Published on Web 06/04/2009
Molecularly Imprinted Tunable Binding Sites Based on
Conjugated Prosthetic Groups and Ion-Paired Cofactors
Kohei Takeda, Atsushi Kuwahara, Kohei Ohmori, and Toshifumi Takeuchi*
Graduate School of Engineering, Kobe UniVersity, Nada-ku, Kobe 657-8501, Japan
Received January 20, 2009; E-mail: takeuchi@gold.kobe-u.ac.jp
Abstract: A molecular imprinting technique was applied to the construction of prosthetic group-coupled
tunable binding cavities for bisphenol A (BPA). A novel template molecule, with a structure consisting of
BPA covalently conjugated with two allyl(4-carboxyphenyl)disulfides through ester bonds (BPA-D), was
designed. After copolymerization of BPA-D with styrene and divinylbenzene, the BPA di(4-mercaptoben-
zoate) moieties were removed by reductive cleavage of the disulfide bonds, resulting in apo-type molecularly
imprinted cavities bearing two thiol residues. 4-Mercaptobenzoic acid was introduced into the apo-type
cavities as a prosthetic group through a disulfide bond by addition of 4,4′-dithiodibenzoic acid, which
transformed the apo-type cavities into holo-type cavities with two carboxylic acid residues for binding BPA.
When pyridyl prosthetic groups were introduced instead of 4-mercaptobenzoic acid by using 4,4′-
dithiodipyridine, BPA recognition ability was maintained but with improved selectivity. The binding affinity
was successfully altered several times by attaching and detaching these prosthetic groups, which showed
that the apo-type scaffold could be reused. Furthermore, noncovalent-type ion-paired cofactors could be
introduced, when the two thiol groups in the apo-type cavities were oxidized to sulfonic acid groups. When
1,2-diaminoethane (DAE) was added to the oxidized apo-type scaffold as a noncovalent-type cofactor, the
binding activity was regulated successively, depending upon the concentrations of DAE added. By using
various prosthetic groups and cofactors, the binding properties of the holo-type cavities could be tuned in
a similar way to those found in biological systems.
Introduction
binding sites inside the imprinted cavities. The apo-type
binding sites are precursors that require the presence of the
Conjugated proteins such as hemoproteins1and lipopro-
teins,2 are known to have prosthetic groups in their structures
which are conjugated by covalent bonds. Without these
prosthetic groups the molecules have no biological activity,
and the integration of such groups into conjugated proteins
allows the development of sophisticated molecular recogni-
tion-based functions in biological systems. Thus, such
prosthetic groups are considered to function as biological
switching systems, in which bioinformation is transferred by
attachment and detachment of the groups. If such systems
can be mimicked using synthetic polymer-based artificial
receptors with prosthetic groups conjugated at the binding
sites, this may lead to the development of unique bioinspired
materials. A diverse range of artificial receptors have been
reported;3 however, polymer-based molecular recognition
cavities bearing prosthetic groups have not yet been synthe-
sized. In this work, we propose artificial polymer receptors
bearing prosthetic group- or cofactor-coupling apo-type
cavities prepared by molecular imprinting, which show
predetermined molecular recognition ability through covalent
conjugation of designed prosthetic groups with apo-type
corresponding prosthetic groups or cofactors to form the
complete binding sites, which are the functioning binding
sites (holo-type binding sites). In addition, noncovalently
coupled cofactors for apo-type binding sites were also
investigated. It was expected that the binding affinity of such
conjugated sites could be tuned by replacing the prosthetic
groups or cofactors bearing alternative groups that have
different binding mode or affinity with the target molecule.
Molecular imprinting has a reputation as a promising
strategy for the preparation of tailor-made receptors for target
molecules.4 Molecular templates-often target molecules or
their derivatives-are used to assemble the functional mono-
mers surrounding them into complementary orientations, and
are then subjected to coimmobilization using cross-linkers.
After removal of the templates, tailor-made binding sites for
the target molecules are left within the synthetic polymer
matrix. Thus, the binding cavities of imprinted polymers are
constructed by self-assembly on the basis of the shapes and
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(1) Hemoglobin and Myoglobin in Their Reactions with Ligands; Antonini,
E., Brunori, M. Eds.; North-Holland: Amsterdam, 1971.
(2) Braun, V.; Wu, H. C. In New ComprehensiVe Biochemistry; Elsevier:
Amsterdam, 1995; Vol. 27, pp 319-341.
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Willey VCH: Weinheim, 2005. (b) Hof, F.; Craig, S. L.; Nuckolls, C.;
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10.1021/ja9004317 CCC: $40.75 2009 American Chemical Society
J. AM. CHEM. SOC. 2009, 131, 8833–8838 8833