Zinc and calcium are ubiquitous intracellular metals, and while a variety of quantitative probes have been developed for measuring intracellular changes in calcium concentration, the same is not true of zinc. We describe here the design, synthesis, and properties of the benzoxazole-based, ratiometric zinc probe, Zinbo-5. This bright fluorescent reporter has a quantum yield of 0.1 in the zinc-form, exhibits a Kd for Zn2+ in the nanomolar range, and shows significant changes in both excitation and emission maxima upon zinc binding. The utility of this cell permeable probe is demonstrated in fluorescence microscopy emission ratio imaging experiments on mammalian cells. We further show that Zinbo-5 is well suited for two-photon excitation microscopy ratio imaging and can readily reveal changes in intracellular zinc concentration within optical planes of single cells. To the best of our knowledge, this is the first example of two-photon excitation microscopy applied to ratio imaging of zinc. These methods can be applied to real-time emission or excitation ratio imaging studies of zinc physiology in living cells.

The first systematic studies on the oxidation of neutral phenols (ArOH) by the mu-eta(2):eta(2)-peroxo)dicopper(II) complex (A) and the bis(mu-oxo)dicopper(III) complex (B) supported by the 2-(2-pyridyl)ethylamine tridentate and didentate ligands L(Py2) and L(Py1), respectively, have been carried out in order to get insight into the phenolic O-H bond activation mechanism by metal-oxo species. In both cases (A and B), the C-C coupling dimer was obtained as a solely isolable product in approximately 50% yield base on the dicopper-dioxygen (Cu(2)/O(2)) complexes, suggesting that both A and B act as electron-transfer oxidants for the phenol oxidation. The rate-dependence in the oxidation of phenols by the Cu(2)/O(2) complexes on the one-electron oxidation potentials of the phenol substrates as well as the kinetic deuterium isotope effects obtained using ArOD have indicated that the reaction involves a proton-coupled electron transfer (PCET) mechanism. The reactivity of phenols for net hydrogen atom transfer reactions to cumylperoxyl radical (C) has also been investigated to demonstrate that the rate-dependence of the reaction on the one-electron oxidation potentials of the phenols is significantly smaller than that of the reaction with the Cu(2)O(2) complexes, indicative of the direct hydrogen atom transfer mechanism (HAT). Thus, the results unambiguously confirmed that the oxidation of phenols by the Cu(2)O(2) complex proceeds via the PCET mechanism rather than the HAT mechanism involved in the cumylperoxyl radical system. The reactivity difference between A and B has also been discussed by taking account of the existed fast equilibrium between A and B.

Tyrosinase is a copper monooxygenase that catalyzes oxygenation of phenols to catechols (phenolase activity) and the subsequent two-electron oxidation of catechols to the corresponding o-quinones (catecholase activity). 1 Chemical and spectroscopic studies have indicated that the enzyme has a dinuclear copper active site nearly identical to that found in hemocyanin, 1,2 where a side-on type (µ-η 2 :η 2 ) peroxo species 3 is generated by the reaction of the reduced dicopper(I) form and O 2 . 1 As a pioneering work by Karlin and co-workers in Cu/O 2 chemistry, aromatic ligand hydroxylation in a dinuclear Cu(I) complex by O 2 was first reported in early 1980s. 4 The mechanistic studies have indicated that the aromatic ligand hydroxylation reaction involves an electrophilic attack on the arene ring by a (µ-η 2 :η 2 -peroxo)-dicopper(II) intermediate. 5 After their finding, several examples of the aromatic ligand hydroxylation have been reported using similar type of m-xylyl dinucleating ligands. 6 With respect to the intermolecular reactions between phenols and the peroxo intermediate, however, most of the reactions so far reported afford a C-C coupling dimer as a major product. 7 Casella and co-workers have recently reported the first synthetic (µ-η 2 :η 2 -peroxo)dicopper-(II) complex which can react with an exogenous phenolate to yield the corresponding catechol. 8,9 Unfortunately, the low yield of the product (20% based on the dicopper complex) has precluded the kinetic and mechanistic investigation on the reaction between the peroxo intermediate and the phenolate. 8 As such, the mechanism for the catechol formation via intermolecular reactions between the peroxo intermediate and phenol derivatives has yet to be clarified. 10 We report herein that efficient conversion of phenol derivatives to the corresponding catechols is achieved for the first time by intermolecular reactions of a (µ-η 2 :η 2 -peroxo)dicopper(II) complex, supported by tridentate ligand L Py2Bz (N,N-bis[2-(2-pyridyl)-ethyl]-R,R-dideuteriobenzylamine), 11 with lithium salts of phenols. The mechanistic studies on the catechol formation have been performed to provide valuable mechanistic insight into the phenolase activity of the enzyme.Treatment of the copper(I) complex, [Cu I (L Py2Bz )](PF 6 ), with dioxygen in anhydrous acetone at -94°C afforded a brown color solution which exhibited a strong absorption band at 364 nm ( ) 26400 M -1 cm -1 ) together with a small one at 530 nm (1500 M -1 cm -1 ) and a resonance Raman band at 737 cm -1 that shifted to 697 cm -1 upon 18 O-substitution. 12,13 The frozen acetone solution of the intermediate was ESR silent at 77 K, and a Cu:O 2 ) 2:1 stoichiometry was obtained for formation of the intermediate by manometry. These results unambiguously indicate that the oxygenated intermediate is a (µ-η 2 :η 2 -peroxo)dicopper(II) complex as suggested previously by Karlin et al. 11 This compound is quite stable (no self-decomposition) at the low-temperature enabling us to examine the reaction with external subst...