Process for producing optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane

Abstract

A process for producing an optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane (2), comprising a step of asymmetric hydrolysis of 2-alkyl-1,1,3-trialokoxycarbonylpropane (1) by using an enzyme capable of selectively hydrolyzing an ester moiety of either one enantiomer of 2-alkyl-1,1,3-trialkoxycarbonylpropane (1), or by using a culture of a microorganism capable of producing the enzyme or a treated object thereof.

Claims

1. A process for producing an optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane of Formula (2): ##STR00003## wherein: R.sub.1, R.sub.2 and R.sub.3, which may be the same or different, are a C1-C4 alkyl group; and * represents an asymmetrical center; comprising hydrolyzing a 2-alkyl-1,1,3-trialokoxycarbonylpropane of Formula (1): ##STR00004## wherein R.sub.1, R.sub.2 and R.sub.3 are as defined above, by contacting the 2-alkyl-1,1,3-trialkoxycarbonylpropane represented by Formula (1) with a culture of a microorganism, wherein the microorganism comprises an enzyme capable of selectively hydrolyzing an ester moiety of either one enantiomer, wherein the enzyme comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, to provide the optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane of Formula (2).

2. The process according to claim 1, wherein R.sub.1 is methyl.

3. The process according to claim 1, wherein R.sub.2 is methyl.

4. The process according to claim 1, wherein R.sub.3 is methyl.

5. The process according to claim 1, wherein both R.sub.1 and R.sub.2 are methyl.

6. The process according to claim 1, wherein both R.sub.2 and R.sub.3 are methyl.

7. The process according to claim 1, wherein R.sub.1, R.sub.2 and R.sub.3 are methyl.

8. The process according to claim 1, wherein the enzyme comprises the amino acid sequence of SEQ ID NO: 1.

9. The process according to claim 1, wherein the enzyme comprises the amino acid sequence of SEQ ID NO: 3.

Description

EXAMPLES

(1) In the following, the present invention will be explained more specifically by way of examples; however, the present invention is not limited to these examples.

Examples 1 to 14

(2) To 35 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane and each of various enzymes shown in Table 1 respectively weighed in the amount shown in Table 2, 2 mL of 100 mM potassium phosphate buffer (pH 7.0) was added. The resultant solution was stirred at 25 C. for 20 hours, and then 2.5 mL of acetonitrile was added thereto and the mixture was drawn through a membrane filter. The optical purity of the resultant filtrate was analyzed by high performance liquid chromatography [column: CHIRALCEL (registered trademark) OB-H, 4.6 mm15 cm, 5 m (produced by Daicel Chemical Industries, Ltd.)], and the chemical purity was analyzed by high performance liquid chromatography [column: SUMIPAX ODS D-21OFF, 4.6 mm15 cm, 3 m (available from Sumika Chemical Analysis Service, Ltd.)]; and thus, yield and enantiomer excess of the obtained optically active 2-methyl-1,1,3-trimethoxycarbonylpropane were determined. The results are shown in Table 2.

Examples 15, 16

(3) To 70 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane and an enzyme shown in Table 1 weighed in the amount shown in Table 2, 4 mL of 100 mM potassium phosphate buffer (pH 7.0) was added. The resultant solution was stirred at 25 C. for 3.5 hours, and then 5 mL of acetonitrile was added thereto and the mixture was drawn through a membrane filter. The filtrate was analyzed in the same method as in Examples 1 to 14, and yield and enantiomer excess of obtained optically active 2-methyl-1,1,3-trimethoxycarbonylpropane were determined. The results are shown in Table 2.

(4) TABLE-US-00001 TABLE 1 Enzyme manufacturer Preparation (commercially method of Example Name of enzyme Origin of enzyme available enzyme) enzyme 1 Cholesterol Candida Roche Diagnostics Esterase cylindracea 2 CHIRAZYME E-3, lyo Thermophilic Roche Diagnostics micro-organism 3 Cholesterol esterase, lyo Candida Roche Diagnostics cylindracea 4 PLE-A Pig liver Amano Enzyme 5 ChiroCLEC-CR Candida rugosa Altus Biologics 6 CHIRAZYME L-3 Candida rugosa Roche Diagnostics 7 Lipase OF Candida Meito Sangyo cylindracea 8 Lipase-MY Candida Meito Sangyo cylindracea 9 Esterase originated from Arthrobacter Prepared Arthrobacter strain globiformis according to the SC-6-98-28 method described in JP 3151893 10 CHIRAZYME E-4, Lyo. Thermophilic Roche Diagnostics micro-organism 11 CHIRAZYME P-1, Bacillus Roche Diagnostics Lyo. (Subtilisin) licheniformis 12 Purafect 4000E Bacillus subtilis GENENCOR 13 SP-525 Candida antactica Novozymes Japan 14 -Chymotrypsin Bovine pancreas SIGMA 15 Esterase Chromobacterium Prepared 160S189F363term chocolatum according to the originated from method described Chromobacterium strain in JP 3486942 SC-YM-1 16 Esterase Chromobacterium Prepared 160A189Y363term chocolatum according to the originated from method described Chromobacterium strain in JP 3486942 SC-YM-1

(5) TABLE-US-00002 TABLE 2 Amount of Enantiomer Excess enzyme Yield excess optical Example (mg) (%) (% ee) isomer 1 2.2 9.6 100 (R)-isomer 2 2.1 9.6 100 (R)-isomer 3 2.1 5.6 100 (R)-isomer 4 2.1 2.8 100 (R)-isomer 5 2.0 24.4 51.5 (R)-isomer 6 2.0 77.0 7.3 (R)-isomer 7 2.2 61.3 18.7 (R)-isomer 8 2.4 77.5 8.2 (R)-isomer 9 74.8 14.4 100 (S)-isomer 10 2.0 46.9 46.5 (S)-isomer 11 2.1 53.0 36.3 (S)-isomer 12 10.2 62.6 31.3 (S)-isomer 13 1.4 53.8 14.0 (S)-isomer 14 2.1 71.2 6.6 (S)-isomer 15 7.0 37.5 95.0 (R)-isomer 16 7.0 36.5 96.5 (R)-isomer

Examples 17, 18

(6) To 70 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane and 7.0 mg of esterase derived from Chromobacterium strain SC-YM-1 (160S189F363term), 4 mL of 100 mM potassium phosphate buffer either at pH 5 or pH 9 was added. The resultant solution was stirred at 25 C. for 3.5 hours, and then 5 mL of acetonitrile was added thereto and the mixture was drawn through a membrane filter. The filtrate was analyzed in the same method as in Examples 1 to 14, and yield and enantiomer excess of the obtained optically active 2-methyl-1,1,3-trimethoxycarbonylpropane were determined. The results compared with Example 15 (pH7) are shown in Table 3.

(7) TABLE-US-00003 TABLE 3 Enantiomer Excess Yield excess optical Example pH (%) (% ee) isomer 17 9 36.2 100.0 (R)-isomer 15 7 37.5 95.0 (R)-isomer 18 5 78.0 21.0 (R)-isomer

Examples 19, 20

(8) To 70 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane and 7.0 mg of esterase derived from Chromobacterium strain SC-YM-1 (160S189F363term), 4 mL of 100 mM potassium phosphate buffer (pH7.0) was added. The resultant solution was stirred at 10 C. or 0 C. for a time described in Table 4, and then 5 mL of acetonitrile was added thereto and the mixture was drawn through a membrane filter. The filtrate was analyzed in the same method as in Examples 1 to 14, and yield and enantiomer excess of the obtained optically active 2-methyl-1,1,3-trimethoxycarbonylpropane were determined. The results compared with Example 15 (25 C.) are shown in Table 4.

(9) TABLE-US-00004 TABLE 4 Enantiomer Excess Temperature Time Yield excess optical Example ( C.) (hr) (%) (% ee) isomer 15 25 3.5 37.5 95.2 (R)-isomer 19 10 8.0 47.3 91.8 (R)-isomer 20 0 21.0 42.3 100.0 (R)-isomer

Examples 21-29

(10) E. coli strain JM105 was transformed with a plasmid including each esterase gene shown in Table 5. The obtained transformant was inoculated onto sterile LB (1% Bacto-Triptone, 0.5% Bacto-Yeast extract, 1% sodium chloride) culture medium (100 ml) containing 0.1 mM IPTG and 50 g/ml of ampicillin, and cultured under shaking (37 C., 24 hours). The obtained culture liquid was centrifuged, to obtain about 0.6 g of wet bacterial cells. About 0.6 g of wet bacterial cells were suspended in 5 mL of 0.1 M potassium phosphate buffer (pH 7.0), 5 g of glass beads of 0.1 mm in diameter was added, and then disrupted by a Multi-beads shocker (produced by Yasui Kikai Corporation, 2500 rpm, 20 minutes). The obtained disrupted liquid was centrifuged (10000 rpm, 4 C., 10 minutes), and the supernatant was provided as a crude enzyme liquid.

(11) To 70 mg of 2-methyl-1,1,3-trimethoxycarbonylpropane, the crude enzyme liquid was added in the enzyme amount shown in Table 6, and further 4 mL of 170 mM potassium phosphate buffer (pH 7.0) was added. The solution was stirred at 25 C. for 5 hours, 2 mL of acetonitrile containing biphenyl (internal standard substance) was added thereto, and the mixture was drawn through a membrane filter. The filtrate was analyzed in the same method as in Examples 1 to 14, and yield and enantiomer excess of the obtained optically active 2-methyl-1,1,3-trimethoxycarbonylpropane were determined. The results are shown in Table 6.

(12) TABLE-US-00005 TABLE 5 Example Name of enzyme Origin of enzyme Plasmid 21 Esterase N43SA363term Chromobacterium pCCN43SA363term originated from chocolatum (see JP2000-78988A) Chromobacterium strain SC-YM-1 22 Esterase Chromobacterium pCC160S189Y363term 160S189Y363term chocolatum (see JP3486942) originated from Chromobacterium strain SC-YM-1 23 Esterase Chromobacterium pCC160A189H363term 160A189H363term chocolatum (see JP3486942) originated from Chromobacterium strain SC-YM-1 24 Esterase Chromobacterium pCC160S189H363term 160S189H363term chocolatum (see JP3486942) originated from Chromobacterium strain SC-YM-1 25 Esterase Chromobacterium pCC160A189F363term 160A189F363term chocolatum (see JP3486942) originated from Chromobacterium strain SC-YM-1 26 Esterase V325I originated Chromobacterium pCCV325I from Chromobacterium chocolatum (see JP2000-78988A) strain SC-YM-1 27 Esterase originated from Chromobacterium pCC363term Chromobacterium strain chocolatum (see JP3486942) SC-YM-1 28 Esterase Chromobacterium pCC160A189Y363term 160A189Y363term chocolatum (see JP3486942) originated from Chromobacterium strain SC-YM-1 29 Esterase T240AV288A Chromobacterium pCCT240AV288A originated from chocolatum (see JP2000-78988A) Chromobacterium strain SC-YM-1

(13) TABLE-US-00006 TABLE 6 Amount of Enantiomer Excess enzyme Yield excess optical Example (mg) (%) (% ee) isomer 21 34.6 40.9 100 (R)-isomer 22 34.9 18.1 100 (R)-isomer 23 35.3 23.8 100 (R)-isomer 24 35.3 10.9 100 (R)-isomer 25 35.2 6.2 100 (R)-isomer 26 34.6 9.5 100 (R)-isomer 27 34.7 20.9 100 (R)-isomer 28 34.9 12.2 100 (R)-isomer 29 35.1 6.3 100 (R)-isomer

Example 30-1

Enzymatic Hydrolysis

(14) To 11.37 g of 2-methyl-1,1,3-trimethoxycarbonylpropane and 68.4 g of 100 mM potassium phosphate buffer (pH 7.0), 1.0 g of a crude enzyme liquid prepared in the same method as in Examples 21 to 29 from a transformant of E. coli strain JM105 containing plasmid (pCCN43SA363term) was added. The resultant solution was stirred at 0 C. for 23 hours. During stirring, pH of the solution was kept at 7 by dropping 10 wt % of aqueous sodium hydroxide solution. After stirring the solution, 20 g of tert-butyl methyl ether was added, and the obtained mixture was drawn through a glass filter. The filtrate was separated into an organic phase and an aqueous phase. To the aqueous phase, 20 g of tert-butyl methyl ether was added and a phase separation gave 81.8 g of an aqueous phase. The obtained organic phases were combined and washed with 5.1 g of 5 wt % of aqueous sodium bicarbonate solution. The washed organic phase was concentrated under reduced pressure, to obtain 4.49 g of (R)-2-methyl-1,1,3-trimethoxycarbonylpropane as a yellow oily substance. Yield: 47.7%, Enantiomer excess: 100% ee.

Example 30-2

Collection of Hydrolysate

(15) To 79.8 g of the aqueous phase obtained in Example 30-1, 3.12 g of 35 wt % of hydrochloric acid was added to adjust pH at 2.0. After adding 20.0 g of ethyl acetate and 25.0 g of sodium chloride and stirring the same, the obtained mixture was subjected to liquid separation. 25.0 g of ethyl acetate was added to the obtained aqueous phase, and extracted. The obtained organic phases were combined and washed with 25.0 g of 25 wt % of aqueous sodium chloride solution. The washed organic phase was concentrated under reduced pressure to obtain 5.1 g of a yellow-brown oily substance.

Example 30-3

Esterification

(16) 1.0 g of the yellow-brown oily substance obtained in Example 30-2 was dissolved in 20.0 g of methanol. The obtained solution was cooled to 0 C., and 1.0 g of trimethylsilyl chloride was added dropwise over about 10 minutes. After dropping, temperature of the obtained reaction solution was raised to 20 to 25 C., and kept at this temperature for 45.5 hours. The obtained reaction solution was diluted with 32 g of tert-butyl methyl ether. After adding 40 g of 5 wt % of aqueous sodium bicarbonate solution and stirring the same, liquid separation was conducted to obtain 36.2 g of an organic phase and 57.8 g of an aqueous phase. For each of the obtained organic phase and aqueous phase, the chemical purity was analyzed by high performance liquid chromatography, and for the organic phase, the optical purity was analyzed by high performance liquid chromatography, and the yield and enantiomer excess of the obtained optically active 2-methyl1,1,3-trimethoxycarbonylpropane were determined. The chemical purity and the optical purity were analyzed in the same method as in Examples 1 to 14. Yield of 2-methyl-1,1,3-trimethoxycarbonylpropane contained in the organic phase and aqueous phase was 46.8%, and enantiomer excess of 2-methyl-1,1,3-trimethoxycarbonylpropane contained in the organic phase was 86.8% ee (S isomer).

Example 31-1

Enzymatic Hydrolysis

(17) To 150 g of 2-methyl-1,1,3-trimethoxycarbonyl propane and 1035 g of 100 mM potassium phosphate buffer (pH 7.0), 17.0 g of a crude enzyme liquid prepared in the same method as in Examples 21 to 29 from a transformant of E. coli strain JM105 containing plasmid (pCC160S189F363term) was added. The resultant solution was stirred at 0 C. for 42 hours. During stirring, the pH of the solution was kept at 7 by dropping 10 wt % of aqueous sodium hydroxide solution. The solution after end of the stirring was added with 613 g of ethyl acetate, and the obtained mixture was drawn through a glass filter. The filtrate was separated into an organic phase and an aqueous phase, and the aqueous phase was separated by adding 750 g of ethyl acetate. The obtained organic phases were combined, to obtain 1500 g of an organic phase and 1321 g of an aqueous phase. The organic phase was further washed with 150 g of 5 wt % of aqueous sodium bicarbonate solution. The washed organic phase was concentrated under reduced pressure, to obtain 67.6 g of (R)-2-methyl-1,1,3-trimethoxycarbonylpropane as a yellow oily substance. Yield: 39.1%, Enantiomer excess: 100% ee.

Example 31-2

Collection of Hydrolysate

(18) To 1315 g of the aqueous phase obtained in Example 31-1, 61 g of 35 wt % hydrochloric acid was added to adjust pH at 2.0. After adding 300 g of ethyl acetate and 415 g of sodium chloride and stirring the same, the obtained mixture was subjected to liquid separation. To the obtained aqueous phase, 375 g of ethyl acetate was added and extracted. The obtained organic phases were combined and washed with 375 g of 25 wt % of aqueous sodium chloride solution. The washed organic phase was concentrated under reduced pressure to obtain 105 g of a yellow-brown oily substance.

Example 31-3

Esterification

(19) 1.0 g of the yellow-brown oily substance obtained in Example 31-2 was dissolved in 20.1 g of methanol. The obtained solution was cooled to 0 C., and 1.0 g of trimethylsilyl chloride was added dropwise over about 10 minutes. After dropping, temperature of the obtained reaction solution was raised to 20 to 25 C., and kept at this temperature for 45 hours. The obtained reaction solution was diluted with 32 g of tert-butyl methyl ether. After adding 40 g of 5 wt % of aqueous sodium bicarbonate solution and stirring the same, liquid separation was conducted to obtain 37.0 g of an organic phase and 57.3 g of an aqueous phase. When analyzing in the same method as in Example 30-3, yield of 2-methyl-1,1,3-trimethoxycarbonylpropane contained in the organic phase and aqueous phase was 51.1%, and enantiomer excess of 2-methyl-1,1,3-trimethoxycarbonylpropane contained in the organic phase was 70.6% ee (S isomer).

Industrial Applicability

(20) According to the method of the present invention, it is possible to produce an optically active 2-alkyl-1,1,3-trialkoxycarbonylpropane with high optical purity without using a low temperature condition.