Study on Inhibitors of Methionine Synthase Ⅷ: Synthesis of 2,5-Diamino-4-oxo-6- (3-butenyl) pyrimidine

Cobalamin-dependent methionine synthase plays a crucial role in folate metabolism and such would appear to be an excellent target for rational antifolate drug design. However, to date, no anticancer agents directed at this enzyme are available, but the enzyme is efficiently and specifically inhibited by N2O and this has proven invaluable for evaluating the biochemical consequence of enzyme inhibition and for mechanistic studies. [1,2] 2,5-Diamino-4-oxo-6-(3-butenyl) pyrimidine, a key intermediate in synthetic inhibitors of methionine synthase, was first synthesized using γbutenyl-β-ketoester and guanidine carbonate (Scheme 1). [3]     

Adducts of N-terminal valines in hemoglobin with isoprene diepoxide, a metabolite of isoprene

Isoprene (2-methylbuta-1,3-diene) is a multi-site carcinogen in rodents. To evaluate the role of the diepoxide metabolite (1,2:3,4-diepoxy-2-methylbutane) in carcinogenesis, measurements of in vivo doses of the diepoxide are needed. The in vivo dose may be inferred from levels of reaction products with hemoglobin (Hb adducts). This report presents in vitro studies of the adduct formation by the diepoxide of isoprene with valinamide and oligopeptides as model compounds of N-terminal valines in hemoglobin (Hb). In the reaction with valinamide it was shown that isoprene diepoxide forms as the main product a ring-closed adduct, which is a pyrrolidine derivative [N,N-(2,3-dihydroxy-2-methyl-1,4-butadiyl)valinamide, MPyr-Val]. The analysis was performed by gas chromatography/mass spectrometry (GC/MS) (EI and PICI) after acetylation. The ring-closed adduct was also identified by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) as the main product in the reaction between isoprene diepoxide and standard hepta- or (2H8)octapeptides, corresponding to the N-terminal peptides of the alpha-chains in mouse and rat Hb. These peptides, alkylated with isoprene diepoxide, to be used as internal standards and calibration standards for quantification of MPyr-adduct levels in vitro and in vivo, were analyzed with respect to the degree of MPyr-alkylation by two independent methods, amino acid analysis and HPLC-UV; similar results were obtained using these methods. A method for measurement of Hb adducts as modified peptides, used earlier to measure a similar adduct to N-terminal valines in Hb from the diepoxide of 1,3-butadiene, has in the present work been tested for application to isoprene diepoxide. The method is based on tryptic degradation of globin and LC/ESI-MS analysis of N-terminal Pyr-heptapeptides of the Hb alpha-chain enriched by HPLC. MPyr-adduct levels in isoprene diepoxide alkylated hemolysate from mouse erythrocytes incubated with different concentrations of isoprene diepoxide (2 and 10 mM) for 1 h were quantified. The adduct level was about 50 nmol/g alpha-chain Hb per mM x h. From the adduct levels the rate constant of isoprene diepoxide for reaction with N-terminal valine was calculated to be about 1.6 times faster than for diepoxybutane.     

Fully Protected Glycosylated Zinc (II) Phthalocyanine Shows High Uptake and Photodynamic Cytotoxicity in MCF‐7 Cancer Cells

Phthalocyanine photosensitizers are effective in anticancer photodynamic therapy (PDT) but suffer from limited solubility, limited cellular uptake and limited selectivity for cancer cells. To improve these characteristics, we synthesized isopropylidene‐protected and partially deprotected tetra β‐glycosylated zinc (II) phthalocyanines and compared their uptake and accumulation kinetics, subcellular localization, in vitro photocytotoxicity and reactive oxygen species generation with those of disulfonated aluminum phthalocyanine. In MCF‐7 cancer cells, one of the compounds, zinc phthalocyanine {4}, demonstrated 10‐fold higher uptake, 5‐fold greater PDT‐induced cellular reactive oxygen species concentration and 2‐fold greater phototoxicity than equimolar (9 μm ) disulfonated aluminum phthalocyanine. Thus, isopropylidene‐protected β‐glycosylation of phthalocyanines provides a simple method of improving the efficacy of PDT.     

Photosensitization of Staphylococcus aureus with malachite green isothiocyanate: inactivation efficiency and spectroscopic analysis

The potential of malachite green isothiocyanate as a photosensitizer for the inactivation of bacteria has been evaluated. Samples of Staphylococcus aureus are treated with the dye and exposed to continuous-wave red light from a filtered xenon lamp. Reduction in cell viability is seen to increase with radiation dose, whilst non-photosensitized samples are largely unaffected with exposure. The mechanism of photosensitization and the subsequent inactivation is addressed. UV-Vis and Fourier-transform infrared absorption spectrometry have been applied to this biological system, revealing the rapid hydrolysis of the isothiocyanate group of the dye and the transition to the colourless carbinol base when in solution. On binding to Staphylococcus aureus via a complexation mechanism, the dye is seen to be stabilized in its cationic form. Involvement of the excited triplet state of the photosensitizer is suggested and identification of reduced dye photoproducts is made following irradiation.     

Impact of oil type on nanoemulsion formation and Ostwald ripening stability

The formation of stable transparent nanoemulsions poses two challenges: the ability to initially create an emulsion where the entire droplet size distribution is below 80 nm, and the subsequent stabilization of this emulsion against Ostwald ripening. The physical properties of the oil phase and the nature of the surfactant layer were found to have a considerable impact on nanoemulsion formation and stabilization. Nanoemulsions made with high viscosity oils, such as long chain triglycerides (LCT), were considerably larger ( D = 120 nm) than nanoemulsions prepared with low viscosity oils such as hexadecane ( D = 80 nm). The optimization of surfactant architecture, and differential viscosity eta D/eta C, has led to the formation of remarkably small nanoemulsions. With average sizes below 40 nm they are some of the smallest homogenized emulsions ever reported. What is more remarkable is that LCT nanoemulsions do not undergo Ostwald ripening and are physically stable for over 3 months. Ostwald ripening is prevented by the large molar volume of long chain triglyceride oils, which makes them insoluble in water thus providing a kinetic barrier to Ostwald ripening. Examination of the Ostwald ripening of mixed oil nanoemulsions found that the entropy gain associated with oil demixing provided a thermodynamic barrier to Ostwald ripening. Not only are the nanoemulsions created in this work some of the smallest reported, but they are also thermodynamically stable to Ostwald ripening when at least 50% of the oil phase is an insoluble triglyceride.