Biocatalytic deuterium- and hydrogen-transfer using over-expressed ADH-'A': Enhanced stereoselectivity and 2H-labeled chiral alcohols

Article abstract of DOI:10.1039/b602487d

Employing the over-expressed highly organic solvent tolerant alcohol dehydrogenase ADH-'A' from Rhodococcus ruber DSM 44541, versatile building blocks, which were not accessible by the wild type catalyst, were obtained in > 99% e.e.; furthermore, employing d₈-2-propanol as deuterium source, stereoselective biocatalytic deuterium transfer was made feasible to furnish enantiopure deuterium labeled sec-alcohols on a preparative scale employing a single enzyme. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b602487d

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COMMUNICATION
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Biocatalytic deuterium- and hydrogen-transfer using over-expressed
2
ADH-‘A’: enhanced stereoselectivity and H-labeled chiral alcohols{
Klaus Edegger,ab Christian C. Gruber,a Tina M. Poessl,a Sabine R. Wallner,ab Iva´n Lavandera,ab
Kurt Faber,ab Frank Niehaus,c Juergen Eck,c Reinhold Oehrlein,d Andreas Hafnerd and Wolfgang Kroutil*ab
Received (in Cambridge, UK) 20th February 2006, Accepted 30th March 2006
First published as an Advance Article on the web 11th April 2006
DOI: 10.1039/b602487d
for this substrate in the WT. Interestingly, the regioisomer
a-tetralone 2a was neither accepted by the WT nor by E. coli/
ADH-‘A’. Encouraged by the selectivity enhancement for
substrate 1a, other previously moderate substrates like 3a–6a were
reduced, and again the corresponding alcohols (R)-3b, (R)-4b,
Employing the over-expressed highly organic solvent tolerant
alcohol dehydrogenase ADH-‘A’ from Rhodococcus ruber
DSM 44541, versatile building blocks, which were not
accessible by the wild type catalyst, were obtained in > 99%
e.e.; furthermore, employing d8-2-propanol as deuterium
source, stereoselective biocatalytic deuterium transfer was made
feasible to furnish enantiopure deuterium labeled sec-alcohols
on a preparative scale employing a single enzyme.
Stereoselective reduction of ketones is one of the most widely
employed methods for the synthesis of non-racemic chiral alcohols
in organic synthesis.1 In this context, biocatalytic approaches for
ketone reduction employing alcohol dehydrogenases (ADHs) have
very recently gained increasing importance.2 However, most of the
identified alcohol dehydrogenases need a further ADH or a
sophisticated set-up for co-factor recycling.2d,3 Only very few
ADHs allow the most simple approach, namely the simultaneous
recycling of the co-factor and the reduction of the desired ketone
by a single enzyme in the coupled substrate approach (Scheme 1).4
Recently, we have successfully employed whole cells of
Rhodococcus ruber DSM5 44541 in this approach6 and have
demonstrated that a single alcohol dehydrogenase named ADH-
‘A’ is catalyzing both reactions in a hydrogen transfer like fashion.
Partially purified ADH-‘A’ showed impressive operational stability
in the presence of high concentrations of organic compounds, for
instance it is active in 2-propanol up to 80% v/v, thereby allowing a
shift in the equilibrium to the desired product and simultaneously
solubilizing the substrate in the aqueous phase.7
Scheme 1 Biocatalytic hydrogen transfer using E. coli/ADH-‘A’.
Table 1 Comparison of selectivity and conversion for over-expressed
ADH-‘A’ in E. coli with the wild type catalyst, i.e. whole cells of
Rhodococcus ruber DSM 44541
Employing lyophilized cells of E. coli containing the over-
expressed ADH-‘A’, selected substrates, which were initially
transformed by the wild type-catalyst (WT) with low stereo-
selectivity, were reinvestigated. For instance, employing the WT-
catalyst reduction of b-tetralone 1a furnished (S)-b-tetralol 1b in
only 83% e.e. To our delight, when employing E. coli/ADH-‘A’
enantiopure (S)-b-tetralol (S)-1b with e.e. > 99% was found8
(Table 1, Scheme 1), which indicates that further ADHs competed
R. ruber DSM 44541 WT
E. coli/ADH-‘A’
Substr. t/h Conv. (%) e.e. (%)
t/h Conv.e (%) e.e. (%)
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
11a
12a
13a
a
22
22
24
24
22
24
48
48
24
20a
,1a
21b
82b
78a
8
83 (S) 19
89
, 1
. 99
. 99
76
. 99 (S)
n.d.
n.d.
19
2
2
1
2
43 (R)
73 (R)
97 (S)
85 (S)
. 99 (R)f
. 99 (R)f
. 99 (S)
. 99 (S)
. 99 (R)f
. 99 (R)f
. 99 (R)f
. 99 (R)f
. 99 (S)
. 99 (S)
. 99 (R)f
aDepartment of Chemistry, Organic and Bioorganic Chemistry,
University of Graz, Heinrichstrasse 28, A-8010, Graz, Austria.
E-mail: wolfgang.kroutil@uni-graz.at; Fax: +43(0)316 380 9840;
Tel: +43(0)316 380 5350
81
7
89 (R) 48 . 99
n.d. 48 . 99
2
99b
99 (R)
. 99 (R)
. 99 (S)
. 99 (S)
n.d.
2
2
1
2
2
. 99
. 99
66
98
. 99
e
24 .99b
bResearch Centre Applied Biocatalysis, Petersgasse 14, A-8010, Graz,
Austria
96
48
24
67c
97d
cBRAIN AG, Darmstaedter Strasse 34, D-64673, Zwingenberg,
Germany
,1b
dCIBA SC Inc., K-420.2.20, CH-4002, Basel, Switzerland
{ Electronic supplementary information (ESI) available: synthesis of 7–8,
protocol of biotransformation, NMR of 14c–16c, [a]D20, analytics and
preparation of the biocatalyst. See DOI: 10.1039/b602487d
b
c
d
Ref. 13a. Ref. 13c. Ref. 6. Ref. 13d. Substrate concentration
y 15 g l21, 16% v v21 2-propanol. Switch in CIP priority. n.d.:
not determined, due to low conversion.
f
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(S)-5b and (S)-6b were obtained with absolute stereoselectivity (>
99% e.e.) using E. coli/ADH-‘A’ (Table 1). The most significant
enhancement (43% e.e. for the WT to > 99% e.e. with E. coli/
ADH-‘A’) was observed for 2-chloro-1-phenylethanol 3b.
Derivatives of 3b are quite frequently employed as intermediates
in the synthesis of pharmaceuticals, e.g. for the treatment of
obesity and depression,9 or for the synthesis of (R)-Salmeterol,
employed to treat asthma and chronic bronchitis.10
Encouraged by the higher activity and higher enantioselectivity
for more sterically demanding ketones, we tested the reduction of
sterically demanding a-azido ketones, e.g. 2-azido-1-phenyletha-
none 7a and its para-hydroxy derivative 8a. The corresponding
azido alcohol (R)-7b is a building block for selective b2-
adrenoceptor agonists like KUR-124611 or denopamine, a b1-
receptor agonist effective in the treatment of congestive heart
failure.12 Indeed both ketones 7a–8a could be stereoselectively
reduced to the corresponding enantiopure (R)-alcohols with > 99%
e.e. at complete conversion.
All substrates recently investigated13 which were converted by
the wild type catalyst Rhodococcus ruber DSM 44541 (WT) were
transformed by lyophilized cells of E. coli/ADH-‘A’ with excellent
e.e. > 99%, too, however now at significantly reduced reaction
times. For instance, reaction times of one to two hours were
required instead of 24 hours or more for substrates 9a–12a
(Table 1). Furthermore, dione 12a was reduced regioselectively at
the (v21)-position to the corresponding hydroxy ketone, (S)-2-
hydroxy-4-octanone (S)-12b, while the oxo-moiety at carbon C-4
remained untouched.
Scheme 2 Preparation of deuterium labeled enantiopure alcohols via
biocatalytic deuterium transfer.
mg lyophilized cells. The host E. coli Tuner2 (DE3) gave the best
results, reaching under optimized reaction conditions, 2.0 U mg21
cell dry weight for the coupled reduction of acetophenone and
recycling of the cofactor. For comparison, employing wild type
cells of R. ruber just 0.01–0.03 U mg21 were obtained, which
corresponds to an improvement up to 200-fold. It has to be
emphasized that the increase of activity, as for instance observed
for the shorter reaction times, resulted from the higher expression
level in E. coli and was not due to an improved enzyme.
In summary, we could access for the first time enantiopure
secondary alcohols deuterium labeled on the chiral center by
biocatalytic deuterium transfer from d8-2-propanol using a single
enzyme. Lyophilized E. coli cells containing the over-expressed
ADH-‘A’ were shown to be an excellent catalyst for biocatalytic
deuterium and hydrogen transfer, allowing the synthesis of
versatile building blocks which were not accessible by the wild
type catalyst.
The (R)-alcohol (R)-13b is an important precursor for
adrenergic b-blockers.14 Unfortunately, the corresponding ketone
13a was not reduced by the WT-catalyst. Surprisingly, E. coli/
ADH-‘A’ did display excellent activity and selectivity forming (R)-
13b in > 99% e.e. The reason for this discrepancy requires further
investigation. Overall, employing E. coli/ADH-‘A’ a broad variety
of pharmaceuticals and related intermediates became accessible
in optically pure form, which could not be obtained with the
WT-catalyst.
This research was performed within the Spezialforschungs-
bereich ‘Biokatalyse’ and the Research Centre Applied
Biocatalysis. Financial support by CIBA SC (Basel), TIG, FFG,
the Province of Styria and the City of Graz is gratefully
acknowledged.
To analyze drug metabolism or to elucidate reaction mechan-
isms, isotopic deuterium labeling of (bioactive) compounds is a
common strategy. Biocatalytic recycling of the labeled cofactor
NAD(P)D has already been successfully demonstrated with
d2-formate DCOOD and 1,1-d2-ethanol for the preparation of
primary alcohols by two enzymes.15 To the best of our knowledge,
a biocatalytic deuterium transfer approach has never been
employed before for the synthesis of secondary alcohols deuterium
labeled at the chiral center16 by a single enzyme. We envisaged that
d8-2-propanol might serve as deuterium donor in an analogous
fashion to 2-propanol (Scheme 2). Indeed, enantiopure (e.e. >
99%) deuterium labeled (S)-alcohols 14c–16c were prepared
starting from 300 mg of the corresponding ketones 14a–16a on a
preparative scale in 70 to 89% isolated yield. When comparing the
reaction rate of the reduction of acetophenone 14a with labeled
and with unlabeled 2-propanol, an apparent kinetic isotope effect17
of 3.3 was measured, as expected for a primary isotope effect.
For the identification of a suitable E. coli/ADH-‘A’ catalyst,
which was employed in the experiments described above as a
lyophilized powder, various E. coli hosts were tested employing a
pET-vector (pET22b+) and the cultivation conditions were
thoroughly optimized to reach the highest apparent activity per
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8 General protocol for biocatalytic hydrogen transfer: Lyophilized cells of
E. coli TunerTM (DE3)/pET22b+-ADH-‘A’ (in general 2–5 mg, 15 mg
for substrates 7a and 8a), stored at 4 uC, were rehydrated in Tris-HCl
buffer (0.5 ml, 50 mM, pH 7.5, 1 mM NADH) for 60 min at 30 uC and
130 rpm on a rotary shaker in an Eppendorf vial (1.5 ml). Substrate
(10 ml) and 2-propanol (100 ml) were added followed by shaking the
reaction mixture at 30 uC and 130 rpm for the appropriate time
(see Table 1). The reaction was stopped by extraction with ethyl acetate
(2 6 0.5 ml), the organic layer was separated from the cells by
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2404 | Chem. Commun., 2006, 2402–2404
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