Structure of the first parallel DNA quadruplex-drug complex

The first crystal structure of a drug (daunomycin) bound to a parallel-stranded intermolecular telomeric G4 quadruplex (d(TGGGGT)4) has been determined to high resolution. A planar assemblage of three daunomycin molecules stacks onto the 5' end of the G4 column, with the daunosamine substituents occupying three of the four quadruplex grooves. The surface area of the terminal G-quartet in this parallel DNA quadruplex, presently occupied by three daunomycins, is sufficiently large that it could easily accommodate other potential telomerase inhibitors such as substituted porphyrins or telomestatin.     

Interaction of Cationic Manganese Porphyrins with DNA. A Binding Model

The DNA cleavage properties of two cationic manganese porphyrins possessing different peripheral substituents were compared. The identical nature of the strand scission patterns of the porphyrins on a 139 base pair restriction fragment of pBR-322 DNA, along with other evidence, suggests that the porphyrin is end-on bound via the minor groove in a melted or partially melted region of DNA. This unusual binding mode underscores the potential of outisde binding cationic metalloporphyrins as probes for low melting regions of DNA     

Ligand Selectivity by Inserting GCGC-Tetrads into G-Quadruplex Structures

G-Quadruplexes (G4s) assembled from tandem G-rich repeat sequences exhibit significant biological functions and applications, which may well depend on their structural features, such as the planar arrangement of G-tetrads and flexibility of loop regions. It has been found that cytosine-intercalated G-repeat sequences also assemble to be quadruplex structures, involving the formation of nonplanar GCGC-tetrads. Herein, to investigate the effect of GCGC-tetrads on structural properties of G4s, some previously studied quadruplexes with or without GCGC-tetrads were selected, and were used to interact with various developed G4 ligands. Our data show that stacked G-tetrads in quadruplexes are important for the π–π stacking interactions, thus promoting the combination with end-stacking ligands, such as porphyrins or planar small molecules. This is confirmed by the observation that the quadruplex formed by d(GGGCT₄GGGC) with two internal G-tetrads and two external GCGC-tetrads can bind to planar ligands in the presence of specific G4-stabilizing cations, including K⁺ and Pb²⁺, and can realize the sensitive detection of Pb²⁺. However, the quadruplex composed of two external G-tetrads and two internal GCGC-tetrads formed by d(GCGGT₃GCGG) facilitates the binding of nonplanar ligands, such as triphenylmethane (TPM) dyes, owing to the structural flexibility induced by internal GCGC-tetrads. This work provides new insights into the interaction between DNA quadruplexes and specific ligands, which is beneficial to the development of quadruplex-based biosensors and the design of anticancer drugs.     

金属卟啉催化下环己烷羟基化反应中的Hammett关系

合成了40个卟啉环上具有不同取代基的单铁、锰卟啉和u-氧桥连双铁、锰卟啉,研究了这些金属卟啉模拟细胞色素P－450单充氧酶在温和条件下催化环己烷羟基化的反应.首次用线性自由能关系对金属卟啉仿生催化反应和金属卟啉自氧化反应进行相关分析,获得一些新的结果和规律.在此基础上,对金属卟啉仿生催化反应的可能机理进行了探讨.     

Effect of loop orientation on quadruplex-TMPyP4 interaction

G-quadruplexes are believed to be potential targets for therapeutic intervention and this has resulted in designing of various quadruplex interacting ligands. Moreover, reports about existence of quadruplex forming sequences across the genome have propelled greater interest in understanding their interaction with small molecules. An intramolecular quadruplex sequence can adopt different conformations, owing to different orientation of loops in the structure. The differences in the loop orientation can affect their molecular recognition. Herein, we have studied the interaction of 5,10,15,20-tetrakis(1-methyl-4-pyridyl)-21H, 23H-porphine (TMPyP4), a well-known G quadruplex binding ligand with three DNA quadruplexes differing in loop orientations. Results obtained from UV, ITC, and SPR studies have coherently revealed that the TMPyP4 molecule shows preferential binding to parallel G-quadruplex ( c-myc and c-kit) over its antiparallel counterpart (human telomeric). The binding affinity for parallel quadruplex was (10(7)) 1 order of magnitude higher than that for antiparallel DNA quadruplex (10 ). The study shows two binding modes, stronger binding (10(7)) of TMPyP4 involving end stacking and a weaker external binding (10 ), while TMPyP4 shows only one binding mode with duplex with a binding affinity of the order of 10(6). Overall, the study emphasizes that differences in the loop orientation give rise to different conformations of quadruplex, which in turn govern its binding to small molecules, and thereby play a pivotal role in molecular recognition.     

A G‐Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

A hemin‐binding DNA G‐quadruplex (also known as a hemin aptamer or DNAzyme) has been previously reported to be able to enhance the peroxidase activity of hemin. In this work, we described a DNAzyme structure that had an effector‐recognizing part appearing as a single stranded DNA linkage flanked by two split G‐quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G‐quadruplex halves and thus efficiently suppressed the enzymatic activity of the G‐quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2′‐azinobis(3‐ethylbenzthiazoline)‐6‐sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re‐engineered G‐quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G‐quadruplex is an especially useful module to design low‐cost and label‐free sensors toward various biologically or environmentally interesting targets.     

Selective recognition of G-qQuadruplex telomeric DNA by a bis(quinacridine) macrocycle

The interaction of G-quadruplex DNA with the macrocyclic compound BOQ1, which possesses two dibenzophenanthroline (quinacridine) subunits, has been investigated by a variety of methods. The oligonucleotide 5'-A(GGGT(2)A)(3)G(3), which mimics the human telomeric repeat sequence and forms an intramolecular quadruplex, was used as one model system. Equilibrium binding constants measured by biosensor surface plasmon resonance (SPR) methods indicate a high affinity of the macrocycle for the quadruplex conformation (K > 1 x 10(7) M(-)(1)) with two equivalent binding sites. The affinity of BOQ1 for DNA duplexes is at least 1 order of magnitude lower. In addition, the macrocycle is more selective than the monomeric control compound (MOQ2), which is not able to discriminate between the two DNA structures (K(duplex) approximately K(quadruplex) approximately 10(6) M(-)(1)). Strong binding of BOQ1 to G4 DNA sequences was confirmed by fluorometric titrations with a tetraplex-forming oligonucleotide. Competition dialysis experiments with a panel of different DNA structures, from single strands to quadruplexes, clearly established the quadruplex binding specificity of BOQ1. Fluorescence resonance energy transfer (FRET) T(m) experiments with a doubly labeled oligonucleotide also revealed a strong stabilization of the G4 conformation in the presence of BOQ1 (DeltaT(m) = +28 degrees C). This DeltaT(m) value is one of the highest values measured for a G-quadruplex ligand and is significantly higher than observed for the monomer control compounds (DeltaT(m) = +10-12 degrees C). Gel mobility shift assays indicated that the macrocycle efficiently induces the formation of G-tetraplexes. Strong inhibition of telomerase was observed in the submicromolar range (IC(50) = 0.13 microM). These results indicate that macrocycles represent an exciting new development opportunity for targeting DNA quadruplexes.     

Detection of structured single‐strand DNA via solid‐state nanopore

Nanopore is a single‐molecule analysis method which also employed electrophoresis has achieved promising single‐molecule detections. In this study, we designed two kinds of confined spaces by fabricating solid‐state nanopores with desirable diameters to study the structured single‐strand DNA of C‐rich quadruplex. For the nanopore whose diameter is larger than the quadruplex size, the DNA molecule could directly translocate through the nanopore with extremely high speed. For the nanopore whose diameter is smaller than the quadruplex size, DNA molecule which is captured by nanopore could return to the solution without translocation or unzip the quadruplex structure into single‐strand and then pass the nanopore. This study certifies that choosing a suitable sensing interface is the vital importance of observing detailed single‐molecule information. The solid‐state nanopores hold the great potential to study the structural dynamics of quadruplex DNA molecule.     

Selective G-quadruplex DNA stabilizing agents based on bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (～0.2-0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.     

Formation of Human Telomeric G-quadruplex Structures Induced by the Quaternary Benzophenanthridine Alkaloids:Sanguinarine,Nitidine,and Chelerythrine

The ligands which can facilitate the formation and stabilize G‐quadruplex structures have attracted enormous attention due to their potential ability of inhibiting the telomerase activity and halting tumor cell proliferation. It is noteworthy that the abilities of the quaternary benzophenanthridine alkaloids (QBAs), the very important G‐quadruplex binders, in inducing the formation of human telomeric DNA G‐quadruplex structures, have not been reported. Herein, the interaction between single‐strand human telomeric DNA and three QBAs: Sanguinarine (San), Nitidine (Nit) and Chelerythrine (Che), has been investigated. Although these molecules are very similar in structure, they exhibit significantly different abilities in inducing oligonucleotide d(TTAGGG)₄ (HT4) to specific G‐quadruplex structures. Our experimental results indicated that the best ligand San could convert HT4 into antiparallel G‐quadruplex structure completely, followed by Nit, which could transform to mixed‐type or hybrid G‐quadruplex structure partially, whereas Che could only transform to antiparallel G‐quadruplex structure in small quantities. The relative QBAs' inducing abilities as indicated by the CD data are in the order of San>Nit>Che. Further investigation revealed that the G‐quadruplex structures from HT4 induced by QBAs are of intramolecular motif. And only sequences with certain length could be induced by QBAs because of their positive charges which could not attract short chain DNA molecules to close to each other and form intermolecular G‐quadruplex. In addition, the factors that affect the interaction between HT4 and QBAs were discussed. It is proposed that the thickness of the molecular frame and the steric hindrance are the primary reasons why the subtle differences in QBAs' structure lead to their remarkable differences in inducing the formation of the G‐quadruplex structures.     

Catalytic Electron‐Transfer Oxygenation of Substrates with Water as an Oxygen Source Using Manganese Porphyrins

Manganese(V)–oxo–porphyrins are produced by the electron‐transfer oxidation of manganese–porphyrins with tris(2,2′‐bipyridine)ruthenium(III) ([Ru(bpy)₃]³⁺; 2 equiv) in acetonitrile (CH₃CN) containing water. The rate constants of the electron‐transfer oxidation of manganese–porphyrins have been determined and evaluated in light of the Marcus theory of electron transfer. Addition of [Ru(bpy)₃]³⁺ to a solution of olefins (styrene and cyclohexene) in CH₃CN containing water in the presence of a catalytic amount of manganese–porphyrins afforded epoxides, diols, and aldehydes efficiently. Epoxides were converted to the corresponding diols by hydrolysis, and were further oxidized to the corresponding aldehydes. The turnover numbers vary significantly depending on the type of manganese–porphyrin used owing to the difference in their oxidation potentials and the steric bulkiness of the ligand. Ethylbenzene was also oxidized to 1‐phenylethanol using manganese–porphyrins as electron‐transfer catalysts. The oxygen source in the substrate oxygenation was confirmed to be water by using ¹⁸O‐labeled water. The rate constant of the reaction of the manganese(V)–oxo species with cyclohexene was determined directly under single‐turnover conditions by monitoring the increase in absorbance attributable to the manganese(III) species produced in the reaction with cyclohexene. It has been shown that the rate‐determining step in the catalytic electron‐transfer oxygenation of cyclohexene is electron transfer from [Ru(bpy)₃]³⁺ to the manganese–porphyrins.     

Magnetic properties and electronic structure of manganese(III) porphyrins

The average magnetic susceptibility (1.2-100 K) and magnetisation (100–15000 Oe at 4.2 K) of two perchlorato manganese(III) porphyrins establish them to be high-spin, in contrast to the “anomalous” behaviour of analogous iron(III) porphyrins. An explanation of the origin of the zero-field splitting in high-spin manganese(III) porphyrins is presented.     

Dinuclear Ruthenium(II) Complexes That Induce and Stabilise G‐Quadruplex DNA

A series of dinuclear ruthenium(II) complexes were synthesised, and the complexes were determined to be new highly selective compounds for binding to telomeric G‐quadruplex DNA. The interactions of these complexes with telomeric G‐quadruplex DNA were studied by using circular dichroism (CD) spectroscopy, fluorescence resonance energy transfer (FRET) melting assays, isothermal titration calorimetry (ITC) and molecular modelling. The results showed that the complexes 1 , 2 and 4 induced and stabilised the formation of antiparallel G‐quadruplexes of telomeric DNA in the absence of salt or in the presence of 100 mM K⁺‐containing buffer. Furthermore, complexes 1 and 2 strongly bind to and effectively stabilise the telomeric G‐quadruplex structure and have significant selectivity for G‐quadruplex over duplex DNA. In comparison, complex 3 had a much lesser effect on the G‐quadruplex, suggesting that possession of a suitably sized plane for good π–π stacking with the G‐quadruplets is essential for the interaction of the dinuclear ruthenium(II) complexes with the G‐quadruplex. Moreover, telomerase inhibition by the four complexes and their cellular effects were studied, and complex 1 was determined to be the most promising inhibitor of both telomerase and HeLa cell proliferation.     

Platinum phenanthroimidazole complexes as G-quadruplex DNA selective binders

Complexes that bind and stabilize G-quadruplex DNA structures are of significant interest due to their potential to inhibit telomerase and halt tumor cell proliferation. We here report the synthesis of the first Pt(II) G-quadruplex selective molecules, containing pi-extended phenanthroimidazole ligands. Binding studies of these complexes with duplex and quadruplex d(T(4)G(4)T(4))(4) DNA were performed. Intercalation to duplex DNA was established through UV/Vis titration, CD spectroscopy, and thermal denaturation studies. Significantly stronger binding affinity of these phenanthroimidazole Pt(II) complexes to G-quadruplex DNA was observed by UV/Vis spectroscopy and competitive equilibrium dialysis studies. Observed binding constants to quadruplex DNA were nearly two orders of magnitude greater than for duplex DNA. Circular dichroism studies show that an increase in pi-surface leads to a significant increase in the thermal stability of the Pt(II)/quadruplex DNA complex. The match in the pi-surface of these phenanthroimidazole Pt(II) complexes with quadruplex DNA was further substantiated by molecular modeling studies. Numerous favorable pi-stacking interactions with the large aromatic surface of the intermolecular G-quadruplex, and unforeseen hydrogen bonds between the ancillary ethylenediamine ligands and the quadruplex phosphate backbone are predicted. Thus, both biological and computational studies suggest that coupling the square-planar geometry of Pt(II) with pi-extended ligands results in a simple and modular method to create effective G-quadruplex selective binders, which can be readily optimized for use in telomerase-based antitumor therapy.     

Towards the Development of Structure‐Selective G‐Quadruplex‐Binding Indolo[3,2‐b]quinolines

The interaction of phenyl‐substituted indolo[3,2‐b]quinolines with DNA G‐quadruplexes of different topology were studied by using a combination of spectroscopic and calorimetric methodologies. N5‐Methylated indoloquinoline derivatives (^MePIQ) with an aminoalkyl side chain exhibit high affinities for the parallel‐stranded MYC quadruplex and a (3+1)‐hybrid structure combined with an excellent discrimination against the antiparallel thrombin‐binding aptamer (TBA) and the human telomeric (HT) quadruplexes. Dissociation constants for the binding of the ligand to the MYC quadruplex are in the submicromolar range, being below the corresponding dissociation constants for the antiparallel‐stranded quadruplexes by about one order of magnitude. Competition experiments with double‐helical DNA reveal the impact of indoloquinoline structural features on the selectivity for the parallel quadruplex relative to duplex DNA. Based on a calorimetric analysis binding to MYC is shown to be equally driven by favorable enthalpic and entropic contributions with no significant impact on the type of cation present.     

Axial Ligand Effects on the Redox Reactions of Manganese Porphyrins

A systematic study for the effect of axially coordinated monovalent anions on the electrode reactions of several manganese porphyrins in acetonitrile is presented. Potential shifts of the metal-centered reduction with changes in counterion were related to the degree of Mn(III)-counterion interaction. In the electrochemically induced ligand exchange, perchlorate anion replaces the other anions as axial ligand coordinated to Mn(III) at oxidation potential less than the first oxidation of manganese porphyrins. Formation constants for axial ligation of OH^? are calculated. One-electron oxidation of dihydroxide coordinated manganese porphyrins generate oxomanganese(IV) porphyrin complexes electrochemically. O=Mn^IVOEP(OH) is more thermodynamically stable than O=Mn^IVTPP(OH), while O=Mn^IVTpFPP(OH) cannot be generated electrochemically. In the presence of styrene or cyclohexene, the absorption spectra of oxomanganese(IV) porphyrins are changed to form manganese(III) porphyrins gradually, which indicates the oxygen atom transfer from oxomanganese(IV) porphyrins to the substrates.     

位阻型金属卟啉的合成及其催化烷烃基化的反应

本文用平衡法制备了中位-四(3,5-二叔丁基-4-甲氧基等基)卟啉(T~D~T~B~M~OPP), 并制备了该卟啉的铁、锰、钴、锌、铜、镍的金属配合物。经红外光谱、电子光谱、核磁共振谱, 元素分析等确认了这些均未见报道的金属卟啉。考察了在温和条件下, 以T~D~T~B~M~O PP Fe^III C和T~D~T~B~M~O PP Mn^III Cl为了催化剂, PhIo为氧化剂, 在CH~2Cl~2中氧化n-C~6H~1~4的反应, 并将结果与别的催化剂进行了比较。     

Oxazole-based peptide macrocycles: a new class of G-quadruplex binding ligands

Herein we report on the synthesis and DNA binding properties of a new class of water soluble oxazole-based peptide macrocycles that bind selectively to quadruplex DNA, with no detectable binding to duplex DNA. We have recently identified one quadruplex in the proto-oncogene c-kit that is suspected to act as a regulatory element for the expression of the c-kit gene. Here we provide the first example of a ligand binding to and stabilizing the c-kit quadruplex. Moreover, we show that these macrocycles show a preference for the c-kit quadruplex as compared to the human telomeric quadruplex.     

Template‐Assembled Synthetic G‐Quadruplex (TASQ): A Useful System for Investigating the Interactions of Ligands with Constrained Quadruplex Topologies

A new biomolecular device for investigating the interactions of ligands with constrained DNA quadruplex topologies, using surface plasmon resonance (SPR), is reported. Biomolecular systems containing an intermolecular‐like G‐quadruplex motif 1 (parallel G‐quadruplex conformation), an intramolecular G‐quadruplex 2 , and a duplex DNA 3 have been designed and developed. The method is based on the concept of template‐assembled synthetic G‐quadruplex (TASQ), whereby quadruplex DNA structures are assembled on a template that allows precise control of the parallel G‐quadruplex conformation. Various known G‐quadruplex ligands have been used to investigate the affinities of ligands for intermolecular 1 and intramolecular 2 DNA quadruplexes. As anticipated, ligands displaying a π‐stacking binding mode showed a higher binding affinity for intermolecular‐like G‐quadruplexes 1 , whereas ligands with other binding modes (groove and/or loop binding) showed no significant difference in their binding affinities for the two quadruplexes 1 or 2 . In addition, the present method has also provided information about the selectivity of ligands for G‐quadruplex DNA over the duplex DNA. A numerical parameter, termed the G‐quadruplex binding mode index (G4‐BMI), has been introduced to express the difference in the affinities of ligands for intermolecular G‐quadruplex 1 against intramolecular G‐quadruplex 2 . The G‐quadruplex binding mode index (G4‐BMI) of a ligand is defined as follows: G4‐BMI=K_D^intra/K_D^inter, where K_D^intra is the dissociation constant for intramolecular G‐quadruplex 2 and K_D^inter is the dissociation constant for intermolecular G‐quadruplex 1 . In summary, the present work has demonstrated that the use of parallel‐constrained quadruplex topology provides more precise information about the binding modes of ligands.