Defense Date


Document Type


Degree Name

Doctor of Philosophy



First Advisor

Matthew C.T. Hartman


The discovery of a method by which proteins of interest can selectively be labeled with a probe of choice intracellularly is a longstanding goal in chemical biology research. Conventional labeling techniques have utilized large domain tags but despite the development of small labeling molecules there have been no short peptide sequences known to covalently label a small molecule without the aid of an enzymatic process or metal chelation. We aimed to find a sequence of nucleophilic peptides that reacted covalently and specifically with electrophilic small labeling molecules using mRNA display. Our goal was to show that an electrophilic small labeling molecule that is brought in proximal distance to a protein of interest via affinity can result in nucleophilic attack by a neighboring nucleophilic amino acid to covalently label the protein of interest. Utilizing affinity between a small labeling molecule and a protein of interest to bring them spatially close to one another maximizes the chance that a covalent reaction can take place and provides selectivity between two components in a complex mixture. Towards this goal, we developed several electrophilic fluorescent small molecules. Covalent labeling was achieved using electrophilic bait in the form of sulfonate esters, a polyethylene oxide linker provided structural flexibility, and a fluorescent affinity tag containing a rhodamine backbone served as the potential binding site to a key peptide sequence encoded within a protein of interest. The synthetic routes to access our electrophilic rhodamine B and sulforhodamine 101 fluorophores were optimized. Key intermediates were produced and served as flexible points of modification to make various analogs of our desired electrophilic fluorophores. The affinity between proteins containing the peptide sequence and the fluorescent electrophiles were determined by fluorescence polarization. Covalent labeling was determined to be both time and concentration dependent. The expected published affinity between the peptides and fluorophore was not high enough to produce selective labeling. However, our small labeling molecules were found to be effective at labeling various proteins in vitro. In addition, our electrophilic fluorophores have been found superior to sulforhodamine 101 in live cell imaging of astrocytes.


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