JPH09165399A - New cyclic peptide - Google Patents

Abstract

(57) [Summary] The following formula I: (In the formula, R represents a benzyl group or an isobutyl group.) And a use thereof. According to the present invention, a novel cyclic peptide is provided. The cyclic peptide of the present invention is useful in that it can inhibit the growth of legume pests.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel cyclic peptide and a pesticidal insect growth inhibitor containing the cyclic peptide as an active ingredient.

[0002]

2. Description of the Related Art Azuki weevil is known as a storage pest that damages the seeds of legumes such as adzuki beans, cowpea and mung beans (mung bean). The adzuki beetle hatches from eggs laid on the surface of ripened pods or ripe seeds, turns into larvae, and then invades and erodes these seeds. Adults that have emerged from the seeds can lay eggs immediately after mating. Therefore, if the temperature conditions are appropriate, they will repeatedly proliferate in the stored adzuki bean, causing great damage to the adzuki bean.

[0003] Mungbean (Vigna radiata) is a kind of legumes that are attacked by the adzuki beetle, and it is desired to develop a substance that is resistant to such pests. So far, TC1 is a direct ancestral line of mung bean.
966 is known to be resistant to Azuki weevil, and as a result of genetic analysis of the progeny of the cross with Mungbean, it is known that the resistance is controlled by a single dominant gene (R) ( Kitamura, K. et al., Japan.J. Breed., 38:
459-464 (1988)). In a line having a homozygous (R / R) resistance gene derived from TC1966, a finch and a Brazilian weevil cannot grow (Fujii, K. et al., Appl.
Entomol.Zool., 24: 126-132 (1989)), and also shows a strong growth inhibition against soybean beetles, which are important pests of soybean. Are also considered to contain substances of high utility value.

[0004]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a novel cyclic peptide and a pesticidal insect growth inhibitor containing the peptide as an active ingredient.

[0005]

Means for Solving the Problems As a result of intensive studies based on the above-mentioned problems, the present inventor has found that a cyclic strain having an inhibitory effect on the growth of pests from seeds of a line incorporating the resistance of TC1966, a mungbean wild species. The peptide was successfully isolated, and the present invention was completed. That is, the present invention relates to the following formula I:

[0006]

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(Wherein R represents a benzyl group or an isobutyl group). Further, the present invention provides a compound of formula II:

[0008]

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A pest growth inhibitor for beans containing the cyclic peptide represented by the formula (1) as an active ingredient. Examples of the pest include azuki weevil. Hereinafter, the present invention will be described in detail.

[0010] 1. Cyclic peptide The cyclic peptide of the present invention can be produced by the following method. First, obtain incorporating TC1966 resistance to a susceptible cultivar Osaka mungbean as recurrent parent resistance (R / R) system, for example, the BC ₂₀ F ₄ seed powder. Here, BC ₂₀ F ₄ is a line created by repeating backcrossing and selection for resistance 20 times, using TC1966 as a parent once and Osaka Mungbean as a recurrent parent, followed by selfing 3 times. .

In the present invention, "susceptibility" refers to the property of being harmed or susceptible to pests such as adzuki weevil, and "resistance" refers to the property of not being susceptible or susceptible to harm.

After the obtained seed powder is degreased with acetone, 80
Extract with% ethanol. Next, the 80% ethanol extract is fractionated by partitioning between water and butanol to obtain a butanol layer and an aqueous layer, and the inhibitory activity is measured according to the method for measuring growth inhibitory activity. The fraction of the butanol layer in which the inhibitory activity was recognized was dried under reduced pressure, dissolved in a small amount of 80% ethanol, and analyzed by HPLC (high performance liquid chromatography). HPLC
For example, it can be performed under the following conditions. That is,
Tosoh ODS80T equilibrated with 45% acetonitrile solution
Inject 250 µl of the above sample into an _M column (7.8 × 300 mm), continuously elute with 45% acetonitrile, and detect the elution peak with 225 nm ultraviolet light. Then, the same method was used as a control, using Osaka mung bean sensitive to Azuki weevil as a control, and the pattern of the chromatogram was compared with the target extract.

Next, in order to concentrate a large amount of a substance eluted in a characteristic peak not found in the sensitive Osaka mung bean, the 80% ethanol extract was equilibrated with 30% ethanol to obtain a C18 reverse phase. Fractionated by open column (30 x 250 mm), non-adsorbed (30% ethanol elution) fraction and 60%
Prepare the ethanol elution fraction. Inhibitory activity was observed
The 60% eluted fraction is concentrated to dryness, dissolved in a small amount of 80% ethanol, and the eluate of the peak characteristically observed in the resistant strain by HPLC is collected to elute a large amount of eluted substance (25 to 30 m
g) By purifying, the cyclic peptide of the present invention can be prepared.

[0014] The purified cyclic peptide can be highly purified by fractionating the substance eluted in the peak collected several times again by HPLC again. The chemical structure of the purified peptide can be determined by a commonly used method such as mass spectrometry and NMR.

2. When the cyclic peptide of the present invention obtained as described above is used as a pest growth inhibitor, the type of beans used is not particularly limited. For example, it can be used for a specific purpose to inhibit the growth of pests in mung bean, adzuki bean, cowpea, soybean and the like.

Pests that inhibit growth are
For example, adzuki beetle, beetle beetle, scorpion beetle, and cutworm beetle are exemplified. The growth inhibitory activity is measured by a bioassay using artificial beans.
In other words, the freeze-dried powder of each fraction was added to Osaka Mung Beef powder for 0.
Add 1-20%, mix well, add a small amount of water to make artificial beans, and dry overnight in a desiccator. Azuki weevil is released on the dried artificial beans and allowed to lay eggs for 2 to 3 days, and then placed in a 30 ° C. incubator at 70% humidity with saturated saline. After a lapse of at least 15 days, the growth of the larvae is examined under a stereoscopic microscope, and the growth inhibitory activity of each fraction is examined by comparison with the growth of Osaka mung bean flour on artificial beans (control).

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.

Example 1 Preparation of Cyclic Peptide T using Osaka Mungbean (obtained from Iwate University) as a repetitive parent
By growing by backcrossing method azuki bean weevil resistant C1966, resistant strain (R / R; BC ₂₀ F
₍₄ lines). Acetone defatted seed powder (200 g) of the resistant line was extracted with 5000 ml of 80% ethanol, concentrated to dryness under reduced pressure using a rotary evaporator, dispersed in a small amount of water, and freeze-dried. The freeze-dried product was mixed with Osaka mung bean flour at 10%, lightly kneaded by adding a small amount of water, then rounded to the size of a little fingertip, and left overnight in a desiccator to obtain artificial beans.

Next, the obtained mixture was subjected to a growth test of Azuki weevil larvae by the following method. That is, azuki weevil was released on the dried artificial beans and laid eggs for 2 to 3 days, and then placed in a 30 ° C. incubator at 70% humidity with saturated saline. After a lapse of 15 days, the growth of the larva was examined under a stereoscopic microscope, and the growth inhibitory activity was examined by comparing the growth of Osaka mung bean flour with artificial beans.

As a control, a test was carried out in the same manner as described above, using a mixture obtained by mixing 10% of an extract of Osaka Mungbean. As a result, when a mixture of the above-mentioned ethanol extract and artificial beans was used, the growth of Azuki weevil larvae was remarkably inhibited (Table 1, Nos. 4, 7, etc.). On the other hand, when a mixture of 10% of the extract of Osaka Mungbean was used, no growth inhibition was observed (Table 1, No. 1).

Therefore, the concentrated and dried product of the 80% ethanol extract of the R / R system was dispersed in 250 ml of 30% ethanol, centrifuged to remove a small amount of precipitate, and then C18 reverse phase equilibrated with a 30% ethanol solution. (Bondapak® HC18 HA 125
(Millipore) open column. The fractionation was performed at room temperature at a flow rate of 30 to 50 ml / hr.

A non-adsorbed (30% ethanol eluted) fraction and a 60% ethanol eluted fraction fractionated from the above column were prepared, and each preparation was tested for the growth inhibitory activity of Azuki weevil in the same manner as described above. Was. As a result, no inhibition was observed when the 30% -eluted fraction was mixed with artificial beans at 20% (Table 1, No. 9), but a strong inhibitory activity was observed in the 60% ethanol-eluted fraction (Table 1). , No. 10).

[0023]

[Table 1]

In Table 1, "OR" represents Osaka Mungbean and "R"
“/ R” means a resistant strain of BC ₂₀ F ₄ generations, and “extract” means 80% ethanol extracted and dried product of seed flour. In addition, among the growth results, “○” indicates that the larvae grew well and led to emergence, “△” indicates that the larvae were poorly grown and could not emerge, and “×” indicates that the larva could not grow, and It means those who died at the age of two.

Next, the inhibitory activity was concentrated in the butanol layer of the 80% ethanol extract in water-butanol two-partition.
This fraction was evaporated to dryness under reduced pressure, and dissolved in a small amount of 80% ethanol.
C (TOSOH _{ODS 80 M (9.2 × 250mm)} , detection 225 nm) analyzed,
Compared.

As a result, upon elution with 45% acetonitrile, the resistant line (R / R) gave two characteristic peaks that were not observed in the sensitive Osaka mung (Fig. 1, arrow; Osaka mungbean, B is HPLC pattern of BC ₂₀ F ₄ lines). Since the two peaks are clearly different between the resistance and the sensitivity, it was convinced that the substance eluted in these peaks was the target substance.

Therefore, the amount of the substance eluted in the two peaks is quantitatively large (the amount is such that the growth inhibitory activity on artificial beans can be investigated, and the final substance amount is at least about 5 to 10 mg).
And highly concentrated. The concentration method is as follows: 80% of the dried product under reduced pressure of the 60% ethanol eluted fraction fractionated by the open column.
The ethanol lysate was fractionated by HPLC to give the characteristic 2
This was done by purifying the two peaks to a single peak each.

As a result, 50 mg of a concentrate (purified substance) of the substance eluted in the main peak was obtained as a lyophilized substance. 0.3 to 0.4% of the substance eluted in the main peak is mixed into the artificial beans,
The growth inhibition test of Azuki beetle larvae was performed in the same manner as described above.

As a result, the growth of adzuki beetle was 1-2.
It was completely inhibited by age. From the above results, the substance eluted in the main peak was determined to be the main insect-resistant substance derived from TC1966, and was determined as GIF-1.

On the other hand, in artificial beans mixed with 0.6 to 1.0% of a substance (referred to as GIF-2) eluted in the other peak, Azuki weevil larvae grew smoothly and reached eclosion. Furthermore, GIF-1 and GIF-2 were fractionated by HPLC to purify them into a single complete peak, and the chemical structure was analyzed using mass spectrometry and NMR. Table 2 shows the result of NMR, and Table 3 shows the result of mass spectrometry.

[0031]

[Table 2]

[0032]

[Table 3]

As a result, GIF-1 has a structure in which three amino acids (hydroxyleucine, phenylalanine and tyrosine) containing two aromatic amino acids and one oxyacid form an amide bond and are cyclized, and :

[0034]

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It was found to be a cyclic peptide represented by GIF-2 contains 3 aromatic amino acids.
Formula III having a structure in which two amino acids (leucine, hydroxyleucine and tyrosine) and one oxyacid form an amide bond and are cyclized.

[0036]

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The cyclic peptide was found to be: The cyclic peptide G represented by the above formula III
IF-2 (in formula I, wherein R is an isobutyl group)
It can also be used as an intermediate when synthesizing GIF-1 represented by the above formula II (where R is a benzyl group in formula I).

[0038]

According to the present invention, a novel cyclic peptide is provided. The cyclic peptide of the present invention is useful in that it can inhibit the growth of legume pests.

[Brief description of the drawings]

FIG. 1 shows the results of HPLC.

Claims (3)

[Claims] 1. The following formula I: (In the formula, R represents a benzyl group or an isobutyl group.) 2. The following formula II: A pest growth inhibitor for beans containing the cyclic peptide represented by the formula (1) as an active ingredient. 3. The method of claim 2, wherein the insect pest is azuki weevil.