Cascade biotransformations via enantioselective reduction, oxidation, and hydrolysis: Preparation of (R)-δ-lactones from 2-alkylidenecyclopentanones

Article abstract of DOI:10.1021/cs400101v

The first cascade biotransformation involving enantioselective reduction of a C=C double bond, Baeyer-Villiger oxidation, and lactone hydrolysis was developed as a green and sustainable tool for synthesizing enantiopure δ-lactones. One-pot cascade biotransformations were achieved with Acinetobacter sp. RS1 containing a novel enantioselective reductase and an enantioselective lactone hydrolase and Escherichia coli coexpressing cyclohexanone monooxygenase and glucose dehydrogenase, converting easily available 2-alkylidenecyclopentanones 1-2 into the corresponding valuable flavors and fragrances (R)-δ-lactones 5-6 in high ee. The one-pot synthesis is better than the reported two-step preparation. This concept is useful in developing other redox cascades with the substrates containing C=C double bond.

Full text of DOI:10.1021/cs400101v

Letter
pubs.acs.org/acscatalysis
Cascade Biotransformations via Enantioselective Reduction,
Oxidation, and Hydrolysis: Preparation of (R)‑δ-Lactones from
‑Alkylidenecyclopentanones
2
†
,†
Ji Liu and Zhi Li*
†Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576
*
S Supporting Information
ABSTRACT: The first cascade biotransformation involving
enantioselective reduction of a CC double bond, Baeyer−
Villiger oxidation, and lactone hydrolysis was developed as a
green and sustainable tool for synthesizing enantiopure δ-
lactones. One-pot cascade biotransformations were achieved
with Acinetobacter sp. RS1 containing a novel enantioselective
reductase and an enantioselective lactone hydrolase and
Escherichia coli coexpressing cyclohexanone monooxygenase
and glucose dehydrogenase, converting easily available 2-
alkylidenecyclopentanones 1−2 into the corresponding
valuable flavors and fragrances (R)-δ-lactones 5−6 in high
ee. The one-pot synthesis is better than the reported two-step
preparation. This concept is useful in developing other redox
cascades with the substrates containing CC double bond.
KEYWORDS: cascade biotransformation, bioreduction, Baeyer−Villiger oxidation, lactone hydrolysis, enantioselective synthesis,
δ-lactone
onducting cascade reactions in one pot has become a
useful tool in sustainable chemical synthesis because it
could avoid time-consuming and yield-reducing isolation and
Scheme 1. One-Pot Reduction−Oxidation−Hydrolysis
Cascade for the Preparation of (R)-δ-Lactones 5−6 from 2-
Alkylidenecyclopentanones 1−2
C
purification of intermediates, minimize waste generation and
1
−4
energy consumption, and reduce production cost.
In
comparison with chemical reactions that are often performed
at different conditions, enzymatic conversions are more suitable1
for one-pot cascades because of the similar reaction conditions.
Moreover, enzymatic reactions are environmentally friendly
and often enantioselective. Therefore, a number of cascade
biotransformations have been developed for non-nature
5
−11
synthesis;
however, there is increasing demand to invent
new types of cascade biotransformations to broaden the
synthetic application scope.
We have been interested in developing novel one-pot cascade
9
−11
biotransformations for enantioselective syntheses.
Here, we
report the first enantioselective reduction−oxidation−hydrol-
ysis cascade for the synthesis of (R)-2-alkyl-δ-lactones 5-6 from
the corresponding 2-alkylidenecyclopentanones 1−2 (Scheme
The enantioselective bioreduction of 1−2 is the key step in
the reduction−oxidation−hydrolysis cascade. Thus far, bio-
catalytic redox cascades rely mainly on the use of alcohol
dehydrogenase as the reducing enzyme. Despite big achieve-
ments of biocatalysis in past decades, enantioselective
1
). δ-Lactones 5−6 are useful flavor and fragrance materials,
and they are produced chemically as a result of the low
1
1
2,13
concentrations in nature.
It was found that different
14
enantiomers of δ-lactones gave different odors. In the only
known enantioselective synthesis, optically active 5−6 were
produced chemically from the easily available starting materials
bioreduction of the CC double bond still remains a
1
−2 via catalytic hydrogenation and Baeyer−Villiger (BV)
Received: February 6, 2013
Revised: April 1, 2013
Published: April 8, 2013
oxidation involving the use of high pressure H ; a toxic and
expensive metal catalyst; and a toxic oxidant, m-CPBA.
2
14
©
2013 American Chemical Society
908
dx.doi.org/10.1021/cs400101v | ACS Catal. 2013, 3, 908−911

ACS Catalysis
Letter
Table 1. Individual Catalytic Step Involved in the Reduction−Oxidation−Hydrolysis Cascade
a
b
sub.
concn (mM)
catalyst (g cdw/L)
prod
spec. act. (U/g cdw)
time (h)
conv (%)
ee (%)
s
ee (%)
p
1
5
9
12 (cat. A)
12 (cat. A)
12 (cat. A)
12 (cat. A)
2.4 (cat. B)
10 (cat. B)
2.4 (cat. B)
10 (cat. B)
4 (cat. A)
(R)-3
(R)-3
(R)-4
(R)-4
(S)-5
(±)-5
(S)-6
(±)-6
7
8.8
8.5
4.7
4.5
28
2
100
100
100
100
39
87
80
92
88
97
1
2
2
5
5
2
7
5
(
(
(
(
(
(
±)-3
5
0.5
1
53
57
±)-3
±)-4
±)-4
±)-5
±)-6
5
100
42
5
30
0.5
1
85
5
100
55
10
10
22
22
1
79
73
4 (cat. A)
8
1
55
a
Cat. A: Acinetobacter sp. RS1; Cat. B: E. coli (CHMO−GDH). Biotransformation was performed in 5 mL Tris buffer (50 mM, pH7.5) containing 20
b
mg/mL glucose at 30 °C, 300 rpm. Specific activity was measured for the first 30 min of the biotransformation.
significant challenge. 5−18 The reductions of 2-methylidenecy-
clopentanone and 2-propylidenecyclopentanone to the corre-
sponding (S)-enantiomers of the products were reported with
1
Acinetobacter sp. RS1 and E. coli (CHMO−GDH) were
combined to perform the cascade reduction−oxidation−
hydrolysis in one pot. Initial experiments showed that E. coli
(CHMO−GDH) had also unexpected oxidation activity on
substrates 1 and 2 (Supporting Information); thus, the one-pot
17,19
Baker’s yeast and a Synechococcus strain, respectively,
but
with long reaction time, low activity and conversion, and
unsatisfactory enantioselectivity. No enzyme has been reported
for the reduction of 1−2 to 3−4, respectively. We initially
examined the reduction of 1−2 with the well-known old yellow
1
cascades were carried out in a subsequent manner. Reduction
of 6 mM 1 with resting cells of Acinetobacter sp. RS1, followed
by the oxidation with resting cells of E. coli (CHMO−GDH)
gave 83% (R)-5 in 98% ee, and the cascade biotransformation
of 5.5 mM 2 afforded 66% (R)-6 in 97% ee.
The courses of the cascade biotransformations were
investigated in detail. As shown in Figure 1a, substrate 1 was
1
6−18
enzymes;
however, no products could be detected.
Screening of microorganisms was then conducted to discover
an appropriate enzyme for the reduction of 1−2. Of 200
alkane-, benzene-, or toluene-degrading strains collected in our
2
0,21
lab,
9 isolates were identified with the desired activity to
reduce 1 and 2. Among them, Acinetobacter sp. RS1 showed
good activity, fast cell growth, and high enantioselectivity
(Supporting Information). Biotransformation of 5 mM 1 and 2
with the resting cells of the strain gave a specific activity of 8.8
U/g cdw and 4.7 U/g cdw, respectively (1 U = 1 μmol
product/min). Reactions for 2 h afforded (R)-3 in 87% ee and
(R)-4 in 92% ee, both with 100% conversion (Table 1).
Obviously, Acinetobacter sp. RS1 contains a unique reductase
that catalyzes the enantioselective reduction of the CC
double bond. Reduction of 9 mM 1 and 7 mM 2 also gave
100% conversion, with slightly lower product ee. Acinetobacter
sp. RS1 was, hence, chosen for the reduction step in the
designed redox cascades. Its R selectivity allows for the access
22
to (R)-δ-lactones, even with nonselective BV reaction.
Recombinant Escherichia coli strains expressing cyclohex-
anone monooxygenase (CHMO) or cyclopentanone mono-
22−25
oxygenase (CPMO), two well-known BVMOs,
engineered for the Baeyer−Villiger (BV) oxidation of 3−4 to
−6. Although no activity from E. coli (CPMO) was observed,
were
5
Figure 1. Time course of the subsequent one-pot cascade reduction−
oxidation−hydrolysis for the preparation of (R)-5 from 1. (a)
Bioreduction of 1 to 3 by cells of Acinetobacter sp. RS1. (b)
Simultaneous oxidation of 3 to 5 by cells of E. coli (CHMO−GDH)
and hydrolysis of 5 by cells of Acinetobacter sp. RS1.
E. coli (CHMO) showed the desired oxidation activity with S
enantioselectivity. E. coli strain coexpressing CHMO and
glucose dehydrogenase (GDH) from Bacillus subtilis was then
engineered as a more efficient whole-cell catalyst for the BV
26,27
oxidations through intracellular cofactor recycling.
As
shown in Table 1, oxidation of 5 mM racemic 3 and 4 with
cells (2.4 g cdw/L) of E. coli (CHMO−GDH) in the presence
of glucose for 0.5 h afforded 39% (S)-5 in 97% ee and 42% (S)-
rapidly reduced with the resting cells of Acinetobacter sp. RS1
within 2 h to give 95% (R)-3 in 83% ee. In the subsequent
oxidation with resting cells of E. coli (CHMO−GDH), both
enantiomers of 3 were converted to 5. The small amount of
6
in 85% ee. Incubation for 1 h at a cell density of 10 g cdw/L
gave 100% conversion in both cases. This indicated a
reasonable activity of E. coli (CHMO−GDH) for the oxidation
of (R)-3 and (R)-4. Since no R-selective BVMOs could be
found for the oxidation of 3−4, CHMO was chosen as the
second step enzyme in the redox cascade.
(
S)-3 generated in the first step was preferentially oxidized to
(S)-5 as a result of the S selectivity of CHMO, leading to a low
ee of (R)-5 at the early stage of the oxidation shown in Figure
1b. Later on, (R)-5 was continuously produced from (R)-3. At
1.5 h, the intermediate 3 was nearly fully converted, giving 91%
22
9
09
dx.doi.org/10.1021/cs400101v | ACS Catal. 2013, 3, 908−911

ACS Catalysis
Letter
Table 2. Cascade Reduction, Oxidation, And Hydrolysis for the Preparation of (R)-δ-Lactones 5−6 from 2-
Alkylidenecyclopentanones 1−2
a
b
sub. concn (mM)
catalyst A
vol (mL) time (h)
catalyst B
vol (mL) time (h)
prod
yield (%)
76
ee (%)
1
1
2
2
6
Acinetobacter sp. RS1
Acinetobacter sp. RS1
Acinetobacter sp. RS1
5
50
5
3
3
3
3
E. coli (CHMO−GDH)
E. coli (CHMO−GDH)
E. coli (CHMO−GDH)
E. coli (CHMO−GDH)
15
150
15
3
(R)-5
(R)-5
(R)-6
(R)-6
98
98
97
97
c
6
1.5
3
83 (56)
5.5
5.5
51
c
Acinetobacter sp. RS1
50
150
1.5
66 (41)
a
b
c
Cell density of 12 g cdw/L was used. Catalyst added after the completion of the first step. Cell density of 10 g cdw/L was used. Numbers in
parentheses represent the isolated yield.
(
R)-5 in 98% ee. Continuing the reaction to 3 h decreased the
synthetic application of this new type of cascade will also be
explored with other substrates. The concept is useful in
developing other types of redox cascades with CC double
bond-containing substrates.
yield to 76% while a high ee value was retained. A similar
phenomenon was also observed for the cascade transformation
of 2 to (R)-6 (Supporting Information).
Because E. coli (CHMO−GDH) showed S enantioselectivity
for the BV oxidation of 3−4, the ee values of the products (R)-
ASSOCIATED CONTENT
■
5
and (R)-6 in the redox cascade could not exceed those of the
*
S
Supporting Information
intermediates (R)-3 and (R)-4 obtained from the first step
reaction. In both cascade cases, however, the ee values of the
products (R)-5−6 were higher than those of (R)-3−4,
indicating the possible enantioselective degradation of the
lactones 5−6 to improve the ee values. To examine this
possibility, racemic 5−6 was incubated with resting cells of
Acinetobacter sp. RS1. S-Enantioselective hydrolysis of 5−6 was
observed, with an E of 8−11 (Table 1). The enzymatic
degradation products were confirmed to be the hydroxyacids
Experimental procedures including microbial screening, genetic
engineering, single step biotransformation, and one-pot cascade
biotransformation; analytic methods; chiral GC, chiral HPLC,
and LC−MS chromatograms. This information is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
*
Corresponding Author
E-mail: chelz@nus.edu.sg.
7
−8 by LC−MS analysis and by comparison with the products
from chemical hydrolysis (Supporting Information). It is also
Notes
worth mentioning that the enzymatic hydrolysis of δ-lactones is
The authors declare no competing financial interest.
2
8−30
rare,
and Acinetobacter sp. RS1 showed much higher
hydrolysis activity (22 U/g cdw) toward the racemic 5−6 than
ACKNOWLEDGMENTS
■
28
the previously reported enzymes.
This work was supported by the Science & Engineering
Research Council of A*STAR, Singapore, through a research
grant (Project No. 1021010026).
Preparative cascade biotransformations were carried out
(Table 2). The cascade catalysis was started with 50 mL of cell
suspension of Acinetobacter sp. RS1 in Tris buffer (12 g cdw/L)
containing 40 mg 1−2 and 20 mg/mL glucose. After 3 h of
reaction, 150 mL of Tris buffer containing cells of E. coli
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