Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Utility Patent
1999-02-18
2001-01-02
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S156000, C435S190000, C435S236000, C435S280000
Utility Patent
active
06168935
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a method of manufacturing an optically active alcohol substituted with one or more halogen atoms which are important as an intermediate in pesticide synthesis, and in particular to a method of manufacturing optically active 4-halo-3-hydroxybutyrate esters which are useful as an intermediate in the synthesis of carnitine.
2. Description of the Related Arts
In the prior art, examples of methods of manufacturing optically active alcohols substituted with one or more halogen atoms, such as optically active 4-halo-3-hydroxybutyrate esters, include a method using an asymmetric reductive reaction of microorganisms such as baker's yeast (disclosed for example in Japanese Patent Laid-Open Publication No. Sho 61-146191 and Japanese Patent Laid-Open Publication No. Hei 6-38776), a method of asymmetric synthesis (disclosed in Japanese Patent Laid-Open Publication No. Hei 1-211551), a method of asymmetric reduction using alcohol dehydrogenase derived from 3&agr;-hydroxysteroid dehydrogenase (Japanese Patent Laid-Open Publication No. Hei 1-277494), glycerol dehydrogenase (Tetrahedron Lett. 29,2453-2454 (1988)), alcohol dehydrogenase derived from
Thermoanaerobium brockii
or horse liver (J.Am.Chem.Soc. 107, 4028-4031 (1985)), or
Sulfolobus solfataricus
MT-4(Biotechnol. Lett.13, 31-34 (1991)), or Pseudomonas sp.(PED, J. Org. Chem. 57, 1526-1532 (1992)), and methods using a reductase dependent on nicotinamide adenine dinucleotide phosphoric acid (hereafter abbreviated as “NADPH”) such as reductase from baker's yeast (Bull. Chem. Soc. Jpn. 67, 524-528 (1994) or aldehyde reductase derived from
Sporobolomyces salmonicolor
(Japanese Patent No. 2566960).
However, the alcohol produced by these methods had low optical purity and/or its concentration was low.
SUMMARY OF THE INVENTION
The inventor studied the reaction conditions required to synthesize an optically active alcohol substituted with one or more halogen atoms such as optically active 4-halo-3-hydroxybutyrate esters, using the asymmetric reduction of a halogen-substituted ketone such as 4-haloacetoacetate esters and the ability of secondary alcohol dehydrogenase to renature NAD
+
(oxidized nicotinamide adenine dinucleotide) to regenerate NADH (reduced nicotinamide adenine dinucleotide) which accompanies the asymmetric reductive reaction.
As a result, it was found that the toxicity and denaturing effects on enzymes of ketones comprising a halogen substituent such as 4-haloacetoacetate esters and optically active alcohols comprising a halogen substituent such as optically active 4-halo-3-hydroxybutyrate esters as substrates, could be diminished by performing the reaction at low temperature, and that by so doing, decomposition of the substrate was also suppressed. Consequently, the amount of optically active alcohols with halogen substituents which accumulated such as optically active 4-halo-3-hydroxybutyrate esters remarkably increased, and this discovery led to the present invention.
The secondary alcohol dehydrogenase of this specification is an alcohol dehydrogenase which acts as a catalyst in the oxidation of alcohols dependent on NAD
+
or NADP
+
or the reductive reaction of ketones dependent on NADH or NADPH, and it refers to secondary alcohol dehydrogenase which more preferably acts on isopropanol than n-propanol.
Specifically this invention, which uses a stereo-selective secondary alcohol dehydrogenase and an alcohol as a renaturing substrate for reduced nicotinamide adenine dinucleotide (hereafter abbreviated as NADH), provides a method of manufacturing a halogen-substituted optically active alcohol such as optically active 4-halo-3-hydroxybutyrate esters by asymmetric reduction of a halogen-substituted ketone such as 4-haloacetoacetate esters and is characterized in that it is performed at a low temperature preferably not exceeding 30° C.., more preferably not exceeding 25° C. and still more preferably not exceeding 20° C.
DETAILED DESCRIPTION OF THE INVENTION
This invention will now be described in further detail.
The stereo-selective secondary alcohol dehydrogenase of this invention may be any enzyme regardless of the source, provided it is a stereo-selective secondary alcohol dehydrogenase which has the ability to asymmetrically reduce a halogen-substituted ketone such as 4-haloacetoacetate esters, and that the amount of a halogen-substituted optically active alcohol such as 4-halo-3-hydroxybutyrate esters increases due to performing the reaction at a low temperature not exceeding 30° C. and preferably not exceeding 25° C. The secondary alcohol dehydrogenase having the following properties (disclosed in Japanese Patent Laid-Open Publication No. Hei 7-231789) are particularly useful.
(a) Functions
Oxidizes alcohols with NAD
+
(oxidized nicotinamide adenine dinucleotide) as coenzyme to form ketones or aldehydes, and reduces ketones or aldehydes with NADH (reduced nicotinamide adenine dinucleotide) as coenzyme to form alcohols.
(b) Substrate Specificity
Acts on aliphatic alcohols comprising aromatic substituents as substrates in an oxidative reaction, has a greater effect on secondary alcohols than on primary alcohols, preferentially oxidizes the S-form of 2-butanol, and acts on aldehydes or aliphatic ketones comprising aromatic substituents as substrates in a reductive reaction.
(c) Molecular Weight
The molecular weight is approximately 40,000 when measured by SDS-PAGE.
Herein, SDS-PAGE is an abbreviation of SDS polyacrylamide gel electrophoresis, and SDS is sodium dodecyl sulfate.
The secondary alcohol dehydrogenase derived from the Candida genus, in particular
Candida parapsilosis
, may be given as an example of a secondary alcohol dehydrogenase with these properties.
Also, the secondary alcohol dehydrogenase having the following properties (disclosed in Japanese Patent application No. Hei 8-279092) is particularly useful.
(a) Effects
Oxidizes alcohols with NAD
+
(oxidized nicotinamide adenine dinucleotide) as coenzyme to form ketones or aldehydes, and reduces ketones or aldehydes with NADH (reduced nicotinamide adenine dinucleotide) as coenzyme to form alcohols.
(b) Substrate Specificity
Acts on aliphatic alcohols comprising aromatic substituents as substrates in an oxidative reaction, has a greater effect on secondary alcohols than on primary alcohols, preferentially oxidizes the S-form of 1-phenylethanol, and acts on aldehydes or aliphatic ketones comprising aromatic substituents as substrates in a reductive reaction.
(c) Molecular Weight
The molecular weight is approximately 51,000 when measured by SDS-PAGE, and approximately 107,000 when measured by gel filtration.
The secondary alcohol dehydrogenase derived from the genus Geotrichum, in particular
Geotrichum candidum
, may be cited as an example of a secondary alcohol dehydrogenase having such properties.
These secondary alcohol dehydrogenases can be used in various forms such as that of the microorganism which originally contained the enzymes, the partially purified enzyme, or the purified enzyme.
It is furthermore possible to use a enzyme produced by cloning and expressing the gene which codes secondary alcohol dehydrogenase, in
Escherichia coli
, the genus Bacillus, lactic acid bacteria, coryneform bacteria, yeast such as baker's yeast, the genus Pichia or the genus Hansenula, and mold such as the genus Aspergillus. For example, recombinant cells obtained by cloning the secondary alcohol dehydrogenase gene (hereafter abbreviated as CpADH2) derived from
Candida Parapsilosis
, and culturing
Escherichia coli
bearing the expression plasmid pKK-CPA1 comprising CpADH2 gene functionally located downstream of tac promoter (disclosed in Japanese Patent Laid-Open Publication No. Hei 7-231785), is particularly to be preferred.
The one or more halogen atoms in the halogen-substituted ketone may for example be chlorine, bromine or iodine. The 4-haloacetoacetate esters mentioned herein may be a straight-chain, branched or aromatic-substituted alcohol e
Daicel Chemical Industries Ltd.
Lilling Herbert J.
Oliff & Berridg,e PLC
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