Stereoselective approach to (2R,3S)- and (2R,3R)-1,2-(cyclohexylidenedioxy) hept-6-en-3-ol by microbial reduction

Article abstract of DOI:10.1016/j.molcatb.2012.05.017

Among twelve incubated whole-cell yeast strains, two were found to selectively reduce (R)-1,2-(cyclohexylidenedioxy)hept-6-en-3-one, which was derived from d-mannitol. Pichia minuta JCM 3622 and Rhodotorula mucilaginosa NBRC 0889 afforded (2R,3S)-form (97

Full text of DOI:10.1016/j.molcatb.2012.05.017

Journal of Molecular Catalysis B: Enzymatic 82 (2012) 8–11
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Journal of Molecular Catalysis B: Enzymatic
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Letter
Stereoselective approach to (2R,3S)- and (2R,3R)-1,2-(cyclohexylidenedioxy)hept-6-en-3-ol by microbial reduction
a r t i c l e i n f o
a b s t r a c t
Keywords:
Whole-cell biocatalyst
Yeast
Among twelve incubated whole-cell yeast strains, two were found to selectively reduce (R)-1,2-
(cyclohexylidenedioxy)hept-6-en-3-one, which was derived from d-mannitol. Pichia minuta JCM 3622
and Rhodotorula mucilaginosa NBRC 0889 afforded (2R,3S)-form (97% diastereomeric purity) and
(2R,3R)-form (89% diastereomeric purity) of 1,2-(cyclohexylidenedioxy)hept-6-en-3-ols, respectively. As
discussed above, the complementary yeast-mediated reduction provided the two diastereomeric glycerol
derivatives in high enantiomeric excess.
Reduction
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Resource Center; Department of Biotechnology, National Institute
of Technology and Evaluation, 2-5-8 Kazusakamatari, Kisarazu-shi,
Stereocontrolled forms of substituted 1,2,3-triols such as 1 have
been recognized as versatile starting materials for natural product
syntheses [1]. Originally, isopropylideneglycerol derivatives from
d-mannitol had often been employed for natural product syntheses
such as azaspiracid-1 [2] and brevenal [3]. As the common precur-
sor of isopropylideneglycelaldehyde [(R)-2a] is volatile, the heavier
cyclohexylideneglycelaldehyde [(R)-2b] [4] was later developed,
which has the advantage on handling in evaporation under reduced
pressure (Scheme 1).
Cyclohexylidene derivative 2b was used for zinc-mediated ally-
lation and propargylation to afford the corresponding alcohols even
in aqueous medium [5]. The stereoselectivity of alkylation with
other organometallic reagents such as homoallylmagnesium bro-
mide, however, was not high (Scheme 1) [6]. We embarked on the
specific approach to each diastereomer based on whole-cell yeast-
catalyzed stereoselective reduction of the corresponding ketone
(R)-3 [7] (Scheme 1). Some compounds, which have ketones with
protected oxygen functionalities on the adjacent position, have so
far been substrates for yeast-catalyzed stereoselective reduction
[8–12]. The substrate [(R)-3], however, has a bulky cyclic sub-
stituent in its skeleton, we compared several incubated whole-cell
yeast strains [13,14] to find out the proper biocatalysts.
Chiba 292-0818, Japan.
2.1. (2R,3S)-1,2-(Cyclohexylidenedioxy)hept-6-en-3-ol (1a) and
(2R,3R)-1,2-(cyclohexylidenedioxy)hept-6-en-3-ol (1a)
To magnesium turnings (602 mg, 24.8 mmol) were added a cat-
alytic amount of solid iodine and a solution of 4-bromo-1-butene
(219 ␮L, 2.02 mmol) in anhydrous tetrahydrofuran (THF, 2 mL)
dropwise under argon atmosphere, to prevent the invasion of mois-
ture. After the reaction initiated, a solution of 4-bromo-1-butene
(2.08 mL, 19.2 mmol) in anhydrous THF (19 mL) was added drop-
wise. The reaction mixture was stirred under reflux for 30 min
and then cooled to −78 ◦C. To the mixture was added a solution
of freshly prepared (R)-2b [4] (1.20 g, 7.07 mmol) in anhydrous
THF (14 mL). The mixture was stirred for 30 min at that temper-
ature and warmed to 0 ◦C. The reaction was then quenched by
the addition of saturated aqueous NH4Cl solution and the mix-
ture was extracted with AcOEt. The organic layer was washed with
brine, dried over anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(24 g). Elution with hexane/AcOEt = 30:1–10:1 afforded a mixture
of (2R,3S)- and (2R,3R)-1a (1.10 g, yield: 58%) as colorless oil. The
NMR spectrum was identical with that reported previously [6]. The
ratio between (2R,3S)- and (2R,3R)-1a was estimated to be 74:26,
by comparing the area of signals at ı 3.78 [m, 1H, H3 for (2R,3S)-
1a] and 3.48 [m, 1H, H3 for (2R,3R)-1a]. TLC: Rf for (2R,3S)-1a: 0.33;
(2R,3R)-1a: 0.43 (developed with hexane/AcOEt = 3:1).
2. Experimental
1H NMR spectra were measured in CDCl3 at 400 MHz, on a VAR-
IAN 400-MR spectrometer. HPLC data were recorded on SHIMADZU
SPD-M20A multi-channel detector. Silica gel 60 N (spherical and
neutral, 100–210 ␮m, 37560-79) of Kanto Chemical Co. was used
for column chromatography. Peptone and yeast extract were pur-
chased from Kyokuto Pharmaceutical Co., for the cultivation of
microorganism. Yeast strains are available at Japan Collection
of Microorganisms; Riken Bioresource Center, Planning Section,
Research Promotion Division, RIKEN Tsukuba Institute, 3-1-1 Koy-
adai, Tsukuba, Ibaraki 305-0074, Japan, and to NITE Biological
To a suspension of CuI (11.4 mg, 0.060 mmol) and (R)-2 (203 mg,
1.19 mmol) in THF (6.5 mL) was added a solution of homoal-
lylmagnesium bromide from 4-bromo-1-butene (3.58 mmol) in
THF (3.5 mL) at −78 ◦C. The mixture was gradually allowed to
warm to 0 ◦C and further stirred for 12 h at that temperature. The
similar workup and purification afforded a mixture (2R,3S)- and
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.05.017

Letter / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 8–11
9
Table 1
Whole-cell yeast-catalyzed reduction of (R)-3, to find out the “stereoselective”
strains.
O
O
Microorganism
Conversion (%)
Product ratio of 1a
(2R,3S):(2R,3R)
O
O
CHO
CHO
Pichia angusta (JCM 3620)
Pichia minuta (JCM 3622)
14
93
34
59
73
94
23
98
92
94
100
93
100
97
85
82
61
47
40
29
21
18
17
11
0
3
(R)-2b
(R)-2a
Saccaromyces cerevisiae (JCM 2214)
Torulaspora delbrueckii (NBRC 10921)
Trichosporon cutaneum (JCM 1534)
Candida nitratophila (JCM 9856)
Rhodotolura minuta (JCM 8105)
Candida kefyr (JCM 21874)
Yarrowia lypolytica (JCM 21884)
Williopsis californica (JCM 3600)
Pichia farinosa (NBRC 10896)
Rhodotolura mucilaginosa (NBRC 0889)
15
18
39
53
60
71
79
82
83
89
O
O
OH
a
Both of the conversion and product ratio were determined by NMR analysis of the
crude product. For the detail, see Section 2.1.
(2R,3S)-1a
(R)-2b
O
of the reduction was monitored by a TLC analysis as in Section
2.1. For each test tube, the mixture was then saturated with NaCl,
filtered through a pad of Celite and extracted with AcOEt twice.
The combined organic layer was washed with brine, dried over
anhydrous Na2SO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography (0.4 g). Elution with
hexane/AcOEt = 30:1–20:1 afforded (2R,3S)- and (2R,3R)-1a, whose
ratio was judged by NMR as in Section 2.1. The result was summa-
rized in Table 1.
O
OH
(2R,3R)-1a
b
O
O
2.4. Preparation of (2R,3S)-1a by the reduction with Pichia
minuta
O
(R)-3
P. minuta JCM 3622 was incubated in the medium (Section 2.3,
200 mL) in two 500 mL baffled Erlenmeyer cultivating flasks. The
flasks were shaken on a gyratory shaker (180 rpm) for 52 h at 30 ◦C.
The wet cells were harvested by centrifugation as in Section 2.3 for
15 min. The weight of combined wet cells was ca. 8.4 g from 200 mL
of the broth.
The harvested cells (4.2 g) were re-suspended with phosphate
buffer solution (pH 6.5, 0.1 M, 50 mL). To the mixture was added
glucose (1.0 g) and ketone (R)-3 (100 mg, 0.446 mmol) and the
resulting mixture was vigorously stirred for 24.5 h at 30 ◦C. The
broth was centrifuged for 15 min. The precipitated cells were
extracted with acetone twice and concentrated in vacuo. The super-
natant was saturated with NaCl and extracted with AcOEt four
times. The combined extract was washed with brine, dried over
anhydrous Na2SO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography (2.0 g). Elution with
hexane/AcOEt = 30:1–10:1 afforded 88.3 mg of a mixture of (2R,3S)-
and (2R,3R)-1a [yield: 87%; (2R,3S):(2R,3R) = 97:3].
Scheme 1. The addition of 3-butenylmagnesium bromide on (R)-2b and the deriva-
tion to (R)-3. Reagents and conditions: (a) BrMg(CH2)2CH CH2, THF (58%) and (b)
SO3-pyridine, DMSO, Et3N, CH2Cl2 (83%).
(2R,3R)-1a (152 mg, yield: 56%), whose ratio was 39:61, as judged
by its NMR spectrum.
2.2. (R)-1,2-(Cyclohexylidenedioxy)hept-6-en-3-one (3)
To a solution of 1a (153 mg, 0.681 mmol), triethylamine (470 ␮L,
3.40 mmol), and anhydrous DMSO (1.1 mL) in anhydrous CH2Cl2
(1.1 mL) was added sulfur trioxide pyridine complex (279 mg,
1.75 mmol) and the mixture was stirred for 1.5 h at 0 ◦C under
argon atmosphere. The reaction was quenched by the addition of
saturated aqueous NH4Cl solution and the mixture was extracted
with AcOEt. The organic layer was washed with brine, dried over
anhydrous Na2SO4 and concentrated in vacuo. The residue was
purified by silica gel column chromatography (2.8 g). Elution with
hexane/AcOEt = 30:1–20:1 afforded (R)-3 (127 mg, yield: 83%) as a
colorless oil. Its NMR spectrum was identical with that reported
previously [7].
Further chromatographic purification yielded pure (2R,3S)-1a.
A small portion (9.9 mg, 0.044 mmol) was dissolved in anhydrous
pyridine (500 ␮L) and to the mixture were added benzoyl chloride
(15 ␮L, 0.127 mmol) and catalytic amount of DMAP. The resulting
mixture was stirred for 8.5 h at room temperature. The reaction
was quenched by the addition of saturated aqueous NH4Cl solution
and the mixture was extracted with AcOEt twice. The organic layer
was washed with brine, dried over anhydrous Na2SO4 and con-
centrated in vacuo. The residue was purified by silica gel column
chromatography (0.2 g). Elution with hexane/AcOEt = 30:1–20:1
afforded (1S,1ꢀR)-1b (9.3 mg, yield: 64%) as colorless oil. HPLC [col-
umn, Daicel Chiralcel AD-3, 0.46 cm × 25 cm; hexane/i-PrOH = 10:1,
flow rate 0.5 mL/min, detected at 230 nm], tR (min) = 11.5 [(1S,1ꢀR)-
, >99.9%], 12.2 [(1R,1ꢀS)-, <0.1%]; >99.9% ee. For the confirmation,
the authentic racemic mixture of (1R*,1ꢀS*)-1b was prepared from
2.3. Screening of the yeasts for the reduction of (R)-3
The microorganisms from stock culture samples were incu-
bated in a medium [glucose (5%), peptone (2%), yeast extract (0.5%),
KH2PO4 (0.3%), K2HPO4 (0.2%) and pH 6.5, total 20 mL] in a test
tube (2.5 cm ␾ × 20 cm) for 48 h at 30 ◦C with reciprocal shaking
at 210 cpm (counts per min). The wet cells were harvested by
centrifugation (5000 rpm, 1200 × g) for 15 min and re-suspended
in phosphate buffer solution (pH 6.5, 0.1 M, 10 mL) in the same
test tube, together with (R)-3 (20 mg, 0.089 mmol). The test tubes
were further shaken at 210 cpm for 48 h at 30 ◦C. The progress
(
)-2. 1H NMR: ı 1.47–1.60 (m, 10H), 1.76–1.90 (m, 2H), 2.08–2.21

10
Letter / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 8–11
(m, 2H), 3.88 (dd, J1 ,2 a = 6.3 Hz, J2 a,2 b = 8.4 Hz, 1H, H2ꢀa), 4.04
ꢀ
ꢀ
ꢀ
ꢀ
(dd, J1 ,2 b = 6.3 Hz, 1H, H2ꢀb), 4.22 (ddd, J1,1 = 5.8 Hz, 1H, H1 ),
4.95 (ddt, J5a,5b = 1.7 Hz, J4,5a = 10.1 Hz, J3,5a = 1.7 Hz, 1H, H5a), 5.01
(ddt, J4,5b = 17.0 Hz, J3,5b = 1.7 Hz, 1H, H5b), 5.25 (ddd, J1,2a = 4.1 Hz,
J1,2b = 8.3 Hz, 1H, H1), 5.79 (ddt, J3,4 = 6.6 Hz, 1H,ꢀꢀH4),ꢀꢀ7.43 (ddd,
ꢀ
ꢀ
ꢀ
ꢀ
O
O
OR
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
,4
J3
= 1.2 Hz, J3
=ꢀ7ꢀ .6 Hz, J3
= 7.6 Hzꢀ,ꢀ 2H,ꢀꢀH3 , H5 ), 7.55 (tt,
,6
,2
ꢀꢀ ꢀꢀ
J2
= 1.5 Hz, 1H, H4 ), 8.03 (ddd, 2H, H2 , H6 ).
(2R,3S)-1a: R = H
(1S,1'R)-1b: R = Bz
,4
b
a
2.5. Preparation of (2R,3R)-1a by the reduction with Rhodotorula
mucilaginosa
(R)-3
R. mucilaginosa NBRC 0889 was incubated for 50 h at 30 ◦C
in a similar manner. The weight of combined wet cells was ca.
10.4 g from 200 mL of the broth. Ketone (R)-3 (102 mg, 0.454 mmol)
was treated with the harvested cells (2.5 g) in the same manner
as in Section 2.4 for 48 h at 30 ◦C. The workup and purification
yielded 43.3 mg of a mixture of (2R,3S)- and (2R,3R)-1a [yield: 42%;
(2R,3S):(2R,3R) = 11:89].
O
O
OR
(2R,3R)-1a: R = H
(1R,1'R)-1b: R = Bz
b
Pure (2R,3R)-isomer was also obtained by chromatographic sep-
aration. In the similar manner in Section 2.4, this sample (11.2 mg,
0.049 mmol) was treated with benzoyl chloride and DMAP in pyri-
dine. The workup and purification yielded (1R,1ꢀR)-1b (12.3 mg,
76%) as colorless oil. HPLC [Daicel Chiralcel AD-3, 0.46 cm × 25 cm;
hexane-i-PrOH (100:1), flow rate 0.5 mL/min, detected at 230 nm]:
tR (min) = 20.2 [(1S,1ꢀS)-, 0.3%], 21.7 [(1R, 1ꢀR)-, 99.7%]; 99.4% ee.
In the similar manner as described in Section 2.4, the authen-
tic racemic mixture (1R*,1ꢀR*)-1b was prepared from ( )-2, by
regioselective benzylation of glycerol on the terminal alcohol, sub-
sequent cyclohexylidene acetal formation on remaining 1,2-diol,
deprotection of benzyl group and finally, the oxidation of resulting
primary alcohol. 1H NMR: ı 1.50–1.62 (m, 10H), 1.76–1.92 (m, 2H),
Scheme 2. Yeast-mediated stereoselective reduction and the subsequent acylation.
Reagents and conditions: (a) whole-cell yeasts, see Table 1 and (b) BzCl, DMAP,
pyridine.
(R)-2. In our hand, the diastereofacial selectivity on the addition
of homoallylmagnesium bromide to (R)-2 was really moder-
ate [(2R,3S): (2R,3R) = 74:26]. Among the reported organometallic
species [5,16], organocopper changed the stereochemical prefer-
ence [(2R,3S):(2R,3R) = 39:61]. The extent of selectivity, however,
was not remarkable.
Major products derived from the yeast-catalyzed reduction
showed very high enantiomeric excess [>99.9% ee for (2R,3S)- and
99.4% for (2R,3R)-1a], which was confirmed by the HPLC analysis
after converting to the corresponding benzoates (1b) compared
with racemic samples, (1R*,1ꢀS*)- and (1R*,1ꢀR*)-1b. By virtue of
very mild conditions for whole-cell biocatalyst-mediated reduc-
tion, almost no racemization at C-2 position in (R)-3 occurred
throughout the operations. Thanks to the development of two
complementary yeast strains, both of (2R,3S)- and (2R,3R)-1a iso-
mers became available. This preparation method is superior to
a separation of diastereomeric mixture of alcohols, by means
of lipase-catalyzed kinetic resolution. A substrate with a similar
alkynyl side chain, instead of 3-butenyl group in 1, had been sub-
mitted for that reaction [17]. Such approach, however, is essentially
accompanied by the formation of byproduct with undesired stere-
ochemistry.
2.11–2.17 (m, 2H), 3.78 (dd, J1 ,2 a = 6.3 Hz, J2 a,2 b = 8.4 Hz, 1H, H2ꢀa),
ꢀ
ꢀ
ꢀ
ꢀ
4.02 (dd, J1 ,2 b = 6.4 Hz, 1H, H2ꢀb), 4.28 (ddd, J1,1 = 4.7 Hz, 1H, H1 ),
4.95 (ddt, J5a,5b = 1.8 Hz, J4,5a = 10.0 Hz, J3,5a = 1.5 Hz, 1H, H5a), 5.00
(ddt, J4,5b = 17.2 Hz, J3,5b = 1.5 Hz, 1H, H5b), 5.23 (ddd, J1,2a = 4.5 Hz,
J1,2b = 9.0 Hz, 1H, H1), 5.80 (ddt, J3,4 = 6.6 Hz, 1H,ꢀꢀH4),ꢀꢀ7.44 (ddd,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
ꢀꢀ ꢀꢀ
,4
J3
= 1.2 Hz, J3
=ꢀ7ꢀ .6 Hz, J3
= 7.6 Hzꢀ,ꢀ 2H,ꢀꢀH3 , H5 ), 7.55 (tt,
,6
,2
ꢀꢀ ꢀꢀ
J2
= 1.5 Hz, 1H, H4 ), 8.04 (ddd, 2H, H2 , H6 ).
,4
3. Results and discussion
Incubated whole-cell biocatalysts of twelve yeast strains were
applied for the reduction of (R)-3, which was prepared by
Parikh–Doering oxidation of 1a (Scheme 2). The results were sum-
marized in Table 1. Five strains showed re-facial selectivity to give
(2R,3S)-1a, while other seven showed the reverse si-facial selec-
tivity to give (2R,3R)-1a. The facial selectivity was very various
in biocatalytic reduction. In contrast, only “syn” stereoselective
chemical reduction by means of LiBH(sec-Bu)3 (l-selectride) was
reported [15], which provides (2R,3R)-form. To our knowledge,
however, no “anti” selective chemical reductions to afford (2R,3R)-
form were reported.
After considering both of stereoselectivity and catalytic activity,
two strains of P. minuta JCM 3622 and R. mucilaginosa NBRC 0889
were further applied under scaled-up conditions. The selectivity
was reproducible, that is, (2R,3S):(2R,3R) = 97:3 in the former, and
11:89 in the latter. Although the starting materials were consumed,
in both cases the yields were 87 and 42%, respectively. In the case
of R. mucilaginosa, some extent of degradation of either substrate
or product is suspected.
4. Conclusion
We found two complementary biocatalysts, P. minuta JCM 3622
and R. mucilaginosa NBRC 0889, for a diastereofacially selective
reduction. The stereocontrol in newly created chiral center effec-
tively worked with the pre-installed one including in carbohydrate
template. The increased availability of (2R,3S)- and (2R,3R)-1a, and
their reliability in regard to the stereochemical purity for both
diastereomers which was qualified through this study, would pro-
mote the further utilization of the products as the chiral starting
materials for natural product synthesis.
Acknowledgements
The predominant formation of each diastereomer facilitated to
avoid the tedious chromatographic separation between (2R,3S)-
and (2R,3R)-1a (ꢀRf = 0.1). In this standpoint, yeast-mediated
reduction of (R)-3 was much advantageous to the diastere-
oselective addition of homoallylmetallic species on aldehyde
This work was supported both by a Grant-in-Aid for Scien-
tific Research (No. 23580152) and formation of a research center
in cell signaling drug discovery for molecular targeting therapies:
matching fund subsidy 2009–2013 from the Ministry of Education,

Letter / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 8–11
11
Culture, Sports, Science and Technology, Japan, and acknowledged
with thanks.
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[17] F. Compostella, L. Franchini, G.B. Giovenzana, L. Panza, D. Prosperi, F. Ronchetti,
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Takeshi Sugai∗
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