JPS63214198A - Production of optically active 4-penten-2-ol derivative - Google Patents

Abstract

PURPOSE:To industrially obtain an optically active 4-penten-1-ol derivative useful for medicines and agricultural chemicals at a low cost, by optically resolving a specific ester with a microorganism-derived enzyme having stereoselective esterase activity. CONSTITUTION:An enzyme (e.g. lipase), derived from Chromobacterium viscosum and having stereoselective esterase activity is added to a reaction solution prepared by suspending 2-60wt./vol.% ester expressed by formula I (R is 1-7C aliphatic hydrocarbon group) so as to provide 1:1-1,000 weight ratio of the enzyme to the above-mentioned ester. The resultant reaction solution is then subjected to asymmetric hydrolytic reaction at 10-45 deg.C while adjusting the pH to 6-7.5 with NaOH to carry out optical resolution and form an optically active alcohol (4-penten-1-ol) expressed by formula II (* indicates asymmetric carbon) and an optically active ester expressed by formula III in the reaction solution. The resultant product is then extracted with methylene chloride, concentrated and subjected to separation treatment by silica gel column chromatographic operation to collect the aimed optically active 4-penten-2-ol derivative.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a compound represented by the general formula (RS)-1 (wherein, 几 represents an aliphatic hydrocarbon group having 1 to 7 carbon atoms). Ester (RS)-1 is asymmetrically hydrolyzed to produce an optically active alcohol (4-penten-2-ol) represented by the general formula 2*H (where * represents an asymmetric carbon). *2 By acting on an enzyme derived from a microorganism having stereoselective esterase activity,
From 1, alcohol 2*, which is a hydrolyzate, and ester 18, represented by the general formula 11 (wherein R is the same as above), which is an unreacted product, are produced, and each optically active substance is separated and collected. Optically active 4-pentene-2- by characteristic biochemical resolution
The present invention relates to a method for producing an all derivative.

These optically active 4-penten-2-ol derivatives are important compounds that serve as starting materials for pharmaceuticals, agricultural chemicals, and the like.

[Conventional technology and problems]

Optically active 4-penten-2-ol derivatives can be synthesized by, for example, (1) a method in which 4-penten-2-ol racemate is esterified with phthalic anhydride and then optically resolved using brucine [Literature: 8ih, J.

C1, Prostaglandins
ins), 18 (5), 881 (1977)), (2
) A method for asymmetrically synthesizing 4-penten-2-ol by reacting optically active allylborane with acetaldehyde [Literature: H, O, Brown et al., Journal of the American Chemical Society (J.
Am.

Ohem, 8oc, ), 105.2092 (198
8)) is known.

However, the above method requires complicated operations or requires the use of expensive reagents, making it unsuitable for production on an industrial scale.

[Means and effects for solving the problem] The present inventors screened for an enzyme that asymmetrically hydrolyzes the acyl group at the 2-position of the ester (RS)-1 represented by the general formula (RS)-1. went. As a result, for example Chromobacterium (Chromobacterium
An enzyme derived from a microorganism belonging to the genus Terium (RS
)-j is asymmetrically hydrolyzed to produce (R)-2((R)-4-
Penten-2-ol \〆\ri〕♂H and (S)-1((S)-4-penten-2-acyle) were identified. Also, (S)-1 can be chemically hydrolyzed as necessary. By doing so, the alcohol (S)-2 generated 1 and 2 can be easily separated by silica gel column chromatography or, if the carbon chain of the 2-position acyl group is long, by distillation, and the respective optically active forms can be separated. Can be collected.

The present invention will be explained in more detail below.

The substituent R of the ester 1 represented by the general formula n used as the substrate of the present invention includes, for example, an aliphatic hydrocarbon group having 1 to 7 carbon atoms. Furthermore, those in which some of the substituents are substituted with halogen groups, such as chloromethyl groups, can also be used.

Raw material (RS)-1 can be synthesized, for example, by the following route.

C0R (RS)-1 (RS)-1 is asymmetrically hydrolyzed to produce optically active *
* Any enzyme may be used as long as it has a stereoselective esterase activity to produce l and 2, and examples thereof include enzymes belonging to the genus Chromobacterium. More specifically, for example, Chromobacterium viscosum (Ohr
omobacterium viscosum). As a commercially available product of this enzyme, Lipase Toyo (
Toyo Jozo■%), lipase (C!albio-chem
(manufactured by a company), etc., and can be used.

The asymmetric hydrolysis reaction reduces (RS)-1 of the substrate by 2 to 60% (
w/v) in the reaction solution, add an appropriate amount of enzyme, for example, at a weight ratio of enzyme to substrate of 1:l to 1:t, ooo, and at a temperature in the range of 10°C to 45°C, preferably The reaction may be carried out at a temperature ranging from 30°C to 40°C. The pH range during the hydrolysis reaction is from 4.0 to 8.5, preferably from 6.0 to 7.5, but as the hydrolysis reaction progresses, the pH in the reaction solution may become more acidic. Since it tilts, it must be carried out in a buffer solution, or it can be purified with a neutralizing agent such as NaOH aqueous solution.
It is desirable to maintain H in the range of 6.0 to 7.5.

The time course of the asymmetric hydrolysis reaction can be determined from the amount of NaOH used to adjust the pH.

It can also be determined by extracting a portion of the reaction solution with ether and performing gas chromatography analysis.

Furthermore, the above-mentioned asymmetric hydrolysis reaction can be repeated by, for example, adsorbing and immobilizing the enzyme on a hydrophobic resin or the like.

After hydrolysis, methods for separating 1* and 21 in the reaction solution include, for example, methylene chloride and ether.

Both 1* and 21 are extracted with an organic solvent such as hexane or ethyl acetate, concentrated, and then separated by silica gel column chromatography, or if the acyl group of *1 has a long carbon chain, By utilizing the boiling point difference, 1* and 2'' can be easily separated and collected by distillation.

〔Example〕

EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but the present invention is not limited to these Examples.

Substrate production example (1) Preparation of (RS)-4-penten-2-ol To a solution containing 15 Jf of magnesium powder and 36 txt of anhydrous ether, add 85.1 F of allyl bromide and 260 ml of anhydrous ether.
A Grignard reagent is prepared by gradually adding a solution containing g/g/, dropwise over about 8 hours, and heating for an additional 2 hours.

Acetaldehyde 15.8F was added dropwise to this liquid over 1 hour under water cooling. After the dropwise addition, stirring was continued for 2 to 3 hours at room temperature to complete the reaction. After the reaction, it was washed with 200g of ice water. After dehydrating the organic layer with anhydrous magnesium sulfate, distillation is performed to obtain high-purity (RS)-4-pentene-2.
-All to, of (yield 40%) was obtained.

t) pH 6-118℃/760wmKglHNMR
(90MHz, 0DO1a) δ (ppm): 1
．． 17 (2H, d, J=6.6f-1z, 0Ha
-), 2.07-2.45 (8H,m, -0H(O
H)-0H2-), 8.63-4.02(IH,m,-
OH-), 5.06 (2H, d, J=14.9t,
0H2=OH-), 5.58-6.07 (I H, m
, 0H2=OH) (2) (RS)-4-penten-2-acetate (RS)-4-penten-2-ol 8.6f, triethylamine 12. 12. Add acetic anhydride to the solution containing Of and 50zt of ether. Of at room temperature, 80
Drip over several minutes. The reaction was further carried out for 10 hours under reflux. After the reaction, add 5011 t of ice water twice, then saturated NaHC
Water washing was performed once with 50 g of Os aqueous solution. After dehydrating the organic layer with anhydrous magnesium sulfate, distillation was performed to obtain 9.2f of high-purity (几5)-4-pentene-2-acetate (
A yield of 72% was obtained.

bp78°C/ 68 mHg 'HNMR (90M) tz, (:!DC1g) δ(
ppm)21.21 (8H, d, J=6.8an,
0H80H(0-)-), 2.01 (8H,s,
CHg0O), 2.83 (2H,t, J = 6
．． 81. -0H(0-)-C! H2-), 4-75 6
．． 08 (4H1m, 0H(0-)-0H20H=
CH2) (3) (RS)-4-penten-2-butyrate (RS)-4-penten-2-ol 8.6 da,
Triethylamine 12. Acetic acid chloride 12.8F was added dropwise to the solution containing Of and ether 5 (Jxtt) over 30 minutes under water cooling. The reaction was further carried out at room temperature for 5 hours.

After the reaction, twice with 50 g of ice water and then with saturated NaHC! O
Washing was carried out once with 50 g/s aqueous solution. After dehydrating the organic layer with anhydrous magnesium sulfate, distillation was performed to obtain high-purity (RS)-4-pentene-2-butyrate 9.2F (
A yield of 59% was obtained.

bp 57°O/ 12 tmHy'HNM几(90MHz, CDC71g) δ(
ppm): 0.94 (8H, t, J=7.8 output,
0H80H20H2CjO-), 1.21 (8H, d
, J = (5,9)1z, C! Ha(:!H(0-
) -), 1, 42-2.50 (6H-m e OH
B cH2cH2co-, OH(0-) C3H5-)
, 4.78-6.08 (4H,m, -CH(0-)
-C3H5-CIj=CjH2) Example 1 Enzyme screening substrate (RS) -4-pentene-2-butyrate 0, 1 each
0.01150 each of enzymes), 2 each of 0.1 M phosphate buffer (pH 7, 2), and Owl reaction solution were placed in test tubes to make two tubes of each. Seal the top and shake on a shaker at 80°C. Extraction was carried out using 4 ml of ether at each time point of 24 hours and 48 hours, and the ether layer was dehydrated with anhydrous sodium sulfate and then subjected to gas chromatography to determine the reaction rate. Six species with reaction rates ranging from 20% to 80% at each time point of 24 hours and 48 hours were selected and subjected to secondary screening.

Example 2 Secondary screening and purification substrate (RS)-
4-pentene-2-butyrate each 4, Of, 0.1M-
Add 0.1f each of the 6 enzymes selected in the secondary screening to 40ml each of phosphate buffer (pH 7, 2) and incubate at 35°C.
The mixture was stirred with a stirrer, and the pH was maintained at 7.2 with a 2 N-NaOH aqueous solution to carry out an asymmetric hydrolysis reaction. Na
The reaction was terminated when the amount of OH consumed reached 50% of the theoretical amount. After cooling, it was extracted twice with 40 ml of ether each.

This extract was dehydrated with anhydrous sodium sulfate and then distilled to produce (R)-4-penten-2-ol and (S)
The quality of the -4-pentene-2-butyrate fraction was evaluated by collecting the fraction and measuring its specific rotation.

Table 1 shows the results of the two enzymes for which asymmetric hydrolysis ability was found.

Example 8 (RS)-4-pentene-2-butyrate 1 as substrate
5.6f, Lipase Toyo 1.56F, 1'40'' in 100gl of 0.1M phosphate buffer (pH 7,2)
An asymmetric hydrolysis reaction is carried out while C1 pH is maintained at 7.2 with a 2 N-NaOH aqueous solution. Reaction was carried out for 8 hours,
The reaction was terminated when the amount of NaOH consumed reached 60% of the theoretical amount. After cooling, it was extracted twice with 100 m/each of ether. This extract was dehydrated with anhydrous sodium sulfate and then distilled to obtain (R)-4-penten-2-ol and (S)-4-penten-2-butyrate.

(R)-4-penten-2-ol: yield 68% (/Z
) D-8,9° (c = 9, EtgO) (S)-4
-Pentene-2-butyrate: Yield 53% [α]D
I 8.8° (c=91.Et20) obtained (S)-
About 5 times the molar amount of 5 N -NaOH aqueous solution was added to 4-pentene-2-butyrate, and the mixture was stirred at room temperature with a stirrer all day and night. After confirming that the water has completely decomposed, (8)-
4-penten-2-ol was extracted with ether, the ether layer was dehydrated with anhydrous sodium sulfate, and then collected by distillation.

(S)-4-penten-2-ol: 75% yield from (S)-4-penten-2-butyrate, [α)”+8
．． 7° (C=1.BtzO) Reference value: H, O, Brown, Journal of Organic Chemistry (J, org, Ohem, )
49, p. 4089 (1984), (S) -4-penten-2-ol: [α) +9.88° (c=9
．． 18, BtzO) 8 bodies [α) 28-9.75 (c=9.16, Btz
O) Example 4 (RS)-4-pentene-2-acetate 6 as substrate
．． 4f, Lipase Toyo 0.64F 10.1M phosphate buffer (pH 7,2) 50g/40℃, pH 2N
The asymmetric hydrolysis reaction was carried out while maintaining the temperature at 7.2 with a -NaOH aqueous solution. The reaction was carried out for 48 hours, and the reaction was terminated when the consumption amount of NaOH reached 50% of the theoretical amount. After cooling, it was extracted twice with 50 g+/- each of ether. This extract was dehydrated with anhydrous sodium sulfate, and then only ether was distilled off at normal pressure to obtain a concentrated solution. Approximately 10xl of this concentrated solution was subjected to silica gel column chromatography (column: silica gel C-100,1,9cIII x 50m,
(R) with developing solution hexane:acetone=15:1).
The -4-penten-2-ol and (S)-4-penten-2-acetate fractions were concentrated and distilled to obtain the desired product.

Claims (3)

[Claims] (1) General formula (RS)-■ (RS)-▲There are mathematical formulas, chemical formulas, tables, etc.▼・・・(
RS)-■ (wherein R represents an aliphatic hydrocarbon group having 1 to 7 carbon atoms) is asymmetrically hydrolyzed to form the general formula ■^* ▲ There are mathematical formulas, chemical formulas, tables, etc.▼・・・■^* (In the formula, * represents an asymmetric carbon) A steric that produces optically active alcohol (4-penten-2-ol)■^* By acting with an enzyme derived from a microorganism that has selective esterase activity, (RS)-■ is converted to an optically active alcohol■^* and the general formula■^* ▲Mathematical formulas, chemical formulas, tables, etc.▼・・・■＾ Optical resolution by a biochemical resolution method characterized by optically resolving an optically active ester represented by * (wherein R and * are the same as above) and separating and collecting each optically active form. Method for producing active 4-penten-2-ol derivatives. (2) The alcohol ■^* of the hydrolysis product is (R)-4
-Penten-2-ol [▲There are mathematical formulas, chemical formulas, tables, etc.▼], and the unreacted ester ■^* is the general formula (S) -■ (S)▲There are mathematical formulas, chemical formulas, tables, etc.▼ ...(S)
-■ (wherein R is the same as above). (3) The production method according to claim 1 or 2, wherein the enzyme derived from a microorganism is derived from the genus Chromobacterium.