The design, synthesis and validation of new tools to study conformationally dynamic processes in molecular biology

This thesis is composed of two parts describing independent projects that have led to the design, synthesis and validation of new techniques to initiate conformationally dynamic processes in peptides and proteins. In Part 1 the evolution of a small molecule antagonist of HIV-1 viral entry into host cells is described. This research was conducted in collaboration with a number of collaborators to design and biologically characterize the inhibitors that were synthesized. Part 2 comprises the synthesis and photophysical validation of tetrazine-based phototriggers that can be incorporated with peptides and utilized to initiate conformational changes on the sub-nanosecond time scale for investigation by two-dimension infrared spectroscopy. Photophysical characterization of the newly developed phototriggers was conducted by Robin Hochstrasser and colleagues.;Small molecule CD4 mimetics were discovered that can initiate the viral entry process in the absence of the native protein-protein interaction. The design, synthesis, and structure activity relationships of a pilot library of related congeners was initially utilized to identify small molecules with enhanced binding affinity for the HIV-1 protein target, gp120. One of the affinity-enhanced small molecule CD4 mimetics proved useful in defined premature allosteric activation, a new method to release the energy stored within the HIV-1 viral entry protein complex prior to cellular encounter. Unfortunately, the compounds described in this chapter also enhance viral entry in to certain host-cell models (i.e., the small molecules are viral entry agonists). Through virtual screening, design, and synthesis in conjunction with detailed biological and structural characterization of inhibitor-protein interactions, the HIV-1 viral entry agonists were converted to antagonists utilizing interactions at two hotspots in the native protein-protein interaction.;In Part 2 the design, synthesis, and photochemical validation of the S,S-tetrazine phototrigger is described. The S,S-tetrazine chromophore photolyzes with sub-nanosecond time constants upon laser flash photolysis in the condensed phase. A method to inscribe the S,S-tetrazine phototrigger between the two cysteine residues in the peptide oxytocin, both in solution and during solid-phase peptide synthesis, was developed.