CN111675632A - Fluorescent molecular probe for detecting genotoxin Colibactin through visual imaging as well as preparation method and application of fluorescent molecular probe - Google Patents
Fluorescent molecular probe for detecting genotoxin Colibactin through visual imaging as well as preparation method and application of fluorescent molecular probe Download PDFInfo
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Abstract
The invention discloses a fluorescent molecular probe for detecting a gene toxin Colibactin through visual imaging, a preparation method and application thereof, wherein the structural formula of the probe is shown as (1):
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a preparation method and application of a fluorescent molecular probe for detecting a gene toxin Colibactin through visual imaging.
Background
The intestinal flora is a normal microbial flora of human intestinal, can synthesize various vitamins necessary for human growth and development, can synthesize essential amino acids by using protein residues, participates in the metabolism of saccharides and proteins, and can promote the absorption of mineral elements such as iron, magnesium, zinc and the like. On the other hand, the intestinal flora is also used as a part of the tumor microenvironment and participates in the generation and development of most of digestive tract malignant tumors. Studies have shown that a gene toxin named Colibactin produced in E.coli can cause tumors by destroying the DNA of intestinal epithelial cells, thereby greatly increasing the incidence of colorectal cancer (Science, 2006,313(5788): 848-851; Science,2018,359(6375): 592-597). Therefore, the detection of the Colibactin has important significance and practical value for the research on the colorectal cancer.
Due to the complex structure of the Colibactin, no method for purifying the Colibactin and determining the exact structure of the Colibactin exists so far, and as a result, the detection of the Colibactin is limited. On the other hand, the peptidase ClbP involved in the biosynthesis of Colibactin is stably present, which produces Colibactin by hydrolysis of biologically inactive Colibactin precursor compounds to which a protecting group N-myristoyl D-asparagine is attached (Journal of the American chemical society, 2013,135(9): 3359-3362). Therefore, a sensitive, simple and good-selectivity method is established to detect or monitor ClbP on line through the characteristic that ClbP can catalyze and hydrolyze N-myristoyl D-asparagine and the combination of a prodrug strategy, so that the rapid detection of Colibactin and related research are feasible.
Disclosure of Invention
The invention aims to provide a preparation method and application of a fluorescent molecular probe for visual imaging detection of a gene toxin Colibactin, which has good selectivity, high sensitivity and high response speed.
In order to achieve the purpose, the invention adopts the following technical scheme:
a fluorescent molecular probe for detecting a gene toxin Colibactin through visual imaging is characterized in that the molecular probe has the following chemical structural formula:
the invention also provides application of the fluorescent molecular probe in preparation of a genotoxin Colibactin and an upstream peptidase ClbP detection reagent thereof.
Preferably, the detection reagent is prepared by dissolving a fluorescent molecular probe into a mixed solvent, wherein the mixed solvent is N, N-dimethylformamide: 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid ═ 2 (7-9).
Preferably, the concentration of the fluorescent molecular probe in the detection reagent is 4-6. mu.M. A further preferred concentration is 5. mu.M.
The invention also provides application of the fluorescent molecular probe in preparing a visual imaging reagent, wherein the visual imaging reagent comprises visual imaging reagents of living bodies, tissues, cells and bacteria.
The invention also provides a preparation method of the fluorescent molecular probe for detecting the gene toxin Colibactin through visual imaging, and the preparation method of the fluorescent molecular probe comprises the following steps:
(1) raw material a: N-Boc-D-asparagine, starting material b: (E) -2- (3- (4-aminostyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile, starting material c: stirring 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate and a catalytic amount of 4-dimethylaminopyridine in an anhydrous pyridine solution at room temperature for 6-8 hours, quenching the reaction mixture by adding a saturated aqueous sodium bicarbonate solution, extracting with ethyl acetate, drying and vacuum-concentrating the combined extracts to obtain a crude product, and purifying the crude product to obtain an intermediate 1: tert-butyl (S, E) - (3-cyano-1- ((4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) amino) -1-oxopropan-2-yl) carbamate;
(2) dissolving intermediate 1 in CH2Cl2Mixing with trifluoroacetic acid, stirring at room temperature for 0.5-3 hr, adding water to remove trifluoroacetic acid, extracting with ethyl acetate, washing organic phase with large amount of water, removing organic phase by rotary evaporation under reduced pressure, and passing through SiO2Silica gel column purityAnd (3) carrying out chemical reaction for 1-6 hours to obtain an intermediate 2: (S, E) -2-amino-3-cyano-N- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) propionamide;
(3) stirring the solution of the intermediate 2, myristic acid and the raw material c in pyridine at room temperature for 5-7 hours, stopping the reaction, pouring the reaction solution into a large amount of saturated sodium bicarbonate aqueous solution to generate solid, filtering, dissolving the filter residue with ethyl acetate, and dissolving with Na2SO4Drying and vacuum concentrating to obtain crude product, passing through SiO2Column chromatography purification of the crude product gave intermediate 3: (S, E) -N- (3-cyano-1- ((4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) amino) -1-oxopropan-2-yl) tetradecanamide;
(4) dissolving the intermediate 3 in acetic acid, adding TiCl4And H2O, stirring for 1-3 hours at room temperature, introducing saturated sodium bicarbonate aqueous solution to quench the reaction mixture, extracting with ethyl acetate, drying the combined extracts and concentrating in vacuum to obtain a crude product, and purifying the crude product to obtain the fluorescent molecular probe (S, E) -N1- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) -2-tetradecanoylaminosuccinamide.
Preferably, in the step (1), the molar ratio of the raw material a to the raw material b to the raw material c is as follows: 2, (1-3) and (1-6); the catalytic amount of the 4-dimethylaminopyridine is 10mg-100 mg.
Preferably, CH in step (2)2Cl2And trifluoroacetic acid, and the volume ratio of the trifluoroacetic acid to the trifluoroacetic acid is 1 (1-2), and the preferable scheme is 1 (0.8-1.2). The molar mass of the intermediate 1 and the volume ratio of the mixed solvent are as follows: 1mM (10-12 mL). The reaction time is more preferably 0.8 to 1.2 hours, and the purification time is preferably 1 to 3 hours.
Preferably, the molar ratio of the intermediate 2 to the myristic acid and the raw material c in step (3) is: the intermediate 2 is myristic acid, and the raw material c is (1-3) and (1-6). Further preferably, the molar ratio of the intermediate 2 to the myristic acid and the raw material c in step (3) is: the intermediate 2 is myristic acid, and the raw material c is (1-2) and (3-5).
Preferably, step (4) dissolves intermediate 3 in 5mL-20mL of acetic acid,adding TiCl4And 20 μ L-200 μ LH2O, wherein the intermediate 3 is reacted with TiCl4The molar ratio of (A) to (B) is 1 (1.2-5). Further preferred, the intermediate 3 is reacted with TiCl4The molar ratio of (1) to (2-3).
The room temperature described in the specification is 20 ℃ to 30 ℃.
The fluorescent molecular probe for detecting the gene toxin Colibactin has the characteristics of good selectivity, high sensitivity, high response speed and the like, can avoid the interference of biological autofluorescence because the emission is in a near infrared region, and can be well applied to the visual imaging analysis of the gene toxin Colibactin in organisms.
Drawings
FIG. 1 is a schematic diagram of the synthesis of a fluorescent molecular probe according to the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of the fluorescent molecular probe of the present invention;
FIG. 3 is a graph showing the change of fluorescence spectrum of a target bacterial lysate into which the fluorescent molecular probe of the present invention has been added;
FIG. 4 is a graph showing the selectivity of the fluorescent molecular probe of the present invention for detection of different bacterial lysates, wherein the abscissa numerals 1 to 8 represent the following: 1. escherichia coli (BL21), 2. salmonella typhimurium, 3. salmonella paratyphi a, 4. pseudomonas aeruginosa, 5. staphylococcus aureus, 6. escherichia coli (K88), 7. salmonella enteritidis, 8. salmonella choleraesuis;
FIG. 5 is a diagram showing the selectivity of the fluorescent molecular probe of the present invention for a common small molecule (A) and ion (B), wherein the abscissa numerals 1 to 28 in the diagram (A) represent the following substances: ClbP,2.K+,3.Na+,4.Mn2+,5.Fe3+,6.Ba2+,7.Cu2+,8. Zn2+,9.Al3+,10.Cd2+,11.Co2+,12.Mg2+,13.NH4 +,14.F-,15.Cl-,16.Br-,17.I-,18.NO3 -,19. NO2 -,20.S2O3 -,21.SO3 2-,22.SO4 2-,23.HCO3 -,24.CO3 2-,25.N3 -,26.HSO3 -,27.S2-,28. CH3COO-.
(B) The abscissa numbers 1 to 30 of the graph represent the following substances: 1, ClbP,2, glycine Gly,3, histidine His,4, methionine Met,5, aspartic acid Asp,6, threonine Thr,7, lysine Lys,8, tyrosine Try,9, glutamic acid Glu, 10, cysteine Cys,11, homocysteine Hcy,12, glutathione GSH,13, ClO-T-butyl hydroperoxide TBHP,15.1O2,16.ONOO-17. OH,18. oxy tert-butyl. OtBu,19.O2 -20 urea,21.H2O222 acid phosphatase ACP,23 choline oxidase ChOD,24 β -galactosidase β -Gal,25 phosphodiesterase PDE,26 Trypsin,27 horseradish peroxidase HRP,28 glucose oxidase GOD,29 acetylcholinesterase AchE,30 alkaline phosphatase ALP;
FIG. 6 is a confocal fluorescent image of a target bacterium with a fluorescent molecular probe of the present invention.
Detailed Description
The invention will be further elucidated with reference to the drawings and specific embodiments. These examples are only for explaining and illustrating the present invention and do not limit the scope of the present invention.
Name notation interpretation:
HATU: 2- (7-benzotriazole oxide) -N, N' -tetramethyluronium hexafluorophosphate;
DMAP: 4-dimethylaminopyridine;
HEPES (high efficiency particulate air): 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid
Example 1: preparation of intermediate 1
(1) N-Boc-D-asparagine (140mg, 600. mu. mol), (E) -2- (3- (4-aminostyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile (127mg, 300. mu. mol), HATU (457mg, 1.2mmol) and a catalytic amount of 4-dimethylaminopyridine (30mg, 250. mu. mol) in dry pyridine (0.5mL) stirred at 25 ℃ for 7 h, the reaction mixture was quenched by addition of saturated aqueous sodium bicarbonate solution (10mL), the resulting mixture was extracted with ethyl acetate (10mL × 3), the combined extracts were extracted with Na2SO4Drying and vacuum concentratingTo obtain a crude product. By SiO2The crude product was purified by column chromatography to afford intermediate 1 in 54% yield.
Example 2: preparation of intermediate 2
Intermediate 1(291mg, 600. mu. mol) was dissolved in 3mL CH2Cl2And 3mL of trifluoroacetic acid. Stirring at 25 deg.C for 1h, adding water to remove trifluoroacetic acid, extracting with ethyl acetate, washing organic phase with large amount of water, removing organic phase by rotary evaporation under reduced pressure, and rapidly purifying by column chromatography for 2 hr. Intermediate 2 was obtained in 67% yield.
Example 3: preparation of intermediate 2
Intermediate 1(291mg, 600. mu. mol) was dissolved in 3mL CH2Cl2And 3mL of trifluoroacetic acid. Stirring at 25 deg.C for 4 hr, adding water to remove trifluoroacetic acid, extracting with ethyl acetate, washing organic phase with large amount of water, removing organic phase by rotary evaporation under reduced pressure, and quickly purifying by column chromatography for 2 hr. The target yield is not obtained, and the target product may be decomposed due to an excessively long reaction time.
Example 4: preparation of intermediate 2
Intermediate 1(291mg, 600. mu. mol) was dissolved in 3mL CH2Cl2And 6mL trifluoroacetic acid. Stirring at 25 deg.C for 1h, adding water to remove trifluoroacetic acid, extracting with ethyl acetate, washing organic phase with large amount of water, removing organic phase by rotary evaporation under reduced pressure, and rapidly purifying by column chromatography for 2 hr. Intermediate 2 was obtained in 20% yield.
Example 5: preparation of intermediate 2
Intermediate 1(291mg, 600. mu. mol) was dissolved in 6mL CH2Cl2And 3mL of trifluoroacetic acid. Stirring at 25 deg.C for 1h, adding water to remove trifluoroacetic acid, extracting with ethyl acetate, washing organic phase with large amount of water, removing organic phase by rotary evaporation under reduced pressure, and rapidly purifying by column chromatography for 2 hr. Intermediate 2 was obtained in 25% yield.
Example 6: preparation of intermediate 2
Intermediate 1(291mg, 600. mu. mol) was dissolved in 3mL CH2Cl2And 3mL of trifluoroacetic acid. Stirring at 25 deg.C for 1 hr, adding water to remove trifluoroacetic acid, and extracting with ethyl acetateWashing the organic phase with a large amount of water, removing the organic phase by rotary evaporation under reduced pressure, and purifying by column chromatography for 6 hours. Intermediate 2 was obtained in 30% yield.
Example 7: preparation of intermediate 3
A solution of intermediate 2(231mg, 600. mu. mol), myristic acid (205mg, 900. mu. mol) and HATU (608mg, 1.6mmol) in pyridine (5mL) was stirred at 25 ℃ for 6 h. After the reaction is stopped, the reaction liquid is poured into a large amount of saturated sodium bicarbonate aqueous solution to generate solid, filter residue is dissolved by ethyl acetate after suction filtration, and Na is used for dissolving2SO4Dried and concentrated in vacuo to give the crude product. By SiO2The crude product was purified by column chromatography to afford intermediate 3 in 43% yield.
Example 8: preparation of Probe
Intermediate 3(595mg, 1mmol) was dissolved in 5mL of acetic acid and TiCl was added4(187mg, 1mmol) and 50. mu. L H2And O. Stirring at 25 deg.C for 2 hr, introducing saturated sodium bicarbonate water solution to quench the reaction mixture, extracting with ethyl acetate, drying the combined extracts and vacuum concentrating to obtain crude product, and purifying by column chromatography to obtain fluorescent molecular probe with yield of 43%.
Example 9: preparation of Probe
Intermediate 3(595mg, 1mmol) was dissolved in 5mL of acetic acid and TiCl was added4(374mg, 2mmol) and 50. mu. L H2And O. Stirring at 25 deg.C for 2 hr, introducing saturated sodium bicarbonate water solution to quench the reaction mixture, extracting with ethyl acetate, drying the combined extracts and vacuum concentrating to obtain crude product, and purifying by column chromatography to obtain fluorescent molecular probe with yield of 68%.
Example 10: preparation of Probe
Intermediate 3(595mg, 1mmol) was dissolved in 5mL of acetic acid and TiCl was added4(561mg, 3mmol) and 50. mu. L H2And O. Stirring at 25 deg.C for 2 hr, introducing saturated sodium bicarbonate water solution to quench the reaction mixture, extracting with ethyl acetate, drying the combined extractive solutions, vacuum concentrating to obtain crude product, purifying by column chromatography to obtain fluorescent molecular probe,the yield was 84%.
Example 11: preparation of Probe
Intermediate 3(595mg, 1mmol) was dissolved in 5mL of acetic acid and TiCl was added4(935mg, 5mmol) and 50. mu. L H2And O. Stirring at 25 deg.C for 2 hr, introducing saturated sodium bicarbonate water solution to quench the reaction mixture, extracting with ethyl acetate, drying the combined extracts and vacuum concentrating to obtain crude product, and purifying by column chromatography to obtain fluorescent molecular probe with yield of 53%.
Example 12
Dissolving the molecular fluorescent probe in DMF to prepare a probe solution of 1 mmol/L; the probe solution was added to DMF and HEPES (pH 7.4) buffer to prepare a 10 μ M (organic phase: HEPES aqueous phase: 2:8, v/v) solution, 10 μ L, 50 μ L, 100 μ L and 200 μ L of bacterial lysate expressing ClbP were added, and after incubation at 37 ℃ for 1h, the fluorescence spectrum change was measured at 470nm as the excitation wavelength. FIG. 2 shows that the fluorescence intensity of the probe molecule at 660nm increases with increasing ClbP content.
Example 13:
dissolving the molecular fluorescent probe in DMF to prepare a probe solution of 1 mmol/L; the probe solution was added with DMF and HEPES (pH 7.4) buffer to prepare a 10 μ M (organic phase: HEPES aqueous phase: 2:8, v/v) solution, different types of bacterial lysates were added, and after incubation at 37 ℃ for 1h, the fluorescence spectrum change was measured at 470nm as the excitation wavelength. FIG. 3 shows that the probes are very selective for detecting ClbP expressing bacteria.
Example 14:
dissolving the molecular fluorescent probe in DMF to prepare a probe solution of 1 mmol/L; the probe solution was added with DMF and HEPES (pH 7.4) buffer to prepare a 10 μ M solution (organic phase: HEPES aqueous phase: 2:8, v/v), and then a common small molecule and ion were added, and after incubation at 37 ℃ for 1h, the fluorescence spectrum change was measured at 470nm as the excitation wavelength. FIG. 4 shows that the probes are very selective for detecting ClbP expressing bacteria.
Example 15:
the molecular probe is added into a cell culture medium and is co-cultured with a bacterium capable of expressing ClbP for 2 hours, and then fluorescence confocal imaging is carried out. As can be seen in FIG. 5, the bacteria to which the probe was added exhibited red fluorescence, indicating that the probe can be used for imaging identification of bacteria.
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. Accordingly, the scope of the invention is defined by the appended claims.
Claims (10)
1. A fluorescent molecular probe for detecting a gene toxin Colibactin through visual imaging is characterized in that the molecular probe has the following chemical structural formula:
2. the fluorescent molecular probe of claim 1 in preparing a detection reagent for the gene toxin Colibactin and the upstream peptidase ClbP thereof.
3. The method for detecting the fluorescence of the nucleic acid molecule of the invention is characterized in that the detection reagent is prepared by dissolving a fluorescent molecular probe into a mixed solvent, wherein the mixed solvent is a mixed solvent of N, N-dimethylformamide: 4- (2-hydroxyethyl) -1-piperazine ethanesulfonic acid ═ 2 (7-9) in a volume ratio.
4. The use according to claim 3, wherein the concentration of the fluorescent molecular probe in the detection reagent is 4 to 6. mu.M.
5. Use of the fluorescent molecular probe of claim 1 in the preparation of a visualization imaging agent, including visualization imaging agents for living organisms, tissues, cells and bacteria.
6. The preparation method of the fluorescent molecular probe for detecting the gene toxin Colibactin through visual imaging as claimed in claim 1, wherein the preparation method of the fluorescent molecular probe comprises the following steps:
(1) raw material a: N-Boc-D-asparagine, starting material b: (E) -2- (3- (4-aminostyryl) -5, 5-dimethylcyclohex-2-en-1-yl) malononitrile, starting material c: stirring 2- (7-benzotriazole oxide) -N, N, N ', N' -tetramethyluronium hexafluorophosphate and a catalytic amount of 4-dimethylaminopyridine in an anhydrous pyridine solution at room temperature for 6-8 hours, quenching the reaction mixture by adding a saturated aqueous sodium bicarbonate solution, extracting with ethyl acetate, drying and vacuum-concentrating the combined extracts to obtain a crude product, and purifying the crude product to obtain an intermediate 1: tert-butyl (S, E) - (3-cyano-1- ((4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) amino) -1-oxopropan-2-yl) carbamate;
(2) dissolving intermediate 1 in CH2Cl2Mixing with trifluoroacetic acid, stirring at room temperature for 0.5-3 hr, adding water to remove trifluoroacetic acid, extracting with ethyl acetate, washing organic phase with large amount of water, removing organic phase by rotary evaporation under reduced pressure, and passing through SiO2Silica gel column purification for 1-6 hours to obtain intermediate 2: (S, E) -2-amino-3-cyano-N- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) propionamide;
(3) stirring the solution of the intermediate 2, myristic acid and the raw material c in pyridine at room temperature for 5-7 hours, stopping the reaction, pouring the reaction solution into a large amount of saturated sodium bicarbonate aqueous solution to generate solid, filtering, dissolving the filter residue with ethyl acetate, and dissolving with Na2SO4Drying and vacuum concentrating to obtain crude product, passing through SiO2The crude product was purified by column chromatography to afford intermediate 3: (S, E) -N- (3-cyano-1- ((4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) amino) -1-oxopropan-2-yl) tetradecanamide;
(4) dissolving the intermediate 3 in acetic acid, adding TiCl4And H2O, stirring for 1-3 hours at room temperature, and introducing saturated sodium bicarbonate water solutionAfter quenching the reaction mixture, extraction with ethyl acetate was performed, and the combined extracts were dried and concentrated in vacuo to give a crude product, which was purified to give fluorescent molecular probe (S, E) -N1- (4- (2- (3- (dicyanomethylene) -5, 5-dimethylcyclohex-1-en-1-yl) vinyl) phenyl) -2-tetradecanoylaminosuccinamide.
7. The method for preparing the fluorescent molecular probe for detecting the gene toxin Colibactin through visual imaging according to claim 6, wherein the molar ratio of the raw material a to the raw material b to the raw material c in the step (1) is as follows: 2, (1-3) and (1-6); the catalytic amount of the 4-dimethylaminopyridine is 10mg-200 mg.
8. The method for preparing the fluorescent molecular probe for detecting the gene toxin Colibactin through visual imaging according to claim 6, wherein CH in the step (2)2Cl2And trifluoroacetic acid at a volume ratio of 1 (1-2).
9. The method for preparing the fluorescent molecular probe for detecting the gene toxin Colibactin through visual imaging according to claim 6, wherein the molar ratio of the intermediate 2 to the myristic acid to the raw material c in the step (3) is as follows: the intermediate 2 is myristic acid, and the raw material c is (1-3) and (1-6).
10. The method for preparing the fluorescent molecular probe for visually imaging and detecting the gene toxin Colibactin as claimed in claim 6, wherein the intermediate 3 is dissolved in 5mL-20mL of acetic acid in the step (4), and TiCl is added4And 20 μ L-200 μ LH2O, wherein the intermediate 3 is reacted with TiCl4The molar ratio of (A) to (B) is 1 (1.2-5).
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114539275A (en) * | 2020-11-26 | 2022-05-27 | 南京碳硅人工智能生物医药技术研究院有限公司 | Based on camptothecin on H2O2Design and synthesis of responsive fluorescently labeled prodrugs |
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| CN115286534B (en) * | 2022-04-26 | 2023-08-15 | 安徽工程大学 | Specific detection Hg 2+ Near infrared fluorescence chemical sensor of (2), preparation method and application thereof |
| CN115385868A (en) * | 2022-10-10 | 2022-11-25 | 湖南师范大学 | Synthesis and application of ClbP fluorescent probe with high selectivity recognition |
| CN115490654A (en) * | 2022-10-18 | 2022-12-20 | 湖南师范大学 | Synthesis and application of fluorescent probe for detecting Clb toxin-carrying escherichia coli |
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