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 3phosphonofluorescein 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.

Voltage imaging with fluorescent indicators offers a powerful complement to traditional electrode or Ca2+-imaging approaches for monitoring electrical activity.

Since its discovery in 1887, rhodamines have become indispensable fluorophores for biological imaging. Recent studies have extensively explored heteroatom substitution at the 10' and a variety of substitution patterns on the 3', 6' nitrogens. Although 3-carboxy-substituted rhodamines were first reported in 1887 and their 3-sulfonated derivatives in 1896, the 3-phosphono analogues have never been reported. We recently reported the synthesis of 3-phosphono fluoresceins, which possessed nearly identical spectral properties to the parent carboxy dyes, but showed enhanced water solubility. However, synthesis and purification were difficult, and yields were low (<17%). Here, we report a mild, generalizable, and scalable synthetic route to 3-phosphonorhodamines. We explore the substrate scope and investigate mechanistic details of the acid-free condensation. Tetramethyl-3-phosphonorhodamine (pTMR) derivatives can be accessed on the 1.5 mmol scale in up to 98% yield (2 steps). Phosphonorhodmines show a 12- to 500-fold increase in water solubility over 3-carboxy and 3-sulfonorhodamine derivatives and have excellent chemical stability. Phosphonates allow for derivatization, and esterification of pTMR allows intracellular delivery with localization profiles that differ from 3-carboxyrhodamines. The free phosphonate can be incorporated into a molecular wire scaffold to create a phosphonated rhodamine voltage reporter, phosphonoRhoVR. PhosRhoVR 1 can be synthesized in just 6 steps, with an overall yield of 37% to provide >400 mg of material, compared to a 6-step, ~2% yield for the previously reported RhoVR 1. PhosRhoVR 1 possesses excellent voltage sensitivity (37% ΔF/F) and a 2-fold increase in cellular brightness compared to RhoVR 1.