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Trotz signifikanter Strukturanderungen beim Dehydratisieren des dreidimensionalen molekularen Gerusts aus Hexaaquacobalt-Kationen und redoxaktiv funktionalisierten Tetrathiafulvalen-Anionen (siehe Bild, der Bereich der mit Wassermolekulen besetzbaren Kanale ist grau dargestellt) bleibt die Verbindung mikroporos und einkristallin. Rontgenstrukturanalysen zufolge fuhrt eine einzigartige Kombination von Wasserstoffbruckenbindungen und π-π-Wechselwirkungen innerhalb des Gerusts zu dieser Strukturflexibilitat, die man von Zeolithen und deren Analoga nicht kennt.

A newly discovered bifunctional antibiotic resistance enzyme from Serratia marcescens catalyzes adenylation and acetylation of aminoglycoside antibiotics. The structure assignment of the enzymic products indicated that acetylation takes place on the 6'-amine of kanamycin A and the adenylation on 3''- and 9-hydroxyl groups of streptomycin and spectinomycin, respectively. The adenyltransferase domain appears to be highly specific to spectinomycin and streptomycin, while the acetyltransferase domain shows a broad substrate profile. Initial velocity patterns indicate that both domains follow a sequential kinetic mechanism. The use of dead-end and product inhibition, the solvent isotope effect, and the solvent viscosity effect reveals that the adenyltransferase domain catalyzes the reaction by a Theorell-Chance kinetic mechanism, where ATP binds to the enzyme prior to the aminoglycoside and the modified antibiotic is the last product to be released. The acetyltransferase domain follows an ordered bi-bi kinetic mechanism, in which the antibiotic is the first substrate that binds to the active site and CoASH is released prior to the modified aminoglycoside. The merging of two genes to create bifunctional resistance enzymes with expanded profiles has now been documented in four instances, including the subject of study in this report, which suggests a new trend in the emergence of resistance to aminoglycoside antibiotics among pathogens.

Glycopeptide antibiotics, including vancomycin, form complexes via a set of five hydrogen bonds with the acyl-l-Lys-d-Ala-d-Ala portion of the peptidyl stems of the bacterial cell wall peptidoglycan. This complexation deprives the organism from the ability to cross-link peptidyl stems of the peptidoglycan, leading to bacterial cell death. Four synthetic fragments as surrogates of the components of the bacterial cell wall have been prepared in our lab in multistep syntheses. These synthetic samples were used in investigations of the thermodynamics properties (DeltaG degrees , DeltaH degrees , and TDeltaS degrees ) for the complexation with vancomycin by isothermal titration calorimetry (ITC). Complexation with the glycopeptide analogues is largely enthalpy-driven (formation of five hydrogen bonds), and in the analogues with a single peptidyl stem, the complexation is 1:1. The complexation is more complicated with an approximately 2 kDa cell wall surrogate (compound 4), which possesses two peptidyl stems. The data were suggestive of interactions between the two vancomycin molecules, with an entropic penalty attributable to restriction of molecular movements within the complex due to restriction of motion of the highly mobile acyl-d-Ala-d-Ala moiety of the peptidyl stems. These data were reconciled with the recently determined NMR solution structure for the peptidoglycan fragment 4 and its implications for the larger cell wall.

Peptidic and peptidomimetic hydroxamates are increasingly being developed as potential pharmaceutical agents in targeting metalloenzymes. For a number of practical considerations, the hydroxamate moiety is often introduced in the synthesis onto valuable advanced synthetic precursors. The approach entails activation of the carboxylate of the synthetic precursor by any number of methods for the preparation of the hydroxamate. We report herein that this widely used approach in preparation of an entire class of enzyme inhibitors is problematic as documented by the formation of an undesirable cyclization product (characterized in this report), which could at times be the exclusive outcome of the carboxylate activation process.

Nitrocefin is a key reagent for high and low throughput assays of the activities of penicillin-binding proteins (PBPs) and beta-lactamases, the former used for discovery of antibiotics and the latter for inhibitors of resistance determinants for beta-lactam antibiotics. This compound is commercially available but is prohibitively expensive because of the circuitous routes to its synthesis. We describe herein a three-step synthesis of nitrocefin that gives an overall yield of 44%. This is a practical route to the synthesis of this key reagent for drug discovery.

Cell wall is indispensable for survival of bacteria. This large molecular “mesh” encases the entire cytoplasm of bacteria, and it is comprised of repeating backbone units of N-acetyl-glucosamine (NAG)-N-acetyl-muramic acid (NAM). A pentapeptide is attached to each of the lactyl units of the N-acetyl-muramic acid. The cell wall has both cross-linked and non-cross-linked components. In the present paper, we have devised a synthetic route for the preparation of a fragment of the cell wall comprised of a tetrasaccharide (NAG-NAM-NAG-NAM), along with the two appended peptides. We also report the syntheses of three glycosyl donors (compounds 5, 7, and 9) and three glycosyl acceptors (compounds 4, 6, and 8) based on the d-glucosamine structure as a building unit. The synthetic strategy that is disclosed is generally useful in construction of other natural products containing the d-glucosamine as a building block.

The major constituent of the bacterial cell wall, peptidoglycan, is comprised of repeating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) with an appended peptide. Penicillin-binding proteins (PBPs) are involved in the final stages of bacterial cell wall assembly. Two activities for PBPs are the cross-linking of the cell wall, carried out by dd-transpeptidases, and the dd-peptidase activity, that removes the terminal d-Ala residue from peptidoglycan. The dd-peptidase activity moderates the extent of the cell wall cross-linking. There exists a balance between the two activities that is critical for the well-being of bacterial cells. We have cloned and purified PBP5 of Escherichia coli. The membrane anchor of this protein was removed, and the enzyme was obtained as a soluble protein. Two fragments of the polymeric cell wall of Gram-negative bacteria (compounds 5 and 6) were synthesized. These molecules served as substrates for PBP5. The products of the reactions of PBP5 and compounds 5 and 6 were isolated and were shown to be d-Ala and the fragments of the substrates minus the terminal d-Ala. The kinetic parameters for these enzymic reactions were evaluated. PBP5 would appear to have the potential for turnover of as many as 1.4 million peptidoglycan strands within a single doubling time (i.e., generation) of E. coli.

Irradiation of rac-[Ru(bpy)(2)(PhP(OMe)(2))(Cl)]Cl (2) at lambda460 nm results in the photochromic generation of a new atropisomer and chirality inversion, via rotation of the PhP(OMe)(2) moiety around the Ru-P bond. However, since the energetic barrier to rotation is low resulting in racemization, it was found that the formation of a supramolecular complex between 2 and gamma-cyclodextrin (gamma-CDx) facilitated the stabilization of the new atropisomeric conformation. On irradiation the bisignate signals in the circular dichroism spectrum of the 2:gamma-CDx complex were converted to an entirely new and distinct circular dichroism spectrum, as a result of a different spatial orientation of the phenyl electronic transition in the PhP(OMe)(2) moiety (the active circular dichroism spectra were found to arise from different g factor values of the Delta-2:gamma-CDx and Lambda-2:gamma-CDx complexes). The new atropisomer formation and subsequent thermally induced interconversion could be further detected by conventional and variable temperature (1)H NMR studies. Determination of the conformation of the new atropisomer was achieved by combining analysis of the changes in the circular dichroism spectra by exciton coupling theory with molecular modeling and DFT calculations.

The bacterial enzyme AmpD is an early catalyst in commitment of cell wall metabolites to the recycling events within the cytoplasm. The key internalized metabolite of cell wall recycling, beta-D-N-acetylglucosamine-(1-->4)-1,6-anhydro-beta-N-acetylmuramyl-L-Ala-gamma-D-Glu-meso-DAP-D-Ala-D-Ala (compound 1), is a poor substrate for AmpD. Two additional metabolites, 1,6-anhydro-N-acetylmuramyl-peptidyl derivatives 2a and 2c, served as substrates for AmpD with a k(cat)/K(m) of >10(4) M(-1) s(-1). The enzyme hydrolytically processes the lactyl amide bond of the 1,6-anhydro-N-acetylmuramyl moiety. The syntheses of these substrates and other ligands are reported herein, which made the characterization of the enzymic reaction possible. Furthermore, it is documented that the enzyme is specific for both the atypical peptide stem of the cell wall fragments and the presence of the sterically encumbered 1,6-anhydro-N-acetylmuramyl moiety; hence it is a peptidase with a unique function in bacterial physiology. The implications of the function of this catalyst for the entry into the cell wall recycling events and the reversal of induction of the production of beta-lactamase, an antibiotic resistance determinant, are discussed.

New isomeric upper-rim bis(cobaltocenium)calix[4]arene receptors L1, L2 and a novel upper-rim cobaltocenium-bridged calix[4]arene derivative L3 have been synthesized and shown to selectively complex and electrochemically sense a variety of anionic guest species. Single-crystal X-ray structures of the chloride complexes of L1 and L3 emphasize the importance of hydrogen bonding and the topological nature of the upper-rim anion recognition site to the anion recognition process in the solid state. Proton NMR titration studies in deuterated DMSO solutions reveal these receptors form strong and remarkably selective complexes with MeCO2 and H2PO4- anions dependent upon the positioning of lower-rim tosyl substituents. Cyclic voltammetric investigations have demonstrated these receptors to electrochemically recognize carboxylate, H2PO4-, and Cl- anions.

Methicillin-resistant Staphylococcus aureus (MRSA) is a global bacterial scourge that has become resistant to many classes of antibiotics, and treatment options for MRSA infections are limited. The cause of MRSA resistance to all commercially available beta-lactam antibiotics is the acquisition of the gene mecA, which encodes penicillin-binding protein 2a (PBP 2a). PBP 2a is a transpeptidase, which in contrast to the other transpeptidases of S. aureus does not experience inhibition by beta-lactam antibiotics. The lack of inhibition is due to a closed conformation for the active site for PBP 2a, which opens up only in the course of the catalytic function of the protein. Here we show that two new anti-MRSA antibiotics now undergoing clinical trials, ceftaroline and ME1036, are able to inhibit PBP 2a effectively, a process that is enhanced in the presence of a cell wall structural surrogate. It is likely that in the course of bacterial growth the occupancy of the allosteric site for the cell wall is co-opted by these antibiotics, and under these conditions the second-order rate constant for the encounter of the antibiotic and PBP 2a approaches the clinically useful value of 10(4)-10(5) M-1 s-1. These compounds are potent inhibitors of PBP 2a as well as PBPs from other species, and have potential as therapeutic agents for treatment of serious infections by MRSA and other resistant bacterial pathogens.

The crystal structure of [(UO 2) 2L 2] (L = 1-acid 3-diethyl amide substituted calix[4]arene) has been determined; the uranyl, lanthanide and heavy metal extraction properties of L and of a recyclable polymeric calix[4]arene based resin material are also described.

Despite significant structural rearrangement upon desolvation of a three-dimensional molecular framework of hexaaquacobalt cations and redox-active functionalized tetrathiafulvalene anions (see the picture; the area filled with water molecules is shown in gray), monocrystallinity and microporosity are retained. X-ray analyses show that a unique combination of hydrogen bonds and π⋅⋅⋅π interactions within the framework gives this material a structural flexibility not seen in zeolites or their analogues.

Retention of the void space upon loss of the solvent template is directly demonstrated using single-crystal X-ray diffraction for molecular frameworks constructed using both hydrogen and co-ordinate bonds. While the co-ordination polymer remains rigid upon desolvation, the extra flexibility in the hydrogen-bonded framework, due both to the large number of redundant hydrogen-bonding donor and acceptor groups and the possibility of significant φ-φ stacking interactions within the framework, lead to a significant structural and symmetry change while retaining significant void volume. Molecular modelling techniques are used to understand and predict the guest uptake chemistry of these host frameworks.

Methicillin-resistant Staphylococcus aureus (MRSA) has acquired a unique penicillin-binding protein (PBP), PBP 2a, which has rendered the organism resistant to the action of all available beta-lactam antibiotics. The X-ray structure of PBP 2a shows the active site in a closed conformation, consistent with resistance to inhibition by beta-lactam antibiotics. However, it is known that PBP 2a avidly cross-links the S. aureus cell wall, which is its physiological function. It is shown herein that synthetic fragments of the bacterial cell wall bind in a saturable manner to PBP 2a and cause a conformational change in the protein that makes the active site more accessible to binding to a beta-lactam antibiotic. These observations and measurements point to a novel strategy by nature to keep the active site of PBP 2a sheltered from the inhibitory activity of the antibiotics, yet it becomes available to the polymeric cell wall by a requisite conformational change for the critical cell wall cross-linking reaction.

New ferrocene and cobaltocenium acetal and aldehyde containing compounds have been prepared. The acid catalysed condensations of both metallocene bis(aldehydes) ( 5, 6 ) with 1,3-dihydroxybenzene yielded complex products. The new metallocene cyclic monoaldehydes ( 7, 8 ), differed markedly in their reactions with 1,3-dihydroxybenzene with the ferrocene derivative 8 producing [1+1] and [1+2] bisphenol: aldehyde adducts, whereas the cobaltocenium analogue gave a cyclic diene compound ( 16 ). A single crystal X-ray structure of 16 is also described and the electrochemical properties of some of the new metallocene derivatives reported.

A new tetrathiafulvalene tetra(benzo-15-crown-5)ether ligand (4) has been prepared and polymetallic alkali metal complexes of 4·4Na+ and 4·2K+ stoichiometries isolated. Relatively small anodic perturbations of the redox-active ligand's redox couples were observed in the presence of either alkali metal cation.

Exotic calix[4]arene mono- and di-topic anion receptors containing one and two ruthenium(II) bipyridyl moieties have been prepared and shown by 1H NMR and CV to bind and electrochemically recognise halide, dihydrogen phosphate and hydrogen sulphate anions. The monotopic calix[4]arene anion receptor 8 selectively electrochemically recognises H2PO 4 - in the presence of tenfold excess amounts of HSO 4 - and Cl-.

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New acyclic tripodal mono- and 1,1′-bis-substituted amide-linked cobaltocenium ligands have been prepared. Proton NMR spectroscopic and cyclic voltammetric anion co-ordination investigations reveal that the combination of a positively charged cobaltocenium unit together with an amide N-H group are the essential components for the molecular and electrochemical recognition of anionic guest species. Correlations were found to exist between Hammett σ p values of electron-donating and -withdrawing substituents of monosubstituted aryl amide cobaltocenium derivatives and relative magnitudes of halide-anion induced perturbations of the amide proton NMR chemical shift and cathodic shift of the respective cobaltocenium-cobaltocene reduction redox couple.

A novel calix[4]arene ditopic anion receptor molecule containing two cobalticinium moieties has been prepared and shown by 1H NMR and cyclic voltammetry to coordinate and electrochemically recognise anions including the dicarboxylate dianion adipate.

Ligand selective monosubstitution with complete enantiomeric retention in Δ and Λ-cis-[Ru(bpy)2(DMSO)(Cl)]PF6 has been achieved photochemically. Irradiation in the presence of various ligands, incl...

New acyclic redox responsive anion receptors containing amide linked cobalticinum moieties are prepared; preliminary anion coordination studies reveal that the combination of a positively charged cobalticinium unit together with an amide N–H group are the essential components for the molecular electro-chemical recognition of anionic guest species.

The bacterial cell wall is recycled extensively during the course of cell growth. The first recycling event involves the catalytic action of the lytic transglycosylase enzymes, which produce an uncommon 1,6-anhydropyranose moiety during separation of the muramyl residues from the peptidoglycan, the major constituent of the cell wall. This product, an N-acetyl-beta-D-glucosamine-(1--4)-1,6-anhydro-N-acetyl-beta-D-muramylpeptide, is either internalized to initiate the recycling process or diffuses into the milieu to cause stimulation of the pro-inflammatory responses by the host. We report the total syntheses of N-acetyl-beta-D-glucosamine-(1--4)-1,6-anhydro-N-acetyl-beta-D-muramyl-L-Ala-gamma-D-Glu-meso-DAP-D-Ala-D-Ala (compound 1, the product of lytic transglycosylase action on the cell wall of gram-negative bacteria) and N-acetyl-beta-D-glucosamine-(1--4)-1,6-anhydro-N-acetyl-beta-D-muramyl-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (compound 2, from lytic transglycosylase action on the cell wall of gram-positive bacteria). The syntheses were accomplished in 15 linear steps. Compound 1 is shown to be a substrate of the AmpD enzyme of the gram-negative bacterium Escherichia coli, an enzyme that removes the peptide from the disaccharide scaffold in the early cytoplasmic phase of cell wall turnover.

The preparation of ruthenium bis(bipyridine) sulfoxides has been carried out under thermal or photochemical conditions, leading to good yields of products with trans or cis configurations, and the configuration of the starting material has also been observed to influence the preferred structure of the final product.

The new chiral ruthenium bis(bpy) sulfoxide reagent, Δ-cis-[Ru(bpy)2(dmso)Cl]PF6 (100% ee following HPLC resolution) is used to prepare Δ-[Ru(bpy)2(dmbpy)][PF6]Cl in excellent yield (97%) with almost complete retention of absolute configuration (96.8%), as confirmed by X-ray crystallographic studies on a single enantiomer of [Ru- (bpy)2(dmbpy)][PF6]Cl (obtained without resolution or chromatographic methods) and by analysis of the optical properties of both the precursor and product.

A new upper-rim cobaltocenium bridged calix[4]arene receptor L is synthesised and by virtue of its unique topology exhibits remarkable carboxylate anion selectivity.

Starting from the corresponding 8-membered cyclic 1,2-diones, spiro-bridged cyclic dimers were prepared in moderate to good yields via barium oxide/hydroxide mediated coupling, followed by in situ methylation with dimethyl sulfate.

A new ferrocenyl ionophore L1 selectively complexes, electrochemically recognises and responds to, via an extraordinary cathodic perturbation of the metallocene redox centre, the potassium guest cation in the presence of equimolar amounts of sodium ions.

The eukaryotic-type serine/threonine kinase StkP from Streptococcus pneumoniae is an important signal-transduction element that regulates the expression of numerous pneumococcal genes. We have expressed the extracellular domain of this protein (C-StkP), elaborated a three-dimensional structural model and performed a spectroscopical characterization of its structure and stability. Biophysical experiments show that C-StkP binds to synthetic samples of the cell wall peptidoglycan and to β-lactam antibiotics, which mimic the terminal portions of the peptidoglycan stem peptide. This is the first experimental report on the recognition of a minimal peptidoglycan unit by a PASTA-containing kinase, suggesting that non-crosslinked peptidoglycan may act as a signal for StkP function and pointing to this protein as an interesting target for β-lactam antibiotics.

A complex link exists between cell-wall recycling/repair and the manifestation of resistance to β-lactam antibiotics in many Enterobacteriaceae and Pseudomonas aeruginosa. This process is mediated by specific cell-wall-derived muropeptide products. These muropeptides are internalized into the cytoplasm and bind to the transcriptional regulator AmpR, which controls the cytoplasmic events that lead to expression of β-lactamase, an antibiotic-resistance determinant. The effector-binding domain (EBD) of AmpR was purified to homogeneity. We document that the EBD exists exclusively as a dimer, even at a concentration as low as 1 μM. The EBD binds to the suppressor ligand UDP-N-acetyl-β-D-muramyl-L-Ala-γ-D-Glu-meso-DAP-D-Ala-D-Ala and binds to two activator muropeptides, N-acetyl-β-D-glucosamine-(1→4)-1,6-anhydro-N-acetyl-β-D-muramyl-L-Ala-γ-D-Glu-meso-DAP-D-Ala-D-Ala and 1,6-anhydro-N-acetyl-β-D-muramyl-L-Ala-γ-D-Glu-meso-DAP-D-Ala-D-Ala, as assessed by non-denaturing mass spectrometry. The EBD does not bind to 1,6-anhydro-N-acetyl-β-D-muramyl-L-Ala-γ-D-Glu-meso-DAP. This binding selectivity revises the dogma in the field. The crystal structure of the EBD dimer was solved to 2.2 Å resolution. The EBD crystallizes in a >closed> conformation, in contrast to the >open> structure required to bind the muropeptides. Structural issues of this ligand recognition are addressed by molecular dynamics simulations, which reveal significant differences among the complexes with the effector molecules.