Concurrent oxidations with tandem biocatalysts in one pot: Green, selective and clean oxidations of methylene groups to ketones

Article abstract of DOI:10.1039/c0cc04706f

A novel tandem-biocatalysts system consisting of a monooxygenase-containing microorganism and an alcohol dehydrogenase is developed for the concurrent oxidations of methylene groups to ketones in one pot, providing green, clean and simple access to valuable ketones with high yield, excellent selectivity and efficient cofactor recycling.

Full text of DOI:10.1039/c0cc04706f

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COMMUNICATION
Concurrent oxidations with tandem biocatalysts in one pot: green,
selective and clean oxidations of methylene groups to ketonesw
Wei Zhang,ab Weng Lin Tang,a Daniel I. C. Wangbc and Zhi Li*ab
Received 30th October 2010, Accepted 8th January 2011
DOI: 10.1039/c0cc04706f
A novel tandem-biocatalysts system consisting of a monooxygenase-
containing microorganism and an alcohol dehydrogenase is
developed for the concurrent oxidations of methylene groups to
ketones in one pot, providing green, clean and simple access
to valuable ketones with high yield, excellent selectivity and
efficient cofactor recycling.
of methylene groups to carbonyl groups may be achieved by
using enzymes as green catalysts and molecular oxygenase as
green oxidant. However, all such biotransformations reported
thus far are based on the use of a single microorganism as
catalyst and gave incomplete reaction with a mixture of alcohol
and ketone as the product,6 possibly due to an undesired ratio of
the necessary enzymes, side-reactions and complicated co-factor
balance in cells. Here, we report a novel tandem biocatalysts
system for green, selective and clean oxidations of methylene
groups to carbonyl groups via concurrent biooxidations in one
pot. This system consists of a monooxygenase-containing micro-
organism to catalyze the selective hydroxylation of a methylene
group to an alcohol and an alcohol dehydrogenase to catalyze
the subsequent oxidation of the alcohol to the corresponding
ketone with co-factor recycling (Scheme 1), where the suitable
biocatalysts for each step are selected and arrayed at an
appropriate ratio for the efficient concurrent biooxidations.
To prove the concept, the selective benzylic oxidation of
tetralin 1a to 1-tetralone 3a was selected as a model reaction
(Scheme 2a). 1-Tetralone 3a is an important pharmaceutical
intermediate,7 and its synthesis via chemical or biological
oxidation of tetralin 1a was unsatisfactory.8,9 For concurrent
biooxidations, Pseudomonas monteilii TA-510 was chosen to
catalyze the benzylic hydroxylation of 1a to 1-tetralol 2a.
While the monooxygenase is difficult to be practically used
in the cell-free form, cells of TA-5 strain were used as the
hydroxylation catalyst. Based on the screening of several
commercially available alcohol dehydrogenases for the oxidation
of 2a to 3a, Lactobacillus kefir alcohol dehydrogenase
(LKADH) was selected as the second catalyst. LKADH
accepts a broad variety of substrates,11 thus the method
developed here could be generally useful. To be practical,
Tandem biocatalysis in one pot enables green and selective
cascade reactions without the time-consuming, energy-intensive
and yield-reducing isolation of intermediates,1 thus being an
important tool for sustainable chemical and pharmaceutical
synthesis.2 While microorganisms can metabolize natural
substrates to produce useful compounds via tandem biocatalysis,
it is difficult to use non-natural substrates for microbial synthesis
in this way due to the lack or the undesired ratio of the necessary
enzymes in the microorganism. Alternatively, one-pot non-
natural cascade transformations may be achieved by using
tandem biocatalysts which are selected for an individual reaction
and combined in a desired form and at a desired ratio. Several
examples have been reported, including the deracemization of a
secondary alcohol3a,b or a-hydroxy acid3c using an oxidase and a
reductase and the asymmetric dihydroxylation of an aryl olefin
using a monooxygenase and an epoxide hydrolase.3d
We are interested in developing a novel tandem-biocatalysts
system for concurrent biooxidations in one pot for organic
synthesis. Selective oxidation of methylene groups (C–H bonds)
to carbonyl groups is chosen as the target reaction, since it is a
potentially very useful reaction in chemical syntheses but remains
as a significant challenge in classic chemistry. Several transition
metals were reported to catalyze the oxidation of non-activated
methylene groups to ketones,4 which suffered from poor
selectivity and generated a mixture of several products. Better
results were obtained for the oxidation of activated methylene
groups such as benzylic C–H bonds,5 but requiring the use of
toxic oxidants and metals. On the other hand, selective oxidation
a Department of Chemical and Biomolecular Engineering,
National University of Singapore, 4 Engineering Drive 4,
Singapore 117576. E-mail: chelz@nus.edu.sg; Fax: +65 6779 1936;
Tel: +65 6516 8416
b Singapore-MIT Alliance, 4 Engineering Drive 3, Singapore 117576
c Department of Chemical Engineering, Massachusetts Institute of
Technology, Cambridge, MA 02139, USA
w Electronic supplementary information (ESI) available: Experimental
procedures and analytical methods. See DOI: 10.1039/c0cc04706f
Scheme 1 Green, clean and selective oxidations of methylene groups
to ketones with tandem biocatalysts in one pot.
c
3284 Chem. Commun., 2011, 47, 3284–3286
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acetone in Tris–HCl buffer (Table 1, entry 5). As shown in the
time course of the reaction (see Fig. 2 in ESIw), the concen-
tration of 2a quickly reached maximum at 3 h and decreased
afterwards, while the concentration of 3a continuously
increased during the whole reaction period. This indicates
clearly concurrent biooxidations. At 30 h, 4.98 mM 3a was
obtained as the only product, giving rise to a clean reaction
with 83% yield and 499% regioselectivity. The TTN for
recycling NADP+ reached 4100, which is high enough for
practical application. The final product was identified by
LC-MS analysis and compared with the commercially
available standard 3a.
The same tandem biocatalysts system was also used for the
concurrent biooxidations of indan 1b to 1-indanone 3b
(Scheme 2a), an important intermediate in the synthesis of
serotonin reuptake inhibitors.12 Cells of P. monteilii TA-5
catalyzed the hydroxylation of 1b to (R)-1-indanol 2b with
an activity of 14 U gÀ1 cdw, 499% regioselectivity and good
enantioselectivity. LKADH showed an activity of 3.5 U gÀ1
protein for the oxidation of (R)-2b to 3b, and the enzymatic
reduction of 3b to 2b was eliminated by adding acetone.
Concurrent biooxidations of 1b with tandem biocatalysts in
one pot under the best conditions gave clean conversion
to 3b, with 87% yield, 499% regioselectivity and 4200 times
recycling of NADP+ (Table 1, entry 7).
Scheme
2 Concurrent oxidations of benzylic or non-activated
methylene groups to ketones with tandem biocatalysts in one pot.
acetone was used as the coupled substrate for LKADH to
regenerate the co-factor NADP+ for the oxidation of 2a.
The activity of the biocatalyst for each reaction step was
separately examined. Resting cells of P. monteilii TA-5 showed
an activity of 12 U gÀ1 cdw for the hydroxylation of tetralin 1a
to (R)-2a with 499% regioselectivity and good enantio-
selectivity. These cells catalyzed also the oxidization of
(R)-2a to 3a, but the activity is rather low (0.41 U gÀ1 cdw).
LKADH demonstrated an activity of 4.5 U gÀ1 protein for the
oxidization of (R)-2a to 3a and an activity of 250 U gÀ1
protein for the reduction of acetone to iso-propanol. The huge
activity difference allows for the efficient regeneration of
NADP+ during the oxidation of 2a to 3a. LKADH was also
found to catalyze the reduction of 3a to 2a with an activity of
0.80 U gÀ1 protein. However, this side reaction was successfully
eliminated by adding acetone as the coupled substrate.
The generality of the novel concept was demonstrated in a
much more challenging reaction: the selective oxidation of
non-activated methylene groups to ketones. The oxidation of
N-benzyl-piperidine 4 to N-benzyl-4-piperidone 6 were chosen
as the target reaction (Scheme 2b). Compound 6 is an
important intermediate for the production of several drugs13
and prepared via multi-step syntheses with low overall yield.14
The selective concurrent oxidations of 4 could give a simple
access to 6, which has not yet been reported. To set up the
tandem biocatalysts system, resting cells of Escherichia coli
(P450pyr) expressing the monooxygenase of Sphingomonas sp.
HXN-20015 was chosen as the hydroxylation catalyst, showing
an activity of 8.0 U gÀ1 cdw for the regioselective hydroxylation
of 4 to N-benzyl-4-hydroxy-piperidine 5 without the further
oxidation to 6 (Table 2, entry 1). NAD+-dependent alcohol
dehydrogenase RDR, known to catalyze the oxidation of
3-hydroxypyrrolidine to the corresponding pyrrolidinone,15b,16
was selected as the second catalyst for the oxidation of 5 to 6.
Histag-RDR was easily produced by E. coli (RDR) expressing
Concurrent biooxidations of 1a to 3a with a tandem-
biocatalysts system containing resting cells of P. monteilii
TA-5, LKADH, NADP+, and acetone were investigated at
different conditions, and the results are summarized in
Table 1. By adjusting the ratio of two catalysts, full conversion
of 1a to 3a was achieved with 3a as the sole product (Table 1,
entry 3 vs. 2). The TTN for NADP+ recycling, which was
calculated after deduction of the concentration of 3a directly
produced from TA-5 cells, was increased by changing the
amount of initially added NADP+ and LKADH (Table 1,
entries 3–5). The best result was obtained in the oxidations of
6 mM tetralin 1a with 15 g cdw LÀ1 of P. monteilii TA-5, 3.5 g
protein LÀ1 of LKADH, 0.001 mM NADP+, and 0.5% (v/v)
Table 1 Concurrent oxidations of tetralin 1a to 1-tetralone 3a and of indan 1b to 1-indanone 3b with tandem biocatalysts in one pot
TA-5/
g cdw LÀ1
LKADH/
g pro. LÀ1
NADP+
mM
/
Acetone
(v/v %)
3b/
mM
(R)-2b/
mM
1b/
mM
Regioselectivityc
(%)
Yield
(%)
Entrya
Substrate
TTNd
1
2
3
4
5
6
7
1a
1a
1a
1a
1a
1b
1b
10 + 5e
10 + 5e
10 + 5e
10 + 5e
10 + 5e
10 + 5e
10 + 5e
—
2
3
—
—
0.92
5.06
5.25
4.75
4.98
0.98
5.20
3.92
0.16
0
0.20
0
0.36
499
499
499
499
499
499
499
15
84
88
79
83
16
87
—
0.002
0.002
0.001
0.001
—
0.5
0.5
0.5
0.5
—
0
0
0
0
0
0
2100
2200
3800
4100
—
3
3.5
—
3.5
4.32
0
0.001
0.5
4200
a
All reactions were carried out with 6 mM 1 in Tris–HCl (100 mM, pH 7.0) buffer containing 1 mM MgCl2 at 30 1C and 250 rpm.
Concentrations of different compounds in the reaction mixture at 30 h. The regioselectivity is referred to the ratio of 1-tetralone 3a to 2-tetralone or
b
c
d
the ratio of 1-indanone 3b to 2-indanone. The concentration of 3 produced by TA-5 cells alone in the control reaction (entries 1 and 6) was deducted in
the calculation of TTN. Additional TA-5 cell pellets were added at 2 h to increase the cell density from 10 g cdw LÀ1 to 15 g cdw LÀ1
e
.
c
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Chem. Commun., 2011, 47, 3284–3286 3285

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Table 2 Concurrent oxidations of N-benzyl-piperidine 4 to N-benzyl-4-piperidone 6 with tandem biocatalysts in one pot
Entrya P450pyr/g cdw LÀ1 RDR/g pro. LÀ1 NAD+/mM Acetone (v/v %) 6b/mM 5b/mM 4b/mM Regioselectivityc (%) Yield (%) TTN
1
2
3
4
5
5
10
10
—
4
4
—
—
0
4.52
0.12
0
0
0.72
0
499
499
499
499
0
72
86
80
—
0.002
0.002
0.001
0.5
0.5
0.5
3.61
4.28
4.02
1800
2100
4000
4
0
0
a
All reactions were carried out with 5 mM 4 in KP (100 mM, pH 8.0) buffer containing 0.5% (w/v) glucose at 30 1C and 250 rpm.
Concentrations of different compounds in the reaction mixture at 25 h. The regioselectivity is referred to the ratio of 6 to N-benzyl-3-piperidone
or N-benzyl-2-piperidone.
b
c
histag-RDR15b and purified by using a Ni-NTA column (see
ESIw). The enzyme showed an activity of 54 U gÀ1 protein for
the oxidation of 5 to 6 and an activity of 480 U gÀ1 protein for
the reduction of acetone to iso-propanol, thus being suitable
for biooxidation of 5 to 6 with NAD+ recycling using acetone
as the coupled substrate. Similar to the case of LKADH,
histag-RDR did not catalyze the reduction of product 6 back
to 5 in the presence of 0.5% (v/v) of acetone.
cofactor balance, and the inefficient elimination of the reduction
of ketone to alcohol.
The financial support by the Ministry of Education of
Singapore through an AcRF Tier 1 Grant (Project No.:
R-279-000-239-112) and Singapore-MIT-Alliance through a
Flagship Research Program is gratefully acknowledged.
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3286 Chem. Commun., 2011, 47, 3284–3286
This journal is The Royal Society of Chemistry 2011