Bacterial lantibiotics: strategies to improve therapeutic potential

Lantibiotics are ribosomally-synthesised antimicrobial peptides produced by Gram-positive bacteria that are characterised by the presence of lanthionine and/or methyllanthionine residues. Other unusual post-translationally modified amino acids, most frequently dehydroalanine and dehydrobutyrine, can also be present. While it has been frequently suggested that these peptides have the potential to be utilised in a wide range of medical applications, to date no actual therapeutic applications have been convincingly described. More recently, however, they have been the focus of much attention as a consequence of improved biotechnological capabilities, an improved understanding of lantibiotic biosynthesis and mode of action, and their high specific activity against multi-drug resistant bacteria. This review concerns the fundamental analyses that have revealed the importance of individual amino acids in these peptides and has permitted the implementation of rational mutagenesis strategies ('intelligenetics') to alter individual residues with a view to ultimately widening the active pH range, improve stability, and enhance binding to cell wall targets with the ultimate aim of optimising their antimicrobial activity. It is hoped that as a consequence of this improved knowledge the most suitable application of individual lantibiotics will become apparent. It should also prove possible, in the near future, to generate tailor-made lantibiotics and utilise biosynthetic enzymes to incorporate modified amino acids into non-lantibiotic peptides. In the shorter term, the extensive characterisation of lantibiotics will be instrumental in reassuring drug industry regulators of their safety and facilitate the widespread application of these novel antimicrobial agents in medicine.     

Ab initio studies of the electronic structure of Be93, Be105, Be111, and Be123 clusters

Ab initio self-consistent-field calculations are reported for electronic states of beryllium clusters comprised of 93, 105, 111, and 123 atoms. The respective clusters correspond to coordination shells 12-15 of a central Be atom with internuclear separations derived from the lattice constants of the bulk metal. Ab initio effective core potentials have been employed to replace the 1 s electrons, thereby reducing the complexity of the calculations. In addition, use of the full D_3h point group symmetry of the clusters results in a substantial reduction of the numbers of two-electron integrals that must be computed and processed. Binding energies, orbital energies, electric field gradient, nuclear-electrostatic potential, diamagnetic shielding constant, second moments, and Mulliken populations are calculated for selected electronic states. Calculated binding energies when compared among the different clusters as well as to smaller and larger fragments from earlier studies provide evidence for the onset of convergence to the Hartree–Fock limit of the bulk. Lowest-state ionization potentials are consistently above and agree to within 14% of the experimental workfunction. The net charge on the central beryllium atom decreases toward zero. The variability of observed bulklike behavior for the different properties indicates that the transition between cluster and bulklike behavior is not sharp and depends on the quantity of interest. © 1995 John Wiley & Sons, Inc.     

Is it biologically relevant to measure the structures of small peptides in the gas-phase?

Recent developments in sample introduction of biologically relevant molecules have heralded a new era for gas-phase methods of structural determination. One of the biggest challenges is to relate gas-phase structures, often measured in the absence of water and counter ions, with in vivo biologically active structures. An advantage of gas-phase based techniques is that a given peptide can be analysed in a variety of different forms, for example, as a function of charge state, or with additional water molecules. Molecular modelling can provide insight into experimental findings and help elucidate the differences between structural forms. Combining experiment and theory provides a thorough interrogation of candidate conformations. Here two important naturally occurring peptide systems have been examined in detail and results are assessed in terms of their biological significance.The first of these is gonadotropin-releasing hormone (GnRH), a decapeptide which is the central regulator of the reproductive system in vertebrates. We have examined several naturally occurring variants of this peptide using Ion Mobility Mass Spectrometry and Electron Capture Dissociation (ECD) in conjunction with Fourier Transform Ion Cyclotron Mass Spectrometry (FT-ICR-MS). Candidate conformations are modelled using the AMBER force field. Single amino acid changes, for example Gly6 → Ala6, or Ala6 → D-Ala6, have observable effects on the gas phase structure of GnRH. It has been shown that evolutionary primary sequence variations are key to the biological activity of GnRH, and it is thought that this is due to different binding affinities at target receptors. This work provides strong evidence that this activity is structurally based. The second system examined is the relationship between the quaternary structure and activity of two novel β-defensins. FT-ICR mass spectrometry has been employed to characterize di-sulphide bridging and dissociation based experiments utilised to investigate their structural core. Defr1, with five cysteines, exists as a covalently bound disulphide linked dimer; Defr1 Y5C with six cysteines also is observed as a dimer, but non-covalently bound, suggesting that this defensin has a tendency to aggregate. The activity of Defr1 is 10 times higher than that of Defr1 Y5C when tested against the pathogen Pseudomonas aeruginosa. The results from these studies could inform future design of novel GnRH type ligands and anti-microbial agents, and illustrate the power of gas-phase based techniques for solving peptide structures.     

Mechanism of the palladium-catalyzed intramolecular hydroalkylation of 7-octene-2,4-dione

Cyclization of (E)-7,8-dideuterio-7-octene-2,4-dione [(E)-1-7,8-d(2)] catalyzed by PdCl(2)(CH(3)CN)(2) (2) formed cis-2-acyl-3,4-dideuteriocyclohexanone (cis-3-3,4-d(2)) in 64% yield as the exclusive isotopomer. This experiment, in conjunction with additional deuterium labeling experiments, was in accord with a mechanism for the conversion of 1 to 3 catalyzed by 2 involving attack of the enol carbon atom on a palladium-complexed olefin followed by palladium migration and protonolysis from a palladium enolate complex.     

Self-assembled monolayers of cobalt(II)- (4-tert-butylphenyl)-porphyrins: the influence of the electronic dipole on scanning tunneling microscopy images

Self-assembled monolayers (SAMs) of cobalt(II) 5,10,15,20-tetrakis(4-tert-butylphenyl)-porphyrin, a promising material for optical, photoelectrochemical, and chemical sensor applications, were prepared on Au(111) via axial ligation to 4-aminothiophenol, and studied by several surface science techniques. Scanning tunneling microscopy (STM) and spectroscopy (STS) measurements showed the apparent topology of the Au(111) herringbone structure reconstruction, but with bias-dependent contrast images and asymmetric I/V characteristics. Photoelectron spectroscopy confirmed the presence of metalloporphyrins on the surface, whereas near-edge X-ray absorption (NEXAFS) measurements revealed that the porphyrin ring was tilted by about 70 degrees with respect to the surface plane. The above effects are ascribed to the presence of oriented molecular dipole layers between the metal and the organic material as confirmed by a comparison with first-principles density-functional theory calculations. The measured bias-dependent STM profiles have been reproduced by a simple monodimensional tunneling model.     

Enantiomeric and isomeric separation of herbicides using cyclodextrin-modified capillary zone electrophoresis

Cyclodextrin-modified capillary zone electrophoresis (CD-CZE) was applied successfully to the enantiomeric and isomeric separation of three herbicides (imazaquin, diclofop and imazamethabenz). Commercially available cyclodextrins were evaluated for separation of the enantiomers and isomers of the three herbicides having varied molecular structures. The enantiomers of imazaquin and diclofop, and the isomers of imazamethabenz could be resolved with a resolution of ≥1.5. The resolution was found to depend on pH of the run buffer, cyclodextrin type and cyclodextrin concentration. By employing mixed cyclodextrins in the running buffer, the three herbicides were simultaneously separated in a single run. In addition, rapid (less than 3 min) enantiomeric separation is demonstrated using imazaquin as a model herbicide. The reported capillary electrophoresis (CE) methods are simple, rapid, efficient and reproducible and our results demonstrate that CE provides a powerful analytical tool for enantiomeric and isomeric separation of herbicides.     

Palladium-catalyzed intramolecular oxidative alkylation of unactivated olefins

Reaction of 5,5-dimethyl-8-nonene-2,4-dione catalyzed by PdCl2(CH3CN)2 (5 mol %) in the presence of CuCl2 (2.5 equiv) at room temperature for 3 h formed 2-acetyl-3,6,6-trimethyl-2-cyclohexenone in 96% isolated yield. Palladium-catalyzed intramolecular oxidative alkylation tolerated a range of substitution and was applicable to the synthesis of spirobicyclic compounds and to the cyclization of zeta-alkenyl beta-keto esters.     

The significance of monoisotopic and carbon-13 isobars for the identification of a 19-component dodecapeptide library by positive ion electrospray Fourier transform ion cyclotron resonance mass spectrometry

Harnessing the ultra high resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) and positive ion electrospray, we have demonstrated the significance and utility of cumulative mass defect high resolution mass separation stable isotope distribution, exact mass measurement and elemental formula as a means of simultaneously identifying 19 components of the dodecapeptide library Ac-ANKISYQS[X]STE-NH(2). With an instrument resolution of 275 000 (average), isobaric multiplets attributed to monoisotopic and carbon-13 components of peptides: Ac approximately SLS approximately NH(2); Ac approximately SNS approximately NH(2); Ac approximately SOS approximately NH(2); Ac approximately SDS approximately NH(2); within the mass window of 1380-1385 Da, and Ac approximately SQS approximately NH(2); Ac approximately SKS approximately NH(2); Ac approximately SES approximately NH(2); Ac approximately SMS approximately NH(2), within the mass window 1395-1400 Da, were mass resolved, accurately mass measured and identified from the computed molecular formulas. This experimental procedure enabled the separation of monoisotopic and carbon-13 isobars yielding enhanced selectivity and specificity and serves to illustrate the significance of monoisotopic and carbon-13 isobars in final product analysis. Chromatographic separation (HPLC) was of limited utility except for monitoring the overall extent of reaction and apparent product distribution. Positive ion electrospray-FTICR-MS and fast atom bombardment (FAB) MS were used to assess final product quality and apparent component distribution.     

The direct determination of cadmium in biological samples by selective volatilization and graphite tube reservoir atomic absorption spectrometry

The utilization of modifications of the rate of metal-catalyzed reactions (mainly redox reactions involving organic dyes of relatively high molar absorptivities) by other organic species (ligands) is outlined. This extension of catalytic reaction-rate methods in solution is of recent development and suggests the possibility of further analytical procedures. Three main types of modifications, (a) inhibition, (b) true metal complex catalysis, and (c) promotion, are distinguished, and their applications to analytical determinations are discussed.     

Condensation of ammonium ion with acetone

Recrystallization of ammonium anthranilate or ammonium salicylate from acetone-hexane results in conversion of the ammonium salts to diacetoneammonium salts. The reaction is general for the ammonium salts of amino- and hydroxy-substituted benzoic acids, all of which are converted at least in part to diacetoneammonium salts when treated with acetone. Under similar conditions ammonium benzoate is converted to the triacetoneammonium salt. These reactions constitute convenient laboratory procedures for the preparation of diacetoneammonium and triacetoneammonium salts, and explain the frequent incursion of diacetoneamine and triacetoneamine as artifacts when natural systems are extracted with acetone.