PhD Studentship Vacancy: CATION CHANNEL CONTROL BY COMPLEX POLYAMMONIUM IONS
Objectives: This proposed study is an investigation of the basicity of amine residues found in biologically important complex polyammonium ions. We aim to determine how basic (or even acidic) an amine or its conjugate ammonium ion can be under physiological conditions with respect to cation channel modulation. Therefore, we are continuing our studies with compounds containing spermine or an isomeric tetraamine e.g. thermospermine. This is a newly established and profitable line of research for us, determining by potentiometric titration and/or NMR spectroscopy, together with deconvolution, how polyammonium ions are protonated under physiological conditions. This is complex as these poly-cation species are cooperative. This is both a physiologically important project for control of excitable cells and their signalling, and an intellectually demanding one. Therefore, this fundamental research might lead to more applied studies with respect to cell-signalling research [1].
Background: Polyamines have recently been shown to have fundamental importance in cell-cell signalling. They mediate by both potentiation (at pM concentrations) and then channel block (at nM concentrations) the movement of key cations, e.g. sodium, potassium, calcium across the membranes of excitable cells. These channels are typically associated with Glu-receptors and voltage gated K+ channels) [1]. In addition to these important roles in cellular communication, polyamines are intimately involved in stabilisation of chromatin and in cell growth and division [1]. It is now widely recognised that the detailed molecular mechanisms of channel modulation and ultimately channel block are not well understood. We will therefore design and synthesise molecular tools, small molecule probes that will assist in determining the molecular basis of cation channel block in cell signalling [1].
The pKa’s along an unsymmetrical polyamine are hard to predict, indeed the literature is often wrong on this point, and therefore there are analytical and physicochemical elements to the project which are potentially suitable for publication. The project is a suitable training ground for a PhD student studying the molecular aspects of cooperative complex multi-charged species. In recent preliminary studies, we have prepared pilot quantities of unsymmetrical polyamines and related polyamine amides [2, 3]. We are sure that we can scale up the project at the beginning, therefore the student can begin confidently with a small piece of research work that will be a success. Synthetic polyamines that are unsymmetrically substituted are a synthetic challenge. Therefore, there is also a novel chemical element to this project.
Methods and design/techniques and approaches: Polyamines and their conjugates have many modes of binding to protein receptors and to polynucleic acids. We will investigate, by physicochemical and spectroscopic methods, the pKa’s of polyamines which are biologically important. Secondary amines, unlike primary amines, are not necessarily protonated under physiological conditions. It is a current problem in biological chemistry to predict how cooperative charges will be distributed along unsymmetrical polyamines. We have made a preliminary assessment of the pKa’s of certain polyamines and their conjugates and considered their many protonation states across a physiological pH range [4].
Experimental measurements: The pKa values for spermine are 11.50, 10.95, 9.79 and 8.90. The student will prepare and then measure the pKa’s of spermine, its regioisomer thermospermine and polyamine containing wasp and spider toxins (argiotoxins) which block glutamate-gated cation channels. Synthetic medicinal chemistry will be used to prepare gram quantities of these toxins and their analogues. Polyamine pKa’s are co-operative. Therefore, the student will find it a challenge to deconvolute the pKa’s and to correlate their predicted (calculated) values with those obtained experimentally by potentiometric titration and by NMR spectroscopy. These values are important as they will dictate the access of polyamines to cells via the polyamine transporter as well as through cation channels. These results are therefore of direct relevance to the selection of glutamate receptor ion channels as targets in potential therapy for neurodegeneration especially stroke and epilepsy.
Suitability and challenge/questions which will be addressed: The student will find an intellectual challenge in rationalising the progression of co-operative pKa’s along a tri-, tetra- or pentaamine as a function of changes in pH. As well as the training in general synthetic methods which this medicinal chemistry project provides, the student will be able to think about how charges are distributed across small molecules and how the reversibility of those charges is relevant to biological transport and channel block. The interplay with both biochemical and physiological control will be particularly important and the student will have to consider the problems of ionic strength and physiological buffers in determining detailed NMR spectra. A successful outcome can be measured by an accurate predictor of charge distribution and by a working model of channel blockade using polyamines. The simple belief that charges are localised (even to heteroatoms) will be challenged by this project which will form an interdisciplinary training in aspects of ion channel control.
In year one, we will use our recently published protocol [2, 3] for the efficient homologation of symmetrical polyamines (reductive alkylation of a suitably protected aldehyde) to prepare homologated, unsymmetrical spermine analogues. The significance of a stepwise increase in the cationic charge will then be determined [4]. The analysis will be by NMR spectroscopic studies using physiological conditions (pH and ionic strength) in the NMR assay (years 1-2).
In summary, our strategy for the preparation of these molecular probes, tools designed to be useful for studying cooperative charge distribution along polyammonium ions, is to introduce the student to this area of current research with an efficient synthesis of thermospermine (year 1), following from our preliminary studies [2, 3]. Then, across years 1-2, we will prepare our novel unsymmetrical polyamines (3.4.3.x) by reductive alkylation with chemical control of the distances between the nitrogen atoms [2, 3]. This is biologically important and will certainly modulate the pKa’s, and therefore the biological activity. We will investigate positive charge distribution along the polymethylene backbone in order to determine binding [4], and move to related, complex natural product polyamine targets in year 3. These important factors require the detailed attention of a medicinal chemist and this project will form an ideal training for a PhD student in chemistry with emphasis on cooperative complex polyammonium ions as ion channel blockers, as well as the novel chemical aspects of controlled polyamines synthesis.
Send your application with the names and addresses of two referees to Dr. Ian S. Blagbrough to arrive by June 30th, 1999.
Further information about current research interests and recent output can be found within ISB's personal web-pages