Phosphonofluoresceins: Synthesis, Spectroscopy, and Applications

Abstract

Xanthene fluorophores, like fluorescein, have been versatile molecules across diverse fields of chemistry and life sciences. Despite the ubiquity of 3-carboxy and 3-sulfonofluorescein for the last 150 years, to date, no reports of 3-phosphonofluorescein exist. Here, we report the synthesis, spectroscopic characterization, and applications of 3-phosphonofluoresceins. The absorption and emission of 3-phosphonofluoresceins remain relatively unaltered from the parent 3-carboxyfluorescein. 3-Phosphonofluoresceins show enhanced water solubility compared to 3-carboxyfluorescein and persist in an open, visible light-absorbing state even at low pH and in low dielectric media while 3-carboxyfluoresceins tend to lactonize. In contrast, the spirocyclization tendency of 3-phosphonofluoresceins can be modulated by esterification of the phosphonic acid. The bis-acetoxymethyl ester of 3-phosphonofluorescein readily enters living cells, showing excellent accumulation (>6x) and retention (>11x), resulting in a nearly 70-fold improvement in cellular brightness compared to 3-carboxyfluorescein. In a complementary fashion, the free acid form of 3-phosphonofluorescein does not cross cellular membranes, making it ideally suited for incorporation into a voltage-sensing scaffold. We develop a new synthetic route to functionalized 3-phosphonofluoresceins to enable the synthesis of phosphono-voltage sensitive fluorophores, or phosVF2.1.Cl. Phosphono-VF2.1.Cl shows excellent membrane localization, cellular brightness, and voltage sensitivity (26% ΔF/F per 100 mV), rivaling that of sulfono-based VF dyes. In summary, we develop the first synthesis of 3-phosphonofluoresceins, characterize the spectroscopic properties of this new class of xanthene dyes, and utilize these insights to show the utility of 3-phosphonofluoresceins in intracellular imaging and membrane potential sensing.

Figure 1.

Spectroscopic characterization of the pH dependence of dichlorofluoresceins. Normalized absorbance spectra and corresponding plots of normalized absorbance vs. pH at λ_max for carboxy- (a, b), sulfono- (d, e) and phosphono- (g, h) dichlorofluoresceins. Spectra were recorded in 10 mM buffered solutions (see supporting information) containing 150 mM NaCl from pH 2.4 (red) to 9.8 (magenta), and intermediate values of 3.1, 3.8, 4.3, 4.8, 5.3, 5.5, 6.1, 6.6, 7.1, 7.5, 8.2, 8.7 and 9.2 at a dye concentration of 2 μM. Titration curves fit to sigmoidal dose response curves (solid black) enabled pK_a determination (dashed red). Error bars represent ± SEM for n = 3 independent determinations and if not visible are smaller than the marker. Summary of pH equilibria with determined pK_a values for carboxy- (c), sulfono- (f) and phosphono- (i) dichlorofluoresceins.

Figure 2.

Cell permeability of phosphonofluoresceins. Widefield fluorescence (a–c) and DIC (d–f) images of HEK cells stained with 500 nM 4 (a,d), 4-AM closed (b,e) and 4-AM open (c,f) for 20 min at 37 °C. Cells were washed once with HBSS prior to imaging. Scale bar is 50 μM.

Figure 3.

Cellular retention of fluoresceins. (a) Comparison of the relative brightness of fluorescein AMs in HEK cells. (b) normalized intensity and (c) fluorescence images of 4-AM Open and FCl-AM Closed loaded onto HEK cells at 500 nM in HBSS for 20 min. Cells were sequentially washed with fresh HBSS and changes in fluorescence intensity were measured by means of fluorescence microscopy. All dyes were loaded at 500 nM in HBSS for 20 min at 37 °C. Error bars in (a) and (b) are ±SEM for n=4 coverslips. Scale bar is 50 μm. (d) Chemical structures of carboxyfluorescein AMs.

Figure 4.

Cellular and in vitro characterization of phosphonated VoltageFluors. (a) Normalized absorbance (solid line) and fluorescence emission (dashed line) spectra of para phosVF2.1Cl (17) in 0.1 M NaOH_(aq). (b) HEK cells stained with 250 nM para phosVF2.1Cl (17). Scale bar is 40 μm. (c) Plot of the fractional change in fluorescence of para phosVF2.1Cl (17) vs time for 100 ms hyper- and depolarizing steps (±100 mV in 20 mV increments) for single HEK cells under whole-cell voltage-clamp mode. (d) plot of ΔF/F vs final membrane potential, revealing a voltage sensitivity of approximately 26% per 100 mV. Error bars are ±SEM for n=17 cells. If not visible, error bars are smaller than the marker.

Scheme 1.

Unique properties of phosphonofluorescein

Scheme 2.

Synthesis of phosphonofluoresceins

Scheme 3.

Synthesis of phosphonofluorescein acetoxy methyl esters and ethers.

Scheme 4.

Synthesis of phosphono-VoltageFluors.