Lanthanide Chelates Research Articles

Samarium-153 and Lutetium-177 Chelation Properties of Selected Macrocyclic and Acyclic Ligands

We describe a simple in vitro characterization of chelation that is useful when choosing an appropriate ligand-metal combination for clinical applications. These properties include the effect of concentration on chelation efficiency, time to maximum chelation, and stability in acidic and serum environments. The macrocyclic ligands nitro-DOTA and nitro-PADOTA, the acyclic ligands nitro-CHX-A-DTPA, nitro-MX-DTPA, DTPA, and a novel terpyridine ligand, TMT-amine, were evaluated as chelate complexes of both intermediate energy β-emitting lanthanides lutetium-177 and samarium-153. The data were compared to results obtained in a previously published study with yttrium-90. Acid lability, time to achieve maximum chelation, and stability in human serum are properties unique to each ligand-metal combination and should be evaluated prior to choosing an appropriate combination for therapeutic applications. Concentration dependence and duration of chelation are general properties of lanthanide and yttrium chelation that can be applied to an appropriate ligand-metal combination to achieve optimum chelation efficiencies.

Preparation and Testing of Diffusible Metal Chelate Fungicides

Water soluble chelates were formed by reacting copper, zinc, nickel, cobalt, chromium and rare earth oxides, hydroxides and carbonates with carboxylic acids, natural and synthetic amino acids, hydroxy acids, peptides, acetylacetone and sodium tripolyphosphate. The ligands were selected to give either electrically neutral or negatively charged metal complexes in order to minimise the coordinate bonding between the metal chelate and the timber substrate. Eighteen of the 38 chelates prepared were found to be effective fungicides, which readily diffused through the heartwood of high density eucalypt construction timbers. There was no apparent correlation between the physical properties of the chelates and fungitoxic efficacy, although the majority of the copper and rare earth chelates had good biocidal properties. The amount of metal ion bonded to the timber substrate could be varied by selecting ligands of different stability constants, chelates with low stability constants giving a greater extent of metal ion-wood bonding than chelates with high stability constants.

Application of the solvation parameter model to the characterisation of gas chromatographic stationary phases containing tris[3-(trifluoromethylhydroxymethylene)camphorato] derivatives of lanthanides

The solvation parameter model proposed by Abraham has been applied to retention factors, k, of over fifty different solutes on stationary phases containing tris[3-(trifluoromethylhydroxymethylene)camphorato] derivatives of Pr, Eu, Dy, Er and Yb dissolved in poly(dimethylsiloxane). Application of the solvation equation, log k=c+rR 2+sπ H 2+a Σα H 2+b Σβ H 2+l log L 16 at 393 K showed that the stationary phases studied had significant values of the descriptors ` a' and ` b' which describe the reversible complexation between lanthanide chelates and nucleophilic solutes. Increasing the atomic number of the lanthanide led to an increase of the ` a' descriptor and a simultaneous decrease of the ` b' descriptor. This behaviour was correlated with changes of the Lewis acidity of the lanthanide ions studied.

Seven-Color Time-Resolved Fluorescence Hybridization Analysis of Human Papilloma Virus Types

Identification of human papilloma virus (HPV) types is important in order to determine the risk of cervical carcinoma in women. This requires a technique to probe individual samples for multiple virus specificities. Here we describe simultaneous multicolor analysis of amplification products for any of seven amplified HPV types 16, 18, 31, 33, 35, 39, and 45, associated with cancer of the cervix. A seminested polymerase chain reaction was performed in a single tube using a biotinylated inner primer. Sets of amplification products, immobilized on a 96-pronged manifold solid support, were rendered single stranded and probed with a mix of seven type-specific, differentially labeled oligonucleotides. These probes contained 10 or 20 lanthanide chelates at the 5′ ends with seven distinct combinations of europium, terbium, and samarium ions. The seven viral strains were correctly identified by time-resolved fluorescence measurement of the specifically hybridized probes. Using this assay format, simultaneous detection of any of seven or even more target variants is possible.

Simple triple-label detection of seven cystic fibrosis mutations by time-resolved fluorometry

We describe a simple hybridization assay performed in microtitration wells with use of DNA probes labeled with three different lanthanide chelates for detection of seven mutations that cause cystic fibrosis. The assay is based on DNA amplification of four fragments containing the mutations (delta F508, G1717-->A, G542X, R553X, 3905 insertion T, W1282X, and N1303K) by PCR, followed by hybridization with short, allele-specific oligonucleotide probes labeled with europium, terbium, or samarium chelates. Because the technology makes it possible to hybridize three DNA probes simultaneously in one reaction, all 14 mutation-related alleles were detected in a total of five reaction wells. Blood spot specimens, obtained from children with cystic fibrosis, their parents, and their siblings, have been assayed, and for all the probes the positive signal-to-noise ratios are > 10. Solution hybridization utilizing triple-label time-resolved fluorometry combined with PCR is a suitable procedure for large-scale screening and automation.

Thiol-Reactive, Luminescent Europium Chelates: Luminescence Probes for Resonance Energy Transfer Distance Measurements in Biomolecules

Lanthanide chelates have recently been shown to be extremely promising luminescence probes for distance measurements in biomolecules using luminescence resonance energy transfer measurements [P. R. Selvin, T.M. Rana, and J.E. Hearst (1994)J. Am. Chem. Soc.116, 6029–6030; P.R. Selvin, and J.E. Hearst (1994)Proc. Natl. Acad. Sci. USA91, 10024–10028]. In this work we describe simple procedures for preparing highly fluorescent thiol-reactive europium chelates. These new compounds contain a uv-absorbing coumarin group which sensitizes europium emission, diethylenetriaminepentaacetic acid or triethylenetetraaminehexaacetic acid groups which provide europium chelating function, and a pyridyl disulfide group which allows specific modification of thiol groups. These reagents can be used to label proteins at Cys residues or synthetic oligonucleotides which contain thiol groups. Modification can be reversed easily by treatment with a reducing agent (dithiothreitol). Luminescence energy transfer between these new chelates and CY5 fluorochrome attached to the opposite ends of 15-bp double-stranded DNA was measured to test their usefulness for distance measurements in macromolecules. The distance measured between the chelate (donor) and CY5 (acceptor) was in the range expected for the length of 15-bp DNA. The stability of europium chelates and their conjugates with a protein, the precision of distance measurements using these chelates, possible errors due to intramolecular energy transfer, and the modulation of theR0value with deuterium oxide were tested. The results obtained fully confirmed the great potential of these new probes for sensitive, simple, and precise distance measurements in biomolecules using luminescence resonance energy transfer.

Macrocyclic polyamino carboxylate complexes of Gd(III) as magnetic resonance imaging contrast agents

Lanthanides are being used in medicine, diagnostics and therapeutics. A major area of their use is in magnetic resonance imaging (MRI) as contrast agents. In the present article, a summary of the studies on lanthanide chelates is presented which is useful in the design of MRI contrast agents with enhanced relaxivity, tolerance and tissue specificity. The properties of four potential hepatobiliary MRI contrast agents, which are being used clinically and preclinically, are also presented.

Development of luminescent lanthanide chelate labels for diagnostic assays

Luminescence properties of some of the lanthanide ions form the basis of highly sensitive diagnostic methods which employ their chelates as probes and time resolved fluorometry in the detection. In addition to being highly luminescent, i.e., having an efficient light collection and energy transfer, photostability, good quantum yield and long excited state lifetime, an ideal luminescent chelate label must be small and highly hydrophilic and contain a functional group enabling its gentle coupling to the bioreagent. Certain qualifications have to be fulfilled when luminescent chelate labels are to be made, such as the optimal position of ligand triplet states, position and type of the binding arm, insulation of the coupling group from the energy absorbing moiety, a critical distance between the luminescent moiety and protein surface (in case of Tb) and elimination of all possible quenching routes associated with O-H and C-H stretching (in case of Eu). Lanthanide chelates have already proven to be very useful in highly sensitive immunoassays and simplified homogeneous assays. In addition, the new chelates developed permit materialization of numerous future ideas, such as miniaturized assay devices able to perform multi-parametric assays on sub-microliter volumes of samples.

Rigidified macrocyclic lanthanide chelates for magnetic resonance imaging

New macrocyclic lanthanide chelates that are potential contrast agents for magnetic resonance imaging have been synthesized. The ligands are derived from 1,4,7,10-tetraazacyclododecane-N,NN″,N″′-tetraacetic acid (DOTA) and feature one or two substituting groups directly grafted onto the tetraaza ring. The rigidity of the chelates as seen in the NMR spectra of paramagnetic species depends directly on the steric preferences of the macrocyclic unit. The solution structures of the rigid species are deduced from a quantitative analysis of the paramagnetic shifts.

1,4,7,10,13,16,19,22-Octaazacyclotetracosane-1,4,7,10,13,16,19,22-octaacetic Acid (H8OTEC) and 1,4,7,10,14,17,20,23-Octaazacyclohexacosane- 1,4,7,10,14,17,20,23-octaacetic Acid (H8OHEC): Synthesis and Characterization of Two Large Macrocyclic Polyamine Polycarboxylic Ligands and Some of Their Copper(II) and Lanthanide(III) Complexes

The optimized synthesis of two new macrocyclic polyamine polycarboxylic ligands, 1,4,7,10,14,17,20,23-octaazacyclohexacosane-1,4,7,10,14,17,20,23-octaacetic acid (H8OHEC) (10) and 1,4,7, 10,13,16,19,22-octaazacyclotetracosane-1,4,7,10, 13,16,19,22-octaacetic acid (H8OTEC) (12), is presented. The key step in the synthesis of both is the high yield carboxymethylation of the corresponding macrocyclic amines using tert-butyl bromoacetate followed by acidic hydrolysis of the acetate protecting groups. The molecular structures of the intermediates 1,4,7,10,14,17,20,23-octaazacyclohexacosane (OHEC-amine) (8), and octa-tert-butyl 1,4,7,10,13,16,19,22- octaazacyclotetracosane-1,4,7,10,13,16,19,22-octaacetate (OTEC-ester) (11) are determined by X-ray crystal structure analysis. OHEC-amine 8 reacts with 2 equiv. of CuSO4 yielding the dinuclear complex [Cu2(OHEC-amine)](SO4)2 (13). Complex 13 crystallizes with 16 molecules of water. 13 · 16 H2O contains two copper atoms, which are coordinated in a strongly distorted octahedral fashion by four nitrogen atoms, one oxygen atom from the sulfate dianion and one oxygen atom from a water molecule. The new ligands 10 and 12 are fully characterized by 1D- and 2D-NMR spectroscopy. Both ligands form dinuclear lanthanide(III) chelates (LnY, Sm, Eu, Gd, Yb, Lu), which are stable and highly water soluble. With lanthanum(III) only mononuclear complexes are formed.

Contrast agents in functional MR imaging.

Contrast agents have greatly expanded the role of MR imaging (MRI) to allow assessment of physiologic, or "functional," parameters. Although activation mapping generally does not require contrast agents, other forms of functional MRI, including mapping of cerebral hemodynamics (eg, perfusion imaging), are best done with the use of contrast agents. Serial echo planar images are obtained after bolus injection of lanthanide chelates. Application of susceptibility contrast physics and standard tracer kinetic principles permits generation of relative cerebral blood volume maps. Deconvolution of cerebral blood flow and mean transit time parameters is also possible within technical limitations. By using diffusion and perfusion pulse sequences, an imaging correlate to the ischemic penumbra can be identified. Functional MRI perfusion imaging of intraaxial tumors is analogous to positron emission tomography for delineation of metabolic activity, yet may be even more sensitive to neovascularity and possesses improved image quality. Clinical applications include biopsy site selection and postirradiation follow-up. Further improvements in data analysis and map generation techniques may improve diagnostic accuracy and utility.

Lanthanide‐based susceptibility contrast agents: Assessment of the magnetic properties

The T2* contrast efficacy of paramagnetic contrast agents is dependent on their magnetic properties. Vibrating sample magnetometry (VSM) and the Live Chan NMR method have been used to evaluate the influence of ligand structure on the bulk magnetic susceptibility (BMS) of low-molecular weight (LMW) lanthanide chelates. VSM was also used for the BMS assessment of LMW lanthanide chelates covalently attached to cross-linked starch particles. The ligand structure had no influence on the BMS of the gadolinium (Gd) and dysprosium (Dy) chelates. The mean BMS value of the Dy-chelates was 1.8 fold higher than that of the Gd-chelates. The holmium (Ho) DTPA-BMA chelate had a similar BMS to that of Dy-DTPA-BMA while the lowest BMS was found for europium (Eu(III)) DTPA-BMA. The covalent attachment of Gd-DTPA and Dy-DTPA to a cross-linked starch particle had no impact on their intrinsic magnetic properties. The BMS data were in good accordance with those obtained for non-particulate bound LMW Dy- and Gd-chelates. The magnetic susceptibility of the Gd-DTPA labeled particles was described by the Curie law, indicative of no magnetic interactions between Gd-DTPA molecules. The magnetic susceptibility of the Dy-DTPA labeled particles followed the Curie-Weiss law with a Curie-Weiss temperature of about-2 K, indicating magnetic interactions. The magnetic susceptibility of Dy-DTPA will, however, not be affected by such magnetic interactions at physiological temperatures.

Complexation of Thorium(IV) and Uranium(IV) by a Hexaacetic Hexaaza Macrocycle: Kinetic and Thermodynamic Topomers of Actinide Chelates with a Large Cavity Ligand.

The solution behavior of the Th(IV) chelate with a polyaza polycarboxylic ligand, HEHA (1,4,7,10,13,16-hexaazacyclooctadecane-N,N ',N ",N "',N "",N ""'-hexaacetic acid), is investigated by one- and two-dimensional NMR spectroscopy. [Th(HEHA)](2)(-) exhibits a very unusual irreversible topomerization process from a kinetically stable topomer of C(2) symmetry to a thermodynamically stable topomer of remarkably high symmetry (S(6)). The metal ion appears to be 12-coordinated in both geometries with a pseudoicosahedral arrangement of the chelating groups. The activation energy of the irreversible topomerization is exceedingly high (199 kJ.mol(-)(1)), and the slow kinetics of conversion is assigned to a complete reorganization of the chelate. U(IV) forms directly a stable entity of C(2) symmetry with HEHA and no reorganization of the complex is observed. The unusual topomerization phenomenon undergone by HEHA, is thus strongly dependant on the ionic radius of the encapsulated metal ion. The corresponding lanthanide chelates are totally asymmetric.

Self-assembled heterodinuclear europium(III)–lanthanide(III) chelates of 2,6-bis[N,N-bis(carboxymethyl)aminomethyl]-4-benzoylphenol and their radiative5D0→7Fjtransitions of EuIII

A photochemical study has been conducted (i) to shed light on the intramolecular T1-relaxation pathways of 2,6-bis[N,N-bis(carboxymethyl)aminomethyl]-4-benzoylphenol (L) to initiate the radiative 5D0→7Fj transitions of EuIII and (ii) to elucidate the effects of different aqua-bridged non-radiative LnIII cations on these EuIII-specific radiative transitions, using the self-assembled heterodinuclear LEuIIILnIIIL chelates where the EuIII and LnIII cations are coordinated to each other by the aqua bridge inside the chelating cage consisting of two deprotonated phenolic hydroxy groups and four partially deprotonated 2,2′-(methylenenitrilo)bis(acetic acid) moieties. Depending on its non-radiative LnIII cation, the encapsulated aqua-bridged EuIIILnIII pair may be (i) as in the case of LnIII= ErIII, HoIII, NdIII or PrIII, a double-nuclear triplet relaxation centre where the T1-relaxation occurs mainly to the excited state of LnIII, (ii) as in the case of LnIII= GdIII or YIII, a single-nuclear triplet relaxation centre where the T1-relaxation is energetically capable of occurring only to the excited state 5Dj of EuIII, so that the resulting radiative 5D0→7Fj transitions of EuIII are to some extent enhanced and, finally, (iii) as in the case of LnIII= YbIII, a single-nuclear triplet relaxation centre where the T1-relaxation occurs only to the excited state of EuIII, so that the resulting excited state 5D0 of EuIII is strongly destabilized by the aqua-bridged YbIII: however, our present experimental results do not exclude the possibility of the aqua-bridged YbIII cation occurring also as the T1-relaxation centre despite the large energy gap ΔE(T1–2F7/2)= 11 560 cm–1.

Mr contrast due to intravascular magnetic susceptibility perturbations

A particularly powerful paradigm for functional MR imaging of microvascular hemodynamics incorporates paramagnetic materials that create significant image contrast. These include exogenous (lanthanide chelates) and endogenous (deoxygenated hemoglobin) agents for mapping cerebral blood volume and neuronal activity, respectively. Accurate interpretation of these maps requires an understanding of the biophysics of susceptibility-based image contrast. The authors developed a novel Monte Carlo model with which the authors quantified the relationship between microscopic tissue parameters, NMR imaging parameters, and susceptibility contrast in vivo. The authors found vascular permeability to water and the flow of erythrocytes to be relatively unimportant contributors to susceptibility-induced delta R2. However, pulse sequence, echo time, and concentration of contrast agent have profound effects on the vessel size dependence of delta R2. For a model vasculature containing both capillaries and venules, the authors predicted a linear volume fraction dependence for physiological volume changes based on recruitment and dilation, and a concentration dependence that is nonlinear and pulse sequence dependent. Using the model, the authors demonstrated that spin echo functional images have greater microvascular sensitivity than gradient echo images, and that the specifies of the volume fraction and concentration dependence of transverse relaxivity change should allow for robust mapping of relative blood volume. The authors also demonstrated excellent agreement between the predictions of their model and experimental data obtained from the serial injection of superparamagnetic contrast agent in a rat model.

Luminescent Polyaminocarboxylate Chelates of Terbium and Europium: The Effect of Chelate Structure

We have synthesized and spectrally characterized a series of new luminescent lanthanide complexes based on linear and macrocycle polyaminocarboxylate chelates, covalently joined to an organic sensitizer 7-amino-4-methyl-2(1H)-quinolinone (carbostyril 124). These complexes are luminescent with both terbium and europium and have millisecond lifetime, sharply spiked emission spectra ( 150 nm), excellent solubility, moderate absorption (12000 M{sup -1} cm{sup -1} at 327 nm), and high quantum yields for lanthanide emission. These characteristics make them useful alternatives to radioactive probes, to fluorescent dyes, and as donors in energy transfer experiments. A comparison of luminescence, intensity, spectra, lifetime, and number of coordinated waters is made. Net charge varies from -2 to neutral and water coordination number from 0.2 to 1.2. By comparison of lanthanide emission lifetimes with lanthanide chelates without sensitizer, it is shown that the sensitizer does not lead to nonradiative de-excitation and that the quantum yield for lanthanide emission is likely close to unity. These chelates are the most efficient energy transfer donors yet synthesized. In imaging, they may make possible two-color detection with no spectral overlap and a single excitation wavelength, as well as the ability to discriminate against short-lived autofluorescence background. 42 refs., 3 figs.,more » 2 tabs.« less

Bifunctional fusion proteins consisting of a single-chain antibody and an engineered lanthanide-binding protein.

The combination of an antibody fragment with a lanthanide chelating protein has desirable characteristics for fluorescence-based immunoassays and tumor radioimmunotherapy. As a model for this design, a fusion protein consisting of a single-chain antibody linked to an engineered version of oncomodulin, a protein with two Ca(2+)-binding motifs (the CD and EF loops), was produced by secretion from Escherichia coli in good yield. The single-chain antibody was specific for a Salmonella O-polysaccharide. The CD loop of oncomodulin had been redesigned to bind lanthanide ions with high affinity. The fusion protein was shown to have antigen-binding activity that was comparable to that of the unfused single-chain antibody, to bind Tb3+ with very high affinity and to give strong, sensitized Tb3+ luminescence via excitation of the tryptophan residue in the CD loop. A second fusion protein containing a 30-residue helix-loop-helix motif as the lanthanide-binding component was also prepared, but showed considerably lower solubility. Competition for Tb3+ binding by a series of metal chelators indicated that the affinities of the oncomodulin and 30 residue fusions for Tb3+ were approximately 10(11) M-1 and 10(7) M-1, respectively. Time-resolved lanthanide luminescence photography of electrophoresis gels demonstrated that the helix-loop-helix Ca(2+)-binding could be used to specifically visualize the scFv fragment.

Nucleic acid labelling employing lanthanide chelates

Labelling of nucleic acids with lanthanide(III) chelates is described. DNA can be labelled either enzymatically or chemically. Enzymatic labelling of DNA can be performed with Eu-dCTP. For chemical labelling of DNA, a two-phase protocol can be used whereby it is possible to incorporate Eu(III), Sm(III) and Tb(III) chelates in DNA. In the chemical labelling of oligonucleotides, an amino-modified phosphoramidite is used to introduce amino groups to oligonucleotides. These amino groups can be labelled with lanthanide chelates.

Luminescent lanthanide chelates—a way to more sensitive diagnostic methods

The unique luminescence properties of certain lanthanides are valuable features in aiming at more sensitive label technologies for clinical diagnostic use. The use of these properties in bioanalytical assays requires development of specific chelate technologies for labelling, i.e. binding of the ion to bioaffinity reagents, carrying it through the assay procedure, creating the optimal environment for emission and so forth. So far the most successful lanthanide label technology is composed of sequential use of two different chelate systems: a bifunctional polyaminopolycarboxylate derivative used for labelling of bioaffinity reactants with the lanthanide ion and a β-diketone-TOPO chelate optimized for time-resolved fluorometric determinations. This technology has achieved an established position as a highly sensitive method in in vitro clinical immunodiagnostics. Subsequently, a number of alternative approaches have been developed, including stable and highly luminescent chelates, which can be directly coupled to bioaffinity reactants to form luminescent tracers. These chelates are invaluable in studies aiming at sensitive and quantifiable localizations of analytes directly in situ, e.g. on tissue sections or cell surfaces.

Synthesis and luminescence properties of some Eu(III) and Tb(III) chelate labels having 2,2′:6′,2″-terpyridine as an energy absorbing part

A series of luminescent 2,2′:6′,2″-terpyridine lanthanide chelates, in which the group needed for coupling to bioactive molecules is situated in the chelating part of the molecule, was synthesized. The luminescence properties of the labels both as free activated chelates and after coupling to proteins were also studied.