A substituent directed regioselective synthesis of aryl/pyronyl pendant unusual adipate and tetrahydronaphthalene

An efficient regioselective synthesis of pyronyl pendant ethyl methylthiocarbonylalkanoates 5 has been delineated from the base catalyzed reaction of suitably functionalized 2-pyranone 1 and 2-carbethoxycycloalkanones 2, 6 through successive substitution and regioselective ring opening by in situ generated mercaptide ion. To assess the effect of C-4 substituent on regioselectivity, reactions of 6-aryl-3-cyano-4-(piperidin-1-yl)-2-oxopyran 8 with 2-carbethoxycyclohexanone 6a and 2-carbethoxy-2-methylcyclohexanone 6b were carried out separately under analogous reaction conditions but the compounds isolated were identical and characterized as 4-aryl-8-methyl-2-piperidin-1-yl-5,6,7,8-tetrahydronaphthalene-1-carbonitriles 9. Ethyl 2-(5-amino-4′-bromo-4,6-dicyanobiphenyl-3-yl)-5-methylsulfanylcarbonylpentanoate 10 has also been prepared through base catalyzed ring transformation of ethyl 2-[6-(4-bromophenyl)-3-cyano-2-oxo-2H-pyran-4-yl]-5-methylsulfanylcarbonylpentanoate 5d by malononitrile in DMF.     

An unusual Mannich type reaction of tertiary aromatic amines in aqueous micelles

An efficient, unusual Mannich type reaction of tertiary aromatic amines, formaldehyde and 1,3-dicarbonyl compounds is described in aqueous micelles catalyzed by boric acid to afford dialkylaminoarylated 1,3-dicarbonyls. In this unusual Mannich type reaction, tertiary aromatic amines react with formaldehyde to generate an N-alkyl-N-(4-methylenecyclohexa-2,5-dienylidene)alkylaminium intermediate (aza quinone methide), which undergoes nucleophilic addition with 1,3-dicarbonyl compounds. The reaction is highly regioselective, and exclusively para functionalized products are formed in high yields.     

An improved synthesis of pyrimidine- and pyrazole-based acyclo-C-nucleosides as carbohybrids

The synthesis of pyrimidine- and pyrazole-based acyclo-C-nucleosides as carbohybrids was optimized and developed. The synthesis of acyclic polyol-fused pyrimidines was achieved in good to excellent yield with high purity by the cyclocondensation of 2-C-formyl glycals and various amidines using K₂CO₃ as inorganic base in co-solvent system. In comparison, the syntheses of pyrazole-based acyclo-C-nucleosides were accomplished simply by the cyclocondensation of 2-C-formyl glycals with hydrazine hydrate at room temperature and with phenyl hydrazine at refluxing temperature in ethanol.     

A polymerase chain reaction-restriction fragment length polymorphism method based on the analysis of a 16S rRNA/tRNA(Val) mitochondrial region for species identification of commercial penaeid shrimps (Crustacea: Decapoda: Penaeoidea) of food interest

A simple PCR-RFLP method has been developed for the identification of 19 penaeid shrimp species of food interest belonging to the superfamily Penaeoidea. Preliminary amplification, sequencing and alignment of a 960 bp fragment of the 16S rRNA/tRNA(Val)/12S rRNA mitochondrial region allowed the design of 16Scru4/16Scru4 primers, constructed on well-conserved mitochondrial sequences of the penaeid shrimp species considered. Such primers afforded the amplification of an internal 515-535 bp region of the 16S rRNA/tRNA(Val) genes that, when subjected to cleavage with AluI, TaqI and HinfI, provided species-specific restriction patterns. Moreover, the proposed method also allowed the definition of different intraspecific restriction types between different populations of Litopenaeus vannamei, Farfantepenaeus notialis, Fenneropenaeus merguiensis, Metapenaeus sp., Melicertus latisulcatus and Pleoticus muelleri of different origins. The method described here was also successfully applied for the identification of penaeid shrimps in complex processed precooked foods, where this type of shellfish is used as an added-value food ingredient. Sequencing analysis provided new information about the genetic relationships among shrimps not only at the levels of species and genus, but also among different populations at intraspecific level. The 16S rRNA/tRNA(Val) fragment considered in this study seems to be accurate for shrimp species identification in raw and processed foodstuffs and for phylogenetic analysis among penaeid shrimp species.     

Nanoscale degradation of polypyrrole films under oxidative stress: An atomic force microscopy study and review

Atomic force microscopy (AFM) is employed to monitor the surface morphology of polypyrrole (PPy) films grown on vitreous carbon substrates during the catalytic reduction of Cr(VI) to Cr(III). The morphology of freshly-prepared films depends on substrate characteristics. Upon reaction, uniform nodules of aggregated PPy clusters appear. No significant differences in surface morphology are found between its oxidized and reduced forms. Loss of catalytic activity after 8-9 oxidation/reduction cycles of exposure to the chromate solution (oxidation) and electrochemical recharging of the film at negative potentials (reduction) correlates well with the observed polymer film dissolution/detachment from the carbon substrate. Formation of well-defined circular features (PPy rings) at different stages leads to a model for the film degradation process that includes formation of Cl₂ gas inside the polymer matrix. In the final stages, the bulk of the film typically fractures and detaches from the electrode. A catalytically inactive, ultrathin PPy layer remains on the substrate even after prolonged exposure to the target solution. A review of techniques for the study of PPy aging/degradation is given.     

Quantification of fenitrothion and its main metabolites in poplar leaves by isotope dilution gas chromatography-mass spectrometry coupled with solid-phase microextraction

A sensitive and specific method for determining fenitrothion and its main metabolites, 3-methyl-4-nitrophenol and fenitrooxon, in poplar leaves using deuterated isotopes as the internal standard is described. The analytes and the labeled isotopes were extracted from leaves by solid-phase microextraction and subsequently analysed by gas chromatography coupled with mass spectrometry. The method had a chromatographic run time of 17.0 min and good linearity over the range 0.01-10 mg kg(-1). The detection limits ranged between 2.5 and 0.6 microg kg(-1). The isotopic dilution technique allowed improving significantly the repetitivity even using different fibers with the same coating (RSD<5.1%). The method was applied successfully to study the persistence of fenitrothion in forestal matrices in a poplar forest after cannon spray application of the insecticide.     

Flavylium chromophores as species markers for dragon's blood resins from Dracaena and Daemonorops trees

A simple and rapid liquid chromatographic method with diode-array UV-vis spectrophotometric detection has been developed for the authentication of dragon's blood resins from Dracaena and Daemonorops trees. Using this method it was discovered that the flavylium chromophores, which contribute to the red colour of these resins, differ among the species and could be used as markers to differentiate among species. A study of parameters, such as time of extraction, proportion of MeOH and pH, was undertaken to optimise the extraction of the flavyliums. This method was then used to make extracts from samples of dragon's blood resin obtained from material of known provenance. From the samples analysed 7,6-dihydroxy-5-methoxyflavylium (dracorhodin), 7,4'-dihydroxy-5-methoxyflavylium (dracoflavylium) and 7,4'-dihydroxyflavylium were selected as species markers for Daemonorops spp., Dracaena draco and Dracaena cinnabari, respectively. The chromatograms from these samples were used to build an HPLC-DAD database. The ability to discriminate among species of dragon's blood using the single marker compounds was compared with a principal components analysis of the chromatograms in the HPLC-DAD database. The results from the HPLC-DAD method based on the presence of these flavylium markers was unequivocal. The HPLC-DAD method was subsequently applied to 37 samples of dragon blood resins from the historical samples in the Economic Botany Collection, Royal Botanic Gardens, Kew. The method identified anomalies in how samples in this collection had been labelled. It is clear that the method can be used to evaluate the provenance of samples used in different areas of cultural heritage. It also could be used to monitor the trade of endangered species of dragon's blood and the species being used in complex formulations of traditional Chinese medicine.     

Iminium salts of omega-dithiafulvenylpolyenals: an easy entry to the corresponding aldehydes and doubly proaromatic nonlinear optic-phores

A short, high-yielding route to omega-dithiafulvenylpolyenals (1) via the corresponding iminium salts (2) and starting from trimethyl-1,3-dithiolium tetrafluoroborate is reported. The Knoevenagel reactions of either 1 or 2 with isoxazolone-containing acceptors afford merocyanines 7 and 9, in a process that is often accompanied by a vinylene-shortening side reaction. Experimental and theoretical studies reveal that compounds 7 and 9, featuring two proaromatic end groups, are strongly polarized and show good second-order nonlinear optical responses.     

Development of a highly sensitive imaged cIEF immunoassay for studying AAV capsid protein charge heterogeneity

Post-translational modifications (PTMs) of adeno-associated virus (AAV) capsid proteins tune and regulate the AAV infective life cycle, which can impact the safety and efficacy of AAV gene therapy products. Many of these PTMs induce changes in protein charge heterogeneity, including deamidation, oxidation, glycation, and glycosylation. To characterize the charge heterogeneity of a protein, imaged capillary isoelectric focusing (icIEF) has become the gold standard method. We have previously reported an icIEF method with native fluorescence detection for denatured AAV capsid protein charge heterogeneity analysis. Although well suited for final products, the method does not have sufficient sensitivity for upstream, low-concentration AAV samples, and lacks the specificity for capsid protein detection in complex samples like cell culture supernatants and cell lysates. In contrast, the combination of icIEF, protein capture, and immunodetection affords significantly higher sensitivity and specificity, addressing the challenges of the icIEF method. By leveraging different primary antibodies, the icIEF immunoassay provides additional selectivity and affords a detailed characterization of individual AAV capsid proteins. In this study, we describe an icIEF immunoassay method for AAV analysis that is 90 times more sensitive than native fluorescence icIEF. This icIEF immunoassay provides AAV stability monitoring, where changes in individual capsid protein charge heterogeneity can be observed in response to heat stress. When applied to different AAV serotypes, this method also provides serotype identity with reproducible quantification of VP protein peak areas and apparent isoelectric point (pI). Overall, the described icIEF immunoassay is a sensitive, reproducible, quantitative, specific, and selective tool that can be used across the AAV biomanufacturing process, especially in upstream process development where complex sample types are often encountered.