A simple non-extractive green method for the spectrophotometric sequential injection determination of copper(ii) with novel thiazolylazo dyes.

This work introduces two new thiazolylazo dyes 1-[(5-benzyl-1,3-thiazol-2-yl)diazenyl]naphthalene-2-ol (BnTAN) and 1-[5-(4-sulfonamidobenzyl-4-methyl-1,3-thiazol-2-yl)diazenyl]naphthalen-2-ol (SABnMeTAN) as possible reagents for spectrophotometric determination of copper(ii) in water samples in the form of a chelate complex. The substances were synthesized for the first time and characterized using the NMR technique. The main characteristics of ligands, such as molar absorption coefficients and protonation constants, were evaluated spectrophotometrically. A non-extractive sequential injection spectrophotometric method for the determination of copper(ii) in the presence of Triton X-100 was developed. The linear range of the calibration plot was observed from 0.063 to 1.270 mg L-1 and the detection limit was 0.044 mg L-1. The SIA method was compared with the respective manual method and applied for the determination of copper(ii) in tap and river water.

Acridin-3,6-dialkyldithiourea hydrochlorides as new photosensitizers for photodynamic therapy of mouse leukemia cells.

Acridin-3,6-dialkyldithiourea hydrochlorides (AcrDTUs) have been evaluated as a new group of photosensitizers (PSs) for photodynamic antitumor therapy (PDT). Mouse leukemia cells L1210 were used for testing of AcrDTUs as the new PSs. The irradiation (UV-A light (365 nm), 1.05 J/cm(2)) increased cytotoxicity of all derivatives against L1210 cells more than ten times. The highest photocytotoxicity was found for propyl-AcrDTU with IC50=0.48±0.03 µM after 48 h incubation. A generation of the superoxide radical anion upon UV-A irradiation of propyl-AcrDTU was confirmed by in situ photochemical EPR experiments. To explain a mechanism of photocytotoxic action of AcrDTUs, an intracellular distribution of propyl-AcrDTU has been studied. It was found that AcrDTU in non-irradiated cells was not present in their nucleus but in the lysosomes and partly in the mitochondria, and sequestration of propyl-AcrDTU was dependent on pH in lysosomes. After irradiation, the cell death was induced by oxidative damage of lysosomal and mitochondrial membranes. Concerning the cell cycle, flow cytometry after PDT with propyl-AcrDTU showed a significant increase of the cells in the subG0 phase. Observed signs of necrosis, apoptosis, and autophagy indicate that PDT/AcrDTU leads to multiple cell death types (caspase independent apoptosis, necrosis, and autophagy).

Dipeptide interactions with Zn(II)-cyclen artificial model for molecular recognition.

The Zn(II)-cyclen-dipeptide ternary systems (where cyclen is abbreviated as L and dipeptide is glycylglycine (HL(1)) or glycyl-(S)-alanine (HL(2))) were investigated by potentiometry applying both "out-of-cell" and direct titrations and by (1) H NMR spectroscopy. Especially, the (1)H NMR study was found to be very efficient to estimate speciation in the systems. The results obtained under full equilibria indicated two main species, [Zn(L)(HL(1,2))](2+) and [Zn(L)(L(1,2))](+), in both the systems. In the [Zn(L)(HL(1,2))](2+) complex, presence of carbonyl-carboxylate chelate was confirmed, and in the [Zn(L)(L(1,2))](+) species, the peptide coordination is re-organized to carbonyl-amine chelate or only terminal amino group is coordinated. Equilibrium constants describing [Zn(L)](2+)-dipeptide interaction are relatively low, log K = 3.4 for Gly-Gly and 4.1 for Gly-(S)-Ala, respectively. Nevertheless, the values are slightly higher than stability constants for interaction of Zn(II) with the dipeptides (i.e. [Zn(L(1,2))](+) species) where a chelate formation is expected. It indicates that interaction between Zn(II) ion in [Zn(L)](2+) and the dipeptides should be supported by some additional interactions. Potentiometry carried out under non-equilibrum condition showed different species where these additional stabilizing forces play more important role.

Intracellular distribution of 3,6-bis(3-alkylguanidino)acridines determines their cytotoxicity.

UNLABELLED: Cytotoxicity of two derivatives of 3,6-bis(3-alkylguanidino)acridines (GNDAs; pentyl- and hexyl-GNDA) was determined against three cell lines: a murine immortalized fibroblast cell line NIH-3T3, a human ovarian carcinoma cell line A2780, and a human neuroblastoma cell line SH-SY5Y. We found out that these GNDAs were cytotoxic against A2780 and NIH-3T3 cells but they showed only a marginal cytotoxicity against neuroblastoma cells SH-SY5Y. To explain differences in cytotoxicity, intracellular distribution of GNDAs was monitored. GNDAs were accumulated in A2780 and NIH-3T3 cells in the nuclei (fluorescence microscopy). In contrast to these cell lines, in SH-SY5Y cells, GNDAs were localized outside of the nuclei, at the plasma membrane and surroundings, extending also to the cytosol. This distribution of GNDAs was confirmed by an ImageStream Flow Cytometer. Acetylcholinesterase (AChE) activity in the SH-SY5Y cells decreased upon incubation with GNDAs. Kinetic studies showed that GNDAs were able to inhibit AChE by the same mode as tacrine (9-amino-1,2,3,4-tetrahydroacridine), a known inhibitor of AChE. A low cytotocity of GNDAs against SH-SY5Y cells could be caused by their affinity to AChE (the enzyme is localized mainly at the plasma membrane). The interaction of GNDAs with AChE may affect their intracellular distribution and consequently the cytotoxicity. KEYWORDS: acetylcholinesterase, acridine, neuroblastoma cell line SH-SY5Y.

Novel 3,6-bis(imidazolidine)acridines as effective photosensitizers for photodynamic therapy.

The photoeffect of new proflavine derivatives with DNA-binding and antitumour activities, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was studied to evaluate them as potential photosensitizers for photodynamic antitumor therapy. EPR measurements showed that superoxide radical anion and singlet oxygen were produced upon irradiation of AcrDIMs with UV-A light (>300nm) in the presence of molecular oxygen. This indicates that AcrDIMs may act as photosensitizers. The most active pentyl-AcrDIM and hexyl-AcrDIM displayed photocytotoxic effect toward the mouse lymphocytic leukemia cell line L1210 and human ovarian cancer cells A2780. Antitumor activity of pentyl-AcrDIM increased as high as about 12 times (72h incubation) after irradiation of A2780 cells (365nm, 1.05J/cm(2)). The photocytotoxicity seems to be associated with oxidative stress. Concerning the cell cycle, flow cytometry showed an arrest in the S-phase already 4h after irradiation. In a comet assay, no genotoxicity of AcrDIMs was found. Typical morphologic changes and formation of DNA-ladders indicated induction of apoptotic cell death, though no activation of caspase-3 was observed. Investigation of intracellular localization of pentyl-AcrDIM confirmed its partial accumulation in mitochondria and lysosomes. After irradiation of the A2780 cells, colocalization of pentyl-AcrDIM with monodansylcadaverine, a lysosomal dye, was proven, suggesting that lysosomes in the irradiated cells may be involved in the cell death.

Subcellular localization of proflavine derivative and induction of oxidative stress--in vitro studies.

Acridines have been studied for several decades because of their numerous biological effects, especially anticancer activity. Recently, cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was confirmed for leukemic cell lines [Bioorg. Med. Chem.2011, 19, 1790]. The mechanism of action of the most cytotoxic hexyl-AcrDIM was studied in this paper focusing attention on a subcellular distribution of the drug. Accumulation of hexyl-AcrDIM in mitochondria was confirmed after labeling mitochondria with MitoRED using ImageStream Imaging Flow Cytometer. The derivative significantly decreased intracellular ATP level (reduction of ATP level was decreased by vitamin E), and induced oxidative stress (ROS production detected by DHE assay) as well as cell cycle arrest in the S-phase (flow cytometry analysis) already after short-time incubation and induction of apoptosis. Cytotoxicity of hexyl-AcrDIM is closely connected with induction of oxidative stress in cells.

Determination of the binding affinities of plasmid DNA using fluorescent intercalators possessing an acridine skeleton.

Novel acridine derivatives, wherein the steric factor has been varied systematically through substitution at the 9 position of the acridine ring, were evaluated as convenient fluorescent probes for nucleic acid detection. The binding affinities of N-(9-acridinylthiocarbamoyl)amino acids (ATA) with plasmid DNA (pUC 19) were investigated using UV-vis spectrophotometry, fluorometric titration and quantum chemical calculations (AM1). From spectrofluorometric analysis, the binding constants for the DNA-ATA complexes were determined. To elucidate its DNA intercalation, the most preferable tautomeric structure of ATA was established by means of AM1 calculations.

Multiplex sequencing of 1.5 Mb of the Mycobacterium leprae genome.

The nucleotide sequence of 1.5 Mb of genomic DNA from Mycobacterium leprae was determined using computer-assisted multiplex sequencing technology. This brings the 2.8-Mb M. leprae genome sequence to approximately 66% completion. The sequences, derived from 43 recombinant cosmids, contain 1046 putative protein-coding genes, 44 repetitive regions, 3 tRNAs, and 15 tRNAs. The gene density of one per 1.4 kb is slightly lower than that of Mycoplasma (1.2 kb). Of the protein coding genes, 44% have significant matches to genes with well-defined functions. Comparison of 1157 M. leprae and 1564 Mycobacterium tuberculosis proteins shows a complex mosaic of homologous genomic blocks with up to 22 adjacent proteins in conserved map order. Matches to known enzymatic, antigenic, membrane, cell wall, cell division, multidrug resistance, and virulence proteins suggest therapeutic and vaccine targets. Unusual features of the M. leprae genome include large polyketide synthase (pks) operons, inteins, and highly fragmented pseudogenes.