Highly enantioselective bioreduction of ethyl 3-oxohexanoate

Article abstract of DOI:10.1016/j.tetlet.2011.09.023

Seven wild-type microorganism strains were used to reduce ethyl 3-oxohexanoate to ethyl (R)-3-hydroxyhexanoate. Free cells of Kluyveromyces marxianus and Aspergillus niger led to higher than 99% of conversion with higher than 99% ee. After immobilization in calcium alginate spheres, cells of K. marxianus exhibited high enantioselectivity (>99% ee) and conversion level (99%) within 24 h even if substrate was added at concentration of 10 g/L (62 mM).

Full text of DOI:10.1016/j.tetlet.2011.09.023

Tetrahedron Letters 52 (2011) 6127–6129
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Highly enantioselective bioreduction of ethyl 3-oxohexanoate
Aline de Souza Ramos a, Joyce Benzaquem Ribeiro b, , Raquel de Oliveira Lopes b,
⇑
Selma Gomes Ferreira Leite c, Rodrigo Octavio Mendonça Alves de Souza b
a Farmanguinhos, Fundação Oswaldo Cruz, Rua Sizenando Nabuco 100, Manguinhos, Rio de Janeiro, RJ 21041-250, Brazil
b Instituto de Química, Universidade Federal do Rio de Janeiro, CT Bloco A, Cidade Universitária, Rio de Janeiro, RJ 21949-909, Brazil
c Escola de Química, Universidade Federal do Rio de Janeiro, CT Bloco E, Cidade Universitária, Rio de Janeiro, RJ 21949-909, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven wild-type microorganism strains were used to reduce ethyl 3-oxohexanoate to ethyl (R)-3-
hydroxyhexanoate. Free cells of Kluyveromyces marxianus and Aspergillus niger led to higher than 99%
of conversion with higher than 99% ee. After immobilization in calcium alginate spheres, cells of K. marxi-
anus exhibited high enantioselectivity (>99% ee) and conversion level (99%) within 24 h even if substrate
was added at concentration of 10 g/L (62 mM).
Received 9 August 2011
Revised 30 August 2011
Accepted 6 September 2011
Available online 10 September 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
b-Ketoester
Biotransformation
Immobilization
Kluyveromyces marxianus
Chiral reduction
Chiral b-hydroxyesters are widely used as building blocks for
the synthesis of fine chemicals, pharmaceuticals, and natural prod-
ucts.1,2 Ethyl 3-hydroxyhexanoate is a key intermediate in the syn-
thesis of (+)-neopeltolide, a bioactive marine macrolide with
potent antiproliferative activity against cancer cell lines. The cellu-
lar target for this class of substance is the inhibition of cytochrome
bc1 complex.3
Many efforts have been made in the search for highly selec-
tive methods to obtain chiral b-hydroxyesters, as ethyl
3-hydroxyhexanoate. Asymmetric reduction of prochiral ketoest-
ers is an alternative route. Bioreductions are attractive methods,
mainly due to high enantioselectivity, mild and safe reaction
conditions, and lower environmental impact compared to conven-
tional reactions in organic chemistry.4–7
Isolated enzymes and whole cells can be used as biocatalysts in
enantioselective reductions. Reductase enzymes require reduced
nicotinamide cofactors and when isolated enzymes are used, a sec-
ond catalytic cycle is necessary to regenerate cofactors and sustain
catalytic activity.4–7 So, whole cells are frequently preferred
because of their own cofactor regeneration system even if compet-
ing enzymes within cells could decrease stereoselectivity. Reac-
tions conditions and suitable microorganisms should be
investigated to improve yields.4–9
microorganism strains were employed in the asymmetric reduc-
tion of ethyl 3-oxohexanoate to ethyl (R)-3-hydroxyhexanoate
(Fig. 1). Some yeasts were also tested after immobilization in
calcium alginate spheres.
In the first step of this work, free cells of five yeasts
(Saccharomyces cerevisiae 40, Hansenula sp., Geotrichum candidum,
Kluyveromyces marxianus, and Rhodorotula rubra) and two filamen-
tous fungi (Trichoderma harzianum and Aspergillus niger) were
tested (Table 1).12 All the strains were able to catalyze the reaction
with excess of the (R)-hydroxyester (determined by the measure-
ment of optical rotation)13 with an excellent conversion level
(>99%) within 24 h. The enantiomeric excess varied from 9% (with
T. harzianum) to higher than 99% (with K. marxianus and A. niger).
Examples of chromatograms obtained are shown in Figure 2.
In previous studies, these microorganisms were successfully
used in the reduction of methyl acetoacetate,10 ethyl acetoace-
tate,2,10 ethyl 2-methylacetoacetate,10 ethyl benzoylacetate,8,11
and ethyl 4-chloroacetoacetate.9 However, excess of the
(S)-hydroxyester was obtained in almost all of the cases. Only K.
marxianus, T. harzianum and A. niger led to the excess of the
(R)-hydroxyester in the reduction of ethyl acetoacetate (67% ee,
51% ee, and 19% ee, respectively) and A. niger maintained the
In recent years we have been investigating the microbial
reduction of ketoesters.2,8–11 In the present work, seven wild-type
biocatalyst
O
O
OH O
O
O
⇑
Corresponding author. Tel.: +55 21 2562 7248.
E-mail address: joyce_benzaquem@yahoo.com.br (J.B. Ribeiro).
Figure 1. Bioreduction of ethyl 3-oxohexanoate to ethyl 3-(R)-hydroxyhexanoate.
0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2011.09.023

6128
A.S. Ramos et al. / Tetrahedron Letters 52 (2011) 6127–6129
Table 1
Bioreduction of ethyl 3-oxohexanoate (5 g/L) to ethyl (R)-3-hydroxyhexanoate by free
cells. Incubation: 30 °C, 150 rpm, 24 h
Microorganism
Conversiona (%)
ee (%)
Saccharomyces cerevisiae 40
Hansenula sp.
Geotrichum candidum
Kluyveromyces marxianus
Rhodotorula rubra
>99
>99
>99
>99
>99
>99
>99
97.5
87.2
85.7
>99
90
Trichoderma harzianum
Aspergillus niger
9
>99
a
Determined by GC analysis.
(R)-enantioselectivity in the reduction of methyl acetoacetate (45%
ee). So, enantioselectivity depends critically on the length of the
carbon chain, as also observed by other researchers.4,14
Some authors described such enantioselectivity and conversion
level employing recombinant microorganisms15 and isolated
enzymes.6,16,17 Rodríguez et al.15 showed that it was possible to
obtain both (S)- and (R)-hydroxyesters with higher than 98% ee
by using genetic engineering of baker’s yeast, although lower sub-
strate concentration (10 mM) had been used in comparison with
the present work (31 mM). Kaluzna et al.16 also achieved high
(S)-enantioselectivity (>99% ee) by using isolated S. cerevisiae
reductases overproduced in Escherichia coli, while Zhu et al.17 used
recombinant ketoreductases to reach both isomers with higher
than 99% ee. However, wild-type microorganism strains are gener-
ally preferred than recombinant strains because of their robust-
ness.2 Moreover, whole cells do not require the addition of
expensive cofactors, which is an advantage over the use of isolated
enzymes, besides the lower cost.18
Among the wild-type microorganisms, S. cerevisiae was most
frequently assayed in the reduction of ethyl 3-oxohexanoate.
North19 obtained higher than 98% ee of the (S)-hydroxyester and
conversion of 55% with S. cerevisiae in petrol; Rotthaus et al.20
employed S. cerevisiae in the reduction using different solvents
and obtained 100% conversion and 86% ee of the (R)-isomer when
the reaction was conducted in water. Rodriguez et al.15 achieved
98% ee of the same isomer when S. cerevisiae was used as the bio-
catalyst, substrate at 12.6 mM, and glucose or galactose as the car-
bon source. Dahl et al.21 achieved 99% ee of the (R)-hydroxyester
using baker’s yeast but they added allyl alcohol to the reaction
medium, which is a carcinogenic and volatile enzymatic inhibitor
not recommended for industrial processes.22 Buisson et al.22 de-
scribed that the yeast G. candidum afforded the same isomer with
98% ee and 90% of conversion within 24 h. In the present work,
as can be seen in Table 1, free cells of K. marxianus and A. niger car-
ried out in the (R)-hydroxyester production with more than 99% ee
and more than 99% conversion in medium containing substrate at
5 g/L or 31 mM.
In order to improve the method, some yeasts (Hansenula sp., K.
marxianus, and R. rubra) were immobilized in calcium alginate
spheres and tested for their ethyl (R)-3-oxohexanoate reduction
ability in two cycles (Table 2).23 This entrapment technique makes
the product recovery much easier and the biocatalyst can be read-
ily reused. However, the immobilization can influence enantio-
meric excess and conversion level.2,8,9,11,24,25
Figure 2. Typical chromatograms showing the selectivities achieved. Chiral GC
m), at 90 °C (23 min):
(A) ethyl 3-oxohexanoate (substrate); (B) ethyl 3-hydroxybutanoate (racemate
obtained via NaBH4 reduction); (C) Trichoderma harzianum reduction; (D) Hansen-
ula sp. reduction; (E) Kluyveromyces marxianus reduction. Retention times (tR): tR
(substrate) = 15.3 min, tR ((S)-enantiomer) = 18.8 min, tR ((R)-enantiomer) = 19.3 min.
analysis on column Beta DEX325 (30 m ꢀ 0.25 mm ꢀ 0.25
l
As shown in Table 2, the immobilized cells of K. marxianus
exhibited the best results because they led to the high enantiose-
lectivity (>99% ee) and conversion level (>99%) after immobiliza-
tion in the two cycles, with storage of 12 days between the
cycles. The yeast Hansenula sp. gave high conversion level in the
two cycles, but with decrease in enantioselectivity. In the first
use of immobilized cells of R. rubra, a little decrease was observed
in conversion level and, in the second cycle, still lower conversion
was obtained.
High substrate concentration is important for an industrial
biotransformation process24,26 but it can decrease conversion level
and enantioselectivity.27 This effect was previously observed in the
reduction of ethyl 4-chloroacetoacetate to ethyl (S)-4-chloro-3-
hydroxybutanoate by the immobilized cells of K. marxianus.9 In
the reactions catalyzed by whole cells, substrate toxicity is an
aggravating factor.28 Immobilization could reduce toxic effects by
a diffusion barrier that protects cells and it could allow the
addition of substrate at higher concentrations.24 To evaluate

A.S. Ramos et al. / Tetrahedron Letters 52 (2011) 6127–6129
6129
Table 2
11. Ribeiro, J. B.; Ramos, M. C. K. V.; Aquino Neto, F. R.; Leite, S. G. F.; Antunes, O. A.
C. Catal. Commun. 2005, 6, 131–133.
Bioreduction of ethyl 3-oxohexanoate (5 g/L) to ethyl (R)-3-hydroxyhexanoate by
immobilized cells. Incubation: 30° C, 150 rpm, 24 h
12. Microorganisms, media, growth conditions, and biotransformation with free
cells. Saccharomyces cerevisiae, Hansenula sp., Geotrichum candidum,
Kluyveromyces marxianus, Rhodotorula rubra, Aspergillus niger, and
Trichoderma harzianum belong to the collection of the ‘Departamento de
Engenharia Bioquímica, Escola de Química, Universidade Federal do Rio de
Janeiro (Cidade Universitária, CT Bloco E, Rio de Janeiro, Brazil, e-mail
selma@eq.ufrj.br)’ and are freely available upon request. Cells were allowed
to grow for 48 h, under 150 rpm at 30 °C in a medium containing 1% glucose,
0.5% yeast extract, 0.5% peptone, 0.1% (NH4)2SO4, and 0.1% MgSO4ꢁ7H2O. After
that period, they were harvested by centrifugation, re-suspended in water, and
used for the reaction. After centrifugation, the cells (4 g/L, dried weight) were
added to the reduction medium containing: glucose (5%), MgCl2 (0.1%) in a
final volume of 100 mL. After 30 min of addition of the microorganisms, the
substrate (0.5 g diluted in 1 mL of ethanol 96%) 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 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 Na2SO4), filtered, and concentrated under
vacuum. Conversions and enantiomeric excesses were determined by (chiral)
gas chromatography (GC), on column Beta Dex325 (30 m ꢀ 0.25 mm ꢀ
Microorganism
First cycle
Second cycle (after 12 d)
Conversiona (%)
ee (%)
Conversiona (%)
ee (%)
K. marxianus
Hansenula sp.
R. rubra
>99
>99
96.5
>99
75.1
80.6
>99
>99
61.1
>99
60.8
80.5
a
Determined by GC analysis.
whether substrate concentration could influence catalytic activity,
the cells of K. marxianus were immobilized again and used in the
reduction of ethyl 3-oxohexanoate at substrate concentrations of
7.5 g/L and 10 g/L. This biocatalyst also showed the same conver-
sion level (>99%) and enantioselectivity (>99% ee) observed before.
In conclusion, the filamentous fungus A. niger and the yeast K.
marxianus led to the high conversion of ethyl 3-oxohexanoate to
ethyl (R)-3-hydroxyhexanoate (>99%) with extremely high
enantioselectivity (>99% ee). To our knowledge, this is the first
Letter on the use of these two microorganisms in the reduction of
ethyl 3-oxohexanoate. The high conversion level, high enantio-
meric excess, tolerance to substrate and the possibility of immobi-
lization, and reuse make K. marxianus a promising biocatalyst for
industrial applications.
0.25 lm), at 90 °C (23 min). The elution order was: ethyl (S)-3-hydroxy-
hexanoate (tR = 18.8 min) followed by ethyl (R)-3-hydroxyhexanoate
(tR = 19.3 min). Substrate was eluted at 15.3 min. The reaction product was
characterized by nuclear magnetic resonance (NMR) and mass spectroscopy.
13.
½
a 2D5
ꢂ
–30.8° (c 1 g/100 mL, CHCl3); literature: ½a D25
–26.9° (c 1.14 g/100 mL,
ꢂ
CHCl3). Optical rotations were measured from CHCl3 solutions using a JASCO
DIP-370 polarimeter at the sodium
D line (589 nm) operating at room
temperature and compared to literature: Roche, C.; Desroy, N.; Haddad, M.;
Phansavath, P.; Genet, J. P. Org. Lett. 2008, 10, 3911–3914.
14. (a) Ema, T.; Moriya, H.; Kofukuda, T.; Ishida, T.; Maehara, K.; Utaka, M.; Sakai, T.
J. Org. Chem. 2001, 66, 8682–8684; (b) Ishihara, K.; Yamaguchi, H.; Nakajima, N.
J. Mol. Catal. B 2003, 23, 171–189.
15. Rodríguez, S.; Kayser, M. M.; Stewart, J. D. J. Am. Chem. Soc. 2001, 123, 1547–
1555.
Acknowledgments
16. Kaluzna, I. A.; Matsuda, T.; Sewell, A. K.; Stewart, J. D. J. Am. Chem. Soc. 2004,
126, 12827–12832.
17. Zhu, D.; Mukherjee, C.; Rozzell, J. D.; Kambourakis, S.; Hua, L. Tetrahedron 2006,
62, 901–905.
18. Yang, W.; Xu, J. H.; Xie, Y.; Xu, Y.; Zhao, G.; Lin, G. Q. Tetrahedron: Asymmetry
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Financial support from CAPES and CNPq-BRAZIL is acknowl-
edged. Analytical support from DQO and DQI-IQ-UFRJ is also
acknowledged.
19. North, M. Tetrahedron Lett. 1996, 37, 1699–1702.
20. Rotthaus, O.; Krüger, D.; Demuth, M.; Schaffner, K. Tetrahedron 1997, 53, 935–
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Supplementary data
21. Dahl, A. C.; Fjeldberg, M.; Madsen, J. Ø. Tetrahedron: Asymmetry 1999, 10, 551–
559.
22. Buisson, D.; Azerad, R.; Sanner, C.; Larchevêque, M. Tetrahedron: Asymmetry
1991, 2, 987–988.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.tetlet.2011.09.023.
23. Immobilization of cells (Kluyveromyces marxianus, Rhodotorula rubra, and
Hansenula sp.) in calcium alginate and biotransformation. Cells grown during
48 h in the medium described before12 were centrifuged and 0.4 g (dry weight)
was re-suspended in 3 mL of distilled water to obtain a cell-suspension. A 1.5%
sodium alginate aqueous solution (20 mL) was added and this mixture (cell-
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