Tyrosine-selective protein modification with pi-allylpalladium complexes

Chapter 1 provides a general overview of current methods to modify the aromatic amino acids of proteins. Brief mention was given to bioconjugation reactions involving lysine, cysteine, and the carboxylates, but the focus was on developments of complementary modification strategies that target tyrosine and tryptophan residues.;Chapter 2 discusses the adaptation of pi-allyl palladium reaction conditions for protein compatibility. A catalyst system derived from palladium acetate and triphenylphosphine trisulfonate (TPPTS) was shown to activate allylic acetates and carbonates towards nucleophilic addition by p-cresol (a tyrosine mimic). Angiotensin I, a small peptide that contains a single tyrosine residue, as well as several other potentially cross-reactive functional groups, was also modified under these conditions. The selectivity of the peptide modification reaction for tyrosine was determined by MS fragmentation analysis.;Chapter 3 focuses on the modification of protein substrates and further reaction development. A fluorescent rhodamine allyl acetate was developed for high throughput screening of protein reaction conditions. Through tryptic digest of a modified protein and MALDI-TOF analysis of the resulting peptide fragments, the site of allylation was narrowed to the tyrosine-171 containing fragment. The protein scope of the reaction was investigated, and it was determined that many tyrosine containing proteins participate, with particularly high levels of reactivity being achieved for chymotrypsinogen-A and GFP. It was also confirmed that the reaction required basic pH conditions.;Chapter 4 discusses the application of the allylic alkylation reaction to protein lipidation. Farnesyl and C16/C17 alkyl chains were derivatized through the introduction of a taurine carbamate moiety to achieve water solubility. These molecules were found to be activated by the water-soluble palladium catalyst and were used successfully to alkylate tyrosine residues on model substrate chymotrypsinogen A. Lipidated chymotrypsinogen A was shown to associate with lipid bilayers.;The final chapter describes the development of a fluorescent lipidation substrate based on pyrene, and its application to the modification of the soluble portion of a transmembrane protein, H-NOX.