CN110698493B

CN110698493B - Fluorescent probe for detecting zinc ions and pH, and preparation method and application thereof

Info

Publication number: CN110698493B
Application number: CN201910968509.8A
Authority: CN
Inventors: 唐波; 李平; 白晓艺; 王昕�; 张雯
Original assignee: Shandong Normal University
Current assignee: Shandong Normal University
Priority date: 2019-10-12
Filing date: 2019-10-12
Publication date: 2020-09-29
Anticipated expiration: 2039-10-12
Also published as: CN110698493A

Abstract

The present disclosure belongs to the field of fluorescent probe synthesizing technologyRelates to a fluorescent probe for detecting zinc ions and pH, a preparation method and application thereof. Zn²⁺Of great importance for maintaining biological function, current studies indicate that the severity of depression is directly proportional to serum zinc levels. In addition, there are related studies that indicate that venous blood pH is elevated and statistically significant in patients with depression. The present disclosure provides a method for simultaneously detecting Zn in brain²⁺And a fluorescent probe compound of pH, wherein the DPA group, the coumarin and the naphthalene fluorescent group are coupled through covalent bonds. Through animal model and living body imaging verification, the fluorescent probe can break through the existence of blood brain barrier, shows good detection signals under complex biological background in brain, and is expected to be used as a reliable depression detection probe.

Description

Fluorescent probe for detecting zinc ions and pH, and preparation method and application thereof

Technical Field

The disclosure belongs to the technical field of fluorescent probe synthesis, and particularly relates to a fluorescent probe compound of zinc ions and pH, and a preparation method and application of the compound.

Background

The information in this background section is only for enhancement of understanding of the general background of the disclosure and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.

Depression, as a group of mental diseases, has extremely high morbidity and mortality, and brings serious harm to human health. However, the pathophysiology of depression is currently poorly understood, and researchers must have a thorough understanding of the development and progression of depression in order to provide effective prevention and treatment of depression. The fluorescence imaging technology is an important imaging method for researching the occurrence and development of biological events and diseases in cells and living bodies on a molecular level due to the advantages of real time, visibility, small damage to organisms and the like.

As the second most abundant transition metal ion, Zn, in the human body²⁺Is important for maintaining biological functions, including regulation of biological redox systems, cell signaling, and the like; inside the cell H⁺Plays a crucial role in many cellular activities such as cell growth, apoptosis, ion transport, autophagy, enzyme activity, homeostasis and other cellular processes. Many assays for Zn have been developed in recent years²⁺Or pH, but these probes are used for single-component detection and mostly for imaging at the cellular level, detecting Zn in vivo due to the complex biological background of the brain and the presence of the blood-brain barrier²⁺And pH have certain design difficulties.

Disclosure of Invention

Against the above research background, the present disclosure is directed to detecting Zn in the brain of an organism²⁺Fluorescent probe compounds with pH were investigated, coupling DPA (Zn) by covalent bond²⁺Recognition group), coumarin and naphthalene fluorescein (pH recognition group) constitute a fluorescent probe compound. Animal experiments prove that the fluorescent probe compound can indicate Zn in vivo²⁺And the content change of pH, and has important significance when being applied to the detection of depression.

In order to achieve the technical effects, the present disclosure provides the following technical solutions:

in a first aspect of the disclosure, there is provided a compound having the structure shown in formula i below:

in a second aspect of the present disclosure, there is provided a process for the preparation of a compound of the first aspect, which compound is synthesized from compound a and compound B; the compound A has a structure shown as the following formula A,

the compound B has a structure shown as a formula B,

preferably, the preparation method comprises the following specific steps:

adding solution of compound A and compound B into 1-Hydroxybenzotriazole (HOBT), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), N-Dimethylformamide (DMF) and CH₂Cl₂And adding piperazine, mixing and reacting at room temperature to obtain the compound.

Further preferably, the preparation method further comprises an elution purification step.

In a third aspect of the disclosure, there is provided a compound of the first aspect and use thereof as a fluorescent probe.

Preferably, the fluorescent probe is used for detecting Zn in the biological sample²⁺Fluorescent probe of pH.

In a fourth aspect of the present disclosure, there is provided a depression detection kit, comprising a compound of the first aspect.

In a fifth aspect of the present disclosure, a method for detecting depression is provided, wherein the compound of the first aspect is injected into a subject to be detected, and the brain fluorescence signal intensity of the subject to be detected is detected.

Compared with the prior art, the beneficial effect of this disclosure is:

because blood brain barrier exists in the brain of an organism and the biological background information is relatively complex, Zn is aimed at in the prior art²⁺And pH, and detection in vivo has not been achieved. The fluorescent probe compound provided in the present disclosure is capable of penetrating the blood brain barrier and clearly showing intracerebral Zn in vivo²⁺And the fluorescence signal of pH, has good popularization significance when being applied to depression detection, and also provides corresponding basis for depression mechanism research.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is a mass spectrum of a fluorescent probe compound of example 1;

FIG. 2 is a graph showing the results of fluorescence detection of the fluorescent compound in example 2;

wherein, FIG. 2(a) is a fluorescence absorption spectrum of the fluorescent probe compound in example 1;

FIG. 2(b) is the fluorescence absorption intensity of the probe compound at 390nm excitation;

fig. 2(c) shows the fluorescence absorption intensity of the probe compound under 610nm excitation at pH 7.4.

FIG. 3 shows the fluorescent compound in Zn in example 2²⁺Fluorescence imaging under the influence conditions;

FIG. 3A is a fluorescent micrograph of control PC12 cells;

FIG. 3B is ZnSO₄Fluorescence micrographs of (50. mu.M) treated PC12 cells;

FIG. 3C is ZnSO₄Fluorescence micrograph of PC12 cells of + TPEN-treated group

FIG. 3D shows control group, ZnSO₄Treatment group and ZnSO₄Fluorescence intensity profile for + TPEN treated group.

FIG. 4 is a graph of fluorescence imaging of the fluorescent compound of example 2 under pH-affected conditions;

wherein, FIG. 4A is a graph of fluorescence intensity of a fluorescent compound at pH 6.5;

FIG. 4B is a graph of the fluorescence intensity of fluorescent compounds at pH 7.0;

FIG. 4C is a graph of the fluorescence intensity of fluorescent compounds at pH 7.5;

FIG. 4D is a plot of the fluorescence intensity of fluorescent compounds at pH 8.0;

FIG. 4E is a fluorescence intensity histogram of FIGS. 4A, 4B, 4C, and 4D.

FIG. 5 is an image of a living body in example 1;

wherein, FIG. 5A shows mouse brain Zn²⁺Imaging result graph;

FIG. 5B is a graph showing the result of pH measurement of mouse brain;

FIG. 5C shows Zn in brains of control mice and depressed mice²⁺A fluorescence intensity histogram;

FIG. 5D is the fluorescence intensity histogram of the pH detection of the brains of the control group of mice and the depressed group of mice.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

As described in the background, Zn²⁺Is important for maintaining biological function, and is also essential for the maturation and function of the brain, and existing studies indicate that the severity of depression is directly proportional to serum zinc levels. In addition, there are related studies that indicate that venous blood pH is elevated and statistically significant in patients with depression. The present disclosure provides a method for simultaneously detecting Zn in brain²⁺And the fluorescent probe compound with pH can break through the existence of blood brain barrier and display good detection signals under the complex biological background in the brain.

the compound B has a structure shown as a formula B,

preferably, the preparation method comprises the following specific steps:

adding the solution of compound A and compound B into HOBT, EDC, DMF and CH₂Cl₂And adding piperazine, mixing and reacting at room temperature to obtain the compound.

In some embodiments, the compound A, the compound B, the HOBT, the EDC and the piperazine are added in a molar ratio of 0.8-1.2: 1.2-1.7: 1-1.4.

In some specific embodiments, the DMF, CH₂Cl₂The volume ratio of (A) to (B) is 0.8-1.2: 3.8-4.2.

In this series of examples, the ratio of compound A to DMF is 0.8 to 1.2 mmol: 1.8-2.2 mL.

In some specific embodiments, the product after the reaction is purified by thin layer chromatography.

In this series of examples, CH is employed₂Cl₂/CH₃OH as eluent.

Preferably, the compound A is synthesized by reacting a dimer amine with 7-hydroxycoumarin.

In some specific embodiments, the specific synthetic steps for the compounds are as follows:

adding formaldehyde aqueous solution into acetonitrile solution of the diamine, heating for reaction for a period of time, adding acetonitrile solution of 7-hydroxycoumarin, and continuing the reaction to obtain the compound A.

In this series of examples, the volume fraction of the aqueous formaldehyde solution is 35 to 39%, more preferably 37%.

In the series of embodiments, the heating reaction temperature is 58-62 ℃ and the heating time is 0.4-0.6 hours.

In the series of examples, the reaction was continued for 0.8 to 1.2 hours after adding the acetonitrile solution of 7-hydroxycoumarin.

Preferably, the compound B is synthesized by 1, 6-dihydroxynaphthalene and 1,2, 4-benzoic anhydride.

In some specific embodiments, the specific synthetic steps for compound B are as follows:

dissolving 1, 6-dihydroxynaphthalene and 1,2, 4-benzoic anhydride in methanesulfonic acid to obtain a mixture, and stirring for a period of time after heating; then, the mixture is cooled to room temperature and filtered under ice bath conditions to obtain a solid part, the solid part is added into an alkali solution, and the pH is adjusted to be slightly acidic by stirring reaction for a period of time, and the solid part is the product compound B.

In this series of examples, the molar ratio of the 1, 6-dihydroxynaphthalene to the 1,2, 4-benzoic anhydride is 7-9: 3-5.

In the series of embodiments, the mixture is heated to 99-102 ℃ and stirred for 18-22 hours.

In the series of embodiments, the alkali solution is a sodium hydroxide solution, and the mass fraction of the alkali solution is 18-22%.

In this series of embodiments, the synthetic method further comprises purifying the product by column chromatography; further, the eluent is methanol/dichloromethane.

In a third aspect of the disclosure, there is provided the use of a compound of the first aspect as a fluorescent probe.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific examples and comparative examples.

EXAMPLE 1 Synthesis and characterization of fluorescent Probe Compounds

7-hydroxycoumarin: 2, 4-dihydroxybenzaldehyde (0.62g, 3.08mmol), diethyl malonate (0.62g, 3.08mmol), 25mL absolute ethanol, 0.5mL piperidine, 2d glacial acetic acid heated under reflux for 8 h. And adding cold water to precipitate after the reaction is finished to obtain the coumarin ethyl ester. 4g of coumarin ethyl ester, 3g of NaOH,20mL of absolute ethyl alcohol and 100mL of water are heated and dissolved for 15min, after the mixture is cooled slightly, the reaction product is poured into 10mL of concentrated hydrochloric acid and 50mL of water, and light yellow crystals are separated out, namely the product.

A compound A: to a solution of the dimer amine (0.62g, 3.08mmol) in 30mL acetonitrile was added aqueous formaldehyde (37%) (0.27mL, 3.08 mmol). After heating at 60 ℃ for 0.5 h, 7-hydroxycoumarin (0.50g, 3.08mmol) in 30mL acetonitrile was added. The reaction was continued for 1 hour and monitored by TLC.

Carboxynaphthalene fluorescein (compound B): 1, 6-dihydroxynaphthalene (1.3g, 8.0mmol) and 1,2, 4-benzoic anhydride (0.78g, 4.0mmol) were dissolved in methanesulfonic acid and the mixture was stirred at 100 ℃ for 20 hours. After that, the mixture was cooled to room temperature and slowly poured into ice water. Then, the resulting mixture was filtered under reduced pressure, and the solution was dissolved in 20% sodium hydroxide solution after washing three times with water. The dark green solution was stirred at room temperature for 30 minutes and the pH was further adjusted to slightly acidic with hydrochloric acid. The crude product was obtained by filtration, which was purified by column chromatography using methanol/dichloromethane (1/9, v/v, 0.4% glacial acetic acid) as eluent, the product being a dark red solid (0.83g, 1.7mmol, 40% yield).

And (3) final product: a solution of Compound A (0.416g, 1.0mmol) and Compound B (0.476g, 1.0mmol) was added to HOBT (0.203g, 1.5mM), EDC (0.288g, 1.5mM) and 2mL DMF, 8mL CH₂Cl₂In (1). Piperazine (0.010g, 1.2mM) was then added. After completion, the mixture was stirred at room temperature overnight. The crude product was purified by TLC using CH₂Cl₂/CH₃OH (10: 1) as eluent gave a red product (DNP).

Example 2 characterization information and applications of fluorescent Probe Compounds

The compound prepared in example 1 is verified for structure by nuclear magnetic resonance and mass spectrometry, wherein the mass spectrometry of the fluorescent probe compound described in example 1 is shown in figure 1.

The nuclear magnetic data are as follows:¹H NMR(400MHz,DMSO-d₆)8.52(d,J＝4.0Hz, 2H),8.10(d,J＝4.0Hz,1H),7.97(s,1H),7.95(d,J＝1.2Hz,2H),7.74(d, J＝1.6Hz,1H),7.71(d,J＝4.0Hz,2H),7.69(s,2H),7.52(d,J＝0.8Hz,2H), 7.50(t,J＝0.8Hz,2H)7.48(d,J＝0.8Hz,2H),7.42(s,1H),7.40(d,J＝0.8 Hz,2H),7.37(t,J＝0.8Hz,2H),7.36(d,J＝0.8Hz,1H),6.88(d,J＝3.6Hz, 1H),5.27(t,J＝4.4Hz,2H),4.74(d,J＝15.2Hz,1H),3.95(s,4H),3.46(t, J＝6.0Hz,4H),3.42(s,2H),3.40(t,J＝5.2Hz,4H).¹³C NMR(101MHz, DMSO-d₆)172.51,168.99,162.88,162.73,158.39,157.92,157.75, 157.66,156.36,155.60,155.05,154.33,151.10,150.24,149.45,148.81, 148.77,146.66,144.40,144.28,143.27,137.61,137.26,136.53,135.53, 130.80,129.94,128.28,127.50,126.68,124.82,124.28,123.67,123.45, 123.14,122.99,119.80,119.53,119.48,117.81,114.44,113.77, 110.11,109.94,109.59,47.53,46.10,45.79,41.39,36.19,31.19,21.65, 21.47 hrms (esi) data, C₅₆H₄₁N₅O₁₀The mass-to-charge ratio hydrogenation peak of (A) should be [ M + H ]]⁺944.2966, find 944.2926.

EXAMPLE 3 fluorescent Properties of fluorescent Probe Compounds

The fluorescent probe compound (DNP) prepared in example 1 shows an absorption band at about 390nm (coumarin moiety), 580nm (naphthalene fluorescein moiety). (FIG. 2 (a)). As shown in FIGS. 2(b) and 2(c), the fluorescence intensity of DNP at 460nm under 390nm excitation is weak, and 10. mu.M Zn is added²⁺The fluorescence intensity of 460nm later is obviously enhanced, and the fluorescence intensity is more than 3.5 times of the initial fluorescence intensity. Similarly, the fluorescence intensity of DNP at 680nm was weak under 610nm excitation at pH 7.4, and when pH was increased to 9.0, the fluorescence intensity at 680nm was significantly increased, which was 4 times or more the initial fluorescence intensity.

Example 4 cell experiments

By using ZnSO₄The PC12 cells were incubated and,increasing intracellular Zn²⁺The concentration of (c). As shown in FIG. 3, ZnSO was used in comparison with the control group₄The (50. mu.M) treated PC12 cells showed intense blue fluorescence. To determine the change in fluorescence is from Zn²⁺Induced by the change, in this example, cells were treated with N, N, N ', N' -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, 50. mu.M), a membrane permeable metal ion chelator, which scavenges Zn²⁺After that, the blue fluorescence intensity was significantly decreased. Next, this embodiment uses a high K⁺Concentration buffer and ionophore nigericin to change intracellular pH levels. FIG. 4 shows that the fluorescence intensity of PC12 cells increased with increasing pH (6.5-8.0). The imaging results show that DNP can observe Zn in living cells²⁺And a transient change in pH.

Example 5 in vivo experiments

In the embodiment, the probe is injected into the abdominal cavity to image the brain of the mouse, and the fluorescence intensity of the experimental group is obviously weakened, which shows that Zn in the brain of the mouse with depression²⁺A significant decrease in pH (figure 5). Through the above experiments, this example first analyzed Zn in the brain of depressed mice by fluorescence imaging²⁺The pH level was reduced to a different extent than in normal mice. The result is Zn²⁺The negative correlation between pH level and extent of depression provides direct evidence.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. A compound having the structure shown in formula i:

2. a process for the preparation of a compound according to claim 1, wherein the compound is synthesized by compound a and compound B; the compound A has a structure shown as the following formula A,

the compound B has a structure shown as a formula B,

the preparation method comprises the following specific steps:

adding the solution of compound A and compound B into HOBT, EDC, DMF and CH₂Cl₂Adding piperazine, mixing and reacting at room temperature to obtain the compound;

the adding proportion of the compound A, the compound B, the HOBT, the EDC and the piperazine is 0.8-1.2: 1.2-1.7: 1-1.4 by mole ratio;

the DMF and CH₂Cl₂The volume ratio of (A) to (B) is 0.8-1.2: 3.8-4.2;

the ratio of the compound A to DMF is 0.8-1.2 mmol: 1.8-2.2 mL.

3. The method of claim 2, further comprising an elution purification step.

4. A process for the preparation of a compound according to claim 3, wherein the product of the reaction is purified by thin layer chromatography.

5. A process for the preparation of a compound according to claim 3, wherein CH is used₂Cl₂/CH₃OH as eluent.

6. Use of a compound according to claim 1 for the preparation of a fluorescent probe.

7. The use of claim 6, wherein the fluorescent probe is for detecting Zn in a biological sample²⁺Fluorescent probe of pH.

8. A depression detection kit comprising a compound of claim 1.