阅读说明：本技术 一种新型的Hg2+离子探针的合成及应用 (Novel Hg2+Synthesis and application of ion probe ) 是由 朱海亮 齐鹏飞 徐镜 李红林 严晓强 于 2018-06-20 设计创作，主要内容包括：一种含氧杂蒽的Hg~(2+)离子探针,它具有如下通式I所示的结构。本发明公开了一种新型的含氧杂蒽的Hg~(2+)离子探针得制备方法与在水中和活细胞中检测Hg~(2+)离子中的应用。(Hg containing oxyanthracene 2+ The ion probe has a structure shown in the following general formula I. The invention discloses a novel Hg of oxygen-containing heteroanthracene 2+ Preparation method of ion probe and detection of Hg in water and living cells 2+ Application in ions.)

1. Hg containing xanthene²⁺A selective fluorescent probe, known in chinese as 4- (1, 3-dithian-2-yl) -2, 3-dihydro-xanthene, characterized in that it has the structure shown below:

2. a process for the preparation of 4- (1, 3-dithian-2-yl) -2, 3-dihydro-xanthene according to claim 1, characterized in that it consists of the following steps:

3. a method for synthesizing the compound 2 as claimed in claim 2

Dissolving phosphorus tribromide at 0 ℃, adding the dissolved phosphorus tribromide into a round-bottom flask, stirring for 1h, adding the dissolved cyclohexanone, stirring for 18-24h at room temperature, wherein the reaction ratio of the phosphorus tribromide to the cyclohexanone is 3: 1-2: 1, tracking and detecting the reaction progress by TLC (thin layer chromatography) until the cyclohexanone is completely reacted, pouring the reaction liquid into ice water, adjusting the pH of the reaction liquid to be neutral, extracting the reaction liquid for three times by using an organic solvent, taking an organic phase, drying by using anhydrous sodium sulfate, evaporating the liquid of the organic phase by using a vacuum rotary evaporator to dryness, and finally obtaining a yellow oily substance, namely a compound 2.

4. A method for synthesizing the compound 3 according to claim 2

Weighing cesium carbonate and 2-hydroxybenzaldehyde, dissolving the cesium carbonate and the 2-hydroxybenzaldehyde into a round-bottom flask, slowly adding a compound 2 into a reaction bottle, stirring for 24-48h at room temperature, wherein the reaction ratio of the 2-hydroxybenzaldehyde to the compound 2 is 1: 1-1: 2, tracking and detecting the reaction progress by TLC until the compound 2 completely reacts, pouring the reaction liquid into water, extracting the reaction liquid for three times by using an organic solvent, taking an organic phase, drying the organic phase by using anhydrous sodium sulfate, evaporating the organic phase by using a vacuum rotary evaporator to dryness, and separating and purifying a crude product by using 200-mesh 300-mesh silica gel column chromatography to finally obtain a yellow solid, namely.

5. Hg as claimed in claim 2²⁺Synthesis method of selective fluorescent probe compound XL-Hg

Weighing a compound 3 and 1, 3-propanedithiol, dissolving the compound and the 1, 3-propanedithiol in a round-bottom flask, dropwise adding boron trifluoride etherate into a reaction solution, stirring the reaction solution for 10-24 hours at 25 +/-5 ℃ by using nitrogen to protect the reaction solution, wherein the reaction ratio of the compound 3 to the 1, 3-propanedithiol is 1: 2-1: 3, detecting that the reaction is complete by TLC (thin layer chromatography), evaporating the reaction solution by using a vacuum rotary evaporator, and separating and purifying a crude product by using 200-mesh and 300-mesh silica gel column chromatography to finally obtain a yellow solid, namely a compound XL-Hg.

6. The ultraviolet absorbing activity of compound XL-Hg of claim 1, the compound XL-Hg being in water to Hg²⁺The ions have a fluorescent response.

7. The compound of claim 1 XL-Hg vs Hg²⁺The fluorescence response time characteristic of compound XL-Hg in water to Hg²⁺The time stability of the ionic fluorescence response is that the fluorescence intensity changes violently within 0-1 hour, and the fluorescence intensity keeps stable within 1-12 hours.

8. The compound of claim 1 XL-Hg for Hg²⁺The compound XL-Hg is selective to metal ions in vitro, and the in vitro fluorescence property is only selective to Hg²⁺The change is not influenced by other metal ions.

9. The method of claim 1Detection of Hg in living cells by Compound XL-Hg²⁺The use of Hg as an addition to living cells²⁺And a compound XL-Hg, the cell shows fluorescence, and the compound XL-Hg is used for Hg in the cell²⁺Has detection capability.

Technical Field

The invention belongs to the field of biochemistry, and relates to novel Hg containing oxyanthracene²⁺The synthesis and application of the ion probe in water and cells.

Background

Hg²⁺Is one of the most dangerous metal ions and has significant harm to the environment and human health. Hg due to its high affinity for imine or sulfhydryl groups in nucleic acids and proteins²⁺Can destroy cell function and cause severe damage to brain, intestine, kidney and immune system. Hg in human body²⁺May lead to various physiological disorders, such as water preferentially.

In recent years, various methods for detecting Hg have been developed²⁺Such as electrochemical, inductive coupling, plasma mass spectrometry, high performance liquid chromatography, and atomic emission spectrometry. Although it is used forThese conventional methods have a good detection range, but they are difficult to be widely used due to expensive equipment or professional operation skills. Compared with the above technology, the fluorescence method has the advantages of high selectivity, sensitivity, agility, low cost, non-invasive detection and the like, so that the fluorescent probe based on the fluorescence method is widely applied to detecting metal ions and other small molecules. According to the fluorescence property of the xanthene, the xanthene is modified by adding 1, 3-propanedithiol to obtain a fluorescent probe XL-Hg containing the xanthene, and the fluorescent activity and intracellular activity of the fluorescent probe XL-Hg are tested.

Disclosure of Invention

The invention relates to Hg of oxygen-containing heteroanthracene²⁺Preparation of ion probe and detection of Hg²⁺Application in ions.

The technical scheme of the invention is as follows:

an oxa-heteroanthracene derivative XL-Hg, named as 4- (1, 3-dithiane-2-yl) -2, 3-dihydro-xanthene in Chinese, has the following chemical structure:

a synthetic method of a derivative XL-Hg containing oxa-anthracene is characterized by comprising the following steps:

and step i, slowly adding phosphorus tribromide into a flask containing a certain volume of organic solvent under proper conditions, uniformly mixing, stirring at proper temperature, adding a proper amount of chloroform-dissolved cyclohexanone, stirring under certain conditions, and tracking and detecting the reaction progress by TLC (thin layer chromatography) until the cyclohexanone is completely reacted. And pouring the reaction liquid into ice water, adjusting the pH of the reaction liquid to be neutral, extracting, and taking an organic phase to obtain a crude product. After silica gel column chromatography, compound 2 is finally obtained as yellow oil.

Step ii, weighing a certain proportion of cesium carbonate and 2-hydroxybenzaldehyde into a flask, dissolving with DMF, slowly adding the compound 2 dissolved with DMF into a reaction bottle, stirring at 25 ℃ for reaction for a period of time, and tracking and detecting the progress of the reaction by TLC until the compound 2 is completely reacted. And pouring the reaction solution into water, extracting, taking an organic phase, evaporating to obtain a crude product, and separating and purifying the crude product by using silica gel column chromatography to obtain a yellow solid, namely the compound 3.

Step iii, weighing the compound 3 and the 1, 3-propanedithiol into a flask, dissolving the compound in dichloromethane, dropwise adding the boron trifluoride etherate into the reaction solution, stirring the reaction solution at 25 ℃ for a period of time by using nitrogen protection, and detecting the completion of the reaction by TLC tracking. Evaporating the reaction liquid by using a vacuum rotary evaporator, and separating and purifying a crude product by using silica gel column chromatography to obtain a yellow solid, namely the compound XL-Hg.

The silica gel column chromatography in the step adopts 200-300-mesh silica gel column, and the eluent is dichloromethane and methanol, anhydrous ethyl acetate and petroleum ether in a certain proportion.

The xanthene fluorescent probe provided by the invention can be used for detecting Hg in vitro and in cells²⁺The ions have better fluorescent properties. Therefore, the fluorescent probe containing the oxa-anthracene can effectively detect Hg²⁺The concentration of the ions.

Drawings

FIG. 1 is a graph showing the UV absorption activity of 4- (1, 3-dithian-2-yl) -2, 3-dihydro-xanthene according to the present invention.

FIG. 2 is a graph showing the fluorescence response time of 4- (1, 3-dithian-2-yl) -2, 3-dihydro-xanthene according to the present invention.

FIG. 3 is a graph showing the metal ion selectivity of 4- (1, 3-dithian-2-yl) -2, 3-dihydro-xanthene according to the present invention.

FIG. 4 detection of Hg in cells by 4- (1, 3-dithian-2-yl) -2, 3-dihydro-xanthene according to the invention²⁺Fluorescence imaging of (1).

Detailed Description

The present invention is further illustrated in detail by the following examples, but the scope of the present invention is not limited by these examples at all.