阅读说明：本技术 一种异佛尔酮类硫化氢荧光探针及其制备方法与应用 (Isophorone hydrogen sulfide fluorescent probe and preparation method and application thereof ) 是由 刘毅 庞意鹏 李鑫蕊 徐婷 刘娇 余佩全 程媛 于 2019-09-12 设计创作，主要内容包括：本发明公开了一种异佛尔酮类硫化氢荧光探针及其制备方法与应用,该荧光探针的结构如式I所示。先将异佛尔酮、丙二腈、哌啶加入反应瓶中,惰性气体保护下加热回流,提纯,然后将得到的纯品、对羟基苯甲醛、哌啶加入反应瓶中,惰性气体保护下加热回流,提纯,再将得到的纯品、乌洛托品、三氟乙酸加入反应瓶中,搅拌,最后将得到的纯品、2-噻吩甲酰氯、三乙胺加入反应瓶中,搅拌,提纯后得到荧光探针。本发明的荧光探针具有大的stokes位移(224nm),较长的荧光发射波长(662nm),检测限低(48nM),响应时间短(15min),能特异性检测硫化氢,还具有良好的生物膜通透性和低细胞毒性；合成路线简单,产率高,实用价值大。<Image he="488" wi="689" file="RE-DDA0002269128560000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention discloses an isophorone hydrogen sulfide fluorescent probe and a preparation method and application thereof. Adding isophorone, malononitrile and piperidine into a reaction bottle, heating and refluxing under the protection of inert gas, purifying, then adding the obtained pure product, p-hydroxybenzaldehyde and piperidine into the reaction bottle, heating and refluxing under the protection of inert gas, purifying, then adding the obtained pure product, urotropine and trifluoroacetic acid into the reaction bottle, stirring, finally adding the obtained pure product, 2-thiophenecarbonyl chloride and triethylamine into the reaction bottle, stirring, and purifying to obtain the fluorescent probe. The fluorescent probe has large stokes shift (224nM), longer fluorescence emission wavelength (662nM), low detection limit (48nM), short response time (15min), capability of specifically detecting hydrogen sulfide, and good performanceBiofilm permeability and low cytotoxicity; the synthesis route is simple, the yield is high, and the practical value is high.)

1. An isophorone hydrogen sulfide fluorescent probe is characterized in that the structure is shown as formula I:

2. the preparation method of the isophorone-type hydrogen sulfide fluorescent probe of claim 1, which is characterized by comprising the following steps:

(1) preparation of the compound of formula II: under the protection of inert gas, adding isophorone, malononitrile and piperidine into a reaction bottle, using ethanol as a solvent, heating, stirring and refluxing, tracking by TLC (thin layer chromatography) until the reaction is finished, cooling the reaction liquid to room temperature, pouring the reaction liquid into ice water to precipitate, performing suction filtration, and performing recrystallization treatment on filter residues by using n-hexane to obtain a compound of a formula II; wherein the molar ratio of isophorone to malononitrile is 1: 1-1: 2;

(2) preparation of the compound of formula III: under the protection of inert gas, adding a compound shown in a formula II, p-hydroxybenzaldehyde and piperidine into a reaction bottle, taking ethanol as a solvent, heating, stirring and refluxing, tracking by TLC (thin layer chromatography) until the reaction is finished, cooling the reaction liquid to room temperature, concentrating the reaction liquid, and purifying by silica gel column chromatography to obtain a compound shown in a formula III; wherein the molar ratio of the compound shown in the formula II to the p-hydroxybenzaldehyde is 1: 1-2: 1;

(3) preparation of the compound of formula IV: under the protection of inert gas, adding a compound shown in the formula III and urotropine into a reaction bottle, using trifluoroacetic acid as a solvent, heating, stirring and refluxing, tracking by TLC (thin layer chromatography) until the reaction is finished, cooling the reaction solution to room temperature, pouring the reaction solution into 6M HCl, stirring for 10min, extracting with dichloromethane, washing with saturated saline solution for 2-3 times, taking a dichloromethane layer, drying with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain a compound shown in the formula IV; wherein the molar ratio of the compound shown in the formula III to the urotropine is 1: 1-1: 2;

(4) preparation of a Compound of formula I: adding the compound shown in the formula IV, 2-thiophenecarbonyl chloride and triethylamine into a reaction bottle, taking dichloromethane as a solvent, stirring at room temperature for reaction, tracking by TLC (thin layer chromatography) until the reaction is finished, concentrating the reaction solution, and purifying by silica gel column chromatography to obtain a pure product of the fluorescent probe shown in the formula I; wherein the molar ratio of the compound shown in the formula IV to the 2-thiophenecarbonyl chloride is 1: 1-1: 2.

3. The method for preparing an isophorone-type hydrogen sulfide fluorescent probe according to claim 2, wherein a developing solvent used for TLC in step (1) is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5: 1; the developing solvent used for TLC in the step (2) is a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of 30: 1; the developing solvent used for TLC in the step (3) is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 2: 1; the developing solvent used for TLC in step (4) is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 3: 1.

4. The method for preparing isophorone-type hydrogen sulfide fluorescent probe according to claim 2, wherein the eluent used for silica gel column chromatography in step (2) is a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of 50: 1; the eluent used for silica gel column chromatography in the step (3) is a mixed solvent of petroleum ether and ethyl acetate with the volume ratio of 10: 1; and (4) eluting with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 8: 1.

5. The use of the isophorone-type hydrogen sulfide fluorescent probe of claim 1 in detecting biological hydrogen sulfide.

Technical Field

The invention relates to a hydrogen sulfide fluorescent probe, in particular to an isophorone hydrogen sulfide fluorescent probe and a preparation method and application thereof, and belongs to the field of organic luminescent materials.

Background

Hydrogen sulfide (H)₂S) is an important gas signal transmission medium in organisms, and exists mainly in two forms in the bodies (1/3H)₂S, 2/3NaHS), is in dynamic equilibrium, maintaining the normal pH range of the body. Hydrogen sulfide is the third atmospheric signal molecule in the human body following Nitric Oxide (NO) and carbon monoxide (CO), and in vivo, hydrogen sulfide participates in intracellular redox reactions and various signaling processes including vasodilation, myocardial contraction, neurotransmission, insulin secretion, etc. And when intracellular hydrogen sulfide is at abnormal levels, it will cause a series of physiological diseases, such as alzheimer's disease, liver cirrhosis, gastric mucosal injury, arterial and pulmonary hypertension, etc. Therefore, effective detection or monitoring of hydrogen sulfide in biological or environmental samples has been a research hotspot in related fields in recent years.

The micromolecule hydrogen sulfide fluorescence probe method is used for detecting by utilizing the principle that a fluorescence probe and hydrogen sulfide are subjected to specific identification, the probe can release a fluorescence mother nucleus to generate fluorescence after reacting with hydrogen sulfide molecules, a fluorescence emission spectrum is displayed under the excitation of a fluorescence spectrophotometer, and then the fluorescence intensity and the concentration of the hydrogen sulfide are analyzed. The method is rapid, sensitive and accurate, and can be combined with a laser confocal imaging technology to realize real-time and in-situ hydrogen sulfide detection and display spatial distribution information.

The near-infrared hydrogen sulfide probe has larger emission wavelength, so that the autofluorescence phenomenon of a plurality of biological tissues can be effectively avoided, the background interference is eliminated, and the sensitivity and the accuracy of the near-infrared hydrogen sulfide probe are greatly enhanced. A qualified near-infrared probe needs to have an emission wavelength in a near-infrared region, large Stokes shift (strong tissue penetration capacity and small light damage), high fluorescence quantum yield (more than 0.1) and low detection limit, and meets the detection of the content of hydrogen sulfide in a certain concentration range inside and outside a body. Therefore, the design of a fluorescent probe which is rapid and sensitive and has a relatively long detection wavelength is of great significance for detecting hydrogen sulfide.

Disclosure of Invention

The invention aims to provide an isophorone hydrogen sulfide fluorescent probe which has relatively longer fluorescence emission wavelength, low detection limit and short response time.

In order to achieve the purpose, the structure of the isophorone hydrogen sulfide fluorescent probe provided by the invention is shown as a formula I:

the invention also aims to provide a preparation method of the isophorone hydrogen sulfide fluorescent probe shown in the formula I, which has the advantages of short synthetic route and simple reaction conditions.

In order to achieve the purpose, the preparation method of the isophorone hydrogen sulfide fluorescent probe provided by the invention comprises the following steps:

(1) preparation of the compound of formula II: under the protection of inert gas, adding isophorone (3,5, 5-trimethyl-2-cyclohexenone), malononitrile and piperidine into a reaction bottle, using ethanol as a solvent, heating, stirring and refluxing, carrying out TLC (thin layer chromatography) tracking until the reaction is finished, cooling the reaction liquid to room temperature, pouring the reaction liquid into ice water, precipitating, carrying out suction filtration, and carrying out recrystallization treatment on the filter residue by using n-hexane to obtain a compound shown in a formula II; wherein the molar ratio of isophorone to malononitrile is 1: 1-1: 2;

(2) preparation of the compound of formula III: under the protection of inert gas, adding a compound shown in a formula II, p-hydroxybenzaldehyde and piperidine into a reaction bottle, using ethanol as a solvent, heating, stirring and refluxing, tracking by TLC (thin layer chromatography) until the reaction is finished, cooling the reaction liquid to room temperature, concentrating the reaction liquid, and purifying by silica gel column chromatography to obtain a compound shown in a formula III; wherein the molar ratio of the compound shown in the formula II to the p-hydroxybenzaldehyde is 1: 1-2: 1;

(3) preparation of the compound of formula IV: under the protection of inert gas, adding a compound of a formula III and urotropine (hexamethylenetetramine) into a reaction bottle, heating, stirring and refluxing trifluoroacetic acid serving as a solvent, tracking by TLC (thin layer chromatography) until the reaction is finished, cooling a reaction solution to room temperature, pouring the reaction solution into 6M HCl, stirring for 10min, extracting with dichloromethane, washing with saturated saline solution for 2-3 times, taking a dichloromethane layer, drying with anhydrous sodium sulfate, concentrating to obtain a crude product, and purifying by silica gel column chromatography to obtain a compound of a formula IV; wherein the molar ratio of the compound shown in the formula III to the urotropine is 1: 1-1: 2;

(4) preparation of a Compound of formula I: adding the compound shown in the formula IV, 2-thiophenecarbonyl chloride and triethylamine into a reaction bottle, taking dichloromethane as a solvent, stirring at room temperature for reaction, tracking by TLC (thin layer chromatography) until the reaction is finished, concentrating the reaction solution, and purifying by silica gel column chromatography to obtain a pure product of the fluorescent probe shown in the formula I; wherein the molar ratio of the compound shown in the formula IV to the 2-thiophenecarbonyl chloride is 1: 1-1: 2.

The reaction route is as follows:

preferably, the developing solvent used for TLC in the step (1) is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 5: 1; the developing solvent used for TLC in the step (2) is a mixed solvent of dichloromethane and ethyl acetate in a volume ratio of 30: 1; the developing solvent used for TLC in the step (3) is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 2: 1; the developing solvent used for TLC in step (4) is a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 3: 1.

Preferably, the eluent used for the silica gel column chromatography in the step (2) is a mixed solvent of dichloromethane and ethyl acetate with a volume ratio of 50: 1; the eluent used for silica gel column chromatography in the step (3) is a mixed solvent of petroleum ether and ethyl acetate with the volume ratio of 10: 1; and (4) eluting with a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 8: 1.

The invention also aims to provide an application of the isophorone hydrogen sulfide fluorescent probe shown in the formula I in detection of biological hydrogen sulfide.

The method is realized by the following steps: adding a fluorescent probe shown in a formula I into a system to be detected, enabling the final concentration of the fluorescent probe to be 10 mu M, incubating for 1 hour at 37 ℃, and detecting the fluorescence intensity of the system; selecting plasma, homogenate or cell culture medium for the system to be detected, and recording fluorescence intensity by a fluorescence spectrophotometer when the system to be detected is plasma or homogenate; and when the system to be detected is a cell culture medium, observing the fluorescence imaging of the living cells by using a laser confocal microscope.

Compared with the prior art, the invention has the following beneficial effects:

(1) the probe has larger fluorescence emission wavelength (662nm), can effectively avoid the interference from the background fluorescence of large biological molecules, has larger Stokes shift (224nm), and has strong tissue penetration capability and small light damage;

(2) the probe has no fluorescence, only has fluorescence after reacting with hydrogen sulfide, has low detection limit (48nM), high sensitivity and short response time (15 min);

(3) the method has excellent selectivity, is not interfered by substances such as amino acids in a living body, and can specifically detect the hydrogen sulfide in organisms;

(4) the probe has good stability and light stability under different pH conditions, and can be stored and used for a long time;

(5) can enter HT22 cells autonomously, has good biological membrane permeability and low cytotoxicity, and is suitable for detecting hydrogen sulfide in living cells;

(6) the fluorescent probe has the advantages of short synthetic route, mild reaction conditions, high yield and great practical value.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a fluorescent probe SFP-CHO-THP of the invention;

FIG. 2 is a nuclear magnetic resonance carbon spectrum of the fluorescent probe SFP-CHO-THP of the present invention;

FIG. 3 is a high-resolution mass spectrum of the fluorescent probe SFP-CHO-THP of the present invention;

FIG. 4 is a schematic diagram of the mechanism of the reaction of the fluorescent probe of the present invention with hydrogen sulfide;

FIG. 5 shows the selectivity of the fluorescent probe of the present invention for some sulfur-containing amino acids in a living body;

FIG. 6 shows the selectivity of the fluorescent probe of the present invention for some non-sulfur-containing amino acids in a living body;

FIG. 7 is a graph of the selectivity of the fluorescent probe of the present invention for other ions in a living organism;

FIG. 8 is a graph showing the change in fluorescence intensity and hydrogen sulfide concentration of the fluorescent probe of the present invention;

FIG. 9 is a linear plot of the change in fluorescence intensity and hydrogen sulfide concentration for the fluorescent probe of the present invention;

FIG. 10 is a graph showing the change in fluorescence intensity and hydrogen sulfide reaction time of the fluorescent probe of the present invention;

FIG. 11 is a linear graph of the change in fluorescence intensity and hydrogen sulfide reaction time of the fluorescent probe of the present invention;

FIG. 12 is a graph showing the relationship between the fluorescence intensity and the pH change of the fluorescent probe of the present invention;

FIG. 13 is a graph of Stokes' shift before and after reaction of a fluorescent probe of the present invention with hydrogen sulfide;

FIG. 14 is a graph showing the measurement of the photostability of the fluorescent probe according to the present invention;

FIG. 15 is a graph showing the cell viability of the fluorescent probes of the present invention after 24h of cell culture.

Detailed Description

The invention is described in further detail below with reference to the figures and specific examples.