Selective recognition of cyclic GMP using a fluorescence-based molecularly imprinted polymer

Guanosine 3′,5′-cyclic monophosphate (cGMP) plays a role as a second messenger in many different biological systems. Given the ubiquitous nature of cGMP, a simple method of detecting cGMP is of interest. To that end a fluorescent polymer with recognition sites for cGMP has been prepared. Its selectivity and sensitivity were investigated and a dose-dependant decrease in fluorescence of the polymer in the presence of cGMP was observed. In contrast, virtually no effect was detected upon application of the structurally similar molecules, guanosine 5′-monophosphate (GMP) and adenosine 3′,5′-cyclic monophosphate (cAMP), thus demonstrating the high selectivity of this polymer. The association constant for the binding of cGMP to the imprinted polymer was determined in order of 3 × 10 5 M -1. A fluorescent, molecularly imprinted polymer that selectively recognises cGMP may have a useful application as a fluorescent chemosensor for cGMP detection in biological samples.

Organic reactions in ionic liquids: N-alkylation of cyclic imides with alkyl halides promoted by potassium fluoride

An ionic liquid based on 1-butyl-3-methylimidazolium hexafluorophosphate is used as an efficient reusable reaction medium in the N-alkylation of cyclic imides with alkyl halides promoted by fluoride ion.

The cationic ring-opening polymerization of cyclic ethers

The kinetics and mechanisms of ring opening polymerization and copolymerizntion of different cyclic ethers were studied using mainly a cationic system of iinitiation. BF30Et2/ethanediol. The cyclic ethers reacted differently showing that ring strain and basicity are the main driving forces in cationic ring opening polymerizaion. In most cases it was found that the degree of polymerization is controlled kinetically via terminations with the counterion and the monomers, and that the contribution of each type of reaction to the overall termination differs markedly. The Gel permeation chromatography studies showed that the molecular weight distribution of the samples of polyoxetanes were bimodal. This was in accordance with previous work establishing that the cyclic tetramer is found in much higher proportions than any of the other cyclic oligomers. However the molecular weight distribution of the copolymers made from oxetane and THF or from oxetane and oxepane were shown to be unimodal. These observations could be explained by a change in the structure of the growing end involved in the cationic polymerization. In addition crown ethers like dibenzo-crown-6 and compounds such as veratrole are believed to stabilise the propagating end and promote the formation of living polymers from oxetane.

Studies of the cationic polymerisation of vinylic and energetic cyclic ether monomers

The cationic polymerisation of various monomers, including cyclic ethers bearing energetic nitrate ester (-ON02) groups, substituted styrenes and isobutylene has been investigated. The main reaction studied has been the ring-opening polymerisation of 3- (nitratomethyl)-3-methyl oxetane (NIMMO) using the alcohol/BF3.0Et2 binary initiator system. A series of di-, tri- and tetrafunctional telechelic polymers has been synthesised. In order to optimise the system, achieve controlled molecular weight polymers and understand the mechanism of polymerisation the effects of certain parameters on the molecular weight distribution, as determined by Size Exclusion Chromatography, have been examined. This shows the molecular weight achieved depends on a combination of factors including -OH concentration, addition rate of monomer and, most importantly, temperature. A lower temperature and OH concentration tends to produce higher molecular weight, whereas, slower addition rates of monomer, either have no significant effect or produce a lower molecular weight polymer. These factors were used to increase the formation of a cyclic oligomer, by a side reaction, and suggest, that the polymerisation of NIMMO is complicated with endbiting and back biting reactions, along with other transfer/termination processes. These observations appear to fit the model of an active-chain end mechanism. Another cyclic monomer, glycidyl nitrate (GLYN), has been polymerised by the activated monomer mechanism. Various other monomers have been used to end-cap the polymer chains to produce hydroxy ends which are expected to form more stable urethane links, than the glycidyl nitrate ends, when cured with isocyanates. A novel monomer, butadiene oxide dinitrate (BODN), has been prepared and its homopolymerisation and copolymerisation with GL YN studied. In concurrent work the carbocationic polymerisations of isobutylene or substituted styrenes have been studied. Materials with narrow molecular weight distributions have been prepared using the diphenyl phosphate/BCl3 initiator. These systems and monomers are expected to be used in the synthesis of thermoplastic elastomers.

The ring opening polymerization of ring strained cyclic ethers

The kinetics and mechanisms of the ring-opening polymerization of oxetane were studied using cationic and coordinated anionic catalysts. The cationic initiators used were BF30Et2!/ethanol, BF30Et2!/ethanediol and BF30Et2/propantriol. Kinetic determinations with the BF30Et2/diol system indicated that a 1: 1 BF3:0H ratio gave the maximum rate of polymerization and this ratio was employed to detenmne the overall rates of polymerization. An overall second-order dependence was obtained when the system involved ethanediol or propantriol as co-catalyst and a 3/2-order dependence with ethanol, in each case the monomer gave a first-order relationship. This suggested that two mechanisms accounted for the cationic polymerization. These mechanisms were investigated and further evidence for these was obtained from the study of the complex formation of BF30Et2 and the co-catalysts by 1H NMR. Molecular weight studies (using size-exclusion chromatography) indicated that the hydroxyl ion acted as a chain transfer reagent when the [OH] > [BF3]. A linear relationship was observed when the number average molecular weight was plotted against [oxetane] at constant [BF3:0H], and similarly a linear dependency was observed on the BF3:0H 1:1 adduct at constant oxetane concentration. Copolymerization of oxetane and THF was carried out using BF30Et2/ethanol system. The reactivity ratios were calculated as rOXT = 1.2 ± 0.30 and rTHF = 0.14 ± 0.03. These copolymers were random copolymers with no evidence of oligomer formation. The coordinated anionic catalyst, porphinato-aluminium chloride [(TPP)AICl], was used to produce a living polymerization of oxetane. An overall third-order kinetics was obtained, with a second-order with respect to the [(TPP)AICl] and a first-order with respect to the [oxetane] and a mechanism was postulated using these results. The stereochemistry of [(TPP)AlCl] catalyst was investigated using cyclohexene and cyclopentene oxide monomers, using extensive 1H NMR, 2-D COSY and decoupling NMR techniques it was concluded that [(TPP)AlCl] gave rise to stereoregular polymers.

Hydrogels containing linear and cyclic polyethers

Hydrogels are a unique class of polymer which swell, but do not dissolve in, water. A range of 2-hydroxyethyl methacrylate based copolymer hydrogels containing both cyclic and linear polyethers have been synthesised and are described in this thesis. Initially, cyclic polyethers were occluded within the polymer matrix and the transport properties investigated. The results indicated that the presence of an ionophore can be used to modulate ion transport and that ion transport is described by a dual-sorption mechanism. However, these studies were limited due to ionophore loss during hydration. Hence, the synthesis of a range of acrylate based crown ether monomers was considered. A pure sample of 4-acryolylaminobenzo-15-crown-5 was obtained and a terpolymer containing this monomer was prepared. Transport studies illustrated that the presence of a `bound' ionophore modulates ion transport in a similar way to the occluded systems. The transport properties of a series of terpolymers containing linear polyethers were then investigated. The results indicated that the dual-sorption mechanism is observed for these systems with group II metal cations while the transport of group I metal cations, with the exception of sodium, is enhanced. Finally, the equilibrium water contents (EWC) surface and mechanical properties of these terpolymers containing linear polyethers were examined. Although subtle variations in EWC are observed as the structure of the polyether side chain varies, generally EWC is enhanced due to the hydrophilicity of the polyether side chain. The macroscopic surface properties were investigated using a sessile drop technique and FTIR spectroscopy. At a molecular level surface properties were probed using an in vitro ocular spoilation model and preliminary cell adhesion studies. The results indicate that the polyethylene oxide side chains are expressed at the polymer surface thus reducing the adhesion of biological species.

Domain and nucleotide dependence of the interaction between Saccharomyces cerevisiae translation elongation factors 3 and 1A

Eukaryotic translation elongation factor 3 (eEF3) is a fungal-specific ATPase proposed to catalyze the release of deacylated-tRNA from the ribosomal E-site. In addition, it has been shown to interact with the aminoacyl-tRNA binding GTPase elongation factor 1A (eEF1A), perhaps linking the E and A sites. Domain mapping demonstrates that amino acids 775-980 contain the eEF1A binding sites. Domain III of eEF1A, which is also involved in actin-related functions, is the site of eEF3 binding. The binding of eEF3 to eEF1A is enhanced by ADP, indicating the interaction is favored post-ATP hydrolysis but is not dependent on the eEF1A-bound nucleotide. A temperature-sensitive P915L mutant in the eEF1A binding site of eEF3 has reduced ATPase activity and affinity for eEF1A. These results support the model that upon ATP hydrolysis, eEF3 interacts with eEF1A to help catalyze the delivery of aminoacyl-tRNA at the A-site of the ribosome. The dynamics of when eEF3 interacts with eEF1A may be part of the signal for transition of the post to pre-translocational ribosomal state in yeast.

Second messenger pathways in the action of cachectic factors

Cachexia in cancer is characterised by progressive depletion of both adipose tissue stores and skeletal muscle mass. Two catabolic factors produced by cachexia-inducing tumours have the potential for inducing these changes in body composition: (i) proteolysis-inducing factor (PIF) which acts on skeletal muscle to induce both protein degradation and inhibit protein synthesis, (ii) lipid-mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. Administration of lipid-mobilising factor (LMF) to mice produced a specific reduction in carcass lipid with a tendency to increase non-fat carcass mass. Treatment of murine myoblasts, myotubes and tumour cells with tumour-produced LMF, caused concentration dependent stimulation of protein synthesis, within a 24hr period. It produced an increase in intracellular cyclic AMP levels, which was linearly related to the increase in protein synthesis. The observed effect was attenuated by pretreating cells with the adenylate cyclase inhibitor, MDL12330A and was additive with stimulation produced by forskolin. Both propranolol and a specific 3 adrenergic antagonist SR59230A, significantly reduced the stimulation of protein synthesis induced by LMF. LMF also affected protein degradation in vitro, as demonstrated by a reduction in proteasome activity, a key component of the ubiquitin-dependent proteolytic pathway. These effects were opposite to those produced by PIF which caused both a decrease in the rate of protein synthesis and an elevation on protein breakdown when incubated in vitro.Incubation of LMF with a fat cell line produced alterations in the levels of guanine-nucleotide binding proteins (G proteins). This was also evident in adipocyte plasma membranes isolated from mice bearing the tumour model of cachexia, MAC16 adenocarcinoma and from patients with cancer cachexia. Progression through the cachectic state induced an upregulation of stimulatory G proteins paralleled with a downregulation of inhibitory G proteins. These changes would contribute to the increased lipid mobilisation seen in cancer cachexia.

Cyclic and acyclic modifications of 5-aminoimidazole-4-carboxamide

The Introduction gives a brief resume' of the biologically important aspects of 5 -aminoimidazole -4 -carbozamide (1) and explores., in-depth, the synthetic routes to this imidazole. All documented reactions of 5 -aninoimidanole-4 -carboxamide are reviewed in detail, with particular emphasis on the preparation and subsequent coupling reactions of 5 –diazo-imidazole-4 -carboxamide (6). A series of thirteen novel amide 5-amino-2-arylazoimidazole-4-carboxamide derivatives (117-129) were prepared by the coupling of aryldiazonium salts with 5-aminoimidazole-4-carboxamide. Chemical modification of these azo-dyes resulted in the preparation of eight previously unknown acyl derivatives (136-143) Interaction of 5-amino-2-arylazoimidazole-4-carboxides with ethyl formate in sodium ethoxide effected pyrimidine ring closure to the novel 8-arylazohypoxanthines (144 and 145). Several reductive techniques were employed in an effort to obtain the elusive 2,5-diaminoimidazole-4-carboxamide (71),a candidate chemotherapeutic agent, from the arylazoiridazoles. No success can be reported although 5-amino-2-(3-aminoindazol-2-yl) imidazole-4-carboxamide (151) was isolated due to a partial reduction and intramolecular cyclisation of 5-amino72-(2-cyanaphenylazo)imidazole-4-carboxamide (122) .Further possible synthetic approaches to the diaminoimidazole are discussed in Chapter 4. An interesting degradation of a known unstable nitrohydrazone is described in Chapter 5.This resulted in formation of 1, 1-bis(pyrazol--3-ylazo)-1-nitroethane (164) instead of the expected cyclisation to a bicyclic tetrazine N-oxide. An improved preparation of 5-diazoinidazole-4-carboxamide has been achieved, and the diazo-azole formed cycloadducts with isocyanates to yield the hitherto unknown imidazo[5,1-d][1,2,3,5]tetrazin-7(6H)-ones. Eleven derivatives (167-177) of this new ring-system were prepared and characterised. Chemical and spectroscopic investigation showed this ring-system to be unstable under certain conditions, and a comparative study of stability within the group has been made. "Retro-cycloaddition" under protic and photolytic conditions was an unexpected property of 6-substituted imidazo[5,1-d][1,2,3,5]tetrazin--7(0)-ones.Selected examples of the imidazotetrazinone ring-system were tested for antitumour activity. The results of biological evaluation are given in Chapter 7, and have culminated in a Patent application by the collaborating body, May and Baker Ltd. One compound,3-carbamoyl-6-(2-chloro-ethyl)imidazo[5,1-d][1,2,3,5jtetrazin-7(6H)-one (175),shows striking anti-tumour activity in rodent test systems.