Candida antarctica lipase B-catalyzed the unprecedented three-component Hantzsch-type reaction of aldehyde with acetamide and 1,3-dicarbonyl compounds in non-aqueous solvent

Article abstract of DOI:10.1016/j.tet.2011.01.045

A direct approach to 1,4-dihydropyridines by lipase-catalyzed unprecedented three-component Hantzschtype reaction of aldehyde with 1,3-dicarbonyl compounds and acetamide in non-aqueous solvent has been developed. Some control experiments have been perform

Full text of DOI:10.1016/j.tet.2011.01.045

Tetrahedron 67 (2011) 2689e2692
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Candida antarctica lipase B-catalyzed the unprecedented three-component
Hantzsch-type reaction of aldehyde with acetamide and 1,3-dicarbonyl
compounds in non-aqueous solvent
*
Jun-Liang Wang, Bo-Kai Liu, Cui Yin, Qi Wu, Xian-Fu Lin
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A direct approach to 1,4-dihydropyridines by lipase-catalyzed unprecedented three-component Hantzsch-
type reaction of aldehyde with 1,3-dicarbonyl compounds and acetamide in non-aqueous solvent has been
developed. Some control experiments have been performed to demonstrate the specific catalytic effect of
CAL-B. Acetamide was utilized as a novel ammonia source in the Hantzsch-type reaction for the first time.
An array of 1,4-dihydropyridines was successfully synthesized through this methodology.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 1 September 2010
Received in revised form 13 January 2011
Accepted 18 January 2011
Available online 22 January 2011
Keywords:
CAL-B
Hantzsch-type reaction
Acetamide
1,4-dihydropyridines
Non-aqueous solvent
Enzyme-catalyzed
1. Introduction
activity of biocatalyst to achieve multicomponent reactions becomes
particularly fascinating and remains a great challenge.
Enzyme catalysts are efficient biotransformation tools in both
organic and bioorganic synthesis.1 The seminal work by Klibanov in
the early 1980s has initiated the non-aqueous enzymology.2 Re-
cently a growing number of enzymes have been found to be capable
of catalyzing secondary reactions besides their primary function.3
This kind of catalytic promiscuity largely enriches the application
of biocatalysts in organic synthesis. For example, lipase and acylase
can catalyze the formation of CeC, CeN, CeO, and CeS through
1,4-dihydropyridines (1,4-DHPs), which possess important
pharmacological activity and chemical activity in medicinal and
organic chemistry, were usually obtained from the classic examples
of the MCRs Hantzsch reaction.8 In the development of Hantzsch
reaction, 1,4-dihydropyridines were commonly prepared by using
ammonium acetate and liquid ammonia as an ammonia source.
More recently, Bridgwood et al. reported magnesium nitride could
also be used as a new source of ammonia.9 In this paper, we wish to
report that under the catalysis of CAL-B, acetamide was firstly
employed as a novel source of ammonia undergoing with alde-
hydes and 1,3-dicarbonyl compounds to furnish 1,4-DHPs in non-
aqueous solvent.
Michael addition and Markovnikov addition,4 racemase can cata-
5a
lyze PLP-dependent aldol additions,
and arylmalonate decar-
boxylase can catalyze aldol additions.5b In spite of the great efforts
dedicated to this field, the catalytic promiscuity of biocatalysts was
mainly focused on two-component reactions by now.
Multicomponent reactions (MCRs), which can form complex bio-
active molecules from simple starting materials have attracted in-
creasing attention in recent years.6 Efforts to explore the efficient
catalysts have continued to increase in popularity. Amino acid de-
rivativeshaveexhibitedpromisingcatalyticabilityinmulticomponent
reactions. Lately, Wang et al. have reported a novel lipase-catalyzed
direct three-component reactions.7 Exploiting the promiscuous
2. Results and discussion
At the outset, the reaction of 4-nitrobenzaldehyde 1a, acetyla-
cetone 2a, and acetamide 3 was carried out using the mixture of
acetylacetone/methyl tert-butyl ether (MTBE) as the reaction
media. Several hydrolases were chosen as the catalysts and the
results were summarized in Table 1. However, five candidates, such
as alkaline protease from Bacillus subtilis (Subtilis), lipase from hog
pancreas (HPL), acylase ‘Amano’ from Aspergillus oryzae (AA),
immobilized penicillin G acylase from Escherichia coli (PGA) and
* Corresponding author. Fax: þ86 571 87952618; e-mail address: llc123@zju.
edu.cn (X.-F. Lin).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.01.045

2690
J.-L. Wang et al. / Tetrahedron 67 (2011) 2689e2692
Table 1
MTBE was found to be the most appropriate reaction medium
Screening of different catalysts
(entry 4 vs entries 1e3 and 5e6, Table 2). Further screening on the
ratio of the mixed solvent led us to find that the best ratio between
MTBE and acetylacetone was 6:4 (entries 4 vs entries 9 and 10,
Table 2). Next, the influence of reaction temperature on the enzy-
matic three-component reaction was also considered. As de-
creasing the reaction temperature from 50 ꢀC to 25 ꢀC, a noticeable
decrease in the yield of the product was observed (entry 4 vs en-
tries 7 and 8, Table 2). Then, the effect of the molar ratio between
two substrates was also tested (entries 4 and 11e13, Table 2). The
best result was obtained by using 4-nitrobenzaldehyde and acet-
amide in a molar ratio of 1:4. Finally, the enzyme concentration was
optimized. As the data shown in Table 2, the yield of the product
was improved greatly by increasing the enzyme concentration from
50 to 100 mg/ml and reached a plateau after 100 mg/ml (entries
14e16, Table 2).
On the basis of the previously optimized reaction conditions, the
scope of this three-component reaction was evaluated. As seen from
Table 3,1,4-dihydropyridines were obtained as the major product in
all cases. For aryl aldehydes, substitution with electron-withdraw-
ing groups could enhance the reactivity of the substrate, compared
with benzaldehyde (entries 1, 6, 7 and 8, Table 3). Conversely, elec-
tron-donating substituents, such as p-methyl and p-methoxyl sub-
stitutions caused a significant loss in the yield (entries 11e12, Table
3). On the other hand, the position of the nitro group on the aromatic
ring of aryl aldehydes was tolerated with yield ranging from 85 to
90% (entries 1, 4 and 5, Table 3). Employing methyl acetoacetate and
ethyl acetoacetate in place of acetylacetone, the reaction also pro-
ceeded smoothly (entries 2 and 3, Table 3). In addition, satisfactory
results were also observed from heterocyclic aldehydes and ali-
phatic aldehydes (entries 13e15, Table 3).
NO
O
O
O
O
50mg enzyme
H
O
O
NH
0.5ml MTBE,
50 C, 3 day
O N
1a
2a
3
N
H
4a
Entry
Catalyst
Yielda(%)
1
2
3
4
5
6
7
8
9
Subtilis
HPL
<1
<1
5
<1
<1
25
0
AA
DA
PGA
CAL-B
CAL-Bb
BSA
0
0
Blank
a
Experimental conditions: 0.5 M 4-nitrobenzaldehyde 1a, 0.5 ml acetylacetone
2a, 0.5 M acetamide 3, 0.5 ml methyl tert-butyl ether (MTBE), 50 mg enzyme, 50 ꢀC,
3day. All yields were detected by HPLC.
b
Enzyme predenatured with urea at 100 ꢀC for 24 h.
D
-aminoacylase from E. coli (DA) (entries 1e5, Table 1) showed no
or low activity towards this reaction. Candida antarctica lipase B
(CAL-B) seemed slightly superior to other hydrolases, and the cor-
responding product was obtained in 25% yield (entry 6, Table 1).
When the reactants were incubated with denatured CAL-B or the
bovine serum albumin (BSA) (entries 7 and 8, Table 1), no product
was formed, ruling out the possibility that the polymeric support or
the similar amino acid distribution on the protein surface promoted
the process. The control experiments (entries 7e9, Table 1) sug-
gested that the specific active sites and the tertiary structure of
CAL-B were responsible for the three-component reaction.
Table 3
Substrate scopea
O
R1
O
In order to improve the activity of enzyme, reaction conditions
including solvents, temperature, molar ratio, and enzyme concen-
tration were investigated. Examination of the results from different
solvents revealed that solvent played an important role in gov-
erning the activity of CAL-B. Among the solvents tested in Table 2,
O
O
O
O
100mg CAL-B
R2
R2
R1
H
R2
NH2
0.6ml MTBE,
50 oC, 3 day
N
H
1
2
3
4
Entry
R1
R2
Product
Yield(%)b
Table 2
1
2
3
4
5
6
7
8
p-NO2C6H4
p-NO2C6H4
p-NO2C6H4
o-NO2C6H4
m-NO2C6H4
p-ClC6H4
CH3
OCH3
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
90
82
76
85
90
82
86
89
39
44
22e
17e
93
92
86
Optimization for CAL-B-catalyzed three-component reactiona
c
d
OC2H5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Entry
Solvent
T/ꢀC
Catalyst/mg
Yield(%)b
1
2
3
4
5
6
7
8
DMSO
Acetonitrile
THF
50
50
50
50
50
50
37
25
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
50
75
100
125
0
4
4
25
8
0
5
0
p-FC6H4
MTBE
p-CF3C6H4
p-OHC6H4
C6H5
p-CH3C6H4
p-OCH3C6H4
3-Pyridyl
2-Furyl
Acetylacetone
n-Hexane
MTBE
MTBE
MTBE
MTBE
MTBE
MTBE
MTBE
9
10
11
12
13
14
15
9
28c
27d
55c,e
69c,f
60c,g
85c,f
91c,f
92c,f
10
11
12
13
14
15
16
(CH3)2CH
a
All the reactions were performed with aldehyde 1 (0.125 mmol), acetamide
(0.5 mmol), and 100 mg CAL-B in 1 ml mixed solvent (acetylacetone/organic sol-
vent¼0.4:0.6) at 50 ꢀC for 72 h.
MTBE
MTBE
MTBE
b
Yield of isolated products except entries 11 and 12.
Using methyl acetoacetate instead of acetylacetone as the solvent.
Using ethyl acetoacetate instead of acetylacetone as the solvent.
Determined by HPLC analysis.
c
a
d
Unless otherwise noted, the reaction was performed by employing 4-nitro-
benzaldehyde 1a (0.5 mmol), acetamide 3 (0.5 mmol), and CAL-B (50 mg) in 1 ml
mixed solvent (acetylacetone/organic solvent¼1:1 by vol) at 50 ꢀC for 72 h.
e
b
Determined by HPLC analysis.
The volume ratio of MTBE and acetylacetone in 1 ml mixed solvent was 6:4.
The volume ratio of MTBE and acetylacetone in 1 ml mixed solvent was 7:3.
Compound 1a (0.25 mmol) was used.
Compound 1a (0.125 mmol) was used.
Compound 1a (0.0625 mmol) was used.
c
In order to further understand the reaction mechanism, an ad-
ditional control reaction to determine the role of CAL-B was carried
out. We investigated the hydrolysis ability of CAL-B on acetamide in
MTBE. The results showed that acetamide couldn’t be hydrolyzed
d
e
f
g

J.-L. Wang et al. / Tetrahedron 67 (2011) 2689e2692
2691
by CAL-B under the specified conditions and no free ammonia
3. Conclusion
generated in our reaction system. It is well accepted that the cat-
alytic triad Asp-His-Ser in the active site of CAL-B can contribute to
the promiscuous activity.4b,10 Based on the common mechanistic
pathway of the Hantzsch reaction, we proposed a tentative mech-
anism of lipase-catalyzed Hantzsch-type reaction of aldehyde with
acetamide and 1,3-dicarbonyl compounds and showed them in
Scheme 1. CAL-B was hypothesized to play different roles during
the catalytic process. Firstly, the Asp-His dyad and the oxyanion
In conclusion, a novel three-component reaction yielding 1,4-
dihydropyridines in a one-pot procedure catalyzed by CAL-B in
non-aqueous solvent has been developed. This approach allowed
acetamide as an ammonia source for Hantzsch-type reaction. The
catalytic promiscuity of CAL-B was demonstrated by the combi-
nation of different control experiments. The influence of reaction
conditions on the three-component reaction, including solvents,
His
His
Asp
O
His
Asp
O
His
Asp
Asp
O
O
O
O
N
N
O
H
N
N
N
N
H
O
O
H
N
O
N
H
H
H
H
H
OH
O
R2
H
O
O
NH
N
NH
N
R2
R2
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
O
O
O
H
H
O
Asp
H
O
Asp
O
Asp
N
N
N
H
O
N
N
N
Ser
His
His
His
Ser
Ser
H2O
O
O
O
OH
O
O
O
H2N
O
H2N
R2
N
R2
R2
H
5
6
OH
His
H
H
H
H
H
H
H
H
H
His
His
His
Asp
Asp
O
Asp
Asp
O
O
O
O
O
O
N
N
R2
N
N
N
N
N
N
H
H
R2
O
H
O
R2
H
O
R1
H
H
H
R2
H
O
O
O
H
O
O
OH
R1
R
2
R1
R1
O
O
O
O
O
H
H
H
H
H
H
7
H
H
H
H
H
H
N
His
His
His
His
Asp
Asp
O
Asp
O
Asp
O
O
O
O
N
N
N
N
N
N
O
H
N
H
H
O
H
H
H
H
O
R2
NH
H
NH
O
R2
O
R2
-H2O
R2
O
The product
NH2
R1
R1
R1
R1
R
R2
R2
2
R2
NH
OH
O
O
O
O
O
O
O
H
H
H
H
H
H
H
H
H
H
H
H
Scheme 1. Proposed mechanism of lipase-catalyzed Hantzsch-type reaction of aldehyde with acetamide and 1,3-dicarbonyl compounds.
hole in the active site stabilized the substrate acetamide. 1,3-
dicarbonyl compounds reacted with activated acetamide through
a condensation reaction to afford the intermediate 5. Then, the
intermediate 5 was hydrolyzed by the nature activity of CAL-B and
the enamine intermediate 6 was formed. Meanwhile, a CAL-B-
catalyzed Knoevenagel condensation reaction of 1,3-dicarbonyl
temperature, and molar ratio of substrate was systematically in-
vestigated. A series of 1,4-dihydropyridines were obtained in
moderate to excellent yields. Further study on the synthetic ap-
plications as well as scope and limitations of the present reaction
are currently being conducted in our laboratory.
compound with the aldehyde generated another intermediate
a
,b
-
4. Experimental
unsaturated carbonyl compound 7. Subsequently, the intermediate
7 was stabilized by the Asp-His dyad and the oxyanion hole and
underwent Michael addition and an intramolecular condensation
with the intermediate 6 to give the final product 1,4-dihydro-
pyridine 4.
4.1. Materials
Alkaline protease from B. subtilis (10 U/mg, 1 U corresponds to
the amount of enzyme, which liberates 1 mmol folin-positive amino

2692
J.-L. Wang et al. / Tetrahedron 67 (2011) 2689e2692
acids and peptides per minute at pH 7.5 and 37 ꢀC) was obtained
from Wuxi Enzyme Co. Ltd. (Wuxi, PR China). Lipase from hog
pancreas (2.4 U/mg, 1 U is the amount of immobilized enzyme,
which forms 1% octyl laurate from 0.5 mmol lauric acid and
1.0 mmol 1-octanol in 10 ml water-saturated isooctane in 1 h at
20 ꢀC) was purchased from Fluka (Switzerland). Immobilized
penicillin G acylase from E. coli (EC 3.5.1.11, immobilized on acrylic
beads) was purchased from Hunan Flag Biotech Co. Lipase immo-
bilized on acrylic resin from C. antarctica (ꢁ10 000 U/g, recombi-
nant, expressed in A. oryzae) was purchased from Sigma
contained fractions were combined, concentrated, and dried to give
the product 1,4-dihydropyridine.
Acknowledgements
The financial support from the National Natural Science Foun-
dation of China (No. 20704037, 20872130, 20874086) and the
Zhejiang Provincial Natural Science Foundation (Project no. 2009-
Y4080198, 2010-Z4090225) is gratefully acknowledged.
(Steinheim, Germany).
1 U is defined as enzyme quantity, which produces 1
-Amino acid per 30 min) and Acylase ‘Amano’ (AA) from A. oryzae
(ꢁ30 000 U/g, 1 U is defined as enzyme quantity, which produces
mol of -Amino acid per 30 min) were purchased from Amano
D
-aminoacylase from E. coli (10 000 U/mg,
Supplementary data
mmol of
D
Supplementary data related to this article can be found online at
doi:10.1016/j.tet.2011.01.045. These data include MOL files and
InChiKeys of the most important compounds described in this article.
1
m
L
Enzyme Inc (Japan). All solvents were analytical grade and were
ꢀ
dried by storing over activated 3 A molecular sieves for 24 h prior to
use. All other reagents were used as received.
References and notes
1. (a) Hult, K.; Berglund, P. Curr. Opin. Biotechnol. 2003,14, 395; (b) Panke, S.; Held, M.;
Wubbolts, M. Curr. Opin. Biotechnol. 2004,15, 272; (c) Kopp, F.; Marahiel, M. A. Curr.
Opin. Biotechnol. 2007, 18, 513; (d) Woodley, J. M. Trends Biotechnol. 2008, 26, 321.
2. (a) Zaks, A.; Klibanov, A. M. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3192; (b) Kli-
banov, A. M. CHEMTECH 1986, 16, 354.
3. (a) O’Brien, P. J.; Herschlag, D. Chem. Biol. 1999, 6, 91; (b) Copley, S. D. Curr. Opin.
Chem. Biol. 2003, 7, 265; (c) Bornscheuer, U. T.; Kazlauskas, R. J. Angew. Chem.,
Int. Ed. 2004, 43, 6032; (d) Khersonsky, O.; Roodveldt, C.; Tawfik, D. S. Curr.
Opin. Chem. Biol. 2006, 10, 498; (e) Hult, K.; Berglund, P. Trends Biotechnol. 2007,
25, 231; (f) Taglieber, A.; Hobenreich, H.; Carballeira, J. D.; Mondiere, RJ. G.;
Reetz, M. T. Angew. Chem., Int. Ed. 2007, 46, 8597.
4. (a) Kitazume, T.; Ikeya, T.; Murata, K. J. Chem. Soc., Chem. Commun. 1986, 1331; (b)
Torre, O.; Alfonso, I.; Gotor, V. Chem. Commun. 2004,1724; (c) Svedendahl, M.; Hult,
K.; Berglund, P. J. Am. Chem. Soc. 2005, 127,17988; (d) Carlqvist, P.; Svedendahl, M.;
Branneby, C.; Hult, K.; Brinck, T.;Berglund, P. ChemBioChem2005, 6, 331;(e)Wu, W.
B.; Wang,N.;Xu,J.M.;Wu,Q.;Lin,X. F. Chem.Commun. 2005,18, 2348;(f)Wu,W. B.;
Xu, J. M.; Wu, Q.; Lv, D. S.; Lin, X. F. Adv. Synth. Catal. 2006, 348, 487; (g) Xu, J. M.;
Zhang, F.; Liu, B. K.; Wu, Q.; Lin, X. F. Chem. Commun. 2007, 20, 2078; (h) Lou, F. W.;
Liu, B. K.; Wu, Q.; Lv, D. S.; Lin, X. F. Adv. Synth. Catal. 2008, 350, 1959.
4.2. Analytical methods
The 1H and 13C NMR spectra were recorded with TMS as internal
standard using a Bruker AMX-400 MHz spectrometer. Chemical
shifts were expressed in parts per million and coupling constants (J)
in hertz. Analytical HPLC was performed using an Agilent 1100 series
with a reversed-phase Shim-Pack VP-ODS column (150ꢂ4.6 mm)
and a UV detector (210 nm). IR spectra were measured with a Nicolet
Nexus FTIR 670 spectrophotometer. Melting points were determined
using XT-4 apparatus and were not corrected. All the known prod-
1
ucts were characterized by comparing the H NMR data with those
reported in the literature. The structures of new compounds were
confirmed by 1H, 13C NMR, and HRMS.
5. (a) Seebeck, F. P.; Hilvert, D. J. Am. Chem. Soc. 2003, 125, 10158; (b) Terao, Y.;
Miyamoto, K.; Ohta, H. Chem. Lett. 2007, 36, 420.
€
6. For recent reviews, see: (a) Domling, A.; Ugi, I. Angew. Chem., Int. Ed. 2000, 39,
3168; (b) Zhu, J. Eur. J. Org. Chem. 2003, 1133; (c) Ulaczyk-Lesanko, A.; Hall, D. G.
€
Curr. Opin. Chem. Biol. 2005, 9, 266; (d) Domling, A. Chem. Rev. 2006, 106, 17; (e)
4.3. General procedure for the synthesis of 1,4-
dihydropyridines derivatives
Tejedor, D.; García-Tellado, F. Chem. Soc. Rev. 2007, 36, 484; (f) D’Souza, D. M.;
Muller, T. J. J. Chem. Soc. Rev. 2007, 36, 1095; (g) Isambert, N.; Lavilla, R. Chem.d
Eur. J. 2008, 14, 8444; (h) Hulme, C.; Dietrich, J. Mol. Diversity 2009, 13, 195; (i)
€
Ganem, B. Acc. Chem. Res. 2009, 42, 463.
Aldehyde (0.125 mmol), acetamide (0.5 mmol), and CAL-B
(100 mg) in the mixed solvent (1 ml, 1,3-dicarbonyl compounds/
methyl tert-butyl ether¼4:6, by vol) at 50 ꢀC for 72 h. The reaction
was terminated by filtering off the enzyme. The crude residue
was purified by silica gel column chromatography with an eluent
consisting of petroleum ether/ethyl acetate (1/1 v/v). Product-
7. Li, K.; He, T.; Li, C.; Feng, X. W.; Wang, N.; Yu, X. Q. Green Chem. 2009, 11, 777.
8. For recent reviews on Hantszch 1,4-DHP synthesis, see: (a) Langer, P. Chem.d
Eur. J. 2001, 7, 3858; (b) Langer, P. Synthesis 2002, 441; (c) Simon, C.; Con-
stantieux, T.; Rodriguez, J. Eur. J. Org. Chem. 2004, 4957.
9. Bridgwood, K. L.; Veitch, G. E.; Ley, S. V. Org. Lett. 2008, 10, 3627.
10. Branneby, C.; Carlqvist, P.; Magnusson, A.; Hult, K.; Brinck, T.; Berglund, P. J. Am.
Chem. Soc. 2003, 125, 874.