Modulation of imprinting efficiency in nanogels with catalytic activity in the Kemp elimination

Article abstract of DOI:10.1002/jmr.2180

The interactions between the template and the functional monomer are a key to the formation of cavities in the imprinted nanogels with high molecular recognition properties. Nanogels with enzyme-like activity for the Kemp elimination have been synthesized

Full text of DOI:10.1002/jmr.2180

Special Issue Article
Received: 18 July 2011,
Revised: 11 January 2012,
Accepted: 11 February 2012,
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jmr. 2180
Modulation of imprinting efficiency in nanogels
†
with catalytic activity in the Kemp elimination
Paolo Bonomi, Ania Servant and Marina Resmini*
The interactions between the template and the functional monomer are a key to the formation of cavities in the
imprinted nanogels with high molecular recognition properties. Nanogels with enzyme-like activity for the Kemp
elimination have been synthesized using 4-vinylpyridine as the functional monomer and indole as the template.
The weak hydrogen bond interaction in the complex is shown to be able to induce very distinctive features in the
cavities of the imprinted nanogels. The percentage of initiator used in the polymerisation, ranging from 1% to 3%,
although it does not have a substantial effect on the catalytic rate, reduces considerably the imprinting efficiency.
The alteration of the template/monomer ratio is also investigated, and the data show that there is considerable
loss of imprinting efficiency. In terms of substrate selectivity, a number of experiments have been performed using
5
-Cl-benzisoxazole as substrate analogue, as well as 5-nitro-indole as template analogue for the preparation of a
different set of nanogels. All the kinetic data demonstrate that the chemical structure of the template is key to
the molecular recognition properties of the imprinted nanogels that are closely tailored and able to differentiate
among small structural changes. Copyright © 2012 John Wiley & Sons, Ltd.
Keywords: imprinted polymers; enzyme mimic; kemp elimination; catalytic nanogels; enzyme-like catalysis; imprinting efficiency
weak hydrogen bond interactions between the template and
INTRODUCTION
monomer,shown in Figure 1, can successfully lead to imprinted
nanogels able to function as catalysts in water and that the
addition of surfactant like Tween 20 has an effect on the catalytic
activity as well as the morphology of the nanoparticles.
Enzymes are nature’s tools that have evolved to maximize their
ability to catalyse a chemical reaction with high efficiency (Kirby,
1
996; Flavin and Resmini, 2009). There have been considerable
efforts to develop systems able to mimic the activity of enzymes,
not only to gain a better understanding of their mechanisms but
also to prepare novel catalysts that can complement enzymes’
vast repertoire (Meekel et al., 1996; Wulff, 2002). Molecular
imprinting (Wulff and Sarhan, 1972; Carter and Rimmer, 2002),
where a template is used in combination with monomers in a
casting-type procedure to generate recognition sites, has been
successfully used as a viable approach for the generation of
polymers with enzyme-like activities (Wulff, 2002; Alexander
et al., 2006). The application of this approach in recent years to
microgels and nanogels, polymeric materials with a cross-linked
but flexible matrix (Graham and Cameron, 1998), has increased
the potential for applications (Markowitz et al., 2000; Ye and
Mosbachm, 2001; Spegel et al., 2003; Fernandez-Barbero
et al., 2009; Lyon et al., 2009). These materials have been
shown to catalyse not only simple reactions, such as ester
and carbonate hydrolysis (Maddock et al., 2004; Pasetto
et al., 2005; Pasetto et al., 2009), but also more energetically
challenging reactions such as the aldol condensation (Carboni
et al., 2008). In all these cases, the key template–monomer
interaction was based on strong ionic bonds or reversible co-
valent interactions (Wulff et al., 1977; Liu and Wulff, 2004;
Mayes and Whitcombe, 2005).
The Kemp elimination is the base-catalysed isomerization of
1,2-benzisoxazole (1) resulting in the formation of a single
chromophoric product 2-cyanophenol (3), occurring via a single
concerted one-step mechanism.
This reaction is not catalysed by enzymes, and a number of
different approaches have been used to develop novel catalysts,
such as synzimes, antibodies, and natural coils (Kemp et al.,
1975). Our initial work focussed on the synthesis of nanogels,
which were never used before for this reaction, and on the use
of surfactants to enhance the catalytic activity. Despite the
interesting results obtained thus far, further work is necessary
to fully understand how molecular recognition and catalytic
efficiency can be tuned for the required applications. There is a
clear need to understand how the different parameters influence
the morphology and characteristics of the nanogels and
ultimately the catalytic changes in activity. It is important to
underline that although considerable work has been carried
* Correspondence to: M. Resmini, School of Biological and Chemical Sciences,
Queen Mary University of London, Mile End Road, London E1 4NS, UK.
E-mail: m.resmini@qmul.ac.uk
†
This article is published in Journal of Molecular Recognition as part of the
We recently reported the synthesis and characterisation of
water-soluble imprinted nanogels with enzyme-like activity in
the Kemp elimination reaction of 1,2-benzisoxazole, using indole
as the template and 4-vinylpyridine (Ikeda et al., 1983; Yilmaz
and Kucukyavuz, 1993) as the functional monomer, as shown
in Scheme 1 (Servant et al., 2011). We were able to show that
special issue on MIP2010: The Future of Molecular Imprinting, edited by David
A. Spivak (Louisiana State University) and Kenneth J. Shea (University of
California, Irvine).
P. Bonomi, A. Servant, M. Resmini
School of Biological and Chemical Sciences, Queen Mary University of London,
Mile end road, London E1 4NS, UK
J. Mol. Recognit. 2012; 25: 352–360
Copyright © 2012 John Wiley & Sons, Ltd.

IMPRINTED NANOGELS AS ENZYME MIMICS
B
H
H
B
X
X
X
BH
N
O
N
N
O
O
1
2
: X = H
: X = Cl
3
4
: X = H
: X = Cl
Scheme 1. Formation of products 3 and 4 from compounds 1 and 2 through the Kemp elimination reaction.
solvents used for polymerizations or analytical experiments
were of analytical grade. Sodium carbonate was purchased
from BDS. 1,2-Benzisoxazole (98%) was purchased from
Aldrich Chemical Co. (Gillingham, Dorset, UK).
1
13
Proton ( H NMR) and carbon ( C NMR) were recorded at
00 MHz on a Brucker Avance III 400 spectrometer. Chemical
4
N
shifts (d) are given in part per million (ppm), and coupling
constants (J) are given in Hertz (Hz). UV–vis samples were ana-
lysed using a Varian Cary 300 BIO UV–vis spectrophotometer,
equipped with an internal thermostat.
H
N
Figure 1. Hydrogen bond interaction between the template indole and
the functional monomer 4-vinylpyridine.
Interactions between templates (indole and 5-NO -indole)
and d5-pyridine
2
Stock solutions of indole and 5-nitroindole were prepared
in different deuterated solvent systems such as d6-dimethylsulfoxide
out with imprinted polymers with binding properties (Spivak,
(
DMSO), d3-acetonitrile, CD Cl , CDCl , d8-toluene, and D O at a
2 2 3 2
2
005), the recognition characteristics and properties of catalytic
concentration of 85.4 mM. Spectra of indole in these different
solvents were recorded. Increasing quantities of d5-pyridine from
zero to seven equivalents were added to the indole solutions,
and the spectra of indole and d5-pyridine at different equivalents
imprinted polymers have yet to be fully studied and that the
conclusions obtained in one field cannot be directly transferred
to the other one.
In the present work, we report our results on the optimisation
and in-depth characterisation of the catalytic activity of
imprinted nanogels in the Kemp elimination. Our data indicate
that the cross-linker concentration and the template/monomer
ratio can influence the catalytic activity and the imprinting effi-
ciency of the nanogels. The extensive and detailed kinetic stud-
ies using substrate analogues and template analogues with
imprinted and non-imprinted nanogels demonstrate that even
when using weak hydrogen bonds for the imprinting complex,
the chemical structure of the template plays a key role in deter-
mining the recognition characteristics and the catalytic efficiency
of the polymeric matrix.
H
were recorded. The chemical shift variation Δd of the proton
bound to the indole nitrogen was calculated and plotted against
deuterated pyridine concentration. The data obtained could be
fitted using Sigma plot 8 software into a hyperbola, and the bind-
ing constant was then determined using the ligand binding
equation as follows:Determination of the binding constant by
NMR titration:
A ꢀ x
Δd ¼
H
KD þ B ꢀ x
where ΔdH is the variation of chemical shift of the nitrogen
proton of indole, x is the concentration of d5-pyridine, KD is
dissociation constant of the complex formation, and A and B
are constants.
EXPERIMENTAL SECTION
Chemicals and materials for substrate and nanogel synthesis
4
%
-Vinylpyridine (%), ethylene glycol dimethacrylate (EGDMA, 98
), indole (98%) and 5-nitroindole (98%), hydroxylamine
The same procedure as for indole was applied to 5-nitroindole.
hydrochloride (98%, ACS reagent) were purchased from Aldrich
Chemical Co. (Gillingham, Dorset, UK). 4-Vinylpyridine and
EGDMA were purified by vacuum distillation before use. 2,2′-
Azobisisobutyronitrile (AIBN, 98%) was purchased from Acros
Fisher Scientific UK (Loughbrough, Leicestershire) and recrystal-
lized from methanol before use. 2-Hydroxy-5-nitrobenzaldehyde
Synthesis of the substrate 5-chloro-benzisoxazole
Synthesis of 5-Cl-salicylaldoxime
–
3
Aqueous sodium acetate (1.2 ml, 5.75 ꢁ 10 mol, 1.9 eq.) was
added to a warm solution of 5-chlorosalicyladehyde (472 mg,
–
3
3.05 ꢁ 10 mol, 1.0 eq.) and hydroxylamine hydrochloride
–3
(97%) was purchased from Fluka (Gillingham, Dorset, UK).
(418 mg, 6.1 ꢁ 10 mol, 2.0 eq.) to a solution of acetonitrile/water
(60% acetonitrile, 6 ml). The clear solution was heated at reflux for
3 hr. The clear orange solution was cooled down to room temper-
ature, and 3 ml of water was added to the mixture. The solution
Anhydrous 1,2-dichloroethane (99%) was purchased from
Aldrich Chemical Co. (Gillingham, Dorset, UK). Dialysis mem-
branes were purchased from Medicell International Ltd, 3500/2
size, 22.0-mm diameter, molecular weight cut-off 3500 Da.
All the deuterated solvents for NMR studies were pur-
chased from Cambridge Isotope Laboratories. Inc. Other
ꢂ
was then kept at 4 C in an ice bath. The formation of light yellow
needles was observed. The solid was filtered off, washed with wa-
ter, and then freeze dried. The yield was 76.4%.
J. Mol. Recognit. 2012; 25: 352–360
Copyright © 2012 John Wiley & Sons, Ltd.
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P. BONOMI ET AL.
1
H-NMR (400 MHz, CDCl ): d 6.93 (d, J = 8.8 Hz, 1H), 7.16
where Ve1 and Ve2 are the volume at the first and second
equivalent point, respectively, and V is the volume of the poly-
mer solution before titration.
3
(
8
d, J = 2.3, 1H), 7.25 (dd, J = 8.8 Hz, J = 2.3 Hz, 1H), 7.6 (br, 1H),
.16 (s, 1H), 9.75 (br, 1H)
1 2
Synthesis of 5-chlorobenzisoxazole
RESULTS AND DISCUSSION
The oxime (410 mg, 2 mmol) was dissolved in anhydrous tetrahy-
drofurane (THF) (5 ml), and 2 ml of a solution of trichloroacetyl
isocyanate in THF (2 M, 4 mmol) was added under anhydrous
conditions. The solution was stirred for 10 min before anhydrous
potassium carbonate (286mg, 2.7mmol) was added. The mixture
was stirred for 30 min before adding 7 ml of 0.1M HCl. The solution
was stirred for an additional 10 min. THF was then evaporated, and
the aqueous phase was extracted with dichloromethane. The com-
bined organic phases were dried with magnesium sulphate. After
evaporation of the dichlormethane, a light yellow solid was recov-
ered. The compound was recrystallized twice with acetonitrile and
water. A mass of 163.2 mg of the product as a white solid was re-
Our previous work demonstrated that the choice of pH and the
percentage of organic solvent in the reaction solutions, together
with the addition of surfactants (Ashbaugh et al., 2000; Barreiro-
Iglesias et al., 2001; Barreiro-Iglesias et al., 2003), had a direct
impact on the imprinting efficiency of the catalytic nanogels,
defined as the ratio between the rate constants for the imprinted
nanogels and those for the non-imprinted nanogels, and on the
morphology of the nanoparticles (Servant et al., 2011). This
prompted further studies aiming to investigate the influence of
polymerisation parameters such as initiator content and
functional monomer/template ratio on the properties of the
nanogels. It was expected that these parameters would have a
direct effect on the polymer morphology and on the concentra-
tion of active site (Resmini et al., 2000) and therefore would alter
the catalytic activity of the nanoparticles. There is very little
knowledge and detailed information on how the efficiency and
molecular recognition properties of these materials can be mod-
ulated to obtain novel catalysts with enzyme-like activities.
covered. The yield was 44%.
1
H-NMR (400 MHz, CDCl ): d 7.53 (dd, J = 8.9 Hz, J = 1.8 Hz,
3
1
2
1
H), 7.57(d, J = 8.9 Hz, 1H), 7.72 (d, J = 1.8, 1H), 8.68 (s, 1H).
1
Synthesis of the 5-chloro-2-cyanophenol
A solution of 5-chlorobenzisoxazole (0.3 g, 1.95mmol) in 5 ml of
acetonitrile and 5 ml of water was mixed with 15ml of a solution
of 3 M NaOH and was allowed to stir for 10 min. Concentrated
HCl was added drop-wise to the latter solution to bring down the
pH to 1. The solution was extracted three times with 10-ml portions
of dichloromethane, and the extracts were combined and dried
over magnesium sulphate. The solvent was evaporated, and the
solid residue was recrystallized with acetonitrile. The recrystallized
solid was dried under vacuum overnight and stored in foil-
wrapped vials in the freezer. A UV–vis spectrum was recorded to
test the purity of the compound. Maximum absorbance was
recorded at a wavelength of 339 nm in agreement with the
literature.
Study of the effect of initiator content
The first part of the work focussed on the evaluation of the effect
of changing the concentration of initiator in the polymerisation
reaction. The nanogels were prepared using high-dilution
polymerisation using AIBN as initiator, following an established
protocol (Maddock et al., 2004) and the experimental conditions
that had been previously shown to be optimal, that is, total
M
monomer concentration C 0.5% in mass and 80% of EGDMA
as cross-linker in DMSO (Maddock et al., 2004). In each case,
two nanogels preparations were synthesized, both containing
the functional monomer 4-vinylpyridine, one imprinted (MIP)
and one non-imprinted (NIP). Three sets of polymers were pre-
pared with initiator content equal to 1%, 2%, and 3% of the
quantity of double bonds in the pre-polymerisation mixture. To
obtain meaningful comparison of kinetic data for the polymer
preparations, it was necessary to estimate the concentration of
functional groups available. This was done by back-titration of
the pyridinium ions using NaOH. The titration curves for MIP
AS232 and MIP AS234 are shown in Figure 2. Similar curves were
obtained for MIP AS230, NIP AS231, NIP AS233, and NIP AS235.
The concentration of pyridine moieties available in the
nanogels was obtained from the difference in NaOH volume
required to reach the two different equivalence points. The data,
in Table 1, regarding active site concentrations, show that in each
of the three sets, there is no significant difference between the
imprinted and non-imprinted nanogels, which indicates that the
presence of the template does not have an effect on the
polymerisation process and in the rate of incorporation of the
functional monomer 4-vinylpyridine. When comparing all six
nanogels, it appears that although the preparations with 1% and
2% initiator show similar results, the one with 3% appears to have
a higher incorporation of functional monomer. These data have
been consistently obtained in repeat experiments and can be
possibly explained by the overall higher rate of polymerisation
due to the higher initiation content.
1
UV–vis: lmax in acetonitrile: 339 nm; H-NMR (400MHz, acetoni-
trile (ACN)-d3): d 6.98(dd, J = 8.9, 1H), 7.44 (dd, J = 10 Hz,
1
1
J = 2.6 Hz, 1H), 7.56 (d, J = 2.6, 1H), 7.9 (br, 1H).
2
1
Titration of nanogels for the determination of pyridine
residues
A 20-ml solution of imprinted nanogels was prepared at a concen-
–1
tration of 1 mg ml in 20% acetonitrile in distilled water. To the lat-
–
3
ter was added 15 ml of HCl solution at a concentration of 4 ꢁ 10 M.
The mixture was then allowed to stir for a few minutes, and the pH of
the solution was recorded. The resulting nanogel solution was
–3
titrated using a NaOH solution at a concentration of 1.5 ꢁ 10 M.
The pH was recorded every milliliter and half milliliter around the
equivalent points area, and the values were plotted against the
volume of NaOH solution. The equivalent points were determined
manually, and the volume at the equivalence was determined as
the mean from the three measurements. The exact same procedure
was applied to the non-imprinted nanogel. The concentration of
pyridine moieties CPy was determined by the volume difference
between the two equivalent points and the knowledge of the
titrant (NaOH) concentration CNaOH as explained in the following
equation:
ðVe2 ꢃ Ve1Þ ꢁ CNaOH
CPy ¼
V
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IMPRINTED NANOGELS AS ENZYME MIMICS
a)
b)
1
1
2
0
8
6
4
2
10
9
pH versus V
(ml)
pH versus V
(ml)
8
7
6
5
4
3
2
0
10
20
30
40
0
10
20
30
40
50
V (ml)
V (ml)
–
3
Figure 2. Titration curves of (a) MIP AS232 and (b) MIP AS234 using a solution of NaOH at a concentration of 1.5 ꢁ 10 M.
These polymers were then used to carry out kinetic experiments
using 1,2-benzisoxazole (1) as the substrate, at pH 9.4 (0.05 M
carbonate buffer) with 10% ACN and 1 mg/ml of nanogels, and
monitoring product formation at l = 325 nm using UV–vis spec-
Evaluation of the effect of the template/monomer ratio on
the catalytic efficiency
In an effort to further optimize the preparation of the imprinted
nanogels with enhanced activity and imprinting efficiency, it was
decided to alter the ratio template/monomer and to study the
effect on the catalytic activity and on the imprinting efficiency to
evaluate whether there was any significant effect. The idea was that
the change in the template/monomer ratio could favour the associ-
ation of these two molecules toward complex formation, therefore
creating more specific cavities. This was done by synthesising the
nanogels using an excess of template in the first instance and then
an excess of functional monomer and evaluating the results.
Two sets of imprinted and non-imprinted nanogels (MIP AS250/
NIP AS251 and MIP AS252/NIP AS253) were prepared, under iden-
tical conditions, with two and five equivalents of indole compared
with 4-vinylpyridine, respectively, and the catalytic activities were
compared with the preparations MIP AS230 and NIP AS231, which
were prepared with a 1:1 ratio of template and functional mono-
mer. As previously, the number of active sites was determined
via back-titration and used to calculate the kinetic parameters.
The data regarding the active site concentration, shown in
Table 2 for all six nanogel preparations, are all in a very similar
order of magnitude. Therefore, the addition of a large excess of
template does not appear to have a substantial effect in the
incorporation of 4-vinylpyridine.
i
troscopy. Initial rates (v) for both imprinted and non-imprinted
nanogels were obtained and corrected for the uncatalysed rate
and divided by the active site concentration to give the apparent
rate constant (k’app) that was used to obtain some qualitative indi-
cation of trends.
The kinetic data showed that the initiator content appeared to
have a noticeable effect on the catalytic properties of the nanogels.
An initial analysis showed that NIP AS233, MIP AS234, and NIP
AS235 did not display a saturation curve in the range of substrate
concentrations investigated; it was therefore not possible to deter-
mine kinetic parameters such as the rate constant kcat and catalytic
M m
efficiency kcat/K . This was most likely due to higher values of K
for the non-imprinted polymers, outside the range of the substrate
concentration being evaluated.
To compare the nanogels with different AIBN content, the
values of apparent rate constant k′app and the apparent imprinting
factor were calculated for a single-substrate concentration (2 mM),
and the values are gathered in Table 1. This substrate concentration
was selected because at this value a significant difference between
imprinted and non-imprinted polymers was observed and a
saturation curve was obtained for MIP AS230 and MIP AS232. The
data clearly show that as the concentration of initiator is increased
from 1% to 3%, the catalytic efficiency of the nanogels
decreases, and, more significantly, the imprinting efficiency is
also reduced, and in the case of the 3% initiator, there is no
relevant difference between the imprinted and non-imprinted
nanogels. This would suggest that although higher initiator
content results in higher incorporation of functional monomer,
especially with 3%, the apparent imprinting efficiency, measured
at a single substrate concentration, is lowered.
The kinetic experiments, carried out at 2 mM substrate concen-
tration, show that the nanogels prepared with 1:1 template/mono-
mer ratio have the highest catalytic activity, as indicated by the
value of the apparent rate constant. The excess of indole was
expected to favour the template–monomer equilibrium toward
the complex formation, increasing the amount of specific cavities
formed during the imprinting process. This does not appear to
be the case. The polymers MIP AS250 and MIP AS252 did not show
any significant change in imprinting efficiency compared with the
Table 1. Kinetics parameters for MIP AS230, AS232, AS234 and NIP AS231, AS233, and AS235 (at pH 9.4, 10% ACN, 1 mg/ml of
nanogels)
–
1
–1
–1
Polymers
AIBN content
v
i
(mM min )
Active site (mmol mg )
k′app (min )
k′app,MIP/k′app,NIP
MIP AS230
NIP AS231
MIP AS232
NIP AS233
MIP AS234
NIP AS235
1
1
2
2
3
3
0.61
0.30
0.49
0.37
0.70
0.73
0.24
0.19
0.28
0.26
0.52
0.60
2.54
1.58
1.75
1.42
1.34
1.22
1.61
—
1.23
—
1.1
—
J. Mol. Recognit. 2012; 25: 352–360
Copyright © 2012 John Wiley & Sons, Ltd.
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P. BONOMI ET AL.
Table 2. Kinetics parameters for MIP AS230, MIP AS250, MIP AS252 and NIP AS231, NIP AS251, and NIP AS253 (pH 9.4, 10% ACN,
1
mg/ml nanogels)
–1
–1
–1
Polymers
Template equivalents
v (mM min )
Active site (mmol mg )
k’app (min )
k’app,MIP/k’app,NIP
i
MIP AS230
NIP AS231
MIP AS250
NIP AS251
MIP AS252
NIP AS253
1
1
2
2
5
5
0.61
0.30
0.42
0.23
0.48
0.38
0.24
0.19
0.58
0.45
0.38
0.45
2.54
1.58
0.72
0.51
1.26
0.84
1.6
—
1.4
—
1.5
—
one exhibited by MIP AS230 (nanogels synthesized with one equiv-
alent of template).
imprinting efficiency. A similar effect, although related to
binding polymers, was also reported by Andersson et al. in the
preparation of binding “bulk” nicotine-imprinted polymers
(Andersson et al., 1999), where an excess of methacrylic acid led
to a decrease of the selectivity of the polymers.
Work on the evaluation of template effect on the selectivity
of imprinted polymers was reported in previous studies by
Mosbach et al. for the generation of binding “bulk” nicotine-
imprinted polymers (Andersson et al., 1999). In this work, it was
demonstrated that an excess of template during the polymerisation
process was clearly unfavourable with regard to selectivity, leading
to the generation of cavities with high heterogeneity, poor binding
activity, and low imprinting efficiency. Other groups have also inves-
tigated and reviewed this issue in the context of binding polymers
Molecular recognition and substrate selectivity
Having evaluated the effect of important polymerisation para-
meters, such as initiator content and template/monomer ratios,
on the activity of the nanogels, the next step focussed on the
study of the molecular recognition properties in relation to catal-
ysis. An important criterion in the development of enzyme
mimics is the ability of these tailor-made catalysts to specifically
recognize a substrate molecule and, in a similar way as enzymes,
to form a complex and generate the product of the targeted
reaction. One of the advantageous features of the molecular
imprinting approach is the possibility of forming a tailored site
complementary to a target molecule into a polymeric network,
which allows the specific recognition of this molecule similarly
to the “lock-and-key” system (Li et al., 2008). Some early work
in the field was carried out by Shea’s group, where imprinted
polymers with esterolytic activity were shown to have a degree
of stereoselectivity, as a result of hydrogen bonding in the bind-
ing step (Sellergren and Shea, 1994). The work presented here
was focussed on two different yet complementary strategies. In
the first instance, the MIP AS230 and NIP AS231, imprinted with
indole and non-imprinted, were evaluated as catalysts for the
Kemp elimination using a substrate analogue, 5-Cl-benzisoxazole
(Manesiotis et al., 2004; Sellergren and Allender, 2005; Spivak, 2005;
Yoshimatsu et al., 2011).
The highest catalytic activity obtained in our case with a 1:1 ratio
would indicate that the excess of template does not favour an
increased selectivity and specificity in the catalytic polymers; how-
ever, the imprinting efficiency as determined by the ratio of the
apparent catalytic constants is not affected, and this could be the re-
sult of using catalytic parameters to evaluate the imprinting effect,
which differs completely for how it is done with binding polymers.
In the second approach, the effect of using an excess of
functional monomer on the kinetic parameters was studied.
One set of nanogels was prepared using seven equivalents of
4
M
-vinylpyridine and C of 0.5% and 80% of EGDMA as cross-
linker, and MIP AS242 and NIP AS243 were synthesized using
the standard protocol of high-dilution radical polymerisation.
Active site titration for the nanogels was carried out, and the
results showed that although these polymers contained 15%
more pyridine units compared with MIP AS230 and NIP AS231,
this did not correlate significantly with the much higher amount
of functional monomer used in the preparation, indicating a
very low incorporation. Analysis of the kinetic data shows that
although both nanogel preparations, MIP AS242 and NIP
AS243, demonstrated catalytic activity in the Kemp elimination
when compared with the background reaction (kuncat was found
(2), a molecule that differs from the cognate substrate benzisoxazole
for the presence of the chlorine atom in the para position. In the
second instance, new nanogels were synthesized, using
5
-nitroindole as the template analogue, and the catalytic activ-
ity toward 1,2-benzisoxazole (1) and 5-chloro benzisoxazole (2)
was evaluated and compared.
The structures of 5-nitroindole and 5-chlorobenzisoxazole are
shown in Figure 3.
The choice of using 5-Cl-benzisoxazole (2) as substrate analogue,
with an electron-withdrawing group like the chlorine atom in para
to the oxygen atom, was expected to enhance the rate of the
–
4
–21
–1
to be 8.18 ꢁ 10 ꢄ 1.03 ꢁ 10 min ), the data could not be
fitted to a Michaelis–Menten saturation curve but only to a
linear regression. In addition, there was no significant difference
between the reaction rates for the imprinted and non-imprinted
nanogels. This suggests that although the percentage of func-
tional monomer incorporated is only marginally increased, the
imprinting efficiency evaluated using the kinetic parameters
has changed dramatically. The fact that the kinetic data could
not be fitted with a saturation curve is a clear indication that
the substrate range evaluated was well below the value of KM.
This indicates a much lower binding affinity between the cavity
and substrate, therefore leading to a possible explanation being
that the higher amount of monomer used has got a significant
effect on the polymerisation process leading to a loss of
Cl
O N
2
N
O
N
H
5-Cl-benzisoxazole
5-NO -indole
2
2
Figure 3. Structure of 5-Cl-benzisoxazole and 5-NO -indole.
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J. Mol. Recognit. 2012; 25: 352–360

IMPRINTED NANOGELS AS ENZYME MIMICS
reaction due to the change in electron environment and distribu-
tion (Casey et al., 1973). The removal of the proton is more favoured
in the presence of electron-withdrawing groups in that position.
To have accurate kinetic data, calibration curves for both pro-
ducts were obtained to give extinction coefficient values: The
The experimental data for the activity of both polymers toward
substrate 2, shown in Figure 4, were used to obtain the kinetic
–7
parameters, and these were found to be Vmax 6.30 ꢁ 10 (SE ꢄ 4.0
–
8
–1
–4
–5
ꢁ 10 ) M min , K 4.87 ꢁ 10 (SE ꢄ 6.50 ꢁ 10 ) M, for MIP
M
AS230. For NIP AS231, the values obtained were Vmax of 4.75 ꢁ
–
1
–7
–8
–1
–4
value of e for 5-Cl-cyanophenol (4) was found to be 56140 cm
10 (SE ꢄ 4.04 ꢁ 10 ) M min and K
M
of 6.16 ꢁ 10 (SE ꢄ 1.50
–
1
–4
M , whereas for 2-cyanophenol (3), the extinction coefficient
ꢁ 10 ) M, also reported in Table 3. The first interesting
observation is that when the concentration of active sites is
taken into account, comparison of the values of the catalytic
rate constant shows that the imprinted nanogel AS230 has
higher activity and displays a catalytic efficiency equal to 2,
despite the substrate analogue 2 having both steric and elec-
tronic differences compared with the cognate substrate
–
1
–1
(
e) was 5940 cm M . The reactions were all carried out under
the same experimental conditions (carbonate buffer 50 mM pH
.4 with a 10% of acetonitrile and 0.5% of Tween 20), and experi-
9
ments were carried out with both substrates in the absence of
any nanogels to evaluate the uncatalysed reaction rates.
–
4
The kuncat for 1,2-benzisoxazole (1) was found to be 4.30 ꢁ 10
–5
(
(
SE ꢄ 5.20 ꢁ 10 ), whereas in the case of 5-Cl-benzisoxazole
M
1. The values of the K unfortunately carry a significant stan-
–4
–4
2), the kuncat registered was 9.80 ꢁ 10 (SE ꢄ 2 ꢁ 10 ) showing
dard deviation value ranging between 13% and 25%, as a result
of not being able to increase the substrate concentration high
enough, therefore limiting any possible discussion. This is an
interesting result that indicates that the imprinting approach,
even when using weak hydrogen bonding, is certainly sensitive
to the microenvironment and that the molecular recognition
properties induced in the active sites are specific and depen-
dent on the structure of the template used.
the expected increase as a result of the substituent. All reactions
were carried out using a fixed concentration of nanogels
–1
(
0.02mg ml ), whereas substrate concentration varied between
.2 mM and 1.2mM. Initial rates were determined by monitoring
product formation at 339 nm for 5-Cl-2-cyanophenol (4) and
25 nm for 2-cyanophenol (3) and were all corrected for the unca-
0
3
talysed reaction.
Because MIP AS230 and NIP AS231 displayed a saturation
curve in the range of the substrate concentration investigated,
it was possible to fit the data using the hyperbola equation, in
agreement with the Michaelis–Menten model (Figure 4).
When the kinetic data for substrate 2 are compared with the
ones for 1,2-benzisoxazole (1), also reported in Table 3, it
becomes apparent that (i) the imprinted nanogels MIP AS230
are catalytically more efficient when working on the cognate
substrate, as indicated by the higher value of kcat, and (ii) more
significantly, the imprinting factor when using the cognate
substrate 1 is two times higher than with the substrate ana-
logue 2.
5
4
3
2
1
e-7
e-7
e-7
e-7
e-7
0
The cavities in the MIPs are specifically tailored for the tem-
plate used in the polymerization process, and for this reason,
AS230 is showing a higher affinity for a molecule, like substrate
1
, that is a very close mimic, from a structural and chemical point
of view, of the indole template. The variation in the catalytic
efficiency can be explained by a combination of electronic and
steric differences between the chemical structures of substrate
MIP AS230
NIP AS231
2
and the indole template.
Having evaluated the specificity and molecular recognition
properties, coupled with specific catalytic activities, of the
indole-imprinted polymers, it was decided to investigate further
the selectivity by preparing new nanogels imprinted with a
different template. 5-Nitrobenzisoxazole was selected as the
template, as the molecule contains the nitro functional group,
which increases the steric hindrance of the template and also
provides important changes in the electronic effect of the
0
,0000
0,0002
0,0004
0,0006
0,0008
0,0010
[substrate] (M)
Figure 4. Initial rates versus substrate concentration plots for MIP AS230
–
1
and NIP AS231 on 5-Cl-benzisoxazole ([nanogel] = 0.02 mg ml , pH 9.4,
0 mM carbonate buffer, 10% ACN, 0.5% Tween 20). All initial rates are
corrected for the uncatalysed reaction.
5
Table 3. Active site concentrations and kinetics parameters for MIP(s) AS230, AS238 and NIP(s) AS231, AS239 reacting with
cognate substrate 1 and analogue 2
–
1
Catalyst
Substrate
V
max (mM
min )
K
M
(M)
k
cat (min )
Active site in the reaction (mM)
k
catMIP/kcatNIP
–
1
–
–
–
–
–
–
–
–
4
4
3
3
4
4
3
4
AS230
AS231
AS230
AS231
AS238
AS239
AS238
AS239
2
2
1
1
2
2
1
1
0.63
0.47
1.54
0.69
0.53
0.26
0.90
0.20
4.87 ꢁ 10
6.16 ꢁ 10
1.58 ꢁ 10
1.83 ꢁ 10
4.17 ꢁ 10
7.60 ꢁ 10
1.00 ꢁ 10
5.36 ꢁ 10
1.13
0.56
2.75
0.82
1.33
0.72
2.25
0.56
0.56
0.84
0.56
0.84
0.40
0.36
0.40
0.36
2
—
3.4
1.8
—
4
—
J. Mol. Recognit. 2012; 25: 352–360
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/jmr

P. BONOMI ET AL.
6
5
4
3
2
1
e-7
e-7
e-7
e-7
e-7
e-7
0
MIP AS238
NIP AS239
0
,0000 0,0002 0,0004 0,0006 0,0008 0,0010 0,0012 0,0014
[substrate] (M)
Figure 5. Plot of the variation of chemical shifts Δd
bonded to the N of the template, corrected from the value of the chem-
ical shift of H of 5-nitroindole alone versus the concentration of
-pyridine.
H
of the proton
Figure 6. Plot of initial rates versus substrate concentration for nanogels
MIP AS238 and NIP AS 239 on 1,2 benzisoxazole (1) ([nanogel] = 0.02 mg
ml , pH 9.4, 50 mM carbonate buffer, 10% ACN, 0.5% Tween 20). All
initial rates are corrected for the uncatalysed reaction.
–
1
i
d
5
molecule. In addition, given the high polar nature of the group,
there is also the possibility that additional interaction points
may arise as a result of hydrogen bonding between the template
5e-7
and the monomers/cross-linker. The pK of the nitrogen proton
4e-7
a
of 5-nitroindole is found to be around 14.75, a value very close
to that one of indole (pK 16). The presence of the strong elec-
a
3
2
1
e-7
e-7
e-7
0
tron-withdrawing and sterically bulky substituent was expected
to provide significant effects to the catalytic activity and molec-
ular recognition properties of the nanogels.
It was not possible to evaluate the catalytic activity of
these new nanogels with the corresponding cognate sub-
strate 5-nitro-benzisoxazole due to the need to work at a
much lower pH to be able to carry out kinetic studies. There
was clear concern that this could impact the morphology of
the nanogels, therefore preventing any comparison to be
MIP AS238
NIP AS239
0
,0000
0,0002
0,0004
0,0006
0,0008
0,0010
0,0012
[substrate] (M)
1
21carried out. Instead, it was decided to test the activity
Figure 7. Plots of intial rates versus substrate concentration for MIP
AS238 and NIP AS239 on 5-Cl-benzisoxazole (2) ([nanogel] = 0.02 mg
ml , pH 9.4, 50 mM carbonate buffer, 10% ACN, 0.5% Tween 20). All ini-
tial rates are corrected for the uncatalysed reaction.
on two substrate analogues to evaluate any differences in
molecular recognition.
–
1
The first step, toward the synthesis of the nanogels, was to
study the interaction between 5-nitroindole and the functional
monomer 4-vinylpyridine. A non-covalent hydrogen bonding
between the proton on the nitrogen of the indole derivative
and the lone pair of the nitrogen of 4-vinylpyridine was expected
to be similar to that one exhibited in the case of the non-
substituted indole. The interaction studies were carried out in a
similar way using H-NMR spectroscopy, studying the variation
of chemical shift of the proton on the nitrogen atom of the tem-
plate (Wilcox et al., 1992).
substituent. A set of imprinted and non-imprinted nanogels,
MIP AS238 and NIP AS239, were prepared with 5-nitroindole
as template molecule, using the standard protocol of high-
dilution radical polymerisation (Graham and Cameron, 1998).
The determination of the active site number was performed
using the standard procedure of back-titration of pyridine
residues described in the experimental session. The active
site number obtained for MIP AS238 was found to be
1
A solution of 5-nitroindole was prepared at a concentration of
–
6
–1
–6
–1
85.4 mM in CD
2
Cl
2
, and increasing equivalents of d5-pyridine
0.20 ꢁ 10 mmol mg
and 0.18 ꢁ 10 mmol mg . These
–2
–1
ranging from 0.2 (1.7ꢁ 10 M) to 7 equivalents (5.9 ꢁ 10 M)
values were found to be within the range of active site
concentrations obtained with previous polymers preparations.
A detailed kinetic study of the catalytic activity of MIP AS238
and NIP AS239 with both substrates 5-chlorobenzisoxazole (2)
and 1,2-benzisoxazole (1) was carried out. All the reactions were
performed under the same conditions previously described,
using 0.02 mg ml of nanogels in 50 mM carbonate buffer, pH
9.4, 10% ACN, and 0.5% Tween 20. The initial rates, corrected
for the background reactions, were plotted versus substrate
concentration as shown in Figure 6.
were added to the solution. The spectra of indole alone and for
H
each equivalents of pyridine were recorded. The values of d were
collected and plotted against the concentration of d5-pyridine, as
shown in Figure 5.
A variation of the chemical shift of the proton from d 8.76
to d 12.3 was noticed, which confirmed the interaction
via non-covalent hydrogen bond between 5-nitroindole and
–1
4
-vinylpyridine. The association constant K
was found to
ass
–
1
be around 0.42 M . The interactions between 5-nitroindole
and pyridine appeared to be stronger than in the case of
indole, and this is clearly the result of the presence of the
Both nanogels MIP AS238 and NIP AS239 demonstrated cata-
lytic activity for the reaction toward the substrates, and, in all
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Copyright © 2012 John Wiley & Sons, Ltd.
J. Mol. Recognit. 2012; 25: 352–360

IMPRINTED NANOGELS AS ENZYME MIMICS
cases, the data could be fitted into a Michaelis–Menten satura-
tion curve to provide the kinetic parameters.
considered as real. This provides once more evidence that
although the nanogels were imprinted with a different template,
the imprinted and non-imprinted cavities show different kinetic
behaviour toward the substrate analogue.
In particular, for MIP AS238 tested on 1,2-benzisoxazole (1),
–
7
we obtained the following values: Vmax = 9.06 ꢁ 10 (SE ꢄ 7.034
–
8
–1
–3
–4
ꢁ
10 ) M min , K
M
= 1.00 ꢁ 10 (SE ꢄ 1.424 ꢁ 10 ) M,
The interesting finding is related to the fact that when the
activities of the 5-nitroindole-imprinted nanogels for the two
different substrate analogues are compared, the data indicate
that the 1,2-benzisoxazole is a better substrate, giving rise to
higher catalysis and imprinting efficiency. The affinity of the
5-nitroindole-shaped cavities of MIP AS238 for a smaller
molecule as 1,2-benzisoxazole is higher than in the case of
a bigger compound as 5-Cl-benzisoxazole; once again, the
size of the molecule seems to play an important role on
the diffusion of the substrate into the three-dimensional
active sites of the nanogels.
–
1
k
cat = 2.25 min . For NIP AS239, the values obtained were as
–
7
–8
–1
follows: Vmax = 2.03 ꢁ 10 (SE ꢄ 1.25 ꢁ 10 ) M min ,
–
4
–5
–1
K
M
= 5.36 ꢁ 10 (SE ꢄ 7.98 ꢁ 10 ) M, kcat = 0.56 min .
These data indicate that MIP AS238 is a better catalyst than
NIP AS239 due to the higher values of Vmax and kcat, but, if
compared with the data obtained using the indole-imprinted
polymer AS230, the catalytic activity of the indole-imprinted
nanogels is still higher. Given that substrate 1 is sterically less
hindered than the 5-nitroindole, it is still able to access the
imprinted cavities of MIP AS238, and the smaller size may
M
explain the smaller value of K compared with AS230.
The imprinting efficiency of the two sets of polymers, when eval-
uated against substrate 1, is fairly similar. It is important to note
that in this case, the kinetic characterisation was carried out using
a full set of data, instead of a single-substrate concentration, and
therefore, the imprinting efficiency is determined by the ratio of
the values of catalytic constants for the MIP and NIP.
To compare the selectivity of these new catalysts (MIP AS238
and NIP AS239) with the indole-imprinted nanogels (MIP AS230
and NIP AS231) and to better investigate the effect of the size
of the substrate on the affinity of the catalysts, we tested the
CONCLUSION
The potential of the molecular imprinting approach for the
development of novel catalysts with enzyme-like activity is yet
to be achieved. Although very significant results have been
obtained thus far, these have frequently relied on very strong
template–monomer interactions, which prove to be limiting in
terms of wider applicability. The use of the more flexible nanogel
matrix coupled with a better understanding of the factors that
contribute to the molecular recognition properties of the cavities
promises important advances. We have showed that it is possi-
ble to develop imprinted nanogel catalysts using weak hydrogen
bond interactions, and in this work, we have presented a very
detailed investigation of the effects of the different polymerisa-
tion parameters on the molecular recognition characteristics
and on the imprinting efficiency. The data demonstrate that
the chemical structure of the template plays a key role in
determining the binding and catalytic properties of the cavities,
which are then able to differentiate between minor structural
differences in the substrates. The fine-tuning of the molecular
recognition characteristics of the imprinted cavities together
with the strategic design of the template and the functional
monomer will provide new opportunities for novel catalysts.
5
-nitroindole-imprinted nanogels in the Kemp elimination using
another substrate analogue, 5-Cl-benzisoxazole, a molecule that
has a large substituent in the same position as in the template
and is also electron-withdrawing.
A set of reactions were carried out using the same conditions
reported before: carbonate buffer 50 mM, pH 9.4 with a 10% of
acetonitrile and 0.5% of Tween 20, fixed concentration of the
–
1
catalysts (0.02 mg ml ), and varying the concentration of
-Cl-benzisoxazole (2) between 0.2 and 1 mM. The kinetic data,
5
shown in Figure 7, were fitted to the hyperbola equation in
agreement with the Michaelis–Menten model, and the kinetic
–
7
parameters were found to be Vmax = 5.35 ꢁ 10 (SE ꢄ 4.25 ꢁ
–
8
–1
–4
–5
1
k
=
(
0 ) M min , K
M
= 4.17 ꢁ 10 (SE ꢄ 7.65 ꢁ 10 ) M,
–1
cat = 1.34 min . For NIP AS239, the values obtained were Vmax
–
7
–8
–1
–4
2.61 ꢁ 10 (SE ꢄ 2.47 ꢁ 10 ) M min , K
M
= 7.60 ꢁ 10
–
4
–1
SE ꢄ 1.3 ꢁ 10 ) M, and kcat = 0.725 min .
Acknowledgements
As demonstrated by the kinetic parameters, MIP AS238 is a
superior catalyst than NIP AS239 showing higher kcat whilst
having a lower K . In this case, the standard deviation errors
associated with the calculations of K are smaller than the differ-
The authors acknowledge the financial support of the European
Commission via the Marie Curie actions (MCRTN-2006-033873
and MCIAPP-2009-251307) and Queen Mary University of
London.
M
M
ence between the two estimated values and therefore can be
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