Near-IR oxazine dyes are reported that contain sulfonate esters which are rapidly cleaved by esterase activity to unmask highly polar anionic sulfonates. Strategies for the synthesis of these dyes included the development of milder dye condensation conditions with improved functional compatibility, and the use of an alkyl halide that allows for the introduction of esterase-labile sulfonates without the need for sulfonation of the target molecule.

Sulfonated molecules exhibit high water solubility, a property which is valuable for many biological applications but often complicates their synthesis and purification. Here we report a sulfonate protecting group that is resistant to nucleophilic attack but readily removed with trifluoroacetic acid (TFA). The use of this protecting group improved the synthesis of a sulfonated near-IR fluorophore and the mild deprotection conditions allowed isolation of the product without requiring chromatography.

Potassium (K+) is constantly exiting electrically excitable cells during normal and pathophysiological activity. Currently, potassium-sensitive electrodes and electrical measurements are the primary tools to detect K+ efflux even though small molecule, visible-light K+ sensors exist. To utilize state-of-the-art in vivo imaging techniques, a fluorescent sensor should absorb and emit at long wavelengths (> 650 nm) and be easily derivatized for implementation in a variety of biological systems. Here we describe a palladium-mediated synthesis of a near-IR, oxazine fluorescent K+ sensor (KNIR-1) with a dissociation constant that is suited for detecting changes in intracellular and extracellular potassium concentrations. KNIR-1 treatment of cells expressing voltage-gated K+ channels enabled the visualization of intracellular potassium depletion upon K+ channel opening and restoration of cytoplasmic K+ after channel closing. Given the commercial-availability of fluorophores bearing alkynes, the synthetic methodology described herein will facilitate the synthesis of near-IR fluorescent sensors to enable the visualization of intracellular and extracellular K+.

RNA is the most mercurial of all biomacromolecules. In contrast to DNA, where the predominant role is the storage of genetic information, the biological role of RNA varies; ranging from a template-based intermediary in gene expression to playing a direct role in catalysis. Their high turnover and metabolic lability makes the detection of specific sequences particularly challenging. This review describes the latest synthetic biological developments that enable the direct imaging of RNA both in vitro and in their native cellular environment.

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