Fast and Stable Photochromic Oxazines for Fluorescence Switching

Abstract

The stringent limitations imposed by diffraction on the spatial resolution of fluorescence microscopes demand the identification of viable strategies to switch fluorescence under optical control. In this context, the photoinduced and reversible transformations of photochromic compounds are particularly valuable. In fact, these molecules can be engineered to regulate the emission intensities of complementary fluorophores in response to optical stimulations. On the basis of this general design logic, we assembled a functional molecular construct consisting of a borondipyrromethene fluorophore and a nitrospiropyran photochrome and demonstrated that the emission of the former can be modulated with the interconversion of the latter. This fluorophore-photochrome dyad, however, has a slow switching speed and poor fatigue resistance. To improve both parameters, we developed a new family of photochromic switches based on the photoinduced opening and thermal closing of an oxazine ring. These compounds switch back and forth between ring-closed and -open isomers on nanosecond-microsecond timescales and tolerate thousands of switching cycles with no sign of degradation. In addition, the attachment of appropriate chromophoric fragments to their switchable oxazine ring can be exploited to either deactivate or activate fluorescence reversibly in response to illumination with a pair of exciting beams. Specifically, we assembled three dyads, each based on either a borondipyrromethene or a coumarin fluorophore and an oxazine photochrome, and modulated their fluorescence in a few microseconds with outstanding fatigue resistance. The unique photochemical and photophysical properties of our fluorophore-photochrome dyads can facilitate the development of switchable fluorophores for superresolution imaging and, ultimately, provide valuable molecular probes for the visualization of biological samples on the nanometer level.