阅读说明：本技术 一种可视化成像检测基因毒素Colibactin荧光分子探针及其制备方法和应用 (Fluorescent molecular probe for detecting genotoxin Colibactin through visual imaging as well as preparation method and application of fluorescent molecular probe ) 是由 吴萃艳 李海涛 李雅倩 卢求钧 张友玉 于 2020-06-22 设计创作，主要内容包括：本发明公开了一种可视化成像检测基因毒素Colibactin荧光分子探针及其制备方法和应用,该探针的结构式如(1)所示：<Image he="494" wi="700" file="DDA0002549964760000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>本发明设计合成了一种基于分子内电荷转移的小分子荧光探针,能够专一性检测识别产基因毒素Colibactin大肠杆菌,不受其他生物分子、离子、细菌等干扰。同时,本探针具有近红外荧光发射,能够应用于产基因毒素Colibactin大肠杆菌的激光共聚焦成像。(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): the invention designs and synthesizes a micromolecular fluorescent probe based on intramolecular charge transfer, can specifically detect and identify escherichia coli producing genotoxin Colibactin, and is not interfered by other biomolecules, ions, bacteria and the like. Meanwhile, the probe has near-infrared fluorescence emission and can be applied to laser confocal of escherichia coli producing genotoxin ColibactinLike this.)

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).

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.S^2-,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