Urea-Based Imprinted Polymer Hosts with Switchable Anion Preference

Article abstract of DOI:10.1021/jacs.0c00707

The design of artificial oxyanion receptors with switchable ion preference is a challenging goal in host-guest chemistry. We here report on molecularly imprinted polymers (MIPs) with an external phospho-sulpho switch driven by small molecule modifiers. The polymers were prepared by hydrogen bond-mediated imprinting of the mono-or dianions of phenyl phosphonic acid (PPA), phenyl sulfonic acid (PSA), and benzoic acid (BA) using N-3,5-bis-(trifluoromethyl)-phenyl-?-4-vinylphenyl urea (1) as the functional host monomer. The interaction mode between the functional monomer and the monoanions was elucidated by 1H NMR titrations and 1H-1H NMR NOESY supported by molecular dynamic simulation, which confirmed the presence of high-order complexes. PPA imprinted polymers bound PPA with an equilibrium constant Keq = 1.8 × 105 M-1 in acetonitrile (0.1percent 1,2,2,6,6-pentamethylpiperidine) and inorganic HPO42- and SO42- with Keq = 2.9 × 103 M-1 and 4.5 × 103 M-1, respectively, in aqueous buffer. Moreover, the chromatographic retentivity of phosphonate versus sulfonate was shown to be completely switched on this polymer when changing from a basic to an acidic modifier. Mechanistic insights into this system were obtained from kinetic investigations and DSC-, MALDI-TOF-MS-, 1H NMR-studies of linear polymers prepared in the presence of template. The results suggest the formation of template induced 1-1 diad repeats in the polymer main chain shedding unique light on the relative contributions of configurational and conformational imprinting.

Full text of DOI:10.1021/jacs.0c00707

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Article
Urea-based imprinted polymer hosts with switchable anion preference
Sudhirkumar Shinde, Anil Incel, Mona Mansour, Gustaf D Olsson,
Ian A Nicholls, Cem Esen, Javier Urraca, and Börje Sellergren
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 19 May 2020
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Urea-based imprinted polymer hosts with switchable anion preference
†,‡
†,
ɸ,
Sudhirkumar Shinde†,ɸ,§,‡ Anil Incel , Mona Mansour , Gustaf D. Olsson⊥, Ian A. Nicholls⊥, Cem Esen
,
∥
∇
Javier Urracaɸ,#, Börje Sellergren*,†,ɸ
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†
Department of Biomedical Sciences, Faculty of Health and Society, Malmö University, 20506 Malmö, Sweden.
ɸ
Faculty of Chemistry, Technical University of Dortmund, Otto-Hahn-Str. 6, 44227, Dortmund, Germany.
⊥
Bioorganic & Biophysical Chemistry Laboratory, Linneaus University Center for Biomaterials Chemistry, Department of Chemistry
& Biomedical Sciences, Linnaeus University, 39182 Kalmar, Sweden.
KEYWORDS (Word Style “BG_Keywords”). If you are submitting your paper to a journal that requires keywords, provide significant key-
words to aid the reader in literature retrieval.
ABSTRACT: The design of artificial oxyanion receptors with switchable ion preference is a challenging goal in host-guest chemistry. We here
report on molecularly imprinted polymers (MIPs) with an external phospho-sulpho switch driven by small molecule modifiers. The polymers
were prepared by hydrogen bond-mediated imprinting of the mono- or di-anions of phenyl phosphonic acid (PPA), phenyl sulphonic acid
(PSA) and benzoic acid (BA) using N-3,5-bis (trifluoromethyl) phenyl-N´-4-vinylphenyl urea (1) as functional host monomer. The interaction
mode between the functional monomer and the mono-anions was elucidated by H-NMR titrations and H-1H-NMR NOESY supported by
dynamic simulations which confirmed the presence of high order complexes. PPA imprinted polymers bound PPA with an equilibrium constant
Keq = 1.8 x 105 M-1 in acetonitrile (0.1% 1,2,2,6,6-pentamethylpiperidine) and inorganic HPO42- and SO4 2- with Keq = 2.9 x 103 M-1 and 4.5 x 103
M-1 respectively in aqueous buffer. Moreover, the chromatographic retentivity of phosphonate versus sulfonate was shown to be completely
switched on this polymer when changing from a basic to an acidic modifier. Mechanistic insights into this system were obtained from kinetic
1
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1
investigations and DSC-, MALDI-TOF-MS-, H-NMR- studies of linear polymers prepared in presence of template. The results suggest the
formation of template induced 1-1 diad repeats in the polymer main chain shedding unique light on the relative contributions of configurational
and conformational imprinting.
Discrimination between phosphate and sulfate and other isosteric
oxyanions (e.g. arsenate, carbonate) occurs in the sulfate and phos-
INTRODUCTION
The molecular constituents of a living cell are predominantly water
soluble molecules carrying a net negative charge, e.g. cofactors, en-
zyme substrates, nucleic acids, the lipid double layer and the gly-
cocalix.1,2,3 As a consequence, evolution has resulted in highly refined
receptors capable of recognizing anions in water. One of the most
impressive examples are the proteins designed to selectively bind
and/or transport either sulfate or phosphate ions. These two pyram-
idal anions are nearly isosteric with similar molecular volumes and
central atom-oxygen bond lengths whereas they differ with respect
to Lewis basicity and hydrophilicity (Table 1).
phate binding proteins predominantly through multiple comple-
mentary hydrogen bonding (H-bond) interactions involving main
chain amides (nests) in a water poor microenvironment with minor
involvement of charge complementary residues.1 Notably, no
charged residue is involved in the sulfate binding protein binding site
which binds sulfate specifically with an equlibrium binding constant
Keq = 106 M-1 in water (5.0 ≤ pH ≤ 8.0), an impressive feat given the
strong hydration of this anion. As a consequence, this overturns the
Hofmeister series of the salting out tendency for anions which oth-
erwise increases in the order; CH3COO- < HPO4 < SO4
.
2-
2-
Inspired by this remarkable performance, much effort has been de-
voted to the development of neutral sulfate/phosphate binding
hosts.4-5 In contrast to the more common approach based on charged
receptors, neutral hosts can achieve higher selectivity but are chal-
lenging to construct since H-bonding is considerably weaker in polar
Table 1. Physical properties of sulfate and phosphate anions
Anion
pKa 1 a
pKa 2 a
pKa 3 a
Molecular
Volumeb
(Å3)
Anion
hydration
energyb
(kJ/mol)
solvents. Nevertheless, both macrocyclic e.g. pyrrole, saphyrine,6
3-
PO4
2.1
-3
7.2
1.9
10.9
-
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51
-498
peptide7 and acyclic podand hosts e.g. ureas,
have been reported showing high sulfate affinity. However, these
hosts rarely overturn the Hofmeister solvation governed series of an-
ion affinity especially with respect to the relative preference for phos-
squaramides12-14
8-11
2-
SO4
-1130
a) pKa of conjugate acid
b) Values for PO43- and SO4
.
2- 3
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phate/sulfate.6 Moreover, these anion receptors are seldomly capa-
ble of switching the anion preference or of binding and releasing the
guest upon external stimuli.13
in a cyclic H-bonded complex. Also, several structural and composi-
tional parameters can be readily tuned in order to enhance HG asso-
ciation strengths (Scheme 2).
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This highlights a need of neutral hosts with tunable microenviron-
ment capable of alternating phosphate/sulfate recognition. This can
in principle be achieved by embedding designed low molecular hosts
in a polymer scaffold imparting a lower dielectric microenviron-
ment.1 Alternatively, a macromolecular host can be designed from
simpler building blocks by molecular imprinting.15-20 Monomers are
here chosen or designed in order to complement functional groups
of a template molecule. Polymerizing the monomer-template com-
plexes into a crosslinked polymer matrix and removal of the tem-
plate, leaves behind sites capable of recognizing the template. This
approach benefits from spontaneous self-assembly guiding the bind-
ing groups to their positions in the receptor site. Thus, the structure
of the final binding site is a priori unknown.
Notable examples of MIPs for anion recognition in water have been
reported using both charged, e.g. imidazolium21-23, amidinium,24-27
and neutral host monomers e.g. urea,28-31 thiourea,32 squaramide33
and others34. Some time ago we introduced urea based host mono-
mers acting as potent H-bond donors for stoichiometric imprinting
of oxyanions or other H-bond acceptors.35 Receptors for beta-lactam
antibiotics,36 phospho-peptides,28 peptide biomarkers,37 phospholip-
ids,22 sugar acids31 and sulfated peptides29 were demonstrated.
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Scheme 2. Structural and compositional parameters for tuning host-
guest association strength. EWG=electron withdrawing group.
The affinity for the guest increases with the acidity of the urea pro-
tons (donor capacity, R3, R4, R5 = electron withdrawing groups
(EWGs)) and the basicity of the oxyanion (acceptor capacity), but
this can be offset by the ability of the host to self-associate and host
deprotonation.40 The latter leads to nondirectional electrostatic in-
teractions and is promoted by increased solvent polarity.41 This is an
undesired effect since it undermines the structural integrity of the
hydrogen bond connected HG complex – hence a compromise be-
tween host acidity and guest basicity has to be found that maximizes
the complex strength. In this context, no deprotonation has so far
been observed for the host monomer, 1, used in the current study
(Scheme 1). The nature of the counter-cation also influences the
HG complex stability. Complexation can be easily induced by pro-
ton transfer to amine bases but this causes undesirable competition
between the urea host and the protonated amine for binding the
guest. Stronger complexation is therefore induced by the use of
bulky tertiary amines (e.g. pentamethylpiperidine, PMP) or quater-
nary ammonium ions like tetrabutylammonium (TBA), which have
been shown to produce pronounced counterion memory effects.28, 42
Another factor is the level of host preorganization, which impacts on
the size of the entropic penalty upon guest complexation due to the
need to freeze internal rotors. For mono-urea host monomers this
can in principle be enhanced by introducing two polymerizable
groups (crosslinkers) in the host thereby increasing host stiffness
(R4= vinyl in Scheme 2).41 For ternary HG complexes formed from
phosphate dianions and urea hosts (2:1 complex in Scheme 3), host
preorganization has been enhanced by introducing the cleft-like host
monomer 2 (1:1 complex in Scheme 3).28
To gain insight into the nature of these imprinted receptors at a mo-
lecular level we here report an in-depth investigation of MIPs for
simple oxyanions (Scheme 1).
Scheme 1. Plausible structural formulas of H-bonded complex between
oxyanions and 1,3-diaryl urea monomer 1 (left) and structures of
oxyacids used in the study (right). PPA=phenylphosphonic acid;
PSA=phenylsulfonic acid; BA=benzoic acid; PPrA=phenylphosphoric
acid.
With this we aimed to answer the following questions: 1) What level
of oxyanion selectivity and affinity can be achieved with MIPs in or-
ganic and aqueous media? 2) Can MIPs recognize inorganic anions
in water? 3) Can the Hofmeister preference be overturned? 4) Can
we switch oxyanion preference using the same MIP? 5) What are the
mechanisms underlying the imprinting process?
2
1
1
Based on combined physical characterization, modelling, spectro-
scopic studies of prepolymerization complexes, and studies of the
polymerization kinetics we here offer answers to the aforementioned
questions.
N
H
O
O
N
H
H
O
H
O
N
H
O
O
N
H
H
H
N
N
CF3
O
P
N
F3C
N
O
P
O
O
TBA
TBA
TBA
TBA
CF3
CF3
RESULTS AND DISCUSSION
2:1 complex
1:1 complex
Monomer/template complex formation. 1,3-Disubstituted ureas
have long been exploited as neutral hosts (H) for complexing oxyan-
ion guests (G).38-39 They provide a twofold H-bond donor which
with the acceptor (carboxylate, phosphate, or sulfonate ions) results
Scheme 3. Anticipated ternary or binary monomer-template complexes
formed using mono- (left) or di- (right) functional urea host monomers.
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Considering oxyanion-oxourea complexation, the stability has been
shown to increase in the order of increasing acceptor basicity i.e.
Table 3. H-bond analysis results*
1
2
3
4
5
6
Ta
PMP
---
PMPH+
0.00
1
EGDMA
0.00
THF
0.00
0.01
9.50
-
-
-
2
PhSO3 < PhOP(OH)O2 < PhP(OH)O2 ≤ PhCOO- < PhPO3 - ≤
2- 43 To verify this trend we decided to investigate 1 with re-
PP1
PMPH+
1
0.00
64.54
24.26
0.11
0.00
0.11
PhOPO3
.
spect to its complex stability with model anions in the form of TBA-
hydroxide salts of PPA, PPrA, BA and PSA. Interaction strength and
P1C
---
0.00
0.00
---
0.00
18.27
1
complex stoichiometries were investigated through H-NMR titra-
tion or isothermal titration calorimetry (ITC) (see the supporting
information (SI)). Titrating 1 with PPA·TBA in DMSO-d6 and
PPA·2PMP in THF-d8 resulted in strong downfield shifts of urea
protons Ha and Hb and more moderate shifts of the aromatic pro-
tons Hc-f (Fig. S1). The moderate downfield shifts of Hf and upfield
shift of He are in agreement with previous NMR titration study of
1,3-bis(4-nitrophenyl)urea with acetate.44 1H-NMR studies using
Jobs method of continuous variation confirmed a preferred 1:1 stoi-
chiometry. Complex stability constants were calculated using the in-
duced downfield shifts of the urea protons of 1 (Fig. S1). With re-
spect to monoanions, 1 formed more stable complexes with benzo-
ate anion BA (Keq=8820 M-1) followed by PPA (Keq=7005 M-1) and
PPrA (Keq=676 M-1), whereas the complex with the sulfonate PSA
was too weak to be detected (Table S1). This agrees with the trend
determined by Kelly et al.43 Comparing with monoanions, the dian-
ions of PPA and PPrA interact more strongly with 1.28 The relative
affinity of the two dianions is reflected in the downfield shifts (Fig.
1, 2) where the latter (Fig. 1c) is more pronounced than that of the
former (Fig. 1b), again in line with the literature results.
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PPA
77.27
13.42
0.00
0.00
---
---
0.04
0.10
46.69
25.93
18.41
12.96
P1N
P2C
1
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PMPH+
23.42
74.37
---
---
0.00
0.00
0.00
0.27
0.00
0.00
7.55
1
15.11
PPA
38.96
33.57
0.00
0.00
---
---
0.09
0.50
77.59
48.62
14.66
24.68
P2N
P3C
1
PMPH+
75.69
47.39
---
---
0.00
0.00
0.00
0.05
0.00
0.13
6.02
1
11.84
P3N
P4C
P4N
PSA
0.08
1.06
0.00
0.00
---
---
0.02
0.36
2.80
2.00
1
29.18
14.78
PMPH+
80.58
85.22
0.00
0.00
0.00
0.00
0.00
0.19
0.00
0.01
7.15
1
16.85
To gain more insight we performed molecular dynamics (MD) sim-
ulations of the prepolymerization mixtures. MD-simulations offer
unique insights into the nature of the interactions between polymer-
ization mixture components providing diagnostic/prognostic corre-
lation to polymer performance.46,47 Compositions of simulated and
evaluated systems and chemical structures are presented in Table 2
and Fig. S15.
PSA
0.08
1.50
0.00
0.00
---
---
0.05
0.39
2.76
2.00
1
30.18
15.69
* This table presents the summarized average interactions, as percent of
total simulation time, between all atom pairs of indicated molecular spe-
cies. See Fig. S15 for molecular representations and defined abbrevia-
tions. All analyses involving templates were averaged against the number
of templates (20 in each system). All interactions including the urea
monomer 1 but not the template was averaged against the number of
urea monomer molecules in each system (see Table 2). Crosslinker in-
teractions with solvent and bases were averaged against the number of
crosslinkers (800 in all systems) and, finally, base self-interaction and
solvent interaction were averaged against the number of base molecules
in each system (20 in P1, P3 N/C systems and P4C, 40 in P2 N/C and
P4N systems). a Refers to interactions with the template in the analyzed
system.
Table 2. Composition of systems simulated and evaluated
**
P1N
PPA
20
P1C
PP1
---
P2N
PPA
40
P2C
PP2
---
P3N
PSA
20
P3C
PS1
---
P4N
PSA
40
P4C
PS1
20
T *
PMP
PMPH+
1 *
*
---
20
---
40
---
20
---
20
20
20
40
40
20
20
40
40
EGDMA
THF
800
2760
800
2760
800
2760
800
2760
800
2760
800
2760
800
2760
800
2760
However, the monoanionic PSA template (PS1) performs as well,
even slightly better than PP2 considering complex formation with 1
(Tables S8, S9 and S10). A rank order of affinity for 1 was observed
where anionic neutral PSA < neutral PPA < anionic PP1 < dianionic
PP2 < PS1 (Table 3 and Tables S8-S10). This obviously disagrees
with the NMR results where no interactions between PS1 and 1
could be detected. The systems differ though with respect to solvent
polarity, the base, presence of crosslinker and the concentration of
all species which we believe can account for the contrasting results
(vide infra).45-48
To gain further insight into the nature of the urea complexes be-
tween 1 and the two anions we performed 2D-NOESY NMR exper-
iments and molecular modelling as reported by others.11 As shown
in Fig. 3a the free 1 shows strong cross-peaks with the residual water
signal at ca 2.9 ppm indicating water protons close in space to the
urea protons. In addition, weaker cross-peaks are observed between
The table presents numbers of virtual molecular models of each mole-
cule included in performed simulations of systems. See Figure S15 for
molecular representations and defined abbreviations.
* T=template (20 eq.), PP1 = PPA monoanion, PP2 = PPA dianion, PS1
= PSA monoanion, PMP = 1,2,2,6,6-pentamethylpiperidine, EGDMA =
ethylenglycoldimethacrylate.
**PXN/C, Polymer system X, neutral (N) or charged (C).
Full system all-atom simulations were performed using the same
stoichiometries employed for polymer synthesis. The results overall
confirmed the presence of stable interactions between the neutral,
mono- and di-valent anions of PPA and 1 (Table 3, Tables S8-S10
and Fig. S15) with the dianion of PPA (PP2) (PPrA not investigated
using MD) displaying a higher degree of H-bonding than the
monoanion (PP1) or neutral PPA template.
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the two-urea protons Ha and Hb resulting from the Z, Z-urea con- As expected, the degree of complexation increases with the template
1
2
3
4
5
6
7
8
former with the two protons juxtaposed (Fig. 3e). The addition of
half an equivalent of PPA●2PMP (Fig. 3b) and PPrA●2PMP (Fig.
3c) to 1 gave rise to a stronger NOE between these protons and an
interesting crosspeak between the more acidic Ha proton and the ex-
changeable PMPH+ proton, visible as a broad signal at 3.5 ppm.
charge, e.g. P2C (T= PPA dianion) display clusters involving any
part of 6 molecules of 1 within 5 Å from a template molecule,
whereas only half of this number is seen in P1C. Generally, PMPH+
was found in the simulations to be in close proximity to the tem-
plates and engaged in extensive H-bonding with charged templates
(Table 3). These results support the observed solubilization of the
template PPA·2PMP in the presence of 1 in THF (Scheme 4).
Whereas the ammonium phosphonate salt was poorly soluble in this
solvent, the solution became completely clear after addition of two
equivalents of 1. This provides unequivocal visual proof for the pres-
ence of prepolymerization complexes between 1 and PPA·2PMP.
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Polymer preparation and characterization. Imprinted and non-
imprinted polymers were thereafter prepared and characterized (see
SI and Tables S2-S4) using the urea host monomer 1 in a 2:1 and 1:1
stoichiometric ratio to the templates: PPA, PSA or BA in their
monoanionic or dianionic forms (Scheme 4). Non-imprinted poly-
mers (PN) were prepared identically to the imprinted polymers
though in the absence of template. Fig. S2 shows the 25-36 µm par-
ticle fraction obtained after crushing and sieving of the polymer
monoliths and template removal by solvent extraction. These were
subjected to physical characterization as follows. C, H, N elemental
analysis of imprinted and non-imprinted polymers after removal of
template matched calculated values after correction for water up-
take. This is reflected in the agreement between calculated and
found N/C ratios (Table S3). Thermogravimetric analysis (TGA)
of the polymers produced weight loss curves typical for highly cross-
linked networks with onset above 200°C (Fig. S3) and complete
weight loss obtained at 450°C. Meanwhile, the transmission FTIR
spectra (Fig. S4) showed all characteristic bands with no apparent
difference between imprinted and nonimprinted polymers all in all
indicating a stoichiometric monomer incorporation and successful
template removal from the imprinted polymers.
Figure 1. 1H-NMR spectra of a) FM1 and b) 2:1 ratio of 1:PPA·2PMP
and (c) 2:1 ratio of 1:PPrA·2PMP in THF-d8. [1]=30mM.
The former reflects stabilization of the Z,Z-conformer whereas the
latter a close contact between 1 and PMP, this providing an indirect
support for the existence of the anticipated ternary complex between
1, PMP and the dianions (Scheme 3a) This is in agreement with pre-
vious reports on urea- and squaramide- based sulfate receptors.12, 49
Results from MD simulations also support the argument for higher
order complexes observed in cameo images from the production run
(Fig. S16 and S17) and in the radial distribution function (RDF)
analyses (Table 4).
Table 4. Numbers of molecules within a 5.05 Å cutoff from
each template*
EGDMA
PMPH+
13
PMP
---
0
T
0
5
0
2
1
0
0
0
THF
15
17
7
1
3
1
6
3
4
1
6
1
T-P1C
T-P1N
T-P2C
T-P2N
T-P3C
T-P3N
T-P4C
T-P4N
8
12
5
---
33
---
0
13
8
---
17
14
21
14
20
14
---
0
12
8
---
11
0
12
---
1
* Values presented assuming the template as the solute molecule with an
average number of molecules within the cutoff for an average template.
Figure 2. 1H-NMR chemical shift changes of urea host monomer 1
complexation with PPA·2PMP or PPrA·2PMP. Δδ = δ (in the pres-
ence of anion) – δ (in the absence of anions), induced by addition of
1/2 equivalent of different anions to receptor 1.
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54
55
56
57
58
59
60
c)
d)
e)
Figure 3. 1H-1H-NMR NOESY spectra of 1: a) and b) (close-up), c) 2:1 ratio of 1:PPA●2PMP and (d) 2:1 ratio of 1:PPrA●2PMP in THF-d8 and (e)
anion induced conformational stabilization detected by an increase H-1H-NMR NOESY between Ha and Hb. [1]=30mM. The arrows indicate the
1
urea-water (a) and urea Ha,Hb cross peaks (b-d).
This contrasted with the results from nitrogen sorption analysis and
swelling tests, both reflecting porous and structural properties of the
polymers (Fig. S5, Table S4). All polymers exhibited mesoporous
N
H
O
O
N
H
H
N
H
H
morphology with surface areas exceeding 200 m2g-1 and average pore
diameters of roughly 4 nm. However, in agreement with our previ-
ous report,28 the imprinted polymers showed a lower surface area
and a higher swelling factor than the non-imprinted polymers. We
tentatively attributed this template-induced difference in morphol-
ogy to two effects of the divalent anion, 1) its action as a physical
crosslinking agent and 2) its action as a good solvent for the growing
chains leading to delayed phase separation and a more gel-like mor-
phology. Below we will invoke a third alternative explanation to the
observed differences based on the dianions ability to coordinate two
monomer units of 1 in a geometry favoring cyclopolymerization
(vide infra).
H
N
CF3
N
F3
C
N
O
P
O
O
PMPH
CF3
PMPH
CF3
EGDMA
ABDV, 50 o
THF
C
CF3
O
N
H
N
H
O
H
H
F3C
N
N
CF3
CF3
CF3
CF3
Scheme 4. Solubilization of the bis PMP-salt of PPA by the addition of
two equivalents of urea host monomer 1 in THF and procedure for
imprinting.
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Template binding studies. To evaluate the anion recognition prop- than P1/PN1 and correlates with results derived from MD simula-
1
2
3
4
5
6
7
8
erties of the materials we first assessed their ability to rebind their
corresponding templates under static conditions. The polymers
were incubated in acetonitrile in presence or absence of base (PMP)
with the anion templates (Fig. 4) followed by quantification of un-
bound template by HPLC.
tions. The non-imprinted polymers (NIPs) displayed consistently
less binding than the MIPs with the corresponding binding curves
rapidly diverging in the low concentration interval of 0-0.5 mM.
This appears clearly in the plots of the differential uptake in Fig. 5c
reflecting MIP binding corrected for binding to the NIP (assuming
the latter to reflect the nonspecific binding contribution). Both of
these curves fitted with a Langmuir mono-site model and gave rise
to nearly identical Bmax values reflecting a similar imprinting effi-
ciency of ca 30% (given identical template concentration in the pre-
polymerization mixture) and with P2 showing a markedly higher af-
finity than P1 (Fig. 5d). The latter is reflected in the higher equilib-
rium constant of P2 (Keq=1.9x105 M-1) versus P1 (Keq=3.1x104 M-1).
NIP
MIP
+ PMP
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
+ PMP
- PMP
- PMP + PMP
- PMP
150
100
50
150
100
50
a)
b)
P3 P4 P3 P4
P5 P6 P5 P6
P1 P2 P1 P2
PPA
PSA
BA
Figure 4. Absorbed amount in percent of PPA (a), PSA (b) and BA (c)
by anion imprinted and non-imprinted polymers in the absence and
presence of 0.1% PMP modifier.
0
0
0.0
0.5
1.0
1.5
40
0.0
0.5
1.0
1.5
F (mM)
F (mM)
c)
Fig. 4 shows the degree of template binding to the polymers in pres-
ence or absence of PMP. MIPs generally bound more of the tem-
plates than NIPs with the largest uptake shown by the polymers pre-
pared from a 2:1 monomer/template ratio and with a significant in-
crease observed upon PMP initiated deprotonation. These results
are all in line with the anticipated binding model and both observa-
tions correlate with the MD simulation results, whereby increasing
the amount of functional monomer in the prepolymerization mix-
tures increases the extent of template H-bonding (Table 3). Further-
more, template complexation with PPA increases with the degree of
deprotonation (neutral < monoanion < dianion). Increasing the
amount of PMP will increase the degree of template deprotonation,
which increases the degree of interaction with 1. Ranking the poly-
mers in order of decreasing uptake showed that only PPA, and to a
lesser extent BA, led to polymers exhibiting these effects whereas the
PSA imprinted polymers lacked significant affinity for the template.
This agrees with the weak basicity of the corresponding anions and
weak interactions observed for this anion in aprotic solvents. The
poor performance of the BA-MIPs P5 and P6 however, disagrees
with the basicity trend. To understand the reason for this, the differ-
encies in anion size and functionality should be considered. PPA
monoanion is larger and features an additional H-bond donor in
d)
40
20
0
200
100
0
20
0
0.0
0.4
0.8
P1
P2
F (mM)
Figure 5. Equilibrium binding isotherms of PPA adsorbed to P1 (blue
circles) and PN1 (red squares) (a) and P2 (blue circles) and PN2 (red
squares) (b) in acetonitrile (0.1% PMP). In (c) the binding to P1 (red
squares) and P2 (blue circles) corrected for binding to the
corresponding non-imprinted polymers is shown. The isotherms were
fitted to a Langmuir binary-site (a, b) or mono-site (c) models resulting
in the binding parameters listed in Table 5. In c, the data points above
X=0.2 have been excluded from the fitting of the data for P2. (d) shows
the binding parameters obtained from Fig.5c.
This difference can be attributed to the state of template complexa-
tion prior to polymerization and is supported by the results obtained
from MD simulations. Again, both H-bond analysis results (Table
3) and the RDF-analyses results (Table 4) indicate a higher degree
of complexation of 1 with the template when increasing the amount
of monomer.
-
PhP(OH)O2 which can interact with the polymer. This is lacking in
-
PhCO2 . In view of the observed trends, we chose to characterize the
PPA imprinted polymers in more detail.
Based on mass balance equations and assuming the solution com-
plexation constants in Table S1, we predict that PPA is present in the
form of a 1:1 complex whereas for P2, ternary complexes are domi-
nating. Turning to the RDF-results (Table 4), the calculated number
of molecules within the given cutoff varies with the cutoff and order
of the selected solvent/solute pairs. However, in the neutrally tem-
plated PPA system (P1N) there is one molecule of 1 within 5 Å of
any average template. This number increases to three when either
increasing the amount of 1 or deprotonating the template PPA to
monoanionic PPA. This offers support for the suggested complexa-
tion model that indicates the formation of ternary complexes. In-
creasing the amount of base further leads to dianionic PPA for which
RDF results (Table 4) show six molecules of 1 in close proximity to
Adsorption isotherms and binding parameters. The binding-en-
ergy distribution of the polymers was obtained from single-compo-
nent adsorption isotherms determined by a batch equilibrium bind-
ing experiment.50 The binding curves of PPA on P1/PN1 and P2/PN2
in acetonitrile buffered with PMP are seen in Fig. 5a and 5b. Com-
paring different binding equations (Fig. S6) showed that the data
were best fitted to the Langmuir binary site model. The associated
binding parameters are given in Table 5. The curves exhibited satu-
ration behavior with P2/PN2 taking up approximately two times
more PPA at a given free concentration. This is in line with the nom-
inal capacity of the materials, which is two times higher for P2/PN2
6
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PPA
PSA
BA
PPrA
any template. The results obtained moving from 1:1, through 3:1 to
6:1 stoichiometries of 1 and template complexes, in association with
the base driven increase in degree of deprotonation, adds support for
the formation of ternary complexes.
Assuming that the structures of the prepolymerization complexes
are carried into the polymer scaffold, the 1:2 complex will result in a
cleft-like receptor featuring four H-bond donors whereas the 1:1
complex will yield a site with a two-fold H-bond donor. Accounting
for the entropic gain, the resulting binding constant can exceed that
of the 1:1 site with 2-3 orders of magnitude, which is clearly not the
case here. Plausible explanations for this disagreement will be dis-
cussed below.
1
2
3
4
5
6
7
8
100
50
0
100
50
0
a)
b)
TEA
none
TFA
TEA
none
TFA
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Percent bound (TEA, none) and retention factor (k x 10)
(TFA) for PPA, PSA, BA and PPrA on columns packed with P2 (a) and
PN2 (b) using acetonitrile with different modifiers as mobile phases as
follows: TEA: Acetonitrile:Water/90:10 (0.1% TEA); TFA:
Acetonitrile:Water/95:5 (0.1% TFA) and none: 100 % Acetonitrile.
Table 5. Equilibrium constants (Keq) and binding capacities
(Bmax) of PPA imprinted polymers
Polymer Site
class
Keq
(x 103 M-1
Bmax
R2
IE a
(%)
)
(µmol/g)
A detailed look at the ionization states of the acids may offer clues to
the origin of the turn on/off effect. Table S5 lists the pKa values and
anticipated charges in presence of the two modifiers of the acids in
the study. In presence of base, PPA and PPrA carry a net charge of -
2 whereas the monovalent acids are negatively charged -1. In this
state both PPA and PPrA can bind to the P2 site while benefitting
from 4 complementary H-bond donors. The PPA dianion is the
most strongly retained anion which agrees with the order of basici-
ties where this dianion- is the most basic of all of the studied anions.
The weak retention of the monovalent anion of PSA we ascribe to its
weak basicity and its hydrophilicity, whereas planar BA lacks steric
complementarity with the PPA templated site. In presence of TFA
on the other hand (pKa = 0.5), PPrA (pKa=0.9) and PPA (pKa=1.8)
with pKa values in vicinity are partially or fully protonated weaken-
ing their interactions with the ureas of the stationary phase. PSA
(pKa= -2.8) should be fully ionized in water but less so in 95% ace-
tonitrile in presence of TFA. As a consequence, PSA is less hydrated
under these conditions and can more easily partition into the sta-
tionary phase. With only one of the three oxygen acceptors blocked
by protonation PSA, as a crude mimic of the PPA dianion, is effec-
tively recognized by the urea binding site.
P1
HI
265 107
4.4 0.8
24 22
23
63
14
56
3
2
8
5
0.99
0.99
0.99
0.99
0.97
0.97
0.99
0.99
0.96
0.96
18
50
-
LO
HI
PN1
P2
LO
HI
1.1 0.6
110 52
0.4 1.2
53 30
-
70 14
56
260
-
LO
HI
326 804
PN2
24
8
LO
2
0.5
2
115
5
-
b
b
P1corr
P2corr
31
36 0.4
38 0.3
29
31
194 16
The binding parameters were obtained by fitting of the binding data in
Fig. 5 to a Langmuir mono-site (P1corr, P2corr) or binary site binding
model.
a) Imprinting efficiency IE = 100 x Bmax/Bmax*, where Bmax* = 125
µmol/g = nominal capacity assuming quantitative template re-
moval and reoccupancy.
b) Fitting parameters for isotherms corrected for nonspecific bind-
ing to nonimprinted polymer (e.g. ∆B=BP1-BPN1).
Switchable oxyanion selectivity. The crushed polymer monoliths
of P1/PN1 and P2/PN2 were sieved and packed in columns for chro-
matographic characterization of their retentive properties for model
oxoacids (Scheme 1). The acids were injected in an acetonitrile rich
mobile phase as such or buffered with either triethylamine (TEA) or
trifluoroacetic acid (TFA). Basic conditions will promote deproto-
nation leaving the oxyanions to interact with the MIP via H-bond-
ing.28 The decisive role of this factor became obvious in the context
of the MD simulations of the imprinting process. Hence, increasing
the extent of deprotonation of PPA strongly influences template
complexation with both 1 and PMPH+.
The MIP exhibited strong affinity for its template (PPA) in this mo-
bile phase (Fig. S7, Fig. 6) with ca 75% of injected analyte remaining
stuck on the column. Meanwhile, with the exception of PPrA, none
of the other acids were retained whereas PN2 exhibited no affinity for
any of the analytes. This contrasts with the retention results in the
TFA-buffered mobile phase. Here we note a complete switch of an-
ion preference with PSA being the only retained analyte. In absence
of modifier both PPA and PSA are retained by P1 and P2 with no
retention observed by non-imprinted PN1 and PN2.
Some insight is provided by the MD simulations. Regarding tem-
plate rebinding in absence of modifier, it is clear from the H-bond
analysis results (Table 3) that with respect to neutral templates, 1
will have a higher degree of interaction with PPA than it does with
PSA and should hence increase selective adsorption of template on
this polymer. These experimental results are based on investigations
of polymer system P2, which included higher amounts of 1 and PMP
(Table 2, Fig. S15). Interestingly, the H-bond analysis results (Table
3) revealed that increasing the amount of 1 promotes template in-
teraction while barely affecting involvement of PMPH+. Further, in-
creasing the degree of deprotonation of template reduces template-
template interactions, which is reasonable given the increased
charge repulsion. Strong support for the formation of higher order
complexes is offered by the RDF-results. The degree of aggregation
is noticeable when inspecting the simulated systems visually (Fig. 7,
S16 and S17). There is a markedly higher presence of PMPH+ (two-
fold) in the vicinity of PPA in the P2C system (Table 4) than in all
the other systems. This supports the presence of ternary complexes
and their possible influence on polymer structure and morphology
(vide infra).50
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40
20
0
40
a)
b)
1
2
3
4
20
0
5
0
1
2
0
1
2
6
F (mM)
F (mM)
7
8
9
20
10
0
6
4
2
0
c)
d)
20
10
0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
0
1
2
A
PP
4
4
SO
PSA
F (mM)
2
Na
NaHPO
Figure 8. Equilibrium binding isotherms of Na2HPO4 (black curve,
triangles), PPA (brown curve, circles), PSA (red curve, diamonds) and
Na2SO4 (blue curve, squares) on polymer P7 (a) and PN7 (b) in 0.1 M
sodium bicarbonate buffer pH=9.0 (c) Binding isotherms of P7
corrected for binding to the nonimprinted polymer PN7. (d)
Figure 7. Example of an observed ternary complex in the P2C system,
here involving three molecules of 1 (grey) engaged in H-bonding
interactions with the template (PP2) (purple/magenta) and two non-
H-bonding base molecules (PMPH+) (yellow). H-bonds are indicated
with dashed lines.
Equilibrium constants (Keq, black bars) and binding capacities (Bmax
,
grey bars) for the indicated anions interacting with P7 in buffer pH 9.0
The binding parameters were obtained by fitting of the corrected
binding data in Fig. 8c to a Langmuir mono-site binding model.
Binding of inorganic phosphate and sulfate in water. Given the
strong and switchable ion binding shown by the MIP in water poor
media, our next goal was to investigate whether the polymers would
cross-react with inorganic anions in buffer. Several synthetic recep-
tors have been developed for this purpose to address various water
processing or sensing applications,6 but so far only few receptors
have shown effective anion recognition in water and even more rare
are those functioning in high ionic strength media.1, 51 A key problem
is that both phosphate and sulfate are strongly hydrated in water
(Table 1) which has a destabilizing effect on the interactions with
the imprinted site.
Table 6. Equilibrium constants (Keq) and binding capacities
(Bmax) of PPA imprinted polymers
Polymer Anion
Keq
Bmax
R2
(x 103 M-1
)
(µmol/g)
P7
NaHPO4
Na2SO4
PPA
1.5 0.3
1.1 0.2
2.2 0.6
1.0 0.3
0.5 0.2
0.4 0.2
1.5 0.5
0.3 0.2
3.9 0.5
4.6 1.0
3.1 0.1
3.4 0.5
53
25
34
24
5
3
3
3
0.973
0.976
0.966
0.959
0.940
0.970
0.950
0.940
0.990
0.973
0.999
0.990
PSA
PN7
NaHPO4
Na2SO4
PPA
51 18
To increase particle wettability, we prepared a new set of polymers
as P2/PN2 but using the hydrophilic crosslinker PETA as crosslinker.
Ion binding was measured by conductometry in buffer pH=9.0
where both anions carry a net 2-fold negative charge (Fig. 8). To our
surprise clear imprinting effects were observed with P7 exhibiting a
consistently higher uptake of both phosphate and sulfate compared
to nonimprinted PN7. In agreement with the Hofmeister series, de-
scribing the salt effect on protein solubility, phosphate is the most
strongly bound ion followed by PPA, sulfate and PSA, the trend be-
ing weaker on PN7 compared to P7 (Table 6). The effect of imprint-
ing appears clearly in the plots of the differential uptake in Fig 8c re-
flecting MIP binding corrected for binding to the NIP (assuming the
latter to reflect the nonspecific binding contribution). Fitting these
data with the Langmuir mono-site model resulted in the data shown
in Fig. 8d. Interestingly, in spite of the higher uptake of phosphate,
30
18
8
2
PSA
27 11
19 0.6
5.8 0.3
18 0.1
6.1 0.3
P7corr
NaHPO4
Na2SO4
PPA
PSA
The binding parameters were obtained by fitting of the binding data in
Fig. 8 to a Langmuir mono-site binding model.
Investigation of imprinting mechanism. Having proven the bind-
ing performance of the anion imprinted receptors, further refine-
ments require a fundamental understanding of the imprinting mech-
anism and the structural nature of the imprinted sites. An overlooked
issue in molecular imprinting concerns the effect of template on the
early stages of polymerization.52-55 Is the template capable of induc-
ing a preferred sequence or stereoregularity in the polymer main
chain that may in turn influence its molecular recognition proper-
ties?
sulfate binds with
a slightly higher equilibrium constant
(Keq=4.6x103 M-1) than phosphate (Keq=3.9x103 M-1). These values
exceed the anion affinity of most reported neutral receptors with re-
spect to anion binding in buffered media.4-5 To investigate a possible
pH dependent anion preference, we also measured ion binding at
lower pH-values (Fig. S8). Binding of both PPA and PSA decrease
with decreasing pH. Although an overturned binding preference
could not be observed, the increase in the BPSA/BPPA ratio shows that
the preference for PPA decreases.
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
Figure 9. Alternative routes to the build-up of recognitive sites in network polymers. (A) Strong monomer-template interactions lead to template
induced host monomer diads in the main chain that are subsequently stabilized by crosslinking. (B) Weak monomer-template interactions lead to sites
formed by multidentade interactions involving preformed polymer chains.
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We raised this question in view of the ternary monomer-template
complexes anticipated to be present during polymerization, the pres-
ence of which finds strong support in results from the MD simula-
tions (Tables 3-4 and Figs 7, S16 and S17). Such an arrangement
may place the reactive double bonds of two monomers in close vi-
cinity (see the left two molecules of 1 in Fig. 7). One consequence of
this arrangement may be a template induced incorporation of two
urea groups juxtaposed in the same chain (Fig. 9, route A) akin to
the established template polymerization mechanism.55-58 The role of
the template is here to bring two or more polymerizable groups close
in space to achieve rate enhancement combined with stereo- and se-
quence- controlled monomer incorporation. The template can be
covalently or noncovalently attached to the monomers and is
cleaved off after polymerization to free up pendent functional
groups.56 This results in high molecular weight copolymers with con-
trolled tacticity and regularity.
To investigate whether monomer reactivity in our system is tem-
plate controlled or the monomers are randomly incorporated, we
used differential scanning calorimetry (DSC), NMR and MALDI-
TOF mass spectrometry to study the initial stages of polymerization.
Crosslinked polymers P1 and P2 were compared with linear copoly-
mers of 1 and methylmethacrylate (MMA). All polymers were pre-
pared in absence or presence of template, the latter in the form of
mono- or bis-TBA salts of PPA. First, we studied the curing process
by DSC monitoring the heat generation upon double bond conver-
sion. The onset and peak maxima temperatures of the curing ex-
otherm were registered (Fig. 10, Fig. S9-S10) and the double bond
conversion was estimated from the total heat generated per unsatu-
ration divided by the literature value for the MMA double bond en-
thalpy (∆H0=13.1 kcal/mol) (Table S6). All co-polymer curing sys-
tems experienced a lowering of the peak maxima temperatures upon
added template, the effect being more pronounced for PPA●2TBA
than for PPA●TBA (Fig. 10a). This contrasted with the effect of
template addition on the curing of MMA alone (Fig. S10) where a
slight increase in the onset temperature as well as a significantly
lower conversion was observed. This indicates that PPA acts as an
inhibitor in absence of the host monomer. Overall, the template had
no significant effect on the monomer conversion, which was 40-50%
in all cases. We cautiously interpret these results as being due to a
template assisted monomer incorporation with PPA●2TBA prear-
ranging two molecules of 1 leading to a catalyzed propagation. The
molecular weight distribution of p-(1-co-MMA) determined by
MALDI-TOF-MS supports this explanation (Fig. 10b; Fig. S11).
This technique provides detailed information on the molar mass dis-
tribution, end groups and repeat units of linear polymers and in the
tandem mode, confirmation of their structural identity.59
Figure 10. (a) Exotherm peak maxima temperatures upon curing of a
mixture of 1 and MMA (1/MMA: 1/5 mole/mole) in the presence of
PPA●2TBA (red circles) and PPA●TBA (blue squares). (b) MALDI-
TOF-MS spectra of p-1-co-MMA prepared in presence of PPA●TBA
(black spectrum) and PPA●2TBA (red spectrum). (c) Equilibrium
constants of PPA●2TBA binding to imprinted and nonimprinted p-(1-
co-MMA). Data obtained from the binding curves in Fig. S13 with
fitting parameters listed in Table S7.
The polymers were isolated from the crude reaction mixtures by pre-
cipitation in methanol and deposited together with the MALDI ma-
trix on the target plate followed by recording of the spectra. As seen
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in Fig. 10b and Fig. S11 the oligomer distributions were found in the broadening which confirm the expected template-polymer interac-
1
2
3
4
5
6
ranges m/z 100-1500 p-MMA; 500-4000 p-(1-co-MMA); 700-5000
p-(1-co-MMA) (template: PPA-TBA); 800-7000 p-(1-co-MMA)
(template: PPA-2TBA), with the signal mass separations corre-
sponding to the expected repeat units i.e. 100.1 g/mol. The corre-
sponding number (Mn) and weight (Mw) average molecular
weights and polydispersity index (PDI) are listed in Table 7.
tions. The CIS plots for all polymers are seen in Fig. S13 with the
binding parameters listed in Table S7. The increase of the average
equilibrium constants from Keq = 139 M-1 for the non-templated pol-
ymer to Keq = 198 M-1 for the templated polymer we ascribe to an
enhanced number of 1-1 diads, predisposed to bind the template via
quadrupole H-bonds in the polymer main chain.
7
8
9
From these results we conclude that polymerization in presence of
the doubly charged template leads to a pronounced increase in the
average molecular weight of the oligomers, and a significant reduc-
tion in polydispersity, all in agreement with the anticipated mecha-
nism.
We therefore conclude that a template memory can be induced by
control of the monomer sequence in the polymer main chain alone.
So, what is the role of the crosslinking agent? Judging from the equi-
librium constants for adsorption, the affinity of the crosslinked P2
for PPA (Keq=1.9x105 M-1) exceeds by far that of the linear polymer.
The contribution of crosslinking to the polymer affinity for the tem-
plate is hence obvious. The importance of the crosslinking monomer
is further highlighted in the results obtained from MD simulations.
Looking at the neutral templated systems (PXN), crosslinker and 1
form H-bonds between 30-50% of the total simulation time (Table
3). Even in the PPA templated systems extensive H-bonding be-
tween the template and the crosslinking monomer is observed, 60-
80%. There is a high presence of crosslinker around templates and
competition for access to functional groups on templates. The inter-
actions are also stable, often more so than interactions between tem-
plate and 1 or PMP/PMPH+. Hydrogen bond interactions with tem-
plate are lower in PSA systems than in PPA systems, even if the pres-
ence of crosslinker is similar. As observed in several other systems,445-
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1
Finally, we analyzed all polymers by H-NMR spectroscopy. This
technique has a broad information value in polymer chemistry.60
From integral ratios of incorporated monomers, the copolymer
composition can be estimated and the technique can be used to es-
timate the number average molecular weight,56, 61 and polymer ste-
reo- and sequence- regularity.55 Of particular relevance in our case is
whether the signals from pendent urea groups are visible and if yes,
whether their affinity vis-à-vis the template can be determined.
Table 7. Properties of linear copolymers prepared in presence
of the templates indicated.
a
b
b
Polymer
Template
∆ Tmax
Mw
Mn
PDIb
MMA/1c
48
the presence of crosslinker in close vicinity to the template is ob-
served in prepolymerization mixtures and is observed to influence
template-polymer rebinding.
(°C)
(g/m
ol)
(g/m
ol)
p-MMA
PPA·2TBA
-
0
-
1114
1025
1.09
1.27
-
p-(1-co-MMA)
1493
1174
1.74
FINAL DISCUSSION
p-(1-co-MMA)
p-(1-co-MMA)
PPA·TBA
-1.0
-2.8
1642
2700
1319
1926
1.24
1.40
2.36
1.57
Charge neutral receptors interacting with ligands via H-bonding
have for long been associated with poor compatibility with aqueous
media. The presence of water and other protic solvents effectively
disrupt the host-guest interactions in these systems.1 Some excep-
tions to this rule have recently been reported although neutral recep-
tors displaying affinity for phosphate and sulfate in pure water or
buffer are still rare. Inspiration from naturally occurring receptors
can be used to move forward. These display high affinity and selec-
tivity in physiological conditions by shielding the anion from the sur-
rounding solvent molecules and thereby freeing it to interact by H-
bonding in a nest like site.1, 5 To mimic this principle, we have de-
signed oxyanion imprinted polymers incorporating charge neutral
urea groups in a hydrophobic scaffold. The polymers exhibit an un-
precedented switchable anion binding behavior as well as strong ion
affinity in buffered media comprising both phosphate and the
strongly hydrated sulfate anion. This performance has a practical rel-
evance in view of the broad need for robust anion binders. As we pre-
viously showed, this behavior may be exploited in bioseparations for
fractionation of phosphorylated and sulfated peptides or saccha-
rides.29 Another classical problem concerns sulfate separation from
nitrate-rich radioactive mixtures which has been a long term goal in
nuclear waste remediation.6 Several synthetic receptors have been
developed for this purpose but so far only few receptors offer effec-
tive anion recognition in water and even more rare are those func-
tioning in high ionic strength media. Through further improvements
of the imprinted receptors, such as incorporation of dual receptors62-
63 or other scaffolds, we hope to sufficiently boost affinity and selec-
tivity for the MIPs to offer a viable alternative host for these pur-
poses.
PPA·2TBA
a) Change in exotherm peak maxima calculated from the data in Fig. S9,
S10 and Fig. 10a.
b) Number (Mn) and weight (Mw) average molecular weights and poly-
dispersity index (PDI) calculated as described in the supporting infor-
mation.
c) Calculated from the integrals of the signals corresponding to the -
OCH3 protons of MMA (d=3.6 ppm) and the aromatic protons Hc of 1
(d=8.2 ppm).
Fig. S12a shows a representative solution spectrum (DMSO-d6) of
p-(1-co-MMA) prepared in absence of template. We gratefully
noted that protons characteristic for both monomers were clearly
visible i.e. the urea protons Ha and Hb and the aromatic protons Hc
of 1 and the -OCH3 protons of MMA. The integrals of the Hc and -
OCH3 signals were used to estimate the stoichiometry of incorpo-
rated monomers. This is expressed as the ratio MMA/1 and the val-
ues are listed in Table 7. For all polymers the ratios are lower than
5/1 indicating an enhanced incorporation of monomer 1 in the
chains. More interesting however is the low ratio observed for the
template induced polymers, most notably for the polymer prepared
in presence of the doubly charged template. This agrees with the an-
ticipated catalytic action of PPA●2TBA enhancing the reactivity of
1. We thereafter monitored the complexation induced shifts of the
urea protons upon addition of PPA●2TBA. As seen in Fig. S12 titra-
tion was accompanied by pronounced downfield shifts and signal
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We also provide detailed molecular level insight into the anion re-
NOTES
1
2
3
4
5
6
7
8
cognitive sites. Comparing stoichiometric imprinting based on 1:1
or 1:2 phosphate anion – urea monomer ratios shows the superiority
of the ternary complex imprinting to generate high fidelity binding
Present Addresses
§ School of Chemistry and Chemical Engineering, Queen`s University
Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, North-
ern Ireland, United Kingdom
1
sites. In spite of the stoichiometric complexation as proven by H-
NMR 2D-NOESY and CIS plots, the imprinting efficiency did not
exceed 30% for the high affinity sites. This is likely related to the
amorphous nature of this class of MIPs,64 supported by the hetero-
geneity of observed complexes in these mixtures (Fig. S16 and Fig
S17). MIPs prepared by free radical polymerization belong to the
thermoset class of materials characterized by extensive and irreversi-
ble crosslinking and insoluble end products. The heterogeneity of
these materials ranges from the molecular level to the micro scale
which precludes molecular level insights into the structural and dy-
namic features of the binding sites. This stands in contrast to the
range of available tools for characterizing biomacromolecules. Nev-
ertheless, reports focusing on well characterized monomer template
systems employing advanced techniques such as solid state NMR
∥
Analysis and Evaluation Department, Egyptian Petroleum Research
Institute, 1 Ahmed el Zomor Street, Nasr City, Cairo, Egypt
∇
Aydin Adnan Menderes University, Faculty of Arts and Sciences, De-
partment of Chemistry, 09010 Aydin, Turkey.
9
# Analytical Chemistry Department, Faculty of Chemistry, Universidad
Complutense of Madrid, 28040. Madrid, Spain.
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60
Author Contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
‡These authors contributed equally.
Funding Sources
68
with isotopically enriched templates,65-67 infrared69-70 or fluores-
Marie Skłodowska-Curie Actions H2020-MSCA-ETN-2016, 722171;
Swedish Vetenskapsrådet (VR) under grant number 2014-3794; Swe-
den Knowledge- Foundation (KKS) for research under project number
20150086.
cence spectroscopy42, 71-72 have increased our knowledge in this re-
gard. As discussed above, the propagation rate versus the on/off rate
of the template-monomer interaction is an indicator for the tem-
plates ability to induce repeat units in the main chain.73 Very few
studies on how the template influences the initial stages of the
polymerization have been reported.54-55 This warrants further studies
in this regard. As we have shown in this report, a solution binding
constant for the host monomer template interaction of 10000 M-1 is
sufficient for achieving this. This contrasts with situations where the
rate of propagation is slow with respect to the on/off rate. Here the
template will have time to dissociate before the next monomer addi-
tion. In this case, imprinting follows the mechanism outlined in Fig.
9b where preformed polymer chains are conformationally stabilized
by the template via multidentate interactions. Although this is likely
the dominating mechanism in most noncovalent imprinting sys-
tems,16-20 studies of corresponding linear polymers can provide fur-
ther important insights into the imprinting process.
ACKNOWLEDGMENT
We gratefully acknowledge financial support from the Marie Skłodow-
ska-Curie Actions (H2020-MSCA-ITN-2016, 722171), Veten-
skapsrådet (VR) under grant number 2014-3794 and the Sweden
Knowledge- Foundation (KKS) for research under project number
20150086. CE is grateful for the postdoctoral research fellowship from
the Council of Higher Education in Turkey (YÖK) and for the visiting
academic funding from the German Academic Exchange Service
(DAAD).
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