Enantioselective reduction of propiophenone formed from 3-chloropropiophenone and stereoinversion of the resulting alcohols in selected yeast cultures

Article abstract of DOI:10.1016/j.tetasy.2014.08.006

Biotransformation of 3-chloro-1-phenylpropan-1-one 1 in sixteen selected cultures of yeast strains has been carried out. For most of the biocatalysts studied the substrate was fully consumed after 1-9 h of transformation, with the exception of the culture of Saccharomyces brasiliensis KCh 905, in which after 24 h trace amounts of the substrate were still visible (2%). However, apart from the expected enantiospecific reduction of the substrate to 3-chloro-1-phenylpropan-1-ol 3, the main biotransformation products comprised of a dehalogenation product - propiophenone 2 and the product of its reduction - 1-phenylpropan-1-ol 4. It was only in the cultures of five strains: Saccharomyces brasiliensis KCh 905, Rhodotorula marina KCh 77, Candida parapsilosis KCh 909, Candida viswanathii KCh 120, and Saccharomyces cerevisiae KCh 464 that 3-chloro-1-phenylpropan-1-ol 3 was observed in amounts of more than 10% of the product mixture. (S)-3-Chloro-1-phenylpropan-1-ol with ee = 91% was identified after 9 h of biotransformation in the culture of Candida viswanathii KCh 120, whereas (R)-3-chloro-1-phenylpropan-1-ol with ee = 28% was found in the culture of Aphanocladium album KCh 417. 1-Day biotransformation of propiophenone 2 in the cultures of Rhodotorula strains gave (S)-1-phenylpropan-1-ol 4 with a very high ee (95-99%) and 85-99% of substrate conversion, whereas transformation of this substrate in the cultures of Candida viswanathii KCh 120 and Candida parapsilosis KCh 909 led to (R)-1-phenylpropan-1-ol with ee = 98% and 97%, respectively. During biotransformation of propiophenone the percent composition of the reaction mixtures changed with time. Employment of the racemic mixture of 1-phenylpropan-1-ol 4 as a substrate for biotransformations allowed us to observe that the biocatalysts tested were capable of enantioselective oxidation of (S)-1-phenylpropan-1-ol. An exception was the culture of Rhodotorula glutinis KCh 242, in which after one day of biotransformation (S)-1-phenylpropan-1-ol was obtained with ee = 96%.

Full text of DOI:10.1016/j.tetasy.2014.08.006

Tetrahedron: Asymmetry 25 (2014) 1264–1269
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Enantioselective reduction of propiophenone formed from
3-chloropropiophenone and stereoinversion of the resulting
alcohols in selected yeast cultures
⇑
Tomasz Janeczko , Edyta Kostrzewa-Susłow
Department of Chemistry, Wrocław University of Environmental and Life Sciences, Norwida 25, 50-375 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Biotransformation of 3-chloro-1-phenylpropan-1-one 1 in sixteen selected cultures of yeast strains has
been carried out. For most of the biocatalysts studied the substrate was fully consumed after 1–9 h of
transformation, with the exception of the culture of Saccharomyces brasiliensis KCh 905, in which after
24 h trace amounts of the substrate were still visible (2%). However, apart from the expected enantio-
specific reduction of the substrate to 3-chloro-1-phenylpropan-1-ol 3, the main biotransformation
products comprised of a dehalogenation product—propiophenone 2 and the product of its reduction—
1-phenylpropan-1-ol 4. It was only in the cultures of five strains: Saccharomyces brasiliensis KCh 905,
Rhodotorula marina KCh 77, Candida parapsilosis KCh 909, Candida viswanathii KCh 120, and Saccharomy-
ces cerevisiae KCh 464 that 3-chloro-1-phenylpropan-1-ol 3 was observed in amounts of more than 10% of
the product mixture. (S)-3-Chloro-1-phenylpropan-1-ol with ee = 91% was identified after 9 h of
biotransformation in the culture of Candida viswanathii KCh 120, whereas (R)-3-chloro-1-phenylpro-
pan-1-ol with ee = 28% was found in the culture of Aphanocladium album KCh 417. 1-Day biotransforma-
tion of propiophenone 2 in the cultures of Rhodotorula strains gave (S)-1-phenylpropan-1-ol 4 with a very
high ee (95–99%) and 85–99% of substrate conversion, whereas transformation of this substrate in the
cultures of Candida viswanathii KCh 120 and Candida parapsilosis KCh 909 led to (R)-1-phenylpropan-1-
ol with ee = 98% and 97%, respectively. During biotransformation of propiophenone the percent
composition of the reaction mixtures changed with time. Employment of the racemic mixture of
1-phenylpropan-1-ol 4 as a substrate for biotransformations allowed us to observe that the biocatalysts
tested were capable of enantioselective oxidation of (S)-1-phenylpropan-1-ol. An exception was the cul-
ture of Rhodotorula glutinis KCh 242, in which after one day of biotransformation (S)-1-phenylpropan-1-ol
was obtained with ee = 96%.
Received 14 June 2014
Accepted 4 August 2014
Available online 10 September 2014
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
of fluoxetine.5 A known fact is that (S)-norfluoxetine is over 20-fold
more potent than the (R)-enantiomer as a selective inhibitor of
1-Chloropropan-1-ol obtained from 3-chloropropiophenone is a
building block for synthesis of fluoxetine (introduced to the market
in 1998 under the brand name Prozac), which is a psychotropic
drug that belongs to the class of selective serotonin reuptake
inhibitors (SSRI). Fluoxetine is produced as a racemic mixture,1–3
(R)-fluoxetine is a drug approved for the treatment of attention-
deficit hyperactivity disorder (ADHD) in teenagers and young
adults, and in special cases in children under the age of six.4
However, many differences have been reported in the literature
with regard to the pharmacological potencies of the enantiomers
serotonin uptake both in vitro and in vivo.6 (R)-Tomoxetine is
two-fold more potent than the racemate and nine-fold more
potent than the (S)-enantiomer.2
The key step in the synthesis of (R)-fluoxetine, as well as other
psychoactive derivatives (tomoxetine, nisoxetine, fenfluramine,
and dapoxetine) is to obtain enantiomerically pure 3-substituted
1-arylpropan-1-ols.7–10 1-Chloro-1-phenylopropan-1-ol can be
obtained with a high enantiomeric excess by the enantiospecific
reduction of 3-chloropropiophenone using chiral chemical cata-
lysts,4,11–14 or with the help of lipases—via esterification15–17 or
hydrolysis.18
A convenient method to obtain enantiomerically pure alcohols
is an enantiospecific reduction of ketones with the help of biocata-
lysts, such as those from plants, filamentous fungi, and yeasts.19–21
⇑
Corresponding author. Tel.: +48 (71) 3205252; fax: +48 (71) 3284124.
E-mail address: janeczko13@interia.pl (T. Janeczko).
http://dx.doi.org/10.1016/j.tetasy.2014.08.006
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.

T. Janeczko, E. Kostrzewa-Susłow / Tetrahedron: Asymmetry 25 (2014) 1264–1269
1265
However, the biotransformation of 3-chloropropiophenone using
2. Results and discussion
live biocatalysts is accompanied with
a competitive side
reaction—dehalogenation.7,22,23
The biotransformations of selected substrates were carried out
in fifteen cultures of yeast strains. The strains were selected based
on their proven ability to reduce a carbonyl group in prochiral
Fronza et al. 1991 employed baker’s yeast as a biocatalyst to
produce (S)-3-chloro-l-phenylpropan-l-ol and 3-chloropropiophe-
none in 58% and 30% yields, respectively. The authors used
isotopically labeled compounds which allowed them to prove
the mechanism of the reductive dehalogenation taking place in
the culture of the biocatalyst.7 When whole cells of the
white-rot fungus Merulius tremellosus ono991 were used,
1-phenylpropan-1-ol (60%) was obtained as a main product,
except for 3-chloro-l-phenylpropan-l-ol and 3-chloropropiophe-
none.22 Biotransformations of 3-chloropropiophenone were also
carried out in the cultures of Daucus carota and five endophytic
strains. After a 2-day incubation of the substrate not only was
the corresponding chiral 3-chloro-l-phenylpropan-l-ol obtained,
but also unsubstituted alcohols and ketones were isolated. The
product distribution was highly dependent on the strain used,
suggesting the participation of both carbonyl reductases and
enoate reductases.23
In this work we submitted 3-chloropropiophenone 1 to bio-
transformation using the cultures of ten-odd yeast strains. Our
aim was to study the dynamics of the two competitive processes:
reduction and dehalogenation and to establish whether 1-phenyl-
propan-1-ol is formed by the dehalogenation followed by the
reduction, or the opposite: the reduction occurs first, and is fol-
lowed by dehalogenation.
ketones.17,18,21,24 3-Chloro-1-phenylpropan-1-on
1 underwent
effective transformation in cultures of all the yeast strains tested.
After 24 h of transformation only in the culture of Saccharomyces
brasiliensis KCh 905 was a small amount of the unreacted substrate
observed (2%). For the rest of biocatalysts tested 3-chloro-1-phe-
nylpropan-1-on 1 was fully consumed after 1-9 h. However, in a
few of the tested strains the expected reduction product:
3-chloro-1-phenylpropan-1-ol 3 was observed in the reaction
mixture (Scheme 1).
In the culture of the strain Candida viswanathii KCh 120 after 6 h
of the transformation (S)-3-chloro-1-phenylpropan-1-ol 3 was
observed in 47% of the reaction mixture and with ee = 87%
(Table 1). The amount of product 3 and its enantiomeric excess
decreased with biotransformation time. There was also (S)-1-phe-
nylpropan-1-ol 4 identified in the reaction mixture in an amount of
41% and ee >99% after 3 h. However, the amount of the (S)-enantio-
mer of 4 was considerably decreased with time (9% after 9 h and 2%
after 3 days), whereas the amount of (R)-1-phenylpropan-1-ol 4
was increased, to reach 28% and ee >99% after 6 days.
In order to confirm that the inversion of configuration of alcohol
4 is the result of an enantioselective oxidation of the (S)-enantio-
mer, we subjected to the biotransformation the product of
OH
O
O
OH
Cl
Cl
Cl
1
2
(R)-3
(S)-3
OH
OH
(R)-4
(S)-4
Scheme 1. Course of the transformation of 3-chloro-1-phenylpropan-1-one 1, 1-phenylpropan-1-one 2, and 1-phenylpropan-1-ol 4 by the selected microorganisms.
Table 1
Microbial transformation of 3-chloro-1-phenylpropan-1-one 1
Microorganism
Reaction time (days)
1 (%)
Product 3
Product 2 (%)
Product 4
%
ee
Absolute configuration
%
ee
Absolute configuration
Yarrowia lipolytica 1-101-1,31-K1
Yarrowia lipolytica KCh 71
Yarrowia lipolytica KCh 72
Yarrowia lipolytica ATCC 32-338 A
Candida parapsilosis KCh 909
Candida viswanathii KCh 120
9
3
3
3
3
0.25
6
1
9
0.25
1
0.25
1
3
6
1
1
6
1
0
0
0
0
0
0
0
0
0
0
0
64
0
0
0
0
0
0
0
0
0
—
—
—
—
—
—
—
(R)
(S)
(S)
(S)
—
—
—
—
—
(S)
—
8
14
22
58
33
23
57
21
32
60
9
36
15
42
0
21
33
33
61
21
92
36
63
70
26
92
95
>99
85
35
>99
91
—
94
94
90
86
65
67
33
63
(R)
(R)
(R)
(R)
(R)
(S)
(R)
(S)
(S)
(S)
(S)
—
(S)
(S)
(S)
(S)
(S)
(S)
(S)
(R)
86
78
42
29
30
28
72
68
34
91
0
85
54
100
70
57
15
2
0
—
38
47
15
7
0
6
0
0
0
4
0
8
10
51
33
7
12
87
28
>99
—
0
—
—
—
Rhodotorula rubra KCh 4
Rhodotorula rubra KCh 82
Rhodotorula glutinis KCh 242
Rhodotorula marina KCh 77
63
—
Zygosaccharomyces bailii KCh 907
Saccharomyces pastorianus KCh 906
Saccharomyces cerevisiae KCh 464
Saccharomyces brasiliensis KCh 905
Aphanocladium album KCh 417
45
74
81
75
29
(S)
(S)
(S)
(S)
(R)
4
0
6
72

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T. Janeczko, E. Kostrzewa-Susłow / Tetrahedron: Asymmetry 25 (2014) 1264–1269
Table 2
Microbial transformation of 1-phenylpropan-1-one 2 and 1-phenylpropan-1-ol 4
Microorganism
Transformation of 2
Transformation of 4
% of 4
ee
Absolute configuration
% of 4
ee
Absolute configuration
Yarrowia lipolytica 1-101-1,31-K1
Yarrowia lipolytica KCh 71
Yarrowia lipolytica KCh 72
Yarrowia lipolytica ATCC 32-338 A
Candida parapsilosis KCh 909
Candida viswanathii KCh 120
Rhodotorula rubra KCh 4
94
94
60
94
70
77
98
83
85
76
70
65
58
50
82
66
76
50
35
97
98
68
94
95
76
97
46
81
8
(R)
(R)
(R)
(R)
(R)
(R)
(S)
(S)
(S)
(S)
(R)
(S)
(S)
(R)
(R)
75
97
98
94
85
87
93
97
92
94
98
93
82
97
97
24
65
1
35
>99
>99
97
70
96
8
0
8
31
2
27
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(S)
(R)
—
Rhodotorula rubra KCh 82
Rhodotorula glutinis KCh 242a
Rhodotorula marina KCh 77
Zygosaccharomyces bailii KCh 907
Saccharomyces pastorianus KCh 906
Saccharomyces cerevisiae KCh 464
Saccharomyces brasiliensis KCh 905
Aphanocladium album KCh 417
(R)
(R)
(R)
(R)
84
Results after nine days of biotransformation.
a
Results obtained after one day of biotransformation.
dehalogenation of 1, that is, 1-phenylpropan-1-one 2 and also a
racemic mixture of 1-phenylpropan-1-ols 4 (Table 2). Incubation
of 2 in the culture of Candida viswanathii KCh 120 showed that
the reduction proceeded with low enantioselectivity (ee = 8%
((R)-4) and 29% substrate conversion after 1 day of the biotransfor-
mation). The enantiomeric excess of (R)-1-phenylpropan-1-ol 4
considerably increased with time, to reach over 98% after 9 days
of biotransformation. The unreacted substrate 2 was still visible
in the reaction mixture (23% after nine days, Fig. 1). Using
compound 4 as a substrate confirmed the capability of the strain
Candida viswanathii KCh 120 for the effective enantiospecific
oxidation. After only 3 days of incubation of 4 we obtained 38%
of 1-phenylpropan-1-one 2 and 62% of (R)-1-phenylpropan-1-ol 4
with ee = 90%. After six days of transformation product (R)-4 was
obtained with an ee >99%.
the same strain. After 3 days (S)-1-phenylpropan-1-ol 4 was iden-
tified with 57% conversion and 66% enantiomeric excess. However,
after 6 days the (R)-enantiomer predominated (ee = 73%), to reach
an ee of 97% after nine days. Incubation of the racemic mixture of
alcohols 4 showed that Candida parapsilosis KCh 909, similarly to
Candida viswanathii KCh 120, was capable of effective oxidation
of the (S)-alcohol, which afforded phenylpropan-1-ol 4 with an
ee >99% (Table 2).
In the culture of Saccharomyces cerevisiae KCh 464 high stability
of (S)-1-phenylpropan-1-ol 4 was observed. During biotransforma-
tion of 1 product 4 was present in the reaction mixture in 53% after
one day of biotransformation and 50% after 9 days (ee = 81%). Note
that the content of propiophenone 2 was changing, as well as the
enantiomeric excess of (S)-1-phenylpropan-1-ol 4 (ee = 74% after
1 day and ee = 45% after 9 days). Propiophenone 2 underwent
Figure 1. Time dependence of transformation of: A—1-phenylpropan-1-one 2 and B—1-phenylpropan-1-ol 4 in the culture of Candida viswanathii KCh 120.
When substrate 1 was incubated in the culture of the strain
Candida parapsilosis KCh 909, after 9 days of biotransformation
the reaction mixture contained 33% of (R)-3-chloro-1-phenylpro-
pan-1-ol 3, however with a very low ee (3%). At the same time in
the reaction mixture we observed also 1-phenylpropan-1-one (2)
(4%) and (R)-1-phenylpropan-1-ol (4) (63%, ee = 75%). The enantio-
meric excess of 4 changed with biotransformation time (from
ee = 5% for the (S)-enantiomer to ee = 75% for the (R-)one). The
same tendency was observed for the biotransformation of 2 by
effective reduction to (S)-alcohol 4 with ee = 98% and 66% conver-
sion. However, both the enantiomeric excess and the content of
(S)-4 in the reaction mixture decreased with biotransformation
time (Table 2). Using racemic 4 as a substrate confirmed the capa-
bility of the strain Saccharomyces cerevisiae KCh 464 for selective
oxidation of the (S)-alcohol.
In a biotransformation using Saccharomyces pastorianus KCh 906
after 9 days of incubation of 1 the reaction mixture consisted of 8%
of halogenated alcohol (S)-3 (ee = 72%), 14% of propiophenone 2,

T. Janeczko, E. Kostrzewa-Susłow / Tetrahedron: Asymmetry 25 (2014) 1264–1269
1267
Figure 2. Time dependence of transformation of: A—1-phenylpropan-1-one 2 and B—1-phenylpropan-1-ol 4 in the culture of Yarrowia lipolytica KCh 71.
and as much as 78% of (S)-1-phenylpropan-1-ol 4 (ee = 59%).
Comparing these results with data presented in Table 1 (after
1 day of transformation) one can see that longer biotransformation
time resulted only in a loss of concentration of 2 and an increase in
enantiomeric excess of (R)-1-phenylpropan-1-ol 4 was observed
during the biotransformation time. Employment of compounds 2
and 4 as substrates confirmed the capability of the strains Yarrowia
lipolytica for the (R)-specific reduction of prochiral ketones and
enantioselective oxidation of the (S)-alcohols (Fig. 2).
content of 4. Employment of propiophenone as
a substrate
has shown that this strain has a weak ability to reduce the sub-
strates tested. After 3 days of biotransformation there was still
76% of the unreacted ketone observed. The reduction process
became faster as late as after 9 days (Table 2). This strain also dem-
onstrated a low capability to oxidize alcohol 4 (Table 2). Similar
results were observed for Zygosaccharomyces bailii KCh 907 (Tables
1 and 2).
In the culture Saccharomyces brasiliensis KCh 905 during incuba-
tion of ketone 2 and also the racemic mixture of alcohols 4, in both
cases low substrate conversion and low enantiomeric excesses of
the respective alcohols were observed (Table 2). However, when
compound 1 was used as a substrate, there was (S)-3-chloro-1-
phenylpropan-1-ol 3 identified in the reaction mixture with
ee = 75% (Table 1). After 6 days there was still 29% of 3 with an
ee = 74% and 52% of propiophenone 2 identified, along with 19%
of (S)-1-phenylpropan-1-ol 4 with an ee = 42%. The considerably
higher enantiomeric excess of 4 observed when 1 was used as a
substrate was the result of dehalogenation of (S)-3-chloro-1-phe-
nylpropan-1-ol 3, which was formed in this process with a high
enantiospecificity.
In biotransformations by the strain Aphanocladium album KCh
417 we also observed the reversible reduction of compound 2, with
a stereospecificity similar to Yarrowia lipolytica KCh 71. There was
also a high degree of similarity between these cultures noticed in
the case of biotransformation of racemic 1-phenylpropan-1-ol 4.
However, during incubation of 1 in the culture of Aphanocladium
album KCh 417 we identified (R)-3-chloro-1-phenylpropan-1-ol 3,
even after 9 days of the reaction (2%, ee = 36%). In the culture of
the strain Rhodotorula marina KCh 77 after one day of incubation
of 1, (S)-1-phenylpropan-1-ol 4 was identified (59%, ee >99%).
However, after 3 days both the content and the enantiomeric
excess of this compound decreased (Table 1). A similar biotransfor-
mation course was observed during the incubation of 2 in the cul-
ture of this strain (Fig. 3). When racemic alcohol 4 served as a
substrate, there was only oxidation of (S)-1-phenylpropan-1-ol 4
to ketone 2 observed (Table 2).
Surprising results were found for biotransformations of the sub-
strates in the culture of the strain Rhodotorula glutinis KCh 242.
After one day of transformation of substrates 1, 2, and 4, (S)-1-phe-
nylpropan-1-ol 4 was identified as a product, with an enantiomeric
excess of 94–96% (Tables 1 and 2). However, after 3 days of the
experiment with each of the three substrates, neither unreacted
substrates, nor the reaction products were observed in the reaction
mixture.
All cultures of the strain Yarrowia lipolytica used as biocatalysts
performed a very effective dehalogenation. After 1 h of biotransfor-
mation neither substrate 1 nor 3-chloro-1-phenylpropan-1-ol 3 was
visible, whereas, a considerable increase in concentration and
Figure 3. Time dependence of transformation of 1-phenylpropan-1-one 2 in the cultures of A—Rhodotorula marina KCh 77 and B—Rhodotorula rubra KCh 82.

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T. Janeczko, E. Kostrzewa-Susłow / Tetrahedron: Asymmetry 25 (2014) 1264–1269
During biotransformations of 3-chloro-1-phenylpropan-1-on 1
3. Conclusion
in cultures of the strains Rhodotorula rubra KCh 4 and KCh 82 there
were significant differences in the amounts and enantiomeric
excess of compound 3 observed. For the strain KCh 4, after 24 h,
compound 3 was still visible in 7% and with an ee >99%, whereas
for the strain KCh 82 after 1 h there was only 5% observed with
ee ꢀ 0, and after 9 h it had completely disappeared. Except for alco-
hol 3, for both strains there was also propiophenone 2 identified
after 1 h, in 74% of the reaction mixture for KCh 4 and 89% for
KCh 82. Longer biotransformation times resulted in very similar
results for both strains. Compound 2 underwent effective enantio-
selective reduction to (S)-1-phenylpropan-1-ol 4 (Table 1), but the
enantiomeric excess of the (S)-alcohol decreased with time in both
cultures. Analogous results have been found when compound 2 was
used as a substrate for the strains tested of Rhodotorula rubra
(Fig. 3). In the culture of Rhodotorula rubra KCh 4 after one day of
biotransformation (S)-1-phenylpropan-1-ol 4 was identified with
99% conversion and ee >99%, whereas for Rhodotorula rubra KCh
82 with 96% of conversion and also ee >99%. The enantiomeric
excess of (S)-4 was decreasing with time (Fig. 4). Using racemic
mixture of alcohols 4 confirmed that enzymatic systems of the Rho-
dotorula rubra strains tested performed enantioselective oxidation
of (S)-1-phenylpropan-1-ol (4), and after 9 days of biotransforma-
tion we obtained (R)-1-phenylpropan-1-ol 4 (Table 2 and Fig. 4).
Based on the experiments carried out in this study we can draw a
conclusion that the majority of biocatalysts tested performed effec-
tive dehalogenation of 3-chloro-1-phenylpropan-1-on 1. In all
strains tested of Yarrowia lipolytica the reaction was complete after
1 h. In the cultures of Rhodotorula rubra, Rhodotorula marina KCh
77, and Zygosaccharomyces bailii KCh 907 we did not identify com-
pound 1, but the presence of 3-chloro-1-phenylpropan-1-ol 3
proved that there were two competitive reactions: dehalogenation
and reduction. In the cultures of Rhodotorula rubra, Rhodotorula mar-
ina KCh 77, and Candida viswanathii KCh 120 (in this particular case
after 6 days) a decrease in content of compound 3 during biotrans-
formation time was observed. For the rest of the biocatalysts the
concentration in the reaction mixture and enantiomeric excess of
3-chloro-1-phenylpropan-1-ol 3 was constant. When we used pro-
piophenone 2 as a substrate, we obtained (S)-1-phenylpropan-1-ol
4 with a high enantiomeric excess in the cultures of Rhodotorula
rubra, Rhodotorula glutinis KCh 242, and Rhodotorula marina KCh
77, and (R)-1-phenylpropan-1-ol 4 in the cultures of Candida visw-
anathii KCh 120 and Candida parapsilosis KCh 909. In the cultures
of all tested microorganisms a longer biotransformation time
resulted in a drop in percent content of the (S)-alcohol, along with
an increase in the percent content of the (R)-alcohol. This
Figure 4. Time dependence of transformation of: A—1-phenylpropan-1-one 2 and B—1-phenylpropan-1-ol 4 in the culture of Rhodotorula rubra KCh 4.
The results obtained indicate that the substrate, 3-chloro-1-
phenylpropan-1-on 1, in the cultures of the biocatalysts tested
undergoes the following transformations: at first, elimination of
HCl takes place to give unsaturated ketone (phenyl vinyl ketone),
which is then hydrogenated to propiophenone 2. The next transfor-
mation is reduction of ketone 2 to 1-phenylpropan-1-ol 4 with the
spatial configuration depending on the biocatalyst used. The pro-
cess of the elimination followed by the hydrogenation was
described in detail by Fronza et al. 1991. In order to confirm this
mechanism the authors used an isotopically labeled substrate
time-dependence of product concentration was observed for micro-
organisms that possessed either (S)-specific or (R)-specific dehydro-
genases. Employment of racemic 4 as a substrate confirmed these
findings. We obtained (R)-1-phenylpropan-1-ol 4 with a high enan-
tiomeric excess in the cultures of Candida viswanathii KCh 120 and
Candida parapsilosis KCh 909, and also in the culture of the strain
Rhodotorula rubra KCh 82, which have (S)-specific dehydrogenases.
An exception was the strain Rhodotorula glutinis KCh 242, which
after one day of transformation afforded (S)-1-phenylpropan-1-ol
4 with an ee = 96%.
(a,a-dideuterio-3-chloropropiophenone), which was added to the
culture of bakers’ yeast.7 As a result, an enantiomerically pure
monodeuteriopropiophenone was obtained, which unequivocally
proved that the proposed course of biotransformation was correct.
The competition between elimination and reduction of the car-
bonyl group was also observed for some other biocatalysts.22,23 Our
results prove that the composition of the reaction mixture depends
on the activity of a few competing enzymes, which are specific for
each of the species. We have also demonstrated that the composi-
tion of the product mixture undergoes dynamic changes during the
biotransformation time.
4. Experimental
4.1. Materials
The substrates: racemic 1-phenylpropan-1-ol 4 was prepared
by reduction of ketone 2 with sodium borohydride in methanol;
3-chloro-1-phenylpropan-1-one 1 and 1-phenylpropan-1-one (2)
were purchased from Sigma–Aldrich. The microorganisms (fifteen
strains of yeast) were obtained as follows: Yarrowia lipolytica
1-101-1,31-K1 from the Department of Biotechnology and Food

T. Janeczko, E. Kostrzewa-Susłow / Tetrahedron: Asymmetry 25 (2014) 1264–1269
1269
Microbiology, Wrocław University of Environmental and Life
Sciences, Yarrowia lipolytica ATCC32-338A from the American Type
Culture Collection, thirteen: Yarrowia lipolytica KCh 71, Yarrowia
lipolytica KCh 72, Candida parapsilosis KCh 909, Candida viswanathii
KCh 120, Rhodotorula rubra KCh 4, Rhodotorula rubra KCh 82,
Rhodotorula glutinis KCh 242, Rhodotorula marina KCh 77, Aphanoc-
ladium album KCh 417, Saccharomyces cerevisiae KCh 464, Saccharo-
myces brasiliensis KCh 905, Saccharomyces pastorianus KCh 906, and
Zygosaccharomyces bailii KCh 907 from the Department of Chemis-
try of Wrocław University of Environmental and Life Sciences. All
the strains were cultivated on a Sabouraud agar consisting of
aminobac (5 g), peptone K (5 g), glucose (40 g), and agar (15 g) dis-
solved in 1 l of distilled water, at 28 °C and pH 5.5 and stored in a
refrigerator at 4 °C.
extracted with CHCl3 (3 Â 300 ml), dried (MgSO4), and concen-
trated in vacuo. The transformation products were separated by
column chromatography and analyzed (TLC, GC, 1H NMR and also
confirmed by 13C NMR and correlation spectroscopy).
4.5. Spectroscopic data of the isolated metabolites
4.5.1. (S)-3-Chloro-1-phenylpropan-1-ol 3
A
one-day transformation of 3-chloropropiophenone
1
(200 mg) in the culture of C. viswanathii KCh 120 yielded 84 mg
of compound (S)-3 (colorless oil); [
a
]
23 = À20.6 (c 1.1, CHCl3)
D
(91% ee) (lit. [
a]
25 = À23 (c 1.93, CHCl3), 96% ee).11 1H and 13C
D
NMR spectra were in agreement with those previously published.11
4.5.2. (S)-1-Phenylpropan-1-ol 4
4.2. Analytical methods
A one-day transformation of propiophenone 2 (200 mg) in the
culture of R. rubra KCh 82 yielded 171 mg of compound (S)-4
The course of biotransformation was controlled by means of TLC.
Analytical TLC was carried out on silica gel G 60 F254 plates (Merck).
Chromatograms were developed using hexane/acetone mixture(3:1
v/v) as the eluent. Compounds were detected by spraying the plates
with 1% Ce(SO4)2 and 2% H3[P(Mo3O10)4] in 10% H2SO4. The products
were separated by column chromatography using silica gel (SiO2,
(colorless oil);
[
a
]
23 = À47.6 (c 2.95, CHCl3) (99% ee) (lit.
D
[a
]
D
25 = À50.8 (c 1.03 CHCl3), >99% ee).25 1H and 13C NMR spectra
were in agreement with those previously published.26
Acknowledgements
Kieselgel 60, 230–400 mesh, 40–63
l
m, Merck) and hexane/acetone
This research was supported financially by the European Union
within the European Regional Development Fund (Grant No.
POIG.01.03.01-00-158/09).
mixture (3:1, v/v) as the developing system. Compositions of
biotransformation mixtures were established by gas chromatogra-
phy (GC) on an Agilent Technologies 7890 A GC instrument, fitted
with a flame ionization detector (FID) and a chiral column
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