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Covalent organic frameworks (COFs) have been used in cell imaging, but very rarely for imaging specific cell conditions. Herein, a β-ketoenamine-based fluorescent COF was post-synthetically modified to incorporate a hypoxia-targeting molecule. Fluorescence microscopy imaging shows that the material discriminates between HeLa cells grown under hypoxia and those cultured under normoxia.

Biocompatible polysaccharide scaffolds with controllable pore size, good mechanical properties and without hazardous chemical crosslinkers, are desirable for long-term tissue engineering applications but their production is highly challenging. Herein, we fabricated three-dimensional (3D) scaffolds using a polysaccharide composite ink composed of nanofibrillated cellulose, carboxymethyl cellulose and citric acid, featuring strong shear thinning behavior and adequate printability. Highly porous and mechanically stable scaffolds were produced by combining direct ink writing 3D printing, freeze-drying and dehydrothermal heat-assisted crosslinking techniques. The last heat-assisted step induces reaction of citric acid, which was chosen as it is a non-hazardous and green crosslinker. Degree of crosslinking was controlled by varying the concentration of citric acid (2.5 – 10 wt.%) to tune the chemical, surface, swelling and degradation properties of the scaffolds in the dry and hydrated states. The scaffolds with the highest porosity (86%) and interconnected pores (100-450 µm) were acquired using the lowest citric acid concentration; and the porosity was significantly reduced at higher citric acid concentration as well as at longer hydration time. The compressive strength, elastic modulus and the shape recovery behavior of the crosslinked scaffolds were increased significantly with increasing crosslinker concentration. The prepared crosslinked scaffolds promoted clustered cell adhesion and showed no cytotoxic effects, as determined by a cell viability assay and live/dead staining with human bone tissue derived osteoblast cells. The water-based and non-hazardous crosslinking method reported here can be extended to all polysaccharide-based materials to develop cell-friendly scaffolds with tailormade properties suitable for various tissue engineering applications in general.

Succinylation of proteins is a commonly encountered reaction in biology and introduces negatively charged carboxylates on previously basic primary amine groups of amino acid residues. In analogy, this work investigates the succinylation of primary amines of the synthetic polyelectrolyte polyallylamine (PAA). It investigates the influence of the degree of succinylation on the cytotoxicity and antibacterial activity of the resulting polymers. Succinylation was performed in water with varying amounts of succinic anhydride and at different pH values. The PAA derivatives were analyzed in detail with respect to molecular structure using nuclear magnetic resonance and infrared absorbance spectroscopy. Polyelectrolyte and potentiometric charge titrations were used to elucidate charge ratios between primary amines and carboxylates in the polymers. The obtained materials were then evaluated with respect to their minimum inhibitory concentration againstStaphylococcus aureusandPseudomonas aeruginosa. The biocompatibility was assessed using mouse L929 fibroblasts. The degree of succinylation decreased cytotoxicity but more significantly reduced antibacterial efficacy, demonstrating the sensitivity of the fibroblast cells against this type of ampholytic polyelectrolytes. The obtained polymers were finally electrospun into microfiber webs in combination with neutral water-soluble polyvinyl alcohol. The resulting non-woven could have the potential to be used as wound dressing materials or coatings.

Funding Information: The authors acknowledge the financial support for this study received from the Slovenian Research Agency (G. no.: P2-0118 and J4-1764) and the Austrian Research Promotion Agency (FFG no. 846065). They also acknowledge Dr. Silvo Hribernik, Dr. Matej Bračič, Dr. Irena Ban, and Sabina Markuš (University of Maribor, Slovenia) for their support regarding the potentiometric charge titration, scanning electron microscopy, and thermogravimetic analysis, as well as Prof. Dr. Cornelia Kasper (University of Natural Resources and Life Sciences, Austria) for her support regarding the biocompatibility testing. Dr. Brigitte Bitschnau from Graz University of Technology, Austria, is also acknowledged for her support regarding XRD measurements. Publisher Copyright: © 2021 The Authors. Published by American Chemical Society. As one of the most abundant, multifunctional biological polymers, polysaccharides are considered promising materials to prepare tissue engineering scaffolds. When properly designed, wetted porous scaffolds can have biomechanics similar to living tissue and provide suitable fluid transport, both of which are key features for in vitro and in vivo tissue growth. They can further mimic the components and function of glycosaminoglycans found in the extracellular matrix of tissues. In this study, we investigate scaffolds formed by charge complexation between anionic carboxymethyl cellulose and cationic protonated chitosan under well-controlled conditions. Freeze-drying and dehydrothermal heat treatment were then used to obtain porous materials with exceptional, unprecendent mechanical properties and dimensional long-term stability in cell growth media. We investigated how complexation conditions, charge ratio, and heat treatment significantly influence the resulting fluid uptake and biomechanics. Surprisingly, materials with high compressive strength, high elastic modulus, and significant shape recovery are obtained under certain conditions. We address this mostly to a balanced charge ratio and the formation of covalent amide bonds between the polymers without the use of additional cross-linkers. The scaffolds promoted clustered cell adhesion and showed no cytotoxic effects as assessed by cell viability assay and live/dead staining with human adipose tissue-derived mesenchymal stem cells. We suggest that similar scaffolds or biomaterials comprising other polysaccharides have a large potential for cartilage tissue engineering and that elucidating the reason for the observed peculiar biomechanics can stimulate further research.

We report here a one-step aqueous method for the synthesis of isolated and purified polysaccharide-amino acid conjugates. Two different types of amino acid esters: glycine methyl ester and L-tryptophan methyl ester, as model compounds for peptides, were conjugated to the polysaccharide carboxymethylcellulose (CMC) in water using carbodiimide at ambient conditions. Detailed and systematic pH-dependent charge titration and spectroscopy (infrared, nuclear magnetic resonance: 1H, 13C- DEPT 135, 1H- 13C HMBC/HSQC correlation), UV–vis, elemental and ninhydrin analysis provided solid and direct evidence for the successful conjugation of the amino acid esters to the CMC backbone via an amide bond. As the concentration of amino acid esters increased, a conjugation efficiency of 20–80% was achieved. Activated charcoal aided base-catalyzed deprotection of the methyl esters improved the solubility of the conjugates in water. The approach proposed in this work should have the potential to tailor the backbone of polysaccharides containing di- or tri-peptides.

d-cycloserine is a broad-spectrum antibiotic that is currently being used as a secondary choice in the treatment of tuberculosis. In recent years, it has become more popular, due to its effect on the nervous system. In this current study, we provide evidence that The International Pharmacopoeia HPLC&ndash, UV method for d-cycloserine impurity profiling is not repeatable due to the variable response of cycloserine dimer, one of d-cycloserine impurities. Therefore, we introduced the DOSY (diffusion ordered spectroscopy) NMR (nuclear magnetic resonance) technique to determine the levels of d-cycloserine impurities in pharmaceutical dosage forms. The DOSY NMR technique allowed separation of d-cycloserine, its degradation products, and key process impurities in concentrations below pharmacopoeial specification limits. The proposed DOSY NMR method allowed accurate identification and quantification of the cycloserine dimer, which was not possible through the use of the pharmacopoeial HPLC method. The current method has the potential for practical use in analytical laboratories of the pharmaceutical industry.

Three-dimensional scaffolds (3D) with controlled shape, dual porosity and long-term mechanical and dimensional stability in biofluids are of interest as biotemplates in tissue engineering. Herein, self-standing and lightweight cellulose-based biogenic scaffolds with a spatially structured morphology, macropores and interconnected micropores were fabricated using a combination of direct ink writing 3D printing and freeze-drying techniques. This was achieved by developing a water-based and low-cost bicomponent ink based on commercially available nanofibrillated cellulose (NFC) and carboxymethyl cellulose (CMC). Physical cross-linking through dehydrothermal treatment significantly increased the surface hardness, indentation modulus, compression strength, as well as the dimensional stability of the scaffolds in biofluids, in comparison to untreated materials. However, no differences in the spectra of solid state nuclear magnetic resonance or infrared were observed for dehydrothermal treated samples, suggesting that the increase of mechanical properties and dimensional stability is based on the physical cross-linking of functional groups both at the interface between NFC and CMC. The supramolecular structure of the polymers was well-preserved as disclosed by X-ray diffraction measurements. The cross-linked scaffolds showed high proliferation, viability, and attachment of human bone tissue derived osteoblast cells (hFOB). The simple and straightforward avenue proposed here for the design of cellulose-based fibrous inks and dual porous scaffolds from the commercially available materials and without the need of any additional cross-linkers should pave the way for the development of implantable, degradable scaffolds and cell-laden biomaterials for bone tissue regeneration and 3D bioprinting applications.

During sample preparation and analysis, samples are coming in contact with different labware materials. By four unrelated analytical (phytochemical and pharmaceutical) case-studies and employing different analytical techniques, we demonstrated the potential misinterpretation of analytical results due to the use of contaminants-leaching labware during sample handling. Oleamide, a common polymer lubricant and a bioactive compound, was identified as a main analytical interference, leaching from different labware items into solvents, recognised as chemically compatible with the tested polymer material. Moreover, anti-inflammatory effect of oleamide at 100 μg mL−1 and considerable pro-inflammatory effect of the plastic syringe extractables (containing oleamide) at the same level were shown in a TLR4-based bioassay. Taking these results into account, together with the fact that oleamide can be a compound of natural origin, we would like to notify the professional public regarding the possible erroneous oleamide-related analytical and bioassay results due to the use of oleamide-leaching labware. Researchers are alerted to double check the real source of oleamide (labware or natural extract), which will prevent further reporting of false results. Analysis of procedural blanks with de-novo developed UHPLC-ESI-MS method is, among some other strategies, proposed for detection of oleamide interference and avoidance of misleading results of certain analyses.

In this study, nuclear magnetic resonance (1H NMR) spectroscopic profiling was used to provide a more comprehensive view of microbial metabolites associated with poor reactor performance in a full-scale 4 MW mesophilic agricultural biogas plant under fully operational and also under inhibited conditions. Multivariate analyses were used to assess the significance of differences between reactors whereas artificial neural networks (ANN) were used to identify the key metabolites responsible for inhibition and their network of interaction. Based on the results of nm-MDS ordination the subsamples of each reactor were similar, but not identical, despite homogenization of the full-scale reactors before sampling. Hence, a certain extent of variability due to the size of the system under analysis was transferred into metabolome analysis. Multivariate analysis showed that fully active reactors were clustered separately from those containing inhibited reactor metabolites and were significantly different. Furthermore, the three distinct inhibited states were significantly different from each other. The inhibited metabolomes were enriched in acetate, caprylate, trimethylamine, thymine, pyruvate, alanine, xanthine and succinate. The differences in the metabolic fingerprint between inactive and fully active reactors observed in this study resembled closely the metabolites differentiating the (sub) acute rumen acidosis inflicted and healthy rumen metabolomes, creating thus favorable conditions for the growth and activity of pathogenic bacteria. The consistency of our data with those reported before for rumen ecosystems shows that 1H NMR based metabolomics is a reliable approach for the evaluation of metabolic events at full-scale biogas reactors.

High-performance thin-layer chromatography (HPTLC) is a powerful separation technique which is often overlooked. In this study, we comprehensively assessed the applicability, ease, and performance of HPTLC in combination with densitometry and mass spectrometry (MS) to characterize physalins — relatively polar secondary metabolites from Physalis alkekengi L. HPTLC silica gel plates were evaluated in combination with 14 developing solvents (13 published in the literature). Bonded stationary phases (HPTLC RP-18, RP-18 W, CN F254S) were also tested. Four detection reagents (sulfuric acid, anisaldehyde, 4-dimethylaminocinnamaldehyde (DMACA), and molybdatophosphoric acid) were compared to ascertain which one is the most suitable. For all chromatographic analyses, a commercial standard physalin L and a P. alkekengi L. crude extract were used. In some cases, physalin L standard appeared as two clearly resolved bands on silica plates, but only after derivatization, where sulfuric acid reagent provided the best sel...

Water-soluble azobenzene derivatives containing amino[bis(ethanesulfonate)] groups are demonstrated as colorful pH indicators in water and on filter paper. Vibrant color changes were observed from yellow/orange to pink between pH 1 and 4, which are attributed to an intramolecular charge-transfer mechanism. The pKas of the indicators range from 2.1 to 2.6. 1H/1H–15N NMR studies in deuterium oxide reveal that the protonation of the azobenzene pH indicators occurs predominantly at the β-azo nitrogen atom, in agreement with the density functional theory calculations. Excellent selectivity for protons was confirmed in water over common biologically relevant metal ions. Studies in methanol, however, indicate that the pH indicator with a methoxy group ortho to the amino[bis(ethanesulfonate)] group facilitates the selective coordination of Cu2+ with a binding constant pβCu2+ of 4.6 ± 0.1. The indicators complement the existing library of azobenzene indicator dyes and may be useful for measuring the environmental pH at higher proton concentrations., peer-reviewed

Due to their ability to inhibit viral DNA or RNA replication, nucleoside analogues have been used for decades as potent antiviral therapeutics. However, one of the major limitations of nucleoside analogues is the development of antiviral resistance. In that regard, flexible nucleoside analogues known as &ldquo, fleximers&rdquo, have garnered attention over the years due to their ability to survey different amino acids in enzyme binding sites, thus overcoming the potential development of antiviral resistance. Acyclic fleximers have previously demonstrated antiviral activity against numerous viruses including Middle East Respiratory Syndrome coronavirus (MERS-CoV), Ebola virus (EBOV), and, most recently, flaviviruses such as Dengue (DENV) and Yellow Fever Virus (YFV). Due to these interesting results, a Structure Activity Relationship (SAR) study was pursued in order to analyze the effect of the pyrimidine functional group and acyl protecting group on antiviral activity, cytotoxicity, and conformation. The results of those studies are presented herein.

The macrocycles 3,7-bis(3-nitrophenyl)-1,5,3,7-dioxadiazocane 1 and 3,7-bis(4-nitrophenyl)-1,5,3,7-dioxadiazocane 2 were synthesised by a one-pot reaction with substituted nitroanilines and formaldehyde under acidic conditions. The same reaction with 2-nitroaniline yielded N, N’-(oxybis(methylene))bis(2-nitroaniline) 3 rather than 3,7-bis(2-nitrophenyl)-1,5,3,7-dioxadiazocane 4. The yellow powders 1–3 were characterised by 1H/13C/15N NMR, FTIR and HRMS. The photophysical properties were studied by UV-visible absorption and steady-state fluorescence spectroscopy. Acid dissociation constants were determined by UV-visible absorption titrations in 1:1 (v/v) acetonitrile/water to be 0.80 and 3.1 for the meta- and para-substituted compounds 1 and 2, respectively. Compounds 1 and 2 were discovered to fluoresce in the solid-state, yellow and green, respectively, but not in solution. Density functional theory (DFT) calculations for 1–4 provide insight into the frontier molecular orbital energy levels. These compounds represent a new class of fluorescent heterocyclic building blocks for organic and supramolecular applications.

A methodology based on off-line multidimensional thin-layer chromatography was developed for isolation of several secondary metabolites from bark of Japanese knotweed (Fallopia japonica Houtt.) rhizomes. Successive fractionation steps using PLC silica gel and HPTLC silica gel or HPTLC cellulose plates in combination with various developing solvents enabled isolation of (+)-catechin, (-)-epicatechin, (-)-epicatechin gallate, procyanidin B1, procyanidin B2, procyanidin B3, proanthocyanidin B dimer gallate, emodin, emodin-8-O-glucoside and emodin-8-O-malonyl-glucoside. Their identity was confirmed by HPTLC, HPTLC-MSn and for most of them also by 1H NMR and 2D NMR analyses. To the best of our knowledge emodin-8-O-malonyl-glucoside, procyanidins B1 and B2 were for the first time isolated from this plant material. HPTLC and HPTLC-MSn analyses were also performed as support of fractionation/isolation process, leading to first detection of some compounds in bark of Japanese knotweed rhizomes and Japanese knotweed rhizomes in general: procyanidins B1 and B2, methyl derivatives of emodin bianthrone and emodin bianthrone-hexose, resveratrol-malonyl-hexoside and taxifolin derivatives. Characterization of flavan-3-ols and proanthocyanidins was facilitated by post-chromatographic derivatization of the corresponding chromatographic zones with 4-dimethylaminocinnamaldehyde (DMACA) detection reagent.

Conformational features of pyridine- and pyrimidine-based bistriazolyl anion receptors dissolved in acetonitrile-d3 were assessed by multidimensional, heteronuclear NMR spectroscopy. NOESY correlation signals suggested preorganization of both host molecules in solution in the absence of anions. In addition, only a single set of signals was observed in the 1H NMR spectra, which suggested a symmetrical conformation of anion receptors or their conformational exchange that is fast on the NMR time-scale. Furthermore, the predominant conformations of the pyridine- and pyrimidine-based anion receptors are preserved upon addition of chloride, bromide, and acetate anions. Chemical shift changes observed upon addition of anions showed that the NH (thio)urea and triazole protons are involved in anion-receptor interactions through hydrogen bonding.

A series of thiosemicarbazide anion chemosensors substituted with naphthalene and 4-nitrophenyl or phenyl units were synthesized. The molecules were characterized by using 1H,13C DEPT and 15N NMR spectroscopy. The anion binding properties of compounds 1–4 were investigated by UV/Vis absorption and fluorescence spectroscopy in DMSO, DMSO/H2O (9:1 v/v), and acetonitrile with hydroxide, fluoride, acetate, hypophosphate, and chloride anions. Striking color changes were observed with nitro-containing chemosensors 2–4 with all anions, with the exception of chloride, which was attributed to an internal charge-transfer mechanism. 1H/13C/15N NMR titration studies and DFT and charge distribution calculations support a mechanism involving deprotonation of the central N–H bond. Compound 4 with a methylene spacer between the naphthalene and thiosemicarbazide moieties was deliberately designed as a fluorescent photoinduced electron-transfer anion (PET) sensor. However, the methylene spacer did not prevent delocalization of the HOMO orbital between the planar aromatic ring and the thiosemicarbazide unit as expected on the basis of the “fluorophore–spacer–receptor” PET model.

Novel halogenated purines and pseudopurines with diverse aryl-substituted 1,2,3-triazoles were prepared. While p-(trifluoromethyl)-substituted 1,2,3-triazole in N-9 alkylated purine and 3-deazapurine was critical for strong albeit unselective activity on pancreatic adenocarcinoma cells CFPAC-1,1-(p-fluorophenyl)-1,2,3-triazole derivative of 7-deazapurine showed selective cytostatic effect on metastatic colon cancer cells SW620. Importantly, 1-(p-chlorophenyl)-1,2,3-triazole-tagged benzimidazole displayed the most pronounced and highly selective inhibitory effect in nM range on non-small cell lung cancer A549. This compound revealed to target molecular processes at the extracellular side and inside the plasma membrane regulated by GPLD1 and growth factor receptors PDGFR and IGF-1R leading to the inhibition of cell proliferation and induction of apoptosis mediated by p38 MAP kinase and NF-κB, respectively. Further optimisation of this compound as to reduce its toxicity in normal cells may lead to the development of novel agent effective against lung cancer.

Keeping the potential synergy of biological activity of synthetic anomalous derivatives of deazapurines and l-ascorbic acid (l-AA) in mind, we have synthesized new 3-, 7- and 9-deazapurine derivatives of l-ascorbic (1–4, 8–10, 13–15) and imino-l-ascorbic acid (5–7, 11, 12, 16–19). These novel compounds were evaluated for their cytostatic and antiviral activity in vitro against a panel of human malignant tumour cell lines and normal murine fibroblasts (3T3). Among all evaluated compounds, the 9-deazapurine derivative of l-AA (13) exerted the most potent inhibitory activity on the growth of CEM/0 cells (IC50 = 4.1 ± 1.8 μM) and strong antiproliferative effect against L1210/0 (IC50 = 4.7 ± 0.1 μM) while the 9-deazahypoxanthine derivative of l-AA (15) showed the best effect against HeLa cells (IC50 = 5.6 ± 1.3 μM) and prominent effect on L1210/0 (IC50 = 4.5 ± 0.5 μM). Furthermore, the 9-deazapurine derivative disubstituted with two imino-l-AA moieties (18) showed the best activity against L1210/0 tumour cells (IC50 = 4.4 ± 0.3 μM) and the most pronounced antiproliferative effects against MiaPaCa-2 cells (IC50 = 5.7 ± 0.2 μM). All these compounds showed selective cytostatic effect on tumour cell lines in comparison with embryonal murine fibroblasts (3T3). When evaluating their antiviral activity, the 3-deazapurine derivative of l-AA (3) exhibited the highest activity against both laboratory-adapted strains of human cytomegalovirus (HCMV) (AD-169 and Davis) with EC50 values comparable to those of the well-known anti-HCMV drug ganciclovir and without cytotoxic effects on normal human embryonal lung (HEL) cells., Graphical abstract, Highlights • Deazapurine derivatives of l-ascorbic and imino-l-ascorbic acid were synthesized. • Compound 13 exerted strong activity on the growth of CEM/0 and L1210/0 cells. • Compound 15 showed the best cytostatic effect against HeLa cells. • Compound 18 showed the best cytostatic activity against L1210/0 and MiaPaCa-2. • Compound 3 exhibited highest effect against HCMV comparable to ganciclovir.

Novel conformationally restricted pyrimidine derivatives with a 1,2,3-triazolyl scaffold bound via Z- and E-2-butenyl spacers were prepared by Cu(I)-catalyzed click chemistry via a one-pot, multi-step reaction under microwave irradiation, while 1,5- and 2,5-disubstituted tetrazoles were synthesized by convenient, environmentally friendly click synthesis and subsequently by N-alkylation of 5-substituted 1H-tetrazoles. Among all the tested compounds, the N-1,N-3-disubstituted olefinic uracil derivative showed the highest antiproliferative effects.

A kinetic approach was used to determine the radical scavenging activities of sinapic acid and its derivatives: sinapine, 4-vinylsyringol, syringic acid, syringaldehyde, and ethyl, propyl and butyl sinapate. The responses were expressed as rates of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH$^·$) scavenging (R$_S$), superoxide radical (O$_2$$^{·−}$) scavenging (R$_{FF}$), and β-carotene bleaching in the emulsion system (R$_B$). For R$_S$ and R$_B$, the esters of sinapic acid showed the highest responses while, for R$_{FF}$, this was seen for syringic acid. The effectiveness of the selected compounds for scavenging these free radicals was also determined at a fixed endpoint. The early response parameters were demonstrated to be good discriminators in assessing differences for antioxidants with comparable fixed endpoint activity. The primary feature that ranks the kinetic data and the endpoint determinations is interpreted in terms of the mechanisms of the reactions involved in each of the assays conducted.

Super-statins are synthetic inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, which is the rate-limiting enzyme responsible for the biosynthesis of cholesterol. All of the super-statins with a C=C double bond spacer between the heterocyclic and the dihydroxycarboxylic moiety that are currently on the market exist as E-isomers. To extend the understanding of conformational and thermodynamic preferences of Z-isomeric super-statin analogues, this study focused on analyzing pitavastatin and its lactonized derivatives via NMR spectroscopy and ab initio calculations. Z-isomeric pitavastatin analogues exist in solution as a pair of interconverting rotamers, where the Gibbs free energies between the major and minor rotamers are within 0.12 and 0.25 kcal mol−1 and the rotational energy barriers are between 15.0 and 15.9 kcal mol−1. The analysis of long-range coupling constants and ab initio calculations revealed that rotation across the C5'–C7 single bond is essential for generating a pair of atropisomers. The overall comparison of the results between Z-isomeric pitavastatin and rosuvastatin analogues demonstrated that the former are to some extent more flexible to attain numerous conformations. Demonstrating how structural differences between super-statin analogues induce distinctive conformational preferences provides important insight into the super-statins’ conformational variability and may well improve future drug design.

Most of the super-statins contain a CC double bond spacer between the heterocyclic and the chiral dihydroxycarboxylic moieties. The known drugs are E-geometric isomers, whereas very little is known about their Z-isomeric analogues. This study explains the unusual resonance line broadening observed in 1H NMR spectra of Z-isomeric rosuvastatin analogues at room temperature, which originates from dynamic exchange between different conformers. Conformational equilibria and intrinsic preferences of Z-isomeric rosuvastatin analogues provide valuable insight into conformational variability that is important for studying potential interactions within the binding site of the enzyme.

Background It had been demonstrated that sugars from various plants can act as potent agents, which induce apoptosis of cancer cells. Methods Using HPLC, we fractionated a mixture of two plant extracts from the plant family Solanaceae, namely Capsicum chinense and the plant family Amaryllidaceae namely Allium sativum. We evaluated the effect of different fractions on apoptosis of HepG2 cell line. The most effective fraction was further studied to determine its molecular composition using mass spectrometry (MS) and NMR. We further evaluated the effect of determined molecular composition found in the selected fraction by using a mixture of commercially available substances, which were found in the fraction and tested its pro-apoptotic effect on HepG2 cells. To get some insight into potential apoptotic mechanisms we studied caspase-3 activity and mitochondrial integrity in treated cells. Results Out of 93 fractions obtained by HPLC from the plant extract we found HPLC fraction 10 (10 min elution) was the most effective. MS and NMR studies revealed high presence of cellobiose together with vitamin C, sulphur (S) and trace amounts of selenium (Se). HPLC fraction 10 triggered apoptosis of HepG2 within 3 h in the 0.01–1.0 mg/mL concentration range. Furthermore, a mixture of pure cellobiose, vitamin C, S and Se (complex cellobiose/C/S/Se) had a very similar capacity in inducing apoptosis of HepG2 cells compared to HPLC fraction 10. Complex cellobiose/C/S/Se was capable of inducing caspase-3 activity and led to loss of mitochondrial integrity. The capacity of cellobiose alone to induce apoptosis of HepG2 was approximately 1000-fold lower compared to complex cellobiose/C/S/Se. Conclusion In this study we present the highly synergistic effect of a unique complex consisting of cellobiose, vitamin C, sulphur and selenium on triggering the apoptosis of human hepatocellular carcinoma (HepG2) cell line.

Two small molecules showed intriguing properties of analytical multipurpose probes, whereby one chromophore gives different signal for many different DNA/RNA by application of several highly sensitive spectroscopic methods. Dyes revealed pronounced fluorescence ratiomeric differentiation between ds-AU-RNA, AT-DNA and GC-DNA in approximate order 10:8:1. Particularly interesting, dyes showed specific fluorimetric response for poly rA even at 10-fold excess of any other ss-RNA, and moreover such emission selectivity is preserved in multicomponent ss-RNA mixtures. The dyes also showed specific chiral recognition of poly rU in respect to the other ss-RNA by induced CD (ICD) pattern in visible range (400–500 nm), which was attributed to the dye-side-chain contribution to binding (confirmed by absence of any ICD band for reference compound lacking side-chain). Most intriguingly, minor difference in the side-chain attached to dye chromophore resulted in opposite sign of dye-ICD pattern, whereby differences in NMR NOESY contacts and proton chemical shifts between two dye/oligo rU complexes combined with MD simulations and CD calculations attributed observed bisignate ICD to the dimeric dye aggregate within oligo rU.

Several novel 1,2,4-triazole and imidazole L-ascorbic acid (1, 2, 3, 5, 6 and 9) and imino-ascorbic acid (4, 7 and 8) derivatives were prepared and evaluated for their inhibitory activity against hepatitis C virus (HCV) replication and human tumour cell proliferation. Compounds 6 and 9 exerted the most pronounced cytostatic effects in all tumour cell lines tested, and were highly selective for human T-cell acute lymphoblastic leukaemia cells (CEM/0) with IC(50)s of 10 ± 4 and 7.3 ± 0.1 μM, respectively. Unlike compound 9, compound 6 showed no toxicity in human diploid fibroblasts. One of the possible mechanisms of action of compound 6 accounting for observed cytostatic activity towards haematological malignancies might be inhibition of inosine monophosphate dehydrogenase (IMPDH) activity, a key enzyme of de novo purine nucleotide biosynthesis providing the cells with precursors for DNA and RNA synthesis indispensable for cell growth and division, which has emerged as an important target for antileukemic therapy. In addition, this compound proved to be the most potent inhibitor of the hepatitis C virus replication as well. However, observed antiviral effect was most likely associated with the effect that the compound exerted on the host cell rather than with selective effect on the replication of the virus itself. In conclusion, results of this study put forward compound 6 as a potential novel antitumor agent (IMPDH inhibitor) for treating leukaemia. Its significant biological activity and low toxicity in human diploid fibroblasts encourage further development of this compound as a lead.

In this study, the antioxidant activities of Camelina sativa methanolic extracts were evaluated by different chemical assays: reducing power, 2,2-diphenyl-1-picrylhydrazyl (DPPH·) assay, the β-carotene bleaching method and the metal chelating activity assay. An LC–MS profiling method was used for a comprehensive study of the phenolic compounds and their representation in camelina seeds, cake and oil. For this purpose, 4-vinyl derivatives of hydroxycinnamic acids were synthesized by thermal decarboxylation of the corresponding phenolic acids and sinapine was isolated from kale ( Brassica oleracea ) applying a new method and confirmed by NMR. The results revealed that besides the total phenolic content and antioxidant activity, seeds and cake also possess a similar phenolic profile. In addition to sinapine and 4-vinyl derivatives, other antioxidants were successfully identified: ellagic acid, protocatechuic acid, p -hydroxybenzoic acid, sinapic acid, salicylic acid, catechin, rutin, quercetin and quercetin glucoside. Since after oil pressing most of the phenolic compounds remain in the seed residues, only a few compounds were identified in the oil. Camelina cake was found to have the best reducing power and radical scavenging activity, whereas camelina oil, with a relatively low phenolic content, exhibited the highest iron-chelating capacity and the best inhibitory action against β-carotene discolouration in an emulsified system.

Since Chinese lantern (Physalis alkekengi L.) represents a rich source of various bioactive secondary metabolites, there is an urge for its detailed characterization. Non-polar flavonoid aglycones represent one of the few bioactive species found in plant's cuticular waxes. The separation of flavonoids is already extensively covered in the literature, but methods dedicated to separation and identification of methylated flavonoids are rather scarce. In the present study a non-targeted approach for the separation, isolation and identification of methylated flavonoids present in P. alkekengi L. var. franchetii cuticular waxes was established. A rapid and simple separation on HPTLC silica gel was developed for preliminary screening of flavonoids. Fast HPLC-UV-MS(n) and HPLC-UV methods using a C6-Phenyl and a C18 stationary phase were also developed, respectively. In both cases, the right combination of temperature and tetrahydrofuran, as a mobile phase modifier, were shown to be crucial for a baseline separation of all studied compounds. By employing a semi-preparative analog of the C18 column, a simultaneous isolation of pure unknown analytes was achieved. Using these developed methods in combination with NMR, four 3-O-methylated flavonols were detected and identified in P. alkekengi L. var. franchetii cuticular waxes: myricetin 3,7,3'-trimethyl ether, quercetin 3,7-dimethyl ether, myricetin 3,7,3',5'-tetramethyl ether and quercetin 3,7,3'-trimethyl ether. Moreover, the simple and fast isocratic HPLC-UV-MS(n) method (under 8min) should prove useful in quality control of P. alkekengi L. var. franchetii by enabling chromatographic fingerprinting of external methylated flavonols. Finally, a rationale for the mechanism of separation of these metabolites by HPLC is also given, which establishes a foundation for future development of chromatographic methods for methylated flavonols and related compounds.

The conformational properties of 1,3-diindolylureas and thioureas were studied by a combination of heteronuclear NMR spectroscopy and quantum mechanics calculations. NOE experiments showed that the anti–anti conformer along the C7–N7α bonds was predominant in DMSO-d6 solution in the absence of anions. Anion-induced changes in the 1H and 15N chemical shifts confirm the weak binding of chloride anions with negligible conformational changes. Strong deshielding of ureido protons and moderate deshielding of indole NH was observed upon the addition of acetate, benzoate, bicarbonate and dihydrogen phosphate, which indicated that the predominant hydrogen bond interactions occurred at the urea donor groups. Binding of oxo-anions caused conformational changes along the C7–N7α bonds and the syn–syn conformer was preferred for anion–receptor complexes. The conformational changes upon anion binding are in good agreement with energetic preferences established by ab initio calculations.

A new series of conformationally restricted pyrimidine derivatives bearing C-6 isobutenyl side-chain (2–9) has been prepared. The novel fluoroalkenyl pyrimidine nucleoside mimetic 3 as model compound for development of tracer molecule in positron emission tomography (PET) was synthesized by fluorination reaction of methoxytritylated pyrimidine derivative using diethylaminosulfur trifluoride (DAST). Conversion of one hydroxyl group to methoxytritylated, fluorinated, mesylated and acetylated pyrimidine derivatives (2, 3, 5–7 and 9) afforded a mixture of Z- and E-isomers in which Z-isomers were predominant. Conformational study of 1, and its fluorinated structural congeners 3 and 4 by the use of NOE experiments revealed predominant conformation of compounds where vinyl H-10 proton is spatially close to N-1 methyl and H-30b methylene protons and on the other hand H-30a methylene protons are close to C-5 methyl protons. The stereostructure of 1, 3-dihydroxyisobutenyl N- methyl thymine 1 was unambiguously confirmed by X- ray crystal structure analysis.

We report on the synthesis of 4-hydroxycoumarin dimers 1-15 bearing an aryl substituent on the central linker and fused benzopyranocoumarin derivatives 16-20 and on their in vitro broad anti-DNA and RNA virus activity evaluations. The chemical identities and structure of compounds 1-20 were deduced from their homo- and heteronuclear NMR measurements whereas the conformational properties of 5, 14 and 20 were assessed by the use of 1D difference NOE enhancements. Unequivocal proof of the stereostructure of compounds 7, 9, 16 and 18 was obtained by single crystal X-ray diffraction method. The X-ray crystal structure analysis revealed that two 4-hydroxycoumarin moieties in the 4-trifluoromethylphenyl- and 2-nitrophenyl derivatives (compounds 7 and 9, respectively) are intramolecularly hydrogen-bonded between hydroxyl and carbonyl oxygen atoms. Consequently, the compounds 7 and 9 adopt conformations in which two 4-hydroxy-coumarin moieties are anti-disposed. Antiviral activity evaluation results indicated that the 4-bromobenzylidene derivative of bis-(4-hydroxycoumarin) (compound 3) possesses inhibitory activity against HSV-1 (KOS), HSV-2 (G), vaccinia virus and HSV-1 TK- KOS (ACVr) at a concentration of 9-12 μM and at a minimum cytotoxic concentration (MCC) greater than 20 μM. Compounds 4-6, 8, and 20 were active against feline herpes virus (50% effective concentration, EC(50) = 5-8.1 μM), that is at a 4-7-fold lower concentration than the MCC. ispartof: Molecules vol:16 issue:7 pages:6023-40 ispartof: location:Switzerland status: published

Novel N-methoxymethylated (MOM) pyrimidine (4-13) and pyrimidine-2,4-diones (15-17) nucleoside mimetics in which an isobutyl side-chain is attached at the C-6 position of the pyrimidine moiety were synthesized. Synthetic methods via O-persilylated or N-anionic uracil derivatives have been evaluated for the synthesis of N-1- and/or N-3-MOM pyrimidine derivatives with C-6 acyclic side-chains. A synthetic approach using an activated N-anionic pyrimidine derivative afforded the desired N,N-1,3-diMOM and N-1-MOM pyrimidines 4 and 5 in good yield. Introduction of fluorine into the side-chain was performed with DAST as the fluorinating reagent to give a N,N-1,3-diMOM pyrimidine 13 with a 1-fluoro-3-hydroxyisobutyl moiety at C-6. Conformational study of the monotritylated N-1-MOM pyrimidine 12 by the use of the NOE experiments revealed the predominant conformation of the compound to be one where the hydroxymethyl group in the C-6 side-chain is close to the N-1-MOM moiety, while the OMTr is in proximity to the CH3-5 group. Contrary to this no NOE enhancements between the N-1-MOM group and hydroxymethyl or fluoromethyl protons in 13 were observed, which suggested a nonrestricted rotation along the C-6 side-chain. Fluorinated N,N-1,3-diMOM pyrimidine 13 emerged as a model compound for development of tracer molecules for non-invasive imaging of gene expression using positron emission tomography (PET). ISSN:1420-3049

Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP(6) ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P-O-Fe-O-P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)-InsP(6) aggregates. (31)P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T(1) relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP(6) molecule results in an unstable heterogeneous system.