KR100591082B1 - The method of making optically active ethyl 3-hydroxy-3-phenylpropionate by microbial deracemization - Google Patents

Abstract

The present invention relates to optically active ethyl 3-hydroxy-3 in racemic ethyl 3-hydroxy-3-phenylpropionate using microorganisms having deracemization ability. It relates to a method for producing phenylpropionate. More specifically, a method of preparing optically ethyl 3-hydroxy-3-phenylpropionate by deracemizing racemic ethyl 3-hydroxy-3-phenylpropionate by enzymes produced by microorganisms. It is about.

In the method for preparing optically active ethyl 3-hydroxy-3-phenylpropionate, the deracemization using microorganisms is a new process that has not been reported before, and the racemic ethyl 3-hydroxy-3-phenylpropionate is known. It is theoretically possible to prepare 100% yield of (R)-or (S) -ethyl 3-hydroxy-3-phenylpropionate from cypionate.

Scheme 1

Ethyl 3-hydroxy-3-phenylpropionate, deracemation, microorganism

Description

The method of making optically active ethyl 3-hydroxy-3-phenylpropionate by microbial deracemization}

The present invention relates to a process for preparing optically active ethyl 3-hydroxy-3-phenylpropionate from racemic ethyl 3-hydroxy-3-phenylpropionate by microorganisms. More specifically, screening microorganisms having a desalamification capacity, and using this, racemic ethyl 3-hydroxy- represented by general formulas (R) -1 and (S) -1 in [Scheme 1] A method for producing 3-phenylpropionate with (S) -ethyl 3-hydroxy-3-phenylpropionate represented by general formula (S) -1. In the same manner, preparation of (R) -ethyl 3-hydroxy-3-phenylpropionate is possible.

(S) -Ethyl 3-hydroxy-3-phenylpropionate is a compound used as an intermediate of fluoxetine, an antidepressant, and can also be used as an intermediate such as tomoxetine and nisoxetine. The range of use is very wide.

The conventional method for preparing optically active ethyl 3-hydroxy-3-phenylpropionate by microorganisms is carried out by reducing ethyl benzoyl acetate as a precursor using microorganisms having alcohol dehydrogenase production ability ( reduction) to prepare optically active ethyl 3-hydroxy-3-phenylpropionate. Kumar et al. (Tetrahedron Letters, 1991, 32 (16), 1901-1904) (B) have 85% ee by reduction in ethyl benzoyl acetate using Bakers' yeast as a biocatalyst in aqueous glucose solution. Ethyl 3-hydroxy-3-phenylpropionate was synthesized. In addition, Chenevert et al. (Tetrahedron, 1992, 48 (33), 6769-6776), using the Geotrichum candidum (CBS 233-76) strain, showed reduction in ethyl benzoyl acetate with 64% yield and 98% ee or more. ) -Ethyl 3-hydroxy-3-phenylpropionate was synthesized.

On the other hand, a process for preparing optically active alcohol compounds from racemic alcohols by microorganisms has been developed. Chadha and Baskar (Tetrahedron: Asymmetry, 2002, 13 , 1461-1464) use racemic ethyl 2 using Candida parapsilosis (ATCC 7330). 99% ee of (S) -ethyl 2-hydroxy-4-phenylbutyrate was prepared in -hydroxy-4-phenylbutyrate. Hasegawa et al. (Agricultural and Biological Chemistry, 1990, 54 (7), 1819-1827) also used the same strain to have 71-98% ee in racemic 1,2-pentanediol (1,2-pentanediol). S) -1,2-pentanediol was prepared. According to Carnell (Advances in Biochemical Engineering / Biotechnology, 1999, 63, 57-72), the enzymes involved in deracemization are NAD ⁺ -linked dehydrogenase and NADPH-linked dehydrogenase. The optically active alcohol compound can be prepared from the racemic alcohol compound.

Therefore, the present inventors have been reported that the process of de-semination of diol (diol) or alpha-hydroxy ester (alpha-hydroxy ester) by the microorganism, but racemic ethyl 3-hydroxy-3-phenylpropionate Taking into account that there is no process for de-assemising, the (S) -ethyl 3-hydroxy-3-phenylpropionate can be obtained with high optical purity in racemic ethyl 3-hydroxy-3-phenylpropionate. The manufacturing method was developed.

Therefore, the object of the present invention is that unlike the conventional reduction process mentioned above, (R) -ethyl 3-hydroxy-3-phenylpropionate present in 50% of the racemic compound using the deracemizing microorganism as a biocatalyst. To provide a process for converting cypionate into (S) -ethyl 3-hydroxy-3-phenylpropionate to produce (S) -ethyl 3-hydroxy-3-phenylpropionate of high optical purity. have. On the contrary, the use of a microorganism acting as a biocatalyst converts (S) -ethyl 3-hydroxy-3-phenylpropionate to (R) -ethyl 3-hydroxy-3-phenylpropionate (R). -Ethyl 3-hydroxy-3-phenylpropionate can be prepared.

The method of the present invention for achieving the above object is screened and cultured microorganisms having optically active ethyl 3-hydroxy-3-phenylpropionate production in racemic ethyl 3-hydroxy-3-phenylpropionate And reacting it using a catalyst.

Hereinafter, the present invention will be described in more detail. As described above, the present invention relates to a process for preparing optically active ethyl 3-hydroxy-3-phenylpropionate from microacetic ethyl 3-hydroxy-3-phenylpropionate using microorganisms.

Microorganisms of various microorganisms used in the present invention having the Kluyveromyces genus microorganism, or the like Candida parapsilosis and Candida with Candida spp, including rugosa such as Geotrichum candidium, Kluyveromyces lactis and Kluyveromyces marxianus, Saccharomyces cerevisiae, etc. Tallahassee semi Huaneng species This includes, but is not limited to.

Racemic ethyl 3-hydroxy-3-phenylpropionate and optically active (R)-and (S) -ethyl 3-hydroxy-3-phenylpropionate were subjected to gas chromatography (Donan Instruments, Model DS 6200). ) And the reaction solution was extracted with ethyl acetate and analyzed by the following method.

Racemic ethyl 3-hydroxy-3-phenylpropionate is heated by capturing BP-1 column (SGE Co., Ltd., 0.53mm × 30m) at 70 ° C for 5 minutes and then raising it by 10 ° C per minute to 220 ° C. And kept at 220 ° C. for 15 minutes. As a carrier gas, helium gas was flowed at a rate of 2 ml / min and detected using a flame ionization detector (FID) at 230 ° C. Racemic ethyl 3-hydroxy-3-phenylpropionate was detected at 19.1 min.

Optically active (R)-and (S) -ethyl 3-hydroxy-3-phenylpropionate was subjected to gas chromatography equipped with chiral column G-TA (Astec, 0.32 mm x 30 m). Quantification by Analytical conditions for (R)-and (S) -ethyl 3-hydroxy-3-phenylpropionate heated the column at 120 ° C. for 30 minutes and then raised the temperature to 50 ° C. per minute to 170 ° C., followed by 15 minutes at 170 ° C. Maintained. Helium gas was used as the carrier and was detected using FID at 170 ° C. while maintaining the column head pressure at 8 psi. (R) -ethyl 3-hydroxy-3-phenylpropionate was detected at 36.5 minutes and (S) -ethyl 3-hydroxy-3-phenylpropionate was detected at 36.7 minutes, respectively.

In addition, the synthesized ethyl 3-hydroxy-3-phenylpropionate was confirmed by FT-NMR (Burker, model DPX300), the analysis results are as follows.

1 H-NMR δ = 7.37-7.28 (m, 5H), 5.13-5.08 (m, 1H), 4.14-4.11 (q, 2H), 3.51 (s, 1H), 2.57-2.54 (t, 2H), 1.23- 1.21 (t, 3 H)

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to the following Examples.

Example 1-9

After dissolving 30 g of ethyl benzoyl acetate in 50 ml of ethanol at 0 ° C., 50 ml of ethanol in which 2 g of sodium borohydride was dissolved was added dropwise for 45 minutes at 0 ° C. After stirring for 15 minutes, 100 ml of ice water was added and titrated to pH 7.0 with 10% HCl. The mixture was extracted with ethyl acetate, followed by water removal, and ethyl acetate was evaporated to remove 27.6 g of racemic ethyl 3-hydroxy-3-phenyl propionate. At this time, the yield was 91%, and the material could be confirmed by FT-NMR.

The microorganisms shown in Table 1 were incubated in 200 ml of GYP medium solution in a 1 L flask for 40 hours, and then the cultured cells were centrifuged. In a 15 ml vial, 1.78 ml of 50 mM phosphate buffer (pH 7.0), 0.2 ml of 1 M glucose and 20 μl of racemic ethyl 3-hydroxy-3-phenylpropionate were added, followed by separation of the cells. After the reaction was carried out at 30 ℃. After reaction for a certain time, the reaction solution was extracted with ethyl acetate and analyzed according to the above analysis method. Optical purity of (S) -ethyl 3-hydroxy-3-phenylpropionate is shown in Table 1 below.

Table 1

Example Strain name Response time (hours) Optical purity (% ee) One C 4 andida parapsilosis IFO 0708 24 99.5 2 Candida parapsilosis KCTC 7653 50 99.0 3 Geotrichum candidum IFO 4597 160 15.5 4 Geotrichum candidum IFO 5767 160 10.3 5 Kluyveromyces lactis KCTC 7133 168 23.6 6 Kluyveromyces marxianus KCTC 7524 160 22.6 7 Saccharomyces cerevisiae KCTC 1218 46 99.0 8 Saccharomyces cerevisiae KCTC 1552 46 99.0 9 Saccharomyces cerevisiae KCTC 7917 46 33.9

As can be seen in Example 1-9, microorganisms having deracemization ability can be used in preparing (S) -ethyl 3-hydroxy-3-phenylpropionate, and if an appropriate microorganism is selected, High optical purity of (R)-or (S) -ethyl 3-hydroxy-3-phenylpropionate can be prepared from racemic ethyl 3-hydroxy-3-phenylpropionate. In addition, the manufacturing process is simple and can be used in the actual production process as well as environmentally friendly in that it uses microorganisms.

Claims (7)

delete A microorganism having a deracemization capacity is cultivated, and an optically active ethyl 3-hydroxy-3-phenylpropionate is prepared from racemic ethyl 3-hydroxy-3-phenylpropionate using the catalyst as a catalyst. Method for preparing optically active ethyl 3-hydroxy-3-phenylpropionate using microorganisms The microorganism of claim 2, wherein the microorganisms are Candida parapsilosis , Geotrichum candidium, Kluyveromyces microorganisms and Saccharomyces cerevisiae. The manufacturing method to make. delete delete delete delete