Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
1999-03-04
2001-01-16
Rotman, Alan L. (Department: 1612)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S027000, C435S135000, C435S136000, C435S264000, C549S373000, C562S566000
Reexamination Certificate
active
06174707
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method for producing an L-allysine acetal. More particularly, the present invention relates to a method for producing L-allysine ethylene acetal. L-allysine ethylene acetal is of use as a synthetic intermediate in the production of medicinal substances.
BACKGROUND ART
Never known heretofore is a production technology for L-allysine acetals. All that is known is a process for producing the racemic compound D,L-allysine ethylene acetal which comprises 8 reaction steps starting with dihydropyran (Bioorganic & Medicinal Chemistry, (3)9, 1237 (1995)). However, this racemic compound cannot be utilized as a synthetic intermediate in the production of medicinal substances.
SUMMARY OF THE PRESENT INVENTION
In view of the above state of the art, the present invention has for its object to provide a method for producing an L-allysine acetal which involves a fewer steps and is efficient.
The present invention is directed to a method for producing an L-allysine acetal which comprises;
converting a D,L-allysine acetal of the following general formula (1):
(wherein R
1
and R
2
are the same or different, and each of them represents an alkyl group having 1 to 8 carbon atoms, or they combinedly form a ring and represent an alkylene group having 2 to 8 carbon atoms) to a mixture of a 2-oxo-6,6-dialkoxyhexanoic acid of the following general formula (2):
(wherein R
1
and R
2
are as defined above) and an L-allysine acetal of the following general formula (3):
(wherein R
1
and R
2
are as defined above) by reacting in the presence of an enzyme capable of stereoselective oxidative deamination of D-amino acids and;
isolating said L-allysine acetal after said converting.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
The present invention is now described in detail.
The D,L-allysine acetal for use in the present invention is a compound of the following general formula (1).
Referring to the D,L-allysine acetal of the above general formula (1), R
1
and R
2
are the same or different, and each of them represents an alkyl group having 1 to 8 carbon atoms, or they combinedly form a ring and represent an alkylene group having 2 to 8 carbon atoms. The alkyl group mentioned above is not particularly restricted but includes lower alkyl groups such as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, t-butyl, s-butyl, hexyl, and octyl. The alkylene group mentioned above is not particularly restricted, either, but includes lower alkylene groups such as ethylene, trimethylene, tetramethylene, ethylethylene, butylethylene, and hexylethylene. In the present invention, use of D,L-allysine ethylene acetal in which R
1
and R
2
represent ethylene is preferred.
The D,L-allysine acetal of the above general formula (1) can be synthesized from dihydropyran, which is commercially available, in 8 steps by the method described in Bioorganic & Medicinal Chemistry, (3)9, 1237 (1995). In the alternative process disclosed in Japanese Kokai Publication Sho-48-39416, the objective compound can be easily produced by synthesizing glutaraldehyde monoethylene acetal from glutaraldehyde and ethylene glycol, both of which are commercially available, reacting said glutaraldehyde monoethylene acetal with potassium cyanide and ammonium carbonate in accordance with the well-known Bucherer method, and hydrolyzing the reaction product.
An enzyme capable of stereoselective oxidative deamination of D-amino acids for use in the present invention is not restricted but includes enzymes available from various sources, for example the D-amino acid oxidase derived from a strain of microorganism of the genus Trigonopsis, porcine kidney, and the like.
The above-mentioned D-amino acid oxidase of the Trigonopsis origin may for example be to “D-AOD Immobilized” (Boehringer Mannheim) and the like and the D-amino acid oxidase derived from porcine kidney includes “D-Amino Acid Oxidase” (Sigma).
The L-allysine acetal production method for the present invention can be typically carried into practice in the following manner.
The D,L-allysine acetal is dissolved in a buffer of pH 5 to 10, preferably pH 6 to 9, and concentration of 1 mM to 1 M, preferably 10 mM to 100 mM, at a substrate concentration of 0.1 to 50 w/v %, preferably 1 to 20 w/v %. To this solution is added 0.001 to 10 parts by weight, preferably 0.01 to 2 parts by weight, based on D,L-allysine acetal, of D-amino acid oxidase, and the D-selective oxidative deamination reaction is conducted under stirring at a reaction temperature of 5 to 80° C., preferably 10 to 50° C., for 1 to 100 hours, preferably 1 to 20 hours. After completion of the reaction, the D-amino acid oxidase is recovered with filtration or centrifugation and the pure L-allysine acetal is isolated by crystallization and the like technique.
The buffer mentioned above is not particularly restricted but includes a phosphate buffer and the like.
The solvent for said crystallization is not particularly restricted but includes alcoholic solvents such as methanol, ethanol, propanol, etc., water, and solvent mixtures thereof.
In the above oxidative deamination reaction, hydrogen peroxide usually is formed as a byproduct because molecular oxygen is the hydrogen acceptor of D-amino acid oxidase. It is known that accumulation of hydrogen peroxide inactivates enzymes and triggers decomposition of 2-oxo-6,6-dialkoxyhexanoic acids. The hydrogen peroxide produced in the course of catalysis by the D-amino acid oxidase can be eliminated by any of several alternative procedures known to those skilled in the art.
The first procedure involves use of the enzyme catalase. Thus, in the L-allysine acetal production method for the present invention, the disproportionation reaction of hydrogen peroxide to molecular oxygen and water can be catalyzed by conducting the reaction in the presence of a catalase.
As the catalase mentioned above, commercial products derived from mammalian livers,
Aspergillus niger
and the like are available.
The second procedure involves use of a metal oxide. Thus, in the L-allysine acetal production method for the present invention, the hydrogen peroxide can be decomposed by conducting the reaction in the presence of a metal oxide.
The above-mentioned metal oxide is not particularly restricted but includes manganese oxides such as manganese dioxide. The manganese oxides lead enzymes inclusive of said D-amino acid oxidase and catalase to stabilize and, as such, can be used with advantage.
In accordance with the present invention, said enzyme capable of stereoselective oxidative deamination of D-amino acids and said catalase may respectively be used in the form of whole cells containing them, crude disrupted cell preparations, partially purified cellular fractions, or purified enzymes. The enzymes may also be used either as they are or as immobilized beforehand.
REFERENCES:
patent: 4376864 (1983-03-01), Drauz et al.
L.J. Fowler et al ; In vitro studies on enzymic biosynthesis of the collagen crosslinks; Biochemical and Biophysical Research Communications vol. 41(1); pp. 251-259, Sep. 7, 1970.
Caplus Abstract 1971:430105, Biochemistry, 1971 vol. 10(21); Nimni Marcel et al., Dec. 1971.
Inoue Kenji
Matsumoto Takehiko
Taoka Naoaki
Desai Rita
Kaneka Corporation
Pollock Vande Sande & Amernick
Rotman Alan L.
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