The ability to attach synthetic functional groups to specific locations on protein targets has become essential to the field of chemical biology. The resulting bioconjugates can be used for a multitude of applications, including the study of biological function and the creation of new materials. This work outlines the development of a site-specific N-terminal transamination reaction that is mediated by the biological cofactor pyridoxal 5'-phosphate (PLP).

Using a variety of peptide and protein substrates, PLP was shown to mediate a transamination reaction at the N-terminus. The resulting ketone or aldehyde products were further elaborated with functionalized alkoxyamines to form stable oxime linkages. Optimization of the reaction conditions indicated that this transformation could take place under mild conditions that would not disrupt the folded structure or function of the target. Even in the presence of internal lysine residues, PLP-mediated transamination led to the formation of a single product that was specific to the N-terminus.

This site-specific strategy was applied to the regioselective labeling of antibody substrates. It was possible to modify a variety of antibody targets using PLP followed by several types of functionalized alkoxyamines. ELISA confirmed that this transformation occurred under conditions mild enough to maintain antigen-binding function. Additionally, immuno-dot blot analysis established that modified antibodies were able to bind their intended target.

In certain cases, PLP has been shown to form covalent N-terminal adducts with the target protein. A careful look at the proposed mechanism for this transformation indicated that the use of alternative aldehydes might lead to improved yields of the desired products with minimal byproduct formation. Derivatives of two different classes of aldehydes were synthesized and used to mediate N-terminal transamination.

Further characterization of the reaction products was necessary for optimization of PLP-mediated transamination. A twenty-member tetrapeptide library was constructed in order to study the effect of the N-terminal residue on the product distribution. Many N-terminal residues led to high yields of the desired products, and only a small number led to significant amounts of PLP adducts. These studies have increased our predictive power for the reaction, both in terms of improving conversion and suppressing reaction byproducts. Furthermore, these studies have led to the development of a platform that will be useful for either screening alternative aldehydes or identifying amino acid sequences that lead to excellent conversion with PLP.