CN115716803B - Naphthalimide fluorescent probe and application thereof in polarity and viscosity detection - Google Patents

Abstract

The invention provides a naphthalimide fluorescent probe, which has a chemical structural formula of. The probe can simultaneously show different spectral properties under the change of polar and viscous solvents with different gradient concentrations, can monitor qualitative and quantitative changes of cell polarity and viscosity in real time, and has good sensitivity. The probe has the advantages of good chemical stability, good light stability, low cytotoxicity, rapid concentration change distinguishing and easy synthesis.

Description

Naphthalimide fluorescent probe and application thereof in polarity and viscosity detection

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a naphthalimide fluorescent probe and application thereof in polarity and viscosity detection.

Background

Polarity and viscosity are important parameters affecting the biological microenvironment, and metabolic processes in the living body are closely related to the polarity and viscosity. Physiological and pathological processes of organisms are often closely related to changes in intracellular polarity and viscosity, which can affect cellular metabolism and normal function of the cells, further triggering micro-environmental disorders and leading to the development of some diseases. Such as: cystic kidney disease, malignant tumor, alzheimer's disease, parkinson's disease, etc. Therefore, the technology for detecting the change of polarity and viscosity in living cells has important significance in the fields of biology, medicine and the like, and has an indispensable position for treating, preventing and controlling certain serious diseases. Although methods for detecting the polarity and viscosity of biological microenvironments have been diverse and rapidly developed in recent years, precise and comprehensive techniques remain lacking.

The current methods for detecting the viscosity include a vibrating disk viscometer, a capillary viscometer, a vibrating string viscometer and the like, and the methods for detecting the polarity include a molecular imprinting polymer selective adsorption method, a high performance liquid chromatography and the like, but the detection methods require complex early sample preparation, damage the sample, are only suitable for detecting large samples, have complex operation, detect single sample and the like, and are not suitable for in-situ detection of the intracellular viscosity and the polarity. Compared with the methods, the fluorescent probe technology has the advantages of low cost, convenience, high sensitivity, good selectivity, excellent stability and the like, and particularly, the fluorescent probe can realize in-situ detection of target substances in living cells. The polarity and viscosity fluorescent probes reported at the present stage are all detected singly, and no fluorescent probes capable of detecting the polarity and the viscosity effectively at the same time exist at present. Considering that the change of the polarity and viscosity of the microenvironment of the cells in the organism can cause some physiological or pathological abnormalities, the development of the fluorescent probe for simultaneously detecting the polarity and viscosity of the cells has strong application value.

Disclosure of Invention

Aiming at the problem that probes for simultaneously detecting polarity and viscosity are lacking in the prior art, the invention provides a novel naphthalimide fluorescent probe which is sensitive to polarity and viscosity, good in light stability and chemical stability and low in toxicity.

It is another object of the present invention to provide the use of the above fluorescent probe for detecting viscosity and polarity in a solution or within a biological cell.

In order to achieve the above purpose, the present invention adopts the following technical scheme.

A naphthalimide fluorescent probe has a chemical name of 2- (2- (dimethylamino) ethyl) -6- (2- (4-nitrobenzyl) hydrazino) -1H-benzo [ de ] isoquinoline-1, 3 (2H) -dione, which is called YZN for short, and has a chemical structural formula shown in a formula (I):

formula (I).

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

(1) Reacting 4-bromo-1, 8-naphthalic anhydride and N, N-dimethylethylenediamine in absolute ethanol, cooling the reaction solution, filtering, washing, and drying to obtain a product 1 (name: 6-bromo-2- (2- (dimethylamino) ethyl) -1H-benzo [ de ] isoquinoline-1, 3 (2H) -dione):

；

(2) Reacting the compound 1 with 80% hydrazine hydrate in absolute ethanol, cooling the reaction liquid, filtering, washing, and drying to obtain a product 2 (name: 2- (2- (dimethylamino) ethyl) -6-hydrazino-1H-benzo [ de ] isoquinoline-1, 3 (2H) -dione):

；

(3) Reacting the compound 2 with 4-nitrobenzyl bromide in absolute ethyl alcohol, cooling the reaction liquid, carrying out suction filtration, washing and drying to obtain a fluorescent probe, namely YZN:

。

in the step (1), the molar ratio of the 4-bromo-1, 8-naphthalene dicarboxylic anhydride to the N, N-dimethyl ethylenediamine is 1:1.

In step (1), the reaction temperature was 95 ℃.

In the step (2), the molar ratio of the compound 1 to 80% of hydrazine hydrate is 1:1.

In step (2), the reaction temperature was 95 ℃.

In step (2), the purification step is to wash the solid with absolute ethanol.

In the step (3), the molar ratio of the compound 2 to the 4-nitrobenzyl bromide is 2:5.

In step (3), the reaction temperature was 95 ℃.

In the step (3), the separation and purification step is to cool the reaction liquid to room temperature, filter, wash the filter cake with a little ethanol for 3 times and then dry.

The fluorescent probes described above can be used to detect the polarity and viscosity of a solution, cell or organism.

The mechanism of the invention is as follows:

When YZN is in a low-polarity environment, the YZN interacts with the surrounding solvent weakly due to small ionization, and electron donating amino groups close to the benzene ring and electron pushing amide groups on the benzene ring form a electron-push system, so that the charge transfer process from electron donating groups to electron withdrawing groups in the molecule, namely the intramolecular charge transfer (Photo-induced CHARGE TRANSFER, ICT effect), is enhanced, and strong fluorescence is emitted, so that the fluorescence intensity is enhanced. Meanwhile, p-nitrobenzyl bromide is introduced, so that the generation of photoinduced electron transfer (Photoinduced Electron Transfer, PET effect) is inhibited, and the phenomenon of fluorescence quenching is prevented. However, when it is in a highly polar environment, the excited state energy of the probe molecule is rapidly dissipated due to the increase of the dipole moment, so that the ICT effect of the molecule is gradually weakened, and the fluorescence emission intensity is greatly reduced.

Since the nitrobenzyl hydrazine group of the probe molecule has high space rotation capability, in a solvent with low viscosity, an intramolecular torsion charge transfer function (Twisted Intramolecular CHARGE TRANSFER, TICT) exists, and a torsion intramolecular charge transfer state (TICT state) is formed, so that fluorescence emission is inhibited. However, as the viscosity of the system increases, the spatial rotation ability of the molecules is gradually inhibited, internal transition of the molecules is hindered, and the fluorescence intensity is changed, so that a strong near infrared fluorescence signal is observed.

The invention has the following advantages:

The fluorescent probe YZN is designed and synthesized based on naphthalimide fluorophor, so that double detection of cell polarity and viscosity by using a spectrometry can be realized, and the limitation of the traditional detection technology in the aspect of in-situ detection is eliminated. The probe can simultaneously show different spectral properties under the change of polar and viscous solvents with different gradient concentrations, can monitor qualitative and quantitative changes of cell polarity and viscosity in real time, and has good sensitivity. The probe has the advantages of good chemical stability, good light stability, low cytotoxicity, rapid concentration change distinguishing and easy synthesis.

Drawings

FIG. 1 is a hydrogen spectrum of fluorescent probe YZN;

FIG. 2 is a carbon spectrum of fluorescent probe YZN;

FIG. 3 is a mass spectrum of fluorescent probe YZN;

FIG. 4 is a fluorescence emission spectrum (a) and an ultraviolet absorption spectrum (b) of YZN (5. Mu. Mol/L) in different solvents, wherein 1 represents 1, 4-dioxane, 2 represents dimethyl sulfoxide, 3 represents acetone, 4 represents glycerin, 5 represents methanol, 6 represents ultrapure water, 7 represents ethanol, and 8 represents acetonitrile;

FIG. 5 is a graph of the fluorescence intensity change (b) of YZN (5 [ mu ] mol/L) at different volume ratios of the fluorescence intensity curves (a) and 460 nm for a 1, 4-dioxane-water hybrid test system;

FIG. 6 is a graph showing the change (b) in fluorescence intensity of YZN (5. Mu. Mol/L) at different volume ratios of the fluorescence intensity curves (a) and 460 nm for a glycerol-water mixture test system;

FIG. 7 is a graph of the light stability performance test of YZN (5. Mu. Mol/L);

FIG. 8 is a graph showing the change in fluorescence intensity of YZN (5. Mu. Mol/L) test systems at different pH values (4.0-10.0);

FIG. 9 is a fluorescence emission spectrum of YZN (5. Mu. Mol/L) in different analytes;

FIG. 10 is the viability of Hela cells at different YZN concentrations (0 [ mu ] mol/L,1.0 [ mu ] mol/L,5.0 [ mu ] mol/L,10.0 [ mu ] mol/L,15.0 [ mu ] mol/L,20.0 [ mu ] mol/L,30.0 [ mu ] mol/L,40.0 [ mu ] mol/L).

Detailed Description

The present invention will be further described with reference to examples and drawings, but the present invention is not limited to the examples.

Example 1 Synthesis of fluorescent probes

(1) 4-Bromo-1, 8-naphthalic anhydride (0.1652 g,0.6 mmol), and N, N-dimethylethylenediamine (0.052 g,0.6 mmol) were added to a 50 mL round bottom flask, then 15 mL absolute ethanol was added to the flask, 12 h was refluxed at 95 ℃ with stirring, after the reaction was complete, cooled to room temperature, suction filtered, the filter cake was washed with a small amount of ethanol, and dried to give compound 1 (0.2041 g, 98%):

；

(2) Compound 1 (0.205 g,0.6 mmol) and 80% hydrazine hydrate (0.5 mL) were added to a 50mL round bottom flask with 20 mL absolute ethanol as solvent and stirred at 95 ℃ under reflux for 12 h, the reaction solution was cooled to room temperature, filtered, the filter cake washed with a small amount of ethanol and dried to give compound 2 (0.1277 g, 72.4%) as an orange solid:

；

(3) Compound 2 (0.1277 g,0.4 mmol) and 4-nitrobenzyl bromide (0.2010 g,1 mmol) were added to a round-bottomed flask of 50 mL, then 20 mL absolute ethanol was added, the mixture was stirred under reflux at 95 ℃ for 12 h, the reaction solution was cooled to room temperature, filtered, and the filter cake was washed 3 times with a small amount of ethanol to give an orange-red solid, namely fluorescent probe YZN (0.1923 g, 96.5%):

；

The hydrogen spectrum, carbon spectrum and mass spectrum of the product are shown in figures 1-3 ：¹H NMR (500 MHz, DMSO-d₆) δ 9.30 (s, 1H), 8.67 (dd, J₁= 8.0 Hz, J₂ = 1.0 Hz, 1H), 8.46 (dd, J₁= 7.5 Hz, J₂ = 1.5 Hz, 1H), 8.37 (d, J = 8.5 Hz, 2H), 8.32 (d, J = 8.0 Hz, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.67 (m, 1H), 7.28 (d, J = 8.0 Hz, 1H), 4.85 (s, 2H), 4.74 (s, 2H), 4.57 (t, J = 6.0 Hz, 2H), 3.63 (t, J = 7.5 Hz, 2H), 3.17 (s, 6H). ¹³C NMR (125 MHz, DMSO-d₆) δ 164.32, 163.17, 154.02, 149.10, 135.37, 135.13, 131.37, 130.05, 129.31, 124.63, 124.26, 121.82, 118.90, 107.06, 104.66, 65.68, 61.33, 49.95, 33.61. HR-MS (ESI) calcd. for C₂₃H₂₃N₅O₄ m/z 434.1830, found 434.1830.

Example 2 fluorescence spectra and ultraviolet spectra of fluorescent probes in different solvents

The fluorescent probe obtained in example 1 was prepared into a mother solution using dimethyl sulfoxide as a solvent, the mother solution of the probe was mixed with different solvents (ultrapure water, dimethyl sulfoxide, acetonitrile, methanol, ethanol, acetone, 1, 4-dioxane, glycerol), and the ultraviolet absorption spectrum of the probe (5 μm) to the different solvents was measured, and the result is shown in fig. 4 a: with 440 nm as excitation wavelength, the maximum absorption wavelength in ultrapure water is 446 nm, the absorption intensity is 0.046, the maximum absorption wavelength in 1, 4-dioxane solution is 434.5 nm, and the absorption intensity is 0.042. It is shown that the probe can judge the polarity of the solution more remarkably by utilizing the absorption spectrum. The maximum absorption peak of probe YZN in pure glycerol solution shows a red shift compared to the other solvents (non-viscous); this is because the nitrobenzylhydrazine group of YZN has a high steric rotation ability, but the steric rotation ability of the molecule is suppressed and the degree of conjugation increases with an increase in viscosity.

Subsequently, the fluorescence emission spectra of probe YZN (5 μm) in different solvents (ultrapure water, dimethyl sulfoxide, acetonitrile, methanol, ethanol, acetone, 1, 4-dioxane, glycerol) were determined, the results are shown in fig. 4 b: the probe has the highest fluorescence intensity in the weak-polarity 1, 4-dioxane, and has weaker fluorescence intensity in the strong-polarity ultrapure water, which indicates that the intensity of the probe can be changed along with the change of the polarity, and has higher fluorescence intensity in the weak-polarity environment. This is because when YZN is in a low polarity environment, it will interact less with the surrounding solvent due to less ionization, and thus will fluoresce more strongly. This shows that the fluorescent probe has a significant effect of identifying the polarity, and can identify the polarity change of the solvent to a certain extent.

Example 3 response of fluorescent probes to different polarities

The fluorescence emission spectra of YZN were measured at 440 nm as excitation wavelength in a polar system of different proportions of 1, 4-dioxane and water, the results are shown in FIG. 5: YZN has the strongest fluorescence intensity at 540 nm in solutions with different percentages of 1, 4-dioxane, and blue shift of emission wavelength occurs (FIG. 5 a), and the percentage of 1, 4-dioxane in the solvent is proportional to the fluorescence intensity (FIG. 5 b). The fluorescence intensity of YZN is related to the polarity of the solvent, the polarity of the solvent is reduced, and the fluorescence intensity is enhanced.

Example 4 response of fluorescent probes to different viscosities

The fluorescence emission spectra of YZN were measured at 440 nm for excitation wavelength in a polar system of different proportions of glycerin and water, and the results are shown in fig. 6: YZN has the strongest fluorescence intensity at 540 nm in glycerol solutions with different percentages, blue shift of emission wavelength occurs (FIG. 6 a), and the percentage of 1, 4-dioxane in the solvent is proportional to the fluorescence intensity (FIG. 6 b). The fluorescence intensity of YZN was related to the viscosity of the solvent, which increased, and the fluorescence intensity increased.

Example 5 stability of fluorescent probes

The photostability of the probe is a key indicator of the performance of fluorescent probes. The photostability of probe YZN was tested at ambient temperature at the excitation wavelength (440 nm) in pure PBS buffer, 50% 1, 4-dioxane solution, 50% glycerol solution, respectively. That is, at the excitation wavelength of 440 nm, the probe YZN was continuously scanned for 30min under the illumination condition, and as shown in FIG. 7, the fluorescence intensity of the probe YZN (5. Mu.M) was not significantly changed in the range of 0-1800 s. The probe was shown to detect changes in polarity and viscosity in cells in an illuminated environment. Thus, the probe has good light stability under 440 nm light conditions.

The pH value can influence the growth and apoptosis of cells, and is one of influencing factors of whether the probe can be applied in the cells. Thus, the probe was tested for fluorescence intensity changes at different pH values (4.0-10.0) in three test solvents, PBS buffer, 50% 1, 4-dioxane solution and 50% glycerol solution. As shown in FIG. 8, the fluorescence intensity of the probe YZN has no obvious change in the weak polar environment, which indicates that the probe can detect the polarity in different acid-base environments. This demonstrates that the probe has pH stability in a weak polar environment.

Example 6 specificity of fluorescent probes

The cellular environment in the organism is complex, and many components can interfere with the detection of the polarity and viscosity of the probe. The probe YZN (5 mu M) is mixed with different amino acids or ions (glutathione, glucose, homocysteine, cysteine 、Cd²⁺、Mg^{2 +}、Na⁺、Zn²⁺、Fe³⁺、Cu²⁺、Fe²⁺、Mn²⁺、Al³⁺、1,4- dioxane and glycerol), a fluorescence emission spectrum chart in coexistence is measured, the volume of Fe ³⁺ added in an experiment is 10 mu L, the volume of 1, 4-dioxane and glycerol are 495 mu L, and the rest are 100 mu L, and the result is shown in figure 9: the fluorescence intensity of other analytes except 1, 4-dioxane and glycerol is hardly changed, which indicates that the probe YZN has selectivity on polarity and viscosity, and the probe YZN is not interfered by components in a cell environment when the polarity and the viscosity are detected in an organism.

Example 7 toxicity of fluorescent probes to cells

100. Mu.L of Hela cell suspension was added to a 96-well plate (5000/well), and the mixture was incubated in an incubator for 24h to adhere the cells. After the incubation, the culture medium was aspirated, washed with PBS solution, probes of different concentrations (1.0-40. Mu.M) were added, after 24-h incubation, the culture medium was aspirated, and then 100. Mu.L of CCK-8+DMEM (1:10) mixed solution was added to each well and placed into an incubator for incubation, after the incubation was completed, washed with PBS, and after 30: 30 min cells were tested for viability. The results are shown in FIG. 10: after the probes are incubated for 24h ℃ within the concentration range of 1.0-40 mu M, the cell viability is over 95%, which shows that the probes have low cytotoxicity, can be used in living cells of complex organisms, and ensures the application safety of the probes.

Claims (6)

1. A naphthalimide fluorescent probe has a chemical structural formula shown in a formula (I):

formula (I).

2. A method of preparing a fluorescent probe according to claim 1, comprising the steps of:

(1) Reacting 4-bromo-1, 8-naphthalene dicarboxylic anhydride with N, N-dimethyl ethylenediamine in absolute ethyl alcohol, cooling the reaction liquid, filtering, washing and drying to obtain a product 1:

；

(2) Reacting the compound 1 with 80% hydrazine hydrate in absolute ethyl alcohol, cooling reaction liquid, carrying out suction filtration, washing and drying to obtain a product 2:

；

(3) Reacting the compound 2 with 4-nitrobenzyl bromide in absolute ethyl alcohol, cooling the reaction liquid, carrying out suction filtration, washing and drying to obtain a fluorescent probe:

。

3. the process according to claim 2, wherein in step (1), the molar ratio of 4-bromo-1, 8-naphthalic anhydride to N, N-dimethylethylenediamine is 1:1;

in the step (2), the molar ratio of the compound 1 to 80% of hydrazine hydrate is 1:1;

in the step (3), the molar ratio of the compound 2 to the 4-nitrobenzyl bromide is 2:5.

4. The process according to claim 2, wherein in the steps (1), (2) and (3), the reaction temperature is 95 ℃.

5. The process according to claim 2, wherein in step (2), the purification step is washing the solid with absolute ethanol;

In the step (3), the separation and purification step is to cool the reaction liquid to room temperature, filter, wash the filter cake with a little ethanol for 3 times and then dry.

6. Use of a fluorescent probe according to claim 1 for the preparation of a polar or viscous reagent for detecting a solution, a cell or an organism.