Zinc biosensing with multiphoton excitation using carbonic anhydrase and improved fluorophores

Previously, we had shown that the zinc-dependent binding of certain fluores cent aryl sulfonamide inhibitors could be used with apocarbonic anhydrase I I to transduce the level of free zinc as a change in the fluorescence of th e inhibitor. While inhibitors such as dansylamide, ABD-M, and ABD-N made po ssible quantitation of free zinc in the picomolar range with high selectivi ty, they have only modest absorbance which limits their utility. We describ e here the synthesis and properties of two new probes, Dapoxyl(TM) sulfonam ide and BTCS, and their use in zinc biosensing. Dapoxyl sulfonamide exhibit s a dramatic increase and blue shift in its emission upon binding to holoca rbonic anhydrase II, as well as a 20-fold increase in lifetime: it is thus well suited for quantitating free Zn(II) down to picomolar ranges. The anis otropy of BTCS increases fivefold upon binding to the holoprotein, making t his probe well suited for anisotropy-based determination of zinc. BTCS and ABD-N are efficiently excited with two photon excitation using 1.5 ps pulse s from a titanium sapphire laser, and exhibit the increased zinc-dependent anisotropy response anticipated on the basis of photoselection. (C) 2000 So ciety of Photo-Optical Instrumentation Engineers. [S1083-3668(00)00201-X].