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

Abstract

In order to discover new 31P NMR markers for probing subtle pH changes (Kas (ranging 6.28–6.97), chemical shifts not overlapping those of phosphorus metabolites, and spectroscopic sensitivities (i.e., chemical shifts variation Δδab 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 pKas and both the 2D structure and pyramidalization at phosphorus, showing that steric crowding around the phosphorus is crucial for modulating Δδab. Finally, the hit 31P NMR pH probe 1b bearing an unsubstituted 1,3,2-dioxaphosphorinane ring, which is moderately lipophilic, nontoxic on A549 and NHLF cells, and showing pKa = 6.45 with Δδab = 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.

Keywords

• α-aminophosphonates
• pH probes
• ³¹P NMR
• phophorus pyramidalization angle
• X-ray crystallography
• trans-sarcolemmal pH gradients