THE DESIGN OF MACROCYCLIC LIGANDS FOR MONITORING MAGNESIUM IN TISSUE BY P-31 NMR

A series of ligands based upon 1,4,7-triazacyclononane, containing var iable numbers of acetate vs methylene-phosphonate ester or methyleneph osphinate sidechains, has been synthesized and characterized as possib le P-31 NMR probes for monitoring [Mg2+](free). The diacetate monophos phinate and diacetate monophosphonate ester mixed ligands proved to ha ve conditional (dissociation) constants at pH7.4 in an appropriate ran ge for measuring typical levels of intracellular [Mg2+](free), and bin ding selectivities for Mg2+ over Ca2+ that ranged from 1.4 to 6.8. The P-31 resonances of these ligands were well downfield of typical tissu e phosphate metabolite resonances, and addition of Mg2+ to any one of these ligands resulted in well-resolved resonances for HL and MgL (whe re L=ligand), in slow chemical exchange. The chemical shift difference s 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 sensi tive to pH in the physiological range, due to protonation of a single macrocyclic nitrogen at high pH (log K-1 values ranged from 10 to 12). However, given conditions that allow an independent assessment of pH (i.e., from the P-31 chemical shift of p(i)), we show that accurate K- D values can be estimated from the known thermodynamic stability const ants (K-MgL) and ligand protonation constants (K-1). This makes these ligands potentially useful for monitoring [Mg2+](free) by P-31 NMR Ove r a variety of solution conditions, even during conditions when the pH may be changing.