Synthesis of nonluminescent lanthanide(III) chelates tethered to an aminooxy group and their applicability to biomolecule derivatization

Synthesis of nonluminescent lanthanide(III) chelates tethered to an aminooxy group (i.e., 1-[4-(6-aminooxyhexamido)benzyl]diethylenetriaminetetraacetic acid lanthanides(III), 6a-d, where Ln(3+) is Eu, Dy, Sm, and Tb) is described. Their applicability to biomolecule derivatization is demonstrated by allowing them to react with a synthetic oligopeptide, a protein, two synthetic drugs, and a steroid. The oligopeptide and protein were linked to 6 after preoxidation of their N-terminal serine residues, while the drugs and the steroid reacted via their ketone functionality. Also some application data is included.     

Introduction of lanthanide(III) chelates to oligopeptides on solid phase

The synthesis of oligopeptide building blocks for the introduction of nonluminescent and luminescent lanthanide(III) chelates to the oligopeptide structure on the solid phase is described. The oligopeptide conjugates synthesized were used in DELFIA-based receptor binding assay (motilin) as well as in LANCE time-resolved fluorescence quenching assay (caspase-3).     

Solid-phase synthesis of oligonucleotides labeled with luminescent lanthanide(III) chelates

The synthesis of phosphoramidite building blocks that allow introduction of luminescent europium(III), terbium(III), dysprosium(III), and samarium(III) chelates to oligonucleotides on the solid phase is described. Several labeled oligonucleotides using these building blocks were prepared, and the photophysical properties of these bioconjugates were investigated.     

Convenient synthesis of maleimido-derivatized lanthanide(III) chelates and their use in mercapto group conjugation

Simple synthesis of luminescent europium(III) and terbium(III) chelates tethered to a maleimido function (7, 8) is described. The method is based on the following: (i) synthesis of protected ligands tethered to a maleimido function and their purification on silica gel; (ii) deprotection by acidolysis; (iii) conversion of the deprotected ligands to the corresponding lanthanide(III) chelates by passing them through a column of strong cation exchange resin loaded with the appropriate lanthanide(III) ions. According to this procedure, large quantities of mercapto-selective biomolecule-labeling reactants of high purity can be prepared.     

The effect of regioisomerism on the coordination chemistry and CEST properties of lanthanide(III) NB-DOTA-tetraamide chelates

Chemical exchange saturation transfer (CEST) offers many advantages as a method of generating contrast in magnetic resonance images. However, many of the exogenous agents currently under investigation suffer from detection limits that are still somewhat short of what can be achieved with more traditional Gd³⁺ agents. To remedy this limitation we have undertaken an investigation of Ln³⁺ DOTA-tetraamide chelates (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that have unusually rigid ligand structures: the nitrobenzyl derivatives of DOTA-tetraamides with (2-phenylethyl)amide substituents. In this report we examine the effect of incorporating hydrophobic amide substituents on water exchange and CEST. The ligand systems chosen afforded a total of three CEST-active isomeric square antiprismatic chelates; each of these chelates was found to have different water exchange and CEST characteristics. The position of a nitrobenzyl substituent on the macrocyclic ring strongly influenced the way in which the chelate and Ln³⁺ coordination cage distorted. These differential distortions were found to affect the rate of water proton exchange in the chelates. But, by far the greatest effect arose from altering the position of the hydrophobic amide substituent, which, when forced upwards around the water binding site, caused a substantial reduction in the rate of water proton exchange. Such slow water proton exchange afforded a chelate that was 4.5 times more effective as a CEST agent than its isomeric counterparts in dry acetonitrile and at low temperatures and very low presaturation powers.     

Comparative study of glutamine synthetase bound lanthanide(III) ions using NMR relaxation and lanthanide(III) luminescence techniques

Changes in the intrinsic fluorescence intensity of glutamine synthetase induced by lanthanide(III) ion binding demonstrate the existence of three types of sites for these ions. The sites are populated sequentially during titrations of the enzyme, and the first two have a stoichiometry of 1 per enzyme subunit. The number of water molecules coordinated to Eu(III) bound to the first site was determined by luminescence lifetime techniques to be 4.1 +/- 0.5. The hydration of Gd(III) bound to the same site was studied by magnetic field dependent water proton longitudinal relaxation rate measurements, and by water proton and deuteron relaxation measurements of one sample at single magnetic fields. The magnetic resonance techniques also yield a value of 4 for the hydration number.     

Spectroscopic study of lanthanide(III) complexes with aliphatic dicarboxylic acids

The complexation of trivalent lanthanides with aliphatic dicarboxylic acids (malonic, succinic, glutaric and adipic) were studied at 25°C and 0.1 M (NaClO₄) ionic strength by luminescence and absorption spectroscopy and luminescence lifetime measurements. The luminescence spectra and decay constants indicate that ML and ML₂ complexes were formed. The stability constants of Eu(III) complexes with the dicarboxylic acids were calculated from the changes of the ⁵D₀←⁷F₀ excitation spectra of Eu(III). For the four dicarboxylic acids studied, both the stability constant and the number of water molecules released from the inner sphere of Eu(III) upon complexation decrease from malonate to adipate for both the ML and ML₂ complexes. The results are interpreted as reflecting an increasing tendency from chelation to monodentation as the carbon chain length increases between carboxylate groups. The trend in the oscillator strength in the hypersensitive transition of the Nd(III)and Ho(III) complexes is the same as that in the ligand basicity.     

Phosphorescence of gadolinium(III) chelates under ambient conditions

The gadolinium(III) chelates Gd(dtpaH₂), Gd(hfac)₃, Gd(tta)₃ and Gd(qu)₃ with dtpa=1,1,4,7,7-diethylenetriaminepentaacetate, hfac=hexafluoroacetylacetonate, tta=thenoyltrifluoroacetonate and qu=8-quinolinolate (or oxinate) show a phosphorescence under ambient conditions. While the UV emission of Gd(dtpaH₂) at λ_max=312 nm comes from a metal-centered ff state, the bluish (λ_max=462 nm), green (λ_max=505 nm) and red (λ_max=650 nm) luminescence of Gd(hfac)₃, Gd(tta)₃ and Gd(qu)₃, respectively, originates from the lowest-energy intraligand triplets.     

Characterization of lanthanide (III) ion binding to calmodulin using luminescence spectroscopy

Pulsed dye laser excitation spectroscopy of the 7F0----5D0 transition of Eu(III) reveals only a single peak as this ion is titrated into apocalmodulin. A titration based on the intensity of this transition shows that the first two Eu(III) ions bind quantitatively to two tight sites, followed by weaker binding (Kd = 2 microM) to two additional sites under conditions of high ionic strength (0.5 M KC1). This excitation experiment is also shown to be a general method for measuring contaminating levels of EDTA down to 0.2 microM in proton solutions. Experiments with Tb(III) using both direct laser excitation and indirect sensitization of Tb(III) luminescence through tyrosine residues in calmodulin also give evidence for two tight and two weaker binding sites (Kd = 2-3 microM). The indirect sensitization results primarily upon binding to the two weaker sites, implying that Tb(III) binds first to domains I and II, which are remote from tyrosine-containing domains III and IV. The 7F0----5D0 excitation signal of Eu(III) was used to measure the relative overall affinities of the tripositive lanthanide ions, Ln(III), across the series. Ln(III) ions at the end of the series are found to bind more weakly than those at the beginning and middle of the series. Eu(III) excited-state lifetime measurements in H2O and D2O reveal that two water molecules are coordinated to the Eu(III) at each of the four metal ion binding sites. Measurements of F?rster-type nonradiative energy-transfer efficiencies between Eu(III) and Nd(III) in the two tight sites were carried out by monitoring the excited-state lifetimes of Eu(III) in the presence and absence of the energy acceptor ion Nd(III).(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuropharmacological actions of some binuclear lanthanide(III) complexes

Binuclear lanthanide(III) compounds are of great interest because of the potential of their mutual Ln(3+)-Ln(3+) electronic couplings to produce unusually sharp images in magnetic resonance and fluorescence imaging of biological tissue. The toxicity and neuropharmacological properties of the water soluble and stable neutral binuclear complex [La(api)](2) were compared with those of binuclear complexes with lower water stability, and the components used in their syntheses. The order of the 24-h LD(50) (mg/kg body wt.) of the compounds in mice was: salicylaldehyde (2.24)160). These compounds induced convulsions, urination and defecation in mice. Due to the relatively very low toxicity of [La(api)](2), its mode of action was explored. Its proconvulsant action may possibly involve an interaction of undissociated complex with muscarinic receptors, and is reversed by atropine.     

Structure and NIR luminescence of lanthanide(III) complexes with an organic tridentate ligand

Some lanthanide (Ln) complexes (Ln = Er, Nd, Yb) with an organic ligand, 6-diphenylamine carbonyl 2-pyridine carboxylic acid (HDPAP), have been synthesized. The crystal structure and near infrared luminescence of these complexes (Er-DPAP, Nd-DPAP and Yb-DPAP) have been investigated. The results showed that the lanthanide complexes have electroneutral structures and the near infrared (NIR) emission exhibits characteristic narrow emission of the lanthanide ions. The energy transfer mechanisms in the lanthanide complexes were discussed.     

Qualitative determination of N-terminal amino acids of peptides and proteins with cobalt(III) chelates

1. A method of N-terminal peptide-bond hydrolysis with the cis-beta-hydroxyaquo(triethylenetetramine)cobalt(III) ion, i.e. beta-[Co(trien)(OH)(OH(2))](2+), is reported. The method has been demonstrated with 22 small peptides and ten proteins. 2. The procedure is rapid (an N-terminal amino acid determination can be made easily in one day), it involves no acid hydrolysis step and thus no destruction of labile amino acids, and it involves the use of easily prepared inexpensive reagents. 3. The released N-terminal amino acids can be identified as their cobalt(III) derivatives, or directly as the amino acid or as their dansylated derivatives. 4. The method is to treat 1 mumol of peptide or protein with beta-[Co(trien)(OH)(OH(2))](2+) reagent at pH8.0, 45 degrees C for 3h. Addition of 0.5m-phosphate buffer, pH10.5 at 45 degrees C for 10min cleaves the N-terminal bidentate amino acid-cobalt complex, which can be identified directly. For greater sensitivity with 10nmol of peptide) the free amino acid is prepared from the complex by treatment (with NaCN (0.1m, 40 degrees C, 30min), or H(2)S or NaBH(4) (25 degrees C, 5min), dried, dansylated and the dansyl-amino acid identified by high-voltage electrophoresis. The method is unaffected by the presence of 4-8m-urea, but will not cleave blocked N-terminal acids.     

Structural and in vivo studies of metal chelates of Ga(III) relevant to biomedical imaging

The solution chemistry and structure of the complex of the triazamacrocyclic ligand NOTP (1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonate)) with Ga3+ in D2O have been investigated by 1H, 71Ga and 31P NMR spectroscopy. These NMR results show the presence of a 1:1 Ga(NOTP)3- complex, with a highly symmetrical, pseudo-octahedral geometry, possibly with a C3 axis. The 1H spectrum shows that the triazamacrocyclic chelate ring is very rigid, with all the ring protons non-equivalent. The complex is stable in aqueous solution in a wide pH range. Its high thermodynamic stability agrees well with previous results from biodistribution and gamma imaging studies in Wistar rats with 67Ga3+ chelates of triaza macrocyclic ligands, which showed that the neutral chelates 67Ga(NOTA) (where NOTA is 1,4,7-triazacyclononane-1,4,7-triacetate) and 67Ga(NOTPME) (where NOTPME is 1,4,7-triazacyclononane-1,4,7-tris(methylenephosphonate monoethylester)) have similar in vivo behaviour, with high stability and rapid renal excretion, but the high negatively charged 67Ga(NOTP)3- has a considerably slower kidney uptake and elimination.     

Quantitative determination of N-terminal amino acids of peptides and proteins with cobalt(III) chelates.

Quantitative N-terminal peptide-bond hydrolysis with the cis-beta-hydroxyaquo(triethylenetetramine) cobal (III) ion, i.e. beta-[Co(trien)(OH)(OH2)]2+, is reported. The method has been demonstrated with 20 small peptides, a hexapeptide, bradykinin, insulin A chain (oxidized), glucagon and insulin. The procedure involves no acidic hydrolysis step and thus no destruction of labile amino acids.     

Structure and luminescence of sodium and lanthanide(III) coordination polymers with pyridine-2,6-dicarboxylic acid

A series of coordination polymers constructed by sodium, lanthanide(III), and pyridine-2,6-dicarboxylate (dipic),NaLn(dipic)₂ · 7H₂O (Ln = Eu, Gd, Tb), have been prepared under a hydrothermal condition. The crystal structures of the three compounds which are isostructual were determined by single-crystal X-ray diffraction. The two-dimensional layers found in the compounds are built up from six-folded {NaO₆} polyhedra and nine-folded {LnN₂O₇} polyhedra, these being edge-shared each other along the c axis and bridged by carboxylate groups of dipic along the b axis, respectively. This two-dimensional framework provides cavities inside the layer and interlayer spaces outside the layer for accommodation of the two dipic molecules coordinated to a lanthanide(III) ions. The dehydrated materials obtained by heating the as-synthesized crystals at 200 °C held their crystal structure, and absorbed the same amounts of water molecules as those of the as-synthesized crystals upon the exposure of 100% relative humidity at room temperature. The Eu and Tb compounds showed strong red and green emissions, respectively, due to an energy transfer from dipic molecules to trivalent emission ions.     

Inactivation of (Na+ + K+)-ATPase by chromium(III) complexes of nucleotide triphosphates

(Na+ + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na+ + K+)-ATPase activity was accompanied by a parallel decrease of the associated K+-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na+-dependently, a phosphointermediate from [gamma-32P]ATP. The kinetics of inactivation and of phosphorylation with [gamma-32P]CrATP and [alpha-32P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: formula: (see text). The dissociation constant of the CrATP complex of the pig kidney enzyme at 37 degrees C was 43 microM. The inactivation rate constant (k + 2 = 0.033 min-1) was in the range of the dissociation rate constant kd of ADP from the enzyme of 0.011 min-1. The phosphoenzyme was unreactive towards ADP as well as to K+. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na+ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 +/- 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na+-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 microM) as well as low concentrations of K+. CrATP was a competitive inhibitor of (Na+ + K+)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na+ + K+)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent proteinkinase reaction as the phosphate acceptor site.     

Metal chelates with biological activity. Part 4. Solution properties of iron(III)-histidinehydroxamic acid

Reactions of carbon monoxide with iron(II) diethyldithiocarbamate and iron(II) ethylxanthate were followed using solution IR spectroscopy. In DMF and CH₃CN solutions, the only Fe—dithiocarbamate—carbon monoxide complex observed was cis-[Fe(CO)₂(dedtc)₂]. This complex formed rapidly and appeared to be very stable, resisting displacement of the coordinated CO molecules by other ligands. Fe(exa)₂ showed very little coordination of CO in DMF solution, but in CH₃CN solution formed the complex cis-[Fe(CO)₂(exa)₂] rapidly via the monocarbonyl intermediate [Fe(CO)(exa)₂CH₃CN]. In CHCl₃ solution, in the presence of CO and added bases, a series of complexes, [Fe(CO)(exa)₂L], where L = pyridine, pyrrolidine, diethylamine and triphenylphosphine, was formed. However, with the exception of [Fe(CO)(exa)₂(ø₃P)], these monocarbonyl complexes were unstable with respect to disproportionation to cis-[Fe(CO)₂(exa)₂] and [Fe(exa)₂L₂]. No mixed-ligand monocarbonyl complexes were observed with Fe(dedtc)₂.     

Synthesis, structure and magnetic properties of lanthanide(III) complexes with new imidazole-substituted imino nitroxide radical

Synthesis, characterization and magnetic properties of new lanthanide-radical complexes, [Ln^III(hfac)₃(IM2imH)] (Ln = Gd, Tb; IM2imH = 2-(2-pyridyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy), are described. The molecular structure of the [Tb(hfac)₃(IM2imH)] has been determined by the X-ray diffraction. The magnetic susceptibility data for [Gd(hfac)₃(IM2imH)] show that the Gd-IM2imH magnetic interaction is antiferromagnetic with an exchange coupling constant J = −2.59 cm⁻¹ in contrast to the ferromagnetic interaction in most of Gd(III) complexes containing paramagnetic center, which will be examined in connection with planarity of the IM2imH chelate.     

Iron(III)--phosphoprotein chelates: stoichiometric equilibrium constant for interation of iron(III) and phosphorylserine residues of phosvitin and casein.

Estimates of the strength of iron binding to model phosphoproteins were obtained from equilibrium dialysis experiments. Iron-free phosvitin (chicken and frog) or alpha sl-casein (cow) was dialyzed against the iron(III) chelates of nitrilotriacetate (NTA), )ethylenedinitrilo)tetraacetate (EDTA), or citrate. Protein-bound metal was measured at equilibrium; competition of chelator and phosphoprotein for iron(III) was determined by reference to comprehensive equilibrium equations presented in the Appendix. Analysis of the iron-binding data for phosvitin suggested that clusters of di-O-phosphorylserine residues (SerP.SerP) were the most probable iron-binding sites. A stoichiometric equilibrium constant of 10(18.0) was calculated for the formation of the Fe3+(SerP.SerP) chelate. When comared on the basis of phosphate content, casein bound iron more weakly than phosvitin. However, if the stoichiometric equilibrium constant for the formation of the casein Fe3+(SerP.SerP) chelate (10(17.5) was adjusted to account for the fact that a smaller percentage of casein phosphoserines occurs in di-O-phosphorylserine clusters, the affinity of casein and phosvitin for iron was very similar. A theoretical comparison showed that the "strengths" of the ferric chelates can be ranked: EDTA greater than phosphoprotein di-O-phosphorylserine greater than citrate greater than NTA.