Chemoselective screening for the reduction of a chiral functionalised (±)-2-(phenylthio)cyclohexanone by whole cells of Brazilian micro-organisms

Article abstract of DOI:10.1016/j.tetasy.2008.10.003

The use of whole cells of micro-organisms to bring about the biotransformation of an organic compound offers a number of advantages, but problems caused by enzymatic promiscuity may be encountered upon with substrates bearing more than one functional group. A one-pot screening method, in which whole fungal cells were incubated with a mixture of 4-methylcyclohexanone 1 and phenyl methyl sulfide 2, has been employed to determine the chemoselectivity of various biocatalysts. The hyphomycetes, Aspergillus terreus CCT 3320 and A. terreus URM 3571, catalysed the oxidation of 2 accompanied by the reduction of 1 to 4-methylcyclohexanol 1a and, for strain A. terreus CCT 3320, the Baeyer-Villiger oxidation of 1. The Basidiomycetes, Trametes versicolor CCB 202, Pycnoporus sanguineus CCB 501 and Trichaptum byssogenum CCB 203, catalysed the oxidation of 2 and the reduction 1, but no Baeyer-Villiger reaction products were detected. In contrast, Trametes rigida CCB 285 catalysed the biotransformation of 1 to 1a, exclusively, in the absence of any detectable sulfide oxidation reactions. The chemoselective reduction of (±)-2-(phenylthio)cyclohexanone 3 by T. rigida CCB 285 afforded exclusively the (+)-cis-(1R,2S) and (+)-trans-(1S,2S) diastereoisomers of 2-(phenylthio)cyclohexan-1-ol 3a in moderate yields (13% and 27%, respectively) and high enantiomeric excesses (>98%). Chemoselective screening for the reduction of a ketone and/or the oxidation of a sulfide group in one pot by whole cells of micro-organisms represents an attractive technique with applications in the development of synthesis of complex molecule bearing different functional groups.

Full text of DOI:10.1016/j.tetasy.2008.10.003

Tetrahedron: Asymmetry 19 (2008) 2385–2389
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Chemoselective screening for the reduction of a chiral functionalised
( )-2-(phenylthio)cyclohexanone by whole cells of Brazilian micro-organisms
Leandro Piovan a, Edna Kagohara a, Luis C. Ricci a, Artur F. Keppler a, Marina Capelari b, Leandro H. Andrade a,
João V. Comasseto a, André L. M. Porto a,c,
*
a Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, CEP 05508-900, São Paulo, SP, Brazil
b Seção de Micologia e Liquenologia, Instituto de Botânica de São Paulo, Av. Prof. Miguel Stéfano, 3687, CEP 04301-902, São Paulo, SP, Brazil
c Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador São-carlense, 400, CEP 13560-970, São Carlos, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The use of whole cells of micro-organisms to bring about the biotransformation of an organic compound
offers a number of advantages, but problems caused by enzymatic promiscuity may be encountered upon
with substrates bearing more than one functional group. A one-pot screening method, in which whole
fungal cells were incubated with a mixture of 4-methylcyclohexanone 1 and phenyl methyl sulfide 2,
has been employed to determine the chemoselectivity of various biocatalysts. The hyphomycetes, Asper-
gillus terreus CCT 3320 and A. terreus URM 3571, catalysed the oxidation of 2 accompanied by the reduc-
tion of 1 to 4-methylcyclohexanol 1a and, for strain A. terreus CCT 3320, the Baeyer-Villiger oxidation of 1.
The Basidiomycetes, Trametes versicolor CCB 202, Pycnoporus sanguineus CCB 501 and Trichaptum byssog-
enum CCB 203, catalysed the oxidation of 2 and the reduction 1, but no Baeyer–Villiger reaction products
were detected. In contrast, Trametes rigida CCB 285 catalysed the biotransformation of 1 to 1a, exclu-
sively, in the absence of any detectable sulfide oxidation reactions. The chemoselective reduction of
( )-2-(phenylthio)cyclohexanone 3 by T. rigida CCB 285 afforded exclusively the (+)-cis-(1R,2S) and (+)-
trans-(1S,2S) diastereoisomers of 2-(phenylthio)cyclohexan-1-ol 3a in moderate yields (13% and 27%,
respectively) and high enantiomeric excesses (>98%). Chemoselective screening for the reduction of a
ketone and/or the oxidation of a sulfide group in one pot by whole cells of micro-organisms represents
an attractive technique with applications in the development of synthesis of complex molecule bearing
different functional groups.
Received 23 August 2008
Accepted 9 October 2008
Available online 5 November 2008
Ó 2008 Published by Elsevier Ltd.
1. Introduction
this context, we have recently obtained biotransformation prod-
ucts with high enantiomeric excesses following a biocatalytic pro-
Enantiomerically pure alcohols, ketones, sulfides or amines are
commonly required as starting materials in the synthesis of chiral
compounds.1 Alcohols with high enantiomeric purities can be
readily formed in quantitative yields of around 100% through
chemical or biocatalytic reduction of prochiral ketones.2 Alterna-
tively, the kinetic resolution of a racemic alcohol with one stereo-
genic centre can be achieved, for example, by lipase-catalysed
transesterification,3 although the maximum quantitative yield of
the desired enantiomer is only 50%. Biotransformation reactions
involving racemic ketones as substrates are not usually employed
in biocatalytic methods.4
tocol involving whole fungal cells in respect of Mannich
eliminations of amine salt,5 the kinetic resolution of seleno-alco-
hols by biomethylation,6 the stereo-inversion of racemic alcohols7
and the stereoselective reduction of prochiral ketones.8 For sub-
strates bearing several functional groups, however, the use of
whole cells may give rise to problems caused by enzymatic
promiscuity.9
Herein, whole cells of micro-organisms have been selectively
screened with regard to their ability to carry out a specific bio-
transformation of a substrate with more than one functional group.
In a preliminary study, cells of six strains of the fungal genera
Aspergillus, Trametes, Pycnoporus and Trichaptum were subjected
to a one-pot screening against a substrate mixture consisting of
4-methylcyclohexanone 1 and methyl phenyl sulfide 2. Under
the reaction conditions employed, only Trametes rigida CCB 285
was able to promote the bioreduction of ketone 1 without the con-
comitant oxidation of the sulfide group of 2. The application of
whole cells of this micro-organism in the biocatalytic reduction
The use of whole cells of micro-organisms to bring about the
biotransformation of an organic compound offers a number of
advantages over processes involving isolated enzyme systems. In
* Corresponding author. Tel.: +55 16 3373 8103; fax: +55 16 3373 9952.
E-mail address: almporto@iqsc.usp.br (A. L. M. Porto).
0957-4166/$ - see front matter Ó 2008 Published by Elsevier Ltd.
doi:10.1016/j.tetasy.2008.10.003

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L. Piovan et al. / Tetrahedron: Asymmetry 19 (2008) 2385–2389
of the synthetic racemic substrate ( )-2-(phenylthio)cyclohexa-
none 3, which contains a ketone and a sulfide function in the same
molecule, was subsequently investigated.
with a high conversion (Table 1, entries 16–18). The microbial sul-
foxidation of phenyl methyl sulfide 2 by A. terreus CCT 3320 has
already been reported.11 Whole cells of A. terreus URM 3571 affor-
ded 2a and 2b from 2 with conversion values similar to those
obtained with strain A. terreus CCT 3320; however, in this case,
the Baeyer–Villiger oxidation of 1 to form lactone 1b was the main
biotransformation reaction of the ketone, and only low concentra-
tions of the reduction product 1a were formed (Table 1, entries 13–
15). The biotransformations of cyclic ketones by strains of A. terreus
have previously been reported.12 The screening experiments indi-
cated that the enzymes present in whole cells of T. rigida CCB
285 were highly chemoselective since the ketone group of 1 was
reduced to yield 1a exclusively, and no reactions occurred at the
sulfide group of 2. This strain of T. rigida was therefore considered
to be the micro-organism of choice in the study of the biocatalytic
transformations of the functionalised ( )-2-(phenylthio)cyclohexa-
none 3, a compound containing both ketone and sulfide groups in
the same molecule. Racemic 3 was synthesised by the reaction of
the nucleophilic thiolate anion (prepared in situ by treating com-
mercial thiophenol with potassium carbonate) with 2-chlorocyclo-
hexanone (Scheme 1).13
The product was purified by column chromatography (CC) over
silica gel with n-hexane–ethyl acetate (8:2) as eluent and was
obtained in good yield. Racemic diastereoisomers of the b-hydroxy
sulfide 3a were prepared for use as standards in the subsequent
HPLC analysis by the reduction of 3 with NaBH4 in the presence
of SiO2–H2O (10:3) (Scheme 2).14 Racemic diastereomers of 3a
were obtained in excellent yields in the form of a mixture with a
cis:trans ratio of 75:25. The diastereomers were separated by CC
over silica gel eluted with n-hexane–ethyl acetate (10:1), and were
identified by NMR, IR and HRMS analyses. The spectroscopic data
of the cis- and trans-diastereoisomers of 3a were in agreement
2. Results and discussion
The biotransformation of a mixture of 4-methylcyclohexanone
1 and phenyl methyl sulfide 2 by whole cells of the Basidiomycetes
Trametes versicolor CCB 202, T. rigida CCB 285, Pycnoporus sanguin-
eus CCB 501 and Trichaptum byssogenum CCB 203), and of the
Hyphomycetes Aspergillus terreus CCT 3320 and A. terreus URM
3571 was studied in a one-pot system. Table 1 shows the progress
of the reduction and/or oxidation reactions as a function of incuba-
tion time, together with the percentage yields of the products
formed. One-pot screening methods using whole cells and several
organic substrates have recently been reported for monitoring
oxidoreductases.10
Under the described conditions, whole cells of T. versicolor CCB
202, P. sanguineus CCB 501 and T. byssogenum CCB 203 promoted
the reduction of ketone 1 to 4-methylcyclohexanol 1a and the oxi-
dation of sulfide 2 to sulfoxide 2a and sulfone 2b, all in high con-
versions (Table 1, entries 1–9). Although these micro-organisms
were able to catalyse the oxidation of the sulfide, the Baeyer–Vil-
liger oxidation reaction of 1 to yield 5-methylheptalactone 1b
was not observed. When cells of T. rigida CCB 285 were employed
as a biocatalyst, ketone 1 was reduced exclusively to the corre-
sponding alcohol 1a in high conversion (Table 1, entries 10–12),
but no oxidation products of the sulfide 2 were detected.
With respect to the strains of A. terreus studied, enzymes pres-
ent in whole cells of A. terreus CCT 3320 catalysed the reduction of
1 to exclusively afford alcohol 1a in very high conversion, while the
oxidation of 2 to yield the sulfoxide 2a and the sulfone 2b occurred
Table 1
Selective one-pot screening for reduction and/or oxidation reactions catalysed by whole cells of micro-organismsa
O
OH
O
O
S
O
O
S
S
O
Fungi
Fungi
+
+
1b
1
1a
2
2a
2b
Entry
Micro-organism
Incubation time (days)
Product concentrationb (%)
Alcohol 1a
Lactone 1b
Sulfoxide 2a
Sulfone 2b
Basidiomycetes
1
2
3
4
5
6
7
8
9
Trametes versicolor CCB 202
1
4
6
1
4
6
1
4
6
1
4
6
82
77
78
68
56
44
71
69
70
9
—
—
—
—
—
—
—
—
—
—
—
—
74
90
81
60
45
21
32
42
38
—
7
10
16
3
11
27
61
58
62
—
Pycnoporus sanguineus CCB 501
Trichaptum byssogenum CCB 203
Trametes rigida CCB 285
10
11
12
76
69
—
—
—
—
Hyphomycetes
13
14
15
16
17
18
Aspergillus terreus URM 3571
Aspergillus terreus CCT 3320
1
4
6
1
4
6
36
24
24
100
100
100
64
76
76
—
—
—
16
9
10
14
12
8
84
91
90
86
88
92
a
Reaction conditions: 1.0 g (wet weight) of fungal mycelia and 20 lL each of 4-methylcyclohexanone 1 and phenyl methyl sulfide 2 in 50 mL of Na2HPO4/KH2PO4 buffer
(pH 7) incubated at 32 °C on an orbital shaker (160 rpm).
b
Concentration determined by GC–MS using an achiral column.

L. Piovan et al. / Tetrahedron: Asymmetry 19 (2008) 2385–2389
2387
O
OH
OH
1) K2CO3, THF
30 min, rt
1) SiO2:H2O (10:3)
2) NaBH4
S
S
S
SH
+
O
Cl
2)
rac-3
( )-trans-3a
( )-cis-3a
(75 %)
(20 %)
(70 %)
THF, 24h, rt
Scheme 1. Synthesis of ( )-2-(phenylthio)cyclohexanone 3 and ( )-2-(phenylthio)cyclohexan-1-ol 3a.
OH
O
OH
S
S
S
T. rigida CCB 285
+
pH = 7, 32 oC,
160 rpm, 144 h
3a
(+)-cis-(1R,2S)-
3a
(+)-trans-(1S,2S)-
3
rac-
yield = 17 %
e.e. > 98 %
yield = 27 %
e.e. > 98 %
Scheme 2. Chemoselective reduction of ( )-2-(phenylthio)cyclohexanone by whole cells of Trametes rigida.
Table 2
Chemoselective biotransformation of (RS)-2-(phenylthio)cyclohexanone 3 by whole cells of Trametes rigida CCB 285a
Entry
Incubation time (h)
Ketone 3
c (%)b
(+)-cis-(1R,2S)-3a
(+)-trans-(1S,2S)-3a
c (%)b
ee (%)
c (%)b
ee (%)
1
2
3
48
96
144
—
—
—
39
31
35
—
61
69
68
—
—
>98
—
>98c
c
a
Reaction conditions: 1.0 g (wet weight) of fungal mycelia and 20 lL of 3 in 50 mL of Na2HPO4/KH2PO4 buffer (pH 7) incubated at 32 °C on an orbital shaker (160 rpm).
Concentration of product (%) determined by chiral HPLC analysis.
Enantiomeric excess (%) determined by chiral HPLC analysis of products isolated in the quantitative study.
b
c
with those reported in the literature.15–19 The racemic diastereo-
mers of 3a could be resolved by HPLC analysis using a Chiralcel
OD-H column and n-hexane–2-propanol (99:1).
A qualitative study of the biotransformation of ( )-2-(phenyl-
thio)cyclohexanone 3 was performed using cells of T. rigida CCB
285 as the source of alcohol dehydrogenase and cofactors required
to bring about a chemo- and stereo-selective reduction of the
ketone group. The results presented in Table 2 show that the bio-
reduction of the ketone group of 3 led to the preferential formation
of trans-b-hydroxysulfide 3a with 32% diastereoisomeric excess. In
this case, the diastereoisomeric ratio obtained by biocatalytic
reduction was the opposite of that obtained by chemical reduction.
This finding is in agreement with the results recently obtained in
our laboratory employing the same fungus in the reduction of
seleno-ketones.20
A quantitative study of the bioreduction of 3 by whole cells of T.
rigida CCB 285 was performed in which alcohols 3a were isolated
from the reaction mixture after 144 h of incubation, purified and
subsequently characterised by spectroscopic analyses. The yields
of cis-3a and trans-3a were 13% and 27%, respectively, and both
diastereoisomers were obtained in high (>98%) enantiomeric puri-
ties (Scheme 2, Fig. 1). The absolute configurations of the products
were assigned by comparison with the values in the literature.16–18
Figure 1. HPLC chromatograms of the chemoselective biotransformations of ( )-2-
(phenylthio)cyclohexanone 3 by T. rigida CCB 285.
3. Conclusion
technique employing different substrates described herein repre-
sents an important biocatalytic methodology with application in
the development of syntheses of molecules bearing multiple stereo-
genic centres in high enantiomeric excesses. Using this screening
The successful chemoenzymatic biotransformation of an organ-
ic compound containing several reactive centres by whole fungal
cells is not trivial. In this context, the one-pot selective screening

2388
L. Piovan et al. / Tetrahedron: Asymmetry 19 (2008) 2385–2389
method, T. rigida CCB 285 was selected to study the chemoselective
reduction of the functionalised (RS)-2-(phenylthio)cyclohexanone
3. The biotransformation reaction afforded the chiral sulfide-alco-
hols (+)-cis-(1R,2S)-3a and (+)-trans-(1S,2S)-3a in high enantio-
meric excesses (ee >98%) and in moderate yields.
MgSO4ꢁ7H2O 0.5 g/L, KH2PO4 1 g/L, NaCl 0.06 g/L) in a refrigerator
at 4 °C. Slices of agar (0.5 ꢀ 0.5 cm) containing mycelia were inoc-
ulated into 1 L of liquid medium with same composition as the
solid medium contained in a 2 L Erlenmeyer flask, and incubated
at 32 °C for 8 days on an orbital shaker (160 rpm). Stock cultures
of Hyphomycetes were stored on solid medium in a refrigerator
at 4 °C. Slants of the cultures were inoculated into 1 L of Oxoid malt
extract medium (20 g/L) contained in a 2 L Erlenmeyer flask, and
incubated at 32 °C for 6 days on an orbital shaker (160 rpm). Fol-
lowing incubation, fungal cells were harvested by vacuum filtra-
tion, and 1.0 g aliquots of the wet cells were suspended in 50 mL
portions of Na2HPO4/KH2PO4 buffer (pH 7) contained in separate
Erlenmeyer flasks (250 mL). Biotransformation reactions were ini-
4. Experimental
4.1. General methods
4-Methylcyclohexanone, benzethiol and 2-chlorocyclohexa-
none were purchased from commercial sources and used as sup-
plied, all other reagents and solvents were previously purified
and/or dried where necessary using methods described in the liter-
ature.21 Micro-organisms were manipulated in a Veco laminar flow
cabinet, and all incubation experiments were carried out using
sterile materials. Technal TE-421 or Superohm G-25 orbital shakers
were employed in the biocatalysed transformations.
tiated by the addition to the cell suspensions of 20
methylcyclohexanone 1 and phenyl methyl sulfide 2 (all fungal
strains), or of 20 L of ( )-2-(phenylthio)cyclohexanone 3 (T. rigida
lL each of 4-
l
CCB 285), and the reaction mixtures were incubated at 32 °C for 6
days on an orbital shaker (160 rpm). Progress of the biotransforma-
tion was monitored every 2 days by GC–MS using an achiral col-
umn (for substrates 1 and 2) or by HPLC using a chiral column
(for substrate 3). In order to determine the yields of (+)-cis-
(1R,2S)-3a and (+)-trans-(1S,2S)-3a formed from 3 by the cells of
T. rigida CCB 285, appropriate reaction mixtures were set up
exactly as described above in 10 separate Erlenmeyer flasks
(250 mL) and incubated for 6 days. After this time, the reaction
mixtures were filtered, the aqueous phases were bulked and
extracted with ethyl acetate, the organic phase was dried over
MgSO4 and the solvent removed under vacuum. Alcohols 3a were
isolated by CC from the resulting residue and identified by NMR,
IR and HRMS analyses.
Compound purification was carried out by column chromato-
graphy over silica gel (230–400 mesh) eluted with mixtures of
n-hexane and ethyl acetate. Column effluents were monitored by
TLC on pre-coated Silica Gel 60 F254 layers (aluminium-backed:
Merck) eluted with mixtures of n-hexane and ethyl acetate, and
visualised by spraying with p-anisaldehyde/sulfuric acid reagent
followed by heating at ca. 120 °C. Products of the enzyme-cata-
lysed reactions were analysed using a Shimadzu model GC-17A
gas chromatograph equipped with a flame ionisation detector
(FID) and a J & W Scientific HP5 column (30 m ꢀ 0.25 mm i.d.;
0.25 lm). The GC conditions were oven temperature initially at
50 °C and increased at a rate of 10 °C/min; run time 20 min; injec-
tor temperature 230 °C; FID temperature 250 °C; injector split ratio
1:20; hydrogen carrier gas pressure 100 kPa. GC–MS analyses were
performed on a Shimadzu model QP5050A instrument equipped
with a capillary column DB-5 (J & W Scientific DB-5 column,
4.4. Characterisation of alcohols 3a
4.4.1. (+)-cis-(1R,2S)-2-(Phenylthio)-1-cyclohexanol cis-3a
Isolated as a yellow oil; yield: 13%; ½a D25
¼ þ19:7 (c 0.42,
ꢂ
30 m ꢀ 0.25 mm i.d.; 0.25
lm) with helium as the carrier gas. HPLC
CH2Cl2); ee >98%; IR (film)
t
/cmꢃ1: 3403, 2942, 2917, 2852,
analyses were carried out using a Shimadzu model SPD-10Av
equipped with UV–vis detector and a Chiralcel OD-H column
(25 ꢀ 0.46 cm i.d.) eluted with n-hexane–2-propanol (99:1).
1H and 13C NMR spectra were measured on a Bruker DRX 500
spectrometer (500 MHz, 1H; 125 MHz, 13C) with samples dissolved
in CDCl3: chemical shifts are presented in ppm with respect to tet-
ramethylsilane (TMS). IR spectra were recorded in nujol mulls
using a Bomem MB 100 spectrometer. HRMS analyses were per-
formed on a Bruker Daltonics Esquire 3000 Plus instrument
equipped with an ion trap detector. Optical rotation values were
determined with a Jasco DIP-378 polarimeter using a 1 dm cuvette,
and reported values refer to the Na-D line.
1579, 1481, 1435, 1066, 733; 1H NMR (CDCl3, 500 MHz):
d = 7.45–7.44 (m, 2H), 7.31–7.23 (m, 3H), 3.78–3.76 (m, 1H),
3.56–3.33 (m, 1H), 1.86–1.65 (m, 6H), 1.53–1.48 (m, 1H), 1.41–
1.34 (m, 2H); 13C NMR (CDCl3, 125 MHz): d = 134.2, 132.2, 129.1,
127.3, 67.0, 54.4, 31.7, 27.7, 24.9, 20.9; HRMS (ESI) [M+Na]+ calcd
for C12H16NaOS 231.0820. Found: 231.0811.
4.4.2. (+)-trans-(1S,2S)-2-(Phenylthio)-1-cyclohexanol trans-3a
Isolated as a yellow oil; yield: 27%; ½a D25
¼ þ61:0 (c 0.44,
ꢂ
CH2Cl2); ee >98%; IR (film)
t
/cmꢃ1: 3392, 2934, 2925, 2851,
1582, 1482, 1354, 1087, 732; 1H NMR (CDCl3, 500 MHz):
d = 7.51–7.46 (m, 2H), 7.32–7.25 (m, 3H), 3.36–3.30 (m, 1H), 2.97
(m, 1H), 2.81–2.75 (m, 1H), 2.17–2.00 (m, 2H), 1.76–1.67 (m,
2H), 1.38–1.21(m, 4H); 13C NMR (CDCl3, 125 MHz): d = 133.8,
132.5, 128.9, 127.7, 72.0, 56.6, 33.8, 32.7, 26.2, 24.3; HRMS (ESI)
[M+Na]+, calcd for C12H16NaOS: 231.0820. Found: 231.0815.
4.2. Synthesis of standard racemic alcohols15
2-(Phenylthio)cyclohexanone 3 (1 mmol, 206 mg) and NaBH4
(1.1 mmol, 42 mg) were added to a stirred mixture of SiO2
(100 mg) and H2O (30 mg) contained in a 5 mL two-necked
round-bottomed flask equipped with a magnetic stirrer, and stir-
ring was continued for 5 min at room temperature. After work-
up with CH2Cl2, the solvent was removed under vacuum and the
residue purified by CC (silica gel; n-hexane–ethyl acetate, 9:2) to
afford the racemic alcohols 3a. The spectroscopic data of these
compounds were in agreement with those reported in the litera-
ture.15–19
Acknowledgements
Fellowships from FAPESP (Proc. 05/52941-7 to LP) and CNPq (to
L.C.R. and A.F.K.) are gratefully acknowledged. The authors wish to
thank FAPESP (Proc. 03/04189-9) and CNPq (Proc. 472663/2004-6
to ALMP) for financial support for the project.
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