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

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, can specifically spin trap HO* in homogeneous solutions, the ionic nature of nitrone at physiologic pH prevents its entry into cells. We hypothesized that conversion of carboxyl-bearing spin probes such as nitrone 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. 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. Moreover, our studies show that aminoxyl 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.