Proteins are ubiquitous in numerous pharmaceutical, technological, and industrial applications. However, these biomolecules are prone to stability issues due to internal and/or external stressors. One way to overcome such challenges is through the conjugation of polymers onto the surface of proteins. While protein-polymer conjugates have shown increased in vivo half-life and increased immunogenicity, reduction in activity has been observed due to either steric hindrance of polymers near the active site or changes to the protein’s secondary structure. The use of traceless linkers to form protein-polymer conjugates allow for recovery of native protein, and thus restored protein activity upon exposure to a trigger. In this dissertation, syntheses of protein-polymer conjugates and other drug systems incorporating such reversible linkages are presented. Much work has been carried out over the years with the use of traceless linkages. In chapter 1, a mini literature review covering recent publications of the use of traceless linkages for protein and peptide applications is presented. These updates from the past few years will be incorporated into a comprehensive review the Maynard group plans to publish in Bioconjugate Chemistry. Chapters 2-4 will discuss the work carried out to synthesize protein-polymer conjugates via a heterobifunctional photocleavable linker, a type of traceless linker that responds to UV irradiation. In chapter 2, a new method to prepare noncovalent enzyme nanogels that utilizes a photocleavable monomer as a hybrid approach is reported. Photoirradiation cleaved the linkage between the polymer and protein to afford the poly((polyethylene glycol) methacrylate)-based noncovalent nanogels and enhanced enzyme activity. In chapter 3, the preparation of poly((polyethylene glycol) acrylate)-lysozyme (pPEGA-Lys) conjugates via a photocleavable linkage is presented. The conjugates were made by both grafting-to and grafting-from approaches. This work provided further evidence that reversing conjugation is successful for activity recovery for ortho-nitrobenzyl linked protein-polymer conjugates. Finally, in chapter 4, preliminary work towards the development of pNIPAM-based noncovalent enzyme nanogels and protein-polymer conjugates via a photocleavable linkage is discussed. This work aimed to demonstrate the versatility of the systems developed in Chapters 2 and 3.
The incorporation of traceless linkages has been applied to other applications beyond proteins and peptides. In chapter 5, the work towards scaling up an optimized abuse-deterrent opioid prodrug sequence is reported. Preparation of this material was carried out to allow for further biological studies.