Enzymatic resolution of (±)-glycidyl butyrate in aqueous media. Strong modulation of the properties of the lipase from Rhizopus oryzae via immobilization techniques

Article abstract of DOI:10.1016/j.tetasy.2004.03.003

The enantioselectivity of the lipase from Rhizopus oryzae for the small yet highly interesting (±)-glycidyl butyrate have been greatly modulated by the use of different immobilization techniques. Thus, the enzyme immobilized by interfacial adsorption on an octyl-agarose support presented a very low enantioselectivity (E=2). This value could be improved up to an E=17 by covalently immobilizing the enzyme on the cyanogen bromide agarose. However, the best results were achieved by immobilizing the enzyme via ionic adsorption on supports coated with dextran sulfate, with an E value of 51 (ee99% at 55% conversion).

Full text of DOI:10.1016/j.tetasy.2004.03.003

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 15 (2004) 1157–1161
Enzymatic resolution of (± ±)-lyciꢀyl butyrate in aqueous meꢀia.
Stron- moꢀulation of the properties of the lipase from
Rhizopus oryzae via immobilization techniques
Jose M. Palomo, Rosa L. Segura, Gloria Fernandez-Lorente, Jose M. Guis ꢀa n*
and Roberto Fernandez-Lafuente*
Departamento de Biocat ꢀa lisis, Instituto de Cat ꢀa lisis (CSIC), Campus UAM, Cantoblanco, 28049 Madrid, Spain
Received 19January 2004; revised 17 February 2004; accepted 2 March 2004
Abstract—The enantioselectivity of the lipase from Rhizopus oryzae for the small yet highly interesting (ꢀ)-glycidyl butyrate have
been greatly modulated by the use of different immobilization techniques. Thus, the enzyme immobilized by interfacial adsorption
on an octyl-agarose support presented a very low enantioselectivity (E ¼ 2). This value could be improved up to an E ¼ 17 by
covalently immobilizing the enzyme on the cyanogen bromide agarose. However, the best results were achieved by immobilizing the
enzyme via ionic adsorption on supports coated with dextran sulfate, with an E value of 51 (ee >99% at 55% conversion).
ꢀ
2004 Elsevier Ltd. All rights reserved.
1
. Introꢀuction
1). This compound is a structurally unique triterpene
alcohol and thought to be a defensive allomone of
P. testudinarius, because it is ichthyotoxic against the
fish, Gambusia affinis. Another example is the use of (R)-
1 as the starting material in the synthesis of enantio-
Enantiomerically pure epoxy alcohols and their esters
are very attractive intermediates for the preparation of
optically active beta-blockers and a wide range of other
1
3
products. In this way, (R)-glycidol 1 has been used as a
2
merically pure amino-anhydro-alditols (Scheme 1).
These are a class of compound used as monosaccharide
synthon in the synthesis of (þ)-testudinariol A (Scheme
H
H
O
O
H
H
OH
HO
O
H
Testudinariol A
OH
(
R)-Glycidol 1
TrO
OR
NH2
O
NH2
O
OH
OH
amino-1,4-anhydro-ara
bino-pentitol
amino-1,5-anhydro-3,4-dide
oxy-arabino-hexitol
Scheme 1. Drugs synthesized from (R)-glycidol.
*
0
Corresponding authors. Tel.: +34-91-585-48-09; fax: +34-91-585-47-60; e-mail addresses: jmguisan@icp.csic.es; rfl@icp.csic.es
957-4166/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.03.003

1158
J. M. Palomo et al. / Tetrahedron: Asymmetry 15 (2004) 1157–1161
4
14
mimetic structures and amino acid surrogates, as well
as the precursor of several organic molecules. In this
sense, it has been used as the key intermediate in the
conformation, where the active site is secluded from
the reaction medium by an oligopeptide chain named as
‘lid’, in a certain equilibrium with an open and active
conformation, where the lid is displaced permitting the
accessibility of substrates to the active site. However,
upon exposure to a hydrophobic interface, the lipase is
adsorbed onto it provoking a conformational change
shifting the equilibrium to the open form, a phenome-
5
synthesis of some natural products, protease inhibi-
6
7
tors, and isonucleosides. Furthermore, (R)-glycidyl
butyrate 2 has been used to introduce a stereogenic
8
center in the synthesis of Linezolid (Scheme 2). This
substrate is currently marketed for the treatment of
multidrug resistant Gram-positive infections such as
nosocomial, community-acquired pneumonia, and skin
infections. In this manner, the resolution of glycidyl
ester derivatives has constituted an important target in
15
non known as Interfacial Activation.
9
biotransformations. For instance, an enzymatic process
has been described using crude porcine pancreas lipase
2. Results anꢀ ꢀiscussion
10
for the resolution of (ꢀ)-glycidyl butyrate, process
The resolution of (ꢀ)-2 catalyzed by different immobi-
lized purified preparations of a lipase from R. oryzae
11
now performed on a multitonne scale, although it was
necessary to reach high conversions to obtain adequate
enantiomeric excesses. In recent years, the resolution of
this compound has been tried with many enzymes acting
(
ROL) in aqueous media at 25 ꢁC and pH 7 was per-
formed (Scheme 3). The lipase was first immobilized by
interfacial activation on a hydrophobic support (octyl-
9;10;12
as biocatalysts.
However, it has been very difficult
16
agarose). This strategy of immobilization showed very
low enantioselectivity value (E ¼ 2) with an enantio-
meric excess (ee) of 25% at 50% conversion (Table 1).
When the lipase was immobilized on agarose activated
with cyanogen-bromide (CNBr), where the enzyme was
attached mainly by the terminal amino group, ROL
showed an increase in enantioselectivity (E ¼ 17) with
to find an enzyme, which presents high enantiomeric
excess against this compound, probably due mainly to
its small size, which reduces any steric hindrance to the
adsorption to the active center of the enzyme.
O
O
7
7% ee at 50% conversion (Table 1). Most interestingly,
O H
O
O
N
N
this lipase, after immobilization by ionic adsorption on
amino-Sepabeads coated with very large dextran sulfate
(DS) presented an even higher E value (E ¼ 51). These
results show that ROL can present very different selec-
tivities toward this compound, depending on the
immobilization strategy used.
O
NHAc
F
(
R)-Glycidyl Butrate 2
Linezolid
Scheme 2. (R)-Glycidyl butyrate as the key intermediate in the syn-
thesis of Linezolid.
a
Table 1. Resolution of (ꢀ)-2 catalyzed by different immobilized
b
Recently, it has been reported that some good results
have been obtained using lipases produced by Rhizopus
preparations of ROL
c
d
Entry Support
Time
h)
Conversion Ee (%)
(%)
E value
12
oryzae. Herein, we report an improvement on those
results using a new strategy based on the use of different
immobilization techniques involving different areas of
the enzyme surface, giving a different rigidity to the
enzyme structure or generating different microenviron-
ments surrounding the enzyme to alter the exact shape
of the open form of lipase. This strategy is denominated
as ‘conformational engineering’ and has successfully
been used in the modulation of the behavior of other
(
1
2
3
Octyl-agarose
CNBr-agarose
DS-Sepabeads
0.5
3
50
50
50
25
77
2
17
6
8951
a
The substrate concentration was 10 mM.
The immobilized preparations of ROL contained 1 mg protein /g
support.
b
17
c
Enantiomeric excess (ee) was determined by Chiral HPLC.
Enantioselectivity (E) was calculated by using the equation resulted
18
by Chen et al.
d
13
lipases. This technique has demonstrated that the
properties of the same lipase immobilized on different
supports could be completely different.
Figure 1 shows the evolution of the enantiomeric excess
ee (%) of substrate and product versus conversion in the
hydrolytic reaction catalyzed by ROL immobilized on
DS or octyl-agarose supports. The enantiomeric excess
This strategy is based on the drastic conformational
changes that lipases undergo during catalysis. In aque-
ous media, they exist mainly in a closed and inactive
O
O
O
O
Lipase (ROL, R.oryzae)
O
+
O
H
o
Water, pH 7.0, 25 C
OH
H
H
O
(±)-2
(
R)-1
(R)-2
Scheme 3. Enzymatic resolution of (ꢀ)-2 catalyzed by R. oryzae lipase.

J. M. Palomo et al. / Tetrahedron: Asymmetry 15 (2004) 1157–1161
1159
1
00
(University of Pavia) (Milan, Italy). Other reagents and
solvents used were of analytical or HPLC grade.
8
6
4
2
0
0
0
0
0
4.2. Enzymatic activity assay ꢀetermination
This assay was performed by measuring the increase in
the absorbance at 348 nm produced by the release of
p-nitrophenol in the hydrolysis of 0.4 mM pNPP in
25 mM sodium phosphate buffer at pH 7 and 25 ꢁC. To
initialize the reaction, 0.05 mL of lipase solution or
suspension was added to 2.5 mL of substrate solution.
One international unit of pNPP activity is defined as the
amount of enzyme that is necessary to hydrolyze 1 lmol
of pNPP per minute (IU) under the conditions described
above.
4
0
50
60
Conversion (%)
Fi-ure 1. Evolution of enantiomeric excess (ee) versus conversion in
the hydrolysis of (ꢀ)-2 catalyzed by different ROL immobilized
preparations. Ee of the remaining ester (R)-2 (r), ee of the release acid
(
R)-1 (j). DS-ROL preparation (––), octyl-ROL (- - -).
4
.3. Purification of ROL
of the remaining ester in the reaction catalyzed by octyl-
ROL preparation remained low even at high degrees of
conversion, showing an ee of 30% at 58% conversion.
Nevertheless, after adsorption of the lipase on amino-
Sepabeads coated with dextran sulfate, the enzyme was
much more selective toward the (S)-enantiomer,
obtaining >90% ee at conversions slightly higher than
Commercial lipase extract was dissolved at 25 mg
extract/mL in 5 mM sodium phosphate, then submitted
to gentle stirring for 2 h at 4 ꢁC and pH 7, and lastly
centrifuged at 12,000 rpm for 30 min.
The lipase preparation was then offered to octyl-agarose
16
5
0%; thus, >99% ee was achieved at 55% conversion,
following the procedure previously described (100 mg
extract per g of support). Periodically, the activity of
suspensions and supernatants were assayed using the
pNPP assay. In order to desorb the enzyme, the
adsorbed lipase preparations were washed with 1%
Triton X-100 in 5 mM sodium phosphate buffer at pH 7
and 4 ꢁC.
conditions where enantiomerically pure (R)-2 can be
obtained. Furthermore, (R)-1 could also be obtained
with high ee, by stopping the reaction at 44% conversion
(
>90% ee of product).
These results compete with those published with lipase
from porcine pancreas and improve upon those using
10
12
this lipase. Moreover, this biocatalyst can be used in at
least 30 reaction cycles while maintaining more than
4
.4. Immobilization of lipases on ꢀifferent supports
9
5% of the catalytic activity.
Three different immobilized preparations were produced
following the procedures previously described:
•
•
•
Interfacial activation of the enzyme on octyl-aga-
rose as described above.
Immobilization on CNBr-activated support using the
protocol from Pharmacia (at pH 7).
Ionically adsorbed lipase on Sepabeads support with
amino groups coated with dextran sulfate (DS) (anio-
nic microenvironment surrounding large areas of the
protein). The immobilization of the protein was per-
formed at pH 5 and 25 ꢁC.
3
. Conclusion
16
In conclusion, by using a suitable immobilization tech-
nique (adsorption on dextran sulfate coated Sepabeads),
an enzymatic resolution of glycidyl butyrate has been
found with >99% ee at 55% conversion catalyzed by a
lipase from R. oryzae.
In all cases, enzyme loading was 1 mg protein/mL of
support (that is approx. 1–2% of the maximum load) in
order to prevent diffusion problems and more than 95%
of the lipase becoming immobilized on all supports
offered. The immobilization was followed by the assay
described above. Protein concentration was determined
4
. Experimental
4
.1. General
The lipase from R. oryzae (ROL) was from Fluka.
Octyl-agarose 4BCL and cyanogen bromide (CNBr-
activated Sepharose 4BCL) were purchased from Phar-
macia Biotech (Uppsala, Sweden). Amino-Sepabeads
were kindly donated by Resindion Srl (Mitsubishi Chem
Coorp, Milan, Italy). Dextran sulfate (DS) (Mr 5000),
p-nitrophenyl propionate (pNPP) were from Sigma. (ꢀ)-
Glycidyl butyrate was kindly donated by Dr. Terreni
17
by the Bradford method.
4.5. Enzymatic hyꢀrolysis of ester
The activities of different ROL immobilized prepara-
tions on the hydrolysis reaction of compound 2 were

1160
J. M. Palomo et al. / Tetrahedron: Asymmetry 15 (2004) 1157–1161
performed with the addition of 0.01 g of immobilized
preparation to 10 mL of 10 mM of substrate at pH 7 and
4. Kim, B. M.; Bae, S. J.; Seomoon, G. Tetrahedron Lett.
998, 39, 6921–6922.
1
5
. Aminocyclitol antibiotics: Damels, P. L. G. In Drug
Action and Resistance in Bacteria; Mitsuhashl, S., Ed.;
University Park Press: Tokyo, 1975; p 75.
2
5 ꢁC.
During the reaction, the pH value was maintained at a
constant using a pH-stat Mettler Toledo DL50 graphic.
The degree of hydrolysis was analyzed by reverse-phase
HPLC (Spectra Physic SP 100 coupled with an UV
detector Spectra Physic SP 8450). For these assays, a
Kromasil C18 (25 · 0.4 cm) column was used, mobile
phase acetonitrile–10 mM ammonium phosphate buffer
at pH 2.95 (35:65, v/v) at 1.5 mL/min and UV detection
was performed at 225 nm.
6
. (a) Fenwick, A. E.; Gribble, A. D.; Ife, R. J.; Stevens, N.;
Witherington, J. J. Bioorg. Med. Chem. Lett. 2001, 11,
1
99–202; (b) Marquis, R. W.; Ru, Y., et al. J. Med. Chem.
001, 44, 725–736; (c) Zook, S. E.; Busse, J. K.; Borer,
B. C. Tetrahedron Lett. 2000, 41, 7017–7021.
2
7. (a) Montgomery, J. A.; Thomas, H. J. J. Org. Chem. 1978,
43, 541–544; (b) Nair, V.; Nuesca, Z. M. J. Am. Chem.
Soc. 1992, 114, 7951–7953; (c) Katefuda, A.; Shuto, S.;
Nagahata, T.; Seki, J.; Sasaki, T.; Matsuda, A. Tetrahe-
dron 1994, 50, 7747–7764.
8
. (a) Brickner, S.; Hutchinson, D.; Barbachyn, M.; Manni-
nen, P.; Ulanowicz, D.; Garmon, S.; Grega, K.; Hendges,
S.; Toops, D.; Ford, C.; Zurenko, G. J. Med. Chem. 1996,
4.6. Determination of enantiomeric excess anꢀ enantio)
selectivity
39, 673; (b) Weidner-Wells, M. A.; Boggs, C. M.; Foleno,
B. D.; Melton, J.; Bush, K.; Goldschmidt, R. M.; Hlasta,
D. J. Bioorg. Med. Chem. 2002, 10, 2345–2351.
The enantiomeric excess (ee) of the remaining ester was
determined on the organic phase obtained by extracting
9. Martins, J. F.; deCarvalho, T. C.; de Sampaio, T. C.;
Barreiros, S. Enzyme Microb. Technol. 1994, 16, 785.
0. Ladner, W. E.; Whitesides, G. M. J. Am. Chem. Soc. 1984,
0
.2 mL of aqueous phase with 0.2 mL hexane and ana-
1
1
1
lyzed by Chiral Phase HPLC. The column was a
Chiracel OD (10 lm 250 · 4.6 mm), the mobile phase an
isocratic mixture of isopropanol and hexane (2:98, v/v)
at a flow of 0.4 mL/min with UV detection was per-
formed at 225 nm. The retention times of the enantio-
mers: (S)-2 (17,32), (R)-2 (18.60).
1
06, 7250–7251.
1. Crosby, J. Chirality in Industry; Wiley: Chichester, 1992;
p 1.
2. (a) Wang, Z.; Quan, J.; Weng, L.; Gou, X.-j.; Ma, J.;
Zhang, G.; Cao, S. Jilin Daxue Xuebao, Lixueban 2003,
4
1, 213–216; (b) Jia, S.; Xu, J.; Li, Q.; Yu, J. Appl.
Biochem. Biotech. 2003, 104, 69–79; (c) Liu, Z.; Wang, Z.;
Yang, S.; Zheng, L.; Weng, L.; Li, N.; Han, S.; Cao, S.
Jilin Daxue Ziran Kexue Xuebao 2001, 4, 78–80; (d) Qiu,
Y.; Xu, J. Yingyong Yu Huanjing Shengwu Xuebao 2001, 7,
The enantiomeric ratio (E) was calculated in all cases
using the equation reported by Chen et al.
18
6
9–473.
1
3. (a) Palomo, J. M.; Fernandez-Lorente, G.; Mateo, C.;
Fuentes, M.; Fernandez-Lafuente, R.; Guisan, J. M.
Tetrahedron: Asymmetry 2002, 13, 1337–1345; (b) Palomo,
J. M.; Fernandez-Lorente, G.; Mateo, C.; Ortiz, C.;
Fernandez-Lafuente, R.; Guisan, J. M. Enzyme Microb.
Technol. 2002, 31, 775–783; (c) Palomo, J. M.; Fernandez-
Lorente, G.; Mateo, C.; Fernandez-Lafuente, R.; Guisan,
J. M. Tetrahedron: Asymmetry 2002, 13, 2375–2381; (d)
Fernandez-Lorente, G.; Fernandez-Lafuente, R.; Palomo,
J. M.; Mateo, C.; Bastida, A.; Coca, J.; Haramboure, T.;
Hernandez-Ortiz, O.; Terreni, M.; Guisan, J. M. J. Mol.
Catal. B: Enzym. 2001, 11, 649–656; (e) Palomo, J. M.;
Fernandez-Lorente, G.; Mateo, C.; Fuentes, M.; Guisan,
J. M.; Fernandez-Lafuente, R. Tetrahedron: Asymmetry
Acknowleꢀ-ements
This work was supported by the Spanish CICYT (pro-
ject BIO2001-2259). Authors thanks to Hispanagar SA
for the gift of glyoxyl-agarose. We also thank Resindion
Srl for donation of the amino-Sepabeads, Dr. Terreni
for the kind supply of compound 2 and Angel Berenguer
for interesting suggestions.
References anꢀ notes
2
002, 13, 2653–2659; (f) Palomo, J. M.; Fernandez-
1
. (a) Caron, M.; Sharpless, K. B. J. Org. Chem. 1985, 50,
Lorente, G.; Mu n~ oz, G.; Mateo, C.; Fuentes, M.; Guisan,
J. M.; Fernandez-Lafuente, R. J. Mol. Catal. B: Enzym.
2003, 21, 201–210; (g) Palomo, J. M.; Fernandez-Lorente,
G.; Rua, M. L.; Guisan, J. M.; Fernandez-Lafuente, R.
Tetrahedron: Asymmetry 2003, 14, 3679–3687.
1560–1563; (b) Hanson, R. M. Chem. Rev. 1991, 91, 437–
475; (c) Tomata, Y.; Sasaki, M.; Tanino, K.; Miyashita,
M. Tetrahedron Lett. 2003, 44, 8975–8977; (d) Dehoux, C.;
Fontaine, E.; Escudier, J. M.; Baltas, M.; Gorrichon, L.
J. Org. Chem. 1998, 63, 2601–2608; (e) Liu, D. G.; Wang,
B.; Lin, G. Q. J. Org. Chem. 2000, 65, 9114–9119; (f)
Vidal-Ferran, A.; Moyano, A.; Pericas, M. A.; Riera, A.
J. Org. Chem. 1997, 62, 4970–4982; (g) Morimoto, Y.;
Shirahama, H. Tetrahedron 1996, 52, 10631–10652; (h)
Iida, H.; Yamazaki, N.; Kibayashi, C. J. Org. Chem. 1987,
14. (a) Ghosh, D.; Wawrzak, Z.; Pletnev, V. Z.; Li, N.; Kaiser,
R.; Pangborn, W.; Jomvall, H.; Erman, M.; Duax, W. L.
Structure 1995, 3, 279–288; (b) Uppenberg, J.; Ohmer, N.;
Norin, M.; Hult, K.; Kleywegt, G. J.; Patkar, S.; Waagen,
V.; Anthonsen, T.; Jones, T. A. Biochemistry 1995, 34,
16838–16851; (c) Pernas, M. A.; Lopez, C.; Rua, M. L.;
Hermoso, J. FEBS Lett. 2001, 501, 87–91.
15. (a) Sarda, L.; Desnuelle, P. Biochim. Biophys. Acta 1958,
30, 513–521; (b) Brady, L.; Brzozowski, A. M.; Derew-
enda, Z. S.; Dodson, E.; Dodson, G.; Tolley, S.; Turken-
burg, J. P.; Christiansen, L.; Huge-Jensen, B.; Norskov,
L.; Thim, L.; Menge, U. Nature 1990, 343, 767–770;
5
2, 3337–3342.
. Takikawa, H.; Yoshida, M.; Mori, K. Tetrahedron Lett.
001, 42, 1527–1530.
2
3
2
. Aragones, S.; Bravo, F.; Diaz, Y.; Matheu, M. I.;
Castillon, S. Tetrahedron: Asymmetry 2003, 14, 1847–
1856.

J. M. Palomo et al. / Tetrahedron: Asymmetry 15 (2004) 1157–1161
1161
(
2
c) Derewenda, Z. S.; Derewenda, U. J. Mol. Biol. 1992,
27, 818–839; (d) Ampon, K.; Basri, M.; Salleh, A. B.;
Wan Yunus, W. M. Z.; Razak, C. N. A. Biocatalysis 1994,
0, 341–351; (e) Grochulski, P.; Li, Y.; Schrag, J. D.;
16. Bastida, A.; Sabuquillo, P.; Armisen, P.; Fernandez-
Lafuente, R.; Huguet, J.; Guisan, J. M. Biotechnol.
Bioeng. 1998, 58, 486–493.
17. Bradford, M. M. Anal. Biochem. 1976, 72, 248–254.
18. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am.
Chem. Soc. 1982, 104, 7294–7299.
1
Bouthillier, F.; Smith, P.; Harrison, D.; Rubin, B.; Cygler,
M. J. Biol. Chem. 1993, 268, 12843–12847.