Olefin metathesis of phospholipids by Ruthenium-based catalysts in solution and on liposomes under biologically...

The last decade has seen a lot of interest in classical organometallic catalysis performed intracellularly, or at least under biological conditions (37 °C, air, water). One such classical reaction is olefin metathesis (OM), which is extremely common in preparative organic chemistry, however under non-biological conditions (esp. organic solvents). For in vivo applications, both prokaryotic and eukaryotic applications would require the passage of the OM catalyst through the cell membrane, which already contains unsaturated olefins and thus potential substrates for OM. This work focuses on the question whether OM is catalysed in membranes containing unsaturated phospholipids. Initial experiments with the 2nd generation Hoveyda-Grubbs catalyst (HGII) showed self-metathesis of the membrane-forming phospholipids POPE, POPC, and POPG, while approximating biological conditions in solution (37 °C, air, neutral pH) with substrate conversions up to 58%. Subsequent experiments with DOPC in large unilamellar vesicles (LUVs) and in solution included a PEGylated (featuring increased water solubility) and a palmitoylated (with increased membrane mobility) HGII derivative. Both were successfully synthesised beforehand and comprehensively characterized. Membrane localisation of the catalysts was evaluated via size exclusion chromatography (SEC) followed by ICP-MS. Product analysis of OM was carried out after one hour and 24 h, respectively, via NMR, TLC, and LC-MS. The intra- and intermolecular OM products of DOPC were identified. First, substrate conversion was significantly diminished in the vesicles, however, the equilibrium state in vesicles was reached in much less time compared to solution (1 h in vesicles vs. 24 h in solution). To our surprise, a distinct difference in product selectivity between OM in solution and in vesicles was observed in that the intramolecular OM is much favoured in solution, while in LUVs, the main products are the result of intermolecular OM reactions. Our results prove that OM is readily catalysed in vesicles and indicate that the milieu of the lipid bilayer has a major impact on the product selectivity, reaction time and substrate conversion. While previous work has focused on the cytotoxicity of Hoveyda-Grubbs catalysts and their interaction with biomolecules, our present work provides valuable insights on what will happen when an OM catalyst like HGII is exposed to membranes in organisms. Moreover, using the results from this work, it may be possible to selectively modify membrane properties, and thereby cellular responsiveness to outside stress, by olefin metathesis in the future.