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PONy Dyes – Fluorescent Dyes with Phosphorus Substituents

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Background

Fluorescent dyes are widely used as indispensable markers in biology, optical microscopy, and analytical chemistry. In particular, the sensitive and stable imaging of cellular components depends on the favourable combination of chemical, biological and physical factors. The availability and proper choice of fluorescent dyes is a key factor to success in the entire labelling and imaging procedure. Due to their superior brightness and photostability, synthetic dyes represent an attractive alternative to fluorescent proteins.
Dyes with compact structures and a zero net charge (neutral or zwitterionic molecules with a short charge separation distance) are known to penetrate the outer plasma membrane of living cells and may be used as fluorescent labels in biology, optical microscopy and material science. Fluorescent dyes with increased Stokes shifts offer an advantage of using more flexible imaging schemes. In this case two fluorescent labels with small and large Stokes shifts can be combined in one experiment and imaged separately. Therefore, the discovery of new labels is a vital task in modern biology related natural science.

Technology

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 phosphorus(III) reagents (see figure 1) to form phosphonylated leuco bases. Upon oxidation, these intermediates provide new fluorophores (PONydyes) 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).

Figure 1: Addition of neutral or anionic P(III) nucleophiles to cationic or highly polarized fluorophores results in „PONy“ dyes with „grown-up“ Stokes shift and red-shifted adsorption and emission bands.

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.

Figure 2: Examples illustrating the structural diversity of stable PONy dyes synthesised according to the reaction scheme in figure 1; A1: substituted acridinium salt, C7: substituted coumarin, P1: substituted pyronin.

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.

Summary

Patent Information

EP patent application filed in February 2017.
US patent application filed in February 2018.

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PONy Dyes – Fluorescent Dyes with Phosphorus Substituents

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