title: 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, 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.
authors.affiliation: Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, and Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD 21201, USA.
authors.forename: Joseph P Y
authors.initials: JP
authors.lastname: Kao
authors.forename: Gerald M
authors.initials: GM
authors.lastname: Rosen
mesh_terms: D004950:Esterases / Q000378:metabolism*; D004952:Esters / Q000737:chemistry; D006801:Humans; D017665:Hydroxyl Radical / Q000032:analysis*; D019169:Jurkat Cells; D011759:Pyrrolidines / Q000737:chemistry* / Q000378:metabolism; D013113:Spin Labels
chemical_list: C488837:3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl; D004952:Esters; D011759:Pyrrolidines; D013113:Spin Labels; D017665:Hydroxyl Radical; D004950:Esterases