JPS6362505B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application] The present invention provides novel optically active bicyclo [2.2.1]
It relates to compounds and methods for producing the same. [Prior Art] Hitherto, 2-endo-hydroxy-, 2-exo-hydroxy- or 2-oxo-bicyclo[2.2.1]hept-7-anti-carboxylic acid and its methyl ester are known, and diasmine perfume oil It can be used as a raw material for the synthesis of methyl diasmonate, which is one of the aroma components and is useful as a perfume preparation (Japanese Patent Application Laid-Open No. 89657/1989). However, since these compounds have conventionally been produced in the form of racemates, they have not been able to be used as raw materials for producing optically active methyl diasmonate having a natural absolute steric structure. In order to obtain natural levorotatory methyl diasmonate, it is necessary to use as a raw material an optically active form of the bicyclo [2.2.1] compound that is rich in enantiomers whose absolute coordination at the 1st position is R. , such a compound has not been previously known. Furthermore, 2-oxo-7-benzyloxymethyl-bicyclo[2.2.1]hept-5-ene, which is a key intermediate for synthesizing prostaglandins, which are useful pharmaceuticals, has not been known. ing. In order to produce pure natural optically active prostaglandins, the intermediate 2-oxo-7-benzyloxymethyl-bicyclo[2.2.1]hept-5-ene is It must be an optically active substance enriched in enantiomers whose absolute configuration is R. Methods for producing such optically active substances include a method using racemic resolution ("Hung.Teljes 8006") and a method using asymmetric reaction ("J.Am.Chem.Soc., 97, 6908 (1975 )"), but in the former case, the yield is low, at most 50%, while in the latter case, although a 100% yield can be theoretically expected, it is difficult to use as a reagent. It cannot be called an industrial method because it requires expensive terpene derivatives and requires many reaction steps to introduce the asymmetric partial structure. On the other hand, conventionally, 2-oxo-bicyclo [2.2.1]
Hept-5-ene-7-carboxylic acid is known, and when this is used, the prostaglandin intermediate can be obtained by changing the carboxylic acid group at the 7-position to a benzyloxymethyl group. However, the conventionally known 2-oxo-bicyclo[2.2.1]hept-5-ene-7-carboxylic acid is a racemate, and even if this is used, it is not possible to obtain an optically active product. Can not. [Problem to be solved] As mentioned above, all of the above-mentioned bicyclo [2.2.1] compounds are only known as racemates [Helv.Chim.Acta., 66, 1991]
(1983); Tetrahedron, 37, Suppl. No. 1, 411
(1981); J.Am.Chem.Soc., 95, 7522 (1973)],
No enantiomer-rich enantiomer whose absolute configuration at the 1st position is R is known, and, as a matter of course, no method for producing it is known either. In terms of application of conventional technology, these racemates can be optically resolved in the form of diastereomeric salts or complexes using fractional crystallization, or as diastereomeric derivatives and then chromatographically. Optically active forms can be obtained by methods such as optical resolution using enzymes or selectively changing one of the enantiomers using enzymes or microorganisms. However, all of these operations are complicated and have the drawback that the theoretical yield is limited to 50%. Therefore, the optically active bicyclo[2.2.1]heptane- or hept-5-ene-rich enantiomer which has a hydroxyl group or an oxo group at the 2nd position and whose absolute configuration at the 1st position is R
There is a need for the development of a method for easily synthesizing 7-carboxylic acid or its ester. [Means for solving the problem] In order to obtain the optically active bicyclo [2.2.1] compound as described above, the raw material must have a prochiral structure, and the reagents and catalysts used must be asymmetric molecules. The condition is that it has a structure. From this point of view, the present inventors used bicyclo [2.2.1] as a raw material.
Using heptane- or hept-5-ene-7-carboxylic acid or its methyl ester,
If the carbon at the 2nd position is stereoselectively hydroxylated by the action of microorganisms or further oxidized, the desired 2nd carbon can be obtained.
Optically active bicyclo[2.2.1]heptane or hepto-5- having a hydroxyl group or an oxo group in the position
Focusing on the fact that ene-7-carboxylic acid or its methyl ester can be produced, we diligently searched for microorganisms suitable for this purpose, and as a result, we found microorganisms of the genus Penicillium, Aspergillus, Absidia, Vieuberia, Cunninghamella, and Mucor. The inventors have found that filamentous fungi of the genus or Chaetomium, actinomycetes of the genus Streptomyces, or bacteria of the genus Bacillus are suitable for the purpose, and have completed the present invention. The elements for carrying out the present invention are raw materials,
There are four types: microorganisms, culture conditions for obtaining microbial cells, and conversion reaction conditions for raw materials using the microorganisms, and these will be described in detail next. [Raw materials] The raw materials used in the present invention are selected from bicyclo[2.2.1]heptane-7-carboxylic acid, bicyclo[2.2.1]hept-5-ene-7-carboxylic acid, and their methyl esters. and is expressed by the following formula.

【formula】 [Microorganisms]

The microorganisms used in the present invention include the genus Penicillium, the genus Aspergillus, the genus Absidia,
Cunninghamella sp., Mucor sp., Chaetomimun
filamentous fungi of the genus Beauveria, actinomycetes of the genus Streptomyces,
and bacteria of the genus Bacillus. Among these strains that are effectively used, Abcidia coerulea (Abcidia coerulea) IFO 4011, Vieuberia
Beauveria bassiana IFO 4848, Cunninghamella blakesleeana IFO 6156, Mucor pusillus IFO 4578,
Chaetomium cochliodes
cochliodes) IFO 6308, Streptomyces albofaciens
IFO 12833 and Bacillus thuringiensis IFO 3951 are both preserved strains of the Fermentation Research Institute. Another strain, Penicillium funiculosum (FERM P-
8051), Aspergillus awamori
awamori) (FERM P-8052), and Aspergillus niger (FERM P
-8053) showed the following mycological properties, so it was concluded that “A Manual of the Penicillia
(Raper and Thom, 1949)”, “The genus”
The taxonomic position was determined with reference to the taxonomy of Aspergillus (Raper and Fennell, (1965)) and the Illustrated Encyclopedia of Fungi (Tsubaki and Udagawa, 1978).
The strain has been deposited with the above number at the Institute of Microbial Technology, Academy of Industrial Science. Penicillium funiculosum
funiculosum) FERM P-8051: Forms white-green colonies on potato dextrose agar or Czapek agar, and secretes a slight red pigment on the underside. The optimal growth temperature is 30℃. No ascosal generation is formed, and the penicillium consists of metre and phialide;
Whorled symmetrically. Conidiophores are generally solitary. A rope-like structure may be formed on the surface of the colony, and the shape of sclerotia can also be observed in some areas. Aspergillus awamori
awamori) FERM P-8052: Separated from Awamori Koji, it shows rapid growth at 30°C on potato dextrose agar medium and produces dense brown to blackish brown conidia in 2 to 3 days. The conidiophore is erect and short, less than a few mm, and the apical sac is spherical with a diameter of approximately 50 μm. Conidia are formed at the tips of the retrograde rays of metres and phialides. Aspergillus niger
niger) FERM P-8053: Grows vigorously on potato dextrose agar medium at 30°C, producing dense black conidia in 2 to 3 days. The back side of the village is pale yellow. Conidiophores rise from the colony but are short, less than a few mm. The apical sac is spherical with a diameter of approximately 50 μm, has metres and phialides, and the conidia are stored at the tips of the ridges. [Bacterial cell production conditions and microbial conversion reaction] The bacterial cell production conditions and microbial conversion reaction method used to perform the microbial conversion reaction are as follows. However, the present invention is not limited to these. For filamentous fungi: Usually cultured in a liquid medium. Its ingredients include glucose, 0.5-5%, yeast extract 0.1-1
%, malt extract 0.1-1%, and heptone 0.1-1
% is basically included, and magnesium salts, calcium salts, copper salts and other trace elements are added as necessary. The pH should be in the range of 4-6. Cultivation is performed under aerobic conditions such as shaking culture or aeration-stirring culture, and the temperature is 28 to 30°C. The culture time is closely related to the expression of activity of the conversion reaction, and preliminary studies are required to find the optimal value each time depending on the amount of inoculated bacteria or spores, medium composition, shaking method, etc. , the values listed in Example 1 below serve as a guide. Next, in order to carry out the conversion reaction, it is possible to add a substrate to the culture and continue culturing, but it is more preferable to separate the bacterial cells by a method such as filtration, and then add the cells to an aqueous solution of the substrate. Alternatively, it is preferable to add it to an aqueous suspension and stir or shake it. The solution used in this case is
It can be a 0.05-0.1M phosphate buffer or a suitable alternative buffer (pH in the neutral range of 6-8). Furthermore, the conversion yield can be increased by adding 0.1 to 5% glycerol, xylose, sucrose, or glucose.
In addition, 0.1 to 10mM Mo ²⁺ , Cu ²⁺ ,
Heavy metal salts such as Co ²⁺ are added as activation. It is appropriate that the amount of substrate added is 10 to 100 mg per 100 ml of the reaction solution. The reaction temperature is 25 to 35°C, and the reaction time varies depending on the amount of bacterial cells used, but is generally within 72 hours. For bacteria and actinomycetes: Cultivate in liquid medium. Its components basically include glucose 0.1-1%, glycerol 0.1-1%, yeast extract 0.1-0.5%, meat extract 0.1-0.5%, peptone 0.1-1%, NaCl 0.1-0.5%,
Add malt extract, liver extract, Mg, other inorganic salts and trace elements as necessary. PH is 7~
The range is 8. Culture is performed under aerobic conditions such as shaking culture or aeration-stirring culture, and the temperature is 30-30°C.
The temperature shall be 37℃. The culture time is closely related to the expression of activity of the conversion reaction, and preliminary studies are required to find the optimal value depending on the case, but it is usually 6 to 10 minutes.
It's time. Next, in order to carry out the conversion reaction, it is also possible to newly suspend the bacterial cells collected by centrifugation etc. in a phosphate buffer solution, add the substrate and xylose, etc. to this, and stir. In the case of Bacillus thuringiensis IFO 3951, which is the strain used in the present invention, the strain was grown during the culture (approximately 7 days after the start).
The conversion yield was higher when the substrate was added to the culture at 100 hr) and the culture was continued for an additional 24 to 48 hours. In this case, the amount of substrate added is 100ml
It is appropriate to set the amount to 5 to 20 mg per day. After the conversion reaction, whether it is filamentous fungi, actinomycetes, or bacteria, the bacterial cells are removed by filtration or centrifugation, and the desired product is extracted from the filtrate or centrifuged product with an organic solvent such as ether or ethyl acetate. do. [Product] The product obtained by the reaction using the microorganism is represented by the following structural formula in which a hydroxyl group is introduced at the 2-position of the bicyclo[2.2.1] compound used as a raw material, and the hydroxyl group is introduced at the 1-position. It is enriched in enantiomers whose absolute configuration is R. In this case, "enriched in enantiomers whose absolute configuration at position 1 is R" means that among stereoisomers consisting of R form and S form, R
This means that the proportion of the body exceeds 50%. Also,
In this specification, the meanings of the symbols R and S used to indicate the absolute configuration are as follows:
Index Guide/Appendix
P.170 (1984).

【formula】

【formula】

【formula】

[Formula] (In the above formula, R is hydrogen or a methyl group) Depending on the microbial reaction, the main product is a bicyclo [2.2.1] compound with a hydroxyl group introduced at the 2-position, but Depending on the microorganism, a product with a ketone group introduced at the 2-position may also be produced, but this usually does not constitute the main product. Therefore, in order to obtain a bicyclo[2.2.1] compound containing an oxo (ketone group) at the 2-position, the above-obtained product containing a hydroxyl group at the 2-position is oxidized. The oxidation treatment in this case can be carried out by a conventionally known method, such as a chromic acid oxidation method or a permanganate oxidation method. The product obtained by this oxidation treatment is represented by the following formula.

【formula】 [Action/effect of the invention]

The bicyclo[2.2.1] compounds represented by formulas (), (), and () according to the present invention can be used as raw materials for synthesizing methyl diasmonate, which is important in the fragrance industry, as a natural optically active substance. Bicyclo [2.2.1] compounds represented by formulas (), (), and () above can be used as raw materials for synthesizing prostaglandins, which are useful pharmaceuticals, from their natural forms as optically active forms. be able to. According to the present invention, all of these compounds can be obtained with high optical purity through an extremely simple one-step operation of bringing a raw material, which is a prochiral substrate, into contact with a microorganism. That is, among the above strains, the conversion reaction product by Bacillus thuringiensis IFO 3951 (R=CH ₃ in the above formulas () and ())
is hydrolyzed to form a free acid, and then chemically oxidized to obtain a compound (in the above formula (), R
=H) has an optical purity of almost 100% ee, and another transformation product (in formulas () and () above, R
=CH ₃ ) to form a free acid and then chemically oxidized, the optical purity of the compound (R=H in the above formula ()) is as high as 82%ee. or other strains of Aspergillus awamori
According to FERM P-8052, the above formula () (R=
The compound represented by H) was obtained as a crystal with an optical purity of 85% ee, which was chemically oxidized and then purified by a recrystallization operation. The optical purity of the compound (R=H in the above formula ()) is It reaches 92% ee.
The crude yield of the conversion reaction is as shown in Example 1 below. [ _2.2.1 ]heptane-7 by Aspergillus awamori _FERM P-8052. -Carboxylic acid [in the above formula (A), R=H) is produced from its alcohol form (in the above formulas () and (), R=H) is 63% in total, both of which are 50% or more. . Also, bicyclo [2.2.1] hept-5-
Aspergillus awamori for ene-7-syn-carboxylic acid (R=H in the above formula (B))
When FERM P-8052 is applied, its alcohol form (in the above formulas () and (), R=
The total yield of H) is 36%, and bicyclo[2.2.1]
When methyl hept-5-ene-7-syn-carboxylate (R=CH ₃ in the above formula (B)) is reacted with Mucor pusillus IFO4578, its alcohol form (formulas () and ()) In, R
= _CH3 ) total yield is 42%. In order to obtain natural methyl diasmonate and prostaglandins using the optically active compounds represented by the above formulas () to () of the present invention, reactions that have already been developed for racemates and known methods can be combined. That's fine. [Example] Next, the present invention will be explained in more detail with reference to Examples, but first, standard compounds necessary for identifying conversion products by microorganisms will be explained. As mentioned above, the target substance has the formula () to
In the structural formula shown in parentheses, it is an optically active substance rich in enantiomers with the absolute configuration of R at the 1st position. Ketone bodies (formulas () and ()) are more preferable than ()), and ketone bodies can be easily obtained by chemically oxidizing alcohol bodies. Furthermore, the ketone bodies represented by the above formulas () (R=H) and () (R=H) have excellent crystallinity. Therefore, from these points, the above formulas () (R=H) and ()
A ketone body represented by (R=H) is a key compound for identification. Next, a method for synthesizing the standard compound will be described. In this case, the raw material is anti-5-carboxytricyclo[ ^2.2.1.0.2,6 ]heptan-3-one (,
racemate) by the method of Grieco et al. (J.
Med.Chem., 23, 1072 (1980)) to obtain a dextrorotatory optically active form ((+)-). This one has mp ((+)-) 142~143℃, [α] ²³ _D = +
It showed physical properties of 91.7° (C=1.0, dioxane). This optically active form ((+)-) is, as shown by Bindra et al. (J. Am. Chem.
Soc., 95, 7522 (1973)), and has a steric structure that can lead to a natural prostaglandin, and therefore, the absolute configuration of the 1st position is R. After adding HBr to the cyclopropane ring of this optically active substance ((+)-) according to the method of Beeley et al. (Tetrahedron, 37, Suppl. No. 1, 411 (1981)), , 2-oxo- obtained by reduction with Zn/AcOH
Bicyclo[2.2.1]heptane-7-anti-carboxylic acid (in the above formula (), R=H) is dextrorotatory, mp141-142.5℃, [α] ²⁵ _D = +18° (C=
0.87, methanol). This was methylated with diazomethane to form a methyl ester (R=CH ₃ in the above formula ()), and then reduced with NaBH ₄ in an isopropanol solvent to form the corresponding endo-anti-hydroxy ester (in the above formula ()). R=CH ₃ ) and exo-anti-
Hydroxy ester (in the above formula (), R
=CH ₃ ) as a mixture in a ratio of approximately 95:5.
The formula () (R=CH ₃ ) obtained as above,
Compounds represented by ()(R= _CH3 ) and ()(R= _CH3 ) are used for identification of microbial transformation products by GC and GC-MS. The gas chromatographic properties of these compounds are summarized in Table 1 below. In addition, "GC" shown in this specification indicates gas chromatography, and GC-MS means gas chromatography coupled mass spectrometry. Next, the optically active substance ((+)-
), after adding hydrogen iodide (HI) to the cyclopropane ring by the method of Beeley et al.
The free carboxylic acid was converted into a methyl ester using diazomethane, and then the 2-position oxo group was ketalized according to the method of Grieco et al. This ketal compound is treated with 1,5-diazabicyclo(5.4.0]unde-5-cene to eliminate hydrogen iodide, and then hydrolyzed to convert the ester group at the 7-position into a carboxylic acid group. 2-
2-oxo-bicyclo[2.2.1]hept-5-ene-7-anti-carboxylic acid (in the above formula (), R=
H) was obtained. This one shows levorotation and mp152
~153°C, [α] ²⁵ _D = -644° (C = 0.30, MeOH). This compound was further methyl esterified with diazomethane and then ₂
The corresponding endo-anti-hydroxy ester (R=CH ₃ in the above formula ()) and exo-anti-hydroxy ester (R=CH ₃ in the above formula ()) are reduced by reducing the oxo group at
Obtained as a 92:8 mixture. The compounds represented by the formulas ()(R=CH ₃ ), ()(R=CH ₃ ) and ()(R=CH ₃ ) obtained as above were analyzed by GC and
Used for identification of microbial transformation products by GC-MS.
Their chromatographic properties are shown in Table 1 below.
are summarized in Table 1 shows the bicyclo[2.2.1]heptane used as a raw material for microbial conversion.
Gas chromatographic properties of methyl 7-carboxylate (A) (R=CH ₃ ) and methyl bicyclo[2.2.1]hept-5-ene-7-syn-carboxylate (B) (R=CH ₃ ) Also shown. The types of columns shown in Table 1 and their usage conditions are as follows. (1) 5CP (2 mm I.D. x 1.5 m glass column) Stationary phase: Sillar 5CP manufactured by Gascro Industries Co., Ltd.
5% Chromosolve W AW 80-100 mesh Carrier gas (He) flow rate: 15 ml/min (2) SP-1000 (2 mm I.D. x 1.5 m glass column) Stationary phase: manufactured by Gascro Kogyo Co., Ltd. SP-1000 5
%, Chromosolve W AW 80-100 mesh Flow rate of carrier gas (He)......15 ml/min (3) CDMS (3 mm I.D. x 1.5 m stainless steel column) Stationary phase......Cyclohexane manufactured by Gascro Kogyo Co., Ltd. Dimethanol succinate (CDMS) 15
%, Chromosolve W AW CDMS 80-100
Mesh Carrier gas (N ₂ ) flow rate: 60 ml/min Temperature conditions: Compound (A) (R=CH ₃ ), ()
(R=CH ₃ ), ()(R=CH ₃ ), ()(R=
160℃ for CH ₃ ), and for other
180℃ (4) OV-1 (OV-1 chemically bonded silica capillary column manufactured by Gascro Industries Co., Ltd., 0.2 mm I.D. x 25
m) Carrier gas (He) flow rate: 70 ml/min Temperature: 120°C (5) OV-1701 (OV-1701 chemically bonded silica capillary column manufactured by Gascro Industries Co., Ltd., 0.25 mmI.
D. x 25 m) Carrier gas (He) flow rate: 70 ml/min Temperature: 120°C (6) BP-20 (BP-20 chemically bonded silica capillary column manufactured by SGE, 0.22 mm I.D. 25 m) Carrier gas (He) flow rate: 60 ml/min Temperature: 175°C In the above, the carrier gas flow rate when using a capillary column is given as the value indicated by the gas chromatograph meter.

[Table] Next, the formulas ()(R=CH ₃ ) and ()(R
Of the 2-hydroxy form, which is the NaBH ₄ reduction product of the compound represented by =CH ₃ ), each minor component (small amount component) has an exo relative configuration.
It was confirmed as follows. That is, 9-borabicyclo[3.3. 1] Perform hydroboration with nonane and decompose the added water with hydrogen peroxide to obtain methyl exo-anti-2-hydroxy-bicyclo[2.2.1]hept-5-ene-7-carboxylate (the above formula In (), R=CH ₃ ) was stereoselectively obtained.
Next, this product is catalytically reduced to hydrogenate its double bond to form methyl 2-exo-hydroxy-bicyclo[2.2.1]heptane-7-carboxylate (R=CH ₃ in the above formula ()). did. The exo form () (R=CH ₃ ) and () obtained in this way
(R=CH ₃ ) is the compound () (R=
GC and GC-MS coincided with the minor components of the NaBH ₄ reduction products of CH ₃ ) and ( ) ((R=CH ₃ ). The optical purity of the microbial conversion product was determined by using a sufficient amount of sample. In the case, ketonic acid () (R=
H) and () (R=H) and measure their optical rotations, but in the case of small amounts, they can be induced into diastereomers with a chiral reagent and then converted into diastereomers.
This is done by separating and quantifying using GC. here,
Hydroxylation product () (R=CH ₃ ) () (R=CH ₃ ),
()(R=CH ₃ ) and ()(R=CH ₃ ) are
A diastereomer is induced using a urethanization reaction (hereinafter, this reaction is abbreviated as NEI reaction) using (R)-(-)-1-(1-naphthyl)ethyl isocyanate. Ketone product () (R=H)
and ()(R=H) are converted into acid chlorides with thionyl chloride and subjected to a condensation reaction with (R)-(+)-1-methylbenzylamine (hereinafter, this reaction is abbreviated as MB reaction) to form diastereomerism. Make it a tough amide. The ketoester type products ()(R=CH ₃ ) and ( )(R=CH ₃ ) are hydrolyzed to the free acids and then converted to (R)-(+)-1-methyl in the same manner as above. Condensate with benzylamine. Standard compounds ()(R=CH ₃ ) and ()(R=
CH ₃ ), ()(R=CH ₃ ) and ()(R
＝CH ₃ ) mixture, compounds ()(R=H) and ()(R=H), the racemic form and optically active form (1R- form) thereof are each treated as described above.
Table 2 shows retention time data in gas chromatography separation of diastereomers obtained by NEI reaction or MB reaction. The diastereomer obtained above is expressed by the compound code, the absolute configuration at the 1st position, and the type of reaction to obtain the diastereomer, for example, (1S)-(R=CH ₃ ). *(R)-NEI,
It was expressed as (IR)-V(R=H)*(R)-MB. In this case, the former is 2-exo-hydroxy-bicyclo[2.2.1]- whose absolute configuration at position 1 is S.
In methyl heptane-7-anti-carboxylate, it refers to the diastereomer obtained by performing an NEI reaction on the hydroxyl group at the 2-position; on the other hand, the latter is
In 2-oxo-bicyclo[2.2.1]heptane-7-anti-carboxylic acid whose absolute configuration at the 1st position is R, it means a diastereomer obtained by subjecting the carboxyl group to an MB reaction. Furthermore, the analytical conditions for the diastereomer separation experiment by gas chromatography are as follows. (1) Column: OV-1 chemically bonded silica capillary column, 0.25mmI.D. x 50m (2) Flow rate of carrier gas (He): 100ml/min (3) Temperature: Experiment No. 1 to 8... 250℃ Experiment No.9-12……200℃ (4) Detector: FID (5) Measurement of peak area: BPR G manufactured by Shimadzu Corporation
-1 Depends on the data processing device.

【table】

[NEI reaction]

EtOAc solution containing 0.1-0.7 mg of desired product (hereinafter
0.1 ml of (EtOAc shall represent ethyl acetate)
Place in a small ampoule and remove the solvent under reduced pressure. Reagent 20μ (=Toluene 17.8μ + (R)-(-)-1-
(1-naphthyl)ethyl isocyanate 2μ+
Add N,N-dimethylethanolamine (0.2μ), seal and heat at 90-100°C for 12 hours. After cooling, add 30-100μ of methanol to dilute appropriately, and then use for GC. [MB Reaction] When the desired product is in the form of a free acid, it is immediately reacted with thionyl chloride, but when it is in the form of a methyl ester, it is first hydrolyzed. Place 0.15 ml of EtOAc solution containing 0.1-0.2 mg of methyl ester in a small ampoule and evaporate the solvent under reduced pressure. methanol
Add 0.2ml and 0.1ml of 6N NaOH and keep at 30℃ for 1 hour. Acidify with 6N HCl and perform ether extraction under salt-saturated conditions (0.5ml x 2). The ether layers were combined, washed with saturated brine, and dried over Na ₂ SO ₄ .
Add 50μ of thionyl chloride to the residue after distilling off the ether, and let stand at room temperature for 1 hour. Excess thionyl chloride is removed under reduced pressure, 0.1 ml of toluene is added, and the mixture is concentrated again. A mixed solution of 85μ of toluene, 5μ of (R)-(+)-1-methylbenzylamine, and 10μ of pyridine is added, and the mixture is left at room temperature for several hours, diluted appropriately with methanol, and subjected to GC. Next, the composition of the medium used in the following examples is shown. Medium A (g/): glucose, 10; yeast extract,
3; Malt extract, 3; Hepton, 5;
MgSO ₄ 7H ₂ O, 0.3; CaCl ₂ 2H ₂ 0, 0.1;
NaCl, 0.1; Trace element solution, 1 ml; PH5.6 Medium B: 0.05 M phosphate buffer containing 3% glycerose (PH7.5) Medium C (g/): Yeast extract, 2; Liver exudate,
2; Meat extract, 2; Glucose, 3; Glycerol, 3; Hepton, ₅ ; NaCl, 3; Malt extract, 2; _MgSO4.7H2O , 0.3; _CaCl2 .
2H ₂ O, 0.1; Trace element liquid, 1 ml; PH7.4 Trace element composition (g/): H ₃ BO ₃ , 0.3; MnCl ₂ .
4H ₂ O, 0.2; ZnCl ₂ , 0.75; CuSO ₄ 5H ₂ O,
0.2; FeCl ₃・6H ₂ O, 2.5; (NH ₄ ) ₆ Mo ₇ O ₂₄・
4H ₂ O, 0.1; CoSO ₄ ·7H ₂ O, 0.15 In the following examples, the shaking speed of the reciprocating shaker was 120 rpm, and that of the rotary type was 170 rpm. Wako gel C-200 manufactured by Wako Pure Chemical Industries, Ltd. was used as a packing material for silica gel column chromatography for product purification. Example 1 The composition of the substrate solution used in this example is as follows. [Substrate solution] S1: 0.05M phosphate buffer (PH7.5) containing 5 mg/ml of bicyclo[2.2.1]heptane-7-carboxylic acid [(A) (R=H)]. S2: Bicyclo[2.2.1]methyl heptane-7-carboxylate [(A) (R=CH ₃ )] uniformly dispersed in 0.5M phosphate buffer (PH7.5) using ultrasound.
The concentration is 5 mg/ml as its free acid. S3: Bicyclo[2.2.1]hept-5-ene-7-
5 mg/ml of syn-carboxylic acid [(B) (R=H)]
0.05M phosphate buffer (PH7.5) containing S4: Bicyclo[2.2.1]hept-5-ene-7-
Syn-methyl carboxylate [(B) (R=CH ₃ )]
Dispersion in 0.05M phosphate buffer (PH7.5).
The content is 5mg/ml as its free acid. Furthermore, silica gel column chromatography used for product purification was performed under the following conditions. Column size: 1cmφ x 20~23cm Eluent: EtOAc concentration in benzene 15, 20, 25,
25 ml each of 30, 35, and 40% solutions. 150 ml of medium A was placed in nine 500 ml shoulder flasks each, and the flasks were sterilized with cotton stoppers. Spores were taken from the storage slant of Aspergillus awamori FERM P-8052 and inoculated into one of the flasks.
Shaking culture was carried out on a reciprocating shaker for 24 hours at 28°C.
15 ml of this culture (pulp-like) was aseptically transplanted into eight other mediums and cultured with shaking at 28°C for 24 hours (main culture). On the other hand, 100 ml of medium B was placed in each of four 500 ml batch-filled Erlenmeyer flasks in advance.
A silicone vent plug was attached to the container, and the container was sterilized. The cultures in the two flasks with shoulders were combined into one set (four sets in total), and the bacterial cells collected by suction filtration were placed in each of the Erlenmeyer flasks. Substrate solutions S1, S2,
1 ml each of S3 and S4 was added and shaken on a rotary shaker at 30°C for 48 hours to perform a conversion reaction. The bacterial cells were separated by filtration, washed with a small amount of water, the filtrate and washing liquid were combined, and the pH was adjusted to 1.5 with 6N HCl. Each of the four types of reaction solutions obtained in this way was saturated with NaCl,
Extracted with 150 ml and 100 ml of EtOAc. The EtOAc layers were combined, dried over Na ₂ SO ₄ and concentrated on a rotary evaporator. After methylation by adding excess diazomethane/ether solution, concentrate to 15%
Dissolved in 1 ml of EtOAc/benzene. This was refined using silica gel column chromatography. The 15-20% and 25-35% EtOAc/benzene eluates were combined, all solvent was evaporated and the resulting residue was dissolved in 0.2 ml of EtOAc. The target product in this sample solution was identified and quantified using GC. The analytical conditions are the same as those described with respect to Table-1. The calibration curve for quantitative determination is based on the standard compound () (R=CH ₃ ) and compound () (R
=CH ₃ ) and compound () (R=CH ₃ ). The production of the target product was also confirmed by GC-MS. Next, 1/
6 to 1/2 was taken and subjected to MB reaction or NEI reaction for measuring optical activity purity, and the obtained diastereomeric derivative was separated and quantified by GC. The analytical conditions are the same as those described with respect to Table-2. The results are listed in Table 3. In addition, experiments similar to those described above were also conducted on seven other types of filamentous fungi. The time (h) for preculture, main culture, and conversion reaction are shown in this order after the bacterial name:
Aspergillus niger FERM P-8053 (24h,
24h, 48h), Penicillium funiculosum FERM
P-8051 (24h, 25h, 48h), Absidia coerulea IFO4011 (24h, 27h, 46h), Vieuberia bassiana IFO 4848 (24h, 27h, 46h), Cunninghamera brachesleana IFO6156 (48h, 26h,
69h), Mucor Pucils IFO 4578 (19h, 26h,
42h), and Chaetomium cochlioides IFO 6308
(48h, 26h, 69h). Next, the conversion reaction between bacteria and actinomycetes was investigated as described below. i.e. 5 bottles of 500ml
100 ml of medium C was placed in each Erlenmeyer flask with a cap, a ventilated silicone stopper was attached, and the flask was sterilized. Bacillus thyuringiensis IFO 3951
A small amount of bacterial cells was taken from a storage slant, inoculated into one flask, and cultured with shaking on a rotary shaker at 30°C for 12 hours. Transfer 20 ml each of this preculture to the other four culture media aseptically and store at 30°C for 7 days.
Shaking culture was performed for hours (main culture). Next, the substrate solution
1 ml each of S1, S2, S3, and S4 was added separately to four main cultures, and shaking was continued for 49 hours. Remove the bacterial cells by centrifugal sedimentation, and soak each supernatant at 6N.
The pH was adjusted to 1.5 with HCl, NaCl was added to saturation, and the mixture was extracted with EtOAc (150 ml + 100 ml). The subsequent operations were performed in the same manner as in the case of filamentous fungi. Also Streptomyces albofaciens IFO
A similar experiment was conducted using 12833. The results are in the table-
The values in the "Amount produced (mg)" and "Optical purity (%ee)" columns shown in Table 3 are summarized in Table 3 when the substrate is [A] (bicyclo[2.2.1]heptane-7- carboxylic acid or its methyl ester), the values are shown for the corresponding compounds (), ()(), while if the substrate is [B](bicyclo[2.2.1]hept-5-ene-7 - syn-carboxylic acid or its methyl ester), the values for the corresponding compound (), (), () are shown. In addition, when the substrate is an acid, the amount produced as an acid and the substrate methyl When it is an ester, the amount produced as a methyl ester is given.

【table】

[Table] Example 2 Spores of Aspergillus awamori FERM P-8052 were inoculated into 150 ml of medium A (sterilized, 12 bottles) in a 500 ml shaking flask, and incubated on a reciprocating shaker at 28°C for 31 hours. Cultured with shaking. For inoculation of spores, 6 ml of medium A was placed in a storage slant to prepare a spore suspension, and 1 ml of the suspension was used. All the bacterial cells were collected by filtration and placed in equal amounts into 12 Erlenmeyer flasks with rounded ends. In the Erlenmeyer flask, medium B was added with 1mM Mo ²⁺ in advance.
100ml each, and sterilized with a silicone vent plug. 20 mg of substrate [A] (R=H) dissolved in 2 ml of water was placed in each of these Erlenmeyer flasks.
was added (total amount used 20 x 12 = 240 mg). Shake on a rotary shaker at 30°C for 64 hours. Filter the bacterial cells, combine the filtrates, and PH with 6N NaOH.
After adjusting the concentration to 7.5, it was concentrated to about 300 ml using an evaporator. Adjust the pH to 1.5 with 6N HCl, add NaCl to saturation, then add 300ml, 200ml, 200ml of EtOAc and
Extracted repeatedly with 100 ml. Combine EtOAc layer
After drying with Na ₂ SO ₄ , it was concentrated using an evaporator.
The concentrate was purified by silica gel column chromatography (column: 2.5 cmφ x 26 cm; elution:
Stepwise elution performed by increasing the EtOAc concentration in 5% increments of the EtOAc/benzene mixture (300 ml each).
Combine and concentrate EtOAc 35-40% fractions,
161 mg of 2-
Endo-hydroxybicyclo[2.2.1]heptane-7-anti-carboxylic acid was obtained as needles (yield 57%). This one is mp140~141℃, [α]
²⁵ _D = -13.4° (C = 0.97, methanol), MS (m/
e): 156 (M ⁺ ), IR (KBr, cm ^-1 ); 3350 (OH),
It showed physical properties of 1700 (COOH). In addition, the retention time of methyl ester in GC is the compound [] (R=
CH ₃ ) agreed with the data of the standard product. Furthermore, from GC of the diastereomer obtained by performing NEI reaction on this methyl ester, the optical purity of the (1R) form was calculated to be 84.7% ee. For confirmation, the above crystal was oxidized to a keto acid as follows. That is, 140 mg of the above crystals were dissolved in 2 ml of acetone, and Johns reagent was added under ice cooling.
0.35 ml was added and stirred for 30 minutes. isopropanol
Add 0.5 ml and 2-3 ice cubes, add NaCl until saturated, and add ether (20 ml x 2) and EtOAc (15 ml x
2). After combining the organic layers and drying with Na ₂ SO ₄ , silica gel column chromatography (column, 1.2 cmφ x 17 cm; 15-35% in 50 ml portions)
Purified with EtOAc/benzene). 20-30
The combined fractions of %EtOAc were concentrated and crystallized from benzene/hexane to give 96 mg of leaflets (69% yield). mp136-141℃, [α] ²⁷ _D = +
16゜(C=0.89, methanol), MS (m/e): 154
(M ⁺ ), IR (KBr, cm ^-1 ): 3400 (OH), 1740 (oxo group), 1710 (COOH). MS, IR and ^1H -HMR
The entire pattern of was consistent with the spectrum of the standard product (+)-V (R=H). The optical purity of the (1R) isomer is 89% ee from the [α] _D data, but it was calculated to be 92.2% ee from the more accurate GC analysis of the diastereomer (GC analysis of the MB reaction product). . Example 3 Spores of Aspergillus awamori FERM P-8052 were placed in a 500 ml shake flask medium A (150 ml,
20 plants) and shaken on a reciprocating shaker at 28°C for 31 hours. Filter and collect bacterial cells, 500 with 10 bottles
ml Erlenmeyer flask (1mM Mo ²⁺ per flask)
100 ml of medium B supplemented with (sterilized), and then the substrate [B] (R=H)
10 mg of each was added as a 2 ml aqueous solution to each flask (total amount used 10 x 10 = 100 mg). The mixture was shaken at 30°C for 64 hours with a silicone vent plug attached. After separating the bacterial cells by filtration, combining the filtrates and adjusting the pH to 7.5 with 6N NaOH,
It was concentrated to about 300ml using an evaporator. 6N HCl
Adjust the pH to 1.5, add NaCl until saturated,
Extracted four times with 300ml, 200ml, 200ml and 100ml of EtOAc. The organic layers were combined, dried over Na ₂ SO ₄ and concentrated using an evaporator. This was purified by silica gel column chromatography (column:
2cmφ×30cm; 100ml each of EtOAc/benzene mixture with increasing concentration of EtOAc by 5% eluent), 35
The ~40% EtOAc fractions were combined and concentrated. The oily residue was dissolved in 0.5 ml of acetone, and Johns reagent (175μ) was added under ice cooling, followed by stirring at 4°C for 1 hour. Add 0.2 ml of isopropanol and 2 ice cubes to saturate with NaCl, and extract with EtOAc (8 ml x
2+3ml). The organic layers were combined, dried over Na ₂ SO ₄ , concentrated using an evaporator, and then purified by silica gel column chromatography (column:
1 cmφ x 25 cm, 5% stepwise elution of EtOAc/benzene). The 25-30% EtOAc fractions were then combined, concentrated and crystallized from benzene/hexane to give 19 mg of compound ( ) (R=H) as lobes (17% yield). mp139～143℃〔α〕 ²⁵ _D =
-431° (C=0.16, methanol). MS (m/e):
152 (M ⁺ ), IR (KBr, cm ^-1 ); 1740 (>=O),
1700 (COOH). The MS, IR, and ¹ H-NMR spectra matched those of the (-)-(R=H) standard product. The optical purity of the 1R isomer is [α] 67%ee from the _D data, and from the GC analysis of the MB reaction.
The ee was 71.4%. Example 4 Medium C in 14 Erlenmeyer flasks with 500ml capacity
100ml each, and sterilized with a silicone vent plug. Bacillus thuringiensis in each
Aseptically inoculate 10 ml of IFO 3951 preculture (100 ml x 2, cultured with shaking at 28°C for 12 hours) and inoculate at 30°C.
The mixture was shaken in a rotary shaker for 5 hours. Ultrasonic dispersion of substrate [A] (R=CH ₃ ) [11 mg/ml-0.5M phosphate buffer (PH7.5)] was added to each culture.
1 ml of was added and shaking was continued at 30°C (total amount of substrate used was 11 x 14 = 154 mg). 45 hours after adding the substrate, remove the bacterial cells by centrifugation, combine the supernatant,
The pH was adjusted to 1.5 with 6N HCl. Divide into 3 equal parts and add NaCl to each
Saturated with EtOAc (600ml, 300ml and 200ml)
Extracted with. The organic layers were combined, dried over Na ₂ SO ₄ and concentrated using an evaporator. This was purified by silica gel column chromatography (column: 1.5cm
The fractions containing the target product (EtOAc 25%) were combined and concentrated using an evaporator. This oil (88 mg) was dissolved in 0.35 ml of methanol, 0.27 ml of 6N NaOH was added, and the mixture was kept at 30°C for 2 hours for hydrolysis. Add 0.5ml of water and PH with 6N HCl
2 and saturated with NaCl, EtOAc (8 ml x 2+
3 ml). The organic layers were combined and concentrated, then dissolved in 0.5 ml of acetone, and oxidized by adding 200 µ of Johns reagent while cooling to 4°C. After post-treatment and extraction in the same manner as in Example 3 and purification by silica gel column chromatography, the target product () (R=H) was crystallized from benzene/hexane to obtain 35 mg as leaf-like crystals. (Compound [A]
(23% yield from R=CH ₃ ). mp138~142℃,
[α] ²⁵ _D = +19° (C = 0.43, methanol), MS (m
/
e): 154 (M ⁺ ), IR (KBr, cm ^-1 ): 1740 (C>=
0), 1710 (COOH). The MS, IR, and H-NMR spectra matched those of the (+)-()(R=H) standard. The optical purity of the (1R) body is [α] _D
The data showed that the ee was approximately 100%, and the GC analysis of the MB reaction product showed that the ee was 94.4%. Example 5 100 ml of medium C was placed in 12 500 ml shoulder flasks each, and the flasks were sterilized with cotton stoppers. Bacillus for each
Preculture of P. thuringiensis IFO 3951 (100ml
10 ml of each culture was aseptically inoculated and shaken on a reciprocating shaker at 28°C for 6.5 hours. Substrate [B] for each of these cultures
Add 1 ml of ultrasonic dispersion (11 mg/ml - 0.5 M phosphate buffer (PH7.5)) of (R=CH ₃ ) and further at 28°C.
Shaking was continued for 44 hours (total amount of substrate used was 11
×12=132mg). The bacterial cells were removed by centrifugation, the supernatants were combined, and the pH was adjusted to 1.5 with 6N HCl. Divide into two and each
Saturate with NaCl, EtOAc (800 ml, 500 ml and 300 ml)
ml). The organic layers were combined, dried over Na ₂ SO ₄ and concentrated using an evaporator. This was purified by silica gel column chromatography (column: 1.5 cmφ x 40 cm; solvent = 100 ml each of 0-40% EtOAc/benzene). Fractions (25% EtOAc) containing the target product were combined and concentrated using an evaporator. 49 mg of this oil was dissolved in 0.2 ml of methanol, 0.2 ml of 6N NaOH was added, and the mixture was kept at 30°C for 2 hours for hydrolysis. Add 0.5 ml of water, adjust the pH to approximately 2 with 6N HCl, and saturated with NaCl, EtOAc (8 ml x 2 + 3 ml).
Extracted with. After combining the organic layers and concentrating, add 0.5 acetone.
ml, cooled to 4°C, Johns reagent
Oxidation was carried out by adding 175μ. After post-treatment and extraction in the same manner as in Example 3 and purification by silica gel column chromatography, the target product was crystallized from benzene/hexane to yield 11 mg of (-)-
( ) (R=H) was obtained as leaf-like crystals (yield: 8% from compound [B] (R=CH ₃ )). mp144~148
°C, [α] ²⁵ _D = -481° (C = 0.09, MeOH). M.S.
(m/e): 152 (M ⁺ ), IR (KBr, cm ^-1 ): 1730 (oxo group), 1700 (COOH). MS, IR and 1H−
The NMR spectrum matched that of the standard product of (-)-() (R=H). Optical purity is [α] _D
The GC analysis of the MB reactant showed 81.9% ee.

Claims (1)

[Scope of Claims] 1 Bicyclo[2.2.1]heptane- or hept-5-ene- having a hydroxyl group or oxo group at the 2-position
It consists of a bicyclo[2.2.1] compound selected from 7-carboxylic acids and methyl esters thereof, and the absolute configuration of the 1-position of the bicyclo[2.2.1] compound is R.
An optically active bicyclo[2.2.1] compound that is enantiomerically enriched. 2 Bicyclo [2.2.1] The carbon atom at the 2-position of a bicyclo [2.2.1] compound selected from heptane- or hept-5-ene-7-carboxylic acid and its methyl ester, Penicillium sp., Aspergillus sp. Absidia spp., Byouveria spp.
Bicyclo having a hydroxyl group at the 2-position [2.2.1], which is characterized by being hydroxylated using filamentous fungi of the genus Cunninghamella, Mucor, or Chaetomium, actinomycetes of the genus Streptomyces, or bacteria of the genus Bacillus [2.2.1]
Consists of a bicyclo[2.2.1] compound selected from heptane or hept-5-ene-7-carboxylic acid and its methyl ester, and the absolute configuration of the 1-position of the bicyclo[2.2.1] compound is R. A method for producing enantiomerically enriched optically active bicyclo[2.2.1] compounds. 3 Bicyclo[2.2.1] The carbon atom at the 2-position of a bicyclo[2.2.1] compound selected from heptane- or hept-5-ene-7-carboxylic acid and its methyl ester, Penicillium spp., Aspergillus spp. Absidia spp., Byouveria spp.
Bicyclo having an oxo group at the 2-position [2.2. 1] Heptane or heptane
An optically active compound consisting of a bicyclo[2.2.1] compound selected from 5-ene-7-carboxylic acid and its methyl ester, and rich in enantiomers in which the absolute configuration of the 1-position of the bicyclo[2.2.1] compound is R. Bicyclo [2.2.1] Method for producing compounds.