Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Resolution of optical isomers or purification of organic...
Reexamination Certificate
2001-05-31
2002-09-24
Saucier, Sandra E. (Department: 1657)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Resolution of optical isomers or purification of organic...
Reexamination Certificate
active
06455302
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method for optically resolving a racemic &agr;-substituted heterocyclic carboxylic acid (hereinafter referred to as “&agr;-HCCA”). More particularly, the present invention pertains to a method for optically resolving a racemic &agr;-HCCA using an enzyme catalyst with enantioselectivity.
2. Description of the Prior Art
Divided into optical isomers, R- and S-forms, tetrahydro-2-furoic acid (hereinafter referred to as “THFA”), a kind of &agr;-HCCA, is an important chiral building block which has various applications in chemistry. Of the optical isomers, R-(+)-THFA is used as a side chain intermediate for the synthesis of penem type antibiotics while S-(−)-THFA is useful as a chiral intermediate for organic synthesis. Therefore, THFA is different in use in R form and S form thereof. However, because THFA is obtained in the form of a racemic mixture when chemically synthesized, additional processes are required to separate THFA into enantiomers thereof: R and S forms.
Optical resolution has been usually used to divide racemic THFA into R- and S-forms thereof. In 1983, Belanger successfully separated THFA racemate into enantiomers thereof by use of brucine and ephedrine as resolving agents (Can. J. Chem., 61, 1383 (1983)). However, the resolving agents are not economical because of their being very expensive. Another problem with this process is that its products are low in enantiomeric excess value.
Japanese Pat. Laid-Open Publication No. 89-216983 discloses the use of a chiral amine (1-(4-halogenophenyl)ethylamine) as a resolving agent, in which diastereomer salts are prepared from R,S-THFA and optically resolved. This method is also economically unfavorable owing to the high price of the chiral amine. Additionally, only low production yields can be obtained because the amount of R,S-THFA to be added in the early reaction is limited to as low as 4 mmol. Furthermore, the chiral THFA finally obtained is poor in enantiomeric excess value.
A method different from optical resolving methods is found in Japanese Pat. Laid-Open Publication. No. 97-71576 which refers to synthesizing R- or S-THFA by treating R- or S-THFA salts with hydrogen halide.
It has been well known for some time that optical resolution of racemates could be achieved by use of enzymes, such as esterases, lipases, and proteases, as enzyme catalysts to enantioselectively hydrolyze one of the two enantiomers present. For example, U.S. Pat. No. 5,928,933 discloses an enzyme with an enantiomeric excess of 95% as a result of extensive experiments for reaction specificity of 44 enzymes, including proteases, lipases and esterases. The enzyme catalyst is very useful for the separation of enantiomeric racemates, but because the selectivity for enantiomers and the optical purity of products may vary depending on the choice of enzymes and chemical structures of their substrates, intensive efforts are required to find combinations of enzymes suitable for substrates. Especially, nowhere is found a method for optical resolution of &agr;-HCCA using an enzyme.
Therefore, there remains a need for an enzymatic optical resolution method that can divide racemic &agr;-HCCA into R- and S-form economically and easily.
SUMMARY OF THE INVENTION
Leading to the present invention, the intensive and through research on the optical resolution of &agr;-HCCA, conducted by the present inventors aiming to develop optically highly pure &agr;-HCCA by an economical procedure, resulted in the finding that some of microorganism- or animal-derived hydrolyzing enzymes may enantioselectively hydrolyze the ester functionality of particular optical isomers of &agr;-HCCA at high efficiency.
Therefore, it is an object of the present invention to overcome the above problems encountered in prior arts and to provide a method for optically resolving a racemic &agr;-HCCA using an enzyme, which is economically favorable.
In one aspect of the present invention, there is provided a method for optically resolving a racemic &agr;-HCCA, comprising the steps of:
reacting a racemic &agr;-HCCA with alcohol to give a racemic &agr;-HCCA ester represented by the following chemical formula 1:
wherein R
1
is selected from the group consisting of substituted or unsubstituted alkyl or alkenyl containing 1 to 6 carbon atoms, benzyl, cycloalkyl containing 3 to 6 carbon atoms, substituted or unsubstituted arylalkyl, and substituted or unsubstituted heteroarylalkyl, X represents O, S or N—H, and n is an integer of 1 to 3;
optically resolving the racemate of the formula 1 by use of an enzyme with enantioselectivity to hydrolyze either R-form or S-form of the ester racemate thereby producing a R-form or S-form of &agr;-HCCA and a counter enantiomeric form of &agr;-HCCA ester thereto, said enzyme existing as a powder or an aqueous solution;
extracting the unhydrolyzed &agr;-HCCA ester with an organic solvent; and
subjecting the extracted &agr;-HCCA ester in an organic solvent to hydrogenation under a constant hydrogen partial pressure at a constant temperature in the presence of a palladium catalyst on carbon (Pd/C).
In another aspect of the present invention, there is provided a method for optically resolving a racemic &agr;-HCCA, comprising the steps of:
reacting a racemic &agr;-HCCA with alcohol to give a racemic &agr;-HCCA ester represented by the chemical formula 1;
optically resolving the racemate of the formula 1 by use of an enzyme with enantioselectivity to hydrolyze either R-form or S-form of the ester racemate, thereby producing a R-form or S-form of &agr;-HCCA and a counter enantiomeric form of &agr;-HCCA ester thereto, said enzyme existing as a powder or an aqueous solution;
extracting the unhydrolyzed &agr;-HCCA ester with an organic solvent; and
treating the extracted &agr;-HCCA ester with a non-enantioselective enzyme in an aqueous solution at a constant pH and temperature, said enzyme existing as a powder or an aqueous solution.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is characterized by the enantioselective hydrolysis of esters of racemic &agr;-HCCA by an enzyme to produce a certain enantiomeric form of &agr;-HCCA and a counter enantiomeric form of the esters of &agr;-HCCA, at once. The separation of the hydrolyzed &agr;-HCCA and the remaining esters of &agr;-HCCA can be achieved by use of an organic solvent. The unhydrolyzed enantiomeric form of &agr;-HCCA ester can be hydrolyzed in the presence of non-enantioselective enzyme or be hydrogenated in the presence of a palladium catalyst on carbon (Pd/C) to obtain the corresponding &agr;-HCCA thereto.
In detail, a racemic mixture of &agr;-HCCA is reacted with an alcohol at an equivalent amount to produce a racemic mixture of an &agr;-HCCA ester, which is then enantioselectively hydrolyzed at a constant temperature and pH in an aqueous solution in the presence of an enzyme with enantioseletivity. As a result, the reaction produces an R- or S-form &agr;-HCCA, along with the ester of &agr;-HCCA which has an enantiomeric form counter to that of the hydrolyzed &agr;-HCCA. After completion of the enantioselective hydrolysis, addition of an organic solvent extracts the ester of &agr;-HCCA thereinto, leaving the &agr;-HCCA in the aqueous phase only. Removal of the organic solvent from the organic phase results in acquisition of an optically pure S- or R-form of &agr;-HCCA ester. Poor in optical purity, the &agr;-HCCA remaining in the aqueous solution may be increased in purity through a purification process using, for example, a column, or may be reused as a starting material in the present invention.
In accordance with a preferred embodiment of the present invention, THFA, which belongs to an (&agr;-HCCA, is used as a starting material and after optical resolution, R- or S-form of THFA can be obtained at a high enantiomeric excess. Aside from THFA, all materials falling within the scope of &agr;-HCCA, for example, proline and tetrahydrothiopen-2-carboxylic acid can be optically resolved in accordance with the pres
Lim Jong-Ho
Lim Sang-Chul
Uhm Ki-Nam
Abelman ,Frayne & Schwab
Afremova Vera
Saucier Sandra E.
SK Corporation
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