Development of copper-free click chemistry and application to imaging glycans in living systems.

Imaging biomolecules within living systems requires a means to distinguish the target from the surrounding components using a spectroscopic probe.

For proteins, genetically encoded tags such as GFP are routinely employed.

Although proteins continue to be central targets of cellular imaging, there is growing interest in visualizing biomolecules that are not amenable to genetic modification (e.g., glycans and lipids).

The bioorthogonal chemical reporter strategy offers a new avenue for labeling and visualizing many classes of biomolecules in vivo without the requirement of genetic manipulation.

In this approach, the cell's metabolic machinery is used to install into target biomolecules a bioorthogonal functional group (the chemical reporter), which is then covalently labeled in a second step with a probe.

The azide is the most widely used chemical reporter due to its small size, metabolic stability, and lack of reactivity with natural biofunctionality.

Accordingly, much work in recent years has been devoted to the development of bioorthogonal reagents capable of selectively tagging azides in biological systems, including phosphines via the Staudinger ligations and activated alkynes via a [3+2] cycloaddition.

This thesis describes the development of copper-free click chemistry, a bioorthogonal reaction between azides and fluorinated cyclooctyne reagents, and its application to the imaging of azide-labeled glycans in live cells and zebrafish embryos.

Chapter 1 provides an overview of various bioorthogonal reactions involving azides, important groundwork that set the stage for copper-free click chemistry in its current incarnation.

Chapter 2 describes the development of copper-free click chemistry, entailing both the synthesis of novel fluorinated cyclooctyne reagents and their kinetic evaluation in model reactions with azides.

Chapters 3--5 contain various applications of copper-free click chemistry, combined with azidosugar-based metabolic labeling techniques, to the in vivo imaging of glycans.

Chapter 3 describes the imaging of glycan trafficking in cultured cells.

Chapters 4 and 5 outline the application of copper-free click chemistry to the imaging of membrane-associated glycans in developing zebrafish larvae (chapter 4) and during zebrafish early embryogenesis (chapter 5).