Improving the toolbox of bioreductions by the use of continuous flow systems

Article abstract of DOI:10.5935/0103-5053.20150009

Packed bed reactors can be used as an interesting alternative on the bioreduction of β-ketoesteres mediated by immobilized microorganisms. Here in, we report our results on the bioreduction of ethyl 3-oxohexanoate by immobilized Kluyveromyces marxianus cells and tert-butyl 3-oxobutanoate by immobilized Rhodotorula rubra cells under continuous flow conditions leading the desired β-hydroxy esters corresponding in high yields and enantiomeric excess.

Full text of DOI:10.5935/0103-5053.20150009

http://dx.doi.org/10.5935/0103-5053.20150009
J. Braz. Chem. Soc., Vol. 26, No. 3, 550-554, 2015.
Printed in Brazil - ©2015 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
A
Improving the Toolbox of Bioreductions by the Use of Continuous Flow Systems
Raquel O. Lopes,a Simon Grimm,a Joyce B. Ribeiro,a Ivana C. R. Leal,b
Leandro S. M. Mirandaa and Rodrigo O. M. A. de Souza*,a
aLaboratório de Biocatálise e Síntese Orgânica, Instituto de Química and
bFaculdade de Farmácia, Universidade Federal do Rio de Janeiro,
22941-909 Rio de Janeiro-RJ, Brazil
Packed bed reactors can be used as an interesting alternative on the bioreduction ofβ-ketoesteres
mediated by immobilized microorganisms. Here in, we report our results on the bioreduction of ethyl
3-oxohexanoate by immobilized Kluyveromyces marxianus cells and tert-butyl 3-oxobutanoate
by immobilized Rhodotorula rubra cells under continuous flow conditions leading the desired
β-hydroxy esters corresponding in high yields and enantiomeric excess.
Keywords: whole cell bioreduction, packed bed reactor, continuous flow, and asymmetric
reduction.
Introduction
catalyzed by whole cells microorganisms usually have long
reactions times, high diluted media needed and sensibility
to harsh reaction conditions (Figure 1).
Enantiosselective reduction reaction is an important
transformation in organic synthesis to afford enantiomerically
pure compounds used as synthons for important applications
such as pharmaceutical intermediates. Different enzyme types
can catalyze these reactions with high enantioselectivity,
providing a good alternative to chemical catalysis.1
Bioreductions have already been studied for several
years and recently, due to the advent of recombinant
DNA technology, more easily introduced in many
industrial processes for the synthesis of chiral alcohols,
a and β-hydroxy acids and aminoacids.2-4 In general, the
enzymes that catalyze such reductions need the presence
of a cofactor. Today, the developments of more efficient
coupled systems for cofactor regeneration have upgraded
the status of such methodology because the feasibility of
the entire process depends on the efficient and economic
supply of the expensive nicotinamide cofactor NAD(P)H
which is required in stoichiometric amounts.3,4
Whole
cell
Substrate
Product
NAD(P)+
Glucose
NAD(P)H
Metabolities
Figure 1. Whole cell biotransformation.
The use of packed bed reactors on biocatalyzed
reactions or biotransformations is not new, but just a few
examples can be found over literature about the use of this
approach to the bioreduction of ketones.7-12 In addition
to the stated in the previous paragraphs concerning the
use of whole cell, a key issue when working with these
cells in packed bed reactors is the immobilization of the
microorganism since the use of the free cell has several
disadvantages, being the reduced recyclability of the
system the most important one. The most simple and
efficient way of immobilizing microorganisms is the
calcium alginate entrapment.13
Alternatively, the use of whole cells does not require
cofactor regeneration but some improvements are still
necessary. In this type of methodology, the living organism
can metabolize substrate or products, the existence of
parallel competing metabolic pathways is an issue, and
the selective activation of the enzymatic system for the
desired reaction must be reached.5,6 Besides that, reactions
Recently, the continuous flow approach has attracted
much attention from the organic chemistry community
and in our continuous efforts towards the development of
continuous flow biocatalyzed process here in we report the
*e-mail: souzarod21@gmail.com

Vol. 26, No. 3, 2015
Lopes et al.
551
results on the bioreduction of acetoacetates derivatives for
the production of chiral alcohols.14-23
GC-MS analysis
Ethyl 3-oxohexanoate (1): conversions and
enantiomeric excesses (e.e.) were determined by (chiral)
gas chromatography (GC), on column Beta Dex325
(30 m × 0.25 mm × 0.25 µm) at 90 °C (23 min). The
elution order was: ethyl (S)-3-hydroxyhexanoate (3b)
(Rt = 18.8 min) followed by ethyl (R)-3-hydroxyhexanoate
(3a) (Rt = 19.3 min). Substrate (1) was eluted at 15.3 min.
The reaction product was characterized by NMR.
tert-Butyl 3-oxobutanoate (2): conversions and
enantiomeric excesses were determined by (chiral)
gas chromatography (GC), on column RTx-5MS
(30 m × 0.25 mm × 0.25 µm) at 90 ºC (11 min). The
elution order was: substrate (2) (Rt = 8.1 min), tert-butyl
(S)-3-hydroxybutanoate (4a) (Rt = 8.6 min) and tert-butyl
(R)-3-hydroxybutanoate (4b) (Rt = 9.0 min). The reaction
product was characterized by NMR.
Experimental
Materials
Ethyl3-oxo-hexanoate(1)andtert-butyl3-oxobutanoate(2)
were purchased from Sigma and racemates were obtained by
NaBH4 reduction. The products were analyzed by 1H and 13C
nuclear magnetic ressonance (NMR).
NMR analysis
rac-Ethyl 3-hydroxyhexanoate (3): 1H NMR (200 MHz,
CDCl3, TMS) δ (ppm) 0.92 (t, 3H, H6), 1.26 (t, 3H,
COOCH2CH3), 1.43 (m, 4H, H4 and H5), 2.44 (m,
2H, H2), 3.15 (s, 1H, OH), 3.99 (m, 1H, H3), 4.16 (m,
2H, COOCH2CH3); 13C NMR (50 MHz, CDCl3, TMS)
δ (ppm) 14.1 (C6), 14.3 (COOCH2CH3), 18.8 (C5), 38.8
(C4), 41.5 (C2), 60.8 (COOCH2CH3), 68.0 (C3), 173.3
(COOCH2CH3); DEPT 135 (50 MHz, CDCl3, TMS)
δ (ppm) 14.1 (C6), 14.3 (COOCH2CH3), 18.8 (C5), 38.8
(C4), 41.5 (C2), 60.8 (COOCH2CH3), 68.0 (C3).
Optical rotation
25
Ethyl (R)-3-hydroxyhexanoate (3a): [a]D –30.8°
25
(c 1 g per 100 mL, CHCl3); literature: [a]D –26.9°
(c 1.14 g per 100 mL, CHCl3).24 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.24
1
rac-tert-Butyl 3-hydroxybutanoate (4): H NMR
(200 MHz, CDCl3, TMS) δ (ppm) 1.20 (d, 3H, J 6 Hz, H4),
1.74 (s, 9H, COOC(CH3)3), 2.39 (d, 2H, J 4 Hz, H2), 3.17
(s, 1H, OH), 4.15 (m, 1H, H3); 13C NMR (50 MHz, CDCl3,
TMS) δ (ppm) 22.5 (C4), 28.3 (COOC(CH3)3), 44.0 (C2),
64.5 (C3), 81.4 (COOC(CH3)3), 172.6 (COOC(CH3)3);
DEPT 135 (50 MHz, CDCl3, TMS) δ (ppm) 22.5 (C4),
28.3 (COOC(CH3)3), 44.0 (C2), 64.5 (C3).
25
tert-Butyl (S)-3-hydroxybutanoate (4a): [a]D +33.5°
(c 1.64 g per 100 mL, CHCl3, 95% optical purity); literature:
25
[a]D +26.3° (c 1.64 g per 100 mL, CHCl3).25 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.25
Microorganisms
Continuous flow reaction procedure
Media, growth conditions and immobilization of cells
in calcium alginate. Kluyveromyces marxianus belong
to the collection of the ‘Departamento de Engenharia
Bioquímica, Escola de Química, UFRJ’. Cells were
allowed to grow for 48 hours, under 150 rpm and 30 °C
in a medium containing 1% glucose, 0.5% yeast extract,
0.5% peptone, 0.1% (NH4)2SO4 and 0.1% MgSO4.7H2O.
After that period, the cells were centrifuged and 0.6 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-suspension sodium alginate aqueous solution) was
dropped in a CaCl2 aqueous solution (0.1 mol L-1) to
form calcium alginate spheres. Spheres were filtered and
washed with distilled water. Then, the column was filled
with these spheres.
A solution of 5 g of glucose in 100 mL of distilled water
was prepared and ethyl 3-oxohexanoate (1) was added to
the solution (0.013 mol L-1 or 2 g L-1; 0.019 mol L-1 or
3 g L-1; 0.025 mol L-1 or 4 g L-1; 0.032 mol L-1 or 5 g L-1).
The starting mixture was stirred for 5 min, while the Asia
(Syrris) instrument was equipped with the packed bed
reactor containing immobilized cells from Kluyveromyces
marxianus (volume: 12.3 mL). The temperature (30 °C)
and 2 mL min-1 flow rate were selected on the flow reactor,
and processing was started, whereby only pure solvent
(glucose 5%) was pumped through the system until the
instrument had achieved the desired reaction parameters
and stable processing was assured.After that, the substrate
was added to the glucose’s solution and it was passed
through the column with recycle. Aliquots were collected

552
Improving the Toolbox of Bioreductions by the Use of Continuous Flow Systems
J. Braz. Chem. Soc.
in different times. The reaction mixture was extracted with
ethyl acetate. The organic phase was dried (anhydrous
Na2SO4), filtered, and concentrated under vacuum. Products
were analyzed by (chiral) gas chromatography (GC). Asia
Flow Reactor: a solution of 5 g of glucose in 100 mL of
distilled water was prepared and the β-ketoester [ethyl
3-oxohexanoate (1) or tert-butyl 3-oxobutanoate (2)]
were added to the solution (0.025 mol L-1 or 4 g L-1). The
starting mixture was stirred for 5 min while the instrument
Asia Flow Reactor was equipped with Omnifit column
(volume: 12.3 mL) containing the immobilized cells
from Kluyveromyces marxianus [for the bioreduction of
ethyl 3-oxohexanoate (1)] and Rhodotorula rubra [for
the bioreduction of tert-butyl 3-oxobutanoate (2)]. The
temperature (30 ºC) was selected on the flow reactor
and for each flow tested (0.2 mL min-1, 0.1 mL min-1
and 0.075 mL min-1), first only the pure solvent (glucose
5%) was pumped through the system. At this point,
the reaction mixture [ethyl 3-oxohexanoate (1) or
tert-butyl 3-oxobutanoate (2)] was pumped through the
system and aliquots were collected in different times
depending on the flow rate tested (0.2 mL min-1 = 62 min;
0.1 mL min-1 = 123 min; 0.075 mL min-1 = 164 min). The
reaction mixture was extracted with ethyl acetate. The
organic phase was dried (anhydrous Na2SO4), filtered, and
concentrated under vacuum. Products were analyzed by
(chiral) gas chromatography (GC).
Figure 2. Packed bed reactor working on a loop system.
Table 1. Bioreduction of ethyl 3-oxohexanoate (1) to ethyl (R)-3-
hydroxyhexanoate (3a) by immobilized K. marxianus cells
Concentration / (g L-1)
Conversion / %
e.e. / %
> 99 (R)
> 99 (R)
> 99 (R)
> 99 (R)
2
3
4
5
> 99
94
91
59
Reaction conditions: a solution of 5 g of glucose in 100 mL of distilled
water was prepared and ethyl 3-oxohexanoate (1) was added to the
solution (0.2; 0.3; 0.4 or 0.5 g). Conditions of flow reactor: volume of the
column (12.3 mL); temperature (30 °C); flow rate (2 mL min-1); pressure
(10 bar); time (24 h).
Results and Discussion
Based on previous results obtained by our group on the
bioreduction of β-ketoester under batch conditions using
K. marxianus and Rhodotorula rubra cells,26,27 we decided
to move forward towards a continuous flow methodology
for such transformation. For such, microorganisms were
immobilized on calcium alginate and packed on a glass
column for the bioreduction of ethyl 3-oxohexanoate (1)
and tert-butyl 3-oxobutanoate (2) under continuous flow
conditions.
We began our work evaluating the effect of substrate
concentration on the bioreduction of ethyl β-ketoester (1)
and the results are shown in Table 1. In this first evaluation
the packed bed worked in a loop system, the final operation
time was set to 24 hours and the substrates pumped at a rate
of 2 mL min-1 (Figure 2). This setup was chosen for in a
previous report from our group on the bioreduction under
batch conditions it demonstrated to be a very low process.
In general, the substrate concentration affects the
conversion of the bioreduction process. The enantiomeric
excess was up to 99% in all concentrations tested, however
the conversion decreased drastically with concentrations
higher than 4 g L-1 and the optimal substrate concentration
was found to be in the range between 2-4 g L-1.
When working on the loop system, we have followed
the reaction conversion through time and observed that
in order to achieve high conversions, starting material
solutions needs to pass through the packed bed for several
times (Figure 3) since at 2 mL min-1 the residence time
is very short (ca. 6 minutes). As shown in Figure 3,
with substrate concentration 4 g L-1, after approximately
24 hours recirculating the solution through the system, the
conversion arrives at the highest value (91%).
Aiming to obtain higher productivity and a more strain
forward strategy towards the desired alcohol, we decided
to reduce the flow rates used in the previous experiments
and try to work in a system without the loop which means
that the starting solution will pass through the packed bed
reactor only once. Different flow rates were tested in order
to find the best reaction condition, in Table 2 are shown the
best results obtained. In this study we decided to use the

Vol. 26, No. 3, 2015
Lopes et al.
553
Table 2. Bioreduction of ethyl 3-oxohexanoate (1) by immobilized K. marxianus cells and tert-butyl 3-oxobutanoate (2) by immobilized Rhodotorula
rubra cells
β-Ketoester
Flow rate / (mL min-1)
Conversion / %
e.e. / %
> 99 R
> 99 R
> 99 R
94 S
0.2
0.1
58
86
98
35
57
82
Ethyl 3-oxohexanoate (1)
0.075
0.2
tert-Butyl 3-oxobutanoate (2)
0.1
96 S
0.075
97 S
Reaction condition: a solution of glucose 5% in distilled water was prepared and the substrate (1 or 2) was added to the solution (0.4 g L-1). Conditions
of flow reactor: volume of the column (12.3 mL); temperature (30 °C); pressure (10 bar); residence time: 0.2 mL min-1 (62 min), 0.1 mL min-1 (123 min),
0.075 mL min-1 (164 min).
100
immobilized cells of K. marxianus or Rhodotorula rubra
90
was reported under continuous flow conditions. The reaction
80
time was reduced significantly compared to the batch process
70
60
and high yields of the corresponding β-hydroxyesthers in
50
concentrations up to 4 g L-1. High enantiomeric excess was
40
30
20
10
0
observed and the absolute stereochemistry could be controlled
varying β-ketoester structure and the microorganism
immobilized.
0
2
4
6
8
10
12
14
16
18
20
22
24
time / h
Supplementary Information
Figure 3. Conversion × time, on reaction loop system. Bioreduction
of ethyl 3-oxohexanoate (1) to ethyl (R)-3-hydroxyhexanoate (3a) by
immobilized K. marxianus cells. Reaction conditions: a solution of
5 g of glucose in 100 mL of distilled water was prepared and ethyl
3-oxohexanoate (1) was added to the solution (0.4 g). Conditions of flow
reactor: volume of the column (12.3 mL); temperature (30 °C); flow rate
(2 mL min-1); pressure (10 bar).
Supplementary data are available free of charge at
http://jbcs.sbq.org.br as PDF file.
Acknowledgements
4 g L-1 starting material solution in order to maximize the
amount of product formed.
As shown in Table 2, increasing residence times (lower
flow rate) lead to an almost quantitative reduction of the
ethyl 3-oxohexanoate (1) with high e.e., while the tert-
butyl 3-oxobutanoate (2) reach’s 80% of conversion with
also high e.e.
We thanks CAPES (Coordenação deAperfeiçoamento
de Pessoal de Nível Superior), CNPq (Conselho Nacional
de Desenvolvimento Científico e Tecnológico), FAPERJ
(Fundação Carlos Chagas Filho de Amparo a Pesquisa
do Estado do Rio de Janeiro) and FINEP (Agência
Financiadora de Estudos e Projetos) for financial
support.
Conclusions
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