Novel Sterically Crowded and Conformationally Constrained α-Aminophosphonates with a Near-Neutral p K a as Highly Accurate 31 P NMR pH Probes. Application to Subtle pH Gradients Determination in Dictyostelium discoideum Cells.

In order to discover new 31 P NMR markers for probing subtle pH changes (<0.2 pH unit) in biological environments, fifteen new conformationally constrained or sterically hindered α-aminophosphonates derived from diethyl(2-methylpyrrolidin-2-yl)phosphonate were synthesized and tested for their pH reporting and cytotoxic properties in vitro. All compounds showed near-neutral p K a s (ranging 6.28-6.97), chemical shifts not overlapping those of phosphorus metabolites, and spectroscopic sensitivities (i.e., chemical shifts variation Δ δ ab between the acidic and basic forms) ranging from 9.2-10.7 ppm, being fourfold larger than conventional endogenous markers such as inorganic phosphate. X-ray crystallographic studies combined with predictive empirical relationships and ab initio calculations addressed the inductive and stereochemical effects of substituents linked to the protonated amine function. Satisfactory correlations were established between p K a s and both the 2D structure and pyramidalization at phosphorus, showing that steric crowding around the phosphorus is crucial for modulating Δ δ ab . Finally, the hit 31 P NMR pH probe 1b bearing an unsubstituted 1,3,2-dioxaphosphorinane ring, which is moderately lipophilic, nontoxic on A549 and NHLF cells, and showing p K a = 6.45 with Δ δ ab = 10.64 ppm, allowed the first clear-cut evidence of trans-sarcolemmal pH gradients in normoxic Dictyostelium discoideum cells with an accuracy of <0.05 pH units.