A series of improved fluorescent dyes which are specific for complexing Ca2+ will be synthesized and evaluated for their absorption and emission characteristics as well as their ability to bind Ca2+. The proposed 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 [Beta-amino ethyl ether N,N,N',N'-tetraacetic acid]) structure which is known to have great specificity for Ca2+ over Mg2+ and other divalent metal ions. The proposed Ca2+ indicators will thus have improved optical characteristics over currently used quin2 and fura-2, which they resemble structurally except for the fluorophore. The indicators will be 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, [Ca2+]i, as a function of time in response to various perturbing influences. Intracellular calcium is currently the subject of widespread interest as a second messenger that triggers and regulates cell activation.
