Site-specific incorporation of unnatural amino acids into proteins, both in vivo and in vitro, is a promising technology with tremendous potential to advance studies in protein structure and function. The technique allows the incorporation of a vast diversity of functional groups that extends beyond the conventional mutagenesis of the twenty common amino acids. The rapid development of orthogonal PylRS/PylT pairs has resulted in an increasing number of novel unnatural amino acids that can be site-specifically introduced into proteins. This dissertation presents the syntheses of several unnatural amino acids for use in protein labeling and activation.;A bipyridine lysine was synthesized for the assembly of metal-binding proteins and its genetic encoding is shown. In addition, unnatural amino acids bearing reactive functionalities for bioconjugation reactions for the selective labeling of proteins were assembled. These include a variety of lysine analogs that can be applied to carbonyl/aminooxy condensations, thiol-ene reactions, and Diels-Alder cycloadditions. Moreover, bioorthogonal reaction partners such as aminooxy dyes, thiol, and tetrazine probes for subsequent protein labeling were prepared.;For the photoregulation of protein activity in live cells, several caged tyrosine analogs were synthesized. The assembly of caged phosphoryl tyrosines to study tyrosine phosphorylation on proteins by light activation is presented; as well as, the synthesis of tyrosine derivatives bearing ortho-nitrobenzyl caging groups to improve decaging kinetics and bioavailability of photocaged tyrosines. In addition, an isotope labeled, photolabile tyrosine was synthesized as a biophysical probe to study protein structure and dynamics by infrared spectroscopy.;Lastly, for the light-triggered regulation of oligonucleotides and gene expression, a caged thymidine phosphoramidite bearing a norbornene functional group was synthesized. This synthetic monomer can be incorporated into oligonucleotides and enable the dual functions of selectively modifying an oligonucleotide post-synthetically and have precise control over oligonucleotide activation by the use of light.
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