Asymmetric trans-dihydroxylation of cyclic olefins by enzymatic or chemo-enzymatic sequential epoxidation and hydrolysis in one-pot

Article abstract of DOI:10.1039/c1gc15501f

Novel and efficient one-pot enzymatic and chemo-enzymatic synthetic methods are developed for the asymmetric trans-dihydroxylations of cyclic olefins 1a and 1bvia sequential epoxidation and hydrolysis. The Novozym 435 -mediated epoxidation of cyclohexene 1a and subsequent hydrolysis of the intermediate cyclohexene oxide 2a with resting cells of Sphingomonas sp. HXN-200 in one-pot gave (1R,2R)-cyclohexane diol 3a in 84% ee and 95% conversion. trans-Dihydroxylation of N-benzyloxycarbonyl 3-pyrroline 1b with the same enzymatic system gave the corresponding (3R,4R)-N- benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 3b in 93% ee and 94% conversion. In the one-pot chemo-enzymatic system, epoxidation of N-benzyloxycarbonyl 3-pyrroline 1b by m-CPBA and subsequent hydrolysis of epoxide intermediate 2b with resting cells of Sphingomonas sp. HXN-200 gave the trans-diol (3R,4R)-3b in 92% ee and 94-97% conversion. While the trans-dihydroxylation of cyclohexene 1a to (1R,2R)-cyclohexane diol 3a is reported for the first time, the trans-dihydroxylation of N-benzyloxycarbonyl 3-pyrroline 1b to (3R,4R)-3b with such an enzymatic or chemo-enzymatic system afforded a much higher product concentration than the same reaction with the system using a microorganism containing the two necessary enzymes. The developed one-pot enzymatic and chemo-enzymatic systems for the asymmetric trans-dihydroxylation of olefins are new, easy to prepare, adjust and operate, are high yielding, complementary to Sharpless asymmetric dihydroxylation and particularly useful for the asymmetric synthesis of cyclic trans-diols.

Full text of DOI:10.1039/c1gc15501f

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PAPER
Asymmetric trans-dihydroxylation of cyclic olefins by enzymatic or
chemo-enzymatic sequential epoxidation and hydrolysis in one-pot†
Yi Xu, Aitao Li, Xin Jia and Zhi Li*
Received 4th May 2011, Accepted 7th June 2011
DOI: 10.1039/c1gc15501f
Novel and efficient one-pot enzymatic and chemo-enzymatic synthetic methods are developed for
the asymmetric trans-dihydroxylations of cyclic olefins 1a and 1b via sequential epoxidation and
R
hydrolysis. The Novozym 435ꢀ-mediated epoxidation of cyclohexene 1a and subsequent
hydrolysis of the intermediate cyclohexene oxide 2a with resting cells of Sphingomonas sp.
HXN-200 in one-pot gave (1R,2R)-cyclohexane diol 3a in 84% ee and 95% conversion.
trans-Dihydroxylation of N-benzyloxycarbonyl 3-pyrroline 1b with the same enzymatic system
gave the corresponding (3R,4R)-N-benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 3b in 93% ee and
94% conversion. In the one-pot chemo-enzymatic system, epoxidation of N-benzyloxycarbonyl
3-pyrroline 1b by m-CPBA and subsequent hydrolysis of epoxide intermediate 2b with resting cells
of Sphingomonas sp. HXN-200 gave the trans-diol (3R,4R)-3b in 92% ee and 94–97% conversion.
While the trans-dihydroxylation of cyclohexene 1a to (1R,2R)-cyclohexane diol 3a is reported for
the first time, the trans-dihydroxylation of N-benzyloxycarbonyl 3-pyrroline 1b to (3R,4R)-3b with
such an enzymatic or chemo-enzymatic system afforded a much higher product concentration
than the same reaction with the system using a microorganism containing the two necessary
enzymes. The developed one-pot enzymatic and chemo-enzymatic systems for the asymmetric
trans-dihydroxylation of olefins are new, easy to prepare, adjust and operate, are high yielding,
complementary to Sharpless asymmetric dihydroxylation and particularly useful for the
asymmetric synthesis of cyclic trans-diols.
appropriate, active and selective enzymes for the desired multi-
Introduction
step reaction. The appropriate enzymes for individual reaction
The preparation of fine chemicals and pharmaceuticals often
steps may be selected and combined to enable the multi-step
involves complex multi-step syntheses, which require expensive
biotransformation of non-natural substrates in one-pot. Up to
isolation and purification of intermediates, and generate a
now, there have been only a few such examples of enzymatic
large amount of waste.1 A potential economic and sustainable
multi-step syntheses from non-natural substrates in one-pot.2–3
solution is to perform multi-step reactions in one-pot. A key
criterion for such a process is the compatibility of the individual
We have been interested in developing the asymmetric trans-
dihydroxylation of olefins via one-pot sequential epoxidation
reactions. Chemical reactions are often carried out under differ-
and hydrolysis for the preparation of optically active vici-
ent conditions, thus being difficult to be performed in one-pot.
nal diols, which are useful and valuable fine chemicals and
On the other hand, enzymatic transformations, which are non-
pharmaceutical intermediates. This type of transformation is
toxic and often highly selective, have similar reaction conditions
unique and different from asymmetric cis-dihydroxylation with
and therefore are preferred for one-pot reactions. Multi-step
Sharpless’ catalyst4 or a dioxygenase.5 Rapid dihydroxylation
enzymatic reactions are common in microbial cells during the
of alkenes was achieved by using a lipase and hydrogen
metabolism of natural compounds by living microorganisms.
peroxide under microwave conditions,6 which was elegant but
However, when non-natural compounds are used as substrates
for chemical synthesis, a microorganism often does not have the
not enantioselective. Previously, we discovered a bacterial strain
Sphingomonas sp. HXN-200 containing the necessary monooxy-
genase and epoxide hydrolase for the trans-dihydroxylation
of specific olefins with high enantioselectivity.7 However, the
efficiency and generality of using a wild-type microorganism for
this type of transformation may be limited by the availability
of the appropriate monooxygenase and epoxide hydrolase at
the desired ratio within the cells. Recently, we developed 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
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1gc15501f
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Scheme 1 The asymmetric trans-dihydroxylation of cyclohexene 1a and N-benzyloxycarbonyl 3-pyrroline 1b via a one-pot sequential epoxidation
and hydrolysis: (a) enzymatic system and (b) chemo-enzymatic system.
novel and more general approach for the asymmetric di-
hydroxylation of styrenes to prepare the corresponding (S)-
vicinal diols in high ee and good yield by using a tandem
biocatalyst system combining styrene monooxygenase and
epoxide hydrolase from different microorganisms.8 Here, we
report our recent success on the development of asymmetric
trans-dihydroxylations of cyclic olefins via sequential epoxi-
dation and hydrolysis in one-pot by two new systems: an
enzymatic one, with lipase-mediated epoxidation and epoxide
hydrolase-catalyzed hydrolysis, and a chemo-enzymatic one,
with epoxidation by m-CPBA and hydrolysis via epoxide
hydrolase.
Lipase-mediated epoxidation of cyclohexene 1a
A high yield of cyclohexene oxide 2a can be achieved by
the lipase-mediated epoxidation of cyclohexene 1a in toluene,
chloroform or dichloromethane.11a However, these solvents are
very toxic to microbial cells and thus are not suitable for
the subsequent enzymatic hydrolysis step. The lipase-mediated
epoxidation of 1a was then investigated in acetonitrile11b since
it has some compatibility with the enzymatic system. Lauric
acid (LA), which showed a high reaction rate in the formation
of peroxy acid,11c was selected as the perhydrolysis substrate.
R
The easily available Novozym 435ꢀ (Candida Antarctica lipase
B immobilized on macroporous acrylic resin) was chosen as the
enzyme. The epoxidation of cyclohexene 1a was thus performed
by using a different amount of cyclohexene 1a, H2O2, Novozym
Results and discussions
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435ꢀ and LA in acetonitrile for 24 h (Table 1). Excellent yields of
cyclohexene oxide 2a were obtained in all cases (Table 1, entries
1–5). The epoxidation of 0.5 M of 1a with 0.25 M LA and 1.25 M
Cyclohexene 1a and N-benzyloxycarbonyl 3-pyrroline 1b were
selected as model substrates for trans-dihydroxylations (Scheme
1) since they represent two different types of cyclic alkene and
the corresponding trans-diols, (1R,2R)-3a and (3R,4R)-3b, are
useful pharmaceutical intermediates that are difficult to make.
As starting cyclic alkenes 1a and 1b are symmetric, Sharpless
dihydroxylations of these compounds would give achiral cis-
diols. On the other hand, the trans-dihydroxylation of 1a and
1b via epoxidation and hydrolysis affords trans-vicinal diols 3a
and 3b, respectively. Since the epoxidation step does not involve
any enantioselectivity, efficient but less enantioselective oxida-
tion systems such as lipase-mediated oxidation9 and chemical
oxidation with m-CPBA were selected to produce achiral meso-
epoxides 2a and 2b. The ee of the final products 3a and 3b
was determined by the enantioselectivity of the hydrolysis step,
thus the enantioselective epoxide hydrolase of Sphingomonas sp.
HXN-200 was selected for the catalytic hydrolysis.7,10 The epox-
idation and hydrolysis were firstly investigated separately and
then coupled in one-pot for the desired trans-dihydroxylation of
alkenes 1a and 1b.
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H2O2 in the presence of 25 g L-1 Novozym 435ꢀ gave nearly a
100% yield of cyclohexene oxide 2a (Table 1, entry 5).
Lipase-mediated epoxidation of N-benzyloxycarbonyl
3-pyrroline 1b
The lipase-mediated epoxidation of N-containingolefins is much
more difficult, possibly due to the existence of basic nitrogen
groups.12 Epoxidation of N-benzyloxycarbonyl 3-pyrroline 1b
using the same conditions optimized for the epoxidation of
cyclohexene 1a afforded only 37% of 2b after 48 h, and increasing
the reaction time to 96 h only improved the conversion to
55% (Table 1, entries 6 and 7). Further increasing the acid
concentration would increase the conversion. However, it was
impossible to do so with lauric acid due to its limited solubility
in acetonitrile. Other organic acids such as 6-hydroxy caporic
acid (HCA), octanoic acid (OA) and phenyl acetic acid (PAA)
were thus used at 1.0 M, respectively. While the first two acids did
not show any significant improvement (Table 1, entries 8–11),
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Table 1 Lipase-mediated epoxidation of cyclohexene 1a and N-benzyloxycarbonyl 3-pyrroline 1b
Entry Sub.a
Sub. conc./M
H2O2/M
Novozym 435ꢀR /g L-1
Organic acidb
Acid conc./M
Time/h
Prod.
Conv. (%)
1
2
3
4
5
6
7
8
1a
1a
1a
1a
1a
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
1b
0.25
0.25
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.50
0.625
0.625
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25
1.25
100
100
100
50
25
25
25
25
25
25
25
25
25
25
LA
LA
LA
LA
LA
LA
LA
HCA
HCA
OA
0.125
0.25
0.25
0.25
0.25
0.25
0.25
1.0
1.0
1.0
1.0
1.0
24
24
24
24
24
48
96
48
96
72
144
72
144
72
2a
2a
2a
2a
2a
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
2b
97
99
99
99
99
37
55
29
46
50
69
70
82
81
88
96
9
10
11
12
13
14
15
16
OA
PAA
PAA
PAA
PAA
PAA
1.0
1.5
1.5
1.5
1.25
1.25
25
144
144
1.25 + 0.50c
25 + 10c
a A mixture of cyclic olefin 1a or 1b, H2O2 (50% aqueous solution), organic acid and Novozym 435ꢀR in acetonitrile (1 mL) was shaken at 300 rpm
and 30 ◦C. b LA: lauric acid; HCA: 6-hydroxy caproic acid; OA: octanoic acid; PAA: phenyl acetic acid. c Another portion of H2O2 and Novozym
435ꢀR was added after 72 h.
Table 2 Epoxidation of N-benzyloxycarbonyl 3-pyrroline 1b by m-CPBA
Entry
Sub.a
Sub. conc./M
m-CPBA/M
Reaction medium
Time/h
Prod.
Conv. (%)
1
2
3
4
5
6
7
1b
1b
1b
1b
1b
1b
1b
0.10
0.10
0.10
0.10
0.10
0.20
0.04
0.20
0.20
0.20
0.20
0.15
0.30
0.06
Acetonitrile
Acetonitrile
Hexane
Hexane
KPBb
KPB
KPB
4.0
72
2.5
4.0
4.0
4.0
6.0
2b
2b
2b
2b
2b
2b
2b
46
99
92
98
99
99
99
a A mixture of cyclic olefin 1b and m-CPBA in a 1 mL reaction medium was shaken at 300 rpm and 30 ◦C. b KPB: potassium phosphate buffer
(50 mM, pH 8.0).
the last one demonstrated a higher conversion (Table 1, entries
12 and 13). Increasing the PAA concentration to 1.5 M afforded
epoxide 2b in 82% conversion after 72 h and 88% after 144 h
(Table 1, entries 14 and 15). Under the same initial conditions,
the conversion was further increased to 96% after 144 h by the
addition of a second portion of H2O2 and enzyme at 72 h (Table
1, entry 16). To the best of our knowledge, this is the first example
of a lipase-mediated epoxidation of an N-containing olefin.
and excess hydrogen peroxide were used in the enzymatic
epoxidation of 1a and 1b, the effects of acetonitrile and hydrogen
peroxide on the hydrolysis of the corresponding meso-epoxides
2a and 2b with the epoxide hydrolase of Sphingomonas sp. HXN-
200 were studied. When the hydrolysis of 2a was carried out with
resting cells of Sphingomonas sp. HXN-200 in the presence of
50 mM of H2O2, no inhibitory effect on enzyme activity by H2O2
was observed (Table 3, entries 1, 2, 4 and 5). Later on, we found
that whole cells of Sphingomonas sp. HXN-200 could decompose
some of the H2O2, indicating the possible existence of catalase in
the cells. While no inhibition of 2% acetonitrile on the enzymatic
hydrolysis was observed (Table 3, entries 3 and 1), the presence
of 5% acetonitrile resulted in some inhibition of the enzymatic
hydrolysis of 2a and nearly no inhibition of the enzymatic
hydrolysis of 2b (Table 3, entries 4 and 8). A big inhibition
was observed with 10% acetonitrile (Table 3, entries 6 and 9)
Epoxidation of N-benzyloxycarbonyl 3-pyrroline 1b by m-CPBA
Chemical epoxidation with m-CPBA as the oxidant is usually
carried out in dichloromethane.13 However, dichloromethane is
very toxic to the second step enzymatic hydrolysis. Thus, the
epoxidation of 1b with m-CPBA was examined at different ratios
in other reaction media, including hexane, acetonitrile and an
aqueous buffer. As listed in Table 2, the oxidations with m-
CPBA were faster than the lipase-mediated examples in all cases.
Hexane and aqueous buffer were much better solvents for the
oxidation. Complete conversion of 0.2 M 1b was achieved with
0.3 M m-CPBA in 50 mM aqueous potassium phosphate buffer
(pH = 8.0) in only 4 h.
Asymmetric trans-dihydroxylation of cyclohexene 1a and
N-benzyloxycarbonyl 3-pyrroline 1b by a one-pot enzymatic
sequential epoxidation and hydrolysis
To perform the sequential epoxidation and hydrolysis of cyclo-
hexene 1a in one-pot, the epoxidation of 1a was first carried out
by lipase-mediated epoxidation under the established conditions
(Table 1, entry 5). A mixture of cyclohexene 1a (0.5 mmol),
H2O2 (50% aqueous solution, 1.25 mmol), LA (0.25 mmol) and
Enantioselective hydrolysis of alicyclic meso-epoxides 2a/b with
the epoxide hydrolase of Sphingomonas sp. HXN-200
Hydrolytic reactions7,10 were investigated with the view of
coupling them with the first epoxidation step. Since acetonitrile
2454 | Green Chem., 2011, 13, 2452–2458
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Table 3 Enantioselective hydrolysis of cyclic meso-epoxides 2a and 2b with resting cells of Sphingomonas sp. HXN-200
Entry
Sub.a
Sub. conc./mM
HXN-200/g cdw L-1
ACN (%)
H2O2/mM
Time/h
Product
Conv. (%)
ee (%)
1
2
3
4
5
6
7
8
9
2a
2a
2a
2a
2a
2a
2b
2b
2b
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
15
15
15
0
0
2
5
5
10
2
5
10
0
50
0
0
50
0
0
0
0
48
48
48
48
48
48
28
28
28
(1R,2R)-3a
(1R,2R)-3a
(1R,2R)-3a
(1R,2R)-3a
(1R,2R)-3a
(1R,2R)-3a
(3R,4R)-3b
(3R,4R)-3b
(3R,4R)-3b
99
99
99
75
76
26
99
97
2.2
85
85
85
85
85
85
93
93
93
a A mixture of cyclic meso-epoxide 2a or 2b and resting cells of Sphingomonas sp. HXN-200 in 10 mL potassium phosphate buffer (100 mM, pH 8.0)
was shaken at 300 rpm and 25 ◦C for 2a, and 30 ◦C for 2b, respectively.
Table 4 The asymmetric trans-dihydroxylation of cyclohexene 1a and N-benzyloxycarbonyl 3-pyrroline 1b by one-pot enzymatic or chemo-enzymatic
sequential epoxidation and hydrolysis
H2O2/Novozym
Conc./
M
Vol./ 435ꢀR /acid
m-CPBA/ Time/ HXN-200
Total
Time/
h
Conc./Conv. ee
Prod. mM (%) (%)
Entry Sys.a Sub.
Sol. mL (M/g L-1/M)
M
h
cellsb/g L-1 Sol. vol./mL
1
2
3
4
5
6
EE
EE
CE
CE
CE
CE
1a
1b
1b
1b
1b
1b
0.50
0.50
0.10
0.20
0.02
0.04
ACN 1.0
ACN 1.0
KPB 1.0
KPB 1.0
KPB 5.0
KPB 5.0
1.25/25/0.25
1.75/35/1.5
24
144
4
4
4
24
15
15
25
21
30
KPBc 50
KPB 50
KPB 10
KPB 10
KPB 10
KPB 10
48
24
24
45
16
20
3a
3b
3b
3b
3b
3b
9.5
9.4
9.6
18.8 94
9.4 94
19.4 97
95
94
96
84
93
92
92
92
92
0.20
0.30
0.03
0.06
6
a EE: enzymatic system; CE: chemo-enzymatic system. b Cell suspension was added after the finishing of the first reaction, resulting in an increase in
the total reaction volume. For CE, sodium sulfite was added before the addition of the cell suspension. c KPB: potassium phosphate buffer (50 mM,
pH 8.0).
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Novozym 435ꢀ (25 mg) in acetonitrile (1 mL) was shaken at
300 rpm and 30 ◦C for 24 h. The conversion of cyclohexene
oxide 2a was over 99%. Then, a cell suspension of Sphingomonas
sp. HXN-200 (49 mL, 24 g cdw ◦L-1) was added, and the
mixture shaken at 300 rpm and 25 C. After 30 h of reaction,
the concentration of (1R,2R)-cyclohexane 1,2-diol 3a reached
9.5 mM, corresponding to an overall conversion of 95% based on
the cyclohexene 1a initially added (Table 4, entry 1). The ee value
of (1R,2R)-3a was 84%, which was nearly the same as that (85%
ee) obtained in the single step hydrolysis of cyclohexene oxide
2a with resting cells of Sphingomonas sp. HXN-200. The ee of
3a might be further improved by using other epoxide hydrolases
with higher enantioselectivities.14
Recycling of biocatalysts in the asymmetric
trans-dihydroxylation of cyclohexene 1a by a one-pot enzymatic
sequential epoxidation and hydrolysis
R
To reduce the cost of the immobilized enzyme (Novozym 435ꢀ)
and to explore the reuse of whole cells, catalyst recycling
was performed in the asymmetric trans-dihydroxylation of
cyclohexene 1a. The oxidation of cyclohexene (0.5 mmol) with
50% hydrogen peroxide (1.25 mmol), lauric acid (0.25 mm) and
R
Novozym 435ꢀ (25 mg) in acetonitrile (1 mL) was performed
at 300 rpm and 30 ◦C for 12 h, followed by the addition of a
cell suspension of Sphingomonas sp. HXN-200 (49 mL, 24 g
cdw L-1) for the hydrolysis reaction for another 48 h. After
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this time, Novozym 435ꢀ was separated by removal of the cell
Similarly, the trans-dihydroxylation of N-benzyloxycarbonyl
3-pyrroline 1b in one-pot was performed by the epoxidation of
suspension, and washed with de-ionized water and then cold
acetone. After drying, the recovered Novozym 435ꢀ was used for
the next reaction cycle. From the cell suspension, the cells were
separated from the supernatant after centrifugation, washed
with de-ionized water and re-used in the next reaction cycle.
As shown in Fig. 1a, the recovered Novozym 435ꢀ gave nearly
the same high conversion for the epoxidation of cyclohexene 1a
to cyclohexene oxide 2a in each cycle and remained at 98% of the
original productivity after four cycles of trans-dihydroxylation
experiments. The results demonstrate that the immobilized
enzyme is stable in the current one-pot reaction system, and that
such an efficient recycling and effective reuse can significantly
reduce its cost. The results also increase the greenness of the
R
R
1b (0.5 M) using Novozym 435ꢀ (15 + 10 g L-1), H2O2 (1.25
+ 0.50 M) and PAA (1.5 M) for 144 h, and the subsequent
addition of a cell suspension of Sphingomonas sp. HXN-200
(15 g cdw L-1) for a 24 h hydrolysis reaction (Table 4, entry
2). (3R,4R)-N-Benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 3b
was obtained in 9.4 mM, corresponding to a 94% total
conversion and in 93% ee, the same ee value as that achieved
by the enzymatic enantioselective hydrolysis of meso-epoxide
2b with resting cells of Sphingomonas sp. HXN-200. Simple
purification by chromatography on a short silica gel column
gave 82 mg (3R,4R)-3b in 98% purity with a 71% isolated
yield.
R
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Conclusions
Asymmetric trans-dihydroxylations of cyclic olefins 1a and 1b
were successfully achieved by a one-pot enzymatic or chemo-
enzymatic sequential epoxidation and hydrolysis. The Novozym
R
435ꢀ-mediated epoxidation of cyclohexene 1a and subsequent
hydrolysis of the intermediate cyclohexene oxide 2a with resting
cells of Sphingomonas sp. HXN-200 in one-pot gave (1R,2R)-
cyclohexane diol 3a in 85% ee and 95% conversion. The trans-
dihydroxylation of N-benzyloxycarbonyl 3-pyrroline 1b with the
same enzymatic system gave (3R,4R)-N-benzyloxycarbonyl-3,4-
dihydroxy-pyrrolidine 3b in 93% ee and 94% conversion. The
compatibility problem caused by the organic solvent used for
the oxidation was successfully solved by the efficient epoxidation
of the substrate at a high concentration in a small amount of
Fig. 1 Recycling and reuse of biocatalysts in the asymmetric trans-
dihydroxylation of cyclohexene 1a to 3a: (a) Novozym 435ꢀR and (b)
cells of Sphingomonas sp. HXN-200.
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solvent. Novozym 435ꢀ was efficiently recycled and effectively
reused, with no significant decrease of productivity after 4 cycles.
On the other hand, cells of Sphingomonas sp. HXN-200 were
also recycled and reused, retaining 60% of their productivity
from the previous cycle. In the one-pot chemo-enzymatic
system, the epoxidation of N-benzyloxycarbonyl 3-pyrroline
1b by m-CPBA and subsequent hydrolysis of epoxide 2b with
resting cells of Sphingomonas sp. HXN-200 gave (3R,4R)-N-
benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 3b in 92% ee and
94–97% conversion. Both reactions were carried out in the same
aqueous buffer system, and the compatibility problem caused
by the unreacted m-CPBA was easily solved by the addition of
Na2SO3. While the trans-dihydroxylation of cyclohexene 1a to
(1R,2R)-cyclohexane 1,2-diol 3a is reported for the first time,
the trans-dihydroxylation of N-benzyloxycarbonyl 3-pyrroline
1a to (3R,4R)-N-benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine
3b with either an enzymatic or chemo-enzymatic system af-
forded much higher product concentrations than the same
reaction with a bacterial strain containing the two necessary
enzymes. Compared with the two-step synthesis procedure, the
one-pot approach avoids the isolation of the epoxide inter-
mediate.
current method. On the other hand, the recovered cells of
Sphingomonas sp. HXN-200 could also be recycled and reused
to some extent. As shown in Fig. 1b, the cells showed 60% of the
productivity achieved in the previous cycle for the hydrolysis of
cyclohexene oxide 2a to the corresponding diol 3a. The decrease
of activity is understandable, since cells are generally less stable
than immobilized biocatalysts. To achieve full conversion, some
new cells could be added. As cells are easily available and cheap,
the use of cells as biocatalysts remains practical. Of course, it
would be interesting to further develop the immobilized epoxide
hydrolase as a second biocatalyst for one-pot asymmetric trans-
dihydroxylations.
Asymmetric trans-dihydroxylation of N-benzyloxycarbonyl
3-pyrroline 1b by a one-pot chemo-enzymatic sequential
epoxidation and hydrolysis
There are only limited reports on chemo-enzymatic reactions
in one-pot,15 possibly due to the compatibility problem. Simply
coupling of the two reactions by the epoxidation of 0.10 M N-
benzyloxycarbonyl 3-pyrroline 1b with 0.20 M m-CPBA in 1 mL
KP buffer for 4 h, followed by hydrolysis with the addition of a 9
mL cell suspension (15 g L-1) of Sphingomonas sp. HXN-200 for
24 h, did not give any diol product 3b. The enzymatic activity
was possibly destroyed by the unreacted m-CPBA remaining
in the reaction system. To solve this problem, Na2SO3 was
added to remove the unreacted m-CPBA after completion
of the epoxidation reaction, and cells of Sphingomonas sp.
HXN-200 were then added to start the second hydrolysis
reaction. By following this procedure, the trans-diol (3R,4R)-N-
benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 3b was obtained
as the only product in 96% conversion and 92% ee (Table 4, entry
3). When the initial concentration of 1b was doubled (Table 4,
entry 4), (3R,4R)-3b was obtained in 92% ee and 94% conversion,
corresponding to a final product concentration of 20 mM,
which is 10 times higher than that previously reported with a
bacterial strain containing the two necessary enzymes.7 When
the volumetric ratio of the two sequential reactions was changed
from 1 : 10 to 1 : 1 (Table 4, entries 5 and 6), similar results were
obtained: (3R,4R)-3b was obtained in 94–97% conversion and
92% ee.
The developed one-pot enzymatic and chemo-enzymatic
systems are novel for the asymmetric trans-dihydroxylation of
olefins. They are better than the only other known system
comprising a bacterial strain containing the two necessary
enzymes, with easy selection and combination of the appropriate
systems for the desired individual reactions, and easy adjust-
ment of the individual systems for complete conversion. The
asymmetric trans-dihydroxylation methods reported here are
complementary to the Sharpless asymmetric dihydroxylation,
being particularly useful for the asymmetric synthesis of cyclic
trans-diols. The principles and knowledge obtained here could
be generally useful for the development of asymmetric trans-
dihydroxylations of other types of olefins by the selection
and combination of appropriate tandem enzymes or chemo-
R
enzymatic systems. While oxidations with Novozym-435ꢀ or
m-CPBA work with a variety of olefins, the substrate scope
for the enantioselective hydrolysis could be broadened by using
different epoxide hydrolases. In addition to the existing ones,
new and appropriate epoxide hydrolases could be obtained by
enzyme engineering. It would also be interesting to develop the
immobilized epoxide hydrolase as the second biocatalyst for one-
pot asymmetric trans-dihydroxylations.
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different time intervals and mixed with 198 mL ethyl acetate
containing 1 mM dodecane. After centrifugation at 15000 rpm
for 10 min, the supernatant was analyzed by GC to quantify the
conversion of 1a to 2a and of 1b to 2b, respectively.
Experimental
Materials
R
Novozym 435ꢀ (Candida Antarctica lipase B immobilized on
macroporous acrylic resin with a specific activity of 10 000 U
g-1 catalyst for the synthesis of propyl laurate from lauric acid
and 1-propanol) was purchased from Novozymes. Ethyl acetate
(>99%) was purchased from Merck. Cyclohexene (>99.5%), cy-
clohexene oxide (98%), N-benzyloxycarbonyl 3-pyrroline (90%),
lauric acid (98%), phenylacetic acid (>99%) and octanoic acid
(98%) were purchased from Sigma-Aldrich. H2O2 (50% w/w)
was obtained from Analar. 3-Chloroperoxybenzoic acid (m-
CPBA) (ca. 70%) was purchased from Fluka. 6-Hydroxycaproic
acid (95%) was bought from Alfa Aesar. Acetonitrile (>99%)
was purchased from Fisher. Hexane (95%) was purchased from
Tedia. Sodium sulfite (98%) was obtained from Acros.
General procedure for the enantioselective hydrolysis of
cyclohexene oxide 2a and N-benzyloxycarbonyl 3,4-epoxy
pyrrolidine 2b with resting cells of Sphingomonas sp. HXN-200
The hydrolysis of epoxide 2a or 2b (10 mM) was performed
with frozen/thawed cells (10–15 g cdw L-1) of Sphingomonas
sp. HXN-200 in 10 mL 100 mM KP buffer (pH 8.0) at 300
rpm. The reaction temperature was 25 ◦C for 2a and 30 ◦C
for 2b, respectively. For GC analysis of the conversions of 2a
to 3a, as well as the ee of 3a, 200 mL aliquots were taken at
predetermined time points, followed by removal of the cells via
centrifugation and extraction with ethyl acetate (1 : 1) containing
2 mM n-dodecane as an internal standard. For HPLC analysis
of the conversion of 2b to 3b, an analytic sample was prepared
by taking 100 mL aliquots, removing cells by centrifugation and
mixing 20 mL of the supernatant with 180 mL ethanol containing
1 mM benzyl alcohol as an internal standard. For chiral HPLC
analysis of the ee of 3b, the sample was prepared by taking
200 mL aliquots, removing the cells via centrifugation, adding
sodium chloride and extracting with ethyl acetate (1 : 1).
Analytical methods
GC analysis was performed by using an Agilent 7890A gas
chromatograph with an HP-5 column (30 m ¥ 0.32 mm ¥
0.25 mm). Temperature program: 4◦0 ◦C for 1 min, then to 140 ◦C
◦
◦
at 12 C min-1 and finally to 280 C at 50 C min-1. Retention
times: 3.6 min for 1a, 5.6 min for 2a, 8.2 min for 3a and 9.3
min◦for n-dodecane (internal◦standar◦d). Temperature program:
150 C for 1 min, then to 280 C at 10 C min-1. Retention times:
2.8 min for n-dodecane (internal standard), 7.0 min for 1b, 8.6
min for 2b and 11.0 min for 3b.
The asymmetric trans-dihydroxylation of cyclohexene 1a and
N-benzyloxycarbonyl 3-pyrroline 1b by enzymatic sequential
epoxidation and hydrolysis in one-pot
HPLC analysis was carried out by using a ShimadzuTM
Prominence HPLC with a Hypersil BDS-C18 column (4.0 ¥
125 mm, 5 mm) and UV detection at 210 nm. Mobile phase: 5%
ACN/95% water from 0 to 5 min, changed to 55% ACN/45%
water from 5 to 10 min and remaining at 55% ACN/45% water
from 10 to 16 min; flow rate, 1.0 mL min-1. Retention time: 11.3
min for benzyl alcohol (internal standard), 12.4 min for 3b, 13.7
for 2b and 14.9 min for 1b.
The asymmetric trans-dihydroxylation of cyclohexene 1a to
prepare (1R,2R)-cyclohexane 1,2-diol 3a. A mixture of cyclo-
hexene 1a (0.5 mmol), H2O2 (50% aqueous solution, 1.25 mmol),
R
lauric acid (0.25 mmol) and Novozym 435ꢀ (25 mg) in acetoni-
trile (1 mL) was shaken at 300 rpm and 30 ◦C for 24 h. A 49 mL
suspension of frozen/thawed cells of Sphingomonas sp. HXN-
200 (24 g cdw L-1) in 100 mM KP buffer (pH 8.0) was added to
the reaction mixture. The reaction was incubated at 300 rpm and
25 ◦C for 48 h. (1R,2R)-Cyclohexane 1,2-diol 3a was formed in
84% ee, as determined by GC analysis with a chiral column, and
in 95% overall conversion, as measured by GC analysis.
The ee of 3a was determined by GC analysis with a chiral
column (Supelco, b-DEXTM 120, 30 m ¥ 0.25 mm ¥ 0.25 mm).
◦
Temperature program:◦from 100 ◦C to 157 ◦C at 3 C min-1,
then to 200 ◦C at 30 C min-1. Retention time: 17.4 min for
(1S,2S)-3a and 17.7 min for (1R,2R)-3a.
The ee of 3b was determined by HPLC analysis with a chiral
column (Chiralpak AS, 250 mm ¥ 4.6 mm). Mobile phase:
n-hexane–isopropanol (95 : 5), flow rate: 1.0 mL min-1,UV
detection at 210 nm. Retention time: 53.2 min for (3R,4R)-3b
and 66.4 min for (3S,4S)-3b.
The asymmetric trans-dihydroxylation of N-benzyloxycarbonyl
3-pyrroline 1b to prepare (3R,4R)-N-benzyloxycarbonyl-3,4-
dihydroxy-pyrrolidine 3b. A mixture of N-benzyloxycarbonyl
3-pyrroline 1b (0.5 mmol), H2O2 (50% aqueous solution,
R
1.25 mmol), phenyl acetic acid (1.5 mmol) and Novozym 435ꢀ
(25 mg) in acetonitrile (1 mL) was shaken at 300 rpm and 30 ◦C
for 72 h. Another portion of H2O2 (50% aqueous solution,
Bacterial strain and growth medium
The strain Sphingomonas sp. HXN-200 was cultivated in E2
media using n-octane as the carbon source according to a
published procedure.16
R
0.5 mmol) and Novozym 435ꢀ (10 mg) was added to the
reaction mixture, and the reaction continued for another 72 h.
After adjusting the pH to 8 by adding an Na2CO3 aqueous
solution (1 M), a 49 mL suspension of frozen/thawed cells of
Sphingomonas sp. HXN-200 (15 g cdw L-1) in 50 mM KP buffer
(pH 8.0) was added to the reaction mixture. The reaction was
performed at 300 rpm and 30 ◦C for 24 h, which gave (3R,4R)-N-
benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 3b in 93% ee and
94% overall conversion, as analysed by HPLC with a chiral
column and a C18 column, respectively.
General procedure for the lipase-mediated epoxidation of
cyclohexene 1a and N-benzyloxycarbonyl 3-pyrroline 1b
A mixture of olefin 1a or 1b (0.5 mmol), H2O2 (50% aqueous
solution, 1.25 mmol), organic acid (0.125–1.5 mmol) and
R
Novozym 435ꢀ (10–250 mg) in different organic solvents (1 mL)
was shaken at 300 rpm and 30 ◦C. 2 mL samples were taken at
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To purify the product, cells were removed from the biotrans-
formation mixtures via centrifugation, and the product extracted
into ethyl acetate. The organic phase was separated, dried over
MgSO4 and the solvent removed by evaporation. Purification by
column chromatography on silica gel (Rf = 0.27, ethyl acetate)
afforded 81.8 mg (70.7%) of (3R,4R)-3b in 93% ee and 98%
purity.
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A mixture of N-benzyloxycarbonyl 3-pyrroline 1b (0.2 mmol)
and m-CPBA (0.3 mmol, 1.5 equiv.) in KP buffer (pH 8.0, 1 mL)
was shaken at 300 rpm and 30 ◦C for 4 h. After adding 1 M
Na2SO3 aqueous solution (1 equiv.), the mixture was stirred for
20 min to remove the unreacted m-CPBA. 9 mL suspension of
frozen-thawed cells of Sphingomonas sp. HXN-200 (25 g cdw
L-1) in 50 mM KP buffer (pH 8.0) was then added. The mixture
was incubated at 300 rpm and 30 ◦C for 45 h. This gave (3R,4R)-
N-benzyloxycarbonyl-3,4-dihydroxy-pyrrolidine 3b in 92% ee,
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Acknowledgements
Financial support from the Ministry of Education of Singapore
through an AcRF Tier 1 Grant (Project no. R-279-000-239-112)
is gratefully acknowledged.
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2458 | Green Chem., 2011, 13, 2452–2458
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The Royal Society of Chemistry 2011
©