Peptide having stabilized alpha helical structure, and peptide stabilization method

Abstract

The present invention relates to a method for stabilizing the alpha helical structure of a peptide and, more specifically, to a method for stabilizing a peptide by linking the position of the α-carbon of any α-amino acid in the peptide to the positions of the α-carbon of the third, fourth or seventh α-amino acid therefrom. According to the present invention, the alpha-helical structure can be stabilized and, at the same time, water solubility can be improved through amine-bearing hydrocarbon cross-links.

Description

Peptides having a stabilized alpha helical structure and methods for stabilizing peptides

[0001] The present invention relates to a peptide having a stabilized alpha helical structure and a method for producing the same, and more particularly, to a method for stabilizing a peptide through amine-bearing cross-links and a peptide produced thereby.

[0002] To maintain life, various proteins within the body interact with other biopolymers, such as protein-protein, protein-RNA, and protein-DNA interactions, thereby playing a crucial role in the body's signaling system. Abnormally activated interactions between specific proteins are often found to contribute to the development and maintenance of specific diseases. In many cases, these interactions between polymers are achieved through specific secondary structures of proteins, such as alpha-helices and β-chains.

[0003] The alpha-helix, one of the most well-known secondary structures of proteins, accounts for more than 30% of the secondary structure found in globular proteins. This alpha-helix structure plays a key role in the interactions between these biopolymers. Furthermore, because the peptides that make up proteins are short, chemically simple to synthesize and manipulate, and possess specific activities depending on their structure and shape, active research is being conducted on methods to control protein-protein interactions using the secondary structure of proteins.

[0004] Meanwhile, while peptides are stable within proteins, when separated from proteins and exist as monomeric peptides, they are thermodynamically unstable. Consequently, secondary structures, such as helical structures, are rarely maintained. Instead, they form disordered structures, making it difficult to achieve high affinity and selectivity for their targets. To address these issues and enable their application as drugs capable of modulating diverse interactions in living organisms, technologies capable of stabilizing peptides into specific secondary structures are increasingly important.

[0005] The alpha helix structure is unstable in solution or difficult to stabilize due to its short peptide length.

[0006] Accordingly, the problem to be solved by the present invention is to provide a method for further stabilizing an alpha-helix structure as a helical secondary structure.

[0007] In addition, another problem to be solved by the present invention is to provide a peptide structure that improves protease resistance, cell permeability, and biological activity by reducing the hydrophobicity of a stapled peptide and increasing its solubility.

[0008] In addition, another problem that the present invention seeks to solve is to provide a peptide having a more stabilized alpha-helix structure by introducing amine-bearing cross-links of hydrocarbons.

[0009] In addition, another problem that the present invention seeks to solve is to propose a peptide structure with diverse water solubility and chemical properties by introducing a cross-linking of a hydrocarbon containing an amine.

[0010] The tasks of the present invention are not limited to the tasks mentioned above, and other tasks not mentioned will be clearly understood by those skilled in the art from the description below.

[0011] In order to solve the above-mentioned problem, the present invention is a method for stabilizing the alpha helix structure of a peptide, wherein the method is characterized in that the position of the α-carbon of any α-amino acid in the peptide and the α-carbon of the third, fourth or seventh α-amino acid therefrom are linked, and the linkage is linked by an amine-bearing cross-link.

[0012] According to another feature of the present invention, the connection can be made by a ring-closing olefin metathesis reaction (RCM).

[0013] According to another feature of the present invention, the hydrocarbon containing the amine includes a secondary amine group, and the length of the hydrocarbon connected in front and behind the secondary amine group may be 1 to 10.

[0014] According to another feature of the present invention, the hydrocarbon containing the amine may include a tertiary amine group substituted with another functional group or a drug or protein, etc.

[0015] According to another feature of the present invention, the hydrocarbon may contain a double bond.

[0016] According to another feature of the present invention, any α-amino acid may comprise an alkenyl group linked to the α-carbon, and the third or fourth or seventh α-amino acid may comprise a side chain amino group in which the alkenyl group is substituted on the nitrogen atom.

[0017] According to another feature of the present invention, said arbitrary α-amino acid may be alkenyl alanine ((S)- or (R)-α-alkenyl alanine), and said third or fourth or seventh α-amino acid may be lysine containing a lysyl group in which an alkenyl group is substituted on the nitrogen atom.

[0018] According to another feature of the present invention, any α-amino acid comprises a side chain amino group in which an alkenyl group is substituted on a nitrogen atom, and the third or fourth or seventh α-amino acid may comprise an alkenyl group linked to the α-carbon.

[0019] According to another feature of the present invention, the arbitrary α-amino acid may be lysine containing a lysyl group substituted with an alkenyl group on the nitrogen atom, and the third or fourth or seventh α-amino acid may be alkenyl alanine ((S)- or (R)-α-alkenyl alanine).

[0020] According to another feature of the present invention, the peptide may have 4 to 35 amino acids.

[0021] In addition, a peptide according to one embodiment of the present invention is represented by the following chemical formula 1 and may have a stabilized alpha helix structure.

[0022] [Chemical Formula 1]

[0023]

[0024] [Chemical Formula 2]

[0025]

[0026] In the above chemical formulas 1 and 2, m is an integer from 1 to 7, and n is an integer from 1 to 6.

[0027] The above x is an integer from 0 to 31, the above y is an integer from 2 to 6, the above z is an integer from 0 to 31, and the sum of the above x and z is 6 to 35.

[0028] The above R1 is hydrogen, deuterium, substituted or unsubstituted C1-C 20 Alkyl group, substituted or unsubstituted C1-C 20 Aryl group, substituted or unsubstituted C1-C 20selected from the group consisting of an acyl group, a benzylcarbamoyl group, and one or more common amino acids,

[0029] The above R2, R3 and R4 are each independently hydrogen, deuterium, substituted or unsubstituted C1-C 20 Alkyl group, substituted or unsubstituted C1-C 20 It can be selected from the group consisting of an aryl group and a side chain of a general amino acid.

[0030] In the above chemical formulas 1 and 2, the stereochemistry of the α-carbon of all amino acids can be (R) or (S).

[0031] In the above chemical formula 1 or chemical formula 2, all amino acids may be L- or D-amino acids.

[0032] According to another feature of the present invention, the N-terminus may be hydrogen or a substituted or unsubstituted alkyl, aryl, acyl, benzylcarbamoyl having 1 to 20 carbon atoms, or a fluorescent substance for observing intracellular trends to add a new function, a functional group for facilitating separation, a functional group for immunoprecipitation reaction, etc., and various functional groups generally known for adding a function to the peptide may be added, and a drug exhibiting a therapeutic effect may be bound to it.

[0033] According to another feature of the present invention, in the chemical formula 1 or 2, y may be an integer of 3, m may be an integer of 1 to 3, and n may be an integer of 1 to 4.

[0034] According to another feature of the present invention, in the chemical formula 1 or 2, y may be an integer of 2, m may be an integer of 1, and n may be an integer of 3 to 4.

[0035] According to another feature of the present invention, in the chemical formula 1 or chemical formula 2, y may be an integer of 6, m may be an integer of 3 to 6, and n may be an integer of 3 to 4.

[0036] Hereinafter, the present invention will be described in more detail through examples. However, these examples are provided merely to illustrate the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

[0037] The present invention can provide a peptide having a stable alpha helical structure by introducing amine-bearing cross-links of hydrocarbons containing amine.

[0038] In addition, the present invention can improve water solubility and dissolution by stapling a peptide with a hydrocarbon containing an amine, unlike peptides stapled with conventional hydrocarbons.

[0039] The effects according to the present invention are not limited to those exemplified above, and more diverse effects are included in this specification.

[0040] Figure 1 is a diagram schematically showing a peptide synthesis process according to one embodiment of the present invention.

[0041] Figure 2 is a diagram schematically showing a peptide synthesis process according to another embodiment of the present invention.

[0042] Figure 3 is a schematic diagram showing a peptide synthesis process according to another embodiment of the present invention.

[0043] Figure 4 is a diagram schematically showing a peptide synthesis process according to another embodiment of the present invention.

[0044] Figure 5 is a diagram schematically showing a peptide synthesis process according to another embodiment of the present invention.

[0045] Figure 6 is a diagram schematically showing a peptide synthesis process according to another embodiment of the present invention.

[0046] Figure 7 is a diagram schematically showing a peptide synthesis process according to another embodiment of the present invention.

[0047] Figure 8 is a schematic diagram showing the process of biotinylation and guanidination from a peptide manufactured according to one embodiment of the present invention.

[0048] Figure 9 is a table describing the composition and helix content of a peptide manufactured according to one embodiment of the present invention.

[0049] Figure 10 is a table evaluating the metathesis reaction conversion rate of a peptide manufactured according to another embodiment of the present invention.

[0050] Figure 11 is a drawing showing the results of LC / MS analysis of a peptide manufactured according to one embodiment of the present invention.

[0051] Figure 12 is a drawing showing the results of LC / MS analysis of a peptide manufactured according to one embodiment of the present invention.

[0052] Figure 13 is a graph evaluating the water solubility of a peptide manufactured according to one embodiment of the present invention.

[0053] The advantages and features of the present invention, and the methods for achieving them, will become clearer with reference to the embodiments described in detail below together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms. These embodiments are provided solely to ensure that the disclosure of the present invention is complete and to fully inform those skilled in the art of the scope of the invention, and the present invention is defined solely by the scope of the claims.

[0054] In this document, the expressions "has," "may have," "includes," or "may include" indicate the presence of a feature (e.g., a number, function, operation, or component such as a part), but do not exclude the presence of additional features.

[0055] In this document, the expressions "A or B," "at least one of A and / or B," or "one or more of A or / and B" can include all possible combinations of the listed items. For example, "A or B," "at least one of A and B," or "at least one of A or B" can all refer to cases where (1) at least one A is included, (2) at least one B is included, or (3) at least one A and at least one B are included.

[0056] The expression "configured to" as used in this document can be used interchangeably with, for example, "suitable for," "having the capacity to," "designed to," "adapted to," "made to," or "capable of."

[0057] The term “cross-link” as used herein refers to the formation of a macrocyclic structure by linking the α-carbon (i) of any α-amino acid within a peptide with the α-carbon of the α-amino acid at the third position (i+3), fourth position (i+4), or seventh position (i+7). In this case, the linkage may be by a ring-closing olefin metathesis reaction, but is not limited thereto. In addition, the linkage may also mean that a covalent bond (cross-link) is formed by stapling.

[0058] Here, “stapling” refers to a technique that can stabilize the secondary structure of the alpha helix structure of a peptide by introducing a cross-link of a hydrocarbon to the peptide.

[0059] The terms used in this document are used only to describe specific embodiments and may not be intended to limit the scope of other embodiments. The singular expression may include the plural expression unless the context clearly indicates otherwise. Terms used herein, including technical or scientific terms, may have the same meaning as commonly understood by those of ordinary skill in the art described in this document. Terms defined in general dictionaries among the terms used in this document may be interpreted as having the same or similar meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined in this document. In some cases, even if a term is defined in this document, it cannot be interpreted to exclude the embodiments of this document.

[0060] The individual features of the various embodiments of the present invention can be partially or wholly combined or combined with each other, and as can be fully understood by those skilled in the art, various technical connections and operations are possible, and each embodiment can be implemented independently of each other or can be implemented together in a related relationship.

[0061] As used herein, the term “or” means “and / or” unless otherwise stated.

[0062] Hereinafter, with reference to FIGS. 1 to 7, a method for stabilizing the alpha helix structure of a peptide according to one embodiment of the present invention will be specifically described.

[0063] Figure 1 is a diagram schematically showing a peptide synthesis process according to one embodiment of the present invention.

[0064] First, a method for stabilizing an alpha helix structure of a peptide according to one embodiment of the present invention is characterized by linking the position of an α-carbon of any α-amino acid (i) in the peptide and the α-carbon of the third (i+3), fourth (i+4), or seventh (i+7) α-amino acid therefrom.

[0065] At this time, the linkage is connected by a ring-closing olefin metathesis reaction (RCM). Specifically, the linkage can be connected by an amine-bearing hydrocarbon cross-link. An amine-bearing hydrocarbon cross-link refers to a structure in which the ith α-amino acid and the i+3, i+4, or i+7th α-amino acid are stapled by an amine-bearing hydrocarbon.

[0066] First, Figure 1 illustrates a specific example of connecting the ith α-amino acid and the (i+4)th α-amino acid. Figure 1 illustrates an example in which the ith α-amino acid includes an alkenyl group linked to the α-carbon, and the (i+4)th α-amino acid includes a side-chain amino group in which the alkenyl group is substituted on the nitrogen atom.

[0067] Referring to Figure 1, in step (a), a peptide is prepared from an amino acid. Rink Amide MBHA resin is used to prepare a C-terminal aminated peptide. In step (a), (S)-α-alkenyl alanine (S) is used as the amino acid at position 4. m ) is used, and the amino acid located at position 8 is (S)-(N-allyl-No-nitrobenzenesulfonylamino)alkyl glycine,No-Ns-L n ) is used. During the peptide synthesis process, 4-methyltrityl (Mtt) protects the amine group.

[0068] (b) The side chain amine group is arylated in step 1. The 4-methyltrityl (Mtt) of the L-lysine analogue in the peptide is removed and replaced with an o-nosyl group through an orthogonal deprotection-protection process to facilitate N-allylation via the Fukuyama sulfonamide alkylation method.

[0069] (c) A peptide metathesis reaction is performed in step (c). Through the peptide metathesis reaction, the ith α-amino acid and the (i+4)th α-amino acid form a structure stapled with an amine-bearing hydrocarbon. Specifically, a ring-closing olefin metathesis (RCM) reaction is performed using a catalyst. Considering the possibility of steric hindrance or electronic deactivation of the catalyst by the o-nosyl group, the ring-closing olefin metathesis (RCM) reaction is performed under more stringent conditions using a second-generation Hoveyda-Grubbs catalyst. Through this, a metathesis reaction occurs between the alkenyl group linked to the alanine at the 4-position and the aryl group substituted on the amino group of the lysine group linked to the glycine at the 8-position, forming a double bond. Thus, the α-amino acid at the 4-position and the α-amino acid at the 8-position are linked by an amine-bearing hydrocarbon cross-link.

[0070] (d) In step 1, the o-nosyl group is removed. By removing the o-nosyl group substituted on the amine, a structure can be formed in which the α-carbon of the α-amino acid (i) at position 4 and the α-carbon of the α-amino acid (i+4) at position 8 are linked by a hydrocarbon containing an amine. This can further stabilize the alpha-helix structure as a helical secondary structure.

[0071] Additionally, step (e) forms a product in which the ring-closing metathesis reaction (RCM) of step (c) does not proceed and only the o-nosyl group is removed. In step (e), the formed byproduct may have a structure in which the side chain amine group linked to lysine is arylated.

[0072] Figure 2 illustrates another example of linking the ith α-amino acid and the (i+4)th α-amino acid. Unlike Figure 1, Figure 2 illustrates an example in which the ith α-amino acid comprises a side chain amino group in which an alkenyl group is substituted on the nitrogen atom, and the (i+4)th α-amino acid comprises an alkenyl group linked to the α-carbon.

[0073] Referring to Figure 2, compared to Figure 1, the amino acid located at position 4 is (S)-(N-allyl-No-nitrobenzenesulfonylamino)alkyl glycine, No-Ns-L n ) is used, and the amino acid located at position 8 is (S)-α-alkenyl alanine (S m ) is used. The subsequent process is the same as steps (c) and (d) of Fig. 1.

[0074] Next, Figure 3 illustrates a specific example of connecting the ith α-amino acid and the (i+7th) α-amino acid. Figure 3 illustrates an example in which the ith α-amino acid includes an alkenyl group linked to the α-carbon, and the (i+7th) α-amino acid includes a side chain amino group in which the alkenyl group is substituted on the nitrogen atom.

[0075] Referring to Figure 3, in step (a), a peptide is prepared from an amino acid. Rink Amide MBHA resin is used to prepare a C-terminal aminated peptide. In step (a), (R)-α-alkenyl alanine (R) is used as the amino acid at position 5.m ) is used, and the amino acid located at position 12 is (S)-(N-allyl-No-nitrobenzenesulfonylamino)alkyl glycine,No-Ns-L n ) is used. During the process of synthesizing the peptide, 4-methyltrityl (Mtt) protects the amine group. Since steps (b), (c), and (d) are the same as in Figure 1, redundant description is omitted.

[0076] Figure 4 illustrates another example of connecting the i-th α-amino acid and the (i+7th) α-amino acid. Unlike Figure 3, in Figure 4, (S)-α-alkenyl alanine (S) is used as the amino acid at position 5. m ) is used. Other than that, the process is the same as steps (b), (c), and (d) of FIG. 3.

[0077] Figure 5 illustrates another example of linking the ith α-amino acid and the (i+7th) α-amino acid. Unlike Figure 4, Figure 5 illustrates an example in which the ith α-amino acid comprises a side chain amino group in which an alkenyl group is substituted on the nitrogen atom, and the (i+7th) α-amino acid comprises an alkenyl group linked to the α-carbon.

[0078] Referring to Figure 5, compared to Figure 4, the amino acid located at position 5 is (S)-(N-allyl-No-nitrobenzenesulfonylamino)alkyl glycine, No-Ns-L n ) is used, and the amino acid located at position 12 is (S)-α-alkenyl alanine (S m ) is used. The subsequent process is the same as steps (b), (c), and (d) of Fig. 3.

[0079] Next, Figure 6 illustrates a specific example of connecting the ith α-amino acid and the (i+3)th α-amino acid. Figure 6 illustrates an example in which the ith α-amino acid includes an alkenyl group linked to the α-carbon, and the (i+3)th α-amino acid includes a side chain amino group in which the alkenyl group is substituted on the nitrogen atom.

[0080] Referring to Figure 6, in step (a), a peptide is prepared from an amino acid. Rink Amide MBHA resin is used to prepare a C-terminal aminated peptide. In step (a), (R)-α-alkenyl alanine (R) is used as the amino acid at position 4. m ) is used, and the amino acid located at position 7 is (S)-(N-allyl-No-nitrobenzenesulfonylamino)alkyl glycine,No-Ns-L n ) is used. During the process of synthesizing the peptide, 4-methyltrityl (Mtt) protects the amine group. Since steps (b), (c), and (d) are the same as in Figure 1, redundant description is omitted.

[0081] Figure 7 illustrates another example of linking the ith α-amino acid and the (i+3)th α-amino acid. Unlike Figure 6, Figure 6 illustrates an example in which the ith α-amino acid includes a side chain amino group in which an alkenyl group is substituted on the nitrogen atom, and the (i+3)th α-amino acid includes an alkenyl group linked to the α-carbon.

[0082] Referring to Figure 7, compared to Figure 6, the amino acid located at position 4 is (S)-(N-allyl-No-nitrobenzenesulfonylamino)alkyl glycine, No-Ns-L n) is used, and the amino acid located at position 7 is (S)-α-alkenyl alanine (S m ) is used. The subsequent process is the same as steps (b), (c), and (d) of Fig. 6.

[0083] A peptide according to one embodiment of the present invention can be manufactured through the above-described method.

[0084] Accordingly, a peptide according to one embodiment of the present invention may be a compound having a stabilized alpha helix structure represented by the following chemical formula 1 or chemical formula 2.

[0085] [Chemical Formula 1]

[0086]

[0087] [Chemical Formula 2]

[0088]

[0089] In the above chemical formula 1 or chemical formula 2, x is an integer from 0 to 31, y is an integer from 2 to 6, z is an integer from 0 to 31, the sum of x, y and z is 6 to 35, m is an integer from 1 to 7, and n is an integer from 1 to 6. When y is 3, m may be an integer from 1 to 3, and n may be an integer from 1 to 4, preferably m is 2, and n may be 3 or 4. In addition, when y is 6, m may be an integer from 3 to 6, and n may be an integer from 3 to 4, preferably m is 5, and n may be 3 or 4. In addition, when y is 2, m may be 1, and n may be an integer from 3 to 4. The alpha helix structure can be further stabilized when the number of carbon atoms on both sides of the nitrogen element is similar.

[0090] The above R1 is hydrogen, deuterium, substituted or unsubstituted C1-C 20Alkyl group, substituted or unsubstituted C1-C 20 Aryl group, substituted or unsubstituted C1-C 20 is selected from the group consisting of an acyl group, a benzylcarbamoyl group, and one or more common amino acids. In addition, R1 can be various functional groups generally known to add functionality to peptides, such as a fluorescent substance for observing intracellular trends, a functional group for facilitating separation, a functional group for immunoprecipitation reaction, etc. to add new functions.

[0091] R2 to R4 are each independently hydrogen, deuterium, substituted or unsubstituted C1-C 20 Alkyl group, substituted or unsubstituted C1-C 20 It can be selected from the group consisting of an aryl group of a compound, or a side chain of an amino acid.

[0092] In the above chemical formula 1 or chemical formula 2, the stereochemistry of the α-carbon of all amino acids can be (R) or (S).

[0093] In the above chemical formula 1 or chemical formula 2, all amino acids may be L- or D-amino acids.

[0094] At this time, the peptide represented by chemical formula 1 or 2 may be composed of an amino acid sequence of 35 or fewer.

[0095] The N-terminus contains hydrogen, deuterium, substituted or unsubstituted C1-C 20 Alkyl group, substituted or unsubstituted C1-C 20 Aryl group, substituted or unsubstituted C1-C 20The functional group may be substituted with an acyl group, a benzylcarbamoyl group, or one or more common amino acids. Furthermore, it may be a variety of functional groups generally known to add functionality to the peptide, such as a fluorescent substance for observing intracellular trends, a functional group for facilitating separation, a functional group for immunoprecipitation reaction, etc. to add new functions, and a drug that exhibits a therapeutic effect may be bound.

[0096] A stapled peptide according to one embodiment of the present invention may be composed of any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 below.

[0097] Sequence number 1: EWAX1TAAX2KFLAAHA

[0098] Sequence number 2: EWAEX1AAAKFLX2AHA

[0099] Sequence number 3: EWAX1TAX2AKFLAAHA

[0100] Sequence number 1 is a peptide in which one amino acid position and the amino acid position 4th thereafter are stapled with a hydrocarbon containing an amine, preferably the 4th amino acid position and the 8th amino acid position may be stapled.

[0101] Sequence number 2 is a peptide in which one amino acid position and the 7th amino acid position thereafter are stapled with a hydrocarbon containing an amine, preferably the 5th amino acid position and the 12th amino acid position may be stapled.

[0102] Sequence number 1 is a peptide in which one amino acid position and the amino acid position 3rd thereafter are stapled with a hydrocarbon containing an amine, preferably the 4th amino acid position and the 7th amino acid position may be stapled.

[0103] Here, X1 and X2 represent mutually stapled positions with hydrocarbons containing amines. The amino acids that can be placed at X1 and X2 are not limited to specific amino acids as long as the hydrocarbons containing amines can be substituted. For example, X1 and X2 can be amino acid derivatives substituted with substituents containing hydrocarbons, alkenyl groups, or amines, and specifically, can be, but are not limited to, alanine (Ala) derivatives, glycine (Gly) derivatives, or lysine derivatives substituted with substituents containing alkenyl groups or amines.

[0104] Hereinafter, peptides according to one embodiment of the present invention will be described in more detail through the following examples. However, these examples are provided merely to aid understanding of the present invention, and the present invention is not limited to the following examples.

[0105] The peptide synthesis process is schematically illustrated in Figure 1. In addition, information on intermediate substances and final products in the peptide synthesis process is specifically presented in the table in Figure 9.

[0106] [Example]

[0107] Example 1: Preparation Process

[0108] Commercially available solvents and reagents were used according to the manual. N-Fmoc-(S)-α-methyl,α-allylglycine (Fmoc-S1-OH), N-Fmoc-(S)-α-methyl,α-butenylglycine(Fmoc-S2-OH), and N-Fmoc-(S)-α-methyl,α-petenylglycine (Fmoc-S3-OH) were purchased from Okeanos Tech Co., Ltd. Fmoc-protected α-amino acids, allyl iodide, N,N'-bis(tert-butoxy)thiourea, bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride (Grubbs first generation catalyst), 2-chloro-1-methylpyridinium iodide (Mukaiyama reagent), (1,3-bis-(2,4,6-trimethylphenyl)-2-imidazolidinylidene)dichloro(o-isopropoxyphenylmethylene)ruthenium (Hoveyda-Grubbs second generation catalyst), 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino)]uranium hexafluorophosphate (COMU), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 2-mercaptoethanol (2-ME), 1,2-dichloroethane (DCE), N,N-diisopropylethylamine (DIPEA), dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP), 2-nitrobenzenesulfonyl chloride (o-NsCl), piperidine, Rink Amide MBHA Resin, trifluoroacetic acid (TFA), triisopropylsilane (TIS), 2,4,6-trimethylpyridine (2,4,6-collidine), and trimethylamine were purchased from Merck Korea.

[0109] Example 2: Peptide Preparation

[0110] (1) Peptide synthesis

[0111] To prepare 2a to 2g as intermediates (substrate peptides) containing (S)-α-alkenyl alanine (Sm) at position 4 in the sequence and (S)-(N-allyl-No-nitrobenzenesulfonylamino) alkylglycine (No-Ns-Ln) at position 8, Rink Amide MBHA resin was used (loading capacity = 0.6 mmol / g). After the dried resin (50 mg, 30 μmol) was swelled in NMP for 10 min, N α -Fmoc protecting group was removed by treating the resin with 25% piperidine in NMP (2 x 10 min). For amino acid coupling, N α -Fmoc amino acid (5 equiv), COMU (4.8 equiv) and DIPEA (10 equiv) were added as a mixture to NMP and treated for 30 min. In the case of coupling α-methyl, α-alkenyl glycines, the reaction proceeded as follows: N α-Fmoc amino acid (3 equiv), COMU (2.85 equiv), and DIPEA (6 equiv) were used for 2 h. After extension, and final deprotection of the Fmoc protecting group, the resin was treated with acetic anhydride (10 equiv) in the presence of DIPEA (20 equiv) in NMP for 2 h at room temperature. After each coupling or deprotection reaction, the resin was thoroughly washed with dichloromethane (DCM) (2 x 1 min), NMP (2 x 1 min), DCM (2 x 1 min), and NMP (2 x 1 min).

[0112] The peptide synthesis process is roughly shown in step (a) of Figure 1.

[0113] Meanwhile, the intermediate material manufactured in this step is manufactured by changing the m of the starting material (S)-α-alkenyl alanine (Sm) and the n of S)-(N-allyl-No-nitrobenzenesulfonylamino) alkylglycine (No-Ns-Ln). The specific contents of the specific products 2a to 2g formed in this step will be described in detail later in the table of Fig. 9.

[0114] (2) Side chain N-allylation

[0115] The resin was treated with 1% TFA in DCM for 1 min each ten times to selectively remove the 4-methyltrityl (Mtt) protecting group of the L-lysine analogue in the resin-bound peptide. After draining the reaction solution, the resin was washed with DCM (2 x 1 min), NMP (2 x 1 min), DCM (2 x 1 min), and NMP (2 x 1 min). The resin was then treated with 2-nitrobenzenesulfonyl chloride (5 equiv) and 2,4,6-trimethylpyridine (10 equiv) in NMP seven times for 15 min each. After draining the reaction solution, the resin was washed thoroughly with DCM (2 x 1 min), NMP (2 x 1 min), DCM (2 x 1 min), and NMP (2 x 1 min). Afterwards, the resin was suspended in NMP in the presence of DBU (3 equiv) for 3 min, and a solution of allyl iodide (20 equiv) in NMP was added. The resulting mixture was allowed to react for 30 min. This process was repeated once more, after which the reaction solution was drained. The resin was carefully washed with DCM (3 x 1 min), NMP (3 x 1 min), DCM (3 x 1 min), and diethyl ether (3 x 2 min), and dried under vacuum overnight.

[0116] The side chain N-allylation process is roughly represented in step (b) of Figure 1.

[0117] (3) Ring closure metathesis reaction and deprotection of o-nosyl group

[0118] The dried resin was swelled in DCM for 10 min. After draining the DCM, the resin was washed with degassed DCE (3 x 1 min). Subsequently, after draining the DCM, the resin was treated with a solution containing 20 mol% of a second-generation Hoveyda-Grubbs catalyst in degassed DCE at 60°C for 2 h. This process was repeated with freshly prepared catalyst solution as needed. Upon completion of the reaction, the solution was drained, and the resin was washed with DCE (3 x 1 min), DCM (3 x 1 min), and NMP (3 x 1 min). For deprotection of the 2-nitrobenzenesulfonyl group, the resin was treated with 2-mercaptoethanol (10 equiv) and DBU (5 equiv) in NMP twice for 10 min each, then washed with DCM (3 x 1 min), NMP (3 x 1 min), DCM (3 x 1 min), diethyl ether (3 x 2 min) and dried under vacuum overnight. This formed a stabilized peptide with a linked structure.

[0119] The ring closure metathesis reaction and the deprotection process of the o-nosyl group are roughly represented in steps (c) and (d) of Fig. 1. Meanwhile, as a control, the capping process of a peptide that did not undergo a ring closure metathesis reaction is roughly represented in step (e) of Fig. 1. The specific contents of peptides 1a to 1j, which are the specific products formed in this step, will be described in detail later in Fig. 9.

[0120] Meanwhile, peptides were manufactured using the methods illustrated in FIGS. 2 to 7, with different starting amino acid materials and amino acid sequences. The specific details of the resulting products, peptides 6a to 10b, are described in FIG. 10.

[0121] (4) Biotinylation of the linkage structure

[0122] Previously, after step (c) where the ring closure metathesis reaction occurs as illustrated in Figure 1, the o-nosyl group deprotection process, step (d), was not performed, and the prepared peptide was swelled in NMP for 10 min and then treated with a solution containing biotin (8 equiv), COMU (7.6 equiv), and DIEA (16 equiv) in NMP at room temperature for 2 h. After draining the reaction solution, the resin was carefully washed with DCM (2 x 1 min), NMP (2 x 1 min), DCM (2 x 1 min), and diethyl ether (3 x 1 min), and then dried under vacuum overnight. The biotinylation process is roughly represented in step (f) of Figure 8.

[0123] (5) Guanidination of the connection structure

[0124] As previously described, after step (c) where the ring closure metathesis reaction occurs as illustrated in Figure 1, the deprotection process of the o-nosyl group, step (d), was not performed, and the prepared peptide was dried and allowed to swell in DCM for 10 min. After draining, the resin was treated with N,N'-bis(tert-butoxy)thiourea (3 equiv) and trimethylamine (4 equiv) in DMF for 15 min. 2-Chloro-1-methylpyridinium iodide (Mukaiyama's reagent) was added to this mixture, and the resulting mixture was allowed to react at room temperature for 3 h. After draining the reaction solution, the resin was washed with DCM (3 x 1 min), NMP (3 x 1 min), DCM (3 x 1 min), and diethyl ether (3 x 1 min), and then dried under vacuum overnight. The biotinylation process is roughly represented in step (g) of Fig. 8.

[0125] The specific details of 5i to 5j, which are specific products formed in this step, will be described in detail later in the table of Fig. 9.

[0126] (6) Cleavage and purification of peptides

[0127] To cleave the peptide bound to the resin, the dried resin was treated with a 95:2.5:2.5 mixture of TFA, TIS, and water for 2 h. The cleaved peptide was precipitated by adding a 1:1 mixture of n-pentane and diethyl ether. The precipitated material was collected by centrifugation and dried for 2 h. The peptide material was then dissolved in a 1:1 mixture of acetonitrile and water, and the resin was filtered. The filtrate was then purified by reversed-phase high-performance liquid chromatography (HPLC) using a Zorbax C18 column (Agilent, 5 μm, 9.4 × 250 mm).

[0128] Example 3: LC / MS analysis of manufactured peptides

[0129] Various peptides prepared according to Example 2 were analyzed using LC / MS (Shimadzu CMS-2020).

[0130] Peptide 1b (C 79 H 117 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 832.95, measured 832.90.

[0131] Peptide 1c (C 80 H 119 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 839.95, measured 840.40.

[0132] Peptide 1d (C 79 H 117 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 832.95, measured 833.00.

[0133] Peptide 1e (C 80 H 119 N21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 839.95, measured 839.95.

[0134] 1g of peptide (C 80 H 119 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 839.95, measured 840.10.

[0135] Peptide 1h (C 79 H 117 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 832.95, measured 833.00.

[0136] Peptide 1i (C 81 H 119 N 21 O 21 [M+2H] 2+ / 2) ESIMS calculated m / z 862.30, measured 861.95.

[0137] Peptide 1j (C 82 H 121 N 21 O 21 [M+2H] 2+ / 2) ESIMS calculated m / z 869.35, measured 868.96.

[0138] Peptide 1s (C 81 H 120 N 20 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 839.46, measured 839.90.

[0139] Peptide 1w (C 75 H 110 N 20 O 21 [M+2H] 2+ / 2) ESIMS calculated m / z 814.41, measured 814.70.

[0140] Peptide 3b (C 81 H 121 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 846.97, measured 847.65.

[0141] Peptide 3c (C 82 H 123 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 853.98, measured 854.05.

[0142] Peptide 3h (C 81 H 121 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 846.97, measured 847.35.

[0143] Peptide 4i (C 91 H 133 N 23 O 23 S [M+2H] 2+ / 2) ESIMS calculated m / z 974.99, measured 975.65.

[0144] Peptide 4j (C 92 H 135 N 23 O 23 S [M+2H] 2+ / 2) ESIMS calculated m / z 982.00, measured 982.70.

[0145] Peptide 5b (C 80 H 119 N 23 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 853.96, measured 853.95.

[0146] Peptide 5c (C 81 H 121 N 23 O 19[M+2H] 2+ / 2) ESIMS calculated m / z 860.97, measured 861.05.

[0147] Peptide 5i (C 82 H 121 N 23 O 21 [M+2H] 2+ / 2) ESIMS calculated m / z 883.30, measured 882.96.

[0148] Peptide 5j (C 83 H 123 N 23 O 21 [M+2H] 2+ / 2) ESIMS calculated m / z 890.30, measured 889.97.

[0149] Peptide 6a (C 82 H 121 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 860.96, measured 861.45.

[0150] Peptide 6b (C 83 H 123 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 867.97, measured 868.50.

[0151] Peptide 6c (C 84 H 125 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 874.98, measured 875.45.

[0152] Peptide 6d (C 85 H 127 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 881.99, measured 882.50.

[0153] Peptide 6e (C 86 H 129 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 888.99, measured 889.45.

[0154] Peptide 7c (C 85 H 127 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 881.99, measured 882.40.

[0155] Peptide 7d (C 86 H 129 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 888.99, measured 889.50.

[0156] Peptide 8a (C 83 H 123 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 867.97, measured 868.25.

[0157] Peptide 8b (C 84 H 125 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 874.98, measured 875.25.

[0158] Peptide 8c (C 85 H 127 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 881.99, measured 882.30.

[0159] Peptide 8d (C 86 H 129 N 21 O 20 [M+2H] 2+ / 2) ESIMS calculated m / z 888.99, measured 889.45.

[0160] Peptide 9a (C 79 H 117 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 832.95, measured 833.2.

[0161] Peptide 9b (C 80 H 119 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 839.96, measured 840.3.

[0162] Peptide 10a (C 79 H 117 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 832.95, measured 833.2.

[0163] Peptide 10b (C 80 H 119 N 21 O 19 [M+2H] 2+ / 2) ESIMS calculated m / z 839.96, measured 840.3

[0164] As shown in the above results, it was confirmed that the calculated and measured values ​​were almost similar, and through this, it was confirmed that the expected peptides were normally manufactured.

[0165] Example 4: Evaluation of the composition and stability of the manufactured peptide material

[0166] The structure of the linking portion of the peptide manufactured in Example 2 was shown above, and the conversion rate and stapled degree at each step were evaluated. At this time, “S” is shown in the table of Figure 9 with respect to the linking structure composition. i,i+4It is written as “L(aNb)”, where a is the number of carbon atoms from the center of the nitrogen atom to the α-carbon of the amino acid at the 4th position in the cross-linked structure of the hydrocarbon containing an amine, and b is the number of carbon atoms from the center of the nitrogen atom to the α-carbon of the amino acid at the 8th position in the cross-linked structure of the hydrocarbon containing an amine.

[0167] Referring to the table in Figure 9, it can be seen that peptides 1b, 1c, 1d, and 1e undergo ring-closing metathesis reactions, but peptide 1a has a low carbon number and is hindered by the o-nosyl group, making the metathesis reaction difficult. In addition, peptide 1f also has a short carbon number and b is 1, so the allyl group is located close to the peptide backbone, making the reaction difficult.

[0168] Meanwhile, in the case of Comparative Example 1, since it does not have both an alkenyl group and an amine group, it can be confirmed that stapling through a hydrocarbon between amino acids is difficult, resulting in a low helix content.

[0169] In addition, the conversion rate was evaluated during the synthesis of a peptide in which the 5th α-amino acid and the 12th α-amino acid were linked in the manners illustrated in FIGS. 3 to 5 and a peptide in which the 4th α-amino acid and the 7th α-amino acid were linked in the manners illustrated in FIGS. 6 and 7. Referring to the table in FIG. 10, when the position of the α-carbon of any α-amino acid and the α-carbon of the seventh α-amino acid from it are linked, the conversion rate according to the metathesis reaction may be somewhat low in the case of peptide 6a due to insufficient carbon number. On the other hand, peptides 7a and 7b have insufficient carbon number compared to peptides 7c and 7d, and the reaction may not proceed due to steric hindrance of the allylic group.

[0170] Example 5: LC / MS analysis of manufactured peptides using circular dichroism spectrometry.

[0171] The peptide prepared according to Example 2 was dissolved in potassium phosphate buffer (25 mM, pH 6.5), and the concentration of the peptide sample was measured by absorbance spectroscopy at 280 nm using the extinction coefficient of tryptophan (λ280 = 5690 / cm). A Chirascan HP dual polarization circular dichroism spectrometer was used for circular dichroism. Circular dichroism (CD) spectra were recorded on a Chirascan CD spectrometer equipped with a temperature controller using a 0.1 cm path length cell. The measurement parameters were set to 1 nm bandwidth, 1 nm step resolution, 3 accumulations, and 0.5 s response.

[0172] The results are shown in Figures 11 and 12.

[0173] Referring to (a) and (b) of Fig. 11, it was confirmed that both the cross-linked structures (ABX) of the hydrocarbons containing amines, when composed of 8 atoms (peptides 1b, 1d) and 9 atoms (peptides 1c, 1e, 1g), had the characteristics of a right-rolling alpha helix. In addition, in Fig. 11 (a), it can be seen that peptide 1b, which has more symmetrical carbon numbers on the left and right sides with respect to nitrogen in the cross-linked structure (ABX) of the hydrocarbons containing amines, has better helical stabilization than peptide 1d, which is more asymmetrical. Similarly, in Fig. 11 (b), it can be seen that peptide 1c, which has symmetrical carbon numbers on the left and right sides with respect to nitrogen with 4 carbon atoms on the left and right sides with respect to nitrogen, has better helical stabilization than peptide 1e, which has an asymmetrical structure with 5 and 3 carbon atoms on the left and right sides with respect to nitrogen.

[0174] Referring to (d) of FIG. 11, it was confirmed that peptide 1b, in which alkenyl alanine is placed at position 4 and lysine is placed at position 8, forms a more stable staple than peptide 1h, in which lysine is placed at position 4 and alkenyl alanine is placed at position 8, and thus the arrangement structure is opposite.

[0175] Referring to (a) and (b) of Fig. 12, peptides 5b and 5c, in which the secondary amine moiety of the cross-linked structure (ABX) of a hydrocarbon containing an amine was guanidinated, exhibited somewhat lower helicity compared to peptides 1b and 1c. At this time, peptide 5b, which was guanidinated from the cross-linked structure (ABX) of a hydrocarbon containing an 8-membered amine, showed a slightly more pronounced decrease in helicity than peptide 5c, which was guanidinated from the cross-linked structure (ABX) of a hydrocarbon containing a 9-membered amine. This shows that the cross-linked structure (ABX) of a hydrocarbon containing a 9-membered amine is slightly more stable and flexible in cross-linking.

[0176] Example 6: Trypsin digestion assay

[0177] Twenty-five microliters of trypsin solution (0.5 μM, Sigma) in digestion buffer (0.1 M NH4HCO3 buffer, pH 8.0) was added to a mixture containing 250 μL of peptide solution (80 μM) in the same buffer and 5 μL of tryptophan solution (4 mM) (used as an internal standard) in the same buffer (enzyme / peptide = 1 / 1,600). The resulting mixture was incubated at room temperature with rapid mixing (600 rpm). At intervals of 0, 10, 20, 30, and 60 min, a 50-μL aliquot of the digestion mixture was withdrawn and quenched with 2 μL of trifluoroacetic acid. Residual substrate and tryptophan were quantified using LC-based peak detection at 280 nm. Peptide stability to trypsin was assessed by calculating the percentage of substrate remaining after each time period. Each experiment was performed in duplicate. The results are shown in (c) and (d) of Fig. 11.

[0178] Referring to (c) and (d) of FIG. 12, Comparative Example 1 (1w) without a binding structure was almost completely decomposed with a half-life of 8.4 minutes. However, under the same conditions, approximately 90% of peptides 1i, 1j and their guanidinated analogs peptides 5i and 5j maintained their structures. That is, it was shown that the resistance to trypsin digestion was improved by 40 times on average. Therefore, it was confirmed that the peptide stabilized with the helical structure of the present invention had improved resistance to protein degradation due to structural reinforcement.

[0179] Example 7: Solubility Test

[0180] According to Example 2, 3 mg of the freeze-dried peptide sample was resuspended in 100 μL of phosphate buffer (25 mM, pH 6.5) at 20ºC. The peptide sample was mixed by vortexing for 1 minute and then sonicated in a bath sonicator for 10 minutes. This process was repeated three times to ensure sample homogeneity. After mixing, the suspension was centrifuged at 3,000 rpm for 5 minutes. 10 μL of the supernatant was recovered and diluted 2-50 times with the same phosphate buffer. The peptide concentration of the diluted solution was measured using a NanoDrop TM The extinction coefficient (λ) of tryptophan was measured using a 2000 spectrophotometer (Thermo Fisher Scientific). 280 = 5690 / cm) was determined by measuring the absorbance at 280 nm. Each experiment was repeated three times. The results are shown in Fig. 13.

[0181] First, referring to FIG. 9, Comparative Example 2 (1S), in which alkenyl alanine is positioned at both positions 4 and 8, has a stapled bond structure and thus exhibits a higher helix content compared to Comparative Example 1 (1w), which does not have a bond structure. However, referring to FIG. 13, Comparative Example 2 (1S) exhibits lower water solubility than Comparative Example 1 (1w), which does not have a bond structure, indicating that Comparative Example 2 (1S), which has a stapled bond structure, has strong hydrophobicity (1.01 vs. 0.08 mg / mL). However, the water solubility of peptide 1b and peptide 1c exceeded 24 mg / mL, confirming that the water solubility was greatly improved.

[0182] Although the embodiments of the present invention have been described in more detail with reference to the attached drawings, the present invention is not necessarily limited to these embodiments, and various modifications may be implemented without departing from the technical spirit of the present invention. Therefore, the embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain it, and the scope of the technical spirit of the present invention is not limited by these embodiments. Therefore, it should be understood that the embodiments described above are exemplary in all aspects and not restrictive. The protection scope of the present invention should be interpreted by the claims below, and all technical ideas within a scope equivalent thereto should be interpreted as being included in the scope of the rights of the present invention.

[0183] [National Research and Development Project Supporting This Invention]

[0184] [Project ID] 1711184577

[0185] [Assignment Number] 2021R1F1A1057327

[0186] [Ministry Name] Ministry of Science and ICT

[0187] [Name of Project Management (Specialist) Institution] National Research Foundation of Korea

[0188] [Research Project Name] Basic Research Project in Science and Engineering

[0189] [Research Project Title] Development of a NEMO-Inhibiting Anti-Inflammatory Peptide Drug for the Treatment of Severe COVID-19 Patients

[0190] [Name of Project Performing Organization] Dongguk University

[0191] Research Period: June 1, 2021 - February 29, 2024

Claims

1. A method for stabilizing the alpha helix structure of a peptide, The above method connects the position of the α-carbon of any α-amino acid in the peptide and the α-carbon of the third, fourth or seventh α-amino acid therefrom, A method for stabilizing a peptide, characterized in that the above linkage is linked by amine-bearing cross-links.

2. In paragraph 1, A method for stabilizing a peptide, wherein the above linkage is linked by ring-closing olefin metathesis (RCM).

3. In paragraph 1, A method for stabilizing a peptide, wherein the hydrocarbon containing the above amine includes a secondary amine group, and the lengths of the hydrocarbons connected in front and behind the secondary amine group are each 1 to 10.

4. In paragraph 3, A method for stabilizing a peptide, wherein the hydrocarbon contains a double bond.

5. In paragraph 1, A method for stabilizing a peptide, wherein the hydrocarbon containing the above amine contains a tertiary amine group.

6. In paragraph 1, Any of the above α-amino acids comprises an alkenyl group linked to the α-carbon, A method for stabilizing a peptide, wherein the third or fourth or seventh α-amino acid comprises a side chain amino group in which an alkenyl group is substituted on the nitrogen atom.

7. In paragraph 1, Any of the above α-amino acids is alkenyl alanine ((S)- or (R)-α-alkenyl alanine), A method for stabilizing a peptide, wherein the third, fourth or seventh α-amino acid is lysine containing a lysyl group substituted with an alkenyl group at the nitrogen atom.

8. In paragraph 1, Any of the above α-amino acids contains a side chain amino group in which an alkenyl group is substituted on the nitrogen atom, A method for stabilizing a peptide, wherein the third or fourth or seventh α-amino acid comprises an alkenyl group linked to the α-carbon.

9. In paragraph 1, Any of the above α-amino acids is lysine containing a lysyl group substituted with an alkenyl group on the nitrogen atom, A method for stabilizing a peptide, wherein the third or fourth or seventh α-amino acid is alkenyl alanine ((S)- or (R)-α-alkenyl alanine).

10. In paragraph 1, A method for stabilizing a peptide, wherein the peptide has 4 to 35 amino acids.

11. A peptide having a stabilized alpha helix structure, represented by the following chemical formula 1 or chemical formula 2. [Chemical Formula 1] [Chemical Formula 2] In the above chemical formula 1 or chemical formula 2, x is an integer from 0 to 31, y is an integer from 2 to 6, z is an integer from 0 to 31, and the sum of x to z is from 6 to 35, The above m is an integer from 1 to 7, the above n is an integer from 1 to 6, The above R1 is hydrogen, deuterium, substituted or unsubstituted C1-C 20 Alkyl group, substituted or unsubstituted C1-C 20 Aryl group, substituted or unsubstituted C1-C 20 selected from the group consisting of an acyl group, a benzylcarbamoyl group, and one or more common amino acids, The above R2, R3 and R4 are each independently hydrogen, deuterium, substituted or unsubstituted C1-C 20 Alkyl group, substituted or unsubstituted C1-C 20 is selected from the group consisting of an aryl group and a side chain of a common amino acid.

12. In paragraph 11, A peptide having a stabilized alpha helix structure, wherein in the above chemical formula 1 or chemical formula 2, y is an integer of 2, 3, or 6.

13. In paragraph 11, In the above chemical formula 1 or chemical formula 2, A peptide having a stabilized alpha helix structure, wherein y is an integer of 3, m is an integer of 1 to 3, and n is an integer of 1 to 4.

14. In paragraph 11, In the above chemical formula 1 or chemical formula 2, A peptide having a stabilized alpha helix structure, wherein y is an integer of 2, m is an integer of 1, and n is an integer of 3 to 4.

15. In paragraph 11, In the above chemical formula 1 or chemical formula 2, A peptide having a stabilized alpha helix structure, wherein y is an integer of 6, m is an integer from 3 to 6, and n is an integer from 3 to 4.

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