Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
2001-03-15
2002-09-10
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S917000, C435S942000
Reexamination Certificate
active
06448051
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the invention
The present invention relates to a process for the preparation of 4(R)-hydroxycyclopent-2-en-1(S)-acetate of the formula 2 below.
More particularly the present invention relates to a process for the enzymatic preparation of 4-(R)-hydroxycyclopent-2-en-(S)acetate of the formula 2 from meso-cyclopent-2-en-1,4-diacetate of the formula 1 below.
2. Description of Related Art
4-(R)-hydroxycyclopent-2-en-1(S)-acetate is an important intermediate in the synthesis of biologically active cyclopentenoid natural products such as prostaglandins, prostacyclins, thromboxane (Harre, M., Raddatz. P., Walenta, R, Winterfeldt, E. Angew. Chem; 1982, 94, 496).
Other known processes for the preparation of 4-(R)-hydroxycyclopent-2-en-1(S)acetate involves the enantioselective hydrolysis of meso-cyclopent-2-en-1,4-diacetate using Pig Liver Esterase (PLE) (Y. F. Wang, C. S. Chen, G. Girdankas, C. J. Sih; J. Am. Chem. Soc. 1984, 106, 3695), and the trans esterification of cyclopent-2-ene-1,4-diol to 4(R)-hydroxycyclopent-2-en-1(S)-acetate (Johnson, Braun, Organic Synthesis, 1995, 73, 36). The known processes described above use costlier pig liver esterase (PLE).
The main object of the present invention is to provide a new process for the preparation of 4-(R)-hydroxycyclopent-2-en-1(S)-acetate which obviates the drawbacks of the prior known processes by using a more easily available microbial whole cell enzyme.
Another object is to provide a new process for the enantioselective hydrolysis of meso-cyclopent-2-en-1,4-diacetate using micro-organisms as whole cell enzyme from the National Collection of Industrial Micro-organisms (NCIM), NCL Pune.
BRIEF SUMMARY OF THE INVENTION
Accordingly, the present invention provides a process for the preparation of 4-(R)hydroxycyclopent-2-en-1(S)-acetate of the formula 2
which comprises:
reacting meso-cyclopent-2-en-1,4-diacetate of formula 1
with a whole cell enzyme in a mixture of a buffer and an organic solvent, wherein the buffer to organic solvent ratio being in the range of from 4:1 to 24:1;
stirring the reaction mixture at a temperature ranging between 25-30° C. for a period ranging from 10-16 hrs;
filtering the reaction mixture to remove the enzyme;
extracting the resultant compound with an organic solvent; and
removing the solvent to obtain the desired product.
In one embodiment of the invention the buffer used is selected from phosphate buffer and citrate buffer.
In a further embodiment of the invention the concentration of the buffer is in the range of 0.05M-0.1M.
In yet another embodiment of the invention the pH of the buffer is in the range of 6.0-8.0.
In another embodiment of the invention the organic solvent used for the reaction is selected from the group consisting of methanol, ethanol, propanol, butanol, isopropanol, acetone, dimethylsulpoxide (DMSO) and dimethylformamide (DMF).
In another embodiment of the invention the organic solvent used for the extraction of the product is selected from the group consisting of ether, ethyl acetate, chloroform and any mixtures thereof.
In a further embodiment of the invention, the whole cell enzyme used is selected from the group consisting of
Aspergillis oryzae, Aspergillus oryzae, Aspergillus sojae, Aspergillus oryzae, Aspergillus parasiticus, Trichosporon beigelii, Trichosporon beigeii
, Trichosporon sp., Trichosporon sp. and
Trichosporon capitatum.
In another embodiment of the invention, the chemical yield of 4-(R)hydroxycyclopent-2-en-1(S)-acetate is 83% and its optical purity is 99%.
DETAILED DESCRIPTION OF THE INVENTION
4-(R)-hydroxycyclopent-2-en-1(S)-acetate of the formula 2
is prepared by reacting meso-cyclopent-2-en-1,4-diacetate of formula 1
with a whole cell enzyme obtained from NCIM, Pune in a mixture of a buffer and an organic solvent, wherein the buffer to organic solvent ratio being in the range of from 4:1 to 24:1. The reaction mixture is then shrred at a temperature ranging between 25-30° C. for a period ranging from 10-16 hrs, and the reaction mixture is subsequently filtered to remove the enzyme. The resultant compound is then extracted with an organic solvent and the solvent removed.
The buffer used is selected from phosphate buffer and citrate buffer, wherein the concentration of the buffer being in the range of 0.05 M-0.1 M. The pH of the buffer is in the range of 6.0-8.0. Preferably, the pH of the phosphate buffer is in the range of 6.0 to 8.0 and that of the citrate buffer is in the range of 6.0 to 7.5.
The organic solvent used for the reaction is selected from methanol, ethanol, propanol, butanol, isopropanol, acetone, dimethylsulpoxide (DMSO) and dimethylformamide (DMF). The organic solvent used for the extraction of the final product is selected from the group consisting of ether, ethyl acetate, chloroform and mixtures thereof.
The whole cell enzyme used is selected from the group consisting of
Aspergillus oryzae, Aspergillus oryzae, Aspergillus sojae, Aspergillus oryzae, Aspergillus parasiticus, Trichosporon beigelli, Trichosporon beigeii
, Trichosporon sp. and
Trichospoon capitalum
. (NCIM Nos. 634, 637, 639, 929,1212, 3326 (ATCC Patent Deposit Designation PTA-3079), 3404, 3369, 3404 and 3412). This group is available to the public.
The chemical yield of 4-(R)-hydroxycyclopent-2-en-1(S)-acetate is 83% and its optical purity is 99%.
The merits of the process of the present invention are the use of easily accessible microorganisms from NCIM, Pune and a high optical purity of the product. The cost of whole cell enzyme (micro-organism) compared to PLE is much less.
REFERENCES:
patent: 4008125 (1977-02-01), Kurozumi et al.
patent: 4618690 (1986-10-01), Schneider et al.
Computer Caplus Abstract 1976:541335 Miura et al DE2552871 PUB Nov. 1975.*
ACS Registry RN-60176-77-4 2001.*
Computer BIOTECHDS Abstract 2000-11907 Kalkote et al Tetrahedron Asymmetry (2000) 11, 14 2965-70.
Bastawade Kulbhussan Balwant
Ghorpade Sandeep Raghunath
Gokhale Digambar Vitthal
Joshi Rohini Ramesh
Kalkote Uttam Ramrao
Council of Scientific and Industrial Research
Ladas & Parry
Lilling Herbert J.
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