The goal of protein modification is to take advantage of the structural complexity and bindingspecificity of a protein and pair it with the additional properties that a synthetic molecule can provide. Many strategies have been developed that target native amino acids present on the protein surface, such as lysine, but these modification methods tend to produce heterogeneous mixtures of products since there are often multiples copies of the residue displayed. To better control the level of modification, the N-terminus has become a popular target since each protein has only one. Our lab has recently developed an N-terminal modification strategy that uses 2-pyridinecarboxaldehyde (2PCA) to form an imidazolidine product. The reaction proceeds through an imine intermediate, which is then cyclized with the second amino acid residue to form the product. The reaction can be performed with all 20 native amino acids at the N-terminus, and high levels of conversion can be obtained. Despite this generality, we have found that over time the imidazolidinone conjugate with 2PCA can reverse to liberate the free, unmodified protein, and that the rate of reversal is dependent on the identity of the N-terminal residue. To better understand the factors that influence product stability, we undertook a mechanistic study of the reaction, using NMR kinetics and DFT calculations, to explore the most likely pathway leading to product formation. Through these studies, we found that N-terminal proline residues create the most stable product, and that glycine at position two can also have a secondary stabilizing effect. DFT calculations supported these findings and allowed for structural analysis of the reactive species and transition states that contributed to this stabilization. With a better understanding of how the reaction proceeds, future goals will be toward the development of 2PCA derivatives that can capitalize on these effects to form irreversible conjugates.

In addition to N-terminal modification, our lab has also developed several methods forsite-selective tyrosine modification using a tyrosinase enzyme. Tyrosinase is able to oxidize the phenol of tyrosine to a reactive quinone intermediate, which can then be captured by a variety of nucleophiles. Our goal was to use this chemistry and apply it toward the synthesis of cyclic peptides. We have demonstrated that linear peptides bearing a tyrosine and cysteine residue are able to be oxidized with tyrosinase, and the subsequently formed quinone can be coupled with the cysteine thiol to form a cyclic product. In addition to peptide substrates, we have also demonstrated that this chemistry works on peptide sequences displayed at the N- or C-terminus of a protein. Initial work has shown that the level of tyrosinase activity and selectivity is heavily influenced by the identity of the amino acids next to the tyrosine residue, and future work will be focused on engineering new tyrosinase variants that are less sequence dependent.