JPH1142094A - Production of 9-hydroxyfluorene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To selectively obtain the subject compound useful as a material for resists, thermally resistant resins, pharmaceuticals and agrochemicals and as a reagent for separation and analysis, etc., by making a microbe, belonging to the genus bacillus, act on fluorenones to subject the fluorenones to reductive reaction. SOLUTION: This 9-hydroxyfluoren is selectively obtained by making a microbe, belonging to the genus bacillus [e.g. bacillus brevis (deposit No. FERM P-14525, etc.)], act on fluorenones in a 2-20 fold dituted medium in an non- oxidative atmosphere consisting of methane, hydrogen, nitrogen, etc., so as to subject the fluorenones to reductive reaction and collecting 9hydroxyfluorene from the reaction liquid so as to selectively the objective 9-hydroxyfluorene. This 9-hydroxyfluorene is useful as a material for synthesis of polymer compounds, such as resists and thermally resistant resins, as a reaction intermediate for synthesis of pharmaceuticals and agrochemicals, and as a reagent for separation and analysis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for producing 9-hydroxyfluorenes. 9-Hydroxyfluorenes obtained by the present invention are useful substances that can be used as raw materials for synthesizing polymer compounds such as resists and heat-resistant resins, synthetic intermediates for pharmaceuticals and agricultural chemicals, and reagents for separation and analysis.

[0002]

2. Description of the Related Art As a method for producing 9-hydroxyfluorenes, a method of treating fluorenones with a basic alcohol (Chem. Ber., 62 <1929>, p. 1333; Huntress; Seike)
l, J. Amer. Chem. Soc., 61 vol. <1939> pp. 816, 818), a method of reducing fluorenones in a basic atmosphere in the presence of a catalyst such as Raney nickel (Forrest; Tucker, J. Chem. .So
c., 1948, p. 1137, p. 1140) is common. But,
In these methods, the former is disadvantageous in handling because it uses a strong base and is low in yield, and the latter is uneconomical in terms of equipment because it is a reaction under conditions of high temperature and high pressure.

[0003] On the other hand, reactions using microorganisms and enzymes are economically advantageous because the microorganisms and enzymes act as catalysts, and can be industrially useful means by selecting the type of microorganism. However, 9-
There is no known example of producing hydroxyfluorenes.

[0004]

An object of the present invention is to provide a method for selectively producing 9-hydroxyfluorenes using a microorganism.

[0005]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, have found that the above-mentioned object can be achieved by allowing a microorganism belonging to the genus Bacillus to act on fluorenones and the like. Was.

That is, the present invention provides a process for producing hydroxyfluorenes, which comprises a step of causing a microorganism belonging to the genus Bacillus to act on a fluorenone to reduce the fluorenone, and a step of collecting 9-hydroxyfluorene from the reaction solution. About the law.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The fluorenone used as a substrate in the present invention includes, in addition to fluorenone, a fluorenone derivative having an alkyl substituent at at least one of the 1- to 8-positions of fluorenone. As the fluorenone derivative, for example, 1-methyl-9
-Fluorenone, 3-ethyl-7-methyl-9-fluorenone and the like.

The microorganism which can be used in the present invention is a gram-positive spore-forming bacillus belonging to the genus Bacillus of the family Bacillaceae, preferably Bacillus brevis and Bacillus cereus.
cillus cereus), particularly preferably the deposit number FERM P-14525 deposited with the Institute of Biotechnology and Industrial Technology on September 7, 1994 and the Biotechnology and Industrial Technology on March 7, 1994. Accession number FERM P-14210, deposited at the institute. Bacillus brevis and Bacillus cereus are facultative anaerobic bacteria resident in soil.They are distributed as spores in all soils, including arable land, rivers, parks, and forests. And oval spores are present in the center or slightly in the center of the cells.

In culturing the microorganism, a liquid medium containing a nutrient used for culturing the microorganism can be used.
As the nutrient source, meat extract, peptone and the like are used as a carbon source and a nitrogen source. Further, for example, inorganic substances such as phosphorus, magnesium, iron, manganese, and sodium chloride, vitamins, and the like may be appropriately mixed. Examples of such a medium include a liquid medium obtained by dissolving fish (bonito) meat extract, polypeptone, and sodium chloride in distilled water. The medium needs to be sterilized.
Sterilization can be performed by ordinary high-pressure steam sterilization or the like.

Before the microorganisms are used in the production method of the present invention, they are used under ordinary conditions, for example, about pH 5 to 9 (the lower limit is preferably pH 6.5 and the upper limit is preferably pH 7.5).
The pre-culture may be performed at a temperature of about 15 to 40 ° C. (a lower limit is preferably 25 ° C. and an upper limit is preferably 35 ° C.) for about 12 to 48 hours until the desired turbidity is obtained. The reaction may be carried out by directly adding the substrate to the pre-cultured culture, or the culture may be added in an appropriate amount to the reaction solution containing the substrate, or centrifuged after the pre-culture. May be collected and used for the reaction.

The type of liquid used in the reaction is not particularly limited, but distilled water, the above-mentioned liquid medium and the like may be used.

In the present invention, the term "diluted medium" and "diluted medium" mean a medium obtained by diluting a liquid medium containing a commonly used amount of a nutrient source. It is contained less than the amount given. Preferably 1/2 to 1/20 amount (upper limit is 1/20
5 volumes, preferably 1/10 volume as the lower limit). Medium 1000 of the liquid medium
The nutrient content in ml is, for example, fish meat (bonito) extract (manufactured by Wako Pure Chemical Industries, fish meat extract (manufactured by bonito), moisture 3
10 g of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd., total nitrogen 12.5 to 14.5%, amino acid nitrogen 5.0 to 6.5)
%) And 2 g of sodium chloride. The diluted liquid medium is preferably about 2 to 20 times (the lower limit is 5 times, and the upper limit is more preferably 10 times) diluted with sterilized water.

The amount of cells of the microorganism in the reaction solution is preferably selected so that the cells can appropriately contact the substrate and the reaction solution. 1 to 10 g cells / 100 ml reaction solution (the lower limit is 2 g cells / 100 ml reaction solution, 5 g cells /
(100 ml reaction solution is preferred). If the amount of the cells is small, the efficiency of contact with the substrate is low, and if the amount of the cells is too large, the efficiency of the reaction by the cells decreases.

As the cells, the cells pre-cultured may be used as they are, but the cells immobilized on a support can be used repeatedly, and the produced 9-hydroxyfluorenes and the like can be used repeatedly. Can be removed from the reaction solution by a circulation method, a batch method (batch method) or a semi-batch method, and a large amount of a substrate such as a fluorenone can be continuously reacted. Examples of the support material include various gelling bases including polysaccharides obtained from seaweed, such as carrageenan, alginic acid, and agar, or gelling ability of polyacrylamide, polyvinyl alcohol, polyacrylic acid (salt), and the like. And a water-absorbing polymer having: Among these support materials, it is particularly preferable to use carrageenan and alginic acid. The shape of the support is not particularly limited, but is preferably spherical. For example, as a procedure for immobilizing cells with gelation,
The liquid containing the support material is heated and dissolved, and then cooled, and then the cells are added at the time of gelation. After the support material is made into a solution, the cells are added, and the solution is added to a metal salt solution such as a sodium salt or a calcium salt. To form a gel by dropping the mixture. In the above, it is preferable that after the liquid containing the material for the support is dissolved by heating, immobilization is performed at 30 to 55 ° C. in consideration of the influence of the heat on the cells. When the procedure using carrageenan as the support material is performed, the immobilization may be performed according to the following method. 1.0 to 2.kappa.-carrageenan per 120 ml of distilled water.
0 g (lower limit: 1.2 g, upper limit: 1.6 g)
g is preferable), and the mixture is dissolved at 45 to 55 ° C., and 2 to 5 g of bacterial cells are added. Using a syringe equipped with a 21 to 25 gauge, preferably 21 to 22 gauge injection needle, the solution is 500 to 1000 ml of a 2 to 3% aqueous potassium chloride solution.
The maximum diameter is 1 to 5 mm (the lower limit is 2 m
m, the upper limit of which is preferably 4 mm, particularly preferably about 3 mm).

When the microorganism is reacted with the fluorenone as a substrate, a crushed fluorenone may be added as it is, but a small amount of an organic solvent is used (1 to 10 ml).
Organic solvent / 100 ml reaction solution, 1-50 mg substrate / ml organic solvent,
(Preferably 4 to 5 mg substrate / ml organic solvent). The type of the organic solvent is not particularly limited, and for example, acetone, dimethyl sulfoxide and the like are preferable.
The substrate may be added all at once or in portions at the beginning of the reaction. Substrate is about 1% (w / v) or less in the reaction solution (the lower limit is 0.01% (w / v), and the upper limit is 0.1% (w / v)).
% (W / v) is preferable).
If the added substrate concentration is too high, the reaction rate will decrease. The reaction is carried out using, for example, an L-shaped tube or a Sakaguchi flask at about 15 to 40 ° C. (the lower limit is preferably 25 ° C., and the upper limit is preferably 35 ° C.) until the reaction product amount reaches a certain value. It is performed for about an hour (preferably 12 as a lower limit and 24 hours as an upper limit). When the immobilized cells are used, the reaction is carried out for about 48 to 96 hours (preferably 60 hours as a lower limit and 80 hours as an upper limit).

The pH during the reaction is preferably adjusted to about 6 to 9 (the lower limit is preferably pH 6.5 and the upper limit is preferably 8). At a pH lower than 6, the reaction does not proceed sufficiently,
Running at lower pH significantly reduces the efficiency of the reaction.
The reaction is usually performed with shaking or stirring.

The atmosphere during the reaction is not particularly limited, and the reaction can be performed in various gas atmospheres. For example, air,
Gas atmospheres such as nitrogen, hydrogen, helium, methane, and oxygen can be used. In the present invention, since the microorganism to be used is facultatively anaerobic, it is desirable to perform the reaction in a non-oxygen atmosphere in that the yield of 9-hydroxyfluorenes is high, and in particular, an atmosphere of methane, hydrogen or nitrogen is more preferable. desirable. In particular, an atmosphere of methane and hydrogen is desirable.

After completion of the reaction, a general isolation method can be employed to collect 9-hydroxyfluorenes and the like from the reaction solution. That is, after removing the bacterial cells from the reaction solution by centrifugation as necessary, extraction is performed about 2 to 3 times using ethyl ether, ethyl acetate, or the like, followed by drying and concentration under reduced pressure. The obtained extract is purified by applying to a column, preferably a silica gel column, to isolate 9-hydroxyfluorenes and the like. When purifying with a silica gel column, it is preferable to use a benzene-ethanol solvent (gradient addition (gradient) method in which the amount of ethanol is gradually increased while developing with benzene). The isolation can also be performed using a separation membrane. The purified and isolated reaction products were analyzed by NMR, mass spectrometry, elemental analysis and HPLC.
It can be analyzed by a normal analysis method such as. Quantification of the isolated product can be performed using HPLC or the like.

[0019]

According to the present invention, 9% of fluorenones can be obtained.
-Hydroxyfluorenes can be produced with high selectivity.

[0020]

Next, the present invention will be described in more detail with reference to examples, but these examples are not intended to limit the scope of the present invention. In addition,% of a product is mol% with respect to a substrate.

Example 1 In a Sakaguchi flask (500 ml), 10 g of fish meat extract (manufactured by skipjack), 10 g of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) and 2 g of sodium chloride were added to 1000 ml of distilled water.
dissolve in water, adjust to pH 7.0, and add
10 ml of a medium sterilized by high-pressure steam sterilization, and deposited in Bacillus Brevis (Deposited with the Research Institute of Biotechnology, Institute of Biotechnology, Japan, on September 7, 1994 (Accession No. FERM P-1)
4525)), 1 ml of a culture solution in which the cells were sufficiently grown by inoculation, and then cultured at 30 ° C. for 24 hours with shaking. A solution prepared by dissolving 2.5 mg (28 micromol) of fluorenone in 0.025 ml of acetone was added, and methane gas was introduced from the inlet of the flask to completely replace the air in the flask. For 24 hours to perform a reduction reaction. After completion of the reaction, a silica gel column (Kieselgel 60)
20 mm i. d. × 30cm Eluent: benzene-
(Ethanol gradient addition) to separate fluorenone and 9-hydroxyfluorene. 1m of methanol
l, and the methanol solution was subjected to HPLC (column
C-18, 4 mm φ × 150 mm, solvent: methanol / water (40/60 to 95/5) gradient method, flow rate 1
ml / min, detection wavelength 230 nm), a reagent manufactured by Aldrich was identified as a standard sample, and 9-hydroxyfluorene was quantified by an internal standard method.
It was confirmed that the yield of 9-hydroxyfluorene was 96%.

Examples 2 to 5 The same operation as in Example 1 was performed, except that at least one of the type of microorganism and the atmosphere of the reaction system was changed as shown in Table 1, 9 in the same way as
-Hydroxyfluorene was identified. Table 1 shows the yield of 9-hydroxyfluorene. The Bacillus cereus used in Example 5 was deposited on March 7, 1994 at the Research Institute of Biotechnology, Industrial Technology Institute of Japan (Accession No. F
ERMP-14210) was used.

Example 6 In a Sakaguchi flask (500 ml), 10 g of fish meat extract (manufactured by skipjack), 10 g of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) and 2 g of sodium chloride were added to 1000 ml of distilled water.
dissolve in water, adjust to pH 7.0, and add
400 ml of medium sterilized by high-pressure steam
Cereus (deposited on March 7, 1994 with the Research Institute of Biotechnology, Institute of Biotechnology, Japan (Accession No. FERM P-1
4210)), 1 ml of a culture solution in which the cells were sufficiently grown by inoculation, and then cultured at 30 ° C. for 24 hours with shaking. After adding 100 mg (556 μmol) of fluorenone dissolved in 1 ml of acetone, methane gas was introduced from the inlet of the flask to completely replace the air in the flask, and then shaken at 30 ° C. for 24 hours while slowly flowing methane gas. Then, a reduction reaction was performed. After completion of the reaction, a silica gel column (Kieselgel 60 20 m
mi. d. × 30 cm eluent: benzene-ethanol gradient addition) to isolate fluorenone and 9-hydroxyfluorene. This was dissolved in 1 ml of methanol, and this methanol solution was subjected to HPLC (column C-1).
8, 4 mm φ × 150 mm, solvent: methanol / water (40/60 to 95/5) gradient method, flow rate 1 ml
/ Min, detection wavelength 230 nm), a reagent manufactured by Aldrich was identified as a standard sample, and 9-hydroxyfluorene was quantified by an internal standard method. As a result, 96.9 mg of 9-hydroxyfluorene (532
Micromol), and the yield was confirmed to be (55%).

Examples 7-14 In Example 6, the type of microorganism, the amount of fluorenone added as a substrate, the preculture time, the reaction time, the presence or absence of shaking,
The same operation as in Example 1 was performed except that at least one of the medium volumes was changed as shown in Table 1, and 9-hydroxyfluorene was confirmed in the same manner as in Example 1. Table 1 shows the yield of 9-hydroxyfluorene.

Comparative Example 1 In Example 1, 10 g of the fish meat extract ((bonito), Wako Pure Chemical Industries, Ltd.) used in Example 1 was used instead of the culture solution.
10 g of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) and 2 g of sodium chloride were dissolved in 1000 ml of distilled water to obtain a pH of 7.0.
Medium 10 prepared in advance and sterilized by autoclaving at 120 ° C. for 20 minutes.
The same operation as in Example 4 was carried out except that no bacterial cells were used during the reduction reaction. As a result, 9-hydroxyfluorene could not be confirmed.

Comparative Example 2 In Example 6, instead of 5 ml of the culture solution,
Extract ((Katsuo), Wako Pure Chemical Industries) 1
0 g, 10 g of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.) and 12 g of sodium chloride were dissolved in 1000 ml of distilled water.
The same operation as in Example 1 was performed except that 5 ml of a medium adjusted to H7.0 and sterilized by high-pressure steam at 120 ° C. for 20 minutes was added so that no cells were present during the reduction reaction. As a result, 9-hydroxyfluorene could not be confirmed.

[0027]

[Table 1]

Claims (6)

[Claims] 1. A method for producing 9-hydroxyfluorenes, comprising a step of causing a microorganism belonging to the genus Bacillus to act on a fluorenone, and a step of collecting the 9-hydroxyfluorene from the reaction solution. . 2. The method according to claim 1, wherein the microorganism is Bacillus brevis and / or Bacillus cereus. 3. The method according to claim 1, wherein the reduction reaction is performed in a diluted medium. 4. The method according to claim 3, wherein said medium is a medium diluted 2 to 20 times. 5. The production method according to claim 1, wherein the reduction reaction is performed in a non-oxygen atmosphere. 6. The method according to claim 5, wherein the non-oxygen atmosphere is an atmosphere composed of at least one selected from methane, hydrogen and nitrogen.