Synthesis and 31P NMR characterization of new low toxic highly sensitive pH probes designed for in vivo acidic pH studies Original Research Article

With the aim to provide sensitive 31P NMR probes of intra- and extracellular pH gradients that may reach cellular acidic compartments in biological systems, new α-aminophosphonates were designed to meet basic requirements such as a low pKas and a great chemical difference (Δδab) between the limiting 31P NMR chemical shifts in acidic (δa) and basic (δb) media. A series of six phosphorylated pyrrolidines and linear aminophosphonates were synthesized using aminophosphorylation reactions and were screened for cytotoxicity on cultured Müller cells. Among the compounds not being toxic under these conditions, three molecules were selected since they displayed the best in vitro (in several phosphate buffers and in a cytosol-like solution) properties as 31P NMR acidic pH markers, that is 3, 5 and 9, having the pKa values of 3.63, 5.89 and 5.66, respectively. The Δδab values of these pH markers were at least 3 times larger than that of standard 31P NMR probes, with a low sensitivity to ionic strength changes. From these data, it was proposed that 3, 5 and 9 could be used as reporting probes of subtle proton movements in acidic compartments, an area that still remains poorly investigated using non invasive 31P NMR methods.