Synthesis of Glycosidic (ß-1''→6, 3' and 4') Site Isomers of Neomycin B and their Effect on RNA and DNA Triplex Stability.

Glycosidic (ß-1''→6, 3' and 4') site isomers of neomycin B (i.e., neobiosamine (ß-1''→6, 3' and 4') neamines) have been synthesized in a straightforward manner. Peracetylated neomycin azide was used as a common starting material to obtain neobiosamine glycosyl donor and 6, 3',4'-tri-O-acetyl neamine azide that after simple protecting group manipulation was converted to three different glycosyl acceptors (i.e., 5,6,4'-, 5,3',4'- and 5,6,3'-tri-O-acetyl neamine azide). Glycosylation between the neobiosamine glycosyl donor and the neamine-derived acceptors gave the protected pseudo-tetrasaccharides, which were converted, via global deprotection (deacetylation and reduction of the azide groups), to the desired site isomers of neomycin. The effect of these aminoglycosides on the RNA and DNA triplex stability was studied by UV-melting profile analysis.

Evaluation of neomycin analogues for HIV-1 RRE RNA recognition identifies enhanced activity simplified neamine analogues.

Synthetic neamine mimetics have been evaluated for binding to the HIV-1 Rev response element. Modified neamine derivatives, obtained from reductive amination of neamine, led to identification of new 6-amino modified neamine-type ligands with HIV-1 RRE binding affinity up to 20× that of neamine and up to 6× that of the more complex neomycin itself. This provides a noteworthy structure-activity increase and a useful lead to simplified, chemically accessible mimetics.

Towards Catalytic Antibiotics: Redesign of Aminoglycosides To Catalytically Disable Bacterial Ribosomes.

The emergence of multidrug-resistant pathogens that are resistant to the majority of currently available antibiotics is a significant clinical problem. The development of new antibacterial agents and novel approaches is therefore extremely important. We set out to explore the potential of catalytic antibiotics as a new paradigm in antibiotics research. Herein, we describe our pilot study on the design, synthesis, and biological testing of a series of new derivatives of the natural aminoglycoside antibiotic neomycin B for their potential action as catalytic antibiotics. The new derivatives showed significant antibacterial activity against wild-type bacteria and were especially potent against resistant and pathogenic strains including Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus. Selected compounds displayed RNase activity even though the activity was not as high and specific as we would have expected. On the basis of the observed chemical and biochemical data, along with the comparative molecular dynamics simulations of the prokaryotic rRNA decoding site, we postulate that the rational design of catalytic antibiotics should involve not only their structure but also a comprehensive analysis of the rRNA A-site dynamics.

Broad-spectrum antibacterial amphiphilic aminoglycosides: A new focus on the structure of the lipophilic groups extends the series of active dialkyl neamines.

Amphiphilic aminoglycosides (AAGs) constitute a new class of antibacterial compounds targeting the bacterial membranes. We have identified the 3',6-dinonyl neamine 9 as a broad spectrum antibacterial AAG. Here, we report on the synthesis, antibacterial activity and eukaryotic cytotoxicity of new 3',6-dialkyl neamines designed in order to finely delineate the structure-activity relationships relating their activity to a lipophilicity window. New broad-spectrum antibacterial derivatives were obtained carrying two identical linear or branched alkyl groups or two different linear alkyl groups. Two fluorescent antibacterial 3',6-heterodialkyl neamines carrying a pyrenylbutyl fluorophore were also identified as potential tools for mechanistic study. Homodialkyl and heterodialkyl neamines appeared to be more active on Gram-negative bacteria than dinaphthylalkyl neamines. However, branched dialkyl neamines or heterodialkyl derivatives were found to be more cytotoxic on mammalian cells than 9. The exposure of P. aeruginosa over one month to half-MIC of one of the most active derivatives 9 demonstrated the high difficulty of resistance emergence to AAGs.

Chiral phosphoric acid-catalyzed desymmetrizative glycosylation of 2-deoxystreptamine and its application to aminoglycoside synthesis.

This work describes chiral phosphoric acid (CPA)-catalyzed desymmetrizative glycosylation of meso-diol derived from 2-deoxystreptamine. The chirality of CPA dictates the outcome of the glycosylation reactions, and the use of enantiomeric CPAs results in either C4-glycosylated (67 : 33 d.r.) or C6-glycosylated (86 : 14 d.r.) 2-deoxystreptamines. These glycosylated products can be converted to aminoglycosides, and the application of this strategy to the synthesis of protected iso-neamine and iso-kanamycin B with inverted connection at the C4 and C6 positions is described.

New Broad-Spectrum Antibacterial Amphiphilic Aminoglycosides Active against Resistant Bacteria: From Neamine Derivatives to Smaller Neosamine Analogues.

Aminoglycosides (AGs) constitute a major family of potent and broad-spectrum antibiotics disturbing protein synthesis through binding to the A site of 16S rRNA. Decades of widespread clinical use of AGs strongly reduced their clinical efficacy through the selection of resistant bacteria. Recently, conjugation of lipophilic groups to AGs generated a novel class of potent antibacterial amphiphilic aminoglycosides (AAGs) with significant improved activities against various sensitive and resistant bacterial strains. We have identified amphiphilic 3',6-dialkyl derivatives of the small aminoglycoside neamine as broad spectrum antibacterial agents targeting bacterial membranes. Here, we report on the synthesis and the activity against sensitive and resistant Gram-negative and/or Gram-positive bacteria of new amphiphilic 3',4'-dialkyl neamine derivatives and of their smaller analogues in the 6-aminoglucosamine (neosamine) series prepared from N-acetylglucosamine.

Effects of 5-O-Ribosylation of Aminoglycosides on Antimicrobial Activity and Selective Perturbation of Bacterial Translation.

We studied six pairs of aminoglycosides and their corresponding ribosylated derivatives synthesized by attaching a ß-O-linked ribofuranose to the 5-OH of the deoxystreptamine ring of the parent pseudo-oligosaccharide antibiotic. Ribosylation of the 4,6-disubstituted 2-deoxystreptamine aminoglycoside kanamycin B led to improved selectivity for inhibition of prokaryotic relative to cytosolic eukaryotic in vitro translation. For the pseudodisaccharide aminoglycoside scaffolds neamine and nebramine, ribosylated derivatives were both more potent antimicrobials and more selective to inhibition of prokaryotic translation. On the basis of the results of this study, we suggest that modification of the 5-OH position of the streptamine ring of other natural or semisynthetic pseudodisaccharide aminoglycoside scaffolds containing an equatorial amine at the 2' sugar position with a ß-O-linked ribofuranose is a promising avenue for the development of novel aminoglycoside antibiotics with improved efficacy and reduced toxicity.

Nucleobase modified neamines with a lysine as a linker, their inhibition specificity for TAR-Tat derived from HIV-1.

Nucleobase modified neamines with a lysine as the linker (NbK-neamines) were synthesized and their binding toward hairpin RNAs derived from HIV-1 activator region were studied. NbK-neamines were bind those RNAs with micro molar level of binding affinities and compete with corresponding activator peptide for TAR RNA, but not for RRE RNA. GbK-neamine denotes the highest binding affinity with TAR RNA, three to five times higher than other three NbK-neamines. GbK-neamine could be a candidate of potential inhibitor for TAR-Tat.

New amphiphilic neamine derivatives active against resistant Pseudomonas aeruginosa and their interactions with lipopolysaccharides.

The development of novel antimicrobial agents is urgently required to curb the widespread emergence of multidrug-resistant bacteria like colistin-resistant Pseudomonas aeruginosa. We previously synthesized a series of amphiphilic neamine derivatives active against bacterial membranes, among which 3',6-di-O-[(2"-naphthyl)propyl]neamine (3',6-di2NP), 3',6-di-O-[(2"-naphthyl)butyl]neamine (3',6-di2NB), and 3',6-di-O-nonylneamine (3',6-diNn) showed high levels of activity and low levels of cytotoxicity (L. Zimmermann et al., J. Med. Chem. 56:7691-7705, 2013). We have now further characterized the activity of these derivatives against colistin-resistant P. aeruginosa and studied their mode of action; specifically, we characterized their ability to interact with lipopolysaccharide (LPS) and to alter the bacterial outer membrane (OM). The three amphiphilic neamine derivatives were active against clinical colistin-resistant strains (MICs, about 2 to 8 µg/ml), The most active one (3',6-diNn) was bactericidal at its MIC and inhibited biofilm formation at 2-fold its MIC. They cooperatively bound to LPSs, increasing the outer membrane permeability. Grafting long and linear alkyl chains (nonyl) optimized binding to LPS and outer membrane permeabilization. The effects of amphiphilic neamine derivatives on LPS micelles suggest changes in the cross-bridging of lipopolysaccharides and disordering in the hydrophobic core of the micelles. The molecular shape of the 3',6-dialkyl neamine derivatives induced by the nature of the grafted hydrophobic moieties (naphthylalkyl instead of alkyl) and the flexibility of the hydrophobic moiety are critical for their fluidifying effect and their ability to displace cations bridging LPS. Results from this work could be exploited for the development of new amphiphilic neamine derivatives active against colistin-resistant P. aeruginosa.

CuAAC-mediated diversification of aminoglycoside-arginine conjugate mimics by non-reducing di- and trisaccharides.

Di- and triguanidinylation of trehalose, sucrose, and melizitose has been achieved via a Huisgen-cycloaddition approach. They can serve as aminoglycoside-arginine conjugate mimics, which has been demonstrated by their biological profiles in assays against Bacillus subtilis. For comparative studies, tetraguanidinylated neamine and kanamycin derivatives have also been synthesized and evaluated.

Synthesis and antibacterial activities of amphiphilic neomycin B-based bilipid conjugates and fluorinated neomycin B-based lipids.

Investigating the effect of lipid hydrophobicity on the activity of amphiphilic neomycin B conjugates, six polycationic amphiphiles (PAs) were created. Four of the new compounds incorporated either palmitic or arachidic di-lipid lysine tails, while two had single fluorinated undecanoic acid tails. The basicity of half of the compounds was increased through the incorporation of six guanidine moieties, in order to assess the effect of base strength on antimicrobial activity. A panel of ten bacteria was used for the testing, with seven strains obtained from the American Type Culture Collection series and three clinical isolates from Canadian Intensive Care Units. When compared to previous results with hydrocarbon monolipids the PAs all compounds were found to have reduced activity, though the hemolytic activity of the compounds with fluorinated tails was sharply reduced, with only a moderate reduction in antimicrobial activity.

Evaluation of amphiphilic aminoglycoside-peptide triazole conjugates as antibacterial agents.

The solid- and solution-phase synthesis of amphiphilic aminoglycoside-peptide triazole conjugates (APTCs) accessed by copper(I)-catalyzed 1,3-dipolar cycloaddition reaction between a hydrophobic and ultrashort peptide-based alkyne and a neomycin B- or kanamycin A-derived azide is presented. Antibacterial evaluation demonstrates that the antibacterial potency is affected by the nature of the peptide component. Several APTCs exhibit superior activity against neomycin B- and kanamycin A-resistant strains when compared to their parent aminoglycoside while displaying reduced activity against neomycin B- and kanamycin A-susceptible strains.

Synthesis and antimicrobial evaluation of amphiphilic neamine derivatives.

The aminoglycoside antibiotics bind to the 16S bacterial rRNA and disturb the protein synthesis. One to four hydroxyl functions of the small aminoglycoside neamine were capped with phenyl, naphthyl, pyridyl, or quinolyl rings. The 3',4'- (6), 3',6- (7a), and the 3',4',6- (10a) 2-naphthylmethylene derivatives appeared to be active against sensitive and resistant Staphylococcus aureus strains. 10a also showed marked antibacterial activities against Gram (-) bacteria, including strains expressing enzymes modifying aminoglycosides, efflux pumps, or rRNA methylases. 7a and 10a revealed a weak and aspecific binding to a model bacterial 16S rRNA. Moreover, as compared to neomycin B, 10a showed a lower ability to decrease (3)H leucine incorporation into proteins in Pseudomonas aeruginosa. All together, our results suggest that the 3',4',6-tri-2-naphthylmethylene neamine derivative 10a should act against Gram (-) bacteria through a mechanism different from inhibition of protein synthesis, probably by membrane destabilization.

Design, synthesis, and evaluation of novel fluoroquinolone-aminoglycoside hybrid antibiotics.

A series of new hybrid structures containing fluoroquinolone (ciprofloxacin) and aminoglycoside (neomycin) antibiotics linked via 1,2,3-triazole moiety were designed and synthesized, and their antibacterial activities were determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The nature of spacers in both the ciprofloxacin and neomycin parts greatly influenced the antibacterial activity. The majority of hybrids was significantly more potent than the parent neomycin and overcame most prevalent types of resistance associated with aminoglycosides. Selected hybrids inhibited bacterial protein synthesis with the potencies similar to or better than that of neomycin and were up to 32-fold more potent inhibitors than ciprofloxacin for the fluoroquinolone targets, DNA gyrase and toposiomerase IV, indicating a balanced dual mode of action. Significant delay of resistance formation was observed in both E. coli and B. subtilis to the treatment with ciprofloxacin-neomycin hybrid in comparison to that of each drug separately or their 1:1 mixture.

Synthesis and evaluation of novel neamine derivatives effectively targeting to RNA.

Three new derivatives of neamine, 3 (NE), 6 (NEA) and 9 (NEL), were synthesized by connecting arginine or lysine to 5-hydroxyl group of neamine using ethylenediamine as a linker. The binding affinities of these derivatives to A site of 16S RNA and TAR RNA indicate that the modification on 5-hydroxyl of neamine by amino acid can enhance the binding affinity of neamine. Compound 9 (NEL) shows some antibacterial activities. These results demonstrate that modification on 5-hydroxyl group of neamine may provide a promising way for the development of potential candidates effectively targeting to RNAs.

Synthesis of a phosphonate-linked aminoglycoside-coenzyme a bisubstrate and use in mechanistic studies of an enzyme involved in aminoglycoside resistance.

Just five steps! The synthesis of a phosphonate-linked aminoglycoside-coenzyme A derivative (see scheme) that includes a Michael addition in water has been realized in just five steps. Aminoglycoside N-6'-acetyltransferases (AAC(6')s) are important determinants of antibiotic resistance. A good mechanistic understanding of these enzymes is essential to overcome aminoglycoside resistance. We have previously reported the synthesis of amide- and sulfonamide-linked aminoglycoside-coenzyme A conjugates, which were useful mechanistic and structural probes of AAC(6')s. We report here the synthesis of a phosphonate-linked aminoglycoside-coenzyme A variant, which is expected to be a superior mimic of the tetrahedral intermediate proposed for catalysis by AAC(6')s. This synthetic target is especially challenging for a number of reasons, including the presence of multiple functional groups, the water solubility of both starting materials, and incompatibility of P(III) chemistry with water. We have overcome these challenges by adding the expensive coenzyme A in the last step by means of an elegant Michael-type addition onto a vinylphosphonate in water. Overall, a single protection step was needed. The decreased inhibitory potency of this bisubstrate compared to that of the amide-linked analogue suggests that Enterococcus faecium AAC(6')-Ii may not stabilize the proposed tetrahedral intermediate, and may act mainly through proximity catalysis.

Chiral ligand-exchange chromatography of amino acids using porous graphitic carbon coated with a dinaphthyl derivative of neamine.

In this paper, we describe the preparation and the evaluation of a porous graphitic carbon (PGC) column coated with a new dinaphthyl derivative of neamine for chiral ligand-exchange (LE) chromatography. It was shown that the graphitic surface/dinaphthyl anchor system efficiently (1.15 micromol/m(2)) and stably (three months of intensive use) adsorbs the neamine template onto the chromatographic support. The resulting coated PGC stationary phase showed appreciable LE-based enantioselective properties towards several native amino acids.

Studies on the mechanism of inhibition of bacterial ribonuclease P by aminoglycoside derivatives.

Ribonuclease P (RNase P) is a Mg2+-dependent endoribonuclease responsible for the 5'-maturation of transfer RNAs. It is a ribonucleoprotein complex containing an essential RNA and a varying number of protein subunits depending on the source: at least one, four and nine in Bacteria, Archaea and Eukarya, respectively. Since bacterial RNase P is required for viability and differs in structure/subunit composition from its eukaryal counterpart, it is a potential antibacterial target. To elucidate the basis for our previous finding that the hexa-arginine derivative of neomycin B is 500-fold more potent than neomycin B in inhibiting bacterial RNase P, we synthesized hexa-guanidinium and -lysyl conjugates of neomycin B and compared their inhibitory potential. Our studies indicate that side-chain length, flexibility and composition cumulatively account for the inhibitory potency of the aminoglycoside-arginine conjugates (AACs). We also demonstrate that AACs interfere with RNase P function by displacing Mg2+ ions. Moreover, our finding that an AAC can discriminate between a bacterial and archaeal (an experimental surrogate for eukaryal) RNase P holoenzyme lends promise to the design of aminoglycoside conjugates as selective inhibitors of bacterial RNase P, especially once the structural differences in RNase P from the three domains of life have been established.

Synthesis and antibacterial activity of novel neamine derivatives.

Synthesis and activity of derivatives at the O5 or O6 positions of 1-N-((S)-4-amino-2-hydroxybutyryl)-3',4'-dideoxyneamine, which is the neamine moiety of arbekacin, were reported. Among these results, the 5-O-aminoethylaminocarbonyl derivative showed effective activity against Staphylococcus aureus expressing a bifunctional aminoglycoside-modifying enzyme AAC(6')-APH(2'').