Understanding Sulfur Based Redox Biology Through Advancements In Chemical Biology PDF Download

Two decades of research on the signaling capacity of hydrogen sulfide (H2S) in biological systems has led to parallel investigations of other reactive sulfur species (RSS) such as persulfides (RSSH) and polysulfides (RSS[subscript n]R, n>1). The purpose of this dissertation was to develop and assess new investigative tools that selectively detect or effectively deliver RSS under biologically relevant conditions. These chemical tools are of significant interest to the redox and molecular signaling communities and can be used to further define the prevalence, signaling capacity, and therapeutic effects of RSS, representing a significant advancement within the field. In chapter II, we describe the design, synthesis, and examination of an innovative, reaction-based fluorescent probe that exploits the double nucleophilic character of H2S by undergoing an intramolecular cyclization to release a fluorescent reporter. Specifically, we employed a highly unique selenosulfide moiety as a trigger for H2S detection. Given the distinct chemical differences between selenium and sulfur, we reasoned that the selenium half would provide access to chemical probes with high reactivity towards and selectively for H2S. Meanwhile, a suitable handle for improving the physicochemical properties of the probe, including its water solubility, cell permeability, and tissue specificity, could be supplied by the sulfide half. Using this novel design strategy, we successfully produced and assessed two H2S-selective probes: AL1, which displayed good water solubility, cell permeability, and overall biocompatibility, and AL2, which displayed cancer cell specificity. Concomitant to detection, chapter III discusses the synthesis and application of a self-reporting donor (ZL47), sensitive to reactive oxygen species (ROS). To accomplish this, we appended an aryl boronate ester to 7-hydroxycoumarin via an S-alkyl thiocarbonate linker allowing for the simultaneous release of COS, an H2S precursor, and a fluorescent reporter in response to hydrogen peroxide. In addition, we determined that ZL47 was a useful bifunctional tool with an ability to both image and alleviate oxidative stress in live cells, highlighting its potential as a theranostic. Chapter IV describes a novel ROS-activated persulfide donor (RAH393) that operates through a unique mechanistic pathway that avoids the production of a 1,4-quinone methide as a toxic byproduct, a major setback for ZL47 and related donors that employ a 1,6-elimination. In addition to confirming that its lactone byproduct was nontoxic, we were able to demonstrate the impressive cellular protection provided by RAH393 against high concentrations of ROS in live HeLa cells. Because this donor proved to be a valuable tool for generating persulfides in response to ROS, in chapter V, we investigated the therapeutic effects of an analogue that targets mitochondria (Mito-PD), the primary producers of ROS and a known site of RSS oxidation. To accomplish this, the mixed disulfide of RAH393 was modified and equipped with a lipophilic cation to facilitate mitochondrion uptake. Using confocal laser microscopy, we confirmed Mito-PD’s preferential release of persulfides within mitochondria from co-localization studies using AL1 (an H2S-selctive probe) and Mitotracker Red. In addition, we demonstrated that Mito-PD protects cardiomyoblast cells in culture (H9C2) from doxorubicin (DOX)-induced cardiotoxicity. Subsequent studies with JC-1, a fluorescent probe that reports on mitochondrial membrane potential, indicate that Mito-PD effectively preserves the membrane potential of DOX treated cells, increasing their viability. These initial studies highlight the therapeutic benefits of mitochondria-targeting RSS donors and their potential for mitigating DOX-induced cardiotoxicity.