Solvent-dependent reactivity in porcine pancreatic lipase (PPL)-catalyzed hydrolysis

Article abstract of DOI:10.1016/j.tetasy.2008.05.021

The solvent-dependent enzyme reactivity of porcine pancreatic lipase (PPL)-catalyzed hydrolysis was investigated using trans-3 and cis-(3-(benzyloxymethyl)oxiran-2-yl)methyl acetate 4 as substrates. The conversion efficiency and enantioselectivity of the

Full text of DOI:10.1016/j.tetasy.2008.05.021

Tetrahedron: Asymmetry 19 (2008) 1647–1653
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Solvent-dependent reactivity in porcine pancreatic lipase
(PPL)-catalyzed hydrolysis
Liu-Lan Shen a, Fang Wang a, Han-Seo Mun a, Myungkoo Suh c, Jin-Hyun Jeong a,b,
*
a College of Pharmacy, Kyung Hee University, #1 Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Republic of Korea
b Kyung Hee Institute of Age-related and Brain Disease, Kyung Hee University, #1 Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, Republic of Korea
c Institute of Basic Science, Sungkyunkwan University, Suwon 440-746, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 April 2008
Accepted 19 May 2008
The solvent-dependent enzyme reactivity of porcine pancreatic lipase (PPL)-catalyzed hydrolysis was
investigated using trans-3 and cis-(3-(benzyloxymethyl)oxiran-2-yl)methyl acetate 4 as substrates. The
conversion efficiency and enantioselectivity of the hydrolysis of these compounds were measured in
three different types of enzymatic media: neat organic solvents, organic–aqueous mixture solvent sys-
tems, and an aqueous buffer solution. Comparison of the catalytic hydrolysis that occurred in 12 different
organic solvents with log P values in the range of ꢀ1.10 to 3.50 revealed that the reactivity and selectivity
of PPL-catalyzed hydrolysis toward (+)- and (ꢀ)-isomers were strongly affected by the solvent system. In
neat organic solvents and organic–aqueous biphasic systems, (+)-cis oxirane acetate (+)-4 was selectively
hydrolyzed by PPL over the (ꢀ)-cis isomer. In contrast, hydrolysis of the (+)-trans substrate (+)-3 was pre-
ferred in monophasic organic–aqueous systems and buffer solutions. The addition of water to the organic
solvent increased the overall hydrolysis rate. However, hydrolysis in the aqueous buffer solution was
considerably retarded since the organic substrates were insoluble in water. Therefore, control of the sol-
vent system can result in the successful kinetic resolution of oxirane esters and their corresponding
alcohols.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
pure compounds.7,9 However, in addition to transesterifications,
concurrent catalytic hydrolysis of ester compounds occurs sub-
Organic solvents have been used as enzyme reaction media to
achieve various enzymatic transformations in organic synthesis
applications.2–4 The substrate selectivities of enzymes in organic
media, including enantio-, prochiral, regio-, and chemoselectivi-
ties, differ profoundly in different solvents.2–6 Numerous studies
have shown that the catalysis properties of enzymes are different
in organic media than in water, forming the basis of product
selectivity.1,5,6 Among the enzymes used for organic synthesis
applications, porcine pancreatic lipase (PPL) has been utilized to
catalyze a wide range of substrate hydrolysis and transesterifica-
tion steps in monophase and multiphase solvent systems with high
enantioselectivity.4,7 Medium engineering (the optimization of en-
zyme selectivity in organic solvents) of PPL-catalyzed hydrolysis
has also been attempted, with substrate specificities such as chain
length, regio-, and enantioselectivity investigated in various med-
ia.3,7–9
stantially even in organic solvents, since water molecules remain-
ing on the enzyme surface10 take part in the hydrolysis reaction.
This hydrolysis reaction prevents transesterification reactions from
completing and impedes their use in kinetic resolutions. Unlike
transesterification reactions, catalytic hydrolysis requires water
as a reactant and generally proceeds to completion in aqueous
media. However, most organic substrates are insoluble in aqueous
solutions and therefore enzyme-catalyzed hydrolysis is commonly
carried out in aqueous–organic mixed-solvent systems to increase
the solubility of the substrates. The minimum amount of water
required to maintain enzyme structure and flexibility in organic
media is about 0.05–3%.11,12 Since the amount of water in the enzy-
matic media plays an important role in enzyme structure and reac-
tivity, the enantioselectivity of enzymatic hydrolysis is also
expected to depend on the water content in the reaction media
and on the characteristics of the organic solvent. Therefore, herein
we explored the enantioselectivity of PPL-catalyzed hydrolysis as a
function of the water content and the organic solvent. The catalytic
hydrolysis of racemic esters containing trans and cis oxirane alco-
hol units rac-1 and rac-2 (Fig. 1) was carried out in three different
types of media: neat organic solvents, aqueous–organic systems
(1:1 volume ratio), and aqueous solutions. A range of organic
Lipase-catalyzed transesterification reactions can be efficiently
carried out in organic solvents, making them useful in preparative
organic chemistry to attain asymmetric access to enantiomerically
* Corresponding author. Tel.: +82 2 961 0368; fax: +82 2 959 0368.
E-mail address: jeongjh@khu.ac.kr (J.-H. Jeong).
0957-4166/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2008.05.021

1648
L.-L. Shen et al. / Tetrahedron: Asymmetry 19 (2008) 1647–1653
O
2. Results and discussion
HO
O
HO
O
O
2.1. Preparation of substrates for enzymatic hydrolysis
rac-1
rac-2
trans- and cis-Monobenzylated oxirane dimethanol esters rac-3
and rac-4 were prepared from monobenzylated cis-butene-1,4-
diol, as shown in Scheme 2. Epoxidation of 5 and 7 was performed
using m-CPBA followed by acetylation to produce the monobenzy-
lated oxiranes rac-4 and rac-3, respectively. For the trans ester rac-
Figure 1.
solvents with log P values in the range of ꢀ1.10 to 3.50 was used.
The conversion efficiency and enantioselectivity with respect to
the substrate structures were investigated in these media.
trans and cis 3-(Benzyloxymethyl)oxirane-2yl methanol, 1 and
2, are composed of fully functional four-carbon units with two ste-
reogenic centers. These compounds are essential synthetic building
blocks of biologically active natural products.13 Enantiomerically
pure compounds 1 and 2 are versatile intermediates for total syn-
thesis via regio- and stereoselective ring opening, methathesis, or
asymmetric aldol condensation.14 For example, the aldehyde
obtained by oxidation of 2 was used as the starting material in
the total synthesis of cyclic guanidine sugars15 and 3-methylated
2 acted as a key intermediate in the construction of the tricyclic
core of phomactin A.16 Compounds 1 and 2 were also used as
precursors in the development of methodologies, such as the
Baylis–Hillman reaction17 and Mukaiyama aldolization.18 The
kinetic resolution of racemic oxirane compounds with biocatalysts
can provide a simple, yet efficient synthetic method for oxirane
enantiomers. However, only a few cases have been reported of
the kinetic resolution of small molecular oxiranes using PPL.19 Faigl
et al. reported that the kinetic resolution of racemic cis-3-((benzyl-
oxymethyl)oxiran-2-yl)methanol rac-2 by PPL-catalyzed acetyla-
tion was achieved by the preferential reaction of a cis-(+)-oxirane
methanol compound ((+)-2) in organic solvents.20 Moseley and
Staunton described the monohydrolysis of meso-oxiranedimetha-
nol diesters by porcine-derived lipases, and also suggested a trans-
formation to generate cis-(+)-epoxide methanol intermediates.21
Here, the reactions that were monitored are shown in Scheme 1.
In these reactions, 1 and 2 are produced by PPL-catalyzed hydro-
lysis from their acetate esters 3 and 4, respectively. The reactivity
and enantioselectivity of the reactions were investigated in
various media.
3, compound 5 was converted to trans-a,b-unsaturated olefin com-
pound 6 due to conjugation during the Dess–Martin periodinane22
oxidation. Next, compound 6 was reduced to the allylic alcohol 7
with a high yield.
2.2. Enzyme selectivity of regioisomers in different media
A major advantage of using organic media in enzyme-catalyzed
reactions is improved substrate solubility. Substrates that are
poorly soluble in water can have a greater chance of interacting
with the enzyme in organic solvents at the expense of enzyme
reactivity, which often accompanies the structural changes that
occur in organic solvents. Partial replacement of the organic sol-
vent with aqueous media might be preferable for the structural
stability of the enzyme, while keeping the hydrophobic substrates
dissolved in the solution. Denaturation of the enzyme can be
avoided in this mixed system and the catalytic activity of the
enzyme can be enhanced, particularly for hydrolases, because
water provides another reactant for the hydrolysis.1 Here, two
different kinds of solvent environments should be considered:
polar organic solvents miscible with water, and non-polar organic
solvents that are partially miscible or immiscible when mixed with
water, resulting in a biphasic system. For PPL-catalyzed hydrolysis,
in a homogeneous solvent system composed of water and a water-
miscible organic solvent, the probability of substrates encounter-
ing each other is improved, since both the enzyme and substrates
are dissolved in the solution. However, the reactivity and specific-
ity of the enzyme cannot be easily predicted. The active-site con-
formation and structural rigidity of the enzyme are altered in
these mixed solvents and even small structural changes can have
O
O
enzymatic media, 25 o
C
AcO
AcO
O
HO
O
O
O
O
rac-3
(+)-1
(-)-3
O
O
O
enzymatic media, 25 oC
OAc
O
OAc
O
HO
(-)-4
rac-4
(+)-2
Scheme 1. Enzymatic hydrolysis reactions investigated in this study.
HO
O
d
c
O
HO
O
O
7
5
6
a,b
a,b
AcO
O
O
O
AcO
O
rac-3
rac-4
Scheme 2. Synthesis of substrates 3 and 4. Reagents and conditions: (a) mCPBA, NaHCO3, CH2Cl2, 0 °C to rt, 18 h, 78%; (b) acetic anhydride, acetic acid, rt, 12 h, 95%; (c) Dess–
Martin periodinane, CH2Cl2, rt, 20 h, 84%; (d) NaBH4, MeOH, 0 °C to rt, 20 min, 98%.

L.-L. Shen et al. / Tetrahedron: Asymmetry 19 (2008) 1647–1653
1649
large effects on the reactivity and specificity. In contrast, in a
water–organic biphasic solvent system, PPL can adopt the native
structure that it has in aqueous media.5 However, the opportuni-
ties for interactions with substrates are significantly reduced, since
the reaction only takes place at the interface.23 In aqueous media,
the encounters of PPL with substrates are expected to be even less
frequent due to the decreased interfacial area.
enzyme tends to denature, resulting in a very rigid conformation.1
This low flexibility of the enzyme structure diminishes the enzyme
activity in neat organic solvents, even though crystalline enzymes
essentially retain their native structures.4
The results of PPL-catalyzed hydrolysis in various organic media
are summarized in Table 1 and the enantiomeric ratios obtained
using rac-3 and rac-4 as substrates and two reaction times are
compared in Figure 2. Note that in nearly anhydrous organic sol-
vents, a substantial amount of enantiomeric catalytic activity of
PPL was observed for the hydrolysis of rac-4, whereas that for
rac-3 was negligible. Both oxirane esters, 3 and 4, were hydrolyzed
slowly and inefficiently, probably due to the limited amount of
reactant water available in the anhydrous media. With rac-3, as
2.2.1. PPL-catalyzed hydrolysis in neat organic solvents
In general, the catalytic activities of enzymes in neat organic
solvents are thought to be far lower than in water.2 In anhydrous
organic solvents, lyophilized or freeze-dried enzymes lose their
lubricating agent, water, which covers the enzyme surface. The
Table 1
PPL-catalyzed hydrolysis in pure organic solventsa
Entry
Substrate
Solventb
logPc
3 h
48 h
Product
Conversiond (%)
eep (%)
Ef
Conversiond (%)
eep (%)
Ef
e
e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
1,4-Dioxane
Methanol
Acetonitrile
Ethanol
ꢀ1.10
ꢀ0.76
ꢀ0.33
ꢀ0.24
ꢀ0.23
0.49
0.60
0.85
2.50
3.10
14.8
6.9
34.0
50.8
22.6
54.6
35.1
44.5
23.0
12.9
38.8
28.0
12.0
23.9
12.1
16.3
10.9
35.2
69.3
61.1
63.7
9.2
2.04
3.08
1.70
4.30
2.06
2.69
1.60
1.31
2.33
1.83
1.29
1.70
1.33
1.45
1.31
2.40
5.73
4.26
4.83
1.26
2.23
1.78
7.95
6.80
59.7
7.7
3.8
44.2
3.4
0.6
1.5
3.6
15.3
3.2
4.5
2.9
1.17
2.60
1.10
1.14
1.07
1.16
1.44
1.07
1.11
1.10
1.07
1.05
1.77
1.67
2.42
1.29
12.67
13.77
17.24
1.18
2.84
67.50
16.66
8.69
(+)-1
(+)-1
(+)-1
(ꢀ)-1
(ꢀ)-1
(+)-1
(+)-1
(ꢀ)-1
(+)-1
(+)-1
(ꢀ)-1
(ꢀ)-1
(+)-2
(+)-2
(+)-2
(ꢀ)-2
(+)-2
(+)-2
(+)-2
(+)-2
(+)-2
(+)-2
(+)-2
(+)-2
10.5
47.9
12.2
21.6
17.7
28.1
20.1
19.7
18.2
29.0
27.6
19.4
16.6
56.7
22.0
22.1
28.9
33.1
23.7
37.7
23.5
23.0
39.5
78.8
69.2
62.3
58.2
68.1
33.2
40.6
27.2
26.0
72.0
24.8
80.0.
95.4
42.8
47.5
84.3
76.2
59.7
59.5
67.2
42.4
Acetone
Tetrahydrofuran
Dichloromethane
Ethyl ether
Toluene
Xylene
Cyclohexane
Hexane
1,4-Dioxane
Methanol
Acetonitrile
Ethanol
3.20
3.50
2.3
1.8
ꢀ1.10
ꢀ0.76
ꢀ0.33
ꢀ0.24
ꢀ0.23
0.49
0.60
0.85
2.50
3.10
20.1
25.7
27.5
0.3
82.7
83.4
79.3
0.7
39.8
95.9
83.3
76.0
Acetone
Tetrahydrofuran
Dichloromethane
Ethyl ether
Toluene
Xylene
Cyclohexane
Hexane
37.8
26.7
76.6
73.3
3.20
3.50
a
The hydrolysis procedure is described in Section 4.
Organic solvents were prepared according to the general procedure described in Section 4.
Ref. 1.
b
c
d
e
Conversion rates were determined based on HPLC analysis using a chiral column.
Enantiomeric excess values were determined based on HPLC analysis using a chiral column.
h
h
i
i
1ꢀee
s
ln
ln
1þðee =ee
Þ
Þ
s
p
f
E ¼
.
1ꢀee
s
1þðee =ee
s
p
70
70
3 hours
48 hours
3 hours
48 hours
60
20
60
20
10
0
10
0
E
E
*
*
*
*
*
*
Figure 2. Trends in E (enantiomeric ratio) values in the hydrolysis of rac-3 (a) and rac-4 (b) by PPL in organic solvents. Asterisks indicate that the opposite enantiomer is in
excess.

1650
L.-L. Shen et al. / Tetrahedron: Asymmetry 19 (2008) 1647–1653
the reaction proceeded, the percentage of conversion to (+)-1 in-
creased, but the enantiomeric excess (ee) value for (+)-1 decreased,
resulting in small E values (Fig. 2a). This racemization of products
indicates that the conversion rate to (+)-1 is higher than that to
(ꢀ)-1, but the differences in their rates were not great enough to
result in kinetic resolution. With rac-4, enantioselectivity was
higher than for rac-3 in most non-polar organic solvents. The gen-
eral conversion rate to (+)-2 was much higher than that to (ꢀ)-2,
resulting in high ee% values. However, the absolute stereochemis-
try was not confirmed experimentally, even though (ꢀ)-1 was ob-
tained. The stereochemistry of (+)-2 has been reported.20 The
conversion percentage in the 12 organic solvents ranged from 3
to 48 h, with little change in the ee% (enantiomeric excess) values.
Particularly in xylene, rac-4 was enantioselectively converted to
(+)-2 by PPL-catalyzed hydrolysis with a high E value, 67.5, at
48 h. This high selectivity demonstrates that PPL maintains enan-
tioselective reactivity even in neat organic solvents.
2.2.2. PPL-catalyzed hydrolysis in aqueous buffer and organic
solvents
The reaction solvent gradually acidifies as the monobenzylated
epoxy esters 3 and 4 are hydrolyzed to the corresponding alcohols
and acids. Giner et al. described in a biomimetic study that trans
epoxy alcohols can be converted to 2-methyl-D-erythritol via
[2,2,1]-bicyclic orthoester intermediate rearrangements under
acidic conditions.24 Accordingly, we designed a solvent system in
Table 2
PPL-catalyzed hydrolysis in a mixture of buffer (pH 7.2, phosphate) and organic solvent (1:1, v/v)a
Entry
Substrate
Solventb
logPc
3 h
48 h
Product
Conversiond (%)
eep (%)
Ef
Conversiond (%)
eep (%)
Ef
e
e
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
3
3
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
1,4-Dioxane
Methanol
Acetonitrile
Ethanol
ꢀ1.10
ꢀ0.76
ꢀ0.33
ꢀ0.24
ꢀ0.23
0.49
0.60
0.85
2.50
3.10
88.6
84.4
94.8
89.9
82.2
28.2
86.6
88.8
81.1
61.4
88.7
67.2
16.4
32.6
11.2
24.9
33.7
11.9
28.0
76.7
89.9
89.8
91.9
88.7
44.7
68.7
60.4
40.2
16.8
64.5
19.9
17.0
89.6
12.9
73.9
1.1
23.7
58.5
18.5
57.5
63.5
22.7
39.5
97.0
93.4
95.3
96.1
89.0
3.07
6.41
11.26
4.85
1.52
2.62
1.82
1.56
32.55
1.53
15.40
1.19
1.75
4.46
1.40
4.15
5.37
1.62
2.57
84.1
60.7
72.4
46.4
82.8
31.3
96.5
98.2
94.0
89.4
99.3
99.4
20.9
52.1
8.5
26.8
50.5
8.9
45.9
91.3
69.6
82.6
90.2
89.8
15.9
66.6
84.1
41.5
13.2
67.6
15.9
14.9
7.8
67.3
0.8
0.6
47.2
79.4
2.7
69.7
83.6
18.3
76.3
82.2
81.9
98.0
99.5
96.2
1.73
4.82
15.84
4.40
1.50
7.99
1.55
2.38
1.57
12.22
1.08
1.07
3.12
12.14
1.10
6.53
(+)-1
(ꢀ)-1
(ꢀ)-1
(ꢀ)-1
(+)-1
(+)-1
(+)-1
(+)-1
(ꢀ)-1
(+)-1
(+)-1
(+)-1
(+)-2
(+)-2
(ꢀ)-2
(+)-2
(+)-2
(ꢀ)-2
(+)-2
(+)-2
(+)-2
(+)-2
(+)-2
(+)-2
Acetone
Tetrahydrofuran
Dichloromethane
Ethyl ether
Toluene
Xylene
Cyclohexane
Hexane
1,4-Dioxane
Methanol
Acetonitrile
Ethanol
3.20
3.50
ꢀ1.10
ꢀ0.76
ꢀ0.33
ꢀ0.24
ꢀ0.23
0.49
0.60
0.85
2.50
3.10
Acetone
15.20
1.49
Tetrahydrofuran
Dichloromethane
Ethyl ether
Toluene
Xylene
Cyclohexane
Hexane
10.22
26.55
17.86
153.91
>200
129.95
99.73
58.32
48.64
103.27
34.67
3.20
3.50
a
The hydrolysis procedure is described in Section 4.
Organic solvents were distilled according to the general procedure described in Section 4 prior to mixing with buffer.
Ref. 1.
b
c
d
e
Conversion rates were determined based on HPLC analysis using a chiral column.
Enantiomeric excesses values were determined based on HPLC analysis using a chiral column.
h
h
i
i
1ꢀee
s
ln
1þðee =ee
Þ
s
p
f
E ¼
.
1ꢀee
s
ln
1þðee =ee
Þ
s
p
200
200
3 hours
48 hours
100
60
100
60
3 hours
48 hours
40
20
0
40
20
0
*
*
*
*
*
*
Figure 3. Trends in E (enantiomeric ratio) values in the hydrolysis of rac-3 (a) and rac-4 (b) by PPL in phosphate buffer (pH 7.2) and a 1:1 (v/v) organic solvent mixture.
Asterisks indicate that the opposite enantiomer is in excess.

L.-L. Shen et al. / Tetrahedron: Asymmetry 19 (2008) 1647–1653
1651
which the acid product could be neutralized immediately to avoid
further cleavage of the oxirane ring in the substrates. Here, a phos-
phate buffer (pH 7.2) was introduced and mixed with different or-
ganic solvents in a 1:1 volume ratio for optimization of the PPL-
catalyzed hydrolysis reactions at 25 °C.
organic solvents. We discovered that longer reaction times, for
example, 120 h, can produce an efficient kinetic resolution with
rac-3, as shown in Fig. 4. The steep slope of rac-4 indicates that
PPL slowly, enantioselectively hydrolyzes the trans-isomers in neat
buffer solutions. Under these circumstances, the enzyme selectiv-
ity for the cis-compound rac-4 was lower than that for the trans-
compound, although the conversion rate slowly increased from 3
h to 120 h. The enantioselective excess values and the conversion
rates of rac-3 hydrolysis improved steadily until the E value
reached 87.6 at 120 h. Therefore, we conclude that in aqueous
media,trans-compounds are better substrates for PPL, and the
enzyme can distinguish enantiomers of the trans compounds
better than those of cis compounds.
Figure 3 shows the improved kinetic resolution that occurred in
aqueous buffer–organic solvents. As summarized in Table 2, both
rac-3 and rac-4 showed more efficient conversions than in neat
organic solvents. The conversion efficiency of rac-3 in the aque-
ous–organic solutions was higher, with larger ee% values than in
organic solvents. In homogeneous mixed-solvent systems, such
as 1,4-dioxane, methanol, acetonitrile, ethanol, and acetone, the
conversion efficiencies of rac-3 were even better than for rac-4 in
the same solvent systems. Nevertheless, the enantioselectivity for
rac-3 was not high enough for an efficient kinetic resolution in
these solvent systems (Fig. 3a). In biphasic solvent systems (aque-
ous buffer mixed with dichloromethane, ethyl ether, toluene,
xylene, cyclohexane, or hexane), the conversion of rac-3 was also
highly effective at 3 and 48 h, but the ee% was much lower at
48 h, except in the xylene biphasic system. In the xylene buffer sys-
tem, the conversion percentage and ee% increased steadily through
48 h.
In contrast, the reactivity of PPL toward rac-4 was significantly
different from that of rac-3. In mixed-solvent systems containing
relatively polar organic solvents, whose log P values were lower
than 1.0, the conversion of rac-4 to (+)-2 was less efficient than
the conversion of rac-3 to (+)-1. However, in solvent systems con-
taining the non-polar organic solvents ethyl ether and hexane, the
conversion efficiency and ee% of rac-4 were both large, resulting in
large E values (Fig. 3b). Among these non-polar solvents, the sol-
vent system containing cyclohexane exhibited the largest E value,
>200, at 48 h. Hence, the results indicate that PPL enantioselective-
ly catalyzes the hydrolysis of rac-4 in an organic buffer mixed-sol-
vent system with a highly non-polar organic solvent. These solvent
mixtures are ideal environments for the kinetic resolution of rac-4.
3. Conclusions
We have found that PPL has a discriminating hydrolysis reactiv-
ity toward substrates depending on the enzymatic medium, even
when the substrates have very similar molecular skeletons. In neat
organic solvent and organic–aqueous systems, the reactivity of PPL
is more suited for the hydrolysis of the rac-4 (cis) compound than
the rac-3 (trans) compound. The kinetic resolution of rac-4 and its
corresponding alcohol (+)-2 was obtained efficiently in an organic
buffer solvent system, in particular cyclohexane, hexane, and
xylene biphasic systems. In contrast, in aqueous buffer media
(pH 7.2), the reactivity of PPL toward rac-3 was ideal for the kinetic
resolution of alcohol (+)-1. In view of these results, we conclude
that in the presence of organic solvents, PPL adopts a structure that
is more suitable for the hydrolysis of rac-4, but in aqueous buffer
media, the enzyme retains its native structure, which is better
for the hydrolysis of rac-3. Hence, the extent of the aqueous nature
of the medium can be an important factor in choosing hydrolysis
enzymatic media for kinetic resolution. By manipulating the water
content in the enzymatic media, a more efficient kinetic resolution
can be achieved.
2.2.3. PPL-catalyzed hydrolysis in buffer aqueous solutions
The enantioselective hydrolysis of the racemic oxirane com-
pounds rac-3 and rac-4 by PPL was performed in the phosphate
buffer (pH 7.2) at 25 °C (Fig. 4). In aqueous media, hydrophobic
substrates drift as microscopic granules until they encounter en-
zymes. Therefore, the hydrolysis of organic compounds in water
generally proceeds very slowly. As anticipated, the observed reac-
tion rates were much slower than in solvent systems containing
4. Experimental
4.1. General
Chemical reagents and PPL were obtained from Aldrich (Mil-
waukee, WI, USA) and Sigma (St. Louis, MO, USA), respectively.
Tetrahydrofuran and ethyl ether were distilled over sodium metal
and benzoquinone under argon; dichloromethane, acetonitrile, and
toluene were distilled over calcium hydride. Other solvents were
purchased from Aldrich, J. T. Baker (Phillipsburg, NJ, USA), and Fish-
er (Hampton, NH, USA) as absolute grades. Enzymatic reactions
were performed in a Jeio Tech (Seoul, Korea) SI-300R incubatory
orbital shaker at 160 rpm and 25 °C. Conversion rates and enantio-
meric excess values were determined by chiral HPLC analysis
(1200 LC; Hewlett–Packard, Palo Alto, CA, USA), using a Chiracel
OD column (250 ꢁ 4.6 mm i.d.; Daicel Chemical Industries Ltd.,
Osaka, Japan) and a general mobile phase of a mixture of hexane
and isopropanol (86:14) with a 1 ml/min flow rate. Optical rota-
tions were measured using a digital polarimeter (DIP-1000; Jasco,
Tokyo, Japan). NMR spectra were recorded on a spectrometer
(Avance 400; Bruker, Bremen, Germany; NMR 500; Varian, Palo
Alto, CA, USA), using CDCl3 as the solvent. Chemical shifts are given
in ppm relative to the tetramethylsilane signal. Coupling constants
are given in Hertz. IR spectra were taken on a Jasco 420 FT-IR
spectrometer. Thin-layer chromatography was performed using
pre-coated silica gel plates (Merck 60 F254; Merck, Darmstadt,
Germany). Column chromatography was performed on Silica Gel
60 (0.040–0.063 mm, 230–400 mesh; Merck), employing hexane
and ethyl acetate as developing solvents.
100
rac-3
rac-4
80
60
40
20
0
E
0
20
40
60
80
100
120
140
hours
Figure 4. PPL-catalyzed hydrolysis of rac-3 and rac-4 in buffer solution (pH 7.2).

1652
L.-L. Shen et al. / Tetrahedron: Asymmetry 19 (2008) 1647–1653
4.2. Preparation of (cis-3-(benzyloxymethyl)oxirane-2-
yl)methyl acetate rac-4
CDCl3) d: 137.86, 132.37, 128.49, 128.36, 127.88, 72.53, 65.68,
and 58.80.
A CH2Cl2 (40 ml) solution of 5 (2.425 g, 13.61 mmol) was added
to NaHCO3 (2172 mg, 25.85 mmol) at 0 °C in one portion and stir-
red for 5 min. Then, mCPBA (2818 mg, 16.33 mmol) was added to
the suspension and stirred for 3 h, during which time the temper-
ature rose to room temperature. The solution was quenched with
saturated aqueous NaHCO3 (25 ml) and diluted with CH2Cl2. The
organic layer was separated and the aqueous layer extracted three
times with CH2Cl2. The combined organic layer was dried with
brine and anhydrous MgSO4. The solvent was evaporated in vacuo,
and the residue was separated by column chromatography (3:1 n-
hexane/EtOAc) to afford the oxirane compound as a colorless
oil, with a 78% yield. The CH2Cl2 solution (40 ml) of the oxirane
compound (2062 mg, 10.61 mmol) was pyridine (1.28 ml,
15.94 mmol), DMAP (259.64 mg, 2.12 mmol), and (CH3CO)2O
(1.30 ml, 12.74 mmol) at 0 °C, and it was stirred for 6 h at room
temperature. Then, the mixture was diluted with dichloromethane
(40 ml) and washed with water. The organic layer was combined,
the solvent was evaporated in vacuo, and the residue was sepa-
rated by column chromatography (4:1 n-hexane/EtOAc) to afford
1.318 g acetylated racemic epoxide 4 in 99% yield. 1H NMR
(400 MHz, CDCl3) d: 7.31 (m, 5H, –C6H5), 4.03 (q, 2H, –O–CH2–
4.5. Preparation of (trans-3-(benzyloxymethyl) oxiran-2-yl)-
methyl acetate rac-3
A CH2Cl2 (40 ml) solution of 7 (2.425 g, 13.61 mmol) was added
to NaHCO3 (2172 mg, 25.85 mmol) at 0 °C in one portion and stir-
red for 5 min. Then, mCPBA (2.818 g, 16.33 mmol) was added to
the suspension and stirred for 3 h, during which time the temper-
ature rose to room temperature. The solution was quenched with
saturated aqueous NaHCO3 (25 ml) and diluted with CH2Cl2. The
organic layer was separated and the aqueous layer extracted three
times with CH2Cl2. The combined organic layer was dried with
brine and anhydrous MgSO4, the solvent was evaporated in vacuo,
and the residue was separated by column chromatography (3:1 n-
hexane/EtOAc) to afford 2.062 g of the oxirane compound with 78%
yield as a colorless oil. The CH2Cl2 solution (40 ml) of the oxirane
compound (2.062 g, 10.61 mmol) was pyridine (1.28 ml,
15.94 mmol), DMAP (259.64 mg, 2.12 mmol), and (CH3CO)2O
(1.30 ml, 12.74 mmol) at 0 °C and was stirred for 6 h at room tem-
perature. Then, the mixture was diluted with dichloromethane
(40 ml) and washed with water. The organic layer was combined,
the solvent evaporated in vacuo, and the residue was separated
by column chromatography (4:1 n-hexane/EtOAc) to afford
1.318 g of acetylated racemic epoxide 3 with 99% yield. 1H NMR
(500 MHz, CDCl3) d: 7.35 (m, 5H, –C6H5), 4.59 (q, 2H, –O–CH2–
4
2
C6H5, Jam = 12.3 Hz, Jaa = 33.1 Hz), 4.32 (q, 1H, –H(O)C–CH2–O–,
3
4Jam = 12.3 Hz, Jab-trans = 3.8 Hz), 4.03 (q, 1H, –H(O)C–CH2–O–,
3
4Jam = 12.3 Hz, Jab-cis = 7.0 Hz), 3.71 (q, 1H, –O–CH2–C(O)H–,
3
4
2
3Jab-trans = 4.1 Hz), 3.59 (q, 1H, –O–CH2–C(O)H–, Jab-cis = 6.1 Hz),
C6H5, Jam = 12.3 Hz, Jaa = 33.1 Hz), 4.33 (q, 1H, –H(O)C–CH2–O–,
3
3
3
3.27 (m, 2H, –C(O)H–C(O)H–, Jab-epoxide = 5.1 Hz, Jab-cis = 6.1 Hz,
4Jam = 12.3 Hz, Jab-trans = 3.83 Hz), 4.03 (q, 1H, –H(O)C–CH2-O–,
3Jab-trans = 4.1 Hz, Jab-cis = 7.0 Hz, Jab-trans = 3.8 Hz), 2.09 (t, 3H,
CH3); 13C NMR (100 MHz, CDCl3) d: 170.69, 137.60, 129.82,
128.49, 127.90, 127.81, 126.85, 71.52, 67.83, 62.60, 54.73, 53.02,
and 20.73.
4Jam = 12.3 Hz, Jab-cis = 6.98 Hz), 3.72 (q, 1H, –O–CH2–C(O)H–,
3
3
3
3Jab-trans = 4.14 Hz), 3.60 (q, 1H, –O–CH2–C(O)H–, Jab-cis = 6.06 Hz),
3
3.28 (m, 2H, –C(O)H–C(O)H–, 3Jab-epoxide = 5.14 Hz, 3Jab-cis = 6.06 Hz,
3Jab-trans = 4.14 Hz, Jab-cis = 6.98 Hz, Jab-trans = 3.83 Hz), 2.10 (t,
3H, CH3); 13C NMR (125 MHz, CDCl3) d: 171.01, 128.75,
128.72, 128.16, 128.08, 128.03, 73.64, 68.08, 66.31, 54.96, 52.97,
and 21.02.
3
3
4.3. Preparation of trans-4-(benzyloxy)but-2-enal 6
A 75-ml CH2Cl2 solution of 5 (1.964 g, 11.02 mmol) was added
to Dess–Martin periodinane22 (5.610 g, 13.22 mmol) and stirred
at room temperature for 20 h. After filtering the reaction mixture,
the solvent was evaporated and the residue separated immediately
by column chromatography with a mixture of n-hexane and ethyl
acetate (10:1) as a developing solvent. Then, 1.63 g of a colorless
liquid product was obtained with 84% yield. 1H NMR (400 MHz,
4.6. General procedure for PPL-catalyzed hydrolysis
Racemic compound 3 or 4 (1.066 g, 8.49 mmol) was dissolved in
solvent (25 ml) and fixed in an incubatory orbital shaker at 25 °C.
PPL (1066 mg) was added to the solution and rotated at 160 rpm.
The solution was filtered with a Celite-packed glass filter to
remove the PPL, and the organic layer was separated. The organic
layer was washed with 20 ml of distilled water and then dried with
brine and anhydrous magnesium sulfate. Then, the solvent was
evaporated in vacuo and separated by chromatography (3:1 n-hex-
ane/EtOAc) to afford chiral epoxide 1 or 2.
3
CDCl3) d: 9.56 (d, 1H, O@CH, Jab = 7.8 Hz), 7.34 (m, 5H, –C6H5),
3
3
4
6.86 (m, 1H, –CH@CH–O–, Jab-trans = 15.8 Hz, Jab = 7.8 Hz, Jam
=
4.0 Hz), 6.41 (m, 1H, O@C(H)–CH@CH–, 3Jab-trans = 15.8 Hz,
3Jab = 4.0 Hz), 4.60 (s, 2H, –O–CH2–C6H5), 4.30 (q, 2H, @CH–CH2–
O–, Jab = 4.0 Hz, Jam = 4.0 Hz); 13C NMR (125 MHz, CDCl3) d:
193.64, 147.09, 137.36, 131.72, 129.00, 128.53, 127.99, 127.90,
127.70, 72.88, and 68.52.
3
4
For 1: 1H NMR (400 MHz, CDCl3) d: 7.33 (m, 5H, –C6H5), 4.55 (q,
4
2
2H, –O–CH2–C6H5, Jam = 11.9 Hz, Jaa = 34.0 Hz), 3.64 (m, 4H,
3
3
–H(O)C–CH2–OH, –H(O)C–CH2–O–, Jab = 5.4 Hz, Jab = 6.0 Hz),
3.26 (m, 1H, –C(O)H–C(O)H–, Jab-epoxide = 9.1 Hz, Jab = 5.4 Hz),
3
3
4.4. Preparation of trans-4-benzyloxy-but-2-en-1-ol 7
3
3
3.19 (m, 1H, –C(O)H–C(O)H–, Jab-epoxide = 9.1 Hz, Jab = 6.0 Hz),
2.83 (s, 1H, –OH); 13C NMR (100 MHz, CDCl3) d: 137.49, 131.64,
129.80, 128.53, 127.98, 127.89, 73.42, 68.06, 60.58, 55.90, 54.92.
Aldehyde compound 6 (624 mg, 3.55 mmol) was dissolved in
methanol (12 ml), and NaBH4 (268.5 mg, 7.1 mmol) was added to
the solution at 0 °C. Then, the solution was stirred for 20 min at
room temperature. The solvents were evaporated and the residue
was diluted with ether. The crude solution was extracted with
NaHCO3 (5 ml), distilled water (5 ml), and brine (5 ml). The organic
extracts were dried with MgSO4, evaporated in vacuo, and chro-
matographed (3:1 n-hexane/EtOAc), with an 82% yield. 1H NMR
(400 MHz, CDCl3) d: 7.34 (m, 5H, –C6H5), 7.26 (s, 1H, –OH), 5.81
ee% 92.3%; ½a 2D4
¼ þ21:65 (c 0.56, EtOAc), HPLC chiral column
ꢂ
retention time, 25.2 min.
For 2: 1H NMR (400 MHz, CDCl3) d: 7.35 (m, 5H, –C6H5), 4.55 (q,
4
2
2H, –O–CH2–C6H5, Jam = 11.8 Hz, Jaa = 34.6 Hz), 3.64 (m, 4H,
3
3
–H(O)C–CH2–OH, –H(O)C–CH2–O–, Jab = 5.0 Hz, Jab = 6.0 Hz),
3.29 (m, 1H, –C(O)H–C(O)H–, Jab-epoxide = 17.0 Hz, Jab = 5.0 Hz),
3.23 (m, 1H, –C(O)H–C(O)H–, Jab-epoxide = 17.0 Hz, Jab = 6.0 Hz),
2.11 (s, 1H, –OH); 13C NMR (100 MHz, CDCl3) d: 137.37, 128.58,
128.05, 127.91, 73.52, 68.09, 60.76, 54.74, and 54.26. ee% 99.5%;
3
3
3
3
3
3
(m, 1H, –CH2–CH@CH–CH2–, Jab-trans = 17.8 Hz, Jab = 6.0 Hz), 5.76
3
3
(m, 1H, –CH2–CH@CH–CH2–, Jab-trans = 17.8 Hz, Jab = 6.2 Hz), 4.53
(s, 2H, –O–CH2–C6H5), 4.18 (d, 2H, HO–CH2–CH=, Jab = 6.2 Hz),
½
a 2D4
18.7 min.
ꢂ
¼ þ20:5 (c 0.45, EtOAc), HPLC chiral column retention time,
3
3
4.10 (d, 2H, @CH–CH2–O–, Jab = 6.0 Hz); 13C NMR (100 MHz,

L.-L. Shen et al. / Tetrahedron: Asymmetry 19 (2008) 1647–1653
1653
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Acknowledgments
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This study was supported by the Seoul R&BD Program, a Grant
from the Brain Korea 21 Project, and Kyung Hee University.
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