DD216734A1 - PROCESS FOR PREPARING JASMONIC ACID ANALOGUE - Google Patents

Abstract

In order to provide Jasmonsaeureanaloga of formula I as plant growth regulators as well as in the form of their lower esters as perfumes available, a fermentative process for the preparation of these substances has been developed. The strains Botryodiplodia theobromae D 7/2, A 82 / 168-2 and A 82/260, their mutants or variants are used. In aerobic submersed or emersed culture using liquid nutrient media containing an assimilable source of carbon and nitrogen as well as mineral salts, the fungi are cultured at 20-35 degrees C and pH 4-6.5. Depending on the nutrient solution, the stems form a substance mixture of approx. 1 mg / ml culture filtrate in 4-10 days.

Description

Process for the preparation of jasmonic acid analogs

The invention relates to a process for the preparation of analogous jasmonic acid by culturing strains of the species Botryodiplbdia theobromae.

Field of application of the invention

After application of Jasmonsäureanaloga the general formula I, these are in plants in growth, development and substance switching processes "regulatory" effect · The lower esters of these compounds are as easy going, fragrant substances for the perfume industry of interest.

Characteristic of the known technical solutions

Acids of the general formula I have hitherto not been found in nature. Only 2-epi-Jasmonsäuremethylester (= 2-iso-Jasmonsäuremethylester), an ester of the acid of formula Ib, as part of a Pherombngemisches the oriental fruit moth Grapholitha molesta (Nishida, E., et al, J. of Chemical Ecology 8, 947-59 (1982)). However, 2-iso-jasmonic acid (compound Ib) and 2-iso-dihydrojasmonic acid (compound Ic) are used as intermediates in the synthesis of jasmonic acid and dihydrojasmonic acid and their methyl esters, respectively (Tanaka, H., Torli, S., J. Org. Chem. 40, 462-65 (1975) I Stevens, EV, Hrib, H., Tetrahedron Lett. 22, 4791-94 (1981)) which are used as perfumes in the perfume industry. Jasmonic acid and dihydrojasmonic acid are stereoisomers of compounds Ib and Ic.

Already in 1968, a fermentative process for the production of jasmonic acid has been proposed (Broadbent, D., et al.,

GB patent 1 286 266), a strain of the fungus Laslo has been added to this. , '4i ^ h ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ so far, not possible to chemically convert jasmonic acid into 2-iso-jasmonic acid.

Object of the invention

It is the object of the invention to economically produce compounds of the general formula I by a simple process and to make them available to agriculture as a basis for active substances for influencing growth, development and metabolism of plants and the perfume industry for the production of fragrances.

Explanation of the essence of the invention .

The object of the invention is to develop a process which makes it possible to produce substances of the general formula I by cultivating a fungus strain. According to the invention for the preparation of compounds of general formula I with E = s 2,4-pentadienyl, 2-pentenyl or n-pentyl, the strains Botryodiplodia theobromae D 7/2, A 82 / 168-2 or A 82/260 or variants thereof or mutants used. The strains Botryodiplodia theobromae D 7/2, A 82 / 168-2 and A 82/260 are registered at the Central Institute for Microbiology and Experimental Therapy of the Academy of Sciences of the DDE in Jena under the names ZIMET, ZIMET and

ZIMET deposited / These are highly resistant strains characterized by good fermentation properties, extremely favorable fast mycelium growth and stable production of the above mentioned compounds with favorable time * yield.

The culture for the characterization of the strains Botryodiplodia theobromae p 7/2, A 82 / 168-2 and A 82/260 was carried out on agar medium with subsequent emersed or submerged culture in various nutrient media (see Table 1). The stems have the following macroscopic appearance:

Strain P 7/2

Age of agar culture: 4 weeks Agar medium: medium NL 4

White aerial mycelium with gutation droplets, cultures older than / 4 weeks blackening

Main ku It ua ?: NL 2, 28 ⁰ C, emerse culture

2-4 days: Initially extremely fast-growing white mycelium, which densifies like a grass

15 days: as mycelium becoming pink-black Main culture: NL 1, 28 ⁰ O, submerse culture, 2 days: fine-fibrous white mycelium, slime-forming 10 days: fine-grained white mycelium with dark mycelium islands, mucilage ildenci,

Strain A 82 / 168- ^ 2

Age of agar culture: 4 weeks

Agar medium: medium NL 4

Initially white mycelium, subsequently black lawn with gray aerial mycelium, agar, hilly colonies.

Main culture: NL 2, 28 ⁰ O, emerse culture 2 days: fine-grained white, fast-growing mycelium 10 days: fine-grained black mycelium, bases.

Strain A 82/26 , 0 '

Age of agar culture: 4 weeks

Agarmediöm: medium NL 4

Initially white mycelium, followed by black bases with gray aerial mycelium, agar, coral-like outgrowths on the surface

main culture: NL 3, 28 ⁰ O, submerse culture I days: fine-grained? white mycelium, sphumous 10 days: fine-grained black mycelium, slimy

For all three strains, the low sporulation after multiple inoculation is characteristic. However, they are very easy to-'ßilfe * pj | ^ || p | Jbyuj3h: | ^ C; ken 'vfriiw ^ s · Dl ^ ^-: EJppre ^ -;. j formation in pycnida ^ Mli durofc ZwjLS <hjniipi ^ eii a »f Ajc ^ elsine ^ ^ 'becoming; $ § $ '? ; ''<''';' . '';'' y ^ ': '. - ·; '"'v; · ':

The aerobic fermentation of the fungus for the production of. Compounds Ia, Ib and Ic are optionally in submerged or emersed culture. The nutrient media contain an assimilable C source such as glucose, sucrose, mannitol, sorbitol, glycerin, citric acid, an assimilable N source such as ammonia, asparagine, glycine, ammonium salts, and mineral salts. Particularly advantageous are complex organic media in an aqueous mixture, such as soybean meal, citrus pulp residues, corn steep liquor or milk. The temperature is variable between 20 and 35 ⁰ C and the pH ¹ of 4.0 to 6.5 for a culture period of 4-15 days. Preferably, however, one works at temperatures of 25-3O ⁰ O, pH values between 5 »0 and 6.0 and a culture time of 5-10 days.

The fermentation product is composed of the compounds Ia, Ib and Io, of which the 2-iso-dehydro-jasmonic acid (compound Ia) is new.

In emerser, aerobic culture to 1 mg substance mixture / ml culture medium are obtained, including about 90 % of 2-iso-jasmonic acid (compound Ib) and 0.03-0.1 mg of 2-iso-dihydrojasmonic acid (compound Ic) and 2- Iso-dehydrojasmonic acid (compound Ia) in approximately equal proportions. In submerged culture, the strains produce 0.8 mg mixture / ml of culture solution having the same percentage composition.

The isolation of the substance mixture is carried out by extraction by means of organic solvents, e.g. Ether, chloroform, ethyl acetate, and subsequent removal of the acids e.g. with weak alkaline bases. Often, the substance mixture can be used without separation into the components, e.g. for influencing growth, development and metabolism of plants in agriculture, horticulture or forestry. For this purpose, the substances may not even need to be isolated from the culture medium. However, if the individual substances are to be obtained, this is possible with the customary methods of partition chromatography; Particularly suitable is the reverse chromatography on silylated adsorbents.

The process according to the invention is a simple and, due to its favorable time efficiency, economically advantageous fermentation process.

The following examples are intended to illustrate the invention. The nutrient media used are shown in Table 1 *

Execute tmp; see games Beisgiel ^

2 ml of a suspension of the mycelium of the strain Botryodiplodia theobromae D 7/2 in water (500 ml capacity) distributed in standing round bottom flask with each "100'ml culture medium NL 1 and C on a Bundsohwingschüttelmasohine (U / ₄ i90 min) shaken at 3O ⁰ After 5 days, the flasks contain about 0.8 mg / ml mixture of the compounds of the formulas Ia, Xb and Ic.

2 ml of a suspension of the mycelium of the strain Botryodiplodia theobromae D 7/2 in water to 500 ml Pernbachkolben filled with 100 ml of culture medium NL 2, and incubated for 10 days at 28 ⁰ C in surface culture. Thereafter, 1 ml of substance mixture of the compounds of the formulas Ia, Ib and Ic can be isolated per ml of nutrient solution.

Beisp_iel_2 "

Each 2 ml of a suspension of the mycelium of the strain Botryodiplodia theobromae A 82/260 in water (500 ml capacity) distributed in standing round bottom flask in 100 ml of culture medium NL 3 and on a Bundsohwingschüttelaaschine at 3O ⁰ C (19Q Ü / min) shaken. After 4 days, the flasks contain about 0.8 mg / ml mixture of the compounds of the formulas Ia, Ib and Ic.

2 ml of a suspension of the mycelium of the strain Botryodiplodia theobromae A 82 / 168-2 in water are distributed on 500 ml Ferribaohkolben filled with 100 ml of culture medium NL 2, and the flasks incubated at 28 ⁰ C in surface culture for 10 days · 1 ml of substance mixture (compounds of the formulas Ia, Ib and Ic) can be isolated per ml of nutrient solution, including 0.9 mg of substance according to formula Ib (R. 2-pentenyl).

To isolate the substance mixture, the mycelium is separated off by filtration or centrifuging, the culture filtrate is acidified to pH 3 with HCl, extracted with chloroform, and all the organic acids are removed by means of sodium bicarbonate solution. By acidification with hydrochloric acid and extraction with chloroform, a Bohsubstanzgemisch is obtained which contains 60 % of the compounds prepared of the formulas Ia, Tb and Ic. A quantitative determination can be carried out by thin-layer chromatography on silica gel with the solvent system n-hexane: ethyl acetate: glacial acetic acid = 60: 40j1 against test substance jasmonic acid in the concentration range 0.2-1 / ug after visualization of the compounds by means of anisaldehyde sulfuric acid reagent at 11O ⁰ C. To isolate the individual substances, the compound mixture can be separated by column chromatography on silylated silica gel by means of the eluent system methanol: water / acetic acid = 50 s50 * 5. The substances are eluted in the following order:

a) j ~ 3-0xo-2- (2,4-pentadienyl) cyclopentyl] -1-acetic acid

b) "3-Oxo-2- (2-pentenyl) -oyclopentyl"] -1-acetic acid and

c) [2-pentyl-eycloperityl-1-acetic acid.

Identification is by GC-MS as methyl ester. - =

a) 222 (M ⁺ ), 204, 193, 191, 167, 149 (100%), 130, 107, Ö3, 79 (100%)

b) 224 (M ⁺ ), 206, 195, 193, 156, 151, 135, 133, 121, 109, 83 (100 %)

c) 226 (M ⁺ ), 194, 156, 153, 137, 109, 83 (100%), 82

Table 1 Composition of the nutrient media used per liter

HL 1

NL2

NL3

NIA

sucrose 50g 30g 50g 20g soy flour 5g 5g 5g .-. , , · '. Corn steep liquor - ' 15ml - - Citrus extract ¹ ' 200ml - 100ml 500ml glycine i - 10g - KH ₂ PO ₄ 0.005 g 0.5g 0.005 g - MgSO ₄ ν 7 H ₂ O 0,003g 0.3g 0,003g   - '' - ' PeSO ₄ .7H ₂ O o _t oöpig 10ng 0.0001 ZnSO ₄ . 7H ₂ O O ₉ 000088g 8,8mg 0,000088g .- Agar Agar - ' 10g Aqua dest ad. 1000 1000 1000 1000 pH before the Sterilize 5Λ 5.4 5.4 6.0 pH adjustment with NaOH NaOH NaOH NaOH autoclaving each uniform at 120 ⁰ C for 20 min

1) Citrus extract: Filtrate of an aqueous slurry of Preßrüokständen of peeled citrus fruits, particularly advantageously of an aqueous slurry of citrus albedo ·

H ₂ C

H ₂ CH R

COOH

Formula I α) R = CH ₂ -CH = CH-CH = CH ₂

b) R = CH ₂ -CH = CH-CH ₂ -CH ₃

C J

Claims (5)

Claim 1. Process for the Preparation of Jasmonsäureanaloga the general! Formula I, characterized in that one of the strains Botryodiplodia theobromae D 7/2, A 82 / 168-2, A 82/260 or their mutants or variants are used. 2. The method of item 1, characterized in that _f is a mixture of compounds of general formula I is prepared in which the compounds Ia (E s 2,4-pentadienyl), Ib (β E 2-pentenyl) and to (B s n Pentyl). 3. The method according to item 1 and 2, characterized in that one of the strains Botryodiplodia theobromae D 7/2, A 82 / 168-2 or A 82/260, ZIMET V.3> 13, ZIMET * /? ^ Or ZIMET <ϊ3Ί-Ί2 \ .η aerobic fermentation using a nutrient medium containing an assimilable C source, an assimilable N source and mineral salts in submerged or outer culture at a temperature of 20-35 ⁰ O and a pH of 4.0-6.5 for a period of 4-15 days and isolated from the nutrient medium, the compounds of general formula I. 4. Method according to item 1 to 3 », characterized in that the nutrient medium contains a complex organic medium. ' · 5. Method according to item 1 to 4, characterized in that the F <
becomes. the fermentation was carried out at 25-3O ⁰ O and pH 5.0-6.0 For this 1 page formulas