A Simple Entropic-Driving Separation Procedure of Low-Size Silver Clusters, Through Interaction with DNA.

Synthesis and purification of metal clusters without strong binding agents by wet chemical methods are very attractive for their potential applications in many research areas. However, especially challenging is the separation of uncharged clusters with only a few number of atoms, which renders the usual techniques very difficult to apply. Herein, we report the first efficient separation of Ag2 and Ag3 clusters using the different entropic driving forces when such clusters interact with DNA, into which Ag3 selectively intercalates. After sequential dialysis of the samples and denaturalizing the DNA-Ag3 complex, pure Ag2 can be found in the dialysate after extensive dialysis. Free Ag3 is recovered after DNA denaturation.

Removal of 1,2-benzanthracene via the intercalation of 1,2-benzanthracene with DNA and magnetic bead-based separation.

In this study, DNA-functionalize-magnetic beads were investigated as sorbent materials for effective removing 1,2-benzanthracene (BaA) from water. In order to reveal the removal mechanism, the interaction mode between BaA and DNA was evaluated by using various characterization tools such as UV-visible and circular dichroism spectroscopy, fluorescence and resonance scattering spectroscopy, and agarose gel electrophoresis. In the presence of BaA, the melting temperature of DNA increased from 76.2 °C to 82.3 °C, which closely related to the intercalating of BaA. It was found that a part of the ethidium bromide (EB) binding sites to DNA were occupied by BaA in EB competing study. The results indicated that a new complex appeared between hsDNA and BaA, and the number of the binding sites (n) and the binding constants (KA) at different temperatures were obtained. DNA binding saturation value (≈0.80) was obtained by resonance scattering spectra study. BaA could be enriched and removed by DNA-functionalize-magnetic beads via the intercalation, and the removal efficiency was 97.73% when the initial concentration was 2.45 x10-6 mol·L-1 (559.31 µg/L).

Molecular Networking Reveals Two Distinct Chemotypes in Pyrroloiminoquinone-Producing Tsitsikamma favus Sponges.

The temperate marine sponge, Tsitsikamma favus, produces pyrroloiminoquinone alkaloids with potential as anticancer drug leads. We profiled the secondary metabolite reservoir of T. favus sponges using HR-ESI-LC-MS/MS-based molecular networking analysis followed by preparative purification efforts to map the diversity of new and known pyrroloiminoquinones and related compounds in extracts of seven specimens. Molecular taxonomic identification confirmed all sponges as T. favus and five specimens (chemotype I) were found to produce mainly discorhabdins and tsitsikammamines. Remarkably, however, two specimens (chemotype II) exhibited distinct morphological and chemical characteristics: the absence of discorhabdins, only trace levels of tsitsikammamines and, instead, an abundance of unbranched and halogenated makaluvamines. Targeted chromatographic isolation provided the new makaluvamine Q, the known makaluvamines A and I, tsitsikammamine B, 14-bromo-7,8-dehydro-3-dihydro-discorhabdin C, and the related pyrrolo-ortho-quinones makaluvamine O and makaluvone. Purified compounds displayed different activity profiles in assays for topoisomerase I inhibition, DNA intercalation and antimetabolic activity against human cell lines. This is the first report of makaluvamines from a Tsitsikamma sponge species, and the first description of distinct chemotypes within a species of the Latrunculiidae family. This study sheds new light on the putative pyrroloiminoquinone biosynthetic pathway of latrunculid sponges.

A new DNA-intercalative cytotoxic allylic xanthone from Swertia corymbosa.

Phytochemical investigation of the CHCl3 fraction of Swertia corymbosa resulted in the isolation of a new 3-allyl-2,8-dihydroxy-1,6-dimethoxy-9H-xanthen-9-one (1), along with four known xanthones, gentiacaulein (3), norswertianin (4), 1,3,6,8-tetrahydroxyxanthone (5), and 1,3-dihydroxyxanthone (6). Structure of compound 1 was elucidated with the aid of IR, UV, NMR, and MS data, and chemical transformation via new allyloxy xanthone derivative (2). Compounds 1-6 exhibited various levels of antioxidant and anti-α-glucosidase activities. Absorption and fluorescence spectroscopic studies on 1-6 indicated that these compounds could interact with calf thymus DNA (CT-DNA) through intercalation and with bovine serum albumin (BSA) in a static quenching process. Compound 1 was found to be significantly cytotoxic against human cancer cell lines HeLa, HCT116, and AGS, and weakly active against normal NIH 3T3 cell line.

Fused aryl-phenazines: scaffold for the development of bioactive molecules.

Fused aryl phenazine derivatives (benzo[a]phenazine, pyrido[a]phenazine, benzo[a]phenazine diones, tetrahydropyrido[a]phenazine (dermacozines), etc) are important heterocyclic compounds, which exhibit various pharmacological activities, prominently in cancer cell lines. These compounds significantly intercalate between DNA base pairs and inhibit the activities of topoisomerase I and II enzymes (Topo I and II). XR11576, XR5944, NC-190 and NC-182 belong to phenazine/fused aryl phenazine category and are under clinical studies. Several fused aryl phenazine dione compounds such as pyridazino[4,5-b]phenazine-5,12-diones, 6,11-dihydro-pyrido[2,3-b]phenazine-6,11-diones, 6,11-dihydrobenzo[2,3-b]phenazine-6,11-diones, tetrahydropyrido[a]phenazine, etc possessed anticancer activities on various cancer cell lines. Benzo[a]phenazine diimine and various other fused aryl phenazine compounds form coordination complex with the metal ions (Ru, Rh, Zn and Pt) that intercalate with the DNA and are used for the treatment of cancer. These molecules have influence on MDR cancer cells and serve as anticancer agents in MDR cancer cells. The structure activity relationship of the fused aryl phenazine derivatives revealed that the occurrence of four or more nitrogen atoms in the compounds has better anticancer activity than those molecules with less number of nitrogen atoms. Phenazine antibiotics derived from marine microbes are used for the treatment of microbial and worm diseases. Recent patents on these scaffolds showed that the benzo[a]phenazine derivatives have inhibitory activity on topoisomerase enzymes (Topo I and II) and that act as anticancer agents.

Recent developments in bisintercalator natural products.

The bisintercalator natural products are a family of nonribosomal peptides possessing a range of biological properties that include antiviral, antibiotic, and anticancer activities. The name bisintercalator is derived from the ability to directly bind to duplex DNA through two planar intercalating moieties. Although 19 members of this family of compounds have been identified over the past 50 years, the biosynthetic genes responsible for the formation of four of these molecules (thiocoraline, SW-163, triostin A, and echinomycin) were identified only recently. This recent progress opens an avenue towards understanding how Nature produces these bisintercalating products and provides the potential to develop and identify novel potent analogous lead compounds for clinical applications. This review discusses the mode of action of bisintercalators and summarizes recent genetic and biochemical insights into their biosynthetic production, analog formation, and possible mechanisms by which resistance to these compounds is achieved by their producing organisms.

Selective accumulation of harmful compounds by the DNA-inorganic hybrid-immobilized glass bead.

Previously, we reported the DNA-inorganic hybrid material including double-stranded DNA by mixing the aqueous DNA solution and silane coupling reagents. Here, we immobilized the DNA-inorganic hybrid material onto the glass bead and prepared the DNA-immobilized glass bead column. The DNA-immobilized glass beads were stable in water and the amount of eluted DNA from the DNA-glass beads did not change for more than 1 week. Additionally, this DNA-immobilized column selectively accumulated the harmful compounds with the planar structure, such as dioxin- and polychlorinated biphenyl (PCB)-derivatives, and these accumulation percentages were 50-70%. Furthermore, the DNA-immobilized glass bead was recycled nine times by the application of ethanol solution and the accumulative ratio was maintained at more than 60% and did not appear to be decreasing. Therefore, these DNA-columns might have a potential for the selective removal and separation of DNA-intercalating molecules and harmful compounds with the planar structure from experimental or industrial drainages.

Preparation of DNA-adsorbed TiO2 particles with high performance for purification of chemical pollutants.

Photocatalysis using semiconductors such as titanium dioxide (TiO2) has been studied and applied to the treatment of wastewater and purification of air, because of its ability to decompose organic contaminants. However, there are still problems associated with the practical application of photocatalytic reactions, one of which is that contact between the reactants and catalysts is absolutely required, because the reaction occurs atthe surface of the catalysts. This restrictsthe purification of pollutants on a large scale. In this study, we developed novel DNA-adsorbed TiO2 particles (DNA-TiO2) to solve the problem. Because DNA has an unique double-stranded structure and interacts with several chemicals, DNA-TiO2 can accumulate chemicals on the surface of TiO2. DNA intercalators (Methylene Blue and ethidium bromide), small amounts of which exist in large-volume solutions, were instantaneously trapped in DNA-TiO2 and degraded under ultraviolet (UV) light rapidly, compared to nonadsorbed TiO2. The efficiency of removal and photocatalytic degradation was dependent on the amount of DNA adsorbed on the surface of TiO2 and was independent of the size of DNA. Even if the pH (2-10) and temperature (approximately 56 degrees C) of the solution were changed, DNA remained stable on TiO2, and the ability to remove intercalators was also maintained. DNA-TiO2 could accumulate other pigments such as Acridine Orange, Orange II, Neutral Red, Brilliant Green, and Crystal Violet. These results suggested that DNA-TiO2 is beneficial for the removal and degradation of chemicals having affinity for DNA and dispersing in a large field.

[Effect of alkaloids from celandine on calcium accumulation and oxidative phosphorylation in mitochondria depending on their DNA intercalating properties].

The effect of principal alkaloids (sanguinarine, chelerythrine, coptisine, chelidonine) of greater celandine Chelidonium majus L., as well as the alkaloids from Colchicum autumnale L. (colchicine and colchamine) on calcium accumulation and oxidative phosphorylation in rat liver mitochondria has been studied. The obtained data were compared with DNA intercalating properties of alkaloids detected by the method of thermodenaturation (DNA melting curve plots). It was found that chelerythrine and sanguinarine blocked absorption and accumulation of calcium cations and inhibited oxidative phosphorylation, while the coptisine significantly diminished those indices. Chelidonine, colchicines and colchamine had no influence on the studied characteristics. The effect of alkaloids upon mitochondria functional state correlated tightly with their DNA intercalating properties: chelerythrine and sanguinarine were strong intercalators, while coptisine was a weak one, and chelidonine, colchicine and colchamine did not interact with DNA and caused no changes in its melting point. Correlation coefficient between the intercalating properties of alkaloids and their inhibition of calcium accumulation was 0.89, and with their oxidative phosphorylation inhibition - 0.93. It is suggested that the effect of studied alkaloids upon functional properties of mitochondria can be mediated by mtDNA.

Time-resolved electrophoretic analysis of mobility shifts for dissociating DNA ligands.

Intercalative binding of ligands to DNA can be demonstrated by helix unwinding, monitored by gel electrophoresis of supercoiled DNA, as electrophoretic mobility is sensitive to the topological DNA state. However, we show that an apparent lack of unwinding in an electrophoretic assay could be due to dissociation of the (intercalated) ligand during the analysis, rather than evidence for a nonintercalative mode of binding to DNA. Repetitive scanning during the electrophoresis ensures that release of the ligand during electrophoresis does not affect the measured degree of unwinding, based on the electrophoretic velocity being determined as a function of time. We use this assay to establish intercalation as a mode of binding to DNA for the cyanine dyes YO, YO-PRO as well as two enantiomeric forms of the ruthenium complexes [(phen)2 Ru(tatpp)Ru(phen)2]4+, and to support groove-binding for the new unsymmetrical cyanine dyes BOXTO and BOXTO-PRO. Groove-binding could be concluded from a lack of unwinding, because we could rule out that it is caused by release of the dye during the electrophoresis. The gel electrophoresis has the advantage over hydrodynamic techniques that much smaller sample amounts are required, and our time-resolved approach can be employed in all mobility-shift assays when applied to dissociating complexes.

Pyrroloquinoline and pyridoacridine alkaloids from marine sources.

Marine organisms are a rich source for natural products. Pyrrolo[4,3, 2-de]quinolines and pyrido[4,3,2-mn]acridines are of major interest as metabolites in sponges and ascidians. Many of these compounds have generated interest both as challenging problems for structure elucidation and synthesis as well as for their cytotoxicities. The isolation, structure proof, biological activities, chemical properties and synthesis have attracted the attention of chemists, biologists and pharmacists. The principal structural feature of these alkaloids is the core of a planar iminoquinone moiety which can intercalate into DNA and cleave the DNA double helix or inhibit the action of topoisomerase II. Of the makaluvamines, makaluvamine F and A are the most cytotoxic to the HCT 116 cell line. The enhanced toxicity of the makaluvamines towards xrs-6 cells shows that all of the makaluvamines, except makaluvamine B, act like m-AMSA and etoposide in inhibiting topo iso merases via cleavable complex formation, or via the direct induction of DNA double-strand breaks. They are also amongst the most potent inhibitors of topoisomerase II. Both makaluvamine A and C can decrease tumor size in a solid human tumor model. Discorhabdin A and C in contrast are of high cytotoxicity, but they exhibit no inhibition of topoisomerase II. As representatives of the derivatives of pyrido[4,3,2-mn]acridine, cystodytins, kuanoniamines and diplamine are the most potent to inhibit HCT replication. Eilatin, as a 1,10-phenanthroline derivative, can form complexes with metal ions. It has been shown that these metal complexes can bind to DNA by intercalation. The new members of the pyrrolo[4,3,2-de]quinolines and pyrido[4,3, 2-mn]acridines, such as veiutamine, discorhabdin G, tsitsikammamines, epinartins, arnoamines as well as sagitol are reviewed. Some successful syntheses of pyrrolo[4,3,2-de]quinoline ring system and pyrido[4,3,2-mn]acridine ring system are also reviewed in this article.

[Sanguinarine and ellipticine cytotoxic alkaloids isolated from well-known antitumor plants. Intracellular targets of their action]. / Sangvinarin i elliptitsin: tsitotoksicheskie alkaloidy, vydelennye iz izvestnykh protivoopukholevykh rastenil, i vnutrikletochnye misheni ikh delstviia.

Common molecular and cellular targets for alkaloids sanguinarine and ellipticine, isolated from well-known antitumor plants (as well as from their various natural and synthetic derivatives), have been studied and described. Sanguinarine and ellipticine are characterized by significant biological activities including a high antitumor potential. Among the important targets of their action the following are to be noted. 1. DNA and other double helical polynucleotides. Due to the ability of DNA-intercalation sanguinarine, ellipticine and some of their derivatives can modify the double helical structures and topological forms of polynucleotides. The results of these modifications in intercalative complexes manifest themselves in the inhibition of numerous enzymatic reactions, dependent on the structures and topological forms of DNA and other polynucleotides. 2. ATP synthesis in mitochondria. Most of DNA-intercalators, including sanguinarine and ellipticine, belong to a group of penetrating (hydrophobic) cations, which are accumulated near the external side of inner mitochondrial membranes during the membrane energization. They neutralize negative charges, arising just as the inner mitochondrial membranes become energized. By this neutralization of membrane charges the ATP synthesis in inhibited and the oxidative phosphorylation renders to be uncoupled. All studied DNA-intercalators under certain conditions uncouple the mitochondrial oxidative phosphorylation. Apparent correlation between the agents' ability for DNA-intercalation and for mitochondrial ATP synthesis inhibition seems to be determined by the importance for both types of reactions of molecule hydrophobicity and positive charges. 3. Cholinesterase systems. Sanguinarine, ellipticine and some of their derivatives, like other DNA-intercalators studied, inhibit also the enzymatic activities of cholinesterase systems due to hydrophobicity and positive charges of their molecules. 4. Sanguinarine (and chelerythrine), are also capable of inhibiting the biological activity of SH-dependent enzymes and proteins. Due to the reactivity of iminium groups in sanguinarine and chelerythrine molecules with nucleophilic reagents, e.g. thiol groups of enzymes and other proteins, the activities of SH-enzymes and proteins are inhibited. In particular, sanguinarine and chelerythrine inhibit enzymatic activity of some SH-dependent ATPases, including membrane-bound cation-transport ATPases. The earlier accumulated experience of the application in medicine of plant saps and extracts containing these alkaloids, and of the treatment of many diseases (including benign and malignant tumors) by isolated alkaloids may be explained, to a certain extent, by the inhibition of activities of the above mentioned cellular targets. The selective toxicity of these alkaloids for the number of transformed cells can be explained in the same manner.

[Genotoxicity resulting from a complex mixture of environmental origin using 2 bacterial systems]. / Estudio de la genotoxicidad resultante de una mezcla compleja de origen ambiental mediante dos sistemas bacterianos.

The genotoxicity of environmental complex mixtures was tested with Ames mutagenicity assay and with Bacillus subtilis rec assay for estimation of primary DNA damage. Soil samples obtained from an area contaminated with petrochemical wastes were sequentially extracted with ethyl ether and methanol. After evaporation to dryness the extracts were resuspended in dimethylsulfoxide for their use in biological tests. No mutagenic response was detected with the Ames test. Ether extract from parcel 1 showed an unusually high toxicity for Salmonella typhimurium TA100 and induced DNA damage in Bacillus subtilis with a clear relationship dose-genotoxic response. The analysis of the results obtained in both assays and the nature of the samples indicate that the genotoxicity detected in the complex mixture is compatible with the one caused by DNA intercalating compounds.

Binding to DNA and cytotoxic evaluation of ascididemin, the major alkaloid from the Mediterranean ascidian Cystodytes dellechiajei.

The isolation of ascididemin from the Mediterranean ascidian Cystodytes dellechiajei is described. This alkaloid consists of a planar pentacyclic chromophore which was investigated for its DNA-binding and cytotoxic properties. Spectroscopic measurements provided evidence that the drug intercalates into DNA. DNase I footprinting assays indicated that the binding of ascididemin to GC-rich sequences is favoured over binding to AT-rich and mixed sequences. Chemical probes were used to detect ligand-induced structural changes in DNA. The alkaloid induces a hyper-reactivity of the DNA towards potassium permanganate, but not towards diethylpyrocarbonate, just as is the case with ethidium bromide; it has little effect on the catalytic activities of topoisomerases I and II. Ascididemin exhibits marked cytotoxicity towards human leukaemic cells in vitro and appears to be practically equally toxic for drug-sensitive and multidrug-resistant cell lines. The results suggest that DNA, but not topoisomerases, may represent the critical cellular target at which this marine alkaloid exhibits its potent cytotoxic properties in vitro.