New Calcium‐Selective Smart Contrast Agents for Magnetic Resonance Imaging

Calcium plays a vital role in the human body and especially in the central nervous system. Precise maintenance of Ca²⁺ levels is very crucial for normal cell physiology and health. The deregulation of calcium homeostasis can lead to neuronal cell death and brain damage. To study this functional role played by Ca²⁺ in the brain noninvasively by using magnetic resonance imaging, we have synthesized a new set of Ca²⁺‐sensitive smart contrast agents (CAs). The agents were found to be highly selective to Ca²⁺ in the presence of other competitive anions and cations in buffer and in physiological fluids. The structure of CAs comprises Gd³⁺‐DO3A (DO3A=1,4,7‐tris(carboxymethyl)‐1,4,7,10‐tetraazacyclododecane) coupled to a Ca²⁺ chelator o‐amino phenol‐N,N,O‐triacetate (APTRA). The agents are designed to sense Ca²⁺ present in extracellular fluid of the brain where its concentration is relatively high, that is, 1.2–0.8 mM . The determined dissociation constant of the CAs to Ca²⁺ falls in the range required to sense and report changes in extracellular Ca²⁺ levels followed by an increase in neural activity. In buffer, with the addition of Ca²⁺ the increase in relaxivity ranged from 100–157 %, the highest ever known for any T₁‐based Ca²⁺‐sensitive smart CA. The CAs were analyzed extensively by the measurement of luminescence lifetime measurement on Tb³⁺ analogues, nuclear magnetic relaxation dispersion (NMRD), and ¹⁷O NMR transverse relaxation and shift experiments. The results obtained confirmed that the large relaxivity enhancement observed upon Ca²⁺ addition is due to the increase of the hydration state of the complexes together with the slowing down of the molecular rotation and the retention of a significant contribution of the water molecules of the second sphere of hydration.     

Conformational Changes in Calcium‐Sensor Proteins under Molecular Crowding Conditions

Fundamental components of signaling pathways are switch modes in key proteins that control start, duration, and ending of diverse signal transduction events. A large group of switch proteins are Ca²⁺ sensors, which undergo conformational changes in response to oscillating intracellular Ca²⁺ concentrations. Here we use dynamic light scattering and a recently developed approach based on surface plasmon resonance to compare the protein dynamics of a diverse set of prototypical Ca²⁺‐binding proteins including calmodulin, troponin C, recoverin, and guanylate cyclase‐activating protein. Surface plasmon resonance biosensor technology allows monitoring conformational changes under molecular crowding conditions, yielding for each Ca²⁺‐sensor protein a fingerprint profile that reflects different hydrodynamic properties under changing Ca²⁺ conditions and is extremely sensitive to even fine alterations induced by point mutations. We see, for example, a correlation between surface plasmon resonance, dynamic light scattering, and size‐exclusion chromatography data. Thus, changes in protein conformation correlate not only with the hydrodynamic size, but also with a rearrangement of the protein hydration shell and a change of the dielectric constant of water or of the protein–water interface. Our study provides insight into how rather small signaling proteins that have very similar three‐dimensional folding patterns differ in their Ca²⁺‐occupied functional state under crowding conditions.     

Free Ca<Superscript>2+</Superscript> as an early intracellular biomarker of exposure of cyanobacteria to environmental pollution

Calcium functions as a versatile messenger in a wide variety of eukaryotic and prokaryotic cells. Cyanobacteria are photoautotrophs which have a great ecological impact as primary producers. Our research group has presented solid evidence of a role of calcium in the perception of environmental changes by cyanobacteria and their acclimation to these changes. We constructed a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 that constitutively expresses the calcium-binding photoprotein apoaequorin, enabling in-vivo monitoring of any fluctuation in the intracellular free calcium concentration of the cyanobacterium in response to any environmental stimulus. The “Ca²⁺ signature” is the combination of changes in all Ca²⁺ signal properties (magnitude, duration, frequency, source of the signal) produced by a specific stimulus. We recorded and analyzed the Ca²⁺ signatures generated by exposure of the cyanobacterium to different groups of environmental pollutants, for example cations, anions, organic solvents, naphthalene, and pharmaceuticals. We found that, in general, each group of tested chemicals triggered a specific calcium signature in a reproducible and dose-dependent manner. We hypothesize that these Ca²⁺ signals may be related to the cellular mechanisms of pollutant perception and ultimately to their toxic mode of action. We recorded Ca²⁺ signals triggered by binary mixtures of pollutants and a signal induced by a real wastewater sample which could be mimicked by mixing its main constituents. Because Ca²⁺ signatures were induced before toxicity was evident, we propose that intracellular free Ca²⁺ may serve as an early biomarker of exposure to environmental pollution.     

Cyclic Adenosine 5′‐Diphosphoribose (cADPR) Mimics Used as Molecular Probes in Cell Signaling

Cyclic adenosine 5′‐diphosphate ribose (cADPR) is a second messenger in the Ca²⁺ signaling pathway. To elucidate its molecular mechanism in calcium release, a series of cADPR analogues with modification on ribose, nucleobase, and pyrophosphate have been investigated. Among them, the analogue with the modification of the northern ribose by ether linkage substitution (cIDPRE) exhibits membrane‐permeate Ca²⁺ agonistic activity in intact HeLa cells, human T cells, mouse cardiac myocytes and neurosecretory PC12 cell lines; thus, cIDPRE and coumarin‐caged cIDPRE are valuable probes to investigate the cADPR‐mediated Ca²⁺ signal pathway.     

Aryl‐Phosphonate Lanthanide Complexes and Their Fluorinated Derivatives: Investigation of Their Unusual Relaxometric Behavior and Potential Application as Dual Frequency 1H/19F MRI Probes

A series of low molecular weight lanthanide complexes were developed that have high ¹H longitudinal relaxivities (r₁) and the potential to be used as dual frequency ¹H and ¹⁹F MR probes. Their behavior was investigated in more detail through relaxometry, pH‐potentiometry, luminescence, and multinuclear NMR spectroscopy. Fitting of the ¹H NMRD and ¹⁷O NMR profiles demonstrated a very short water residence lifetime (<10 ns) and an appreciable second sphere effect. At lower field strengths (20 MHz), two of the complexes displayed a peak in r₁ (21.7 and 16.3 mM ^?1 s^?1) caused by an agglomeration, that can be disrupted through the addition of phosphate anions. NMR spectroscopy revealed that at least two species are present in solution interconverting through an intramolecular binding process. Two complexes provided a suitable signal in ¹⁹F NMR spectroscopy and through the selection of optimized imaging parameters, phantom images were obtained in a MRI scanner at concentrations as low as 1 mM . The developed probes could be visualized through both ¹H and ¹⁹F MRI, showing their capability to function as dual frequency MRI contrast agents.     

Excimer‐Based On‐Off Bis(pyreneamide) Macrocyclic Chemosensors

A series of bis(pyreneamide) macrocycles, synthesized in two steps from THF, THP, oxepane and 1,4‐dioxane, are tested as chemosensors for a large range of mono‐, di‐ and trivalent cations. In their native states, these macrocycles exhibit a strong excimer fluorescence that is quenched upon the addition of the metal ions (alkaline, alkaline earth, p‐, d‐, and f‐block metals). UV‐Vis spectrophotometric titrations, cyclic voltammetry, excimer fluorescence quenching, and transient absorption spectroscopy experiments helped characterize the On‐Off changes occurring upon binding and demonstrate that the highest stability constants are obtained with divalent cations Ca²⁺ and Ba²⁺ specifically.     

A Highly Selective Fluorescence Turn‐On Sensor for Extracellular Calcium Ion Detection

A highly water‐soluble, fluorescence turn‐on sensor for Ca²⁺ is reported. The sensor affords high selectivity in sensing Ca²⁺ over other biologically important metal cations. The dissociation constant of the sensor in binding Ca²⁺ is 0.92 mm . Fluorescence microscopy experiments demonstrate that the sensor is cell‐impermeable and capable of detecting extracellular Ca²⁺.     

Direct Observation of Ca2+‐Induced Calmodulin Conformational Transitions in Intact Xenopus laevis Oocytes by 19F NMR Spectroscopy

The Ca²⁺‐mediated conformational transition of the protein calmodulin (CaM) is essential to a variety of signal transduction pathways. Whether the transition in living cells is similar to that observed in buffer is not known. Here, we report the direct observation by ¹⁹F NMR spectroscopy of the transition of the Ca²⁺‐free and ‐bound forms in Xenopus laevis oocytes at different Ca²⁺ levels. We find that the Ca²⁺‐bound CaM population increased greatly upon binding the target protein myosin light‐chain kinase (MLCK) at the same Ca²⁺ level. Paramagnetic NMR spectroscopy was also exploited for the first time to obtain long‐range structural constraints in cells. Our study shows that ¹⁹F NMR spectroscopy can be used to obtain long‐range structural constraints in living eukaryotic cells and paves the way for quantification of protein binding constants.     

Unanimous Model for Describing the Fast Bioluminescence Kinetics of Ca2+‐regulated Photoproteins of Different Organisms

Upon binding their metal ion cofactors, Ca²⁺‐regulated photoproteins display a rapid increase of light signal, which reaches its peak within milliseconds. In the present study, we investigate bioluminescence kinetics of the entire photoprotein family. All five recombinant hydromedusan Ca²⁺‐regulated photoproteins—aequorin from Aequorea victoria, clytin from Clytia gregaria, mitrocomin from Mitrocoma cellularia and obelins from Obelia longissima and Obelia geniculata—demonstrate the same bioluminescent kinetics pattern. Based on these findings, for the first time we propose a unanimous kinetic model describing the bioluminescence mechanism of Ca²⁺‐regulated photoproteins.     

Specific Detection and Imaging of Enzyme Activity by Signal‐Amplifiable Self‐Assembling 19F MRI Probes

Specific turn‐on detection of enzyme activities is of fundamental importance in drug discovery research, as well as medical diagnostics. Although magnetic resonance imaging (MRI) is one of the most powerful techniques for noninvasive visualization of enzyme activity, both in vivo and ex vivo, promising strategies for imaging specific enzymes with high contrast have been very limited to date. We report herein a novel signal‐amplifiable self‐assembling ¹⁹F NMR/MRI probe for turn‐on detection and imaging of specific enzymatic activity. In NMR spectroscopy, these designed probes are “silent” when aggregated, but exhibit a disassembly driven turn‐on signal change upon cleavage of the substrate part by the catalytic enzyme. Using these ¹⁹F probes, nanomolar levels of two different target enzymes, nitroreductase (NTR) and matrix metalloproteinase (MMP), could be detected and visualized by ¹⁹F NMR spectroscopy and MRI. Furthermore, we have succeeded in imaging the activity of endogenously secreted MMP in cultured media of tumor cells by ¹⁹F MRI, depending on the cell lines and the cellular conditions. These results clearly demonstrate that our turn‐on ¹⁹F probes may serve as a screening platform for the activity of MMPs.     

Simultaneous Fenton‐like Ion Delivery and Glutathione Depletion by MnO2‐Based Nanoagent to Enhance Chemodynamic Therapy

Chemodynamic therapy (CDT) utilizes iron‐initiated Fenton chemistry to destroy tumor cells by converting endogenous H₂O₂ into the highly toxic hydroxyl radical (^.OH). There is a paucity of Fenton‐like metal‐based CDT agents. Intracellular glutathione (GSH) with ^.OH scavenging ability greatly reduces CDT efficacy. A self‐reinforcing CDT nanoagent based on MnO₂ is reported that has both Fenton‐like Mn²⁺ delivery and GSH depletion properties. In the presence of HCO₃^?, which is abundant in the physiological medium, Mn²⁺ exerts Fenton‐like activity to generate ^.OH from H₂O₂. Upon uptake of MnO₂‐coated mesoporous silica nanoparticles (MS@MnO₂ NPs) by cancer cells, the MnO₂ shell undergoes a redox reaction with GSH to form glutathione disulfide and Mn²⁺, resulting in GSH depletion‐enhanced CDT. This, together with the GSH‐activated MRI contrast effect and dissociation of MnO₂, allows MS@MnO₂ NPs to achieve MRI‐monitored chemo–chemodynamic combination therapy.     

The Role of Equilibrium and Kinetic Properties in the Dissociation of Gd[DTPA‐bis(methylamide)] (Omniscan) at near to Physiological Conditions

[Gd(DTPA‐BMA)] is the principal constituent of Omniscan, a magnetic resonance imaging (MRI) contrast agent. In body fluids, endogenous ions (Zn²⁺, Cu²⁺, and Ca²⁺) may displace the Gd³⁺. To assess the extent of displacement at equilibrium, the stability constants of DTPA‐BMA^3? complexes of Gd³⁺, Ca²⁺, Zn²⁺, and Cu²⁺ have been determined at 37 °C in 0.15 M NaCl. The order of these stability constants is as follows: GdL≈CuL>ZnL?CaL. Applying a simplified blood plasma model, the extent of dissociation of Omniscan (0.35 mM [Gd(DTPA‐BMA)]) was found to be 17 % by the formation of Gd(PO₄), [Zn(DTPA‐BMA)]^? (2.4 %), [Cu(DTPA‐BMA)]^? (0.2 %), and [Ca(DTPA‐BMA)]^? (17.7 %). By capillary electrophoresis, the formation of [Ca(DTPA‐BMA)]^? has been detected in human serum spiked with [Gd(DTPA‐BMA)] (2.0 mM ) at pH 7.4. Transmetallation reactions between [Gd(DTPA‐BMA)] and Cu²⁺ at 37 °C in the presence of citrate, phosphate, and bicarbonate ions occur by dissociation of the complex assisted by the endogenous ligands. At physiological concentrations of citrate, phosphate, and bicarbonate ions, the half‐life of dissociation of [Gd(DTPA‐BMA)] was calculated to be 9.3 h at pH 7.4. Considering the rates of distribution and dissociation of [Gd(DTPA‐BMA)] in the extracellular space of the body, an open two‐compartment model has been developed, which allows prediction of the extent of dissociation of the Gd^III complex in body fluids depending on the rate of elimination of the contrast agent.     

A Synthetic Fluorescent Mitochondria‐Targeted Sensor for Ratiometric Imaging of Calcium in Live Cells

Ca²⁺ handling by mitochondria is crucial for cell life and the direct measure of mitochondrial Ca²⁺ concentration in living cells is of pivotal interest. Genetically‐encoded indicators greatly facilitated this task, however they require demanding delivery procedures. On the other hand, existing mitochondria‐targeted synthetic Ca²⁺ indicators are plagued by several drawbacks, for example, non‐specific localization, leakage, toxicity. Here we report the synthesis and characterization of a new fluorescent Ca²⁺ sensor, named mt‐fura‐2, obtained by coupling two triphenylphosphonium cations to the molecular backbone of the ratiometric Ca²⁺ indicator fura‐2. Mt‐fura‐2 binds Ca²⁺ with a dissociation constant of ≈1.5 μm in vitro. When loaded in different cell types as acetoxymethyl ester, the probe shows proper mitochondrial localization and accurately measures matrix [Ca²⁺] variations, proving its superiority over available dyes. We describe the synthesis, characterization and application of mt‐fura‐2 to cell types where the delivery of genetically‐encoded indicators is troublesome.     

Activatable 19F MRI Nanoparticle Probes for the Detection of Reducing Environments

¹⁹F magnetic resonance imaging (MRI) probes that can detect biological phenomena such as cell dynamics, ion concentrations, and enzymatic activity have attracted significant attention. Although perfluorocarbon (PFC) encapsulated nanoparticles are of interest in molecular imaging owing to their high sensitivity, activatable PFC nanoparticles have not been developed. In this study, we showed for the first time that the paramagnetic relaxation enhancement (PRE) effect can efficiently decrease the ¹⁹F NMR/MRI signals of PFCs in silica nanoparticles. On the basis of the PRE effect, we developed a reduction‐responsive PFC‐encapsulated nanoparticle probe, FLAME‐SS‐Gd³⁺ (FSG). This is the first example of an activatable PFC‐encapsulated nanoparticle that can be used for in vivo imaging. Calculations revealed that the ratio of fluorine atoms to Gd³⁺ complexes per nanoparticle was more than approximately 5.0×10², resulting in the high signal augmentation.     

Hydro­gen bonding in calcium–tri­fluoro­methane­sulfonate–1,3‐di‐4‐pyridyl­urea–methanol (1/2/2/4)

The structure of the supramolecular complex calcium–tri­fluoro­methane­sulfonate–1,3‐di‐4‐pyridyl­urea–methanol (1/2/2/4), Ca²⁺·2CF₃SO₃⁻·2C₁₁H₁₀N₄O·4CH₄O, is presented. The Ca²⁺ ion lies on an inversion centre and is octahedrally coordinated by four methanol mol­ecules and two tri­fluoro­methane­sulfonate counter‐ions. The molecular packing is dominated by hydrogen‐bonded sheets in the (110) plane which contain R(32) rings; in these rings, significant π–π interactions are observed between inversion‐related 1,3‐di‐4‐pyridyl­urea mol­ecules.     

Dual‐Modal Magnetic Resonance/Fluorescent Zinc Probes for Pancreatic β‐Cell Mass Imaging

Despite the contribution of changes in pancreatic β‐cell mass to the development of all forms of diabetes mellitus, few robust approaches currently exist to monitor these changes prospectively in vivo. Although magnetic‐resonance imaging (MRI) provides a potentially useful technique, targeting MRI‐active probes to the β cell has proved challenging. Zinc ions are highly concentrated in the secretory granule, but they are relatively less abundant in the exocrine pancreas and in other tissues. We have therefore developed functional dual‐modal probes based on transition‐metal chelates capable of binding zinc. The first of these, Gd ?1 , binds Zn^II directly by means of an amidoquinoline moiety (AQA), thus causing a large ratiometric Stokes shift in the fluorescence from λ_em=410 to 500 nm with an increase in relaxivity from r₁=4.2 up to 4.9 mM ^?1 s^?1. The probe is efficiently accumulated into secretory granules in β‐cell‐derived lines and isolated islets, but more poorly by non‐endocrine cells, and leads to a reduction in T₁ in human islets. In vivo murine studies of Gd ?1 have shown accumulation of the probe in the pancreas with increased signal intensity over 140 minutes.     

The calcium‐binding properties of pamidronate,a bone‐resorption inhibitor

The title compound, calcium bis(3‐ammonio‐1‐hydroxy­propyl­idene‐1,1‐bis­phospho­nate) dihydrate, Ca²⁺·2C₃H₁₀N­O₇P₂^?·2H₂O, consists of calcium octahedra arranged in columns along the c axis and coordinated by hydrogen‐bonded molecular anions. The Ca²⁺ cation lies on a twofold axis. Pamidronate adopts a twisted conformation of the hydroxy­alkyl­amine backbone that enables the formation of an intramolecular N—H?O hydrogen bond. The molecular anion is chelating monodentate as well as bidentate, with an O?O bite distance of 3.0647 (15) Å.     

Spectroscopic Observation of Calcium‐Induced Reorientation of Cellobiose Dehydrogenase Immobilized on Electrodes and its Effect on Electrocatalytic Activity

Cellobiose dehydrogenase catalyzes the oxidation of various carbohydrates and is considered as a possible anode catalyst in biofuel cells. It has been shown that the catalytic performance of this enzyme immobilized on electrodes can be increased by presence of calcium ions. To get insight into the Ca²⁺‐induced changes in the immobilized enzyme we employ surface‐enhanced vibrational (SERR and SEIRA) spectroscopy together with electrochemistry. Upon addition of Ca²⁺ ions electrochemical measurements show a shift of the catalytic turnover signal to more negative potentials while SERR measurements reveal an offset between the potential of heme reduction and catalytic current. Comparing SERR and SEIRA data we propose that binding of Ca²⁺ to the heme induces protein reorientation in a way that the electron transfer pathway of the catalytic FAD center to the electrode can bypass the heme cofactor, resulting in catalytic activity at more negative potentials.     

Lanthanum Chloride Promoted Proliferation with Enhanced S‐phase Entry and Inhibited Potassium Currents of NIH 3T3 Cells

The effects of La³⁺ on proliferation, cell cycles, apoptosis and ion channels were investigated in mouse embryo fibroblast NIH 3T3 cells and its possible mechanisms were explored. Our data showed that La³⁺ promoted cell proliferation with increased S‐phase entry and inhibited the outward potassium currents in a concentration‐dependent manner in NIH 3T3 cells. La³⁺ and Ca²⁺ had synergistic effect on cell proliferation and cell cycles. It showed that Ca²⁺ was needed for La³⁺‐promoted cell cycle progression. Using the whole‐cell voltage‐clamp technique, we found that La³⁺ blocked the outward potassium current in a concentration‐dependent manner in NIH 3T3 cells. Lanthanum ions can increase intracellular Ca²⁺ concentration through inhibition of potassium currents, which induce a series of physiological changes and improve proliferation of cells. This may be one of the molecular mechanisms of lanthanum ions induced cell proliferation. The present work provides a new perspective for understanding the biological and toxicological effects of lanthanum.     

Synthesis of Novel Dibenzo‐18‐crown‐6‐ ether‐Functionalized Benzimidazoles and its Applications in Colorimetric Recognition to Hg2+ and as Antifungal Agents

New crown ether‐functionalized benzimidazoles was designed and synthesized via formylation of dibenzo‐18‐crown‐6 followed by condensation with different o‐phenylene diamines. The complexation properties of crown ether‐functionalized benzimidazoles with various metals (K⁺, Ca²⁺, Ba²⁺, Co²⁺, Hg²⁺) were examined using UV–vis spectroscopy. Hg²⁺ showed a well‐defined peculiar absorption maximum at 366 nm exclusively. All these newly synthesized compounds were screened for antifungal activity against Aspergillus niger and Aspergillus oryzae, respectively.