Oxygen quenching of sensitized terbium luminescence in complexes of terbium with small organic ligands and proteins

Oxygen does not quench the luminescence of either free Tb or of Tb bound to dipicolinate. However, sensitized Tb luminescence in complexes of that ion with elastase, thermolysin, and alpha-amylase is quenched by oxygen at rates that far exceed that with which the intrinsic fluorescence of the proteins is quenched. We infer that this more rapid quenching of Tb luminescence indicates a major role for energy transfer from tryptophan moieties in a triplet excited state.     

Synthesis and luminescence properties of pyrazolone derivatives and their terbium complexes

Seven novel pyrazolone derivatives were synthesized and characterized by ¹H NMR and ¹³C NMR spectra, mass spectra, infrared spectra and elemental analysis. Their terbium complexes were prepared and characterized by elemental analysis, EDTA titrimetric analysis, UV/vis spectra, infrared spectra and molar conductivity, as well as thermal analysis. The fluorescence properties and fluorescence quantum yields of the complexes were investigated at room temperature. The results indicated that pyrazolone derivatives had good energy‐transfer efficiency for the terbium ion. All the terbium complexes emitted green fluorescence characteristic of terbium ions, possessed strong fluorescence intensity, and showed relatively high fluorescence quantum yields. Cyclic voltammograms of the terbium complexes were studied and the highest occupied molecular orbital (HOMO) and lowest occupied molecular orbital (LUMO) energy levels of these complexes were estimated. Copyright © 2014 John Wiley & Sons, Ltd.     

Advances in the study of luminescence probes for proteins

Spectral probes (or labels) have been widely used for the investigation and determination of proteins and have made considerable progress. Traditional luminescence probes include fluorescent derivatizing reagents, fluorescent probes and chemiluminescence probes which continue to develop. Of them, near infrared (NIR) fluorescent probes are especially suitable for the determination of biomolecules including proteins, so their development has been rapid. Novel luminescence probes (such as nanoparticle probes and molecular beacons) and resonance light scattering probes recently appeared in the literature. Preliminary results indicate that they possess great potential for ultrasensitive protein detection. This review summarizes recent developments of the above-mentioned probes for proteins and 195 references are cited.     

Copper chelates as probes of biological systems: stable copper complexes with a macrocyclic bifunctional chelating agent

Monoclonal antibodies tagged with chelated metal ions have numerous potential applications. Here we report the synthesis of a new "bifunctional" metal chelator, 6(p-bromoacetamidobenzyl)-1,4,8,11-tetraazacyclotetradecane- N,N',N",N"' -tetraacetic acid, which can be covalently attached to proteins and which binds copper stably in human serum under physiological conditions. In contrast, other reagents based on ethylenediaminetetraacetic acid or diethylenetriaminepentaacetic acid rapidly lose copper to serum albumin under the same conditions.     

Lanthanide(III) complexes with phosphoryl containing 1,8-naphthyridine: Crystal structures and vibrational spectra

The complexes of 2-[2-(diphenylphosphoryl)prop-2-yl]-1,8-naphthyridine (L) with lanthanide nitrates Ln(NO₃)₃ (Ln = Nd, Eu, Lu) were investigated to elucidate the coordination ability of a novel type of potentially tridentate ligands - phosphorylalkyl substituted naphthyridines. The X-ray crystal structures of [NdL₃]³⁺ · 3(NO₃)⁻ · MeCN (1), [EuL₃]³⁺ · 3(NO₃)⁻ · [Eu(NO₃)₃ · 4H₂O] · MeCN (2), and [LuL₃]³⁺ · 3(NO₃)⁻ · [Lu(NO₃)₃ · 3H₂O] · 2 MeCN · 0.5 H₂O (3) are reported together with their IR and Raman spectra. All the compounds studied contain isostructural [LnL₃]³⁺ cations and three NO₃⁻ counterions. Coordination of each L appears to be O,N,N tridentate-cyclic and coordination number of Ln is nine. Vibrational spectra of 1-3 are also compared with that of free ligand and model compounds.     

Lanthanide complexes of aminophosphonates as shift reagents for 7Li and 23Na NMR studies in biological systems.

A systematic NMR characterization of various Dy(III) complexes of linear and macrocyclic aminophosphonates as 7Li and 23Na NMR shift reagents for biological systems was undertaken. Their efficacy as shift reagents (SR) was tested under constant aqueous solution ionic strength conditions at pH 7.5 as a function of rho = [SR]/[M+]. Further characterization of the two best SRs, Dy(PcPcP)2(7-) and Dy(DOTP)5-, led to the conclusion that, although quite sensitive to solution pH and the presence of alkali metal ions and Mg2+ and Ca2+, these complexes were stable towards hydrolysis by phosphatases. The lack of precipitation of its solutions in the presence of Ca2+, allowed the choice of Dy(DOTP)5- as the best overall SR for biological studies. Other SRs, like Dy(TTHA)3-, although less sensitive to pH and to divalent ions, require significantly higher concentrations to yield the same shifts, leading to large bulk susceptibility artifacts in perfused tissues and organs.     

Fitting fluorescence emission spectra of probes bound to biological membranes

We show that fluorescence emission spectra for molecules containing the dansyl fluorophor can be accurately described as skewed Gaussians, and that spectra for dansyl probes bound to biological membranes can be resolved using least-squares techniques into two components, representing probe bound to the lipid and protein sites in the membrane.     

Comparison of terbium (III) luminescence enhancement in mutants of EF hand calcium binding proteins.

The luminescent isomorphous Ca2+ analogue, Tb3+, can be bound in the 12-amino acid metal binding sites of proteins of the EF hand family, and its luminescence can be enhanced by energy transfer from a nearby aromatic amino acid. Tb3+ can be used as a sensitive luminescent probe of the structure and function of these proteins. The effect of changing the molecular environment around Tb3+ on its luminescence was studied using native Cod III parvalbumin and site-directed mutants of both oncomodulin and calmodulin. Titrations of these proteins showed stoichiometries of fill corresponding to the number of Ca2+ binding loops present. Tryptophan in binding loop position 7 best enhanced Tb3+ luminescence in the oncomodulin mutant Y57W, as well as VU-9 (F99W) and VU-32 (T26W) calmodulin. Excitation spectra of Y57F, F102W, Y65W oncomodulin, and Cod III parvalbumin revealed that the principal Tb3+ luminescence donor residues were phenylalanine or tyrosine located in position 7 of a loop, despite the presence of other nearby donors, including tryptophan. Spectra also revealed conformational differences between the Ca2+- and Tb(3+)-bound forms. An alternate binding loop, based on Tb3+ binding to model peptides, was inserted into the CD loop of oncomodulin by cassette mutagenesis. The order of fill of Tb3+ in this protein reversed, with the mutated loop binding Tb3+ first. This indicates a much higher affinity for the consensus-based mutant loop. The mutant loop inserted into oncomodulin had 32 times more Tb3+ luminescence than the identical synthetic peptide, despite having the same donor tryptophan and metal binding ligands. In this paper, a ranking of sensitivity of luminescence of bound Tb3+ is made among this subset of calcium binding proteins. This ranking is interpreted in light of the structural differences affecting Tb3+ luminescence enhancement intensity. The mechanism of energy transfer from an aromatic amino acid to Tb3+ is consistent with a short-range process involving the donor triplet state as described by Dexter (Dexter, D. L. (1953) J. Chem. Phys. 21, 836). This cautions against the use of the F?rster equation in approximating distances in these systems.     

Lanthanide(III) complexes of pyridine-tetraacetic acid-glycoconjugates: synthesis and luminescence studies of mono and divalent derivatives

A potent lanthanide chelate, fulfilling the requirements for the development of MRI contrast agents or luminescent probes, was armed with alkyne groups. We then implemented a click methodology to graft the bifunctional ligand to azide-containing glucoside and maltoside scaffolds. The resulting hydrophilic glycoconjugates retained the ligand binding capacity for Eu(3+) or Tb(3+) ion as evidenced by the number of bound water molecules to the lanthanide ion. Divalent Eu(3+) and Tb(3+) complexes were shown to double the brightness of the emitted fluorescent signal compared to its monovalent derivatives. Designing multivalent lanthanide luminescent probes would enable the fluorescent signal of labeled biomolecules to be enhanced.     

Gold complexes with benzimidazole derivatives: synthesis,characterization and biological studies

Synthesis, characterization, DFT studies and biological assays of new gold(I) and gold(III) complexes of benzimidazole are reported. Molecular and structural characterizations of the compounds were based on elemental (C, H and N) and thermal (TG–DTA) analyses, and FT-IR and UV–Visible spectroscopic measurements. The structures of complexes were proposed based DFT calculations. The benzimidazole compounds (Lig1 and Lig2) and the gold complexes were tested against three Leishmania species related to cutaneous manifestations of leishmaniasis. The free benzimidazole compounds showed no leishmanicidal activity. On the other hand, the gold(I and III) complexes have shown to possess significant activity against Leishmania in both stages of parasite, and the gold(III) complex with Lig2 exhibited expressive leishmanicidal activity with IC₅₀ values below 5.7 μM. Also, the gold complexes showed high leishmania selectivity. The gold(I) complex with Lig1, for example, is almost 50 times more toxic for the parasite than for macrophages. Besides the leishmanicidal activity, all complexes exhibited toxic effect against SK-Mel 103 and Balb/c 3T3, cancer cells.     

Time-resolved europium(III) luminescence excitation spectroscopy: characterization of calcium-binding sites of calmodulin

Pulsed-dye laser excitation and lifetime spectroscopy of the 7F0----5D0 transition of Eu3+ reveals details of the binding of this ion to the calcium-binding sites of calmodulin (labeled I-IV, starting at the N-terminus). For 10 microM calmodulin Eu3+ binds quantitatively at sites I and II and more weakly at sites III and IV with Kd values of approximately 0.5 microM and 1.0 microM at the latter sites. In D2O solution the time course of luminescence emission of Eu3+-loaded calmodulin can be separated into three exponential components with lifetimes of 2.50 (sites I and II) and 1.70 and 0.63 ms (sites III and IV). This finding permits the time resolution of the excitation spectrum by determination of the amplitudes of the three components as the excitation wavelength is scanned across the spectral profile in 0.1-nm increments. The amplitudes (intensities at time t = O) are plotted as a function of wavelength and the results fitted to three Lorentzian peaks centered at 579.20, 579.40, and 579.32 nm in order of decreasing lifetimes. In H2O solution only two exponential luminescence decay components are resolvable with lifetimes of 0.41 and 0.27 ms, corresponding to sites I and II and sites III and IV, respectively. These results indicate that two water molecules are coordinated to the Eu3+ ions at sites I and II and at either site III or site IV, with three water molecules at the remaining site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytotoxic dinuclear titanium-salan complexes: structural and biological characterization

Controlled hydrolysis of donor-substituted titanium-salan complexes led to the formation of well-defined dinuclear complexes. Structure determination by means of X-ray and NMR-studies revealed the presence of a single μ-oxo bridge and one labile alkoxide ligand per titanium center. Concomitant cytotoxicity assays of the isolated dinuclear complexes showed cytotoxicities in the low micro-molar region, surpassing in this respect even their monomeric ancestors, thus making them possible highly active metabolites of titanium-salan anti-cancer drugs.     

Circularly polarized luminescence from terbium(III) as a probe of metal ion binding in calcium-binding proteins.

A number of different experimental techniques have been used to probe the details of structural changes on the binding of Ca(II) to the large number of known calcium-binding proteins. The use of luminescent lanthanide(III) ions, especially terbium(III) and europium(III), as substitutional replacement for calcium(II), has led to a number of useful experiments from which important details concerning the metal ion coordination sites have been obtained. This work is concerned with the measurement of the circularly polarized luminescence (CPL) from the 5D4----7F5 transition of Tb(III) bound to the calcium binding sites of bovine trypsin, bovine brain calmodulin, and frog muscle parvalbumin. It is demonstrated that it is possible to make these polarization measurements from very dilute solutions (less than 20 microM) and monitor structural changes as equivalents of Tb(III) are added. It is shown that the two proteins that belong to the class of "EF-hand" structures (calmodulin and parvalbumin) possess quite similar CPL line shapes, whereas Tb(III) bound to trypsin has a much different band structure. CPL results following competitive and consecutive binding of Ca(II) and Tb(III) bound to calmodulin are also reported and yield information concerning known differences between the sequence of binding of these two species.