KR20230050889A - Tripeptide containing histidine derivative and antibacterial composition containing the same - Google Patents

Abstract

The present invention relates to a tripeptide containing a histidine derivative and an antibacterial composition containing the same, and more particularly, to a tripeptide containing a histidine derivative having antibacterial activity against Vibrio and an antibacterial composition containing the same.
The tripeptide containing the histidine derivative of the present invention has an excellent antibacterial effect against Gram-negative bacteria, particularly Vibrio bacteria, and excellent stability in the body when applied to the human body, so it is very useful in industrial fields such as pharmaceuticals.

Description

Tripeptide containing histidine derivative and antibacterial composition containing the same {Tripeptide containing histidine derivative and antibacterial composition containing the same}

The present invention relates to a tripeptide containing a histidine derivative and an antibacterial composition containing the same, and more particularly, to a tripeptide containing a histidine derivative having antibacterial activity against Vibrio and an antibacterial composition containing the same.

Peptide is a core material of bio and is the smallest functional unit of protein, and consists of 2 to 50 amino acids. Not only does it show excellent efficacy in small amounts, but it is also widely used as a major raw material for medicines, foods, and cosmetics because it is non-toxic. In particular, the use of peptide cosmetic materials that are safe and effective for the human body is increasing day by day, and the development of new materials is very important in the industry, such as import substitution effect.

In addition, Peptidomimetic is an analog of peptide, and is a key next-generation biomaterial that can dramatically overcome the in vivo stability, which is a disadvantage of peptides, while maintaining high efficacy, which is an advantage of peptides.

Since 2000, with the development of synthetic technology, commercialization and research on peptides, which had not been commercialized until now, began to be conducted in earnest, and research on new drugs based on this has also steadily increased every year.

On the other hand, Vibrio parahaemolyticus is a food poisoning bacterium that lives in seawater and is mainly detected in seawater, tidal flats, and fish and shellfish in coastal waters. It attaches to the intestines and causes food poisoning to those who ingest it. In general, Vibrio begins to proliferate when the seawater temperature is higher than 15 ° C, and increases to 1 million times in 3 to 4 hours at 20 ~ 37 ° C. Vibrio bacteria live in fish and shellfish in the summer, so when eaten raw, they become infected and cause sepsis and death within a very short period of time, but the development of drugs to treat this is insufficient.

The Vibrio bacteria that causes sepsis is 'Vibrio vulnificus', and Vibrio sepsis causes symptoms throughout the body, such as chills and fever, as well as diarrhea, abdominal pain, and lower extremity pain. Vibrio sepsis is easily infected by people with weakened immune function due to chronic diseases such as chronic liver disease, and when fish and shellfish are eaten raw or undercooked, or when Vibrio bulnificus bacteria in fish and shellfish, seawater, and tidal flats come into contact with skin wounds. become infected when

Korean Patent No. 2037383 discloses a bacteriophage having antibacterial activity against Vibrio and an antibiotic composition containing the same, and Korean Patent No. 1151878 discloses a fatty acid tripeptide salt and an antibacterial composition containing the same. There have not been many studies on peptides with excellent antibacterial activity.

Therefore, as mentioned above, the present inventors, in the process of researching and developing various antibacterial peptidomimetics containing histidine derivatives, found that tripeptides containing histidine derivatives, which are peptidomimetics having specific sequences, have excellent antibacterial activity against Vibrio bacteria. It was confirmed that it has, and the present invention was completed.

An object of the present invention is to provide a tripeptide containing a histidine derivative that acts specifically on Vibrio bacteria and an antibacterial composition containing the tripeptide.

Another object of the present invention is to provide a method for producing a tripeptide containing a histidine derivative that acts specifically on Vibrio bacteria.

In order to achieve the above object, the present invention provides a tripeptide containing a histidine derivative having the structure of Formula I below.

[Formula I]

The present invention also provides an antibacterial composition containing, as an active ingredient, a tripeptide containing a histidine derivative having the structure of Formula I.

In the present invention, the antimicrobial composition is characterized in that it has an antibacterial activity against Vibrio bacteria.

The present invention also, (a) obtaining Fmoc-His (mono-cyclohexylpropyl) -NH-resin represented by Formula 1 by a solid-phase synthesis method; (b) Fmoc-His (mono-cyclohexylpropyl) -OH and Fmoc-Arg (protecting group) -OH are sequentially bonded to the resin obtained in step (a) to which the protected peptide represented by Formula 2 is bonded. Obtaining; (c) obtaining a non-purified tripeptide represented by Formula 3 by simultaneously removing the protecting group of the resin and amino acid from the resin to which the protected peptide obtained in step (b) is bound; and (d) purifying the unpurified tripeptide obtained in step (c) to obtain a tripeptide represented by Formula I.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula I]

In the present invention, the resins are Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, and Rink Amide RAM Resin. ) It is characterized in that selected from the group consisting of.

In the present invention, the acidic solution that simultaneously removes the protecting group of the resin and amino acid is TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/ 5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA/phenol (95 /5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/water (95/ 5), TFA/DCM/indole (70/28/2) and TFA/TIPS/water (95/2.5/2.5).

The tripeptide containing the histidine derivative of the present invention has an excellent antibacterial effect against Gram-negative bacteria, particularly Vibrio bacteria, and excellent stability in the body when applied to the human body, so it is very useful in industrial fields such as pharmaceuticals.

1 is a process diagram for synthesizing a tripeptide including a histidine derivative according to an embodiment of the present invention.
2 is a result of molecular weight measurement (MALDI-TOF MS) of a tripeptide containing a histidine derivative according to the present invention.
3 is an HPLC chromatogram result of measuring the purity of a tripeptide containing a histidine derivative according to the present invention.
4 is a picture of a Disc Diffusion assay evaluating the antibacterial activity of the tripeptide containing histidine derivatives against Vibrio according to the present invention.
5 is an Alamar blue assay result of evaluating the growth inhibitory effect of the tripeptide containing a histidine derivative according to the present invention on Vibrio bacteria.

Unless otherwise indicated herein, the abbreviations used for the designation of amino acids and protecting groups are based on terms recommended by the Commission of Biochemical Nomenclature of IUPAC-IUB ( Biochemistry, 11:1726-1732(1972)). ( Pure & Appl. Chem., Vol. 56, No. 5, pp. 595-624, 1984).

As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by a peptide bond.

Abbreviations for solvents, reagents, protecting groups and amino acids used herein are as follows:

DCM: Dichloromethane

DMSO: Dimethylsulfoxide

DTT: Dithiolthreitol

EDT: 1,2-Ethanedithiol

ACN: Acetonitrile

Arg: Arginine

His: Histidine

HPLC: High Performance Liquid Chromatography

Fmoc: 9-Fluorenyloxycarbonyl

Trt: Triphenylmethyl or Trityl

Pbf: 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl)

PMC: 2,2,5,7,8-pentamethylchroman-6-sulfonyl (2,2,5,7,8-Pentamethylchroman-6-sulfonyl)

Mtr: 4-methoxy-2,3,6-trimethylphenyl-sulfonyl (4-Methoxy-2,3,6-trimethylphenyl-sulfonyl)

Tos: para-Toluenesulfonyl

TES: Triethylsilane

TFA: Trifluoroacetic acid

TIPS: Triisopropylsilane

In the present invention, a tripeptide containing a histidine derivative, which is a peptidomimetic having a specific sequence, was prepared in the course of research and development of various antibacterial peptidomimetics including histidine derivatives. It was confirmed that it has activity.

Accordingly, in one aspect, the present invention relates to a tripeptide comprising a histidine derivative having the structure of Formula I below.

[Formula I]

The present invention also relates to an antibacterial composition containing, as an active ingredient, a tripeptide containing a histidine derivative having the structure of Formula I.

The antimicrobial composition has antibacterial activity against Gram-negative bacteria, and Gram-negative bacteria are Vibrio spp., Salmonella spp. etc. can be exemplified, but is not limited thereto. Examples of the Vibrio bacteria include Vibrio vulnificus , Vibrio alginolyticus , and Vibrio parahaemolyticus .

When the antibacterial composition of the present invention is used as a pharmaceutical composition, it can be used for skin, injection, subcutaneous injection, oral preparation, and the like.

The content of the tripeptide containing the histidine derivative having the structure of formula (I) included in the antibacterial composition of the present invention can be appropriately adjusted according to the use, application form, purpose of use and desired effect, and considering the effect of the content, For example, it may be used within 0.0001 to 99.9% by weight based on the total weight of the composition.

The antibacterial composition of the present invention can be administered orally and parenterally, and can be administered orally, transdermally, subcutaneously, and intravenously, and can also be applied in a topical application method by applying and applying transdermally.

The present invention also, (a) obtaining Fmoc-His (mono-cyclohexylpropyl) -NH-resin represented by Formula 1 by a solid-phase synthesis method; (b) Fmoc-His (mono-cyclohexylpropyl) -OH and Fmoc-Arg (protecting group) -OH are sequentially bonded to the resin obtained in step (a) to which the protected peptide represented by Formula 2 is bonded. Obtaining; (c) obtaining a non-purified tripeptide represented by Formula 3 by simultaneously removing the protecting group of the resin and amino acid from the resin to which the protected peptide obtained in step (b) is bound; and (d) purifying the unpurified tripeptide obtained in step (c) to obtain a tripeptide represented by Formula I.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula I]

Hereinafter, the method for preparing the tripeptide containing the histidine derivative of the present invention will be described in each step.

Step (a): Fmoc-His (mono-cyclohexylpropyl) -NH-resin represented by Formula 1 is obtained by a solid-phase synthesis method.

As the resin, Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, Rink Amide RAM Resin, etc. can be used. can

Solvents used in the present invention include dichloromethane, N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrroly N-Methylpyrrolidone, Chloroform, 1,2-Dichloroethane, Tetrahydrofurane, 1,4-Dioxane, Methanol ), ethanol, isopropanol, ethylene glycol, methyl acetate, ethyl acetate, and the like, but are not limited thereto.

Step (b): Fmoc-His (mono-cyclohexylpropyl) -OH and Fmoc-Arg (protecting group) -OH are sequentially bonded to the resin obtained in step (a) to obtain a protected peptide represented by Formula 2. obtain resin.

The step (b) may further include (i) removing the amino acid protecting group (N-terminal amino group) bound to the resin by treating it with a base and washing the resin; (ii) reacting and binding the newly bound protected amino acid to the washed resin.

In Formula 2 of the present invention, the protecting group is a methoxymethyl group, a benzyloxymethyl group, a triphenylmethyl group, a tert -butyldimethylsilyl group, a tert-butyldiphenylsilyl group ( tert -Butyldiphenylsilyl) group, phenyldimethylsilyl group, triphenylsilyl group, triethylsilyl group, triisopropylsilyl group, para-methoxybenzyl ( para -Methyloxybenzyl) group, tetrahydropyran group, tetrahydrofuran group, tert -butyl group, diphenylmethyl group, 2-chlorotrityl group, benzyl ( Benzyl) group , o-Nitrobenzyl group, p-nitrobenzyl group, 9-phenylxanthyl group, methoxytrityl group, allyl group, methyl group, methoxyethoxymethyl group, ethoxyethyl group, methylthiomethyl group, 2-naphtylmethyl group, tert-butyloxy Carbonyl ( tert -Butyloxycarbonyl) group, benzyloxycarbonyl (Benzyloxycarbonyl), acetyl (Acetyl) group, benzoyl (Benzoyl) group, para-toluenesulfonyl ( para -Toluenesulfonyl) group, 2,4,6-trimethylphenyl group phonyl (2,4,6-Trimethylphenylsulfonyl, mesityl-2-sulfonyl) group, 4-methoxy-2,3,6-trimethylphenylsulfonyl (4-Methoxy-2,3,6-trimethylphenylsulfonyl) group, Pmc ( 2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group, MIS (1,2-dimethylindole-3-sulfonyl) Group, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl) group, NO₂ (Nitro) group, etc. can be exemplified, and para-Toluenesulfonyl ( para -Toluenesulfonyl) group, 2,4,6-trimethylphenyl Sulfonyl (2,4,6-Trimethylphenylsulfonyl, mesityl-2-sulfonyl) group, 4-methoxy-2,3,6-trimethylphenylsulfonyl (4-methoxy-2,3,6-trimethylphenylsulfonyl) group, Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group, MIS (1,2-dimethylindole-3-sulfonyl) group ) group, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl) group, NO₂ (Nitro) group is preferred, and Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group, MIS (1,2-dimethylindole-3-sulfonyl) group, Mtr (4-methoxy-2,3,6-trimethylphenylsulfonyl) group, NO₂ It is more preferable to use a (Nitro) group, and it is most preferable to use Pbf (2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) as the amide protecting group, but is not limited thereto.

Bases that remove the amino acid protecting group (N-terminal amino group) include piperidine, 4-methylpiperidine, pyrrolidine, piperazine, hydrazine hydrate (Hydrazine hydrate), DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene), 4-Methylpiperidine, 1-methyl-3-butylimidazolium tetrafluoro Borane (1-methyl-3-butyl imidazolium BF4), Ethanolamine, Cyclohexylamine, Dicyclohexylamine, Tris(2-aminoethyl)amine ), 1,3-Dicyclohexanebis-(methylamine) (1,3-Dicyclohexanebis-(methylamine)), 1,4-bis-(3-aminopropyl)piperazine (1,4-Bis-(3 -aminopropyl)piperazine), diethylamine, 4-dimethylaminopyridine, or organic bases such as Lithium hydroxide, Sodium Hydroxide, Calcium Hydroxide, Hydroxide Inorganic bases such as potassium (Potassium Hydroxide) may be used.

In the present invention, reagents used when reacting and binding protected amino acids to the washed resin include DCC (N,N-Dicyclohexylcarbodiimide), DIC (N,N-Diisopropylcarbodiimide), BOP (Benzotriazole-1-yl- oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), PyBrOP (Bromo-tripyrrolidino-phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate) , PyOxim (Ethyl cyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate), HBTU (O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), HCTU (2-(6 -Chloro-1H-benzotriazol-1-yl)-N,N,N',N',-tetramethylaminium hexafluorophosphate), HDMC (N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino] -uronium hexafluorophosphate N-oxide), TBTU (O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU (2-(1H-7-Azabenzotriazol-1-yl)-1 ,1,3,3-tetramethyluroniumhexafluorophosphatemethanaminium), TATU (2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminium), COMU (1-[1-(Cyano-2- ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate), TOTT (2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethyl-isothiouronium tetrafluoroborate), EDCㅇ HCl (N-(3-dimethylaminopropyl)-N`-ethylcarbodiimide hydrochloride), TFFH (Tetramethylfluoroformamidinium hexafluorophosphate), EEDQ (N-Ethoxycarbony-2-ethoxy-1,2-dihydro-quinoline), T3P (2-propanephosphonic acid anhydride) , DEPBT (3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma (Ethyl cyanohydroxyiminoacetate), HOBt (1-Hydroxybenzotriazole), HOOBt (HODhbt, Hydroxy-3,4-dihydro- 4-ox-1,2,3-benzo-triazine), BTC (bis-Trichloromethylcarbonate or Triphosgene), CDI (1,1'-Carbonyldiimidazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole), HOSu (N-Hydroxysuccinimide), and the like can be exemplified.

Step (c): From the resin to which the protected peptide obtained in step (b) is bound, the protecting groups of the resin and the amino acid are simultaneously removed to obtain a non-purified tripeptide represented by Chemical Formula 3.

The process of simultaneously removing the protecting groups of the resin and the amino acid is performed in the presence of an acidic solution, which is TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5/5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2 ), TFA/phenol (95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5) , TFA/water (95/5), TFA/DCM/indole (70/28/2) and TFA/TIPS/water (95/2.5/2.5) solutions.

Step (d): The tripeptide represented by Formula I is obtained by purifying the crude tripeptide obtained in step (c).

The purification may be performed by a method commonly used for peptide purification, and normal phase or reverse phase high performance liquid chromatography (HPLC) may be used. The solvent used for purification may be selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, purified water, and a mixture thereof, and trifluoroacetic acid , acids such as acetic acid and formic acid may be used together.

Hereinafter, the present invention will be described in more detail by way of examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

[Example]

Throughout this specification, "%" used to indicate the concentration of a particular substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and (weight/volume) % for solid/solid, and liquid /liquid is (volume/volume) %.

Example 1: Preparation of H-His (mono-cyclohexylpropyl) -NH-resin (Rink Amide)

Linkamide AM resin (substitution rate = 0.70 mmol/g, 100 mmole) and N,N-dimethylformamide (1000 ml, hereinafter referred to as DMF) were put in a solid-phase synthesis reactor equipped with a filtration membrane, and the resin was allowed to boil for 10 minutes. After swelling, the solvent was removed through a filtration membrane under reduced pressure. Fmoc-His(mono-cyclohexylpropyl)-OH (molecular weight = 501.62 g/mol) (150.49 g, 300 mmol, 3.0 equiv) and Oxyma (molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) were dissolved in 1000 ml of DMF. then put in. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution, and the mixture was slowly stirred at room temperature for 5 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of a 20% piperidine/DMF solution was added and stirred for 15 minutes, then the reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-His(mono-cyclohexylpropyl)-NH-resin (Rink Amide) was obtained (substitution rate: 0.7 mmol/g, yield >99%).

Example 2: Preparation of H-His (mono-cyclohexylpropyl) -His (mono-cyclohexylpropyl) -NH-resin (Rink Amide)

Fmoc-His(mono-cyclohexylpropyl)-OH (molecular weight = 501.62 g/mol) (150.49 g, 300 mmol, 3.0 equivalent) and Oxyma (molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution, and the mixture was slowly stirred at room temperature for 5 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of a 20% piperidine/DMF solution was added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, the resin was treated once more in the same manner, and the resin was washed 6 times with 1000 ml of DMF in total, followed by H-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-resin (Rink Amide) was obtained (yield >99%).

Example 3: Preparation of H-Arg(pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-Resin (Rink Amide)

Fmoc-Arg(Pbf)-OH (molecular weight = 648.8 g/mol, 194.64 g , 300 mmole) and Oxyma (molecular weight = 142.11 g/mol, 46.90 g, 330 mmole) were dissolved in 1000 ml of DMF and added thereto. DIC (molecular weight = 126.2 g/mol, 41.7 g, 330 mmole) was added dropwise to the mixed reaction solution, and the mixture was slowly stirred at room temperature for 5 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of a 20% piperidine/DMF solution is added and stirred for 15 minutes. The reaction solution was removed by filtration under reduced pressure, and the resin was treated once more in the same manner. After washing the resin a total of 6 times with 1000 ml of DMF, H-Arg(Pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-resin (Rink Amide) was obtained (yield >99%).

Example 4: Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ Manufacturing

H-Arg(Pbf)-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH-resin (Rink Amide) was put into the reactor and cooled TFA/TIPS/water (95/2.5/2.5) solution 3 L was slowly poured into the mixture and stirred at room temperature for 3 hours. The reaction solution was slowly added dropwise to 12 L of cooled diethyl ether to precipitate the peptide. After stirring at room temperature for 30 minutes and then filtering to recover the peptide, the solvent was removed under reduced pressure. Dry in a vacuum dryer for 5 hours to obtain white Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ 80.03 g was obtained. (Molecular weight 696.15 g/mol, crude peptide purity 65.7%, yield 115.0%)

Example 5: Preparation of H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂

[Formula I]

Crude H-Arg-His(mono-cyclohexylpropyl)-His(mono-cyclohexylpropyl)-NH₂ obtained in Example 4 80.03 g was dissolved in purified water and filtered through a 0.46 μm membrane. After repeated injection of the filtrate by industrial HPLC (230 nm, 500 ml/min, 10 micron C18 column, initial concentration of acetonitrile in 0.1% TFA increased from 30% to 65% within 20 minutes), the main fraction was fractionated and chemical formula 29.45 g of the tripeptide represented by I (yield: 36.8%, purity: 99.21%) was obtained.

The molecular weight and purity of the obtained tripeptide containing histidine derivatives were confirmed, and the results are shown in FIGS. 2 and 3 .

Experimental Example 1: Disk Diffusion Assay

A disk diffusion assay was performed to confirm the antibacterial activity of the antibacterial peptide prepared in Example 5 against Vibrio.

More specifically, the tripeptide represented by Formula I prepared in Example 5 (represented as WP-Vib1) (2 mM), DMSO, H ₂ O, and gentamicin (2 mM) as a control, 20 μL, respectively, were absorbed into a 6 mm paper disc. then dried. Next, 1.5 × 10 ⁸ cfu of Vibrio parahaemolyticus (Biological Resources Center, KCTC 2471) was spread on the LB Agar medium, and then the dried paper disc was placed on the medium and maintained at 37.5 ° C for 18 hours. cultured for a while.

As a result, as shown in Figure 4, the tripeptide represented by Formula I prepared in Example 5 (represented by WP-Vib1) is similar to gentamicin, which is a positive control, for Vibrio parahaemolyticus . It was found that it had antibacterial activity and inhibited the growth of bacteria.

Experimental Example 2: Alamar blue assay

In order to confirm the antibacterial activity of the antibacterial peptide prepared in Example 5 against Vibrio, alamar blue assay was performed.

10 ⁶ Vibrio parahaemolyticus (Center for Biological Resources, KCTC 2471) 80 μL of culture medium and 10 μL of Alamar Blue (10x) were put into each well of a 96 well plate, and cultured in a shaking incubator for 1 hour. After incubation, 10 μL of the sample was treated in each well, and fluorescence was measured at 15-minute intervals under emission conditions at 530 nm and excitation at 590 nm using a fluorescence plate reader (Victor3, PerkinElmer).

At this time, the sample is gentamicin (concentration: 0.016 ~ 2mM), tripeptide (concentration: 0.016 ~ 2mM) (represented by WP-Vib1), tripeptide represented by Formula I prepared in Example 5 (concentration: 0.016 ~ 2mM) (denoted as WP-Vib1) was used.

Effective concentration (EC ₅₀ ) values were calculated using Graphpad prism software (version 4.0), and the results are shown in FIG. 5 .

As shown in Figure 5, the tripeptide represented by Formula I prepared in Example 5 (concentration: 0.016 ~ 2 mM) (represented as WP-Vib1) is similar to the positive control gentamicin, Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) was found to have antibacterial activity.

As above, specific parts of the present invention have been described in detail, and it will be clear to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

A tripeptide comprising a histidine derivative having the structure of Formula I below.
[Formula I]

An antibacterial composition containing a tripeptide containing a histidine derivative having the structure of claim 1 as an active ingredient.
The antimicrobial composition according to claim 2, wherein the antimicrobial composition has antibacterial activity against Vibrio bacteria.
(a) obtaining Fmoc-His (mono-cyclohexylpropyl) -NH-resin represented by Formula 1 by a solid-phase synthesis method;
(b) Resin obtained by sequentially combining Fmoc-His (mono-cyclohexylpropyl) -OH and Fmoc-Arg (protecting group) -OH to the resin obtained in step (a) to which the protected peptide represented by Formula 2 is bound Obtaining;
(c) obtaining a non-purified tripeptide represented by Chemical Formula 3 by simultaneously removing the protecting group of the resin and amino acid from the resin to which the protected peptide obtained in step (b) is bound; and
(d) Purifying the non-purified tripeptide obtained in step (c) to obtain a tripeptide represented by formula (I).

[Formula 1]

[Formula 2]

[Formula 3]

[Formula I]

The method of claim 4, wherein the resins are Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, and Rink Amide RAM Resin. Resin) method for producing a tripeptide, characterized in that selected from the group consisting of.
The acidic solution of claim 4, which simultaneously removes the protecting groups of the resin and amino acids, is TFA/phenol/water/TIPS (88/5/5/2), TFA/phenol/water/thioanisole/EDT (82.5/5/5 /5/2.5), TFA/phenol/water/thioanisole/1-decanethiol (82.5/5/5/5/2.5), TFA/DTT/water/TIPS (88/5/5/2), TFA/phenol ( 95/5), TFA/phenol/Methanesulfonic acid (95/2.5/2.5), TFA/thioanisole/EDT/anisole (90/5/3/2), TFA/TES (95/5), TFA/water (95 /5), TFA / DCM / indole (70/28/2) and TFA / TIPS / water (95 / 2.5 / 2.5) method for producing a tripeptide, characterized in that selected from the group consisting of.

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