Promiscuous acylase-catalyzed aza-Michael additions of aromatic N-heterocycles in organic solvent

Article abstract of DOI:10.1016/j.tetlet.2007.06.164

A novel and efficient enzymatic promiscuous protocol for aza-Michael addition of aromatic N-heterocycles to α,β-unsaturated compounds has been described. The reactions were catalyzed by promiscuous zinc-active-site acylase in organic solvent at 50 °C. The strategy works with a broad range of N-heterocycles to afford the corresponding Michael adduct with good yields in several hours (0.5-6 h). This catalytic promiscuity is the first example of metal-active-site enzyme-catalyzed aza-Michael addition for aromatic N-heterocycles.

Full text of DOI:10.1016/j.tetlet.2007.06.164

Tetrahedron Letters 48 (2007) 6100–6104
Promiscuous acylase-catalyzed aza-Michael additions of
aromatic N-heterocycles in organic solvent
Chao Qian, Jian-Ming Xu, Qi Wu, De-Shui Lv and Xian-Fu Lin*
Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China
Received 30 April 2007; revised 28 June 2007; accepted 29 June 2007
Available online 6 July 2007
Abstract—A novel and efficient enzymatic promiscuous protocol for aza-Michael addition of aromatic N-heterocycles to a,b-unsat-
urated compounds has been described. The reactions were catalyzed by promiscuous zinc-active-site acylase in organic solvent at
50 ꢀC. The strategy works with a broad range of N-heterocycles to afford the corresponding Michael adduct with good yields in
several hours (0.5–6 h). This catalytic promiscuity is the first example of metal-active-site enzyme-catalyzed aza-Michael addition
for aromatic N-heterocycles.
ꢁ 2007 Elsevier Ltd. All rights reserved.
The synthesis of biologically interesting compounds is
an area of growing interest in organic and in some of
bio-organic chemistry. N-Substituted aromatic N-het-
erocyclic compounds, such as imidazoles, triazoles, pyr-
azoles, pyrimidines and their derivatives obtained
through aza-Michael addition are pharmacologically
active and may be applied as potential therapeutic
alternatives.1
In our previous work, we examined the aza-Michael
addition of aromatic N-heterocycles catalyzed by
serine-active-site hydrolases5 and found the reaction
usually requires one or more days. In this Letter, we
surveyed the aza-Michael additions of zinc-active-site
acylases and surprisingly found that two zinc-active-site
acylase, D-aminoacylase ‘Amano’ from Escherichia coli
(DA) and acylase ‘Amano’ from Aspergillus oryzae
(AA), could catalyze the reaction of aromatic N-hetero-
cycles in several hours. This catalytic promiscuity is the
first example about metal-active-site enzyme-catalyzed
aza-Michael addition for aromatic N-heterocycles.
Under the optimal conditions, Michael derivatives of
diversified pentacyclic N-heterocycles and pyrimidines
were successfully obtained under mild conditions.
In general, the aza-Michael addition requires basic con-
ditions or acidic catalysts that may lead to environmen-
tally hazardous residues or undesirable side-products.2
To avoid typical disadvantages resulting from the pres-
ence of such catalysts, a great number of alternatives
have been studied in the past few years, such as CeCl3,3a
InCl3,3b Yb(OTf)3,3c Bi(OTf)3,3d Cu(OTf)2,3e LiClO4,3f
b-cyclodextrin,3g polyethylene glycol,3h heterogeneous
solid acids,3i ionic liquids,3j–l and water.3m Recently,
several serine-active-site hydrolases were found to be
able to catalyze aza-Michael additions. Wild-type4a
and the Ser105Ala mutant of CAL-B4b could catalyze
the Michael-type addition of thiol and amine nucleo-
philes to a range of a,b-unsaturated carbonyl com-
pounds. Unfortunately, these works were focused on
the enzymatic Michael addition of aliphatic amines
and the related reports about aromatic N-heterocycles
were rare.
Acylase-catalyzed Michael addition of imidazole and
a,b-unsaturated carbonyl compounds in organic med-
ium has been achieved and it is shown in Scheme 1.
Eighteen derivatives of aromatic N-heterocycles have
been obtained in moderate to high yields. The results
are summarized in Tables 3 and 4. The Michael addition
of 4-nitro-imidazole (1a) and methyl acrylate (2a) was
first examined. When 0.2 mmol 1a and 2 equiv of 2a
were added to 2 ml DMSO containing 10 mg DA, a sin-
gle product was prepared in 95% isolated yield at 50 ꢀC
after 24 h. A similar result was observed for AA. The
structure of the additional compounds was confirmed
by IR, 1H NMR, 13C NMR, and ESI-MS. We also mon-
itored the formation of the product by HPLC and it is
worthwhile to mention that no byproduct was detected
*
Corresponding author. Tel.: +86 571 87951588; fax: +86 571
87952618; e-mail: llc123@zju.edu.cn
0040-4039/$ - see front matter ꢁ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.06.164

C. Qian et al. / Tetrahedron Letters 48 (2007) 6100–6104
Table 2. Solvents screening for enzymatic Michael additiona
6101
2
R
N
Solvents
logP
Yield (%) of
imidazoleb
Yield (%) of
4-nitro-imidazoleb
R1
R1
EWG
N
R
EWG
Zinc acylase
N
DMSO
DMF
À1.3
À1.0
À0.5
0.46
0.65
1.9
94
92
55
70
60
69
77
93
97
97
+
NH
R2
3
4
DMSO, 50 oC
0.5-6 h
3
4
R
R
R
Dioxane
THF
Pyridine
Isopropyl ether
Toluene
n-Hexane
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
R1,R3,R4=H, CH3; R2=H, CH3, NO2;
EWG=COOMe, COOEt, COOBu, COOCH=CH2, CN.
2.6
3.9
Scheme 1. Acylase-catalyzed Michael addition of imidazole and a,b-
unsaturated carbonyl compounds in organic solvent.
a Experimental conditions: 0.3 M imidazole (4-nitro-imidazole), 0.6 M
methyl acrylate, 15 mg AA, 1 ml solvents, 50 ꢀC, 6 h.
b All yields were determined by HPLC. n.d. means no reaction was
found.
resulting from acylation reaction, hydrolytic reaction or
other reactions.
We designed some control experiments to demonstrate
the catalytic specificity of DA and AA. The reaction
yields and the initial reaction rates were compared,
which are summarized in Table 1. From this study, the
addition without any catalyst led to a very low yield
(<0.5%) even after 24 h. In contrast, more than 95%
yield was obtained in the presence of 10 mg DA or
AA. The initial reaction rates of these cases were up to
333-fold and 638-fold faster than the process in absence
of a biocatalyst, respectively. The initial rate is practi-
cally proportional to the enzyme amount.
ing with serine-active-site hydrolyses such as Candida
antarctica lipase B (CAL-B), alkaline protease from
Bacillus subtilis (PA) and Amano Lipase M from Mucor
javanicus (MJL), zinc-active-site acylase could catalyze
the reaction more efficiently. Among the two acylase,
AA could catalyze the enzymatic Michael addition more
efficiently. Thus AA was selected for the next steps.
Reaction media is an important factor in the enzymatic
reaction. We examined the enzymatic Michael addition
in different organic solvents with logP value ranging
from À1.3 to 4.9 and the results are shown in Table 2.
The logP value of the solvents is the widely used para-
meter to describe solvent polarity and their possible
effects on the enzyme activity, where P is the partition
coefficient of a given solvent between water and n-octa-
nol in a two-phase system.7 It was found that the reac-
tion only took place in DMSO and DMF. No obvious
reaction was detected in other solvents. This may be
attributed to the low solubility of 4-nitro-imidazole. In
order to exclude the influence, we examined the Michael
addition of imidazoles (1b) and 2a. The results are also
shown in Table 2. Similar results were observed in
DMF and DMSO. In other solvents, the Michael ad-
ducts could also be obtained in moderate to high yield.
To our surprise, those solvents with a higher logP value
exhibited higher conversion. The reaction proceeded
smoothly and good yield was obtained in non-polar sol-
vents such as n-hexane. Based on the good yield and
substrate solubility, DMSO was selected in the following
reactions.
When the enzyme (AA) is pre-treated with urea at
100 ꢀC in order to completely denaturalize the protein,
the rate is practically equal to the bovine serum albumin
(BSA), suggesting that the tertiary structure of the bio-
catalyst is necessary to catalyze the reaction. Then con-
trol reactions were run with inhibited DA and AA by
adding 50 mM non-competitive inhibitor ZnCl2.6 The
inhibited enzyme did not show any acylase activity to
catalyze the hydrolysis of N-acetyl-D-methionine, and
the specific activity for the Michael addition was as that
of BSA protein. All these results suggest that the tertiary
structure and the specific active sites of zinc-acylase are
responsible for the Michael addition reaction. Compar-
Table 1. Michael addition between 4-nitro-imidazole (1a) and methyl
acrylate (2a) in the presence of different catalysts
Catalyst
Time (h) Yielda (%) V0 (M m minÀ1
)
Vrb
No catalyst
AA
24
6
6
0.4
98.2
97.5
92.7
8.7
6.7
6.0
66.8
73.6
53.4
4.2 · 10À5
0.027
0.014
0.007
1.0
638
333
165
13
10.9
1.2
95
DA
DAc
The scope of this method was investigated with a variety
of structurally diverse imidazoles and a,b-carbonyl com-
pound under Michael additions to generate the corre-
sponding derivatives in moderate to good yields. The
results are summarized in Table 3. The reactivity
decreased with increasing chain length of the acceptor,
the longer chain the acrylates had, the lower the yield
was obtained. Apart from acrylate ester, acrylonitrile
could also react with imidazoles in good yields. Vinyl
acrylate and butenone showed higher reactivity than
methyl acrylate and ethyl acrylate. In order to extend
the scope of this method, a,b-substituted Michael accep-
tors like methyl a-methylacrylate and methyl crotonate
were tested under the same conditions. Both of the
6
DA denaturedd 24
5.1 · 10À4
4.6 · 10À4
5.0 · 10À5
0.004
BSA
24
24
24
24
24
DA inhibitede
CAL-B
MJL
4.6 · 10À3
110
63
PA
2.6 · 10À3
a Experimental conditions: 0.3 M imidazole (4-nitro-imidazole), 0.6 M
methyl acrylate, 10 mg enzyme, 2 ml DMSO, 50 ꢀC, 6 h. All yields
were detected by HPLC.
b Relative initial reaction rate to the reaction in the absence of enzyme.
c DA 2.5 mg mlÀ1
.
d Enzyme predenatured with urea at 100 ꢀC for 24 h.
e Enzyme inhibited by 50 mM ZnCl2.

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C. Qian et al. / Tetrahedron Letters 48 (2007) 6100–6104
Table 3. Michael addition of imidazoles to a,b-carbonyl compound promoted by AAa
Entry
1
Imidazole
a,b-Carbonyl compound
Time (h)
3
Yieldb (%)
NO2
N
CO Me
2
(3a) 96
N
H
NO2
N
2
3
4
5
3
24
24
24
(3b) 90
(3c) 19
(3d) 16
(3e) 37
CO2Et
N
H
NO2
N
CO But
2
N
H
NO2
N
CO2Me
N
H
NO2
N
CO2Me
N
H
NO2
N
6
3
(3f) 90
CN
N
H
NO2
N
O
7
8
9
0.5
0.5
3
(3g) 85
(3h) 82
(3i) 83
O
N
H
NO2
N
O
N
H
N
CO Me
2
N
H
N
10
11
3
6
(3j) 75
CN
N
H
NO2
N
N
(3k) 68
CO Me
2
N
H
12
13
6
(3l) 82
CO Me
2
N
H
N
6
(3m) 83
CO Me
2
N
H
a Reactions were carried out on 0.3 mmol scale of substrate with 2 equiv acceptor in 1 ml DMSO at 50 ꢀC, 15 mg AA was added.
b Isolated yields.

C. Qian et al. / Tetrahedron Letters 48 (2007) 6100–6104
6103
Table 4. Michael addition of other aromatic N-heterocycles with methyl acrylate catalyzed by AAa
Entry
Substrate
Product
Time (h)
3
Yieldb (%)
O
O
N
NH
1
(5a) 95
(5b) 56
N
N
OCH3
OCH3
N
N
N
NH
N
N
2
3
3
3
O
O
F
F
HN
HN
(5c) 97
O
N
O
O
O
N
H
OCH3
O
Br
Br
O
HN
HN
4
3
(5d) 91
N
O
O
O
N
H
OCH
3
O
HN
HN
5
6
3
3
(5e) 93
(5f) 90
O
O
O
N
O
O
N
H
OCH
OCH
3
O
CH3
HN
HN
O
N
O
N
H
3
a Reactions were carried out on 0.3 mmol scale of substrate with 2 equiv acceptors in 1 ml DMSO at 50 ꢀC, 15 mg AA was added.
b Yields was determined by HPLC.
acceptors showed rather lower activity because of the
strong steric hindrance. To our disappointment, no
enantioselectivity could be observed for the above tested
reactions with the catalysis of DA or AA. The reason for
this will be further investigated.
obtained in high yield. Among the examined pyrimi-
dines, fluorouracil, and bromouracil reacted faster than
uracil and thymine. It is worthwhile to mention that
only N-1 adducts were observed (monitored by 1H
NMR and HPLC). This result reveals that AA exhibited
higher regioselectivity compared to the chemical
transformation.
Comparable behavior was observed using imidazole as
substrate. The four substituted imidazoles examined
underwent Michael addition with methyl acrylate favor-
ably and all substituted imidazoles could be obtained
from moderate to good yields. The reactivity decreased
by the following order: 4-nitro-imidazole, imidazole,
and 4-methyl-imidazole. The reactivity was in accor-
dance with the nucleophilicity of imidazole derivatives.
Substituted imidazoles with strong steric hindrance,
such as 2-methyl-4-nitro-imidazole, underwent Michael
addition much more slowly.
In this study, a facile and an efficient biotransformation
method to perform aza-Michael addition of aromatic N-
heterocycles has been developed by utilizing two promis-
cuous zinc-active-site acylases as biocatalysts. The struc-
tural influence of the Michael donor and acceptor was
systematically studied. This strategy has expanded the
application of catalytic promiscuities in organic syn-
thesis. The obtained N-heterocyclic derivatives may be
potentially bioactive and could be used as pharmaco-
logical alternatives.
Having obtained favorable results with imidazoles, we
then examined the addition of other N-heterocycles to
methyl acrylate. Other five membered N-heterocycles
such as pyrazole and triazole also exhibited good
Michael addition activity. Triazole reacted faster due
to its stronger nucleophilicity than pyrazole. More com-
plicated N-heterocycles, such as pyrimidines, can also be
used as a substrate to obtain the corresponding Michael
adducts. All the substituted pyrimidines derivatives were
Acknowledgments
This investigation has enjoyed financial support from
the Natural Science Foundation of China (No.
20572099) and Ph.D. Programs Foundation of Ministry
of Education of China (20060335031).

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C. Qian et al. / Tetrahedron Letters 48 (2007) 6100–6104
Spencer, J. B. Tetrahedron Lett. 2002, 43, 3891–3894; (f)
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