A Protocol for the Computational Design of High Affinity Molecularly Imprinted Polymer Synthetic Receptors

Molecularly imprinted polymer (MIP) nanoparticles, commonly referred to as ‘plastic antibodies’ or synthetic receptors, are polymeric materials with strong affi nity and selectivity for a particular chemical target. MIPs are regularly produced for use in sensors for monitoring food quality and environmental pollutants, and in the design of robust and affordable replacements for biological receptors, enzymes and antibodies in drug testing and assays. More recently the easy production of MIP nanoparticles has also permitted research relating to possible in vivo applications, primarily in drug delivery systems, toxin sequestration and pathogen inhibition. The strength of the interaction between the target and the polymer binding site is dependent on the particular monomers selected in synthesis of the MIP, and the relative concentrations of these in the pre-polymerization mixture. While computational approaches have been used to aid in MIP design previously, the methods adopted are often slow and simplistic, centring on observations of the template structure with a couple of functional monomers presumed to be appropriate. We present here an automated method of rapidly screening numerous functional monomers and effectively determining appropriate monomer ratios, while accounting for spatial discrimination in selection and dynamic parameters in optimization. Example are then given of effect MIP synthesis resulting from the protocol, and the benefi ts of this approach over competing methods are discussed. Short Communication A Protocol for the Computational Design of High Affi nity Molecularly Imprinted Polymer Synthetic Receptors Kal Karim*, Todd Cowen, Antonio Guerreiro, Elena Piletska, Michael J. Whitcombe and Sergey A. Piletsky Department of Chemistry, University of Leicester, Leicester, LE1 7RH, UK Dates: Received: 13 March, 2017; Accepted: 13 April, 2017; Published: 14 April, 2017 *Corresponding author: Kal Karim, Department of Chemistry, University of Leicester, Leicester, LE1 7RH, UK, Tel: +44 116 294 4668; Fax: +44 116 252 3789; E-mail: https://www.peertechz.com


Introduction
While computational methods have long been used in the design of synthetic receptors, a comprehensive approach has largely been unavailable for use as a standard protocol in the production of high affi nity biomimetic materials. Molecularly imprinted polymer (MIP) synthetic receptors or 'plastic antibodies' are prepared by the formation of a crosslinked polymer in the presence of a molecular template. The self-assembly of functional monomers with complementary functional groups to those of the template results in formation of a pre-polymerization complex, which is stabilized by crosslinking during polymer formation. Appropriate selection of functional monomers with strong interaction energies with the template will favour the associated state, resulting in the formation of MIPs with high affi nity and specifi city for the target. In the absence of a rational approach to the design of MIPs, monomer selection is often made on the basis of previous experience or chemical intuition; in many cases methacrylic acid is used as the sole functional monomer due to its importance in the history of molecular imprinting.
Laboratory-based approaches to the optimization of monomer compositions centre on combinatorial synthesis and screening [1,2], an approach limited by the large number of different polymers required to account for the many potentially suitable monomers.
With thousands of functional monomers commercially available or readily synthesized, a more rational approach  [3][4][5][6], molecular dynamics (MD) [7][8][9], and quantum mechanics (QM) [10,11], based molecular modelling techniques, but these are still limited by the sequential screening of each monomer manually. monomers. The validation of the described computational protocol as a means of rapid and reliable MIP design is provided by reference to many published examples of high-affi nity MIPs for a diverse range of targets prepared according to this design strategy.

Incentives
The foundational principle of computational MIP design is that the stability of the template-monomer complex is directly related to the quality of imprinted sites created in the polymer after cross-linking. Much of the contemporary use of molecular modelling in the design of MIPs therefore is centred on the equation: Where E C , E T and E M are the lowest calculable energies of the template-monomer complex, template and monomer respectively. Comparison of the different values of ΔE gives an indication of the relative stability of different components in the system, and thus provides an appropriate guide to the selection of an appropriate monomer [12][13][14], to fi nd the most suitable template-monomer ratio [15][16][17], or both [18][19][20]. This method has become popular along with the use of QMbased (predominantly DFT) techniques in MIP design as a result of advances in hardware making greater computer power available. However, this approach is still time consuming and computationally demanding, and QM is for this reason associated with the screening of a relatively small number of monomers (typically fi ve or fewer). There are rare exceptions to this in which 20 or more functional monomers, along with a number of cross-linkers, have been ranked against a particular template [21][22][23], but the time presumably invested in this does not lend to this being an appropriate general model of MIP design.
While QM has advantages in accuracy over the alternatives which makes it desirable in the comparison of different polymerization constituents, either directly via the above equation, or by frontier orbital analysis [24][25][26], which can be used as an indicator of kinetic stability [27], MM and MD have the power to perform hundreds of tasks simultaneously, such as the simulation of pre-polymerization systems consisting of thousands of molecules, and provide an analysis of interactions occurring between molecules over many nanoseconds [28]. These models are also continually being revisited, often with the adoption of techniques that are new to MIP design, such as analysis by determination of the radial distribution functions (RDFs) of atoms likely to be involved in hydrogen bond formation. RDF methods provide the distances between atoms and allows selection of appropriate chemical components in the polymerization system (monomers, solvents, etc.), when used as a tool in predicting the likelihood of successful template complexation and polymer synthesis [29][30][31].
The protocol described herein began development in 2000 [32], and an early form of the procedure was employed for the fi rst time to design MIPs for creatinine [33], ephedrine [34], and microcystin-LR [35]. Dozens of papers have since been published describing the use of this protocol for a broad range of templates, with the technique being continually modifi ed to provide a reliable method of designing high affi nity imprinted polymers. Here can be seen the incremental advancements describing how molecular modelling techniques can be used to rapidly screen large databases of functional monomers in order to identify suitable candidate monomers for MIP preparation. The computational time and resources required for performing these MM and MD calculations of monomertemplate interactions are modest and can produce results within a few hours. The method represents a generic procedure for the selection of monomer mixtures for the imprinting of virtually any template.

Experimental
All calculations and procedures were carried out on a desktop PC running RHEL 3.0 or later (Linux platform), executing the software package SYBYL 7.3 (Tripos Inc.). The protocol described was developed using the SYBYL software but can be adapted for application in other programs. Standard procedures are followed regarding preparation of the selected template, which may be either the whole molecule (as is typical in smaller structures) or an appropriate epitope may be used to represent the binding region of a biochemical macromolecule. These structures are often obtained from online sources such as PubChem [36], ZINC [37,38], or RCSB PDB [39], when possible to ensure the correct appropriate template geometries are presented in screening.

Automatic monomer screening
Templates constructed manually may be minimized and processed by simulated annealing using any available force fi eld, but for greater compatibility with the LeapFrog protocol the Tripos force fi eld and Gasteiger-Hückel charges are preferred. All structures must be available in a mol2 fi le format.
The monomer library can be constructed by a number of approaches. Using the SYBYL software a large number of molecules can be saved under one fi le name or retained in one folder easily, facilitating the writing of a script which sequentially loads a monomer, records the total internal energy of the monomer and template in isolation, forms a complex by energy minimization, and records the energy of the new arrangement before restarting with a new monomer. This process can be easily automated using a simple algorithm written in SYBYL Programming Language (SPL), and can be easily adapted for use in other software. Here however we emphasize the benefi ts of adapting LeapFrog for use in the screening process; Leapfrog includes a function to add an observed structure to a database ('add fragments'), or a large number of monomer can be automatically added with simple SPL algorithms (An example script is given in Appendix 1).
Once the library is established the screening can be initiated by launching the Leapfrog program. Using the 'dream' mode allows greater freedom to modify parameters and ensuring the 'calculate' option is enabled and set to 'all atoms' allows observation of the whole template as opposed to the binding cavity of a macromolecule. In the 'tradeoff' between quality and variety the former must be maximized in the 'tradeoff'

Application of the method
The protocol will be of interest to researchers involved in the design and synthesis of MIPs in any format (e.g. micro-and nano-particles, fi lms or monoliths), and suitable for the design of high affi nity MIPs for diverse templates including clinical targets (drugs), environmental/food targets (e.g. toxins) and for MIPs to be used in extreme environments. This protocol is particularly suitable for use with low molar mass templates and where the development of high affi nity MIPs is required: such as (i) in the separation and purifi cation of high-value products; (ii) analytical sample pre-treatment and solid-phase extraction;   the successful design of MIPs for a broad range of templates [40,41], some of which are listed in table 2 [33][34][35][42][43][44][45][46][47][48][49][50][51][52]. The protocol has been advanced through its initial use by refi nement of the parameters and the introduction of further functional monomers. A number of these new additions are more specialist compounds that must fi rst be synthesized and cannot be readily obtained commercially, but are useful in controlling target affi nity and selection for certain polymer properties.
In the case of RDPs the choice of monomer selected via the screening process has been shown to be suffi cient for the synthesis of high affi nity materials [43,44]. For the synthesis of these materials further refi nement is not required, and so the whole design procedure can be completed in under an hour.
Typically however a stoichiometric ratio must be determined for effective complexation in the pre-polymerization mixture for an imprinted polymer, and thus the MD protocol must be followed. The examples in table 2 range from (i) good imprinting factors [33,35]; (ii) high recovery of template from using solid phase extraction (SPE) [42,43,50,51]; (iii) controlled release [46,47]; (iv) dissociation constant in nM [35]; and (v) industrial applications [44,48]. This procedure therefore can be observed to produce excellent results with minimal time requirements, making this whole process highly effi cient in comparison with alternative approaches.

Refi nement using MD simulations
The appropriate ratio of polymerization mixture components is determined by performing MD simulations.
This may involve the use of a single monomer species, or if the screening shows that two different monomers interact with different regions of the template, then these monomers may act synergistically in the imprinting of that template and both will then be used.
A pre-computed box of fi xed dimensions is prepared by saturating the space around the template with the monomer selected from the results obtained during the screening. Figure   3 shows

Protocol development
The procedure was fi rst demonstrated in a simple form some time ago for the design of a MIP for creatinine [34]. Since that time dozens of papers have been published describing Figure 3: Pre-computed box of a small organic template (in red) surrounded by itaconic acid monomers. By performing the molecular dynamics simulation followed by energy minimization, an appropriate ratio of functional monomers to template can be determined from the complex formed in the model.
Tylosin [48] The polymer capacity for tylosin was estimated as 6.4 mg/g for MIP, which was suitable for practical applications Curcumin [49] Comparison of the batch analysis of the MIP-and NIP-grafted nanoparticles shows superior MIP binding to curcumin (μg per g particles) Kukoamine A [50] Kukamine can be purifi ed (90%) from potato extract using MIP Artemisinin [50] Quantitative recovery of artemisinin (87%) N-acyl-homoserine lactones [52] Computationally designed polymers could sequester a signal molecule of V. fi scheri bacteria