<p>Xanthene fluorophores, like fluorescein, have been versatile
molecules across diverse fields of chemistry and life sciences. Despite the
ubiquity of 3-carboxy and 3-sulfuonofluorescein 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), rivalling that of sulfono-based
VF dyes. In sum, 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. </p>
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