(2,2'-dihydroxy-4,4'-dimethoxybenzophenone), in high yield and purity without chromatographic separation. While several protecting groups 2,21 were evaluated for acidic phenol groups of 3, TBS-protected compound 4a was the substrate of choice for the preparation of unsubstituted sulfonefluoresceins 5a,b and sulfone-fluorone 5c (Scheme 4, a). The photophysical properties of these fluorophores are compiled in Table and Figures . Both dyes 5a,b have absorption maxima within 620-630 nm range (nearly optimal for excitation with a 630-640 nm pulsed laser) and are characterized with far-red emission, however their quantum yields in aqueous buffer are modest. The fluorescent forms of sulfone-fluoresceins and sulfone-fluorones undergo protonation closing into the colorless spirolactones (for 5a, with pKa ~8) or form triarylmethanol water adducts (for 5b,c, with corresponding pKa ~5-6); in particular, the color of the anionic form of 5c quickly fades in solution. This electrophilic reactivity of the sulfone-fluorone core also accounts for lower than expected values of molar attenuation coefficients for sulfone-fluoresceins (ε = 3×10 3 M -1 cm -1 at pH 9 for 5a vs. ε = 9×10 4 M -1 cm -1 at pH 10 for fluorescein For the preparation of the fluorophores tagged with a free carboxylic acid (suitable for conversion into targeted labels for fixed or live-cell imaging via attachment to a suitable high-affinity ligand), di-O-benzyl protected 4e was chosen for the technical ease of separation and maintained orthogonality to the tert-butyl ester group. As a test ligand, the bioorthogonal ωchloroalkane/HaloTag protein system was selected for its extremely high reaction rates with triarylmethane dye-derived probes (kapp = 1.0×10 6 M -1 s -1 for fluorescein ligand and approaching 10 8 M -1 s -1 for certain rhodamines ), leading to rapid and irreversible covalent linking within the living cell environment. The benzyl-protected sulfone-fluorescein carboxylic acids 6a-c were coupled to the HaloTag(O2) amine, and the target fluorescent probes 7a-c were liberated by hydrogenolysis followed by sequential treatment with TFA and DDQ (Scheme 4, b). Test labeling was performed in living U2OS-Vim-Halo cells engineered using the CRISPR-Cas technology, which were preferred for their high cell-to-cell reproducibility as opposed to transient transfection methods. HaloTag-fused vimentin (a cytoskeletal structural protein) was stained with 7a-Halo (500 nM overnight in complete cell-growing medium) followed by two washing steps (30 min each). Cells were imaged live (Figure ) or after fixation with paraformaldehyde (Figure ). Relatively long integration times (line or frame accumulation, see Table for detailed imaging parameters) were used to compensate for the fraction of markers in non-fluorescent spirolactone form of 7a at the cytosolic pH. The imaging results demonstrated cell membrane permeability of 7a-Halo, selective staining of the intermediate filaments and good compatibility of the label with stimulated emission depletion (STED) superresolution fluorescence imaging technique. The attainable resolution was limited by low molecular brightness of the fluorophore 5a and the dynamics of intermediate filaments in living cells, despite the distinct fluorogenic response of the dye upon binding to HaloTag7 protein (Figure ). Therefore, live-cell labeling with post-fixation, permitting longer total imaging time on immobile structures, was attempted and indeed demonstrated improved resolution (Figure ). The samples stained with 7b,c-Halo were characterized with significantly inferior quality of labeling making the imaging impracticable. In summary, we have developed an original entry into the synthesis of sulfone-fluorescein fluorophores, performed their photophysical characterization and preliminary evaluation as livecell compatible far-red emitting fluorescent labels. The proposed synthetic approach to thioxanthone 10,10-dioxides increases the availability of these building blocks for organophotocatalysis, as well as for photovoltaics, electroluminescence and other material science applications. Furthermore, we are currently exploring variations of the reported reactivity towards the synthesis of other sulfone-embedded heterocycles.