Microwave-Assisted Catalytic Method for a Green Synthesis of Amides Directly from Amines and Carboxylic Acids.

Amide bonds are among the most interesting and abundant molecules of life and products of the chemical pharmaceutical industry. In this work, we describe a method of the direct synthesis of amides from carboxylic acids and amines under solvent-free conditions using minute quantities of ceric ammonium nitrate (CAN) as a catalyst. The reactions are carried out in an open microwave reactor and allow the corresponding amides to be obtained in a fast and effective manner when compared to other procedures of the direct synthesis of amides from acids and amines reported so far in the literature. The amide product isolation procedure is simple, environmentally friendly, and is performed with no need for chromatographic purification of secondary amides due to high yields. In this report, primary amines were used in most examples. However, the developed procedure seems to be applicable for secondary amines as well. The methodology produces a limited amount of wastes, and a catalyst can be easily separated. This highly efficient, robust, rapid, solvent-free, and additional reagent-free method provides a major advancement in the development of an ideal green protocol for amide bond formation.

Crystal Structure of Mistletoe Lectin I (ML-I) from Viscum album in Complex with 4-N-Furfurylcytosine at 2.85 Å Resolution.

BACKGROUND: Viscum album (the European mistletoe) is a semi-parasitic plant, which is of high medical interest. It is widely found in Europe, Asia, and North America. It contains at least three distinct lectins (i.e. ML-I, II, and III), varying in molecular mass and specificity. Among them, ML-I is in focus of medical research for various activities, including anti-cancer activities. To understand the molecular basis for such medical applications, a few studies have already addressed the structural and functional analysis of ML-I in complex with ligands. In continuation of these efforts, we are reporting the crystal structure of ML from Viscum album in complex with the nucleic acid oxidation product 4-N-furfurylcytosine (FC) refined to 2.85 Å resolution. FC is known to be involved in different metabolic pathways related to oxidative stress and DNA modification. METHODS: X-ray suitable hexagonal crystals of the ML-I/FC complex were grown within four days at 294 K using the hanging drop vapor diffusion method. Diffraction data were collected up to a resolution of 2.85 Å. The ligand affinity was verified by in-silico docking. RESULTS: The high-resolution structure was refined subsequently to analyze particularly the active site conformation and a binding epitope of 4-N-furfurylcytosine. A distinct 2Fo-Fc electron density at the active site was interpreted as a single FC molecule. The specific binding of FC is achieved also through hydrophobic interactions involving Tyr76A, Tyr115A, Glu165A, and Leu157A of the ML-I A-chain. The binding energy of FC to the active site of ML-I was calculated as well to be -6.03 kcal mol-1. CONCLUSION: In comparison to other reported ML-I complexes, we observed distinct differences in the vicinity of the nucleic acid base binding site upon interaction with FC. Therefore, data obtained will provide new insights in understanding the specificity, inhibition, and cytotoxicity of the ML-I A-chain, and related RIPs.

[Origins of molecular life sciences. Polish context]. / Poczatki molekularnych nauk o zyciu - Kontekst Polski

Different scientific disciplines such as physics, genetics or biochemistry crossed over into molecular biology in the last century. The Polish state didn't existed at the beginning of XX century, but the territory for a large number of scientists was not a limitation in delineating new routes, making fundamental discoveries or training the new generation of distinguished people of sciences. We want to tell the story of roots of molecular biology from the Polish perspective and outline its importance, by bringing closer the most essential discoveries of elite scientists in different fields of life science, associated with Poland and its territory.

Selected small molecules as inducers of pluripotency.

The general idea of regenerative medicine is to fix or replace tissues or organs with live and patient-specific implants. Pluripotent stem cells are capable of indefinite self-renewal and differentiation into all cell types of the body. An easily accessible source of induced pluripotent stem cells (iPSCs) may allow obtaining and culturing tissues in vitro. Many approaches in the methods leading to obtain iPSCs have been tested in order to limit immunogenicity and tumorigenesis, and to increase efficiency. One of the approaches causing pluripotency is usage of small molecule compounds. It would be of great importance to assess their specific properties and reveal their new capacity to induce pluripotent stem cells and to improve reprogramming efficiency. Identification of the epigenetic changes during cellular reprogramming will extend our understanding of stem cell biology and many therapeutic applications. In this paper we discuss mainly the nucleotide derivatives, already proven or for now only putative inducers of the cells' pluripotency, that modulate the epigenetic status of the cell.

Non-Nucleosidic Analogues of Polyaminonucleosides and Their Influence on Thermodynamic Properties of Derived Oligonucleotides.

The rationale for the synthesis of cationic modified nucleosides is higher expected nuclease resistance and potentially better cellular uptake due to an overall reduced negative charge based on internal charge compensation. Due to the ideal distance between cationic groups, polyamines are perfect counterions for oligodeoxyribonucleotides. We have synthesized non-nucleosidic analogues built from units that carry different diol structures instead of sugar residues and functionalized with polyamines. The non-nucleosidic analogues were attached as internal or 5'-terminal modifications in oligodeoxyribonucleotide strands. The thermodynamic studies of these polyaminooligonucleotide analogues revealed stabilizing or destabilizing effects that depend on the linker or polyamine used.

Polyaminooligonucleotide: NMR structure of duplex DNA containing a nucleoside with spermine residue, N-[4,9,13-triazatridecan-1-yl]-2'-deoxycytidine.

BACKGROUND: The nature of the polyamine-DNA interactions at a molecular level is not clearly understood. METHODS: In order to shed light on the binding preferences of polyamine with nucleic acids, the NMR solution structure of the DNA duplex containing covalently bound spermine was determined. RESULTS: The structure of 4-N-[4,9,13-triazatridecan-1-yl]-2'-deoxycytidine (dCSp) modified duplex was compared to the structure of the reference duplex. Both duplexes are regular right-handed helices with all attributes of the B-DNA form. The spermine chain which is located in a major groove and points toward the 3' end of the modified strand does not perturb the DNA structure. CONCLUSION: In our study the charged polyamine alkyl chain was found to interact with the DNA surface. In the majority of converged structures we identified the presumed hydrogen bonding interactions between O6 and N7 atoms of G4 and the first internal -NH2(+)- amino group. Additional interaction was found between the second internal -NH2(+)- amino group and the oxygen atom of the phosphate of C3 residue. GENERAL SIGNIFICANCE: The knowledge of the location and nature of a structure-specific binding site for spermine in DNA should be valuable in understanding gene expression and in the design of new therapeutic drugs.

Novel method of synthesis of 5''-phosphate 2'-O-ribosyl-ribonucleosides and their 3'-phosphoramidites.

Synthesis of 5''-phosphate 2'-O-ribosylribonucleosides [Nr(p)] of four common ribonucleosides, and 3'-phosphoramidites of 5''-phosphate 2'-O-ribosyladenosine and 2'-O-ribosylguanosine using the H-phosphonate chemistry is described. An additional ring protected by benzoyl groups was incorporated into the main ribosyl ring in the reaction with 1-O-acetyl-2,3,5-tri-O-benzoyl-ß-D-ribofuranose in the presence of SnCl4. The obtained 2'-O-ribosylribonucleosides (Nr) were applied in the subsequent transformations with selective deprotection. Ethanolamine was applied as a very convenient reagent for selective removal of benzoyl groups. Additionally, the tetraisopropyldisiloxane-1,3-diyl (TIPDSi) group was found to be stable under these deprotection conditions. Thus, the selectively deprotected 5''-hydroxyl group of Nr was transformed into an H-phosphonate monoester which was found to be stable under the following conditions: the removal of the TIPDSi group with triethylammonium fluoride and the dimethoxytritylation of the 5''-hydroxyl function. The 5''-H-phosphonate of Nr precursors was easily transformed to the corresponding dicyanoethyl 5''-O-phosphotriesters before phosphitylation, which gave 3'-phosphoramidite units of Nr(p) in high yield. The derived phosphoramidite units were used in an automated oligonucleotide synthesizer to produce dimer Ar(p)T via the phosphoramidite approach. The obtained products were fully deprotected under standard deprotection conditions giving dimers with a 5''-phosphate monoester function. Application of an alkaline phosphatase to prove the presence of an additional phosphate group was described.

Convenient and efficient syntheses of N(6)- and N(4)- substituted adenines and cytosines and their 2'-deoxyribosides.

Convenient and efficient methods of the synthesis of N(6)- and N(4)-substituted derivatives of adenine and cytosine and their 2'-deoxyribosides were developed. The reactions of either unprotected nucleobases (adenine, cytosine) or unprotected 2'-deoxyribosides with aryl or alkyl aldehydes give corresponding Schiff bases that can be reduced to the target title compounds with high overall yields. In the case of aryl aldehydes the imine derivatives are obtained in the presence of methoxides in methanol and reduced with sodium borohydride. The corresponding reactions with alkyl aldehydes require the use of acetic acid and borane dimethyl sulfide complex instead.

New cytosine derivatives as inhibitors of DNA methylation.

DNA cytosine methylation catalyzed by DNA methyltransferase 1 (DNMT1) is an epigenetic method of gene expression regulation and development. Changes in methylation pattern lead to carcinogenesis. Inhibition of DNMT1 activity could be a good strategy of safe and efficient epigenetic therapy. In this work, we present a novel group of cytosine analogs as inhibitors of DNA methylation. We show new methods of synthesis and their effect on in vitro reaction of DNA methylation. Almost all of analyzed compounds inhibit DNA methyltransferase activity in the competitive manner. K(i) values for the most potent compound 4-N-furfuryl-5,6-dihydroazacytosines is 0.7 µM. These compounds cause also a decrease of 5-methylcytosine (m(5)C) level in DNA of mammalian HeLa and HEK293 cells.

Application of click chemistry to the production of DNA microarrays.

The copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was applied as the novel method of DNA immobilization on a modified solid support. The CuAAC click reaction enables the covalent binding of DNA modified with pentynyl groups at its 5'-end to azide-loaded slides. Click microarrays were produced using this approach and successfully employed in biological/model experiments.

[Cytosine methylation in DNA and its role in cancer therapy]. / Metylacja cytozyny w DNA i jej znaczenie w terapii przeciwnowotworowej.

DNA methyltransferase 1 (DNMT1) is one of three enzymes independently coded in mammalian cells. It catalyses postreplicative synthesis of 5-methylcytosine in DNA. The aim of this modification is regulation of gene expression characteristic for the given organism. DNMT1 maintains methylation pattern by copying it from maternal to daughter stand. S-adenosylo-L-methionina is a donor of methyl group in his process. Disturbance in methylation level results in changes in cell differentiation and finally to tumor transformation. Hypermethylation of promotor or first exon of tumor suppression gene results in silencing of its transcription. While hypomethylation of regulatory sequence of protooncogene and retrotransposon make them transcriptionally active. DNMT1 as a key enzyme in maintaing of proper methylation pattern is a attractive target in anti-tumor therapy.

Atmospheric structure and dynamics as the cause of ultraviolet markings in the clouds of Venus.

When seen in ultraviolet light, Venus has contrast features that arise from the non-uniform distribution of unknown absorbers within the sulphuric acid clouds and seem to trace dynamical activity in the middle atmosphere. It has long been unclear whether the global pattern arises from differences in cloud top altitude (which was earlier estimated to be 66-72 km), compositional variations or temperature contrasts. Here we report multi-wavelength imaging that reveals that the dark low latitudes are dominated by convective mixing which brings the ultraviolet absorbers up from depth. The bright and uniform mid-latitude clouds reside in the 'cold collar', an annulus of cold air characterized by approximately 30 K lower temperatures with a positive lapse rate, which suppresses vertical mixing and cuts off the supply of ultraviolet absorbers from below. In low and middle latitudes, the visible cloud top is located at a remarkably constant altitude of 72 +/- 1 km in both the ultraviolet dark and bright regions, indicating that the brightness variations result from compositional differences caused by the colder environment rather than by elevation changes. The cloud top descends to approximately 64 km in the eye of the hemispheric vortex, which appears as a depression in the upper cloud deck. The ultraviolet dark circular streaks enclose the vortex eye and are dynamically connected to it.

[Chemical biology--a new branch of science]. / Biologia chemiczna--nowa dyscyplina nauki.

Chemical biology is a new branch of science characteristic with global description of biological processes occurring within the cell. It covers synthesis of new chemical compounds for systems biology studies and search for natural small molecular weight chemical compounds with high cellular regulatory potential. Chemical biology power is based on 3 pillars as chemical synthesis, computer calculations and systems biology.

Dealing with repetitions in sequencing by hybridization.

DNA sequencing by hybridization (SBH) induces errors in the biochemical experiment. Some of them are random and disappear when the experiment is repeated. Others are systematic, involving repetitions in the probes of the target sequence. A good method for solving SBH problems must deal with both types of errors. In this work we propose a new hybrid genetic algorithm for isothermic and standard sequencing that incorporates the concept of structured combinations. The algorithm is then compared with other methods designed for handling errors that arise in standard and isothermic SBH approaches. DNA sequences used for testing are taken from GenBank. The set of instances for testing was divided into two groups. The first group consisted of sequences containing positive and negative errors in the spectrum, at a rate of up to 20%, excluding errors coming from repetitions. The second group consisted of sequences containing repeated oligonucleotides, and containing additional errors up to 5% added into the spectra. Our new method outperforms the best alternative procedures for both data sets. Moreover, the method produces solutions exhibiting extremely high degree of similarity to the target sequences in the cases without repetitions, which is an important outcome for biologists. The spectra prepared from the sequences taken from GenBank are available on our website http://bio.cs.put.poznan.pl/.

Multivalent display system on filamentous bacteriophage pVII minor coat protein.

The systems for display of foreign peptides and polypeptides on filamentous bacteriophage have exploited genetic fusion to all of the five coat proteins. Multivalent display systems allowing selection of low affinity antibody fragments have been devised for fusions to gene III. However, since pIII has to interact with the bacterial receptors during the infection process, reduced infectivity can be observed. Alternative display systems utilizing other coat protein have been examined. These, however, take advantage of phagemid systems, in which a mixture of fusion and non-fusion coat proteins becomes displayed, thus preventing multivalent display. In this paper, we describe genetically stable fusion of scFv fragments to gene VII directly in the phage genome, thus giving rise to a multivalent display system where infectivity is not comprised. A hundred-fold enrichments factor can be obtained in model selection. Our results demonstrate that the small size of pVII (33 amino acids) is not structurally compromised by fusion of scFv antibody fragments at their N-terminus, thus demonstrating the feasibility of utilizing pVII as a fusion partner.

Evidence from the Mars Express High Resolution Stereo Camera for a frozen sea close to Mars' equator.

It is thought that the Cerberus Fossae fissures on Mars were the source of both lava and water floods two to ten million years ago. Evidence for the resulting lava plains has been identified in eastern Elysium, but seas and lakes from these fissures and previous water flooding events were presumed to have evaporated and sublimed away. Here we present High Resolution Stereo Camera images from the European Space Agency Mars Express spacecraft that indicate that such lakes may still exist. We infer that the evidence is consistent with a frozen body of water, with surface pack-ice, around 5 degrees north latitude and 150 degrees east longitude in southern Elysium. The frozen lake measures about 800 x 900 km in lateral extent and may be up to 45 metres deep--similar in size and depth to the North Sea. From crater counts, we determined its age to be 5 +/- 2 million years old. If our interpretation is confirmed, this is a place that might preserve evidence of primitive life, if it has ever developed on Mars.

Tabu search algorithm for DNA sequencing by hybridization with isothermic libraries.

In this paper, a problem of isothermic DNA sequencing by hybridization (SBH) is considered. In isothermic SBH a new type of oligonucleotide libraries is used. The library consists of oligonucleotides of different lengths depending on an oligonucleotide content. It is assumed that every oligonucleotide in such a library has an equal melting temperature. Each nucleotide adds its increment to the oligonucleotide temperature and it is assumed that A and T add 2 degrees C and C and G add 4 degrees C. The hybridization experiment using isothermic libraries should provide data with a lower number of errors due to an expected similarity of melting temperatures. From the computational point of view the problem of isothermic DNA sequencing with errors is hard, similarly like its classical counterpart. Hence, there is a need for developing heuristic algorithms that construct good suboptimal solutions. The aim of the paper is to propose a heuristic algorithm based on tabu search approach. The algorithm solves the problem with both positive and negative errors. Results of an extensive computational experiment are presented, which prove the high quality of the proposed method.

"Action-at-a distance" of a new DNA oxidative damage product 6-furfuryl-adenine (kinetin) on template properties of modified DNA.

N(6)-furfuryladenine (kinetin, K) was shown to have cytokinin activity and antiageing effects. It also appears to protect DNA against oxidative damage mediated by the Fenton reaction. Kinetin was identified as a natural component of DNA in plant extract, calf thymus DNA, fresh DNA preparations from human cell culture, as well as in human urine. A proposed mechanism of kinetin synthesis includes furfural, the oxidative damage product of a 2-deoxyribose moiety of DNA, which reacts with an adenine residue to form N(6)-furfuryladenine at DNA level. The identification of kinetin in plant cell extracts, as well as human urine, suggests its excision from DNA by repair mechanisms. Since such a bulky modification as kinetin induces conformational changes of DNA, this could lead to mutations. Therefore, it was interesting to analyze an effect of kinetin on coding properties of DNA. Chemically synthesized oligodeoxynucleotide (20-mer) containing kinetin AAAACTGCCGTCCTGAKGAT was used as a primer. It was elongated in a polymerase chain reaction (PCR) on a template plasmid pEW1 harboring a 210-bp fragment of DNA derived from the 5' end of HIV mRNA. The PCR product of that length containing kinetin in position 17 from the 5' end was isolated and sequenced. Interestingly, DNA polymerase correctly incorporates thymine opposite of kinetin (an adenine derivative) on the complementary strand, but the misincorporations occur in a vicinity of the modified base.