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A. Riise Moen et al. / Tetrahedron: Asymmetry 15 (2004) 1551–1554
OH
O
O
OH
O
Cl
OR
HO
OEt
2
4
A
C
OH
O
N
C
B
OEt
E
1
O
OH
O
D
O
OH
O
H2N
OEt
EtO
OEt
5
3
Figure 2. Retrosynthetic analysis for the synthesis of enantiopure target molecule 1 by biocatalysis. Route A, resolution of racemic chlorohydroxy
ester 2; route B, enzyme catalyzed asymmetrization by ‘half’-hydrolysis of diethyl ester 3, followed by route C, conversion of the enantiopure
monoester 4 to nitrile 1; route D ammonolysis of prochiral diester 3 to give enantiopure amide 5, followed by route E, conversion of amide 5 to target
nitrile 1.
2. Results and discussion
values (ꢀ50%). It has previously been reported that the
(R)-monoethyl ester is predominant in hydrolysis cata-
lyzed by chymotrypsin, and moreover, that the reaction
gives the product with very high ee.12 This reported high
ee-value and configuration is based on comparison with
a previously reported specific rotation value for the
monomethyl ester produced by classical resolution via
the cinchonidine salt.13 It has been reported that grow-
ing cells of Acinetobacter lwoffii gives predominance of
the (R)-enantiomer when used for hydrolysis of diethyl
3-hydroxyglutarate with an ee of 80%.14 In our hands
the same organism (ATCC-17925) gave the (S)-enan-
tiomer and with low ee (56%).
We analyzed the possibilities of making 1 by biocatalysis
based on the retrosynthetic scheme shown in Figure 2.
Our first approach was to make 1 from racemic chlo-
rohydroxy ester 2 by enzyme catalyzed kinetic resolution
(route A).6 The drawback with this resolution is that
only 50% of the right enantiomer could be obtained.
This may not be serious since quantitative conversion to
one single enantiomer is possible using the technique of
stereoinversion.7;8 We started a systematic approach by
varying the R-groups, solvents, acyl donors and en-
zymes. The highest E-value of >100 was obtained for the
tert-butyl ester with vinyl propanoate as the acyl donor
and lipase from Rhizomucor miehei as the catalyst.
However, the synthesis of the ester substrate was not
trivial starting with the diketene. Moreover, target ni-
trile 1 is an ethyl ester and a transesterification would be
necessary in order to convert the tert-butyl ester to the
ethyl ester. Hence we looked for an alternative method.
By asymmetric synthesis instead of resolution, a theo-
retical yield of 100% of one enantiomer may be obtained
directly, however, with enzyme catalysis, it is not guar-
anteed that this will be the wanted enantiomer.
Since the specific rotation of the monoethyl ester 4 is a
very small numerical value, we wanted to check the
absolute configuration of the monoester produced by
CALB not only by comparison of the ½aꢁ values, but
D
with a more reliable method. The monoethyl ester pro-
duced by CALB was co-crystallized with (R)-phenyl-
ethylamine with the crystal structure determined to be as
shown in Figure 3. This structure determination shows
conclusively that the monoester produced by CALB is
the (S)-enantiomer and so we consider it safe to assume
that amide 5 has the same configuration. Based on very
reliable chiral GLC analyses we also find that chymo-
trypsin gives a predominance of the (R)-enantiomer 4.
However, in our hands the ee values obtained were
much lower than previously reported12 both for the
diethyl and dimethyl esters.11 Recently, a patent for a
process for producing the monoethyl ester of diethyl 3-
acetoxyglutarate using chymotrypsin, has been reported.
The ee-values are very high and the configuration (R) as
wanted for synthesis of 1.15
An obvious starting material for the asymmetric syn-
thesis would be diethyl 3-hydroxyglutarate 3, a prochiral
substrate. Enantiopure monoester 4 could be obtained
by route B using enzymatic hydrolysis and this in turn
could be converted to the nitrile using chlorosulfony-
lisocyanate (route C).9 An even simpler method would
be enzyme catalyzed ammonolysis via route D to the
enantiopure monoamide 5, which in turn could be
converted to the target nitrile 1 using 1-(3-dimethyla-
minopropyl)-3-ethylcarbodiimide (route E).10
After use in the first hydrolysis of diethyl and dimethyl
3-hydroxyglutarate, the immobilized catalyst CALB was
re-used more than 10 times with retention of high
activity and enantioselectivity. In fact, the activity of the
enzyme increased significantly from the first to the sec-
ond batch of hydrolysis of 3 (Fig. 4).
All of the mentioned strategies were tried using a range
of enzymes.11 By far the highest ee-values were obtained
using lipase B from Candida antarctica (CALB). Indeed,
the ammonolysis reaction gave ee ¼ 98% and 95% yield.
Chymotrypsin catalyzed hydrolysis gave much lower ee-