Chemical Tools to Interrogate Protein Sulfenylation by Fluorescence Microscopy and NMR.

The cellular redox environment is a complex milieu of reactive oxygen, nitrogen, sulfur, carbon, and lipid species (ROS, RNS, RSS, RCS, RLS respective; collectively, RxS) that all have functional group interplay. Nowhere is this more apparent than on cysteine, which is readily oxidized to a wide range of functional groups. One group, the sulfenic acid, is the first oxidative step forward into this web. It has been studied extensively with biochemical and proteomic tools based upon the active methylene compound dimedone, but study of the localization of sulfenic acids has been limited to fixed cells due to the use of constitutively active dyes and antibodies to image. Herein we propose a design for a chemical ‘switch’ that would change the photoelectronics of an attached dye upon conjugation to a sulfenic acid, which he have developed in Chapter 2. This is based upon the observation that only one of the two α-protons is necessary for the reaction. We have successfully synthesized an aminonaphthalene-based dye with a fluorodimedone, F-DiNap, that allows for ratiometric imaging of live cells on TIRF and confocal microscopy. We have demonstrated F-DiNaps utility as a live-cell probe by collecting images before and after stimulation, as well as distinguishing established models of cancer malignancy with the cell lines that have been transfected with the transcription factor Snail. Critically, we have shown that our probe both increases selectivity over aldehydes, which are post-translational modifications that are formed from the same oxidative stimuli as sulfenic acids; and that the rate of these probes is dramatically enhanced by over a thousand-fold by the addition of the fluorine. Serendipitously, 19F is a minimalist reporter group for nuclear-spin instrumentation such as NMR and MRI. In Chapter 3 we have leveraged this capacity to study sulfenic acids in NMR, synthesizing several probes based on minimalist fluorobarbituic acids, or F-Barbs. We have made preliminary studies into profiling these probes rates of conjugation, selectivity over other functional groups, and reactivity with biological samples, including purified proteins and lysates. The probes will be a large step towards imaging and interrogating the biological role of sulfenic acids in vivo.PhDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/133267/1/ctchem_1.pd