| Chronic exposure to arsenic leads to a variety of cardiovascular diseases, diabetes, and increased cancer risk, while acute poisoning results in painful death. Conversely, arsenic is used as a homeopathic medicine, and has been approved for use in certain blood cancers. Despite the established physiological responses to arsenic exposure, very little is known about the actual molecular targets of this metalloid beyond the knowledge that As(III) binds preferentially to dithiol motifs in proteins. To aid in the study of arsenic effects on dithiol proteins, we have designed and evaluated several arsenic-based molecular probes, and synthesized dicysteine peptides as models of specific secondary structures. Some arsenic probes are UV-silent to allow changes in peptide structure to be evaluated by circular dichroism. UV-absorbing probes allow for accurate measurement of binding stability and kinetics. This work demonstrates that arsenic binds rapidly, stoichiometrically, and with high affinity little influenced by the secondary structure and cysteine arrangements of peptide dithiols. Several fluorescent arsenicals were also synthesized to visualize the proteins and thiol-rich surfaces to which arsenic binds. The concept of "masked" fluorophores was developed, where the emission of a fluorescent arsenical is diminished via conjugation with a dithiol quencher moiety, but is restored when the target dithiol (e.g. protein) displaces the quencher. Several probes based on this concept were synthesized and evaluated.; To keep dithiol proteins in the reduced state necessary for arsenic binding, a new class of phosphine reductants was devised. The methyl-esters of tris-(2-carboxyethyl)phosphine (TCEP), proved to be more reactive at lower pH values than TCEP. One of them, trimethyl-TCEP, can cross model biological membranes more rapidly than dithiothreitol. To aid in the characterization of these reductants, a dicysteine peptide was iteratively designed from the thioredoxin active site. The peptide shows a significant increase in its tryptophan fluorescence upon reduction.; The tools, techniques, and results described herein will add to the understanding of arsenic-protein interactions, aid future studies of redox-active proteins, and provide the means for both reduction and oxidation of dicysteine peptides under acidic conditions. |
