Ratio fluorescent probe for high-selectivity detection of colorectal cancer marker homocysteine, preparation method of ratio fluorescent probe, and method for detecting homocysteine and imaging homocysteine by using ratio fluorescent probe

Abstract

The invention provides a ratiometric fluorescent probe for high-selectivity detection of colorectal cancer marker homocysteine, a preparation method of the ratiometric fluorescent probe, and a method for detecting homocysteine and imaging homocysteine by using the ratiometric fluorescent probe, and relates to the field of analytical chemistry. The ratio fluorescent probe adopts a double binding site strategy, nitryl is introduced into a coumarin fluorophore to realize specific detection of homocysteine, and meanwhile, a chromophore with strong green fluorescence is connected through piperazine to realize ratio detection of homocysteine. Potential interference of background fluorescence can be avoided through the ratio of red fluorescence to green fluorescence, so that the sensitivity and precision of homocysteine detection are improved. In addition, the probe has the advantages of good selectivity, good biocompatibility and the like when being used for detecting homocysteine, and has a huge application prospect in the technical fields of analytical chemistry, life science, biomedical treatment and the like.

Description

Technical Field

[0001] The present application relates to the field of analytical chemistry, and in particular to a ratio fluorescent probe for highly selectively detecting homocysteine, a marker of colorectal cancer, and a preparation method thereof, and a method for detecting homocysteine ​​and imaging homocysteine ​​using the ratio fluorescent probe. Background Art

[0002] Homocysteine ​​(Hcy) is a key intermediate in the methionine cycle and cysteine ​​synthesis. Abnormal methylation / transsulfurization metabolism will lead to an increase in Hcy content, resulting in hyperhomocysteinemia. The total serum Hcy level directly reflects the state of methylation and transsulfurization metabolism in the body. Excessive Hcy (>15μM) can damage cells, tissues, and organs. It is an independent risk factor or an important risk factor for many chronic diseases (Diabetes and Vascular Disease Research 2007, 4, 143-149), and has become the most accurate independent health indicator after hypertension, hyperlipidemia, and hyperglycemia (International Journal of Molecular Sciences 2016, 17, 1733). In various epidemiological and clinical correlation analyses, elevated serum total Hcy levels are closely associated with major diseases such as cardiovascular disease, Alzheimer's disease, dementia, Parkinson's disease, pregnancy syndrome, habitual abortion, osteoporosis, and cancer (Food Science & Nutrition 2020, 8, 4696-4707; Metabolites, 2021, 11, 37). Homocysteine ​​is an important indicator of chronic inflammation, and its elevated levels promote the occurrence or progression of colorectal cancer through inflammatory mechanisms (Autoimmunity Reviews, 2007, 503–509). In patients with inflammatory bowel disease, elevated homocysteine ​​levels are very common and are closely associated with colorectal cancer. At the same time, serum homocysteine ​​levels are also one of the effective markers of dietary inflammation, and dietary inflammation is closely related to the recurrence of colorectal adenomas (Cancer Epidemiology, Biomarkers & Prevention, 2010, 19, 1441–1452). As is known, ratiometric fluorescent probes are able to measure emission intensities at two different wavelengths, thus providing built-in correction for environmental effects (Chemical Society Reviews 2018, 47, 2873-2920; Chemical Society Reviews 2020, 49, 143-179). Compared with single-emission fluorescent probes, this type of fluorescent probe eliminates errors caused by various factors, such as the concentration of the probe molecule itself, and increases the dynamic range of fluorescence measurements.

[0003] At present, a variety of methods have been developed to detect Hcy and viscosity, such as liquid chromatography-mass spectrometry (LCMS), high performance liquid chromatography, gas chromatography-mass spectrometry (GCMS) and fluorescence spectroscopy. Among these detection methods, fluorescent probe analysis is generally favored due to its ability of rapid response and high sensitivity, as well as spatial resolution and satisfactory biocompatibility (Angew. Chem. Int. Ed. 2017, 56, 16611–16615; Anal. Chem. 2016, 76, 166–181). Some fluorescent probes for detecting Hcy have been reported (Angew. Chem. Int. Ed. 2018, 57, 4991; Analyst, 2022, 147, 2470), but it is difficult to detect homocysteine ​​by ratio. And homocysteine ​​and cysteine ​​are similar in structure and difficult to distinguish. Therefore, it is particularly difficult to detect highly selective ratios without interference from other substances, which is one of the difficulties in current research. Summary of the invention

[0004] The purpose of the present application is to provide a ratio fluorescent probe for highly selectively detecting homocysteine, a marker of colorectal cancer, and a method for preparing the same, as well as a method for detecting homocysteine ​​and imaging homocysteine ​​using the same, so as to solve the above-mentioned problems.

[0005] To achieve the above objectives, this application adopts the following technical solutions:

[0006] A ratiometric fluorescent probe for highly selective detection of homocysteine, a marker of colorectal cancer, wherein the structural formula of the ratiometric fluorescent probe is:

[0007]

[0008] The present application also provides a method for preparing the ratio fluorescent probe for highly selectively detecting homocysteine, a marker of colorectal cancer, comprising:

[0009] 11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine is added, and after stirring, 1-BOC-piperazine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added to carry out a first reaction, and after the reaction, a first separation and purification is carried out to obtain 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazine-1-carboxylic acid tert-butyl ester;

[0010] The tert-butyl 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazine-1-carboxylate is added to anhydrous dichloromethane, and then trifluoroacetic acid is added to carry out a second reaction. After the reaction is completed, the solvent is removed, and a second separation and purification is carried out to obtain 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate;

[0011] (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine is added to carry out a third reaction, and then the 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate is added, and after stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added to carry out a fourth reaction. After the reaction is completed, the solvent is removed, and a third separation and purification is carried out to obtain the ratio fluorescent probe.

[0012] Preferably, the molar ratio of the (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid and the 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetic acid is 1:1.

[0013] Preferably, the first reaction, the second reaction, the third reaction and the fourth reaction are all carried out at room temperature.

[0014] Preferably, the first separation and purification, the second separation and purification, and the third separation and purification are all performed by column chromatography.

[0015] The present application also provides a method for detecting homocysteine ​​using the ratio fluorescent probe for highly selectively detecting homocysteine, a colorectal cancer marker, comprising:

[0016] The ratio fluorescent probe is dissolved in a solvent and reacted with a homocysteine-containing system, the red fluorescence intensity at 650 nm and the green fluorescence intensity at 505 nm are measured, and the ratio of the red fluorescence intensity at 650 nm to the green fluorescence intensity at 505 nm is calculated to obtain the homocysteine ​​content in the homocysteine-containing system.

[0017] Preferably, the solvent is a mixed solution of dimethyl sulfoxide and PBS.

[0018] Preferably, in the mixed solution, the volume ratio of the dimethyl sulfoxide to the PBS is 5:5.

[0019] Preferably, the measurement is performed under an excitation wavelength of 450 nm.

[0020] The present application also provides a method for imaging homocysteine ​​using the ratio fluorescent probe for highly selectively detecting homocysteine, a colorectal cancer marker, comprising:

[0021] adding the ratio fluorescent probe to cells, tissues or living bodies containing homocysteine, and then culturing;

[0022] The homocysteine ​​was imaged under a confocal fluorescence microscope using a green channel of 490-550 nm and a near-infrared fluorescence channel of 580-700 nm.

[0023] Compared with the prior art, the beneficial effects of this application include:

[0024] The ratio fluorescent probe for highly selective detection of colorectal cancer marker homocysteine ​​provided by the present application adopts a dual binding site strategy, and introduces a nitro group in the coumarin fluorophore to achieve specific detection of homocysteine, and simultaneously connects a chromophore with strong green fluorescence through piperazine to achieve ratio detection of homocysteine. The probe combines two coumarin derivatives by piperazine, and the coumarin derivative with a nitro moiety can quickly and highly selectively detect homocysteine ​​from various bioactive substances, while another coumarin derivative emits strong green fluorescence to act as a ratio signal, which can overcome the interference of fluorescence self-quenching and background signals. In addition, the probe has the advantages of fast real-time response, high sensitivity, and good chemical stability. When excited at 430nm, the probe itself shows a strong green fluorescence of 505nm. When reacting with homocysteine, the probe not only shows a green fluorescence of 540nm, but also emits near-infrared fluorescence at 650nm, and the potential interference of background fluorescence can be avoided by the ratio of red fluorescence to green fluorescence, thereby improving the sensitivity and accuracy of detecting homocysteine. The ratio of fluorescence at 650nm and 505nm can be used to accurately and quantitatively detect homocysteine, with a detection limit of as low as 28.4nM. In addition, the probe has good selectivity and good biocompatibility in detecting homocysteine, and has great application prospects in technical fields such as analytical chemistry, life sciences, and biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0026] Figure 1 The nuclear magnetic resonance hydrogen spectrum of the ratiometric fluorescent probe provided in this application;

[0027] Figure 2 The ultraviolet and fluorescence spectra of the ratiometric fluorescent probe provided in the present application in response to homocysteine;

[0028] Figure 3 A fluorescence quantitative analysis diagram of the ratiometric fluorescent probe provided in the present application in response to homocysteine;

[0029] Figure 4 The present application provides a cellular map of endogenous homocysteine ​​in cells using dual-channel imaging of the ratiometric fluorescent probe. DETAILED DESCRIPTION

[0030] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0031] Example

[0032] This embodiment provides a ratiometric fluorescent probe for highly selective detection of homocysteine, a marker of colorectal cancer, and the preparation method thereof is as follows:

[0033] 1. Add 500.0 mg (1.75 mmol) of 11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxylic acid to 15 mL of anhydrous dichloromethane, then add 21.4 mg of dimethylaminopyridine (DMAP), stir for 5 min, then add 391.7 mg (2.10 mmol) of 1-BOC-piperazine and 502.2 mg (2.62 mmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was added and the reaction was stirred at room temperature overnight. After the reaction was complete, column chromatography was used for separation and purification to obtain 645.1 mg of tert-butyl 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazine-1-carboxylate with a yield of 81.45%.

[0034] 2. Add 500.0 mg (1.1 mmol) of tert-butyl 4-(11-oxo-2,3,6,7-tetrahydro-1H, 5H, 11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazine-1-carboxylate to 8 mL of anhydrous dichloromethane, and then add 2 mL of trifluoroacetic acid. Stir overnight at room temperature. After the reaction is complete, spin dry the reaction system, separate and purify by column chromatography to obtain 464.0 mg of 4-(11-oxo-2,3,6,7-tetrahydro-1H, 5H, 11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate with a yield of 90.05%.

[0035] 3. Add 100.0 mg (172.81 μmol) of (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid to 6 mL of anhydrous dichloromethane, and then add 10.5 mg 4-Dimethylaminopyridine (DMAP) was reacted at room temperature for 5 minutes, and then 80.8 mg (172.81 μmol) of 4-(11-oxo-2,3,6,7-tetrahydro-1H, 5H, 11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate was added and stirred for 5 minutes. Then, 49.7 mg (259.22 μmol) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added and stirred at room temperature overnight. After the reaction was complete, the reaction system was dried by rotary evaporation, and 80.0 mg of the target ratio fluorescent probe was obtained by column chromatography purification and separation with a yield of 50.64%.

[0036] The reaction equation is as follows:

[0037]

[0038] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the ratiometric fluorescent probe provided in this application.

[0039] Experimental Example 1 - Ratiometric fluorescent probe for detection of homocysteine ​​in vitro

[0040] This experimental example is a ratiometric fluorescence probe spectral property experiment, as follows:

[0041] The bifunctional probe obtained above was dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1mM, and the concentration of homocysteine ​​was 10mM. The specific test method is: take 20μL of 1mM probe solution, add 20μL of 10mM analyte solution, and finally add 980μL of analytical pure DMSO and 980μL PBS. The volume ratio of organic phase to aqueous phase is kept at 5:5 for all tests (the total volume of each test sample is 2mL). For example, when the fluorescence intensity of homocysteine ​​with a concentration of 100μM is required to be tested, the sample preparation is: take 20μL of 1mM probe solution, 20μL of 10mM homocysteine ​​aqueous solution, and then add 980μL of analytical pure DMSO and 980μL of PBS buffer solution in a 2mL sample tube. After shaking at room temperature for 30 minutes, the fluorescence emission intensity can be measured with an excitation wavelength of 450nm. Other test operations are similar to the above steps.

[0042] Figure 2The ultraviolet and fluorescence spectra of the ratiometric fluorescent probe provided in the present application in response to homocysteine; Figure 3 This is a fluorescence quantitative analysis diagram of the ratiometric fluorescent probe provided in this application in response to homocysteine.

[0043] Experimental Example 2-Dual-channel fluorescence imaging analysis of homocysteine ​​in HepG2 (liver cancer cell) cells

[0044] HepG2 cells were passaged into a confocal dish cell culture medium, and after culturing for 24 hours under standard growth conditions, an appropriate amount of probe (5 μM) was added and continued to be cultured under standard growth conditions for 30 minutes, and then photographed under a confocal fluorescence microscope, and homocysteine ​​in the HepG2 cells was imaged using a 490-550 nm green channel and a 580-700 nm near-infrared fluorescence channel, respectively. The fluorescent probe of the present invention can emit fluorescence of different wavelengths in the cells, indicating that the probe can detect homocysteine ​​in the cells by dual-channel ratio, and the ratio fluorescence imaging analysis of homocysteine ​​in the cells is successfully achieved.

[0045] Figure 4 The present application provides a cellular map of endogenous homocysteine ​​in cells using dual-channel imaging of the ratiometric fluorescent probe.

[0046] The present application provides a ratio fluorescent probe for highly selective detection of homocysteine, a marker of colorectal cancer. Two coumarin derivatives are combined by piperazine. The coumarin derivative with a nitro moiety can quickly and highly selectively detect homocysteine ​​from various biologically active substances, while the other coumarin derivative emits strong green fluorescence as a ratio signal. When reacting with homocysteine, it emits 505nm green fluorescence and 650nm near-infrared fluorescence at an excitation wavelength of 430nm. The ratio of the fluorescence signals at the two locations is used to detect homocysteine. The probe has the advantages of good water solubility, fast response speed, large Stokes shift, etc., and has great practical application value in the fields of biochemistry, analytical detection, etc.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A ratiometric fluorescent probe for highly selective detection of homocysteine, a marker for colorectal cancer, characterized in that: The structural formula of the ratiometric fluorescent probe is:

2. A method for preparing a ratiometric fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to claim 1, characterized in that: include: 11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine is added, and after stirring, 1-BOC-piperazine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride are added to carry out a first reaction, and after the reaction, a first separation and purification is carried out to obtain 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazine-1-carboxylic acid tert-butyl ester; The tert-butyl 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazine-1-carboxylate is added to anhydrous dichloromethane, and then trifluoroacetic acid is added to carry out a second reaction. After the reaction is completed, the solvent is removed, and a second separation and purification is carried out to obtain 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate; (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid is added to anhydrous dichloromethane, and then 4-dimethylaminopyridine is added to carry out a third reaction, and then the 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetate is added, and after stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added to carry out a fourth reaction. After the reaction is completed, the solvent is removed, and a third separation and purification is carried out to obtain the ratio fluorescent probe.

3. The method for preparing a ratiometric fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to claim 2, characterized in that: The molar ratio of the (E)-2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-yl)-1-cyanovinyl)benzo[d]thiazole-6-carboxylic acid and the 4-(11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carbonyl)piperazin-1-ium 2,2,2-trifluoroacetic acid is 1:

1.

4. The method for preparing a ratiometric fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to claim 2, characterized in that: The first reaction, the second reaction, the third reaction and the fourth reaction are all carried out at room temperature.

5. The method for preparing a ratiometric fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to any one of claims 2 to 4, characterized in that: The first separation and purification, the second separation and purification, and the third separation and purification are all performed by column chromatography.

6. A method for detecting homocysteine ​​using the ratiometric fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to claim 1, characterized in that: include: The ratio fluorescent probe is dissolved in a solvent and reacted with a homocysteine-containing system, the red fluorescence intensity at 650 nm and the green fluorescence intensity at 505 nm are measured, and the ratio of the red fluorescence intensity at 650 nm to the green fluorescence intensity at 505 nm is calculated to obtain the homocysteine ​​content in the homocysteine-containing system.

7. The method for detecting homocysteine ​​using the ratio fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to claim 6, characterized in that: The solvent is a mixed solution of dimethyl sulfoxide and PBS.

8. The method for detecting homocysteine ​​using the ratio fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to claim 7, characterized in that: In the mixed solution, the volume ratio of the dimethyl sulfoxide to the PBS is 5:

5.

9. The method for detecting homocysteine ​​using the ratio fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to any one of claims 6 to 8, characterized in that: The measurement was performed at an excitation wavelength of 450 nm.

10. A method for imaging homocysteine ​​using the ratiometric fluorescent probe for highly selectively detecting homocysteine, a marker for colorectal cancer, according to claim 1, characterized in that: include: adding the ratio fluorescent probe to cells, tissues or living bodies containing homocysteine, and then culturing; The homocysteine ​​was imaged under a confocal fluorescence microscope using a green channel of 490-550 nm and a near-infrared fluorescence channel of 580-700 nm.

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