Process for the preparation of amino alcohols and...

Details Process for the preparation of amino alcohols and... Process for the preparation of amino alcohols and...

2001-11-14

2004-09-07

Saucier, Sandra E. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

C435S252500, C435S253300

Reexamination Certificate

active

06787347

ABSTRACT:

The invention relates to a novel process for the preparation of (1R,4S)- or (1S,4R)-1-amino-4-(hydroxymethyl)-2-cyclopentene of the formulae
and/or of (1S,4R)- or (1R,4S)-amino alcohol derivatives of the general formulae
and to novel microorganisms which are able to utilize a cyclopentene derivative of the general formula
as sole nitrogen source, as sole carbon source or as sole carbon and nitrogen source.
The invention further relates to enzyme extracts and enzymes having N-acetylamino-alcohol hydrolase activity obtainable from these microorganisms.
(1R,4S)-1-Amino-4-(hydroxymethyl)-2-cyclopentene of the formula I is an important intermediate for the preparation of carbocyclic nucleosides such as, for example, Carbovir® (Campbell et al., J. Org. Chem. 1995, 60, 4602-4616).
Processes for the preparation of (1R,4S)-amino-4-(hydroxymethyl)-2-cyclopentene are described by Campbell et al. (ibid) and by Park K. H. & Rapoport H. (J. Org. Chem. 1994, 59, 394-399).
The precursor used in these processes is either D-glucono-&dgr;-lactone or D-serine, and about 15 synthesis stages are necessary to form (1R,4S)-N-tert-butoxycarbonyl-4-hydroxymethyl-2-cyclopentene, which is then deprotected to give (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene. These two processes are costly, elaborate and cannot be implemented industrially.
WO 93/17020 describes a process for the preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene, wherein (1R,4S)-4-amino-2-cyclopentene-1-carboxylic acid is reduced with lithium aluminium hydride to the desired product.
The disadvantage of this process is, on the one hand, that the double bond of the cyclopentene ring is also reduced, the lithium aluminium hydride is difficult to handle, and, on the other hand, that it is too costly.
Taylor, S. J. et al. (Tetrahedron: Asymmetry Vol. 4, No. 6, 1993, 1117-1128) describe a process for the preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene starting from (±)-2-azabicyclo [2.2.1]hept-5-en-3-one as precursor. In this case, the precursor is converted by means of microorganisms of the species
Pseudomonas solanacearum
or
Pseudomonas fluorescens
into (1R,4S)-2-azabicyclo[2.2.1]hept-5-en-3-one, which is then converted with di-tert-butyl dicarbonate into (1R,4S)-N-tert-butoxycarbonyl-2-azabicyclo [2.2.1]hept-5-en-3-one, which is reduced with sodium borohydride and trifluoroacetic acid to the desired product. This process is much too costly.
In addition, Martinez et al. (J. Org. Chem. 1996, 61, 7963-7966) describe a 10-stage synthesis of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene starting from diethyl dialkylmalonate. This process also has the disadvantage that it is elaborate and cannot be implemented industrially.
It was an object of the present invention to provide a simple process for the preparation of (1R,4S)-1-amino-4-(hydroxymethyl)-2-cyclopentene.
This object is achieved with the microorganisms of the invention according to claim
1
, and enzyme extracts therefrom, with the enzymes of the invention according to claim
4
and with the process of the invention according to claim
7
.
The microorganisms of the invention can be isolated from soil samples, sludge or wastewater with the assistance of conventional microbiological techniques.
The microorganisms are isolated according to the invention by cultivating them in a nutrient medium containing one or more cyclopentene derivatives of the general formula
in which R
1
denotes C
1
-C
4
-alkyl, C
1
-C
4
-alkoxy, aryl or aryloxy,
as sole carbon and nitrogen source
as sole nitrogen source with a suitable carbon source or
as sole carbon source with a suitable nitrogen source,
in a conventional way.
It is possible to use as C
1
-C
4
-alkyl for example methyl, ethyl, propyl, isopropyl or butyl. It is possible to use as C
1
-C
4
-alkoxy for example methoxy, ethoxy, propoxy, isopropoxy, butoxy or tert-butoxy. It is possible to use as aryl for example phenyl or benzyl. Benzyl is preferably used. It is possible to use as aryloxy for example benzyloxy or phenoxy. Accordingly, the following examples are suitable as cyclopentene derivative of the general formula VII:
1-acetylamino-4-hydroxymethyl-2-cyclopentene, 1-butyrylamino-4-hydroxymethyl-2-cyclopentene or 1-phenylacetylamino-4-hydroxymethyl-2-cyclopentene.
It is expedient to select from the culture obtained by cultivation those which utilize the (1R,4S) isomer of the cyclopentene derivative of the formula VII as sole nitrogen source, as sole carbon source or as sole carbon and nitrogen source.
The microorganisms can use as suitable nitrogen source, for example, ammonium, nitrates, amino acids or ureas as substrate for growth. The microorganisms can use as suitable carbon source, for example, sugars, sugar alcohols, C
2
-C
4
-carboxylic acids or amino acids as substrate for growth. Hexoses such as glucose or pentoses can be used as sugars. Glycerol, for example, can be used as sugar alcohol. Acetic acid or propionic acid can be used, for example, as C
2
-C
4
-carboxylic acids. Leucine, alanine, asparagine can be used, for example, as amino acids.
The selection medium and culture medium which can be used are those conventional among those skilled in the art, such as, for example, the one described in Table 1 or a complete medium (medium containing yeast extract), preferably using the one described in Table 1.
During the culturing and selection, the active enzymes of the microorganisms are expediently induced. The cyclopentene derivatives of the general formula VII can be used as enzyme inducer.
The culturing and selection normally takes place at a temperature from 20° C. to 40° C., preferably from 30° C. to 38° C. and at a pH between 5.5 and 8.0, preferably between 6.8 and 7.8.
Preferred microorganisms are those of the genus Rhodococcus, Gordona, Arthrobacter, Alcaligenes, Agrobacterium/Rhizobium, Bacillus, Pseudomonas or Alcaligenes/Bordetella, in particular of the species
Rhodococcus erythropolis
CB 101 (DSM 10686), Alcaligenes/Bordetella FB 188 (DSM 11172), Arthrobacter sp. HSZ 5 (DSM 10328), Rhodococcus sp. FB 387 (DSM 11291),
Alcaligenes xylosoxydans
ssp.
denitrificans
HSZ 17 (DSM 10329), Agrobacterium/Rhizobium HSZ 30, Bacillus simplex K2,
Pseudomonas putida
K32, or Gordona sp. CB 100 (DSM 10687) and their functionally equivalents variants and mutants. Deposition in accordance with the Budapest Treaty at the Deutsche Sammlung von Mikro-organismen und Zellkulturen GmbH (DSMZ), Mascheroderweg 1b, D-38124 Braunschweig, took place on 20.05.1996 for the microorganisms DSM 10686 and 10687, on 6.11.1995 for the microorganisms DSM 10328 and DSM 10329, on 8.10.1996 for the microorganism DSM 11291 and on 20.09.1996 for the microorganism DSM 11172.
“Functionally equivalent variants and mutants” mean microorganisms having essentially the same properties and functions as the original microorganisms. Variants and mutants of this type can be produced by chance, for example by UV radiation.
Taxonomic description of Alcaligenes/Bordetella FB 188 (DSM 11172)
Cell form
rods
Width &mgr;m
0.5-0.6
Length &mgr;m
1.0-2.5
Motility
+
Flagellation
peritrichous
Gram reaction
−
Lysis by 3% KOH
+
Aminopeptidase (Cerny)
+
Spores
−
Oxidase
+
Catalase
+
ADH (alcohol dehydrogenase)
−
NO
2
from NO
3
−
Denitrification
−
Urease
−
Hydrolysis of gelatin
−
Acid from (OF test):
Glucose
−
Fructose
−
Arabinose
−
Adipate
+
Caprate
+
Citrate
+
Malate
+
Mannitol
−
Taxonomic Description of
Rhodococcus erythropolis
CB 101 (DSM 106 86)
1. Morphology and color of the colonies: short branched hyphae which, when old, disintegrate into rods and cocci, colonies glistening and partly confluent, beige with pink tinge, RAL 1001;
2. Diagnosed amino acid of the peptidoglycan: mesodiaminopimelic acid;
3. Mycolic acids: Rhodococcus mycolic acids; determination of the mycolic acid chain length (C
32
-C
44
) and comparison of the data with the entries in the DSM mycolic acid data bank revealed very

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