Promiscuous zinc-dependent acylase-mediated carbon-carbon bond formation in organic media

Article abstract of DOI:10.1039/b700327g

A zinc-dependent acylase, d-aminoacylase from Escherichia. Coli, displays a promiscuous activity to catalyze the carbon-carbon bond formation reaction of 1,3-dicarbonyl compounds to methyl vinyl ketone in organic media. The Royal Society of Chemistry.

Full text of DOI:10.1039/b700327g

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Promiscuous zinc-dependent acylase-mediated carbon–carbon bond
formation in organic media{
Jian-Ming Xu, Fu Zhang, Bo-Kai Liu, Qi Wu and Xian-Fu Lin*
Received (in Cambridge, UK) 9th January 2007, Accepted 1st February 2007
First published as an Advance Article on the web 22nd February 2007
DOI: 10.1039/b700327g
addition activity of metallo-acylases is of practical significance in
expanding the application of enzymes and in the evolution of new
biocatalysts.9
A zinc-dependent acylase, D-aminoacylase from Escherichia.
Coli, displays a promiscuous activity to catalyze the carbon–
carbon bond formation reaction of 1,3-dicarbonyl compounds
to methyl vinyl ketone in organic media.
We first examined the Michael addition of ethyl acetoacetate
(1b) and methyl vinyl ketone (2) (Scheme 1) in a solventless system.
When 6 mmol 1b was added to a solution containing 10 mg
D-aminoacylase and 8.4 mmol 2 at 50 uC for 24 h, a single product
was prepared in 35% isolated yield after flash chromatography.
The structure of this compound was confirmed by IR, 1H NMR,
13C NMR and MS. In order to improve the activity of enzyme, we
carried out the reaction in organic solvent. Some conventional
organic solvents with different log P values were screened for the
enzymatic Michael addition reaction and the results are shown in
Table 1. In those solvents such as toluene, chloroform or isopropyl
ether, the Michael additions proceeded very slowly and the yields
were less than 10% (entries 2–4, Table 1). The result in hexane was
almost the same as that in the solvent-free reaction (entries 1,
Table 1). In polar solvents such as THF and dioxane, the yields are
far from satisfactory (entries 7 and 8, Table 1). D-Aminoacylase
showed higher Michael addition activity in some tertiary alcohol
solvents (entries 5 and 6, Table 1). The Michael addition in
2-methyl-2-butanol leads to 82% yield in 24 h. Thus, 2-methyl-2-
butanol was chosen as solvent in the following experiments.{
Three types of 1,3-dicarbonyl compounds, ethyl acetoacetate,
acetylacetone and diethyl malonate, were tested as nucleophiles for
the enzymatic Michael addition reaction in 2-methyl-2-butanol.
The progress curves were shown in Fig. 1 and the initial reaction
rates were calculated accordingly. The Michael addition of
acetylacetone with methyl vinyl ketone proceeded fastest and the
initial reaction rate was up to 60.4 mM h21. The reaction of 1b
also underwent smoothly leading to the corresponding Michael
adduct in 82% yield in 24 h. Diethyl malonate exhibited rather
low Michael addition activity and only about 1% yield was
obtained even after 24 h. This may be attributable to the weaker
acidity of a-protons of diethyl malonate or steric hindrance.
Taking into account these results, we performed some control
experiments focusing on demonstrating the specific catalytic effect
The seminal work by Klibanov in the early 1980s initiated non-
aqueous enzymology.1 In organic media, many enzymes have
demonstrated their activities with non-natural substrates.2 During
the exploration of new activities of enzymes, a growing number of
them have been found to be capable of catalyzing not only their
‘‘natural’’ reactions but also one or more alternative reactions.3
This catalytic promiscuity of enzymes expands largely the
application of biocatalysts in synthetic chemistry.4 Research in
this heating area has attracted much attention of chemists and
biochemists in recent years.
The Michael addition reaction is among the most fundamental
types of reactions in organic synthesis. However, there were only
rare reports about enzymes which are able to catalyze the Michael
addition reactions. Kitazume et al. used hydrolytic enzymes to
catalyze the Michael-type addition of thiols and amines to
triflourinate a,b-unsaturated carbonyl compounds in buffer
solution.5 Our group reported that an alkaline protease from
Bacillus subtilis showed a remarkable activity to catalyze the
Michael addition of N-nucleophiles to acrylates in organic
solvents.6 The wild-type and the Ser105Ala mutant of CAL B
could catalyze the Michael-type addition of thiol and amine
nucleophiles to a range of a,b-unsaturated carbonyl compounds.7
However, the relevant report was only limited in the formation of
carbon–nitrogen and carbon–sulfur bonds. Recently, Berglund
and co-workers reported the unprecedented carbon–carbon bond
formation by an engineered mutant of CAL B.8 This led us to
believe that other types of hydrolase could also catalyze this
addition reaction to form carbon–carbon bonds.
Here, we surprisingly found that a zinc-binding metallo-acylase,
D-aminoacylase from Escherichia coli, which naturally catalyzes
the hydrolysis of N-acyl-D-amino acids, also possesses the
promiscuous activity to catalyze the Michael addition of 1,3-
dicarbonyl compounds to methyl vinyl ketone. The catalytic
specificity of acylases was demonstrated by the combination of
different control experiments. According to the control experi-
ments and the observations, a tentative mechanism for the acylase-
mediated Michael addition was proposed. This novel Michael
Department of Chemistry, Zhejiang University, Hangzhou 310027,
People’s Republic of China. E-mail: llc123@zju.edu.cn;
Fax: 86 571 87952618; Tel: 86 571 87951588
{ Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/b700327g
Scheme 1 Michael addition of 1,3-dicarbonyl compounds to methyl
vinyl ketone catalyzed by D-aminoacylase.
2078 | Chem. Commun., 2007, 2078–2080
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Acylase ‘‘Amano’’ from Aspergillus oryzae, which is also a zinc-
dependent acylases, did not show any Michael addition activity.
Three widely used hydrolases, Lipase from Candida cylindracea
(CCL), CAL B and Amano lipase M, can not accelerate the
reaction efficiently in 2-methyl-2-butanol either (entries 7–9,
Table 2). All these results suggest that the tertiary structure and
the specific spatial conformation of D-aminoacylase are responsible
for the Michael addition reaction of 1,3-dicarbonyl compounds to
methyl vinyl ketone.
Table 1 The influence of solvents on the enzymatic Michael addition
reactiona
Entry
Solvent
log P
Yield (%)
1
2
3
4
5
6
7
8
a
n-Hexane
Toluene
3.9
2.6
2.0
1.9
1.10
0.79
0.46
20.5
31.0
2.8
1.1
7.0
82.1
55.5
17.6
16.8
Chloroform
Isopropyl ether
2-Methyl-2-butanol
2-Methyl-2-propanol
THF
It was also observed that the D-aminoacylase-mediated Michael
addition reaction was strongly affected by the order of adding
reagents. When acetylacetone was added to the solutions contain-
ing enzyme and methyl vinyl ketone, the reaction proceeded
Dioxane
Reaction conditions: D-aminoacylase 10 mg, ethyl acetoacetate
1 mmol, methyl vinyl ketone 2 mmol, solvent 1 mL, 50 uC for 24 h.
rapidly and the initial reaction rate was about 60.0 mM h21
.
However, only slow reaction rate was observed when methyl vinyl
ketone was added to the solution of enzyme and acetylacetone; its
initial reaction rate was only about 20.0 mM h21. It is worthwhile
to note that D-aminoacylase could also catalyze the Michael
addition of acetylacetone and 2-cyclohexene-1-one, although the
reaction rate was much slower.
The proposed mechanism in Scheme 2 might explain the
observed addition order activity. The generally accepted zinc-
dependent acylase mechanism usually involves the polarization of
a carbonyl group and nucleophile water by the zinc ion in the
active sites. A highly conserved Asp plays a key role in the proton
transfer from the nucleophile water to the leaving group.10 The
proposed mechanism would start with the accommodation of
addition acceptor (methyl vinyl ketone) and nucleophile (acetyl-
acetone) in the active site. The interaction of the tightly bound zinc
ion with the carbonyl group of methyl vinyl ketone would increase
the electrophilic ability of Michael acceptor. On the other hand,
one of the carbonyl groups of acetylacetone would coordinate with
the zinc ion, thus rendering the acetylacetone more nucleophilic.
Then, Asp366 deprived the C3-H of acetylacetone and the
nucleophile would add simultaneously to the b-carbon of methyl
vinyl ketone to form the enolate intermediate, which could be
stabilized by the zinc ion in the active site. Finally, the Asp, now
functioning as a general acid, would deliver the proton to complete
the reaction.
Fig. 1 Progress curve of Michael addition of 1,3-dicarbonyl compounds
to methyl vinyl ketone catalyzed by D-aminoacylase in 2-methyl-2-
butanol.
of the D-aminoacylase (Table 2). The reaction of acetylacetone
with methyl vinyl ketone in the absence of enzyme led to the
Michael adduct in very low yield (3.7%) even after 24 h. In
contrast, the reactions in the presence of D-aminoacylase was up to
40-fold faster (entries 1 and 2, Table 2). Besides, the initial reaction
rate is practically proportional to the enzyme amount, also
suggesting the catalytic effect of the enzyme (entries 2 and
3, Table 2). When the reaction was incubated with denatured
D-aminoacylase or bovine serum albumin (BSA), both of the
initial rates were almost equal to the background reaction (entries
5 and 6, Table 2), ruling out the possibility that the similar amino
acid distribution on the protein surface has promoted the process.
This tentative mechanism was based on the proposal that the
zinc ion in the active site coordinated with methyl vinyl ketone and
Table 2 Initial rates (Vo) of the Michael addition between acetyl-
acetone (1 M) and methyl vinyl ketone (2 M)
Entry
Catalyst
Amount/mg
V0/mM h21
Vra
1.0
1
2
—
—
10
1.5
60.4
40.3
D-Aminoacylase
3
5
30.8
20.5
D-Aminoacylase
Acylase ‘‘Amano’’
BSA
4
5
6
7
8
9
a
10
10
10
10
10
10
2.2
2.7
2.3
6.2
7.2
2.1
1.5
1.8
1.5
4.1
4.8
1.4
denaturedb
CAL B
Amano Lipase M
CCL
b
Relative initial rate to the reaction in absence of enzyme. Pre-
Scheme 2 Proposed mechanism of D-aminoacylase-catalyzed Michael
treated with urea at 100 uC for 6 h.
addition.
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Notes and references
{ Typical experimental procedure: In a conical flask containing 10 mg
D-aminoacylase, a solution of internal standard (dodecane) and methyl
vinyl ketone (2 mmol) in 1 mL 2-methyl-2-butanol was incubated at 50 uC
and 250 rpm (orbitally shaken) for 20 min. Then, 1,3-dicarbonyl compound
(1 mmol) was added in order to initiate the reaction. Samples were
withdrawn from the reaction and directly analysed using a GC (SE-54
capillary column, FID detection; oven temperature: from 60 to 200 uC, rate
of heating 20 uC min.21). All the compounds were spectroscopically
characterized (IR, 1H, 13C NMR and MS) and analytically compared (GC)
with authentic samples prepared by conventional methods.
Scheme 3 Possible interactions of reactants with zinc ion in the active
site of enzyme.
1 A. Zaks and A. M. Klibanov, Proc. Natl. Acad. Sci. U. S. A., 1985, 82,
3192; A. M. Klibanov, CHEMTECH, 1986, 16, 354.
one of the carbonyl oxygens of acetylacetone. In fact, the
interactions of zinc ion in the active site with reactants can occur
in three possible ways as shown in Scheme 3. If both reactants
coordinated simultaneously to the zinc ion in the active site, the
Michael acceptor should adopt a s-cis conformation as shown in I.
However, the fact that D-aminoacylase could catalyze the Michael
addition of acetylacetone and 2-cyclohexene-1- excluded auto-
matically the possibility of I. On the other hand, because the zinc
ion in the active site have already tightly bound with Cys96,
His220 and His250,10 the three carbonyl oxygens of acetylacetone
and methyl vinyl ketone could not simultaneously coordinate to
the zinc ion in the active site. Therefore, only two carbonyl
oxygens of the reactants could coordinate with the zinc ion in the
active site to form II or III. The experiment results of different
orders of adding reagents are inclined to support the formation of
III. Thus the proposed mechanism was based on the formation of
III.
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In conclusion, a facile biotransformation path to perform
Michael additions between 1,3-dicarbonyl compounds to
methyl vinyl ketone has been developed by utilizing promiscuous
D-aminoacylase as biocatalyst. The catalytic promiscuity of
D-aminoacylase was demonstrated by the combination of different
control experiments. Based on the experiments, a tentative
mechanism was proposed.
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This investigation has enjoyed financial support from the
Natural Science Foundation of China (NO.20572099).
2080 | Chem. Commun., 2007, 2078–2080
This journal is ß The Royal Society of Chemistry 2007