A photochromic green fluorescent protein chromophore derivative

Abstract

The application relates to the technical field of organic solid fluorescent photochromic materials, and particularly discloses a green fluorescent protein chromophore derivative capable of photochromism, which comprises three green fluorescent protein chromophore (HBI) derivatives, and is characterized in that the benzene ring and the imidazolinone part of the HBI are respectively modified by a cyano group and an ethyl group; the p-CBIE, the m-CBIE and the m-DCBIE have the advantages of good biocompatibility (low cytotoxicity).

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organic solid fluorescent photochromic materials, and particularly relates to a green fluorescent protein chromophore derivative capable of photochromism. BACKGROUND

[0002] Traditional fluorophores emit bright fluorescence in a solvent state. However, when they are aggregated, the emitted light is completely quenched due to strong intermolecular Pi-Pi stacking. This phenomenon is called the "aggregation-caused quenching (ACQ)" effect. This effect seriously hinders their practical application in material science. So far, people have made great efforts to solve this problem, but the effect is not satisfactory. Until 2001, Tang Benzhong's research group found that a series of organic molecules with twisted conformation solve the defects of traditional fluorescent materials, such as hexaphenylsilole (HPS) and tetraphenylethylene (TPE), which do not emit light in a molecular solution state, but strongly emit fluorescence in an aggregated state. This phenomenon is contrary to traditional fluorophores and is called the "aggregation induced emission (AIE)" effect.

[0003] Stimulus-responsive organic fluorescent color-changing materials are a new generation of smart materials. When subjected to various specific stimuli (light, heat, force, temperature, electricity, magnetism, pH, and ultrasound), they can cause changes in molecular interactions and energy, or changes in polymer chain structures between molecules, thereby causing color changes and fluorescence changes. Stimulus-responsive materials have shown great potential in the fields of light-sensitive anti-counterfeiting, information storage, nanomaterials, robot engineering, and biomedicine. The reversible photoconversion of a chemical substance between two forms with different absorption spectra, accompanied by a significant color change, is called the photochromic phenomenon. Currently widely studied photochromic materials include spiropyrans, diarylethene, azobenzene, hydrazone, and captax, which have the advantages of strong contrast solid-state emission, sensitive stimulus response, recyclable color change process, and high light stability. However, these materials have problems such as multiple synthesis steps, difficulty in synthesis, high cost, and difficulty in mass production. Therefore, in order to improve the applicability of photochromic materials, a photochromic fluorescent material with the advantages of traditional photochromic fluorescent materials and the characteristics of fewer synthesis steps, low cost, simple synthesis, and easy mass production must be developed. SUMMARY

[0004] The purpose of the present application is to provide a green fluorescent protein chromophore derivative capable of photochromism to solve the problems presented in the background.

[0005] To achieve the above-mentioned purpose, the present application provides the following technical solutions.

[0006] A photochromic green fluorescent protein chromophore derivative, including three green fluorescent protein chromophore (HBI) derivatives, the benzene ring and imidazolinone part of HBI are modified by cyano and ethyl respectively, and the structural formula is as follows:

[0007] ;

[0008] The three green fluorescent protein chromophore derivatives all have photochromic properties.

[0009] The chemical names are respectively:

[0010] (Z)-5-(4-cyanobenzylidene)-2-ethyl-3-methylimidazolinone, (Z)-5-(3-cyanobenzylidene)-2-ethyl-3-methylimidazolinone, (Z)-5-(3,5-dicyanobenzylidene)-2-ethyl-3-methylimidazolinone, are marked as p-CBIE, m-CBIE, m-DCBIE respectively.

[0011] Preferably, the synthesis method of the photochromic green fluorescent protein chromophore derivative is as follows:

[0012] ;

[0013] Preferably, the application of the green fluorescent protein chromophore derivative in preparing photochromic fluorescent materials;

[0014] The green fluorescent protein chromophore derivative has photochromic fluorescence performance, and can be applied to photoelectric switches and data storage, biomolecules and bionics, imaging and detection, surface functionalization, catalysis, ion sensing, and drug delivery and photopharmacology and many other fields as photochromic fluorescent materials.

[0015] Preferably, the application of the green fluorescent protein chromophore derivative in cell imaging;

[0016] The three derivatives provided by the application have the advantages of good biocompatibility (low cytotoxicity), and the photochromic fluorescent characteristics of the series can be well used for fluorescent labeling and imaging of cells. Therefore, the application of the above-mentioned derivatives in cell fluorescent imaging is provided.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] 1. The synthesis of p-CBIE, m-CBIE and m-DCBIE is very simple, environmentally friendly, low in cost and high in yield.

[0019] 2. p-CBIE, m-CBIE, m-DCBIE have strong contrast solid-state emission, stimulus response sensitivity, color change process can be recycled, high stability to light and other characteristics.

[0020] 3. p-CBIE, m-CBIE, m-DCBIE have good biocompatibility (low cytotoxicity). BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 : The photos of the assembly of the derivatives before (A) and after (B) light irradiation, the assembly conditions from left to right are: water / acetonitrile V / V = 1 / 1, water / acetonitrile V / V = 7 / 3, water / acetonitrile V / V = 1 / 1; the substances from left to right are: p-CBIE, m-CBIE, m-DCBIE, 1 mg / mL.

[0022] Figure 2 : The photochromic reversibility of p-CBIE assembly at different temperatures, from top to bottom, the temperatures are: 40℃, 70℃, 100℃, among which A1 from left to right are respectively before ultraviolet irradiation, after ultraviolet irradiation, the sample recovered in a 40℃ environment.

[0023] Figure 3 : The photochromic reversibility of m-CBIE assembly at different temperatures, from top to bottom, the temperatures are: 40℃, 70℃, 100℃, among which A2 from left to right are respectively before ultraviolet irradiation, after ultraviolet irradiation, the sample recovered in a 70℃ environment.

[0024] Figure 4 : The photochromic reversibility of m-DCBIE assembly at different temperatures, from top to bottom, the temperatures are: 40℃, 70℃, 100℃, among which A3 from left to right are respectively before ultraviolet irradiation, after ultraviolet irradiation, the sample recovered in a 100℃ environment.

[0025] Figure 5 : The fluorescence emission spectra of the assembly of the derivatives in water / tetrahydrofuran V / V = 8 / 2 before and after light irradiation, from left to right are: p-CBIE, m-CBIE, m-DCBIE, 1 mg / mL.

[0026] Figure 6 : The results of cell dark toxicity of the derivatives in SK-OV-3 cells, the three derivatives from left to right are m-DCBIE, p-CBIE, m-CBIE.

[0027] Figure 7 : The laser scanning confocal microscope imaging diagram of the derivatives after incubation with SK-OV-3 cells; from left to right are the bright field, DAPI light before irradiation, DAPI light after irradiation, the superimposed diagram of the bright field and DAPI light after irradiation of the cells. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1: Preparation of HBI derivatives p-CBIE, m-CBIE, and m-DCBIE

[0030] 1. Preparation of p-CBIE, m-CBIE, and m-DCBIE

[0031] Take a 50 mL pear-shaped flask, add 3-cyanobenzaldehyde (1.7138 g, 13 mmol) and ethylamine solution (1150 μL, 17 mmol), using anhydrous ethanol (20 mL) as solvent, stir at room temperature and in the dark under a nitrogen atmosphere for 15 h, then add (Z)-2-((1-ethoxyethylidene)amino)methyl acetate (2.4870 g, 14 mmol), and continue stirring for 20 h. Remove the solvent using a rotary evaporator, and separate and purify by column chromatography (petroleum ether:ethyl acetate = 10:1, 6:1, 4:1, 2:1, 2:3). After removing the solvent again using a rotary evaporator, a pale yellow solid m-CBIE (0.9122 g) is obtained. 1H NMR (400 MHz, DMSO) δ 8.64(s, 1H), 8.52 (d, J = 7.8 Hz, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.67 (t, J = 7.7Hz, 1H), 7.02 (s, 1H), 3.62 (dd, J = 13.8, 6.7 Hz, 2H), 2.43 (s, 3H), 1.16(t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 169.84, 166.13, 140.83, 136.45,135.80, 135.19, 133.17, 130.38, 122.23, 118.79, 112.03, 35.59, 15.90, 14.63.

[0032] p-CBIE was obtained analogously. 1H NMR (400 MHz, DMSO-d6) δ 8.38 (d, J = 8.2 Hz, 2H), 7.91 (d, J = 8.2 Hz, 2H), 7.03 (s, 1H), 3.57 (t, J = 7.3 Hz, 2H), 2.41 (s, 3H), 0.90 - 0.83 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.98, 166.72, 141.50, 139.11, 132.83 (2C), 132.56 (2C), 122.46, 119.24, 111.81, 35.44, 15.93, 14.61.

[0033] m-DCBIE was obtained analogously. 1H NMR (400 MHz, DMSO-d6) δ 8.21 (d, J = 7.4 Hz, 2H), 7.46 - 7.42 (m, 2H), 6.97 (s, 1H), 3.61 (q, J = 7.2 Hz, 2H), 2.40 (s, 3H), 1.17 - 1.12 (m, 3H). 13C NMR (101 MHz, Chloroform-d) δ 169.74, 166.25, 142.22, 138.37 (2C), 137.17, 134.67, 120.04 (2C), 116.57, 114.46 (2C), 35.67, 15.79, 14.57.

[0034] Example 2: Application of HBI derivatives p-CBIE, m-CBIE, m-DCBIE in preparing photochromic fluorescent materials.

[0035] 1. Fluorescent color change of HBI derivative assemblies before and after illumination

[0036] Each of 1 mg of the three HBI derivatives was dissolved in 500 μL of acetonitrile, and then 500 μL of water was added, and the mixture was allowed to stand for 2 h. 10 μL of the sample was taken onto a glass slide, and after the solvent was evaporated, the assembly was obtained.

[0037] As shown in Figure 1 , the fluorescent color of the assembly p-CBIE changed from green to blue after UV irradiation, the fluorescent color of the assembly m-CBIE changed from dark to blue after UV irradiation, and the fluorescent color of the assembly m-DCBIE changed from green to blue after UV irradiation. The above results show that p-CBIE, m-CBIE, and m-DCBIE have photochromic properties.

[0038] 2. Reversibility of photochromism in HBI derivative assemblies at different temperatures

[0039] Accurately weigh 1 mg of each of the three derivatives, dissolve them in 100 μL of acetonitrile, then add 900 μL of water and let stand for 2 hours to assemble. Take 10 μL of sample on a glass slide, and after the solvent evaporates, test the photoreversibility of the sample.

[0040] like Figure 2 As shown, after UV irradiation, the fluorescence color of the p-CBIE assembly changed from green to blue at different temperatures. At 40℃, the time for fluorescence to recover from blue to green was: 4 h for the first time, 24 h for the second time, 24 h for the third time, and 24 h for the fourth time. With increasing cycle number, it became increasingly difficult to recover to the green fluorescence state. After heating to 70℃, the time for fluorescence to recover from blue to green was: 4 h for the first time, 6 h for the second time, 10 h for the third time, and 10 h for the fourth time. After four cycles, the photochromic reversibility was gradually lost. After heating to 100℃, the time for fluorescence to recover from blue to green was: 4 h for the first time, 4 h for the second time, 12 h for the third time, and 9 h for the fourth time. After four cycles, the photochromic reversibility was gradually lost, and with increasing cycle number, the sample gradually turned yellowish-brown and completely lost its color-changing properties.

[0041] The above results demonstrate that p-CBIE possesses reversible photochromic properties, and that increasing the temperature can reduce the time it takes for the fluorescence to recover from blue to green. However, excessively high temperatures can cause the sample to lose its inherent properties.

[0042] like Figure 3 As shown, after UV irradiation, the fluorescence color of the m-CBIE assembly changed from green to blue at different temperatures. At 40℃, the time for fluorescence to recover from blue to green was: 4 h for the first time, 24 h for the second time, 24 h for the third time, and 24 h for the fourth time. With increasing cycle number, it became increasingly difficult to recover to the green fluorescence state. After heating to 70℃, the time for fluorescence to recover from blue to green was: 4 h for the first time, 6 h for the second time, 10 h for the third time, and 10 h for the fourth time. After four cycles, the photochromic reversibility was gradually lost. After heating to 100℃, the time for fluorescence to recover from blue to green was: 4 h for the first time, 4 h for the second time, 12 h for the third time, and 9 h for the fourth time. After four cycles, the photochromic reversibility was gradually lost, and with increasing cycle number, the sample gradually turned yellowish-brown, completely losing its color-changing properties.

[0043] The above results demonstrate that m-CBIE possesses reversible photochromic properties, and that increasing the temperature can reduce the time it takes for fluorescence to recover from blue to green. However, excessively high temperatures can cause the sample to lose its inherent properties.

[0044] As shown in Figure 4 m-DCBIE also has reversible photochromic properties. The fluorescence color of the assembly m-DCBIE can change from green to blue at different temperatures after UV irradiation. At 40°C, the fluorescence recovery time from blue to green is 2 h for the first time, 4 h for the second time, 5 h for the third time, and 24 h for the fourth time. After four cycles, the photochromic reversibility gradually disappears. After heating to 70°C, the fluorescence recovery time from blue to green is 2 h for the first time, 3 h for the second time, 5 h for the third time, and 12 h for the fourth time. After four cycles, the photochromic reversibility gradually disappears, and it takes about 100 h to recover the photochromic reversibility. After heating to 100°C, the fluorescence recovery time from blue to green is 2 h for the first time, 3 h for the second time, 5 h for the third time, and 12 h for the fourth time. After four cycles, the photochromic reversibility gradually disappears, and the sample gradually turns yellow with increasing cycle number.

[0045] The above results show that m-DCBIE has reversible photochromic properties, and increasing temperature can reduce the time for fluorescence to recover from blue to green. However, too high a temperature can cause the sample to lose its original properties.

[0046] 3. Fluorescence spectra of HBI derivative assemblies before and after light irradiation

[0047] Each of the three derivatives was accurately weighed at 1 mg, dissolved in 100 μL of tetrahydrofuran, and then 900 μL of water was added. After standing for 2 h, the assembly was filtered, and the fluorescence emission spectra before and after light irradiation were measured.

[0048] As shown in Figure 5 Before light irradiation, p-CBIE had obvious emission peaks at 453 nm, 515 nm, and 556 nm, and after light irradiation, p-CBIE had an obvious emission peak at 456 nm. It can be seen that there is a significant blue shift and a significant increase in fluorescence intensity before and after light irradiation. Before light irradiation, m-CBIE had obvious emission peaks at 505 nm, 471 nm, and 438 nm; after light irradiation, m-CBIE had an obvious emission peak at 459 nm. It can be seen that there is a significant blue shift and a significant increase in fluorescence intensity before and after light irradiation. Before light irradiation, m-DCBIE had obvious emission peaks at 550 nm, 509 nm, and 448 nm; after light irradiation, m-CBIE had an obvious emission peak at 457 nm. It can be seen that there is a significant blue shift and a significant increase in fluorescence intensity before and after light irradiation.

[0049] The above results show that the three derivatives have obvious photochromic properties.

[0050] Example 3: Application of HBI derivatives p-CBIE, m-CBIE, m-DCBIE in cell imaging.

[0051] 1. Cell toxicity experiment of HBI derivatives

[0052] Cell viability experiment of p-CBIE, m-CBIE or m-DCBIE was detected by MTT assay. First, SK-OV-3 cells were seeded in 24-well plates at a density of 70000 cells per well and incubated for 12 h. Different concentrations (6, 8, 12 mM) of p-CBIE, m-CBIE, m-DCBIE were added, 25 μL / well, and only DMSO group was added to the cells as a control group (at this time the concentration of the derivative was set to 0 mM).

[0053] The whole process was in the dark and light-free conditions, and continued to incubate for 4 h. At the time point, PBS was used to wash to remove dead cells and derivatives that did not enter the cells, 1 mL of medium and 50 μL / well of MTT detection reagent were added and incubated for 2 h. 200 μL / well was removed to a 96-well plate, and the absorbance at 450 nm was recorded by a microplate reader (Spark Control Tecan, USA) to detect the cell viability. As shown in Figure 6 The cell toxicity of p-CBIE, m-CBIE, m-DCBIE at a concentration in the range of 6-12 mM was very low (cell viability was greater than 80%). The experimental results showed that p-CBIE, m-CBIE and m-DCBIE had very low dark toxicity.

[0054] 2. Application of HBI derivatives in cell imaging

[0055] SK-OV-3 cells were seeded in culture dishes, after 12 h of culture, 12 mM concentration of p-CBIE, m-CBIE, m-DCBIE was added and incubated for 4 h, and confocal laser scanning microscope (CLSM) was used to study the fluorescence properties of p-CBIE, m-CBIE and m-DCBIE in cells. Figure 7From left to right, the pictures are bright field, DAPI light before, DAPI light after, overlay of bright field and DAPI light after of SK-OV-3 cell imaging of three derivatives respectively. It can be seen that before light, p-CBIE, m-CBIE and m-DCBIE can target to the nucleus of tumor cells for imaging, and the imaging can be regulated by DAPI light. However, the intensity of imaging is low. After light, the intensity of imaging of three derivatives is higher than before light. It is predicted that p-CBIE, m-CBIE and m-DCBIE will have good application prospects in the field of biomedical applications.

[0056] While embodiments of the present application have been shown and described with reference to particular embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the application. The scope of the application is defined by the appended claims and their equivalents.

Claims

1. A photochromic green fluorescent protein chromophore derivative, the structural formula of which is shown below:

2. The method for synthesizing a photochromic green fluorescent protein chromophore derivative according to claim 1, the synthetic route of which is shown below:

3. The application of the green fluorescent protein chromophore derivative according to claim 1 in the preparation of photochromic fluorescent materials.

4. The application of a green fluorescent protein chromophore derivative according to claim 1 in the preparation of cell imaging agents.

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