Synthesis and Application of a Fluorescent Probe for Visual Imaging and Online Monitoring of Escherichia coli Carrying Clb Toxin

Abstract

The present invention discloses the synthesis of a fluorescent probe for detecting Escherichia coli carrying Clb toxin, and an application method for visual imaging and long-term online monitoring of Escherichia coli carrying Clb toxin. The chemical structural formula of the fluorescent probe is shown as follows:. The present invention patent designs and constructs a fluorescent probe for detecting Escherichia coli carrying Clb toxin. The probe can achieve highly selective and sensitive fluorescence turn-on detection of Escherichia coli carrying Clb toxin ( λ ex / λ em = 400 / 520 nm); it can perform visual imaging analysis on Escherichia coli carrying Clb toxin, and at the same time can achieve long-term online monitoring of Escherichia coli carrying Clb toxin. This result provides a solid foundation for further interpreting the role of Escherichia coli carrying Clb toxin in colorectal cancer, and has great application prospects in technical fields such as analytical chemistry, life science, and biomedicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting Escherichia coli carrying Clb toxin, a synthesis method of the probe, visual imaging, and long-term on-line monitoring and analysis. Background Art

[0002] There is a genotoxin called Colibactin (Clb) in Escherichia coli subtype B2, which can enter intestinal epithelial cells, damage intracellular DNA, cause genetic damage, and increase the incidence of colorectal cancer. However, due to the complex structure of Clb toxin, there is currently no way to purify and determine its exact structure. Research shows that Clb toxin is encoded and synthesized by the clb gene cluster, and there is a key coenzyme (ClbP) involved in the biosynthesis of Clb toxin, which can catalyze the hydrolysis of the inactive Precolibactin precursor compound to produce the biologically active substance Clb toxin; compared with Colibactin, ClbP can exist stably ( Science . 2006, 313, 848-851; Science . 2012, 338, 52-53; Science . 2019,363, 7785; Nat. Microbiol . 2016, 1, 15009; Nat. Prod. Rep . 2015, 32, 1534-1540; Nat. Chem. Biol . 2017, 13, 1059-1061; J. mol. Biol. 2012, 424, 203-214;).

[0003] Currently, chromatography is mainly used for quantitative analysis of N N-myristoyl-D-asparagine residue ( N N-myr-Asn) generated during the synthesis of the biologically active substance Clb toxin, so as to indirectly detect Clb toxin. However, this method is complex in operation, time-consuming, and requires sample pretreatment; in addition, current research on ClbP mainly focuses on the detection and analysis part, and there is very little imaging research on ClbP and Escherichia coli carrying Clb toxin. Therefore, it is of great significance to develop a detection method that can quickly detect Clb toxin and realize on-line and visual analysis of Clb toxin, and further explore the pathway between it and colorectal cancer. Summary of the Invention

[0004] In view of the above situation, to overcome some of the deficiencies of the prior art, the purpose of the present invention is to provide a fluorescent probe with good selectivity, high sensitivity, non-invasiveness, and capable of online monitoring and visual imaging of ClbP, which can achieve the detection of Escherichia coli carrying Clb toxin, and the long-term online monitoring imaging of Escherichia coli carrying Clb toxin, and has important significance for the study of the mechanism of action of Clb toxin and colorectal cancer.

[0005] The specific technical solution adopted by the present invention to solve the problem is the synthesis and imaging analysis of a fluorescent probe for the detection of Escherichia coli carrying Clb toxin, and the chemical structural formula of the probe is as follows:

[0006] The synthesis of a fluorescent probe for the detection of Escherichia coli carrying Clb toxin is characterized in that the preparation method of the fluorescent probe includes the following steps.

[0007] Step: Synthesize (R)-N 1 -(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)-2-tetradecanamidosuccinimide

[0008] . Add an appropriate amount of 4-phenyl-2-(trifluoromethyl)quinolin-7-amine, N -Boc- D -asparagine, HATU and DIPEA to a dichloromethane solution, and react at room temperature for 2 - 10 h; after the reaction is complete, quench with water, extract with ethyl acetate, rotary evaporate the solvent, and purify by column chromatography to obtain a gray solid;

[0009] . Add an appropriate amount of the obtained medium gray solid to dichloromethane, dropwise add an appropriate amount of trifluoroacetic acid, and react at room temperature for 0.5 - 10 h; neutralize, extract with ethyl acetate, rotary evaporate the solvent, and quickly purify by column chromatography to obtain an off-white solid;

[0010] . Add an appropriate amount of the obtained off-white solid, myristic acid and HATU to pyridine, and react at room temperature for 1 - 10 h; neutralize, extract with ethyl acetate, rotary evaporate the solvent, and purify by column chromatography to obtain a white solid;

[0011] . Add an appropriate amount of the obtained white solid to concentrated hydrochloric acid, and react at 20 - 80 °C for 1 - 10 h; neutralize, extract with ethyl acetate, rotary evaporate the solvent, and recrystallize to obtain the probe (R)-N 1 -(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)-2-tetradecanamidosuccinimide.

[0012] The present invention also provides an application capable of achieving fluorescence-on response detection for Escherichia coli carrying Clb toxin.

[0013] The present invention also provides an application for long-term online monitoring and imaging analysis of Escherichia coli carrying Clb toxin. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 . 1H NMR spectrum of the fluorescent probe of the present invention.

[0015] Figure 2 . In the system of N,N-dimethylformamide and HEPES buffer (volume ratio 3:7), the change of the fluorescence emission spectrum of the fluorescent probe of the present invention with the volume of Escherichia coli-containing bacterial solution. The abscissa is the wavelength and the ordinate is the fluorescence intensity.

[0016] Figure 3 . Visualization of Escherichia coli containing ClbP by the fluorescent probe of the present invention.

[0017] Figure 4 . Long-term online monitoring of Escherichia coli containing ClbP by the fluorescent probe of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will be further described in conjunction with the following formula.

[0019] The synthesis route of the fluorescent probe of the present invention is shown in the following formula.

[0020]

[0021] Example 1. Synthesis of tert-butyl (R)-(3-cyano-1-oxo-1-((4-phenyl-2-(trifluoromethyl)quinolin-7-yl)amino)propan-2-yl)carbamate

[0022] . N -Boc- D- Asparagine (23.0 mg, 99.0 μmol), 4-phenyl-2-(trifluoromethyl)quinolin-7-amine (25.0 mg, 83.0 μmol), 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (76.0 mg, 200.0 μmol) and N,N-diisopropylethylamine (DIPEA) (14.5 μL, 200.0 μmol) were reacted in dichloromethane solution at room temperature for 7 h. Then, saturated sodium bicarbonate solution (10 mL) was added to quench the reaction. The mixture was extracted with ethyl acetate (10 mL×3). The combined organic phases were dried over anhydrous Na2SO4, the solvent was evaporated, and the crude product was obtained. After purification by column chromatography, 25.0 mg of tert-butyl (R)-(3-cyano-1-oxo-1-((4-phenyl-2-(trifluoromethyl)quinolin-7-yl)amino)propan-2-yl)carbamate was obtained with a yield of 61.9%.

[0023] Example 2. Synthesis of (R)-2-amino-3-cyano-N-(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)propanamide

[0024] Tert-butyl (R)-(3-cyano-1-oxo-1-((4-phenyl-2-(trifluoromethyl)quinolin-7-yl)amino)propan-2-yl)carbamate (100 mg, 206 μmol) was dissolved in 4.0 mL of dichloromethane. 2.0 mL of trifluoroacetic acid was slowly added dropwise. After reacting at room temperature for 0.5 h, the reaction was neutralized with saturated sodium bicarbonate solution. The mixture was extracted with ethyl acetate (20 mL×3), dried over anhydrous Na2SO4, the solvent was evaporated, and the product was purified by flash column chromatography (completed within 4 h) to obtain 25.0 mg of (R)-2-amino-3-cyano-N-(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)propanamide with a yield of 31.6%.

[0025] Example 3. Synthesis of (R)-N-(3-cyano-1-oxo-1-((4-phenyl-2-(trifluoromethyl)quinolin-7-yl)amino)propan-2-yl)tetradecanamide

[0026] .(R)-2-Amino-3-cyano-N-(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)propanamide (100.0 mg, 250.0 μmol), myristic acid (63.0 mg, 276.0 μmol) and HATU (210.0 mg, 552.0 μmol) were dissolved in 5 mL of pyridine, reacted at room temperature for 3 h, neutralized with dilute hydrochloric acid, extracted with ethyl acetate (20 mL×3), dried over anhydrous Na2SO4, the solvent was evaporated, and purified by column chromatography to obtain 95 mg of (R)-N-(3-cyano-1-oxo-1-((4-phenyl-2-(trifluoromethyl)quinolin-7-yl)amino)propan-2-yl)tetradecanamide, with a yield of 24.6%.

[0027] Example 4. Synthesis of (R)-N 1 -(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)-2-tetradecanamidosuccinamide

[0028] .(R)-N-(3-cyano-1-oxo-1-((4-phenyl-2-(trifluoromethyl)quinolin-7-yl)amino)propan-2-yl)tetradecanamide (200.0 mg, 337.0 μmol) was dissolved in 5.0 mL of concentrated hydrochloric acid, reacted at 45 °C for 2 h, neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate (20 mL×3), dried over anhydrous Na2SO4, the solvent was evaporated, and recrystallized from ethyl acetate to obtain 50.0 mg of the probe (R)-N 1 -(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)-2-tetradecanamidosuccinamide, with a yield of 24.3%.

[0029] Example 5. Detection of Escherichia coli solution containing ClbP by fluorescence probe

[0030] Spectral property experiment of the fluorescence probe of the present invention for detecting Escherichia coli solution containing ClbP: The probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1 mM, and the concentration of Escherichia coli containing ClbP was prepared with PBS at OD 600It is 0.3. The specific test method is as follows: Take 10 μL of 1 mM probe solution, 590 μL of analytical pure DMF, the required amount of bacterial solution and the required amount of HEPES buffer solution in a 2 mL sample tube. All test samples maintain a volume ratio of organic phase to aqueous phase of 3:7 (the total volume of each test sample is 2 mL). For example, when preparing a sample: Take 10 μL of 1 mM probe solution, 590 μL of analytical pure DMF, 100 μL of bacterial solution, and 1300 μL of HEPES buffer solution in a 2 mL sample tube. After shaking and incubating at 37 °C for 2 h, measure its fluorescence emission intensity at an excitation wavelength of 400 nm. This probe realizes a fluorescence turn-on response to the Escherichia coli solution containing ClbP, has good water solubility, a relatively fast response speed, and can rapidly and highly selectively detect the Escherichia coli solution containing ClbP.

[0031] Example 6. Visualization imaging analysis of a fluorescence probe for Escherichia coli containing ClbP

[0032] The imaging analysis of Escherichia coli containing ClbP is carried out as follows: Place the cultured Transetta-pGEX4T-1-ClbP Escherichia coli in LB medium. After culturing at 37 °C for 24 h, add an appropriate amount of the probe (10 μM) and co-incubate for 40 minutes, then observe under a confocal fluorescence microscope and perform fluorescence imaging in the green fluorescence channel. Figure 3 It can be seen that there are obvious green fluorescence signals in the Escherichia coli co-incubated with the probe, indicating that the fluorescence probe described in the present invention has successfully realized the visualization imaging analysis of Escherichia coli containing ClbP.

[0033] Example 7. Long-term online monitoring imaging analysis of a fluorescence probe for Escherichia coli containing ClbP

[0034] The implementation method of long-term online monitoring imaging analysis is as follows: Place the cultured Transetta-pGEX4T-1-ClbP Escherichia coli in LB medium. After culturing at 37 °C for 24 h, add an appropriate amount of the probe (10 μM) and co-incubate, and place it under confocal imaging for online monitoring. The results show that no green fluorescence signal was observed within the first 25 min; obvious green fluorescence signals appeared starting from the 30th min; at 40 min and 50 min, they still existed in the Escherichia coli and maintained obvious green fluorescence signals.

[0035] The synthesis of the fluorescent probe described in the present invention and its application in detecting Escherichia coli solution containing ClbP have developed an efficient, simple and low-cost application for detecting Escherichia coli solution containing ClbP; at the same time, it has also realized the on-line detection and long-time continuous imaging analysis of Escherichia coli containing ClbP, providing a solid foundation for exploring the mechanism of action between Clb toxin and colorectal cancer. It has great practical application value in the fields of biochemistry, analytical detection, etc. Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After those skilled in the art have read the above content, various modifications and substitutions of the present invention will be obvious. Therefore, the application methods of fluorescent probes for detecting and imaging analysis of Escherichia coli carrying Clb toxin with similar technical features described herein all fall within the protection scope of this patent.

Claims

1. Use of a fluorescent probe in the preparation of a visualization imaging reagent for on-line monitoring of Escherichia coli carrying Clb toxin, characterized in that, The chemical structural formula of the fluorescent probe is as follows: 。 2. The application of the fluorescent probe according to claim 1, characterized in that, The synthesis method of the fluorescent probe comprises the following steps: Step: Synthesis of (R)-N 1 -(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)-2-tetradecanamidosuccinamide I. Add an appropriate amount of 4-phenyl-2-(trifluoromethyl)quinolin-7-amine, N -Boc- D -asparagine, 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and N,N-diisopropylethylamine (DIPEA) to a dichloromethane solution, and react at room temperature for 2 - 10 h; after the reaction is complete, quench with water, extract with ethyl acetate, evaporate the solvent under reduced pressure, and purify by column chromatography to obtain a gray solid; II. Dissolve the medium gray solid obtained in I in dichloromethane, add an appropriate amount of trifluoroacetic acid dropwise, and react at room temperature for 0.5 - 10 h; neutralize, extract with ethyl acetate, spin-dry the solvent, and purify by flash column chromatography to obtain an off-white solid; III. Dissolve the appropriate amount of off-white solid obtained in II, myristic acid, and HATU in pyridine, and react at room temperature for 1 - 10 h; neutralize, extract with ethyl acetate, spin-dry the solvent, and purify by column chromatography to obtain a white solid; IV. Dissolve the appropriate amount of the white solid obtained in III in concentrated hydrochloric acid, and react at 20 - 80 °C for 1 - 10 h; neutralize, extract with ethyl acetate, rotary evaporate the solvent, and recrystallize to obtain the probe (R)-N 1 -(4-phenyl-2-(trifluoromethyl)quinolin-7-yl)-2-tetradecanamidosuccinimide.

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