JP3149265B2 - Method for producing optically active 3,5-dihydroxy fatty acid ester derivative - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for reducing an optically active S- or R-5-hydroxy-3-keto fatty acid ester derivative using a microorganism to obtain an optically active (3R) compound having a syn-type configuration. ) A method for producing a 3,5-dihydroxy fatty acid ester derivative. Optically active 3,5-dihydroxy fatty acid ester derivatives are important as intermediates for pharmaceutical synthesis. In particular, it has a syn-type configuration (3
3,5-dihydroxy fatty acid ester derivatives such as (R, 5S) -3,5,6-trihydroxyhexanoate derivative and (2S, 4R) -2,4-dihydroxyadipate ester derivative are hydroxymethylglutaryl- CoA (H
It is known to be useful as an intermediate for MG-CoA) reductase inhibitors.

[0002]

2. Description of the Related Art In order to obtain an optically active 3,5-dihydroxy fatty acid ester derivative, as a method for reducing a 5-hydroxy-3-oxo fatty acid ester derivative, a method using triethylborane [Chemistry Letters (Che)
m. Lett.), 1923-1926 (1981) and Tetrahedron Lett., 28, 1
55 (1987)], a method for catalytic hydrogen reduction using an asymmetric catalyst (JP-A-2-28937) and a method for reduction using titanium isopropoxide [Journal of Organic Chemistry (J. Org. Chem.)]. .), 56, 4
050 (1991)].

[0003]

The above-mentioned chemical synthesis methods require expensive reagents, complicated preparation of the catalyst, and the need to carry out the reaction at an ultra-low temperature or at a high temperature and a high pressure. The method also has points to be improved as an industrial production method. Accordingly, an object of the present invention is to provide a method capable of producing an optically active 3,5-dihydroxy fatty acid ester derivative having a syn-type configuration under mild conditions without using an expensive reagent. is there.

[0004]

SUMMARY OF THE INVENTION Optically active S- or R-
Using certain microorganisms for the reduction of 5-hydroxy-3-oxo fatty acid ester derivatives, under mild conditions,
Optical activity (3R, 5S)-having a syn-type configuration
It was found that (3R, 5R) -3,5-dihydroxy fatty acid ester derivatives can be stereospecifically produced.

That is, the present invention provides a compound represented by the general formula (1):

Embedded image [Wherein, R ¹ represents lower alkoxycarbonyl group, a carboxyl group, a cyanomethyl group, a protected or unprotected hydroxymethyl group, or a halomethyl group, R ² represents H or general alcohol protecting group, R ³ Represents a lower alkyl group. Optically active 5-hydroxy-3-
Keto fatty acid ester derivative, Candida
A general formula, wherein the keto group at the 3-position is stereospecifically reduced using a microorganism selected from the group consisting of the genus Debaryomyces, the genus Hansenula and the genus Trigonopsis.
(2):

Embedded image [Wherein, R ¹ , R ² and R ³ are as defined above. Optically active (3R) -3,5 having a syn-type configuration represented by
The present invention relates to a method for producing a dihydroxy fatty acid ester derivative.

Compounds (1) and (2) are (5R) -hydroxy when R ¹ is a cyanomethyl group, and are (5S) -hydroxy when R ¹ is any other group.

In the above formulas (1) and (2), the lower alkoxycarbonyl group represented by R ¹ is methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxy. Examples include a carbonyl group and a t-butoxycarbonyl group, preferably an ethoxycarbonyl group and an isopropoxycarbonyl group. Examples of the protecting group for the hydroxymethyl group represented by R ¹ include a lower alkyl group such as a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group and a t-butyl group, an aralkyl group such as a benzyl group, an acetyl group, Acyl groups such as benzoyl group, t-butyldiphenylsilyl group, t-butyldimethylsilyl group,
A silyl group such as a trimethylsilyl group; and a sulfonyl group such as a tosyl group and a mesyl group. Examples of the halomethyl group represented by R ¹ include a chloromethyl group, a bromomethyl group, an iodomethyl group, and the like. Examples of the lower alkyl group represented by R ³ include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl and the like, preferably t
-Butyl group.

[0008] The microorganism producing the 3R-hydroxy form in the method of the present invention is a microorganism belonging to the genus Candida, the genus Debaryomyces, the genus Hansenula and the genus Trigonopsis.

The starting material of the present invention, 5-hydroxy-3-
The keto fatty acid ester derivative (1) can be synthesized using various methods depending on the structure. For example, in the general formula (1), R ¹ is an isopropoxycarbonyl group, R ² is H, R
^In the case where 1 is 1-isopropyl 6-t-butyl 2-hydroxy-4-oxoadipate in which ³ is a t-butyl group, malic acid is acetonidized, then subjected to a 2 carbon-carbon enrichment reaction, and esterified. Can be synthesized.

Embedded image

In the method of the present invention, the microorganism used is:
Any form such as a culture obtained by culturing the strain in a liquid medium, cells isolated from the culture solution, or dried cells obtained by treating the cells or the culture, or immobilized cells Can also be used. Operation is batch type, semi-batch type,
Alternatively, it can be performed in any of a continuous system.

One preferred embodiment of the method of the present invention is described below. First, 10 ml of a liquid culture medium having a pH of 3 to 8 is inoculated with a seed bacterium from a slant culture medium, aerobically shake-cultured at 10 to 50 ° C for 1 to 5 days, and then centrifuged to obtain cells. Then
A 4% glucose buffer (pH 3 to 8) was added to the cells.
After adding 5 ml and pre-incubating at 10 to 50 ° C. for 1 hour,
Hydroxy-3-keto fatty acid ester derivatives (1) 1-1
Add 00 g / l and shake at 10-50 ° C for several hours to 5 days. After completion of the reaction, 3,5
-The dihydroxy fatty acid ester derivative (2) is recovered by an operation such as extraction with an organic solvent.

[0012] The medium contains glucose as a carbon source,
Carbohydrates such as sucrose, alcohols such as ethanol and glycerol, fatty acids such as oleic acid and stearic acid and esters thereof, oils such as rapeseed oil and soybean oil, and ammonium sulfate, sodium nitrate, peptone and casamino acids as nitrogen sources , Corn steep liquor, bran, yeast extract, etc., inorganic salts such as magnesium sulfate, sodium chloride, calcium carbonate, potassium monohydrogen phosphate,
Malt extract, meat extract and the like may be contained as other nutrient sources such as potassium dihydrogen phosphate.

As a result of the reduction of the 5-hydroxy-3-keto fatty acid ester derivative (1) by the microorganism, the reduced coenzyme in the cells is reduced, and the reaction is eventually stopped. To continue the reaction, it is desirable to regenerate the reduced form coenzyme. For that purpose, usually, the above-mentioned carbon source may be added.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[0015]

【Example】

Examples 1-4 Glucose (40 g), yeast extract _{(3g), KH 2 PO 4} (0.
7g), (NH ₄ ) ₂ HPO ₄ (1.3 g), MgSO ₄ .7H ₂ O
_{(0.8g), ZnSO 4 · 7H} 2 O (0.06g), FeSO 4 ·
_{7H 2 O (0.09g), CuSO} 4 · 7H 2 O (0.005g),
A 10 ml portion of a liquid medium (pH = 6.8) consisting of MnSO ₄ (0.01 g), NaCl (0.1 g) and water (1 L) was dispensed into a test tube and heated at 121 ° C. for 20 minutes in an autoclave. Sterilized. Each medium was inoculated with each of the various strains described in Table 1 from a slant medium, and cultured at 30 ° C. for 1 to 2 days with aerobic shaking. After the culture, the cells are collected by centrifugation, and 0.1
After washing with M phosphate buffer (pH = 7.0), 5 ml of 0.1 M phosphate buffer (pH = 7.0) containing 4% of glucose was added, and the mixture was cultured at 30 ° C. for 1 hour. Subsequently, 0.5% of 1-isopropyl 6-t-butyl 2 (S) -hydroxy-4-ketoadipate was added, and the mixture was shaken at 30 ° C for 24 hours.

After the reaction, the cells are removed by centrifugation, followed by high pressure liquid chromatography [Finepak].
C ₁₈₁₅ , eluent acetonitrile / water = 1/1, flow rate 1 ml
/ Min, detection wavelength 210 nm], the production amount of 1-isopropyl 6-t-butyl 2,4-dihydroxyadipate in each reaction solution was measured. 1-isopropyl 2,4-dihydroxyadipate 6 extracted from the reaction solution with hexane
The optical purity of t-butyl was determined by high pressure liquid chromatography [Chiralcel OD, eluent hexane / isopropanol = 20/1, flow rate 2 ml / min, detection wavelength 2
10%] to determine the isomer excess [% de: {(4R hydroxy form% -4S hydroxy form%) / (4R hydroxy form% + 4S hydroxy form%)} × 100%]. The results were as shown in Table 1.

Table 1 Example Strain Yield Isomer excess Number (%) (% de) 1 Candida guilliermondii 98 84 (Candida guilliermondii) IFO 0454 2 Debaryomyces sp. 88 92 (Debaryomyces sp. Glist et Guiel) IFO 0064 3 Hansenula saturnus 100 80 (Hansenula saturnus) IFO 0809 4 94 16 (Trigonopsis variabilis) IFO 0671

Example 5 According to the same procedure as in Examples 1 to 4, using Hansenula Saturnus IFO 0809, 2 (S) -hydroxy-
1-ethyl 6-t-butyl 4-ketoadipate is converted to 2,4-
Converted to 1-ethyl 6-t-butyl dihydroxyadipate. Yield = 87% Isomer excess [{(4R hydroxy form% -4S hydroxy form%) / (4R hydroxy form% + 4S hydroxy form%)}} ×
100%] = 74% de

Example 6 According to the same procedure as in Examples 1-4, using Hansenula Saturnus IFO 0809, 6-benzoyloxy-
T-Butyl 5 (S) -hydroxy-3-ketohexanoate was converted to t-butyl 6-benzoyloxy-3,5-dihydroxy-hexanoate. Yield = 15% Isomer excess [{(3R hydroxy form% -3S hydroxy form%) / (3R hydroxy form% + 3S hydroxy form%)} ×
100%] = 60% de

[0019]

Industrial Applicability According to the method of the present invention, it is possible to efficiently produce an optically active 3,5-dihydroxy fatty acid ester derivative having a syn-type configuration which is extremely useful as an intermediate of an HMG-CoA reductase inhibitor. it can.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ identification code FI (C12P 7/62 C12R 1:78) (58) Investigated field (Int.Cl. ⁷ , DB name) C12P 7/62 CA ( STN) REGISTRY (STN)

Claims (6)

(57) [Claims] [Claim 1] General formula (1): [Wherein, R ¹ represents lower alkoxycarbonyl group, a carboxyl group, a cyanomethyl group, a protected or unprotected hydroxymethyl group, or a halomethyl group, R ² represents H or general alcohol protecting group, R ³ Represents a lower alkyl group. Optically active 5-hydroxy-3-
A general formula (2) characterized in that the keto fatty acid ester derivative is stereospecifically reduced at the 3-position keto group using a microorganism selected from the group consisting of Candida, Debaryomyces, Hansenula and Trigonopsis. ): [Wherein, R ¹ , R ² and R ³ are as defined above. Optically active (3R) -3,5 having a syn-type configuration represented by
-A method for producing a dihydroxy fatty acid ester derivative. 2. The method according to claim 1, wherein R ¹ is a hydroxymethyl group protected by a lower alkyl group, an aralkyl group, an acyl group or a silyl group. 3. The method according to claim 1, wherein R ¹ is a hydroxymethyl group protected with a benzoyl group. 4. The method according to claim 1, wherein R ¹ is a lower alkoxycarbonyl group. 5. The method according to claim 1, wherein R ¹ is an isopropoxycarbonyl group. 6. The method according to claim 1, wherein R ³ is a t-butyl group.