Radioactive Photoreactive Fatty Acid Analogues: Synthesis, Biological Utilization and Tools for the Study of Fatty Acid Transport

Abstract

<p>The present study focused on the development, synthesis and investigation of the biological activity of a radioactive photoreactive homologous series of fatty acid analogues The carbene precursor m-diazirinophenol was chosen because of its known photolytic properties. In the course of its synthesis according to published procedures, it was found that chlorinated isomers were produced. The latter contributed in lowering the yield of the desired product. Addition of pyridine in the t-butyl hypochlorite oxidation step generated higher yields of the desired protected m-diazirinophenol, free of chlorine.</p> <p>A synthesis of an homologous series of omega tritiated omega hydroxy fatty acids was developed based on the oxidation of commercially available olefins with catalytic amounts of OsO₄ in presence of NaIO₄ to produce the precursor semialdehydes. Reduction of the latter with [³H]NaBH₄ afforded the corresponding omega hydroxy fatty acids. Subsequently, the pure m-diazirinophenol was coupled to the omega hydroxy group to yield the corresponding radioactive photoreactive fatty acid analogues.</p> <p>The photoreactive homologous series was found to be biologically active both in vitro and in vivo. Using rat liver microsomes it was possible to compare Michaelis-Menten constants with those observed for normal substrates for the production of acyl-CoA derivatives. Furthermore, these derivatives were converted to phosphatidylcholine and phosphatidylethanolamine when the corresponding lysophospholipids were present in the incubation mixture. In vivo investigations consisted of incubating L-cells, a fibroblastic cell line, in culture with the radioactive photoreactive fatty acid analogues. Analyses of the lipid fraction of these cells showed that these analogues were present mostly in phosphatidylcholine and in phosphatidylethanolamine. These phospholipids form the major components of L-cells membranes. The photolysis at 360 nm of cells grown in presence of these analogues resulted in the radioactive labelling of a large number of proteins presumed to be integral membrane proteins. In a collaborative effort, it was found that upon infection of these cells with vesicular stomatitis virus (VSV), followed by isolation and photolysis of the virions, the viral membrane glycoprotein G was intensely labelled. Prior to photolysis it was demonstrated that radioactivity attached to the G protein corresponded to the non-photolyzed fatty acid.</p> <p>Use of these fatty acids as photoaffinity fatty acid analogues, permitted the identification of an Escherichia coli 30 kD membrane protein with high affinity for [11-³H]11-diazirinophenoxyundecanoate. When the photolytically labelled 30 kD protein was boiled in SDS its polyacrylamide gel electrophoresis pattern shifted and it behaved as a 43 kD protein.</p> <p>Finally, attempts were made to develop a fatty acid diffusion assay consisting of encapsulating BSA inside lipid vesicles as a trapping agent. Its potential suitability as a fatty acid transport reconstitution assay is discussed.</p>