Conformational analysis of the natural iron chelator myo-inositol 1,2,3-trisphosphate using a pyrene-based fluorescent mimic

myo-Inositol phosphates possessing the 1,2,3-trisphosphate motif share the remarkable ability to completely inhibit iron-catalysed hydroxyl radical formation. The simplest derivative, myo-inositol 1,2,3-trisphosphate [Ins(1,2,3)P₃], has been proposed as an intracellular iron chelator involved in iron transport. The binding conformation of Ins(1,2,3)P₃ is considered to be important to complex Fe³⁺ in a 'safe' manner. Here, a pyrene-based fluorescent probe, 4,6-bispyrenoyl-myo-inositol 1,2,3,5-tetrakisphosphate [4,6-bispyrenoyl Ins(1,2,3,5)P₄], has been synthesised and used to monitor the conformation of the 1,2,3-trisphosphate motif using excimer fluorescence emission. Ring-flip of the cyclohexane chair to the penta-axial conformation occurs upon association with Fe³⁺, evident from excimer fluorescence induced by π-π stacking of the pyrene reporter groups, accompanied by excimer formation by excitation at 351 nm. This effect is unique amongst biologically relevant metal cations, except for Ca ²⁺ cations exceeding a 1:1 molar ratio. In addition, the thermodynamic constants for the interaction of the fluorescent probe with Fe³⁺ have been determined. The complexes formed between Fe ³⁺ and 4,6-bispyrenoyl Ins(1,2,3,5)P₄ display similar stability to those formed with Ins(1,2,3)P₃, indicating that the fluorescent probe acts as a good model for the 1,2,3-trisphosphate motif. This is further supported by the antioxidant properties of 4,6-bispyrenoyl Ins(1,2,3,5)P₄, which closely resemble those obtained for Ins(1,2,3)P₃. The data presented confirms that Fe³⁺ binds tightly to the unstable penta-axial conformation of myo-inositol phosphates possessing the 1,2,3-trisphosphate motif.