Water-Soluble BODIPY Dyes for Membrane Potential Imaging

Fluorophores based on 4,4-difluoro-4-bora-3a,4a,-diaza-s-indacene (BODIPY) are used widely as biological probes and labeling agents because of their brightness and highly modifiable scaffold. This dissertation describes the design, synthesis, and characterization of a variety of BODIPY-based probes aimed towards membrane potential imaging. We first synthesized a probe based on a zwitterionic BODIPY, but found its synthesis to be challenging and not generalizable. We then designed and synthesized new water-soluble BODIPYs featuring an ortho-sulfonated meso-aromatic pendant ring and a range of 2,6-substituents: ethyl, hydrogen, carboxylate, amide, and cyano. The condensation methodology we developed is high-yielding (29-61% over three steps), installs the water-solubilizing sulfonate moiety in the same step the fluorophore is formed, and is amenable to pyrrole building blocks of a wide solubility and nucleophilicity range. This new BODIPY scaffold is water-soluble without the need for added detergent and displayed impressive quantum yields of fluorescence in the ϕfl = 0.70–0.99 range. These BODIPYs were functionalized with a lipophilic photo-induced electron transfer (PeT) donor to act as membrane-localized voltage-sensitive dyes, or VoltageFluors. Altering the 2,6-substituents allowed the voltage-sensitivity to be tuned, and in general we found the order of voltage sensitivity to be ethyl < hydrogen < carboxy < amide > cyano. 2,6-amido based VoltageFluor amidemH is the most voltage-sensitive BODIPY probe to date, with a 48% ΔF/F per 100 mV. Two other BODIPY VoltageFluors, TMmOMe and carboxymOMe, display voltage sensitivities of 33 and 24% ΔF/F and were used to obtain real-time membrane potential dynamics from neurons and cardiomyocytes. In addition to these BODIPY VoltageFluors, we also report on additional strategies to increase the hydrophilicity of BODIPY, 2,6-chlorination methodologies, and alternate routes towards the VoltageFluor scaffold. Forming monoalkoxy BODIPYs by functionalizing the boron with an alcohol or adding a second ortho-sulfonate to the meso-pendant ring both increased hydrophilicity of BODIPY VoltageFluors and improved membrane localization. We found 2,6-chlorination of 1,3,5,7-tetramethyl BODIPY with the ortho-sulfonated meso-pendant ring could be accomplished with N-chlorosuccinimide or 1-chloro-1,2-benziodoxol-3-one, but the resulting 2,6-chloro BODIPY was highly susceptible to decomposition when exposed to aqueous conditions or slightly acidic conditions, such as silica gel chromatography. Finally, we developed two alternate routes to the VoltageFluor scaffold that complement the typical Heck coupling route. The first hinged on replacing the Heck coupling with a Suzuki coupling, and we synthesized two different boronate ester molecular wire Suzuki coupling partners. The second was a linear, bottom-up route, in which the entire VoltageFluor scaffold except the fluorophore is assembled, and then the fluorophore condensation is performed. Both routes are generalizable to a wide range of BODIPY and xanthene fluorophores. Together, these projects add valuable synthetic routes to a range of highly water-soluble BODIPY dyes that can be applied directly to voltage-sensitive dyes or more broadly for biological imaging.

Advisor: Miller, Evan W