Synthesis, characterization and use of new fluorescein-based fluorescent sensors for Zn2+ and nitric oxide.

Abstract

A new family of fluorescent probes for Zn2+ has been prepd. and their phys. properties evaluated. Zinpyr-1 and Zinpyr-2 are first generation probes that install Zn2+ binding ligands on a fluorescein framework. Zn2+ binding to the amine functionality of these mols. inhibits a photoinduced electron transfer (PET) quenching pathway, resulting in enhanced fluorescence emission. Zinpyr-4 shares many structural features with Zinpyr-1 and Zinpyr-2 like the use of fluorescein as a reporting group and di-(2-picolyl)amine (DPA) as the metal-binding unit; however, Zinpyr-4 is the product of an improved synthetic route and incorporates an aniline nitrogen that endows this second generation probe several with advantages over its predecessors. All of the Zinpyr sensors display a Zn2+-specific fluorescent response, have excitation and emission wavelengths in the visible range (∼500 nm) and dissocn. consts. (Kd) for Zn2+ of <1 nM. These mols. have been used in biol. investigations including the imaging of Zn2+-contg. vesicles in rat brain slices. The ability of these sensors to undergo two-photon excitation also has been demonstrated. Two-photon microscopy (TPM) affords higher resoln. and sensitivity in imaging and improves the ability to study biol. tissues. In addn., a new class of rhodafluor sensors has been prepd. as sensors for Zn2+ and NO. The first rhodafluor sensor for Zn2+ utilizes a cyclen macrocycle as the metal ion receptor. Rhodafluor derivs. contg. a metal-binding moiety also may provide the fluorescent component of a NO sensor that incorporates a metalloporphyrin. A coordinating atom has the potential to occupy one of the axial sites of the metalloporphyrin, quenching the fluorescence. Upon exposure to NO, a five-coordinate mononitrosyl species is formed resulting in dissocn. of the fluorophore. The released rhodafluor unit would exhibit an increase in fluorescence. This work was supported by the McKnight Foundation for the Neurosciences, NSF, and MIT. [on SciFinder(R)]