Candida antarctica lipase B-catalyzed reactions of β-hydroxy esters: Competition of acylation and hydrolysis

Article abstract of DOI:10.1016/j.molcatb.2013.09.024

The ester function of ethyl cis-(±)-2-hydroxycyclopentane-1- carboxylate [(±)-1] and ethyl (±)-5-hydroxycyclopent-1- enecarboxylate [(±)-2] was demonstrated to undergo hydrolysis, as a side-reaction, during asymmetric (E > 200) O-acylation with Candida antarctica lipase B (CAL-B) as catalyst and vinyl acetate as acyl donor in t-BuOMe at 30 C. This competition of acylation and undesirable hydrolysis draws attention to CAL-B-catalyzed non-hydrolytic resolutions where the substrates contain any hydrolysable functions. Enantiomerically enriched cis-2-hydroxycyclopentane-1-carboxylic acid (ee = 90%) and 5-hydroxycyclopent-1- enecarboxylic acid (ee = 47%) were prepared through de novo CAL-B-catalyzed hydrolysis of (±)-1 and (±)-2 with added H₂O in t-BuOMe at 30 C.

Full text of DOI:10.1016/j.molcatb.2013.09.024

Journal of Molecular Catalysis B: Enzymatic 98 (2013) 92–97
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Candida antarctica lipase B-catalyzed reactions of ˇ-hydroxy esters:
Competition of acylation and hydrolysis
Eniko˝ Forró∗, Zsolt Galla, Ferenc Fülöp∗
Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös u 6, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The ester function of ethyl cis-( )-2-hydroxycyclopentane-1-carboxylate [( )-1] and ethyl ( )-5-
hydroxycyclopent-1-enecarboxylate [( )-2] was demonstrated to undergo hydrolysis, as a side-reaction,
during asymmetric (E > 200) O-acylation with Candida antarctica lipase B (CAL-B) as catalyst and vinyl
acetate as acyl donor in t-BuOMe at 30 ◦C. This competition of acylation and undesirable hydrolysis draws
attention to CAL-B-catalyzed non-hydrolytic resolutions where the substrates contain any hydrolysable
functions. Enantiomerically enriched cis-2-hydroxycyclopentane-1-carboxylic acid (ee = 90%) and 5-
hydroxycyclopent-1-enecarboxylic acid (ee = 47%) were prepared through de novo CAL-B-catalyzed
hydrolysis of ( )-1 and ( )-2 with added H2O in t-BuOMe at 30 ◦C.
Received 10 July 2013
Received in revised form
24 September 2013
Accepted 25 September 2013
Available online 14 October 2013
Keywords:
Acylation
CAL-B
© 2013 Elsevier B.V. All rights reserved.
Hydrolysis
ˇ-Hydroxy acid
ˇ-Hydroxy ester
1. Introduction
the O-acylated enantiomer was obtained with a yield of 35% [2d].
That relatively modest yield is not surprising if hydrolysis of the C1
Candida antarctica lipase B (CAL-B), with broad substrate speci-
ficity [1], is an excellent catalyst for a wide range of regio- and
enantioselective transformations through various types of reac-
tions such as acylation [2], transesterification [3], hydrolysis [4],
desymmetrization of prochiral compounds [1f,5], polymerization
[6], etc. Some of the reported acylation substrates contain a COOR
function next to the reactive OH or NH group [2b,2d,2g], and
this COOR might possibly undergo hydrolysis as a side-reaction
under certain conditions, although no such observations have been
noticed.
Our earlier results on the Burkholderia cepacia lipase (PSIM)-
and CAL-B-catalyzed reactions of carbocyclic [7] and acyclic [8] ˇ-
amino esters and hydroxylated ˇ-amino esters [9] suggested the
possibility that these, above-mentioned ˇ-hydroxy and ˇ-amino
esters underwent hydrolysis. This supposition also emerges from
the results published by Gotor et al. [2d] on the lipase-catalyzed
enantioselective O-acylation of cyclic cis- and trans-ˇ-hydroxy
esters. As a particular case, excellent enantioselectivity (E > 200)
was observed when the R-selective acylation of ethyl cis-( )-
2-hydroxycyclopentane-1-carboxylate was performed with vinyl
acetate (VA) in the presence of CAL-B in t-BuOMe at 30 ◦C, whereas
ester function occurs as a side-reaction.
We set out to obtain definite answers through the use of
examples of pharmacological importance. The earlier-mentioned
ethyl cis-( )-2-hydroxycyclopentane-1-carboxylate and (in view
of the pharmacological importance of its unsaturated homo-
logue, 5-hydroxycyclopent-1-enecarboxylic acid) ethyl
( )-5-
hydroxycyclopent-1-enecarboxylate were selected as model com-
pounds for the enzymatic experiments. It may be mentioned that
5-hydroxycyclopent-1-enecarboxylic acid is of considerable syn-
thetic potentiality as a versatile chiral building block, e.g. for
the synthesis of the naturally occurring (+)-mitsugashiwalactone
and (−)-dolichodial [10], or an intermediate of (+)-vincamine
[11].
The enzymatic preparation of optically pure ethyl 5-
hydroxycyclopent-1-enecarboxylate has been described through
both O-acylation of the racemic ˇ-hydroxy ester and enantio-
selective hydrolysis of the racemic O-acetylated ester. Excellent
enantioselectivities (E > 200) were obtained both when the R-
selective acylation was carried out with Pseudomonas cepacia
lipase PS on celite as catalyst and VA as acyl donor, and when
the reaction was performed in t-BuOMe at room temperature
(RT), or when the lipase PS-catalyzed R-selective hydrolysis was
performed in 0.1 M phosphate buffer:acetone (9:1) at RT [12].
It is noteworthy that no hydrolysis of the C1 ester function was
reported during either the acylation or the hydrolysis carried out
with lipase PS.
∗
Corresponding authors. Tel.: +36 62 545564; fax: +36 62 545705.
E-mail addresses: Forro.Eniko@pharm.u-szeged.hu (E. Forró),
fulop@pharm.u-szeged.hu (F. Fülöp).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.09.024

E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 92–97
93
Scheme 1. CAL-B-catalyzed O-acetylation of ( )-1 and ( )-2.
In the present work, the primary challenge was to demon-
strate the occurrence of hydrolysis of the C1 ester function (which
might occur due to the presence of water in the organic sol-
vent or on the surface of the enzyme) during the O-acetylation
of ethyl cis-( )-2-hydroxycyclopentane-1-carboxylate ( )-1 and
ethyl ( )-5-hydroxycyclopent-1-enecarboxylate ( )-2 with CAL-
B as catalyst. Although exact data have not been given, it is well
known that the water content at the surface of the differently
immobilized forms of CAL-B (Novozyme 435 from Novo Nordisk,
Chirazyme L-2 from Roche, Lipolase from Sigma–Aldrich, etc.) is in
the range 2–5%. This information might be of considerable impor-
tance as regards the hydrolytic processes. If the above-mentioned
hydrolysis could be successfully demonstrated, we hoped to devise
an adequate enzymatic strategy for the preparation of cis-2-
hydroxycyclopentane- and 5-hydroxycyclopent-1-enecarboxylic
acid enantiomers through the enzyme-catalyzed hydrolyses of ( )-
1 and ( )-2.
30 ◦C. After a reaction time of 30 min, besides the O-acetylated
product [(1S,2R)-3, ee > 98%] and the unreacted ˇ-hydroxy ester
[(1R,2S)-1, ee ∼ 35%,] a considerable amount of hydroxy acid
[(1S,2R)-5, ee > 98%] was detected (Fig. 1). The conversion (∼30%)
was calculated [15] through the use of n-heptadecane (retention
time: 23.18 min) as an internal standard.
Next,
a small-scale acetylation of ( )-2 (Scheme 1) was
performed under the above-mentioned conditions (CAL-B, VA, t-
BuOMe, 30 ◦C). The derivatized (CH2N2) enzymatic sample, taken
after a reaction time of 1 h, was analyzed by GC [Chrompack
Chiralsil-Dex CB column (25 m) [80 ◦C for 10 min → 190 ◦C (tem-
perature rise 10 ◦C min−1), 0.9 mL min−1; retention times (min);
(R)-6: 16.36 (antipode: 16.26), (S)-2: 17.04 (antipode: 17.10);
(R)-4: 18.96 (antipode: 18.85), internal standard (1S,6R)-7-
azabicyclo[4.2.0]oct-3-en-8-one [16]: 22.07]. In this case, the
hydrolysis of the C1 ester group proved to be the main reaction;
after a reaction time of 1 h, the conversion reached 30%, but rela-
tively low ee values were obtained (ee2 ∼ 20% and ee6 = 39.8%), with
the exception of that for the O-acylated product (ee4 > 98%) (Fig. 2).
When VA was used in a higher excess (3, 5, 10, 25 or 50 equiv.),
the hydrolysis of ( )-2 could be suppressed (after a reaction time
of 1 h, the hydrolysis conversion was ∼9% at 3 equiv. and <1% at
5 equiv. vs. ∼30% at 2 equiv.) [17].
2. Results and discussion
Ethyl cis-( )-2-hydroxycyclopentane-1-carboxylate [( )-1]
was synthetized from ethyl 2-oxocyclopentane-1-carboxylate by
NaBH4 reduction, followed by separation of the resulting cis and
trans diastereomers, according to a literature method [13]. Ethyl
Since the CAL-B-catalyzed asymmetric acetylation of ( )-2 has
not been described to date, a preparative-scale acetylation of ( )-2
was performed with 5 equiv. of VA (Table 5 and Section 4).
Absolute configurations were proved by comparing the [␣]
value of the unreacted 5-hydroxycyclopent-1-enecarboxylate
{[␣]D25 = −34 (c = 0.31; CHCl3)} with the literature value [10]
for (S)-5-hydroxycyclopent-1-enecarboxylate {[a]D31 = −34.5
(c = 1.10; CHCl3}; thus, the CAL-B-catalyzed acetylation of ( )-2
displayed R selectivity.
(
)-5-hydroxycyclopent-1-enecarboxylate [( )-2] was also syn-
thetized by a literature method, from succinaldehyde and triethyl
phosphonoacetate in aqueous potassium carbonate solution [12].
2.1. CAL-B-catalyzed acetylation of ethyl
cis-( )-2-hydroxycyclopentane-1-carboxylate [( )-1] and ethyl
(
)-5-hydroxycyclopent-1-enecarboxylate [( )-2]
Before the O-acetylation of ( )-1 under the reaction conditions
described by Gotor et al. (CAL-B, VA, t-BuOMe, 30 ◦C), an appro-
priate analytical method was required with which to follow not
only the progress of the acylation, but also the possible hydrolysis
(Scheme 1).
2.2. Enzyme-catalyzed hydrolysis of ethyl
cis-( )-2-hydroxycyclopentane-1-carboxylate [( )-1] and ethyl
(
)-5-hydroxycyclopent-1-enecarboxylate [( )-2]
Our earlier results on derivatization with CH2N2 (esterification
of the carboxyl function) [14] led us to apply GC with a Chrompack
Chiralsil-Dex CB column (25 m) [100 ◦C for 10 min → 190 ◦C (tem-
perature rise 5 ◦C min−1), 1.4 mL min−1; retention times (min);
(1S,2R)-5: 11.19 (antipode: 10.88), (1R,2S)-1: 12.51 (antipode:
12.73); (1S,2R)-3: 16.82 (antipode: 16.92)] (Supplementary data).
With the GC method for the enantioseparation of the desired
compounds, a small-scale CAL-B (30 mg mL−1)-catalyzed acety-
lation of ( )-1 was carried out with VA (2 equiv.) in t-BuOMe at
The results presented above led us to start preliminary experi-
ments in order to optimize the CAL-B-catalyzed hydrolyses of ( )-1
and ( )-2, the latter with stereocentre remote from the reaction
centre (Scheme 2).
First, besides CAL-B, several lipases, such as PSIM (B. cepacia), AK
(Pseudomonas fluorescens), AY (Candida rugosa), PPL (porcine pan-
creatic lipase) and CAL-A (C. antarctica lipase A), were also tested
for the hydrolysis of ( )-1 with 4 equiv. of added H2O in t-BuOMe
at 45 ◦C (Table 1). All the lipases tested exhibited some activity,

94
E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 92–97
Fig. 1. GC illustration of the competition of the acylation and hydrolysis of ( )-1.
Fig. 2. The competition of the acylation and hydrolysis of ( )-2.
OH
The moderately enantioselective hydrolysis of ( )-1 was found to
be relatively slow in n-hexane, THF and 1,4-dioxane (entries 6–8),
but slightly faster in toluene (entry 5) and CH2Cl2 (entry 9). The
reactions were fastest in ether solvents (entries 1 and 4). In spite
of the lower enantioselectivity (E = 36 vs. 40), t-BuOMe was chosen
for use in further experiments as a green solvent. As the amount of
enzyme was increased from 30 to 50 and then 75 mg mL−1 (entries
1–3), the reaction rate increased slightly, while E progressively
decreased. Thus, 30 mg mL−1 enzyme was used in the subsequent
experiments.
Both the catalytic activity and enantioselectivity of the tested
CAL-B in t-BuOMe at 45 ◦C were affected by the amount of H2O
added to the reaction mixture (Table 3). As the amount of added
H2O was increased from 0 to 15 equivalents, the reaction rate
increased, but E displayed a maximum of 22 at 10 equiv. of H2O,
OH
OH
H2O
CAL-B
COOH
COOEt
COOEt
+
COOEt
organic solvent
(1S,2R)-5
(1R,2S)-1
( )-1
OH
OH
OH
H2O
CAL-B
COOH
COOEt
+
organic solvent
(R)-6
(S)-2
( )-2
Scheme 2. CAL-B-catalyzed hydrolyses of ( )-1 and ( )-2.
but the best enantioselectivity was observed with CAL-B (entry 6),
which was therefore chosen as the catalyst for further experiments.
Next, the effects of solvents and increased amounts of CAL-B
on the enantioselectivity and reaction rate were tested (Table 2).
Table 2
Conversion and enantioselectivity of the hydrolysis of ( )-1 a
b
c
Entry Solvent
CAL-B
ee1 (%) ee5 (%) Conv. (%)
E
Table 1
(mg mL−1
)
Enzyme screening for the hydrolysis of ( )-1.a
1
2
3
4
5
6
7
8
9
t-BuOMe
t-BuOMe
t-BuOMe
Et2O
Toluene
n-Hexane
THF
30
50
75
30
30
30
30
29
31
35
21
8
4
3
3
8
93
91
86
94
83
72
78
85
43
24
25
29
18
9
5
4
3
16
36
28
18
40
12
6
8
13
3
b
c
Entry
Enzyme (30 mg mL−1
)
ee1 (%)
ee5 (%)
Conv. (%)
E
1
2
3
4
5
6
PSIM
AKd
34
15
23
3
5
29
8
35
14
35
49
93
80
30
62
8
9
24
1
2
2
2
3
AYd
PPL
CAL-Ad
CAL-B
36
1,4-Dioxane 30
CH2Cl2 30
a
0.05 M substrate, 4 equiv. of added H2O, t-BuOMe, 45 ◦C, after 20 min.
According to GC (Section 4).
According to GC, after derivatization with CH2N2 (Section 4).
Contains 20% (w/w) lipase adsorbed on celite in the presence of sucrose.
b
c
a
b
c
0.05 M substrate, 4 equiv. of added H2O, t-BuOMe, 45 ◦C, after 20 min.
According to GC (Section 4).
According to GC, after derivatization with CH2N2 (Section 4).
d

E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 92–97
95
Table 3
Conversion and enantioselectivity of hydrolysis of ( )-1.a
Table 4
Optimum temperatures for the CAL-B-catalyzed hydrolysis of ( )-1.a
b
c
b
c
Entry
H2O (equiv.)
ee1 (%)
ee5 (%)
Conv. (%)
E
Entry
Temperature (◦C)
ee1 (%)
ee5 (%)
Conv. (%)
E
1
2
3
4
5
6
7
0
1
2
30
30
31
31
36
36
33
82
84
85
88
88
79
70
27
26
27
26
29
31
32
13
15
16
21
22
12
8
1
2
3
4
5
6
7
2
25
30
45
60
70
80
5
6
90
93
93
93
88
81
63
5
6
19
29
36
33
18
11
5
29
21
17
15
12
24
18
16
16
16
4
10
12
15
a
a
0.05 M substrate, 30 mg mL−1 CAL-B, t-BuOMe, 45 ◦C, after 30 min.
According to GC (Section 4).
According to GC, after derivatization with CH2N2 (Section 4).
0.05 M substrate, 30 mg mL−1 CAL-B, 10 equiv. of added H2O, t-BuOMe, after
b
c
20 min.
b
According to GC (Section 4).
According to GC, after derivatization with CH2N2 (Section 4).
c
and therefore 10 equiv. of added water was planned to be used
for the preparative-scale reaction (entry 5). It is noteworthy that,
in accordance with earlier observations [7a,15], the reaction pro-
ceeded without the addition of H2O (entry 1); the quantity of H2O
present in the reaction medium (<0.1%) or in the enzyme prepara-
tion (2–5%) was still sufficient for the hydrolysis.
Finally, a set of preliminary experiments was performed in order
to determine the effect of temperature in the course of the hydrol-
ysis of ( )-1 (Table 4). As the temperature was increased from 2 to
25 and then 30 ◦C both E and the reaction rate increased (entries
1–3), but a further increase of temperature was accompanied by
decreases in both E and the reaction rate (entries 4–7).
When the hydrolysis of ( )-2 was performed under the con-
ditions optimized for the hydrolysis of ( )-1 (CAL-B, 10 equiv. of
added H2O, t-BuOMe, 30 ◦C) (Fig. 4), similarly modest results were
obtained as in the case of ( )-1 (ee2 = 22% and ee6 = 49%, conv. = 34%,
after 1 h, E = 4) (Fig. 3). In an attempt to improve the enantioselec-
tivity, the hydrolysis of ( )-2 was carried out without the addition
of H2O. Although the enantioselectivity did increase slightly (E = 5
vs. 4), the reaction rate decreased considerably (ee2 = 22% and
ee6 = 49%, conv. = 34%, after 1 h).
The results of preparative-scale hydrolyses of ( )-1 and ( )-2
under the optimized conditions (CAL-B, 10 equiv. of H2O, t-BuOMe,
30 ◦C) are presented in Table 5 and Section 4. Both hydroxy acids
Fig. 3. Hydrolysis of ( )-1, GC [Chrompack Chiralsil-Dex CB column, 80 ◦C for 10 min → 190 ◦C (temperature rise 10 ◦C min−1), 0.9 mL min−1].
Fig. 4. Hydrolysis of ( )-2, GC [Chrompack Chiralsil-Dex CB column, 100 ◦C for 10 min → 190 ◦C (temperature rise 5 ◦C min−1), 1.4 mL min−1].
Table 5
CAL-B (30 mg mL−1)-catalyzed preparative-scale resolution of ( )-1 and ( )-2.a
VA or H2O
T (h)
Conv. (%)
E
Product enantiomer
Unreacted enantiomer
b
25
c
25
Yield (%)
Isomer
eeP (%)
[˛]D
Yield (%)
Isomer
eeS (%)
[˛]D
(
(
(
)-1
)-2
)-2
H2O
VA
H2O
1
1.1
6
22
50
34
24
>200
4
16
46
28
(1S,2R)-5
(R)-4
(R)-6
90
>98
47
−23d
78
43
60
(1R,2S)-1
(S)-2
(S)-2
26
>98
24
+8e
−34g
−6i
+2.5f
+5h
a
b
c
d
e
f
0.05 M substrate, 5 equiv. of VA as acyl donor or 10 equiv. of added H2O as nucleophile, t-BuOMe.
According to GC, after derivatization with CH2N2 (Section 4).
According to GC.
c 0.15, H2O.
c 0.25, EtOH.
c 0.4, CHCl3.
c 0.31, CHCl3.
c = 0.2, EtOH.
c 0.18, CHCl3.
g
h
i

96
E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 92–97
{66 mg, 16%; [␣]D25 = −23 (c 0.15, H2O) ee = 90%}, as light-yellow
[(1S,2R)-5 and (R)-6] proved to be quite unstable (elimination of
water takes place), and we were therefore able to record the 1H
NMR spectrum only for (1S,2R)-5.
oils.
4.3. Preparative-scale resolution of racemic ethyl
5-hydroxycyclopent-1-enecarboxylate [( )-2] through acylation
3. Conclusions
Racemic 2 (100 mg, 0.64 mmol) and VA (0.3 mL, 3.2 mmol) in
The CAL-B-catalyzed enantioselective (E > 200) O-acylation
of ethyl cis-( )-2-hydroxycyclopentane-1-carboxylate [( )-1]
and ethyl ( )-5-hydroxycyclopent-1-enecarboxylate [( )-2] with
2 equiv. of VA in t-BuOMe at 30 ◦C has been demonstrated to be
accompanied by hydrolysis, due to the presence of H2O in the reac-
tion mixture or on the surface of the enzyme.
t-BuOMe (10 mL) were added to lipase CAL-B (0.3 g, 30 mg mL−1
)
and the mixture was shaken at 30 ◦C for 1.1 h. The reaction was
stopped by filtering off the enzyme at 50% conversion. The sol-
vent was evaporated off and the residue was subjected to column
chromatography [EtOAc:hexane (3:1)]. The O-acetylated product
(R)-4 {58 mg, 46%; [␣]D25 = +2.5 (c 0.4, CHCl3), ee > 98%; lit [10]
[␣]D29 = +2.6 (c 1.03, CHCl3), ee > 98%} and the unreacted alco-
hol (S)-2 {43 mg, 43%; [␣]D25 = −34 (c 0.31, CHCl3), ee > 98%; lit
[10] [␣]D31 = −34.5 (c 1.1, CHCl3), ee > 99%} were obtained as pale-
yellow oils.
Although the optimization of the hydrolyses of ( )-1 and
(
)-2 (CAL-B, with 10 equiv. of H2O in t-BuOMe at 30 ◦C) did
not lead to an efficient strategy for the preparation of enan-
tiopure 2-hydroxycyclopentane-1-carboxylic acid [(1S,2R)-5] and
5-hydroxycyclopent-1-enecarboxylic acid [(R)-6] in terms of ee
and efficiency requirements (ee5 = 90%, yield = 16% and ee6 = 47%,
yield = 28%), a new method has been devised for the enantiosepa-
ration of ˇ-hydroxy esters through enzyme-catalyzed hydrolysis.
It should be highlighted that, since many substrates selected for
non-hydrolytic enzymatic transformation contain a hydrolysable
function, the possibility of their hydrolysis needs to be carefully
investigated and as a possibility to avoid the side hydrolysis, the
excess of aimed reagent has also to be experimented.
4.4. Preparative-scale resolution of racemic ethyl
5-hydroxycyclopent-1-enecarboxylate [( )-2] through hydrolysis
Racemic 2 (200 mg, 1.28 mmol) was dissolved in t-BuOMe
(30 mL), and CAL-B (0.9 g, 30 mg mL−1
) and H2O (230 ␮L,
12.80 mmol) were added. The mixture was stirred at 30 ◦C for 6 h.
The reaction was stopped by filtering off the enzyme at 34% conver-
sion. The solvent was evaporated off, and the residue was subjected
to column chromatography. The resulting ˇ-hydroxy ester (S)-
2 was eluted with EtOAc, followed by the ˇ-hydroxy acid (R)-6
with MeOH. Evaporation of the corresponding fractions resulted
in (S)-2 {120 mg, 60%; [␣]D25 = −6 (c 0.18, CHCl3); ee = 24%; lit [10]
[␣]D25 = −34.5 (c 1.1, CHCl3); ee > 99, as a light-yellow oil and (R)-
6 {46 mg, 28%; [␣]D25 = +5 (c 0.2, MeOH); ee = 47%} as an unstable
colourless crystalline product [elimination of H2O (phenomenon
supported by 1H NMR spectra, data not shown)].
4. Experimental
4.1. Materials and methods
VA and lipase AY (C. rugosa) were purchased from Fluka, CAL-B
(lipase B from C. antarctica), produced by the submerged fermen-
tation of a genetically modified Aspergillus oryzae microorganism
and adsorbed on a macroporous resin, and PPL (porcine pancreatic
lipase) were from Sigma–Aldrich. Lipase PSIM (B. cepacia), immobi-
lized on diatomaceous earth was from Amano Enzyme Europe Ltd.
while CAL-A (lipase A from C. antarctica) was purchased from Roche
Diagnostics Corporation. Lipase AK (P. fluorescens) was from Amano
Pharmaceuticals. The solvents were of the highest analytical grade.
In a typical small-scale experiment, ( )-1 or ( )-2 (0.05 M solu-
tion) in an organic solvent (1 mL) was added to the lipase tested (30,
50 or 75 mg mL−1). For hydrolysis H2O (0, 1, 2, 4, 10, 12 or 15 equiv.)
and for acylation VA (2, 3, 5, 10, 25 and 50 equiv.) was added. The
mixture was shaken at the temperature tested (2, 25, 30, 45, 60,
70 or 80 ◦C). The progress of the reaction was followed by taking
samples from the reaction mixture at intervals and analysing them
by gas chromatography, as described in Section 2.
1H NMR (CDCl3, 400 MHz) ␦ (ppm) for (1R,2S)-1: 1.26–1.29 (t,
J = 7.0 Hz, 3H, CH3), 1.60–1.99 (m, 6H, 3xCH2 and br s, 1H, OH over-
lapping), 2.65–2.67 (m, 1H, CHCOOCH2CH3), 4.16–4.21 (q, J = 7.2 Hz,
2H, CH2CH3), 4.28–4.37 (d, J = 3.7 Hz, 1H, CHOH).Anal. calcd for
C8H14O3: C, 60.74; H, 8.92. Anal. found: C, 60.58; H, 9.10.
1H NMR (D2O, 400 MHz) ␦ (ppm) for (1S,2R)-5: 1.73–2.03 (m,
6H, 3xCH2), 2.93–2.98 (m, 1H, CHCOOH), 4.56 (s, 1H, CHOH). Anal.
calcd for C6H10O3: C, 55.37; H, 7.74. Anal. found: C, 55.49; H, 7.55.
1H NMR (MeOH, 400 MHz) ␦ (ppm) for (S)-2: 1.30–1.33 (t, J = 7.2,
3H, CH3), 1.63–2.66 (m, 4H, 2xCH2), 4.2–4.27 (m, 2H, CH2CH3),
4.98–5.00 (d, J = 4.7 Hz, 1H, CHOH), 6.95–6.96 (m, 1H, CH C). Anal.
calcd for C8H12O3: C, 61.52; H, 7.74. Anal. found: C, 61.50; H, 7.56.
GC chromatograms for the enantiomerically enriched hydrolytic
products {(1R,2S)-1, (1S,2R)-5, (S)-2 and (R)-6} are available in the
Supplementary data.
Optical rotations were measured with a Perkin-Elmer 341
polarimeter. 1H NMR spectra were recorded on a Bruker Avance
DRX 400 spectrometer. Melting points were determined on a Kofler
apparatus.
Acknowledgements
The authors acknowledge the receipt of grants OTKA No. NK-
81371, K-108943 and TÁMOP-4.2.2/A-11/1/KONV-2012-0035 for
financial support.
4.2. Preparative-scale resolution of racemic ethyl
2-hydroxycyclopentanecarboxylate [( )-1] through hydrolysis
Racemic 1 (500 mg, 3.16 mmol) was dissolved in t-BuOMe
Appendix A. Supplementary data
(40 mL), and CAL-B (1.2 g, 30 mg mL−1
) and H2O (569 ␮L,
31.60 mmol) were added. The mixture was stirred at 30 ◦C for 1 h.
The reaction was stopped by filtering off the enzyme at 22% conver-
sion. The solvent was evaporated off, and the residue was subjected
to column chromatography. The resulting ˇ-hydroxy ester (1R,2S)-
1 was first eluted with t-BuOMe, and the ˇ-hydroxy acid (1S,2R)-5
was then eluted with H2O. Evaporation of the corresponding frac-
tions resulted in (1R,2S)-1 {390 mg, 78%; [␣]D25 = +8 (c 0.25, MeOH),
ee = 26%; lit [2d] [␣]D25 = +22.1 (c 1, MeOH), ee > 99%} and (1S,2R)-5,
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2013.09.024.
References
[1] (a) U.T. Bornscheuer, R.J. Kazlauskas, Hydrolases in organic synthesis. Regio-
and stereoselective biotransformations, Wiley, Weinheim, 1999;

E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 98 (2013) 92–97
97
(b) A. Ghanem, H.-Y. Aboul-Enein, Tetrahedron: Asymmetry 15 (2004)
3331–3351;
(c) T.A. Paál, E. Forró, A. Liljeblad, L.T. Kanerva, F. Fülöp, Tetrahedron: Asymmetry
18 (2007) 1428–1433;
(c) A. Liljebled, L.T. Kanerva, Tetrahedron 32 (2006) 5831–5854;
(d) E. Busto, V. Gotor-Fernandez, V. Gotor, Chem. Rev. 111 (2011) 3998–4035;
(e) E. Forró, F. Fülöp, Curr. Med. Chem. 19 (2012) 6178–6187;
(f) M. Paravidino, P. Böhm, H. Gröger, U. Hanefeld, in: K. Drauz, H. Gröger, O.
May (Eds.), Hydrolysis and Formation of C O Bonds: Hydrolysis and Formation
of Carboxylic Acid Esters in Enzyme Catalysis in Organic Synthesis, Wiley-VCH
Verlag GmbH, Weinheim, 2012, pp. 249–350.
(d) Z. Cabrera, M.L.E. Gutarra, J.M. Guisan, J.M. Palomo, Catal. Commun. 11 (2010)
964–967;
(e) J.S. Reis, L.H. Andrade, Tetrahedron: Asymmetry 25 (2012) 1294–1300.
[5] E. Garcia-Urdiales, I. Alfonso, V. Gotor, Chem. Rev. 105 (2005) 313–354.
[6] A.R.A. Palmans, A. Heise, Enzymatic Polymerisation, Springer-Verlag, Berlin,
Heidelberg, 2010.
[7] (a) E. Forró, F. Fülöp, Chem. Eur. J. 22 (2007) 6397–6401;
(b) L. Kiss, E. Forró, F. Fülöp, in: A.B. Hughes (Ed.), Synthesis of Carbocyclic ␤-
Amino Acids in Amino Acids, Peptides and Proteins in Organic Chemistry, vol.
1, Wiley-VCH, Weinheim, 2009, pp. 367–409.
[8] (a) G. Tasnádi, E. Forró, F. Fülöp, Tetrahedron: Asymmetry 19 (2008)
2072–2077;
[2] (a) M.D.M. Ferrero, S. Fernández, V. Gotor, Tetrahedron: Asymmetry 13 (2002)
539–546;
(b) M. Solymár, A. Liljeblad, L. Lázár, F. Fülöp, L.T. Kanerva, Tetrahedron: Asym-
metry 13 (2002) 1923–1928;
(c) S. Acherar, G. Audran, N. Vanthuyne, H. Monti, Tetrahedron: Asymmetry 14
(2003) 2413–2418;
(d) L.M. Levy, J.R. Dehli, V. Gotor, Tetrahedron: Asymmetry 14 (2003)
2053–2058;
(e) M. Fitz, K. Lundell, F. Fülöp, L.T. Kanerva, Tetrahedron: Asymmetry 17 (2006)
(b) G. Tasnádi, E. Forró, F. Fülöp, Tetrahedron: Asymmetry 20 (2009)
1771–1777;
(c) G. Tasnádi, E. Forró, F. Fülöp, Org. Biomol. Chem. 8 (2010) 886–895;
(d) E. Forró, F. Fülöp, Tetrahedron: Asymmetry 21 (2010) 637–639;
(e) E. Forró, G. Tasnádi, F. Fülöp, J. Mol. Catal. B Enzym. 93 (2013) 8–14.
[9] E. Forró, L. Schönstein, L. Kiss, A.V. Pen˜aloza, E. Juaristi, F. Fülöp, Molecules 15
(2010) 3998–4010.
1129–1134;
(f) C. Pen˜a, J. González-Sabín, F. Rebolledo, V. Gotor, Tetrahedron: Asymmetry
19 (2008) 751–755;
(g) M.C. Turcu, E. Kiljunen, L.T. Kanerva, Tetrahedron: Asymmetry 18 (2007)
1682–1687;
(h) J. Brem, S. Pilbák, C. Paizs, G. Bánóczi, F.-D. Irimie, M.-I. Tos¸ a, L. Poppe, Tetra-
hedron: Asymmetry 22 (2011) 916–923;
[10] Y. Takahiro, T. Michiyasu, O. Kunio, Synthesis (1994) 444–448.
[11] Y. Takahiro, I. Michiyo, S. Mahito, T. Michiyasu, H. Kou, T. Seiichi, O. Kunio,
Heterocycles 42 (1996) 65–69.
[12] T. Seiichi, Y. Takahiro, T. Michiyasu, O. Kunio, Tetrahedron: Asymmetry 3 (1992)
837–840.
(i) L. Schönstein, E. Forró, F. Fülöp, Tetrahedron: Asymmetry 24 (2013) 202–206.
[3] (a) I. Itabaiana Jr., L.S. de Mariz e Miranda, R.O.M.A. de Souza, J. Mol. Catal. B
Enzym. 85–86 (2013) 1–9;
[13] G. Bernath, G. Gondos, P. Marai, L. Gera, Acta Chim. Acad. Sci. Hung. 74 (1972)
471–478.
(b) J. Oterra, J. Nishikido, Reaction of Alcohols with Carbocyclic acids and Their
Derivatives in Esterification. Methods, Reactions and Applications, Wiley-VCH,
Weinheim, 2010, pp. 52–100.
[14] E. Forró, J. Chromatogr. A 1216 (2009) 1025–1029.
[15] E. Forró, F. Fülöp, Org. Lett. 5 (2003) 1209–1212.
[16] E. Forró, F. Fülöp, Tetrahedron: Asymmetry 15 (2004) 2875–2880.
[17] A similar observation was reported when the transesterification of racemic
ethyl 2-hydroxy-4-phenylbutyrate was performed by using lipase PS: S.-H.
Huang, S.-W. Tsai, J. Mol. Catal. B Enzym. 28 (2004) 65–69.
[4] (a) A. Ghanem, V. Schurig, Tetrahedron: Asymmetry 14 (2003) 57–62;
(b) D. Klomp, J.A. Peters, U. Hanefeld, Tetrahedron: Asymmetry 16 (2005)
3892–3896;