Expanding the Genetic Code to Probe Biological Systems

The chemical modification of proteins has been a longstanding interest in the scientific community. In addition to the natural modifications necessary for life to function, unnatural covalent modifications are particularly useful because they facilitate research efforts that require the precise manipulation of protein, including the installation of fluorescent labels, post-translational modification mimics, and affinity reagents. Historically, appending such modifications onto proteins was achieved by generating covalent adducts onto one of the twenty canonical amino acids. However, such modifications are not site-selective, and may interfere with the native function of the modified protein. Genetic code expansion can overcome the limitations inherent to canonical amino acid modification, especially when bioorthogonal functional groups are incorporated. Using orthogonal aminoacyl-tRNA synthetase-tRNA pairs, one can reliably obtain homogenous samples of modified protein in a site-selective manner. In order to fully understand the steric requirements of a rationally designed pyrrolysyl-tRNA synthetase, several large meta-substituted phenylalanine derivatives were synthesized and incorporated into superfolder green fluorescent protein using this synthetase. All synthesized substrates were incorporated, albeit with differing incorporation efficiencies. Moreover, this synthetase was found to incorporate 3-formyl-phenylalanine, an aldehyde-based amino acid that can be directly installed onto proteins. Prior to this work, only indirect post-translational approaches could install aldehydes, and these methodologies were limited as to where the modification could occur. Aldehyde labeling occurs rapidly at neutral pH, and peptide cyclization has been accomplished using the aldehyde to form a thiazolidine linkage with an N-terminal cysteine, further demonstrating the rich chemistry available to aldehydes. Finally, efforts to optimize azidophenylalanine bioconjugation led to a kinetic investigation of copper-catalyzed click chemistry, which led to a revision of the currently accepted mechanism; it is proposed that copper-catalyzed click chemistry requires two separate copper-chelating events, with one equivalent of copper binding to azide, and another equivalent of copper binding to alkyne. The work presented herein demonstrates that phenylalanine derivatives are useful substrates for probing and manipulating biological systems, as well as providing opportunities for discovery in chemical biology