Highlights d Inositol hexakisphosphate (IP6) and analogs induce TcdB auto-proteolysis in vitro d Analogs retain activity in the presence of lumenal calcium concentration in vitro d Oral gavage of analogs promoted survival in a C. difficile infection mouse model

There is a paucity of chiral phosphoramidite reagents or chiral catalysis methods for the synthesis of biologically relevant inositol phosphates. A new C2-symmetrical chiral phosphoramidite has been developed and successfully applied to the synthesis of a set of chiral inositol bisphosphates. The reagent allowed bis-phosphorylation and chiral resolution, resulting in a concise synthetic route, thus expanding the toolbox available for the preparation of biologically relevant inositol phosphates in high optical purity.