CN112079847B

CN112079847B - Rhodamine imidazole-containing six-membered ring compound and preparation method and application thereof

Info

Publication number: CN112079847B
Application number: CN202010963711.4A
Authority: CN
Inventors: 杨松; 杨林林; 王培义; 唐阿玲; 向红梅; 谭帅
Original assignee: Guizhou University
Current assignee: Guizhou University
Priority date: 2020-09-14
Filing date: 2020-09-14
Publication date: 2022-09-27
Anticipated expiration: 2040-09-14
Also published as: CN112079847A

Abstract

The invention relates to a rhodamine imidazole-containing six-membered ring compound, and a preparation method and application thereof. The compound can improve the hydrolysis rate of metal copper ions, and the complete imidazole compound is introduced into the system to synthesize a series of rhodamine imidazole hexatomic ring-containing compounds, the naked eye recognition capability of the compound on the metal copper ions is 80nM at most, the detection limit is 35pM, the fluorescence quantum yield is 0.63, and the recognition of Cu in adenocarcinoma human alveolar basal epithelial cells is successfully realized ²⁺ 。

Description

Rhodamine imidazole-containing six-membered ring compound and preparation method and application thereof

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a rhodamine imidazole containing hexatomic ring compound and a preparation method and application thereof.

Background

Transition metal ions are indispensable and important components in the living body, and participate in a variety of basic physiological processes in many living bodies. Among them, copper is an extremely important transition metal responsible for the normal functions and metabolic processes of various organs. However, Cu ²⁺ Can lead to growth failure and neurodegenerative disease, while excessive Cu in biological processes ²⁺ Can lead to severe alzheimer's disease, wilson's disease and metabolic disorders. Currently, the fluorescent chemical sensor can effectively and selectively monitor the distribution and dynamic fluctuation of metal ions in living cells and subcellular microenvironments, and the deep understanding of various metal-mediated physiological and pathological processes and disease diagnosis draws extensive attention.

Researchers develop fluorescent small molecular probes taking coumarin, curcumin, pyrene, naphthalimide, anthocyanin, BODIPY, rhodamine and the like as fluorophores as parent bodies. Rhodamine, as represented, has high advantages due to its excellent photophysical properties, such as high extinction coefficient, excellent quantum yield, good photostability, and relatively long emission wavelength. Because rhodamine spironolactone or spironolactone derivatives are non-fluorescent and colorless, the corresponding spironolactone/lactone ring-opening produces intense fluorescence emission and a pink fluorescence-enhanced probe. Based on the above, the proper ligand on the spironolactone ring can cause color change and fluorescence change after metal ions are added, and the ligand becomes the first choice for scientific researchers to design and detect copper ions.

1997, Czarnik group [ Dujols, v.; ford, F.; czarnik, A.W.J.Am.chem.Soc.1997, 119, 7386-]Report that rhodamine B hydrazide ring-opening reaction detects Cu ²⁺ The pioneering work of (1). Rhodamine B hydrazide as Cu ²⁺ Can selectively identify Cu ²⁺ While being Cu ²⁺ Promoting hydrolysis to obtain fluorescent rhodamine B. Experiments show that the system can detect 10nM Cu ²⁺ 。

Swamy et al [ Swamy, k.m.k.; ko, s.k.; kwon, s.k.; lee, h.n.; mao, c.; kim, j.m.; lee, k.h.; kim, j.; shin, I,; yoon, J.chem.Commun.2008, 5915-]Boronic acid-linked copper ion fluorescent and colorimetric chemical probes are reported. Realizes the Cu in different metal ions by a chelation mechanism ²⁺ The naked eye identification capacity of the probe is 300 mu M, the fluorescence quantum yield is 0.45, and the application of the probe in the aspect of detecting copper ions in mammalian cells and organisms is successfully realized.

2012 Kumar et al [ Kumar, m.; kumar, n.; bhalla, v.; sharma, p.r.; kaur, T.org.Lett.2012, 14, 406-409) reports rhodamine hydrazine containing five-membered ring compounds, and the detection limit of the compounds on copper ions is 20nM by a hydrolysis mechanism, and the yield of fluorescence quantum is 0.19. Realizes the imaging of copper ions in human prostate cancer cells.

The traditional rhodamine five-membered ring structure is difficult to improve the detection capability of copper ions. Because, the detection mechanism of copper ions by rhodamine five-membered ring C-N bond is roughly divided into two types: 1) chelating mode and 2) hydrolysis mode, but the probe has low chelating efficiency with copper ions and hydrolysis is incomplete. Meanwhile, the traditional rhodamine five-membered ring lactam structure usually shows an 'unstable' characteristic in an acid solution, thereby bringing uncertainty and limitation to the application of the rhodamine five-membered ring lactam structure. Therefore, the search for a new and advantageous helical toroidal skeleton is a feasible path to improve the copper ion detection capability.

Wu et al [ Wu, C., 2012; bian, q.n.; zhang, b.g.; cai, x.; zhang, s.d.; zheng, h.; yang, s.y.; jiang, Y.B.org.Lett.2012, 14, 4198-4201] reports rhodamine six-membered ring compounds for the first time, the naked eye recognition capability of the probe on copper ions is 10 mu M by participating in the cleavage of C-N bonds and the formation of isothiocyanate groups, and the fluorescence quantum yield is 0.079.

2017, Majumdar et al [ Majumdar, A.; lim, c.s.; kim, h.m.; ghosh, K.ACS Omega 2017, 2, 8167-]Reports that rhodamine six-membered ring compounds can selectively distinguish Cu by C-C bond cleavage and metallic copper ion chelation ²⁺ The detection limit of the probe to copper ions is 0.554 mu M, the fluorescence quantum yield is 0.63, and the application of the probe to the copper ions in cervical cancer cells is successfully realized.

However, there are few reports that probes can simultaneously achieve the detection of copper ions at the nM naked eye recognition and the pM detection level. In order to find ultra-sensitive detection copper ions, the rhodamine compounds are used as the basis, the metal copper ion hydrolysis rate can be possibly improved and recognized, a series of rhodamine imidazole hexatomic ring compounds are synthesized, and the copper ion detection capability of the rhodamine imidazole hexatomic ring compounds is investigated. Opens up a new way for developing a hypersensitive fluorescent probe.

Disclosure of Invention

The purpose of the invention is: provides a six-membered ring compound containing rhodamine imidazole, a preparation method and application thereof, which has good selectivity on metallic copper ions and an economic and simple synthetic method. The invention aims to provide a probe which is simple to prepare, good in selectivity, high in sensitivity and wide in detection range, can be used for identifying copper ions by breaking C-C and C-N to release rhodamine acid through catalytic hydrolysis and opening fluorescence, and can be used for detecting metal copper ions in adenocarcinoma human alveolar basal epithelial cells.

The invention is realized by the following steps: a six-membered ring compound containing rhodamine imidazoles has a structure shown as a general formula (I):

in the formula, R is hydrogen, methyl, amido, benzyl, aminomethyl, thiomethyl and thioethyl.

The preparation method of the six-membered ring containing rhodamine imidazole comprises the following synthetic route;

Application of rhodamine imidazole-containing six-membered ring compound in detecting metal cupric ions.

By adopting the technical scheme, the invention takes the rhodamine compound as the basis, introduces the imidazole compound which can improve the hydrolysis rate of identifying metallic copper ions and completely identify the metallic copper ions into the system to synthesize a series of six-membered ring compounds containing the rhodamine imidazole, and discovers that in the series of compounds, the naked eye identification capacity of one compound to the metallic copper ions is 80nM, the detection limit is 35pM, and the fluorescence quantum yield is 0.63, so that the identification of Cu in the adenocarcinoma human alveolar basal epithelial cells is successfully realized ²⁺ . Opens up a new way for developing a hypersensitive fluorescent probe.

Drawings

FIG. 1: a is the probe 1 (10. mu.M) of the present invention added with 10. mu.M Cu in a mixed solution of acetonitrile and water (3: 7, V/V) ²⁺ And other metal ions (Ca) ²⁺ 、Ni ²⁺ 、Ba ²⁺ 、Cr ³⁺ 、Co ²⁺ 、Hg ²⁺ 、Al ³⁺ 、Fe ³⁺ 、Zn ²⁺ 、Li ⁺ 、Pb ²⁺ 、Cd ²⁺ 、Mg ²⁺ 、K ⁺ 、Na ⁺ 、Ag ⁺ 、Fe ²⁺ ) The fluorescence emission spectrum of (d) (λ ex 556nm, slit 3/3 nm); b is the probe 1 (10. mu.M) of the present invention added with 10. mu.M Cu in a mixed solution of acetonitrile and water (3: 7, V/V) ²⁺ And other metal ions (Ca) ²⁺ 、Ni ²⁺ 、Ba ² ⁺ 、Cr ³⁺ 、Co ²⁺ 、Hg ²⁺ 、Al ³⁺ 、Fe ³⁺ 、Zn ²⁺ 、Li ⁺ 、Pb ²⁺ 、Cd ²⁺ 、Mg ²⁺ 、K ⁺ 、Na ⁺ 、Ag ⁺ 、Fe ²⁺ ) The change of the ultraviolet absorption spectrum of (1); c is the probe 1 (10. mu.M) of the present invention added with 10. mu.M Cu in a mixed solution of acetonitrile and water (3: 7, V/V) ²⁺ And other metal ions (Ca) ²⁺ 、Ni ²⁺ 、Ba ²⁺ 、Cr ³⁺ 、Co ²⁺ 、Hg ²⁺ 、Al ³⁺ 、Fe ³⁺ 、Zn ²⁺ 、Li ⁺ 、Pb ²⁺ 、Cd ²⁺ 、Mg ²⁺ 、K ⁺ 、Na ⁺ 、Ag ⁺ 、Fe ²⁺ ) Color change under fluorescent light.

FIG. 2 shows that 10. mu.M of Cu was added to a mixed solution of probe 1 (10. mu.M) of the present invention in acetonitrile/water (3: 7, V/V) ²⁺ And other metal ions (1) Probe 1(2) Hg ²⁺ ，(3)Pb ²⁺ ，(4)Ni ⁺ ，(5)K ⁺ ，(6)Cd ²⁺ ，(7)Zn ²⁺ ，(8)Ba ²⁺ ，(9)Na ⁺ ，(10)Cr ³⁺ ，(11)Ag ⁺ ，(12)Al ³⁺ ，(13)Co ²⁺ ，(14)Li ⁺ ，(15)Fe ³⁺ ，(16)Mg ²⁺ ，(17)Ca ²⁺ ，(18)Fe ²⁺ ，(19)50μMFe ²⁺ Change of fluorescence emission spectrum intensity (lambda) of competition experiment _ex /λ _em 556nm/578nm, slit 3/3nm)

FIG. 3: a is that the probe 1(10 mu M) of the invention is added with Cu with different concentrations in a mixed solution of acetonitrile and water (3: 7, V/V) ²⁺ (10. mu.M, 7.5. mu.M, 5.0. mu.M, 2.5. mu.M) fluorescence emission spectrum intensityChange of (λ) _ex /λ _em 556nm/578nm, slit 3/3 nm); b is the mixture of probe 1 (10. mu.M) of the present invention in acetonitrile/water (3: 7, V/V) and 2.5. mu.M Cu ²⁺ Fluorescence emission spectrum change condition (lambda) at different time _ex 556nm with slit 3/3 nm).

FIG. 4 shows that Cu is added to a mixed solution of acetonitrile and water (3: 7, V/V) of probe 1 (10. mu.M) of the present invention ²⁺ 10 μ M high resolution mass spectrum (ESI) M/z: [ M ] A] ⁺ Calcd for C ₂₈ H ₃₁ N ₂ O ₃ 443.2329；Found 443.2317。

FIG. 5 shows that the rhodamine (1 μ M) of the invention is added with Cu in the mixed solution of acetonitrile and water (3: 7, V/V) ²⁺ 0.5 μ M HPLC (column EC-C184.6X 100mm, column temperature 35 deg.C, mobile phase methanol: water (65: 35, V/V), flow rate 1mL/min, injection amount 2 μ L, λ _ex /λ _em 556nm/578nm, retention time 4.716 min).

FIG. 6 is a high performance liquid chromatography of probe 1 (1. mu.M) of the present invention in a mixed solution of acetonitrile and water (3: 7, V/V) (column EC-C184.6X 100mm, column temperature 35 ℃, mobile phase methanol and water (65: 35, V/V), flow rate 1mL/min, injection amount 2. mu.L, lambda./V) _ex /λ _em 556nm/578nm, retention time 4.716 min).

FIG. 7 shows that Cu is added to a mixed solution of acetonitrile and water (3: 7, V/V) of probe 1 (1. mu.M) of the present invention ²⁺ 0.5. mu.M, high performance liquid chromatography for 2 minutes of reaction (column EC-C184.6X 100mm, column temperature 35 ℃, mobile phase methanol: water (65: 35, V/V), flow rate 1mL/min, injection amount 2. mu.L, lambda. (V/V)) _ex /λ _em 556nm/578nm, retention time 1-2.330 min, retention time 2-2.878 min, retention time 3-4.717 min).

FIG. 8 shows that the probe 1 (1. mu.M) of the present invention was added with Cu in a mixed solution of acetonitrile and water (3: 7, V/V) ²⁺ 0.5. mu.M, high performance liquid chromatography for 1.5 hours (column EC-C184.6X 100mm, column temperature 35 ℃, mobile phase methanol: water (65: 35, V/V), flow rate 1mL/min, injection amount 2. mu.L,. lambda. _ex /λ _em 556nm/578nm, retention time 1-2.332 minClock, retention time 2-3.119 minutes, retention time 3-4.715 minutes).

FIG. 9 shows that Cu is added to a mixed solution of acetonitrile and water (3: 7, V/V) of probe 1 (1. mu.M) of the present invention ²⁺ 0.5 μ M, high performance liquid chromatography for 3.5 hours (column EC-C184.6X 100mm, column temperature 35 deg.C, mobile phase methanol: water (65: 35, V/V), flow rate 1mL/min, injection amount 2 μ L, λ L _ex /λ _em 556nm/578nm, retention time 4.714 min).

FIG. 10 shows Cu of the present invention ²⁺ A possible mechanism for inducing the catalytic hydrolysis reaction of probe 1 in acetonitrile-water.

FIG. 11 shows that probe 1 (1. mu.M) of the present invention was added to a mixed solution of acetonitrile and water (3: 7, V/V) with Cu of different concentrations ²⁺ Linear relationship of fluorescence emission spectrum intensity after reaction for 60 minutes (. lamda.) _ex /λ _em 556nm/578nm with slit 3/3 nm).

FIG. 12 shows that probe 1 (1. mu.M) of the present invention was added to a mixed solution of acetonitrile and water (3: 7, V/V) with Cu of different concentrations ²⁺ After 60 minutes of reaction, the force diagram was recognized with the naked eye.

FIG. 13 shows Probe 1 (5. mu.M) of the present invention against Cu in adenocarcinoma human alveolar basal epithelial cells ²⁺ Identified fluorescence image wherein (a) ₁ ) Adenocarcinoma human alveolar basal epithelial cell +1 (5. mu.M) brightfield, (a) ₂ ) Adenocarcinoma human alveolar basal epithelial cells +1 (5. mu.M) dark field, (a) ₃ ) Adenocarcinoma human alveolar basal epithelial cells +1 (5. mu.M) brightfield dark field merging, (b) ₁ ) Adenocarcinoma human alveolar basal epithelial cells +1(5 μ M) + Cu ²⁺ (5. mu.M) brightfield, (b) ₁ ) Adenocarcinoma human alveolar basal epithelial cells +1 (5. mu.M) + Cu ²⁺ (5. mu.M) dark field, (b) ₁ ) Adenocarcinoma human alveolar basal epithelial cells +1 (5. mu.M) + Cu ²⁺ (5 μ M) bright field dark field merging, (dark field channel 510-.

Examples

The invention is further illustrated by the following examples. It should be understood that the method described in the examples is only for illustrating the present invention and not for limiting the present invention, and that simple modifications of the preparation method of the present invention based on the concept of the present invention are within the scope of the claimed invention. All starting materials and solvents used in the examples are commercially available in analytical purity.

Example 1: preparation of target Compound 1

Rhodamine B (200.0mg, 0.4mmol) was added to 10mL of a dry 1, 2-dichloroethane solution containing 0.4mL of phosphorus oxychloride, the reaction was stopped after heating and refluxing for 6 hours, desolventizing was carried out, and the reaction was concentrated by evaporation under reduced pressure. The crude product obtained was dissolved in 10mL of anhydrous acetonitrile. Was added dropwise to 8mL of anhydrous acetonitrile containing 2-aminoimidazole (33.2mg, 0.4mmol) and triethylamine (0.5mL) and refluxed for 4 hours. Desolventizing, concentrating by evaporation under reduced pressure, and performing column chromatography (eluent PE: EA is 100: 1-60: 1) to obtain a yellow solid with the yield of 46.2%.

Synthesis of other target compounds reference example 1.

The structure, nuclear magnetic resonance hydrogen spectrum, carbon spectrum data and high-resolution mass spectrum data of the synthesized six-membered ring compound containing rhodamine imidazoles are shown in table 1.

TABLE 1 NMR hydrogen, carbon and high resolution mass spectra of some of the compounds

Test examples:

the technical scheme adopted by the invention is as follows: a copper ion catalytic hydrolysis type fluorescent molecular probe 1 has the following structure:

in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to fig. 1 to 13 of the present specification.

The embodiment is as follows:

1. preparation of Probe 1 solution

0.0051g of probe 1 was weighed into a 10mL volumetric flask, dissolved in acetonitrile solution and fixed to volume to obtain a concentration of 10 ^-3 Stock solution of M Probe 1.

2. Preparation of metal ions

Weighing various metal nitrates with certain mass in a 100mL volumetric flask, dissolving with deionized water and fixing the volume to obtain the metal nitrate with the concentration of 10 ^-3 M metal ion stock solution.

3. Selectivity of Probe 1 to copper ion

In a system of acetonitrile: water (3: 7, V/V), 10. mu.M each of Ca was added to probe 1 (10. mu.M) ²⁺ 、Ni ²⁺ 、Ba ²⁺ 、Cr ³⁺ 、Co ²⁺ 、Hg ²⁺ 、Al ³⁺ 、Fe ³⁺ 、Zn ²⁺ 、Li ⁺ 、Pb ²⁺ 、Cd ²⁺ 、Mg ²⁺ 、K ⁺ 、Na ⁺ 、Ag ⁺ 、Fe ²⁺ After 10 minutes, the system has no obvious fluorescence and ultraviolet absorption change, and 10 mu M Cu is added ²⁺ After 10 minutes, the fluorescence and UV absorption increased significantly, indicating that probe 1 is present for Cu ²⁺ There was a clear selectivity (FIG. 1).

4. Competitive property of other metal ions to copper ions

At 10. mu.M Cu ²⁺ Other metal ion (Ca) ²⁺ 、Ni ²⁺ 、Ba ²⁺ 、Cr ³⁺ 、Co ²⁺ 、Hg ²⁺ 、Al ³⁺ 、Fe ³⁺ 、Zn ²⁺ 、Li ⁺ 、Pb ²⁺ 、Cd ²⁺ 、Mg ²⁺ 、K ⁺ 、Na ⁺ 、Ag ⁺ 、Fe ²⁺ ) In the presence, after 10 minutes, test Probe 1 vs Cu ²⁺ Fluorescence selectivity of (2). By comparing the reaction systems _ex (fluorescence intensity at 578 nm), we found that the fluorescence intensity of the reaction system containing other metal ions is comparable to that of the probe 1 and Cu alone ²⁺ The fluorescence intensity of the reaction system (2) is substantially equivalent. Illustrating other metal ions to Cu ²⁺ No significant competition was shown (FIG. 2), further verifying that the probe1 can identify Cu in complex environment ²⁺ Ability of the probe to detect Cu in a biological environment ²⁺ Providing the possibility of.

5. Probe 1 to Cu ²⁺ Identification mechanism of

The probe 1 is proved to be used for identifying Cu through a catalytic hydrolysis mode by means of fluorescence, high resolution and high performance liquid chromatography ²⁺ (FIGS. 3-10).

6. Probe 1-Cu ²⁺ Sensitivity of (2)

To better illustrate the sensitivity of the probe, the invention selects to add Cu of different concentrations to the probe 1 ²⁺ After 60 minutes, with Cu ²⁺ The fluorescence intensity also increases with increasing concentration. The probe can have good linear response to copper ions between 30nM-1300nM (FIG. 11).

The present invention utilizes a signal-to-noise ratio method (S/N) [ Guo, l.; xu, y.; ferran, a.r.; chen, g.; kim, D.H.J.Am.chem.Soc.2013, 135, 12338-12345]Test Probe 1 vs. Cu ²⁺ The test method comprises the following steps: the probe solution to which no copper ion was added was used as a blank sample, the fluorescence intensity at a specific wavelength was measured 20 times and the average value (average) of the data was calculated _blank ) And Standard Deviation (SD) _blank ) The standard deviation is the noise (N) of the test system; adding a lower amount of copper ions to the probe system with the concentration of the probe constant, measuring the fluorescence intensity at 5 specific wavelengths (corresponding to the above wavelengths) and calculating the average value (average) thereof _sample ) And finally, calculating the signal-to-noise ratio (S/N) of the system according to the following formula.

S/N＝(|average _sample -average _blank |)/SD _blank

And when the value range of S/N is between 3 and 5, the concentration under the condition is the detection limit of the probe. The above method was used to determine the Cu to probe 1 in acetonitrile/water (3: 7, V/V) solution ²⁺ The detection limit of (2) is 35 pM.

6. Adding a certain amount of Cu into a solution of the probe 1 in acetonitrile and water (3: 7, V/V) ²⁺ 60 minutes later, evaluate Probe 1 vs Cu ²⁺ By naked eyesOther capabilities (fig. 12).

7. Probe 1 recognizes Cu in adenocarcinoma human alveolar basal epithelial cells ²⁺ Fluorescent image of

The invention uses a fluorescence microscope to discuss that the probe 1 detects Cu in adenocarcinoma human alveolar basal epithelial cells ²⁺ The possibility of (a). First, 5. mu.M of Probe 1 was added to cultured adenocarcinoma human alveolar basal epithelial cells in ozone (95% air, 5% CO) ₂ ) After incubation at 37 ℃ for 0.5h and rinsing three times with PBS (pH 7.4, 0.1M) buffer, it was imaged under a fluorescence microscope and no significant fluorescence could be observed in the cells. Next, 5. mu.M Cu was added to the cells ²⁺ After incubation for 1.0h under the same conditions, it was rinsed three times with PBS (pH 7.4, 0.1M) buffer and imaged under a fluorescence microscope, and a distinct red fluorescence was observed, indicating Cu ²⁺ Capable of entering cells, and probe 1 and Cu ²⁺ An effect occurs. The experiments prove that the probe 1 can realize the effect of Cu in adenocarcinoma human alveolar basal epithelial cells ²⁺ Detection of (2) (fig. 13).

Claims

1. A six-membered ring compound containing rhodamine imidazole is characterized in that: the compound has a structure shown as a general formula (I):

in the formula, R is hydrogen, methyl, benzyl, aminomethyl, thiomethyl and thioethyl.

2. The rhodamine imidazole containing six-membered ring compound according to claim 1, wherein: and R is methyl.

3. A method for preparing the six-membered ring compound containing rhodamine imidazoles as defined in claim 1, which is characterized in that: the synthetic route is as follows: