Phosphonofluoresceins: Synthesis, Spectroscopy, and Applications.

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.

Palladium(II)-Catalyzed Directed anti-Hydrochlorination of Unactivated Alkynes with HCl.

A regioselective anti-hydrochlorination of unactivated alkynes is reported. The reaction utilizes in situ generated HCl as the source of both the Cl- and H+ and is catalyzed by palladium(II) acetate, with loadings as low as 25 ppm. Removable picolinamide and 8-aminoquinoline bidentate directing groups are used to control the regioselectivity of the chloropalladation step and stabilize the resulting alkenylpalladium(II) intermediate for subsequent protodepalladation. This method provides access to a broad array of substituted alkenyl chlorides in excellent yields and with high regioselectivity. The products from this transformation were successfully derivatized via Stille coupling to a variety of trisubstituted alkene products. Reaction progress kinetic analysis was performed, shedding light on a possible mechanism for this catalytic process.

Tridentate Directing Groups Stabilize 6-Membered Palladacycles in Catalytic Alkene Hydrofunctionalization.

Removable tridentate directing groups inspired by pincer ligands have been designed to stabilize otherwise kinetically and thermodynamically disfavored 6-membered alkyl palladacycle intermediates. This family of directing groups enables regioselective remote hydrocarbofunctionalization of several synthetically useful alkene-containing substrate classes, including 4-pentenoic acids, allylic alcohols, homoallyl amines, and bis-homoallylamines, under Pd(II) catalysis. In conjunction with previous findings, we demonstrate regiodivergent hydrofunctionalization of 3-butenoic acid derivatives to afford either Markovnikov or anti-Markovnikov addition products depending on directing group choice. Preliminary mechanistic and computational data are presented to support the proposed catalytic cycle.

An open and shut case? Chemistry to control xanthene dyes.

Fluorescent dyes are an indispensable part of the scientific enterprise. Xanthene-based fluorophores, like fluorescein and rhodamine, have been in continual use across numerous fields since their invention in the late 19th century. Modern methods to synthesize and expand the scope of xanthene dye chemistry have enabled new colors, enhanced stability, and improved brightness. Modifications to the 3-position of xanthene dyes have been, until recently, less well-explored. Here, we discuss how small changes to the identity of the substituent at the 3-position of fluoresceins and rhodamines can profoundly alter the properties of xanthene dyes, with the potential to unlock new applications at the interface of chemistry and biology.

Mild and scalable synthesis of phosphonorhodamines.

Since their discovery in 1887, rhodamines have become indispensable fluorophores for biological imaging. Recent studies have extensively explored heteroatom substitution at the 10' position and a variety of substitution patterns on the 3',6' nitrogens. Although 3-carboxy- and 3-sulfono-rhodamines were first reported in the 19th century, the 3-phosphono analogues have never been reported. Here, we report a mild, scalable synthetic route to 3-phosphonorhodamines. We explore the substrate scope and investigate mechanistic details of an exogenous acid-free condensation. Tetramethyl-3-phosphonorhodamine (phosTMR) derivatives can be accessed on the 1.5 mmol scale in up to 98% yield (2 steps). phosTMR shows a 12- to 500-fold increase in water solubility relative to 3-carboxy and 3-sulfonorhodamine derivatives and has excellent chemical stability. Additionally, phosphonates allow for chemical derivatization; esterification of phosTMR facilitates 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.

Fluorescence lifetime predicts performance of voltage sensitive fluorophores in cardiomyocytes and neurons.

Voltage imaging with fluorescent indicators offers a powerful complement to traditional electrode or Ca2+-imaging approaches for monitoring electrical activity. Small molecule fluorescent indicators present the unique opportunity for exquisite control over molecular structure, enabling detailed investigations of structure/function relationships. In this paper, we tune the conjugation between aniline donors and aromatic π systems within the context of photoinduced electron transfer (PeT) based voltage indicators. We describe the design and synthesis of four new voltage-sensitive fluorophores (VoltageFluors, or VFs). Three of these dyes have higher relative voltage sensitivities (ΔF/F) than the previously-reported indicator, VF2.1.Cl. We pair these new indicators with existing VFs to construct a library of voltage indicators with varying degrees of conjugation between the aniline nitrogen lone pair and the aromatic π system. Using a combination of steady-state and time-resolved fluorescence spectroscopy, cellular electrophysiology, fluorescence lifetime imaging microscopy (FLIM), and functional imaging in mammalian neurons and human cardiomyocytes, we establish a detailed link between the photophysical properties of VF dyes and their ability to report on membrane potential dynamics with high signal-to-noise. Anilines with intermediate degrees of conjugation to the aromatic π system experience intermediate rates of PeT and possess the highest absolute voltage sensitivities. Measured using FLIM in patch-clamped HEK cells, we find that the absolute voltage sensitivity of fluorescence lifetime (Δτfl per mV), coupled with traditional fluorescence intensity-based metrics like ΔF/F and signal-to-noise ratio (SNR), provides a powerful method to both predict and understand indicator performance in cellular systems.