Enhancements of enantio and diastereoselectivities in reduction of (Z)-3-halo-4-phenyl-3-buten-2-one mediated by microorganisms in ionic liquid/water biphasic system

Article abstract of DOI:10.1016/j.molcatb.2012.08.005

Reductions of (Z)-C₆H₅CHCXC(O)CH₃ (X = Cl, Br) mediated by Saccharomyces cerevisiae, Candida albicans, Rhodotorula glutinis, Geotrichum candidum and Micrococcus luteus gave the corresponding halohydrins through consecutive

Full text of DOI:10.1016/j.molcatb.2012.08.005

Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 61–64
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enhancements of enantio and diastereoselectivities in reduction of
(Z)-3-halo-4-phenyl-3-buten-2-one mediated by microorganisms
in ionic liquid/water biphasic system
Dávila S. Zampieria, Bruno R.S. de Paulaa, Luiz A. Zampieria, Juliana A. Valeb,
J. Augusto R. Rodriguesa, Paulo J.S. Morana,∗
a Institute of Chemistry, University of Campinas, C.P. 6154, 13084-971 Campinas, SP, Brazil
b Universidade Federal da Paraíba, Centro de Ciências Exatas e da Natureza, Departamento de Química, 55081-970 João Pessoa, PB, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 May 2012
Received in revised form 30 July 2012
Accepted 8 August 2012
Available online 30 August 2012
Reductions of (Z)-C6H5CH CXC( O)CH3 (X = Cl, Br) mediated by Saccharomyces cerevisiae, Candida
albicans, Rhodotorula glutinis, Geotrichum candidum and Micrococcus luteus gave the corresponding halo-
hydrins through consecutive reduction reactions of C C and C O bonds. In general, the reactions
performed in the biphasic system water/[(bmim)PF6] gave better diastereoselectivity and enantiose-
lectivity than in pure water.
© 2012 Elsevier B.V. All rights reserved.
Keywords:
Halohydrins
Bioreduction
Saccharomyces cerevisiae
Ionic liquid
Haloenones
1. Introduction
There are very few examples of microorganism inhibition
caused by IL [6], although studies show noninvasive effects of
Enzymatic reactions using ionic liquids (ILs) with both whole
cells and free enzymes were firstly described in the early 2000s and
have recently been reviewed [1]. Ionic liquids, low-temperature
molten salts, have been used as green reaction solvents due to their
physical and chemical properties such as negligible vapor pressure,
good chemical and thermal stability and relative low flammability
and viscosity [2]. In general, ILs are used as solvent or co-solvent for
biocatalytic reactions employing isolated enzymes, whereas whole
cell reactions in aqueous medium usually employ hydrophobic IL as
a second liquid phase [3]. In this biphasic system, the substrate and
products are mostly in the IL and the cells are dispersed in the water
phase. In case of hydrophobic substrates and products, the IL acts as
a substrate reservoir, delivering the substrate to the aqueous phase
and withdrawing the product from it. This implies a decrease of
the concentrations of substrate and product in the aqueous phase,
preventing cellular inhibition by them [3,4]. Moreover, the work
up is very simple since the product is extracted from the IL using a
volatile solvent and after that the IL may be recycled. Nowadays, a
variety of ILs are commercially available in high purity [5].
hydrophobic ILs on cellular membranes, especially those with hex-
afluorophosphate and bis(trifluoromethylsulfonyl)imide anions in
biphasic IL/water systems [7]. 1-(n-Butyl)-3-methylimidazolium
hexafluorophosphate [bmim(PF6)] has been demonstrated to have
a greater biocompatibility with Saccharomyces cerevisiae than n-
hexane, and to be superior to the use of the water miscible
1-(n-butyl)-3-methylimidazolium tetrafluoroborate [bmim(BF4)]
as a co-solvent [8]. The use of hydrophobic IL in reduction of ketones
mediated by whole cells is generally carried out with a second liquid
phase, giving secondary alcohols in good yields and enantiomeric
excesses [4,8,9].
The enones have been investigated as potential substrates for
biocatalytic reductions that can lead to the introduction of one, two
or three new chiral centers into an achiral structure [10], and the
bioreduction of ␣- and ␤-haloenones was recently studied in deep
by Fuganti et al. [11].
In this work, the results of (Z)-3-bromo-4-phenyl-3-buten-
2-one 1a and (Z)-3-chloro-4-phenyl-3-buten-2-one 1b reduction
mediated by five microorganisms in a water/[bmim(PF6)] sys-
tem are reported. The bioreduction of these substrates proceeds
by two consecutive reactions (Scheme 1), beginning with a C
C
bond reduction catalyzed by an enoate reductase, producing the
transient species 2a or 2b, followed by C O bond reduction cat-
alyzed by an alcohol dehydrogenase to produce halohydrins 3a
∗
Corresponding author. Tel.: +55 19 3521 3048; fax: +55 19 3521 3023.
E-mail address: moran@iqm.unicamp.br (P.J.S. Moran).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.08.005

62
D.S. Zampieri et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 61–64
conditions, incubation at 30 ◦C on an orbital shaker (180 rpm) for 1
day just before use. All materials and medium were sterilized in an
autoclave at 121 ◦C before use and the yeast was manipulated in a
laminar flow cabinet.
O
O
OH
X
X
X
2.3. Growth conditions of M. luteus
1a X=Br
1b X=Cl
2a X=Br
2b X=Cl
3a X=Br
3b X=Cl
M. luteus CCT 2283 was cultivated in NB (nutrient broth)
obtained from Oxoid under aseptic conditions, incubation at 30 ◦C
on an orbital shaker (180 rpm) for 1 day just before use. All mate-
rials and medium were sterilized in an autoclave at 121 ◦C before
use and the yeast was manipulated in a laminar flow cabinet.
Scheme 1. Consecutive C C and C O bonds reduction of (Z)-3-halo-4-phenyl-3-
buten-2-one mediated by microorganisms.
or 3b [12]. Our interest is to study the effect of the biphasic sys-
tem water/[(bmim)PF6] in these consecutive reactions mediated
by microorganisms.
2.4. Test of inhibition activity
2. Experimental
Aqueous dispersions of each microorganism (100 ␮L) were
spreaded over a YMA (malt extract, yeast extract, peptone, glucose
and agar) medium, and after that, sterile filter paper disks with
[bmim(PF6)] or 1a or 1b adsorbed (10 ␮L of a 1 mg/mL solution in
ethyl acetate was used) were placed over them. The standards were
fungicide (Cyclopirox amine®) and bactericide (Cloranfenicol®) for
control. After 24 h at 30 ◦C, inhibition halos on the filter paper were
observed for 1a and 1b and no halo of inhibition was observed for
[bmim(PF6)].
2.1. General
The substrates (Z)-3-bromo-4-phenyl-3-buten-2-one 1a and
(Z)-3-chloro-4-phenyl-3-buten-2-one 1b were prepared follow-
ing published methodologies [12]. The [bmim(PF6)] was pre-
pared following
3-methylimidazolium trifluoromethanesulfonate and potassium
hexafluorophosphate as starting materials.
a literature procedure [13] using 1-butyl-
The reduction products 3a–b were analyzed by 1H NMR, 13C
NMR, IR spectra and are identical to those previously published
[12]. 1H NMR spectra were determined at 250 MHz (Varian Gemini
250) or 500 MHz (INOVA 500). 13C NMR spectra were determined
at 62.5 MHz (Varian Gemini 250) or 125.7 MHz (INOVA 500). IR
spectra were recorded on a FT-IR Bomen MB-100 from Hartmann
& Braun. GC–MS analyses were obtained on a QP 5000-Shimadzu
instrument (70 eV) using a DB1 silica capillary column from J&W
Scientific (30 m × 0.25 m ID × 0.25 ␮m film thickness) and helium
as a carrier gas (0.8 mL/min). The split ratio was 1:30. The injector
temperature was at 270 ◦C and the detector was at 280 ◦C. The col-
umn temperature was held at 80 ◦C for 3 min, increased to 290 ◦C
at a rate of 29 ◦C/min and then kept constant for 8 min. One ␮L of
a compound solution or extracted reaction solution (1 mg/mL) in
ethyl acetate was injected and the retention times (min) for each
compound are: 1a (8.87), 2a (8.27), syn-3a (8.43), anti-3a (8.59), 1b
(8.84), 2b (8.93), syn-3b (8.61), anti-3b (8.45). Chiral GC-FID anal-
yses were obtained on an Agilent 6850 Series GC System, using
a Hydrodex chiral capillary column (30 m × 0.25 mm × 0.25 ␮m).
Hydrogen was used as carrier gas (1 mL/min), the injector tem-
perature was 200 ◦C and the detector temperature was 220 ◦C. The
column temperature was held at 80 ◦C for 3 min, increased to 180 ◦C
at a rate of 1 ◦C/min, and then kept constant for 5 min. One ␮L of
a compound solution or extracted reaction solution (1 mg/mL) in
ethyl acetate was injected and the retention times (min) for each
compound are: (2S,3S)-3a (23.10), (2R,3R)-3a (23.28), (2S,3S)-3b
(24.71), (2R,3R)-3b (24.85). Thin-layer chromatographic (TLC) anal-
yses were performed with precoated aluminum sheets (silica gel 60
Merck), and flash column chromatography was carried out on sil-
ica (200–400 mesh, Merck). The ultraviolet spectra were obtained
on an Agilent Spectrophotometer, model 8453, with diode array.
The yeast strains S. cerevisiae CCT 3019, Candida albicans CCT 0776,
Geotrichum candidum CCT 1205, Rhodotorula glutinis CCT 2182 and
Micrococcus luteus CCT 2283 were obtained from the André Tosello
Research Foundation (Campinas, SP, Brazil) [14].
2.5. General procedure for bioreduction of ˛-haloenones 1a and
1b mediated by microorganisms in aqueous media
Compounds 1a or 1b (100 mg) dissolved in 1 mL of ethanol
was added to an Erlenmeyer flask containing 200 mL of the slurry
medium of growing microorganism cells. The reaction mixture was
incubated in an orbital shaker (200 rpm at 30 ◦C) for 120 h. Then, the
product was extracted by a continuous liquid/liquid extractor with
dichloromethane for 2 days. The solvent was dried over sodium
sulfate, evaporated, and the products were purified by preparative
TLC (hexane/ethyl acetate 9:1), analyzed by GC–MS and 1H NMR,
and the ee was determined by chiral GC-FID.
2.6. General procedure for bioreduction of ˛-haloenones 1a and
1b mediated by microorganisms in aqueous media with addition
of ionic liquid
Compounds 1a or 1b (100 mg) dissolved in [bmim(PF6)] (1 mL)
was added to an Erlenmeyer flask containing 200 mL of the
slurry medium of growing microorganism cells. The resulting mix-
ture was stirred in an orbital shaker (400 rpm, 30 ◦C) for 24 h.
Then, the products were extracted by a continuous extractor with
dichloromethane for 2 days. The organic phase was dried over
sodium sulfate, the solvent evaporated, and the products were puri-
fied by preparative TLC (hexane/ethyl acetate 9:1), analyzed by
GC–MS and 1H NMR, and the ee was determined by chiral GC/FID.
2.7. General procedure for detection of intermediaries during
bioreduction of ˛-haloenones 1a and 1b mediated by S. cerevisiae
in aqueous media
Compounds 1a or 1b (10 mg) dissolved in ethanol (0.5 mL) were
added to each of seven Erlenmeyer flasks containing 20 mL of the
slurry medium of growing S. cerevisiae cells. The reaction mixtures
were incubated in an orbital shaker (200 rpm at 30 ◦C) for appro-
priate periods of time. After the flasks were withdrawn from the
shaker, the mixtures were centrifuged and the supernatants free of
cells were submitted to extractions with ethyl acetate (3 × 10 mL).
The extracts were dried over sodium sulfate, the solvent evapo-
rated, and the products were analyzed by GC–MS.
2.2. Growth conditions of C. albicans, R. glutinis, G. candidum and
S. cerevisiae
Microorganisms were cultivated in YM (yeast-malt extract)
nutrient broth obtained from Merck (1000 mL) under aseptic

D.S. Zampieri et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 61–64
63
Table 1
Table 2
Reagent and product percentages after 120 h of bioreduction of 1a in 200 mL of
Reagent and product percentages after 120 h of bioreduction of 1b in 200 mL of
slurries of growing microorganisms.
slurries of growing microorganisms.
Microorganism
System
1a (%)a
2a (%)a
3a (%)a
Microorganism
System
1b (%)a
2b (%)a
3b (%)a
syn (2S, 3S ee)b
anti
syn (2S, 3S ee)b
anti
S. cerevisiae
C. albicans
R. glutinis
G. candidum
M. luteus
H2Oc
–
28
21
32
18
39
12
33
55
58
26
10
18
13
9
14
13
15
5
74 (90)
62 (97)
61 (65)
55 (81)
47 (70)
47 (92)
39 (65)
52 (91)
40 (38)
42 (42)
–
–
–
–
26
–
36
–
–
S. cerevisiae
C. albicans
R. glutinis
G. candidum
M. luteus
H2Oc
13
18
22
49
–
44
38
42
61
76
20
21
8
10
13
8
2
5
6
10
67 (71)
61 (92)
47 (79)
41 (94)
63 (72)
48 (95)
44 (67)
53 (95)
33 (32)
18 (39)
–
–
23
–
24
–
16
–
H2O/ILd
H2Oc
H2O/ILd
H2Oc
H2O/ILd
H2Oc
H2O/ILd
H2Oc
H2O/ILd
H2Oc
H2O/ILd
H2Oc
H2O/ILd
H2Oc
H2O/ILd
H2Oc
–
–
H2O/ILd
–
–
H2O/ILd
a
a
Conversion determined by the relative areas of the chromatographic peaks using
Conversion determined by the relative areas of the chromatographic peaks using
GC–MS.
GC–MS.
b
b
ee were determined by CG-FID using a chiral column.
ee were determined by CG-FID using a chiral column.
c
c
Reaction conditions: 10 mg 1a in 200 mL of growing cell slurry, in an orbital
Reaction conditions: 10 mg 1b in 200 mL of growing cell slurry, in an orbital
shaker at 200 rpm and 30 ◦C.
shaker at 200 rpm and 30 ◦C.
d
d
Reaction conditions: 100 mg of 1a in 1 mL of [bmim(PF6)] and 200 mL of growing
Reaction conditions: 100 mg of 1b in 1 mL of [bmim(PF6)] and 200 mL of growing
cell slurry, in an orbital shaker at 200 rpm and 30 ◦C.
cell slurry, in an orbital shaker at 200 rpm and 30 ◦C.
The same protocol was applied for bioreduction of chloroenone
1b, and the results are in Table 2. When the reduction is mediated
by S. cerevisiae in aqueous media only the isomer (2S,3S)-3b was
formed in 71% ee while in presence of IL the enantiomeric excess
was improved up to 92%, while the conversion was decreased from
67 to 61%. Again, the bireduction of 1b mediated by R. glutinis, G.
candidum and also, in this case, by C. albicans, two diastereoiso-
mers were observed when performed in a monophasic system,
whereas the reaction in the presence of IL gave exclusively (2S,3S)-
3b diastereoisomer in 94–95% ee.
The distribution coefficients of the substrates 1a and 1b in the
two phases of the system water/[bmim(PF6)] were determined as
316 for enone 1a and 257 for enone 1b, this implies in log K of
2.50 and 2.41, respectively. Therefore, in the reactions applying the
biphasic system the substrates are mostly in the IL layer and thus
the effective initial concentration of these compounds in the aque-
ous layer is much lower (2.3–2.5 times) than in the system that
utilizes only water as solvent. This decrease in the concentration
of substrates may be extended to the concentration of intermedi-
ates 2a and 2b since organochlorides with similar structures like
chloroacetophenone, have the similar values of log P [16].
The above mentioned IL effects on the concentration of
substrates and intermediates may be responsible for the diastere-
oselectivity and enantioselectivity of the reduction of 1a and
1b. Therefore, the IL is acting like absorbing resins in an in situ
extractive biocatalysis, controlling the substrate concentration of
hydrophobic substrates and improving enantio-chemoselectivity
[17]. Considering the pull of oxidoredutases present in the microor-
ganism cells, the enzyme with the lowest KM is favored when the
concentration of substrate is decreased. This effect may be occur-
ring in the C C bond reduction of 1a–b and more effectively in the
consecutive C O bond reduction reaction of intermediates 2a–b,
increasing the enantioselectivity and diastereoselectivity when the
bioreduction is performed in presence of IL (Fig. 1).
It was crucial to prove that the intermediates 2a–b have per-
meability through cells membrane of microorganism during the
reduction of 1a–b. Thus, an experiment was designed to detect the
intermediates 2a–b in the aqueous media, just after the cells been
withdrawn by centrifugation, during the reduction of 1a–b medi-
ated by S. cerevisiae using only water as solvent. Fig. 2 clearly shows
the presence of 2a as transient in the aqueous media.
2.8. Determination of the distribution coefficient of ˛-haloenones
1a–b in water/[bmim(PF6)]
The haloenone (100 mg) dissolved in IL (1 mL) was added to
a 250 mL Erlenmeyer flask containing water (100 mL). The mix-
ture was kept under orbital stirring at 400 rpm and 30 ◦C for 24 h.
Then, an aliquot was removed from the aqueous phase. The con-
centration of haloenones in the water phase was determined by
measuring the absorbance at 295 nm and with aid of calibrations
curves (R = 0.9999) for 1a and 1b constructed from standard solu-
tions. The obtained distribution coefficients values were 316 for
enone 1a and 257 for enone 1b.
3. Results and discussion
Bioreductions of 1a and 1b were carried out in aqueous media
and in aqueous/[bmim(PF6)] biphasic systems mediated by the
following microorganisms: S. cerevisiae, R. glutinis, C. albicans, G.
candidum and Microccocus luteus. For all experiments substrate
concentrations of 100 mg in 200 mL of slurry medium of growing
microorganism were used and the biotransformations were carried
out for 120 h and then analyzed by GC–MS to determine the con-
versions and the diastereoselectivity, and by GC-FID using a chiral
column to determine the enantiomeric excesses of 3a and 3b, as
shown in Tables 1 and 2. Samples were withdrawn every 24 h (up
to 120 h), and after 96 h no change was observed in the composition
of the reaction mixture.
Even though the reactions in the presence of [bmim(PF6)]
resulted in slightly lower conversions of 1a into 3a, they gave over-
all better results, since in all biotransformations the corresponding
halohydrins were obtained in higher enantiomeric excesses than
in their monophasic system counterpart (see Table 1). When the
reduction of 1a was mediated by S. cerevisiae in aqueous media
only the syn isomer (2S,3S)-3a is formed in 90% ee, in accordance
with previous work [15]. This reaction in IL improved the enan-
tiomeric excess up to 97% while the conversion was decreased from
74 to 62%. Experiments with C. albicans gave similar results while M.
luteus gave unsatisfactory results. It is also noteworthy that, when
G. candidum and R. glutinis were used as biocatalysts without addi-
tion of IL, both syn and anti diastereoisomers were formed, whereas
in the presence of IL only the syn diastereoisomer (2S,3S)-3a was
observed in higher ee than from the experiments without addition
of [bmim(PF6)].
The toxicities of these ␣-haloenones to the microorganisms
were determined by inhibition assays in Petri dishes measur-
ing the inhibition halo. Table 3 shows the inhibitory effects of

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D.S. Zampieri et al. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 61–64
system water/[bmim(PF6)] gave the corresponding halohydrins
with better enantiomeric excesses and diastereoselectivities than
the reductions accomplished in pure water. This result may be due
to the dramatic reduction of the substrate concentrations in the
aqueous phase due to their better solubility in IL. The inhibitory
effect of substrates on the biocatalysts was decreased in the pres-
ence of IL due to the reduction of substrate concentration in
the aqueous phase. The extractive methodology employing the IL
biphasic system can be of general use in biocatalysis for toxic sub-
strates, as exemplified by the compounds described in this paper.
Fig. 1. The consecutive bioreduction reactions of 1a–b performed in a water/ionic
liquid biphasic system.
Acknowledgements
1a
100
2a
The authors thank CNPq and FAPESP for financial support.
3a
80
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compounds 1a and 1b, fungicide (Cyclopirox amine®) and bac-
tericide (Cloranfenicol®) on the six studied microorganisms. No
formation of inhibition halos was observed for [bmim(PF6)]. On the
other hand, enones 1a and 1b have toxicity at the same level as the
fungicide and bactericide used for comparison. Since the use of the
biphasic system decreases the concentrations of enones 1a and 1b
and as a consequence the toxicity, this is another advantage of the
bioreduction of these substrates being performed in water/IL.
[17] (a) P. D’Arrigo, M. Lattanzio, G.P. Fantoni, S. Servi, Tetrahedron: Asymmetry 9
(1998) 4021–4026;
4. Conclusion
(b) G.J.A. Conceic¸ ão, P.J.S. Moran, J.A.R. Rodrigues, Tetrahedron: Asymmetry 14
(2003) 43–45;
(c) S. Serra, C. Fuganti, F.G. Gatti, Eur. J. Org. Chem. (2008) 1031–1037.
The reductions of 1a and 1b mediated by C. albicans, G. can-
didum, M. luteus, R. glutinis and S. cerevisiae in the two-phase