The hexachlorotechnetate reagent in the synthesis,characterization and biodistribution of two novel lipophilic complexes

The hexachlorotechnetate reagent has been used to synthesize lipophilic complexes of technetium-99m with polydentate ligands, using ligand exchange reactions and acetonitrile as an aprotic solvent. The complexes isolated were partially characterized by chromatographic (paper and HPLC) and electrophoretic methods. Preliminary data on biodistribution studies, carried out in rabbits, are also presented. This work documents that the hexachlorotechnetate reagent is a suitable intermediate for the rapid synthesis of new lipophilic complexes of technetium-99m.     

Synthesis and evaluation of the europium(III) and zinc(II) complexes as luminescent bioprobes in high content cell-imaging analysis

Novel phenanthroline derivatives and their europium(III) and zinc(II) complexes have been prepared in up to 92%. In contrast to the stable zinc complexes, the europium compounds exhibit a strong luminescence in THF solution. However, quenching of the emission is observed in DMSO indicating complete dissociation of the complexes back to free ligands in this solvent. ¹H NMR studies of the Eu(III)-complexes 5 and 6 also confirmed the existence of different states depending on the solvent used. Moreover, it was found that compound 5 is stable in EtOH-PBS solutions; here a strong signal in the emission spectra corresponding to the europium ion was detected. No spectral changes were observed for the zinc(II) complexes, they were shown to be stable in the media. These metal complexes can be used as fluorescence markers for the diagnosis of oesophageal squamous carcinoma (OE21) cells at low concentrations. Cell images were acquired using the compounds 5, 7-9 as luminescent agents. The first images were taken already after 20 min incubation time at a very low concentration range (0.7-1.6 μM).     

稀土离子对磷脂酰胆碱脂质体及人红细胞膜自由基氧化的影响

通过荧光和电泳方法研究了稀土离子对磷脂酰胆碱（PC）脂质体及人红细胞膜脂质过氧化的影响．结果表明稀土离子（除钇外）都能够强烈的抑制膜的脂质过氧化,其作用强度随不同的稀土离子可有较大的差别．稀土离子对分离的人红细胞膜的脂质过氧化的抑制作用比对PC脂质体更强．但是,对完整红细胞用稀土离子处理反而会导致膜的脂质过氧化大大加强．     

阿霉素脂质体的制备及其体外抗肿瘤活性的初步研究

目的:应用超声波分散法制备脂质体阿霉素,并比较脂质体阿霉素与游离性阿霉素抗肿瘤活性。方法:以卵磷脂和胆固醇为原料,将阿霉素包封于脂质体中,采用超声分散法制备脂质体阿霉素,对其在290-700nm范围内进行紫外扫描,用SephedexG-50柱分离脂质体阿霉素并计算其包封率。以昆明种小鼠为载体建立肿瘤模型(S180型肉瘤)和细胞荧光染色法研究脂质体阿霉素的抗肿瘤活性,以ZITA SIZER3000型表面电位与粒度测定仪测定其粒径分布。结果:脂质体阿霉素在480nm处有最大吸收峰值,包封率达91.3%,细胞荧光染色显示,脂质体及游离型阿霉素均对S180细胞有明显的抑制作用。结论:此法制备的脂质体阿霉素包封率高,粒径分布集中,脂质体阿霉素较游离型阿霉素有较强的抗肿瘤活性剂及较低的细胞毒作用,对阿霉素的临床应用有一定的参考价值。     

Monoporphyrinate neodymium (III) complexes stabilized by tripodal ligand: synthesis, characterization and luminescence

A series of monoporphyrinate neodymium (III) complexes stabilized by anionic tripodal ligand (cyclopentadienyl)tris(dimethylphosphito)cobaltate(I) were prepared and characterized by IR, ESI-HRMS, UV-Vis and X-ray diffraction studies. Structural analyses revealed that the Nd³⁺ ion was seven-coordinate, surrounded by four nitrogen atoms from the porphyrinate dianion and three oxygen atoms from the anionic tripodal ligand. Photoluminescence studies showed that the porphyrinate dianion acting as a sensitizer absorbed the light and transferred the energy to the Nd³⁺ center, which then allowed the metal ion to emit efficiently at 885 and 1071 nm. The luminescent intensity of the complexes increases when there are strong electronic donating groups on the porphyrin rings but decreases with increasing polarity of the solvents.     

Syntheses, structural and spectroscopic investigation (IR, NMR and luminescence) of new terbium and europium acylpyrazolonates

The synthesis and characterization of new lanthanide complexes of formulae [M(Q)₃(H₂O)(EtOH)], NBu₄[M(Q)₄] and [M(Q)₃(L)] (M = Eu or Tb; HQ = 1-phenyl-3-methyl-4-R-pyrazol-5-one: R = cyclopentylcarbonyl, HQ = HQ^CP; R = cyclopentylpropionyl, HQ = HQ^EtCP; L = 1,10-phenanthroline (phen) or 4,7-diphenyl-1,10-phenanthroline (bathophen)) are reported. The crystal structure of the tetrakis (β-diketonate) complex [NBu₄][Eu(Q^ETCP)₄] containing an eight-coordinated Eu atom in a distorted square antiprismatic environment has been determined. Luminescence studies have been performed on selected derivatives: the data suggested a strong influence of the nature of the acyl moiety in Q ligands and of Ph groups in bathophen (with respect to phen) on the luminescence properties.     

Structural studies of lanthanide complexes with tetradentate nitrogen ligands

Complexes have been synthesised with bis(2-pyridine carboxaldehyde) ethylenediimine (1) and bis(2-pyridine carboxaldehyde)propylene-1,3-diimine (2) with all of the available lanthanide trinitrates. Crystal structures were obtained for all but one complex with 1 and for all but one complex with 2. Four distinct structural types were established for 1 but only two for 2, although in all cases the structures contained one ligand bound to the metal in a tetradentate fashion. With 1, the four different structures of the lanthanide(III) nitrate complexes included 11-coordinate [Ln(1)(NO₃)₃(H₂O)] for Ln = La; 10 coordinate [Ln(1)(NO₃)₃(H₂O)] with one monodentate and two bidentate nitrates for Ln = Ce, then 10-coordinate [Ln(1)(NO₃)₃] for Ln = Pr-Yb with three bidentate nitrates; and 9-coordinate [Ln(1)(NO₃)₃] with one monodentate and two bidentate nitrates for Ln = Lu. On the other hand for 2 only two distinct types of structure are obtained, the first type with Ln = La-Pr and the second type for Ln = Sm-Lu, although all are 10-coordinate with stoichiometry [Ln(2)(NO₃)₃]. The difference between the two types is in the disposition of the ligand relative to the nitrates. With the larger lanthanides La-Pr the ligand is found on one side of the coordination sphere with the three nitrate anions on the other. In these structures, the ligand is folded such that the angle between the two pyridine rings approaches 90°, while with the smaller lanthanides Sm-Lu, two nitrates are found on one side of the ligand and one nitrate on the other and the ligand is in an extended conformation such that the two pyridine rings are close to being coplanar. In both series of structures, the Ln-N and Ln-O bond lengths were consistent with the lanthanide contraction though there are significant variations between ostensibly equivalent bonds which are indicative of intramolecular hydrogen bonding and steric crowding in the complexes.     

Synthesis, properties and solution speciation of lanthanide chloride complexes of triphenylphosphine oxide

The reaction of Ph₃PO with LnCl₃ · nH₂O (Ln=La-Lu ≠ Pm) in a 3.5:1 ratio in acetone produces [LnCl₃(Ph₃PO)₃], whilst from a 6:1 ratio in ethanol the products are [LnCl₂(Ph₃PO)₄]Cl · n(solvate). In the presence of [NH₄][PF₆] in ethanol solution, [LnCl₂(Ph₃PO)₄]PF₆ can be isolated. The last complexes are stable in solution but the [LnCl₃(Ph₃PO)₃] and [LnCl₂(Ph₃PO)₄]Cl partially interconvert in non-coordinating solvents, the neutral species being preferred by the lighter lanthanides, the cationic tetrakis complexes becoming more favoured towards the end of the series. The complexes have been characterised in the solid state by analysis and IR spectroscopy and in solution by ³¹P{¹H} NMR spectroscopy and conductance measurements. The crystal structures of trans-[LnCl₂(Ph₃PO)₄]Cl · nEtOH (Ln=Tb or Yb) and mer-[LnCl₃(Ph₃PO)₃] · 0.5Me₂CO (Ln=La or Ce) are reported and discussed.     

Design,synthesis and antimycobacterial activity of less lipophilic Q203 derivatives containing alkaline fused ring moieties

We report herein the design and synthesis of a series of less lipophilic Q203 derivatives containing an alkaline fused ring moiety. Most of them show considerable potency against MTB H37Rv strain (MIC?<?0.25?μM). Nine compounds (13, 15, 19, 21, 23, 25, 29, 35, 36) have the same excellent activity against both drug-sensitive and -resistant strains (MIC?<?0.035?μM) as Q203 and PBTZ169. Especially, compound 29 also displays acceptable safety, greater absorption in plasma and aqueous solubility than Q203, suggesting its promising potential to be lead compound for future antitubercular drug discovery.     

Question 7: Biosynthesis of Phosphatidic Acid in Liposome Compartments – Toward the Self-Reproduction of Minimal Cells

Self-reproduction is one of main properties that define living cells. In order to explore the self-reproduction process for the study of early cells, and to develop a research line somehow connected to the origin of life, we have built up a constructive ‘synthetic cells (minimal cells)’ approach. The minimal cells approach consists in the investigation of the minimal number of elements to accomplish simple cell-like processes – like self-reproduction. Such approach belongs to the field of synthetic biology. The minimal cells are reconstructed from a totally reconstituted cell-free protein synthesis system (PURESYSTEM) and liposome compartments as containers. Based on this approach, we synthesized two membrane proteins (enzymes), GPAT and LPAAT, which are involved in the phosphatidic acid biosynthesis in bacteria. Both membrane proteins were successfully synthesized by PURESYSTEM encapsulated inside POPC liposomes. Additionally, the enzymatic activity of GPAT was restored by mixing the expressed enzyme with lipid and by forming liposomes in situ. Through these experimental evidences, here we present a possible model to achieve self-reproduction in minimal cells. Our results would contribute to the idea that early cells could have been built by an extremely small number of genes. Presented at the International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

A comparison of biodistribution of liposomal and soluble IL-2 by a new method based on time-resolved fluorometry of europium

A novel method was developed to determine the pharmacokinetics and biodistribution of cytokines and lymphokines based on time-resolved fluorometry (TRF) of europium (Eu). The comparison of two formulations of IL-2 was used to illustrate the sensitivity and applicability of this method as well as to extend the information on the pharmacokinetics of liposomal IL-2 and soluble IL-2. The blood kinetics and biodistribution of liposomal and soluble IL-2 in lymphoid organs and kidneys as measured by TRF were similar to those determined by the radioisotopic method. In both instances, the formulation of IL-2 into liposomes increased its serum half-life and accumulation in reticuloendothelial and lymphoid organs. The increased sensitivity of the Eu/TRF method permitted the extension of observational time points and the analysis of biodistribution in organs such as lymph nodes and bone marrow. These results suggest that Eu-labelled proteins in conjunction with TRF offer a suitable alternative to radiolabelled proteins for pharmacokinetics and tissue distribution studies in animals. This method offers distinct advantages over traditional techniques employing radioistopes since it has greater sensitivity, no half-life limitations and no radioactive or hazardous waste disposal.     

Structural and magnetic properties of Ln(III) complexes with diimines and crotonato as a bridging ligand

Five new lanthanide complexes displaying crotonato bridges have been prepared: [Gd₂(crot)₆(H₂O)₄] · 4(bpa) (1); [Ho₂(crot)₇]_n · (Hbpa) (2); [Gd₂(crot)₆(bipy)₂] (3); [Ho₂(crot)₆(bipy)₂] (4) and [Nd₂(crot)₆(H₂O)₃]_n (5), where bipy=2,2^′-bipyridine; bpa=di(2-pyridyl)amine; crot=crotonato. The compounds were characterized by magnetic susceptibility measurements and their crystal structures were determined by single crystal X-ray diffraction. These studies showed complexes 1, 3 and 4 to be dimers while structures 2 and 5 are polymeric in nature.     

Liposome destabilization induced by synthetic lipopeptides corresponding to envelope and non-structural domains of GBV-C/HGV virus. Conformational requirements for leakage

Liposomes have been used primarily as a model system for studying biological membranes. Numerous chemical, biochemical and biophysical methods have been used to elucidate the various aspects of the interaction between proteins or peptides and phospholipids. Having in mind the potential use of synthetic lipopeptides as antiviral therapies and aiming for a better understanding of the molecular interaction of the GBV-C/HGV with liposomes as model membranes, epitopes of GBV-C/HGV located at the E2 (99-118) and NS3(440-460) regions were selected. Peptides were modified at the N-terminus with acyl chains of different length (C(14) and C(16)) yielding the corresponding myristoil and palmytoil lipopeptides. The main aim of the present study was to get insight into the membrane-interacting properties of the above-described synthetic lipopeptides and to study their inhibition of the capacity of perturbing model membranes of fusion peptide of HIV-1 using fluorescence spectroscopy. In an attempt to establish a relationship between peptide membrane activity and structure, we use Circular Dichroism (CD) and Fourier-Transform Infrared Spectroscopy (FTIR).     

Antibody-dependent difference in biodistribution of monoclonal antibodies in animal models and humans

 The study was designed to clarify the difference in pharmacokinetics of monoclonal antibodies (mAb) in animal models and humans, and to elucidate the applicability of animal models. ^99mTc-labeled murine mAb – against carcinoembryonic antigen (designated BW431/26), and neural cell adhesion molecule (NE150) – and one chimeric mouse/human mAb against nonspecific cross-reacting antigen (chNCA) were administered i.v. to normal mice and athymic mice (370 kBq, 400 ng) xenografted with human cancer cells expressing antigens, and into patients with tumor (925 MBq, 1 mg). The biodistribution of two of the three mAb (not ^99mTc-BW431/26) differed clearly in mice and patients. ^99mTc-NE150 showed specific uptake in xenografted tumor and otherwise a normal biodistribution; however, clinical examination showed increased uptake in the liver with rapid blood clearance (mean α half-life = 31.1 min) compared with ^99mTc-BW431/26 (28.4 h). ^99mTc-chNCA demonstrated increased blood clearance and renal excretion in both normal and athymic mice, with accumulation in tumors. Clinical examination showed rapid blood clearance (mean α half-life = 6.4 min) and increased uptake in the liver. High-performance liquid chromatographic analysis of ^99mTc-chNCA revealed the immune complex in blood, suggesting uptake of the complex by the reticuloendothelial cells. The biodistribution of radiolabeled mAb in animal and human models was variable and specific for each of the three mAb. The results of animal studies with mAb should be evaluated carefully before being extrapolated to humans, on the basis of the nature of the mAb and interacting substances. Received: 9 April 1997 / Accepted 3 March 1998     

Dynamics of the peptide hormone motilin studied by time resolved fluorescence spectroscopy

Time resolved fluorescence was used to study the dynamics on the nanosecond and subnanosecond time scale of the peptide hormone motilin. The peptide is composed of 22 amino acid residues and has one tyrosine residue in position 7, which was used as an intrinsic fluorescence probe. The measurements show that two rotational correlation times, decreasing with increasing temperature, are needed to account for the fluorescence polarization anisotropy decay data. Viscosity measurements combined with the fluorescence measurements show that the rotational correlation times vary approximately as viscosity with temperature. The shorter rotational correlation time (0.08 ns in an aqueous solution with 30% hexafluoropropanol, HFP at 20°C) should be related to internal movement of the tyrosine side chain in the peptide while the longer rotational correlation time (2.2 ns in 30% HFP at 20°C) describes the motion of the whole peptide. In addition, the interaction of motilin or the derivative motilin (Y7F) –23W (with tyrosine substituted by phenylalanine and with a tryptophan fluorophore added to the C-terminal) with negatively charged phospholipid vesicles (DOPG) was studied. The results show the development of a long anisotropy decay time which reflects partial immobilization of the peptide by interaction with the vesicles.Correspondence to: A. Gräslund     

Synthesis and derivatization of halflanthanidocene aryl(alk)oxide complexes

The reaction of halflanthanidocene aryloxides Cp^R′Ln(OAr^tBu,R)₂ (Ln = Y, La, Lu; Cp^R′ = C₅Me₅, C₄Me₄H; R = H, Me) and halflanthanidocene alkoxides [(C₅Me₅)Ln(OCH₂CMe₃)₂]₂ (Ln = Y, Lu) with trimethylaluminum (TMA) was investigated. Monomeric Cp^R′Ln(OAr^tBu,R)₂, derived from the ortho-tBu-substituted OC₆H₂tBu₂-2,6-R-4 (R = H, Me) ligands, form mono(tetramethylaluminate) complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) for the smaller lanthanide metal centers yttrium and lutetium. Such an [aryloxide] → [aluminate] ligand exchange was not observed at the larger lanthanum metal center. The mobility of the tetramethylaluminate ligands of complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) (Ln = Y, Lu) was examined by variable-temperature (VT) ¹H NMR spectroscopy, revealing two signals for bridging and terminal methyl groups at lower temperatures. The treatment of complexes Cp^R′Ln(OAr^tBu,R)(AlMe₄) with donor solvent d₈-THF gave Cp^R′Ln(OAr^tBu,R)(Me)(d₈-THF)₂ (Ln = Y, Lu) with terminal methyl groups, according to a donor-induced aluminate cleavage reaction. Dimeric [(C₅Me₅)Ln(OCH₂CMe₃)₂]₂ (Ln = Y, Lu) was synthesized from (C₅Me₅)Ln(NiPr₂)₂(THF) and reacted with two equivalents of TMA per Ln center to yield monomeric bis(TMA) adduct complexes (C₅Me₅)Ln(OCH₂CMe₃)₂(AlMe₃)₂(Ln = Y, Lu). VT NMR spectroscopic studies confirmed a high mobility of the Ln(μ-OCH₂CMe₃)(μ-Me)AlMe₂ moieties at an ambient temperature. Both bis(TMA) adduct complexes were characterized by X-ray structure analysis.     

Phosphodiesterolytic activity of lanthanide (III) complexes with α-amino acids

Kinetics of the hydrolysis of BNPP (bis(4-nitrophenyl)phosphate) mediated by lanthanide - samarium (III) and ytterbium (III) - alone and in the presence of various alfa amino acids has been systematically studied at 37.0 °C and I = 0.15 M in NaClO₄, in the pH interval of 7-9. The rate of BNPP cleavage is sensitive to metal ion concentration, pH, and ligand to metal molar ratio. Hydrolysis follows Michaelis-Menten-type saturation kinetics. For both metals, high pH values markedly increase the observed activity. Besides, potentiometric titrations of all these systems under identical conditions allowed us to identify the active coordination compounds towards hydrolysis. The results show that complexes with phosphodiesterolytic activity are monomeric cationic species such as [Ln(aa)₃(OH)]²⁺ or [Ln(aa)₂(OH)₂]⁺. Since phosphodiesterolytic activity is evident above pH 7 and it is increased with increasing pH, hydrolytic reactions of the metals are competitive processes that could lead to their precipitation as Ln(OH)₃(s). In this sense, ligand excess (for example, ligand to metal molar ratio equal to 30) was employed. Furthermore, due to its more extended hydrolysis, ytterbium shows, in general, less activity than samarium under the studied conditions. In general, a good phosphodiesterolytic activity is observed for these complexes under similar conditions to the physiological ones. Amino acids could be easily derivatized without changing their coordinating ability, leading to lanthanide complexes possibly capable of efficiently hydrolyzing the phosphodiester linkages of nucleic acids.     

The time resolved emission spectra of peptide conformers measured by pulsed laser excitation

The fluorescence decays of a number of peptides were measured using pulsed laser excitation and time correlated single photon counting techniques. In all cases double exponential kinetics were observed with the short lifetime component (0.5 – 0.85 ns) predominating over the long lifetime componet (1.62 – 2.21 ns). The lifetimes varied with the peptides measured. The time resolved emission spectra of LysTrpLys shows both components have similar spectral maxima at 345 nm. It is suggested that the dual exponential kinetics originate from different conformers of the peptides.     

Unraveling photoexcited conformational changes of bacteriorhodopsin by time resolved electron paramagnetic resonance spectroscopy

By means of time-resolved electron paramagnetic resonance (EPR) spectroscopy, the photoexcited structural changes of site-directed spin-labeled bacteriorhodopsin are studied. A complete set of cysteine mutants of the C-D loop, positions 100-107, and of the E-F loop, including the first alpha-helical turns of helices E and F, positions 154-171, was modified with a methanethiosulfonate spin label. The EPR spectral changes occurring during the photocycle are consistent with a small movement of helix C and an outward tilt of helix F. These helix movements are accompanied by a rearrangement of the E-F loop and of the C-terminal turn of helix E. The kinetic analysis of the transient EPR data and the absorbance changes in the visible spectrum reveals that the conformational change occurs during the lifetime of the M intermediate. Prominent rearrangements of nitroxide side chains in the vicinity of D96 may indicate the preparation of the reprotonation of the Schiff base. All structural changes reverse with the recovery of the bacteriorhodopsin initial state.     

Detection of mitochondrial defects by laser fluorimetry

The mitochondrial function in skeletal muscle biopsies of three patients with chronic progressive external ophthalmoplegia, having deletions of the mitochondrial DNA, was studied by laser-excited fluorescence measurements of NAD(P)H and flavoproteins in saponin-skinned fibers. We detected substantially elevated steady state redox states of the mitochondrial NAD-system in the muscle fibers of these patients. Moreover, the respiratory chain-linked autofluorescence changes in the muscle fibers of these patients were larger in comparison to controls indicating substantial alterations of the mitochondrial content. These results are in line with the presence of elevated numbers of partially respiratory chain inhibited mitochondria in the skeletal muscle of chronic progressive external ophthalmoplegia patients. (Mol Cell Biochem 174: 97–100, 1997)