JP2641759B2 - New peptides and antibacterial agents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a horseshoe crab (Tachypleusgiga) produced in Malaysia.
The present invention relates to a novel peptide having an antibacterial activity and a salt thereof extracted and isolated from s), and an antibacterial agent containing these.

[Conventional technology]

In recent years, peptides have been attracting attention as useful physiologically active substances. With the development of protein engineering, research and development of not only conventional natural peptides but also synthetic peptides has been actively promoted, and the development of useful peptides has Has achieved considerable achievements in the center.

The development of peptides having an antibacterial action has also been carried out quite aggressively in the past, and a number of reports have been made mainly on relatively low molecular weight oligopeptides. For example, phosphonotripeptides having antibacterial activity against Gram-positive bacteria and Gram-negative bacteria (Japanese Patent Laid-Open No. 57-10 / 1982).
No. 6689), a phosphonodipeptide derivative (JP-A-58-1359)
No. 4), cyclic peptide derivatives (JP-A-58-213744),
Novel peptide comprising 10 amino acid sequences useful as an antibacterial agent and an antiviral agent (JP-A-59-51247), a polypeptide useful as an antibacterial agent against yeasts (JP-A-60-1987)
-130599), glycopeptide derivatives useful as antibacterial agents against gram-positive bacteria (JP-A-60-172998), and novel glycopeptide derivatives (JP-A-61-251699, JP-A-63-44).
No. 598), oligopeptides useful against Gram-positive bacteria (JP-A-62-22798), and peptide antibiotics (JP-A-6-22798).
No. 2-51697, JP-A-63-17897) and many others.

[Problems to be solved by the invention]

However, some of the conventional technologies have not always been put to practical use and have not been out of the scope of basic research, and it is understood that the development of peptide-based antibacterial agents is still under development. In any case, developing new peptides having excellent antibacterial activity and being practically usable as antibacterial agents seems to be extremely important in establishing new application fields of peptides.

[Means for solving the problem]

From such a viewpoint, the present inventors have conducted various searches for useful novel peptides and found that peptides extracted and isolated from blood cells of horseshoe crab (Tachypleus gigas) produced in Malaysia have strong antibacterial activity. To complete the present invention.

That is, the present invention is recognized as including the technical items described in the following (1) and (2).

1) The following structural formula (I) (Wherein and are directly bonded to each other. Arg-NH ₂ indicates that the carboxyl group of arginine is an amide.) And a salt thereof.

2) The following structural formula (I) (Wherein, and are directly bonded to each other. Arg-NH ₂ indicates that the carboxyl group of arginine is an amide.) And a salt thereof as an active ingredient Antibacterial agent.

Thus, an object of the present invention is to provide a novel peptide having a strong antibacterial action, and a salt thereof, and an antibacterial agent containing them as an active ingredient.

In this description, this novel peptide is referred to as GigasinII. In this specification, the amino acids are defined as IUPACIUB commi-ssion on Biolo
Abbreviations based on gical Nomenclature may be indicated, and examples are as follows.

Arg: arginine, Trp: tryptophan, Cys: 1/2 cystine, Ile: isoleucine, Val: valine, Tyr: tyrosine,
Gly: glycine, Lys: lysine The novel gigacin II of the present invention is composed of 17 amino acids and has a disulfide between each pair of cysteine molecules located at the 3rd and 16th positions and at the 7th and 12th positions. It has a bicyclic structure bridged by a bond. Arginine present at the C-terminal has its carboxyl group amidated (-CON
H ₂ ) is present as argininamide. Gigacin II of the present invention can be in the form of a salt, for example, an alkali metal salt, an alkaline earth metal salt ammonium salt, an inorganic base such as an organic amine salt, a salt with an organic base, and trifluoroacetic acid, Methanesulfonic acid, hydrochloric acid, sulfuric acid, nitric acid,
Includes addition salts of organic or inorganic acids such as phosphoric acid.

The novel gigacin II of the present invention and a salt thereof can be produced by the following method.

 That is, the horseshoe crab (Tachypleusgi) from Malaysia
gas) on blood cells, sodium chloride and benzamidine salts
Tris-HCl buffer containing acid salt is added and pulverized.
To obtain a precipitate. Add hydrochloric acid solution to this and pulverize, centrifuge
And obtain the supernatant. This is Sephadex G-50
Elute with hydrochloric acid solution. Measure the absorbance at 280nm
Cosmo Seal Add to 5C18
Dissolve in trifluoroacetic acid solution with varying nitrile concentration
By taking out, the desired Kigacin II fraction is obtained.

Gigacin II of the present invention can also be synthesized by a solid phase method. When this method is applied to the present invention, the α-amino group is protected with a tert-butyloxycarbonyl group (Boc group) for any amino acid, and the hydroxyl group of tyrosine is a 2,6-dichlorobenzyl group (Cl _2). -Bzl group), and the guanidino group of arginine is preferably a tosyl group (Tos group), each of which is preferably protected. First, a protected derivative of arginine at the C-terminal amino acid is introduced into a chloromethyl resin. Thereafter, the amino acid chain is extended to synthesize a protected peptide resin, which is treated with hydrogen fluoride to cleave the protected peptide from the resin. , At the same time deprotect and reduce this,
Hereinafter, a synthetic peptide is obtained according to a conventional method. The resulting crude synthetic peptide is purified by means commonly used for peptide purification such as gel filtration and reverse phase HPLC to obtain high-purity gigacin II. The solid phase method commonly used for peptide synthesis is described in, for example, “Biochemical Experiment Course (I)” edited by The Biochemical Society of Japan, Protein Chemistry, Vol. 4, pp. 208-495, published by Tokyo Kagaku Dojin ( 1977) and Nobuo Izumiya et al., "Peptide Synthesis", published by Maruzen Co., Ltd. (1975), according to the method of Merrifield and the like.

The toxicity of gigacin II of the present invention is extremely low or non-toxic. When this gigacin II is administered as a medical antibacterial agent, an oral administration method or a parenteral administration method, that is, intravenous administration, intramuscular administration, subcutaneous administration and the like are preferable. And the amount of Gigacin II used per dosage unit is 100-1000m
g. Furthermore, Gigacin II of the present invention can be effectively used as a preservative, antibiotic, and antiseptic for paints, in addition to a medical antibacterial agent.

〔The invention's effect〕

The gigacin II of the present invention has a strong antibacterial activity against, for example, Gram-positive bacteria, Gram-negative bacteria, and fungi, as shown in Reference Examples below, and is useful as an inhibitor for these bacteria. Specifically, antibacterial agents for medical use, preservatives, antibiotics, preservatives for paints and the like can be used in a wide variety of fields, and these are all included in the range of the antibacterial agents of the present invention. Needless to say.

〔Example〕

Hereinafter, the present invention will be described in more detail with reference to Examples of the present invention.

Example 1 (1) Production of Gigacin II Malaysian horseshoe crab (Tachypleus gigas) blood cells 15
g was added with 100 ml of 20 mM Tris-HCl buffer (pH 8.0) containing 50 mM sodium chloride and 5 mM benzamidine hydrochloride, and the mixture was pulverized. This operation was repeated twice to obtain a precipitate. 100 ml of a 20 mM hydrochloric acid solution is added to the obtained precipitate and ground. Centrifuge at 4 ° C (7
500 rpm for 40 minutes) to obtain a supernatant. 20 mM hydrochloric acid solution 10 for precipitation
0 ml was added and the mixture was pulverized again and centrifuged at 4 ° C. (7500 rpm for 40 minutes) to obtain a supernatant. The same operation was further performed twice to obtain a supernatant.
The obtained supernatant was collected and freeze-dried.

 Dissolve the above lyophilized product in 20 mM hydrochloric acid solution and centrifuge at 4 ° C.
The supernatant was obtained with the heart (12,000 rpm for 15 minutes). This is Sephadex
G-50 (Pharmacia) column (3 x 85cm)
And eluted with a 20 mM hydrochloric acid solution. This is fractionated by 10ml
While simultaneously measuring the absorbance at 280 nm, fraction number 69
The -86 fraction was collected and lyophilized. (See Figure 1
See). This lyophilized product is dissolved in 10 ml of 20 mM hydrochloric acid solution and
Moshir 5C₁₈(Hansui Chemicals) column (10 × 250m
m) and change the concentration of acetonitrile to 22-30%
Elution was performed with a 0.1% trifluoroacetic acid solution. This solution
The outgoing curve is shown in FIG. Eluted with 28% acetonitrile
Gigacin of the present invention is collected and freeze-dried.
I got II. The yield was 2 mg.

(2) Structure determination The structure of this substance was determined as follows. That is, a sample pyridylethylated with reduced S-vinylpyridine was hydrolyzed using 6N hydrochloric acid to determine the amino acid composition. In addition, this sample was subjected to 16 times Edman degradation using a gas-phase sequencer to determine the types of amino acids from the N-terminal lysine to the 16th cysteine and the binding order.
The aminoethylated sample was treated with lysyl endopeptidase to release the C-terminal residue. The elution time of the phenylthiocarbamyl derivative obtained on reversed-phase high performance liquid chromatography was separately synthesized. Consistent with that of phenylthiocarbamylargininamide.
Furthermore, the mass spectrometry results also showed the mass number with the C-terminal being argininamide, so it was determined that the C-terminal was argininamide.

Example 2 Formulation of Gigacin II Formulation Examples of oral or parenteral formulations containing the gigacin II active ingredient of the present invention are shown below.

(1) 200mg tablet gigacin II 200mg corn starch 40mg lactose 98mg magnesium stearate 8mg talc 4mg (2) 100mg ampoule for injection (qsp ampoule) gigacin II 100mg distilled water for injection qs (3) 500mg capsule gigacin II 500mg lactose 50mg magnesium stearate 5 mg (4) 500 mg tablet Gigacin II 500 mg Corn starch 70 mg Polyvinylpyrrolidone 35 mg Lactose 74 mg Magnesium stearate 14 mg Talc 7 mg The results of examining the antibacterial activity of peptide gigacin II of the present invention are shown below as reference examples.

Reference Examples (1) Resistance to Gram-positive and Gram-negative bacteria
Test Experimental material Test bacteria:Escherichia coli ATCC 11775Salmonella t
yphimurum Staphylococcus aureus ATCC 25923Baci
llus subtilis IAM 1069 medium: Bovine heart leachate 250g Casamino acid 15g L-tryptophan 0.05g Purified water 1000ml Experimental method Nutrient Broth 20 hours in medium (Difico)
After shaking culture, the concentration of the bacteria is reduced to about 0.3 at 660 nm.
And add 0.1 ml to the above medium.
A test solution was used. 300 μg of this substance freeze-dried product in 12 ml of purified water
To obtain a 250 μm / ml solution. Use this as a stock solution 2
12.5, 6.25, 3.13, 1.56 μg / ml samples after performing serial dilution
A liquid was obtained.

Dispense 1 ml of each concentration of the sample solution into a sterilized test tube, add 1 ml of each bacterial solution thereto, mix, and incubate at 37 ° C for 20 hours.
The absorbance at 60 mn was measured. As a control, purified water was used in place of the sample solution, and the minimum production-inhibitory concentration of this substance for each bacterium was determined. Table 1 shows the results.

(2) Antibacterial test against fungi Experimental materials Test bacteria: Aspergillus nigar Psnicillium funiculos
um medium: polypeptone 1 g malt extract 20 g dextrose 20 g agar 20 g purified water 1000 ml Experimental method Dissolve 500 µg of the freeze-dried product of this substance in 2 ml of purified water 250
A sample solution of μg / ml was obtained. This was serially diluted twice, and 125 and 62.
5, a sample solution of 31.3 μg / ml was prepared.

Add 1 ml of each concentration of sample solution to 9 ml of the above medium, and add
Each 5 ml was dispensed into a sterilized test tube to prepare a slant medium.
A straight line was drawn with a platinum loop on which the conidia of the above bacterium was adhered on the slant medium, and cultured at 30 ° C. for 48 hours, and the growth was examined.
Table 2 shows the results.

As the above results indicate, this substance was found to be a unique substance showing strong antibacterial activity against a wide range of microorganisms ranging from gram-positive bacteria, gram-negative bacteria and fungi.

[Brief description of the drawings]

 Fig. 1 shows Sephadex 20 mM hydrochloric acid dissolved in G-50 column
FIG. 2 shows the elution curve of the liquid extract.
5C₁₈Elution of 0.1% trifluoroacetic acid solution on column
Fig. 3 shows the curve. Fig. 3 shows the UV absorption spectrum of Gigacin II.
FIG.

 ────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Shoji Kimura 3-2-9 Tsukishima, Chuo-ku, Tokyo Ocean fishery Inside the Ocean Research Institute Co., Ltd. (56) References JP-A-2-500194 (JP, A) Biochem. , 106, 663-668 (1989) Chem. Pharm, Bull. , 37 (10), 261-2664 (1989)

Claims (2)

(57) [Claims] (1) The following structural formula (I) (Wherein, and are directly bonded to each other. Arg-NH ₂ indicates that the carboxyl group of arginine is an amide.) And a salt thereof. 2. The following structural formula (I) (Wherein, and are directly bonded to each other. Arg-NH ₂ indicates that the carboxyl group of arginine is an amide.) Agent.