JPH0213676B2 - - Google Patents

Description

[Detailed description of the invention]

[Technical Field of the Invention] The present invention relates to a novel phosphorus-containing compound and a method for producing the same, and more specifically, the present invention relates to a novel phosphorus-containing compound and a method for producing the same. The present invention relates to a method for producing a novel phosphorus-containing compound, which is characterized by collecting the compound. [Prior art and its problems] 2-Amino-4-(hydroxy)(methyl)phosphinoyl-butyryl-L-alanyl-L-alanine (hereinafter referred to as SF-1293 substance) has strong herbicidal activity. known as a compound.
(Refer to Japanese Patent Publication No. 51-639 and Japanese Patent Publication No. 59-23282). Traditionally, the compounds have been used in Streptomyces hygroscopicus.
hygroscopicus) SF-1293 strain [Feikoken Bacterium No. 996
No. (FERM-BP130, deposited with the Institute of Fine Technology in June 1970, but subsequently transferred to international deposit);
ATCC Bacteria No. 21705] was cultured and collected from the resulting culture. However, since its production ability was not sufficient, there was a need for an improved method for producing the compound. [Object of the invention] The object of the present invention is to provide a method capable of increasing the production ability of SF-1293 substance or a compound that can be used in the method. [Summary of the Invention] The present inventors have discovered that SF-1293 is useful as a herbicide.
Streptomyces hygroscopicus, a bacterium that produces the SF-1293 substance, is being researched to improve the manufacturing method of the substance.
When fluoroacetic acid or its salt, which is known as an inhibitor of the citric acid cycle, is added to the culture medium during the culture production of SF-1293 substance by strain SF-1293, the bacteria produce a new phosphorus-containing compound, 2-phosphinomethylmalic acid. Furthermore, when 2-phosphinomethylmalic acid was added during production culture of SF-1293 substance using SF-1293 strain,
The present invention was completed by discovering that the yield of SF-1293 substance can be increased. That is, the present invention provides the following formula: 2-phosphinomethylmalic acid or a salt thereof; and 2-phosphinomethylmalic acid-producing bacteria belonging to the genus Streptomyces are cultured, and 2-phosphinomethylmalic acid is collected from the obtained culture. 2-
Features a method for producing phosphinomethylmalic acid. The present invention will be explained in more detail below. Any strain of Streptomyces used in the present invention may be used as long as it has the ability to produce 2-phosphinomethylmalic acid in a sufficient amount to be collected in culture. Examples of such strains of the genus Streptomyces include the aforementioned strain SF-1293 (FERMP996; FERM-BP130;
21705). The mycological properties of Streptomyces hygroscopicus SF-1293 strain are specified in Japanese Patent Publication No. 639/1983. Streptomyces hygroscopicus SF-1293 strain, as seen in the case of other strains of the genus Streptomyces, is susceptible to changes in its properties and can be easily mutated by artificial mutagenesis methods using, for example, ultraviolet rays, X-rays, and chemicals. However, even such mutant strains can be used in the present invention as long as they are Streptomyces strains capable of producing 2-phosphinomethylmalic acid. In the production method of the present invention, strain SF-1293 is cultured in a medium containing nutrients that can be used by ordinary microorganisms. As the nutrient source, conventionally known nutrient sources used for culturing Streptomyces bacteria are used. For example, carbon sources include glucose, starch, glycerin, sucrose, starch syrup, molasses, etc., and these may be used alone or in combination. Nitrogen sources include soybean flour, wheat germ, meat extract, peptone, dried yeast,
Corn staple liquor, ammonium sulfate, ammonium nitrate, etc. may be used alone or in combination. In addition, calcium carbonate, salt,
In addition to adding inorganic salts such as potassium chloride and phosphates, organic or inorganic substances that aid the growth of bacteria and promote the production of 2-phosphinomethylmalic acid may be appropriately added. In particular, the production of 2-phosphinomethylmalic acid is significantly promoted by the addition of fluoroacetic acid and its salts. The most suitable culture method is liquid culture, especially deep culture. Cultivation is carried out under aerobic conditions, and the suitable temperature for cultivation is 25 to 35°C, but in most cases it is cultured at around 28°C. The appropriate number of days for culturing is 3 to 7 days, and more preferably 4 to 6 days. In order to purify and isolate 2-phosphinomethylmalic acid, which is the compound of the present invention, from a culture solution, separation and purification methods commonly used for isolating microbial metabolites from the culture solution can be used. Specifically, since the compound of the present invention, 2-phosphinomethylmalic acid, is a water-soluble acidic substance,
For its purification, Amberlite IR-120,
Cation exchange resin such as Dowex 50W, or Amberlite IRA-400, Dowex 1x
It is preferable to use an appropriate combination of a method using an anion exchange resin such as No. 2 and chromatography using Sephadex, cellulose, silica gel, charcoal powder, etc. For example, Dowex 50W
The method of removing basic impurities by passing the 2-phosphinomethylmalic acid through a resin column (H ⁺ type) is an effective method for purifying 2-phosphinomethylmalic acid. By the above production method, the novel 2-phosphinomethylmalic acid or its salt of the present invention can be obtained. Any salt may be used, but specific examples include sodium and potassium salts. The physical and chemical properties of 2-phosphinomethylmalic acid (free acid) and its sodium salt are described below. Free acid ●Appearance and properties: Colorless oil ●Solubility: Easily soluble in water, but poorly soluble in organic solvents such as ethanol, acetone, ethyl acetate, and ethyl ether. ●[α] ²⁰ _D : -23.1° (C=1, H ₂ O) ●Ultraviolet absorption spectrum: Only shows terminal absorption. ●Thin layer chromatography: Cellulose thin layer chromatography using developing solvent n-butanol:acetic acid:water (2:1:1) gives a single spot at an Rf value of 0.4. ●Elemental analysis: C26.32, H4.99, O55.32, P13.14% ●FD-mass spectrum: m/z: 213 (M ⁺ +1) ● ^1H -nuclear magnetic resonance spectrum: Measured in heavy water ¹ H-NMR spectrum is shown in Figure 1 (DSS
internal standard). ●13C-Nuclear magnetic resonance spectrum: Measured in heavy water using dioxane (δ67.4) as an internal standard.
The 13C-NMR spectra are shown in Table 1 in comparison to citric.

[Table] Sodium salt ●Appearance and properties: Amorphous white powder ●Melting point: 105-107℃ (decomposed) ●Solubility: Easily soluble in water, but in organic solvents such as ethanol, acetone, ethyl acetate, and ethyl ether does not melt easily. ●[α] ²⁰ _D : -18.1°C (C=1, H ₂ O) ●Ultraviolet absorption spectrum: Only shows terminal absorption. ●Infrared absorption spectrum: Figure 2 shows what was measured in a KBr tablet. ●Elemental analysis: C20.35, H2.67, O42.91, P13.39,
Na23.15% ●FD-mass spectrum: m/z: 279 (M ⁺ +1) From the results of elemental analysis and FD-mass spectrum,
The molecular formulas of the measured free acid and sodium salt are thought to be C ₅ H ₉ O ₇ P・H ₂ O and C ₅ H ₆ O ₇ PNa ₃・H ₂ O, respectively, and the results of the above-mentioned nuclear magnetic resonance spectra, etc. Therefore, its structure is as follows: (In the formula, M represents H or Na.) [Effects of the Invention] According to the novel 2-phosphinomethylmalic acid of the present invention, by adding the compound during the cultivation of the SF-1293 substance using the actinomycetes that produce the SF-1293 substance, , the production of SF-1293 substance is dramatically increased. Therefore, 2-phosphinomethylmalic acid is a useful SF- herbicide.
It can be said to be an extremely important compound as a precursor of the 1293 substance. Furthermore, according to the production method of the present invention, the compound can be effectively produced using 2-phosphinomethylmalic acid producing bacteria. [Examples of the Invention] Hereinafter, the present invention will be explained in more detail with reference to Examples and Test Examples. Example Streptomyces hygroscopicus SF-
1293 strain (Feikoken Bacteria No. 996) was precultured with medium (2.0% soluble starch, 1.0% polypeptone, 0.3% meat extract).
%, dipotassium hydrogen phosphate 0.05%; PH7.0). This was cultured with shaking at 28°C for 24 hours, and then subcultured into 80 ml of the same medium and cultured with shaking at 28°C for 24 hours, which was used as a seed mother for a jar fermenter. Jarfurmenter contains 3.9% glycerin, 3.6% wheat germ, 0.5% sungrain, 0.1% potassium dihydrogen phosphate, 0.1% fluoroacetic acid, and cobalt chloride.
The seed mother was inoculated into a 0.0001% composition production medium 4.0, and cultured with aeration and stirring at 28°C. The culture solution cultured for 144 hours was adjusted to pH 2.0, and the bacterial cells were removed by centrifugation to obtain a culture solution with a pH of 2.0. 600 ml of the obtained culture solution was applied to a column filled with 200 ml of Dowex 50W x 2 (H ⁺ type). 700ml of passing portion, 200ml dowex 1x
After adsorption using a column packed with 2 (CH ₃ COO ^- type) and washing with water (approximately 200 ml), the mixture was eluted with 2% NaCl water and fractionated into 10 ml portions. Combine fractions No. 18 to 23 containing 2-phosphinomethylmalic acid, concentrate, separate the precipitated NaCl, and collect approximately 5 ml of the liquid.
The mixture was applied to a column packed with 450 ml of 50W x 2 (H ⁺ type), developed with water, and fractionated into 8 ml portions. Fractions No. 24 to 30 containing 2-phosphinomethylmalic acid
(approximately 55 ml) and layered the concentrate with cellulose powder in the form of a paste on a column (320 ml) packed with cellulose powder suspended in butanol-acetic acid-water (2:1:1). It was eluted with the same mixed solution and fractionated into 6 ml portions. Fraction No.45-50 containing 2-phosphinomethylmalic acid (approx. 35ml)
was concentrated, passed through a column packed with 5 ml of Dowex 50W x 2 (H ⁺ ), and then concentrated again to yield 112 mg of white, oily 2-phosphinomethylmalic acid.
I got it. Dissolve 50 mg of 2-phosphinomethylmalic acid in a small amount of water, add 3 molar equivalents of NaOH, concentrate,
After drying, 64 mg of white powder of 2-phosphinomethylmalic acid sodium salt was obtained. Test example Streptomyces hygroscopicus SF-
1293 strain (Feikoken Bacteria No. 996) was precultured with medium (2.0% soluble starch, 1.0% polypeptone, 0.3% meat extract).
%, dipotassium hydrogen phosphate 0.05%; PH7.0) and cultured at 28°C for 24 hours. This seed culture is 2
%, glycerin 3.9%, wheat germ 3.6%,
Sungrain 0.5%, potassium dihydrogen phosphate 0.1%,
The cells were inoculated into a production medium containing 0.0001% cobalt chloride and cultured at 28°C for 7 days. 2-phosphinomethylmalic acid was added at the concentrations and times shown in Table 2. After making the culture solution acidic, centrifuge it (300 rpm, 15 minutes) to obtain the supernatant.
Amino acid analyzer (Ato MLO-703
The production amount of SF-1293 substance was measured by mold, holding time 14 minutes). The results are shown in Table 2.

[Table] From Table 2, it was found that the addition of 2-phosphinomethylmalic acid increased the productivity of SF-1293 substance.

[Brief explanation of drawings]

FIG. 1 is a ¹ H-NMR spectrum of 2-phosphinomethylmalic acid measured in heavy water.
FIG. 2 is an IR spectrum diagram of 2-phosphinomethylmalic acid sodium salt measured with a KBr tablet.

Claims (1)

[Claims] Primary formula: 2-phosphinomethylmalic acid or a salt thereof. 2. A method for producing 2-phosphinomethylmalic acid, which comprises culturing 2-phosphinomethylmalic acid-producing bacteria belonging to the genus Streptomyces and collecting 2-phosphinomethylmalic acid from the resulting culture. . 3. The manufacturing method according to claim 2, wherein fluoroacetic acid or a salt thereof is added to the culture medium of the 2-phosphinomethylmalic acid producing bacteria.