Synthesis and studies of annelated quinolizinium derivatives as versatile constructs for fluorescent probes and ligands for triple-helical and abasic DNA structures

In the present Thesis, various annelated derivatives of the quinolizinium ion, i.e. polycyclic compounds with a bridgehead quaternary nitrogen atom, were synthesized and investigated as fluorescent probes for the detection of biomacromolecules and as ligands for triple-helical DNA and abasic DNA structures. Along these lines, 9-donor-substituted acridizinium (benzo[<i>b</i>]- quinolizinium) derivatives represent a versatile construct for the development of fluorescent probes. Especially promising are the substituted <i>N</i>-aryl-9-aminoacridizinium salts, the fluorescence of which is susceptible to the microviscosity of the medium. Such compounds may be used for the fluorimetric detection of biomacromolecules, like DNA and proteins. <br /> A series of unsubstituted and methyl-substituted diazoniapenthaphenes, as well as the isomeric diazoniaanthra[1,2-<i>&alpha;</i>]anthracenes and diazoniahexaphene were prepared by the cyclodehydration synthesis. These extended derivatives diazoniapolycyclic salts represent novel lead structures, in which preferential, high-affinity binding to the triple-helical DNA may be achieved in the absence of an additional alkaline side chains. <br /> Two isomeric acridizinium–adenine conjugates, as well as three acridizinium-9-carboxamides, were prepared. Their affinity towards regular and abasic-site containing DNA structures was investigated by thermal denaturation experiments with synthetic oligodeoxyribonucleotides. Acridizinium– adenine conjugates and aminoalkyl-substituted acridizinium-9-carboxamides are capable of selective binding to abasic DNA sites. They readily induce the photo-damage of plasmid DNA, however, without selectivity towards abasic DNA structures.Im Rahmen dieser Arbeit wurden verschiedene Chinoliziniumderivate, d.h. polycyclische Aromaten, die über ein quartäres Stickstoffatom verfügen, synthetisiert und sowohl auf ihre Eignung als Fluoreszenzsensoren zur Detektion von Biomakromolekülen als auch auf ihre Bindungseigenschaften gegenüber Triplex-DNA und DNA mit abasischen Positionen hin überprüft. Zum einen wurde gezeigt, dass Derivate des Acridizinium- (Benzo[<i>b</i>]quinolizinium)-Ions, die in Position 9 einen Donorsubstituenten besitzen, ein viel versprechendes Grundgerüst für die Entwicklung von Fluoreszenzsensoren darstellen. Insbesondere die substituierten <i>N</i>-Aryl-9- aminoacridiziniumsalze erscheinen aussichtsreich, da deren Fluoreszenz besonders empfindlich auf kleinste Veränderungen der Viskosität des umgebenden Mediums reagiert. <br /> Eine Reihe von unsubstituierten und methylsubstituierten Diazoniapentaphen-Derivaten sowie die isomeren Diazoniaanthra[1,2-<i>&alpha;</i>]anthracene und das Diazoniahexaphen wurden durch Cyclodehydratisierung erhalten. Diese polycyclische Diazoniasalze repräsentieren neue Leitstrukturen, die mit hoher Affinität eine selektive Bindung an Triplex-DNA aufweisen, ohne dass zusätzliche Aminoalkylseitenketten notwendig sind. <br /> Zwei isomere Acridizinium–Adenin–Konjugate sowie drei Modellverbindungen, d.h. Acridizinium- 9-carboxamidderivate, wurden ausgehend von den entsprechenden Carboxyacridiziniumsalzen erhalten. Ihre Bindungsaffnität gegenüber DNA, die entweder eine oder keine abasische Stelle aufweist, wurde mit Hilfe von DNA-Schmelzexperimenten mit synthetischen Oligonukleotiden ermittelt. Acridizinium–Adenin–Konjugate und aminoalkylsubstituierte Acridiziniumcarboxamide sind in der Lage, an abasische Positionen in DNA zu binden. Sie können die Photoschädigung von Plasmid-DNA auslösen, jedoch gibt es keine selektive Schädigung von DNA mit abasischen Positionen

Identification of optimal fluorescent probes for G-quadruplex nucleic acids through systematic exploration of mono- and distyryl dye libraries

International audienceA library of 52 distyryl and 9 mono-styryl cationic dyes was synthesized and investigated with respect to their optical properties, propensity to aggregation in aqueous medium, and capacity to serve as fluorescence "light-up" probes for G-quadruplex (G4) DNA and RNA structures. Among the 61 compounds, 57 dyes showed preferential enhancement of fluorescence intensity in the presence of one or another G4-DNA or RNA structure, while no dye displayed preferential response to double-stranded DNA or single-stranded RNA analytes employed at equivalent nucleotide concentration. Thus, preferential fluorimetric response towards G4 structures appears to be a common feature of mono-and distyryl dyes, including long-known mono-styryl dyes used as mitochondrial probes or protein stains. However, the magnitude of the G4-induced "light-up" effect varies drastically, as a function of both the molecular structure of the dyes and the nature or topology of G4 analytes. Although our results do not allow to formulate comprehensive structure-properties relationships, we identified several structural motifs, such as indole-or pyrrole-substituted distyryl dyes, as well as simple mono-stryryl dyes such as DASPMI [2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide] or its 4-isomer, as optimal fluorescent light-up probes characterized by high fluorimetric response (I/I 0 of up to 550-fold), excellent selec-tivity with respect to double-stranded DNA or single-stranded RNA controls, high quantum yield in the presence of G4 analytes (up to 0.32), large Stokes shift (up to 150 nm) and, in certain cases, structural selectivity with respect to one or another G4 folding topology. These dyes can be considered as promising G4-responsive sensors for in vitro or imaging applications. As a possible application , we implemented a simple two-dye fluorimetric assay allowing rapid topological classification of G4-DNA structures

Pattern-Based Sensing of Short Oligodeoxynucleotides with Palladium–Dye Complexes

The colorimetric response of a sensor array composed of palladium–dye complexes can be used to identify different hexadeoxynucleotides and to distinguish mixtures of sequence-isomeric hexadeoxynucleotides

“One Ring to Bind Them All”—Part I: The Efficiency of the Macrocyclic Scaffold for G-Quadruplex DNA Recognition

Macrocyclic scaffolds are particularly attractive for designing selective G-quadruplex ligands essentially because, on one hand, they show a poor affinity for the “standard” B-DNA conformation and, on the other hand, they fit nicely with the external G-quartets of quadruplexes. Stimulated by the pioneering studies on the cationic porphyrin TMPyP4 and the natural product telomestatin, follow-up studies have developed, rapidly leading to a large diversity of macrocyclic structures with remarkable-quadruplex binding properties and biological activities. In this review we summarize the current state of the art in detailing the three main categories of quadruplex-binding macrocycles described so far (telomestatin-like polyheteroarenes, porphyrins and derivatives, polyammonium cyclophanes), and in addressing both synthetic issues and biological aspects

“One Ring to Bind Them All”—Part II: Identification of Promising G-Quadruplex Ligands by Screening of Cyclophane-Type Macrocycles

A collection of 26 polyammonium cyclophane-type macrocycles with a large structural diversity has been screened for G-quadruplex recognition. A two-step selection procedure based on the FRET-melting assay was carried out enabling identification of macrocycles of high affinity (ΔT1/2 up to 30°C) and high selectivity for the human telomeric G-quadruplex. The four selected hits possess sophisticated architectures, more particularly the presence of a pendant side-arm as well as the existence of a particular topological arrangement appear to be strong determinants of quadruplex binding. These compounds are thus likely to create multiple contacts with the target that may be at the origin of their high selectivity, thereby suggesting that this class of macrocycles offers unique advantages for targeting G-quadruplex-DNA

Monitoring DNA–Ligand Interactions in Living Human Cells Using NMR Spectroscopy

International audienceStudies on DNA−ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA−ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA−ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands bindin

Double threading through DNA: NMR structural study of a bis-naphthalene macrocycle bound to a thymine–thymine mismatch

The macrocyclic bis-naphthalene macrocycle (2,7-BisNP), belonging to the cyclobisintercalator family of DNA ligands, recognizes T–T mismatch sites in duplex DNA with high affinity and selectivity, as evidenced by thermal denaturation experiments and NMR titrations. The binding of this macrocycle to an 11-mer DNA oligonucleotide containing a T–T mismatch was studied using NMR spectroscopy and NMR-restrained molecular modeling. The ligand forms a single type of complex with the DNA, in which one of the naphthalene rings of the ligand occupies the place of one of the mismatched thymines, which is flipped out of the duplex. The second naphthalene unit of the ligand intercalates at the A-T base pair flanking the mismatch site, leading to encapsulation of its thymine residue via double stacking. The polyammonium linking chains of the macrocycle are located in the minor and the major grooves of the oligonucleotide and participate in the stabilization of the complex by formation of hydrogen bonds with the encapsulated thymine base and the mismatched thymine remaining inside the helix. The study highlights the uniqueness of this cyclobisintercalation binding mode and its importance for recognition of DNA lesion sites by small molecules

Selective recognition of pyrimidine–pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators

Binding of three macrocyclic bis-intercalators, derivatives of acridine and naphthalene, and two acyclic model compounds to mismatch-containing and matched duplex oligodeoxynucleotides was analyzed by thermal denaturation experiments, electrospray ionization mass spectrometry studies (ESI-MS) and fluorescent intercalator displacement (FID) titrations. The macrocyclic bis-intercalators bind to duplexes containing mismatched thymine bases with high selectivity over the fully matched ones, whereas the acyclic model compounds are much less selective and strongly bind to the matched DNA. Moreover, the results from thermal denaturation experiments are in very good agreement with the binding affinities obtained by ESI-MS and FID measurements. The FID results also demonstrate that the macrocyclic naphthalene derivative BisNP preferentially binds to pyrimidine–pyrimidine mismatches compared to all other possible base mismatches. This ligand also efficiently competes with a DNA enzyme (M.TaqI) for binding to a duplex with a TT-mismatch, as shown by competitive fluorescence titrations. Altogether, our results demonstrate that macrocyclic distance-constrained bis-intercalators are efficient and selective mismatch-binding ligands that can interfere with mismatch-binding enzymes

Strength in Numbers: Development of a Fluorescence Sensor Array for Secondary Structures of DNA

International audienceHigh-throughput assessment of secondary structures adopted by DNA oligonucleotides is currently hampered by the lack of suitable biophysical methods. Fluorescent sensors hold great potential in this respect; however, the moderate selectivity that they display for one DNA conformation over the others constitutes a major drawback to the development of accurate assays. Moreover, the use of single sensors impedes a comprehensive classification of the tested sequences. Herein, we propose to overcome these limitations through the development of a fluorescence sensor array constituted by easily accessible, commercial dyes. Multivariate analysis of the emission data matrix produced by the array allows to explore the conformational preferences of DNA sequences of interest, either by calculating the probability of group membership in the six predefined structural categories (three G-quadruplex groups, double-stranded, and two groups of single-stranded forms), or by revealing their particular structural features. The assay enables rapid screening of synthetic DNA oligonucleotides in a 96-well plate format