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ATP-binding peptide-hydrogel composite synthesized by molecular imprinting on beads

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DOI: 10.1515/molim-2015-0008
Abstract
Molecular imprinting has been recognized as a useful technique to produce synthetic mimics of functional proteins, such as antibodies and enzymes. However, only a few studies have examined peptides as starting materials for synthesizing molecularly imprinted polymers in spite of the expectation that peptides would be suitable materials for realizing water-compatibility and proteinlike functions. In this study, molecular imprinting was performed using a vinyl-end-capped on-beads-peptide as functional monomer to produce an on-beads-peptide hydrogel composite selective for ATP; the on-beadspeptide peptide, of which sequence was designed to possess both an adenine-recognition site and phosphate recognition site, was co-polymerized with NIPAM and BIS in the presence of ATP as a template species. The resultant ATP-imprinted composite showed 14-times higher affinity and an enhanced selectivity towards ATP, suggesting that the peptide conformation, i.e. a mutual orientation of the two binding sites, was pre-organized and immobilized in a manner where the ATP binding is more favored.
Figures
Mol. Impr. 2015; 3: 65–70
interaction provided by the immobilized functional groups
[1]. Molecular recognition phenomena displayed by the
imprinted polymers have successfully been applied for
separation [2], sensing [3,4], catalysis [5] and medication
[6].
For synthesizing highly selective molecularly
imprinted polymers, it is of critical importance to adopt
appropriate starting materials that can be bound firmly
with a given template species. To date, a wide variety of
starting materials have been studied, including vinyl
monomers, inorganic monomers and biopolymers such
as proteins [7]. Recently we have examined a peptide as
a starting material for MI because a peptide is a naturally
occurring molecular recognition element [8,9]; a variety of
peptides and proteins are known to be engaged in highly
precise molecular recognition of biological systems,
taking advantage of a feature that a peptide possesses
various functionalities within a single molecule. Peptides
were considered to be suitable as starting materials of MI
also in an aspect of availability: peptides with affinity to
a given molecule can be obtained by several conventional
approaches such as rational design [10,11] and random
selection (combinatorial chemistry) [12]. With an envision
that a peptide would be allowed by MI to take and preserve
a conformation that is favorable for binding with a target
species, we examined two kinds of strategies of MI with
peptides. One utilized glutaraldehyde, a well-known
cross-linker for
proteins, to cross-link lysine residues of an on-beads-
peptide. The cross-linking was performed in the presence
of a template molecule, ATP, and subsequent removal of
ATP resulted in on-beads peptide showing a 1.75 times-
larger binding constant (J. Matsui et al., unpublished
data). The other utilized bifunctional reagents, such as
dimethyl suberimidate and dimethyl adipimidate, to
cross-link lysine residues; dimethyl adipimidate was
found to possess a more appropriate length of spacer to
form an ATP-fitting binding site [8].
Although the both strategies appeared to be
successful for enhancing the affinity of the ATP-binding
peptide, the cross-linking employed in these studies was
DOI 10.1515/molim-2015-0008
Received August 3, 2015; accepted December 28, 2015
Abstract: Molecular imprinting has been recognized as a
useful technique to produce synthetic mimics of functional
proteins, such as antibodies and enzymes. However, only
a few studies have examined peptides as starting materials
for synthesizing molecularly imprinted polymers in
spite of the expectation that peptides would be suitable
materials for realizing water-compatibility and protein-
like functions. In this study, molecular imprinting was
performed using a vinyl-end-capped on-beads-peptide
as functional monomer to produce an on-beads-peptide
hydrogel composite selective for ATP; the on-beads-
peptide peptide, of which sequence was designed to
possess both an adenine-recognition site and phosphate
recognition site, was co-polymerized with NIPAM and BIS
in the presence of ATP as a template species. The resultant
ATP-imprinted composite showed 14-times higher affinity
and an enhanced selectivity towards ATP, suggesting that
the peptide conformation, i.e. a mutual orientation of the
two binding sites, was pre-organized and immobilized in
a manner where the ATP binding is more favored.
Keywords: peptide, hydrogel, ATP
1 Introduction
Molecular imprinting (MI) is a methodology to pre-
organize and immobilize plural functional groups using
a target molecule as a template, producing polymeric
materials which, after removal of the template, can
selectively capture the target molecule via multiple-point
Short communication Open Access
© 2015 Ayana Takata et al., published by De Gruyter Open.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License.
Ayana Takata, Kenji Usui, Jun Matsui*
ATP-binding peptide-hydrogel composite
synthesized by molecular imprinting on beads
*Corresponding author Jun Matsui, Department of
Nanobiochemistry, FIRST, Konan University, 7-1-20 Minatojima-
minami-machi, Chuo-ku, Kobe 650-0047, Japan,
Email: matsui@konan-u.ac.jp
Ayana Takata, Kenji Usui, Department of Nanobiochemistry, FIRST,
Konan University, 7-1-20 Minatojima-minami-machi, Chuo-ku, Kobe
650-0047, Japan
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66  Ayana Takata, et al.
only cyclization within a peptide or tethering between
peptide chains rather than creating three-dimensional
network. Thus, in this study, a new strategy of peptide-
based MI was examined, whereby an on-beads-peptide
was imbedded in three-dimensional polymer network,
a NIPAM-BIS hydrogel [13], in the presence a template
molecule ATP (Fig. 1).
2 Experimental
2.1 Materials
Solid-phase polymer support for peptide synthesis
(TentaGel S NH2 Resin) was purchased from Rapp
Polymere GmbH (Germany). Fmoc amino acids were
purchased from Watanabe Chemical Industries (Japan).
Acetonitrile, acryloyl chloride, N-methylpyrrolidone
(NMP), piperidine, sodium chloride, trifluoroacetic acid
(TFA), m-cresol, thioanisole, adenosine 5’-triphosphate
disodium salt (ATP), adenosine 5’-diphosphate sodium
salt (ADP), adenosine 5’-monophosphate sodium salt
(AMP), cytidine 5’-triphosphate disodium salt (CTP) and
guanosine 5’-triphosphate disodium salt (GTP) were
purchased form Wako Pure Chemical Industries (Japan).
2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid
(HEPES) and ethylenediamine-N,N,N’,N’-tetraacetic acid
(EDTA), disodium salt, dihydrate were obtained from Dojin
Laboratories (Japan). N-Isopropylacrylamide (NIPAM)
was obtained from Tokyo Chemical Industry (Japan).
2.2 Synthesis of a vinyl-end-capped
on-bead-peptide
The synthesis of an on-bead-peptide (Fmoc-GLYKEGGG-
KGRG-Resin) (Fig. 2) was conducted following a conven-
tional Fmoc synthesis procedure using TentaGel S NH2
Resin [14]. After deprotecting the Fmoc group using 25%
piperidine NMP solution, the resin was treated with 1%
acryloyl chloride NMP solution for a vinyl-end-capping.
After deprotecting the side-chain-protecting groups using
m-cresol, thioanisole and TFA, the resin was washed with
acetonitrile and dried in vacuo.
Figure 1: Schematic representation of molecular imprinting with on-beads-peptide for producing ATP-selective on-beads-peptide-hydrogel
composite. The peptide which bears a vinyl group at N-terminus was synthesized by a conventional Fmoc solid-phase protocol, and (A) was
allowed to form a complex with ATP, and (B) co-polymerized with NIPAM and BIS. After removal of ATP (C), the composite could possess ATP-
binding sites which are expected to show higher ATP affinity due to pre-organized and immobilized peptide conformation.
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ATP-binding peptide-hydrogel composite synthesized by molecular imprinting on beads  67
2.3 Synthesis and evaluation of molecularly
imprinted peptide-hydrogel composite
Into a polymerization mixture, consisting of N-isopropyla-
crylamide (NIPAM, 620 mg), N,N’-methylenebisacrylamide
(BIS, 53 mg) as a cross-linker, ATP (15.2 mg, 30 µmol ) as
a template molecule, and 3.0 mL of 10 mM HEPES buffer
(10 mM NaCl, 1 mM EDTA),µmol), the mixture was sus-
pension immersed the resin (115 mg, 13 µmol) was placed
at 20°C for 12 h. After APS and TEMED were added for
initiating polymerization, the suspension was incu-
bated with a rotary-type mixer at 20°C for further 12 h.
The obtained on-beads-peptide hydrogel composite was
repeatedly washed with 10 mM HEPES buffer including
a higher concentration of salt (1 M NaCl, 1 mM EDTA)
and water to remove the template ATP, dried and roughly
crushed. Another kind of peptide-hydrogel compo-
site was also prepared as a control in a similar manner
without addition of ATP, in which peptide chains were
supposed to be immobilized in random coil.
2.4 Binding test for evaluating the affinity
for ATP
Each xerogel (10 mg) was immersed in 1.0 mL of ATP
solution (0−0.5 mM, 100 mM HEPES, pH 7.0) at 4°C for
12h. Supernatants were analyzed by reversed-phase HPLC
to quantify the free ATP, thus determining the amount of
ATP bound to the on-beads-peptide hydrogel composite.
The saturation binding curves obtained (Fig. 3) was
further analyzed by Scatchard plot to estimate a binding
constant, assuming that all the peptide chains possess the
identical affinity for ATP with stoichiometry of 1:1 [15].
3 Results and Discussion
3.1 Design of a peptide sequence
In this study, ATP was chosen as a target molecule, of which
various materials capable of recognition and sensing have
been devised [16-19]. The sequence of the peptide used in
this study was GLYKEGGGKGRG, as shown in Fig. 2, which
consists of a phosphate-recognizing sequence (KGRG)
and an adenine-recognizing sequence (GLYKE) [8]. The
former was selected from a combinatorial library using a
fluorescent-labeled ATP-based competition assay and the
latter was rationally designed according to a sequence
of a naturally occurring adenine-recognizing protein
[20]. The length of a spacer sequence (GGG) linking the
two recognition sites were optimized for higher affinity
towards ATP [8]. Synthesis of the peptide was conducted
by a conventional Fmoc method except that the end
capping was conducted using acryloyl chloride instead
of conventional acetyl chloride to introduce a vinyl group
Figure 2: Sequence of a peptide used for molecular imprinting in this study, which consists of an adenine-recognition site and a phosphate
recognition site.
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68  Ayana Takata, et al.
at N-terminus for allowing radical polymerization. As a
polymer support for peptide synthesis, TentaGel S NH2
resin was adopted because it is suitable for leaving the
synthesized peptide uncleaved.
3.2 Molecular imprinting with an
on-beads-peptide
MI scheme is shown in Fig. 1: (A) the peptide with a
vinyl group at N-terminus is allowed to bind with ATP
in the pre-polymerization mixture for pre-organizing the
peptide conformation so that it can be more favorable
for ATP binding, and (B) incorporated in hydrogel by
co-polymerization with NIPAM and BIS for preserving
the conformation. In the resultant on-beads-peptide
hydrogel composite, it is expected that a phosphate-
recognition site and an adenine-recognition site are
immobilized with a mutual orientation favorable for
ATP binding. After removal of the template (C), the two
recognition sites are expected to work cooperatively to
capture ATP.
As described in the experimental section, the obtained
on-beads-peptide was directly used for subsequent MI
process without scission from the support beads. This is
because using the isolated peptide resulted in a peptide-
hydrogel composite showing no affinity for ATP regardless
of whether it was imprinted or not. The lack of ATP affinity
of the isolated-peptide-hydrogel composite suggests that
the peptide is required to be located near the solid support
surface, which could provide hydrophobic environment
favorable for electrostatic interaction and hydrogen
bonding between the peptide and ATP [21,22].
3.3 Affinity for ATP
The estimated binding constant (Ka) and theoretical
number of binding sites (Bmax) for ATP are shown in
Fig.3: the ATP-imprinted hydrogel composite (IG) showed
a higher affinity with a fewer number of binding sites, as
compared with the non-imprinted blank one (BG). The
higher affinity could be explained by the possible effects
of template-directed pre-organization and immobilization
of the peptide conformation; the adenine-recognition
site and phosphate-recognition site were located with
appropriate distance and orientation to each other to
cooperatively uptake ATP. Concentration of proton in
the polymerization mixture might also have affected the
binding property of the immobilized peptide chains,
because it is known that the proton can play an important
role in controlling the conformation of cross-linked
polymer [23]. In our ATP-imprinting, the polymerization
mixture for IG was more acidic (pH = 4.74) than that
for BG (pH = 7.97), which could affect the protonation/
deprotonation of acidic moieties, such as Tyr and Glu, and
therefore might have brought the difference in peptide
conformation between IG and BG.
Figure 3: Saturation binding curves for ATP-imprinted on-beads-peptide hydrogel composite (IG) and non-imprinted blank hydrogel compo-
site (BG).
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ATP-binding peptide-hydrogel composite synthesized by molecular imprinting on beads  69
The fewer binding sites suggest that the presence of
the template could also deprive the ATP-binding ability
of on-beads-peptide. Although no clear explanation
can be made with the present data, it would be possible
that complexation with the template, which possesses
a hydrophobic adenine-base, tends to draw the peptide
chains too close to the resin surface, leaving no space
enough for accommodating ATP. Another possible
cause is the formation of a covalent bond between the
template ATP and vinyl-functionalized peptide, such as
Michael reaction of an amino group with a vinyl group
[24]. In the putative resultant conjugate, the peptide
could lose the ATP binding ability due to the covalently
bound ATP that competitively occupies the binding site
of the peptide.
3.4 Selectivity for ATP
Affinity for ADP, AMP, GTP and CTP was also evaluated
on the imprinted and non-imprinted composites (Fig. 4).
For these nucleotides, the ATP-imprinted composite (IG)
showed a slightly lower affinity, as compared with the non-
imprinted composite (BG), supporting the assumption
that the enhanced affinity for ATP can be attributed to the
pre-organized and immobilized peptide.
4 Conclusion
This study is still in a preliminary stage. Therefore, the
number of vinyl groups that are introduced to a peptide
has not been optimized; one vinyl group perpeptide chain
was introduced at the N-terminus. Using an amino acid
with a vinyl group as a side-chain, more vinyl groups can
be introduced to a peptide, and such peptide would enable
more rigid immobilization of a pre-organized conformation.
Thus, optimization of the number of vinyl groups for higher
affinity and selectivity is ongoing in our group.
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Figure 4: Selectivity of ATP-imprinted on-beads-peptide hydrogel composite (IG) and non-imprinted blank hydrogel composite (BG).
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70  Ayana Takata, et al.
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