Steroid-imprinted polymers

Comparable results were presented when the number of Mi-polymers was extended to other templates of similar steroid libraries. The use of a polymer imprinted with 11-deoxycortisol in a separation of 11-a-hydroxyprogesterone, progesterone and 11-deoxycortisol resulted in a chromatogram where the 11-de-oxycortisol eluted last (Fig. 17, bottom). When an 11-a-hydroxyprogesterone imprinted polymer was used, on the other hand, the 11 -a-hydroxyprogesterone was found to elute last (Fig. 17, top) [80]. 

A few years later, in 1993, Akermark et al. reported the preparation of imprinted polymers capable of reducing selectively a 3-17-steroidal diketone (21) to the corresponding alcohol with high control of the stereochemistry [13]. The polymers were prepared attaching a polymerisable unit via an ester linkage to the desired steroidal product in position 17 (Fig. 3). The resulting compound was then used as a... 

Ramstrom, O., Ye, L., Krook, M., Mosbach, K. Screening of a combinatorial steroid library using molecularly imprinted polymers Anal. Comm., 1998, 35, 9-11... 

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. 

Sreenivasan, K. Effect of the type of monomers of molecularly imprinted polymers on the interaction with steroids. J. Appl. Polym. Sci. 1998, 68, 1863-1866. 

Molecularly imprinted polymers in conjunction with QCM have been used in the rapid detection of hormones. Percival et al. [36] has modified an QCM by spin coating a covalently imprinted polymer for the detection of the anabolic steroid, nandrolone. The 4-vinyl phenol carbonate ester of nandrolone (Fig. 8) was copolymerized with ethylenegylcol dimethacrylate and methacrylic acid. 

Examples of this technique are described for artificial receptors for the alkaloid yohimbine binding peptides obtained from a phage display library [57], for the steroid libraries related to lla-hydroxyprogesterone [58], corticosterone [58] (reported in Fig. 12), and cortisol [59]. A molecularly imprinted polymer working as a synthetic receptor for a series of chiral benzodiazepines [47], artificial receptors for the tricyclic antidepressant drug nortriptyline—obtained by covalent and noncovalent molecular imprinting and studied by capillary liquid chromatography with a simulated combinatorial library [60,61]—were also examined. 

Figure 12 Screening of a steroid library, (a) An imprinted polymer prepared for lla-hydro-xyprogesterone. Mobile phase dichloromethane (DCM) - 0.1% acetic acid v/v, Flux 0.5mL/min. (b) An imprinted polymer prepared for lla-hydroxyprogesterone. Gradient elution, 0-25 min, DCM 0.1% acetic acid v/v 25-30 min, DCM 0.1-5% acetic acid v/v 30-40 min, DCM 5% acetic acid v/v 40-45 min, DCM 5-0.1% acetic acid v/v. Flux 0.5mL/min. (c) A control polymer prepared in the absence of template molecule. Isocratic elution, DCM 0.1% acetic acid v/v. Flux 0.5mL/min. Sample component. (1) lla-hydroxyprogesterone, (2) lla-hydroxyprogesterone, (3) 17a-hydroxyprogesterone, (4) progesterone, (5) 4-androsten-3,17-dione, (6) l,4-androstadiene-3,17-dione, (7) corticosterone, (8) cortexone, (9) 11-deoxy-cortisol, (10) cortisone, (11) cortisone-21-acetate, (12) cortisol-21-acetate. Reproduced from Ref. 58, with permission.

A fluorescence sensing method based on a combination of HPLC separation and fluorescence detection was developed for the steroid hormone p-estradiol (Fig. 2, 5), which is fluorescent [22]. The imprinted polymer in this system was prepared by the co-polymerization of MAA and EDMA in the presence of the template molecule (Table 1), p-estradiol. This system was able to rapidly and effectively measure p-estradiol concentrations in the range of 0.1-4.0 pM with satisfactory reproducibility. However, this in essence is a HPLC method with the imprinted polymers used as the stationary phase. 

Four-element arrays, with each element consisting of three Pt electrodes (one modified 30 pm x 30 pm square working, one counter and one Pt pseudoreference), have been fabricated on glass substrates for the detection of the anabolic steroid albuterol, which is a doping concern in athletics and has been used as a food supplement for farm animals [88]. The arrays are based on working electrode modification with a molecularly imprinted polymer that is selective for albuterol over clenbuterol and terbutaline, two closely related compounds. The arrays are reusable (at least 20 times) and linear responses, as differential pulse voltammetric peak currents, for the oxidation of phenolic hydroxyl groups at about +0.45 V, occur over the 1-50 pM concentration range of albuterol. The molecularly imprinted polymer electrode films allow discrimination over similarly functionalized interferants. 

Fig. 16. Screening of a steroid library using (top) MIP prepared against 11-a-hydroxyproges-terone (1), gradient elution using (bottom) non-imprinted control polymer, isocratic elution. Reprinted with permission from Ramstrom O, Ye L, Krook M, Mosbach K (1998) Anal Com-mun 35 9. Copyright 1998 The Royal Society of Chemistry...

In some very remarkable experiments, Bystrom et al. [77] were recently able to demonstrate high regio- and stereoselectivity in reactions inside the imprinted cavity. The steroid 12 was copolymerized as the template monomer, and removed by reduction. The hydroxyl group in the polymer newly formed from the carboxyl group was converted into an active hydride by LiAlH4. With the help of this polymer, androstan-3,I7-dione was reduced to the alcohol exclusively in position 17, whereas in solution or with a polymer with statistically distributed hydride groups it is reduced exclusively in position 3. 

Figure 3.4.1 Model of the binding of the steroidal diketone B to the cavity in a cross-linked polymer formed by the imprint molecule A.

The molecularly imprinted copolymer of 2-hydroxyethyl methacrylate (HEMA) and p-CyD-coupled HEMA was synthesized in chloroform to study its interaction with a pair of steroids cholesterol and testosterone [42]. The molecularly imprinted copolymer was found to absorb the print molecule several times better than an imprinted poly HEMA. Asanuma et al. [44] concluded that cholesterol binding by the HEMA polymer was not due to CyD inclusion because no inclusion of CyD to cholesterol can form in chloroform. Another strategy developed for imprinting using CyD elements is the vinyl CyD (Table 1 and 10.3(A)), which has been successfully applied for imprinting antibodies, oligo-peptides [45], and vancomycin [46[. 

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