Enantioselective desymmetrization of prochiral diesters catalyzed by immobilized Rhizopus oryzae lipase

Article abstract of DOI:10.1016/j.tetasy.2011.11.012

The asymmetric hydrolysis of dimethyl 3-phenylglutarate 1 catalyzed by different immobilized preparations of Rhizopus oryzae lipase (ROL) has been studied. The Lewatit CNP 105 commercial support was activated to aldehyde groups (Lewatit-aldehyde) and used

Full text of DOI:10.1016/j.tetasy.2011.11.012

Tetrahedron: Asymmetry 22 (2011) 2080–2084
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Tetrahedron: Asymmetry
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Enantioselective desymmetrization of prochiral diesters catalyzed
by immobilized Rhizopus oryzae lipase
Zaida Cabrera , Jose M. Palomo b,
a,
⇑
⇑
a
Escuela de Ingeniería Bioquímica. Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2950, Valparaíso, Chile
Departamento de Biocatálisis. Instituto de Catálisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The asymmetric hydrolysis of dimethyl 3-phenylglutarate 1 catalyzed by different immobilized prepara-
tions of Rhizopus oryzae lipase (ROL) has been studied. The Lewatit CNP 105 commercial support was
activated to aldehyde groups (Lewatit-aldehyde) and used as a support for the immobilization of ROL
using different strategies. Thus, the lipase immobilized in the presence of dithiothreitol at pH 7 (ROL-
Lew-pH 7) was the most enantioselective catalyst for the hydrolysis of 1 at pH 7 and 25 °C producing
the (R)-monoester with an E value of 4.2 (ee = 62%) whereas ROL immobilized at pH 10 gave only an E
value of 1.1 (ee = 4%). The medium engineering was also an interesting tool for improving the lipase
selectivity. The addition of a solvent, combined with decreasing temperature improved the E value for
the reaction from 4.2 to 24 (ee = 62 to ee = 92%). Finally, the application of the ROL-Lew-pH 7 preparation
in the presence of 20% dioxane and 5 °C allowed us to obtain the (R)-isomer of the monoester with an E
value of 24 (ee = 92%) in a 97% yield (of monoesters).
Received 19 October 2011
Accepted 22 November 2011
Available online 22 December 2011
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
Medium engineering has been used in many cases as a simple
and successful strategy to improve the features of these en-
8
–11
The development of new biocatalysts with high selectivity,
zymes.
Using this method, some additives, in particular deter-
activity, and stability for the application of different kinds of chem-
ical reactions is of great interest for the implementation of en-
zymes in industrial processes.
gents and solvents have been shown to modulate the properties of
lipases. Detergents may shift the equilibrium between closed and
open structures of the enzyme by coating the large hydrophobic
Currently, lipases are the enzymes mostly used in biocatalysis
pocket that surrounds the active center of lipases, thus greatly
1
,2
13–18
and organic chemistry. In particular, the use of lipases to cata-
lyze asymmetric reactions has attracted great interest3 due to
them being able to accept a broad range of substrates with good
activity, and in many cases high regio- and enantioselectivity and
altering the activity and enantiospecificity of the enzyme.
Sol-
–5
vents in turn, can promote the opening of the hydrophobic pocket
to strengthen the electrostatic interactions, which were often re-
1
3,19,20
lated to the solvent property of logP.
6
,7
specificity. However, when a lipase is used as a biocatalyst for
a given reaction, its application is often hampered by; (a) its diffi-
cult recovery and reuse; (b) its low selectivity toward non-natural
compounds; and (c) its low stability under processing conditions.
Thus, several strategies have been used to improve these draw-
backs. In this context, conformational engineering has been
described as a very interesting approach toward tuning a lipase’s
properties by using different immobilization protocols.8,9 Thus,
immobilization of a particular lipase by different orientations, by
different rigidity, or in the presence of different micro-environ-
ments allows us to generate different biocatalysts from the same
lipase with very different catalytic properties.1
Asymmetric hydrolysis of prochiral compounds, such as the
diesters of phenylglutaric acid, important building blocks for the
synthesis of several biologically active compounds (Scheme 1), is
a simple and noteworthy alternative for the production of chiral
2
1–23,4
compounds.
This strategy has the advantage of permitting
100% conversion to the desired compound, instead of the maxi-
mum 50% in the standard resolution of racemic mixtures. In this
reaction, neither the substrate (diester) nor the final products (dia-
cid) are chiral compounds. However, the monoester, an intermedi-
ate product, is chiral. If the reaction is stopped at the monoester
stage and the enantioselectivity of the process is very high, a
100% yield of an enantiomerically pure compound can be
0–12
2
4,25
produced.
Rhizopus oryzae lipase (ROL) has been broadly used in many bio-
transformations, such as esterifications and trans-esterifica-
⇑
Corresponding authors. Tel.: + 56 32 2274178; fax: +56 32 2273803 (Z.C.); tel.:
34 915854809; fax: +34 915854760 (J.M.P.).
E-mail addresses: zaida.cabrera@ucv.cl (Z. Cabrera), josempalomo@icp.csic.es
+
2
6–29
tions.
However, to date ROL has not been reported as an
(
J.M. Palomo).
enantioselective biocatalyst in asymmetric reactions. Therefore
0
957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.11.012

Z. Cabrera, J. M. Palomo / Tetrahedron: Asymmetry 22 (2011) 2080–2084
2081
O
Cl
S
O
O
N
S
N
-Cl +H3N
COOH
Me
(R)-baclofen
inhibitor HIV-1
Cl
Cl
F
OMe
O
O
O
H
N
O
N
H
(
+)-SCH 54016
(-)-paroxetine
Scheme 1. Biologically active compounds containing a 3-arylglutaric building block.
O
O
O
OCH2CH3
immobilized ROL
OCH CH
OH
OCH2CH3
OH
2
3
+
OCH CH3
2
O
O
O
(
S)-2
1
(R)-2
Scheme 2. Asymmetric hydrolysis of 1.
its use constitutes an important precedent for its application in
such reactions.
Table 1
Specific activity of different immobilized preparations in the hydrolysis of pNPB
Biocatalyst
Activitya
Recovered activityb (%)
Herein, we have studied the potential of ROL to catalyze enan-
tioselective asymmetric reactions, in particular, the hydrolysis of
dimethyl 3-phenylglutarate 1 (Scheme 2) catalyzed by three differ-
ent covalently immobilized preparations on a Lewatit-aldehyde
support.
ROL-Lew-pH 7
ROL-Lew-pH 7/pH 10
ROL-Lew-pH 10
22
13
11
75
50
40
a
ꢀ1
ꢀ1
Activity in
l
mol mg prot min
.
b
After the covalent immobilization.
2
2
. Results and discussion
.1. Immobilization and characterization of the immobilized
preparations
1
00
The immobilization on a Lewatit-aldehyde support, using differ-
ent protocols was studied.
9
5
The immobilization process was relatively quick, after 4 h the
activity of the supernatant was constant. However, the percentages
of activity recovered were quite different and dependent upon the
immobilization strategy used. For example, ROL-Lew-pH 7, recov-
ered 75% of the initial activity offered, while ROL-Lew-pH 7/pH
90
8
8
7
5
0
5
1
0 and ROL-Lew-pH 10 only recovered 50% and 40% of activity,
respectively (Table 1).
The stability of the different preparations immobilized of ROL in
the presence of 40% (v/v) of dioxane was also studied. Figure 1
shows that the stability was very similar for the three immobilized
preparations. However, the enzyme immobilized at pH 10 slightly
improved the enzyme stability compared to the enzyme immobi-
lized at pH 7.
70
0
10
20
30
40
Time (h)
50
60
70
80
Figure 1. Inactivation courses of ROL immobilized preparations on Lewatit-
aldehyde in the presence of 40% (v/v) co-solvent. ROL-Lew-PH 7 (square), ROL-
Lew-PH 7/pH 10 (asterisk), ROL-Lew-pH 10 (triangles).

2
082
Z. Cabrera, J. M. Palomo / Tetrahedron: Asymmetry 22 (2011) 2080–2084
Table 2
Selectivity of different ROL immobilized preparations in the hydrolysis of 1 at pH 7 and 25 °C
Biocatalyst
Activitya
Time (h)
Conversion (%)
eeb (%)
E value (R)/(S)
ROL-Lew-pH 7
ROL-Lew-pH 7/pH 10
ROL-Lew-pH 10
0.036
0.012
0.011
8
22
24
19
17
18
62
7
4
4.2
1.2
1.1
a
ꢀ1 ꢀ1
Activity in
l
mol mg prot
h .
b
Enantiomeric excess of monoester (R)-2 calculated at 15–20% conversion.
2
.2. Asymmetric hydrolysis of 1 catalyzed by different ROL
only reached a value of 6.1 (ee = 72%), (Table 3, entries 1, 3, 6, 9
and 12).
immobilized preparations
The reaction was also carried out in the presence of 40% solvent.
Here, the results were not higher than those previously obtained
(20% (v/v) solvent), although they were higher than those obtained
in the absence of the solvent (E = 4.2, ee = 62%), (Table 3, entries 5,
8, 11 and 14).
The activities and selectivities of the different ROL immobilized
preparations in the hydrolysis of 1 at pH 7 and 25 °C are shown in
Table 2. In all cases, the diester was recognized and the (R)-mono-
methyl ester was obtained as the main product.
When using the ROL-Lew-pH 7 immobilized preparation as the
catalyst, the reaction proceeded more rapidly (initial activity was
threefold higher) than using the ROL-Lew-pH 7/pH 10 or ROL-
Lew-pH 10 preparations (Table 2). The selectivity of ROL was
strongly affected by the immobilization protocol used. Thus, ROL-
Lew-pH 7/pH 10 and ROL-Lew-pH 10 immobilized preparations
exhibited very low enantiomeric ratio (E) values, E = 1.2 (ee = 7%)
and E = 1.1 (ee = 4%), respectively, although this value could be
greatly improved upon by using the ROL-Lew-pH 7 preparation
as the biocatalyst, reaching an E value of 4.2 (ee = 62%) (Table 2).
Thus, the ROL-Lew-pH 7 appeared to be the optimal immobi-
lized preparation exhibiting the best selectivity and activity during
the hydrolysis of 1. Thus, this immobilized preparation was used in
all subsequent experiments.
The enzymatic activity of the ROL immobilized preparation de-
creased in the presence of co-solvents, although it was more dra-
matic when 40% (v/v) of the solvent was used. Thus, the ROL
activity decreased by 70% with 20% (v/v) acetone while with that
obtained with 20% (v/v) of diglyme only 30% activity was lost (Ta-
ble 3, entries 6 and 9).
Detergents can in some cases alter the enantioselectivity of the
lipases. Thus, the effect of SDS, in the presence and absence of co-
solvents on the asymmetric reaction was studied (SDS showed
very good results with the soluble lipase, results not shown). The
enantioselectivity of the ROL immobilized preparation was only
slightly influenced by the presence of SDS, with E values ranging
from 4.2 (ee = 62%) to 5.4 (ee = 69%) without co-solvent (Table 3,
entries 1 and 2) or from 6.1 (ee = 72%) to 7.4 (ee = 76%) with 20%
(
v/v) of DMSO (Table 3, entries 12 and 13). The enzymatic activity
2
7
.2.1. Influence of the experimental conditions on ROL-Lew-pH
in the asymmetric hydrolysis of 1
In order to optimize the selectivity of the reaction, the enzy-
in the presence of a detergent was significantly lower (Table 3).
Finally, the influence of temperature on the activity and stere-
oselectivity of the ROL immobilized preparation was evaluated
(Table 4). Thus, when the reaction was conducted at 5 °C, the
enantioselectivity of the ROL-Lew-pH 7 immobilized preparation
was improved from E = 4.2 (ee = 62%) at 25 °C to E = 5.4
(ee = 69%) at 5 °C (Table 3, entry 1 and Table 4, entry 1).
When the reaction was catalyzed by ROL preparation in the
presence of 20% (v/v) of diglyme, the E value also improved from
9 (ee = 80%) to 12.3 (ee = 85%) when the temperature was de-
creased from 25 to 5 °C (Table 3, entry 6 and Table 4, entry 2).
However, the best result was found when the reaction was per-
formed in the presence of 20% dioxane and at 5 °C where E = 24
matic hydrolysis was performed under different conditions. In all
cases, the main product was the (R)-monoester. The enantioselec-
tivity of the enzyme was strongly altered by the addition of some
organic solvents and additives (Table 3). The presence of 20% (v/v)
of diglyme, acetone, or dioxane, as co-solvent in the reaction media
significantly improved the selectivity of immobilized preparation
with an E value of 4.2 (ee = 62%) in the absence of solvent, which
increased to more than 9 (ee = 80%) when the aforementioned sol-
vent was used. However, in the case of DMSO, enantioselectivity
(
ee = 92%) was achieved (Table 4, entry 3). The selectivity of the
immobilized preparation in the presence of acetone could not be
quantified due to the long reaction time required (results not
shown).
Table 3
Selectivity of ROL-Lew-pH 7 in the presence of different concentrations of solvent and
additives in the hydrolysis of 1 at pH 7 at 25 °C
Under these conditions, the enzymatic activity of the immobi-
lized preparation decreased considerably. However, the ROL
immobilized preparation at 5 °C and in the presence of dioxane
maintained more than 60% of the activity value of that at 5 °C
and without solvent; these were the best conditions to perform
the asymmetric hydrolysis of 1 (Table 4).
Entry Co-solvent (%) Additive
Activitya eeb (%) E value (R)/(S)
1
2
3
4
5
6
7
8
9
0
1
2
3
4
—
—
0.036
0.05% SDS 0.008
0.017
0.05% SDS 0.009
0.004
62
69
80
77
72
80
81
83
80
nd
78
72
76
74
4.2
5.4
9.0
7.7
6.1
9.0
9.5
10.8
9.0
Dioxane
Dioxane
Dioxane
Diglyme
Diglyme
Diglyme
Acetone
Acetone
Acetone
DMSO
DMSO
DMSO
20
20
40
20
20
40
20
20
40
20
20
40
0.025
0.05% SDS 0.013
0.004
Table 4
0.01
Selectivity of ROL-Lew-pH 7 in the presence of different solvents (20% v/v) in the
hydrolysis of 1 at 5 °C and pH 7
0.05% SDS ndc
0.003
c
nd
c
1
1
1
1
1
8.1
6.1
7.4
6.7
Entry
Co-solvent
Activitya
eeb (%)
E value (R)/(S)
0.026
0.05% SDS 0.022
0.005
1
2
3
—
0.012
0.005
0.008
69
85
92
5.4
12.3
24.0
Diglyme
Dioxane
a
b
c
ꢀ1 ꢀ1
Activity in
lmol mg prot
h .
a
ꢀ1 ꢀ1
Enantiomeric excess of (R)-2 calculated at 15–20% conversion.
nd: not determined.
Activity in
l
mol mg prot
h
.
b
Enantiomeric excess of (R)-2 calculated at 15–20% conversion.

Z. Cabrera, J. M. Palomo / Tetrahedron: Asymmetry 22 (2011) 2080–2084
2083
2
.2.2. Effects of co-solvents on the stability of ROL-Lew-pH 7
The stability of the immobilized preparation ROL-Lew-pH 7 in
3. Conclusion
the presence of 40% of different solvents (acetone, dioxane and dig-
lyme) for long incubation times was studied. In all cases, the
immobilized preparation was highly stable, retaining over 80% of
its activity after 15 days (Fig. 2).
The results reported herein suggest that R. oryzae lipase immo-
bilized on Lewatit-aldehyde has an interesting potential to catalyze
enantioselective asymmetric reactions and that the presence of
solvents can significantly improve the enantioselectivity of the
immobilized preparation. Thus, in the presence of dioxane and at
a low temperature, it was possible to obtain the (R)-methyl-3-phe-
nylglutarate with an E value of 24 (ee = 92%) and with a yield in
monoester of 97%. The high stability of the preparation immobi-
lized in the presence of an organic solvent allows the reuse of
the biocatalyst for at least two cycles, while maintaining the selec-
tivity and more than 85% of the activity.
100
9
9
8
8
7
7
5
0
5
0
5
0
4
4
. Experimental
.1. General
0
50
100
150
200
Time (h)
250
300
350
400
R. oryzae lipase (ROL), p-nitrophenyl butyrate (pNPB), DL-dithio-
threitol (DTT), dimethyl 3-phenylglutarate 1, bis(2-methoxyethyl)
ether (diglyme), dioxane, acetone and dimethyl sulfoxide (DMSO)
were obtained from Sigma. Lewatit CNP 105 was obtained from
Lanxess. Lewatit-aldehyde support was prepared as previously de-
Figure 2. Inactivation courses of ROL immobilized on Lewatit-aldehyde at pH 7 in
the presence of 40% solvent. dioxane (triangles), acetone (squares), diglyme
(
asterisk).
3
0
scribed with minor modifications. Other reagents were of analyt-
ical or HPLC grade.
2
.2.3. Reuse of ROL-Lew-pH 7 biocatalyst in the asymmetric
hydrolysis of 1
4
4
.2. Methods
Under the optimal conditions, the total conversion of the sub-
strate was performed and the reuse of the biocatalyst was evalu-
ated. The reaction course and evolution of the selectivity of ROL
immobilized preparation is shown in Figure 3. The selectivity of
ROL-Lew-pH 7 was not altered over the course of the reaction
and an E value >20 (ee >90%) was maintained until the end of
the process. Another excellent result was the final yield, with
.2.1. Lipase activity determination
Activity assay was performed by measuring the increase in
absorbance at 348 nm produced by the releasing of p-nitrophenol
in the hydrolysis of 0.4 mM p-nitrophenyl butyrate in 25 mM so-
dium phosphate at pH 7 and 30 °C, using a thermostatized spectro-
photomer with magnetic stirring. To initialize the reaction, 10 mg
of immobilized enzyme were added to 10 mL of substrate solution.
An international unit of pNPB activity is defined as the amount of
9
7% of 1 being transformed into 2 with 100% conversion.
1
9
00
100
90
80
70
60
50
40
30
20
10
0
enzyme necessary to hydrolyze 1
the conditions described above.
lmol of pNPB/min (IU) under
0
0
0
0
0
0
0
0
0
0
8
7
6
5
4
3
2
1
4
.2.2. Immobilization of ROL on Lewatit-aldehyde support
The different ROL immobilized preparations were prepared fol-
lowing the procedures previously described with minor
3
1
modifications.
(
a) Immobilization at pH 7 (ROL-Lew-pH 7): One gram of sup-
port was added to 10 mL of 25 mM sodium phosphate pH
7, containing 2 mg of protein/mL, in the presence of
50 mM DTT and 0.05% Triton X-100 and kept under gentle
stirring at 25 °C, for 4 h. Immobilization was through the ter-
minal amino group of ROL.
0
5
10
Time (d)
15
20
(
b) Immobilization at pH 7/pH 10 (ROL-Lew-pH 7/pH 10): After
the enzyme immobilization at pH 7 for 4 h, the pH was
adjusted to 10.05 using 1 M sodium bicarbonate and kept
under gentle stirring at 25 °C, for 2 h. ROL was immobilized
by terminal amino group orientation and finally covalent
attachment between lysines on the enzyme and aldehydes
on the support.
Figure 3. Course of the asymmetric hydrolysis of 1 catalyzed by ROL-Lew-PH 7.
Yield (circles), ee (triangles).
At the end of the reaction, the immobilized preparation con-
served over 90% of its initial activity, which permits the realization
of a second production cycle.
When a second cycle was performed, the results obtained were
very similar to those of the first production cycle. Thus, a yield of
over 95% was obtained, while the asymmetry slightly decreased,
E = 19 (ee = 90%). The activity after a second reaction cycle was
over 85% of its initial activity. These results suggest that the immo-
bilized enzyme preparation could be reused for three or more pro-
duction cycles.
(c) Immobilization at pH 10 (ROL-Lew-pH 10): One gram of
support was added to 10 ml of 100 mM sodium bicarbonate
pH 10.05, containing 2 mg of protein/mL, in the presence of
0.05% Triton X-100 and kept under gentle stirring at 25 °C,
for 4 h. ROL was immobilized directly via covalent attach-
ment by the richest area in lysine and aldehydes on the
support.

2
084
Z. Cabrera, J. M. Palomo / Tetrahedron: Asymmetry 22 (2011) 2080–2084
In all cases, the activities of the supernatants were periodically
Acknowledgements
taken, and the activities were assayed by the method described
above. Finally, 10 mg of sodium borohydride were added under
This work has been sponsored by Fondecyt, Chile (Proyect
Fondecyt iniciation 11090321) and CSIC (Intramural Project
200980I133). We also thank the PUCV and Programa Bicentenario
de Ciencia y Tecnología (PBCT, Project PSD-081/2009). The help
and suggestions of Dr. Ángel Berenguer-Murcia (Instituto de Mate-
riales, Universidad de Alicante, Spain) are gratefully recognized.
32
gentle stirring at 25 °C to reduce the imino groups.
0 min, the immobilized enzyme was filtered by vacuum and
washed several times with an excess of distilled water.
After
3
4
.2.3. Inactivation of different ROL preparations
ROL immobilized preparations were incubated in the presence
of different solvents. At different times, samples were withdrawn
and washed 5 times with water. Finally, the residual activity was
measured as described previously.
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.2.5. Reuse of ROL-Lew-pH 7 biocatalyst in the asymmetric
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n½R ꢀ 2ꢂ þ n½S ꢀ 2ꢂ
yield ¼
;
n ¼ enantiomerðmolÞ:
n½1ꢂ
4
0, 997–1000.
2
5. Fern a´ ndez-Lorente, G.; Palomo, J. M.; Cabrera, Z.; Guisan, J. M.; Fern a´ ndez-
Lafuente, R. Enzyme Microb. Technol 2007, 41, 565–569.
4
.2.6. Determination of enantiomeric excess
The enantiomeric excess (ee) of the monoester formed was ana-
26. Haalck, L.; Paltauf, F.; Pleiss, J.; Schmid, R. D.; Spener, F.; Stadler, P. Methods
Enzymol. 1997, 284, 353–376.
27. Hiol, A.; Jonzo, M. D.; Rugani, N.; Druet, D.; Sarda, L.; Comeau, L. C. Enzyme
lyzed by Chiral Reverse Phase HPLC. The column was a Chiracel
OD-R. The mobile phase was acetonitrile (25% v/v) and 10 mM
ammonium phosphate (75% v/v) at pH 3 and the analyses were
performed at a flow of 0.7 mL/min by recording the absorbance
at 225 nm. The enantiomeric excesses were determined at 10–
Microb. Technol. 2000, 26, 421–430.
28. Li, W.; Li, R.-W.; Li, Q.; Du, W.; Liu, D. Process Biochem. 2010, 45, 1888–1893.
2
9. Ghamgui, H.; Karra-Chaâbouni, M.; Gargouri, Y. Enzyme Microb. Technol. 2004,
5, 355–363.
0. Guisan, J. M. Enzyme Microb. Technol. 1988, 10, 375–382.
3
3
31. Bolivar, J. M.; López-Gallego, F.; Godoy, C.; Rodrigues, D. S.; Rodrigues, R. C.;
Batalla, P.; Rocha-Martín, J.; Mateo, C.; Giordano, R. L. C.; Guisan, J. M. Enzyme
Microb. Technol. 2009, 45, 477–483.
2. Mateo, C.; Abian, O.; Bernedo, M.; Cuenca, E.; Fuentes, M.; Fernández-Lorente,
G.; Palomo, J. M.; Grazu, V.; Pessela, B. C. C.; Giacomini, C.; Irazoqui, G.;
Villarino, A.; Ovsejevi, K.; Batista-Viera, F.; Fernández-Lafuente, R.; Guisan, J. M.
Enzyme Microb. Technol. 2005, 37, 456–462.
1
5% conversion. The calculation of the ee and enantiomeric ratio
(E) was performed using the following equations:
3
n½Rꢂ ꢀ n ½Sꢂ
n½Rꢂ þ n½Sꢂ
n½Rꢂ ;
eeð%Þ ¼ ½
ꢂ ꢁ 100; E ¼
n ¼ enantiomerðmolÞ:
n½Sꢂ