Selective recognition of pyrimidine-pyrimidine DNA mismatches by distance-constrained macrocyclic bis-intercalators.
ABSTRACT: 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.
Project description: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.
Project description:To explore the utility of the azinomycin B chromophore as a platform for the development of major-groove binding small molecules, we have prepared a series of 3-methoxy-5-methylnaphthalene derivatives containing diamine, triamine, and carbohydrate linker moieties. All bis- and tris-azinomycin derivatives are intercalators that display submicromolar binding affinities for calf-thymus DNA, as revealed by viscometry measurements and fluorescent intercalator displacement (FID) assays, respectively. Although the tightest binding ligand 1d (Ka = 2.42 × 107 M-1) has similar affinities for sequence diverse polynucleotides, competition binding studies with methylated phage DNA and known major and minor groove binding small molecules suggest that the tether moiety linking the naphthalene chromophores may occupy the major groove of DNA.
Project description:A series of novel acyclic, macrocyclic and mechanically interlocked luminescent anion sensors have been prepared by incorporation of the isophthalamide motif into dipyridylbenzene to obtain cyclometallated complexes of platinum(II) and ruthenium(II). Both the acyclic and macrocyclic derivatives 7?Pt, 7?Ru?PF6 , 10?Pt and 10?Ru?PF6 are effective sensors for a range of halides and oxoanions. The near-infra red emitting ruthenium congeners exhibited an increased binding strength compared to platinum due to the cationic charge and thus additional electrostatic interactions. Intramolecular hydrogen-bonding between the dipyridylbenzene ligand and the amide carbonyls increases the preorganisation of both acyclic and macrocyclic metal derivatives resulting in no discernible macrocyclic effect. Interlocked analogues were also prepared, and preliminary luminescent chloride anion spectrometric titrations with 12?Ru?(PF6 )2 demonstrate a marked increase in halide binding affinity due to the complementary chloride binding pocket of the rotaxane. 1 H?NMR binding titrations indicate the interlocked dicationic receptor is capable of chloride recognition even in competitive 30?% aqueous mixtures.
Project description:An efficient and short synthetic route to a novel decadentate ligand 7-[2-(bis-carboxymethyl-amino)-ethyl]-4,10-bis-carboxymethyl-1,4,7,10-tetraaza-cyclododec-1-yl-acetic acid (DEPA) with both macrocyclic and acyclic binding moieties is reported. A reproducible and scalable synthetic method to a precursor molecule of DEPA, 1,4,7-tris(tert-butoxycarbonylmethyl)tetraazacyclododecane was developed. DEPA was evaluated as a chelator of (177)Lu, (212)Bi, and (213)Bi for potential use in an antibody-targeted cancer therapy, radioimmunotherapy (RIT) using Arsenazo III based spectroscopic complexation kinetics, in vitro serum stability, and in vivo biodistribution studies.
Project description:Non-macrocyclic heteroaryls represent a valuable class of ligands for nucleic acid recognition. In this regard, non-macrocyclic pyridyl polyoxazoles and polyoxadiazoles were recently identified as selective G-quadruplex stabilizing compounds with high cytotoxicity and promising anticancer activity. Herein, we describe the synthesis of a new family of heteroaryls containing oxadiazole and pyridine moieties targeting DNA G-quadruplexes. To perform a structure?activity analysis identifying determinants of activity and selectivity, we followed a convergent synthetic pathway to modulate the nature and number of the heterocycles (1,3-oxazole vs. 1,2,4-oxadiazole and pyridine vs. benzene). Each ligand was evaluated towards secondary nucleic acid structures, which have been chosen as a prototype to mimic cancer-associated G-quadruplex structures (e.g., the human telomeric sequence, c-myc and c-kit promoters). Interestingly, heptapyridyl-oxadiazole compounds showed preferential binding towards the telomeric sequence (22AG) in competitive conditions vs. duplex DNA. In addition, G4-FID assays suggest a different binding mode from the classical stacking on the external G-quartet. Additionally, CD titrations in the presence of the two most promising compounds for affinity, TOxAzaPy and TOxAzaPhen, display a structural transition of 22AG in K-rich buffer. This investigation suggests that the pyridyl-oxadiazole motif is a promising recognition element for G-quadruplexes, combining seven heteroaryls in a single binding unit.
Project description:A series of new 9,9-diethylfluorenes consisting of three side-arms each bearing a heterocyclic, bis(carboxymethyl)amino, bis(carbamoylmethyl)amino, bis(ethoxycarbonylmethyl)amino or an amino group were prepared on the basis of 2,4,7-tris(bromomethyl)-9,9-diethylfluorene. Imidazolyl, benzimidazolyl, pyrazolyl, pyrrolyl, 1,3-dioxoisoindolyl and pyridinium groups were taken into account as heterocyclic units, attached to the aromatic skeleton via -CH<sub>2</sub>-, -CH<sub>2</sub>NHCH<sub>2</sub>- or -CH<sub>2</sub>N=CH- linkers. In addition to the seventeen 2,4,7-trisubstituted 9,9-diethylfluorenes, two macrocyclic compounds were prepared on the basis of 2,7-bis(aminomethyl)-9,9-diethylfluorene. The excellent yield of the macrocyclization reaction is worth a special mention. Both the acyclic and the macrocyclic fluorene-based compounds have, among other things, the potential to act as artificial receptors for different substrates in analogy to the known receptors consisting of a benzene or biphenyl core.
Project description:We have previously demonstrated that polymerases such as telomerase can be inhibited by molecules (e.g., intercalators) that target the key RNA/DNA duplex substrate. In this work we show that this also holds true for reverse transcriptase, and show that the lead intercalators can be modified to increase inhibition efficacy. Specifically, we use the strategy of multiple simultaneous intercalation, by linking two intercalators with a variable linker. The rationale behind this design is that a specific linker has the potential to increase affinity and specificity for the target duplex. We have synthesized a library of 45 ethidium bis-intercalators in which the distance between intercalators is systematically varied. We observe that members of the dimer library have improved telomerase and reverse transcriptase inhibition, relative to the monomeric leads. We show that this improvement in inhibition over mono-intercalators is most prominent when non-productive sites of inhibitor binding are limited in the assay mix. When this is done, a 400-fold increase in inhibition efficacy is observed.
Project description:Two macrocyclic bis-benzimidazolium salts 2 and 4 (23-membered for 2 and 25-membered for 4) were prepared, and their structures were confirmed by X-ray crystallography, 1H NMR and 13C NMR spectroscopy. The research of anion recognitions using 2 or 4 as hosts were carried out with the methods of fluorescence and ultraviolet spectroscopy, 1H NMR titrations, MS and IR spectra. The experiment results show that 2 can detect acetate anion and 4 can detect nitrate anion with favorable selectivity and sensitivity.
Project description:A newly designed cyclic bis-naphthyridine carbamate dimer CMBL4: with a limited conformational flexibility was synthesized and characterized. Absorption spectra revealed that two naphthyridines in CMBL4: were stacked on each other in aqueous solutions. The most efficient binding of CMBL4: to DNA was observed for the sequence 5'-T-3'/5'-GG-3' (T/GG) with the formation of a 1:1 complex, which is one of possible structural elements involved in the higher order structures of (TGG)n repeat DNA triggering the genome microdeletion. Surface plasmon resonance assay also showed the binding of CMBL4: with TGG repeat DNA. Potassium permanganate oxidation studies of CMBL4: -bound duplex containing the T/GG site showed that the CMBL4: -binding accelerated the oxidation of thymine at that site, which suggests the flipping out of the thymine base from a ?-stack. Preferential binding was observed for CMBL4: compared with its acyclic variants, which suggests the marked significance of the macrocyclic structure for the recognition of the T/GG site.
Project description:DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines.