Synthesis and application of fluorescent probe for selective detection of homocysteine
By synthesizing fluorescent probes with specific chemical structures and detecting Hcy in specific solution ratios, the problems of low sensitivity and poor selectivity in existing technologies have been solved, achieving highly sensitive Hcy detection and quantification, suitable for applications in biological samples and the environment.
Patent Information
- Application Number
- CN202211195448.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2022-09-29
- Publication Date
- 2025-02-07
- Estimated Expiration
- 2042-09-29
AI Technical Summary
Existing fluorescent probes suffer from low sensitivity and poor selectivity when detecting homocysteine (Hcy), making it difficult to achieve direct and rapid quantification. Furthermore, existing methods are time-consuming and costly, failing to meet the needs of clinical applications.
A fluorescent probe for selective detection of Hcy was synthesized. Hcy was detected in a mixed solution of organic and aqueous phases using a fluorescent probe with a specific chemical structure. The fluorescent probe was synthesized using a specific chemical reaction and detected in a solution with an organic phase to aqueous phase ratio of 4:6, achieving highly sensitive and selective detection of Hcy.
It achieves highly sensitive and selective detection of Hcy, enabling selective fluorescence imaging in biological samples and direct quantification of total Hcy in serum. It is suitable for quantitative detection of Hcy in the environment and has the advantages of simple operation and low cost.
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Figure CN115490674B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of analytical chemistry, and specifically relates to the synthesis of a class of fluorescent probes for selectively detecting Hcy, and the application of the probes in preparing devices for directly quantifying total Hcy in serum, quantitatively detecting Hcy in the environment, and selectively fluorescently imaging Hcy in biological samples. Background Art
[0002] Homocysteine is a sulfur-containing amino acid, which is a non-essential amino acid for the human body. It is an intermediate product of methionine in the protein intake of the human body metabolized into glutathione and S-adenosylmethionine (SAMe) in the body. Under normal circumstances, Hcy in plasma is at a low level, but when Hcy in the blood increases (≥10μmol / L), it will have a significant impact on human health. In recent years, studies have found that the accuracy of serum Hcy levels in predicting myocardial infarction is almost 40 times that of cholesterol; high concentrations of Hcy are independent risk factors for coronary artery disease, cerebrovascular disease, and peripheral vascular disease. At present, the methods for quantifying Hcy in serum mainly include chromatography, enzyme-linked immunosorbent assay, chemiluminescence, and cyclic enzyme method, but these methods have the disadvantages of being time-consuming, costly, and requiring special instruments. Therefore, the development of efficient detection methods / techniques for direct and rapid quantification of total serum Hcy levels has very important clinical significance and broad application prospects.
[0003] In recent years, fluorescent probes have attracted extensive attention due to their good selectivity, high sensitivity, simple operation, and ability to monitor and visualize biomolecules in living cells. So far, some fluorescent probes have been reported for detecting Hcy. However, the reported Hcy fluorescent probes have problems such as low sensitivity and poor selectivity, making them difficult to be used for direct and rapid quantification of actual samples. Summary of the invention
[0004] In view of the above situation, in order to overcome some deficiencies of the prior art, the present invention aims to provide a type of fluorescent probe for selectively detecting homocysteine, as well as an application method of the probe in preparing a reagent for directly quantifying total Hcy in serum, and the application of the probe in preparing a device for quantitatively detecting Hcy in the environment and selectively fluorescently imaging Hcy in biological samples.
[0005] The present invention also aims to provide a method for synthesizing a fluorescent probe for selectively detecting Hcy with a simple preparation method.
[0006] The specific technical solution adopted by the present invention to solve the problem is to synthesize and apply a fluorescent probe for selectively detecting homocysteine, and the chemical structure formula of the probe is as follows:
[0007] Here, R is a 1-alkylpyridinium salt group, a substituted 1-alkylpyridinium salt group, a 1-(substituted alkyl)pyridinium salt group, or a substituted 1-(substituted alkyl)pyridinium salt group.
[0008] The synthesis of a fluorescent probe for selectively detecting homocysteine is characterized in that the preparation method of the fluorescent probe comprises the following steps:
[0009] Add raw material 1 and raw material 2 containing R substituents into an appropriate amount of organic solvent, stir and react at 40-95°C for 5-48 hours, and purify after the reaction to obtain the fluorescent probe shown above. The synthesis route is as follows: R is defined as above.
[0010] The preparation method of the raw material 1 comprises the following steps:
[0011] Step 1. Synthesis of 4-chloro-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carbaldehyde
[0012] I. At -10--5℃, slowly add an appropriate amount of concentrated sulfuric acid to an equal volume of concentrated nitric acid, then add 4-chloro-7-(diethylamino)-2-oxo-2H-chromene-3-carbaldehyde, and continue stirring to react for 10-60 minutes;
[0013] II. After the reaction is complete, the reaction solution is slowly added to an appropriate amount of ice water, filtered, and the filter cake is purified by column chromatography to obtain 4-chloro-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde;
[0014] Step 2. Synthesis of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carbaldehyde
[0015] a. Add an appropriate amount of 4-chloro-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde to anhydrous dichloromethane, then add an appropriate amount of n-butyl mercaptan and triethylamine, and stir at room temperature for 5-24 hours;
[0016] b. After the reaction is complete, the solvent is dried on a rotary evaporator and purified by column chromatography to obtain a yellow solid product 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carbaldehyde.
[0017] The method of using the fluorescent probe of the present invention to detect Hcy: unless otherwise specified, the probe molecule is usually dissolved in an environment where the volume ratio of the organic phase to the aqueous phase is 4:6 at room temperature for detecting Hcy, the organic phase is dimethyl sulfoxide (DMSO), and the aqueous phase is a phosphate buffer solution (PBS) with a pH of 7.4 and an aqueous solution of the analyte. The specific features are as follows: the fluorescent probe is dissolved in dimethyl sulfoxide (DMSO), and the detection environment for the probe to detect Hcy is a buffer solution with a volume ratio of the organic phase to the aqueous phase of 4:6. The specific test method is: take 20 μL of 1 mM probe solution, 780 μL of analytical grade DMSO, the required amount of PBS buffered aqueous solution and the required amount of 1 mM Hcy aqueous solution in a 2 mL sample tube, and all tests maintain a volume ratio of the organic phase to the aqueous phase of 4:6 (the total volume of each test sample is 2 mL). For example, when the spectral changes after the probe reacts with Hcy when the Hcy concentration is 10μM are required to be tested, the sample preparation is as follows: 20μL of 1mM probe solution, 780μL of analytical grade DMSO, 1180μL of PBS buffer solution and 20μL of 1mM Hcy aqueous solution are placed in a 2mL sample tube, and the spectral changes can be measured after shaking at room temperature. This probe has extremely high selectivity in detecting Hcy, has no obvious response to other common amino acids, metal ions, reactive oxygen and reactive nitrogen species, and has high sensitivity in detecting Hcy. This type of probe can achieve long-wavelength absorption and red / near-infrared fluorescence selective detection of Hcy ( Figure 1 , Figure 2 ). Therefore, the fluorescent probe disclosed in the present invention can achieve highly sensitive and selective detection of Hcy. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 The fluorescent probe synthesized in Example 1 of the present invention detects cysteine (Cys), Hcy, glutathione (GSH), N-acetylcysteine (NAC), NaHSO 3 , sodium hydrosulfide (NaHS) and sodium polysulfide (Na 2 S n )'s UV-visible absorption spectrum.
[0019] Figure 2 The fluorescent probe synthesized in Example 1 of the present invention detects cysteine (Cys), Hcy, glutathione (GSH), N-acetylcysteine (NAC), NaHSO 3 , sodium hydrosulfide (NaHS) and sodium polysulfide (Na 2 S n )'s fluorescence emission spectrum. DETAILED DESCRIPTION
[0020] The synthetic route of the fluorescent probe described in the present invention is as follows:
[0021]
[0022] Example 1. Synthesis of probe 2-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)-1-methylpyridinium-1-iodide. The synthesis method is as follows:
[0023]
[0024] 100.0 mg (264.2 μmol) of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde and 103.1 mg (396.4 μmol) of 2-(cyanomethyl)-1-methylpyridinium-1-iodide were added to 10 mL of anhydrous ethanol, and the mixture was stirred at 80° C. for 24 hours. After the reaction was complete, the mixture was filtered and the filter cake was recrystallized from anhydrous ethanol to obtain 110.0 mg of an orange-yellow solid with a yield of 67.1%. The maximum absorption and emission peaks of the probe for detecting Hcy were 543 nm and 627 nm, respectively.
[0025] Example 2. Synthesis of 3-bromo-4-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)-1-(4-hydroxybutyl)pyridinium-1-iodide. The synthesis method is as follows:
[0026]
[0027] 100.0 mg (264.2 μmol) of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde and 209.8 mg (528.5 μmol) of 3-bromo-4-(cyanomethyl)-1-(4-hydroxybutyl)pyridinium-1-iodide were added to 10 mL of anhydrous ethanol, and the mixture was stirred at 70° C. for 48 hours. After the reaction was complete, the mixture was filtered, and the filter cake was recrystallized from anhydrous ethanol to obtain 98.0 mg of a red-black solid with a yield of 48.9%. The probe can detect Hcy by near-infrared fluorescence.
[0028] Example 3. Synthesis of 3-(2-(4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromen-3-yl)-1-cyanovinyl)-1-(3-carboxypropyl))pyridinium-1-iodide. The synthesis method is as follows:
[0029]
[0030] 100.0 mg (264.2 μmol) of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde and 219.4 mg (660.6 μmol) of 1-(3-carboxypropyl)-3-(cyanomethyl)pyridinium-1-iodide were added to 10 mL of anhydrous ethanol and stirred at 90° C. for 48 hours. After the reaction was complete, 25.0 mg of an orange-red solid was purified by column chromatography with a yield of 13.7%.
[0031] Example 4. Application of the fluorescent probe of the present invention in detecting Hcy
[0032] The spectral property experiment of detecting Hcy by the fluorescent probe described in the present invention: the probe is dissolved in dimethyl sulfoxide (DMSO) to prepare a probe solution with a concentration of 1mM, and an aqueous solution of Hcy with a concentration of 1mM is prepared. The specific test method for detecting Hcy is: take 20μL of the probe solution (1mM), 780μL of analytical grade DMSO, the required amount of 1mM Hcy aqueous solution and the required amount of PBS buffer aqueous solution in a 2mL sample tube, and all tests keep the final volume ratio of the organic phase and the aqueous phase at 4:6 (the total volume of each test sample is 2mL), and after shaking at room temperature, the reaction rate at a specific time or the absorbance or fluorescence intensity of the reaction product per unit time can be measured.
[0033] Example 5. Application of the fluorescent probe of the present invention in direct quantification of total Hcy in serum
[0034] The operation method for directly quantifying total Hcy in serum is as follows: take 0.2 mL of serum, add tris(2-carboxyethyl)phosphine (40 μL, 10 mM) for reduction treatment for 30 minutes. Take 20 μL of probe solution (10 mM, taking the probe synthesized in Example 1 as an example), 780 μL of analytical grade DMSO, 960 μL of PBS buffer, and then add the above serum solution. After reacting at room temperature for 2 minutes, measure its absorption spectrum, and calculate the concentration of total Hcy in serum according to the standard curve. The experimental results of 5 serum samples are shown in the following table:
[0035] Serial number Hcy concentration (μM) 1 7.6 2 12.8 3 20.3 4 27.7 5 36.9 .
[0036] Example 4. Application of endogenous Hcy in fluorescent imaging cells
[0037] The SH-SY5Y 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, taking the probe synthesized as shown in Example 2 as an example) was added and continued to be cultured under standard growth conditions for 30 minutes, and then photographed under a confocal fluorescence microscope. Fluorescence imaging was performed using a near-infrared fluorescence channel, and obvious red fluorescence was observed. The fluorescent probe of the present invention can realize fluorescence imaging analysis of endogenous Hcy in cells, and has great application value in the fields of biochemistry, analytical detection, and environmental science.
[0038] The synthesis and application of the fluorescent probe for selective detection of homocysteine of the present invention reports a class of fluorescent probes for selective detection of Hcy and their synthesis and application methods, which can realize direct and rapid quantification of Hcy, and selective fluorescence imaging analysis of Hcy in biological samples, and can be used to prepare devices for direct quantification of total Hcy in serum, quantitative detection of Hcy in the environment, and selective fluorescence imaging of Hcy in biological samples. Although the content of the present invention has been described in detail through the above preferred embodiments, it should be recognized that the above description should not be considered as a limitation of the present invention. After reading the above content, various modifications and substitutions of the present invention will be obvious to those skilled in the art. Therefore, the synthesis and application of the fluorescent probe for selective detection of homocysteine with the technical features described herein all fall within the scope of protection of this patent.
Claims
1. A fluorescent probe for selectively detecting homocysteine, characterized in that: The chemical structure formula of the fluorescent probe is as follows: Wherein, R is a 1-alkylpyridinium salt group, and the alkyl group in R is an alkyl group with 1 to 10 carbon atoms.
2. The synthesis of the fluorescent probe according to claim 1, characterized in that The preparation method of the fluorescent probe is as follows: Add raw material 1 and raw material 2 containing R substituents into an appropriate amount of organic solvent, stir and react at 40-95°C for 5-48 hours, and purify after the reaction to obtain the fluorescent probe shown in claim 1. The synthesis route is as follows: The definition of R is the same as that in claim 1.
3. The synthesis of the fluorescent probe according to claim 2, characterized in that The preparation method of the raw material 1 comprises the following steps: Step 1. Synthesis of 4-chloro-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde I. Slowly add an appropriate amount of concentrated sulfuric acid to an equal volume of concentrated nitric acid at -10–-5°C, then add 4-chloro-7-(diethylamino)-2-oxo-2H-chromene-3-carboxaldehyde, and continue stirring the reaction for 10–60 minutes; II. After the reaction is complete, the reaction solution is slowly added to an appropriate amount of ice water, filtered, and the filter cake is purified by column chromatography to obtain 4-chloro-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde; Step 2. Synthesis of 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carbaldehyde a. Add an appropriate amount of 4-chloro-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde to anhydrous dichloromethane, then add an appropriate amount of n-butyl mercaptan and triethylamine, and stir at room temperature for 5–24 hours; b. After the reaction is complete, the solvent is dried on a rotary evaporator and purified by column chromatography to obtain a yellow solid product 4-(butylthio)-7-(diethylamino)-6-nitro-2-oxo-2H-chromene-3-carboxaldehyde.
4. The synthesis of the fluorescent probe according to claim 3, characterized in that The molar ratio of the raw material 1 to the raw material 2 is 1:(1-3); the organic solvent is ethanol, methanol or a mixed solvent thereof.
5. The use of the fluorescent probe as claimed in claim 1, characterized in that: The fluorescent probe is used in preparing a reagent for directly quantifying total Hcy in serum.
6. Use of the fluorescent probe according to claim 5 in preparing a reagent for directly quantifying total Hcy in serum, characterized in that: The direct quantitative inclusion method includes the following steps: i. Reduction of oxidized Hcy in serum: Mix serum with an appropriate amount of tris(2-carboxyethyl)phosphine and incubate at 20–37°C for 10–30 minutes; ii. UV detection: Take an appropriate amount of the serum solution in step i and add it to the probe solution of dimethyl sulfoxide and PBS buffer in a volume ratio of 4:
6. After obtaining the test solution, the reaction rate at a specific time or the absorbance of the reaction product per unit time can be measured; iii. Result calculation: Use graphing software to calculate the concentration of total Hcy in serum based on the standard curve prepared in the experiment and the reaction rate or absorbance value detected in step ii.
7. The use of the fluorescent probe as claimed in claim 1, characterized in that: The fluorescent molecular probe can be used to prepare devices for quantitatively detecting Hcy in the environment and for fluorescent imaging of Hcy in biological samples.