Using nonaqueous capillary electrophoresis to analyze several quinolones in pig kidney samples

We show the potential of nonaqueous capillary electrophoresis (NACE) for analyzing enrofloxacin (ENR), ciprofloxacin (CPR), danofloxacin (DAN), difloxacin (DIF), marbofloxacin (MAR), flumequine (FLU), and oxolinic acid (OXA) in pig kidney samples. We have studied the effects of parameters such as the composition of the organic media, the choice of electrolyte, the pH* of the background electrolyte (BGE), the addition of modifiers, and the reversal of electroosmotic flow. Separation was good with 20 mM ammonium acetate, 0.004% polycation hexadimethrine bromide (HDB), and 4% acetic acid (pH* 5.4) in methanol/acetonitrile (50:50 v/v) medium. We used a quick and simple sample preparation method, hydrochloric acid as an extractant and solid-phase extraction (SPE) with Baker C18 cartridges as the cleanup step. Recoveries for all quinolones were over 80%.     

Proteomic analysis of pancreatic ductal carcinoma cells treated with 5-aza-2'-deoxycytidine

A pancreatic adenocarcinoma cell line (PaCa44), which contains, among other alterations, a methylated p16 promoter, was treated with a chemoterapeutic agent, 5-aza-2'-deoxycytidine (DAC), in order to evaluate the effect of this drug on cell growth and protein expression. Cell proliferation was strongly inhibited by a 24 h DAC treatment and this inhibition lasted for at least 10 days. Master maps of control and treated PaCa44 cells were generated by analysis with the PDQuest software. The comparison between such maps showed up- and downregulation of 45 polypeptide chains, of which 32 were downregulated and 13 upregulated, out of a total of 700 spots detected by a medium-sensitivity stain, micellar Coomassie Brilliant Blue. Fingerprinting by mass spectrometry analysis enabled the identification of 36 of these spots. Among the major changes in DAC-treated cells: cofilin and profilin 1 are silenced; coactosin, peptidyl-propyl cis-trans isomerase A and cystatin B are decreased by 22, 16- and 15-fold, respectively; stress-70 protein, superoxide dismutase and protein disulfide isomerase A3 are increased by 13-, 11-, and 5-fold, respectively. The significance of some of these major changes is discussed.     

Selective and environmentally friendly methodologies based on the use of electrochemistry for fine chemical preparation: an efficient synthesis of N,N'-disubstituted ureas

A novel efficient synthesis of N,N'-disubstituted ureas has been developed. Aromatic and aliphatic primary amines undergo oxidative carbonylation under atmospheric pressure of carbon monoxide using Pd(II) catalyst in combination with its anodic recycling at a graphite electrode.     

Raman analysis of prion protein in blood cell membranes from naturally affected scrapie sheep

At present, there are no efficient antemortem diagnostic tests for transmissible spongiform encephalopathys (TSEs), particularly in blood. New strains of prion PrPSc, which causes TSEs, are currently appearing, and researchers remain concerned that if prion variants continue to emerge, some of them may escape detection by existing immunoassay tests. Because a common structural feature of PrPSc strains is their high content of beta sheets, Raman spectroscopy has proven to be a suitable technique to analyze a key membranous fraction of blood containing PrPSc. In this fraction, a significant increase in beta sheets has been correlated with the worsening of this TSE in naturally scrapie-infected animals in comparison with healthy controls. Since sensitivity and specificity were found to be 100% for each, this test may lead to a new and alternative diagnosis for prion diseases.     

Inhibition of immediate type hypersensitivity reaction by combined irradiation with ultraviolet and visible light

Recently we found that ultraviolet B (UVB) irradiation in erythematous doses significantly inhibited the immediate type hypersensibility reaction in the skin. In the present study we investigated the effects of different wavelengths on the skin prick test reaction (SPT). The forearm of ragweed allergic patients was irradiated with increasing doses of ultraviolet A (UVA), visible light (VIS) or combined UVB, UVA and VIS light, referred to as mUV/VIS. SPTs were performed 24 h after irradiation both on irradiated and non-irradiated control skin areas using ragweed extract. UVA and VIS irradiation led to a slight, not significant inhibition of allergen-induced wheal formation. Mixed irradiation with mUV/VIS light resulted in a dose-dependent inhibition of the allergen-induced wheal formation. The inhibition was significant already at suberythematous doses. As there is a good correlation between SPT and the nasal symptoms in patients with hay fever these data suggest that phototherapy with mUV/VIS light might be an effective and safe treatment modality for immediate type hypersensibility reactions in the skin and nasal mucosa.     

Gaussian mapping of chemical fragments in ligand binding sites

We present a new approach to automatically define a quasi-optimal minimal set of pharmacophoric points mapping the interaction properties of a user-defined ligand binding site. The method is based on a fitting algorithm where a grid of sampled interaction energies of the target protein with small chemical fragments in the binding site is approximated by a linear expansion of Gaussian functions. A heuristic approximation selects from this expansion the smallest possible set of Gaussians required to describe the interaction properties of the binding site within a prespecified accuracy. We have evaluated the performance of the approach by comparing the computed Gaussians with the positions of aromatic sites found in experimental protein-ligand complexes. For a set of 53 complexes, good correspondence is found in general. At a 95% significance level, approximately 65% of the predicted interaction points have an aromatic binding site within 1.5 A. We then studied the utility of these points in docking using the program DOCK. Short docking times, with an average of approximately 0.18 s per conformer, are obtained, while retaining, both for rigid and flexible docking, the ability to sample native-like binding modes for the ligand. An average 4-5-fold speed-up in docking times and a similar success rate is estimated with respect to the standard DOCK protocol.     

The Michaelis-Arbuzov rearrangement of anomeric thiocyanates: synthesis and application of S-glycosyl thiophosphates, thiophosphonates and thiophosphinates as glycosyl donors

Reaction of anomeric thiocyanates with triethyl phosphite, dimethyl phenylphosphonite and methyl diphenylphosphinite afforded the corresponding S-glycosyl thiophosphates, thiophosphonates and thiophosphinates in good yields. These derivatives were applied as glycosyl donors in the synthesis of benzyl glycosides and disaccharides with excellent stereoselectivity.     

Investigation of photodegradation products generated after UV-irradiation of five polybrominated diphenyl ethers using photo solid-phase microextraction

In this study, the photoinduced degradation of five polybrominated diphenyl ethers (PBDEs), BDE-47, BDE-100, BDE-99, BDE-154 and BDE-153, is studied using solid-phase microextraction polydimethylsiloxane fibers as photolytic support. PBDEs are extracted from aqueous solutions using SPME fibers that are subsequently exposed to UV irradiation for different times (from 2 to 60 min). Photodegradation kinetics of the five PBDEs, tentative identification and photochemical behavior of the generated photoproducts, as well as photodegradation pathways, have been studied employing this on-fiber approach technique (photo-SPME) followed by gas chromatography-mass spectrometry analysis. Aqueous photodegradation studies have also been performed and compared with photo-SPME. All the photoproducts detected in the aqueous experiments were previously found in the photo-SPME experiments. In this study, reductive debromination by successive losses of bromine atoms is confirmed as the main photodegradation pathway of PBDEs. A large number of PBDEs were obtained as photoproducts of the five target analytes. Other mechanism of photodegradation observed was intramolecular cyclization from the homolytic dissociation of the C-Br bond; thus, polybromo-dibenzofurans were generated. This work contributes to the study of the photodegradation of PBDEs and shows the potential of photo-SPME to evaluate the photo-transformation of organic pollutants.     

Synthesis of substituted pyrrolidines and piperidines from endocyclic enamine derivatives. Synthesis of (±)-laburnamine

Functionalization of the α- and β-positions of readily available endocyclic enamine derivatives provides a convenient method for the formation of substituted pyrrolidines and piperidines. α-Alkoxy-β-iodopyrrolidines are formed by the electrophilic addition of iodine to the endocyclic enamine double bond of an N-substituted 2-pyrroline, and nucleophillic attack by an alcohol on the intermediate iodonium ion. The resultant α-alkoxy-β-iodopyrrolidines can be used in radical cyclization reactions to give bicyclic hemiaminal compounds, which can be further elaborated using N-acyliminium chemistry to form α,β-cis-dialkylsubstituted pyrrolidines. A strategy for the incorporation of amino functionality at the β-position was also established by using iodoamination of the enamine double bond, followed by migration of the amine functionality through an aziridination/methanolysis protocol. An alternative method uses an azidomethoxylation protocol using ceric ammonium nitrate (CAN) in the presence of NaN₃ and methanol. Formation and trapping of the N-acyliminium ions derived from these substrates, afforded the 3-carbamate and 3-azido-2-substituted products with good diastereoselectivity, with the preferential formation of the trans and cis stereoisomers, respectively. Using the sequential iodoamination, aziridination in methanol and N-acyliminium transformation, trans-3-NHCO₂Me-2-allyl-pyrrolidine was prepared, which was used as the key precursor in a synthesis of the natural 1-amidopyrrolizidine alkaloid, (±)-laburnamine.     

Selective Michael-aldol reaction by use of sterically hindered aluminum aryloxides as Lewis acids: an easy approach to cyclobutane amino acids

A formal [2 + 2] cycloaddition of 2-amidoacrylates with monosubstituted donor olefins, including its asymmetric version, is described. The stereoselectivity of this reaction can be modulated by the use of sterically hindered aluminum aryloxides or methylaluminoxane as Lewis acids. The reaction was applied to the synthesis of both stereoisomers of 2-benzyloxycyclobutane-alpha-amino acid, which are protected serine analogues c(4)Ser(OBn).