| Hydrogen peroxide (H2O2) is a ubiquitous reactive oxygen species (ROS) produced by respiring cells and is implicated as a critical mediator in numerous signaling cascades known to affect differentiation, proliferation, and induction of apoptosis. The chemical biology of H 2O2 production, trafficking, and reaction chemistry involves an exceedingly complex network of molecular interactions, in which disparate physiological and pathological consequences may arise depending upon subtle contextual differences. Mechanistic details of H2O2 mediated cell signaling have remained elusive in part because the chemical tools used in current ROS research suffer from vexing limitations. This dissertation presents the design, synthesis, spectroscopic evaluation, and biological testing of new, small molecule fluorescent chemical tools that exhibit unprecedented selectivity for H2O2 over competing cellular ROS. Naphtho-Peroxyfluor-1 (NPF1) and Peroxyresorufin-1 (PR1) respond to H2O2 by an increase in far-red and red fluorescence, respectively and are able to respond to micromolar changes in H2O2 levels in living cells as demonstrated in flow cytometry and confocal microscopy experiments in human cervical cancer (HeLa) and human embryonic kidney (HEK) cells. Highly conjugated Aza-Peroxyfluor-1 (APF1) responds to H2O2 by a ratiometric increase in far-red visible wavelength absorbance and near-infrared fluorescence, qualities favorable for cell signaling experiments that will ultimately take place in vivo. Ratio-Peroxyfluor-1 (RPF1) utilizes fluorescence resonance energy transfer (FRET) to report quantitative changes in H2O2 levels by a ratiometric increase in visible wavelength fluorescence intensity, as demonstrated by experiments performed with purified eukaryotic mitochondria. Monoamine oxidases (MAO) in mitochondria produce H2O2 in their catalytic cycle from oxidative deamination of neurotransmitters. MAO reporter-1, and -2 (MR1, MR2) report MAO activity by an increase in red fluorescence. MR1 and MR2 are competent enzyme substrates with low Michaelis constant (K m) values, and are capable of detailing relative monoamine oxidase activities as demonstrated in nerve growth factor-differentiated human PC12 cells. Fluorescent small molecules possessing a benzylguanylyl (SNAP) targeting moiety may be used to irreversibly label genetically engineered fusions bearing mutated human O6-alkylguanine DNA-alkyltransferase (hAGT). SNAP-Peroxygreen-1 (SPG1) and SNAP-Peroxyfluor-1 (SPF1) respond to H2O2 by an increase in green fluorescence, and shall be used to monitor endogenous H2O2 within defined mammalian subcellular microenvironments. |
