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
1999-02-16
2001-08-21
Saucier, Sandra E. (Department: 1651)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Resolution of optical isomers or purification of organic...
C435S117000, C435S120000, C435S121000
Reexamination Certificate
active
06277626
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to the enzymatic resolution of lactams. The method of the present invention is useful in preparing compounds which may have utility as pharmaceutical, agricultural and veterinary products or starting materials and intermediates for their synthesis.
2. Discussion of the Prior Art
It is known in the art that chiral resolution of compounds can be achieved by using enzymes. Chiral resolution using enzymes such as esterases on aliphatic esters and cyclic compounds containing esters are described in, for example, W. Boland et al.,
Synthesis,
1049-1072, 1991. Chiral resolutions using enzymes in aliphatic methyl esters hydrolysis is described in, for example, H. Ohta et al.,
Chem. Lett.,
657-660, 1992. Chiral resolutions using enzymes in cyclohexanes systems are described in, inter alia, M. Ohno,
Tet. Lett.,
29, 6961-6964, 1988; H. Hemmerle,
Tet Lett.,
28, 3471-3474, 1987; and B. Brion,
Tet Lett.,
33, 4889-4892, 1992. Chiral resolutions using lipases or Acetylcholine esterases on cycloheptanes containing diacetates is found in A. J. Pearson et al.,
JOC,
54, 3882, 1989. Beta-lactams have been reported to be selectively acylated by lipase at the nitrogen function (C. Sih et al.,
JOC,
58, 1068, 1993).
However, there is no prior art for the enzymatic resolution of lactam esters. It is often desired to obtain a single enantiomer of a racemic lactam ester. These compounds can be used as intermediates for preparing compounds which have utility as starting materials and intermediates for the synthesis of pharmaceutical, agricultural and veterinary products. For example, the enanantiomerically pure form of 7-carbomethoxycaprolactam is a useful intermediate in the synthesis of pharmaceutical drug candidates.
SUMMARY OF THE INVENTION
The present invention is directed to a method of separating enantiomeric lactam esters. The lactam esters are contacted with a biocatalyst, such as an enzyme or a microorganism, in an aqueous solution, an organic solvent, or a mixture of organic and aqueous solvents, wherein only one enantiomer is selectively hydrolyzed to give the optically active isomer of the corresponding acid. The hydrolysis product is then separated from the unreacted lactam esters using standard methods known to those skilled in the art. This invention also discloses a novel method for the recycling and reuse of the enzymes as well as the racemization of either enantiomer of the lactam ester after enzymatic resolution.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention is directed to contacting racemic esters of lactam with a biocatalyst, such as an enzyme or a microorganism, whereby one of the optical isomers is selectively hydrolyzed to give the optically active isomer of the corresponding acid. The optically active products are then isolated/purified using suitable procedures.
For illustrative purposes only, the process of the present invention is demonstrated by the following example of enzymatic cleavage of a racemic 7-carbomethoxy caprolactam wherein the S configuration is converted to its acid:
For convenience, the procedure is described herein using racemic esters of lactam; however, the method of the present invention is not limited to use with the racemic form. The lactam ester may be present in the optical active form or in nonracemic mixtures which have an excess of one of the optical isomers. The method of the present invention allows the lactam mixture to react such that only one of two enantiomeric esters of the lactam is converted to its acid.
The term “stereoselective hydrolysis” refers to the preferential hydrolysis of one enantiomer relative to another. The term “mixture” as used herein in relation to enantiomeric compounds, denotes mixtures having equal (racemic) or nonequal amounts of enantiomers. The term “resolution” denotes partial, as well as, preferably, complete resolution. The term “enzymatic process” or “enzymatic method” or “enzymatic reaction” denote a process or method or reaction of the present invention employing an enzyme or microorganism. The term “enantiomeric excess(es)” is related to the older term “optical purity”. In a mixture of a pure enantiomer (R or S) and a racemate, enantiomeric excess is the percent excess of the enantiomer over the racemate. It can be expressed in the following equation, for example:
Optical
purity
=
percent
enantiomeric
excess
=
[
R
]
-
[
S
]
[
R
]
+
[
S
]
×
100
=
%
R
-
%
S
The enzyme may be any enzyme obtainable from animals, plants, microorganisms, etc. The enzyme may be employed in any conventional form such as in a purified form, a crude form, a mixture with other enzymes, a microbial fermentation broth, a fermentation broth, a microbial body, a filtrate of fermentation broth, and the like, either solely or in combination. In addition, the enzyme or microbial body may be immobilized on a resin.
The activities of the enzymes used in this invention are expressed in “units”. Units are defined as the rate of hydrolysis of p-nitrophenyl proprionate per minutes as expressed in &mgr;mol/min at room temperature.
Specific examples of the enzyme are those obtained from animal and plants such as cow liver esterase, pig liver esterase, pig pancreas esterase, horse liver esterase, dog liver esterase, pig phosphatase, amylase obtainable from barley and potato and lipase obtainable from wheat. Other examples are hydrolases obtained from such microorganisms as Rhodotorula, Trichoderma, Candida, Hansenula, Pseudomonas, Bacillus, Achromobacter, Nocardia, Chromobacterium, Flavobacterium, Rhizopus, Mucor, Aspergillus, Alkaligenes, Pediococcus, Klebsiella, Geotrichum, Lactobaccilus, Cryptococcus, Pichia, Aureobasidium, Actinomucor, Enterobacter, Torulopsis, Corynebacterium, Endomyces, Saccaromyces, Arthrobacter, Metshnikowla, Pleurotus, Streptomyces, Proteus, Gliocladium, Acetobacter, Helminthosporium, Brevibacterium, Escherichia, Citrobacter, Absidia, Micrococcus, Microbacterium, Penicillium and Schizophyllium as well as from lichen and algae.
Specific examples of the microorganisms useful in the present invention include, but are not limited to,
Rhodotorula minuta, Rhodotorula rubra, Candida krusei, Candida cylindracea, Candida tropicalis, Candida utilus, Pseudomonas fragi, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa, Rhizopus chinensis, Mucor pusillus, Aspergillus niger, Alkaligenes faecalis, Torulopsis ernobii, Bacillus cereus, Bacillus subtilis, Bacillus pulmilus, Bacillus subtilis
var.
niger, Citrobacter freundii, Micrococcus varians, Micrococcus luteus, Pediococcus acidlactici, Klebsiella pneumoriae, Absidia hyalospora, Geotrichun candidum, Schizophyllum commune, Nocardia uniformis subtsuyanarenus, Nocardia uniformis, Chromobacterium chocolatum, Hansenula anomala
var.
ciferrii, Hansenula anomala, Hansenula polymorpha, Achromobacter lyticus, Achromobacter parvulus, Achromobacter simplex, Torulopsis candida, Corynebacterium sepedonicum, Endomyces geotrichum, Saccaromyces carrvisial, Arthrobacter globiformis, Streptomyces grisens, Micrococcus luteus, Enterobacter cloacae, Corynebacterium ezui, Lacto bacillus casei, Cryptococcus albidus, Pichia polimorpha, Penicillium frezuentans, Aureobasidium pullulans, Actinomucor elegans, Streptomyces grisens, Proteus vulgaris, Gliocladium roseum, Gliocladium virens, Acetobacter aurantius,
Helminthosporium sp.
Chromobacterium iodinum, Chromobacterium violaceum, Flavobacterium lutescens, Metschnikowia pulcherrima, Pleurotus ostreatus, Brevibacterium ammoniagenes, Brevibacterium divaricatum, Escherichia coli, Rodotolura minuta
var.
texensis, Trichoderma longibrachiatum, Mucor javanicus, Flavobacterium arbonescens, Flavobacterium heparinum,
and
Flavobacterium capsulatum.
Exemplary, commercially available enzymes suitable for use in the present invention include lipases such as Amano PS-30 (
Pseudomonas cepacla
), Amano GC-20 (
Geotrichum candidum
), Amano APF (
Aspergillus n
Awasthi Alok K.
Gapud Rolando E.
Hansen, Jr. Donald W.
Ng John S.
Trivedi Mahima
G.D. Searle & Co.
Saucier Sandra E.
Scrivner Alan
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