Application of preparing ratiometric fluorescent probe for high-selectivity detection of colorectal cancer marker homocysteine for tumor diagnosis
By designing a highly selective ratio fluorescent probe and utilizing dual binding sites and nitrocoumarin fluorophores, the selectivity and interference problems of homocysteine detection were solved, enabling rapid and sensitive homocysteine detection, which is applicable to analytical chemistry and life sciences.
Patent Information
- Application Number
- CN202511548832.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-08
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies are difficult to detect homocysteine, a marker of colorectal cancer, with high selectivity, and it is also difficult to distinguish between homocysteine and cysteine. Interfering substances can also affect the detection process.
A ratiometric fluorescent probe for the highly selective detection of colorectal cancer markers was developed. By employing a dual-binding-site strategy, a nitrocoumarin fluorophore was introduced, and two coumarin derivatives were linked using piperazine to achieve specific detection of homocysteine. Background interference was eliminated by using the red-green fluorescence ratio.
It achieves rapid, sensitive, and accurate detection of homocysteine with a detection limit as low as 28.4 nM, exhibiting good selectivity and biocompatibility, and is suitable for analytical chemistry and life sciences.
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Figure CN121758460A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of analytical chemistry, and in particular to a ratiometric fluorescent probe for the highly selective detection of homocysteine, a biomarker of colorectal cancer, a method for its preparation, a method for using the probe to detect homocysteine, and a method for imaging homocysteine. Background Technology
[0002] Homocysteine (Hcy), a key intermediate in the methionine cycle and cysteine synthesis, is elevated in levels due to abnormal methylation / transsulfurization metabolism, leading to hyperhomocysteinemia. Serum total Hcy levels directly reflect the state of methylation and transsulfurization metabolism in the body. Excessively high Hcy levels (>15 µM) can damage cells, tissues, and organs, and are an independent or important risk factor for many chronic diseases (Diabetes and Vascular Disease Research 2007, 4, 143-149). It 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 homocysteine (Hcy) levels have been closely associated with major diseases such as cardiovascular disease, Alzheimer's disease, dementia, Parkinson's disease, pregnancy complications, recurrent miscarriage, osteoporosis, and cancer (Food Science & Nutrition 2020, 8, 4696-4707; Metabolites, 2021, 11, 37). Homocysteine, as an important indicator of chronic inflammation, promotes the occurrence or progression of colorectal cancer through inflammatory mechanisms (Autoimmunity Reviews, 2007, 503–509). Elevated homocysteine levels are very common in patients with inflammatory bowel disease and are closely associated with colorectal cancer. Furthermore, serum homocysteine levels are also an effective marker of diet-induced inflammation, and dietary inflammation is closely related to the recurrence of colorectal adenomas (CancerEpidemiology, Biomarkers & Prevention, 2010, 19, 1441–1452). As is well known, ratiometric fluorescent probes can measure emission intensities at two different wavelengths, thus providing built-in correction for environmental influences (Chemical Society Reviews 2018, 47, 2873-2920; Chemical Society Reviews 2020, 49, 143-179). Compared to single-emission fluorescent probes, these probes eliminate errors caused by various factors, such as the concentration of the probe molecule itself, and increase the dynamic range of fluorescence measurements.
[0003] Currently, various methods have been developed for the detection of homocysteine (Hcy) and viscosity, such as liquid chromatography-mass spectrometry (LCMS), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GCMS), and fluorescence spectroscopy. Among these methods, fluorescent probe analysis is generally favored due to its rapid response, high sensitivity, spatial resolution, and satisfactory biocompatibility (Angew. Chem. Int. Ed. 2017, 56, 16611–16615; Anal. Chem. 2016, 76, 166–181). Several fluorescent probes for the detection of Hcy have been reported (Angew. Chem. Int. Ed. 2018, 57, 4991; Analyst, 2022, 147, 2470), but ratiometric detection of homocysteine is challenging. Furthermore, homocysteine and cysteine have similar structures and are difficult to distinguish. Therefore, it is particularly difficult to detect them with high selectivity without interference from other substances, which is one of the current research challenges. Summary of the Invention
[0004] The purpose of this application is to provide a ratiometric fluorescent probe for the highly selective detection of homocysteine, a biomarker of colorectal cancer, a method for its preparation, a method for using the probe to detect homocysteine, and a method for imaging homocysteine, so as to solve the above-mentioned problems.
[0005] To achieve the above objectives, this application adopts the following technical solution: A ratiometric fluorescent probe for the highly selective detection of homocysteine, a biomarker for colorectal cancer, wherein the structural formula of the ratiometric fluorescent probe is as follows: .
[0006] This application also provides a method for preparing the highly selective ratiometric fluorescent probe for detecting homocysteine, a marker of colorectal cancer, comprising: 11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxylic acid was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine. After stirring, 1-BOC-piperazine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimine hydrochloride were added for the first reaction. After the reaction was completed, the first separation and purification were performed 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 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 was added to anhydrous dichloromethane, and then trifluoroacetic acid was added for a second reaction. After the reaction was completed, the solvent was removed, and a second separation and purification were performed 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-onium 2,2,2-trifluoroacetate. (E)-2-(2-(4-(butthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazol-6-carboxylic acid was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine. After the third reaction, the 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-onium 2,2,2-trifluoroacetate was added. After stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added for the fourth reaction. After the reaction was completed, the solvent was removed, and the ratiometric fluorescent probe was obtained by the third separation and purification.
[0007] Preferably, the molar ratio of (E)-2-(2-(4-(butthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-yl)-1-cyanovinyl)benzo[d]thiazol-6-carboxylic acid and the molar ratio of 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-onium 2,2,2-trifluoroacetic acid is 1:1.
[0008] Preferably, the first reaction, the second reaction, the third reaction, and the fourth reaction are all carried out at room temperature.
[0009] Preferably, the first separation and purification, the second separation and purification, and the third separation and purification are all performed using column chromatography.
[0010] This application also provides a method for detecting homocysteine using the aforementioned highly selective ratiometric fluorescent probe for detecting homocysteine, a marker of colorectal cancer, comprising: The ratiometric fluorescent probe was 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 were measured, and the ratio of the red fluorescence intensity at 650 nm to the green fluorescence intensity at 505 nm was calculated to obtain the homocysteine content in the homocysteine-containing system.
[0011] Preferably, the solvent is a mixed solution of dimethyl sulfoxide and PBS.
[0012] Preferably, in the mixed solution, the volume ratio of dimethyl sulfoxide to PBS is 5:5.
[0013] Preferably, the measurement is performed at an excitation wavelength of 450 nm.
[0014] This application also provides a method for imaging homocysteine using the aforementioned highly selective ratiometric fluorescent probe for detecting the colorectal cancer marker homocysteine, comprising: The ratiometric fluorescent probe is added to cells, tissues, or living organisms containing homocysteine, and then cultured. The homocysteine was imaged under a confocal fluorescence microscope using the 490-550 nm green channel and the 580-700 nm near-infrared fluorescence channel.
[0015] Compared with the prior art, the beneficial effects of this application include: This application provides a highly selective ratiometric fluorescent probe for the detection of homocysteine, a biomarker for colorectal cancer. This probe employs a dual-binding-site strategy, introducing a nitro group into the coumarin fluorophore to achieve specific detection of homocysteine. Simultaneously, it connects a piperazine-based chromophore with strong green fluorescence to achieve ratiometric detection of homocysteine. The probe binds two coumarin derivatives via piperazine. The coumarin derivative with the nitro group can rapidly and selectively detect homocysteine from various bioactive substances, while the other coumarin derivative emits strong green fluorescence as a ratiometric signal, overcoming fluorescence self-quenching and background signal interference. Furthermore, this probe has advantages such as rapid real-time response, high sensitivity, and good chemical stability. Upon excitation at 430 nm, the probe itself exhibits strong green fluorescence at 505 nm. When reacting with homocysteine, the probe not only displays green fluorescence at 540 nm but also emits near-infrared fluorescence at 650 nm. The ratio of red to green fluorescence avoids potential interference from background fluorescence, thereby improving the sensitivity and accuracy of homocysteine detection. The ratio of fluorescence at 650 nm to 505 nm can be used to accurately quantify homocysteine, with a detection limit as low as 28.4 nM. Furthermore, this probe exhibits good selectivity and biocompatibility for homocysteine detection, showing great promise for applications in analytical chemistry, life sciences, and biomedicine. Attached Figure Description
[0016] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.
[0017] Figure 1 The proton NMR spectrum of the ratiometric fluorescent probe provided in this application; Figure 2 The UV and fluorescence spectra of the ratiometric fluorescent probe provided in this application in response to homocysteine; Figure 3 A fluorescence quantitative analysis diagram of the ratiometric fluorescent probe in response to homocysteine provided in this application; Figure 4 is a cell diagram of endogenous homocysteine in cells obtained by dual-channel imaging with a ratiometric fluorescent probe provided in this application. Detailed Implementation
[0018] The implementation schemes of this application will be described in detail below with reference to specific embodiments. However, those skilled in the art will understand that the following embodiments are only for illustrating this application and should not be regarded as limiting the scope of this application. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used without specified manufacturers are all conventional products that can be purchased commercially. Example
[0019] This embodiment provides a ratiometric fluorescent probe for the highly selective detection of homocysteine, a biomarker of colorectal cancer, and its preparation method is as follows: 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, and then add 391.7 mg (2.10 mmol) of 1-BOC-piperazine and 502.2 mg (2.62 mmol) of [the following is a separate, unrelated sentence:] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was reacted overnight at room temperature with stirring. After the reaction was complete, column chromatography was used to separate and purify the product to obtain 645.1 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)piperazine-1-carboxylic acid tert-butyl ester, with a yield of 81.45%.
[0020] 2. 500.0 mg (1.1 mmol) of 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 was added to 8 mL of anhydrous dichloromethane, followed by 2 mL of trifluoroacetic acid. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was evaporated to dryness, and the product was purified 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)piperazine-1-onium 2,2,2-trifluoroacetate, with a yield of 90.05%.
[0021] 3. 100.0 mg (172.81 µmol) (E)-2-(2-(4-(butthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazol-6-carboxylic acid was added to 6 mL of anhydrous dichloromethane, followed by 10.5 mg of 4-dimethylaminopyridine (DMAP). The mixture was reacted at room temperature for 5 min. Subsequently, 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)piperazine-1-onium 2,2,2-trifluoroacetate was added and stirred for 5 min. Finally, 49.7 mg (259.22) of [the following solution was added] was added. The reaction mixture was stirred overnight at room temperature with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (µmol). After the reaction was complete, the reaction system was evaporated to dryness and purified by column chromatography to obtain 80.0 mg of the target ratio fluorescent probe, with a yield of 50.64%.
[0022] The reaction equation is shown below: .
[0023] Figure 1 The proton NMR spectrum of the ratiometric fluorescent probe provided in this application. Experimental Example 1 - Detection of homocysteine with ratiometric fluorescent probes in vitro
[0024] This experimental example demonstrates the spectral properties of a ratiometric fluorescent probe, as detailed below: The bifunctional probe obtained above was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM probe solution, and a 10 mM homocysteine solution was prepared. The specific testing method was as follows: 20 μL of the 1 mM probe solution was taken, followed by 20 μL of the 10 mM analyte solution, and finally 980 μL of analytical grade DMSO and 980 μL of PBS were added. For all tests, the organic phase to aqueous phase volume ratio was maintained at 5:5 (total volume of each test sample was 2 mL). For example, when testing the fluorescence intensity of homocysteine at a concentration of 100 μM, the sample preparation was as follows: 20 μL of the 1 mM probe solution, 20 μL of the 10 mM homocysteine aqueous solution, and then 980 μL of analytical grade DMSO and 980 μL of PBS buffer solution were added to a 2 mL sample tube. After shaking and mixing at room temperature for 30 minutes, the fluorescence emission intensity could be measured using an excitation wavelength of 450 nm. Other testing procedures were similar to the above steps.
[0025] Figure 2 The UV and fluorescence spectra of the ratiometric fluorescent probe provided in this application in response to homocysteine; Figure 3 The fluorescence quantitative analysis diagram of the ratiometric fluorescent probe provided in this application in response to homocysteine.
[0026] Experimental Example 2 - Analysis of Homocysteine Dual-Channel Fluorescence Imaging in HepG2 (Hepatocellular Carcinoma) Cells HepG2 cells were passaged into confocal cell culture medium and cultured under standard growth conditions for 24 hours. Then, an appropriate amount of probe (5 μM) was added and cultured under standard growth conditions for another 30 minutes. The cells were then photographed under a confocal fluorescence microscope, and homocysteine in HepG2 cells was imaged using the 490-550 nm green channel and the 580-700 nm near-infrared fluorescence channel, respectively. The fluorescent probe of this invention can emit fluorescence at different wavelengths in the cells, indicating that the probe can detect homocysteine in the cells using a dual-channel ratio, and successfully realizes the ratio fluorescence imaging analysis of homocysteine in cells.
[0027] Figure 4 is a cell diagram of endogenous homocysteine in cells obtained by dual-channel imaging with a ratiometric fluorescent probe provided in this application.
[0028] This application provides a ratiometric fluorescent probe for the highly selective detection of homocysteine, a biomarker for colorectal cancer. Two coumarin derivatives are bound together by a piperazine. The coumarin derivative with a nitro group can rapidly and selectively detect homocysteine from various bioactive substances, while the other coumarin derivative emits strong green fluorescence as a ratiometric signal. When reacting with homocysteine, it emits 505 nm green fluorescence and 650 nm near-infrared fluorescence at an excitation wavelength of 430 nm. The ratio of these two fluorescence signals is used to detect homocysteine. This probe has advantages such as good water solubility, fast response speed, and large Stokes shift, and has significant practical application value in biochemistry, analytical detection, and other fields.
[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.
Claims
1. An application of a ratiometric fluorescent probe for preparing a highly selective detection biomarker for colorectal cancer, homocysteine, in tumor diagnosis, characterized in that, The structural formula of the ratiometric fluorescent probe is: ， The fluorescent probe is used in the preparation of biomolecular detection reagents for homocysteine.
2. The application of the ratiometric fluorescent probe according to claim 1 for preparing a highly selective colorectal cancer biomarker, homocysteine, for tumor diagnosis, is characterized in that... The ratiometric fluorescent probe is added to cells, tissues, or living organisms containing homocysteine, and then cultured. The homocysteine was imaged using a confocal fluorescence microscope, employing the 490-550 nm green channel and the 580-700 nm near-infrared fluorescence channel. The method described is not a disease diagnosis method.
3. The application of the ratiometric fluorescent probe according to claim 1 for preparing a highly selective marker for detecting homocysteine in colorectal cancer for tumor diagnosis, characterized in that, The method for preparing the fluorescent probe includes: 11-oxo-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1-ij]quinoline-10-carboxylic acid was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine. After stirring, 1-BOC-piperazine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimine hydrochloride were added for the first reaction. After the reaction was completed, the first separation and purification were performed 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 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 was added to anhydrous dichloromethane, and then trifluoroacetic acid was added for a second reaction. After the reaction was completed, the solvent was removed, and a second separation and purification were performed 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-onium 2,2,2-trifluoroacetate. (E)-2-(2-(4-(butthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazol-6-carboxylic acid was added to anhydrous dichloromethane, followed by the addition of 4-dimethylaminopyridine. After the third reaction, the 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-onium 2,2,2-trifluoroacetate was added. After stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was added for the fourth reaction. After the reaction was completed, the solvent was removed, and the ratiometric fluorescent probe was obtained by the third separation and purification.
4. The application of the ratiometric fluorescent probe according to claim 3 for preparing a highly selective colorectal cancer biomarker, homocysteine, for tumor diagnosis, is characterized in that... The molar ratio of (E)-2-(2-(4-(butthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)benzo[d]thiazol-6-carboxylic acid and the molar ratio of 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-onium 2,2,2-trifluoroacetic acid is 1:
1.
5. The application of the ratiometric fluorescent probe according to claim 3 for preparing a highly selective marker for detecting homocysteine in colorectal cancer for tumor diagnosis, characterized in that, The first reaction, the second reaction, the third reaction, and the fourth reaction are all carried out at room temperature.
6. The application of the ratiometric fluorescent probe according to claims 3-4 for preparing a highly selective marker for detecting homocysteine in colorectal cancer for tumor diagnosis, characterized in that... The first separation and purification, the second separation and purification, and the third separation and purification are all performed using column chromatography. The method is not a diagnostic method for diseases.
7. The application of the ratiometric fluorescent probe according to claim 1 for preparing a highly selective colorectal cancer biomarker, homocysteine, for tumor diagnosis, is characterized in that... Its features include: The ratiometric 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. This method is not a disease diagnosis method.
8. The application of the ratiometric fluorescent probe according to claim 7 for preparing a highly selective marker for detecting homocysteine in colorectal cancer for tumor diagnosis, characterized in that, The solvent is a mixture of dimethyl sulfoxide and PBS, and the method is not a diagnostic method for disease.
9. The application of the ratiometric fluorescent probe according to claim 7 for preparing a highly selective marker for detecting homocysteine in colorectal cancer for tumor diagnosis, characterized in that, In the mixed solution, the volume ratio of dimethyl sulfoxide and PBS is 5:5, and the method is not a disease diagnosis method.
10. The application of the ratiometric fluorescent probe for preparing a highly selective colorectal cancer biomarker homocysteine according to any one of claims 7-9, characterized in that, The measurements were performed at an excitation wavelength of 450 nm, and the method is not a diagnostic method for disease.