Lighting Molecules with Light: Fluorescence Activation with Photochromic Oxazines

Photoactivatable fluorophores switch from a nonemissive to an emissive state upon illumination at an activating wavelength and then emit after irradiation at an exciting wavelength. The interplay of such activation and excitation events can be exploited to switch fluorescence on in a defined region of space at a given interval of time. In turn, the spatiotemporal control of fluorescence translates into the opportunity to implement imaging and spectroscopic schemes that are not possible with conventional fluorophores. Specifically, photoactivatable fluorophores permit the monitoring of dynamic processes in real time as well as the reconstruction of images with subdiffraction resolution. These promising applications can have a significant impact on the characterization of the structures and functions of biomolecular systems. As a result, strategies to implement mechanisms for fluorescence photoactivation with synthetic fluorophores are particularly valuable. In this context, the work described in this thesis explores operating principles to activate the emission of organic chromophores with the aid of photochromic auxochromes. Specifically, I synthesized molecular dyads combining fluorescent and photochromic components within their covalent skeleton. These photoswitchable compounds all have an oxazine as their photochromic component and differ in the nature of their fluorescent component. In particular, I incorporated arene, borondipyrromethene (BODIPY), coumarin, cyanine, and oligothiophene fluorophores in these dyads and characterized their photochemical and photophysical properties with a combination of steady-state and time-resolved spectroscopic measurements. The compound integrating a coumarin-oxazine fluorophore turned out to be the dyad with the most spectroscopic signature for the acquisition of fluorescence images with subdifraction resolution. As a result, I developed also hydrophilic derivatives of this compound as well as a supramolecular strategy to impose hydrophilic character on it. The latter protocol is based on the encapsulation of the photoactivatable compound within the hydrophobic interior of polymer nanoparticles. Indeed, in collaborative studies, we demonstrated that these nanoscaled assemblies can be resolved in space even when they are separated by subdifraction distances on the basis of fluorescence photoactivation