Stereoselective bioreduction of bulky-bulky ketones by a novel ADH from Ralstonia sp.

Article abstract of DOI:10.1021/jo800849d

(Figure Presented) Ketones with two bulky substituents, named bulky-bulky ketones, as well as less sterically demanding ketones were successfully reduced to the corresponding optically highly enriched alcohols using a novel identified recombinant short-chain alcohol dehydrogenase RasADH from Ralstonia sp. DSM 6428 overexpressed in E. coli.

Full text of DOI:10.1021/jo800849d

Stereoselective Bioreduction of Bulky-Bulky
Ketones by a Novel ADH from Ralstonia sp.
at least one small substituent, thus a sterically nondemanding
group like methyl, ethyl, azido-, cyano-, or chloromethyl. Very
recently, purified or overexpressed ADHs have been applied
1
†
‡
§
for the reduction of bulky-bulky ketonessnoncyclic sterically
Iv a´ n Lavandera, Alexander Kern, Bianca Ferreira-Silva,
2
‡
|
impeded ketones swhereby mainly activated bulky-bulky ke-
Anton Glieder, Stefaan de Wildeman, and
3
,
§
tones bearing R- or ꢀ-ketoesters have been transformed.
Wolfgang Kroutil*
However, the stereoselective reduction of bulky aryl alkyl
ketones has been scarcely studied. For instance, Zhu and Hua
Research Centre Applied Biocatalysis c/o Department of
4
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
4/2, 8010 Graz, Austria, Department of Chemistry, Organic
and Bioorganic Chemistry, UniVersity of Graz,
Heinrichstrasse 28, 8010 Graz, Austria, and DSM
Pharmaceutical Products, P.O. Box 18,
described a NADPH-dependent carbonyl reductase from Sporobo-
lomyces salmonicolor able to reduce aryl long-chain alkyl or
5
cyclopropyl derivatives. Besides the limited knowledge about
ADHs accepting bulky-bulky ketones another limitation is the
low ketone concentration generally applied and the commonly
moderate stereoselectivities obtained for these subset of substrates.
In a screening, we identified the wild-type strain Ralstonia
sp. DSM 6428 capable of transforming sterically hindered
ketones. Here, we report the identification of the involved
enzyme and a study of its substrate spectrum using recombinant
enzyme overexpressed in Escherichia coli.
1
6
160, MD Geleen, The Netherlands
wolfgang.kroutil@uni-graz.at
ReceiVed April 18, 2008
For the identification of the corresponding ADH a gene library
6
was constructed and screened on filter paper for increased
7
NADPH fluorescence during oxidation of 1-phenyl-1-propanol
+
in the presence of NADP . A putative short-chain dehydroge-
nase gene was identified (arbitrarily called RasADH), amplified
8
by PCR, and cloned into the plasmid pEamTA. The enzyme
RasADH was subsequently overexpressed in E. coli DH5R
which was applied as lyophilized powder for biocatalytic
transformations (denoted as E. coli/RasADH).
In biocatalytic reductions, the reducing equivalents can be
provided by mainly two approaches: (i) in an “enzyme-coupled”
approach a second (and preferably irreversible) enzymatic
reaction is employed additionally to the alcohol dehydrogenase
Ketones with two bulky substituents, named bulky-bulky
ketones, as well as less sterically demanding ketones were
successfully reduced to the corresponding optically highly
enriched alcohols using a novel identified recombinant short-
chain alcohol dehydrogenase RasADH from Ralstonia sp.
DSM 6428 overexpressed in E. coli.
1
,9,10
to shift the equilibrium to the desired product;
(ii) in the
“substrate-coupled” or “biocatalytic hydrogen transfer” approach
a single enzyme catalyzes the reduction of the ketone as well
1
,9,11
as the recycling of the cofactor simultaneously.
Until now,
only two ADHs transforming bulky-bulky ketones have been
3
g,l
described to work via hydrogen transfer.
Testing E. coli/
RasADH we were very pleased to notice that RasADH could
Stereoselective reduction of ketones using alcohol dehydro-
genases (ADHs) has become an important method for industrial
preparation of optical pure alcohols. Unfortunately, most of
the biocatalytically applicable ADHs show a rather narrow
substrate pattern: preferentially ketones are reduced which bear
(
2) Bulky-bulky ketones possess two substituents which are larger than ethyl,
1
azido-, cyano-, or halomethyl groups. ADHs capable of reducing bulky-bulky
ketones are not that commonly found in Nature, in contrast to those which reduce
ketones with at least one small group (ethyl, azido-, cyano-, or halomethyl
groups), the small-bulky ketones.
(
3) (a) Ema, T.; Okita, N.; Ide, S.; Sakai, T. Org. Biomol. Chem. 2007, 5,
1
175–1176. (b) Yang, Y.; Zhu, D.; Piegat, T. J.; Hua, L. Tetrahedron: Asymmetry
†
Research Centre Applied Biocatalysis, University of Graz.
Institute for Molecular Biotechnology, Graz University of Technology.
Department of Chemistry, Organic and Bioorganic Chemistry, University
2007, 18, 1799–1803. (c) Zhu, D.; Stearns, J. E.; Ramirez, M.; Hua, L.
Tetrahedron 2006, 62, 4535–4539. (d) Zhu, D.; Mukherjee, C.; Rozzell, J. D.;
Kambourakis, S.; Hua, L. Tetrahedron 2006, 62, 901–905. (e) Zhu, D.; Yang,
Y.; Hua, L. J. Org. Chem. 2006, 71, 4202–4205. (f) Zhu, D.; Yang, Y.; Buynak,
J. D.; Hua, L. Org. Biomol. Chem. 2006, 4, 2690–2695. (g) Inoue, K.; Makino,
Y.; Itoh, N. Tetrahedron: Asymmetry 2005, 16, 2539–2549. (h) Feske, B. D.;
Kaluzna, I. A.; Stewart, J. D. J. Org. Chem. 2005, 70, 9654–9657. (i) Kaluzna,
I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am. Chem. Soc. 2004, 126,
12827–12832. (j) Yamaguchi, H.; Nakajima, N.; Ishihara, K. Biosci. Biotechnol.
Biochem. 2002, 66, 588–597. (k) Ema, T.; Moriya, H.; Kofukuda, T.; Ishida,
T.; Maehara, K.; Utaka, M.; Sakai, T. J. Org. Chem. 2001, 66, 8682–8684. (l)
Bradshaw, C. W.; Fu, H.; Shen, G.-J.; Wong, C.-H. J. Org. Chem. 1992, 57,
1526–1532.
‡
§
of Graz.
|
DSM Pharmaceutical Products.
(
1) (a) de Wildeman, S. M. A.; Sonke, T.; Schoemaker, H. E.; May, O. Acc.
Chem. Res. 2007, 40, 1260–1266. (b) Goldberg, K.; Schroer, K.; L u¨ tz, S.; Liese,
A. Appl. Microbiol. Biotechnol. 2007, 76, 237–248. (c) Kroutil, W.; Mang, H.;
Edegger, K.; Faber, K. Curr. Opin. Chem. Biol. 2004, 8, 120–126. (d) Faber, K.
Biotransformations in Organic Chemistry, 5th ed.; Springer-Verlag: New York,
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New York, 2000; pp 9-866.
(4) Recently, the reduction of diaryl ketones using a set of S. cereVisiae
reductases has been published. For instance, see: (a) Truppo, M. D.; Pollard,
D.; Devine, P. Org. Lett. 2007, 9, 335–338.
(5) Zhu, D.; Hua, L. J. Org. Chem. 2006, 71, 9484–9486.
1
0.1021/jo800849d CCC: $40.75  2008 American Chemical Society
J. Org. Chem. 2008, 73, 6003–6005 6003
Published on Web 07/03/2008

TABLE 1. Comparison of the Recycling System for the E.
TABLE 2. E. coli/RasADH-Catalyzed Reductions of Several
a
a
coli/RasADH-Catalyzed Reduction of Bulky-Bulky Ketones
Small-Bulky Ketones
b
c
substrate
rel rate (%)
ee (%)
6
7
8
9a
10a
1
1
1
a
a
a
38
30
10
26
11
64
98
51
73 (R)
>99 (S)
82 (S)
98 (S)
98 (S)
cofactor recycling systems
2-propanol 10% v v-
1
glucose/GDH
rel rate (%)
b
b
c
substrate
rel rate (%)
ee (%)
1a
2a
3a
>99 (S)
1a
2a
3a
4a
5a
49
8
7
21
1.8
7
>99 (S)
>99 (S)
>99 (S)
96 (S)
64 (S)
d
>99 (R)
37 (S)
d
d
100
40
9
3.4
a
Substrate concentration: 10 g L-1
substrate converted per min per mg E. coli/RasADH and were calculated
from initial progress curves. Measured by chiral GC. Change in CIP
priority.
b
.
100% corresponds to 32.4 nmol
d
c
d
a
Substrate concentration: 10 g L-1
b
.
100% corresponds to 32.4 nmol
substrate converted per min per mg E. coli/RasADH and were calculated
c
from initial progress curves. Measured by chiral HPLC or GC.
d
Change in CIP priority.
clear that glucose/GDH was more efficient, since in all cases
the measured activities were higher with GDH than with
-propanol: for instance, in the case of 4a and 5a a 10-fold
acceleration was observed using glucose/GDH. In contrast to
the activity, the choice of the recycling system did not have
any influence on the optical purity; thus, ee’s were the same
with both recycling systems. The bulky-bulky aryl alkyl ketones
transform bulky-bulky ketones at the expense of 2-propanol,
thus working via hydrogen transfer. Therefore, both cofactor
recycling approaches can be applied for reduction with this
enzyme.
Subsequently, we compared the two recycling systems,
namely (i) the “enzyme-coupled” approach using glucose with
glucose dehydrogenase (GDH) and (ii) the hydrogen transfer
approach employing just 2-propanol as hydrogen source for the
reduction of various bulky-bulky ketones (Table 1). It became
2
1
a-3a were reduced to optically pure (S)-1b-3b (ee >99%).
For substrates 2a and 3a, this is the highest ee ever obtained
via biocatalytic reduction. For substrates 4a and 5a, a switch
in stereopreference was observed compared to 1a-3a. Obvi-
ously, the R-keto esters 4a and 5a bind in a different mode in
the active site of the enzyme; thus, the aromatic moiety is not
on the same side as in the case of substrates 1a-3a.
(
6) Ausubel, F. M.; Brent, R.; Kingston, R. E.; Moore, D. D.; Seidmann,
J. G.; Smith, J. A.; Struhl, K. Short Protocols in Molecular Biology, 5th ed.;
Wiley-VCH: New York, 2003.
(
7) Reisinger, C.; van Assema, F.; Sch u¨ rmann, M.; Hussain, Z.; Remler, P.;
Schwab, H. J. Mol. Catal. B: Enzym. 2006, 39, 149–155.
8) Balzer, D.; Ziegelin, G.; Pansegrau, W.; Kruft, V.; Lanka, E. Nucleic
Acids Res. 1992, 20, 1851–1858.
9) (a) Ruinatscha, R.; H o¨ llrigl, V.; Otto, K.; Schmid, A. AdV. Synth. Catal.
006, 348, 2015–2026. (b) van der Donk, W. A.; Zhao, H. Curr. Opin.
Biotechnol. 2003, 14, 421–426.
10) (a) Kataoka, M.; Kita, K.; Wada, M.; Yasohara, Y.; Hasegawa, J.;
Since the novel catalyst showed excellent stereoselectivity
for bulky-bulky ketones, we wondered how the stereoselectivity
would change for the reduction of small-bulky ketones, ketones
bearing a methyl, ethyl, or chloromethyl substituent. Since
bulky-bulky ketones like 3a were transformed, we expected that
this catalyst should possess a rather spacious active site allowing
small-bulky ketones to bind in various modes and conforma-
tions. Therefore, we anticipated that smaller ketones would be
reduced with lower stereoselectivity. Although this proved to
be true for various substrates like alkyl methyl ketone 8a or
chloromethyl-substituted ketone 13a (Table 2), we were aston-
ished to notice that other small-bulky substrates like acetophe-
none 7a or ω-chloroacetophenone 12a were reduced with perfect
stereoselectivity. As an example, the reduction of 12a was
performed on 60 mg scale, yielding the corresponding optically
pure (R)-alcohol (ee > 99%) at >99% GC conversion with 91%
isolated yield. Ketones bearing a bulky group and an ethyl
moiety (9a-11a) were also reduced with excellent stereose-
lectivity. The absolute configuration obtained for the open-chain
(
(
2
(
Shimizu, S. Appl. Microbiol. Biotechnol. 2003, 62, 437–445. (b) Bommarius,
A. S.; Schwarm, M.; Drauz, K. J. Mol. Catal. B: Enzym. 1998, 5, 1–11. (c)
Bommarius, A. S.; Drauz, K.; Hummel, W.; Kula, M. R.; Wandrey, C.
Biocatalysis 1994, 10, 37–47.
(
11) For some recent examples, see: (a) Musa, M. M.; Ziegelmann-Fjeld,
K. I.; Vieille, C.; Zeikus, J. G.; Phillips, R. S. Angew Chem., Int. Ed. 2007, 46,
091–3094. (b) de Gonzalo, G.; Lavandera, I.; Faber, K.; Kroutil, W. Org. Lett.
007, 9, 2163–2166. (c) Bisel, P.; Walter, L.; Nieger, M.; Hummel, W.; M u¨ ller,
3
2
M. Tetrahedron: Asymmetry 2007, 18, 1142–1144. (d) Hildebrand, F.; L u¨ tz, S.
Tetrahedron: Asymmetry 2006, 17, 3219–3225. (e) Edegger, K.; Gruber, C. C.;
Poessl, T. M.; Wallner, S. R.; Lavandera, I.; Faber, K.; Niehaus, F.; Eck, J.;
Oehrlein, R.; Hafner, A.; Kroutil, W. Chem Commun. 2006, 2402–2404. (f)
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(
1
4
g) Eckstein, M.; Villela, M.; Liese, A.; Kragl, U. Chem Commun. 2004, 1084–
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6
004 J. Org. Chem. Vol. 73, No. 15, 2008

1
2
alcohols 7b-12b correlated in all cases with Prelog’s rule,
General Protocol for the Biocatalytic Reduction of Ketones
Employing Lyophilized Cells of E. coli/RasADH Using GDH.
Lyophilized cells of E. coli/RasADH (20 mg) were rehydrated in
Tris-HCl buffer (600 µL, 50 mM, pH 7.5, 1 mM NADPH) for 30
except for the ꢀ-ketoester 13a. For this type of substrate, again
a switch in stereopreference was observed as already noticed
for 4a and 5a. These results showed that the novel alcohol
dehydrogenase RasADH transforms different types of substrates
with high stereoselectivity including bulky-bulky as well as
small-bulky ketones.
min at 30 °C and 120 rpm on a rotary shaker in an Eppendorf vial
1
(
1.5 mL). Then, the corresponding ketone (10 g L- ), glucose (5
equiv), and GDH (1 U) were added. Reactions were shaken at 30
C and 120 rpm for 24 h and stopped by extraction with ethyl
°
In summary, we have reported the identification and over-
expression of a novel short chain dehydrogenase originated from
Ralstonia sp. DSM 6428 which accepts bulky-bulky R-keto ester
derivatives as well as small-bulky ketones, and is one of the
first examples that transforms bulky-bulky alkyl aryl ketones
showing very high stereoselectivities in most of the cases
studied. Thus, for the reduction of such sterically demanding
substrates like 2a and 3a perfect ee’s (>99%) were achieved,
acetate (2 × 0.5 mL). The organic layer was separated from the
cells by centrifugation (2 min, 13000 rpm) and dried (Na SO ).
2
4
Conversions and enantiomeric excesses of the corresponding
alcohols were determined by GC or HPLC analysis on a chiral
stationary phase (see the Supporting Information).
General Protocol for the Biocatalytic Reduction of Ketones
Employing Lyophilized Cells of E. coli/RasADH Using 2-Pro-
panol. Lyophilized cells of E. coli/RasADH (20 mg) were rehy-
drated in Tris-HCl buffer (600 µL, 50 mM, pH 7.5, 1 mM NADPH)
for 30 min at 30 °C and 120 rpm on a rotary shaker in an Eppendorf
5,13
which was never obtained before via biocatalytic means. The
obtained chiral alcohols are precursors for valuable products:
e.g., enantiopure halohydrin 12b has been used as a precursor
-1
vial (1.5 mL). Then, 2-propanol (67 µL, 10% v v ) and the
-1
corresponding ketone (10 g L ) were added. Reactions were shaken
1
4
of several pharmaceutical compounds like for the synthesis
at 30 °C and 120 rpm for 24 h and stopped by extraction with
ethyl acetate (2 × 0.5 mL). The organic layer was separated from
1
5
16
of (R)-salmeterol, fluoxetine, or nisoxetine.
2 4
the cells by centrifugation (2 min, 13000 rpm) and dried (Na SO ).
Conversions and enantiomeric excesses of the corresponding
alcohols were determined by GC or HPLC analysis on a chiral
stationary phase (see the Supporting Information).
Experimental Section
General methods. Ralstonia sp. DSM 6428 is commercially
available from the German culture collection DSMZ. Ketones
Acknowledgment. Financial support by DSM, FFG, and
Province of Styria is gratefully acknowledged. B.F.-S. acknowl-
edges the FAPSEP (Funda c¸ a˜ o de Amparo a Pesquisa do Estado
de S a˜ o Paulo) and the CAPES (Funda c¸ a˜ o Coordena c¸ a˜ o de
Aperfei c¸ oamento de Pessoal de N ´ı vel Superior).
1
5
a-13a and racemic alcohols 4b, 6b-11b, (R)-1b, (S)-1b, (R)-
b, (S)-5b, and 2-propanol were commercially available. Racemic
compounds 2b, 3b, 12b, and 13b were synthesized by conventional
reduction from the corresponding ketones (NaBH , MeOH, room
4
1
7
temperature). GDH from Bacillus megaterium was purchased
from J u¨ lich Fine Chemicals. All other reagents and solvents were
of the highest quality available.
Supporting Information Available: Procedures of molecular
biology, analytics, and experimental procedures are described.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(
(
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