Coupled chemoenzymatic transfer hydrogenation catalysis for enantioselective reduction and oxidation reactions

Article abstract of DOI:10.1016/j.tetasy.2005.09.026

Stereoselective reductions of prochiral ketones were performed using a new thermophilic, NAD-dependent alcohol dehydrogenase from Thermus sp. (TADH). The enzyme was produced on 2L-scale from recombinant Escherichia coli and purified by a simple, one-step heat treatment procedure yielding 220 mg of pure enzyme. Regeneration of NADH was catalyzed by the organometallic complex [Cp*Rh(bpy)(H₂O)]²⁺ using formate as a reducing agent. The catalytic performance of [Cp*Rh(bpy)(H₂O)] ²⁺ in terms of total number of catalytic cycles and number of catalytic cycles per hour achieved herein (up to 1500 and more than 400 h ^-1, respectively), are the highest reported for a non-enzymatic nicotinamide regeneration system so far. Chemoenzymatic reduction reactions in a two liquid phase setup were performed on a gramme-scale, for example, 1.3 g of enantiopure (1S,3S)-3-methylcyclohexanol was obtained after purification. The volumetric productivity reached up to 3.9 mM h^-1 with an average of 2.6 mM h^-1 (5.3 g L^-1 d^-1) over 10 h. In addition, chemoenzymatic oxidations utilizing the same catalyst set and molecular oxygen as a terminal electron acceptor were performed. Thus, the preparative value of chemoenzymatic transfer hydrogenations with [Cp*Rh(bpy)(H₂O)]²⁺ as a regeneration catalyst coupled especially to thermophilic ADHs was demonstrated.

Full text of DOI:10.1016/j.tetasy.2005.09.026

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 3512–3519
Coupled chemoenzymatic transfer hydrogenation catalysis
for enantioselective reduction and oxidation reactions
à
§
*
Frank Hollmann, Andreas Kleeb, Katja Otto and Andreas Schmid
Institute of Biotechnology, Swiss Federal Institute of Technology, 8093 Zurich, Switzerland
Received 8 September 2005; accepted 20 September 2005
Available online 14 November 2005
Abstract—Stereoselective reductions of prochiral ketones were performed using a new thermophilic, NAD-dependent alcohol dehy-
drogenase from Thermus sp. (TADH). The enzyme was produced on 2L-scale from recombinant Escherichia coli and purified by a
simple, one-step heat treatment procedure yielding 220 mg of pure enzyme. Regeneration of NADH was catalyzed by the organo-
2
+
2+
metallic complex [Cp*Rh(bpy)(H
2 2
O)] using formate as a reducing agent. The catalytic performance of [Cp*Rh(bpy)(H O)] in
1
ꢀ
terms of total number of catalytic cycles and number of catalytic cycles per hour achieved herein (up to 1500 and more than 400 h
,
respectively), are the highest reported for a non-enzymatic nicotinamide regeneration system so far. Chemoenzymatic reduction
reactions in a two liquid phase setup were performed on a gramme-scale, for example, 1.3 g of enantiopure (1S,3S)-3-methylcyclo-
ꢀ
1
ꢀ1
hexanol was obtained after purification. The volumetric productivity reached up to 3.9 mM h with an average of 2.6 mM h
ꢀ
1
ꢀ1
(
5.3 g L
d ) over 10 h. In addition, chemoenzymatic oxidations utilizing the same catalyst set and molecular oxygen as a terminal
electron acceptor were performed. Thus, the preparative value of chemoenzymatic transfer hydrogenations with
2
+
[
Cp*Rh(bpy)(H
2
O)] as a regeneration catalyst coupled especially to thermophilic ADHs was demonstrated.
ꢀ
2005 Elsevier Ltd. All rights reserved.
1
. Introduction
Alternatively, alcohol dehydrogenases (ADHs) are
attractive catalysts because of their remarkable chemo-,
regio- and stereoselectivities, their impressive catalytic
efficiency and their reactivity in aqueous media. Early
problems with ADH catalysis, such as the sometimes
narrow substrate spectrum of a given ADH or exclusive
preference for one product stereoisomer, can now be
addressed with standard molecular biology tools, such
There is an increasing need for enantiopure biologically
active compounds and their precursors. Among them,
chiral alcohols are important building blocks used for
pharmaceuticals, agrochemicals, and flavours. As a
result, much effort has been made in the development
of stereoselective processes for the preparation of enan-
tiopure compounds. Today, transition metal-based
hydrogenations and transfer hydrogenations of ketones,
as well as hydrolytic kinetic resolution of the racemic
alcohols, are the approaches most preferred by industry
1
2
3
5
,6
as directed evolution. ADH catalysis however necessi-
tates stoichiometric amounts of costly and unstable nic-
otinamide cofactors, which so far has severely impaired
the preparative usefulness of ADHs for enantioselective
catalysis. Stoichiometric use of these cofactors is prohib-
itive, leaving in situ regeneration as one of few alterna-
tives. Many specialized regeneration concepts based on
4
and organic chemists.
7
,8
9
enzymatic, electro-, and photochemical approaches
have been reported. All these regeneration catalysts how-
ever share the major drawback of being very specific for
one form of the nicotinamide cofactor (phosphorylated
or non-phosphorylated as well as oxidized or reduced).
Furthermore, the optimal reaction conditions of a given
ADH do not necessarily show a large overlap with those
from the putative regeneration catalyst in terms of pH,
temperature and ionic strength of the reaction medium,
often resulting in poor compromises under which the full
potential of each catalyst cannot be exploited.
*
Corresponding author at present address: Chair of Chemical
Biotechnology, University of Dortmund and Institute for Analytical
Sciences, 44227 Dortmund, Germany. Tel.: +49 231 7557380; fax:
+49 231 7557382; e-mail: andreas.schmid@bci.uni-dortmund.de
Present address: Max-Planck-Institute for Coal Research, 45470
M u¨ lheim/Ruhr, Germany.
Present address: Laboratory of Organic Chemistry, Swiss Federal
Institute of Technology, 8093 Zurich, Switzerland.
à
§
Present address: Chair of Chemical Biotechnology, University of
Dortmund, 44227 Dortmund, Germany.
0957-4166/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2005.09.026

F. Hollmann et al. / Tetrahedron: Asymmetry 16 (2005) 3512–3519
3513
Over a decade ago, Steckhan et al. introduced [Cp*Rh-
the conversion of cyclic ketones and alcohols.14 Both
catalysts are productively coupled via the nicotinamide
cofactor to perform enantioselective reductions of
ketones (Fig. 1).
2
+
(
bpy)(H O)] as a catalyst for the catalytic regeneration
2
1
0–12
of NADH.
Its advantages over the aforementioned
regeneration approaches are its versatility with respect
to the source of reducing equivalents, its high activity
over a broad temperature and pH-range, as well as its
identical activity towards phosphorylated and non-phos-
2. Results and discussion
2.1. Catalyst preparation
1
3
phorylated nicotinamides. Furthermore, there are indi-
cations that the same catalyst might also be applicable
for the catalytic regeneration of oxidized nicotinamides.
2
+
Considering these features, [Cp*Rh(bpy)(H O)]
A prerequisite for the practical applicability of the
chemoenzymatic reaction sequence is the facile and inex-
pensive preparation of the catalysts. [Cp*Rh(bpy)Cl]Cl
was prepared in one step, starting from commercially
available [(Cp*Rh) Cl ] and bipyridine in near quantita-
2
appears to be an ideal catalyst to promote ADH-
catalyzed enantiospecific reduction reactions as well as
oxidative kinetic resolutions. However, a demonstration
of its preparative applicability is still missing.
2
2
1
5
tive yield.
Aqueous stock solutions containing
Herein, we report the practical usefulness of
[Cp*Rh(bpy)Cl]Cl readily hydrolyzed and yielded the
catalytically active complex; these were stored for sev-
eral months at ambient temperature without detectable
loss of activity.
2
+
[
Cp*Rh(bpy)(H O)]
for this purpose. As biocata-
2
lyst we used a secondary ADH recently isolated from
Thermus sp. (TADH), which is particularly useful for
TADH was prepared in a similarly facile fashion using
well-established Escherichia coli BL21 (DE3). From
16
O
OH
1
1
a: H
b: 2-CH3
2a: H
2b: 2-CH3
one 2L lab-scale fermentation, approximately 3.6 g cell
dry weight with more than 2.2 g of total protein were
obtained (Table 1). To purify TADH from the host pro-
teins, heat denaturation of the mesophilic E. coli pro-
teins (80 ꢁC for 15 min) proved to be a facile and
efficient procedure yielding more than 220 mg of essen-
tially pure TADH (Fig. 2). Interestingly, heat treatment
even increased the total TADH activity. It was specu-
lated that if under the mesophilic growth conditions of
E. coli, TADH did not fold into in its native, catalyt-
ically most active, ternary- and quaternary structure
and that increasing the temperature would allow refold-
ing into a catalytically more active structure; alterna-
tively host proteins present in the crude cell lysate
might inhibit TADH. Further investigations to clarify
this phenomenon are currently in progress.
1
1
c: 3-CH3
d: 4-CH3
2c: 3-CH3
2d: 4-CH3
R
R
O
OH
3
3
a: 1-Decalone
b: 2-Decalone
4a: Decahydro-1-naphthol
4b: Decahydro-2-naphthol
5
5
5
5
5
5
a: C H5
6a: C H5
2
2
b: C H7
OH
3
6b: C H7
O
3
c: C H9
6c: C H9
4
4
R'
R'
d: C H
6d: C H
5
11
5
11
e: C H
6e: C H
6
13
6
13
f: CO H
5f: CO H
2
2
(
a)
5g: benzyl
5g: benzyl
organic phase: octane
O
OH
Table 1. Purification characteristics of TADH from Escherichia coli
BL21(DE3) (pASZ2)
Crude
extract
Supernatant after
heat denaturation
O
OH
TADH
Total protein (mg)
Total activity (U)
Specific activity (U mg
Purification factor (-fold)
Recovery (%)
2244
673
0.3
1
228
1072
4.7
15.6
159
a
ꢀ1
)
NAD+
100
NADH
Recombinant E. coli were grown in standard LB-medium supple-
mented with 0.5% (w/v) glucose at 37 ꢁC to a final cell density of 1.8 g
2+
ꢀ1
CDW L , TADH expression was induced with 1 mM isopropyl-b-D-
N
N
ꢀ1
CO2
HCOOH
-
thiogalactopyranoside at a cell concentration of 0.24 gCDW L
.
Rh
a
The TADH activity was determined spectrophotometrically based on
OH2
ꢀ1
ꢀ1
the formation of NADH (k = 340 nm, e = 6220 M cm ) from
+
NAD (1mM) during the oxidation of 1-butanol (100 mM) at 60 ꢁC
(
b)
aqueous phase
and pH 9.0; 1 international unit (U) corresponds to the formation of
1
lmol product per minute.
Figure 1. (a) Substrates and products used for chemoenzymatic
reduction in a two-liquid-phase system as shown in (b). The nomen-
clature is used throughout the text. (b) Chemoenzymatic reduction
TADH was stored at ꢀ20 ꢁC at a concentration of
ꢀ
1
1
.9 mg mL , where it retained full activity over at least
of various ketones in
a two-liquid-phase system catalyzed by
2+
2
TADH coupled to [Cp*Rh(bpy)(H O)] -promoted regeneration of
NADH.
5 months. Initial characterization of TADH revealed
its suitability for the conversion of cyclohexanone

3514
F. Hollmann et al. / Tetrahedron: Asymmetry 16 (2005) 3512–3519
1
1
20
00
160
1
1
1
40
20
00
8
6
4
2
0
0
0
0
80
6
4
2
0
0
0
0
0
20
40
60
Temperature [ C]
80
100
o
Figure 2. Native PAGE analysis of TADH-purification. Lines 1,1a:
cell crude extracts from non-induced recombinant Escherichia coli;
Figure 3. Influence of temperature on the TADH-activity (Ç) and
TADH-stability (m) during the oxidation of 1-butanol compared to
2,2a: heat-treated cell crude extracts from non-induced recombinant
2+
13
2
the activity of [Cp*Rh(bpy)(H O)] (d) during NADH regeneration.
E. coli; 3,3a: heat-treated cell crude extracts from induced recombinant
E. coli. Lines 1–3 Coomassie brilliant blue staining, lines 1a–3a
General conditions: Assays were performed in potassium phosphate
ꢀ1
17
buffer (50 mM, pH 9.0), c(TADH) = 0.19 lM (30.3 lg mL ),
qualitative in situ ADH-activity assay. General conditions: Gel-
electrophoresis under non-denaturating conditions was performed
using a PhastGel PAGE-System (Amersham Pharmacia, Freiburg,
Germany) (native gradient gel 8–25%) following the recommendations
of the supplier. In situ alcohol dehydrogenase activity was determined
qualitatively by immersion of the native gradient PAGE gels for 5 min
+
c(NAD ) = 1 mM, c(1-butanol) = 100 mM, activity calculations are
based on the initial 30 s of the reaction. Stability assays of TADH were
ꢀ
1
performed by incubating the enzyme (1.9 mg mL
in potassium
phosphate buffer) at the indicated temperature. At intervals samples
were withdrawn and analyzed at 60 ꢁC.
ꢀ1
at 60 ꢁC in a solution containing 0.2 mg mL blue nitrotetrazolium-
ꢀ
1
chloride, 0.08 mg mL
N-methylphenazoniummethylsulfate, 1 mM
+
17
NAD and 1% (v/v) ethanol in 100 mM Tris (pH 8).
Interestingly, none of the experiments gave full conver-
sion of the starting material even though from a thermo-
dynamic point of view the equilibrium should be far on
the side of the products (>99.5% conversion as calcu-
derivatives, a detailed study on its substrate range and
catalytic properties will soon be reported elsewhere.
1
8,19
lated from the group contribution method
). This
Overall, TADH was obtained in pure form and free
from competing NAD-dependent enzymes present in
E. coli following a simple and scalable protocol. Its
preparation was completed within 3 days from inexpen-
sive and non-toxic starting materials without tedious
chromatographic steps.
could be explained by assuming that TADH is subject
to product inhibition, which is quite common amongst
2
0,21
ADHs.
The significantly decreased ÔinitialÕ rates of
the chemoenzymatic reactions (determined after 15 min
and up to 20% conversion) compared to the rates deter-
mined by steady state kinetic measurements further sup-
port this assumption (Table 2).
2
.2. Optimization of chemoenzymatic reductions
In fact, kinetic studies revealed a pronounced product
inhibition, for example, for 2c (isolated from prepara-
tive-scale chemoenzymatic reactions, vide infra)
In the first set of experiments, we investigated the influ-
ence of temperature on stability and activity of the cat-
alysts. As shown in Figure 3, TADH activity
exponentially increased with temperature, which was
counteracted by the exponentially decreasing half-life
of the biocatalyst. Therefore, we chose an operation
temperature of 60 ꢁC as at this temperature the product
of TADH activity and TADH half life was at its highest
(K = 8.7 ± 1.4 mM). Furthermore, TADH stability
i
was significantly impaired in the presence of substrate
(data not shown). Any inhibitory effects of the reactants
2
+
(alcohols and/or ketones) on [Cp*Rh(bpy)(H O)] was
2
excluded (data not shown).
(
Fig. 3). pH 7 was chosen as a compromise between
With the goal of efficiently shifting the transfer hydroge-
nation equilibrium to the side of the desired products we
evaluated the influence of in situ NADH regeneration
TADH activity and stability of the nicotinamide cofac-
tor. In the reductive direction, TADH activity was high-
est at pH 6 with approximately 80% of this activity at
pH 7. NADH, on the other hand had a half life of 38
and 139 min at pH 6 and pH 7 in 50 mM phosphate buf-
fer, respectively (data not shown).
2
+
(catalyzed by [Cp*Rh(bpy)(H O)] ) and in situ product
2
removal on the reaction progress (Fig. 4). In the case of
stoichiometric NADH addition (Fig. 4j) only 1.9 mM
of product were formed corresponding to 38% of the
theoretical yield [exclusively considering that exclusively
2
+
Under these conditions, [Cp*Rh(bpy)(H O)] exhibited
a specific activity of approximately 11 U mg with a
half life exceeding the time-scale of TADH stability (at
(S)-3-methyl cyclohexanone is converted by TADH
2
ꢀ
1
2+
at significant rates]. [Cp*Rh(bpy)(H O)] -catalyzed
2
regeneration of the reduced nicotinamide coenzyme
had a positive but not very pronounced effect on the
yield (2.9 mM product formed, corresponding to 58%
of the theoretical yield, Fig. 4m). If however, an organic
phase (here heptane) was added, TADH turnover
increased drastically (Fig. 4Ç).
1
3
8
0 ꢁC a half life of over 15 days was determined).
To evaluate the scope of the chemoenzymatic reaction,
we performed catalytic reduction reactions as indicated
in Figure 3; results are shown in Table 2.

F. Hollmann et al. / Tetrahedron: Asymmetry 16 (2005) 3512–3519
Table 2. Chemoenzymatic reduction of the ketones 1, 3 and 5
3515
ꢀ1
a
2+ e
)
b
Substrate
Initial rate (U mg
)
Yield (%)
TN ([Cp*Rh(bpy)(H
2
O)]
ee-value (%)
b
c
After 15 min
3.6
Initial rate studies
1
a
b
6.2
1.0
57.3
Traces
36.4
—
40.6
19.2
27.9
Traces
228
n.d.
110
—
—
76
112
n.d.
—
n.d.
99.6
—
n.d.
14.9
n.d.
n.d.
d
d
d
1
n.d.
g
rac-1c
R)-1c
2.0
—
3.4
1.0
1.7
12.4
0.2
12.4
3.5
3.1
0.2–0.9
(
f
d
rac-1c
1
3
5
d
d
b
d
d
d
a–d
n.d.
+
General reaction conditions: 1 mL-scale, 20 mM Tris–HCl buffer (pH 7), T = 60 ꢁC, c(substrate) = 20 mM, c(NAD ) = 5 mM, c([Cp*Rh(bpy)-
2+
2 2
(H O)] ) = 0.05 mM, c(NaHCO ) = 150 mM.
a
b
c
d
e
f
Determined after 3.5 h reaction time with residual reactant concentration.
Determined from chemoenzymatic reactions via GC.
From initial rate studies determined spectrophotometrically.
Not determined.
Calculated from the amount of product formed.
ꢀ1
Formate dehydrogenase [20 U mL (at 25 ꢁC)] was used for NADH-regeneration.
g
ꢀ1
Determined according to (Vrac ꢀ V
S
) · (Vrac ꢀ V
R
)
[due to the low activity with (R)-1c, we approximated values determined for the racemate
solely to the (S)-enantiomer].
1
1
6000
2000
mechanically demanding reaction conditions, necessary
for forming an emulsion. When the reaction was mixed
by shaking (e.g., Fig. 4Ç), TADH activity was lost after
approximately 3 h. If, however, stirring was necessary
(e.g., to form emulsions on larger scale, vide infra)
already after 30 min stirring no residual TADH activity
was detected. To stabilize TADH it was covalently
attached to Eupergit C . Immobilization of TADH,
however, resulted in a dramatically decreased biocata-
lyst activity, which we could assign to an increase of
8
4
000
000
0
TM 22
0
1
2
3
4
5
Time [h]
the apparent K -value for NADH by at least a factor
M
of 500 (Fig. 5). However, we accepted this drawback
in the light of the expected stability increase for TADH.
Currently, we are evaluating various alternative
approaches to stabilize TADH, without impairing its
catalytic efficiency.
Figure 4. Influence of NADH-regeneration and in situ product
removal on the reduction of 3-methylcyclohexanone. General condi-
tions: 50 mM potassium phosphate buffer pH 6.0, T = 60 ꢁC, c(NaH-
2
CO ) = 150 mM. The reactions were performed in 2 mL Eppendorf
cups in a thermoshaker (1500 rpm) with a total volume of 1 ml; (j)
Stoichiometric NADH: c(TADH) = 0.484 lM, c(NADH) = c(1c) =
+
2+
To estimate the potential of [Cp*Rh(bpy)(H O)] -catal-
yzed regeneration of NADH, chemoenzymatic reduc-
1
0
0 mM; (m) NADH-regeneration: c(TADH) = 0.484 lM, c(NAD ) =
.5 mM, c([Cp*Rh(bpy)(H O)] ) = 0.1 mM, c(3-methylcyclohexa-
2
2
2+
none) = 10 mM; (Ç) in situ-product removal: aqueous concentrations
tion reactions with immobilized TADH were performed
+
2+
c(TADH) = 0.242 lM, c(NAD ) = 0.5 mM, c([Cp*Rh(bpy)(H
2
O)] ) =
2+
under [Cp*Rh(bpy)(H O)] -limited conditions: a total
2
0
.05 mM, c(3-methylcyclohexanone) = 250 mMheptaneffi19 mMaq; phase
2+
turnover number of [Cp*Rh(bpy)(H O)] of approxi-
2
ratio heptane:buffer = 1.5. Due to the varying TADH concentrations
applied, different product formation rates were obtained. In order to
make the experiments comparable, normalization on the turnover
number of TADH [TN(TADH)] were performed.
mately 1500 was achieved at turnover frequencies of
up to 475 ± 6 turnovers per hour. Compared to litera-
ture values, these represent an increase of at least one
order of magnitude (Table 3).
Despite the high catalytic turnover of [Cp*Rh(bpy)-
2
+
The second organic phase extracts the product formed,
and thus decreases the effect of product inhibition on
the enzymatic reduction reaction. Therefore, we concen-
trated on the biphasic reaction media. Among the sol-
vents tested with respect to their compatibility with
TADH octane was found to be the most suitable organic
phase (data not shown). Compounds 1c and 2c parti-
tioned between octane and the buffer (phase ratio 0.5)
at 13:1 and 10:1, respectively. Emulsions were formed
to assure a sufficiently high transfer rate of substrates
and products over the phase interface. Unfortunately,
TADH turned out to be very sensitive towards the
(H O)] observed (Table 3), it did not reach the values
2
ꢀ1
determined in the absence of TADH (up to 1000 h ).
We assigned this to an inhibitory effect of TADH on
[Cp*Rh(bpy)(H O)] . It is possible that nucleophilic
residues present in TADH can coordinate to
[Cp*Rh(bpy)(H O)] , thereby gradually decelerating
2
+
2
2
+
2
the rate of the formate-driven regeneration reaction.
2
3
Similar effects have been observed previously. This
phenomenon was attenuated by the addition of nucleo-
philic buffer components such as Tris. Principally, the
interaction between TADH and [Cp*Rh(bpy)(H O)]
can be eliminated completely by more nucleophilic addi-
2
+
2

3516
F. Hollmann et al. / Tetrahedron: Asymmetry 16 (2005) 3512–3519
5
4
3
2
1
00
00
00
00
00
0
15000
tives making electrochemical regeneration of the active
hydridorhodium species an option. Further investiga-
tions are currently underway.
1
3
1
1
7
5
2
0
2500
0000
500
2.3. Gramme-scale preparation of (1S,3S)-3-methyl-
cyclohexanol ((1S,3S)-2c)
Preparative-scale reactions (750 mL-scale emulsion
process at 2:1 ratio of organic:aqueous phase) were
performed. Reaction parameters from reductions of 1c
and 3b are listed in Table 4.
000
500
The NMR spectrum of purified 2c indicated at least
0
2
4
6
8
10
9
7.2% enantiomeric excess for the trans-substituted
+
[
NAD ] / mM
isomer (the diastereomeric ratio was determined by the
+
integrals of the carbinol protons, d 3.45 and 3.97 ppm
Figure 5. Comparison of the influence of NAD concentration on the
[
2
8
2+
for the cis and trans, respectively). Considering the
high preference of TADH for (3S)-1c, it can be asserted
that (1S,3S)-2c is the predominant product from the
chemoenzymatic reduction reaction. As shown in Table
4, respectable catalytic performances of both catalysts
2
Cp*Rh(bpy)(H O)] -catalyzed NADH regeneration rate (j) and on
the rate of the chemoenzymatic reduction of 3-methylcyclohexanone in
terms of turnover rate of the immobilized biocatalyst (Ç). General
2
conditions: 20 mM Tris buffer (pH 7), T = 60 ꢁC, c(NaHCO ) =
2+
150 mM; TF([Cp*Rh(bpy)(H
2
O)] ) (5 lM) was determined via
2
+
NADH formation (k = 340 nm).
were obtained, especially for [Cp*Rh(bpy)(H O)] out-
2
numbering previous results by far (Table 3). Also, the
mass-based yield for both catalysts is promising.
2+
2
Table 3. Catalytic parameters determined for [Cp*Rh(bpy)(H O)]
However, comparably high concentrations of the nico-
tinamide cofactor (10 mM) were necessary to guarantee
high activity of the immobilized biocatalyst. Currently,
we are evaluating alternatives to Eupergit in order to
stabilize the biocatalyst under the mechanically demand+-
ing conditions. Thus, a significant decrease in the NAD
from previous studies
2+
c([Cp*Rh(bpy)(H
mM)
2
O)]
)
TNmax
TFmax
Scale
(ml)
ꢀ1
(
(h
)
ꢀ
1
2
5
5
5
5
1
1
g
0.5
0.21
0.5
0.5
50
30
14
200
1500
10
10
2
g
1.25 10
6
2.5
470
11
f
50
n.d.
Up to 750
demand is expected as the K -value of diffusible TADH
M
2
6
a
towards NADH is only about 10 lM (data not shown),
which will further increase the economic attractiveness
of the chemoenzymatic approach. Overall, we have for
a
c
0.001–0.1
a
This study.
a
Table 4. Gram-scale chemoenzymatic reduction reactions of 1c and 3b
1
c
3b
Product
Final concentration in octane (mM)
Enantiomeric excess (% ee)
21.4
>97
1.3 (95% pure)
3.9 (0.445)
18.5
n.d.
1.8 (95% pure)
2.1 (0.354)
b
d
d
Isolated product (g)
Maximal productivity [mmol L
c
ꢀ1 ꢀ1
ꢀ1 ꢀ1
h
] ([g L h ])
TADH
ꢀ1
TFmax (h
TN
)
4300
2210
28,000
0.012
24,200
0.011
ꢀ1e
g g
2+
[
Cp*Rh(bpy)(H O)]
2
ꢀ1
TFmax (h
TN
)
120
95
233
0.035
146
0.035
ꢀ1e
g g
General conditions: 250 mL Tris/phosphate-buffer (20/50 mM; pH60 = 7.0), T = 60 ꢁC, c(NaHCO
2
) = 150 mM, c(TADH) = 1.53 lM (60 mg
O)] ) = 5 lM; 500 mL octane, c(1c) = 250 mM, c(3a) = 1.4 M, respectively,
+
2+
immobilized on Eupergit C), c(NAD ) = 10 mM, c([Cp*Rh(bpy)(H
2 0
2+
2
c(dodecane) = 2 mM (internal standard for GC analysis); [Cp*Rh(bpy)(H O)] was continuously added to the reaction to compensate the gradual
2
+
ꢀ1
[
2
a
Cp*Rh(bpy)(H
3 lM h ).
2
O)] -inhibition (1.15 mL h of a 5 mM stock-solution corresponding to an approximate increase of the aqueous concentration of
ꢀ
1
28
Determined via NMR.
b
c
After chromatographic purification.
Based on the organic phase.
As determined via GC-analysis.
d
e
3
ꢀ1
ꢀ1
) = 483 g mol )
Mass-based ratio: mass of catalyst consumed per gramme of product (M(TADH) = 47 · 10 g mol ; M([Cp*Rh(bpy)(H
2
O)]Cl
2
per gramme of product.

F. Hollmann et al. / Tetrahedron: Asymmetry 16 (2005) 3512–3519
3517
the first time applied chemoenzymatic catalysis using
3
2.5
2
0.6
0.5
0.4
0.3
0.2
0.1
0
an organometallic and an enzymatic catalyst for
preparative production of chiral alcohols. Though
the results obtained so far are already promising,
further significant increases of the catalytic activity of
especially NAD is expected by optimized reaction
conditions (i.e., non-heterogeneous stabilization of the
1.5
2
4
biocatalyst).
1
2.4. Chemoenzymatic oxidation of racemic 3-methyl-
cyclohexanol rac-2c
0.5
0
0
0.5
1
1.5
2
2.5
3
From a synthetic point-of-view, it may be more desirable
to produce enantiopure alcohols via kinetic resolution of
the racemate than by enantioselective reduction of the
prochiral ketone. In this case, an additional driving force
Time [h]
Figure 7. Oxidation of racemic 3-methylcyclohexanol (mixture of all
four stereoisomers) in the system TADH/[Cp*Rh(bpy)(H O)] /FAD/
. General conditions: 1 mL of 50 mM potassium phosphate buffer
(pH 9.0), c(TADH) = 0.242 lM (38 lg mL ), c(NADH) = 5 mM,
2
+
2
+
is required, since the NAD -promoted oxidation of alco-
O
2
1
8,19
ꢀ1
hols is thermodynamically unfavourable.
Recently,
2+
2
c([Cp*Rh(bpy)(H O)] ) = 50 lM, c(FAD) = 0.2 mM, c(catalase) =
we reported evidence that a system consisting of
2
+
0.64 lM; (Ç) 1c, (j) ratio of 2c-enantiomers (alcohols 1 and 2, see
Section 4) detected by GC. The reactions were performed in Eppendorf
cups placed in a thermoshaker. Air input was performed by rotating
the reaction vessels at 1400 rpm.
[
Cp*Rh(bpy)(H O)] and flavin in an O -containing
2 2
+
media might be suitable for regenerating NAD(P) .
The very high oxidation potential of the O /H O couple
2
2
2
is exploited to shift the redox equilibrium NAD(P)H/
+
13
NAD(P) to the side of the desired products. Studies
on chemoenzymatic kinetic resolution of rac-2c were per-
formed on mL-scale using soluble TADH (Fig. 7).
+
Overall, we have shown for the first time that an NAD
regeneration system based on [Cp*Rh(bpy)(H O)] /fla-
2
+
2
vin/O can be successfully applied to a dehydrogenase-
2
The chemoenzymatic oxidation following the reaction
cascade proposed in Figure 6 was shown to be feasible.
Direct oxidation of the alcohols via [Cp*Rh(bpy)-
catalyzed kinetic resolution of alcohols.
2
+
(
H O)] could be excluded since no ketone formation
2
3. Conclusion
was observed in the absence of the biocatalyst. Further-
more, the oxidation reaction preceded stereoselectively
Herein, we have demonstrated the synthetic practicabil-
ity of chemoenzymatic redox reactions. [Cp*Rh(bpy)-
(
Fig. 7) indicating that the oxidation step was catalyzed
by TADH. Approximately 2 mM 1c was formed after
.5 h. This corresponds to 80% of the theoretical yield
2
+
(H O)] is an efficient and versatile regeneration cata-
2
2
lyst for reduced and oxidized nicotinamide cofactors.
Especially coupled to thermophilic ADHs, it can deploy
its high catalytic potential. The use of thermophilic
ADHs bears further practical advantages such as very
easy production and purification. Overall, we believe
that the chemoenzymatic approach will render biocata-
lytic procedures more attractive to preparative organic
chemists thus becoming a new tool for enantioselective
synthesis.
assuming an even distribution of the four stereoisomers
within the substrate and a high preference of TADH
towards (1S,3S)-2c. The initial oxidation rate under
ꢀ
1
ꢀ1
non-optimized conditions was 1.92 mM h
and 38.5 h
(2.2 s
ꢀ
1
2+
for TADH and [Cp*Rh(bpy)(H O)] ,
2
respectively). Further characterization of this reaction
system is expected to lead to optimized catalyst
performances.
2
+
O
O
R
N
H
N
O
NH2
N
N
Rh
NH
O2
H O
2
N
H
N
R
OH2
O
TADH
Catalase
OH
O
+
R
N
N
O
O
H O
2
NH2
2
N
N
+
NH
Rh
N
R
N
OH2
Figure 6. Chemoenzymatic oxidation.

3518
F. Hollmann et al. / Tetrahedron: Asymmetry 16 (2005) 3512–3519
4. Experimental
tadt, Germany) and incubated for 48 h at room
temperature. Afterwards, the beads were washed with
50 mM potassium phosphate buffer (pH 7.0) and
stored in the same buffer at 4 ꢁC. Eleven milligrammes
of TADH per gramme Eupergit were completely
immobilized (determined from the protein content in
the supernatant) after 2–3 days of incubation at
room temperature. Catalytic activity of immobilized
TADH was determined using a thermostatted flow
through glass column (volume 2.5 mL), containing
250 mg of TADH–Eupergit. The specific activity of
immobilized TADH for cyclohexanone conversion
(c(cyclohexanone) = 7 mM, c(NADH) = 5 mM, resi-
4
.1. Chemicals, bacterial strains and plasmids
Chemicals were purchased from Fluka (Buchs, Switzer-
land) or Aldrich (Steinheim, Germany) in the highest
quality available and used without further purification.
{
Cp*RhCl(l-Cl)} was a gift from DEGUSSA, Hanau,
2
Germany. Appropriate safety precautions were taken,
especially when working with rhodium and organic sol-
vents at elevated temperatures. [Cp*Rh(bpy)(H O)]
was synthesized according to literature methods by
addition of 2 equiv of 2,2 -bipyridine to a suspension
2
+
2
1
5
0
ꢀ
1
of {Cp*RhCl(l-Cl)} in methanol. [Cp*Rh(bpy)Cl]Cl
dence time 1 min) was determined to be 17.2 U g
2
ꢀ
1
was precipitated upon addition of diethyl ether. Aque-
ous stock solutions were stored at room temperature
for several months without detectable loss of transfer
hydrogenation activity.
(Eupergit) and 1.5 U mg (TADH) corresponding to
25% of the specific activity of soluble TADH with
cyclohexanone.
4
.2. Analytical methods
Unless indicated otherwise, all pH values were adjusted
to 60 ꢁC.
Initial catalyst activities were determined spectrophoto-
metrically in a thermostable Carey 1E UV-spectro-
photometer (Varian, Switzerland) and were based
on accumulation or depletion of NADH (e =
E. coli BL21(DE3)16 was used as a host organism for the
overexpression of the TADH gene from derivatives of
plasmid pET11aEco (a derivative of pET11a, Novagene,
carrying an additional EcoRI site (5 -AAGAA-
GGAGAATTCCATATG-3 ) between the NdeI site
and the RBS of pET11a). In previous work, the TADH
gene was localized on the chromosome of Thermus sp.
and subcloned into a pUC20-vector. From this vector
it was now amplified by polymerase chain reaction
ꢀ1
ꢀ1
6230 M cm ). All components of the reaction (except
the catalysts) were incubated for 2 min at the indicated
temperature. Assays were started by adding TADH
0
0
2
+
and [Cp*Rh(bpy)(H O)] , respectively. Protein concen-
2
trations were determined according to the method of
2
7
Bradford. Affinity constants (K -values) were calcu-
M
lated using the Enzfitter software (Elsevier-Biosoft,
1987) by weighted linear regression.
(
PCR) using the DyNazyme EXT kit (Finnzymes) and
0
the primers 5 -GAAGGAATTCCATGCGCGCAGT-
GGTTTTTGA-3 and 5 -ATGCTCCTCGGATCCCA-
GAAGTA-3 . The forward primer was designed to
introduce an EcoRI site whereas the reverse primer
contained a BamHI site. The resulting PCR product
was digested with EcoRI and BamHI and ligated
into the EcoRI and BamHI digested expression vector
pET11aEco to give pASZ2.
0
0
For gas chromatography (GC), aqueous samples were
extracted with an aliquot of diethyl ether containing
2 mM dodecane as internal standard. The organic phase
was dried over anhydrous sodium sulfate and analyzed.
GC analysis was done using a GC (Fisons Instruments,
Milano, Italy) equipped with a Lipodex A capillary
column (25 m; inner diameter 0.25 mm) from Mach-
ery-Nagel (D u¨ ren, Germany) with split injection (1:20)
and hydrogen as carrier gas. The following temperature
profiles were applied. Cyclohexanones: 3 min isotherm
0
4
.1.1. Production of TADH. For overexpression of the
TADH genes recombinant E. coli BL21(DE3) carrying
pASZ2 were grown in Luria-Bertani (LB) medium (1%
w/v Bacto Tryptone, 0.5% w/v yeast extract, 1% NaCl,
pH 7.5) containing chloramphenicol (20 lg/mL) and
ampicillin (200 lg/mL) at 37 ꢁC. At OD = 0.8 (optical
ꢀ
1
at 60 ꢁC, from 60 to 120 ꢁC at 5 ꢁC min ; decalones:
2 min isotherm at 60 ꢁC, from 60 to 160 ꢁC at
ꢀ
1
5 ꢁC min . Compounds were detected using a flame
ionization detector.
4
50
density at k = 450 nm) 1 mM isopropyl b-D-thiogalacto-
side (IPTG) was added to induce protein expression. The
cells were grown until the late exponential phase (OD450
about 9), harvested by centrifugation and stored at
Resolution of isomers (retention time (min)): 3-methyl-
cyclohexanol: 10.03 and 10.34 (ratio: 1:1.95); 3-methyl-
cyclohexanone 8.98; 2-decalone: 14.03 and 15.1 (ratio:
1:3.3); 2-decahydronaphthol: 15.3, 16.2, and 16.3 (ratio:
ꢀ
80 ꢁC after shock-freezing using liquid nitrogen.
1
:1.2:2.9).
4
.1.2. Purification and immobilization of TADH. Frozen
cells were resuspended in 20 mM Tris buffer (pH 7.5) and
disrupted by two passages through a French press (1200
psi). After centrifugation (150,000g at 4 ꢁC for 30 min)
the supernatant was incubated at 80 ꢁC for 15 min.
Subsequent centrifugation (150,000g at 4 ꢁC for 30 min)
resulted in essentially pure TADH in the supernatant.
Chemoenzymatic reactions: 1–2 mL scale chemoenzy-
matic reduction and oxidation reactions were performed
in Eppendorf reaction tubes in an Eppendorf 5436
thermomixer (Vaudaux-Eppendorf, Sch o¨ nenbuch, Swit-
zerland). Unless indicated otherwise (especially for
chemoenzymatic oxidation reactions), the reaction mix-
tures were degassed by nitrogen purging and equili-
For immobilization, 12 mL TADH solution (1.9
mg mL ) was added to 2 g Eupergit C (R o¨ hm, Darms-
brated at 60 ꢁC. Reactions were started by addition of
ꢀ
1
2+
[Cp*Rh(bpy)(H O)] .
2

F. Hollmann et al. / Tetrahedron: Asymmetry 16 (2005) 3512–3519
3519
Biotransformation reactions were performed under non-
sterile conditions on a 750 mL scale in a stirred tank
reactor with two Rushton turbine impellers, four baffles
and a total volume of 3 L. The reactor, containing
5. Reetz, M. T.; Bocola, M.; Carballeira, J. D.; Zha, D.;
Vogel, A. Angew. Chem., Int. Ed. 2005, 44, 4192–4196.
6
7
8
9
. Reetz, M. T. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
716–5722.
. van der Donk, W. A.; Zhao, H. Curr. Opin. Biotechnol.
003, 14, 421–426.
. Liese, A. Adv. Biochem. Eng. Biotechnol. 2005, 92, 197–
24.
5
2
50 mL buffer and 500 mL octane (2 mM dodecane),
was heated to 60 ꢁC and degassed by purging for
5 min with nitrogen. After supplementation with 5 g
TADH–Eupergit (corresponding to approximately
2
1
2
. Hollmann, F.; Schmid, A. Biocatal. Biotransform. 2004,
22, 63–88.
6
0 mg TADH), the reactions were started by addition
2
+
of [Cp*Rh(bpy)(H O)]
5
(aqueous concentration:
lM). [Cp*Rh(bpy)(H O)] was added continuously
2
1
10. Ruppert, R.; Herrmann, S.; Steckhan, E. J. Chem. Soc.,
Chem. Commun. 1988, 17, 1150–1151.
2
2
+
ꢀ
1
1
1
1
1. Ruppert, R.; Herrmann, S.; Steckhan, E. Tetrahedron
Lett. 1987, 28, 6583–6586.
2. Westerhausen, D.; Herrmann, S.; Hummel, W.; Steckhan,
E. Angew. Chem., Int. Ed. Engl. 1992, 31, 1529–1531.
3. Hollmann, F.; Witholt, B.; Schmid, A. J. Mol. Catal. B
Enzym. 2002, 19–20, 167–176.
4. Otto, K. Characterization of a thermostable alkane oxidiz-
ing system, PhD thesis, Technical University of Hamburg,
Harburg.
(
1.2 ml h of a 5 mM stock solution). After 24 h, the
phases were separated by settling and the organic phase
was removed under reduced pressure. The product was
purified chromatographically on silica using chloroform
as the eluent (Rf (3-methylcyclohexanol) = 0.3, Rf
(
2-decalol) = 0.35).
4
.2.1. Characterization of the product from preparative-
1
scale chemoenzymatic reduction of 1c. H NMR
250 MHz, CDCl ), d (ppm): 4.0–3.94 (m, 1H), 1.83–
15. Lo, H. C.; Leiva, C.; Buriez, O.; Kerr, J. B.; Olmstead, M.
(
M.; Fish, R. H. Inorg. Chem. 2001, 40, 6705–6712.
3
1
1
6. Sambrook, J.; Fritsch, E. F.; Maniatis, T. Molecular
Cloning, 2nd ed.; Cold Spring Harbor Laboratory Press:
Cold Spring Harbor, 1989.
7. Heeb, M. J.; Gabriel, O. Enzyme Localization in Gels. In
Methods in Enzymology; Academic Press: Orlando, 1984;
Vol. 104, pp 416–439.
1
1
6
.69 (m, 1H), 1.62–1.50 (m, 4H), 1.48–1.35 (m, 2H),
.21–1.10 (m, 1H), 0.97–0.85 (m, 1H), 0.81 (d, J =
5
89
.8 Hz, 3H); [a] = +2.2 (neat).
1
1
2
2
8. Mavrovouniotis, M. L. Biotechnol. Bioeng. 1990, 36,
Acknowledgements
1
070–1082.
9. Mavrovouniotis, M. L. J. Biol. Chem. 1991, 266, 14440–
4445.
0. Hummel, W.; Kula, M.-R. Eur. J. Biochem. 1989, 184, 1–
3.
The authors express their gratitude to Dr. Bruno B u¨ hler
and Prof. Dr. Richard Fish for critical reading of the
manuscript and helpful discussion. We thank B a¨ rbel
Pierstorff for assistance with the NMR measurements,
Dr. Jan van Beilen for providing pET11aEco, and Karin
Hofstetter for excellent assistance during cloning. This
work was financially supported by the ETH research
grant TH 24/99-3.
1
1
1. Kula, M.-R.; Kragl, U. Dehydrogenases in the Synthesis
of Chiral Compounds. In Stereoselective Biocatalysis;
Patel, R. N., Ed.; Marcel Dekker: New York, 2000; pp
839–867.
22. Katchalski-Katzir, E.; Kraemer, D. M. J. Mol. Catal. B
Enzym. 2000, 10, 157–176.
2
3. Lutz, J.; Hollmann, F.; Ho, V. T.; Schnyder, A.; Fish, R.
H.; Schmid, A. J. Organomet. Chem. 2004, 689, 4783–
4790.
References
2
4. Hofstetter, K.; Lutz, J.; Lang, I.; Witholt, B.; Schmid, A.
Angew. Chem., Int. Ed. 2004, 43, 2163–2166.
1
. Stinson, S. C. Chem. Eng. News 1999, 77, 57.
2
. Ramos Tombo, G. M.; Blaser, H. U. In Pesticide
Chemistry and Bioscience; Brooks, G. T., Roberts, T. R.,
Eds.; Royal Society of Chemistry: Cambridge, 1999; Vol.
25. Lo, H. C.; Fish, R. H. Angew. Chem., Int. Ed. 2002, 41,
478–481.
26. Delecouls-Servat, K.; Basseguy, R.; Bergel, A. Chem. Eng.
Sci. 2002, 57, 4633–4642.
2
2, p 360.
3
4
. Noyoyri, R. ChemTech 1992, 22, 360.
. Blaser, H.-U.; Malan, C.; Pugin, B.; Spindler, F.; Steiner,
H.; Studer, M. Adv. Synth. Catal. 2003, 345, 103–151.
27. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
28. Moberg, C.; R a´ kos, L. J. Organomet. Chem. 1987, 335,
125–131.