%0 Generic
%A Heynck, Lukas
%C Heidelberg
%D 2024
%F heidok:32463
%R 10.11588/heidok.00032463
%T Design and synthesis of anionic and photoactivatable fluorophores for super-resolution microscopy
%U https://archiv.ub.uni-heidelberg.de/volltextserver/32463/
%X Small-molecule fluorophores have been recognized as efficient fluorescent reporters  in the visualisation of living cells by super-resolution fluorescence microscopy. Much  of the ongoing research focuses on positively charged fluorophores derived from rhodamine  cores, which show good photostability, live cell permeability and brightness,  but suffer from undesired off-targeting and accumulation in certain cell compartments,  most typically in mitochondria. Furthermore, their derived photoactivatable labels often  rely photocleavable caging groups as bulky nitrobenzene-type groups or α-diazoketones,  which may generate toxic or non-fluorescent by-products.  Negatively charged fluorophores derived from fluorescein present a valuable alternative  with no off-targeting and accumulation. Fluorescein, one of the oldest synthetic  fluorophores, is a brightly emitting fluorophore, but suffer from low photostability and  cell membrane-impermeability compared to rhodamines. Therefore, the rational design  of negatively charged small-molecule fluorophores with improved photostability and cell  membrane-permeability is desired. Furthermore, photoactivatable fluorescein analogs  may rely on photocleavable groups less bulky than the nitrobenzene-types, which favor  the generation of non-toxic by-products, and hence useful for super-resolution microscopy.  Within this thesis, the chemical design and synthetic methods to access  these fluorophores are discussed and the photophysical properties and application in  live-cell super- resolution fluorescence microscopy examined.  As a result of this work, N-cyanorhodamines have been identified as bathochromically  shifted fluorescein analogs with improved photostability and cell-permeability.  Three different synthetic methods have been developed to access these fluorophores  from known aryl triflates or iodides. Unlike tetramethylrhodamine, N-cyanorhodamines  demonstrate no off-targeting affinity. Based on these fluorophores, fluorescent ligands  for self-labeling protein tags HaloTag and SNAP-tag have been prepared and applied  in combination with commercially available labels in multi-color confocal and stimulated  emission depletion microscopy.  The second part of this thesis explores several different strategies to access photoactivatable  fluorescein analogs bearing no or smallest possible photocleavable group. Protection  of negatively charged fluorophores with photocleavable groups resulted in Ntetrazinylrhodamines  and mercaptofluorans as unstable or non-photoactivatable compounds.  On the other hand, a synthetic route towards 3,6-dihydroxyxanthones on previously  described caging-group-free photoactivatable PaX dyes has been established.  Efficient photoactivation of these compounds indicated their potential for future application  in single-molecule localisation imaging and as enzymatic sensor activity probes.  In conclusion, it was demonstrated that negatively charged fluorescein analogs represents  a valuable addition to the toolkit of live cell-compatible fluorescent dyes. Within  this work, N-cyanorhodamines as highly photostable and cell membrane-permeable  fluorescein derivatives and hydroxy analogs of PaX dyes as photoactivatable analogs  of fluoresceins have been prepared. The application of such dyes as fluorophores in  live cell imaging has been demonstrated.