Title of Invention

"PROCESS FOR PREPARING OPTICALLY ACTIBE BETA HYDROXYLBUTYL ESTER DERIVATIVES AND THEIR ACIDS DERIVATIVES"

Abstract The present invention relates to process for the preparing of optically active ester derivatives and their acid derivatives which are used intensively as important chiral intermediates from racemic p- hydroxybutyl ester derivatives. In more detail, this invention relates to the process for preparing optically active p-hydroxybutyl ester derivatives and their acid derivatives by stereospecific hydrolysis of racemic p-hydroxybutyl ester derivatives using lipases or lipase- producing microorganisms in the aqeous phase or organic phase including aqeous solvent. The method of making optically active ester derivatives and their acid derivatives by hydrolysis of ß-hydroxybutyl ester derivatives represented by the general formula 1 in scheme 1 is easier and more economical comparing to the conventional methods and the products have high optical purity. Also separation of ester derivatives from acid derivatives is easy after reaction. Thus this method is a useful process on the industrial scale.
Full Text [Technical Field]
The present invention relates to a process for the preparation of optically active p-hydroxybutyl ester derivatives and their acids derivatives. In more detail, this invention relates to the process for preparing optically active p-hydroxybutyl ester derivatives and their acid derivatives by the hydrolysis of racemic p-hydroxybutyl ester derivatives represented by the general formula 1 in scheme 1 using lipases or lipase-producing microorganisms.
The above-mentioned optically! active p-hydroxybutyl ester derivatives and their acid derivatives can be used intensively as important chiral intermediates. Also}, p-hydroxybutyl ester derivatives and their acid derivatives produced by this invention have high optical purity and this method can be used in practical process because separation and recovery of the products are easy. Therefore this invention can be used on the industrial scale. [Scheme 1]
(Scheme Removed) (R=CnH2n+1.(n=l~8)) (X=H, N3, CN, F, C1, Br. I)
According to a report, ethyl (R)-3+hydroxybutyrate is an intermediate for an anti-glaucoma drug(Chirality in industry II. Chichester, UK:Wiley, 1997, 245-262) and (b)-3-hydroxybutyrate is used for synthesizing pheromones(Tertahedron, 1989, 45:3233-3298) and carbapenems(Journal of the Chemical Society. Perk in Transaction, 1999, l: 2489-2494).
And ethyl (R)-4-chloro-3-hydroxybutyrate is used for synthesizing L-carnitine(Journal of the American Chemical Society, 1983. 105:5925-5926), (R)-4-amino-3-hydroxybutyric acid(GABOB) and (R)-hydroxy-2-pyrrolidone. Ethyl (S)-4~chloro-3-hydroxybutyrate is a valuable synthon for the production of hydroxymethylglutaryl CoA(HMG-CoA) reductase inhibitorOournal of Medicinal Chemistry, 1990,33:2952-2956).
[Background Art]
There are several methods to prepare optically active p-hydroxybutyl ester derivatives. Ethyl (S)-3~hydroxybutyrate is synthesized by asymmetric hydrogenation of ethylacetoacetate using B1NAP-coordinated Ru(II) complexs(journal of the American Chemical

Society, 1987, 109:5856-5858)

However, this method has

disadvantages of high pressure during the reaction and high cost of metal catalyst.

using
reduction of 3-oxo-esters
et
al. (Tetrahedron Lettes, 1993,
Another method is the microorganisms. Jayasinghe
34:3949-3950) obtained ethyl (S)-3-hydroxybutyrate(58 % yield, 94 %e.e) by the reduction of ethyl acetoacetate using freeze-dried yeast in petroleum ether and Medson et al.(Tetrahedron:Asymmetry, 1997, 8:1049-1054) obtained ethyl (S)-3-hydroxybutyrate(yield 69 %, 99 %e.e) from ethyl acetoacetate by the reduction using yeast in
organic solvent. Chin-Joe et al.((Biotechnology and Bioengineering, 2000, 69:370-376) obtained ethyl (S)-3-hydroxybutyrate(99 %e.e) at 85 % conversion by the reduction of ethyl acetoacetate using Baker's yeast. However, these methods have disadvantages of low yield and purification problem after reaction.
On the other hand, Sugai et al.(Agricultural and Biological Chemistry,
1989, 53:2009-2010) obtained ethyl (S)-3-
hydroxybutyrate(99.4 %e.e) by transesterification of racemic ethyl 3-hydroxybutyrate using vinyl butanoate as an acylating agent and porcine pancreatic lipase as a catalyst. Fishman et al.(Biotechnology and Bioengineering, 2001, 74:256-263) obtained ethyl (S)-3~ hydroxybutyrate(40 % yield, 99-4 %e.e) using CALlMCandida antartica) lipase and vinyl acetate as an acyl donor.
In another case, ethyl (R)-3-hydroxybutyrate can be prepared by acidic alcoholysis of Poly-(R)-3-hydroxybutyrate accumulated by microorganisms(Enzyme and Microbial Technology, 2000, 27:33-36).
Optically active ethyl 4-chloro-3-hydroxybutyrate can be produced by reduction of ethyl 4-chloroacejtoacetate. Matsuyama et al.(Japan Kokai Tokkyo Koho, 06-209782, Aug.2, 1994) obtained ethyl (S)-4-chloro-3-hydroxybutyrate(97 % yield, 98 %e.e) using Kluyveromyces lactis NR1C 1329. Kataoka et al.(Applied microbiology and Biotechnology, 1999, 51:486-490) obtained ethyl (R)-4-chloro-3-hydroxybutyrate(94 % yield, 92 % e.e) using recombinant microorganism, which coexpress both the alcohol reductase I gene from Sporobolomyces salmonicolor and the glucose1 dehydrogenase gene from Bacillus megaterium. Yamamoto et al.(Bioscience Biotechnology and Biochemistry. 2002, 66(2):481~483) produced ethyl (R)-4-chloro-3-hydroxybutyrate(95.2 % conversion. 99 %e.e) using recombinant microorganism expressing secondary alcohol dehydrogenase from Candida par&psilosis. However, these methods have disadvantage of long reaction time.
On the other hand, Hoff et al.(Tetrahedron'-Asymmetry, 1999,10:1401-
1412) obtained ethyl (S)-4-chlifo-3-hydroxybutyrate(24 % yield, 86 %e.e) by transesterifying for 5 days using Rhizomucor miehei lipase(RML) in organic phase(benzene).
Suzuki et al.(Enzyme Microbiology and Technology. 1999,24:13-20) produced ethyl (R)-4-chloro-3-)hydroxybutyrate(99.8 %e.e) using dechlorinase-producing microorgan sm.
As previously stated, optically active p-hydroxybutyl ester derivatives can be prepared by the stereoselective reduction of keto esters or the enzymatic transesterification. However, these methods are not suitable due to their disadvantages including low enantiomeric excess, low yield or difficulties in the separtation of products and reactants after reaction. For solving these problems, there is hydrolysis of (3-hydroxybutyl ester derivatives. Santaniello et al.(Gazzetta Chemica Italiana, 1989,119:581-584) obtained ethyl (R)-4-chloro-3-hydroxybutyrate and (S)-their acid by hyrolysis of ethyl 4~chloro-3-hydroxybutyrate using pig liver esterase. However, this method has disadvantages of low yield(23 %) and low enantiomeric excess(16 %e.e) and is not suitable- for industrial use.
[Disclosure]
[Technical Problem]
The process for preparing of (3-hydroxy butyl ester derivatives and their acid derivatives of high optical purity was developed from racemic f3~hydroxybutyl ester derivatives represented by the general formula 1 in scheme 1 by stereospecific hydrolysis using lipases or lipase-producing microorganims.
This method is simple and ester derivatives and their acid derivatives of higher optical purity can be obtained comparing to the conventional methods.
Accordingly, the objective of this invention is to provide the method of preparing optically active esters and their acids from racemic p-hydroxybutyl ester derivatives using enzymes or microorganisms.
For the above objectives, the present invention consists of the process for preparing high optically acive p-hydroxybutyl ester derivatives and their acids from racemic p-hydroxybutyl ester derivatives by stereospecific hydrolysis using lipases or lipase-producing microorganisms as biocatalysts in aqeous phase or organic phase including aqeous solvent.
[Technical Solution]
This invention is explained in more detail as follows. As mentioned above, this invention relates to the process for preparing optically active p-hydroxybutyl ester derivatives and their acid derivatives by stereospecific hydrolysis of raeemic p-hydroxybutyl ester derivatives using lipases or lipase-producing microorganisms as biocatalysts in aqeous phase or organic phase including aqeous solvent.
In this invention, methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate, butyl 3-hydroxybutyrate, ethyl 3-azido-3-hydroxybutyrate, ethyl \-chloro-3-hydroxybutyrate, ethyl I 4~bromo-3-hydroxybutyrate and ethyl 4-cyano-3-hydroxybutyrate| are used as racemic p-hydroxybutyl esters, but the reactant is not restricted to them. In the general formula 1 in scheme 1, X :s H, CN. N3. F, CI, Br or I and R is CnH2n+1 (n=l~8).
Non-limiting examples of the commercially available lipases include PS lipase from Amano Inc., Candida rugosa lipase and Novozyme 435 and non-limiting examples of the lipase-producing microorganism include Candida rugosa and Rhodococcus butanica.
After reaction, optically active est|ers and their acids are seperated
respectively by solvent extraction method or column chromatography.
In this invention, racemic compounds were determined by gas chromatography(Donam Instrument Inc. Model 6200) equipped with HP-FFAP(Agilent, Inc., 30 mm X 0.53 m) column. The oven temperature was maintained initially at 70 V for 5 rnin and then raised at the rate of 10 °C/min to 220V, and maintained for 10 minutes. Helium gas is used as carrier at the rate of 2 ml/min, and compounds were detected using FID detector. The typical retention time of the components in this invention were as follows:
racemic methyl 3-hydroxybutyrate -15.48 min racemic ethyl 3-hydroxybutyrate-14.32 min racemic butyl 3-hydroxybutyrate-17.16 min racemic ethyl 4-azido-3-hydroxybutyrate-22.50 min racemic ethyl 4-chloro-3-hydroxyqutyrate-20.31 rnin
Racemic ethyl 4~bromo-3-hydroxybutyrate and racemic ethyl 4-cyano-3-hydroxybutyrate were analyzed using the same method used in the analysis of racemic methyl 3-hydroxybutyrate except that oven temperature was increased at 20 °C/min. in this condition racemic ethyl 4-bromo-3-hydroxybutyrate and racemic ethyl 4-cyano-3-hydroxybutyrate are detected at 11.7 min and 14.07 min respectively. Optically active methyl 3-hydroxybutyrate, ethyl 3-hydroxybutyrate. butyl 3-hydroxybutyrate and ethyl 4-azido-3-hydroxybutyrate were determined by HPLC(Waters, Inc., Model 1525) equipped with chial column OD-H(Daicel, 0.46 cm X 25 cm) using hexane and isopropyl alcohol mixture(90:10) as mobile phase. The absorbance was 220 nm and flow rate was 0.7 ml/min. The typical retention time of the components in this invention was as follows:
methyl (R)-3-hydroxybutyrate-10.28 min methyl (S)-3-hydroxybutyrate-12.44min ethyl (R)-3-hydroxybutyrate-12.77 min ethyl (S)-3-hydroxybutyrate-11.43 rnin butyl (R)-3-hydroxybutyrate~9.4 min
butyl (S)-3-hydroxybutyrate-10.64 min ethyl (R)-4-azido-3-hydroxybutyrate-8.7 min ethyl (S)-4-azido-3-hydroxybutyrate-10.86 min
Optically active ethyl 4-cyano~3-hydroxybutyrate was determined by a gas chromatography(Donam Instrument Inc. Model 6200) equipped with chiral column G-TA(Astec, 30 mm X 0.32 m). The oven temperature was maintained initialily at 100 °C for 5 min and then raised to 170 °C at the rate of 10 °C/min. and maintained for 20 minutes. Helium gas was used as carrier gas and column head pressure was maintained at 10 psi, and compounds were detected using FID detector. In this condition, the typical retention time of ethyl (R)-4-cyano-3-hydroxybutyrate and ethyl (S)-4-cyano-3-hydroxybutyrate was 16.75 min anq1 16.53 min respectively.
Optically active ethyl 4-chloro-3~hiydroxybutyrate was determined by a HPLC(Lab Alliance,Model 201) equipped with chial column OB-HCDaicel, 0.46 cm X 25 cm) using hexane and isopropyl alcohol mixture(95:5) as mobile phase. The flow rate was 0.7 ml/min and absorbance was 215 nm. The typical retention time of ethyl (R)-4-chloro-3-hydroxybutyrate and ethyl (S)-4-chloro-3-hydroxybutyrate was 14.42 min and 15.38 min, respectively.
Optically active ethyl 4-bromo-3-hydroxybutyrate was determined by a HPLC(Lab Alliance,Model 201) equipped with chial column AD-IKDaicel, 0.46 cm X 25 cm) using hexane and isopropyl alcohol mixture(90:l0) as mobile phase. Tjhe flow rate was 0.7 ml/min and absorbance was 220 nm. In this condition, the typical retention time of ethyl (R)-4-bromo-3-hydroxybutyrate and ethyl (S)-4-bromo-3-hydroxybutyrate was 12.23 min and 11.24 min. respectively.
And racemic compounds were confirmed by FT-NMR(Burker, Model DRX300 or JEOL.Model AR400) andj the results are as follows:
ethyl 4-azido-3~hydroxybutyrate :
'H-NMR (CDC13, 300MHz) 5(ppm)=1.28 (t.3H), 2.53(m,2H), 3.28(d,lH),
3.34(m,2H), 4.21(q,2H)
ethyl 4-chloro-3-hydroxybutyrate:
1H-NMR (CDC13, 400MHz) 4(ppm)=1.28 (t,3H), 2.62(d,2H),
3.53(br,lH), 3.60(d,2H), 4.20(q.2H), 4.33(m.lH)
ethyl 4-bromo-3-hydroxybutyrate:
1H-NMR (CDCi3, 400MHz) 5(ppm)=1.28 (t,3H), 2.7(m,2H), 3.48(dd,lH),
3.51(dd,lH), 4.17(q.2H), 4.20(m,lH)
ethyl 4-cyano~3-hydroxybutyrate:
1H-NMR (CDC13, 300MHz) 8(p[mi)=1.26 (t,3H). 2.5~2.7(m,4H).
4.18(q,2H), 4.32(m,lH)
A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.
Example 1
Racemic ethyl 3-hydroxybutyrate(l %, v/v) was added to the vial
containing 5 mi potassium phosphate bufer(pH 8.0. 0.1 M) and
Novozyme 435 (4 %, w/v). The reaction was carried out at 30 °C for
2 hours. The reaction mixture was extracted with ethyl acetate and
analyzed by above-mentioned method. Ethyl (S)~3-
hydroxybutyrate(97 %e.e) was obtained from organic solvent at 55 % conversion. The aqueous solution was acidified with hydrochloric acid and extracted with organic solvent. After esterification, ethyl (R)-3-hydroxybutyrate with optical purity of 80.4 % e.e was obtained.
Example 2-3
Instead of lipase used in Example 1, whole cells(20 %(w/v)) were used. Lipase-producing microorganisms were isolated by cultivating in a medium containing 1 % tributyrin. Isolated strains assimilated tributyrin and made clear zone. The strains were grown in LB

medium or GYP medium including glucose, and microorganisms were harvested by centrifuge and used as biocatalysts. The results are shown in Table 1.(Table Removed)
Example 4-5
Instead of ethyl 3-hydroxybutyrate used in Example 1,1% methyl 3-hydroxybutyrate and 5 % butyl; 3-hydroxybutyrate were used as reactants. The reaction was carried out with novozyme 435 lipase and the results are shown in Table} 2. .(Table Removed)
For Example 6
instead of ethyl 3-hydroxybutyrate used in Example 1, ethyl 4-azido-3-hydroxybutyrate was used The reaction was carried out for 1 hour and ethyl (S)-4-azido-3-hydroxybutyrate(80.2 %e.e) was obtained at 83.5 % conversion.
Example 7-8
Instead of ethyl 3-hydroxybutyrate used in Example 1, ethyl 4-chloro-3-hydroxybutyrate was used as a reactant and lipases were used as biocatalysts. The results are shown in Table 3. .(Table Removed)
Example 9
Instead of ethyl 3-hydroxybutyralte used in Example 1, ethyl 4-bromo-3-hydroxybutyrate(l %, w/v) was used as a reactant and novozyme 435 lipase was used as a biocatalyst. After reaction for 1 hour 40 minutes, ethyl (R)-4-bromo-3-hydroxybutyrate(99 %e.e) was obtained at 88.3 % conversion,.
Example 10
Instead of ethyl 3-hydroxybutyrate used in Example 1. ethyl 4-cyano-3-hydroxybutyrate(l %, w/v) was used as a reactant and novozyme 435 lipase was used as; a biocatalyst. After reaction for 3 hours, ethyl (R)-4-cyano-3-hydifoxybutyrate(99 %e.e)was obtained at 57.3 % conversion.
Example 11-13
Instead of ethyl 3-hydroxybutyra:e used in Example 2, ethyl 4-chloro-3-hydroxybutyrate was used as a reactant and microorganisms in Table 4 were used as biocatalysts. The results are shown in Table 4.
.(Table Removed) [Industrial Applicability] In accordance with Examples 1-113, optically active p-hydroxybutyl ester derivatives can be produced easily by hydrolysis of this invention. With appropriate lipases jor microorganisms, p-hydroxybutyl ester derivatives of high optical purity can be produced. Also, it is easy to seperate optically active esters from their acids after reaction. Therefore, this method is a useful process on the industrial scale.






We claim:
1. A process for preparing optically active p-hydroxybutyl ester
derivatives and their acids derivatives from racemic p-hydroxybutyl
ester derivatives represented by the general formula 1 in scheme 1
by lipases or lipase-producing microorganisms as biocatalysts in the
aqeous phase or organic phase including aqeous solvent.
[Scheme 1](Scheme Removed)
In the scheme 1, R is CnH2n+1(n=l~8) and X is H, N3, CN, F, CI,Br, I.
2. A process for preparing optically active p-hydroxybutyl ester
derivatives and their acids derivatives as claimed in claim 1, wherein
microorganisms were Candida rugosa KCTC 7292, Candida rugosa
KCCM 50521 and Rhodococcus butanica ATCC 21197


Documents:

3252-delnp-2005-abstract.pdf

3252-delnp-2005-claims.pdf

3252-delnp-2005-complete specification(granted).pdf

3252-delnp-2005-correspondenc-others..pdf

3252-delnp-2005-correspondenc-po.pdf

3252-delnp-2005-description (complete).pdf

3252-delnp-2005-form-1.pdf

3252-delnp-2005-form-18.pdf

3252-delnp-2005-form-2.pdf

3252-delnp-2005-form-26.pdf

3252-delnp-2005-form-3.pdf

3252-delnp-2005-form-5.pdf

3252-delnp-2005-pct-304.pdf

3252-delnp-2005-petition-137.pdf

3252-delnp-2005-petition-138.pdf


Patent Number 245939
Indian Patent Application Number 3252/DELNP/2005
PG Journal Number 06/2011
Publication Date 11-Feb-2011
Grant Date 07-Feb-2011
Date of Filing 22-Jul-2005
Name of Patentee ENZYTECH, LTD
Applicant Address 217-2, SINSEONG-DONG, YUSEONG-GU, DAEJEON 305-805 (KR).
Inventors:
# Inventor's Name Inventor's Address
1 HWANG, SOON OOK 09-1402 EXPO APT JEOMIN-DONG, YUSEONG-GU, DAEJEON 305-761 (KR).
2 CHUNG, SUN HO 512-302 CHEONGSOL APT, SONGGANG-DONG, YUSEONG-GU, DAEJEON 305-752 (KR)
PCT International Classification Number C08K 3/38
PCT International Application Number PCT/KR2005/001213
PCT International Filing date 2005-04-27
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10-2004-0029791 2004-04-29 Republic of Korea