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

Abstract

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 Ca²⁺ 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 Fe³⁺ 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.