JPH0480039B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel phosphorus-containing peptide compounds and salts thereof that are useful as herbicides, and also relates to methods for producing these novel compounds. Streptomyces hygroscopicus SF-1293 strain described in the Japanese Patent Publication No. 51-639 (Patent No. 827768) (Feikoken Bokuyori No. 996, since June 1970, in the FIKEN) It has been deposited, and it has been transferred to international deposit and FERM
−Deposited as BP130 and also by the US ATCC
It has also been deposited with ATCC deposit number 21705.
It is already known that a substance represented by the following general formula () can be produced by culturing these substances (available for distribution from depositories)
59-23282 and JP-A-60-222498).

[Table] It is also known that these substances are useful as herbicides (JP-A-54-67026 and JP-A-60-222498). Recently, the present inventors have discovered that when the SF-1293 strain described above is cultured, a phosphorus-containing peptide compound (hereinafter sometimes referred to as bialaphos dimer) having the following structural formula () is produced in the culture solution together with these substances. They discovered that this compound is a novel substance and found that it is effective as a herbicide. Therefore, according to the first invention, the following formula () A novel phosphorus-containing peptide compound (bialaphos dimer) represented by the following formula and a salt thereof are provided. Examples of salts of the phosphorus-containing peptide compound (bialaphos dimer) of formula () include mono-, di-, tri- or tetra-alkali metal salts of this compound;
For example, there are metal salts such as sodium salts; potassium salts; ammonium salts; alkaline earth metal salts; and also salts with various bases, such as alkylamines, and salts with choline. Furthermore, according to the second invention, the following formula () belonging to the genus Streptomyces Provided is a method for producing a phosphorus-containing peptide compound (bialaphos dimer) of the formula (), which comprises culturing a producing bacterium of the phosphorus-containing peptide compound represented by the formula () and collecting the compound of the formula () from the culture. . An example of a bacterium producing the compound of formula () used in the method of the present invention is Streptomyces hygroscopicus SF described in Japanese Patent Publication No. 51-639.
There are -1293 strains (FERM-P996; FERM-BP130). The mycological properties and culture method of this SF-1293 strain are as described in Japanese Patent Publication No. 1983-639. When culturing the SF-1293 strain, the compound of the present invention of formula () can be used as SF-
123 substance (the above formula) and substances (Ia) to (If) described in Japanese Patent Publication No. 60-222498, it is produced in the culture solution, so it was separated together with these substances, and isolation from them was included. Refining is required. Usually, the SF-1293 strain is cultured and the culture solution is used.
The compound of the present invention can be isolated from the SF-1293 substance by combining cation or anion exchange resin, thin layer chromatography, high performance liquid chromatography, chromatography using a porous resin such as Diaion HP-20, etc. can be collected. In the method of the present invention, a microorganism producing the compound of the present invention, for example strain SF-1293, is cultured in a medium containing nutrients that can be used by common microorganisms. As the nutrient source, any known nutrient source that has been conventionally used for culturing Streptomyces bacteria can be used. For example, glucose, starch, glycerin, sucrose, starch syrup, molasses, etc. can be used as carbon sources. In addition, soybean flour, wheat germ, meat extract, peptone,
Dried yeast, corn steep liquor, ammonium sulfate, sodium nitrate, etc. may be used. In addition to adding inorganic salts such as calcium carbonate, common salt, potassium chloride, and phosphates as necessary, organic and inorganic substances that aid the growth of bacteria and promote the production of the compound of the present invention may be appropriately added. As for the culture method, the liquid culture method, especially the deep culture method, is most suitable, as is the case with general antibiotic production methods.
Cultivation using the SF-1293 strain is performed under aerobic conditions, and the appropriate temperature for cultivation is 25-35°C, but in most cases it is cultured at around 28°C. Thus, the production of the compound of the present invention takes 5 to 8 days for both shaking culture and tank culture.
Reaches maximum in days. Substances SF-1293, substances (Ia) to (If), and the compound of the present invention (bialaphos dimer) mainly exist in the liquid part of the culture, but as shown in the physical and chemical properties below, they are water-soluble amphoteric substances. For extraction from culture solution, Amberlite IR-120,
Adsorb using a cation exchange resin such as Dowex 50W or an anion exchange resin such as Amberlite IRA-400, IR-45, IR-4B, and elute using an appropriate acid, alkali or salt solution. can do. For example, an effective extraction method is to pass the culture filtrate through a resin column made of cation exchange resin Dowex 50W (H type), adsorb the active ingredients onto the resin, and elute this with aqueous ammonia. The coarse powder containing the SF-1293 substance and the compound of the present invention obtained by such a method was
When subjected to chromatography using resin or cellulose, silica gel, alumina or Cephadex, SF-1293 substances and (Ia) to (If)
The compound of the present invention can be separated from substances. The compound of the present invention thus separated was further subjected to chromatography using Sephadex G-10 and Dowex 1x2 resins, chromatography using nonionic adsorption resins, cellulose thin layer chromatography, and high performance liquid chromatography. Target substances can be isolated and purified by combining graphi. This compound was found to have excellent herbicidal activity. Therefore, the compound of the present invention (bialaphos dimer) of the formula () can be used as a herbicide containing it as an active ingredient, and this herbicide can contain carriers and adjuvants commonly used in agricultural chemicals. Example (a) Streptomyces hygroscopicus SF
-1293 strain (FERM-BP130) with 2.0% starch, 1.0% peptone, 0.3% meat extract, dipotassium phosphate
Inoculated into liquid medium 15 with 0.05%, PH7.0, and incubated at 28°C.
Culture with aeration and agitation for 24 hours, and use this as a seed mother. Glucose 3.0%, starch syrup 1.0%, wheat germ 2.5
% Soluble Vegetable Protein
The seed mother was inoculated into a liquid medium 200 containing 0.5% soybean oil, 0.1% soybean oil, 0.1% yeast extract, 0.001% ferrous sulfate, 0.0001% nickel chloride, 0.0001% cobalt chloride, and PH7.0, and incubated at 28°C for 96 hours. Culture was carried out with aeration and stirring. Pass the culture solution at PH3 to obtain a solution of 150 (culture titer 100 mcg/ml). The liquid is passed through a column (7.5) packed with activated carbon and washed with 30 g of water. Combine the passing liquid and washing liquid (180), and use Dowex 50W x 2 (50
-100 mesh) (H type) 9 resin tower to adsorb the active ingredient. The resin tower is 0.05N after washing with water.
Elute with aqueous ammonia. The active fraction (45) was concentrated under reduced pressure to obtain 50g of pale yellow crude powder (potency 200mc).
g/ml). (b) 10 g of the above crude powder was dissolved in 50 ml of water and passed through a column packed with 1 x 2 Dowex (acetic acid form) to adsorb the target substance, and the column was washed with 2 parts of water. Then, SF-1293 substance and substances (Ia) to (Ie) were completely eluted with 0.3N acetic acid 6. Thereafter, when eluted with a 0.3-2.0N acetic acid concentration gradient, AMPB and bialaphos dimer (If) substances are eluted in this order, overlapping each other. Thin layer chromatography using cellulose plate [Developing agent: n-butanol-acetic acid-water (2:1:1)]
showed Rf0.54, and high performance liquid chromatography (JASCO Tri-rotor, column: Asahi Kasei)
ASAHIPACK ES-502N, mobile phase: 68.8mM
A fraction containing a large amount of a substance exhibiting a retention time of 9.7 minutes (NH ₄ Cl, detection: 210 nm) (however, it did not contain any (If) substance) was designated as a bialaphos dimer fraction. The bialaphos dimer fractions were collected and concentrated to dryness to obtain 0.8 g of powder. This powder was dissolved in 5 ml of water, passed through a column packed with 400 ml of Cephadex G-10, and developed with water.
Since it is eluted in this order, it is fractionated into 10 ml portions, collected by thin layer chromatography using a cellulose plate and high performance liquid chromatography to collect fractions containing only bialaphos dimer, and concentrated to dryness to obtain a white powder of bialaphos dimer. Got 100mg.
This substance showed a retention time of 9.7 minutes in high performance liquid chromatography analysis. Mass spectrum (SIMS): m/z629 [M+1] ^{+ 1} H-NMR spectrum (400MHz, D ₂ O) δH
ppm: 1.27 (3H, d, J = 13.6Hz), 1.30 (3H,
d, J = 13.6Hz), 1.37 (3H, d, J = 7.2Hz),
1.42 (9H, m), 1.62 (2H, m), 1.68 (2H, m),
1.98 (2H, m), 2.12 (2H, m), 4.08 (1H, t, J
= 6.5Hz), 4.16 (1H, q, J = 7.2Hz), 4.35 (4H,
m) ¹³ C-NMR spectrum (100MHz, D ₂ O) δC
ppm: 180.9 (s), 175.9 (s), 175.2 (s), 174.
Five
(s), 174.0 (s), 170.3 (s), 55.5 (d) ( ³ Jc-p
=
15.9Hz), 54.4(d) ( ³ Jc−p=14.3Hz), 52.0(d),
50.7(d)d), 50.6(d)d), 50.5(d)d), 28.6(t)
( ¹ Jc
-p=92.2Hz), 27.7(t) ( ¹ Jc-p=90.6Hz),
25.9(t), 25.8(t), 18.6(q), 17.7(q), 17
.6
(q)×2, 16.3(q) ( ¹ Jc−p=93.8Hz), 16.2
(q) ( ¹ Jc-p=92.2Hz) Constituent amino acid analysis: After degassing in 6N hydrochloric acid under reduced pressure and hydrolyzing at 110℃ for 18 hours, AMPB
Only two amino acids, alanine and alanine, were released (amino acid analysis, thin layer chromatography).
The molar ratio of AMPB to alanine was 1:2. Amino acid sequence analysis: The sequence order of each amino acid by an amino acid sequence analyzer is from the N-terminus to
The order was Ala→Ala→X→Ala→Ala. X indicates an unidentifiable amino acid. Properties: White powder, water-soluble Color reactions: Positive for ninhydrin, Lydon Smith, and Harness reactions. Actual elemental analysis values:
C42.08%, H6.75%, N13.40%, O28.03% Theoretical value as molecular formula C ₂₂ H ₄₂ N ₆ O ₁₁ P ₂ :
C42.04%, H6.73%, N13.37%, O28.00% Test example 1 Weeding test for crabgrass. Crabgrass seeds were sown in plastic pots with a diameter of 5 cm filled with field soil, and when the plant height was 3.5 cm.
An aqueous solution of Bialaphos dimer at a predetermined concentration (shown in Table 1) was applied by spraying it over the entire stems and leaves. The amount of aqueous solution sprayed is 10/are,
As a spreading agent, polyoxyethylene (20) sorbitan monolaurate was added to the aqueous solution at a concentration of 0.05%. The herbicidal effect was investigated by observation on the 7th day after treatment.
It was calculated as a percentage of the control (untreated). The test results are shown in Table 1.

[Table] Test example 2. Weed killing test for Japanese knotweed. Seeds of Japanese knotweed were sown in plastic pots with a diameter of 5 cm filled with field soil, and when the plant height was 2.5 cm, a spray solution containing a predetermined concentration of Bialaphos dimer was applied as an aqueous solution (shown in Table 2). was applied by spraying it over the entire stem and leaves. The amount of aqueous solution sprayed was 10/R, and polyoxyethylene (30) sorbitan monolaurate was added as a spreading agent to the aqueous solution at a concentration of 0.05%. The herbicidal effect was investigated by observation on the 7th day after treatment.
It was calculated as a percentage of the control group (untreated). The test results are shown in Table 2.

【table】

Claims (1)

[Claims] Linear formula () A phosphorus-containing peptide compound and a salt thereof. 2 The following formula () belonging to the genus Streptomyces 1. A method for producing a phosphorus-containing peptide compound of formula (), which comprises culturing a bacterium that produces a substance represented by formula () and collecting a substance of formula () from the culture.