Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof
阅读说明：本技术 基于自组装的可检测透明质酸酶的卟啉衍生物、其制备方法及应用 (Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof ) 是由 曾荣今 王胜兰 张崇华 张培盛 陈建 成奋民 于 2020-08-11 设计创作，主要内容包括：本发明公开了一种基于自组装的可检测透明质酸酶的卟啉衍生物、其制备方法及应用,该卟啉衍生物的制备方法通过以下步骤完成：卟啉母体先与二溴烷烃反应,所得产物再和三乙胺反应,得到一种卟啉衍生物。该卟啉衍生物与带负电荷的透明质酸链通过静电自组装的方法形成了一种新型的荧光纳米传感器。本发明的荧光纳米传感器能在水溶液中实现对透明质酸酶的高选择性检测,相比于现有的检测技术,本发明得到的荧光纳米传感器具有良好的生物相容性,制备方法简单,选择性高,发射波长位于近红外能有效扣除生物背景荧光,具有良好的工业发展前景,在分析化学、生命科学等技术领域有着巨大的应用前景。(The invention discloses a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, a preparation method and application thereof, wherein the preparation method of the porphyrin derivative is completed by the following steps: the porphyrin parent is firstly reacted with dibromoalkane, and the obtained product is then reacted with triethylamine to obtain the porphyrin derivative. The porphyrin derivative and the hyaluronic acid chain with negative charges form a novel fluorescent nano sensor by a static self-assembly method. Compared with the existing detection technology, the fluorescence nano-sensor obtained by the invention has good biocompatibility, simple preparation method and high selectivity, can effectively subtract biological background fluorescence when the emission wavelength is in the near infrared region, has good industrial development prospect, and has huge application prospect in the technical fields of analytical chemistry, life science and the like.)
1. A porphyrin derivative based on self-assembly and capable of detecting hyaluronidase is characterized in that the general formula of the porphyrin derivative is as follows:
wherein R1, R2 and R3 are respectively H or O (CH)2)nN(CH2CH3)3 +(ii) a The O (CH)2)nN(CH2CH3)3 +N =2~ 24.
2. A preparation method of a porphyrin derivative capable of detecting hyaluronidase based on self-assembly is characterized by sequentially carrying out the following steps:
adding SM1 into dry N, N-dimethylformamide, starting stirring, adding anhydrous potassium carbonate, stirring at room temperature, adding SM2 after stirring and mixing uniformly, refluxing and stirring for 24-48 h under the protection of inactive gas, detecting by thin layer chromatography, cooling to room temperature after reaction, washing with water to remove the N, N-dimethylformamide, purifying the obtained solid material by column chromatography to obtain SM3, wherein the chemical reaction equation is as follows:
wherein, X1，X2，X3Are respectively H or OH; at X1=X2=X3When = H, A1=A2=A3= H; at X1=X2=H，X3When OH is not zero, A1=A2=H，A3=O（CH2）nBr; at X1=H，X2=X3When OH is not zero, A1=H，A2=A3=O（CH2）nBr at X1=X2=X3When OH is not zero, A1=A2=A3= O（CH2）nBr; the O (CH)2）nN = 2-24 in Br;
adding SM3 into dry N, N-dimethylformamide, adding excessive triethylamine, refluxing and stirring for 10-20 hours under the protection of inactive gas, monitoring the reaction by thin-layer chromatography, cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4, namely the porphyrin derivative, wherein the reaction equation is as follows:
wherein A is1，A2，A3Are each H or O (CH)2）nBr; in A1=A2=A3When H, R1=R2=R3= H; in A1=A2=H，A3= O（CH2）nBr is, R1=R2=H，R3= O(CH2)nN(CH2CH3)3 +(ii) a In A1=H，A2=A3=O（CH2）nBr is, R1=H，R2=R3= O(CH2)nN(CH2CH3)3 +(ii) a In A1=A2=A3= O（CH2）nBr is, R1=R2=R3= O(CH2)nN(CH2CH3)3 +(ii) a The O (CH)2）nBr and O (CH)2)nN(CH2CH3)3 +N =2~ 24.
3. The preparation method of the porphyrin derivative based on self-assembly detectable hyaluronidase according to claim 2, characterized in that the reflux temperature is 110-140 ℃.
4. The method for preparing a porphyrin derivative based on self-assembly detectable hyaluronidase according to claim 3, the SM 1: SM 2: the molar ratio of the anhydrous potassium carbonate is 1: 15-25: 15-30.
5. The preparation method of the porphyrin derivative based on self-assembly detectable hyaluronidase according to any one of claims 2-4, characterized in that the thin layer chromatography liquid is a mixed liquid of petroleum ether and dichloromethane, wherein the volume ratio of petroleum ether to dichloromethane is 1-10: 1; the column chromatography eluent is a mixed solution of petroleum ether and dichloromethane, wherein the volume ratio of the petroleum ether to the dichloromethane is 1-5: 1.
6. The preparation method of the porphyrin derivative capable of detecting hyaluronidase based on self-assembly as claimed in claim 5, wherein the molar ratio of SM3 to triethylamine is 1: 20-40.
7. Use of a porphyrin derivative based on self-assembly for detecting hyaluronidase, characterized in that a fluorescent nanoorgan for detecting hyaluronidase is prepared by mixing the porphyrin derivative as described in claim 1 with a hyaluronic acid chain.
8. The application of the porphyrin derivative capable of detecting hyaluronidase based on self-assembly as claimed in claim 7 is characterized in that a tetrahydrofuran solution of the porphyrin derivative and an aqueous solution of a hyaluronic acid chain are placed in a centrifuge tube, a HEPES buffer solution with pH of 7-8 is added for dilution, and after ultrasonic treatment is performed for 1-2 min, the mixed solution is continuously reacted for 5-8 h, so as to obtain the fluorescent nano-sensor capable of detecting hyaluronidase.
9. The use of a porphyrin derivative self-assembly based detectable hyaluronidase according to claim 7, wherein the hyaluronan chain has the formula:
wherein m =100~ 1000.
10. The use of the self-assembly based porphyrin derivative detectable hyaluronidase according to claim 8, wherein the mass ratio of the porphyrin derivative to the hyaluronan chain is 2-6: 1.
Technical Field
The invention belongs to the field of chemical materials and analysis and detection, and particularly relates to a preparation method and application of a porphyrin derivative for detecting hyaluronidase.
Background
Hyaluronic Acid (HA) contains multiple repeating glucuronic acid and N-acetylglucosamine disaccharide units, is negatively charged, HAs good water solubility, and is a main component constituting extracellular matrix (ECM) and intercellular matrix (ICM). Hyaluronidase (HAase) is a specific hydrolase of HA, is an endoglycosidase, can directly hydrolyze beta-1, 4 glycosidic bonds of hyaluronic acid, and the final product is mainly a metabolite of tetrasaccharide, and is used as an auxiliary drug for chemotherapy for many years to enhance the permeability of drugs. HAase has been reported to be involved in many physiological and pathological processes, and is highly expressed in malignant tumors such as bladder cancer, prostate cancer, brain cancer, and rectal cancer. Therefore, HAase has attracted extensive attention as a novel tumor marker, and the design and synthesis of a sensor capable of detecting HAase is of great significance for clinical diagnosis and treatment of early cancers.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a porphyrin derivative capable of detecting hyaluronidase based on self-assembly and a preparation method thereof, wherein the porphyrin derivative is used as a fluorescent chromophore of a fluorescent sensor to solve the problem of biological background fluorescence interference; the invention also relates to an application of the porphyrin derivative based on self-assembly and capable of detecting hyaluronidase, and the porphyrin derivative and the negatively charged hyaluronidase chain are prepared into a fluorescent nano sensor so as to achieve the purpose of quickly and effectively identifying hyaluronidase.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention relates to a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, which has a general formula as follows:
wherein R1, R2 and R3 are respectively H or O (CH)2)nN(CH2CH3)3 +(ii) a The O (CH)2)nN(CH2CH3)3 +N =2~ 24.
A preparation method of porphyrin derivative based on self-assembly and capable of detecting hyaluronidase comprises the following steps in sequence:
adding SM1 into dry N, N-dimethylformamide, starting stirring, adding anhydrous potassium carbonate, stirring at room temperature, adding SM2 after stirring and mixing uniformly, refluxing and stirring for 24-48 h under the protection of inactive gas, detecting by thin layer chromatography, cooling to room temperature after reaction, washing with water to remove the N, N-dimethylformamide, purifying the obtained solid material by column chromatography to obtain SM3, wherein the chemical reaction equation is as follows:
wherein, X1，X2，X3Are respectively H or OH; at X1=X2=X3When = H, A1=A2=A3= H; at X1=X2=H，X3When OH is not zero, A1=A2=H，A3=O（CH2）nBr; at X1=H，X2=X3When OH is not zero, A1=H，A2=A3=O（CH2）nBr at X1=X2=X3When OH is not zero, A1=A2=A3=O（CH2）nBr; the O (CH)2）nN = 2-24 in Br;
adding SM3 into dry N, N-dimethylformamide, adding excessive triethylamine, refluxing and stirring for 10-20 hours under the protection of inactive gas, monitoring the reaction by thin-layer chromatography, cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4, namely the porphyrin derivative, wherein the reaction equation is as follows:
wherein A is1，A2，A3Are each H or O (CH)2）nBr; in A1=A2=A3When H, R1=R2=R3= H; in A1=A2=H，A3= O（CH2）nBr is, R1=R2=H，R3= O(CH2)nN(CH2CH3)3 +(ii) a In A1=H，A2=A3=O（CH2）nBr is, R1=H，R2=R3= O(CH2)nN(CH2CH3)3 +(ii) a In A1=A2=A3= O（CH2）nBr is, R1=R2=R3= O(CH2)nN(CH2CH3)3 +(ii) a The O (CH)2）nBr and O (CH)2)nN(CH2CH3)3 +N =2~ 24.
As a limitation to the preparation method of the invention, the reflux temperature is 110-140 ℃.
As a further limitation to the above preparation method of the present invention, the SM 1: SM 2: the molar ratio of the anhydrous potassium carbonate is 1: 15-25: 15-30.
The thin-layer chromatography liquid is a mixed liquid of petroleum ether and dichloromethane, wherein the volume ratio of the petroleum ether to the dichloromethane is 1-10: 1; the column chromatography eluent is a mixed solution of petroleum ether and dichloromethane, wherein the volume ratio of the petroleum ether to the dichloromethane is 1-5: 1.
As another limitation to the preparation method of the invention, the molar ratio of the SM3 to the triethylamine is 1: 20-40.
The invention also provides an application of the porphyrin derivative based on self-assembly and capable of detecting hyaluronidase, and the porphyrin derivative and the hyaluronan chain are mixed to prepare the fluorescent nano-device capable of detecting hyaluronidase.
As a limitation of the invention relating to the use of porphyrin derivatives, the hyaluronic acid chain is
Wherein m =100~ 1000.
As another limitation of the application of the porphyrin derivative, the mass ratio of the porphyrin derivative to the hyaluronic acid chain is 2-6: 1.
Due to the adoption of the technical scheme, compared with the prior art, the porphyrin derivative and the corresponding fluorescence sensor have the following beneficial effects:
(1) according to the invention, a functional hydroxyl porphyrin matrix is synthesized by triethyl quaternary ammonium salt to obtain a porphyrin derivative (Mito TPP) with positive charge as a near infrared fluorescence chromophore, so that a sensor is targeted by mitochondria;
(2) the invention is a fluorescence nano-sensor which is formed by self-assembling Mito TPP with positive charges and hyaluronic acid chain with negative charges, the hyaluronic acid further increases the biocompatibility of the nano-sensor and reduces the adverse reaction generated after the fluorescence nano-sensor is injected into the body;
(3) compared with the traditional organic small molecule fluorescence sensor, the fluorescence nano sensor prepared by the porphyrin derivative provided by the invention has the advantages that the water solubility of the sensor is improved, so that the use of organic solvents is reduced, and the toxicity of the sensor is reduced;
(4) the emission spectrum of the porphyrin derivative is near 650nm, and compared with the traditional fluorescent nano sensor, the emission spectrum of the porphyrin derivative can effectively solve the problem of interference generated in the detection process of biological background fluorescence on hyaluronidase dissolved in water.
In conclusion, the invention provides a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, and a preparation method and application thereof. The synthesis route of the porphyrin derivative is simple, the whole process is easy to operate, the steps are short, and the cost investment is low; the prepared fluorescent nano sensor has excellent water solubility and biocompatibility, weak biological background fluorescence and low cytotoxicity, and can directly realize specific recognition on hyaluronidase.
The method is suitable for detecting hyaluronidase and is used for specifically targeting the hyaluronic acid receptor overexpressed on the surface of the cancer cell.
Drawings
The invention is described in further detail below with reference to the figures and the embodiments.
FIG. 1 is a schematic diagram of hyaluronidase recognition by the prepared fluorescent nanosensor;
FIG. 2 is a nuclear magnetic data plot of 5- (4- (triethylamine) -butyloxyphenyl) -10,15, 20-triphenylporphyrin;
FIG. 3 is a particle size distribution diagram of a fluorescent nanosensor;
FIG. 4 is a graph showing the change of fluorescence emission spectrum of the fluorescent nanosensor with the addition time of hyaluronic acid (excitation wavelength: 425 nm);
FIG. 5 is a graph showing the change of fluorescence emission spectra of fluorescence nanosensors when HAase was added at different concentrations;
FIG. 6 is a graph of a fitted curve corresponding to the change in the fluorescence intensity value with the change in the hyaluronidase concentration and a function corresponding to the curve;
FIG. 7 is a graph of interference contrast data for fluorescence recovery of various ions on a fluorescent nanosensor;
FIG. 8 is a graph of interference contrast data for various ions on the fluorescence intensity of a fluorescent nanosensor;
FIG. 9 is a graph of HeLa cell viability data for fluorescent nanoprobes of different concentrations.
Detailed Description
Preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the description of the preferred embodiment is only for purposes of illustration and understanding, and is not intended to limit the invention.
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