JP2641744B2 - 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 comprising 10 amino acid sequences useful as antibacterial agents and anti-wealth 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 antibacterial agents is still under development. In any case, developing 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 viewpoint, the present inventors have searched various useful novel peptides, and as a result, have found that the horseshoe crab (Limulu)
s polyphemus), and found that the peptide extracted and isolated from the blood cells had a strong antibacterial action, and completed 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 specification, this novel peptide is referred to as Polyphemusin II. Also, in this specification, the amino acids are defined as IUPACIUB commissio
Abbreviations based on n on Biological Nomenclature may be used, 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 polyfemcin II 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 II of the present invention can be in the form of a salt, for example, a salt with an organic base such as an inorganic salt such as an alkali metal salt, an alkaline earth metal salt ammonium salt, an organic amine salt, and trifluoroacetic acid. And addition salts of organic acids or inorganic acids such as methanesulfonic acid, hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid.

The novel polyphemsin II 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 and pulverize it.
Get clear. This is Sephadex Add to G-50 and dissolve in hydrochloric acid
Elute with liquid. Measured absorbance at 280nm and collected
Elution category, Cosmoseal Acetonitrile added to 5C18
Elution with trifluoroacetic acid solutions with varying concentrations of
As a result, a desired polyphemsin II fraction is obtained.

Further, the polyfemsin II 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 resulting crude synthetic peptide is purified by gel filtration, reverse phase HPLC, or other means commonly used for peptide purification to obtain high-purity polyphemsin II. The solid phase method commonly used for peptide synthesis is described in, for example, “Chemical Chemistry Laboratory Course (I)” edited by The Biochemical Society of Japan, Protein Chemistry, Vol. 4, pp. 208-495, published by Tokyo Chemical Doujin Co., Ltd. 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 II of the present invention is extremely low or non-toxic. In the case of administering this polyfemsin II 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 polyphemsin II used per administration unit is about 100 to 1000 mg. Further, the polyfemsin II of the present invention is a preservative, antibiotic,
It can be effectively used as a preservative for paint.

〔The invention's effect〕

As shown in the following Reference Examples, the polyfemsin II of the present invention has a strong antibacterial action 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 polyphemusin II 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. The precipitate was added with 100 ml of a 20 mM hydrochloric acid solution, 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-mentioned dried product in a 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 outgoing curve is shown in FIG. Use this column with 27-28% acetonitrile.
Elute with trill, collect the extracted fractions and freeze-dry
As a result, the polyfimsin II of the present invention was obtained. Yield 20
mg.

The infrared absorption spectrum of this polyfemsin II is the third
As shown, absorption is at 3,000, 1650, 1540, 1420, 1240, and 740 cm ^-1 . The ultraviolet absorption spectrum is as shown in FIG. The amino acid composition of this polyfemsin II is shown in Table 1.

(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 elution time of the phenylthiocarbamyl derivative obtained by treating the aminoethylated sample with lysyl endopeptidase to release the C-terminal residue on a reversed-phase high-performance liquid chromatography was separately calculated. Phenylcarbamyl argininamide. Further, the mass spectrometry results indicated that the C-terminus was argininamide, and the mass number was indicated.

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

(1) 200mg tablet Polyfemcin II 200mg corn starch 40mg lactose 98mg magnesium stearate 8mg talc 4mg (2) 100mg ampoule for injection (qsp ampoule) polyfemsin II 100mg distilled water for injection qs (3) 500mg capsule polyfemsin II 500mg lactose 50mg magnesium stearate 5 mg (4) 500 mg tablet Polyphemsin 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 the peptide polyfemsin II 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 materials Test bacteria: Esoherichia coli ATCC 11775 Salmonella typhimurium Staphylococcus aureus ATCC 25923 Bacillus 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: Polyppetone 1 g Malt extract 20 g Glucose 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 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, cultured at 30 ° C. for 48 hours, and the production was examined.
Table 3 shows the results.

As the above results indicate, this substance was found to be a good 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
The figure showing the curve, and FIG. 3 shows the infrared absorption spectrum of polyphemsin II.
Fig. 4 shows the ultraviolet absorption spectrum of polyphemsin II.
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.) And a salt thereof as an active ingredient Agent.