Abstract
Inositol pyrophosphates (PP-IPs) are densely phosphorylated messenger molecules involved in numerous biological processes. PP-IPs contain one or two pyrophosphate group(s) attached to a phosphorylated myo-inositol ring. 5PP-IP5 is the most abundant PP-IP in human cells. To investigate the function and regulation by PP-IPs in biological contexts, metabolically stable analogs have been developed. Here, we report the synthesis of a new fluorinated phosphoramidite reagent and its application for the synthesis of a difluoromethylene bisphosphonate analog of 5PP-IP5 . Subsequently, the properties of all currently reported analogs were benchmarked using a number of biophysical and biochemical methods, including co-crystallization, ITC, kinase activity assays and chromatography. Together, the results showcase how small structural alterations of the analogs can have notable effects on their properties in a biochemical setting and will guide in the choice of the most suitable analog(s) for future investigations.