Our group has previously described large chelation-enhanced fluorescence (“CHEF”) effects upon the binding of metal ions, phosphates, and carboxylates to conjugate probes, providing large, readily measurable “signals” to these molecular recognition events. In understanding the structural requirements for CHEF, it is now possible to use the vast body of information on selective binding by azacrowns and cryptands in the synthesis of selective fluorescence probes. For example, a conjugate probe that allows for the selective, simultaneous assay of Zn(II) and Cd(II) ions has been synthesized and will be describe. In the homologous series of anthrylazamacrocycles that demonstrate chelation-enhanced fluorescence (CHEF) upon Zn(II) or Cd(II) binding in water, the pentacyclen derivative uniquely complexes Cd(II) with perturbation of the emission spectrum. The binding of anions such as phosphate and citrate give rise to fluorescence enhancements as large as 6-fold; an observed pH dependence on the magnitude of fluorescence enhancements upon phosphate binding points to intracomplex protonation of the benzylic nitrogen by the HPO42- ion as the origin of this CHEF effect. Anthrylpolyamine conjugate probes yield large (up to 80-fold) changes in fluorescence upon binding to biological polyanions (e.g., DNA, Heparin, and polyglutamate) at μM concentrations. These fluorescence changes have been used as the basis of a fluorometric assay of heparinase activity.
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