JPS647079B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to the herbicide 2-amino-4-(hydroxy)(methyl)phosphinoyl-butyric acid (Unexamined Japanese Patent Publication No.
54-92628) and SF-1293 substance (Japanese Patent Application Laid-open No. 1983
2-amino-4-(hydroxy)phosphinoyl-butyric acid (hereinafter referred to as MP-101), which is useful as a synthetic intermediate for
and 2-amino-4-(hydroxy)phosphinoyl-butyryl-L-alanyl-L-alanine (hereinafter referred to as MP-102), and a method for producing the same. To explain in more detail, the general formula, (R is -OH or

[Formula] Group) New compounds MP-101 and MP-102 substances, and MP-101 and MP- belonging to actinomycetes
102-producing bacteria are cultured in a nutrient medium supplemented with ordinary carbonate sources, nitrogen sources, and other inorganic salts, and from the culture
The objective is to obtain MP-101 and MP-102 substances. The present inventors used a strain belonging to actinomycetes to
When searching for a substance with herbicidal activity, a new substance was found in a cobalt-free culture of a strain of the genus.
We found that the substances MP-101 and MP-102 were produced, and based on the physical and chemical properties shown below, we determined that they were respectively 2-amino-4-(hydroxy)phosphinoyl-butyric acid and a tripeptide containing this phosphorus amino acid, It was identified as 2-amino-4-(hydroxy)phosphinoyl-butyryl-L-alanyl-L-alanine, and the present invention was completed. MP
-101 and MP-102 substances themselves have weak herbicidal activity and cannot be put to practical use, but 2-amino-4-(hydroxy)(methyl)phosphinoyl-butyric acid and
It is an important compound as a raw material for the synthesis of SF-1293 substance. MP-101 and MP-102, compounds of the present invention
The physical and chemical properties of the substance are shown below. MP-101 substance is a white crystal with a melting point of 221-222℃ (decomposition), and is easily soluble in water, but poorly soluble in organic solvents such as ethanol, acetone, ethyl acetate, chloroform, and benzene. The specific optical rotation is [α] ²⁰ _D =
At +28.9° (c1, 1N hydrochloric acid), ultraviolet absorption only shows terminal absorption. The infrared absorption spectrum of such a substance is shown in FIG. The results of elemental analysis are
C28.96, H6.06, N8.21, O37, 90, P18.23%,
Since an M+1 peak is observed at M/E168 in the FD-mass spectrum, the molecular formula of this substance is
It is C ₄ H ₁₀ NO ₄ P (molecular weight 167). The color reaction is
The ninhydrin and potassium permanganate reactions were positive, and the Mauritsch and Anthrone reactions were negative. MP-101 substance is developing solvent n-butanol:
Acetic acid:water (2:1:1) silica gel thin layer chromatography gives a single spot at Rf 0.18, and cellulose thin layer chromatography gives a single spot at Rf 0.49. Based on the above physical and chemical properties and the results of separate research
The chemical structure of MP-101 substance is shown below.
It was identified as 2-amino-4-(hydroxy)phosphinoyl-butyric acid. MP-102 substance is a white crystal with a melting point of 226-228℃ (decomposition), and is well soluble in water, but poorly soluble in organic solvents such as ethanol, acetone, chloroform, ethyl acetate, and benzene. The specific optical rotation is [α] ²¹ _D = -
At 37.7° (c1, 1N hydrochloric acid), ultraviolet absorption only shows edge absorption. The infrared absorption spectrum of such a substance is shown in FIG. Elemental analysis value is C39.02,
H6.62, N12.98, O31.17, P9.73%, an M+1 peak was observed at M/E310 in the FD-mass spectrum, and the molecular formula of this substance is C ₁₀ H ₂₀ N ₃ O ₆ P (molecular weight 309) is given. Regarding the color reaction, the reaction with ninhydrin and potassium permanganate is positive, and the reaction with Anthrone is negative. The MP-102 substance was analyzed by silica gel thin layer chromatography using a developing solvent of n-butanol:acetic acid:water (2:1:1) to obtain a single spot at Rf0.25, and by cellulose thin layer chromatography at Rf0.67. show. MP-102 substance was hydrolyzed in 6N hydrochloric acid at 110°C for 20 hours to produce 1 mol of MP-101 substance and 2 mols of L-alanine. Based on the above physical and chemical properties and other research results, the chemical structure of substance MP-102 was identified as L-2-amino-4-(hydroxy)phosphinoyl-butyryl-L-alanyl-L-alanine. Chemical induction of the compounds MP-101 and MP-102 of the present invention into 2-amino-4-(hydroxy)(methyl)phosphinoylbutyric acid and SF-1293 is carried out by the steps shown below. (Process 1) (R ₁ is an amino group, R ₂ is a carboxyl group and a protecting group for the phosphorous acid moiety) (Step 2) (R ₁ is an amino group, R ₂ is a carboxyl group and a protecting group for the phosphorous acid moiety) In other words, compounds derived from MP-101 and MP-102 substances and benzene, toluene, xylene, N,N-dimethylformamide, etc. When methyl iodide or methyl bromide is reacted in the presence of a base such as metallic sodium, sodium hydride or lithium isopropylamide in an organic solvent, a reaction product with a methyl group introduced into the phosphorous acid moiety is produced. do. The reaction temperature is 50-110°C and the reaction time is 2-6 hours. The amount of base used is 1 to 1.5 equivalents based on the raw material compound, and the amount of methyl iodide and methyl bromide added is 1 to 5 equivalents. As protecting groups for amino groups, formyl group, acetyl group, trifluoroacetyl group, benzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, t-butoxycarbonyl group, tosyl group, etc. are used, and carboxyl group and phosphorous acid group are used. As the protective group for the group, protective groups used in ordinary peptide synthesis such as lower alkyl groups such as methyl, ethyl, and t-butyl, benzyl group, p-methoxybenzyl group, and benzhydryl group can be used as they are. By deprotecting the reaction intermediates thus obtained by means such as acid, alkali or catalytic reduction, the desired compound 2-amino-4-(hydroxy)(methyl)-phosphinoyl-butyric acid [ ] and SF-1293 substance ( ) are obtained.The details of the reaction are specifically described in the reference examples.Next, the method for producing the compounds MP-101 and MP-102 substances of the present invention will be explained. MP−101
All actinomycetes that produce and accumulate MP-101 and MP-102 substances can be used as the actinomycetes that produce and accumulate MP-102 substances. Among these actinomycetes, an example of a strain of the genus Streptomyces is strain SF-1293, which was isolated from soil and was identified and named Streptomyces hygroscopicus SF-1293 (Feikoken Bibori No. 996). ) can be given. The mycological properties of Streptomyces hygroscopicus SF-1293 are published in Japanese Patent Publication No. 48-22688.
This is clearly stated in the Publication No. 51-639. Streptomyces hygroscopicus SF−
As seen in the case of other strains of the genus Streptomyces, the 1293 strain is susceptible to changes in its properties and can be mutated by artificial mutagenic means using, for example, ultraviolet rays, X-rays, radio frequencies, radiation, chemicals, etc. , even such mutant strains MP-101 and MP-102
All Streptomyces strains capable of producing substances can be used in the method of the present invention. In the cultivation of the bacteria producing the MP-101 and MP-102 substances of the present invention, the presence of cobalt ions may significantly inhibit the production of the MP-101 and MP-102 substances. It is important to culture in a medium that has been removed as much as possible. Examples of carbon sources for culturing include glucose, starch, glycerin, sucrose, starch syrup, and molasses, either alone or in combination. Inorganic and organic nitrogen sources include ammonium sulfate, ammonium nitrate, ammonium chloride, sodium nitrate, urea, soybean flour, wheat germ, meat extract, peptone, dried yeast, corn staple liquor, casamino acids, soluble vegetable protein, etc. alone or in combination. It is used as In addition to adding inorganic salts such as calcium carbonate, common salt, potassium chloride, and phosphates as necessary, the growth of the bacteria used and MP-
101, MP-102 Appropriate organic and inorganic substances can be added to promote the production of substances. The most suitable culture method is a liquid culture method, especially a deep culture method. Cultivation is carried out under aerobic conditions;
A suitable temperature for culturing is 25-35°C. MP-102 substance is produced and accumulated in the culture solution when the culture is carried out in liquid culture for usually 3 to 6 days. When the culture time is further extended, the alanyl-alanine part of the produced MP-102 substance is hydrolyzed, and the MP-102 substance is converted into MP-
The conversion reaction to 101 substances progresses. The number of culture days when the amount of MP-101 substance produced reaches the maximum is 8 to 11 days, and the presence of MP-102 substance is hardly recognized in the culture solution at this stage. Therefore, when purifying MP-101 and MP-102 substances, the time when the production amount in the culture medium reaches the maximum (MP-102
It is most effective to stop the culture after 5 to 6 days (usually 5 to 6 days for MP-101 substances, 8 to 9 days for MP-101 substances), and extract and isolate each substance from the culture solution. MP-101, the compound of the present invention, from the culture solution
In order to purify and isolate MP-102 substances, separation and purification methods commonly used for isolating microbial metabolites from their culture solutions can be used. Specifically, since the compounds MP-101 and MP-102 of the present invention are water-soluble amphoteric substances, their extraction and
For purification, it is adsorbed onto a cation exchange resin such as Amberlite 1R-120 or Dowex 50W, or an anion exchange resin such as Amberlite 1RA-400 or Dowex 1x2, and then mixed with an appropriate acid, alkali or salt solution. A method of elution using
Chromatography using DEAE-Sephadex, charcoal powder, cellulose, silica gel, etc. can be carried out in an appropriate combination. Next, the present invention will be explained with reference to Examples, but the present invention is not limited to these in any way. Example 1 Streptomyces hygroscopicus SF-1293 strain was cultured in a preculture medium (2.0% soluble starch, 1.0% polypeptone).
%, meat extract 0.3%, dipotassium phosphate 0.05%, PH7.0). This at 28℃
Cultured with shaking for 24 hours, and subcultured into 80 ml of the same medium for 28 hours.
The seedlings cultured at ℃ for 24 hours were used as seeds for jar fermenters. Jiahua Armenter contains 4.4% glucose, 2.25% sungrain, 3.5% wheat germ,
The above seed mother was inoculated into production medium 4.0 containing 0.1% monopotassium phosphate, and cultured with aeration and stirring at 28°C.
A culture solution cultured with aeration and stirring for 144 hours was adjusted to pH 2.0, and bacterial cells were removed by centrifugation to obtain a culture solution with pH 2.0. This culture solution was developed with water using a column packed with 300 ml of Dowex 50W x 2 (H ⁺ type). The fraction containing the MP-102 substance was then washed with water using a column packed with 200 ml of Dowex 1x2 (CH ₃ COO ^- type) and eluted with 1N acetic acid. When the eluent fraction containing MP-102 substance was concentrated, crystals were precipitated. When this material was taken, 2.4 g of white crystals of substance MP-102 were obtained. Example 2 Under the same conditions as in Example 1, aerated agitation culture was carried out, and the culture solution was extracted over time and analyzed using an amino acid analyzer (Model MLC-703 manufactured by ATTO, retention time 11 minutes).
The amount of MP-101 substance produced was measured. The results are shown in the table.

[Table] A culture solution cultured with aeration and stirring for 216 hours was adjusted to pH 2.0, and bacterial cells were removed by centrifugation to obtain a culture solution with pH 2.0. Amino acid analysis revealed that the amount of MP-101 substance produced was 1320 μg/ml. The obtained culture solution was developed with water using a 300 ml Dowex 50W x 2 (H ⁺ type) column. Add the fraction containing MP-101 substance to 200 ml of Dowex 1 x 2.
After washing with water using a (CH ₃ COO ^- type) column,
Eluted with 0.3N acetic acid. When the eluent fraction containing MP-101 substance was concentrated, white crystals were precipitated. This material was taken, and 1.13 g of white crystals of MP-101 substance was obtained. Reference example 1 610 mg of MP-101 substance was suspended in 20 ml of water,
It was dissolved at pH 7.5 with 1N NaOH. To this was added 1300 mg of di-t-butyl dicarbonate, and the mixture was allowed to react under stirring for 7 hours while maintaining the pH at 7.5 to 8.0 at room temperature. The reaction solution was extracted with 20 ml of ethyl acetate to remove excess reagent, and the aqueous layer was extracted with 2N hydrochloric acid under cooling.
The pH was adjusted to 2.0 and the mixture was directly freeze-dried. The residue was extracted with 30 ml of acetone, and after the extraction, an ether solution of diazomethane was added and left at 10°C for 2 hours. The reaction solution was dried to give 2-t-butoxycarbonylamino-4
810 mg of an oily substance of -(methoxy)phosphinoyl-butyric acid methyl ester was obtained.・Elemental analysis value: C44.53, H7.58, N4.17, P9.76% ・Theoretical value as molecular formula C ₁₁ H ₂₂ NO ₆ P:
C44.75, H7.46, N4.75, O32.54, P10.51%.・ NMR (CDCl ₃ ) δppm: 1.47 (9H, s, -
COOC( _CH3 ) ₃ ), 1.7-2.2(4H, m, _H3 , _H4 ),
3.78 (3H, s, −COOC H ₃ ), 3.80 (3H, d, J
= 11Hz, P-OC H ₃ ), 7.1 (1H, d, J = 536
Hz, P- H ). 400 mg of this product was dissolved in 6 ml of toluene, 34 mg of sodium hydride was added, and the mixture was stirred at room temperature for 30 minutes and at 70°C for 1 hour. Then, 400 mg of methyl iodide was added, and the mixture was vigorously stirred at 60 to 70°C for 3 hours. After cooling, the reaction solution was neutralized with acetic acid and concentrated under reduced pressure. The residue was dissolved in 6 ml of 3N hydrochloric acid, hydrolyzed at 70°C for 2 hours, concentrated to dryness, dissolved in 5 ml of water, and 3N
After adjusting the pH to 2.5 with caustic soda, dowex 1
×2 (AC type) 0.5N applied to a column packed with 30ml
It was developed with acetic acid. When the fraction containing the target product was concentrated and crystallized from water-ethanol, 2-amino-4-(hydroxy)(methyl)
130 mg of white crystals of phosphinoyl-butyric acid were obtained. Reference example 2 620 mg of MP-102 substance was suspended in 30 ml of water, dissolved with 1N caustic soda to pH 8, and di-t-butyl-
Add 600mg of dicarbonate and adjust the pH to 8 at room temperature.
The reaction was allowed to proceed for 5 hours while stirring. The reaction solution was washed with 30 ml of ethyl acetate to remove excess reagent, and the aqueous layer was adjusted to pH 2 and extracted twice with 30 ml of a mixture of n-butanol and ethyl acetate (1:3).
After concentrating the extract under reduced pressure, the residue was mixed with 5 ml of methanol.
Add an ethereal solution of diazomethane to 15
It was left at ℃ for 3 hours. When the reaction solution was dried, 2-t-butoxycarbonylamino-4-
(methoxy)phosphinoyl-butyryl-L-
780 mg of white powder of alanyl-L-alanine methyl ester was obtained.・Elemental analysis value: C46.23, H7.46, N9.35, P6.92% ・Theoretical value as molecular formula C ₁₇ H ₃₂ N ₃ O ₈ P:
C46.68, H7.32, N9.61, O29.29, P7.09%.・ NMR (CDCl ₃ ) δppm: 1.45 (9H, s, -
COOC( _CH3 ) ₃ ), 1.6-2.2(4H, m, _H3 , _H4 ),
3.73 (3H, s, −COOC H ₃ ), 3.82 (3H, d, J
= 11.2Hz, P-OC H ₃ ), 7.2 (1H, d, J = 538
Hz, P- H ) 440 mg of this product was dissolved in 5 ml of N,N-dimethylformamide, 30 mg of sodium hydride was added, and the mixture was stirred at room temperature for 40 minutes and at 60°C for 2 hours. Next, 700 mg of methyl iodide was added, and the mixture was heated at 50 to 60℃ for 2 hours.
Stir vigorously for hours. After cooling, the reaction solution was neutralized with acetic acid and concentrated under reduced pressure. Dissolve the residue in 5 ml of acetonitrile and add trimethylsilyl iodide.
After adding 600 mg and stirring at 50°C for 2 hours, the mixture was concentrated to dryness under reduced pressure. This product was then dissolved in 5 ml of trifluoroacetic acid, left at room temperature for 1 hour, dried again, dissolved in 5 ml of water, and diluted with 3N caustic soda to pH 2.
The column was eluted with 0.3N acetic acid using a column packed with 40ml of Dowex 1x2 (AC type). When the fraction containing the target product was concentrated,
153 mg of SF-1293 substance was obtained. Reference Example 3 600 mg of MP-101 substance was suspended in 30 ml of methanol, and the same molar amount of pyridine was added and dissolved. Add 2 ml of trifluoroacetic acid methyl ester to this,
The mixture was stirred at room temperature for 15 hours while maintaining the liquid slightly alkaline with pyridine. The reaction solution was dried, dissolved in 5 ml of water, adjusted to pH 2 with 2N hydrochloric acid, and directly freeze-dried. The residue was extracted with 25 ml of acetone, and an ether solution of diazomethane was added to the extract and left at 15°C for 3 hours. The reaction solution was dried, dissolved in 30 ml of ethyl acetate, and washed with 10 ml of water. The ethyl acetate layer was concentrated to obtain 790 mg of an oily substance of 2-trifluoroacetylamino-4-(methoxy)phosphinoyl-butyric acid methyl ester.・Elemental analysis value: C32.75, H4.53, N4.67, P9.23% ・Theoretical value as molecular formula C ₈ H ₁₃ NO ₅ PF ₃ :
C32.99, H4.47, N4.81, O27.49, P10.65% ・NMR (CDCl ₃ ) δppm: 1.65-2.3 (4H, m,
H ₃ , H ₄ ), 3.76 (3H, s, −COOC H ₃ ), 3.81
(3H, d, J = 11Hz, P-OC H ₃ ), 7.08 (1H,
d, J=540Hz, P- H ). Then, 360 mg of this product was treated in the same manner as in Example 1, resulting in 2-amino-4-(hydroxy)
White crystals of (methyl)phosphinoyl-butyric acid 83
mg was obtained.

[Brief explanation of drawings]

FIG. 1 is an infrared absorption spectrum diagram of the MP-101 substance, and FIG. 2 is an infrared absorption spectrum diagram of the MP-102 substance.

Claims (1)

[Claims] 1. General formula (R is -OH or [Formula] group) 2-amino-4-(hydroxy)phosphinoyl-butyric acid and 2-amino-4-(hydroxy)phosphinoyl-butyryl-L-
Alanyl-L-alanine. 2 2-Amino-4 belonging to the genus Streptomyces
-(Hydroxy)phosphinoyl-butyric acid and 2-amino-4-(hydroxy)phosphinoyl-butyryl-L-alanyl-L-alanine producing strains were cultured in a nutrient medium under aerobic conditions, and the culture was extracted with 2- Amino-4-(hydroxy)phosphinoyl-butyric acid and 2-amino-4-(hydroxy)phosphinoyl-butyryl-L-
A method for producing 2-amino-4-(hydroxy)phosphinoyl-butyric acid and 2-amino-4-(hydroxy)phosphinoyl-butyryl-L-alanyl-L-alanine, which comprises collecting alanyl-L-alanine. 3. The manufacturing method according to claim 2, which uses a nutrient medium with a low cobalt ion content.