CN106749523B - Method for forming nanotube by utilizing self-assembly of stapler polypeptide - Google Patents

Abstract

The invention provides a method for forming a nanotube by utilizing self-assembly of stapler polypeptide, which comprises the step of synthesizing-Fmoc-protected unnatural amino acid with chiral carbon R at the 2-position of a side chain; connecting unnatural amino acids with resin by adopting a solid-phase polypeptide synthesis method, continuing to connect 3 amino acids, connecting cysteine, sealing the amino end of the cysteine with acetyl, removing a sulfhydryl protecting group of the cysteine, and performing intramolecular sulfhydryl-alkene reaction to obtain a polypeptide compound with a side chain modified by 2-carbon chirality; shearing the polypeptide from the resin, purifying and freeze-drying to obtain a white powdery solid; dispersing the white powdery solid with ultrapure water, and performing ultrasonic treatment to obtain the self-assembled polypeptide nanotube. The invention provides a novel polypeptide self-assembly nanotube, which is characterized in that the basic unit of the nanotube is a spiral structure, and the polypeptide spiral is realized by regulating and controlling the accurate chiral center of a side chain.

Description

Method for forming nanotube by utilizing self-assembly of stapler polypeptide

The technical field is as follows:

the invention belongs to the field of bioengineering, relates to a nanotube, and particularly relates to a method for forming a nanotube by utilizing self-assembly of stapler polypeptide.

Background art:

the nanomaterial refers to a material having at least one dimension in a three-dimensional space in a nanometer size (0.1 to 100nm) or composed of them as a basic unit. Due to the unique structural characteristics, nanomaterials have physical properties that are significantly different from those of macroscopic materials, including: surface and interface effects, small-scale effects, quantum-size effects, and macroscopic quantum tunneling effects. In recent decades, research into nanoscience has made tremendous progress, and nanoscience and technology have penetrated all aspects of our research and life. Nanotechnology has become one of the most important technologies to alter human life.

The nano material represented by graphene and carbon nano tube has been well applied and popularized in various subject fields such as materials science, chemistry, biology, medicine and the like. Although nanotechnology has made great progress, development of new nanomaterials and development of the application depth and breadth of nanotechnology are still the targets of common efforts of scientists all over the world. In contrast to the dramatic leap of inorganic nanomaterials achieved in the last thirty years of review, the development of biological nanomaterials is dwarfed. The reasons are manifold. First, biological materials are more fragile to handle than inorganic materials. Biological materials are generally composed of molecules such as proteins, nucleic acids, or carbohydrates, which are less capable of withstanding extreme conditions. Secondly, biomaterials are more difficult to synthesize. Therefore, it is difficult to obtain sufficient quality for the study of the properties. Third, retention of the activity of a biomaterial is often associated with specific conditions, and changes in conditions can cause structural changes, resulting in changes in properties.

For the above reasons, research on biological nanomaterials is not always available. However, the bio-nanomaterial has properties completely different from those of an inorganic material. The biological nano material has a complex spatial structure, unique electrical and optical properties, and good biocompatibility and degradability, so that the biological nano material can be widely developed in a plurality of fields such as photocatalysis, electricity, biomedicine, bionic materials, life science and the like.

Among the common biological nanomaterials, materials represented by polypeptides are most valued. Polypeptides are a class of biomolecules linked by amino acids through amide bonds. Due to the abundance of amino acids and the diversity of polypeptide modifications, the composition of polypeptides is exceptionally abundant. Polypeptide molecules often contain abundant donors and acceptors of hydrogen bonds, and side chains contain conjugated electron systems, and meanwhile, salt bridges formed by acid and amino groups enable the polypeptide molecules to spontaneously assemble into supramolecules, so that the supramolecular form is quite common. The system comprises amphiphilic polypeptide, beta folding polypeptide, D and L alternating polypeptide, collagen system composed of spiral, dipeptide FF and the like. Currently, such polypeptide self-assembly nano materials have been widely applied in the above fields.

How to construct more complex nano-materials with more special properties based on polypeptide frameworks becomes a target of the efforts of scientists. Through slight structural change, the time and space control is accurately carried out on the material assembly, the uniform and controllable effect of the structure is obtained, and the method is a key point and a difficult point in the field of polypeptide self-assembly.

The invention content is as follows:

aiming at the technical problems in the prior art, the invention provides a method for forming a nanotube by utilizing self-assembly of stapler polypeptide, and the method for forming the nanotube by utilizing self-assembly of stapler polypeptide aims to solve the technical problems that the method for preparing the nanotube by adopting a biological method in the prior art is difficult and the property of the nanotube is unstable.

The invention provides a method for forming a nanotube by utilizing self-assembly of stapler polypeptide, which is characterized by comprising the following steps of:

1) a step of synthesizing an unnatural amino acid chiral at carbon R at position 2 of the-Fmoc-protected side chain, wherein the structural formula of the unnatural amino acid chiral at carbon R at position 2 of the-Fmoc-protected side chain is shown as follows,

wherein X is

2) Connecting the unnatural amino acid in the step 1) with resin by adopting a solid-phase polypeptide synthesis method, then continuing to connect 3 amino acids, wherein the amino acid is any natural amino acid, connecting cysteine, and sealing the amino end of the cysteine by acetyl;

3) removing a protecting group on cysteine sulfydryl from the product obtained in the step 2), and obtaining a polypeptide compound with a side chain modified by carbon chirality at the 2-position through intramolecular sulfydryl-alkene reaction, wherein the position of the carbon chirality side chain coupling amino acid is i/i + 4;

4) shearing the polypeptide from the resin, and purifying by high performance liquid chromatography;

5) freeze-drying the purified polypeptide sample on a freeze dryer to obtain a white powdery solid;

6) dispersing a white powdery polypeptide sample by ultrapure water, and placing the white powdery polypeptide sample in an ultrasonic instrument for ultrasonic treatment to obtain the self-assembled polypeptide nanotube.

Further, the reaction equations of step 2), step 3) and step 4) are as follows:

wherein, Y₁、Y₂、Y₃、Y₄Selected from any one natural amino acid or modified non-natural amino acid

In the side chain of (A), X is

The basic unit polypeptide for forming the nano tube is polypeptide with a stable helical structure, stable stapler polypeptide is used as a monomer, pentapeptide with a side chain having a carbon-terminal unnatural amino acid gamma-site R-type chiral center is synthesized by a solid phase synthesis method through the regulation and control of a precise chiral center of a polypeptide side chain, the polypeptide is dispersed by ultrapure water (2mg/ml) and placed in an ultrasonic instrument for ultrasonic treatment for 10 minutes, the polypeptide nano tube with uniform size and a special structure is simply and efficiently obtained, and the polypeptide nano tube in a solution phase can be further subjected to solvent removal in the modes of freeze-drying, volatilization in the air and the like to obtain the required polypeptide nano tube solid powder.

Compared with the prior art, the invention has remarkable technical progress. The invention uses the stapler polypeptide as the basic material to simply and efficiently prepare the tubular compound with the nanometer property, and the prepared novel polypeptide nanotube has wide application in the aspects of materials science, biomedicine and the like.

Description of the drawings:

FIG. 1 is a flow chart of the present invention for nanotube formation using self-assembly of stapler polypeptides.

FIG. 2 is a scanning electron microscope topography of the polypeptide obtained in example 1.

FIG. 3 is a morphology under a transmission electron microscope of the polypeptide obtained in example 1. A and B are the surface appearances of the polypeptide nanotubes under different magnifications respectively.

FIG. 4 is a topography (A) under an atomic force microscope and a measured height (B) of the polypeptide obtained in example 1.

FIG. 5 is a graph showing a distribution of particle sizes of the polypeptide obtained in example 1 measured by dynamic light scattering.

FIG. 6 is the infrared characterization data of the polypeptide nanotubes obtained in example 1. A and B respectively represent the infrared absorption of the polypeptide nanotubes in different wavenumber regions.

Fig. 7 is raman spectrum data of the polypeptide nanotubes obtained in example 1.

FIG. 8 is the solid powder diffraction data of the polypeptide nanotubes obtained in example 1.

FIG. 9 is the mass spectrometric data of the polypeptide nanomaterials obtained in example 1.

FIG. 10 is the circular dichroism spectrum data of the polypeptide nanotubes obtained in example 1.

The specific implementation mode is as follows:

the invention provides a method for forming a nanotube by utilizing self-assembly of stapler polypeptide, which comprises the following steps:

synthesizing an unnatural amino acid chiral at carbon R at position 2 of the side chain of the-Fmoc protection; the structural formula is as follows:

wherein X is a benzene ring or a group shown below;

(ii) using solid phase synthesis of polypeptide method, connecting the unnatural amino acid to the resin, continuing to connect 3 amino acids and then connecting cysteine and closing the amino terminal of the polypeptide with acetyl;

the acetylation sealing reagent consists of acetic anhydride, N-Diisopropylethylamine (DIEA) and N-methylpyrrolidone (NMP), and the mass percentages of the acetic anhydride and the N, N-Diisopropylethylamine (DIEA) and the N-methylpyrrolidone (NMP) are respectively 4.25%, 15.75% and 80%;

(iii) removing the thiol protecting group from cysteine from the product of step (ii), and then performing intramolecular thiol-ene reaction to obtain a polypeptide compound with 2-position carbon chiral modification of a side chain, wherein the position of the carbon chiral side chain coupling amino acid is i/i + 4; the reaction process is as follows:

wherein X is a benzene ring or a group shown below, Y₁、Y₂、Y₃、Y₄Including the side chains of 20 natural amino acids or modified unnatural amino acids.

The reagent for removing the mercapto protecting group on cysteine consists of trifluoroacetic acid (TFA), Triisopropylsilane (TIS) and dichloromethane, wherein the mass percent of the trifluoroacetic acid (TFA) is 3%, the mass percent of the Triisopropylsilane (TIS) is 5%, and the mass percent of DCM (dichloromethane) is 92%.

The intramolecular mercapto-alkene reaction conditions were: p-Methoxyacetophenone (MAP) (1.0eq), 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone (MNP) (1.0eq) and anhydrous Dimethylformamide (DMF) as a solvent were reacted for 3h under the condition of 365nm ultraviolet light.

(iv) cleaving the polypeptide from the resin and purifying by high performance liquid chromatography.

(v) The purified polypeptide sample was lyophilized on a lyophilizer to give a white powdery solid.

(vi) Dispersing a white powdery polypeptide sample by ultrapure water (2mg/ml), and placing the white powdery polypeptide sample in an ultrasonic instrument for 10 minutes to obtain the self-assembled polypeptide nanotube.

The novel polypeptide nanotube prepared by using the stable stapler polypeptide has a specific structure and potential application in biomedicine and nano photoelectricity, and the structure of the nanotube is characterized in detail by a Scanning Electron Microscope (SEM), a Transmission Electron Microscope (TEM), an Atomic Force Microscope (AFM), an infrared spectrum (FTIR), a Raman spectrum (Raman), a solid powder diffraction technology (XRD) and a Dynamic Light Scattering (DLS) characterization technology.

Example 1

The invention provides stapler cyclic peptides Ac-cyclo (1,5) -CAAAS₅(2-phenyl)-NH₂A method for preparing a polypeptide nano tube formed by self-assembly,

r-configuration Fmoc-protected unnatural amino acid S₅The structural formula of the (2-phenyl) is as follows:

firstly, synthesizing NH by Fmoc solid-phase polypeptide synthesis method₂-CAAAS₅The (2-phenyl) -MBHA resin is prepared by the following specific route:

the specific operation is as follows:

1. grafting with the first amino acid: weighing 1.0g of MBHA resin into a 100ml peptide connecting tube, adding 20ml of N-methylpyrrolidone (NMP) and blowing nitrogen for swelling for 30 min; filtering off the solvent, adding NMP solution of 25% morpholine by volume, blowing nitrogen for 30min, and washing; and (3) connection reaction: Fmoc-S5(2-phenyl) -OH (0.4M in NMP) solution, HCUT (0.38M in NMP) and DIEA were added thereto at 5.0ml/5.0ml/0.71ml, mixed uniformly, and the mixture was purged with nitrogen for 120min, and the reaction solution was filtered off. Washing: the solvent in the linker tube was drained and the resin was washed three times with NMP (10ml x 3) for one minute each;

2. and (3) inoculating a second amino acid: deprotection: adding NMP solution of 25% morpholine by volume, blowing nitrogen for 30min, and washing; and (3) connection reaction: uniformly mixing the prepared Fmoc-Ala-OH (0.4M in NMP) solution, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea Hexafluorophosphate (HCUT) (0.38M in NMP) and DIEA according to the volume of 7.5ml/7.5ml/1ml, adding the mixture into resin, and blowing nitrogen for 50 min; the reaction solution was filtered off, washed and then subjected to the next operation.

3. And (3) inoculating a third amino acid: the procedure was as 2 followed by third Ala.

4. And (4) grafting a fourth amino acid: the procedure was identical to 2 after the fourth Ala. Deprotection: adding NMP solution of 25% morpholine, bubbling nitrogen for 30min, and washing for 3 times.

5. Next to the fifth amino acid Cys: deprotection: adding NMP solution of 25% morpholine by volume, blowing nitrogen for 30min, and washing; and (3) connection reaction: uniformly mixing the prepared Fmoc-Cys (Trt) -OH (0.4M in NMP) solution, 6-chlorobenzotriazole-1, 1,3, 3-tetramethylurea Hexafluorophosphate (HCUT) (0.38M in NMP) and DIEA according to the volume ratio of 7.5ml/7.5ml/1ml, adding the mixture into resin, and blowing nitrogen for 50 min; the reaction solution was filtered off, washed and then subjected to the next operation.

N-terminal acetylation blocking: deprotection: adding NMP solution of 25% morpholine by volume, blowing nitrogen for 30min, and washing; n-terminal acetylation and sealing: mixing the prepared acetylation blocking reagent (acetic anhydride: DIEA: NMP 4.25%: 15.7%: 80%) uniformly, adding into resin, and blowing nitrogen for 120 min; the reaction solution was filtered off, washed and then subjected to the next operation.

7. Removing thiol-Trt protecting group on cysteine: uniformly mixing the prepared reagent (3% TFA, 5% TIS and 92% DCM) for removing the-Trt group, adding the mixed reagent into the resin, bubbling nitrogen for 20min, filtering out the reaction liquid, washing, adding the reagent for removing the-Trt group again, bubbling nitrogen for 20min, filtering out the reaction liquid, washing, and then carrying out the next operation.

8. The reaction was filtered off and the resin was washed alternately with NMP (10ml), Dichloromethane (DCM) (10ml), methanol (MeOH) (10ml) and stored under suction or used for the next reaction.

Side chain construction is accomplished by a thiol-ene reaction (mercapto-ene reaction).

The specific operation is as follows: weighing 1.0g AcHN-CAAAS₅(2-phenyl) -MBHA resin was put in a 100ml flask, and 70mg of MAP,105mg of MNP and 50ml of DMF were added in this order; purging with argon for three times to remove oxygen in the solvent; placing the flask in a photoreactor to react for 3 hours under stirring; then transferring the reaction resin into a peptide grafting tube, filtering reaction liquid, washing with DMF (10ml) and DCM (10ml) alternately, and pumping to dry to obtain Ac-cyclo (1,5) -CAAAS₅(2-phenyl)-NH₂And (3) resin.

The polypeptide was cleaved from the resin using a cleavage solution (trifluoroacetic acid: triisopropylsilane: water: 95: 2.5: 2.5), the resin was filtered off, and the cleavage solution was washed with N₂The shear is dried, precipitated with cooled (ether: n-hexane ═ 1: 1), the precipitate is dissolved with water and acetonitrile and purified by HPLC, 460nm x 2.5mm C18 reverse phase chromatography, liquid a: 0.1% trifluoroacetic acid/water, liquid B: 0.1% trifluoroacetic acid/acetonitrile; solvent gradient: 5-15% of 0-10 min; 15-55% for 10-30 min; rt 26.00 min. And (3) after MS detection, freeze-drying on a freeze dryer to obtain a white powdery solid, wherein the structural formula of the white powdery solid is as follows:

the molecular formula is: c₂₇H₄0N₆O₆S。

9. And dispersing the freeze-dried white powdery polypeptide sample by using ultrapure water (2mg/ml), and placing the mixture in an ultrasonic instrument for ultrasonic treatment for 10 minutes to obtain the self-assembled polypeptide nanotube.

10. And sucking the polypeptide out, uniformly coating the polypeptide on the silicon wafer, placing the silicon wafer in a well-ventilated environment, and forming a uniform thin film layer on the silicon wafer by the polypeptide nanotube after the solvent is volatilized.

11. The morphology of the polypeptide nanotubes is characterized using scanning electron microscopy or the above mentioned technical means.

Example 1 preparation and characterization data for polypeptide nanotubes were obtained:

the preparation flow chart of the polypeptide nanotube is shown in figure 1. FIG. 2 shows the polypeptide nanotubes under a scanning electron microscope, wherein the polypeptide nanotubes have a square tubular structure. The structure of the polypeptide was further characterized by transmission electron microscopy (as shown in FIG. 3), as well as atomic force microscopy. From the analysis of atomic force microscope (as shown in FIG. 4), the height of the polypeptide is about 200nm, the width is about 1-2um, and the length is 10-100 um. (as shown in FIG. 4), dynamic light scattering resulted in a particle size distribution of the polypeptide, indicating that the average particle size of an aqueous solution of the polypeptide was 1um (as shown in FIG. 5). FIG. 6 is the infrared characterization data of the polypeptide nanotubes obtained in example 1. A strong infrared absorption at 1654cm-1 indicated that the polypeptide was helical (A). The absorption peaks at 3266cm-1 and 3319cm-1 indicated a strong hydrogen bonding network (B) inside the polypeptide tube. FIG. 7 is Raman spectrum data of the polypeptide nanotubes obtained in example 1, and as shown in the figure, the polypeptide nanomaterial has strong Raman absorption at 1652cm-1, further illustrating that the polypeptide has a helical structure. Fig. 8 is solid powder diffraction data of the polypeptide nanotubes obtained in example 1, and shows that the polypeptide nanotubes have a very regular internal structure. FIG. 9 shows circular dichroism data of the polypeptide obtained in example 1. As shown in the figure, the absorption is stronger at 205nm and 220nm, which shows that the polypeptide has a spiral structure. FIG. 10 is the mass spectrometric data of the polypeptide nanomaterials obtained in example 1. Mass spectrometry data indicated that the molecular weight of the polypeptide was 576 g/mol. The molecular formula is shown as follows: c₂₇H₄0N₆O₆S。

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Claims (2)

1. A method for forming nanotubes by utilizing self-assembly of stapler polypeptides is characterized by comprising the following steps:

1) a step of synthesizing an unnatural amino acid chiral at carbon R at position 2 of the-Fmoc-protected side chain, wherein the structural formula of the unnatural amino acid chiral at carbon R at position 2 of the-Fmoc-protected side chain is shown as follows,

wherein X is

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2) Connecting the unnatural amino acid in the step 1) with resin by adopting a solid-phase polypeptide synthesis method, then continuing to connect 3 amino acids, wherein the amino acid is alanine, connecting cysteine, and sealing the amino end of the cysteine by acetyl;

3) removing a protecting group on cysteine sulfydryl from the product obtained in the step 2), and obtaining a polypeptide compound with a side chain modified by carbon chirality at the 2-position through intramolecular sulfydryl-alkene reaction, wherein the position of the carbon chirality side chain coupling amino acid is i/i + 4;

4) shearing the polypeptide from the resin, and purifying by high performance liquid chromatography;

5) freeze-drying the purified polypeptide sample on a freeze dryer to obtain a white powdery solid;

6) dispersing a white powdery polypeptide sample by ultrapure water, and placing the white powdery polypeptide sample in an ultrasonic instrument for ultrasonic treatment to obtain the self-assembled polypeptide nanotube.

2. The method of claim 1, wherein the reaction equations of step 2), step 3) and step 4) are as follows:

wherein, Y₁、Y₂、Y₃Is the side chain of alanine, Y₄Is acetyl, X is

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