Microbiological bio-reduction of prochiral carbonyl compounds by antimycotic agent Boni Protect

Article abstract of DOI:10.1016/j.catcom.2017.07.021

The selective properties of the fungus Aureobasidium pullulans, in the antifungal agent Boni Protect, were investigated in the fermentative bioreduction of selected carbonyl compounds. Catalyzed by oxidoreductases contained in the microorganism Aureobasidium pullulans highly enantioselective biotransformation of prochiral ketones provides the secondary alcohols when the reaction is done in the presence of specific additives. Aureobasidium pullulans has also proved to be an effective bioreagent in the reduction of α- and β-keto esters. Optically pure hydroxy esters were obtained under fermentation conditions without the use of additives.

Full text of DOI:10.1016/j.catcom.2017.07.021

Accepted Manuscript
Microbiological bio-reduction of prochiral carbonyl compounds
by antimycotic agent Boni Protect
Renata Kołodziejska, Renata Studzińska, Marcin Kwit, Maciej
Jelecki, Agnieszka Tafelska-Kaczmarek
PII:
S1566-7367(17)30322-9
doi: 10.1016/j.catcom.2017.07.021
CATCOM 5140
DOI:
Reference:
To appear in:
Catalysis Communications
Received date:
Revised date:
Accepted date:
2 July 2017
24 July 2017
25 July 2017
Please cite this article as: Renata Kołodziejska, Renata Studzińska, Marcin Kwit, Maciej
Jelecki, Agnieszka Tafelska-Kaczmarek , Microbiological bio-reduction of prochiral
carbonyl compounds by antimycotic agent Boni Protect. The address for the corresponding
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ACCEPTED MANUSCRIPT
Microbiological bio-reduction of prochiral carbonyl compounds by antimycotic agent
Boni Protect
a,
b
c
c
Renata Kołodziejska *, Renata Studzińska , Marcin Kwit , Maciej Jelecki , Agnieszka Tafelska-
d
Kaczmarek
a
Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine,
Department of Biochemistry, Karłowicza 24, 85–092 Bydgoszcz, Poland
b
Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Pharmacy,
Department of Organic Chemistry, Jurasza 2, 85–089 Bydgoszcz, Poland
c
Adam Mickiewicz University, Department of Chemistry, Umultowska 89B, 61-614 Poznań, Poland
d
Nicolaus Copernicus University in Toruń, Department of Chemistry, Gagarina 7, 87-100 Toruń
Corresponding author:
Renatakol@poczta.fm, Karłowicza 24, 85–092 Bydgoszcz, Poland
Phone: + 48 52 585–37–55, fax: (52) 585–39–43
ABSTRACT
The selective properties of the fungus Aureobasidium pullulans, in the antifungal agent Boni Protect,
were investigated in the fermentative bioreduction of selected carbonyl compounds. Catalyzed by
oxidoreductases contained in the microorganism Aureobasidium pullulans highly enantioselective
biotransformation of prochiral ketones provides the secondary alcohols when the reaction is done in
the presence of specific additives. Aureobasidium pullulans has also proved to be an effective
bioreagent in the reduction of α- and β-keto esters. Optically pure hydroxy esters were obtained under
fermentation conditions without the use of additives.
Keywords: biocatalysis; microbiological reduction; stereochemistry; antifungal agent; Saccharomyces
cerevisiae; Aureobasidium pullulans

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1
. Introduction
Bio-reduction of the prochiral ketones is one of the methods for the synthesis of optically active
secondary alcohols, versatile as building blocks in the synthesis of important biologically active
compounds. Applying chemical methods for the asymmetric reduction requires the use of, e.g. chiral
boron compounds [1, 2], borates with transition metal complexes containing the chiral P- or N-donor
ligands [3-6], molecular hydrogen in the presence of Ru and Rh catalysts with chiral phosphine
ligands [7, 8], and others hydrogen sources (e.g. HCOOH) in the presence of chiral complexes [9-12].
Often, the use of aforementioned methods does not lead to the satisfactory results (low
enantioselectivity), is expensive, and contributes to environmental pollution. In this context, an
interesting alternative is the application of bioreagents - living organisms in the form of tissue cultures,
whole microbial cells, or isolated enzymes. The reduction methods utilizing the biocatalysts potential
are cheap and simple means to obtain alcohols of high enantiomeric purity. The use of whole cell
microorganisms suspended in the nutrient solutions is particularly economical because it does not
require the regeneration of cofactors, necessary in the reduction reaction with oxidoreductases. Hence,
a great interest in searching new, cheap, and easily accessible bioreagents, mainly among fungi that
can be used in whole cell forms in the asymmetric bio-reduction of prochiral carbonyl compounds [13-
16].
Aureobasidium pullulans, known as a black-yeast, is a commonly occurring oligotrophe, living in
temperate as well as tropical climate, and also in saline water. It is used in biotechnology, especially
for production of the polysaccharide called pullulan and the antimycotic aureobasidin A, and as a
biocontrol agent in agriculture. Its antagonistic activity against a number of phytopathogenic fungi is
used in fruit orchards to combat white mold [17-18].
In this work, the selective properties of the fungus Aureobasidium pullulans in the reduction of
selected unsymmetrical ketones, and α- and β-keto esters are presented. For this purpose, a widely
available antimycotic agent Boni Protect containing living cells of Aureobasidiumpullulans was used.

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2
2
. Experimental Section
.1. Asymmetric reduction by Aureobasidium pullulans
-
4
For a typical experiment, 0.14 g glucose (7.6x10 mol) was added to a suspension of Boni Protect (2
g) in 30 mL of potassium phosphate buffer (pH 7.0) and the resulting suspension was stirred at 37 C
-
5
-4
for 30 minutes. Then an additive compound (1.25x10 mol; see Table 2) and substrate (1.25x10 mol
in 0.5 mL EtOH) were added and stirring was continued at the same temperature. The reaction
progress was monitored by TLC (the solvent system used was hexane:ethyl acetate 3:1). After the
reaction, hyflo-super celit and ethyl acetate were added and the mixture was filtered. The celit was
washed with ethyl acetate and combined filtrates were extracted with ethyl acetate (5x20 mL). The
organic portion was dried with MgSO . The solvent was evaporated under reduced pressure. The crude
4
product was purified by PLC (Preparative Layer Plate) using hexane:ethyl acetate (3:1) as eluent. The
enantiomeric ratios were determined on an HPLC system using a chiral column.
2
.2. Asymmetric reduction by Saccharomyces cerevisiae
A suspension of baker’s yeast (3.75 g) in 20 mL potassium phosphate buffer (pH 7.0) and 0.9 g
-
3
(
2.6x10 ) sucrose was put into a 250 mL Erlenmeyer flask. The resulting mixture was stirred at 37 C.
-
4
After this stage of fermenting yeast (30 minutes), the solution of ketones/ketoesters (1.25x10 mol in
0.5 mL EtOH) was added. The reaction progress was monitored by TLC. After 24 h the yeast cells
were removed by filtration of celit filter. The yeast cells were washed with 2x25 mL water. The filtrate
was saturated with NaCl and then extracted with 5x20 mL portions of ethyl acetate. The combined
organic layers were dried over MgSO and the solvent was evaporated under reduced pressure to leave
4
a residue of the crude product, which was purified by PLC using hexane:ethyl acetate (3:1) as eluent.
The enantiomeric ratios were determined on an HPLC system using a chiral column.
3
. Results and discussion
As we previously reported, the enantioselective microbiological reduction of prochiral ethyl (9-
anthryl)glyoxylate in the presence of Aureobasidium pullulans (Boni Protect) afforded an appropriate

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hydroxy ester with 99% ee [19]. The method applied to obtain the derivative which is used in the
synthesis of chiral derivatizing agent, is simple, economical, and does not require an additional
cultivation of the microbial cell.
Herein we report the microbiological reduction of selected ketones with the use of A. pullulans (Figure
1).
O
OH
R2
Boni Protect
R1
R2
R1
potassium phosphate buffer; pH 7.0
1- 4
1'- 4'
R1
R2
O
O
1
2
3
4
-CH2Br
-CH3
-CH2Br
O N
2
-CH3
Br
Figure 1. Bio-reduction of prochiral ketones 1-4 by Aureobasidium pullulans.
Fermentative bio-reduction allowed for obtaining the corresponding secondary alcohols in high yields.
However, the reduction was either non-selective or with moderate selectivity. Oxidoreductases,
contained in the microorganism, act competitively to each other by transferring the hydride (pro-S or
pro-R) from the NAD(P)H cofactor to one of the sides of the prochiral carbonyl group (face si or re).
The final enantiomeric excess of product results from the reactions occurring at different rates, carried
out by a set of oxidoreductases. Furthermore, the selectivity of reduction depends on the substrate
structure, particularly on the presence of additional functional groups or heteroatoms in it [20, 21].
Ketone 3 was reduced with the highest enantioselectivity 65%, while the reduction of 1 at the presence
of A. pullulans occurred at a high rate but completely non-selectively (Table 1). The microbiological
reduction of compounds 1-4, catalyzed by Saccharomyces cerevisiae was also performed and
compared with the fermentation method in the presence of A. pullulans. Alcohols 1’ and 4’ were
obtained with higher enantiomeric excesses (45% and 72%, respectively) than compounds 2’ and 3’

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(36% and 62%, respectively). In each case, the product of the same configuration was preferentially
produced, similarly to the reaction catalyzed by A. pullulans.
In order to improve the selectivity of the reaction, optimization of the process towards the reduction of
enzyme incompatibility is necessary. The best known microbial reduction is the synthesis of alcohols
from the corresponding ketones with the use of baker’s yeast (Saccharomyces cerevisiae). The wide
range of baker’s yeast activity is conditioned by the presence of dehydrogenases of different
stereopreferences and high substrate tolerance. Therefore, both in the fermentation method and in
organic solvents, additional substances are used to improve the enantioselectivity, i.e. additives which
inhibit oxidoreductases with a specific stereopreference [22, 23].
In the reduction with A. pullulans, ethyl chloroacetate, allyl bromide, and allyl alcohol were used as
the additives which had already been applied earlier in the baker’s yeast reduction [22, 23]) and also
ionic liquid - [BMIM][PF ], keto esters - ethyl 3-oxovalerate, ethyl (9-anthryl)glyoxylate (Table 2).
6
The use of the additives improved the microorganism selectivity in the reduction of tested ketones.
Reduction of bromoketone 1 in the presence of ethyl (9-anthryl)glyoxylate gave the best results. The
appropriate bromohydrin was obtained in quantitative yield and with 55% ee, whereas an optically
pure 2’ was afforded in the presence of the following inhibitors: allyl alcohol, ethyl 3-oxovalerate,
ethyl (9-anthryl)glyoxylate. The reactions were carried out within 72 h and alcohol 2’ was obtained in
3
9.5-46.9% yields. Ketone 3 was also selectively reduced (99% ee) and obtained in the highest yield
79.9%) when [BMIM][PF ] was applied. As it can be seen from the Table 2, each of the additives
allows for the preparation of 3’ with a high enantiomeric excess (74-99%). 4’-Bromoacetophenone (4)
was reduced with very high enantioselectivity (99% ee) using [BMIM][PF ], ethyl chloroacetate, and
(
6
6
ethyl 3-oxovalerate, but in comparison to the reduction of 3, these reactions occurred in yields less
than 10%. The only reduction of 4 in the presence of ethyl (9-anthryl)glyoxylate provided alcohol 4’
with high enantioselectivity and in high yield (93% and 74%, respectively).
The absolute configuration of the obtained alcohols depends on the structure of starting prochiral
ketones. In the case of ketones with a bulky substituent (benzofuran ring), the hydride attacks the
carbonyl group preferentially from the re face. Hence, from ketones 1 and 2, the alcohols of the

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configuration R and S, respectively, were obtained. While the reduction of 3 and 4 with less sterical
hindrance occurred from the si face and giving predominantly (S)-3’ and (R)-4’.
In general, the use of the antifungal agent Boni Protect for the bio-reduction of ketones 2-4, in the
presence of inhibitors, allows receiving the appropriate alcohols with high enantiomeric excesses. The
results are comparable to those obtained in chemical reduction methods. For example, alcohols 2’ and
4
’ were afforded by the asymmetric transfer hydrogenation with 96.9% ee and 93% ee, respectively [3,
10, 24]. The compound 3’ was prepared with 73-92% ee in the borane-mediated asymmetric reduction
[
(
4, 5, 6]. However, the chemical reduction of ketone 1 gave a product with higher enantiomeric excess
91%) [12] than in bioreduction.
In the next step, the selected α- and β-keto esters were subjected to the reduction with Boni Protect
fungicide (Figure 2).
O
OH
R2
Boni Protect
R1
R2
R1
potassium phosphate buffer; pH 7.0
5-10
5'-10'
R1
R2
R1
R2
5
-CO2CH3
8
-CH2CO2C2H5
Cl
6
-CH2CO2C2H5
-CO2C2H5
9
-CO2C2H5
7
10
-CH2CO2C2H5
Cl
Figure 2. Bio-reduction of prochiral ketoesters 5-10 by Aureobasidium pullulans.
The results obtained from the reduction of keto esters by A. pullulans and from the fermentation
method (reduction in the presence of S. cerevisiae) are summarized in Table 3.
The reduction of methyl 2-oxo-2-phenylacetate (5) in the presence of baker’s yeast leads to the
appropriate hydroxy ester of R configuration (according to the literature [25]). In the applied
fermentative method, without the additive (methyl vinyl ketone), the product 5’ was obtained with
63% ee. While in the bio-reduction reaction with Boni Protect, the same compound was formed in the

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opposite configuration with 99% ee. Similarly, ethyl 3-oxo-3-phenylpropanoate (6) was selectively
reduced in the presence of A. pullulans to β-hydroxy ester 6’ of S configuration. In contrast, the bio-
reduction by baker’s yeast allowed for the formation of (R)-product with 84% ee. The presence of
chlorine in the para position of phenyl ring did not affect the selectivity of the fungus A. pullulans.
From both, - and β-keto esters (7 and 8) the appropriate optically pure hydroxy esters of R and S
configuration, respectively, were obtained. Using baker’s yeast under fermentation conditions, without
the use of dehydrogenase inhibitors of a specific stereopreference, the corresponding hydroxy esters
were formed with lower enantioselectivity. However, for the reduction of compound 9, baker’s yeast
occurred to be the best biocatalyst. The reaction in the presence of S. cerevisiae gave 9’ with 98% ee
whereas in the presence of A. pullulans this product was afforded with lower enantiomeric excess
(77%). The β-keto ester 10 was reduced in the reaction catalyzed by A. pullulans, alike the
aforementioned -keto ester 9, to the product of S configuration. The hydroxy ester 10’ was formed in
high chemical yield, as well as optical yield. In contrast, in the presence of baker’s yeast, 10’ was
produced in trace amounts with 69% ee.
Antimycotic agent Boni Protect shows high selectivity in the reduction of α- and β-keto esters. In the
majority of tested compounds, the hydride ion selectively attacks the prochiral carbon atom on the re
face giving predominantly the hydroxy esters of S configuration. Compared to the reduction by S.
cerevisiae, the fermentative bio-reduction in the presence of A. pullulans leads to the optically pure
hydroxy esters in high yields and does not require the use of additives to improve the reaction
selectivity.

-Keto esters 5, 7, and 9 were reduced by chemical methods with comparable high enatioselectivity,
respectively compound 5 with 90.9-96.3% ee [8, 26, 27], compound 7 with 69.9-96.1% ee [8, 27], and
compound 9 with 83.7-98.35% ee [27]. Similarly, β-keto esters 6 and 8 were reduced by the
asymmetric hydrogenation to the optically pure hydroxy esters (compound 6 with 86-99% ee [7, 11,
28-30]; compound 8 with 95-99.9% ee [11, 28-30]). As it can be seen from the above comparison, the
biochemical methods can compete with typical chemical methods. These reactions are highly selective
and more ecological, inexpensive and simple.

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4
Conclusion
Aureobasidium pullulans as an antifungal formulation Boni Protect was successfully used for the
enantioselective bio-reduction of selected ketones, and α- and β-keto esters. Chiral alcohols and
hydroxy esters were obtained in high optical purity. The reduction method is simple, economical, and
does not require the cultivation of the bioreagent.
Acknowledgments
This work has been supported by Nicolaus Copernicus University, Collegium Medicum in Bydgoszcz.
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Table 1 Reduction of 1-4 by Aureobasidium pullulans and Saccharomyces cerevisiae.
[a]
[a]
[a]
[a]
[a]
[a]
[a]
[a]
Microorganism
1’ [%]
ee [%]
2’ [%]
ee [%]
3’ [%]
ee [%]
4’ [%]
ee [%]
[
b]
[b]
[b]
[c]
Aureobasidium pullulans
Saccharomyces cerevisiae
100
rac
92.9
76.8
61 (S)
36 (S)
75.5
65.0
65 (S)
62 (S)
68.3
47.9
51 (R)
72 (R)
[
c]
[c]
[b]
[c]
96.5
45 (R)
[
a] The ee and yield were determined by HPLC. [b] 24h. [c] 72h.

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Table 2 Reduction of 1-4 by Aureobasidium pullulans with additives.
[a]
[a]
[a]
[a]
[a]
[a]
[a]
[a]
Additives
1’ [%]
ee [%]
2’ [%]
ee [%]
3’ [%]
ee [%]
4’ [%]
ee [%]
[
b]
[b]
[b]
[d]
[
BMIM][PF
6
]
100
98.6
78.7
99.6
rac
33
43
21
55
12
8.9
77
69
-
79.9
65.3
74.7
84.0
75.3
66.8
99
99
99
74
96
99
9.0
99
99
76
52
93
99
[
b]
[b]
[b]
[d]
Ethyl chloroacetate
Allyl bromide
26.4
-
1.5
[
c]
[b]
[b]
[b]
[b]
[d]
55.8
33.7
74.0
[
c]
[d]
[d]
Allyl alcohol
46.9
40.8
39.5
99
99
99
[c]
[d]
[d]
[d]
Ethyl (9-anthryl)gloxylate
Ethyl 3-oxovalerate
100
100
[c]
[d]
7.8
[
a] The ee and yield were determined by HPLC. [b] 24h. [c] 2h. [d] 72h.

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Table 3 Reduction of 5-10 by Aureobasidium pullulans and Saccharomyces cerevisiae.
Microorganism
5’
6’
7’
8’
9’
10’
5.0
[a]
Saccharomyces cerevisiae
Yield [%]
100
100
20.0
25.0
100
[
a]
ee [%]
63 (R)
100
84 (R)
100
60 (R)
100
29 (S)
80.0
98 (S)
100
69 (S)
100
[
a]
Aureobasidium pullulans
Yield [%]
[a]
ee [%]
99 (S)
99 (S)
99 (R)
98 (S)
77 (S)
99 (S)
[
a] The ee and yield were determined by HPLC.

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Graphical abstract

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Highlights



Antimycotic agent Boni Protect was used for the enantioselective bio-reduction.
The microbiological reduction of ketones, and keto esters were investigated.
Chiral alcohols and hydroxy esters were obtained in high optical purity.