Analysis of heparan sulfate oligosaccharides with ion pair-reverse phase capillary high performance liquid chromatography-microelectrospray ionization time-of-flight mass spectrometry

Heparan sulfate, a cell surface bound glycosaminoglycan polysaccharide, has been implicated in numerous biological functions. Heparan sulfate molecules are highly complex and diverse, yet deceivingly look simple and similar, rendering structure--function correlation tedious. Current chromatographic and mass spectrometric techniques have limitations for analyzing glycosaminoglycan samples that are in low abundance and that are large in size, due to their highly acidic nature arising from a large number of sulfate and of carboxylate groups. A new methodology was developed using capillary ion-paired reverse-phase C18 HPLC directly coupled to ESI-TOF-MS to address the above issues. On the basis of HS disaccharide analysis, dibutylamine was found to be the best suited for HS analysis among many ion-pairing agents investigated. Next, analysis of oligosaccharides derived from heparosan, the precursor for heparan sulfate, was undertaken to demonstrate its greater applicability in a more complex structural analysis. The established chromatographic conditions enabled the characterization of heparosan oligosaccharides of sizes up to tetracontasaccharide with high resolution in a single run and were amenable to negative ion electrospray MS in which sodium adduction and fragmentation were avoided. To date, these are the largest nonsulfated HS precursor oligosaccharides to be characterized by LC/MS. Finally, the current methodology was applied to the characterization of the biologically important ATIII binding pentasaccharide and its precursors, which differ from each other by sulfation pattern and/or degree of sulfation. All of these pentasaccharides were well-resolved and characterized by the LC/MS system with (34)SO(4) as a mass spectral probe. This newly developed methodology facilitates the purification and rapid characterization of biologically significant HS oligosaccharides, and will thus expedite their synthesis. These findings should undoubtedly pave the way in deciphering multiple functional arrangements, ascribed to many biological activities, which are predictably embedded in a single large chaotic, yet well-organized HS polysaccharide chain. Development of newer techniques for HS oligosaccharide analysis is greatly needed in the postgenome era as attention shifts to the functional implications of proteins and carbohydrates in general and HS in particular.     

Synthesis and Structural Characterisation of Derivatives of Tricyclo[5.2.1.02,6]Dec‐8‐Ene‐3,5‐Dione with an Expected Antimicrobial Activity

Anhydrides, imides, N‐ethylimides, N‐hydroxyimides and N‐aminoimides of 1,4,5,6‐tetramethyl‐bicyclo[5.2.1.0^2,6]hept‐5‐ene‐2,3‐dicarboxylic acid, 1,4,5,6,7‐pentamethyl‐bicyclo[5.2.1.0^2,6]hept‐5‐ene‐2,3‐dicarboxylic acid and 7‐ethyl‐1,4,5,6‐tetramethyl‐bicyclo[5.2.1.0^2,6]hept‐5‐ene‐2,3‐dicarboxylic acid were obtained. Antimicrobial activity of the newly obtained derivatives was tested against selected Gram‐positive and Gram‐negative bacteria and fungi of the Candida species. The structures of obtained compounds and their antimicrobial activity were compared. Structure of 1b, 2b and 1e were determined by an X‐ray analysis.     

Epitaxial Growth of α-Mercuric Iodide

Mercuric iodide has been grown for the first time heteroepitaxially on a substrate. α-HgI₂ is deposited as a single crystalline layer which is azimuthally completely oriented. The orientation of the grown layer has been confirmed with texture X-ray diffractometry, reflection powder diffractometry and Atomic Force Microscopy.     

Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5’ cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3’-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.     

Crystal structure, spectroscopy and thermodynamic properties of MVWO6(M – Li, Na)

In the present work lithium (sodium) vanadium tungsten oxides with brannerite structure is refined by the Rietveld method (space group C2/m, Z=2). IR and Raman spectroscopy was used to assign vibrational bands and determine structural particularities. The diffuse reflectance spectra allow to calculate bandgap for M^IVWO₆(M^I – Li, Na). The temperature dependences of heat capacity have been measured first in the range from 7 to 350 K for these compounds and then between 330 and 640 K, respectively, by precision adiabatic vacuum and dynamic calorimetry. The experimental data were used to calculate standard thermodynamic functions, namely the heat capacity C_p^o(T), enthalpy H^o(T)−H^o(0), entropy S^o(T)−S^o(0) and Gibbs function G^o(T)−H^o(0), for the range from T→0 to 640 K. The differential scanning calorimetry was applied to measure decomposition temperature of compounds under study.     

Spectroscopic properties and photodynamic effects of new lipophilic porphyrin derivatives: efficacy, localisation and cell death pathways

Photodynamic therapy (PDT) and photodynamic diagnostics (PDD) of cancer are based on the use of non-toxic dyes (photosensitisers) in combination with harmless visible light. This paper reports physicochemical properties, cell uptake, localisation as well as photodynamic efficiency of two novel lipophilic porphyrin derivatives, suitable for use as PDT sensitisers. Both compounds are characterised by high quantum yield of singlet oxygen generation which was measured by time-resolved phosphorescence. Photodynamic in vitro studies were conducted on three cancer cell lines. Results of cell survival tests showed negligible dark cytotoxicity but high phototoxicity. The results also indicate that cell death is dependent on energy dose and time following light exposure. Using confocal laser scanning microscopy both compounds were found to localise in the cytoplasm around the nucleus of the tumour cells. The mode of cell death was evaluated based on the morphological changes after differential staining. In summary, good photostability, high quantum yield of singlet oxygen and biological effectiveness indicate that the examined lipophilic porphyrin derivatives offer quite interesting prospects of photodynamic therapy application.     

On the Nature of Extra-Framework Aluminum Species and Improved Catalytic Properties in Steamed Zeolites

Steamed zeolites exhibit improved catalytic properties for hydrocarbon activation (alkane cracking and dehydrogenation). The nature of this practically important phenomenon has remained a mystery for the last six decades and was suggested to be related to the increased strength of zeolitic Bronsted acid sites after dealumination. We now utilize state-of-the-art infrared spectroscopy measurements and prove that during steaming, aluminum oxide clusters evolve (due to hydrolysis of Al out of framework positions with the following clustering) in the zeolitic micropores with properties very similar to (nano) facets of hydroxylated transition alumina surfaces. The Bronsted acidity of the zeolite does not increase and the total number of Bronsted acid sites decreases during steaming. O₅Al(VI)-OH surface sites of alumina clusters dehydroxylate at elevated temperatures to form penta-coordinate Al₁O₅ sites that are capable of initiating alkane cracking by breaking the first C-H bond very effectively with much lower barriers (at lower temperatures) than for protolytic C-H bond activation, with the following reaction steps catalyzed by nearby zeolitic Bronsted acid sites. This explains the underlying mechanism behind the improved alkane cracking and alkane dehydrogenation activity of steamed zeolites: heterolytic C-H bond breaking occurs on Al-O sites of aluminum oxide clusters confined in zeolitic pores. Our findings explain the origin of enhanced activity of steamed zeolites at the molecular level and provide the missing understanding of the nature of extra-framework Al species formed in steamed/dealuminated zeolites.     

Diurnal variability of δ13C and δ18O of atmospheric CO2 in the urban atmosphere of Kraków,Poland

This article presents the results of measurements of the isotopic composition and concentration of atmospheric carbon dioxide, performed on air samples from Kraków (Southern Poland) in different seasons of the year. A simple isotope mass balance model has been applied to determine the contributions of different sources of CO₂ to the urban atmosphere of Kraków city: the latitudinal/regional background, biospheric contributions and anthropogenic emissions. The calculations show that during the summer and early autumn the dominant contribution to local CO₂ peaks is the biosphere, making up to 20% of atmospheric CO₂ during the nocturnal temperature inversion in the lower troposphere. During early spring and winter, anthropogenic emissions are the main local source.     

Variable high-pressure and concentration study of cyclohexane pyrolysis at high temperatures

A high-pressure and temperature cyclohexane pyrolysis shock tube study was completed with the goal of extending the experimental cyclohexane pyrolysis data to pressures relevant to current and future combustors and to investigate whether ring contraction products observed in high-pressure, supercritical phase cyclohexane and cycloalkane pyrolysis experiments can form at matching, and higher, pressures in the gas phase. The experiments in the current work were completed over a range of 950–1650 K and at nominal pressures of 40, 100, and 200 bar. No alkylcyclopentanes, possible ring contraction products, were observed to form under the conditions of the current study. The production of methylenecyclopentane and 1,3-cyclopentadiene, and the other three cyclic species quantified: cyclohexene, benzene, and toluene, increased significantly with a substantial increase in the initial fuel concentration. Two sets of experimental data obtained at 200 bar were compared with a literature and laboratory-generated model. Both models had difficulty capturing the propadiene and propyne profiles, and the literature model significantly overestimated the benzene observed in the set of experiments completed with the more dilute fuel mixture. The literature model was able to better predict propadiene, propyne, and benzene product profiles in the 200 bar set of experiments, which used a higher concentration of fuel in the test gas. These results suggest that despite both cyclohexane and benzene being well-studied and important species in combustion chemistry, their reaction pathways and reaction rates would benefit from further refinement.     

Experimental investigation on using the piezoelectric and electromagnetic vibration absorbers in milling

The paper presents an active control system that counteracts the development of chatter vibration. The vibration amplitude depends on the dynamic properties of the machine tool, cutting tool and work-piece. In the paper we analyze the case when the loss of machining stability is caused by the work-piece. The proposed active control system employs electromagnet or piezoelectric actuator to suppress vibration during milling. The active control introduces damping into the system, thereby raising the critical depth of cut and reducing forced vibration amplitude. It enables stable cutting under a much wider range of cutting parameters that for the uncontrolled system. Cutting tests are performed on JAFO FYN-50 machine with mill DIN 845 B-25 K-N HSS to demonstrate an effectiveness of the proposed systems.