JP2641742B2 - New peptides and antibacterial agents - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION INDUSTRIAL APPLICATION The present invention relates to a North American horseshoe crab (Limulus polyphemus).
The present invention relates to a novel peptide having an antibacterial activity and a salt thereof, extracted and isolated from the same, and an antibacterial agent comprising 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, and many reports have been made so far, 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. 57-10668).
9), phosphonodipeptide derivatives (JP-A-58-13594),
Cyclic peptide derivatives (JP-A-58-213744), novel peptides consisting of 10 amino acid sequences useful as antibacterial agents and antiviral agents (JP-A-59-51247), polypeptides useful as antibacterial agents against yeasts ( JP-A-60-130599),
Glycopeptide derivatives useful as antibacterial agents against Gram-positive bacteria (JP-A-60-172998), novel glycopeptide derivatives (JP-A-61-251699, JP-A-63-44598), oligos useful for Gram-positive bacteria Peptides (JP-A-62-2279)
8), peptide antibiotics (JP-A-62-51697, JP-A-63
−17897).

[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 that have excellent antibacterial activity and can be practically used as antibacterial agents seems to be extremely important in establishing a new application field of peptides.

[Means for solving the problem]

From such a point of view, the present inventor has conducted various searches for useful novel peptides and, as a result, found that the horseshoe crab (Limulus
polyphemus), extracted and isolated from the blood cells,
The inventors have found that the present invention has a strong antibacterial action, and have completed the present invention.

That is, the present invention is recognized as including the descriptive matters 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 specification, this novel peptide is referred to as Polyphemusin I. In addition, in this specification, the IUPACIUB commission
Abbreviations based on on Biological Nomenclature may be displayed, but examples are as follows.

Arg: arginine, Trp: tryptophan, Cys: 1/2 cystine, Phe: phenylalanine, Val: valine, Tyr: tyrosine, Gly: glycine, Lys: lysine The novel polyfemcine I of the present invention is composed of 18 amino acids, It has a bicyclic structure bridged by disulfide bonds between each pair of cysteine molecules present at positions 4 and 17 and at positions 8 and 13. In addition, arginine present at the C-terminal has its carboxyl group amidated (−
CONH ₂ ) is present as argininamide.
The polyfemsin I of the present invention can be in the form of a salt, for example, an inorganic base such as an alkali metal salt, an alkaline earth metal salt ammonium salt, an organic amine salt, a salt with an organic base, and trifluoroacetic acid. Addition salts of organic or inorganic acids such as methanesulfonic acid, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like are included.

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

 That is, horseshoe crab (Limulus polyphemus) from North America
Blood cells with sodium chloride and benzamidine hydrochloride
Tris-HCl buffer solution was added and crushed.
Obtain a sediment. Add hydrochloric acid solution to this, pulverize, centrifuge and supernatant
Get. This is Sephadex Hydrochloric acid solution added to G-50
Elute at The solution collected by measuring the absorbance at 280 nm
Cosmoseal Acetonitrile added to 5C18
Elution with trifluoroacetic acid solutions with varying concentrations of
As a result, a desired polyphemsin I fraction is obtained.

In addition, the polyfemsin I of the present invention can be synthesized by a solid phase method. When this method is applied to the present invention, α
The amino group is protected with a tert-butyloxycarbonyl group (Boc group) for any amino acid, the tyrosine hydroxyl group is a 2,6-dichlorobenzyl group (Cl ₂ -Bzl group),
The guanidino group of arginine is preferably a tosyl group (Tos group), 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. And deprotection at the same time and reducing the same to obtain a synthetic peptide according to a conventional method. The obtained crude synthetic peptide is purified by means commonly used for peptide purification such as gel filtration and reverse phase HPLC to obtain high-purity polyphemsin I. 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 the polyfemsin I of the present invention is extremely low or non-toxic. In the case of administering this polyfemsin I as a medical antibacterial agent, an oral administration method or a parenteral administration method, that is, an intravenous administration, an intramuscular administration, a subcutaneous administration and the like are preferable. The amount of polyfemsin I used per administration unit is about 100 to 1000 mg. Further, the polyphemsin I of the present invention may contain preservatives, antibiotics,
It can be effectively used as a preservative for paint.

〔The invention's effect〕

As shown in Reference Examples below, the polyphemsin I of the present invention exhibits strong antibacterial activity against, for example, Gram-positive bacteria, Gram-negative bacteria, and fungi, and is useful as an inhibitor for these bacteria. Specifically, medical antimicrobial agents, preservatives,
It can be used in a wide variety of fields such as antibiotics and preservatives for paints, and it goes without saying that all of them are included in the scope of the antibacterial agent of the present invention.

〔Example〕

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

Example 1 (1) Production of polyphemsin I 5 g of 37 g of Limulus polyphemus blood cells from North America
Contains 0 mM sodium chloride and 5 mM benzamidine hydrochloride
Add 100 ml of 20 mM Tris-HCl buffer (pH 8.0) and pulverize.
The precipitate was obtained by centrifugation at 7,500 rpm (40 minutes). This operation was repeated twice to obtain a precipitate. 20 in the resulting precipitate
Add 100 ml of mM hydrochloric acid solution and pulverize. Centrifuge at 4 ° C (7500rpm
40 minutes) to obtain a supernatant. To the precipitate, 100 ml of a 20 mM hydrochloric acid solution 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 66
The -87 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 eluate is shown in FIG. Use this column with 28-30% acetonitrile.
Eluting with trill, collecting the resulting elution fractions and freeze-drying
As a result, polyfimsin I of the present invention was obtained. Yield 20
mg.

The infrared absorption spectrum of this polyphemsin I is the third
As shown, there are absorptions at 3,300, 1650, 1540, 1420, 1240, and 740 cm ^-1 . The ultraviolet absorption spectrum is as shown in FIG. Table 1 shows the amino acid composition of this polyfemsin I.

(2) Structure determination The structure of this substance was determined as follows. That is, the disulfide bond is cleaved using dithiothreitol, and 4
-6N of pyridylethylated sample with vinylpyridine
Hydrolysis was performed using hydrochloric acid to determine the amino acid composition. Also,
This sample was subjected to 17 times of Edman degradation using a gas-phase sequencer to determine the types of amino acids from the N-terminal arginine to the 17th 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 where the C-terminal was defined as arginine and amide, so it was determined that the C-terminal was arginine amide.

Example 2 Formulation of Polyfemcin I Formulation Examples of a formulation for oral or parenteral administration containing the active ingredient of polyfemsin I of the present invention are shown below.

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

Reference example (1) Antibacterial activity against Gram-positive bacteria and Gram-negative bacteria
Test Experimental material Test organism: Escherichia coli ATCC 11775 Salmonela typhimurium Staphylococcus aureus ATCC 25923 Bicillus subtilis IAM 1069 Medium: Bovine heart exudate 250 g Casamino acid 15 g L-tryptophan 0.05 g Purified water 1000 ml 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 give a 250 μg / 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 nm 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 2 shows the results.

(2) Antibacterial test against fungi Experimental materials Test bacteria: Aspergillus nigar Psnicillium funiculosum 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 lyophilized product of this substance in 2 ml 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 infrared absorption spectrum of polyphemsin I.
Fig. 4 shows the ultraviolet absorption spectrum of polyphemsin I.
It is a figure showing a spectrum.

 ──────────────────────────────────────────────────続 き Continuation of front page (56) References 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.