Organic compounds -- part of the class 532-570 series – Organic compounds – Cyclopentanohydrophenanthrene ring system containing
Reexamination Certificate
2000-07-31
2002-06-25
Marx, Irene (Department: 1651)
Organic compounds -- part of the class 532-570 series
Organic compounds
Cyclopentanohydrophenanthrene ring system containing
C552S546000, C552S554000, C552S582000, C435S052000, C435S252100
Reexamination Certificate
active
06410759
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for hydroxylating cholesterol by the action of microorganisms, and more specifically to a method for preparing one or more of either 25-hydroxycholesterol, 17,25-dihydroxycholesterol or 25,26-dihydroxycholesterol from cholesterol. The invention also relates to the aforementioned new dihydroxycholesterols.
BACKGROUND ART
For a biological method, in particular, a method for preparing hydroxy-derivatives of steroids including cholesterol by means of microorganisms, a method by which cholesterol is converted into 25-hydroxycholesterol using microorganisms of the genus Streptomyces has been disclosed in Japanese Laid-Open Patent Publication No. 1,23997/95. Also, in the biological conversion, which is interesting, of compounds other than steroids, there are known the methods for preparing 25-hydroxyvitamin D compounds by the hydroxylation of vitamin D compounds using microorganisms, for example,
Nocardia autotroihica, Streptomyces roseosporus, Amycolata saturnea, Amycolata autotrophica
, Sphingomonas sp. (Japanese Laid-Open Patent Publication Nos. 166090/92, 241197/95).
It is known that cholesterol may be, for example, an intermediate on the chemical synthesis of various kinds of vitamin D compounds (Yuki Gosei Kagaku (Organic Synthetwic Chemistry) 37, 809-829 (1979)), and compounds wherein one or more of the specific sites of cholesterol is/are hydroxylated beforehand may be expected for their use as intermediates on the synthesis of various hydroxylated vitamin D compounds. Indeed, according to the conversion method using microorganisms described in the abovementioned Japanese Laid-Open Patent Publication No. 123997/95, it has been published that cholesterol can be selectively hydroxylated at the 25-position which is preferable in relation to activated vitamin D.
However, the hydroxylation efficiency, according to the method described in said patent publication, is not always satisfactory. From the view point of improving the water-solubility of final compounds derived from cholesterol, for example, vitamin D compounds, it would be also desired to provide not only mono-hydroxylated cholesterols but also further hydroxylated cholesterols, for example, dihydroxylated cholesterols.
The purpose of the present invention is therefore to provide a method for efficiently preparing mono-hydroxylated cholesterols, in particular 25-hydroxycholesterol, and a method for preparing further hydroxylated dihydroxycholesterols, and new dihydroxycholesterols per se.
DISCLOSURE OF INVENTION
In the course of intensive study for the accomplishment of the above purpose, the present inventors have found that microorganisms of genera other than the genus Streptomyces described in the abovementioned Japanese Laid-Open Patent Publication No. 123997/95 hydroxylate cholesterol not only at the 25-position but also at the 17- or 26-position.
Thus, the above purpose can be achieved by providing a method, by which cholesterol having formula (I)
is biologically converted into hydroxylated cholesterols of formula (II)
(in which R
1
is a hydroxyl radical, and R
2
and R
3
are a hydroxyl radical and a hydrogen atom, respectively, or a hydrogen atom and a hydroxyl radical, respectively), for preparing hydroxylated cholesterols having the formula (II), according to.the invention, comprising
(A) a step in which the aforementioned biological conversion can be carried out, and in which cholesterol having the formula (I) is treated by incubation in the presence of a microorganism, chosen from those that belong to the genus Amycolata and the genus Sphingomonas, or its preparation from cultures and oxygen, and
(B) a step in which at least one of the hydroxylcholesterols having the formula (II) is recovered from the incubation-treated solution.
Among the hydroxylated cholesterols having the formula (II), dihydroxycholesterols of formula (II-b)
(in which R
2
and R
3
are a hydroxyl radical and a hydrogen atom, respectively, or a hydrogen atom and a hydroxyl radical, respectively) can be also prepared by another method in which 25-hydroxycholesterol is substituted for cholesterol as a starting material in the above method.
Furthermore, dihydroxycholesterols having the above formula (II-b), that is, 17,25-dihydroxycholesterol and 25,26-dihydroxycholesterol, are compounds that have not been published in literature in the prior art. Therefore, according to the present invention, new compounds, dihydroxycholesterols having the formula (II-b), are also provided.
DETAILED DESCRIPTION OF THE INVENTION
In the biological conversion according to the present invention, microorganisms and their preparations from cultures can be used, regardless of the kinds of species and strains, provided that they are microorganisms belonging to the genus Amycolata and the genus Sphingomonas and having the capacity to convert cholesterol of the above formula (I) into hydroxylated cholesterols of the formula (II). Mention can be made, as preferred microorganisms,
Amycolata saturnea
having the abovementioned conversion capacity, in particular, the microorganisms that have been deposited at the National Institute of Bioscience and Human Technology, the Agency of Industrial Science and Technology, the Ministry of International Trade and Industry in Japan with the deposition numbers of FERM BP-5544 (deposited Aug. 7, 1995) and FERM BP-2307 (deposited Feb. 27, 1989), and
Amycolata autotrophica
, in particular, the strain that has been deposited at American Type Culture Collection in the United States with the deposition number of ATCC 33796 (on deposit since 1996).
Species that belong to the genus Sphingomonas,
mali
IFO 15500,
paucimobilis
IFO 13935,
parapaucimobilis
IFO 15100,
vanoikuyae
IFO 15102,
adhaesiva
IFO 15099,
capsulata
IFO 12533,
sanguis
IFO 13937,
macrogoltabidus
IFO 15033 and
terrae
IFO,15098, although they are inferior in conversion capacity compared to that of microorganisms belonging to the above-described genus Amycolata, can be also used. Here, IFO is a deposition number in the Institute for Fermentation in Japan.
According to the invention, cholesterol (the compound of formula (I)) and/or 25-hydroxycholesterol (the compound of formula (II-a)) being a starting material (or a substrate) will be treated by incubation in the presence of any of said strains or their mycelia from cultures and oxygen. This treatment can be carried out by adding a substrate, at the time of the cultivation of the above strain under the aerobic conditions, into a culture solution, or optionally by adding a substrate into a suspension of, for example, the mycelia as such or the homogenized preparations obtained from cultures of the above strains, followed by incubation with oxygen, for example, with air. The addition of a substrate into a culture solution may be performed either before the cultivation or at a certain period of time after the cultivation. The above mycelia can be prepared by inoculating any of the above strains into a medium containing nutrient sources, followed by aerobic cultivation.
The cultivation of a strain to obtain such bacterial preparation from cultures or the cultivation of a strain carried out with the addition of a substrate can be performed, in principle, in accordance with cultivation methods for general microorganisms, but it is usually preferable to be carried out under aerobic conditions such as shaking liquid culture, aerated and stirred culture, etc.
The media used for the cultivation may be those containing nutrient sources which can be utilized by microorganisms belonging to the genus Amycolata and the genus Sphingomonas, and any of various kinds of synthetic or semi-synthetic media, natural media and the like can be used. For the medium compositions, glucose, maltose, xylose, fructose, sucrose and the like can be used alone or in combination as carbon sources. For nitrogen sources, organic nitrogen sources such as peptone, meat extract, soybean meal, casein, amino acids, yeast extract, urea and the like, and inorganic nitrogen sources such as so
Dobashi Kazuyuki
Takeda Koji
Terasawa Tadashi
Yoshioka Takeo
Marx Irene
Mercian Corporation
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