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 methylene‐phosphonate ester or methylenephosphinate sidechains, has been synthesized and characterized as possible ³¹P NMR probes for monitoring [Mg²⁺ ]_free. The diacetate monophosphinate 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 Mg²⁺‐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 P_i), we show that accurate K_D values can be estimated from the known thermodynamic stability constants (K_MgL) and ligand protonation constants (K₁). 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.