New methods for the synthesis and characterization of protein bioconjugates

The synthesis of protein bioconjugates and the analysis of those products has been an important area of research in the fields of medicine, materials and biology. This thesis will address the progress I have made in the development of new reactions for the transition-metal catalyzed synthesis of protein bioconjugates, the characterization of complex products formed by a three-component, Mannich type reaction on proteins, and efforts towards a photo-labile protecting group for amino-oxy compounds.;An efficient method for the reductive alkylation of proteins has been developed, which is catalyzed by [pentamethylcyclopentadienyl iridium (III) (4,4'-dimethoxy-2,2'-bipyridine)]2+ complex. The catalyst complex uses formate as the terminal reductant. The iridium hydride complex is a water-stable reductant that efficiently reduces Schiff bases formed on proteins at neutral pH and room temperature. As aldehydes are readily available from primary alcohols, the modification of lysozyme with poly(ethylene glycol) using the reaction has been demonstrated.;The rhodium(II) acetate catalyzed alkylation of tryptophan residues by vinyl diazo esters had previously been reported by the Francis lab. I have developed a strategy for the facile synthesis of substituted vinyl diazo esters, which now allows for the attachment of many different functional groups to tryptophan residues. In addition, I have demonstrated that high-temperature denaturation of hen egg white lysozyme enables alkylation of tryptophan residues, providing further evidence that the reaction is highly sensitive to the solvent accessibility of the side chains. Finally, I have shown that oligomerization of the diazo compound can occur in reaction solutions at pH 6.0, resulting in multiple modifications on a single tryptophan side chain.;One of the most significant challenges associated with the development of new bioconjugation reactions is the analysis of the protein products. I have developed a nuclear magnetic resonance based method for the characterization of proteins modified with isotopically enriched reagents. As a result, I confirmed the structure of the product of the three-component, Mannich type reaction on proteins as well as identified an unexpected product formed on tryptophan residues. Other residues were found to not form stable products with formaldehyde or the aniline reagents, except for a reduced disulfide, which formed a dithioacetal.;The final chapter outlines progress in the development of a photo-labile protecting group suitable for alkoxy-amines, which is also compatible with protein bioconjugation reactions.