The application of template selectophores for the preparation of molecularly imprinted polymers

Article abstract of DOI:10.3390/molecules200917601

Molecularly imprinted polymers are versatile materials with wide application scope for the detection, capture and separation of specific compounds present in complex feed stocks. A major challenge associated with their preparation has been the need to sacrifice one mole equivalent of the template molecule to generate the complementary polymer cavities that selectively bind the target molecule. Moreover, template molecules can often be difficult to synthesise, expensive or lack stability. In this study, we describe a new approach, directed at the use of synthetic selectophores, chosen as readily prepared and low cost structural analogues with recognition groups in similar three-dimensional arrangements as found in the target molecule. To validate the approach, a comparative study of selectophores related to the polyphenolic compound (E)-resveratrol has been undertaken using traditional and green chemical synthetic approaches. These molecular mimic compounds were employed as polymer templates and also as binding analytes to interrogate the recognition sites associated with the molecularly imprinted polymers. Importantly, the study confirms that the use of selectophores has the potential to confer practical advantages, including access to more efficient methods for selection and preparation of suitable template molecules with a broader range of molecular diversity, as well as delivering imprinted polymers capable of recognizing the target compound and structurally related products.

Full text of DOI:10.3390/molecules200917601

Molecules 2015, 20, 17601-17613; doi:10.3390/molecules200917601
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
The Application of Template Selectophores for the Preparation
of Molecularly Imprinted Polymers
Basil Danylec, Lachlan J. Schwarz, Simon J. Harris, Reinhard I. Boysen
and Milton T. W. Hearn *
Centre for Green Chemistry & Australian Centre for Research on Separation Science (ACROSS),
School of Chemistry, Monash University, Melbourne, VIC 3800, Australia
* Author to whom correspondence should be addressed; E-Mail: milton.hearn@monash.edu;
Tel.: +61-3-9905-4547; Fax: +61-3-9905-8501.
Academic Editor: Kei Saito
Received: 11 July 2015 / Accepted: 28 August 2015 / Published: 23 September 2015
Abstract: Molecularly imprinted polymers are versatile materials with wide application
scope for the detection, capture and separation of specific compounds present in complex feed
stocks. A major challenge associated with their preparation has been the need to sacrifice
one mole equivalent of the template molecule to generate the complementary polymer
cavities that selectively bind the target molecule. Moreover, template molecules can often be
difficult to synthesise, expensive or lack stability. In this study, we describe a new approach,
directed at the use of synthetic selectophores, chosen as readily prepared and low cost
structural analogues with recognition groups in similar three-dimensional arrangements as
found in the target molecule. To validate the approach, a comparative study of selectophores
related to the polyphenolic compound (E)-resveratrol has been undertaken using traditional
and green chemical synthetic approaches. These molecular mimic compounds were employed
as polymer templates and also as binding analytes to interrogate the recognition sites associated
with the molecularly imprinted polymers. Importantly, the study confirms that the use of
selectophores has the potential to confer practical advantages, including access to more
efficient methods for selection and preparation of suitable template molecules with a broader
range of molecular diversity, as well as delivering imprinted polymers capable of recognizing
the target compound and structurally related products.
Keywords: selectophore; template; molecularly imprinted polymer; resveratrol

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1. Introduction
Historically, waste disposal and recycling of chemicals present at the terminal stages of manufacture
within the agricultural and food processing industries have often been perceived as burdens rather than
opportunities. In particular, the value-add derived from the recovery and purification of low abundance,
but nevertheless very valuable chemicals has rarely been captured as part of integrated unit operations
at the level of process efficiencies that are needed to comprehensively address overall supply-chain and
productivity considerations. However, within the context of green chemical and engineering technologies,
opportunities now exist for industry to deploy new approaches for the separation and recovery of
commercially important chemicals from agriculture and food bio-waste sources using non-traditional
processes. Some of these considerations have been addressed in our previous investigations, through the
development of novel immobilised catalysts and separation materials, which take into account more atom
efficient reactions, and through the application of new technologies, which are less resource or energy
intensive, for the separation and purification of chemical or biological products.
Within this context, the application of green chemical methodologies for the efficient extraction
and separation of naturally occurring bioactive molecules, as an alternative to their chemical synthesis,
has considerable potential. For example, in recent studies we have documented a new approach for the
separation and purification of bioactive polyphenolic compounds from bio-waste sources, such as
grape must and pomace, or peanut husks, using molecularly imprinted polymers (MIPs), with a focus
on the phytosterol and polyphenolic compounds, and other valuable plant-derived chemicals [1–4].
(E)-Resveratrol, 1, is an archetypical example of a phytochemical compound, which arises as an early
product from the biosynthesis of phenylalanine. (E)-Resveratrol is found in many foodstuffs and is
associated with various, important anti-oxidant protective effects [5,6]. Moreover, (E)-resveratrol is an
intermediate in the biosynthesis of structurally more complicated polyphenols and flavonoids [7].
Based on our previous synthetic studies, analogues of (E)-resveratrol were identified as appropriate
compounds to validate the concept of selectophores [8] in the generation of molecularly imprinted
polymers. Selectophores, as defined by Widstrand et al. [8], are template molecules with similar
chemical functionalities and geometries—all fitting a similar set of specific properties embodied in a
“selectophore” structure, which will bind to the same MIP because of a similar three-dimensional
arrangement of interacting functional groups. (E)-Resveratrol has a well-defined geometry around the
alkene bond, which together with the planar aromatic ring systems, defines the three dimensional
orientations and locations occupied by the three hydroxyl groups. These hydroxyl groups function as
key sites on this molecule for molecular imprinting, and are recognized by the functional monomers as
they self-assemble to form a non-covalent complex as part of the preparation of selective MIPs. The
resultant non-covalent complex can then be polymerised with a cross linker to generate the MIP. Upon
removal of the template molecule, the imprinted polymer contains complementary cavities that should
be able to selectively bind to the original template molecule, or other structurally similar analogues.
From the perspective of green and efficient laboratory practice, the use of a target compound, e.g.,
(E)-resveratrol 1, as the template to generate the corresponding MIP represents a sacrificial stoichiometric
loss, a circumstance which would not be acceptable for large scale MIP-based manufacturing processes,
when toxic, high cost or difficult to prepare template molecules are involved. Although the preparation
of (E)-resveratrol can be achieved, albeit in low yield, by traditional multi-step synthetic methods from

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available petrochemically-derived precursors, larger template molecules of greater structural complexity
often are synthetically more challenging and much more expensive to synthesise. When the issues of
time, cost and reagent conservation are taken into account, we were attracted to explore whether template
mimics or selectophores [8,9] could be more readily synthesised, and efficiently used, to provide
effective substitutes for the target compound of interest. Conceptually, this approach is similar to the
use of pharmacophores encountered in rational drug design [10] with the additional advantage that the
application of close structural analogues of the parent template for imprinting may overcome the
problem of template “bleeding” [11] and the associated uncertainty in trace level analysis after solid-phase
extraction with MIPs [12–14]. Here, we report the synthesis of the imine 2 and amide 3 selectophores
of (E)-resveratrol. By utilising green chemical synthetic strategies, these selectophores were more
easily and efficiently prepared than (E)-resveratrol, with both compounds synthesised in very high
yield and purity by “single-pot” reactions. We also report the performances of these new polyphenolic
selectophore-imprinted polymers relative to the characteristics found for the corresponding
(E)-resveratrol-imprinted polymer.
2. Results and Discussion
2.1. Selectophore Preparations
(E)-Resveratrol 1 is a polyphenolic compound with an alkene constraint. Several analogues were
chosen for investigation from the perspective of their ability to replicate this constraint and function as
suitable selectophores. The criteria employed were based on their conformational and configurational
similarities, as assessed inter alia by molecular modelling calculations performed using PM3 geometry
optimisation software, and their perceived ease of synthesis. Based on these considerations, the imine,
5-[(4-hydroxy-phenylimino)methyl]-benzene-1,3-diol 2, and the amide, 3,5-dihydroxy-N-(4-hydroxy-
phenyl)benzamide 3, (Figure 1) were selected as potentially useful selectophores. All three compounds
have two resorcinol hydroxyls, which are able to occupy the same 3-dimensional spaces, whilst the
third phenolic hydroxyl group can exist in a closely aligned overlap position.
OH
OH
OH
O
N
H
HO
HO
HO
N
OH
OH
OH
H
H
Alkene 1
Imine 2
Amide 3
Figure 1. (E)-Resveratrol 1 and the polyphenolic selectophores, imine 2 and amide 3.
In our previous studies, (E)-resveratrol was employed as a template molecule for the preparation of
several MIPs [2–4,15,16]. A variety of traditional methods have been reported [17–22] for the synthesis
of (E)-resveratrol. These methods were not based on the principles of Green Chemistry [23,24], nor did
they take into account the requirements of modern industrial practicality. All of these previous synthetic
methods invariably employed multistep procedures with significant losses of materials, along with the
need for extensive purification processes, repetitive handlings of hazardous reagents, the use of several
protection/deprotection steps and significant energy consumption to drive the reactions. Representative of
these earlier procedures is the synthesis of (E)-resveratrol described by Andrus et al. [17] where traditional

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synthetic protocols are used to transform 3,5-dihydroxybenzoic acid 4 to (E)-resveratrol 1 (Scheme 1).
As reported in the literature [17–22] and replicated in our hands, this multistep procedure for the
synthesis of 1 returned a poor atom economy value (i.e., an AEV < 27% if the contributions from all
solvents and purification reagents are excluded, or an AEV < 4.3% if the contributions from all chemical
components but not the carbon equivalent of the energy used was included), low reaction mass
efficiency (2.8% or 0.03%, respectively) and a high E-factor (>26.2 or >3895 respectively), with the
desired product obtained in only 29% overall yield. At this laboratory scale typically >5 days were
required to complete this synthesis, with handling of a variety of corrosive or hazardous chemicals, as
well as the generation of significant waste from the mandatory chromatographic purification steps.
OH
OAc
OAc
Ac2O, pyr, DMAP, EtOAc
SOCl2, DMF, toluene
100o C, 3 h
0o C, 1 h then
room temp, overnight
72%
HO2C
OH
Cl
HO2C
OAc
C
O
OAc
4
2% Pd(OAc)2,
NEM, toluene
reflux, overnight
51%
AcO
OH
OAc
OAc
1) KOH, MeOH
reflux, 1 h
H
AcO
H
OH
OAc
2) aq. HCl,
79%
OAc
H
HO
H
O
AcO
Alkene 1
Scheme 1. Preparation of the alkene, (E)-resveratrol 1.
In contrast, the imine selectophore 5-[(4-hydroxy-phenyl-imino)-methyl]-benzene-1,3-diol 2 could be
prepared from commercially available 4-aminophenol 5 and 3,5-dihydroxybenzaldehyde 6 as shown in
Scheme 2, with an atom economy value of 58%, a reaction mass efficiency of 43.4% and an excellent
E-factor of 1.3. By utilising this simple and rapid condensation reaction, nearly quantitative yields of the
pure product were rapidly achieved after a simple filtration workup. The stability of the imine 2 was
confirmed by 1H-NMR spectroscopy studies in d6-DMSO and CD3CN, which showed that this compound
remained unchanged in solution at room temperature for at least a 6 day period. This observation was
concordant with previous studies [25] with this compound. This imine selectophore was found to also
remain intact when used as a template molecule under the conditions employed for MIP preparations,
and when used as an analyte to evaluate the binding properties of the various MIPs.
OH
OH
NH2
O
N
anhyd. Na2SO4, CH2Cl2
room temp, 3 h, 100%
OH
OH
+
H
H
HO
HO
6
Imine 2
5
Scheme 2. Preparation of the imine 5-[(4-hydroxy-phenylimino)-methyl]-benzene-1,3-diol 2.
The second selectophore examined was the amide, 3,5-dihydroxy-N-(4-hydroxyphenyl)benzamide
3. An authentic sample was initially prepared by the multistep procedure (Scheme 3) as adapted from

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a method briefly outlined as a patent application embodiment by Kim et al. [26]. Full experimental
details for this multistep synthesis have been included as Electronic Supplementary Information. The
conventional multistep preparation of the amide 3 clearly suffered from the same constraints of poor
green chemistry metrics with a low atom economy value (i.e., <19.8%) and a high E-factor (in excess
of 140) similar to that noted above for the traditional synthesis of (E)-resveratrol 1 shown in Scheme 1.
Due to our ongoing requirement for larger amounts of this amide 3, an alternative improved synthetic
process was developed, the basis of which is summarized in Scheme 4. Workup of the reaction mixture
by removal of the solvent followed by trituration and washing with water rapidly returned the pure
amide 3 in 76% yield with an atom economy value of 53.2% and an E-factor of 1.77. The advantage
of this carbodiimide-activated coupling procedure is that the same starting reagents were used as for
the reported multistep synthesis, yet the amide could be produced in improved overall yield, using a
single-pot method, and did not require additional energy, functional group protection and deprotection steps
or chromatographic purification.
HO
AcO
O
S
AcO
Ac2O, pyr, DMAP, EtOAc
CH3SO2Cl, Et3N, THF
0 o
O
O
CH3
CO2H
CO2H
O
0o C/1 h then
room temp overnight,
72%
C
HO
AcO
AcO
4
H2N
OH
AcO
HN
OAc
0o C, 4 h, 82%
1) KOH, MeOH,
reflux, 2 h,
2) aq. HCl, 60%
O
AcO
Ac2O, DMAP, pyr, EtOAc,
reflux, 2 h, 76%
HO
AcO
AcO
HN
OH
HN
O
OH
O
1) KOH, MeOH, reflux, 75 min,
2) aq. HCl, 55%
HO
Amide 3
Scheme 3. Conventional synthetic method used for the preparation of the amide
3,5-dihydroxy-N-(4-hydroxyphenyl)benzamide 3.
Scheme 4. Alternative method for the preparation of the amide 3,5-dihydroxy-N-(4-
hydroxyphenyl)benzamide 3.
2.2. Molecularly Imprinted Polymers (MIPs)
(E)-Resveratrol 1 and the two selectophores, 2 and 3, were used as templates for the preparation of the
corresponding MIPs, and as analytes to interrogate the binding characteristics of the newly prepared
polymers. The protocols employed for the preparation of such molecularly imprinted polymers (MIPs)
have been described in detail elsewhere [2] with the optimal composition of the pre-polymerisation

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complex obtained using a combination of molecular docking computational and 1H-NMR spectroscopy
titration procedures. The syntheses of these MIPs, in brief, involved the thermally initiated free radical
polymerization of a mixture of the selectophore template, the functional monomer 4-vinylpyridine
(4-VP) and the crosslinker in a porogen such as acetonitrile/EtOH (5:1 v/v). (E)-Resveratrol imprinted
polymers (MIPRES) of similar performance were generated when acetone was used as porogen, with all
other variables kept constant. The resultant solid materials were ground, appropriately sized particles
recovered by sieving and then washed with methanolic acetic acid to desorb the template. Washings
were monitored using RP-HPLC with UV-Vis detection, and repeated until no traces of the template
were detected. Non-imprinted polymers (NIPs) were prepared in an analogous manner in the absence
of template molecule. Static binding studies were performed using the MIPRES and the corresponding
NIP with individual analytes. In these studies, the polymers were incubated with a freshly prepared
solution of the analyte, the supernatant removed, the concentration of the free analyte in the supernatant
determined by RP-HPLC measurements, and the amount of analyte bound (B) by the polymer determined.
MIP blanks were examined without any added analytes present and demonstrated no template bleed.
The results are summarized in Figure 2.
14
NIP
MIP
12
10
8
6
4
2
0
2
3
1
Figure 2. Single analyte binding of (E)-resveratrol 1, or the selectophores, the imine 2, and
the amide 3, to the MIPRES. The standard error of the mean (SEMs) for these binding
capacity results was less that ±5% (n = 3).
As evident from these results, the MIPRES displayed similar binding capacities and comparable
selectivities (BMIP − BNIP) for the alkene, the amide and the imine molecules. The similarity of these
binding parameters suggests that the MIPRES displays a generic three-point binding cross-reactivity
towards each of these polyphenolic compounds. This result is concordant with all three molecules having
similar conformations and configurations in solution and within the micro-environment of the MIP
cavity. Moreover, it is interesting to note that although the two selectophores, 2 and 3, are likely to
possess similar electronic characteristics as the parent (E)-resveratrol, due to the nature of the differences
between the three bridging linkages (alkene, imine and amide), inductive variations appear to be moderated
by both the distance from the aromatic hydroxyl groups and the electronic nature of the aromatic π
clouds. Consequently, although these selectophores appear to bind in a similar manner with MIPRES at
the same cavity recognition sites as (E)-resveratrol, differences in affinity occur, as manifested by the
subtle variation in their binding capacities.
Based on these results, the suitability of the amide and imine selectophores as (E)-resveratrol template
mimics was then assessed using similar static binding assays. Thus, both of these compounds were

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employed as templates for the preparation of new MIPs (i.e., MIPAMIDE and MIPIMINE respectively) using
similar protocols to that employed for the preparation of MIPRES. The ability of these MIPs to recognise
(E)-resveratrol is shown in Figure 3.
14
MIP NIP
12
10
8
6
4
2
0
Figure 3. Recognition of (E)-resveratrol by MIPRES, MIPAMIDE and MIPIMINE. The standard
error of the mean (SEMs) for these binding capacity results was less that ±5% (n = 2).
As evident from Figure 3, the selectophore-imprinted MIPAMIDE and MIPIMINE both displayed
enhanced affinity for (E)-resveratrol compared to the corresponding NIPs, but with the structural rigidity
of this compound due to the alkene constraint reduced capacities compared to MIPRES. Despite the
fact that all three polyphenolic compounds may have similar conformations in solution, these findings
indicate that the stereo-topological spaces occupied by the pyridyl nitrogens of the 4-VP monomer
units within the polymers, despite acting as complementary functional binding sites for hydrogen bond
formation during the imprinting process and for the subsequent molecular recognition phenomenon
with the polyphenols, must differ slightly for all three MIPs. These differences results in slightly lower
binding capacities and affinities for both MIPAMIDE and MIPIMINE with (E)-resveratrol, a finding concordant
with the conformation of (E)-resveratrol being more constrained due to the presence of its alkene
moiety and less able to be fully accommodated in the selectophore-generated binding cavities
3. Experimental Section
3.1. Reagents and Equipment
Reagents and other chemicals were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia) or
Merck (Kilsyth, VIC, Australia). AR solvents were used as obtained from the manufacturer except for
dimethylformamide (DMF), which was dried over 4 Å molecular sieves, toluene was dried over sodium
wire, and pyridine and triethylamine were dried over KOH pellets. Milli-Q distilled water was used for
aqueous solutions. Solvent mixtures are expressed as volume/volumes. Solvent extracts were dried over
anhydrous sodium sulfate, filtered and then rotary evaporated to dryness at low pressures (≥30 mbar) at
30–35 °C. Reaction mixtures were sonicated in a 37 kHz/150 W Elmasonic S100 ultrasonic bath.
Polymers were ground using a Retsch PM 200 planetary ball mill (Haan, Germany) and sized through
sieves on a Retsch AS 200 sieve shaker. Analytical thin layer chromatography (TLC) was conducted
on silica gel using Merck® 1.05554.001 plates (Merck, Darmstadt, Germany). The components were
visualised by: (i) fluorescence at 254 nm; and (ii) ethanolic phosphomolybdic acid solution dip and
char. Silica gel column chromatography was conducted using Merck® 1.09385.1000. Nuclear magnetic

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13
resonance spectra were recorded on a Bruker DPX-300 spectrometer (1H at 300 MHz and C JMOD
at 75 MHz) (Bruker, Billerica, MA, USA). Deuterated solvents were used as indicated and the residual
solvent peaks used for internal reference. Coupling constant J values are reported in Hz (hertz). Low
resolution electrospray ionisation mass spectra (ESI) were recorded on a Micromass platform II API
QMS electrospray mass spectrometer in both positive (ESI+) and negative (ESI−) polarity modes.
High-resolution electrospray mass spectra (HRMS) were recorded on a Bruker BioApex 47e Fourier
Transform mass spectrometer. Melting points were determined using a Büchi melting point B-545
apparatus with a digital thermometer by open glass capillary method and are uncorrected.
3.2. Syntheses of Polyphenolic Selectophores
3.2.1. 3,5-Diacetoxybenzoic Acid
A suspension of 3,5-dihydroxybenzoic acid (15.40 g, 0.100 mol) in ethyl acetate (220 mL) was
cooled in an ice-bath. Acetic anhydride (24.52 mL, 0.2421 mol), pyridine (16.16 mL, 0.1998 mol) and
4-(dimethylamino)pyridine (100 mg, 0.8186 mmol) were added and the reaction stirred at 0 °C for
60 min and then at room temperature overnight. Formic acid (5.12 mL, 0.1357 mmol) was added and
the reaction poured onto ice (ca. 500 g). The volume was increased by the addition of further ethyl
acetate (300 mL) and the organic phase separated and washed with water (2 × 200 mL), sat. aq. NaHCO3
(100 mL), further water (2 × 200 mL), and then dried and evaporated to a white solid. Recrystallization of
the product from 5:1 (v/v) EtOAc/hexane (120 mL) gave 2 crops of 3,5-diacetoxybenzoic acid (combined
weight 17.07 g, 72%) as a white powder. Rf 0.20 (1:1 EtOAc/hexane), 0.39 (3:1 EtOAc/hexane); mp
161–162 °C (lit. [27] mp: 157–159 °C); δH (CDCl3) 2.29 (s, 6H, 2 × OAc), 7.18 (pseudo t, 1H,
J = 2.1 Hz, 4-H) and 7.70 (pseudo d, 2H, J = 2.1 Hz, 2-H, 6-H); δC (CD3OD) 18.43, 118.94, 119.02,
131.70, 150.19, 165.46 and 168.17; m/z (ESI) 261 ([M + Na]+, 100%).
3.2.2. (E)-3,4′,5-Triacetoxystilbene
A suspension of 3,5-diacetoxybenzoic acid (8.022 g, 33.71 mmol) in a mixture of toluene (130 mL),
DMF (500 μL) and thionyl chloride (16.00 mL, 220.6 mmol) was heated at 100 °C for three hours under
an argon gas atmosphere. The solvents were removed by vacuum distillation and the residue re-suspended
in toluene (85 mL) and sonicated under vacuum to remove dissolved gases. 4-Acetoxystyrene (5.74 mL,
37.5 mmol), N-ethylmorpholine (4.31 mL, 33.9 mmol) and palladium diacetate (35 mg, 0.16 mmol,
0.46 mol %) were added and the reaction heated to reflux for 2 h. Further palladium diacetate (116 mg,
0.52 mmol, 1.54 mol %) was added and the reaction left to reflux overnight. On return to room
temperature, ethyl acetate (500 mL) was added, the solution washed with 0.1 M aq. HCl (2 × 300 mL)
and water (300 mL) and then dried and evaporated to return a brown solid. Purification with column
chromatography (isocratically eluted with 2:1 Et2O/hexane) gave 7.888 g of a white solid, shown by
1H-NMR spectroscopy to be predominantly the desired adduct. Further chromatography (gradient
eluted starting with 4:1 hexane/EtOAc and finishing with 2:1 hexane/EtOAc) returned pure (E)-3,4′,5-
triacetoxystilbene (6.071 g, 51%) as a white solid. Rf 0.29 (2:1 hexane/EtOAc); mp 112.5–113.0 °C
(lit. [17] mp 116 °C); δH (CDCl3) 2.27 (s, 9H, 3 × OAc), 6.80 (pseudo t, 1H, J = 2.1 Hz, 4′-H), 6.93 (d,
1H, J = 16.3 Hz, Htrans), 7.03 (d, 1H, J = 16.3 Hz, Htrans), 7.04–7.09 (m, 4H, 3-H, 5-H, 2′-H, 6′-H) and

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7.44–7.47 (m, 2H, 2-H, 6-H); δC (CDCl3) 20.07, 113.39, 115.88, 120.88, 126.19, 126.64, 128.64,
133.45, 138.53, 149.46, 150.34, 167.91 and 168.30; m/z (ESI) 377 ([M + Na]+, 100%), 378 (21%).
3.2.3. (E)-Resveratrol, 1
The synthesis was conducted under an argon gas atmosphere. Potassium hydroxide (22 mg,
0.3922 mmol) was dissolved in methanol (3.0 mL) and this solution added to a suspension of
(E)-3,4′,5-triacetoxystilbene (113 mg, 0.319 mmol) in methanol (10 mL). The solid immediately dissolved
and the clear solution was gently heated to reflux for 60 min, with an associated progressive darkening
in colouration. The volume was then reduced to half by rotary evaporation and the remaining solution
acidified (pH 2) with 1 M aq. HCl. Ethyl acetate (150 mL) was added and the reaction washed with
saturated brine (3 × 20 mL), dried and evaporated to return a dark red solid. Purification with column
chromatography (isocratically eluted with 100% EtOAc) gave (E)-resveratrol (58 mg, 79%) as a pale
beige coloured solid. Rf 0.65 (EtOAc); mp 261.0–263.0 °C (lit. [18] mp 255–260 °C); δH (CD3OD)
6.13 (pseudo t, 1H, J = 2.2 Hz, 4-H), 6.41–6.42 (m, 2H, 2-H, 6-H), 6.71–6.79 (m, 3H, Htrans, 3′-H, 5′-H),
6.93 (d, 1H, J = 16.3 Hz, Htrans) and 7.29–7.36 (m, 2H, Jortho = 8.6 Hz, 2′-H, 6′-H); δC (CD3OD) 100.30,
103.47, 114.12, 124.64, 126.43, 127.06, 128.07, 138.97, 155.89 and 157.20; m/z (ESI) 229 ([M + H]+,
100%), 230 (23%).
3.2.4. 5-[(4-Hydroxy-phenylimino)-methyl]-benzene-1,3-diol, 2
A mixture of 3,5-dihydroxybenzaldehyde (300 mg, 2.174 mmol), 4-aminophenol (237 mg,
2.174 mmol), anhydrous sodium sulphate (309 mg, 2.176 mmol) and dichloromethane (15 mL) was
vigorously stirred at room temperature for 3 h. Further anhydrous sodium sulphate (309 mg, 2.176 mmol)
was added and the stirring continued for an additional one hour. Dichloromethane was removed by
rotary evaporation and the white residue re-suspended in boiling ethanol (10 mL) and filtered whilst hot.
The solid was washed in the funnel with further hot ethanol (10 mL). The clear filtrates were combined
and rotary evaporated to dryness to give 5-[(4-hydroxy-phenylimino)-methyl]-benzene-1,3-diol (501 mg,
100%) as a pale pink solid. Rf 0.48 (2:1 EtOAc/hexane); darkens with heating with mp >340 °C; δH
(d6-DMSO) 6.36 (pseudo t, 1H, J 2.2, 4-H), 6.80–6.85 (m, 4H, 2-H, 6-H, 3′-H, 5′-H), 7.16–7.22 (m, 2H,
Jortho = 8.8 Hz, 2′-H, 6′-H), 8.43 (s, 1H, imine-H), 9.47 (bs, 3H, 3 × phenolic-OH); δC (d6-DMSO)
106.27, 107.41, 116.70, 123.42, 139.35, 143.61, 157.16, 158.38 and 159.62; m/z (ESI) 230 ([M + H]+,
100%), 231 (13%); m/z (HRESI) 252.0633 ([M + Na]+, C13H11NO3Na+ requires 252.0637).
3.2.5. 3,5-Dihydroxy-N-(4-hydroxyphenyl)benzamide, 3
The reaction was conducted under an argon gas atmosphere. 3,5-Dihydroxybenzoic acid (2.310 g,
15.0 mmol) and 4-aminophenol (1.962 g, 18.0 mmol) were dissolved in DMF (90.0 mL). N-Ethyl-N′-
(3-dimethylaminopropyl)carbodiimide hydrochloride (3.450 g, 17.997 mmol) was added and the reaction
stirred at room temperature overnight. The solvent was removed by rotary evaporation under high
vacuum and the oily residue co-evaporated with water until a solid formed. This solid was triturated
with cold 0.01 M aq. HCl (50 mL), filtered, and the pale purple coloured product washed with cold
water (4 × 20 mL). The solid was dried over desiccant under vacuum to give pure 3,5-dihydroxy-N-(4-

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hydroxyphenyl)benzamide (2.791 g, 76%); Rf 0.39 (4:1 EtOAc/hexane); mp 266.0–266.5 °C; δH
(CD3OD) 6.47 (pseudo t, 1H, Jmeta 2.2, 4-H), 6.77–6.82 (m, 4H, 3′-H, 5′-H, 2-H, 6-H), 7.43–7.46 (m, 2H,
Jortho = 8.9 Hz, 2′-H, 6′-H); δH (d6-DMSO) 6.42 (pseudo t, 1H, Jmeta = 2.2 Hz, 4-H), 6.72–6.78 (m, 4H,
3′-H, 5′-H, 2-H, 6-H), 7.51–7.56 (m, 2H, Jortho = 8.9 Hz, 2′-H, 6′-H), 9.20 (s, 1H), 9.51 (s, 2H) and 9.83
(s, 1H); δC (CD3OD) 104.41, 104.67, 113.92, 122.13, 129.10, 136.08, 153.26, 157.41 and 166.70; m/z
+
(ESI) 244 ([M − H]−, 100%), 489 ([2M − H]−, 29%); m/z (HRESI) 246.0764 ([M + H]+, C13H12N1O4
requires 246.0766).
3.3. Preparation of Molecularly Imprinted Polymers
Molecularly imprinted polymers (MIPs) were prepared by procedures described previously using
an optimised composition for the pre-polymerisation complex, calculated from the average energy of
formation for the template, monomer 4-vinylpyridine (4-VP) clusters and monomer-template clusters,
using semi-empirical equilibrium geometry level theory and PM3 force field calculations [28,29].
1
The reliability of these determinations was then confirmed using H-NMR spectroscopy titration
1
experiments by monitoring the H-NMR spectrum of the template molecule (0.1 mM), dissolved in
trideuteroacetonitrile (CD3CN) and titrated with increasing molar equivalents of 4-VP. Spectra were
recorded after each addition. This process was continued until the aromatic –OH signal was no longer
detectable due to peak broadening. Thus, in brief, the selectophore template 2 (1 mmol) was dissolved
in the porogen (5:1 acetonitrile/EtOH, 6 mL) in a disposable glass test tube. For the amide 3,
acetone (6 mL) was the preferred porogen due to the lower solubility of this selectophore in 5:1
acetonitrile/EtOH. The functional monomer 4-VP (322 μL, 3 mmol) was added and the mixture sonicated
for 10 min. Ethylene glycol dimethacrylate (2.314 mL, 15 mmol) was then added as cross-linker, and
2,2′-azobisisobutyronitrile (51 mg, 0.31 mmol) added as radical initiator. This pre-polymerisation
mixture was sparged with nitrogen gas for 5 min before being heated in a thermostatic water bath at
50 °C for 24 h. The solid polymers were removed from the reaction tubes, crushed with a mortar and
pestle and finely ground using a Retsch 200 ball mill. The powder was sieved and the 63–100 μm size
particles recovered. These particles were repeatedly suspended in ethanol or acetone and the supernatant
decanted to remove fines. The bound template was then removed from the polymer by repeated washings
with methanolic 10% acetic acid until the washings were free of template (monitored by UV-Vis
spectroscopy at 321, 270 and 280 nm for compounds 1, 2 and 3, respectively). Non-imprinted control
polymers (NIPs) were prepared using the same procedure in the absence of a template molecule.
3.4. Evaluation of Molecularly Imprinted Polymer
Static binding experiments were conducted with both the MIPs and NIPs in parallel under identical
conditions. The analyte solutions (1.5 mL, 0.5 mM in acetonitrile) were added to the polymers (30 mg)
in Eppendorf tubes. This suspension was mixed at 40 rpm for 18 h and the polymer pelleted by
centrifugation for 15 min at 13,000 rpm. A 200 μL aliquot of the supernatant was removed and analysed
by RP-HPLC with UV detection at 321, 270 and 280 nm for compounds 1, 2 and 3, respectively. The
concentration of the non-bound analyte was determined by comparison with a linear 5-point calibration
curve. The amount of analyte bound (B) was then determined as the difference between this non-bound
value and the initial value, and expressed in units of μmol analyte bound/g polymer. MIP blanks, using

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1.5 mL of acetonitrile without any added analyte present to assess the extent of template bleeding [11],
were also employed as controls.
4. Conclusions
This study has documented that structural analogues with spatially defined functionalities similar to a
parent compound can be used as selectophores or template mimics for the preparation of molecularly
imprinted polymers (MIPs). Using the polyphenol (E)-resveratrol as exemplar, such polymeric materials
were found to display comparable, but not identical, binding behaviour towards this representative
polyphenolic compound. These findings indicate that by mimicking the structural and stereo-electronic
properties of a parent compound, appropriate selectophores can be designed and employed as suitable
templates, enabling the convenient preparation of generic MIPs which are capable of binding not only
to the parent molecule, but also structurally similar compounds. This approach thus provides an avenue
to readily explore the binding characteristics of new MIPs, particularly when the availability, structural
complexity or instability of the parent molecule limits its use as a template. One such example where
the utilisation of these concepts would be beneficial is with the structurally more complex, but highly
valued, flavonoid and phytosterol compounds present in low abundance in plant waste materials. Since
in many cases the possibility exists for such selectophores to be more easily and conveniently prepared
than the target compounds using single-pot methods, or with other green chemical synthetic procedures,
increased application of selectophores in the development of MIP technologies in molecular sensing
devices, compound purification and waste stream processing can thus be anticipated.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/09/17601/s1.
Acknowledgments
This work was supported by the Australian Research Council and the Centre for Green Chemistry at
Monash University, Australia. Michael Clarke assisted with the chemistry metrics tools. The financial
support of the CSIRO National Research Food Futures Flagship is gratefully acknowledged.
The current address of the author Lachlan J. Schwarz is National Wine and Grape Industry Centre,
Charles Sturt University, NSW 2650, Australia.
Author Contributions
Basil Danylec carried out the synthesis of the (E)-resveratrol and the selectophores; Lachlan Schwarz
performed the molecular modelling, polymer synthesis and binding studies; Simon Harris,
Reinhard Boysen and Milton Hearn were responsible for the analysis and interpretation of the data. All
authors contributed to the writing of the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.
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