Analyte separation by OMNiMIPs imprinted with multiple templates

Changes detected in the imprinting effect by OMNiMIPs imprinted with multiple templates appear to be a function of the maximum template loading. Below the maximum template loading, the polymers imprinted with multiple compounds provide molecular recognition close to the polymers imprinted with single compounds, for each template compound tested. However, template loading past this point can result in significant lowering of the imprinting effect. For example, 1,1′-bi-2-naphthol enantiomers showed nearly a 60% loss in enantioselectivity on OMNiMIP 8 (imprinted with four templates); yet still maintained a separation factor of 3.7 allowing baseline separation of enantiomers. Similar behavior was seen for the other three template molecules, although losses in enantioselectivity were less severe. The multi-analyte imprinted OMNiMIP 8 was shown to be capable of separating a template molecule individually from a mixture, including enantiomers, but not all four concurrently. With respect to physical properties of the different OMNiMIPs, gradual trends in porosity and surface area correlated to the concentration of the templates, independent of their molecular structure.     

Crown ethers as a tool for the preparation of molecularly imprinted polymers

Molecularly imprinted polymers have been prepared against aniline and a bis-aniline compound, making use of a crown ether (18-crown-6) to solubilize the monomer-template complexes. Subsequent chromatographic rebinding studies in the absence of crown ether revealed regioselectivity for the templates in the respective polymers. This study indicates that crown ethers can be potentially useful in conjunction with molecular imprinting to solubilize and imprint organic solvent-insoluble compounds.     

Some new developments and challenges in non-covalent molecular imprinting technology

The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules via the use of templates. Molecularly imprinted polymers have been applied in an increasing number of applications where molecular binding events are of interest. These include the use of molecularly imprinted polymers as tailor-made separation materials, antibody and receptor binding site mimics in recognition and assay systems, enzyme mimics for catalytic applications and as recognition elements in biosensors. The stability and low cost of molecularly imprinted polymers make them advantageous for use in analysis as well as in industrial-scale production and application.     

Unusual regioselectivity and active site topology of human cytochrome P450 2J2

The oxidation of six derivatives of terfenadone by recombinant human CYP2J2 (CYP = cytochrome P450) was studied by high-performance liquid chromatography coupled to mass spectrometry (MS) using tandem MS techniques and by 1H NMR spectroscopy. CYP2J2 exhibited a surprising regioselectivity in favor of the hydroxylation of the substrate terminal chain at the weakly reactive homobenzylic position. In contrast, hydroxylation of the same substrates by CYP3A4 mainly occurred on the most chemically reactive sites of the substrates (N-oxidation and benzylic hydroxylation). A 3D homology model of CYP2J2 was constructed using recently published structures of CYP2A6, CYP2B4, CYP2C8, CYP2C9, and CYP2D6 as templates. In contrast with other CYP2 structures, it revealed an active site cavity with a severely restricted access of substrates to the heme through a narrow hydrophobic channel. Dynamic docking of terfenadone derivatives in the CYP2J2 active site allowed one to interpret the unexpected regioselectivity of the hydroxylation of these substrates by CYP2J2, which is mainly based on this restricted access to the iron. The structural features that have been found to be important for recognition of substrates or inhibitors by CYP2J2 were also interpreted on the basis of CYP2J2-substrate interactions in this model.     

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.     

Design and synthesis of molecularly imprinted polypyrrole based on nanoreactor SBA-15 for recognition of ascorbic acid

Molecular imprinting is an attractive technique for preparing mimics of natural and biological receptors. Nevertheless, molecular imprinting for aqueous systems remains a challenge due to the hydrogen bonding between templates and functional monomers destroyed in the bulk water. The hydrogen bonding between templates and monomers are the most crucial factor governing recognition, particularly in non-covalent molecularly imprinted polymers. Using mesoporous materials for molecular imprinting is an effective approach to overcome this barrier and to remove the limitations of the traditional molecularly imprinted polymers which include incomplete template removal, small binding capacity, slow mass transfer, and irregular materials shape. Here, SBA-15 was used as a mesoporous silica material for synthesis of molecularly imprinted polypyrrole. The pyrrole monomers and template molecules were immobilized onto the SBA-15 hexagonal channels, and then polymerization occurred. The resulting nanocomposites were characterized by Fourier transform infrared (FT-IR) analysis, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) methods. In batch rebinding tests, the imprinted nanocomposites reached saturated adsorption within 100min and exhibited significant specific recognition toward the ascorbic acid (AA) with high adsorption capacity (83.7mgg(-1)). To further illustrate the recognition property of the imprinted nanocomposites, binary competitive and non-competitive adsorption experiments were performed with ascorbic acid, dopamine, paracetamol and epinephrine. The imprinting factors for these compounds in non-competitive adsorption experiments were 3.2, 1.5, 1.4 and 1.3, respectively. The results showed that the imprinted nanocomposites exhibited significant adsorption selectivity for the ascorbic acid against the related compounds.     

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.     

A brief review of coarse-grained and other computational studies of molecularly imprinted polymers

Molecular imprinting is an established method for the creation of artificial recognition sites in synthetic materials through polymerization and cross-linking in the presence of template molecules. Removal of the templates leaves cavities that are complementary to the template molecules in size, shape, and functionality. In recent years, various theoretical and computational models have been developed as tools to aid in the design of molecularly imprinted polymers (MIPs) or to provide insight into the features that determine MIP performance. These studies can be grouped into two general approaches-screening for possible functional monomers for particular templates and macromolecular models focusing on the structural characterization of the imprinted material. In this review, we pay special attention to coarse-grained models that characterize the functional heterogeneity in imprinted pores, but also cover recent advances in atomistic and first principle studies. We offer a critical assessment of the potential impact of the various models towards improving the state-of-the-art of molecular imprinting.     

Systematic cross-selectivity study of the factors influencing template receptor interactions in molecularly imprinted nitrogen heterocycles

A systematic cross-selectivity study involving a series of structurally related N-methylated and non-methylated substituted pyridines was performed with the aim of evaluating the parameters responsible for template receptor binding in molecularly imprinted polymers. Variation in binding of substrate structure permitted evaluation of the steric restraints of the imprinted cavity. The electrostatic effects, primarily hydrogen-bonding, were investigated through rebinding in chloroform and acetonitrile. All species were non-covalently imprinted in thermoinduced methacrylic acid-ethylene glycol dimethacrylate co-polymers. Evaluation of template properties indicate that a correlation exists between non-specific binding and template basicity for a series of structural isomers. A correlation between non-specific binding and hydrophobicity was also identified for templates increasing in alkyl character. However no overall correlation was observed, as it was speculated that these factors may be competing. All species imprinted, with the exception of 2-dimethylaminopyridine, produced a selective response for the template species. Varying degrees of cross-selectivity were observed for each imprinted polymer. Polymers imprinted with templates of higher basicity demonstrated a greater degree of cross-selectivity relative to those of lower basicity. While overall binding was reduced in acetonitrile relative to chloroform, specificity was increased. This highlights the intrinsic difference in binding sites within imprinted and non-imprinted sites of the polymer. Finally, while the ability of the template species to form a co-operative interaction may be advantageous in producing a selective imprint it is not a prerequisite. For species based on this co-operative interaction the steric environment in the immediate proximity to the binding functionalities are critical to recognition. Steric hindrance of non-functionally active groups can dramatically impair the formation of interactions.     

Thermochemical study of amino acid imprinted polymer films

Molecularly imprinted polymers provide an alternative to traditional methods of amino acid analysis. The imprinted polymers are more robust and significantly less expensive than, for example, ELISA analysis. Amino acid imprinted nylon‐6 thin films were studied by differential scanning calorimetry and scanning electron microscopy. Endothermic peaks were observed for imprinted films at temperatures higher than that for pure nylon, indicating the formation of a more‐ordered, hydrogen bonded polymer. Removal of the amino acid from the imprinted film resulted in reversion to the peak observed for pure nylon‐6. Additives, β‐cyclodextrin and multiwalled carbon nanotubes, were added to the imprinted polymer solutions as a means to increase the porosity of the films. These studies resulted in alternative morphologies and calorimetric results that provide additional functionalities and applications for imprinted polymers. Copyright © 2015 John Wiley & Sons, Ltd.     

CYP2D6.10 present in human liver microsomes shows low catalytic activity and thermal stability

Comparing bufuralol 1'-hydroxylase activity among liver microsomes prepared from individuals whose CYP2D6 genotypes had been determined, we found that the activity tended to decrease depending on the number of the CYP2D6*10 allele. Pre-incubation of liver microsomes from individuals homozygous for the CYP2D6*10 allele resulted in a decrease in the enzyme activity more rapidly than those from individuals homozygous for the CYP2D6*1, suggesting that not only the catalytic activity but also the thermal stability of the enzyme appeared to be affected by the genetic polymorphism. To confirm this hypothesis, the kinetic parameters of CYP2D6.1 and CYP2D6.10 were compared for bufuralol 1'-hydroxylation and dextromethorphan O-demethylation using microsomes prepared from yeast transformed with plasmids carrying CYP2D6 cDNAs (*1A and *10B). Kinetic studies of these CYP2D6 forms indicated clear differences in the metabolic activities between the wild (CYP2D6.1) and the mutant enzymes (CYP2D6.10). Bufuralol 1(')-hydroxylase activity in microsomes of yeast expressing CYP2D6.10 was rapidly decreased by heat treatment, supporting the idea that the thermal stability of the enzyme was reduced by amino acid replacement from Pro (CYP2D6.1) to Ser (CYP2D6.10). These data strongly suggest that the thermal instability together with the reduced intrinsic clearance of CYP2D6.10 is one of the causes responsible for the known fact that Orientals show lower metabolic activities than Caucasians for drugs metabolized mainly by CYP2D6, because of a high frequency of CYP2D6*10 in Orientals.     

Characterization of variant alleles of cytochrome CYP2D6 in a Spanish population

The CYP2D6 gene codes for a P450 monooxygenase which is involved in the biotransformation of a large number of commonly prescribed drugs. Adverse drug effects and therapeutic failure can be related to abnormal CYP2D6 activity. We investigated the allele and genotype frequencies of cytochrome P4502D6 in a Spanish population to predict the prevalence of ultra-rapid and poor metabolizer phenotypes in our population and to design a feasible CYP2D6 genotyping protocol. The study included 105 healthy unrelated Spanish Caucasian volunteers. CYP2D6 genotyping was performed by a combination of long-PCR, direct sequencing and allele-specific real-time PCR. The frequency of the wild-type CYP2D6*1 allele was 31%. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity showed frequencies of 40.47, 2.38 and 1.90%, respectively. Frequencies of defective alleles *3, *4, *5 and *6 were 0.95, 13.8, 3.33 and 0.95%, respectively. The defective CYP2D6 alleles *7, *8, *12, *14, *15 and *21 were not found. Duplicated CYP2D6 alleles were detected at a frequency of 4.27%. Our protocol allows the identification of the four inactive CYP2D6 alleles (*3, *4, *5 and *6) and the detection of alleles with CYP2D6 *1, CYP2D6 *2 and CYP2D6*4 gene duplications. Testing for this reduced CYP2D6 allele set would facilitate its use in clinical practice by assisting in the development of individualized pharmacotherapy.     

Controlling size and uniformity of molecularly imprinted nanoparticles using auxiliary template

Molecularly imprinted nanomaterials are gaining substantial importance. As a simple and efficient synthetic method, precipitation polymerization has been used to prepare uniform molecularly imprinted microspheres for numerous template compounds. Despite of its general applicability, the difficulty of obtaining uniform particles for some difficult templates by precipitation polymerization has been reported. In this work, we attempted to produce uniform atrazine-imprinted nanoparticles using propranolol as an auxiliary template under standard precipitation polymerization condition. When propranolol was added in the prepolymerization mixture for atrazine imprinting, it displayed a significant effect on particle size and size distribution of atrazine-imprinted polymers. The molecular binding characteristics of the molecularly imprinted polymer (MIP) nanoparticles were found to be dependent on the relative ratios of the two templates. Under an optimal template propranolol-atrazine ratio of 1:3 mol/mol, very uniform imprinted nanoparticles (d(H) =?106?nm) with a polydispersity index of 0.07 were obtained. The loading of the auxiliary template (propranolol) could be reduced to as low as 5% without sacrificing the uniformity of the MIP nanoparticles. The uniform MIP nanoparticles could be easily encapsulated into polyethylene terephthalate nanofibers using a simple electrospinning technique. The composite nanofibers containing the MIP nanoparticles maintained specific molecular binding capability for both atrazine and propranolol.     

The decreased in vivo clearance of CYP2D6 substrates by CYP2D6*10 might be caused not only by the low-expression but also by low affinity of CYP2D6

CYP2D6 exhibits genetic polymorphism with interindividual differences in metabolic activity. We have found a significant influence on the pharmacokinetics of venlafaxine by the CYP2D6*10 allele in a Japanese population. CYP2D6.10, which is translated from CYP2D6*10, has two amino acid substitutions: Pro34 --> Ser and Ser486 --> Thr. In this study, CYP2D6.10 was expressed in Saccharomyces cerevisiae and its catalytic activity for CYP2D6 substrates was investigated. The CYP2D6*10B- and *10C-associated cDNA were isolated from human lymphocyte genotyped as CYP2D6*10. In addition, three forms of CYP2D6, Pro34/Thr486 (PT), Ser34/Ser486 (SS), and Pro34/Ser486 (wild type, CYP2D6.1), were constructed by PCR-site mutagenesis to clarify the effects of the two amino-acid substitutions. The expression of CYP2D6 protein was confirmed by immunoblotting using CYP2D antibody. The absorbance at 450 nm was measured by CO-reduced difference spectra from five all microsome preparations. The CYP2D6 forms with Pro34 --> Ser amino acid substitution were at a lower expression than CYP2D6.1 from the findings of immunoblotting and spectral analysis. The apparent K(m) values of CYP2D6.1, CYP2D6.10A, and CYP2D6.10C were 1.7, 8.5, and 49.7 microM, respectively, for bufuralol 1'-hydroxylation, and 9.0, 51.9, and 117.4 microM, respectively, for venlafaxine O-demethylation, respectively. The V(max) values were not significantly different among the three variants. These findings suggest that the decreased in vivo clearance by CYP2D6*10 was caused not only by low expression of but also the increased K(m) value of CYP2D6.     

A simple approach to prepare molecularly imprinted polymers from nylon‐6

A convenient and simple approach for the preparation of molecularly imprinted polymers (MIPs) based on polyamide (nylon‐6) was developed. The polymer matrix formation occurred during the transition of nylon from dissolved to solid state in the presence of template molecules in the initial solution. 2,2,2‐Trifluoroethanol (TFE) was chosen as a main solvent for the polyamide. It provides a high solubility of nylon and does not significantly change the structure of biopolymers. The alteration of the polymer matrix structure after the addition of different types of porogens in the nylon/TFE solution was investigated. The structured polymers in the form of films and microparticles were prepared in the chosen optimal conditions. Different model biomolecular templates (of low‐ and high‐molecular weight) were used for the preparation of MIPs, which were shown to specifically recognize these molecules upon binding experiments. The binding of the template molecules to MIPs was monitored using spectrophotometric, radioisotopic, or fluorometric detection. The selectivity coefficients of the MIPs were estimated to be 1.4–4.6 depending on the type of templates and conditions of the polymer matrix formation. Copyright © 2013 John Wiley & Sons, Ltd.     

Evolution of the CYP2D gene cluster in humans and four non-human primates

The human cytochrome P450 2D6 (CYP2D6) is a primary enzyme involved in the metabolism of about 25% of commonly used therapeutic drugs. CYP2D6 belongs to the CYP2D subfamily, a gene cluster located on chromosome 22, which comprises the CYP2D6 gene and pseudogenes CYP2D7P and CYP2D8P. Although the chemical and physiological properties of CYP2D6 have been extensively studied, there has been no study to date on molecular evolution of the CYP2D subfamily in the human genome. Such knowledge could greatly contribute to the understanding of drug metabolism in humans because it makes us to know when and how the current metabolic system has been constructed. The knowledge moreover can be useful to find differences in exogenous substrates in a particular metabolism between human and other animals such as experimental animals. Here, we conducted a preliminary study to investigate the evolution and gene organization of the CYP2D subfamily, focused on humans and four non-human primates (chimpanzees, orangutans, rhesus monkeys, and common marmosets). Our results indicate that CYP2D7P has been duplicated from CYP2D6 before the divergence between humans and great apes, whereas CYP2D6 and CYP2D8P have been already present in the stem lineages of New World monkeys and Catarrhini. Furthermore, the origin of the CYP2D subfamily in the human genome can be traced back to before the divergence between amniotes and amphibians. Our analyses also show that reported chimeric sequences of the CYP2D6 and CYP2D7 genes in the chimpanzee genome appear to be exchanged in its genome database.     

Functional characterization of a first avian cytochrome P450 of the CYP2D subfamily (CYP2D49)

The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other animal models, research concerning the chicken CYP2Ds is limited. In this study, a cDNA encoding a novel CYP2D enzyme (CYP2D49) was cloned from the chicken liver for the first time. The CYP2D49 cDNA contained an open reading frame of 502 amino acids that shared 52%-57% identities with other CYP2Ds. The gene structure and neighboring genes of CYP2D49 are conserved and similar to those of human CYP2D6. Additionally, similar to human CYP2D6, CYP2D49 is un-inducible in the liver and expressed predominantly in the liver, kidney and small intestine, with detectable levels in several other tissues. Metabolic assays of the CYP2D49 protein heterologously expressed in E. coli and Hela cells indicated that CYP2D49 metabolized the human CYP2D6 substrate, bufuralol, but not debrisoquine. Moreover, quinidine, a potent inhibitor of human CYP2D6, only inhibited the bufuralol 1'-hydroxylation activity of CYP2D49 to a negligible degree. All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile. Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein. The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.     

Rat CYP2D2, not 2D1, is functionally conserved with human CYP2D6 in endogenous morphine formation

The assumption that CYP2D1 is the corresponding rat cytochrome to human CYP2D6 has been revisited using recombinant proteins in direct enzyme assays. CYP2D1 and 2D2 were incubated with known CYP2D6 substrates, the three morphine precursors thebaine, codeine and (R)-reticuline. Mass spectrometric analysis showed that rat CYP2D2, not 2D1, catalyzed the 3-O-demethylation reaction of thebaine and codeine. In addition, CYP2D2 incubated with (R)-reticuline generated four products corytuberine, pallidine, salutaridine and isoboldine while rat CYP2D1 was completely inactive. This intramolecular phenol-coupling reaction follows the same mechanism as observed for CYP2D6. Michaelis-Menten kinetic parameters revealed high catalytic efficiencies for rat CYP2D2. These findings suggest a critical evaluation of other commonly accepted, however untested, CYP2D1 substrates.