The cellular phosphoinositides, including the phosphatidylinositols and their mono- and polyphosphate derivatives, are critical in intracellular signaling and related cascades. We have developed methods for synthesizing analogs of the cellular series, and the synthetic products are now established as important in vitro reagents. The available synthetic phosphoinositides are not consistently membrane-permeant. Heptakis(acetoxymethyl) derivatives of pbosphatidylinositol-3,4,5-trisphospbate (Ptdlns-3,4,5-P3) enter cells and display activity of Ptdlns-3,4,5-P3. However, polykis-acetoxymethylated phosphoinositides have serious inherent drawbacks as reagents because these suffer extensive phosphate shifts resulting in mixtures of isomers and provide a variable indeterminate amount of the unmodified phosphoinositide inside cells. The overall objective of this investigation is to develop membrane-permeant functional analogs of the phosphoinositides. During Phase I, an approach integrating the design, synthesis and evaluation of novel fonnulations will be tested to assess internalization of synthetic phosphatidylinositol-4,5-bisphosphate (Ptdlns4,5-P2) analogs in cells. During Phase II, the approach will be optimized for Ptdlns-4,5-P2 and extended to a complete range of phosphoinositide analogs. PROPOSED COMMERCIAL APPLICATION: This research will provide a reliable means for introducting quantitatively controlled amounts of exogenous phosphoinositides into intact live cells, and a complete range of novel membrane-permeant phosphoinositide products for use as in vivo research reagents for studies ranging from cell physiology to delivery of phosphoinositide-based therapeutics. These products are in demand but not currently available from any commercial source, and are suitable for transfer directly from production in a laboratory setting to the market.
