Heterocycles 35. CaL-B mediated synthesis of enantiomerically pure (R)- and (S)-ethyl 3-(2-arylthiazol-4-yl)-3-hydroxypropanoates

Article abstract of DOI:10.1016/j.tetasy.2014.01.013

Herein we present the lipase catalyzed synthesis of four new enantiomerically pure (R)- and (S)-ethyl 3-(2-arylthiazol-4-yl)-3- hydroxypropanoates and their butanoates by enzymatic enantioselective acylation of the racemic alcohols rac-1a-d and by ethanolysis of the corresponding racemic esters rac-2a-d mediated by lipase B from Candida antarctica (CaL-B) in organic solvents. In terms of stereoselectivity and activity, both procedures, the acylation and alcoholysis, are successful (50% conversion, E ? 200). The absolute configuration of the resolution products was determined by a detailed ¹H NMR study of the Mosher's derivatives of (S)-1a.

Full text of DOI:10.1016/j.tetasy.2014.01.013

Tetrahedron: Asymmetry 25 (2014) 298–304
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Heterocycles 35. CaL-B mediated synthesis of enantiomerically pure
(R)- and (S)-ethyl 3-(2-arylthiazol-4-yl)-3-hydroxypropanoates
Annamária Varga a, Mara Ana Naghi b, Melinda Füstös b, Gabriel Katona b, Valentin Zaharia a,
⇑
a Iuliu Haßtieganu University of Medicine and Pharmacy, Department of Organic Chemistry, Ro-400012 Cluj-Napoca, Victor Babeßs 41, Romania
b Faculty of Chemistry and Chemical Engineering, Babeßs-Bolyai University, 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:
Received 11 October 2013
Accepted 22 January 2014
Herein we present the lipase catalyzed synthesis of four new enantiomerically pure (R)- and (S)-ethyl
3-(2-arylthiazol-4-yl)-3-hydroxypropanoates and their butanoates by enzymatic enantioselective acyla-
tion of the racemic alcohols rac-1a–d and by ethanolysis of the corresponding racemic esters rac-2a–d
mediated by lipase B from Candida antarctica (CaL-B) in organic solvents. In terms of stereoselectivity
and activity, both procedures, the acylation and alcoholysis, are successful (50% conversion, E ꢀ 200).
The absolute configuration of the resolution products was determined by a detailed 1H NMR study of
the Mosher’s derivatives of (S)-1a.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Recently, the lipase catalyzed kinetic resolution of various hetero-
aryl-b-hydroxy esters such as benzofuranyl- and benzothiophenyl-
Thiazoles and their derivatives can be found associated with
various antibacterial, antifungal, and anti-inflammatory activities.1
Some thiazole compounds, such as ritonavir, show anti-inflamma-
tory effects,2 imidacloprid is used as a pharmaceutical product,
while others are agrochemical products.3
b-hydroxy esters10 or phenothiazinyl-b-hydroxy esters11 has been
studied. Moreover, the opposite enantiomer preference of CaL-A
and CaL-B in the enantioselective acylation of furan-2-yl- and
furan-3-yl-, thiophene-2-yl-, and thiophene-3-yl-b-hydroxy esters
was reported.12 Brem et al. demonstrated that CrL catalyzed
esterification with fatty acid vinyl esters can significantly enhance
the enantiopurity of the resolution products.13 The influence of
the pretreatment of the lipases, the substrate concentration, and
the reaction conditions upon the stereochemical outcome of the
enzymatic reaction was also reported on.14 Furthermore, using
adsorption, cross linked enzyme aggregate (CLEA) technology,
and sol–gel encapsulation methods, a tailor made enzyme immobi-
lization for the selective O-acylation of the aryl b-hydroxy esters
was also successfully realized.15
Encouraged by these factors, we have developed a lipase cata-
lyzed kinetic resolution of racemic 1-(2-arylthiazol-4-yl)-b-
hydroxypropanoates rac-1a–d and their corresponding butanoates
rac-2a–d (Scheme 1). The absolute configuration of the novel enan-
tiopure compounds was then determined by a detailed 1H NMR
study of the Mosher’s derivatives of (S)-1a.
Enantiopure b-hydroxy esters bearing various aromatic moie-
ties are versatile chiral synthons4 in organic synthesis and are
widely used as starting materials or intermediates for the synthesis
of many pharmaceutical products such as L
-carnitine5 or fluoxe-
tine.6 In addition to chemical methods,7 milder enzymatic methods
have been developed for the synthesis of enantiomerically pure
b-hydroxy esters. Biocatalytic approaches for the synthesis of these
target molecules broadly involve: (1) enantioselective reduction of
the corresponding b-ketoesters; (2) kinetic or dynamic kinetic res-
olution of b-hydroxy esters; and (3) deracemization of b-hydroxy
esters.8
The enzymatic kinetic resolution of a racemate with a suitable
hydrolytic enzyme is one of the most common biocatalytic meth-
ods used nowadays. Enantioselectivity, high stability, and good
commercial availability in free and immobilized forms have made
lipases (EC 3.1.1.3) attractive kinetic resolution catalysts either
through the acylation of a nucleophilic (for example an alcohol)
functionality, or through the deacylation (hydrolysis, alcoholysis,
or interesterification) of an ester functionality in a molecule.9
2. Results and discussion
2.1. Chemical synthesis
Racemic ethyl 3-(2-arylthiazol-4-yl)-3-hydroxypropanoates
rac-1a–d were prepared by the Reformatsky reaction starting from
⇑
Corresponding author. Tel.: +40 264 594 148; fax: +40 264 597 257.
E-mail address: vzaharia@umfcluj.ro (V. Zaharia).
http://dx.doi.org/10.1016/j.tetasy.2014.01.013
0957-4166/Ó 2014 Elsevier Ltd. All rights reserved.

A. Varga et al. / Tetrahedron: Asymmetry 25 (2014) 298–304
299
O
OH
O
O
O
O
OH
O
I.
CaL-B
O
C3H7
R
R
O
O
R
O
R
O
+
+
toluene,
molecular sieves
1a-d
(S)-
1a-d
II.
2a-d
(R)-
rac-
O
O
O
OH
O
O
O
O
CaL-B
R
O
R
R
O
O
+
OH
+
R
O
DIPE,
molecular sieves
2a-d
(S)-
1a-d
(R)-
rac-2a-d
OH
O
CaL-B
OH
+
O
O
O
DIPE,
molecular sieves
1a-d
(R)-
R
O
2a-d
(R)-
N
N
N
N
S
S
S
S
R:
Cl
d
c
a
b
I. Ethyl 2-bromoacetate, Zn, THF, reflux; II. CH2Cl2, butanoic anhydride, triethylamine, DMAP
Scheme 1. The chemical synthesis of rac-1a–d and rac-2a–d and the enzymatic route for the preparation of enantiomerically pure arylthiazol-b-hydroxy esters and their
corresponding diesters.
the corresponding 2-arylthiazol-4-carbaldehydes.16 By the chemi-
cal acylation of rac-1a–d, with butanoic anhydride in the presence
of triethylamine and a catalytic amount of 4-N,N-dimethylamino-
pyridine (DMAP) in dichloromethane the preparation of the race-
mic diesters rac-2a–d was also performed as shown in Scheme 1,
routes I and II.
Celite (AK on Celite) were tested as suitable biocatalysts in dry or-
ganic solvents. The experiments were performed in the presence
of molecular sieves, since even small traces of water could promote
hydrolytic reactions, thus yielding undesired by-products or caus-
ing a decrease in the enantiopurity of the products.
The nature of the solvent and the nucleophile can significantly
influence the activity and selectivity of the enantioselective enzy-
matic acylation. Thus, the solvents used were selected and tested
as they are commonly accepted by lipases. Moreover, solvents
should be chosen from those frequently used in industry, they have
to be easy to evaporate and must dissolve both the substrates and
products. Among the enzymes tested for the O-acylation of our
model compound rac-1a using vinyl acetate and vinyl butanoate,
lipase AK on Celite and Pseudomonas cepacia lipase (LPS free) were
catalytically inactive in all of the solvents tested, while CrL,
depending on the acylation agent and the solvent used, showed
higher or lower selectivity and activity. For the CrL mediated acyl-
ation using vinyl acetate in methyl tert-butyl ether (MTBE), diiso-
propyl ether (DIPE), hexane and toluene, both the reaction rate
and the selectivity were low (Table 1, entries 10–13). When using
vinyl butanoate, the reaction gave low selectivity in MTBE, DIPE,
and hexane (Table 2, entries 6–8). By performing the CaL-A on
Celite mediated O-acylation of rac-1a using vinyl acetate in various
ethers such as MTBE and DIPE or nonpolar solvents such as hexane,
toluene, and acetonitrile, the reactions showed moderate
enantioselectivities (E = 5–14, Table 1, entries 1–5). When, in the
same experiments, vinyl butanoate was used as the acyl donor, a
considerably decreased enzyme activity was detected in MTBE
(Table 2, entry 1), while for the rest of the solvents tested, CaL-A
2.2. Enzymatic synthesis
In order to investigate the stereoselectivity of the reactions
involving chiral 3-(2-arylthiazol-4-yl)-3-hydroxypropanoates and
their diesters, the chromatographic separation of their enantio-
mers was first established. The base-line separation of the enanti-
omers of rac-1, 2a–d was performed using a Chiralpak IB HPLC
column as shown in Section 4.1.
2.2.1. Analytical scale enzymatic acylation of rac-1a–d
In an effort to obtain highly enantiomerically enriched resolution
products, commercially available free or immobilized lipase prepa-
rations (25 mg/ml) were screened in various organic solvents at
room temperature, for the enantioselective acylation using vinyl
acetate and vinyl butanoate as irreversible acyl donors (0.1 M) of
the racemic 3-hydroxy-3-(2-phenylthiazol-5-yl)propanoate rac-1a
(0.025 M), which was used as a model compound. Lipase A from
Candida antarctica immobilized by adsorption on Celite (CaL-A on
Celite), CaL-B (Novozyme 435), lipase B from Candida antarctica
immobilized by adsorption on single wall carbon nanotube
(CaL-B-SWCNT), lipase from Candida rugosa (CrL), free Pseudomonas
cepacia lipase (LPS free) and lipase AK immobilized by adsorption on

300
A. Varga et al. / Tetrahedron: Asymmetry 25 (2014) 298–304
Table 1
those found for CaL-A on Celite. However, a major improvement
in selectivity was obtained for the CaL-B catalyzed O-acylation
with vinyl butanoate using hexane and toluene as solvents (Table 2,
entries 4–5), yielding highly enantiomerically enriched resolution
products at approximately 50% conversion. CaL-B-SWCNT proved
to be completely inactive for the present purpose, although it did
show high activity and selectivity for the acylation of rac-1-
phenylethanol.
Lipase and solvent screen for the selective O-acylation of racemic substrate rac-1a
using vinyl acetate, after 17 h
Entry
Lipase
Solvent
c (%)
ee(R)-2a
ee(S)-1a
E
1
2
3
4
5
6
7
8
CaL-A on Celite
CaL-A on Celite
CaL-A on Celite
CaL-A on Celite
CaL-A on Celite
CaL-B
CaL-B
CaL-B
CaL-B
CrL
MTBE
DIPE
Acetonitrile
Hexane
Toluene
MTBE
35.3
78.3
68
72.4
29.6
35.6
32.3
53.7
24.5
5.2
64
26
45
38
64
47
65
62
74
36
40
47
68
35
94
96
6
5
9
>99
14
6
27
26
31
72
24
2
15
24
10
3.5
2.2.2. Analytical scale enzymatic alcoholysis of rac-2a–d
Lipases usually retain the enantiopreference found in the stere-
oselective acylation of chiral alcohols including the hydrolysis or
alcoholysis of the ester counterparts. Consequently, such reactions
should result in the opposite enantiomeric forms of the enantiome-
rically enriched ethyl 3-(2-arylthiazol-4-yl)-3-hydroxypropanoates
1a–d and their diesters 2a–d as those found in the enzymatic
acylation of rac-1a–d. The enzymatic alcoholysis of rac-2a, used
as the model compound, was investigated. The experiments were
carried out using the same enzymes in all of the solvents tested
for the enzymatic acylation by adding 5 equiv of ethanol or 1-buta-
nol into the reaction mixture. Ethanolysis mediated by CaL-B
showed the highest selectivity and activity when MTBE (Table 3,
entry 5) and DIPE (Table 3, entry 6) were used as reaction media.
Due to the higher activity of the enzyme in DIPE, this solvent was
used in further experiments.
DIPE
6.3
8.9
8.4
2
2.7
3.5
5.8
Hexane
Toluene
MTBE
DIPE
Hexane
Toluene
9
10
11
12
13
CrL
CrL
CrL
27.2
33.8
12.8
Table 2
Lipase and solvent screen for the selective O-acylation of racemic substrate rac-1a
using vinyl butanoate, after 17 h
Entry
Lipase
Solvent
c (%)
ee(R)-2a
ee(S)-1a
E
1
2
3
4
5
6
7
8
9
CaL-A on celite
CaL-B
CaL-B
CaL-B
CaL-B
CrL
CrL
CrL
LPS free
MTBE
MTBE
DIPE
Hexane
Toluene
MTBE
DIPE
81.9
51.6
52.6
52.8
50
13
91
89
89
59
97
99
>99
>99
8
23
50
73
2
89
89.7
127.8
>99
ꢀ200
2.2.3. Preparative scale enzymatic acylation of rac-1a–d
Using the optimal conditions found for the analytical scale bio-
transformations of both rac-1a and rac-2a, the CaL-B mediated pre-
parative scale acylation of rac-1a–d with vinyl butanoate in
toluene (Table 4, entries 1–4) and the ethanolysis of rac-2a–d in
DIPE (Table 4, entries 5–8) was next performed with good reactiv-
ity and selectivity (E ꢀ 200, 99% ees for the most of the resolution
products at approximately 50% conversion). All of the dilutions,
substrate/biocatalyst ratios and reaction conditions were the same
as in the analytical scale reactions. The yields of the isolated and
chromatographically purified resolution products were in the
range of 45–48%, calculated related to the starting racemic
substrates and are given in Table 4, together with specific rotations
of the enantiopure products.
20
32
35
41
80
2
2.5
3.8
39.6
54.9
47.7
Hexane
Hexane
19.5
Table 3
Alcohol, lipase, and solvent screen for the selective alcoholysis of racemic diester rac-
2a, after 14 h
Entry Alcohol Lipase Solvent
c (%) ee(R)-1a ee(S)-2a
E
1
2
3
4
5
6
7
8
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
BuOH
BuOH
BuOH
BuOH
BuOH
BuOH
CaL-A
CaL-A
CaL-A
CaL-A
CaL-B
CaL-B
CaL-B
CrL
CaL-A
CaL-B
CaL-B
CaL-B
CrL
MTBE
DIPE
Acetonitrile
Hexane
MTBE
11.1
32.2
8.3
19.3
49.7
48.4
6.6
9.8
3
83
64
59
77
71
97
>99
>99
55
>99
19
8
28
7
17
96
93
7
6
3
94
46
17
8
4.9
5
8.2
7
>200
DIPE
ꢀ200
2.2.4. Reuse of the catalysts
Toluene
Hexane
Acetonitrile
DIPE
Hexane
Toluene
DIPE
>200
An important consideration for the catalytic reaction is the
reusability of the catalyst. In order to test the recycling capacity
of CaL-B, we used rac-1a as the substrate for the O-acylation and
rac-2a as the substrate for the alcoholysis. Graphic 1 presents the
initial activity of CaL-B as a function of the number of cycles and
it can be observed that after 10 cycles, for both the acylation and
alcoholysis, the activity of the enzyme decreases only slightly.
Every reaction was allowed to proceed up to 50% conversion before
the catalyst was subjected to the next cycle. Between cycles, the
catalyst was washed with dry solvent and then immediately
3.6
9
>200
4
10
11
12
13
14
39.3
16.6
13.1
14.5
71
85
53
47
9
14
3.5
3
CrL
Hexane
8
on Celite was inactive. In terms of activity and selectivity (E = 3–9,
Table 1, entries 6–9) for the CaL-B catalyzed O-acylation using vi-
nyl acetate in all solvents tested, similar results were obtained to
Table 4
Preparative scale enzymatic acylation of rac-1a-d and ethanolysis of rac-2a–d
Entry
Substrate
Time (h)
Products
E
a Da
ꢁ
½ ꢁ
a Da
ee(R)-2
Yield (%)
ee(S)-1
99
>99
99
>99
ee(S)-2
Yield (%)
½
1
2
3
4
rac-1a
rac-1b
rac-1c
rac-1d
19
19
19
17
99
98
99
99
47
45
45
+78.5
+75.2
+72.8
+86.2
46
47
45
ꢂ56.8
ꢂ53.5
ꢂ51.3
ꢂ60.4
ꢀ200
ꢀ200
ꢀ200
ꢀ200
46
45
a Da
ꢁ
½
a Da
ꢁ
ee(R)-1
Yield (%)
Yield (%)
½
5
6
7
8
rac-2a
rac-2b
rac-2c
rac-2d
17
48
48
17
>99
98
>99
>99
48
46
45
45
+57.2
+49.8
+51.9
+59.8
98
>99
>99
99
48
45
45
46
ꢂ76
ꢀ200
ꢀ200
ꢀ200
ꢀ200
ꢂ74.1
ꢂ68.5
ꢂ83

A. Varga et al. / Tetrahedron: Asymmetry 25 (2014) 298–304
301
reused. During the recycling process, there was a small decrease in
reaction rate, but the enantiomeric ratio remained high (E >200).
2.3. Absolute configuration
The absolute configuration of ethyl 3-hydroxy-3-(2-phen-
ylthiazol-4-yl)propanoate (ꢂ)-1a was determined by 1H NMR using
both enantiomers of
a-methoxy-a-(trifluoro-methyl)phenylacetic
acid (MTPA), of already known absolute configuration.17 Thus, the
enantiomerically pure alcohol (ꢂ)-1a, recovered during the CaL-
B-mediated acylation, was esterified with both (R)-MTPA and (S)-
MTPA. The chemical shifts of the methoxy groups of the two diaste-
reomeric MTPA esters were used as reference signals in order to
determine the absolute configuration of (ꢂ)-1a. The chemical shift
difference of the methoxy signals appeared because in one of
the diastereomers, the methoxy group and the phenylthiazole
ring of the hydroxy ester are on the same side of the MTPA
plane, while in the other diastereomer they are opposite to each
other. The methoxy signals for the two diastereomers appeared
at d 3.47 for the (S)-MTPA-(ꢂ)-1a ester and d 3.57 for the
(R)-MTPA-(ꢂ)-1a ester (Fig. 1). This proves that the unknown enan-
tiomerically pure (ꢂ)-1a obtained from the O-acylation reaction of
rac-1a is the (S)-enantiomer. The results showed that as expected,
in the acylation reaction of the racemic alcohols, all of the enzymes
followed Kazlauskas’ rule, which predicted the (R)-enantiomer
preference of the lipases. The configurations of all of the unreacted
enantiomers in the CaL-B catalyzed acylations were assigned as (S)
on the basis of the same sign of their specific rotations.
Graphic 1. Variation of CaL-B activity in both acylation and alcoholysis reactions
during 10 reuse cycles.
S
S
O
O
N
N
O
O
O
O
Ph
(R)
H3CO
OCH3
Ph
(S)
O
O
CF3
CF3
Figure 1. Overlapped H NMR spectra of –OCH3 in (S,S) MTPA-1a (blue trace) and (R,S) MTPA-1a (green trace) esters.

302
A. Varga et al. / Tetrahedron: Asymmetry 25 (2014) 298–304
Table 5
tion of CaL-B-SWCNT was purchased from Chiralvision. The ethyl
The retention times of the enantiomers of rac-1–2a–d
3-(2-arylthiazol-4-yl)-3-hydroxypropanoates were synthesized
from the corresponding aldehydes using the Reformatsky
reaction.11
Compound
tr (min)
Compound
tr (min)
(S)-1a
(R)-1a
(S)-1b
(R)-1b
(S)-1c
(R)-1c
(S)-1d
(R)-1d
7.6
10.3
12.4
17.5
12.4
17
(S)-2a
(R)-2a
(S)-2b
(R)-2b
(S)-2c
(R)-2c
(S)-2d
(R)-2d
5.2
5.6
6.5
7
6.2
6.7
5.9
6.4
4.3. Synthesis of racemic compounds
4.3.1. Synthesis of the racemic ethyl 3-(2-arylthiazol-4-yl)-3-
hydroxypropanoates rac-1a–d
10.8
15.5
At first, b-hydroxy esters rac-1a–d were prepared by adding a
solution of ethyl bromoacetate (8.4 g, 5.6 ml, 0.05 mol) in tetrahy-
drofuran (10 ml) in small portions with slight warming into a mix-
ture of zinc powder (4 g, 0.061 mol) and one of the corresponding
aldehydes (0.06 mol) in tetrahydrofuran (10 ml). The aldehyde in
THF and zinc powder had been previously refluxed for 30 min.
After completion of the reaction (approximately 1.5 h), the mixture
was cooled to room temperature and filtered to remove the unre-
acted zinc. The solvent was evaporated 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) with vigorous stirring. After separation,
the aqueous layer was extracted with CH2Cl2 (3 ꢃ 100 ml). The
combined organic layers were washed with water (50 ml), dried
over anhydrous magnesium sulfate, and evaporated in vacuo. The
crude product was purified by preparative vacuum chromatogra-
phy using hexane:ethyl acetate 3:1 (v/v) as the eluent.
3. Conclusion
An efficient enzymatic procedure for the synthesis of enantio-
merically pure ethyl 3-(2-arylthiazol-4-yl)-3-hydroxypropanoates
has been described (ee 99%). By using an enzymatic kinetic
resolution, both enantiomerically pure stereoisomers of four ethyl
3-(2-arylthiazol-4-yl)-3-hydroxypropanoates 1a–d and four but-
anoates 2a–d were synthesized with high yields. CaL-B proved to
be the optimal biocatalyst for both the acylation of rac-1a–d with
vinyl butanoate in toluene and the ethanolysis of rac-2a–d in DIPE.
CaL-B has also been shown to be efficiently reusable in up to ten
cycles, since its activity and stereoselectivity remain unaltered.
4. Experimental
4.1. Analytical methods
4.3.1.1. rac-Ethyl 3-hydroxy-3-(2-phenylthiazol-4-yl)propano-
ate rac-1a.
Yield: 92%; yellow solid; mp = 48 °C; 1H NMR
The 1H NMR and 13C NMR spectra were recorded on a Bruker
spectrometer operating at 300 MHz and 75 MHz respectively, at
25 °C. Electron impact mass spectra (EI-MS) were taken on a VG
7070E mass spectrometer operating at 70 eV. High Performance Li-
quid Chromatography (HPLC) analyses were conducted using an
Agilent 1200 instrument (Table 5). The enantiomeric separation
(300 MHz, CDCl3): d =1.25 (t, J = 7.5 Hz, 3H), 2.97 (ddd, J = 3 Hz,
J = 9 Hz, J = 15 Hz, 2H), 3.86 (s, 1H), 4.18 (q, J = 9 Hz, 2H), 5.3 (dd,
J = 3 Hz, J = 9 Hz, 1H), 7.24 (d, J = 3 Hz, 1H), 7.37–7.45 (m, 3H),
7.88–7.94 (m, 2H); 13C NMR (75 MHz, CDCl3): d = 14.2, 41.5, 60.9,
67.6, 114.0, 126.5, 128.9, 130.1, 133.5, 158.1, 168.4, 172.4; IR:
m
= 3019, 2985, 2881, 2826, 2772, 2330, 1777, 1384, 1326, 1275,
of rac-1,2a was performed on
a
Chiralpak IB column
1185, 1033, 762, 751; MS: M+ found (M+ calculated for C14H15NO3-
S): 277.0752 (277.0773); MS m/z: 278(M+1, 12), 277(M, 29),
214(13), 204(100), 190(52), 188(17), 162(18), 121(13), 104(18),
77(15), 59(22), 29(16).
(4.6 ꢃ 250 mm) and a mixture of n-hexane and 2-propanol 90:10
(v/v) as eluent; rac-1–2b,c,d were separated on Chiralpak IB col-
umn with a mixture of n-hexane and 2-propanol 95:5 (v/v) as
eluent.
Thin layer chromatography (TLC) was carried out using Merck
Kieselgel 60F254 sheets. Spots were visualized by treatment with
5% ethanolic phosphomolybdic acid solution and heating. Prepara-
tive chromatographic separations were performed using column
4.3.1.2. rac-Ethyl 3-(2-(4-chlorophenyl)thiazol-4-yl)-3-hydroxy-
propanoate rac-1b.
Yield: 91%; yellow solid; mp = 49 °C; 1H
NMR (300 MHz, CDCl3): d = 1.24 (t, J = 6 Hz, 3H), 2.94 (ddd,
J = 3 Hz, J = 9 Hz, J = 15 Hz, 2H), 3.90 (d, J = 3 Hz, 1H), 4.16 (q,
J = 6 Hz, 2H), 5.25–5.30 (m, 1H), 7.25 (s, 1H), 7.35 (d, J = 9 Hz,
2H), 7.81 (d, J = 9 Hz, 2H); 13C NMR (75 MHz, CDCl3): d = 14.2,
41.5, 60.9, 67.5, 114.4, 127.7, 129.1, 132.0, 136.0, 159.0, 167.0,
chromatography on Merck Kieselgel 60 (63–200
lm). Melting
points were determined by hot plate method and are
a
uncorrected. Optical rotations were determined on a Bellingham-
Stanley ADP 220 polarimeter. The determination of E was based
on the equation E = ln[(1 ꢂ c)(1 ꢂ eeS)]/ln [(1 ꢂ c)(1 + eeS)], with
c = eeS/(eeS + eeP).18
172.3; IR:
m = 3162, 3116, 1792, 1457, 1395, 1275, 1186, 1092,
835, 751; MS: M+ found (M+ calculated for C14H14ClNO3S):
311.0407 (311.0383); MS m/z: 314(M+1, 37Cl, 2), 313(M, 37Cl, 10),
312(M+1, 35Cl, 6), 311(M, 35Cl, 30), 240(37Cl, 32), 238(35Cl, 100),
226(37Cl, 20), 224(35Cl, 60), 198(37Cl, 5), 196(35Cl, 15), 157(37Cl,
4), 155(13), 59(25), 43(13), 29(15).
4.2. Reagents, solvents, and biocatalysts
All reagents were purchased from Aldrich or Fluka and used as
received. Solvents and acyl donors for the enzymatic reactions
were stored over molecular sieves unless otherwise stated. Lipases
from Pseudomonas fluorescens (AK) immobilized by adsorbtion on
Celite and Pseudomonas cepacia (Burkholderia cepacia, LPS) were
purchased from Amano England. Candida rugosa lipase (CrL) was
purchased from Fluka. Lipase B from Candida antarctica (CaL-B,
Novozyme 435) was purchased from Novozyme, Denmark. Lipase
A from Candida antarctica immobilized by adsorbtion on Celite
(CaL-A) was a gift from Prof. Liisa T. Kanerva, University of Turku,
Finland. CaL-B-SWCNT was prepared in our laboratories. Single-
walled carbon nanotubes were purchased from Organic Chemicals
Co. Ltd. The microcrystalline CaL-B powder used for the prepara-
4.3.1.3. rac-Ethyl 3-hydroxy-3-(2-p-tolylthiazol-4-yl)propano-
ate rac-1c.
Yield: 95%; yellow semisolid; 1H NMR (300 MHz,
CDCl3): d = 1.25 (t, J = 7.5 Hz, 3H), 2.37 (s, 3H), 2.96 (ddd, J = 3 Hz,
J = 9 Hz, J = 15 Hz, 2H), 3.84 (d, J = 6 Hz, 1H), 4.17 (q, J = 7.5
Hz, 2H), 5.26-5.32 (m, 1H), 7.21 (d, J = 6 Hz, 3H), 7.79 (d,
J = 9 Hz, 2H); 13C NMR (75 MHz, CDCl3): d = 14.2, 21.4, 41.6, 60.8,
67.6, 113.5, 126.4, 129.6, 130.9, 140.3, 158.6, 168.6, 172.4; IR:
m
= 3371, 3328, 3101, 3037, 2990, 1727, 1517, 1461, 1185, 1034,
756; MS: M+ found (M+ calculated for C15H17NO3S):
291.0897(291.0929); MS m/z: 292(M+1, 27), 291(M, 35), 274(11),
219(15), 218(100), 204(52), 202(19), 200(12), 176(16), 135(13),
118(18), 91(12), 29(16).

A. Varga et al. / Tetrahedron: Asymmetry 25 (2014) 298–304
303
4.3.1.4. rac-Ethyl 3-hydroxy-3-(2-m-tolylthiazol-4-yl)propano-
ate rac-1d.
Yield: 94%; yellow solid; mp = 40 °C; 1H NMR
4.3.2.4. rac-2-(Ethoxycarbonyl)-1-(2-m-tolylthiazol-4-yl)ethyl
butanoate rac-2d.
Yield: 91%; yellow oil; 1H NMR
(300 MHz, CDCl3): d = 1.29 (t, J = 6 Hz, 3H), 2.43 (s, 3H), 2.99
(ddd, J = 3 Hz, J = 9 Hz, J = 15 Hz, 2H), 3.69 (d, J = 6 Hz, 1H), 4.21
(q, J = 6 Hz, 2H), 5.35–5.29 (m, 1H), 7.23–7.36 (m, 3H), 7.71–7.78
(m, 2H); 13C NMR (75 MHz, CDCl3): d = 14.3, 21.4, 41.5, 60.9,
67.7, 113.9, 123.8, 127.1, 128.9, 131.0, 133.5, 138.8, 158.6, 168.7,
(300 MHz, CDCl3): d = 0.95 (t, J = 7.5 Hz, 3H), 1.26 (t, J = 7.5 Hz,
3H), 1.69 (overlapped tq, J1 ꢄ J2 ꢄ 7.2 Hz, 2H), 2.36 (t, J = 7.5, 2H),
2.44 (s, 3H), 3.16 (d, J = 6 Hz, 2H), 4.17 (q, J = 6 Hz, 2H), 6.41 (t,
J = 7.5 Hz,1H), 7.24–7.36 (m, 3H), 7.73 (d, J = 9 Hz, 1H), 7.79 (s,
1H); 13C NMR (75 MHz, CDCl3): d = 13.7, 14.2, 18.5, 21.4, 36.3,
39.3, 60.8, 68.2, 116.4, 123.9, 127.2, 128.9, 131.0, 133.4, 138.8,
172.6; IR:
m= 3571, 1725, 1312, 1286, 1245, 1185, 1029, 798,
752, 733, 687; MS: M+ found (M+ calculated for C15H17NO3S):
291.0975 (291.0929); MS m/z: 292(M+1, 8), 291(M, 29), 219(15),
218(100), 204(46), 202(11), 176(12), 135(8), 118(11), 91(10),
59(11), 43(11), 29(7).
154.7, 168.7, 170.1, 172.7; IR: m = 2968, 1738, 1462, 1371, 1286,
1243, 1173, 1033, 798, 751, 706; MS: M+ found (M+ calculated
for C19H23NO4S): 361.1337 (361.1348); MS m/z: 362(M+1, 2),
361(M, 4), 290(20), 289(100), 243(71), 227(16), 203(15), 201(70),
71(16), 58(9), 43(31), 29(15).
4.3.2. Synthesis of racemic 2-(ethoxycarbonyl)-1-(2-arylthiazol-
4-yl)ethyl butanoate rac-2a–d
4.4. Enzyme mediated biotransformation of rac-1–2a–d on an
To a solution of the b-hydroxyester rac-1a–d (50 mg) in dichlo-
analytical scale
romethane (1.5 ml), butanoic anhydride (1.1 equiv, 32.16
ll),
triethylamine (1.1 equiv, 27.6 l) and catalytic amount of
l
a
4.4.1. Analytical scale enzymatic acylation of rac-1a–d
For each reaction, the enzyme (12.5 mg) and molecular sieves (2
pieces) were added into a solution of the substrate rac-1a–d
4-N,N-dimethylamino-pyridine (0.028 equiv, 0.6 mg) were added.
After stirring for 3 h at room temperature, the solvent was
evaporated and the crude product was separated using column
chromatography and a 6:4 mixture of hexane/ethyl acetate as the
eluent.
(0.025 M) in 500 ll of solvent, followed by the addition of the acyl-
ation agent (0.1 M). The reaction mixture was shaken at 1350 rpm
at room temperature for 17 h. The reactions were then verified on
TLC after 2 and 17 h, respectively using a hexane/ethyl acetate 6:4
mixture as the eluent. The spots were visualized by treatment with
a 5% ethanolic phosphomolybdic acid solution and heating.
4.3.2.1.
butanoate rac-2a.
rac-2-(Ethoxycarbonyl)-1-(2-phenylthiazol-4-yl)ethyl
Yield: 92%; yellow oil; 1H NMR
(300 MHz, CDCl3): d = 0.93 (t, J = 6 Hz, 3H), 1.23 (t, J = 7.5 Hz, 3H),
1.66 (overlapped tq, J1 ꢄ J2 ꢄ 7.2 Hz, 2H), 2.33 (t, J = 7.5 Hz, 2H),
3.13 (d, J = 6 Hz, 2H), 4.14 (q, J = 6 Hz, 2H), 6.39 (t, J = 6 Hz, 1H),
7.25 (d, J = 3 Hz, 1H), 7.40–7.45 (m, 3H), 7.90–7.94 (m, 2H); 13C
NMR (75 MHz, CDCl3): d = 13.6, 14.2, 18.5, 36.2, 39.3, 60.8, 68.2,
116.5, 126.6, 128.9, 130.2, 133.5, 154.7, 168.4, 170.0, 172.7; IR:
4.4.2. Analytical scale enzymatic alcoholysis of rac-2a–d
Into a solution of rac-2a–d (5 mg) in each solvent (500
ll), the
alcohol (2.4 l) and the lipase (12.5 mg) were added. The hydroly-
l
sis was monitored by TLC and HPLC and was stopped at approxi-
mately 50%. For HPLC analysis, samples taken from the reaction
m
= 2975, 1737, 1489, 1286, 1243, 1172, 1089, 1026, 774, 728,
mixture (10
90:10 (v/v) and filtered before injection.
ll) were diluted to 500 ll with hexane/2-propanol
684; MS: M+ found (M+ calculated for C18H21NO4S): 347.1182
(347.1191); MS m/z (%): 348(M+1, 3), 347(M, 5), 277(19),
276(100), 231(13), 230(87), 214(18), 190(17), 188(71), 71(15),
58(11), 43(25), 29(16).
4.4.3. Determination of the reusability of CaL-B
For the O-acylation, CaL-B (12.5 mg) and molecular sieves (2
pieces) were added into a solution of the substrate rac-1a–d
4.3.2.2. rac-2-(Ethoxycarbonyl)-1-(2-(4-chlorophenyl)thiazol-4-
yl)ethyl butanoate rac-2b.
(0.018 M) in 500 ll of toluene, followed by the addition of vinyl
butanoate (0.075 M). The reaction mixture was then shaken at
Yield: 90%; light yellow oil; 1H
NMR (300 MHz, CDCl3): d = 0.92 (t, J = 7.5 Hz, 3H), 1.22
(t, J = 7.5 Hz, 3H), 1.60 (overlapped tq,, J1 ꢄ J2 ꢄ 7.8 Hz, 2H), 2.31
(t, J = 7.6 Hz, 2H), 3.11 (d, J = 9 Hz, 2H), 4.13 (q, J = 6 Hz, 2H), 6.38
(t, J = 6 Hz, 1H), 7.24 (s, 1H), 7.36 (d, J = 8.6 Hz, 2H), 7.8 (d,
J = 9 Hz, 2H); 13C NMR (75 MHz, CDCl3): d = 13.6, 14.2, 18.4, 36.2,
39.3, 60.7, 68.1, 115.9, 126.5, 129.5, 130.8, 140.4, 154.5, 168.5,
1350 rpm at room temperature. For HPLC analysis, samples taken
from the reaction mixture (10 ll) were diluted to 500 ll with hex-
ane/2-propanol 90:10 (v/v) and injected. When the reaction
reached 50% conversion, the enzyme was filtered and thoroughly
washed with the solvent and was ready to be reused. This proce-
dure was repeated by using the same CaL-B in order to determine
its recyclability. By quantifying the transformed substrate, we were
able to determine the enzyme’s activity.
For the alcoholysis, into a solution of rac-2a–d (0.015 M) in each
solvent (500 ll), the alcohol (0.075 M) and the lipase (12.5 mg)
were added. The hydrolysis was monitored by TLC and HPLC and
was stopped at approximately 50% conversion. The enzyme was fil-
tered and washed several times with solvent, then reused. For
170.0, 172.6; IR:
m = 2975, 1737, 1518, 1462, 1372, 1286, 1243,
1173, 1027, 825; MS: M+ found (M+ calculated for C18H20ClNO4S):
381.0793 (381.0802); MS m/z: 383(M, 37Cl, 2); 381(M, 35Cl, 5);
311(37Cl, 32); 309(35Cl, 100); 265(37Cl, 24); 263(35Cl, 74);
249(37Cl, 8); 247(35Cl, 25); 223(37Cl, 22); 221(35Cl, 69); 71(29);
58(16); 43(59); 29(31).
4.3.2.3.
butanoate rac-2c.
rac-2-(Ethoxycarbonyl)-1-(2-p-tolylthiazol-4-yl)ethyl
Yield: 92%; light yellow oil; 1H NMR
HPLC analysis, samples taken from the reaction mixture (10
ll)
were diluted to 500 l with hexane/2-propanol 90:10 (v/v) and
l
(300 MHz, CDCl3): d = 0.92 (t, J = 7.5 Hz, 3H), 1.22 (t, J = 7.5 Hz,
3H), 1.60 (overlapped tq,, J1 ꢄ J2 ꢄ 7.8 Hz, 2H), 2.27–2.39 (m, 5H),
3.11 (d, J = 9 Hz, 2H), 4.13 (q, J = 6 Hz, 2H), 6.38 (t, J = 6 Hz, 1H),
7.21 (d, J = 6 Hz, 3H), 7.8 (d, J = 9 Hz, 2H); 13C NMR (75 MHz,
CDCl3): d = 13.6, 14.2, 18.4, 21.4, 36.2, 39.3, 60.7, 68.1, 115.9,
126.5, 129.5, 130.8, 140.4, 154.5, 168.5, 170.0, 172.6; IR:
filtered before injection. This procedure was repeated, using the
same CaL-B in order to determine its recyclability.
4.5. Preparative scale enzyme mediated biotransformation of
rac-1–2a–d
m
= 2975, 1737, 1518, 1462, 1372, 1286, 1243, 1173, 1027, 825;
The enzyme (750 mg) and molecular sieves (5 pieces) were
added into a solution of substrate rac-1a–d (300 mg, 0.025 M) in
30 ml of toluene, followed by the addition of the vinyl butanoate
(0.1 M). The reaction mixture was shaken at 1350 rpm at room
temperature for 17 h. When the reaction reached 50% conversion,
MS: M+ found (M+ calculated for C19H23NO4S): 361.1379
(361.1348); MS m/z: 362(M+1, 20), 361(M, 82), 290(35),
289(100), 273(79), 243(98), 227(45), 203(25), 201(92), 71(28),
43(69), 29(34).

304
A. Varga et al. / Tetrahedron: Asymmetry 25 (2014) 298–304
the enzyme was filtered, the solvent was evaporated, and the crude
products were separated by column chromatography using a hex-
ane/ethyl acetate 6:4 mixture as the eluent. Next, into a solution of
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filter (pore size 0.2
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Bradford method. The obtained CaL-B-SWCNT (protein content
ꢄ1%) was freeze-dried and stored under argon.
Acknowledgements
This work was supported by the Romanian Grant PN-II-ID-PCE-
2011-3-0346. Support to A.V. from the European Social Fund POS-
DRU number 107/1.5/S/78702 is also acknowledged.