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

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Abstract
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.