Selectivity by Design: Bioorthogonal Surface Functionalization and Fluorogenic Probes for Nucleic Acid Structure

Many problems in chemical biology require a molecule to act on one partner among many. This dissertation addresses that requirement in two settings: bioactive molecules attached to material surfaces by a bioorthogonal reaction needing no transition metal (Chapter 2), and fluorogenic probes that emit only when a bound nucleic acid restricts their rotation (Chapters 3 and 4). Auramine O links the last two: selected in Chapter 3, its derivatives are the probes of Chapter 4. Chapter 2 develops a surface functionalization route that uses no free transition metal, because the copper in existing catecholamine coatings is cytotoxic. A ʟ-DOPA derivative bearing a 1,2,4,5-tetrazine remains a tyrosinase substrate, and enzymatic oxidation deposits an adherent coating on titanium, silicon, glass and polymers. Molecules carrying trans-cyclooctene, from a small fluorophore to a 160 kDa enzyme, were grafted onto it by tetrazine ligation. Grafted enzymes kept native Michaelis constants over ten cycles, and grafted vancomycin inhibited Staphylococcus aureus. The method was extended to titanium bone screws and evaluated in a rabbit model. Chapter 3 selects the fluorophore for a split-aptamer microRNA platform, where it must stay dark in biological media unless the aptamer assembles. Two dapoxyl fluorophores were synthesized and compared with Auramine O. Both fluoresced strongly in cell medium, serum and plasma with no aptamer present, and serum albumin was the cause. A background independent of the aptamer is independent of the analyte, so Auramine O was selected instead. Chapter 4 builds a fluorescence displacement assay for human telomeric G-quadruplex ligands from an Auramine O derivative, because standard affinity methods are too slow to guide synthesis. Ten derivatives were screened against twenty G-quadruplex-forming sequences, yielding two probe-target pairs. The telomeric pair gave an assay whose inhibition constants reproduce published affinities to within about a factor of two. It reduced a focused library to one hit, EN01, and guided its Mitsunobu derivatization to P12A15, a fivefold gain in apparent potency. Circular dichroism confirmed target engagement independently of fluorescence. Screening the purchased building blocks before synthesis placed the lead in a shortlist of four after twenty-four measurements; docking ranked EN01 last of 210.