KR101829453B1 - Probes for Detecting Drug-Resistant Bacteria in a Sample and Uses Thereof - Google Patents

Abstract

The present invention relates to a probe for detecting a drug-resistant bacteria in a sample, a composition comprising the same, a kit, and a detection method, and a compound according to one aspect and a probe containing the same can detect β- Is capable of clinical detection of antibiotic resistant bacteria that are not only applicable to a variety of biochemical studies, but which are not possible with conventional pH indicator factor-based methods, and that can be used for molecular diagnosis and purposes of antibiotic- The antibiotic-resistant bacteria can be analyzed with high sensitivity from the sample, so that the present invention can be effectively applied to medical applications such as in vitro diagnosis.

Description

[0001] The present invention relates to a probe for detecting a drug-resistant bacteria in a sample, and to a probe for detecting the same.

To a probe for detecting a drug-resistant bacteria in a sample, a composition containing the same, a kit, and a detection method.

The emergence of antibiotic resistant bacteria has posed a threat to public health. One of the most common mechanisms of resistance is the expression of [beta] -lactamase (Bla), which is capable of cleaving the [beta] -lactam ring of antibiotics, which results in the loss of activity of the drug. Moreover, it has been found that a significant number of the above-mentioned bacteria classified as extended spectrum beta-lactamase (ESBL) producers exhibit resistance to a variety of beta-lactam-based antibiotics as well as to one type of drug. While the development of next generation antibiotics to treat the drug resistant bacteria is a time-consuming and costly process, identification of the infected individual based on initial detection and sensitivity detection is an optional way of dealing with the relevant situation, It is possible to prevent widespread infections caused by drug-resistant bacteria. There are two approaches to quickly and efficiently detecting the bacteria: (1) pH sensor-based probes and (2) fluorescent substrate probes. The hydrolysis of most β-lactam antibiotics can be easily monitored with a pH indicator to lower the pH of the solution and to detect the drug resistance of the bacteria. However, the pH indicator factor-based Bla assay often provides false-negative results because the pH of the solution can vary greatly depending on the reaction environment. In particular, the hydrolysis of cephalazidime (CAZ), a cephalosporin-derived β-lactam antibiotic by Bla, does not induce acidity of the medium due to the buffering effect of pyridine released due to the enzymatic degradation of the drug. In order to directly monitor the hydrolysis of CAZ, a fluorescent substrate of Bla can be used instead of an acidic sensor. The substrates described above can be prepared by labeling antibiotics with fluorophores and a quencher or by converting them into fluorescent moieties. In general, previously reported pro-fluorophores in fluorescent substrates do not share any structural similarity with CAZ and can result in selective signaling to CAZ-resistant strains.

In order to overcome the above-mentioned disadvantages, there is a urgent need in the art to more accurately and efficiently detect the presence of ESBL-expressing antibiotic-resistant bacteria.

One aspect is a compound represented by Formula 1 below:

[Chemical Formula 1]

(Core-structure-L-Z).

Another aspect of the present invention is to provide a probe for detecting an antibiotic-resistant bacteria comprising the compound represented by the above formula (1).

Another aspect of the present invention is to provide a reagent composition for detecting an antibiotic-resistant bacteria comprising the compound represented by the above formula (1).

Another aspect of the present invention is to provide an antibiotic-resistant bacterial infection diagnosis kit comprising the compound represented by the above formula (1).

Another aspect is to provide a method for detecting an antibiotic-resistant bacteria comprising the step of contacting a compound represented by the above formula (1) with a biological sample.

An aspect provides a compound represented by the following formula (1).

[Chemical Formula 1]

(Core-structure-L-Z).

,

,

또는

이고, R_L은 단일결합이거나, C_1-6 알킬렌기, C_2-6 알케닐렌기, 또는 C_2-6 알키닐렌기이고, R₃ 및 R₄는 각각 독립적으로 C_1-6 알킬기, C_2-6 알케닐기, 또는 C_2-6 알키닐기이고, R₅는 단일결합이거나, C_1-6 알케닐기이고, R₆ 및 R₇는 각각 독립적으로, 수소 또는 C_1-6 알킬기, C_2-6 알케닐기, 또는 C_2-6 알키닐기이고, n은 1 내지 4의 정수이고, m은 1 내지 9의 정수이고, *는 코어 구조와의 결합 부위이고, *'는 Z와의 결합 부위일 수 있다. Wherein R < ₁ > may be selected from substituents at position 7 of the core structure of the cephalosporin antibiotic. In addition, A may be selected from the group consisting of S, O, SO, SO ₂ , and CH ₂ . The L may be a linker connecting the core structure and Z. Specifically, L

,

,

or

And, R _L is either a single bond, C _1-6 alkylene, C _2-6 alkenylene or C _2-6 alkynylene group, R ₃ and R ₄ are each independently C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl group, R ₅ is a single bond, a C _1-6 alkenyl group, R ₆ and R ₇ are, each independently, hydrogen or C _1-6 alkyl group, C ₂ and _-6 alkenyl group, or C _2-6 alkynyl group, n is an integer from 1 to 4, m is an integer from 1 to 9, * is a binding site of the core structure, * 'is Z with one binding site .

Z is a fluorescent moiety.

,

,

,

,

,

,

,

,

,

,

,

,

,

,

, 및

로 이루어진 군으로부터 선택되는 것일 수 있다. Wherein R ₁ is selected from the group consisting of H, -NH ₂ , and -NHCO-R ', R' is selected from the group consisting of -CH ₂ -CN, -CH ₂ S-CF ₃ ,

,

,

,

,

,

,

,

,

,

,

,

,

,

,

, And

≪ / RTI >

In one embodiment,

이고, 상기 식 중 R_L, *, 및 *'는 상기에서 정의한 바와 같다. 예를 들면, 상기 R_L은 단일결합이거나, 메틸 또는 에틸일 수 있다. The L

, Wherein R _L , *, and * are as defined above. For example, the R _L may be a single bond or may be methyl or ethyl.

In the present specification, a C ₁ -C ₆ alkyl group means a linear or branched aliphatic hydrocarbon monovalent group having 1 to 6 carbon atoms. Specific examples thereof include a methyl group, an ethyl group, a propyl group, an isobutyl group, a sec-butyl group , a tert-butyl group, a pentyl group, an iso-amyl group, or a hexyl group. The specification of the C ₁ -C ₆ alkylene group may refer to a divalent (divalent) group having the same structure as the C ₁ -C ₆ alkyl group.

In the present specification, the C ₁ -C ₆ alkenyl group has a structure containing at least one carbon double bond at the middle or end of the C ₁ -C ₆ alkyl group, and specific examples thereof include an ethynyl group, a propenyl group, a butenyl group, . The C ₁ -C ₆ alkenylene group in the present specification may mean a divalent group having the same structure as the C ₁ -C ₆ alkenyl group.

In the present specification, the C ₁ -C ₆ alkynyl group has a structure containing at least one carbon triple bond at the middle or end of the C ₁ -C ₆ alkyl group, and specific examples thereof include ethynyl or propynyl ), And the like. The C ₁ -C ₆ alkynylene group in the present specification may mean a divalent group having the same structure as the C ₁ -C ₆ alkynyl group.

As used herein, the term "fluorescent moiety" refers to a fluorescent molecule or derivative or conjugate thereof that absorbs light of a particular frequency (e.g., UV light) can do. For example, the fluorescent moiety may be a quencher dye. Examples of fluorescent moieties include phenolic dyes (dyes) such as umbelliferone, fluorescein and resorufin; Aromatic amines, and other compounds such as rhodamine, and the like. Also, for example, the fluorescent moiety may comprise coumarin and related dyes; Xanthene dyes such as fluorescein, rodol, and rhodamine; Resorcinol; Cyanine dyes; Bimain dyes; Acridine; Isoindole; Dansyl dyes; Aminophthalic hydrazides such as luminol and isoleuminol derivatives; Aminophthalimide; Aminonaphthalimide; Aminobenzofuran; Aminoquinoline; Dicyanohydroquinone; BODIPY; And europium and terbium complexes and related compounds. Wherein the BODIPY is selected from the group consisting of pyridyl BODIPY, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, BODIPY 558/568, BODIPY 564/570, It may be selected.

The fluorescent moiety may further include a free carboxyl group, an ester (for example, an N-hydroxysuccinimide (NHS) ester) or a maleimide derivative, and may further include streptavidin, biotin, paloyidine, amine, azide Or an iodoacetamide conjugate.

In one embodiment, the fluorescent moiety may be attached to the cephalosporin compound via the linker. Therefore, the fluorescent moiety according to one embodiment is separated from the above-mentioned Formula 1 by β-lactamase or extended spectrum β-lactamase (ESBL) together with L (linker) .

In one embodiment, the compound of Formula 1 may be a compound of Formula 2;

(2)

.

.

The compound of formula (1)

Replacing the functional group (B) at the 3-carbon position of the cephalosporin antibiotic represented by the following formula (3) with a linker; And binding the fluorescent moiety to the core structure of the cephalosporin antibiotic via the linker.

For example, the compound of Formula 1 may be prepared by binding a linker to a fluorescent moiety to prepare a linker-fluorescent moiety conjugate; And replacing the functional group (B) at the 3-carbon position of the cephalosporin antibiotic represented by the following formula (3) with a complex of the linker-fluorescent moiety.

(3)

For example, the compound of formula 1 may be prepared as disclosed in FIG.

As shown in FIG. 1, a compound according to one embodiment emits a high fluorescence signal (for example, fluorescence of 2.5 times or more) in the presence of? -Lactamase or ESBL in a sample, The present invention can efficiently detect the presence of antibiotic-resistant bacteria expressing? -Lactamase or ESBL as compared with the control, and can provide an antibiotic-resistant bacterial detection compound, a reagent composition, an antibiotic-resistant bacterial infection diagnosis kit, an antibiotic- Can be usefully used for the diagnosis of resistant bacterial infection.

Hereinafter, the use of the above formula (1) will be described in detail.

Another aspect provides a probe for detecting an antibiotic-resistant bacteria comprising the compound represented by the above formula (1).

Another aspect is to provide a reagent composition or an assay composition for detecting an antibiotic-resistant bacteria comprising the compound represented by the above formula (1).

Yet another aspect provides the use of a compound represented by Formula 1 for the preparation of a probe, an assay composition, or a reagent composition for detecting an antibiotic-resistant bacteria.

The compound represented by the formula (1) is as described above.

In one embodiment, the antibiotic-resistant bacteria may refer to bacteria that are not effectively antibacterial by antibiotics, including beta-lactam. The antibiotic-resistant bacterium may be a bacterium expressing? -Lactamase or extended-spectrum? -Lactamase (ESBL). Examples of the bacterium expressing the? -Lactamase or ESBL include Enterobacter spp . ), Escherichia species spp . ), Salmonella species (Samonella spp.), Proteus species (Proteus spp . ), Citrobacter spp. , Morganella spp. spp . ), Bacteroides spp . ), Providencia species (Providencia spp . ), Serratia species ( Serratia spp ), Klebsiellaspp. , Shigella spp . ), Pseudomonas species (Pseudomonas spp.), Acinetobacter species (Acinetobacter spp . ) Or Kluyvera ( Kluyvera spp . ). Also for example, examples of such bacteria include E. cloacae , E. aerogenes , E. coli , Salmonella enterica ( S. enterica), Salmonella typhimurium (S. typhi), Salmonella typhimurium Farah (S. paratyphi), Proteus vulgaris (P. vulgaris), Proteus Billy's mum (P. mirabilis), bakteo Bra Aki (C. braaki) into a sheet, the sheet a frame bakteo undi (C.freundii), bakteo di bereususeu a sheet (C. diversus), do not know the Nella know going (M.morganii), plastic foil teroyi des Gillis (B. fragilis), watermelon teroyi death No tooth ( B.bulgatus), Providencia inlet Gary (P. rettgeri), Providencia Stuttgart are Tea (P.stuartii), Serratia sense Marseille (S. marcescens), Serratia Ponti cola (S.fonticola), Serratia Pacifico liquor Enschede (S. liquefaciens), keulrep when Ella pneumoniae (K.pneumoniae), keulrep when Ella oxy Saturday (K. oxytoca), sh Gela disen Terry Ke (S.dysenteriae), sh Gela Small Naples (S. sonnei), Pseudomonas her rugi Labor (P. aeruginosa), Acinetobacter baumannii (A. baumannii), Cluj Vera Ascorbata , K. ascorbata , and K. georgiana .

In one embodiment, the bacterium-resistant antibiotic may include an antibiotic having a beta-lactam ring, for example, a penicillin antibiotic, or a cephalosporin antibiotic. Examples of such penicillin-based agents can generally include amino-penicillins (e.g., amoxicillin, ampicillin, epicatechin, etc.) or carboxy-penicillins (e.g., carbenicillin, temocillin, etc.). Examples of such cephalosporin antibiotics include 1-generation cephalosporins (e.g., cephazoline, cephalothin, cephapirin, etc.) and 2-generation cephalosporins (e.g., cephalocor, sephalanol, cephinox, etc.) (Such as ceftazidime, cytotoxic, cetearmic, cell dejim, aztreonam, etc.) and 4-generation cephalosporins (such as ceftipim, cephiprom, Ceftioxane, cephalosporin, etc.).

Another aspect provides an antibiotic-resistant bacterial infection test kit comprising the compound represented by the above formula (1).

Yet another aspect provides a method of detecting an antibiotic resistant bacteria comprising contacting a compound represented by Formula 1 with a biological sample.

Yet another aspect provides a method of diagnosing an antibiotic resistant bacterial infection comprising contacting a compound of formula 1 with a biological sample.

Yet another aspect provides the use of a compound represented by Formula 1 for the preparation of an antibiotic resistant bacterial infection diagnostic kit.

The compound represented by the above formula (1) and the antibiotic resistant bacteria are as described above.

Fluorescence generated from the compound represented by formula (1) according to one aspect can be an indicator of the presence of ESBL or bacteria containing it in the sample, and thus can be used for diagnosis of an infectious disease caused by the bacteria .

The term "infectious disease " as used herein refers to a disease or condition related to the presence of a subject or an organism (infectious agent) in contact therewith, particularly a bacterial infectious disease, " For example, "beta-lactam antibiotic resistant bacterial infectious disease" may refer to an antibiotic resistant bacterial infectious disease that is not effectively treated by beta-lactam containing antibiotics.

The term "diagnosing" is used herein to refer to determining the susceptibility of an individual to a particular disease or disorder, determining whether an object currently has a particular disease or disorder, (E.g., identifying an infectious disease or condition, determining responsiveness to treatment of the disease, and determining the effect thereof), or determining therametrics (e.g., to provide information about therapeutic efficacy) Monitoring the state of the object). The subject may be a mammal, including a human.

In one embodiment, the method of detecting or diagnosing comprises contacting the compound of Formula 1 with a target sample; And detecting fluorescence generated from the compound of Formula 1 in the target sample.

The contacting step may include adding the biological sample or the target sample that has been pretreated to the composition comprising the compound of Formula 1. The pretreatment of the sample can be suitably carried out according to the intended use of the ordinary person skilled in the art.

By analyzing the final signal intensity by measuring the fluorescence emission upon hydrolysis of the compound of formula (1) by contacting (reacting) with the objective sample, the infection disease caused by the antibiotic resistant bacteria can be diagnosed or the antibiotic resistant bacteria can be detected. The fluorescence signal analysis can be performed using various methods known in the art and is read and processed by a suitable apparatus available in the art. For example, protocols and procedures known in the art can be used including fluorescence analyzers, microplate readers, automated processing using robotic devices, laser scanning systems.

The sample may be a biological sample obtained from a lysate of bacterial cells or a supernatant of a bacterial cell culture liquid or an animal organs or cells such as blood, saliva, sputum, cerebrospinal fluid, secretions, lymph, dialysate, sap, , Urine, feces, etc.).

According to one aspect of the present invention and a probe containing the same, it is possible to detect β-lactamase or ESBL with high sensitivity, so that it can be applied not only to various biochemical studies but also to a conventional pH indicator factor-based method , It is possible to perform clinical detection of antibiotic-resistant bacteria which is not possible in the present invention and to analyze the antibiotic-resistant bacteria with high sensitivity from the molecular diagnosis of the antibiotic-resistant bacterial infectious disease and the objective sample, There is an effect that can be.

Figure 1 is a chemical reaction showing the hydrolysis of ceftazidime (top panel) and BODIPY-based fluorescent substrate (bottom panel) by [beta] -lactamase.
2 is a view showing a process of synthesizing a probe compound according to one embodiment. _{a) i) CF 3 CO 2} H, CH 2 Cl 2 / EtOH (14: 1), room temperature, 72 hours; And ii) 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) at room temperature for 4 hours; b) i-Pr ₂ NEt, BF ₃ OEt ₂ , CH ₂ Cl ₂ , room temperature, 12 h, 21% (2 steps); c) i) MSTFA, CH ₂ Cl ₂ , 40 ° C, 2 h; ii) TMSI, CH ₂ Cl ₂ , 0 ° C to room temperature, 1h; And iii) 2, CH ₃ CN, at room temperature, 12 hours, 33%. DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone / MSTFA = N-methyl-N- (trimethylsilyl) trifluoroacetamide / iodotrimethylsilane.
FIG. 3A illustrates a probe (1 [mu] M) according to one embodiment when the probe according to one embodiment excites at 490 nm before the reaction with TEM-1 [beta] -lactamase (black line) Lt; / RTI > fluorescence emission profile; An inset image shows the probe solution under the absence (left) and presence (right) of the enzyme;
Figure 3b shows the signal increase in the enzymatic degradation of probe (200 nM) according to one embodiment according to the concentration of TEM-1 [beta] -lactamase.
Figure 4A is a graph showing the time-dependent fluorescence signal increase in the enzyme reaction for a probe (2 [mu] M) according to one embodiment.
Figure 4b is a graph showing Michaelis-Menton plots for the enzymatic degradation of 4;
5A is a graph showing fluorescence signal results measured at 30, 60 and 90 minutes after treatment of a probe according to one embodiment with a CAZ-resistant bacterial lysate; Signals observed from bacteria treated with MIC <0.5 mg / mL or MIC ≥ 0.5 mg / mL CAZ were expressed as black bars or gray bars, respectively.
Figure 5b is a graph showing the fluorescence intensity measured in response to a CAZ-resistant bacterial lysate of a probe according to one embodiment, plotted against MIC of CAZ; The fluorescence intensity is the intensity at 90 min minus the intensity at 0 min.
6A is a graph showing fluorescence signal results measured at 30, 60 and 90 minutes after treatment of the probes according to one embodiment with CAZ and CTX resistant bacterial lysates; Signals observed from bacteria treated with (MIC _CAZ + MIC _CTX ) <1 ug / ml or (MIC _CAZ + MIC _CTX )> 1 ug / ml <0.5 mg / mL or MIC ≥ 0.5 mg / mL CAZ, Or red bars; ESBL production strains are marked with an asterisk.
FIG. 6B is a graph showing the fluorescence intensities measured in response to CAZ and CTX resistant bacterial lysates of probes according to one embodiment, plotted against MIC of CAZ and CTX; FIG. The fluorescence intensity is the intensity at 90 min minus the intensity at 0 min; ESBL producers are represented by triangles, and each data value is the mean value of two independent experiments; The triangle enclosed in the circle represents the CTX-M-9 strain.
Figure 7 is a graph showing the fluorescence intensity measured in the reaction of a probe according to one embodiment with a CAZ and / or a CTX resistant bacterial lysate; S and R each represent susceptibility and resistance to antibiotics; The fluorescence intensity is the intensity at 90 min minus the intensity at 0 min.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Reference Example

TEM-1 [beta] -lactamase was purchased from Prospec (USA). Unless otherwise stated, all materials were purchased from Sigma-Aldrich (USA) and used without further purification. UV absorption spectra and profiles of fluorescence emission spectra were recorded on a Libra S22 UV / Vis spectrophotometer (Biochrom, UK) and an F-7000 fluorescence spectrometer (Hitachi, Japan), respectively. The fluorescence intensities of the synthesized probes used in pH-dependent experiments and enzyme assays were measured with an Appliskan (TM) multimode microplate reader (Thermo Scientific, USA).

Example  1. For antibiotic-resistant bacteria detection Of the probe  Produce

A BODIPY-based fluorescent probe was synthesized as shown in FIG. According to the previously reported procedure (J. Bartelmess and WW Weare, Dyes Pigm., 2013, 97, 1.), condensation of 4-pyridinecarboxaldehyde and 2,4-dimethylpyrrole in the presence of trifluoroacetic acid Followed by oxidation with DDQ to synthesize non-spiro pyridine (compound 1) first. Treatment of compound 1 with boron trifluoride diethyl etherate and Hunig's base provided the BODIPY derivative 2 as a dark red powder. Next, Brown et al. (RF Brown, MD Kinnick, JM Jr. Morin, RT Vasileff, FT Counter, EO Davidson, PW Ensminger, JA Eudaly, JS Kasher, AS Katner, J. Med. ) Was carried out in the order of the three steps according to the method reported by K.-K. Silylation of compound 3 with MSTFA yielded the corresponding TMS ester, which was then reacted with iodotrimethylsilane dissolved in dichloromethane to produce the 3'-iodo intermediate product. Finally, substitution of iodine with BODIPY 2 produced a red powder probe (Compound 4). The above process will be described in detail as follows.

1.1. 1,3,5,7-tetramethyl-8- (4-pyridyl) -4,4'-di-fluoroboradiazine -4,4'-di-fluoroboradiazaindacene) (2)

2,4-Dimethylpyrrole (300 μL, 2.91 mmol) and 4-pyridinecarboxaldehyde (137 μL, 1.46 mmol) were added to an oxygenated 1:14 (v / v) mixture of ethanol / Melted. Several portions of trifluoroacetic acid were added and the reaction mixture was stirred at ambient temperature for 72 hours. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (330 mg, 1.46 mmol) was added to the solution. The reaction mixture was stirred for an additional 4 hours. The solvent was then removed under reduced pressure and the resulting residue was dissolved in dry dichloromethane (30 mL). N, N-Diisopropylethylamine (1.49 mL, 8.32 mmol) and boron trifluoride diethyl etherate (1.59 mL, 12.85 mmol) were then added and the reaction mixture was stirred for 12 h. The resulting dark red precipitate was filtered off and the filtrate was concentrated in vacuo. The crude material was purified by column chromatography on silica gel (dichloromethane / ethyl acetate, 4: 1 (v / v)) to yield compound 2 (100 mg, 21%) as a dark red powder.

Melting Point (MP): 235.3-236.2 [deg.] C. ^{1 H NMR (400 MHz, CDCl} 3) δ 8.78 (d, J = 4.4, 1.6 Hz, 2H), 7.30 (dd, J = 4.3, 1.6 Hz, 2H), 6.01 (s, 2H), 2.56 (s, 6H), 1.41 (s, 6H). ^{13 C NMR (100 MHz, CDCl} 3) δ 156.9, 150.7, 143.6, 142.7, 137.7, 130.4, 123.3, 121.8, 14.62, 14.58.

1.2. (E) -10- (1 - ((7- (2- (2-aminothiazol-4-yl) -2- (methoxyimino) acetamido) -2-carboxy- 4-yl) -5,5-difluoro-1,3,7,9-tetrahydro-naphthalen- (E) -10 &lt; / RTI &gt; &lt; RTI ID = 0.0 &gt; - (1 - ((7- (2- (2-aminothiazol-4-yl) -2- (methoxyimino) acetamido) -2-carboxy-8-oxo- 5- thia- 1-azabicyclo [4.2.0] oct Pyridin-1-yl) -4,5-difluoro-1,3,7,9-tetramethyl-5H-pyrrolo [1,2- , 1'-f] [1,3,2] diazaborinin-4-ium-5-uide iodide) (4)

The cell tubercan (Compound 3) (80 mg, 0.18 mmol) was suspended in dichloromethane (5 mL) under a nitrogen atmosphere. N-Methyl-N- (trimethylsilyl) trifluoroacetamide (127 μL, 0.68 mmol) was added and the mixture was allowed to warm to 40 ° C for a while. A homogeneous solution of cephemtrimethylsilyl ester was cooled to room temperature and then trimethylsilyl iodide (77 L, 0.54 mmol) was added. The reaction mixture was stirred for 1 hour and then the solvent was evaporated in vacuo to give 3'-iodomethyl cephem as an oil. The residue was taken up in acetonitrile (4 mL) and tetrahydrofuran (64 L, 0.8 mmol) was added to destroy excess trimethylsilyliodide. The pyridine-substituted BODIPY 2 (59 mg, 0.18) was dissolved in acetonitrile (1 mL) and a portion was added to the reaction solution. The reaction was stirred at room temperature for 12 hours and then added to 5% methanolic acetone (20 mL) to hydrolyze the trimethylsilyl group. The crude product isolated with hydrogen iodide salt was filtered off, washed with ethyl acetate / dichloromethane and dried under vacuum. The resulting solid (insoluble in dichloromethane) was purified by hot filtration to yield compound 4 as a red powder (50 mg, 33%).

MP: 107.0-110.0 [deg.] C (decomp). 2H), 6.80 (s, 1H), 6.07 (s, 2H), 5.80 (d, J = 6.8 Hz, 2H) (d, J = 5.2 Hz, 1H), 5.79 (d, J = 14.4 Hz, 1H), 5.44 J = 18.4 Hz, 1 H), 2.41 (s, 6H), 1.37 (s, 6H). HRMS-ESI (m / z) : It was the [M] ⁺ was 721.1997 calculated for _{C 32 H 32 BF 2 N 8} O 5 S 2 + 721.1998.

1.3. Determination of quantum yield

The fluorescence quantum yield (? F) of the synthesized probe was calculated from the known value of pyridyl BODIPY (Compound 2) (? Ref = 0.3 in dichloromethane) (Bartelmess, J., Weare, WW, Dyes Pigm. 2013, 97 (1), 1.).

Where? Is the refractive index of the solvent. I is the integrated fluorescence intensity, and OD is the absorbance at the excitation wavelength. The integrated fluorescence domain was obtained from the emission spectrum of the compound using an F-7000 fluorescence spectrometer. As a result, the relative quantum yield (? Fl) of the probe synthesized in Example 1 was determined to be 0.09 relative to the quantum yield (? Fl = 0.3) of pyridyl BODIPY.

Experimental Example  1. β- Lactamasee  About Of the probe Signaling  Character analysis

Enzyme kinetics analysis was performed to characterize the signaling properties of the probe to beta-lactamase.

Specifically, 0.1 to 2.0 U / μL of TEM-1 Bla was added to a 96-black well to a fluorescent probe (2 to 150 μM) present in 100 mM Tris-HCl (pH 7). Immediately after enzyme treatment, fluorescence intensity was measured and signal intensity was continuously monitored for 60 minutes at 5 minute intervals in a microplate reader and the results are shown in FIG. The control group was not treated with TEM-1 Bla. In order to determine the catalytic constant (kcat) and the Michaelis constant (Km) of the decomposition reaction of the probe by the enzyme, the increased intensities were plotted according to the probe concentration, and the results are shown in FIG. The parameters were then obtained by non-linear fitting of the curve.

As shown in FIG. 3A, when the probe was reacted with an enzyme, the fluorescence intensity at 512 nm was increased to 2.5-fold. In addition, the turn-on green signal was visible even under the naked eye under a light irradiator (490 nm).

In addition, as shown in FIG. 3B, the sensitivity of the probe to the detection of TEM-1 was found to be as low as 0.03125 U / mL. As a result, it can be seen that the probe can successfully detect the enzyme activity of Bla.

As shown in FIG. 4A, when the signal was measured over time, the fluorescence intensity rapidly increased at the start time and reached a saturation value after 45 minutes.

In addition, as shown in Fig. 4B, the intensity measurements in the probes at various concentrations provided kcat of 12 + 1 s &lt; ^-1 &gt; and Km of 51 + 12 [mu] M. The enzyme efficiency of TEM-1 estimated by the value of kcat / Km was 230,000 s-1M-1, which is due to the fluorescent substrate CC1 (kcat = 52 ± 1 s ^-1 and Km = 70 ± 7 μM) And the previously reported values for the hydrolysis of CCF ₂ (kcat = 29 ± 1 s ^-1 and Km = 23 ± 1 μM) (W. Gao, B. Xing, RY Tsien and J. Rao, J Am Chem Soc. , 2003, 125, 11146.).

Experimental Example  2. Bacteria In the melt CAZ  And / or CTX - Detection of resistant bacteria

The practical utility of the fluorescent substrate was further tested by attempting to use the probe synthesized in Example 1 for the detection of CAZ and / or CTX-resistant bacteria.

Specifically, in the detection of extended-spectrum beta-lactamase (ESBL), CAZ and / or CTX-resistant bacteria were used to evaluate the performance of fluorescent tests with dual-reactive probes. The strains were obtained from several Korean hospitals or Dr. Nordmann (Swizterland). The above-mentioned strains were screened for ESBL production using the disk strengthening method (W. Song, IK Bae, YN Lee, CH Lee, SH Lee and SH Jeong, J Clin Microbiol, 2007, 45, 1180) and blaCTX-M Were characterized for the content of beta-lactamase at the molecular level through multiplex PCR and sequencing.

The strain was isolated from Muller-Hinton AGA (Asan, Korea) and incubated at 37 ° C for 16-24 hours before performing the fluorescence test. Extraction of bacterial proteins was performed as previously described (P. Nordmann, L. Poirel and L. Dortet, Emerg. Infect. Dis., 2012, 18, 1503.). Briefly, the test strains of one calculated inoculation loop (10 μL) were replicated in 150 μL of 200 mM Tris-HCl lysis buffer (B-PERII, bacterial protein extraction reagent; Thermo Scientific Pierce, Rockford, , Vortexed for 1 min and further reacted at room temperature for 30 min. These bacterial suspensions were centrifuged at 10,000 x g for 5 minutes at room temperature. 30 μL of the supernatant was mixed with 100 μL of the fluorescent probe (100 nM, 100 mM Tris-HCl, pH = 8) prepared in Example 1 in the 96-well tray.

Fluorescence signals were measured using a Infinite F200pro (Tecan Croup Ltd., Mannedorf, Switzerland) microplate reader in power mode. The excitation and emission wavelengths were set to 485 nm (bandwidth 20 nm) and 535 nm (bandwidth 25 nm), respectively. A polystyrene black 96 well bottom microplate (Greiner bio-one, Frickenhausen, Germany) with a maximum capacity of 130 μL was used in each well. The fluorescence signal of each test sample was measured for 90 minutes every 5 minutes, and the results are shown in Figs. 5-7. A phenol red assay was performed as a positive control. Specifically, for the phenol red assay, 30 μL of the supernatant as reported previously (P. Nordmann, L. Poirel and L. Dortet, Emerg. Infect. Dis., 2012, 18, 1503) (pH 7.8), and the results are shown in Table 1. The results are shown in Table 1 below.

As shown in Figures 5a and 6a, the CAZ and / or CTX resistant bacteria are clearly distinguishable from drug-sensitive bacteria having a minimum inhibitory concentration (MIC) of less than 0.5 mg / ml or 1 ug / ml .

Also, as shown in Figures 5b and 6b, it can be seen that the increased signal is somewhat dependent on the minimum inhibitory concentration of CAZ and / or CTX on the bacteria, which is the specificity of the probe for CAZ and / or CTX hydrolysis activity .

In addition, as shown in Fig. 7, the fluorescence intensity of the strains resistant to CAZ, CTX-sensitive, CAZ-sensitive, CTX-resistant, and CAZ and CTX were increased, but CAZ and CTX It can be seen that fluorescence intensity did not increase in strains susceptible to both. As a result, the probe according to one embodiment can detect a cephalosporin antibiotic resistant strain.

bacteria Phenol red The probe of Example 1 CTX-M-14 (4) voice positivity CTX-M-15 + SHV-12 (128) voice positivity SHV-28 + TEM-1 (64) voice positivity CTX-M-9 (0.25) voice voice BL1560 (8) voice positivity BL1679 (8) voice positivity BL2161 (4) voice positivity BL2514 (8) voice positivity BL2750 (0.125) voice voice BL2780 (0.125) voice voice BL2784 (0.5) voice voice BL2788 (0.06) voice voice

Note: (a) Numbers in parentheses, MIC of CAZ; (b) BL series such as BL1560, BL is a blood-derived strain; And (c) the signal is detected by a positive, corresponding probe (phenol red or the probe of Example 1) (i.e., no signal is generated in the probe).

As shown in Table 1, it can be seen that the conventional phenol red method can not effectively detect antibiotic resistant bacteria, while probes according to one embodiment can effectively detect antibiotic resistant bacteria.

Experimental Example  3. Detection of antibiotic-resistant bacteria in clinical samples

3.1. Evaluation of Antibiotic Resistant Bacteria in Blood Samples

A total of 185 blood samples were received from Kangnam St. Mary's Hospital. Among them, the number of patients was 119, of which 126 samples were obtained by cultivating the samples from 63 patients under aerobic or anaerobic culture conditions, and one sample was analyzed by two bacteria each in aerobic and anaerobic culture conditions And from 55 patient samples were obtained by culturing bacteria (aerobic-21 / anaerobic-28) only in one of the anaerobic or aerobic culture conditions. As a result, 96 samples were obtained in anaerobic culture condition, and 93 samples were obtained under aerobic culture conditions. Of the total 185 samples, 44 were ESBL positive and 141 were ESBL negative. The classification of these species is shown in Tables 2 to 5 below.

ESBL-positive strains No. Escherichia coli 27 Klebsiella pneumoniae 16 Citrobacter freundii, Kluyvera cryocrescens One Total 44

ESBL negative strains No. Achromobacter denitrificans One Acinetobacter baumannii 3 Citrobacter braakii, Escherichia coli 2 Citrobacter freundii One Enterobacter aerogenes 6 Enterobacter cloacae 5 Escherichia coli 78 Klebsiella oxytoca 3 Klebsiella pneumoniae 34 Proteus mirabilis One Pseudomonas aeruginosa One Salmonella, non-typhi One Serratia marcescens One Escherichia coli,
Klebsiella oxytoca 2 Klebsiella pneumoniae,
Escherichia coli,
Enterobacter cloacae, 2 total 141

The 185 samples used in the experiment Probes Tests 185 Species of the sample No. Achromobacter denitrificans One Acinetobacter baumannii 3 Citrobacter freundii One Enterobacter aerogenes 6 Enterobacter cloacae 5 Escherichia coli 105 Klebsiella oxytoca 3 Klebsiella pneumoniae 50 Proteus mirabilis One Pseudomonas aeruginosa One Salmonella, non-typhi One Serratia marcescens One Citrobacter freundii, Kluyvera cryocrescens One Escherichia coli, Klebsiella oxytoca 2 Citrobacter braakii, Escherichia coli 2 Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae 2 Total 185

Blood sample type 185 Aerobic blood culture 63 Anaerobic blood culture 126 ESBL positivity 44 voice 141 Number of patients 119 Aerobic, anaerobic sample simultaneously 62 * Aerobic samples only 32 ** Only anaerobic samples 25 * - 1 patient has aerobic, anaerobic 2set 4 samples
** - Two patients had only two anaerobic samples

Four strains of Citrobacter freundii and Kluyvera cryocrescens, Escherichia coli and Klebsiella oxytoca, Citrobacter braakii and Escherichia coli, Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae were grown together.

Treatment of anaerobic samples was performed as follows. The 10 mL blood culture was centrifuged and the supernatant was removed except for the needle. After washing three times with 10 ml of saline, 0.5 ml of saline was added to the needle to suspend the needle, and then transferred to two 1.5 ml tubes in an amount of 100 ul and 400 ul. After centrifugation, 400 μl of the supernatant was discarded, and 300 μl of B-PER was added, followed by vortexing at 4 ° C. for 30 minutes. The lysed sample was then centrifuged and the supernatant was used for fluorescence intensity measurement. After centrifugation, the supernatant was removed and 100 μl of DW was added. The tube was heated at 10 ° C for 10 minutes, centrifuged, and the supernatant was used for PCR.

Treatment of aerobic samples was performed as follows. The 10 mL blood culture was centrifuged, and the supernatant was removed. 1 ml of ammonium chloride and 5 ml of 5% saponin solution were mixed with the needle and left for 10 minutes. After centrifugation, the supernatant was removed, washed three times with 10 ml of saline, and treated in the same manner as the anaerobic sample.

The fluorescence was measured using the probe prepared in Example 1 as an experimental group and phenol red was used as a positive control. 30 μl of the sample, which had been pretreated, was mixed in a 96-well tray into 10 μl of the fluorescent solution containing 10 nM of the probe according to one embodiment. The phenol red assay was carried out as described in Experimental Example 1 above. Fluorescent signals were measured in a powered mode using Infinite F200pro (Tecan Croup Ltd., Mannedorf, Switzerland) microplate reader. The excitation and emission wavelengths were set to 485 nm (bandwidth 20 nm) and 535 nm (bandwidth 25 nm), respectively. The fluorescence intensity was measured by subtracting the fluorescence value of the well containing the sample from the fluorescence value of the control well (sample containing only B-PER) containing no sample.

The ROC curve was confirmed for the above results. As a result, the sensitivity and specificity of the probe according to one embodiment were calculated as the value of AUC of 10 minutes at 0.967 because the best result was obtained.

The specificity and sensitivity of bacterial detection in each test method were calculated through genuine positive results, and the results are shown in Table 6 below. The genotype was confirmed by PCR using the Vitek2 (manufacturer: bioMérieux) ESBL test, and the positive strain was identified by performing a phenotype test against PCR negative strains.

ESBL detection Detection of CTX or CAZ resistance Test method responsiveness Specificity responsiveness Specificity The probe of Example 1 90.9%
(40/44) 87.9%
(123/141) 84.2%
(48/57) 92.2%
(118/128) Phenol red 88.6%
(39/44) 99.3%
(140/141) 68.4%
(39/57) 99.2%
(127/128)

In addition, the sensitivity and specificity of probes according to one embodiment of the ESBL genotypes were further analyzed, and the results are shown in Table 7 below.

In order to confirm whether the ESBLs could be distinguished in a case where the CTX / CAZ resistance was higher than a certain level, the fluorescence intensity of a strain having a specific MIC was analyzed while expressing ESBL, and the results are shown in Table 8 below.

The probe of Example 1 Phenol red beta-lactamase type sensitivity (%) specificity (%) sensitivity (%) specificity (%) only CTX-M 96.30 97.83 96.30 100.00 including CTX-M 96.67 97.83 96.67 100.00 only TEM 42.86 97.83 0.00 100.00 including TEM 55.56 97.83 22.22 100.00 only SHV 100.00 97.83 0.00 100.00 including SHV 100.00 97.83 11.11 100.00 cAmpC 100.00 97.83 64.71 100.00 DHA 100.00 97.83 0.00 100.00 CIT 100.00 97.83 0.00 100.00 CMY 100.00 97.83 0.00 100.00 Total 93.51 97.83 45.45 100.00

beta-lactamase type Bell No. of
isolates Positivity of assays Resistant rate of MIC
by EUCAST The probe of Example 1 CTX [2 < CAZ [4 < ESBL CTX-M Escherichia coli 14 13 92.9% 13 92.9% 8 57.1% Serratia marcescens 5 5 100.0% 5 100.0% 4 80.0% Klebsiella pneumoniae 3 3 100.0% 3 100.0% 2 66.7% Citrobacter freundii 3 3 100.0% 3 100.0% 2 66.7% Enterobacter cloacae One One 100.0% One 100.0% One 100.0% Samonella typhimurium One One 100.0% One 100.0% One 100.0% CTX-M total 27 26 96.3% 26 96.3% 18 66.7% TEM Escherichia coli 5 2 40.0% 2 40.0% 3 60.0% Proteus mirabilis One One 100.0% One 100.0% 0 0.0% Providencia stuartii One 0 0.0% 0 0.0% One 100.0% TEM total 7 3 42.9% 3 42.9% 4 57.1% SHV Escherichia coli 3 3 100.0% 3 100.0% 3 100.0% Klebsiella pneumoniae 2 2 100.0% 2 100.0% One 50.0% Serratia marcescens One One 100.0% One 100.0% One 100.0% SHV total 6 6 100.0% 6 100.0% 5 83.3% CTX-M + TEM Citrobacter freundii One One 100.0% One 100.0% One 100.0% CTX-M + SHV Escherichia coli One One 100.0% One 100.0% One 100.0% Enterobacter cloacae One One 100.0% One 100.0% One 100.0% SHV + TEM Klebsiella pneumoniae One One 100.0% One 100.0% One 100.0% AmpC cAmpC Escherichia coli 6 6 100.0% 6 100.0% 6 100.0% Citrobacter freundii 6 6 100.0% 6 100.0% 6 100.0% Serratia marcescens 5 5 100.0% 5 100.0% 2 40.0% cAmpC total 17 17 100.0% 17 100.0% 14 82.4% DHA Klebsiella pneumoniae 7 7 100.0% 2 28.6% 6 85.7% CIT Escherichia coli 4 4 100.0% One 25.0% 3 75.0% CMY Escherichia coli 2 2 100.0% 2 100.0% One 50.0% Proteus mirabilis 2 2 100.0% 2 100.0% 2 100.0% MOX Klebsiella pneumoniae One One 100.0% One 100.0% One 100.0% beta-lactamase
gun 77 72 93.5% 64 83.1% 58 75.3% Negative Escherichia coli 44 One 2.3% 0 0.0% 0 0.0% Klebsiella pneumoniae 2 0 0.0% 0 0.0% 0 0.0% gun 123 73 59.3% 64 52.0% 58 47.2%

3.2. Assessment of Antibiotic Resistant Bacteria Ability in Urine Samples

Urine samples were supplied from Kangnam St. Mary 's Hospital. Escherichia coli strains were identified as Escherichia coli coli were 43, and Klebsiella pneumoniae were 10, ESBL negative strains were 51 in total, Escherichia coli is 43, Klebsiella 5 pneumoniae , and 3 Klebsiella oxytoca .

The pretreatment of urine samples was performed as follows. 7 ml of urine was centrifuged at 4000 g, and the supernatant was discarded. The pellet was washed twice with 7 ml of PBS. In the final wash step, the pellet was dissolved in 350 μl of PBS and then 70 μl was used for DNA isolation. 180 μl was centrifuged again and 200 μl of B-PER solution was added and vortexed for 30 minutes.

Thereafter, fluorescence measurement and phenol red test were carried out in the same manner as in Experimental Example 3.1. As a result, the sensitivity and specificity of the probes according to one embodiment were calculated as the values of the time points of ESBL detection and the 90 minutes AUC of the ROC curve according to the CTX or CAZ resistance were 0.725 and 0.739, respectively, The results are shown in Table 9 below.

ESBL detection Detection of CTX or CAZ resistance Test method responsiveness Specificity responsiveness Specificity The probe of Example 1 63.0%
(34/54) 80%
(40/50) 64.8%
(35/54) 82%
(41/50) Phenol red 31.5%
(17/54) 100%
(50/50) 31.5%
(17/54) 100%
(50/50)

As shown in Tables 6 to 9, probes according to one embodiment can detect ESBL-expressing bacteria and antibiotic-resistant bacteria at high sensitivity and specificity even in actual clinical samples.

As a result, probes according to one embodiment can detect beta -lactamase or ESBL with high sensitivity, so that they can be applied to various biochemical studies as well as in a conventional pH indicator factor-based method Resistant bacteria can be clinically detected, and the molecular diagnosis of antibiotic resistant bacterial infectious diseases and antibiotic resistant bacteria can be analyzed with high sensitivity from the target sample.

Claims (18)

A compound represented by Formula 1 below;
[Chemical Formula 1]

(Core-structure-LZ)
Wherein R ₁ is selected from the group consisting of -NH ₂ and -NHCO-R ', R' is selected from the group consisting of -CH ₂ S-CF ₃ ,

,

,

,

,

,

,

,

,

,

,

,

,

,

, And

&Lt; / RTI &gt;
R ₂ is H,
A is selected from the group consisting of S, O, SO, SO ₂ , and CH ₂ ,
L is

,

or

And, R _L is either a single bond, C _1-6 alkylene, C _2-6 alkenylene or C _2-6 alkynylene group, R ₃ and R ₄ are each independently C _1-6 alkyl, C _2-6 alkenyl, or C _2-6 alkynyl group, R ₅ is a single bond, a C _2-6 alkenyl group, R ₆ and R ₇ are, each independently, hydrogen or C _1-6 alkyl group, C _{2 -6} alkenyl group, or C _2-6 alkynyl group, in the case where the structure of the ring L of 6-ring and n is an integer from 1 to 4 and m is an integer from 1 to 9, when the ring is 5-ring n is an integer of 1 to 4, m is an integer of 1 to 7, * is a bonding site with the core structure, * 'is a bonding site with Z, and
Z is a fluorescent moiety, BODIPY (boron-dipyrromethene). delete The method according to claim 1,
The L

Wherein R _L is a single bond, methyl or ethyl, * is a bonding site with the core structure, and * 'is a bonding site with Z. delete The method of claim 1, wherein the BODIPY is selected from pyridyl BODIPY, BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY 581/591, BODIPY TR, BODIPY 630/650, BODIPY 650/665, BODIPY 558/568, BODIPY 564/570, &Lt; / RTI &gt; The compound according to claim 1, wherein the compound represented by Formula 1 is a compound represented by Formula 2 below:
(2)

. [Claim 2] The compound according to claim 1, wherein Z in Formula 1 is separated from the Formula 1 by β-lactamase or extended spectrum β-lactamase (ESBL) together with L to exhibit fluorescence. A probe for detecting an antibiotic-resistant bacteria comprising the compound of any one of claims 1, 3 and 5 to 7. 9. The probe of claim 8, wherein the antibiotic-resistant bacteria is a bacteria expressing beta-lactamase or extended-spectrum beta-lactamase (ESBL). The method according to claim 9, bacteria expressing the ESBL has Enterobacter species (Enterobacter spp.), S-cyano cherry species (Escherichia spp . ), Salmonella species (Samonella spp.), Proteus species (Proteus spp . ), Citrobacter spp. , Morganella spp. spp . ), Bacteroides spp . ), Providencia species (Providencia spp . ), Serratia species ( Serratia spp ), Klebsiellaspp. , Shigella spp . ), Pseudomonas species (Pseudomonas spp.), Acinetobacter species (Acinetobacter spp . ) And Kluyvera spp . ). &Lt; / RTI &gt; 9. The probe of claim 8, wherein the antibiotic is a cephalosporin antibiotic. 12. The probe of claim 11, wherein the cephalosporin antibiotic is cytotoxic or ceftazidime. A reagent composition for detecting antibiotic-resistant bacteria comprising the compound of any one of claims 1, 3, and 5 to 7. A kit for the diagnosis of an antibiotic-resistant bacterial infection comprising the compound of any one of claims 1, 3, and 5 to 7. 15. The kit of claim 14, wherein the bacterial infection is a bacterial infection expressing an ESBL. [Claim 15] The method according to claim 15, wherein the disease caused by the bacterial infection expressing the ESBL is at least one selected from the group consisting of skin and soft tissue infections, febrile neutropenia, airway tube infections, catheter infections, bronchiolitis, pneumonia (pathogenic), sepsis, meningitis, , Brucellosis, acute enteritis, community-acquired respiratory infections, trachoma, neonatal inclusive conjunctivitis, bourllnumin food poisoning, acute food poisoning, diarrhea, hemorrhagic colitis, diarrhea, hemorrhagic colitis, infectious sinusitis, peritonitis, anthrax, , Keratosis pilaris, acne, harlequin lupus, pigmented scleroderma, keratosis, eczema, and brain damage (including acne), bronchitis, gastric ulcer, endocarditis, salmonellosis, gastroenteritis, rash, opportunistic infections, otitis media, sinusitis, pharyngitis, Keratitis, and fasciitis. Contacting the compound of any one of claims 1, 3, and 5 to 7 with a sample of interest; And
And detecting fluorescence generated from the compound of any one of claims 1, 3, and 5 to 7 in the target sample. 18. The method of claim 17, wherein the sample is at least one selected from the group consisting of cell culture fluids, blood, saliva, sputum, cerebrospinal fluid, urine feces, and combinations thereof.

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