Silica particles containing surface-bound free radical initiators have been used as supports for the grafting of thin films of molecularly imprinted polymers (MIPs). This technique offers a means of fine-tuning the layer thickness for improved kinetic properties or enhanced capacity in chromatographic or sensor applications. Thus prepared MIPs imprinted with L-phenylalanine anilide, have been characterized by FT-IR spectroscopy, thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), elemental analysis, fluorescence microscopy, and scanning electron microscopy (SEM), providing evidence concerning the reproducibility in each step and the quantity and quality of the grafted films. The chromatographic properties of the materials have been investigated with respect to the average layer thickness of the polymer on the surface, the solvent, the support pore diameter, the cross-linker concentration, and the composition of the mobile phase. For the porous particles, the column efficiency depended strongly on the amount of grafted polymer. Thus, polymers grafted as thin films (ca. 0.8 nm as average film thickness) on silica with 10 nm average pore diameter showed the highest column efficiency giving plate numbers (N) for the imprinted enantiomer of ca. 700 m -1 and for the antipode ca. 24 000 m -1 . This resulted in baseline resolutions on a 33 mm long column in less than 5min. On the other hand the sample load capacity and separation factor increased up to 7.0 nm layer thickness and dropped again for larger amounts of grafted polymer. A support with an average pore diameter of 100 nm and a 3.8 nm layer thickness showed a far higher saturation capacity than values previously determined for the conventional monolithic materials.

Hyperphosphorylation at tyrosine is commonly observed in tumor proteomes and, hence, specific phosphoproteins or phosphopeptides could serve as markers useful for cancer diagnostics and therapeutics. The analysis of such targets is, however, a challenging task, because of their commonly low abundance and the lack of robust and effective preconcentration techniques. As a robust alternative to the commonly used immunoaffinity techniques that rely on phosphotyrosine(pTyr)-specific antibodies, we have developed an epitope-imprinting strategy that leads to a synthetic pTyr-selective imprinted polymer receptor. The binding site incorporates two monourea ligands placed by preorganization around a pTyr dianion template. The tight binding site displayed good binding affinities for the pTyr template, in the range of that observed for corresponding antibodies, and a clear preference for pTyr over phosphoserine (pSer). In further analogy to the antibodies, the imprinted polymer was capable of capturing short tyrosine phosphorylated peptides in the presence of an excess of their non-phosphorylated counterparts or peptides phosphorylated at serine.

A series of urea-based vinyl monomers was synthesized and investigated for their ability to function as polymerizable hosts for the molecular imprinting of N-Z-D- or L-glutamic acid in polar media (DMSO or DMF). The monomers were synthesized in one step from a polymerizable isocyanate and a nonpolymerizable amine or vice versa, with yields typically over 70%. Prior to polymerization their solution binding properties vis-a-vis tetrabutylammonium benzoate in DMSO were investigated by 1H NMR, UV-vis and fluorescence monitored titrations. The affinities of the urea monomers for benzoate depended upon the substitution pattern of the urea, with all diaryl ureas exhibiting high affinity. EDMA-based imprinted polymers prepared in DMF or DMSO against Z-D-(or L)-glutamic acid using 2 equiv of the urea monomer and 2 equiv of base were able to recognize the imprinted dianion as well as larger molecules containing the glutamic acid substructure. The affinity, reflected in liquid chromatography retention data, correlated with the solution binding properties of the corresponding monomers.

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Wide pore silica (D P ~100 nm) and gel-type or macroporous (12% nominal crosslinking density) Merrifield resins were modified with iniferter groups for grafting of crosslinked molecularly imprinted or non-imprinted polymer layers through quasi-living polymerisation. Prior to iniferter coupling, the silica supports were premodified by silanisation with p-(chloromethyl)phenyl trimethoxysilane. The iniferter groups were then introduced by reacting the resin-bound chloromethyl groups with sodium N,N-diethyldithiocarbamate. It was shown that the coupling yield, measured as the conversion of the chloromethyl groups, could be varied between 5 and 85% through kinetic control, with the fastest conversions observed for the macroporous resins. This allows the density of radical generating groups to be finely adjusted. Ultraviolet light-initiated copolymerisations of ethyleneglycol dimethacrylate and methacrylic acid in toluene resulted in grafting of 0.2-1.9 g of polymer per gram of support, where the grafted amount increased with reaction time, iniferter content and monomer concentration. The dry-state texture of the composite beads prepared from the gel-type resin depended strongly on the amount of grafted polymer. According to the scanning electron micrographs, the beads with the lower grafted amounts (0.4 g polymer per g support) were deformed exhibiting a peculiar folded structure, whereas the beads containing more grafted polymer (1.6 g polymer per g support) were spherical, with an appearance similar to the precursor particles. None of these materials exhibited permanent porosity. Only the composites obtained from the porous precursor particles also exhibited porosity after grafting. Among these, the silica-based composites also showed recognition for their templates when assessed in the chromatographic mode, whereas no imprinting effects could be demonstrated for the polystyrene-supported materials.

Two approaches to synthesize molecularly imprinted polymers with affinity for folic acid and other substituted pteridines have been compared. In the first approach, the folic acid analogue methotrexate was used as template and functional monomers capable of generating selective binding sites were searched in a miniaturized screening system based on binding assessment in the batch mode. Highest selectivity was seen using 2-vinylpyridine as functional monomer, which was confirmed in the chromatographic mode for a batch synthesized on a gram scale. However, the retentivity and selectivity of this phase were insufficient for anticipated applications. In a second approach, using methacrylic acid as the functional monomer, organic soluble inhibitors for the enzyme dihydrofolate reductase were used to develop sites complementary toward the pteridine substructure. This resulted in materials showing enhanced selectivity for substituted pteridines when evaluated by HPLC. Thus, methotrexate and leucovorine were selectively retained in mobile phases of either low or high aqueous content, thus showing the typical bimodal retention behavior of previously reported MIPs. In organic mobile-phase systems, the inhibitor used as template had an influence on the retentivity and selectivity of the MIP. The polymer imprinted with trimethoprim retained all folic acid analogues strongly and showed the highest selectivity among the MIPs in an organic mobile-phase system. This was supported by Scatchard analysis resulting in biphasic plots and a quantitative yield of high-energy binding sites. All templates were shown to associate strongly with MAA in CDCl(3), the strength of association correlating roughly with the template basicity and the selectivity observed in chromatography. Nonparallel complexation-induced shifts indicated formation of 1:2 template monomer complexes at concentrations corresponding to those of the prepolymerization solutions.

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The crude products resulting from solid-phase peptide synthesis can be used as epitope templates to generate surface-confined sites for the template and larger peptides containing the template motif. This offers a facile route to robust affinity stationary phases for the chromatographic separation of peptides.