A Simple Methodology for the Synthesis of Heterotelechelic Protein-Polymer-Biomolecule Conjugates

A synthetic protocol for the preparation of hetero-biofunctional protein-polymer conjugates is described. A chain transfer agent, SS-bis (alpha,alpha'-dimethyl-alpha ''-acetic acid) trithiocarbonate was functionalized with alpha,omega-pyridyl disulfide (PDS) groups, Subsequently, one of the PDS groups was covalently attached to bovine serum albumin (BSA) at the specific free thiol group on the cysteine residue through a disulfide linkage. The second PDS group remained intact, as it was found to be inaccessible to further BSA functionalization. The BSA-macro-reversible addition-fragmentation chain transfer (RAFT) agent was then used to prepare BSA-polymer conjugates via in situ polymerization of oligo (ethyleneglycol) acrylate and N-(2-hydroxypropyl) methacrylamide using an ambient temperature initiator, 4,4'-azobis [2,9-imidazolin-2-ethyl)propane] dihydrochloride in an aqueous medium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that the in situ polymerization occurred at the protein surface where the RAFT agent was attached and the molecular weights of the BSA-polymer conjugates were found to increase concomitantly with monomer conversion and polymerization time. After polymerization the remaining terminal PDS groups were then utilized to attach thiocholesterol and a flurophore, rhodamine B to the protein-polymer conjugates via disulfide coupling. UV-Vis and fluorescence analyses revealed that similar to 80% of the protein conjugates were found to retain integral PDS end groups for further attachment to free thiol-tethered precursors. (C) 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1399-1405, 2010