SYNTHESIS OF FLUORESCENT INDICATORS FOR CYTOSOLIC CA2+

A SERIES OF IMPROVED FLUORESCENT DYES SPECIFIC FOR COMPLEXING CA2+ WILL BE SYNTHESIZED AND EVALUATED FOR THEIR ABSORPTION AND EMISSION CHARACTERISTICS AND THEIR ABILITY TOBIND CA2+. THESE CHELATORS INCORPORATE BENZOCOUMARIN LASER DYE FLUOROPHORES, WHICH HAVE DESIRABLE HIGH-QUANTUM YIELDS AND VISIBLE WAVELENGTH ABSORPTION AND EMISSION. THE FLUOROPHORE IS ATTACHED TO AN EGTA-LIKE (ETHYLENEGLYCOL BIS [B-AMINO ETHYL ETHER N,N,N',N'-TETRAACETIC ACID]) STRUCTURE THAT IS KNOWN TO HAVE GREAT SPECIFICITY FOR CA2+ OVER MG2+ AND OTHER DIVALENT METAL IONS. THE PROPOSED CA2+ INDICATORSWILL HAVE IMPROVED OPTICAL CHARACTERISTICS OVER THOSE CURRENTLY USED (QUIN 2 AND FURA-2). THE NEW INDICATORS WILLBE CELL MEMBRANE PERMEABLE WHEN THE ACETATE GROUPS ARE ESTERIFIED, AND RECONVERTIBLE TO THE ACETATE FORM BY CYTOSOLIC ESTERASES INSIDE THE CELL. THUS THE TRAPPED CHELATOR CAN BE USED TO FOLLOW CONCENTRATION CHANGES IN INTRACELLULAR CALCIUM CONCENTRATION AS A FUNCTION OF TIME INRESPONSE TO VARIOUS PERTURBING INFLUENCES. INTRACELLULAR CALCIUM IS CURRENTLY THE SUBJECT OF WIDESPREAD INTEREST AS ASECOND MESSENGER THAT TRIGGERS AND REGULATES CELL ACTIVATION. THIS PHASE I WORK INVOLVES SYNTHESIS OF THE SUBSTITUTED BENZOCOUMARIN PORTION OF THE OVERALL STRUCTURE. IN PHASE II, THE FLUOROPHORES WILL BE LINKED TO THE EGTA- LIKE COMPLEXING AGENT. THE RESULTING SERIES OF FLUORESCENT CA2+ COMPLEXING AGENTS WILL BE EVALUATED FOR THEIR ABSORPTION AND EMISSION WAVELENGTHS AND FLUORESCENCE QUANTUM YIELD IN THE FREE AND CA2+-BOUND STATES. IN ADDITION, THEIR CA2+ COMPLEX STABILITY CONSTANTS WILL BE DETERMINED. THE MOST PROMISING INDICATORS WILL BE TESTED FOR CA2+ SPECIFICITY AND USEFULNESS INDICATORS OF INTRACELLULAR CONCENTRATION.