Anton Granzhan

Connection of Metallamacrocycles via Dynamic Covalent Chemistry: A Versatile Method for the Synthesis of Molecular Cages

A modular approach for the synthesis of cage structures is described. Reactions of [(arene)RuCl(2)](2) [arene = p-cymene, 1,3,5-C(6)H(3)Me(3), 1,3,5-C(6)H(3)(i-Pr)(3)] with formyl-substituted 3-hydroxy-2-pyridone ligands provide trinuclear metallamacrocycles with pendant aldehyde groups. Subsequent condensation reactions with di- and triamines give molecular cages with 3, 6, or 12 Ru centers in a diastereoselective and chemoselective (self-sorting) fashion. Some of the cages can also be prepared in one-pot reactions by mixing [(arene)RuCl(2)](2) with the pyridone ligand and the amine in the presence of base. The cages were comprehensively analyzed by X-ray crystallography. The diameter of the largest dodecanuclear complex is ∼3 nm; the cavity sizes range from 290 to 740 Å(3). An amine exchange process with ethylenediamine allows the clean conversion of a dodecanuclear cage into a hexanuclear cage without disruption of the metallamacrocyclic structures.

Combining Metallasupramolecular Chemistry with Dynamic Covalent Chemistry: Synthesis of Large Molecular Cages

Ru-built cube: By combining metallasupramolecular chemistry with dynamic covalent chemistry, complex nanostructures can be formed. Large cages are synthesized by reaction of trinuclear metallamacrocycles containing pendant aldehyde groups.

Asymmetric Distyrylpyridinium Dyes as Red‐Emitting Fluorescent Probes for Quadruplex DNA

The interactions of three cationic distyryl dyes, namely 2,4-bis(4-dimethylaminostyryl)-1-methylpyridinium (1a), its derivative with a quaternary aminoalkyl chain (1b), and the symmetric 2,6-bis(4-dimethylaminostyryl)-1-methylpyridinium (2a), with several quadruplex and duplex nucleic acids were studied with the aim to establish the influence of the geometry of the dyes on their DNA-binding and DNA-probing properties. The results from spectrofluorimetric titrations and thermal denaturation experiments provide evidence that asymmetric (2,4-disubstituted) dyes 1a and 1b bind to quadruplex DNA structures with a near-micromolar affinity and a fair selectivity with respect to double-stranded (ds) DNA [K(a)(G4)/K(a)(ds)=2.5-8.4]. At the same time, the fluorescence of both dyes is selectively increased in the presence of quadruplex DNAs (more than 80-100-fold in the case of human telomeric quadruplex), even in the presence of an excess of competing double-stranded DNA. This optical selectivity allows these dyes to be used as quadruplex-DNA-selective probes in solution and stains in polyacrylamide gels. In contrast, the symmetric analogue 2a displays a strong binding preference for double-stranded DNA [K(a) (ds)/K(a) (G4)=40-100), presumably due to binding in the minor groove. In addition, 2a is not able to discriminate between quadruplex and duplex DNA, as its fluorescence is increased equally well (20-50-fold) in the presence of both structures. This study emphasizes and rationalizes the strong impact of subtle structural variations on both DNA-recognition properties and fluorimetric response of organic dyes.

Ligand-Induced Conformational Changes with Cation Ejection upon Binding to Human Telomeric DNA G-Quadruplexes

The rational design of ligands targeting human telomeric DNA G-quadruplexes is a complex problem due to the structural polymorphism that these sequences can adopt in physiological conditions. Moreover, the ability of ligands to switch conformational equilibria between different G-quadruplex structures is often overlooked in docking approaches. Here, we demonstrate that three of the most potent G-quadruplex ligands (360A, Phen-DC3, and pyridostatin) induce conformational changes of telomeric DNA G-quadruplexes to an antiparallel structure (as determined by circular dichroism) containing only one specifically coordinated K(+) (as determined by electrospray mass spectrometry) and, hence, presumably only two consecutive G-quartets. Control ligands TrisQ, known to bind preferentially to hybrid than to antiparallel structures, and L2H2-6M(2)OTD, known not to disrupt the hybrid-1 structure, did not show such K(+) removal. Instead, binding of the cyclic oxazole L2H2-6M(2)OTD was accompanied by the uptake of one additional K(+). Also contrasting with telomeric G-quadruplexes, the parallel-stranded Pu24-myc G-quadruplex, to which Phen-DC3 is known to bind by end-stacking, did not undergo cation removal upon ligand binding. Our study therefore evidences that very affine ligands can induce conformational switching of the human telomeric G-quadruplexes to an antiparallel structure and that this conformational change is accompanied by removal of one interquartet cation.

“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.

Macrocyclic DNA‐Mismatch‐Binding Ligands: Structural Determinants of Selectivity

A collection of 15 homodimeric and 5 heterodimeric macrocyclic bisintercalators was prepared by one- or two-step condensation of aromatic dialdehydes with aliphatic diamines; notably, the heterodimeric scaffolds were synthesized for the first time. The binding of these macrocycles to DNA duplexes containing a mispaired thymine residue (TX), as well as to the fully paired control (TA), was investigated by thermal denaturation and fluorescent-intercalator-displacement experiments. The bisnaphthalene derivatives, in particular, the 2,7-disubstituted ones, have the highest selectivity for the TX mismatches, as these macrocycles show no apparent binding to the fully paired DNA. By contrast, other macrocyclic ligands, as well as seven conventional DNA binders, show lesser or no selectivity for the mismatch sites. The study demonstrates that the topology of the ligands plays a crucial role in determining the mismatch-binding affinity and selectivity of the macrocyclic bisintercalators.

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.

Studies of the fluorescence light-up effect of amino-substituted benzo[b]quinolizinium derivatives in the presence of biomacromolecules

A comparative study of the ability of amino-substituted benzo[b]quinolizinium derivatives to act as DNA- or protein-sensitive fluorescent probes is presented. Spectrophotometric titrations, DNA denaturation studies and viscometric titrations showed that all tested aminobenzo[b]quinolizinium derivatives intercalate into DNA with binding constants K(b) = 10(4)-10(5) M(-1). The intense fluorescence of the 9-aminobenzo[b]quinolizinium (Φ(fl) = 0.41) as well as the intrinsically very weak emission of the 7-aminobenzo[b]quinolizinium (Φ(fl) < 0.005) are quenched by the addition of DNA, most likely caused by a photoinduced electron transfer (PET) between the excited intercalated ligand and the DNA bases. The 6-aminobenzo[b]quinolizinium (1b) and the 6-amino-9-bromobenzo[b]quinolizinium (1c) exhibit very low fluorescence intensity in water (Φ(fl) < 0.005). However, in water-glycerol mixtures the emission intensity increases by factors of 56 (1b) and 27 (1c) with increasing glycerol content of the solution (0-100 wt%), which indicates the radiationless deactivation of the excited state of 1b and 1c due to a torsional relaxation, i.e. rotation about the exocyclic C(ar)-NH(2) bond. In the case of the bromo-substituted derivative 1c, a viscosity-independent heavy-atom-effect of the bromo substituent leads to additional quenching. The association of 1b and 1c with ds DNA leads to a restricted conformational flexibility of the intercalated ligand and results in an increase of fluorescence intensity. This effect is particularly strong in the presence of poly[dA-dT]-poly[dA-dT]. Upon association with ct DNA or poly[dG-dC]-poly[dG-dC] only very small enhancement of emission intensity (1b) or even a slight quenching (1c) of the fluorescence was observed because of the interfering PET reaction with the guanine residues. Preliminary experiments reveal that the 6-aminobenzo[b]quinolizinium derivatives 1b and 1c may also be employed as protein-sensitive probes, because their emission intensity increases upon association with selected albumins.

Recognition of G-Quadruplex DNA by Triangular Star-Shaped Compounds: With or Without Side Chains?

We report the synthesis of two new series of triangular aromatic platforms, either with three aminoalkyl side chains (triazatrinaphthylene series, TrisK: six compounds), or without side chains (triazoniatrinaphthylene, TrisQ). The quadruplex-DNA binding behavior of the two series, which differ essentially by the localization of the cationic charges, was evaluated by means of FRET-melting and G4-FID assays. For the trisubstituted triazatrinaphthylenes (TrisK), the length of the substituents and the presence of terminal hydrogen-bond-donor groups (NH(2)) were shown to be crucial for ensuring a high quadruplex affinity (ΔT(1/2) values of up to 20 °C at 1 μM for the best candidate, TrisK3-NH) and selectivity versus duplex DNA. Subsequently, comparison of data collected on both the telomeric- and c-myc-quadruplex showed that the nonsubstituted TrisQ is even more efficient than TrisK3-NH, both in terms of quadruplex affinity (ΔT(1/2)=26 °C in K(+) buffer) and selectivity versus duplex DNA. Structural considerations conducted with the c-myc quadruplex indicate that both TrisK3-NH and TrisQ stack well onto the G-quartet but in an offset position, which might be influenced by the formation of multiple hydrogen bonds with the target in the former case. Finally, the nonsubstituted TrisQ displays a binding profile very similar to some of the best quadruplex binders, BRACO-19 and bisquinolinium 360A, used herein as references, and thereby represents a highly promising novel molecular design for quadruplex recognition.

“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.