A series of 436 Munro database chemicals were studied with respect to their corresponding experimental LD50 values to investigate the possibility of establishing a global QSAR model for acute toxicity. Dragon molecular descriptors were used for the QSAR model development and genetic algorithms were used to select descriptors better correlated with toxicity data. Toxic values were discretized in a qualitative class on the basis of the Globally Harmonized Scheme: the 436 chemicals were divided into 3 classes based on their experimental LD50 values: highly toxic, intermediate toxic and low to non-toxic. The k-nearest neighbor (k-NN) classification method was calibrated on 25 molecular descriptors and gave a non-error rate (NER) equal to 0.66 and 0.57 for internal and external prediction sets, respectively. Even if the classification performances are not optimal, the subsequent analysis of the selected descriptors and their relationship with toxicity levels constitute a step towards the development of a global QSAR model for acute toxicity.

The interactions between each component of the pre-polymerisation mixtures used in the synthesis of molecularly imprinted polymers (MIP) specific for 1,2,3,4,5-pentachlorobenzene (1) and 1,2,3-trichlorobenzene (2) were examined in four molecular dynamics simulations. These simulations revealed that the relative frequency of functional monomer template (FM T) interactions was consistent with results obtained by the synthesis and evaluation of the actual MIPs. The higher frequency of 1 interaction with tri-methylstyrene (TMS; 54.7%) than 1 interaction with pentafluorostyrene (PFS; 44.7%) correlated with a higher imprinting factor (IF) of 2.1 vs. 1.7 for each functional monomer respectively. The higher frequency of PFS interactions with 2 (29.6%) than TMS interactions with 2 (1.9%) also correlated well with the observed differences in IF (3.7) of 2 MIPs imprinted using PFS as the FM than the IF (2,8) of 2 MIPs imprinted using TMS as the FM. The TMS-1 interaction dominated the molecular simulation due to high interaction energies, but the weaker TMS-2 resulted in low interaction maintenance, and thus lower IF values. Examination of the other pre-polymerisation mixture components revealed that the low levels of TMS-2 interaction was, in part, due to interference caused by the cross linker (CL) ethyleneglycol dimethylacrylate (EGDMA) interactions with TMS. The main reason was, however, attributed to MeOH interactions with TMS in both a hydrogen bond and perpendicular configuration. This positioned a MeOH directly above the it-orbital of all TMS for an average of 63.8% of MD2 creating significant interference to pi-pi stacking interactions between 2 and TMS. These findings are consistent with the deviation from the 'normal' molecularly imprinted polymer synthesis ratio of 1 : 4 : 20 (T : FM : CL) of 20 : 1 : 29 and 15 : 6 : 29 observed with 2 and TMS and PFS respectively. Our molecular dynamics simulations correctly predicted the high level of interference from other MIP synthesis components. The effect on PFS-1 interaction by MeOH was significantly lower and thus this system was not adversely affected.

Molecular imprinting is a technique used to design polymeric recognition materials with selectivity for a predetermined structure. The molecular imprinting process generates cavities in the polymer matrix that are complementary in size, shape and functionality to the template-structure. The recognition properties of molecularly imprinted polymers (MIPs) are comparable to those of antibodies and enzymes, which make MIPs utilizable in a wide range of application areas including biomimetic assays and biosensors [1]. Previous studies have shown that the prepolymerization step is central for the establishment of high affinity binding sites in MIPs [2]. However, our understanding of the physical mechanisms underlying MIP formation and template recognition is still limited. With the rapid increase of computational power and the development of suitable software molecular dynamics (MD) simulation methods have become a valuable theoretical tool to aid our understanding of the molecular imprinting process, and even in the development of rational design strategies [2]. Recently the first simulation of a complete prepolymerization mixture was presented [3].

Here we present 10 ns MD simulations of a series of all-component prepolymerization mixtures. The simulated systems were assembled with different molar ratios using the local anaesthetic bupivacaine as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the crosslinker, 2,2’-azobis-(2-methylpropionitrile) (AIBN) as the initiator and toluene as the solvent. The simulations were performed using the AMBER (v. 10.0 UCSF, San Francisco, CA) suite of programs (4) and the GAFF [6] force field. Molecular trajectories were evaluated with radial distribution functions and hydrogen bond analysis.

References

1. Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O´Mahony, J.; Whitcombe, J., J. Mol. Recognit. (2006), 19, 106-180
2. Nicholls, I. A.; Andersson, H. S.; Charlton, C.; Henschel, H.; Karlsson, B. C. G.; Karlsson, J. G.; O´Mahony, J.; Rosengren, A. M.; Rosengren, K. J.; Wikman, S. Biosens. Bioelectron. (2009), 25, 543-552
3. Karlsson, B. C. G.; O´Mahony, J.; Karlsson, J. G.; Bengtsson, H.; Eriksson, L. A.; Nicholls, I. A. J. Am. Chem. Soc. (2009), 131, 13297-13304
4. Case, D. A.; Cheatham, T. E.; Darden, T.; Gohlke, H.; Luo, R.; Merz, K. M.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. J. Comput. Chem. (2009), 26, 1668-1688
5. Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A. J. Comput. Chem. (2004), 25, 1157-1174

Molecularly imprinted polymers (MIPs) are polymeric receptors with selectivity for a predetermined structure. The molecular imprinting process generates cavities in a synthetic polymer matrix that are complementary in size, shape and functionality to the template. MIPs exhibit recognition properties analogous to their biological counterparts, such as antibodies, and can be utilized in a wide range of application areas [1]. Nonetheless, the physical mechanisms underlying MIP formation and template recognition are still poorly understood. Molecular dynamics (MD) based computer simulations are a valuable theoretical tool which may be used to aid our understanding of the molecular imprinting process, and even for the development of rational design strategies [2]. Recently the first MD simulation of a complete prepolymerization mixture was presented [3].

In the present work, MD simulations of a series of all-component prepolymerization mixtures were performed, using the local anaesthetic bupivacaine as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the crosslinker, 2,2’-azobis-(2-methylpropionitrile) (AIBN) as the initiator and toluene as the solvent. The simulated systems differed in the molar fraction of MAA. Systems were evaluated with radial distribution functions and hydrogen bond analyses. By correlating the results with the rebinding behaviour of a series of synthesized MIPs the importance of the stoichiometry between template, functional monomer and crosslinker was highlighted. The analysis of the MD simulations revealed strong competition for hydrogen bonding between the carbonyl oxygen’s of MAA and EGDMA and the amide proton of bupivacaine. Moreover, the hydrogen bonding contact between EGDMA and bupivacaine remained nearly unaffected by the varied molar fraction MAA in the different systems demonstrating the role of the crosslinker being more important as generally accepted.

References

[1]             Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O´Mahony, J.; Whitcombe, J., J. Mol. Recognit., 19, 106-180 (2006)

[2]            Nicholls, I. A.; Andersson, H. S.; Charlton, C.; Henschel, H.; Karlsson, B. C. G.; Karlsson, J. G.; O´Mahony, J.; Rosengren, A. M.; Rosengren, K. J.; Wikman, S. Biosens. Bioelectron., 25, 543-552 (2009)

[3]            Karlsson, B. C. G.; O´Mahony, J.; Karlsson, J. G.; Bengtsson, H.; Eriksson, L. A.; Nicholls, I. A. J. Am. Chem. Soc., 131, 13297-13304 (2009)

Through the rapid increase in computational power and the development of suitable software, molecular dynamics (MD) has become a promising tool for use in the development of molecularly imprinted polymers (MIPs).¹ MD is a computational method based on Newtonian mechanics, which enables the simultaneous simulation of thousands of discrete molecules, and can be used to establish the states of the molecular species present in MIP-prepolymerization mixtures. As detailed understanding of the molecular basis for formation of high affinity MIP sites is still lacking and the physical mechanism underlying specific recognition is still a matter of debate, the use of MD as a tool to investigate MIP-prepolymerization mixtures is highly motivated.¹ Recently the first MD simulation of an all-component prepolymerization mixture was presented, which gave a detailed picture of the underlying monomer-template interactions important for the “molecular memory” in MIPs.²

Here, we present results obtained from a series of MD simulations representing all-component MIP/REF prepolymerization mixtures assembled with differences in stoichiometries of functional and crosslinking monomer. In these mixtures, the local anaesthetic drug bupivacaine was used as a template, methacrylic acid as the functional monomer, ethylene dimethacrylate as crosslinking monomer, 2,2’-azobis-(2-methylpropionitrile) as the initiator and toluene as the solvent. Bupivacaine complexation in each system was evaluated with radial distribution functions and hydrogen bond analyses. By correlating the results with the rebinding behaviour of a series of synthesized bupivacaine-MIPs, the relationship between the degree of crosslinking and MIP-performance was highlighted.

[1] Nicholls, I. A.; Andersson, H. S.; Charlton, C.; Henschel, H.; Karlsson, B. C. G.; Karlsson, J. G.; O´Mahony, J.; Rosengren, A. M.; Rosengren, K. J.; Wikman, S. Biosens. Bioelectron., 25, 543-552 (2009)

[2] Karlsson, B. C. G.; O´Mahony, J.; Karlsson, J. G.; Bengtsson, H.; Eriksson, L. A.; Nicholls, I. A. J. Am. Chem. Soc., 131, 13297-13304 (2009)

A combination of theoretical and experimental studies has provided correlations between molecularly imprinted polymer composition, morphology, and recognition behavior obtained using a series of bupivacaine-imprinted methacrylic acid (MAA)–ethylene glycol dimethacrylate copolymers differing in molar ratios of the respective monomers. Results extracted from analysis of molecular dynamics (MD) trajectory data demonstrated that stability and frequency of interactions between bupivacaine and the monomers in the prepolymerization phase were strongly affected by minor changes in polymer composition, which in turn affected binding site affinity and heterogeneity of the imprinted polymers. Moreover, through the characterization of polymer morphology, we show that higher molar fractions of MAA resulted in polymeric materials with increased pore size, a feature that enhanced the binding capacity of the polymers. Furthermore, the results presented point at the strength of MD for predicting MIP-template binding capacity and affinity.

In this report, principal component analysis (PCA) has been used to explore the influence of template complexation in the pre-polymerization phase on template molecularly imprinted polymer (MIP) recognition and polymer morphology. A series of 16 bupivacaine MIPs were studied. The ethylene glycol dimethacrylate (EGDMA)-crosslinked polymers had either methacrylic acid (MAA) or methyl methacrylate (MMA) as the functional monomer, and the stoichiometry between template, functional monomer and crosslinker was varied. The polymers were characterized using radioligand equilibrium binding experiments, gas sorption measurements, swelling studies and data extracted from molecular dynamics (MD) simulations of all-component pre-polymerization mixtures. The molar fraction of the functional monomer in the MAA-polymers contributed to describing both the binding, surface area and pore volume. Interestingly, weak positive correlations between the swelling behavior and the rebinding characteristics of the MAA-MIPs were exposed. Polymers prepared with MMA as a functional monomer and a polymer prepared with only EGDMA were found to share the same characteristics, such as poor rebinding capacities, as well as similar surface area and pore volume, independent of the molar fraction MMA used in synthesis. The use of PCA for interpreting relationships between MD-derived descriptions of events in the pre-polymerization mixture, recognition properties and morphologies of the corresponding polymers illustrates the potential of PCA as a tool for better understanding these complex materials and for their rational design.

This report demonstrates that the diversity of hydrogen bond interactions present in molecularly imprinted polymer pre-polymerization mixtures, typically associated with binding-site heterogeneity, can also contribute to morphological characteristics that may influence polymer–template recognition. Comparisons have been made between a series of bupivacaine molecularly imprinted methacrylic acid (MAA)–ethylene glycol dimethacrylate (EGDMA) copolymers and a series of analogous methyl methacrylate (MMA)–EGDMA copolymers using comprehensive molecular dynamics studies of the respective pre-polymerization mixtures, template–polymer binding studies and detailed BET surface area and BJH porosity analyses. The role of the carboxylic acid functionality of MAA, and in particular the acidic proton, in generating morphological features conducive to analyte access (slit-like rather than ink bottle-like structures) and recognition is discussed.

Warfarin is one of the most commonly used drugs in anticoagulent therapy. Notwithstanding its wide use, achieving correct dosage is often a major challenge due to its narrow therapeutic window.[1] The bioavailability of warfarin is believed to be greatly influenced by the environment-dependent composition of the ensemble of isomers present. While the different structures of warfarin have been discussed in earlier publications,[2] details of the mechanism underlying the formation of the cyclic hemiacetal (Figure 1) had not yet been investigated.

Figure 1. Cyclization reaction of warfarin.

Figure 2. Transition state in presence of one water molecule.

We have now studied the reaction by means of density functional calculations. Comparison of results from calculations performed on the isolated warfarin molecule and in presence of water molecules (compare Figure 2) highlight the importance of intermolecular interactions in the key proton transfer step for the reaction to proceed. A viable model for the mechanism underlying the isomerisation shall be presented.

References

[1]             J. Ansell, J. Hirsh, L. Poller, H. Bussey, A. Jacobsen and E. Hylek, Chest, 126, 204S (2004).

[2]            B. C. G. Karlsson, A. M. Rosengren, P. O. Andersson and I. A. Nicholls, J. Phys. Chem. B, 111,10520 (2007).

The anticoagulent drug warfarin exhibits chameleon-like isomerism, where the environment-dependent composition of the ensemble of structures greatly influences its bioavailability. Here, the mechanism of conversion between the major isomeric forms is studied. The dramatic differences in transition state energies, as determined by density functional calculations, highlight the necessity for the involvement of intermolecular interactions in the key proton transfer step. A viable model for the mechanism underlying the isomerization reactions is presented.

Whisky is distilled to around 70% alcohol by volume (vol-%) then diluted to about 40 vol-%, and often drunk after further slight dilution to enhance its taste. The taste of whisky is primarily associated with amphipathic molecules, such as guaiacol, but why and how dilution enhances the taste is not well understood. We carried out computer simulations of water-ethanol mixtures in the presence of guaiacol, providing atomistic details on the structure of the liquid mixture. We found that guaiacol is preferentially associated with ethanol, and, therefore, primarily found at the liquid-air interface in mixtures that contain up to 45 vol-% of ethanol. At ethanol concentrations of 59 vol-% or higher, guaiacol is increasingly surrounded by ethanol molecules and is driven to the bulk. This indicates that the taste of guaiacol in the whisky would be enhanced upon dilution prior to bottling. Our findings may apply to other flavour-giving amphipathic molecules and could contribute to optimising the production of spirits for desired tastes. Furthermore, it sheds light on the molecular structure of water-alcohol mixtures that contain small solutes, and reveals that interactions with the water may be negligible already at 89 vol-% of ethanol.

The role of the structural diversity of the widely used anticoagulant drug warfarin on its distribution in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer membranes was investigated using a series of both restrained (umbrella sampling) and unrestrained molecular dynamics simulations. Data collected from unrestrained simulations revealed favorable positions for neutral isomers of warfarin, the open side chain form (OCO), and the cyclic hemiketal (CCO), along the bilayer normal close to the polar headgroup region and even in the relatively distant nonpolar lipid tails. The deprotonated open side chain form (DCO) was found to have lower affinity for the DOPC bilayer membrane relative to the neutral forms, with only a small fraction interacting with the membrane, typically within the polar headgroup region. The conformation of OCO inside the lipid bilayer was found to be stabilized by intramolecular hydrogen bonding thereby mimicking the structure of CCO. Differences in free energies, for positions of OCO and CCO inside the bilayer membrane, as compared to positions in the aqueous phase, were −97 and −146 kJ·mol^–1. Kinetic analysis based on the computed free energy barriers reveal that warfarin will diffuse through the membranes within hours, in agreement with experimental results on warfarin’s accumulation in the plasma, thus suggesting a passive diffusion mechanism. We propose that this membrane transport may be an isomerization-driven process where warfarin adapts to the various local molecular environments encountered under its journey through the membrane. Collectively, these results improve our understanding of the influence of warfarin’s structural diversity on the drug’s distribution and bioavailability, which in turn may provide insights for developing new formulations of this important pharmaceutical to better address its narrow therapeutic window.

Warfarin is an oral anticoagulant drug used to prevent thrombolic disorders such as myocardial infarction and stroke by inhibiting the active site of vitamin-K dependent epoxide reductase (VKOR) [1]. Despite being in widespread use and having a narrow therapeutic window, its mechanisms of action are not yet fully understood and incorrect warfarin dosage often leads to severe side effects. A factor limiting our understanding of warfarin’s bioavailability is warfarin’s structural diversity, which has been shown to be strongly affected by the nature of molecular environment e.g. solvent polarity and pH [2-7]. One of the major factors contributing to a drug’s biological effect is membrane transport, a process involving exposure of warfarin to environments of quite different character. Since a drug’s transport across membrane may include both active transport by carriers as well as diffusion-controlled processes, it may be envisaged that in order to fully predict warfarin’s anticoagulant effect these mechanisms must be carefully elucidated.

Molecular dynamics (MD) simulations have previously been performed in order to obtain detailed information on static equilibrium as well as dynamic properties of small organic drugs in biomembranes. One of the most studied lipids in cell membrane simulations has been dipalmitoylphosphatidylcholine (DPPC) which is the most abundant phospholipid in cell membranes. Here we present lipid bilayer membrane transport properties for a series of warfarin structures previously reported in the literature using a fully solvated DPPC membrane model. Data extracted from simulations shed light on differences in membrane partioning as well as mobilities of warfarin isomers studied and a mechanism by which warfarin permeates through membranes in vivo is presented.

References

1. Landefeld, C.; Beyth, R. Am. J. Med. 1993, 95, 315-328.
2. Karlsson, B. C. G.; Rosengren, A. M.; Andersson, P. O.; Nicholls, I. A. J. Phys. Chem. B 2007, 111, 10520-10528.
3. Karlsson, B. C. G.; Rosengren, A. M.; Andersson, P. O.; Nicholls, I. A. J. Phys. Chem. B 2009, 113, 7945-7949.
4. Karlsson, B. C. G.; Rosengren, A. M.; Näslund, I.; Andersson, P. O.; Nicholls, I. A. Submitted 2010.
5. Rosengren, A. M.; Karlsson, B. C. G.; Näslund, I.; Andersson, P. O.; Nicholls, I. A. Submitted 2010.
6. Nicholls, I. A.; Karlsson, B. C. G.; Rosengren, A. M.; Henschel, H. J. Mol. Recognit. 2010, In press.
7. Henschel, H.; Karlsson, B. C. G.; Rosengren, A. M.; Nicholls, I. A. Submitted 2010.

We here present the first simulation of a complete molecularly imprinted polymer prepolymerization system. Molecular dynamics studies were performed for a system comprising a total of 1199 discrete molecules, replicating the components and concentrations employed in the corresponding polymer synthesis. The observed interactions correlate well with results obtained from (1)H NMR spectroscopic studies. Comparison with simulations performed in the absence of cross-linking agent (ethylene dimethacrylate) demonstrated its significance in the formation of ligand recognition sites. Moreover, the influence of events such as template-template (bupivacaine) and monomer-monomer (methacrylic acid) self-association, porogen-template interactions, and template conformational variability was revealed. The template recognition capacity of the modeled polymer system was verified by synthesis of imprinted and reference polymers and subsequent radioligand binding Analysis. Collectively, through a series of statistical analyses of molecular trajectories in conjunction with spectroscopic data it was demonstrated that an ensemble of complex structures is present in the prepolymerization mixture and that this diversity is the basis for the binding site heterogeneity observed in molecularly imprinted polymers (MIPs) prepared using the noncovalent strategy.

A series of steady-state fluorescence anisotropy experiments has been performed to demonstrate the presence of a deprotonated open side chain form of warfarin in organic environments. We explain the observed emission-wavelength-dependent anisotropy of warfarin in ethanol, 2-propanol, and acetonitrile due to the coexistence of neutral isomers and deprotonated open side chain forms displaying different fluorescence decay kinetics. To investigate solvent-solute interactions in more detail, a series of molecular dynamics simulations was performed to study warfarin solvation and to predict the time scale of rotational diffusion displayed by this compound. Predictions obtained provide an explanation for the nonzero values in anisotropy observed for neutral isomers of warfarin associated with the short fluorescence lifetime (tau < 0.1 ns) and for an approximately zero anisotropy observed for the deprotonated open side chain form, which is associated with the longer fluorescence lifetime (tau = 0.5-1.6 ns). Finally, we address the potential use of fluorescence anisotropy for an increased understanding of the structural diversity of warfarin in protein binding pockets.

Warfarin is a clinically important drug widely used in the treatment of thrombolic disorders e.g. myocardial infarction and stroke.¹ When administered, 99% of the drug present in blood is bound to the transport protein human serum albumin (HSA).² On account of the fact that HSA demonstrates polymorphism and warfarin has a narrow therapeutic index, careful monitoring of the effect of drug-dosage must be performed.

Currently, warfarin’s anticoagulant effect is measured by an indirect method in which the clotting time is measured and correlated to the amount of warfarin present. As current methods for self-monitoring are limited, the development of alternative robust and more sensitive methods is desirable.

In this study, we have developed a non-covalent molecularly imprinted polymer³ (MIP) system with selectivity for warfarin.⁴ The HSA-like binding properties of this MIP were established in previous efforts to develop polymers capable of HSA-like binding of warfarin.⁵

In principle, the fluorophoric nature of warfarin should allow for the fluorescence spectroscopy-based detection of the drug. Recent efforts by us,^6-8 using a series of theoretical and spectroscopic studies have highlighted the complex nature of warfarin. In particular, the medium dependent isomerization of this drug illustrates why spectroscopy based methods for the direct detection of the drug has not been forthcoming. Results from these studies have been used to develop a method for the in situ detection of warfarin using time resolved fluorescence spectroscopy.

(1)      Landefeld, C.; Beyth, R. Anticoagulant-related bleeding - epidemiology, prediction and prevention. Am. J. Med. 1993, 95, 315-328.

(2)      Yacobi, A.; Udall, J. A.; Levy, G. Comparative pharmacokinetics of coumarin anticoagulants.18 Serum-protein binding as a determinant of warfarin body clearance and anticoagulant effect. Clin. Pharmacol Ther. 1976, 19, 552-558.

(3)      Alexander, C.; Andersson, H. S.; Andersson, L. I.; Ansell, R. J.; Kirsch, N.; Nicholls, I. A.; O'Mahony, J.; Whitcombe, M. J. Molecular imprinting science and technology: A survey of the literature for the years up to and including 2003. Journal of Molecular Recognition 2006, 19, 106-180.

(4)      Rosengren, A. M.; Karlsson, B. C. G.; Näslund, I.; Andersson, P. O.; Nicholls, I. A. Time resolved fluorescence spectroscopic detection of the anticoagulant warfarin: A sensor-based method for direct detection in blood plasma. 2010, Submitted.

(5)      Karlsson, B. C. G.; Rosengren, A. M.; Näslund, I.; Andersson, P. O.; Nicholls, I. A. Synthetic Human Serum Albumin Sudlow I binding site mimics. 2010, Submitted.

(6)      Karlsson, B. C. G.; Rosengren, A. M.; Andersson, P. O.; Nicholls, I. A. The Spectrophysics of Warfarin: Implications for Protein Binding J. Phys. Chem. B 2007, 111, 10520-10528.

(7)      Karlsson, B. C. G.; Rosengren, A. M.; Andersson, P. O.; Nicholls, I. A. Molecular Insights on the Two Fluorescence Lifetimes Displayed by Warfarin from Fluorescence Anisotropy and Molecular Dynamics Studies. J. Phys. Chem. B 2009, 113, 7945-7949.

(8)      Nicholls, I. A.; Karlsson, B. C. G., Rosengren, A. M.. Henschel, H. Warfarin: an Environment-Dependent Switchable Molecular Probe. J. Mol. Recognit. 2010, in press.

Here, we report the design, synthesis, and characterization of molecularly imprinted polymer (MIP) derived mimics of the human serum albumin (HSA) Sudlow I site-the binding site for the anticoagulant warfarin. MIP design was based upon a combination of experimental (H-1 NMR) and computational (molecular dynamics) methods, Two MIPs and corresponding nonimprinted reference polymers were synthesized and characterized (scanning electron microscopy; nitrogen sorption; and Fourier transform infrared spectroscopy). MIP-ligand recognition was examined using radioligand binding studies, where the largest number of selective sites was found in a warfarin-imprinted methacrylic acid ethylene dimethacrylate copolymer (MAA-MIP). The warfarin selectivity of this MIP was confirmed using radioligand displacement and zonal chromatographic studies. A direct comparison of MIP-warfarin binding characteristics with those of the HSA Sudlow I binding site was made, and similarities in site population (per gram polymer or protein) and affinities were observed. The warfarin selectivity of the MIP suggests its potential for use as a recognition element in a MIP-based warfarin sensor and even as a model to aid in understanding and steering blood-plasma protein-regulated transport processes or even for the development of warfarin sensors.

The further evolution of molecularly imprinted polymer science and technology necessitates the development of robust predictive tools capable of handling the complexity of molecular imprinting systems. A combination of the rapid growth in computer power over the past decade and significant software developments have opened new possibilities for simulating aspects of the complex molecular imprinting process. We present here a survey of the current status of the use of in silico-based approaches to aspects of molecular imprinting. Finally, we highlight areas where ongoing and future efforts should yield information critical to our understanding of the underlying mechanisms sufficient to permit the rational design of molecularly imprinted polymers. 

In principle, molecularly imprinted polymer science and technology provides a means for ready access to nano-structured polymeric materials of predetermined selectivity. The versatility of the technique has brought it to the attention of many working with the development of nanomaterials with biological or biomimetic properties for use as therapeutics or in medical devices. Nonetheless, the further evolution of the field necessitates the development of robust predictive tools capable of handling the complexity of molecular imprinting systems. The rapid growth in computer power and software over the past decade has opened new possibilities for simulating aspects of the complex molecular imprinting process. We present here a survey of the current status of the use of in silico-based approaches to aspects of molecular imprinting. Finally, we highlight areas where ongoing and future efforts should yield information critical to our understanding of the underlying mechanisms sufficient to permit the rational design of molecularly imprinted polymers.

The development of in silico strategies for the study of the molecular imprinting process and the properties of molecularly imprinted materials has been driven by a growing awareness of the inherent complexity of these systems and even by an increased awareness of the potential of these materials for use in a range of application areas. Here we highlight the development of theoretical and computational strategies that are contributing to an improved understanding of the mechanisms underlying molecularly imprinted material synthesis and performance, and even their rational design.

The need for materials with predetermined ligand-selectivities for use in sensing and separation technologies, e.g. membranes and chromatography, has driven the development of molecularly imprinted polymer science and technology. Over recent years, the need to develop robust predictive tools capable of handling the complexity of molecular imprinting systems has become apparent The current status of the use of in silica techniques in molecular imprinting is here presented, and we highlight areas where new developments are contributing to improvements in the rational design of molecularly imprinted polymers.

The complex nature of the structure of the anticoagulant warfarin is reflected in the diversity of binding modes observed in warfarin–protein recognition systems. A series of theoretical, ¹H-NMR and steady state and time resolved fluorescence spectroscopic studies, have been used to establish correlations between the molecular environment provided by various solvent systems and the relative concentrations of the various members of warfarin's ensemble of isomers. A consequence of these observations is that the judicious choice of solvent system or molecular environment of warfarin allows for manipulation of the position of the equilibrium between isomeric structures such as the hemiacetal and open phenol-keto forms, the latter even possible in a deprotonated form, where in each case unique spectroscopic properties are exhibited by the respective structures. Collectively, warfarin's capacity to adapt its structure as a function of environment in conjunction with the fluorescence behaviours of the various isomers together provide an environment-dependent molecular switch with reporter properties, which allows for the simultaneous detection of warfarin in different states with lifetimes spanning the range < 0.10–5.5 ns. These characteristics are here used to examine warfarin binding domains in a series of materials (solvents, protein, inorganic matrix and synthetic polymer). Moreover, these studies demonstrate the potential for using warfarin, or other switchable analogues thereof, as a tool for studying molecular level characteristics, for example local dielectricity. Copyright © 2010 John Wiley & Sons, Ltd.

Theoretical and computational studies of molecular imprinting have helped provide valuable insights concerning the nature of the molecular-level events underlying the recognition characteristics of molecularly imprinted materials. Here, we first present an overview of a thermodynamic treatment of factors governing the behaviour of these functional materials, and then a summary of the development and current status of the use of computational strategies for studying aspects of molecular imprinting and the resulting material properties.

The risks to society associated with the spread of new strains of influenza with human pathogenicity, or with impact on agricultureare significant. Our capacity to challenge the threat of the virus is dependent upon our ability to develop new vaccines, and upon ouraccess to effective virus-targeted small molecule pharmaceuticals. The current primary small molecule weapons oseltamivir(Tamiflu) and zanamivir (Relenza) currently form our last line of defence against this virus. More recently, the identification ofstrains resistant to (in particular) drugs targeting neuraminidase has awoken serious concern. Equally as worrying is the clearevidence of the presence of these substances in the World’s water systems which has now come forth. Collectively, this makes thedevelopment of techniques giving us better insight into the virus and antiviral agents a priority. Robust methods for the rapid andsensitive determination of these substances are required, especially as the monitoring methods should be able to withstand therigours of environments not normally conducive to biomacromolecules (temperature, toxic substances etc) e.g. antibodies.Advanced materials fulfilling these requirements can be obtained by Molecular Imprinting, which is a technique forproducing highly selective synthetic receptors for biochemical and chemical structures in synthetic polymers. The polymerscontain nano-structured cavities that are of complementary functional and structural character to predetermined target.The technique entails the judicious selection of a monomer or monomer mixture with chemical functionality comple-mentary to that of the imprint species (template). The complementary interacting functionalities (reversible covalent ornon-covalent) form predictable solution structures, which after polymerisation in the presence of a suitable cross linkingagent and removal of the template lead to the defining of recognition sites of complementary steric and functionaltopography to the template molecule. These sites give selective recognition of the template. Furthermore, by analogy tocatalytic antibody production, using transition state analogues as templates yields synthetic enzymes.Synthetic polymers with neuraminidase-like behaviour have been designed through the screening of candidate polymersystems using a combination of molecular dynamics and NMR studies. The characterisation of the resulting materials hasdemonstrated systems with selectivity for the targeted antiviral agents. Our studies illustrate the potential of these uniquenanostructured materials for the monitoring of these antiviral agents in the environment, which is an important aspect inefforts aimed at limiting the development of resistant strains, and as a tool for policy makers.

The 71-82 fragment of the non-amyloid-beta component (NAC) region of the Parkinson's disease (PD) and dementia with Lewy bodies (DLB) related protein alpha-Synuclein, has been reported to be important during protein misfolding. Although reports have demonstrated the importance of this fragment for the aggregation properties of the full-length protein, its exact role in pre-fibrillar oligomerisation, fibrillar growth and morphology has not yet been fully elucidated. Here, we provide evidence that fibrils prepared from an acetylated and methyl amidated peptide of the NAC 71-82 amino acid stretch of alpha-Synuclein are amyloid and contain, in addition to the cross-beta structure detected in the full-length protein fibrils, a cross-beta structure previously observed in prion proteins. These results shed light on the aggregation propensity of the NAC 71-82 amino acid stretch of the full-length protein but also the roles of the N- and C-terminal domains of alpha-Synuclein in balancing this aggregation propensity. The results also suggest that early aggregated forms of the capped NAC 71-82 peptide generated structures were stabilised by an anti-parallel and twisted beta-sheet motif. Due to its expected toxicity, this beta-sheet motif may be a promising molecular target for the development of therapeutic strategies for PD and DLB.

Alpha-synucleinopathies are featured by fibrillar inclusions in brain cells. Although α-synuclein fibrils display structural diversity, the origin of this diversity is not fully understood. We used molecular dynamics simulations to design synthetic peptides, based on the NAC 71-82 amino acid fragment of α-synuclein, that govern protofilament contacts and generation of twisted fibrillar polymorphs. Four peptides with structures based on either single or double fragments and capped or non-capped ends were selected for further analysis. We determined the fibrillar yield and the structures from these peptides found in the solution after fibrillisation using protein concentration determination assay and circular dichroism spectroscopy. In addition, we characterised secondary structures formed by individual fibrillar complexes using laser-tweezers Raman spectroscopy. Results suggest less mature fibrils, based on the lower relative β-sheet content for double- than single-fragment peptide fibrils. We confirmed this structural difference by TEM analysis which revealed, in addition to short protofibrils, more elongated, twisted and rod-like fibril structures in non-capped and capped double-fragment peptide systems, respectively. Finally, time-correlated single-photon counting demonstrated a difference in the Thioflavin T fluorescence lifetime profiles upon fibril binding. It could be proposed that this difference originated from morphological differences in the fibril samples. Altogether, these results highlight the potential of using peptide models for the generation of fibrils that share morphological features relevant for disease, e.g., twisted and rod-like polymorphs.

Although Lewy bodies and Lewy neurites are hallmarks of Parkinson's disease (PD) and dementia with Lewy bodies (DLB), misfolded α-synuclein oligomers are nowadays believed to be key for the development of these diseases. Attempts to target soluble misfolded species of the full-length protein have been limited so far, probably due to the fast aggregation kinetics and burial of aggregation prone segments in final cross-β-sheet fibrils. A previous characterisation study of fibrils prepared from a capped peptide of the non-amyloid β-component (NAC) 71-82 amino acid stretch of α-synuclein demonstrated an increased aggregation propensity resulting in a cross-β-structure that is also found in prion proteins. From this, it was suggested that capped NAC 71-82 peptide oligomers would provide interesting motifs with a capacity to regulate disease development. Here, we demonstrated, from a series of circular dichroism spectroscopic measurements and molecular dynamics simulations, the molecular-environment-sensitive behaviour of the capped NAC 71-82 peptide in a solution phase and the formation of β-sheet oligomeric structures in the supernatant of a fibrillisation mixture. These results highlighted the use of the capped NAC 71-82 peptide as a motif in the preparation of oligomeric β-sheet structures that potentially could be used in therapeutic strategies in the fight against progressive neurodegenerative disorders, such as PD and DLB.

In this study, computational methods were employed in efforts to elucidate physical mechanisms underlying the ligand selectivity of polymeric sorbents produced through the molecular imprinting of 17-beta-estradiol. Previous computational and experimental studies had identified candidate systems applicable to the development of synthetic polymeric receptors for the detection and possible removal of pollutants with endocrine-disrupting properties. Here we present a series of comprehensive molecular dynamics studies of candidate molecular imprinting prepolymerization systems. The results from the studies highlight the role of the cross-linker and the importance of the interplay between functionalities of the various monomers employed in template complexation. The significance of these results for future studies is discussed.

A series of molecular dynamics simulations of prepolymerization mixtures for phenylalanine anilide imprinted co-(ethylene glycol dimethacrylate-methacrylic acid) molecularly imprinted polymers have been employed to investigate the mechanistic basis for template selective recognition in these systems. This has provided new insights on the mechanisms underlying template recognition, in particular the significant role played by the crosslinking agent. Importantly, the study supports the occurrence of template self-association events that allows us to resolve debate between the two previously proposed models used to explain this system's underlying recognition mechanisms. Moreover, the complexity of the molecular level events underlying template complexation is highlighted by this study, a factor that should be considered in rational molecularly imprinted polymer design, especially with respect to recognition site heterogeneity.

It is now widely accepted that the recognition properties of a MIP are derived from molecular level events present during the prepolymerization stage.¹ Studies regarding the nature and extent of template complexation during this stage should therefore yield valuable information regarding the template recognition properties of the final MIP. One method of great potential for illuminating molecular level details in this area of MIP research is molecular dynamics (MD).² MD simulations enable studies of molecular-level events in MIP prepolymerization mixtures.

Phenylalanine anilide (PA) is a molecule that has been extensively used as a template in a series of seminal molecular imprinting studies.^3-5 In an effort to elucidate the origin to the imprinting effect, Sellergren, Lepistö and Mosbach proposed that selective high-affinity sites in the PA-MIP were based on functional monomer-template complexation of a 2:1 stoichiometry.³ In a follow-up study, Katz and Davis presented results that revealed further information regarding the origin of recognition in PA-MIPs.⁵ It was suggested that the template recognition sites were based on functional monomer-template complexes of 1:1 stoichiometry, and also that the formation of higher order template-template complexes has important effects on the final PA-MIP recognition properties. In light of this conjecture and several more recent studies highlighting the diversity of template complexation mechanisms in prepolymerization mixtures, have pointed at the complexity and diversity in the ensemble of complexes leading to the final “molecular memory”.

Here we present the novel insights into the molecular basis for PA-MIP template recognition derived from a series of MD simulations of the PA-MIP prepolymerisation systems. Data support the presence of PA-PA complexes and that the most statistically prevalent stoichiometry functional monomer-PA complexes was 1:1. The role of cross-linker is also discussed. This study highlights the potential of all component MD studies for rational MIP design.

(1)      Alexander, C.; Andersson, H.S.; Andersson, L.I.; Ansell, R.J.; Kirsch, N.; Nicholls, I.A.; O'Mahony, J.; Whitcombe, M.J.  Molecular imprinting science and technology: A survey of the literature for the years up to and including 2003. Journal of Molecular Recognition 2006, 19, 106-180.

(2)      Nicholls, I.A.; Andersson, H.S.; Charlton, C; Henschel, H.; Karlsson, B.C.G.; Karlsson, J.G.; O’Mahony, J.; Rosengren, A.M.; Rosengren, J.K.; Wikman, S. Theoretical and computational stratgies for rational molecularly imprinted polymer design. Biosensors and Bioelectronics 2009, 25, 543-552

(3)      Sellergren, B.; Lepistoe, M.; Mosbach, K.. Highly enantioselective and substrate-selective polymers obtained by molecular imprinting utilizing noncovalent interactions. NMR and chromatographic studies on the nature of recognition. Journal of American Chemical Society 1988, 110, 5853-5860

(4)      Sellergren, B.. Molecular imprinting by noncovalent interactions: Tailor-made chiral stationary phases of high selectivity and sample load capacity. Chirality 1989, 1, 63-68

(5)      Katz, A.; Davis, M.E. Investigations into the mechanism of molecular recognition with imprinted polymers. Macromolecules 1999, 32, 4113-4121

In this work, classical molecular dynamics (MD) simulations have been used to provide unique insights on the nature and extent of intermolecular interactions present in a phenylalanine anilide (PA) molecularly imprinted polymers (MIP) prepolymerization mixture.

Molecular Imprinting is a technique for producing highly selective synthetic receptors for a predetermined molecular structure, and involves the formation of cavities in a synthetic polymer matrix that are of complementary functional and structural character to a template molecule.¹ It is generally accepted that the recognition properties of a MIP is a product of the interactions between monomers and template during the prepolymerization stage. Accordingly, studies of the nature and extent of the interactions present in prepolymerization mixtures, in patricular those involving template, should yield information which can be related to the observed recognition properties of the final MIP.

Phenylalanine anilide MIPs have been the subject of a significant number of studies aimed at producing an understanding of the mechanisms underlying the recognition processes. Interestingly, two different models have been proposed to explain the behaviour of PA-MIPs. Studies by Sellergren et al. proposed that template selectivity, was a consequence of  the presence of a functional monomer-template complexes of 2:1 stoichiometry.² Later, however, Katz and Davis proposed an alternative model,³ where the template (PA) recognition sites in the MIP were suggested to arise from functional monomer-template complexes of 1:1 stoichiometry in combination with the presence of higher order template-template complexes.

To resolve this conjecture, we performed a series of MD studies, the results of which demonstrated both the presence of PA-PA self association complexes, and that the most statistically prevalent monomer-PA complex stoichiometry was of a 1:1 nature, though differetn in character from that proposed by Katz and Davis.  Moreover, the role of cross-linker in forming recognition sites was apparnet in these studies, a fact not previously considered.

References

1. Alexander C, Andersson HS, Andersson LI, Ansell RJ, Kirsh N, Nicholls IA, O’Mahony J, Whitcombe MJ. Molecular imprinting science and technology: A survey of the literature for the years up to and including 2003. Journal of Molecular Recognition 2006;19:106-180
2. Sellergren B, Lepistö M, Mosbach K. Highly enantioselective and substrate selective polymers obtained by molecular imprinting utilizing noncovalent interactions. NMR and chromatographic studies on the nature of recognition. Journal of the American Chemical Society 1988;110:5853-5860
3. Katz A, Davis ME. Investigations into the mechanisms of molecular recognition with imprinted polymers. Macromolecules 1999;32:4113-4121

The molecular imprinting technique has received significant attention due to its utility in the production of synthetic polymeric materials with predetermined ligand recognition properties [1].

It is generally accepted that the recognition properties of a molecularly imprinted polymer (MIP) is established during the prepolymerization stage. Previous investigations on the nature and extent of template prepolymerization complexation in a phenylalanine anilide (PA) MIP pointed at the complexity and diversity in the ensemble of complexes leading to the final “molecular memory”. In particular, conflicting models have been used to explain the observed molecular memory. Sellergren, Lepistö and Mosbach [2] proposed that selective, high-affinity sites in the final MIP were based on functional monomer-PA complexation of a 2:1 stoichiometry. Later, Katz and Davis [3] proposed that the template recognition sites arose due to a 1:1 functional monomer-template complex stoichiometry and that the effect of template dimerization is critical for the observed PA-MIP recognition properties.

In this study, we have attempted to shed new light on this as yet unresolved conflict using a series of molecular dynamics (MD) simulations. Results demonstrated the presence of PA-PA complexes and that the most statistically prevalent stoichiometry of functional monomer-PA complexes was of 1:1.

[1]             Alexander C, Andersson HS, Andersson LI, Ansell R, Kirsch N, Nicholls IA et al. Molecular imprinting science and technology: a survey of the literature for the years up to and including 2003, Journal of Molecular Recognition, 19, 106-180 (2006).

[2]            Sellergren B, Lepistö M, Mosbach K. Highly enantioselective and substrate selective polymers obtained by molecular imprinting utilizing noncovalent interactions. NMR and chromatographic studies on the nature of recognition, Journal of the American Chemical Society, 110, 5853-5860 (1988).

[3]             Katz A, Davis ME. Investigations into the mechanisms of molecular recognition with imprinted polymers, Macromolecules, 32, 4113-4121 (1999).