2-O-(2-Aminoethyl)-Ins(1,4,5)P-3 (5), a novel derivative of the Ca2--mobilising second messenger D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3], was synthesised from myo-inositol. 5 was found to be a potent mobiliser of intracellular Ca2-, and an Ins(1,4,5)P-3 affinity matrix synthesised from 5 was effective at selectively binding N-terminal fragments of the Ins(1,4,5)P-3 receptor containing the intact Ins(1,4,5)P-3 binding site. The microprotonation scheme for 5 was resolved and the related constants were determined in comparison with Ins(1,4,5)P-3 and another reactive Ins(1,4,5)P-3 analogue 1-O-(2-aminoethyl-1-phospho)-Ins(4,5)P-2 (2a) by potentiometric and NMR titration methods. The P-31 and H-1 NMR titration curves for compound 5 and Ins(1,4,5)P-3 are remarkably close, indicating analogous acid-base properties and intramolecular interactions for the two compounds. The 1-phosphate-modified Ins(1,4,5)P-3 derivative 2a, on the contrary, behaves as a bisphosphorylated rather than a trisphosphorylated inositol. Thus, 5 is a new reactive Ins(1,4,5)P-3 analogue of considerable potential for investigation of the chemical biology of Ins(1,4,5) P-3-mediated cellular signalling. (C) 2004 Elsevier Inc. All rights reserved.