Kinetic resolution of (±)-1-phenylethanol in [Bmim][PF6] using high activity preparations of lipases

Article abstract of DOI:10.1016/j.bmcl.2006.11.057

Lipases from two different sources Candida rugosa (CRL) and Burkholderia cepacia (BCL) were formulated as enzyme precipitated and rinsed with organic solvents, organic solvent rinsed enzyme preparation, cross-linked enzyme aggregates (CLEAs) and protein coated micro-crystals (PCMCs). These various enzyme formulates were evaluated for the kinetic resolution of (±)-1-phenylethanol in ionic liquid [Bmim][PF₆] by transesterification with vinyl acetate. Of all the enzyme forms evaluated EPRP and PCMC in the case of CRL showed the best results with 26 % (E value = 153) and 53% (E value = 79) conversion, respectively, at 35 °C in 24 h. Carrying out this conversion with PCMC at lower temperature of 25 °C further improved the E value to 453 (with 44% conversion in 12 h). For BCL the acetone-rinsed enzyme preparation (AREP), CLEA and PCMC performed equally well with % conversion of 50 and 99 ee_p (%) (E value >1000) in just 2 h, whereas, the free lipase gave only 8% conversion.

Full text of DOI:10.1016/j.bmcl.2006.11.057

Bioorganic & Medicinal Chemistry Letters 17 (2007) 921–924
Kinetic resolution of ( )-1-phenylethanol in [Bmim][PF6]
using high activity preparations of lipases
Shweta Shah and Munishwar N. Gupta*
Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India
Received 20 September 2006; revised 16 November 2006; accepted 18 November 2006
Available online 22 November 2006
Abstract—Lipases from two different sources Candida rugosa (CRL) and Burkholderia cepacia (BCL) were formulated as enzyme
precipitated and rinsed with organic solvents, organic solvent rinsed enzyme preparation, cross-linked enzyme aggregates (CLEAs)
and protein coated micro-crystals (PCMCs). These various enzyme formulates were evaluated for the kinetic resolution of ( )-1-
phenylethanol in ionic liquid [Bmim][PF6] by transesterification with vinyl acetate. Of all the enzyme forms evaluated EPRP and
PCMC in the case of CRL showed the best results with 26 % (E value = 153) and 53% (E value = 79) conversion, respectively,
at 35 °C in 24 h. Carrying out this conversion with PCMC at lower temperature of 25 °C further improved the E value to 453 (with
44% conversion in 12 h). For BCL the acetone-rinsed enzyme preparation (AREP), CLEA and PCMC performed equally well with
% conversion of 50 and 99 eep (%) (E value >1000) in just 2 h, whereas, the free lipase gave only 8% conversion.
Ó 2006 Elsevier Ltd. All rights reserved.
The ionic liquids (ILs) are fast emerging as a useful reac-
tion medium for enantioselective biocatalysis.1 In many
cases, especially with lipase-catalyzed transesterification
reactions; higher reaction rates and greater enantioselec-
tivity have been reported as compared to the corre-
sponding performances in anhydrous organic media.2
Most such studies have been carried out with pH-tuned
lyophilized enzyme powders, either in free or immobi-
lized forms.2,3 In the last few years, it has become clear
that structural changes during lyophilization are largely
responsible for relatively low catalytic rates shown by
enzymes in nearly anhydrous organic solvents.4 Conse-
quently, many enzyme formulations which ‘dry’ en-
zymes for use in such media have been described. Such
enzyme forms include propanol-rinsed enzyme prepara-
tions (PREP),5 cross-linked enzyme aggregates
(CLEAs)6 and protein coated micro-crystals (PCMCs).7
PREP refers to propanol-rinsed immobilized enzymes,
the corresponding free enzyme preparations rinsed with
propanol (EPRP) also give higher reaction rates than
pH-tuned lyophilized powders in low water-containing
organic solvents.8 Studies with such preparations in
ILs have begin to be described.1a The present work
shows that these enzyme preparations, which have prov-
en efficiency in organic solvents, work well in a water-
immiscible IL, 1-butyl-3-methylimidazolium hexafluoro-
phosphate ([Bmim][PF6]). The reaction chosen is the
kinetic resolution of ( )-1-phenylethanol (1) by transe-
sterification with vinyl acetate (2) (Scheme 1); this reac-
tion has been often chosen for evaluating the reaction
rates and enantioselectivity with various lipases.2,3 The
Candida rugosa lipase (CRL) is reported to give worst
result in [Bmim][PF6]3b (for kinetic resolution of ( )-1-
phenylethanol). The lipase from Burkholderia cepacia
(BCL) has been found to give very poor reaction rates
in [Bmim][PF6] for transesterification of ( )-1-phenyl-
ethanol.2b,c Hence these two enzymes were converted
into above-mentioned formulations and these enzyme
forms were evaluated for their comparative performance
in the ionic liquid.
The alcoholysis between ( )-1-phenylethanol (1 mmol)
and vinyl acetate (1 mmol) was carried out in 1 ml ionic
liquid (Acros Organics, USA; the purity of [Bmim][PF6]
as specified by the vendor was 99.6% (by HPLC) with
water content of 0.05% v/v by Karl-Fischer) using differ-
ent preparations of CRL/BCL (containing 10 mg of lip-
ases) at a fixed temperature as mentioned in the legend
with constant shaking at 250 rpm. CRL (lipase AYS
‘Amano’) and BCL (lipase PS ‘Amano’) were a kind gift
from Amano, Nagoya, Japan. For analysis, 50 ll of the
reaction mixture was extracted with 500 ll n-hexane/
Keywords: Cross-linked enzyme aggregates; Enantioselectivity; Enzy-
me precipitated and rinsed with propanol; Ionic liquids; Lipases;
Propanol-rinsed enzyme preparation; Protein coated microcrystals.
*
Corresponding author. Tel.: +91 11 2659 1503/91 11 2659 6568; fax:
+91 11 2658 1073; e-mail: appliedbiocat@yahoo.co.in
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.11.057

922
S. Shah, M. N. Gupta / Bioorg. Med. Chem. Lett. 17 (2007) 921–924
O
OH
OH
O
O
Lipase
CH3CHO
+
+
+
O
Preparation
1
2
3
4
Scheme 1.
propan-2-ol (97.5:2.5) mixture. The extract was ana-
lyzed by HPLC using Chiracel OD-RH column (Diacel,
Japan). The eluent consisted of 96.5% (v/v) n-hexane,
3% (v/v) propan-2-ol and 0.5% (v/v) ethanol with a flow
rate of 1 ml minÀ1 and detection was carried out with a
UV detector at 254 nm.3b
It was seen that EPRP9 (of CRL) did perform much bet-
ter than pH-tuned lyophilized powder10 (entries 1–4, Ta-
ble 1). Longer time period of 24 h (entry 4, Table 1)
resulted in only marginal increase in eep (%) but sub-
stantially improved E (E value was calculated by using
Chen’s equation).11 The PREP12 (of CRL) gave higher
conversion than EPRP but poorer eep (%) and E value
(entries 5–6, Table 1). CLEAs13 prepared from free
CRL did not show any dramatic improvement in % con-
version (entries 7–8, Table 1). The % conversion was
better than free enzyme but worse than both EPRP
and PREP. Table 1 shows that eep (%) was higher than
both pH-tuned lyophilized enzyme and PREP but lower
than EPRP. PCMC14 gave highest % conversion (entries
9–10, Table 1); eep (%) was quite good while ees (%) was
highest of all enzyme preparations. The E value, howev-
er, was lower than that obtained with EPRP. Lowering
of temperature reduces reaction rates but is often associ-
ated with improved enantioselectivity in organic sol-
vents.15 On the other hand, in the case of ILs, no
significant difference in E with lowering of temperature
has been reported.16 Table 1 (entries 11–12) in the pres-
ent instance shows that lowering of temperature to
25 °C further improved enantioselectivity of PCMC.
The ideal time period was 12 h with 44% conversion,
99 eep (%) and E value of 453.
Figure 1 shows the steps involved in the various prepa-
rations of biocatalysts. All these preparations involve
simple protocol and each involves either precipitation
or washing with organic solvent for drying. All these
preparations essentially avoid drying by lyophilization,
which is now known to affect the secondary structure
of proteins/enzymes.4 Such changes are reversible when
lyophilized enzymes are used in the conventional water-
rich media, that is, aqueous buffers. However, in low
water media such as nearly anhydrous media, these
structural changes are not reversed and are an important
factor for low enzyme activities.4,5
It is noteworthy that while PCMCs have shown en-
hanced catalytic activity in organic solvents, no change
in enantioselectivity (E) was observed by others who
compared the PCMC (prepared from 10 different lip-
ases) with respective free enzymes for kinetic resolution
of ( )-1-phenylethanol.7
In the case of BCL, the precipitation with propanol
resulted in substantial loss of activity (up to 60%) as
evaluated by hydrolysis of p-nitrophenylpalmitate.17
However, substitution of propanol with acetone gave
full recovery. Hence for BCL, enzyme precipitated and
rinsed with acetone (EPRA) and acetone-rinsed enzyme
preparation (AREP) were the correct formulations. For
preparation of PCMC as well (of BCL), acetone was
used as the solvent for precipitation.
The relative catalytic performances of EPRA, AREP,
CLEA and PCMC of BCL were evaluated by a standard
transesterification assay in [Bmim][PF6].1b The initial
rates obtained during the assay (Table 2) showed that
AREP was the best catalyst followed by PCMC >
CLEA > EPRA > pH tuned.
After this, these preparations were studied for kinetic
resolution of ( )-1-phenylethanol. Again, the perfor-
Figure 1. General protocol for the preparation of EPRP (a); PREPs
(b); CLEAs (c); PCMCs (d).

S. Shah, M. N. Gupta / Bioorg. Med. Chem. Lett. 17 (2007) 921–924
923
Table 1. Kinetic resolution of 1-phenylethanol in [Bmim][PF6] catalyzed by different preparations of Candida rugosa lipase
a
eep (%)
Entry
Lipase preparations
Time (h)
Conversiona (%)
eesa (%)
Eb
11
1
2
pH tuned
pH tuned
EPRP
12
24
12
24
12
24
12
24
12
24
6
5
7
80
83
97
98
80
79
91
95
87
88
99
99
5
6
12
123
153
21
3
22
26
49
51
10
12
51
53
34
44
28
34
78
82
10
12
93
99
51
77
4
EPRP
PREP
PREP
5
6
22
30
7
CLEA
CLEA
PCMC
PCMC
PCMC
PCMC
8
69
53
9
10
11
12
79
283
453
12
All reactions corresponding to entries 1–10 were carried, at 35 °C, whereas reactions corresponding to entries 11 and 12 were carried out at 25 °C.
a Conversion and ee’s (%) were estimated by HPLC.
b E = ln[1 À c(1 + eep)]/ln[1 À c(1 À eep)], where c = ees/(ees + eep).
Table 2. Initial transesterification rates exhibited by different preparations of Burkholderia cepacia lipase in [Bmim][PF6]
S. No.
Lipase preparations
Initial ratesa (lmol minÀ1 mgÀ1
)
1
2
3
4
5
pH tuned
EPRA
AREP
8
68
168
100
120
CLEA
PCMC
a Reaction conditions: ethylbutanoate (60 mM), 1-butanol (120 mM), different lipase preparations (prepared from 10 mg lipase) in [Bmim][PF6]
(0.5 ml) at 40 °C with constant shaking at 200 rpm. The substrate and product were extracted by hexane and hexane phase was analyzed by Agilent
6890 N GC with a flame ionization detector using EquityTM -5 fused silica capillary column (Supelco, USA).6b
Table 3. Kinetic resolution of 1-phenylethanol in [Bmim][PF6] catalyzed by different preparations of Burkholderia cepacia lipase at 25 °C
Entry
Lipase preparations
Time (h)
Conversion (%)
eep (%)
ees (%)
E
1
2
pH tuned
pH tuned
EPRA
1
2
1
2
1
2
1
2
1
2
5
8
99
99
99
99
99
99
99
99
99
99
5
8
114
187
3
31
49
44
50
41
50
40
50
45
96
78
99
69
99
66
99
314
736
4
EPRA
AREP
5
415
>1000
432
6
AREP
CLEA
7
8
CLEA
PCMC
PCMC
>1000
405
>1000
9
10
mance of all the four preparations (EPRA, AREP,
CLEA and PCMC) in case of BCL exceeded well be-
yond the pH-tuned lipase (Table 3). The EPRA gave
49% conversion, 99 eep (%) and the E value of 736 in
2 h. The AREP, CLEA and PCMC performed equally
well with % conversion of 50 and 99 eep (%) (E value
>1000) (entries 6, 8 and 10, Table 3) in just 2 h, whereas
the free lipase gave only 8% conversion (entry 2, Table
3) in agreement with the earlier works which reported
the slow conversion rates in [Bmim][PF6] with BCL.2b,c
Park and Kazalausks2b achieved ꢀ30% conversion in
96 h in [Bmim][PF6] with BCL.
water-miscible (more hydrophilic) ionic liquid was ade-
quately fast.2b,c It may be, however, added that in the
case of subtilisin catalyzed transesterification, EPRP
was found to give much higher rates in both [Bmim]
[PF6] and [Bmim][BF4] as compared to pH-tuned lyoph-
ilized enzyme.18
ILs are perceived as green solvents in view of their low
vapor pressure. These are also called designer solvents,
as different combinations of cations and anions can pro-
duce novel reaction media with desirable properties.1,2
In order to fully exploit these advantages, it is necessary
that the best ways of biocatalyst preparation are identi-
fied and tested with large number of systems. This work
shows that with CRL (which did not show promising re-
sults when used as lyophilized powder), PCMC of the
enzyme gives high enantioselectivity and conversion,
The present work was limited to [Bmim][PF6] which is a
water-immiscible ionic liquid. This is because lipases are
reported to give poor rates for this reaction in
[Bmim][PF6].2b,c The reaction when carried out in

924
S. Shah, M. N. Gupta / Bioorg. Med. Chem. Lett. 17 (2007) 921–924
7. Kreiner, M.; Moore, B. D.; Parker, M. C. Chem. Commun.
2001, 1096.
8. Roy, I.; Gupta, M. N. Bioorg. Med. Chem. Lett. 2004, 14,
2191.
whereas all the four preparations were found to be high-
ly active in the case of BCL.
9. Preparation of EPRP; CRL (10 mg mlÀ1, 50 mM sodium
phosphate buffer, pH 7.0) was added to dry chilled 1-
propanol at 4 °C. The precipitate was rinsed two times
with 1 ml of ice-cold 1-propanol at 4 °C.8 Before using,
EPRP was washed two times with 1 ml of chilled vinyl
acetate. A similar procedure was adopted for BCL where
acetone was used in place of 1-propanol.
10. Preparation of pH-tuned lipase; CRL/BCL (10 mg) was
dissolved in 1 ml of 0.02 M sodium phosphate buffer, pH
7.0, and was immediately frozen at À20 °C and lyophilized
for 24 h.
Acknowledgments
The funds provided by Council of Scientific and Indus-
trial Research (Extramural Division and Technology
Mission on Oilseeds), Department of Science and Tech-
nology (DST), and Department of Biotechnology
(DBT), all of which are Government of India Organiza-
tions, are gratefully acknowledged.
11. Chen, C.-S.; Fujimoto, Y.; Girdauskas, G.; Sih, C. J.
J. Am. Chem. Soc. 1982, 102, 7294.
Supplementary data
12. Preparation of PREP; CRL powder (10 mg) was dissolved
in 200 ll of 50 mM sodium phosphate buffer, pH 7.0, and
mixed with 100 mg of Celite and rinsed with dry chilled 1-
propanol.5 Before using PREP was washed two times with
1 ml of chilled vinyl acetate. In the case of BCL acetone
was used in place of 1-propanol.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2006.11.057.
13. Preparation of CLEA: CRL/BCL (10 mg) along with
bovine serum albumin (2.5 mg) was dissolved in 1 ml of
50 mM phosphate buffer, pH 7.0. This aqueous mixture
was then added dropwise to a centrifuge tube containing
4 ml of acetone. After 30 min, glutaraldehyde (25% v/v in
water) was added so that the final concentration of
glutaraldehyde was 25 mM. The mixture was kept at 4 °C
for 3 h with constant shaking at 300 rpm. The CLEAs
formed were washed three times with 1 ml of acetone.6b
Before using, CLEAs were washed two times with 1 ml of
chilled vinyl acetate.
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