Thesis advisor: Abhishek Chatterjee / This dissertation explores the development and applications of bioorthogonal chemistries, particularly focusing on genetically encoded methods for site-specific protein modification. Advances in genetic code expansion (GCE), such as the development of efficient aminoacyl tRNA synthetase (aaRS) enzymes, enable the incorporation of noncanonical amino acids (ncAAs) with bioorthogonal handles into proteins, allowing precise chemical modifications with diverse applications in basic biological research and therapeutic development. Here, I present new methodologies for chemoselective modification at encoded 5-hydroxytryptophan (5HTP) residues under mild oxidative conditions. I demonstrate that such reactions allow up to three distinct modifications at a single protein residue, facilitating the creation of functional multicomponent bioconjugates. This work also explores the use of our aaRS enzymes in nascent proteome tracking, allowing cell-type-specific labeling and time-resolved tracking of newly synthesized proteins in complex biological systems. Further, I demonstrate that genetically encoded biosynthesis and proteome-wide incorporation of 5HTP allows selective enrichment from mixed bacterial and mammalian environments when using our mild oxidative conditions to label the residues. These new bioorthogonal chemistries and enzymes for their proteomic incorporation enable the creation of antibody-drug conjugates, protein-protein conjugates, and enable access to new methods of nascent proteome labelling and enrichment. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.