CN115385868A - Synthesis and application of ClbP fluorescent probe with high selectivity recognition - Google Patents
Synthesis and application of ClbP fluorescent probe with high selectivity recognition Download PDFInfo
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- 238000003786 synthesis reaction Methods 0.000 title claims abstract description 14
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- 241000588724 Escherichia coli Species 0.000 claims abstract description 20
- 239000000126 substance Substances 0.000 claims abstract 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 36
- 125000005605 benzo group Chemical group 0.000 claims description 17
- 125000000437 thiazol-2-yl group Chemical group [H]C1=C([H])N=C(*)S1 0.000 claims description 17
- 238000001035 drying Methods 0.000 claims description 16
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- 238000000034 method Methods 0.000 claims description 11
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- 238000004440 column chromatography Methods 0.000 claims description 9
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- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 claims description 3
- 235000021360 Myristic acid Nutrition 0.000 claims description 3
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 claims description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims description 2
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- 238000001514 detection method Methods 0.000 abstract description 15
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- 230000035945 sensitivity Effects 0.000 abstract description 2
- 238000011895 specific detection Methods 0.000 abstract 1
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 20
- 239000000243 solution Substances 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 8
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical group OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 6
- 239000007995 HEPES buffer Substances 0.000 description 6
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 5
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- ZWKHDAZPVITMAI-ROUUACIJSA-N colibactin Chemical compound C[C@H]1CCC(=N1)C1=C(CC(=O)NCC(=O)c2csc(n2)C(=O)C(=O)c2csc(CNC(=O)CC3=C(C(=O)NC33CC3)C3=N[C@@H](C)CC3)n2)C2(CC2)NC1=O ZWKHDAZPVITMAI-ROUUACIJSA-N 0.000 description 5
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- 238000006243 chemical reaction Methods 0.000 description 3
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- 238000011160 research Methods 0.000 description 3
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- 229960000549 4-dimethylaminophenol Drugs 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- LVTJOONKWUXEFR-FZRMHRINSA-N protoneodioscin Natural products O(C[C@@H](CC[C@]1(O)[C@H](C)[C@@H]2[C@]3(C)[C@H]([C@H]4[C@@H]([C@]5(C)C(=CC4)C[C@@H](O[C@@H]4[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@@H](O)[C@H](O[C@H]6[C@@H](O)[C@@H](O)[C@@H](O)[C@H](C)O6)[C@H](CO)O4)CC5)CC3)C[C@@H]2O1)C)[C@H]1[C@H](O)[C@H](O)[C@H](O)[C@@H](CO)O1 LVTJOONKWUXEFR-FZRMHRINSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- LFKDJXLFVYVEFG-UHFFFAOYSA-N tert-butyl carbamate Chemical compound CC(C)(C)OC(N)=O LFKDJXLFVYVEFG-UHFFFAOYSA-N 0.000 description 2
- OSLFUNIZLWQZCC-UHFFFAOYSA-N 4-phenyl-2-(trifluoromethyl)quinolin-7-amine Chemical compound C1(=CC=CC=C1)C1=CC(=NC2=CC(=CC=C12)N)C(F)(F)F OSLFUNIZLWQZCC-UHFFFAOYSA-N 0.000 description 1
- KXDHJXZQYSOELW-UHFFFAOYSA-M Carbamate Chemical compound NC([O-])=O KXDHJXZQYSOELW-UHFFFAOYSA-M 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 240000001929 Lactobacillus brevis Species 0.000 description 1
- 235000013957 Lactobacillus brevis Nutrition 0.000 description 1
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- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
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Abstract
The invention discloses synthesis and application of a ClbP fluorescent probe with high selectivity identification, wherein the chemical structural formula of the fluorescent probe is as follows:
. The invention designs and constructs a fluorescent probe for detecting ClbP based on an intramolecular charge transfer mechanism. The probe can realize high-selectivity fluorescence-activated detection on ClbP (a)λ ex /λ em =340/440 nm), high sensitivity and strong interference resistance; the probe can also realize specific fluorescence detection of ClbP in harmful escherichia coli liquid, provides a new specific detection idea for distinguishing harmful escherichia coli in intestinal bacteria, and belongs to the technical fields of analytical chemistry, life science, biomedical treatment and the likeHas great application prospect.
Description
Technical Field
The invention belongs to the technical field of fluorescent probe analysis, and particularly relates to synthesis and application of a fluorescent probe for ClbP detection in harmful escherichia coli.
Background
The intestinal bacteria play an indispensable great role in regulating and controlling the intestinal microenvironment. Among them, escherichia coli plays an important role in the maintenance of the intestinal environment. Research shows that the pathogenesis of colorectal cancer patients is closely related to the Colibactin genotoxin secreted by harmful escherichia coli in the intestinal microenvironment. A key peptidase ClbP exists in the biosynthesis of the gene toxin Colibactin, and the gene toxin Colibactin with biological active substances is produced by hydrolyzing a Colibactin precursor compound without biological activity. Therefore, the detection of the Colibactin is realized through the specific hydrolysis of the ClbP, and further the high-selectivity differentiation of harmful escherichia coli in colorectal cancer is realized, so that the method has great feasibility. Therefore, a simple and efficient detection method/technology is developed for sensitively identifying ClbP, so that the harmful escherichia coli can be distinguished with high selectivity, the effect analysis of the harmful escherichia coli between colorectal cancers can be further explored, and the method has very important clinical value and wide application prospect.
Therefore, by utilizing the characteristic that harmful escherichia coli secretes colibacin and combining the specificity of ClbP, a new direction is hopefully provided for high-selectivity distinguishing of harmful escherichia coli in colorectal cancer. At present, in the differentiation detection of intestinal bacteria, the differentiation detection is mainly performed through macroscopic genomics analysis, and the differentiation detection has the defects of complex operation, need of expensive equipment, long sample pretreatment time, high cost, long time consumption and the like; the fluorescent probe detection method has the advantages of high selectivity, high sensitivity, flexibility, simplicity, easiness in processing and the like, and has been widely concerned and applied in the field of analysis and detection.
Disclosure of Invention
In view of the above circumstances, the present invention overcomes some deficiencies of the prior art, and aims to provide a fluorescent probe for ClbP detection in harmful escherichia coli, in particular to a synthetic method of the fluorescent probe, and a spectrum property research; the specific ClbP can be detected, so that the harmful escherichia coli can be identified with high selectivity, and different intestinal bacteria can be selectively explored; the invention also aims to provide a synthesis and application method of the fluorescent probe, which has the advantages of simple preparation method and low cost.
The invention solves the problem by adopting the specific technical scheme that the synthesis and application of the fluorescent probe for identifying ClbP with high selectivity are as follows:
the synthesis of the ClbP fluorescent probe with high selectivity recognition 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- (benzo [ d ])]Thiazol-2-yl) phenyl) -2-tetradecylamido-succinamide
I, adding proper amount of 4- (benzo [ d ]]Thiazol-2-yl) aniline,N-Boc-Dadding asparagine, HATU and DMAP into acetonitrile, and reacting for 2-10h at room temperature; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and purifying by column chromatography to obtain a white solid;
II, dissolving a white solid obtained by the proper amount of the I in dichloromethane, dropwise adding a proper amount of trifluoroacetic acid, and reacting at room temperature for 0.5-10 h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and purifying by column chromatography to obtain a compound C: a white solid;
III, dissolving a proper amount of the compound C obtained in the step II, myristic acid and HATU in pyridine, and reacting at room temperature for 1-18 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- (benzo [ d ]]Thiazol-2-yl) phenyl) -2-tetradecylamido-succinamide.
The invention provides an application of a fluorescent probe capable of being used for ClbP open type detection in harmful escherichia coli.
The method for detecting ClbP by the fluorescent probe comprises the following steps: the probe is used for detecting ClbP in an environment system with the volume ratio of an organic phase to an aqueous phase of 2:8, wherein the organic phase is Acetone (ACN), and the aqueous phase is HEPES buffer solution with pH = 7.4 and a bacterium solution of ClbP. The probe was dissolved in dimethyl sulfoxide (DMSO), and a ClbP bacterial solution was prepared with PBS, and its ClbP bacterial concentration OD 600nm 0.3, dissolving the probe in a system with the volume ratio of organic phase to aqueous phase of 2:8, reacting with ClbP bacterial liquid at 37 ℃ for 1.5 h, and exciting at 340 nmThe fluorescence signal of 440 nm was detected at the emission wavelength.
The invention also provides application of the fluorescent probe for identifying and distinguishing harmful escherichia coli with high selectivity.
Drawings
FIG. 1 shows the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe of the present invention.
FIG. 2 shows that in an environment system of acetone and HEPES buffer solution (volume ratio of 2:8), the fluorescence emission intensity of the fluorescent probe changes with the volume of ClbP bacterial liquid, the abscissa is wavelength, and the ordinate is fluorescence intensity.
FIG. 3 shows the response of fluorescence emission intensity of the fluorescent probe according to different bacterial liquids in different bacterial lysates (1, transetta-pGEX4T-1-ClbP Escherichia coli liquid; 2, lactobacillus brevis; 3, lactobacillus pentosus; 4, lactobacillus plantarum; 5, lactobacillus lactis; 6, staphylococcus aureus), with different bacterial liquids on the abscissa and fluorescence intensity on the ordinate.
Detailed Description
The invention is further described in connection with the following figures.
The synthetic route of the fluorescent probe of the invention is shown in the following figure.
Example 1 Synthesis of tert-butyl (R) - (1- ((4- (benzo [ d ] thiazol-2-yl) phenyl) amino) -3-cyano-1-oxopropan-2-yl) carbamate;
4-phenyl-2- (trifluoromethyl) quinolin-7-amine (20.0 mg, 83.0. Mu. Mol),N-Boc-Dasparagine (27.0 mg, 99.0. Mu. Mol), HATU (79.0 mg, 200.0. Mu. Mol) and DMAP (30 mg, 280.0. Mu. Mol) in acetonitrile at room temperature after reaction of 7 h, quenching the reaction by addition of saturated sodium bicarbonate solution (10 mL), extraction with ethyl acetate (10 mL X3), combining the organic phases, anhydrous Na 2 SO 4 Drying, spin-drying solvent to obtain crude product, and purifying by column chromatography to obtain (R) - (1- ((4- (benzo [ d ])]Thiazol-2-yl) phenyl) amino) -3-cyano-1-oxopropane-2-yl) carbamic acid tert-butyl ester 23.0 mg in 56% yield.
EXAMPLE 2 Synthesis of (R) -2-amino-N- (4- (benzo [ d ] thiazol-2-yl) phenyl) -3-cyanopropanamide
(R) - (1- ((4- (benzo [ d ]))]Thiazol-2-yl) phenyl) amino) -3-cyano-1-oxopropan-2-yl) carbamic acid tert-butyl ester (110 mg, 206. Mu. Mol) was dissolved in 4.0 mL dichloromethane, 1.5mL trifluoroacetic acid was slowly added dropwise, after reaction at room temperature of 0.8 h, saturated sodium bicarbonate solution was neutralized, ethyl acetate (20 mL. Times.3) was extracted, anhydrous Na 2 SO 4 Drying, spin-drying solvent, and purifying by column chromatography to obtain (R) -2-amino-N- (4- (benzo [ d ] b]Thiazol-2-yl) phenyl) -3-cyanopropanamide 40.0 mg, 43.6% yield.
EXAMPLE 3 Synthesis of (R) -N- (1- ((4- (benzo [ d ] thiazol-2-yl) phenyl) amino) -3-cyano-1-oxopropan-2-yl) tetradecanamide
(R) -2-amino-N- (4- (benzo [ d ]]Thiazol-2-yl) phenyl) -3-cyanopropanamide (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 5mL pyridine, reacted at room temperature for 3 h, neutralized, extracted with ethyl acetate (20 mL X3), anhydrous Na 2 SO 4 Drying, spin-drying solvent, and purifying by column chromatography to obtain white solid (R) -N- (1- ((4- (benzo [ d ] b)]Thiazol-2-yl) phenyl) amino) -3-cyano-1-oxopropan-2-yl) tetradecanamide 69 mg in 41.6% yield.
EXAMPLE 4 Synthesis of (R) -N 1 - (4- (benzo [ d ]]Thiazol-2-yl) phenyl) -2-tetradecylamido-succinamides
(R) -N- (1- ((4- (benzo [ d ]))]Thiazol-2-yl) phenyl) amino) -3-cyano-1-oxopropan-2-yl) tetradecanamide (200.0 mg, 337.0. Mu. Mol) was dissolved in 5.0 mL concentrated hydrochloric acid, reacted at 45 ℃ for 2h, 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 to obtain white solid probe (R) -N 1 - (4- (benzo [ d ])]Thiazol-2-yl) phenyl) -2-tetradecylamido-succinamide 80.0 mg in 44.3% yield.
Example 5 fluorescent probe realizes ClbP bacterial liquid open type fluorescence detection
The fluorescent probe of the invention is used for detecting ClbPThe implementation method of the bacterial liquid comprises the following steps: the probes were dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution having a concentration of 1 mM, and a bacterial suspension of ClbP was prepared using PBS, and its bacterial concentration OD of ClbP was obtained 600 Is 0.3, and the specific test method comprises the following steps: 10 μ L of 1 mM probe solution, 390 μ L of analytically pure acetone, the required amount of bacterial solution and the required amount of HEPES buffer were taken in a sample tube of 2 mL, all test samples were kept in a total volume of 2 mL, with a volume ratio of organic and aqueous phases of 2:8. for example, the sample was formulated as follows: 10 μ L of 1 mM probe solution, 390 μ L of analytically pure acetone, 50 μ L of bacterial solution, 1550 μ L of HEPES buffer solution in 2 mL sample tube, shaking up to incubate 1.5 h at 37 deg.C, and collecting the fluorescence signal at 440 nm under 340 nm excitation.
Example 6 fluorescent probes highly selectively distinguish between harmful E.coli
The high-selectivity implementation method of the fluorescent probe comprises the following steps: selecting the concentration OD of bacteria 600 0.3, cleaved for subsequent spectroscopic testing.
The specific implementation mode is as follows: 10 μ L of 1 mM probe solution, 390 μ L of analytically pure acetone, the required amount of corresponding bacterial solution and the required amount of HEPES buffer were taken in a 2 mL sample tube, all test samples were kept in a total volume of 2 mL, with a volume ratio of organic phase to aqueous phase of 2:8. for example, the sample is formulated as follows: 10 μ L of 1 mM probe solution and 390 μ L of analytically pure acetone were taken, 100 μ L of bacterial liquid and 1500 μ L of HEPES buffer solution were put in a sample tube of 2 mL, incubated with shaking at 37 ℃ for 1.5 h, and fluorescence signals at 440 nm were observed under excitation of 340 nm.
The invention provides a fluorescent probe for detecting ClbP in harmful escherichia coli, and researches the spectral performance, thereby realizing a method for detecting ClbP in harmful escherichia coli with high selectivity; and different intestinal bacteria are selectively distinguished, harmful escherichia coli is identified with high selectivity, a simple, reliable, rapid and low-cost new method is provided for complex intestinal colony distinguishing detection screening, a kit for intestinal bacteria distinguishing detection can be further developed, and the kit 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, fluorescent probes with the technical characteristics described herein and similar ClbP detection are all within the scope of this patent.
Claims (3)
1. The synthesis and application of the fluorescent probe for highly selectively recognizing the ClbP are characterized in that the chemical structural formula of the fluorescent probe is as follows:
2. the method for synthesizing a fluorescent probe according to claim 1, wherein the method for synthesizing a fluorescent probe comprises the following steps:
step 1: synthesis of (R) -N 1 - (4- (benzo [ d ]]Thiazol-2-yl) phenyl) -2-tetradecylamido-succinamides
I, adding proper amount of 4- (benzo [ d ]]Thiazol-2-yl) aniline,N-Boc-Dadding asparagine, 2- (7-azabenzotriazole) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) and 4-Dimethylaminopyridine (DMAP) into acetonitrile, and reacting at room temperature for 2-10h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and purifying by column chromatography to obtain a white solid;
II, dissolving a white solid obtained by the proper amount of the I in dichloromethane, dropwise adding a proper amount of trifluoroacetic acid, and reacting at room temperature for 0.5-10 h; neutralizing, extracting with ethyl acetate, spin-drying the solvent, and purifying by column chromatography to obtain a white solid;
III, dissolving the white solid obtained by the proper amount of II, myristic acid and HATU in pyridine, and reacting 1-18 h at room temperature; 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; in (1)And extracting with ethyl acetate, spin-drying the solvent, and recrystallizing to obtain the probe (R) -N 1 - (4- (benzo [ d ])]Thiazol-2-yl) phenyl) -2-tetradecylamido-succinamide.
3. The use of the fluorescent probe according to claim 1, wherein the fluorescent probe can detect ClbP in E.coli as a harmful substance with high selectivity.
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| US20060199242A1 (en) * | 2005-03-01 | 2006-09-07 | Ching-Ying Cheung | Chemical probe compounds that become fluorescent upon reduction, and methods for their use |
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