Polypeptide supermolecule chiral filler for high performance liquid chromatography, preparation method and application
阅读说明：本技术 高效液相色谱用多肽超分子手性填料及制备方法与应用 (Polypeptide supermolecule chiral filler for high performance liquid chromatography, preparation method and application ) 是由 王跃飞 齐崴 范玉琪 苏荣欣 于 2020-07-31 设计创作，主要内容包括：本发明公开了高效液相色谱用多肽超分子手性填料及制备方法与应用,制备方法为：(1)合成<Sub>L</Sub>-Phe-<Sub>L</Sub>-Phe-<Sub>L</Sub>-Lys,在其N-端接枝具有π-π堆积效应的9-芴基甲氧基羰基作为保护基团；(2)取步骤1)获得的产物,加入超纯水中,搅拌分散均匀,调节pH,静置反应至少24h,离心去除上清,得到多肽组装体,即为高效液相色谱用多肽超分子手性填料。本发明以对多肽分子进行理性设计,设计合成了数种多肽衍生物,继而通过自组装方法合成得到多肽组装体,即为高效液相色谱用多肽超分子手性填料,本发明的填料对手性化合物有良好的拆分效果,可以实现手性化合物,特别是手性药物的高效快速分离。(The invention discloses a polypeptide supermolecule chiral filler for high performance liquid chromatography, a preparation method and application thereof, wherein the preparation method comprises the following steps: (1) synthesis of L ‑Phe‑ L ‑Phe‑ L -Lys, to whose N-terminus 9-fluorenylmethoxycarbonyl group having a pi-pi stacking effect is grafted as a protecting group; (2) adding the product obtained in the step 1) into ultrapure water, uniformly stirring and dispersing, adjusting pH, standing for at least 24h, centrifuging to remove supernatant, and thus obtaining a polypeptide assembly, namely the polypeptide supramolecular chiral filler for the high performance liquid chromatography. The invention designs and synthesizes a plurality of polypeptide derivatives by rational design of polypeptide molecules, and then synthesizes a polypeptide assembly by a self-assembly method, namely the polypeptide supermolecule chiral filler for the high performance liquid chromatography.)
1. The preparation method of the polypeptide supermolecule chiral filler for the high performance liquid chromatography is characterized by comprising the following steps:
(1) synthesis ofL-Phe-L-Phe-L-Lys inL-Phe-L-Phe-L-9-fluorenylmethoxycarbonyl having a pi-pi stacking effect is N-terminally grafted with Lys as a protecting group;
(2) taking 2-10mmol of the product obtained in the step 1) according to the proportion, adding 1mL of ultrapure water, stirring and dispersing uniformly, adjusting the pH to 3-12.6, standing and reacting at 20-35 ℃ for at least 24h, centrifuging and removing the supernatant to obtain the polypeptide supramolecular assembly, namely the polypeptide supramolecular chiral filler for the high performance liquid chromatography.
2. The method of claim 1, wherein the step of preparing the composition is carried out inL-Phe-L-Phe-LC-terminal of-Lys is further linked toL-Lys orL-Asp。
3. The supramolecular chiral polypeptide filler for high performance liquid chromatography prepared by the preparation method of claim 1 or 2.
4. The supramolecular chiral polypeptide filler for high performance liquid chromatography as claimed in claim 3, wherein the microstructure of the chiral filler is nanosphere, nanobelt, nanofiber or nanosheet.
5. Use of the polypeptide supramolecular chiral filler for high performance liquid chromatography according to claim 3 or 4 for resolving racemic chiral compound mixtures.
Technical Field
The invention belongs to the technical field of high performance liquid chromatography chiral packing materials and preparation thereof, and particularly relates to a polypeptide supermolecule chiral packing for high performance liquid chromatography, a preparation method and application thereof.
Background
In recent years, chiral drugs have been the leading field of the pharmaceutical industry. There are many chiral compounds in nature, which have two enantiomers. When a chiral compound enters a living organism, its two enantiomers often exhibit different biological activities. For chiral drugs, one isomer may be effective, while the other isomer may be ineffective or even harmful. Therefore, a fast and efficient chiral separation method is established, and the method has important significance on pharmacokinetic research and chiral drug product quality. At present, the application of High Performance Liquid Chromatography (HPLC) in chiral drug resolution is the most extensive and is an important means for pharmacological and toxicological research of drug quality control and stereoselectivity. The high performance liquid chromatography chiral resolution can be divided into a chiral derivation method, a chiral mobile phase method and a chiral stationary phase method according to the principle. The chiral stationary phase method (CSP) requires a packing material for the chiral stationary phase, which directly splits the chiral enantiomer, wherein the separated enantiomer forms a temporary diastereomeric complex with the chiral stationary phase due to the energy difference. The chiral stationary phase method has higher chiral resolution capability and good durability, can be used for quickly and efficiently separating more drug enantiomers, and has wide application prospect. The filling materials commonly used as the chiral stationary phase at present comprise polysaccharide derivatives, macrocyclic antibiotics, molecularly imprinted polymers, cyclodextrins, proteins and the like. However, no report of adopting polypeptide supramolecular assembly as chiral filler exists at present. The polypeptide supermolecule self-assembly is a process that the polypeptide forms a supermolecule chiral nano structure with a microstructure highly ordered through a short peptide self-assembly way from top to bottom. Due to the inherent chirality of the polypeptide supermolecule nano-structure and the existence of a plurality of binding sites, the difference of the binding capacity of the polypeptide supermolecule nano-structure and different chiral drugs is realized, so that the polypeptide supermolecule nano-structure has the capacity of efficiently and quickly separating the chiral drugs.
Disclosure of Invention
The invention aims to provide a polypeptide supermolecule chiral filler for high performance liquid chromatography, which has high separation efficiency and large application potential.
The second purpose of the invention is to provide a preparation method of the polypeptide supermolecule chiral packing for high performance liquid chromatography.
The second purpose of the invention is to provide the application of the polypeptide supermolecule chiral packing for high performance liquid chromatography.
The technical scheme of the invention is as follows:
the preparation method of the polypeptide supermolecule chiral packing for the high performance liquid chromatography comprises the following steps:
(1) synthesis ofL-Phe-L-Phe-L-Lys inL-Phe-L-Phe-L-9-fluorenylmethoxycarbonyl having a pi-pi stacking effect is N-terminally grafted with Lys as a protecting group;
(2) taking 2-10mmol of the product obtained in the step 1) according to the proportion, adding 1mL of ultrapure water, stirring and dispersing uniformly, adjusting the pH to 3-12.6, standing and reacting at 20-35 ℃ for at least 24h, centrifuging and removing the supernatant to obtain the polypeptide supramolecular assembly, namely the polypeptide supramolecular chiral filler for the high performance liquid chromatography.
Can also be used inL-Phe-L-Phe-LC-terminal of-Lys is further linked toL-Lys orL-Asp。
The polypeptide supermolecule chiral filler for the high performance liquid chromatography prepared by the preparation method.
The microstructure of the chiral filler is nanospheres, nanobelts, nanofibers or nanosheets.
The polypeptide supermolecule chiral filler for the high performance liquid chromatography is applied to the resolution of a racemic chiral compound mixture.
The invention has the advantages that:
the invention designs and synthesizes a plurality of polypeptide derivatives by rational design of polypeptide molecules, and then synthesizes a polypeptide supermolecule assembly body, namely the polypeptide supermolecule chiral filler for the high performance liquid chromatography by a self-assembly method.
Drawings
Fig. 1 is a Scanning Electron Microscope (SEM) image of the polypeptide supramolecular assembly prepared in example 1.
FIG. 2 is a liquid chromatogram of the resolution of the polypeptide supramolecular chiral filler for the high performance liquid chromatography prepared in example 1 on R/S-alpha-methylbenzylamine.
Fig. 3 is an SEM image of the polypeptide supramolecular assembly prepared in example 3.
FIG. 4 is a liquid chromatogram of the resolution of the polypeptide supramolecular chiral filler for the high performance liquid chromatography prepared in example 3 on R/S-2-phenylpropionic acid.
Fig. 5 is an SEM image of the polypeptide supramolecular assembly prepared in example 5.
FIG. 6 is a liquid chromatogram of the polypeptide supramolecular chiral filler prepared in example 5 for R/S-1-phenylethanol resolution.
Fig. 7 is an SEM image of the polypeptide supramolecular assembly prepared in example 7.
FIG. 8 is a liquid chromatogram of the resolution of the polypeptide supramolecular chiral filler for high performance liquid chromatography prepared in example 7 on R/S-alpha-methylbenzylamine.
Fig. 9 is an SEM image of the polypeptide supramolecular assembly prepared in example 9.
FIG. 10 is a liquid chromatogram of the resolution of the polypeptide supramolecular chiral filler for high performance liquid chromatography prepared in example 9 on R/S-2-phenylpropionic acid.
Fig. 11 is an SEM image of the polypeptide supramolecular assembly prepared in example 11.
FIG. 12 is a liquid chromatogram of the resolution of the polypeptide supramolecular chiral filler for R/S-1-phenylethanol for high performance liquid chromatography prepared in example 11.
Detailed Description
Fmoc is an abbreviation for 9-fluorenylmethoxycarbonyl.
Fmoc-L-Phe-L-Phe-L-Lys is abbreviated as Fmoc-FFK and has the structure as formula (I):
Fmoc-L-Phe-L-Phe-L-Lys-L-Lys is abbreviated as Fmoc-FFKK and has the structure as formula (II):
Fmoc-L-Phe-L-Phe-L-Lys-Lasp is abbreviated as Fmoc-FFKD and has the structure shown as the formula (III):
Fmoc-FFK, Fmoc-FFKK and Fmoc-FFKD were prepared by committing GL Biochem Ltd. (Shanghai, China).
The invention will be further illustrated with reference to specific examples, without limiting the scope of the invention thereto. The equivalent replacement of the present disclosure, or the corresponding improvement, still falls into the protection scope of the present invention.