Bright Dyes Bring Biology into Focus

Major advances in microscopy have revolutionized our view of the cell, allowing us to see what was previously not visible, now with unprecedented resolution. This would not be possible without an impressive portfolio of bright, modular, and photostable fluorescent dyes. Among these are xanthene-based dyes which includes fluorescein, a versatile FDA-approved reagent first synthesized more than a century ago. Since then, coarse-tuning by substitution of the endocyclic xanthene oxygen with carbon, silicon, and phosphorus has expanded this family to include members with emission wavelengths spanning the entire visible spectrum. However, fine-tuning of specific photophysical properties remains a profound challenge owing to lengthy, nonmodular, and low-yielding synthetic approaches. In this issue, a group led by Lavis, from Janelia Research Campus, has established a powerful divergent strategy to enable the assembly of an assortment of xanthene dyes with unique properties primed to further push the boundaries of molecular imaging (Figure 1, Figure 2). Previously, Lavis reported the synthesis of Janelia Fluor 646 (JF646), 2 a bright silicon-containing xanthene dye that was immediately adopted by the field for super-resolution microscopy. However, its synthesis was low-yielding (4.6% over 11 steps) which hampered further development. The major bottleneck was indeed the preparation of a key silicon−xanthone building block since it accounts for a majority of the synthesis and involves chemistry that is typically difficult to reproduce (at least in our hands). This was followed by metal−halogen exchange chemistry using an appropriate aryl halide precursor to assemble the final dye scaffold. However, the electrophile and nucleophile are often electronically mismatched resulting in another low-yielding step (Figure 2). Lavis’s innovative solution drew inspiration from the Friedel−Crafts reaction, which was originally used to synthesize fluorescein. Starting from bis(bromoaryl)silane, a sequence involving lithium−bromide exchange, magnesium transmetalation, and electrophilic addition onto anhydrides or esters directly yields the Si-xanthene scaffold with high overall conversions (30−60% yield).