DE1768985A1 - Optically active unsaturated steroids - Google Patents

Description

PATENT LAWYERS

DR.-ING. BY KREISLER DR.-ING. SCHÖNWALD 1768985 DR.-IN6. TH. MEYER DR. FUES

COLOGNE 1, DEICHMANNHAUS

Cologne, July 11, 1968 AvK / Ax

Takeda Chemical Industries, Ltd.,

27, Doshomachi 2-chome, Higashi-ku, Osaka (Japan)

Visually, active unsaturated steroids

The invention relates to a method for producing optically active unsaturated steroids, especially for the preparation of optically active compounds that have a 17-hydroxy-8,14-secogonapolyen-14-one nucleus substituted on the 1313 carbon atom contain (hereinafter referred to as "compounds (II)"), from compounds which have one on the 13β-carbon atom substituted 8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione nucleus contain (hereinafter referred to as "compounds (I)"), using microorganisms.

Recently, methods for the total synthesis of 19-nor steroids have been developed (see, for example, Miki and coworkers "Proceedings of the Chemical Society" 1963, page 139, Crispin and coworkers, ibid, 1963, page 22, Windholz and coworkers "Journal of the Organic Chemistry ", 28 , page 1092 and Smith et al." Bxperienta ", 19, page 394).

However, these methods have disadvantages when used for large-scale production: When a connection that has a 8,14-Secogona-polyene-14,17-dione nucleus substituted on the 13β-carbon atom contains, a ring closure reaction to

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speaking 13β-carbon-substituted gonapolyene-17-one nucleus a racemate arises with respect to the 13-substituent, a yield being obtained the best half of the stoichiometric yield amounts to. Furthermore, the natural-type steroid (in which the 13-substituent is β-oriented) is only visualized Get splitting.

In addition, two chemical processes have been found for the preparation of compounds which substituted one on the 13 [beta] carbon atom 17a-hydroxy-8,14-secogona-1,3,5 (10), 9 (11) -tetraen-14-one nucleus included, by Miki (one of the inventors) and his co-workers and by CH. Kuo and co-workers (Chemistry and Industry 1340 (1966)).

The compounds obtainable by this process are, however, always 5O: 5O mixtures of a compound which has an am 13β-carbon atom substituted 17a-hydroxy-8,14-secogona-1,3,5 (10), 9 (11) -tetraen-14-one-nucleus contains, with their enantiomers. In order to convert these compounds into natural estrone and the like, they must undergo optical splitting be subjected at any suitable stage.

The main object of the invention is a new process for producing the compounds which can be carried out on an industrial scale (II) directly from compound (I) in good yield. The method is characterized in that one Microorganism belonging to the genera Candida, Debaryomyces, Kloeckera, Pichia, Rhodotorula, Schizosaccharomyoes or Hansenula is one of, cultivated, a redox enzyme system of the so obtained culture with a compound that an 8,14-secogona-1,3,5 (10), 9 (1i), 15-pentaene-14,17-dione nucleus substituted on the 13β-carbon atom contains, and separates the desired compound from the reaction mixture.

As a substituent on the 13-carbon atom, for example an alkyl radical, e.g. methyl, ethyl, propyl, butyl, an aryl radical, e.g. a phenyl radical, an aralkyl radical, e.g. benzyl,

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Phenethyl, phenylpropyl, etc., stand.

The compound (i) used as a starting material, the for example according to the method of the Dutch patent application 6 612 205 (designed 1.3ο 1967) can, carries a substituent on the 13-carbon atom and any in the 1-, 2-, 3-, 4-, 6-, 7-, 11-, 12-, 15- or 16-position 1 to 3 other substituents from the group consisting of oxygen groups, Halogen groups and lower alkyl groups.

Examples of oxygen groups are hydroxyl groups, lower acyloxy radicals with 1 to 4 carbon atoms and ™

Alkoxy radicals having 1 to 4 carbon atoms and, for example, fluorine, chlorine and bromine as halogen atoms. as lower alkyl radicals come, for example, straight-chain radicals with 1 to 4 carbon atoms or three-membered or six-membered rings including those mentioned above as substituents Radicals that contain one or more oxygen groups, in question.

Examples of starting compounds for the process in accordance with the invention include:

8,14-Secoöstra-1,3,5 (10), 9 (11), 15-pentaen-14,17-dione, 3-methoxy-8,14-secoöstra-1,3,5 (10), 9 ( 11) _f 15-pentaen-14,17- _Λ dione I

3-ethoxy-8,14-secoestra-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-methoxy-13-ethyl-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-ethoxy-13-ethyl-8,14-s ecogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-methoxy-13-isopropyl-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-ethoxy-13-isopropyl-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-benzyloxy-8,14-secoestra-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

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3-methoxy-13-benzyl-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-ethoxy-13-benzyl-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-methoxy-13-phenyl-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-ethoxy-13-phenyl-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

2,3-dimethoxy-13-ethyl-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione

3-methoxy-6-methyl-8,14-secoestra-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione and

3-methoxy-6,6-dimethyl-8,14-secoestra-1,3,5 (1θ), 9 (11), 15-pentaene-14,17-dione

The desired compounds (II) belong to the following groups:

1) Compounds with one substituted on the 13β-carbon atom 17β-hydroxy-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaen-14-one nucleus, those of microorganisms such as Debaryomyoes vini, Debaryomyces vanriji, Pichia wickerhamii, Pichia pijperi, Hansenula holstii and Hansenula capsulata, are formed,

2) Compounds that have one substituted on the 13β carbon atom Ha-Hydroxy-SjU-secogona-i, 3.5 (10), 9 (11), 15-pentaen-14-one nucleus and are formed by microorganisms such as Kloeckera Magna.

3) Compounds that have one substituted on the 13β carbon atom 17β-Hydroxy-8,14-secogona-1,3,5 (10), 9 (11) -tetraen-14-one nucleus and by microorganisms such as Candida utilis, Debaryomyces nicotianae, Pichia etchellsii, Rhodotorula rubra, Schizosaccharomyces pombe, Hansenula saturnus, Hansenula holstii.

The nutrient media for the growth of these microorganisms contain carbon and nitrogen sources derived from the Microorganisms are and can be assimilable and usable

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Containing inorganic salts, various vitamins and amino acids.

Particularly suitable carbon sources are, for example, glucose, sucrose, dextrin, glycerin, etc. and al3 Nitrogen sources for example inorganic nitrogenous materials such as peptone, meat extract, casein, Corn steep liquor, dry yeast and yeast extract, and inorganic nitrogen compounds such as ammonium nitrate, ammonium phosphate and ammonium sulfate.

Examples of the above-mentioned inorganic salts are sodium chloride, potassium sulfate and magnesium sulfate. The nutrients% favoring the growth of the microorganisms used in suitable proportions in a culture medium "

The cultivation can be as shaking culture, stationary culture and submerged culture with movement and ventilation take place.

The starting compound (i) can be added either at the beginning of the cultivation or in a suitable phase in the course of the cultivation, continuously or at intervals or all at once. The final concentration of the compound (i) in the medium is preferably about 0.05 to 0.65 o (weight / volume). (J

The compound (I) can be in the form of a powder or as a solution or suspension in a suitable solvent, e.g., B _e acetone, methanol, ethanol, ethylene glycol, propylene glycol, dimethylformamide or dioxane, with or without addition of a surface active agent, dispersing agent, etc. . be used.

When the microorganism after separation from the culture liquid is used, the microbial cells in a buffer solution of suitable ρττ value and suitable Ionic strength or suspended in water and then with the

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Starting compound (i) are merged to this to convert.

The reaction usually takes place at a p "value of about

ο 2 to 11 and at a temperature between about 10 and 50 C,

preferably between about 25 and 40 ^° C. for a period of about 2 days. The optimal conditions, however, vary with factors such as the starting compounds and the microorganisms, and it is appropriate to select the optimal conditions in each individual case.

That formed and enriched in this way in the reaction medium End product (II) can be separated therefrom by various known methods, for example by adsorption on a suitable adsorbent, e.g. silica gel, and subsequent elution with a suitable solvent, e.g. a benzene-acetone mixture or a chloroform-acetone mixture, or by taking advantage of the difference in partition coefficient between two liquid phases, for example with countercurrent distribution or according to customary chromatographic methods.

The optically active compounds containing a 17-hydroxy-8 _f H-secogona-pentaen-H-one nucleus substituted on the 13β-carbon atom are converted into the corresponding compound using the enzyme system of a yeast, for example Candida solani, by the method according to the invention Tetraene transferred.

The optically active compounds prepared in the manner described above which have a carbon atom on the 13β ~ substituted IT-Hydroxy-ejH-secogona-tetraen-H-one nucleus contain, for example, according to the method which is described in "Chemistry and Industry" 1966, page 1340, converted into estrone and other 19 nar steroids.

Presently preferred embodiments of the invention are described in the following examples in which the Percentages are based on weight / volume. Parts by weight

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relate to parts of space as grams to cubic centimeters.

The microorganisms which were used in the experiments described in the examples "are deposited with the Institute for Fermentation, Osaka, Japan under the deposit numbers which are named in the examples as" IIO-Iunans ". Some microorganisms which are identified by ¹¹ NRRL "marked with a number are deposited with the Northern Regional Research Laboratory, USA.

example 1

Contains per 40 parts by volume of a liquid medium (p _H 5 »8) $ 0.3% yeast extract, 0.5i & polypeptone, 0.2 $ corn steep liquor, 5 $ glucose and sucrose 5fő be with a seven day agar slant in one of the Table 1 mentioned microorganisms vaccinated. The inoculated medium is ^{kept at 28 °} C. for 2 days. After this time a solution of 0.02 parts by weight of 3-methoxy-8, H-secoöstra-1,3,5 (10), 9 (11), 15-pentaen-14,17-dione (hereinafter referred to as "Substrate (i)" denotes) added in 0.8 parts by volume of ethanol. The cultivation is continued for another 2 days. The resulting broth is then mixed in two portions with a total of the same amount of ethyl acetate. The transferred into the Äthylacetatphase reaction products are washed with water, dehydrated ä Siert and concentrated to dryness.

Column chromatography is performed using silica gel carried out. The reaction products are isolated from the fraction treated with a 9: 1 mixture of benzene and Acetone has been eluted. The products are determined according to their rotational dispersion curves in ethanol. Fig. 1 (a) to (g) shows the spectra of these compounds and other closely related steroids:

a) 3-methoxy-8,14-secoöstra-1,3 »5 (1O) _f 9 (11) -tetraen-17β-ol-14-one (designated in the table as ββ);

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b) 3-methoxy-8,14-secoestra-1,3,5 (10), 9 (11), 15-pentaen-17βol-14-one (referred to in the table as Δ-ββ);

c) 3-methoxy-13a-methyl-8,14-secogona-1,3,5 (10), 9 (11) -tetraen-17β-ol-14-one;

d) 3-Kethoxy-13a-methyl-8 _f 14-secogona-1,3,5 (10), 9 (11), 15-pentaen-17β-ol-14-onej

e) Mixture of 3-methoxy-8,14-secoestra-i, 3,5 (10), 9 (11) -tetraen-17β-ol-14-one and 3-methoxy-8,14-secoöstra-1, 3,5 (1OX 9 (11), 15-pentaen-17β-ol-14-one.

f) mixture of 3-methoxy-13a-methyl-8,14-secogona-1,3,5 (10), 9 (11) -tetraen-17β-ol-14-one and 3-methoxy-13a-methyl-8,14-secogona-1,3,5 (10), 9,15-pentaen-17β-ol-14- on

g) 3-methoxy-8,14-secoöstra-1,3,5 (J0), 9 (11), 15-pentaene-17aol-14-one (in the table as _{Δ'5_β} α hereinafter).

The samples that were shown in FIG. 1 did not differ from one another can be, e.g. 3-methoxy-8,14-secoöstra-1,3,5 (10), 9 (11), 15-pentaen-17ß-ol-14-one and 3-methoxy-13a-methyl-8,14-secogona-1,3,5 (10) , 9 (11), 15-pentaen-17β-ol-r14-one, are given in Methanol together with a small amount of p-toluenesulfonic acid left to stand at room temperature and on the formation of 3-methoxy-9,14-oxido-8,14-secoöstra-1,3,5 (10), 15-tetraen-17-one examined by thin-layer chromatography (a 9 »14 oxide only occurs in the case of the latter compound on). In this way it is determined whether the steric orientation between the 13-methyl group and the 17-hydroxyl group is cis or trans.

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- y - (1214) (0980) ßß,
NRRL-Y-2155 1768985 sales
Weight # Microorganism IPO no. ·
NRRL - No. (1285) (0984)
FRRL Y-1842 product 72 Candida utilis (1086) (0868) (0993) ßß 75 Debaryomyces nicotianae (0855) (1278) Example 2 ßß 68 Debaryomyces vini (1283) 70 Debaryomyces vanriji (1290) ^15- ßß 75 Kloeckera magna (0891) Δΐ5_ _β β 65 Pichia wickerhamii Schizosaccharomyces pombe (0362 Δ ¹⁵ -ββ 70 Pichia etchellsii Hansenula holstii ßß 80 Pichia pijperi Hansenula capsulata Δΐ5_ _ββ 75 Rhodotorula rubra Hansenula saturnus ßß 78 ßß 78 Δ ¹⁵ -ββ 70 ^Δΐ5 " ^ββ 75 ßß

1000 parts by volume of a liquid medium (p "5.8) containing 0.3 $ yeast extract, 0.5> proteose peptone (Difco), 0.2 / ί corn steep liquor, 5 $ glucose and 5 $ sucrose, are with a culture of Debaryomyces nicotianae IPO-0855 inoculated, the microorganism is ^{cultured at 28 0} O for 2 days. Then a solution of 1 part of the substrate (I) in 40 parts by volume of ethanol is added to the culture, which is then incubated for a further 2 days. The reaction product is extracted twice with 1000 parts by volume of ethyl acetate each time and, after the solvent has been distilled off, purified by column chromatography on silica gel with a benzene-acetone mixture as the eluent. The solvent is distilled off from the fraction containing the product. The residue is recrystallized from ethanol, where .0 »42 wt.

texraen

Parts of 3-methoxy-8,14-secoöstra-1,3,5 (1O), 9 (11) -17ß-ol-14-one as colorless plates, melting point 112-113 ⁰ O are obtained. The compound shows the negative cotton effect curve

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and a specific rotation / ö_7-n of -39 (O = 0.5 $ in Dioxane).

Example 3

In the manner described in Example 2, a solution is obtained from 1 part by weight of the substrate (I) in 40 parts by volume of ethanol to 1000 parts by volume of a two-day culture of Pichia etchellsii (IFO-1283), which is then incubated for two more days will. The reaction product is extracted with ethyl acetate ^ purified by column chromatography and crystallization left from ethanol, with 0.52 parts by weight

ρ 3-Methoxy-8, H-secoöstra-1,3,5 (10), 9 (11) -tetraen-17β-ol-14-one can be obtained as colorless flakes with a melting point of 111-113 ° C.
JJJ ² Q -59 ° (C = I ₁ OJi dioxane).

Example 4

8 parts by volume of a 2.5 $ solution of the substrate (I) in ethanol to 200 parts by volume of a two-days culture of Hansenula holstii which is then incubated at 28 ⁰ C are added (IFO-0980) in the manner described in Example 2. Sampling takes place 12, 24, 36 and 48 hours after the addition of the substrate (I). The samples are each extracted with ethyl acetate. The extracts are purified by column chromatography on silica gel and dissolved in ethanol.

The rotation dispersion of the ethanol solution is measured. The results are shown in Figure 2 (a, b, c and d).

These rotational dispersion curves vary with the cultivation (£ teaction) time For example, the curves are correct for the products that have a cultivation time of 12, 24, 36 and 48 hours correspond, with the curves for 3-methoxy-8,14-secoö8tra-1,3,5 (10), 9 (11), 15-pentaen-17ö-ol-14-one, a

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Mixture of 3-methoxy-8,14-secoestra-1,3,5 (10), 9 (11), 15-pentaen-17β-ol-14-one and 3-methoxy-8,14-secoöstra-1,3 »5 (10), 9 (11) -tetraen-17β-ol-14-one or 3-methoxy-8,14-secoestra-1 »3.5 (10), 9 (11) -tetraen-17β-ol-14-one match.

While the product, which corresponds to the reaction time of 12 hours, does not crystallize, its absorption spectrum recorded in a chloroform solution shows the absorption for hydroxyl (3620 cm ") and α, β-unsaturated ketone (1680 cm" ¹ ).

The rotary dispersion of this product, shown in Fig.2a is shown, gives the negative Cottone effect curve with "one foot at 360 µm. Since v / eder 3-methoxy-9,14-oxido-8,14-secoöstra-1,3,5 (10), 9 (11) - pentaen-17-on nor its enantiomer is formed when this product is treated with a small amount of p-toluenesulfonic acid in benzene, it is evident that this product contains the compound 3-methoxy-8,14-secoöstra-1,3,5 (10), 9 (11), 15-pentaen-17β-ol-14-one is.

The product, which is obtained after a cultivation time of 36 or 48 hours, is crystallized from ethanol in the form of colorless flakes with a melting point of 111-113 ° C. (yield 78% by weight). The specific rotation £ ~ clJj7 λ and the infrared spectra of the products agree completely with those of 3-methoxy-8,14-secoestra-1,3,5 (10), 9 (11) -tetraen-17βol-14-one.

From the foregoing it can be concluded that Hansenula holstii IFO-0980 has the ability to first perform the 17-carbonyl group of the substrate (I) to form 3-Hethoxy-8,14-secoöstra-1,3,5 (10), 9 (11), 15-pentaen-17β-ol-

Una to reduce on 14-1 5 then Δ of about 3-methoxy-8,14-secoöstra-1,3,5 (10), 9 (11) -tetraene-17-ol-14-one to reduce the latter.

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Example 5

In the manner described in Example 2 ", a solution of 0.5 wt _e parts by the substrate (l) in 40 parts by volume of ethanol is added to 1,000 parts of a two-days culture of Debaryomyces vanriji IFO-1285, which is then cultured for 2 days, the reaction product is extracted with ethyl acetate and the extract purified by column chromatography in the manner described in Example 2, 0.35 parts of 3-methoxy-8, H-seoogona-1,3,5 (10), 9 (11), 15-pentaene -17ß-ol-14-one can be obtained as an oil.

ψ The rotary dispersion of the oily product dissolved in ethanol solution is identical to the rotary dispersion shown in Fig. 1 (b). No 9,14 oxide occurs when the product is treated with p-toluenesulfonic acid.

Example 6

In the manner described in Example 2, 40 parts by volume of a 2.5% solution of the substrate (i) in ethanol are added to 1000 parts by volume of a two-day culture of Kloeckera magna IFO-0868 and the reaction is then allowed to proceed at 28 ^° C. for 2 days. After this time the product is extracted with ethyl acetate.

™ The extract is purified by column chromatography on silica gel in the manner described in Example 2, using 3-methoxy-8,14-secoöstra-1,3,5 (10), 9,15-pentaen-17a-ol-14-one is obtained as an oil (yield 75 wt. - $>),

The nuclear magnetic resonance spectrum of the product in CDCl, (D; deuterium) shows peaks that are responsible for the Δ-17-ol structure:

17-H (5.52, doublet), 16-H (T 2.62 quartet) and 15-H (T 3.89 quartet) are characteristic. The rotational dispersion of the product, measured in an ethanol solution, gives the posi-

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tive cotton effect curves. The first peak occurs at 360 mu on · This product gives upon treatment with p-toluenesulfonic acid in benzene a 9,14 oxide.

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Claims (8)

Claims 1) Optically active compounds that have one on the 13ß-carbon atom substituted 17β-hydroxy-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaen-14-one core included. 2) Optically active compounds that have one on the 13ß-carbon atom substituted 17a-hydroxy-8,14-secogona-1f3,5 (10), 9 (11), 15-pentaen-14-one nucleus contain. 3) 3-methoxy-8,14-secoestra-1,3,5 (10), 9 (11), 15-pentaen-17βol-14-one. 4) 3-methoxy-8,14-secoestra-1,3,5 (10), 9 (1i), 15-pentaen-17aol-14-one. 5) Process for the preparation of optically active compounds which have one substituted on the 13β-carbon atom Contain 17-hydroxy-8,14-secogona-polyen-14-one core, characterized in that compounds containing an 8,14-Secogona-1t3,5 (1O), 9 (11), 15-pentaene-14,17-dione nucleus substituted on the 13ß-carbon atom contain, the action of an enzyme system from Candida utilis, Debaryomyoes vini, Debaryomyces vanriji, Debaryomyces nicotinanae, Kloeokera magna, Pichia wickerhamii, Pichia pijperi, Pichia etchellsii, Rhodotorula rubra, Schizosaccharomyces pombe, Hanaenula capsulata, Hansenula holstii or Hansenula saturnus and the desired Product wins from it. 6) The method according to claim 5 for the preparation of compounds containing a substituted on the 13ß-carbon atom 17ß-hydroxy-8,14-eeeogona-1,3,5 (1O), 9 (11), 15-pentaen-14-one- contain a core, characterized in that compounds which contain an 8,14-Secogona-1,3 »5 (1O), 9 (11) -pentaene-14,17-dione nucleus substituted on the 13ß-carbon atom are exposed to the action an enzyme system from Debaryomyoes vini IPO 1214, Debaryomyoes vanriji IFO 1285, Pichi * wickerhAJBii IFO 1278, Piohia pijperi IPO 1290, Haneenul * oapwlata IPO 0984 (NRRL Y-1842) or Haiuenul * holetii IFO 0980 (MRRL weft Y-Sl. 109882/1787 7) The method according to claim J for the production of compounds, the one substituted on the 13β-carbon atom 17ct-hydroxy-8,14-secogona-1,3,5 (10), 9 (11), 15-pentaen-14-one-kern contain, characterized in that one is compounds which substituted one on the 13ß-carbon atom 8,14-Secogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione-kernel contained, subjected to the action of an enzyme system from Kloeckera magna ΓΕΌ 0868. 8) The method according to claim J for the preparation of compounds which anthalten a substituted on the 13ß-carbon atom 17ß-hydroxy-8,14-secogona-1,3,5 (10), 9 (11) -tetraen-14-one nucleus , characterized in that compounds which contain an 8,14-üecogona-1,3,5 (10), 9 (11), 15-pentaene-14,17-dione nucleus substituted on the 13ß-carbon atom are exposed to the action of an enzyme system from Candida utilis IPO 1086, Debaryomyces nicotianae IPO 0855, Pichia etchellsii Ii ¹ O 1283, Rhodotorula rubra IPO 0891, Schizosaccharomyces pombe IPO 0362, Hansenula saturnus IFO 0993 or Hansenula holstii IPO 0980 (NRRL Y-2155). 109882/1787 Le he