Biocatalytic reduction of a racemic selenocyclohexanone by Brazilian basidiomycetes

Article abstract of DOI:10.1016/j.tetasy.2007.05.036

An efficient synthesis of the chiral cyclic secondary alcohols, trans-2-(phenylseleno)cyclohexanol 1a and cis-2-(phenylseleno)cyclohexanol 1a, was obtained by enzymatic reduction of 2-(phenylseleno)cyclohexanone 1 using whole fungal cells. Five strains of white-rot basidiomycetes were examined; Irpex lacteus CCB 196, Pycnoporus sanguineus CCB 196, Trametes rigida CCB 285, Trametes versicolor CCB 202 and Trichaptum byssogenum CCB 203. Cells of T. rigida CCB 285 gave alcohols cis-(RS)-1a and trans-(SS)-1a in high enantiomeric excesses (ca. 99%).

Full text of DOI:10.1016/j.tetasy.2007.05.036

Tetrahedron: Asymmetry 18 (2007) 1398–1402
Biocatalytic reduction of a racemic selenocyclohexanone
by Brazilian basidiomycetes
Leandro Piovan,a Marina Capelari,b Leandro H. Andrade,a
a
a,c,
*
´
Joao V. Comasseto and Andre L. M. Porto
˜
aInstituto de Quı´mica, Universidade de Sa˜o Paulo, Av. Prof. Lineu Prestes, 748, CEP 05508-900 Sa˜o Paulo, SP, Brazil
bInstituto de Botanica, Sec¸a˜o de Micologia e Liquenologia, Av. Miguel Este´fano, 3687, CEP 04301-902 Sa˜o Paulo, SP, Brazil
cInstituto de Quı´mica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Av. Trabalhador Sa˜o-carlense, 400, CEP 13566-590 Sa˜o Carlos-SP, Brazil
Received 10 April 2007; accepted 24 May 2007
Abstract—An efficient synthesis of the chiral cyclic secondary alcohols, trans-2-(phenylseleno)cyclohexanol 1a and cis-2-(phenyl-
seleno)cyclohexanol 1a, was obtained by enzymatic reduction of 2-(phenylseleno)cyclohexanone 1 using whole fungal cells. Five strains
of white-rot basidiomycetes were examined; Irpex lacteus CCB 196, Pycnoporus sanguineus CCB 196, Trametes rigida CCB 285, Trametes
versicolor CCB 202 and Trichaptum byssogenum CCB 203. Cells of T. rigida CCB 285 gave alcohols cis-(RS)-1a and trans-(SS)-1a in high
enantiomeric excesses (ca. 99%).
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
selenoalcohols, the bioreduction of a selenoketone by some
Brazilian basidiomycetes has been investigated. Previously
we have employed white-rot basidiomycetes as biocatalysts
in the oxidation of sulfides into sulfoxides,10 and recently
the bioreduction of ketones by basidiomycetes has been re-
ported.11 As a result, we herein report the preparation of
enantiomerically pure cyclic secondary alcohols 1a via the
biocatalytic reduction of the racemic selenocyclohexanone
1.
Chiral alcohols are important intermediates in organic syn-
theses in the food, agrochemical and, most importantly,
pharmaceutical industries.1–7 Reactions that are catalysed
by micro-organisms and isolated enzymes are amongst
the best methods currently available for the preparation
of enantiomerically pure compounds.1,2 In this context,
the microbial reduction of carbonyl compounds represents
a particularly convenient route for the preparation of chiral
alcohols. In nature, oxidoreductase enzymes, such as alco-
hol dehydrogenases, facilitate the interconversion between
alcohols and aldehydes or ketones. Such dehydrogenases
catalyse the removal of hydrogen from a substrate and
the transfer of the hydrogen to an acceptor, either
NAD+/NADP+ or a flavin enzyme, in an oxidation–reduc-
tion reaction.3
2. Results and discussion
(R,S)-2-(Phenylseleno)cyclohexanone 1 was prepared by
the treatment of cyclohexanone with lithium diisopropyl-
amide (LDA), followed by the addition of a solution of
PhSeBr in THF (Scheme 1).12 Selenoketone 1 was obtained
in 48% yield following purification by column chromato-
graphy (CC) over silica gel eluted with n-hexane/EtOAc
(10:1).
We have recently demonstrated that enzymes from fungal
strains8 and carrot root9 can be employed as excellent bio-
catalysts in the syntheses of chiral organoselenium and
organosulfur compounds. In order to extend this biocata-
lytic protocol to the preparation of enantiomerically pure
O
OLi
O
Se
PhSeBr
LDA
THF, -78 oC
1 (48%)
*
Corresponding author. Tel.: +55 16 3373 8103; fax: +55 16 3373 9952;
e-mail: almporto@iqsc.usp.br
Scheme 1. Synthesis of 2-(phenylseleno)cyclohexanone 1.
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.05.036

L. Piovan et al. / Tetrahedron: Asymmetry 18 (2007) 1398–1402
1399
OH
OH
O
Se
Se
Se
SiO2/H2O- 30%
+
NaBH4, 5 min
rac-cis-1a
rac-1 (48%)
rac-trans-1a
yield 80%
Scheme 2. Synthesis of racemic cis-1a and trans-1a alcohols.
In order to prepare the racemic diastereoisomers of
b-hydroxyselenides 1a, selenoketone 1 was reduced using
a mixture of NaBH4 and SiO2/H2O (30%) (Scheme 2).13
A mixture of cis-1a and trans-1a isomers was purified by
CC eluted with n-hexane/EtOAc (5:1) to afford the alcohols
in 80% yield (cis-1a 68%; trans-1a 12%) for use as standards
in subsequent GC and HPLC analyses. The alcohols were
identified by their NMR, IR and HRMS data, which were
in agreement with those reported in the literature (see Sec-
tion 4.4).14–16
It is noteworthy that the chemical reduction of 1 led to the
preferential formation of the cis-1a isomer (cis:trans ratio
85:15), since this result complements the methodology for
the preparation of b-hydroxyselenides involving the ring
opening of epoxides by nucleophilic selenium species to
yield alcohols with a trans-configuration.14
Figure 1. HPLC chromatograms of: (A) ( )-cis-1a and ( )-trans-1a; and
(B) the bioreduction of ( )-ketone 1 by Trametes rigida CCB 285 (96 h).
The results obtained following the biotransformation of
selenoketone 1 promoted by whole cells of the fungal spe-
cies Irpex lacteus CCB 196, Pycnoporus sanguineus CCB
501, Trametes rigida CCB 285, Trametes versicolor CCB
202 and Trichaptum byssogenum CCB 203 are summarised
in Table 1. In each experiment, the enantiomeric purities of
alcohols 1a produced were determined by HPLC analyses
using a chiral column (Fig. 1).
As can be seen from Table 1, the bioreduction reactions
promoted by different basidiomycetes varied significantly,
especially with respect to the stereoselectivity. The fungus
I. lacteus CCB 196 biotransformed 1 to alcohols cis-1a
(ee 73%) and trans-1a (ee 68%) with moderate enantiomeric
Table 1. Reduction of 2-(phenylseleno)cyclohexanone 1 by whole cells of Brazilian basidiomycetes
OH
O
OH
R
S
Se
Se
basidiomycetes
Se
S
+
S
cis 1a
cis-1a
rac-1 (48 %)
trans 1a
Entry
Microorganism
Time (h)
1 ca (%)
trans-1a
eeb (%)
(PhSe)2 ca (%)
ca (%)
eeb (%)
ca (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Irpex lacteus
CCB 196
48
96
144
48
96
144
48
96
144
48
96
144
48
10
10
7
30
22
18
30
34
34
20
21
26
—
8
—
—
73
—
—
99
—
—
77
—
—
—
—
—
68
55
45
42
50
51
46
37
38
44
—
5
—
—
68
—
—
99
—
—
62
—
—
—
—
—
79
10
23
33
—
—
8
—
—
—
—
—
—
—
—
14
Trametes rigida
CCB 285
20
15
12
43
41
30
100
88
83
31
19
19
Trichaptum byssogenum
CCB 203
Trametes versicolor
CCB 202
8
5
Pycnoporus sanguineus
CCB 501
29
29
30
40
40
37
96
144
a Concentration (%): determined by GC analysis using an achiral column.
b Enantiomeric excess (%): determined by HPLC analysis using a chiral column.

1400
L. Piovan et al. / Tetrahedron: Asymmetry 18 (2007) 1398–1402
excess (entry 3). This was accompanied, however, by the
formation of a small amount of diphenyldiselenide after a
48 h incubation (entry 1), the concentration of which in-
creased appreciably after 96 and 144 h incubation (entries
2 and 3). The formation of (PhSe)2 may occur via com-
plex-enzymatic oxidoreduction reactions of the seleno-
compound 1.17 An investigation of these mechanistic
sequences is currently underway in our laboratory.
high stereoselectivity (ee >99%). It can be concluded that
the stereospecific reduction of 1, which contains a selenium
atom and a ketone group in some structure, can be accom-
plished using whole fungal cells.
4. Experimental
4.1. General methods
The microbial reduction of selenoketone 1 by whole cells of
T. rigida CCB 285 led to the formation of alcohols cis-1a
(ee >99%) and trans-1a (ee >99%) with high stereoselec-
tivity and in yields, following CC purification (see Section
4.3), of 15% and 27%, respectively, (Fig. 1). In this case,
only a small amount of diphenyldiselenide was obtained
after 144 h of incubation (entry 6). The low isolated yields
of selenoalcohols 1a were caused by their degradation over
time. The absolute configuration of trans-1a alcohol was
assigned by comparison of the sign of the measured specific
rotation with those reported in the literature.14–16
1H and 13C NMR spectra were recorded on a Bruker DRX
1
500 (500 MHz, H; 125 MHz, 13C) spectrometer. All spec-
tra were measured in CDCl3 and the chemical shifts d are
quoted with respect to the internal standard tetramethyl-
silane (TMS). IR spectra of the product oils were deter-
mined on a Bomem MB 100 spectrometer. HRMS
analyses were performed using a Bruker Daltonik Esquire
3000 Plus instrument equipped with an ion trap detector.
Optical rotations were measured in a Jasco DIP-378 polar-
imeter using a 1 dm cuvette and are referenced to the Na–D
line value.
Selenoalcohol cis-1a was transformed into the correspond-
ing allylic alcohol 2 by selenoxide syn-elimination using
hydrogen peroxide as the oxidant12 (Scheme 3). The abso-
lute stereochemistry of 2 was assigned as (R) by compari-
son of its specific rotation with published data18 As a
consequence of the cis relation-ship of the substituents in
1a, the absolute configuration of the selenium containing
stereogenic centre was assigned as S.
Substrates, reagents and solvents were purchased from
commercial sources and purified and/or dried, where neces-
sary, by methods described in the literature.19 Stock cul-
tures of basidiomycetes were stored on solid culture
medium (agar 20 g/L, peptone 5 g/L, yeast extract 2 g/L,
MgSO4Æ7H2O 0.5 g/L, KH2PO4 1 g/L, NaCl 0.06 g/L) in
Petri dishes maintained at 4 ꢁC. All manipulations involv-
ing micro-organisms were carried out under sterile condi-
tions in a Veco laminar flow cabinet. Technal TE-421 or
Superohm G-25 orbital shakers were employed in the bio-
catalysed transformation experiments. Purification of the
products of the reduction reactions was carried out by
CC over silica gel (230–400 mesh) eluted with mixtures of
n-hexane and EtOAc. The column effluent was monitored
by TLC using pre-coated silica gel 60 F254 layers (alumin-
ium-backed: Merck) eluted with n-hexane/EtOAc and vis-
ualised by spraying with p-anisaldehyde/sulfuric acid
reagent followed by heating at ca. 120 ꢁC. Reaction prod-
ucts were analysed using a Shimadzu model GC-17A
(FID) gas chromatograph equipped with a J&W Scientific
HP5 column (30 m · 0.25 mm i.d.; 0.25 lm). The chro-
matographic conditions were: oven temperature initially
at 50 ꢁC and increased at 10 ꢁC/min; run time 20 min;
injector temperature 230 ꢁC; detector temperature 250 ꢁC;
injector split ratio 1:20; hydrogen carrier gas at a pressure
of 100 kPa. The enantiomeric excesses of the alcohols were
determined by HPLC analyses performed using a Shima-
dzu model SPD-10Av instrument with UV–vis detector
and equipped with a Chiralcel OD-H column (25 ·
0.46 cm) eluted with n-hexane/2-propanol (99:1). For ( )-
trans-2-(phenylseleneo)-1-cyclohexanol 1a the retention
times of the (1S,2S)- and (1R,2R)-isomers were 8.266 and
10.170 min, respectively. In the case of ( )-cis-2-(phenylsel-
eneo)-1-cyclohexanol 1a the retention times of the stereo-
isomers were 7.318 and 8.06 min.
OH
OH
R
Se
CH2Cl2, 35 o
C
H2O2, 20 min.
2
cis-1a
ee > 99%
(R)-(+)-cyclohex-2-en-1-ol
yield 68%
[α]D = +105.7 (c 0.51, CHCl3)
Scheme 3. Selenoxide syn elimination of cis-1a by reaction with H2O2.
When the fungus Trichaptum byssogenum CCB 203 was
used to promote the biotransformation of 1, trans-1a was
obtained as the main product. HPLC analysis showed that
cis-1a and trans-1a were produced in enantiomeric excesses
of 77 and 62%, respectively (Table 1, entry 9). Similar re-
sults were obtained following the biotransformation of 1
with P. sanguineus CCB 501, but the bioreduction was
not as selective and the alcohols showed only modest enan-
tiomeric excesses (cis-1a, ee 68% ee; trans-1a, ee 79%; Table
1, entry 15). The reaction with T. versicolor CCB 202 was
attempted but with little success since cis-1a and trans-1a
were obtained only in very small amounts (Table 1, entries
11 and 12).
3. Conclusion
Whole cells of some Brazilian basidiomycetes efficiently
catalyse the reduction of racemic selenoketone 1 to the cor-
responding alcohols 1a. T. rigida CCB 285 afforded alco-
hols cis-1a and trans-1a with two stereogenic centres in
4.2. Synthesis of standard racemic alcohols7
Selenocyclohexanone 1 (1 mmol, 254 mg) and NaBH4
(1.1 mmol, 42 mg) were added to a stirred mixture of silica

L. Piovan et al. / Tetrahedron: Asymmetry 18 (2007) 1398–1402
1401
(100 mg) and H2O (30 mg), contained in a 5 mL two-
4.5. Characterisation of 2-(phenylseleno)cyclohexanone 1
necked round-bottomed flask equipped with a magnetic
stirrer, and the whole mixture stirred for 5 min at room
temperature. Following work-up with CH2Cl2, the solvent
was removed under vacuum and the residue was purified by
CC over silica gel eluting with n-hexane/EtOAc (9:1) to
afford the racemic alcohols 1a. The spectral data of
these compounds were in agreement with those previously
reported (see Section 4.4).14–16
Yellow oil; isolated yield 48%. 1H NMR (500 MHz,
CDCl3) d 7.5 (m, 2H), 7.2 (m, 3H), 3.9 (t, 1H), 2.9 (m,
1H), 2.3–1.7 (m, 7H). 13C NMR (125 MHz, CDCl3) d
207.8, 134.6, 129.2 (2C), 128.1 (2C), 51.6, 38.5, 34.0, 26.8,
22.9. IR (film) m/cmꢁ1 3054, 2940, 2859, 1700. HRMS
(ESI) [M+Na]+ calcd for C12H14ONaSe: 277,0196. C,
56.92; H, 5.57. Found: C12H14ONaSe 277.0099; [M+1]+
calcd for C12H14OSe: 255.021. Found: 255.0281.
4.3. Biotransformation of 1 by basidiomycetes
4.6. Selenoxide syn elimination of cis-1a
Small slices of solid medium (0.5 · 0.5 cm) bearing a myce-
lia of the basidiomycete were cut from the stock culture
and inoculated into liquid culture medium (1 L) contained
in Erlenmeyer flasks (2 L). The fungal cells were incubated
at 32 ꢁC for 8 days on a rotatory shaker (160 rpm), har-
vested by filtration and suspended in a buffer solution con-
tained in Erlenmeyer flasks (250 mL). Initially, reactions
were carried out with 3.0 g (wet weight) of cells and
20 lL of selenoketone 1 in a phosphate buffer (Na2H-
PO4/KH2PO4, 40 mL, pH 7), and the mixture was incu-
bated for 6 days on an orbital shaker at 32 ꢁC. The
progress of the reaction was assessed every 2 days by GC
using an achiral column (Table 1). In further experiments,
cells of the most efficient biocatalyst (T. rigida CCB 285)
were transferred, together with 20 lL of selenoketone 1,
to 10 Erlenmeyer flasks (250 mL) and incubated under
the above conditions for 96 h. After this time, the reaction
mixtures were filtered and the combined aqueous phases
extracted with EtOAc (5 · 30 mL). The yellow organic
phase was dried over MgSO4. The solvent removed under
vacuum and the residue purified by CC over silica gel to
yield the cis and trans alcohols 1a.
To a 25 mL one necked round bottomed flask was added
cis-alcohol 1a (77 mg, 0.3 mmol), 0.05 mL of H2O2 (30%
v/v) and 10 mL of CH2Cl2. The reaction mixture was stir-
red for 20 min at 35 ꢁC, after which, the reaction was ex-
tracted with ethyl acetate (2 · 10 mL). The combined
organic phases were dried over magnesium sulfate and then
filtered. The organic solvent was evaporated under reduced
pressure and the residue purified by silica gel column chro-
matography using hexane/ethyl acetate (8:2) as eluent.
25
½aꢀD ¼ þ105:7 (c 0.51, CHCl3).18 Yield: 20 mg (68%).
1
The H NMR and 13C NMR spectra of cyclohex-2-en-1-
ol was in agreement with that reported in the literature.20
Acknowledgements
The authors are grateful to E. Kagohara for the technical
biological support. L.P. thanks FAPESP for a fellowship
(Proc. 05/52941-7); L.H.A., J.V.C. and A.L.M.P. thank
FAPESP and CNPq for the support (Proc. 472663/2004-6).
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25
Yellow oil; isolated yield 27%; ½aꢀD ¼ þ44:8 (c 0.53,
CHCl3); ee 99%. 1H NMR (500 MHz, CDCl3) d 7.56–
7.65 (m, 2H), 7.26–7.36 (m, 3H), 3.30–3.39 (1H), 2.87–
2.95 (m, 1H), 2.11–2.21 (m, 2H), 1.71–1.75 (m, 1H),
1.61–1.65 (m, 1H), 1.36–1.45 (m, 1H), 1.19–1.34 (m, 4H).
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4.4.2.
cis-(1R,2S)-2-(Phenylseleno)-1-cyclohexanol
1a.
25
Yellow oil; isolated yield 15%; ½aꢀD ¼ þ10:1 (c 0.42,
CHCl3); ee 99%. 1H NMR (500 MHz, CDCl3) d 7.51–
7.63 (m, 2H), 7.18–7.37 (m, 3H), 3.70–3.73 (1H), 3.50–
3.53 (m, 1H), 1.95–2.06 (m, 2H), 1.83–1.88 (m, 1H),
1.64–1.77 (m, 3H), 1.59–1.62 (m, 1H), 1.36–1.45 (m, 1H).
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53.6, 32.5, 33.4, 29.1, 25.2, 25.2, 20.9. IR (film) m/cmꢁ1
3429, 2991, 2855, 1758, 1476, 1442, 1066, 740, 693. HRMS
(ESI) [M+Na]+ calcd for C12H16ONaSe: 279,0265. C,
56.47; H, 6.32. Found: C12H16ONaSe: 279.0254.
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