JPS589677B2 - Novel bicyclodecadiene compound and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel bicyclodecadiene compound and a method for producing the same.

The present inventors have previously discovered that a substance that significantly induces sporulation of filamentous fungi is present in a culture of Sclerotinia S-1 bacteria isolated from rotting plum fruit infected with Monilia disease. is 5-isopropyl-8-methyl-bicyclo (5.3.0) from various analytical data.
It was identified as deca-2,8-diene-2-carboxylic acid (SF-1 substance) (Japanese Patent Application No. 116139/1989).
(See Japanese Unexamined Patent Publication No. 54-52795)).

While conducting further research on the culture, the present inventors discovered that in addition to the above-mentioned carboxylic acid compound, another useful new substance was present in the culture, and this substance (SF-
As a result of isolating and purifying 2 substances), 5 having the following structural formula was obtained.
-isobrobyl-8-methyl-bicycloC 5.3.0
) It was confirmed that it was deca-2,8-diene-2-carbaldehyde.

The new compounds of formula I have the following physicochemical properties.

(1) Color and shape: Colorless oil (2) Molecular formula: C15H220 Molecular weight: (Calculated from the measured value of high resolution mass spectrum) Calculated value = 218.1671 Actual value = 218.1672 (3) Elemental analysis value: CHO Calculated value (φ) 82.52 10.16 7.3
3 Actual measurement value (related) 82.48 10.16 7,
36(4) Mass spectrum: Shown in FIG.

m/e (relative intensity ratio): 218 (molecular ion M+, 10), 203 (2), 20
0 (4), 189 (5), 1 7 5 (57), 162
(Reference Pidoku 100), 157 (21), 1 4 9 (
62), 133a■, 119(2υ, 117(22),
1 0 7 (80), 9 1 (87), 81 (23),
71 (27), 69 (23), 59 (26), 5 7 (
56), 4 3 (39), 41 (20), 29 (4) (5) Proton nuclear magnetic resonance spectrum: The spectrum measured at 100 MHz in deuterated chloroform is shown in FIG.

δ: 0.8 4 (3H.d.J=7.
1Hz) 0.93 (3H, d.J=7.1Hz)
1.2-2.4 (9H) 1.69 (.3H, S) 2.6 0 (IH.d.J=1 2.4H
z) 5.52 (IH. Broad S, half width 8 Hz) 6.
80 (IH.t.J=5.1Hz)9.40(
IH, s) (6) Ultraviolet absorption spectrum: The spectrum measured in n-hexane is shown in FIG.

λmax 2 2 8 nm (molecular extinction coefficient ε: 97
60) (Force Circular Dichroism: “Experimental Chemistry Structure” Volume 11 (
Complex Salt Chemistry) pp. 317-334 (1956), published by Maruzen Co., Ltd. When measured in n-hexane, [2
25 nm: shows a circular dichroism cotton effect of -48.000.

(8) Solubility: Soluble in methanol, ethanol, acetone, diethyl ether, ethyl acetate, n-hexane, chloroform, carbon tetrachloride, methylene chloride, and benzene, but insoluble in water.

(9) R4 value: Silica gel 60 PF254 thin layer chromatography [layer thickness - 0. 5 Dragon, developing solvent ethyl monoacetate/n-hexane (1:9), developing distance = 16
cm, development temperature = room temperature], Rf value 10. It is 53.

(10) Gas chromatography: 3% silicon O
■ Column with diameter 3 and length 1 m filled with 11 (carrier gas chromium Q), carrier gas - nitrogen, flow rate = 50.
1 A'/min, pressure = 0. It elutes at a retention time of 5.8 minutes under the following conditions: 6 kg/cit, column salinity -130°C, detector = hydrogen flame ionization method.

The novel compound I has the following chemical reactivity.

(1) When 0.5% vanillin-sulfuric acid is sprayed on a thin layer of silica gel 60PF254 and heated, the color initially appears reddish-purple and then changes to blue-purple.

(2) Lithium aluminum hydride (L
Reduction with tAIH4 ) produces an alcohol compound (C15 H24 0 ).

(3) Mr. Jones's reagent in acetone (see L.F. and M. Feeser, Re-Aging for Organic Synthesis, published by John Wiley & Sons, Vol. 1, p. 142, 967) When oxidized using
202).

This carboxylic acid compound reacts with diazomethane to produce methyl ester ('C 16 H24 02).

In addition, the compound I of the present invention has the property of significantly promoting (inducing) sporulation of various microorganisms, particularly filamentous fungi.

Compound (1) can be produced by culturing a bacterium belonging to the genus Sclerotinia that has the ability to produce this compound, and collecting Compound I from the culture.

Although any bacteria can be used as long as it has the ability to produce Compound I, Sclerotinia si bacteria is particularly preferred.

Sclerotinia S-1 is a new strain that the present inventors isolated from rotten plum fruits infected with monilia disease in a fruit orchard in Nagano Prefecture, and is the 42nd strain of Sclerotinia S-1 that was submitted to the Institute of Fine Technology.
14, and American Type Culture.
It has been deposited in the collection (ATCC) as ATCCA20497.

Sclerotinia S-1 bacteria has the following mycological properties.

Growth status in medium (both were cultured in the dark at 25°C for 7 days at pH 5.3) 1. Color of colony on wort agar medium: The overall surface color is light brown and no pigment is formed.

Shape of the village: Smooth overall with ripples associated with growth.

Strong spore adhesion was observed in areas corresponding to high waves. I can't understand both the front and back.

Growth: Fairly good (35-45 mm in diameter) with little growth of hyphae.

Spores: All sporulations are segmented spores, 6-7.5μ in diameter.
x 12-15 μ oval, wort agar medium (pH 5
．． FIG. 4 shows a copy of the micrograph (450x magnification) of Sclerotinia S-1 bacteria cultured in the dark at 25C for 7 days in 3).

As is clear from this figure, the tip of the hyphae is constricted, Formation of segmented spores was observed. It will be done.

2. Zurbeck agar medium Colonial color: White, no pigments are formed.

Shape of the colony: The central part is white, but due to poor growth, it looks like a thin layer of cotton. Growth: Very poor (5-10 mm in diameter).

Spores: Due to its poor growth, only a few segmented spores are observed.

3. Color of colony on Zaburo agar medium: The whole colony is light brown, but the hyphae grow well and the central part is colored.

No pigment is produced.

Shape of the colony: The growth of the bacteria is uneven and there are no wrinkles on either the front or back.

The whole thing feels like cotton dust.

Growth: Good (diameter 25-451 Itm).

Spores: All sporulations are segmental spores.

Egg-shaped with a diameter of 6-7.5μ x 12-15μ be.

■ Physiological and ecological properties ■ Growth temperature = 10-45℃ 2. Optimum temperature for growth: 25-26°C 3. Growth pH: 3. o~9.0 4. Optimum pH for growth: 5. o~6.0 5 Other notable properties: Vigorously forms segmental spores in the dark.

Regarding the Sclerotinia S-1 bacterium, based on the above-mentioned mycological properties, the state of growth on the surface of rotting plums, and the results of microscopic observations, the following was published in "Morphology and Taxonomy of Funghi" by E. A. Betsey. (1952) and Makoto Hiura's "Explanation of Plant Pathogenic Fungi"
A comparative study was conducted according to the classification method described in (1967).

According to the results, this strain forms a disc-shaped ascocarp at the tip of a long stalk that arises from the sclerotium, and as the ascocarp does not have a lid, it belongs to the family Sclerotiniaceae, and it also produces spores. Since no formation was observed, it was determined that it belonged to the genus Sclerotinia in the family Sclerotiniaceae.

The genus Moniliani, which belongs to the family Sclerotiniaceae, is similar to the genus Sclerotinia, but since the genus Monilinia forms spores of the Deuteromycetes genus Monilia type, it is clearly distinguished from the genus Sclerotinia.

Therefore, this strain is clearly determined to be a new strain belonging to the genus Sclerotinia.

Compound I can also be produced by synthesis, but in order to produce it by fermentation, Sclerotinia S-1 bacteria is cultured by solid culture or preferably by liquid culture.

As the medium, one containing a nitrogen source and a carbon source is used.

Nitrogen sources include, for example, meat extract, pebtone, corn steep liquor, gluten, casein, yeast extract, urea, amino acids, etc. Carbon sources include, for example, sugar, glucose, maltose, lactose, starch syrup, juice, starch, Bagasse, wheat bran, molasses, glycerin, etc. may be used alone or in combination of two or more.

In addition, examples of inorganic substances include ammonium sulfate, potassium phosphate, magnesium chloride, salt, iron, manganese,
In addition to molybdenum, vitamins, oils and fats, etc. can be added.

Generally, it is preferable to culture at a temperature of about 20 to 32° C. and a medium with a substantially neutral initial pH for about 5 to 10 days.

It is of course possible to use artificial and natural mutant strains of Sclerotinia S-1 as well.

To isolate and purify Compound I of the present invention from a culture, the culture is first extracted using an organic solvent in a conventional manner.

As the extraction solvent, for example, ethyl acetate, acetone, benzene, diethyl ether, chloroform or a mixture thereof is used.

Then, by operating an appropriate combination of, for example, fractional precipitation distillation, adsorption, thin layer chromatography, column chromatography, countercurrent distribution method, etc., the SF-1 substance is separated and pure Compound I is obtained.

For the fractional precipitation method, for example, methanol, ethanol, acetone, dioxane or a mixture thereof is used as the solvent.

In chromatography, for example, silica gel, silicic acid, magnesium silicate, diatom, activated carbon, alumina, etc. are used as adsorbents.

In addition, by appropriately selecting and using methods such as the sodium sulfite method using a sulfonyl reagent, the Girard reagent method, and the phenylhydrazine method, various derivatives of Compound I can be obtained by separating the SF-1 substance. Pure Compound I can be obtained by treatment according to a conventional method, for example, Organic Compound Identification Method, Vol. 1, pp. 325-624 (Yokendo, 1954).

The compound of the present invention has the effect of significantly promoting spore formation of various microorganisms, especially filamentous fungi, and is important for the industrial cultivation of filamentous fungi.

For example, when the present compound is used, the ability to produce antibiotics such as cephalosporins and enzymes such as protease and amylase can be dramatically improved.

Example 8 liters of distilled water and 1600 g of potatoes were boiled for 1 hour,
Filter the boiling liquid through gauze.

Add 160 g of sugar and 160 g of agar to 8 liters of this filtrate,
After sterilizing in an autoclave, 20 ml each is dispensed into 400 Petri dishes.

Then, Sclerotinia S-1 was placed on the surface of the solidified plate medium.
Bacteria (Feikoken Bacteria No. 4214, ATCC Boiled 20497)
Mycelium was uniformly inoculated under sterile conditions and incubated in an incubator for 30 min.
C. for 7 days in the dark.

After the culture is finished, the culture is crushed into small pieces, and then acetone 11 is added.
The extract was concentrated under reduced pressure to remove acetone and a portion of water to give 3 g.

Further, this concentrate is thoroughly shaken together with ethyl acetate 5e to transfer it to the ethyl acetate layer.

Next, the ethyl acetate layer was dried with Glauber's salt and concentrated, and the obtained ethyl acetate extract was mixed with n-hexane/ethyl acetate (
3:7) as a developing solvent, and the active fraction with Rf=0.8 to 1.0 is collected and eluted with ethyl acetate to obtain a crude product.

The crude product obtained is purified as follows.

This crude product was mixed with 100% n-hexane and 1%.
2%. 3%. n- containing 4% and 5% ethyl acetate
Silica gel column chloratography was performed using 1 liter of hexane as a developing solvent, and the resulting 1% ethyl acetate/n-hexane fractions were collected and concentrated under reduced pressure.

This concentrate was then mixed with ethyl acetate/n-hexane (1:
Silica gel 60 PF254 thin layer chromatography (layer thickness 0.5 mm, 2
0 x 20cm) and Rf20.50~0
．． The active fraction of 55 is collected and eluted with ethyl acetate.

Furthermore, this active fraction was similarly subjected to silica gel thin layer chromatography using 3% ethyl acetate/n-hexane as the developing solvent, and after collecting the R42 fraction of 0.5 to 0.6, it was eluted with ethyl acetate. Removal of the solvent under reduced pressure yields pure 5-isobrobyl-8-methyl-bicyclo (5.
3.0) 7'-2,8-diene-2-carbaldehyde is obtained.

This compound has the properties described above. The results of the sporulation induction test for the compound thus obtained are shown below.

A potato-sugar agar medium prepared in the same manner as in the example was dispensed into test tubes, plugged with cotton plugs, and solidified to prepare a slant medium.

Next, 30 μe of ethyl acetate solutions of various concentrations of the active substance compound I) were uniformly applied to the surface of the slant medium, and then Sclerotinia S- was placed in the center of the slant medium.
One platinum loopful of one bacterium (same as in Example) was inoculated, plugged with cotton, and cultured in an incubator at 30° C. for 3 days in a bright place under fluorescent lamp illumination at 7500 lux.

The number of spores formed within a circle with a diameter of 5 mm on the hyphal growth surface was counted under a microscope.

As a control, 30 μl of ethyl acetate containing no active substance
After applying E, inoculation and culture were carried out in the same manner as above.

As a control, the entire test tube was wrapped in aluminum foil and tested in the same manner in the dark.

All operations were performed aseptically.

The results obtained are summarized in the table below.

As is clear from this result, when cultured in the light without adding the compound of the present invention, almost no sporulation occurs, but when this compound is added, sporulation is markedly increased even under light culture conditions. The effect of inducing sporulation also increased as the amount added increased.

Furthermore, as is clear from the results of culturing the control in the dark, Sclerotinia S-1 bacteria is extremely active in spore formation in the dark.

[Brief explanation of drawings]

Figure 1 shows the mass spectrum of the compound of the present invention, Figure 2 shows the proton nuclear magnetic resonance spectrum, Figure 3 shows the ultraviolet absorption spectrum, and Figure 4 shows the wort agar medium (pH 5.3).
Fig. 2 is a replica of a micrograph (450x magnification) of Sclerotinia S-1 bacteria cultured in the dark at 25°C for 7 days.

Claims (1)

[Scope of Claims] 5-isopropyl-8-methyl-bicyclo(5.3.0)deca-2,8-diene-2-carbaldehyde having the chemical structure of the following formula: 2.5 having the chemical structure of the following formula, characterized by culturing a bacterium belonging to the genus Sclerotinia that has the ability to produce the bicyclodecadiene compound represented by the above formula, and collecting this bicyclodecadiene compound from the culture. -Production of isopropyl-8-methyl-bisic(5.3.0)deca-2,8-diene-2-carpaldehyde.