We developed a metal-based fluorescent probe for H(2)O(2) called MBFh1, which has an iron complex as a reaction site for H(2)O(2) and a 3,7-dihydroxyphenoxazine derivative as the fluorescent reporter unit. The iron complex reacts quickly with H(2)O(2) to form oxidants, and then the oxidants convert the closely appended nonfluorescent 3,7-dihydroxyphenoxazine moiety to resorufin in an intramolecular fashion. The quick response to H(2)O(2) allows us to plot the enzymatic evolution of H(2)O(2). A combination of N-acetyl-3,7-dihydroxyphenoxazine and horseradish peroxidase has been frequently used to detect enzymatically generated H(2)O(2), but this method has interference with phenol derivatives. The use of MBFh1 overcomes this drawback.

A metal-based fluorescent probe for H2O2, named MBFh2, releases a highly fluorescent resorufin in the seconds time scale even in the presence of 5 μM H2O2. The use of MBFh2 enabled the visualization of intracellular H2O2 that was generated after stimulation of the epidermal growth factor.

A mononuclear iron(III) complex of a noncyclic tetradentate monoamido ligand, Fe(III)mpaq, catalyses the oxidation of Orange II, guaiacol, ABTS and Amplex Red with H2O2 in aqueous solutions at neutral pH. Under identical conditions, other structurally related nonheme iron complexes showed only negligible activities.

We previously reported the synthesis of the metal-based fluorescent probes MBFh1 and MBFh2 for hydrogen peroxide (H2O2) imaging, which release the red-emitting fluorophore resorufin, whose response rate is significantly faster than that of other nonmetal-based H2O2 probes, upon reacting with H2O2. In this study, we have synthesized the new fluorescent probe MBFh3 1 and its ester derivatives 2 and 3, in which a mononuclear nonheme iron(III)–monoamido complex and a 3′-O-methyldihydrofluorescein derivative are connected via a propane linker. Probes 2 and 3 are methyl and 1-(ethoxycarbonyloxy)ethyl ester derivatives of 1, respectively. These probes rapidly react with H2O2 to release green-emitting 3′-O-methylfluoresceins. Fluorescent microscopy studies using HeLa and A431 cells showed that intracellular H2O2 can be visualized using probes 1 and 2, but not 3.