Synthesis of polymer bioconjugates using controlled radical polymerization

Covalent attachment of synthetic polymers to proteins provides materials with enhanced properties for a wide-range of applications in the fields of medicine, biotechnology and nanotechnology. New methods to prepare well-defined polymers for site-specific bioconjugation are described in this dissertation. Traditional approaches and recently developed techniques are described in Chapter 1.;Controlled radical polymerizations such as atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain transfer (RAFT) polymerization have emerged as powerful techniques to synthesize well-defined functional polymers. Chapters 2 and 3 describe the synthesis of pyridyl disulfide and aminooxy functionalized ATRP initiators for the polymerization of biocompatible and thermoresponsive polymers. The resulting polymers contained pyridine disulfide or aminooxy end-groups for conjugation to thiol or synthetically introduced ketone groups of proteins, respectively. Chemospecific bioconjugation to bovine serum albumin (BSA) was performed, and in the absence of the requisite functional groups, bioconjugates were not formed. Bioconjugation was characterized by gel electrophoresis.;Chapters 4 and 5 describe the synthesis of pyridyl disulfide and biotinylated chain transfer agents (CTAs) for RAFT polymerization. The functional CTAs mediated the polymerization of poly(ethylene glycol) acrylate (PEGA) and N-isopropylacrylamide (NIPAAm). The pyridyl disulfide alpha-functional p(PEGA) was conjugated to a thiol-modified short interfering ribonucleic acid (siRNA) through an asymmetric disulfide bond. Described in Chapter 5, biotin-maleimide heterotelechelic pNIPAAm was synthesized to prepare a streptavidin-BSA heterodimer. Protein immobilization is demonstrated and confirmed using enzyme-linked immunosorbent assay (ELISA).;Chapter 6 describes a method to form bioconjugates in-situ by polymerizing directly from a protein. BSA and V131C T4 lysozyme (T4L) were modified with ATRP initiators through disulfide or thioether bonds on Cys-34 and Cys-131, respectively. The proteins initiators were used to polymerize both NIPAAm and PEG methacrylate (PEGMA). UV-Vis and fluorescence activity assays of the T4L macroinitiator and resultant conjugate were performed and bioactivity was retained.