阅读说明：本技术 一种用于检测锌离子的近红外荧光探针及其制备方法和应用 (Near-infrared fluorescent probe for detecting zinc ions and preparation method and application thereof ) 是由 许志红 周起航 杨莉 刘高兵 杨涵熙 许泗林 许锐杰 高志颖 段颖颖 潘明霞 于 2021-08-24 设计创作，主要内容包括：本发明公开一种用于检测锌离子的近红外荧光探针及其制备方法和应用,属于有机合成技术领域,所述荧光探针以罗丹明半花菁为荧光团,7-羟基-4-甲基香豆素醛为识别基团,实现了对锌离子的特异性识别响应,具体结构式如下：；探针本身荧光极弱,与锌离子反应后荧光增强,溶液由无色变为绿色,可裸眼识别,且不受干扰离子的影响,可用于锌离子的检测,同时在生物领域也具有广阔的应用场景。(The invention discloses a near-infrared fluorescent probe for detecting zinc ions, a preparation method and application thereof, and belongs to the technical field of organic synthesis, wherein the fluorescent probe takes rhodamine hemicyanine as a fluorophore and 7-hydroxy-4-methylcoumarin aldehyde as an identification group, realizes specific identification response to the zinc ions, and has the following specific structural formula:)

1. A near-infrared fluorescent probe for detecting zinc ions is characterized in that the molecular formula of the probe is C₄₈H₄₇O₅N₄The structural formula is as follows:

。

2. the method for preparing the near-infrared fluorescent probe for detecting zinc ions as claimed in claim 1, characterized in that the synthetic route is as follows:

the method specifically comprises the following steps:

(1) dissolving cyclohexanone in concentrated sulfuric acid, adding LF-1, stirring at 92-98 ℃ until the reaction is complete, cooling to room temperature, adding perchloric acid, performing suction filtration, washing with water, and drying to obtain a compound LF-2;

(2) adding acetic anhydride into LF-2 and Fischer aldehyde under the protection of nitrogen, stirring at 50 +/-5 ℃ until the reaction is complete, then adding ice water into the mixed solution, extracting with dichloromethane and water, extracting the organic phase with saturated sodium bicarbonate solution, finally washing the organic phase with deionized water, drying to obtain a crude product, and separating the obtained crude product by using column chromatography to obtain a pure compound LF-3;

(3) under the protection of nitrogen, dichloromethane is added into a compound LF-3, a Kate condensing agent (BOP) is dissolved in dichloromethane and added into methane solution of LF-3, then hydrazine hydrate is added into reaction liquid, the reaction liquid is stirred at room temperature until the reaction is completed, dichloromethane and water are used for extraction, drying is carried out, a crude product LF-4 is obtained, and the obtained crude product is separated by column chromatography to obtain a pure compound LF-4;

(4) adding compounds LF-4 and 7-hydroxy-4-methyl-coumarin aldehyde into ethanol, stirring and refluxing at 100 +/-5 ℃ until the reaction is complete, separating out solids after the reaction is finished, washing with absolute ethanol, and drying to obtain the fluorescent probe.

3. The method for preparing a near-infrared fluorescent probe for detecting zinc ions according to claim 2, wherein the molar ratio of LF-1, cyclohexanone and perchloric acid in the step (1) is 1 (2-3) to (10-16).

4. The method for preparing the near-infrared fluorescent probe for detecting the zinc ions according to claim 2, wherein the molar ratio of the compound LF-2 to the Fischer aldehyde in the step (2) is 1 (1.1-1.2).

5. The method for preparing a near-infrared fluorescent probe for detecting zinc ions according to claim 2, wherein the molar ratio of the compound LF-3 to BOP to hydrazine hydrate in the step (3) is 1 (1.1-1.3) to (5-12).

6. The preparation method of the near-infrared fluorescent probe for detecting zinc ions according to claim 2, wherein the molar ratio of the compound LF-4 to the 7-hydroxy-4-methyl-coumarin aldehyde in the step (4) is 1 (1.1-1.3).

7. The near infrared fluorescent probe according to claim 1, which is used for fluorescence detection, visual qualitative detection and cell imaging detection of zinc ion content.

Technical Field

The invention belongs to the technical field of organic synthesis, and particularly relates to a near-infrared fluorescent probe for detecting zinc ions, and a preparation method and application thereof.

Background

People pay more and more attention to their health problems while our living standard is increasing day by day. The metal ions not only participate in various physiological activities in the body but also maintain various balances in the body. However, the problem of metal contamination is becoming more serious, and the problem of biological diseases caused by abnormal metal ion content is also increasing due to the complexity of the biological chain. Therefore, the detection of the ion content in the organism and the understanding of the pathogenesis of the disease by doctors are very important, so that the occurrence of the disease is prevented in advance, and convenience is provided for subsequent treatment.

The near-infrared fluorescent probe has excellent detection characteristics in biological tissues or organisms, for example: excitation and emission are in a near infrared region, most of near infrared can penetrate into biological tissues during detection, autofluorescence interference of the biological tissues can be eliminated, the biological tissues can be stably located in cells, and the biological tissues are low-toxic or nontoxic to cells, and can detect micro-changes of organisms.

Zn²⁺Is one of the ions which play a vital role in various physiological activities in the living body and is the second most abundant metal ion in the human body. Zn²⁺Present throughout the life cycle of the cell. Such as Zn in ER (endoplasmic reticulum)²⁺Plays an important role, Zn²⁺Causes a corresponding stress response of the ER (unfolded or misfolded proteins are synthetically accumulated in the endoplasmic reticulum), thereby causing the corresponding disease: neurodegenerative diseases, Parkinson's disease, diabetes and the like. In the biological field, the biological method is used,with Zn²⁺Transient fluctuations in localization to regulate some large-scale physiological processes, such as: systemic immune response, nerve signaling, etc. Therefore, how to simply and rapidly detect Zn²⁺The method is a valuable research applied in the fields of medicine, biology and the like.

Currently detecting Zn²⁺The fluorescence sensor of (2) is relatively free of other metal ions and is mostly not in the near infrared region but in the ultraviolet visible region. It is well known that sensors with excitation and emission in the near infrared region mostly have a series of excellent properties: high sensitivity, good stability (chemical stability and light stability), small interference of biological background, no toxicity or low toxicity to cells. Development and design of near-infrared detection Zn²⁺ Fluorescent probes, and their successful application to fluorescence imaging of biological organisms, are a necessary study.

All intermediates and target products herein were characterized. The research shows that: in the buffer solution, the compound can identify zinc ions with high selectivity, and other ions hardly cause any interference to the identification process.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention provides a near-infrared fluorescent probe for detecting zinc ions, which realizes specific recognition response to the zinc ions, is not influenced by interfering ions, and can judge whether the zinc ions exist or not by direct visual observation.

The invention also provides a preparation method and application of the near-infrared fluorescent probe.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a near-infrared fluorescent probe for detecting zinc ions, the molecular formula of the probe is C₄₈H₄₇O₅N₄The structural formula is as follows:

。

the synthesis route for detecting zinc ions is as follows:

the preparation method specifically comprises the following steps:

(1) dissolving cyclohexanone in concentrated sulfuric acid, adding LF-1, stirring at 92-98 ℃ until the reaction is complete, cooling to room temperature, adding perchloric acid, performing suction filtration, washing with water, and drying to obtain a compound LF-2;

(2) adding acetic anhydride into LF-2 and Fischer aldehyde under the protection of nitrogen, stirring at 50 ℃ until the reaction is complete, then adding ice water into the mixed solution, extracting with dichloromethane and water, extracting the organic phase with saturated sodium bicarbonate solution, finally washing the organic phase with deionized water, drying to obtain a crude product, and separating the crude product by using column chromatography to obtain a pure compound LF-3;

(3) under the protection of nitrogen, dichloromethane is added into a compound LF-3, a Kate condensing agent (BOP) is dissolved in dichloromethane and added into methane solution of LF-3, then hydrazine hydrate is added into reaction liquid, the reaction liquid is stirred at room temperature until the reaction is completed, dichloromethane and water are used for extraction, drying is carried out, a crude product LF-4 is obtained, and the obtained crude product is separated by column chromatography to obtain a pure compound LF-4;

(4) adding compounds LF-4 and 7-hydroxy-4-methyl-coumarin aldehyde into ethanol, stirring and refluxing at 100 +/-5 ℃ until the reaction is complete, separating out solids after the reaction is finished, washing with absolute ethanol, and drying to obtain the fluorescent probe.

Preferably, the molar ratio of LF-1, cyclohexanone and perchloric acid in the step (1) is 1 (2-3) to (10-16).

Preferably, the molar ratio of the compound LF-2 to the Fischer aldehyde in the step (2) is 1 (1.1-1.2).

Preferably, in the step (3), the molar ratio of the compound LF-3 to BOP to hydrazine hydrate in the step (3) is 1 (1.1-1.3) to (5-12).

Preferably, the molar ratio of the compound LF-4 and the 7-hydroxy-4-methyl-coumarin aldehyde in the step (4) is 1 (1.1-1.3).

The near-infrared fluorescent probe is applied to the fluorescent detection of zinc ions, and is particularly used for the fluorescent detection, visual qualitative detection and cell imaging detection of the content of the zinc ions.

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

1. after the probe is added with zinc ions, the fluorescence intensity is obviously enhanced and is accompanied with obvious eye color change, the selected interference ions and the like almost have no influence on the detection effect, and the specific identification response to the zinc ions is realized.

2. The detection limit of the near-infrared fluorescent probe can reach 0.066 mu M.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of the fluorescent probe prepared in example 1;

FIG. 2 is a nuclear magnetic carbon spectrum of the fluorescent probe prepared in example 1;

FIG. 3 is a fluorescence spectrum of the fluorescent probe prepared in example 1 after reacting with various ions;

FIG. 4 is a graph showing fluorescence intensities of the fluorescent probes prepared in example 1 after reacting with different ions;

FIG. 5 is a fluorescence spectrum of the reaction of the fluorescent probe prepared in example 1 with zinc ions as a function of time;

FIG. 6 shows fluorescent probes prepared in example 1 and different Zn concentrations²⁺Fluorescence spectra after ion response;

FIG. 7 shows the fluorescence intensity of a solution with Zn²⁺A linear relationship between ion concentrations;

FIG. 8 shows the interaction of the fluorescent probe prepared in example 1 with Zn in different pH system solutions²⁺Fluorescence spectra after ion response.

Detailed Description

The invention is further illustrated, but not limited, by the following examples and the accompanying drawings.

Example 1

The preparation method of the fluorescent probe comprises the following steps:

(1) preparation of Compound LF-2

8 mL of concentrated sulfuric acid (commercially available 98% concentrated sulfuric acid) was placed in a 50 mL pear-shaped flask and stirred at 0 ℃ for 20 min. Slowly injecting cyclohexanone (0.2941 g, 3.0 mmol) into a bottle, stirring for 15-20 min, adding LF-1 (0.4691 g, 1.5 mmol) into the reaction solution in small amount for multiple times, controlling the addition within 30 min, gradually changing the color of the solution from colorless to red, heating at 92 ℃ for 4 h after the solid is completely dissolved, stopping heating after the reaction is completely finished, cooling to room temperature, slowly pouring the reaction solution into a beaker filled with 240 mL of ice water, stirring, and dropwise adding 1.5 mL of HClO₄(generally 10 to 16 times the molar amount of LF-1), accompanied by precipitation of a red solid, was filtered off with suction, washed with cold water, and dried. 0.5462 g of a red solid was finally obtained (yield: 95.6%), which was directly subjected to the next step without purification;

(2) preparation of Compound LF-3

LF-2 (0.3262 g, 0.8 mmol), Fisher's aldehyde (0.1812 g, 0.9 mmol) were placed in a 50 mL two-necked pear-shaped flask, nitrogen was added, acetic anhydride (5 mL) was added, stirring was carried out at 50 ℃ for 2 hours, ice water (typically 2-3 times the volume of acetic anhydride) was added to the mixture, extraction was carried out with dichloromethane and water, the organic phase was extracted three times with saturated sodium bicarbonate solution, washed three times with deionized water, and Na was added₂SO₄Drying to obtain a crude product, and separating and purifying by using a chromatographic column, wherein an eluent is dichloromethane: methanol = 20:1, solvent was spin dried, dried to yield 0.2564 g of green solid (55.3% yield).¹H NMR (400 MHz, CDCl₃ ) δ 8.24- 8.15 (m, 1H), 8.10 (d, J = 13.2 Hz, 1H), 7.55-7.44 (m, 2H), 7.28 (s, 1H), 7.03 (q, J = 6.3, 4.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 6.55 (d, J = 9.2 Hz, 1H), 6.45 (s, 1H), 5.63 (d, J = 13.2 Hz, 1H), 5.29 (s, 1H), 3.45 (t, J = 7.3 Hz, 4H), 3.36 (s, 3H), 2.58 (tq, J = 16.3, 9.7, 7.5 Hz, 2H), 2.42 (dt, J = 14.1, 5.4 Hz, 1H), 2.10 (dd, J = 16.3, 6.3 Hz, 1H), 1.98 (s, 1H), 1.84 (dd, J = 13.4, 6.4 Hz, 1H), 1.23 (t, J = 7.1 Hz, 6H).

(3) Preparation of Compound LF-4

LF-3 (0.2018 g, 0.36 mmol) was placed in a two-necked flask, nitrogen blanketed, 4 mL of methylene chloride was injected into the flask, the Kate condensing agent (BOP, 0.43 mmol) was dissolved in methylene chloride, slowly injected into the flask, and stirred for 20-30 minutes, then 3mL of hydrazine hydrate was injected into the flask via syringe, and stirred vigorously (1800 + 2500 rpm) at room temperature. The solution changed from green to yellow or red with the addition of hydrazine hydrate, stirred at room temperature for 3 hours, extracted three times with dichloromethane and water, and then Na is added₂SO₄Drying to obtain crude product, and separating and purifying with chromatographic column to obtain eluent only using dichloromethane. The product was collected and dried to yield 0.1624 g of a yellow solid (76.18% yield).¹H NMR (400 MHz, CDCl₃) δ 7.93-7.85 (m, 1H), 7.56-7.38 (m, 3H), 7.23-7.11 (m, 3H), 6.65-6.58 (m, 1H), 6.40-6.23 (m, 3H), 5.37 (d, J = 12.6 Hz, 1H), 3.65 (s, 2H), 3.35 (q, J = 7.1 Hz, 4H), 3.15 (s, 3H), 2.65-2.41 (m, 2H), 2.04 (s, 1H), 1.71 (d, J = 6.4 Hz, 6H), 1.37 (d, J = 1.1 Hz, 1H), 1.26 (t, J = 7.1 Hz, 2H), 1.18 (t, J = 7.0 Hz, 6H)。

(4) Preparation of Probe LFX

Compound LF-4 (0.0501 g,0.0844 mmol) was weighed into a 50 mL pear-shaped bottle, then 7-hydroxy-4-methyl-coumarinal aldehyde (0.0189 g, 0.0928 mmol) was added to the bottle, and 5 mL absolute ethanol was added, which was refluxed at 100 ℃ for 5 hours. A yellow solid precipitated, which was filtered off with suction and washed with ethanol. The solid was collected and dried. The product, 0.0657 g (80.71% yield), was a yellow solid whose hydrogen and carbon spectra are shown in FIGS. 1 and 2,¹H NMR (400 MHz, CDCl₃) δ 12.23 (s, 1H), 9.45 (s, 1H), 7.97 (d, J = 7.5 Hz, 1H), 7.62-7.54 (m, 2H), 7.50 (td, J = 7.4, 1.1 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H), 7.17 (dd, J = 8.4, 7.2 Hz, 2H), 6.88 – 6.77 (m, 2H), 6.60 (d, J = 7.7 Hz, 1H), 6.52 (d, J = 2.6 Hz, 1H), 6.43 (d, J = 8.8 Hz, 1H), 6.28 (dd, J = 8.9, 2.6 Hz, 1H), 6.04 (d, J = 1.4 Hz, 1H), 5.36 (d, J = 12.6 Hz, 1H), 3.34 (q, J = 7.1 Hz, 4H), 3.13 (s, 3H), 2.32 (d, J = 1.2 Hz, 3H), 1.75 (d, J = 7.0 Hz, 6H), 1.62 (s, 6H), 1.17 (t, J = 7.1 Hz, 6H),¹³C NMR (100 MHz, CDCl3) δ 164.26, 162.14, 158.06, 152.71, 149.77, 149.03, 148.76，146.43, 145.33, 133.50, 129.82, 128.64, 127.85, 127.60, 126.83, 123.75, 123.55, 121.57, 120.22, 119.96, 119.28, 113.77, 111.82, 111.44, 108.52, 106.75, 105.63, 102.36, 98.35, 92.04, 77.23, 68.44, 45.56, 44.34, 29.10, 28.50, 28.12, 25.27, 23.02, 22.16, 18.78, 12.57.

the application test of the near-infrared fluorescent probe prepared by the experiment is as follows:

(1) preparation of stock solution for detection

a. The concentration is 1.00X 10^-3Preparing mol/L probe LFX stock solution: 7.6 mg of the probe LFX is weighed with a ten-thousandth electronic balance, the weighed probe LFX is then dissolved in 10 mL of acetonitrile, the solution is heated and shaken well and sonicated at 500W power for 5 minutes.

b. Various cations (Zn)²⁺、Mg²⁺、Fe²⁺、Al³⁺、Co²⁺、Cd²⁺、Cr³⁺、Cu²⁺、Ca²⁺、Ni²⁺、Ag⁺、Li⁺、Ba²⁺、Mn²⁺、K⁺、Hg²⁺) Are all prepared into 1.00 multiplied by 10 by deionized water^-2mol/L solution, and diluting to 1.00X 10^-3 mol/L。

Preparation of hepes solution (10 nM, pH = 7.4): pouring 2.38 g of solid HEPES (4-hydroxyethyl piperazine ethanesulfonic acid) weighed by balance into a 250 mL conical flask, adding purified water subjected to secondary distillation, stirring to dissolve the solid, transferring the solution into a washed 1000 mL volumetric flask, washing the conical flask with deionized water subjected to secondary distillation for 3-4 times, draining the washing solution into the volumetric flask through a glass rod, and then fixing the volume to 1000 mL. The HEPES solution concentration at this time was 1.00X 10^-2 mol/L。

The buffers used in the following assays were all mixed solutions of HEPES solution (10 nM, pH = 7.4) and absolute ethanol at a volume ratio of 4: 6.

(2) Detection assay

3mL of buffer was added to a concentration of 15uLIs 1.0X 10^-3The fluorescence spectra of the probe stock solutions of mol/L were measured by adding 10 equivalents of the above ion stock solutions, and the results are shown in FIG. 3, where the fluorescence parameters were set to 5nm for the excitation slit width, 2.5nm for the emission slit width, and Ex = 690nm for the excitation wavelength. As can be seen from FIG. 3, Zn is added²⁺The fluorescence spectrum shows that the fluorescence intensity of the probe is obviously enhanced, and the fluorescence intensity is not obviously changed after other ions are added, so that the probe is used for Zn²⁺There is good selectivity for ions, but no specific selectivity for other ions. The fluorescence intensity of the fluorescent probe after reacting with different ions is shown in FIG. 4. As can be seen from FIG. 4, the fluorescent probe reacts with Zn²⁺The fluorescence intensity after the reaction is obviously higher than that of the blank sample and other interfering ions, so that the probe can be used for detecting Zn with high selectivity²⁺The fluorescence-enhanced near-infrared probe of (1).

3mL of the buffer was added to a solution containing 15uL of 1.0X 10^-3mol/L of the probe stock solution, 15uL of 1.0X 10^-2mol/L of Zn²⁺Detecting the time variation of fluorescence spectrum in the solution, wherein the excitation slit width is 5nm, the emission slit width is 2.5nm, the excitation wavelength is Ex = 690nm, and the probes LFX and Zn are shown in FIG. 5²⁺The response was rapid, changing from colorless to green, the fluorescence intensity reached the maximum almost instantaneously, and the reaction was substantially complete in 1 minute. The fluorescence intensity does not change with time, and the probes LFX and Zn are proved²⁺The reaction is rapid and stable.

3mL of the buffer was added to a solution containing 15uL of 1.0X 10^-3Adding Zn in different equivalents (0.1 eq, 0.2eq, 0.3 eq, 0.4 eq, 0.5 eq, 0.6 eq, … … 3.5.5 eq, 3.6 eq, 3.7 eq, 3.8 eq, 3.9 eq, 4.0 eq, respectively, based on the molar amount of the probe) into the probe stock solution of mol/L²⁺Shaking the solution evenly, and detecting the change of fluorescence intensity. As shown in fig. 6, with Zn²⁺The fluorescence intensity gradually increased with increasing concentration. When Zn²⁺The concentration is 0-2 × 10^-2mol/L, fluorescence intensity of the solution and Zn²⁺The ion concentrations have a good linear relationship, and as a result, as shown in fig. 7, by linear fitting, a one-dimensional linear equation with y =72.045+482 can be obtained.3276x, the square of the correlation coefficient (R) can be obtained by calculation²) 0.99139, indicating a good fit. The LOD detection limit is finally calculated by the formula LOD =3 σ/k (σ is the standard deviation of 22 pure probes LFX tested consecutively), and the detection limit is 6.594 × 10^-8 mol/L。

Adjusting the pH value of the HEPES solution by using 0.01mol/L NaOH solution or 0.01mol/L HCl solution to be 1-12, and mixing the HEPES solution with the pH value of 1-12 with absolute ethyl alcohol according to a volume ratio of 4:6 to prepare a buffer solution; 3mL of the above buffer solution with different pH was added to a concentration of 1.0X 10^-3mol/L Probe stock solution, 15uL of 1.0X 10^-2mol/L of Zn²⁺And (4) dissolving, and detecting the change of the fluorescence spectrum. The results are shown in FIG. 8. As can be seen from fig. 8, the fluorescence spectrum probes for Zn between pH =7-9²⁺The response of the ions has good stability, so that the probe can detect Zn under physiological environment²⁺Ions.