KR101032922B1 - Nanostructure having stabilized multiple alpha-helical structure, self-assembling macrocyclic compound for forming the nanosructure, and the method of preparing the compound - Google Patents

Abstract

The present invention relates to a nanostructure having a stabilized multiple alpha-helix, a cyclic self-assembled compound forming the nanostructure, and a method for preparing the same, and more particularly, to an alpha-helix forming fragment and a rod ( cyclic self-assembled compound and stabilized multi-alpha-helix nanostructures formed by self-assembly therefrom.

Alpha-helix, rod, beta-sheet, self-assembled, cyclic compounds

Description

Nanostructure having stabilized multiple alpha-helical structure, self-assembling macrocyclic compound for forming the nanosructure, and the method of preparing the compound}

The present invention relates to a nanostructure having a stabilized multiple alpha-helix, a cyclic self-assembled compound forming the nanostructure, and a method for preparing the same, and more particularly, to an alpha-helix forming fragment and a rod ( A cyclic self-assembled compound characterized in that it comprises a rod) forming fragment and a stabilized multi-alpha-helix nanostructure formed by self-assembly.

Alpha-helix, the secondary structure of proteins, is essential for maintaining the secondary structure of proteins, and is essential for various biological recognition processes inside and outside cells, such as protein-DNA, protein-RNA, and protein-protein interactions. This interaction is very important in maintaining and regulating various physiological functions of cells and living things.

Accordingly, studies are being actively conducted to develop drugs that can control or inhibit various interactions of living organisms by making only peptides that are easy to handle and synthesize, rather than using large proteins as they are. However, since alpha-helix structures are well stabilized only when they exist in their own state, alpha-helix forming fragments as peptide fragments separated from proteins have little helical structure due to their unique thermodynamic instability.

Since maintaining the helical morphology of peptides plays an important role in the inherent function of proteins, numerous studies have been conducted to produce peptides with stabilized alpha-helix structures. Mainly, chemical modification of the peptide is employed. Examples of studies for stabilizing the helix structure of the peptide include covalent crosslinking, hydrogen bond replacement, and metal bonding of amino acids located on the same plane as alpha-helix. Complexes, salt bridge formation, helix nucleation, helix capping, and artificial helix receptors. These studies have shown beneficial results in terms of simplicity and cost efficiency. However, there is a drawback that the bioactivity of the peptide may be reduced during such chemical changes, and the interaction in living organisms often increases the avidity by participating in multiple alpha-helix structures simultaneously. There is a fundamental limitation that the stabilization method cannot be effectively applied to complex multivalent biological interactions.

The first problem to be solved by the present invention is characterized in that it comprises an alpha-helix (α-helix) forming fragment and a rod forming fragment at the same time, the ring-shaped magnetic to form a stabilized multi-alpha-helix nanostructure It is to provide an assembly compound.

The second problem to be solved by the present invention is to provide a method for producing the cyclic self-assembled compound.

A third object of the present invention is to provide a stabilized multi-alpha-helix nanostructure formed through the self-assembly process of the cyclic self-assembled compound.

A third object of the present invention is to provide a method of stabilizing a peptide including an alpha-helix structure without undergoing chemical denaturation by using a cyclic self-assembled compound.

In order to achieve the first object, the present invention is characterized in that it comprises an alpha-helix (α-helix) forming fragment and a rod (rod) fragment at the same time, the ring to form a stabilized multi-alpha-helix nanostructure It provides a type self-assembled compound.

According to one embodiment of the invention, the rod-forming fragment of the cyclic self-assembled compound is for example in β-sheet rod, α-helical rod, polyproline rod, p -phenylene, p -phenylene vinylene In one embodiment, the cyclic self-assembled compound according to the present invention may further include a linker in addition to the alpha-helix forming fragment and the rod forming fragment.

According to another embodiment of the present invention, the cyclic self-assembled compound forming the stabilized multi-alpha-helix nanostructures may be selected from cyclic compounds represented by the following formulas (1) to (3):

     (1) (2) (3)

In order to achieve the second object, the present invention provides a method for producing a cyclic self-assembled compound comprising an alpha-helix forming fragment and a rod forming fragment at the same time, the alpha-helix forming fragment and the rod forming fragment at the same time It provides a method for producing a cyclic self-assembled compound having a stabilized multi-alpha-helix structure comprising the step of preparing an acyclic compound, and cyclizing the acyclic compound through an intramolecular reaction. do.

In the method for preparing a cyclic self-assembled compound according to an embodiment of the present invention, the rod-forming fragment is β-sheet rod, α-helical rod, polyproline rod, p -phenylene, p -phenylene vinylene It may be selected from the group consisting of.

According to another embodiment of the present invention, preparing the acyclic compound as a precursor may include combining the alpha-helix forming fragment and the rod forming fragment using a linker, wherein the linker is Examples of oligoethylene glycol based linkers include N- (Fmoc-8-amino-3,6-dioxaoctyl) succinic acid.

In the method for producing a cyclic self-assembled compound according to an embodiment of the present invention, the acyclic compound as a precursor for forming a cyclic compound is, for example, in a cyclic compound represented by the following formulas (4) to (6) Can be chosen:

(4)

(5)

(6)

According to one embodiment of the invention, the intramolecular cyclization step for cyclizing the acyclic compound is preferably carried out by diisopropylethylamine (DIPEA).

In order to achieve the third object, the present invention provides a nanostructure having a stabilized multi-alpha-helix structure, characterized in that the cyclic self-assembled compound is formed by self-assembly.

In order to achieve the fourth object, the present invention provides a peptide comprising a multi-alpha-helix structure by preparing a cyclic self-assembled compound comprising a peptide fragment of the alpha-helix structure having a biological activity and a rod forming fragment at the same time It provides a method of stabilizing.

According to one embodiment of the invention, the peptide having biological activity may be, for example, Rev peptide derived from HIV-1.

According to the present invention, by providing a cyclic self-assembled compound comprising an alpha-helix forming fragment and a rod forming fragment at the same time, and a method for preparing the same, the multi-alpha-helix structure in the self-assembled nanostructure is stably It could be maintained. Nanostructures with these stabilized multi-alpha-helix structures are expected to contribute significantly to biotechnology and medical research involving proteins, and are likely to be used for the treatment of various diseases such as cancer, immune diseases, and infectious diseases. . In particular, the self-assembling compound according to the present invention can go beyond the mono-molecular alpha helix structure, not only can the multi-molecular alpha helix structure remain unmodified, but also can be chemically synthesized to meet the purpose, and thus has been developed. There is greater potential than material.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms.

As a simple but effective supramolecular approach, the present inventors have developed a method of stabilizing alpha helix structure and simultaneously expressing a plurality of alpha helix structures on the surface of nanostructures. The basic idea of the present invention is to provide a cyclic compound having a peptide portion (potential alpha-helix segment) that can form an alpha helix structure and a self-assembling segment at the same time. Within the annular structure, the molecules are constrained, and the alpha helix structure is stabilized to some degree. The inventors of the present invention, when the macrocyclic compound is self-assembled to make a nanostructure, the self-assembled peptide portion is more rigid, the molecule is more constrained, and the alpha helix structure is reduced by reducing the structural entropy of the entire alpha helix structure portion. Was found to stabilize. In conclusion, the self-assembly of macrocyclic compounds stabilized the alpha-helix structure and, at the same time, succeeded in producing nanostructures in which a large number of alpha-helix structures were expressed on the surface.

In particular, bottom-up self-assembly of supramolecular formation is a method for constructing a multi-functional structure having biological activity, and is cost effective. To achieve this object, the peptide former according to the present invention has been designed in such a way that both alpha-helix peptide forming fragments and self-assembled fragments are located within a single macrocyclic compound (FIG. 2, peptide 4).

We have selected beta-sheet forming peptides that are known for their predictable and well-assembled properties as an example of rod forming fragments. Alpha-helix forming fragments selected alanine based peptides. Lysine in the stretch of alanine increases water solubility and can prevent aggregation. Beta-sheet fragments are hydrophobic and charged (+ or-) repeats of amino acids. This combination of amino acids appears to promote beta-sheet hydrogen bonding and thus self-assembly into fiber-like nanostructures that are double ribbons. Oligoethylene glycol based linker fragments are positioned between alpha-helix and beta-sheet forming fragments so that both fragments do not bind directly.

Macrocyclic peptides will first stabilize the helical structure by first reducing the conformational entropy of the unfolded ring structure, and secondly, coil-to-rod transition induced by self-assembly limits and stabilizes the helical structure. This cyclization reaction allows peptides treated with protecting groups to perform pseudo-dilution effects and reduce entropy disadvantages associated with intramolecular cyclization reactions. This is done while letting it do.

First, the inventors investigated the secondary structure of helix forming peptide 1 (FIG. 2), an alanine-based fragment, by circular dichroism spectroscopy. The alpha-helix structure maintains the maximum helix structure at low temperatures, and as the temperature increases, the helix structure is released, and thus the temperature dependency is very high. Even at low temperatures (1 ° C.), Peptide 1 showed a strong negative band at 196 nm, meaning that the peptide formed an almost random coil (FIG. 6A). The formation of the alpha-helix structure of peptide 1 can be stabilized only at low temperatures even when helix stabilizer 2,2,2-trifluoroethanol (TFE) is used as a cosolvent. This can be seen through the negative band and the strong positive band around 190nm. A slight deviation from the nature of the perfect alpha-helix structure is presumed to mean that the helix is in fact only partially stabilized. As can be seen in FIG. 6B, the temperature-induced helix-coil transition shows an apparent isodichroic point at 201 nm, providing evidence that the two states, alpha-helix structure and random coil structure, are in equilibrium. These results also indicate that peptide 1 lacks the ability to form helix structures. Thus, it can be seen that some change is necessary to stabilize the helix structure of peptide 1.

Secondly, we examined the secondary structure of peptide 2, which is only a beta-sheet forming fragment. The CD spectrum shows a strong negative band at 212 nm, a strong positive band at 197 nm, and an intersection at 203 nm, indicating that the peptide mainly forms beta-sheets (FIG. 6A). Beta-sheet formation of peptide 2 was found to be stable even at high temperatures (possibly up to 81 ° C.).

The inventors then examined the secondary structure of peptide 3, including both alpha-helix forming fragments and beta-sheet forming fragments in a linear structure. The results showed that peptide 3 mainly forms beta-sheets. Peptide 4 is a cyclic peptide prepared from acyclic precursor peptide 3 via a cyclization reaction.

In stark contrast to peptide 3, which is acyclic, the CD spectrum of cyclic peptide 4 has a strong positive band near 190 nm (alpha-helix), a strong negative band at 204 and 207 nm (alpha-helix), at 214 nm (beta-sheet). A strong negative band, 223nm (alpha-helix) weak shoulders, indicating the coexistence of alpha-helix and beta-sheet formation.

Considering the fact that peptide 1, which has a partial helix structure only in the presence of TFE, exhibits a negative band at 204 nm, the band at 204 nm shown in peptide 4 is the result of forming a partially stabilized alpha-helix structure or other type. It suggests that it is an intermediate with a helix structure of. These results clearly show that the cyclic compounds according to the present invention can stabilize the alpha-helix structure.

It is also shown that the alpha-helix structures according to the invention do not aggregate. Therefore, the formation of the coiled-coil alpha-helix bundle structure is not expected to be the cause of the stabilization of the alpha-helix structure according to the present invention. Considering the fact that acyclic peptide 3 mainly forms a beta sheet, the fact that two fragments are present in the cyclic compound of alpha-helix structure plays a very important role in effectively stabilizing through the beta-sheet mediated self-assembly process. It can be seen that. In particular, it should be noted that the peptide 4 according to the present invention showed substantially the same CD profile even after several months of incubation. This indicates that the compound having an alpha-helical structure according to the present invention is a thermodynamically stable structure. According to the spectrum results, the cyclic peptide 4 according to the present invention shows that a part of the helical structure exists even at a high temperature, which can be interpreted that the thermal stability of the alpha-helix structure is rapidly increased by self-assembly.

To more closely investigate the stabilization of alpha-helix structures mediated by beta-sheet self-assembly, we synthesized peptide 5 (FIG. 4). Peptide 5 comprises the same alpha-helix forming fragment as peptide 4. However, mutations (Trp-> Gly and Glu-> Lys) were performed on beta sheet fragments to make peptides in which beta-sheet structures could not be formed. CD analysis showed that there were more unstructured portions than those that formed alpha-helix and beta-sheet structures (FIG. 7A), which suggests that beta-sheet induced self-assembly plays a critical role in stabilizing the helix structure. To show.

Meanwhile, cyclic peptide 7 was prepared to further exclude the possibility of forming a coiled-coil helix bundle to stabilize the alpha-helix structure (FIG. 4). Peptide 7 replaced the hydrophobic Ala residue in the helix-forming fragment of peptide 4 with a hydrophilic Glu residue to inhibit the possibility of coiled-coil formation (FIG. 4). Glu residues are located at i and i + 4 spacing of Glu and Lys residues. In this type of arrangement, the Glu and Lys residues lie on the same side as the alpha-helix, thus facilitating salt bridge formation between oppositely charged ions. Research shows that peptide helix structures formed by this type of amino acid configuration do not aggregate. Peptide 7 also showed bands at 208 nm (alpha-helix), 215 nm (beta-sheet), and 221 nm (alpha-helix), similar to peptide 4, the cyclic compound according to the invention, which was beta-sheet mediated. It indicates that the alpha-helix structure was stabilized by self-assembly, which proves that the helix structure stabilization of the cyclic self-assembled compound according to the present invention is not due to coiled-coil formation.

On the other hand, TEM investigation, peptide 2 was self-assembled in the form of twisted fibers (twisted fibers) was a little thick, twisted (Fig. 13a). It is difficult to accurately measure the length of nano-fibers beyond the TEM boundaries in these high-resolution images, but it can be seen that they are more than a few nanometers. This result is consistent with previous reports that beta-sheet peptides are laterally bound to form a hierarchy when no hydrophilic fragments such as PEG or hydrophilic peptides are attached. In contrast, acyclic peptide 3, in which a hydrophilic alpha-helix fragment is attached to a beta-sheet forming fragment with an oligoethylene glycol based linker, mostly forms broken nano-fibers of 100 nm or more in length (FIG. 13B).

In contrast to the formation of one-dimensional bodies in acyclic peptide 3, cyclic peptide 4 forms a two-dimensional sphere with a few one-dimensional bodies (Fig. 13C). The diameter of the sphere is about 10 nm. Typically, the diameter of alpha-helix is about ˜1.2 nm, and the distance between strands between beta-strands is ˜0.47 nm. Given the fact that alpha-helices are bulkier than beta-strands as described above, helices that are filled side by side in the sheet may have helix rotated relative to each other, thus facilitating spherical formation. (FIG. 1B). This structure appears to be similar to the protein folds found in alpha / beta barrels. Similar to alpha / beta barrels, hydrophobic Trp residues on one side of the beta-tape seem to form the hydrophobic center of the globular. Considering the linker length (~ 2 nm), the size of the sphere can be considered appropriate: 10 nm (sphere diameter) = 1.2 nm x 2 (helix diameter) + 2 nm x2 (linker length) + additional from beta-strand The length and center of the sphere.

The formation of one-dimensional fiber materials has also been found. The formation of ancillary one-dimensional materials is thus estimated. During the self-assembly process, alpha-helix structures are formed by the formation of double-layered beta-ribbons, which can be thought to be dynamically frozen by the combination of strong interstrand beta-sheets and intersheets, and the hydrophobic reaction is widely It can be said. In this case, the alpha-helix forming fragment may be considered to have a helix structure partially or unstructured in the one-dimensional body.

Dynamic light scattering (DLS) investigation showed that the R _H distribution of the peptide 4 nano-aggregates according to the present invention was smaller than the R _H distribution of the peptide 3 nano-aggregates, indicating that smaller spherical material was formed from peptide 4. It supports the view. The results of the FT-IR spectra show that peptide 3 and peptide 4 both form anti-parallel beta-sheet structures, with peptide 3 at 1625 and 1695 cm ⁻¹ and peptide 4 at 1620 and 1689 cm ⁻¹ amide I bands. Is evidenced by the fact that (Fig. 12).

Although the spectra overlap considerably due to the formation of alpha-helix, a major difference between the spectra of peptide 3 and peptide 4 can be found in the amide I frequency of the random coil (~ 1640-1650 cm ^-1). ). The IR spectrum of Peptide 3 shows a strong random coil band around 1651 cm ⁻¹ , which is due to another two dimensional structure.

In contrast, a random coil band near 1654 cm ⁻¹ decreases the intensity in the spectrum of peptide 4, and this result can be interpreted as a modification of the random coil from the cyclic peptide nano-aggregates to other structures (alpha-helix). .

We investigated whether the process of cyclization of macromolecules could be applied to stabilize multiple alpha-helix structures of biologically active peptides. As an example of a peptide having biological activity, Rev peptide derived from human immunodeficiency virus type I (HIV-1) was selected. Rev peptide is an arginine rich peptide that binds deeply in the major groove of the Rev response element (RRE) RNA of HIV-1. It is well known that alpha-helix formation of Rev peptides is well associated with specific binding to RRE RNA and that peptides are monomeric and do not aggregate with each other. In order to stabilize the alpha-helix structure of Rev peptide and to make nanostructures having multiple Rev alpha-helix through self-assembly, Rev peptide sequence (14 amino acids) and beta-sheet forming fragments which are essential for binding to RRE RNA Macrocyclic peptide 11 and other control peptides were simultaneously synthesized (Fig. 5). The same experiments with peptides 1-7 above show that Rev peptide alpha-helix structures can be stabilized by mediated self-assembly through the beta-sheets of macrocyclic peptide 11, which approach By means of which a multivalent protein consisting of alpha-helices which are biologically active can be produced.

According to the present invention, a well-designed compound comprising fragments self-assembled in rod form, such as alpha-helix and beta-sheet, within a single macrocyclic compound will be self-assembled into a plurality of alpha-helix coated nanostructures. The present invention is very useful because it is possible to stabilize the multiple alpha-helix structure without chemical modification by using the cyclic self-assembled compound.

According to the fact that alpha-helices outside of proteins is one of the important mediators of biological recognition processes, the basic principles of the present invention are able to regulate molecular recognition processes mediated by alpha-helix structures, which occur in a multivalent form. It is a good starting point for developing synthetic proteins. In addition, the degree of stabilization and biological activity of alpha-helix structures should be further studied considering chemical and structural variables of alpha-helix, self-assembled fragments, and linker fragments.

Example: Preparation of Cyclic Self-Assembly Compound According to the Present Invention

Peptides were synthesized on Rink Amide MBHA resin using the Applied Biosystems Model 433A Peptide Synthesizer according to standard Fmoc protocol. Standard amino acid protecting groups were used, except for cysteine, which used acid-labile methoxytrityl. N- (Fmoc-8-amino-3,6-dioxaoctyl) succinic acid (Fmoc-PEG2-Suc-OH), an oligoethylene glycol based linker available from Anspec, was used. Peptide-attached resin (20 mmol N-terminal amino group) in N-methyl-2-pyrrolidone (NMP) was swollen for 30 minutes.

To carry out the cyclization reaction, bromoacetic acid was first bound to the N-terminus of the peptide immobilized on the resin. Then, before adding to the resin, bromoacetic acid (28 mg, 200 mmol) and N, N-diisopropylcarbodiimide (15.5 mL, 100 mmol) were allowed to stand in N-methyl-2-pyrrolidone for 10 minutes, The carboxyl group was activated. The reaction was continued in a 6 mL polypropylene tube (Restek) equipped with frit at room temperature and shaken for 1 hour. Then washed sequentially with N-methyl-2-pyrrolidone and dichloromethane. To remove the protecting group at the orthogonal position of acid-labile methoxytrityl from cysteine, the resin was treated several times with 1% trifluoroacetic acid (TFA) in dichloromethane ((1 min min-5).

The intramolecular cyclization reaction was then performed by shaking overnight at room temperature in 3 mL of 1% diisopropylethylamine (DIPEA) in N-methyl-2-pyrrolidone. Thereafter, the mixture was washed sequentially using N-methyl-2-pyrrolidone and acetonitrile, followed by vacuum drying. The dried resin was treated with a cleavage mixture (TFA: 1,2-ethanedithiol: thioanisole; 95: 2.5: 2.5) for 3 hours, precipitated with tert-butyl methyl ether, The peptide was purified by reversed phase HPLC (water-atetonitrile with 0.1% TFA). The molecular weight was confirmed by a MALDI-TOF mass spectrometer. HPLC analysis showed that the purity of the peptide was 95% or more. On the other hand, the concentration was spectroscopically determined in 8M urea using a molar extinction coefficient (5,500M ^-1 cm ^-1 ) at 280nm.

Experimental Example 1: Circular dichroism

CD spectra were measured using a JASCO model J-810 spectropolarimeter with thermostat. Spectra were recorded between 250 nm and 190 nm using a cuvette with a path-length of 0.1 cm, and the scan was repeated several times to average. We also calculated molar ellipticity per amino acid residue. Peptide concentration was 20 mM. Peptides were prepared in 75 mM KF solution unless otherwise indicated. The sample solution was incubated for at least two days before the measurement. Even after prolonging the incubation period, the measurements yielded substantially identical CD spectra, indicating thermodynamic equilibrium. The same results were also obtained when the peptide was treated in phosphate buffer solution (PBS), which indicates that the present invention can be applied under physiological conditions.

Figure 6 is a graph showing the secondary structure analysis results by CD analysis of peptides 1 to 4, a) peptide 1 (red) and peptide 2 (blue) in 1 ℃ 75mM KF, b) 1 ℃ 75mM KF Peptide 1 in c / 30% TFE, c) Peptide 3 (light blue) and Peptide 4 (purple) and d) in 1 ° C. 75 mM KF are graphs of the temperature change of peptide 4 in 75 mM KF.

7 is a graph showing the CD spectrum of Peptide 2 at various temperature changes.

8 is a graph showing the results of secondary structure analysis by CD analysis of peptides 5 to 7, a) peptide 5 in 1 ° C 75 mM KF, b) peptide 6 (red) and 1 in 1 ° C 75 mM KF. Peptide 6 (blue) in 75 ° C. 75 mM KF / 30% TFE, c) Graph of peptide 7 in 1 ° C. 75 mM KF.

Figure 9 is a graph showing the secondary structure analysis of the peptide by CD analysis, a) peptide 1 (red) in 1 ℃ 75mM KF / 30% TFE and peptide 4 (blue) in 1 ℃ 75mM KF. , b) is a graph of peptide 6 (red) in 1 ° C. 75 mM KF / 30% TFE and peptide 7 (blue) in 1 ° C. 75 mM KF.

10 is a graph showing the secondary structure analysis results of the peptides by CD analysis, a) is an analysis graph of peptide 8 (red) and peptide 9 (blue) in 1 ° C 75mM KF, and b) is 1 ° C 75mM KF, analytical graph of peptide 8 in 60% TFE, c) is analytical graph of peptide 10 (light blue) and peptide 11 (purple) in 25 ° C. 75 mM KF.

Experimental Example 2: IR Measurement

For FT-IR measurements, 100 mL of sample (300 mM in 75 mM KF) in solution was separated and transferred to the ZnSe window. 20,000 scans were obtained on a Bruker Equinox 55 FT-IR spectrometer.

11 a b c d shows the MALDI-TOF MS spectrum of peptides 1 to 4 (Matrix: a-cyano-4-hydroxycinnamic acid).

12 is a b c d shows the MALDI-TOF MS spectrum of peptides 5 to 7 (Matrix: a-cyano-4-hydroxycinnamic acid).

Figure 13 a b c d shows the MALDI-TOF MS spectrum of peptides 8 to 11 (Matrix: a-cyano-4-hydroxycinnamic acid).

Experimental Example 3: Transmission Electron Microscope

2 mL of sample was placed on a carbon coated copper grid and completely dried. Then 2 mL of 2% (w / v) uranyl acetate solution was added for 1 minute, then excess solution was removed with filter paper. Sample concentrations were about 5-20 mM in 75 mM KF. Samples were observed at 120 kV using JEOL-JEM 2010 and the images were recorded with an SC 1000 CCD camera (Gatan). Data was analyzed with Digital Micrograph software.

14 is a TEM image of peptides 2-4. Specifically, FIG. 14a shows TEM images of peptides 2, 14b of peptide 3 and 14c of peptide 4. Detailed description of each image is as described above.

15 is a TEM image of peptide 7 nano-aggregates. Similar to peptide 4, peptide 7 also has a spherical shape with most particles about 10 nm in diameter.

16 a and b are TEM images of peptides 9 and 11. Particles in the shape of spheres are indicated by arrows.

Experimental Example 4: Dynamic light scattering (DLS)

DLS experiments were performed at 632.8 nm at room temperature using an LV / CGS-3 Compact Goniometer System equipped with a He-Ne laser. Samples were centrifuged at 16,110 × g for 20 minutes prior to measurement to precipitate impurity particles. Sample concentrations were about 5-20 mM in 75 mM KF. The size distribution was determined by the constrained regularization method.

17 a and b are R _H distributions of peptides 3 (green) and 4 (red) by DLS, and c is the FT-IR spectrum of peptides 3 and 4. FIG.

FIG. 18 is a distribution of R _H of peptide 11 by DLS. FIG.

1 is a schematic diagram showing a structure in which a cyclic peptide is self-assembled in a form having a multi-alpha-helix structure according to an embodiment of the present invention, and is stabilized in a multi-nanostructure state on a self-assembly of a beta-sheet fragment. Show alpha-helix structure.

Figure 2 is a ring having a stabilized multiple alpha-helix structure, characterized in that it comprises at the same time alpha-helix (α-helix) forming fragments and beta-sheet (β-sheet) forming fragments in accordance with an embodiment of the present invention The type self-assembled compound is represented by the chemical formula, and the chemical formulas of peptides 1 to 4 are represented.

3 is a structural formula of the peptides 1 to 4 to the linker moiety.

4 shows the chemical formulas of peptides 5, 6, 7 and FIG. 5 shows the chemical formulas of peptides 8, 9, 10 and 11.

FIG. 6 is a graph showing secondary structure analysis results by CD analysis of each peptide, a) peptide 1 and peptide 2 in 1 ° C 75 mM KF, and b) peptide 1 in 1 ° C 75 mM KF, 30% TFE. c) Peptide 3 and peptide 4 in 1 mM 75 mM KF, d) Peptide 4 in 75 mM KF.

7 is a graph showing the CD spectrum of Peptide 2 at various temperature changes.

Fig. 8 is a graph showing the results of secondary structure analysis by CD analysis of peptides 5 to 7, a) peptide 5 in 1 ° C 75 mM KF, b) peptide 6 and 1 ° C 75 mM KF in 1 ° C 75mM KF. Peptide 6 in / 30% TFE, c) Peptide 7 in 1 ° C 75 mM KF.

9 is a graph showing the secondary structure analysis results of the peptides by CD analysis, a) is peptide 1 in 1 ° C 75mM KF / 30% TFE and peptide 4 in 1 ° C 75mM KF, and b) is 1 ° C Graphs of peptide 6 in 75 mM KF / 30% TFE and peptide 7 in 1 ° C. 75 mM KF.

10 is a graph showing the secondary structure analysis results of the peptide by CD analysis, a) is an analysis graph of peptide 8 and peptide 9 in 1 ℃ 75mM KF, b) in 1 ℃ 75mM KF, 60% TFE Is an analytical graph of peptide 8, c) is an analytical graph of peptide 10 and peptide 11 in 25 ° C. 75 mM KF.

11, a, b, c, and d show MALDI-TOF MS spectra of peptides 1 to 4 (Matrix: a-cyano-4-hydroxycinnamic acid).

12 shows a, b, c, and d show MALDI-TOF MS spectra of peptides 5 to 7 (Matrix: a-cyano-4-hydroxycinnamic acid).

FIG. 13 shows a, b, c and d showing MALDI-TOF MS spectra of peptides 8 to 11 (Matrix: a-cyano-4-hydroxycinnamic acid).

14 is a TEM image of peptides 2-4. Specifically, FIG. 14a shows TEM images of peptides 2, 14b of peptide 3 and 14c of peptide 4. Detailed description of each image is as described above.

15 is a TEM image of peptide 7 nano-aggregates. Similar to peptide 4, peptide 7 also has a spherical shape with most particles about 10 nm in diameter.

16 a and b are TEM images of peptides 9 and 11. Particles in the shape of spheres are indicated by arrows.

17 a and b are R _H distributions of peptides 3 and 4 by DLS, and c is the FT-IR spectrum of peptides 3 and 4. FIG. .

FIG. 18 is a distribution of R _H of peptide 11 by DLS. FIG.

Claims (13)

A cyclic self-assembled compound, characterized in that it comprises an alpha-helix (α-helix) forming fragment and β-sheet rod (beta-sheet rod) at the same time, to form a stabilized multi-alpha-helix nanostructure. delete The cyclic self-assembled compound of claim 1, further comprising a linker in addition to the alpha-helix forming fragment and the β-sheet rod. The cyclic self-assembled compound according to claim 1, which is selected from cyclic compounds represented by the following formulas (1) to (3).

     (1) (2) (3) The nanostructure having a stabilized multi-alpha-helix structure, characterized in that the cyclic self-assembly compound according to any one of claims 1, 3 or 4 is formed by self-assembly. In the method for producing a cyclic self-assembled compound comprising an alpha-helix forming fragment and β-sheet rod at the same time, Preparing an acyclic compound comprising the alpha-helix forming fragment and the β-sheet rod simultaneously; Method for producing a cyclic self-assembled compound having a stabilized multi-alpha-helix structure comprising the step of cyclizing the acyclic compound through an intramolecular reaction. delete The cyclic self-assembled compound of claim 6, wherein the preparing of the acyclic compound comprises combining the alpha-helix forming fragment and the β-sheet rod by using a linker. Manufacturing method. The method of claim 8, wherein the linker is an oligoethylene glycol-based linker. The method for producing a cyclic self-assembled compound according to claim 6, wherein the acyclic compound is selected from the following formulas (4) to (6).

(4)

(5)

     (6) The method for preparing a cyclic self-assembled compound according to claim 6, wherein the intramolecular cyclization step for cyclizing the acyclic compound is carried out by diisopropylethylamine (DIPEA). A method of stabilizing a peptide comprising multiple alpha-helix structures by preparing a cyclic self-assembled compound comprising a peptide fragment of an alpha-helix structure having a biological activity and a β-sheet rod simultaneously. The method of claim 12, wherein the biologically active peptide is a Rev peptide derived from HIV-1.

Images