The design of macrocyclic ligands for monitoring magnesium in tissue by 31P NMR.

A series of ligands based upon 1,4,7-triazacyclononane, containing variable numbers of acetate vs methylenephosphonate ester or methylenephosphonate sidechains, has been synthesized and characterized as possible 31P NMR probes for monitoring [Mg2+]free. The diacetate monophosphonate and diacetate monophosphonate ester mixed ligands proved to have conditional (dissociation) constants at pH 7.4 in an appropriate range for measuring typical levels of intracellular [Mg2+]free, and binding selectivities for Mg2+ over Ca2+ that ranged from 1.4 to 6.8. The 31P resonances of these ligands were well downfield of typical tissue phosphate metabolite resonances, and addition of Mg2+ to any one of these ligands resulted in well-resolved resonances for HL and MgL (where L = ligand), in slow chemical exchange. The chemical shift differences between the HL and MgL species varied from 2.1 to 2.6 ppm for three different ligands. The conditional Mg(2+)-L binding constants were sensitive to pH in the physiological range, due to protonation of a single macrocyclic nitrogen at high pH (log K1 values ranged from 10 to 12). However, given conditions that allow an independent assessment of pH (i.e., from the 31P chemical shift of Pi), we show that accurate KD values can be estimated from the known thermodynamic stability constants (KMgL) and ligand protonation constants (Ki). This makes these ligands potentially useful for monitoring [Mg2+]free by 31P NMR over a variety of solution conditions, even during conditions when the pH may be changing.