Development of bioorthogonal reactions and their applications for protein labeling
Lim, Reyna Koreen V.
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Bioorthogonal chemistry offers an exciting new strategy for the study of biomolecular dynamics and function in living systems. Compared to ligand-based approach where binding is noncovalent in nature, bioorthogonal chemistry relies upon specific, covalent attachment of probe molecules to the biomolecule of interest. A number of bioorthogonal chemistries were featured in Chapter 1 highlighting the key attributes of each reaction, with a particular emphasis on the reaction mechanism and kinetic studies. Chapter 2 proceeds to outline the development of a new photoinducible bioorthogonal reaction, termed 'azirine ligation' – a 1,3-dipolar azirine-alkene cycloaddition reaction between p -nitrophenylazirine and dimethyl fumarate that provides a rapid (∼2 mins) and highly selective route to protein conjugation at neutral pH and room temperature in biological medium. Chapters 3 to 5 describe the development of sequence-specific bioorthogonal reactions using phage display. In Chapter 3, the feasibility of displaying homoallylglycine (HAG) and homopropargylglycine (HPG) on phage surface was demonstrated. Efforts toward the development of sequence-specific photoclick chemistry and a new efficient strategy to incorporate the unnatural amino acids, HAG and HPG, on the surface of a phage via methionine biosynthesis inhibition (a metabolic approach) were summarized in Chapter 4. Chapter 5 describes the development of sequence-specific copper-free Sonogashira cross-coupling reaction and its utility in functionalizing a metabolically encoded alkyne-containing proteins in aqueous medium, in bacterial cells, and in live mammalian cells. Specifically in this chapter, a short peptide was identified through Cu-free Sonogashira cross-coupling reaction-based selection that led to rate enhancement and lower palladium complex loading.