Synthesis of optically active dihydrocarveol via a stepwise or one-pot enzymatic reduction of (R)- and (S)-carvone

Article abstract of DOI:10.1016/j.tetasy.2012.05.019

A recombinant enoate reductase LacER from Lactobacillus casei catalyzed the reduction of (R)-carvone and (S)-carvone to give (2R,5R)-dihydrocarvone and (2R,5S)-dihydrocarvone with 99% and 86% de, respectively, which were further reduced to dihydrocarveols by a carbonyl reductase from Sporobolomyces salmonicolor SSCR or Candida magnolia CMCR. For (R)-carvone, (1S,2R,5R)-dihydrocarveol was produced as the sole product with >99% conversion, while (1S,2R,5S)-dihydrocarveol was obtained as the major product, but with a lower de when (S)-carvone was used as the substrate. The one-pot reduction was performed at a 0.1 M substrate concentration, indicating that it might provide an effective synthetic route to this type of chiral compound.

Full text of DOI:10.1016/j.tetasy.2012.05.019

Tetrahedron: Asymmetry 23 (2012) 734–738
Contents lists available at SciVerse ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of optically active dihydrocarveol via a stepwise or one-pot
enzymatic reduction of (R)- and (S)-carvone
⇑
Xi Chen, Xiuzhen Gao, Qiaqing Wu, Dunming Zhu
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao,
Tianjin Airport Economic Area, Tianjin 300308, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 March 2012
Accepted 15 May 2012
A recombinant enoate reductase LacER from Lactobacillus casei catalyzed the reduction of (R)-carvone and
(S)-carvone to give (2R,5R)-dihydrocarvone and (2R,5S)-dihydrocarvone with 99% and 86% de, respectively,
which were further reduced to dihydrocarveols by a carbonyl reductase from Sporobolomyces salmonicolor
SSCR or Candida magnolia CMCR. For (R)-carvone, (1S,2R,5R)-dihydrocarveol was produced as the sole prod-
uct with >99% conversion, while (1S,2R,5S)-dihydrocarveol was obtained as the major product, but with a
lower de when (S)-carvone was used as the substrate. The one-pot reduction was performed at a 0.1 M sub-
strate concentration, indicating that it might provide an effective synthetic route to this type of chiral
compound.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Carvone and related compounds are important components in
the flavor and fragrance industry.1 Dihydrocarvones are potential
inhibitors of bacterial and fungal growth, as well as prospective in-
sect repellents.2 For example, they have shown insecticidal activity
against the rice weevil Sitophilus oryzae (L.), one of the most wide-
spread insect pests of stored cereals.2,3 Recently, dihydrocarvones
have also been used as important renewable building blocks in
the synthesis of functional materials such as shape memory poly-
esters.4 Dihydrocarveol is a fragrance ingredient used in decorative
cosmetics, fine fragrances, shampoos, soaps and other toiletries as
well as in household products such as cleaners and detergents.5
Carvone is currently extracted from caraway, dill, and spearmint
seeds.1 While transformations of carvone into dihydrocarvones
and dihydrocarveol have been widely studied, the chemo-, regio-
and stereospecificity of these transformations still present a chal-
lenging task. (R)- or (S)-Carvone (Fig. 1), which possesses three
unsaturated bonds and a C-5 stereogenic center, possesses many
selectivity problems: (1) chemoselectivity between the C@C and
C@O functional groups; (2) regioselectivity between the conju-
gated and isolated C@C bond; and (3) cis versus trans diastereose-
lectivity with respect to the C-5 substituent in the C@O and C@C
bond reductions. With no control over selectivity, 17 different
hydrogenation products are possible.6 Bogel-Łukasik reported that
at least 10 products were generated in the hydrogenation of carv-
one catalyzed by Pd, Rh, or Ru supported on alumina as the catalyst
in supercritical carbon dioxide.7
O
O
(S)-carvone
(R)-carvone
Figure 1.
As a result, biocatalytic transformation of carvone has recently
been the focus of several papers.8–17 The bioreduction of (R)-carv-
one and (S)-carvone using whole cells of bacteria,13 fungi,12
yeast,17 plant cell cultures,15 and marine microalgae14 has already
been reported. The microbial reduction of these compounds usu-
ally gives a mixture of saturated ketone, saturated alcohol, and
occasionally the allylic alcohol, indicating that both enoate reduc-
tase and carbonyl reductase may catalyze the reduction of C@C and
C@O double bonds competitively. The use of isolated enzymes of-
fers several advantages including the elimination of undesirable
side product formation mediated by contaminating enzymes in
the whole cell system, the possibility of achieving a high substrate
load, easy downstream product separation, and easy handling for
organic chemists without microbiological knowledge. In recent
years, isolated reductase enzymes have been demonstrated to be
highly effective catalysts for the enantioselective reduction of a
wide range of substrates.18
In this context, isolated enoate and carbonyl reductases
were used in an attempt to address the chemo-, regio- and
stereoselectivity in the transformations of carvone into dihydro-
carvone, and then into dihydrocarveol.9,10 Herein we report the
⇑
Corresponding author. Tel.: +86 22 84861962; fax: +86 22 84861996.
E-mail address: zhu_dm@tib.cas.cn (D. Zhu).
0957-4166/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetasy.2012.05.019

X. Chen et al. / Tetrahedron: Asymmetry 23 (2012) 734–738
735
CMCR
LacER
O
O
HO
NADP+
NAD+
NADPH
NADH
(R)-carvone
cofactor recycling
cofactor recycling
LacER
SSCR
O
O
HO
NADP+
NAD+
NADPH
NADH
(S)-carvone
cofactor recycling
cofactor recycling
Scheme 1. The tandem reduction of carvone by enoate reductase and carbonyl reductase.
Table 1
two-step enzymatic reduction of (R)- and (S)-carvone to dihydro-
Stepwise enzymatic reduction of (R)-carvone to dihydrocarveol
carveol (Scheme 1). For the first hydrogenation step, carbon–carbon
double bond reduction, enoate reductase LacER from Lactobacillus
casei was used; for the second step, the carbonyl reduction, a reduc-
tase from Sporobolomyces salmonicolor SSCR or Candida magnolia
CMCR was employed. The reaction was evaluated by adding the
two enzymes sequentially or simultaneously, with the aim of devel-
oping a more effective process for the conversion of carvones into
enantiomerically pure dihydrocarveols.
Entry
Enzymea
Conversionb (%)
dec (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CMCR
SSCR
Q245P
Q245L
Q245H
M242Y
M242C
M242D
99
73
42
81
73
73
68
80
88
99
93
99
94
—
>99
98
93
99
66
99
99
99
99
99
97
98
96
—
M242Q
2. Results and discussion
M242CQ245L
M242LQ245T
M242FQ245T
M242LQ245P
Ymr226c
Gre2
2.1. Enzymatic reduction of (R)- and (S)-carvone to
dihydrocarvone
—
—
We have recently reported that LacER catalyzed the reduction
of (R)-carvone to give (2R,5R)-dihydrocarvone with 99% diastereo-
selective excess (de), which is one of the best results obtained so
far.19 When (S)-carvone was used as the substrate for this enzyme,
the configuration of newly formed C-2 stereomeric center of dihy-
drocarvone was also (R), with the de value being 86%. The results
showed that the C-5 configuration did not have much impact on
the stereochemistry of the hydride transfer in the LacER-catalyzed
reduction of carvone. This is consistent with the observations for
other enoate reductases such as pentaerythritol tetranitrate reduc-
tase PETNR from the anaerobic microorganism Enterobacter cloa-
cae st. PB2,9 a thermophilic ‘ene’ reductase (TOYE) isolated from
Thermoanaerobacter pseudethanolicus E3911 and Saccharomyces
pastorianus old yellow enzyme.10 Stewart et al. reported that
(2S,5S)-dihydrocarvone was obtained in 88% de from the reduction
a
b
c
See Section 4 for the source of these enzymes.
The conversion was measured by GC analysis.
The de values were measured by chiral GC analysis.
carbonyl reductases Ymr226c and Gre2 from Baker’s yeast showed
no detectable activity toward (2R,5R)-dihydrocarvone.
In the case of (S)-carvone as the substrate, it was reduced to
give both (2S,5S)-dihydrocarvone (7%) and (2R,5S)-dihydrocarvone
(93%). After the reaction was completed, the resulting mixture was
treated with the carbonyl reductase and a NADPH regeneration
system. The conversion for the carbonyl reduction and the de value
for the reduction of (2R,5S)-dihydrocarvone (major product) and
(2S,5S)-dihydrocarvone (minor product) are summarized in Table 2.
From Table 2, it can be seen that SSCR and its mutant enzymes
showed higher activity than CMCR, Ymr226c, and Gre2. In all
cases, the stereoselectivity for the reduction of (2R,5S)-dihydro-
carvone was higher than that for (2S,5S)-dihydrocarvone. For
example, when CMCR was used as the catalyst, the product
(1S,2R,5S)-dihydrocarveol was obtained in 99% de for the reduction
of (2R,5S)-dihydrocarvone, but was obtained in only 48% de for the
reduction of (2S,5S)-dihydrocarvone. SSCR and some mutant en-
zymes also catalyzed the reduction of (2R,5S)-dihydrocarvone to
give (1S,2R,5S)-dihydrocarveol in 99% de.
of (S)-carvone catalyzed by
a mutant enzyme (W116I) of
Saccharomyces pastorianus OYE.10
2.2. Stepwise enzymatic reduction of (R)- and (S)-carvone to
dihydrocarveol
(R)-Carvone was first reduced to (2R,5R)-dihydrocarvone by using
LacER as the catalyst. To the resulting reaction mixture,
D
-glucose dehydrogenase, NADP+, and the carbonyl reductase were
added to reduce the carbonyl group in dihydrocarvone, and the
results are presented in Table 1. As shown in Table 1, CMCR, SSCR,
and its mutant enzymes catalyzed the reduction of (2R,5R)-dihydro-
carvonetogive(1S,2R,5R)-dihydrocarveolwithhighstereoselectivity.
In the cases of CMCR and SSCR mutant M242CQ245L, (1S,2R,5R)-
dihydrocarveol was obtained with 99% conversion and 99% de. The
2.3. One-pot enzymatic reduction of (R)- and (S)-carvone to
dihydrocarveol
The C@C bond reduction of (R)-carvone and (S)-carvone by La-
cER and the following C@O reduction by carbonyl reductases were

736
X. Chen et al. / Tetrahedron: Asymmetry 23 (2012) 734–738
Table 2
Table 4
Stepwise enzymatic reduction of (S)-carvone to dihydrocarveol
One-pot enzymatic reduction of (S)-carvone to dihydrocarveol
Entry
Enzyme
Conversiona (%)
deb (%)
dec (%)
Entry
Enzyme
Conversiona (%)
deb (%)
dec (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CMCR
SSCR
Q245P
Q245L
Q245H
M242Y
M242C
M242D
68
99
80
96
83
97
94
99
97
99
99
85
99
7
99
99
87
79
82
99
99
97
98
94
78
91
89
76
18
48
75
68
4
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CMCR
SSCR
Q245P
Q245L
Q245H
M242Y
M242C
M242D
36
87
43
56
53
29
58
34
66
76
86
27
72
6
99
99
86
57
73
92
55
85
91
87
72
66
86
75
22
32
76
84
32
83
74
75
75
77
87
61
79
61
—
65
50
46
20
28
24
10
41
21
M242Q
M242Q
M242CQ245L
M242LQ245T
M242FQ245T
M242LQ245P
Ymr226c
Gre2
M242CQ245L
M242LQ245T
M242FQ245T
M242LQ245P
Ymr226c
Gre2
d
—
—
d
26
17
—
a
a
The conversion for the carbonyl reduction was measured by GC analysis.
The de values for the reduction of (2R,5S)-dihydrocarvone catalyzed by carbonyl
The conversion was for the carbonyl reduction of dihydrocarvone to dihydro-
b
carveol and measured by GC analysis. No (S)-carvone was detected after reaction.
b
reductase.
The de values were for the reduction of (2R,5S)-dihydrocarvone by carbonyl
c
The de values for the reduction of (2S,5S)-dihydrocarvone catalyzed by carbonyl
reductase.
reductase, and measured by chiral GC analysis.
c
The de values were for the reduction of (2S,5S)-dihydrocarvone by carbonyl
d
The conversion was so low that the de value was not determined.
reductase, and measured by chiral GC analysis.
also performed in a mode in which LacER, carbonyl reductase, and
the cofactor regeneration system were added into the reaction
mixture at the same time. LacER required NADH for the (R)- or
(S)-carvone reduction, while carbonyl reductase needed NADPH
for the dihydrocarvone reduction; hence GDH and glucose were
used for the regeneration of both NADH and NADPH. Since the
optimal pH for LacER was 8.0 and the pH was not adjusted after
the C@C bond reduction in the stepwise procedure described
above, while the following C@O reduction proceeded smoothly,
the one-pot reaction was thus performed at pH 8.0. The results
are presented in Tables 3 and 4. For both (R)-carvone and (S)-carv-
one, the C@C bond reduction proceeded faster than the carbonyl
reduction, as evidenced by the fact that both (R)-carvone and (S)-
carvone were converted into the dihydrocarvones in about 4 h.
The stereoselectivity for the C@C bond reduction was the same
as that in the stepwise reaction, and was not affected by the reac-
tion mode. The conversions in Tables 3 and 4 are for the carbonyl
reduction of dihydrocarvone to dihydrocarveol.
When LacER was combined with CMCR as the catalysts, (R)-carv-
one was exclusively converted into (1S,2R,5R)-dihydrocarveol. For
SSCR and its mutants, the product dihydrocarveol was obtained
with a lower de than those in stepwise reactions, implying that
the reaction mode had some effects on the stereoselectivity of
the carbonyl reduction of dihydrocarvone to dihydrocarveol cata-
lyzed by SSCR and its mutants. The carbonyl reductases Ymr226c,
and Gre2 showed no activity toward (2R,5R)-dihydrocarvone in the
one-pot reaction mode (Table 3, entries 14 and 15).
When (S)-carvone was used as the substrate, it was first re-
duced to (2R,5S)-dihydrocarvone and (2S,5S)-dihydrocarvone with
a ratio of about 93:7. The results in Table 4 show that the conver-
sions were generally lower than those in the stepwise reaction
(Table 2), while the stereoselectivity for the carbonyl reduction
was also influenced by the reaction mode for some carbonyl reduc-
tases. For example, with CMCR as the reductase, the product dihy-
drocarveol was obtained in only 36% conversion while the de for
the reduction of (2S,5S)-dihydrocarvone decreased from 48% to
32%. In the case of SSCR, while the product dihydrocarveol was ob-
tained with 87% conversion compared to the 99% conversion in the
stepwise reaction, the stereoselectivity was not affected. The (S)-
carvone could not be completely transformed into dihydrocarveol
via a one-pot reaction even with larger amount of the glucose,
GDH, SSCR, or CMCR.
As shown in Table 3, (R)-carvone was reduced to give
(1S,2R,5R)-dihydrocarveol and (1R,2R,5R)-dihydrocarveol with the
former being the major stereoisomer with de values of 52–99%.
Table 3
One-pot enzymatic reduction of (R)-carvone to dihydrocarveol
Entry
Enzyme
Conversiona (%)
deb (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CMCR
SSCR
Q245P
Q245L
Q245H
M242Y
M242C
M242D
>99
83
67
83
88
90
80
87
99
89
96
98
99
—
>99
94
88
93
67
75
74
69
52
88
75
66
82
—
2.4. Synthesis of (1S,2R,5R)-dihydrocarveol
The concomitant reduction of the C@O group was observed in
the reduction of (R)- and (S)-carvone by whole cells of Nicotiana
tabacum, Baker’s yeast, and other strains, but the conversions and
stereoselectivity were low.8,16,17,20 As described above, the one-
pot reduction of (R)-carvone by combining the enoate reductase
LacER and the carbonyl reductase CMCR generated exclusively
(1S,2R,5R)-dihydrocarveol with >99% conversion. This reaction
should be very useful for the synthesis of (1S,2R,5R)-dihydrocarve-
ol from the readily available (R)-carvone. The reaction was thus
carried out on a 30 mL scale with the substrate concentration being
0.02 M. (R)-Carvone was thus completely transformed into the
product. The substrate concentration was then increased to
0.1 M, the substrate was converted completely, while (1S,2R,5R)-
dihydrocarveol was isolated in 93% yield and with >99% de.
M242Q
M242CQ245L
M242LQ245T
M242FQ245T
M242LQ245P
Ymr226c
Gre2
—
—
a
The conversion was for the carbonyl reduction of dihydrocarvone to dihydro-
carveol and measured by GC analysis. No (R)-carvone was detected after reaction.
b
The de values were measured by chiral GC analysis.

X. Chen et al. / Tetrahedron: Asymmetry 23 (2012) 734–738
737
The enzymatic asymmetric reduction of (R)- or (S)-carvone to
dihydrocarvone has been previously reported, and many reports
have described the enzymatic asymmetric reduction of ketones
to the corresponding enantiomerically pure alcohols. To the best
of our knowledge, only one report is available with regard to the
combination of the asymmetric hydrogenation of a carbon–carbon
double bond and a carbon–oxygen double bond using isolated en-
zymes.21 Wada et al. reported that (4R,6R)-4-hydroxy-2,2,6-trim-
ethylcyclohexanone was produced at a concentration of 9.5 mg/
ml by a two-step enzymatic reduction of ketoisophorone with
94% enantiomeric excess in the presence of glucose, NAD+, and glu-
cose dehydrogenase. In that case, the enzymes must be added
stepwise because ketoisophorone could be reduced by levodione
reductase; the resulting 4-hydroxy-2,6,6-trimethyl-2-cyclohexe-
none would not be converted into actinol if the levodione reduc-
tase was added with the old yellow enzyme at the same time.21
Herein, the enoate and carbonyl reductases could be added at the
same time to allow the conversion of (R)- or (S)-carvone into dihy-
drocarveols. (R)-Carvone was completely converted into
(1S,2R,5R)-dihydrocarveol at a substrate concentration of 0.1 M in
13 h. Under the same conditions, if the enoate and carbonyl reduc-
tases were added in two steps, the reaction was finished after 24 h.
This suggests that the ‘one-pot’ reduction is more effective than the
stepwise procedure.
13.5 min; (2S,5S)-dihydrocarvone, 13.3 min. The absolute configu-
rations of the product alcohols were identified by comparing the
chiral GC data with the standard samples and the 1H NMR data
with those in the literature.20 (2R,5S)-Dihydrocarvone 1H NMR
(CDCl3, 400 MHz) d (ppm) 4.82 (s, 1H), 4.68 (s, 1H), 2.59–2.52
(m, 2H), 2.41–2.37 (m, 2H), 1.86–1.82 (m, 3H), 1.72 (s, 3H), 1.62–
1.58 (m, 1H), 1.08 (d, J = 4.8 Hz, 3H).
4.3. Stepwise enzymatic reduction of (R)- and (S)-carvone to
dihydrocarveol
The general experimental procedure was as follows: a (R)-carv-
one or (S)-carvone solution in DMSO (100
in a potassium phosphate buffer (0.9 mL, 100 mM, pH 8.0). Next,
-glucose (24 mg),
-glucose dehydrogenase (2 mg), NAD+ (1 mg),
and the enoate reductase (LacER, 2 mg) were added. After the mix-
ture was shaken at 37 °C for 4 h, -glucose dehydrogenase (2 mg),
lL, 0.20 M) was mixed
D
D
D
NADP+ (1 mg), and the carbonyl reductase (2 mg) were added. The
mixture was shaken at 37 °C for another 24 h. The mixture was ex-
tracted with methyl tert-butyl ether (1 mL). The organic extract
was dried over anhydrous sodium sulfate and then subjected to chi-
ral GC analysis to determine the diastereomeric excess. (2R,5R)-
dihydrocarvone and (2R,5S)-dihydrocarvone were reduced by
NaBH4 to give two sets of diastereomer mixtures, which served as
the standard samples for GC analysis. Their retention times were
as follows: (1S,2R,5R)-dihydrocarveol, 16.6 min; (1R,2R,5R)-dihy-
3. Conclusion
drocarveol,
17.2 min;
(1S,2R,5S)-dihydrocarveol,
20.0 min;
(1R,2R,5S)-dihydrocarveol, 19.3 min. The absolute configurations
of the product alcohols were identified by comparing the chiral GC
data with the standard samples and the 1H NMR spectra with those
in the literature.20 (1S,2R,5R)-Dihydrocarveol 1H NMR (CDCl3,
400 MHz) d (ppm) 4.69 (s, 2H), 3.88 (s,1H), 2.29 (t, J = 8 Hz, 1H),
1.93–1.90 (dd, J = 1.6 Hz, 1H), 1.77–1.75 (m,1H), 1.71 (s, 3H), 1.50–
1.40 (m, 5H), 1.84 (m,1H), 0.97 (d, J = 4.8 Hz, 3H). 13C NMR (CDCl3,
100 MHz) d (ppm) 150.26, 108.38, 70.99, 38.69, 37.82, 36.08,
31.41, 28.14, 20.96, 18.29. (1S,2R,5S)-Dihydrocarveol 1H NMR
(CDCl3, 400 MHz) d (ppm) 4.69 (d, J = 2.8 Hz, 2H), 3.78 (m, 1H),
2.07 (m, 1H), 1.96(m, 1H), 1.71(s, 3H), 1.70–1.62 (m, 2H), 1.55–
1.49 (m, 1H), 1.44–1.38 (m, 2H), 1.34–1.27 (m, 1H), 0.93–0.92 (d,
J = 4.8 Hz, 3H). 13C NMR (CDCl3, 100 MHz) d (ppm) 149.48, 108.55,
72.70, 44.02, 34.05, 33.68, 30.58, 24.59, 20.88, 10.69.
(R)- and (S)-Carvone were reduced by LacER to (2R,5R)-dihy-
drocarvone and (2R,5S)-dihydrocarvone with de values of 99%
and 86%, respectively. The resulting dihydrocarvones were reduced
stereoselectively to give dihydrocarveols by carbonyl reductases,
CMCR, SSCR, or its mutant enzymes. In particular, (1S,2R,5R)-dihy-
drocarveol was produced in high yield by combining the C@C and
C@O reductions in one-pot reactions, in which LacER and CMCR
were added at the same time or stepwise. The one-pot reduction
was carried out at a 0.1 M substrate concentration, thus demon-
strating its applicability in the synthesis of (1S,2R,5R)-dihydrocar-
veol from renewable (R)-carvone.
4. Experimental
4.1. General
4.4. One-pot enzymatic reduction of (R)- and (S)-carvone to
dihydrocarveol
LacER, CMCR, SSCR, and its mutants, Gre2, Ymr226c, and GDH
were prepared as previously reported.19,22–25 NAD+, NADH, NADP+,
and NADPH were obtained from Codexis (USA). (R)-Carvone,
(S)-carvone, (+)-(2R,5R)-dihydrocarvone, and (1R,2R,5R)-dihydro-
carveol were obtained from Sigma–Aldrich Chemical Co. The GC
analyses were performed on an Agilent 7890 gas chromatography
The NADH and NADPH regeneration system containing NAD+,
NADP+, GDH, and glucose was used for the reduction of carvone
and dihydrocarvone. An (R)-carvone or (S)-carvone solution in
DMSO (100
lL, 0.20 M) was mixed in a potassium phosphate buffer
(0.9 mL, 100 mM, pH 8.0), after which
D
-glucose (24 mg), -glucose
D
with CD-Chiral-DEX CB (30 m  0.25 mm  0.25
lm) column,
dehydrogenase (4 mg), NADP+ (1 mg), NAD+ (1 mg), the enoate
reductase (LacER, 2 mg), and the carbonyl reductase (CMCR,
2 mg) were added. The mixture was then shaken at 37 °C for
24 h. The mixture was extracted with methyl tert-butyl ether
(1 mL). The organic extract was dried over anhydrous sodium sul-
fate and then subjected to chiral GC analysis to determine the dia-
stereomeric excess. The absolute configurations of the product
alcohols were identified as described above.
detector temperature 220 °C, split ratio 20:1. Programme: 70 °C,
hold for 2 min, 5 °C/min to 120 °C, hold for 12 min.
4.2. Reduction of (S)-carvone to dihydrocarvone catalyzed by
LacER
The general procedure was as follows:
cose dehydrogenase (2 mg), NAD+ (1 mg), the enoate reductase
(LacER, 2 mg), and (S)-carvone solution in DMSO (100 L, 0.20 M)
D-glucose (12 mg), D-glu-
l
4.5. Synthesis of (1S,2R,5R)-dihydrocarveol
were mixed in a potassium phosphate buffer (0.9 mL, 100 mM,
pH 8.0) and the mixture was shaken at 37 °C for 4 h. The mixture
was extracted with methyl tert-butyl ether (1 mL). The organic
extract was dried over anhydrous sodium sulfate and then sub-
jected to chiral GC analysis to determine the conversion and
diastereomeric excess. Retention times: (2R,5S)-dihydrocarvone,
AtÀ1first,
D
-glucose (110 g LÀ1),
D-glucose dehydrogenase
(2 g L ), NAD+ (1 g LÀ1), NADP+ (1 g LÀ1), enoate reductase enzyme
(2 g LÀ1), and carbonyl reductase (2 g LÀ1) were dissolved in potas-
sium phosphate buffer (100 mM, pH 8.0, 27 mL). The resulting solu-
tion was mixed with 3 mL of (R)-carvone solution in DMSO (1.0 M).

738
X. Chen et al. / Tetrahedron: Asymmetry 23 (2012) 734–738
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The reaction mixture was shaken at room temperature for 24 h. The
reduction was completed as shown by GC analysis. The mixture was
extracted with methyl tert-butyl ether (3 Â 30 mL). The organic ex-
tract was dried over anhydrous sodium sulfate and removal of the
solvent gave a yellow oil (429.5 mg, 93% yield), which was identi-
fied as (1S,2R,5R)-dihydrocarveol with >99% de.
Acknowledgments
This work was financially supported by the National Basic
Research Program of China (973 Program, No. 2011CB710801),
Chinese Academy of Sciences and Tianjin Municipal Science &
Technology Commission (10 ZCZDSY06600).
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