This dissertation covers the development of combinatorial peptide library methodology to screen for short peptides with enhanced and sequence-specific reactivity; the design, synthesis, and screening of small molecule fluorescent probes for short peptide tags; and the identification of peptides with enhanced and sequence-specific reactivity.

Chapter 1 covers short peptides identified from a one-bead one-compound (OBOC) library that become fluorescent under photooxidative conditions. Only one peptide (AKPWGGDA) reproduced its fluorogenic reactivity in solution phase and the fluorescent photoproduct of this peptide was identified as a 4-aminoquinoline-2-carboxylic acid, which was presumably derived from tryptophan. Chapter 2 reviews the literature on peptide tags used for fluorescently labeling proteins in living cells with a specific emphasis on the biarsenical tetracysteine system. Chapter 3 reviews the literature on the sequence-specific structure and reactivity of short peptides. It also presents elements that may be applicable to enhanced and sequence-specific reactivity between short peptide tags and small molecule probes.

Chapter 4 covers OBOC and SPOT peptide library methodology developed in order to screen for the sequence-specific reactivity of short peptides with small molecule fluorescent probes. These methodological advancements were primarily in the synthesis, screening, and tandem mass spectrometry sequencing of OBOC peptide libraries, and, in particular, cysteine-rich OBOC libraries. Chapter 5 covers the design, synthesis, and screening of small molecule fluorescent probes that contained the dansyl fluorophore and esters that target lysine residues in short peptide tags. Short peptides were discovered that exhibited enhanced and sequence-specific reactivity with these fluorescent probes, and, in particular, with a tetrahydroisoquinoline N-hydroxysuccinimide ester probe in an OBOC library, in a SPOT library, and in solution phase. However, the sequence-specific reactivity of the short peptide tags is reduced outside of the bead environment. Chapter 6 covers the design, synthesis, and screening of small molecule fluorogenic probes. These fluorogenic probes consisted of FlAsH, amino coumarin alkyl esters, and benzoyl quinoline carboxyaldehydes. Short peptides were discovered that exhibited enhanced and sequence-specific reactivity for FlAsH and the amino coumarin alkyl ester probes.