Developing chemo-proteomics methods for tyrosine conjugation and studies of organometallic complexes of gold.

Abstract

**Please note that the full text is embargoed until 05/14/2025** ABSTRACT: The incredible changes in MS instrumentation since the late 1980s with the advent of electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) has revolutionized protein chemistry. Mass spectrometry coupled with chemical crosslinking is a powerful tool to decipher protein structure and protein-protein interactions. A majority of commercially available crosslinkers target only few amino acid residues such as lysine, arginine and cysteine, thereby limiting the utility of this technology. The prospect of using chemical reagents to target amino acids is challenging since covalent labeling experiments are performed under physiological aqueous conditions that may be detrimental to the stability of these reagents. The second chapter explores the utility of a chemical reagent, N-phenyl-1,2,4-triazoline-3,5-dione (PTAD) to selectively label tyrosine residues using tandem mass spectrometry. While this reagent has been used previously to label tyrosine residues in standard proteins like bovine serum albumin (BSA), the utility of PTAD to covalently label tyrosine in complex protein samples has not been previously investigated. This work evaluates the performance of PTAD to label tyrosine residues in complex protein samples like cell lysates. The fragmentation characteristics of these labeled proteins have been investigated using tandem mass spectrometry. The third chapter expands on the utility of PTAD to develop a new chemical crosslinker that is capable of capturing protein-protein interactions by selectively targeting tyrosine residues. The efficiency of this novel crosslinker was studied by tandem mass spectrometry experiments involving samples of varying complexity ranging from standard proteins to cell lysate. In addition, we have also studied the products obtained from the reaction of this crosslinker with the SARS-CoV-2 S1 spike protein to provide a complementary structural biology perspective on this important protein that is implicated in the spread of the global COVID-19 pandemic. The fourth chapter focuses on developing a methodology to selectively isolate zinc binding proteins using immobilized metal affinity chromatography. A zinc nitrilotriacetic acid column containing a sepharose stationary phase was designed and evaluated using proteins that are well known to exhibit an affinity to zinc such as bovine serum albumin and carbonic anhydrase. The experimental conditions to facilitate these binding interactions were optimized by adopting a pH- based elution approach dependent on the pKa of amino acids like cysteine and histidine that are usually present in the zinc binding motif in proteins. This methodology was used to investigate the zinc binding site of carbonic anhydrase by tandem mass spectrometry experiments. The capability of this zinc nitrilotriacetic acid column to separate protein mixtures was evaluated by introducing samples of the HeLa cell to selectively isolate potential zinc binding proteins using this immobilized metal affinity chromatography approach. The fifth chapter of this work describes an investigation of the synthesis of organic azide complexes of copper(I) and silver(I) supported by N-heterocyclic carbene ancillary ligands. Auxiliary ligand free coinage metal N-heterocyclic carbene complexes [(SIPr)M][SbF6] have been synthesized though metathesis reactions of (SIPr)MCl (M= Cu, Ag, Au) with AgSbF6. The structure and bonding properties of these coinage metal complexes were investigated by various spectroscopic techniques and X-ray crystallography, The final chapter of this work focuses on the study of gold complexes of olefins like ethylene and propylene supported by bidentate ancillary ligands with electron donating substituents. The π-back bonding interactions and its influence on olefin binding properties exhibited in these rare gold(I) complexes are discussed using results from X-ray crystallography and NMR experiments.