Effect of the cross-linker on the general performance and temperature dependent behaviour of a molecularly imprinted polymer catalyst of a Diels-Alder reaction

Article abstract of DOI:10.1016/j.molcatb.2011.06.006

Here we present a series of molecularly imprinted polymers capable of catalysing the Diels-Alder reaction between benzyl 1,3-butadienylcarbamate (1) and N,N-dimethyl acrylamide (2). The polymer systems studied here demonstrated an unusual cross-linker and temperature dependent behaviour, namely that polymer catalysis of the Diels-Alder reaction was lower at elevated temperature, in contrast to the solution reaction. Furthermore, not only was the catalytic activity significantly influenced by the choice of cross-linker, but in a similar fashion also the extent of the temperature effect, indicating a close relationship between catalysis and the observed inhibition. Molecular dynamics simulations of both the polymer systems studied were used to provide insight into the molecular background of transition state stabilisation, and differences in properties of the systems based on different cross-linkers.

Full text of DOI:10.1016/j.molcatb.2011.06.006

Journal of Molecular Catalysis B: Enzymatic 72 (2011) 199–205
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Effect of the cross-linker on the general performance and temperature dependent
behaviour of a molecularly imprinted polymer catalyst of a Diels–Alder reaction
Henning Henschela, Nicole Kirscha, Jimmy Hedin-Dahlströma, Michael J. Whitcombeb,
Susanne Wikmana, Ian A. Nichollsa,c,∗
a Bioorganic & Biophysical Chemistry Laboratory, School of Natural Sciences, Linnæus University, SE-391 82 Kalmar, Sweden
b Cranfield Health, Cranfield University, Cranfield, Bedfordshire MK43 0AL, UK
c Department of Biochemistry & Organic Chemistry, Uppsala University, SE-751 23 Uppsala, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Here we present a series of molecularly imprinted polymers capable of catalysing the Diels–Alder reac-
tion between benzyl 1,3-butadienylcarbamate (1) and N,N-dimethyl acrylamide (2). The polymer systems
studied here demonstrated an unusual cross-linker and temperature dependent behaviour, namely that
polymer catalysis of the Diels–Alder reaction was lower at elevated temperature, in contrast to the solu-
tion reaction. Furthermore, not only was the catalytic activity significantly influenced by the choice of
cross-linker, but in a similar fashion also the extent of the temperature effect, indicating a close rela-
tionship between catalysis and the observed inhibition. Molecular dynamics simulations of both the
polymer systems studied were used to provide insight into the molecular background of transition state
stabilisation, and differences in properties of the systems based on different cross-linkers.
© 2011 Elsevier B.V. All rights reserved.
Received 1 March 2011
Received in revised form 19 May 2011
Accepted 8 June 2011
Available online 15 June 2011
Keywords:
Diels–Alder reaction
Molecularly imprinted polymer
Molecular dynamics simulation
Plastic antibody
Transition state analogue
1. Introduction
polymer can – just as a catalytic antibody – be expected to sta-
bilise the transition state, catalysing the corresponding reaction
The Diels–Alder reaction is one of the most powerful synthetic
tools for the creation of carbon–carbon bonds. It is widely used
in the preparation of physiologically active compounds [1]. This
makes it an important target for the development of catalytic sys-
tems [2–4]. One approach towards the rational design of catalysts
is the production of antibodies against a transition state analogue
(TSA) as demonstrated for the Diels–Alder reaction by the Janda
group [5,6].
An approach analogous to the production of catalytic antibodies
can be seen in the preparation of catalytic molecularly imprinted
polymers. Molecular imprinting is carried out by polymerisation of
commonly two types of monomer (one functional monomer and
one cross-linker) in presence of a template, giving, after removal
of the template, a polymer with selective recognition sites for the
template – essentially a plastic antibody [7–14]. If a structure sim-
ilar to a certain transition state is used as template, the resulting
[13]. While a growing number of catalytic polymers have been
designed and synthesised for a number of different reaction types,
e.g. ester hydrolysis [15–25], transamination [26], ␤-elimination
[27,28], only a few reports deal with C–C bond formation, e.g. aldol
[29,30], Suzuki [31], and Diels–Alder reactions [32–34].
We recently described a previously unreported temperature
dependent behaviour of catalytic molecularly imprinted polymers
[35], where a polymer system, based on methacrylic acid (MAA) as
functional monomer and divinylbenzene (DVB) as cross-linker, was
designed to catalyse the Diels–Alder reaction shown in Fig. 1. The
template used in this study was adapted from the transition state
analogue developed by Gouverneur et al. [5] in the production of
catalytic antibodies for this [4 + 2] cycloaddition. While success-
fully enhancing the rate of the reaction up to 20-fold compared
to the solution reaction at room temperature, it was noted, how-
ever, that at elevated temperature the polymers were shown to
lower the rate of the reaction instead. Here further insights into this
phenomenon have been obtained through a combination of stud-
ies using polymers synthesised using an alternative cross-linking
agent, and including one additional temperature step in the evalu-
ation, while keeping the same functional monomer and template,
granting direct comparability to the results presented previously.
Moreover, a series of molecular dynamics studies was conducted,
providing insight into which interactions are of importance for tem-
plate, as well as rationalizing the effect of the change in cross-linker.
Abbreviations: ACHN, azobis(cyclohexanecarbonitrile); BHT, 3,5-di-tert-butyl-
4-hydroxy-toluene; DVB, divinylbenzene; EGMA, ethylene glycol dimethacrylate;
MAA, methacrylic acid; MD, molecular dynamics; TSA, transition state analogue.
∗
Corresponding author at: Bioorganic & Biophysical Chemistry Laboratory, School
of Natural Sciences, Linnæus University, SE-391 82 Kalmar, Sweden.
Tel.: +46 480 44 62 58; fax: +46 480 44 62 44.
E-mail addresses: ian.nicholls@lnu.se, ian.nicholls@hik.se (I.A. Nicholls).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.06.006

200
H. Henschel et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 199–205
Fig. 1. The Diels–Alder reaction studied in Ref. [35] and this article.
2. Materials and methods
(5/6), 0.0979 g (1.14 mmol; 20 mol%) MAA, and 0.921 g (4.55 mmol,
80 mol%) EGMA for the synthesis of 1 g polymer. Polymerization
was performed in 2 mL toluene, with ACHN (1 mol% relative to
double bonds) as initiator. To remove the template and additional
fines, Soxhlet extraction was carried out in acetone. More complete
template removal from the polymer was achieved by packing the
polymer into a stainless steel HPLC column and subjecting it to
extensive washing under a variety of conditions, as described else-
where [36]. Control polymers were prepared in the same manner
but in the absence of template.
2.1. General
Reagents were purchased from Aldrich (Germany). Inhibitors
from ethylene glycol dimethacrylate (EGMA) were removed by
extraction with 0.1 M sodium hydroxide solution. The EGMA was
subsequently dried over magnesium sulfate and stored at 4 ◦C.
Treatment of other substances prior to their use is described in Ref.
[35].
2.2. Synthesis of Diels–Alder products and TSAs
2.4. Batch binding studies
The endo- and exo-transition state analogues (5 and 6) were
synthesized in six steps with an overall yield of 35 and 11%,
respectively, as summarized in Fig. 2. A detailed description of the
synthesis can be found in Ref. [35].
A solution of 5 or 6 in toluene (0.5 mL; 0.1 and 1 mM, respec-
tively) was shaken with 5 0.1 mg polymer overnight. The polymer
was filtered off and the concentration of the TSA remaining in solu-
tion determined by HPLC performed on a HP1050 or a HP1090
system using a Beckman silica column (250 mm × 4.6 mm i.d.).
Polymer suspensions were filtered through 2 ␮m PTFE membrane
filter cartridges prior to HPLC analysis. Elution was carried out in
90% heptane containing 0.1% acetic acid and 10% isopropanol at a
flow rate of 1 mL/min with UV detection at 220 nm.
2.3. Polymer synthesis
Polymers were prepared, as previously described for DVB-
based polymers [35], using 0.0933 g (0.284 mmol, 5 mol%) TSA
Fig. 2. Synthesis of the endo- (5) and exo- (6) transition state analogues (TSAs).

H. Henschel et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 199–205
201
2.5. Swelling studies
of the template are most relevant for interactions with the func-
tional monomer (Fig. 3). From the representations of the density
grids it becomes obvious that there are two main interaction sites
at the template for the formation of hydrogen bonds involving the
MAA proton, namely the oxygen atom of the amide group and the
doubly bound oxygen atom of the carbamate group. Significant dif-
ferences between the spatial arrangements relative to the different
diastereomers are, however, not observed.
Polymer (1 mL) was weighed into a 5 mL glass cylinder, and
3 mL of toluene were added. The cylinder was sealed with a glass
stopper and left standing for 12 h. The increase in polymer vol-
ume (swelling, SW), was recorded and the specific swelling (SWsp
)
calculated according to Eq. (1)1.
SW
m
SWsp
=
(1)
These interactions, as well as possible interactions involving the
carbamate proton, were studied more closely◦in terms of hydrogen
˚
bonds analysis with a 3.0 A distance and a 90 angle cut-off for the
2.6. Kinetics studies
interaction to count as a hydrogen bond. The results of this analysis
are summarised in Table 1.
A
solution was
prepared
containing
benzyl
1,3-
One rather clear trend that can be found upon moving from
DVB as cross-linker to EGMA is that the total relative occupation
of the hydrogen bond to the carbamate oxygen atom is consid-
erably lowered even though the strength of the bond (in terms of
lifetime) does not change. For the hydrogen bond to the amide oxy-
gen atom no such clear trend is to be found. In case of the endo-TSA
the occupancy is only lowered slightly in the presence of EGMA as
cross-linker as compared to the system with DVB as cross-linker.
However, in case of the exo-TSA the occupancy is lowered by more
than half. In both cases these changes are accompanied by similar
relative changes in the occupancy of the hydrogen bond between
the carbamate proton of the template and the doubly bond oxygen
atom of MAA. This can safely be assumed to be due to the close
proximity of the two groups in the structure of the TSA (cf. Fig. 3),
leading to a cooperative effect. This cooperative effect can also be
the background to the rather large difference in occupancy of these
two hydrogen bonds of the different TSA structures in the presence
of EGMA. In case of the exo-TSA the much lower binding to MAA is
contrasted by a higher occupancy of the hydrogen bond between
the carbamate proton and one of the doubly bound oxygen atoms of
EGMA. For these two occupancies the difference between the sys-
tems is similar (but differently signed), thus, the total binding of the
carbamate proton is similar in all studied systems. In the systems
with EGMA as cross-linker the proton binds in part to the EGMA,
which – due to the cooperative effect – also lowers the binding of
MAA protons to the amide oxygen atom. The difference in the rela-
tive distribution of the bonds of the carbamate proton is assumedly
due to differences in the randomized starting geometries favouring
one of the two similar binding modes over the other.
butadienylcarbamate (1, 20 mM), N,N-dimethyl acrylamide
(20 mM) and BHT (5 mg/10 mL solution) in toluene. Portions
(3 mL) of this solution were added to pressure tubes containing
100 mg polymer (no polymer in case of the solvent reaction). The
tubes were purged with nitrogen and heated to the respective
temperature (120, 40, and 20 ◦C) using an oil bath. At appropriate
intervals reaction was stalled by cooling on ice and samples of 1 mL
were taken. The samples were centrifuged for 5 min (10 000 rcf,
20 ◦C), the supernatant filtered, and HPLC analyses performed in
duplicate to determine the amount of product formed. Elution
was carried out with heptane containing 0.1% acetic acid and
15% isopropanol at a flow rate of 1 mL/min with UV detection at
210 nm. Afterwards, the polymer was re-suspended, the mixture
returned to the pressure tube, the tube was purged with nitrogen,
sealed and heating was resumed. Studies using inhibitor were
performed accordingly.
2.7. Molecular dynamics simulations
All calculations were performed using the AMBER10 [37] pro-
gram package. Studies of the prepolymerization mixture were set
up and conducted as has been described by Karlsson et al. [38].
Models of the respective prepolymerization mixtures were set up
containing 20 molecules of the respective TSA, 80 molecules of
MAA, 320 molecules representing the cross-linker (divinylbenzene
was modelled as a 1:1 mixture between para- and meta-isomer),
1320 toluene molecules and 4 molecules of ACHN. Simulations
˚
were run for 20 ns at 310 K using a 9.0 A cut-off for non-bonded
interactions.
3. Results
3.2. Polymer synthesis and evaluation
The subject of this study is the influence of an imprinted polymer
system with EGMA as cross-linker at different temperatures on the
Diels–Alder reaction shown in Fig. 1. This reaction has been the
subject of a recent study in which structures 5 and 6 (Fig. 2) were
used as transition state analogues in the preparation of imprinted
polymers based on DVB as cross-linker.
Imprinted polymers were prepared with a composition of
20 mol% functional monomer (methacrylic acid, MAA) and 80 mol%
cross-linker (ethylene glycol dimethacrylate, EGMA) in the pres-
ence of 5 mol% equivalents template (either endo- (5) or exo- (6)
TSA). Thus, the polymers have the same molar composition as those
used in our previous study. Physical and elemental characterisation
of the obtained polymers is to be found in Table 2.
3.1. Molecular dynamics simulations
After work-up of the polymers, batch binding studies with the
endo- or exo-TSA at 0.1 and 1 mM, respectively, were carried out
using the imprinted polymers and their controls in toluene. Table 3
shows the summary of the specific binding.
The specific binding exhibited by the EGMA polymers is much
higher than that of the corresponding DVB polymers (19–39% as
compared to 3–12%). While the larger surface area could lead to
the expectation of higher non-specific binding, the higher specific
binding shows that this effect is more than balanced by the forma-
tion of additional specific binding sites. Selectivity for the template
utilized in its preparation could, however, only be observed for
In order to gain better understanding of the differences
and similarities of the interactions between template and func-
tional monomer in the presence of different cross-linkers,
molecular dynamics simulations were performed, modelling the
pre-polymerization mixture over a timeframe of 20 ns. The sim-
ulations were analysed in terms of densities of the MAA protons
relative to the template molecules in order to identify which sites
1
In ref. [35] the corresponding formula is wrongly written with density d instead
P(EGMA)ENDO
.
of mass m. Results, however, are correct.

202
H. Henschel et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 199–205
Fig. 3. Grid density analysis of the distribution of MAA protons relative to the time-averaged structures of the two different TSAs in the presence of the two different
cross-linkers, respectively. Each colour represents the grid surface at 15 ps residence time for a single copy of the template.
Table 1
3.3. Assay
Occupancy and mean lifetime of hydrogen bonds in the systems modelled.
In order to investigate the catalytic properties of the
polymers, i.e. P(EGMA), the reaction between diene (1) and N,N-
dimethylacrylamide (2) in toluene was performed in the presence
of each of the different polymers prepared. The formation of the
products was monitored by HPLC.
When the reaction was carried out at room temperature the
imprinted polymers increased the rate of formation of the endo-
product approximately 6-fold, while the non-imprinted polymer
gave a 4.5-fold increase as compared to the solvent reaction
Cross-linker
TSA
EGMA
DVB
endo
exo
endo
exo
Oamide–HMAA
Occ.
ꢀ [ps]
Occ.
ꢀ [ps]
Occ.
ꢀ [ps]
Occ.
51.7%
5.4 3.5
13.6%
4.5 2.4
20.7%
0.7 0.5
6.5%
26.3%
6.2 3.7
12.7%
4.8 2.5
8.1%
0.7 0.5
15.0%
59.8%
4.9 3.6
22.5%
4.6 2.6
24.0%
0.7 0.4
59.9%
4.7 3.1
25.5%
4.5 2.7
22.0%
0.8 0.4
Ocarbamate–HMAA
OMAA–Hcarbamate
OEGMA–Hcarbamate
ꢀ [ps]
0.6 0.5
0.6 0.5
Table 3
Table 2
Elemental composition and physical analysis of polymers.
Specific binding of the endo or exo TSA (c = 1 mM or 0.1 mM) in toluene to the dif-
ferent polymers. The uptakes are the specific uptakes, i.e. the binding to the control
subtracted from the binding to the MIP.
Polymer code
TSA
Specific
swelling
Surface area
Anal. Calcd. [%]a
[m2 g−1
]
[ml g−1
]
Specific uptake [%]
1 mM
Ligand
Polymer
0.1 mM
P(EGMA)ENDO
P(EGMA)EXO
P(EGMA)REF
endo
exo
–
6.70
7.01
6.57
316.61
277.11
280.53
C: 57.8, H: 7.25, N: <0.3
C: 57.55, H: 7.3, N: <0.3
C: 57.15, H: 7.2, N: <0.3
endo TSA
endo TSA
exo TSA
exo TSA
P(EGMA)ENDO
P(EGMA)EXO
P(EGMA)ENDO
P(EGMA)EXO
24.31 0.74
21.65 1.91
19.51 2.55
22.69 0.83
38.60 2.24
30.47 2.55
30.18 3.28
31.35 7.87
a
Theoretical calculations (based on complete incorporation initiator radicals): C:
60.51, H: 7.16, N: 0.28.

H. Henschel et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 199–205
203
0.25
0.2
0.6
a
solvent [endo]
solvent [endo]
P
[endo]
[endo]
[endo]
P
[endo]
[endo]
[endo]
REF
REF
P
P
ENDO
ENDO
P
0.5
0.4
0.3
0.2
0.1
0
P
EXO
EXO
0.15
0.1
0.05
0
0
100 200 300 400 500 600 700 800 900
t [h]
0
50
100
150
200
t [h]
0.05
0.04
0.03
0.02
0.01
0
b
solvent [exo]
Fig. 4. Formation of 3 in the presence of PREF, PENDO, and PEXO and solvent only in
toluene at room temperature.
P
[exo]
[exo]
[exo]
REF
P
ENDO
P
EXO
(Fig. 4). As for the exo-product, a significant increase in its for-
mation could not be observed as concentrations remain below
the detection limit. However, diastereoselectivity of the catalysis
could not be observed, i.e. there is no observable difference in endo-
product formation between the polymers imprinted with endo- and
exo-TSAs. This, in turn, is in agreement with the results obtained
from the batch binding studies where no significant diastereose-
lectivity of binding of the TSAs was observed.
In a second study the reaction was carried out at 120 ◦C.
While formation of the endo-product is initially (t < 20 h) slightly
0
50
100
150
200
t [h]
Fig. 6. Formation of 3 and 4 in the presence of PREF, PENDO, and PEXO and solvent only
in toluene at 40 ◦C.
4
a
solvent [endo]
P
[endo]
[endo]
[endo]
REF
3.5
3
P
ENDO
EXO
P
faster in presence of imprinted polymer as compared to both
solvent reaction and control polymer, at longer reaction times
its concentration is lower if polymer is present (Fig. 5). In case
of the exo-product the concentration in presence of polymer is
at all sample times lower than for the solution reaction. Fur-
thermore, the concentration of the exo-product is lower for the
imprinted polymers than for the non-imprinted control polymer
(the same phenomenon can be observed for the endo-product at
reaction times >60 h, but there the trend is not conclusive). As for
the room temperature study, stereoselectivity in terms of prod-
uct formed in the presence of different polymers could not be
observed.
To further investigate the temperature dependence of the reac-
tion, another series of experiments was conducted at 40 ◦C. At
this temperature the formation of the endo-product is up to 9.5
times faster in presence of polymer than in its absence (Fig. 6).
Remarkably, the difference between imprinted and non-imprinted
polymers is far less distinguished in this study than at room
temperature. This is especially true for the formation of exo-
product, where there is (with respect to experimental error) no
difference to be found between the different polymers’ perfor-
mance. Nonetheless, a 2-fold enhancement of reaction rate can be
observed.
2.5
2
1.5
1
0.5
0
0
20
40
60
80
100
t [h]
b
solvent [exo]
P
[exo]
[exo]
[exo]
2.5
2
REF
P
ENDO
P
EXO
1.5
1
0.5
0
Another reaction assay was performed at room temperature in
presence of 20 mM endo-TSA (5) as inhibitor. The result of this
experiment was essentially the same as for the DVB-based poly-
mers in our previously published study [35], i.e. the reaction rate
was reduced by approximately 30% compared to the non-inhibited
reaction.
0
20
40
60
80
100
t [h]
Fig. 5. Formation of 3 and 4 in the presence of PREF, PENDO, and PEXO and solvent only
in toluene at 120 ◦C.

204
H. Henschel et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 199–205
4. Discussion
polymers. This could be explained by sites having high catalytic
activity being also more reactive towards the inhibiting reaction,
resulting in a stronger decrease for the imprinted polymers than
for the non-imprinted.
4.1. Influence of cross-linker
The first interesting finding in this study is the specific binding of
template to the imprinted polymers, which is substantially higher
for each of the EGMA polymer systems than for the comparable
DVB-based polymers. This is especially to be highlighted as it is
despite a larger surface area, which could be expected to lead to a
larger extent of non-specific binding.
Interestingly the higher specific binding observed in the batch
binding studies does not lead to higher reaction rates. Even though
a fundamental enhancement of the rate of product formation
is observed at room temperature, the DVB-based polymers fea-
tured a more pronounced effect. Furthermore, the relative effect
of inhibitor is similar for both systems, which implies similar ratios
between the number of reactions taking place on the polymer sur-
face and inside binding sites.
In the results of the molecular dynamics studies it can be seen
that the interactions between MAA and the template are similar in
the presence of both cross-linkers – the strength of the different
interactions in terms of lifetime is essentially identical. In the case
of EGMA as cross-linker, however, there is competition between
the template and EGMA as acceptors of hydrogen bonds from MAA
on one hand, and between MAA and EGMA as acceptors of hydro-
gen bonds from the TSA on the other hand. The larger diversity of
interactions with the template in the system containing EGMA can
easily explain the higher total binding observed as compared to the
system containing DVB. Simultaneously, the higher specific binding
observed for the EGMA system can be explained by the competi-
tive binding of the different functionalities of functional monomer
and cross-linker, leading to a situation in which functional groups
that otherwise could lead to non-specific binding are involved in
interactions between MAA and EGMA.
A common characteristic of the two different polymer systems
is the lack of diastereoselectivity in terms of product formed in their
presence. The fact that the choice of cross-linker does not have
an impact on diastereoselectivity supports our previously made
argument that the placement of a single double bond within the
template structure is not sufficient to induce selective recognition.
Nonetheless, the employment of EGMA-based polymers allowed
for observation of exo-product formation in the presence of poly-
mer. The formation over time parallels that of the endo-product –
at a significantly lower rate.
A higher reactivity of the specific binding sites towards the side
reaction is also supported by the data obtained at 40 ◦C. While
the reaction rate is – as expected – much higher than at room
temperature, the differences between the imprinted and reference
polymers are much less distinct. This suggests a diminished effect
of the specific binding sites, which in turn can be explained by
the integrity of the binding sites being destroyed by side reactions,
while on the polymer surface higher temperatures are required in
order for this process to take place.
Both the fact, that the unusual temperature dependent
behaviour is more distinct for the polymer systems showing higher
catalytic activity (i.e. the DVB-based polymers), and that the effect is
more distinct for the imprinted than non-imprinted polymers, sug-
gests that the process diminishing the polymers’ catalytic activity
is similar to the catalysed reaction itself, e.g. a reaction of substrates
and/or product with residual double bonds in the polymer.
5. Conclusions
In this study a series of molecularly imprinted polymers was
designed with the help of analogues for two possible transition
states of the Diels–Alder reaction of 1 and 2. The polymers show
specific binding of the template employed in their synthesis and
significant catalytic activity for the reaction studied.
Importantly, at high temperature (120 ◦C) significantly less
product was formed in the presence of polymer than in its absence.
This is analogous to a temperature-dependent behaviour that – by
our group – only recently was reported for the first time for another
imprinted polymer system. Furthermore, in this study we investi-
gated the temperature dependence of the polymer’s performance
in more detail by performing the reaction assay at an additional
temperature. The additional information obtained from this new
polymer system not only supports our provisional theory that at
higher temperature the polymer participates in the reaction, it also
suggests that this reaction preferentially takes places within the
catalytically active sites.
Finally, the equilibria in the pre-polymerization mixtures were
studied by means of molecular dynamics simulations. The results
illustrate the importance of different interactions of the tem-
plate with functional monomer and cross-linker molecules, as
well as interactions between functional monomer and cross-
linker. Insights from these simulations were successfully applied
to explain the different properties of the different polymer sys-
tems. Thisdemonstratesonceagaintheimportanceof theprocesses
taking place in the pre-polymerization mixture for the imprint-
ing process and ultimately for the performance of the resulting
polymer.
4.2. Influence of temperature
Interestingly, the unprecedented temperature dependent
behaviour of polymer activity, reported in our previous study [35],
could be reproduced in spite of the fundamental change in the
polymers’ composition (the cross-linker makes up 80 mol% of the
reaction mixture). Even though the effect is less pronounced than
for the DVB-based polymer, a considerable decline in reaction rate
compared to the solution reaction could be observed at 120 ◦C.
However, due to the lesser extent of the effect, a more detailed
analysis is possible. Amongst others, it can be observed that the
rate of formation of the endo-product initially (t < 20 h) is slightly
higher in the presence of imprinted polymers than in solvent or
with the reference polymer. This indicates that the polymers, even
at 120 ◦C, have some catalytic activity from the start which then,
under the course of the experiment, is lost. This supports our the-
ory that additional reactions of substrates and/or product of the
Diels–Alder reaction with the polymer occur, blocking the specific
binding sites and obstructing further reactions with its surface.
Another finding is that the activity of the imprinted polymers
at longer reaction times becomes lower than that of the reference
Acknowledgements
We gratefully acknowledge the financial support of the Swedish
Research Council (VR), the Swedish Knowledge Foundation (KKS),
Graninge Foundation and the Linnæus University. We are indebted
to Dr. Sacha Legrand and Dr. Jesper G. Wiklander (both Linnæus
University) for the recording of 2D NMR spectra.
References
[1] K.C. Nicolaou, S.A. Snyder, T. Montagnon, G. Vassilikogiannakis, Angew. Chem.
Int. Ed. 41 (2002) 1668–1698.
[2] E.J. Corey, Angew. Chem. Int. Ed. 41 (2002) 1650–1667.
[3] U. Pindur, G. Lutz, C. Otto, Chem. Rev. 93 (1993) 741–761.
[4] M.R. Trembley, T.J. Dickerson, K.D. Janda, Adv. Synth. Catal. 343 (2001) 577–585.

H. Henschel et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 199–205
205
[5] V.E. Gouverneur, K.N. Houk, B. de Pascual-Teresa, B. Beno, K.D. Janda, R.A.
Lerner, Science 262 (1993) 204–208.
[6] J.T. Yli-Kauhaluoma, J.A. Ashley, C.-H. Lo, L. Tucker, M.M. Wolfe, K.D. Janda, J.
Am. Chem. Soc. 117 (1995) 7041–7047.
[7] C. Alexander, H.S. Andersson, L.I. Andersson, R.J. Ansell, N. Kirsch, I.A. Nicholls,
J. O’Mahoney, M.J. Whitcombe, J. Mol. Recogn. 19 (2006) 106–180.
[8] D. Batra, K.J. Shea, Curr. Opin. Chem. Biol. 7 (2003) 434–442.
[9] K. Haupt, K. Mosbach, Chem. Rev. 100 (2000) 2495–2504.
[10] M. Komiyama, T. Takeuchi, T. Mukawa, H. Asanuma, Molecular Imprinting:
From Fundamentals to Applications, Wiley-VCH, Weinheim, 2002.
[11] S. Piletsky, A. Turner (Eds.), Molecular Imprinting of Polymers, Landes Bio-
science, Georgetown, 2006.
[12] B. Sellergren (Ed.), Molecularly Imprinted Polymers: Man-Made Mimics of Anti-
bodies and Their Applications in Analytical Chemistry, Elsevier, Amsterdam,
2001.
[13] G. Wulff, Chem. Rev. 102 (2002) 1–28.
[14] M. Yan, O. Ramström (Eds.), Molecularly Imprinted Materials: Science and
Technology, Dekker, New York, 2005.
[23] B. Sellergren, R.N. Karmalkar, K.J. Shea, J. Org. Chem. 65 (2000) 4009–4027.
[24] A.G. Strikovsky, D. Kasper, M. Grün, B.S. Green, J. Hradil, G. Wulff, J. Am. Chem.
Soc. 122 (2000) 6295–6296.
[25] G. Wulff, T. Gross, R. Schönfeld, Angew. Chem., Int. Ed. Engl. 36 (1997)
1962–1964.
[26] J. Svenson, N. Zheng, I.A. Nicholls, J. Am. Chem. Soc. 126 (2004) 8554–8560.
[27] J.V. Beach, K.J. Shea, J. Am. Chem. Soc. 116 (1994) 379–380.
[28] R. Müller, L.I. Andersson, K. Mosbach, Makromol. Chem., Rapid Commun. 14
(1993) 637–641.
[29] J. Hedin-Dahlström, J.P. Rosengren-Holmberg, S. Legrand, S. Wikman, I.A.
Nicholls, J. Org. Chem. 71 (2006) 4845–4853.
[30] J. Matsui, I.A. Nicholls, I. Karube, K. Mosbach, J. Org. Chem. 61 (1996) 5414–5417.
[31] A.N. Cammidge, N.J. Baines, R.K. Bellingham, Chem. Commun. (2001)
2588–2589.
[32] X.-C. Li, K. Mosbach, Macromol. Rapid Commun. 18 (1997) 609–615.
[33] B.P. Santora, A.O. Larsen, M.R. Gagné, Organometallics 17 (1998) 3138–3140.
[34] A. Viesnjevsky, R. Schomäcker, E. Yilmaz, O. Brüggemann, Catal. Commun. 6
(2005) 601–606.
[15] Y. Kawanami, T. Yunoki, A. Nakamura, K. Fujii, K. Umano, H. Yamauchi, K.
Masuda, J. Mol. Catal. A 145 (1999) 107–110.
[35] N. Kirsch, J. Hedin-Dahlström, H. Henschel, M.J. Whitcombe, S. Wikman, I.A.
Nicholls, J. Mol. Catal. B: Enzym. 58 (2009) 110–117.
[16] J.M. Kim, K.D. Ahn, G. Wulff, Macromol. Chem. Phys. 202 (2001) 1105–1108.
[17] J.-Q. Liu, G. Wulff, J. Am. Chem. Soc. 130 (2008) 8044–8054.
[18] K. Ohkubo, Y. Urata, S. Hirota, Y. Funakoshi, S. Usui, K. Yoshinaga, J. Mol. Catal.
A 101 (1995) L111–L114.
[19] K. Ohkubo, Y. Funakoshi, Y. Urata, S. Usui, T. Sagawa, J. Chem. Soc., Chem.
Commun. (1995) 2143–2144.
[20] D.K. Robinson, K. Mosbach, J. Chem. Soc., Chem. Commun. (1989) 969–970.
[21] T. Sagawa, H. Ihara, E.K. Mitamura, K. Ohkubo, Recent Res. Dev. Pure Appl. Chem.
3 (1999) 57–71.
[36] J.G. Karlsson, L.I. Andersson, I.A. Nicholls, Anal. Chim. Acta 435 (2001)
57–64.
[37] D.A. Case, T.A. Darden, T.E. Cheatham III, C.L. Simmerling, J. Wang, R.E. Duke,
R. Luo, M. Crowley, R.C. Walker, W. Zhang, K.M. Merz, B. Wang, S. Hayik, A.
Roitberg, G. Seabra, I. Kolossváry, K.F. Wong, F. Paesani, J. Vanicek, X. Wu, S.R.
Brozell, T. Steinbrecher, H. Gohlke, L. Yang, C. Tan, J. Mongan, V. Hornak, G.
Cui, D.H. Mathews, M.G. Seetin, C. Sagui, V. Babin, P.A. Kollman, AMBER 10,
University of California, San Francisco, 2008.
[38] B.C.G. Karlsson, J. O’Mahony, J.G. Karlsson, H. Bengtsson, L.A. Eriksson, I.A.
Nicholls, J. Am. Chem. Soc. 131 (2009) 13297–13304.
[22] B. Sellergren, K.J. Shea, Tetrahedron: Asymmetry 5 (1994) 1403–1406.