KR102163568B1 - Fatty acid antifungal peptide and antifungal composition comprising the same - Google Patents

Abstract

The present invention relates to a fatty acid antimicrobial peptide and an antimicrobial composition containing the same. According to the present invention, there is no skin irritation with strong antibacterial/antifungal activity against various bacteria including Staphylococcus strain, and antibacterial for pharmaceutical use or cosmetic use for the prevention or treatment of diseases caused by various bacteria There is an effect of providing a composition.

Description

Fatty acid antifungal peptide and antifungal composition comprising the same}

The present invention has strong antibacterial/antifungal activity against various bacteria including fatty acid antibacterial peptide and Staphylococcus strain containing the same and does not have skin irritation, and uses pharmaceuticals for the prevention or treatment of diseases caused by various bacteria , To an antibacterial composition for cosmetic use.

Infectious diseases are diseases caused by pathogenic microorganisms, and sometimes bacterial growth and infection occur even in a healthy immune system. As a result, bacteria cause typical bacterial infections by releasing toxins that both threaten the body and damage cells and tissues. Infectious diseases have been controlled and prevented by antimicrobial drugs developed since the 1970s, known as the era of antibiotics, but due to the emergence of new diseases and resistance to antibiotics caused by mutations of bacteria, it is necessary to develop new antimicrobial agents that are less resistant and safe for the human body. In addition to pharmaceuticals, safety issues have arisen in antimicrobial agents currently used in quasi-drugs such as cosmetics or pharmaceutical ointments applied to the skin.

Currently, mycin, triclosan, and salicylic acid are used as antibacterial ingredients in pharmaceuticals and quasi-drugs, but because of strong skin toxicity and skin side effects and tolerance problems, they are prescribed by doctors or are prescribed for some topically used products. In cosmetics, parabens such as methyl paraben, ethyl paraben, propyl paraben, and butyl paraben, and antibacterial agents such as phenoxy ethanol, dichlorophene, and methyl isothiazolinone were used.However, a product that is applied due to the rise of human toxicity problems (Leave-on product) The use of paraben and methyl isothiazolinone is prohibited by the Ministry of Food and Drug Safety, and plant extracts, 1,2-hexanediol, and caprylyl glycol are used as substitute raw materials for preservatives. There are disadvantages such as giving changes, so the development of new antibacterial agents is urgently needed.

Peptides are substances in which more than 20 amino acids exist in nature through one or more peptide bonds, and are known as substances that control most of the signal transduction in the human body. Peptides have high efficacy even at low concentrations when they have a specific sequence, and are known to have significantly less side effects on the human body.In the case of pharmaceuticals, the development of indicator substances using peptides or new drugs using peptides or derivatives is being actively conducted. It is composed of and is decomposed by enzymes in the human body after it has fulfilled its necessary role, so that the human body is more safe than other materials.

Antibacterial peptides for application to pharmaceuticals or cosmetics can be produced in large quantities and should have excellent antibacterial effects (refer to previous papers in Documents 1 and 2 below). In particular, long-sequence peptides (oligo peptides) with a large number of amino acids are difficult to commercially apply because of high production cost due to high raw material cost and low yield when a general chemical synthesis method is applied, whereas peptides with short amino acids have the potential to induce immunity. It is suitable for the application of pharmaceuticals, quasi-drugs and cosmetics because it is safe for human body and is easy to use commercially at cost.

Prior art literature

[Document 1] Mohammad R, Andreas V, Appl Microbiol Biotechnol (2015) 99:8847-8855

[Document 2] Sanadhya I, Durve A, International Journal of pharmacy and pharmaceutical science, (2014) Vol 6, Issue 6

Accordingly, the present inventors constructed an antibacterial activity test system using two types of gram-positive bacteria, two gram-negative bacteria, and two fungi, which are the most representative antibacterial and antifungal dipeptides with the maximum number of amino acids, and then minimal inhibition of the bacteria by the active ingredient. The concentration and the minimum sterilization concentration were measured, the antibacterial activity was confirmed, the skin stability was confirmed through a human intelligence test, and the present invention was completed.

That is, the main object of the present invention is to provide a fatty acid dipeptide salt that is harmless to the human body, has excellent antibacterial/antifungal effects, and is excellent in skin penetration and stability, and an antibacterial composition containing the same.

According to an embodiment of the present invention, a fatty acid dipeptide represented by the following formula 1 is provided:

[Formula 1]

Ne-X ₁ -X ₂ -Ce

(In the above formula, at least one selected from X ₁ and X ₂ is lysine (K), and the remaining X ₁ or X ₂ not selected as lysine (K) is 19 amino acids excluding lysine (K) (lysine (K) Except) is selected from (preferably histidine (H) or arginine (R)), and at least _one of the side chains of X ₁ and X ₂ forms a salt with HCl or R ¹ COOH, wherein R ¹ Is selected from CH ₃ , CF ₃ , or CCl ₃ ,

Ne corresponds to the N-terminus of the peptide and is selected from the group consisting of hydrogen, acetyl, ethanoyl, octanoyl, tetradecanoyl, hexadecanoyl and octadecanoyl, and Ce corresponds to the C-terminus of the peptide, carboxylic acid, It is selected from the group consisting of amide, hydroxyamic acid, methyl ester and ethyl ester.

According to another embodiment of the present invention, there is provided an antimicrobial composition comprising the fatty acid dipeptide as an active ingredient.

According to the present invention, dipeptide bound with fatty acids exhibits excellent antibacterial activity not only against gram-positive bacteria and gram-negative bacteria, but also fungi, and thus, pharmaceutical compositions for preventing or treating diseases caused by various bacteria, antibacterial quasi-drug compositions and cosmetics, etc. It can be widely used.

1 shows the minimum bactericidal concentration for Candida Albicans and Aspergilus niger at effective concentrations of MIP_2, MIP_3, MIP_4, MIP_6, and MIP_7 synthesized according to the present invention by spreading on a solid medium. This is the result of confirmation.

Hereinafter, the present invention will be described in more detail.

Fatty acid dipeptide according to the present invention is a long sequence peptide (oligopeptide) with a large number of amino acids in consideration of the fact that it is difficult to commercially apply because the raw material cost is high and the production cost is low due to low yield when a general chemical synthesis method is applied. It was conceived, as the number of amino acids was short and the possibility of inducing immunity was low, so it is safe for the human body and it is easy to commercially use in terms of cost, so it is suitable for application to various fields such as pharmaceuticals, quasi-drugs and cosmetics.

That is, the fatty acid dipeptide according to the present invention has an amino acid sequence selected from the group consisting of KK, KH, KR, HK, RK, etc., mainly of polar amino acids, and may be represented by the following Formula 1 as an example:

[Formula 1]

Ne-X ₁ -X ₂ -Ce

(In the above formula, at least one selected from X ₁ and X ₂ is lysine (K), and the remaining X ₁ or X ₂ not selected as lysine (K) is 19 amino acids excluding lysine (K) (lysine (K) Except), and at least _one of the side chain of X _{1 and} the side chain of X ₂ forms a salt with HCl or R ¹ COOH, wherein R ¹ is selected from CH ₃ , CF ₃ or CCl ₃ ,

Ne corresponds to the N-terminus of the peptide and is selected from the group consisting of hydrogen, acetyl, ethanoyl, octanoyl, tetradecanoyl, hexadecanoyl and octadecanoyl, and Ce corresponds to the C-terminus of the peptide, carboxylic acid, It is selected from the group consisting of amide, hydroxyamic acid, methyl ester and ethyl ester.

The fatty acid dipeptide according to the present invention may have an amino acid sequence selected from the group consisting of KK, KH, KR, HK, RK, etc., mainly polar amino acids, but is not limited thereto. The peptide can insert, replace, or delete amino acids within a range that does not significantly lower the stability of the peptide and similar antimicrobial performance, and this also falls within the scope of the present invention. Ne corresponding to the N-terminus of the peptide may be a hydrogen or an acetyl group without acylation or acylation with a fatty acid. In the case of acylating the N-terminus, the N-terminus close to hydrophilicity is changed to hydrophobic properties, so that the N-terminus of various proteases present in vivo is recognized and decomposed by enzymes that cleave proteins or peptides. It is possible to induce stability in vivo by inhibiting it.

In a specific example, Ne is hydrogen or acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, octanoyl, nonanoyl, decanoyl, dodecanoyl, tetradecanoyl, hexadecanoyl, octadecanoyl, etc. Acyl groups may be included. It is preferable to be selected from hydrogen, acetyl, octanoyl, decanoyl, didecanoyl, tetradecanoyl, hexadecanoyl or octadecanoyl, and in the case of acetyl, didecanoyl, tetradecanoyl, and hexadecanoyl, the antibacterial effect is It is most preferable because it is more excellent. Ne may also be an alkenyl group containing at least one double bond.

In addition, Ce corresponding to the C-terminus of the peptide may be carboxylic acid (COOH), amide (CONH ₂ ), hydroxylamic acid, methyl ester, ethyl ester, and the like.

In addition, the antimicrobial peptide according to the present invention may further include a cell permeable peptide that promotes intracellular migration of the peptide. For example, the cell-penetrating peptide may be a TAT peptide (RKKRRYRRR) and a Tat-PETD peptide (GRKKRRQRRR: Tat PTD), but is not limited thereto, and cell-penetrating peptides known in the art are the peptides according to the present invention. Anything within the range that does not inhibit the antimicrobial activity falls within the scope of the present invention.

On the other hand, the peptide of the present invention may exist in the form of a salt, and the form of the salt that can be used at this time may be made during final separation and purification of the compound or by reacting an amino group with an appropriate acid. For example, as an acid addition salt, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorrate, camposulfonate, digluconate, glycerophosphate, hemi Sulfate, heptanoate, hexanoate, formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, mesylenesulfonate, methanesulfonate, Naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, Trichloroloacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, para-toluenesulfonate, and undecanoate may be used, but are not limited thereto. In addition, examples of the acid that can be used to form the acid addition salt may be inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid, or organic acids such as oxalic acid, maleic acid, succinic acid, and citric acid, but are not limited thereto. .

The alkyl group R ¹ of the fatty acid is selected from CH ₃ , CF ₃ or CCl ₃ , and such a salt can be synthesized by a solid phase method using a conventional peptide synthesizer.

Typically, amino acids used in solid-phase peptide synthesis are used in a form in which the N-terminus is protected and a form in which the reactive side chain is protected. As a protecting group protecting the N-terminal, a 9-fluorenylmethyloxycarbonyl group (Fmoc) is generally used. As a protecting group for protecting the reactive side chain, triphenylmethyl, butyloxycarbonyl, t-butyl ester, pentamethylchroman-6-sulfonyl, and the like are used. Lysine, an amino acid used in the present invention, may be protected at the N-terminus with a 9-fluorenylmethyloxycarbonyl group (Fmoc), and an amine group on the side chain may be protected with t-butyloxycarbonyl. The resin used for the solid phase reaction is made to recover the peptide C-terminus in the form of an amide, for example, trialkoxybenzhydrylamine (Rink amide), methylbenzhydrylamine (MBHA), or 5,4-amino Methyl-3,3-dimethoxyphenoxy valeric acid (PAL) resin can be used.

At this time, the peptide is synthesized from the C-terminus to the N-terminus. Upon coupling, the carboxyl group of the amino acid is activated with an active agent, and after coupling, the N-terminal protecting group is removed with piperidine, and the next amino acid coupling is performed. Carboxyl activators include HATU (Azabenzotriazole Tetramethyl Uronium Hexafluorophosphate) and 1-hydroxy-7-azabenzotriazole (HOAt) or HBTU (N-[(1H-benzotriazol-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate) and 1-hydroxybenzotriazole (HOBt) can be used with DIEA (N,N-diisopropylethylamine), and N,N'-diisopropyl Carbodiimide (DIC) and 1-hydroxy-7-azabenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt) may be used together as an additive adjuvant.

In the present invention, after two amino acids are coupled, the N-terminal amine group is an acyl activator having a desired alkyl group, for example, acyl anhydride and DMAP (4-(N,N-dimethylamino)pyridine)) together. Reaction to form an amide bond at the N-terminus or fatty acid (Fatty acid) and N,N'-diisopropylcarbodiimide (DIC), 1-hydroxy-7-azabenzotriazole (HOAt) or 1-hydroxy Roxybenzotriazole (HOBt) can be used together to form amide bonds.

Then, the peptide derivative synthesized by the cleavage solution is separated from the solid resin, and the protecting group bound to its side chain is also removed. At this time, when a cleavage solution containing trifluoroacetic acid (TFA) is used, the cleaved peptide is typically recovered in the form of a TFA salt, and in the present invention, it was confirmed that the peptide TFA salt was formed by the characteristics of the lysine residue. . This is the same even when acetic acid or trichloroacetic acid is used. At this time, both side chain amine groups in the lysine of the peptide derivative according to the present invention can form a salt, and all salts of 1 to 2 side chain amine groups can be formed according to pH adjustment, and all salt forms are antibacterial Have activity

Accordingly, it can be understood that the fatty acid peptide derivative according to the present invention includes not only a form in which both side chains of the two amino acids of the peptide form a salt with a fatty acid, but also a form in which one of the two amino acids forms a salt.

The antimicrobial peptide according to the present invention has a broad antibacterial spectrum and can be included in the antibacterial composition. In the present invention, the antibacterial composition includes a cosmetic composition as a composition exhibiting antibacterial or antifungal activity, and may also include a pharmaceutical composition. In addition, since the antibacterial peptide according to the present invention is less irritating and has almost no side effects, it can provide many advantages when applied to a skin treatment composition.

The antimicrobial composition according to the present invention may contain one or two or more of the peptides represented by Formula 1, and further include a previously known antibacterial agent within a range that does not interfere with the antibacterial efficacy of the peptide according to the present invention. I can.

The composition according to the present invention may additionally contain conventional additives known in the art for stabilization, formulation, or in vivo delivery of a novel antimicrobial peptide compound, for example, a sugar chain compound, a lipid compound, a nucleic acid compound, a peptide Or it may include a protein or the like. For example, lipid compounds include dipalmitoylphosphatidylcholine (DPPC), palmitoyl oleylphosphatidylglycerol (POPG), phosphatidylglycerol (PG), C ₁₈ saturated fatty acid, C ₁₆ unsaturated fatty acid, and C ₁₈ unsaturated fatty acid.

The composition according to the present invention comprising the novel antimicrobial peptide of the present invention as an active ingredient may be in the form of a general pharmaceutical formulation. Parenteral preparations may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried agents, and pharmaceutically acceptable carriers; Orally administered preparations may contain a binder, a lubricant, a disintegrant, an excipient, a solubilizing agent, a dispersing agent, a stabilizer, a suspending agent, a colorant, a fragrance, and the like; Injectables may include buffers, preservatives, painless agents, solubilizers, isotonic agents, stabilizers, and the like; Formulations for topical administration may include base agents, excipients, lubricants, preservatives, and the like.

The pharmaceutical formulation of the composition according to the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above, 　For example, when administered orally, tablets, troches, capsules, elisir, suspension, It can be prepared in the form of syrup, wafer, or the like, and in the case of injection, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. It is administered through the usual route of introducing a predetermined substance to the patient by an appropriate method of administration. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered, but are not limited thereto. However, when administered orally, since the peptide is digested, the oral composition is preferably coated with an active agent or formulated to be protected from degradation in the stomach. In addition, the composition according to the present invention can be administered by any device capable of moving the active substance to the target cell. Preferred modes of administration and formulations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, drop injections and the like. Injectables include aqueous solvents such as physiological saline and Ringel's solution, vegetable oils, higher fatty acid esters (e.g., oleic acid ethyl, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). It can be prepared using, and stabilizers for preventing deterioration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, and for inhibiting microbial growth Pharmaceutical carriers such as preservatives (eg, mercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.

The peptide of the present invention or a composition comprising the same is administered in a pharmaceutically effective amount. According to factors well known in the medical field, such as type of disease, patient's age, weight, health, sex, patient's sensitivity to drugs, route of administration, method of administration, frequency of administration, duration of treatment, and drugs used in combination or concurrently. Can be easily determined by

For example, the bio-administration dose of the antimicrobial peptide according to the present invention is not limited as long as it is sufficient to show antibacterial or skin treatment performance, and those skilled in the art can appropriately select the dosage amount for exhibiting the desired effect. Meanwhile, according to the present invention, the antimicrobial peptide of the present invention can be used not only in humans, but also in livestock such as cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, cats, etc. that may exhibit similar bioimmune reactions.

For example, the compound of Formula 1 is preferably contained in an antibacterial cosmetic or pharmaceutical composition in an amount of 0.0001 to 20.0% by weight, and more preferably 0.001 to 15.0% by weight. When the content is less than 0.0001% by weight, the antibacterial effect is insignificant, and when the content is more than 20.0% by weight, there is a high tendency to lack economical efficiency according to the amount used.

Hereinafter, the present invention will be described in more detail through examples, but these examples are for illustrative purposes only, and the scope of the present invention is not construed as being limited by these examples. In the following examples and throughout the specification, abbreviations and notations have the following meanings. The stereochemistry of the α-carbon of amino acids and aminoacyl residues in the peptides described throughout the specification may be in the form of'L' or'D'. The stereochemistry of the chiral center in a specific acyl substituent at the N-terminus of the peptide of the present invention can be indicated by using the names Kahun-Ingold-Prelog'R' and'S'. This nomenclature is described in R.S. Cahn, et al., Angew. Chem. Int. Ed. Engl., (1966) 5, 385-415]. All peptide sequences were described according to generally accepted conventions with the N-terminal amino acid residue on the left and the C-terminal on the right.

As used herein, the'N-terminal' refers to the free α-amino group of an amino acid in the peptide, and the term'C-terminal' refers to the free carboxylic acid end of the amino acid in the peptide. Most of the time, as used herein, the nomenclature for naturally occurring and non-naturally occurring aminoacyl moieties is the IUPAC Committee on Naming Organic Chemistry and literature ['Nomenclature of α-Amino Acids (Recommendations, 1974)' Biochemistry, ( 1975) 14(2)] in accordance with the nomenclature convention proposed by the IUPAC-IUB Committee on Biochemical Nomenclature. The names and abbreviations of naturally occurring or non-naturally occurring amino acids used in this specification and the appended claims are indicated as follows. Ala, A: alanine; Arg, R: arginine; Asn, N: asparagine; Asp, D: aspartic acid; Cys, C: cysteine; Glu, E: glutamic acid; Gin, Q: glutamine; Gly, G: glycine; His, H: histidine; Ile, I: isoleucine; Leu, L: leucine; Lys, K: lysine; Met, M: methionine; Nle: noleucine; Phe, F: phenylalanine; Pro, P: proline; Ser, S: serine; Thr, T: threonine; Trp, W: tryptophan; Tyr, Y: tyrosine; Val, V: valine.

If not indicated above as an abbreviation, nomenclature and abbreviations are described in Calbiochem-Novabiochem Corp. 1999 Catalog and Peptide Synthesis Handbook or the Chem-Impex International, Inc. Tools for Peptide amp; Solid Phase Synthesis 1998-1999 Catalog] can be referred.

[Example]

<Example 1: Synthesis of Compounds 01 to 15>

Sample preparation (synthesis and separation and purification)

Merrifield's SPPS (Solid Phase Peptide Synthesis) method (J. Am. Chem. Soc.,) to synthesize 15 types of MIP series of compounds of the present invention shown in Table 1 below as fatty acid dipeptides of the present invention (J. Am. Chem. Soc., 85(14), 2149-2154) was used.

Specifically, Rink amide resin (trialkoxybenzhydrylamine resin) was used to synthesize a peptide having an amide form at the carboxyl terminal, and Fmoc (9-fluorenylmethoxy carbonyl)-amino acid was used as HBTU (N-[(1H-benzotriazol-1-yl). -1,1,3,3-tetramethyluronium hexafluorophosphate) and HOBt (1-hydroxybenzotriazole) and DIEA (N,N-diisopropylethylamine) were used together to perform coupling.

From the Fmoc-amino acid in the subsequent step, DIEA (N,N-diisopropylethylamine) in HATU (Azabenzotriazole Tetramethyl Uronium Hexafluorophosphate) and 1-hydroxy-7-azabenzotriazole (HOAt) or HBTU and 1-hydroxybenzotriazole (HOBt). ) Was used together to extend the chain of the peptide by performing coupling.

At this time, the synthesis proceeds from the C-terminus to the N-terminus, and after coupling the Fmoc-amino acid to the amino terminal, the Fmoc group is removed with a 20% piperidine solution (Solid-Phase Synthesis: A Practical Guide (1 ed.). CRC Press. 848), washed several times with DMF (Dimethylforamide), and then coupled.

Finally, after coupling the Fmoc-amino acid, remove the Fmoc group with a 20% piperidine solution, and the fatty acid or carboxylic acid with N,N'-diisopropylcarbodiimide (DIC), 1-hydroxy-7-azabenzotria A sol (HOAt) was used together to couple an acyl group, and then washed several times with DMF and dried.

TFA (Trifuloroacetic acid)-H ₂ O-Triisopropylsilane (95:2.5:2.5, vol./vol.) solution was added and reacted for 2 to 3 hours to remove the protecting group, and the peptide was separated from the resin, followed by diethyl ether ( diethyl ether) to precipitate the peptide.

The crude peptide obtained by this method was a gradient solvent composition of acetonitrile containing 0.1% TFA and water, and the purity was checked using RP-HPLC (2996 Detector, 515 Pump, Waters), and the molecular weight was confirmed by Mass.

At this time, when the C-terminus was a carboxyl compound, 2-chlorotrityl chloride (CTC) resin was used, and coupling was performed in the same manner from the next step except for the first coupling method when synthesizing a peptide using Rink amide resin.

In the case of methyl ester, the final product was recovered from the resin, methanol was added to a solvent, esterified under an acid catalyst, and then a protecting group was removed and purified. Like compound MIP_1, 15 compounds of MIP_15 were synthesized from compound MIP_1.

division order MIP_1 C14-EK-NH ₂ MIP_2 C14-HK-NH ₂ MIP_3 C14-KK-NH ₂ MIP_4 C14-RK-NH ₂ MIP_5 C14-KE-NH ₂ MIP_6 C14-KH-NH ₂ MIP_7 C14-KR-NH ₂ MIP_8 C14-KK-OMe MIP_9 C14-KK-OH MIP_10 C16-KK-NH ₂ MIP_11 C2-KK-NH ₂ MIP_12 C2-KR-NH ₂ MIP_13 C10-KK-NH ₂ MIP_14 C12-KK-NH ₂ MIP_15 C14-KKK-OH

<Comparative Example 1: Synthesis of Compounds 313 and 315>

Sample preparation (synthesis and separation and purification)

To contrast with the fatty acid dipeptide of the present invention, the tripeptide was synthesized as follows.

For the synthesis of tripeptides having an amide carboxyl terminus, synthesis was started using Rink amide resin (Trialkoxybenzhydrylamine resin), and Fmoc (9-fluorenylmethoxy carbonyl)-amino acid was used with HATU and HOAt or HBTU and HOBt with DIEA. The chain of the peptide was extended by a method of performing coupling.

At this time, synthesis from the C-terminus to the N-terminus, after coupling Fmoc-amino acid to the amino terminal, and then removing the Fmoc group with a 20% piperidine solution (Solid-Phase Synthesis: A Practical Guide (1 ed.). CRC Press. 848), washed several times with DMF (Dimethylforamide), and then subjected to coupling.

Finally, after coupling the Fmoc-amino acid, the Fmoc group was removed with a 20% piperidine solution, and an acyl group was coupled by using a fatty acid or carboxylic acid, N,N'-diisopropylcarbodiimide (DIC), and HOAt together. Then, it was washed several times with DMF and dried.

TFA (Trifuloroacetic acid)-H2O-Triisopropylsilane (95:2.5:2.5, vol./vol.) solution was added and reacted for 2 to 3 hours to remove the protecting group and to separate the peptide from the resin. The peptide was precipitated with diethylether.

The crude peptide obtained by the above method was confirmed for purity by using RP-HPLC (2996 Detector, 515 Pump, Waters) with a composition of acetonitrile containing 0.1% TFA and a gradient solvent of water, and the purity was 95%. The following peptides were separated and purified by Prep-LC to obtain a peptide of 95% or more. In order to confirm the exact component of this peptide, the molecular weight was confirmed by mass. The obtained two kinds of peptide substances are shown in Table 2 below.

division order Compound 313 C14-KKK-NH ₂ Compound 315 C16-KKK-NH ₂

<Example 2> Measurement of antibacterial activity

(1) Strain and bacterial culture

The experimental strains were S. aureus, S. epidermidis, E. coli, P. aeruginosa, a total of 4 species, and the 15 species of Example 1 and the 2 species of Comparative Example 1 were each cultured under the conditions shown in Table 3 below. .

Strain badge Medium concentration Incubation time Staphylococcus Aureus Trypton Soy Broth (TSB) 30g/L 24h Staphylococcus Epidermidis Nutrient Broth (NB) 8g/L 48h Escherichia Coli Trypton Soy Broth (TSB) 30g/L 24h Pseudomonas Aeruginosa NB+0.5% NaCl 8g/L + 1g NaCl 24h

(2) Antimicrobial activity (Minimun Inhibitory Concentration, MIC) measurement

The strain was cultured one day before the MIC test test, and the OD600 value was measured with a spectrophotometer to confirm CFU. Prepare synthetic samples in each well of a 96-well plate to a final concentration of 2, 4, 8, 16, 32, 64, 128, 256 ppm, and then add the bacteria shown in Table 2 to 5x10 ⁵ CFU/ml. After inoculation, the culture was stationary in an incubator.

The confirmed antimicrobial activity results were measured at 600 nm with an ELISA reader and converted into% based on the control, and the MIC values (unit ppm) of each strain are shown in Table 4 below.

division order Microbial growth minimum inhibitory concentration (MIC, ppm) S. Aures S. epidermis E. Coli P. aeruginosa MIP_1 C14-EK-NH ₂ >200 >200 >200 >200 MIP_2 C14-HK-NH ₂ 64 16 32 64 MIP_3 C14-KK-NH ₂ 4 2 32 32 MIP_4 C14-RK-NH ₂ 4 2 32 32 MIP_5 C14-KE-NH ₂ >200 >200 >200 >200 MIP_6 C14-KH-NH ₂ 8 4 32 128 MIP_7 C14-KR-NH ₂ 2 2 32 32 MIP_8 C14-KK-OMe 4 4 32 64 MIP_9 C14-KK-OH 64 32 128 128 MIP_10 C16-KK-NH ₂ 64 32 128 128 MIP_11 C2-KK-NH ₂ 8 16 128 128 MIP_12 C2-KR-NH ₂ 8 16 128 128 MIP_13 C10-KK-NH ₂ 16 4 64 128 MIP_14 C12-KK-NH ₂ 16 4 64 128 MIP_15 C14-KKK-OH 32 8 128 128 Compound 313 C14-KKK-NH ₂ 4 2 32 64 Compound 315 C16-KKK-NH ₂ 4 4 8 16

As shown in Table 4, it was confirmed that all of the compounds (MIP_1, MIP_5) to which glutamic acid (E) is bound have good antibacterial activity.

<Example 3> Measurement of antifungal activity (Minimun Bactericidal Concentration, MBC)

When some of the synthetic samples were dissolved in Yeast Peptone Dextrose and Potato Dextrose Broth medium, it was confirmed that they exhibit turbidity or color, and antibacterial inhibition experiments by absorbance such as MIC may have poor objectivity. Antifungal activity was confirmed by confirming the concentration that did not occur.

(1) Strain and bacterial culture

The experimental strains were C.albicans and A.niger, a total of 2 species, and the 15 species of Example 1 and the 2 species of Comparative Example 1 were cultured under the conditions shown in Table 5 below.

Strain badge Medium concentration Incubation time Candida Albicans Yeast Peptone Dextrose (YPD) 50g/L 24h Aspergillus Niger Potato Dextrose Broth (PDB) 24g/L 48h

(2) Measurement of antifungal activity

The strains specified in [Table 5] were cultured before the test and the OD600 value was measured with a spectrophotometer to confirm the number of bacteria. Synthetic samples were prepared in a 15 ml tube to obtain a final concentration of 50, 100, 200, 300, 400, and 500 ppm, respectively, and then the bacteria were inoculated into each tube so as to be ⁵ ×10 ⁵ CFU/ml, followed by stationary culture. After 24 hours incubation, the samples collected from each tube were spread on a solid medium plate, and the concentration (unit ppm) at which colonies did not occur after the culture were shown in Table 6 below.

division order Minimum sterilization concentration (MBC, ppm) C.albicans A.niger MIP_1 C14-EK-NH ₂ >500 >500 MIP_2 C14-HK-NH ₂ 300 0 MIP_3 C14-KK-NH ₂ 300 300 MIP_4 C14-RK-NH ₂ 300 300 MIP_5 C14-KE-NH ₂ >500 >500 MIP_6 C14-KH-NH ₂ 300 300 MIP_7 C14-KR-NH ₂ 300 300 MIP_8 C14-KK-OMe 300 300 MIP_9 C14-KK-OH 400 400 MIP_10 C16-KK-NH ₂ 300 300 MIP_11 C2-KK-NH ₂ 300 300 MIP_12 C2-KR-NH ₂ 400 400 MIP_13 C10-KK-NH ₂ 400 400 MIP_14 C12-KK-NH ₂ 400 400 MIP_15 C14-KKK-OH 300 300 Compound 313 C14-KKK-NH ₂ 300 300 Compound 315 C16-KKK-NH ₂ 300 300

As shown in Table 6, it was confirmed that among the synthesized compounds, all compounds except for the compounds to which glutamic acid (E) is bound (MIP_1, MIP_5) had excellent antifungal activity at 500 ppm or less.

Among them, the antifungal effects against A. niger and C. albicans at effective concentrations of MIP_2, MIP_3, MIP_4, MIP_6, and MIP_7 are shown in FIG. 1. As shown in Figure 1, it was possible to confirm the improved antifungal effect.

<Example 4> Skin patch test result of the active compound

In order to measure the skin stability tests of the active compounds MIP_2, MIP_3, MIP_4, MIP_6, and MIP_7 obtained in Example 1, a human patch test was performed to perform a primary irritation test on the human body. This test was conducted by a domestic clinical institution, and in 31 women in their 30s and 50s, the reaction was observed 30 minutes, 24 hours and 120 hours after applying 0.1% of the active compound to the skin. The degree of irritation was classified according to the evaluation criteria of the International Contact Dermatitis Research Group (ICDRG) [Table 7]. After calculating the mean score according to the formula for calculating the average skin reactivity, the presence or absence of irritation was determined according to the skin patch test result table [Table 8].

The skin patch test results of the active compound are shown in Table 9 below.

Sample name Number of responders Reactivity Average skin reactivity 30 min 24hr 120hr MIP_2 0 0.0 0.0 0.0 0.00 MIP_3 0 0.0 0.0 0.0 0.00 MIP_4 0 0.0 0.0 0.0 0.00 MIP_6 0 0.0 0.0 0.0 0.00 MIP_7 0 0.0 0.0 0.0 0.00

As shown in Table 9, no response was observed in all participants with a reactivity of 0.00, and according to the criteria, it was confirmed that there was no stimulation of the active compound.

According to the present invention, a peptide having a shorter amino acid number than a long sequence peptide (oligo peptide) with a large number of amino acids was synthesized and the antibacterial/antifungal activity thereof was confirmed.Since the possibility of inducing immunity is low, it is safe and cost-effective for commercial use. It can provide a composition suitable for the application of pharmaceuticals, quasi-drugs and cosmetics.

Claims (1)

C ₁₄ -histidine-lysine-NH ₂
C ₁₄ -lysine-lysine-NH ₂
C ₁₄ -Arginine-lysine-NH ₂
C ₂ -lysine-lysine-NH ₂
C ₁₀ -lysine-lysine-NH ₂ or
Contains a fatty acid dipeptide represented by C ₁₂ -lysine-lysine-NH ₂ as an active ingredient,
Antimicrobial composition comprising the active ingredient in 0.0001 to 15% by weight based on the total weight of the composition.

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