JPS5939298A - Preparation of 12beta-hydroxyandrostan-4-ene-3,17-dione - Google Patents

Abstract

PURPOSE:To prepare 12beta-hydroxyandrostan-4-ene-3,17-dione, by cultivating a bacterium belonging to Pseudomonas putida in a medium containing deoxycholic acid or its salt. CONSTITUTION:A bacterium such as Pseudomonas putida D4014-A 1615 (FREM- P 6572) belonging to the genus Pseudomonas, capable of producing 12beta-hydroxyandrostan-4-ene-3,17-dione using deoxycholic acid or its salt as a substrate is inoculated into a medium containing deoxycholic acid or its salt, and cultivated under aerobic conditions at 25-35 deg.C for 10 hours -7 days, and 12beta-hydroxyandrostan-4-ene-3,17-dione is collected from the culture solution.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides the production of 12β-hydroxyandrost-4-ene-3 by microorganisms from deoxycholic acid and/or its salts.
, 17-dione, specifically, using deoxycholic acid and/or its salt as a substrate to produce 12β-hydroxyandrost-4-ene-3,17-dione using bacteria belonging to Sulpseudomonas putida, 12 by culturing in a medium containing deoxycholic acid and/or its salts.
1 characterized in that it produces β-hydroxyandrost-4-ene-3,17-dione and collects it.
2β-hydroxyandrost-4-ene-3,f7-
This invention relates to a method for producing dione.

Conventionally, 12β-
The following report has been found as a method for obtaining hydroxyandrogter-4-ene-3°17-dione (hereinafter referred to as % 12β-hydroxy 4AD). First of all, Barness (P,
Pseudomonas sp. N0IB 10590 (Pseudomonas sp.
nas sp, N0IB 10590) strain has been reported (Tetrahedron
, 32 volumes.

See pages 89-93 (1976)]. In this method, 1 strain of Pseudomonas sp.
After 4 hours of aerobic incubation, deoxycholic acid 10 to 12
β-hydroxyandrosta-1,4-diene-3,1
7-dione 260 da and 12α-hydroxypregna
1,4-dien-3-one-20α-carboxylic acid 657
The main product is 12β, and one of the minor components is 12β.
- It merely confirms the existence of hydroxy-4AD.

Regarding the Pseudomonas sp. N0IB 10590 strain used in this method, nothing has been reported other than that this strain is a bacterium belonging to the genus Pseudomonas collected from animal feces. Also, Bacteroides-7-7Gy XF 23 (Bacteroides
des fragilisXF25. ) 8 strains or Bacteroides fragilis saph sp.
ilis 5ubsp+thetaiotaomicr
onE59) strain from deoxycholic acid.
-Hydroxy 4AD, 12α-hydroxyandrosta-1,4-diene-3,17-dione, 12β-hydroxyandrosta-1,4-diene-3°17-dione and 12α-hydroxypregna-1,4- Diene-3
Method for obtaining -one-20α-carboxylic acid (Bioche
m, Soc, Trans, vol. 5, 1711-17.
13 (1977)], but all of these methods use anaerobic bacteria, require low concentrations of substrates, require long cultivation times, and are difficult to collect the target product. There are various problems in industrial fermentation production, such as low yield.

The present inventors have conducted a long-term search for microorganisms that produce 12β-hydroxy 4AD gold using deoxycholic acid and/or its salts as a substrate. The present inventors have discovered that 12β-hydroxy 4AD can be produced with high selectivity and yield using salt as a substrate, and have completed the present invention.

The bacteria belonging to Pseudomonas putida obtained by the present inventors include Pseudomonas putida D4014-
A1615 (Pseudomonas putida
D4014-A1615) strain (Feikoken Bacteria No. 657
No. 2). This strain was collected from soil and was collected from Pseudomonas D4014 (Pseudomo)-y, -Pseudomo
This is a mutant strain obtained by subjecting the nas putida D4014 strain (Feikoken Bibori No. 6325) to mutation treatment.

Pseudomonas putida D4014 strain and Pseudomonas fu”tida D4014-AI61 obtained by the present inventors
The following table lists the mycological properties of the five strains.

/ Based on the mycological properties shown in the table above, Pseudomonas putida D4014 strain and Pseudomonas putida D
The 4014-A1615 strain was identified. Pseudomonas putida D4o*4 strain? i.Being a bacillus;
Microscopic findings such as having polar flagella, negative Durham staining, positive oxidase reaction and catafuse reaction, and being aerobic;
The result of the 0-F test is oxidative (C) oxidative
Based on the physiological properties such as ), it was identified as a bacterium belonging to the genus Pseudomonas based on the Purgation Manual of Determinative Bacteriology, 7th and 8th editions. In addition, Pseudomonas putida strain D4014 belongs to the Pseudomonas putida species because of the fact that the culture solution has a fluorescent color, that it does not liquefy gelatin, that it does not grow at 37°C, and that it produces arginine dihydrolase. It was identified that Additionally, mutant strains are generally considered to belong to the same species as their parent strain, and Pseudomonas putida D4014-
The A1615 strain was determined to be a bacterium belonging to the species Putida of the genus Pseudomonas.

The production of 12β-hydroxy 4AD by the method of the present invention is carried out using deoxycholic acid and/or its salt as a substrate.
This is carried out by culturing bacteria belonging to Pseudomonas putida that produce β-hydroxy 4AD in a medium containing deoxycholic acid and/or its salt. Specific examples of the salts of deoxycholic acid include salts of deoxycholic acid with alkali metals such as sodium and potassium. The concentration of deoxycholic acid and/or its salt may normally be in the range of 1 to 100 g/#, but the
- About 2 to 50 g/l from an economical point of view, such as the yield of hydroxy 4AD, culture conditions, and operability! A range of is preferred.

The culture method is basically the same as that used for aerobic culture of general mimic organisms.

Usually, a shaking culture method or an aerated agitation culture method using a liquid medium is used. The medium may contain a nutrient source that can be assimilated and utilized by bacteria belonging to Pseudomonas putida that produce 12β-hydroxy 4AD using the above deoxycholic acid and/or its salt as a substrate. Although deoxycholic acid and/or its salt may be used as the sole carbon source, it is preferable to use deoxycholic acid and/or its salt in combination with crucose, glycerin, peptone, meat extract, malt extract, yeast extract, etc. It is better.

As the nitrogen source, for example, an inorganic nitrogen source such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, sodium nitrate, potassium nitrate, or an organic nitrogen source such as polypeptone, peptone, meat extract, etc. can be used. In addition, this ability includes dipotassium hydrogen phosphate and dibasic phosphate.
Inorganic salts such as potassium hydrogen and magnesium sulfate are added.

Although there are no specific culture conditions, shaking culture or aerated agitation culture is usually performed at 25 to 55°C for 10 hours to 7 days.

In order to separate and collect the 12β-hydroxy 4AD accumulated in the culture solution in this way, the culture solution is made alkaline and then an organic solvent that dissolves the 12β-hydroxy 4AD and phase separates from water, such as ethyl acetate, is used. , chloroform, a mixture of chloroform and methanol, etc. are used for extraction. This extraction with an organic solvent may be performed on the culture fluid containing the bacterial cells, or the insoluble components of the bacterial cells in the culture fluid may be separated and removed by filtration or centrifugation in advance, and the resulting culture The filtrate or supernatant may be used. The extract thus obtained was collected and the solvent was distilled off.
Almost the entire amount of 2β-hydroxy 4AD can be recovered. The obtained extract contains 12β-hydroxy 4AD
It contains almost no residual substrate deoxycol and/or its salts and by-products, and highly pure 12β-hydroxy 4AD can be easily obtained by recrystallizing from ethyl acetate, dichloromethane/methanol, etc. can be obtained.

12β-hydroxy 4AD obtained by the method of the present invention
By chemically removing the hydroxyl group at the 12-position, 4-androstene-3,1 is produced, which is a raw material for the synthesis of steroids such as subironofucton, which is useful as a diuretic.
7-dione. For example, 12β-
Hydroxy 4AD to 2-methy)v-2-ethyl 1v-L3
- By reacting with K, 12β-hydroxy-5-androstene-3,17-sion-3,17-bis(ethylene aceter) v) in which the keto groups at positions 3 and 17 are protected is obtained (G , Rosenkranz
et al., J.

Am, Ohem, Soc, + 76 volumes 5024-5
See page 026 (1954)]. This aceter ~ is p-
The 12-position hydroxyl group is tosylated by treatment with toluenesulfonyl chloride, and then reduced with lithium triethylborohydride to eliminate the 12-position tosyloxy group, or after tosylation, sodium iodide is applied. The tosyloxy group was replaced with iodine, then catalytically reduced with metallic zinc, and then treated with mineral acid/acetone to replace the 3- and 1-positions.
By removing the protecting group at the 7-position, cy-4-androstene-5+17-dione can be obtained.

The present invention will be explained in more detail below with reference to Examples and Reference Examples.

Reference example Pseudomonas futida D4014-AI615 strain? ! Method Medium 1 (composition: deoxycholic acid o, s
aA, sodium hydroxide 0.05%, peptone 0.5
Medium 2 (composition: Deoxycholic acid 2%, sodium hydroxide 0.2%, ammonium nitrate 0.2, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%
, magnesium sulfate heptahydrate 0.02% and yeast extract 0.02%), and incubated at 30°C for 14 to 30 minutes.
The culture was carried out with shaking for 15 hours. This culture! Medium 5 (composition: deoxycholic acid 0
．． 5%, sodium hydroxide 0.05-, glucose o,
1%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0.1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, and yeast equinu 0.02%
) and cultured at 30°C for 8 to 9 hours.

Next, the cells in the logarithmic growth phase were collected by aseptic filtration using a 0.45μ membrane filter, washed with 0.1M phosphate buffer (pH near, 0) 20*, and then washed with the same buffer 251WIK. suspended. Add to this a final concentration of 50μf/
Mutation treatment was carried out by adding N-methyl-N'-nitro-N-nitrosoquanidine so as to obtain me and leaving it for 3 to 4 minutes. The mutant-treated bacterial cells were collected by filtration using a 0.45μ membrane filter, and the cells were collected using a 0.45μ membrane filter.
After washing with 20 ml of 1M phosphate buffer (pH: 7.0), it was suspended in 20 ml of the same buffer. The obtained bacterial suspension was diluted with sterile physiological saline and mixed with medium 4 (composition:
Deoxycholic acid 0.5%, sodium hydroxide 0.05
%, ammonium nitrate 0.2%, potassium dihydrogen phosphate 0
．． 1%, dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02%, yeast extract 0.02%, and agar 1.5%). 500 to 1000 colonies appeared on an agar plate. After applying it in such a way that it
It was cultured for 4 days. After isolating a microscopic colony among the colonies that appeared on a slant of medium 1, a platinum loop thereof was prepared in a test tube in advance in medium 5 (composition: 0.0% deoxycholic acid).
2%, sodium hydroxide 0.02%, glucose 0.1
%, ammonium nitrate a, 2%, potassium dihydrogen phosphate 0
．． 1-1 dipotassium hydrogen phosphate 0.6%, magnesium sulfate heptahydrate 0.02% and yeast extract 0.02%)
The cells were inoculated into 10*/cells and cultured with shaking at 30°C for 24 hours. The products in each of the obtained culture solutions were assayed by thin layer chromatography, and one strain was found that selectively accumulated the target 12β-hydroxy 4AD, and this was isolated from Pseudomonas putida D4014-AI615. It was named.

Example 1 Pseudomonas putida D4014-A1615 strain (
Microtechnical Research Institute No. 6572) was cultured using the following method.

Deoxycholic acid 0.5g, glucose 0.1g, ammonium nitrate 0.2y, fJQ acid potassium dioxygen hydrogen 09. Add 50 ml of tap water to 0.6 g of dipotassium hydrogen phosphate, 0.02 g of magnesium sulfate heptahydrate, and 0.02 g of yeast extract, and adjust the pIl of this aqueous solution to 8.4 with 1N aqueous sodium hydroxide solution. After adjusting the volume, tap water was further added to make the volume 100 m/bin, and this was used as a culture medium. This medium was placed in a 500 me capacity Sakaro flask and steam sterilized at 120°C for 15 minutes. 10 ml of the inoculum grown in advance in the same medium as the above medium for 24 hours in a test tube shaker was added to the above 500 capacity Sakaguchi flask, and cultured with shaking at 30°C for 2 hours. After culturing, collect this culture solution, remove bacterial cells and insoluble matter using a centrifuge,
The pH of the culture supernatant was adjusted to around 12 by adding a 1N aqueous sodium hydroxide solution to the culture supernatant. Then, the culture supernatant was diluted with 300 liters of ethyl acetate.
#/, and remove ethyl acetate from the extract using a rotary tube.
By distilling it off with an evaporator, 332 volumes of 12β-hydroxy 4AD were obtained.

The bacterial cells and insoluble matter removed from the above culture solution were mixed with methanol/
L/50 ml to dissolve the insoluble matter, and the resulting methanol solution was centrifuged in an I11 machine to obtain a methanol supernatant. By distilling methanol off from this supernatant using a rotary evaporator,
2α-Hydroxylignar 4-en-3-one-20-
48.1% I of carbaldehyde was obtained.

Take a part of the obtained 12β-hydroxy 4AD, add methanol to it to make a 2% solution, and add 25 μl of this solution.
High performance liquid chromatography (HLO-
GPO-244). pH 4 as mobile phase,
A mixed solution of water/methanol with a volume ratio of 50,770 adjusted to 0 was flowed at a flow rate t me of 7 minutes, and detection was performed using a refractive index method. The area ratio of each peak in the obtained liquid chromatograph was determined using an integrator (Shimadzu Chromatopack C-RIA, manufactured by Shimadzu Corporation), and the purity of the above 12β-hydroxy, sAD was determined from this area ratio, and it was found to be 93%. Met.

In addition, 12β-hydroxy 4AI) obtained above and human 2α-hydroxybrebnar-4-en-3-one-
20-carbaldehyde was confirmed by the following method.

12β-hydroxy 4AD Melting point: 185-186°C Mass spectrum/l/m/Z: 302 (M)
-287(M-CI(3).284(M-H2C)J.) Furthermore, the presence of m/Z' = 124 and cy-3-keto-4-ene was confirmed.

cDOg5゜, NMR spectrum yv (90MI(z)δHM
8'0.86 (3H,s) 1B-CH5
1,16(sl(,S) 19-0Hs4.12 (1
1:I, 5) 12β-0■5.72 (IH,s) 4
-H Melting point: 179-181°C Mass tl' nl, /Z: 344 (M)
-326(M-H2O) 316 (M-CO) Also, the presence of 3-keto-4-ene was confirmed from m/Z = 124.

NMR spectrum/I/('90MHz) δoDo1
3゜HM8.

0.72 (3H, 8) 18-0Hs1.10 (
5[L d) 21-CH31,15(3H,s)
19-CH53,95 (11i, 5) 12β-1t 5.70 (11(,S) 4-ii9.40
(1]: L, d,) 22-C■0 Patent applicant
RiRashi Co., Ltd. Agent Patent Attorney Ken Honda

Claims (1)

[Claims] 12β using deoxycholic acid and/or its salt as a substrate
- a bacterium belonging to Pseudomonas putida that produces hydroxyandrost-4-ene-3,17-dione,
12β-Hydroxyandroster-4-ene! 1
．． 12β-hydroxyandrost-4-ene, which is characterized by producing 17-dione tl and collecting it! 1.17-Dione production method.