PONy Dyes – Fluorescent Dyes with Phosphorus Substituents

While searching for new fluorophores suitable for nanoscale imaging of intracellular targets, various organic dyes with electrophilic conjugated systems were found to react with nucleophilic phos-
phorus(III) reagents (see figure 1) to form phos-phonylated leuco bases. Upon oxidation, these intermediates provide new fluorophores (PONy dyes) with red-shifted absorption and emission
maxima and increased Stokes shifts as compared to the precursor dyes. The versatility of phosphorus addition at the sp²-carbon, combined with the wide availability of functionally substituted P(III) reagents, offers an extended array of conceivable PONy dyes with broadly varying properties (for examples, see table 1).

Comparison of the UV-vis spectra indicates that PONy dyes absorb and emit at longer wavelengths and possess larger Stokes shifts than the parent dyes. Additionally, PONy dyes are particularly attractive due to their low molecular masses (typically M < 500 Da) and orange to near-infrared emission. These dyes can be prepared in cationic or zwitterionic forms, making them promising candidates for the development of cell-permeant fluorescent markers for living cells. The fluorescence lifetimes of PONy dyes vary and generally do not exceed 3 ns. Their sensitivity to the nature of the solvent may allow using the PONy-derived probes in fluorescence lifetime imaging or as polarity sensors. Example structures for different PONy dye classes are given in figure 2.

The versatility of the proposed transformation is demonstrated by the facile functionalizations of the commercial fluorophores Atto 495 and Pyronin Y. Using the phosphinylation/oxidation chemistry, Atto 495 was converted into an orange emitting dye A1 applicable in immunolabelling (figure 2). Pyronin Y gave dye P1. Additionally, the red-emitting coumarin dye C7 with huge Stokes shift was prepared. The transformations required only few synthetic steps and provided functional derivatives of the PONy dyes (e. g., A1 and P1) in zwitterionic form with a very short charge separation distance – which is known to favour intact membrane permeability in living cells.

• neutral or zwitterionic dyes with a very short charge separation
• low molecular mass and compact structures
• bathochromic and bathofluoric shifts of absorption and emission bands
• possibility to introduce additional functional groups
• increased Stokes shifts with sufficient emission efficiency

EP patent application filed in February 2017.

US patent application filed in February 2018.