Biochimica et Biophysica Acta, 1014(1989) 211-218 Elsevier 211 BBAMCR 12572 Development of nitroxides for selective localization inside cells Huping Hu 1, George Sosnovsky 2, Shu Wen Li 2, Nuti Urea Maheswara Rao 2, Philip D. Morse II 1, and Harold M. Swartz 1 s University of Illinois College of Medicine and ESR Research Center, Urbana, IL and 2 Department of Chemistr); University of Wisconsin-Milwaukee, Milwaukee, WI (U.S.A.) (Received 27 March 1989) Key words: ESR; Oxygea,intracellular; Selectivelocalization; Nitroxide; Aminoxyl;Nitroxyl The use of nitroxides to measure intracellular phenomena, especially oxygen concentrations, is a new and potentially important approach to a number of physiological and pathophysiological studies. This study provides data indicating the feasibility of developing nitroxides that localize selectively in the intracellu|ar compartment; it is based on the use of readily hydrolysed ester linkages, such that the nitroxides become converted intracellularly to ionic derivatives that do not cross cell membranes readily. Up to |20-fold increased concentrations of intracellular nitroxides (and their one electron reduction product, the hydroxylamines) were obtained. The ESR spectra of the intracellular nitroxides were consistent with their conversion to the ionic species. Preliminary studies indicate that these nitroxides have the properties needed for their use as probes of intracellular concentrations of oxygen and that it should be feasible to synthesize nitroxides that will be even more effective for this purpose. Introduction The use of nitroxides to measure oxygen concentra- tions and related redox metabolism in cells and tissues has been demonstrated recently and seems to have considerable potential for providing useful data on physiological and pathophysiological processes [1-5]. The studies made with nitroxides include the measure- ment of intracellular oxygen concentrations and recent results indicate that this type of measurement may be very useful because significant differences between intracellular and extracellular oxygen concentrations have been demonstrated in cell suspensions under some conditions [6]. The current methods to measure intracellular oxygen with nitroxides require the use of an extracellular paramagnetic broadening agent to suppress the ESR signal from the extracellular compartment, and, while this approach has been effective, it has several draw- backs. The concentrations of paramagnetic ions that are * Present address: Department of Chemistry, Illinois State Univer- sity, Normal, IL 61761, U.S.A. Correspondence: Harold M. Swartz, 190 Medical Sciences Building, 506 South MathewsAvenue,Urbana, IL 61801, U.S.A. needed are relatively high (50-100 raM) and therefore potentially can alter the system in obvious or, perhaps more problematically, in subtle ways that may lead to misinterpretation of the results. Also, the need for an extracellular broadening agent makes it difficult to ex- tend this important new measurement to tissues and intact animals; this is particularly unfortunate because recent developments in ESR methodology make ESR measurements in vivo feasible [7-9]. In this report we present the preliminary results of an approach to overcome the problem of the methods that require an extracellular broadening agent to measure intracellular oxygen concentrations by nitroxides. It was developed in analogy with the methods used to obtain some selective intracellular measurements of calcium [10-12]. We have synthesized a series of nitroxides that have potentially labile ester bonds covering one or more carboxyl groups. Our expectation is that the ester forms of the nitroxides will penetrate readily into cells but, after intracellular hydrolysis to their respective acid anions, they will not be able to leave the cells (Fig. 1). In this report we present data on 14 such nitroxides, plus four analogs whose acid groups are not esterified (Fig. 2). Our aim is to demonstrate that under experi- mental condi0ons that would be used in typical experi- ments some of these nitroxides can be localized selec- tively within cells and that they have other properties 0167-4889/89/$03.50 © 1989 ElsevierScience Publishers B.V.(Biomedical Division)