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Abstract: Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagine–lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively 15N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1–H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5–1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo. [Copyright &y& Elsevier]

Photohydrates are formed in high yield when uridine (Urd), 2′-deoxyuridine ( dUrd), cytidine (Cyd) and 2′-deoxycytidine ( dCyd) are irradiated with UVC in aqueous solution. The thermal reactions of the photohydrates of Urd with amines at pH values near pH 7.5 have been studied using UV spectroscopy, HPLC, mass spectrometry and, in some cases, NMR. It has been found that a number of amines ( i.e. ethylenediamine, N, N′-dimethylethylenediamine, glycine, glycinamide, glycylglycine, glycylgylcylglycine, putrescine, spermidine and spermine) react thermally with such hydrates to form products with UV spectra characteristic of opened ring uridine-amine adducts. In general, these products display a strong absorption peak with λmax in the range between 288 and 310 nm. Mass spectral studies of a number of the products indicate that they contain one molecule of parent nucleoside and one molecule of reactant amine. Upon standing in water these products revert to parent hydrate, while heating produces parent nucleoside. Less comprehensive studies indicate that photohydrates of dUrd and dCyd undergo analogous thermal reactions. Preliminary results suggest that UV-irradiated polyuridylic acid and polycytidylic acid undergo similar reactions. These results may have relevance for obtaining a complete understanding of the biological effects of producing Urd and dCyd photohydrates in a cellular environment. [ABSTRACT FROM AUTHOR]

Abstract: 3-Substituted, 2,6-dichloropyridines have featured in the syntheses of small molecule inhibitors of a wide variety of biological targets. Hence, the regioselective displacement of the chlorines is of significant interest. Through conducting an extensive solvent study, we have found that non-polar, aprotic solvents of low hydrogen bond basicities favour substitution of the chlorine ortho to the 3-substituent by alkali metal alkoxides. We present convincing evidence that coordination of the alkali metal counter-ion to the 3-substituent (nitro, ester, amide) is the origin of the ortho-selectivity to give a cyclic, six-membered transition state. Excellent ortho-selectivities (⩾98:2) for secondary and tertiary alkoxides were realized with the sodium counter-ion, whereas the more reactive primary alkoxides required the harder, more Lewis acidic lithium counter-ion. [Copyright &y& Elsevier]

Investigates the bovine cytochrome b[sub 5]-horse cytochrome c complex using high-resolution multidimensional nuclear magnetic resonance spectroscopy. Nucleotide and amino acid sequence of the synthetic cytochrome b[sub 5] gene; Resonance assignment strategy; Assignment of backbone nuclei in free ferrous cytochrome b[sub 5].

During optimization of the synthesis of the mixed μ opioid agonist/δ opioid antagonist 5-(hydroxymethyl)oxymorphone (UMB425) for scale-up, it was unexpectedly discovered that the 4,5-epoxy bridge underwent rearrangement on treatment with boron tribromide (BBr3) to yield a novel opioid with a little-studied pyranomorphinan skeleton. This finding opens the pyranomorphinans for further investigations of their pharmacological profiles and represents a novel drug class with the dual profile (μ vs δ) predicted to yield lower tolerance and dependence. The structure was assigned with the help of 1D, 2D NMR and the X-ray crystal structure. [ABSTRACT FROM AUTHOR]

Tristetraprolin (TTP) is a nonclassical CCCH zinc finger (ZF) that plays a crucial role in regulating inflammation. TTP regulates cytokine mRNAs by specific binding of its two conserved ZF domains (CysX8CysX5CysX3His) to adenylate-uridylate-rich sequences (AREs) at the 3ʹ-untranslated region, leading to degradation of the RNA. Dysregulation of TTP in animal models has demonstrated several cytokine-related syndromes, including chronic inflammation and autoimmune disorders. Exposure to Pb(II), a prevalent environmental toxin, is known to contribute to similar pathologies, in part by disruption of and/or competition with cysteine-rich metalloproteins. TTP's role during stress as a ubiquitous translational regulator of cell signaling (and dysfunction), which may underpin various phenotypes of Pb(II) toxicity, highlights the importance of understanding the interaction between TTP and Pb(II). The impact of Pb(II) binding on TTP's fold and RNA-binding function was analyzed via UV–Vis spectroscopy, circular dichroism, X-ray absorption spectroscopy, nuclear magnetic resonance spectroscopy, and fluorescence anisotropy. A construct containing the two ZF domains of TTP (TTP-2D) bound to Pb(II) with nanomolar affinity and exhibited a different geometry and fold in comparison to Zn2-TTP-2D. Despite the altered secondary structure, Pb(II)-substituted TTP-2D bound a canonical ARE sequence more selectively than Zn2-TTP-2D. Taken together, these data suggest that Pb(II) may interfere with proper TTP regulation and hinder the cell's ability to respond to inflammation. [ABSTRACT FROM AUTHOR]

The photo-induced ring opening reactions of thymine (Thy) and 1-methylthymine (MeT) with ammonia and with methylamine (MA) at basic pH, and the subsequent ring closure reactions of the resulting adducts, have been studied. In the photo-induced reaction of Thy with ammonia, the dominant product is the Eform of N-carbamoyl-3-amino-2-methylacrylamide (IIIa). Heating or acid treatment of aqueous IIIaresults in rapid formation of Thy as final product, while allowing IIIato stand at 4 °C produces Thy and two isomeric Thy hydrates, namely trans- and cis-6-hydroxy-5,6-dihydrothymine (Iaand IIa). The main products in the other reactions have analogous structures and undergo analogous ring closure reactions. Incubation of IIIa(and similar adducts) in phosphate buffer near pH 7 significantly enhances the rate of hydrate formation; other weak acid/conjugate base buffer systems also increase the rate of hydrate formation. A mechanism leading from opened ring adduct (e.g.IIIa) to hydrates (e.g.Iaand IIa) is proposed; ring closure leads initially to a dihydrothymine intermediate containing a 6-amino moiety, while further reaction with water produces the observed hydrates. The results described may be relevant to understanding the fate of cross-links generated by photo-induced reaction of thymine moieties in DNA with lysine residues in nuclear proteins (e.g.histones) when they are formed in a cellular environment. Decomposition of such cross-links, catalyzed by cellular phosphate, could lead to production of thymine hydrates attached to lysine residues contained in the protein partner and concurrent generation of an apyrimidinic site in the DNA partner. [ABSTRACT FROM AUTHOR]

Abstract: A catalyst-free Knoevenagel type synthesis of isatinylidenerhodanines is reported starting from substituted isatins and rhodanines. The reaction proceeds spontaneously at room temperature in dimethyl sulfoxide (DMSO), employing a unique macrocyclic intermediate formed through an intramolecular H-bonding. Various substituted isatinylidenerhodanines could be synthesized in good yields. [Copyright &y& Elsevier]