What Is Best Strategy for Water Soluble Fluorescence Dyes? – a Case Study Using Long Fluorescence Lifetime DAOTA Dyes

The applications of organic fluorophores in biological sciences rely heavily on their properties in aqueous solution. The lipophilic nature of virtually all such chromophores provides several challenges to adapt them to biologically relevant conditions. In this work we investigate three different strategies for achieving water-solubility of the diazaoxatriangulenium (DAOTA⁺) chromophore: hydrophilic counter ions, aromatic sulfonation of the chromophore core, and attachment of cationic or zwitterionic side chains. The long fluorescence lifetime (FLT, τ_f » 20 ns) of DAOTA⁺ makes it a sensitive probe for changes in the rate of non-radiative deactivation and for aggregation leading to multi exponential decay profiles. Direct sulfonation of the chromophore, as applied in several Alexa dyes, does indeed increase solubility drastically, but at the cost of greatly reduced quantum yields (QY) due to enhanced non-radiative deactivation processes. The introduction of either cationic (4) or zwitterionic side chains (5), however, brings the FLT (τ_f = 18 ns) and QY (φ_f = 0.56) of the dye to the same level as the parent chromophore in acetonitrile. For these derivatives time-resolved fluorescence spectroscopy also reveals a high resistance to aggregation and non-specific binding in a high loading of bovine serum albumin (BSA). The results clearly show that addition of charged flexible side chains is preferable to direct sulfonation of the chromophore core.