Microbial synthesis of (S)- And (R)-benzoin in enantioselective desymmetrization and deracemization catalyzed by aureobasidium pullulans included in the blossom protect agent

Article abstract of DOI:10.3390/molecules26061578

In this study, we examined the Aureobasidium pullulans strains DSM 14940 and DSM 14941 included in the Blossom Protect agent to be used in the bioreduction reaction of a symmetrical dicarbonyl compound. Both chiral 2-hydroxy-1,2-diphenylethanone antipodes were obtained with a high enantiomeric purity. Mild conditions (phosphate buffer [pH 7.0, 7.2], 30 ^?C) were successfully employed in the synthesis of (S)-benzoin using two different methodologies: benzyl desymmetrization and rac-benzoin deracemization. Bioreduction carried out with higher reagent concentrations, lower pH values and prolonged reaction time, and in the presence of additives, enabled enrichment of the reaction mixture with (R)-benzoin. The described procedure is a potentially useful tool in the synthesis of chiral building blocks with a defined configuration in a simple and economical process with a lower environmental impact, enabling one-pot biotransformation.

Full text of DOI:10.3390/molecules26061578

molecules
Article
Microbial Synthesis of (S)- and (R)-Benzoin in Enantioselective
Desymmetrization and Deracemization Catalyzed by
Aureobasidium pullulans Included in the Blossom
Protect™ Agent
1
,
2
, Agnieszka Tafelska-Kaczmarek 3 , Hanna Pawluk 1,
Renata Kołodziejska * , Renata Studzi n´ ska
Dominika Mlicka 1 and Alina Wo z´ niak 1
1
Department of Medical Biology and Biochemistry, Faculty of Medicine, Nicolaus Copernicus University
in Toru n´ , Collegium Medicum in Bydgoszcz, 24 Karłowicz Street, 85-092 Bydgoszcz, Poland;
hannapawluk1@wp.pl (H.P.); mikaa0901@gmail.com (D.M.); alina-wozniak@wp.pl (A.W.)
Department of Organic Chemistry, Faculty of Pharmacy, Nicolaus Copernicus University in Toru n´ ,
Collegium Medicum in Bydgoszcz, 2 Jurasz Street, 85-089 Bydgoszcz, Poland; rstud@cm.umk.pl
Department of Organic Chemistry, Faculty of Chemistry, Nicolaus Copernicus University in Toru n´ ,
2
3
7
Gagarin Street, 87-100 Toru n´ , Poland; tafel@umk.pl
*
Correspondence: Renatakol@poczta.fm
Abstract: In this study, we examined the Aureobasidium pullulans strains DSM 14940 and DSM 14941
included in the Blossom Protect agent to be used in the bioreduction reaction of a symmetrical
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
™
ꢀ
ꢁꢂꢃꢄꢅꢆ
dicarbonyl compound. Both chiral 2-hydroxy-1,2-diphenylethanone antipodes were obtained with a
high enantiomeric purity. Mild conditions (phosphate buffer [pH 7.0, 7.2], 30 C) were successfully
Citation: Kołodziejska, R.;
◦
Studzi n´ ska, R.; Tafelska-Kaczmarek,
A.; Pawluk, H.; Mlicka, D.; Wo z´ niak,
A. Microbial Synthesis of (S)- and
employed in the synthesis of (S)-benzoin using two different methodologies: benzyl desymmetriza-
tion and rac-benzoin deracemization. Bioreduction carried out with higher reagent concentrations,
lower pH values and prolonged reaction time, and in the presence of additives, enabled enrichment
of the reaction mixture with (R)-benzoin. The described procedure is a potentially useful tool in the
synthesis of chiral building blocks with a defined configuration in a simple and economical process
with a lower environmental impact, enabling one-pot biotransformation.
(R)-Benzoin in Enantioselective
Desymmetrization and Deracemization
Catalyzed by Aureobasidium pullulans
Included in the Blossom Protect™
Agent. Molecules 2021, 26, 1578.
https://doi.org/10.3390/
Keywords: desymmetrization; bioreduction; microbial synthesis; asymmetric; microorganism;
α-hydroxy ketones
molecules26061578
Academic Editor: Maciej Szaleniec
Received: 9 February 2021
Accepted: 10 March 2021
Published: 12 March 2021
1
. Introduction
Derivatives of
synthesis. They are extremely valuable components of more architecturally complex com-
pounds, offering a wide spectrum of applications. Optically active -hydroxy ketones
also called acyloins) are considered useful building blocks in the preparation of various
pharmaceuticals and chemicals thanks to their functional properties. Synthetic -hydroxy
ketones with the R configuration are used as precursors in the synthesis of some antide-
pressants (bupropion), selective inhibitors of amyloid- protein production used in the
treatment of Alzheimer’s disease, and as inducers of apoptosis in oral tumors and anti-
fungal agents [ ]. Acyloins are also found as structural subunits of natural compounds
used in pharmacotherapy, for example in kurasoins A and B, protein farnesyltransferase
inhibitors produced by Penicillium sp. FO-3929 [ ], and in natural antitumor antibiotics,
such as chromomycin A3, epothilones, mithramycin and olivomycin A [5,6].
,2-Diaryl-2-hydroxyethanone structures (benzoins) are useful as urease inhibitors [
α
-hydroxy ketones play an important role in asymmetric organic
α
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
(
α
β
1–3
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4
1
2],
as building blocks for the synthesis of heterocycles and multifunctional compounds such
as diols, diamines, amino alcohols and epoxides [7–9].
There are many strategies, both chemical and biochemical, leading to the synthesis
4
.0/).
of enantiomerically enriched
α
-hydroxy ketones. Hydrobenzoins may be prepared by
Molecules 2021, 26, 1578. https://doi.org/10.3390/molecules26061578
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 1578
2 of 15
chemical means using oxidation of prochiral enolates in a reaction catalyzed by chiral oxi-
dants, dihydroxylation of silyl enol ethers, RuO -catalyzed reaction of ketohydroxylation
4
of olefins, mono-oxidation of diols using a chiral catalyst; e.g., copper, or oxidation of
racemic benzoins by a chiral iron or cobalt complex [10
can be obtained by direct -oxygenation of ketones in the presence of chiral amino acids
L-proline, L-alanine) and by benzoin condensation catalyzed by chiral thiazolium or tri-
azolium salts (chiral metallophosphites) [18 22]. Other strategies leading to formation
–17]. In addition, α-hydroxyketones
α
(
–
of optically active benzoins involve biocatalysis, which uses natural tools–catalytic pro-
teins. For instance, thiamine diphosphate-dependent lyases (ThDP-lyases) catalyze the
umpolung carboligation of aldehydes to yield chiral α-hydroxy ketones. In addition to
ThDP-lyases employed as bioreagents, catalysts, such as pyruvate decarboxylase (PDC),
benzoylformate decarboxylase (BFD), and benzaldehyde lyase (BAL) [23] are included.
Hydrolase-catalyzed kinetic resolutions or chemo-enzymatic dynamic kinetic resolutions
of racemate have proven to be an efficient method of obtaining enantiopure
ketones. Several kinetic resolutions of racemic hydrobenzoins have been described using
different lipases and esterases [24]. Enantiomerically enriched -hydroxy ketones can
α-hydroxy
α
also be successfully produced using the catalytic properties of oxidoreductases in the
following reactions: reduction of diketones, oxidation of vicinal diols and deracemization
(
stereoinversion) of hydroxy ketones [25,26].
Biotransformation based on the reduction reaction of symmetrical diaryl diketones is
carried out mainly in the presence of whole cells of microorganisms or isolated oxidore-
ductases. Biocatalytic reduction of benzils to chiral benzoin has been achieved by using
different microorganisms, leading to the desired
tion continues and yields the corresponding -diol. The first attempts at microbiological
reduction of benzil to benzoin were carried out with low or moderate selectivity [27 29].
α-hydroxy ketone. In some cases, the reac-
α
–
Saccharomyces cerevisiae has been used for the production of benzoin with the R configura-
tion with an enantiomeric excess (ee) of up to 50%, while reduction of other derivatives,
4
-Me-Ph and furyl, was achieved with 36% and 82% ee, respectively [28]. Benzil and a
series of para-substituted benzils have been reduced using Cryptococcus macerans to obtain
S)-benzoins with enantiomeric excesses of 20–30% [29]. Selection of strains of microorgan-
(
isms has had a significant impact on the enantioselectivity of symmetrical diaryl diketone
bioreduction. T. Saito et al. tested several different Bacillus cereus (bacterial) strains towards
the selective reduction of a diaryl diketone. Of the strains examined, only wild-type Bacillus
cereus Tim-r01 selectively reduced benzil to (S)-benzoin with a high 94% ee (92% yield) [30].
A. S. Demir et al. evaluated the ability of four different species of fungi (Rhizopus oryzae
(
(
ATCC 9363), Rhizopus oryzae (72465), Rhizomucor miehei (72460) and Rhizomucor pusillus
72561)) to convert benzil into benzoin and hydrobenzoin. Rhizopus oryzae (ATCC 9363)
allows obtaining (R)-benzoin with >99% ee, while Rhizomucor pusillus (72561) yielded
S)-benzoin with 73% ee [31]. J. Konishi et al. obtained optically pure (R)-benzoin using
(
Xanthomonas oryzae IAM 1657, which was selected via screening tests from multiple types
of cultures [32]. Another group of fungi tested for selective properties in the biotransforma-
tion of benzils were Aspergillus oryzae and Fusarium roseum strains. Of the strains examined,
Aspergillus oryzae OUT5048 and Fusarium roseum OUT4019 were found to be effective biocat-
alysts. Benzil and ortho-, meta-, and para-substituted derivatives of benzil were reduced to
their corresponding benzoins by A. oryzae OUT5048 with up to 94% ee (benzil 94% ee) and
by F. roseum OUT4019 with up to 98% ee (benzil 95% ee) [
7]. Monoreduction of different
1
,2-diaryl-1,2-diketones was carried out in a reaction catalyzed by lyophilized whole cells
of Pichia glucozyma CBS 5766, selected among the following yeasts tested: Pichia minuta
CBS 1708, Pichia fermentans DPVPG 2770, Pichia glucozyma CBS 5766, Pichia etchellsii CBS
2
011, Candida utilis CBS 621 and Kluyveromyces marxianus CBS 397. The optical yield of
benzoins from symmetrical benzils was 54–99% ee, and 1,2-diphenylethanone was reduced
with a 75% enantiomeric excess and 99% conversion [ ]. Using the same microorganism
9
P. glucozyma CBS 5766, Maria Caterina (M.C). Fragnelli et al. successfully conducted benzil
biotransformation in water/organic solvent two-liquid-phase systems. An increase in

Molecules 2021, 26, 1578
3 of 15
enantioselectivity was observed: use of n-heptane:water at 1:1 v/v yielded (S)-benzoin with
a 90% conversion and 99% ee. The presence of the organic solvent influences the redox
systems implicated in the reactions, preventing the formation of the corresponding diols.
Under optimized conditions, other diaryl benzoins were obtained with 85–99% ee [33].
Oda et al. conducted asymmetric reduction of benzil to (S)-benzoin by Penicillium claviforme
IAM 7294 in a two-phase system in a liquid–liquid interface bioreactor (L-L IBR) using a
unique polymeric material, ballooned microsphere (MS). The L-L IBR showed superior
performance, yielding 14.4 g/L-organic phase of (S)-benzoin (99.0% ee) [34].
Reduction of diketones with isolated enzymes is definitely less popular due to the need
for expensive cofactors. R. Maruyama et al. used isolated recombinant benzil reductase
from live B. cereus cells overexpressed in Escherichia coli. B. cereus benzil reductase enables
selective reduction of the distal ketone group to a benzene ring, yielding optically pure
(S)-benzoin [35].
Herein we report selective desymmetrization of symmetrical diaryl diketones using
the antifungal agent Blossom Protect
™
. Previously, we described an effective biotransfor-
2
mation of prochiral sp hybridized compounds using the Boni Protect preparation as a
catalyst. Chiral secondary alcohols and α- and β-hydroxy esters were obtained with high
optical purity [36–39].
2. Results and Discussion
Several approaches to asymmetric biotransformation aiming to obtain high optical
purity products are available. One can select the optimal bioreagent among several tested
to perform the desired biotransformation with a high ee or to modulate the conditions of a
reaction catalyzed by a microorganism yielding a moderate ee so as to improve the optical
purity of the product. Optimization of the bioprocess parameters is one of the strategies
affecting the enantioselectivity of biotransformation alongside site-directed-mutagenesis.
Conversions and stereoselectivity can be improved by using enzyme inhibitors (high con-
centration of substrate, additives), thermal deactivation (temperature effect on selectivity)
and organic cosolvents.
Aureobasidium pullulans strains DSM 14940 and DSM 14941 included in the Blossom
Protect™
agent were tested in the bioreduction reaction of a symmetrical diketone—benzil
(Scheme 1). The purpose of biotransformation catalyzed by Blossom Protect
™
was to
obtain both chiral 2-hydroxy-1,2-diphenylethanone (benzoin) antipodes in an effective
bioconversion reaction characterized by a high catalytic activity of A. pullulans and a high
enantioselectivity.
Scheme 1. Biotransformation of a symmetrical diketone catalyzed by Blossom Protect™ containing
live A. pullulans cells.
2
.1. Enantioselective Bioreduction Reaction of Benzil to the Corresponding (S)-Benzoin
We first carried out the reduction of benzil ( ) catalyzed by an antifungal agent
containing live A. pullulans cells in a phosphate buffer solution at different pH values
1

Molecules 2021, 26, 1578
4 of 15
(pH 5.0 to 8.0). We used glucose as the source of carbon. The reaction was started by adding
compound
1
, previously dissolved in ethanol at various concentrations as described in
◦
the Experimental section. The mixture was incubated at 30 C for 24 h. The results are
summarized in Table 1.
Table 1. Microbial Bioreduction of Benzil (
1
) to (S)-2-Hydroxy-1,2-Diphenylethanone (2) in Phosphate Buffer at Different
pH Values and Various Concentrations of 1.
Amount of Substrate × 10− (mol)
5
Conv. (%)/
pH
ee (%)
× 10−1
5
1
2
3
4
5
Conv. (%)/
ee (%)
Conv. (%)/
ee (%)
Conv. (%)/
ee (%)
Conv. (%)/
ee (%)
Conv. (%)/
ee (%)
Conv. (%)/
ee (%)
Conv. (%)/
ee (%)
Conv. (%)/
ee (%)
74.4
74 (S)
81.3
83 (S)
83.7
80 (S)
80.8
76 (S)
86.7
80 (S)
84.7
75 (S)
81.0
73 (S)
77.0
67 (S)
79.8
86 (S)
89.3
91 (S)
86.4
90 (S)
83.2
89 (S)
88.1
90 (S)
87.6
92 (S)
75.3
91 (S)
89.1
77 (S)
75.5
58 (S)
87.4
83 (S)
81.1
87 (S)
82.2
82 (S)
86.4
87 (S)
86.3
87 (S)
87.9
92 (S)
90.2
70 (S)
72.7
21 (S)
74.8
32 (S)
80.9
81 (S)
82.4
74 (S)
84.8
84 (S)
93.5
88 (S)
91.6
86 (S)
95.5
67 (S)
52.1
11 (R)
66.7
8 (R)
66.1
6 (S)
80.6
67 (S)
89.8
82 (S)
90.2
89 (S)
89.3
84 (S)
89.9
62 (S)
76.9
21 (R)
63.9
28 (R)
79.1
8 (R)
93.3
66 (S)
94.4
77 (S)
91.8
87 (S)
93.8
82 (S)
89.4
60 (S)
5.0
5.5
6.0
6.5
7.0
7.2
7.5
8.0
The effectiveness of bioconversion was strongly affected by both the concentration
of substrate and pH of the reaction medium. The highest (up to 92%) ee of (S)-benzoin
−
5
was obtained for 1
×
10 mol of 1 in a solution at pH 5.5, 6.0, 7.0, 7.2 and 7.5. At a
lower pH (5.0–6.0), a gradual decrease in the optical efficiency of the (S)-enantiomer was
observed with increasing substrate concentration. The phosphate buffer system at pH
5
.0, 5.5 and 6.0 showed a slight advantage of the opposite enantiomer with 8–28% ee for
(R)-benzoin. These results suggest that the activities of different dehydrogenases contained
in strains DSM 14940 and DSM 14941 of A. pullulans strongly depend on two parameters:
pH of the solution and the concentration of the starting reagent. Putatively, in a neutral or
slightly alkaline solution (pH 7.2 and 7.5), (S)-stereopreference dehydrogenases selectively
reduce the symmetrical diaryl diketone, yielding a significant proportion of the (S)-isomer
irrespective of the substrate concentration. In most cases, the degree of benzil conversion
after 24 h of reaction was high (up to 95.5%).
The addition of other sources of carbon, fructose and sucrose, at the pre-incubation
◦
stage was also examined. The reaction was carried out at 30 C. Optimal results were
obtained with glucose. It seems that the presence of fructose as a carbon source increases
the follow-up reaction, reducing benzoin to the corresponding diol. As a consequence, in
most cases, it reduces the enantiomeric purity of hydroxy ketone and its contribution to
the post-reaction mixture (Table 2).
Table 2. The Efficiency and Enantioselectivity of the Bioconversion of
1
by A. pullulans Strain DSM 14940 and DSM 14941
with Fructose and Sucrose as Energy Source.
Conditions
pH
◦
−5
3
0 C; 1 × 10
(S)-2
5
.0
5.5
6.0
6.5
7.0
7.2
7.5
8.0
mola of 1
glucose
fructose
sucrose
Conv. (%)/ee (%)
Conv. (%)/ee (%)
Conv. (%)/ee (%)
79.8/86
82.7/89
78.2/87
89.3/91
76.9/73
82.5/88
86.4/90
76.5/81
80.7/88
83.2/89
78.7/76
89.2/86
88.1/90
85.8/72
88.8/82
87.6/92
63.4/72
69.5/75
75.3/91
86.1/69
89.5/82
89.1/77
86.5/60
87.6/71

Molecules 2021, 26, 1578
5 of 15
Temperature often has a significant impact on bioprocess selectivity, especially in the
presence of isolated enzymes, for example hydrolases, or in simple oxidation–reduction
models [40
–
42]. Decreasing the temperature usually results in an improvement in enan-
44].
tioselectivity, but may have an adverse effect on the degree of substrate conversion [43
,
M.C. Fragnelli et al. observed a completely different relationship: reduction of benzil to the
corresponding hydroxy ketone, along with a decrease in temperature, was characterized
by a slight decrease in the enantiomeric purity of the product with a simultaneous increase
in process efficiency. The reduced temperature provides cells with decreased reducing
activity towards diol [33].
The bioreduction of
1 was carried out at two additional temperatures (Table 3). Based
on previous experiments, glucose was selected as an energy source. It seems that the
◦
◦
temperature of 30 C is optimal, as along with a decrease in temperature to 28 C, a slight
decrease in chemical and optical efficiency and a greater percentage of diol in the post-
reaction mixture were observed. A similar observation was made at a higher temperature,
and a particular decrease in enantioselectivity was noticed at lower pH (5.5, 6.0).
◦
◦
◦
Table 3. The Efficiency and Enantioselectivity of the Bioconversion of
4940 and DSM 14941.
1
at 28 C, 30 C and 32 C by A. pullulans Strain DSM
1
pH
Conditions Glucose;
(
S)-2
−5
1
× 10 mola of 1
5.0
5.5
6.0
6.5
7.0
7.2
7.5
8.0
◦
2
3
3
8 C
Conv. (%)/ee (%)
Conv. (%)/ee (%)
Conv. (%)/ee (%)
80.0/86 81.1/87 77.9/87 80.8/85 83.0/85 84.0/87 80.7/87 81.5/81
79.8/86 89.3/91 86.4/90 78.7/89 88.1/90 87.6/92 87.9/92 89.1/77
78.5/67 70.0/49 63.0/41 78.5/76 78.2/72 73.6/86 86.0/73 85.3/62
◦
0 C
◦
2 C
Attempts have been made to improve the enantioselectivity of biotransformation with
the so-called additives. Compounds used as additives can act as hydrogen donors for
cofactor regeneration (alcohols), act as inhibitors of enzymes with a particular stereoprefer-
ence or increase the availability of the substrate for the enzyme (surfactants), and perform
chemical modification of the enzyme, increasing its activity (sulfur compounds) [7,45–47].
The following potential inhibitors were used in the benzil biotransformation reaction: allyl
alcohol, ethyl chloroacetate, cysteine, (9-antryl)glyoxylate (AMA-1), 3-methylbutan-2-one,
4-methylpentan-2-one. The results are shown in Table 4.
Table 4. Microbial Bioreduction of 1 in Phosphate Buffer at Different pH Values with Additives.
Additives
Conv. (%)/
ee (%)
pH
Ethyl
Chloroacetate
3-Methylbutan- 4-Methylpentan-
Allyl Alcohol
Cysteine
AMA-1
2-one
2-one
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
24.0
44 (R)
10.5
48 (R)
23.9
44 (R)
35.9
46 (R)
45.7
24 (R)
42.4
46 (R)
56.5
26 (R)
56.8
4 (R)
35.5
42 (R)
9.2
56 (R)
20.8
48 (R)
25.2
55 (R)
16.5
54 (R)
19.6
51 (R)
15.0
52 (R)
41.2
48 (R)
58.3
43 (R)
41.9
51 (R)
29.3
41 (R)
52.3
18 (R)
85.7
88 (S)
86.0
73 (S)
73.4
83 (S)
84.9
82 (S)
22.3
41 (R)
27.3
51 (R)
42.0
46 (R)
59.1
9 (R)
79.6
75 (S)
70.2
41 (S)
79.3
89 (S)
73.3
10 (R)
62.2
40 (R)
33.6
50 (R)
50.2
48 (R)
50.4
43 (R)
48.2
46 (R)
62.7
17 (R)
48.3
45 (R)
53.8
22 (R)
54.0
34 (R)
43.9
51 (R)
53.8
16 (R)
30.5
38 (R)
44.9
33 (R)
49.6
40 (R)
64.7
31 (R)
67.5
1 (R)
5.0
5.5
6.0
6.5
7.0
7.2
7.5
8.0

Molecules 2021, 26, 1578
6 of 15
Surprisingly, all additives used—to a greater or lesser extent—inhibited the effect of
dehydrogenases with (S)-stereopreference. The magnitude of these changes depended on
the pH of the reaction medium. In the solution at pH 5.0–6.5 in the presence of additives,
we observed enrichment of the mixture with the (R)-isomer. The highest excess of this
isomer, regardless of the pH of the solution, was observed in the biotransformation reaction
with the addition of ethyl chloroacetate. In a system with a phosphate buffer at pH 5.5,
the optical purity (ee) of the product was 56%. Interestingly, in this solution, the biore-
duction reaction proceeded with approximately 50% ee in the presence of each additive,
indicating the phosphate buffer solution at pH 5.5 as optimal for the conversion of benzil
to (R)-benzoin.
Another parameter examined in the benzil bioreduction reaction was the effect of
organic solvents on the efficiency and selectivity of the bioprocess. Biocatalysis in a two-
phase system is often more effective than in water or polar organic solvents, especially
for hydrolases. Enzyme selectivity is conditioned by conformational rigidity, which in-
creases in a more hydrophobic medium. A hydrophobic solvent decreases biocatalyst
lability, which prevents binding of a structurally mismatched substrate to the active site
of an enzyme [48–50]. Water/organic solvent two-liquid-phase systems were successfully
used by M.C. Fragnelli et al. and S. Oda et al. in the synthesis of enantiomerically pure
(
S)-benzoin [33,34]. The enantioselectivity of the reaction was increased in hydrophobic
solvents such as hexane, heptane, and isooctane. The presence of a cosolvent promoted
the deracemization reaction without the simultaneous occurrence of reduction activities
towards benzoin [33]. We bioreduced benzil with added cosolvents using hexane, cyclohex-
ane, tert-butyl methyl ether (TBME), tetrahydrofuran (THF), acetonitrile (AcCN), and ionic
liquids (Bmim)(BF ) and (Bmim)(PF ) (Table 5). No increase in the optical efficiency of the
4
6
(S)-isomer was observed; however, after 24 h in a solution of phosphate buffer mixed with
organic solvents at a 10:1 ratio (v/v), the presence of TBME, AcCN, and THF promoted
the growth of (R)-benzoin in the mixture. The highest excess of the (R)-enantiomer was
observed in the buffer: THF and buffer: AcCN mixtures at pH 5.5 and 6.0, respectively.
Table 5. Bioreduction of 1 Catalyzed by A. pullulans Strain DSM 14940 and DSM 14941 in a Two Liquid-Phase Systems.
Organic Solvent: Phosphate Buffer 1:10 v/v
Conv. (%)/
ee (%)
pH
Hexane
Cyclohexane
TBME
(Bmim)(PF6)
(Bmim)(BF4)
THF
AcCN
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
59.9
81 (S)
63.7
83 (S)
69.3
86 (S)
64.7
84 (S)
70.6
81 (S)
75.5
86 (S)
66.1
81 (S)
71.3
44 (S)
67.8
74 (S)
64.8
67 (S)
59.4
73 (S)
69.4
82 (S)
83.4
75 (S)
92.5
77 (S)
91.2
69 (S)
89.0
36 (S)
12.9
43 (R)
8.3
45 (R)
9.6
46 (R)
19.9
42 (R)
25.4
40 (R)
31.9
41 (R)
33.1
33 (R)
39.7
35 (R)
34.3
6 (R)
70.4
9 (R)
22.4
3 (R)
37.9
29 (S)
36.1
40 (S)
46.4
41 (S)
51.2
42 (S)
47.6
21 (S)
74.9
13 (R)
47.9
45 (R)
76.8
26 (S)
73.7
80 (S)
77.6
81 (S)
78.6
41 (S)
76.7
55 (S)
41.9
26 (R)
11.4
52 (R)
11.0
56 (R)
17.3
42 (R)
11.7
40 (R)
16.4
40 (R)
11.4
25 (R)
17.2
35 (R)
14.3
32 (R)
14.9
48 (R)
12.2
48 (R)
23.4
55 (R)
27.7
52 (R)
29.0
52 (R)
18.5
35 (R)
17.1
26 (R)
11.5
7 (R)
5.0
5.5
6.0
6.5
7.0
7.2
7.5
8.0
For the evaluation of the enantioselective properties of the microorganism in benzil
reduction, a time profile was established by determining the composition of the reaction
mixture over time. The reaction was stopped after 2 and 4 h. Optimal conditions were
◦
−5
selected, incubation at 30 C with glucose as energy source and 1
×
10 mol of substrate.
The results are shown in Table 6. Both in the second and fourth hour, the reaction was
practically non-selective. After two hours, the highest ee of (S)-benzoin was obtained

Molecules 2021, 26, 1578
7 of 15
in solutions at pH 7.0 and 7.5: 61% and 66%, respectively. However, after 4 h, in each
case a slight increase in the (R)-isomer content in the mixture was observed, similarly
as after 2 h in a solution with pH 5.0–6.0. This result is in apparent contradiction to the
−
5
previous hypothesis that for 1
selectively reduces the symmetrical diaryl diketone. It seems that only stereoinversion
deracemization) of the stereogenic benzoin atom can explain the situation. Stereoinversion
×
10 mol of 1, dehydrogenase with (S)-stereopreference
(
of secondary alcohols can occur by concurrent tandem biocatalytic oxidation and reduction.
If one of the enantiomers is selectively oxidized to carbonyl and then there is a selective
reduction to the opposite enantiomer, enantiomeric enrichment of the mixture with the
specified enantiomer can be observed. For this reason, a significant contribution of the
(S)-isomer is observed after 24 h.
Table 6. Time Profile of Bioreduction of
1 Catalyzed by A. pullulans Strain DSM 14940 and DSM 14941
in Phosphate Buffer at Different pH Values.
Conv. (%)
1
Conv. (%)
2
Conditions
pH
Time (h)
ee (%)
(S)
(R)
5.0
5.5
6.0
6.5
7.0
7.2
7.5
8.0
5.0
5.5
6.0
6.5
7.0
7.2
7.5
8.0
2
2
2
2
2
2
2
2
4
4
4
4
4
4
4
4
28.0
43.7
39.6
23.4
13.3
19.3
10.9
38.4
79.7
68.3
47.9
46.6
34.2
50.6
44.3
65.9
27.0
18.9
24.5
42.1
72.1
54.7
71.5
23.2
5.6
10.1
21.6
23.8
26.4
19.3
27.5
14.2
45.0
37.4
35.9
34.4
14.5
25.9
17.5
38.3
14.7
20.2
29.3
28.2
36.3
30.2
28.2
19.3
25 (R)
33 (R)
19 (R)
10 (S)
66 (S)
36 (S)
61 (S)
25 (S)
45 (R)
33 (R)
15 (R)
8 (R)
11 (R)
22 (R)
1 (R)
15 (R)
This is confirmed by data in the literature, which show that during the reduction of
benzil using microbiological methods, selective oxidation of (R)-benzoin to benzil occurs.
Increased oxidative activity was observed by M.C. Fragnelli et al. in a two-liquid-phase
system [33] and by A. S. Demir et al. in a phosphate buffer at different pH values [31]. The
process of stereoinversion might be caused by the activity of different dehydrogenases:
one of them selectively oxidizes (R)-benzoin to diketone and the other—(S)-selective
dehydrogenase with a high affinity towards benzil—reduces it to the (S)-isomer. In order to
determine how stereoinversion affects the optical purity of the product, we biotransformed
rac-benzoin over time (Table 7). After 2 h, there was practically no reduction to diol, only
deracemization which led to a significant percentage of the (S)-enantiomer (up to 95% ee),
due to the selective oxidation of the isomer of the opposite configuration. Kinetic resolution
of the racemate through enantioselective oxidation was observed. In the fourth hour, the
(
S)-enantiomer contribution was modulated by three competitive reactions: oxidation
of benzoin to benzil, reduction of benzil to benzoin, and bioconversion of benzoin to
hydrobenzoin. An increased percentage of benzil and (R,R)-hydrobenzoin (pH 5.0, 6.0–7.0,
7
.5, and 8.0) or (S,S)-hydrobenzoin (pH 5.5 and 7) was observed. However, after 24 h, the
situation was further complicated due to the presence of an additional oxidation reaction
of the previously obtained hydrobenzoin to benzoin. The optical purity of benzoin was a
result of all these competitive reactions. The highest ee of (S)-enantiomer was obtained in a
solution with pH 6.5 (84%).

Molecules 2021, 26, 1578
8 of 15
Table 7. Deracemization of Rac-
Different pH Values.
2
Catalyzed by A. pullulans Strain DSM 14940 and DSM 14941 in Phosphate Buffer at
Yield (%)
1
Yield (%)
2
Yield (%)
Hydrobenzoin
(S,S) Meso
Conditions
pH
Time (h)
ee (%)
(
S)
(R)
(R,R)
5
5
6
6
7
7
7
8
5
5
6
6
7
7
7
8
5
5
6
6
7
7
7
8
.0
.5
.0
.5
.0
.2
.5
.0
.0
.5
.0
.5
.0
.2
.5
.0
.0
.5
.0
.5
.0
.2
.5
.0
2
2
2
2
2
2
2
2
4
4
4
4
4
4
4
4
24
24
24
24
24
24
24
24
18.5
13.6
13.3
11.5
12.6
12.9
11.7
42.0
43.2
49.7
24.1
29.8
29.1
52.7
44.5
51.9
31.3
37.4
74.6
53.1
48.4
48.7
52.2
55.4
87.0
68.5
80.6
78.0
85.7
74.3
70.8
44.1
15.8
8.4
4.1
4.8
4.4
7.6
16.4
18.1
49.3
39.7
9.3
33.1
26.3
24.7
19.6
19.4
8.6
11.4
6.1
2.2
2.2
2.2
3.6
6.2
3.0
4.0
1.0
1.8
2.0
3.2
7.1
10.2
7.9
8.4
5.5
2.9
6.5
8.1
8.4
10.9
82 (S)
71 (S)
86 (S)
94 (S)
95 (S)
94 (S)
90 (S)
75 (S)
68 (S)
35 (S)
61 (S)
45 (S)
38 (S)
41 (S)
40 (S)
28 (S)
72 (S)
65 (S)
26 (S)
84 (S)
60 (S)
51 (S)
40 (S)
28 (S)
7.4
4.5
-
0.4
0.4
-
1.4
1.5
-
0.8
1.5
2.0
3.2
2.6
-
-
-
-
-
6.7
2.8
8.1
10.3
0.5
13.1
26.1
14.3
17.2
22.4
26.0
9.5
2.6
4.0
8.1
9.9
4.8
4.3
4.3
5.4
2.2
0.7
1.5
0.5
0.5
0.2
34.9
11.7
56.5
46.4
42.1
10.4
11.6
5.9
1.4
0.7
0.4
0.3
0.1
0.2
0.2
0.2
-
10.9
11.2
6.1
5.6
0.3
5.8
14.3
13.9
13.1
11.9
2.2. Enantioselective Bioreduction Reaction of Benzil to the Corresponding (R)-Benzoin
The increase in substrate concentration contributed to the reduced proportion of (S)-
benzoin in the mixture after 24 h of reaction, especially for lower pH values (Table 1). In
order to obtain an excess of the R-configuration enantiomer, we decided to bioreduce the
prochiral diketone in a solution with pH 5.0 and 5.5 for higher reagent concentrations. The
results are shown in Table 8.
Table 8. Microbial Bioreduction of
and Various Concentrations of 1.
1
to (R)-2-Hydroxy-1,2-Diphenylethanone (
2
) in Phosphate Buffer at Different pH Values
pH
Amount of Substrate
5.0
5.5
−
5
×
10 (mol)
Conv. (%)
ee (%) of (R)-2
Conv. (%)
ee (%) of (R)-2
a
d
a
d
a
d
a
d
6
7
8
9
57.7 /67.6
21 /13
69.0 /83.5
23 /4
a
c
d
d
a
c
d
d
a
c
d
d
a
c
d
d
37.8 /90.7 /69.1
35 /73 /61
51.8 /84.0 /85.8
36 /79 /59
35 /65 /76
a
c
a
c
a
c
a
c
37.8 /78.6 /70.6
29 /66 /17
24.9 /80.8 /47.2
a
b
c
d
a
b
c
d
a
b
c
d
d
a
b
c
d
24.5 /18.3 /48.0 /53.8
38 /33 /71 /66
22.0 /41.3 /69.4 /57.7
36 /8 /71 /74
a
c
d
a
c
d
a
b
c
a
b
c
d
10
22.6 /47.0 /15.6
30 /68 /93
25.1 /26.9 /62.2 /36.9
37 /47 /75 /63
a
4 h; b 48 h; c 120 h; d 168 h.
2
The change in stereobias with increasing concentration of substrate could be explained
taking into account the possible presence of more than one alcohol dehydrogenase with
different enantioselectivity inside the cell. Oxidoreductases, contained in the microorgan-
ism, act competitively to each other by transferring the hydride (pro-S or pro-R) from the
nicotinamide adenine dinucleotide (NAD(P)H) cofactor to one of the sides of the prochiral
carbonyl group (face si or re). The final ee of the product results from the reduction reactions

Molecules 2021, 26, 1578
9 of 15
occurring at different rates, carried out by a set of oxidoreductases, and the hydroacetone
deracemization reaction. We observed that with the increase of reagent concentration,
the reduction reaction proceeded more slowly, so that after the prolonged reaction time,
another dehydrogenase could start acting. Under favorable conditions, with a correspond-
ingly higher substrate concentration and prolonged reaction time, the (S)-isomer of benzil
can be deracemized. The diketone can then be converted by (R)-dehydrogenase to the
corresponding (R)-benzoin, despite the (S)-selective dehydrogenase showing a higher affin-
ity for benzil. The effectiveness of bioconversion is usually strongly affected by substrate
concentrations. A decrease in the catalytic activity of the microorganism is observed to
increase as the concentration of the reagent increases. It can be presumed that too high a
concentration of the substrate may be inhibitory to dehydrogenases, especially those with
(
S)-stereopreference, resulting in a decrease in their chemical efficiency and an increased
contribution of the enantiomer of the R configuration. Finally, after 5 days, up to 79% ee
was obtained for (R)-benzoin in a solution at pH 5.5. However, the highest ee of 93% was
−
4
obtained after 7 days in a solution at pH 5.0 for 1 × 10 mol of 1.
−
5
For 5 10 of , benzil reduction was carried out in the presence of potential
×
1
inhibitors (Table 9). Compared with the results presented in Table 4, a significant effect of
reagent concentration on the enantioselectivity of the bioprocess was observed. In each
case, the reaction mixture was enriched by the (R)-isomer, although with a fairly low degree
of conversion.
Table 9. Microbial Bioreduction of
1
Catalyzed by A. pullulans Strain DSM 14940 and DSM 14941 in Phosphate Buffer at
Different pH Values with Additives.
Additives
Conv. (%)/
ee (%)
pH
Allyl
Alcohol
Ethyl
Chloroacetate
3-Methylbutan- 4-Methylpentan-
Cysteine
AMA-1
2-one
2-one
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
Conv. (%)
ee (%)
0.9
78 (R)
15.3
39 (R)
4.9
75 (R)
10.0
54 (R)
2.7
56 (R)
4.9
60 (R)
26.7
63 (R)
32.2
48 (R)
2.8
22 (R)
25.9
52 (R)
30.4
54 (R)
18.9
53 (R)
0.2
45 (R)
1.9
46 (R)
2.3
29 (R)
24.4
64 (R)
8.4
57 (R)
1.8
56 (R)
13.5
51 (R)
54.5
29 (R)
9.5
57 (R)
22.1
52 (R)
30.8
53 (R)
49.1
2.8
59 (R)
2.0
60 (R)
31.2
58 (R)
39.6
53 (R)
18.9
59 (R)
6.6
58 (R)
18.1
61 (R)
30.3
34 (S)
0.7
59 (R)
13.6
41 (R)
12.4
46 (R)
18.7
54 (R)
4.5
63 (R)
10.2
55 (R)
7.9
65 (R)
11.1
0.6
54 (R)
5.0
42 (R)
30.8
51 (R)
46.5
43 (R)
5.9
60 (R)
16.9
53 (R)
19.6
68 (R)
14.7
5.0
5.5
6.0
6.5
7.0
7.2
7.5
8.0
45 (R)
57 (R)
54 (R)
In the most promising cases with ee above 60%, the diketone bioreduction reaction
was performed using selected (S)-dehydrogenase inhibitors under the same conditions and
with the reaction time prolonged to 5 days. Results are shown in Table 10.
As a result, in solutions with a pH value of 7.0 to 7.5, selectivity and biotransformation
efficiency were improved in the presence of selected additives. Optimal results were
obtained in a phosphate buffer solution of pH 7.0, using 4-methylpentan-2-one as selective
inhibitor. Ee reached 91%.
The effect of additives on the selectivity of rac-benzoin bioconversion was subsequently
determined. As the highest enantiomeric excess, in most cases (except cysteine and ethyl
chloroacetate), was obtained in a solution with a pH value of 7.5, the reaction under these
−
5
−5
10 mol (Table 11).
conditions was carried out for both amount of rac-
2: 1
×
10 and 5
×

Molecules 2021, 26, 1578
10 of 15
Table 10. Bioreduction of
1
Catalyzed by A. pullulans Strain DSM 14940 and DSM 14941 in Specific
Conditions with Selected Additives.
Conditions pH
Additives
Conv. (%)
ee (%)
5
6
7
7
7
7
7
7
5
7
8
.0
.0
.2
.5
.0
.0
.5
.5
.5
.5
.0
Allyl alcohol
Allyl alcohol
Allyl alcohol
7.9
9.7
32 (R)
72 (R)
83 (R)
88 (R)
91 (R)
89 (R)
87 (R)
84 (R)
37 (R)
83 (R)
41 (R)
60.9
45.9
71.1
88.5
82.9
48.9
3.8
Allyl alcohol
4-Methylpentan-2-one
3-Methylbutan-2-one
4-Methylpentan-2-one
3-Methylbutan-2-one
AMA-1
AMA-1
Ethyl chloroacetate
43.1
50.8
Table 11. Deracemization of Rac-Benzoin (2) and Bioreduction Catalyzed by A. pullulans Strain DSM 14940 and DSM 14941 in Phosphate
Buffer with Additives.
Conv. (%)
1
Conv. (%)
2
Conv. (%)
Conditions
pH
Additives
ee (%) of 2
3
(
S)
(R)
(R,R)
(S,S)
meso
7.5
7.5
7.0
7.5
7.5
8.0
7.5
7.5
7.0
7.5
7.5
8.0
Allyl alcohol
AMA-1
Cysteine
41.9
38.1
51.1
35.1
54.4
65.3
38.9
49.9
55.6
26.5
23.5
45.9
24.6
29.8
26.6
33.1
22.8
20.4
7.6
9.3
10.4
1.2
30.2
27.9
20.5
25.9
22.9
10.6
6.1
8.2
9.3
12.1
13.7
3.3
10 (R)
3 (S)
13 (S)
12 (S)
rac
32 (S)
11 (S)
6 (S)
0.8
3.4
1.4
1.6
-
1.9
-
2.5
0.7
0.4
4.2
-
0.9
11.0
6.6
15.5
-
-
-
-
-
-
3-Methylbutan-2-one
4-Methylpentan-2-one
Ethyl chloroacetate
Allyl alcohol
AMA-1
Cysteine
3-Methylbutan-2-one
4-Methylpentan-2-one
Ethyl chloroacetate
0.9
36.4
25.9
9.3
17.5
14.6
39.7
-
-
6 (S)
82 (R)
1 (S)
42.6
34.1
-
14.0
4.5
-
6.6
15 (S)
Surprisingly, the bioconversion reaction of rac-benzoin at a reagent amount of
−
5
1
× 10 mol was not selective. A significant proportion of diketone in the mixture was
found, which suggests promoting oxidation rather than reduction. On the other hand, for
a higher concentration of the reagent, besides the non-selective deracemization reaction, an
increase in the bioreduction reaction rate was observed. In the presence of 3-methylbutan-
2
-one and 4-methylpentan-2-one, diol with the (R,R) configuration was obtained as the
main product, pointing out the occurrence of the reduction activity of dehydrogenases. The
R,R): meso ratio was 2.5:1 and 2.3:1 for 3-methylbutan-2-one and 4-methylpentan-2-one,
(
respectively. The (S,S)-isomer was detected using reaction with cysteine ((S,S): meso 1.7:1).
In other cases, the predominant isomer obtained was the meso compound. The highest
composition of this isomer was accumulated in the reaction medium using ethyl chloroac-
etate as additive ((S,S): meso 1:6). Based on the results obtained, it can be assumed that by
performing a more detailed optimization of the reaction conditions, it will be possible to
obtain a diol with the desired configuration.

Molecules 2021, 26, 1578
11 of 15
3
3
3
. Experimental Section
.1. Materials and Methods
.1.1. General Informations
The enantiomeric excess of the chiral products was determined by a chiral station-
ary phase of high-performance liquid chromatography (HPLC). HPLC analyses were
performed on a Shimadzu SCL-10A VP (Kyoto, Japan).
®
Compound 1, (S)-
Column 250
and propan-2-ol (90:10 v/v), at the flow rate of 0.6 mL per min. The retention times of 1,
2
and (R)-
2
was separated on a column Lux 5
µ
Cellulose-1, LC
×
4.6 mm, Phenomenex (Warsaw, Poland). The mobile phase was n-hexane
(
S)-2 and (R)-2 were 9.2 min, 20.1 min and 30.0 min, respectively.
Compound (R,R)- , (S,S)- and (S,R)- were separated on a column Lux 5
, LC Column 250 4.6 mm, Phenomenex (Poland). The mobile phase was n-hexane and
®
3
3
3
µ Cellulose-
3
×
propan-2-ol (90:10 v/v), at the flow rate of 0.6 mL per min. The retention times of (R,R)-
S,S)-3 and meso-3 were 21.6 min, 28.4 min and 26.2 min, respectively.
The samples were incubated in an orbital shaker (VORTEMP 1550 S2050; Equimed,
Cracow, Poland).
The absolute configurations of (S)-2, (R)-
based on the configuration of standards purchased from Merck (Darmstadt, Germany).
3,
(
2, (R,R)-3, (S,S)-3 and meso-3 were assigned
3.1.2. Reagents and Solvents
The chemical substances of analytical grade were commercially available and they
included: sucrose, glucose, fructose, ethyl acetate, ethanol, n-hexane, cyclohexane, tert-
butyl methyl ether (TBME), acetonitrile (AcCN), tetrahydrofuran (THF), allyl alcohol,
ethyl chloroacetate, MgSO , Na HPO , NaH PO , n-hexane for HPLC, propan-2-ol for
4
2
4
2
4
HPLC from POCH (Gliwice, Poland), benzil, rac-benzoin, (S)-benzoin, (R)-benzoin, (R,R)-
hydrobenzoin, meso-hydrobenzoin, cysteine, 3-methylbutan-2-one, 4-methylpentan-2-one,
1
-butyl-3-methylimidazolium tetrafluoroborate ((Bmim)(BF ) from Merck (Darmstadt, Ger-
4
many). 1-butyl-3-methylimidazolium hexafluorophosphate ((Bmim)(PF ), Fluka, Switzer-
6
land). Blossom Protect
™ from Koppert Biological Systems (Wien, Austria) contains ger-
minating fungal Aureobasidium pullulans strains DSM 14940 and 14941 (500 g per 1 kg of
the preparation).
3
.2. Bioreduction of 1 by Blossom Protect™ Contains Aureobasidium pullulans Strains DSM
1
4940 and 14941
For a typical experiment, to a suspension of Blossom Protect
™
(1 g) in 7.5 mL of
−
4
potassium phosphate buffer was added 1
×
10 mol glucose (Tables 1, 3–6 and 8–10) or
fructose, sucrose (Table 2) and the resulting suspension was incubated in an orbital shaker
◦
(
350 rpm) for 30 min at 30 C.
3
.2.1. I
10 6, 1 × 10
−
−5
−5
,
,
After pre-incubation, benzil
1
was added in the following amounts: 5
×
−
5
−5
−5
−5
−5
−5
2
9
×
10 , 3
× 10 , 1
×
10 , 4
×
10 , 5
×
10 moles (Table 1); 6
×
10 , 7
×
10 , 8
×
10
−
5
−5
×
10 moles (Table 8). Substrate was dissolved in 0.5 mL of ethanol and added
to the suspension of biocatalyst in phosphate buffer at the following pH values: 5.0, 5.5,
.0, 6.5, 7.0, 7.2, 7.5, 8.0 (Table 1) or 5.0 and 5.5 (Table 8). Next Falcon tubes were stirred at
same temperature at different intervals: 24 h (Table 1/Table 8); 48 h, 120 h, 168 h (Table 8).
6
3
.2.2. II
After pre-incubation, 1
added to the suspension of biocatalyst in phosphate buffer at the following pH values: 5.0,
−
5
×
10 mole of benzil dissolved in 0.5 mL of ethanol were
◦
◦
5
.5, 6.0, 6.5, 7.0, 7.2, 7.5, 8.0. Next Falcon tubes were stirred 24 h at 28 C, 32 C (Table 3) or
◦
0.5 h, 2 h and 4 h (Table 6) 24 h (Table 2) at 30 C.

Molecules 2021, 26, 1578
12 of 15
3
.2.3. III
After pre-incubation, 1
added to biphasic system composed by phosphate buffer at the following pH values: 5.0,
.5, 6.0, 6.5, 7.0, 7.2, 7.5, 8.0 and organic solvent: n-hexane, cyclohexane, TBME, THF, AcCN,
Bmim)(PF ), (Bmim)(BF ) (buffer:organic solvent 10:1 v/v). Next Falcon tubes were stirred
−
4
×
10 mole of benzil dissolved in 0.5 mL of ethanol was
5
(
6
4
◦
24 h at 30 C (Table 5).
3
.2.4. IV
−
5
−5
10 (Tables 9 and 10) mole of benzil
After pre-incubation, 1
×
10 (Table 4) or 1
×
dissolved in 0.5 mL of ethanol, additives: allyl alcohol/ethyl (9-antryl)glyoxylate (AMA-
−
5
1)/cysteine/3-methyl-2-butanone/4-methyl-2-pentanone/ethyl chloroacetate (1.25
×
10
)
were added to the suspension of biocatalyst in phosphate buffer at the following pH
values: 5.0, 5.5, 6.0, 6.5, 7.0, 7.2, 7.5, 8.0. Next Falcon tubes were stirred 24 h at 30 C
◦
(
Tables 4, 9 and 10).
The reaction progress was monitored by TLC (the solvent system used was n-hexane:
ethyl acetate 4:1 v/v). After the reaction was completed, microorganism was filtered off the
supernatant, washed with distilled water. The solid residue was washed with ethyl acetate
and the combined aqueous phases were extracted with ethyl acetate (3
×
20 mL). The
collected organic layer was dried over anhydrous MgSO and the solvent was evaporated
4
in a vacuum. The conversion degrees of the substrates and enantiomeric ratios of the
products were determined on HPLC system using a chiral column.
3
.3. Biotransformation of Rac-2 by Blossom Protect™ Contains Aureobasidium pullulans Strains
DSM 14940 and 14941
Biotransformation reaction was preceded by half an hour pre-incubation on a ro-
◦
tary shaker at 350 revolutions per minute (rpm) at 30 C. The composition of the pre-
−
4
incubation mixture was as follows: 1
×
10 mol glucose, 1 g Blossom Protect
™
,
7.5 mL
−
5
phosphate buffer (pH 5.0, 5.5, 6.0, 6.5, 7.0, 7.2, 7.5, 8.0). After pre-incubation, 1 × 10
−
5
(
Table 7/Table 11) or 5
×
10 (Table 11) moles of rac-benzoin dissolved in 0.5 mL of
ethanol were added to the mixture and optionally additives (Table 11). After completion of
the reaction, the mixture was filtered. Then, the filtrate and the solid phase were extracted
four times with ethyl acetate (25 mL) and the organic layer was evaporated. The conversion
degrees of the substrates and enantiomeric ratios of the products were determined on
HPLC system using a chiral column.
3
.4. Blossom Protect-Mediated Synthesis of (S)-2/(R)-2—Semi-Preparative-Scale
Compounds (S)- and (R)- were obtained on a semi-preparative scale using previously
optimized reaction conditions.
2
3
.4.1. A. Synthesis of (S)-2
A suspension of Blossom ProtectTM agent (40 g) and glucose (1.44 g) in phosphate
◦
buffer (pH 7.2, 300 mL) was maintained for 30 min at 30 C with stirring (350 rpm). After
this time, to the fermenting mixture, a solution of
1
(0.4 mmol; 95.2 mg) in EtOH (10 mL)
◦
was added and the resulting mixture was vigorously stirred at 30 C using laboratory
orbital shaker (350 rpm) under anaerobic conditions for 24 h. The rest of the reaction
work-up were performed in analogy to the previously described analytical-scale reactions
with obvious preservation of the appropriate the reaction parameters. The crude products
have been purified by column chromatography using mixture of n-hexane/AcOEt (7:3,
v/v) as an eluent to afford the respective (S)-2 (49.5 mg, 0.24 mmol, 85% conv., 52% yield;
90% ee).
3
.4.2. B. Synthesis of (R)-2
A suspension of Blossom ProtectTM agent (40 g) and glucose (1.44 g) in phosphate
◦
buffer (pH 7.0, 300 mL) was maintained for 30 min at 30 C with stirring (350 rpm). After

Molecules 2021, 26, 1578
13 of 15
pre-incubation, ethanolic solution of
1
(EtOH 10 mL) was added in the amount of 0.4 mmol
(
95.2 mg) and 4-methylpentan-2-one (63
µ
L). The resulting mixture was vigorously stirred
at 30 C using laboratory orbital shaker (350 rpm) under anaerobic conditions. After 2 days
biomass was removed by filtration. The bio-reduction product ((R)- ) was eluted from
◦
2
the aqueous phase and the recovered biomass with ethyl acetate. The rest of the reaction
work-up were performed in analogy to the previously described analytical-scale reactions
with obvious preservation of the appropriate the reaction parameters. The crude products
have been purified by column chromatography using mixture of n-hexane/AcOEt (7:3,
v/v) as an eluent to afford the respective (R)-2 (34.3 mg, 0.24 mmol, 68% conv., 36% yield;
90% ee).
4. Conclusions
In conclusion, this work shows that slight modifications of the reaction conditions
grant the opportunity to obtain 2-hydroxy-1,2-diphenylethanone with defined chirality
on an asymmetric carbon atom. In the enzymatic bioreduction applying the catalytic
properties of the antifungal agent, both enantiomers can be obtained with good optical
efficiency.
The product with S configuration ((S)-
proximately 90%) was received by reducing
2
1
) with the highest enantiomeric excess (ap-
in a phosphate buffer solution at pH 5.5–7.5
◦
−5
for 24 h at 30 C with 1
×
10 mol of substrate, as well as after a two-hour incubation of
−
5
◦
1
× 10 mol rac-2 at 30 C in an aqueous medium with a pH value of 6.5–7.5.
Benzil was effectively reduced to (R)-2 with good stereoselectivity up to 93% optical
−
5
purity in a biotransformation of 10
×
10 moles of 1 conducted for 168 h in a solution
◦
of slightly acidic pH 5.0 at 30 C. (R)-isomer with high enantiomeric excess was also
obtained in phosphate buffer pH 7.0–7.5 with the use of additives (allyl alcohol, AMA-1,
−
5
3
-methylbutan-2-one, 4-methylpentan-2-one) for 5
×
10 moles of substrate. After 120 h
TM
of incubation with 4-methylpentan-2-one, Blossom Protect -mediated biotransformation
of 1 was carried out with good stereoselectivity, allowing for obtaining an enantiomeric
excess of up to 91%.
The performed studies made it possible to formulate the conclusion that the synthesis
of (S)-benzoin is possible thanks to the enzymatic desymmetrization of benzil and der-
acemization of benzoin if the reaction is carried out in mild reaction conditions: neutral
◦
environment at 30 C for 24 h. Reagent concentration and the additives (potential inhibitors,
co-solvents) affect change of selective properties of the microorganism. Along with the
increase in substrate concentration, decreased pH value, prolonged reaction time and addi-
tion of substances “poisoning” the protein catalyst with defined chirality, preferentially an
enantiomer with the opposite configuration, was obtained. The substrate, like the additive,
acts as an inhibitor, selectively inhibiting the action of S-dehydrogenases.
Author Contributions: Conceptualization, R.K.; Methodology, R.K.; Validation, R.K., R.S. and D.M.;
Investigation, R.K., R.S., D.M., A.T.-K., H.P.; Resources, R.K., H.P.; Data Curation, R.K., D.M. Writing–
Original Draft Preparation, R.K.; Writing–Review & Editing, R.S., A.T.-K., H.P., A.W.; Visualization,
R.K. and R.S.; Supervision, R.K. and A.T.-K.; Project Administration, R.S.; Funding Acquisition, R.K.,
H.P., A.W. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Data Availability Statement: Not applicable.
Acknowledgments: This work has been supported by Nicolaus Copernicus University, Collegium
Medicum as part of the statutory research project in 2021, No. 166. The authors also wish to thank
Bio-ferm GmbH and Technical Director Christina Donat for the gift of Blossom ProtectTM
.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds (S)-2 (90% ee), (R)-2 (90% ee) are available from
the authors.

Molecules 2021, 26, 1578
14 of 15
References
1
.
Wallace, O.B.; Smith, D.W.; Deshpande, M.S.; Polson, C.; Felsenstein, K.M. Inhibitors of A
β
production: Solid-phase synthesis
and SAR of α-hydroxycarbonyl derivatives. Bioorganic Med. Chem. Lett. 2003, 13, 1203–1206. [CrossRef]
Tanaka, T.; Kawase, M.; Tani, S. α-Hydroxyketones as inhibitors of urease. Bioorganic Med. Chem. 2004, 12, 501–505. [CrossRef]
Hoyos, P.; Sinisterra, J.-V.; Molinari, F.; Alcántara, A.R.; De María, P.D. Biocatalytic Strategies for the Asymmetric Synthesis of
α-Hydroxy Ketones. Accounts Chem. Res. 2010, 43, 288–299. [CrossRef] [PubMed]
2
3
.
.
4
.
Uchida, R.; Shiomi, K.; Inokoshi, J.; Sunazuka, T.; Tanaka, H.; Iwai, Y.; Takayanagi, H.; Omura, S. Andrastins A-C, New Protein
Farnesyltransferase Inbibitors Produced by Penicillium sp. FO-3929. II. Structure Elucidation and Biosynthesis. J. Antibiot. 1996
9, 418–424. [CrossRef] [PubMed]
,
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
.
.
.
.
.
Slavik, M.; Carter, S.K. Chromomycin A3, Mithramycin, and Olivomycin: Antitumor Antibiotics of Related Structure. Stud. Surf.
Sci. Catal. 1975, 12, 1–30. [CrossRef]
Gozari, M.; Bahador, N.; Mortazavi, M.S.; Eftekhar, E.; Jassbi, A.R. An “olivomycin A” derivative from a sponge-associated
Streptomyces sp. strain SP 85. 3 Biotech 2019, 9, 1–11. [CrossRef]
Demir, A.S.; Ayhan, P.; Demirta s¸ , U.; Erkılıç, U. Fusarium roseum and Aspergillus oryzae-mediated enantioselective reduction of
benzils to benzoins. J. Mol. Catal. B: Enzym. 2008, 55, 164–168. [CrossRef]
Wildemann, H.; Dünkelmann, P.; Muller, M.; Schmidt, B. A Short Olefin Metathesis-Based Route to Enantiomerically Pure
Arylated Dihydropyrans and α,β-Unsaturated δ-Valero Lactones. J. Org. Chem. 2003, 68, 799–804. [CrossRef]
Hoyos, P.; Sansottera, G.; Fernández, M.; Molinari, F.; Sinisterra, J.V.; Alcántara, A.R. Enantioselective monoreduction of different
1,2-diaryl-1,2-diketones catalysed by lyophilised whole cells from Pichia glucozyma. Tetrahedron 2008, 64, 7929–7936. [CrossRef]
0. Plietker, B. New oxidative pathways for the synthesis of α-hydroxy ketones—the α-hydroxylation and ketohydroxylation.
Tetrahedron Asymmetry 2005, 16, 3453–3459. [CrossRef]
1. Davis, F.A.; Clark, C.; Kumar, A.; Chen, B.-C. Asymmetric Synthesis of the AB Ring Segments of Daunomycin and 4-
Demethoxydaunomycin. J. Org. Chem. 1994, 59, 1184–1190. [CrossRef]
2. Gore, M.P.; Vederas, J.C. Oxidation of enolates by dibenzyl peroxydicarbonate to carbonates of.alpha.-hydroxy carbonyl com-
pounds. J. Org. Chem. 1986, 51, 3700–3704. [CrossRef]
3. Adam, W.; Mueller, M.; Prechtl, F. Dimethyldioxirane Oxidation of Titanium Enolates: Diastereoselective.alpha.-Hydroxylations.
J. Org. Chem. 1994, 59, 2358–2364. [CrossRef]
4. Plietker, B. The RuO4-Catalyzed Ketohydroxylation, Part II:A Regio-, Chemo- and Stereoselectivity Study. Eur. J. Org. Chem. 2005
005, 1919–1929. [CrossRef]
5. Onomura, O.; Arimoto, H.; Matsumura, Y.; Demizu, Y. Asymmetric oxidation of 1,2-diols using N-bromosuccinimide in the
presence of chiral copper catalyst. Tetrahedron Lett. 2007, 48, 8668–8672. [CrossRef]
6. Muthupandi, P.; Alamsetti, S.K.; Sekar, G. Chiral iron complex catalyzed enantioselective oxidation of racemic benzoins. Chem.
Commun. 2009, 3288–3290. [CrossRef] [PubMed]
7. Alamsetti, S.K.; Muthupandi, P.; Sekar, G. Chiral Cobalt-Catalyzed Enantiomer-Differentiating Oxidation of Racemic Benzoins by
Using Molecular Oxygen as Stoichiometric Oxidant. Chem.-A Eur. J. 2009, 15, 5424–5427. [CrossRef] [PubMed]
8. Bøgevig, A.; Sundén, H.; Córdova, A. Direct Catalytic Enantioselective
,
2
α
-Aminoxylation of Ketones: A Stereoselective Synthesis
0
ofα-Hydroxy and α,α -Dihydroxy Ketones. Angew. Chem. Int. Ed. 2004, 43, 1109–1112. [CrossRef]
9. Hayashi, Y.; Yamaguchi, J.; Sumiya, T.; Hibino, K.; Shoji, M. Direct Proline-Catalyzed Asymmetric α-Aminoxylation of Aldehydes
and Ketones. J. Org. Chem. 2004, 69, 5966–5973. [CrossRef] [PubMed]
0. Enders, D.; Kallfass, U. An Efficient Nucleophilic Carbene Catalyst for the Asymmetric Benzoin Condensation. Angew. Chem. Int.
Ed. 2002, 41, 1743–1745. [CrossRef]
1. Martí, J.; López-Calahorra, J.C.A. Introduction to a rational design of chiral thiazolium salts. Tetrahedron Lett. 1993, 34,
5
21–524. [CrossRef]
2. Linghu, X.; Potnick, J.R.; Johnson, J.S. Metallophosphites as Umpolung Catalysts: The Enantioselective Cross Silyl Benzoin
Reaction. J. Am. Chem. Soc. 2004, 126, 3070–3071. [CrossRef]
3. Müller, M.; Gocke, D.; Pohl, M.; Rother, D. Thiamin diphosphate in biological chemistry: Exploitation of diverse thiamin
diphosphate-dependent enzymes for asymmetric chemoenzymatic synthesis. FEBS J. 2009, 276, 2894–2904. [CrossRef]
4. Hoyos, P.; Domínguez de María, P.; Sinisterra, J.V.; Alcántara, A.R. Hydrolase-Based Synthesis of Enantiopure α-Hydroxy Ketones:
From Racemic Resolutions to Chemo-Enzymatic Dynamic Kinetic Resolutions, Biotechnology Research: Technology and Applications;
Richter, F.W., Ed.; Nova Science Publishers, Inc.: New York, NY, USA, 2008; pp. 97–119.
2
2
2
2
5. Carballeira, J.; Quezada, M.; Hoyos, P.; Simeó, Y.; Hernaiz, M.; Alcantara, A.; Sinisterra, J. Microbial cells as catalysts for
stereoselective redox reactions. Biotechnol. Adv. 2009, 27, 686–714. [CrossRef]
6. Gamenara, D.; De María, P.D. Candida spp. redox machineries: An ample biocatalytic platform for practical applications and
academic insights. Biotechnol. Adv. 2009, 27, 278–285. [CrossRef]
7. Chênevert, R.; Thiboutot, S. Baker’s yeast reduction of 1,2-diketones. Preparation of pure (S)-(-)-2-hydroxy-1-phenyl-1-propanone.
Chem. Lett. 1988, 17, 1191–1192. [CrossRef]
8. Mahmoodi, N.O.; Mohammadi, H.G. Enantio-, Regio-, and Chemoselective Reduction of Aromatic ?-Diketones by Baker?s Yeast.
Mon. Chem.-Chem. Mon. 2003, 134, 1283–1288. [CrossRef]

Molecules 2021, 26, 1578
15 of 15
2
3
3
3
3
9. Imuta, M.; Ziffer, H. Microbial reduction of a series of substituted benzils. Optical properties and nuclear magnetic resonance
spectra of products. J. Org. Chem. 1978, 43, 3319–3323. [CrossRef]
0. Saito, T.; Maruyama, R.; Ono, S.; Yasukawa, N.; Kodaira, K.-I.; Nishizawa, M.; Ito, S.; Inoue, M. Asymmetric Reduction of Benzil
to (S)-Benzoin With Whole Cells of Bacillus cereus. Appl. Biochem. Biotechnol. 2003, 111, 185–190. [CrossRef]
1. Demir, A.S.; Hamamci, H.; Ayhan, P.; Duygu, A.N.; Igdir, A.C.; Capanoglu, D. Fungi mediated conversion of benzil to benzoin
and hydrobenzoin. Tetrahedron Asymmetry 2004, 15, 2579–2582. [CrossRef]
2. Konishi, J.; Ohta, H.; Tsuchihashi, G.-I. Asymmetric Reduction of Benzil to Benzoin Catalyzed by the Enzyme System of a
Microorganism. Chem. Lett. 1985, 14, 1111–1112. [CrossRef]
3. Fragnelli, M.C.; Hoyos, P.; Romano, D.; Gandolfi, R.; Alcántara, A.R.; Molinari, F. Enantioselective reduction and deracemisation
using the non-conventional yeast Pichia glucozyma in water/organic solvent biphasic systems: Preparation of (S)-1,2-diaryl-2-
hydroxyethanones (benzoins). Tetrahedron 2012, 68, 523–528. [CrossRef]
3
3
3
4. Oda, S.; Isshiki, K. Asymmetric Reduction of Benzil to (S)-Benzoin with Penicillium claviforme IAM 7294 in a Liquid-Liquid
Interface Bioreactor (L-L IBR). Biosci. Biotechnol. Biochem. 2008, 72, 1364–1367. [CrossRef] [PubMed]
5. Maruyama, R.; Nishizawa, M.; Itoi, Y.; Ito, S.; Inoue, M. The enzymes with benzil reductase activity conserved from bacteria to
mammals. J. Biotechnol. 2002, 94, 157–169. [CrossRef]
6. Kołodziejska, R.; Wroblewski, M.; Studzinska, R.; Karczmarska-Wódzka, A.; Grela, I.; Augustynska, B.; Modzelewska-
Banachiewicz, B. Aureobasidium pullulans as a key for the preparation of optical purity (R)-2-(anthracen-9-yl)-2-methoxyacetic
acid–The chiral auxiliary reagent in determination of absolute configuration. J. Mol. Catal. B Enzym. 2015, 121, 28–31. [CrossRef]
7. Kołodziejska, R.; Studzi n´ ska, R.; Kwit, M.; Jelecki, M.; Tafelska-Kaczmarek, A. Microbiological bio-reduction of prochiral carbonyl
compounds by antimycotic agent Boni Protect. Catal. Commun. 2017, 101, 81–84. [CrossRef]
3
3
8. Kołodziejska, R.; Studzi n´ ska, R.; Pawluk, H.; Karczmarska-Wódzka, A.; Wo z´ niak, A. Enantioselective Bioreduction of Prochiral
Pyrimidine Base Derivatives by Boni Protect Fungicide Containing Live Cells of Aureobasidium pullulans. Catalysts 2018, 8,
2
90. [CrossRef]
3
9. Kołodziejska, R.; Studzi n´ ska, R.; Tafelska-Kaczmarek, A.; Pawluk, H.; Stasiak, B.; Kwit, M.; Wo z´ niak, A. Effect of chemical struc-
ture of benzofuran derivatives and reaction conditions on enantioselective properties of Aureobasidium pullulans microorganism
contained in Boni Protect antifungal agent. Chirality 2019, 32, 407–415. [CrossRef]
4
4
0. Gotor, V.; Alfonso, I.; Garcia-Urdiales, E. Asymmetric Organic Synthesis with Enzymes, 1st ed.; Wiley-VCH Verlag GmbH & Co.
KGaA: Weinheim, Germany, 2008; pp. 1–340. [CrossRef]
1. Kołodziejska, R.; Studzi n´ ska, R.; Pawluk, H. Lipase-catalyzed enantioselective transesterification of prochiral 1-((1,3-
dihydroxypropan-2-yloxy)methyl)-5,6,7,8-tetrahydroquinazoline-2,4(1H,3H)-dione in ionic liquids. Chirality 2018, 30, 206–214.
[CrossRef] [PubMed]
4
4
2. Kołodziejska, R.; Studzi n´ ska, R.; Tafelska-Kaczmarek, A.; Pawluk, H.; Kwit, M.; Stasiak, B.; Wo z´ niak, A. The application of safe for
humans and the environment Polyversum antifungal agent containing living cells of Pythium oligandrum for bio-transformation
of prochiral ketones. Bioorg. Chem. 2019, 92, 103204. [CrossRef]
3. Sakai, T. ‘Low-temperature method’ for a dramatic improvement in enantioselectivity in lipase-catalyzed reactions. Tetrahedron:
Asymmetry 2004, 15, 2749–2756. [CrossRef]
4
4
4. Sakai, T. Temperature control of the enantioselectivity in the lipase-catalyzed resolutions. Future Dir. Biocatal. 2007, 21–50.
5. Fujisawa, T.; Tanaka, S.; Onogawa, Y.; Shimizu, M. Enantiocontrol in the bakers’ yeast reduction of trifluoroacetylbiphenyl
derivatives. Tetrahedron Lett. 1999, 40, 1953–1956. [CrossRef]
4
4
4
4
5
6. Hayakawa, R.; Nozawa, K.; Shimizu, M.; Fujisawa, T. Control of Enantioselectivity in the Bakers’ Yeast Reduction of β-Keto Ester
Derivatives in the Presence of a Sulfur Compound. Tetrahedron Lett. 1998, 39, 67–70. [CrossRef]
˙
7. Brzezi n´ ska-Rodak, M.; Zyma n´ czyk-Duda, E.; Kafarski, P.; Lejczak, B. Application of Fungi as Biocatalysts for the Reduction of
Diethyl 1-Oxoalkylphosphonates in Anhydrous Hexane. Biotechnol. Prog. 2002, 18, 1287–1291. [CrossRef] [PubMed]
8. Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Rules for Optimization of Biocatalysis in Organic Solvents. Biotechnol. Bioeng. 2009, 102,
1
–8. [CrossRef]
9. Gardossi, L.; Poulsen, P.B.; Ballesteros, A.; Hult, K.; Švedas, V.K.; Vasi c´ -Ra cˇ ki, Ð.; Carrea, G.; Magnusson, A.; Schmid, A.;
Wohlgemuth, R.; et al. Guidelines for reporting of biocatalytic reactions. Trends Biotechnol. 2010, 28, 171–180. [CrossRef]
0. Liese, A.; Seelbach, K.; Wandrey, C. Industrial Biotransformations, 2nd ed.; Wiley-VCH Verlag GmbH & Co. KGaA: Weinheim,
Germany, 2006; pp. 1–428. [CrossRef]