CN115490635A - Synthesis and application of visualized imaging and online monitoring Clb toxin-carrying escherichia coli fluorescent probe - Google Patents

Synthesis and application of visualized imaging and online monitoring Clb toxin-carrying escherichia coli fluorescent probe Download PDF

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CN115490635A
CN115490635A CN202211142017.1A CN202211142017A CN115490635A CN 115490635 A CN115490635 A CN 115490635A CN 202211142017 A CN202211142017 A CN 202211142017A CN 115490635 A CN115490635 A CN 115490635A
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escherichia coli
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李海涛
赵奎成
程佩
陈盛游
张友玉
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Abstract

The invention discloses a synthesis method of a fluorescent probe for detecting Escherichia coli carrying Clb toxin, a visual imaging method and an application method of long-time online monitoring for Escherichia coli carrying Clb toxin, wherein the chemical structural formula of the fluorescent probe is as follows:
Figure 100004_DEST_PATH_IMAGE001
. The invention discloses a fluorescent probe for detecting Escherichia coli carrying Clb toxin. The probe can realize high-selectivity and high-sensitivity fluorescence-opening detection on Escherichia coli carrying Clb toxin (λ ex /λ em =400/520 nm); can be carried to each otherClb toxin Escherichia coli carries out visual imaging analysis, can carry out the long-time on-line monitoring to Clb toxin Escherichia coli simultaneously. The result provides a solid foundation for further explaining the research on the effect of the Clb toxin-carrying escherichia coli in colorectal cancer, and has great application prospects in the technical fields of analytical chemistry, life science, biological medicine and the like.

Description

Synthesis and application of visualized imaging and online monitoring Clb toxin-carrying escherichia coli fluorescent probe
Technical Field
The invention belongs to the technical field of analytical chemistry, and particularly relates to a fluorescent probe for detecting Escherichia coli carrying Clb toxin, a synthetic method of the probe, visual imaging and long-time online monitoring analysis.
Background
Escherichia coli B2 subtype has a genotoxin named Colibactin (Clb), and can enter intestinal epithelial cells, destroy intracellular DNA, cause genetic damage and increase the incidence rate of colorectal cancer. However, because of the complex structure of Clb toxin, there is no way to purify and determine its exact structure. Research shows that Clb toxin is coded and synthesized by clb gene cluster, wherein a key coenzyme (ClbP) exists, participates in Clb toxin biosynthesis, and can catalyze and hydrolyze biologically inactive Precolibactin precursor compounds to generate Clb toxin with biological activity; (ClbP can stably exist relative to Colibactin: (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；）。
At present, the biologically active substance Clb toxin is synthesized mainly by chromatographyN-myristoyl-D-asparagine residue (N-myr-Asn) to realize indirect detection of Clb toxin. However, the method is complex in operation, takes a long time, and requires pretreatment of the sample; in addition, at present, more researches on ClbP are focused on a detection analysis part, and imaging researches on ClbP and Escherichia coli carrying Clb toxin are few, so that the development of a detection method capable of rapidly detecting Clb toxin and realizing online and visual analysis on Clb toxin and further researching a path between the detection method and colorectal cancer are of great significance.
Disclosure of Invention
In view of the above circumstances, the present invention overcomes the shortcomings of some prior arts, and an object of the present invention is to provide a fluorescent probe with good selectivity, high sensitivity, and non-invasive property, and capable of online monitoring and visualization of ClbP, which can realize detection of escherichia coli carrying Clb toxin, and long-time online monitoring and imaging of escherichia coli carrying Clb toxin, and has important significance for research on the mechanism of action of Clb toxin and colorectal cancer.
The invention solves the problem by adopting the specific technical scheme that the synthesis and imaging analysis of the fluorescent probe for detecting the Escherichia coli carrying Clb toxin are carried out, and the chemical structural formula of the probe is as follows:
the synthesis of the fluorescent probe for detecting the Escherichia coli carrying Clb toxin is characterized in that the preparation method of the fluorescent probe comprises the following steps.
The method comprises the following steps: synthesis of (R) -N 1 - (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) -2-tetradecylamido-succinamide
Figure 100002_DEST_PATH_IMAGE001
The appropriate amount of 4-phenyl-2- (trifluoromethyl) quinolin-7-amine,N-Boc-Dadding asparagine, HATU and DIPEA into a dichloromethane solution, and reacting at room temperature for 2-10h; after the reaction is completed, adding water for quenching, extracting by ethyl acetate, spin-drying the solvent, and purifying by column chromatography to obtain a gray solid;
Figure DEST_PATH_IMAGE002
mixing the right amount
Figure 121960DEST_PATH_IMAGE001
Dissolving the obtained intermediate grey solid in dichloromethane, dropwise adding a proper amount of trifluoroacetic acid, and reacting at normal temperature for 0.5-10 h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and purifying by flash column chromatography to obtain off-white solid;
Figure DEST_PATH_IMAGE003
mixing the appropriate amount
Figure 101417DEST_PATH_IMAGE002
Dissolving the obtained off-white solid, myristic acid and HATU in pyridine, and reacting at room temperature for 1-10 h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and purifying by column chromatography to obtain a white solid;
Figure DEST_PATH_IMAGE004
mixing the right amount
Figure 694204DEST_PATH_IMAGE003
Dissolving the obtained white solid in concentrated hydrochloric acid, and reacting at 20-80 ℃ for 1-10 h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and recrystallizing to obtain the probe (R) -N 1 - (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) -2-tetradecylamido-succinamide.
The invention also provides application of the fluorescent turn-on response detection to the Escherichia coli carrying Clb toxin.
The invention also provides application of the long-time online monitoring imaging analysis on the Escherichia coli carrying the Clb toxin.
Drawings
FIG. 1 shows the NMR chart of the fluorescent probe of the present invention.
FIG. 2 shows that in a system of N, N-dimethylformamide and HEPES buffer solution (volume ratio of 3:7), fluorescence emission spectra of the fluorescent probe changes with the volume of Escherichia coli-containing bacteria liquid, the abscissa is wavelength, and the ordinate is fluorescence intensity.
FIG. 3 shows the visualization of ClbP-containing E.coli using the fluorescent probe of the present invention.
FIG. 4 shows that the fluorescent probe of the present invention can be used for long-term on-line monitoring of Escherichia coli containing ClbP.
Detailed Description
The invention is further illustrated in connection with the following figures.
The synthetic route of the fluorescent probe of the invention is shown in the following figure.
Figure DEST_PATH_IMAGE005
EXAMPLE 1 Synthesis of tert-butyl (R) - (3-cyano-1-oxo-1- ((4-phenyl-2- (trifluoromethyl) quinolin-7-yl) amino) propan-2-yl) carbamate
Figure 151730DEST_PATH_IMAGE001
．N-Boc-DAsparagine (23.0 mg, 99.0. Mu. Mol), 4-phenyl-2- (trifluoromethyl) quinolin-7-amine (25.0 mg, 83.0. Mu. Mol), 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) (76.0 mg, 200.0. Mu. Mol) and N, N-Diisopropylethylamine (DIPEA) (14.5. Mu.L, 200.0. Mu. Mol) in dichloromethane solution at room temperature for 7 h, after which saturated sodium bicarbonate solution (10 mL) is added to quench the reaction, ethyl acetate (10 mL. Times.3) is extracted, the organic phases are combined, anhydrous Na 2 SO 4 Drying, spin-drying the solvent to obtain a crude product, and purifying by column chromatography to obtain (R) - (3-cyano-1-oxo-1- ((4-phenyl-2- (trifluoromethyl) quinoline-7-yl) amino) propan-2-yl) carbamic acid tert-butyl ester 25.0 mg with the yield of 61.9%.
EXAMPLE 2 Synthesis of (R) -2-amino-3-cyano-N- (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) propionamide
Figure 966102DEST_PATH_IMAGE002
Tert-butyl (R) - (3-cyano-1-oxo-1- ((4-phenyl-2- (trifluoromethyl) quinolin-7-yl) amino) propan-2-yl) carbamate (100 mg, 206. Mu. Mol) was dissolved in 4.0 mL dichloromethane, 2.0 mL trifluoroacetic acid was slowly added dropwise, after reaction at room temperature of 0.5 h, the saturated sodium bicarbonate solution was neutralized, ethyl acetate (20 mL X3) was extracted, anhydrous Na 2 SO 4 Drying, spin-drying the solvent, and flash column chromatography purification (4 h internal excess) to obtain (R) -2-amino-3-cyano-N- (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) propionamide 25.0 mg with a yield of 31.6%.
EXAMPLE 3 Synthesis of (R) -N- (3-cyano-1-oxo-1- ((4-phenyl-2- (trifluoromethyl) quinolin-7-yl) amino) propan-2-yl) tetradecanamide
Figure 788565DEST_PATH_IMAGE003
(R) -2-amino-3-cyano-N- (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) propanamide (100.0 mg, 250.0. Mu. Mol), myristic acid (63.0 mg, 276.0. Mu. Mol) and HATU (210.0 mg, 552.0. Mu. Mol) were dissolved in 5mL pyridine, reacted at room temperature 3 h, dilute saltAcid neutralization, ethyl acetate (20 mL × 3) extraction, anhydrous Na 2 SO 4 Drying, spin-drying the solvent, and purifying by column chromatography to obtain (R) -N- (3-cyano-1-oxo-1- ((4-phenyl-2- (trifluoromethyl) quinoline-7-yl) amino) propan-2-yl) tetradecanamide 95 mg with a yield of 24.6%.
EXAMPLE 4 Synthesis of (R) -N 1 - (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) -2-tetradecylamido-succinamide
Figure 868647DEST_PATH_IMAGE004
(R) -N- (3-cyano-1-oxo-1- ((4-phenyl-2- (trifluoromethyl) quinolin-7-yl) amino) propan-2-yl) tetradecanamide (200.0 mg, 337.0. Mu. Mol) was dissolved in 5.0 mL concentrated hydrochloric acid, reacted for 2h at 45 ℃, neutralized with saturated sodium bicarbonate solution, extracted with ethyl acetate (20 mL X3), anhydrous Na 2 SO 4 Drying, spin-drying the solvent, and recrystallizing with ethyl acetate to obtain probe (R) -N 1 - (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) -2-tetradecylamido-succinamide 50.0 mg in 24.3% yield.
Example 5 detection of ClbP-containing E.coli liquid by fluorescent Probe
The fluorescence probe of the invention is used for detecting the spectrum property experiment of the ClbP-containing escherichia coli liquid: the probes were dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution having a concentration of 1 mM, and then PBS was added to prepare a ClbP-containing Escherichia coli OD 600 Is 0.3, and the specific test mode is as follows: 10. Mu.L of 1 mM probe solution, 590. Mu.L of analytically pure DMF, the required amount of bacterial suspension and the required amount of HEPES buffer solution were taken in a sample tube of 2 mL, all samples were kept with a volume ratio of organic phase to aqueous phase of 3:7 (total volume of each test sample is 2 mL). For example, the sample was formulated as follows: 10 μ L of 1 mM probe solution, 590 μ L of analytically pure DMF, 100 μ L of bacterial liquid, 1300 μ L of HEPES buffer solution were incubated in a 2 mL sample tube at 37 ℃ with shaking and shaking to incubate 2h, and then the fluorescence emission intensity was measured at an excitation wavelength of 400 nm. The probe realizes fluorescent open-type response to the ClbP-containing escherichia coli bacterial liquid, has good water solubility and high response speed, and can realize quick and high selection of the ClbP-containing escherichia coli bacterial liquidAnd (5) detecting the blood sample.
Example 6 visual imaging analysis of ClbP-containing Escherichia coli by fluorescent Probe
Coli imaging assays containing ClbP were performed as follows: the cultured Escherichia coli Transetta-pGEX4T-1-ClbP is placed in an LB culture medium, cultured at 37 ℃ for 24 h, added with a proper amount of probe (10 mu M) for co-incubation for 40 minutes, observed under a confocal fluorescence microscope, and subjected to fluorescence imaging in a green fluorescence channel. As can be seen from FIG. 3, the Escherichia coli co-incubated with the probe has an obvious green fluorescence signal, which indicates that the fluorescent probe successfully realizes the visual imaging analysis of the Escherichia coli containing ClbP.
Example 7 Long-time on-line monitoring and imaging analysis of ClbP-containing Escherichia coli by fluorescent Probe
The long-time online monitoring imaging analysis implementation mode is as follows: placing the cultured Transetta-pGEX4T-1-ClbP escherichia coli in an LB (lysogeny broth) culture medium, culturing 24 h at 37 ℃, adding a proper amount of probe (10 mu M) for co-incubation, and performing on-line monitoring under confocal imaging, wherein the result shows that no green fluorescent signal is observed in the first 25 min; at the beginning of 30 min, an obvious green fluorescence signal appears; at 40 min,50 min, it still exists in E.coli and a clear green fluorescence signal is maintained.
The synthesis of the fluorescent probe and the application of the fluorescent probe in the detection of the ClbP-containing escherichia coli liquid develop the application of the ClbP-containing escherichia coli liquid with high efficiency, simplicity and low cost; meanwhile, the on-line detection and long-time continuous imaging analysis of the ClbP-containing Escherichia coli are realized, and a solid foundation is provided for exploring an action mechanism between Clb toxin and colorectal cancer. Has great practical application value in the fields of biochemistry, analysis and detection and the like. While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Therefore, the application of the fluorescent probe with the technical characteristics similar to those of Clb toxin-carrying escherichia coli detection and imaging analysis, which are described herein, fall into the protection scope of the present patent.

Claims (3)

1. The synthesis and application of the fluorescent probe for on-line monitoring of Escherichia coli carrying Clb toxin are characterized in that the chemical structural formula of the fluorescent probe is as follows:
Figure DEST_PATH_IMAGE001
。
2. the method of synthesizing a fluorescent probe according to claim 1, wherein the method of synthesizing a fluorescent probe comprises the following steps.
The method comprises the following steps: synthesis of (R) -N 1 - (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) -2-tetradecylamido-succinamide
I, adding a proper amount of 4-phenyl-2- (trifluoromethyl) quinoline-7-amine,N-Boc-Dadding asparagine, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) and N, N-Diisopropylethylamine (DIPEA) into a dichloromethane solution, and reacting at room temperature for 2-10h; after the reaction is completed, adding water for quenching, extracting by ethyl acetate, spin-drying the solvent, and purifying by column chromatography to obtain a gray solid;
II, dissolving the intermediate grey solid obtained by the proper amount of the I in dichloromethane, dropwise adding a proper amount of trifluoroacetic acid, and reacting at normal temperature for 0.5-10 h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and purifying by flash column chromatography to obtain off-white solid;
III, dissolving the off-white solid obtained by the proper amount of II, myristic acid and HATU in pyridine, and reacting at room temperature for 1-10 h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and purifying by column chromatography to obtain a white solid;
IV, dissolving a proper amount of white solid obtained from the III into concentrated hydrochloric acid, and reacting at the temperature of between 20 and 80 ℃ for 1 to 10h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and recrystallizing to obtain the probe (R) -N 1 - (4-phenyl-2- (trifluoromethyl) quinolin-7-yl) -2-tetradecylamido-succinamide.
3. The use of the fluorescent probe of claim 1, wherein the fluorescent probe is capable of fluorescence-activated detection, visual imaging analysis and long-term online monitoring of Escherichia coli harboring Clb toxin.
CN202211142017.1A 2022-09-20 2022-09-20 Synthesis and application of visualized imaging and online monitoring Clb toxin-carrying escherichia coli fluorescent probe Pending CN115490635A (en)

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