Systematic Tuning of Rhodamine Spirocyclization

Fluorescence microscopy is a powerful method to examine cellular structures and biological processes with high spatial and temporal resolution. The development of novel microscopy techniques and labeling systems has expanded the limits of cellular imaging. However, this progress also relies on the availability of suitable fluorescent probes, thereby creating a need for new synthetic strategies that enable systematic modification of fluorophores. Rhodamine derivatives are widely used fluorophores, which exist in an equilibrium between a non-fluorescent, cell-permeable spirocyclic form and a fluorescent zwitterion. This spirocyclization equilibrium affects crucial properties of rhodamines regarding their applicability in cellular imaging, including fluorogenicity, cell-permeability and blinking behavior. Strategies that provide control over this equilibrium therefore hold the potential to generate suitable fluorescent probes for different microscopy techniques and labeling systems. This thesis describes the development and application of a synthetic strategy, which allows systematic tuning of the spirocyclization equilibrium of rhodamines. To this end, the ortho-carboxy group of various rhodamine derivatives was transformed into amides with different substituents. Introduction of substituted acyl benzenesulfonamides enables to control the spirocyclization equilibrium with unprecedented precision and to specifically optimize the fluorogenicity for HaloTag and SNAP-tag labeling. The resulting probes show suitable properties for live-cell, no-wash confocal and stimulated emission depletion (STED) microscopy. Replacing the ortho-carboxy group with more electron-rich amides strongly shifts the position of the equilibrium toward the spirocyclic state and results in spontaneous blinking. This led to the development of fluorophores for single molecule localization microscopy (SMLM). The generality of this synthetic strategy allowed conversion of various rhodamine-based scaffolds into highly fluorogenic HaloTag and SNAP-tag probes as well as spontaneously blinking dyes. In addition to HaloTag and SNAP-tag labeling, fluorogenic probes for Escherichia coli dihydrofolate reductase (eDHFR), tubulin and human immunodeficiency virus-1 (HIV-1) protease were generated. The ortho-carboxy group was also used as a handle for further functionalization of rhodamines, thereby introducing a zinc ligand to form a localizable and fluorogenic zinc indicator. Altogether, differently colored fluorescent probes optimized for various microscopy techniques and labeling systems were developed. These results demonstrate the value of systematic modification of rhodamines for expanding the fluorophore palette in cellular imaging