Enzymatic method for preparing synthesis intermediates

Details Enzymatic method for preparing synthesis intermediates Enzymatic method for preparing synthesis intermediates

2000-12-15

2003-04-22

Marx, Irene (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...

C435S108000

Reexamination Certificate

active

06551816

ABSTRACT:

This application was filed under 35 USC 371 as the national phase of PCT/FR99/01098 filed May 10, 1999.
FIELD OF THE INVENTION
A The present invention relates to a novel method for preparing optically active synthesis intermediates for the manufacture of chemical compounds. More particularly, the present invention relates to a method for the enantiomeric enrichment of &agr;-phenylalanine derivatives.
PRIOR STATE OF THE ART
In many fields, such as for example pharmacy and agrochemistry, active chemical compounds are increasingly complex and very frequently comprise one or more asymmetric centres. Most of these commercial products are marketed in racemate form, that is to say that all the enantiomeric and/or diastereoisomeric forms are present in the active material. However, in a very large number of cases, only one of the optically active forms possesses the desired activity, whereas the other optical isomers are inactive or can even act against the desired effect and even have risks for mammals and/or the environment. It is therefore important to be able to have methods of chemical or biochemical (enzymatic for example) synthesis which lead to the sole isomer desired. Such methods are for example asymmetric syntheses or, when the starting material is a mixture of enantiomers, the so-called enantiomeric enrichment methods.
Enzyme-catalysed enantiomeric enrichment methods are known in the literature. Among them, there may be mentioned for example the publication by B. Kaptein et al. (
Tetrahedron Asymetry,
4(6), (1993), 1113-1116) in which the hydrolysis of an ethyl ester by a pig liver lipase is described; however, the selectivity is very poor, that is to say that the enantiomeric excess of the product is not satisfactory.
Patent WO-A-96/12035 discloses the access, by enzyme catalysis, to carboxylic acid esters possessing a high enantiomeric excess. The catalyst used is chosen from bacteria, lipases and proteases. Here again, the enantiomeric excesses observed are relatively low, and in particular in the case of the S enantiomers.
U.S. Pat. No. 5,552,318 describes a method of enriching the ethyl ester of DL-homophenylalanine with the aid of enzymes, but the enantiomeric excesses observed are highly variable. Finally patent application EP 178,553 describes an enantiomeric enrichment of amino acids by the action of &agr;-chymotrypsin.
One subject of the present invention consists in providing an enzyme-catalysed enantiomeric enrichment method leading to a compound having a high enantiomeric excess.
One subject of the present invention consists in providing an enzyme-catalysed method of preparing a substantially enantiomerically pure compound.
Another subject of the present invention consists in providing an enzyme-catalysed method of preparing, from a mixture of enantiomers, substantially pure S enantiomer, or substantially pure R enantiomer.
Another subject of the present invention consists in providing an enzyme-catalysed method of enantiomeric enrichment from a mixture of enantiomers of esters.
Another subject of the invention consists in providing an enzyme-catalysed method of enantiomeric enrichment of a mixture of enantiomers of esters in substantially enantiomerically pure compounds, with a high yield.
It has now been discovered that all these aims can be achieved fully or partially by means of a method according to the invention, whose description is presented below.
BRIEF DESCRIPTION OF THE INVENTION
The present invention provides a novel method for the enantiomeric enrichment of &agr;-phenylalanine esters, characterized in that a mixture of enantiomers of &agr;-phenylalanine esters is brought into contact with an enzyme catalyst comprising unpurified or partially or completely purified enzymes. These may be used free or absorbed or immobilized on organic or inorganic supports according to methods well known to a person skilled in the art.
The substantially enantiomerically pure compounds thus obtained can serve as synthesis intermediates in the preparation of chiral active materials useful in therapy or in agriculture. By way of example, the enantiomers of &agr;-phenylalanine esters are used as intermediates in tne preparation of certain fungicidal 2-imidazolin-5-ones and 2-imidazoline-5-thiones described ip patent EP-A-O 629 616.
DETAILED DESCRIPTION OF THE INVENTION
The present invention therefore relates to a novel method for preparing substantially enantiomerically pure &agr;-phenylalanine esters of formulae (I) and (II)
in which Alk represents a linear or branched alkyl radical containing from 1 to 6 carbon atoms, such as for example the methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, isopropyl, isobutyl, isopentyl, isohexyl, tert-butyl, tert-pentyl, tert-hexyl, neopentyl or neohexyl radical,
which method is characterized in that a mixture of enantiomers of &agr;-phenylalanine esters is brought into contact with a lipase, an esterase or a protease other than PLE (pig liver esterase), in free, absorbed or immobilized form.
The expression mixture of enantiomers is understood to mean a mixture in equal or different proportions of the S isomer of formula (I) and of the R isomer of formula (II) of the &agr;-phenylalanine ester of formula (A):
in which Alk represents a linear or branched alkyl radical containing 1 to 6 carbon atoms, as defined in the formulae (I) and (II), and the asterisk indicates the asymmetric centre of the ester.
The method according to the invention makes it possible to obtain substantially pure enantiomers, either of S configuration (ester of formula (I)), or of R configuration (ester of formula (II)).
The term “substantially pure” means that the enantiomeric excess of the enantiomer considered is greater than 80%, more particularly greater than 90%. Some enzymes used in the method according to the invention also lead to an enantiomeric excess of 100%; that is to say that, in this case, the enantiomer obtained is pure, the other enantiomeric form not being detectable.
The expression enantiomeric excess is understood to mean the ratio of the excess of the desired enantiomer relative to the undesired enantiomer.
This ratio is calculated according to one of the following equations:
%
⁢
 
⁢
e
.
e
.
(
S
)
=
[
S
]
-
[
R
]
[
R
]
+
[
S
]
×
100
%
⁢
 
⁢
e
.
e
.
(
R
)
=
[
R
]
-
[
S
]
[
R
]
+
[
S
]
×
100
in which:
% e.e. (S) represents the enantiomeric excess in the S isomer,
% e.e. (R) represents the enantiomeric excess in the R isomer,
[S] represents the concentration of S isomer, and
[R] represents the concentration of R isomer.
The method of the invention uses enzymes, in particular lipases, esterases and proteases.
The proteases make it possible to obtain the S enantiomer of formula (I) defined above; the lipases or esterases make it possible to obtain the R enantiomer of formula (II) defined above.
The appropriate enzymes for the method of the invention are more particularly chosen from the following enzymes which are commercially available;
Enzyme
Supplier
Candida Antardca (immob.)
Novo
L1754
Sigma
Lipase A Rhizopus
Lipase CE
Amano
Lipase Chirazyme E1
Boehringer
Lipase Chirazyme L1
Boehringer
Lipase Chirazyme L2
Boehringer
Lipase Chirazyme L3
Boehringer
Lipase Chirazyme L4
Boehringer
Lipase Chirazyme L5
Boehringer
Lipase Chirazyme L6
Boehringer
Lipase Chirazyme L7
Boehringer
Lipase Chirazyme L8
Boehringer
Lipase GC
Amano
Lipase L3001
Sigma
Lipase L3126
Sigma
Lipase, pancreatic L115P
Sigma
Lipase PLE
Amano
Liver Acetone powder L9627
Sigma
Liver porcine L8521
Sigma
MAP 10
Amano
Protease Biofeed
Novo
Protease Prozyme
Amano
Protease Subtilisin
Boehringer
Protease Subtilisin A
Novo
Protease Trypsin
Sigma
PS
Amano
Protease P5147
Sigma
Protease P4032 (aspergillus)
Sigma
Among the proteases, proteases from aspergillus (such as Prozyme for Amano or the protease P4032 from Sigma), Subtilisin from Boehringer of from Novo, Biofeed from Novo and trypsin.
Among the lipases and estrases, the lipascs Chirazyme L2, L5, L7 and E1 from Boehringer and the lipase PLE from Amono will be more particularly prefe

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