JPH04356190A - Production of vitamin ds - Google Patents

Abstract

PURPOSE:To provide a method for directly introducing hydroxyl groups into the 24-, 25- and/or 26-positions of vitamin Ds according to easier operation. CONSTITUTION:The subject method is to produce 24,25-dihydroxyvitamin Ds, 25,26-dihydroxyvitamin Ds or 24,25,26-trihydroxyvitamin Ds characterized as follows. Vitamin Ds having hydrogen atoms at the 24-, 25- and/or 26-positions are added into a solution containing microbial cells of an actinomyces capable of hydroxylating the vitamins Ds or an enzyme produced thereby to convert the hydrogen atoms into hydroxyl groups.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention provides hydroxyvitamin D
This invention relates to a manufacturing method using microorganisms of the following types.

0002

[Prior art] Two types of vitamin D are produced by organic chemical methods.
It is extremely difficult to directly introduce a hydroxyl group into the 4- or 26-position, and no such example has been reported yet. As an enzymatic chemical method using microorganisms, vitamin D is ranked 1st and 2nd.
A method of directly introducing a hydroxyl group into the 5-position has been reported (JP-A-2-469). However, no method has been reported so far for directly introducing a hydroxyl group into the 24th, 25th, and/or 26th positions of vitamin D by an enzymatic chemical method using microorganisms.

On the other hand, it has hitherto been possible to directly introduce a hydroxyl group into the 24th and/or 26th positions of the raw material vitamin D by an enzymatic chemical method using animal organs. That is, a method using an enzyme extracted from a kidney homogenate fraction of an animal such as a chicken [introduction of a hydroxyl group at the 24-position:
Biochemistry, Vol. 13, p. 1543 (1974), etc.; Introduction of 26-position hydroxyl group; Biochemical and Biophysical Research Communication (Biochem)
．． Biophys. Res. Commun. ), No. 83
Volume, page 7 (1978), etc.] are known.

0004

[Problems to be Solved by the Invention] However, the enzymatic chemical method using animal organs requires a large amount of animal kidney, and its preparation is time-consuming, making it inefficient and not a practical production method. . An object of the present invention is to provide a method for directly introducing a hydroxyl group into the 24th, 25th, and/or 26th positions of vitamin Ds through easier operations.

[0005]

[Means for Solving the Problems] The present inventors have discovered that the above object can be achieved by utilizing a specific microorganism, and have completed the present invention. The present invention involves adding vitamin D having hydrogen atoms at the 24th, 25th, and/or 26th positions to a solution containing actinomycete cells or enzymes produced by actinomycetes that hydroxylate vitamin D. This is a method for producing 24,25-dihydroxyvitamin D, 25,26-dihydroxyvitamin D, or 24,25,26-trihydroxyvitamin D, which is characterized by converting hydrogen atoms into hydroxyl groups.

[0006] The production method of the present invention is a method for producing 24 vitamin Ds.
In this method, a hydroxyl group is introduced directly into the 24th, 25th, and/or 26th positions in one step, and any substituent other than the 24th, 25th, or 26th position (for example, a halogen atom such as fluorine, a hydroxyl group, or a lower alkyl group, etc.). Therefore, in the present invention, vitamin Ds are compounds of the vitamin D2, D3, D4, D5, D6, and D7 series, and these include, for example, vitamin D2, vitamin D3,
, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, 24,25-dihydroxyvitamin D3,
23,25-dihydroxyvitamin D3, 25,26-
Dihydroxyvitamin D3, 23,24,25-trihydroxyvitamin D3, 24,24-difluoro-25
-Hydroxy-26,27-dimethylvitamin D3,2
5-hydroxy-26,26,26,27,27,27
-Hexafluorovitamin D3, etc.

The actinomycetes used in the present invention are actinomycetes that have the ability to introduce hydroxyl groups into the 24th, 25th and/or 26th positions of vitamin D compounds. They are e.g. Actinomyces spp., Arcanobacterium spp.
Genus, Arthrobacter, Micrococcus, Renibacterium, Rothia, Stoma
tococcus), Brevibacterium (Brev)
ibacterium), Cellulomonas (Cellu
romonas) genus, Honesia (Jonesia) genus,
Genus Oerskovia, Genus Promicromonospora,
Genus Dematophilus, Genus Blasstococcus, Genus Microbacterium,
Genus Agromyces, Genus Aureobacterium, Genus Clavibacter, Genus Curtobacterium, Genus Actinoplanes, Genus Ampullariella. , Dactylosporangium Genus, Micromonospora
Genus, Pilimeria, Microtetraspora, Streptosporanium
gium) genus, Nonomuria genus,
Planobispora genus, Planomonospora genus, Pseudonocardia genus, Actinopolyspora genus
a) Genus, Amycolatopsis
s) genus, Micropolyspora
ra), Saccharopolyspora
Genus lyspora, Genus Frankia, Actinosporanium
Corynebacterium spp.
cterium), Joldermatophilus (Geo
dermatophilus genus, Mycobacterium genus, Nocardia (Nocardia) genus
cardia), Rhodococcus
us) genus, Streptomyce
s), genus Streptosporandium (Streptosporandium)
porangium), Thermomonospora (Ther
genus momonospora, Actinomadura (Ac
tinomadura), Saccharomonospora (Sa
ccharomonospora), Spirillospora (Spirillospora), Kitasatospora (
Genus Kitasatospora, Genus Saccharothrix, Genus Catenuloplanes, Genus Elytrosporangium, Genus Microellobospora.
), Kineospora, Microstreptospora
a) Genus, Actinoall
oteichus), Amorphosporandium (Am
genus orphosporangium, genus Spirillospora, genus Actinopycnidium, genus Chainia, genus Amycola
ta), Streptoverticillium (Strept.
overticillium), Nocardioides (
Genus Nocardioides, Nocardiopsis (N
It belongs to the genus (Ocardiopsis), etc.
Preferably Streptomyces sclerotiarus
FERM BP-1370, Amycolata autotrophica ATCC 13181, Amycolata autotrophica ATCC 19727, Amycolata autotrophica ATCC 33794, Amycolata autotrophica ATCC 33795, Amycolata autotrophica ATCC 33 796, Amycolata autotrophica ATCC 33797, Amycolata saltunae ATCC 15778 and Amycolata hydrocarbonoxy Dance ATCC
It is 15104.

[0008] Streptomyces sclerotiarus
FERM BP-1370 is a preserved strain of the Institute of Microbiology, Agency of Industrial Science and Technology, and its bacteriological properties have already been clarified (Japanese Patent Application Laid-Open No. 2-469).

[0009] Also, the above-mentioned Amycolata autotrophica, Amycolata saltunae ATCC 15778
and Amycolata hydrocarbonoxidans A
TCC 15104 is part of the American Type Culture Collection.
It is a preserved strain of the Lture Collection, and its mycological properties have already been clarified.

The method of the present invention is carried out by reacting the substrate vitamin D under aerobic conditions in a solution containing actinomycete cells or enzymes produced by the actinomycetes. In order to obtain the actinomycete cells necessary for the reaction, the bacteria are cultured in a medium under aerobic conditions. The medium is mainly a liquid medium, and the carbon sources are glucose, maltose, dextrose, starch, and arabinose. Xylose is used alone or in combination. As the nitrogen source, polypeptone, casamino acid, yeast extract, meat extract, corn steep liquor, soy peptone, soy bean meal, etc. are used alone or in combination. In addition, organic substances and inorganic salts that aid the growth of this strain and promote the production of vitamin Ds having a hydroxyl group introduced at the 24th, 25th, and/or 26th positions may be added as necessary. Suitable culture methods include aerobic culture such as shaking culture and aerated agitation culture.
Culture at pH 5-9, 20-37°C for 1-10 days.

[0011] A solution containing bacterial cells obtained by this culture is used in the production method of the present invention. That is, the culture solution containing the bacterial cells being cultured is used as is, or after the cultivation is completed, a solution in which the bacterial cells separated by centrifugation or filtration are suspended is used. Alternatively, a solution obtained by crushing the cells obtained after culturing and removing the cells by centrifugation or the like is used. Furthermore, the bacterial cells are made of a photocrosslinkable resin prepolymer, such as ENT3.
400 [trade name; manufactured by Kansai Paint Co., Ltd.], urethane prepolymers such as PU-9 [trade name; manufactured by Toyo Tire & Rubber Co., Ltd.], etc., or polysaccharides such as κ-carrageenan, and then fixed in a solution. May be added to.

[0012]Furthermore, freeze-dried products of the above-mentioned bacterial cells can also be used in the same manner as above. The solution used in the present invention is the above-mentioned medium, or tris-acetic acid, tris-hydrochloric acid, sodium succinate-succinic acid, potassium succinate-succinic acid, sodium citrate-citric acid,
Buffers such as phosphoric acid-sodium phosphate, sodium cacodylate-hydrochloric acid, imidazole-hydrochloric acid, and boric acid-borax may be used alone or in combination. In addition, surfactants, organic substances, and inorganic salts that promote the production of the target vitamin Ds may be added to the buffer solution as necessary.

The production method of the present invention is suitably carried out under aerobic conditions by subjecting the solution containing the bacterial cells to shaking operations, aeration stirring operations, etc., and pH 5 to 9, 20 to 37.
Stir and shake at <RTIgt;C</RTI> for 5 minutes to 96 hours. Moreover, this reaction can be carried out under an oxygen stream. Add an appropriate amount of the substrate vitamin D at the beginning of stirring and shaking. In addition, when using a culture solution containing bacterial cells that are being cultured, the main reaction is carried out by adding the substrate vitamin D and then culturing for 5 minutes to 96 hours under the same conditions.

[0014] When vitamin D having hydrogen atoms at the 24, 25, and 26 positions is used as a raw material, the reaction time is determined by confirming the product by high performance liquid chromatography, etc. described later, and the 24, 25, 26 -Trihydroxyvitamin D
can produce similar products.

[0015] Vitamin D produced by these reactions
In order to isolate vitamin Ds, it may be carried out according to a general method for collecting vitamin Ds from blood. For example, after the reaction is completed, the reaction solution is extracted with an organic solvent such as ethyl acetate, methylene chloride, or chloroform, and concentrated to dryness. This 2
- After dissolving in a suitable solvent such as n-hexane containing propanol and removing insoluble matter by centrifugation, high performance liquid chromatography using a silica gel normal layer column (e.g. Zorbax SIL, manufactured by DuPont, USA) or silica gel Reverse phase columns (e.g. Zorbax ODS
, manufactured by DuPont, USA), or Sephadex LH-
The target hydroxyvitamin D can be isolated by subjecting it to partition chromatography using 20 (manufactured by Pharmacia, Sweden) or silica gel chromatography using a Rover column (manufactured by Merck, Switzerland). . In this case, the retention time in each chromatography is determined by measuring the ultraviolet absorption spectrum (maximum ultraviolet absorption 265 nm) of each fraction.

[0016]

[Effect of the invention] By the method of the present invention, two types of vitamin D can be obtained.
It became possible to directly introduce a hydroxyl group into the 4, 25 and/or 26 positions. That is, the method using microorganisms of the present invention requires no time and effort to prepare microorganisms, reaction solutions, etc., and can be carried out in one step in a short time, making it extremely easy and efficient.

[0017]

[Examples] The present invention will be explained in more detail below with reference to Examples.

Example 1 A sterile liquid medium containing 1.5% glucose, 1.5% Bactosoitone (trade name; manufactured by Difco), 0.5% corn steep liquor, 0.5% sodium chloride, and pH 7.0 (hereinafter referred to as A platinum loop of Streptomyces sclerothiarus FERN BP-1370 strain was inoculated into each of five 500 ml Erlenmeyer flasks containing 50 ml of medium A, and cultured with stirring and shaking at 28° C. for 48 hours. After the culture is completed, the culture solution is centrifuged to collect the bacterial cells, which are then added to a pH 7.4 buffer (hereinafter abbreviated as buffer B) consisting of 15 mM Tris-acetic acid, 25 mM sodium succinate, and 2 mM magnesium acetate. Suspend in 200ml, 5
40 ml each was dispensed into five 00 ml Erlenmeyer flasks and kept at 28° C. for 5 minutes. 5 mg dissolved in 250 ml of ethanol in each of the five Erlenmeyer flasks.
Add the substrate 25-hydroxyvitamin D3 and heat at 28°C.
The mixture was stirred and shaken for 45 minutes. The reaction liquid in each Erlenmeyer flask was collected, extracted with 400 ml of methylene chloride, the methylene chloride layer was dried under reduced pressure at 40°C or lower, and then immediately dissolved in 2 ml of a mixed solvent of 2-propanol:n-hexane = 1:9.
It was left at 20°C overnight. This was centrifuged to remove the insoluble fraction to obtain a supernatant. This supernatant liquid was concentrated under reduced pressure at 40°C or lower, and subjected to high-performance liquid rest chromatography [Zorvax S
IL, manufactured by DuPont, USA].

Column size: 4.6 mmφ x 25 cm Elution solvent: 2-
Propanol: n-hexane = 1:9 Column temperature: 25
°C, Elution rate: 1.5 ml/min Photodiode array detector (Waters M990
, manufactured by Nippon Waters Co., Ltd.).

After elution, a fraction of a peak around a retention time of 5.2 minutes (hereinafter referred to as peak 1) and a peak around a retention time of 7.2 minutes (hereinafter referred to as peak 2) coincide with the ultraviolet absorption pattern of vitamin D. ) was collected. Then peak 1 is 4
Concentrate under reduced pressure at below 0°C and perform high performance liquid chromatography [
Zorbax ODS, manufactured by DuPont, USA].

Column size: 4.6 mmφ x 25 cm Elution solvent: Water:
Methanol = 1:9 Column temperature: 40°C Elution rate: 1.0 ml/min Photodiode array detector (Waters M990
, manufactured by Nippon Waters Co., Ltd.).

After elution, fractions with a peak at a retention time of around 3.8 minutes, which matched the ultraviolet absorption pattern of vitamin D compounds, were collected. This was concentrated to dryness under reduced pressure at 40° C. or below while purging with nitrogen gas to obtain 150 ml of 24,25-dihydroxyvitamin D3. This is commercially available 24(R)
, 25-dihydroxyvitamin D3 (manufactured by Dufur,
The retention time (Zorbax SIL), ultraviolet absorption spectrum, and mass spectral cleavage pattern in liquid chromatography completely matched those of the standard product from the Netherlands.

Maximum ultraviolet absorption: λmax=265 nm (ethanol) EI-MS (
m/z): 416 (M+), 398 ([M-H2O]+), 380
([M-2H2O]+),271,253,145,1
36,118,59,55

Next, peak 2 was concentrated under reduced pressure at a temperature below 40°C and subjected to high performance liquid chromatography [Zorbax ODS,
DuPont, USA].

Column size: 4.6 mmφ x 25 cm Elution solvent: Water:
Methanol = 2:23, column temperature: 40°C, elution rate: 1.0 ml/min, measured with a photodiode array detector (Waters M990, manufactured by Nippon Waters Co., Ltd.).

After elution, fractions with a peak at a retention time of around 5.0 minutes, which matched the ultraviolet absorption pattern of vitamin D compounds, were collected. This was concentrated to dryness under reduced pressure at 40° C. or lower while purging with nitrogen gas to obtain 400 ml of 25,26-dihydroxyvitamin D3. This is a standard specimen of the metabolite 25,26-dihydroxyvitamin D3 obtained by homogenizing the kidney or liver of a chicken chick (white leghorn, 4 weeks old) [Citation: Methods in Enzymology] Enzy
mology), Volume 67, Page 370 (1980)
and Biochemical and Biophysical Research Communication (Biochem).
．． Biophys. Res. Commun. ), No. 83
Vol., page 7 (1978)] and the retention time of liquid chromatography [Zorbax SIL], ultraviolet absorption spectrum, and mass spectral cleavage pattern completely matched.

Maximum ultraviolet absorption: λmax=265 nm (ethanol) EI-MS (
m/z): 416 (M+), 398 ([M-H2O]+), 380
([M-2H2O]+) , 271,253,1
45,136,118,109,75

Example 2 Streptomyces sclerotiarus FERM was added to one 500 ml Erlenmeyer flask containing 50 ml of medium A.
BP-1370 strain was inoculated one platinum loop at a time and incubated at 28℃ for 7 days.
Culture was carried out with stirring and shaking for 2 hours. Next, 50 ml of the seed culture solution was inoculated into 3 liters of medium A using a culture jar with a capacity of 5 liters, and cultured with aeration at 28° C. for 48 hours. 3300 mg of the substrate 25-hydroxyvitamin D dissolved in 10 ml of ethanol was added to this 48-hour culture solution, and this was again added to 25-hydroxyvitamin D.
Aerated culture was performed at 8°C for 24 hours. After the culture is completed, the culture solution is centrifuged to separate the bacterial cells and the filtrate, and the bacterial cells are separated by Bligh and Dyer (
The filtrate was extracted using Amberlite XAD-7 [trade name: Organo Co., Ltd.].
After adsorption to 50 ml of the product, the mixture was eluted with methanol. The bacterial cell extract fraction and Amberlite
This was dissolved in 100 ml of water and subjected to Sephadex LH-20 (manufactured by Pharmacia, Sweden) chromatography. Elution solvent; methylene chloride: n-hexane = 7:3 (elution volume: approximately 3 L) Column volume: 400 ml

The product was confirmed by thin layer chromatography using silica gel (Kieselgel 60 F254, manufactured by Merck & Co.). 24(R), 25- commercially available as standard products
A preparation of dihydroxyvitamin D3 (manufactured by Dufer, Netherlands) and the metabolite 25,26-dihydroxyvitamin D3 obtained by homogenizing the kidney or liver of a chicken chick (white leghorn, 4 weeks old) was used for this purpose. . Developing solvent: chloroform:methanol = 20:1 Spots were detected by ultraviolet absorption.

After elution, fractions containing 24,25-dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3 were collected. After concentrating these to dryness under reduced pressure at 40°C or lower, immediately 2-propanol: n-hexane = 3:
A silica gel column prepared with the same mixed solvent (Rover column size B25) was prepared using the same mixed solvent.
It was adsorbed onto a 310 mm (mmφ×310 mm) LicroPrep Si60 (trade name, manufactured by Merck & Co., Ltd.).

This silica gel column was eluted with 1 ml of the same mixed solvent with which the column was prepared, and the eluted fractions of 24,25-dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3 were eluted with silica gel (Kieselgel 60).
This was confirmed by thin layer chromatography using F254 (manufactured by Merck & Co.), and each eluted fraction was collected and concentrated to dryness.

1 to each of these concentrated and dried fractions.
．． After adding 0 ml of n-hexane and further sealing with nitrogen gas, it was left to stand at 4°C for 3 days, and 5 mg of 24,25-dihydroxyvitamin D3 (white needle-shaped crystals) and 25,26
-310 mg of dihydroxyvitamin D was obtained. These are commercially available 24(R),25-dihydroxyvitamin D3 (manufactured by Dufer, Netherlands) and the metabolite 25, which is obtained by homogenizing kidney or liver of chicken chicks (white leghorn, 4 weeks old). The retention time of liquid chromatography [Zorvax SIL and Zorbax ODS, manufactured by DuPont, USA], ultraviolet absorption spectrum, mass spectral cleavage pattern, and 13C-NMR spectrum completely matched with the standard sample of 26-dihydroxyvitamin D3.

24,25-dihydroxyvitamin D3;
Maximum ultraviolet absorption: λmax=265 nm (ethanol) EI-MS (
m/z): 416 (M+), 398 ([M-H2O]+), 380
([M-2H2O]+),271,253,145,1
36,118,59,55 13C-NMR (100MHz, CD3OD, δ)
33.5 (C-1), 122.3 (C-6), 118.
7 (C-7), 12.4 (C-18), 112.5 (C
-19), 19.2 (C-21), 28.6 (C-23
), 79.4 (C-24), 73.6 (C-25), 2
5.0 (C-26), 25.4 (C-27)

0034
]25,26-dihydroxyvitamin D3; maximum ultraviolet absorption: λmax=265 nm (ethanol) EI-MS (
m/z): 416 (M+), 398 ([M-H2O]+), 380
([M-2H2O]+),271,253,145,1
36,118,109,75 13C-NMR (100MHz, CD3OD, δ) 33
．． 5 (C-1), 122.4 (C-6), 118.8 (
C-7), 12.4 (C-18), 112.5 (C-1
9), 19.4 (C-21), 21.0 (C-23),
39.4 (C-24), 73.5 (C-25), 70.
3 (C-26), 23.6 (C-27)

Example 3 In the same manner as in Example 2, 24,25,26-trihydroxyvitamin D3 was obtained from 24,25-dihydroxyvitamin D3. Column size: 4.6 mmφ x 25 cm Elution solvent: 2-
Propanol: n-hexane = 7:43 Column temperature: 2
5°C, elution rate: 1.5 ml/min Measured with a photodiode array detector (Waters M990, manufactured by Nippon Waters Co., Ltd.) Retention time of product sample: 5.7 minutes

Maximum ultraviolet absorption: λmax=265 nm (ethanol) EI-MS (
m/z): 432 (M+), 414 ([M-H2O]+), 396
([M-2H2O]+),271,253,136,1
18,59

Trimethylsilyl ether derivatives are obtained by treating the 24,25,26-trihydroxyvitamin D3 obtained above with N,O-bis-trimethylsilyltrifluoroacetamide containing 1% trimethylsilyl chloride in pyridine at 30-50°C. and subjected it to mass spectrometry. EI-MS (m/z): 720 (M+
), 487, 397, 307, 208, 113, 118

Example 4 Amycolata autotrophica ATCC33797 was added to one 500 ml Erlenmeyer flask containing 50 ml of medium A.
One platinum loopful of each strain was inoculated and cultured with stirring and shaking at 28°C for 48 hours. Next, 50 ml of the above seed culture solution was inoculated into 3 liters of medium A using a culture jar with a 5 liter capacity, and
Cultured with stirring and shaking for hours. 3600 mg of the substrate 25-hydroxyvitamin D dissolved in 15 ml of ethanol and Tween 80 were added to the culture of Amycolata autotrophica ATCC 33797 which was undergoing logarithmic growth, and the culture was again cultured with stirring and shaking at 28° C. for 96 hours. After culturing, this culture solution is ▲filtered, and the bacterial cells are ▲filtered.
Divide into liquid and remove bacterial cells using Bligh & Dyer.
400 ml of a chloroform extracted fraction was obtained. In addition, ▲The filtrate is 150ml of Amberlite XAD-7 [trade name; manufactured by Organo Co., Ltd.]
After adsorption, it was eluted with 1 L of methanol. After collecting the chloroform extraction fraction and methanol elution fraction,
The solvent was removed by concentration under reduced pressure under a stream of nitrogen gas. This is 1
Dissolved in n-hexane containing 0% 2-propanol,
It was subjected to silica gel chromatography. Elution solvent; methylene chloride: n-hexane = 7:3 (elution volume: approximately 500 ml) Column volume: 50 ml

The product was confirmed by thin layer chromatography using silica gel (Kieselgel 60 F254, manufactured by Merck & Co.). 24(R), 2 commercially available as a standard product
5-dihydroxyvitamin D3 (manufactured by Dufar, Netherlands) and 25-hydroxyvitamin D3 (manufactured by Dufar, Netherlands) were added to chicken chicks (white leghorn, 4
A preparation of the metabolite 25(S),26-dihydroxyvitamin D3 obtained by reacting with a kidney or liver homogenate of 18 weeks of age was used for this purpose. Developing solvent: 2-propanol:n-hexane=1:5 Spots were detected by ultraviolet absorption.

After elution, fractions containing 24,25-dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3 were collected. After concentrating these to dryness under reduced pressure at 40°C or lower, immediately dissolving them in 1 ml of methanol, using a reversed-phase silica gel column (Rover column size B 25 m
mφ×310 mm, trade name, manufactured by Merck & Co.). Elution solvent; acetonitrile:water = 13:7 (elution amount approximately 1
L)

After elution, the fractions containing 24,25-dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3 were each concentrated to dryness.

1 to each of these concentrated and dried fractions.
．． After adding 0 ml of n-hexane and further sealing with nitrogen gas, it was left to stand at 4°C for 3 days, and 2 mg of 24,25-dihydroxyvitamin D3 (white needle-shaped crystals) and 25,26
-3 mg of dihydroxyvitamin D3 (white needle-like crystals) was obtained. These are commercially available 24(R),25-dihydroxyvitamin D3 (manufactured by Dufer, Netherlands).
and 25-hydroxyvitamin D3 (Duffar, Netherlands) to chicken chicks (white leghorn, 4 weeks old).
The retention time, ultraviolet absorption spectrum, mass spectral cleavage pattern, and 13C-NMR spectrum of the metabolite 25(S),26-dihydroxyvitamin D3 obtained by reacting with renal or liver homogenate in liquid chromatography are as follows. It was a perfect match.

24,25-dihydroxyvitamin D3;
Maximum ultraviolet absorption: λmax=265 nm (ethanol) EI=MS(
m/z): 416 (M+), 398 ([M-H2O]+), 380
([M-2H2O]+),271,253,145,1
36,118,59,55 13C-NMR (100MHz, CD3OD, δ) 33
．． 5 (C-1), 122.3 (C-6), 118.7 (
C-7), 12.4 (C-18), 112.5 (C-1
9), 19.2 (C-21), 28.6 (C-23),
79.4 (C-24), 73.6 (C-25), 25.
0 (C-26), 25.4 (C-27)

[0044]2
5,26-dihydroxyvitamin D3; maximum ultraviolet absorption: λmax=265 nm (ethanol) EI-MS (
m/z): 416 (M+), 398 ([M-H2O]+), 380
([M-2H2O]+),271,253,145,1
36,118,109,75 13C-NMR (100MHz, CD3OD, δ) 33
．． 5 (C-1), 122.4 (C-6), 118.8 (
C-7), 12.4 (C-18), 112.5 (C-1
9), 19.4 (C-21), 21.0 (C-23),
39.4 (C-24), 73.5 (C-25), 70.
3 (C-26), 23.6 (C-27)

Example 5 24,25-dihydroxyvitamins D3 and 25 were prepared in the same manner as in Example 4, using the strains listed in Table 1.
, 26-dihydroxyvitamin D3 was obtained. 24, 25
The production amounts of -dihydroxyvitamin D3 and 25,26-dihydroxyvitamin D3 are shown in Table 1 along with the strains used.

[0046]

[Table 1]

Claims (5)

[Claims] Claim 1: In a solution containing actinomycete cells or enzymes produced by actinomycetes that hydroxylate vitamin D, 24th position,
24,25-dihydroxyvitamin D or 25, which is characterized by adding vitamin D having a hydrogen atom at the 25th or 26th position and converting the hydrogen atom into a hydroxyl group.
, 26-dihydroxyvitamin D production method. Claim 2: In a solution containing actinomycete cells or enzymes produced by actinomycetes that hydroxylate vitamin D, 24th position,
A method for producing 24,25,26-trihydroxyvitamin D, which comprises adding vitamin D having hydrogen atoms at the 25th and 26th positions and converting the hydrogen atoms into hydroxyl groups. [Claim 3] The actinomycetes are Chainia
), the genus Nocardia, the genus Streptomyces, and the genus Amycolata.
The manufacturing method described in. 4. The actinomycete is Streptomyces sclerothiarus.
tialus). 5. The actinomycetes are Amycolata autotrophica, Amycolata autotrophica, and Amycolata autotrophica.
aturnea) and Amycolata autotrophica (
Amycolata hydrocarbonoxi
3. The manufacturing method according to claim 1 or 2, wherein