Molecular imprinting of nitrophenol and hydroxybenzoic acid isomers: effect of molecular structure and acidity on imprinting

Three nitrophenol isomer-imprinted polymers were prepared under the same conditions using 4-vinylpyridine as a functional monomer. Different recognition capacities for template molecules were observed for the three polymers. Another imprinting system with stronger acidity than nitrophenol isomers, 2-hydroxybenzoic acid (salicylic acid) and 4-hydroxybenzoic acid, was imprinted using 4-vinylpyridine or acrylamide as functional monomer respectively. Both 4-hydroxybenzoic acid-imprinted polymers using the two monomers showed recognition ability for the template molecule. However, when acrylamide was chosen as functional monomer, the salicylic acid-imprinted polymer showed very weak recognition for the template molecule, whereas strong recognition ability of the resultant polymer for salicylic acid was observed with 4-vinylpyridine as functional monomer. It seems that the structure and acidity of template molecules is responsible for the difference in recognition, by influencing the formation and strength of interaction between template molecule and functional monomer during the imprinting process. An understanding of the mechanism of molecular imprinting and molecular recognition of MIPs will help to predict the selectivity of MIPs on the basis of template molecule properties.     

Study on the recognition of templates and their analogues on molecularly imprinted polymer using computational and conformational analysis approaches

A simplified computational model was proposed to simulate the synthesis of molecularly imprinted polymers (MIP), removal of template and recognition of the template and its analogues by MIP. The MIPs with nicotinamide and iso-nicotinamide as templates were prepared using methacrylic acid as functional monomer. Based on our computational model, the interaction energies between the monomer and the template or its analogues were calculated, which were well correlated with the retention factors and imprinting factors obtained on HPLC columns packed with the corresponding MIP particles. The imprinting effects of the template and its analogues were also investigated from the viewpoint of conformational analysis. The computational data were successfully used to predict the chromatographic behaviour of some chemicals in separation on HPLC columns. We believe that the computational method will find application in designing monomers for MIP synthesis and in studying recognition of templates and their analogues on MIP.     

Insights into the origins of binding and the recognition properties of molecularly imprinted polymers prepared using an amide as the hydrogen-bonding functional group

Molecularly imprinted polymers (MIPs) prepared using an amide hydrogen-bonding functional monomer (acrylamide) exhibited efficient enantiomeric recognition properties in both organic and aqueous media in the HPLC mode. The results indicate that the amide functional groups formed strong hydrogen-bonding interactions with the template molecule, and specific recognition sites were created within the polymer matrix during the imprinting process. When Boc-L-Trp was used as the template, an MIP prepared in a polar organic solvent (acetonitrile) using acrylamide as the functional monomer showed better enantiomeric recognition of Boc-Trp than the MIPs prepared in the same solvent using an acidic (methacrylic acid) or a basic (2-vinylpyridine) functional monomer or a combination of an acidic and a basic functional monomer (methacrylic acid + 2-vinylpyridine). Our results indicate that in organic media the degree of retention of the sample molecule on the imprinted polymer was controlled by hydrogen-bonding interactions between the sample molecule and the polymer, while in aqueous media it was determined to a considerable extent by hydrophobic interactions. In both media the shape, size and the nature of the hydrogen-bonding groups of the sample molecules were all important factors in determining the enantiomeric and substrate selectivity. In the aqueous media, however, the hydrophobicity of the sample molecules was also found to play an important role.     

Chiral separation using molecularly imprinted heteroaromatic polymers

Novel molecularly imprinted polymer systems utilizing 4-vinylpyridine and 1-vinylimidazole as functional monomers have been developed for enantioselective recognition of carboxylic and N-protected amino acids. Non-covalent interactions between the functional monomers and the template molecules were the source of the subsequent recognition sites in the resultant polymers. The capacity of the polymers for molecular recognition was investigated by using them as stationary phases in the HPLC mode. Polymers prepared with 4-vinylpyridine were found to be more efficient in racemic resolution than those prepared with 1-vinylimidazole. When applying a racemic mixture of the template molecule, the polymers showed highest affinity for the enantiomer used as template. Imprints of a racemic template molecule, as expected, did not exhibit enantioselectivity. The optimal molar ratio of 4-vinylpyridine to the template Cbz-L -Asp-OH in the polymerization mixture was determined to be 12:1. In addition to enantioselectivity, the investigated polymers demonstrated ‘ligand selectivity’ e.g., a Cbz-L -Asp-OH-imprinted polymer was able to separate Cbz-D ,L -Asp-OH, but was unable to separate Cbz-D ,L -Glu-OH.     

Molecularly imprinted polymer using-p-hydroxybenzoic acid, p-hydroxyphenylacetic acid and p-hydroxyphenylpropionic acid as templates.

Molecularly imprinted polymers (MIPs) using p-hydroxybenzoic acid (p-HB), p-hydroxyphenylacetic acid (p-HPA) and p-hydroxyphenylpropionic acid (p-HPPA) as templates were synthesized. The performance of the templates and their analogues on polymer-based high performance liquid chromatography (HPLC) columns was studied. The imprinting effect of the MIP using p-HB as template is more obvious than that of MIP using either p-HPA or p-HPPA as template, and the mixture of p-HB and p-HPA can be well separated on the MIP using p-HB as template, but not on the blank. Interestingly, the recognition of MIP (p-HB as the template) to p-HB showed a synergistic effect. The retention factor of p-HB is not the sum of those of phenol and benzoic acid. We also found that the imprinting effect decreased when increasing the concentration of acetic acid in mobile phase. The possible reason is that acetic acid molecules occupied the binding sites of the polymer, thereby decreasing the concentration of binding sites. Furthermore, polymers, which showed specificity to 3,4-dihydroxybenzoic acid, can be prepared with p-HB as template. It is thus possible to synthesize a specific polymer for a compound that is either expensive or unstable by using a structurally similar compound as template.     

Acrylic polymeric nanospheres for the release and recognition of molecules of clinical interest

Cross-linked poly(methylmethacrylate-co-methacrylic acid) nanospheres were imprinted with theophylline through template radical polymerisation in diluted acetonitrile solution. This study will focus on the effect of functional monomer nature used (methylmethacrylate and/or methacrylic acid) in the recognition and in the release of template in order to develop a material with combined properties of drug delivery and rebinding for clinical applications. After template extraction the nanospheres showed satisfactory recognition properties (up to 1mg template/g of polymer). Moreover polymers prepared selectively removed theophylline with a theophylline rebinding of 5.1 times higher than that of caffeine, a compound of similar structure. Drug release properties were also satisfactory (up to 95% of loaded theophylline in 7 days).     

Virtual imprinting as a tool to design efficient MIPs for photosynthesis-inhibiting herbicides

Molecular modelling and computational screening were used to identify functional monomers capable of interacting with several different photosynthesis-inhibiting herbicides. The process involved the design of a virtual library of molecular models of functional monomers containing polymerizable residues and residues able to interact with the template through electrostatic, hydrophobic, Van der Waals forces and dipole-dipole interactions. Each of the entries in the virtual library was probed for its possible interactions with molecular models of the template molecules. It was anticipated that the monomers giving the highest binding score would represent good candidates for the preparation of affinity polymers. Strong interactions were computationally determined between acidic functional monomers like methacrylic acid (MAA) or itaconic acid (IA) with triazines, and between vinylimidazole with bentazone and bromoxynil. Nevertheless, weaker interactions were seen with phenylureas. The corresponding blank polymers were prepared using the selected monomers and tested in the solid phase extraction (SPE) of herbicides from chloroform solutions. A good correlation was found between the binding score of the monomers and the affinities of the corresponding polymers. The use of computationally designed blanks can potentially eliminate the need for molecular imprinting, (adding a template to the monomer mixture to create specific binding sites). Data also showed that some monomers have a natural selectivity for some herbicides, which can be further enhanced by imprinting. Thus, in regard to retention on the blank polymer, we can estimate if the resulting imprinted polymer will be effective or not.     

Synthesis of imprinted beads by aqueous suspension polymerisation for chiral recognition of antihistamines

A novel non-stabilised aqueous suspension polymerisation methodology for the preparation of spherical molecularly imprinted polymers is described with chlorpheniramine (CP), d-chlorpheniramine (d-CP), brompheniramine (BP) and d-brompheniramine (d-BP) as the templates, respectively. Using this rapid and simple technique, controlled polymer beads in the low micron range with narrow size distributions were generated by photo-polymerisation. The use of agitation speed as a method of controlling bead size distribution was demonstrated. Enantioselective properties of the imprinted beads were examined and the polymers prepared using d-chlorpheniramine and d-brompheniramine were capable of discriminating between the enantiomers of the template. Cross-selectivity studies were performed by batch rebinding with the influence of template size and functional group orientation of analytes on the recognition properties of the imprinted polymers investigated. Physical characteristics of all polymers were studied by nitrogen sorption porosimetry, particle size analysis and scanning electron microscopy (SEM) in order to gain an insight into the role of such properties on retention behaviour.     

An innovative approach to molecularly imprinted capillaries for polar templates by grafting polymerization

Molecularly imprinted polymers have been successfully used as selective stationary phases in capillary electrophoresis. Notwithstanding, this technique suffers from several drawbacks as the loss of molecular recognition properties in aqueous media and the lack of feasibility for imprinted systems directed towards highly polar templates soluble in aqueous environments only. Thus, the preparation of imprinted polymers for highly polar, water-soluble analytes, represents a challenge. In this work, we present an innovative approach to overcome these drawbacks. It is based on a surface molecular imprinting technique that uses preformed macromonomers as both functional recognition elements and cross-linking agents. A poly-2-hydroxyethyl-co-methacrylic acid linear polymer was grafted from the surface of silica capillaries. The grafted polymer was exhaustively esterified with methacrylic anhydride to obtain polyethylendimethacrylate-co-methacrylic acid linear chains. Then, as a proof of concept, an adequate amount of a very polar template like penicillin V was added in a hydro-organic mixture, and a thin layer of imprinted polymer was obtained by cross-linking the polymer linear chains. The binding behaviour of the imprinted and non-imprinted capillaries was evaluated in different separation conditions in order to assess the presence of template selectivity and molecular recognition effects. The experimental results clearly show that this innovative kind of imprinted material can be easily obtained in very polar polymerization environments and that it is characterized by enhanced molecular recognition properties in aqueous buffers and good selectivity towards the template and strictly related molecules.     

Molecular imprinting and solid phase extraction of flavonoid compounds

Molecularly imprinted polymers (MIPs) for quercetin have been successfully prepared by a thermal polymerization method using 4-vinylpyridine (4-VP) and ethylene glycol dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The obtained molecularly imprinted polymers were evaluated by HPLC using organic eluents, with respect to their selective recognition properties for quercetin and related compounds of the flavonoid class. Two equivalent control polymers, a blank polymer and a polymer imprinted with a structural analogous template, were synthesized, in order to confirm the obtained results. Furthermore, preliminary experiments confirm the applicability of the prepared MIPs for solid phase extraction (SPE), as rapid and facile clean-up of wine samples for HPLC analysis is an envisaged field of application. The successful preparation of molecularly imprinted polymers for flavones provides an innovative opportunity for the development of advanced separation materials, with applications in the field of wine and fermentation analysis.     

N-isopropylacrylamide as a functional monomer for noncovalent molecular imprinting

Although N-isopropylacrylamide (NIPAM) has previously been used in molecular imprinting, it has mostly been considered as an 'inert' monomer, or included for its temperature-responsive nature, rather than as a functional monomer responsible for the interactions with the template at the recognition site. A comparative study of NIPAM and other traditional, functional monomers for the imprinting of a hydrogen bond donor template, bisphenol A (BPA), is reported. Nuclear magnetic resonance titration data suggest that NIPAM forms a stronger complex with BPA than either acrylamide or methacrylic acid but a weaker complex than vinylpyridine. Molecular imprinted polymers (MIPs) were prepared using each functional monomer and compared as stationary phases for the separation of BPA from structural analogues. The NIPAM-containing MIP bound BPA with better selectivity than those prepared using acrylamide or methacrylic acid. Using NIPAM also reduces the nonspecific binding, which is found with MIPs using vinylpyridine as functional monomer.     

New biomedical devices with selective peptide recognition properties. Part 1: Characterization and cytotoxicity of molecularly imprinted polymers

Molecular imprinting is a technique for the synthesis of polymers capable to bind target molecules selectively. The imprinting of large proteins, such as cell adhesion proteins or cell receptors, opens the way to important and innovative biomedical applications. However, such molecules can incur into important conformational changes during the preparation of the imprinted polymer impairing the specificity of the recognition cavities. The "epitope approach" can overcome this limit by adopting, as template, a short peptide sequence representative of an accessible fragment of a larger protein. The resulting imprinted polymer can recognize both the template and the whole molecule thanks to the specific cavities for the epitope. In this work two molecularly imprinted polymer formulations (a macroporous monolith and nanospheres) were obtained using the protected peptide Z-Thr-Ala-Ala-OMe, as template, and Z-Thr-Ile-Leu-OMe, as analogue for the selectivity evaluation, methacrylic acid, as functional monomer, and trimethylolpropane trimethacrylate and pentaerythritol triacrylate (PETRA), as cross-linkers. Polymers were synthesized by precipitation polymerization and characterized by standard techniques. Polymerization and rebinding solutions were analyzed by high performance liquid chromatography. The highly cross-linked polymers retained about 70% of the total template amount, against (20% for the less cross-linked ones). The extracted template amount and the rebinding capacity decreased with the cross-linking degree, while the selectivity showed the opposite behaviour. The PETRA cross-linked polymers showed the best recognition (MIP 2-, alpha=1.71) and selectivity (MIP 2+, alpha'=5.58) capabilities. The cytotoxicity tests showed normal adhesion and proliferation of fibroblasts cultured in the medium that was put in contact with the imprinted polymers.     

Development of molecularly imprinted polymers for the binding of nitrofurantoin

Novel molecularly imprinted polymers (MIPs) for the recognition of nitrofurantoin (NFT) were prepared by photoinitiated polymerisation in polar solvent using 2,6-bis(methacrylamido) pyridine (BMP) as the functional monomer and carboxyphenyl aminohydantoin (CPAH) as the analogue of the template. The binding constants of the complex between BMP and nitrofurantoin or CPAH in DMSO were determined with 1H NMR titration to be 630 ± 104 and 830 ± 146 M⁻¹, respectively. To study the influence of the functional monomer, two polymer compositions were prepared containing the template, the functional monomer and the crosslinker in the molar ratio 1:1:12 for MIP1 and 1:4:20 for MIP2, respectively. The imprinting factor at saturation concentration of nitrofurantoin, which is the ratio of the amount bound to the MIP and the non-imprinted control polymer (NIP), was determined to be 2.47 for MIP1 and 2.49 for MIP2. The cross reactivity of the imprinted polymers seems to be determined by the ability to form hydrogen bonds to the functional monomer while the shape of the molecule has no real influence.     

Surface imprinting polymers for the recognition of nucleotides

A highly selective polymer has been prepared for the selective separation of nucleotides by the surface imprinting polymerization. A dialkyl quaternary ammonium chloride was effective as the functional molecule for recognizing the difference in the structure of nucleotides. Adsorptive behavior of the ionic species of the structural analogues, inosine-5'-monophosphoric acid (IMP) and guanosine-5'-monophosphoric acid (GMP), could be controlled by changing the pH condition. Surface imprinting polymers were prepared under different pH conditions; pH 9.0 and pH 8.5. The IMP-imprinted polymers exhibited higher template effect for IMP than for a structural analogue, GMP. A reference polymer prepared without the imprint molecule neither exhibit any selectivity to IMP nor to GMP. The adsorption behavior was quantitatively evaluated by the binding constants for the IMP-imprinted polymer. The imprinting polymer was found to recognize a small structural difference in nucleotides.     

Modeling and prediction of the mixed-mode retention mechanisms for puerarin and its analogues on n-octylamine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monoliths

n-Octylamine modified poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (poly(GMA-co-EGDMA)) monoliths were prepared for the rapid screening and determination of puerarin content of a crude extract Radix puerariae. The mixed-mode retention mechanisms for puerarin and its analogues on n-octylamine modified monoliths were investigated using a variety of solvent systems, chromatographic evaluation and molecular dynamics (MDs) modeling. The equilibrated conformations between cross-linked polymers and target molecules were obtained from MD modeling. Both the polymer skeleton and functional groups played important roles in the recognition process. The cross-linker formed a structural network skeleton, in which recognition cavities were formed surrounded by functional groups. The polymer network structures provided good interaction access for isoflavones. The active groups recognized isoflavones by both intermolecular hydrogen bonding and hydrophobic interaction. The interaction energies and retention factors between polymers and target molecules were also evaluated and compared. A higher value of interaction energy corresponded to a higher value of retention factor. The potential of using modeling technology for predicting the chromatographic performances of target molecules was explored.     

Supported imprinted nanospheres for the selective recognition of cholesterol

The preparation of innovative polymeric systems using molecular imprinting technology for application in extracorporeal blood purification is described. Membranes based on a methylmethacrylate-co-acrylic acid copolymer, produced through the phase inversion method, were modified introducing into their structure specific binding sites for cholesterol molecule by adding molecularly imprinted nanoparticles in the membrane matrix. Membranes prepared are intended to selectively remove cholesterol from the blood by using interactions at a molecular level, between the membrane/nanoparticles devices and the template, created during the preparation of polymers. Three polymeric systems in form of nanoparticles were prepared differing in the polymerisation solvent (a mixture of acetonitrile and ethanol (1:1) or pure ethanol), and the molar ratio between the functional monomer and the cross-linker (2.3:1 and 1:1). Two out of three of the prepared polymers showed a very good template rebinding capacity both in phosphate buffer solution (pH 6.9) and in ethanol. In particular the nanoparticles rebound 115.4 mg cholesterol/g polymer in buffer solution, and 57 mg cholesterol/g polymer in ethanol.

The deposition of the nanoparticles on the surface of the phase inversion membranes produced devices with interesting rebinding performances towards cholesterol in buffer solution: a specific recognition of 14.09 mg cholesterol/g system (membrane and nanoparticles) was detected, indicating maintained binding capacity of supported particles as well.     

Biodegradable molecularly imprinted polymers based on poly(ε-caprolactone)

Novel biodegradable molecularly imprinted polymers (MIPs) based on poly(ε-caprolactone) (PCL) were prepared by combining two important properties required of ideal biomaterials, biodegradability (with biocompatibility) and molecular recognition properties. Acrylate or methacrylate end-capped PCL macromers were synthesized through the reaction of PCL diol or triol with acryloyl or methacryloyl chloride. The synthesis of acrylate or methacrylate end-capped macromers was confirmed using FT-IR and H NMR spectroscopic techniques. These macromers were used to prepare biodegradable crosslinked networks by photopolymerization with functional monomer (acrylic acid) and a model template (theophylline). The theophylline-imprinted polymer showed higher binding capacity for theophylline compared with non-imprinted polymer (NIP), and also showed selectivity for theophylline over caffeine (similar structure molecules). PCL-based MIP degraded 8% of the initial weight in 30 days in phosphate-buffered saline (PBS) solution (pH 7.4) and over 90% of the initial weight within 24 h in 1 N NaOH at 37°C.     

A molecular recognition strategy towards tetra-chlorinated dibenzo-p-dioxins, TCDDs

Uniformly sized polymeric separation media were prepared using o- or p-xylene as porogenic template to investigate chromatographic selectivity towards tetra-chlorinated dibenzo-p-dioxins (TCDDs). TCDDs having chlorine atoms at ortho positions of phenyl rings were selectively retained on stationary phase prepared with o-xylene as porogenic template, while TCDDs having chlorine atoms at para positions of phenyl ring were found to be retained selectively on the stationary phase imprinted by the porogenic template, p-xylene. Slightly longer cross-linking agent afforded chromatographically selective retention for larger TCDD isomers. It was also found that positional relationship between substituted chlorine atoms was also important for chromatographic recognition.     

A molecularly imprinted receptor for separation of testosterone and epitestosterone, based on a steroidal cross-linker

A series of molecularly imprinted polymers have been prepared and investigated as stationary phases in high performance liquid chromatography for the separation of testosterone and epitestosterone using non-polar mobile phases. The polymers were imprinted using 5α-dihydrotestosterone as template, and all retain testosterone more strongly than its 17α-OH epimer. The best polymer was prepared using trifluoromethylacrylic acid as functional monomer (interacting with the template via hydrogen bonds), divinylbenzene as ‘inert’ cross-linker, and chloroform as porogen. It also included a steroid-based cross-linker, which may interact with the template via van der Waals interactions to lend additional ‘shape selectivity’. A 250 × 4.6 mm column packed with this polymer gave baseline resolution of testosterone and epitestosterone (15 μg each) in under 20 min. Preparation of the steroid based cross-linker included the selective reduction of 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) to the 3α,17β-diol using K-selectride.     

Molecular recognition in synthetic polymers. Enantiomeric resolution of amide derivatives of amino acids on molecularly imprinted polymers

Molecular imprints were prepared using L-phenylalanine anilide as the print molecule and methacrylic acid as the functional monomer. Methacrylic acid interacts ionically with the primary amine of the print molecule and via hydrogen bonding with the amide function. In the HPLC mode such polymers were shown to exhibit efficient enantiomeric resolution of a racemic mixture of the original print molecule. Enantiomeric resolution was shown to be dependent on the ratio of methacrylic acid to print molecule in the pre-polymerization mixture and specific for the presence of both print molecule and functional monomer. Further analyses showed the importance of both the primary amino and amide functions in the correct stereochemistry for recognition and enantiomeric resolution of compounds on such polymers. Other amide derivatives of amino acids including p-nitroanilides, beta-naphthylamides and amides were recognized by such polymers, and enantiomeric resolution was obtained for amide derivatives of amino acid ranging from alanine to tryptophan on a single polymer. The implications of these findings with respect to the mechanism of recognition and the ability to predict enantiomeric resolution of molecules on molecularly imprinted polymers will be discussed.