Phosphoinositide recognition by binding proteins and lipid-metabolizing enzymes involves specific interactions with the head group and diacylglycerol backbone, which vary significantly from protein to protein. When a fluorescent probe is introduced into the inositol head group, binding and metabolism are severely attenuated or abrogated entirely. When a fluorescent probe is introduced into the acyl groups, many of the resulting derivatives fail to show adequate Km and Vmax values as substrates for lipid kinases and phosphatases as well as binding to cognate PIP-binding proteins. Recently, Echelon Biosciences has developed a new approach to make phospholipid-phophoinositide hybrids based on a four-carbon threitol backbone. These new materials, known as Pea-PIPs TM, have a phosphatidylethanolamine head group that permits biochemical or chemical probes to be attached in a position spatially orthogonal to the phosphoinositide head group and the two acyl chains. In this Phase i grant, we will first synthesize a full repertoire of all eight Pea-PIPns TM. Second, we will prepare two Pea-PIPs TM with four different hydrophilic linker arms to optimize probe-head group distances. Third, we will establish the feasibility of using Pea- PIPs TM as in vitro substrates for lipid kinases, lipid phosphatases, and lipid binding proteins.