Photophysics of Benzoxazole and Dicyano Functionalised Diketopyrrolopyrrole Derivatives: Insights into Ultrafast Processes and the Triplet State.

Diketopyrrolopyrrole (DPP) functionalised with an electron donating unit acts as a donor-acceptor molecules that have shown potential for application in dyes and photovoltaics. These molecules offer broad absorption/emission properties and structure-dependent dynamics. In this study, we used femtosecond pump-probe spectroscopy to investigate the photo-initiated dynamics of thiophene linked DPP derivatives. The thio-DPPs are further functionalised by different electrons withdrawing terminal groups, namely benzoxazole and thiophene dicyanide. The benzoxazole derivative is strongly emissive and directly relaxes directly to the ground state chloroform solution. Thiophene dicyanide derivative exhibits distinct spectral evolution in the first 10 ps, associated with structural and vibronic process. Later, it crosses over to the triplet state with a yield of 20 %. In the solid-state (thin film), we observed a signal that resembles singlet fission. However, upon careful analysis of temperature-dependent steady state absorbance spectra, we conclude that these features are due to laser-induced thermal artifacts. We describe a simplified excited state evolution in the thin film that does not include any additional excited states. These findings have significant implications for the analysis of triplet formation, which plays a major role in the photophysics of many organic materials.

Preparation and characterization of novel double-decker rare-earth phthalocyanines substituted with 5-bromo-2-thienyl groups.

BACKGROUND: A series of rare-earth bisphthalocyanines of praseodymium, samarium and gadolinium bearing 5-bromo-2-thienyl substituents were prepared for the first time. RESULTS: Three bis[octakis(5-bromo-2-thienyl)] rare-earth metal(III) bisphthalocyanine complexes (Pr, Sm, Gd) were synthesized for the first time. The new compounds were characterized by UV-vis, NIR, FT-IR, mass spectroscopy and thermogravimetry as well as elementary analysis and electrochemistry. Production of singlet oxygen was also estimated using 9,10-dimethylanthracene method. CONCLUSIONS: The bromine substituent causes significant changes in molecule paramagnetism, singlet oxygen production, HOMO position and spectral characteristics. The compounds in solutions exist in two forms (neutral and/or reduced) depending on the solvent and rare-earth metal. Moreover, the compounds exhibit much increased stability under acid conditions compared with non-brominated derivatives. Graphical abstract Prepared compounds for the study of their chemical and other properties.

Enzyme-linked electrochemical DNA ligation assay using magnetic beads.

DNA ligases are essential enzymes in all cells and have been proposed as targets for novel antibiotics. Efficient DNA ligase activity assays are thus required for applications in biomedical research. Here we present an enzyme-linked electrochemical assay based on two terminally tagged probes forming a nicked junction upon hybridization with a template DNA. Nicked DNA bearing a 5' biotin tag is immobilized on the surface of streptavidin-coated magnetic beads, and ligated product is detected via a 3' digoxigenin tag recognized by monoclonal antibody-alkaline phosphatase conjugate. Enzymatic conversion of napht-1-yl phosphate to napht-1-ol enables sensitive detection of the voltammetric signal on a pyrolytic graphite electrode. The technique was tested under optimal conditions and various situations limiting or precluding the ligation reaction (such as DNA substrates lacking 5'-phosphate or containing a base mismatch at the nick junction, or application of incompatible cofactor), and utilized for the analysis of the nick-joining activity of a range of recombinant Escherichia coli DNA ligase constructs. The novel technique provides a fast, versatile, specific, and sensitive electrochemical assay of DNA ligase activity.

The use of trigonometry in bloodstain analysis.

Bloodstain pattern analysis (BPA) is a valid forensic method which belongs to the category of biological methods using trigonomic models. Despite its development through the years, the method has been re-formulated a standard one and globally used, recognized in standard sheets. This method permits exact analysis of the dynamic and characteristic properties of bloodstains after impact on surfaces such as floors, walls, and ceilings, various exterior and interior items, and clothes. It is also possible to determine the characteristics of blood from the outer part of the body. According to the presence of blood and its quantity, it is also possible to use this method for verification of reconstruction of criminal acts, while being tested for its validity with primary conditions of preserved and readable traces of blood. Even though this method is not considered as the major one or the only one information obtained in this way can be used for judicial. In our research, we tested the validity of this method in an experimental model using firearms. We compared measurements of the lengths of trajectory of impact and the height of the blood sprayed upwards from a distance of 1, 3, 5 and 10 meters. The experiment was based on two main presumptions. The first was the knowledge of the value of the distance and the angle of impact of the bloodstain, the second, the ability of the blood to reach a certain height and the angle of its impact. In accordance with trigonometric formulas, both the impact of the selected distance of drops of blood, and the height of the selected bloodstain could be determined without any verification of the flight trajectory and the distance of bloodstains. The results indicate that the method for these requirements differs from the real values, while increasing the measurements with the indicated spot of the shot. Aside from the unique values which were calculated, other results of the impact of the distance of drops of bloodstain were considered of lower value, and the values concerning the height of the bloods stains after the shot higher than real values. In spite of the lack of total accuracy, we recommend using this method widely and more often for investigation and verification of individual acts in criminal and forensic practice.

Extracellular matrix rigidity controls podosome induction in microvascular endothelial cells.

BACKGROUND INFORMATION: Podosomes are actin-based structures involved in cell adhesion, migration, invasion and extracellular matrix degradation. They have been described in large vessel endothelial cells, but nothing is known concerning microvascular endothelial cells. Here, we focussed on liver sinusoidal endothelial cells (LSECs), fenestrated microvascular cells that play major roles in liver physiology. Liver fibrosis induces a dedifferentiation of LSECs leading notably to a loss of fenestrae. Because liver fibrosis is associated with increased matrix stiffness, and because substrate stiffness is known to regulate the actin cytoskeleton, we investigated the impact of matrix rigidity on podosome structures in LSECs. RESULTS: Using primary LSECs, we demonstrated that microvascular endothelial cells are able to form constitutive podosomes. Podosome presence in LSECs was independent of cytokines such as transforming growth factor-ß or vascular endothelial growth factor, but could be modulated by matrix stiffness. As expected, LSECs lost their differentiated phenotype during cell culture, which was paralleled by a loss of podosomes. LSECs however retained the capacity to form active podosomes following detachment/reseeding or actin-destabilising drug treatments. Finally, constitutive podosomes were also found in primary microvascular endothelial cells from other organs. CONCLUSIONS: Our results show that microvascular endothelial cells are able to form podosomes without specific stimulation. Our data suggest that the major determinant of podosome induction in these cells is substrate rigidity.

Anthraquinone as a redox label for DNA: synthesis, enzymatic incorporation, and electrochemistry of anthraquinone-modified nucleosides, nucleotides, and DNA.

Modified 2'-deoxynucleosides and deoxynucleoside triphosphates (dNTPs) bearing anthraquinone (AQ) attached through an acetylene or propargylcarbamoyl linker at the 5-position of pyrimidine (C) or at the 7-position of 7-deazaadenine were prepared by Sonogashira cross-coupling of halogenated dNTPs with 2-ethynylanthraquinone or 2-(2-propynylcarbamoyl)anthraquinone. Polymerase incorporations of the AQ-labeled dNTPs into DNA by primer extension with KOD XL polymerase have been successfully developed. The electrochemical properties of the AQ-labeled nucleosides, nucleotides, and DNA were studied by cyclic and square-wave voltammetry, which show a distinct reversible couple of peaks around -0.4 V that make the AQ a suitable redox label for DNA.

Alkylsulfanylphenyl derivatives of cytosine and 7-deazaadenine nucleosides, nucleotides and nucleoside triphosphates: synthesis, polymerase incorporation to DNA and electrochemical study.

Aqueous Suzuki-Miyaura cross-coupling reactions of halogenated nucleosides, nucleotides and nucleoside triphosphates derived from 5-iodocytosine and 7-iodo-7-deazaadenine with methyl-, benzyl- and tritylsufanylphenylboronic acids gave the corresponding alkylsulfanylphenyl derivatives of nucleosides and nucleotides. The modified nucleoside triphosphates were incorporated into DNA by primer extension by using Vent(exo-) polymerase. The electrochemical behaviour of the alkylsulfanylphenyl nucleosides indicated formation of compact layers on the electrode. Modified nucleotides and DNA with incorporated benzyl- or tritylsulfanylphenyl moieties produced signals in [Co(NH(3))(6)](3+) ammonium buffer, attributed to the Brdicka catalytic response, depending on the negative potential applied. Repeated constant current chronopotentiometric scans in this medium showed increased Brdicka catalytic response, which suggests the deprotection of the alkylsulfanyl derivatives to free thiols under the conditions.

Tail-labelling of DNA probes using modified deoxynucleotide triphosphates and terminal deoxynucleotidyl transferase. Application in electrochemical DNA hybridization and protein-DNA binding assays.

A simple approach to DNA tail-labelling using terminal deoxynucleotidyl transferase and modified deoxynucleoside triphosphates is presented. Amino- and nitrophenyl-modified dNTPs were found to be good substrates for this enzyme giving 3'-end stretches of different lengths depending on the nucleotide and concentration. 3-Nitrophenyl-7-deazaG was selected as the most useful label because its dNTP was efficiently incorporated by the transferase to form long tail-labels at any oligonucleotide. Accumulation of many nitrophenyl tags per oligonucleotide resulted in a considerable enhancement of voltammetric signals due to the nitro group reduction, thus improving the sensitivity of electrochemical detection of the tail-labelled probes. We demonstrate a perfect discrimination between complementary and non-complementary target DNAs sequences by tail-labelled hybridization probes as well as the ability of tumour suppressor p53 protein to recognize a specific binding site within tail-labelled DNA substrates, making the methodology useful in electrochemical DNA hybridization and DNA-protein interaction assays.

Direct voltammetric analysis of DNA modified with enzymatically incorporated 7-deazapurines.

Nucleic acids studies use 7-deazaguanine (G*) and 7-deazaadenine (A*) as analogues of natural purine bases incapable of forming Hoogsteen base pairs, which prevents them from being involved in DNA triplexes and tetraplexes. Reduced propensity of the G*- and/or A*-modified DNA to form alternative DNA structures is utilized, for example, in PCR amplification of guanine-rich sequences. Both G* and A* exhibit significantly lower potentials of their oxidation, compared to the respective natural nucleobases. At carbon electrodes, A* yields an oxidation peak which is by about 200-250 mV less positive than the peak due to adenine, but coincides with oxidation peak produced by natural guanine residues. On the other hand, oxidation signal of G* occurs at a potential by about 300 mV less positive than the peak due to guanine, being well separated from electrochemical signals of any natural DNA component. We show that enzymatic incorporation of G* and A* can easily be monitored by simple ex situ voltammetric analysis of the modified DNA at carbon electrodes. Particularly G* is shown as an attractive electroactive marker for DNA, efficiently incorporable by PCR. While densely G*-modified DNA fragments exhibit strong quenching of fluorescence of SYBR dyes, commonly used as fluorescent indicators in both gel staining and real time PCR applications, the electrochemical detection provides G*-specific signal suitable for the quantitation of the amplified DNA as well as for the determination of the DNA modification extent. Determination of DNA amplicons based on the measurement of peak G*(ox) is not affected by signals produced by residual oligonucleotide primers or primary templates containing natural purines.

Determination of the level of DNA modification with cisplatin by catalytic hydrogen evolution at mercury-based electrodes.

Electrochemical methods proved useful as simple and inexpensive tools for the analysis of natural as well as chemically modified nucleic acids. In particular, covalently attached metal-containing groups usually render the DNA well-pronounced electrochemical activity related to redox processes of the metal moieties, which can in some cases be coupled to catalytic hydrogen evolution at mercury or some types of amalgam electrodes. In this paper we used voltammetry at the mercury-based electrodes for the monitoring of DNA modification with cis-diamminedichloroplatinum (cisplatin), a representative of metallodrugs used in the treatment of various types of cancer or being developed for such purpose. In cyclic voltammetry at the mercury electrode, the cisplatin-modified DNA yielded catalytic currents the intensity of which reflected DNA modification extent. In square-wave voltammetry, during anodic polarization after prereduction of the cisplatinated DNA, a well-developed, symmetrical signal (peak P) was obtained. Intensity of the peak P linearly responded to the extent of DNA modification at levels relevant for biochemical studies (rb = 0.01-0.10, where rb is the number of platinum atoms bound per DNA nucleotide). We demonstrate a correlation between the peak P intensity and a loss of sequence-specific DNA binding by tumor suppressor protein p53, as well as blockage of DNA digestion by a restriction endonuclease Msp I (both caused by the DNA cisplatination). Application of the electrochemical technique in studies of DNA reactivity with various anticancer platinum compounds, as well as for an easy determination of the extent of DNA platination in studies of its biochemical effects, is discussed.

Molecular modeling of surface modification of Wyoming and Cheto montmorillonite by methylene blue.

The surface area of various types of montmorillonites (MMT) with different values of layer charge plays a very important role in surface arrangement of methylene blue cations (MB). Photoluminescence measurements can be strongly or partially influenced by this surface arrangement of cations. For these reasons and on the basis of our previous results, molecular simulations were performed for various types of montmorillonites covered with methylene blue cations. Adsorption of methylene blue cations on Na-Wyoming MMT surface is different from Ca-Cheto MMT. In the case of Wyoming with a lower layer charge, MB cations lie parallel to the silicate layer for all investigated samples. On the other hand, Cheto surface is covered with a higher amount of MB cations. The results obtained from molecular modeling indicate that MB lies parallel to low loading case and become tilted with respect to layer for a higher loading. Moreover, a higher amount of MB cations covering the silicate layer are much less energy-stable. A higher loading of MB cations leads to aggregates but at low loading MB cations degrade to monomers.

Base-modified DNA labeled by [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes: construction by polymerase incorporation of modified nucleoside triphosphates, electrochemical and luminescent properties, and applications.

Modified 2'-deoxynucleoside triphosphates (dNTPs) bearing [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) complexes attached via an acetylene linker to the 5-position of pyrimidines (C and U) or to the 7-position of 7-deazapurines (7-deaza-A and 7-deaza-G) have been prepared in one step by aqueous cross-couplings of halogenated dNTPs with the corresponding terminal acetylenes. Polymerase incorporation by primer extension using Vent (exo-) or Pwo polymerases gave DNA labeled in specific positions with Ru(2+) or Os(2+) complexes. Square-wave voltammetry could be efficiently used to detect these labeled nucleic acids by reversible oxidations of Ru(2+/3+) or Os(2+/3+). The redox potentials of the Ru(2+) complexes (1.1-1.25 V) are very close to that of G oxidation (1.1 V), while the potentials of Os(2+) complexes (0.75 V) are sufficiently different to enable their independent detection. On the other hand, Ru(2+)-labeled DNA can be independently analyzed by luminescence. In combination with previously reported dNTPs bearing ferrocene, aminophenyl, and nitrophenyl tags, the Os-labeled dATP has been successfully used for "multicolor" redox labeling of DNA and for DNA minisequencing.

Novel base-functionalized DNA. Efficient methodology for construction and bioanalytical applications.

A novel efficient two-step methodology for the construction of base-functionalized DNA is based on direct aqueous cross-coupling reactions of unprotected nucleoside triphosphates followed by polymerase incorporation. Preliminary applications of the modified DNA in electrochemical detection and bioanalysis are outlined.

Osmium tetroxide, 2,2'-bipyridine: electroactive marker for probing accessibility of tryptophan residues in proteins.

A complex of osmium tetroxide with 2,2'-bipyridine (Os,bipy) has been applied as a chemical probe of DNA structure as well as an electroactive DNA label. The Os,bipy has been known to form covalent adducts with pyrimidine DNA bases. Besides the pyrimidines, electrochemically active covalent adducts with Os,bipy are formed also by tryptophan (W) residues in peptides and proteins. In this paper we show that Os,bipy-treated proteins possessing W residues (such as avidin, streptavidin, or lysozyme) yield at the pyrolytic graphite electrode (PGE) a specific signal (peak alphaW) the potential of which differs from the potentials of signals produced by free Os,bipy or by Os,bipy-modified DNA. No such signal is observed with proteins lacking W (such as ribonuclease A or alpha-synuclein). Subpicomole amounts of W-containing proteins modified with Os,bipy can easily be detected using adsorptive transfer stripping voltammetry with the PGE. Binding of biotin to avidin interferes with Os,bipy modification of the protein, in agreement with the location of W residues within the biotin-binding site of avidin. These Ws are accessible for modification in the absence of biotin but hidden (protected from modification) in the avidin-biotin complex. The Os,bipy-modified avidin is unable to bind biotin, and its quarternary structure is disrupted. Analogous effects were observed with another biotin-binding protein, streptavidin. Our results demonstrate that modification of proteins with Os,bipy under conditions close to physiological, followed by a simple electrochemical analysis, can be applied in the microanalysis of protein structure and interactions.