The ability to study biomolecules within their naturally occurring context is often complicated by the complexity of biological systems. To overcome this obstacle, methods to selectively tag the biomolecule of interest with a fluorophore or affinity handle have been developed; once tagged, the desired biomolecule has a unique signal that allows it to be identified and studied within the complex biological setting. Ideally, these tags are small and non-perturbing so as not to modify the behavior of the biomolecule. However, while these design rules for bioorthogonal reagents seem simple, translating the rules into useful tools is challenging.;In the first chapter of this dissertation, I outline current methods to label biomolecules with small tags. I begin by describing genetically-encoded tags that are useful for protein-based studies. Next, the field of bioorthogonal chemistry and the ability to tag non-proteinaceous biomolecules is introduced. An overview of various chemical reporters used for live cell- and whole organism- studies is presented, and the future requirements for the field are discussed.;In Chapter 2, I describe a novel class of molecule for azide-based bioorthogonal tagging, the thiacycloalkyne. By introducing an endocyclic sulfur atom, the strain of the cycloalkynes can be fine-tuned. Taking advantage of this strategy, a previously reported thiacycloheptyne, TMTH, is shown to be exceptionally reactive with azides and to have promising qualities as a bioorthogonal reagent. In Chapter 3, I extend the endocyclic large heteroatom strategy to the design of dibenzoselenacycloheptynes. However, while these compounds can be trapped with azides in situ, they behave as triplet diradicals in the absence of a trap.;In Chapter 4, I focus on the development of a new strategy to bioorthogonally label glycans on specific cell-types. By creating trivalent reagents that comprise of a targeting aptamer, an azide-reactive cyclooctyne, and a fluorophore, I show that cell-selective glycan labeling can be achieved. These simple-to-use, modular reagents will be helpful in studying the geneses and outcomes of cell-specific glycosylation changes.;Finally, in Chapter 5, I present work towards the development of a novel method for single-step, non-toxic protein tagging. This method is based on the reaction between 1,2-aminothiols and cyanobenzothiazole, which has previously been shown to be selective, bio-friendly, and fast. Both phage display and rational peptide design are implemented in attempts to find a short peptide tag that can covalently react with cyanobenzothiazole, but only high-affinity binding and reversible reactions are achieved.
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