The use of polymerizable hosts in anion imprinting has led to powerful receptors with high oxyanion affinity and specificity in both aqueous and non-aqueous environments. As demonstrated in previous reports, a carefully tuned combination of orthogonally interacting binding groups, for example, positively charged and neutral hydrogen bonding monomers, allows receptors to be constructed for use in either organic or aqueous environments, in spite of the polymer being prepared in non-competitive solvent systems. We here report on a detailed experimental design of phenylphosphonic and benzoic acid-imprinted polymer libraries prepared using either urea-or thiourea-based host monomers in the presence or absence of cationic comonomers for charge-assisted anion recognition. A comparison of hydrophobic and hydrophilic crosslinking monomers allowed optimum conditions to be identified for oxyanion binding in non-aqueous, fully aqueous, or high-salt media. This showed that recognition improved with the water content for thiourea-based molecularly imprinted polymers (MIPs) based on hydrophobic EGDMA with an opposite behavior shown by the polymers prepared using the more hydrophilic crosslinker PETA. While the affinity of thiourea-based MIPs increased with the water content, the opposite was observed for the oxourea counterparts. Binding to the latter could however be enhanced by raising the pH or by the introduction of cationic amine-or Na+-complexing crown ether-based comonomers. Use of high-salt media as expected suppressed the amine-based charge assistance, whereas it enhanced the effect of the crown ether function. Use of the optimized receptors for removing the ubiquitous pesticide glyphosate from urine finally demonstrated their practical utility.

Molecularly imprinted polymers (MIPs) are artificial receptors with template tailored recognition sites complementary to the targets. The versatility of this molecular imprinting technique has been hampered by the lack of practical synthetic procedures to prepare highly selective MIP nanoparticles targeting phospholipids, which are challenging to be imprinted due to their amphiphilic structure. Here, a novel sedimentation-based solid phase imprinting strategy is introduced relying on polymerization in the presence of template-modified silica nanospheres (SNs). To demonstrate this concept, the sphingosine-1-phosphate receptor agonist fingolimod phosphate (FP) was coupled to SNs which were dispersed in the prepolymerization medium consisting of the fluorescent functional monomer 1,8-bis(N-vinylimidazol-N'-methyl)anthracene bromide and the crosslinking monomer ethyleneglycol dimethacrylate. High dilution polymerization of the dispersion under agitation followed by simple sedimentation-based separation of the SN template resulted in the isolation of surface imprinted fluorescent MIP nanoparticles (FMIP NPs) in a high yield (17 %). The FMIP NPs displayed fluorescence enhancement in response to the template with a high imprinting factor (IF=9) under the experimental conditions and good specificity, and could recognize FP in human serum with recoveries of 68-74 %. Moreover, the template-modified SNs could be recycled for reuse. Such molecular imprinting strategy opens a new approach to produce highly selective artificial receptors targeting phospholipids.

Glycosylation plays an important role in various pathological processes such as cancer. One key alteration in the glycosylation pattern correlated with cancer progression is an increased level as well as changes in the type of sialylation. Developing molecularly-imprinted polymers (MIPs) with high affinity for sialic acid able to distinguish different glycoforms such as sialic acid linkages is an important task which can help in early cancer diagnosis. Sialyllactose with alpha 2,6 ' vs. alpha 2,3 ' sialic acid linkage served as a model trisaccharide template. Boronate chemistry was employed in combination with a library of imidazolium-based monomers targeting the carboxylate group of sialic acid. The influence of counterions of the cationic monomers and template on their interactions was investigated by means of H-1 NMR titration studies. The highest affinities were afforded using a combination of Br- and Na+ counterions of the monomers and template, respectively. The boronate ester formation was confirmed by MS and H-1/B-11 NMR, indicating 1 : 2 stoichiometries between sialyllactoses and boronic acid monomer. Polymers were synthesized in the form of microparticles using boronate and imidazolium monomers. This combinatorial approach afforded MIPs selective for the sialic acid linkages and compatible with an aqueous environment. The molecular recognition properties with respect to saccharide templates and glycosylated targets were reported.

Protein histidine phosphorylation (pHis) is involved in molecular signaling networks in bacteria, fungi, plants, and higher eukaryotes including mammals and is implicated in human diseases such as cancer. Detailed investigations of the pHis modification are hampered due to its acid-labile nature and consequent lack of tools to study this post-translational modification (PTM). We here demonstrate three molecularly imprinted polymer (MIP)-based reagents, MIP1-MIP3, for enrichment of pHis peptides and subsequent characterization by chromatography and mass spectrometry (LC-MS). The combination of MIP1 and β-elimination provided some selectivity for improved detection of pHis peptides. MIP2 was amenable to larger pHis peptides, although with poor selectivity. Microsphere-based MIP3 exhibited improved selectivity and was amenable to enrichment and detection by LC-MS of pHis peptides in tryptic digests of protein mixtures. These MIP protocols do not involve any acidic solvents during sample preparation and enrichment, thus preserving the pHis modification. The presented proof-of-concept results will lead to new protocols for highly selective enrichment of labile protein phosphorylations using molecularly imprinted materials.

The design of hosts for either cations or anions is complicated due to the competition for binding by the host or guest counterions. Imprinting relying on self-assembly offers the possibility to stabilize the guest and its counterion in a favorable geometry. We here report on a comprehensive supramolecular approach to anion receptor design relying on concurrent recognition of both anion and cation. This was achieved by high order complex imprinting of the disodium salt of phenyl-phosphonic acid in combination with neutral urea and sodium ion selective 18-crown-6 monomers. The polymers displayed enhanced affinity for the template or inorganic phosphate or sulfate in competitive aqueous buffers, with affinity and selectivity increasing with increasing ionic strength. The presence of engineered sites for both ionic species dramatically increases the salt uptake in strongly competitive media such as brine.

Reversible protein phosphorylation on serine, threonine, and tyrosine residues is essential for fast, specific, and accurate signal transduction in cells. Up to now, the identification and quantification of phosphorylated amino acids, peptides, and proteins continue to be one of the significant challenges in contemporary bioanalytical research. In this paper, a series of surface grafted monoliths in the capillary format targeting phosphorylated serine has been prepared by first synthesizing a monolithic core substrate material based on trimethylolpropane trimethacrylate, onto which a thin surface-imprinted layer was established by oriented photografting of a variety of mono- and bis-imidazolium host monomers at subzero temperature, using six different continuous or pulsed UV light sources. The imprinted monolith capillaries were evaluated in a capillary liquid chromatographic system connected to a mass spectrometer in order to test the specific retention of phosphorylated peptides. Site-specific recognition selectivity and specificity for phosphorylated serine was demonstrated when separating amino acids and peptides, proving that the optimized materials could be used as novel trapping media in affinity-based phosphoproteomic analysis.

We report on the design and characterization of imprinted cationic host polymers for selective trapping of phosphoserine and phosphotyrosine peptides. A series of imidazolium host monomers were synthesized and characterized with respect to binding affinity and stoichiometry of interaction with salts of phenylphosphonic acid. The strongest binders were subsequently used for the preparation of imprinted polymers in the form of crushed monoliths, using Fmoc-phosphotyrosine-ethyl ester or Fmoc-phosphoserine-ethyl ester as templates in combination with a hydrophilic crosslinking monomer. The polymers were compared with respect to binding and its dependence on solvent, and whether charged or uncharged host monomers were used. The recipes were subsequently implemented in the capillary monolith format for evaluation by micro-liquid chromatography in both buffered and organic media. Results from both tested formats reveal that the cationic host polymers displayed enhanced recognition in polar and buffered media, in contrast to neutral urea-based hosts which showed best results in acetonitrile rich mobile phases.

Two templating approaches to produce imprinted phosphotyrosine capture beads with a controllable pore structure are reported and compared with respect to their ability to enrich phosphopeptides from a tryptic peptide mixture. The beads were prepared by the polymerization of urea-based host monomers and crosslinkers inside the pores of macroporous silica beads with both free and immobilized template. In the final step the silica was removed by fluoride etching resulting in mesoporous polymer replicas with narrow pore size distributions, pore diameters approximate to 10 nm and surface area > 260 m(2) g(-1). The beads displayed pronounced phosphotyrosine affinity and selectivity in binding tests using model peptides in acetonitrile rich solutions with a performance surpassing solution polymerized bulk imprinted materials. Tests of the beads for the enrichment of phosphopeptides from tryptic digests of twelve proteins revealed both pY/pS and pY/Y selectivity. This was reflected in a nearly 6-fold increase in the enrichment factor of a 23-mer pY-peptide and pY/pS normalized intensity ratios up to 1.5, when comparing the template mesoporous beads with the bulk materials.

Protein phosphorylation is a reversible post-translational modification (PTM)playing a central role in numerous biological events including disease pathogenesis.Thus, the analysis of phosphoproteome is crucial for understandingcellular regulation processes and can facilitate the development of new diagnosticand therapeutic tools.Phosphoproteins are typically analyzed using liquid chromatography coupledwith mass spectrometry (LC-MS) after proteolytic processing. However,phosphopeptides are notoriously difficult to analyze by LC-MS due their lowabundance and transient nature. This creates a need for effective enrichmenttools for phosphorylated proteins and peptides prior to mass spectrometryanalysis.The work presented in this thesis is focused on development and validationof methods and tools for enrichment of phosphopeptides with the use of molecularimprinting technology. In particular, the targeted PTMs include phosphorylationon tyrosine (pTyr) and histidine (pHis).The key recognition element employed in developed synthetic receptors was1,3-diaryl urea functional monomer FM1. This monomer is a potent hydrogenbond donor forming strong cyclic hydrogen bonds with oxyanions such asphosphates. The bias of the imprinted urea-based receptor towards differentphosphorylated residues can be programmed by selection of the template. Thus, the N, C-protected phosphotyrosine and phosphonotriazolylalaninewere used as templates to generate phosphotyrosine (pTyr MIP) and phosphohistidine(pHis MIP) selective molecularly imprinted polymers, respectively.The application of previously reported pTyr MIP for phosphoproteomicstudies was validated on complex biological samples of the mouse brain lysatedigest spiked with standard peptides and HeLa cells digested proteins. Furthermore,the pTyr MIP was developed in the format of microspherical porous beads characterized by uniformly sized and shaped particles with increasedsurface area and pore size as well as improved binding affinity and selectivityfor larger pTyr peptides (2-3 kDa). This opens the way to generation of capturematerials suitable for middle-down phosphoproteomics.In response to the lack of adequate tools and methods for enrichment of acid-labile phosphohistidine peptides a pHis MIP-based approach is proposed asa solution. The method involving selective dephosphorylation ofphosphoserine (pSer) peptide by alkali treatment of the sample, followed byextraction of base-stable pHis peptides with MIP was demonstrated on thesample of bovine serum albumin digest spiked with standard pSer and pHispeptides.The last part of this thesis is focused on improving the recognition ofphosphopeptides in aqueous media – the natural environment of biologicalsamples. Guided by the principles of supramolecular chemistry, novel cationichost monomers were introduced for binding phosphates by ionic hydrogenbonds. These were used to synthesize MIPs showing enhanced binding ofphosphopeptides in aqueous media.

Herein we explore phospholipid imprinting as a means to design receptors for complex glycolipids comprising the toxic lipopolysaccharide endotoxin. A series of polymerizable bis-imidazolium and urea hosts were evaluated as cationic and neutral hosts for phosphates and phosphonates, the latter used as mimics of the phospholipid head groups. The bis-imidazolium hosts interacted with the guests in a cooperative manner leading to the presence of tight and well defined 1:2 ternary complexes. Optimized monomer combinations were subsequently used for imprinting of phosphatidic acid as an endotoxin dummy template. Presence of the aforementioned ternary complexes during polymerization resulted in imprinting of lipid dimers - the latter believed to crudely mimic the endotoxin Lipid A motif. The polymers were characterized with respect to template rebinding, binding affinity, capacity and common structural properties, leading to the identification of polymers which were thereafter subjected to an industrially validated endotoxin removal test. Two of the polymers were capable of removing endotoxin down to levels well below the accepted threshold (0.005 EU/mg API) in pharmaceutical production.