KR102521421B1 - Heterochiral peptide-complex and composition for measuring nuclear magnetic resonance spectroscopy residual dipolar coupling containing self-assembled intermediates of Heterochiral peptide-complex - Google Patents

Abstract

The present invention relates to a heterochiral peptide complex, a self-assembly intermediate thereof, an orientation mediator composition used for measuring residual dipole bonds (NMR-RDC), a type of nuclear magnetic resonance spectroscopy including the same, and a method for NMR measurement of biomolecules using the same. , Beta sheet-based peptides have excellent structural stability to environmental factors such as heat, acidity, and ion concentration, and thus have the advantage of being usable as an orientation medium for RDC measurement for a wide range of biomolecules.

Description

Heterochiral peptide-complex and composition for measuring nuclear magnetic resonance spectroscopy residual dipolar coupling containing self-assembled intermediates of Heterochiral peptide-complex}

The present invention relates to a heterochiral peptide complex and its self-assembly intermediate, an alignment mediator composition used for measuring residual dipole bonds (NMR-RDC), a type of nuclear magnetic resonance spectroscopy including the same, and a NMR measurement method of biomolecules using the same. .

The identity of the peptide and its formation mechanism are very important information for the development of customized materials. However, most studies have focused on analyzing the final form of self-assembly rather than the mechanism. In contrast, many studies have been conducted on protein folding. Since protein folding is performed rapidly in seconds, special techniques such as dynamics and structural studies based on nuclear magnetic resonance (NMR) spectroscopy or other special spectroscopic methods have been developed and used, but it is difficult to clearly identify actual intermediates.

RDC is a part of NMR structural analysis and can analyze the structure of a target material more specifically than NMR analysis. Specifically, when the sample is present in aqueous conditions, it becomes difficult to measure dipolar couplings due to randomized molecular motion. Therefore, in order to measure RDC, it is necessary to limit the direction of the dipole to have anisotropy. As conventional alignment media, nematic liquid crystal molecules, compressed or stretched polymer gel, Pf1 filamentous phage, and bicelles are known. The use of the RDC alignment medium is limited because it has specific conditions for temperature, pH, solvent, and state of charge of the alignment medium. Therefore, there is an urgent need to develop a new orientation medium that can be widely applied.

(Reference 1.) Israelachvili, J. N., Intermolecular and surface forces. 3rd ed.; Academic Press: Burlington, MA, 2011; p 535-561.

An object of the present invention is to provide a peptide complex comprising a hydrophobic segment and a hydrophilic segment composed of a hydrophilic polymer.

Another object of the present invention is to provide a self-assembly intermediate in which a peptide complex is formed through self-assembly.

Another object of the present invention is to provide an orientation medium composition for measuring residual dipole bonds (RDC) including self-assembly intermediates.

Another object of the present invention is to provide a novel use of a peptide complex or self-assembling intermediate thereof for NMR spectrum measurement.

The present invention designed various heterochiral peptide complexes, and among them, it was confirmed that the self-assembly process proceeds significantly slower than the homochiral peptide complex in the case of racemic or asymmetric heterochiral peptide complexes. Self-assembly intermediates of racemic or asymmetric heterochiral peptide complexes with strong anisotropy are prepared, and can be used as an alignment medium used in NMR-RDC measurement, a type of nuclear magnetic resonance spectroscopy. As confirmed, the present invention was completed.

The present invention is a peptide represented by the following general formula 1 or 2 in order to solve the above object; And a compound represented by Formula 1 bonded to the N-terminus of the peptide; provides a peptide complex comprising a.

[Formula 1]

[Y1] _a -{-[X1] _b -[Y2] _c } _d -[X2] _e

[Formula 2]

[X1] _f -{-[Y1] _g -[X2] _h } _i -[Y2] _j

In Equations 1 and 2 above

X1 and X2 are the same or different from each other, and are each independently any one selected from D-Trp, D-Phe, D-Tyr, D-Val, D-Leu, and D-Ile;

Y1 and Y2 are the same or different from each other, and are each independently any one selected from L-Trp, L-Phe, L-Tyr, L-Val, L-Leu, and L-Ile,

Wherein a, f, e and j are each independently any one integer selected from 0 to 2, b, c, g and h are each independently any one integer selected from 1 to 5, and d and i Each is an integer independently selected from 1 to 10.

[Formula 1]

In Formula 1, n and m are each independently an integer ranging from 1 to 12.

In Formulas 1 and 2, X1 and X2 are the same or different from each other, each independently any one selected from D-Trp, D-Val and D-Phe, Y1 and Y2 are the same or different from each other, and each It may be any one independently selected from L-Trp, D-Val and L-Phe.

In Formulas 1 and 2, X1 may be the same as X2, and Y1 may be the same as Y2.

In Formulas 1 and 2, a, f, e, and j are each independently an integer selected from 0 to 2, and b, c, g, and h are each independently selected from 1 to 3. It is an integer, and d and i may each independently be any one integer selected from 1 to 4.

The peptides may be represented by SEQ ID NOs: 1 to 12 below.

[SEQ ID NO: 1]

(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp

[SEQ ID NO: 2]

(D)Val-(L)Val-(D)Val-(L)Val-(D)Val-(L)Val

[SEQ ID NO: 3]

(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe

[SEQ ID NO: 4]

(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp

[SEQ ID NO: 5]

(L)Val-(D)Val-(L)Val-(D)Val-(L)Val-(D)Val

[SEQ ID NO: 6]

(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe

[SEQ ID NO: 7]

(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp

[SEQ ID NO: 8]

(L)Val-(D)Val-(L)Val-(D)Val-(L)Val

[SEQ ID NO: 9]

(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe

[SEQ ID NO: 10]

(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp

[SEQ ID NO: 11]

(D)Val-(L)Val-(D)Val-(L)Val-(D)Val

[SEQ ID NO: 12]

(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe

In the peptide conjugate, the compound may be represented by Formula 2 below.

[Formula 2]

In Formula 2, m is an integer ranging from 1 to 12.

The peptide complex may be any one selected from those represented by Chemical Formulas 3 to 6 below.

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

In Chemical Formulas 3 to 6, m is an integer ranging from 1 to 5.

The peptide complex slowly forms a self-assembly in solution, but in Formulas 1 and 2, when a, f, e and j are 0, a right-handed fashion (P-helix) over time ) and in the left-handed fashion (M-helix) twisted fiber structure, the helical portion of the fiber is laterally extended (fattened) to form an intermediate of a flat ribbon fiber structure, and finally a flat ribbon crystal structure. It may be to form a self-assembly of.

The peptide complex slowly forms a self-assembly in solution, but in Formulas 1 and 2, when a, f, e, and j are not 0 at the same time, a right-handed fashion (P -helix) and fibers composed of variable pitch helices twisted in a left-handed fashion (M-helix); and sausage strings due to superhelix fibers formed by excessive twisting and entanglement of the fibers ( string); and finally, the peptide complex is twisted in a right-handed direction (P-helix) and a left-handed fashion (M-helix) at a variable pitch ( It may be a peptide complex characterized in that it is formed as a self-assembly of a porous superhelical network structure by intertwining and overtwisting fibers composed of variable pitch helices.

The present invention provides an orientation mediator composition for measuring residual dipole bonds (RDC) comprising a peptide complex in order to solve the above another object.

In order to solve the above another object, the present invention provides a method for performing NMR spectrum analysis of a biomolecule comprising the following steps.

(a) mixing the peptide complex in a mixed solvent;

(b) adding a biomolecule to be measured to the mixed solution; and

(c) performing NMR measurement on the biomolecule-peptide complex mixture.

The NMR measurement may be performed by any one selected from the group consisting of ¹ H, ¹ H edit, and 2D NMR.

The mixed solvent may be a mixture of a deuterium solvent and a solvent.

In order to solve the above another object, the present invention provides a drug delivery system containing the peptide complex.

In order to solve the above another object, the present invention provides a scaffold for cell culture comprising the peptide complex.

The heterochiral peptide complex of the present invention includes a polyethylene glycol moiety, thereby maintaining affinity with biomolecules and minimizing interaction with analytes. In addition, the heterochiral peptide complex of the present invention has excellent structural stability to environmental factors such as heat, acidity, and ion concentration, including beta-sheet-based peptides, and can be used as an orientation medium for RDC measurement for a wide range of biomolecules. have

1 is a diagram showing the three-dimensional structure of the heterochiral peptide complex of the present invention. The drawing on the right of FIG. 1A shows the structure of a homochiral peptide (Comparative Examples 1 and 2), and the drawing on the left of FIG. 1A shows the structure of a heterochiral peptide (Example 1) (L-type amino acid and D-type amino acid are alternately combined structure).
Figure 1b specifically shows the chemical structure of the amphiphilic peptide of the present invention, W (capital letter) means L-type tryptophan, and w (lower case letter) means D-type tryptophan.
2 is a view showing the chemical structures of the heterochiral peptide complexes prepared in Examples 1 to 4 and the homochiral peptide complexes prepared in Comparative Examples 1 and 2;
3 is a graph of MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry results of heterochiral peptide complexes prepared from Examples 1 to 4, and FIG. Figure 3b is a homochiral peptide complex prepared from Comparative Example 2, Figure 3c is a heterochiral peptide complex prepared from Example 1, Figure 3d is a heterochiral peptide complex prepared from Example 2, Figure 3e The heterochiral peptide complex prepared from Example 3, Figure 3f is the heterochiral peptide complex prepared from Example 4.
Figure 4 is a graph of HPLC analysis results of heterochiral peptide complexes prepared from Examples 1 to 4, Figure 4a is a homochiral peptide complex prepared from Comparative Example 1, and Figure 4b is a homochiral peptide prepared from Comparative Example 2 Figure 4c is a heterochiral peptide complex prepared from Example 1, Figure 4d is a heterochiral peptide complex prepared from Example 2, Figure 4e is a heterochiral peptide complex prepared from Example 3, Figure 4f is Example 4 It is a heterochiral peptide complex prepared from
5 is an observation of different self-assembly processes of the homochiral peptide complexes of Comparative Examples 1 and 2 and the heterochiral peptide complexes of Examples 1 to 4.
Figure 5a is a CD spectrum result graph of homochiral peptide complexes of Comparative Example 1 and Comparative Example 2, which are enantiomers, Figure 5b is an AFM image of the homochiral peptide complex of Comparative Example 1, and Figure 5c is Comparative Example 2 It is an AFM image of a homochiral peptide complex.
6 is an AFM image of the homochiral peptide complex prepared from Comparative Example 1 in a global minimum state (crystallized form).
7 is an AFM image of the homochiral peptide complex prepared from Comparative Example 2 in a global minimum state (crystallized form).
8 is a CD spectrum graph showing the self-assembly process of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) of Example 1 over time in solution.
Figure 9a is a CD spectrum result graph of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) of Example 1 after 6 weeks, and Figure 9b is a heterochiral (racemic) peptide complex of Example 2 after 6 weeks. ) CD spectrum result graph of the peptide complex (T-(Ww) ₃ ).
Figure 10a is an AFM image of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 hours, and Figure 10b is an AFM image of the heterochiral (racemic) peptide complex (T-(wW) ₃ after 2 days). ), and FIG. 10c is an AFM image of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 weeks.
11 is an AFM image of a heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 hours.
12 is an AFM image of a heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 days.
13 is an AFM image of a heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 weeks.
Figure 14a shows the synchrotron WAXS of the heterochiral peptide complex (T-(wW) ₃ ) prepared from Example 1 and the homochiral peptide complexes (T-W6 and T-w6) of Comparative Examples 1 and 2 data, and FIG. 14b is an MD simulation result of (wW) ₃ peptides in the heterochiral peptide complex of Example 1.
The numbers mentioned in FIG. 14 represent distances in angstroms.
15 is a diagram schematically showing the self-assembly pathway of the heterochiral peptide complex and the homochiral peptide complex of the present invention.
16 is a result of measuring the structures of the asymmetric heterochiral peptide (ee) complexes (TW(wW) ₂ and Tw(Ww) ₂ ) of Examples 3 and 4 in the initial stage of self-assembly, and FIG. 16a shows Example 3 and An AFM image measured at the initial stage of self-assembly (2 hours) of the asymmetric heterochiral peptide (ee) complex of 4 (TW(wW) ₂ and Tw(Ww) ₂ ), and FIG. 16B is an enlarged image of FIG. 16A (Enlarged images) ), and FIG. 16c is a diagram showing the unique helical structures of the asymmetric heterochiral peptide (ee) complexes (TW(wW) ₂ and Tw(Ww) ₂ ) of Examples 3 and 4.
17 is an AFM image of the asymmetric heterochiral peptide (ee) complex (TW(wW) ₂ ) of Example 3 in the early stage of self-assembly (2 hours).
18 is an AFM image of the asymmetric heterochiral peptide (ee) complex (Tw(Ww) ₂ ) of Example 4 in the early stage of self-assembly (2 hours).
19 is an AFM image of the asymmetric heterochiral peptide (ee) complex (TW(wW) ₂ ) of Example 3 at the final stage of self-assembly (2 weeks, kinetically confined state).
20 is an AFM image of the asymmetric heterochiral peptide (ee) complex (Tw(Ww) ₂ ) of Example 4 at the final stage of self-assembly (2 weeks, kinetically confined state).
21 is a deuterium NMR spectrum of the heterochiral peptide complex of Example 1 and the homochiral peptide complex of Comparative Example 1 having various assembly states at 10% (v/v) D ₂ O, 298K. All spectra were obtained using a 700 MHz NMR spectrometer.
22 is a graph of HPLC analysis results of homochiral peptide complexes prepared in Comparative Examples 3 and 4 and heterochiral peptide complexes prepared in Examples 5 to 7, and FIG. 22a is a homochiral peptide complex prepared in Comparative Example 3. , Figure 22b is a homochiral peptide complex prepared from Comparative Example 4, Figure 22c is a heterochiral peptide complex prepared from Example 5, Figure 22d is a heterochiral peptide complex prepared from Example 6, Figure 22e is Example 7 It is a heterochiral peptide complex prepared from
Figure 23 is a graph of HPLC analysis results of the heterochiral peptide complexes prepared from Examples 8 to 11, Figure 23a is the heterochiral peptide complex prepared from Example 8, Figure 23b is the heterochiral peptide complex prepared from Example 9, Figure 23c is a heterochiral peptide complex prepared from Example 10, Figure 23d is a heterochiral peptide complex prepared from Example 11.
Figure 24 is a graph of HPLC analysis results of heterochiral peptide complexes prepared from Examples 12 to 16, Figure 24a is a heterochiral peptide complex prepared from Example 12, Figure 24b is a heterochiral peptide complex prepared from Example 13, Figure 24c is a heterochiral peptide complex prepared from Example 14, Figure 24d is a heterochiral peptide complex prepared from Example 15, Figure 24e is a heterochiral peptide complex prepared from Example 16.
Figure 25 is a graph of HPLC analysis results of heterochiral peptide complexes prepared from Examples 17 to 20, Figure 25a is a heterochiral peptide complex prepared from Example 17, Figure 25b is a heterochiral peptide complex prepared from Example 18, 25c is a heterochiral peptide complex prepared in Example 19, and FIG. 25d is a heterochiral peptide complex prepared in Example 20.
26a is an AFM image of the heterochiral peptide complex (T-(vV) ₃ ) of Example 5, and FIG. 26b is an AFM image of the heterochiral peptide complex (T-(fF) ₃ ) of Example 6, 26C is an AFM image of the heterochiral peptide complex (T-(fF) ₄ ) of Example 7.
27a is an AFM image of the heterochiral peptide complex (Tw ₃ -W ₃ ) of Example 9, and FIG. 27b is an AFM image of the heterochiral peptide complex (T-(wW) ₂ -w ₂ ) of Example 10. 27c is an AFM image of the heterochiral peptide complex (TW ₂ -(wW) ₂ ) of Example 11, and FIG. 27d is an AFM image of the heterochiral peptide complex ((PEG3-PA) ₂ -(wW) of Example 15). ₂ ) is an AFM image for.
28a is an AFM image of the heterochiral peptide complex of Example 17 ((PEG10-PA)-V-(vV) ₂ ), and FIG. 28b is an AFM image of the heterochiral peptide complex of Example 18 ((PEG10-PA)-f ₃ -F ₃ ), and FIG. 28c is an AFM image of the heterochiral peptide complex ((PEG10-PA)-F-(fF) ₂ ) of Example 19.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

Until now, observation of the entire self-assembly process of peptides has been very limited. In the case of conventional NMR spectroscopy, it is difficult to measure multimolecular and large objects due to slow tumbling rates and short signal relaxation time. Furthermore, research and development on the self-assembly pathway of peptides is a field that has recently attracted attention, and most studies on peptide misfolding-related diseases such as amyloid β (Aβ) diseases are the majority.

Until the middle of the 20th century, unnatural D-amino acids were considered unnatural, that is, isomers that did not exist in nature. However, through various studies, it has been confirmed that it is a useful material in various fields. In particular, D-amino acids are mainly used for the development of protease-resistant peptides. In the present invention, heterochiral peptides were designed by combining these D-amino acids alternately with L-amino acids, which can obtain more diverse dihedral angles, Φ and Ψ, for the backbone than homochiral peptides. , can be folded or assembled in a unique way. For example, there is gramicidin having D-amino acid and L-amino acid, which are natural peptides obtained from microorganisms.

The forms of the self-assembled objects of the peptides are mostly limited to a few typical structures, specifically showing final self-assembled forms such as spherical micelles, cylindrical micelles, vesicles, planar membranes and nanotubes. Amyloid β (Aβ) and misfolded peptides also mostly assemble into typical shapes such as twisted ribbons, multi-stranded filaments, helical ribbons, nanotubes and crystals. Despite various studies on misfolding peptides, their morphological transitions and self-assembly intermediates are still in the early stages of research.

The present inventors have tried to develop a material capable of overcoming the limitations of a method for observing the self-assembly process of a self-assembly. As a result, a new peptide complex was derived, its function was identified, and the present invention was completed by confirming that there was a clear alignment effect when treated with an orientation mediator for RDC measurement.

One aspect of the present invention is a peptide represented by the following general formula 1 or 2; And a compound represented by Formula 1 bonded to the N-terminus of the peptide; It relates to a peptide complex comprising a.

[Formula 1]

[Y1] _a -{-[X1] _b -[Y2] _c } _d -[X2] _e

[Formula 2]

[X1] _f -{-[Y1] _g -[X2] _h } _i -[Y2] _j

In Equations 1 and 2 above

X1 and X2 are the same or different from each other, and are each independently any one selected from D-Trp, D-Phe, D-Tyr, D-Val, D-Leu, and D-Ile;

Y1 and Y2 are the same or different from each other, and are each independently any one selected from L-Trp, L-Phe, L-Tyr, L-Val, L-Leu, and L-Ile,

Wherein a, f, e and j are each independently any one integer selected from 0 to 2, b, c, g and h are each independently any one integer selected from 1 to 5, and d and i Each is an integer independently selected from 1 to 10.

[Formula 1]

In Formula 1, n and m are each independently an integer ranging from 1 to 12.

In Formula 1, n may be appropriately selected according to the length of the peptide for solubilization of the peptide complex, preferably, n may be an integer in the range of 1 to 5, more preferably an integer in the range of 1 to 3, , most preferably n may be 3.

The abbreviations used in the designation of amino acids and protecting groups used herein are based on terms recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB (Biochemistry, 11:1726-1732 (1972)). 'D-' used in the invention means an unnatural amino acid (UAA), D-Trp is D-Tryptophan, D-Phe is D-Phenylalanine, D-Tyr is D-Tyrosine, D-Val is D-Valine, D-Leu is D-Leucine, and D-Ile is D-Isoleucine ( D-Isoleucine). 'L-' means natural amino acid, L-Trp is L-Tryptophan, L-Phe is L-Phenylalanine, L-Tyr is L-Tyrosine ), L-Val is L-Valine, L-Leu is L-Leucine, and L-Ile is L-Isoleucine.

The term "peptide" as used herein refers to the full length of a peptide consisting of one or more amino acid residues according to the present invention. As a preferred embodiment, the term "peptide" includes isolated peptides and peptides prepared by recombinant methods, such as by isolation and purification from a sample, by protein synthesis by conventional methods, all of the above methods It is known to those skilled in the art. Preferably, the entire peptide or part thereof can be synthesized by a conventional synthesis method such as a solid-phase peptide synthesis method (Merrifield, R. B., J. Am. Chem. Soc., 85:2149-2154 ( 1963)). A single amino acid is sometimes referred to herein as an “amino acid residue or amino acid”.

Since the peptide of the present invention is synthesized at the C-terminus through solid-phase peptide synthesis, coupling between amino acid residues is completed to synthesize the peptide, and in this process, the compound represented by Formula 1 can also be coupled with the peptide. .

The peptides of the present invention exist as stereoisomers or mixtures of stereoisomers, for example, they may be formed by alternately repeating bonding of L-amino acids and D-amino acids. Preferably, the peptides of the present invention can be formed by alternately repeating bonds independently of each other, and depending on the number of asymmetric carbon atoms in which the isomers or isomeric mixtures exist, not only isomeric mixtures, but also racemates or diastereomeric mixtures, or diastereomers Alternatively, peptides having an enantiomer or asymmetric heterochiral structure may be obtained. Preferred structures of the peptides of Formulas 1 and 2 are heterochiral peptides in a racemic form or an asymmetric form.

A peptide of the invention is contemplated to contain at least one functional group attached to its N-terminus or C-terminus. Said at least one functional group may be attached to the peptide by any means known in the art, preferably covalently. In a preferred embodiment, the peptide may include one functional group covalently linked to its N-terminus and one functional group covalently linked to its C-terminus.

The functional group (preferably one N-terminal portion) bonded to the at least one N-terminus is H, acetyl (hereinafter referred to as Ac), butanoyl, hexanoyl, 2-methylhexanoyl, cyclohexanecarboxyl . However, since the compound represented by Formula 1 is bound to the N-terminus of the peptide in the present invention, the functional group binding site may be omitted.

The functional group (preferably one C-terminus) bonded to the at least one C-terminus is selected from H, -NR ₁ R ₂ - , -OR ₁ and -SR ₁ , wherein R ₁ and R ₂ are independently as H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclyl, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or It may be any one selected from unsubstituted aralkyl. In a most preferred embodiment, an NH ₂ functional group may be attached to at least one C-terminus (preferably one C-terminal site).

Therefore, more preferably, the peptide of the present invention comprises one site linked to the N-terminus and one site linked to the C-terminus, wherein the N-terminus is butanoyl or Ac, and the C-terminus is NH. is ₂ .

In the present invention, rare helical intermediates from slowly assembling species were discovered and identified for conformational space that crosses the chirality barrier of self-assembly of the peptides.

The present invention has confirmed the comprehensive content of the energy environment of peptide self-assembly, that is, when the peptide complex is present in the solution phase, it self-assembles slowly while being fixed to the structure of the final self-assembly and has a strong anisotropy. form Based on this information, a transformable NMR alignment medium was developed. The design concept of the slowly self-assembling heterochiral peptide complex newly developed in the present invention is as follows.

The heterochiral peptide complex of the present invention includes a racemic peptide or an asymmetric racemic peptide composed of alternating D- and L-amino acids, and a compound represented by Formula 1 at one end of the peptide.

1 is a diagram showing the three-dimensional structure of the heterochiral peptide complex of the present invention. As shown in the right diagram of FIG. 1A, conventional linear homochiral peptides (all composed of L or D-type amino acids) , a conformationally stable state is achieved when the side chains are placed on opposite sides of the peptide backbone, with alternating amide bonds at 180° to each other.

As shown in the left diagram of FIG. 1A, in the case of linear heterochiral peptides in which D/L-amino acids are alternately bonded, the molecule will be in a distorted form to relieve torsional strain from adjacent side chains. Likely, it has a conformation that is less optimized for efficient non-covalent bond formation (particularly hydrogen bonds) than its homochiral counterparts. Due to this morphological difference, homochiral peptides form aggregates with stronger anisotropy than heterochiral peptides.

Based on the foregoing, the present invention considered that self-assembly intermediates could be identified when heterochiral peptides aggregate slowly enough to be observed directly under a microscope. Accordingly, the present invention designed the peptide complex, specifically, the peptide complex has the structure of an amphiphilic building block, and the peptide represented by the following general formula 1 or 2 containing heterochiral amino acids forms a beta sheet. It is a hydrophobic segment, and the compound represented by Formula 1 bonded to one end of the peptide may be referred to as a hydrophilic segment (FIG. 1b). Therefore, the peptide complex with the peptide and the compound of the present invention is self-assembled in solution by the peptide having a beta sheet structure through several hydrogen bonds between chains, and the helical structure arrangement in the M and P directions increases and becomes rigid. It is bound and has thermal and physical stability.

In the preferred embodiment, in Formulas 1 and 2, X1 and X2 are the same or different from each other, and are each independently any one selected from D-Trp, D-Val, and D-Phe, and Y1 and Y2 are They may be the same or different from each other, and may be any one selected from L-Trp, L-Val, and L-Phe each independently.

More preferably, in Formulas 1 and 2, X1 and X2 are the same or different from each other, each independently any one selected from D-Trp, D-Val and D-Phe, and Y1 and Y2 are the same or different from each other. different, and may be any one selected from L-Trp, L-Val, and L-Phe each independently.

In a more preferred embodiment, X1 may be the same as X2, and Y1 may be the same as Y2.

For solubilization of the peptide complex, the ratio of the hydrophobic segment to the hydrophilic segment may be determined, and a preferred structure of the peptide complex of the present invention considering solubilization is as follows. In Formulas 1 and 2, a, f, e, and j are each independently an integer selected from 0 to 2, and b, c, g, and h are each independently selected from 1 to 3. It is an integer, and d and i may each independently be any one integer selected from 1 to 4.

In the peptide complex, the peptide may be any one selected from the heterochiral peptide sequences represented by SEQ ID NOs: 1 to 36, more preferably any one selected from the heterochiral peptide sequences represented by SEQ ID NOs: 1 to 12 It may be, more preferably any one selected from the heterochiral peptide sequences represented by 1, 2, 4, 5, 7, 8, 10, 11, most preferably 1, 4, 7, It may be any one selected from the heterochiral peptide sequence represented by 10.

[SEQ ID NO: 1]

(D)Trp-(L)Trp-(D)Trp-(L)Tre-(D)Trp-(L)Trp

[SEQ ID NO: 2]

(D)Val-(L)Val-(D)Val-(L)Val-(D)Val-(L)Val

[SEQ ID NO: 3]

(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe

[SEQ ID NO: 4]

(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp

[SEQ ID NO: 5]

(L)Val-(D)Val-(L)Val-(D)Val-(L)Val-(D)Val

[SEQ ID NO: 6]

(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe

[SEQ ID NO: 7]

(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp

[SEQ ID NO: 8]

(L)Val-(D)Val-(L)Val-(D)Val-(L)Val

[SEQ ID NO: 9]

(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe

[SEQ ID NO: 10]

(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp

[SEQ ID NO: 11]

(D)Val-(L)Val-(D)Val-(L)Val-(D)Val

[SEQ ID NO: 12]

(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe

In the peptide sequence, D means 'D-amino acid residue' and L means 'L-amino acid residue'.

In the peptide conjugate, the compound may be represented by Formula 2 below.

[Formula 2]

In Formula 2, m is an integer ranging from 1 to 12.

Therefore, the most preferred example of the peptide complex according to the present invention may be any one selected from those represented by Formulas 3 to 6 below.

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

In Formulas 3 to 6, m is an integer in the range of 1 to 5, more preferably an integer in the range of 2 to 5.

The peptides of the present invention may be synthesized and produced by any means known in the art. For example, they can be synthesized and produced by chemical synthesis (preferably by solid phase peptide synthesis) expressing the peptides in cell culture or by transgenic production of the peptides in plants or animals. In addition, the peptides of the present invention can be purified by any means known in the art.

As is clear from the examples included below, the peptide complex of the present invention forms self-assembly slowly in solution, and self-assembles in solution through the same principle as the structure formation principle of proteins, so through this, conventional peptides or proteins or The overall process of self-assembly of amyloid can be inferred. In addition, as can be derived from the examples included below, the peptide complex of the present invention is biocompatible, easy to prepare, has excellent structural stability, and is maintained as a self-assembly intermediate for a long time, so that the RDC of NMR spectroscopy It has a direct effect as an orientation medium at the time of measurement. Therefore, the self-assembly intermediate of the peptide complex of the present invention has strong anisotropy, which can improve alignment properties, and has excellent structural properties against environmental factors such as heat, acidity, and ion concentration because it is based on a beta sheet structure. Additional or alternative peptide complexes may be provided that may provide stability.

The peptide complex may gradually form a self-assembly in a solution, pass through an intermediate self-assembly having a specific crystal structure, and finally form a self-assembly having a flat ribbon crystal structure or a porous superhelical network structure. The intermediates of the self-assembly and the structure of the self-assembly vary depending on the peptide sequence constituting the peptide complex and the chain length of the compound.

For example, the peptide complex slowly forms a self-assembly in solution, but in Formulas 1 and 2, when a, f, e, and j are 0, a right-handed fashion over time (P-helix) and in the left-handed fashion (M-helix) twisted fiber structure, the helix portion of the fiber is laterally extended (fattened) to form an intermediate of a flat ribbon fiber structure, and finally It may be formed of a self-assembly of a flat ribbon crystal structure.

In Formulas 1 and 2, when a, f, e, and j are not 0 at the same time, a right-handed fashion (P-helix) and a left-handed fashion ( A self-assembling intermediate consisting of fibers composed of variable pitch spirals twisted into M-helix; and fibers having a structure similar to a sausage string due to superhelical fibers formed by excessive twisting and entanglement of the fibers. It may be to form a self-assembly of a porous superhelical network structure formed by extensive intertwining and overtwisting.

The structure of the self-assembly intermediate or the final self-assembly formed by the peptide complex of the present invention may be different, but since all of the peptide complexes of the present invention gradually form self-assembly intermediates, and all of the self-assembly intermediates have strong anisotropy, subsequent NMR analysis It can act as an alignment medium enabling accurate measurement of residual dipole bonds (NMR-RDC). That is, the peptide complex of the present invention facilitates accurate measurement of NMR-RDC, making it feasible to determine the structure of a wide range of biomedical important molecules that are difficult to characterize. In addition, it can be used as a drug delivery system and a scaffold for cell culture.

Another aspect of the present invention relates to a self-assembly intermediate formed through self-assembly of the peptide complex.

The peptide complexes are slowly self-assembled in solution, and self-assembly intermediates can be identified. Specifically, according to the heterochiral peptide of the peptide complex, the self-assembling intermediate of the peptide complex is twisted in a right-handed fashion (P-helix) and a left-handed fashion (M-helix). In the fiber structure, the helical portion of the fiber is fattened to form an intermediate of a flat ribbon fiber structure or a right-handed fashion (P-helix) and a left-handed fashion (M-helix). Having a self-assembling intermediate composed of fibers composed of variable pitch helices twisted into a helix; and fibers having a structure similar to a sausage string due to superhelical fibers formed by excessive twisting and entanglement of the fibers. characterized by At this time, the P-helical type and the M-helical type were present in almost the same ratio.

Another aspect of the present invention relates to an orientation medium composition for experimental analysis of residual dipolar coupling constant (RDC) including the peptide complex.

In addition, the present invention provides a new use for the peptide complex as an orientation mediator for residual dipolar coupling constant (RDC) experimental analysis.

The advantage of the NMR method is that no crystals are required and data can be obtained in solution. Like X-ray crystallography, the NMR method cannot directly obtain the structure of biomolecules such as proteins, but determines its tertiary structure through computational simulation from experimental data. Specifically, in the NMR experiment, protons and neutrons in the nucleus of an atom constituting a biomolecule have a spin angular momentum of S = 1/2, so each has a unique magnetic moment. If the number of protons or neutrons is odd, the magnetic moment of the entire nucleus has a value other than zero, and energy levels are split by an external magnetic field. This energy split occurs in external electromagnetic waves (mainly radio waves). ) and resonance. Using this phenomenon, in particular, the spectrum of hydrogen (H) is measured, and by analyzing it, information on the distance between the various hydrogen atoms constituting the protein and the dihedral angle of the protein backbone and side-chain are obtained. Information is obtained as a constraint. In particular, the distance information between hydrogen atoms that plays a central role in determining the structure is called a Nuclear Overhauser Effect (NOE) distance restraint.

Although various experiments are used in the NMR experiment to determine the three-dimensional structure of a biomolecule, in the case of a biomolecule of less than 20 kDa, since there is a limitation in ROSY (transverse relaxation-optimized spectros) analysis, the biomolecule backbone, side -Residual Dipolar Coupling Constant (RDC) measurement experiments that can determine the three-dimensional orientation between chains and each domain are used.

To measure RDC, when a lipid such as a bicell is inserted between biomolecules in an aqueous solution and a magnetic field is applied, the biomolecules tend to align along the alignment of the bicells according to the magnetic field. Through this, the nuclear spins of two atoms located in each bond are constantly aligned, resulting in dipole coupling, and energy divergence is observed due to resonance according to the frequency of the external magnetic field. This is called Residual Dipolar Coupling (RDC), and through this, information about the direction between protein domains is obtained.

As mentioned above, conventionally, as an alignment medium, lipids such as bicell, nematic liquid crystal molecules, compressed or stretched polymer gel, and Pf1 filamentous phage is known. However, the alignment mediators have problems such as limitations in conditions such as specific temperature, pH, and solvent, low structural stability, and difficulty in manufacturing.

In contrast, the peptide complex of the present invention is easy to prepare, biocompatible, independent of temperature, pH and solvent conditions, and has structural stability. In addition, the peptide complex of the present invention forms an intermediate having strong anisotropy through self-assembly in a solution without a separate process, thereby inducing orientation stabilization of biomolecules.

In addition, the peptide complex of the present invention is based on peptides having a beta sheet structure and is tightly bound, so it has high thermal and physical stability, excellent solubility in solvents used for NMR measurement, and excellent orientation with biomolecules. . This is a remarkably superior effect that cannot be achieved in homochiral peptide-based complexes (Comparative Examples 1 and 2) having the same final self-assembling structure as the peptide complex of the present invention.

Therefore, the self-assembling intermediate of the peptide complex of the present invention can effectively act as an alignment medium enabling accurate measurement of NMR analysis, particularly RDC.

Another aspect of the present invention is to provide a method for performing NMR analysis of a biomolecule comprising the following steps.

(a) mixing the peptide complex in a solution phase;

(b) adding a sample containing a biomolecule to be measured to the mixed solution; and

(c) performing NMR measurement on the mixture of the sample and the peptide complex;

The method of the present invention is a method for performing NMR, particularly RDC analysis of biomolecules. The method induces the orientation of biomolecules by mixing a peptide complex in a solution and forming a self-assembling intermediate having anisotropy through this. , By performing NMR spectroscopy on this, it is possible to analyze the three-dimensional structure of biomolecules.

The NMR measurement may be performed with any one selected from the group consisting of ¹ H, ¹ H edit, and 2D NMR, but is not particularly limited to the NMR, and any commonly used one may be used without limitation.

The 2D NMR may also be commonly used, preferably from the group consisting of residual dipolar coupling (RDC), 1H-1H COZY (Correlated Spectroscopy) and 1H-13C HSQC (Heteronuclear Single Quantum Coherence spectroscopy) It may be one or more selected ones.

To perform the NMR, the peptide complex and the biomolecule are sequentially mixed in a mixed solvent, and the mixed solvent may be a mixture of a deuterium solvent and a solvent. The deuterium solvent is Chloroform-d (CDCl3), Deuterium Oxide (D ₂ O), DMSO-d6 (Dimethyl Sulfoxide-d6), DMF (N, N-dimethyl-foramide) -d7, NMP (N-methylpyrrolidone) -d9 And it may be any one or more selected from mixtures thereof, and distilled water or methanol may be used as the solvent.

The mixing volume ratio of the deuterium solvent and the solvent in the mixed solvent may be 1: 1-10, preferably 1: 4-10, more preferably 1: 6-10, most preferably It may be mixed in a volume ratio of 1:9.

Biomolecules may be sampled using the mixed solvent. The sampling means pretreatment, and NMR experiments can be performed by adding the peptide complex of the present invention together with the sample.

The order of addition to the mixed solvent is not particularly limited, and the peptide complex and the biomolecule may be added sequentially or simultaneously.

The biomolecule may be any one or more selected from the group consisting of peptides, proteins, nucleic acids, and polysaccharides, and is not particularly limited thereto. For example, nucleic acids may be selected from the group consisting of DNA, RNA, PNA, LNA and hybrids thereof, and proteins may be selected from the group consisting of enzymes, substrates, antigens, antibodies, ligands, aptamers and receptors, A peptide refers to a polymer in the form of a chain formed by binding a plurality of amino acid residues to each other by peptide bonds, and is interchangeable with 'polypeptide', 'oligopeptide' and 'protein'.

Another aspect of the present invention relates to a drug delivery system comprising the peptide complex.

In addition, the present invention provides a new use for the peptide conjugate as a delivery system for drug delivery.

The drug delivery system of the present invention stably entraps the drug in the beta sheet and the three-dimensional network structure through the self-assembly process of the peptide complex, or entraps the hydrophobic drug in the hydrophobic inner region of the self-assembly body, It prevents aggregation or denaturation, can maintain the activity of the drug more stably for a long period of time, and has the effect of structurally stabilizing the drug and increasing the half-life.

In the present invention, "hydrophobic" refers to a property that does not easily bind to water molecules, does not dissolve well in water, or is non-polar. The term “hydrophobic” may be used interchangeably with the term “lipophilic”. Hydrophobic substances can be classified as follows according to water solubility. Slightly soluble: 1 to 10 mg/mL; very slightly soluble: 0.1 to 1 mg/ml; Practically insoluble: <0.1 mg/mL.

In the present invention, the drug is not particularly limited thereto, but is preferably a hydrophobic drug, and the hydrophobic drug may include a chemical substance or biodrug having a water solubility of about 10 mg/ml or less. For example, the hydrophobic drug may be an anthracycline-based substance, a hydrophobic glucocorticoid, a steroid-based substance, a taxane-based drug, a cyclic peptide-based drug, or a combination thereof. The anthracycline-based substance may be doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, or a combination thereof. Hydrophobic glucocorticoids include, for example, dexamethasone, trimacinolone, beclomethasone diproprionate, triamcinolone acetonide, triamcinolone diacetate, betamethasone diproprionate, testosterone (Testosterone), budesonide, 17α-ethynylestradiol, levonorgestrel, fluticasone proprionate, or a combination thereof. For example, hydrophobic drugs include sorafenib, paclitaxel, docetaxel, doxorubicin, cyclosporine A, amphotericin B, indinavir ), Rapamycin, Doxorubicin, Coenzyme Q 10, Ursodeoxycholic acid, Ilaprazole, Imatinib Mesilate, Tanespimycin ( Tanespimycin), or a combination thereof. The contrast agent (imaging agent or contrast media) increases the contrast of the image by artificially increasing the X-ray absorption difference of each tissue so that tissues or blood vessels can be seen well during examinations such as magnetic resonance imaging or computed tomography. refers to matter The contrast agent may be, for example, a transition element or a chelate complex of a transition element.

The drug delivery system according to the present invention has structural stabilization and half-life enhancement effects of protein or peptide drugs, is a sustained release type that slowly releases the drug, and has biocompatibility and biodegradability.

The concentration of the drug entrapped in the drug delivery system is not particularly limited, and is preferably higher than the concentration capable of exerting an effect in vivo when released from the drug delivery system of the present invention. It is preferably below the concentration at which excessive aggregation or inactivation occurs. The concentration of such a drug may vary depending on the specific type, and may be, for example, 0.01 mg/ml to 1000 mg/ml. In particular, since there is a possibility of forming drug-to-drug aggregates during preparation of an aqueous drug solution, it is preferable to dissolve the drug slowly without stirring.

Compared to the case where the drug was encapsulated in the peptide complex of Comparative Examples 1 and 2, which self-assembles immediately instead of slowly self-assembling like the peptide complex according to the present invention, when a drug delivery system was prepared using the peptide complex of the present invention , can have a better drug weight ratio and drug encapsulation rate. In the process of self-assembly of the peptide complex, the internal drug may be protected, thereby minimizing drug loss.

Another aspect of the present invention relates to a scaffold for cell culture comprising the peptide complex.

In addition, the present invention provides a new use for the peptide complex as a scaffold for cell culture.

In this specification, "scaffold" is a term used in tissue engineering, and refers to a structure that uses a combination of cells and various materials by placing living cells therein, and a bio-scaffold is a biological structure, that is, It refers to a structure (cast) that leaves only the outline of the microstructure and organ after removing cells by decellularization of living organs. In the present invention, in particular, the peptide complex may be configured in the form of a beta sheet or a three-dimensional network formed by self-assembly.

The peptide complex according to the present invention forms a beta sheet through self-assembly in a solution and forms a three-dimensional network structure therethrough, thereby providing a biodegradable structure that can preferably provide a place for transplanting and growing biological cells. Therefore, it can have an excellent effect as a scaffold.

The peptide conjugate according to the present invention is not cytotoxic and can easily induce proliferation without killing living cells. Therefore, it can be used as a scaffold, etc. for making biological tissue for treatment using living cells. Furthermore, it can be used as a scaffold for the production of various artificial organs such as artificial skin for transplantation, artificial hair, and artificial kidney.

In the present invention, "regeneration" generally refers to an action of restoring a part of a tissue or organ to an original state or restoring its function when a part of a tissue in an organism is damaged or loses its function. Due to the nature of the present invention, the regeneration may include all processes of restoring a biological tissue to its original state or restoring its function when a biological tissue is damaged or loses its function.

In addition, the peptide complex as described above may be used by being coated on the surface of an artificial organ, such as an artificial joint.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are intended to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

<Test material>

Protected Fmoc amino acid derivatives and all D- and L-forms of 12-(Fmoc-amino)-4,7,10-trioxadodecanoic acid (Fmoc-PEG3-propionic acid) or Fmoc-amino-PEG10-propionic acid are Anaspec ( USA) and AAPPTec (USA) were purchased and used. Rink Amide MBHA resin LL was purchased from Novabiochem (Germany) and used.

1-Hydroxybenzotriazole (HOBt) and O-(6-chlorobenzotriazol-1-yl)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HCTU) were purchased from Anaspec and AAPPTec. All other common chemicals including N-methyl-2-pyrrolidone (NMP) and dimethylformamide (DMF) were purchased from Thermo Fisher Scientific (USA) and Samchun (Korea).

<Experiment method>

1. CD(Circular Dichroism)

The CD spectrum of the peptide complex in aqueous solution was measured using a Chirascan TM CD spectrometer. The peptide complex to be measured was adjusted to a concentration of 20 μM in DI distilled water, and then incubated at room temperature for 2 hours. The CD spectrum was measured in the range of 250 nm to 190 nm using a 2 mm path-length cuvettefmf under the condition of 25 °C. A total of three measurements were taken, the average was obtained, and the spectrum was corrected and measured using distilled water without any background as a background value.

2. Atomic Force Microscopy (AFM)

Each peptide complex sample to be measured was diluted to 20 μM, and 10 μl was dropped onto a freshly cleaved mica surface. After incubation for 1 minute, the mica plate was washed with 100 μl of doubly distilled water, and then the remaining liquid was gently removed using a filter paper. After complete drying at room temperature, AFM was measured. The AFM scan was performed in a noncontact mode (NCM) using a Park NX10 instrument (Park Systems, Korea). Scanning was performed under conditions of a scan speed of 0.3 Hz and a Z-gain of 1.

3. Wide-angle X-ray Scattering (WAXS)

Wide-angle X-ray scattering analysis was performed using the 4C SAXS II beamline (BL) of Pohang Accelerator Laboratory (PAL, Korea). As the beam source, a vacuum undulator 20 of the Pohang Light Source II storage ring was used, and a vertical toroidal focusing mirror was used to deliver an X-ray beam of 0.675 Å. A 100 μL sample solution was measured by mounting it in a quartz microcapillary tube with a thickness of 10 μm and a length of 80 mm. Samples were irradiated at room temperature for an exposure time of 5 seconds. At this time, the concentrations of samples such as Comparative Examples 1 and 2 and Example 1 (T-W6, T-w6 and T-(wW) ₃ ) were all used at 3.0 mg/mL (other samples to be measured were also 3.0 mg/mL). It was used after dilution or concentration to mg/ml). X-ray scanning patterns were collected using a charge-coupled detector (Mar USA, Inc.) (0.25 Å-1 < q < 2.00 Å-1) positioned 20 cm away from the sample.

Data were captured in 6 consecutive frames of 0.1 min each to minimize damage. Each 2D scattering pattern was circularly averaged at the beam focus and normalized to the intensity of the transmitted X-ray beam. Transmitted beam intensity was monitored with a scintillation counter. The scattering pattern of water was used as a background value.

4. Nuclear Magnetic Resonance Spectroscopy (NMR)

² H-NMR spectrum was measured using a Bruker System 700 Ascend equipped with a TXI-Probe (5 mm) with a z-gradient ( ² H:107.48 MHz) at 298 K. All measurements were performed in an aqueous environment with 10% (v/v) D ₂ O. The parameters of the specific NMR measurement are as follows:

Time Domain (TD) = 65536

Number of Scans (NS) = 1

Spectral Width (SW) = 24 [ppm]

Acquisition time (AQ) = 12.7 [sec]

Nucleus = 2H

Transmitter Freq. offset = 516.86 [Hz] / 4.809 [ppm]

Chemical shifts (δ) are reported in parts per million (ppm) and are relative to residual solvent protons. Each sample measured over the 0-24 ppm (SW) range. Coupling constants (J) were calculated in Hertz (Hz). All samples were concentrated or diluted to a concentration of 8 mg/ml.

All samples were heated at 95 °C for 1 hour and slowly cooled in a water bath overnight (annealing process). Samples of Comparative Examples 1 and 2 and Example 1 (T-W6 and T-(wW) ₃ ) were cultured for 2 hours or 2 weeks and measured.

5. Molecular Modeling and Mechanistic Analysis

(1) 3D modeling and optimization (Model Systems and Energy Minimization)

For 3D modeling and optimization, Maestro 11 software (Schrodinger, LLC) was used. (wW) ₃ ((D)Trp-(L)Trp-(D)Trp-(L)Tre-(D)Trp-(L)Trp: SEQ ID NO: 1) The three-dimensional model of the peptide was prepared by the following two methods. Confirmed.

The first simulation model fixed the peptide structure of (wW) ₃ ((D)Trp-(L)Trp-(D)Trp-(L)Tre-(D)Trp-(L)Trp: SEQ ID NO: 1) without restriction. The dihedral angles (Φ = -180°, ψ = -180°) were arbitrarily extended. The second model used the structure of a DL-substituted poly(tyrosine) (Protein Data Bank entry 1UNO) to produce a three-dimensional structure in which all side chains at each position were replaced with D- and L-Trp residues. The replacement created a 3D structure.

All shapes were energetically optimized by the Polak-Ribiere conjugate gradient (PRCG) method and the truncated Newton conjugate gradient (TNCG) method. The process was repeated until the gradient was converged using AMBER as the force field (convergence threshold of 0.05) at each step.

(2) Molecular Dynamics (MD)

Using two strands of the energy-optimized structure, a group of structures similar to parallel strand formation such as β-sheets was roughly generated based on 1D-WAXS distance information (interstrand distance of 6 Å). The simulation was performed three times for a total of 50 ns for each model. Measurements were made with an absolute solvent system using AMBER as the force field. The energy for each interaction was calculated using the generalized Born model. In the Born calculation, the dielectric constants of the inside and outside of the molecule were set to 1.0 and 78.5, respectively.

A volume-based continuum solvation model (GB/SA model) used a polar component. At this time, the bond length for hydrogen was limited to the equilibrium length using the SHAKE component. Cut-off distance parameters for non-covalent interactions such as van der Waals, electrostatic interactions and hydrogen bonding were set to 8.0 Å, 20.0 Å and 4.0 Å, respectively. The time integration step was 1.0 fs, and the interval was set to 5.0 ps for equilibrium in all simulation processes. Calculations were performed at 298 K without any symmetry restrictions or constraints to make the sample state constant at 25 °C.

<Example 1. Heterochiral peptide complex, T-(wW) ₃ synthesis>

The PEGylated poly-Trp peptide ((D)Trp-(L)Trp-(D)Trp-(L)Tre-(D)Trp-(L)Trp) (SEQ ID NO: 1) was synthesized according to the standard Fmoc protocol. Specifically, the peptide complex was synthesized by extending from the surface of Rink amide MBHA resin LL as a solid support (resin substitution 0.36 mmol/g, synthesis scale 0.1 mmol). Before starting the synthesis, the rink amide resins were soaked in NMP for 30 minutes to preswollen. All Fmoc groups were deprotected by treatment with NMP containing 20% piperidine for 20 minutes. Before starting the coupling reaction, all amino acid derivatives and Fmoc-PEG3-PA (Fmoc-PEG3-propionic acid) were mixed with 4.5 equivalents (relative to the reaction scale) of HCTU and HOBt and 10 equivalents of diisopropylethylamine (DIPEA). ) for 5 minutes with a DMF solvent containing, and activated. Each coupling step was performed for 30 minutes, and each amino acid and PEG3-PA or PEG10-PA were sequentially coupled and synthesized according to the design of the peptide complex. At this time, (PEG3-PA) ₃ was also expressed as 'T' as shown in FIG. 2 . After performing the final deprotection procedure, the peptide was treated with a cleavage cocktail mixture (trifluoroacetic acid (TFA): triisopropylsilane (TIS): water = 92.5: 2.5: 2.5) at room temperature for 4 hours. Through this, the peptide was separated from the peptidyl resin. The separated product was triturated with tert-butylmethylether (TBME), and then purified by RP-HPLC chromatography using a C4 (Waters) column with a 0.1% TFA-H2O/CH3CN (ACN) solvent system. (FIG. 4c). The purified heterochiral peptide complex (T-(wW) ₃ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. shown in

The heterochiral peptide complex (T-(wW) ₃ ) was lyophilized and stored until the experiment.

<Example 2. Heterochiral peptide complex, T-(Ww) ₃ synthesis>

PEGylated (L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D) except that the sequence was designed and synthesized as follows in Example 1. ) Trp (SEQ ID NO: 4) was prepared. The prepared heterochiral peptide complex (T-(Ww) ₃ ) was purified by RP-HPLC chromatography in the same manner as in Example 1 (FIG. 4d). The purified heterochiral peptide complex (T-(Ww) ₃ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. shown in

The heterochiral peptide complex (T-(Ww) ₃ ) was lyophilized and stored until the experiment.

<Example 3. Heterochiral peptide complex, T-W-(wW) ₂ synthesis>

PEGylated (L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp (SEQ ID NO: 7) except that the sequence was designed and synthesized as follows in Example 1. ) was prepared. The prepared heterochiral peptide complex (TW-(wW) ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1 (FIG. 4e). The purified heterochiral peptide complex (TW-(wW) ₂ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. shown in

The heterochiral peptide complex (TW-(wW) ₂ ) was lyophilized and stored until the experiment.

<Example 4. Heterochiral peptide complex, T-w-(Ww) ₂ synthesis>

PEGylated (D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp (SEQ ID NO: 10) except that the sequence was designed and synthesized as follows in Example 1. ) was prepared. The prepared heterochiral peptide complex (Tw-(Ww) ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1 (FIG. 4f). The purified heterochiral peptide complex (Tw-(Ww) ₂ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. shown in

The heterochiral peptide complex (Tw-(Ww) ₂ ) was lyophilized and stored until the experiment.

<Example 5. Heterochiral peptide complex, T-(vV) ₃ synthesis>

PEGylated (D)Val-(L)Val-(D)Val-(L)Val-(D)Val-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Val (SEQ ID NO: 2) was prepared. The prepared heterochiral peptide complex (T-(vV) ₃ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (T-(vV) ₃ ) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 22c.

The heterochiral peptide complex (T-(vV) ₃ ) was lyophilized and stored until the experiment.

<Example 6. Heterochiral peptide complex, T-(fF) ₃ synthesis>

PEGylated (D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Phe (SEQ ID NO: 3). The prepared heterochiral peptide complex (T-(fF) ₃ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (T-(fF) ₃ ) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 22D.

The heterochiral peptide complex (T-(fF) ₃ ) was lyophilized and stored until the experiment.

<Example 7. Heterochiral peptide complex, T-(fF) ₄ synthesis>

PEGylated (D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L) except that the sequence was designed and synthesized as follows in Example 1. )Phe-(D)Phe-(L)Phe (SEQ ID NO: 15) was prepared. The prepared heterochiral peptide complex (T-(fF) ₄ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (T-(fF) ₄ ) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 22e.

The heterochiral peptide complex (T-(fF) ₄ ) was lyophilized and stored until the experiment.

<Example 8. Heterochiral peptide complex, T-w-(W) ₂ -(w) ₂ -W synthesis>

PEGylated (D)Trp-(L)Trp-(L)Trp-(D)Trp-(D)Trp-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Trp (SEQ ID NO: 16) was prepared. The prepared heterochiral peptide complex (Tw-(W) ₂ -(w) ₂ -W) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (Tw-(W) ₂ -(w) ₂ -W) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 23a. showed up

The heterochiral peptide complex Tw-(W) ₂ -(w) ₂ -W) was lyophilized and stored until the experiment.

<Example 9. Heterochiral peptide complex, T-w ₃ -W ₃ synthesis>

PEGylated (D)Trp-(D)Trp-(D)Trp-(L)Trp-(L)Trp-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Trp (SEQ ID NO: 19) was prepared. The prepared heterochiral peptide complex (Tw ₃ -W ₃ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (Tw ₃ -W ₃ ) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 23B.

The heterochiral peptide complex (Tw ₃ -W ₃ ) was lyophilized and stored until the experiment.

<Example 10. Heterochiral peptide complex, T-(wW) ₂ -w ₂ synthesis>

PEGylated (D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp-(D) except that the sequence was designed and synthesized as follows in Example 1. )Trp (SEQ ID NO: 22) was prepared. The prepared heterochiral peptide complex (T-(wW) ₂ -w ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (T-(wW) ₂ -w ₂ ) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 23c.

The heterochiral peptide complex (T-(wW) ₂ -w ₂ ) was lyophilized and stored until the experiment.

<Example 11. Heterochiral peptide complex, TW ₂ - (wW) ₂ synthesis>

PEGylated (L)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Trp (SEQ ID NO: 25) was prepared. The prepared heterochiral peptide complex (TW ₂ -(wW) ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (TW ₂ -(wW) ₂ ) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 23D.

The heterochiral peptide complex (TW ₂ -(wW) ₂ ) was lyophilized and stored until the experiment.

<Example 12. Heterochiral peptide complex, (PEG3-PA) ₄ -(wW) ₃ synthesis>

PEGylated (D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Trp (SEQ ID NO: 1) was prepared. The prepared heterochiral peptide complex ((PEG3-PA) ₄ -(wW) ₃ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ((PEG3-PA) ₄ -(wW) ₃ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 24a. .

In the present invention, '(PEG3-PA) ₄ ' means {(CH2CH2O) ₃ CH2CH2C(O)} ₄ *, and specifically means a compound in which n is 3 and m is 4 in Formula 1.

The heterochiral peptide complex ((PEG3-PA) ₄ -(wW) ₃ ) was lyophilized and stored until the experiment.

<Example 13. Heterochiral peptide complex, (PEG3-PA) ₄ -(wW) ₄ synthesis>

PEGylated (D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L) except that the sequence was designed and synthesized as follows in Example 1. )Trp-(D)Trp-(L)Trp (SEQ ID NO: 13). The prepared heterochiral peptide complex ((PEG3-PA) ₄ -(wW) ₄ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ((PEG3-PA) ₄ -(wW) ₄ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 24B. .

The heterochiral peptide complex (T ₄ -(wW) ₄ ) was lyophilized and stored until the experiment.

<Example 14. Heterochiral peptide complex, (PEG3-PA) ₄ -W-(wW) ₃ synthesis>

PEGylated (L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp in the same manner as in Example 1 except that the sequence was designed as follows. -(L)Trp (SEQ ID NO: 28) was prepared. The prepared heterochiral peptide complex ((PEG3-PA) ₄ -W-(wW) ₄ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ((PEG3-PA) ₄ -W-(wW) ₄ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 24c. showed up

The heterochiral peptide complex ((PEG3-PA) ₄ -W-(wW) ₄ ) was lyophilized and stored until the experiment.

<Example 15. Heterochiral peptide complex, (PEG3-PA) ₂ -(wW) ₂ synthesis>

PEGylated (D)Trp-(L)Trp-(D)Trp-(L)Trp (SEQ ID NO: 31) was prepared in the same manner as in Example 1 except that the sequence was designed and synthesized as follows. The prepared heterochiral peptide complex ((PEG3-PA) ₂ -(wW) ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ((PEG3-PA) ₂ -(wW) ₂ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 24D. .

The heterochiral peptide complex ((PEG3-PA) ₂ -(wW) ₂ ) was lyophilized and stored until the experiment.

<Example 16. Heterochiral peptide complex, (PEG3-PA) ₂ -w ₂ -W ₂ synthesis>

PEGylated (D)Trp-(D)Trp-(L)Trp-(L)Trp (SEQ ID NO: 34) was prepared in the same manner as in Example 1 except that the sequence was designed and synthesized as follows. The prepared heterochiral peptide complex ((PEG3-PA) ₂ -w ₂ -W ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ((PEG3-PA) ₂ -w ₂ -W ₂ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 24e. was

The heterochiral peptide complex ((PEG3-PA) ₂ -w ₂ -W ₂ ) was lyophilized and stored until the experiment.

<Example 17. Heterochiral peptide complex, (PEG10-PA)-V-(vV) ₂ synthesis>

PEGylated (L) Val- (D) Val- (L) Val- (D) Val- (L) Val (SEQ ID NO: 8 ) was prepared. The prepared heterochiral peptide complex ((PEG10-PA)-V-(vV) ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ( ( PEG10-PA)-V-(vV) ₂ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 25a. was

In the present invention, '(PEG10-PA)' means {(CH2CH2O) ₁₀ CH2CH2C(O)}*, and specifically means a compound in which n is 10 and m is 1 in Formula 1.

The heterochiral peptide complex ((PEG10-PA)-V-(vV) ₂ ) was lyophilized and stored until the experiment.

<Example 18. Heterochiral peptide complex, (PEG10-PA)-f ₃ -F ₃ synthesis>

PEGylated (D)Phe-(D)Phe-(D)Phe-(L)Phe-(L)Phe-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Phe (SEQ ID NO: 21). The prepared heterochiral peptide complex ((PEG10-PA)-f ₃ -F ₃ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ((PEG10-PA)-f ₃ -F ₃ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 25B. .

The heterochiral peptide complex ((PEG10-PA)-f ₃ -F ₃ ) was lyophilized and stored until the experiment.

<Example 19. Heterochiral peptide complex, (PEG10-PA)-F-(fF) ₂ synthesis>

PEGylated (L) Phe-(D) Phe-(L) Phe-(D) Phe-(L) Phe (SEQ ID NO: 9) except that the sequence was designed and synthesized as follows in Example 1. ) was prepared. The prepared heterochiral peptide complex ((PEG10-PA)-F-(fF) ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ((PEG10-PA)-F-(fF) ₂ ) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and the results are shown in FIG. 25C. was

The heterochiral peptide complex ((PEG10-PA)-F-(fF) ₂ ) was lyophilized and stored until the experiment.

<Example 20. Heterochiral peptide complex, (PEG10-PA)-(fF) ₂ synthesis>

PEGylated (D)Phe-(L)Phe-(D)Phe-(L)Phe (SEQ ID NO: 33) was prepared in the same manner as in Example 1 except that the sequence was designed and synthesized as follows. The prepared heterochiral peptide complex ((PEG10-PA)-(fF) ₂ ) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex ((PEG10-PA)-(fF) ₂ ) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 25D.

The heterochiral peptide complex ((PEG10-PA)-(fF) ₂ ) was lyophilized and stored until the experiment.

<Comparative Example 1. Preparation of T-W6, homochiral peptide complex>

PEGylated (L)Trp-(L)Trp-(L)Trp-(L)Trp-(L)Trp-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Trp (Comparative Example 1) (SEQ ID NO: 37) was prepared. The prepared heterochiral peptide complex (T-W6) was purified by RP-HPLC chromatography in the same manner as in Example 1 (FIG. 4a). The purified heterochiral peptide complex (T-W6) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and its structure is shown in FIG. 2a, and the results are shown in FIG. 3a. showed up

The heterochiral peptide complex (T-W6) was lyophilized and stored until the experiment.

<Comparative Example 2. Preparation of T-w6, homochiral peptide complex>

PEGylated (D)Trp-(D)Trp-(D)Trp-(D)Trp-(D)Trp-(D)Trp in the same manner as in Example 1 except that the sequence was designed as follows. (Comparative Example 2) (SEQ ID NO: 38) was prepared. The prepared heterochiral peptide complex (T-w6) was purified by RP-HPLC chromatography in the same manner as in Example 1 (FIG. 4b). The purified heterochiral peptide complex (T-w6) was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry, and its structure is shown in FIG. 2b, and the result is shown in FIG. 3b. .

The heterochiral peptide complex (T-w6) was lyophilized and stored until the experiment.

<Comparative Example 3. Preparation of T-v6, homochiral peptide complex>

PEGylated (D)Val-(D)Val-(D)Val-(D)Val-(D)Val-(D) except that the sequence was designed and synthesized as follows in Example 1. ) Val (Comparative Example 3) (SEQ ID NO: 39). The prepared heterochiral peptide complex (T-v6) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (T-w6) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 22a.

The heterochiral peptide complex (T-v6) was lyophilized and stored until the experiment.

<Comparative Example 4. Preparation of T-V6, homochiral peptide complex>

PEGylated (L)Val-(L)Val-(L)Val-(L)Val-(L)Val-(L) except that the sequence was designed and synthesized as follows in Example 1. ) Val (Comparative Example 4) (SEQ ID NO: 40). The prepared heterochiral peptide complex (T-V6) was purified by RP-HPLC chromatography in the same manner as in Example 1. The purified heterochiral peptide complex (T-V6) was confirmed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, and the results are shown in FIG. 22B.

The heterochiral peptide complex (T-V6) was lyophilized and stored until the experiment.

<Experimental Example 1. Induction of self-assembly formation>

The lyophilized heterochiral peptide complex powder obtained from Examples 1 to 4 and the lyophilized homochiral peptide complex powder obtained from Comparative Examples 1 and 2 were rehydrated with double distilled water. , water/acetonitrile (1:1) solution, and then the concentration was measured spectrophotometrically using the molar extinction coefficient of 280 nm tryptophan (5,690 M ⁻¹ cm ⁻¹ ).

All peptide samples were diluted to 1 mM and divided into 1 mL aliquots. The aliquots were heated at 95° C. for 1 hour and slowly cooled in a water bath overnight (annealing process). Thereafter, the self-assembly formation process was analyzed through CD, AFM, WAXS, NMR, etc. over time at room temperature, and the results are the same as the experimental examples described later.

<Experimental Example 2. Self-assembly structure analysis of homochiral peptide complex>

Figure 5a is a CD spectrum result graph of homochiral peptide complexes of Comparative Example 1 and Comparative Example 2, which are enantiomers, Figure 5b is an AFM image of the homochiral peptide complex of Comparative Example 1, and Figure 5c is Comparative Example 2 It is an AFM image of a homochiral peptide complex.

6 is an AFM image (crystallization form) of the homochiral peptide complex prepared from Comparative Example 1 in the global minimum state, and FIG. 7 is the homochiral peptide complex prepared in Comparative Example 2 in the global minimum state. AFM image of the ral peptide complex (crystallized form).

As shown in FIG. 5A, the CD of the homochiral peptide complex (T-W6) of Comparative Example 1 composed only of L-type tryptophan and the homochiral peptide complex (T-w6) of Comparative Example 2 composed only of D-type tryptophan. As a result of spectroscopic analysis, it can be confirmed that the spectra are mirror images of each other. That is, it can be seen that the peptides of Comparative Examples 1 and 2 have an enantiomeric relationship with each other.

As shown in FIGS. 5B, 6, and 7, the homochiral peptide complexes of Comparative Examples 1 and 2 are flat and rectangular, such as misfolding peptides or proteins (eg, amyloid peptides). ) form of nanostructures (crystals), it was confirmed that they were quickly self-assembled.

In addition, through the CD spectrum for confirming the secondary structure and the AFM result for confirming the morphology, the homochiral peptide complexes of Comparative Examples 1 and 2 showed little change in structure and morphology regardless of dissolution time. , which means that rapid self-assembly occurs at the global free energy minimum.

<Experimental Example 3. Self-assembly structure analysis of heterochiral peptide complexes>

8 is a CD spectrum graph showing the self-assembly process of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) of Example 1 over time in solution.

Figure 9a is a CD spectrum result graph of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) of Example 1 after 6 weeks, and Figure 9b is a heterochiral (racemic) peptide complex of Example 2 after 6 weeks. ) CD spectrum result graph of the peptide complex (T-(Ww) ₃ ).

As shown in FIG. 8, the heterochiral peptide complex (T-(wW) ₃ ) of Example 1, which is a heterochiral racemic peptide composed of alternately bonded D- and L-tryptophan, is the same as Comparative Examples 1 and 2. It can be seen that the self-assembly rate is significantly slower than that of the mochiral peptide complex.

After the heterochiral (racemic) peptide complex (T-(wW) ₃ ) of Example 1 was dissolved in distilled water, there was no CD spectrum change for some time, and most of the CD spectrum was maintained at a level similar to background water. Through this, it can be seen that the self-assembly is not immediately formed and the initial structure is maintained.

As time passed (from 2 days to 2 weeks), the CD signal gradually increased in the heterochiral (racemic) peptide complex (T-(wW) ₃ ) of Example 1. After 2 weeks, the increase in CD signal almost ceased, indicating that the self-assembly formation process of the heterochiral peptide complex (T-(wW) ₃ ) of Example 1 reached an equilibrium state.

The heterochiral peptide complex (T-(Ww) ₃ ) of Example 2, which is an enantiomer of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) of Example 1, also exhibited the same slow assembly kinetics. As in 9, the heterochiral (racemic) peptide complex of Example 1 (T-(wW) ₃ ) and the heterochiral peptide complex of Example 2 (T-(Ww) ₃ ) are mirror images in self-assembly equilibrium. -image) CD spectrum was observed.

Figure 10a is an AFM image of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 hours, and Figure 10b is an AFM image of the heterochiral (racemic) peptide complex (T-(wW) ₃ after 2 days). ), and FIG. 10c is an AFM image of the heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 weeks. 11 is an AFM image of a heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 hours, and FIG. 12 is an AFM image of a heterochiral (racemic) peptide complex (T-(wW) ₃ after 2 days). ), and FIG. 13 is an AFM image of a heterochiral (racemic) peptide complex (T-(wW) ₃ ) after 2 weeks.

In order to investigate whether the heterochiral peptide complex of Example 1 (T-(wW) ₃ ) of the present invention can confirm the morphology of each step of the self-assembly pathway, AFM images were taken over time.

10a and 11, the heterochiral peptide complex (T-(wW) ₃ ) of Example 1 self-assembles into 1-dimensional (1D) helical fibers for the first 2 hours did Some fibers were twisted in a right-handed fashion (P-helix) and others were twisted in a left-handed fashion (M-helix). The P-helix and M-helix were present in almost the same ratio.

Referring to FIGS. 10B and 12, it can be seen that on the second day, the helices are fattened and connected laterally to form a flat ribbon-shaped fiber. At this stage, the lengths of the ribbons are different.

Referring to FIGS. 10C and 13 , in the last two weeks, crystals were formed in an equilibrium or total energy minimization state, and the length of each flat ribbon crystal was the same. Therefore, since the self-assembly process of the heterochiral peptide complex of Example 1 is performed over a total of 2 weeks, it can be seen that the self-assembly kinetics are sufficiently slow to be observed (slow-motion self-assembly).

That is, the self-assembly pathway of the heterochiral peptide complex (T-(wW) ₃ and T-(Ww) ₃ ) of the present invention has many similarities with the self-assembly pathway of amyloid peptide. Specifically, one of the most common morphological modifications of amyloid peptides is known as:

Twisted ribbon -> flattening of the twisted ribbon -> crystal

It can be seen that the twisted ribbons of amyloid peptide are similar to the helical fibers of the heterochiral racemic peptide complexes of the present invention. The crystalline form of the amyloid peptide is considered to be the total energy minimum in the amyloid peptide, which is determined at equilibrium or total energy minimization in the heterochiral peptide complexes of the present invention (T-(wW) ₃ and T-(Ww) ₃ ). It can be seen that this is consistent with the formation of

In other words, taking the above results together, it was confirmed that the self-assembly pathways between the peptides were consistent, even though they had different sequences and chirality, as in Comparative Examples 1 and 2 and Examples 1 and 2. In other words, the self-assembly process of the slowly self-assembling heterochiral peptide complex of the present invention (T-(wW) ₃ ) and the homochiral peptides of Comparative Examples 1 and 2 (T-W6, T-w6) differs only in speed. , it can be seen that self-assembly proceeds through the same path.

<Experimental Example 4. WAXS analysis>

Through the above-described experiments, the overall crystal shape of the final self-assembly of the heterochiral peptide complex (T-(wW) ₃ ) prepared from Example 1 was obtained from the homochiral peptide complexes (T-W6 and It was confirmed that the crystal shape was identical to that of T-w6).

Even if the crystal shape of the final self-assembly is the same, the detailed molecular arrangement of the peptides constituting it may be different. Therefore, wide-angle X-ray scattering (WAXS) of the colloidal crystal solution was performed to accurately confirm.

Figure 14a shows the synchrotron WAXS of the heterochiral peptide complex (T-(wW) ₃ ) prepared from Example 1 and the homochiral peptide complexes (T-W6 and T-w6) of Comparative Examples 1 and 2 data, and FIG. 14b is an MD simulation result of (wW) ₃ peptides in the heterochiral peptide complex of Example 1. The numbers mentioned in FIG. 14 represent distances in angstroms.

As shown in FIG. 14, the reflections for the homochiral peptide complexes (T-W6 and T-w6) (d = 4.7, 5.8 and 17.5 Å) of Comparative Example 1 and Comparative Example 2 were the same. This is a natural result since the homochiral peptide complexes (T-W6 and T-w6) of Comparative Example 1 and Comparative Example 2 have the same molecular structure except that they are enantiomers.

On the other hand, looking at the reflections of the heterochiral peptide complex (T-(wW) ₃ ) (d = 4.7, 5.6 and 17.0 Å) prepared from Example 1, the heterochiral peptide complex of Comparative Examples 1 and 2 and 5.6 It was confirmed that there is a difference between Å and 17.0 Å.

Reflections at 4.7 Å were all found in the complexes of Example 1, Comparative Example 1 and Comparative Example 2, and it can be seen that this is the distance between strands of the β-sheet by the peptide of the present invention.

In addition, the d-spacings of the heterochiral peptide complex (T-(wW) ₃ ) prepared from Example 1 is 17.0 Å, and the homochiral peptide complexes of Comparative Examples 1 and 2 (T- The d-spacings of W6 and T-w6) are 17.5 Å. Their d-spacings are larger than typical values for β-sheets, but represent the distance between sheets.

Molecular dynamics (MD) simulation analysis was performed to gain insight into the structure of the heterochiral peptide complex (T-(wW) ₃ ) prepared in Example 1 as it self-assembles. To simplify the simulation, only the (wW) ₃ peptide portion involved in self-assembly was used for analysis. The first simulation results show a randomly generated D-Trp/L-Trp repeating peptide with fixed dihedral angles (Φ = -180° and Ψ = -180°). started in The second was to replace tyrosine with tryptophans from poly(D-/L-tyrosine) before starting the MD simulations.

Originally, poly(D-/L-tyrosine) (D-/L-tyrosine) was known to have a helical form of an antiparallel double-stranded β-helix, but tyrosine was converted to tryptophan. It was confirmed that it had a similar shape even when it was replaced. That is, it was confirmed that the shape of the (wW) ₃ peptide portion in Example 1 was similar to the antiparallel double-stranded β-helix of poly(D-/L-tyrosine) (FIG. 14 b).

More specifically, in Example 1, the (wW) ₃ peptide part is more bent than the normal β-sheet structure, but unlike poly(D-/L-tyrosine), the peptide length is shorter, so it does not show a perfect helix. This is consistent with the WAXS data that the distance between the β strands of the heterochiral peptide complex (T-(wW) ₃ ) prepared from Example 1 was 4.56-4.92 Å.

Taken together, the homochiral peptide complexes (T-W6 and T-w6) of Comparative Example 1 and Comparative Example 2 and the heterochiral peptide complex (T-(wW) ₃ ) prepared from Example 1 have chiral configuration and molecular structure. is different, but it was confirmed that the final self-assembled structure is almost the same (FIG. 15).

<Experimental Example 5. Structural analysis of asymmetric heterochiral (ee) peptide complexes>

To further study the structural space of heterochiral peptides, another structure of heterochiral peptides was designed in which one of the tryptophan stereoisomers is an enantiomeric excess (Fig. 2e, f) (Fig. 2e, f). heterochiral at 15 ee)

At this time, among the asymmetric heterochiral peptides (ee) (TW(wW) ₂ and Tw(Ww) ₂ ) prepared from the designed Examples 3 and 4, one of the stereoisomers is more resistant to the self-assembly process than the other. expected to provide contributions.

16 is a result of measuring the structures of the asymmetric heterochiral peptide (ee) complexes (TW(wW) ₂ and Tw(Ww) ₂ ) of Examples 3 and 4 in the initial stage of self-assembly, and FIG. 16a shows Example 3 and An AFM image measured at the initial stage of self-assembly (2 hours) of the asymmetric heterochiral peptide (ee) complex of 4 (TW(wW) ₂ and Tw(Ww) ₂ ), and FIG. 16B is an enlarged image of FIG. 16A (Enlarged images) ), and FIG. 16c is a diagram showing the unique helical structures of the asymmetric heterochiral peptide (ee) complexes (TW(wW) ₂ and Tw(Ww) ₂ ) of Examples 3 and 4.

17 is an AFM image of the asymmetric heterochiral peptide (ee) complex (TW(wW) ₂ ) of Example 3 at the initial stage of self-assembly (2 hours), and FIG. 18 is an AFM image of Example at the early stage of self-assembly (2 hours). An AFM image of the asymmetric heterochiral peptide (ee) complex (Tw(Ww) ₂ ) of 4, and FIG. 19 is an asymmetric heterochiral peptide (ee) of Example 3 in the final stage of self-assembly (2 weeks, kinetically confined state) ) AFM image of the complex (TW(wW) ₂ ), and FIG. 20 is an asymmetric heterochiral peptide (ee) complex (Tw(Ww) ₂ of Example 4 in the final stage of self-assembly (2 weeks, kinetically confined state) ) is an AFM image of

As shown in FIGS. 16 to 20, the asymmetric heterochiral peptide (ee) complexes (TW(wW) ₂ and Tw(Ww) ₂ ) of Examples 3 and 4 are the heterochiral (racemic) peptide complexes of Examples 1 and 2. It was confirmed that it self-assembled slowly enough to be similar to In particular, it was confirmed that significant twisting of the one-dimensional helical fibers occurs in the initial stage of self-assembly. Furthermore, unique self-assembling intermediates containing fibers composed of helices of variable pitch twisted in right-handed fashion (P-helix) and left-handed fashion (M-helix) It was confirmed that was created.

Referring to FIG. 16B, in the initial stage of self-assembly of the asymmetric heterochiral peptide (ee) complexes (TW(wW) ₂ and Tw(Ww) ₂ ) of Examples 3 and 4, a variable pitch twisted into an MP-helix In addition to the spiral fibers, short fibers of a swirling shape similar to sausage strings were connected, and it was confirmed that a sausage-like fiber structure also existed as an intermediate. The sausage-like intermediate is believed to be formed by excessive twisting of one-dimensional helical fibers. The twisted one-dimensional helical fibers were intertwined to form a superhelix (FIG. 16c). It can be seen that all these unique structures are induced by the formation of misalignment of asymmetric peptide building blocks by combining the slow and dynamic characteristics of heterochiral self-assemblies.

After 2 weeks of self-assembly, the asymmetric heterochiral peptide (ee) complex (TW(wW) ₂ ) of Example 3 undergoes extensive intertwining and overtwisting, resulting in a porous superhelical network. ) was confirmed to be formed (Figs. 19 and 20). The porous superhelical network state can be referred to as a kinetically trapped state because conversion to a crystal is kinetically inhibited by the formation of an intertwined superhelical network.

As in the present invention, the heterochiral peptide complex forms a slow-motion self-assembly, which is an alignment medium for NMR and residual dipolar coupling (RDC) measurements, in a one-dimensional helical form. It can be seen that it can be used as a new intermediate with RDC can provide important information about the long-range orientation restraints of internuclear vectors in solution NMR spectroscopy. In particular, distance restraints induced from the nuclear Overhauser effect (NOE) are difficult to obtain for proteins larger than 15 kDa in size.

That is, RDC can provide information capable of improving the quality of NMR structural analysis. Specifically, when the sample is present in aqueous conditions, dipolar couplings are degraded due to randomized molecular motion. Therefore, in order to measure RDC, it is necessary to limit the direction of the dipole to have anisotropy. Accordingly, the alignment media of the present invention can be utilized as angular restraints according to the degree of increase in arrangement. As such alignment media, nematic liquid crystal molecules, compressed or stretched polymer gel, Pf1 filamentous phage, and bicelles have been developed. . However, the orientation medium of the RDC has a limitation in that it has unique characteristics with respect to temperature, pH, solvent, and state of charge of the orientation medium. Therefore, there is an urgent need to develop a new orientation medium that can be widely applied.

Accordingly, since various peptide complexes, including the heterochiral peptide complex (T-)wW)3) of Example 1 according to the present invention completed, self-assemble slowly in solution and form intermediates having strong anisotropy, RDC measurement It can be seen that it can act sufficiently as a culture medium for In addition, it can be seen that the heterochiral peptide complexes of Examples 1-4 form a helical one-dimensional self-assembly intermediate, which is similar in structure to the Pg1 filamentous phage having a one-dimensional structure, and thus can sufficiently act as an orientation medium for RDC measurement. can

<Experimental Example 6. ² H-NMR analysis results>

When the peptide complex of the present invention was used as an alignment medium, the ordering of water molecules was measured. For this purpose, ² H-NMR was used, and its analysis conditions were measured as follows: 1-pulse experiments were performed to measure and evaluate the quadrupole coupling of midpoints in D ₂ O. It was measured under the conditions described in the NMR test method, and in this experiment, in order to confirm signal splitting from the NMR spectrum, a general 10% (v / v) D ₂ O aqueous solution (H ₂ O / D ₂ O (90 / 10;

The homochiral peptide complex of Comparative Example 1 (crystal), the heterochiral peptide complex of Example 1 (T-(wW) ₃ ) (mature; crystal) and the heterochiral peptide complex of Example 1 (T-(wW) ₃ ) (intermediate; one-dimensional self-assembly intermediate) was evaluated, and the results are shown in FIG. 21 .

21 is a deuterium NMR spectrum of the heterochiral peptide complex of Example 1 and the homochiral peptide complex of Comparative Example 1 having various assembly states at 10% (v/v) D ₂ O, 298K. All spectra were obtained using a 700 MHz NMR spectrometer.

As shown in Figure 21. Quadrupolar splitting of the deuterium peak was measured for each sample containing different peptides under 10% (v/v) D ₂ O conditions. At this time, the concentration of all peptide complexes was 8 mg/ml.

The homochiral peptide complex (T-W6) of Comparative Example 1 formed planar supramolecular structures (crystals), and the heterochiral peptide complex (T-(wW) ₃ ) of Example 1 was a self-assembling intermediate. In , a spiral one-dimensional self-assembly form was formed, and in the final stage of self-assembly, a crystalline form was formed. Since the one-dimensional self-assembling intermediate of the heterochiral peptide complex (T-(wW) ₃ ) of Example 1 is an anisotropic self-assembled nanostructure, it is similar to a nematic liquid crystal molecule. It can be seen that they are aligned (or oriented) as From the above results, it can be seen that quadrupolar splitting of the deuterium peak is very effective for the heterochiral peptide complex (T-(wW) ₃ ) of Example 1, which is a one-dimensional self-assembly intermediate state.

One-dimensional self-assembly of the heterochiral peptide complex (T-(wW) ₃ ) of Example 1 was obtained by ² H quadrupole splitting at 1.74 Hz at 8 mg/ml. At this time, the relatively small peak of the heterochiral peptide complex (T-(wW) ₃ ) of Example 1 limits the degree of alignment (or orientation) of the heterochiral peptide complex (T-(wW) ₃ ) of Example 1. because the concentration was so low.

In contrast, in the case of the homochiral peptide complex of Comparative Example 1 (crystal) and the heterochiral peptide complex of Example 1 (final state: crystal), no quadrupole coupling was observed. This is believed to be due to differences in the degree of alignment depending on the supramolecular morphology and dimension. That is, the heterochiral peptide complex (T-(wW) ₃ ) of Example 1 changes the degree of quadrupole splitting depending on the degree of molecular self-assembly. It was confirmed that it can be used as a new alignment medium. To date, no orientation medium for NMR measurement showing a change in quadrupole splitting with time has been reported. Only two different samples, with and without an orientation medium, were prepared for RDC measurements. However, the heterochiral peptide complex (T-(wW) ₃ ) of Example 1 of the present invention has a great advantage that only one sample is required to measure RDC.

Summarizing the experiments of the present invention, the present invention identified a self-assembling intermediate of a peptide that had not been observed before by developing a heterochiral peptide complex in which a new self-assembly process proceeds slowly. It was confirmed that the heterochiral peptide complex self-assembles in solution to form a self-assembling intermediate having a specific structure with strong anisotropy, thereby inducing orientation stability of a sample for NMR measurement, in particular, RDC analysis. In addition, it can be used as a drug delivery system that entraps and delivers hydrophobic drugs to the hydrophobic inner region of the self-assembly, and can also be used as a scaffold for 3-dimensional culture of cell tissues such as artificial skin.

<Experimental Example 7. Structural analysis of various heterochiral (ee) peptide complexes>

Various heterochiral peptide complexes were prepared, and AFM analysis was performed to confirm the structure thereof.

26a is an AFM image of the heterochiral peptide complex (T-(vV) ₃ ) of Example 5, and FIG. 26b is an AFM image of the heterochiral peptide complex (T-(fF) ₃ ) of Example 6, 26C is an AFM image of the heterochiral peptide complex (T-(fF) ₄ ) of Example 7.

27a is an AFM image of the heterochiral peptide complex (Tw ₃ -W ₃ ) of Example 9, and FIG. 27b is an AFM image of the heterochiral peptide complex (T-(wW) ₂ -w ₂ ) of Example 10. 27c is an AFM image of the heterochiral peptide complex (TW ₂ -(wW) ₂ ) of Example 11, and FIG. 27d is an AFM image of the heterochiral peptide complex ((PEG3-PA) ₂ -(wW) of Example 15). ₂ ) is an AFM image for.

28a is an AFM image of the heterochiral peptide complex of Example 17 ((PEG10-PA)-V-(vV) ₂ ), and FIG. 28b is an AFM image of the heterochiral peptide complex of Example 18 ((PEG10-PA)-f ₃ -F ₃ ), and FIG. 28c is an AFM image of the heterochiral peptide complex ((PEG10-PA)-F-(fF) ₂ ) of Example 19.

26 to 28, it was confirmed that the heterochiral peptide complexes of Examples 5 to 20 also gradually self-assemble similarly to the heterochiral peptide complexes of Examples 1 to 4, and that unique self-assembly intermediates were produced depending on the peptides. did

Therefore, in addition to the heterochiral peptide complexes of Examples 1 to 4 in the present invention, other heterochiral peptide complexes are also self-assembled in solution to form self-assembly intermediates of specific structures with strong anisotropy, thereby performing NMR measurement, in particular, RDC analysis. It can be seen that it can be used to induce the orientation stability of the sample.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Heterochiral peptide-complex and composition for measuring nuclear magnetic resonance spectroscopy residual dipolar coupling containing self-assembled intermediates of heterochiral peptide-complex <130> HPC9759 <150> KR 10-2020-0008499 <151> 2020-01-22 <160> 40 <170> KoPatentIn 3.0 <210> 1 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the third and the fifth tryptophan are D-tryptophan which is D-enantiomer. <400> 1 Trp Trp Trp Trp Trp Trp 1 5 <210> 2 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the third and the fifth valine are D-valine which is D-enantiomer. <400> 2 Val Val Val Val Val Val 1 5 <210> 3 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the third and the fifth phenylalanine are D-phenylalanine which is D-enantiomer. <400> 3 Phe Phe Phe Phe Phe Phe 1 5 <210> 4 <211> 6 <212> PRT <213> artificial sequence <220> <223> The second, the fourth and the sixth tryptophan are D-tryptophan which is D-enantiomer. <400> 4 Trp Trp Trp Trp Trp Trp 1 5 <210> 5 <211> 6 <212> PRT <213> artificial sequence <220> <223> The second valine, the fourth valine, and the sixth valine are D-valine, D-enantiomer of valine. <400> 5 Val Val Val Val Val Val 1 5 <210> 6 <211> 6 <212> PRT <213> artificial sequence <220> <223> The second, the fourth and the sixth phenylalanine are D-phenylalanine which is D-enantiomer. <400> 6 Phe Phe Phe Phe Phe Phe 1 5 <210> 7 <211> 5 <212> PRT <213> artificial sequence <220> <223> The second and the fourth tryptophan are D-tryptophan which is D-enantiomer. <400> 7 Trp Trp Trp Trp Trp 1 5 <210> 8 <211> 5 <212> PRT <213> artificial sequence <220> <223> The second and the fourth valine are D-valine which is D-enantiomer. <400> 8 Val Val Val Val Val 1 5 <210> 9 <211> 5 <212> PRT <213> artificial sequence <220> <223> The second and the fourth phenylalanine are D-phenylalanine which is D-enantiomer. <400> 9 Phe Phe Phe Phe Phe 1 5 <210> 10 <211> 5 <212> PRT <213> artificial sequence <220> <223> The first, the third and the fifth tryptophan are D-tryptophan which is D-enantiomer. <400> 10 Trp Trp Trp Trp Trp 1 5 <210> 11 <211> 5 <212> PRT <213> artificial sequence <220> <223> The first, the third and the fifth valine are D-valine which is D-enantiomer. <400> 11 Val Val Val Val Val 1 5 <210> 12 <211> 5 <212> PRT <213> artificial sequence <220> <223> The first, the third and the fifth phenylalanine are D-phenylalanine which is D-enantiomer. <400> 12 Phe Phe Phe Phe Phe 1 5 <210> 13 <211> 8 <212> PRT <213> artificial sequence <220> <223> The first, the third, the fifth and the seventh tryptophan are D-tryptophan which is D-enantiomer. <400> 13 Trp Trp Trp Trp Trp Trp Trp Trp 1 5 <210> 14 <211> 8 <212> PRT <213> artificial sequence <220> <223> The first, the third, the fifth and the seventh valine are D-valine which is D-enantiomer. <400> 14 Val Val Val Val Val Val Val Val 1 5 <210> 15 <211> 8 <212> PRT <213> artificial sequence <220> <223> The first, the third, the fifth and the seventh phenylalanine are D-phenylalanine which is D-enantiomer. <400> 15 Phe Phe Phe Phe Phe Phe Phe Phe 1 5 <210> 16 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the fourth and the fifth tryptophan are D-tryptophan which is D-enantiomer. <400> 16 Trp Trp Trp Trp Trp Trp 1 5 <210> 17 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the fourth and the fifth valine are D-valine which is D-enantiomer. <400> 17 Val Val Val Val Val Val 1 5 <210> 18 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the fourth and the fifth phenylalanine are D-phenylalanine which is D-enantiomer. <400> 18 Phe Phe Phe Phe Phe Phe 1 5 <210> 19 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the second and the third tryptophan are D-tryptophan which is D-enantiomer. <400> 19 Trp Trp Trp Trp Trp Trp 1 5 <210> 20 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the second and the third valine are D-valine which is D-enantiomer. <400> 20 Val Val Val Val Val Val 1 5 <210> 21 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the second and the third phenylalanine are D-phenylalanine which is D-enantiomer. <400> 21 Phe Phe Phe Phe Phe Phe 1 5 <210> 22 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the third, the fifth and the sixth tryptophan are D-tryptophan which is D-enantiomer. <400> 22 Trp Trp Trp Trp Trp Trp 1 5 <210> 23 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the third, the fifth and the sixth valine are D-valine which is D-enantiomer. <400> 23 Val Val Val Val Val Val 1 5 <210> 24 <211> 6 <212> PRT <213> artificial sequence <220> <223> The first, the third, the fifth and the sixth phenylalanine are D-phenylalanine which is D-enantiomer. <400> 24 Phe Phe Phe Phe Phe Phe 1 5 <210> 25 <211> 6 <212> PRT <213> artificial sequence <220> <223> the third and the fifth tryptophan are D-tryptophan which is D-enantiomer. <400> 25 Trp Trp Trp Trp Trp Trp 1 5 <210> 26 <211> 6 <212> PRT <213> artificial sequence <220> <223> the third and the fifth valine are D-valine which is D-enantiomer. <400> 26 Val Val Val Val Val Val 1 5 <210> 27 <211> 6 <212> PRT <213> artificial sequence <220> <223> the third and the fifth phenylalanine are D-phenylalanine which is D-enantiomer. <400> 27 Phe Phe Phe Phe Phe Phe 1 5 <210> 28 <211> 7 <212> PRT <213> artificial sequence <220> <223> The second, the fourth and the sixth tryptophan are D-tryptophan which is D-enantiomer. <400> 28 Trp Trp Trp Trp Trp Trp Trp 1 5 <210> 29 <211> 7 <212> PRT <213> artificial sequence <220> <223> The second, the fourth and the sixth valine are D-valine which is D-enantiomer. <400> 29 Val Val Val Val Val Val Val 1 5 <210> 30 <211> 7 <212> PRT <213> artificial sequence <220> <223> The second, the fourth and the sixth phenylalanine are D-phenylalanine which is D-enantiomer. <400> 30 Phe Phe Phe Phe Phe Phe Phe 1 5 <210> 31 <211> 4 <212> PRT <213> artificial sequence <220> <223> The first and the third tryptophan are D-tryptophan which is D-enantiomer. <400> 31 Trp Trp Trp Trp One <210> 32 <211> 4 <212> PRT <213> artificial sequence <220> <223> The first and the third valine are D-valine which is D-enantiomer. <400> 32 Val Val Val Val One <210> 33 <211> 4 <212> PRT <213> artificial sequence <220> <223> The first and the third phenylalanine are D-phenylalanine which is D-enantiomer. <400> 33 Phe Phe Phe Phe One <210> 34 <211> 4 <212> PRT <213> artificial sequence <220> <223> The first and the second tryptophan are D-tryptophan which is D-enantiomer. <400> 34 Trp Trp Trp Trp One <210> 35 <211> 4 <212> PRT <213> artificial sequence <220> <223> The first and the second valine are D-valine which is D-enantiomer. <400> 35 Val Val Val Val One <210> 36 <211> 4 <212> PRT <213> artificial sequence <220> <223> The first and the second phenylalanine are D-phenylalanine which is D-enantiomer. <400> 36 Phe Phe Phe Phe One <210> 37 <211> 6 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 37 Trp Trp Trp Trp Trp Trp 1 5 <210> 38 <211> 6 <212> PRT <213> artificial sequence <220> <223> all of tryptophan is D-tryptophan which is D-enantiomer. <400> 38 Trp Trp Trp Trp Trp Trp 1 5 <210> 39 <211> 6 <212> PRT <213> artificial sequence <220> <223> Artificial Sequence <400> 39 Val Val Val Val Val Val 1 5 <210> 40 <211> 6 <212> PRT <213> artificial sequence <220> <223> all of valine is D-valine which is D-enantiomer. <400> 40 Val Val Val Val Val Val 1 5

Claims (15)

A peptide represented by the following general formula 1 or 2; And a compound represented by Formula 1 bonded to the N-terminus of the peptide; a peptide complex comprising:
[Formula 1]
[Y1] _a -{-[X1] _b -[Y2] _c } _d -[X2] _e
[Formula 2]
[X1] _f -{-[Y1] _g -[X2] _h } _i -[Y2] _j
In Equations 1 and 2 above
X1 and X2 are the same or different from each other, and are each independently any one selected from D-Trp, D-Phe, D-Tyr, D-Val, D-Leu, and D-Ile;
Y1 and Y2 are the same or different from each other, and are each independently any one selected from L-Trp, L-Phe, L-Tyr, L-Val, L-Leu, and L-Ile,
Wherein a, f, e and j are each independently any one integer selected from 0 to 2, b, c, g and h are each independently any one integer selected from 1 to 5, and d and i Each is an integer independently selected from 1 to 10.
[Formula 1]

In Formula 1, n and m are each independently an integer ranging from 1 to 12. According to claim 1,
In Formulas 1 and 2 above,
X1 and X2 are the same or different from each other, and are each independently any one selected from D-Trp, D-Val, and D-Phe;
Y1 and Y2 are the same or different from each other, and each independently selected from L-Trp, D-Val and L-Phe peptide complex, characterized in that any one selected. According to claim 1,
In Formulas 1 and 2 above,
Wherein X1 is the same as X2, and Y1 is the same as Y2. According to claim 1,
In Formulas 1 and 2 above,
Wherein a, f, e and j are each independently any one integer selected from 0 to 2, b, c, g and h are each independently any one integer selected from 1 to 3, and d and i Is each independently an integer selected from 1 to 4 peptide complex, characterized in that. According to claim 1,
The complex, characterized in that the peptide is any one selected from the heterochiral peptide sequences represented by SEQ ID NOs: 1 to 12:
[SEQ ID NO: 1]
(D)Trp-(L)Trp-(D)Trp-(L)Tre-(D)Trp-(L)Trp
[SEQ ID NO: 2]
(D)Val-(L)Val-(D)Val-(L)Val-(D)Val-(L)Val
[SEQ ID NO: 3]
(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe
[SEQ ID NO: 4]
(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp
[SEQ ID NO: 5]
(L)Val-(D)Val-(L)Val-(D)Val-(L)Val-(D)Val
[SEQ ID NO: 6]
(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe
[SEQ ID NO: 7]
(L)Trp-(D)Trp-(L)Trp-(D)Trp-(L)Trp
[SEQ ID NO: 8]
(L)Val-(D)Val-(L)Val-(D)Val-(L)Val
[SEQ ID NO: 9]
(L)Phe-(D)Phe-(L)Phe-(D)Phe-(L)Phe
[SEQ ID NO: 10]
(D)Trp-(L)Trp-(D)Trp-(L)Trp-(D)Trp
[SEQ ID NO: 11]
(D)Val-(L)Val-(D)Val-(L)Val-(D)Val
[SEQ ID NO: 12]
(D)Phe-(L)Phe-(D)Phe-(L)Phe-(D)Phe According to claim 1,
In the peptide conjugate, the compound is a peptide conjugate, characterized in that represented by the following formula (2).
[Formula 2]

In Formula 2, m is an integer ranging from 1 to 12. According to claim 1,
The peptide complex is characterized in that any one selected from those represented by the following formulas 3 to 6:
[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

In Chemical Formulas 3 to 6, m is an integer ranging from 1 to 5. According to claim 1,
The peptide complex slowly forms a self-assembly in solution,
In Formulas 1 and 2, when a, f, e, and j are 0, the right-handed fashion (P-helix) and the left-handed fashion (M- In the fiber structure twisted into a helix, the helical portion of the fiber is laterally fattened to form an intermediate of a flat ribbon fiber structure, and finally formed into a self-assembly of a flat ribbon crystal structure. A peptide complex. According to claim 1,
The peptide complex slowly forms a self-assembly in solution,
In Formulas 1 and 2, when a, f, e and j are not 0 at the same time,
The peptide complex is formed by intertwining and overtwisting of fibers composed of variable pitch helices twisted in a right-handed fashion (P-helix) and a left-handed fashion (M-helix). A peptide complex characterized in that it is formed as a self-assembly of a porous superhelical network structure. An orientation mediator composition for measuring residual dipole bonds (RDC) comprising the peptide complex according to claim 1. (a) mixing the peptide complex according to claim 1 in a mixed solvent;
(b) adding a biomolecule to be measured to the mixed solution; and
(c) performing NMR measurement on the mixture of the biomolecule and peptide complex; According to claim 11,
The method of performing NMR spectrum analysis of biomolecules, characterized in that the NMR measurement is carried out by any one selected from the group consisting of ¹ H, ¹ H edit and 2D NMR. According to claim 11,
The method of performing NMR spectrum analysis of biomolecules, characterized in that the mixed solvent is a mixed solution of a deuterium solvent and a solvent. A drug delivery system comprising the peptide complex according to claim 1. A scaffold for cell culture comprising the peptide complex according to claim 1.

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