L.-H. Zhou et al. / Journal of Molecular Catalysis B: Enzymatic 109 (2014) 170–177
171
Scheme 1. Lipase-catalyzed direct vinylogous Michael addition reaction.
that the Michael-type additions of thiols to ␣,-unsaturated car-
bonyl compounds are most likely catalyzed in the active-site of
Ser105Ala mutant CAL-B on the basis of docking and molecular-
dynamics simulations. Recently, the authors also discovered that
the CAL-B Ser105Ala mutant possessed an increased ability to
harbor substrates close to a catalytically competent conformation
using docking studies and molecular dynamics simulations [29].
In 1935, Fuson formulated the concept of vinylogy, which is the
propagation of electronic density through conjugated bonds, has
been applied to provide a better understanding of the reactivity
of some unsaturated compounds and explain how the influence
of a functional group in the molecule when it is attached to
conjugated unsaturated linkages [30]. However, direct vinylogous
Michael reactions are more applicable when considering the devel-
opment of green chemistry and atom economy. To the best of
our knowledge, there have been no investigations on enzyme-
catalyzed direct vinylogous Michael addition reactions. Herein, we
report the successful development of lipase-catalyzed direct viny-
logous Michael reactions of ␣,␣-dicyanoolefins (Scheme 1), which
simultaneously generate multi-functional compounds that can be
used to prepare complex organic targets. The nitro group in these
compounds can easily be converted into a series of new func-
tionalities, such as amines and nitrile oxides [31,32]. Oxidation or
activities (Table 1, entry 3), which may be because that the immo-
bilized enzyme can improve the stability and adaptability of the
RML. Two ␣-amylases (Table 1, entries 4 and 5) also demonstrated
the ability to catalyze direct vinylogous Michael additions. Several
other lipases expressed in Candida (Table 1, entries 6–8) and the
non-enzyme protein bovine serum albumin (BSA) (Table 1, entry
9) exhibited low activities in this reaction. Notably, CAL-B previ-
ously reported to catalyze Michael-type additions, however, was
incapable of catalyzing the model reaction, which may be because
the proton from vinyl malononitrile 1a to Histidine cannot trans-
fer efficiently. To further explain the different catalytic efficiencies
amongst the enzymes, docking studies on the amino acids in the
active sites of the substrates were performed.
Additional control experiments were conducted to confirm the
specific catalytic effect of MML. When the reactions were incubated
with no enzyme (Table 1, entry 10) or with denatured MML (Table 1,
entry 11), no products were detected. All the results demonstrated
that the catalysis was not simply due to nonspecific amino acid
residues on the surface of the enzyme and that the specific cat-
alytic site and spatial conformation of the natural lipases were
responsible for its activity during its reaction.
2.2. The influence of reaction media
reduction of the [
C C(CN) ] moiety is also possible [33–35]. Like-
2
wise, as important core structural units and frequently employed
synthetic precursors of various bioactive molecules, they also play
vital roles in medicinal chemistry [36]. In addition, various struc-
turally diverse nitroalkenes and vinyl malononitriles were used in
direct vinylogous Michael additions to create their correspond-
ing products with moderate to high yields in the presence of
Selecting an appropriate reaction medium due to its potentially
great effects on enzyme performance and substrate properties is
very important [39]. However, quantifying these effects remains
challenging due to the complexity of the solvent–protein inter-
actions. Several organic solvents were investigated for the model
reaction (Table 2), and the results revealed that the solvent had
a great effect on the reaction process. The reaction in acetonitrile
(Table 2, entry 5) afforded the highest yield of 84% after 48 h. The
®
MML (Lipozyme , immobilized lipase from Mucor miehei) and with
excellent diastereoselectivities in all reactions under optimized
conditions. Docking studies were performed to further illuminate
the differences between various enzymes and substrates. Lastly,
to illustrate the synthetic potential of the products, a chemo-
and diastereoselective oxidation reaction of the double bond was
developed for the combination of CAL-B and neutral ionic liquid
Table 1
The direct vinylogous Michael addition reaction between vinyl malononi-
trile 1a and trans--nitrostyrene 2a in the presence of various enzymes.a
[
Hmim]PF for metal-free H O activation (see in Supporting Infor-
6 2 2
mation).
.
2
. Results and discussion
Entry
Enzyme
Yieldb [%]
2
.1. The catalytic activities of different enzymes
1
2
3
4
5
6
7
8
9
Lipozyme®,immobilized, from Mucor miehei (MML)
84
70
36
14
29
2
2
2
6
N.R
N.R
Amano lipase M from Mucor javanicus (MJL)
Lipase from Rhizomucor miehei (RML)
␣-Amylase from Aspergillus oryzae (AOA)
␣-Amylase from hog pancreas (HPA)
Lipase B from Candida antarctica (CAL-B)
Lipase from Candida rugosa (CRL)
We initially used vinyl malononitrile 1a derived from 1-
tetralone and trans--nitrostyrene 2a as a model reaction. To
select the appropriate biocatalyst, a series of commercially avail-
able enzymes were screened (Table 1). We found that Lipozyme ,
immobilized, from Mucor miehei (MML) (Table 1, entry 1) is dis-
tinctly preferred for the model reaction with an 84% yield. Amano
lipase M from Mucor javanicus (MJL) (Table 1, entry 2) also showed
moderate catalytic activities, which may be because they are all
expressed in the same fungi (Mucor) and possess some degree
of homology with MML. Lipase from Rhizomucor miehei (RML)
which was the free form of MML [37,38], exhibited relatively lower
®
Lipase from Candida cylindracea (CCL)
Bovine Serum Albumin (BSA)
1
1
0
1
—
Denatured MMLc
a
Reaction conditions: 1a (0.1 mmol), 2a (0.1 mmol), enzyme (10 mg), acetonitrile
◦
(
1 mL) were shaken at 200 rpm at 30 C for 48 h.
b
Determined by HPLC.
c
◦
Heated at 200 C for 8 h.