Hanna Krawczyk

Characterisation of the 1H and 13C NMR spectra of N-acetylaspartylglutamate and its detection in urine from patients with Canavan disease

1H and 13C NMR spectra of N-acetylaspartylglutamate (NAAG) have been recorded and interpreted. The values of the 1H chemical shifts and 1H-(1)H coupling constants at different pH were obtained by iterative computer fitting of 1-D 1H NMR spectra. This provided information on the solution conformation of the investigated molecule. Proton-decoupled high resolution 13C NMR spectra of NAAG have been measured in a series of dilute water solution of various acidity. These data have provided a basis for unequivocal determination of the presence of NAAG in the urine sample of a patient suffering from Canavan disease. NMR spectroscopy provides a possibility of detecting NAAG in body fluids.

13C NMR spectroscopy: a convenient tool for detection of argininosuccinic aciduria.

Proton decoupled high resolution 13C NMR spectra of argininosuccinic acid have been measured in a series of dilute water solutions of various acidity. These data have provided a basis for unequivocal determination of the presence of this metabolite in the investigated sample. The method additionally enables simultaneous rough estimation of the metabolite concentration. In order to check the practicability of the usage of this spectroscopy for diagnostic purposes, the spectra of several unprocessed urine samples have been recorded including three from patients with argininosuccinic aciduria. It has been concluded that 13C NMR spectroscopy can be a convenient method of recognising the above syndrome and probably many other inborn metabolic errors which manifest themselves with the excretion of the marker metabolite in amounts comparable to (or larger than) creatinine.

Cytotoxicity Studies of Novel Combretastatin and Pterostilbene Derivatives

We synthesised seven 2-aminestilbenes with methoxy substitents in reactions of dinitrostilbenes with sodium azide. In order to study the positioning of the nitro groups, the optimum structure of obtained stilbenes using the DFT B3LYP/6-311++G(2d,p) method was calculated. Very interesting aspect of this regioselectivity reaction is the fact that in all substrates and synthetized compounds the nitro groups in position 2 were not coplanar whereas the para-nitro groups were coplanar with respect to the benzene ring. Due to unique features of stilbene derivatives, such as antitumor agents, we undertook the studies on the biological properties of new stilbene derivatives. Using five cancer cell lines, we investigated the effects of 2-aminestilbenes with methoxy substitents on cell growth.

Production of uremic toxin methylguanidine from creatinine via creatol on activated carbon.

The mixture of creatinine, activated charcoal and water was stirred. As a result the conversion of creatinine into two products was observed. (1)H, (13)C NMR and HMBC spectra were recorded and the chemical shifts assigned. Two uremic toxins: creatol and N-methylguanidine were identified. To interpret the NMR data obtained, the optimum structure of creatol, which can exist in the forms of seven tautomers, has been calculated using the DFT B3LYP/6-311G(2d,p) method. The influence of the solvent was described by the polarizable continuum model (PCM). The calculated energy of the most energetically stable tautomeric form A is lower by 12.2, 16.9, 33.8, 81.5, 106.3, 130.4kJ/mol in water than that of the tautomers B-G, respectively, which suggests that the A form of creatol should prevail in solution. In DMSO, the calculated energy of the most energetically stable tautomeric form A is lower than that of both D and B and the remaining tautomeric forms (C, E-G) are less energetically stable. Subsequently, we sought the correlations between the experimental and the calculated chemical shifts of protons and carbons-13 for the forms -A, B (in water) and A, B, D (in DMSO) - of creatol. The population of the A tautomer is predominant in both H(2)O and DMSO. We have also recorded the spectra of creatol and N-methylguanidine at different pH. Our data are complete enough to be used in the analysis of body fluids.

Novel non-covalent stable supramolecular ternary system comprising of cyclodextrin and branched polyethylenimine

The synthesis of a novel supramolecular system comprising of branched polyethylenimine and cyclodextrin, is presented. The synthesis route is based on the self-assembly phenomena with the inclusion of solvent molecules. The systems are formed by a hydrogen-bonding network and host–guest type interactions between the building blocks. It was found that the native cyclodextrin and polyethylenimine are able to form stable systems when the reaction medium constitutes a polar solvent forming host–guest type complexes with cyclodextrin. A special consideration was paid on the detailed spectroscopic analyses of the obtained water-soluble constructs, including ROESY and diffusion-ordered (DOSY) NMR spectroscopy studies. The versatility and significance of DOSY technique for the analysis of the cyclodextrin complexes and its non-covalent systems with branched polymers, were presented. It was also found that the guest molecules that were incorporated in the complexes exhibited enhanced thermal stability. The morphological details in the solid state were obtained by scanning electron microscope.Graphical Abstract

Evaluation of anti-cancer activity of stilbene and methoxydibenzo[b,f]oxepin derivatives

BACKGROUND Stilbenes, 1,2-diphenylethen derivatives, including resveratrol and combretastatins, show anticancer features especially against tumor angiogenesis. Fosbretabulin, CA-4, in combination with carboplatin, is in the last stages of clinical tests as an inhibitor of thyroid cancer. The mode of action of these compounds involves suppression of angiogenesis through interfering with tubulin (de)polymerization. OBJECTIVE We have previously synthesized five E-2-hydroxystilbenes and seven dibenzo [b,f]oxepins in Z configuration, with methyl or nitro groups at varied positions. The aim of the present work was to evaluate the anticancer activity and molecular mechanism(s) of action of these compounds. RESULTS Two healthy, EUFA30 and HEK293, and two cancerous, HeLa and U87, cell lines were treated with four newly synthetized stilbenes and seven oxepins. Two of these compounds, JJR5 and JJR6, showed the strongest cytotoxic effect against cancerous cells tested and these two were selected for further investigations. They induced apoptosis with sub-G1 or S cell cycle arrest and PARP cleavage, with no visible activation of caspases 3 and 7. Proteomic differential analysis of stilbene-treated cells led to the identification of proteins involved almost exclusively in cell cycle management, apoptosis, DNA repair and stress response, e.g. oxidative stress. CONCLUSION Among the newly synthesized stilbene derivatives, we selected two as potent anticancer compounds triggering late apoptosis/necrosis in cancerous cells through sub-G1 phase cell cycle arrest. They changed cyclin expression, induced DNA repair mechanisms, enzymes involved in apoptosis and oxidative stress response. Compounds JJR5 and JJR6 can be a base for structure modification(s) to obtain even more active derivatives.

Uraemic Toxins Generated in the Presence of Fullerene C60, Carbon-Encapsulated Magnetic Nanoparticles, and Multiwalled Carbon Nanotubes

Uraemic toxins—creatol and N-methylguanidine—are generated in conversion of creatinine in water in the presence of various forms of carbon such as fullerene C60, carbon-encapsulated magnetic nanoparticles, and multiwalled carbon nanotubes and oxygen. The conversion degree for creatinine was different for fullerene C60, CEMNPs, and MWCNTs and was 9% (3.6% creatol, 5.4% N-methylguanidine), 35% (12% creatol, 23% N-methylguanidine), and 75% (16% creatol, 59% N-methylguanidine), respectively.

Synthesis and investigation of new cyclic molecules using the stilbene scaffold

A new approach to the synthesis of asymmetrical cyclic compounds using a stilbene scaffold has been developed. The use of boron trifluoride diethyl etherate as the catalyst, both with and without paraformaldehyde, allows us to obtain new substituted dioxanes, oxanes, cyclic compounds or dimer. The analysis of products was run using experimental and theoretical methods.

Marking of metabolites in the diagnostics of metabolic diseases and in the investigation of xenobiotics metabolism using NMR spectroscopy.

There are currently no sound estimates of the number of children born with a serious congenital disorder attributable to genetic or environmental causes (World Health Organization) but there is a supposed number of babies born with birth defects per year: in the world approximately 7.9 million children (6% of births). There is conducted population-based screening by the individual countries. The specialised methods are used when it is not possible to diagnose disease in screening. In recent years in the diagnostics of these disorders the methods of Magnetic Resonance Spectroscopy of the brain (in vivo1H-MRS) and high resolution NMR spectroscopy gain in importance. The manuscript focused on developing the method of marking the metabolic diseases markers of various origins using NMR spectroscopy (including synthesis of markers). Considering the disorders occurring among children, according to Hoffman, Zschocke, Nyhan, there are three following groups of inherited metabolic diseases: disorders of intermediary metabolism, disorders of the biosynthesis and breakdown of complex molecules and neurotransmitter defects and related disorders. The presented investigation is focused on: a study of selected compounds that cause disorders of intermediary metabolism, a study of compounds that cause disorders of the biosynthesis and breakdown of complex molecules and a study of compounds that cause neurotransmitter defects and related disorders. In the subsequent chapter of manuscript there are presented the results of investigation concerning the metabolism of xenobiotics that could potentially be used in therapy of inherited metabolic diseases, basing on stilbene derivatives. In the last chapter there are presented the results of experiments with creatinine- the metabolite produced in muscles.