Stimuli-responsive polymer-protein conjugates prepared by reversible addition-fragmentation chain transfer polymerization

Recently, there has been significant attention dedicated to developing routes for the conjugation of synthetic polymers to biomacromolecules. Particularly, the immobilization of stimuli-responsive polymers onto proteins, peptides, and nucleic acids can lead to intelligent bioconjugates with many potential applications, including the recovery of enzymes from complex solutions, molecular sensing, and the enhanced control of protein binding. While the conjugation of biologically-relevant molecules with synthetic polymers has been an important area of research for many years, more recently there has been significant interest in preparing well-defined synthetic polymer components with controlled molecular weights, low polydispersities, complex chain architecture, and enhanced functionality that gives rise to responsive behavior. Due to their ability to provide many of these characteristics, controlled radical polymerization (CRP) methods have significantly advanced synthetic capabilities in the field of "smart" polymer-biomacromolecule conjugate. Of the various CRP techniques, reversible addition-fragmentation chain transfer (RAFT) polymerization offers considerable benefits in this area because of its ability to be conducted in aqueous, non-denaturing media (e.g., buffer), the absence of a transition metal catalyst, and the wide range of monomers that can be successfully polymerized. This dissertation describes various routes by which RAFT polymerization can be successfully applied for the facilitated synthesis of thermoresponsive, polymer-protein conjugates.Three different synthetic routes were investigated. Initially, copper-catalyzed azide-alkyne "click" cycloaddition was employed to couple a preformed responsive polymer to a protein. A model protein, bovine serum albumin (BSA), was functionalized with an alkyne moiety by reaction of its free cysteine residue with propargyl maleimide.Conjugation of thermoresponsive polymers was also accomplished via a route that capitalized on the sulfur chemistry inherent to the RAFT process. End group activation of RAFT-generated poly(N-isopropylacrylamide) (PNIPAM) was accomplished by conversion of the thiocarbonylthio end groups moieties to thiols and subsequent reaction with excess of a bismaleimide. The maleimido end groups allowed near-quantitative coupling with model low molecular weight thiols or dienes by Michael addition or Diels-Alder reactions, respectively. Grafting of the maleimide-activated PNIPAM to another thiol-terminated polymer proved to be an efficient means of preparing block copolymers by a modular coupling approach. A similar reaction with free thiols in BSA or ovalbumin led to thermoresponsive polymer-protein conjugates.In another approach, the potential limitations of the grafting-to approach were overcome by grafting PNIPAM directly from a protein. BSA was functionalized with a RAFT agent, and subsequent room temperature polymerization of N-isopropylacrylamide (NIPAM) in aqueous buffer led to BSA-PNIPAM conjugates. Since the thiocarbonylthio portion of the RAFT agent remained on the free end group of the immobilized polymer, the chain extension by copolymerization with N,N-dimethylacrylamide (DMA) resulted in the formation of block copolymer-protein conjugates.Successful syntheses of the various conjugates by the above-mentioned methods were confirmed by combinations of UV-Vis, FT-IR, NMR spectroscopies, size exclusion chromatography, and polyacrylamide gel electrophoresis. In most cases, the conjugation procedures did not lead to significant reduction in bioactivity of the protein component. The thermoresponsive solution behaviors of the conjugates were also investigated. In aqueous solution at high temperatures, the BSA-PNIPAM bioconjugates formed stable nanoaggregates composed of dehydrated polymer and hydrophilic protein, as determined by dynamic light scattering.