Cellular cations play fundamental roles in hormonal signaling, and are also involved in the mediation of cell injury. The development of intracellular indicators for cytosolic cations and other parameters of interest has had a major impact on the field of cell biology. Our goals in this area have included the development of more sensitive intracellular calcium indicators, and more selective intracellular magnesium indicators. During the past year, we have worked on the development of a new and general route towward the synthesis of fluorescent chelators related to BAPTA [bis(o-aminophenoxy)ethane-N,N,N?,N?-tetraacetic acid] or APTRA (o-aminphenol-N,N,O-triacetic acid), which are used for determining calcium and magnesium ion levels in cells. The key step is a palladium-catalyzed coupling of the chelating moiety to the fluorophore. Using this method, it becomes possible to more rapidly synthesize and screen indicators with a range of potential fluorophores. Coupling of triflate [4-(CF3SO3)C6H3NO2-2-(OBenzyl)] with several polyaromatic boron and tin species proceeds with excellent yields. Further elaboration of the 2-benzyloxy-nitrophenyl ring (reduction, followed by triple alkalyation with BrCH2COOMe and saponification) gives the desired APTRA chelators. The fluorescent response to magnesium of several of these molecules has been evaluated. In addition to the above studies, we have recently succeeded in developing a more magnesium selective chelator than the APTRA molecules referred to above. These fluorescent molecules exhibit an emission shift upon magnesium complexation, and are relatively insensitive to calcium ions. Derivatives more suitable for loading into cells are currently in preparation. In parallel with the probe development work, physiological studies utilizing NMR have continued in collaboration with Dr. Murphy?s group at NIEHS and Dr. Steenbergen?s group at Duke University. These studies have been aimed at understanding the ionic changes which occur during periods of stress, and the role that these changes play in causing irreversible injury. During the last year, NMR studies performed on perfused rat hearts indicated that differences in intracellular pH observed between pre-conditioned and non pre-conditioned hearts are not due to increased H+ extrusion in the former. Studies on the transport of the fluorinated nucleoside: 2'-fluoro-5-methyl-beta-L-arabinofuranosyl uracil (L-FMAU), were recently concluded.
