Therapeutic agents including small molecules and proteins compose the majority of marketed pharmaceutics and are indispensible to modern medicine. Although many of these therapeutic agents have proven to be excellent drug products, others possess limitations that have detracted their full potential. In such cases, strategic covalent modification can be employed to reversibly or irreversibly modify a known drug substance in order to improve one or more of its therapeutic properties. Due to the broad variability and availability of functional handles on any given therapeutic molecule, covalent modification strategies vary substantially from one drug to another and must be rationally tailored to meet the needs of the desired application. This dissertation provides several unique examples in which strategic bioconjugation was employed with the goal of enhancing the therapeutic potential of small molecules and proteins to address unmet challenges in diverse therapeutic areas.Biomacromolecules such as proteins, peptides, and nucleic acids represent important therapeutic agents that have become indispensable for the treatment of a broad array of diseases ranging from hemophilia to cancer. The practice of covalently attaching the polymer polyethylene glycol (PEG) to these biologics, known as PEGylation, has further enabled the development and regulatory approval of at least 15 distinct biotherapeutics by extending circulation time and reducing immunogenicity. Despite its overwhelming success over the last 40 years, evidence from a number of independent research groups has revealed that antibodies can be generated in animals and humans that specifically bind to PEG and can compromise the safety and efficacy of PEGylated therapeutics. More concerning are the increasingly frequent reports of high incidences of pre-existing anti-PEG antibodies in healthy individuals who have never received PEGylated medicines. Chapter 1 discusses the clinical reports of immune responses against PEGylated biomolecules, how anti-PEG antibodies can affect the safety and efficacy of these therapeutics, as well as the incidences and effects of pre-existing antibodies. Additionally, Chapter 1 examines what is known about the induction of antibodies against PEG, factors that may contribute to this immune stimulation, and methods for the detection of anti-PEG antibodies. Finally, possible strategies for overcoming PEG immunogenicity are considered and perspectives are offered on the future of PEGylated biotherapeutics.Human vault nanoparticles are naturally occurring, uniform, barrel-shaped protein nanostructures that have demonstrated aptitude as drug delivery vehicles due to their large size, abundant and easily modifiable side chains, and biocompatibility. Vaults are readily internalized by over 90% of CD4+ T-cells, which are the key immune cells targeted by Human Immunodeficiency Virus (HIV). Chapter 2 explores the utility of these protein nanoparticles as a targeting vehicle for the delivery of three covalently attached antiretroviral drugs to vulnerable cell populations. Drugs were conjugated to vault lysine residues, by first modifying each drug with a protein reactive group in such a way that the active drug could be released in its unmodified form following hydrolytic or enzymatic cleavage of an ester linkage between the protein and drug. Good drug loading was achieved, and the conjugation efficiencies were found to correlate with the hydrophobicity of each drug. All vault-drug conjugates performed similarly to free drug in in vitro infection assays, and are expected to demonstrate enhanced targeting to CD4+ T-cells in vivo. Prescription opioids, though often necessary for pain management, are highly addictive and are consequently abused with increasing frequency. Over the last 20 years, deaths involving opioids have increased dramatically. There are few FDA approved abuse deterrent opioid formulations available, and even fewer are robust enough to deter motivated users. Chapter 3 outlines the design and synthesis of a dual enzyme responsive prodrug platform for oxycodone. This prodrug is designed to only release the active drug upon ingestion and yields no active drug following injection, snorting, nor when these methods of administration are preceded by physical tampering such as acidification, grinding, microwaving, etc. Various chemistries were investigated to provide relevant and stable, but reversible covalent linkages. These linkages were assessed for hydrolytic stability over a broad pH range to simulate accelerated release conditions that could be employed by drug users. The prodrugs showed resistance to hydrolytic degradation and were determined to release only in the presence of gastric enzymes trypsin and chymotrypsin. Synthetic efforts and observation of unexpected side-products are also discussed.Regulation of human growth hormone (GH) signaling has important applications in the remediation of several diseases including acromegaly and cancer. Growth hormone receptor (GHR) antagonists currently provide effective means for suppression of GH signaling. However, these small 22 kDa recombinantly engineered GH analogs exhibit short plasma circulation times. To improve clinical viability, between 4-6 molecules of 5 kDa PEG are nonspecifically conjugated to the 9 amines of the GHR antagonist designated as B2036 in the FDA-approved therapeutic pegvisomant. PEGylation increases the molecular weight of B2036 and considerably extends its circulation time, but also dramatically reduces its bioactivity, contributing to high dosing requirements and increased cost. As an alternative to nonspecific PEGylation, Chapter 4 reports the use of genetic code expansion technology to site-specifically incorporate the unnatural amino acid propargyl tyrosine (pglY) into B2036 with the goal of producing site-specific protein-polymer conjugates. Substitution of tyrosine 35 with pglY yielded a B2036 variant containing an alkyne functional group without compromising bioactivity, as verified by a cellular assay. Subsequent conjugation of 5, 10, and 20 kDa azide-containing PEGs via the copper catalyzed click reaction yielded high purity, site-specific conjugates with >89% conjugation efficiencies. Site-specific attachment of PEG to B2036 is associated with substantially improved in vitro bioactivity values compared to pegvisomant, with an inverse relationship between polymer size and activity observed. Notably, the B2036-20 kDa PEG conjugate has a comparable molecular weight to pegvisomant, while exhibiting a 12.5 fold improvement in half-maximal inhibitory concentration in GHR-expressing Ba/F3 cells (103.3 nM vs. 1289 nM). This straightforward route to achieve site-specific GHR antagonists is expected to be useful for GH signal regulation.Given the potential issues associated with the use of PEG in protein-polymer conjugates as outlined in Chapter 1, there is significant interest in the development of structural analogs to PEG that offer added functionality while retaining the beneficial properties of the polymer. In addition to its immunogenic concerns, PEG is not biodegradable and can also exhibit significant heterogeneity in molecular weight. Lack of biodegradability has been linked with safety concerns such as vacuole formation in liver and kidney tissue following repeated dosing. Additionally, the heterogeneity inherent to the molecular weight of PEG could potentially lead to variability in efficacy when conjugated to therapeutic proteins. Chapter 5 details efforts to overcome these obstacles by exploring the synthesis of degradable and uniform PEG analogs. Degradable PEG analogs were synthesized from unsaturated crown-ether monomers by ring-opening metathesis polymerization (ROMP) and found to readily depolymerize in aqueous conditions in the presence of Grubbs III catalyst. These polymers contained aldehyde end groups that were conjugated to the model enzyme lysozyme. Depolymerization of the polymer from the protein was demonstrated and the effect of the reaction on the activity of the conjugate was evaluated. Chapter 5 also outlines and discussed efforts toward the synthesis of high molecular weight, uniform PEG analogs prepared by iterative monomer addition of heterobifunctional thiol-vinyl ether PEG oligomers.