Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2001-07-10
2002-06-04
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
C435S134000, C435S135000, C435S136000, C435S146000, C435S190000, C435S280000
Reexamination Certificate
active
06399339
ABSTRACT:
The present invention relates to a method for the enantioselective reduction of 3,5-dioxo-carboxylic acids, their salts and their esters.
Homochiral 3,5-dihydroxycarboxylic acid derivatives having Formula 1 are intermediates in the synthesis of numerous natural and active substances.
wherein X stands for a component from the group consisting of hydrogen, halogen, alkyl, aryl, CH═CHR
2
, C≡CR
3
, (wherein R
2
=R, except for metal cation, and R
3
═R); &Sgr; stands for H or for a protective group for the hydroxyl function; R stands for H, metal cation, for an alkyl, aryl, aralkyl or cycloalkyl radical.
Depending on the absolute configuration at the stereo centers C-3 and C-5, they can be systematically employed in the synthesis of chiral natural substances, such as mevic acids, or synthetic HMG-CoA-reductase inhibitors.
Other natural or active substances call for different configurations of the stereogenic centers in position C-3 and C-5. Consequently, there is great interest in the preparation of all possible stereoisomers of 3,5-dihydroxycarboxylic acid derivatives according to Formula 1 in an optically pure form. An advantageous method for the preparation of these compounds is the catalytic enantioselective reduction of the prochiral 3,5-dioxocarboxylic acid derivatives according to Formula 2.
When this method is employed, there is no need for costly and environmentally burdensome separation procedures for the racemate, scalemate or diastereomer. This avoids the binding and cleavage of a stoichiometric quantity of a homochiral auxiliary group that are necessary in a diastereoselective synthesis. Moreover, the carbon skeleton of the 3,5-di-hydroxycarboxylic acid ester according to Formula 1 is already complete in the initial compounds according to Formula 2, that is to say, the stereocenters are only introduced into the overall synthesis sequence at a later point in time, as a result of which the loss of homochiral material is kept low.
European Patent Application No. 0,569,998 A2 discloses an enantioselective microbial process to reduce di-ketoesters that are oxyalkyl-substituted and oxyaralkyl-substituted in position 6.
WO 97/00968 discloses a process to reduce 3-oxo-5-hydroxy-carboxylic acid esters by means of reductases of Beauveria, Candida, Kluyveromycis, Torulaspora or Pichia.
DE 196 10 984 A1 discloses a stable microbial enzyme with alcohol-dehydrogenase activity, a process to obtain it as well as its use for the enantioselective reduction/oxidation of organic keto compounds/hydroxy compounds whereby, depending on the type of initial compounds, either R-hydroxy or S-hydroxy compounds are obtained.
The publication titled “Enantioselective microbial reduction of 3,5-dioxo-6-(benzyloxyl)hexanoic acid ethyl ester” in Enzyme Microb. Technol. (1993), 15 (12), 1014-21 ff. shows the reduction of 3,5-dioxo-6-(benzyloxy)hexanoic acid ethyl ester by means of a reductase.
As far as the terminology is concerned, a definition of the term will be introduced here as a working term that is to be valid within the disclosure:
In Formula 3, the OH group projects from the paper plane in position 5, whereas the chains with the carboxylic acid function or with the ester function as well as with the radical X lie in the paper plane. Therefore, the hydrogen atom on C-5 recedes behind the paper plane. In accordance with the CIP (RS) nomenclature, as a function of the priority of the chain that is substituted with X, an R-designation or an S-designation would be employed for the same spatial configuration of the OH group projecting from the paper plane towards the front. As defined by the invention, the designation r-configuration will be used for an arrangement of the substituent used in which the OH group in position 5 projects forward from the paper plane, in which the hydrogen atom in position 5 extends below the paper plane and in which the side chain provided with the carboxylic acid function or with the ester function lies on the right-hand side of the fifth carbon atom while the side chain provided with the substituent X lies on the left-hand side of the fifth carbon atom. For those cases where the right-hand side chain provided with the carboxylic acid function or with the ester function has a higher priority than the left-hand side chain provided with the substituent X, this corresponds to the classic R-configuration. If the priorities of the above-mentioned chains are reversed (for example, by selecting X=halogen), then the classic S-configuration would be ascribed to the target compound. Both cases, however, should be encompassed by the definition of an r-configuration.
The objective of the invention is to regioselectively introduce an r-configuration of the hydroxyl group in position 5 during the enzymatic reduction of 3,5-dioxocarboxylic acid derivatives.
Another object of the invention is to provide a new method for preparing enantiomerpure 3,5-dihydroxycarboxylic acid derivatives, in other words, to create a process with which a syn-reduction or an anti-reduction of the keto group can systematically take place in position 3 towards position 5, so that the 3,5-dihydroxycarboxylic acid derivative has a tailor-made, absolute configuration with respect to C-3 which can be prepared as desired.
Another objective of the invention is to create an improved method for the synthesis of 3,5-dioxocarboxylic acid esters according to Formula 4 that can be used as educts for the enzymatic reduction.
On the basis of the generic part of claim
1
, the objective is achieved according to the invention by means of the features indicated in the characterizing part of claim
1
.
With the method according to the invention, it is now possible to reduce 3,5-dioxo carboxylic acids as well as their esters in a highly enantioselective manner and thus to obtain compounds that can then be employed in a synthesis of natural and active substances that have a defined absolute configuration in the 3,5-dihydroxycarboxylic acid structural element.
Advantageous embodiments of the invention are described in the subordinate claims.
The invention will be described below in a general manner.
According to the invention, a compound according to Formula 4
wherein R
1
stands for a component from the group consisting of alkyl, alkenyl, cyclo-alkyl, cycloalkenyl, aryl, aralkyl, cycloalkylalkyl, hydrogen or metal cation, and X stands for a component from the group consisting of hydrogen, halogen, alkyl, aryl, CH═CHR
2
, C≡CR
3
(wherein R
2
═R
1
, except for metal cation, and R
3
═R
1
) is reacted by means of an alcohol dehydrogenase with the addition of NADPH or of another co-factor.
The term alkyl refers to straight-chain as well as to branched saturated carbon chains. Examples of these are methyl, ethyl, n-propyl, i-propyl, t-butyl, pentyl, i-pentyl, n-hexyl, i-hexyl. The term alkenyl relates to straight-chain and branched unsaturated hydro-carbons, examples of which are vinyl, 1-propenyl, allyl, butenyl, i-butenyl. The term cycloalkyl encompasses saturated, ring-shaped hydrocarbon chains consisting of three, four, five, six or seven carbon atoms. Cycloalkenyl designates unsaturated, ring-shaped hydrocarbons having 5, 6, 7 or 8 carbon atoms. Aryl refers to aromatic systems, enclosed heteroaromatic compounds and substituted aromatic systems, such as phenyl, p-tolyl, furanyl. Aralkyl refers to aryl radicals that are bonded via alkyl groups such as, for instance, a benzyl radical. The term cycloalkylalkyl comprises cycloalkyl radicals that are bonded via alkyl groups. Halogen preferably refers to fluorine and chlorine.
The alcohol dehydrogenase is preferably recombinant and stems from Lactobacillus, especially
Lactobacillus brevis
(recLBADH). The particularly advantageous aspect of this enzyme is that it can be recombinantly over-expressed and thus can be made available in large quantities. This also allows its use on a large, technical scale. The reaction can take place in an aqueous medium by means of an enzyme as well as on the intracellular level in a microorganism. I
Hummel Werner
Müller Michael
Wolberg Michael
Connolly Bove & Lodge & Hutz LLP
Forschungszentrum Julich GmbH
Lilling Herbert J.
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