DE4215738A1 - Novel microorganism Streptomyces albus R2374 - is e.g 5-0-sulphamoyl-2-chloro adenosine which can be used as a herbicide - Google Patents

Abstract

(A) Streptomyces albus R2374 and its mutants capable of preparing 5'-O-sulphamoyl-2-chloroadenosine of formula (I) are new. (B) The use of (I) as a herbicide is new. USE/ADVANTAGE - (I) is a known nucleoside antibiotic which is now found to be effective as a herbicide. (I) can be used on seeds and on the plants. In examples, (I) was found to be completely effective against Sinapis alba and Stellaria media in both a pre-emergent test using 4kg/ha.

Description

The present invention relates to the novel microorganism Streptomyces albus R 2374 and derived mutants, process for its preparation and its Use for the preparation of 5'-O-sulfamoyl-2-chloroadenosine of the formula (I)

the use of the compound of formula I as a herbicide and new herbicidal agents.

Derivatives of the antibacterially active 5'-O-sulfamoyl-2-chloradenosine (I) are by chemical synthesis has been made, and a natural derivative has been found under the Asnamycin described (JP-A-62 1 08 896, JP-A-62 0 56 500 and JP-A-62 1 08 897). 5'-O-sulfamoyl-2-chloroadenosine (I) is in a fermentative process of a culture of Streptomyces rishiriensis and as a nucleoside antibiotic AT-265 (E. Takahashi Journal of Antibiotics (1982) 35, 939-947).

5'-O-sulfamoyl-2-chloroadenosine (I) is also obtained by chemical synthesis from chloroadenosine and chlorosulfonic acid amide (H.A. Maguire et al., Journal of Medicinal Chemistry (1978) 21 520-525).

It has now been found that 5'-O-sulfamoyl-2-chloradenosine (I) from a hitherto unknown, from a soil sample from Jaipur (India) isolated, Streptomyces albus R 2374; from now on also called trunk R 2374) is produced. Due to the taxonomic analysis ensures that it is in the inventive Strain R 2374 to one different from the previously known Streptomyces rishiriensis  Tribe acts.

The new strain R 2374 is available from the Deutsche Sammlung für Mikroorganismen, Mascheroderweg 1B, D-3300 Braunschweig, Federal Republic of Germany on 5 February 1991 under the accession number DSM 6407 under the name Streptomyces albus R 2374 deposited.

It has been found that 5'-O-sulfamoyl-2-chloroadenosine of the formula (I)

herbicidally active. The invention thus also relates to the use of 5'-O-sulfamoyl-2-chloroadenosine (I) as a herbicide method of combating Harmful plants and new herbicidal compositions containing this compound.

5'-O-sulfamoyl-2-chloroadenosine (I) can be prepared by cultivating the new microorganism Streptomyces albus R 2374 in a nutrient medium under aerobic Conditions. The following is an example of the preparation of 5'-O-sulfamoyl 2-chloroadenosine (I) indicated. This method relates to the fermentation of the strain R 2374 under aerobic conditions in an aqueous environment with assimilable Nitrogen and carbon sources. The isolation of 5'-O-sulfamoyl-2-chloroadenosine (I) from the culture is also described.

originating Description

The new strain R 2374 has the following microbiological characteristics:

Morphological features

There is abundant aerial mycelium and spores formed on media such as oatmeal agar and Yeast-malt agar. The aerial mycelium is simply branched and does not form any tangles. Closed, compact spirals are formed at the tips of the aerial hyphae. The Electron microscopic examination shows that the spore surface is smooth  the spores have no processes, and the spore chains are tight, closed Spirals are arranged. The spores have an elliptical to oval shape, and 10 or more spores are usually linked. What the color of the mycelium is concerned, it is after about 10 days, a white-gray aerial mycelium and a light brown Substrate mycelium firm.

Physiological features pigmentation

Formation of soluble pigments is neither synthetic nor organic Observed media, that is, the strain R 2374 is not chromogenic. On Peptone Iron Agar (PIA) strain R 2374 does not form melanin.

C source utilization

Strain R 2374 grows well and sporulates using the following C sources: D-fructose, D-glucose, D-maltose, mannitol, D-sucrose, sorbitol, raffinose.

With L-arabinose as the C source, strain R 2374 grows poorly and hardly spores.

With DL-xylose as the C source, strain R 2374 does not grow.

Antibiotic resistance

In Waksman's plate diffusion test, solutions become more antibiotically active Agents (concentration: 1 mg / ml, 0.3 mg / ml, 0.1 mg / ml) 20 μl on paper rondels with 6 mm diameter pipetted. The paper rondelles are placed on the surface of yeast Malt agar plates seeded with strain R 2374 are placed. After hatching the Plates for 4-5 days at 28 ° C, the growth inhibition in the form of inhibitors farms are observed.

The growth of strain R 2374 at 0.1 mg / ml is inhibited by: novobiocin, Tobramycin.

A partial Wachtumhibemmung the strain R 2374 cause: chloramphenicol, Kanamycin, tetracycline.

The growth of strain R 2374 at 1 mg / ml is not inhibited by: ampicillin,  Carbonicillin, gentamycin, neomycin, spectinomycin, streptomycin, sulfametazine, Trimethoprim.

metabolite production

In addition to the herbicidal active ingredient CGA 251734, the strain R 2374 also still the metabolites 2-chloroadenosine and Albonoursin ago.

taxonomy

If one compares the above features of the strain R 2374 with those of the species Cluster of Streptomyces albus, as described by Hütter in "Systematics of Streptomycetes" R.Hütter, Bibliotheca Microbiologica Fasc.6 (S.Karger, Basel / New York 1967) ben, it can be seen that five features apply to this species cluster:

• 1. The oval spores have a smooth surface.
• 2. The aerial mycelium is white-gray (type niveus).
• 3. The spore chains form tight spirals (Spira type A).
• 4. No melanin is formed on PI agar.
• 5. The substrate mycelium is light brown.

In other features, such as. B. C-source utilization and metabolite production under However, R 2374 differs from Hütter's description. That's why we call R 2374 as Streptomyces albus R 2374. The strain R 2374 has properties that are quite may vary, as commonly observed in other streptomycetes. So can the Strain R 2374 produce a variant or a mutant when mixed with different Mutagens such as ultraviolet radiation, X-rays, radioactive radiation, high-frequency electromagnetic fields and chemical mutagens. Any natural or artificial variant or mutant of strain R 2374 may be in accordance with this invention for the preparation of 5'-O-sulfamoyl-2-chloroadenosine Formula (I) can be used.  

fermentation

A stock strain of strain R 2374 is used for inoculation of 12 500 ml shake flask with puncture consisting of 100 ml of aqueous medium consisting of Glucose 22 g / liter, Oxoid Lab. Lemco 5 g / liter, Peptone-C 5 g / liter, yeast extract 0.5 g / liter, Casitone 3 g / liter, sodium chloride 1.5 g / liter contained, at a pH of 7.3 (before sterilization). The fermentation of these precultures takes place on the shaking table 250 rpm, 28 ° C during 48h. These precultures were used for inoculation from a fermenter containing 30 liters of aqueous medium consisting of mannitol 20 g / liter and defatted soya flour 20 g / liter, at a pH of 7.5 (before sterilization). The fermentation is carried out at 28 ° C, aeration rate of 1 fermenter volume per Minute and a stirrer speed of 200 rpm for 116 h.

Work-up and isolation

After each work-up and separation step, the resulting fractions become herbicidal activity against Nasturtium officinalis and Agrostis tenuis tested. Both Plants are sown on vermiculite, which is previously soaked in sample solutions had been. The assessment took place 10 days after sowing.

27 liters of culture broth are adjusted to pH 7.0 with 5N H ₂ SO ₄ , stirred with 1.5 kg of washed diatomaceous earth (Hyflo Superflow) and filtered on a flow-through centrifuge (Heine-703). The clear culture filtrate is first concentrated by vacuum distillation of water in a rotary evaporator at a water bath temperature of 45 ° C to 2 liters and then lyophilized. 85 g of herbicidal solid material are obtained. This material is taken up in a mixture of 400 ml of methanol and 100 ml of water and extracted three times with 500 ml of heptane. The heptane phases are discarded, as well as the resulting during the filtration of the methanol phase filter residue. The filtrate is then gently evaporated in vacuo. 68 g of herbicidal solid material are isolated. This is taken up in a little methanol and chromatographed on a column, 7.5 cm x 78 cm (3.5 liters) with 80% aqueous methanol coated separation material, Sephadex LH-20 (Pharmacia, Uppsala, Sweden) with 80% aqueous methanol, wherein a herbicidal Fraction of 1.7 g is obtained. 700 mg of this material are then chromatographed on a column, 2.5 cm x 82 cm (400 ml) methanol-grown Sephadex LH-20, with methanol. This is isolated 210 mg herbicidal material. This material is separated again on the same column under analogous conditions. The available herbicidal material of 165 mg is further purified by high-pressure liquid chromatography (HPLC), on a reversed-phase separation material Lichroprep RP-8 (15-25 μ) (E. Merck, Darmstadt, Germany) in a steel column (20 mm · 250 mm). The eluent used is a gradient of 100% water to 100% methanol. In this case, 100 mg of highly enriched, herbicidal material are obtained. From this it is possible to obtain by HPLC on reversed-phase separation material Lichroprep RP-18 (15-25 μ) (from E. Merck) in analogous steel columns and under analogous elution conditions 92 mg of pure herbicidal active ingredient CGA 251734.

The compound of the formula I is herbicidally active. The active ingredient is used as herbicides in usually with application rates of 0.001 to 5 kg / ha, in particular 0.05 to 4 kg / ha, used successfully. The dosage required for the desired effect can be determined by experiments. It depends on the type of effect that Stage of development of crop and weed, as well as application (location, Time, procedure) and can, due to these parameters, within wide ranges vary.

At lower application rates, the compound of formula I is characterized by selective herbicidal properties that make them excellent for use in crops of Crops, especially in cereals, cotton, soy, rapeseed, corn and rice enable.

Furthermore, the compound of formula I is also suitable for use in the treatment of seed. Here, both the seed before sowing with the active ingredient or a treated or stained the formulation containing the active substance, as well as the Active ingredient be applied during sowing in the seed furrow.

i) seed dressing

a) seed dressing with a formulated as a wettable powder by shaking in a container until evenly distributed on the seed surface (dry dressing). It uses up to 4 g of the active ingredient of the formula I (in a 50% Formulation: up to 8.0 g of spray powder) per 1 kg of seed.

b) dressing the seeds with an emulsion concentrate of the active ingredient or with a aqueous solution of formulated as a wettable powder active ingredient of the formula I after Method a) (wet pickling).  

c) dressing by dipping the seed in a broth containing up to 1000 ppm of the active ingredient Formula I for 1 to 72 hours and optionally subsequent drying of Seeds (dip dressing, seed soaking).

The dressing of the seeds or the treatment of the germinated seedling are natural the preferred methods of application, because the drug treatment full is always focused on the target culture. As a rule, 4.0 g to 0.001 g are used Active substance per 1 kg of seed, depending on the methodology, which includes the additive other active substances or micronutrients, from the may vary up or down (repeat stain).

The compound of formula I is in unchanged form, as from the synthesis or the Fermentation available, or preferably with the usual in formulation technology Tools used and are therefore z. B. emulsifiable concentrates, directly sprayable or dilutable solutions, dilute emulsions, wettable powders, soluble powders, dusts, granules, and encapsulations in z. B. polymers Materials processed in a known manner. The application methods such as spraying, Nebulizing, dusting, wetting, scattering or pouring will be the same as the type of Means chosen according to the desired goals and the given circumstances.

The formulations, d. H. the active ingredient of the formula I and optionally one or several solid or liquid additives containing agents, preparations or Compositions are prepared in known manner, e.g. B. by intimate Ver mix and / or grinding the active ingredients with extenders, such. B. with solution agents, solid carriers and optionally surface-active compounds (Surfactants).

Suitable solvents are: Aromatic hydrocarbons, in particular the fractions C₈ to C₁₂, such as mixtures of alkylbenzenes, z. B. xylene mixtures or alkylated naphthalenes; aliphatic and cycloaliphatic hydrocarbons such Paraffins, cyclohexane or tetrahydronaphthalene; Alcohols, such as ethanol, propanol or butanol; Glycols and their ethers and esters, such as propylene glycol or dipropylene glycol ether, Ketones such as cyclohexanone, isophorone or diacetone alcohol, strong polar Solvents such as N-methyl-2-pyrrolidone, dimethyl sulfoxide or water; vegetable oils and their esters, such as rapeseed, castor oil or soybean oil; optionally also silicone oils.  

As solid carriers, for. As for dusts and dispersible powders are in the Usually natural minerals used, such as calcite, talc, kaolin, montmorillonite or attapulgite. To improve the physical properties can also be high disperse silica or highly dispersed absorbent polymers are added. When granulated, adsorptive granules are porous types such. B. pumice, brick break, sepiolite or bentonite, as non-sorptive carrier materials, for. B. calcite or sand in Question. In addition, a variety of pregranulated inorganic materials or organic nature such as in particular dolomite or crushed plant residues be used.

As surface-active compounds come, depending on the nature of the formulated Active ingredient of the formula I nonionic, cationic and / or anionic surfactants with good Emulsifying, dispersing and wetting properties into consideration. Among surfactants are also To understand surfactant mixtures.

Suitable anionic surfactants can be both so-called. Water-soluble soaps such as water Liche synthetic surface-active compounds.

As soaps are the alkali, alkaline earth or optionally substituted ammonium salts of higher fatty acids (C ₁₀ -C ₂₂ ), such as. As the sodium or potassium salts of the oil or stearic acid, or called by natural fatty acid mixtures, the z. B. can be obtained from coconut or tallow oil. Furthermore, the fatty acid methyl taurine salts should be mentioned.

More often, however, so-called synthetic surfactants are used, especially fatty alcohol sulfonates, fatty alcohol sulfates, sulfonated benzimidazole derivatives or alkylarylsulfonates.

The fatty alcohol sulfonates or sulfates are generally available as alkali, alkaline earth or optionally substituted ammonium salts and have an alkyl radical with 8 to 22 carbon atoms, wherein alkyl also includes the alkyl moiety of acyl radicals, e.g. B. the Na or Ca salt of lignosulfonic acid, dodecylsulfuric acid ester or a fatty alcohol sulfate mixtures made from natural fatty acids. Also belong here the salts of sulfuric acid esters and sulfonic acids of fatty alcohol-ethylene oxide Adducts. The sulfonated benzimidazole derivatives preferably contain 2 sulfonic acid groups and a fatty acid residue with 8-22 carbon atoms. Alkylarylsulfonates are z. For example, the na  Ca or triethanolamine salts of dodecylbenzenesulfonic acid, the dibutylnaphthalene sulfonic acid, or a naphthalenesulfonic acid-formaldehyde condensation product.

Furthermore, corresponding phosphates such. B. salts of phosphoric acid ester a p-nonylphenol (4-14) -Ethylenoxid adduct or phospholipids in question.

Nonionic surfactants are primarily polyglycol ether derivatives of alipha or cycloaliphatic alcohols, saturated or unsaturated fatty acids and Alkylphenols in question, the 3 to 30 glycol ether groups and 8 to 20 carbon atoms in the (aliphatic) hydrocarbon radical and 6 to 18 carbon atoms in the alkyl radical of May contain alkylphenols.

Other suitable nonionic surfactants are the water-soluble, 20 to 250 ethylene glycol ether groups and 10 to 100 propylene glycol ether groups, containing poly ethylene oxide adducts with polypropylene glycol, ethylene diaminopolypropylene glycol and Alkylpolypropylene glycol having 1 to 10 carbon atoms in the alkyl chain. The gene The compounds usually contain from 1 to 5 ethylene per propylene glycol unit glycol units.

Examples of nonionic surfactants are nonylphenolpolyethoxyethanols, castor oil polyglycol ethers, polypropylene-polyethylene oxide adducts, tributylphenoxypolyethoxy ethanol, polyethylene glycol and octylphenoxypolyethoxyethanol mentioned.

Further, fatty acid esters of polyoxyethylene sorbitan such as polyoxyethylene also come sorbitan trioleate.

The cationic surfactants are mainly quaternary ammonium salts, which contain as N-substituent at least one alkyl radical having 8 to 22 carbon atoms and as further substituents lower, optionally halogenated alkyl, benzyl or having lower hydroxyalkyl radicals. The salts are preferably in the form of halides, Methyl sulfates or ethyl sulfates, e.g. For example, the stearyltrimethylammonium chloride or Benzyldi (2-chloroethyl) ethylammonium bromide.

The surfactants commonly used in formulation technology are u. a. in the following public described:

• "McCutcheon's Detergents and Emulsifiers Annual", McPublishing Corp., Glen Rock, New Jersey, 1988.
• M. and J. Ash, "Encyclopedia of Surfactants", Vol. I-III, Chemical Publishing Co., New York, 1980-1981.
• - Dr. Helmut Stache "Surfactant Paperback", Carl Hanser Verlag, Munich / Vienna 1981.

The pesticidal preparations usually contain 0.1 to 99%, in particular 0.1 to 95%, active ingredient of the formula I, 1 to 99% of a solid or liquid additive and 0 to 25%, especially 0.1 to 25% of a surfactant.

While as a commodity rather concentrated funds are preferred, uses the Consumers usually diluted funds.

The agents may also contain other additives such as stabilizers z. B. optionally Epoxidized vegetable oils (epoxidized coconut oil, rapeseed oil or soybean oil), defoamers, z. Silicone oil, preservatives, viscosity regulators, binders, adhesives as well as fertilizers or other active ingredients for achieving special effects.

In particular, preferred formulations are composed as follows: (% = Weight percent)

Emulsifiable concentrates: Active ingredient: 1 to 90%, preferably 5 to 20 Surfactant: 1 to 30%, preferably 10 to 20% liquid vehicle: 5 to 94%, preferably 70 to 85%

dusts: Active ingredient: 0.1 to 10%, preferably 0.1 to 1% solid carrier: 99.9 to 90%, preferably 99.9 to 99%

Suspension concentrates Active ingredient: 5 to 75%, preferably 10 to 50% Water: 94 to 24%, preferably 88 to 30% Surfactant: 1 to 40, preferably 2 to 30%

Wettable powders: Active ingredient: 0.5 to 90%, preferably 1 to 80% Surfactant: 0.5 to 20%, preferably 1 to 15% solid carrier material: 5 to 95%, preferably 15 to 90%

granules: Active ingredient: 0.5 to 30%, preferably 3 to 15% solid carrier: 99.5 to 70%, preferably 97 to 85%

formulation Examples

Example F1: Formulation Examples of the Active Ingredient of the Formula I.

(% = Weight percent)

The active ingredient is mixed well with the additives and in a suitable mill well ground. One receives spraying powders, which become with water to suspensions each dilute the desired concentration.

From this concentrate, by diluting with water emulsions of any desired Concentration are produced.

Ready-to-use dusts are obtained by mixing the active ingredient with the carrier and grinding on a suitable mill.

The active ingredient is mixed with the additives, ground and moistened with water. This mixture is extruded and then dried in a stream of air.

e) serving granules Active substance of the formula I | 3% Polyethylene glycol (MG 200) 3% kaolin 94%

The finely ground active substance is mixed with polyethylene glycol in a mixer moistened kaolin applied evenly. In this way one contains dust-free Coated granules.  

The finely ground active substance is intimately mixed with the additives. You get like that a suspension concentrate from which by dilution with water suspensions Any desired concentration can be produced.

g) saline Active substance of the formula I | 5% isopropylamine 1% Octylphenol polyethylene glycol ether (78 moles of AeO) 3% water 91%

H. Herbicidal activity of 5'-O-sulfamoyl-2-chloroadenosine (I) example H1 Pre-emergent herbicidal action

In the greenhouse, immediately after sowing, the test plants are placed in seed trays the earth's surface with an aqueous spray mixture corresponding to an application rate of 4 kg of active substance / hectare treated. The seed trays are in the greenhouse at 22-25 ° C and 50-70% relative humidity maintained.

After 3 weeks, the herbicidal action with a nine-stage (1 = complete Damage, 9 = no effect) Boniturschema compared with an untreated Control group evaluated. The individual results are summarized in Table 1:  

Table 1: Herbicidal action of 5'-O-sulfamoyl-2-chloroadenosine (I) at an application rate of 4 [kg / ha] plant Boniturnote Sinapis alba 1 Stellaria media 1

Example H2 Post-emergent herbicidal action (contact herbicide)

A number of weeds, both monocots and dicotyledons, are released after emergence (in the 4- to 6-leaf stage) with an aqueous active ingredient dispersion in a dosage of 4 kg of active substance per hectare sprayed on the plants and this at 24 ° -26 ° C and 45-60% relative humidity maintained. 15 days after the treatment will be the Herbicidal action with a nine-step (1 = complete damage 9, = no Effect) rating scheme compared with an untreated control group. The individual results are summarized in Table 2:

Table 2: Herbicidal action of 5'-O-sulfamoyl-2-chloroadenosine (I) at an application rate of 4 [kg / ha] plant Boniturnote Sinapis alba 1 Stellaria media 1

Structure elucidation of 5'-O-sulfamoyl-2-chloroadenosine (I)

The elucidation of the structure of the isolated herbicidal active ingredient by means of its spectroscopic properties, notably by evaluation of Mass spectra, NMR spectra, UV spectrum and IR spectrum.

mass spectroscopy

The FAB mass spectrum of 5'-O-sulfamoyl-2-chloroadenosine (I) shows, originating from the two chlorine isotopes 35 and 37, the molecular weights 380 and 382.  

NMR spectroscopy

The test results of 5'-O-sulfamoyl-2-chloradenosine (I) with respect to its 1H NMR properties (500 MHz, methanol-d4) are summarized in Table 1. Where:

δ = chemical shift in ppm
J = coupling in Hz
s = singlet d = doublet t = triplet m = multiplet

Table 1

Infrared Spectroscopy

The IR spectrum of 5'-O-sulfamoyl-2-chloroadenosine (I) is recorded in KBr and shows at 3400 cm ^-1 a broad, strong band indicating the presence of several OH groups. Further, the three bands are 1650 cm ^-1 (strong), 1600 cm ^-1 (strong) and 1570 cm ^-1 (medium) conspicuous, and 1355 cm ^-1 (strong) and 1165 cm ^-1 (strong).

UV spectroscopy

5'-O-Sulfanloyl-2-chloroadenosine (I) shows in water an absorption maximum at 264 nm (ε = 1.3 × 10 ⁴ ).

Claims (10)

1. Streptomyces albus R 2374 and its for the preparation of 5'-O-sulfanloyl-2-chloroadenosine of the formula (I) enabled mutants. 2. Streptomyces albus R 2374 according to claim 1. 3. The use of Streptomyces albus R 2374 or its for the preparation of 5'-O-sulfamoyl-2-chloroadenosine of the formula (I) capable mutants for the preparation of 5'-O-sulfamoyl-2-chloradenosin of the formula (I). 4. Process for the preparation of 5'-O-sulfamoyl-2-chloroadenosine of the formula (I) characterized in that a strain of the microorganism Streptomyces albus R 2374 or a strain derived therefrom according to claim 1 or 2 is cultured under anerobic conditions and the 5'-O-sulfamoyl-2-chloroadenosine of the formula (I) is isolated from the culture broth. 5. A herbicidal composition containing 5'-O-sulfamoyl-2-chloroadenosine of the formula (I) and an adjuvant or carrier. 6. A method for controlling undesired plant growth, characterized in that the harmful plant or its habitat is a herbicidally effective amount of 5'-O-sulfamoyl-2-chloradenosin of the formula (I) allow to act. 7. The method of claim 6 for the selective control of weeds in Crops of useful plants. 8. The use of 5'-O-sulfamoyl-2-chloroadenosine of the formula (I) for controlling harmful plants. The use of claim 8 for the selective control of weeds in Crops of useful plants. 10. Seed of useful plants, which is treated with a herbicidally effective amount of 5'-O-sulfamoyl-2-chloroadenosine of the formula (I) has been treated.