High-affinity ligands for the asialoglycoprotein receptor, which is uniquely localized on the parenchymal liver cell and recognizes oligoantennary galactosides, might be utilized as homing device to specifically target drugs or genes to parenchymal liver cells. In the present study, the synthesis of galactose-terminated triantennary glycosides, provided with various spacers between the beta-galactopyranosyl moieties and the branching point of the dendrite, is described. N-[Tris[[(methylthio)methoxy]methyl]methyl]-N alpha-[1-(6- methyladipy)]glycinamide (3b) was glycosylated with monogalactosyl derivatives, containing propanediol or ethylene glycol units as hydrophilic spacer moieties, to yield the corresponding cluster galactosides. To determine the affinity of the cluster galactosides for the asialoglycoprotein receptor, we have performed competition studies of [125I]ASOR binding, a specific ligand for the asialoglycoprotein receptor, to isolated parenchymal cells. The affinity for the asialoglycoprotein receptor significantly increased with increasing spacer length. N-[[[Tris-O-(beta-D-galactopyranosyl)-3,6,9-trioxaunde- canoxy]methoxy]methyl]-N-alpha-[1-(6-methyladipyl)]glycinami de (4e), a cluster galactoside provided with a 20 A spacer, possessed an at least 2000-fold higher affinity for the receptor than N-[[tris-O-(beta-D-galactopyranosyl)methyl]methyl]-N alpha-[1-(6- methyladipyl)]glycinamide (4a), a cluster galactoside lacking the spacer. It is concluded that vicinal galactosyl moieties within a cluster galactoside are more optimal recognized by the galactose binding sites of the asialoglycoprotein receptor upon proper spacing. The most potent galactoside, TG(20A), may constitute an attractive targeting device for the specific delivery of drugs and/or genes to the parenchymal liver cell.
DNA strand scission by bleomycin in the presence of Cu and Fe was further characterized. It was found that DNA degradation occurred readily upon admixture of Cu(I) or Cu(II) + dithiothreitol + bleomycin, but only where the order of addition precluded initial formation of Cu(II)--bleomycin or where sufficient time was permitted for reduction of the formed Cu(II)--bleomycin to Cu(I)--bleomycin. DNA strand scission mediated by Cu + dithiothreitol + bleomycin was inhibited by the copper-selective agent bathocuproine when the experiment was carried out under conditions consistent with Cu chelation by bathocuproine on the time scale of the experiment. Remarkably, it was found that the extent of DNA degradation obtained with bleomycin in the presence of Fe and Cu was greater than that obtained with either metal ion alone. A comparison of the sequence selectivity of bleomycin in the presence of Cu and Fe using 32P-end-labeled DNA duplexes as substrates revealed significant differences in sites of DNA cleavage and in the extent of cleavage at sites shared in common. For deglycoblemycin and decarbamoylbleomycin, whose metal ligation is believed to differ from that of bleomycin itself, it was found that the relative extents of DNA cleavage in the presence of Cu were not in the same order as those obtained in the presence of Fe. The bleomycin-mediated oxygenation products derived from cis-stilbene were found to differ in type and amount in the presence of added Cu vs. added Fe. Interestingly, while product formation from cis-stilbene was decreased when excess Fe was added to a reaction mixture containing 1:1 Fe(III) and bleomycin, the extent of product formation was enhanced almost 4-fold in reactions that contained 5:1, as compared to 1:1, Cu and bleomycin. The results of these experiments are entirely consistent with the work of Sugiura [Sugiura, Y. (1979) Biochem. Biophys. Res. Commun. 90, 375-383], who first demonstrated the generation of reactive oxygen species upon admixture of O2 and Cu(I)--bleomycin.
This work describes the first automated solid-phase synthesis of metal derivatives of peptide nucleic acid (PNA) oligomers and their interaction with DNA and PNA. PNA constitutes a relatively young and very promising class of DNA analogues with excellent DNA and RNA binding properties. However, PNA lacks a suitable handle that would permit its sensitive detection on its own as well as when hybridized with complementary oligonucleotides. Metal complexes, on the other hand, offer high potential as markers for biomolecules. In this paper, we describe the synthesis of PNA heptamers (tggatcg-gly, where gly is a C-terminal glycine carboxylic acid amide) with two covalently attached metal complexes at the PNA N-terminus, namely a ferrocene carboxylic acid derivative and a tris(bipyridine)ruthenium(II) derivative. We show how all synthesis steps may be carried out with high yield on a DNA synthesizer, including attachment of the metal complexes. The conjugates were characterized by HPLC (>90% purity) and ESI-MS. Binding studies of the purified Ru-PNA heptamer to complementary DNA and PNA and comparison to the isosequential metal-free acetyl PNA heptamer proves that the attached metal complex has an influence on the stability (UV-T(m)) and structure (CD spectroscopy) of the conjugates, possibly by disruption of the nearby A:T base pair.
[structure: see text]. The cyclooctenol derivative 1 can be transformed into the nine-membered ring lactone 3, as well as the amino-containing carbocycles 4 and 5. The corresponding ketone 2 gives access to the conformationally locked azasugar 6.
All lipo-chitin oligosaccharides identified from Rhizobium leguminosarum carry an O-acetyl moiety on C6 of the nonreducing terminal N-acetylglucosamine residue. Previously, we have shown that purified NodL protein, using acetyl-CoA as acetyl donor, in vitro acetylates N-acetylglucosamine, chitin oligosaccharides, and lipo-chitin oligosaccharides. In this paper, the enzymatic properties and substrate specificity of NodL protein were analyzed, using a spectrophotometric assay to quantify NodL transacetylating activity. NodL functions optimally under alkaline conditions. Transacetylating activity has a broad temperature optimum between 28 and 42 degrees C. NodL protein is stable for at least 15 min up to 48 degrees C. Glucosamine, chitosan oligosaccharides, terminally de-N-acetylated chitin derivatives, and cellopentaose were identified as acetyl-accepting substrates for NodL protein. Quantitative substrate specificity studies show that chitin derivatives with a free amino group on the nonreducing terminal residue are the preferred substrates of the NodL protein. Our results strongly indicate that the nonreducing terminally de-N-acetylated chitin oligosaccharides produced by the NodC and NodB enzymes are the in vivo acetyl-accepting substrates for NodL protein.
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