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This study presents the validation of a sensitive method for the determination of 6‐nitrodopa, 6‐nitrodopamine, 6‐nitroadrenaline and 6‐cyanodopamine in Krebs–Henseleit solution by LC–MS/MS with ESI+. HRMS was used to precisely characterize the structures of the fragment ions. The method was applied to investigate the catecholamine basal release from rabbit isolated atria and ventricles. The atria and ventricles were suspended separately in a 5 ml organ bath containing Krebs–Henseleit solution with ascorbic acid (3 mM), gassed (95%O2/5%CO2) at 37°C for 30 min. Strata‐X 33 μm SPE cartridges were employed for the extraction of the catecholamines and the internal standard 6‐nitrodopamine‐d4. The catecholamines were separated employing a 150 × 3 mm Shim‐pack GIST C18‐AQ (3 mm particle size), placed in an oven at 40°C and perfused by 65% of mobile phase A (MeCN/H2O; 90/10, v/v) + 0.4% CH3COOH and 35% mobile phase B (deionized H2O) + 0.2% CH2O2 at 320 μl/min in isocratic mode. The method was linear at 0.1–20 ng/ml. The method was used to identify for the first‐time basal release of the three nitrocatecholamines mentioned above and a member of a novel class of catecholamines, the cyanocatecholamines. [ABSTRACT FROM AUTHOR]

Copper-catalyzed azide-alkyne cycloadditions (CuAAC or click chemistry) are convenient methods to easily couple various pharmacophores or bioactive molecules. A new series of 1,2,3-triazole-linked nucleoside-amino acid conjugates have been designed and synthesized in 57-76% yields using CuAAC. The azido group was introduced on the 50-position of uridine or the acyclic analogue using the tosyl-azide exchange method and alkylated serine or proparylglycine was the alkyne. Modeling studies of the conjugates in the active site of LpxC indicate they have promise as antibacterial agents. [ABSTRACT FROM AUTHOR]

An unnatural base-pair architecture with base pairs 2.4 Å larger than the natural DNA-based genetic system (xDNA) is evaluated for its ability to function like DNA, encoding amino acids in the context of living cells. xDNA bases are structurally analogous to natural bases but homologated by the width of a benzene ring, increasing their sizes and resulting in a duplex that is wider than native B-DNA. Plasmids encoding green fluorescent protein were constructed to contain single and multiple xDNA bases (as many as eight) in both strands and were transformed into Escherichia coli. Although they yielded fewer colonies than the natural control plasmid, in all cases in which a modified plasmid (containing one, two, three, or four consecutive size-expanded base pairs) was used, the correct codon bases were substituted, yielding green colonies. All four xDNA bases (xA, xC, xG, and xT) were found to encode the correct partners in the replicated plasmid DNA, both alone and in longer segments of xDNA. Controls with mutant cell lines having repair functions deleted were found to express the gene correctly, ruling out repair of xDNA and confirming polymerase reading of the unnatural bases. Preliminary experiments with polymerase deletion mutants suggested combined roles of replicative and lesion-bypass polymerases in inserting correct bases opposite xDNA bases and in bypassing the xDNA segments. These experiments demonstrate a biologically functioning synthetic genetic set with larger-than-natural architecture. [ABSTRACT FROM AUTHOR]

The design of smart nonviral vectors for gene delivery is of prime importance for the successful implementation of gene therapies. In particular, degradable analogues of macromolecules represent promising targets as they would combine the multivalent presentation of multiple binding units that is necessary for achieving effective complexation of therapeutic oligonucleotides with the controlled degradation of the vector that would in turn trigger drug release. Toward this end, we have designed and synthesized hybrid polyacylhydrazone-based dynamic materials that combine bis-functionalized cationic monomers with ethylene oxide containing monomers. Polymer formation was characterized by (1) H and DOSY NMR spectroscopy and was found to take place at high concentration, whereas macrocycles were predominantly formed at low concentration. HPLC monitoring of solutions of these materials in aqueous buffers at pH values ranging from 5.0 to 7.0 revealed their acid-catalyzed degradation. An ethidium bromide displacement assay and gel electrophoresis clearly demonstrated that, despite being dynamic, these materials are capable of effectively complexing dsDNA in aqueous buffer and biological serum at N/P ratios comparable to polyethyleneimine polymers. The self-assembly of dynamic covalent polymers through the incorporation of a reversible covalent bond within their main chain is therefore a promising strategy for generating degradable materials that are capable of establishing multivalent interactions and effectively complexing dsDNA in biological media., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)