Lipases A and B from Candida antarctica in the enantioselective acylation of ethyl 3-heteroaryl-3-hydroxypropanoates: Aspects on the preparation and enantiopreference

Article abstract of DOI:10.1016/j.tetasy.2011.01.027

The preparative scale kinetic resolution of racemic ethyl 2- and 3-furyl- and 2- and 3-thienyl-3-hydroxypropanoates has been performed by Candida antarctica lipases A and B with vinyl esters. A study based on the present work together with the literature has been carried out in terms of lipase enantiopreference and substrate structure. We also discuss the excellent behavior of the lipase A in O-acylations of secondary alcohols with respect to enantiopreference.

Full text of DOI:10.1016/j.tetasy.2011.01.027

Tetrahedron: Asymmetry 22 (2011) 315–322
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Lipases A and B from Candida antarctica in the enantioselective acylation
of ethyl 3-heteroaryl-3-hydroxypropanoates: aspects on the preparation
and enantiopreference
Jürgen Brem a,b, Arto Liljeblad a, Csaba Paizs b, Monica Ioana Toßsa b, Florin-Dan Irimie b, Liisa T. Kanerva a,
⇑
a Institute of Biomedicine, Department of Pharmacology, Drug Development and Therapeutics/Laboratory of Synthetic Drug Chemistry and Department of Chemistry,
University of Turku, FIN-20014, Finland
b Department of Biochemistry and Biochemical Engineering, Babeßs-Bolyai University of Cluj-Napoca, Ro-400028 Cluj-Napoca, Arany János 11, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
The preparative scale kinetic resolution of racemic ethyl 2- and 3-furyl- and 2- and 3-thienyl-3-hydroxy-
propanoates has been performed by Candida antarctica lipases A and B with vinyl esters. A study based on
the present work together with the literature has been carried out in terms of lipase enantiopreference
and substrate structure. We also discuss the excellent behavior of the lipase A in O-acylations of second-
ary alcohols with respect to enantiopreference.
Received 21 December 2010
Accepted 20 January 2011
Available online 14 March 2011
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
racemization catalyst in one-pot.11,12 The choice of an enzyme is
determined by the reactive functional groups of the racemate. High
b-Hydroxy esters are versatile chiral intermediates in the
synthesis of several pharmaceuticals and fine chemicals. For in-
stance, (R)-3-hydroxybutanoate esters serve as starting materials
for b-lactam antibiotics, including carbapenems and penems with
broad antimicrobial spectra, and for dorzolamide, a topically active
human carbonic anhydrase II inhibitor.1–7 Amongst aryl-substi-
tuted b-hydroxy esters, ethyl (S)-3-hydroxy-3-(thiophen-2-yl)-
propanoate (S)-2a is a precursor for the synthesis of duloxetine
(a serotonin-norepinephrine re-uptake inhibitor) and ethyl
(R)-3-hydroxy-3-phenylpropanoate (R)-2e for that of atomoxetine
(a norepinephrine re-uptake inhibitor).8–10 These examples indi-
cate that an active drug molecule may have either an (R)- or (S)-
configuration. More generally, the preparation of both enantiomers
of a drug and its key intermediates is necessary to recognize the
physiological properties of these molecules.
With regards to green chemistry, biocatalysts have been
increasingly included and used by modern synthetic chemists in
addition to expensive and often complicated chemical asymmetric
catalysts and natural products used in traditional resolution. The
enzymatic kinetic resolution of a racemate with a suitable hydro-
lytic enzyme is one of the most common biocatalytic methods used
today. In the best case, both enantiomers (one as the less reactive
enantiomer and the other as a new product) can be obtained in
one-pot in a single reaction. Kinetic resolution can be further
transformed into a dynamic kinetic resolution (DKR) by combin-
ing an enzyme as an enantioselective catalyst with a suitable
stability, enantioselectivity, and good commercial availability in
free and immobilized forms have made lipases (EC 3.1.1.3) espe-
cially attractive kinetic resolution catalysts via the acylation of a
nucleophilic (such as alcohol) functionality and through the
deacylation (hydrolysis, alcoholysis or interesterification) of an
ester functionality in the molecule.13 Thus, b-hydroxy esters enable
a variety of possibilities as both alcohols and esters for kinetic
resolutions with lipases.
Lipase-catalyzed methods have previously been used for the
kinetic resolution of racemic ethyl 3-benzofuranyl-, 3-benzo[b]-
thiophenyl-, and 3-(10-alkyl-10H-phenothiazin-3-yl)-3-hydroxy-
propanoates,14,15 and for the DKR of rac-2e.16 Herein our aim was
to study the chemoenzymatic possibility of preparing the enantio-
mers of ethyl 3-heteroaryl-3-hydroxypropanoates via chemical
synthesis of rac-2a–e followed by enantioselective lipase-catalyzed
O-acylation of racemic mixtures in organic solvents (Scheme 1).
Compound 2e, although not heteroaromatic, was included in the
initial screens for comparison. We also wanted to study the enan-
tiopreferences of lipases A (CAL-A) and B (CAL-B) from Candida ant-
arctica; the two lipases were thoroughly studied in the kinetic
resolution of various b-amino esters.17 Some other commercial li-
pases were also screened for the sake of comparison. Many lipases
have been shown to produce the (R)-ester in the O-acylation of sec-
ondary alcohols. For such (R)-enantioselectivity, the large group at
the stereogenic center must owe CIP priority over the medium size
group as with (R)-1-phenylethanol.18 Interestingly, an opposite
enantiopreference in O- and N-acylations was sometimes detected
with the title enzymes CAL-A and CAL-B (Fig. 1), and we have
illustrated this phenomenon with rac-2a–e. The value of the
⇑
Corresponding author. Tel.: +358 2 3336773; fax: +358 2 3337955.
E-mail address: lkanerva@utu.fi (L.T. Kanerva).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.01.027

316
J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 315–322
O
R
R1
O
O
OH
O
(b)
(a)
R
O
R
OEt
rac-3a-e
OEt
1a-e
rac-2a-e
R1CO2R2
Lipase
O
S
R =
O
R
b
a
R1
O
OH
R
S
O
+
CO2Et
CO2Et
c
d
e
(S)- or (R)-3a-e
(S)- or (R)-2a-e
Scheme 1. Chemoenzymatic route to the enantiomers of 3-heteroaryl-3-hydroxypropanoates. Reagents and conditions: (a) Zn, BrCH2CO2Et, THF, reflux; (b) R0COCl, Et3N,
DMAP, CH2Cl2, rt or (R0CO)2O, CoCl2, CH3CN, reflux.
XH
R3
X = O or NH
XCOR1
R3
XH
R3
CAL-A or CAL-B
acyl donor, solvent
+
R2
R2
R2
NHCOR1
CO2Et
OCOR1
CO2Me
OCOR1
CO2Et
rac-2a-e
rac-5
rac-4
MeO2C
R
R
vinyl butanoate/CH3CN
2,2,2-trifluoroethyl butanoate/DIPE
CAL-A:34,37 R=Me, Et, Pr, Ph, iPr,
cyclohexyl, CH2(Et)2, 2- and 3-thienyl and 2- and 3-
furyl; E=6-510
neat vinyl acetate
CAL-A:35 E=40
CAL-A: R=Ph; E=7, 2- and 3-thienyl
and 2 and 3-furyl; E=15-54
OCOR1
CAL-B:29,37 R=Me, Et, iPr; E not determined
OCOR1
CO2Me
MeO2C
CO2Et
vinyl butanoate/CH3CN
R
NHCOR1
CAL-B:35 E=2
vinyl butanoate/DIPE
CO2Et
CAL-B: R=2- and 3-thienyl and
2 and 3-furyl; E>200
OCOR1
R
rac-7
neat ethyl butanoate
CAL-B:34 2- and 3-thienyl and 2- and 3-furyl; E=2-10
OCOR1
rac-6
neat vinyl butanoate
Ph
Si
Ph
CAL-A: E= 2
O
OCOR1
O
2,2,2-trifluoroethyl butanoate/TBME
CAL-A:41 E>300
CAL-B:41 no reaction
isopropenyl acetate/toluene
CAL-B:36 E>>200
Figure 1. Enantiodiscrimination in O- and N-acylations by CAL-A (on Celite) and CAL-B (Novozym 435). O-Acylated product enantiomers are presented by drawing the
aromatic ring always on the left side of the stereogenic center.
enantiopreference mapping resides in finding lipases with an
opposite enantiopreference for DKR purposes.
a GGGX-motif at the active site, whereas most lipases consist of
a GX-motif.22 The excellent properties of CAL-A with regards to
its ability to hydrolyze amides have also been reported.23 Along
with its peculiar catalytic behavior, the use of CAL-A is now rapidly
increasing. Summaries for CAL-A17,24–26 exist but the catalytic
properties of CAL-A toward secondary alcohols are still rela-
tively scattered in the literature. Herein we integrate the obtained
results for rac-2a–e to the existing knowledge of CAL-A related to
secondary alcohols.
Since the 1990s, CAL-B used as Novozym 435 preparation (CAL-
B adsorbed on a divinylbenzene-crosslinked, hydrophobic macro-
porous polymer based on methyl and butyl methacrylic esters)
has become the most common lipase used in synthetic organic
chemistry due to its ability to effectively catalyze various highly
enantioselective acyl transfers in polar as well as in non-polar sol-
vents. In our previous studies, CAL-B indicated low chemoselectiv-
ity for the N-acylation of various b-amino esters leading to
competitive interesterification with an acyl donor.17 On the other
hand, excellent chemoselectivity was reported for CAL-A in the
N-acylation of the same b-amino esters.17 The high chemoselectiv-
ity has been explained by recent studies which proved the highly
restricted acyl binding pocket of CAL-A.19–21 Moreover, the ability
of CAL-A to catalyze the acylation of bulky nucleophilic sub-
strates is noteworthy. This has been explained by the presence of
2. Results and discussion
2.1. Synthesis of the racemic starting materials
In principle, various chemical methods exist for the preparation
of racemic ethyl 3-hydroxy-3-arylpropanoates. Amongst them, the

J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 315–322
317
Reformatsky reaction is a straightforward method which was pre-
viously successful in the preparation of 3-hydroxy-3-heteroaryl-
propanoates from the corresponding aldehydes.14,15 Accordingly,
the method was used to transform aldehydes 1a–e into rac-2a–e
with ethyl bromoacetate in tetrahydrofuran in the presence of zinc
dust (Scheme 1, route a). Compounds rac-2a and rac-2e were ob-
tained with acceptable yields (92% and 72%, respectively). With
rac-2b the yield was improved from 5% to 28% by performing the
reaction without solvent (THF). With rac-2b and rac-2d, the low
yields (28% and 17%, respectively) obtained were possibly due to
the instability of the furan ring under acidic conditions and to
the interference of the zinc with the ring oxygen. Efforts to synthe-
size the hydroxy esters through the corresponding b-keto esters
which were first prepared by condensation of aldehydes 1b–d with
ethyl diazoacetate proved to be impractical as low keto ester yields
(35–40%) discouraged us from continuing with reduction into rac-
2b–d.27
a side reaction with CAL-B, vinyl acetate was chosen as a com-
monly used and irreversible acyl donor. Vinyl acetate is more reac-
tive an acyl donor than the substrate ester rac-2a or rac-2e and also
prevents another possible side reaction, the enzymatic dimeriza-
tion of the substrate. With CAL-A, interesterification and dimeriza-
tion are less likely due to the restricted acyl binding pocket as
already mentioned.
The results with the model substrates show two interesting fea-
tures. Firstly, the S-enantiomers reacted with CAL-A and Rhizopus
oryzae lipase (Table 1, entries 1–4 and 20–22), whereas the other
lipases indicated (R)-enantioselectivity (entries 5–19). Secondly,
enantioselectivity in terms of enantiomer ratio (E) was clearly dif-
ferent with the phenyl (rac-2e) and heteroaryl (rac-2a) substrates.
Thus, lipases from Burkholderia cepacia and Pseudomonas fluores-
cens were favorable with rac-2e (entries 7–16), whereas the other
lipases favored rac-2a (entries 1–6 and 17–22). The performance of
lipase Burkholderia cepacia preparations with rac-2e is also in
accordance with the excellent enantioselectivity observed in the
DKR of rac-2e in the presence of (pentaphenylcyclopentadie-
nyl)ruthenium complex.16 Clearly, CAL-B (entries 5 and 6) along
with various CAL-A preparations, especially when adsorbed on Cel-
ite in the presence of sucrose (entries 2 and 3),32 gave fast and
enantioselective reactions with rac-2a but not with rac-2e. In addi-
tion to the lipases shown in Table 1, lipases from porcine pancreas,
Mucor miehei, Rhizopus arrhizus, Mucor javanicus, Aspergillus niger,
Candida lipolytica, and Penicillium camemberti were practically inac-
tive under the screening conditions.
To obtain racemic resolution products rac-3a–e (R1 = Me, Et or
Pr) for analytical identification, the racemates rac-2a–e were ester-
ified (route b) with the corresponding acyl chloride in dichloro-
methane in the presence of triethylamine and a catalytic amount
of 4-N,N-dimethylaminopyridine, or with the corresponding acid
anhydride in the presence of anhydrous cobalt chloride in acetoni-
trile, leading to good yields as described in the Section 4.
2.2. Enzymatic kinetic resolution
Extensive screening with various lipase preparations for the O-
acylation of rac-2a and rac-2e as model compounds in neat vinyl
acetate started our analytical scale studies (Scheme 1). The forma-
tion of an acyl-enzyme intermediate, an ester intermediate be-
tween the serine hydroxyl at the active site of the lipase and an
acyl donor, is pivotal for lipase catalysis. This means that a b-hy-
droxy ester as an added alcohol nucleophile binds enantioselec-
tively in the nucleophile binding site before reacting with the
intermediate, whereas as an acyl donor, it enantioselectively binds
in the acyl binding site and forms the intermediate by itself.
Accordingly, care is needed when choosing an achiral acyl donor
for enzymatic O-acylation due to the dual nature of the substrates
present. For CAL-B catalysis competitive interesterification may be
an especially serious side reaction28,29 as it might also be exploit-
able in various applications.30,31 To prevent interesterification as
As shown in Table 1, CRL and Rhizopus oryzae lipase seem to
have potential for the kinetic resolution of rac-2a with E values
of 71 and 124, respectively (entries 17 and 20). However, CRL gave
considerable ester hydrolysis as a side reaction and was not
studied further. Rhizopus oryzae lipase kept low activity even after
tedious optimization studies. Accordingly, attention was paid to
CAL-A and CAL-B, two fascinating lipases, which have been previ-
ously exploited, for instance in the N-acylation of b-amino
esters.28–30,33–37 The acylation of rac-2a in neat vinyl acetate, pro-
panoate, and butanoate (used as acyl donors and solvents) revealed
the opposite effects of CAL-A and CAL-B on the E-values (Table 2).
With CAL-A, E = 54 (entry 1) was the highest in neat vinyl acetate,
whereas the reaction with CAL-B in vinyl butanoate gave excellent
enantioselectivity (E >200, entry 3). Solvent effects were studied
further for the acylation of rac-2a with CAL-A using vinyl acetate
Table 1
Acylation of rac-2a and rac-2e (0.050 M) with enzymes (50 mg/mL) in neat CH3CO2CH@CH2 at room temperature
Entry
Substrate
Lipase
Time (h)
Conv. (%)
Reactive enantiomer
E
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
rac-2a
rac-2a
rac-2e
rac-2a
rac-2a
rac-2e
rac-2a
rac-2e
rac-2a
rac-2e
rac-2a
rac-2e
rac-2a
rac-2e
rac-2a
rac-2e
rac-2a
rac-2e
rac-2a
rac-2a
rac-2e
rac-2a
CAL-A (lyo.)
CAL-A on Celite
CAL-A on Celite
CAL-A CLEA
1
1
1
25
50
10
38
7
(S)
(S)
(S)
(S)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(R)
(S)
(S)
(S)
7
54
7
10
40
5
2
34
5
104
10
276
12
33
7
37
71
12
50
124
39
2
1
CAL-B (Novozym 435)
CAL-B (Novozym 435)
Burkholderia cepacia (lyo.)
Burkholderia cepacia (lyo.)
Burkholderia cepacia-C II
Burkholderia cepacia-C II
Burkholderia cepacia-D
Burkholderia cepacia-D
Pseudomonas fluorescens (lyo.)
Pseudomonas fluorescens (lyo.)
Pseudomonas fluorescens on Celite
Pseudomonas fluorescens on Celite
CRL
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
20
48
4
21
70
41
55
35
25
31
20
8
35
40
29
7
CRL
CRL sol-gel
Rhizopus oryzae lipase
Rhizopus oryzae lipase
Rhizopus oryzae lipase IMMARO-T2-150
2
6

318
J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 315–322
Table 2
Table 4
CAL-A- and CAL-B-catalyzed (50 mg/mL) acylations of rac-2a (0.25 M) in neat
Acylations of rac-2a–d (0.25 M) with CAL-A (50 mg/mL) in neat R1CO2CH@CH2
(R1 = Me), and with CAL-B (50 mg/mL) and R1CO2CH@CH2 (R1 = Pr, 5 equiv) in MTBE
R1CO2CH@CH2 at room temperature
Entry
R1
CAL-A on Celitea
Novozym 435
Conv. (%)
Entry
Substrate
CAL-A on Celitea
Novozym 435
Conv. (%)
Conv. (%)
E
E
Conv. (%)
E
E
1
2
3
CH3
CH3CH2
CH3CH2CH2
50
62
51
54
19
13
5
15
45
6
74
>200
1
2
3
4
rac-2a
rac-2b
rac-2c
rac-2d
50
49
50
46
54
33
14
22
50
51
50
48
>200
>200
>>200
>>200
Reaction time 1 h (CAL-A) and 60 h (CAL-B).
20% (w/w) of CAL-A and 12% (w/w) of sucrose on Celite.32
Reaction time 1 h (CAL-A) and 60 h (CAL-B).
a
20% (w/w) of CAL-A and 12% (w/w) of sucrose on Celite.32
a
and with CAL-B using vinyl butanoate as an acyl donor in organic
solvents (Table 3). With both lipases the reactions in the neat vinyl
ester were acceptable (entry 12). In addition, reactivity with CAL-B
in ethers and in hydrophobic solvents was good and in most cases
the reactions proceeded with excellent enantioselectivities (E
>200) (entries 1–6 and 9). As vinyl butanoate is relatively expen-
sive and its boiling point is high, the work with CAL-B was contin-
ued using vinyl butanoate in tert-butyl methyl ether (MTBE) as a
low-boiling solvent.
N-acylations (Figs.
1
and 2)5,14,15,29,34–37,40–49 reveals some
interesting facts about the enantiodiscrimination of CAL-A and
fundamental differences of lipases CAL-A and CAL-B. Lipase CAL-B
catalyzes the O-acylation of a series of simple secondary alcohols
with excellent enantioselectivity. Examples include the O-acyla-
tion of 1-phenylethanol rac-7, a frequently used model compound,
and 3-hydroxybutanoate rac-8 (E >200).5,36 This is based on a so-
called stereospecificity pocket delimited by Thr42, Ser47, and
Trp104 at the active site of CAL-B. The pocket is able to accommo-
date groups smaller than propyl.50–52 Also the enantiopreference of
the reactions is predictable. Thus, with rac-7, rac-8, and rac-16 the
methyl group (the medium-sized group) at the asymmetric center
fits into the stereospecificity pocket, leading to the excellent R-
enantiopreference with CAL-B. With all other substrates showing
reaction with CAL-B, the faster reacting enantiomer has the same
stereostructure around the asymmetric center with the aforemen-
tioned substrates, although the medium-sized group exceeds the
size of propyl rac-4, -5, -12, -14, and -15 or the group is electron
rich such as CN (rac-9–11). With these compounds, CAL-B consid-
erably lost its enantioselective character. The present substrates
rac-2a–d with CH2CO2Et and substrate rac-15 (X = S, E = 108) with
CH2CN as medium-sized groups are exceptions with excellent
enantioselectivity. However, either of the groups, heteroaryl or
CH2CO2Et/CH2CN, evidently does not fit into the stereospecificity
pocket. Common to these substrates, a five-membered heteroaryl
ring and a group with a lone-pair of electrons are located next to
the asymmetric center. The interactions of these groups with
CAL-B stay currently unclear.
Table 3
CAL-A- and CAL-B-catalyzed (50 mg/mL) acylations of rac-2a (0.25 M) with
R1CO2CH@CH2 (R1 = Me) and R1CO2CH@CH2 (R1 = Pr) as acyl donors (5 equiv),
respectively, in different solvents at room temperature
Entry
Solvent
CAL-A on Celitea
Novozym 435
Conv. (%)
Conv. (%)
E
E
1
2
3
4
5
6
7
8
9
n-Octane
n-Hexane
Toluene
Benzene
MTBE
21
20
14
12
19
31
9
1
21
3
8
8
7
10
10
9
15
3
16
5
44
46
49
46
45
50
8
3
8
6
9
94
>200
>200
>200
>200
>200
>200
>200
27
Diethyl ether
THF
1,4-Dioxane
Acetonitrile
Chloroform
Dichloromethane
Neat acyl donor
10
11
12
>200
>200
>200
7
50
15
54
45
Reaction time 1 h (CAL-A) and 60 h (CAL-B).
a
20% (w/w) of CAL-A and 12% (w/w) of sucrose on Celite.32
Finally, the acylation of rac-2a–d was tested with CAL-A in neat
vinyl acetate and with CAL-B with vinyl butanoate in MTBE
(Scheme 1, Table 4). The results clearly indicated excellent (R)-
enantioselectivity in the CAL-B-catalyzed O-acylations (E >200),
whereas CAL-A displayed moderate (S)-selectivity (E = 14–54). Fi-
nally, the preparative scale kinetic resolutions of rac-2a–d were
performed successfully using CAL-B in MTBE as described in Sec-
tion 4, allowing the preparation of the (R)-esters 3a–d (R1 = Pr)
and the unreacted (S)-2a–d in highly enantiomerically enriched
forms with ee values between 95% and 99% (Table 5). In spite of
low enantioselectivities (E 14–54), the preparative scale kinetic
resolutions were also performed with CAL-A in neat vinyl acetate.
In accordance with the opposite enantiodiscriminations of CAL-A
Lipase CAL-A, on the other hand, has a wide nucleophile binding
site,19 and the O-acylation of rac-7 proceed with low enantioselec-
tivity (E = 2), even though the reaction is otherwise very fast. There
are a lot of data concerning the kinetic resolution of 1-phenyleth-
anol but studies with CAL-A seem to be not published. The results
given in Figure 1 were performed using vinyl butanoate as an acyl
donor. Based on the crystal structure of CAL-A and molecular mod-
eling, it has been proposed that upon substrate binding, CAL-A
undergoes a conformational change whereby the active-site ‘flap’
(Gly426–Gly440) moves around the active site, widening the avail-
able space essentially infinitely.19,20 This can explain why CAL-A is
active and shows enantioselectivity in the O-acylation of highly
sterically hindered secondary alcohols, such as rac-6, rac-10–14,
rac-17, and rac-18, whereas CAL-B, or some other lipase, hardly
catalyzes the reactions. As an interesting feature, amines rac-5
(E = 75–510)34,37 display much higher enantioselectivity when
compared to the corresponding alcohols rac-2a–e (E = 7–54).
As can be seen in Figures 1 and 2, CAL-A may change the enan-
tiopreference in O- (and N-)acylation according to the substrate
structure, whereas CAL-B always prefers the same stereostructure
with the substrates considered. Thus, when the bulkiness of the
heteroaromatic rings increases from thienyl and furyl [rac-2a–d,
(S)-enantiopreference] to benzothiophenyl and benzofuranyl (rac-
12 and rac-14), or the ring is substituted with another aromatic
substituent (rac-9, rac-11, and rac-15), the enantiopreference of
and CAL-B, the signs of the specific rotations [
materials 2 changed, as did those of products 3. The absolute con-
figurations of the present resolution products are based on the [
a] of the starting
D
a]
D
values in Table 5 and on the literature values +19 (c 1.7, CHCl3,
ee = 84%)38 for (R)-2a, +18 (c 1.6, CHCl3, ee = 54)38 and +15 (c 0.9,
CHCl3, ee = 75%)39 for (R)-2b and ꢀ94.2 (c 1.3, CHCl3, ee = 90%)39
for (R)-3b (R1 = Pr).
2.3. Enantiopreference of CAL-A and CAL-B
Integration of the results obtained for the O-acylation of
rac-2a–e (Tables 4 and 5) with the previously published O- and

J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 315–322
319
Table 5
Kinetic resolution of rac-2a–d, characterization of the isolated products
Novozym 435
CAL-A on Celite
a
a
a
a
Product
Yield (%)/ee (%)/[
a
]
D
Product (R1 = Pr)
Yield/ee /[
a
]
D
Product
Yield (%)/ee (%)/[
a
]
D
Product (R1 = Me)
Yield/ee/[a]
D
(S)-2a
(S)-2b
(S)-2c
(S)-2d
46/99/ꢀ17
47/99/ꢀ16
46/99/ꢀ44
46/97/ꢀ29
(R)-3a
(R)-3b
(R)-3c
(R)-3d
48/96/+88
48/97/+85
48/99/+49
47/99/+52
(R)-2a
(R)-2b
(R)-2c
(R)-2d
45/68/+11
47/63/+10
47/51/+21
46/60/+16
(S)-3a
(S)-3b
(S)-3c
(S)-3d
44/94/ꢀ63
45/90/ꢀ60
40/81/ꢀ47
41/88/ꢀ58
a
10ꢀ1 deg cm2 gꢀ1; c 1.0, CHCl3, T = 25 °C. Isolated yields based on the maximum theoretical recovery from the racemic starting material.
OH
R3
OCOR1
R3
OH
R3
CAL-A or CAL-B
acyl donor, solvent
+
R2
R2
R2
OCOR1
CO2Et
OCOR1
CN
R''
rac-8
EtO2C
rac-11
rac-14
S
N
O
OCOR1
x
R'
vinyl acetate/DIPE
CAL-A:40 ee=64%
neat vinyl acetate
CAL-B:5 E=150
vinyl acetate/CH3CN
vinyl butanoate/benzene
CAL-A:44 R'=H Cl, Me or H,
R''=H, H, H or Cl; E=14-64
neat vinyl acetate
CAL-A:14 X=O, E=87; X=S: E=65
CAL-B:14 reaction stops at 10%
conversion
CAL-B:44 R'=R''=H; E=3
OCOR1
OCOR1
rac-15
rac-9
OCOR1
CN
rac-12
CN
x
CO2Et
O
Br
x
vinyl butanoate/TBME
CAL-A:46,47 X=O, E=5; X=S,
racemic product
vinyl butanoate/cyclohexane
CAL-A:14 X=O, X=S; E>200
CAL-B:14 X=O, X=S; E<7
vinyl butanoate/DIPE
CAL-A:43 racemic product
CAL-B:43 E=10
CAL-B:46,47 X=O, E=30; X=S, E=108
OCOR1
CN
OCOR1
CO2Et
rac-16
OCOR1
rac-10
rac-13
S
S
S
N
N
N
R´
Et
R'
vinyl acetate/MeCN
neat vinyl acetate
CAL-A:45 E=30
CAL-B:45 E>200
vinyl acetate/MeCN
CAL-A:42R'=Me, Et, Pr, Bu,
(CH2)4CH3 ; E=23 - >100
CAL-B:42 R'=Pr; E=13
CAL-A:15 R'=Me, Et, Pr, Bu,
(CH2)4CH3 ; E=42 - >200
CAL-B:15 no reaction
OCOR1
rac-17
rac-18
OCOR1
CO2Me
Cl
vinyl acetate/toluene/hexane
CAL-A:48 E= 67
vinyl propanoate/toluene
CAL-A:49 E=16
CAL-B:48 no reaction
CAL-B:49 no reaction
Figure 2. Enantiodiscrimination in O-acylations by CAL-A (on Celite) and CAL-B (Novozym 435). O-Acylated product enantiomers are presented by drawing the aromatic ring
always on the left side of the stereogenic center.
CAL-A reverses. Reversal from an (S)- to an (R)-enantiopreference is
also seen when the phenyl in rac-2e becomes more bulky (in rac-
13) and in compounds rac-10 and rac-16–18. The (S)-enantiopre-
lost), yet functional, CAL-A.23 The presence of two catalytically ac-
tive forms of CAL-A may have an effect not only on the enantiose-
lectivity but also on the enantiopreference of the reactions.
ference is also evident with rac-4, both the a- and b-hydroxy dies-
ters. It is possible that the heteroaryl ring is connected to
enantiopreference and high enantioselectivity in the O-acylation
of heteroaryl-substituted secondary alcohols. These observations
are rather difficult to explain without modeling. It is also worth
remembering that all the given results were obtained with com-
mercially available CAL-A, which not only contains contaminant
enzymes, but also decomposed (evidently the active-site flap was
3. Conclusions
An enantioselective O-acylation method has been developed to
resolve ethyl 3-hydroxy-3-(thiophenyl/furanyl)propanoates rac-
2a–d with vinyl butanoate in MTBE by using CAL-B (Novozym
435) as an enantioselective catalyst. The highly enantioselective

320
J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 315–322
(E >200) reactions allowed the separation of the enantiomers in al-
most theoretical yields. Interestingly, CAL-A (on Celite) showed an
opposite enantiopreference to that obtained with CAL-B. Although
the enantioselectivities with CAL-A were more or less moderate
(E = 14–54) the resolution products (R)-2a–d and (S)-3a–d
(R1 = Me) were separated, and the specific rotations were deter-
mined to witness the opposite specific rotations to the (S)-2a–d
and the butanoate esters (R)-3a–d (R1 = Pr) obtained with CAL-B
catalysis.
it (5 g) was adsorbed on Celite (17 g) in the presence of sucrose
(3 g).32 CAL-A CLEA was from Fluka. Lipase B from C. antarctica
(CAL-B, Novozym 435) and lipase from Thermomyces lanuginosus
(Lipozyme TL IM) were purchased from Novozymes, Denmark. Li-
pase from Candida rugosa (CRL) immobilized on sol-gel was a gift
from Professor László Poppe, Budapest University of Technology
and Economics, Hungary.
4.3. Synthesis of racemic compounds
We have shown that with regards to the enantiodiscrimination,
the behavior of CAL-A and CAL-B depends on the substrate struc-
ture. Using literature data in addition to the present results it is
clear that in CAL-B catalysis the more reactive enantiomer in the
O-acylation of secondary alcohols has always similar predictable
stereostructure around the stereogenic center. With CAL-A hetero-
aryl analogs seems to be favored and enantiopreference is changed
by changing the size and substitution of an aromatic ring.
4.3.1. Synthesis of racemic b-hydroxy esters rac-2a–e
A solution of ethyl bromoacetate (84 g, 56 mL, 0.50 mol) in tet-
rahydrofuran (100 mL) was added in small portions under slight
warming into a mixture of zinc powder (40 g, 0.61 mol) and one
of the aldehydes 1a–e (0.60 mol) in tetrahydrofuran (10 mL) which
was previously refluxed for 30 min. After completion of the reac-
tion (approx. 1.5 h), the mixture was cooled to room temperature
and filtered to remove the unreacted zinc. The solvent was evapo-
rated in vacuo and the crude semisolid product was dissolved in
CH2Cl2 (20 mL). The solution was cooled in an ice bath and treated
with saturated ammonium chloride solution (50 mL) under vigor-
ous stirring. After separation, the aqueous layer was extracted with
CH2Cl2 (3 ꢁ 100 mL). The combined organic layer was washed with
water (50 mL), then dried over anhydrous magnesium sulfate, and
evaporated in vacuo. The crude product was purified by prepara-
tive vacuum-chromatography using hexane/EtOAc (3:1, v/v) as
an eluent. Spectroscopic data are in accordance with the literature
data.38,39
4. Experimental
4.1. Analytical methods
1H and 13C NMR spectra were recorded on Bruker spectrometers
operating at 500 or 300 MHz with tetramethylsilane (TMS) as an
internal standard. Analytical data for compounds 2 and 3a,b,e corre-
spond to the literature data.14,15 HRMS were measured in ESI+ mode
with a Bruker Avance microOTOF-Q quadrupole-TOF spectrometer.
The optical rotations were measured with a PerkinElmer 341 or a
Bellingham-Stanley ADP 220 polarimeter, and [a]D values are given
in units of 10ꢀ1 deg cm gꢀ1. Thin layer chromatography (TLC) was
carried out using Merck Kieselgel 60F254 sheets. Spots were visual-
ized by treatment with 5% ethanolic phosphomolybdic acid solution
and heating. Preparative chromatographic separations were per-
formed using column chromatography on Merck Kieselgel 60 Å
4.3.1.1. Ethyl 3-hydroxy-3-(thiophen-2-yl)propanoate rac-2a.
Yield: 92%; 1H NMR: (300 MHz, CDCl3): d = 1.27 (t, J = 7.2 Hz, 3H);
2.86 (ddd, J = 5.4, J = 8.7 Hz, J = 15.6 Hz, 2H); 4.19 (q, J = 7.2 Hz,
2H); 5.37 (dd, J = 5.5 Hz, J = 8.5 Hz, 1H); 6.95–7.01 (m, 2H); 7.27–
7.31 (m, 1H); 13C NMR: (75 MHz, CDCl3): d = 14.1; 43.2; 61.0;
66.5; 123.6; 124.8; 126.7; 146.3; 171.9. HRMS: calcd for
C9H12NaO3S [M+Na]+ 233.0399, found 233.0394.
(63–200 lm). The determination of the E-value was based on the
equation E = ln[(1 ꢀ c)(1 ꢀ eeS)]/ln[(1 ꢀ c)(1 + eeS)].53
The enantiomeric separations of 2c,e and 3c,e were performed
by GC (HP1090 gas chromatograph) equipped with Cyclosil-B
4.3.1.2. Ethyl 3-hydroxy-3-(furan-2-yl)propanoate rac-2b.
Yield: 28%; 1H NMR: (300 MHz, CDCl3): d = 1.25 (t, J = 7.2 Hz, 3H);
2.84 (ddd, J = 4.5 Hz, J = 8.4 Hz, J = 16.2 Hz, 2H); 4.17 (q, J = 7.2 Hz,
2H); 5.12 (dd, J = 4.5 Hz, J = 8.4 Hz, 1H); 6.25–6.35 (m, 2H); 7.35–
7.39 (m, 1H); 13C NMR: (75 MHz, CDCl3): d = 14.0; 40.0; 60.9;
64.0; 106.1; 110.2; 142.1; 154.9; 171.7. HRMS: calcd for
C9H12NaO4 [M+Na]+ 207.0627, found 207.0617.
(30 ꢁ 0.32 mm ꢁ 0.25
lm) column and those of 2a,d and 3a,d
Chrompack CP-Chirasil-Dex CB column (25 ꢁ 0.25 mm). The fol-
lowing oven temperatures were used: 2a and 3a 125 °C, 2b,c and
3b,c 140 °C, 2d and 3d 110 °C, and 2e and 3e 125 °C. The retention
times for the enantiomers are shown in Table 6.
4.2. Reagents and solvents
4.3.1.3. Ethyl 3-hydroxy-3-(thiophen-3-yl)propanoate rac-2c.
Yield: 27%; 1H NMR: (500 MHz, CDCl3): d = 1.24 (t, J = 7.5 Hz, 3H);
2.75 (ddd, J = 5.5 Hz, J = 8.5 Hz, J = 15.5 Hz, 2H); 4.15 (q, J = 7.5 Hz,
2H); 5.18 (dd, J = 5.5 Hz, J = 8.5 Hz, 1H); 7.08–7.07 (m, 1H); 7.20–
7.22 (m, 1H); 7.27–7.30 (m, 1H); 13C NMR: (126 MHz, CDCl3):
d = 14.1; 42.6; 60.8; 66.7; 120.9; 125.5; 126.2; 144.0; 172.2. HRMS:
calcd for C9H12NaO3S [M+Na]+ 233.0399, found 233.0392.
All reagents were purchased from Aldrich or Fluka and used as
received. Solvents and acyl donors for enzymatic reactions were
stored over molecular sieves unless otherwise stated. Lipases from
Aspergillus niger, Pseudomonas fluorescens, Burkholderia cepacia, Rhi-
zopus oryzae, Rhizopus arrhizus, Penicillium camemberti, and Mucor
javanicus were products of Amano. Immobilized Rhizopus oryzae li-
pase (IMMARO-T2-150) was from ChiralVision. Lipases from Can-
dida rugosa (CRL), Candida lipolytica, Mucor miehei, and porcine
pancreas (PPL) were purchased from Fluka. Lipase A from C. antarc-
tica (CAL-A) was the product from Roche (Chirazyme L-5, lyo.), and
4.3.1.4. Ethyl 3-hydroxy-3-(furan-3-yl)propanoate rac-2d.
Yield: 17%; 1H NMR: (500 MHz, CDCl3): 1.26 (t, J = 7.5 Hz, 3H);
2.73 (ddd, J = 5.5 Hz, J = 8.5 Hz, J = 15.5 Hz, 2H); 4.17 (q, J = 7.5 Hz,
Table 6
Retention times [(S)/(R)] of the enantiomers of 2 and 3 (R1 = Me and R1 = Pr)
Compound
tr (min)
Compound (R1 = Pr)
tr (min)
Compound (R1 = Me)
tr (min)
2a
2b
2c
2d
2e
78.8/80.8
6.3/14.3
32.9/34.2
60.3/57.2
50.1/52.8
3a
3b
3c
3d
3e
127.4/124.4
4.2/4.5
69.3/65.5
118.8/114.0
72.3/68.7
3a
3b
3c
3d
3e
50.9/48.8
5.2/6.2
31.3/29.9
47.4/45.8
46.2/44.9

J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 315–322
321
2H); 5.09 (dd, J = 5.5 Hz, J = 8.5 Hz, 1H); 6.38–6.43 (m, 1H); 7.36–
7.42 (m, 2H); 13C NMR: (126 MHz, CDCl3): d = 14.1; 42.1; 60.9;
63.4; 108.4; 127.4; 139.0; 143.4; 172.2. HRMS: calcd for
C9H12NaO4 [M+Na]+ 207.0627, found 207.0620.
3H); 7.38–7.42 (m, 1H); 13C NMR: (75 MHz, CDCl3): d = 13.5;
14.1; 18.3; 36.0; 37.7; 60.8; 64.6; 108.8; 110.3; 142.7; 151.3;
169.4; 172.4. HRMS: calcd for C13H18NaO5 [M+Na]+ 277.1046,
found 277.1095.
4.3.1.5. Ethyl 3-hydroxy-3-phenylpropanoate rac-2e. Yield:
72%; 1H NMR: (300 MHz, CDCl3): 1.26 (t, J = 7.2 Hz, 3H); 2.72
(ddd, J = 4.2 Hz, J = 8.7 Hz, J = 16.2 Hz, 2H); 4.18 (q, J = 7.2 Hz, 2H);
5.13 (dd, J = 4.2 Hz, J = 8.7 Hz, 1H); 7.25–7.41 (m, 5H); 13C NMR:
(75 MHz, CDCl3): d = 14.1; 43.4; 60.8; 70.3; 125.7; 127.7; 128.5;
142.6; 172.3. HRMS: calcd for C11H14NaO3 [M+Na]+ 217.0835,
found 217.0841.
4.3.2.5. Ethyl 3-acetoxy-3-(thiophen-3-yl)propanoate rac-3c
(R1 = Me). Yield: 90%; 1H NMR: (500 MHz, CDCl3): d = 1.22 (t,
J = 7.5 Hz, 3H); 2.04 (s, 3H); 2.88 (ddd, J = 5.5 Hz, J = 8.5 Hz,
J = 15.5 Hz, 2H); 4.13 (q, J = 7.5 Hz, 2H); 6.30 (dd, J = 5.5 Hz,
J = 8.5 Hz, 1H); 7.08–7.09 (m, 1H); 7.28–7.30 (m, 2H); 13C NMR:
(126 MHz, CDCl3): d = 14.1; 21.0; 40.7; 60.7; 67.8; 122.9; 125.9;
126.2; 140.0; 169.6; 169.8. HRMS: calcd for
C11H14NaO5S
[M+Na]+ 265.0505, found 265.0511.
4.3.2. Synthesis of racemic diesters rac-3a–e
Method A. At first, Et3N (6.17 mmol, 624 mg, 860
l
L), acetyl
4.3.2.6. 3-Ethoxy-3-oxo-1-(thiophen-3-yl)propyl butyrate rac-3c
(R1 = Pr). Yield: 86%; 1H NMR: (500 MHz, CDCl3): d = 0.90 (t,
J = 7.5 Hz, 3H); 1.22 (t, J = 7.0 Hz, 3H); 1.63 (overlapped tq,
J1 ꢂ J2 ꢂ 7.5 Hz, 2H); 2.27 (t, J = 7.5 Hz, 2H); 2.88 (ddd, J = 5.5 Hz,
J = 8.5 Hz, J = 15.5 Hz, 2H); 4.12 (q, J = 7.5 Hz, 2H); 6.31 (dd,
J = 5.5 Hz, J = 8.5 Hz, 1H); 7.07–7.09 (m, 1H); 7.27–7.31 (m, 2H);
13C NMR: (126 MHz, CDCl3): d = 13.5; 14.1; 18.4; 36.2; 40.8;
60.7; 67.6; 122.8; 125.9; 126.2; 140.2; 169.9; 172.4. HRMS: calcd
for C13H18NaO5S [M+Na]+ 293.0818, found 293.0818.
chloride (6.17 mmol, 485 mg, 440 L), and DMAP (0.16 mmol,
l
20 mg) were added into a solution of racemic 2a–e (5.61 mmol)
in dry CH2Cl2 (15 mL). The mixture was stirred at room tempera-
ture for 2–3 h and then quenched with water (15 mL). The isolated
organic layer was dried over anhydrous sodium sulfate and the sol-
vent was distilled off by rotatory evaporation. The crude product
was purified by vacuum-chromatography on silica gel using hex-
ane–EtOAc (3:1, v/v) as eluent to give the product as a semisolid
with 60–70% yield.
Method B. A mixture of racemic 2a–e (50 mmol), acid anhydride
(60 mmol, 9.5 g, 9.55 ml), and anhydrous cobalt (II) chloride
(32.4 g, 250 mmol) in acetonitrile (100 mL) was refluxed overnight.
After cooling at room temperature, the cobalt (II) chloride was fil-
tered off. The filtrate was evaporated in vacuo and the crude prod-
uct was purified by preparative vacuum-chromatography on silica
gel using hexane/EtOAc (3:1, v/v) as an eluent to give the product
as a semisolid with 80–90% yield.
4.3.2.7. Ethyl 3-acetoxy-3-(furan-3-yl)propanoate rac-3d
(R1 = Me). Yield: 81%; 1H NMR: (500 MHz, CDCl3): 1.23 (t,
J = 7.5 Hz, 3H); 2.03 (s, 3H); 2.85 (ddd, J = 5.5 Hz, J = 8.5 Hz,
J = 15.5 Hz, 2H); 4.14 (q, J = 7.5 Hz, 2H); 6.20 (dd, J = 5.5 Hz,
J = 8.5 Hz, 1H); 6.41–6.42 (m, 1H); 7.36–7.38 (m, 1H); 7.46–7.48
(m, 2H); 13C NMR: (126 MHz, CDCl3): d = 14.1; 21.0; 40.0; 60.7;
64.7; 108.7; 123.8; 140.5; 143.3; 169.5; 169.8. HRMS: calcd for
C
11H14NaO5 [M+Na]+ 249.0733, found 249.0726.
4.3.2.1. Ethyl 3-acetoxy-3-(thiophen-2-yl)propanoate rac-3a
(R1 = Me). Yield: 91%; 1H NMR: (300 MHz, CDCl3): d = 1.23 (t,
J = 7.2 Hz, 3H); 2.03 (s, 3H); 2.96 (ddd, J = 5.4 Hz, J = 8.7 Hz,
J = 15.6 Hz, 2H); 4.15 (q, J = 6.9 Hz, 2H); 6.47 (dd, J = 5.4 Hz,
J = 8.7 Hz, 1H); 6.94–6.97 (m, 1H); 7.09 (d, J = 3,3 Hz, 1H); 7.27
(d, J = 5,1 Hz, 1H); 13C NMR: (75 MHz, CDCl3): d = 14.1; 21.0;
41.4; 60.9; 67.3; 125.8; 126.2; 126.7; 141.8; 169.3; 169.7. HRMS:
calcd for C11H14NaO5S [M+Na]+ 265.0505, found 265.0501.
4.3.2.8. 3-ethoxy-1-(furan-3-yl)-3-oxopropyl butyrate rac-3d
(R1 = Pr). Yield: 85%; 1H NMR: (500 MHz, CDCl3): d = 0.91 (t,
J = 7.5 Hz, 3H); 1.23 (t, J = 7.0 Hz, 3H); 1.62 (overlapped tq,
J1 ꢂ J2 ꢂ 7.5 Hz, 2H); 2.26 (t, J = 7.5 Hz, 2H); 2.85 (ddd, J = 5.5 Hz,
J = 8.5 Hz, J = 15.5 Hz, 2H); 4.13 (q, J = 7.5 Hz, 2H); 6.21 (dd,
J = 5.5 Hz, J = 8.5 Hz, 1H); 6.40–6.42 (m, 1H); 7.36–7.38 (m, 1H);
7.45–7.47 (m, 2H); 13C NMR: (126 MHz, CDCl3): d = 13.5; 14.1;
18.4; 36.2; 40.1; 60.8; 64.5; 108.8; 124.0; 140.5; 143.3; 169.6;
172.5. HRMS: calcd for C13H18NaO5 [M+Na]+ 277.1046, found
277.1058.
4.3.2.2. 3-Ethoxy-3-oxo-1-(thiophen-2-yl)propyl butyrate rac-
3a (R1 = Pr). Yield: 87%; semisolid; 1H NMR: (300 MHz, CDCl3):
d = 0.91 (t, J = 7.5 Hz, 3H); 1.24 (t, J = 7.0 Hz, 3H); 1.63 (overlapped
tq, J1 ꢂ J2 ꢂ 7.5 Hz, 2H); 2.28 (t, J = 7.5 Hz, 2H); 2.97 (ddd, J = 5.4 Hz,
J = 9 Hz, J = 16.2 Hz, 2H); 4.14 (q, J = 7.2 Hz, 2H); 6.49 (dd,
J = 5.4 Hz, J = 9 Hz, 1H); 6.94–6.97 (m, 1H); 7.08–7.11 (m, 1H);
7.26–7.29 (m, 1H); 13C NMR: (75 MHz, CDCl3): d = 13.5; 14.1;
18.3; 36.1; 41.4; 60.8; 67.1; 125.6; 126.0; 126.6; 141.9; 169.3;
172.3. HRMS: calcd for C13H18NaO5S [M+Na]+ 293.0818, found
293.0818.
4.3.2.9. Ethyl 3-acetoxy-3-phenylpropanoate rac-3e (R1 = Me).
Yield: 88%; 1H NMR: (300 MHz, CDCl3): 1.23 (t, J = 7.2 Hz, 3H); 2.03
(s, 3H); 2.88 (ddd, J = 5.0 Hz, J = 9 Hz, J = 15.6 Hz, 2H); 4.14 (q,
J = 7.2 Hz, 2H); 6.21 (dd, J = 5.1 Hz, J = 9 Hz, 1H); 7.29–7.41 (m,
5H); 13C NMR: (75 MHz, CDCl3): d = 14.1; 21.0; 41.6; 60.7; 71.8;
126.4; 128.3; 128.5; 139.4; 169.6; 169.8. HRMS: calcd for
C
13H16NaO4 [M+Na]+ 259.0941, found 259.1030.
4.4. Enzymatic acylations
4.3.2.3. Ethyl 3-acetoxy-3-(furan-2-yl)propanoate rac-3b
(R1 = Me). Yield: 81%; 1H NMR: (300 MHz, CDCl3): d = 1.24 (t,
J = 7.2 Hz, 3H); 2.05 (s, 3H); 3.00 (ddd, J = 6 Hz, J = 8.1 Hz,
J = 15.6 Hz, 2H); 4.15 (q, J = 7.2 Hz, 2H); 6.25–6.38 (m, 3H); 7.39–
7.42 (m, 1H); 13C NMR: (75 MHz, CDCl3): d = 14.1; 20.9; 37.6;
60.8; 64.7; 109.0; 110.3; 142.7; 151.1; 169.4; 169.8. HRMS: calcd
for C11H14NaO5 [M+Na]+ 249.0733, found 249.0730.
4.4.1. Analytical scale kinetic resolution of rac-2a and rac-2e
One of the lipases (50 mg/mL) and an acyl donor (vinyl acetate
or butanoate, 5 equiv) were added into a solution of substrate (rac-
2a or rac-2e, 0.25 M) in an organic solvent (1 mL) or in neat vinyl
acetate. The reaction mixture was shaken (300 rpm) at room tem-
perature (23–24 °C). For HPLC or GC analysis, the samples taken
from the reaction mixture (10
lL) were diluted to 500 lL with 2-
4.3.2.4. 3-Ethoxy-1-(furan-2-yl)-3-oxopropyl butyrate rac-3b
(R1 = Pr). Yield: 85%; 1H NMR: (300 MHz, CDCl3): d = 0.91 (t,
J = 7.5 Hz, 3H); 1.23 (t, J = 7.2 Hz, 3H); 1.63 (overlapped tq,
J1 ꢂ J2 ꢂ 7.5 Hz, 2H); 2.27 (t, J = 7 Hz, 2H); 2.98 (ddd, J = 6 Hz,
J = 9 Hz, J = 15.9 Hz, 2H); 4.14 (q, J = 7.2 Hz, 2H); 6.26–6.38 (m,
propanol or TBME and filtered before injection
4.4.2. Preparative scale kinetic resolution of rac-2a–d
4.4.2.1. General procedure with CAL-A. At first, CAL-A on Celite
(250 mg) was added into a solution of one of the substrates

322
J. Brem et al. / Tetrahedron: Asymmetry 22 (2011) 315–322
18. Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia, L. A. J. Org. Chem.
1991, 56, 2656–2665.
19. Ericsson, D. J.; Kasrayan, A.; Johansson, P.; Bergfors, T.; Sandström, A. G.;
Bäckvall, J.-E.; Mowbray, S. L. J. Mol. Biol. 2008, 376, 109–119.
20. Engström, K.; Nyhlén, J.; Sandström, A. G.; Bäckvall, J.-E. J. Am. Chem. Soc. 2010,
132, 7038–7042.
21. Naik, S.; Basu, A.; Saikia, R.; Madan, B.; Paul, P.; Chaterjee, R.; Brask, J.;
Svendsen, A. J. Mol. Catal. B: Enzym. 2010, 65, 18–23.
22. Henke, E.; Pleiss, J.; Bornscheuer, U. T. Angew. Chem., Int. Ed. 2002, 41, 3211–3213.
23. Liljeblad, A.; Kallio, P.; Vainio, M.; Niemi, J.; Kanerva, L. T. Org. Biomol. Chem.
2010, 8, 886–895.
24. Domínguez de María, P.; Carboni-Oerlemans, C.; Tuin, B.; Bargeman, G.; Van
der Meer, A.; Van Gemert, R. J. Mol. Catal. B: Enzym. 2005, 37, 36–46.
25. Nielsen, T. B.; Ishii, M.; Kirk, O. Lipases A and B From the Yeast Candida
Antarctica. In Biotechnological Application of Cold-Adapted Organisms; Margesin,
R., Schinner, S., Eds.; Springer: Berlin-Heidelberg, Texas, 1999; pp 49–61.
26. Kirk, O.; Christensen, M. W. Org. Process. Res. Dev. 2002, 6, 446–451.
27. Balaji, B. S.; Chanda, B. M. Tetrahedron 1998, 54, 13237–13252.
28. Gedey, S.; Liljeblad, A.; Fülöp, F.; Kanerva, L. T. Tetrahedron: Asymmetry 1999,
10, 2573–2581.
29. Gedey, S.; Liljeblad, A.; Lázár, L.; Fülöp, F.; Kanerva, L. T. Can. J. Chem. 2002, 80,
565–570.
30. Li, X.-G.; Kanerva, L. T. Org. Lett. 2006, 8, 5593–5596.
31. Liljeblad, A.; Kavenius, H.-M.; Tähtinen, P.; Kanerva, L. T. Tetrahedron:
Asymmetry 2007, 18, 181–191.
rac-2a–d (1.25 mmol) in vinyl acetate (5 mL). The reaction mix-
tures were shaken (300 rpm) for 1 h at room temperature to reach
the conversion and enantiomeric composition of the products
shown in Table 4. The enzyme was removed by filtration and the
excess vinyl acetate by distillation. The crude products were puri-
fied by column chromatography on silica gel using hexane/EtOAc
(3:1, v/v) as an eluent to give the enantiomerically enriched prod-
ucts as semisolids with the isolated yields shown in Table 5.
NMR and MS spectra of the resolution products were indistin-
guishable from those of the racemates.
4.4.2.2. General procedure with CAL-B. Vinyl butanoate (5 equiv)
and CAL-B (250 mg) were added into a solution of one of the sub-
strates rac-2a–d (approx. 250 mg, 1.25 mmol) in MTBE (5 mL). The
reaction mixture was shaken (300 rpm) for 60 h at room tempera-
ture to reach the conversions and enantiomeric compositions
shown in Table 4. Work-up as above gave the products shown in
Table 5.
32. Kanerva, L. T.; Sundholm, O. J. Chem. Soc., Perkin Trans. 1 1993, 2407–2410.
33. Solymár, M.; Liljeblad, A.; Lázár, L.; Fülöp, F.; Kanerva, L. T. Tetrahedron:
Asymmetry 2002, 13, 1923–1928.
34. Solymár, M.; Fülöp, F.; Kanerva, L. T. Tetrahedron: Asymmetry 2002, 13, 2383–
2388.
35. Liljeblad, A.; Kanerva, L. T. Tetrahedron: Asymmetry 1999, 10, 4405–4415.
36. Mavrynsky, D.; Päiviö, M.; Lundell, K.; Sillanpää, R.; Kanerva, L. T.; Leino, R. Eur.
J. Org. Chem. 2009, 1317–1320.
Acknowledgments
J.B. thanks The Sectoral operational program human resources
development, Contract POSDRU 6/1.5/S/3—‘Doctoral studies:
through science toward society’ and the grant from the Center of
International Mobility (CIMO) in Finland for financial support.
37. Gedey, S.; Liljeblad, A.; Lázár, L.; Fülöp, F.; Kanerva, L. T. Tetrahedron:
Asymmetry 2001, 12, 105–110.
38. Fernández-Ibáñez, Á. M.; Maciá, B.; Minnaard, A. J.; Feringa, B. L. Angew. Chem.,
Int. Ed. 2008, 47, 1317–1319.
References
1. Bush, K.; Macielag, M.; Weidner-Wells, M. Curr. Opin. Microbiol. 2004, 7, 466–
476.
2. Kobayashi, R.; Konomi, M.; Hasegawa, K.; Morozumi, M.; Sunakawa, K.;
Ubukata, K. Antimicrob. Agents Chemother. 2005, 49, 889–894.
3. Dalhoff, A.; Janjic, N.; Echols, R. Biochem. Pharmacol. 2006, 71, 1085–1095.
4. Berks, A. H. Tetrahedron 1996, 52, 331–375.
5. Turcu, M. C.; Kiljunen, E.; Kanerva, L. T. Tetrahedron: Asymmetry 2007, 18,
1682–1687.
6. Hung, H.; Pan, X.; Tan, N.; Zeng, G.; Ji, C. Eur. J. Med. Chem. 2007, 42, 365–372.
7. Turcu, M. C.; Rantapaju, M.; Kanerva, L. T. Eur. J. Org. Chem. 2009, 5594–5600.
8. Zhang, S.; Norrlöw, O.; Wawrzynczyk, J.; Dey, E. S. Appl. Environ. Microbiol.
2004, 70, 6776–6782.
39. Xu, C.; Yuan, C. Tetrahedron 2005, 61, 2169–2186.
40. Fishman, A.; Eroshov, M.; Dee-Noor, S. S.; van Mil, J.; Cogan, U.; Effenberger, R.
Biotechnol. Bioeng. 2001, 74, 256–263.
41. Pihko, A. J.; Lundell, K.; Kanerva, L.; Koskinen, A. M. P. Tetrahedron: Asymmetry
2004, 15, 1637–1643.
42. Paizs, C.; Tähtinen, P.; Tosßa, M.; Majdik, C.; Irimie, F.-D.; Kanerva, L. T.
Tetrahedron 2004, 60, 10533–10540.
43. Paizs, C.; Tähtinen, P.; Lundell, K.; Poppe, L.; Irimie, F.-D.; Kanerva, L. T.
Tetrahedron: Asymmetry 2003, 14, 1895–1904.
44. Paizs, C.; Tosßa, M.; Majdik, C.; Tähtinen, P.; Irimie, F.-D.; Kanerva, L. T.
Tetrahedron: Asymmetry 2003, 14, 619–627.
ˇ
45. Brem, J.; Toßsa, M. I.; Paizs, C.; Munceanu, A.; Matkovíc-Calogovic´, D.; Irimie, F.-
9. Collins, A. N.; Sheldrake, G. N.; Crosby, J. Chirality in Industry II; John Wiley &
Sons, 1997.
10. Ratovelomanana-Vidal, V.; Girard, C.; Touati, R.; Tranchier, J. P.; Ben Hassine,
B.; Genêt, J. P. Adv. Synth. Catal. 2003, 345, 261–274.
D. Tetrahedron: Asymmetry 2010, 21, 1993–1998.
46. Turcu, M. C.; Perkiö, P.; Kanerva, L. T. Arkivoc 2009, iii, 251–263.
47. Turcu, M. C.; Perkiö, P.; Kanerva, L. T. Tetrahedron: Asymmetry 2010, 21, 739–
745.
11. Turner, N. J. Curr. Opin. Chem. Biol. 2010, 14, 115–121.
12. Lee, J. H.; Han, K.; Kim, M.-J.; Park, J. Eur. J. Org. Chem. 2010, 999–1015.
13. Kanerva, L. T.; Liljeblad, A. Transesterification—Biological, Wiley Online Library,
Encyclopedia of Catalysis 2010, doi:10.1002/0471227617.eoc197.
14. Brem, J.; Paizs, C.; Tosßa, M. I.; Vass, E.; Irimie, F. D. Tetrahedron: Asymmetry
2009, 20, 489–496.
48. Tjosås, F.; Anthonsen, T.; Jacobsen, E. E. Arkivoc 2008, vi, 81–90.
49. Uhm, K.-N.; Lee, S.-J.; Kim, H.-K.; Kang, H.-Y.; Lee, Y. J. Mol. Catal. B: Enzym.
2007, 45, 34–38.
50. Uppenberg, J.; Hansen, M. T.; Patkar, S.; Jones, T. A. Structure 1994, 2, 293–308.
51. Rotticci, D.; Hæ´ ffner, F.; Orrenius, C.; Norin, T.; Hult, K. J. Mol. Catal. B: Enzym.
1998, 5, 267–272.
52. Uppenberg, J.; Öhrner, N.; Norin, M.; Hult, K.; Kleywegt, G. J.; Patkar, S.;
Waagen, V.; Anthonsen, T.; Jones, T. A. Biochemistry 1995, 34, 16838–16851.
53. Chen, C.-S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104,
7294–7299.
15. Brem, J.; Toßsa, M. I.; Paizs, C.; Vass, E.; Irimie, F. D. Tetrahedron: Asymmetry
2010, 21, 365–373.
16. Huerta, F. F.; Bäckvall, J.-E. Org. Lett. 2001, 3, 1209–1212.
17. Liljeblad, A.; Kanerva, L. T. Tetrahedron 2006, 62, 5831–5854.