A pocket of resistance: We have analysed the molecular determinants for nucleotide recognition by the resistance enzyme ANT(4′). Our results demonstrate that its binding epitope is restricted to the inorganic triphosphate fragment. Strikingly, this, together with longer polyphosphate oligomers, can be employed as cosubstrates in aminoglycoside inactivation (see figure), implying a change in the normal activity of the enzyme. These results have implications in the design of specific enzymatic inhibitors. (Figure Presented) © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

The sequence information available for homeodomains reveals that salt bridges connecting pairs 19/30, 31/42, and 17/52 are frequent, whereas aliphatic residues at these sites are rare and mainly restricted to proteins from homeotherms. We have analyzed the influence of salt and hydrophobic bridges at these sites on the stability and DNA binding properties of human Hesx-1 homeodomain. Regarding the protein stability, our analysis shows that hydrophobic side chains are clearly preferred at positions 19/30 and 31/42. This stabilizing influence results from the more favorable packing of the aliphatic side chains with the protein core, as illustrated by the three-dimensional solution structure of a thermostable variant, herein reported. In contrast only polar side chains seem to be tolerated at positions 17/52. Interestingly, despite the significant influence of pairs 19/30 and 31/42 on the stability of the homeodomain, their effect on DNA binding ranges from modest to negligible. The observed lack of correlation between binding strength and conformational stability in the analyzed variants suggests that salt/ hydrophobic bridges at these specific positions might have been employed by evolution to independently modulate both properties. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

The most significant mechanism of bacterial resistance to aminoglycosides is the enzymatic inactivation of the drug. Herein, we analyze several key aspects of the aminoglycoside recognition by the resistance enzyme ANT(4′) from Staphylococcus aureus, employing NMR complemented with site-directed mutagenesis experiments and measurements of the enzymatic activity on newly synthesized kanamycin derivatives. From a methodological perspective, this analysis provides the first example reported for the use of transferred NOE (trNOE) experiments in the analysis of complex molecular recognition processes, characterized by the existence of simultaneous binding events of the ligand to different regions of a protein receptor. The obtained results show that, in favorable cases, these overlapping binding processes can be isolated employing site-directed mutagenesis and then independently analyzed. From a molecular recognition perspective, this work conclusively shows that the enzyme ANT(4′) displays a wide tolerance to conformational variations in the drug. Thus, according to the NMR data, kanamycin-A I/II linkage exhibits an unusual anti-Ψ orientation in the ternary complex, which is in qualitative agreement with the previously reported crystallographic complex. In contrast, closely related, kanamycin-B is recognized by the enzyme in the syn-type arrangement for both glycosidic bonds. This observation together with the enzymatic activity displayed by ANT(4′) against several synthetic kanamycin derivatives strongly suggests that the spatial distribution of positive charges within the aminoglycoside scaffold is the key feature that governs its preferred binding mode to the protein catalytic region and also the regioselectivity of the adenylation reaction. In contrast, the global shape of the antibiotic does not seem to be a critical factor. This feature represents a qualitative difference between the target A-site RNA and the resistance enzyme ANT(4′) as aminoglycoside receptors. © 2008 American Chemical Society.

A series of 3-spirocyclopropanedihydro- and -tetrahydropyrid-4-ones have been synthesized by nitrone cycloaddition to 1,1′-bicyclopropylidene (BCP), chemoselective reduction of the N-O bond of the isoxazolidine ring, and PdII-catalyzed cascade rearrangement of the β-aminocyclopropanol derivatives into the final products. The new tetrahydropyridone derivatives were also prepared by thermal rearrangement of the isoxazolidines. © Wiley-VCH Verlag GmbH & Co. KGaA, 2008.

The Ugi four-component condensation between anilines, aromatic aldehydes, isocyanides, and α-oxoacids afforded the expected adducts which were cleaved in mild conditions to give 2,N-diarylglycines in high yields. © 2008 Elsevier Ltd. All rights reserved.

A complete characterisation of the protonation equilibrium that accompanies the molecular recognition of neomycin-B by a specific RNA receptor has been achieved by employing simple NMR measurements. © The Royal Society of Chemistry.

Aminoglycosides are clinically relevant antibiotics that participate in a large variety of molecular recognition processes involving different RNA and protein receptors. The 3-D structures of these policationic oligosaccharides play a key role in RNA binding and therefore determine their biological activity. Herein, we show that the particular NH2/NH3 +/OH distribution within the antibiotic scaffold modulates the oligosaccharide conformation and flexibility. In particular, those polar groups flanking the glycosidic linkages have a significant influence on the antibiotic structure. A careful NMR/theoretical analysis of different natural aminoglycosides, their fragments, and synthetic derivatives proves that both hydrogen bonding and charge-charge repulsive interactions are at the origin of this effect. Current strategies to obtain new aminoglycoside derivatives are mainly focused on the optimization of the direct ligand/receptor contacts. Our results strongly suggest that the particular location of the NH 2/NH3+/OH groups within the antibiotics can also modulate their RNA binding properties by affecting the conformational preferences and inherent flexibility of these drugs. This fact should also be carefully considered in the design of new antibiotics with improved activity. © 2007 American Chemical Society.

The synthesis of enantiopure cyclic nitrones has become a frequently addressed topic in discussions of their usefulness in the production of natural products and biologically active compounds. This emphasis has stimulated the development of a variety of synthetic approaches that are described in this review, organized on the basis of the size of the nitrone ring. 1 Introduction 2 Four-Membered Cyclic Nitrones 3 Five-Membered Cyclic Nitrones 3.1 Oxidation of Hydroxylamines, Amines, and Imines 3.2 Condensation of Ketones with Hydroxylamines 3.3 N-Alkylation of Oximes 4 Six-Membered Cyclic Nitrones 4.1 Oxidation of Amines and Imines 4.2 N-Alkylation of Oximes 4.3 Condensation of Carbonyl Compounds with Hydroxylamines 4.4 [4+2]-Cycloaddition Reactions 4.5 Hydroxylamine-Alkyne Cyclizations 5 Seven-Membered Cyclic Nitrones 6 Conclusion. © Georg Thieme Verlag Stuttgart.

The use of streptidine as a decoy acceptor allows the antibiotic activity of streptomycin to recover against the Escherichia coli strain overexpressing the aminoglycoside-modifying enzyme 6-O-adenyl transferase. © The Royal Society of Chemistry.