Phosphonofluoresceins: Synthesis, Spectroscopy, and Applications

10.26434/chemrxiv.13665083 ◽

2021 ◽

Author(s):

Joshua Turnbull ◽

Brittany Benlian ◽

Ryan Golden ◽

Evan Miller

Keyword(s):

Water Solubility ◽

Free Acid ◽

Spectroscopic Characterization ◽

Spectroscopic Properties ◽

Voltage Sensitivity ◽

Acetoxymethyl Ester ◽

Xanthene Dyes ◽

New Class ◽

Low Dielectric ◽

Free Acid Form

<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> <p> </p>

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Calcium ion imaging in plant cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100146503 ◽

1993 ◽

Vol 51 ◽

pp. 132-133

Author(s):

Peter K. Hepler ◽

Dale A. Callaham

Keyword(s):

Plant Cell Wall ◽

Cytoplasmic Membrane ◽

Fluorescent Dyes ◽

Free Acid ◽

Methyl Esters ◽

Free Calcium ◽

Local Concentration ◽

Ion Imaging ◽

Membrane Compartments ◽

Free Acid Form

Calcium ions (Ca) participate in many signal transduction processes, and for that reason it is important to determine where these ions are located within the living cell, and when and to what extent they change their local concentration. Of the different Ca-specific indicators, the fluorescent dyes, developed by Grynkiewicz et al. (1), have proved most efficacious, however, their use on plants has met with several problems (2). First, the dyes as acetoxy-methyl esters are often cleaved by extracellular esterases in the plant cell wall, and thus they do not enter the cell. Second, if the dye crosses the plasma membrane it may continue into non-cytoplasmic membrane compartments. Third, even if cleaved by esterases in the cytoplasm, or introduced as the free acid into the cytoplasmic compartment, the dyes often become quickly sequestered into vacuoles and organelles, or extruded from the cell. Finally, the free acid form of the dye readily complexes with proteins reducing its ability to detect free calcium. All these problems lead to an erroneous measurement of calcium (2).

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BODIPY Fluorophores for Membrane Potential Imaging

10.26434/chemrxiv.8219057.v1 ◽

2019 ◽

Author(s):

Jenna Franke ◽

Benjamin Raliski ◽

Steven Boggess ◽

Divya Natesan ◽

Evan Koretsky ◽

...

Keyword(s):

Mammalian Cells ◽

Water Solubility ◽

Water Soluble ◽

Voltage Sensitivity ◽

Chemical Transformations ◽

Direct Modification ◽

Biological Compatibility ◽

Meso Position ◽

Ionizable Groups ◽

Boron Center

Fluorophores based on the BODIPY scaffold are prized for their tunable excitation and emission profiles, mild syntheses, and biological compatibility. Improving the water-solubility of BODIPY dyes remains an outstanding challenge. The development of water-soluble BODIPY dyes usually involves direct modification of the BODIPY fluorophore core with ionizable groups or substitution at the boron center. While these strategies are effective for the generation of water-soluble fluorophores, they are challenging to implement when developing BODIPY-based indicators: direct modification of BODIPY core can disrupt the electronics of the dye, complicating the design of functional indicators; and substitution at the boron center often renders the resultant BODIPY incompatible with the chemical transformations required to generate fluorescent sensors. In this study, we show that BODIPYs bearing a sulfonated aromatic group at the meso position provide a general solution for water-soluble BODIPYs. We outline the route to a suite of 5 new sulfonated BODIPYs with 2,6-disubstitution patterns spanning a range of electron-donating and -withdrawing propensities. To highlight the utility of these new, sulfonated BODIPYs, we further functionalize them to access 13 new, BODIPY-based voltage-sensitive fluorophores. The most sensitive of these BODIPY VF dyes displays a 48% ΔF/F per 100 mV in mammalian cells. Two additional BODIPY VFs show good voltage sensitivity (≥24% ΔF/F) and excellent brightness in cells. These compounds can report on action potential dynamics in both mammalian neurons and human stem cell-derived cardiomyocytes. Accessing a range of substituents in the context of a water soluble BODIPY fluorophore provides opportunities to tune the electronic properties of water-soluble BODIPY dyes for functional indicators.

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Effects of acidosis on resting cytosolic and mitochondrial Ca2+ in mammalian myocardium.

The Journal of General Physiology ◽

10.1085/jgp.102.3.575 ◽

1993 ◽

Vol 102 (3) ◽

pp. 575-597 ◽

Cited By ~ 26

Author(s):

G Gambassi ◽

R G Hansford ◽

S J Sollott ◽

B A Hogue ◽

E G Lakatta ◽

...

Keyword(s):

Lactic Acid ◽

Ventricular Myocytes ◽

Free Acid ◽

Left Ventricular ◽

Ruthenium Red ◽

Acetoxymethyl Ester ◽

Cytosolic Ph ◽

Bicarbonate Buffer ◽

Adult Rat ◽

Mammalian Myocardium

Acidosis increases resting cytosolic [Ca2+], (Cai) of myocardial preparations; however, neither the Ca2+ sources for the increase in Cai nor the effect of acidosis on mitochondrial free [Ca2+], (Cam) have been characterized. In this study cytosolic pH (pHi) was monitored in adult rat left ventricular myocytes loaded with the acetoxymethyl ester (AM form) of SNARF-1. A stable decrease in the pHi of 0.52 +/- 0.05 U (n = 16) was obtained by switching from a bicarbonate buffer equilibrated with 5% CO2 to a buffer equilibrated with 20% CO2. Electrical stimulation at either 0.5 or 1.5 Hz had no effect on pHi in 5% CO2, nor did it affect the magnitude of pHi decrease in response to hypercarbic acidosis. Cai was measured in myocytes loaded with indo-1/free acid and Cam was monitored in cells loaded with indo-1/AM after quenching cytosolic indo-1 fluorescence with MnCl2. In quiescent intact myocytes bathed in 1.5 mM [Ca2+], hypercarbia increased Cai from 130 +/- 5 to 221 +/- 13 nM. However, when acidosis was effected in electrically stimulated myocytes, diastolic Cai increased more than resting Cai in quiescent myocytes, and during pacing at 1.5 Hz diastolic Cai was higher (285 +/- 17 nM) than at 0.5 Hz (245 +/- 18 nM; P < 0.05). The magnitude of Cai increase in quiescent myocytes was not affected either by sarcoplasmic reticulum (SR) Ca2+ depletion with ryanodine or by SR Ca2+ depletion and concomitant superfusion with a Ca(2+)-free buffer. In unstimulated intact myocytes hypercarbia increased Cam from 95 +/- 12 to 147 +/- 19 nM and this response was not modified either by ryanodine and a Ca(2+)-free buffer or by 50 microM ruthenium red in order to block the mitochondrial uniporter. In mitochondrial suspensions loaded either with BCECF/AM or indo-1/AM, acidosis produced by lactic acid addition decreased both intra- and extramitochondrial pH and increased Cam. Studies of mitochondrial suspensions bathed in indo-1/free acid-containing solution showed an increase in extramitochondrial Ca2+ after the addition of lactic acid. Thus, in quiescent myocytes, cytoplasmic and intramitochondrial buffers, rather than transsarcolemmal Ca2+ influx or SR Ca2+ release, are the likely Ca2+ sources for the increase in Cai and Cam, respectively; additionally, Ca2+ efflux from the mitochondria may contribute to the raise in Cai. In contrast, in response to acidosis, diastolic Cai in electrically stimulated myocytes increases more than resting Cai in quiescent cells; this suggests that during pacing, net cell Ca2+ gain contributes to enhance diastolic Cai.

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An ATP-sensitive conductance in cultured smooth muscle cells from pregnant rat myometrium

AJP Cell Physiology ◽

10.1152/ajpcell.1989.257.2.c297 ◽

1989 ◽

Vol 257 (2) ◽

pp. C297-C305 ◽

Cited By ~ 54

Author(s):

E. Honore ◽

C. Martin ◽

C. Mironneau ◽

J. Mironneau

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Voltage Clamp ◽

Free Acid ◽

Muscle Cells ◽

Monovalent Cation ◽

Acid Form ◽

Pregnant Rat ◽

Free Acid Form ◽

Cultured Smooth Muscle Cells

The whole cell voltage-clamp technique was used to study the effects of extracellular ATP in cultured smooth muscle cells isolated from pregnant rat myometrium. An inward current was elicited by ATP (IATP) in cells held at -70 mV under voltage clamp. The amplitude of IATP was reduced by estrogen pretreatment and by the end of pregnancy. IATP not only did not undergo any desensitization but showed facilitation. The current-voltage relationship of IATP was linear and reversed close to 0 mV. Changing the sodium electrochemical gradient by decreasing extracellular or intracellular sodium resulted in a linear relationship between the reversal potential of IATP and Na equilibrium potential that, however, differed from the predicted curve for a purely sodium conductance. The conductance activated by ATP was monovalent cation selective with little discrimination between potassium, cesium, and sodium ions. IATP was depressed by divalent cations, and the rank order of potency was Co greater than Mg greater than Ca greater than Ba, suggesting that the free-acid form of ATP was the effective ligand. Adenosine, AMP, and ADP were ineffective in eliciting IATP, whereas ATP gamma S and alpha,beta-methylene ATP were capable of mimicking the effects of ATP, although they were less potent. These results are consistent with the free-acid form of ATP activating a monovalent cation-selective and estrogen-sensitive conductance in myometrium.

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EFFECTS OF VARIOUS CHEMICAL AND COLD-HARDENING TREATMENTS ON RESISTANCE OF SUGAR BEET SEEDLINGS TO FREEZING

Canadian Journal of Botany ◽

10.1139/b56-015 ◽

1956 ◽

Vol 34 (1) ◽

pp. 154-158

Author(s):

William G. Corns

Keyword(s):

Sugar Beet ◽

Sodium Salt ◽

Free Acid ◽

Cold Hardening ◽

Chemically Treated ◽

Free Acid Form ◽

Series Of Experiments ◽

The Difference ◽

And Control ◽

Low Temperature Resistance

Either the free acid form or the sodium salt of Dalapon (2,2-dichloropropionic acid) and of TCA (trichloroacetic acid) and the sodium salt of 2,2,3-trichloropropionic acid (free acid not tested) were effective in improving the low temperature resistance of sugar beet seedlings grown in 4- and 8-p.p.m. solutions in the dark at 21 °C., and evaluated by short exposures to −10 °C. Isopropy-N(3-chlorophenyl) carbamate, amino triazole, sodium chloride, and trichlorobenzoic acid were ineffective in similar tests. In a series of experiments involving periodic sampling and freezing of Dalapon-treated illuminated sugar beet seedlings during a 24 day period of storage at 6 °C., the chemically treated plants were again superior to the comparable controls. The "cold-hardening" treatments tended to increase the magnitude of the difference between chemically treated and control plants. The amount of improvement was more variable in the tests with green plants than with those grown in the dark.

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