Phenoxazines with a Phototransferable N-Acetyl Group and Acrylate Linker: Assembly by C-H Activation, Photoconversion...

Photoactivatable (PA) fluorescent dyes with a reactive group are designated markers used in bioimaging techniques, tracking cellular processes, and observing the nanoscale organization of biological specimens with high spatiotemporal resolution. Conventional (non-PA) phenoxazine dyes are widely used in fluorescence microscopy owing to their high brightness, fluorescence emission in the far-red, and outstanding photostability, which allow their detection down to single molecules. So far, there has been no general synthetic route to PA oxazines with various emission colors and a reactive group. By applying metal-catalyzed C-H activation to symmetric and easily available N¹⁰-acetylphenoxazines, we demonstrated that this approach represents a powerful tool for the design of PA dyes. Versatile and relatively short syntheses directly involved 3,7-disubstituted-10-acetylphenoxazines or commercial Resazurin (7-hydroxy-10-oxyphenoxazin-3-one) as starting materials. These underwent site-selective Rh- or Ru-catalyzed C¹-H activation followed by olefination with alkyl acrylates. The presence of the acrylate C═C bond attached to C¹ in the N¹⁰-acetylphenoxazine scaffold results in a red shift (ca. 50 nm) in the absorption spectra, provides a site trapping the acetyl group cleaved off upon irradiation, and enables PA above 400 nm. Red-emitting PA oxazines having a CH═CHCONHR group were prepared and delivered to living and fixed cells. The PA probes incorporating HaloTag or BG-PEG amine (for labeling of Halo- or SNAP-Tags) were found to be cell-permeable and provided good images in (two-color) fluorescence microscopy and nanoscopy techniques, such as PALM (Photoactivation Localization Microscopy) and MINFLUX (MINimal FLUXes), based on the activation and detection of single molecules.