Organic compounds -- part of the class 532-570 series – Organic compounds – Carboxylic acids and salts thereof
Reexamination Certificate
2000-08-04
2002-09-24
Killos, Paul J. (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carboxylic acids and salts thereof
C560S076000
Reexamination Certificate
active
06455730
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to a process for preparing dicarboxylic acid monoesters from cyanocarboxylic acid esters. More particularly, the nitrilase from
Acidovorax facilis
72W catalyzes the conversion of an aliphatic or aromatic cyanocarboxylic acid ester to the corresponding dicarboxylic acid monoester with high chemoselectivity at 100% conversion.
BACKGROUND
Nitriles are readily converted to the corresponding carboxylic acids by a variety of chemical processes, but these processes typically require strongly acidic or basic reaction conditions and high reaction temperatures, and usually produce unwanted byproducts and/or large amounts of inorganic salts as unwanted waste. The reaction conditions used to chemically hydrolyze nitriles will usually also hydrolyze any ester functional groups also present in the molecule. For example, the chemoselective hydrolysis of aromatic or aliphatic cyanocarboxylic acid esters by tetrahalophthalic acids produces the corresponding dicarboxylic acid monoesters in 56% to 65% yields, and the use of an equimolar quantity of tetrahalophthalic acid generated an undesirable reaction byproduct (Rounds et al.,
Tetrahedron Lett
. (1988) 29, 6557). The enzyme-catalyzed hydrolysis of nitrile-containing substrates to the corresponding carboxylic acids is often preferred to chemical methods because the reactions 1) are often run at ambient temperature, 2) do not require the use of strongly acidic or basic reaction conditions, and 3) produce the desired product with high selectivity at high conversion.
A nitrilase enzyme directly converts a nitrile to the corresponding carboxylic acid and ammonia in an aqueous reaction mixture without the intermediate formation of an amide. A stereospecific nitrilase of
Alcaligenes faecalis
has been used to resolve racemic nitrites in the manufacture of chiral carboxylic acids, and the gene encoding the nitrilase has been cloned and expressed (WO 00/23577). A nitrilase has been isolated from the thermophilic bacterium
Bacillus pallidus
strain Dac521 that catalyzed the hydrolysis of aliphatic, aromatic, and heterocyclic nitrites (Almatawah et al.,
Extremophiles
(1999) 3:283-291). A nitrilase from
Rhodococcus rhodochrous
NCIMB 40757 or NCIMB 40833 has been used to convert acrylonitrile to ammonium acrylate (U.S. Pat. No. 5,998,180). Kobayashi et al. (Tetrahedron (1990) 46, 5587-5590
; J. Bacteriology
(1990) 172, 4807-4815) have described an aliphatic nitrilase isolated from
Rhodococcus rhodochrous
K22 that catalyzed the hydrolysis of a variety of aliphatic nitriles to the corresponding carboxylic acid ammonium salts. A nitrilase from
Comamonas testosteroni
has been isolated that can convert a range of aliphatic &agr;,&ohgr;-dinitriles to either the corresponding &ohgr;-cyanonitrilecarboxylic acid ammonium salt or dicarboxylic acid diammonium salt (CA 2,103,616; S. Levy-Schil et al., Gene (1995) 161, 15-20).
Nitrilases are also produced by
Rhodococcus rhodochrous
NCIMB 11216 (C. Bengis-Garber, A. L. Gutman,
Appl. Microbiol. Biotechnol
. (1989) 32, 11; M. L. Gradley, C. J. Knowles,
Biotechnology Lett
. (1994) 16, 41),
Rhodococcus rhodochrous
PA-34 (Bhalla et al.,
Appl. Microbiol. Biotechnol
. (1992) 37, 184),
Fusarium oxysporum
f. sp. melonis (A. Goldlust, Z. Bohak,
Biotechnol. Appl. Biochem
. (1989) 11, 581),
Acinetobacter sp. AK
226 (K. Yamamoto, K. Komatsu,
Agric. Biol. Chem
. (1991) 55, 1459),
Acidovorax facilis
ATCC8750 (Yamamoto et al.,
J. Ferment. Bioeng
. (1992) 73, 425), and
Acidovorax facilis
72W (Gavagan et al.,
J. Org. Chem
. (1998) 63, 4792).
The ability to chemoselectively convert a nitrile functional group to the corresponding carboxylic acid is a powerful tool for the preparation of agrochemicals and pharmaceuticals. These industries have a need to prevent losses in yield from hydrolysis of functional groups other than nitriles. Eliminating protection and deprotection of other hydrolyzable functional groups such as esters, acetals, or epoxides would bring an additional benefit to industry.
Propanedioic acid monoethyl ester (commonly known as ethyl hydrogen malonate or monoethylmalonate) is a useful bifunctional building block (see, for example, Pollett et al.,
Synthesis
(1978) 2, 142-143). Monoethylmalonate has been prepared by hydrolysis of cyanoacetic acid ethyl ester using Corynebacterium, Gordona, or Rhodococcus cells having nitrilase activity (WO 9837219):
G. terrae
MA-1 gave a yield of monoethylmalonate of 89.9%. This method is touted as superior in yield and cost to both the chemical hydrolysis of diethylmalonate and the regioselective enzymatic hydrolysis of diethylmalonate using esterases.
Not all nitrile-hydrolyzing enzymes (either as contained in microbial cell catalysts or as isolated enzymes) can chemoselectively hydrolyze a cyanocarboxylic acid ester nitrile group to produce the corresponding dicarboxylic acid monoester. In the process, the ester may also be hydrolyzed or the chemoselectivity of the enzyme may be specific to the particular cyanocarboxylic acid ester reactant. An immobilized enzyme preparation from Rhodococcus sp. has been examined for the chemoselective hydrolysis of cyanocarboxylic acid esters (de Raadt et al.,
J. Chem Soc. Perkins Trans. I, (
1992) 1, 137-140); 2-cyanoethylpropanedioic acid diethyl ester was chemoselectively hydrolyzed to 1,1,3-propanetricarboxylic acid diethyl ester in 92% yield, whereas under the same conditions 5-cyanopentanoic acid methyl ester was completely converted with no chemoselectivity to adipic acid (complete ester and nitrile hydrolysis). Resting cells of
Rhodococcus rhodochrous
NCIMB 11216 have been examined for the chemoselective hydrolysis of cyanocarboxylic acid esters (Klempier et al.,
Food Technol. Biotechnol
. (1996) 34, 67-70), and with this catalyst 2-cyanoethyl-propanedioic acid diethyl ester was chemoselectively hydrolyzed to 1,1,3-propanetricarboxylic acid diethyl ester in 56% yield (20% 2-cyanoethyl-propanedioic acid monoethyl ester was also produced), whereas under the same conditions 5-cyanopentanoic acid methyl ester was completely converted with no chemoselectivity to 5-cyanovaleric acid (complete ester hydrolysis with no nitrile hydrolysis). For the hydrolysis of 4-cyanobenzoic acid methyl and ethyl esters,
R. rhodochrous
NCIMB 11216 (Klempier et al. supra) produced the corresponding 1,4-benzenedicarboxylic acid esters in 79% and 42% yields at 100% conversion, respectively, with ester hydrolysis products accounting for the remaining reaction byproducts.
U.S. Pat. No. 5,858,736 describes the use of the nitrilase activity of a microbe,
Acidovorax facilis
72W (ATCC 55746), as a catalyst for the hydrolysis of aliphatic &agr;,&ohgr;-dinitriles to the corresponding &ohgr;-cyanocarboxylic acids and ammonia in an aqueous reaction mixture. The nitrilase was found to be highly regioselective, where hydrolysis of an &agr;-alkyl-&agr;,&ohgr;-dinitrile produced only the &ohgr;-cyanocarboxylic acid resulting from hydrolysis of the &ohgr;-nitrile group. U.S. Pat. No. 5,814,508 describes a process whereby heating a suspension of
Acidovorax facilis
72W (ATCC 55746) in a suitable buffer at 50° C. for a short period of time deactivates an undesirable nitrile hydratase and amidase activity of the whole-cell catalyst, without producing a significant decrease in the desired nitrilase activity.
The problem to be solved remains the lack of a facile enzymatic catalyst for chemoselective hydrolysis of nitrile functional groups in the presence of other hydrolyzable functional groups, with high yield and high selectivity and with the added advantages of low temperature requirements and low waste.
SUMMARY OF THE INVENTION
A process is disclosed for preparing aliphatic or aromatic dicarboxylic acid monoesters from aliphatic or aromatic cyanocarboxylic acid esters. The invention has the steps of (a) contacting an aliphatic or aromatic cyanocarboxylic acid ester in an aqueous reaction mixture with an enzyme catalyst characterized by a chemoselective nitrilase activity derived from
Acidovorax fa
Chauhan Sarita
Dicosimo Robert
Fallon Robert D.
Gavagan John E.
Payne Mark S.
E. I. du Pont de Nemours & Company
Killos Paul J.
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