NOTPME: A ³¹P NMR probe for measurementof divalent cations in biological systems

1, 4, 7-Triazacyclononame-N, N′,N′-tris(methylenephosphonate monoethylester) (NOTPME) has been synthesized, characterized and analyzed for use as a ³¹P NMR indicator of intracellular Mg²⁺ and Zn²⁺ ions. The ³¹P NMR spectrum of this chelate in the presence of metal ions shows characteristics resonances for the free chelate, Mg(NOTPME)^-, Zn(NOTPME)^- and Ca(NOTPME)^-. The K_??? values indicate that this chelate has a 10-fold higher affinity for Mg²⁺ than for Ca²⁺ at physiological pH values. In the presence of Mg²⁺, NOTPME is readily loaded into red blood cells. A ³¹P NMR spectrum of red cells taken after several washing shows resonances characteristics of entrapped NOTPME and the Mg(NOTPME)^- complex, the relative areas of which report an intracellular free Mg²⁺ concentration of 0.32 mM. The ³¹P chemical shifts of the free chelate and its metal complexes are far downfield from the typical phosphorus-containing metabolites observed in biological systems, thus making it possible to monitor intracellular cation concentration and cell energetics simultaneously.