KR101558160B1 - Biomimetic scaffold for catalytic redox activity and its use - Google Patents

Abstract

The present invention provides a redox catalyst comprising a nanostructure formed by a peptide having the structure of X ¹ a-Zn-X ² b wherein X ¹ and X ² are each independently a pyrophyll interaction Wherein X ¹ and X ² are the same or different and Z is a D or L-type natural or unnatural amino acid or derivative thereof having possible side chains, and Z is a charged D or L having a charged side chain or nucleophilic side chain capable of cross- A and b are the same or different integers from 1 to 10, and n is an integer from 1 to 10, respectively. The peptide nanostructures according to the present invention form a very regular and dense structure and promote chemical electrochemical reactions by the tyrosine residues in the peptides, which can serve as a platform for the production of large area peptide or hybrid films.

Description

[0001] Biomimetic scaffold for catalytic redox activity and its use [0002]

TECHNICAL FIELD The present invention relates to a biomimetic material, a production method thereof, and a use thereof.

There is a great need to develop new organic electronic materials required for the industry, including organic light emitting diodes. In addition, existing mineral-based materials are difficult to lightweight and large-scale thin, and the cost of raw materials is increasing due to the depletion of rare resources. Especially recently, as touch panels have become the mainstay of the display industry, it is urgent to secure alternative materials for materials used as transparent electrodes.

Indium tin oxide (ITO), which is currently used as a transparent electrode, is expected to be depleted in the next few years (cited in Kato et al., Nat. Commun. 3, 645 (2012), Physics and Advanced Technology June 2012) It is very urgent to replace indium tin oxide with an organic material in the state that it enters the electrode. Currently, organic materials are partially used industrially, but only a small part of them are expected to be replaced with organic materials or polymer materials in the future.

Materials used to store or convert solar energy as an alternative energy source are also ultimately replaced by organic materials due to resource depletion. However, currently developed organic electronic materials have low yield and efficiency, And the production cost is high. Specifically, modification of carbon nanotubes (CNTs) is difficult. In the case of single-wall carbon nanotubes (SW-CNTs), it is difficult to commercialize them due to difficulty in mass production. graphene has emerged as a new material to replace ITO. However, in order to commercialize transparent electrodes, excellent quality, large-area production, and adhesion to substrates are required, and there are many difficulties in meeting these requirements.

Therefore, although efforts are made to make two-dimensional nanostructures using polymers or organic materials, there is no clear result in the commercialization stage. One of them is a nano structure formation technique using a peptide which is a biomaterial.

U.S. Patent No. 7,179,784 relates to surfactant peptide nanostructures and uses thereof, and discloses the use of nanotubes and drug delivery using the oligopeptides having dipolar sequences.

U.S. Patent No. 7,491,699 relates to peptide nanostructures and methods of making the same, and discloses a spherical structure formed using a peptide consisting of four amino acids including aromatic side chains and uses thereof.

However, the level of research on peptide nanostructures is at an early stage and there is a need for the development of various nanostructures using peptides and their application areas, as compared to the degree of understanding of the formation mechanism.

The present invention has been made to solve the above-mentioned problems, and it is intended to provide a peptide nanostructure formed by self-assembly and its use.

In one embodiment, the present invention provides a redox reaction facilitating support comprising a nanostructure formed of a peptide having a structure represented by the following formula (1).

[Chemical Formula 1]

X ¹ a-Zn-X ² b

In the above formula

X ¹ and X ² are each independently a D- or L-type natural or unnatural amino acid or a derivative thereof having a side chain capable of pi-phi interactions, X ¹ and X ² are the same or different,

Z is a D- or L-type natural or unnatural amino acid or derivative thereof having charged side chains or nucleophilic side chains capable of forming a bridge,

a and b are the same or different integers from 1 to 10,

n is an integer of 1 to 10;

The redox reaction promoting support according to the present invention may function as a biomimetic or biomimetic having redox reaction catalytic activity. In this respect, the present invention also provides a biomimetic material having a redox-catalytic activity comprising a nanostructure formed of a peptide having the structure of Formula 1 above.

In one embodiment according to the present invention, the cross-linking included in the support or biomimetic material of the present invention includes a covalent bond or a coordination bond by a metal, and the amino acid capable of coordinating by the metal is cysteine, homocysteine, penicillamine, Salenocysteine, histidine, lysine, ornithine, aspartic acid, glutamic acid, asparagine, or glutamine. Also, the amino acid capable of forming the crosslinking includes amino acid cysteine having an SH residue.

In another embodiment according to the present application, the pi-phi interchangeable amino acid included in the support or mimetic according to the present invention is an amino acid having an aromatic side chain, for example, phenylalanine, tryptophan or tyrosine.

In formula (1), X ¹ a and X ² b have amino acid sequences of the same sequence or symmetrically symmetric with respect to each other about Z, for example, n is 1, and a and b Are each an integer of 2 to 5, which are the same or different from each other.

In one embodiment according to the present disclosure the peptide is selected from the group consisting of YYCYY, YYYCYYY, YYACAYY, YYAACAAYY, YYGCGYY, YFCFY, FYCYF, FFCFF, YYAKAYY, YYAEAYY, CYY, YY, YYY, YYYY, YKCKYY, YYECEYY, YYDCDYY, YYAHAYY, YYHYY, YYAPAYY, YYPYY and derivatives thereof.

The peptide nanostructure according to the present invention can form a film, nanofiber, or sphere. In another aspect, it may be formed as a flat face or face face, in which case it may have a single layer or a multi-layer structure.

The redox reaction promoting or catalytic activity possessed by the support according to the present application is activated by the formation of tyrosyl radical. It may further comprise a metal that can act as a cofactor in the reaction if desired.

In another aspect, the present invention provides a method for producing a polymer film, which comprises using a redox reaction promoting support according to the present invention. The polymer film according to the present invention may exhibit conductivity, anti-conduction or non-conductivity depending on the manufacturing process, and in one embodiment, the polymer film is made from a polymeric material and the polymeric material is selected from alkyne, alkenyl, alkynyl, Phenol or a derivative thereof, pyrrole, aniline, thiophene, or 3,4-ethylenedioxythiophene.

In the method according to the present invention, the compound of Formula 1 and the polymerizable material may be contained in various ratios, and may be contained at a molar ratio of, for example, about 1: 0.001 to 1: 100,000.

In another aspect, the present invention also provides a method for promoting redox reaction characterized by using a redox reaction promoting support according to the present invention.

The peptide nanostructure comprising the compound of Formula 1 according to the present invention is useful as a platform for preparing nanostructured bodies having various functions in water or various organic inorganic buffer solutions, And as a mold necessary for the production of a polymerizable material. In addition, the nanostructure according to the present invention may have various conductivities according to the type of the adopted sequence, and can control the thickness of the nanostructure according to the control of the reaction time, And can be used not only as an organic electronic material but also as a tunable peptide lens for various purposes, a floating hydrophobic scaffold for cell culture, a reactive drug delivery vehicle, an ion selective membrane, a regular conductive polymer, And a biocomponent such as a catalyst based on tyrosine.

Also, the peptide nanostructure according to the present invention forms a very regular and dense structure, promotes a chemical electrochemical reaction by the tyrosine residue in the peptide, particularly promotes the redox reaction, and is useful as a platform for manufacturing a large area peptide or organic hybrid film Can act as a support.

1 shows a peptide nanostructure formed of a peptide of the YYACAYY sequence according to one embodiment of the present invention. (A) is an optical microscope photograph (magnification: 50 times) taken in terms of shape change over time of the dropped peptide solution, and the bright region in the center is due to backlight transmission. A total of 80 μl of YYACAYY solution (2 mM) was dropped onto the slide glass, and a flat facet was formed on the upper surface over time. (B) shows an oblique observation of the dripped peptide solution by an optical microscope, showing a clear boundary formed on the upper surface by the flat surface peptide nanostructure. (C) is a transmission electron microscope (TEM) image of a film formed with a 2 mM concentration of peptide. (D) is a photograph of a peptide film formed in a 10-cm-diameter petri dish with a large and thick film over the entire water / air interface when 10 ml of 2 mM peptide was added. (E) is a diagrammatic representation of the process of forming a planar surface, in which dense horizontal association mediated by the tyrosine of peptides floating at the air / water interface, vertical growth of disulfide bonds and a plurality of thin nanosheets And the formation of a flat plane through the surface.
Figure 2 shows a peptide nanostructure formed of a peptide of the YYCYY sequence according to another embodiment of the present application. (A) shows a flat surface structure formed on the upper surface by dropping 80 of a peptide solution (2 mM) on a slide glass, left for 30 minutes in the air, and observed from the side by an optical microscope. (B) was a TEM image of the peptide nanostructure formed in (A), and the formed film was transferred to a carbon-coated grid and then subjected to a negative amber color with aqueous solution with 2% uranyl acetate. (C) is the same as in (A) but reacted in a non-oxidizing environment (desiccator). Unlike (A), no nanostructure was formed, indicating that the oxygen environment is important for the formation of a flat surface.
FIG. 3 is a photograph of a film nanostructure formed of a peptide having a sequence of YYCYY (A, B) or YYACAYY (C) stained with a fluorescent coloring reagent. The coloring reagent is Nile Red, which binds to a hydrophobic chemical substance, (B) and (C) of thioflavin-T (Th-T) reagent that penetrates into the nanoparticles of the protein or peptide.
Figure 4 shows the effect of pH on the formation of a flat surface. (A) is a graph showing the time taken for 80 占 퐇 of YYACAYY peptide (1 mM) to form a flat surface of 2 mm at different pH values. Under pH 3.3, peptides did not dissolve and white agglutinates were formed. When the pH was above 8, no flat surface was formed, and the most effective flat surface was formed at about pH 5 corresponding to the equipotential point of the peptide used, that is, pH 5.8 .
Figure 5 shows the effect of the volume of solution used for peptide nanostructure formation on the rate of planar surface formation. Different volumes of the solution were dropped onto the glass and the time until the size of the flat surface reached 2 mm was measured. The lower the drop volume, the higher the flatness rate, which is believed to be due to the accelerated evaporation of water at the interface where the film is formed, which suggests that the rate of evaporation of water during film formation affects the rate of film formation This means that the larger the size of the water droplet, the longer it takes to evaporate, so that the rate of film formation is estimated to decrease. On the other hand, when the volume decreases below 20 ㎕, the surface tension is relatively increased and the flat surface formation rate is slowed down.
FIG. 6 is a graph showing the tendency (number of times) of film and nanofiber formation according to peptide concentration and assembly region in which nanostructure is formed using TEM and AFM measurement results. The minimum concentration of film formation was 0.22 mM, and film formation was also increased with increasing concentration. The nanofibers were formed on the bulk at a minimum concentration of 0.33 mM. The flat plane structure was observed at a concentration of 0.44 mM or more.
Figure 7 shows the effect of the number of tyrosine in the peptide on the formation of a flat plane. The relative formation rates of the planar surface were monitored at different concentrations (0.33, 0.55, 1.10, 1.65, and 2.20 mM of peptide (50 mM HEPES, pH 7.4) for each peptide with YCY, YYCYY and YYYCYYY sequences. The relative velocities were determined based on YYCYY (1.1 mM) and compared. YCY did not form a film due to the lack of pi-pi interactions. (B) shows that YYCYY effectively forms a flat plane (single flat plane) at concentrations above 1.1 mM (C) shows that YYYCYYY forms a corrugated flat plane at 1.1 mM. This is due to the pi-pi interactions due to the additional tyrosine present. This indicates that if the functional group of tyrosine is changed, the shape of the final material can be changed by controlling the pi-pi interactions.
Figure 8 shows the chemical and structural analysis results of the formed peptide films. (A) is an AFM image showing a structure in which nanosheets are stacked in layers (film formation for 20 minutes, Z range: 10 nm). (B) shows the vertical growth of the peptide film as a function of time. (C) is a result of XRD analysis after collecting the formed films as powder, and the graph in the graph is an enlarged view of the range of 13 to 56 degrees in the 2θ range. (D) represents a Raman spectrum of a monomeric peptide (black line) and a film (blue line). (*) Represent tyrosine peaks, and 503 cm ^-1 and 2568 cm ^-1 peaks represent -SS-bond and sulfhydryl group (-SH). The Raman signal was magnified in the graph in the range of 460-540 cm ^-1 .
9 is an AFM image of a YYACAYY film having a multilayer structure. (A) is a photograph of a film transferred from the interface to mica after 20 minutes from the start of film formation, and the edge shows a stepped shape. This indicates that the nanosheets of the single layer are continuously stacked to form a multilayered structure with a sharp edge and a flat surface indicating that the nanosheet has a well-ordered structure.
(B) shows that the formed peptide film is uniform, smooth and has a thickness of 320 nm to 365 nm. The film thickness increased with time. 80 [mu] l of peptide solution (2 mM) in silicone-treated glass was left for 30 minutes to form a flat surface, and the formed film was transferred to mica and analyzed.
10 is an AFM image of a thin film formed by a peptide of YYACAYY. The membrane has a thickness of about 1.4 nm (1.1 mM, Z range: 10 nm) and the graph in the image is the cross-sectional analysis of the red line.
Figure 11 is a TEM image of a film formed using YYACAYY peptide at different concentrations (0.11-1.1 mM). (A) is a schematic representation of the location of the sample taken at the air / water interface for TEM analysis. (B) shows images of nanostructures formed at different concentrations, taken 30 minutes after the start of film formation, showing an increase in film size with increasing concentration. The minimum amount required for film formation is about 0.22 mM. It can be seen that the thickness increases as well as the lateral expansion as the concentration increases. At 1.1 mM, the size of the film grows to cover the entire area of the grid (97 μm × 97 μm).
12 is a TEM image of coexisting nanofibers formed using a YYACAYY peptide. (A) is a schematic representation of the location of the sample taken from the bulk phase solution for TEM analysis. (B) are TEM images of the peptides at different concentrations of 0.33, 1.1, and 1.43 mM. This suggests that formation of nanofibers is preferred in the bulk phase, and film formation is preferred in the water-air interface, thereby determining the type of nanostructure formed depending on the position of the peptide. This is because at the interface, the hydrophobic tyrosine residues of the peptide are arranged on one plane due to the presence of the air phase, and the tyrosine positions are different in the bulk phase to form nanofibers. The formation of nanofibers started at a concentration of 0.33 mM and the amount of nanofibers formed as the concentration increased. When the concentration exceeded about 1.43 mM, the twisted nanofibers were tangled and coiled together in a helix form.
Fig. 13 shows the results of X-ray diffraction analysis of YYACAYY peptide. Because the film is not a single crystal and is difficult to analyze at the atomic level, it is matched with the known protein structure and calculated results according to the present invention. The large and strong peak at 13.5 Å represents the thickness of the layer, which is close to the typical thickness of 14.0 Å observed in the thinnest layer by AFM analysis. 6.8 A represents the Cα-Cα 'distance, which is the interval between the cysteine residues of the YYACAYY dimer linked by a disulfide bond (Cα-CH ₂ -SS-CH ₂ -Cα'), similar to the typical interval found in conventional proteins Do. The 4.8 Å spacing is similar to the distance of the face-to-edge structure in the tyrosine-tyrosine bond. The 3.4 Å spacing corresponds to the diameter of the a-helix strand in the backbone direction.
Fig. 14 shows the mechanism of nanostructure formation of a peptide having a YYACAYY sequence. (A) represents the chemical structure of YYACAYY. (B) is a result of a simulation (500-ps molecular dynamics) of the conformation of the peptide with the YYACAYY sequence. The hydrophobic phenol ring (red) and the sulfhydryl group (-SH) Is located. (C) shows the structure of the YYACAYY peptide dimer observed in two different aspects. Indicates that the plane formed by two Tyr2 and two Tyr6 is perpendicular to the plane formed by two Tyr1 and two Tyr7. (D) shows the structure of the YYACAYY peptide assembled transversely, and the four neighboring dimers are associated by tyrosine mediated pi-pi interactions, indicating that nanosheets are continuously deposited. The dotted line is an enlargement of the interaction between the hydroxyl group of the phenol group of Tyr1 and the plane of the phenol group of Tyr7. Tyr2 and Tyr6 were expressed using a ball-and-stick model. (E) is the detailed structure of the YYACAYY peptide grafted horizontally. It is the result of analyzing the geometrical structure of two dimers (YYACAYY - YYACAYY) in order to understand the mechanism of association between peptides in the film formation process. The helix structure is green, the tyrosine residue is red, and the dimer is blue. Each monomer was designated A, B, C, and D, respectively. When the first dimer (left A, B) is closely packed with the second dimer (right C.D), Tyr7 of monomer A is associated with Tyr1 of monomer C through face-to- , And this action is equally applied to Tyr1 of monomer B and Tyr7 of monomer D. Tyr2 and Tyr6 are represented by the ball-and-stick model. (F) is a nanosheet structure in which dimeric peptides associated with pi-pi interactions are successively stacked.
15 shows the results of HPLC-ESI mass spectrometry of YYACAYY monomers and films. (A) shows that the monomer (purity:> 97%) before forming the nanostructure has a peak at 8.78 min and mass data (916.5). (B) showed a new peak at 10.88 min as a result of the formation of the nanostructure by the peptide at the water / air interface, and it was confirmed by mass spectrometry (1830.5) that there was a disulfide bond in the peptide film .
16 shows the results of MALDI-TOF (matrix-assisted laser desorption / ionization time of flight) analysis for YYACAYY monomers and formed films. The appearance of a peak corresponding to the dimer at the water / air interface after film formation indicates that crosslinking was formed by the side chain of cysteine, and this peak disappeared when dithiothreitol (DTT) was added to the peptide solution. Indicating that disulfide bonds are important for the formation of peptide nanostructured films.
17 is a graph showing the time taken to form a flat surface according to the concentration of the reducing agent. After adding various amounts of β-mercaptoethanol to the YYACAYY peptide solution at a concentration of 1.1 mM, the time required to form a flat surface of 2 mm in diameter was compared. As shown in the results, the concentration-dependent increase in the time taken to form a flat plane (the result is the result of three independent experiments, bars represent the standard deviation).
18 is a graph showing the ability of the peptide nanostructure to recover self-damage. (A) shows that the process of forming a peptide nanostructure is photographed with a black-and-white photograph of a close-up camera, indicating that the damage (17 minutes) caused by the surface tension during the formation process is restored (26 minutes). (B) shows that even if part of the peptide nanostructure formed at the interface between water and air is artificially damaged, it is restored to within 10 minutes.
FIG. 19 shows that a film nanostructure formed of a peptide of the YYCYY sequence is transferred to another substrate, which is capable of forming a large-area peptide nanostructure and can be easily transferred onto another substrate.
Figure 20 shows 2D crystals formed by YYACAYY peptide and polypyrrole. (A) is the TEM image of the multilayer sheet on the carbon grid, and (B) is the AFM analysis result. The 2D sheet was prepared by adding ammonium persulfate to the pyrrole and YYACAYY peptide (1.1 mM) solution. The morphology of the resulting film was analyzed in the Height mode of the AFM. Cross-sectional area (red line) The analysis shows that the sheet is very flat and exhibits a homogeneity of 5 nm in thickness (internal graph). (C) is the HRTEM image of the edges of the stacked and folded sheets. The acceleration voltage of the TEM was 300 kV. (D) is a high-power lattice image of a single sheet, indicating that the crystal structure is highly ordered. Represents a hexagonal pattern resulting from the Fourier transform of the image (interior).
Figure 21 shows the generation of tyrosine radicals in the peptide film formed by YYACAYY induced by galvanostatic or Cu (II) in a biomimetic catalytic process and thereby promoting the oxidative polymerization process of pyrrole. a) shows the cyclic voltammetric (CV) polarization curve for the YYACAYY film on the FTO glass in 0.1 M NaCl electrolyte. A broad peak (*) with a maximum current of 0.9 V (versus NHE) was generated due to the electrochemical oxidation of tyrosine ((Tyr → Tyr)), and b) Shows the current density profile of the YACAYY peptide film (red) and the tyrosine monomer (blue) on the FTO substrate during bulk electrolysis, and after 30 minutes of electrolysis as evidenced by discoloration of the substrate (inset) Were observed; c) is a photograph of a polypyrrole / peptide film stably suspended in neutral water. Using a peptide film formed on the surface of water containing 4 mM CuCl ₂ , the pyrrole polymerization reaction could be induced and the atmospheric pressure (7 mmHg ), It was confirmed that the pyrrole existing in the vapor state at the air / peptide interface warmed through the polymerization reaction. d) schematically shows the mechanism associated with the Cu (II) reduction, tyrosine radical production and pyrrole oxidation processes, which can be accomplished by addition of any oxidizing agent due to Cu (II) reduction in distilled water or by addition of a tyrosine radical Tyr) is spontaneously induced, and then pyrrole polymerization is initiated by a tyrosine radical.

In the formation of self-assembling peptide nanostructures, the present invention rapidly forms various nanostructures in various environments through self-assembly of peptides of a specific sequence. Such a structure has a very regular and dense structure and has a structure in the tyrosine residue To promote chemical and electrochemical reactions, in particular to promote redox reactions.

Also, the peptide nanostructure comprising the compound of formula (1) according to the present invention can be easily prepared into various structures in water or various organic-inorganic buffer solutions, particularly in a face-to-face structure, And as supports or molds necessary for the production of polymeric materials.

Accordingly, in one aspect, the present invention relates to biomimetics with redox-catalyzed redox reaction-promoting scaffold or oxidation-reduction catalytic activity formed by the peptide of formula 1 of X ¹ a-Zn-X ² b .

[Chemical Formula 1]

X ¹ a-Zn-X ² b

Wherein X ¹ and X ² are each independently a D- or L-type natural or unnatural amino acid having a side chain capable of pi-phi interactions, X ¹ and X ² are the same or different, and Z is A and b are the same or different integers of 1 to 10, n is an integer of 1 to 10, and n is an integer of 1 to 10, Lt; / RTI > In one embodiment according to the present application, n is 1, and a and b are each an integer of 2 to 5, and may be the same or different. In another embodiment according to the present application, a and b are the same integer between 2 and 5.

As used herein, the term " amino acid " includes 20 natural and non-natural amino acids, amino acids that are modified in vivo after translation, such as phosphocerine and phosphothreonine; And other rare amino acids such as 2-amino adipic acid, hydroxy lysine, nor-valine, nor-leucine; Including those enriched for enhanced cell permeability or in vivo stability, including both enantiomeric D- and L-forms.

As used herein, the term natural and non-natural refers to compounds in the form found in cells, tissues or in vivo, the latter of which means that artificial modifications have been made to these compounds for various purposes.

As used herein, the term " peptide " is intended to include both native amino acids or their degradation products, synthetic peptides, recombinantly produced peptides, peptide mimetics (typically synthetic peptides), and peptoids and semipeptides Peptide analogs and modifications to enhance cell permeability or in vivo stability. Such modifications include, but are not limited to, N-terminal modifications, C-terminal modifications, peptide bond modifications such as CH ₂ -NH, CH ₂ -S, CH ₂ -S═O, CH ₂ -CH ₂ , Side chain modifications. Methods for preparing peptide mimetic compounds are well known in the art and can be found in, for example, Quantitative Drug Design, CA Ramsden Gd., Choplin Pergamon Press (1992).

As used herein, the term " pi-pi interactions " refers to chemical bonds formed by pi electrons resulting from the superposition of two pi orbital functions. Thus, the compounds of formula (1) herein include amino acids having such interactable moieties, for example amino acids with aromatic side chains, such as tyrosine, tryptophan or phenylalanine, and variants, derivatives or analogs thereof.

As used herein, the term " cross-linking " includes covalent bonds or coordination bonds with metals, which can lead to association of molecules by bonding of adjacent peptide molecules. For example, a disulfide bond through an -SH group of an amino acid side chain, and the amino acid capable of coordination bonding with the metal is selected from the group consisting of cysteine, homocysteine, penicillamine, histidine, lysine, ornithine, arginine, aspartic acid, glutamic acid, asparagine , Or glutamine, but are not limited thereto.

In the compound of formula (I) of the present invention, X ¹ a and X ² b have an amino acid sequence of the same sequence or symmetrically symmetric with respect to Z, wherein a and b may be the same. NX ¹ a-Zn-X ² bC, and CX ¹ a-Zn-X ² bN directions, wherein N - means the amino terminus of the peptide and C - means the carboxy terminus.

The compound of formula (1) according to the present invention can form a nanostructure through stable binding through self-assembly among the compounds. In particular, in the tyrosine symmetric structure having a -SH group in the center, the pi bonds have a two-dimensional structure and can provide a bonding angle at which the layer can accumulate (see Figs. 13 to 17, etc.)

The compounds of formula (1) constituting the nanostructure according to the present invention may be the same or different. A peptide having one sequence or a peptide having two or more sequences can be used in the preparation of the peptide nanostructure according to the present invention.

In one embodiment, the peptide of formula 1 herein is selected from the group consisting of YYCYY, YYYCYYY, YYACAYY, YYAACAAYY, YYGCGYY, YFCFY, FYCYF, FFCFF, YYAKAYY, YYAEAYY, YYYY, YYCYYY, YYYYY, YYDCDYY, YYAHAYY, YYHYY, YYAPAYY , Or YYPYY or a derivative thereof, but other peptide sequences forming a nanostructure having the characteristics of the present invention may also be included. In an embodiment of the present invention, the derivative includes an acyl derivative containing an acetyl group at the N-terminus or a derivative having an amide group at the C-terminus. Y is tyrosine, C is cysteine, A is alanine, P is proline, K is lysine, E is glutamic acid, D is aspartic acid, H is histidine, H is a single letter of amino acid, , G is glycine, and F is phenylalanine.

The nanostructure according to the present invention can be self-assembled. As used herein, the term self-assembly refers to the process by which materials such as atoms, molecules or peptides form a regular-shaped structure in a particular environment. As used herein, the term " nanostructure " is intended to include fibers, tubular, spherical, or film, having a diameter or cross section of 1 μm or less, particularly 500 nm, more particularly less than 50 nm and even more particularly less than 5 nm. And in one embodiment according to the present application is between 200 nm and 1 nm. In the case of a tubular or fiber, it means that its length is at least 1 μm, in particular at least 10 nm, more particularly at least 100 nm, even more particularly at least 500 nm.

The peptides of the present invention can be formed into various forms depending on the conditions of nanostructure formation. For example, a peptide having a concentration of 2 mM of the same sequence can be produced in a two-dimensional structure at the interface, in a solvent, or in a fiber or spherical form depending on whether it is an external stimulus (ultrasound) or not. For example, the film structure is formed at the liquid-gas interface, and the nanofibers are formed on a liquid or liquid-liquid bulk (see FIGS. 11 and 12).

Films formed from the peptides herein include facets and planar structures. The planar surface means a film formed by overcoming the surface tension of the peptide solution as shown in Fig. 1 or 2, and the face surface means a film formed along the surface of the solution maintained by surface tension.

The peptide nanostructure according to the present invention can be easily formed into a large area by self-assembly, and the peptide monomer can be structurally modified by a disulfide bond, a coordination bond by a metal ion, an ionic bond, a hydrogen bond and a hydrophobic bond with another peptide monomer It grows in a two-dimensional structure, a nanostructure, that is, a plane. Cross-linkable amino acids associate peptides through covalent bonds with other amino acids and form nanostructures with more firm planar surfaces. These bonds result in the formation of dimeric peptides, while peptide peptides are continuously accumulated due to binding of amino acid side chains capable of pi-phi interaction in the dimeric peptides and side chain edge bonds, resulting in a stable nanostructure through lateral and vertical expansion (See Figs. 13 to 17). Thus, in one embodiment, the film-form nanostructure herein has a single layer or multi-layer structure. In one embodiment, the thickness of the monolayer structure is about 1.4 nm, and if multiple layers are available, various thicknesses are possible, depending on the number of layers, in one embodiment about 50 to 400 nm (see Figures 8, 9,

The compound of Formula 1 contained in the self-assembled nanostructure according to the present invention may vary, and the rate of formation or the structure of the nanostructure may vary depending on the volume of the solution used, the sequence of the peptide, and the concentration 5, 6, and 7). In one embodiment according to the present disclosure, the self-assembled nanostructure is a flat surface and the compound of formula 1 is used at a concentration of at least about 0.1 mM, more particularly at a concentration of about 0.1 mM to 10 mM, especially at a concentration of about 0.2 mM to 10 mM, 0.22 mM. In another embodiment according to the present application, the self-assembled nanostructure is a nanofiber, and the compound of Formula 1 is used at a concentration of at least about 0.1 mM, more particularly at a concentration of about 0.1 mM to 10 mM, especially at a concentration of about 0.2 mM to 10 mM, 0.33 mM.

The nanostructures formed by the peptides according to the present invention are formed on the bulk of the liquid-gas interface or liquid-liquid. The liquid forming the liquid-gas interface according to the present invention is a volatile or non-volatile hydrophobic or hydrophilic solvent and the gas includes but is not limited to air, nitrogen, helium, hydrogen, carbon monoxide, or carbon dioxide. The liquid-liquid bulk phase according to the present invention is formed in a hydrophilic and hydrophobic liquid interface in one embodiment, and the liquid is a volatile or non-volatile hydrophobic or hydrophilic solvent.

Various solvents capable of forming the nanostructure according to the present invention can be used, and for example, the volatile or nonvolatile hydrophobic or hydrophilic solvent constituting the liquid-gas or liquid-liquid bulk phase according to the present invention can be, for example, water But are not limited to, alcohols, hydrocarbons including halogens, dimethylsulfoxide, dimethylformamide, dimethyl formacetamide, N-methylpyrrolidone, acetonitrile, tetrahydrofuran, dioxane, or organic acids .

The interface at which the nanostructure of the present invention is formed may be hydrophilic or hydrophobic. In the case of the peptide compound of the present invention, the lipophilic moiety has a lipophilic moiety, and the moiety having hydrophilicity toward the air is directed toward the aqueous solution and the molecules are aligned. In one embodiment according to the present invention, the compound of formula (I) forms a nanostructure at a pH of about 3 to 8, particularly about isoelectric point, including isoelectric point (see FIG. 4).

In the natural world, a variety of amino acids or peptides can combine with each other to achieve a very rapid chemical reaction using metals that are not catalytically active, such as iron or copper. Until now, these natural phenomena have not been used in real industries. This is because there are no mediators available to facilitate the electron transfer phenomenon, that is, no mediator that can deliver electrons or radicals with a maximum travel distance of only 10 nanometers to the desired location .

The tyrosine-based peptide structure according to the present invention has a structure in which the peptide is self-assembled on the basis of ingrowth, thereby favoring electron transfer. Such self-assembled peptide constructs can act as a radical transporter. Furthermore, since it can be controlled in a desired structure such as wire, tube, sphere, film and thin film, it can be applied to various fields. Also, the use of such tyrosine-based peptide nanostructures lowers the oxidation-reduction transition of commonly used metals such as inexpensive and safe metals such as iron, copper, and zinc, as well as increases chemical catalytic reactivity, Or expensive heavy metal catalysts such as palladium and ruthenium that have been widely used in the industry, and can also be used as an electronic material such as an electron transport layer, which is an essential element for an organic light emitting diode.

The peptide nanostructure according to the present invention functions as a support and functions as a redox reaction enzyme mimetic by promoting a chemical or electrochemical reaction, for example, a redox reaction.

In one embodiment, the nanostructure according to the present invention promotes a redox reaction. As used herein, the term " facilitation " means that the rate of a particular specific reaction is faster or less possible than in the case where the nanostructure according to the present invention is not used. As used herein, the term " catalyst " refers to a substance that rapidly induces a reaction in a reaction such as oxidation-reduction where the former loses electrons through the electron transfer between the substances and the latter is reduced.

The peptide nanostructures according to the present invention also function as "supports", "scaffolds", "platforms" or "templates" due to their regular structure, making them easy to manufacture in water or various organic inorganic buffers in various structures, And can be used as a platform for nanostructure fabrication with adjustable functionality and as a template for the production of polymeric materials.

As used herein, the term "mimetic," "biomimetic," or "biomimetic," refers to a compound that mimics the activity of an enzyme in vivo, including peptides. The mimetics may also include amino acids linked by non-peptide or non-peptide linkages, such as pi bonds (see, for example, Benkirane, N., et al. J. Biol. Chem., 271: 33218-33224 (1996) and US Pat. No. 5,637,677) or crosslinking. In one embodiment according to the present disclosure, the peptide mimetics according to the present invention can form films, nanofibers, or spheres. In another embodiment, the peptide mimetics according to the present invention may form a planar surface or a facing surface, and in one embodiment, the planar surface or facing surface includes a single layer or a multi-layer structure.

As described above, the support according to the present invention has redox catalytic activity, and the support and mimic are used interchangeably in accordance with the context.

While not intending to be limited to this theory, the support or mimic according to the present application is based on an electrochemically generated tylosil radical. Also, though not limited to this theory, the generation of the tirosyl radicals in the peptide nanostructures according to the present invention plays a key role in efficient electron transfer as shown in photosystem II. For example, tirosyl radicals are also capable of cooperating with the citetene residues as observed in ribonucleotide reductase and galactose oxidase. The class I ribonucleotide reductase forms radicals continuously from multiple tyrosine groups with Mn (IV) or Fe (IV) ions and oxidizes the cysteine residues to form sulfhydryl radicals to form deoxyribonucleotides Reduce to the ribonucleotide. Galactose oxidase reduces oxygen to hydrogen peroxide by using a tyrosyl radical (Y ㆍ) produced by Cu (II) ion and converts primary alcohol to aldehyde.

Thus, despite the biological importance of tyrosine, its use in the field of material synthesis is challenging. The reason is that it is difficult to obtain a well-defined structure (macroscopic structure) because it is easily quenched in the reaction medium.

Thus, the peptide nanostructure comprising tyrosine according to the present invention, which forms a very regular dense structure, can be easily grounded with any base electrode and systematically investigate the regular arrangement according to the sequence.

In one embodiment, the peptide nanostructure herein can act as an enzyme-mimicking catalyst support or scaffold with redox-catalyzed ability. Peptide nanostructures according to the present invention catalyze chemical reactions in a manner similar to that found in biological systems.

In one embodiment, the peptide nanostructure of the present invention shows that radicals are readily formed through cyclic voltammetric (CV) polarization curve experiments at 0.9 V (versus NHE) (see FIG. 21 a). In general, polypyrrole is synthesized using an oxidizing agent such as dislocations or Fe (III) ions in excess of 1.0V. In addition, it was difficult to deposit polypyrrole having a macro structure due to a slow reaction rate even in the reaction using 0.9 V potential (see FIG. 21 b). However, as a result of using the peptide nanostructure (film) according to the present invention, it was confirmed that the current increased to 68 μA after 300 seconds, and a black polypyrrole film was produced. In this reaction, the peptide film of the present invention mediates electron transfer from the pyrrole to the electrode in the FTO substrate, thereby lowering the energy barrier in the oxidation polymerization reaction. In another embodiment, in an experiment mimicking galactose oxidase, it has been shown that radicals are produced by a change in the valence of the copper ion, without the need to supply additional voltage. When the pyrrole was polymerized with polypyrrole in the presence of a film formed of YYACAYY according to one embodiment of the present invention, the activity of copper ions with remarkably different characteristics was observed. The peptides were generated in a 4 mM Cu (II) chloride solution and then exposed to pyrrole vapor. As a result, a black polypyrrole film was gradually formed on the peptide nanostructure according to the present invention. After the overnight reaction, the polypyrrole and the peptide nanostructure, A hybrid film capable of being free-standing was formed (see Fig. 21C). On the other hand, when the tyrosine monomer was used, the polypyrrole film could not be formed even after several days in the copper (II) chloride solution. This result indicates that the Cu (II) ion causes pyrrole to be oxidized by the peptide film according to the present invention, which is considered to be the cause of the electron transfer mediated by the tyrosyl radical (see FIG. 21 d). The polypyrrole film was formed at the air / peptide structure interface, indicating that electrons were transferred from the vapor pyrrole to Cu (II) in solution through the YYACAYY film.

Further, the peptide nanostructure according to the present invention can promote various redox reactions as a biomimetic entity, and various metals may be used together as a cofactor depending on the specific reaction involved. Such metals include, but are not limited to, for example, gold, silver, manganese, iron, copper, magnesium, zinc, cobalt, and silicon.

The self-assembled peptide nanostructure according to the present invention can be produced by the above-described description and the following methods, but is not limited thereto. In describing the method of the present invention, the overlapping parts of the above description are omitted in order to avoid the complexity of the description.

As described above, the self-assembled peptide nanostructure according to the present invention can be formed into various structures under various conditions including interfacial conditions formed by various solvents and / or gases, concentration of peptides, and / or pH. 4 to 12, etc.). In one embodiment according to the present disclosure, favorable conditions for the formation of the self-assembled nanostructure are those wherein the compound of formula 1 is dissolved in a liquid-gas or liquid-liquid bulk at a concentration of at least 0.2 mM, Means that the compound is reacted at a pH including the isoelectric point of the compound of formula (1) in an oxidizing atmosphere such as the above. The pH including the isoelectric point means a pH in the vicinity of the isoelectric point, which is about pH 4 with respect to the pI of each peptide used for forming the nanostructure, and is about pH 3 to 8 in one embodiment according to the present invention.

The monolayer of the unit peptide nanostructure according to the present invention has a thickness of about 1.4 nm. As the monomer / dimer remaining in the solution continues to undergo nanostructuring reaction over time, the layer is piled up on the formed nanostructure and thickened do. This process can be confirmed by matching the thickness of the theoretical peptide nanostructure with the thickness of the experimental peptide nanostructure. Therefore, the thickness of the present peptide nanostructure can be adjusted according to the reaction time (see FIGS. 8 to 10)

 It is also possible to control the type of the nanostructure to be formed, as described above. In one embodiment according to the present application, for example, the self-assembled nanostructure is a film or nanofiber structure, the film is formed at a liquid-gas interface, and the nanofiber structure is formed on a bulk of a liquid or liquid-liquid.

In the present application, a rigid nanostructure comprising a planar surface is formed by the above-described mechanism at the liquid-gas, liquid-liquid interface. In this regard, in order to form the nanostructure more quickly, an inorganic substance such as a metal ion including gold, silver, copper, zinc, iron, cobalt, and manganese is added, or an organic substance including various monomolecules or polymers is added The binding between the peptides is increased to promote the formation of the nanostructure.

While not intending to be limited to this theory, in the peptide nanostructure according to the present invention, as the molecular structure of the peptide monomer is stabilized, the amino acid having the hydrophilic group and the amino acid having the hydrophobic group are positioned opposite to each other, As you head toward the water, you will come to the surface. Thus, a peptide nanostructure having a two-dimensional structure is formed at the interface between water and air. Peptides are bound to each other via pi-pi bonding and / or hydrophobic bonding, and crosslinked to form a rigid nanostructure. As the reaction is repeated, a peptide nanostructure having a two-dimensional structure is stacked to form a multi-layered structure (see FIGS. 13 to 17).

In addition, the peptide forms a planar surface between the interface and the interface, especially between water and air, and can form a large-area nanostructure in centimeters.

Further, the present invention can also produce a conductive polymer film using a peptide self-assembled nanostructure formed by the present compound as a template (see FIG. 20). The conductive polymer film according to the present invention is mixed with various polymerizable materials, and is produced through a polymerization process such as oxidation, for example, and has a well-ordered crystal structure.

In another aspect, the present invention also relates to a process for preparing a polymeric film using a catalyst comprising a peptide nanostructure of Formula 1 according to the present invention, and to a molecular film produced thereby.

In addition, the nanostructure formed using the peptide of Formula 1 according to the present invention can be complexed with gold, silver, copper, zinc, iron, cobalt, manganese and the like, and can be simultaneously aligned in a two-dimensional structure. In the case of a peptide nanostructure added with such a metal, it may be a conductor, a semiconductor, or an insulator depending on the kind of the metal.

Therefore, the polymer film according to the present invention may have conductivity, anti-conduction property or non-conductive property depending on the manufacturing process.

The polymer film according to the present invention can be produced from a polymerizable material that can be polymerized by a redox reaction depending on the final properties and uses, and various polymerizable materials can be used. For example, but not limited to, pyrrole, aniline, thiophene, or 3,4-ethylenedioxythiophene. The compound of formula (I) and the polymerizable material may be used in various ratios, for example, but not limited to, a molar ratio of 1: 1.

In one embodiment according to the present disclosure, when the peptide nanostructure according to the present invention comprises tyrosine, it can help to transfer electrons to other objects well. Accordingly, the peptide nanostructure according to the present invention can be used as an electron transfer layer / film or a nanostructure that helps to transfer electrons well. Therefore, when a high-priced metal inorganic material having a specific electrical property is required to be used In comparison with. There is a great advantage in cost. This means that even if a small amount of metallic inorganic material is used in conceptually, it can be provided with superior electrical properties when applied on the nanostructure according to the present invention.

Furthermore, the flat plane between the interfaces seen in the peptide nanostructure according to the present invention is known to be involved in the intercellular fusion reaction in recent years, so that it is very important in biological applications.

In addition, the peptide nanostructure according to the present invention has an advantage that it can be produced conveniently at a relatively low production cost. Unlike, for example, indium tin oxide (ITO), carbon nanotube (CNT), and graphene production, it is possible to produce water or various organic / inorganic buffers without special equipment / equipment. In addition, the addition of metal ions allows rapid formation of solid nanostructures, which can be applied in many materials requiring metal deposition.

In another aspect, the present invention also provides a method for promoting redox reaction characterized by using a redox reaction promoting support according to the present invention. As described above, a person skilled in the art will appreciate that the peptide nanostructures according to the present invention can be used in a variety of ways depending on the kind of specific reaction desired, Method.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

[Materials and Methods]

compound

(100-200 mesh, 1.26 mmol / g) resin, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluoro (HIPt), hydroxybenzotriazole (HOBt) and Fmoc-protected amino acids were purchased from BeadTech (Seoul, Korea), and N, N-diisopropylethylamine (DIPEA), triisopropylsilane ), 3,6-dioxa-1,8-octanedithiol (DODT), 4- (3,6-dimethyl-1,3-benzothiazol- Dimethylaniline chloride (thiopurine), β-mercaptoethanol, HEPES buffer, eurosyl acetate, and anisole were purchased from Aldrich (USA). Dichloromethane (DCM), N, N-dimethylformamide (DMF), methanol, piperidine, diethyl ether and trifluoroacetic acid (TFA), sodium hydroxide (NaOH) (Korea) and hydrogen peroxide (H ₂ O ₂ , 30%) were purchased from Junsei (Japan).

Atomic force microscope (AFM) analysis

A peptide aqueous solution drop (1 mM) was placed on a glass surface-treated glass and allowed to stand at room temperature for 30 minutes to form a peptide two-dimensional structure at the interface, and a peptide two-dimensional structure was transferred to a clean silicon wafer or mica surface to obtain a surface. To remove the salt transferred from the solvent, the peptide nanostructure adsorbed on the silicon wafer or mica surface was rinsed with distilled water and allowed to dry for 10 minutes. To obtain detailed photographs, a scanning electron microscope (Dimension 3100, Veeco Instruments, Woodbury, NY) was scanned at a rate of 0.5 Hz using a can travertip tip with a spring constant value of 21-78 N / m to obtain a peptide nanostructure Respectively.

Transmission electron microscope (TEM) analysis

20 占 퐇 of the peptide nanostructure was dropped on a copper mesh coated with 200 mesh carbon and dried for 5 minutes. The remaining solvent was removed using filter paper, and the peptide nanostructure was stained with 2% Uranyl acetate dyeing reagent (Electron Microscopy Sciences). The grid was then placed in a desiccator to remove even moisture. And a peptide nanostructure was confirmed using an 80 kV transmission electron microscope (JEOL, JEM-1010).

Fluorescence imaging

In order to stain the peptide nanostructure with thiopeptide and confirm with an optical microscope, 2 mM peptide was dissolved in a 20 mM HEPES buffer solution (pH 7.4) at 90 DEG C for 30 minutes, and 250 μM of thiopurine was added thereto. The peptide nanostructure was dropped on the glass surface to form a two-dimensional structure at the interface. The peptide nanostructure was transferred to a clean glass surface and then placed in a desiccator for 2 hours to remove water. Fluorescence-stained peptide nanostructures were confirmed by excitation at 430 ± 10 nm on a fluorescence microscope (Deltavision RT (Applied Precision, USA) microscope, Olympus IX70 camera).

Computer Modeling Research

Computer predictive simulation of all peptide nanostructures was predicted using the Discovery Studio program (Version 3.1, Accelrys Inc., San Diego, Calif.). Each environment was measured by a molecular behavior predictor in a Discovery Studio program called CHARMm (Chemistry at Harvard Macromolecular Mechanics). The peptide molecular structure was heated and stabilized at 0.001 ps for 10 ps at 5-1,000 ° K.

MALDI-TOF mass spectrometry

10 mg of 2,5-dihydroxybenzoic acid was dissolved in 1 ml of a solvent in which ion-free water and acetonitrile containing 0.1% trifluoroacetic acid were mixed in a ratio of 3: 7. The peptide solution (1 mM, 0.5 ㎕) was dropped on a Maltitof plate, and 20 mM dithiothreitol (DTT) (20 mM, 0.5)) and ammonia water (8 mM, 0.5 ㎕) . All results were obtained using an Autoflex MALDI-TOF mass spectrometer (Bruker Daltonics), Smartbeam laser, and FlexControl software (Bruker Daltonics).

Electrospray ionization (ESI) mass spectrometry

The peptide two-dimensional structure was dissolved in methanol containing 0.5% acetic acid and purified by high performance liquid chromatography electrospray ionization mass spectrometry (Thermo-Finnigan LCQ deca XP MS ESI-MS), Phenomenex C4 reverse-phase column , 100 x 3 mm). The molecular weight of acetonitrile was measured by changing the water content of acetonitrile to 10-65% for 21 minutes.

X-ray diffraction (XRD) analysis

Tide nanostructures (1 mM, 1 ml) were formed on the glass surface at the air / solution interface, transferred to a silicon wafer, and allowed to dry for 24 hours. Thereafter, the distance between the peptides was measured at 3 ° to 60 ° using a reflection mode (Rigaku, D / max 2200 VK) at room temperature.

FT-IR (Fourier transform infrared) spectroscopy

Peptide nanostructures (1 mM, 1 ml) were formed on the glass surface at the air / solution interface, transferred to a silicon wafer and allowed to dry for 24 hours. After that, the peptide nanostructures were observed at 300 cm ^-1 to 4,000 cm ^-1 (with the aid of an infrared measuring device (Thermo Nicolet 6700 FT-IR spectrometer, attenuated total reflection (ATR) accessory) resolution, 1.93 cm ^-1 ) was measured by 8 repetitive scans.

Raman Spectroscopy

Peptide nanostructures (1 mM, 1 ml) were formed on the glass surface at the air / water interface, transferred to gold-coated silicon wafers and dried for 24 hours. The measurement range of 200 cm ^{- 1} to 3000 cm- ¹ was then measured for 300 seconds in a Raman analyzer (Horiba Jobin-Yvon / LabRam Aramis Raman spectrometer (diode laser 785 nm, power = 1 mW)).

Example 1 Peptide Synthesis

The peptide was synthesized with 2-chlorotrityl chloride (CTC) resin (1.26 mmol / g) using the Fmoc / tBu method. After the first amino acid was loaded (0.3-0.5 mmol / g), each coupling step consisted of 2 equivalents of Fmoc-amino acid, 2 equivalents of HBTU, 2 equivalents of HOBt and 4 equivalents of DIPEA, For two hours. The solvent was DCM and DMF in a ratio of 1: 1. The Fmoc group was removed by reaction with 20% piperidine / DMF for 20 min. The final peptide synthesized was cleaved in CTC resin by reaction with 93% TFA, 2% TIPS, 2% DODT and 3% The cut mixture was then filtered, washed with DCM and methanol, and the filtrate was concentrated in vacuo. The resulting residue was precipitated with cold (<10 ° C) diethyl ether, then centrifuged and vacuum dried. Peptides were purified, if necessary, by reversed phase HPLC and confirmed by MALDI TOF and ESI mass spectrometry. The purity of all synthesized peptides was over 95%.

1-1. Synthesis of Tyr-Tyr-Cys-Tyr-Tyr (YYCYY)

On a polymeric support Tyrosine  Introduction Tyr - CTC  Resin)

Basically, the peptide synthesis proceeded as described above. 2 mmol of 2-CTC resin (substitution ratio: 1.41 mmol / g), 459.54 mg (1 mmol) of tyrosine derivative Fmoc-Tyr (tBu) -OH and 60 ml of DMF were added and mixed well. Lt; / RTI &gt; The resin was filtered, and the resin was washed three times with DMF and MC. The resin was then washed three times with DMF, MC, and methanol sequentially. Fmoc titration revealed that the desired amino acid was introduced into the resin.

Tyrosine  Introduction: Tyrosine - CTC  Coupling with resin ( Tyr - Tyr - CTC  Resin)

50 ml of 20% piperidine / DMF solution was added to the supernatant containing 2 g of the resin obtained above, and the mixture was allowed to react for 30 minutes. After three washes with DMF, MC, and methanol, the positive results of the Kaiser test confirmed that the Fmoc was lost. To 2 g of the obtained resin, 919.08 mg (2 mmol) of Fmoc-Tyr (tBu) -OH, 270.2 mg (2 mmol) of HOBt and 758.5 mg (2 mmol) of HBTU were added and then 60 ml of DMF was added. 662 [mu] l (4 mmol) of DIEA was added thereto and stirred for 2 hours. After the reaction, the resin was washed three times with DMF, MC, and methanol, and the reaction was terminated by negative results of the Kaiser test. The resin was then treated with 20% piperidine / DMF for 30 minutes to remove Fmoc.

Introduction of cysteine: Tyrosine - Tyrosine - CTC Resin Medicine  Coupled coupling Cys - Tyr -Tyr-CTC resin)

Cysteine was bound and washed using 1171.4 mg (2 mmol) of Fmoc-Cys (Trt) -OH, 270.2 mg (2 mmol) of HOBt, 758.5 mg (2 mmol) of HBTU and 662 μl (4 mmol) of DIEA. The resin thus obtained was treated with 20% piperidine / DMF as described above to remove and wash Fmoc.

Tyrosine - Tyrosine  Introduction: Cysteine- Tyrosine - Tyrosine - CTC  Coupling with resin Tyr - Try - Cys - Tyr - Tyr - CTC  Resin)

(TBu) -Tyr (tBu) -Cys (Trt) -Tyr (tBu) -OH by treating with 20% piperidine / DMF in the same manner as described above, -Tyr (tBu) -CTC resin.

Peptides  Separation from polymer scaffold

Tyr-Tyr-Cys-Tyr-Tyr was then recovered from the CTC resin as follows. Trifluoroacetate in resin: anisole: (triisopropylsilane: 3,6-dioxa-1,8-octanedithiol (93: 3: 2: 2) After removing the protecting group of the side chain and separating the peptide from the resin, the resin was filtered out, the resin was washed with MC and methanol, and the filtrate was collected. The reaction mixture was concentrated, the cold diethyl ether was added to the concentrate, and the residue was placed in a freezer to maximize the amount of precipitate. The precipitate was filtered, washed with diethyl ether four times using a centrifugal separator, The dried peptide in solid form was obtained in 85% yield.

1-2 Tyr - Tyr - Ala - Cys - Ala - Tyr - Tyr  ( YYACAYY ) synthesis

622.68 mg (2 mmol) of Fmoc-Ala-OH, 270.2 mg (2 mmol) of HOBt and 758.5 mg (2 mmol) of HBTU were added to 2 g of the Tyr (tBu) -Tyr (tBu) -CTC resin obtained in Example 1-1 And dissolved in 60 ml of DMF. 662 [mu] l (4 mmol) of DIEA was added thereto and stirred for 2 hours. After the reaction, the resin was washed three times with DMF, MC, and methanol, and the reaction was terminated by the negative result of the Kaiser test. The resin was then treated with 20% piperidine / DMF for 30 minutes to remove Fmoc.

Cysteine was coupled with 1171.44 mg (2 mmol) of Fmoc-Cys (Trt) -OH, 270.2 mg (2 mmol) of HOBt, 758.5 mg (2 mmol) of HBTU and 662 μl And washed. The resin thus obtained was treated with 20% piperidine / DMF as described above to obtain Cys (Trt) -Ala-Tyr (tBu) -Tyr (tBu) -CTC resin.

Tyr (tBu) -Try (tBu) -Ala-Cys (Trt) -Ala-Tyr (tBu) -Tyr (tBu) -CTC resin was obtained by sequential bonding of two alanines and tyrosines. Tyr-Tyr-Ala-Cys-Ala-Tyr-Tyr was recovered from the polymer scaffold by the method of Example 1-1 to obtain the title peptide in 80% yield.

1-3 Tyr - Tyr - His - Tyr - Tyr  ( YYHYY ) synthesis

On a polymeric support Tyrosine  Introduction Tyr - CTC  Resin)

(Loading level: 1.41 mmol / g), 459.54 mg (1 mmol) of a tyrosine derivative Fmoc-Tyr (tBu) -OH and 60 ml of DMF (Dimethylformamide) were added to 2 ml of a 2-CTC resin (Beadtech, , And 331 DI (2 mmol) of DIEA (N, N-diisopropylethylamine) was added thereto, followed by stirring for 2 hours. The resin was filtered and the resin was washed three times with DMF and MC (dichloromethane). The resin was then washed three times with DMF, MC, and methanol. Fmoc titration indicated that the resin was well introduced with tyrosine.

Tyrosine  Introduction: Tyrosine - CTC  Coupling with resin ( Tyr - Tyr - CTC  Resin)

50 ml of 20% piperidine / DMF solution was added to the supernatant containing 2 g of the resin obtained above, followed by reaction for 30 minutes. After washing three times with DMF, MC, and methanol, a positive result of Kaiser test showed that Fmoc was dropped. (2 mmol) of Fmoc-Tyr (tBu) -OH, 270.2 mg (2 mmol) of HOBt (1-Hydroxybenzotriazole) uronium-hexafluoro-phosphate (758.5 mg, 2 mmol) were dissolved in 60 ml of DMF. 662 [mu] l (4 mmol) of DIEA was added thereto and stirred for 2 hours. After the reaction, the resin was washed three times with DMF, MC, and methanol, and the reaction was terminated by a negative result of the Kaiser test. The resin was then treated with 20% piperidine / DMF for 30 min to remove Fmoc.

Introduction of histidine: Tyrosine - Tyrosine CTC  Coupling with resin ( His - Tyr - Tyr -CTC resin)

Histidine was bound and washed using 1171.44 mg (2 mmol) of Fmoc-His (Trt) -OH, 270.2 mg (2 mmol) of HOBt, 758.5 mg (2 mmol) of HBTU and 662 μl (4 mmol) of DIEA in the same manner as above. The resin thus obtained was treated with 20% piperidine / DMF as described above to remove and wash Fmoc.

Tyrosine - Tyrosine  Introduction: Histidine- Tyrosine - Tyrosine - CTC  Coupling with resin Tyr - Try - His - Tyr - Tyr - CTC  Resin)

Tyr (tBu) -His (Trt) -Tyr (tBu) -Tyr (tBu) -Tyr (tBu) -Tyr (tBu) -Tyr (tBu) -CTC resin.

Peptides  Separation from polymer scaffold

Anisole: (triisopropylsilane: 3,6-dioxa-1,8-octanedithiol (46.5: 1.5: 1: 1) was added to the above polymer scaffold. 1) solution was added and the reaction was carried out for 1 hour to remove all protecting groups on the side chain and the peptide was separated from the resin.After filtering the resin and collecting the filtered filtrate, the resin was washed with MC and methanol, The precipitated derivative was allowed to stand in a freezing chamber to obtain a precipitate as much as possible, washed with diethyl ether four times using a centrifugal separator, and washed with diethyl ether (Tyr-Tyr-His-Tyr-Tyr) was obtained in a yield of 70% in the form of a white solid.

Example 2 Formation of Nanostructures under Various Conditions Using Peptides of Various Sequences

2-1 YYCYY  And YYACAYY Peptides  Formation of nanostructures using

2-1-1 Formation of nanostructure on glass slide

0.5 mg, 1 mg, 1.5 mg and 2 mg of each of the peptides of the YYCYY and YYACAYY sequences obtained in Example 1 were dissolved in 50 mM phosphate buffer (pH 7.4) or HEPES buffer (pH 7.4) (4- (2 -hydroxyethyl) -1-piperazine ethane sulfonic acid, pH 7.4) and dissolved at 90 ° C. 20 μl, 40 μl, 60 μl, 80 μl, and 100 μl of this solution were dropped onto the glass surface and left at 25 ° C. in a constant humidity atmosphere to form a nanostructure on the water and air interface (FIG. 1 And Fig. 2).

2-1-2 Petri dish ( petridish ) Formation of nanostructures on top

40 mg of each peptide of the above sequence was added to 20 ml of 50 mM phosphate buffer (pH 7.4) or HEPES buffer (pH 7.4) and dissolved at 90 ° C. Thereafter, the solution was poured into a Petri dish and allowed to stand at 25 ° C in a constant humidity environment to form a nanostructure having a flat surface between water and the atmosphere on the entire surface of the Petri dish (see FIG. 1D).

2-1-3 metal ion Added Peptides  Manufacture of nanostructures

After adding 1 equivalent of iron chloride, zinc chloride, cobalt chloride, cobalt nitrate, cobalt hydrate, manganese hydroxide, or lithium hydroxide to 0.5 mg, 1 mg, 1.5 mg and 2 mg of each of the above peptides, 50 mM phosphate buffer ) Or 1 ml of HEPES buffer (pH 7.4) was added and dissolved at 90 ° C. Then, 10 μl, 20 μl, 40 μl, 60 μl, 80 μl, and 100 μl of each solution were dropped on a glass surface and allowed to stand at 25 ° C in a humidity atmosphere. As a result, Or more.

The results indicate that the addition of metal ions such as zinc, iron, cobalt, etc., can promote the formation of nanostructures due to their interaction with the peptide (results not shown).

2-2 Various Peptides  And Derivatives for the Formation of Nanostructures

The peptides of various sequences in Table 1 were synthesized in the same manner as in Example 1, and each of these peptides was dissolved in 50 mM HEPES (pH 7.4) to a final concentration of 1.1 mM. Next, for each solution of 80 占 퐇, the formation process of nanostructures was examined as in Example 2-1. The results are shown in Table 1. As shown in the following Table 1, when one or more tyrosines and one or more nucleophiles, cysteine, were bonded, it was confirmed that solid and flat solid nanostructures were well formed. Also, it was confirmed that a nanostructure was formed on the surface even when the tyrosine analog chain waveguide was used.

[Table 1]

(Y: tyrosine, C: cysteine, F: phenylalanine, A: alanine, E: glutamic acid, K: lysine, S: serine, DOPA (tyrosine derivative) Fluorenylmethyloxycarbonyl chloride]

In Table 1, the flat surface formation was measured at a room temperature until the diameter reached 2 mm, very fast: formation in 1 minute, fast: 1 to 30 minutes, slow: 30 to 60 minutes, very slow: 60 minutes or more.

Example 3 Characterization of the formed peptide nanostructure

3-1 Surface Analysis of Nanostructures Using Atomic Force Microscopy (AFM)

Each of the peptides YYACAYY and YYCYY obtained in Example 1 was dissolved to a concentration of 2 mM with 50 mM HEPES pH 7.2 buffer. This solution was completely dissolved through ultrasonic treatment (20 minutes) and heat treatment (80 占 폚, 20 minutes). 100 μl of this solution was dropped onto the glass surface and allowed to stand at 25 ° C in a constant humidity atmosphere to induce peptide nanostructure formation on the interface. The surface-cleaned mica was transferred over the peptide nanostructure and allowed to dry for 5 minutes in air. The prepared samples were analyzed by AFM (see Figures 8-10).

Through AFM analysis, the self-assembling phenomenon of the nanostructure is specifically observed, and the self-assembly shape, the relationship between the fault layers, and the thickness or height of a single layer can be known.

FIG. 8A shows a relationship between a single layer formed by partially removing a portion of a peptide nanostructure having a stacked layered structure by using a cellophane tape, and FIG. 9 is a cross-sectional view of a portion of the nanostructure as a cellophane tape And the thickness or height of a single nanostructure was measured through a section obtained by removing the nanostructure.

As a result of the AFM analysis, it was confirmed that the peptide nanostructure has a typical supramolecular structure resulting from self-assembly phenomenon. Specifically, it was confirmed that a thin layer of about 1.4 nm, which corresponds to the height of the YYCYY molecular monomer, was formed on the interface with the layer repeatedly layered repeatedly (see FIGS. 8 to 10). It is believed that the repetitive stacking of thin layers with such regular arrangement is the driving force for water to overcome the strong surface tension seen at the interface with air and to form a flat surface where the nanostructure is visible.

3-2 Through fluorescent dye staining Peptides  Characterization of nanostructures

3-2-1 Nile Red dyeing

Peptide nanostructures were stained with nile red that specifically reacted to hydrophobic environments. 2 mg / ml (2.2 mM) YYCYY, 0.2 mg / ml nile red was prepared by dissolving in 50 mM Hepes buffer pH 7.2 containing 5% DMSO. 100 μl of this solution was dropped on a glass surface and allowed to stand at 25 ° C in a constant humidity atmosphere to induce peptide nanostructure formation on the air / water interface. The formed peptide nanostructure was transferred to a cleaned surface of the glass surface and observed with a fluorescence microscope (see FIG. 3A).

Fluorescent coloring reagents used for fluorescence microscopy were Nile red fluorescent coloring reagents that fluoresce well and bind well to hydrophobic groups. As shown in FIG. 3A, it can be confirmed that the formed nanostructure is a peptide containing at least one tyrosine having a hydrophobic property.

3-2-2 Thioplavin-T (ThT) staining

In general, fluorescence analysis was performed using thioflavin T (ThT), which fluoresces by being inserted between peptide nanostructures. 1 mg / ml YYCYY dissolved in 50 mM Hepes buffer pH 7.2 was reacted with 50 μM ThT. 100 μl of this solution was dropped on a glass surface and allowed to stand at 25 ° C in a constant humidity atmosphere to induce peptide nanostructure formation on the interface. The formed peptide nanostructure was transferred to a cleaned glass surface and observed with a fluorescence microscope (see FIG. 3).

As shown in FIGS. 3B and 3C, it was confirmed that the peptide was one containing at least one tyrosine in which the nanostructure was formed.

Example 4 Characterization of complementary self-defects of formed peptide nanostructures

In the peptide nanostructure formed in Example 2, defects were formed on the surface using sharp tweezers, and self-complementary complementary properties by intermolecular self-assembly were analyzed. The prepared peptide nanostructure was found to have an ability to actively react to damage caused by external stimuli and to immediately heal the defect (see FIG. 18B). This means that the self-healing system allows for immediate self-repair of some damage to the nanostructure.

Therefore, it will be suitable for a scratch-resistant nanostructure, and when a specific molecule having a very important function is encapsulated with the nanostructure according to the present invention, it can be applied to a storage and delivery system of a specific drug by storing the drug well .

Example 5 Transition of a formed peptide nanostructure onto a secondary substrate

Transition techniques on various substrates are needed for high applicability of peptide nanostructures formed on the interface. Herein, the peptide nanostructure obtained in Example 2 is transferred onto the surface of a secondary substrate which has been cleaned. In addition, a secondary substrate is previously placed inside the peptide solution for more stable nanostructure transfer, And the nano structure naturally transfers over the secondary substrate according to the reduction of the total volume (see Figs. 1 and 19).

This is a new immersion coating method that can replace gravure coating, Meyer bar coating, or spin coating, which are widely used. In other words, it means that the nanostructure can be applied to the substrate without specific equipment. In this way, the use of expensive coating equipment and the problems of complex coating processes can be solved.

Example 6 Preparation of Peptide Structure Bonded with Metal Ion

The peptide nanostructure according to the present invention can promote various oxidation-reduction reactions as a biomimetic material. In this case, various metals may be used together as a cofactor according to the specific reaction involved. To this end, Peptide nanostructures can be prepared. The use of the peptide nanostructures according to the present invention can reduce the oxidation-reduction transition of commonly used metals such as inexpensive and safe metals such as iron, copper, and zinc while increasing the chemical catalytic reactivity, It can replace expensive heavy metal catalysts such as palladium or ruthenium.

6-1. Preparation of metal ion added peptide structure

6-1-1. Introduction of metal ions such as zinc, iron, cobalt, copper or silicon to the peptide

One equivalent of iron chloride, zinc chloride, cobalt chloride or copper chloride was added to 0.5 mg, 1 mg, 1.5 mg and 2 mg of the peptide (YYCYY or YYHYY) obtained in Example 1, followed by addition of 50 mM phosphate buffer (pH 7.4) 1 ml of HEPES buffer (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, pH 7.4) was added and dissolved at 40 ° C.

6-1-2. Introduction of copper ion into the peptide

1 equivalent of copper chloride was added to each of 0.5 mg, 1 mg, 1.5 mg and 2 mg of the peptide (YYCYY or YYHYY) obtained in Example 1, and the mixture was diluted with 50 mM phosphate buffer (pH 7.4) or HEPES buffer 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, pH 7.4) was added and dissolved at 90 ° C.

6-1-3. Introduction of other metal ions to peptides

1 equivalent of iron chloride, zinc chloride, cobalt chloride (cobalt nitrate and cobalt hydrate), manganese hydroxide, lithium hydroxide and the like were added to 0.5 mg, 1 mg, 1.5 mg and 2 mg of the peptide (YYCYY) obtained in Example 1, 1 ml of phosphate buffer (pH 7.4) or HEPES buffer (4- (2-hydroxyethyl) -1 -piperazineethanesulfonic acid, pH 7.4) was added and dissolved at 40 ° C.

Example 7. Oxidation-Reduction Using Peptide Structure: Low Redox Transition Confirmation

7-1. Polymerization of pyrrole monomer on ITO glass substrate coated with peptide structure in water

0.5 mg, 1 mg, 1.5 mg and 2 mg of the peptide (YYACAYY, YYCYY or YYHYY) obtained in Example 1 were suspended in 50 mM phosphate buffer (pH 7.4) or HEPES buffer (4- (2-hydroxyethyl ) -1 -piperazineethanesulfonic acid, pH 7.4) was dissolved in the solution at 40 ° C, and the solution was placed on an ITO glass substrate and dried. At this time, the drying was naturally dried or slightly elevated (80 ° C or less) and dried. Then, ITO glass plate coated with the structure of the peptide with the silver electrode as the reference electrode was put into water containing 0.5 mol of pyrrole (0.5 M), which is one of the conductive polymer monomers, and a total voltage of 0.9 V was applied. At this time, since the reference electrode, silver, had a force of 1.2 V, practically only 0.78 V was applied. As a result, it was confirmed that only on the ITO glass substrate coated with the peptide structure, the pyrrole monomer was polymerized into a polypyrrole polymer having conductivity. On the ITO glass substrate without peptide structure, however, it was confirmed that the polymer could not be polymerized with polypyrrole polymer when 0.9 V was applied.

7-2. Polymerization of pyrrole monomers of peptide structure / metal ion with low redox transition

One equivalent of copper chloride was added to 0.5 mg, 1 mg, 1.5 mg and 2 mg of the peptide (YYACAYY, YYCYY or YYHYY) obtained in Example 1, followed by addition of 50 mM phosphate buffer (pH 7.4) or HEPES buffer ) (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, pH 7.4) was added thereto, and the mixture was dissolved at 40 占 폚 and then placed in a Petri dish. Then, only copper chloride was dissolved in the same 1 ml solvent at the same equivalent amount, and then put into a Petri dish. Then, the lid was put in a glass bottle with a solution containing only copper chloride, a peptide / copper chloride solution, and a pyrrole monomer solution. Then, a total of three petri dishes were put together and capped. After 1 hour, we observed that the pyrrole monomers flew into the vapor phase in the petri dish containing the peptide / cupric chloride solution and the polymerization was proceeded to black / polypyrrole on the surface / interface. However, there was no change in the petri dish containing the copper chloride solution.

The results are shown in FIG. The peptide nanostructure of the present invention showed easy radical formation by a cyclic voltammetry (CV) polarization curve test at 0.9 V (versus NHE) (see FIG. 21 a). In general, polypyrrole is synthesized using an oxidizing agent such as dislocations or Fe (III) ions in excess of 1.0V. In addition, even with the reaction using the 0.9 V potential, the polypyrrole deposition with a large structure was difficult due to the slow reaction rate (see FIG. 21 b). However, as a result of using the peptide nanostructure (film) according to the present invention, it was confirmed that the current increased to 68 μA after 300 seconds and a black polypyrrole film was produced.

As an experiment mimicking galactose oxidase, it has been shown that radicals are produced by the change in the valence of copper ions without the need to supply additional voltage. When polymerizing pyrrole with polypyrrole in the presence of a film according to the present invention, very different activities of copper ions were observed. That is, the peptide was produced in a 4 mM Cu (II) Cl ₂ solution and then exposed to pyrrole vapor. As a result, a black polypyrrole film was gradually formed on the peptide nanostructure according to the present invention. A hybrid film of free standing, polypyrrole and peptide nanostructures was formed (see Figure 21c). On the other hand, when the tyrosine monomer was used, the copper chloride could not form a polypyrrole film even after several days.

This result indicates that the Cu (II) ion causes pyrrole to be oxidized by the peptide film according to the present invention, which is considered to be the cause of the electron transfer mediated by the tyrosyl radical (see FIG. 21 d). The polypyrrole film was formed at the air / peptide structure interface, indicating that electrons were transferred from the vapor pyrrole to Cu (II) in solution through the YYACAYY film.

Example 8 Conductive polymer film prepared by YYACAYY peptide and polypyrrole

Pyrrole (or aniline, thiophene or 3,4-ethylenedioxythiophene) and YYACAYY peptide (2 mM) were mixed in 1 ml of 50 mM HEPES at a 1: 1 molar ratio, and 100 μl of the mixture was used as in Example 2 Ten minutes after formation of the nanostructure, a two-dimensional nanostructure was formed at the interface between the solution and air. Aniline, thiophene, or pyrrole trapped between the peptide two-dimensional structures by using ammonium peroxide or iron (III) chloride (2 molar equivalents relative to the monomer) as an oxidizing agent on the pyrrole-containing hybrid flat surface formed at the interface of the air and the solution The 3,4-ethylenedioxythiophene molecule was polymerized as a conductive polymer having a two-dimensional structure. As a result, a well-ordered crystal structure was formed (see Fig. 20). As a result of AFM analysis, the thickness was 5 nm, and as a result of a high-power TEM analysis, it was confirmed that the crystal had a hexagonal structure with a = 0.445 nm (Fig. 20C, D). These results indicate that the metallicity of the polymer film can be increased due to the regular arrangement as described above.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (20)

A redox reaction facilitating support comprising a nanostructure formed of a peptide having the structure of Formula 1:
[Chemical Formula 1]
X ¹ a-Zn-X ² b
In the above formula
X ¹ and X ² are each independently a natural or unnatural pennylalanine, tryptophan or tyrosine of D- or L-form having an aromatic side chain, X ¹ and X ² are the same or different,
Z is a natural or an unnatural covalent bond of a D- or L-type or a cysteine capable of coordinating with a metal, homocysteine, penicillamine, salenocysteine, histidine, lysine, ornithine, aspartic acid, glutamic acid, asparagine Or glutamine,
Wherein X ¹ a and X ² b are amino acid sequences having the same sequence or being symmetrical with respect to each other about the Z.
delete delete delete delete delete delete 2. The scaffold of claim 1, wherein n is 1, a and b are the same as each other, and are an integer of 2 to 5.
The compound of claim 1, wherein the compound of formula (1) is selected from the group consisting of YYCYY, YYYCYYY, YYACAYY, YYAACAAYY, YYGCGYY, YFCFY, FYCYF, FFCFF, YYAKAYY, YYAEAYY, YYY, YYECEYY, YYDCDYY, YYAHAYY, YYHYY, YYAPAYY and YYPYY &Lt; / RTI &gt; or a derivative thereof.
The support for promoting a redox reaction according to claim 1, wherein the nanostructure comprises a film, a nanofiber, or a sphere.
The scaffold of claim 1, wherein the nanostructure is a planar surface or a facing surface.
12. The scaffold of claim 11, wherein the planar surface or the facing surface has a multilayer structure.
2. The redox reaction promoting support according to claim 1, wherein the redox reaction exhibits activity by formation of a tyrosyl radical.
The support for promoting a redox reaction according to claim 1, wherein the support further comprises a metal capable of acting as a cofactor in the redox reaction.
A process for producing a polymer film, characterized by using an oxidation-reduction reaction promoting support according to any one of claims 1 to 8.
16. The method of claim 15, wherein the polymer film is conductive, semi-conductive or nonconductive according to a manufacturing process.
16. The method of producing a polymer film according to claim 15, wherein the polymer film is made from a polymerizable substance.
The method of producing a polymer film according to claim 17, wherein the polymerizable substance is alkene, alkenyl, alkynyl, polyphenol or a derivative thereof, pyrrole, aniline, thiophene or 3,4-ethylenedioxythiophene.
The method of producing a polymer film according to claim 17, wherein the compound of Formula 1 and the polymerizable substance are contained in a molar ratio of 1: 0.001 to 1: 100,000.
14. A method for promoting an oxidation-reduction reaction, which comprises using an oxidation-reduction reaction promoting support according to any one of claims 1 to 8.

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