A tandem Aldol condensation/dehydration co-catalyzed by acylase and N-heterocyclic compounds in organic media

Article abstract of DOI:10.1016/j.molcatb.2010.09.014

A tandem Aldol condensation/dehydration of aldehydes and ketones could be performed under d-aminoacylase and N-heterocyclic compounds used as co-catalyst in organic media. Some control experiments have been designed to demonstrate that either acylase or N-heterocyclic compounds could not catalyze the tandem reaction. The acylase showed the highest activity in the presence of imidazole and has been used to catalyze the tandem Aldol condensation/dehydration between different aldehydes and ketones. This method has provided a new strategy to perform the tandem Aldol condensation/dehydration and expanded the application of biocatalysts.

Full text of DOI:10.1016/j.molcatb.2010.09.014

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 71–76
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
A tandem Aldol condensation/dehydration co-catalyzed by acylase and
N-heterocyclic compounds in organic media
Xiang Chen, Bo-Kai Liu, Hong Kang, Xian-Fu Lin∗
Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 June 2010
Received in revised form
23 September 2010
Accepted 27 September 2010
Available online 1 November 2010
A tandem Aldol condensation/dehydration of aldehydes and ketones could be performed under d-
aminoacylase and N-heterocyclic compounds used as co-catalyst in organic media. Some control
experiments have been designed to demonstrate that either acylase or N-heterocyclic compounds could
not catalyze the tandem reaction. The acylase showed the highest activity in the presence of imi-
dazole and has been used to catalyze the tandem Aldol condensation/dehydration between different
aldehydes and ketones. This method has provided a new strategy to perform the tandem Aldol conden-
sation/dehydration and expanded the application of biocatalysts.
Keywords:
© 2010 Elsevier B.V. All rights reserved.
d-Aminoacylase
Claisen–Schmidt condensation
Co-catalyze
Imidazole
1. Introduction
group of Itoh used ionic liquid to enhance enantioselectivity of
lipase [14].
Biocatalytic promiscuity focuses on the enzyme catalytic
activities with unnatural substrates and alternative chemical
transformations [1a–f]. To the best of our knowledge, several
hydrolase-catalyzed basic reactions, such as Aldol condensa-
tion [2a–c], Mannich reaction [3], Michael additions [4a–c],
Markovnikov additions [5a–c] and Henry reactions [6a–b], have
been reported. Furthermore, a new area was established that
using the single-enzyme catalyzed tandem reactions base on the
multifunction of enzyme. Lipase could catalyze domino kinetic
resolution/intramolecular Diels–Alder reactions [7a–b]. Klaas et
al. have reported a combined multistep process of deprotection,
acetylation and epoxidation catalyzed by CAL-B [8]. Our group has
reported the two-step enzymatic synthesis of N-substituted imida-
zole derivatives containing glucose mediated by protease [9] and
one-pot synthesis of pyrimidine-saccharide complexes catalyzed
by d-aminoacylase [10]. Several methods have been reported to
improve the performance of enzymes in organic solvent. Addi-
tives are quite simple and nearly universally scalable technique.
For example, adding N-methylimidazole caused a significant acti-
vation of the lipase acrylic resin from Candida antarctica (CAL B)
in acylation [11]. Methyl-␤-cyclodextrin improved the activity and
enantioselectivity of subtilisin [12]. Sodium dodecyl sulfate showed
influence on lipase MY catalyzed the hydrolysis of ester [13]. The
Claisen–Schmidt condensation was thought to be a classical
method to prepare ␣, ␤-unsaturated ketones from aromatic alde-
hydes and aliphatic ketones [15]. The products of Claisen–Schmidt
were useful intermediates for further transformations, such
as Diels–Alder reactions, Stetter reactions, Michael additions,
Baylis–Hillman reactions, Juliaˇı–Colonna epoxidations and Robin-
son annulations. The traditional methods employed a relatively
strong base such as metal hydroxide or metal alkoxide, but these
methods suffered from several side reactions and the narrow
substrate diversity [16]. The heterogeneous catalysts have also
been used for the Claisen–Schmidt condensation, including l-
proline – TEA [17], H3PW12O40/SiO2 [18], ionic liquids [19–20],
Zr(HSO4)4/SiO2 [21], micrometer-sized nanostructured magne-
sium oxide [22]. However these methods always used toxic
catalysts and an excess of solvent.
In this paper, a tandem Aldol condensation/dehydration of
aldehydes and ketones catalyzed by d-aminoacylase and N-
heterocyclic compounds in organic media has been discovered. A
novel and effective approach to achieve tandem Aldol condensa-
tion/dehydration was established (Scheme 1). After optimization of
the stepwise process, a number of ␣, ␤-unsaturated ketones were
successfully synthesized.
2. Experimental
∗
1H spectra were recorded on a Bruker AVANCE DMX-400 spec-
trometer at 400 MHz, respectively. Chemical shifts are reported
Corresponding author. Tel.: +86 571 87951588; fax: +86 571 87952618.
E-mail address: llc123@zju.edu.cn (X.-F. Lin).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.09.014

72
X. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 71–76
O
O
O
D-aminoacylase
H
+ R1
+
H2O
+
R
R1
imidazole
O
R=H,P-NO2,M-NO2,O-NO2
Scheme 1. d-aminoacylase and imidazole co-catalyzed tandem Aldol condensation/dehydration between ketones and aldehydes.
in ppm (ı), relative to the internal standard of tetramethylsilane
(TMS). IR spectra were measured with a Nicolet Nexus FTIR 670
spectrophotometer. HPLC was carried out using a Agilent 1100
series column (methanol/water = 32/68, 1.0 ml/min and 274 nm).
d-aminoacylase from Escherichia coli (DA), acylase “Amano” from
Aspergillus oryzae (AA) and lipase from acrylic resin from Candida
antarctica (CAL B) were purchased from Amano Enzyme Inc. (Japan).
Bovine serum albumin (BSA) was obtained from Wuxi Enzyme
Co. Ltd., Wuxi, PR China. Lipase from Candida cylindracea (CCL)
and lipase from Mucor jiavanicus (MJL) and lipozyme immobilized
from Mucor miehei (MML) were purchased from Fluka. All chemi-
cals were obtained from commercial suppliers. For all reactions dry
(molecular sieve), analytical grade solvents were used. Solvents for
column chromatography were not distilled before use.
1H), 6.69 (d, J = 16.0 Hz, 1H), 2.37 (s, 3H); IR (neat): 1671, 1613,
1359, 983.
2.1.7. (3E)-4-(2-chlorophenyl)-3-buten-2-one [23]
1H NMR (400 MHz, CDCl3) ı 7.49 (t, J = 8.8, 4.4 Hz, 1H), 7.48 (t,
J = 8.8, 4.4 Hz, 1H), 7.46 (d, J = 16.4 Hz, 1H), 6.90 (d, J = 8.0 Hz, 1H),
6.90 (d, J = 8.0 Hz, 1H), 6.59 (d, J = 16.4 Hz, 1H), 2.35 (s, 3H); IR (neat):
1673, 1609, 1359, 975.
2.1.8. (3E)-4-(4-hydroxylphenyl)-3-buten-2-one [28]
1H NMR (400 MHz, CDCl3) ı 7.96 (s, 1H), 7.53 (d, J = 16.0 Hz, 1H),
7.44 (d, J = 8.4 Hz, 2H), 6.93 (d, J = 8.8 Hz, 2H) 6.61 (d, J = 16.4 Hz, 1H),
2.40 (s, 3H); IR (neat): 3150, 1666, 1629, 1364, 971.
2.1.9. (3E)-4-(3-hydroxylphenyl)-3-buten-2-one [27]
1H NMR (400 MHz, CDCl3) ı7.65 (s, 1H), 7.47 (d, J = 16.0 Hz, 1H),
7.24 (t, J = 8.0, 15.6, 7.6 Hz, 1H), 7.05-7.08 (m, 2H), 6.95 (d, J = 8.0 Hz,
1H), 6.67 (d, J = 16.0 Hz, 1H), 2.39 (s, 3H); IR (neat): 3170, 1645,
1615, 1356, 997.
2.1. General procedure for the Claisen–Schmidt reaction of
4-nitrobenzaldehyde and acetone
A
suspension of 4-nitrobenzaldehyde (100 mg), imidazole
(50 mg), acetone (1.5 ml) and d-aminoacylase 100 mg in octane
(10 ml) was incubated at 50 ◦C and 200 r.p.m. (orbitally shaken) for
48 h. Then, solvent was evaporated under vacuum to dryness. The
crude residue was purified by flash column chromatography on sil-
ica gel using petroleum/ethylacetate mixtures. Product-containing
fractions were combined, concentrated, and dried to give 1. All the
compounds were spectroscopically characterized (IR, 1H NMR).
2.1.10. (3E)-4-(2-hydroxyphenyl)-3-buten-2-one [28]
1H NMR (400 MHz, CDCl3) ı 7.89 (d, J = 16.8 Hz, 1H), 7.72 (s, 1H),
7.48 (d, J = 7.6 Hz, 1H), 7.26 (t, J = 8.8, 4.4 Hz, 1H), 7.02 (d, J = 16.0 Hz,
1H), 6.94 (t, J = 7.6, 14.8, 7.2 Hz, 2H), 2.43 (s, 3H); IR (neat): 3358,
1673, 1619, 1356, 972.
2.1.11. (3E)-4-(4-methoxyphenyl)-3-buten-2-one [24]
2.1.1. (3E)-4-(4-nitrophenyl)-3-buten-2-one [23]
1H NMR (400 MHz, CDCl3) ı 7.48 (d, J = 8.4 Hz, 2H), 7.46 (d,
J = 16.4 Hz, 1H), 6.90 (d, J = 8.0 Hz, 2H), 6.59 (d, J = 16.4 Hz, 1H), 3.83
(s, 3H), 2.35 (s, 3H); IR (neat): 1663, 1624, 1356, 974.
1H NMR (400 MHz, CDCl3) ı 8.24 (d, J = 8.8 Hz, 2H), 7.68 (d,
J = 8.8 Hz, 2H), 7.53 (d, J = 16.0 Hz, 1H), 6.81 (d, J = 16.4 Hz, 1H), 2.41
(s, 3H); IR (neat): 1677, 1612, 1525, 1346, 974.
2.1.12. (3E)-4-(4-tolyl)-3-buten-2-one [25]
2.1.2. (3E)-4-(3-nitrophenyl)-3-buten-2-one [23]
1H NMR (400 MHz, CDCl3) ı 7.50 (d, J = 16.4 Hz, 1H), 7.45 (d,
J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H), 6.68 (d, J = 16.4 Hz, 1H), 2.39,
(s, 3H), 2.38 (s, 3H); IR (neat): 1679, 1612, 1317, 970.
1H NMR (400 MHz, CDCl3) ı 7.97 (d, J = 8.0 Hz, 1H), 7.84 (s, 1H),
7.34 (t, J = 16.0, 8.0 Hz, 1H), 7.26 (d, J = 8.0 Hz, 1H), 4.38 (d, J = 6.0 Hz,
1H), 4.02 (d, J = 6.4 Hz, 1H), 2.43 (s, 3H); IR (neat): 1660, 1615, 1354.
2.1.13. E-2-(4-nitrobenzylidene)cyclohexanone [26]
2.1.3. (3E)-4-(2-nitrophenyl)-3-buten-2-one [24]
1H NMR (400 MHz, CDCl3) ı 8.18 (d, J = 7.6 Hz, 2H), 7.49 (d,
J = 7.6 Hz, 2H), 5.47 (s, 1H), 2.62 (t, J = 8.8, 16.8, 8.0 Hz, 2H), 2.57 (t,
2H), 2.09 (m, 2H), 1.83 (m, 2H); IR (neat): 1658, 1633, 1359, 973.
1H NMR (400 MHz, CDCl3) ı 7.96 (d, J = 8.0 Hz, 1H), 7.90 (d,
J = 8.0 Hz, 1H), 7.67 (t, J = 16.0, 8.0 Hz, 1H), 7.44 (t, J = 16.0, 8.0 Hz,
1H), 5.68 (d, J = 9.6 Hz, 1H), 3.12 (d, J = 9.6 Hz, 1H), 2.24 (s, 3H); IR
(neat): 1677, 1612, 1525, 1346.
2.1.14. 3-Methyl-5-(4-nitrophenyl)cyclohex-2-enone [29]
1H NMR (400 MHz, CDCl3) ␦ 8.23 (d, J = 8.8 Hz, 2H), 7.43 (d,
J = 8.4 Hz, 2H), 6.03 (s, 1H), 3.47 (m, 1H), 2.71-2.56 (m, 4H), 2.05
(s, 3H); IR (neat): 1661, 1606, 1596, 1521, 1346, 853; GC–MS:
m/z = 231.
2.1.4. (3E)-4-phenyl-3-buten-2-one [24]
1H NMR (400 MHz, CDCl3) ı 7.55 (d, J = 16.0 Hz, 2H), 7.51 (d,
J = 16.0 Hz, 1H), 7.39 (t, J = 6.4, 3.2 Hz, 3H), 6.71 (d, J = 16.0 Hz, 1H),
2.38 (s, 3H); IR (neat): 1659, 1616, 1354, 761, 694.
3. Results and discussion
2.1.5. (3E)-4-(4-chlorophenyl)-3-buten-2-one [23]
1H NMR (400 MHz, CDCl3) ı 7.45 (d, J = 8.0 Hz, 2H), 7.44 (d,
J = 16.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 2H), 6.67 (d, J = 16.0 Hz, 1H), 2.36
(s, 3H); IR (neat): 1652, 1626, 1341, 826.
When 4-nitrobenzaldehyde and acetone was catalyzed by d-
aminoacylase and imidazole in hexane at 50 ◦C, two products were
observed. The structures of these two products were approved by
IR and 1H NMR. Based on the observation, we envisioned that d-
aminoacylase and imidazole could serve as co-catalyst for direct
preparation of the synthetically useful ␣, ␤-unsaturated carbonyl
compounds from aldehydes and ketones.
2.1.6. (3E)-4-(3-chlorophenyl)-3-buten-2-one [25]
1H NMR (400 MHz, CDCl3) ı 7.51 (s, 1H), 7.42 (d, J = 16.0 Hz, 1H),
7.39 (d, J = 8.0 Hz, 1H), 7.34 (t, J = 16.0, 8.0 Hz, 1H), 7.32 (d, J = 8.0 Hz,

X. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 71–76
73
Table 1
Table 3
Direct tandem Aldol condensation/dehydration of 4-nitrobenzaldehyde and acetone
Screening of N-heterocyclic for the tandem Aldol condensation/dehydration of ace-
catalyzed by different enzymes in hexanea.
tone and p-nitrobenzaldehydea.
Entry
Enzyme
Yieldb (%)
E/Z
Entry
N-heterocyclic
Yieldb (%)
E/Z
1/16
c
1
2
3
4
5
6
7
8
9
Blank
Blankd
DA
–
–
1
2
3
4
5
7
None
Imidazole
N-methylimidazole
Benzoimidazole
Pyridine
6.4
67.8
29.9
20.7
3.7
2.2
33.5
6.4
6.0
3.3
–
2.9
1.6
1.8
–
12/1
15/1
16/1
1/5
9/1
–
1/10
1/13
5/1
–
38/1
20/1
10/1
7/1
DAe
DA (denature)f
AA
2-Methyl-5-nitroimidazole
0.6
1/3
BSA
CCL
MJL
MML
CAL B
a
Reaction conditions: d-aminoacylase (10 mg), 4-nitrobenzaldehyde (10 mg),
acetone (0.15 ml), N-heterocyclic (5 mg), octane (1 ml), 50 ◦C, reaction time (48 h).
b
Determined by HPLC.
10
11
a
Reaction conditions: Enzyme (10 mg), 4-nitrobenzaldehyde (10 mg), acetone
(0.15 ml), imidazole (5 mg), hexane (1 ml), 50 ◦C, reaction time (48 h).
Table 3). Considering the high selectivity of the elimination, octane
was chosen as the medium for further investigation.
b
Determined by HPLC.
Not detected.
Without d-aminoacylase.
c
The effect of varieties N-heterocyclic compounds was also eval-
uated (Table 3). The product could be furnished with yields of
20.7% and 29.9%, when N-methylimidazole and benzoimidazole
was added. Other N-heterocyclic compounds, such as pyridine and
2-methyl-5-nitroimidazole, did not exhibit promising result. How-
ever, imidazole showed a unique property and could enhance the
activity of d-aminoacylase for this tandem reaction obviously. The
yield could be up to 67.8%.
d
e
Without imidazole.
f
Pretreated with urea at 100 ◦C for 8 h.
Some control experiments were designed to demonstrate
that the tandem Aldol condensation/dehydration between 4-
nitrobenzaldehyde and acetone was catalyzed by d-aminoacylase
and imidazole. As seen from Table 1, the tandem Aldol conden-
sation/dehydration between 4-nitrobenzaldehyde and acetone did
not take place without d-aminoacylase and imidazole (entry 1,
Table 1). Imidazole could not catalyze tandem reaction efficiently
(entry 2, Table 1). The reaction of 4-nitrobenzaldehyde and ace-
tone with d-aminoacylase, but in the absence of imidazole, led to
very low yield (entry 4, Table 1) after 48 h. When the reactants
were incubated with denatured d-aminoacylase and bovine serum
albumin (BSA), the yield were lower than 7% (entries 5 and 7,
Table 1), ruling out the possibility that the amino acid distribu-
tion on the protein surface promoted the process. Several widely
used hydrolases, such as AA, CCL, MJL, MML and CAL B, could not
accelerate the reaction efficiently in hexane (entries 6 and 8–11,
Table 1). However, the reaction yield was 33.5% under the cataly-
sis of d-aminoacylase and imidazole (entry 3, Table 1). All these
results suggest that the specific catalytic site of d-aminoacylase
and imidazole were responsible for promoting the Claisen–Schmidt
condensation.
The yields with different concentrations of imidazole are shown
in Fig. 1. The amount of imidazole exhibited an obviously influ-
ence on the yield and selectivity of the reaction. In the absence
of imidazole, the reaction did not take place. However, the reac-
tion yield decreased as the dosage of imidazole increasing from 5
to 50 mg ml−1. A similar phenomenon was observed for the reac-
tion selectivity. The best concentration of imidazole was 5 mg ml−1
.
It can be concluded that the imidazole showed dual role in this
enzymatic reaction. One side, imidazole could catalyze the tandem
reaction with d-aminoacylase at the appropriate amount. On the
other side, when excessive imidazole was added, the deactivation
of DA occurred, which has been proved by a further investiga-
tion. As seen from Fig. 2, the reaction was improved as increasing
the d-aminoacylase with a fixed concentration of the imidazole at
the beginning. Then, the plateau was reached after d-aminoacylase
exceeding 10 mg ml−1. It proved that d-aminoacylase could not cat-
alyze this reaction without the assistant of imidazole. Therefore, the
optimum quality ratio of d-aminoacylase and imidazole was 2/1.
Some conventional organic solvents with different log P val-
ues were surveyed for this tandem reaction and the results are
shown in Table 2. In these solvents, such as DMSO, THF, chloro-
form and toluene, the yields of this tandem reaction were less than
10% (entries 1–4, Table 3). In cyclohexane and hexane, the yields
were improved to 32.4% and 33.5% (entries 5 and 6, Table 3). Bet-
ter results were obtained in isooctane or octane (entries 7 and 8,
50
40
30
20
10
0
100
80
60
40
20
0
Yield (%)
E/Z
Table 2
The tandem Aldol condensation/dehydration between 4-nitrobenzaldehyde and
acetone under different solventsa.
Entry
Solvent
Log p
Yieldb (%)
E/Z
c
1
2
3
4
5
6
7
8
DMSO
THF
−1.3
0.56
1.95
2.61
3.35
3.9
–
–
12/1
–
3.3
–
–
32.4
33.5
67.9
67.8
Chloroform
Toluene
Cyclohexane
Hexane
Isooctane
Octane
–
12/1
15/1
16/1
38/1
0
5
10
20
50
imidazole
4.5
4.9
Fig. 1. Effect of different imidazole concentrations on the reaction of tandem Aldol
condensation/dehydration between 4-nitrobenzaldehyde and acetone in octane.
Reaction conditions: d-aminoacylase (10 mg), 4-nitrobenzaldehyde (10 mg), ace-
tone (0.15 ml), octane (1 ml), 50 ◦C, reaction time (48 h). Yields were determined by
HPLC.
a
Reaction conditions: d-aminoacylase (10 mg), 4-nitrobenzaldehyde (10 mg),
acetone (0.15 ml), imidazole (5 mg), solvent (1 ml), 50 ◦C, reaction time(48 h).
b
Determined by HPLC.
Not detected.
c

74
X. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 71–76
40
38
36
34
32
30
28
26
24
22
20
18
16
14
100
80
60
40
20
0
Yield (%)
E/Z
70
Yield (%)
45
E/Z
60
50
30
40
30
20
15
10
0
0
0
5
10
20
50
0
10
20
30
40
50
D-Aminoacylase
4-nitrobenzaldehyde
Fig. 2. Effect of different enzyme concentrations on the reaction of tandem Aldol
condensation/dehydration between 4-nitrobenzaldehyde and acetone in octane.
Reaction conditions: 4-nitrobenzaldehyde (10 mg), acetone (0.15 ml), imidazole
(5 mg), octane (1 ml), 50 ◦C, reaction time (48 h). Yields were determined by HPLC.
Fig. 4. Influence of different substrates concentration on the reaction of
tandem Aldol condensation/dehydration between 4-nitrobenzaldehyde and ace-
tone in octane. Reaction conditions: d-aminoacylase (10 mg), imidazole (5 mg),
n(4-nitrobenzaldehyde)/n(acetone) = 1/30, octane (1 ml), 50 ◦C, reaction time (48 h). Yields
were determined by HPLC.
The influence of catalyst loading on the reaction efficiency
was investigated. The quality concentration of d-aminoacylase
and imidazole in the reaction system were increased in the same
proportion. As shown in Fig. 3, with the concentration of d-
aminoacylase changed from 0 to 10 mg ml−1, the yield increased
from 49.7% to 67.8%, but it decreased remarkably with the con-
curves are shown in Fig. 5. The yield increased as the reaction
time prolonged. 88.9% of 4-(4-nitrophenyl)-3-buten-2-one was
obtained after 72 h with the ratio of E to Z being 1/154.
Having established optimal reaction conditions, we probed the
generality of the process. The tandem reaction between differ-
ent aldehydes and ketones in octane at 50◦C in the presence of
d-aminoacylase and imidazole was conducted. Results are sum-
marized in Table 4. Examination of electron-withdrawing groups
was revealed that a pronounced steric effect is evident in the ortho
position (entries 3 and 7, Table 4) than other positions of aro-
matic aldehydes (entries 1–2 and 5–6, Table 4). The electronic
effect showed slight influence on the reactions (entries1, 4, 5, 11
and 12, Table 4). Compared with benzaldehyde, electron-donating
group could enhance the reactivity of substrates (entries 4, 11 and
12, Table 4). However, electron-withdrawing group showed neg-
ative effect on the reaction (entry 1, Table 4). Slight decrease in
yields was observed. Chlorine had two kinds of electronic effects.
Thus, an excellent result was obtained (entry 5, Table 4). When
the aldehydes, containing hydroxyl group were used as substrates,
the tandem reactions hardly took place except the hydroxyl group
on the ortho position (entries 8–10 and 13–14, Table 4). It may
centration of d-aminoacylase increased from 20 to 50 mg ml−1
.
We found that a by-product was formed with the concentra-
tion of d-aminoacylase at 50 mg ml−1 by TLC. The by-product was
3-methyl-5-(4-nitrophenyl)-cyclohex-2-enone by analyzed IR, 1H
NMR and GC–MS. From Figs. 1–3, it demonstrated that imidazole
and d-aminoacylase can catalyze carbon–carbon double bond for-
mation.
As shown in Fig. 4, when the concentration of p-
nitrobenzaldehyde was changed from
5 , the
to 10 mg ml−1
yield increased from 21.1% to 67.8% and E/Z increased from 18 to
38. Decreased in yields and E/Z were observed as the concentration
continually varied from 20 to 50 mg ml−1
.
The time course of tandem Aldol condensation/dehydration
between 4-nitrobenzaldehyde and acetone catalyzed by d-
aminoacylase and imidazole was tested in octane. The progress
120
100
80
60
40
20
0
100
80
60
40
20
0
100
Yield (%)
E/Z
160
140
80
120
60
100
80
40
60
40
20
20
Yield (%)
E/Z
0
0
-20
0
5
10
20
50
-10
0
10
20
30
40
50
60
70
80
D-aminoacylase
Reaction time (h)
Fig. 3. Co-effect of different enzyme and imidazole concentrations on the reac-
tion of tandem Aldol condensation/dehydration between 4-nitrobenzaldehyde
and acetone in octane. Reaction conditions: 4-nitrobenzaldehyde (10 mg), acetone
(0.15 ml), m(D-aminoacylase)/m(Imidazole) = 2/1, octane (1 ml), 50 ◦C, reaction time (48 h).
Yields were determined by HPLC.
Fig. 5. The progress curve of different time that d-aminoacylase catalyzed 4-
nitrobenzaldehyde and acetone in octane. Reaction conditions: d-aminoacylase
(10 mg), 4-nitrobenzaldehyde (10 mg), acetone (0.15 ml), imidazole (5 mg), octane
(1 ml), 50 ◦C. Yields were determined by HPLC.

X. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 71–76
75
Table 4
be due to the hydrogen bond between the hydroxyl of aldehydes
and the nitrogen of imidazole, which leading to affect the func-
tion of imidazole. For the 2-hydroxylbenzaldehyde, the hydrogen
of hydroxyl can form intramolecular hydrogen bond with carbonyl,
which reduce the hydrogen bond between aldehyde and imidazole.
So the 2-hydroxylbenzaldehyde can react with acetone and got the
yield in 20.4% (entry 10, Table 4). The tandem reaction between p-
nitrobenzaldehyde and cyclohexanone was also investigated and
the high yield was observed (entry 15, Table 4).
Direct tandem Aldol condensation/dehydration between other aromatic aldehydes
and ketonesa.
Entry
1
Aldehydes
Acetones
Yieldb (%)
67.8
E/Z
O
O
38/1
O2N
O
O
2
88.8
224/1
4. Conclusions
NO2
A
facile method to perform tandem Aldol condensa-
O
O
O
O
3
4
5
44.4
74.3
90
1/1
245/1
E
tion/dehydration between aldehydes and ketones has been
developed by d-aminoacylase and N-heterocyclic compounds as
co-catalyst in octane. The relationship of d-aminoacylase and
imidazole was demonstrated by the combination of different
control experiments. To make summarize, the quality ratio of
d-aminoacylase and imidazole should be 2/1 and the tandem
reaction between aldehydes and ketones was carried out in octane
at 50 ◦C. After the optimization of the stepwise process, series
of ␣, ␤-unsaturated ketones were prepared efficiently using d-
aminoacylase and imidazole via tandem condensation/dehydration
reaction.
NO2
O
O
Cl
O
O
O
6
99.6
57/1
Cl
Acknowledgements
O
O
7
8
39
282/1
90/1
This investigation has enjoyed financial support from the
National Science Foundation of China (no. 20572099) and The
Zhejiang Provincial Natural Science Foundation (Project no. 2008-
Y407086)
Cl
O
8.5
HO
Appendix A. Supplementary data
O
O
O
O
9
0.6
E
E
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2010.09.014.
OH
References
10
20.4
OH
[1] (a) P.J. O’Brian, D. Herschlag, Chem. Biol. 6 (1999) 91–105;
(b) U.T. Bornscheuer, R.J. Kazlauskas, Angew. Chem. Int. Ed. 43 (2004)
6032–6040;
O
O
O
11
12
89.8
87.1
19/1
12/1
H3CO
(c) R.J. Kazlauskas, Curr. Opin. Chem. Biol. 9 (2005) 195–201;
(d) P. Berglund, S. Park, Curr. Org. Chem. 9 (2005) 325–336;
(e) K. Hult, P. Berglund, Trends Biotechnol. 25 (2007) 231–238;
(f) P.J. O’Brian, Chem. Rev. 106 (2006) 720–752.
O
[2] (a) C. Li, X.W. Feng, N. Wang, Y.J. Zhou, X.Q. Yu, Green Chem. 10 (2008) 616–618;
(b) C. Branneby, P. Carlqvist, A. Magnusson, K. Hult, T. Brinck, P. Berglund, J. Am.
Chem. Soc. 125 (2003) 874–875;
O
(c) X.W. Feng, C. Li, N. Wang, K. Li, W.W. Zhang, Z. Wang, X.Q. Yu, Green Chem.
11 (2009) 1933–1936.
[3] K. Li, T. He, C. Li, X.W. Feng, N. Wang, X.Q. Yu, Green Chem. 6 (2009) 777–779.
[4] (a) O. Torre, I. Alfonso, V. Gotor, Chem. Commun. 15 (2004) 1724–1725;
(b) M. Svedendahl, K. Hult, P. Berglund, J. Am. Chem. Soc. 127 (2005)
17988–17989;
(c) J.M. Xu, F. Zhang, B.K. Liu, Q. Wu, X.F. Lin, Chem. Commun. 27 (2007)
2078–2080.
[5] (a) For recent examples of the Markovnikov addition, see: W.B. Wu, N. Wang,
J.M. Xu, Q. Wu, X.F. Lin, Chem. Commun. 18 (2005) 2348–2350;
(b) F.W. Lou, B.K. Liu, Q. Wu, D.S. Lv, X.F. Lin, Adv. Synth. Catal. 350 (2008)
1959–1962;
O
Cl
c
H
13
14
–
–
OH
O
O
O
H
–
–
H3CO
O2N
OH
(c) W.B. Wu, J.M. Xu, Q. Wu, D.S. Lv, X.F. Lin, Adv. Synth. Catal. 348 (2006)
487–492.
O
[6] (a) R.C. Tang, Z. Guan, Y.H. He, W. Zhu, J. Mol. Catal. B: Enzym. 63 (2010) 62–67;
(b) J.L. Wang, X. Li, H.Y. Xie, B.K. Liu, X.F. Lin, J. Biotechnol. 145 (2010) 240–243.
[7] (a) S. Akai, K. Tanimoto, Y. Kita, Angew Chem. Int. Ed. 43 (2004) 1407–1410;
(b) S. Akai, T. Naka, S. Omura, K. Tanimoto, M. Imanishi, Y. Takebe, M. Matsugi,
Y. Kita, Chem. Eur. J. 8 (2002) 4255–4264.
[8] M.R.G. Klaas, M. Kunz, S. Warwel, J. Mol. Catal. B: Enzym. 7 (1999) 283–289.
[9] S.P. Yao, D.S. Lv, Q. Wu, Y. Cai, S.H. Xu, X.F. Lin, Chem. Commun. 17 (2004)
2006–2007.
15
70.8
E
a
Reaction conditions: d-aminoacylase (10 mg), aldehydes (10 mg), ketones
(0.15 ml), imidazole (5 mg), octane (1 ml), 50 ◦C, reaction time (48 h).
b
Determined by HPLC.
Not detected.
c
[10] Q. Wu, J.M. Xu, X. Li, J.L. Wang, X.F. Lin, Adv. Synth. Catal. 351 (2009) 1833–1841.
[11] B.K. Liu, Q. Wu, J.M. Xu, X.F. Lin, Chem. Commun. 3 (2007) 295–297.

76
X. Chen et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 71–76
[12] K. Griebenow, Y.D. Laureano, A.M. Santos, I.M. Clemente, L. Rodríguez, M.W.
Vidal, G. Barletta, J. Am. Chem. Soc. 121 (1999) 8157–8163.
[13] S. Mori, H. Yumoto, R. Matsumi, T. Nishigaki, Y. Ebara, S. Ueji, Tetrahedron:
Asymmetr. 16 (2005) 3698–3702.
[21] B.B.F. Mirjalili, A. Bamoniri, M. Alipour, M.A.K. Zarchi, J. Chem. Sci. 63 (2008)
1421–1424.
[22] S.W. Bian, Z. Ma, Z.M. Cui, L.S. Zhang, F. Niu, W.G. Song, J. Phys. Chem. C 112
(2008) 15602–115602.
[14] T. Itoh, S.H. Han, Y. Matsushita, S. Hayase, Green Chem. 6 (2004) 437–439.
[15] A. Yanagisawa, T. Arai, Org. Lett. 6 (2004) 4281–4283.
[16] (a) For recent examples of the Claisen–Schmidt condensation K. Mogilaiah, N.
Vasudeva Reddy, Synth. Commun 33 (2003) 73–78;
(b) M. Hatsuda, T. Kuroda, M. Seki, Synth. Commun. 33 (2003) 427–434;
(c) U. Sensfuss, Tetrahedron Lett. 44 (2003) 2371–2374.
[17] J.F. Wang, M. Lei, Q. Li, Z.M. Ge, X. Wang, R.T. Li, Tetrahedron 65 (2009)
4826–4833.
[18] B.F. Mirjalili, Z. Zaghaghi, J. Chin. Chem. Soc. 55 (2008) 694–697.
[19] Y.Y. Wang, X.X. Gong, L.Y. Dai, Chin. J. Org. Chem. 29 (2009) 1470–
1473.
[20] S.D. Yang, L.Y. Wu, Z.Y. Yan, Z.L. Pan, Y.M. Liang, J. Mol. Catal. A: Chem. 268
(2007) 107–111.
[23] Y.M. Lin, Z.T. Li, V. Casarotto, J. Ehrmantraut, A.N. Nguyen, Tetrahedron Lett. 48
(2007) 5531–5534.
[24] M. McConville, O. Saidi, J. Blacker, J.L. Xiao, J. Org. Chem. 74 (2009) 2692–2698.
[25] W. Pei, L. Sun, Y.S. Shao, Process for the preparation of 4-aryl-1-benzenesulfonyl
-3-buten-2-one [P], Chinese Patent, CN 1583720, 2005-02-23.
[26] U. Das, A. Doroudi, S. Das, B. Bandy, J. Balzarini, E.D. Clercq, J.R. Dimmock, Bioorg.
Med. Chem. 16 (2008) 6261–6268.
[27] D. Fattori, C. Rossi, C.I. Fincham, M. Berettoni, F. Calvani, F. Catrambone, P.
Felicetti, M. Gensini, R. Terracciano, M. Altamura, A. Bressan, S. Giuliani, C.A.
Maggi, S. Meini, C. Valenti, L. Quartara, J. Med. Chem. 49 (2006) 3602–3613.
[28] X.H. Liu, P.Ch. Lv, B. Li, H.L. Zhu, B.A. Song, Aust. J. Chem. 61 (2008) 223–230.
[29] M. Moura, S. Delacroix, D. Postel, A.N. Van Nhien, Tetrahedron 65 (2009)
2766–2772.