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Interfacing chemistry and biology for new approaches to site-selective peptide modification and mycobacteria-specific fluorogenic probes


Selective labeling of biological matter enables not only basic research but also improved diagnostic platforms and therapeutics. Bioorthogonal chemistry provides a set of tools for researchers to study biomolecules of interest by inserting selectively reactive chemical partners into living systems. In the case of proteins, genetic manipulation can allow the introduction of uniquely reactive peptide sequences for small-molecule or enzymatic labeling. For molecules that cannot be genetically manipulated, metabolic probes provide an attractive alternative; by exploiting gaps in substrate specificity, a biomolecule can be modified with a bioorthogonal functional group without the need to extensively engineer the living system. In some cases, these biomolecules are organism- or cell type-specific, allowing them to be used as diagnostics for cancer or bacterial infection.

In Chapter 1 of this dissertation, I provide an overview of commonly used methods for selective protein modification as well as for selective labeling of the bacterial cell wall. In Chapter 2, I describe the computation-guided rational design of a cysteine- and lysine-containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives. Our data show that the cysteine residue reversibly reacts with the nitrile group on the CBT moiety to form an intermediate thioimidate, which undergoes irreversible SN transfer to the lysine residue, yielding an amidine-linked product. The concepts outlined herein lay a foundation for future development of peptide tags in the context of site-selective modification of lysine residues within engineered microenvironments. In Chapter 3, I outline our ongoing efforts to optimize this motif for protein labeling. In Chapter 4, I describe the synthesis and characterization of several mycobacteria-selective environment-sensitive fluorogenic probes as well as their potential applications in studies of host-pathogen interactions and diagnostics.

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