Importance of Co-operative Hydrogen Bonding in the Apramycin-Ribosomal Decoding A-Site Interaction

An intramolecular hydrogen bond between the protonated equatorial 7'-methylamino group of apramycin and the vicinal axial 6'-hydroxy group acidifies the 6'-hydroxy group leading to a strong hydrogen bond to A1408 in the ribosomal drug binding pocket in the decoding A site of the small ribosomal subunit. In 6'-epiapramycin, the trans-nature of the 6'-hydroxy group and the 7'-methylamino group results in a much weaker intramolecular hydrogen bond, and a consequently weaker cooperative hydrogen bonding network with A1408, resulting overall in reduced inhibition of protein synthesis and antibacterial activity

Engineering the rRNA decoding site of eukaryotic cytosolic ribosomes in bacteria

Structural and genetic studies on prokaryotic ribosomes have provided important insights into fundamental aspects of protein synthesis and translational control and its interaction with ribosomal drugs. Comparable mechanistic studies in eukaryotes are mainly hampered by the absence of both high-resolution crystal structures and efficient genetic models. To study the interaction of aminoglycoside antibiotics with selected eukaryotic ribosomes, we replaced the bacterial drug binding site in 16S rRNA with its eukaryotic counterpart, resulting in bacterial hybrid ribosomes with a fully functional eukaryotic rRNA decoding site. Cell-free translation assays demonstrated that hybrid ribosomes carrying the rRNA decoding site of higher eukaryotes show pronounced resistance to aminoglycoside antibiotics, equivalent to that of rabbit reticulocyte ribosomes, while the decoding sites of parasitic protozoa show distinctive drug susceptibility. Our findings suggest that phylogenetically variable components of the ribosome, other than the rRNA-binding site, do not affect aminoglycoside susceptibility of the protein-synthesis machinery. The activities of the hybrid ribosomes indicate that helix 44 of the rRNA decoding site behaves as an autonomous domain, which can be exchanged between ribosomes of different phylogenetic domains for study of function

Oligonucleosides with Integrated Backbone and Base: Foldamers with a Novel Architecture

Dinucleoside and tetranucleoside analogues with integrated backbone and nucleobase represent a novel type of oligonucleotide foldamers. They are characterized by a linker between C(5?) of one nucleoside moiety and C(8) of an adjacent adenosine, or C(6) of an adjacent uridine. Solutions in chloroform or chloroform/DMSO of these partially protected di-, or tetranucleoside analogues associate. The strength of the association depends on the nature of the linker, the presence, or absence of a hydroxymethyl group on the terminal nucleobase, other protecting groups, and intramolecular hydrogen bonds. The thiomethylene-linked dimers were studied in detail; one of them associates strongly enough that a melting temperature can be determined, evidencing base stacking in chloroform solution. Some dimers form organogels. Two 2?,3?-O-isopropylidene protected self-complementary tetramers were prepared. Their duplexes are conformationally homogeneous. One of the tetramers was characterised in detail; it associates via hydrogen bonds of the Watson-Crick type to form a partial A-type helix with all bases in a syn-conformation and a rather large twist angle. Also studied were di- and tetranucleotide analogues possessing an all-carbon linker; they were derived from (hydroxy)propynylene linked dimers by deoxygenation, partial reduction to the (E)- and (Z)-propenylene linked analogues, and further hydrogenation to propanylene-linked analogues. The propargylic hydroxy group forms an intramolecular hydrogen bond to N(3) of the adenine moiety of the same unit; the resulting conformers associate weakly. Deoxygenation leads to foldamers that associate much more strongly. A (Z)-propenylene-linked dimer also associates strongly, its (E) isomer less so, and the propanylene-linked analogues associate weakly. The results show that backbone-base integration defines a novel structural relation between backbone and nucleobases that favours selective pairing and leads to oligonucleotide foldamers

Synthesis, Antibacterial and Antiribosomal Activity of the 3C-Aminoalkyl Modification in the Ribofuranosyl Ring of Apralogs (5-O-Ribofuranosyl Apramycins)

The synthesis and antiribosomal and antibacterial activity of both anomers of a novel apralog, 5-O-(5-amino-3-C-dimethylaminopropyl-D-ribofuranosyl)apramycin, are reported. Both anomers show excellent activity for the inhibition of bacterial ribosomes and that of MRSA and various wild-type Gram negative pathogens. The new compounds retain activity in the presence of the aminoglycoside phosphoryltransferase aminoglycoside modifying enzymes that act on the primary hydroxy group of typical 4,5-(2-deoxystreptamine)-type aminoglycoside and related apramycin derivatives. Unexpectedly, the two anomers have comparable activity both for the inhibition of bacterial ribosomes and of the various bacterial strains tested

Synthesis of monosaccharide-derived spirocyclic cyclopropylamines and their evaluation as glycosidase inhibitors

The glucose-, mannose-, and galactose-derived spirocyclic cyclopropylammonium chlorides 1a–1d, 2a–2d and 3a–3d were prepared as potential glycosidase inhibitors. Cyclopropanation of the diazirine 5 with ethyl acrylate led in 71% yield to a 4 : 5 : 1 : 20 mixture of the ethyl cyclopropanecarboxylates 7a–7d, while the Cu-catalysed cycloaddition of ethyl diazoacetate to the exo-glycal 6 afforded 7a–7d (6 : 2 : 5 : 3) in 93–98% yield (Scheme 1). Saponification, Curtius degradation, and subsequent addition of BnOH or t-BuOH led in 60–80% overall yield to the Z- or Boc-carbamates 11a–11d and 12a–12d, respectively. Hydrogenolysis of 11a–11d afforded 1a–1d, while 12a–12d was debenzylated to 13a–13d prior to acidic cleavage of the N-Boc group. The manno- and galacto-isomers 2a–2d and 3a–3d, respectively, were similarly obtained in comparable yields (Schemes 2 and 4). Also prepared were the differentially protected manno-configured esters 24a–24d; they are intermediates for the synthesis of analogous N-acetylglucosamine-derived cyclopropanes (Scheme 3). The cyclopropylammonium chlorides 1a–1d, 2a–2d and 3a–3d are very weak inhibitors of several glycosidases (Tables 1 and 2). Traces of Pd compounds, however, generated upon catalytic debenzylation, proved to be strong inhibitors. PdClequation image is, indeed, a reversible, micromolar inhibitor for the β-glucosidases from C. saccharolyticum and sweet almonds (non-competitive), the β-galactosidases from bovine liver and from E. coli (both non-competitive), the α-galactosidase from Aspergillus niger (competitive), and an irreversible inhibitor of the α-glucosidase from yeast and the α-galactosidase from coffee beans. The cyclopropylamines derived from 1a–1d or 3a–3d significantly enhance the inhibition of the β-glucosidase from C. saccharolyticum by PdClequation image, lowering the K<SUB>i</SUB> value from 40 μM (PdClequation image) to 0.5 μM for a 1 : 1 mixture of PdClequation image and 1d. A similar effect is shown by cyclopropylamine, but not by several other amines

Synthesis of Gentamicins C1, C2, and C2a and Antiribosomal and Antibacterial Activity of Gentamicins B1, C1, C1a, C2, C2a, C2b, and X2

Complementing our earlier syntheses of the gentamicins B1, C1a, C2b, and X2, we describe the synthesis of gentamicins C1, C2, and C2a characterized by methyl substitution at the 6'-position, and so present an alternative access to previous chromatographic methods for accessing these sought-after compounds. We describe the antiribosomal activity of our full set of synthetic gentamicin congeners against bacterial ribosomes and hybrid ribosomes carrying the decoding A site of the human mitochondrial, A1555G mutant mitochondrial, and cytoplasmic ribosomes and establish structure-activity relationships with the substitution pattern around ring I to antiribosomal activity, antibacterial resistance due to the presence of aminoglycoside acetyl transferases acting on the 6'-position in ring I, and literature cochlear toxicity data