Cholesterol-imprinted polymers

Fig. 7.2. Binding of structural analogues to cholesterol-imprinted polymer. Cholesterol (2), cholestane (3), cholesteryl acetate (4), epicholesterol (cholest-5-ene-3a-ol) (5) and cholest-5-ene-3-one (6), all 2 mM in hexane. Adapted from [9].

In order to demonstrate the imprinting effect, a control polymer, using phenyl (4-vinyl) phenyl carbonate as the template, was prepared under the same condition as the MIP. This was chemically identical to the cholesterol-imprinted polymer after template cleavage, but would have a much smaller imprint cavity associated with the phenol in the recognition site, too small to accommodate the cholesterol molecule. An additional control, prepared in the absence of either template was also made, to test the effect of the hydrolysis conditions on the polymer matrix. Both of these polymers in their hydrolyzed state and the cholesterol-imprinted polymer in its unhydrolyzed state (template still bound in the polymer) were unable to bind cholesterol. 

Figure 8 (a) Binding of cholesterol to cholesterol-imprinted and control polymers, from a 2 mM solution of cholesterol in hexane, as a function of polymer concentration, (b) Binding of cholesterol and various cholesterol analogues (2 mM) to the cholesterol-imprinted polymer, prepared by the sacrificial spacer method. Reprinted with permission from Journal of the American Chemical Society. Copyright 1995 American Chemical Society (Ref. 10). 

One consequence of the uniform binding site distribution and high capacity of these polymers is that they may be rather better suited to the preparation of chromatographic (HPLC) stationary phases than noncovalently imprinted materials. In fact this was the conclusion arrived at by Hwang and Lee [15], who compared the chromatographic performance of cholesterol-imprinted polymers prepared by the semi-covalent (carbonate spacer) method with noncovalent MIPs incorporating... 

Hwang, C.C. Lee, W.C. Chromatographic characteristics of cholesterol-imprinted polymers prepared by covalent and non-covalent imprinting methods. J. Chromatogr. 

Asanuma H, Kakazu M, Shibata M, Hishiya T, Komiyama M. Molecularly imprinted polymer of beta-cyclodextrin for the efficient recognition of cholesterol. Chem Commun 1997 1971-1972. 

Fig. 8 (a) Schematic representation of an ordered assembly of cyclodextrin molecules with each one binding a part of the template, (b) Binding modes of (a) cholesterol, (b) 4-hydroxyazobenzene and (c) 4-phenylphenol to the guest-binding sites in the cholesterol-imprinted beta-cyclodextrin polymer. Reprinted with permission from [89]. Copyright 1999 American Chemical Society... 

In a short communication, Cooper et al. [28] report the utilisation of two novel fluorescent functional monomers (Fig. 20.10) in EDMA-based MlPs. Initially, a polymerisable phenolic compound was treated with cholesterol chloroformate (as per Whitcombe et al. [29]) and used to prepare a covalently imprinted polymer. Details are lacking, but this procedure was not successful both the imprinted and the non-imprinted polymers had a similar affinity for cholesterol and no changes in... 

Essentially, the same basic protocol can be adapted for the preparation of non-covalently imprinted polymers. In this case, the cholesterol template monomer is replaced by the template to be imprinted and additional functional monomer (or monomers) is included in the polymerization mixture, at a predetermined molar ratio with respect to the template. Typical functional monomers might be chosen from amongst methacrylic acid, itaconic acid, vinylpyridine, dimethylaminoethyl methacrylate, acrylamide, hydroxyethyl methacylate, and many more. Typical solvents for non-covalent imprinting Include chloroform, THF, and acetonitrile. Templates are removed from non-covalently imprinted polymers by exhaustive washing with a suitable solvent. 

High selectivity can be obtained by imprinting polymers with neutral molecules.80 In this process, a cross-linked polymer is prepared in the presence of a template. Then the template is removed by solvent extraction. The extracted polymer is then used to pick up the template molecules from other sources. Among the examples in the literature are some that deal with atrazine (an herbicide), cholesterol, other sterols, dipeptides, TV-acetyltryptophane resolution (L-isomer favored by a factor of 6), adenine, and barbiturates.81 The polymerizations in the first two examples, are shown in (7.16) (The cross-linking comonomer with the cholesterol-containing monomer was ethylenebis-methacrylate. The cholesterol was cleaved from the polymer with sodium hydroxide in methanol.)... 

The application of MIPs as the stationary phase in solid-phase extraction (SPE), often referred to as molecularly imprinted polymer solid-phase extraction (MIS P E), is a rapidly growing area [197-199]. With MISPE, highly specific enrichment of substances present at trace levels is possible. The technique has been applied to the analysis of drugs, for example, caffeine [200], scopolamine [201], naproxen [202], tetracycline [203], cholesterol [204] and local anesthetics [205], as well as environmental pollutants, exemplified by organophosphate flame retardants [206-208], triazines in soil and vegetable samples [71] and naphthalene sulfonates in river water [209]. 

Quite some examples have been published in recent years (see review [28] and, e.g. [64,65]). Despite the high association constants of the cyclodextrins, there are still some problems in their application as stoichiometric noncovalent binding site. They are mainly connected with the necessity to use very polar solvents and to get the steroids soluble. Komiyama and coworkers [64] used cholesterol as the template and prepared inclusion complexes with P-cyclodextrin which are cross-linked by toluene 2,4-diisocyanate in DMSO. It is shown that first a 1 1 complex with cholesterol is formed and it is assumed that during the imprinting the stoichiometry changes to 1 2 or even 1 3. The imprinted polymer shows double the uptake of cholesterol compared to a control polymer. 

Figure 7 Sacrificial spacer method, as exemplified by the imprinting of cholesterol (a) cholesteryl (4-vinyl)phenyl carbonate 8 is used as the template monomer to form a covalently imprinted polymer (b) in the polymerization step, the carbonyl group of the carbonate ester holds the functional monomer and template oxygen atoms apart by two bond distances (c) hydrolysis results in loss of the template and loss of the spacer as CO2 (d) rebinding can now occur with the cholesterol ligand occupying essentially the same space as the template cholesteryl group (adapted from Ref. 10).

Sreenivasan, K. Imparting cholesterol recognition sites in radiation polymerized poly(2-hydroxyethyl methacrylate) by molecular imprinting. Polym. Int. 1997, 42,... 

The seed latexes used as the cores of the imprinted particles were prepared from hydrophilic or hydrophobic polymers. The hydrophilic seeds were prepared from methyl methacrylate and methyl methacrylate/ethyleneglycol dimethacrylate copolymers, while the hydrophobic seeds were composed of polystyrene or styre-ne/divinyl benzene copolymers. Hydrophilic- and hydrophobic-imprinted shells were then laid over these cores. It was found that the best cholesterol recognition was obtained with a hydrophilic-imprinted shell and a poly(methyl methacrylate) core. However, the performance deteriorated when the core was lightly cross-linked with ethyleneglycol dimethacrylate. In a second paper [10], imprinted polymers were prepared by the noncovalent approach with cholesterol rebinding relying upon hydrophobic interactions between cholesterol and the imprinted shell. To achieve this, the template was modified to give it the characteristics of a surfactant. The structure of the template surfactant is illustrated in Fig. 2. 

A broader range of sensor devices has been developed, where the sensor response is modulated by the presence of an imprinted polymer, or an imprinted surface. Perhaps the simplest way of preparing an imprinted surface is in the use of self-assembled monolayer (SAM) techniques. A cholesterol-specific SAM was prepared by Piletsky et al. [44], in which a gold electrode was treated with a solution of cholesterol and hexadecanethiol in methanol. After the SAM had been established, the template was removed, leaving cholesterol-shaped cavities in the imprinted surface. Amperometric measurements were then used to investigate the sensitivity of the electrode towards cholesterol. It was found that the unmodified electrode, and the imprinted electrode displayed the same peak current. However, exposure of the imprinted electrode to a solution of template caused a decrease in... 

Asanuma, H. Kakazu, M. Shibata, M. Hishiya, T. Komiyama, M. Synthesis of molecular imprinted polymer of P-cyclodextrin for the efficient recognition of cholesterol. Super Molecule Sci. 1998, 5, 417-421. 

Various novel imprinting techniques have also been presented recently. For instance, latex particles surfaces were imprinted with a cholesterol derivative in a core-shell emulsion polymerization. This was performed in a two-step procedure starting with polymerizing DVB over a polystyrene core followed by a second polymerization with a vinyl surfactant and a surfactant/cholesterol-hybrid molecule as monomer and template, respectively. The submicrometer particles did bind cholesterol in a mixture of 2-propanol (60%) and water [134]. Also new is a technique for the orientated immobilization of templates on silica surfaces [ 135]. Molecular imprinting was performed in this case by generating a polymer covering the silica as well as templates. This step was followed by the dissolution of the silica support with hydrofluoric acid. Theophylline selective MIP were obtained. 

Whitcombe MJ, Rodriguez ME, Villar P, Vulfson EN. A new method for the introduction of recognition site functionality into polymers prepared by molecular imprinting synthesis and characterization of pol3mieric receptors for cholesterol. J Am Chem Soc 1995 117 7105-7111. 

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