 Hz, 3H). HRMS (ESI) m / z calcd for C ₃₁ H ₃₁ BF ₂ N ₃ O ₄ (M-1) ^- 558.2376, found 558.2377.

The 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.

Application Example 1

The use of the fluorescent probe prepared by the invention for detecting carbapenemase and carbapenem-resistant bacteria comprises the following steps:

(1) 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).

It can be seen from Fig. 2 that the fluorescence probe CVB-1 undergoes a significant change in absorption under the action of IMP-1, and finally produces an absorption spectrum of a typical fluoroboron dipyrrole.

It can be seen from Fig. 3 that the fluorescent probe CVB-1 absorbs under the action of IMP-1 to cause a significant color change, from purple to pink.

As can be seen from Fig. 4, the fluorescent probe CVB-1 significantly enhanced the absorption fluorescence under the action of carbapenemase.

(2) Measuring an optical signal change of a compound having optical properties or observing a color change of the fluorescent probe to determine the content or concentration of carbapenemase or carbapenem-resistant bacteria in the sample to be tested.

Application Example 2

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.

The fluorescence change of the fluorescent probe under the action of β-lactamase was measured:

Mix the test sample (β-lactamase or drug-resistant bacteria) in phosphate buffer {1X PBS, pH=7.4, 0.1% surfactant [CHAPS, 3-[3-(cholamidopropyl) dimethyl) Ammonium]-1-propanesulfonic acid inner salt]} was placed in the microplate of the enzyme standard. 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.

The results of the measurement are shown in Figure 5. Figure 5 shows different β-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. It can be seen from Fig. 5 that 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.

Application Example 3

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 β-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 μL). 5 μL of the gradient-diluted bacteria were simultaneously added to each well of the 384-well plate. Then 10 μL of 7.5 μM 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.

The results of the assay are shown in Figure 6 - Fluorescence changes of mixed recombinant β-lactamase E. coli and fluorescent probe CVB-1 for 2 hours. Figure 7 - Relative fluorescence intensity plots containing different clinically resistant bacteria (VIM-27, IMP-1, KPC-3, TEM-1) and wild E. coli enzymes mixed with fluorescent probes for 2 hours.

In Figure 7:

NDM-1-Kp is 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).

It can be seen from Fig. 6 and Fig. 7 that after direct fluorescence imaging, only the clinically resistant bacteria expressing carbapenemase can respond to the fluorescent probe CVB-1, and the fluorescent signal generated is obvious. However, the clinically resistant bacteria of the non-carbapenease and the wild type E. coli in the control group could not respond to the fluorescent probe CVB-1, and they had almost no fluorescence signal.

The results of Application Examples 1 to 3 prove that:

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. Furthermore, the pathogenic drug-resistant bacteria with carbapenemase expression can be quickly detected to guide the rational use of antibiotic drugs in medical clinics. In addition, 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.

Claims (12)

1. 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:

   

   Where: n = 1, 2.

   

   

   In the formula:

   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.
2. The carbapenem-resistant antibiotic pathogen fluorescent probe according to claim 5, wherein, further, the fluorescent probe has the structural formula represented by the formula III:

   

   Wherein 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.
3. The carbapenem antibiotic pathogen fluorescent probe according to claim 5, wherein R ⁸ , R ⁹ , R ¹⁰ , R ¹¹ and R ¹² are also independently selected from lithium or sodium of a carboxyl group or a sulfonic acid group. Potassium, magnesium or calcium salt derivatives.
4. The carbapenem-resistant antibiotic pathogen fluorescent probe according to claim 5, wherein a specific structure of the fluorescent probe is represented by the structural formula i:

   
5. The carbapenem-resistant antibiotic pathogen fluorescent probe according to claim 1, wherein the fluorescent probe is capable of selectively recognizing the biomarker carbapenemase to detect carbapenem-resistant antibiotics. The role of germs.
6. The carbapenem-resistant antibiotic pathogen fluorescent probe according to claim 4, wherein when the dye is naphthalimide, the fluorescent probe has the structural formula represented by the formula IV:

   

   In the formula:

   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.
7. The carbopenem antibiotic pathogen fluorescent probe according to claim 1, wherein the β-lactam ring is opened by a carbapenemase to cause a structural change of the entire fluorescent probe, thereby producing The change in optical properties allows the fluorescent probe to have properties for detecting penicillinase and its resistant bacteria, and the detection principle is expressed as:

   
8. A method for synthesizing a fluorescent probe resistant to a carbapenem antibiotic pathogen, comprising the following steps:

   (1) Preparation of Compound 3

   Compound 1 and Compound 2 were used as initial reactants in a reaction flask, and N,N-dimethylformamide (DMF) and triethylamine (TEA) were added to the reaction flask, and the resulting mixture was frozen- After degassing under vacuum-dissolving for three cycles, palladium acetate (PdOAc ₂ ) and tris(o-methylphenyl)phosphine (P(o-Tol) ₃ were added and degassed twice;

   The 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;

   (2) Preparation of Compound 4

   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. The volume ratio is 1:3;

   Add ammonium hydrogen difluoride (NH ₄ ·HF ₂ ) and react at room temperature;

   After the reaction, the 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;

   (3) Preparation of fluorescent probe

   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 reaction solution was filtered, washed with EtOAc EtOAc (EtOAc)EtOAc.
9. The method for synthesizing a carbapenem-resistant antibiotic pathogen fluorescent probe according to claim 12, wherein

   The synthetic route of the fluorescent probe is as follows:

   
10. The method according to claim 12, wherein the compound 1 is a key intermediate, and the derivative is capable of synthesizing other fluorescent probe compounds.
11. 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.
12. The application according to claim 15, wherein the application comprises the following steps:

    (1) mixing a fluorescent probe of a carbapenem-resistant antibiotic pathogen with a sample to be tested under certain conditions to form a compound having optical properties;

    (2) Measuring optical signal changes of a compound having optical properties, including fluorescence, ultraviolet absorption, or color change, thereby determining the content or concentration of carbapenemase or carbapenem-resistant bacteria in the sample to be tested.

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