Development of molecularly imprinted polymers as tailored templates for the solid-state [2+2] photodimerization

In this study, a molecularly imprinted polymer (MIP) was prepared to selectively template the [2+2] photodimerization of trans-1,2-bis(4-pyridyl)ethylene. First, an MIP selective for rctt-tetrakis(4-pyridyl)cyclobutane, which is the [2+2] photodimerization product of trans-1,2-bis(4-pyridyl)ethylene, was prepared from methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA). The non-covalent MIP showed enhanced affinity for both the templating agent, rctt-tetrakis(4-pyridyl)cyclobutane, and the alkene precursor, trans-1,2-bis(4-pyridyl)ethylene. The solid-state photodimerization reaction proceeded in significantly higher yields in the presence of the MIP. Control reactions carried out in the absence of polymer gave no product, and reactions carried out in the presence of a non-imprinted polymer and an MIP imprinted with a different template, 3-hydroxymethylpyridine, gave much lower yields of the cyclobutane photodimerization product. The outcome of the MIP-templated photodimerization reaction was strongly influenced by the binding site heterogeneity of the non-covalently imprinted polymers. For example, higher yields were observed with decreasing olefin loadings levels on the MIPs. This binding site heterogeneity was characterized via application of the Freundlich binding model to the experimentally measured binding isotherms. These confirmed that the non-covalent MIPs had very few high-affinity binding sites, which greatly limits the capacity and ultimately the utility of these materials as templates in synthetic organic applications.     

Binding cross-reactivity of Boc-phenylalanine enantiomers on molecularly imprinted polymers

A potential problem associated with molecularly imprinted polymer (MIP) sorbents is that of cross-reactivity. In this study three MIPs (imprinted with Boc-L-phenylalanine, Boc-L-alanine, Boc-L-glutamic acid) plus a non-imprinted control were prepared and examined for their ability to bind differentially the enantiomers of boc-protected phenylalanine in an effort to quantify cross-reactivity and to develop a predictive model. Batch rebinding studies showed a degree of predictability for a number of MIP-ligand pairs, but other combinations showed unexpectedly high levels of cross-reactivity. Despite the general acceptance of heterogeneity of MIP binding sites, many previous studies report linear Scatchard plots, which is indicative of binding site homogeneity. The data from this study produced curves, clearly demonstrating heterogeneity. The theoretical and practical implications of this heterogeneity are discussed. Chirality 9:233–237, 1997. © 1997 Wiley-Liss, Inc.     

Refined models of New Delhi metallo-beta-lactamase-1 with inhibitors: an QM/MM modeling study

New Delhi metallo-beta-lactamase 1 (NDM-1) has been identified as a potential target for the treatment of multi-drug resistance bacterial infections. We used molecular docking, normal MD, SIE, QM/MM MD simulations, QM/MM GBSA binding free energy, and QM/MM GBSA alanine-scanning mutagenesis techniques to investigate interactions of the NDM-1 with 11 inhibitors (Tigecycline, BAL30072, D-captopril, Penicillin G, Ampicillin, Carbenicillin, Cephalexin, Cefaclor, Nitrocefin, Meropenem, and Imipenem). From our normal MD and QM/MM simulations, the correlation coefficients between the predicted binding free energies and experimental values are .88 and .93, respectively. Then simulations, which combined QM/MM/GBSA and alanine-scanning mutagenesis techniques, were performed and our results show that two residues (Lys211 and His250) have the strongest impact on the binding affinities of the 11 NDM-1/inhibitors. Therefore, our approach theoretically suggests that the two residues (Lys211 and His250) are responsible for the selectivity of NDM-1 associated inhibitors.     

Towards the rational development of molecularly imprinted polymers: 1H NMR studies on hydrophobicity and ion-pair interactions as driving forces for selectivity

The preparation of molecularly imprinted polymers (MIP) based on non-covalent interactions has become a widely used technique for creating highly specific sorbent materials predominantly used in separation chemistry. A crucial factor in a successful imprinting protocol is the optimisation of the template/functional monomer interaction in the pre-polymerisation mixture, eventually leading to a maximum of high-affinity binding sites in the resulting polymer matrix. In order to develop more efficient preparation technologies for imprinted polymers, two separate pre-polymerisation complexes were investigated by NMR spectroscopic techniques in order to identify the types of interactions occurring in the pre-polymerisation mixture, and their implications for the subsequently formed imprinted polymer. In particular, hydrophobic effects have been followed by NMR spectroscopy and their contribution to the selectivity of the resulting MIP has been investigated. The 2,4-D imprint system is used as an example to fundamentally study whether observations at the pre-polymerisation stage correlate with properties of the finally prepared MIP, and which parameters govern success of an imprinting protocol.     

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

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

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

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

Correlation between biological activity and binding energy in systems of integrin with cyclic RGD-containing binders: a QM/MM molecular dynamics study

We here report a combined quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) study on the binding interactions between the α(V)β(3) integrin and eight cyclic arginine-glycine-aspartate (RGD) containing peptides. The initial conformation of each peptide within the binding site of the integrin was determined by docking the ligand to the reactive site of the integrin crystal structure with the aid of docking software FRED. The subsequent QM/MM MD simulations of the complex structures show that these eight cyclic RGD-peptides have a generally similar interaction mode with the binding site of the integrin to the cyclo(RGDf-N[M]V) analog found in the crystal structure. Still, there are subtle differences in the interactions of peptide ligands with the integrin, which contribute to the different inhibition activities. The averaged QM/MM protein-ligand interaction energy (IE) is remarkably correlated to the biological activity of the ligand. The IE, as well as a three-variable model which is somewhat interpretable, thus can be used to predict the bioactivity of a new ligand quantitatively, at least within a family of analogs. The present study establishes a helpful protocol for advancing lead compounds to potent inhibitors.     

Combinatorial methods in molecular imprinting

Molecular imprinting is a general method for synthesizing robust, network polymers with highly specific binding sites for small molecules. Recently, combinatorial and computational approaches have been employed to select an optimal molecularly imprinted polymer (MIP) formulation for a targeted analyte. The use of MIPs in the combinatorial field, specifically their use for screening libraries of small molecules, has also been developed.     

Molecularly imprinted ligand-exchange recognition assay of glucose by quartz crystal microbalance

Molecular imprinted polymers (MIP) as a recognition element for sensors are increasingly of interest and MIP-quartz crystal microbalance (QCM) have started to appear in the literature. In this study, we have combined quartz crystal microbalance with MIP to prepare a sensor using the ability of glucose to chelate of copper (II) ion of methacrylamidohistidine (MAH) monomer to create ligand exchange (LE) assembled monolayer which is suitable for glucose determination. The study includes the measurement of binding interaction of molecularly imprinted QCM sensor via ligand interaction, investigation of the pH effect on frequency shift and recognition selectivity studies of glucose-imprinted polymer with respect to methyl-alpha-d-glucopyranoside and sucrose. Bmax (number of binding sites) and K(D) (dissociation constant of the metal-chelate copolymer) were also calculated using Scathard plot and the detection limit was found as 0.07 mM. MIP showed higher glucose-binding affinity than a well-known glucose binding protein, conconavalin A.     

Synthesis and evaluation of uniformly sized nalidixic acid-imprinted nanospheres based on precipitation polymerization method for analytical and biomedical applications

For the first time in this work, uniform molecularly imprinted polymer (MIP) nanoparticles were prepared using nalidixic acid as a template. The MIP nanoparticles were successfully synthesized by precipitation polymerization applying methacrylic acid (MAA) as a functional monomer and trimethylolpropane trimethacrylate (TRIM) as a cross-linking monomer at different mole ratios. The morphology, binding, recognition, selectivity, and in vitro release behaviors of obtained particles were studied. The produced polymers were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetric. Furthermore, their morphology was analyzed accurately by scanning electron microscopy, photon correlation spectroscopy, and Brunauer-Emmett-Teller analysis. The nanospheres and microspheres with mean diameter values of 94 nm, 256 nm, and 1.2 μm were obtained using nalidixic acid-MAA-TRIM various mole ratios. Among the MIPs, the product with nalidixic acid-MAA-TRIM mole ratio of 1:12:12 established nanospheres with the lowest polydispersity index (0.003), an average pore diameter (12 nm), and the highest specific surface area (280 m(2) g(-1)) and selectivity factor (10.4). Results from binding experiments demonstrated that the imprinted nanospheres with a 94-nm mean diameter and a binding capacity of 28 mg of nalidixic acid per gram of polymer had higher specific affinity to nalidixic acid in contrast with the other imprinted nanospheres, microspheres, and nonimprinted particles. However, the binding performance of imprinted nanospheres in human serum was estimated using high-performance liquid chromatography analysis (binding approximately 98% of nalidixic acid). In addition, release experiments proved to be successful in the controlled release of nalidixic acid during a long period. The 20% of loaded nalidixic acid was released from the imprinted nanospheres within the first 20 h, whereas the remaining 80% was released in the after 120 h. The nalidixic acid release kinetics from the MIPs was highly affected by properties of the particles.     

Binding site characteristics of 17beta-estradiol imprinted polymers

The variety of applications utilizing molecularly imprinted polymers (MIPs) requires synthetic strategies yielding different MIP formats including films, irregular particles, or spheres, along with precise knowledge on the specific material characteristics, such as binding capacity and binding efficiency of these materials. In response to this demand, MIPs are prepared in different formats by variation of the polymerization methodology. It is commonly agreed that micro- and sub-microspheres are particularly advantageous MIP formats, due to their monodispersity and facile synthesis procedures in contrast to conventional imprinted polymers prepared by bulk polymerization. However, the differences in actual rebinding characteristics of different MIP formats based on molecular interactions under a variety of binding/rebinding conditions have not been studied in detail to date. Consequently, the present work details an analytical strategy generically applicable to MIP systems for rebinding studies including equilibrium binding, non-equilibrium binding, and release experiments enabling more profound understanding on the molecular interactions between the imprinted materials and the template molecules. In this study, three MIP formats were considered for the same template molecule, 17beta-estradiol: irregularly shaped particulate polymers prepared by bulk polymerization and grinding, microspheres, and sub-microspheres. The latter two formats were synthesized via precipitation polymerization using different processing strategies. The morphologies and porosities of the resulting imprinted materials were characterized by scanning electron microscopy (SEM) and Brunauer-Emmett-Teller (BET) analysis, respectively. The obtained results indicate that microspheres prepared by precipitation polymerization provide superior rebinding properties during equilibrium binding in contrast to bulk polymers and sub-microspheres, and that the rebinding properties are different during equilibrium binding versus non-equilibrium binding. The median binding affinity constant determined during non-equilibrium rebinding is higher than the values obtained from equilibrium rebinding. Furthermore, the binding site distribution appears more homogeneous thief derived from non-equilibrium rebinding, as reflected in a heterogeneity index of m=0.725. Moreover, it is hypothesized that the specific interactions between template and monomers are related to the porosity of the imprinted polymers, which implies that the amount of binding sites and the pore sized distribution of the imprinted materials are a critical factor in achieving the desired MIP performance in various analytical applications. The BET results indicate that particles prepared with lower cross-linker-to-template ratio have a reduced surface area. Furthermore, it can be expected that there are less specific binding sites available at particles with reduced surface area and pore volume given similar distribution of the binding sites, as confirmed by the equilibrium binding isotherm studies. The pore size distribution results reveal that control of the pore size in the range of 100-180 A is essential to obtain the desired retention properties and Gaussian peak shape during HPLC analysis of small molecules.     

Characterization of the heterogeneous binding site affinity distributions in molecularly imprinted polymers

Molecularly imprinted polymers (MIPs) are polymers that can be tailored with affinity and selectivity for a molecule of interest. Offsetting the low cost and ease of preparation of MIPs is the presence of binding sites that vary widely in affinity and selectivity. Presented is a review of methods that take into account binding site heterogeneity when calculating the binding properties of MIPs. These include the bi-Langmuir, Freundlich, and Langmuir-Freundlich binding models. These methods yield a measure of heterogeneity in the form of binding site affinity distributions and the heterogeneity index. Recent developments have made these methods surprisingly easy to use while also yielding more accurate measures of the binding properties of MIPs. These have allowed for easier comparison and optimization of MIPs. Heterogeneous binding models have also led to a better understanding of the imprinting process and of the advantages and limitations of MIPs in chromatographic and sensor applications.     

Surface molecular imprinting by atom transfer radical polymerization

Results are presented that demonstrate the successful preparation of ultrathin (< 10 nm), surface-confined, molecularly imprinted polymer (MIP) films on model gold substrates using atom transfer radical polymerization (ATRP). 2-Vinylpyridine (2Vpy) was investigated as the functional monomer, and ethylene glycol dimethacrylate (EGDMA) was the cross-linking monomer. Fluorescently labeled N,N'-didansyl-L-cystine and N,N'-didansyl-L-lysine were used as the template molecules to form the MIPs. Spectroscopic and ellipsometric results are presented that follow film formation and growth rates. Results are also presented from fluorescence experiments used to quantify and compare the adsorption capacities of MIP surface films and nonimprinted (NIP) control films. MIP films exhibited higher binding capacities than the control NIP films at all solution concentrations of N,N'-didansyl-L-cystine and N,N'-didansyl-L-lysine. Furthermore, template removal from these imprinted films appears to be 100% efficient. Selectivity studies showed that the MIPs display some cross-reactivity between these two molecules; nevertheless, MIPs prepared against one template showed selectivity for that template. A selectivity coefficient of 1.13 was achieved for MIP surfaces prepared against N,N'-didansyl-L-lysine; a value of 1.51 was observed for MIP surfaces prepared against N,N'-didansyl-L-cystine.     

Designing of MIP based QCM sensor having thymine recognition sites based on biomimicking DNA approach

Quartz crystal microbalance (QCM) sensors coated with molecular imprinted polymers (MIP) have been developed for the determination of thymine. In this method, methacryloylamidoadenine (MA-Ade) have used as a new monomer and thymine template for inspiration of DNA nucleobases interaction. The thymine can be simultaneously hydrogen binding to MA-Ade and fit into the shape-selective cavities. Thus, the interaction between nucleobases has an effect on the binding ability of the QCM sensors. The binding affinity of the thymine imprinted sensors has investigated by using the Langmuir isotherm. The thymine imprinted QCM electrodes have shown homogeneous binding sites for thymine (K_a: 1.0 × 10⁵ M⁻¹) while heterogeneous binding sites for uracil. On the other hand, recognition selectivity of the QCM sensor based on thymine imprinted polymer toward to uracil, ssDNA and ssRNA has been reported in this work.     

Application of Molecularly Imprinted Polymers to Selective Removal of Clofibric Acid from Water

A new molecularly imprinted polymer (MIP) adsorbent for clofibric acid (CA) was prepared by a non-covalent protocol. Characterization of the obtained MIP was achieved by scanning electron microscopy (SEM) and nitrogen sorption. Sorption experimental results showed that the MIP had excellent binding affinity for CA and the adsorption of CA by MIP was well described by pseudo-second-order model. Scatchard plot analysis revealed that two classes of binding sites were formed in the MIP with dissociation constants of 7.52±0.46 mg L⁻¹ and 114±4.2 mg L⁻¹, respectively. The selectivity of MIP demonstrated higher affinity for CA over competitive compound than that of non-imprinted polymers (NIP). The MIP synthesized was used to remove CA from spiked surface water and exhibited significant binding affinity towards CA in the presence of total dissolved solids (TDS). In addition, MIP reusability was demonstrated for at least 12 repeated cycles without significant loss in performance.     

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.     

A novel lanthanide-chelate based molecularly imprinted cryogel for purification of hemoglobin from blood serum: An alternative method for thalassemia diagnosis

Purification and analyzing of proteins is an essential means for understanding their function and diseases associated with their lack or defect. In this research, a new lanthanide-chelate based molecularly imprinted polymer (MIP) was synthesized for selective separation of Hemoglobin (Hb) from human serum in the presence of various interference molecules. The Hb-imprinted polymer was prepared by using complex functional monomer N-methacryloylamido antipyrine (MAAP)-Ce(III) and 2-Hydroxyethyl methacrylate (HEMA) in accordance of cryopolymerization techniques. The nonimprinted cryogel (NIP) was also prepared at same polymerization conditions in the absence of template Hb molecule. The effects of pH, initial Hb concentration, flow rate, temperature and ionic strength on the binding capacity of both imprinted and nonimprinted cryogels was investigated. The maximum binding capacity for the MIP column was found to be as 79.41 mg g⁻¹ dry cryogel, that is four times higher than the NIP column under the optimum conditions (pH 5.0, flow rate: 1.0 mL min⁻¹, T: 25 °C). Moreover, selectivity experiments were performed by using two interference proteins as myoglobin (Mb) and cytochrome c (Cyt-c) and the relative selectivity coefficients (k') for Hb/Mb and Hb/Cyt-c pairs were determined as 36.59 and 37.22, respectively.     

A computational approach to study functional monomer‐protein molecular interactions to optimize protein molecular imprinting

Molecular imprinting has become a promising approach for synthesis of polymeric materials having binding sites with a predetermined selectivity for a given analyte, the so‐called molecularly imprinted polymers (MIPs), which can be used as artificial receptors in various application fields. Realization of binding sites in a MIP involves the formation of prepolymerization complexes between a template molecule and monomers, their subsequent polymerization, and the removal of the template. It is believed that the strength of the monomer‐template interactions in the prepolymerization mixture influences directly on the quality of the binding sites in a MIP and consequently on its performance. In this study, a computational approach allowing the rational selection of an appropriate monomer for building a MIP capable of selectively rebinding macromolecular analytes has been developed. Molecular docking combined with quantum chemical calculations was used for modeling and comparing molecular interactions among a model macromolecular template, immunoglobulin G (IgG), and 1 of 3 electropolymerizable functional monomers: m‐phenylenediamine (mPD), dopamine, and 3,4‐ethylenedioxythiophene, as well as to predict the probable arrangement of multiple monomers around the protein. It was revealed that mPD was arranged more uniformly around IgG participating in multiple H‐bond interactions with its polar residues and, therefore, could be considered as more advantageous for synthesis of a MIP for IgG recognition (IgG‐MIP). These theoretical predictions were verified by the experimental results and found to be in good agreement showing higher binding affinity of the mPD‐based IgG‐MIP toward IgG as compared with the IgG‐MIPs generated from the other 2 monomers.     

Combinatorial screening of polymer precursors for preparation of benzo[α] pyrene imprinted polymer: an ab initio computational approach

A combinatorial screening procedure was used for the selection of polymer precursors in the preparation of molecularly imprinted polymer (MIP), which is useful in the detection of the air pollution marker molecule benzo[a]pyrene (BAP). Molecular imprinting is a technique for the preparation of polymer materials with specific molecular recognition receptors. The preparation of imprinted polymers requires polymer precursors such as functional monomer, cross-linking monomer, solvent, an initiator of polymerization and thermal or UV radiation. A virtual library of functional monomers was prepared based on interaction binding scores computed using HyperChem Release 8.0 software. Initially, the possible minimum energy conformation of the monomers and BAP were optimized using the semi-empirical (PM3) quantum method. The binding energy between the functional monomer and the template (BAP) was computed using the Hartree-Fock (HF) method with 6-31 G basis set, which is an ab initio approach based on Moller-Plesset second order perturbation theory (MP2). From the computations, methacrylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) were selected for preparation of BAP imprinted polymer. The larger interaction energy (ΔE) represents possibility of more affinity binding sites formation in the polymer, which provides high binding capacity. The theoretical predictions were complimented through adsorption experiments. There is a good agreement between experimental binding results and theoretical computations, which provides further evidence of the validity of the usefulness of computational screening procedures in the selection of appropriate MIP precursors in an experiment-free way.     

Synthesis of a molecularly imprinted polymer for the solid‐phase extraction of betulin and betulinic acid from plane bark

Introduction – Plant extracts are usually complex mixtures of various polarity compounds and their study often includes a purification step, such as solid‐phase extraction (SPE), to isolate interest compounds prior analytical investigations. Molecularly imprinted polymers (MIPs) are a new promising type of SPE material which offer tailor‐made selectivity for the extraction of trace active components in complex matrices. Numerous specific cavities that are sterically and chemically complementary of the target molecules, are formed in imprinted polymers. A molecularly imprinted polymer (MIP) was synthesised in order to trap a specific class of triterpene, including betulin and betulinic acid from a methanolic extract of plane bark. Methodology – Imprinted polymers were synthesised by thermal polymerisation of betulin as template, methacrylic acid (MAA) or acrylamide (AA) as functional monomer, ethylene glycol dimethacrylate as crosslinking agent and chloroform as porogen. Afterwards, MAA‐ and AA‐MIPs were compared with their non‐imprinted polymers (NIPs) in order to assess the selectivity vs betulin and its derivatives. Recovered triterpenes were analysed by HPLC during MIP‐SPE protocol. Results – After SPE optimisation, the MAA‐imprinted polymer exhibited highest selectivity and recovery (better than 70%) for betulin and best affinity for its structural analogues. Thus, a selective washing step (chloroform, acetonitrile) removed unwanted matrix compounds (fatty acids) from the SPE cartridge. The elution solvent was methanol. Finally, the MAA‐MIP was applied to fractionate a plane bark methanolic extract containing betulin and betulinic acid. Conclusion – This study demonstrated the possibility of direct extraction of betulin and its structural analogues from plant extracts by MIP technology. Copyright © 2009 John Wiley & Sons, Ltd.