Aspergillus genus as a source of new catalysts for sulfide oxidation

Article abstract of DOI:10.1016/j.molcatb.2012.05.003

This work extends the present knowledge about the ability of filamentous fungi to selectively transform sulfur-containing compounds. Here, it has been demonstrated that several species of the Aspergillus genus are able to perform chemo- and stereoselectiv

Full text of DOI:10.1016/j.molcatb.2012.05.003

Journal of Molecular Catalysis B: Enzymatic 82 (2012) 32–36
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Aspergillus genus as a source of new catalysts for sulfide oxidation
María Laura Mascottia, Alejandro A. Ordena, Fabricio R. Bisognoa,b, Gonzalo de Gonzaloc,
Marcela Kurina-Sanza,∗
a INTEQUI-CONICET, Facultad de Química Bioquímica y Farmacia, Universidad Nacional de San Luis, Chacabuco y Pedernera, 5700, San Luis, Argentina
b INFIQC-CONICET, Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000, Córdoba, Argentina
c Departamento de Química Orgánica e Inorgánica, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006, Oviedo, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
This work extends the present knowledge about the ability of filamentous fungi to selectively trans-
form sulfur-containing compounds. Here, it has been demonstrated that several species of the Aspergillus
genus are able to perform chemo- and stereoselective oxidation of organic sulfides. Two substrates
were chosen as models, the aliphatic cyclohexyl(methyl)sulfide and the alkyl aryl sulfide, thioanisole.
All the tested strains showed the ability to oxidize cyclohexyl(methyl)sulfide, whereas most of them
oxidized thioanisole. In all cases R-stereoselectivity was observed and full chemoselectivity was veri-
fied since no sulfone was detected. Several biotransformation parameters such as the employment of
growing or resting cell, different head-space volumes, age of the cultures, bio-reaction times and biocat-
alyst/substrate ratios resulted crucial in the optimization of the processes. Thus, when using isopropyl
alcohol as co-solvent in growing cell systems, chemo- and stereoselectivity could be modulated. Among
the studied strains, Aspergillus japonicus ICFC 744/11 was found to be a promising whole cell biocatalyst
to prepare enantioenriched sulfoxides since after optimization, either conversion and optical purity of
(R)-cyclohexyl(methyl)sulfoxide were excellent (c: 100%, ee > 99%).
Received 30 September 2011
Received in revised form 9 April 2012
Accepted 8 May 2012
Available online 24 May 2012
Keywords:
Biotransformation
Aspergillus
Chiral sulfoxide
Stereoselective sulfoxidation
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
as Helminthosporium, Rhizopus, Trichaptum and Trametes species,
Mortierella isabellina, Botrytis cinerea, Trichoderma viride and Eutypa
Non-racemic sulfoxides are recognized as valuable reagents.
They are extensively used as chiral auxiliaries since they present
configurational stability and exert high asymmetric induction [1].
Furthermore, the stereoselective oxidation of organic sulfides has
particular interest since chiral sulfoxides are valuable synthons,
being precursors of bioactive compounds [2], such as omeprazole,
for which optical purity is essential.
A vast number of bioactive sulfur-containing metabolites are
found in plants belonging to Cruciferae and Alliaceae families [3–6].
It is well known that among the fungal genus involved in patho-
genesis of such sulfur-rich plants, one of the most important is
Aspergillus [7–9]. At first glance, it is easy to assume that Aspergilli
contain the enzymatic machineries to detoxify such defense com-
pounds [10,11], transforming them into more hydrophilic and
-often- less toxic metabolites. Such ability to metabolize xenobi-
otic chemicals might be suitably exploited by organic chemists to
generate interesting derivatives [12,13].
lata [14–21].
Aspergillus genus has been historically used to perform biotrans-
formations due to its immense metabolic potential. However, its
ability to carry out sulfoxidation of prochiral sulfides has been
scarcely explored. At the best of our knowledge, only a few pre-
vious works have been reported dealing with Aspergillus capability
to oxidize prochiral sulfides. In the 60–70s, Auret’s group studied
the fungal oxidation of diaryl and aryl alkyl sulfides employ-
ing mainly A. niger with certain success albeit the enantiomeric
excesses (ees) and optical rotation signs resulted rather variable and
substrate over-oxidation (sulfone formation) was always detected
[14,22–24]. More recently, it has been studied the possibility of
performing sulfoxidations using A. terreus [25,26].
Among the enzymes reported to catalyze sulfides oxidation, the
most common are Baeyer-Villiger monooxygenases (BVMOs) [27]
and peroxidases [28]. Their presence in Aspergillus strains, along
with the occurrence of other enzymes competent for sulfoxidation
such as cytochrome P450 monooxygenases [29] make this fungal
genus a good candidate to be investigated in the selective prepara-
tion of enantioenriched sulfoxides.
Some fungi have been tested for the oxidation of sulfides
in order to prepare sulfoxides with good optical purities, such
Hence, in the frame of our studies on the biotransformation of
prochiral compunds [30–33] we have undertaken a survey study
of different Aspergillus strains with the aim of applying them as
∗
Corresponding author. Tel.: +54 02652 439099.
E-mail address: marcelakurina@gmail.com (M. Kurina-Sanz).
1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2012.05.003

M.L. Mascotti et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 32–36
33
O
O
S
S
S
S
Aspergillus strain
Aspergillus strain
1
1a
Fig. 1. Biotransformation of prochiral sulfides by Aspergillus strains.
2
2a
biocatalysts in selective sulfoxidation reactions toward an aliphatic
and an aromatic sulfide as model substrates (Fig. 1).
pre-culture was used, then cells were harvested and re-suspended
in buffer for 2 days and then substrate was added, reaction was
monitored every 24 h for 2 days; C: a 5-day-old pre-culture was
used and biotransformation was monitored every 24 h for 2 days
(Fig. 2).
2. Experimental
To evaluate the effect of co-solvents, biotransformation of 1 with
A. japonicus growing cells was performed. In each case the substrate
(1 mM) was dissolved in n-hexane, toluene, methanol, tert-butanol,
iso-propanol or DMSO (0.5% (v/v) final concentration), and added to
the reaction mixture. To evaluate the effect of increasing substrate
concentration, biotransformation of 1 employing A. japonicus grow-
ing cells was performed. Substrate concentrations were 1, 10, 25, 50
and 100 mM (dissolved in 25 ␮L of DMSO). In both assays reaction
was incubated for 5 days. Blank assays without fungi were carried
out in parallel. Experiments were performed in duplicate.
Sulfides 1 and 2 were purchased from Alfa Aesar. Sulfoxides
and sulfones were prepared by chemical oxidation from the corre-
sponding sulfides (H2O2/MeOH and m-CPBA/CH2Cl2) and exhibited
physical and spectral properties in agreement with those reported
[34].
Microorganisms
Fungal strains were purchased from different collections:
American Type Culture Collection (Aspergillus niger ATCC 11394),
Instituto Malbran collection (Aspergillus terreus INM0 31783), Fac-
ultad de Ciencias Exactas-Universidad de Buenos Aires (Aspergillus
flavus UBA 294), Departamento de Microbiología e Inmunología-
Universidad Nacional de Rio Cuarto (Aspergillus fumigatus RC 78;
Aspergillus nidulans (Emericella) RC 36; Aspergillus ochraceus RC
33), Laboratorio de Micología-Universidad Nacional del Litoral
(Aspergillus candidus LMFIQ 081; Aspergillus clavatus LMFIQ 089;
Aspergillus parasiticus LMFIQ 133), IIB-INTECH Collection of Fungal
Cultures (Aspergillus japonicus ICFC 744/11). Stock cultures of all
fungal strains were stored in Petri dishes on solid PDA or Czapeck
media at 4 ◦C.
2.3. Analytical methods
Biotransformation samples were extracted with ethyl acetate
(3× 15 mL). No internal standard was added. The organic phases
were combined and dried over anhydrous Na2SO4 and sol-
vent removed in vacuo. Conversion and ee of substrate 1 were
determined by chiral GC-FID with a ␤-DEX-column. Tempera-
ture setting: 140 ◦C, 0.5 ◦C/min, 150 ◦C (10 min), 0.5 ◦C/min, 160 ◦C
(10 min); injector: 200 ◦C, carrier gas N2: 25 cm/seg; FID: 300 ◦C; rt
(1) 9.7 min; rt (S)-1a: 50.3 min; rt (R)-1a: 50.8 min; rt 1b: 52.9 min.
Absolute configuration of enantiomers was determined by optical
rotation of the isolated products purified by column chromatogra-
phy (CC) over silica gel (70–230 mesh) eluted with CH2Cl2:CH3OH
mixtures. For substrate 2, conversion was determined by GC-FID
equipped with a 007 methyl 5% phenyl silicone column. Tem-
perature setting: 100 ◦C (2 min), 2 ◦C min, 130 ◦C; injector: 200 ◦C;
carrier gas N2: 25 cm/seg; FID: 300 ◦C; rt 2: 6.2 min; rt 2a: 14.37 min.
The ee was determined by chiral HPLC-DAD with a Chiralcel OD
column; n-hexane: IPA (9:1); 0.8 mL/min; 25 ◦C; 210 nm; rt (R)-2a:
16.8 min; rt (S)-2b: 18.1 min.
2.1. General procedures for biotransformation
2.1.1. Growing cells
A two-step-process was conducted. Spores of Aspergillus strains
were inoculated into 50 mL Czapeck medium in 250 mL baffled
Erlenmeyer flasks and incubated at 28 ◦C for 2 days on orbital shaker
(150 rpm). Following incubation, fungal cells were harvested and
subcultured in 20 mL fresh medium in 50 mL Erlenmeyer flasks.
Substrates 1 and 2 were added dissolved in DMSO and the reac-
tion mixtures were incubated at 28 ◦C for 6 days on orbital shaker
(150 rpm). Biotransformation progress was monitored every 2 days
by GC-FID. Blank assays without substrates and without fungi were
carried out in parallel. Experiments were performed in triplicate.
3. Results and discussion
3.1. Screening procedure
2.1.2. Resting cells
Previous works have reported the sulfoxidation of thioanisole
to (R)-methyl(phenyl)sulfoxide employing fungi such as M. isabel-
lina and T. viride, which furnished optical purities of 65% and 70%,
Pre-cultures of selected Aspergillus strains were prepared as
described above. After incubation, fungal cells were harvested by
centrifugation and washed with phosphate buffer. Cells (1 g per
flask) were re-suspended in phosphate buffer (30 mL, pH 6.0, 0.1 M)
in Erlenmeyer flasks (50 mL). Substrate 1 (1 mM, dissolved in 25 ␮M
of DMSO) was added and bio-reaction was incubated in orbital
shaker (150 rpm) at 28 ◦C for 6 days. Biotransformation progress
was monitored every 2 days by GC-FID analysis. Blank assays
without substrate and without fungi were carried out in parallel.
Experiments were performed in triplicate.
2.2. Optimization of the biotransformation process
To determine best culture ages and bio-reaction times, biotrans-
formation of 1 using A. candidus resting cells was conducted in
three different conditions. A: a 3-day-old pre-culture was used and
biotransformation was monitored for 2 and 4 days; B: a 3-day-old
Fig. 2. Culture age and bio-reaction time using A. candidus resting cells. Arrows
indicate the time when substrate 1 was added. Dotted lines show the day when
samples were taken. c (%) and ee (%) determined by chiral GC analysis.

34
M.L. Mascotti et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 32–36
Table 1
A. fumigatus after 4 days of bio-reaction but, the ees did not remain
constant during the process. Specifically in the case of A. flavus, the
ee decreased during incubation similarly to what occurred with A.
parasiticus although this fungus did not fully transform the sub-
strate (entries 22–24, Table 1). On the contrary, with A. niger and
A. fumigatus the ees increased during the bio-reaction time, more
significantly for the latter (entries 10–12 and 19–21, Table 1). Col-
lado et al. [20] observed the same phenomena by using B. cinerea as
biocatalyst and tried to rationalize this fact by suggesting that one
enantiomer of the sulfoxide is transformed into sulfone. This expla-
nation could not be applied in our system since no over-oxidation
was noticed. Besides, as it happened with substrate 1, when using A.
nidulans, A. candidus and A. clavatus in the oxidation of 2, no enan-
tioselectivity was displayed throughout the process (entries 1–3
and 4–6, Table 1).
Screening results for the oxidation of sulfides (1, 2) employing Aspergillus strains as
biocatalysts.
Entry
Strain
Biotransformation
time (days)
(R)-1a
c (%)
(R)-2a
c (%)
ee (%)
ee (%)
1
2
3
2
4
6
29
71
100
62
58
58
10
55
55
rac
rac
rac
Aspergillus
candidus
4
5
6
2
4
6
29
60
100
22
22
22
2
4
7
rac
rac
rac
Aspergillus
clavatus
7
8
9
2
4
6
31
96
100
66
57
59
33
98
100
17
12
9
Aspergillus
flavus
A. terreus INM 031783 did not show stereoselection. In contrast,
other authors have reported that a Brazilian strain (A. terreus CCT
3320) oxidized 2 into the S-sulfoxide, but with low chemoselec-
tivity, since an appreciable amount of sulfone was reported, either
using free cell or immobilized on chrysotile systems [25].
10
11
12
2
4
6
85
95
100
58
61
62
42
92
100
38
52
72
Aspergillus
fumigatus
13
14
15
2
4
6
87
89
90
60
62
97
80
85
88
50
53
55
Aspergillus
japonicus
Interestingly, A. japonicus resulted an excellent biocatalyst to
perform the oxidation of 1. In this case even before optimiza-
tion, optical purity of the obtained (R)-cyclohexyl(methyl)sulfoxide
was excellent (ee 97%) and conversion reached 90% in this pre-
liminary screening assay. Subsequently, in order to isolate the
product without changing oxygenation conditions, the experi-
ment was performed increasing the number of batches to 12.
The isolated yield obtained after purification by CC was 21%
(97% ee by chiral GC analysis). The [␣]D −27.87 (c = 2.44, ace-
tone) is in agreement with the optical rotation sign, reported
by Capozzi et al., for the R-enantiomer [36]. This experiment
allowed us to isolate and purify the biotransformation product and
unambiguously assign the sulfur absolute configuration. A hypoth-
esis to explain this yield might be the probable degradation of
the substrate and/or product into more polar molecules which
were not extracted from the reaction medium. Consequently the
time-course ee values could also be affected (e.g. entries 13–15,
Table 1).
16
17
18
2
4
6
5
98
100
rac
rac
rac
2
18
64
9
20
15
Aspergillus
nidulans
19
20
21
2
4
6
93
nd
100
32
nd
37
58
95
100
42
54
57
Aspergillus
niger
22
23
24
2
4
6
82
100
100
48
54
55
3
22
36
25
11
rac
Aspergillus
parasiticus
25
26
27
2
4
6
nd
nd
100
rac
rac
rac
nd
nd
100
nd
nd
rac
Aspergillus
terreus
c (%): Conversion of substrate into sulfoxide. In all cases no sulfone formation was
detected. ee (%): enantiomeric excess determined by chiral GC-FID for 1a and by
chiral HPLC-DAD for 2a.
nd: not determined; rac: racemic.
respectively [35]. Conversely, Helmintosporum sp. and B. cinerea
showed a slight preference to the S-enantiomer with ees of 48%
and 50% [19,20]. Also, the basidiomycetes Irpex lacteus, Pycnoporus
sanguineus, Trametes rigida and T. versicolor were able to trans-
form thioanisole and other aromatic sulfides to the S-sulfoxides
[21]. Nevertheless, we consider that the use of a sole substrate
as molecular probe to detect selective sulfoxidation in general
screenings might guide to misleading conclusions, since false neg-
atives may pass through. Thus, by using two substrates of different
chemical nature the chance to detect a biocatalyst will certainly
increase. Therefore a dialkyl and an alkyl aryl sulfide, namely
cyclohexyl(methyl)sulfide (1) and thioanisole (2), were selected as
model substrates (Fig. 1).
In the present work, nine different Aspergillus species as grow-
ing cells were screened for their sulfoxidation ability. At different
biotransformation times (2, 4 and 6 days) samples were withdrawn
and the relative amounts of the corresponding sulfide and sulfox-
ide were measured (Table 1). In cases in which stereoselection
was achieved, the formation of the R-enantiomer was observed.
Remarkably, no sulfone was detected in all the experiments.
Overall it was noticed that 1 was better substrate than 2,
concerning conversion and stereoselection. Conversions of dialkyl
sulfide were almost completed at 6 days of incubation with all
the tested fungi. It is noticeable that some strains display poor or
moderate (if any) stereoselection toward thioanisole but good to
excellent selectivities for sulfide 1. This observation gives a good
reason for the use of at least two different substrates for general
screenings. Thioanisole was totally oxidized by A. flavus, A. niger and
3.2. Optimization of biotransformation conditions
3.2.1. Avoiding non-enzymatic oxidation
Substrate 1 was chosen to optimize biotransformation condi-
tions.
It was observed by the analysis of the blank assays that around
3–10% of the sulfoxide is produced by non-enzymatic oxidation
as already reported [37]. This fact worsens the overall stereose-
lectivity of the process lowering the ee. Therefore, we chose the
sulfoxidation catalyzed by A. flavus to set up conditions in order to
minimize this effect since with this strain the product ee remained
unaltered along the reaction times (entries 7–9, Table 1). The strat-
egy consisted of varying the availability of oxygen, by modifying the
head-space volume of the reaction batch using different volumes
of culture media in Erlenmeyer flasks of the same size. Oxygena-
tion proved to be a critical parameter. As depicted in Table 2,
best conditions resulted with 30 mL of media in 50 mL flasks, since
with smaller volume of medium (entries 1 and 2) non-enzymatic
oxidation was higher, and with less oxygen availability sulfoxide
formation was very poor (entry 5).
3.2.2. Biotransformations with resting cells
An advantage of using resting cells is that the process is gen-
erally cleaner and work-up is much easier. Based on the results
of the growing cell biotranformations, we selected A. flavus, A.
fumigatus, A. parasiticus, A. candidus and A. japonicus to explore
the influence of resting cell on the process (Table 3). Under these

M.L. Mascotti et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 32–36
35
Table 2
Table 4
Effect of head-space volume in non-enzymatic oxidation.
Effect of substrate/biocatalyst ratio in the oxidation of substrate 1 using A. japonicus.
Entry
Culture medium
volume (mL)
c (%)
ee (%)
Entry
[S] (mM)a
c (%)
ee (%)
1
2
3
4
5
1
10
25
50
100
100
99
27
14
<3
97
70
30
30
nd
1
2
3
4
5
6
Biotransformation
Non-biocatalyzed reaction
Biotransformation
Non-biocatalyzed reaction
Biotransformation
Non-biocatalyzed reaction
20
20
30
30
40
40
12
17
24
<3
4
64
rac
76
nd
nd
nd
c (%) and ee (%) determined by chiral GC-FID analysis. nd: not determined.
<3
a
Final substrate concentration. Bio-reaction time 5 days.
Biotransformation of substrate 1 carried out using A. flavus growing cells. Different
volumes of medium were assessed in 50 mL Erlenmeyer flasks. As control non-
biocatalyzed reactions were performed in different volumes of medium.
nd: not determined.
(66%) resulted constant along all reaction times. These results are
comparable to those observed on the sixth day in growing cells
methodology where the optical purity was a little lower (ee 58%)
although conversion was 100%. The conversions and ees decreased
significantly in experiment B compared to experiment A. When
resting cell systems were performed with “old cultures” (experi-
ment C) results were intermediate. In the last two experiments,
conversion increased along with the bio-reaction time, although
maximal values were lower than 60%.
conditions, best results were achieved employing A. candidus as
biocatalyst. After 5 days, (R)-1a was recovered with 66% ee. For
the other Aspergilli tested, complete conversions were obtained
after 5–6 days, while optical purities of (R)-1a were slightly lower.
By using this methodology the expression and/or the activity of
enzymes may vary and consequently, reaction rates as well as stere-
oselection could be significantly affected. This fact is particularly
important in these studies since not only the enzymes responsible
of the sulfide oxidation are involved but also the ones that play a
role in the regeneration of the redox cofactors. Besides, the results
of the screening procedure suggested that the expression and/or
the activity of the enzyme/s involved in the desired reaction depend
on the developmental stage of the fungal culture, since conversions
and ees vary along the incubation times in several cases. By com-
paring these results with those of the growing cell experiments, it
is observed that in general, the sulfoxide ees slightly improved by
using resting cells. Unfortunately, A. japonicus, our best biocatalyst
in growing cell systems, resulted severely affected by this starving
condition, in which the ee of the product dropped drastically from
97% to 57% (entries 13–15, Table 3). A possible explanation of this
fact could be the inhibition/impaired expression of cofactor regen-
eration enzymes that affects the overall sulfoxidation performance,
as it was discussed above.
3.2.4. Substrate/biocatalyst ratio
Since the best results were observed employing A. japonicus as
growing cells toward susbtrate 1, this system was chosen in order
to continue with the process optimization.
To improve the substrate/biocatalyst ratio, increasing substrate
concentrations were evaluated. Working conditions were similar
to those already described for the screening test, varying only the
substrate concentration between 1 and 100 mM. Samples were
withdrawn at the fourth day of bio-reaction. Results are depicted
in Table 4.
For the two lowest concentrations (1 and 10 mM) no signifi-
cant changes were evident in the biomass dry weight at the end
of the experiment, in comparison with blank assays without sub-
strate addition (data not shown). At concentrations higher than
25 mM, the conversion and stereoselectivity decreased, although
the chemoselectivity was not affected since no sulfone was
detected. This fact could be attributed to a toxic effect caused by the
substrate, since there was no biomass development after substrate
addition. These results are in agreement with those reported for a
bacterial strain, Rhodococcus sp. ECU0066. Despite this microorgan-
ism was isolated from soil using substrate 2 as a sole carbon source,
its viability was affected by high substrate concentration [38,39].
3.2.3. Age of the culture and reaction times in resting cell systems
Since A. candidus was the best biocatalyst as resting cells
(Table 3), it was chosen to analyze the influence of using fungal
cultures of different ages and reaction times. Three different exper-
iments (A–C) were run in parallel using A. candidus, as shown in
Fig. 2. In experiment A, conversion rates (100%) and sulfoxide ee
3.2.5. Use of co-solvents
Although in nature enzymes work in aqueous medium, many
substrates of interest for organic chemists are hydrophobic, requir-
ing the addition of organic solvents to increase their solubilities.
Moreover, the use of biphasic systems with whole cell biocatalysts
offers the possibility to xenobiotic substrates and/or their prod-
ucts may be partitioned between the two phases, reducing their
toxic effects on the living biocatalyst and improving the overall
performance of the process. In this regard, it was reported the
use of organic-aqueous biphasic systems to overcome the poor
substrate tolerance and improve enantioselectivity for the above
mentioned Rhodococcus strain in the asymmetric sulfoxidation of
2 [39]. The effect of six different co-solvents in the biooxidation
of 1 was studied (Table 5). Among the most notable results, the
use of DMSO in concentration of 0.5% (v/v) significantly dimin-
ished cell development and viability, being affected the conversion,
as well as the stereoselectivity. Something similar occurred when
toluene was used as co-solvent since the substrate was oxidized in
47% with poor stereoselectivity (ee 25%). With methanol and tert-
butanol, although the conversions were excellent, the ees dropped
significantly and, more importantly, the formation of sulfone was
observed for the first time. Undoubtedly, the best results were
Table 3
Biotransformation of 1 employing Aspergillus resting cells.
Entry
Microorganism
Incubation
time (days)
c (%)
ee (%)
1
2
3
2
5
6
81
100
100
55
58
60
A. fumigatus
4
5
6
2
5
6
83
100
100
48
69
63
A. parasiticus
A. flavus
7
8
9
2
5
6
10
55
99
49
52
56
10
11
12
2
5
6
nd
100
100
–
66
66
A. candidus
A. japonicus
13
14
15
2
3
5
nd
98
99
–
50
57
c (%) and ee (%) determined by chiral GC-FID analysis. nd: not determined.

36
M.L. Mascotti et al. / Journal of Molecular Catalysis B: Enzymatic 82 (2012) 32–36
Table 5
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Entry
Co-solvent
[0.5%, v/v]
Sulfide
1 (%)
Sulfoxide
1a (%)
(R)-1a ee (%)
Sulfone
1b (%)
1
2
3
4
5
6
DMSO
94
0
6
42
0
0
7
0
7
0
iso-Propanol
Methanol
n-Hexane
100
>99
0
93
95
80
40
64
80
77
25
5
tert-Butyl
alcohol
Toluene
13
60
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4. Conclusion
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The ability of several Aspergillus species of performing enantios-
elective sulfoxidations toward an aryl alkyl and a dialkyl sulfide
was screened. All the strains that showed selectivity favored the
(R)-enantioner. Remarkably, no sulfone was detected in the tested
standard conditions, demonstrating the chemoselectivity of the
process. The results of the optimization studies indicated that A.
japonicus presents a huge potential to be used as whole cell biocat-
alyst for symmetric sulfoxidations. In fact its growing cell cultures
with the addition of isopropyl alcohol as co-solvent resulted a good
alternative for the preparation of (R)-cyclohexyl(methyl)sulfoxide
since the conversion (100%) and the optical purity (ee > 99%) were
excellent.
Further studies aimed at the cloning and characterization of the
involved enzyme/s, as well as their use for sulfoxidation of different
substrates, are in progress in our laboratory.
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Acknowledgments
This work was supported by grants from Universidad Nacional
de San Luis 7301, PIP6228 Consejo Nacional de Investigaciones
Científicas y Técnicas (CONICET) and PICT352 Agencia Nacional de
Promoción Científica y Tecnológica (ANPCyT). M.L.M is a doctoral
CONICET fellow. F.R.B is a postdoctoral CONICET fellow. A.A.O and
M.K.S are members of the Research Career of CONICET. We espe-
cially acknowledge Dr. Ivan Lavandera for his helpful contributions.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2012.05.003.