Microbiological transformations. Part 46: Preparation of enantiopure (S)-2-pyridyloxirane via epoxide hydrolase-catalysed kinetic resolution

Article abstract of DOI:10.1016/S0957-4166(00)00285-8

The hydrolytic kinetic resolution (HKR) of 2-pyridyloxirane is described, using the overexpressed epoxide hydrolase from the filamentous fungus Aspergillus niger. This allows the preparation of the (S)-enantiomer of this product in enantiopure form (ee >9

Full text of DOI:10.1016/S0957-4166(00)00285-8

Tetrahedron: Asymmetry 11 (2000) 3041±3044
Microbiological transformations. Part 46: Preparation of
enantiopure (S)-2-pyridyloxirane via epoxide
hydrolase-catalysed kinetic resolution
Yvonne Genzel,a Alain Archelas,a,* Q. B. Broxterman,b Birgit Schulzeb
and Roland Furstossa
a
Â
Groupe Biocatalyse et Chimie Fine, ESA 6111 associee au CNRS, Universite de la Mediterranee,
Faculte de Sciences de Luminy, Case 901, 163 avenue de Luminy, 13288 Marseille Cedex 9, France
Â
Â
Â
Â
bDSM Research. Life Science Chemistry and Catalysis, PO Box 18, 6160 MD Geleen, The Netherlands
Received 12 June 2000; accepted 20 July 2000
Abstract
The hydrolytic kinetic resolution (HKR) of 2-pyridyloxirane is described, using the overexpressed
epoxide hydrolase from the ®lamentous fungus Aspergillus niger. This allows the preparation of the
(S)-enantiomer of this product in enantiopure form (ee >99%), which could not be obtained using con-
ventional chemical methods. # 2000 Published by Elsevier Science Ltd.
1. Introduction
It is currently widely recognised that enantiopure epoxides are highly valuable chiral synthons,
and several chemical methods have recently been developed in order to synthesise these important
building blocks, or their corresponding vicinal diols, in enantiopure form.y,1 3 We (and others) have
recently developed a methodology that enables the preparation of such targets using a biocata-
lysed Hydrolytic Kinetic Resolution approach (HKR) performed by a `new' type of enzyme,
i.e. microbial epoxide hydrolases (EHs) (EC 3.3.2.3).4 9 An application of this methodology to
the synthesis of enantiomerically pure bioactive compounds has recently been illustrated.10 12 Of
course, this strategy may be even more attractive, if it allows the preparation of epoxides which
cannot be obtained in enantiopure form by conventional chemical techniques. This is, for
instance, the case for 2-pyridyloxirane 2, a very valuable chiral synthon,13,14 and we describe here
the preparation of this targetÐin nearly enantiopure formÐusing a resolution process catalysed
by the EH from the ®lamentous fungus Aspergillus niger (Scheme 1).
* Corresponding author. Tel: +33 04 91 82 91 58; fax: +33 04 91 82 91 45; e-mail: archelas@luminy.univ-mrs.fr
y
Several examples of enantioselective epoxidation of prochiral substrates have been described recently, most of them
using heavy metal based catalysts. However, the obtained products rarely show excellent ees (>98%). Only a few
applications of the new chemical HKR strategy have been described up to now. For a direct comparison of such
strategies on one particular compound, see for instance Ref. 3.
0957-4166/00/$ - see front matter # 2000 Published by Elsevier Science Ltd.
PII: S0957-4166(00)00285-8

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Y. Genzel et al. / Tetrahedron: Asymmetry 11 (2000) 3041±3044
Scheme 1. Biocatalysed HKR of rac-2 using the Aspegillus niger epoxide hydrolase
2. Results and discussion
2.1. Chemical asymmetric oxidation
As stated above, some innovative metal-catalysed asymmetric oxidation procedures of
prochiral ole®ns have been developed recently. However, these su€er from various intrinsic
limitations and are therefore not applicable to all types of substrates. For instance, due to: (a) the
fact that the ole®nic precursor of 2 would be a monosubstituted (and therefore electron poor)
ole®n; and (b) the additional inductive electron-withdrawing power of the pyridine nitrogen
atom, it can be predicted that 2-vinylpyridine 1, the prochiral precursor of epoxide 2, should be a
bad substrate for such (electrophilic) catalysts. This was con®rmed by the results we have
obtained using these various chemical methodologies in order to prepare enantiomerically
enriched epoxide 2 (or its corresponding vicinal diol 3). Thus, in our hands: oxidation of 1 using
the commercially available (salen)Mn-catalyst led to a very poor ee (9%) of the epoxide 2,15 the
ADmix b catalysed dihydroxylation of 1 a€orded diol 3 which showed an ee of only 79%16 and
the resolution of racemic 2 using the recently discovered HKR (salen)Co-catalyst was very slow
and led, after 70 h, to epoxide 2 with an ee as low as 4%.17
2.2. Screening for appropriate epoxide hydrolase activity
Epoxide 2 was synthesised following the procedure described by Hanzlik et al.18 In order to
select the most suitable biocatalyst to carry out the resolution of 2, we screened 14 enzyme
extracts from our present collection of EH containing enzyme preparations, i.e. nine originating
from fungal strains and ®ve enzymes from various origins (overexpressed using a baculovirus
system). Analytical scale experiments were pursued in order to determine their overall reaction
pro®le, including their enantio- and regioselectivity. The results obtained throughout this study
showed that the EH from A. niger (AnEH) was the best choice to achieve the preparative scale
resolution of 2.{ We thus performed a more accurate determination of the parameters of this
biocatalysed HKR process by using a crude enzyme extract obtained from an overexpressing
fungal strain.x,19 21
{
These detailed results will be described in a separate paper.
This recombinant strain has been constructed by Professor J. Visser (Wageningen Agricultural University, Section
x
Molecular Genetics of Industrial Microorganisms, Wageningen, 6703H, The Netherlands) by cloning the epoxide
hydrolase gene from the wild strain Aspergillus niger LCP 521 into an appropriate Aspergillus host (unpublished
results, for the corresponding patent applications, see Ref. 19). The enzyme has been previously puri®ed in our
laboratory (see Ref. 20) and further on sequenced and cloned in an Escherichia coli host (see Ref. 22). The X-ray crystal
structure of this enzyme has also been published recently (see Ref. 21).

Y. Genzel et al. / Tetrahedron: Asymmetry 11 (2000) 3041±3044
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2.3. Determination of the enantio- and regioselectivity
Interestingly, the AnEH exhibited a rather high enantioselectivity towards 2, hydrolysing
preferentially the (R)-enantiomer and thus leading to the recovery of the slow reacting antipode
(S)-2. The ee of this epoxide, as well as of the formed diol 3, was determined using chiral GC
analysis (using a chiral capillary column: Chirasil Dex CB; Chrompack). To allow this determination,
3 was ®rst cyclised back into epoxide 2 (without change of its absolute con®guration) following
the procedure previously described by Golding et al.23 The E value of this biohydrolysis was
calculated, on the basis of both the substrate ee and the percentage of conversion, to be about 80
(at a 5 mM substrate concentration).24 The absolute con®guration of 2 and of the corresponding
diol 3 were assigned on the basis of their speci®c rotation sign by comparison with previously
described data.25,26 Since determination of the regioselectivity is also an important factor in this
type of reactions, we have also determined the a(S)/b(S) and a(R)/b(R) ratios by studying,
separately, the stereochemical outcome of the enzymatic hydrolysis of enantiopure (S)-2 and of
racemic 2, as described previously.27 Both these values were about 3:97, indicating that the
enzymatic attack occurred almost exclusively at the b-carbon atom for each enantiomer. As a
result, biohydrolysis essentially led to retention of con®guration for the formed diol 3, an
observation consistent with the fact that the ee of 3 was decreasing throughout the conversion of
the racemic mixture down to 0% at total conversion.
A semi-preparative scale experiment was conducted using a 5 mM concentration of racemic 2
(0.61 g/L). Thus, 610 mg of 2 in 1 L pure water were treated with 990 mg of a crude enzyme
extract of EH. After 97 min, the medium was extracted with chloroform. After normal work-up,
this led to 260 mg (43% yield) of (S)-2 whose ee was shown to be higher than 99%, and to 300 mg
of (R)-3 diol (43% yield) with 62% ee.
3. Conclusion
The biocatalytic hydrolytic kinetic resolution of 2-pyridyloxirane 2 was explored using 14
di€erent epoxide hydrolases. We observed that the EH of the fungus Aspergillus niger GBCF 79
was a very ecient catalyst to achieve the resolution of this substrate, and (S)-2 was obtained in
enantiopure form (ee >99%) and a 43% yield. It has to be emphasised that neither of the two
enantiomers of this epoxide are accessible in good enantiomeric purity using the presently
available conventional chemical procedures based on the use of heavy metal catalysts. Thus, this
very simple `green chemistry' procedure, which uses water as a reactant and solvent, currently
appears to be the best and most direct way to prepare (S)-2 in enantiopure form. Work is in
progress in our laboratory in order to perform the scaling-up of this procedure, as well as to
explore its applicability for other pyridine ring containing epoxides.
Acknowledgements
We are extremely grateful to the following colleagues, who helped us to achieve this work:
Mrs. C. Guitton and Mr. D. Faucher (Rhone-Poulenc Company, Lyon, France) for having
constructed the appropriate true probes for the Aspergillus niger epoxide hydrolase, allowing
further molecular biology work; Professor M. Arand (Institute of Toxicology, Mainz, Germany)

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Y. Genzel et al. / Tetrahedron: Asymmetry 11 (2000) 3041±3044
for having achieved the cloning of the enzyme; Professor J. Visser (University of Wageningen,
Holland) for having performed the overexpression of the wild type A. niger enzyme in an
appropriate Aspergillus host. The DSM Company is greatly acknowledged for ®nancial support
of this work (including a stipend to Y.G.).
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