JPH08176190A - Peptide derivative and its use - Google Patents

Abstract

PURPOSE: To obtain a new peptide derivative having a specific amino acid sequence and excellent suppressing action to active oxygen, free radical and hyperoxidation of lipid, and antibacterial action to bacterium and fungus, etc., and useful for an antioxidant or an antibacterial agent to drug or food, etc. CONSTITUTION: This new peptide derivative is expressed by the formula: R1 -X (R1 is acetyl, acyl or polyethylene glycol; X is a peptide composed of D- or L-configurated amino acid and having an amino acid sequence expressed by formula I to formula III), the formula: X-R2 (R2 is an amino acid, acyl or polyethylene glycol) or the formula: R1 -X-R2 and having excellent suppressing action to active oxygen, free radical and hyperoxidation of lipid, and antibacterial action to microorganism such as bacterium and fungus and useful for an antioxidant or an antibacterial agent to drug or food, etc. The peptide derivative is obtained by adding an acyl group or polyethylene glycol to a peptide synthesized by a peptide synthesizer or hydrolyzing lactoferrins and separating with a liquid chromatography.

Description

Detailed Description of the Invention

[0001]

This invention relates to a peptide derivative,
It is related to its use. More specifically, the present invention relates to a safe peptide derivative that can be used in medicines, quasi drugs and other products.

[0002]

2. Description of the Related Art In recent years, the involvement of free radicals in the pathophysiology of various diseases has become clear. Human beings require oxygen to maintain their lives, and at that time, active oxygen and free radicals are constantly generated in the living body. The generated free radicals cause denaturation of membrane lipids and membrane proteins, damage the cell membrane, and cause denaturation of DNA and enzymes, causing various diseases. In particular, polyunsaturated fatty acids in cell membranes are easily attacked by free radicals, resulting in the production of harmful lipid peroxides and further enhancing cell damage (Modern Medicine, Vol. 25, No. 1).
63, 1993).

Diseases related to active oxygen include burns, inflammation such as arthritis, reperfusion disorder, side effects of anticancer agents, radiation disorders,
It is widely present in peptic ulcer, bacterial shock, cachexia, autoimmune disease and the like. It is considered that suppression of active oxygen, free radicals and lipid peroxides is effective for the prevention and treatment of these diseases (Nikkei Biotech, "Nikkei Bio Yearbook", page 350, Nikkei BP, 1994).
Year).

On the other hand, although many antibacterial agents have been developed and used in the past, infectious diseases never disappear, and the causes and symptoms thereof are becoming more complicated. In particular, the increase in drug resistance on the infecting bacterium side and the emergence of a compromised host on the host side still make it difficult to solve the problem. For this reason, new preventive methods and cures for infectious diseases are desired.

Lactoferrin is one of them. Lactoferrin is an iron-binding protein contained in tears, saliva, exocrine fluid such as milk, and neutrophil granules, and is known to have various physiological activities such as antibacterial activity, antioxidant activity, and immunomodulatory activity. (Archives of Diseases in
Childhood [Archives of Disease in Childhoo
d], 67, 657, 1992). It is also known that lactoferrin degradation products, lactoferrin-derived peptides, and peptides having homology with these have antibacterial and antioxidant effects.
For example, an antibacterial agent containing a lactoferrin degradation product as an active ingredient (JP-A-5-320068) and an antibacterial peptide composed of at least 20 amino acid residues (JP-A-5-9).
2994), an antibacterial peptide consisting of 11 amino acid residues (JP-A-5-78392), an antibacterial peptide consisting of 6 amino acid residues (JP-A-5-148).
297), an antibacterial peptide consisting of 5 amino acid residues (JP-A-5-148296), an antibacterial peptide consisting of 3 to 6 amino acid residues (JP-A-5-14).
No. 8295), a degradation product of lactoferrins, a peptide isolated from degradation products of lactoferrins, and an antioxidant comprising a synthetic peptide having the same amino acid sequence as the peptide isolated from degradation products of lactoferrins as an active ingredient. Agents (JP-A-6-199687).

[0006] Peptide derivatives have been studied for many purposes by taking advantage of the property of simple peptides that they do not have. By derivatizing a peptide, it has been attempted to increase activity, impart resistance to protease, and increase durability in the body.
For example, a metal-peptide complex having antioxidant and anti-inflammatory activity (Japanese Patent Publication No. H05-503939), a tripeptide arginal derivative that suppresses ischemic injury and reperfusion injury (Japanese Patent Laid-Open No. 6-199682). , N-terminal substitution of a quaternary ammonium-derived peptide used as a skin cosmetic (JP-A-4-82822), an amphipathic ion channel-forming peptide having biological activity against target cells, viruses and virus-infected cells Peptides (WO93 / 24138), biologically active amphipathic ion channel-forming peptides having C-terminal substitution peptides (WO92 / 22317), and the like are known.

[0007]

However, regarding the lactoferrin-derived peptide, the antioxidant property of the derivative,
Physiological activity such as antibacterial property has not been known at all, and therefore, neither antioxidant nor antibacterial agent containing these as active ingredients existed. The present invention has been made in view of the above conventional techniques, and provides a safe antioxidant and antibacterial agent containing a lactoferrin-derived peptide derivative as an active ingredient, which is completely different from conventional antioxidants and antibacterial agents. The purpose is to do.

[0008]

In order to solve the above problems, the present invention provides a derivative of a lactoferrin-derived peptide represented by a specific chemical formula, or an antioxidant containing the same as an active ingredient, and an antibacterial agent. provide. Hereinafter, the present invention will be described in detail.

The peptide derivative used in the present invention is synthesized by a known method (for example, a method using an automatic peptide synthesizer) to obtain a target peptide, a peptide synthesized by a peptide synthesizer, and an organic synthetic technique. By adding an acyl group, polyethylene glycol or the like, or by hydrolyzing lactoferrins with an acid or an enzyme, and separating a fraction containing a peptide having a certain amino acid sequence from the mixture of the decomposed products by a separation means such as liquid chromatography. After it is obtained, it can be prepared by a method of adding an acyl group, polyethylene glycol or the like using an organic synthesis technique.

The method for producing a peptide having an acetyl group at the amino terminal and an amide at the carboxyl terminal as an example of the peptide derivative of the present invention is as follows. Peptide synthesizer (Pharmacia LKB Biotechnology Co., LKB Biolynx 4170) is used and a solid phase according to Shepard et al. [Journal of Chemical Society Perkin I, 538, 1981]. It is synthesized as follows based on the peptide synthesis method.

Solid phase resin for C-terminal amide peptide synthesis (Calbiochem-Novabiochem. NovaSyn KR 0.1meq)
Is used to extend the peptide chain by repeating the deprotection group reaction and the condensation reaction according to the above-mentioned peptide synthesizer synthesis program. That is, with 20% piperidine / dimethylformamide (hereinafter referred to as DMF), 9-fluorenylmethoxycarbonyl (hereinafter referred to as an amino-protecting group)
Fmoc) group is cleaved off, washed with DMF, and then Fm
The procedure of reacting oc-amino acid active ester / N-hydroxybenzotriazole (hereinafter referred to as HOBt) and washing with DMF is repeated.

After the condensation reaction, if necessary, a Kaiser test is performed to confirm that the coupling is complete, and the process proceeds to the next step. The amino acid used is Fmoc-Arg (Mt
r) -Opfp, Fmoc-Lys (Boc) -Opfp, Fmoc-Trp (Boc) -Opfp, F
A 0.5 mmol cartridge of moc-Gln-Opfp and Fmoc-Met-Opfp (both Calbiochem-Novabiochem) is used. After completion of all peptide chain extension reactions, 20
The Fmoc group is cleaved with% piperidine / DMF, washed with DMF, and acetylated with 10% acetic anhydride / DMF. After confirming that the acetylation was completed by the Kaiser test, the resin was then treated with DMF, tert-pentyl alcohol, acetic acid and tert.
-Wash well with pentyl alcohol, DMF and diethyl ether in that order and dry under vacuum.

After stirring ethanedithiol and thioanisole to the above-mentioned protected peptide resin at room temperature under an argon stream, the mixture is further stirred under ice cooling. Trifluoroacetic acid is added to this and stirred, and trimethylsilyl bromide is added and stirred. The resin is filtered off with a glass filter, and the filtrate is quickly concentrated under reduced pressure. Precooled diethyl ether is added to the residue to make the peptide a white powder. Transfer to a centrifuge tube, centrifuge, discard the supernatant, add cold diethyl ether newly, stir well, and centrifuge again. This operation is repeated 4 times. The peptide precipitate is vacuum dried, dissolved in water and freeze-dried to obtain a crude peptide.

The above crude peptide is dissolved in water, centrifuged and the supernatant is filtered with a 0.45 mm filter. This solution is subjected to high performance liquid chromatography (HPLC) to purify the peptide. HPLC uses a Gulliver PU-986 high pressure gradient system (Nippon Bunko), and the column is a reverse phase system.
Lichrospher 100RP-18 (e) 250x10mm (Merck) is used. The eluent is 0.1% TFA / water as the solution A and 80% acetonitrile / A solution as the solution B, which is eluted with a linear concentration gradient from solution A to solution B. The peak of the chromatogram is almost single, and the corresponding fractions are collected. Repeat collection several times,
These are lyophilized to obtain a purified peptide.

This is an analytical column (Lichrospher 100RP-1
8 (e) 250x4.6mm. HPLC analysis is performed using Merck) to further confirm that the purified product is single. Also,
To confirm the structure of the purified peptide, conventional amino acid analysis, amino acid sequence analysis, elemental analysis, mass spectrometry, etc. are carried out to confirm that the desired peptide has been obtained. An example of a method for adding polyethylene glycol, an acyl group or the like to a peptide obtained by hydrolysis of lactoferrin is as follows.

CH ₃ (OCH ₂ CH ₂ ) n-OH [manufactured by Polyscience Corporation. Acetic anhydride is allowed to act on monomethoxy polyethylene glycol (hereinafter referred to as mPEG) to obtain mPEG-COOH. N-hydroxysuccinimide and dicyclohexylcarbodiimide are allowed to act on this to synthesize mPEG N-hydroxysuccinimide ester. The peptide fragment derived from lactoferrin is reacted with mPEG succinimide ester to obtain a polyethylene glycol modified product. A fatty acid succinimide ester is obtained from CH ₃ (CH ₃ ) n-COOH by the same method as above. This is allowed to act on a peptide fragment derived from lactoferrin to obtain an acyl derivative. Here, the molecular weights of polyethylene glycol and the acyl group are not particularly limited.

The peptide derivative of the present invention is not only used as a drug, but is also incorporated into the human or animal body such as foods, feeds and cosmetics, or is applied to the body surface,
In general, it can be used by blending it with any product in which active oxygen, free radicals, suppression of lipid peroxidation, suppression of microorganisms such as bacteria and fungi, and sterilization are desired. Raw material can be processed. That is, the peptide derivative of the present invention can be directly administered to humans or animals,
Or pharmaceuticals (eg, preventive agents for various diseases, therapeutic agents for various diseases, antibacterial agents, etc.), foods (eg, formula powder, enteral nutritional supplements, chewing gum, etc.), quasi-drugs (eg, mouthwashes, health drinks etc.) ), Various cosmetics (for example, cream, milky lotion, sunscreen cosmetics, skin aging preventive cosmetics, etc.), raw materials thereof, and other generally active oxygen, free radicals, suppression of lipid peroxidation, bacteria, fungi It may be added, blended, sprayed, attached, coated, impregnated, etc. to all articles for which microbial control such as sterilization or sterilization is desired. In addition, generally, active oxygen / free radicals, suppression of lipid peroxidation, bacteria, fungi. It can be used for the treatment of any article for which the suppression of microorganisms and sterilization are desired. It is also possible to use together with known antioxidants and antibacterial agents.

The present invention will be described in more detail with reference to test examples. Test Example 1 This test was carried out in order to investigate the antioxidant activity of various compounds. 1) Preparation of samples Eight types of samples shown below were prepared. Sample 1: Peptide described in SEQ ID NO: 4 prepared from the enzymatic decomposition of bovine lactoferrin by the method described in Reference Example Sample 2: Amino terminal was acetylated and carboxyl terminal was amidated, and the following prepared by the method described in Example 1 Chemical formula 1 peptide derivative

[0019]

Embedded image

Sample 3: A peptide derivative of the following Chemical formula 2 prepared by the method described in Example 2 in which an acyl group having 18 carbon atoms is bound to the amino terminal and the carboxyl terminal is amidated

[0021]

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Sample 4: A peptide derivative of the following chemical formula 3 prepared by the method described in Example 3 in which an acyl group having 16 carbon atoms is bound to the amino terminus and the carboxyl terminus is amidated

[0023]

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Sample 5: A peptide derivative of the following chemical formula 4 prepared by the method described in Example 4 in which an acyl group having a carbon number of 14 is bound to the amino terminal and the carboxyl terminal is amidated.

[0025]

[Chemical 4]

Sample 6: The following peptide derivative of Formula 5 prepared by the method described in Example 5 in which an acyl group having 10 carbon atoms is bound to the amino terminus and the carboxyl terminus is amidated.

[0027]

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Sample 7: An acyl group having a carbon number of 6 was bound to the amino terminus and the carboxyl terminus was amidated to give Example 6.
The peptide derivative of the following chemical formula 6 prepared by the method described

[0029]

[Chemical 6]

Sample 8: A peptide derivative of the following chemical formula 7 prepared by the method described in Example 7 in which the amino terminus was acetylated and polyethylene glycol (hereinafter referred to as PEG) was bound to the carboxyl terminus.

[0031]

[Chemical 7]

2) Test method Gutteridge's method [Analytical Biochemistry, Vol. 82, No. 76]
[Page, 1977] with some modifications as described below to test the antioxidant effect of each sample. In an eggplant-shaped flask, 10 mM HEPES buffer (pH 7.4) 2
0 ml, egg yolk phospholipid (manufactured by Nacalai Tesque) 100 m
g, and glass beads were added and replaced with nitrogen.
Stir for 5 minutes by vortexing at ° C. After holding at 4 ° C for 1 hour, the supernatant was used as a liposome solution.

0.5 ml of the liposome solution was dispensed into a 10 ml plastic tube, and 100 μl of a 7-fold concentrated sample solution was added to 112 μM FeNH ₄ (SO ₄ ) ₂ ·.
12H ₂ O (manufactured by Nacalai Tesque, Inc.) 50 μl, 532
50 μl of μM ascorbic acid (Nakarai Tesque)
And stir for 5 seconds. Cap the tube and incubate at 37 ° C. for 1 hour. 2.9M H
1% sodium arsenite dissolved in Cl (manufactured by Wako Pure Chemical Industries)
3 ml is added, mixed and centrifuged at 4000 rpm for 15 minutes. Take 3 ml of the clean solution and add 0.05M N
1 ml of 1% thiobarbituric acid (manufactured by Nacalai Tesque, Inc.) dissolved in aOH was added and heated in boiling water for 15 minutes. After cooling, it is filtered with a 0.45 μM filter (manufactured by Advantech). The absorbance of the filtrate was measured at 532 nm to determine the degree of peroxidation of liposome phospholipids. A control solution was prepared by adding 100 μl of distilled water instead of the sample solution.

The oxidation inhibition rate (%) of the sample was calculated by the following formula. Oxidation inhibition rate = [(absorbance of control−absorbance of sample) / absorbance of control] × 100 3) Test results The results of this test are shown in Table 1. As is clear from Table 1, in the concentration of 100 μg / ml, Samples 2 to 8 showed stronger oxidation inhibiting effect than Sample 1. In addition, Sample 3 to Sample 7 are 10 μg / ml, Sample 3
Therefore, Sample 6 exhibited an oxidation inhibitory action even at a low concentration of 1 μg / ml. In addition, although the kind of peptide derivative was changed and the same test was performed, the substantially same result was obtained.

[0035]

[Table 1]

Test Example 2 This test was conducted to examine the antibacterial action of various compounds. 1) Preparation of samples Eight types of samples shown below were prepared. Sample 1: The same as Sample 1 of Test Example 1 Sample 2: The following peptide derivative of Chemical formula 8 prepared by the method described in Example 8 in which the carboxyl end was amidated

[0037]

Embedded image

Sample 3: A peptide derivative of the following chemical formula 9 prepared by the method described in Example 9 in which an acetyl group is bonded to the amino terminus, the carboxyl terminus is amidated, and all amino acids are in the D-form.

[0039]

[Chemical 9]

Sample 4: Same as Sample 3 of Test Example 1 Sample 5: Same as Sample 4 of Test Example 1 Sample 6: Same as Sample 5 of Test Example 1 Sample 7: Sample of Test Example 1 Same as Sample 6 Sample 8: Same as Sample 7 of Test Example 1 2) Test method The above sample was added to sterilized 1% Bactopeptone (manufactured by Difco) at a final concentration of 200, 100, 50, 25, 12 ,
6 and 3 μg / ml, Escheric in log phase
Add hia coli O111 or Escherichia coli IID-861 at a rate of 10 ⁶ cells / ml in the medium, and add 17 at 37 ° C.
Cultured for hours. The minimum concentration at which no bacterial growth was visually observed was defined as the minimum inhibitory concentration (hereinafter referred to as MIC) of the sample. 3) Test results The results of this test are shown in Table 2. As is clear from Table 2, are Sample 2 to Sample 8 equivalent to Sample 1?
Alternatively, it showed a MIC of ½ or less, and a strong antibacterial effect was recognized. In addition, although the kind of peptide derivative was changed and the same test was performed, the substantially same result was obtained.

[0041]

[Table 2]

Test Example 3 This test was conducted to examine the resistance of various compounds to proteases. 1) Preparation of samples Three kinds of samples shown below were prepared. Sample 1: Same as Sample 1 of Test Example 1 Sample 2: Carboxyl terminal was amidated, amino acid at the second residue from the amino terminal was D-form, and other amino acids were all L-form. The peptide derivative of the following chemical formula 10 prepared by the method of 10

[0043]

[Chemical 10]

Sample 3: Same as Sample 3 of Test Example 2) Test method Treatment of peptide derivative with trypsin 100 mM Tris-HCl buffer (pH 8.
In 1), the sample and trypsin (type IX, manufactured by Sigma) were added to final concentrations of 900 and 50 μg / ml, respectively.
And incubate at 37 ° C for 1 hour.
Trypsin was inactivated by heat treatment at 0 ° C. for 10 minutes. Induction of Trichophyton Protease Produced Trichophyton mentagrophytes TIMM-1189 [2%
Keratin powder, 1.2% yeast carbon base (manufactured by Difco), 0.005% inositol, 0.0
It consists of 01% thiamine and 0.001% pyridoxine], and was shake-cultured at 27 ° C. for 10 days, and it was confirmed that protease was induced using bovine albumin as a substrate. It was a liquid. Treatment of Peptide Derivative with Ringworm-Producing Protease Solution A sample was added to the ringworm-producing Protease solution diluted to 1/2 at a final concentration of 500 μg / ml, incubated at 37 ° C. for 1 hour, and at 80 ° C. for 10 minutes. Heat treated,
The Trichophyton produced protease was inactivated. Determination of antibacterial activity of protease-treated peptide derivatives Escherichia of protease-treated peptide derivatives
Antibacterial activity against coli O111 was measured by the same method as in Test Example 2. 3) Test results The results of this test are shown in Table 3. As is clear from Table 3, Sample 1 was completely inactivated by trypsin and Trichophyton produced by Trichophyton, whereas Sample 2 had some residual activity, and Sample 3 was completely retained even after protease treatment. . In addition, although the kind of peptide derivative was changed and the same test was performed, the substantially same result was obtained.

[0045]

[Table 3]

Test Example 4 This test was conducted to examine the acute toxicity of various peptide derivatives. 1) Animals used Six-week-old CD (SD) strain rats (purchased from Japan SLC) were used, and males and females were randomly divided into 4 groups (5 animals per group) and used. 2) Test method A peptide derivative prepared by repeating the same method as in Example 1 at a rate of 1000 and 2000 mg per kg body weight was dissolved in water for injection (Otsuka Pharmaceutical Co., Ltd.) to weigh 100 g.
A single oral gavage was performed using a needle with a metal ball at a rate of 4 ml per time, and acute toxicity was tested. 3) Test results This peptide derivative was treated with 1000 mg / kg body weight and 2
No deaths were observed in the group administered at a rate of 000 mg / kg body weight. Therefore, LD5 of this peptide derivative
0 was 2000 mg / kg body weight or more, and the toxicity was found to be extremely low. Similar tests were conducted for other peptide derivatives, but almost the same results were obtained. Reference Example 50 mg of commercially available beef lactoferrin (manufactured by Sigma) is dissolved in 0.9 ml of purified water, and the pH is adjusted to 2.5 with 0.1N hydrochloric acid.
Adjusted to, and later commercially available pig pepsin (manufactured by Sigma) 1 mg
Was added and hydrolyzed at 37 ° C. for 6 hours. Then 0.1
Adjust the pH to 7.0 with N sodium hydroxide and
Heat for 10 minutes to inactivate the enzyme, cool to room temperature, and
After centrifugation at 5,000 rpm for 30 minutes, a clear supernatant was obtained. 100 μl of this supernatant was added to TSK gel ODS-120.
High performance liquid chromatography using T (manufactured by Tosoh Corporation)
And elute with 20% acetonitrile containing 0.05% TFA (trifluoroacetic acid) for 10 minutes after injection of the sample at a flow rate of 0.8 ml / min, and then for 30 minutes with 0.05% TFA.
Was eluted with a gradient of 20-60% acetonitrile containing 20 to 60%, and the fractions eluted during 24 to 25 minutes were collected and dried under vacuum. This dried product was dissolved in purified water at a concentration of 2% (W / V), and again subjected to high performance liquid chromatography using TSK gel ODS-120T (manufactured by Tosoh Corporation),
0.05% TF for 10 minutes after sample injection at a flow rate of 8 ml / min
Fractions eluting with 24% acetonitrile containing A, then 30 minutes with a gradient of 24-32% acetonitrile containing 0.05% TFA, and eluting between 33.5 and 35.5 minutes were collected. . The above operation was repeated 25 times, and vacuum drying was performed to obtain about 1.5 mg of the peptide.

The above-mentioned lactoferrin-derived peptide was
After hydrolysis with hydrochloric acid, the amino acid composition was analyzed by an ordinary method using an amino acid analyzer. The same sample was used 2 using a gas phase sequencer (manufactured by Applied Biosystems).
The Edman degradation was performed 5 times and the sequence of 25 amino acid residues was determined. Also, a disulfide bond analysis method using DTNB (5,5-dithio-bis (2-nitrobenzoic acid)) [Analytical Biochemistry, Vol. 67, No. 493]
Page, 1975] confirmed the presence of disulfide bonds.

As a result, this peptide consisted of 25 amino acid residues, the 3rd and 20th cysteine residues were disulfide-bonded, and the 3rd cysteine residue was replaced by N
-It was confirmed that 2 amino acid residues at the terminal side and 5 amino acids at the C-terminal side from the 20th cysteine residue have the amino acid sequence described in SEQ ID NO: 4 respectively. It was

Next, the present invention will be described in more detail and concretely by showing examples, but the present invention is not limited to the following examples.

[0050]

【Example】

Example 1 Using a peptide synthesizer (Pharmacia LKB Biotechnology, LKB Biolynx 4170), a method such as Shepard's method [Journal of Chemical Society Perki I (Journal of Chemical Society Perki
n I), p. 538, 1981], the peptide derivative was synthesized as follows.

Solid-phase resin for C-terminal amide peptide synthesis (Calbiochem-Novabiochem. NovaSyn KR 0.1meq)
Using 1 g, the deprotecting group reaction and the condensation reaction were repeated according to the synthesis program of the above peptide synthesizer to extend the peptide chain. That is, the Fmoc group, which is an amino protecting group, is cleaved off with 20% DMF, washed with DMF, and then 0.5 mmol each of Fmoc-amino acid active ester / N-hydroxybenzotriazole (hereinafter referred to as HOBt) is reacted, The operation of washing with DMF was repeated. After the condensation reaction, a Kaiser test was performed as necessary to confirm that the coupling was complete, and the process proceeded to the next step. The amino acids used are Fmoc-Lys (Boc) -Opfp and Fmoc-Trp (Boc) -Op.
A cartridge of fp, Fmoc-Gln-Opfp and Fmoc-Met-Opfp (all manufactured by Calbiochem-Novabiochem) was used at a concentration of 0.5 mmol. After completion of the peptide chain extension reaction, cleave the Fmoc group with 20% piperidine / DMF, and add DM
After washing with F, acetylation was performed with 10% acetic anhydride / DMF. After confirming that the acetylation was completed by the Kaiser test, the resin was treated with DMF, tert-pentyl alcohol,
It was washed well with acetic acid, tert-pentyl alcohol, DMF, and diethyl ether in that order, and dried under vacuum.

To 571 mg of the above protected peptide resin, 1.0 ml of ethanedithiol, 200 μl of m-cresol and 2.4 ml of thioanisole were stirred at room temperature for 15 minutes under an argon stream, and then for 10 minutes under ice cooling. To this, 15 ml of trifluoroacetic acid was added and stirred for 10 minutes, 2.6 ml of trimethylsilyl bromide was added and stirred for 50 minutes. The resin was filtered off with a glass filter, and the filtrate was quickly concentrated under reduced pressure. Precooled diethyl ether was added to the residue to make the peptide a white powder. The procedure was repeated 4 times by transferring to a centrifuge tube, centrifuging (2500 rpm, 5 minutes), discarding the supernatant, newly adding cold diethyl ether, stirring well, and centrifuging again. The peptide precipitate was vacuum dried, dissolved in water and lyophilized to obtain crude peptide 62.
5 mg was obtained.

The above crude peptide was dissolved in water and centrifuged (15000 rpm, 5 minutes) to obtain 0.45 of the supernatant.
After filtering with a mm filter, this solution was subjected to high performance liquid chromatography (HPLC) to purify the peptide.
For the HPLC, a Gulliver PU-986 high pressure gradient system (manufactured by JASCO Corporation) was used, and a commercially available reversed-phase column (manufactured by Merck Ltd., Lichrospher 100RP-18 (e) 250x10 mm) was used as the column. The eluents were 0.1% TFA / water as solution A and 80% acetonitrile / A solution as solution B, which was eluted with a linear concentration gradient from solution A to solution B. The chromatogram was recognized as a single peak, and the corresponding fractions were collected. This preparative operation was repeated several times and freeze-dried to obtain 28.5 mg of purified peptide.

An analytical column (manufactured by Merck. Lichro
HPLC analysis with spher100RP-18 (e) 250x4.6 mm) further confirmed that the purified product was single. Also,
To confirm the structure of the purified peptide, conventional amino acid analysis, amino acid sequence analysis, elemental analysis, mass spectrometry and the like were performed, and it was confirmed that the desired peptide derivative shown in Chemical formula 11 below was obtained.

[0055]

[Chemical 11]

Example 2 The same operation as in Example 1 was carried out, but a peptide derivative was synthesized as follows by a method using succinimide ester as the acyl group-introducing method. Stearic acid [(CH ₃ (CH ₂ ) ₁₆ COOH] 2.8 g was dissolved in dichloromethane, and N-hydroxysuccinimide 1.2 was added thereto.
g of DMF solution and dicyclohexylcarbodiimide 2.3
A dichloromethane solution of g was added and the mixture was stirred at room temperature for 5 hours.
The progress of the reaction was monitored by thin layer chromatography, and it was confirmed that the reaction was completed. By-product dicyclohexylurea was filtered off, concentrated under reduced pressure, diethyl ether was added to the residue, and the mixture was transferred to a centrifuge tube and centrifuged (2500 rpm).
5 minutes). The supernatant was discarded, diethyl ether was newly added, the mixture was stirred well, and the operation of centrifugation was repeated twice. It was filtered with a glass funnel and vacuum dried to obtain 2.0 g of succinimide stearate. A DMF solution of 382 mg of stearic acid succinimide ester was added to a protected peptide resin (N-terminal amino group-free, 0.1 mmol scale) synthesized by a peptide synthesizer and reacted at room temperature for 5 hours. It was confirmed by Kaiser test that it was completely acylated. The yield of acylated protected peptide resin is 1.
It was 13 g. The yield of the crude peptide after deprotection and deresination was 181 mg, and about 98 mg of the purified peptide derivative shown in Chemical formula 12 below was obtained.

[0057]

[Chemical 12]

Example 3 The same operation as in Example 1 was carried out, but the method for introducing an acyl group was as follows using a condensing agent. Palmitic acid (CH ₃ (CH ₂ ) ₁₄ COOH) 154 mg, HBTU [2- (1H-benzotr
iazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorop
Hosphate] 228 mg, HOBt 92 mg are dissolved in DMF, and DIEA is added.
(Diisopropylethylamine) 105 μl was added to the mixed solution to the protected peptide resin (N-terminal amino group-free, 60 μmol scale) synthesized by peptide synthesizer, stirred for 1 hour at room temperature, and then completely acylated by Kaiser test. It was confirmed. The yield of the acylated protected peptide resin was 476 mg and the yield of the crude peptide after deprotection and deresination was 103 mg, and about 46 mg of the purified peptide derivative shown in Chemical formula 13 below was obtained.

[0059]

[Chemical 13]

Example 4 The same operation as in Example 3 was performed. The yield of the acylated protected peptide resin was 444 mg, and the yield of the crude peptide after deprotection and deresination was 92 mg, and about 47 mg of the purified peptide derivative shown in Chemical formula 14 below was obtained.

[0061]

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Example 5 The same operation as in Example 3 was performed. The yield of the acylated protected peptide resin was 493 mg and the yield of the crude peptide after deprotection and deresination was 101 mg, and about 53 mg of the purified peptide derivative shown in Chemical formula 15 below was obtained.

[0063]

[Chemical 15]

Example 6 The same operation as in Example 3 was performed. The yield of the acylated protected peptide resin was 493 mg and the yield of the crude peptide after deprotection and deresination was 105 mg, and about 57 mg of the purified peptide derivative shown in the following Chemical formula 16 was obtained.

[0065]

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Example 7 The same operation as in Example 1 was performed. However, the solid phase resin
TantaGel-NH ₂ [manufactured by Shimadzu Corporation, polyethylene glycol (hereinafter referred to as PEG) -spacer partial molecular weight: 3000) was used. Yield of protected peptide resin is 4
84 mg, the yield of the crude peptide after deprotection and deresination was 311 mg, and about 132 mg of the purified peptide derivative shown in Chemical formula 17 below was obtained.

[0067]

[Chemical 17]

Example 8 The same operation as in Example 1 was performed. The yield of the protected peptide resin was 562 mg, the yield of the crude peptide after deprotection and deresination was 62 mg, and about 29 mg of the purified peptide derivative shown in Chemical formula 18 below was obtained.

[0069]

Embedded image

Example 9 The same operation as in Example 1 was performed. However, for the peptide bond formation reaction, a method using an HBTU / HOBt condensing agent was adopted. Therefore, the amino acid introduction of the peptide synthesizer was performed manually. The amino acid is Fmoc-D-Arg (Mtr)-
OH, Fmoc-D-Lys (Boc) -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-D
-Gln-OH and Fmoc-D-Met-OH (both manufactured by Watanabe Chemical Co., Ltd.) were used. Yield of protected peptide resin is 502 mg, yield of crude peptide after deprotection and deresination is 57 mg.
And the purified peptide derivative of about 3
2 mg was obtained.

[0071]

[Chemical 19]

Example 10 The same operation as in Example 1 was performed. However, 2 from the N end
The amino acid Arg at the th position was introduced in the same manner as in Example 9 using a HBTU / HOBt condensing agent. Amino acids are Fmoc-Arg (Mtr) -Opfp, Fmoc-Trp (Boc) -Opfp, Fmoc-G
ln-Opfp (all manufactured by Calbiochem-Novobiochem) and Fmoc-D-Arg (Mt as the second amino acid from the N-terminal.
r) -OH (manufactured by Watanabe Chemical Industry Co., Ltd.) was used. The yield of the protected peptide resin was 188 mg, the yield of the crude peptide after deprotection and deresination was 14 mg, and about 8 mg of the purified peptide derivative shown in Chemical formula 20 below was obtained.

[0073]

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Example 11 A tablet having the following composition was produced by a conventional method. Lactose 79.45 (%) Peptide derivative of Example 2 0.55 Magnesium stearate 20.00 Example 12 An injection having the following composition was produced by a conventional method.

Peptide derivative of Example 9 1.20 (%) Surfactant 8.00 Physiological saline solution 90.80 Example 13 Chewing gum having the following composition was produced by a conventional method.

Gum base 25.00 (%) Calcium carbonate 2.00 Fragrance 1.00 Peptide derivative of Example 1 0.10 Sorbitol powder 71.90 Example 14 A hand lotion having the following composition was prepared by a conventional method. Manufactured.

Carbowax 1500 9.00 (%) Alcohol 4.00 Propylene glycol 47.00 Fragrance 0.50 Peptide derivative of Example 7 0.40 Distilled water 39.10.

[0078]

As described in detail above, according to the present invention, an antioxidant having an excellent inhibitory effect on active oxygen, free radicals, and peroxidation of lipids, and an excellent antibacterial activity against microorganisms such as bacteria and fungi. An antibacterial agent having an action is provided. These antioxidants and antibacterial agents are extremely safe even when used in medicines and foods.

[0079]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 6 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 2 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 3 Sequence length: 9 Sequence type: Amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 4 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Phe Lys Cys Arg Arg Trp Gln Trp Arg Met Lys Lys Leu Gly Ala Pro 1 5 10 15 Ser Ile Thr Cys Val Arg Arg Ala Phe 20 25

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location A61K 38/00 ADZ AED C07K 14/79 C09K 15/22 (72) Inventor Mitsunori Takase East Zama City, Kanagawa Prefecture 5-1-83 Morinaga Milk Industry Co., Ltd. Nutrition Research Institute (72) Inventor Wayne Bellamy Higashihara 5-183 Wayne Bellamy Morinaga Milk Industry Co., Ltd. Nutrition Science Research Institute (72) Inventor Koichi Kashimoto Zama Kanagawa Prefecture 5-1-83 Ichihitohara Morinaga Milk Industry Co., Ltd., Institute for Biological Sciences (72) Inventor Hiroyuki Wakabayashi Zama City, Kanagawa Prefecture 5-1-883 Morinaga Milk Industry Co., Ltd., Institute for Nutrition Science (72) Inventor Hiroshi Matsumoto Zama, Kanagawa Prefecture 5-1-83 Higashihara, Morinaga Milk Industry Co., Ltd., Institute for Biological Sciences (72) Inventor Hirohiko Nakamura Zama City, Kanagawa Prefecture 5-1-83 Higashihara Morinaga Milk Industry Co., Ltd.

Claims (5)

[Claims] 1. The following general formula a), b) or c) a) R ₁ -X b) X-R ₂ c) R ₁ -X-R ₂ (wherein R ₁ is an acetyl group or an acyl group). Group or polyethylene glycol, X is a peptide having the amino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 3 consisting of D or L amino acids, and R ₂ is an amino group, an acyl group or Peptide derivative represented by polyethylene glycol). 2. The following general formula a), b) or c) a) R ₁ -X b) X-R ₂ c) R ₁ -X-R ₂ (wherein R ₁ is an acetyl group or an acyl group). Group or polyethylene glycol, X is
A peptide derivative having the amino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 3 consisting of D or L amino acids, wherein R ₂ represents an amino group, an acyl group or polyethylene glycol) An antioxidant containing as an active ingredient. 3. The peptide derivative is at least 1 μg /
The antioxidant according to claim 2, which is contained in an amount of ml. 4. The following general formula a), b) or c) a) R ₁ -X b) X-R ₂ c) R ₁ -X-R ₂ (wherein R ₁ is an acetyl group or an acyl group). Group or polyethylene glycol, X is
A peptide having the amino acid sequence of any of SEQ ID NO: 1 to SEQ ID NO: 3 consisting of D- or L-amino acids, wherein R ₂ represents any of an amino group, an acyl group or polyethylene glycol) An antibacterial agent containing a peptide derivative as an active ingredient. 5. The peptide derivative is at least 1 μg /
The antibacterial agent according to claim 4, which is contained in an amount of ml.