Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 µM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

Synthesis, structural determination and spectroscopic analysis of a novel 2D terbium coordination network with ethyleneglycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid.

A novel two-dimensional (2D) mononuclear complex, namely, (enH(2))[Tb(III)(egta)(H(2)O)](2)·6H(2)O (H(4)egta=ethyleneglycol-bis-(2-aminoethylether)-N,N,N',N'-tetraacetic acid and en=ethylenediamine), was successfully synthesized and characterized by infrared spectrum, UV-vis spectrum, fluorescence spectrum, thermal analysis and single-crystal X-ray diffraction techniques. Single-crystal X-ray diffraction analysis reveals that the central Tb(III) ion is nine-coordinate in geometry of pseudo-monocapped square antiprismatic polyhedron. Furthermore, the hydrogen bonds play an important role in the fabrication of layer structure of the complex. Through hydrogen bonds between ethylenediamine cation (enH(2)(2+)) and [Tb(III)(egta)(H(2)O)](-) complex anion, the title complex forms a 2D layer network along [111] crystallographic direction. Particularly, the fluorescent property is also fully investigated, which indicates that the title complex would be a potential candidate as fluorescent materials.

Synthesis and characterization of a new red-emitting Ca2+ indicator, calcium ruby.

Calcium Ruby m-Cl (X = H, Y = Cl) is a visible-light excited red-emitting calcium concentration ([Ca2+]) indicator dye (579/598 nm peak excitation/emission) with a side arm for conjugation via EDC or click chemistry. Its large molar extinction and high quantum yield rank it among the brightest long-wavelength Ca2+ indicators. Calcium Ruby is a promising alternative to existing dyes for imaging [Ca2+] in multicolor fluorescence applications or in the presence of yellow-green cellular autofluorescence.

The nitrodibenzofuran chromophore: a new caging group for ultra-efficient photolysis in living cells.

Photochemical uncaging of bio-active molecules was introduced in 1977, but since then, there has been no substantial improvement in the properties of generic caging chromophores. We have developed a new chromophore, nitrodibenzofuran (NDBF) for ultra-efficient uncaging of second messengers inside cells. Photolysis of a NDBF derivative of EGTA (caged calcium) is about 16-160 times more efficient than photolysis of the most widely used caged compounds (the quantum yield of photolysis is 0.7 and the extinction coefficient is 18,400 M(-1) cm(-1)). Ultraviolet (UV)-laser photolysis of NDBF-EGTA:Ca(2+) rapidly released Ca(2+) (rate of 20,000 s(-1)) and initiated contraction of skinned guinea pig cardiac muscle. NDBF-EGTA has a two-photon cross-section of approximately 0.6 GM and two-photon photolysis induced localized Ca(2+)-induced Ca(2+) release from the sarcoplasmic recticulum of intact cardiac myocytes. Thus, the NDBF chromophore has great promise as a generic and photochemically efficient protecting group for both one- and two-photon uncaging in living cells.

New fluorescent calcium indicators designed for cytosolic retention or measuring calcium near membranes.

A new family of fluorescent calcium indicators has been developed based on a new analog of BAPTA called FF6. This new BAPTA analog serves as a versatile synthetic intermediate for developing Ca2+ indicators targeted to specific intracellular environments. Two of these new Ca2+ indicators, fura-PE3 and fura-FFP18, are described in this report. Fura-PE3 is a zwitterionic indicator that resists the rapid leakage and compartmentalization seen with fura-2 and other polycarboxylate calcium indicators. In contrast to results obtained with fura-2, cells loaded with PE3 remain brightly loaded and responsive to changes in concentration of cytosolic free calcium for hours. Fura-FFP18 is an amphipathic indicator that to binds to liposomes and to cell membranes. Studies to be detailed later indicate that FFP18 functions as a near-membrane Ca2+ indicator and that calcium levels near the plasma membrane rise faster and higher than in the cytosol.

NMR-sensitive fluorinated and fluorescent intracellular calcium ion indicators with high dissociation constants.

A new series of high-dissociation constant (KD) Ca2+ indicators has been developed to reduce perturbations due to buffering of transients, to carry out measurements in cells and organelles with high basal Ca2+ concentrations, and to measure cytosolic Ca2+ levels in the presence of perturbations that may significantly increase these levels. A tetrafluorinated derivative of the chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 1,2-bis(2-amino-5,6-difluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (TF-BAPTA), has a KD of 65 microM and exhibits two fluorine nuclear magnetic resonances, one of which is insensitive to Ca2+ chelation and the second of which shifts by approximately 10 ppm upon Ca2+ binding. TF-BAPTA has pK values of approximately 5.0 and Mg2+ dissociation constants > 50 mM. At a field of 8.5 T, the Ca(2+)-sensitive resonance is in fast-intermediate exchange. Correction factors for the effects of intermediate exchange and for the effect of protonation (pK approximately 5.0) and Mg2+ complexation are discussed. An analogous approach has been used to synthesize 2-[2-(5-carboxyoxazole)]-5-[2-(2-bis(carboxymethyl) amino-5,6-difluorophenoxy)]ethoxy-6-bis(carboxymethyl)aminobenz ofuran (fura F), a structural analogue of fura 2, which exhibits fluorescence characteristics similar to those of fura 2, but has a KD of 20 microM.

Styryl-based wavelength-ratiometric probes: a new class of fluorescent calcium probes with long wavelength emission and a large Stokes' shift.

We describe the chemical synthesis and fluorescence spectral characterization of a styryl-benzothiazole probe which contains the Ca2+ chelating group BAPTA as an integral part of the chromophoric system. The visible absorption spectrum of this probe displays a dramatic shift in the long wavelength maxima from 508 to 407 nm upon complexation with Ca2+, with a Ca2+ dissociation constant of 1.5 microM. The emission maximum centered at 615 nm is well shifted from the absorption. The emission spectrum displays a small blue shift upon binding Ca2+, allowing this probe to possibly be used as an emission wavelength-ratiometric probe using a single-excitation wavelength. This probe is likely to be the first of a series of long-wavelength ratiometric Ca2+ probes whose structure can be modified for improved quantum yield or altered Ca2+ affinity.

Enhancement of the endothelial production of prostacyclin by substituted derivatives of BAPTA-AM.

Our observation that loading of bovine aortic endothelial cells with quin 2 or 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) enhances their release of prostacyclin (PGI2) has been studied in detail. The action of the acetoxymethyl ester (AM) of BAPTA (BAPTA-AM) was biphasic: at high concentrations (50 microM) and after prolonged exposure (60 min or more), it behaved as an inhibitor instead of an amplifier. Since inhibition could be related to calcium chelation, we tested 5,5'-difluoro-BAPTA-AM (5FBAPTA-AM), 4,4'-difluoro-BAPTA-AM (4FBAPTA-AM) and 5,5'-dibromo-BAPTA-AM (5BBAPTA-AM), whose corresponding free acids have a reduced affinity for Ca2+: these compounds were much more active in enhancing PGI2 production than BAPTA-AM itself. The effect of 5BBAPTA-AM was detectable at 0.5 microM and almost maximal at 5 microM (5-fold increase). 5BBAPTA-AM increased the release of free arachidonate induced by ATP but had no effect on the generation of inositol phosphates, the release of choline metabolites, and the accumulation of cAMP. 5BBAPTA-AM had a cytotoxic effect on the endothelial cells only after prolonged exposure to a high (50 microM) concentration. 5BBAPTA-AM inhibited the platelet production of thromboxane stimulated by a high (0.5 U/ml) concentration of thrombin and slightly potentiated the effect of a low (0.005 U/ml) concentration. In conclusion, the effect of 5BBAPTA-AM on the production of PGI2 seems to be rather specific to arachidonate metabolism in endothelial cells.

Synthesis and characterization of 19F NMR chelators for measurement of cytosolic free Ca.

Fluorine 19 nuclear magnetic resonance (NMR) studies of intracellular fluorinated calcium chelators provide a useful strategy for the determination of cytosolic free calcium levels in cells and perfused organs. However, the fluorinated chelator with the highest affinity for calcium ions which has been described to date. 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), exhibits a dissociation constant (Kd) value 5- to 10-fold greater than the intracellular calcium concentration levels in most cell types, thus limiting the ability of fluorine NMR to report these concentrations reliably. We have consequently designed and synthesized several fluorinated calcium chelators with higher affinity for calcium. The best of these, 2-(2-amino-4-methyl-5-fluorophenoxy)-methyl-8 aminoquinidine-N,N,N',N'-tetraacetic acid (quinMF), has a Kd value approximately 10 times lower than that of 5FBAPTA. Several of the newly synthesized indicators have different chemical shifts for the calcium complexed and uncomplexed chelators to allow the simultaneous use of two indicators. In addition to providing information about the level of cytosolic free calcium, chelators containing a quinoline ring exhibit considerable sensitivity to magnesium levels and hence have potential application for the determination of cytosolic-magnesium concentrations. Application of these chelators is illustrated by determination of the cytosolic-free calcium level in erythrocytes. Use of quinMF, the chelator with the lowest Kd value, gives a calcium value of 25-30 nM.

Control of cytoplasmic calcium with photolabile tetracarboxylate 2-nitrobenzhydrol chelators.

This paper introduces nitr-2, a new Ca2+ chelator designed to release Ca2+ upon illumination with near UV (300-400 nm) light. Before illumination nitr-2 has Ca2+ dissociation constants of 160 and 630 nM in 0.1 and 0.3 M ionic strength respectively; after photoconversion to a nitrosobenzophenone the values shift to 7 and 18 microM, high enough to liberate substantial amounts of Ca2+ under intracellular conditions. The speed of release is limited by a dark reaction with rate constant 5 s-1. Aplysia central neurons injected with nitr-2 and exposed to UV light exhibit two separate Ca2+-dependent membrane currents: one carried by potassium ions and one a nonspecific cation current. A quantitative estimate of the spatial distribution of intracellular [Ca2+] changes in large cells filled with a high concentration of nitr-2 and exposed to an intense UV flash is offered.

New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures.

A new family of high-affinity buffers and optical indicators for Ca2+ is rationally designed and synthesized. The parent compound is 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), a relative of the well-known chelator EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] in which methylene links between oxygen and nitrogen are replaced by benzene rings. BAPTA and its derivatives share the high (greater than 10(5)) selectivity for Ca2+ over Mg2+ of EGTA but are very much less affected by pH changes and are faster at taking up and releasing Ca2+. The affinity of the parent compound for Ca2+ (dissociation constant 1.1 x 10(-7) M in 0.1 M KCl) may be strengthened or weakened by electron-releasing or -withdrawing substituents on the aromatic rings. The Ca2+ and Mg2+ affinities may further be altered by replacing the ether oxygens by heterocyclic nitrogen atoms. The compounds described are fluorescent Ca2+ indicators absorbing in the ultraviolet region; the very large spectral shifts observed on binding Ca2+ fit the prediction that complexation should hinder the conjugation of the nitrogen lone-pair electrons with the aromatic rings. Derivatives with quinoline nuclei are notable for their high sensitivity of fluorescent quantum yield to the binding of Ca2+ but not of Mg2+. Preliminary biological tests have so far revealed little or no binding to membranes or toxic effects following intracellular microinjection.