Synthesis and application of a bifunctional fluorescent probe for simultaneous discrimination of mercury ions and amyloid

Abstract

The application relates to a synthesis of a bifunctional fluorescent probe for simultaneously distinguishing mercury ions and amyloid and application thereof. The application discloses a bifunctional fluorescent probe for simultaneously distinguishing mercury ions (Hg 2+ ) and amyloid by using green and red fluorescent emission signals, and the chemical structural formula of the fluorescent probe is as follows: the fluorescent probe can generate different fluorescent substances by using different chemical reactions of the probe with Hg 2+ and amyloid under the same detection condition, thereby emitting green and red fluorescent light of two colors under a specific excitation wavelength, and achieving the purpose of simultaneously distinguishing and detecting Hg 2+ and amyloid. The probe emits green light of 579 nm under the excitation wavelength of 505 nm after the reaction with Hg 2+ , and emits red light of 706 nm under the excitation wavelength of 645 nm after the reaction with amyloid. The probe has the advantages of high sensitivity, good selectivity and large distinguishing degree in the detection of Hg 2+ and amyloid, and has great application prospects in technical fields such as analytical chemistry, life science and environmental science.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to the synthesis of a bifunctional fluorescent probe and its ability to simultaneously distinguish and detect mercury ions (Hg) in the environment. 2+ ) and amyloid protein, mercury ions (Hg) in cells under dual-channel fluorescence imaging 2+ ) and the application of amyloid protein. Background Technology

[0002] Mercury and its derivatives pose a serious threat to the environment and human health due to their persistence, mobility, and high bioaccumulation. Mercury ions (Hg) 2+ Even low-concentration exposure can damage the heart, stomach, intestines, and kidneys. Furthermore, Hg in aquatic ecosystems... 2+ It can be converted into lipophilic, readily absorbed, and poorly excreted organomercury species, affecting many different brain regions and their related functions (Sensor. Actuat B-chem. 2014, 191, 605−611). Therefore, developing selective and sensitive methods for the determination of Hg is crucial. 2+ The methods used are crucial for understanding its distribution and conducting more detailed toxicological studies. Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory impairment, irreversible cognitive decline, and behavioral disabilities. Amyloid proteins, including monomers, oligomers, and fibers, are widely considered to be major biomarkers and therapeutic targets for AD (Anal. Chem. 2021, 93, 12447−12455). Therefore, early diagnosis of amyloid proteins is significant for delaying the progression of AD and slowing irreversible neuronal damage.

[0003] Therefore, for Hg in the body 2+ Simultaneous detection of mercury ions and amyloid is of great significance. While numerous fluorescent probes have been reported for detecting both, most currently only detect one, with very few capable of detecting both, and even then, the degree of differentiation is somewhat lacking. Therefore, simultaneously differentiating between mercury ions and amyloid remains a significant challenge. Summary of the Invention

[0004] In view of the above, and to overcome the shortcomings of the prior art, the object of the present invention is to provide a method for simultaneously distinguishing Hg. 2+ The bifunctional fluorescent probes for amyloid protein provide some analytical detection methods and ideas for fields such as analytical detection and environmental science.

[0005] The present invention also aims to provide a simple method for the synthesis and application of a bifunctional fluorescent probe.

[0006] The specific technical solution adopted by this invention to solve the problem is the synthesis of a bifunctional fluorescent probe and its application in simultaneously distinguishing and detecting mercury ions and amyloid protein. The chemical structural formula of the bifunctional fluorescent probe is as follows:

[0007] A bifunctional fluorescent probe that can simultaneously distinguish between mercury ions and amyloid protein, characterized in that the preparation method of the bifunctional fluorescent probe includes the following steps: 2. The synthesis of the bifunctional fluorescent probe according to claim 1, characterized in that the preparation method of the bifunctional fluorescent probe includes the following steps: Step 1. Synthesis of 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodine a. Dissolve an appropriate amount of 5-carboxy-1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide and 3-(4-(dimethylamino)phenyl)propenal in anhydrous ethanol, add an appropriate amount of piperidine, and stir the reaction at 50 degrees Celsius for 10 hours. b. After the reaction is complete, the mixture is filtered to obtain a blue solid 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodine.

[0008] Step 2. Synthesis of N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracene-3-ylidene)-N-ethylethylamine a. Dissolve an appropriate amount of m-hydroxyphenylpiperazine and 4-diethylaminoketo acid in trifluoroacetic acid, and reflux and stir at 120°C for 24 hours; b. After the reaction is complete, the solvent trifluoroacetic acid is removed under reduced pressure, and the mixture is separated by column chromatography to obtain the red solid N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracene-3-ylidene)-N-ethylethylamine; Step 3. Synthesis of 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one a. Dissolve an appropriate amount of N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracene-3-ylidene)-N-ethylethylamine in anhydrous ethanol, add hydrazine hydrate solution dropwise, and reflux and stir at 80°C for 12 hours. b. After the reaction was complete, the crude product was purified by column chromatography to obtain a milky white oily liquid, 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one.

[0009] Step 6. Synthesize the bifunctional fluorescent probe. The synthesized 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide was dissolved in anhydrous DCM, and 4-dimethylaminopyridine (DMAP) was added. The mixture was stirred for 10-15 minutes, and then 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the crude product was purified by column chromatography to obtain a blue solid, which is the bifunctional fluorescent probe.

[0010] The bifunctional fluorescent probe of this invention can simultaneously distinguish Hg 2+ For use with amyloid: Unless otherwise specified, the probe molecule is usually dissolved at room temperature in an environment with an organic phase and an aqueous phase volume ratio of 5:5 for analysis and detection. The organic phase is dimethyl sulfoxide (DMSO), and the aqueous phase is phosphate buffered solution (PBS) at pH 7.4 and an aqueous solution of the analyte.

[0011] The specific features of the bifunctional fluorescent probe of the present invention are as follows: the molecular fluorescent probe is dissolved in dimethyl sulfoxide (DMSO), and the probe is dissolved in an organic phase and an aqueous phase (5:5, v / v) solution, and reacted with Hg. 2+ After reacting at room temperature for 30 minutes, it emitted strong green fluorescence at 579 nm under an excitation wavelength of 505 nm; after reacting with amyloid protein at room temperature for 30 minutes, it emitted strong red fluorescence at 706 nm under an excitation wavelength of 645 nm. The probe itself did not exhibit obvious fluorescence when detecting Hg. 2+ When Hg is excited, green fluorescence is produced only at 505 nm, while red fluorescence is produced only when excited at 645 nm for the detection of amyloid protein. Therefore, specific excitation and fluorescence emission signals can be used to detect specific analytes. 2+ When both Hg and amyloid are present, they can be effectively distinguished using different excitation and fluorescence emission signals. The fluorescent probe described above enables the simultaneous detection and differentiation of Hg under specific excitation conditions. 2+ And amyloid protein, for NAC, Gly, Ala, His, Met, Thr, Lys, Asp, Glu, Pro, Ser, O2 - , 1O2, ONOO - Amino acids such as TBHP, ·OH, NO, n-Butylamine, and aniline, sulfur-containing derivatives, and other reactive oxygen species showed no significant response. Therefore, the bifunctional fluorescent probe disclosed in this invention can achieve highly sensitive differentiation and detection of both substances. Attached Figure Description

[0012] Figure 1 This is the 1H NMR spectrum of the fluorescent probe of the present invention.

[0013] Figure 2 The fluorescent probe of the present invention is used for the quantitative detection of Hg 2+ In, the fluorescence emission intensity varies with Hg 2+ The changes in concentration are shown on the horizontal axis as wavelength and the vertical axis as fluorescence intensity.

[0014] Figure 3 The graph shows the change in fluorescence emission intensity of the fluorescent probe of the present invention with the concentration of amyloid protein in quantitative detection. The horizontal axis represents wavelength and the vertical axis represents fluorescence intensity.

[0015] Figure 4 The fluorescent probe of this invention simultaneously distinguishes exogenous Hg in HeLa cells for imaging. 2+ And amyloid protein. Detailed Implementation

[0016] The present invention will now be further described with reference to the accompanying drawings.

[0017] The synthetic route of the bifunctional fluorescent probe described in this invention is shown in the following formula:

[0018] Example 1. Synthesis of 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodine a. Dissolve 1.02 g (2.84 mmol) of 5-carboxy-1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide and 676.71 mg (3.86 mmol) of (E)-3-(4-(dimethylamino)phenyl)propenal in 10 mL of anhydrous ethanol, add 30 mg of piperidine, and stir the reaction at 50 °C for 10 hours; b. After the reaction was complete, the mixture was filtered to obtain 1.05 g of blue solid 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodine, with a yield of 71.6%.

[0019] Example 2. Synthesis of N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracene-3-ylidene)-N-ethylethylamine a. Dissolve 3.00 g (16.83 mmol) m-hydroxyphenylpiperazine and 5.27 g (16.83 mmol) 4-diethylaminoketo acid in 50 mL of trifluoroacetic acid and reflux and stir at 120 °C for 24 hours; b. After the reaction was complete, the solvent trifluoroacetic acid was removed under reduced pressure, and the mixture was separated by column chromatography to obtain 3.60 g of red solid N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracene-3-ylidene)-N-ethylethylamine, with a yield of 46.7%.

[0020] Example 3. Step 3. Synthesis of 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one a. Dissolve 500 mg (1.10 mmol) of N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracite-3-ylidene)-N-ethylethylamine in 10 mL of anhydrous ethanol, add 280 mg (5.48 mmol) of hydrazine hydrate solution dropwise, and reflux and stir at 80 °C for 12 hours; b. After the reaction was complete, the crude product was purified by column chromatography to obtain 424 mg of milky white oily liquid 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one, with a yield of 82.5%.

[0021] Step 6. Synthesize the bifunctional fluorescent probe. 95 mg (183.96 μmol) of synthesized 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide was dissolved in 5 mL of anhydrous DCM, and 2.25 mg (18.40 μmol) of 4-dimethylaminopyridine (DMAP) was added. The mixture was stirred for 10–15 minutes, followed by the addition of 86.38 mg (183.96 μmol) of 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one and 52.90 mg (275.94 μmol) of [the following is a separate, unrelated sentence:] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) was stirred at room temperature for 2 hours. After the reaction was complete, the crude product was purified by column chromatography to obtain a blue solid, which is the bifunctional fluorescent probe.

[0022] Example 7. Detection of Hg by a bifunctional fluorescent probe in the in vitro environment 2+ Applications of amyloid protein The bifunctional fluorescent probe of this invention was subjected to the following spectral property experiments: the probe was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mM probe solution, and 10 mM Hg solutions were prepared separately. 2+ And amyloid protein solution. The specific testing method is as follows: Take 20 μL of 1 mM probe solution and 20 μL of analytical grade Hg. 2+ Amyloid protein, 980 μL of DMSO solution, 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 was measured using excitation wavelengths of 505 nm and 645 nm. Other testing procedures were similar to those described above. This bifunctional probe enables the simultaneous detection of Hg using different excitation wavelengths and fluorescence emission signals. 2+ Both amyloid and Hg have high sensitivity. 2+ Both Hg and amyloid have low detection limits, making them ideal for detecting Hg in live cells. 2+ Imaging and analysis of amyloid proteins.

[0023] The present invention provides the synthesis of a bifunctional fluorescent probe and its ability to simultaneously distinguish and detect Hg. 2+ The application method for Hg in amyloid proteins has advantages such as high efficiency, simplicity, low cost, and high accuracy, and can achieve Hg in environmental and biological samples. 2+ Simultaneous differentiation and imaging analysis of Hg and amyloid protein. Although the invention has been described in detail through the above preferred embodiments, it should be understood that the above description should not be considered as limiting the invention. Various modifications and substitutions to the invention will be apparent to those skilled in the art after reading the above. Therefore, the synthesis of a bifunctional fluorescent probe with the technical features described herein and its simultaneous differentiation and detection of Hg... 2+ The application of amyloid protein falls within the scope of protection of this patent.

Claims

1. Simultaneously distinguish Hg 2+ A bifunctional fluorescent probe for amyloid protein, characterized in that, The chemical structure of the bifunctional fluorescent probe is shown in (1): (1).

2. The synthesis of the bifunctional fluorescent probe as described in claim 1, characterized in that, The preparation method of the bifunctional fluorescent probe includes the following steps: Step 1. Synthesis of 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodine a. Dissolve an appropriate amount of 5-carboxy-1-ethyl-2,3,3-trimethyl-3H-indole-1-iodide and 3-(4-(dimethylamino)phenyl)propenal in anhydrous ethanol, add an appropriate amount of piperidine, and stir the reaction at 50 degrees Celsius for 10 hours; b. After the reaction is complete, the mixture is filtered to obtain a blue solid 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodine; Step 2. Synthesis of N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracene-3-ylidene)-N-ethylethylamine a. Dissolve an appropriate amount of m-hydroxyphenylpiperazine and 4-diethylaminoketo acid in trifluoroacetic acid, and reflux and stir at 120°C for 24 hours; b. After the reaction is complete, remove the solvent trifluoroacetic acid under reduced pressure, and separate the product by column chromatography to obtain a red solid N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracene-3-ylidene)-N-ethylethylamine; Step 3. Synthesis of 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one a. Dissolve an appropriate amount of N-(9-(2-carboxyphenyl)-6-(piperazin-1-yl)-3H-anthracene-3-ylidene)-N-ethylethylamine in anhydrous ethanol, add hydrazine hydrate solution dropwise, and reflux and stir at 80°C for 12 hours. b. After the reaction was complete, the crude product was purified by column chromatography to obtain a milky white oily liquid 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one; Step 4. Synthesize the bifunctional fluorescent probe. The synthesized 5-carboxy-2-((1E,3E)-4-(4-(dimethylamino)phenyl)but-1,3-dien-1-yl)-1-ethyl-3,3-dimethyl-3H-indole-1-iodide was dissolved in anhydrous DCM, and 4-dimethylaminopyridine was added. The mixture was stirred for 10-15 minutes, and then 2-amino-3'-(diethylamino)-6'-(piperazin-1-yl)spiro[isoindoline-1,9'-xanthine]-3-one and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were added. The mixture was stirred at room temperature for 2 hours. After the reaction was complete, the crude product was purified by column chromatography to obtain a blue solid, which is the bifunctional fluorescent probe.

3. The application of the bifunctional fluorescent probe as described in claim 1 in device fabrication, characterized in that, The fabricated device can quantitatively detect Hg in the environment. 2+ And amyloid protein, and simultaneously differentiate Hg within cells 2+ And amyloid protein.

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