Le but général du groupe thématique "Composés luminescents de lanthanides, spectroscopie et bioimagerie" est de créer, synthétiser, caractériser et étudier de nouveaux systèmes moléculaires et nanomatériaux pour l’analyse du vivant et l’imagerie biologique optique.
Pour ce faire, le groupe combine des expertises dans les domaines de la synthèse organique, inorganique et de nanomatériaux, de la chimie supramoléculaire, de la spectroscopie (absorbance et luminescence) visible et proche-infrarouge, de la biochimie et de la biophysique, de l’imagerie optique cellulaire et du petit animal.
Nous proposons une recherche fondamentale et transversale qui inclut la conception de systèmes luminescents en tenant compte de l’application finale. L’établissement de preuves de principe de la possibilité d’utilisation de ces composés fait partie de de nos activités de recherche.
Les composés de lanthanide sont très résistants au photoblanchiment qui est souvent un inconvénient majeur des fluorophores organiques, en particulier dans le cas d’illuminations intenses ou répétées. De plus, leurs émissions apparaissent sous forme de bandes très étroites qui permet une discrimination spectrale plus efficace que celle obtenue avec les fluorophores organiques.
Nous avons un intérêt particulier pour les composés de lanthanide émettant dans le proche-infrarouge. En effet, cette gamme spectrale est intéressante pour l’imagerie biologique pour plusieurs raisons 1. D’une part, l’autofluorescence des tissus biologiques est fortement réduite dans le NIR, ce qui augmente la sensibilité de détection. D’autre part, la profondeur de pénétration de la lumière dans les tissus est plus importante (en raison de la diminution de la diffusion), ce qui constitue un atout important pour la détection sur l’animal et en clinique.
Afin de générer la luminescence des cations lanthanide, ces derniers doivent être sensibilisés au moyen d’ « antennes ». Ces entités doivent absorber une grande quantité de lumière et transférer l’énergie résultante aux cations lanthanide. L’un de nos axes de recherche est la quête de nouvelles antennes (synthèse et rationalisation du transfert d’énergie) qui permettront la sensibilisation d’un nombre plus grand de cations lanthanide avec des longueurs d’ondes s’étendant sur une gamme plus large, notamment avec des chromophores absorbant à basse énergie.
Un autre aspect de nos recherches est la création de nouveaux types de rapporteurs permettant la localisation et la quantification d’entités ou d’activités biologiques permettant l’utilisation de ces composés dans des applications pratiques avec un intérêt particulier pour la cellule et le petit animal. Nous nous intéressons également à la création de complexes de lanthanide combinant les fonctions d’agents de luminescence optique et d’agents d’imagerie magnétiques afin de créer des agents multimodaux utilisables en modalités comme l’imagerie optique et l’IRM.
Pour atteindre ces buts, nous nous intéressons à différentes familles de composés incluant des complexes de types petites molécules comme des métallacrowns 2, 3, des systèmes polymétalliques comme des dendrimères 4, 5, et des réseaux-métallo-organiques (MOFs) 6, aussi des nanomatériaux comme des nanocristaux 7, des nanobilles, des micelles et des microémulsions 8.
Quelques exemples de ces composés sont illustrés ci-dessous.
La stratégie des systèmes polymétalliques est basée sur l’hypothèse selon laquelle un grand nombre de cations lanthanide et un grand nombre d’antennes par unité de volume conduira à l’émission d’un plus grand nombre de photons permettant de compenser les faibles rendements quantiques souvent observés pour les lanthanides émettant dans le proche-infrarouge.
Plusieurs systèmes ont été testés pour valider cette approche. Par exemple, des micelles et des liposomes ont été synthétisés qui peuvent incorporer un grand nombre de chromophores et de lanthanides 8.
L’incorporation de lanthanides et de chromophores dans des billes a aussi été testée pour la formation de systèmes polymétalliques par un effort de synthèse minimal tout en assurant de meilleures propriétés photophysiques et une bonne stabilité.
Une autre approche est l’utilisation de réseaux métallo-organiques (« Metal Organic Frameworks », MOF), qui représentent une nouvelle classe de matériaux à la structure bien définie, construits à partir d’ions métalliques et de ligands organiques rigides. Il est possible d’ajuster les MOFs afin d’obtenir des propriétés spécifiques en modulant la taille de leurs pores, la topologie du réseau et les zones de surface d’une manière contrôlée.
Des complexes polymétalliques à base de dendrimères ont été utilisés dans plusieurs projets. Nous développons des méthodologies efficaces et flexibles de fixation de chromophores en leur périphérie. Nous avons établi la première preuve de principe d’utilisation des dendrimères à base de lanthanides pour l’imagerie en visible et en NIR dans les cellules vivantes 4.
Notre recherche implique de nombreuses collaborations nationales (Université d’Orléans, Université de Tours, ICSN à l’Université Paris Saclay, INRA à Nouzilly, synchrotron Soleil et CEA de Grenoble) et internationales (dont les Université de Genève et de Berne en Suisse, les Universités du Michigan, Northwestern et de Pittsburgh aux Etats Unis, l’Université Nationale de Singapour, les Universités de Cracovie et de Poznan en Pologne, les Universités de Coimbra et Aveiro au Portugal, les Universités Thessalonique, Athènes et Ioannina en Grèce, l’Institut Catalan de Nanotechnologie).
Notre groupe est également actif dans l’établissement de brevets (6 brevets depuis 2011) et accueille un projet de maturation financé par la SATT Grand Centre (Société d’accélération de Transfert de Technologie).
Les composés polynucléaires de lanthanides peuvent être avantageusement préparés en utilisant une approche synthétique supramoléculaire 10.
La préparation de systèmes auto-assemblés stables et solubles dans l’eau, où de nombreux facteurs doivent être pris en compte est une tâche difficile. Dans ce contexte, un effort dans la conception et la synthèse de ligands appropriés a été réalisé afin de fournir et de caractériser des précurseurs (ligands polytopiques et leur complexes) pour des applications en imagerie. Des études détaillées de spéciation thermodynamique de complexes supramoléculaires avec différents cations lanthanide sont effectuées par RMN et différentes méthodes spectroscopiques.
De nouvelles familles d’oligonucléotides originaux sont développées en vue d’obtenir des outils pour la recherche fondamentale, le diagnostic in vitro, l’imagerie biologique ainsi que des agents thérapeutiques potentiels.
La réalisation de ces différents objectifs nécessite l’utilisation d’oligonucléotides qui doivent posséder un certain nombre de propriétés. En fonction des applications, les oligonucléotides doivent, en effet, posséder plusieurs des propriétés suivantes : former des complexes spécifiques et stables avec leurs cibles, induire des modifications irréversibles des cibles par pontage ou coupure, produire un signal modifié en présence des cibles utilisable comme preuve de l’interaction. De plus, dans le cas des applications en culture cellulaire et in vivo, ils doivent être stables vis-à-vis des nucléases et être capables d’atteindre leurs cibles à l’intérieur des cellules 11.
Afin de conférer aux oligonucléotides des propriétés adaptées en fonction des applications, nous modifions chimiquement leurs éléments structuraux (bases nucléiques, sucres, liaisons internucleotidiques). A ces oligonucléotides peuvent être fixés différents ligands : molécules intercalantes et/ou réactives, peptides, molecules lipophiles, inhibiteurs de topoisomérases, marqueurs fluorescents détectables dans le visible et NIR….). Le développement de conjugués possédant des propriétés optimales dépend des paramètres de liaison entre les deux entités (positions de liaison sur l’oligonucléotide et le ligand, longueur et nature du bras de liaison). La synthèse chimique de ces nouvelles séries d’oligonucléotides nécessite le développement de nouveaux réactifs et méthodes de synthèse.
Afin de caractériser les composés conçus au sein de l’équipe, nous avons construit un ensemble d’équipements uniques pour les études spectroscopiques des composés luminescents en visible et proche-infrarouge : système de mesure de temps de vie ns-us-ms, à température variable de 10K à ambiante, avec excitation à des longueurs d’onde comprises entre 266 et 700 nm ; analyse spectroscopique haute résolution en excitation et émission visible et proche-infrarouge ; mesure de rendements quantiques visible et proche-infrarouge ; imagerie de luminescence sur cellule (vidéomicroscopie proche infrarouge, confocale) et sur petit animal (macroscope visible et proche-infrarouge).
En plus de cette activité, notre groupe est responsable de la plateforme de spectroscopie optique au CBM. Le but de cette plateforme est de mettre à la disposition des utilisateurs internes et externes des instruments de spectroscopie de pointe et, au besoin, la formation nécessaire à ses utilisateurs.
Fluorescence optical imaging is a highly sensitive and powerful tool with diverse in vitro and in vivo applications for preclinical and clinical studies. This review is focused on the optical imaging in the near-infrared (NIR) region since improved signal-to-noise ratio and deeper penetration of light through tissues could be achieved due to the minimal autofluorescence and reduced light scattering at these wavelengths. In particular, imaging agents absorbing and emitting in the biological diagnostic window (650–1450 nm) are discussed. The photophysical properties and particularities of chemical structures or compositions of four different families of probes : (i) organic fluorophores, (ii) fluorescent proteins (FPs), (iii) semi-conductor nanocrystals (quantum dots) and (iv) lanthanide(III)-based complexes and nanomaterials are presented. Advantages and drawbacks, commercial availability and toxicity as well as selected applications of these probes are discussed. A specific attention is given to lanthanide(III)-based compounds due to their unique optical properties, e.g. sharp emission bands with minimal sensitivity to the microenvironment, large differences between excitation and emission wavelengths and strong resistance toward photobleaching. The use of such probes brings additional perspectives and facilitates developments of novel strategies in optical imaging including real-time experiments and new approaches for diagnostic.
Considered at the beginning of the 21th century as being incompatible with the presence of closely bound high-energy oscillators, lanthanide-centered superexcitation, which is the raising of an already excited electron to an even higher level by excited-state energy absorption, is therefore a very active topic strictly limited to the statistical doping of low-phonon bulk solids and nanoparticles. We show here that molecular lanthanide-containing coordination complexes may be judiciously tuned to overcome these limitations and to induce near-infrared (NIR)-to-visible (VIS)-light upconversion via the successive absorption of two low-energy photons using linear-optical responses. Whereas single-ion-centered excited-state absorption mechanisms remain difficult to implement in lanthanide complexes, the skillful design of intramolecular intermetallic energy-transfer processes operating in multimetallic architectures is at the origin of the recent programming of erbium-centered molecular upconversion.
Luminescent lanthanide(III)-based molecular scaffolds hold great promises for materials science and for biological applications. Their fascinating photophysical properties enable spectral discrimination of emission bands that range from the visible to the near-infrared (NIR) regions. In addition, their strong resistance to photobleaching makes them suitable for long duration or repeated biological experiments using a broad range of sources of excitation including intense and focalized systems such as lasers (e.g., confocal microscopy). A main challenge in the creation of luminescent lanthanide(III) complexes lies in the design of a ligand framework that combines two main features : (i) it must include a chromophoric moiety that possesses a large molar absorptivity and is able to sensitize several different lanthanide(III) ions emitting in the visible and/or in the near-infrared, and (ii) it must protect the Ln3+ cation by minimizing nonradiative deactivation pathways due to the presence of −OH, −NH and −CH vibrations. Herein, a new family of luminescent Ga3+/Ln3+ metallacrown (MC) complexes is reported. The MCs with the general composition [LnGa4(shi)4(C6H5CO2)4(C5H5N) (CH3OH)] (Ln-1, Ln = Sm3+–Yb3+) were synthesized in a one pot reaction using salicylhydroxamic acid (H3shi) with Ga3+ and Ln3+ nitrates as reagents. The molecular structure of [DyGa4(shi)4(C6H5CO2)4(C5H5N) (CH3OH)] was obtained by X-ray analysis of single crystals and shows that the complex is formed as a [12-MCGa(III)shi-4] core with four benzoate molecules bridging the central Dy3+ ion to the Ga3+ ring metals. The powder X-ray diffraction analysis demonstrates that all other isolated complexes are isostructural. The extended analysis of the luminescence properties of these complexes, excited by the electronic states of the chromophoric ligands, showed the presence of characteristic, sharp f–f transitions that can be generated not only in the NIR (Sm, Dy, Ho, Er, Yb) but also in the visible (Sm, Eu, Tb, Dy, Tm). All Ln-1 complexes possess very high quantum yield values with respect to other literature compounds, indicating a good sensitization efficiency of the [12-MCGa(III)shi-4] scaffold. Especially, as of today, the Yb-1 complex exhibits the highest NIR quantum yield reported for a lanthanide(III) complex containing C–H bonds with a value of 5.88(2)% in the solid state. This work is a significant step forward toward versatile, easily prepared luminescent lanthanide(III) complexes suitable for a variety of applications including highly in demand biological imaging, especially in the NIR domain.
Full and congruent crystallization from glass is applied to the SrREGa3O7 melilite family (RE = Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Y). This innovative process enables the synthesis of polycrystalline ceramics exhibiting high transparency both in the visible and near infrared regions, despite tetragonal crystal structures and micrometer scale grain sizes. Moreover, glass crystallization provides an original route to synthesize new crystalline phases which are not accessible via a classic solid state reaction, as demonstrated for SrYbGa3O7 and SrTmGa3O7. To illustrate the potential optical applications of such materials, SrGdGa3O7 transparent polycrystalline ceramics are doped with Dy3+ or Tb3+/Eu3+ in order to generate white light emission under UV excitation. It is foreseen that such transparent melilite ceramic phosphors, prepared via a cost-effective process, can be successfully used in solid state lighting devices of considerable technological interest.
This contribution explores synthetic routes adopted and optimized for the preparation of three new hexatopic tripodal organic ligands designed for Ln(III) complexation. In these ligands, three strands bearing two pyridyldicarbonyl binding moieties are anchored with a small aliphatic triamine. The described synthesis is not straightforward and depends on the nature of the spacer between coordinating moieties. In addition to the characterisation of targeted ligands, the structure of ditopic intermediates is assessed by X-ray crystallography and discussed with respect to the spacer nature.
Most of the existing optical methods for CuII detection rely on a “turn-off” approach using visible lanthanide(III) luminescence. In this work we present an innovative molecular systems where the podands bis(2-hydrazinocarbonylphenyl) ethers of ethylene glycol (L1) and diethylene glycol (L2) have been designed, synthesised and tested with an ultimate goal to create a "turn-on" lanthanide(III)-based molecular probe for the specific detection of CuII ions based on both visible (TbIII, EuIII) and near-infrared (NdIII, YbIII) emission. Quantum yields of the characteristic LnIII emission signals increases by at least two-orders of magnitude upon addition of CuII into water/acetonitrile (9/1) solutions of LnL (L=L1, L2) complexes. A detailed investigation of ligand-centred photophysical properties of water/acetonitrile (9/1) solutions of CuL, GdL and GdCuL complexes revealed that the presence of CuII ions does not significantly affect the energy positions of the singlet (32,260 cm−1) and triplet (25,640–25,970 cm−1) states, but partially or fully eliminates the singlet state quenching through an electron transfer mechanism. This effect increases the probability of intersystem crossing leading to enhanced triplet-to-singlet emission ratio and to longer triplet state lifetimes. The redox activity of hydrazine moieties and their ability to reduce CuII to CuI has been indicated by a qualitative assay with neocuproine. Finally, the probe demonstrates a good selectivity towards CuII over other transition metal ions : the addition of divalent ZnII, CdII, PdII, NiII, CoII or trivalent FeIII, GaIII, InIII ion salts into solutions of TbL either does not affect emission intensity or increases it to a maximum of 2–3 times, while, under similar experimental conditions, the presence of CuII results in a 20- to 30-times lanthanide luminescence enhancement. This new strategy results in a versatile and selective optical platform for the design of efficient “turn-on” sensors for CuII ions based on visible and near-infrared LnIII luminescence.
Herein, we report on the rational design, preparation and characterization of a novel homo-octanuclear helicate, which results from a spatial extension of the central tetranuclear platform. The 3D supramolecular assembly is obtained by complexing europium(III) with a new hexatopic tripodal ligand. The isolated octanuclear helicate is fully characterized by different methods clearly evidencing the structure predicted with molecular modelling. The ligand preorganization plays a crucial role in a successful self-assembly process and induces the formation of a well-defined triple-stranded helical structure. This prototypal octanuclear edifice accommodating functional lanthanides within a 3D scaffold offers attractive perspectives for further applications.
Homoleptic mononuclear nine-coordinate lanthanum(III) and europium(III) tris-complexes [Ln(N(wedge)N(wedge)O)3].nH2O with two tridentate N-benzylbenzimidazole pyridine-2-carboxylates exhibit a rare C3-symmetry of the lanthanide coordination polyhedron in the solid state, as confirmed by luminescence spectroscopy and by X-ray crystallography (the three N(wedge)N(wedge)O ligands are arranged "up-up-up" around the lanthanide ion). The symmetry, however, is changed to the more common C1 upon dissolution of the complexes in dichloromethane, as revealed by luminescence spectroscopy (the three ligands are likely to be arranged "up-up-down"). The new europium complexes emit efficient ligand-sensitized metal-centered luminescence with excited-state lifetimes of 1.56-2.18 ms and quantum yields of 25-41% in the solid and in solution. The change of the symmetry from (a higher) C3 to (a lower) C1 alters the luminescence spectrum, shortens the radiative lifetime, and increases the luminescence efficiency of the europium complexes.
We report on seven new anionic benzimidazole-pyridine carboxylate and tetrazolate tridentate N^N^O and N^N^N ligands that are modified with chromophore (phenyl, biphenyl, naphthyl) and solubilizing groups. The ligands are UV chromophores with the lowest-energy absorption maxima at 312–335nm and with the molar absorption coefficients of (20–25)×10 3 M −1 cm −1 in DMSO solution. The ligands form neutral complexes with trivalent lanthanides and sensitize the red luminescence of europium. The triplet state energies of the deprotonated ligands, which were measured from the phosphorescence spectra of their lanthanum complexes at 77K, are in the range of (18.8–21.1)×10 3 cm −1 . We also describe synthesis of non-symmetric pyridines that are 2,6- and 2,4,6-substituted with hydroxymethyl, carboxaldehyde, and carbonitrile groups.
The development of efficient sensors for the determination of the water content in organic solvents is highly desirable for a number of chemical industries. Presented herein is a Mg2+ metal-organic framework (MOF), which exhibits the remarkable capability to rapidly detect traces of water (0.05-5 % v/v) in various organic solvents through an unusual turn-on luminescence sensing mechanism. The extraordinary sensitivity and fast response of this MOF for water, and its reusability make it one of the most powerful water sensors known.
This work shows that the operation of near-infrared to visible light-upconversion in a discrete molecule is not limited to non-linear optical processes, but may result from superexcitation processes using linear optics. The design of nine-coordinate metallic sites made up of neutral N-heterocyclic donor atoms in kinetically inert dinuclear [GaEr(L1)3]6+ and trinuclear [GaErGa(L2)3]9+ helicates leads to [ErN9] chromophores displaying unprecedented dual visible nanosecond Er(4S3/2—>4I15/2) and near-infrared microsecond Er(4I13/2—>4I15/2) emissive components. Attempts to induce one ion excited-state absorption (ESA) upconversion upon near-infrared excitation of these complexes failed because of the too-faint Er-centred absorption cross sections. The replacement of the trivalent gallium cation with a photophysically-tailored pseudo-octahedral [CrN6] chromophore working as a sensitizer for trivalent erbium in [CrEr(L1)3]6+ improves the near-infrared excitation efficiency, leading to the observation of a weak energy transfer upconversion (ETU). The connection of a second sensitizer in [CrErCr(L2)3]9+ generates a novel mechanism for upconversion, in which the superexcitation process is based on the CrIII-sensitizers. Two successive Cr—>Er energy transfer processes (concerted-ETU) compete with a standard Er-centred ETU, and a gain in upconverted luminescence by a factor larger than statistical values is predicted and observed.
Antisense oligonucleotides are promising medicines for treating various diseases, although their efficiency still requires high doses. Their delivery in the cytosol and nucleus to reach their mRNA targets would increase their efficiency at the same time as reducing the dose.
We conjugated the histidine-rich peptide H5WYG (GLFHAIAHFIHGGWHGLIHGWYG) at the 5’-end of the RNase H-incompetent antisense 2’-O-methyl-phosphodiester oligonucleotide (2’-Ome RNA705) targeting aberrant splicing of luciferase pre-mRNA in HeLa pLuc705 cells. H5WYG was also conjugated with 2’-Ome-RNA705 labelled by fluorescein at the 3’-end. Then, H5WYG-2’-Ome-RNA705 conjugate and 2’-Ome-RNA705 were formulated with lipofectamine to favor their uptake in HeLa pLuc705 cells.
Confocal microscopy showed that, after 4 h and overnight incubation, the presence of fluorescein-labelled 2’-Ome-RNA705 in the cytosol and nucleus was enhanced when the oligonucleotide was conjugated with H5WYG. We found that H5WYG-2’-Ome-RNA705 increased the splicing redirection and restoration of a functional luciferase mRNA. Luciferase activity and luciferase mRNA levels in these cells were 6.6- and two-fold higher, respectively, with H5WYG-2’-Ome-RNA705 than with 2’-Ome-RNA705.
The results of the present study show that the conjugation of 2’-Ome antisense RNA to peptide H5WYG is a good strategy for improving its cytosol delivery, accumulation in the nucleus and antisense activity.
A series of novel pyridine-based Gd3+ complexes have been prepared and studied as potential MRI contrast agents for Zn2+ detection. By independent assessment of molecular parameters affecting relaxivity, we could interpret the relaxivity changes observed upon Zn2+ binding in terms of variations of the rotational motion.
We have proposed recently that the DO3A-N-alpha-(amino) propionate chelator and its amide conjugates are leads to targeted, high relaxivity, safe contrast agents for magnetic resonance imaging. In this work we illustrate further the expeditious nature and robustness of the synthetic methodologies developed by preparing the DO3A-N-(alpha-pyrenebutanamido) propionate chelator. Its Gd3+ chelate retains the optimized water exchange, high stability and inertness of the parent complex. The pyrene moiety imparts concentration- dependent self-assembly properties and aggregation-sensitive fluorescence emission to the Gd3+ complex. The Gd3+ complex displays pyrene-centred fluorescence whilst the Yb3+ and Nd3+ complexes exhibit sensitized lanthanide-centred near-infrared luminescence. The aggregated form of the complex displays high relaxivity (32 mM(-1) s(-1), 20 MHz, 25 degrees C) thanks to simultaneous optimization of the rotational correlation time and of the water exchange rate. The relaxivity is however still limited by chelate flexibility. This report demonstrates that the DO3A-N-(alpha-amino) propionate chelator is a valuable platform for constructing high relaxivity CA using simple design principles and robust chemistries accessible to most chemistry labs.
The synthesis and characterization of two novel DTPA bisamide derivatives DTPA-BC(12)PheA and DTPA-BC(14)PheA functionalized with p-dodecylaniline and p-tetradecylaniline are described. The ligands were coordinated to Gd(III) and Eu(III), resulting in highly paramagnetic and luminescent complexes, respectively. Mixed micelles consisting of Gd/Eu-DTPA-BC(12)PheA and DTPA-BC(14)PheA with a homogeneous size distribution (33-40 nm) were prepared by the assembly of the amphiphilic complexes with phospholipid DPPC and a surfactant Tween 80 (R). Taking into account the sensitivity difference between magnetic resonance and optical imaging techniques, the ratios of Gd and Eu complexes (Gd/Eu) 1 : 1, 2 : 1, 3 : 1, 20 : 1 and 50 : 1 were combined in one single micelle and their optical and relaxometric properties were characterized in detail. Upon excitation at 290 nm, the micelles display characteristic red emission bands due to the D-5(0)-> F-7(J) (J = 0-4) transitions of Eu(III). The number of water molecules in the first coordination sphere of Eu(III) (q(Eu) = 0.1-0.2) was calculated from the lifetime measurements performed in H2O and D2O solutions. Micelles composed of exclusively europium complexes display quantum yields in the range of 1.0%, decreasing with the europium concentration when going from 1 : 1 to 50 : 1 Gd/Eu contents. The ligand-to-lanthanide sensitization efficiency for micelles consisting of Eu-DTPA-BC(12)PheA and Eu-DTPA-BC(14)PheA equals 3.8% and 4.1%, respectively. The relaxivity r(1) per Gd(III) ion at 40 MHz and 310 K reaches a maximum value of 14.2 s(-1) mM(-1) for the Gd-DTPA-BC(12)PheA assemblies and 16.0 s(-1) mM(-1) for the micellar Gd-DTPA-BC(14)PheA assemblies compared to a value of 3.5 s(-1) mM(-1) for Gd-DTPA (Magnevist r). Theoretical fitting of the H-1 NMRD profiles results in tR values of 4.2 to 6.6 ns. The optimal concentration ratio of Gd/Eu compounds in the micelles in order to provide the required bimodal performance has been determined to be 20 : 1. In the search for other bimodal systems, this discovery can be used as a guideline concerning the load of paramagnetic agents with respect to luminescent probes.
The synthesis of a pyrazolo[1,5-a]-1,3,5-triazine C-nucleoside (dA(PT)), designed to form two hydrogen bonds with a complementary dT residue, is reported. Oligonucleotides including this dA nucleoside analogue possess base-pairing properties similar to those of the parent oligonucleotide. This dA nucleoside analogue is more resistant to acid-catalyzed hydrolysis than dA.
In Archaea the two major modes of DNA packaging are wrapping by histone proteins or bending by architectural non-histone proteins. To supplement our knowledge about the binding mode of the different DNA-bending proteins observed across the three domains of life, we present here the first model of a complex in which the monomeric Methanogen Chromosomal protein 1 (MC1) from Euryarchaea binds to the concave side of a strongly bent DNA. In laboratory growth conditions MC1 is the most abundant architectural protein present in Methanosarcina thermophila CHTI55. Like most proteins that strongly bend DNA, MC1 is known to bind in the minor groove. Interaction areas for MC1 and DNA were mapped by Nuclear Magnetic Resonance (NMR) data. The polarity of protein binding was determined using paramagnetic probes attached to the DNA. The first structural model of the DNA-MC1 complex we propose here was obtained by two complementary docking approaches and is in good agreement with the experimental data previously provided by electron microscopy and biochemistry. Residues essential to DNA-binding and -bending were highlighted and confirmed by site-directed mutagenesis. It was found that the Arg25 side-chain was essential to neutralize the negative charge of two phosphates that come very close in response to a dramatic curvature of the DNA.
We report herein the synthesis of a luminescent polynuclear dendritic structure (SmIII-G3P-2,3Nap) in which eight SmIII ions are sensitized by thirty-two 2,3-naphthalimide chromophores. Upon a single excitation wavelength, the dendrimer complex exhibits two types of emission in the visible and in the near-infrared (NIR) ranges. SmIII-G3P-2,3Nap was non-cytotoxic after 24 h of incubation and up to 2.5 μM. The ability of the SmIII-based probe to be taken up by cells was confirmed by confocal microscopy. Epifluorescence microscopy validated SmIII-G3P-2,3Nap as a versatile probe, capable of performing interchangeably in the visible or NIR for live-cell imaging. As both emissions are obtained from a single complex, the cytotoxicity and biodistribution are inherently the same. The possibility for discriminating the sharp SmIII signals from autofluorescence in two spectral ranges increases the reliability of analysis and reduces the probability of artifacts and instrumental errors.
VEGFs are found at high levels in hypoxic tumors. As major components directing pathologic neovascularization, they regulate stromal reactions. Consequently, novel strategies targeting and inhibiting VEGF overproduction upon hypoxia offer considerable potential for modern anticancer therapies controlling rather than destroying tumor angiogenesis. Here, we report the design of a vector expressing the soluble form of VEGF receptor-2 (sVEGFR2) driven by a hypoxia-responsive element (HRE)-regulated promoter. To enable in vivo imaging by infrared visualization, mCherry and IFP1.4 coding sequences were built into the vector. Plasmid construction was validated through transfection into embryonic human kidney HEK293 and murine B16F10 melanoma cells. sVEGFR2 was expressed in hypoxic conditions only, confirming that the gene was regulated by the HRE promoter. sVEGFR2 was found to bind efficiently and specifically to murine and human VEGF-A, reducing the growth of tumor and endothelial cells as well as impacting angiogenesis in vitro. The hypoxia-conditioned sVEGFR2 expression was shown to be functional in vivo : Tumor angiogenesis was inhibited and, on stable transfection of B16F10 melanoma cells, tumor growth was reduced. Enhanced expression of sVEGFR2 was accompanied by a modulation in levels of VEGF-A. The resulting balance reflected the effect on tumor growth and on control of angiogenesis. A concomitant increase of intratumor oxygen tension also suggested an influence on vessel normalization. The possibility to express an angiogenesis regulator as sVEGFR2, in a hypoxia-conditioned manner, significantly opens new strategies for tumor vessel–controlled normalization and the design of adjuvants for combined cancer therapies. Mol Cancer Ther ; 13(1) ; 165–78. ©2013 AACR.
Near-infrared (NIR) luminescent lanthanide complexes hold great promise for practical applications, as their optical properties have several complementary advantages over organic fluorophores and semiconductor nanoparticles. The fundamental challenge for lanthanide luminescence is their sensitization through suitable chromophores. The use of the metallacrown (MC) motif is an innovative strategy to arrange several organic sensitizers at a well-controlled distance from a lanthanide cation. Herein we report a series of lanthanide ?encapsulated sandwich ? MC complexes of the form Ln3+[12-MCZn(II),quinHA-4]2[24-MCZn(II),quinHA-8] (Ln3+[Zn(II)MCquinHA]) in which the MC framework is formed by the self-assembly of Zn2+ ions and tetradentate chromophoric ligands based on quinaldichydroxamic acid (quinHA). A first-generation of luminescent MCs was presented previously but was limited due to excitation wavelengths in the UV. We report here that through the design of the chromophore of the MC assembly, we have significantly shifted the absorption wavelength toward lower energy (450 nm). In addition to this near-visible inter- and/or intraligand charge transfer absorption, Ln3+[Zn(II)MCquinHA] exhibits remarkably high quantum yields, long luminescence lifetimes (CD3OD ; Yb3+, QLnL = 2.88(2)%, τobs = 150.7(2) ?s ; Nd3+, QLnL = 1.35(1)%, τobs = 4.11(3) ?s ; Er3+, QLnL = 3.60(6)·10 ?2%, τobs = 11.40(3) ?s), and excellent photostability. Quantum yields of Nd3+ and Er3+ MCs in the solid state and in deuterated solvents, upon excitation at low energy, are the highest values among NIR-emitting lanthanide complexes containing C ?H bonds. The versatility of the MC strategy allows modifications in the excitation wavelength and absorptivity through the appropriate design of the ligand sensitizer, providing a highly efficient platform with tunable properties.
Background Information Autofluorescence spectroscopy is a powerful tool for molecular histology and for following metabolic processes in biological samples as it does not require labelling. However, at the microscopic scale, it is mostly limited to visible and near infrared excitation of the samples. Several interesting and naturally occurring fluorophores can be excited in the UV and deep UV (DUV), but cannot be monitored in cellulo nor in vivo due to a lack of available microscopic instruments working in this wavelength range. To fulfil this need, we have developed a synchrotron-coupled DUV microspectrofluorimeter which is operational since 2010. An extended selection of endogenous autofluorescent probes that can be excited in DUV, including their spectral characteristics, is presented. The distribution of the probes in various biological samples, including cultured cells, soft tissues, bone sections and maize stems, is shown to illustrate the possibilities offered by this system. In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology. Results To fulfil this need, we have developed a synchrotron-coupled DUV microspectrofluorimeter which is operational since 2010. An extended selection of endogenous autofluorescent probes that can be excited in DUV, including their spectral characteristics, is presented. The distribution of the probes in various biological samples, including cultured cells, soft tissues, bone sections and maize stems, is shown to illustrate the possibilities offered by this system. In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology. Conclusions In this work we demonstrate that DUV autofluorescence is a powerful tool for tissue histology and cell biology.
Two new tridentate(NNO)-bidentate(NN) compartmental ligands, HL5 and HL6, are synthesized from pyridine and benzimidazole synthons. They react in aqueous solution under physiological conditions with ZnII, LnIII, or a mixture thereof, to yield complexes of different stoichiometries, 1:3, 2:2, 2:3, 1:1:3, the speciation of which is established by UV-visible titrations and ESI mass spectrometry. Photophysical studies of the EuIII-containing solutions in Tris-HCl 0.1 M (pH = 7.4) show that lanthanide luminescence arises from a unique N6O3 coordination site with pseudo D3 symmetry. Relevant parameters such as crystal field splitting, lifetime, radiative lifetime and intrinsic quantum yield perfectly match those reported for dinuclear 4f-4f helicates in which the EuIII ion has the same coordination environment.
Diethylenetriaminepentaacetic acid (DTPA)–bisamide derivatives functionalized with p-toluidine, 6-aminocoumarin, 1-naphthalene methylamine and 4-ethynylaniline were synthesized and fully characterized by mass spectrometry, NMR spectroscopy, FTIR spectroscopy and elemental analysis. LnIII complexes (Ln = Gd, Eu, Tb, Y) of the ligands DTPA–bis-p-toluidineamide (DTPA–BTolA), DTPA–bis-6-coumarinamide (DTPA–BCoumA), DTPA–bis-1-naphthylmethylamide (DTPA–BNaphA) and DTPA–bis-4-ethynylphenylamide (DTPA–BEthA) were prepared and studied for their bimodal magnetic resonance imaging/optical properties. EuIII and TbIII derivatives in aqueous solutions exhibit characteristic red and green emission, respectively, with quantum yields of 0.73 % for EuIII–DTPA–BNaphA and 2.5 % for TbIII–DTPA–BEthA. Ligand-centred photophysical properties of the GdIII complexes were investigated to gain insight into energy-transfer processes that take place in these systems. The GdIII complexes were also analyzed by nuclear magnetic relaxation dispersion (NMRD) techniques. The relaxivity (r1) at 20 MHz and 310 K equals 4.1 s–1 mM–1 for Gd–DTPA–BTolA, 5.1 s–1 mM–1 for Gd–DTPA–BCoumA, 6.4 s–1 mM–1 for Gd–DTPA–BNaphA and 5.7 s–1 mM–1 for Gd–DTPA–BEthA. These values are higher than the value of 3.8 s–1 mM–1 for Gd–DTPA (Magnevist). The improved relaxivity is due to the increase in the rotational tumbling time τR with a factor of 1.6 for Gd–DTPA–BTolA, 2.1 for Gd–DTPA–BCoumA, 3.1 for Gd–DTPA–BNaphA and 6.5 for Gd–DTPA–BEthA. In a 4 % human serum albumin solution, the apparent relaxivity at 20 MHz increases to values of 13.9 and 19.1 s–1 mM–1 for Gd–DTPA–BNaphA and Gd–DTPA–BEthA, respectively. All these features assist the search for optimal bimodal optical and magnetic resonance imaging probes.
Light-upconversion via stepwise energy transfer from a sensitizer to an activator exploits linear optics for converting low-energy infrared or near-infrared incident photons to higher energy emission. This approach is restricted to activators possessing intermediate long-lived excited states such as those found for trivalent lanthanide cations dispersed in solid-state matrices. When the activator is embedded in a molecular complex, efficient nonradiative relaxation processes usually reduce excited state lifetimes to such an extent that upconversion becomes too inefficient to be detected under practical excitation intensities. Theoretical considerations presented here predict that the combination of at least two millisecond time scale sensitizers with a central lanthanide activator in supramolecular complexes circumvents this bottleneck by creating a novel upconversion pathway, in which successive excitations are stored on the sensitizers prior to inducing stepwise energy transfer processes. Application of this concept to the chromium/erbium pair demonstrates that strong-field trivalent chromium chromophores irradiated with near-infrared photons produce upconverted green erbium-centered emission in discrete dinuclear and trinuclear triple-stranded helicates.
Calcium plays a vital role in the human body and especially in the central nervous system. Precise maintenance of Ca2+ levels is very crucial for normal cell physiology and health. The deregulation of calcium homeostasis can lead to neuronal cell death and brain damage. To study this functional role played by Ca2+ in the brain noninvasively by using magnetic resonance imaging, we have synthesized a new set of Ca2+-sensitive smart contrast agents (CAs). The agents were found to be highly selective to Ca2+ in the presence of other competitive anions and cations in buffer and in physiological fluids. The structure of CAs comprises Gd3+-DO3A (DO3A=1,4,7-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane) coupled to a Ca2+ chelator o-amino phenol-N,N,O-triacetate (APTRA). The agents are designed to sense Ca2+ present in extracellular fluid of the brain where its concentration is relatively high, that is, 1.2–0.8 mM. The determined dissociation constant of the CAs to Ca2+ falls in the range required to sense and report changes in extracellular Ca2+ levels followed by an increase in neural activity. In buffer, with the addition of Ca2+ the increase in relaxivity ranged from 100–157 %, the highest ever known for any T1-based Ca2+-sensitive smart CA. The CAs were analyzed extensively by the measurement of luminescence lifetime measurement on Tb3+ analogues, nuclear magnetic relaxation dispersion (NMRD), and 17O NMR transverse relaxation and shift experiments. The results obtained confirmed that the large relaxivity enhancement observed upon Ca2+ addition is due to the increase of the hydration state of the complexes together with the slowing down of the molecular rotation and the retention of a significant contribution of the water molecules of the second sphere of hydration.
We have created unique near-infrared (NIR)–emitting nanoscale metal-organic frameworks (nano-MOFs) incorporating a high density of Yb3+ lanthanide cations and sensitizers derived from phenylene. We establish here that these nano-MOFs can be incorporated into living cells for NIR imaging. Specifically, we introduce bulk and nano-Yb-phenylenevinylenedicarboxylate-3 (nano-Yb-PVDC-3), a unique MOF based on a PVDC sensitizer-ligand and Yb3+ NIR-emitting lanthanide cations. This material has been structurally characterized, its stability in various media has been assessed, and its luminescent properties have been studied. We demonstrate that it is stable in certain specific biological media, does not photobleach, and has an IC50 of 100 μg/mL, which is sufficient to allow live cell imaging. Confocal microscopy and inductively coupled plasma measurements reveal that nano-Yb-PVDC-3 can be internalized by cells with a cytoplasmic localization. Despite its relatively low quantum yield, nano-Yb-PVDC-3 emits a sufficient number of photons per unit volume to serve as a NIR-emitting reporter for imaging living HeLa and NIH 3T3 cells. NIR microscopy allows for highly efficient discrimination between the nano-MOF emission signal and the cellular autofluorescence arising from biological material. This work represents a demonstration of the possibility of using NIR lanthanide emission for biological imaging applications in living cells with single-photon excitation.
We describe a novel method for creating luminescent lanthanide-containing nanoparticles in which the lanthanide cations are sensitized by the semiconductor nanoparticle’s electronic excitation. In contrast to previous strategies, this new approach creates such materials by addition of external salt to a solution of fully formed nanoparticles. We demonstrate this post-synthetic modification for the lanthanide luminescence sensitization of two visible emitting lanthanides (Ln), Tb3+ and Eu3+ ions, through ZnS nanoparticles in which the cations were added post-synthetically as external Ln(NO3)3.xH2O salt to solutions of ZnS nanoparticles. The post-synthetically treated ZnS nanoparticle systems display Tb3+ and Eu3+ luminescence intensities that are comparable to those of doped Zn(Ln)S nanoparticles, which we reported previously (J. Phys. Chem. A, 2011, 115, 4031-4041). A comparison with the synthetically doped systems is used to contrast the spatial distribution of the lanthanide ions, bulk versus surface localized. The post-synthetic strategy described in this work is fundamentally different from the synthetic incorporation (doping) approach and offers a rapid and less synthetically demanding protocol for Tb3+:ZnS and Eu3+:ZnS luminophores, thereby facilitating their use in a broad range of applications.
In this work, we studied enzyme-catalyzed oxidation of single-walled carbon nanotubes (SWCNTs) produced by the high-pressure carbon monoxide (HiPco) method. While oxidation via strong acids introduced defect sites on SWCNTs and suppressed their near-infrared (NIR) fluorescence, our results indicated that the fluorescence of SWCNTs was restored upon enzymatic oxidation, providing new evidence that the reaction catalyzed by horseradish peroxidase (HRP) in the presence of H2O2 is mainly a defect-consuming step. These results were further supported by both UV ?vis ?NIR and Raman spectroscopy. Therefore, when acid oxidation followed by HRP-catalyzed enzyme oxidation was employed, shortened (<300 nm in length) and NIR-fluorescent SWCNTs were produced. In contrast, upon treatment with myeloperoxidase, H2O2, and NaCl, the oxidized HiPco SWCNTs underwent complete oxidation (i.e., degradation). The shortened, NIR-fluorescent SWCNTs resulting from HRP-catalyzed oxidation of acid-cut HiPco SWCNTs may find applications in cellular NIR imaging and drug delivery systems.
There is a growing interest in the development of new medical diagnostic tools with higher sensibility and less damage for the patient body, namely on imaging reporters for the management of diseases and optimization of treatment strategies. This article examines the properties of a new class of lanthanide complexes with a tripodal tris-3-hydroxy-4-pyridinone (tris-3,4-HOPO) ligand - NTP(PrHP)3. Among the studies herein performed, major relevance is given to the thermodynamic stability of the complexes with a series of Ln(3+) ions (Ln = La, Pr, Gd, Er, Lu) and to the magnetic relaxation properties of the Gd(3+) complex. This hexadentate ligand enables the formation of (1 : 1) Ln(3+) complexes with high thermodynamic stability following the usual trend, while the Gd-chelates show improved relaxivity (higher hydration number), as compared with the commercially available Gd-based contrast agents (CAs) ; transmetallation of the Gd(3+)-L complex with Zn(2+) proved to be thermodynamically and kinetically disfavored. Therefore, NTP(PrHP)3 emerges as part of a recently proposed new generation of CAs with prospective imaging sensibility gains.
This work demonstrates how minor structural and electronic changes between Ln(NO3)3 and Ln(hfac)3 lanthanide carriers (Ln = trivalent lanthanide, hfac = hexafluoroacetylacetonate) lead to opposite thermodynamic protocols for the metal loading of luminescent polynuclear single-stranded oligomers. Whereas metal clustering is relevant for Ln(hfac)3, the successive fixation of Ln(NO3)3 provides stable microspecies with an alternated occupancy of the binding sites. Partial anion dissociation and anion/ligand bi-exchange processes occur in polar aprotic solvents, which contribute to delay the unambiguous choice of a well-behaved neutral lanthanide carrier for the selective complexation of different trivalent lanthanides along a single ligand strand. Clues for further improvement along this stepwise strategy are discussed.
Near-infrared emitting, magnetic particles for combined optical and MR detection based on liposomes or artificial lipoproteins are presented. They provide a novel strategy for the luminescence sensitization of lanthanide cations (Yb3+, Nd3+) without covalent bonds between the chromophore and the lanthanide, and provide an unambiguous tool for monitoring the integrity of the liponanoparticles, via emission in the NIR region.
2’-O-Neopentyldeoxyuridine (Un) was synthesized and incorporated into a series of oligodeoxyribonucleotides. Single and triple incorporations in various arrangements were performed. The Watson and Crick pairing properties with complementary DNA and RNA were investigated by UV melting curves, CD spectroscopy, and molecular dynamic simulations. The results were compared to those obtained with DNA-DNA and DNA-RNA duplexes involving dU at the same positions. Oligonucleotides containing Un clearly demonstrated their ability to form duplexes with both complementary DNA and RNA but with higher stabilities for the DNA-RNA duplexes similar to the one of the parent DNA-RNA duplex. Investigations into the thermodynamic properties of these 17-base-pair duplexes revealed [capital Delta]G values (37°C) that are in line with the measured Tm values for both the DNA-DNA and DNA-RNA duplexes. CD spectroscopic structural investigations indicated that the conformations of the DNA-DNA and DNA-RNA duplexes involving Un are similar to those of the dT-rA and dU-rA containing duplexes. Only small changes in intensities and weak blue shifts were observed when three Uns were incorporated into the duplexes. The results of the molecular dynamic simulations showed, for the six duplexes involving the modified nucleoside Un, calculated curvatures similar to those of the corresponding unmodified duplexes without base-pair disruption. The neopentyl group is able to be accommodated in the minor grooves of both the DNA-DNA and RNA-DNA duplexes. However, molecular dynamic simulations indicated that the Uns adopt a C2’-exo sugar pucker conformation close to an A-helix type without perturbing the C2’-endo sugar pucker conformations of their 2’-deoxynucleoside neighbours. These results confirm the potential of 2’-O-neopentyldeoxyuridine as a nucleoside surrogate for oligonucleotide based therapeutic strategies.
A ditopic DTPA monoamide derivative containing an 8-hydroxyquinoline moiety was synthesized and the corresponding gadolinium(iii) complex ([Gd(H5)(H2O)]-) was prepared. After adding aluminum(iii), the 8-hydroxyquinoline part self-assembled into a heteropolymetallic triscomplex [(Gd5)3Al(H2O)3]3-. The magnetic and optical properties of this metallostar compound were investigated in order to classify it as a potential in vitro bimodal contrast agent. The proton nuclear magnetic relaxation dispersion measurements indicated that the relaxivity r1 of [Gd(H5)(H2O)]- and [(Gd5)3Al(H2O)3]3- at 20 MHz and 310 K equaled 6.17 s-1 mM-1 and 10.9 s-1 mM-1 per Gd(iii) ion respectively. This corresponds to a relaxivity value of 32.7 s-1 mM-1 for the supramolecular complex containing three Gd(iii) ions. The high relaxivity value is prominently caused by an increase of the rotational tumbling time [small tau]R by a factor of 2.7 and 5.5 respectively, in comparison with the commercially used MRI contrast agent Gd(iii)-DTPA (Magnevist[registered sign]). Furthermore, upon UV irradiation, [(Gd5)3Al(H2O)3]3- exposes green broad-band emission with a maximum at 543 nm. Regarding the high relaxivity and the photophysical properties of the [(Gd5)3Al(H2O)3]3- metallostar compound, it can be considered as a lead compound for in vitro bimodal applications.
Gd-III-containing metallostar contrast agents are gaining increased attention, because their architecture allows for a slower tumbling rate, which, in turn, results in larger relaxivities. So far, these metallostars find possible applications as blood pool contrast agents. In this work, the first example of a tissue-selective metallostar contrast agent is described. This RGD-peptide decorated Ru-II(Gd-III)(3) metallostar is synthesized as an alpha(v)beta(3)-integrin specific contrast agent, with possible applications in the detection of atherosclerotic plaques and tumor angiogenesis. The contrast agent showed a relaxivity of 9.65 s(-1) mM(-1), which represents an increase of 170%, compared to a low-molecular-weight analogue, because of a decreased tumbling rate (tau(R) = 470 ps). The presence of the MLCT band (absorption 375-500 nm, emission 525-850 nm) of the central Ru-II(Ph-Phen)(3)-based complex grants the metallostar attractive luminescent properties. The (MLCT)-M-3 emission is characterized by a quantum yield of 4.69% and a lifetime of 804 ns, which makes it an interesting candidate for time-gated luminescence imaging. The potential application as a selective MRI contrast agent for alpha(v)beta(3)-integrin expressing tissues is shown by an in vitro relaxometric analysis, as well as an in vitro T-1-weighted MR image.
Transfer news : The use of a simple method allows the various sensitization steps in Eu(III) -containing complexes to be deciphered. Incorporation of an increasing number of electron-withdrawing fluorine atoms on the rigid and electronically tunable phenyl spacer between two tridentate binding units (see picture, red O, dark blue N) affects the quantum yield, intersystem crossing, and energy transfer processes in a rational way.
This work illustrates a simple approach for optimizing long-lived near-infrared lanthanide-centered luminescence using trivalent chromium chromophores as sensitizers. Reactions of the segmental ligand L2 with stoichiometric amounts of M(CF(3)SO(3))(2) (M = Cr, Zn) and Ln(CF(3)SO(3))(3) (Ln = Nd, Er, Yb) under aerobic conditions quantitatively yield the D(3)-symmetrical trinuclear [MLnM(L2)(3)](CF(3)SO(3))(n) complexes (M = Zn, n = 7 ; M = Cr, n = 9), in which the central lanthanide activator is sandwiched between the two transition metal cations. Visible or NIR irradiation of the peripheral Cr(III) chromophores in [CrLnCr(L2)(3)](9+) induces rate-limiting intramolecular intermetallic Cr→Ln energy transfer processes (Ln = Nd, Er, Yb), which eventually produces lanthanide-centered near-infrared (NIR) or IR emission with apparent lifetimes within the millisecond range. As compared to the parent dinuclear complexes [CrLn(L1)(3)](6+), the connection of a second strong-field [CrN(6)] sensitizer in [CrLnCr(L2)(3)](9+) significantly enhances the emission intensity without perturbing the kinetic regime. This work opens novel exciting photophysical perspectives via the buildup of non-negligible population densities for the long-lived doubly excited state [Cr*LnCr*(L2)(3)](9+) under reasonable pumping powers.
Due to its extreme kinetic inertness, trivalent chromium, Cr(III), has been rarely combined with labile trivalent lanthanides, Ln(III), to give discrete self-assembled (supra)molecular polynuclear complexes. However, the plethora of accessible metal-centered excited states possessing variable lifetimes and emissive properties, combined with the design of efficient intramolecular Cr(III) ↔ Ln(III) energy transfer processes open attractive perspectives for programming directional light-conversion within these heterometallic molecules. Efforts made to address this exciting challenge for both light-sensitization and light-upconversion are discussed in this article.
Herein, we discuss how, why, and when cascade complexation reactions produce stable, mononuclear, luminescent ternary complexes, by considering the binding of hexafluoroacetylacetonate anions (hfac-) and neutral, semi-rigid, tridentate 2,6-bis(benzimidazol-2-yl)pyridine ligands (Lk) to trivalent lanthanide atoms (LnIII).
The solid-state structures of [Ln(Lk)ACHTUNGTRENUNG(hfac)3] (Ln=La, Eu, Lu) showed that [Ln-ACHTUNGTRENUNG(hfac)3] behaved as a neutral six-coordinate lanthanide carrier with remarkable properties : 1) the strong cohesion between the trivalent cation and the didentate hfac anions prevented salt dissociation ; 2) the electron-withdrawing trifluoromethyl substituents limited charge-neutralization and favored cascade complexation with Lk ; 3) nine-coordination was preserved for [Ln(Lk)-
ACHTUNGTRENUNG(hfac)3] for the complete lanthanide series, whilst a counterintuitive trend showed that the complexes formed with the smaller lanthanide elements were destabilized. Thermodynamic and NMR spectroscopic studies in solution confirmed that these characteristics were retained for solvated molecules, but the operation of concerted anion/ligand transfers with the larger cations induced subtle structural variations.
Combined with the strong red photoluminescence of [Eu(Lk)ACHTUNGTRENUNG(hfac)3], the ternary system LnIII/hfac-/Lk is a promising candidate for the planned metalloading of preformed multi-tridentate polymers.
Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line : CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using ‘‘in situ’’ proximity ligation assay (PLA).
In the objective of developing ligands that simultaneously satisfy the requirements for MRI contrast agents and near-infrared emitting optical probes that are suitable for imaging, three isoquinoline-based polyaminocarboxylate ligands, L1, L2 and L3, have been synthesized and the corresponding Gd(3+), Nd(3+) and Yb(3+) complexes investigated. The specific challenge of the present work was to create NIR emitting agents which (i) have excitation wavelengths compatible with biological applications and (ii) are able to emit a sufficient number of photons to ensure sensitive NIR detection for microscopic imaging. Here we report the first observation of a NIR signal arising from a Ln(3+) complex in aqueous solution in a microscopy setup. The lanthanide complexes have high thermodynamic stability (log K(LnL) =17.7-18.7) and good selectivity for lanthanide ions versus the endogenous cations Zn(2+), Cu(2+), and Ca(2+) thus preventing transmetalation. A variable temperature and pressure (17)O NMR study combined with nuclear magnetic relaxation dispersion measurements yielded the microscopic parameters characterizing water exchange and rotation. Bishydration of the lanthanide cation in the complexes, an important advantage to obtain high relaxivity for the Gd(3+) chelates, has been demonstrated by (17)O chemical shifts for the Gd(3+) complexes and by luminescence lifetime measurements for the Yb(3+) analogues. The water exchange on the three Gd(3+) complexes is considerably faster (k(ex)(298) = (13.9-15.4) × 10(6) s(-1)) than on commercial Gd(3+)-based contrast agents and proceeds via a dissociative mechanism, as evidenced by the large positive activation volumes for GdL1 and GdL2 (+10.3 ± 0.9 and +10.6 ± 0.9 cm(3) mol(-1), respectively). The relaxivity of GdL1 is doubled at 40 MHz and 298 K in fetal bovine serum (r(1) = 16.1 vs 8.5 mM(-1) s(-1) in HEPES buffer), due to hydrophobic interactions between the chelate and serum proteins. The isoquinoline core allows for the optimization of the optical properties of the luminescent lanthanide complexes in comparison to the pyridinic analogues and provides significant shifts of the excitation energies toward lower values which therefore become more adapted for biological applications. L2 and L3 bear two methoxy substituents on the aromatic core in ortho and para positions, respectively, that further modulate their electronic structure. The Nd(3+) and Yb(3+) complexes of the ligand L3, which incorporates the p-dimethoxyisoquinoline moiety, can be excited up to 420 nm. This wavelength is shifted over 100 nm toward lower energy in comparison to the pyridine-based analogue. The luminescence quantum yields of the Nd(3+) (0.013-0.016%) and Yb(3+) chelates (0.028-0.040%) are in the range of the best nonhydrated complexes, despite the presence of two inner sphere water molecules. More importantly, the 980 nm NIR emission band of YbL3 was detected with a good sensitivity in a proof of concept microscopy experiment at a concentration of 10 μM in fetal bovine serum. Our results demonstrate that even bishydrated NIR lanthanide complexes can emit a sufficient number of photons to ensure sensitive detection in practical applications. In particular, these ligands containing an aromatic core with coordinating pyridine nitrogen can be easily modified to tune the optical properties of the NIR luminescent lanthanide complexes while retaining good complex stability and MRI characteristics for the Gd(3+) analogues. They constitute a highly versatile platform for the development of bimodal MR and optical imaging probes based on a simple mixture of Gd(3+) and Yb(3+)/Nd(3+) complexes using an identical chelator. Given the presence of two inner sphere water molecules, important for MRI applications of the corresponding Gd(3+) analogues, this result is particularly exciting and opens wide perspectives not only for NIR imaging based on Ln(3+) ions but also for the design of combined NIR optical and MRI probes.
A series of novel triazole derivative pyridine-based polyamino-polycarboxylate ligands has been synthesized for lanthanide complexation. This versatile platform of chelating agents combines advantageous properties for both magnetic resonance (MR) and optical imaging applications of the corresponding Gd(3+) and near-infrared luminescent lanthanide complexes. The thermodynamic stability constants of the Ln(3+) complexes, as assessed by pH potentiometric measurements, are in the range log K(LnL) =17-19, with a high selectivity for lanthanides over Ca(2+) , Cu(2+) , and Zn(2+) . The complexes are bishydrated, an important advantage to obtain high relaxivities for the Gd(3+) chelates. The water exchange of the Gd(3+) complexes (k(ex) (298) =7.7-9.3×10(6) s(-1) ) is faster than that of clinically used magnetic resonance imaging (MRI) contrast agents and proceeds through a dissociatively activated mechanism, as evidenced by the positive activation volumes (ΔV(≠) =7.2-8.8 cm(3) mol(-1) ). The new triazole ligands allow a considerable shift towards lower excitation energies of the luminescent lanthanide complexes as compared to the parent pyridinic complex, which is a significant advantage in the perspective of biological applications. In addition, they provide increased epsilon values resulting in a larger number of emitted photons and better detection sensitivity. The most conjugated system PheTPy, bearing a phenyl-triazole pendant on the pyridine ring, is particularly promising as it displays the lowest excitation and triplet-state energies associated with good quantum yields for both Nd(3+) and Yb(3+) complexes. Cellular and in vivo toxicity studies in mice evidenced the non-toxicity and the safe use of such bishydrated complexes in animal experiments. Overall, these pyridinic ligands constitute a highly versatile platform for the simultaneous optimization of both MRI and optical properties of the Gd(3+) and the luminescent lanthanide complexes, respectively.
This work illustrates a simple approach for optimizing the lanthanide luminescence in molecular dinuclear lanthanide complexes and identifies a particular multidentate europium complex as the best candidate for further incorporation into polymeric materials. The central phenyl ring in the bis-tridentate model ligands L3-L5, which are substituted with neutral (X = H, L3), electron-withdrawing (X = F, L4), or electron-donating (X = OCH(3), L5) groups, separates the 2,6-bis(benzimidazol-2-yl)pyridine binding units of linear oligomeric multi-tridentate ligand strands that are designed for the complexation of luminescent trivalent lanthanides, Ln(III). Reactions of L3-L5 with [Ln(hfac)(3)(diglyme)] (hfac(-) is the hexafluoroacetylacetonate anion) produce saturated single-stranded dumbbell-shaped complexes [Ln(2)(Lk)(hfac)(6)] (k = 3-5), in which the lanthanide ions of the two nine-coordinate neutral [N(3)Ln(hfac)(3)] units are separated by 12-14 angstrom. The thermodynamic affinities of [Ln(hfac)(3)] for the tridentate binding sites in L3-L5 are average (6.6 <= log(beta(Y,Lk)(2,1)) <=> L3 >> L5), which suggests that the 1,4-difluorophenyl spacer in L4 is preferable, we have developed a novel tool for deciphering the photophysical sensitization processes operating in [Eu(2)(Lk)(hfac)(6)]. A simple interpretation of the complete set of rate constants characterizing the energy migration mechanisms provides straightforward objective criteria for the selection of [Eu(2)(L4)(hfac)(6)] as the most promising building block.
We have created a dendrimer complex suitable for preferential accumulation within liver tumors and luminescence imaging by substituting thirty-two naphthalimide fluorophores on the surface of the dendrimer and incorporating eight europium cations within the branches. We demonstrate the utility and performance of this luminescent dendrimer complex to detect hepatic tumors generated via direct subcapsular implantation or via splenic injections of colorectal cancer cells (CC531) into WAG/RijHsd rats. Luminescence imaging of the tumors after injection of the dendrimer complex via hepatic arterial infusion revealed that the dendrimer complex can preferentially accumulate within liver tumors. Further investigation indicated that dendrimer luminescence in hepatic tumors persisted in vivo. Due to the incorporation of lanthanide cations, this luminescence agent presents a strong resistance against photobleaching. These studies show the dendrimer complex has great potential to serve as an innovative accumulation and imaging agent for the detection of metastatic tumors in our rat hepatic model.
Surgery is currently the best approach for treating either primary or metastatic hepatic malignancies. Because only 20% of hepatic cancers are operable in patients, several types of regional therapy (RT) are emerging as alternate treatment modalities. However, RTs can have their own limitations at controlling tumor growth or may lack the ability to detect such metastases. Additional strategies can be implemented to enhance their efficacy. An animal model of hepatic metastases coupled with a gastroduodenal artery (GDA) cannulation technique may provide a site to apply such therapies. In our study, splenic injections were performed with CC531 adenocarcinoma cells, which generated metastatic hepatic tumors in WAG/RijHsd rats. Cannulation of GDA was achieved via a polyethylene catheter. Infusion of generation 3 polyamidoamine 4-amino-1,8-naphthalimide dendrimer containing 8 europium ions (Eu-G3P4A18N) via the GDA resulted in luminescence of the hepatic metastatic nodules. Imaging of the metastatic hepatic nodules was obtained with the help of a cooled charge coupled device (CCD) camera.
From the clinical editor :
Hepatic malignancies represent a major therapeutic challenge, despite the available surgical and oncologic treatment modalities. In this paper, an animal model of hepatic adenocarcinoma is used in demonstrating successful targeting of spleen metastases with generation 3 polyamidoamine 4-amino-1,8-naphthalimide dendrimer containing 8 europium ions (Eu-G3P4A18N) for luminescence imaging.
This work explores the sensitization of luminescent lanthanide Tb3+ and Eu3+ cations by the electronic structure of zinc sulfide (ZnS) semiconductor nanoparticles. Excitation spectra collected while monitoring the lanthanide emission bands reveal that the ZnS nanoparticles act as an antenna for the sensitization of Tb3+ and Eu3+. The mechanism of lanthanide ion luminescence sensitization is rationalized in terms of an energy and charge transfer between trap sites and is based on a semiempirical model, proposed by Dorenbos and co-workers (Dorenbos, P. J. Phys. : Condens. Matter2003, 15, 8417−8434 ; J. Lumin.2004, 108, 301−305 ; J. Lumin.2005, 111, 89−104. Dorenbos, P. ; van der Kolk, E. Appl. Phys. Lett.2006, 89, 061122-1−061122-3 ; Opt. Mater.2008, 30, 1052−1057. Dorenbos, P. J. Alloys Compd.2009, 488, 568−573 ; references 1−6.) to describe the energy level scheme. This model implies that the mechanisms of luminescence sensitization of Tb3+ and Eu3+ in ZnS nanoparticles are different ; namely, Tb3+ acts as a hole trap, whereas Eu3+ acts as an electron trap. Further testing of this model is made by extending the studies from ZnS nanoparticles to other II−VI semiconductor materials ; namely, CdSe, CdS, and ZnSe.
DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH(-). Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH degrees. The catalytically potent state FADH(-) can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH degrees is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 A distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH degrees, in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329].
We recently reported the design of new fluorescent oligo-2’-deoxyribonucleotides (FODNs) for the detection of terminal mismatches on DNA duplexes in homogeneous assays. We now report the validation of this method in homogeneous assays with other sequences and the feasibility of the detection of terminal mismatches with immobilized FODNs. In all cases studied, the mismatched duplexes were more fluorescent than the perfect ones and results confirmed that the discrimination factor is sequence-dependent.
Cellular autofluorescence was characterized in normal human esophageal cells and in malignant esophageal epithelial cells. The study was performed under excitation at 351 nm where the cell fluorescence is mainly due to the reduced pyridine nucleotides (NAD(P) H) with a very small contribution from the oxidized flavins (FMN, FAD) or lipopigments. The autofluorescence emission of squamous cell carcinoma, adenocarcinoma on Barrett’s mucosa and normal cells was characterized by microspectrofluorimetry on monolayers and by spectrofluorimetry on cell suspensions. The relative contribution of each fluorophore to the fluorescence emission of the different cell types was evaluated by a curve-fitting analysis.
This paper describes the design of terminal-mismatch discriminating fluorescent oligonucleotides (TMDFOs). The method is based on the use of sets of oligo-2’-deoxyribonucleotide probes linked via their 5’-ends, and varying-sized flexible polymethylene chains, to thiazole orange, with the linker being attached to the benzothiazole moiety. The sequence of each set of labelled probes was identical and complementary to the sequence to be analyzed on the single-stranded nucleic acid target except at the interrogation position, located at the 5’-end of the probes in a position adjacent to the attachment site of the label, where each of the four nucleic bases were incorporated. This work allowed the selection of probes showing, upon their hybridization with the target sequence, good discrimination between the matched and the mismatched duplexes under non-stringent conditions, with the mismatched duplexes being more fluorescent than the perfectly matched ones.
Because the influence of the chemical structure of monomethine cyanine-oligo-2’-deoxyribonucleotide (ODN) conjugates on their binding and fluorescence properties has remained largely undetermined, we synthesized and studied a wide range of conjugates with various structural patterns. Different cyanine dyes such as thiocyanine, quinocyanine, and thiazole orange isomers were obtained. In the case of unsymmetrical cyanines, the linker was attached to either the quinoline or the benzothiazole nucleus. The influence of the ODN counterpart was evaluated by linking the cyanines to the 5’-end or to an internucleotidic phosphate. In the first case, the influence of neighboring nucleic bases was studied, whereas in the second, the stereochemical configuration at the phosphorus atom bearing the cyanine was investigated. We report here on relationships between the structures of the dyes and conjugates and some of their properties, such as the stability and fluorescence changes observed on their hybridization with the target sequence. This study provides useful information towards the design of ODN-cyanine conjugates.
Fluorescent oligonucleotides (FONs) are used in a wide variety of areas such as molecular and mechanistics biological studies, molecular diagnostics, therapeutic development, biotechnology and nanotechnology. Ever since the post-genome era, there has been an ever-increasing demand for more rapid and accurate nucleic acid detection and quantification methods. Genetic information analyses require highly sensitive and specific detection of certain sequences, single nucleotide changes, specific structures and varied reactions in different formats in vitro, in living cells and, ultimately in animals and in human beings. Ideally, a unique event Could be detected and quantified using the genomic information without amplification of the nucleic acids to be analyzed. Recent developments enabling detection at the single-molecule (SM) level have opened new perspectives for applications.
Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences ; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities ; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5’-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate : by SPIR measurements at 37 degrees C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life similar to 10 h (50-fold longer compared with the unconjugated TFO/ LNA).
We report here the synthesis of oligo-2’-deoxyribonucleotides (ODNs) conjugated with perylene. Introduction of perylene, coupled either directly or via a propyl linker to the anomeric position of a 2’-deoxyribose residue, induces the formation of two anomers. Single incorporations of each pure anomer of these sugar perylene units have been performed at either the 5’-end or an internal position of a pyrimidic pentadecamer. The binding properties of these modified ODNs with their single- and double-stranded DNA targets were studied by absorption spectroscopy. Double incorporations of the sugar perylene unit most efficient at stabilizing the triplex and duplex structures (the beta-anomer involving the propyl linker) have been performed at both the 5’-end and at an internal position (or both the 5’- and 3’-ends) of the ODN chain.
We report here a novel, simple reagent enabling the chemical incorporation of a thiophospliate or a phosphate group at the 5’-end of oligonucleotides using very mild basic deprotection conditions. This method can be useful in the case of alkali sensitive modified oligonucleotides. This reagent also gives access to the preparation of bifunctional oligonucleotides with either a thiophosphate group at the 5’-end and a phosphate at the 3’-end, or two thiophosphate groups at both the 5’- and the 3’-ends, or a 5’-thiophosphate group and a 3’-amino-containing linker. (C) 2004 Elsevier Ltd. All rights reserved.
DNA binding compounds, such as benzo[e] (BePI) and benzo[g] pyridoindole (BgPI) derivatives, exhibit preferential stabilization of triple helices. We report here the synthesis of a series of pyrimidine triple-helix-forming oligo-2’-deoxyribonucleotides conjugated with these molecules. BePI was coupled to the 5-position of 2’-deoxyuridine via two linkers of different sizes attached to its 11-position and placed at either the 5’-end, inside the sequence, or at both the 5’-end and the internal positions using periodate oxidation of a diol-containing oligonucleotide followed by reductive coupling with amino-linked BePI. The same BePI derivatives were also linked to the oligonucleotide chain via internucleotidic phosphorothiolate or phosphoramidate linkages. A mixture of diastereoisomers was prepared as well as separate pure Rp and Sp isomers.
Single and multiple incorporations of stereochemically pure modified dinucleoside-phosphoramidates involving substituent groups ending with bis-hydroxyethyl and bis-aminoethyl groups have been performed into pyrimidic triple helix-forming oligo-2’-deoxyribonucleotides designed to bind parallel to the purine strand of the DNA target. The ability of these modified oligo-2’-deoxyribonucleotides to form triple helices has been studied by UV-melting curve analyses, and circular dichroism. Only the oligonucleotides involving modified phosphate groups with the Rp configuration formed more stable triple helices than did the parent phosphodiester sequences.
We report here the synthesis and binding properties of oligonucleotides involving a perylene unit linked to the anomeric position of a 2’-deoxyribose residue. Both anomers were separated and incorporated separately at either the 5’-end or the internal position of a pyrimidine sequence. In any case the presence of the perylene unit stabilizes the complexes formed with either the single or the double-stranded target.
The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophillic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2’-deoxyriboadenylate (dA(15)), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp) ; these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (Phi(fl)) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5’[CACT(15)CAC(3’)]) ; Phi(fl) values of between 0.06-0.35 were determined for the double-stranded conjugates. The Phi(fl) value (0.17) of thiazole orange, 1-(N,N’-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)- 2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1 : dA(15)(.)d(5’)[CACT(15)CAC(3’)] (1:1)) is much less than that for some of the hybrids of the conjugates.
Multiple incorporations of 7-chloro-7-deaza-2’-deoxyguanosine in place of 2’-deoxyguanosine have been performed into a triple helix-forming oligodeoxyribonucleotide involving a run of six contiguous guanines designed to bind in a parallel orientation relative to the purine strand of the DNA target. The ability of these modified oligodeoxyribonucleotides to form triple helices in a buffer containing monovalent cations was studied by UV-melting curves analysis, gel shift assay and restriction enzyme protection assay. In the presence of Na+, the incorporation of two, three or five modified nucleosides in the third strand has improved the efficacy of formation of the tripler as compared to that formed with the unmodified oligonucleotide.
Perylene has been covalently linked, via polymethylene tethers. to the 5’- and the 3’-ends of an oligopyrimidine sequence. The presence of the polycyclic ligand stabilizes the duplexes and the triplexes formed by the modified oligonucleotides and their single- and double-stranded DNA targets Lis compared to those formed with the parent unmodified oligonucleotide used as reference. Stabilization of the triplex is at its highest when perylene is linked to the 5’-end of the oligonucleotide via a nine-atom size linker. Stabilization of the duplexes is nearly equivalent whatever the position of the substitution (5’ or 3’) and the linker size used to tether both entities. (C) 2001 Elsevier Science Ltd. All rights reserved.
Thiazole orange label was coupled to the eighth phosphate of a pentadeca-2 ’ -deoxyriboadenylate via a phosphoramidate linkage using different linkers. The stereoisomers were separated, and their absolute configurations were determined. Finally, the thiazole orange moiety was also linked to the tenth phosphate of icosathymidylates in both the a and the ß series via a phosphoramidate linkage. Once again, the thiazole orange-icosathymidylate conjugates were obtained as pure stereoisomers. The binding properties of these oligo-2 ’ -deoxyribonucleotide-thiazole orange conjugates with their complementary sequences were studied by absorption spectroscopy. The covalent attachment of the thiazole orange derivatives to the oligoadenylates stabilizes the complexes formed with both the DNA and RNA targets. On the contrary, when the thiazole orange is tethered to the oligo-a -thymidylate or oligo-ß -thymidylate, no significant stabilization of the duplexes formed with poly r(A) can be observed.
"Light-up" probes, icosa-a -thymidylate-thiazole orange conjugates, for the in situ time-resolved detection of messenger ribonucleic acid (mRNA) in living cells are evaluated. Upon annealing with polyA in aqueous, solutions, the icosa-a -thymidylate-thiazole orange conjugates were shown to be up to 15 times more fluorescent. Microinjection of these probes inta adherent fibroblasts, resulted in high yields of hybridization and fluorescent signals. Incubation of cells in the presence of these probes resulted in facile internalization of the probe and similar painting of the messenger RNA in the nuclear and cytosolic regions.
A new deprotection procedure enables a medium scale preparation of phosphodiester and phosphorothioate oligonucleotides substituted with a protected thiol function at their 5’-ends and an amino group at their 3’-ends in good yield (up to 72 OD units/mu mol for a 19mer phosphorothioate). Syntheses of 3’-amino-substituted oligonucleotides were carried out on a modified support. A linker containing the thioacetyl moiety was manually coupled in two steps by first adding its phosphoramidite derivative in the presence of tetrazole followed by either oxidation or sulfurization to afford the bis-derivatized oligonucleotide bound to the support. Deprotection was achieved by treating the fully protected oligonucleotide with a mixture of 2,2’-dithiodipyridine and concentrated aqueous ammonia in the presence of phenol and methanol.
The use of triple helix-forming oligonucleotides constitutes an attractive strategy to regulate gene expression by inhibition of transcription. Psoralen-oligonucleotide conjugates form, upon irradiation, covalent triplexes and thereby modify the specific target sequence. The processing of such photoproducts on the promoter of the gene coding for the interleukin-2 receptor a chain was investigated in HeLa cells and HeLa nuclear extracts. We demonstrate that psoralen cross-links are not repaired within the cell extracts nor inside cells. The mechanism of repair inhibition was elucidated in vitro : the presence of the third strand oligonucleotide inhibits the incision step of the damaged target by repair endonucleases. These results demonstrate the possibility of using this approach to induce a persistent intracellular DNA damage at a specific site and to afford prolonged transcription inhibition. (C) 2000 Academic Press.
sAmsacrine-4-carboxamide-oligonucleotide conjugates were synthesized and studied for their capacity to form DNA triple helices and to after human topoisomerase II binding and cleavage properties. The intercalating agent was attached to the 3’- or the 5’-end of a 24 nt triple helix-forming oligonucleotide via linkers of different lengths. The stability of these DNA triple helices was investigated by gel retardation and melting temperature studies using a synthetic 70 bp DNA duplex target. The effect of the conjugates on DNA cleavage by topoisomerase II was evaluated using the 70 bp duplex and a 311 bp restriction fragment containing the same triple helix site. The conjugate with the amsacrine derivative linked to the 3’ end of the TFO via a hexaethylene glycol linker modulates the extent of DNA cleavage by topoisomerase II at specific sites.
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