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Molecularly Imprinted Polymers with Strong Carboxypeptidase A-Like Activity Combination of an Amidinium Function with a Zinc-Ion Binding Site in Transition-State Imprinted Cavities.

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Angewandte
Chemie
Enzyme Models
Molecularly Imprinted Polymers with Strong
Carboxypeptidase A-Like Activity: Combination
of an Amidinium Function with a Zinc-Ion
Binding Site in Transition-State Imprinted
Cavities**
Jun-qiu Liu and Gnter Wulff*
Mimicking of natural enzyme systems with catalytically active
arrangements in designed receptors is a challenging topic for
chemists. Notable achievements to date have been obtained
with several model systems, such as synthetic macrocyclic
compounds, molecular assemblies, catalytic antibodies, and
molecularly imprinted polymers.[1] Among these models,
molecular imprinting has been demonstrated to be an
attractive strategy for creating catalytically active binding
sites for enzyme mimetics.[2] This method should give the
opportunity to generate more complicated active sites with a
high similarity to natural systems. For this purpose, to mimic
enzyme behavior, especially esterase activity, numerous
experiments have been undertaken by imprinting with
transition-state analogues (TSAs).[3, 4] Although the earlier
attempts to mimic enzyme behavior using imprinted polymers
showed only limited catalytic efficiency, significant rate
enhancement by catalysis with imprinted polymers has been
obtained in recent years.[4] Evidently, increasing the transition-state binding, as well as correctly incorporating and
positioning the functional groups is essential for the construction of an effective enzyme model.[1–4] In our previous
work on preparing polymer catalysts by molecular imprinting,[4] amidinium functional groups were oriented in
imprinted cavities. These groups acted as anchors for binding
the tetrahedral transition states of basic ester or carbonate
hydrolysis in a similar manner to the catalytic role of
guanidinium moieties in certain catalytic antibodies[1c] and
in carboxypeptidase A.[5] The catalytic action of carboxypeptidase A involves two guanidinium groups and a Zn2+ ion. The
guanidinium moiety of Arg 127 binds the oxyanion generated
in the rate-limiting formation of the tetrahedral transition
[*] Prof. Dr. G. Wulff
Institute of Organic Chemistry and Macromolecular Chemistry
Heinrich-Heine-University D"sseldorf
Universitaetsstrasse 1, 40225 D"sseldorf (Germany)
Fax: (+ 49) 211-811-5840
E-mail: wulffg@uni-duesseldorf.de
Prof. Dr. J.-q. Liu
Key Laboratory of Supramolecular Structure and Materials
Jilin University, Changchun, 130023 (P.R. China)
[**] This research was supported by Deutsche Forschungsgemeinschaft
and Fonds der Chemischen Industrie. J.-q. Liu acknowledges a
fellowship from the Alexander von Humboldt Foundation. Helpful
discussions with Prof. Dr. W. Kl?ui, Institute of Inorganic Chemistry
of our university are greatly acknowledged.
Supporting information (synthetic and kinetic details) for this article
is available on the WWW under http://www.angewandte.org or from
the author.
Angew. Chem. Int. Ed. 2004, 43, 1287 –1287
DOI: 10.1002/anie.200352770
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1287
Communications
state.[6] The zinc ion coordinated to the amino acid residues of
His 69, Glu 72, and His 196 plays a decisive role in the catalysis
of the enzyme. Substrate specificity is brought about by a
hydrophobic pocket and another guanidinium moiety of
Arg 145.[5]
Herein we report on a novel molecularly imprinted
polymer, in which an amidinium group and a Zn2+ center
are oriented in a TSA imprinted cavity, in a similar way to the
active site in carboxypeptidase A.[5] These imprinted polymers exhibited strong rate enhancements for carbonate
hydrolysis.
Our recent studies have clearly shown that the amidinium
group of the functional monomer N,N’-diethyl-4-vinylbenzamidine 1,[4] which is responsible for binding and catalysis, can
be positioned in imprinted cavities by complexation with
carboxyl groups or with a TSA, such as a phosphate or a
phosphonate through stoichiometric noncovalent interaction.[7] A similar amidinium group was incorporated in our
new monomer 2. In this functional monomer an additional
triamine group was introduced with a defined proximity to the
amidinium group. This group gives rise to a strong threefold
coordination of the Zn2+ ion leaving the fourth coordination
site free for other ligands.[8] Coordination of the Zn2+ ion to
the amidinium group is much weaker.[9]
As with previous templates for carbonate hydrolysis (see
for example, 3)[1c, 4b] the new template 4 acts as a stable TSA
for the tetrahedral transition state of the basic carbonate
hydrolysis. One phenyl ring in 3 is replaced by a pyridine ring
in 4. This change gives the possibility of incorporating the
pyridine nitrogen atom as the fourth ligand in the Zn2+
coordination sphere (Scheme 1 a). This additional coordination permits the formation of more stable complexes between
the functional monomer and the template.[10] Furthermore,
the amidinium group and the Zn2+ ion are brought into
defined proximity to each other and to the template molecule
in the cross-linking polymerization during the imprinting
process.
Imprinted polymers were prepared in bulk by radical
initiation at 60 8C from polymerization mixtures consisting of
cross-linker, ethylene dimethacrylate (EDMA), methylme-
Scheme 1. Schematic representation of a) the molecular imprinting with template (T) 4 and monomer 2 in the presence of Zn2+, b) removal of the
template, and c,d) catalysis.
1288
2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
Angew. Chem. Int. Ed. 2004, 43, 1287 –1287
Angewandte
Chemie
thacrylate (MMA), the functional monomer 2, and either
diphenylphosphate (3) or phenyl-2-pyridyl-phosphate (4) as
template molecules in the presence of ZnCl2 in acetonitrile
(MeCN). This procedure corresponds to earlier preparations
(in absence of Zn2+) of imprinted polymers from 1 and 3.[4b]
The templates were removed from the crushed polymer
(particles 45–125 mm) with a solution of 0.1m NaOH and
MeCN (1:1, v/v), to give 73–86 % of free cavities. The zinc
ions bound at the triamine moiety during imprinting were not
removed in the process of template removal.[11] In addition, a
control polymer of identical composition to that of the
imprinted polymer but without the template was prepared.
The catalytic activity of the imprinted polymers and of the
controls was investigated in the hydrolysis of the substrate
diphenylcarbonate (5) in a 1:1 solution of 2-[4-(2-hydroxyethyl)-1-piperazine] ethanesulfonic acid (HEPES) buffer
(pH 7.3; 1 mm 5 in buffer/acetonitrile) and MeCN. To follow
the reaction kinetics, aliquots were taken at regular intervals
and checked by HPLC; phenol as one of the products of
hydrolysis was determined quantitatively. The kinetics were
measured at low conversion and were calculated as pseudo
first-order rate constants.
Table 1 shows the results obtained with different catalysts
prepared by molecular imprinting. Table 2 gives control data
for the kinetics of the hydrolysis either in the same solution
without any added polymer, or with added control polymer,
or with added monomer 2 and Zn2+. As in earlier invesTable 1: Kinetic parameters for the hydrolysis of diphenylcarbonate (5) in
presence of imprinted polymers in buffer (pH 7.3)/MeCN 1:1.[a]
Imprinted polymer[b]
Monomer Template kimpr [min 1] kimpr/
ksoln
P1,3[c]
PZn2,3
PZn2,4
–
Zn2+
Zn2+
1
2
2
3
3
4
2.80 E 10
1.30 E 10
2.35 E 10
4
3
3
455
1806
3264
kimpr/
kcontr
10.7
34.0
61.5
[a] Hydrolysis of diphenylcarbonate (5) in a solution of 50 mm HEPES
buffer (pH 7.3)/MeCN 1:1 at 20 8C. There are 2 mm of available cavities
in the polymer in relation to 1 mm substrate. All k values are the average
of at least three measurements and standard deviations were less than
5 %. kimpr is the pseudo first-order rate constant in presence of the
imprinted polymer at this catalyst to substrate ratio; kcontr the rate
constant in presence of the control polymer; ksoln is the rate constant for
hydrolysis in HEPES buffer (pH 7.3)/MeCN 1:1 solution. [b] The
composition of the monomer mixture for the preparation of the
imprinted polymers consisted of 83.3 wt % of EDMA, 10.4 wt % of
MMA, 6.3 wt % of the monomer-template 1:1 complex, and 1 wt % of
azobis(isobutyronitrile), diluted by the same weight of the porogen
MeCN. The control polymer was prepared accordingly but without
template. For details see Supplemental Material. [c] This data is taken
from ref. [4b] and is measured at 15 8C.
Table 2: Kinetic parameters for control experiments of the hydrolysis of 5
in buffer (pH 7.3)/MeCN 1:1 of and with addition of control substances.
Control
no addition
monomer 2 and Zn2+[a]
control polymer and Zn2+
k [min 1]
7.20 E 10
1.29 E 10
3.82 E 10
2+
[a] The concentration of the monomer and Zn
the available binding sites in the polymer.
Angew. Chem. Int. Ed. 2004, 43, 1287 –1287
k/ksoln
7
5
5
1.00
17.9
53.1
corresponded to that of
tigations[4] the efficiency of the catalyst was expressed by the
ratio of reaction in presence of polymer catalyst to that in neat
HEPES–MeCN solution (kimpr/ksoln). Effects that are only
connected to the imprinting in the polymer (the imprinting
effect)[2a] can be elucidated from the ratio kimpr/kcontr. In our
earlier investigations with a polymer of monomer 1 and
template 3 (P1,3) a rate enhancement of 455-fold compared
to solution and an imprinting effect of 10.7 was obtained.[4b]
Our new polymers with template 3 but with monomer 2 and
Zn2+ (PZn2,3) showed a much stronger rate enhancement of
1800-fold and an improved imprinting factor of 34.0. By using
the template 4 and the monomer 2 under Zn2+ complexation
(PZn2,4) a much better orientation in the cavity during
imprinting is possible (Scheme 1 a). This polymer gives rise to
a further improved catalytic activity of more than 3200-fold
and an imprinting factor of 61.5 (Table 1). This strong rate
enhancement is in accord with the postulated cooperativity
between the amidinium group and the Zn2+ ion. The high
activity can best be explained by the complexation of the
pyridine group of template 4 that stabilizes a favorable
conformation during imprinting. To establish the influence of
the functional groups in the polymer on the catalysis, the
soluble monomer 2 with Zn2+ was investigated in HEPES–
MeCN solution. Their impact on catalysis is only small, a rate
enhancement of 18-fold is observed in comparison to reaction
in neat solution (Table 2).
As with natural enzymes the catalysis of carbonate
hydrolysis by PZn2,4 shows typical Michaelis–Menten kinetics. A plot of initial velocities of the reaction versus the
substrate concentration shows at first an increase in the rate
of reaction with increasing substrate, the rate then levels off.
At higher substrate concentration, when all the active sites
are occupied, the rate remains constant which means it is
zero-order with respect to carbonate concentration (saturation kinetics). From this data the Michaelis constant Km =
2.01 mm and the turnover number kcat = 0.035 min 1 can be
calculated (Table 3). kcat is higher than kimpr which is
Table 3: Data for Michaelis–Menten kinetics of hydrolysis of 5 in
presence of PZn2,4.
Km
2.01 mm
kcat
0.035 min
kcat/Km
1
kcat/ksoln
1
17.0 min m
1
6900
determined for only one ratio of catalyst to substrate. The
ratio kcat/kuncat is used to express the catalytic activity of
antibodies and natural enzymes. In our case, by using ksoln as
kuncat for PZn2,4 a kcat/ksoln value of 6900 is obtained. These are
the highest values that have been obtained for imprinted
polymers of this type. These values are considerably higher
than those for catalytic antibodies for which kcat/kuncat = 810[12]
has been reported for carbonate hydrolysis.
With regard to the mechanism it can be assumed that the
Zn2+ ion is tetrahedrally coordinated by three amine groups
and one water molecule.[13] This Zn2+-bound water molecule
is ionized through base catalysis that is facilitated by the
amidinium group. The Zn2+ bound water is more acidic than
usual water (Scheme 1 b).[13] The pH–rate profile for the
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2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1289
Communications
carbonate hydrolysis by PZn2,4 is shown in Figure 1. The
reaction is strongly dependent on the pH value, it has an
inversion point at pH 7.5 which is in agreement with data for
Zn2+-bound water[14] and is also in reasonable accord with the
pKs value of 7.4 obtained by potentiometric titration of the
catalyst PZn2,4.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
Figure 1. pH profile of the rate of the hydrolysis of 5 in the presence of
1 mm PZn2,4 active sites. Buffer solution/MeCN 1:1. Buffer used were
MES (pH 5.5–6.5), HEPES (pH 7.3–8.1), CHES (pH 8.4–9.5).
k = 10 4 min 1. MES = 2-(4-morpholinyl) ethanesulfonic acid,
CHES = 2-(cyclohexylamino) ethanesulfonic acid.
[9]
The catalysis occurs as follows: After binding the substrate (Scheme 1 c) and polarization of the carbonyl group by
the amidinium ion a nucleophilic attack of the Zn2+-bound
OH ion results, via the transition state, in the tetrahedral
intermediate (Scheme 1 d). The reaction is accelerated by the
preferred binding of the transition state (compared to the
substrate) in the active site of the catalyst (transition-state
stabilization). In this way the activation energy of the reaction
is reduced. After this rate-determining step one molecule of
CO2 (as HCO3 ) and two molecules of phenol are formed in a
number of further faster reaction steps regenerating the
catalyst (Scheme 1 b).
In summary, this novel catalytic system shows a remarkable rate enhancement of kcat/ksoln = 6900. This is the first
example in which molecularly imprinted catalysts are clearly
more efficient than catalytic antibodies. In earlier work[4b] the
efficiency of antibody catalysis[15] could be reached in the case
of carbamate hydrolysis. These new catalysts are very
promising for broad application since in contrast to antibodies
molecularly imprinted polymers show a much higher chemical, mechanical, and thermal stability, can easily be prepared,
and have the potential of simple variation of structure.[16]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
Received: September 2, 2003 [Z52770]
.
Sci. 1994, 19, 9 – 14; f) K. J. Shea, Trends Polym. Sci. 1994, 5,
166 – 173.
For reviews see: a) G. Wulff, Chem. Rev. 2002, 102, 1 – 27; b) O.
Ramstrom, K. Mosbach, Curr. Opin. Chem. Biol. 1999, 3, 759 –
764.
For examples see: a) D. K. Robinson, K. Mosbach, J. Chem. Soc.
Chem. Commun. 1989, 969 – 970; b) B. Sellergren, R. N. Karmalkar, K. J. Shea, J. Org. Chem. 2000, 65, 4009 – 4027; c) K.
Ohkubo, Y. Urata, Y. Honda, Y. Nakashima, K. Yoshinaga,
Polymer 1994, 35, 5372 – 5374.
a) G. Wulff, T. Gross, R. Schoenfeld, Angew. Chem. 1997, 109,
2049 – 2052; Angew. Chem. Int. Ed. Engl. 1997, 36, 1961 – 1964;
b) A. G. Strikovsky, D. Kasper, M. Gruen, B. S. Green, J. Hradil,
G. Wulff, J. Am. Chem. Soc. 2000, 122, 6295 – 6296; c) M.
Emgenbroich, G. Wulff, Chem. Eur. J. 2003, 9, 4106 – 4117.
D. W. Christianson, W. N. Lipscomb, Acc. Chem. Res. 1989, 22,
62 – 69.
M. A. Phillips, R. Fletterick, W. J. Rutter, J. Biol. Chem. 1990,
265, 20 692 – 20 698.
G. Wulff, K. Knorr, Bioseparation 2002, 10, 257 – 276.
For the cooperativity of guanidinium and metal ions in recent
enzyme model studies for small molecule enzyme models, see:
H. Haddou, J. Sumaoka, S. L. Wiskur, J. F. Anderson, E. V.
Anslyn, Angew. Chem. 2002, 114, 4186 – 4188; Angew. Chem. Int.
Ed. 2002, 41, 4014 – 4016. For a macromolecular enzyme model,
see: B.-B. Jang, K.-P. Lee, D.-H. Min, J. Suh, J. Am. Chem. Soc.
1998, 120, 12 008 – 12 016
In case of guanidinium functionalities: S. Aoki, K. Iwaida, N.
Hanamoto, M. Shiro, E. Kimura, J. Am. Chem. Soc. 2002, 124,
5256 – 5257.
The association constant for a 1:1 complex of monomer 2 and
template 3 was determined to be 5.6 H 103 m 1 in CDCl3 by
1
H NMR spectroscopic titration. Zn2+ complexes with the
triamine part of the monomer 2 showed a binding constant of
log K 9.3 determined by potentiometric titration in aqueous
solution. Although we could not directly measure the complexation constant of 2 with 4 in the presence of Zn2+ions, it should
be expected that the complexation of 2 with 4 is stronger than
that of 1 and 3 (K = 4.6 H 103 in MeCN at 25 8C)[7b] owing to the
multiple binding of amidinium ion with phosphate as well as of
the Zn2+ ion with the pyridine ring in template 4.
The amount of zinc ions in the imprinted polymers was
determined by elemental analysis. Only a slight loss of Zn2+
ions was detected during spitting off the template from the
imprinted polymer, for example, in PZn2,4 the zinc-ion content
was 0.84 wt % prior to, and 0.82 wt % after template removal.
J. W. Jacobs, P. G. Schultz, R. Sugasawara, M. Powell, J. Am.
Chem. Soc. 1987, 109, 2174 – 2176.
R. P. Sheridan, L. C. Allen, J. Am. Chem. Soc. 1981, 103, 1544 –
1550.
E. Kimura, T. Shiota, T. Koike, M. Shiro, M. Kodama, J. Am.
Chem. Soc. 1990, 112, 5805 – 5811.
P. Wentworth, A. Datta, S. Smith, A. Marshall, L. J. Partridge,
G. M. Blackburn, J. Am. Chem. Soc. 1997, 119, 2315 – 2316.
In antibodies functional groups can not be easily varied, for
example, it is difficult to introduce a Zn2+ center in defined
orientation by using site-directed mutagenesis.
Keywords: enzyme models · heterogeneous catalysis ·
molecular imprinting · polymers · reaction kinetics
[1] For reviews see: a) A. Kirby, Angew. Chem. 1996, 108, 770 – 790;
Angew. Chem. Int. Ed. Engl. 1996, 35, 707 – 724; b) R. Breslow,
S. D. Dong, Chem. Rev. 1998, 98, 1997 – 2011; c) R. A. Lerner,
S. J. Benkovic, P. G. Schultz, Science 1991, 252, 659 – 667; d) G.
Wulff, Angew. Chem. 1995, 107, 1958 – 1979; Angew. Chem. Int.
Ed. Engl. 1995, 34, 1812 – 1832; e) K. Mosbach, Trends Biochem.
1290
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Angew. Chem. Int. Ed. 2004, 43, 1287 –1290
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site, cavities, carboxypeptidase, ion, molecular, state, transitional, amidinium, zinc, imprinted, like, polymer, strong, activity, function, binding, combinations
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