Immobilized microorganisms in the reduction of ethyl 4-chloro acetoacetate

Article abstract of DOI:10.1016/j.tetasy.2009.09.018

Microorganisms were used to reduce ethyl 4-chloroacetoacetate (CAAE) to ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE]. Mucor ramannianus provided 98% conversion with 84% ee. Free cells of Kluyveromyces marxianus led to 95% conversion with 81% ee. After a fractionary factorial design to study the reaction conditions, calcium alginate immobilized cells of K. marxianus furnished the product with 99% conversion with 91% ee.

Full text of DOI:10.1016/j.tetasy.2009.09.018

Tetrahedron: Asymmetry 20 (2009) 2263–2266
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Immobilized microorganisms in the reduction of ethyl 4-chloro acetoacetate
Joyce Benzaquem Ribeiro a,b, , Aline de Souza Ramos , Sorele Batista Fiaux , Selma Gomes Ferreira Leite ,
a,c
d
a
*
b
b
b
Maria da Conceição Klaus V. Ramos , Francisco Radler de Aquino Neto , Octavio A. C. Antunes
Escola de Química, Universidade Federal do Rio de Janeiro, CT Bloco E, Cidade Universitária, Rio de Janeiro RJ 21949-909, Brazil
Instituto de Química, Universidade Federal do Rio de Janeiro, CT Bloco A, Cidade Universitária, Rio de Janeiro RJ 21949-909, Brazil
Farmanguinhos, Fundação Oswaldo Cruz, Rua Sizenando Nabuco 100, Manguinhos, Rio de Janeiro RJ 21041-250, Brazil
a
b
c
d
Faculdade de Farmácia, Universidade Federal Fluminense, Rua Mário Viana, 523, Santa Rosa, Niterói, Rio de Janeiro RJ 24241-000, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 July 2009
Accepted 23 September 2009
Available online 14 October 2009
Microorganisms were used to reduce ethyl 4-chloroacetoacetate (CAAE) to ethyl (S)-4-chloro-3-hydroxy-
butanoate [(S)-CHBE]. Mucor ramannianus provided 98% conversion with 84% ee. Free cells of Kluyveromy-
ces marxianus led to 95% conversion with 81% ee. After a fractionary factorial design to study the reaction
conditions, calcium alginate immobilized cells of K. marxianus furnished the product with 99% conversion
with 91% ee.
Ó 2009 Elsevier Ltd. All rights reserved.
1
. Introduction
Asymmetric reductions of ketoesters can provide versatile chi-
factor for the metabolism of fatty acids,11 while the (S)-enantiomer
is a key intermediate for HMG-CoA reductase inhibitors, a com-
pound class used in the treatment of hypercholesterolemia.12
A
ral building blocks in organic synthesis. These procedures are pre-
ferred over the kinetic resolution of racemates, due to the
possibility of producing only one enantiomer with high enantio-
meric excess and chemical yields.1 Chiral metal complexes such
as Ru-BINAP and related systems can be used as catalysts in asym-
metric hydrogenations, but these routes employ drastic conditions
such as high temperature and pressure.2 Biocatalytic transforma-
tions are an advantageous alternative, especially because of their
high selectivity (chemo-, regio-, and stereo-selectivity), mild reac-
tion conditions, and environmental aspects.3
racemic mixture of such compounds cannot be used; ethyl 4-
chloro acetoacetate is an inexpensive substrate for asymmetric
reduction, the best way to produce optically active ethyl 4-
13
chloro-3-hydroxybutanoate.
In this study, 12 microorganisms were employed to catalyze the
asymmetric reduction of ethyl 4-chloroacetoacetate (CAAE) to (S)-
ethyl 4-chloro-3-hydroxybutanoate [(S)-CHBE] (Fig. 1) in whole-
cell processes. Some strains were also used immobilized in calcium
alginate spheres. This immobilization technique can influence the
product configuration, enantiomeric excess, and conversion le-
,4
1
4,15
Isolated enzymes and whole cells can be used as biocatalysts
in enantioselective reductions. The use of isolated enzymes
frequently leads to higher enantiomeric excess while avoiding
problems associated with competing catalysts with differing
stereoselectivities. Unfortunately, reduced cofactors must be
regenerated in situ in a second catalytic cycle or provided in
stoichiometric amounts to sustain catalytic activity.5,6 Hence,
whole cells are used more often because of their own cofactor
vel.
Some reaction’s conditions were studied by an experimen-
16
tal design as recommended by Taguchi to enhance conversion.
O
O
OH
O
biocatalyst
Cl
Cl
O
O
Figure 1. Microbial reduction of ethyl 4-chloro acetoacetate (CAAE) to ethyl (S)-4-
chloro-3-hydroxybutanoate (S-CHBE).
7
regeneration system. Saccharomyces cerevisiae has been widely
used to obtain hydroxyesters, due to its low cost, ease of use,
and good prediction for the stereochemistry of the product,
however the desired configuration cannot always be achieved.
Ethyl 4-chloro-3-hydroxybutanoate is a very useful chiral build-
ing block for enantiomerically pure pharmaceuticals. For example,
the (R)-enantiomer can be converted to (R)-carnitine, an essential
2. Results and discussion
8
,10
In the first step of this work, the free cells of twelve microorgan-
isms (nine yeast strains and three filamentous fungi strains) were
tested for CAAE reduction ability (Table 1). All microorganisms
were able to reduce the substrate with excess of (S)-CHBE under
the experimental conditions used.
Only Aspergillus niger gave lower than 50% conversion. Rhodo-
torula rubra and Rhodotorula minuta furnished 100% conversion,
*
Corresponding author. Tel.: +55 21 2562 7248.
E-mail address: joyce_benzaquem@yahoo.com.br (J. B. Ribeiro).
0
957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.09.018

2
264
J. B. Ribeiro et al. / Tetrahedron: Asymmetry 20 (2009) 2263–2266
Table 1
Microbiological reduction of CAAE to S-CHBE with free cells
To evaluate the entrapment effects on microorganisms, S. cere-
visiae 40, Pichia sp., Hansenula sp., Candida sp., K. marxianus, R. ru-
bra were immobilized in calcium alginate spheres and tested again
for CAAE reduction ability (Table 2). After immobilization, Candida
sp. and R. rubra provided lower conversion levels and enantiomeric
excesses in comparison with free yeasts used in the first step of
this study. Immobilized cells of S. cerevisiae 40, Hansenula sp, and
K. marxianus led to higher enantiomeric excess than the free cells,
with a decrease in conversions. The maximum conversion level
was obtained with immobilized cells of R. rubra, which provided
Microorganism
Conversion (%)
ee (%)
Saccharomyces cerevisiae 40
Saccharomyces cerevisiae 60
Saccharomyces cerevisiae 80
Pichia sp.
Hansenula sp.
Candida sp.
62.9
63.8
55.8
66.5
86.1
82.6
94.7
100
81.5
79.5
82.1
75.8
57.5
56.4
80.8
48.1
50.2
82.8
83.7
90.0
Kluyveromyces marxianus
Rhodotorula rubra
Rhodotorula minuta
Trichoderma harzianum
Mucor ramannianus
Aspergillus niger
100
9
9.3% of conversion, but only 31.9% ee. K. marxianus is considered
95.0
98.5
36.1
as the best biocatalyst after immobilization, with 84.2% ee of (S)-
CHBE at moderate conversion level (77.5%) after 24 h.
Incubation: 30 °C, 150 rpm, 24 h.
Table 2
Microbiological reduction of CAAE to S-CHBE with immobilized cells
Microorganism
Conversion (%)
ee (%)
but the enantiomeric excess was moderate (48.1% and 50.2%,
respectively). Mucor ramannianus and Trichoderma harzianum were
considered the best biocatalysts, with high conversion levels
S. cerevisiae 40
Pichia sp.
Hansenula sp.
Candida sp.
K. marxianus
R. rubra
41.7
75.4
51.9
48.8
77.5
99.3
86.2
78.1
62.8
48.6
84.2
31.9
(
98.5% and 95.0%, respectively) and up to 82% ee. Kluyveromyces
marxianus also provided good results, with 94.7% conversion and
0.7% ee. However, when ethyl acetoacetate was used as a sub-
8
strate, K. marxianus, A. niger and T. harzianum produced an excess
of the (R)-hydroxy ester,9 hence these microorganism are sensi-
tive to changes in the substrate’s structure.
Incubation: 30 °C, 150 rpm, 24 h.
,17
In addition to the good enantiomeric excess obtained with K.
marxianus, product recovery was much easier after immobilization
than the product obtained when free cells were used. Hence, a 2
Several microorganisms such as yeasts, molds, bacteria, and
actinomycetes have been found to catalyze this reaction.1 Most
of them predominantly produce the (S)-enantiomer, as we found
in this work. We confirmed the (S)-configuration by using the
8
5-2
fractional factorial design was used to enhance conversion levels
and enantioselectivity of the reaction with this strain.1
6,25
Five
(S)-isomer purchased from Aldrich. It is sometimes difficult to find
2
variables were studied: concentrations of CAAE, MgCl , glucose,
a good microorganism by screening whole cells because the coen-
zyme NADPH is frequently required in this reduction and there is
not always an efficient NADPH-recycling system and an CAAE-
reducing enzyme in the same cell.1
biomass (Cell), and volume of sodium alginate solution (V). Three
central points replicates were accomplished. Experimental domain
and results are shown in Table 3. Response variables were % con-
version and % ee after 18 h-incubation-period. All experimental
runs furnished an excess of (S)-CHBE.
8,19
Numerous approaches have been documented to improve the
stereospecificity of whole-cell bioconversions.6
,8,12,19,20
Better
According to Table 3, in entries 3 and 5 (when the maximum
biomass concentration and minimum substrate concentration
were used) 100% conversion and high enantioselectivity was ob-
tained (95.0% ee and 94.3% ee, respectively). In both conditions,
conversion and enantioselectivity were better than that obtained
with free cells of K. marxianus (94.7% conversion and 80.8% ee Table
1). The maximum biomass/substrate ratio also furnished good re-
sults in the bio-reduction of CAAE by the fungus Aureobasidium
6
results were obtained with the addition of allyl alcohol, higher
biomass concentrations (150–400 g wet weight/L),6 heating
,8
2
0
19
cells, heating acetone dried cells, and the substitution of glu-
12
cose by 2-propanol as a co-substrate. Recombinant microorgan-
isms have also been used with good results.7
,21–23
However,
wild-type strains, such as the ones used in this work, are generally
preferred to industrial processes mainly because of their robust-
2
4
13
ness. Our results are compatible with wild-type strains reported
in the literature, but in our case without toxic inhibitor or cell pre-
treatment. This suggests that some approaches could enhance the
conversion levels and enantiomeric excess.
pullulans. The high stereoselectivity achieved in run 5 with
immobilized cells of K. marxianus is shown in Figure 2.
It was not possible to establish a model to explain the enantio-
meric excess, but the same conditions that furnished the highest
Table 3
Experimental design to improve reaction conditions of CAAE reduction to S-CHBE by immobilized cells of K. marxianus
Entries
Factorsa
Responses (18 h)
Conversion (%)
V (mL)
Cell (gdw/L)
CAAE (mM)
MgCl
2
(g/L)
Glucose (g/L)
ee (%)
1
2
3
4
5
6
7
8
9
30
30
30
30
70
70
70
70
50
50
50
1.9
1.9
5.6
5.6
5.6
5.6
1.9
1.9
3.7
3.7
3.7
35
70
35
70
35
70
35
70
0
2
2
0
0
2
2
0
1
1
1
10
50
50
10
50
10
10
50
30
30
30
38.6
11.8
100
46.2
100
53.6
26.2
10.8
38.4
34.8
36.4
86.3
84.9
94.3
85.5
95.0
86.3
91.0
100
52.5
52.5
52.5
88.4
87.7
88.0
1
1
0
1
Incubation: 30 °C, 150 rpm, 18 h.
a
V: volume of 1.5% w/v sodium alginate solution in 100 mL of medium + cells; Cell (biomass concentration—g dry weight/L).

J. B. Ribeiro et al. / Tetrahedron: Asymmetry 20 (2009) 2263–2266
2265
is rarely obtained with wild microorganisms in the absence of toxic
enzymatic inhibitors. Optimization studies are currently in
progress.
A
B
C
3. Conclusions
Twelve strains of yeast and filamentous fungi were used in the
reduction of CAAE. Some of them provided good enantiomeric ex-
cess of (S)-CHBE with conversion higher than 50% when used as
free cells. M. ramannianus provided 98.5% conversion with 83.7%
ee. Despite providing good and easy recovery of the product,
immobilized cells in calcium alginate spheres led to lower conver-
sion levels in comparison with free cells used in most cases. Good
enantiomeric excess was obtained with K. marxianus and product
recovery was much easier after immobilization than when free
cells were used. A fractionary factorial design to study some reac-
tion conditions with K. marxianus allowed us to improve conver-
sion level to 99% with 91% ee. Further optimization studies can
improve these results.
D
E
10
12
tR (min)
4
. Experimental
Figure 2. Chiral GC analysis on column Lipodex-E (25 m  0.25 mm  0.25
0–130 °C (5 min) at 5 °C/min: (A) CAAE; (B) ethyl 4-chloro-3-hydroxybutanoate
racemate obtained via NaBH reduction); (C) (S)-CHBE (Aldrich); (D) immobilized
K. marxianus reduction (experimental design—entry 5);and (E) co-elution of (B) and
D).
lm), at
7
(
Microorganisms, media, growth conditions, and biotransformation
4
with free cells: Hansenula sp., K. marxianus, Candida sp., Pichia sp., 3
strains of S. cerevisiae, R. rubra, R. minuta and filamentous fungi, A.
niger, T. harzianum and M. ramannianus, belong to the collection of
the ‘Departamento de Engenharia Bioquıímica, Escola de Quıímica,
UFRJ’ and are freely available upon request. Cells were allowed to
grow for 48 h, under 150 rpm at 30 °C in a medium containing
(
Table 4
Results of regression analysis for the fractionary factorial design to response variable,
%
conversion (normalized variables)
1% glucose, 0.5% yeast extract, 0.5% peptone, 0.1% (NH
4 2 4
) SO , and
Terma
0.1% MgSO O. After this period, the cells were harvested by
centrifugation, re-suspended in water and then used for the reac-
tion. After centrifugation, the cells (4 g/L, dried weight) were added
4
Á7H
2
Coefficient
t-Value
p-Value
Intercept
V
Cell
45.4
À0.75
26.6
À17.8
À0.50
7.25
83.4
À1.18
41.8
À27.9
À0.78
11.4
0.0001
0.3606
0.0006
0.0013
0.5151
0.0076
0.0552
0.0664
2
to the reduction’s medium containing: glucose (5%), MgCl (0.1%)
CAAE
in a final volume of 100 mL. After 30 min of addition of the micro-
organisms, the substrate (0.5%) in the aqueous-ethanol was added
to the medium. The reaction was carried out in 500 mL cotton
plugged Erlenmeyer flasks for 24 h at 30 °C and 150 rpm. After
MgCl
2
Glucose
V Â Cell
V Â CAAE
2.60
2.35
4.08
3.69
2
4 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 ), filtered, and concentrated
under vacuum. Products were analyzed by (chiral) gas chromatog-
2
Coefficient of determination, r = 0.96.
V: volume of 1.5% w/v sodium alginate solution in 100 mL of medium; Cell
biomass concentration).
a
2
4
(
raphy (GC), on column Lipodex-E (25 m  0.25 mm  0.25
lm), at
conversions also provided the highest stereoselectivity, except
entry 8 that provided 100% ee and only 10.8% conversion. Then,
only conversion was maximized in the experimental domain stud-
ied. The regression coefficients and t-values of terms are presented
in Table 4. Analysis of variance was performed in order to validate
70 °C to 130 °C (5 min) at 5 °C/min.
Immobilization of cells (S. cerevisiae 40, K. marxianus, R. rubra,
Candida sp., Hansenula sp, and Pichia sp.) in calcium alginate and bio-
transformation: Cells grown for 48 h in the medium described be-
fore were centrifuged and re-suspended in distilled water to
obtain a cell-suspension. Sodium alginate aqueous solution was
added and this mixture (cell-suspension + 1.5% w/v sodium algi-
2
the regression model. The fit of the model is expressed by r , which
was calculated to be 0.96. This indicates that the model explains
9
6% of the variability in the data.
2
nate aqueous solution) was dropped into CaCl aqueous solution
Biomass and glucose had a positive effects on the conversion,
(0.1 M) to form calcium alginate spheres. Spheres were filtered,
washed with distilled water and then added to the medium for
reduction. Reduction was carried out in 500 mL cotton plugged
Erlenmeyer flasks at 30 °C, 150 rpm for 24 h. After that period,
medium was filtered to separate the biocatalyst and the liquid
phase was treated as described above.
while the substrate showed a negative effect. The addition of the
salt MgCl did not affect conversions, as also observed by Houng
2
8
et al. in the same reaction but employing baker’s yeast as biocat-
alyst. The volume of sodium alginate solution and interactions
studied had no significant effect (p-value >0.05) in response. Based
on these results, the following conditions were selected for the bio-
reduction of ethyl 4-chloroacetoacetate by K. marxianus in 100 mL
of medium: volume of 1.5% sodium alginate solution, 30 mL; bio-
mass, 5.6 gdw/L; substrate 35 mM; glucose 50 g/L.
5
-2
fractionary factorial design16 was
Experimental design: A 2
used to study five variables in 8 runs with 3 replicates of the cen-
tral point. Variables and domain were: volume of 1.5% w/v sodium
alginate solution with cells of K. marxianus (V): 30–70 mL in
100 mL of medium; Biomass concentration (Cell): 1.9–5.6 g dry
weight/L CAAE: 35–70 mM; MgCl : 0–2 g/L; Glucose: 10–50 g/L.
2
Response variables were % conversion and % ee. Cells of K. marxi-
anus, obtained as described above, was re-suspended in sodium
Experiments performed in triplicate were conducted in order to
verify the conversion level and enantiomeric excess in the reaction
conditions described above. After 18 h, 99% conversion was ob-
tained (SD = 1.7%) and 91% ee (SD = 2.5%) of (S)-CHBE. Such a result

2
266
J. B. Ribeiro et al. / Tetrahedron: Asymmetry 20 (2009) 2263–2266
alginate solution according to the biomass and total volume of
sodium alginate solution described in the experimental domain.
After 30 min of immobilization, the substrate in aqueous-ethanol
was added to the medium. The reaction was carried out in
8. Houng, J. Y.; Hsu, H. F.; Liu, Y. H.; Wu, J. Y. J. Biotechnol. 2003, 100, 239–250.
9.
Ribeiro, J. B.; Ramos, M. C. K. V.; Aquino Neto, F. R.; Leite, S. G. F.; Antunes, O. A.
C. J. Mol. Catal. B. 2003, 121, 24–25.
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5
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medium for 18 h at 30 °C and 150 rpm. After that period, medium
was filtered to separate the biocatalyst and the liquid phase was
treated as described above. Statistical analyses were performed
using Statistica 6.0 (Statsoft Inc., Tulsa, OK, USA).
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Acknowledgments
Financial support from FAPERJ and CNPq—BRAZIL is acknowl-
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also acknowledged.
17. Ramos, A. S.; Ribeiro, J. B.; Vazquez, L.; Fiaux, S. B.; Leite, S. G. F.; Cruz, R. A.;
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