Prototypes of Lanthanide(III) Agents Responsive to Enzymatic Activities in Three Complementary Imaging Modalities: Visible/Near-Infrared Luminescence, PARACEST-, and T1-MRI.

We report first prototypes of responsive lanthanide(III) complexes that can be monitored independently in three complementary imaging modalities. Through the appropriate choice of lanthanide(III) cations, the same reactive ligand can be used to form complexes providing detection by (i) visible (Tb(3+)) and near-infrared (Yb(3+)) luminescence, (ii) PARACEST- (Tb(3+), Yb(3+)), or (iii) T1-weighted (Gd(3+)) MRI. The use of lanthanide(III) ions of different natures for these imaging modalities induces only a minor change in the structure of complexes that are therefore expected to have a single biodistribution and cytotoxicity.

Lanthanide(III) complexes that contain a self-immolative arm: potential enzyme responsive contrast agents for magnetic resonance imaging.

Enzyme-responsive MRI-contrast agents containing a "self-immolative" benzylcarbamate moiety that links the MRI-reporter lanthanide complex to a specific enzyme substrate have been developed. The enzymatic cleavage initiates an electronic cascade reaction that leads to a structural change in the Ln(III) complex, with a concomitant response in its MRI-contrast-enhancing properties. We synthesized and investigated a series of Gd(3+) and Yb(3+) complexes, including those bearing a self-immolative arm and a sugar unit as selective substrates for ß-galactosidase; we synthesized complex LnL(1), its NH(2) amine derivatives formed after enzymatic cleavage, LnL(2), and two model compounds, LnL(3) and LnL(4). All of the Gd(3+) complexes synthesized have a single inner-sphere water molecule. The relaxivity change upon enzymatic cleavage is limited (3.68 vs. 3.15 mM(-1) s(-1) for complexes GdL(1) and GdL(2), respectively; 37 °C, 60 MHz), which prevents application of this system as an enzyme-responsive T(1) relaxation agent. Variable-temperature (17)O NMR spectroscopy and (1)H NMRD (nuclear magnetic relaxation dispersion) analysis were used to assess the parameters that determine proton relaxivity for the Gd(3+) complexes, including the water-exchange rate (k(ex)(298), varies in the range 1.5-3.9×10(6) s(-1)). Following the enzymatic reaction, the chelates contain an exocyclic amine that is not protonated at physiological pH, as deduced from pH-potentiometric measurements (log K(H)=5.12(±0.01) and 5.99(±0.01) for GdL(2) and GdL(3), respectively). The Yb(3+) analogues show a PARACEST effect after enzymatic cleavage that can be exploited for the specific detection of enzymatic activity. The proton-exchange rates were determined at various pH values for the amine derivatives by using the dependency of the CEST effect on concentration, saturation time, and saturation power. A concentration-independent analysis of the saturation-power-dependency data was also applied. All these different methods showed that the exchange rate of the amine protons of the Yb(III) complexes decreases with increasing pH value (for YbL(3), k(ex)=1300 s(-1) at pH 8.4 vs. 6000 s(-1) at pH 6.4), thereby resulting in a diminution of the observed CEST effect.

Calcium-responsive paramagnetic CEST agents.

The assessment of changes in the extracellular calcium concentration by magnetic resonance imaging would be a valuable biomedical research tool to monitor brain neuronal activity. In this perspective, we report here the synthesis of novel ligands consisting of tetraamide and bisamide derivatives of cyclen, L(1) and L(2), respectively, each bearing imino(diacetate) moieties for Ca(2+) binding. Yb(3+) and Eu(3+) complexes are investigated as chemical exchange saturation transfer (CEST) agents that respond to the presence of Ca(2+). A CEST effect is observed for both YbL(1) and EuL(1) complexes (B=11.7T), originating from the slow exchange of the amide protons and those of the coordinated water, respectively, whilst no CEST is detected for complexes of L(2). Upon calcium binding, the CEST effect decreases considerably (from 60% to 20% for YbL(1) and from 35% to 10% for EuL(1)). A similar variation is observed in the presence of Mg(2+). The affinity constants between the lanthanide complexes and the alkaline earth metal ions have been estimated from the variation of the CEST effect to be K(YbL(1)-Ca)(aff) = 8 ± 2M(-1), K(YbL(1)-Mg)(aff) = 23 ± 3M(-1) and K(EuL(1)-Ca)(aff) = 10 ± 3M(-1). These low values imply the coordination of the alkaline earth ions to a single iminodiacetate arm. Ca(2+)/Mg(2+) binding to the lanthanide complexes slows down the exchange of the amide protons on YbL(1) which is responsible for the diminished CEST effect. This has been evidenced by assessing the proton exchange rates from the dependency of the CEST effect on the saturation time and the saturation power, in the absence and in the presence of Ca(2+) and Mg(2+). The applicability of the PARACEST MRI agents for Ca(2+) detection has been evaluated on a 16T MRI scanner.

Molecular recognition of sialic acid by lanthanide(III) complexes through cooperative two-site binding.

Herein we report two new ligands, 1,4,7-tris(carboxymethyl)-10-[2-(dihydroxyboranyl)benzyl]-1,4,7,10-tetraazacyclododecane (L(1)) and 1,4,7-tris(carboxymethyl)-10-[3-(dihydroxyboranyl)benzyl]-1,4,7,10-tetraazacyclododecane (L(2)), which contain a phenylboronic acid (PBA) function and a 1,4,7,10-tetraazacyclododecane-1,4,7-triacetate cage for complexation of lanthanide ions in an aqueous solution. The pK(a) of the PBA function amounts to 4.6 in [Gd(L(1))] and 8.9 in [Gd(L(2))], with the value of the L(2) analogue being very similar to that of PBA (8.8). These results are explained by the coordination of the PBA function of L(1) to the Gd(III) ion, which results in a dramatic lowering of its pK(a). As a consequence, [Gd(L(1))] does not bind to saccharides at physiological pH. The nuclear magnetic relaxation dispersion profiles recorded for [Gd(L(1))] and [Gd(L(2))] confirm that the phenylboronate function is coordinated to the metal ion in the L(1) derivative, which results in a q = 0 complex. The interaction of the [Gd(L(2))] complex with 5-acetylneuraminic acid (Neu5Ac) and 2-alpha-O-methyl-5-acetylneuraminic acid (MeNeu5Ac) has been investigated by means of spectrophotometric titrations in an aqueous solution (pH 7.4, 0.1 M 3-(N-morpholino)propanesulfonic acid buffer). Furthermore, we have also investigated the binding of these receptors with competing monosaccharides such as D-(+)-glucose, D-fructose, D-mannose, D-galactose, methyl alpha-D-galactoside, and methyl alpha-D-mannoside. The binding constants obtained indicate an important selectivity of [Gd(L(2))] for Neu5Ac (K(eq) = 151) over D-(+)-glucose (K(eq) = 12.3), D-mannose (K(eq) = 21.9), and D-galactose (K(eq) = 24.5). Furthermore, a very weak binding affinity was observed in the case of methyl alpha-D-galactoside and methyl alpha-D-mannoside. An 8-fold increase of the binding constant of [Gd(L(2))] with Neu5Ac is observed when compared to that of PBA determined under the same conditions (K(eq) = 19). (13)C NMR spectroscopy and density functional theory calculations performed at the B3LYP/6-31G(d) level show that this is due to a cooperative two-site binding of Neu5Ac through (1) ester formation by interaction on the PBA function of the receptor and (2) coordination of the carboxylate group of Neu5Ac to the Gd(III) ion. The emission lifetime of the (5)D(4) level of Tb(III) in [Tb(L(2))] increases upon Neu5Ac binding, in line with the displacement of inner-sphere water molecules due to coordination of Neu5Ac to the metal ion.

Densely packed Gd(III)-chelates with fast water exchange on a calix[4]arene scaffold: a potential MRI contrast agent.

A pyridine-N-oxide functionalized DOTA analogue has been conjugated to a calix[4]arene and the corresponding Gd-complex was characterized with respect to its suitability as MRI contrast agent. The compound forms spherical micelles in water with a cmc of 35 microM and a radius of 8.2 nm. The relaxivity of these aggregates is 31.2 s(-1) mM(-1) at 25 degrees C and 20 MHz, which corresponds to a molecular relaxivity of 125 s(-1) mM(-1). The high relaxivity mainly originates from the short tau(M) (72.7 ns) and the size of the micelles. The interaction with bovine serum albumin (BSA) was studied and an observed relaxivity of up to 40.8 s(-1) mM(-1) (163.2 s(-1) mM(-1) per binding place) at 20 MHz and 37 degrees C was found in the presence of 2.0 mM protein.