Esterase-assisted accumulation of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl into lymphocytes

Abstract

The intracellular detection of hydroxyl radical (HO˙) through spin trapping/electron paramagnetic resonance (EPR) spectroscopy has been one of the great challenges in studying free radicals in biology. While 5-carboxy-5-methyl-1-pyrroline N-oxide, [3], can specifically spin trap HO˙ in homogeneous solutions, the ionic nature of nitrone [3] at physiologic pH prevents its entry into cells. We hypothesized that conversion of carboxyl-bearing spin probes such as nitrone [3] into an esterase-hydrolyzable labile ester would permit intracellular localization and accumulation of the spin probes. To test the feasibility of such an approach, we prepared the model compound, 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [7]. This ester enabled ready accumulation of spin label to mM levels in lymphocytes. We suggest that its retention within these cells was the result of intracellular hydrolysis to 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl [6]. Moreover, our studies show that aminoxyl [6] was stable in the intracellular environment. These model studies suggest a viable strategy for detecting intracellular HO˙ by using the acetoxymethyl ester of 5-carboxy-5-methyl-1-pyrroline N-oxide [3].