DE10361201A1 - New biologically active oligopeptides with modified termini, useful as insecticides, acaricides, antibacterials and antitumor agents, especially neoefrapeptins from Geotrichum candidum - Google Patents

Abstract

The present invention relates to new oligopeptides with biological activity, in free form or in salt form, in particular those which have both a modified N-terminal amino acid residue and a modified C-terminal amino acid residue, preferably corresponding pentadecapeptides. The present invention further relates to a method for producing such peptides and their use, biologically active agents whose active ingredient is selected from these peptides, a method for producing such agents and their use, plant propagation material which has been treated with such agents, and a Pest control procedures.

Description

The present invention relates to new oligopeptides with biological activity, in free form or in Salt form, especially those that have both a modified N-terminal amino acid residue as well as a modified C-terminal amino acid residue have, preferably corresponding pentadecapeptides. The present The invention further relates to a method for producing such Peptides and their use, biologically active agents, their Active ingredient selected from these peptides is a process for Production of such agents and their use, plant propagation material, that is treated with such agents, and a method for combating Pests.

ersetzt ist.The oligopeptides according to the invention are, in particular, those pentadecapeptides in which a hydrogen atom of the amino group of the N-terminal amino acid residue is replaced by an acetyl group and in which the hydroxy partial structure of the carboxy group of the C-terminal amino acid residue is replaced by a substituted amino group of the formula

is replaced.

The oligopeptides according to the invention are preferred those pentadecapeptides in which at least one of the 15 amino acid units the α-amino acid is 1-aminocyclopropane carboxylic acid.

The oligopeptides according to the present In particular preferred pentadecapeptides are invention, which have the composition listed in Table 1.

Table 1: Composition of neoefrapeptin A, Neoefrapeptin B, Neoefrapeptin C, Neoefrapeptin D, Neoefrapeptin E, Neoefrapeptin F and Neoefrapeptin I.

The abbreviations used in Table 1 have the following meanings: Ac acetyl Acc 1-aminocyclopropanecarboxylic acid Aib 2-aminoisobutyric acid [α-aminoisobutyric acid] β-Ala β-alanine [3-aminopropionic acid] Gly glycine Iva Isovalin [2-amino-2-methylbutyric acid] Leu leucine pip Pipecolic acid [piperidine-2-carboxylic acid] Say Substituted amino group of formula I.

In Table 1, for each of the first five listed compounds, follow the trivial name given in the first column and the acetyl group listed in the second column (which replaces a hydrogen atom of the amino group of the N-terminal amino acid residue) from left to right Amino acid building blocks of the respective compound, starting with the N-terminal building block and ending with the C-terminal building block, and finally in the last column completed by the substituted amino group of the formula I (which replaces the hydroxy partial structure of the carboxy group of the C-terminal amino acid residue).

Neoefrapeptin A The formula of neoefrapeptin A is given below as an example of the neoefrapeptins A to E listed in Table 1:

Neoefrapeptin A

The formulas of Neoefrapeptin B, Neoefrapeptin C, Neoefrapeptin D and Neoefrapeptin E are derived from the above for Neoefrapeptin A formula indicated by the fact that of the three amino acid building blocks in peptide positions 4, 5 and 10 (which are found in neoefrapeptin A Iva, Aib and Aib are located), counting the peptide positions by definition at the N-terminal amino acid residue begins, either an Aib block by an Iva block (in In the case of neoefrapeptin B and in the case of neoefrapeptin C) or an Iva building block through an Aib building block (in the case of neoefrapeptin D) is replaced or (in the case of neoefrapeptin E) both Aib building blocks in neoefrapeptin A can be replaced by Iva building blocks.

Neoefrapeptin F is an isomer of Neoefrapeptin A. Neoefrapeptin I is an isomer of Neoefrapeptin B.

Neoefrapeptin is preferred according to the invention A. Also according to the invention Neoefrapeptin B is preferred. Also preferred according to the invention is neoefrapeptin C. Neoefrapeptin is also preferred according to the invention D. Also according to the invention Neoefrapeptin E is preferred. According to the invention, it is very particularly preferred is neoefrapeptin A.

Also an object of the present Invention are mixtures of two or more than two of the oligopeptides according to the invention.

Also an object of the present Invention are salt forms of neoefrapeptins, e.g. B. with chloride, Acetate, trifluoroacetate, sulfate, carbonate or other anions.

The for the oligopeptides according to the invention elected Trivial term "Neoefrapeptine" is based on the Trivial name "Efrapeptine" known from the literature, which there for oligopeptides with a certain structural relationship to the oligopeptides according to the invention is used.

In the literature, certain oligopeptides as active ingredients in pesticides proposed. The biological properties of these known compounds capital in the field of pest control, however not fully satisfied, which is why there is a need to connect with pesticidal Properties, especially for the control of insects and representatives of the order Acarina to provide, this task according to the invention by providing the present oligopeptides solved becomes.

Another subject of the present The invention is a process for the preparation of the oligopeptides according to the invention, characterized in that the production strain SID 22780 (microorganism: Geotrichum candidum Link: Fr.) in a culture medium containing carbon sources, Nitrogen sources and mineral salts according to the usual microbiological procedure cultivated aerobically at pH values between 5.5 and 8 and the oligopeptides according to the invention from the fermentation broth by means of usual Process, e.g. B. by extraction and / or chromatography, if appropriate in combination with spectroscopic methods, isolated and if desired an according to the invention available Oligopeptide in free form in a salt form or one obtainable according to the invention Oligopeptide in salt form converted to the free form or in another salt form.

The SID 22780 strain was June 2002 under the number DSM 15068 at “DSMZ - German Collection for Microorganisms and cell cultures "(Mascheroder Weg 1b, D-38124 Braunschweig, Federal Republic of Germany) according to the Budapest Contract for international recognition of the deposit of microorganisms for the Filed for purposes of patent proceedings.

The oligopeptides according to the invention can by means of HPLC, ESI (+) - MS and MS / MS analyzed and as described below be characterized.

HPLC analyzes are done on a waters Alliance 2690 - device with one 996 diode array detector performed under the following experimental Conditions: Pillar: YMC-Pak ODS-AQ 120 Å, 5 μm, 125 mm × 2.0 mm plus guard column 10 mm; mobile phase A: water / TFA 100: 0.01; mobile phase B: Acetonitrile / TFA 100: 0.01; Flow: 0.5 ml / min .; Temperature: 40 ° C; linear Gradient: 0 min. 5% B, 1 min. 45% B, 15 min. 55% B; Injection: 5 μl of a solution in methanol; UV detection: 210 nm.

The ESI (+) - MS of neoefrapeptins gives only two fragments, e.g. B. m / z 943.6 and 689.5 in the case of Neoefrapeptin A. The MS / MS of these two fragments gives spectra with the fragments given below:

Neoefrapeptin A:

HPLC retention time: 8.7 minutes.

HR-MS (Cone voltage: 20 V): 689.503; 816.531 (M + H ²⁺ ); 943.556.MS/MS from 943.6 (cone voltage: 40 V; collision energy: 40 - 50 V):
943.6; 858.5; 705.5; 647.4; 620.5; 534.3; 449.4; 409 ,; 350.3; 296.3; 239.1; 154.1.

MS / MS of 689.6 (cone voltage: 100 V; collision energy: 25 - 35 V):
689.6; 671.6; 578.5; 565.5; 464.4; 436.4; 367.3; 339.3; 323.4; 254.2; 197.2; 169.2; 125.1.

Neoefrapeptin B and Neoefrapeptin C (mixture):

HPLC retention time: 9.8 minutes.

HR-MS (cone voltage: 20 V): 689,500; 823.536 (M + H ²⁺ ); 957.569.

MS / MS of 957.6 (Cone voltage: 40 V; collision energy: 40 - 50 V):
957.6; 872.5; 858.6; 719.6; 661.6; 647.6; 634.5; 548.3; 534.4; 449.3; 350.3; 310.3; 239.1; 154.1.

MS / MS of 689.5 (cone voltage: 100 V; collision energy: 25 - 35 V):
689.5; 671.6; 578.5; 565.5; 464.4; 436.4; 367.3; 339.3; 323.4; 254.2; 197.2; 169.2; 125.1.

Neoefrapeptin C:

MS / MS of 957.6 (cone voltage: 20 V; collision energy: 30 V):
957.6; 858.5; 775.5; 719.5; 647.4; 620.4; 534.3; 449.3; 350.2; 296.2; 239.1; 154.1.

MS / MS of 689.5: (Cone voltage: 20 V; collision energy: 50 V):
689.5; 671.5; 578.4; 464.3; 454.3; 436.4; 367.2; 339.2; 254.2; 197.1; 169.1; 125.1.

Neoefrapeptin D:

HPLC retention time: 8.5 minutes.

HR-MS (Cone voltage: 20 V): 689.504; 809.525 (M + H ²⁺ ); 929.543.

MS / MS of 929.6 (Cone voltage: 40 V; collision energy: 30 - 45 V):
929.6; 844.5; 761.5; 704.5; 691.5; 633.4; 606.4; 520.3; 435.3; 395.3; 350.3; 282.2; 239.2; 154.1.

MS / MS of 689.6 (Cone voltage: 40 V; collision energy: 45 - 60 V):
689.6; 671.6; 578.4; 565.5; 464.4; 436.4; 367.3; 339.3; 323.4; 254.2; 197.2; 169.2; 125.1.

Neoefrapeptin E:

HPLC retention time: 11.2 minutes.

HR-MS (Cone voltage: 20 V): 689.502; 830.546 (M + H ²⁺ ); 971.590.

MS / MS from 971.6 (Cone voltage: 40 V; collision energy: 30 - 45 V):
971.6; 872.5; 789.5; 733.5; 661.5; 634.4; 548.3; 449.3; 423.3; 350.2; 310.2; 239.1; 154.1.

MS / MS of 689.6 (cone voltage: 40 V; collision energy: 45 - 50 V):
689.6; 671.6; 578.5; 565.5; 464.4; 436.4; 367.3; 339.3; 323.4; 254.2; 197.2; 169.2; 125.1.

Neoefrapeptin F:

HPLC retention time: 11.5 minutes.

HR-MS (Cone voltage: 20 V): 689.506; 816.527 (M + H ²⁺ ); 943.560.

MS / MS of 943.6 (Cone voltage: 40 V; collision energy: 30 - 45 V):
943.6; 858.5; 775.5; 718.5; 705.5; 647.4; 620.5; 534.3; 449.4; 409.4; 350.3; 296.3; 239.1; 154.1.

MS / MS of 689.6 (cone voltage: 40 V; collision energy: 45 - 50 V):
689.6; 671.6; 565.5; 464.4; 436.4; 367.3; 339.3; 254.2; 197.2; 169.1; 125.1.

Neoefrapeptin I:

HPLC retention time: 12.8 minutes.

HR-MS (Cone voltage: 20 V): 957.562; 823.538 (M + H ²⁺ ); 689.498.

MS / MS of 957.6 (Cone voltage: 40 V; collision energy: 30 V):
957.6; 872.5; 719.4; 661.4; 634.4; 548.3; 449.3; 350.2; 310.2; 239.1; 154.1.

MS / MS of 689.5 (Cone voltage: 40 V; collision energy: 50 V):
689.5; 671.5; 565.4; 503.4; 464.3; 436.4; 367.2; 339.2; 254.1; 197.1; 169.1; 125.1.

Advantageously isolated or synthesized one each has the more biologically active isomer, e.g. B. enantiomers or Diastereomers, or mixture of isomers, e.g. B. mixture of enantiomers or Diastereomer mixture, provided that the individual components are different possess biological effectiveness.

The oligopeptides according to the invention are on the field of pest control cheaper warm-blooded, fish and plant tolerance preventive and / or even at low application concentrations curative active ingredients with a very favorable biocidal spectrum. The active substances according to the invention are against all or individual stages of development of normally sensitive, but also of resistant, animal pests, such as insects or Representatives of the order Acarina, effective. The insecticidal or acaricidal Effect of the invention Active ingredients can directly, d. H. in killing the pests which immediately or after some time, for example at a molt, occurs, or indirectly, e.g. B. in a reduced egg laying and / or Hatching rate, showing the good effect of a mortality rate of at least Corresponds to 50 to 60%.

The animal pests mentioned include, for example, those described in the European patent application EP-A-0 736 252 Page 5, line 55, to page 6, line 55. The pests mentioned there are therefore included by reference in the present subject matter of the invention.

With the active ingredients according to the invention can be found in particular on plants, especially on useful and ornamental plants in agriculture, horticulture and forestry, or on parts, like fruits, Blossoms, Foliage, stems, tubers or roots, such plants occurring pests of the mentioned Combat type, i.e. contain or destroy, with parts of the plant growing later against these pests protected become.

In particular come as target cultures Cereals such as wheat, barley, rye, oats, rice, corn or sorghum; beets, like sugar beet or fodder beet; Fruit, e.g. B. pome, stone and berry fruits, such as apples, pears, plums, Peaches, almonds, cherries or berries, e.g. B. strawberries, raspberries or blackberries; Legumes, like Beans, lentils, peas or soybeans; Oil fruits, such as rapeseed, mustard, poppy seeds, Olives, sunflowers, coconut, castor, cocoa or peanuts; Cucumber family, like pumpkins, Cucumbers or melons; Fiber plants, like Cotton, flax, hemp or jute; Citrus fruits such as oranges, lemons, Grapefruit or tangerines; Vegetables, such as spinach, lettuce, asparagus, cabbages, carrots, onions, tomatoes, potatoes or peppers; Laurel family, such as avocado, cinnamonium or camphor; as well as tobacco, nuts, coffee, Eggplants, Sugar cane, tea, pepper, grapevines, hops, banana plants, natural rubber plants and Ornamental plants.

Further areas of application of the active compounds according to the invention are the protection of stocks and storage and of material as well as in the hygiene sector in particular the protection of domestic and farm animals from pests of the type mentioned.

The invention therefore also relates Pesticides, how, depending on the desired goals and given circumstances to choose emulsifiable concentrates, suspension concentrates, directly sprayed or dilutable Solutions, spreadable Pastes, diluted Emulsions, wettable powders, soluble Powders, dispersible powders, wettable powders, dusts, Granules or encapsulations in polymeric substances, which at least one of the inventive Contain active ingredients.

The active ingredient is in these funds in pure form, a solid active ingredient e.g. B. in a special grain size, or preferably together with at least one of those customary in formulation technology Excipients such as extenders, e.g. B. solvents or solid carriers, or like surface active Compounds (surfactants) used.

Solid auxiliaries, solvents, stabilizers, "slow release" auxiliaries, dyes and optionally surface-active substances (surfactants) are used as formulation auxiliaries. All substances commonly used in crop protection agents can be used as carriers and auxiliaries. The auxiliaries such as solvents, solid carriers, surface-active compounds, nonionic surfactants, cationic surfactants, anionic surfactants and other auxiliaries in the agents used according to the invention are, for example, the same as those in EP-A-0 736 252 are described; they are included in the present subject matter by reference.

The agents generally contain 0.1 to 99%, in particular 0.1 to 95%, active ingredient and 1 to 99.9%, in particular 5 to 99.9%, of at least one solid or liquid auxiliary, with 0 as a rule up to 25%, in particular 0.1 to 20%, of the agent can be surfactants (% means weight percent in each case). While concentrated agents are preferred as a commercial product, the end user generally uses diluted agents which have significantly lower active substance concentrations. Preferred agents are composed in particular as follows (% = weight percent): Emulsifiable concentrates: Active ingredient: 1 to 95%, preferably 5 to 20% surfactant: 1 to 30%, preferably 10 to 20% Solvent: 5 to 98%, 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: 5 to 99%, preferably 15 to 98% granules: Active ingredient: 0.5 to 30%, preferably 3 to 15% solid carrier: 99.5 to 70%, preferably 97 to 85%

The effect of the compounds according to the invention and the anti-animal pest agent containing them by adding other insecticides, acaricides or nematocides significantly broaden and adapt to existing circumstances. Come as additives for example, representatives of the following classes of active substances: Organic phosphorus compounds, nitrophenols and derivatives, formamidines, ureas, Carbamates, pyrethroids, chlorinated hydrocarbons, neonicotinoids and Bacillus thuringiensis preparations.

Particularly suitable mixing partners are, for example: Azamethiphos; chlorfenvinphos; Cypermethrin, cypermethrin high-cis; cyromazine; diafenthiuron; diazinon; dichlorvos; dicrotophos; dicyclanil; fenoxycarb; fluazuron; furathiocarb; isazofos; Jodfenphos; Kinoprene; Lu fenuron; methacriphos; methidathion; monocrotophos; phosphamidon; profenofos; a compound available from Bacillus thuringiensis strain GC91 or from strain NCTC 11821; pymetrozine; bromopropylate; methoprene; disulfoton; quinalphos; Tau-fluvalinate; thiocyclam; thiometon; aldicarb; Azinphos-methyl; benfuracarb; bifenthrin; buprofezin; carbofuran; Dibutylaminothio; cartap; chlorfluazuron; chlorpyrifos; cyfluthrin; Lambda-cyhalothrin; Alpha-cypermethrin; Zeta-cypermethrin; deltamethrin; diflubenzuron; endosulfan; ethiofencarb; fenitrothion; fenobucarb; fenvalerate; formothion; methiocarb; heptenophos; imidacloprid; isoprocarb; methamidophos; methomyl; mevinphos; parathion; Parathion-methyl; phosalone; pirimicarb; propoxur; teflubenzuron; terbufos; Triazamate; fenobucarb; tebufenozide; fipronil; Beta-cyfluthrin; silafluofen; fenpyroximate; pyridaben; fenazaquin; pyriproxyfen; pyrimidifen; nitenpyram; acetamiprid; Avermectin B1 (abamectin); emamectin; Emamectin benzoate; spinosad; a plant extract that is active against insects; a preparation that contains nematodes and is active against insects; a preparation available from Bacillus subtilis; a preparation that contains fungi and is active against insects; a preparation that contains viruses and is active against insects; chlorfenapyr; acephate; acrinathrin; alanycarbe; alphamethrin; amitraz; Az 60541; Azinphos A; Azinphos M; azocyclotin; bendiocarb; bensultap; Beta-cyfluthrin; BPMC; Brofenprox; Bromophos A; Bufencarb; Butocarboxim; Butylpyridaben; cadusafos; carbaryl; carbophenothion; Chloethocarb; chlorethoxyfos; Chlormephos; Cis-resmethrin; Clocythrin; clofentezine; Cyanophos; cycloprothrin; cyhexatin; Demeton M; Demeton S; Demeton-S-methyl; dichlofenthion; Dicliphos; Diethion; dimethoate; dimethylvinphos; Dioxathion; edifenphos; esfenvalerate; ethion; ethofenprox; ethoprophos; Etrimphos; fenamiphos; Fenbutatin oxides; fenothiocarb; fenpropathrin; fenpyrad; fenthion; fluazinam; flucycloxuron; Flucythrinate; flufenoxuron; flufenprox; fonophos; fosthiazate; fubfenprox; HCH; hexaflumuron; hexythiazox; IKI-220; iprobenfos; isofenphos; isoxathion; Ivermectin; malathion; Mecarbam; Mesulfenphos; metaldehyde; metolcarb; milbemectin; moxidectin; naled; Nc 184; Omethoate; oxamyl; Oxydemeton M; oxydeprofos; permethrin; Phenthoate; phorate; phosmet; phoxim; Pirimiphos M; Pirimiphos E; promecarb; propaphos; prothiofos; Prothoate; Pyrachlophos; pyridaphenthion; Pyresmethrin; pyrethrum; tebufenozide; salithion; Sebufos; Sulfotep; sulprofos; tebufenpyrad; Tebupirimphos; tefluthrin; temephos; terbam; tetrachlorvinphos; thiacloprid; Thiafenox; Thiamethoxam, thiodicarb; thiofanox; Thionazin; thuringiensin; tralomethrine; Triarthene; triazophos; triazuron; trichlorfon; triflumuron; trimethacarb; Vamidothion; xylylcarb; YI 5301/5302; Zetamethrin; DPX-MP062 - indoxacarb; methoxyfenozide; bifenazate; clothianidin; XMC (3,5-xylyl methyl carbamate); or the fungal pathogen metarhician anisopliae; especially fipronil, thiamethoxam or lambda-cyhalothrin.

The agents according to the invention can also more solid or liquid Excipients, such as stabilizers, e.g. B. optionally epoxidized vegetable oils (e.g. epoxidized coconut oil, rapeseed oil or soybean oil), defoamers, z. B. silicone oil, Preservatives, viscosity regulators, Binders and / or adhesives, as well as fertilizers or other active substances to achieve special effects, e.g. B. acaricides, bactericides, Fungicides, nematocides, molluscicides or selective herbicides.

The agents according to the invention are known Made way, in the absence of additives such. B. by Grinding, sieving and / or pressing a solid active substance or mixture of active substances, z. B. to a certain grain size, and in the presence of at least one auxiliary such. B. by intimate mixing and / or grinding of the active ingredient or mixture of active ingredients with the excipient (s). This method of making the invention Agents and the use of the active compounds according to the invention for the preparation these means also form objects of the invention.

The application procedures for the funds that is, the methods of combating of pests of the mentioned Type, how, depending on the desired goals and given circumstances to choose Spraying, fogging, Pollinate, Brushing, pickling, spreading or pouring, and using the Control means of pests of the mentioned Type are other objects the invention. Typical application concentrations are between 0.1 and 1000 ppm, preferably between 0.1 and 500 ppm, active ingredient. The application rates per hectare are generally 1 to 2000 g of active ingredient per hectare, in particular 10 to 1000 g / ha, preferably 20 to 600 g / ha.

A preferred method of use in the field of crop protection is application to the foliage of the plants (leaf application), with application frequency and the application rate to the infestation pressure of the respective pest to let. The active ingredient can also through the root system in the Plants arrive (systemic effect) by looking at the location the plants with a liquid Means soak or the active ingredient in solid form in the location of the plants, z. B. in the ground, z. B. in the form of granules (floor application). In water rice crops, such granules can be found in the flooded rice field metered.

The agents according to the invention are also suitable for the Protection of plant reproductive material, including genetic modified propagation material, e.g. Seeds, such as fruits, tubers or grains, or Plant cuttings, from animal pests. The propagation material can be treated with the agent, seed before application z. B. pickled before sowing. The active compounds according to the invention can also on seeds can be applied (coating) by placing the grains in either a liquid medium steeped or coated them with a solid agent. The remedy can also when spreading the propagation material to the place of spreading, z. B. when sowing in the seed furrow, are applied. This treatment procedure for vegetable Propagation material and the plant propagation material treated in this way are other items the invention.

The oligopeptides according to the invention can also are used as active ingredients in pharmaceutical agents, e.g. B. in antibiotics or anti-tumor agents. Appropriate means whose Production and their use are also objects of the present invention.

The following examples are for explanation the invention. You restrict the invention is not a. Temperatures are in degrees Celsius, mixing ratios of solvents stated in volume fractions.

example 1

Production of the inoculation material: The organism SID 22780 is on HA agar [4 g glucose (Merck), 10 g malt extract (Difco), 4 g yeast extract (Oxoid), 18 g agar (Difco), 1000 ml of demineralized water, pH 7 (with 2N NaOH)] for 10 days Incubated at 25 ° C, until mycelium and spores are well developed. The agar culture is at Stored at 4 ° C. For the Long-term preservation becomes mycelium and spores in aqueous suspension brought and mixed with 10 to 30% glycerol. This suspension is transferred to sterile cryo ampoules and stored at –80 ° C.

Preparation of the preculture: Four 500 ml shake flasks without chicane with TSM-37 medium [20 g oatmeal, 20 g malt extract, 0.5 g calcium carbonate, 1000 ml demineralized water, pH 7 (with 2N NaOH)] are mixed with the strain SID 22780 inoculated. For this purpose, rondelles (∅ r = 5 - 8 mm) from the agar culture gouged. 3 to 5 rondelles are transferred to each shake flask. The shake flasks are incubated on a shaker for 7 days at 25 ° C and 120 revolutions per minute.

Production culture production: For the Ten shake flasks without baffle (volume 2 l) with 400 ml TSM-37 medium [20 g oatmeal, 20 g malt extract, 0.5 g calcium carbonate, 1000 ml demineralized water, pH 7 (with 2N NaOH)] sterilized. The sterilized shake flasks are included 10 volume percent of well-grown pre-culture inoculated. The shake flasks are on a shaker 7 days at 25 ° C and incubated at 120 revolutions per minute.

Example 2

The production of the inoculation material takes place as described in Example 1.

Preparation of the preculture: a 500 ml shake flasks with a baffle with TSM-40 medium [4 g glucose (Merck), 10 g Malt extract (Difco), 4 g yeast extract (Oxoid), 1000 ml demineralized Water, pH 7 (with 2N NaOH)] is inoculated with the SID 22780 strain. For this purpose, rondelles (∅ r = 5 - 8 mm) from the agar culture gouged. 3 to 5 rondelles are transferred to the shake flask. The shaking is on a shaker 7 days at 25 ° C and incubated at 150 rpm.

Production culture production: For the Production of the active substance will be two shake flasks without baffle (volume 2 l) with 400 ml CDY medium [33.4 g Chapex-Dox Broth (Oxoid), 5 g yeast extract (Difco), 1000 ml demineralized water, pH 7 (with 2N NaOH)] sterilized. The sterilized shake flasks are each inoculated with 10 percent by volume of well-grown preculture. The shake flasks are on a shaker 18 days at 25 ° C and incubated at 120 revolutions per minute.

Example 3

The production of the inoculation material and the preculture are carried out as described in Example 2.

Production culture production: The procedure is as described in Example 2, but instead of CDY medium uses a supplemented CDY medium by the CDY medium 2.5 g L-pipecolic acid per liter added before pH adjustment become.

Example 4

4 l culture broth (from 10 shake flasks), manufactured according to Example 1, are on a Beckman centrifuge (J2-21 M Induction Drive centrifuge; Rotor JA-10; 6000 revolutions per minute) at 10 minutes Centrifuged at 4 ° C. The solid cell mass and the culture filtrate are three times with ethyl acetate extracted and the combined organic phases concentrated in vacuo. The solid (5 g) is dissolved in methanol / water (95: 5), the solution extracted three times with heptane and concentrated the methanol phase in vacuo. The precipitation (1.5 g) is dissolved in methanol and the solution on an LH-20 column (Sephadex; 36 mm × 920 mm; Eluent: methanol) chromatographed. All factions that Containing neoefrapeptins are combined and deliver after removal of the solvent 145 mg of crude product. This is determined by means of HPLC (reversed-phase silica gel: Kromasil 100 C18; 7 µm; 250 mm × 50 mm; 70 ml / min) cleaned (gradient containing water / acetonitrile) 0.1% trifluoroacetic acid). You get after removal of the solvent pure neoefrapeptin D (0.5 mg). Another HPLC separations from another Fraction (40 mg) with reversed-phase silica gel under isocratic Conditions (Kromasil 100 C18; 10 μm; 250 mm × 20 mm; 10 ml / min;. Acetonitrile / water / trifluoroacetic acid = 50 : 50: 0.1) after evaporation give neoefrapeptin A (10 mg), Neoefrapeptin E (0.6 mg); Neoefrapeptin F (1.3 mg) and a mixture from neoefrapeptin B and neoefrapeptin C (8 mg).

Formulation examples (% = weight percent)

Mixing the finely ground active ingredient and additives results in an emulsion concentrate that is passed through Dilution with water provides emulsions of desired concentration.

Mixing finely ground active ingredient and additives gives a solution which is suitable for use in the form of tiny drops.

The active ingredient is in dichloromethane solved, the solution on the carrier mixture sprayed and the solvent evaporated in vacuo.

biological Examples

Example B1: Action against Heliothis virescens caterpillars

Young soybeans are grown with one aqueous Emulsion spray liquor, the Contains 400 ppm active ingredient, sprayed. After the spray coating has dried on, the soybean plants are added 10 caterpillars of the first stage of Heliothis virescens populated and in a plastic container given. 6 days later the evaluation takes place. From the comparison of the number of dead caterpillars and the damage to the treated to those to the untreated plants is the percentage reduction in population or the percentage reduction in feeding damage (% effect).

The oligopeptides according to the invention show a good effect against Heliothis virescens in this test.

Example B2 Effect against Plutella xylostella caterpillars

Young cabbages are grown with a aqueous Emulsion spray liquor, the Contains 400 ppm active ingredient, sprayed. After the spray coating has dried on, the cabbages are included 10 caterpillars of the third stage of Plutella xylostella populated and in a plastic container given. 3 days later the evaluation takes place. From the comparison of the number of dead caterpillars and the damage to the treated to those to the untreated plants is the percentage reduction in population or the percentage reduction in feeding damage (% effect).

The oligopeptides according to the invention show a good effect against Plutella xylostella in this test. In particular Neoefrapeptin A shows an effect of over 80%.

Example B3: Action against Spodoptera littoralis

Young soybeans are grown with one aqueous Emulsion spray mixture containing 400 ppm of active ingredient, sprayed, after the spray coating has dried on populated with 10 caterpillars of the first stage of Spodoptera littoralis and then into a plastic container given. From the comparison of the number of dead caterpillars and the feeding damage 3 days between the treated and untreated plants later the percentage reduction in population and the percentage reduction of feeding damage (% effect).

The connections in the tables show a good effect against Spodoptera littoralis in this test.

Claims (16)

A new oligopeptide with biological effectiveness, which is both a modified N-terminal amino acid residue as well as a modified C-terminal amino acid residue has, in free form or in salt form. An oligopeptide according to claim 1, characterized in that that it is a pentadeka peptide. An oligopeptide according to claim 2, characterized in that a hydrogen atom of the amino group of the N-terminal amino acid residue is replaced by an acetyl group and the hydroxy partial structure of the carboxy group of the C-terminal amino acid residue is replaced by a substituted amino group of the formula

is replaced. An oligopeptide according to claim 3, characterized in that that at least one of the 15 amino acid building blocks is the α-amino acid 1-aminocyclopropanecarboxylic acid. An oligopeptide according to claim 4, characterized in that it is one of the in the table below

has compositions listed, the abbreviations used in the table have the meanings listed below Ac acetyl, Acc 1-aminocyclopropanecarboxylic acid, Aib 2-aminoisobutyric acid [α-aminoisobutyric acid], β-Ala β-alanine [3-aminopropionic acid], Gly glycine, Iva Isovalin [2-amino-2-methylbutyric acid], Leu leucine, pip Pipecolic acid [piperidine-2-carboxylic acid], Say Substituted amino group of formula 1 as defined in claim 3, to have. An oligopeptide according to claim 5, characterized in that it is neoefrapeptin A of the formula

is. An oligopeptide according to claim 5, characterized in that that it is neoefrapeptin B. An oligopeptide according to claim 5, characterized in that that it is neoefrapeptin C. An oligopeptide according to claim 5, characterized in that that it is neoefrapeptin D. An oligopeptide according to claim 5, characterized in that that it is neoefrapeptin E. An oligopeptide according to claim 5, characterized in that that it is Neoefrapeptin F. An oligopeptide according to claim 5, characterized in that that it is neoefrapeptin I. An oligopeptide according to claim 1, characterized in that that it has physico-chemical properties like it are described above. An oligopeptide according to claim 1, characterized in that that it is using one of the manufacturing methods described above available is. A method of making an oligopeptide according to Claim 1, characterized in that it is one of the above described manufacturing process. A microorganism of the SID 22780 strain or one Mutant of it.