KR20100127042A - Fluorescein derivatives having selectivity for cyanide and method for in vivo monitoring pseudomonas aeruginosa using the same - Google Patents

Abstract

PURPOSE: A fluorescence sensor containing fluorescein derivative having cyanide ion selectivity and a method for detecting Pseudomonas aeruginosa are provided to detect cyanide ion in vivo without dissection. CONSTITUTION: A fluorescence sensor for detecting Pseudomonas aeruginosa contains a compound of chemical formula 1. Pseudomonas aeruginosa detected by reacting the compound of chemical formula 1 and cyanide ion. The reaction is performed with saliva, sweat, blood, secreted liquid or tissue.

Description

Fluorescence sensor comprising fluorescein derivative having cyanide ion selectivity and detection method for Pseudomonas aeruginosa using the same {Pluorescein derivatives having selectivity for cyanide and method for in vivo monitoring Pseudomonas aeruginosa using the same}

The present invention relates to a fluorescent sensor comprising a fluorescein derivative having cyanide ion selectivity and a method for detecting Pseudomonas aeruginosa using the same, and more particularly, to a fluorescein derivative which selectively binds to cyanide ion and exhibits a green fluorescence enhancement effect. A fluorescent sensor comprising a fluorescein derivative having a cyanide ion selectivity capable of quantitatively or qualitatively analyzing the extent of Pseudomonas aeruginosa infection by detecting in real time cyanide ions resulting from Pseudomonas aeruginosa infection in real time without dissection It relates to a method for detecting Pseudomonas aeruginosa using the same.

Pseudomonas aeruginosa is a pathogenic bacterium that causes refractory infections in patients with chronic or sepsis and cystic fibrosis. If the body's defenses are weakened by surgery or burns, it can lead to death due to chronic infections or sepsis, and especially in patients with cystic fibrosis. In recent years, urethral infections and corneal ulcers of Pseudomonas aeruginosa occur, which can lead to blindness. The risk of Pseudomonas aeruginosa infection is resistance to existing antibiotics, which is associated with cellular mechanisms in which Pseudomonas aeruginosa can grow under limited oxygen supply conditions in vivo. In addition, Pseudomonas aeruginosa produces and releases large amounts of cyanide ions along with various toxic substances in these microenvironments. Cyanide disrupts energy metabolism in the cell, causing cell death, paralyzing the body's oxygen-carrying function, and beginning to make tissues housework.

Cyanide is one of the fastest reactants, particularly harmful to animals, causing vomiting, unconsciousness, and death. Blood cyanide concentrations resulting in death from the toxicity of cyanide are about 23-26 μM.

Diagnosis of Pseudomonas aeruginosa infection is carried out by cultivating and classifying bacteria that extract body fluids such as saliva from patients, or detecting cyanide ions in body fluids (Doring, G., Conway, SP, Heijerman, HGM, Hodson). , ME, Hoiby, N., Smyth, A. & Touw, DJ Eur.Respir.J . 2000, 16, 749-767; Ryall, B., Davies, JC, Wilson, R., Shoemark, A. & Williams , HD Eur.Respir . J. 2008, 32, 740-747). However, these methods only provide information on Pseudomonas aeruginosa infection, but do not provide information on the extent of infection or the site of infection. Furthermore, in view of the toxicity of cyanide ions, there is no information on the concentration of cyanide ions or their toxic effects in vivo resulting from Pseudomonas aeruginosa infection.

It is an object of the present invention to provide a fluorescence sensor capable of detecting Pseudomonas aeruginosa using a fluorescein derivative capable of selectively detecting cyanide ions.

Another object of the present invention is a method for detecting Pseudomonas aeruginosa which can detect cyanide ions generated when Pseudomonas aeruginosa is infected with a living organism using the fluorescein derivative in a living organism and detect whether Pseudomonas aeruginosa is infected, the extent of infection, or the site of infection. To provide.

In order to achieve the above object, the present invention provides a fluorescent sensor for detecting Pseudomonas aeruginosa comprising a compound represented by the following formula (1):

[Formula 1]

Where

R represents hydrogen or aldehyde.

The present invention also provides a method for detecting Pseudomonas aeruginosa comprising reacting a compound represented by the following Chemical Formula 1 with a cyanide ion:

[Formula 1]

Where

R represents hydrogen or aldehyde.

The detection of Pseudomonas aeruginosa is

Culturing the compound represented by Chemical Formula 1 with an organism having motility and sensitivity to chemicals; And

Preferably, the step of measuring the fluorescence intensity generated in the living organism.

In addition, the detection of Pseudomonas aeruginosa may include the step of measuring the fluorescence intensity in the living organism by administering the compound represented by the formula (1).

In addition, the detection of Pseudomonas aeruginosa may include the step of measuring the fluorescence intensity in the tissue sections by treating the tissue fragments of the living organism with the compound represented by Formula 1 in an aqueous solution.

Since the fluorescein derivative of the present invention reacts with cyanide ions in 100% aqueous solution to emit green fluorescence, the fluorescein derivative may be used as an "off-on" type fluorescent sensor. In addition, the present invention can be quantitatively or qualitatively analyzed whether or not the infection of Pseudomonas aeruginosa, the degree of infection, the site of infection, etc. by selectively reacting with cyanide ions generated during Pseudomonas aeruginosa infection using the fluorescein derivative.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a fluorescent sensor for detecting Pseudomonas aeruginosa comprising a compound represented by Formula 1 below:

[Formula 1]

Where

R represents hydrogen or aldehyde.

In the case of Pseudomonas aeruginosa, cyanide ions are generated when the organism is infected, and thus, Pseudomonas aeruginosa may be detected by using a fluorescent sensor having selectivity for cyanide ions.

According to the mechanism of Scheme 1 or 2 below, the fluorescein derivative of Formula 1 in an aqueous solution may react with cyanide ions to generate a compound of Formula 3 that emits green fluorescence as a result of proton migration through the compound of Formula 2. The selectivity for cyanide ions of the fluorescein derivative may be due to the nucleophilicity of the cyanide in aqueous solution.

Scheme 1

Scheme 2

Fluorescein mono or dialdehyde of formula 1 according to the present invention exhibits selective chelate amplification fluorescence effect at excitation wavelength of 520 nm depending on CN ⁻ addition in 100% aqueous solution, thus turning off the fluorescein derivative. Cyanide ions can be detected using a fluorescent probe of the -On "type.

According to one embodiment, no fluorescence change is shown for anions such as NO ₃ ⁻ , but when cyanide ions are reacted with fluorescein mono or dialdehyde of the general formula (1), a large change in concentration is dependent.

In addition, detection of cyanide ions can also be measured through colorimetric change. More specifically, fluorescein mono or dialdehyde in aqueous solution exhibits a color change in which the color saturation increases with increasing concentration of reacted cyanide ions.

Therefore, when the body fluid of a living organism infected with Pseudomonas aeruginosa reacts with fluorescein mono or dialdehyde of Chemical Formula 1, it reacts with cyanide ions generated in the body fluid to show a color change, thereby detecting Pseudomonas aeruginosa.

The method for preparing the compound of Formula 1 of the present invention can be synthesized according to a known method (W. Wang et al. , J. Am. Chem. Soc . 2005, 127, 15949), but is not particularly limited thereto.

According to one embodiment, the fluorescein dialdehyde of Formula 1 is a mixture of fluorescein, chloroform, methanol and 15-crown-5, while maintaining a constant temperature, more specifically 40 to 70 ℃ Carefully add 50% sodium hydroxide solution. The mixture is reacted for 3 to 7 hours in the above temperature range. After the reaction is cooled, the mixture is acidified by addition of a strong acid. The precipitates can be collected, dried and purified to give the fluorescein dialdehyde of formula (1) of the present invention.

The present invention also relates to a method for detecting Pseudomonas aeruginosa comprising reacting a compound represented by the following Chemical Formula 1 with a cyanide ion:

[Formula 1]

Where

R represents hydrogen or aldehyde.

Fluorescein mono or dialdehyde of the formula (1) of the present invention is a "OFF-ON" type sensor that can detect cyanide ions in an aqueous solution state when the cyanide ion is detected in a low fluorescence intensity (OFF) fluorescence The sensor is a type of sensor that senses the fact that the strength of the sensor becomes very large (ON). Since the living body infected with Pseudomonas aeruginosa generates cyanide ions, the fluorescein mono or dialdehyde of the formula (1) can determine whether or not the infection of Pseudomonas aeruginosa body fluid It can be diagnosed in real time on site without separate analysis equipment.

Examples of the body fluids include saliva, sweat, secretion liquids flowing out of blood or living organisms, extracts of biological tissues, and the like.

In addition, the detection of Pseudomonas aeruginosa by the fluorescein mono or dialdehyde of the formula (1)

Culturing the compound represented by Chemical Formula 1 with an organism having motility and sensitivity to chemicals; And

It may include the step of measuring the fluorescence intensity generated in the living being.

With life having a sensitivity to the mobility and chemical may use a ciliate (Ciliate), paramecium (Paramecium), stenter (Stentor), flagellate (flagellate), nematode (Nematode), or bacteria and the like. More specifically, nematodes such as Caenorhabditis elegans may be used.

The fluorescence intensity may be confirmed by real time observation with a stereoscopic mirror, but is not particularly limited thereto.

In addition, the detection of Pseudomonas aeruginosa may be to measure the fluorescence intensity in the organism by administering the compound represented by the formula (1) to the organism.

More specifically, the living body may be a mouse.

According to one embodiment, when the fluorescein mono or dialdehyde of the formula (1) is administered directly to the lungs of Pseudomonas aeruginosa infected mice, the fluorescence of the mouse shows strong fluorescence.

According to another embodiment, when nasal injection of fluorescein mono or dialdehyde of the formula (1) to the mouse, strong fluorescence is observed in the bronchus, lungs and the like.

Formulation form of the fluorescein mono or dialdehyde of the formula (1) to be administered to the living body is preferably a liquid, but is not particularly limited thereto.

In addition, the detection of Pseudomonas aeruginosa may be to measure the fluorescence intensity in the tissue sections by treating the tissue fragments of the living organism with the compound represented by the formula (1) in an aqueous solution.

According to one embodiment, when the fluorescein mono or dialdehyde of the formula (1) is treated in the lung frozen sections of mice infected with K. aeruginosa in aqueous solution, strong fluorescence is observed in the frozen sections.

The aqueous solution may be used a conventional buffer solution is not particularly limited.

From the above results, the fluorescein mono or dialdehyde of the formula (1) of the present invention not only exhibits a strong fluorescence or color change in response to body fluids, tissues, etc. obtained from an organism infected with Pseudomonas aeruginosa, but also strong fluorescence even when administered to the organism itself Since the infection of Pseudomonas aeruginosa, the degree of infection, the site of infection, etc. can be quantitative or qualitative analysis.

Hereinafter, the present invention will be described in more detail through examples according to the present invention and comparative examples not according to the present invention, but the scope of the present invention is not limited by the examples given below.

Example 1 Preparation of Fluorescein Dialdehyde of Formula 1

Fluorescein dialdehyde of Formula 1 may be synthesized according to a known method (W. Wang et al. , J. Am. Chem. Soc . 2005, 127, 15949).

Specifically, fluorescein (4 g, 12 mmol), 10 mL chloroform, 6 mL methanol and 0.06 g of 15-crown-5 were added to a 100 mL flask. 20 g of 50% sodium hydroxide solution was carefully added while maintaining the temperature of the flask at 50 ° C. The mixture was reacted at a temperature of 50 ° C. for 5 hours, then cooled after the reaction, and the solution was acidified with 10 M H ₂ SO ₄ . The precipitates were collected, dried in vacuo and chromatographed using silica gel (5:95 = EtOAc: DCM) to give 142 mg of white fluorescein dialdehyde (yield 3.0%).

¹ H NMR (CDCl ₃ , 250 MHz) δ (ppm): 6.66 (2H, d, J = 9.0 Hz), 6.86 (2H, d, J = 9.0 HZ), 7.11 (2H, d, J = 7.5 Hz) , 7.70-7.59 (2H, m), 8.99 (2H, d, J = 7.5 Hz), 10.59 (2H, s), 12.06 (2H, s).

¹³ C NMR (CDCl ₃ , 62.5 MHz) δ (ppm): 191.80, 168.69, 164.67, 151.87, 151.67, 136.98, 145.66, 130.53, 126.51, 125.55, 123.69, 115.21, 109.48, 109.00, 80.61).

Experimental Example 1 Determination of Selectivity of Cyanide Anion of Fluorescein Dialdehyde of Formula 1

^{CN -, F -, Cl -} , Br -, I -, H 2 PO 4 -, ClO 4 -, NO 3 - HSO 4 - CH 3 COO - nitrile various anions such as acetonitrile -HEPES (1: 2, v / v, 0.01 M, pH 7.4 HEPES) and the fluorescence change generated by reaction with 3 μM fluorescein dialdehyde is measured and shown in FIG. 1 (excitation wavelength: 485 nm, excitation and emission slit width: 1.5 nm).

As shown in FIG. 1, only cyanide ions among the anions reacted with fluorescein aldehyde react with fluorescein aldehyde to show a large change in fluorescence.

Experimental Example 2 Measurement of Fluorescence Intensity According to the Concentration of Cyanide Ion

Fluorescence intensity generated by reacting various concentrations of cyanide with 3 μM of fluorescein dialdehyde in aqueous solution of CH ₃ CN-H ₂ O (1: 2, v / v, 0.01 M, pH 7.4) at room temperature Measured using a quantitative meter is shown in FIG. 2 (excitation wavelength: 485 nm, excitation & light emission slit: 1.5 nm).

As shown in Figure 2, the fluorescence intensity was found to increase as the concentration of cyanide ions reacted with fluorescein aldehyde increased, indicating a saturation point near the wavelength of 520 nm.

Example 2 Detection of Cyanide Ions in Pretty Nematodes Infected with Pseudomonas aeruginosa

C. elegans, a bacterial feeding nematode, was used as an in vivo model to identify cyanide ions caused by Pseudomonas aeruginosa infection with fluorescein dialdehyde. Pretty nematodes have already been frequently used as toxic experimental models of Pseudomonas aeruginosa (Mahajan-Miklos, S., Tan, MW, Rahme, LG & Ausubel, FM Cell 1999, 96, 47-56). Specifically, C. elegans wild type strain N2 was obtained from Caenorhabditis Genetics Center (University of Minnesota, USA). Nematodes were grown on NGM (nematode growth medium) agar plates (0.3% NaCl, 0.25% peptone, 2.5% agar, 5 mg / mL cholesterol, 1 mM CaCl ₂ , 1 mM MgSO) incubated with Escherichia coli OP50 at 25 ° C. ₄ , 25 mM KH ₂ PO ₄ ) were co-cultured according to the alkali-bleaching method. Collect nematodes maintained for one day on E. coli-excluded media for Pseudomonas aeruginosa infection and maintain them on NGM agar plates cultured with Pseudomonas aeruginosa for 4 hours, and then collect nematodes (0.3% NaCl, 5 mg / mL cholesterol). , 1 mM CaCl ₂ , 1 mM MgSO ₄ , 25 mM KH ₂ PO ₄ ). The nematodes were washed three times with NGM buffer and then placed on glass slides with Faramount ™ aqueous mounting medium (Dako, Glostrup, Denmark) in NGM buffer for 20 minutes. Fluorescence photographs of nematodes were magnified 200 times using a confocal laser scanning microscope (LSM510, Carl Zeiss). FIG. 3 is a fluorescence picture of a nematode not exposed to fluorescein dialdehyde (Control), exposed (CN sen.) And to the sensor after Pseudomonas aeruginosa infection (PA14).

As shown in FIG. 3, the unexposed (Control) or exposed (CN sen.) Nematodes did not show any fluorescence, whereas the nematode exposed to the sensor (PA14) after Pseudomonas aeruginosa infection was strong. Fluorescence was shown. It can be seen that fluorescein dialdehyde is very suitable for the in vivo image of cyanide ions generated from Pseudomonas aeruginosa.

Example 3 Detection of Cyanide Ions in Laboratory Mice Infected with Pseudomonas Aeruginosa

In addition to nematode models, experimental mice (BALB / c nude mice weighing 17-19 g, 6 weeks old) were used to confirm cyanide ion detection caused by Pseudomonas aeruginosa infection with fluorescein dialdehyde. PBS buffer solution (pH 7.4) was mixed with 2.5% tryptic soy agar (Sigma-Aldrich) and 50 mL heavy mineral oil (Sigma-Aldrich) at 50 ° C in Pseudomonas aeruginosa (OD0.9) grown in 5 mL medium for Pseudomonas aeruginosa infection. Agar beads containing Pseudomonas aeruginosa at a concentration of 9 × 10 ⁷ CFU / mL were prepared by washing three times. The method of infection was 50 μl (4.5) using a 24 G catheter (BD, CA, USA) with the tongue pulled out of the oral cavity in mice anesthetized with anesthetic (zoletil50; Virbac, Carros, France) injection by injection into the respiratory tract. 10 ⁶ CFU) Pseudomonas aeruginosa aga beads were injected into the respiratory tract (intratracheal instillation). Eighteen hours after infection, the mice were anesthetized again, and 20 μl of 1 mM fluorescein dialdehyde was directly injected into the lung area using a 0.3 mL insulin injector, followed by fluorescence with a small animal fluorescence imaging device (Kodak, Image station IS2000MM). Was observed. 4 is a small animal fluorescence imaging device (Kodak, Image station) for the experimental mouse that is not injected with fluorescein dialdehyde (Control), injected (CN sen.) And the sensor after Pseudomonas aeruginosa infection (PA14) IS2000MM).

As shown in FIG. 4, mice that were not injected with fluorescein dialdehyde (Control) or mice that were injected (CN sen.) Showed no fluorescence, whereas mice injected with sensors after Pseudomonas aeruginosa infection (PA14) were lungs. Strong fluorescence was shown at the site. This suggests that fluorescein dialdehyde is well suited for in vivo imaging of cyanide ions produced by Pseudomonas aeruginosa in the lungs of rats.

For more practical application of fluorescein dialdehyde, a fluorescent sensor was injected through the nasal cavity. Mice infected with Pseudomonas aeruginosa were anesthetized after 18 hours as described above, and then 200 μl of 1 mM fluorescein aldehyde was injected intranasally using a 24 G catheter (BD, CA, USA). 5 is a small animal fluorescence imaging apparatus for fluorescein aldehyde injection (Control), nasal cavity injection (CN sen.) And a laboratory mouse injected with the nasal cavity after Pseudomonas aeruginosa infection (PA14) Picture taken with (Kodak, Image station IS2000MM).

As shown in FIG. 5, mice that did not receive fluorescein dialdehyde (Control) or mice that injected (CN sen.) Showed no fluorescence, whereas the sensor was injected through the nasal cavity after Pseudomonas aeruginosa infection (PA14). Mice showed fluorescence near the bronchus as well as strong fluorescence at the lung site. This suggests that even if fluorescein dialdehyde is injected into the nasal cavity, it can not only detect cyanide ions generated by Pseudomonas aeruginosa in the lungs of mice, but also provide information on other infected sites such as bronchus.

Example 4 Detection of Cyanide Ions in Frozen Sections of Mouse Lung Tissue Infected with Pseudomonas Aeruginosa

Lung frozen sections infected with Pseudomonas aeruginosa were used to confirm that fluorescein dialdehyde could also be used for histological methods. After 18 hours of sacrifice of Pseudomonas aeruginosa infected mice as described above, lungs were removed and a 20 mm thick frozen section was fixed on a glass slide. After fixing with acetone for 30 minutes and washing three times with PBS buffer solution, it was reacted with an antibody that binds to Pseudomonas aeruginosa at a concentration of 10 μg / mL for 1 hour (rabbit polyclonal antibody to Pseudomonas ; Abcam inc. Cambridge, MA, USA). After washing three times with PBS buffer solution, it was reacted with a secondary antibody (goat anti-rabbit IgG-TRITC; Sigma-Aldrich) bound with a red fluorescent dye at a concentration of 2 μg / mL for 30 minutes. After washing three times with PBS buffer solution, reacted with fluorescein dialdehyde at a concentration of 2 mM for 5 minutes, and then washed three times with PBS buffer solution, followed by Faramount ™ aqueous mounting medium (Dako, Glostrup, Denmark). Maintained at. Fluorescence images of frozen sections were magnified 400 times and observed using a confocal laser scanning microscope (LSM510, Carl Zeiss). FIG. 6 is a fluorescence image of frozen sections that did not react with fluorescein dialdehyde and antibodies (Control), reacted (CN sen.), And sensors and antibodies after Pseudomonas aeruginosa infection (PA14).

As shown in FIG. 6, no fluorescence was observed in the control (C) or reacted (CN sen.) Frozen sections that did not react with the fluorescein aldehyde or the antibody, whereas the fluorescein aldehyde or the antibody after Pseudomonas aeruginosa infection. Strong fluorescence was observed in frozen sections reacted with (PA14). It can be seen that fluorescein dialdehyde is well suited for histological methods as well as in vivo images of cyanide ions generated by Pseudomonas aeruginosa.

1 is a graph showing the fluorescence change generated by reacting with fluorescein dialdehyde by adding various anions in a CH ₃ CN-HEPES solution at room temperature.

Figure 2 is a graph measuring the fluorescence intensity generated by reacting various concentrations of cyanide and fluorescein dialdehyde in CH ₃ CN-H ₂ O solution at room temperature.

FIG. 3 is a fluorescence picture of a small nematode not exposed to fluorescein dialdehyde (Control), exposed (CN sen.) And exposed to sensors after Pseudomonas aeruginosa infection (PA14).

4 is a small animal fluorescence imaging device (Kodak, Image station) for the experimental mouse that is not injected with fluorescein dialdehyde (Control), injected (CN sen.) And the sensor after Pseudomonas aeruginosa infection (PA14) IS2000MM), the top of each picture is the image of the fluorescence intensity expressed in black and white contrast, and the picture below is the reconstructed color of the corresponding fluorescence (Kodak 1D Image Analysis Software; Kodak) .

5 is a small animal fluorescence imaging apparatus for fluorescein aldehyde injection (Control), nasal cavity injection (CN sen.) And a laboratory mouse injected with the nasal cavity after Pseudomonas aeruginosa infection (PA14) Photo taken with (Kodak, Image station IS2000MM), the upper one of each photograph is the image of the fluorescence intensity expressed in black and white contrast, and the lower one is the reconstructed color of the corresponding fluorescence (Kodak 1D Image Analysis Software; Kodak).

FIG. 6 is a fluorescence photograph of frozen sections that did not react with fluorescein dialdehyde and antibodies (Control), reacted (CN sen.) And reacted with sensors and antibodies (PA14) after Pseudomonas aeruginosa infection, each photograph. The upper picture is a fluorescence picture after the reaction with fluorescein dialdehyde, the lower picture is a fluorescence picture after the reaction with an antibody containing a red fluorescent substance and an antibody that binds Pseudomonas aeruginosa.

Claims (8)

Fluorescence sensor for detecting Pseudomonas aeruginosa comprising a compound represented by Formula 1 below: [Formula 1]

Where R represents hydrogen or aldehyde. Pseudomonas aeruginosa detection method comprising the step of reacting the compound represented by the formula (1) with cyanide ions: [Formula 1]

Where R represents hydrogen or aldehyde. The method of claim 2, Reaction is a method for detecting Pseudomonas aeruginosa carried out in at least one aqueous solution selected from the group consisting of secretion fluid leaked from saliva, sweat, blood or living organisms and extracts of biological tissues. The method of claim 2, wherein the detection of Pseudomonas aeruginosa is Culturing the compound represented by Chemical Formula 1 with an organism having motility and sensitivity to chemicals; And Pseudomonas aeruginosa detection method comprising the step of measuring the fluorescence intensity generated in the living organism. The method of claim 4, wherein Organism having sensitivity to movement, and the chemical is ciliate (Ciliate), paramecium (Paramecium), stenter (Stentor), flagellate (flagellate), nematode (Nematode) and any one of Pseudomonas aeruginosa selected bacteria from the group consisting of Detection method. The method of claim 2, Detecting Pseudomonas aeruginosa is a method for detecting Pseudomonas aeruginosa by measuring the fluorescence intensity in the organism by administering the compound represented by Chemical Formula 1 to the organism. The method of claim 6, The living organism is a mouse Pseudomonas aeruginosa detection method. The method of claim 2, The detection of Pseudomonas aeruginosa is a method of detecting Pseudomonas aeruginosa in which the compound represented by Chemical Formula 1 in an aqueous solution is treated with a tissue fragment of a living body to measure the fluorescence intensity in the tissue fragment.

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