Design, Synthesis and Biological Evaluation of cell-permeable small molecule probes

Open Access
Mottram, Laurie Frances
Graduate Program:
Doctor of Philosophy
Document Type:
Date of Defense:
August 07, 2007
Committee Members:
  • Blake R Peterson, Committee Chair
  • Philip C Bevilaqua, Committee Member
  • Ken S Feldman, Committee Member
  • Avery August, Committee Member
  • fluorescent probes
  • molecular probes
  • fluorophores
Molecular probes derived from fluorescein are widely used as tools for studies of cellular biology. This classic green fluorophore is particularly suited for cellular analysis by confocal laser scanning microscopy and flow cytometry due to its excitation maximum at 490 nm, closely matching the 488 nm spectral line of the argon-ion laser. Under physiological conditions (pH = 7.4), fluorescein predominantly exists as a highly hydrophilic dianion exhibiting excellent quantum yield. However, protonation of fluorescein to the monoanion is observed with the relatively high pKa of 6.5, rendering this dye much less fluorescent in acidic solutions. Additionally, fluorescein is relatively susceptible to photobleaching. Oregon Green, a more acidic 2’, 7’-difluoro derivative of fluorescein, was developed as a less pH-sensitive fluorophore. The appended fluorine atoms reduce the pKa of this dye to 4.8, substantially improving both photostability and fluorescence at low pH. Another more hydrophobic fluorescein analogue termed Tokyo Green was recently reported that replaces the carboxylate with a methyl group. This structural modification yields a highly fluorescent monoanion. We report a novel fluorophore termed Pennsylvania Green that combines the pH-insensitivity and photostability of Oregon Green with the hydrophobicity of Tokyo Green. To demonstrate the utility of the Pennsylvania Green fluorophore, we compared cellular membrane probes derived from 4-carboxy Tokyo Green and 4-carboxy Pennsylvania Green. Because of its lower pKa, only the Pennsylvania Green-derived probe enables facile visualization of acidic endosomes within living mammalian cells. We also report an improved synthesis of the Pennsylvania Green fluorophore and its related 4-carboxy derivative. 4-Carboxy-Pennsylvania Green was prepared from 4-iodo-3-methyl-benzoic acid methyl ester in three steps and 32% overall yield. This new synthesis is a notable improvement over our original methods. Chinese hamster ovary cells expressing O6-alkylguanine-DNA alkyltransferase fusion proteins were treated with Pennsylvania Green and Oregon Green linked to O6-benzylguanine (SNAP-Tag substrates). Analysis by confocal laser scanning microscopy revealed that Pennsylvania Green derivatives are substantially more cell permeable than analogous Orgeon Green Probes. Using the improved synthetic methods identified for the preparation of Pennsylvania Green, four novel red fluorophores were designed and synthesized: Penn Fluor 550, Penn Fluor 555, Penn Fluor 576 and Penn Fluor 609. The ability of biotinylated derivatives of these new fluorophores to image strepavidin fusion proteins in living mammalian cells was investigated. As a non-fluorescent molecular probe of potential drug targets and the steroid chaperone cycle, we synthesized dimeric molecules design to control estrogen receptor and heat shock protein 90 in living cells. Heat shock protein 90 (Hsp90) is an abundant ATP-dependent cellular chaperone that facilitates the folding of estrogen receptors (ERs) and other oncogenic proteins involved in cancer proliferation. This protein is under intense investigation as a target of anticancer drugs because breast cancer cells contain Hsp90 in an activated high affinity conformation that is particularly susceptible to Hsp90 inhibitors. These inhibitors include the anticancer agent geldanamycin (GDM), which is thought to inhibit the proliferation of cancer cells in part by blocking agonist-induced release of steroid hormone receptors such as ERs from Hsp90. Here, we report the synthesis and biological evaluation of chimeric ligands derived from estrogens and GDM designed to heterodimerize ER and Hsp90 proteins. These compounds were found to enforce interactions between these proteins in vitro and uniquely affect the subcellular localization of ER alpha in a cancer cell line. We also investigated a new class of immunosuppressant drugs called bis(trifluoromethyl)pyrazoles (BTPs). To probe the mechanism of action and molecular target of these new molecules, we designed BTP derivatives useful for affinity chromatography. A biotinylated BTP successfully identified drebrin as a potential target of BTPs. The binding of BTP to drebrin was further confirmed by western blot and confocal microscopy experiments.