| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | October 24, 2000 |
| PATENT TITLE |
Bioresolution of N-acylazetidine-2-carboxylic acids |
| PATENT ABSTRACT |
Process for obtaining an enantiomerically enriched N-acylazetidine-2-carboxylic acid, wherein a racemic N-acylazetidine-2-carboxylic acid ester is contacted with an enzyme that displays enantiospecificity to form enantiomerically enriched N-acylazetidine-2-carboxylic acid. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | January 22, 1998 |
| PATENT CT FILE DATE | July 15, 1997 |
| PATENT CT NUMBER | This data is not available for free |
| PATENT CT PUB NUMBER | This data is not available for free |
| PATENT CT PUB DATE | January 22, 1998 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
C.R. Johnson et al.: "Enzymic asymmetrization of meso-2-cycloalken-1, 4-diols and their diacetates in organic and aqueous media" Tetrahedron Letters, vol. 33, No. 48, 1992, pp. 7287-7290. R.M. Rodebaugh et al.: "Resolution of DL-Azetidine-2-carboxylic acid" Journal of Heterocyclic Chemistry, vol. 6, No. 6, 1969, pp. 993-994. |
| PATENT CLAIMS |
What is claimed is: 1. A process for obtaining an enantiomerically enriched N-acylazetidine-2-carboxylic acid comprising contacting a racemic N-acylazetidine-2-carboxylic acid ester with an enzyme that displays enantiospecificity to form enantiomerically enriched N-acylazetidine-2-carboxylic acid. 2. The process as claimed in claim 1, wherein the acyl group is optionally substituted benzoyl. 3. The process as claimed in claim 2, wherein the acyl group is benzoyl. 4. The process as claimed in claim 1, wherein the ester is a lower alkyl ester. 5. The process as claimed in claim 4, wherein the ester is a methyl ester. 6. The process as claimed in claim 1, the enzyme has enantiospecificity for the (5)-ester. 7. The process as claimed in claim 6, wherein the enzyme has properties characteristic of Candida antarctica lipase. 8. The process as claimed in claim 6, wherein the enzyme has properties characteristic of Aspergillus tamarii esterase. 9. The process according to claim 1, and further comprising crystallizing the N-acylazetidine-2-carboxylic acid from a solvent to increase the enantiomeric enrichment of the N-acylazetidine-2-carboxylic acid. 10. The process as claimed in claim 9, wherein the solvent is ethyl acetate. 11. The process according to claim 1, further comprising racemizing the unwanted enantiomer. 12. The process as claimed in claim 11, wherein the racemization is effected through treatment with a base. 13. The process as claimed in claim 12, wherein the base is sodium methoxide. 14. The process according to claim 1, further comprising the enantiomerically enriched N-acylazetidine-2-carboxylic acid. 15. The process as claimed in claim 14, wherein the deacylation is effected by hydrolysis in the presence of alkali. 16. The process as claimed in claim 15, wherein the alkali is an alkali metal hydroxide. 17. The process as claimed in claim 14, wherein the azetidine-2-carboxylic acid is (S)-azetidine-2-carboxylic acid. 18. A method of selection of an enzyme for use in a process according to claim 1, which method comprises the attempted biotransformation of a racemic N-acylazetidine-2-carboxylic acid ester in the presence of a test enzyme and determining whether or not an enantiomerically enriched N-acylazetidine-2-carboxylic acid is formed. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to a novel resolution method which is useful in the production of enantiomerically-pure azetidine-2-carboxylic acid, especially enantiomerically-pure (S)-azetidine-2-carboxylic acid. BACKGROUND OF THE INVENTION Azetidine-2-carboxylic acid is an unusual amino acid, the (S)-enantiomer of which is known to be useful in the synthesis of inter alia high molecular weight polypeptides, and in particular as an analogue of the well known amino acid proline. This amino acid is of limited availability from natural sources, and in nature is found only as the (S)-enantiomer. The development of an efficient and economic synthetic method for producing both the pure racemic compound and either of the individual (R)- or (S)-single enantiomers is therefore desirable. Previously documented chiral syntheses of (S)-azetidine-2-carboxylic acid include a five step preparation via homoserine lactone, starting from N-tosyl protected L-methionine (see for example Japanese Patent Application N.sup.o 14457/74 and Bull. Chem. Soc. Jpn. (1973) 46, 699) and a five step preparation via L-4-amino-2-chlorobutyric acid, starting from L-2,4-diaminobutyric acid (see Biochem. J. (1956) 64, 323). Previously documented preparations of enantiomerically-pure azetidine-2-carboxylic acid from the racemate involve long and relatively complicated multi-step methodology. For example, a four step preparation involving the protection, resolution and subsequent deprotection of racemic azetidine-2-carboxylic acid is known from J. Heterocyclic Chem. (1969) 6, 993. In this method, N-carbobenzyloxy-protected racemic azetidine-2-carboxylic acid is resolved using L-tyrosine hydrazide as resolution agent, and then isolated before a final deprotection step. This process has the further disadvantage that L-tyrosine hydrazide is expensive. Such methods present the problem that they are typically cumbersome because of the need to recycle the resolving agent and inevitably only produce half of the material as the required isomer. For an economic overall process it thus becomes necessary to find a method for the recycling of the unwanted isomer, and for this to be integrated into a process with the minimum of extra chemical steps. Moreover, racemic azetidine-2-carboxylic acid obtained via chemical synthesis inevitably contains contaminants. Thus a resolution procedure which produces only the required single enantiomer, as well as being more economic, is also expected to facilitate chemical purification of the product. Bioresolution is a procedure which is known to be of use generally in the production of enantiomerically-pure compounds. However, the potential utility and effectiveness of the technique in the resolution of a particular chiral compound is difficult to predict. No biocatalytic resolution method has been previously disclosed for azetidine-2-carboxylic acid. Moreover, no resolution has been disclosed for azetidine-2-carboxylic acid which integrates recycling of the unwanted isomer in an efficient manner and which takes account of impurities arising from the racemate synthesis. We have now surprisingly found that enantiomerically enriched azetidine-2-carboxylic acid may be obtained in an extremely enantiomerically-pure form, and in extremely high yields, via a novel and efficient bioresolution process. DESCRIPTION OF THE INVENTION According to the invention there is provided a process for obtaining an enantiomerically enriched N-acylazetidine-2-carboxylic acid, which process comprises the biotransformation of a racemic N-acylazetidine-2-carboxylic acid ester with an enzyme that displays enantiospecificity (hereinafter referred to as "the process according to the invention"). The term "enantiomerically enriched" when used herein means any mixture of the enantiomers of an N-acylazetidine-2-carboxylic acid in which one enantiomer is present in a greater proportion than the other, for example mixtures with an enantiomeric purity (enantiomeric excess; e.e.) of greater than 50%, preferably at least 70% and more preferably at least 90%. Persons skilled in the art will be aware that the process according to the invention may also be referred to as a process for obtaining an "optically enriched" N-acylazetidine-2-carboxylic acid. The process according to the invention comprises the use of an appropriate enzyme to preferentially hydrolyse one enantiomer of an N-acylazetidine-2-carboxylic acid ester to the corresponding acid, which acid may be readily separated from the other, unwanted, enantiomeric ester and from impurities arising from the synthesis of the racemic N-acylazetidine-2-carboxylic acid ester. Moreover, the remaining ester may be readily recovered, racemized and re-used in the resolution process. Esters of N-acylazetidine-2-carboxylic acids which may be used in the process according to the invention include aryl (e.g. phenyl) or linear or cyclic alkyl (especially lower alkyl (e.g. C.sub.1-6 alkyl)) esters. Particular esters which may be mentioned include propyl, ethyl and especially methyl esters of N-acylazetidine-2-carboxylic acids. N-Acyl groups of N-acylazetidine-2-carboxylic acids, and esters, which may be used in the process according to the invention include linear or cyclic alkanoyl or optionally-substituted benzoyl groups. However, we prefer the N-acyl group to be an optionally substituted N-benzoyl group, and particularly a N-benzoyl group. The process according to the invention may be carried out in the presence of a suitable solvent which does not interfere with the resolution process. Suitable solvents include water, which may be buffered to a suitable pH using a suitable buffer system including those commonly used in biological systems (e.g. buffers such as Tris, MESm, Bis-Tris, ACES, PIPES, MOPSO) and inorganic buffers such as phosphate buffers. The biotransformed acid and ester may be separated in accordance with techniques which are well known to those skilled in the art, for example by solvent extraction. The N-acyl group of the biotransformed enantiomerically enriched acid may subsequently be removed in order to produce enantiomerically pure azetidine-2-carboxylic acid in accordance with techniques which are well known to those skilled in the art, for example by hydrolysis in the presence of alkali. Saponification may be carried out in this way in aqueous media, at between room temperature and 100.degree. C., in the presence of an appropriate alkali (e.g. an alkali metal hydroxide, such as lithium, sodium or potassium hydroxide). We have found, advantageously, that saponification of the enantiomerically enriched N-acylazetidine-2-carboxylic acid (and, in particular, the N-benzoyl derivative) proceeds without racemization. The process according to the invention may thus be used as part of a process to produce enantiomerically enriched azetidine-2-carboxylic acid. According to a further aspect of the invention there is provided a process is for preparing an enantiomerically-pure azetidine-2-carboxylic acid, which comprises carrying out a biotransformation as hereinbefore described, followed by deacylation of the resultant enantiomerically enriched N-acylazetidine-2-carboxylic acid. Although the process according to the invention may be used in the production of either enantiomer of azetidine-2-carboxylic acid with an enantiomeric purity (enantiomeric excess; e.e.) of greater than 80%, by "enantiomerically pure azetidine-2-carboxylic acid" we mean an enantiomer of azetidine-2-carboxylic acid with an e.e. of greater than 50%. Enantiomeric purity may be further improved (for example to greater than 98%) by crystallisation from an appropriate solvent (e.g. ethyl acetate) which at the same time increases chemical purity. Racemization of the remaining, non-biotransformed yet enantiomerically enriched, ester may take place by treatment with an appropriate base (e.g. sodium methoxide) in the presence of an appropriate solvent (e.g. methanol) at, for example, between 20 and 100.degree. C. (depending on the solvent employed). The re-racemized ester may subsequently be re-used in the process according to the invention. By employing a suitable enzyme, the process according to the invention may be used, in conjunction with a deacylation step, to produce enantiomerically pure (R)- or enantiomerically pure (S)-azetidine-2-carboxylic acid. However, in view of the aforementioned utility of the (S)-enantiomer we prefer that the process according to the invention is used in the production of the latter, and that the enzyme has enantiospecificity for the (S)-ester. In particular, we have found that an efficient process to (S)-azetidine-2-carboxylic acid may be effected through bioresolution of a racemic N-benzoylazetidinecarboxylic acid alkyl ester using an appropriately enantiospecific enzyme followed by removal of the N-benzoyl group. Enzymes for use in the process according to the invention may be used in the presence of the organism from which it originates or in an isolated form. The enzyme may be immobilised if desired. The selection of a suitable enzyme system may proceed by way of a suitable protocol comprising the attempted biotransformation of a racemic N-acylazetidine-2-carboxylic acid ester in the presence of a test enzyme, for example as described hereinafter. The term "attempted biotransformation" when used herein means providing a racemic N-acylazetidine-2-carboxylic acid ester in the presence of a suitable quantity of test enzyme, and determining whether or not an enantiomerically enriched N-acylazetidine-2-carboxylic acid (as defined hereinbefore) is formed. Resolution conditions may be varied as described hereinbefore and the enantiomeric purity of the product determined in accordance with techniques which are well known to those skilled in the art, such as those described hereinafter. Thus, according to a further aspect of the invention there is provided a method of selection of an enzyme for use in the process according to the invention which comprises the attempted biotransformation of a racemic N-acylazetidine-2-carboxylic acid ester in the presence of a test enzyme. Examples of suitable enzymes for use in the process according to the invention include those with properties characteristic of (and/or having the same enzymatic activity as) Candida antarctica lipase and Aspergillus tamarii esterase. We have found that such enzymes preferentially hydrolyse the (S)-ester to the (S)-acid, which may be easily separated from the unwanted (R)-ester by extraction, and subsequently saponified a hereinbefore described. By using the term "enzymes with properties characteristic of" an enzyme from an organism, we are including enzymes which originate both directly and indirectly from the original organism, for example enzymes which are expressed from the relevant gene in a suitable heterologous host organism. It will also be clear to those skilled in the art that an alternative route to the production of (S)-azetidine-2-carboxylic acid from racemic N-acylazetidine-2-carboxylic acid ester may involve the enzymatic production of (R)-N-acylazetidine-2-carboxylic acid using an appropriately enantiospecific enzyme (leaving the (S)-ester unconverted), followed by separation as indicated above. The (R)-azetidine-2-carboxylic acid may then be racemized and esterified, in either order, to provide further racemic substrate. The ester and N-acyl groups of the (S)-ester may subsequently be removed in accordance with conventional procedures, in one or two steps, in order to produce to (S)-azetidine-2-carboxylic acid. However, in view of the number of steps involved, this procedure is less preferred than the direct enzymatic hydrolysis of the (S)-ester to the (S)-acid. The process according to the invention has the advantages that, unlike the chemical methods described hereinbefore, it avoids the need to handle stoichiometric amounts of resolving agents or auxiliaries; the products from the biotransformation are easily separable; and it provides the materials in a form where the unwanted enantiomer can be readily recycled. Moreover, the process according to the invention has the advantage that enantiomerically pure azetidine-2-carboxylic acid may be prepared in higher yields, with greater enantiomeric purity, in a manner which involves fewer steps, in less time, more conveniently and at a lower cost than processes previously employed for the production of enantiomerically pure azetidine-2-carboxylic acid. |
| PATENT EXAMPLES | Available on request |
| PATENT PHOTOCOPY | Available on request |
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