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Chemistry Letters Vol.36, No.3 (2007)
The Aldol Type Reaction Catalyzed by Arylmalonate Decarboxylase
—A Decarboxylase can Catalyze an Entirely Different Reaction, Aldol Reaction—
Yosuke Terao, Kenji Miyamoto, and Hiromichi OhtaÃ
Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, Yokohama 223-8522
(Received January 10, 2007; CL-070027; E-mail: hohta@bio.keio.ac.jp)
The catalytic promiscuity of AMDase was demonstrated
enzyme has already been cloned and overexpressed in E. coli,
and the enzyme has also been purified.6 The reaction mechanism
of AMDase has been extensively studied. It has been clarified
that Cys188 is located in the active site as the key amino
acid residue, of which role has been revealed to protonate to
the intermediate enolate form of the resulting carboxylic acid.
Recently, we have succeeded to endow AMDase with a
new catalytic activity, i.e., racemization of carboxylic acids, in
addition to its original decarboxylation activity. It was achieved
by introducing only one mutation in the active site based on
the estimated reaction mechanism and the homology with some
isomerases.7 Thus, it could be said that AMDase also had cata-
lytic promiscuity similarly to lipases and other enzymes. In this
case, the catalytic promiscuity of AMDase became apparent via
the introduction of the mutation into the enzyme itself, i.e., by
the modification of the enzyme. Then, we made another chal-
lenge to draw its catalytic promiscuity by designing the structure
of the substrate. To trap the intermediate enolate resulting from
the decarboxylation of the malonate, an aldehyde group was
introduced at the ortho-position of the aromatic ring.
First, we tried the reaction of compound 3 expecting to
obtain the condensation product 4, which had six-membered ring
(Scheme 2). However, no reaction occurred in this case, prob-
ably because the steric bulkiness of the substrate is too large.
Then, we tried to synthesize a less bulkier substrate 14,
which was expected to give the condensation product with a
five-membered ring. The substrate 14 was synthesized via nine
steps from the commercially available compound 5 as illustrated
in Scheme 3.
using a well-designed substrate based on the consideration of
the reaction mechanism. As the reaction catalyzed by AMDase
is supposed to proceed via an enolate intermediate, we expected
that the enzyme promotes the aldol type reaction when an accep-
tor is properly arranged, which turned out to be true.
Enzymes are becoming more efficient and useful catalysts
not only in biochemistry or agricultural chemistry but also in
organic synthetic chemistry. It had been believed that they could
transform only natural substrates in aqueous solutions and
catalyze only one reaction. However, it has been revealed that
substrate specificities of enzymes are broad and many enzymes
can catalyze the transformation of unnatural substrates. Also,
some enzymes, for example lipases and esterases, were turned
out to be able to catalyze the reactions in organic solvents. These
properties of enzymes are called as enzymatic promiscuity.1 It is
said that the broad substrate specificities of enzymes is called as
the first promiscuity and the flexibility of reaction conditions of
enzymes is called as the second promiscuity. Recently, the third
enzymatic promiscuity has been proposed, i.e., the flexibility of
the reactions catalyzed by enzymes. For example, it was reported
that a lipase was endowed a new catalytic activity, i.e., aldolase
activity by changing an amino acid residue in the active site.2 In
this case, the mutation was introduced into the critical amino
acid residue to change the function of the lipase by considering
the reaction mechanism. Also, there are some reports recently
showing the enzymes’ promiscuities that a single active site of
one enzyme can catalyze more than one distinct chemical trans-
formations.3 Over the last few years, evidences have mounted
that such catalytic promiscuity exists not just among a few en-
zymes but is rather common.4
As expected, the treatment of 14 with AMDase (reaction
conditions: 1.0% substrate, enzyme 100 unit, in 1.0 mL of
0.1 M Tris-HCl buffer, at 35 ꢀC, for 12 h) gave the aldol product
in 35% yield (Scheme 4). The product was identified by compar-
ing the NMR and IR spectra with those of the authentic speci-
men. Because the reaction was taken place in the active site of
the enzyme that generally differentiates the chirality and prochir-
ality of the compounds, we expected that the aldol product
would exhibit some enantio- or diastereomeric excess. However,
In this letter, we would like to describe the promiscuity
of a decarboxylation enzyme, arylmalonate decarboxylase
(AMDase, EC 4.1.1.76).5 AMDase has been isolated as a unique
decarboxylase to catalyze the decarboxylation of malonates to
give optically pure ꢀ-arylpropionates (Scheme 1). In addition
to this original activity, we have found that the enzyme has
aldolase activity to catalyze the aldol-type condensation of the
malonate derivative containing an aldehyde group.
CHO
O
AMDase
AMDase was purified from Alcaligenes bronchisepticus
KU1201, which was isolated from soil. The gene coding the
CO2H
CO2H
CO2
OH
CO2
O
3
R
R
AMDase
CO2
O
H
CO2H
CO2H
*
Ar
Ar
*
OH
CO2H
OH
CO2H
O
1
2
4
Scheme 1. Asymmetric decarboxylation of AMDase.
Scheme 2. Enzymatic aldol reaction catalyzed with AMDase.
Copyright Ó 2007 The Chemical Society of Japan
Terao, Yosuke
