# Phthalocyanine-Based Molecularly Imprinted Polymers as Nucleoside Receptors

Authors: Luigia Longo; Giuseppe Vasapollo
Journal: Metal-Based Drugs
 (2008)

---

## Abstract

A molecularly imprinted polymer (MIP) for
tri-O-acetyladenosine (TOAA), PPM(TOAA), was
prepared by the combined use of methacrylic acid (MAA) and
Zn(II)tetra(4'-methacryloxyphenoxy) phthalocyanine as functional
monomers. This MIP exhibited a higher binding ability for TOAA
compared to the MIP prepared using only MAA, PM(TOAA), in batch
rebinding tests.  Scatchard analysis gave a higher association
constant of PPM(TOAA) for TOAA(2.96×104M−1) than that of
PM(TOAA) (1.48×104M−1). The MIP
prepared using only the zinc-phthalocyanine, PP(TOAA), did
not show any binding capacity for TOAA. This means that the
phthalocyanine in the MIP contributes to higher affinities,
although it barely interacts with TOAA. Since selectivity
for this kind of MIPs is more important than binding
affinity, the binding of TOAA and a structurally related
compound,
tri-O-acetyluridine (TOAU), on the polymers was
investigated. Both PPM(TOAA) and PM(TOAA) exhibited binding
affinities for TOAA while they did not show any binding capacity
for TOAU.


---

## Body

## 1.introduction

Molecularly imprinted polymers (MIPs) have received much
attention recently as new materials capable of molecular recognition [1]. They
have been extensively utilized in solid-phase extraction [2–5], chromatography
[6–8], sensing [9, 10], and catalysis [11–13]. During molecular imprinting,
crosslinked polymers are formed by free-radical copolymerization of functional
monomers with an excess of crosslinker around an analyte that acts as a
template. After polymerization, the template is removed leaving in this way selective
binding sites in the polymer network that are complementary in form and
functionality to the analyte molecules [1].The main advantages of MIPs, over conventional polymers
used as separation material, are the high selectivity and affinity for the target
analytes used in the imprinting procedure. Furthermore, MIPs are characterized
by superior mechanical and thermal stability, as well as better inertness
towards acids, bases, metal ions, and organic solvents compared to enzymes. In
addition, imprinting polymerization is a very inexpensive procedure for the
development of artificial receptors. In the majority of the cases, the price of
an MIP depends entirely on the price of the template used. Moreover, if the
template is expensive, it is possible to recover the template and use it again.
Alternatively, inexpensive template analogues can be used for the preparation
of MIPs [1].In most studies, methacrylic acid (MAA) was used as
functional monomer to synthesize MIPs making the polymer preparation a simple
and facile process [14–16]. MAA can form hydrogen bonds with the template
molecule in porogen prior to polymerization. A more deliberate approach using
synthetically designed functional monomers could enable a better control in the
formation of high-affinity binding sites for each corresponding template minimizing,
at the same time, the inherent nonspecific binding properties common in noncovalent
imprinted polymers.In this paper, we report for the first time the
preparation of MIPs as nucleoside receptors using both methacrylic acid and a
zinc-phthalocyanine peripherally substituted with methacrylic groups (Compound 1, Scheme1)
[17] as functional monomers.
The receptor site is a three-dimensional cavity
around the phthalocyanine plane in crosslinked polymers to which the analyte
molecule could be specifically bound via coordination through the metal of the
phthalocyanine and hydrogen bonding/electrostatic interaction with MAA and the modifiers
linked to the phthalocyanine (Scheme 1). Such cooperative interaction approach
in MIP formation, already proposed for porphyrin derivatives [18–21], was
applied here for the first time to phthalocyanine compounds.Scheme 1
Schematic representation of the molecular imprinting of TOAA using both 1 and MAA as functional monomers.An organic soluble nucleoside derivative, tri-O-acetyladenosine (TOAA), was utilized
in this work as template in the preparation of phthalocyanine-based MIPs. The
development of synthetic receptors that recognize nucleotide bases and their
derivatives is an important area in chemistry today. The literature provides
many examples of artificial receptors for each of the common nucleoside bases [15, 16, 22–25]. Recently, these receptors have provided insights into DNA-DNA and
protein-DNA interactions, and applications are envisioned in the fields of biosensors,
drug therapy, separation science, and genetic engineering. TOAA was selected as
model template in a previous work [17] where the binding affinity and
selectivity of the zinc-phthalocyanine 1 towards different nucleosides were
evaluated by UV-vis titration experiments. Binding experiments showed that zinc-phthalocyanine 1
bounds TOAA most strongly, giving a
binding constant Ka of 1.35×104, 500 times that
of 1 to tri-O-acetyluridine (TOAU), showing in this way a high
selectivity [17]. The presence of the 2-aminopyridine moiety in the structure
of TOAA could be probably responsible for the good binding characteristics of
this nucleoside derivative with compounds functionalized with methacrylic
groups [15].An imprinted polymer receptor for TOAA, namely, PPM(TOAA),
was prepared using both 1 and MAA as functional monomers. Imprinted polymers
were also prepared using either MAA or 1, called PM(TOAA) and PP(TOAA),
respectively, and used as references. Corresponding unimprinted blank polymers,
PPM(BL), PM(BL), and PP(BL), were prepared using the same monomers in the
absence of TOAA. Batch rebinding studies were conducted by UV-vis spectroscopy
and the binding characteristics of the MIPs were examined by Scatchard  analysis. In order to verify the selectivity
of the MIPs, the binding of TOAA and its structurally related compound, TOAU,
on the all prepared polymers was investigated.


## 2. Experimental Section

### 2.1. Materials and Methods

Ethylene glycol dimethacrylate (EGDMA), 2′,3′,5′-tri-O-acetyladenosine
(TOAA), 2′,3′,5′-tri-O-acetyluridine
(TOAU), and acetic acid were purchased from Sigma-Aldrich (Steinheim, Germany).
α-α′-Azoisobutyronitrile (AIBN) and
methacrylic acid (MAA) were supplied from Fluka (Steinheim, Germany).
Analytical grade dichloromethane and methanol were purchased from J. T. Baker
(Deventer, Holland). Zn(II) tetra(4′-methacryloyloxyphenoxy)phthalocyanine (1)
was prepared on the basis of a published method [17]. UV-Vis spectra were
obtained with a Cary 100 Scan UV-vis spectrophotometer.


### 2.2. Polymer Preparation

The preparation of PPM(TOAA) was carried out as follows:
to a solution of TOAA (0,0468 mmol, as template) in dichloromethane
(262μL)
were added phthalocyanine 1 (0.0117 mmol, as the first functional monomer) and
MAA (0.1758 mmol, as the second functional monomer) in a glass tube. After
adding of EGDMA (0.9360 mmol, as cross-linker) and AIBN (0.0112 mmol, as
initiator), the mixture was sonicated for 5 minutes flushing with nitrogen gas and
then polymerized by heating at 60°C for 16 hours. The resultant polymer was
crushed and sieved. The template molecules were removed by washing the polymer first
with methanol/acetic acid (7/3 v/v) and then with methanol until no template
molecules were detected from the recovered solutions with a UV-vis
spectrophotometer. Drying under vacuum afforded particles which were used for
rebinding studies. PM(TOAA) and PP(TOAA) were identically prepared using either
MAA (0.1758 mmol) or 1 (0.0117 mmol) as functional monomer, respectively. The
corresponding blank polymers, PPM(BL), PM(BL), and PP(BL), were prepared in the
same manner in the absence of TOAA.


### 2.3. Batch Rebinding Experiments and Scatchard Analysis

The polymer (20 mg) was added to a dichloromethane
solution (3.5 mL) of TOAA of known concentrations (2.0−8.0×105 M) in vials.
The resulting suspension was shaken
for 16 hours at room temperature, then the polymer was rapidly removed by
filtration and the resulting solution was analyzed by UV-vis spectrophotometer
at 258 nm. The amount of TOAA bound to the polymer, B, was calculated by
subtraction of the concentration of free TOAA, [TOAA], from the initial TOAA
concentration. [TOAA] was determined as an average value of three measurements.
Scatchard analysis was provided by the Scatchard equation, B/[TOAA] =
(Bmax⁡–B) Ka, where Ka is the association constant and Bmax⁡ is the
apparent maximum number of binding sites. Therefore, Ka and Bmax⁡
of the polymer were determined from the slope and the intercept, respectively,
by plotting of B/[TOAA] versus B. Batch rebinding experiments and
Scatchard analysis were performed in a similar manner for PPM(TOAA), PM(TOAA),
PP(TOAA), and the corresponding blank polymers. The rebinding tests were also
carried out incubating the polymers with TOAU in order to verify their binding
selectivity.


## 2.1. Materials and Methods

Ethylene glycol dimethacrylate (EGDMA), 2′,3′,5′-tri-O-acetyladenosine
(TOAA), 2′,3′,5′-tri-O-acetyluridine
(TOAU), and acetic acid were purchased from Sigma-Aldrich (Steinheim, Germany).
α-α′-Azoisobutyronitrile (AIBN) and
methacrylic acid (MAA) were supplied from Fluka (Steinheim, Germany).
Analytical grade dichloromethane and methanol were purchased from J. T. Baker
(Deventer, Holland). Zn(II) tetra(4′-methacryloyloxyphenoxy)phthalocyanine (1)
was prepared on the basis of a published method [17]. UV-Vis spectra were
obtained with a Cary 100 Scan UV-vis spectrophotometer.


## 2.2. Polymer Preparation

The preparation of PPM(TOAA) was carried out as follows:
to a solution of TOAA (0,0468 mmol, as template) in dichloromethane
(262μL)
were added phthalocyanine 1 (0.0117 mmol, as the first functional monomer) and
MAA (0.1758 mmol, as the second functional monomer) in a glass tube. After
adding of EGDMA (0.9360 mmol, as cross-linker) and AIBN (0.0112 mmol, as
initiator), the mixture was sonicated for 5 minutes flushing with nitrogen gas and
then polymerized by heating at 60°C for 16 hours. The resultant polymer was
crushed and sieved. The template molecules were removed by washing the polymer first
with methanol/acetic acid (7/3 v/v) and then with methanol until no template
molecules were detected from the recovered solutions with a UV-vis
spectrophotometer. Drying under vacuum afforded particles which were used for
rebinding studies. PM(TOAA) and PP(TOAA) were identically prepared using either
MAA (0.1758 mmol) or 1 (0.0117 mmol) as functional monomer, respectively. The
corresponding blank polymers, PPM(BL), PM(BL), and PP(BL), were prepared in the
same manner in the absence of TOAA.


## 2.3. Batch Rebinding Experiments and Scatchard Analysis

The polymer (20 mg) was added to a dichloromethane
solution (3.5 mL) of TOAA of known concentrations (2.0−8.0×105 M) in vials.
The resulting suspension was shaken
for 16 hours at room temperature, then the polymer was rapidly removed by
filtration and the resulting solution was analyzed by UV-vis spectrophotometer
at 258 nm. The amount of TOAA bound to the polymer, B, was calculated by
subtraction of the concentration of free TOAA, [TOAA], from the initial TOAA
concentration. [TOAA] was determined as an average value of three measurements.
Scatchard analysis was provided by the Scatchard equation, B/[TOAA] =
(Bmax⁡–B) Ka, where Ka is the association constant and Bmax⁡ is the
apparent maximum number of binding sites. Therefore, Ka and Bmax⁡
of the polymer were determined from the slope and the intercept, respectively,
by plotting of B/[TOAA] versus B. Batch rebinding experiments and
Scatchard analysis were performed in a similar manner for PPM(TOAA), PM(TOAA),
PP(TOAA), and the corresponding blank polymers. The rebinding tests were also
carried out incubating the polymers with TOAU in order to verify their binding
selectivity.


## 3. Results and Discussion

TOAA-imprinted and unimprinted polymers were obtained by
the above method. The binding behavior of the prepared MIPs was evaluated by
batch rebinding tests and the binding data were processed with Scatchard
equation in order to estimate the binding properties of the polymers. Figure1
shows the Scatchard plot for PPM(TOAA). As can be seen, it is a single straight
line, which indicates that there exists one kind of binding sites populated in
the MIP. The Scatchard plot is linear also in the case of PM(TOAA). Similar
Scatchard plots were obtained in a study of Yan et al. [26] with malachite
green-imprinted polymers. This fact is very interesting since a nonlinear
profile was commonly observed in the Scatchard assessment of MIPs indicating
the presence of binding sites that exhibit various affinities to the ligand
[18, 20, 21]. Table 1 shows the values of
Ka and Bmax⁡ for PPM(TOAA) and PM(TOAA).
As shown, the MIP prepared with both functional monomers 1 and MAA,
PPM(TOAA), exhibited higher binding affinities for TOAA compared to PM(TOAA),
prepared solely with MAA. PP(TOAA), prepared only with 1, did not show any
binding capacity for TOAA. This means that phthalocyanine 1 contributes to
higher binding affinities, although 1 itself barely interacts with TOAA. The
effects of the use of both functional monomers strongly suggest that the
imprint that allows the simultaneous multipoint interactions of the template with
the carboxylic residues of MAA and the Zn(II) ion of 1 shows a higher binding
ability for TOAA than that allowing only the individual template/functional
monomer interactions. The corresponding blank polymers, PPM(BL), PM(BL), and PP(BL),
showed no binding affinities for TOAA confirming that the selectivity was due
to the imprinting of the polymer matrix and not to the intrinsic affinity of
the template to the functional monomers alone.Table 1
Association constant (Ka) and maximum number of binding sites
(Bmax) for PPM(TOAA) and PM(TOAA).
PolymerKa(M-1)Bmax (μM g-1)PPM(TOAA)(2.96 ± 0.5)× 1046.53PM(TOAA)(1.48 ± 0.6)× 1041.66Figure 1
Scatchard plot for PPM(TOAA).As for the selectivity (Figure2), both PPM(TOAA) and
PM(TOAA) exhibited binding affinities for TOAA while they did not show any
binding capacity for TOAU. As expected, also PP(TOAA) did not show any binding
capacity for TOAU. The corresponding blank polymers showed no binding affinity
for TOAU too. If we examine the chemical structure of TOAA and TOAU, it is
clear that TOAA has more chances than TOAU to form hydrogen bonds with the MIPs
because of more functional groups that could form hydrogen bonds with the polymers.
Consequently, the polymers PPM(TOAA) and PM(TOAA) showed a good binding affinity
for TOAA and no binding capacity for TOAU. This is not surprising since the
inability of carboxylic acid receptors to bind uracil derivatives appears to be
rather general as evidenced in the literature [15, 27, 28].Figure 2
Binding selectivity test of TOAA and TOAU on the MIPs.The higher binding affinity of PPM(TOAA) for TOAA in
comparison with PM(TOAA) could be explained considering the possibility of coordination
of the Zn(II) ion of 1 with the nitrogen atoms of the 2-amonopyridine moiety of
TOAA. Similar multipoint interactions have already been proposed by Takeuchi et
al. [18] in a study on the preparation of MIPs for cinchonidine by the combined
use of MAA and a vinyl-substituted zinc(II) porphyrin as functional monomers.
The coordination of the Zn(II) ion with the nitrogen atoms of TOAA could be explained
considering that the zinc(II) ion is the acid with moderate hardness and the
nitrogen atom of the 2-aminopyridine moiety is the base with moderate softness
[18]. The absence of this kind of nitrogen atom in the chemical structure of
TOAU could explain the inability of PPM(TOAA) to bind TOAU. On the other hand,
the inability of PP(TOAA) to bind TOAA suggests that the coordination of the Zn(II)
ion of the phthalocyanine with the nucleoside is not sufficient alone to assure
the recognition and subsequent complementary binding between the receptor and
the nucleoside molecule, confirming in this way that the analyte molecule is
specifically bound to the polymer through multipoint interactions.


## 4. Conclusion

A highly specific and selective TOAA-imprinted polymer
was prepared by the combination of MAA and a zinc-phthalocyanine substituted
with methacrylic groups as functional monomers. The effects of the simultaneous
use of the two functional monomers suggest the effective cooperation of the
phthalocyanine-based and carboxylic residues rather than independent operation
for retaining of TOAA.Considering these promising results for easily constructed and highly selective MIPs for nucleosides, new investigations are
now being directed towards the development of MIP-based sensors arrays for
nucleoside discriminations.



---

*Source: 281843-2007-11-21.xml*