CN109908361B - Targeting polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation and preparation method and application thereof - Google Patents

Targeting polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation and preparation method and application thereof Download PDF

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CN109908361B
CN109908361B CN201910292361.0A CN201910292361A CN109908361B CN 109908361 B CN109908361 B CN 109908361B CN 201910292361 A CN201910292361 A CN 201910292361A CN 109908361 B CN109908361 B CN 109908361B
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polypeptide
cucurbituril
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benzyl imidazole
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CN109908361A (en
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刘育
刘江华
张瀛溟
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Nankai University
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Abstract

A targeting polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation, and a preparation method and application thereof. The method is characterized in that: benzyl imidazole is chemically modified on the skeleton of polypeptide, not only retains the capability of polypeptide to target tubulin, but also is benzyl imidazole and CB [8]]Provides an anchor point. Morphological studies show that the self-assembly morphology of microtubules passes
Figure DDA0002025339360000011
Can dramatically transform from fibrous to particulate nanoaggregates. In addition, cell and living body experiments prove that extensive supramolecular cross-linking can induce apoptosis and finally inhibit the proliferation of tumors. The invention has the advantages that: it is demonstrated that the aggregation between microtubules can be effectively regulated by polypeptide-tubulin interactions and polypeptide-cucurbituril supramolecular interactions, which may be developed as a promising therapy for the treatment of many degenerative diseases such as cancer.

Description

Targeting polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation and preparation method and application thereof
Technical Field
The invention relates to the technical field of cancer and tumor treatment, in particular to a novel targeting polypeptide-cucurbituril supramolecular assembly based on microtubule aggregation regulation and potential application thereof in cancer treatment.
Background
The diversity and complexity of macrocyclic-protein supramolecular assemblies thus show great potential in the areas of controlling energy transfer, biocatalysis/sensing, morphological interconversion and regulation of cellular viability, Microtubules (MTs) are key protein filaments of the cytoskeleton, consisting of spherical α/β -tubulin heterodimers in a highly dynamic manner, the polymerization and depolymerization of MTs are closely related to cell division and intracellular transport processes, so microtubules become the subject of molecular targeting and biomolecular assembly for tumor chemotherapy.
Disclosure of Invention
The system is constructed based on host-guest interaction between benzyl imidazole covalently modified antimitotic polypeptide (BP) and CB [8], the antimitotic polypeptide shows microtubule targeting by competitive binding with α -tubulin subunit in microtubules, and CB [8] induces microtubule aggregation by forming a 1: 2 complex with benzyl imidazole, thereby being used as a new strategy for resisting microtubule aggregation related diseases.
The technical scheme of the invention is as follows:
a targeted polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation is disclosed, wherein a structure unit of the targeted polypeptide-cucurbituril supramolecular assembly takes CB [8] as a main body and polypeptide BP covalently modified by benzyl imidazole as an object, and the polypeptide-cucurbituril supramolecular assembly is constructed through interaction of the supramolecular main body and the object, and the chemical structural formula of the structure unit is as follows:
Figure BDA0002025339340000021
a preparation method of a targeting polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation comprises the following steps:
step 1, preparing benzyl imidazole modified antimitotic polypeptide BP;
step 2,
Figure BDA0002025339340000022
Preparing and characterizing a binary polypeptide-cucurbituril supramolecular assembly solution;
step 3,
Figure BDA0002025339340000023
Preparing and characterizing a binary polypeptide-cucurbituril supramolecular assembly to induce microtubule in-vitro aggregation;
step 4,
Figure BDA0002025339340000031
And (3) inducing aggregation of cells and microtubules in vivo by the binary polypeptide-cucurbituril supramolecular assembly.
Wherein the tubulin is derived from pig brain, has purity of more than 99%, and is stored in an environment at 4 deg.C before reuse.
Further, the preparation method of the benzyl imidazole modified antimitotic polypeptide BP in the step 1 comprises the following steps:
1) synthesis of alanine modified benzyl imidazole intermediate BA
The reaction mixture of 0.27mmol, 44.6mg of the compound 2-chloroacetamidopropionic acid and 0.29mmol, 46.3mg of 1-benzylimidazole was dissolved in 1, 4-dioxane, stirred at 110 ℃ for 16 hours, and then the solvent was removed under reduced pressure; dissolving the solid in methanol, and then adding activated carbon; after 16 hours, the activated carbon was removed by filtration; concentrating the filtrate under reduced pressure to obtain solid, and performing reprecipitation purification by using ethyl acetate and methanol to obtain a yellow solid alanine-modified benzyl imidazole intermediate;
2) synthesis of benzylimidazole-modified antimitotic polypeptide BP
BP is synthesized by the reaction of an alanine modified benzyl imidazole intermediate BA and a polypeptide of an antimitotic target tubulin.
Further, wherein in step 2
Figure BDA0002025339340000032
The preparation and characterization of the binary polypeptide-cucurbituril supramolecular assembly solution are as follows:
1)
Figure BDA0002025339340000033
preparation of binary polypeptide-cucurbituril supramolecular assembly solution
BP and CB [8] solid samples were mixed as per 2: 1, dissolving the mixture in deuterated water/secondary water, and performing ultrasonic treatment for 20 minutes to fully dissolve and assemble the mixture;
2)
Figure BDA0002025339340000034
characterization of binary polypeptide-cucurbituril supramolecular assemblies
Will be prepared
Figure BDA0002025339340000035
Performing nuclear magnetic titration characterization and analysis on the binary polypeptide-cucurbituril supramolecular assembly deuterated aqueous solution
Figure BDA0002025339340000036
Assembly mode and bonding constant of binary polypeptide-cucurbituril supramolecular assembly, wherein concentration of BP is fixed to be 1.5 × 10-3M-1,CB[8]Are respectively 0,1.5 × 10-4M-1,3.0×10-4M-1,4.5×10-4M-1,6.0×10-4M-1,7.5×10-4M-1,9.0×10-4M-1And 1.2 × 10-3M-1。
Further, wherein in step 3
Figure BDA0002025339340000041
Preparation and characterization of binary polypeptide-cucurbituril supramolecular assembly for inducing microtubule in-vitro aggregation:
1)
Figure BDA0002025339340000042
binary polypeptide-cucurbituril supermolecule assembly body for inducing microtubule to aggregate in vitroPreparation of
BP and CB [8]]Solid samples were as follows 2: 1 in a conventional tubulin buffer solution, and performing ultrasonic treatment for 20 minutes to fully dissolve and assemble the tubulin buffer solution; dissolving tubulin in a conventional tubulin buffer solution, and reconstructing the tubulin buffer solution to obtain an initial concentration of 10 mg/ml; will be provided with
Figure BDA0002025339340000043
Mixing the binary polypeptide-cucurbituril supramolecular assembly and tubulin buffer solution, diluting to the experimental concentration, and standing at 37 deg.C for 15min to obtain
Figure BDA0002025339340000044
@ MT ternary mixture, the conventional tubulin buffer solution is 8 × 10-2M-1PIPES、6×10-3M-1MgCl2、1×10-3M-1EGTA、5％glycerol、5×10-3M-1GTP, pH 6.9;
2)
Figure BDA0002025339340000045
characterization of binary polypeptide-cucurbituril supramolecular assembly induced microtubule in-vitro aggregation
The TEM characterization comprises dropping 5 μ L sample on carbon supporting film, standing, air drying at room temperature, and characterizing the appearance of microscopic aggregation in vitro, D L S particle size characterization, wherein the sample solution is placed in a scintillation tube, the particle size of the sample solution is measured under the laser of 636nm at 25 deg.C, and the sample solution is placed in a quartz cuvette with 10mm light path at 37 deg.C for testing.
Further, wherein in step 4
Figure BDA0002025339340000046
Experiment of binary polypeptide-cucurbituril supramolecular assembly inducing aggregation of cells and microtubules in vivo:
1)
Figure BDA0002025339340000047
binary polypeptide-cucurbiturilExperiment for inducing microtubule aggregation in cell by supramolecular assembly
A549 lung cancer cells were placed in a 96-well plate, cultured in F12 medium containing 10% FBS for 24h, and then BP and BP were added separately
Figure BDA0002025339340000048
The cells are continuously cultured for 24 hours; cell death was determined by propidium iodide PI staining and fluorescence, the percentage of PI positive cells was calculated by dividing the number of PI positive cells by the total number of cells in each well, and cell viability was determined by the CCK-8 enzymatic method; in addition, the expression of the most important apoptosis-related protease caspase-3 is also evaluated by taking glyceraldehyde phosphate dehydrogenase GAPDH as an internal reference;
2)
Figure BDA0002025339340000051
experiment for inhibiting tumor growth by inducing microtubule aggregation in vivo by binary polypeptide-cucurbituril supramolecular assembly
100 mu L of S180 cell suspension at a concentration of 107cells/m L, subcutaneously inoculated into 4-week-old female BA L B/c mice, and after 7 days of inoculation, 0.06mmol/kg of BP and
Figure BDA0002025339340000052
the assembly is directly injected into the tumor part once every 2 days, after 12 days of treatment, tumor specimens are taken from mice, weighed, fixed by 4% formaldehyde solution, embedded by paraffin, sliced, stained by TUNE L kit and observed under a fluorescence microscope
Figure BDA0002025339340000053
The composition of the assembly was 0.06mmol/kg BP and 0.03mmol/kg CB [8]]。
The invention has the advantages that 1) the polypeptide containing 10 amino acid residues can be competitively combined with α -tubulin subunit, shows antimitotic activity and targeting capability, and 2) the benzyl imidazole part can be used for chemically modifying the polypeptide, can not interfere the original tubulin targeting capability of the polypeptide and can not interfere the original tubulin targeting capability of the polypeptideCan be and CB [8]]Non-covalent binding of (a) provides a functional anchor; 3) BP and CB [8] alone]Are all nontoxic, but the assembly
Figure BDA0002025339340000055
The compound has high cytotoxicity by gathering microtubules; 4) the preparation method of the supermolecule nano assembly is simple, easy to implement and particularly good in effect, so that the supermolecule nano assembly has potential application in the aspects of inhibiting tumor growth and treating cancers.
Drawings
Figure 1 is a synthetic scheme for the alanine-modified benzylimidazole intermediate BA.
FIG. 2 is
Figure BDA0002025339340000054
Schematic representation of the targeting of microtubule aggregation by the @ MT ternary supramolecular assembly.
FIG. 3 is nuclear magnetic hydrogen spectrum of alanine modified benzyl imidazole intermediate BA.
FIG. 4 is a carbon spectrum of an alanine-modified benzyl imidazole intermediate BA.
Fig. 5 is a high resolution mass spectrum of alanine modified benzyl imidazole intermediate BA.
FIG. 6 is a nuclear magnetic hydrogen spectrum of a polypeptide BP modified by benzyl imidazole.
Fig. 7 is a high resolution mass spectrum of the benzyl imidazole modified polypeptide BP.
FIG. 8 is a high performance liquid chromatogram of a benzylimidazole-modified polypeptide BP.
FIG. 9 is a schematic view of
Figure BDA0002025339340000061
A nuclear magnetic hydrogen spectrum characterization diagram of a molecular bonding mode of the binary supramolecular assembly in an aqueous solution.
FIG. 10 shows the NMR spectra of BP with increasing CB [8] concentration.
FIG. 11 is a Job diagram of BP and CB [8 ].
FIG. 12 is a drawing showing
Figure BDA0002025339340000062
@ MT ternary superThe appearance and structure of the molecular assembly in common tubulin buffer solution are characterized.
FIG. 13 is a drawing showing
Figure BDA0002025339340000063
The particle size distribution of the @ MT ternary supramolecular assembly in a common tubulin buffer solution.
FIG. 14 is a drawing showing
Figure BDA0002025339340000064
Graph of optical transmittance of @ MT ternary supramolecular assembly in common tubulin buffer solution.
FIG. 15 is a drawing showing
Figure BDA0002025339340000065
And (3) inducing the aggregation of microtubules in the cells by the supramolecular assembly, so as to inhibit the proliferation of the cells. (a) Confocal fluorescence images of a549 cells; (b) cell viability; (c) (ii) PI positive cell rate; (d) ratio of cleavage caspase-3 to GAPDH; (e)
Figure BDA0002025339340000066
supramolecular complex-induced apoptosis.
FIG. 16 is a drawing showing
Figure BDA0002025339340000067
The experiment that the supermolecular assembly induces microtubule aggregation in vivo so as to inhibit the growth of mouse tumor, (a) a picture of tumor at the end of an in vivo anti-cancer experiment, (b) the weight change of tumor after treatment, (c) a confocal fluorescence image of tumor tissue TUNE L, and (d) the ratio of TUNE L positive cells/total number of cells after treatment.
Detailed Description
The invention is further illustrated by the following examples:
example (b):
a targeted polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation is disclosed, wherein a structure unit of the targeted polypeptide-cucurbituril supramolecular assembly takes CB [8] as a main body and a polypeptide (BP) covalently modified by benzyl imidazole as an object, and the polypeptide-cucurbituril supramolecular assembly is constructed by the interaction of the main body and the object of supramolecular, and the chemical structural formula of the structure unit is as follows:
Figure BDA0002025339340000071
a preparation method of a targeting polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation comprises the following steps:
step 1, preparing benzyl imidazole modified antimitotic polypeptide (BP);
step 2,
Figure BDA0002025339340000072
Preparing and characterizing a binary polypeptide-cucurbituril supramolecular assembly solution;
step 3,
Figure BDA0002025339340000073
Preparing and characterizing a binary polypeptide-cucurbituril supramolecular assembly to induce microtubule in-vitro aggregation;
step 4,
Figure BDA0002025339340000074
And (3) inducing aggregation of cells and microtubules in vivo by the binary polypeptide-cucurbituril supramolecular assembly.
The preparation method of the benzylimidazole modified antimitotic polypeptide (BP) in the step 1 comprises the following steps:
1) referring to figure 1, the synthesis of alanine modified benzylimidazole intermediate BA
A reaction mixture of the compound 2-chloroacetamidopropionic acid (0.27mmol, 44.6mg) and 1-benzylimidazole (0.29mmol, 46.3mg) was dissolved in 1, 4-dioxane, stirred at 110 ℃ for 16 h, then the solvent was removed under reduced pressure. The solid was dissolved in methanol and then activated carbon was added. After 16 hours, the activated carbon was removed by filtration. Concentrating the filtrate under reduced pressure to obtain solid, and performing reprecipitation purification by using ethyl acetate and methanol to obtain a yellow solid alanine-modified benzyl imidazole intermediate;
2) synthesis of benzylimidazole-modified antimitotic polypeptide (BP)
BP is synthesized by the reaction of an alanine modified benzyl imidazole intermediate BA and a polypeptide of an antimitotic target tubulin.
FIG. 3 is nuclear magnetic hydrogen spectrum of alanine modified benzyl imidazole intermediate BA. The figure shows that: the alanine modified benzyl imidazole intermediate BA has a correct structure.
FIG. 4 is a carbon spectrum of an alanine-modified benzyl imidazole intermediate BA. The figure shows that: the alanine modified benzyl imidazole intermediate BA has a correct structure.
Fig. 5 is a high resolution mass spectrum of alanine modified benzyl imidazole intermediate BA. The figure shows that: the alanine modified benzyl imidazole intermediate BA has a correct structure.
FIG. 6 is a nuclear magnetic hydrogen spectrum of a polypeptide BP modified by benzyl imidazole. The figure shows that: the polypeptide BP modified by the benzyl imidazole has correct structure.
Fig. 7 is a high resolution mass spectrum of the benzyl imidazole modified polypeptide BP. The figure shows that: the polypeptide BP modified by the benzyl imidazole has correct structure.
FIG. 8 is a high performance liquid chromatogram of a benzylimidazole-modified polypeptide BP. The figure shows that: the polypeptide BP modified by the benzyl imidazole has correct structure.
In step 2 of the preparation method of the present invention,
Figure BDA0002025339340000081
the preparation and characterization of the binary polypeptide-cucurbituril supramolecular assembly solution are as follows:
1)
Figure BDA0002025339340000091
preparation of binary polypeptide-cucurbituril supramolecular assembly solution
BP and CB [8] solid samples were mixed as per 2: 1, dissolved in deuterated water/secondary water, and subjected to ultrasonic treatment for 20 minutes to fully dissolve and assemble the mixture.
2)
Figure BDA0002025339340000092
Binary polypeptide-cucurbitCharacterization of urea supramolecular assemblies
Will be prepared
Figure BDA0002025339340000093
Performing nuclear magnetic titration characterization and analysis on the binary polypeptide-cucurbituril supramolecular assembly deuterated aqueous solution
Figure BDA0002025339340000094
The assembly mode and the bonding constant of the binary polypeptide-cucurbituril supramolecular assembly, wherein the concentration of BP is fixed to be 1.5 × 10-3M-1,CB[8]Are respectively 0,1.5 × 10-4M-1,3.0×10-4M-1,4.5×10-4M-1,6.0×10-4M-1,7.5×10-4M-1,9.0×10-4M-1And 1.2 × 10-3M-1。
FIG. 9 is a schematic view of
Figure BDA0002025339340000095
A nuclear magnetic hydrogen spectrum representation diagram of a binary polypeptide-cucurbituril supramolecular assembly molecule bonding mode. The figure shows that: benzylimidazole moiety and CB [8] in BP molecule]2 is formed: 1, and the inclusion pattern is determined as drawn in the figure.
FIG. 10 is a nuclear magnetic titration chart of BP with increasing CB [8] concentration. The figure shows that: the benzylimidazole moiety of the BP molecule forms a 2: 1, and determining the inclusion pattern.
FIG. 11 shows BP and CB [8]]Job diagram of (1). The figure shows that: benzylimidazole moiety and CB [8] in BP molecule]A 2: 1 inclusion compound was formed, and a bonding constant Ks of 2.84 × 10 was determined7M–2。
As shown in fig. 2, in step 3 of the preparation method of the present invention,
Figure BDA0002025339340000096
the preparation and characterization of the binary polypeptide-cucurbituril supramolecular assembly for inducing microtubule in-vitro aggregation are as follows:
1)
Figure BDA0002025339340000097
preparation of binary polypeptide-cucurbituril supermolecule assembly for inducing microtubule to aggregate in vitro
BP and CB [8]]The solid sample was dissolved in conventional tubulin buffer (8 × 10) at a stoichiometric ratio of 2: 1-2M-1PIPES,6×10-3M-1MgCl2,1×10-3M-1EGTA,5％glycerol,5×10-3M-1GTP, pH 6.9), sonicated for 20 minutes to allow for sufficient dissolution and assembly. Tubulin was dissolved in conventional tubulin buffer and reconstituted to an initial concentration of 10 mg/ml. Will be provided with
Figure BDA0002025339340000098
Mixing the binary polypeptide-cucurbituril supramolecular assembly and tubulin buffer solution, diluting to the experimental concentration, and standing at 37 deg.C for 15min to obtain
Figure BDA0002025339340000101
@ MT ternary mixture.
2)
Figure BDA0002025339340000102
Characterization of binary polypeptide-cucurbituril supramolecular assembly induced microtubule in-vitro aggregation
And (3) TEM characterization, namely dripping a5 mu L sample on a carbon supporting film, standing, airing at room temperature and characterizing the appearance of microscopic in-vitro aggregation, and D L S particle size characterization, namely placing the sample solution in a scintillation tube, measuring the particle size of the sample solution under the laser of which the lambda is 636nm at the temperature of 25 ℃, and placing the sample solution in a quartz cuvette (the light path is 10mm) for testing at the temperature of 37 ℃.
FIG. 12 is a drawing showing
Figure BDA0002025339340000103
The appearance and structure of the @ MT ternary supramolecular assembly in a common tubulin buffer solution are characterized. The figure shows that: BP alone is not sufficient to significantly alter the morphology of microtubules, but is abundant
Figure BDA0002025339340000104
Crosslinking can dramatically transform the morphology of the microtubules from fibrous to particulate nanoaggregates.
FIG. 13 is a drawing showing
Figure BDA0002025339340000105
The particle size distribution of the @ MT ternary supramolecular assembly in a common tubulin buffer solution. The figure shows that: ternary assemblies compared to BP and BP @ MT alone
Figure BDA0002025339340000106
@ MT has a larger particle size distribution, indicating
Figure BDA0002025339340000107
The @ MT aggregation results in a larger assembly.
FIG. 14 is a drawing showing
Figure BDA0002025339340000108
Graph of optical transmittance of @ MT ternary supramolecular assembly in common tubulin buffer solution. The figure shows that: ternary assemblies compared to BP and BP @ MT alone
Figure BDA0002025339340000109
@ MT has a smaller optical transmittance, explanation
Figure BDA00020253393400001010
The @ MT aggregation results in a larger assembly.
In step 4 of the preparation method of the present invention,
Figure BDA00020253393400001011
the experiment of binary polypeptide-cucurbituril supramolecular assembly inducing cell and in vivo microtubule aggregation is as follows:
1)
Figure BDA00020253393400001012
binary polypeptide-cucurbitExperiment for inducing microtubule aggregation in cells by urea supramolecular assembly
A549 lung cancer cells were placed in a 96-well plate, cultured in F12 medium containing 10% FBS for 24h, and then BP and BP were added separately
Figure BDA0002025339340000111
The cells were cultured for 24 hours. Cell mortality was determined by Propidium Iodide (PI) staining and fluorescence, and the percentage of PI positive cells was calculated by dividing the number of PI positive cells by the total number of cells in each well. Cell viability was determined enzymatically using CCK-8. In addition, the expression of caspase-3, the most important apoptosis-related protease, was also evaluated using glyceraldehyde phosphate dehydrogenase (GAPDH) as an internal reference.
2)
Figure BDA0002025339340000112
Experiment for inhibiting tumor growth by inducing microtubule aggregation in vivo by binary polypeptide-cucurbituril supramolecular assembly
100 μ L suspension of S180 cells (107cells/m L) was inoculated subcutaneously into 4-week-old female BA L B/c mice, 7d after inoculation, BP (0.06mmol/kg) and
Figure BDA0002025339340000113
assembly (0.06mmol/kg BP and 0.03mmol/kg CB [8]]) Direct injection to the tumor site (once every 2 days), 12 days after treatment, tumor specimens were removed from mice, weighed, fixed with 4% formaldehyde solution, paraffin embedded, sectioned, and then stained with the TUNE L kit and viewed under a fluorescent microscope.
FIG. 15 is a drawing showing
Figure BDA0002025339340000114
And (3) inducing the aggregation of microtubules in the cells by the supramolecular assembly, so as to inhibit the proliferation of the cells. (a) Confocal fluorescence images of a549 cells; (b) cell viability; (c) (ii) PI positive cell rate; (d) ratio of cleavage caspase-3 to GAPDH; (e)
Figure BDA0002025339340000115
supramolecular complex-induced apoptosis. The figure shows that:
Figure BDA0002025339340000116
the assembly has high toxicity to A549 lung cancer cells and proves that
Figure BDA0002025339340000117
The complexes enhance the rate of apoptosis in the cellular environment by inducing aggregation between microtubules, ultimately leading to cell death primarily through caspase-dependent pathways.
FIG. 16 is a drawing showing
Figure BDA0002025339340000118
The supramolecular assembly induces microtubule aggregation in vivo, thereby inhibiting tumor growth experiments of mice, (a) a picture of tumors at the end of an in vivo anti-cancer experiment, (b) weight change of the tumors after treatment, (c) a confocal fluorescence image of tumor tissue TUNE L, (d) a ratio of TUNE L positive cells/total number of cells after treatment, which shows that:
Figure BDA0002025339340000119
the anticancer potential of the complexes was further evaluated in a tumor-bearing mouse model,
Figure BDA00020253393400001110
the complex can effectively inhibit the growth of tumors in a tumor-bearing mouse model by inducing the aggregation among microtubules, and can be used as a new strategy for resisting diseases related to microtubule aggregation.

Claims (3)

1. A targeted polypeptide-cucurbituril supramolecular assembly based on regulation and control of microtubule aggregation is characterized in that: the construction unit takes CB [8] as a main body and takes polypeptide BP covalently modified by benzyl imidazole as an object, and constructs a polypeptide-cucurbituril supramolecular assembly through interaction of the supramolecular main body and the object, wherein the chemical structural formula of the construction unit is as follows:
Figure 18113DEST_PATH_IMAGE002
Figure 293237DEST_PATH_IMAGE004
。
2. the preparation method of the targeting polypeptide-cucurbituril supramolecular assembly based on the regulation and control of microtubule aggregation according to claim 1, characterized by comprising the following steps:
step 1, preparing benzyl imidazole modified antimitotic polypeptide BP;
step 2, preparing and characterizing BP i CB [8] binary polypeptide-cucurbituril supermolecular assembly solution;
step 3, BP i CB [8] binary polypeptide-cucurbituril supermolecular assembly induces the preparation and characterization of microtubule external aggregation;
step 4, BP i CB [8] binary polypeptide-cucurbituril supermolecular assembly induces aggregation experiment of cells and microtubules in vivo.
3. The method for preparing the targeting polypeptide-cucurbituril supramolecular assembly based on the regulation and control of microtubule aggregation according to claim 2, wherein the method comprises the following steps: the preparation method of the benzyl imidazole modified polypeptide BP in the step 1 comprises the following steps:
1) synthesis of alanine modified benzyl imidazole intermediate BA
The reaction mixture of 0.27mmol, 44.6mg of the compound 2-chloroacetamidopropionic acid and 0.29mmol, 46.3mg of 1-benzylimidazole was dissolved in 1, 4-dioxane, stirred at 110 ℃ for 16 hours, and then the solvent was removed under reduced pressure; dissolving the solid in methanol, and then adding activated carbon; after 16 hours, the activated carbon was removed by filtration; concentrating the filtrate under reduced pressure to obtain solid, and performing reprecipitation purification by using ethyl acetate and methanol to obtain a yellow solid alanine-modified benzyl imidazole intermediate;
2) synthesis of benzylimidazole-modified antimitotic polypeptide BP
BP is synthesized by the reaction of an alanine modified benzyl imidazole intermediate BA and a polypeptide of an antimitotic target tubulin.
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