Whole cells in enantioselective reduction of tert-butyl acetoacetate

Article abstract of DOI:10.1080/00397911.2012.655685

The b-ketoester tert-butyl acetoacetate was enantioselectively reduced to tertbutyl (S)-3-hydroxybutanoate by seven microorganism strains. The best result using free cells was obtained with the yeast R. rubra, which furnished 97.6% ee and higher than 99% of conversion within 24 h. After immobilization in calcium alginate spheres, R. rubra furnished 96% ee and higher than 99% ee within 24 h, even if substrate concentration was 58mM. Immobilized cells were reused three times without loss of enantioselectivity. Copyright Taylor & Francis Group, LLC.

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Whole Cells in Enantioselective
Reduction of tert-Butyl Acetoacetate
a
b
Aline de Souza Ramos , Joyce Benzaquem Ribeiro , Raquel de
b
b
Oliveira Lopes & Rodrigo Octavio Mendonça Alves de Souza
a
Farmanguinhos, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
Biocatalysis and Organic Synthesis Group, Instituto de Química,
b
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Accepted author version posted online: 17 May 2012.Version of
record first published: 06 Mar 2013.
To cite this article: Aline de Souza Ramos , Joyce Benzaquem Ribeiro , Raquel de Oliveira Lopes &
Rodrigo Octavio Mendonça Alves de Souza (2013): Whole Cells in Enantioselective Reduction of tert-
Butyl Acetoacetate, Synthetic Communications: An International Journal for Rapid Communication of
Synthetic Organic Chemistry, 43:12, 1611-1618
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1
Synthetic Communications , 43: 1611–1618, 2013
Copyright # Taylor & Francis Group, LLC
ISSN: 0039-7911 print=1532-2432 online
DOI: 10.1080/00397911.2012.655685
WHOLE CELLS IN ENANTIOSELECTIVE REDUCTION OF
tert-BUTYL ACETOACETATE
1
Aline de Souza Ramos, Joyce Benzaquem Ribeiro,
2
2
Raquel de Oliveira Lopes, and
2
Rodrigo Octavio Mendon c¸ a Alves de Souza
Farmanguinhos, Funda c¸ a˜ o Oswaldo Cruz, Rio de Janeiro, Brazil
1
2
Biocatalysis and Organic Synthesis Group, Instituto de Qu ı´ mica,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
GRAPHICAL ABSTRACT
Abstract The b-ketoester tert-butyl acetoacetate was enantioselectively reduced to tert-
butyl (S)-3-hydroxybutanoate by seven microorganism strains. The best result using free
cells was obtained with the yeast R. rubra, which furnished 97.6% ee and higher than
9
9% of conversion within 24 h. After immobilization in calcium alginate spheres, R. rubra
furnished 96% ee and higher than 99% ee within 24 h, even if substrate concentration was
8 mM. Immobilized cells were reused three times without loss of enantioselectivity.
5
Keywords Calcium alginate; chiral reduction; immobilization; b-ketoester; R. rubra
INTRODUCTION
Both enantiomers of the hydroxyester tert-butyl 3-hydroxybutanoate are impor-
[
1,2]
tant chiral building blocks in the synthesis of bioactive natural products
and
3
[3]
b -aminoxy peptides. The asymmetric reduction of b-ketoesters to b-hydroxyesters
is very useful for organic synthesis, and much attention has been paid to biotrans-
formation reactions, particularly because of their easy accomplishment, high stereo-
[4–6]
selectivity, and ecofriendly appeal.
For an economically viable bioreduction process, reduced nicotinamide
cofactors must be regenerated in a second catalytic cycle to sustain catalytic activity.
Whole cells have their own cofactor regeneration system and therefore are usually
preferred to purified enzymes. Saccharomyces cerevisiae is often used in the biotrans-
formation of carbonyl compounds because it is easily available. However, the desired
Received November 30, 2011.
Address correspondence to Joyce Benzaquem Ribeiro, Biocatalysis and Organic Synthesis Group,
Instituto de Qu ´ı mica, Universidade Federal do Rio de Janeiro, CT Bloco A, Cidade Universit a´ ria, Rio de
Janeiro, RJ 21949-909, Brazil. E-mail: joyce_benzaquem@yahoo.com.br
1
611

1612
A. DE SOUZA RAMOS ET AL.
Figure 1. Microbial reduction of tert-butyl acetoacetate to tert-butyl (S)-3-hydroxybutanoate.
configuration cannot always be achieved and competing enzymes within cells can lead
[5–8]
to poor stereoselectivity.
Several techniques have been reported to enhance stereo-
specificity of whole-cell bioconversions, such as screening for other microorganisms
[5,7]
besides S. cerevisiae,
[9]
addition of enzymatic inhibitors, biocatalyst immobiliza-
[
10–13]
[14]
[15,16]
tion,
modification of reaction conditions.
Over the past few years, we have been investigating the reduction of various
heating acetone dried cells,
the use of organic solvents,
and the
[6,10,11]
[7,10,11,17–20]
b-ketoesters by microorganisms.
In the present work we report our
studies on the reduction of tert-butyl acetoacetate (Fig. 1) using wild yeasts and
filamentous fungi previously tested on other bioreductions. Some yeast strains were
immobilized in calcium alginate spheres before assays and evaluated for recyclability.
RESULTS AND DISCUSSION
The low cost of the bioreduction process using whole cells and the abundance
of microorganisms in nature encouraged us in the search for microorganisms, in
addition to the well-known Saccharomyces cerevisiae and Aspergillus niger, that
are capable of conducting the reduction of tert-butyl acetoacetate with good conver-
sion and enantioselectivity. Thus, we started this study with a screening step using
free cells of five yeasts (Saccharomyces cerevisiae 40, Hansenula sp., Geotrichum can-
didum, Kluyveromyces marxianus, and Rhodorotula rubra) and two filamentous fungi
(
Trichoderma harzianum and Aspergillus niger). Conversions and enantiomeric
excesses (ee) obtained after 24 h, with substrate concentration at 29 mM, are shown
in Table 1.
All tested strains were able to catalyze the reaction with excess of the
[
21]
(
ation found in ketoester microbial reductions described in the literature.
S)-hydroxyester, the one predict by Prelog’s rule and the most frequent configur-
[
7,8]
The
product configuration was determined by measurement of optical rotation, which
Table 1. Bioreduction of tert-butyl acetoacetate (29 mM) to tert-butyl (S)-3-
hydroxybutanoate by free cells
a
a
Microorganism
Conversion (%)
ee (%)
Saccharomyces cerevisiae 40
Hansenula sp.
57.6
>99
>99
64.2
>99
>99
76.4
73.9
84.1
96.5
51.5
97.6
53.2
46.2
Geotrichum candidum
Kluyveromyces marxianus
Rhodotorula rubra
Trichoderma harzianum
Aspergillus niger
ꢀ
Note. Incubation: 30 C, 150 rpm, 24 h.
a
Conversion and enantiomeric excess (ee) determined by GC analysis.

REDUCTION OF tert-BUTYL ACETOACETATE
1613
2
3
ꢀ
was [a]
[
CHCl3).
þ33.5 (95% ee, c 1.64 g=100 mL, CHCl ), and compared to the literature:
3
D
3
2
3
ꢀ
[22a]
20
and [a]D þ36.0 (c 1.08 g=100 mL,
ꢀ
a]D þ26.3 (c 1.64 g=100 mL, CHCl )
[
22b]
It is worth noting that S. cerevisiae and A. niger, the most used microorganisms
in biotransformations, furnished the reaction product in moderate enantimeric excess
(73.9% ee and 46.2% ee, respectively), which highlights the need to seek new biocata-
lysts for this reaction. Conversions varied from 57.6% (with S. cerevisiae) to more
than 99% (with Hansenula sp., G. candidum, R. rubra, and T. harzianum). Enantio-
meric excesses varied from 46.2% (with A. niger) to 97.6% (with R. rubra). Among
the tested biocatalysts as free cells, the most promising was R. rubra, which led to
the excellent conversion (>99%) and high enantiomeric excess (97.6% ee). Geotrichum
candidum also showed very good results, with more than 99% conversion and 96.5%
ee. Examples of chromatograms obtained are shown in Fig. 2.
Compared to our previous studies, free cells of K. marxianus had greater conver-
[
sion and enantioselectivity in the reduction of ethyl 4-chloroacetoacetate and ethyl
11]
[
20]
3
-oxohexanoate,
strate. However, R. rubra seemed to give better results than K. marxianus on the con-
although product configuration changed according to the sub-
[
10]
version of bulkier substrates, as ethyl benzoylacetate and tert-butyl acetoacetate.
Salvi and Chattopadhyay
[23]
observed in their work on microbial reductions of
ketones by the filamentous fungus Rhizopus arrhizus that the elongation of the alkyl
chain (methyl to tert-butyl) in the alkoxy group led to a gradual increase in yields and
enantioselectivities of the reduced products. In our study, we also observed that free
cells of R. rubra exhibited better conversion and enantioselectivity in the reduction of
tert-butyl acetoacetate compared to the reduction of methyl and ethyl acetoacetate,
but we did not detect such correspondence with other biocatalysts, and even different
[
7]
product configurations were found with K. marxianus and A. niger.
Several microorganisms have already been assayed as biocatalysts in the
[
15]
reduction of tert-butyl acetoacetate. North
>98% ee) and 75% of conversion with S. cerevisiae in a medium containing petrol=
water 25:4, but this author employed very high biomass concentration (approximately
obtained high (S)-enantioselectivity
(
[16]
2
00 g freeze-dried=L). Medson et al.
also used S. cerevisiae in petroleum and
reported isolated yield of 68% and 98% ee for the same isomer when the substrate
concentration was 20 mM. Salvi and Chattopadhyay
Rhizopus arrhizus in the reaction and achieved 71% of isolated yield and 94% ee after
[
23]
used the filamentous fungi
[
24]
4
days, but the substrate concentration in medium was only 0.66 g=L. Bernardi et al.
obtained low to moderate excess of the (S)-hydroxyester when using A. niger (48% ee)
and G. candidum (85% ee), whereas 92% ee of the R configuration was achieved with
the actinomycete Streptomyces sp. as biocatalyst. Acetone powder of G. candidum
was used in the asymmetric reduction of tert-butyl acetoacetate and furnished the
corresponding (S)-alcohol in high enantiomeric purity (>99%) and more than 99%
[
25]
[26]
conversion. Afterward, Matsuda et al. developed a method using ionic liquids
as reaction media for asymmetric reduction by acetone-dried cells of G. candidum
immobilized on water-absorbing polymer containing water. The substrate was
added at 20 mM, and 87% of conversion with 98% ee was achieved for the
(
S)-hydroxyester. In both cases in which dried G. candidum cells were used, it was
necessary to add NADþ and 2-propanol as cosubstrate. Our data obtained with free
cells of R. rubra and G. candidum are comparable with the greatest conversions

1614
A. DE SOUZA RAMOS ET AL.
Figure 2. Typical chromatograms showing the selectivities achieved. Chiral GC analysis on column
ꢀ
RTx-5MS (30 m ꢁ 0.25 mm ꢁ 0.25 mm), at 90 C (11 min): (a) tert-butyl acetoacetate (substrate); (b)
tert-butyl 3-hydroxybutanoate (racemate obtained via NaBH4 reduction); (c) Aspergillus niger reduction;
(
d) Rhodotorula rubra reduction. Retention times (tR): tR (substrate) ¼ 8.1 min, tR ((S)-enantiomer) ¼
8
.6 min, tR ((R)-enantiomer) ¼ 9.0 min.
and enantioselectivities described in the literature for whole cells, but have some
advantages, such as lower biomass concentration (12 g=L dry weight), greater
substrate concentration (29 mM), and no need to add reduced cofators to reaction
medium.
[
27]
In the studies with plant cells, Speicher et al. obtained greater than 95% ee of
tert-butyl (S)-3-hydroxybutanoate with cultured cells of Riccia fluitans, with 70–90%
[
28]
yield within 1–2 days. In another study with plant cells, Kojima et al. carried out
the asymmetric reduction of ketones, including tert-butyl acetoacetate, using
cultured cells of Nicotiana tabacum as biocatalyst and showed that the reduction of

REDUCTION OF tert-BUTYL ACETOACETATE
1615
Table 2. Bioreduction of tert-butyl 3-oxobutanoate (29 mM) to tert-butyl (S)-3-
hydroxybutanoate by immobilized cells
First cycle
Second cycle (after 14 d)
a
a
a
a
Microorganism
Conversion (%)
ee (%)
Conversion (%)
ee (%)
K. marxianus
G. candidum
Hansenula sp.
R. rubra
97.0
97.4
98.0
51.6
90.8
77.5
95.9
>99
97.0
77.5
>99
72.4
95.0
79
>99
94.1
ꢀ
Note. Incubation: 30 C, 150 rpm, 24 h.
a
Conversion and enantiomeric excess (ee) determined by GC analysis.
tert-butyl acetoacetate gave high ee of the corresponding (S)-alcohol (98% ee) under
illumination of fluorescent light and high CO concentration, with 98% conversion.
2
In contrast, under dark conditions, low CO concentration and with the addition
2
of glucose, the (R)-hydroxyester was achieved with 82% ee and 83% yield. Although
interesting, plant cells hardly have been used until now for asymmetric reductions
because of the lack of knowledge about this kind of process, mainly on a large scale.
Microbial transformation processes are better known, but they are difficult to apply
in biotransformations using plant cells.
In a second step of this study, K. marxianus, G. candidum, Hansenula sp., and R.
rubra were immobilized in calcium alginate spheres to evaluate the entrapment effects
on the bioreduction of tert-butyl acetoacetate. This immobilization technique makes
product recovery easier, allows biocatalyst recycling, and makes continuous processes
[
10–13,17,18]
possible. Moreover, cell immobilization in calcium alginate spheres
sometimes improves conversion and enantioselectivity.
recycling, immobilized yeasts were used twice: the first cycle was carried out immedi-
[
10,13]
To evaluate biocatalyst
ately after the immobilization, and the second cycle was performed after a storage
ꢀ
for 14 days in CaCl solution at 4 C (Table 2).
2
Immobilized cells of K. marxianus exhibited better conversion and enantio-
selectivity in the second cycle.
Compared to free cells, the entrapment of K. marxianus increased conversion
level, while the immobilization decreased conversion level and enantioselectivity of
Hansenula sp. (especially in the second cycle) and G. candidum. High conversion
(
>99%) and enantioselectivity (>94% ee) were maintained with immobilized R. rubra
in both cycles, although a slight decrease in enantioselectivity had been observed
compared to the use of free cells. Based on results showed in Tables 1 and 2, R. rubra
was chosen for further studies.
Immobilization of cells in calcium alginate sometimes allows adding substrate
to reaction medium at greater concentrations because of the diffusion barrier, which
[
13]
could protect cells from substrate toxicity.
always desired in industrial biotransformation processes,
As high substrate concentration is
[
13,29]
we evaluated the
catalytic ability of immobilized cells of R. rubra in three cycles when tert-butyl
acetoacetate was added at 58 mM. In the first cycle, the biocatalyst was used immedi-
ately after the immobilization; second and third cycles were performed after storage
ꢀ
at 4 C, at intervals of 7 days (Table 3). In previous studies with other substrates,

1616
A. DE SOUZA RAMOS ET AL.
Table 3. Bioreduction of tert-butyl 3-oxobutanoate (58 mM) to tert-butyl (S)-3-hydroxybutanoate by
immobilized cells of R. rubra
First cycle
Second cycle (after 7 days) Third cycle (after 14 days)
Biomass
(
a
g dry weight=L) Conversion (%) ee (%)
a
a
a
a
a
Conversion (%)
ee (%)
Conversion (%)
ee (%)
1
2
4
>99
>99
95.5
96.0
>99
>99
93.0
96.0
54.5
94.5
96.0
2
>99
ꢀ
Note. Incubation: 30 C, 150 rpm, 24 h.
a
Conversion and enantiomeric excess (ee) determined by GC analysis.
[
10,11]
we verified that biomass concentration can influence the reaction.
In addition,
[16]
Medson et al. observed that the amount of yeast (S. cerevisiae) required to effect
complete consumption of the same amount of starting material increased with the size
of the ester group: Methyl and ethyl acetoacetate were reduced with just 1 g of yeast
whereas the most sterically demanding tert-butyl group required 11 g. So, to evaluate
whether the biomass amount could affect the reaction, cells were employed at two
concentrations: 12 g dry weight=L and 24 g dry weight=L.
In the first two cycles, the biomass concentration did not affect conversion,
which was the same with free cells of R. rubra (>99%). High conversion was main-
tained in the third cycle when biomass was used at 24 g=L, but at 12 g=L only
5
4.5% of conversion was achieved. Enantioselectivity was high in all tested conditions,
especially when biomass was added at 24 g=L (96.0% ee), showing that cells do not
need to be used immediately after cultivation and immobilized biomass could be
reused at least three times. These findings suggest robustness, an important character-
istic for continuous process and industrial applications.
In summary, we described here a search for biocatalysts to be used in the asym-
metric reduction of tert-butyl acetoacetate to tert-butyl (S)-3-hydroxybutanoate. In
the screening step, free cells of seven microorganism strains were tested, and the yeast
R. rubra led to best results. The biocatalytic potential of this yeast was demonstrated
by the high enantioselectivity (96%) and excellent conversion (>99%) obtained within
2
4 h even after immobilization of cells in calcium alginate spheres during up to three
cycles, when substrate concentration was 58 mM and biomass was used at 24 g=L.
This is the first use of R. rubra in this reaction.
EXPERIMENTAL
Saccharomyces cerevisiae, Hansenula sp., Geotrichum candidum, Kluyveromyces
marxianus, Rhodotorula rubra, Aspergillus niger, and Trichoderma harzianum
belonged to the collection of the Departamento de Engenharia Bioqu ´ı mica, Escola
de Qu ´ı mica, Universidade Federal do Rio de Janeiro. Cells were grown for 48 h under
ꢀ
1
50 rpm and 30 C in a medium containing 1% glucose, 0.5% yeast extract, 0.5%
peptone, 0.1% (NH ) SO , and 0.1% MgSO ꢂ 7H O. After that period, they were
4
2
4
4
2
harvested by centrifugation, resuspended in water, and used for the reaction. After
centrifugation, the cells (12 g=L, dry weight) were added to 50 mL of the reduction
medium containing glucose (5%) and substrate (29 mM). The reaction was carried

REDUCTION OF tert-BUTYL ACETOACETATE
1617
ꢀ
out in 500-mL, cotton-plugged Erlenmeyer flasks for 24 h at 30 C and 150 rpm. After
24 h, the medium was centrifuged again to separate the cells, and the liquid phase was
extracted with ethyl acetate. The organic phase was dried (anhydrous Na SO ),
2
4
filtered, and concentrated under vacuum. Conversions and enantiomeric excesses
were determined by (chiral) gas chromatography (GC), on column RTx-5MS
ꢀ
(
(
(
30 m ꢁ 0.25 mm ꢁ 0.25 mm) at 90 C (11 min). The elution order was substrate
tR ¼ 8.1 min), tert-butyl (S)-3-hydroxybutanoate (tR ¼ 8.6 min) and tert-butyl
R)-3-hydroxybutanoate (tR ¼ 9.0 min). The reaction product was characterized by
nuclear magnetic resonance (NMR) and mass spectroscopy. Optical rotations were
measured from CHCl solutions using a Jasco DIP-370 polarimeter at the sodium
3
[
22]
D line (589 nm) operating at room temperature and compared to the literature.
Cells grown during 48 h in the medium described were centrifuged, and 0.6 g
dry weight) was resuspended in 3 mL of distilled water to obtain a cell suspension.
(
A sodium alginate aqueous solution (1.5%, 20 mL) was added, and this mixture
cell-suspension sodium alginate aqueous solution) was dropped in a CaCl aqueous
(
2
solution (0.1 M) to form calcium alginate spheres. Spheres were filtered, washed with
distilled water, and added to 50 mL of medium containing glucose (5%) and substrate
(29 or 58 mM). Reduction was carried out in 500-mL, cotton-plugged Erlenmeyer
flasks, at 30 C and 150 rpm during 24 h. After that period, the medium was filtered
ꢀ
to separate the biocatalyst, and the liquid phase was treated as described previously.
ꢀ
Immobilized cells were washed with distilled water and maintained at 4 C in a CaCl
aqueous solution (0.1 M) for 7 or 14 days. Thus, immobilized cells were incubated in
2
ꢀ
growth medium under 150 rpm and 30 C for 2 h, washed with distilled water again,
and reused under the same reaction conditions.
Compound tert-Butyl-3-oxo-butanoate
1
H NMR (200 MHz; CDCl ): 1.47 [s, 9H, COOC(CH )]; 2.25 (s, 3H, H ); 3.35
3
3
4
(s, 2H, H2).
Compound tert-Butyl (S)-3-Hydroxybutanoate
1
H NMR (200 MHz; CDCl ): 1.19 and 1.22 (d, 3H, H ); 1.74 [s, 9H,
3
4
COOC(CH )]; 2.40 and 2.38 (d, 2H, H ); 3.17 (s, 1H, OH); 4.07, 4.10, 4.13, 4.16,
3
2
1
3
4
.19, 4.23 (m, 1H, H3).
C NMR (50 MHz; CDCl ): 22.54 (C ); 28.34
3
4
[
DEPT 135 (50 MHz; CDCl ): 22.54 (C ); 28.34 [COOC(CH )]; 44.04 (C ); 64.59
COOC(CH )]; 44.04 (C ); 64.59 (C ); 81.45 [COOC(CH )]; 172.63 [COOC(CH )].
3 2 3 3 3
3
4
3
2
(C ). CGMS m=z 57: tert-butyl; m=z 103: RCOO.
3
ACKNOWLEDGMENTS
Financial support from FAPERJ, CAPES, and CNPq-BRAZIL is acknowledged.
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