SYNTHETIC LIPIDS FOR THE STUDY OF MEMBRANE DYNAMICS

Abstract

This thesis describes the production of lipids and sterols which can be used as tools to promote or probe membrane asymmetry, both transverse and lateral.

The first section of this work will detail efforts to synthesise the molecular recognition lipids, BAR and TAP lipid for incorporation into liposomes, in order to produce transversely asymmetric vesicles. The synthesis of TAP lipid proceeded as expected, whilst the production of BAR lipid proved challenging. Efforts to produce BAR lipid by conventional means were met with frustration, low conversion was observed due to residual H2O promoting by-product formation. BAR lipid was resolved chromatographically, but low conversion led to alternative strategies being explored which ultimately proved unsuccessful. An alternative strategy involving a one pot Knoevenagel condensation proved promising on a test substrate, but the aldehyde precursor required for BAR lipid formation was not producible.

Development of a methodology to synthesise dialkyl phosphates and lipids on a solid support is reported. The methodology employs the use of a β-hydroxysulfone linker, which was produced and quantitatively bound to Merrifield resin. The β-hydroxysulfone linker proved successful for the attachment of substrates via the phosphate. In practice the use of a β-hydroxythioether linker, with the desired transformations taking place, followed by on resin oxidation to the sulfone in a penultimate step produced the desired linker in a ‘safety catch’ approach. Development of linker binding, phosphitylation and oxidation through both solid phase and solution phase mimic studies are described. The developed methodology was implemented to synthesise didecyl phosphate in high purity as a proof of concept. The production of this symmetric didecyl phosphate is envisaged to be the first step in producing a solid phase methodology for the production of glycerophospholipids.

Syntheses for the production of heavy oxygen labelled cholesterol, 6-ketocholestanol, 25-hydroxycholesterol, 5α-cholestanol and lanosterol are reported. The compounds were obtained through two routes using 17O and 18O-water as the enrichment source. Sterols were enriched by the hydration of 3,5-cyclosterols and by one pot enrichment and reduction of ketone precursors. Use of expensive labelled H2O was minimised, by optimisation of the reaction conditions, with 5 and 10 equivalents for the 3,5-cyclosterol and direct enrichment routes respectively. Enrichments of 28-38% and 79-94% were obtained for enrichment by 17O and 18O respectively, representing > 77% of the maximum theoretical enrichment. Enrichments were verified by both 13C NMR and GC-MS. In most cases GC-MS and NMR data correlated well, the exception being 6-ketocholestanol, wherein a discrepancy was observed with lower enrichments reported by NMR. A unique keto-enol tautomerism leading to exchange of the label through a 3,5-cyclosterol intermediate was observed in CDCl3, diminishing enrichment. This tautomerism was not observed for samples in DCM by GC-MS.