Functional mimicry of the active site of carboxypeptidase A by a molecular imprinting strategy: Cooperativity of an amidinium and a copper ion in a transition-state imprinted cavity giving rise to high catalytic activity

Article abstract of DOI:10.1021/ja048372l

A model for the natural enzyme carboxypeptidase A was prepared by molecular imprinting in synthetic polymers. An unusually high activity and efficiency for carbonate hydrolysis could be obtained by imprinting with a stable transition-state analogue template and introducing an amidinium group and a Cu²⁺ ion-binding site in a defined orientation to each other into the active site. With substrates having a very similar structure to the template, extraordinarily high enhancements of rates of 110000-fold were obtained of catalyzed to uncatalyzed reaction k_cat/k_uncat. The efficiency k_cat/K_m of the molecularly imprinted catalysts compared to that of the nonimprinted control polymers containing the same functional groups was 790-fold higher, a clear indication of a very efficient imprinting procedure. Copyright

Full text of DOI:10.1021/ja048372l

Published on Web 05/27/2004
Functional Mimicry of the Active Site of Carboxypeptidase A by a Molecular
Imprinting Strategy: Cooperativity of an Amidinium and a Copper Ion in a
Transition-State Imprinted Cavity Giving Rise to High Catalytic Activity
Jun-qiu Liu† and Gu¨nter Wulff*,‡
Institute of Organic and Macromolecular Chemistry, Heinrich Heine UniVersity, 40225 Du¨sseldorf, Germany, and
Key Laboratory of Supramolecular Structure and Materials, Jilin UniVersity, Changchun,130023, P.R. China
Received March 22, 2004; E-mail: wulffg@uni-duesseldorf.de
Designing efficient artificial mimics for naturally occurring
enzymes has been a challenging area to chemists and biologists.1
Inspired by the concept of transition-state stabilization in enzyme
catalysis,2 catalytically quite active antibodies have been raised
against stable transition-state analogues (TSAs) of the corresponding
reaction.3 Similarly, molecularly imprinted polymers4 offer an
excellent possibility to mimic the active site of natural enzymes
since not only can the shape of the transition state be mimicked by
imprinting but also at the same time suitable catalytic groups and
binding sites can be introduced into the active site in a predeter-
mined orientation.5-7 However, only recently has strong catalytic
activity approaching6e or surpassing7 the activity of the catalytic
antibodies been reported.
High activity could be obtained7 in a model of carboxypeptidase
A by a combination of imprinting with a stable transition-state
analogue and introducing an amidinium function and a Zn2+ binding
site in the active cavity in a defined orientation. We report now on
the replacement of Zn2+ by Cu2+ and the use of other substrates
Figure 1. Preparation and Function of Imprinted Catalyst. (a) Molecular
which are more similar in structure to the template. These changes
imprinting with the template 2 and monomer 1 in the presence of Cu2+. (b)
Cavity after removal of the template 2. (c) Substrate 4 bound in the cavity
result in a dramatic increase in catalytic activity. At the same time,
these models show typical enzyme properties such as selectivity,
Michaelis-Menten kinetics, competitive inhibition, etc.
of b. (d) Intermediate in the catalysis. After release of products (phenol,
pyridone, CO2), structure b is regenerated.
In our earlier article on Zn2+-containing catalysts, the optimiza-
tion showed that the use of functional monomer 1 and template 2
in catalysis. In our present model, a Cu2+ center is introduced
instead of the Zn2+, although natural carboxypeptidases do not
contain copper. Cu2+ is expected to form more stable complexes
between the triamine part of 1 and the pyridine nitrogen of the
stable transition-state analogue 2,9 thus providing a better defined
conformation of the complex during the imprinting procedure (see
Figure 1a). More importantly, Cu2+ generates a more nucleophilic
OH- compared to Zn2+ for promoting the catalysis.
The polymers have been prepared in bulk by standard procedures
(see Supporting Information). The templates were removed to about
75% from the crushed polymer particles (45-125 µm). A control
polymer was prepared accordingly but without the template 2. The
catalytic activity of the imprinted polymers was determined by
investigating the rate of hydrolysis of different carbonates in HEPES
buffer (pH 7.3)/MeCN 1:1. A comparison of the catalytic activity
of the corresponding Zn2+ and Cu2+ catalysts with diphenyl
carbonate (3) as the substrate (see Table 1) showed that the best
values of the Zn2+ catalyst show an 3260-fold7 enhancement
compared to buffer solution, whereas the new copper catalyst
showed a considerably stronger enhancement of 8015-fold.
To have substrates that are more similar to the template, the new
substrates phenyl-2-pyridyl-carbonate (4) and di-(2- pyridyl)-
carbonate (5) were used. Both substrates are inherently more
reactive toward water than 3. They are better bound in the cavity
compared to 3 since the pyridyl ring interacts with the Cu2+ center
similarly to the template (see Figure 1c). Investigation of the
catalytic hydrolysis showed much stronger rate enhancements of
resulted in the most active catalysts. The monomer 1 contains a
triamine functionality for binding the metal ion and an amidinium
ion. The amidinium group is designated to bind the tetrahedral
transition state of carbonate hydrolysis similar to the catalytic role
of the guanidinium moiety of Arg 127 in carboxypeptidase A.8 The
enzyme also contains an active Zn2+ ion playing an important role
† Jilin University.
‡ Heinrich Heine University.
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J. AM. CHEM. SOC. 2004, 126, 7452-7453
10.1021/ja048372l CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Pseudo-First-Order Kinetics of the Hydrolyses of
Different Carbonates (3-5) in Presence of Catalytic Imprinted
Polymers
Information). This clearly shows a unique bifunctional nature of
the catalysis similarly as discussed, for example, by Breslow for
cyclodextrin-type catalysts for the enolization of ketones.1c The
maximum rate is obtained when the copper is in the catalytically
active aqua hydroxy form1d (Figure 1b). Suh and co-workers1e,11
investigated carefully similar catalysts with Cu2+ and guanidinium
ions for their peptidase activity. These catalysts correspond to our
nonimprinted control systems such as CPCu1.
The bifunctional catalysis proceeds via a binding as shown in
Figure 1c and an activation of the carbonyl group by the protonated
amidinium ion followed by hydroxyl attack. The reaction is further
accelerated by the preferred binding of the tetrahedral transition
state (compared to the substrate). In summary, catalysts with very
high catalytic activity and efficiency have been obtained. The high
activity and selectivity, together with strong chemical, mechanical,
and thermal stability, give these catalysts a real advantage compared
to catalytic antibodies and also provides a good alternative compared
to natural enzymes.
imprinted polymera
substrate
k
impr(min-1 b
)
kimpr/ksoln
kimpr/kcontr
PZn1,2c
PCu1,2
PCu1,2
PCu1,2
3
3
4
5
0.00235
0.00571
0.41
3264
8015
15700
76570
61.5
49.0
76.9
80.1
13.0
a
The imprinted polymers were prepared from 6.3% of a 1:1 complex
of 1 and 2 in the presence of either Zn2+ or Cu2+, 83.3% of ethylene
dimethacrylate, and 10.4% of methyl methacrylate in the presence of the
b
same volume acetonitrile/DMSO 1:1 (v/v). Hydrolyses of carbonates 3,
4, or 5 in a solution of 50 mM HEPES buffer (pH 7.3)/MeCN 1:1 at 20 °C.
(HEPES ) 2-[4-(2-hydroxy-ethyl)-1-piperazine] ethanesulfonic acid). There
are 2 mM of available active sites in relation to 1 mM substrate. kimpr is the
pseudo-first-order rate constant in the presence of the polymer, kcontr is the
rate constant in the presence of the control polymer, and ksoln is the rate
c
constant in the HEPES buffer (pH 7.3)/MeCN 1:1 solution. Data from
ref 7. For experimental details, see Supporting Information.
Table 2. Comparison of Michaelis-Menten Kinetics of Carbonate
Hydrolyses with Imprinted Polymers and Control Polymer CPCu1
Acknowledgment. This research was supported by Deutsche
Forschungsgemeinschaft and Fonds der Chemischen Industrie and
partly by the National Natural Science Foundation of China
(20174013). J.-q. Liu acknowledges a fellowship from the Alex-
ander von Humboldt Foundation. Helpful discussions with Prof.
Dr. W. Kla¨ui, Institute of Inorganic Chemistry of the Heinrich Heine
University, are greatly acknowledged.
b
kcat
Km
(mM)
k
cat/Km
polymera
substrate
(min-1
)
k
cat/kuncat
(min-1 M-1
)
PCu1,2
CPCu1
PCu1,2
CPCu1
PZn1,2c
5
5
4
4
3
28.0
0.37
2.86
0.035
0.035
0.58
6.10
0.65
4.25
2.01
110000
1450
75700
946
48200
61
4400
8.2
17.4
6900
Supporting Information Available: More data on the synthesis
of monomers and polymers as well as on kinetic investigations. This
material is available free of charge via the Internet at http://pubs.acs.org.
a
The control polymer CPCu1 was prepared in the same manner as
b
PCu1,2, but only the template 2 was omitted. Data of the Michaelis-
Menten kinetics were obtained from a plot of initial velocities of the reaction
versus the substrate concentration (see Supporting Information). Data from
c
ref 7.
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15 700- and 76 570-fold compared to the reaction in buffer/MeCN
solution. Also, the imprinting factors5a (i.e., the ratio of the catalysis
by the imprinted compared to the control polymer) are rather high,
with 76.9 and 80.1 (see Table 1). These catalytic enhancements
are the highest values reported until now for catalysts prepared by
molecular imprinting.
The better catalysis can be explained by better binding of the
substrate and a more efficient catalysis as the data from the
Michaelis-Menten kinetics show (see Table 2). Remarkable
turnover numbers of kcat ) 28.0 (5) and 2.86 (4) min-1 are obtained
for the imprinted polymers. kcat is higher than kimpr, which is
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and natural enzymes; it shows in our case values of up to 110 000,
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imprinted polymers compared to the control polymers. Both effects
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sum up to a much better catalytic efficiency kcat/Km (min-1 M-1
)
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for the imprinted polymer compared to the control by factors of
790 and 536. These differences are remarkable since the control
also contains the catalytic functional group of 1, and the excellent
catalysis relates to a very efficient imprinting procedure.
The pH rate profile for the carbonate hydrolysis in the presence
of PCu1,2 is quite different from that of PZn1,2. The Zn-containing
catalyst shows a strong increase in rate with the pH having an
inversion point at pH 7.5;7 the copper-containing one shows a bell-
shaped profile with an optimum at pH 7.2 (see Supporting
(9) Binding of 1 and 2 in the presence of Cu2+ is rather strong. Copper
complexes are prepared in MeCN/DMSO 1:1 (v/v) for solubility reasons,
though in this solvent lower association of phosphate with the amidinium
ion is seen compared to pure MeCN. The Cu2+ ion complex of 1 showed
a very high association constant of log K 15.8 compared to the Zn2+
complex with log K 9.3.
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