Fungi mediated conversion of benzil to benzoin and hydrobenzoin

Article abstract of DOI:10.1016/j.tetasy.2004.07.010

An enzyme system of four fungi catalyses the reduction of benzil to benzoin, as well as benzoin to hydrobenzoin. Depending on the pH of the medium, both enantiomers of benzoin can be obtained in good yield and high ee starting from benzil via a reduction,

Full text of DOI:10.1016/j.tetasy.2004.07.010

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 2579–2582
Fungi mediated conversion of benzil to benzoin and hydrobenzoin
b,c
Ayhan S. Demir,a,b,* Haluk Hamamci, Peruze Ayhan, A. Nese Duygu,
b
a
a
A. Cigdem Igdir and Doga Capanoglu
b
_
a
Middle East Technical University, Department of Chemistry, 06531 Ankara, Turkey
Middle East Technical University, Department of Biotechnology, 06531 Ankara, Turkey
Middle East Technical University, Department of Food Engineering, 06531 Ankara, Turkey
b
c
Received 16 June 2004; accepted 5 July 2004
Abstract—An enzyme system of four fungi catalyses the reduction of benzil to benzoin, as well as benzoin to hydrobenzoin. Depend-
ing on the pH of the medium, both enantiomers of benzoin can be obtained in good yield and high ee starting from benzil via a
reduction, as well as from rac-benzoin via a novel deracemization reaction. Starting from benzil, (R)-, (S)- and rac-benzoin only
(
R,R)-hydrobenzoin was obtained in high ee and chemical yield.
Ó 2004 Elsevier Ltd. All rights reserved.
1
. Introduction
alternative option for the fungus-mediated synthesis of
both enantiomers of benzoin 2 and (R,R)-hydrobenzoin
3.
Biocatalysis is one of the most important stereoselective
1
preparations of optically active compounds. Among
these methods, the reduction of benzil 1 using biocata-
lysts is highly desirable. Enantiomerically pure hydro-
benzoins has been proven to be very useful chiral
2. Results and discussion
2
3
auxiliaries and ligands for stereoselective organic syn-
4
To assess the ability of selected microorganisms to con-
vert benzil into benzoin and hydrobenzoin, four differ-
ent species of fungi were selected; R. oryzae (ATCC
9363), R. oryzae (72465), Rhizomucor miehei (72460)
and Rhizomucor pusillus (72561). These four species
were preferred due to their kinship and because of the
preliminary work conducted concerning R. oryzae
thesis. These diols are accessible through resolution, di-
5
hydroxylation of olefins, reduction of benzils and
6
through carbon–carbon bond formation. BakerÕs yeast,
which is a widely used biocatalyst, reduces various car-
bonyl compounds such as monoketones, diketones and
7
ketoesters to the corresponding chiral alcohols. How-
ever, in the reduction of benzil, BakerÕs yeast gave only
1
0f,11
(ATCC 9363) mediated bioconversion.
The ease
8
9
racemic benzoin. Inoue et al. and Konishi et al. have
recently reported the reduction of benzil to (S)-benzoin
using the entire cell of Bacillus cereus, but benzoin was
not further reduced to hydrobenzoin. In connection with
and commercial availability of all the possible stereoiso-
meric products of benzoin and hydrobenzoin makes the
analysis of the processes simple. Initially, all four micro-
organisms were screened and shown to produce benzil–
benzoin conversion reactions. After the optimization of
the parameters for microbial conversion, the best results
were obtained using the following conditions.
1
0
our work in the area of benzoins, we have reported in
a short communication the preparation of both enantio-
mers of benzoins via a novel enzymatic deracemization
reaction using the fungus Rhizopus oryzae (ATCC
1
1
9
363). During the deracemization reactions, we also
Microorganisms were inoculated in a medium contain-
ing 0.2% ammonium sulfate, 0.065% monobasic potas-
sium phosphate, 0.025% magnesium sulfate, 0.005%
zinc sulfate, 0.5% glucose and 1.5% agar. The streaked
plates were incubated at 30°C for 3–4days for spore
production and then stored at 4°C until utilized. The
surface of the petri plate containing spores was rubbed
observed a low yield formation of hydrobenzoin. In con-
nection with this work, we report herein an attractive
*
Corresponding author. Address: Middle East Technical University,
Department of Chemistry, 06531 Ankara, Turkey. Tel.: +90-312-
2103242; fax: +90-312-2101280; e-mail: asdemir@metu.edu.tr
0
957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.07.010

2580
A. S. Demir et al. / Tetrahedron: Asymmetry 15 (2004) 2579–2582
with a sterile inoculation loop and then transferred to a
L sterile beaker containing a 400mL growing medium,
in which the organism was grown in a rotary shaker at
5°C for 2days. After 2days, benzil (2mmol) dissolved
in 5mL DMSO was added. Conversions were monitored
by TLC and HPLC [equipped with chiral column using
authentic (R)- and (S)-benzoin, as a reference]. After the
appropriate time, benzoin was isolated as a colourless
solid. All physical and chemical properties of benzoin
were identical with commercially available materials.
The ee values were determined by HPLC using a chiral
column.
In addition to the above-described conditions the con-
version reactions were also carried out at different pH
levels (Table 1) and it was found that the pH of the med-
ium strongly affected the stereochemical outcome of the
products (pH values of the reactions were adjusted using
a 1M NaOH solution and a lactic acid solution). While
R. oryzae (ATCC 9363) and R. oryzae (72465) afforded
at a low pH (4.2–5.0) (S)-enantiomer of benzoin in 80–
85% ee (Table 1, entries 2 and 4), both Rhizomucor mie-
hei (72460) and Rhizomucor pusillus (72561) showed the
same selectivity at a pH of 8.0–8.5 [(S)-benzoin forma-
tion]. However the ee value of benzoin decreased to
1
3
3
3–37% (Table 1 entries 6 and 8).
Under these conditions, the Rhizopus species furnished
(
R)-benzoin in 98 to >99% ee while the Rhizomucor spe-
cies furnished (S)-benzoin in 71–73% ee (Table 1, entries
, 3, 5 and 7). During the conversions it was found that
each microorganism worked at a different pH. R. oryzae
ATCC 9363) and R. oryzae (72465) did not change the
The benzil–benzoin bioconversion reactions, under op-
timized conditions with all four microorganisms, also
furnished hydrobenzoin in a low yield. This reaction
was repeated at different pH levels with prolonged times
without breaking the reaction at the benzoin step, with
the Rhizopus species at a pH of 6.5–8.5 and the Rhi-
zomucor species at a pH of 8.0–8.5 to furnish hydrobenz-
oin as a major product. After work-up and purification,
hydrobenzoin was isolated in 71–77% yields and in 96–
99% ees with 71/29–99/1 dl/meso ratios (Table 2). The
absolute configurations of the products were determined
as (R,R)- by using HPLC with a chiral column. The best
results were obtained with the Rhizopus species.
1
(
pH after the incubation, while both Rhizomucor miehei
and Rhizomucor pusillus first decreased the pH of the
medium to 4.5 for 3days, in which time most of the ben-
zil was converted into (S)-benzoin with 71–73% ee. The
pH then increased to 8.0–8.5 after 5days and remained
constant at this pH for 15days. At this high pH value,
partial racemization of (S)-benzoin occurred (Scheme
1
).
Table 1. Fungi mediated reduction of benzil
Entry
Fungus
Reaction time (days)
pH
(R)-Benzoin
(S)-Benzoin
a
a
Ee (%)
Yield (%)
Ee (%)
Yield (%)
1
2
3
4
5
6
7
8
Rhizopus oryzae (ATCC 9363)
Rhizopus oryzae (ATCC 9363)
Rhizopus oryzae (72465)
6
5
7
5
2
3
3
3
6.5–8.5
4.2–5.0
6.8–8.5
4.1–5.0
4.3–4.6
8.0–8.5
4.3–4.5
8.0–8.5
>99
98
81
83
80
75
b
Rhizopus oryzae (72465)
Rhizomucor miehei (72460)
85
71
33
73
37
78
72
75
77
73
b
Rhizomucor miehei (72460)
Rhizomucor pusillus (72561)
b
Rhizomucor pusillus (72561)
a
ꢀ1
Determined by HPLC analysis using Chiralpak AD column, UV detection at 254nm, eluent: hexane/2-propanol = 9:1, flow 0.80mLmin . t
benzoin: 25.70min, t (S)-benzoin: 32.21min.
R
(R)-
R
b
Obtained from TUBITAK Marmara Research Center, Turkey.
O
O
1
O
OH
O
OH
OH
R,R)-3
OH
(
(S)-2
(
R)-2
O
OH
rac-2
Scheme 1.

A. S. Demir et al. / Tetrahedron: Asymmetry 15 (2004) 2579–2582
Table 2. Fungal conversion of benzil to hydrobenzoin
2581
Entry
Fungus
Reaction time (days)
pH
Hydrobenzoin
a
b
dl/meso
Ee (%)
Yield (%)
1
2
3
4
Rhizopus oryzae (ATCC 9363)
Rhizopus oryzae (72465)
Rhizomucor miehei (72460)
Rhizomucor pusillus (72561)
1
21
18
13
11
pH 6.5–8.5
pH 6.5–8.5
pH 8.0–8.5
pH 8.0–8.5
99/1
99/1
>99
>99
96
77
74
76
71
71/29
79/21
97
a
Determined by H NMR.
b
ꢀ1
Determined by HPLC analysis using a Diacel Chiralcel OJ column, UV detection at 254nm, eluent: hexane/2-propanol = 9:1, flow 0.80mLmin
20°C. t
R
(R,R)-hydrobenzoin: 26.19min, t
R
(S,S)-hydrobenzoin: 32.94min.
O
OH
OH
O
OH
R)-2
OH
(S)-2
(
Scheme 2.
Using both of the Rhizopus species, rac-benzoin was also
used as a substrate and converted to hydrobenzoin.
During this work, we found deracemization activity in
both of the fungi on rac-benzoin. The enzyme system
of these microorganisms catalyzed the inversion of the
of the ligands around the asymmetric centre depends
on the acidity of the medium. We suggest that the pH
dependency is due to some change in the enzyme, or
more likely that the fungal strain contains many differ-
ent enzymes, which are active at different pH values.
1
1
configuration of benzoin. The conversion of rac-benz-
oin was monitored by TLC and LC MS [equipped with a
chiral column using authentic (R)- and (S)-benzoin as
reference]. The disappearance of the (S)-enantiomer
and the increased formation of the (R)-enantiomer were
observed during the reaction while shaking was resumed
until no more change was observed. (R,R)-Hydrobenzo-
in was isolated in a 73% yield, 95% ee [R. oryzae
The mode of interaction between the enzyme and the
substrate is not clear at present. However, it is notewor-
thy that by only changing the pH of the medium a dra-
matic change in the enantioselectivity can be brought
about. In all cases no reaction occurred in the absence
of the biocatalyst.
(
9
72465)], and a 76% yield, 97% ee [R. oryzae (ATCC
363)] with 97/3; 99/1 dl/meso ratio.
3. Conclusion
For the conversion of benzil to hydrobenzoin via benz-
oin, the enzyme system of the microorganisms must first
realize selective reductions and isomerizations. It seems
that many different enzymes of fungal strain participate
in the selective conversion processes.
In conclusion, we have found an enzyme system that
gives both enantiomers of benzoin depending on the
pH of the medium via a novel reduction of benzil and
a deracemization reaction of benzoin. The reduction of
benzoin isomers furnishes (R,R)-hydrobenzoin in high
ee and good yield. This process can be performed under
mild reaction conditions using an extremely simple pro-
cedure and provides good to excellent yields and enan-
tiomeric excesses. Further investigation on the scope
and limitation of these reactions, as well as a mechanis-
tic study is underway.
During the conversion of benzil to hydrobenzoin, the
preferred substrate was benzil. Before the consumption
of all benzil, only a small amount of hydrobenzoin
formed.
Although the mechanism for deracemization is not clear
at present, there are two possible paths for the asymmet-
rization of the substrates. One is the deracemization of
the substrate via the formation of an ene-diol while
the other is the enantioselective degradation of one
enantiomer (Scheme 2). The latter is supposed to be a
minor path, if at all, based on the yield and the ee men-
tioned above.
4. Experimental
4.1. Materials and methods
NMR spectra were recorded on a Bruker DPX 400.
Chemical shifts d are reported in ppm relative to CHCl
3
1
13
13
(
H: d=7.27), CDCl3 ( C: d=77.0) and CCl ( C:
4
However, the deracemization reaction of rac-benzoin
proceeded smoothly and the enantiomeric excess of the
product reached 97% after incubation. Moreover, upon
changing the pH, the absolute configuration of benzoin
also changed. This means that the spatial arrangement
d=96.4) as internal standards. Column chromatography
was conducted on silica gel 60 (40–63lm). TLC was car-
ried out on aluminium sheets pre-coated with silica gel
60F254 (Merck), and the spots visualized with UV light
(k=254nm). Enantiomeric excesses were determined

2582
A. S. Demir et al. / Tetrahedron: Asymmetry 15 (2004) 2579–2582
by HPLC and LC–MS analysis using a Thermo Finni-
gan Surveyor equipped with an appropriate chiral phase
column, as described in the footnotes of the tables. Opti-
cal rotations were measured with an Autopol IV auto-
matic polarimeter.
State Planning Organization (DPT) is greatly appreci-
ated.
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6
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4
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4
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25
D
¨
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25
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22
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Acknowledgements
(
Financial support from the Middle East Technical Uni-
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Research Council of Turkey (TUBITAK), the Turkish
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Academy of Sciences (TUBA), as well as the Turkish