Construction of artificial cyclic amide amidohydrolases using molecular imprinting technique

A general molecular imprinting approach is proposed to synthesize artificial enzymes to mimic the family of cyclic amide amidohydrolases which share similar active site and catalytic mechanism. The artificial enzymes were constructed by co-polymerizing 4(5)-vinylimidazole-Co²⁺-methacrylic acid clusters with divinylbenzene micro-spheres in the presence of corresponding substrates. The artificial enzymes mimicked creatininase and hydantoinase by showing specific affinity towards the corresponding substrates in buffer. The artificial hydantoinase also showed specific affinity towards corresponding substrate in organic solvent, and catalyzed the hydrolysis of hydantoin.     

The molecular reaction vessels for a transesterification process created by molecular imprinting technique

A polymeric catalyst was synthesized by co-polymerizing 4(5)-vinylimidazole and itaconic acid with trimethylpropanol trimethacrylate micro spheres. The catalyst obtained catalysed the transesterification process between p-nitrophenyl acetate and hexanol with maximal initial velocity(nu(max)) of 4.73 +/- 0.47 microM min(-1) mg(-1), and turnover rate (k(cat)) of 8.67 min(-1). Using p-nitrophenyl acetate as template, molecular imprinting process enhanced the nu(max) of the polymeric catalyst 3-fold. Each imprinted site can be considered as a single molecular reaction vessel, and achieved a k(cat) of 169 min(-1) towards the conversion of p-nitrophenyl acetate in hexanol.     

Stereoselectivity of phosphotriesterase with paraoxon derivatives: a computational study

The bacterial enzyme phosphotriesterase (PTE) exhibits stereoselectivity toward hydrolysis of chiral substrates with a preference for the S_p enantiomer. In this work, docking analysis and two explicit-solvent molecular dynamics (MD) simulations were performed to characterize and differentiate the structural dynamics of PTE bound to the S_p and R_p paraoxon derivative enantiomers (R_p-1 and S_p-1) hydrolyzed with distinct catalytic efficiencies. Comparative analysis of the molecular trajectories for PTE bound to R_p-1 and S_p-1 suggested that substrate binding induced conformational changes in the loops near the active site. After 100 ns of MD simulation, the Zn β²⁺ metal ion formed hexacoordinated- and tetracoordinated geometries in the S_p-1-PTE and R_p-1-PTE ensembles, respectively. Simulation results further showed that the hydrogen bond between Asp301 and His254 occurred with a higher probability after S_p-1 binding to PTE (47.5%) than that after R_p-1 binding (22.2%). These results provide a qualitative and molecular-level explanation for the 10 orders of magnitude increase in the catalytic efficiency of PTE toward the S_p enantiomer of paraoxon.     

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.     

Exploring stereochemical specificity of phosphotriesterase by MM-PBSA and MM-GBSA calculation and steered molecular dynamics simulation

Wild-type phosphotriesterase (PTE) prefers the S_P-enantiomers over the corresponding R_P-enantiomers by factors ranging from 10 to 90. To satisfy the binding modes of the PTE of S_P- and R_P-enantiomers, all-atom molecular dynamics simulations were carried out on two paraoxon S_P and R_P derivatives, namely, S_p-1 and R_p-1. Molecular mechanics Poisson–Boltzmann surface area and molecular mechanics generalized Born surface area (MM-PBSA and MM-GBSA) calculations indicated that His230 in S_p-1-PTE had a closer interaction with the substrate than that in R_p-1-PTE and that such interaction increased the catalytic efficiency of PTE for S_p-1. The steered molecular dynamics simulation indicated that, compared with S_p-1, R_p-1 in the unbinding (binding) may hinder some residue displacement, thus requiring more effort to escape the binding pocket of PTE. In addition, Trp131, Phe306, and Tyr309 are deemed important residues for the S_p-1 unbinding pathway via PTE, whereas Tyr309 alone is considered an important residue for the R_p-1 unbinding pathway. These results demonstrate the possibility of dramatically altering the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates by modifying specific residues located within the active site of PTE.     

Determination of phenformin hydrochloride using molecular imprinting technology coupled with flow-injection chemiluminescence

A novel method was developed using molecular imprinting technology (MIT) coupled with flow‐injection chemiluminescence (FI‐CL) for highly sensitive detection of phenformin hydrochloride (PH). The phenformin imprinted polymer was synthesized with methacrylic acid (MAA) as a functional monomer and ethylene glycol dimethacrylate (EGDMA) as a cross‐linker. Newly synthesized molecularly imprinted polymer (MIP) particles were packed into a column as a selective recognition element for determination of PH. A CL method for the determination of PH was developed based on the CL reaction of PH with N‐bromosuccinimide sensitized by eosin Y in basic media. The optimization of detection conditions was investigated. The CL intensity responded linearly to the concentration of PH in the range 0.09–2.0 µg/mL, with a correlation coefficient of 0.9920. The detection limit was 0.031 µg/mL. The relative standard deviation for the determination of 1.0 µg/mL PH solution was 1.0% (n = 11). The method was applied to the determination of PH in urine samples, with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.     

Molecular modelling of a template substitute and monomers used in molecular imprinting for aflatoxin B1 micro-HPLC analysis

The preparation of molecularly imprinted polymers (MIPs) involves the polymerisation of functional monomers in the presence of template molecules. 5,7-Dimethoxycoumarin (DMC) was found to be a structural analogue for aflatoxin B1 (AB1) and serves as its substitute in a grafting solution for the MIP synthesis. It was found that both methacrylic acid and allylamine are functional monomers which could provide a similar binding towards AB1 and DMC.     

SOFT‐MI: A novel microfabrication technique integrating soft‐lithography and molecular imprinting for tissue engineering applications

An innovative approach has been employed for the realization of bioactive scaffolds able to mimic the in vivo cellular microenvironment for tissue engineering applications. This method is based on the combination of molecular imprinting and soft‐lithography technology to enhance cellular adhesion and to guide cell growth and proliferation due to presence of highly specific recognition sites of selected biomolecules on a well‐defined polymeric microstructure. In this article polymethylmethacrylate (PMMA) scaffolds have been realized by using poly(dimethylsiloxane) (PDMS) microstructured molds imprinted with FITC‐albumin and TRITC‐lectin. In addition gelatin, an adhesion protein, was employed for the molecular imprinting of polymeric scaffolds for cellular tests. The most innovative aspect of this research was the molecular imprinting of whole cells for the development of substrates able to enhance the cell adhesion processes. Biotechnol. Bioeng. 2010;106: 804–817. © 2010 Wiley Periodicals, Inc.     

Probing the limits of molecular imprinting: strategies with a template of limited size and functionality

A series of polymers molecularly imprinted with the general anaesthetic propofol were synthesized using both semi- and non-covalent approaches. The polymers were evaluated with respect to template rebinding in both aqueous and organic media. In aqueous media, the observed propofol binding in these polymer systems was largely hydrophobic and non-specific in nature. In non-polar solvents such as hexane, electrostatic (hydrogen bonding) interactions dominate resulting in some selectivity. The implication of these results, in conjunction with those obtained using structures of similar size in other studies, is that propofol, a template possessing limited functionality and size, appears to define the lower limit for template size and degree of functionalization that can be used for the creation of ligand-selective recognition sites in molecularly imprinted polymers. Furthermore, studies with alternative ligands indicate that the steric crowding of a ligand's functionality to the polymer contributes to the extent of polymer-ligand recognition.     

Highly selective determination of phenolphthalein by flow injection chemiluminescence method based on a molecular imprinting polymer

The phenolphthalein‐imprinted polymer was prepared with methacrylic acid as functional monomer and ethylene glycol dimethacrylate as cross‐linker. Taking advantage of the quenching effect of phenolphthalein on the potassium permanganate–HCl–anhydrous alcohol chemiluminescence system, a new model was established to determine phenolphthalein by a highly selective flow injection chemiluminescence method. The traditional flow‐though cell was replaced with a novel flow path using a Y‐shaped polymethyl methacrylate column, through which the three reactants were injected simultaneously. The linear range of this assay was from 1.0 × 10^?8 to 1.0 × 10^?6 g/mL (r = 0.9978). The limit of detection was 8.9 × 10^?9 g/mL. The relative standard deviation for the determination of 1.0 × 10^?8 g/mL phenolphthalein solution was below 2.9% (n = 11). The proposed method was applied to the determination of phenolphthalein in real samples with satisfactory results. Copyright © 2009 John Wiley & Sons, Ltd.     

Insight into molecular imprinting in precipitation polymerization systems using solution NMR and dynamic light scattering

Molecular imprinting is a powerful synthetic technique for generating template-defined binding sites in cross-linked polymers. One scientific challenge in molecular imprinting research is to understand the intermolecular interactions leading to molecular complexation and the process of binding site formation during polymerization. In this work, we present a novel method for studying the molecular imprinting process in precipitation polymerization systems. This method employs solution (1) H NMR and dynamic light scattering (DLS) to investigate the association of template molecules with colloidal particles and the dynamic process of particle growth. Under precipitation polymerization conditions, the colloidal particles formed did not interfere with NMR signals from the soluble components, allowing unreacted monomers and free template to be easily quantified. To examine the process of particle nucleation and growth, DLS was used to measure the hydrodynamic particle size at different reaction times. To corroborate the interpretation of the NMR and DLS results, imprinted nanoparticles were collected at different reaction times and their binding characteristics were evaluated using radioligand-binding analysis. Our experimental results provide new insights into the molecular imprinting process that will be useful in the development of new imprinted nanoparticles.     

Altered product specificity of a cyclodextrin glycosyltransferase by molecular imprinting with cyclomaltododecaose

Cyclodextrin glycosyltransferases (CGTases), members of glycoside hydrolase family 13, catalyze the conversion of amylose to cyclodextrins (CDs), circular α‐(1,4)‐linked glucopyranose oligosaccharides of different ring sizes. The CD containing 12 α‐D‐glucopyranose residues was preferentially synthesized by molecular imprinting of CGTase from Paenibacillus sp. A11 with cyclomaltododecaose (CD₁₂) as the template molecule. The imprinted CGTase was stabilized by cross‐linking of the derivatized protein. A high proportion of CD₁₂ and larger CDs was obtained with the imprinted enzyme in an aqueous medium. The molecular imprinted CGTase showed an increased catalytic efficiency of the CD₁₂‐forming cyclization reaction, while decreased k_cat/K_m values of the reverse ring‐opening reaction were observed. The maximum yield of CD₁₂ was obtained when the imprinted CGTase was reacted with amylose at 40°C for 30 min. Molecular imprinting proved to be an effective means toward increase in the yield of large‐ring CDs of a specific size in the biocatalytic production of these interesting novel host compounds for molecular encapsulations. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Synthetic cinchonidine receptors obtained by cross-linking linear poly(methacrylic acid) derivatives as an alternative molecular imprinting technique

A molecular imprinting approach to construct synthetic receptors was examined, wherein a linear pre-polymer bearing functional groups for intermolecular interaction with a given molecule is cross-linked in the presence of the molecule as a template, and subsequent removal of the template from the resultant network-polymer is expected to leave a complementary binding site. Poly(methacrylic acid) (PMAA) derivatized with a vinylbenzyl group as a cross-linkable side chain was utilized as the pre-polymer for the molecular imprinting of a model template, (-)-cinchonidine. Selectivity of the imprinted polymer was evaluated by comparing the retentions of the original template, (-)-cinchonidine and its antipode (+)-cinchonine in chromatographic tests, exhibiting a selectivity factor up to 2.4. By assessment of the imprinted polymers in a batch mode, a dissociation constant at 20 degrees C for (-)-cinchonidine was estimated to be K (d) = 2.35 x 10(-6) M (the number of binding sites: 4.54 x 10(-6) mol/g-dry polymer). The displayed affinity and selectivity appeared comparable to those of an imprinted polymer prepared by a conventional monomer-based protocol, thus showing that the pre-polymer, which can be densely cross-linked, is an alternative imprinter for developing template-selective materials. (-)-Cinchonidine-imprinted polymers were prepared and assessed using the pre-polymers bearing different densities of the vinylbenzyl group and different amounts of the cross-linking agent to examine the appropriate density of the cross-linking side chain that was crucial for developing the high affinity and selectivity of the imprinted polymers.     

Structural determinants of ligand imprinting: a molecular dynamics simulation study of subtilisin in aqueous and apolar solvents

The phenomenon known as "ligand imprinting" or "ligand-induced enzyme memory" was first reported in 1988, when Russell and Klibanov observed that lyophilizing subtilisin in the presence of competitive inhibitors (that were subsequently removed) could significantly enhance its activity in an apolar solvent. (Russell and Klibanov, J Biol Chem 1988;263:11624-11626). They further observed that this enhancement did not occur when similar assays were carried out in water. Herein, we shed light on the molecular determinants of ligand imprinting using a molecular dynamics (MD) approach. To simulate the effect of placing an enzyme in the presence of a ligand before its lyophilization, an inhibitor was docked in the active site of subtilisin and 20 ns MD simulations in water were performed. The ligand was then removed and the resulting structure was used for subsequent MD runs using hexane and water as solvents. As a control, the same simulation setup was applied using the structure of subtilisin in the absence of the inhibitor. We observed that the ligand maintains the active site in an open conformation and that this configuration is retained after the removal of the inhibitor, when the simulations are carried out in hexane. In agreement with experimental findings, the structural configuration induced by the ligand is lost when the simulations take place in water. Our analysis of fluctuations indicates that this behavior is a result of the decreased flexibility displayed by enzymes in an apolar solvent, relatively to the aqueous situation.     

Fabrication of a cell-adhesive protein imprinting surface with an artificial cell membrane structure for cell capturing

We proposed a new molecular imprinting procedure based on molecular integration for the purpose of cell capture. We selected the cell-adhesive protein fibronectin (FN) as the imprinting protein for preparing templates and evaluated selective cell adhesion on the FN imprinting substrate. Silica beads with a diameter of 15 μm were used as the stamp matrix and FN molecules were adsorbed as a monolayer. The FN recognition sites were constructed by integrating a surfactant as the ligand and immobilizing it with new biocompatible photoreactive phospholipid polymer composed of 2-methacryloyloxyethyl phosphorylcholine (MPC) units. As control substrates, imprinting procedures were carried out using albumin (BSA imprinting substrate) and without imprinting protein (non-imprinting substrate). The binding of FN from the cell culture medium with the fetal calf serum was achieved on the FN imprinting substrate, and induced the cell adhesion. On the other hand, on the non-imprinted and BSA imprinting substrates, the FN scarcely bound from the cell culture medium, and subsequent cell adhesion could not be observed on the substrate. These results indicate that the FN binding sites were well constructed by arranging the ligand surfactant to a suitable position and immobilized by the photoreactive MPC polymer. The MPC polymer prevented the nonspecific adsorption of proteins from the cell culture medium. We concluded that this procedure is convenient and can be potentially used for the preparation of surfaces for cell engineering devices.     

Covalent molecular imprinting of bisphenol A using its diesters followed by the reductive cleavage with LiAlH(4)

Bisphenol A (BPA) recognition materials were synthesized by a covalent imprinting technique using BPA-dimethacrylate or BPA-diacrylate as the template monomer. Binding sites in the polymers consisted of two hydroxyl groups that are generated by reducing the ester bonds of the template monomer with lithium aluminum hydride. The polymers strongly adsorbed BPA and structurally related compounds, however, other endocrine disruptors were hardly adsorbed. The BPA-dimethacrylate-based polymer interacted with the samples more strongly than the BPA-diacrylate-based polymer.     

Molecular imprinting science and technology: a survey of the literature for the years 2004–2011

Herein, we present a survey of the literature covering the development of molecular imprinting science and technology over the years 2004–2011. In total, 3779 references to the original papers, reviews, edited volumes and monographs from this period are included, along with recently identified uncited materials from prior to 2004, which were omitted in the first instalment of this series covering the years 1930–2003. In the presentation of the assembled references, a section presenting reviews and monographs covering the area is followed by sections describing fundamental aspects of molecular imprinting including the development of novel polymer formats. Thereafter, literature describing efforts to apply these polymeric materials to a range of application areas is presented. Current trends and areas of rapid development are discussed. Copyright © 2014 John Wiley & Sons, Ltd.     

Molecular imprinting science and technology: a survey of the literature for the years up to and including 2003

Over 1450 references to original papers, reviews and monographs have herein been collected to document the development of molecular imprinting science and technology from the serendipitous discovery of Polyakov in 1931 to recent attempts to implement and understand the principles underlying the technique and its use in a range of application areas. In the presentation of the assembled references, a section presenting reviews and monographs covering the area is followed by papers dealing with fundamental aspects of molecular imprinting and the development of novel polymer formats. Thereafter, literature describing attempts to apply these polymeric materials to a range of application areas is presented.     

In crystallo capture of a Michaelis complex and product-binding modes of a bacterial phosphotriesterase

The mechanism by which the binuclear metallophosphotriesterases (PTEs, E.C. 3.1.8.1) catalyse substrate hydrolysis has been extensively studied. The μ-hydroxo bridge between the metal ions has been proposed to be the initiating nucleophile in the hydrolytic reaction. In contrast, analysis of some biomimetic systems has indicated that μ-hydroxo bridges are often not themselves nucleophiles, but act as general bases for freely exchangeable nucleophilic water molecules. Herein, we present crystallographic analyses of a bacterial PTE from Agrobacterium radiobacter, OpdA, capturing the enzyme-substrate complex during hydrolysis. This model of the Michaelis complex suggests the alignment of the substrate will favour attack from a solvent molecule terminally coordinated to the α-metal ion. The bridging of both metal ions by the product, without disruption of the μ-hydroxo bridge, is also consistent with nucleophilic attack occurring from the terminal position. When phosphodiesters are soaked into crystals of OpdA, they coordinate bidentately to the β-metal ion, displacing the μ-hydroxo bridge. Thus, alternative product-binding modes exist for the PTEs, and it is the bridging mode that appears to result from phosphotriester hydrolysis. Kinetic analysis of the PTE and promiscuous phosphodiesterase activities confirms that the presence of a μ-hydroxo bridge during phosphotriester hydrolysis is correlated with a lower pK_a for the nucleophile, consistent with a general base function during catalysis.