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
1999-06-28
2001-04-10
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...
C435S121000
Reexamination Certificate
active
06214610
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of producing optically active N-benzyl-3-pyrrolidinol, which is useful as an intermediate for the synthesis of medicinal compounds such as &bgr;-lactam antibiotics and dihydropyridine compounds.
BACKGROUND ART
Optically active N-benzyl-3-pyrrolidinol is useful as an intermediate for the synthesis of medicinal compounds. For the method of producing optically active N-benzyl-3-pyrrolidinol, a technology comprising synthesizing the same from an optically active compound and a technology starting with a prochiral compound and conducting asymmetric synthesis or optical resolution, among others, are known. As such a method, the method disclosed in Japanese Kokai Publication Hei-06-141876 comprises producing optically active N-benzyl-3-pyrrolidinol by stereoselectively reducing N-benzyl-3-pyrrolidinone in the presence of an enzyme capable of catalyzing the stereoselective reduction of N-benzyl-3-pyrrolidinone. However, this method is not suited for practical use, partly because fungi used as an enzyme source are difficult to cultivate on an industrial scale and carry out the process thereafter and partly because the charge concentration of the substrate and the rate of conversion from the substrate to the product are low.
SUMMARY OF INVENTION
In view of the foregoing, it is an object of the present invention to provide an efficient method of producing optically active N-benzyl-3-pyrrolidinol by an enzymatic reaction involving stereoselective reduction of N-benzyl-3-pyrrolidinone.
The present invention consists in a method of producing optically active N-benzyl-3-pyrrolidinol which comprises a step of obtaining a reaction mixture by treating N-benzyl-3-pyrrolidinone with cells or a culture of a microorganism, or a material derived therefrom, and a step of recovering optically active N-benzyl-3-pyrrolidinol from said reaction mixture, in which method said microorganism is a microorganism belonging to the genus Dipodascus, Debaryomyces, Cryptococcus, Pichia, Rhodosporidium , Trichosporon, Micrococcus, Komagataella, Ogataea or Zygosaccharomyces.
DETAILED DESCRIPTION OF THE INVENTION
In the following, the present invention is described in detail.
In the present invention, the substrate N-benzyl-3-pyrrolidinone is first treated with cells or a culture of the microorganism, or a material derived therefrom, to give a reaction mixture.
Said N-benzyl-3-pyrrolidinone can be synthesized by the process disclosed in Japanese Kokai Publication Sho-54-16466. Thus, benzylamine and ethyl acrylate are subjected to Michael addition and the thus-obtained &bgr;-alanine derivative is reacted with ethyl chloroacetate in the presence of a base. The compound obtained is cyclized in the presence of metallic sodium to give N-benzyl-4-carboethoxy-3-pyrrolidone, which is decarboxylated by using hydrochloric acid, whereby N-benzyl-3-pyrrolidinone can be obtained.
In the present invention, a microorganism belonging to the genus Dipodascus, Debaryomyces, Cryptococcus, Pichia, Rhodosporidium, Trichosporon, Micrococcus, Komagataella, Ogataea or Zygosaccharomyces is used as the above-mentioned microorganism. Such a microorganism stereoselectively reduces the 3-position carbonyl group of the above-mentioned N-benzyl-3-pyrrolidinone.
Specific examples of said microorganism include, but are not particularly limited to,
Dipodascus tetrasperma
CBS 765.70
, Debaryomyces hansenii var. hansenii
IFO 0728
, Cryptococcus albidus var. albidus
IFO 0378
, Pichia membranaefaciens
IFO 0189
, Rhodosporidium toruloides
IFO 0413
, Trichosporon fermentans
ATCC 10675
, Micrococcus luteus
IFO 13867
, Komagataella pastoris
IFO 0948
, Ogataea polymorpha
IFO 1476
, Zygosaccharomyces bailii
IFO 0519 and the like.
These microorganisms can be obtained from stock cultures which are readily available or purchasable. They can also be isolated from the natural world. It is also possible to obtain microbial strains having properties advantageous to the present reaction by causing mutation in these microorganisms. Further, those derived from these microorganisms by the genetic engineering or bioengineering techniques, such as recombinant DNA and cell fusion etc., may also be used.
Said microorganisms can be cultivated by using a medium containing nutrient components. A solid medium such as an agar medium or a liquid medium is generally used as said medium. For mass cultivation of said microorganism, a liquid medium is suitably used. The carbon source, for example, saccharides such as glucose, sucrose and maltose, organic acids such as lactic acid, acetic acid and citric acid, alcohols such as ethanol and glycerol, and mixtures of these can be incorporated in the medium, and the nitrogen source, for example, ammonium sulfate, ammonium phosphate, urea, yeast extracts, meat extracts, peptone and the like can be incorporated. Further, other inorganic salts, vitamins and other nutrient sources may also be incorporated optionally.
Said microorganisms can be cultivated generally under ordinary conditions, for example aerobically at pH 4.0 to 9.5 within the temperature range of 20 to 45° C. for 10 to 96 hours.
In treating the above-mentioned N-benzyl-3-pyrrolidinone with the above-mentioned microorganisms, the culture fluid containing said microorganism as obtained can generally be used. In cases, however, a component in the culture fluid interferes the reaction, the use of a suspension obtained by treating said culture fluid by centrifugation, for instance, is preferred.
The materials derived from cells of said microorganisms include, but are not particularly limited to, dried cells, materials obtained from the cells by treatment with a surfactant or an organic solvent, and materials obtained by treating the cells with a lytic enzyme. Furthermore, enzyme preparations prepared by extraction from the cells or cultures can also be used.
The material derived from the culture includes, but is not particularly limited to, concentrated cultures, dried cultures, surfactant- or organic solvent-treated cultures, lytic enzyme-treated cultures and the like. Furthermore, enzyme preparations purified from the cultured cells or cultures may also be used.
In carrying out the reaction by treating the above-mentioned N-benzyl-3-pyrrolidinone with the above-mentioned microbial cells, cultures or materials derived therefrom, said N-benzyl-3-pyrrolidinone may be added all once at initially or in portions. The reaction temperature on that case is generally 15 to 50° C., preferably 20 to 40° C., and the pH is 2.5 to 9.0.
The amount of the cells in the reaction mixture may suitably be selected depending on the catalysing ability of the cells. The substrate concentration is preferably 0.01 to 50% (w/v), more preferably 0.1 to 20% (w/v).
Generally, the reaction is carried out with shaking or with aeration and agitation. The reaction time may suitably be selected depending on the substrate concentration, the amount of microorganism and other reaction conditions. Generally, it is preferred that the respective conditions be selected so that the reaction may be complete in 2 to 168 hours. For promoting the above reaction, an energy source such as glucose can be added to the reaction mixture in an amount of 1 to 5%, whereby good results may be obtained.
Further, the reaction can be promoted by adding a coenzyme component, such as reduced form nicotinamide adenine dinucleotide (NADH) or reduced form nicotinamide adenine dinucleotide phosphate (NADPH), which is generally deemed necessary for reduction reactions in biological processes. In the concrete, either these may be added to the reaction system, or a reaction system capable of forming NADH, NADPH or the like may be added to the reaction system. For example, use may be made of the reaction by which NADH is formed from NAD on the occasion of the formation of carbon dioxide and water from formic acid in the presence of formate dehydrogenase, or the reaction by which NADH/NADPH is formed from NAD/NADP on the occasion of the formation of gluconolactone from glucose in the
Hasegawa Junzo
Yasohara Yoshihiko
Connolly Bove & Lodge & Hutz LLP
Kaneka Corporation
Saucier Sandra E.
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