Self-Assembly and Host-Guest Interactions of Pd3L2 Metallo-cryptophanes with Photoisomerizable Ligands.

New photoswitchable pyridyl-azo-phenyl-decorated tripodal host ligands (Laz) that belong to the cyclotriveratrylene family have been synthesized, and their photoswitching behavior and crystal structures determined. The latter includes a remarkable 7-fold Borromean-weave entanglement of π-π stacked layers. Trigonal bipyramidal {[Pd(en)]3(Laz)2}6+ metallo-cryptophanes (en = ethylenediamine) were formed from these and a previously known pyridyl-azo-phenyl-decorated tripodal host ligand. These coordination cages dissociate at low concentrations and are less robust to photoswitching of the Laz ligands than were previously reported Ir(III)-linked metallo-cryptophanes with similar ligands, reflecting the greater lability of the Pd-N bonds. The {[Pd(en)]3(Laz)2}6+ cages all act as hosts, binding octyl sulfate anions, or N-[2-(dimethylamino)ethyl]-1,8-naphthalimide in a dimethyl sulfoxide solution.

Synthesis and organogelating behaviour of amino acid-functionalised triphenylenes.

Four novel amino acid-functionalised triphenylenes have been prepared with glycine, l-alanine, l-phenylalanine and l-tryptophan ethyl ester side-chains. The glycine derivative is a good gelator of chloroform, the alanine derivative gels ethanol and toluene, and the phenylalanine derivative gels benzene and toluene. The tryptophan derivative does not gel any of the solvents tested, most probably due to its more bulky structure, but forms microspheres by evaporation-induced self-assembly. The self-assembly properties of the π-gelators have been investigated using infrared, UV-absorption and fluorescence spectroscopy, concentration- and temperature-dependent NMR, and X-ray scattering experiments on dried xerogel as well as the wet organogel. The latter experiments suggest the glycine gel in chloroform includes columnar aggregates, with an overall disordered columnar oblique mesophase. These compounds are of interest because of the well-known hole-transporting properties of triphenylene liquid crystals: 1-D columnar assemblies of these compounds may find applications in organic electronic devices.

Characterization of the Binding Properties of Molecularly Imprinted Polymers.

The defining characteristic of the binding sites of any particular molecularly imprinted material is heterogeneity: that is, they are not all identical. Nonetheless, it is useful to study their fundamental binding properties, and to obtain average properties. In particular, it has been instructive to compare the binding properties of imprinted and non-imprinted materials. This chapter begins by considering the origins of this site heterogeneity. Next, the properties of interest of imprinted binding sites are described in brief: affinity, selectivity, and kinetics. The binding/adsorption isotherm, the graph of concentration of analyte bound to a MIP versus concentration of free analyte at equilibrium, over a range of total concentrations, is described in some detail. Following this, the techniques for studying the imprinted sites are described (batch-binding assays, radioligand binding assays, zonal chromatography, frontal chromatography, calorimetry, and others). Thereafter, the parameters that influence affinity, selectivity and kinetics are discussed (solvent, modifiers of organic solvents, pH of aqueous solvents, temperature). Finally, mathematical attempts to fit the adsorption isotherms for imprinted materials, so as to obtain information about the range of binding affinities characterizing the imprinted sites, are summarized.

Encapsulation of sodium alkyl sulfates by the cyclotriveratrylene-based, [Pd(6)L(8)](12+) stella octangula cage.

We have previously described a cyclotriveratrylene (CTV)-based complex, [Pd6L8](12+), that forms a highly symmetric stella octangula cage. Here we report on the ability of this system to host sodium salts of three alkyl sulfates; octyl (SOS), dodecyl (SDS) and tetradecyl (STS). (1)H NMR chemical shift and diffusion coefficient measurements reveal that two molecules of alkyl sulfate reside in the cage, though rapid exchange between cage and bulk solvent is apparent. Host : guest association constants have been determined and support for the internalisation of the sulfates is available from 2D ROESY and NOESY data.

Imprinted polymers for chiral resolution of (±)-ephedrine, 4: packed column supercritical fluid chromatography using molecularly imprinted chiral stationary phases.

(-)-Ephedrine-molecularly imprinted polymers (MIPs) have been successfully used as stationary phases in supercritical fluid chromatography for the separation of (±)-ephedrine enantiomers. This approach combines the simple preparation and predictable elution order of MIP stationary phases with the superior mobile phase diffusivity and low viscosity of supercritical fluid mobile phases. The optimised mobile phase comprised supercritical carbon dioxide with a modifier consisting of MeOH/isopropylamine/H(2)O 93:5:2 (v/v/v). In many cases, better resolution separations were observed compared to when liquid mobile phases were used, and better separations achieved at high sample loads, although interestingly the MIPs which work best in SFC are different from the MIPs that work best in HPLC with an amine modifier. The MIP stationary phases were stable under the conditions employed and the chromatography was reproducible. This work opens the door to exploiting MIP stationary phases in preparative SFC.

N-isopropylacrylamide as a functional monomer for noncovalent molecular imprinting.

Although N-isopropylacrylamide (NIPAM) has previously been used in molecular imprinting, it has mostly been considered as an 'inert' monomer, or included for its temperature-responsive nature, rather than as a functional monomer responsible for the interactions with the template at the recognition site. A comparative study of NIPAM and other traditional, functional monomers for the imprinting of a hydrogen bond donor template, bisphenol A (BPA), is reported. Nuclear magnetic resonance titration data suggest that NIPAM forms a stronger complex with BPA than either acrylamide or methacrylic acid but a weaker complex than vinylpyridine. Molecular imprinted polymers (MIPs) were prepared using each functional monomer and compared as stationary phases for the separation of BPA from structural analogues. The NIPAM-containing MIP bound BPA with better selectivity than those prepared using acrylamide or methacrylic acid. Using NIPAM also reduces the nonspecific binding, which is found with MIPs using vinylpyridine as functional monomer.

A molecularly imprinted receptor for separation of testosterone and epitestosterone, based on a steroidal cross-linker.

A series of molecularly imprinted polymers have been prepared and investigated as stationary phases in high performance liquid chromatography for the separation of testosterone and epitestosterone using non-polar mobile phases. The polymers were imprinted using 5α-dihydrotestosterone as template, and all retain testosterone more strongly than its 17α-OH epimer. The best polymer was prepared using trifluoromethylacrylic acid as functional monomer (interacting with the template via hydrogen bonds), divinylbenzene as 'inert' cross-linker, and chloroform as porogen. It also included a steroid-based cross-linker, which may interact with the template via van der Waals interactions to lend additional 'shape selectivity'. A 250×4.6mm column packed with this polymer gave baseline resolution of testosterone and epitestosterone (15 µg each) in under 20 min. Preparation of the steroid based cross-linker included the selective reduction of 5α-dihydrotestosterone (17ß-hydroxy-5α-androstan-3-one) to the 3α,17ß-diol using K-selectride.

On the interactions of alkyl 2-hydroxycarboxylic acids with alkoxysilanes 2. Complexation and esterification of di- and tricarboxylic acids.

The selective esterifications of L-malic, L-tartaric and citric acids with tetramethoxysilane Si(OMe)4, in methanol (MeOH) have been demonstrated for the first time. The interactions between these acids and Si(OMe)4, and also between oxalic acid and Si(OMe)4, were investigated using 1H, 13C and 29Si solution phase NMR, and electrospray mass spectrometry (ES-MS). Si(OMe)4 acts as a catalyst/reagent in the selective esterification of simple 2-hydroxycarboxylic acids (2HOAs), but with the di- and tri-carboxylic acids more complex selectivities are observed: the esterification of oxalic acid proceeds slower than in MeOH alone, L-malic acid is selectively esterified approximately 1000 times faster at the 2-hydroxy acid, L-tartaric acid is esterified approximately 1000 times faster to the mono- and diester, while citric acid is selectively methylated at the terminal (3-hydroxycarboxyl) groups approximately 1000 times faster than in the absence of Si(OMe)4. Esterification is associated with the condensation of silane units to form oligomers. A mechanism is proposed in which 2HOAs attach to silicon via the alkoxy group and carboxyl groups to form various reactive cyclic intermediates. These intermediates may lead to accelerated esterification via nucleophilic attack of MeOH at the ligated carboxyl group, while a separate reaction pathway leads to condensation of silicon centres leading to oligosilanes. The mechanism has implications for the use of 2HOAs as templates in sol-gel silica preparation.

Fluorescent imprinted polymer sensors for chiral amines.

Molecularly imprinted polymer (MIP) based fluorescent sensors require suitable fluorescent moieties which respond to the binding event with significant fluorescence changes. Two novel polymerisable coumarins: 6-styrylcoumarin-4-carboxylic acid (SCC) and 6-vinylcoumarin-4-carboxylic acid (VCC) have been designed and synthesised. These functional monomers allow for the preparation of fluorescent sensors of chiral amines, an important class of pharmaceutical compounds. MIPs were prepared with SCC and VCC, using (-)-ephedrine as a template and ethylene glycol dimethacrylate as a cross-linker. In MeCN, the polymers exhibited a decrease of fluorescence in response to amines, with some selectivity for the template over its enantiomer (+)-ephedrine and other structural analogues. Interestingly the response of SCC to (-)-ephedrine in the MIP occurs in the opposite direction to the change when recognition occurs in solution. The control polymers (NIPs) exhibited a lesser response to (-)-ephedrine, and no resolving power, suggesting that imprinting has been successful and selective recognition sites exist in the MIPs. Recognition in aqueous buffers at different pHs has also been investigated.

Imprinted polymers for chiral resolution of (+/-)-ephedrine. Part 3: NMR predictions and HPLC results with alternative functional monomers.

The monomers trifluoromethacrylic acid (TFMAA), 2-hydroxyethylmethacrylate (HEMA) and itaconic acid (IA) have been compared for the molecular imprinting of (-)-ephedrine. Data from NMR titrations were fitted using the program HypNMR to obtain association constants for monomer-template (M-T) complexes of different stoichiometries. These were used to predict the speciation in imprinting mixtures with porogen and cross-linker, and molecularly imprinted polymers (MIPs) were fabricated and their ability to bind (-)-ephedrine and its enantiomer were assessed by high performance liquid chromatography (HPLC). TFMAA and IA interact more strongly with ephedrine than does MAA, yet MIPs made with each of these monomers perform worse. With TFMAA, covalent monomer-template adducts and TFMAA oligomers, present in the polymerisation mixture, may detract from the MIP recognition properties. With IA, the relative flexibility of the monomer may be an issue. HEMA interacts more weakly with ephedrine, and HEMA-based MIPs exhibit much worse retention, and poorer recognition, than those based on MAA. It may be useful to use a higher ratio of M : T in the case of HEMA because the monomer interacts with the cross-linker EDMA.

Electrophoretic field gradient focusing with on-column detection by fluorescence quenching.

Native, uncoloured, proteins can be focused in a column containing a fluorescent packing material, using hydrodynamic flow and a counteracting non-linear electric field, and imaged along the length of the channel by fluorescence quenching.

Imprinted polymers for chiral resolution of +/--ephedrine. Part 2: Probing pre-polymerisation equilibria in different solvents by NMR.

A sophisticated analysis of the pre-polymerisation association of monomer and template for the molecular imprinting of (-)-ephedrine (template) using methacrylic acid (monomer) has been performed using NMR titrations and data analysis with the computer program HypNMR. The fitted model takes account of the dimerisation of monomer, and 1 : 1, 2 : 1 and 3 : 1 monomer-template complexes. Values for the association constants and chemical shifts of the different nuclei in the various species are generated quickly and simply. The speciation has been compared in chloroform, toluene and acetonitrile, and in the absence and presence of cross-linking monomers. The 1 : 1 monomer-template interaction is very strong in all three solvents, such that 1 : 1 mixtures contain an almost stoichiometric amount of 1 : 1 complex. Imprinted polymers made with different monomer-template ratios have been used in the enantioseparation of (+/-)-ephedrine and confirm the utility of the 1 : 1 complex for the formation of effective recognition sites.

Electrophoretic field gradient focusing: an investigation of the experimental parameters.

Electrophoretic field gradient focusing has been used to separate the two oxidation states of myoglobin (Mb), and to separate Mb from bromophenol blue (BPB). Polyacrylamide and Sephadex were shown to be suitable packing materials whilst silica led to band broadening with Mb. BPB and Mb could be simultaneously focused apart using either a fixed 21-electrode setup or a dynamic 6-electrode setup. Using a dynamic three-electrode setup either analyte could be focused but not both simultaneously. It was shown that a higher ionic strength buffer in the separation channel compared to the coolant channel enhanced focusing between electrodes due to a conductivity gradient. Different running buffers were investigated and it was found that using a pH 8.6 buffer containing N,N,N-tris(hydroxymethyl)aminomethane (Tris) and phosphate ions the oxidation states of Mb could be separated but the separation of Mb from BPB was not as good as would be hoped for. Using a pH 8.6 buffer containing Tris, N-2-hydroxyethylpiperazine-N'-3-propanesulphonate and chloride ions as running buffer, BPB and Mb could be well separated but the two oxidation states of Mb merged.

On the interactions of alkyl 2-hydroxycarboxylic acids with alkoxysilanes: selective esterification of simple 2-hydroxycarboxylic acids.

The interactions of a range of monocarboxylic acids with tetramethoxysilane Si(OMe)(4) (TMOS), in methanol (MeOH), have been investigated by using (1)H, (13)C and (29)Si solution-phase NMR spectroscopy and electrospray mass spectrometry (ESMS). Si(OMe)(4) acts as a catalyst/reagent in the selective methylation of 2-hydroxycarboxylic acids (2HOAs) in MeOH at room temperature: glycolic acid, lactic acid and 2-hydroxybutyric acid are esterified more than a hundred times faster in MeOH and Si(OMe)(4) than in MeOH alone. No acceleration of methylation is observed for carboxylic acids lacking the 2-hydroxy group. Methylation of the 2HOAs is associated with the condensation of individual siloxane units to form oligomers. A mechanism is proposed in which 2HOAs attach to silicon via the alkoxy group, then subsequently via the carboxyl group in an intramolecular rearrangement to form an unstable and reactive cyclic intermediate. This intermediate may lead to accelerated methylation of the carboxylic acid via nucleophilic attack of MeOH at the carbonyl group, while a separate reaction pathway leads to condensation of silanols and/or alkoxysilanes leading to oligosiloxanes. The mechanism has implications for the use of 2HOAs as templates in sol-gel silica preparation.

A fluorescence polarisation molecular imprint sorbent assay for 2,4-D: a non-separation pseudo-immunoassay.

The first pseudo-immunoassay which employs a molecularly imprinted receptor and a fluorescent probe, and quantifies the bound analyte directly using the fluorescence anisotropy of the polymer-probe-analyte suspension, is described.

Use of fluorescence shift and fluorescence anisotropy to evaluate the re-binding of template to (S)-propranolol imprinted polymers.

The binding of (R)- and (S)-propranolol to an (S)-propranolol imprinted polymer in organic and aqueous solutions has been studied using fluorescence. The amount of propranolol that binds can be measured by separating non-bound propranolol from the polymer by centrifugation, and measuring the fluorescence intensity. However, this work demonstrates that other measurements can indicate how much propranolol has bound without the need to separate bound and non-bound analyte. In toluene + 0.5% AcOH, and in aqueous buffer (25 mM citrate pH 6 + 0.5% Triton X100) the fluorescence anisotropy increases as the fraction of analyte bound to the polymer increases. In aqueous buffer, binding to the polymer is also accompanied by a change in the relative intensities of fluorescence at 322 nm and at 352 nm. These non-separation techniques have been used to show that the imprinted polymer binds more (S)-propranolol than a non-imprinted polymer, and at least in organic solvent, shows selectivity for (S)- over (R)-propranolol.

Molecular imprinting science and technology: a survey of the literature for the years up to and including 2003.

Over 1450 references to original papers, reviews and monographs have herein been collected to document the development of molecular imprinting science and technology from the serendipitous discovery of Polyakov in 1931 to recent attempts to implement and understand the principles underlying the technique and its use in a range of application areas. In the presentation of the assembled references, a section presenting reviews and monographs covering the area is followed by papers dealing with fundamental aspects of molecular imprinting and the development of novel polymer formats. Thereafter, literature describing attempts to apply these polymeric materials to a range of application areas is presented.

Molecularly imprinted polymers for the enantioseparation of chiral drugs.

Molecularly imprinted polymers have been applied for many years in chiral separations and increasingly have been applied to drugs. Separation speed and efficiency in methods such as HPLC and CEC are increasingly competitive with alternative established CSPs. The literature on separation of drug enantiomers using MIPs via HPLC, TLC, SFC and CEC are reviewed. Issues for method development, particularly the monomer-template interactions during the imprinting process, and the mechanism of solute retention on MIP CSPs in chromatography using normal phase or reverse-phase mobile phases are considered.

Imprinted polymers for chiral resolution of (+/-)-ephedrine: understanding the pre-polymerisation equilibrium and the action of different mobile phase modifiers.

A thorough study has been made of the ephedrine-methacrylic acid (MAA) system for molecular imprinting, involving NMR studies of the pre-polymerisation equilibria, modelling and HPLC enantioseparations with different mobile phases. When dimerisation of MAA is accounted for, NMR titrations demonstrate there is a very strong ('stoichiometric', K approximately 10000 M(-1)) interaction between ephedrine and a single MAA monomer. Polymers prepared with a 1:1 monomer:template ratio are capable of enantioseparation, indicating, in combination with the NMR results, that the 1:1 interaction probably involves the carboxylic acid acting as a chelating monomer, forming hydrogen bonds to both the template amine and hydroxyl moieties. Higher monomer:template ratios cause further changes in the NMR signals, suggesting at least one further MAA can interact with the amine group, with a weaker association constant (K approximately 80 M(-1)). Polymers prepared with a 4:1 monomer:template ratio are thus proposed to contain a mixture of 1-monomer binding sites and 2-monomer binding sites, the latter being of enhanced acidity. In HPLC, better results are obtained with the 4:1 polymer using acetic acid as a modifier, while better results are obtained for the 1:1 polymer using butylamine as a modifier. We propose a model whereby acetic acid exerts its effect by reducing binding to the 1-monomer sites, while butylamine works largely through blocking the most acidic, 2-monomer sites. For preparative chromatography we suggest the 1:1 polymer, with a larger population of weaker but more uniform binding sites, is most promising.

Molecularly imprinted polymers in pseudoimmunoassay.

Immunoassays are a class of analytical techniques based on the selective affinity of a biological antibody for its antigen. Competitive binding assays, of which the radioimmunoassay (RIA) was the first example, are based on the competition between analyte and a labelled probe for a limited number of binding sites. Molecularly imprinted polymers (MIPs) have been shown to be suitable replacements for biological antibodies in such techniques. Molecularly imprinted sorbent assays (MIAs) similar to RIA have been developed for a range of analytes of clinical and environmental interest. Limits of detection and selectivities of such assays are often similar to those using biological antibodies. Some assays have been used for measurements directly in biological fluids. The field is reviewed and it is shown that some perceived disadvantages of MIPs do not hinder their application in competitive binding assays: many MIAs have been demonstrated in aqueous solvents, and it has been shown that the quantity of template required to prepare imprinted polymers can be drastically reduced, and that binding site heterogeneity is not a problem as long as the sites which bind the probe most strongly are selective. Finally, recent developments including assays in microtitre plates, the use of enzyme-labelled probes, flow-injection assays and a scintillation proximity MIA are discussed.