CN110003355B

CN110003355B - Glucan derivative with AIE characteristic and synthesis method and application thereof

Info

Publication number: CN110003355B
Application number: CN201910291713.0A
Authority: CN
Inventors: 方敏; 徐倩文; 郭一帆; 李村; 朱维菊
Original assignee: Anhui University
Current assignee: Anhui University
Priority date: 2019-04-12
Filing date: 2019-04-12
Publication date: 2020-12-11
Anticipated expiration: 2039-04-12
Also published as: CN110003355A

Abstract

The invention discloses a glucan derivative with AIE characteristics, a synthesis method and application thereof, wherein the structure of the glucan derivative with AIE characteristicsThe formula is as follows:

wherein x is 59.4 and y is 1.6. The glucan derivative having the AIE characteristics of the present invention is soluble in water and has a low critical micelle concentration of 5X 10^‑3mg/mL, has good aggregation-induced emission characteristics at low concentration, and has concentration range of 1 × 10^‑3mg/mL to 60X 10^‑3mg/mL, has important research value in the fields of cell imaging and related research.

Description

Glucan derivative with AIE characteristic and synthesis method and application thereof

Technical Field

The invention relates to a glucan derivative with AIE characteristics, a synthesis method and application thereof, and belongs to the technical field of fluorescent materials.

Background

In recent years, with the continuous development of biological imaging technology, the nano-carrier with imaging function is receiving attention from researchers. However, the conventional fluorescent probe is affected by the aggregation quenching (ACQ) effect, and the fluorescence efficiency thereof is significantly reduced at a high concentration, so that the detection and analysis means using fluorescence as an output signal is severely restricted. However, researchers have discovered a new fluorescent molecule that emits strong fluorescence intensity in an aggregated state, and this new fluorescent molecule can avoid the phenomenon of fluorescence quenching that occurs in conventional fluorescent molecules at high concentrations, which is called aggregation-induced emission (AIE) effect. The researchers have generated great interest in the fluorescent molecules, and as the research goes into, the researchers have developed systems with aggregation-induced emission effect in the whole visible wavelength range, and these systems with AIE are widely applied to the fields of cell imaging and related research.

However, the AIE systems reported to date have focused primarily on small molecules and organic polymers with pi conjugated groups. These molecules containing pi conjugated groups not only have poor water solubility for biological applications, but also have great cytotoxicity, and these disadvantages severely limit their application in the biomedical field. On the other hand, few researchers report that amphiphilic fluorescent molecules with aggregation-induced emission properties can self-assemble to form nano-micelles with hydrophobic cores and hydrophilic shells in aqueous solution, and form amphiphilic molecules with small molecular fluorescent compounds with AIE characteristics and hydrophilic substances, and structurally, the amphiphilic molecules contain both hydrophobic fluorescent compounds with AIE characteristics and hydrophilic structural units. The glucan molecule can be compatible with biological tissues because the glucan molecule chain contains a large amount of hydrophilic hydroxyl groups and can be degraded under the action of enzyme, and the glucan can be used as a carrier due to the hydrophilicity and biocompatibility of the glucan and is widely applied to the field of biological imaging.

Since researchers do not have long time to recognize aggregation-induced emission, the compounds synthesized to date have limited species and relatively single structures.

Disclosure of Invention

The invention aims to provide a glucan derivative with AIE characteristics, a synthesis method and application thereof. The invention synthesizes a novel compound with aggregation induction characteristic through molecular design, and the compound which forms amphipathy with glucan can be used for cell imaging and related research fields.

The invention relates to a glucan derivative with AIE characteristics, which has the following structural formula:

wherein x is 59.4 and y is 1.6.

The invention relates to a preparation method of a glucan derivative with AIE characteristics, which is characterized in that glucan and a compound 1-N-hydroxyethyl-4- (4-formylphenyl) -1, 8-naphthalimide with AIE characteristics are subjected to esterification reaction through Carbonyl Diimidazole (CDI) to obtain a target product.

The structural formula of the compound 1 is as follows:

the preparation method of the glucan derivative with the AIE characteristic comprises the following steps:

step 1: the reaction apparatus was dried thoroughly in an oven at 120 ℃ and placed in a desiccator to cool to room temperature. 0.194g (1.2mmol) of CDI and 3ml of anhydrous DMSO are added into a round-bottom flask, and the mixture is stirred and uniformly dispersed; accurately weighing 0.345g (1mmol) of compound 1, completely dissolving in 8ml of anhydrous DMSO, slowly dropwise adding the compound into a round-bottom flask by using a constant-pressure funnel, and stirring for reaction for 3 hours at normal temperature;

step 2: 0.31g of dextran was weighed into a completely dry reactor, and a suitable amount of anhydrous DMSO was added in N₂Heating and stirring the mixture under protection until the glucan is completely dissolved, then slowly dripping the reaction solution obtained in the step (1) into the reactor, and stirring the mixture to react for 24 hours at the temperature of 70 ℃; the reaction solution was transferred to a dialysis bag and dialyzed first in DMSO solution for 24h and then in deionized water for 72h, the dialysate was changed once for 8h, and the product was freeze-dried.

The synthetic route of the target product of the invention is as follows:

the application of the glucan derivative with the AIE characteristic is the application of the glucan derivative as a fluorescent probe in the cell imaging process.

The glucan derivative having the AIE characteristics of the present invention is soluble in water and has a low critical micelle concentration of 5X 10^-3mg/mL, has good aggregation-induced emission characteristics at low concentration, and has concentration range of 1 × 10^-3mg/mL to 60X 10^-3mg/mL, has important research value in the fields of cell imaging and related research.

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

the invention selects a novel micromolecule compound with aggregation-induced emission characteristic, forms an amphiphilic glucan derivative with glucan, has good solubility in aqueous solution, can form autofluorescence nano-micelle when the concentration in the aqueous solution is higher than the critical micelle concentration, and can be used as an autofluorescence probe for intracellular imaging.

Drawings

FIG. 1 is an infrared spectrum of a glucan derivative having AIE characteristics according to the present invention.

FIG. 2 shows the nuclear magnetic hydrogen spectrum of the dextran derivative having AIE characteristics according to the present invention.

FIG. 3 shows the fluorescence spectra (. lamda.) of aqueous solutions of different concentrations of dextran derivatives with AIE characteristics_ex＝360nm)。

FIG. 4 is a graph of the fluorescence intensity at 460nm versus the log concentration (lambda) of different concentrations of a dextran derivative with AIE characteristics_ex＝360nm)。

FIG. 5 is a photo-bleaching graph of dextran derivatives with AIE characteristics according to the present invention.

FIG. 6 is a confocal image of HeLa cells. Among them, a fluorescence image (a), a superposition of fluorescence and bright field images (B), and a bright field image (C) of HeLa cells.

Detailed Description

The invention is further illustrated by, but is not limited to, the following examples.

Example 1: preparation of dextran derivatives with AIE characteristics

1. The reaction apparatus was dried thoroughly in an oven at 120 ℃ and placed in a desiccator to cool to room temperature. 0.194g (1.2mmol) of CDI and 3ml of anhydrous DMSO are added into a round-bottom flask, and the mixture is stirred and uniformly dispersed; 0.345g (1mmol) of Compound 1 was accurately weighed out and completely dissolved in 8ml of anhydrous DMSO, and slowly added dropwise to a round-bottomed flask using a constant pressure funnel, and the reaction was stirred at room temperature for 3 hours.

2. Another accurately weighed 0.31g dextran in a completely dry three-necked flask, added with a proper amount of anhydrous DMSO in N₂Heating and stirring under protection until the glucan is completely dissolved, and obtaining the product in the step 1Placing the obtained reaction solution in a constant pressure funnel, slowly dripping the reaction solution into a round-bottom flask, and stirring the reaction solution at the temperature of 70 ℃ for 24 hours; the reaction was transferred to a dialysis bag and first dialyzed in DMSO for about 24h, then in deionized water for 72h, the dialysate was changed once for 8h, and the product was freeze-dried.

FIG. 1 is an infrared spectrum of a Dextran derivative with AIE characteristics (Dextran: Dextran, Dex-CHO: Dextran fluorescent marker). FT-IR (KBr, cm)^-1): 3400(O-H stretching vibration, 2929 (-CH)₂Stretching vibration), indicating that the hydroxyl group of the raw material glucan is reacted by the fact that the O-H peak of the product is weaker than that of the raw material glucan as compared with the raw material. At 1747cm^-1There is a newly formed peak of ester groups, demonstrating successful synthesis of the product.

FIG. 2 is a nuclear magnetic hydrogen spectrum of a glucan derivative having AIE characteristics. The solvent chosen was DMSO-d6, 3.2-4.9 ppm-CH belonging to dextran₂and-OH peaks, 7.8-8.5ppm belonging to hydrogen on naphthalene and benzene rings of Compound 1, 10.1ppm belonging to-CHO peaks of Compound 1, indicating successful synthesis of the product.

Example 2: aggregation-induced emission properties of dextran derivatives with AIE characteristics

Accurately weighing 20mg of target product, completely dissolving the target product in DMSO solvent to prepare a stock solution with a concentration of 25mg/mL, diluting the stock solution with water to obtain a solution to be detected with a concentration of 1mg/mL, putting 4mL of pure water into PE tubes, respectively adding 4 muL, 8 muL, 12 muL, 20 muL, 28 muL, 36 muL, 48 muL and 72 muL of solutions to be detected with a concentration of 1mg/mL into each PE tube, and respectively taking 3.84mL and 3.76mLH₂O adding 160 μ L and 240 μ L of solution to be tested with concentration of 1mg/mL into two PE tubes, shaking to obtain solutions to be tested with different concentrations, measuring fluorescence spectra of the solutions to be tested (as shown in FIG. 3), increasing the concentration of the solution to gradually increase the fluorescence intensity of the dextran derivative with AIE characteristic at λ_ex360 nm. The inset is a concentration of 10 × 10^-3Photograph of mg/mL solution under 365nm UV lamp.

FIG. 4 is the fluorescence emission spectra and concentrations of different concentrations of dextran derivatives with AIE characteristicsLogarithmic graph, CMC is 5X 10^-3mg/mL, indicating that the product has a lower critical micelle concentration.

Example 3: photobleaching experiments with dextran derivatives with AIE characteristics

1.6mL of the 1mg/mL test solution obtained in example 2 was added dropwise to 80mL of water to prepare a 20X 10 concentration solution^-3Stirring mg/mL solution under 365 ultraviolet lamp, and measuring fluorescence intensity (lambda) by taking 3-4mL solution at certain time intervals_em360nm), and its fluorescence intensity I (as shown in fig. 5) was measured over 8h₀The fluorescence intensity of the solution to be detected is represented when the illumination time is 0, and the fluorescence intensity of the solution to be detected is found to slightly decrease and then tend to be stable within 8 hours, which indicates that the solution has good photobleaching resistance.

Example 4: cellular imaging of dextran derivatives with AIE characteristics

FIG. 6 is a confocal laser microscopy image of HeLa cells treated for 30min with the dextran derivative with AIE characteristics, A being a fluorescence field image, B being the superposition of fluorescence and bright field images, and C being a bright field image. As can be seen from FIG. 6A, the dextran derivative emitted a significant blue fluorescence in HeLa cells under 406nm laser excitation. As can be seen from FIG. 6C, the cells co-cultured with the glucan derivative maintained their cell morphology well, indicating that the glucan was very biocompatible.

Claims

1. A dextran derivative having AIE characteristics characterized by the structural formula:

wherein x is 59.4 and y is 1.6.

2. A method of preparing the AIE-characterized glucan derivative of claim 1, wherein:

the target product is obtained by esterification reaction of dextran and compound 1-N-ethoxyl-4- (4-formylphenyl) -1, 8-naphthalimide with AIE characteristic through carbonyl diimidazole;

the structural formula of the compound 1 is as follows:

3. the method of claim 2, comprising the steps of:

step 1: adding 1.2mmol of CDI and anhydrous DMSO into a completely dry round-bottom flask, and uniformly stirring and dispersing; weighing 1mmol of compound 1, completely dissolving in anhydrous DMSO, slowly dropwise adding into a round-bottom flask by using a constant-pressure funnel, and reacting for 3h under stirring at normal temperature;

step 2: 0.31g of dextran was weighed into a completely dry reactor, anhydrous DMSO was added in N₂Heating and stirring the mixture under protection until the glucan is completely dissolved, then slowly dripping the reaction solution obtained in the step (1) into the reactor, and stirring the mixture to react for 24 hours at the temperature of 70 ℃; the reaction solution was transferred to a dialysis bag and dialyzed first in DMSO solution for 24h and then in deionized water for 72h, the dialysate was changed once for 8h, and the product was freeze-dried.

4. Use of the dextran derivative with AIE characteristics according to claim 1, characterized in that: the dextran derivative is used as a fluorescent probe in a cell imaging process.

5. Use according to claim 4, characterized in that:

the dextran derivative is soluble in water at 1 × 10^-3mg/mL to 60X 10^-3Has obvious aggregation-induced luminescence characteristics in the concentration range of mg/mL.

6. Use according to claim 4, characterized in that:

the critical micelle concentration of the dextran derivative is 5 × 10^-3mg/mL。