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AbstractProtein bioconjugation is an interdisciplinary field that integrates advances in chemistry and molecular biology to engineer protein-payload conjugates with great biomedical significance. By selectively modifying a designated site of the target protein, site-specific bioconjugation techniques create homogenous protein conjugates to preserve their physiological function and improve their toxicological profile. In Chapter I we reviewed the contemporary site-specific bioconjugation techniques to modify proteins chemically. We believe that site-specificity can be achieved from chemoselective chemistries that only modify one kind of amino acid residue. Alternatively, site-selectivity can also be achieved by developing chemistries that localize the chemical ligation on proteins' unique molecular and structural motifs. The former strategies, or namely chemoselective site-specific bioconjugation (CSSB), are the most effective when modifying low-abundance amino acids on small or middle-size proteins, while the later strategies, also called regioselective site-specific bioconjugation (RSSB), can achieve site-specificity when modifying larger proteins, such as albumin and immunoglobulins. Having realized the pros and cons of each method, we then developed novel bioconjugation strategies to modify different kinds of proteins.In Chapter II, we discussed utilizing proximity-driven Human Serum Albumin (HSA), the most abundant protein in human blood plasma. It plays a critical role in the innate transportation of numerous drugs, metabolites, nutrients, and small molecules. HSA has been successfully used clinically as a non-covalent carrier for insulin (e.g., Levemir), GLP-1 (e.g., Liraglutide), and paclitaxel (e.g., Abraxane). However, none has been approved for clinical use. Using the one-bead one-compound (OBOC) technology, we generated combinatorial peptide libraries containing myristic acid, a well-known binder to HSA at Sudlow I and II binding pockets. We then used HSA as a probe to screen the OBOC myristylated peptide library for reactive affinity elements (RAEs) that can specifically and covalently ligate to the lysine residue at the proximity of these pockets. Several RAEs have been identified and confirmed to be able to conjugate to HSA covalently. The conjugation can occur at physiological pH and proceed with high yield within 1 hour at room temperature. Tryptic peptide profiling of derivatized HSA has revealed two lysine residues (K225 and K414) as the conjugation sites, which is much more specific than the conventional N-hydroxy succinimide ester lysine labeling strategy. The RAE-driven site-specific ligation to HSA was found to occur even in the presence of other prevalent blood proteins such as immunoglobulin or whole serum. Furthermore, these RAEs are orthogonal to the maleimide-based conjugation strategy for Cys34 of HSA. Together, these attributes make the RAEs promising reagents to derivatize HSA, both in vitro and in vivo, for many biomedical applications.In Chapter III we leveraged proximity-driven bioconjugation strategies as a therapeutic method to cure COVID-19. By May 2022, the COVID-19 pandemic has become a major public health incident in the 21 century. The emerging pandemic demands the rapid development of diagnostic and therapeutic methodology. Based on the One-bead-one-compound (OBOC) high-throughput screening technology, several peptides with nanomolar binding affinity towards wild-type and variant SARS-CoV-2 spike protein, the characteristic surface proteins from the SARS-CoV-2 virus, were successfully discovered. These peptides have been successfully immobilized on porous nanofibers for sensitive SARS-CoV-2 detection. Meanwhile, serving as the warhead, these peptides can form transformable nanoparticles when they are associated with KLVFF-Por transformable sequence, blocking spike protein-ACE2 acceptor interaction to inhibit viral infection. These affinity peptides were further optimized through OBOC libraries to derivatize reactive peptidomimetics that can covalently ligation COVID-19 spike proteins through proximity-induced ligation, which can be potentially used as potent covalent inhibitor.In Chapter IV we discussed the development of universal bioconjugation strategies at N-terminal aspartate and glutamate using the Ugi 3-component reaction. Ligation at N-terminus has become a powerful approach for site-specific protein bioconjugation. Current chemical strategies can modify generic protein N-terminus, as well as specific N-terminal amino acid residue including cysteine, proline, and glycine. However, few methods have been developed for N-terminal specific aspartic/glutamic acid ligation. Herein we wish to report using the 3-component Ugi reaction to selectively modify N-terminal aspartic/glutamic acid at protein and peptide levels associated with isocyanide and aldehyde. The conjugation can be performed under mild conditions and demonstrates N-terminal selectivity. Using this approach, human serum albumin (HSA), which has an aspartic acid moiety at N-terminus, can be efficiently modified. This strategy enriches N-terminal ligation strategies in protein bioconjugation as novel protein-based therapeutic and diagnostic approaches.