Design, Synthesis and Evaluation of Fluorescent Small Molecule Probes of Biological Systems

Open Access
DeGrazia, Michael
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
October 30, 2007
Committee Members:
  • Blake Peterson, Committee Chair
  • Ken S Feldman, Committee Member
  • Ayusman Sen, Committee Member
  • John Patrick Vanden Heuvel, Committee Member
  • PPAR
  • Cholesterol
  • Oregon Green
  • Cell Surface Receptors
  • Membrane Spanning
Fluorescent derivatives of biologically active molecules are essential tools for studying molecular interactions at the cellular level. Fluorescent tags and/or labels have been used to study protein-protein interactions, intracellular localization of biomolecules and organelles, and small molecule-protein interactions. We report here studies of fluorescent ligands of Peroxisome Proliferator Activated Receptors (PPARs), protein targets under intense investigation for the treatment of human metabolic disorders. Such fluorescent ligands have the potential to enable faster identification of novel therapeutics. To pursue this goal, we synthesized a variety of fluorescent small molecule PPAR ligands for high-throughput examination of potential endogenous and exogenous ligands that bind these proteins. We found that our fluorescein labeled PPAR ligands bound PPARγ and PPARα with high selectivity and were good candidates for fluorescence polarization assays. In addition, we also utilized Pennsylvania Green as a more hydrophobic analogue of fluorescein to generate cell-permeable versions of these PPAR probes to develop a novel method for studying PPAR ligand binding in living cells, which can be further extended to other related members of the nuclear hormone receptor family. To extend previously reported studies on 3β-cholesterylamine derivatives that function as non-natural cell surface receptors, we synthesized a series of Oregon Green labeled symmetrical dimers of 3β-cholesterylamine as potential precursors to membrane spanning artificial cell surface receptors. We examined these compounds using confocal microscopy, flow cytometry, and spectroscopy to identify the optimal structural requirements for insertion into membranes of living mammalian cells. Modeled after our best compound, we synthesized an asymmetrical 3β-cholesterylamine dimer bearing Oregon Green as an extracellular fluorescent label and biotin as an intracellular ligand of streptavidin expressed in the cytosol. Examination of this dimer using confocal microscopy showed low cellular association, which could be due to the non-protonatable amide bearing biotin. Further studies to generate synthetic analogues bearing protonatable groups on the intracellular motif could project biotin into the cytosol and recruit streptavidin to the inner-leaflet of the membrane. Through intracellular control of protein localization it is postulated that asymmetrical cholesterylamines of this type could be used to influence internal cell signaling and cellular uptake pathways. Artificial cell surface receptors that span cellular membranes could provide a new strategy for controlling therapeutically relevant intracellular pathways.