Asymmetric anti-Prelog reduction of ketones catalysed by Paracoccus pantotrophus and Comamonas sp. cells via hydrogen transfer

Article abstract of DOI:10.1016/j.tetasy.2008.08.005

A broad range of ketones including methyl-aryl-, methyl-alkyl-, cyclic and sterically hindered ketones were reduced to the corresponding anti-Prelog alcohols with moderate to excellent stereoselectivities by employing lyophilised cells of Paracoccus pantotrophus DSM 11072 and Comamonas sp. DSM 15091 via hydrogen transfer. The reduction equivalents were provided using 2-propanol as a hydride donor. For instance, acetophenone was reduced to the corresponding (R)-enantiomer with >99% ee.

Full text of DOI:10.1016/j.tetasy.2008.08.005

Tetrahedron: Asymmetry 19 (2008) 1954–1958
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Asymmetric anti-Prelog reduction of ketones catalysed by
Paracoccus pantotrophus and Comamonas sp. cells via hydrogen transfer
a
b
b
a
b
Iván Lavandera , Brigitte Höller , Alexander Kern , Ursula Ellmer , Anton Glieder ,
c
a,
*
Stefaan de Wildeman , Wolfgang Kroutil
Research Centre Applied Biocatalysis c/o Department of Chemistry, Organic and Bioorganic Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
Research Centre Applied Biocatalysis c/o Institute for Molecular Biotechnology, Graz University of Technology, Petersgasse 14/2, 8010 Graz, Austria
DSM Pharmaceutical Products, PO Box 18, 6160, MD Geleen, The Netherlands
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
A broad range of ketones including methyl-aryl-, methyl-alkyl-, cyclic and sterically hindered ketones
were reduced to the corresponding anti-Prelog alcohols with moderate to excellent stereoselectivities
by employing lyophilised cells of Paracoccus pantotrophus DSM 11072 and Comamonas sp. DSM 15091
via hydrogen transfer. The reduction equivalents were provided using 2-propanol as a hydride donor.
For instance, acetophenone was reduced to the corresponding (R)-enantiomer with >99% ee.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 2 July 2008
Accepted 5 August 2008
Available online 27 August 2008
1
. Introduction
Biocatalysis has recently gained increasing importance for the
donor for the preparation of ‘anti-Prelog’ alcohols at elevated
substrate concentrations.
preparation of enantiopure compounds required for pharmaceuti-
cal, agrochemical and food industry.1 In this context, enantiopure
alcohols are very frequently needed as chiral synthons. Methods
for their preparation, such as kinetic resolutions or dynamic kinetic
O
Biocatalyst
OH
*
Buffer, 30°C
1
2
1
2
R
R
R
R
2
resolutions, have been widely employed leading to the acylated
1a - 22a
8.5 g L
OH
O
1b - 22b
-
1
alcohol. Reduction of ketones to the corresponding enantiopure
3
alcohols has been achieved by chemical (metal) catalysts or bio-
catalytic methods in order to obtain quantitative yields. Alcohol
dehydrogenases (ADHs) are found in many microbial strains cata-
lysing the stereoselective reduction of the carbonyl group leading
O
O
O
O
1a
2a
3a
4a
4
O
to the corresponding (S)-or (R)-alcohol. However, most of these
N
biocatalysts follow ‘Prelog’s rule’,5 thus the (S)-alcohol is usually
obtained assuming that the smaller substituent of the ketone has
the lower CIP priority. Only a few ‘anti-Prelog’-(R)-specific biocata-
lysts have been described.6
O
O
O
O
7a
5
a
6a
When using cells as the catalyst, the reducing equivalents are
mostly provided by the metabolic pathways by employing fer-
menting cells and glucose, or a huge excess of cells is needed. Both
cases lead to unwanted side-reactions or competing opposite
O
O
11a
9a
O
a
10a
8
selectivities leading to lower ees, since many enzymes are active
O
under these conditions as well.7 Only a few examples have been
O
O
X
O
O
i
OMe
OEt
Pr
reported, where cells are combined with other reducing agents,
such as 2-propanol (Scheme 1).8 An additional advantage of
employing this hydride donor is that many undesired enzymes
are deactivated in the presence of 2-propanol. Herein, we report
two novel bacterial strains that accept 2-propanol as a hydrogen
Cl
O
O
O
12a, X = OH
13a, X = Cl
14a
15a
16a
O
O
7a, m= 4
8a, m= 5
19a, n = 1; 20a, n = 2
n
21a, n = 3; 22a, n = 4
1
1
m
*
Corresponding author. Tel.: +43 (0)316 380 5350; fax: +43 (0)316 380 9840.
E-mail address: wolfgang.kroutil@uni-graz.at (W. Kroutil).
Scheme 1. Asymmetric reduction of ketones via biocatalytic hydrogen transfer.
0
957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.08.005

I. Lavandera et al. / Tetrahedron: Asymmetry 19 (2008) 1954–1958
1955
2
. Results and discussion
100
By testing more than 50 lyophilised cells from bacteria and
8
6
0
0
yeasts for the reduction of acetophenone 1a (Scheme 1) as a model
substrate at 15% v/v of 2-propanol as a co-substrate, six positive
strains leading to the conversion of >5% were identified (Table 1).
However, only Paracoccus pantotrophus DSM9 11072 and Coma-
monas sp. DSM 15091 showed (R)-stereopreference. According to
40
20
0
1
0
the literature, Yarrowia lipolytica usually displays (R)-stereo-pref-
erence as well, but in our screening several Yarrowia strains
showed only low activity (conv. ꢀ1%). Repetition of the experi-
ments with 1% v/v 2-propanol did not lead to significant
improvements.
6
6.5
7
7.5
8
8.5
9
pH
À1
Figure 1. Reduction of 1a (8.5 g L , t = 24 h) with 2-propanol (15% v/v) and
lyophilised cells of Paracoccus pantotrophus DSM 11072 (Ç) and Comamonas sp.
DSM 15091 (h) at varied pHs.
Table 1
À1
Active strains for the reduction of acetophenone (1a, 8.5 g L ) via hydrogen transfer
Biocatalyst
Cosubstratea
cb (%)
eeb (%)
Arthrobacter sp. DSM 7325
Candida parapsilosis DSM 70125
Paracoccus pantotrophus DSM 11072
Pseudomonas sp. DSM 12877
Sphingomonas sp. DSM 11094
Comamonas sp. DSM 15091
Ralstonia sp. DSM 9750
2-Propanol
2-Propanol
2-Propanol
2-Propanol
2-Propanol
2-Propanol
Ethanol
6
7
16
6
5
67
8
11
8
>99 (S)
>99 (S)
>99 (R)
>99 (S)
>99 (S)
>99 (R)
>99 (S)
>99 (S)
>99 (S)
1
00
8
6
4
2
0
0
0
0
0
Pseudomonas sp. DSM 12877
Sphingomonas sp. DSM 11094
Ethanol
Ethanol
a
1
5% v/v for 2-propanol; 10% v/v for ethanol.
b
Measured by GC on a chiral stationary phase.
It was noticed that the stereoselectivity of P. pantotrophus de-
0
5
10
% PrOH (v v )
15
20
pended on the medium used for cell cultivation. For instance, lower
stereoselectivities (90% ee) were obtained in cases where the cells
were grown on DSMZ medium 1 or Luria broth, instead of the com-
i
-1
Figure 2. Reduction of 1a (t = 24 h) at varied 2-propanol concentration in Tris
buffer, pH 7.5, employing Paracoccus pantotrophus DSM 11072 (Ç) and Comamonas
sp. DSM 15091 (h).
plex medium used here which led to >99% ee.
As an alternative hydrogen source, ethanol11 was tested with
the same bacterial strains and yeasts. Using 10% v/v ethanol in-
stead of 2-propanol as a co-substrate, we identified three active
strains showing conversion higher than 5% (Table 1); all of them
transformed acetophenone to the enantiopure (S)-enantiomer (ee
experiments, various methyl ketones were reduced (Table 2): aro-
matic ketone 1a and heteroaromatic ketone 2a, aliphatic ketones
>99%). However, the ‘anti-Prelog’ strains identified with 2-propa-
4
a–6a and a diketone such as 2,5-hexanedione 3a were reduced
nol showed no significant activity with ethanol.
with both strains, yielding the corresponding (R)-alcohols with
good to excellent conversions (69–94%) and stereoselectivities
In a concluding screening, 31 fungi were tested at 15% v/v and
1
% v/v of 2-propanol concentration. Only one active fungus namely
(
58–99%) after 24 h. Comamonas sp. led to higher conversions in
Mortierella alpina ATCC 8978 was identified, which showed a con-
version of just 1%.
most cases, however, P. pantotrophus displayed better stereoselec-
tivities. Diketone 3a was reduced to the corresponding (R,R)-diol,
Therefore, we decided to continue the study with P. pantotro-
phus DSM 11072 and Comamonas sp. DSM 15091 strains, since they
displayed a promising anti-Prelog activity with acetophenone. In
the next step, the pH of the Tris buffer (50 mM) was optimised.
For that purpose, lyophilised cells of both strains were rehydrated
in 50 mM Tris buffer at varied pHs, and assayed with 1a and 2-pro-
panol (Fig. 1). While P. pantotrophus showed an optimum at pH 7.0,
Comamonas sp. showed the best activity between pH 7.0 and 8.0.
The co-substrate 2-propanol fulfils two functions: on the one
hand, it provides the hydrogen equivalents, while on the other
hand, it improves the solubility of the substrate in the aqueous
phase; nevertheless it can also lead to deactivation of the biocata-
lyst. To obtain a rough idea about its effect, the concentration of
Table 2
Reduction of methyl ketones 1a–6a employing lyophilised cells of P. pantotrophus or
Comamonas sp. and 2-propanol (t = 24 h)
Biocatalyst
Substrate
ca (%)
eea (%)
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
1a
2a
3a
4a
5a
6a
1a
2a
3a
4a
5a
6a
69
93
60
>99 (R)
>99 (R)
66 (R,R)c
91 (R)
84 (R)
94 (R)
b
82
79
82
77
94
98 (R)
97 (R)
2
-propanol was varied and the conversion was determined
d
94 (R,R)c
58 (R)
92
(Fig. 2). P. pantotrophus showed the highest conversion at 5% v/v
94
92
92
and Comamonas sp. at 10% v/v, although the latter strain was active
over a broad concentration range of 2-propanol. Obviously, 2-pro-
panol was required for the biotransformations, since without it
almost no conversion was observed.
After optimisation of the reaction parameters, various types of
substrates were transformed with both strains. In the first set of
58 (R)
87 (R)
Comamonas sp.
a
Measured by GC on a chiral stationary phase.
b
c
In addition, 11% of hydroxyketone intermediate 3c was obtained.
No trace of any other diastereomer could be detected, thus >99% de.
No hydroxyketone was detected.
d

1
956
I. Lavandera et al. / Tetrahedron: Asymmetry 19 (2008) 1954–1958
and the corresponding intermediate hydroxyketone12 was only
detectable for P. pantotrophus (11% at 24 h).
13b with good stereoselectivity (94% P. pantotrophus). Derivatives
of 13b are frequently employed as intermediates in the synthesis
of pharmaceuticals, for example, in the treatment of obesity and
In another set of substrates, cyclic ketones 7a–11a were tested
1
3
(
Table 3). It can be concluded that the activity depended on the po-
sition of the carbonyl moiety within the ring. Thus, if the ketone
was at position 1 or of the cyclic system (7a, 9a and 11a), reduc-
depression. Another a-chloro ketone 14a was reduced to the
anti-Prelog product with excellent ee (>99%) and conversion
(99%, P. paracoccus). For Comamonas sp., the biotransformation of
14a was stopped after 4 h since longer reaction times led to the
a
tions proceeded slowly, if at all, leading to low conversion even
after 24 h; however, if the carbonyl group was at position 2 or b
degradation of the alcohol catalysed by the organism. Two
esters 15a–16a were tested, since -hydroxyesters are valuable
for instance, alcohol 16b is an important inter-
a-keto-
(
8a, 10a), conversions were much better and in the case of b-tetra-
lone 10a, the corresponding (R)-10b alcohol was obtained with
9% conversion and excellent enantiomeric excess (98%), employ-
ing Comamonas sp. The low conversion for the ketones at the
a
4
f,g,14
compounds;
mediate for ACE inhibitors. Both ketones were converted with
moderate to good selectivity to the corresponding (S)- -hydroxy-
1
5
9
a
-
a
position could be attributed to steric hindrance compared to the
b-position.
esters (72% for 15b and Comamonas, 85% for 16b with P. pantotro-
phus). In the case of 15a with Comamonas sp., an additional 7% of
the corresponding isopropyl ketoester was formed, probably due to
transesterification caused by the presence of lipase(s) or ester-
ase(s) in the lyophilised cell preparation. The three ethyl ketones
tested, 17a–19a, were converted with good activity and excellent
stereoselectivity to afford enantiomerically pure (R)-17b–19b.
Finally, a set of bulky 1-phenyl-1-alkanones 20a–22a with dif-
ferent alkyl chain lengths were tested to study the influence of
the length of the alkyl chain on the conversion (Fig. 3). For P. pan-
totrophus, a clear decrease of activity was observed when methyl
Table 3
Reduction of cyclic ketones 7a–11a employing lyophilised cells of P. pantotrophus or
Comamonas sp. and 2-propanol (t = 24 h)
ca (%)
eea (%)
Biocatalyst
Substrate
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
7a
8a
9a
10a
11a
7a
8a
9a
0
13
0
63
3
20
61
8
n.d.
—
n.d.
92 (R)
n.d.
n.d.
—
n. d.
98 (R)
n.d.
1
a was exchanged with an ethyl chain 19a, finding no conversion
for substrates possessing a larger alkyl chain 20a–22a. In contrast,
this effect was less pronounced for Comamonas sp., thus substrates
21a and 22a were accepted and reduced to the corresponding (R)-
alcohols. Additionally, a change in stereoselectivity was observed
too. For acetophenone 1a, good conversion and ee were achieved
(Table 2), for propiophenone 19a moderate conversion but good
selectivity was obtained (Table 4), while for bulkier ketones low
conversions and moderate ees were measured (Table 4). This could
be explained by the presence of at least two different ADHs with a
different substrate pattern and stereopreference. Thus, (at least)
one ADH which could accept ‘small-bulky’ ketones with good
anti-Prelog preference could reduce efficiently substrates 1a and
10a
11a
99
3
n.d. not determined due to low conversion.
a
Measured by GC on a chiral stationary phase.
Continuing to study the substrate spectrum of these anti-Pre-
log strains, we tested various ketones with two bulky substituents
(
‘bulky–bulky’ ketones) (Table 4). The acetophenone derivatives
2a and 13a were compared: one with a hydroxy- and one with
a chloro group at the -position. -Chloro acetophenone 13a
1
x
x
1
9a, while another ADH(s) with better affinity for ‘bulky-bulky’
reacted much faster than 12a, to give the corresponding (S)-alcohol
substrates, but worse or even reverse stereoselectivity transforms
substrates 20a–22a.
Table 4
Reduction of ‘bulky–bulky’ ketones 12a–22a employing lyophilised cells of P.
pantotrophus or Comamonas sp. and 2-propanol (t = 24 h)
Biocatalyst
Substrate
ca (%)
eea (%)
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Paracoccus pantotrophus
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
Comamonas sp.
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
12a
13a
14a
15a
16a
17a
18a
19a
20a
21a
22a
4
n.d.
96
99
41
98
71
70
17
1
0
0
29
>99
99
80
98
84
84
53
13
17
19
94 (S)b
>99 (S)
b
b
29 (S)
85 (S)b
>99 (R)
>99 (R)
>99 (R)
n.d.
n.d.
n.d.
Figure 3. Effect of the alkyl chain length on the conversion of 1-phenyl-1-
alkanones (1a, 19a–22a, t = 24 h) in Tris buffer (50 mM) and 2-propanol for:
Paracoccus pantotrophus DSM 11072 (Ç); Comamonas sp. DSM 15091 (h).
n.d.
b
71 (S)
c
93 (S)b
b,d
72 (S)
31 (S)b
>99 (R)
>99 (R)
89 (R)
47 (R)
54 (R)
53 (R)
3
. Conclusions
We have identified two bacterial strains that reduce ketones to
the corresponding anti-Prelog alcohols via biocatalytic hydrogen
transfer using 2-propanol as a hydrogen source, resembling a
Meerwein–Ponndorf–Verley reaction. P. pantotrophus DSM 11072
and Comamonas sp. DSM 15091 usually showed very good activi-
ties and selectivities towards a broad spectrum of ‘small–bulky’,
cyclic and ‘bulky–bulky’ ketones. In general, the first strain showed
a better stereoselectivity than the second one. Purification of the
Comamonas sp.
Comamonas sp.
n.d. not determined.
a
Measured by GC or HPLC on a chiral stationary phase.
Switch in CIP priority.
Conversion at 4 h.
b
c
d
In addition, 7% of the corresponding 2-propyl ketoester was formed.

I. Lavandera et al. / Tetrahedron: Asymmetry 19 (2008) 1954–1958
1957
enzymes responsible for these biotransformations is ongoing in
our laboratories.
1a–22a (6 lL or 6 mg) were added. Reactions were shaken at
30 °C and 120 rpm for 24 h, and stopped by extraction with ethyl
acetate (2 Â 0.5 mL). The organic layer was separated from the
2 4
cells by centrifugation (2 min, 13,000 rpm) and dried over Na SO .
4
4
. Experimental
.1. General
Conversions and enantiomeric excesses of the corresponding alco-
hols were determined by chiral GC or HPLC analysis.
Acknowledgements
Ketones 1a–22a, alcohol 8b, racemic alcohols 1b, 4b–7b, 9b,
0b, 12b and 15b–19b, (R)- and (S)-13b, (R)- and (S)-20b, diol 3b
and hydroxyketone 3c were commercially available, either from
Sigma–Aldrich–Fluka (Vienna, Austria) or from Lancaster (Frank-
furt am Main, Germany). Racemic compounds 2b, 11b, 14b, 21b
1
Financial support by DSM Linz and Geleen, FFG and Province of
Styria is gratefully acknowledged.
and 22b were synthesised by conventional reduction from the cor-
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4
.2.2. Optimisation of pH and 2-propanol concentration
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4
.2.3. General method for the biocatalytic reduction of ketones
employing lyophilised cells of P. pantotrophus DSM 11072
Lyophilised cells of P. pantotrophus (20 mg) stored at 4 °C were
rehydrated in Tris/HCl buffer (600
(
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3
0 °C and 120 rpm on a rotary shaker in an Eppendorf vial (1.5 mL).
Then, 2-propanol (32
lL, 5% v/v) and the corresponding ketones
lL or 6 mg) were added. Reactions were shaken at
1
a–22a (6
3
0 °C and 120 rpm for 24 h, and stopped by extraction with ethyl
7
8
.
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2
4
Conversions and enantiomeric excesses of the corresponding alco-
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.2.4. General method for the biocatalytic reduction of ketones
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