JPH0676B2 - Production method of steroids - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for producing steroids.

(Prior Art) Conventionally, as a method for obtaining 11β, 17α, 21-trihydroxysteroids, various methods of oxidizing a steroid having two hydrogen atoms at the 11-position have been studied.

For example, 17α of 21α, 21-dihydroxypregn-4-ene-3,20-dione (hereinafter referred to as “CS”) is an improved method for producing hydrocortisone.
-By using acetate, it is known that 14α-hydroxylation can be suppressed and a large improvement in yield can be achieved (see Japanese Patent Publication No. 47-13112). (Problems to be solved by the invention However, this method has a drawback that the substrate concentration is low (0.05% in the above publication). It is also known that when a 21-acetate is used as a raw material, only the reaction rate is reduced and by-products are not reduced (see the above publication).

Furthermore, in 16α-hydroxylation using a microorganism belonging to the genus Streptomyces, the Δ ′ · CS derivative is less reactive and the yield is lower than that of the CS derivative (JAC
S., Vol 79, 4818, 1957).

Then, according to the study by the present inventors, similarly, 11β-
In the hydroxylation, it has been confirmed that Δ ′ · CS has a lower reaction rate and lower yield than CS.

(Means for Solving Problems) The present inventors conducted various studies on its 11β-hydroxylation using Δ ′ · CS, which is useful as a raw material for prednisolone and its derivatives, and increased the reaction rate to improve productivity. The present invention has been accomplished by finding a method for producing steroids that improves

That is, the gist of the present invention is the following general formula (I) (In the above formula, X and Y represent a hydrogen atom or an acyl group, and at least one of X and Y is an acyl group.)
A microorganism belonging to the genus Curvularia and having the ability to hydroxylate the hydrogen atom at the 11β position can be cultured in a medium containing an acyloxysteroid represented by: By aerobically acting, the following general formula (II) (In the above formula, X and Y have the same meanings as in the above general formula (I).) The method for producing steroids is characterized by obtaining 11β-hydroxysteroids.

Hereinafter, the present invention will be described in detail.

First, the raw material steroids used in the present invention include the following general formula (I) The acyloxy steroids represented by

In the formula, X and Y each represent a hydrogen atom or an acyl group, and at least one of X and Y is an acyl group, but it is optimal that both X and Y are an acyl group.

As the acyl group, an aliphatic group such as an acetyl group, a propionyl group, a butyryl group is generally used.

In the method of the present invention, a microorganism belonging to the genus Curvularia and capable of hydroxylating the hydrogen atom at the 11β position is cultured in a medium containing the above-mentioned acyloxysteroids. Examples of such microorganisms include Curryularia lunate ATCC 12017 (= IFO6299), 13432, 1
3633, 14595 and C. brachspora (C. brach
yspora) ATCC 12330, 16643 and C. pallescens ATCC 12018.

Further, in the method of the present invention, the objective is also to aerobically act on oxidative enzymes produced by the above-mentioned microorganisms, such as proliferating cells, resting cells, immobilized cells or cell-treated products. Steroids can be obtained.

The above-mentioned culture is usually about 20 to 37 ° C, preferably 24 to 37 ° C.
The pH is selected from 3 to 8 at about 30 ° C, and the culture time is usually 2
It is selected from about 4 hours to 14 days.

The composition of the medium is not particularly limited. That is, in addition to the raw material acyloxysteroids, various carbohydrates as a carbon source, organic acids, etc. may be further added, and as a nitrogen source,
Organic ammonium salts, inorganic ammonium salts, urea and the like can be used.

In addition, if necessary, various phosphates, sulfates and the like can be used as inorganic substances, and various organic nutrients can be added as necessary.

The concentration of the raw material steroid is usually selected from about 0.5 to 10 wt%.

In the method of the present invention, better results can be obtained by allowing the presence of methanol in the medium.

That is, the yield can be further improved by allowing methanol to exist in the medium in an amount of about 0.5 to 10 vol%, preferably about 3 to 8 vol%.

The yield can also be improved by allowing a nonionic activator such as polyoxyethylene sorbitan fatty acid ester to be present in the medium in an amount of 1 to 10 wt%.

Examples of the fatty acid include lauric acid, palmitic acid, stearic acid, oleic acid and the like.

By culturing, the 11β-hydroxysteroids represented by the above general formula (II) can be obtained.

In the present invention, the 11β-hydroxysteroids can be induced into prednisolone by adding 0.1 to 5 wt% of an alkali, for example, a weak alkali such as potassium carbonate, and performing hydrolysis.

Extraction of steroids from the culture can be performed by a conventional method.

For example, preferably, a method of adding methanol, sterilizing and concentrating and then separating, or a method of extracting with an organic solvent such as chloroform or ethyl acetate is adopted.

(Examples) Hereinafter, the present invention will be described in more detail with reference to Examples.

Example 1 Curvlaria lunata ATCC 12017
Seed culture was carried out by a rotary shaker at 27 ° C. for 24 hours using a 500 ml Erlenmeyer flask equipped with a bellows containing 100 ml of a medium having the following composition.

Medium Glucose 10 g / 100 ml Peptone 2 Corn stay liquor 0.5 "Twin 80" (Kao Atlas Co., Ltd., 0.5 registered trademark) (PH5.0) (polyoxyethylene sorbitan monooleate) Next, 2 ml of this culture solution Was added to a 500 ml Erlenmeyer flask equipped with a belly in 100 ml of the same composition as the seed culture, and the same culture was carried out at 27 ° C.

After 1 day of culture, 0.58 mmol of fine powdery Δ ¹ .CS-21 acetate was added, and the culture was continued to react.

After the reaction, 400 ml of methyl alcohol was added and the bacteria were removed by centrifugation. Then, 30 ml of 5% potassium carbonate was added, and the remaining ester was hydrolyzed to produce prednisolone. The results are shown in Table 1.

The analysis was performed using high performance liquid chromatography.

Analytical conditions Column: “Water-Radialback G8” (Waters Co., Ltd. registered trademark) Eluent: Water / methyl alcohol = 4/6 Flow rate: 1 ml / mm Detection: UV 254 nm Example 2 Culture and reaction were carried out in the same manner as in Example 1 except that ¹ -CS-17α acetate was used. The results are shown in Table 1.

Example 3 Incubation and reaction were carried out in the same manner as in Example 1 except that the reaction substrate was changed to Δ ¹ · CS-17α, 21-diacetate. The results are shown in Table 1.

5ml When adding Example 4 reaction substrate △ ¹ · CS-21-acetate
Example 1 except that it is dissolved in the above methyl alcohol and added.
The cells were cultured and reacted in the same manner as in. The results are shown in Table 1.

Except for changing Example 5 reaction substrate △ to ¹ · ^{CS-17α-acetate} was cultured in the same manner as in Example 4 was reacted. The results are shown in Table 1.

Example 6 Incubation and reaction were carried out in the same manner as in Example 4 except that the reaction substrate was changed to Δ ¹ · CS-17α, 21-diacetate. The results are shown in Table 1.

Example 7 Culture and reaction were performed in the same manner as in Example 6 except that "Twin 80" was removed from the medium. The results are shown in Table 1.

Comparative Example 1 Culture and reaction were carried out in the same manner as in Example 1 except that the reaction substrate was changed to Δ ¹ · CS. The results are shown in Table 1.

Comparative Example 2 The reaction substrates were CS, CS-21-acetate, and C, respectively.
The cells were cultured and reacted in the same manner as in Example 1 except that S-17α-acetate and CS-17α, 21-diacetate were used instead. The respective results are shown in Table 1.

(Effect of the Invention) According to the method of the present invention, steroids useful as raw materials for prednisolone and its derivatives can be produced in good yield.

Claims (4)

[Claims] 1. The following general formula (I): (In the above formula, X and Y each represent a hydrogen atom or an acyl group, and at least one of X and Y is an acyl group.) In a medium containing an acyloxysteroid represented by the genus Curvularia, the 11β-position Cultivate a microorganism having the ability to hydroxylate the hydrogen atoms of
Alternatively, a compound represented by the following general formula (II) can be prepared by aerobically reacting the compound (I) with an oxidase produced by the microorganism. (In the above formula, X and Y have the same meanings as in the above general formula (I).) A method for producing steroids, which comprises obtaining 11β-hydroxysteroids. 2. The production method according to claim 1, wherein X and Y are sialic groups. 3. The method according to claim 1 or 2, characterized in that methanol is present in the medium. 4. The method according to any one of claims 1 to 3, wherein a nonionic activator is present in the medium.