Dual-frequency calcium-responsive MRI agents.

Responsive or smart magnetic resonance imaging (MRI) contrast agents are molecular sensors that alter the MRI signal upon changes in a particular parameter in their microenvironment. Consequently, they could be exploited for visualization of various biochemical events that take place at molecular and cellular levels. In this study, a set of dual-frequency calcium-responsive MRI agents are reported. These are paramagnetic, fluorine-containing complexes that produce remarkably high MRI signal changes at the (1)H and (19)F frequencies at varying Ca(2+) concentrations. The nature of the processes triggered by Ca(2+) was revealed, allowing a better understanding of these complex systems and their further improvement. The findings indicate that these double-frequency tracers hold great promise for development of novel functional MRI methods.

Investigation of a calcium-responsive contrast agent in cellular model systems: feasibility for use as a smart molecular probe in functional MRI.

Responsive or smart contrast agents (SCAs) represent a promising direction for development of novel functional MRI (fMRI) methods for the eventual noninvasive assessment of brain function. In particular, SCAs that respond to Ca(2+) may allow tracking neuronal activity independent of brain vasculature, thus avoiding the characteristic limitations of current fMRI techniques. Here we report an in vitro proof-of-principle study with a Ca(2+)-sensitive, Gd(3+)-based SCA in an attempt to validate its potential use as a functional in vivo marker. First, we quantified its relaxometric response in a complex 3D cell culture model. Subsequently, we examined potential changes in the functionality of primary glial cells following administration of this SCA. Monitoring intracellular Ca(2+) showed that, despite a reduction in the Ca(2+) level, transport of Ca(2+) through the plasma membrane remained unaffected, while stimulation with ATP induced Ca(2+)-transients suggested normal cellular signaling in the presence of low millimolar SCA concentrations. SCAs merely lowered the intracellular Ca(2+) level. Finally, we estimated the longitudinal relaxation times (T1) for an idealized in vivo fMRI experiment with SCA, for extracellular Ca(2+) concentration level changes expected during intense neuronal activity which takes place upon repetitive stimulation. The values we obtained indicate changes in T1 of around 1-6%, sufficient to be robustly detectable using modern MRI methods in high field scanners. Our results encourage further attempts to develop even more potent SCAs and appropriate fMRI protocols. This would result in novel methods that allow monitoring of essential physiological processes at the cellular and molecular level.

Towards libraries of luminescent lanthanide complexes and labels from generic synthons.

A synthetic approach is developed to obtain families of luminescent lanthanide complexes and markers from a generic family of precursors built from nonadentate coordination sites. The syntheses of the precursors, based on a directed regioselective nucleophilic aromatic substitution on polyfluoropyridines, are described. Functionalisation of the synthons on the aromatic moieties allowed the introduction of labelling functions and/or the extension of the electronic delocalisation, with concomitant changes in the spectroscopic properties. The synthesis of two such families of ligands and of some of their complexes of Eu(III) and Tb(III) are described, and the photo-physical properties of the complexes were measured, revealing excellent luminescence quantum yields reaching unity in some cases. For some of these complexes, the emphasis was further put on the preparation of an N-hydroxylsuccinimide (NHS) ester as activated function for labelling. The Tb and La complexes in the NHS activated form were synthesized and fully characterized. The labelling was first demonstrated on amino functionalized polymer beads and characterized by time-resolved luminescence microscopy. In a second step, the activated Tb complex was used for the labelling of GFR44 monoclonal antibody, and was applied to the detection of carcinoembryonic antigene (CEA) within the frame of a time-resolved fluoroimmunoassay. Comparison with a commercially available kit based on a europium cryptate as energy donor confirms the efficiency of Tb to act as an energy donor with an unoptimised 35% increase of the detection efficiency.

Divergent approach to a large variety of versatile luminescent lanthanide complexes.

Using a regioselective strategy for nucleophilic aromatic substitution on polyfluoropyridines, a nonacoordinating precursor was designed that is adequately suited for complexation of lanthanide cations. Further functionalizations afforded numerous applications for near-IR emission, two-photon absorption spectroscopy, or the formation of luminescent gels.

Solution structure and dynamics, stability, and NIR emission properties of lanthanide complexes with a carboxylated bispyrazolylpyridyl ligand.

The complexation behavior of the ligand 2,6-bis{3-[N,N-bis(carboxymethyl)aminomethyl]pyrazol-1-yl}-pyridine, L, toward lanthanide cations was investigated throughout the series. Potentiometric titration experiments on L (0.1 M KCl) revealed the presence of four protonation steps in the 2-12 pH domain, associated with the protonation of the two tertiary amine nitrogen atoms and with two of the four carboxylate functions. The stability constants for the formation of the [LnL](-) complexes (Ln = La, Nd, Eu, Ho, and Lu) were determined in water and evidenced a hill-shaped complexation trend along the series, with log K increasing from 14.56(9) (La) to 16.68(2) (Ho) and decreasing to 15.42(2) (Lu). Geometry optimizations showed the [LnL](-) complexes (Ln = La, Nd, Eu, Ho, Yb, and Lu) adopting a C(2) symmetry with the symmetry axis going through the metal atom and the nitrogen atom of the central pyridine ring, leading to the presence of Delta and Lambda helical enantiomers. Analysis of the calculated Ln-O and Ln-N bond lengths showed a marked deviation from the expected values at the end of the series, which accounts for the observed decreased complexation affinity. (1)H and (13)C NMR experiments in D(2)O (room temperature) showed the shortening of the bond distances in [LnL](-) complexes from La to Lu to be accompanied by a rigidification of the structure, leading to a decreased C(2) symmetry for the Lu complex compared to C(2v) for La. This decreased symmetry was attributed to a slow Delta <--> Lambda interconversion that was followed by variable-temperature (13)C NMR experiments on the Lu complex. The activation parameters determined by line broadening analysis for this interconversion process point to an associatively assisted interconversion process. The (1)H NMR spectrum of the paramagnetic [YbL](-) complex was investigated in D(2)O, and a lanthanide induced shift analysis showed good agreement between the observed paramagnetic chemical shifts and those calculated from the DFT optimized structures using a dipolar model, especially when solvent effects are taken into account. The UV-vis absorption and near-infrared luminescence spectra of the Pr, Nd, Er, and Yb complexes were measured in water and showed the complexation pocket provided by the ligand to be well-suited for the protection of the cations, all of them displaying typical Ln-centered emission spectra, the Yb complex having a relatively long lifetime of 3.0 micros in water.

Luminescence properties of heterodinuclear Pt-Eu complexes from unusual nonadentate ligands.

The synthesis of ligand L(1), based on the tetramethyl ester of [4'-ethynyl-(6,6''-bis(aminomethyl))-2,2':6',2''-terpyridine]tetrakisacetate, and its acidic form L(2), are described. Using a Cu-assisted coupling reaction, L(1) was connected to the Pt atom of a [(tpy)Pt] precursor (tpy = 2,2':6',2''-terpyridine) to afford the [(tpy)PtL(1)](BF(4)) metallosynthon, from which a hydrolysis reaction gave Na(3)[(tpy)PtL(2)]. The photophysical properties of the metallosynthons were studied by means of absorption and steady state emission spectroscopic techniques in various solvents, which revealed a dramatic impact of the solvent polarity. DFT and TDDFT calculations (B3LYP) were used to investigate the absorption and emission properties of the [(tpy)PtL(1)](+) system both in vacuo and in different solvents. Spectrophotometric titrations of [(tpy)PtL(1)](+) with EuCl(3) x 6 H(2)O in acetonitrile revealed the formation of complicated mixtures of complexes with different [(tpyPt(1))(x)Eu(y)] stoichiometries, all of which display the typical Eu(III) centred luminescence upon excitation into the Pt centred (1)MLCT absorption band. The energy transfer from the Pt subunit of [(tpy)PtL(2)](3-) to Eu(III) is inefficient in polar solvents, but it is restored in a TFA-CH(2)Cl(2) mixture, together with the observation of a new emission band at 684 nm, likely arising from a charge transfer process involving reduction of Eu(III).

Tuning the coordination sphere around highly luminescent lanthanide complexes.

A series of three ligands designed for the formation of water-soluble luminescent lanthanide complexes is described. All ligands are based on a 6''-carboxy-2,2':6',2''-terpyridine framework linked via a methylene bridge to n-butylamine. The second negatively charged arm consists of a 6-carboxy-2-methylenepyridine for L1, a 6'-carboxy-6-methylene-2,2'-bipyridine for L2, and a 6''-carboxy-6-methylene-2,2':6',2''-terpyridine for L3. The photophysical properties of the Eu and Tb complexes were studied in aqueous solutions by means of absorption spectroscopy and steady-state and time-resolved luminescence spectroscopy. Luminescence excited-state lifetimes were recorded and led to the determination of two water molecules in the first coordination sphere. The europium complexes were characterized by means of (1)H NMR spectroscopy in D 2O and DFT calculations performed at the B3LYP level both in vacuo and in aqueous solution. Finally, the influence of different phosphorylated anions such as HPO 4 (2-), ATP (4-), ADP (3-), and AMP (2-) on the luminescence properties of the [Eu L X (H 2O) 2] (+) complexes ( X = 1-3) was investigated in buffered aqueous solutions (0.01 M TRIS, pH 7.0), showing a significant interaction of ATP (4-) with [Eu( L2)(H 2O) 2] (+). The coordination of anions was understood in terms of partial decomplexation of one arm of the ligands and water displacement, with formation of ternary species, and it was rationalized on the basis of the structural models of the complexes obtained from DFT calculations.

Improving visible light sensitization of luminescent europium complexes.

The synthesis and characterization of the new ligands L(1), L(2) and L(4) are described with the series of four europium complexes of formula [EuL(n)(TTA)(3)] in which TTA refers to 2-thenoyltrifluoroacetonate and L(n) to tridentate ligands with nitrogen containing heterocyclic structure, such as a 2,6-bis(3-methyl-pyrazolyl)-4-(p-toluyl-ethynyl)-triazine for L(1), or terpyridines functionalized at the 4' position by a phenyl-vinylene for L(2), a p-dimethylamino-phenylene for L(3), or a p-aminophenyl-ethynylene for L(4). The spectroscopic properties of the ligands and of the complexes are studied by means of UV-Vis absorption spectroscopy, as well as steady-state and time-resolved luminescence spectroscopy. All complexes display europium centred luminescence upon ligand excitation. Careful examination of the excitation spectra revealed differences in the ligand based sensitization efficiencies. For complexes of L(1) and L(2), excitation of europium is mainly achieved through the TTA moieties and the photo-physical studies on [EuL(1)(TTA)(3)] evidenced a weaker coordination of the bispyrazolyltriazine tridentate ligand, resulting from a partial decomplexation upon dilution. Complexes of L(3) and L(4) display intense excitation through the tridentate units, which extend down to 460 nm in the visible region. In the case of L(3), selective excitation reveals the presence of a ligand-centred emission band at 520 nm which is likely ascribed to a L(3) centred charge transfer state.

Highly efficient blue photoexcitation of europium in a bimetallic Pt-Eu complex.

We report the preparation and characterization of dinuclear Pt-Ln complexes constructed from a square-planar Pt(II) core bearing an ethynyl-terpyridine residue connected to platinum by the ethynyl bond. Complexation of the neutral Eu(hfac)3 (hfac = hexafluoroacetylacetonate) fragment to free terpyridine (terpy) gives a stable bimetallic complex (log beta = 6.7). In the crystal structure, the flat Pt[triple bond]terpy core coordinates to Eu(III), which is nonacoordinated with the three nitrogen atoms of the terpy subunit and six oxygen atoms of the three hfac ligands. These atoms form a distorted monocapped square antiprism with a pseudo-C2 symmetry axis passing through the nitrogen atom of the central pyridine ring and the Eu atom. Spectroscopic measurements showed that irradiation with visible light of wavelength up to 460 nm in the 1MLCT state of the Pt subunit resulted in a quantitative energy transfer to the Eu center, which strongly luminesces in the red with an overall luminescence quantum yield of 38%. The energy-transfer process is quantitative and not sensitive to oxygen, and the complexation of Eu to the Pt metallosynthon allows the recovery of the energy lost due to triplet-oxygen quenching of the 3MLCT state observed in the uncomplexed Pt precursor.