Modulation of bacterial quorum sensing with synthetic ligands: systematic evaluation of N-acylated homoserine lactones in multiple species and new insights into their mechanisms of action

Bacteria use a language of low molecular weight ligands to assess their population densities in a process called quorum sensing. This chemical signaling process plays a pivotal role both in the pathogenesis of infectious disease and in beneficial symbioses. There is intense interest in the development of synthetic ligands that can intercept quorum-sensing signals and attenuate these divergent outcomes. Both broad-spectrum and species-selective modulators of quorum sensing hold significant value as small-molecule tools for fundamental studies of this complex cell-cell signaling process and for future biomedical and environmental applications. Here, we report the design and synthesis of focused collections of non-native N-acylated homoserine lactones and the systematic evaluation of these approximately 90 ligands across three Gram-negative bacterial species: the pathogens Agrobacterium tumefaciens and Pseudomonas aeruginosa; the model symbiont Vibrio fischeri. This study is the first to report and compare the activities of a set of ligands across multiple species and has revealed some of the most potent synthetic modulators of quorum sensing to date. Moreover, several of these ligands exhibit agonistic or antagonistic activity in all three species, while other ligands are only active in one or two species. Analysis of the screening data revealed that at least a subset of these ligands modulate quorum sensing via a partial agonism mechanism. We also demonstrate that selected ligands can either inhibit or promote the production of elastase B, a key virulence factor in wild-type P. aeruginosa, depending on their concentrations. Overall, this work provides broad insights into the molecular features required for small-molecule inhibition or activation of quorum sensing in Gram-negative bacteria. In addition, this study has supplied an expansive set of chemical tools for the further investigation of quorum-sensing pathways and responses.     

The duplicate method of uncertainty estimation: are eight targets enough?

This paper presents methods for calculating confidence intervals for estimates of sampling uncertainty (s(samp)) and analytical uncertainty (s(anal)) using the chi-squared distribution. These uncertainty estimates are derived from application of the duplicate method, which recommends a minimum of eight duplicate samples. The methods are applied to two case studies--moisture in butter and nitrate in lettuce. Use of the recommended minimum of eight duplicate samples is justified for both case studies as the confidence intervals calculated using greater than eight duplicates did not show any appreciable reduction in width. It is considered that eight duplicates provide estimates of uncertainty that are both acceptably accurate and cost effective.     

Functionalized bis(thiosemicarbazonato) complexes of zinc and copper: synthetic platforms toward site-specific radiopharmaceuticals

Two new types of unsymmetrical bis(thiosemicarbazone) proligands and their neutral zinc(II) and copper(II) complexes have been synthesized. These bifunctional ligands both chelate the metal ions and provide pendent amino groups that can be readily functionalized with biologically active molecules. Functionalization has been demonstrated by the synthesis of three water-soluble glucose conjugates of the new zinc(II) bis(thiosemicarbazonato) complexes, and their copper(II) analogues have been prepared in aqueous solution via transmetalation. A range of techniques including NMR, electron paramagnetic resonance, cyclic voltammetry, high-performance liquid chromatography (HPLC), UV/vis, and fluorescence emission spectroscopy have been used to characterize the complexes. Four compounds, including two zinc(II) complexes, have been characterized by X-ray crystallography. The connectivity and conformation of the glucose conjugates have been assigned by NMR spectroscopy. Time-dependent density functional theory calculations have been used to assign the electronic transitions of the copper(II) bis(thiosemicarbazonato) chromophore. Two copper-64-radiolabeled complexes, including one glucose conjugate, have been prepared and characterized using radio-HPLC, and transmetalation is shown to be a viable method for radiolabeling compounds with copper radionuclides. Preliminary cell washout studies have been performed under normoxic conditions, and the uptake and intracellular distribution have been studied using confocal fluorescence microscopy.     

Isolation and seasonal effects on characteristics of fulvic acid isolated from an Australian floodplain river and billabong

Fulvic acids from an Australian floodplain river and billabong were isolated using DEAE and DAX-8 resins, and characterised with the use of size exclusion chromatography and solid-state CP-MAS (13)C NMR spectroscopy. Differences between the two resin isolates were evident. Fulvic acids isolated using DEAE-cellulose had higher apparent M(n) and M(w) values, while the DAX-8 resin showed a slight preference for aliphatic components. Fulvic acids from the river and billabong had the same functional groups present, however, the river fulvic acids had higher apparent M(n) (number average molecular weight) and M(w) values (weight average molecular weight), and were more polydisperse than the billabong fulvic acid. There were no significant changes in the characteristics of the fulvic acid isolated from the river at four sampling times: summer, autumn, winter and spring. In contrast, fulvic acids isolated from a billabong displayed seasonal variation in molecular weights. This work emphasizes the importance in ecological studies of isolation procedure for the operationally defined fulvic acids.     

Synthesis of, and structural assignments to the stereoisomers of bis (2,2')- and tris (2,2',2")-tetrahydrofurans: conformational features and ionic binding capacities of these gateway polycyclic networks

Construction of the polytetrahydrofuranyl building blocks 6-10 from the common bissiloxyacetone precursor 11 is detailed. The approach is concise and, for the bis-(THF) pair, capitalizes on the full retention of configuration observed during the rhodium-promoted decarbonylation of aldehydes 18 and 19. The capability of the title compounds to associate with alkali metal ions in solution and the gas phase has demonstrated a preference for Li+ over Na+ and K+ in all cases, with 6 and 7 exhibiting somewhat higher binding selectivities than 8-10. The relative energy orderings of attainable conformations with the bis-THF and tris-THF series were explored computationally. The various envelope arrangements present in the individual THF units are shown to play a significant role alongside prevailing gauche interactions. The "gauche effect" is shown computationally not to be an accurate predictor of the lowest energy conformer.     

Reactions of cyclopropanone acetals with alkyl azides: carbonyl addition versus ring-opening pathways

The Lewis acid-mediated reactions of substituted cyclopropanone acetals with alkyl azides were found to strongly depend on the structure of the ketone component. When cyclopropanone acetal was treated with alkyl azides, N-substituted 2-azetidinones and ethyl carbamate products were obtained, arising from azide addition to the carbonyl, followed by ring expansion or rearrangement, respectively. When 2,2-dimethylcyclopropanone acetals were reacted with azides in the presence of BF3.OEt2, the products obtained were alpha-amino-alpha'-diazomethyl ketones, which arose from C2-C3 bond cleavage of the corresponding cyclopropanone, giving oxyallyl cations that were captured by azides. Aryl-substituted cyclopropanone acetals, when subjected to these conditions, afforded [1,2,3]oxaborazoles exclusively, which were also the result of C2-C3 bond rupture, azide capture, and then loss of nitrogen. In the reactions of n-hexyl-substituted cyclopropanone acetals with alkyl azides, a mixture of 2-azetidinones and regioisomeric [1,2,3]oxaborazoles was obtained. The reasons for the different behavior of the various systems are discussed.     

An experimental and theoretical study of the asymmetric lithiation of 1,2,3,5,6,7-hexahydro-3a,4a-diazacyclopenta[def]phenanthren-4-one

The inability of bis-N-Boc-protected octahydrophenanthroline to undergo asymmetric lithiation with (-)-sparteine is circumvented by use of a urea functionality as the directing group. Asymmetric lithiation followed by electrophile quench gives products substituted alpha to nitrogen in better yield (17-30%) but slightly lower enantiomeric ratio (er 84:16) than analogous lithiation of N-Boc-piperidine (er 87:13). Computational studies at the MP2/6-316(d)//B3LYP/6-316(d) level indicate that the prochiral equatorial S-hydrogen is removed preferentially over the pro-R hydrogen, with a difference in transition state activation energies of 1.26 kcal/mol, corresponding to a predicted er of 89:11. The predicted stereochemistry of the reaction was confirmed by single-crystal X-ray analysis of an aryl dibromide prepared from the enantiomerically enriched alpha-methyl-substituted product.     

Acid-catalyzed aza-Diels-Alder reactions for the total synthesis of (+/-)-lapatin B

A 5-step total synthesis of microfungal alkaloid (+/-)-lapatin B has been accomplished via a key 2-aza-Diels-Alder reaction. Br?nsted acids catalyze the cycloaddition step and provide improved exo selectivity. This synthetic route has been applied to the construction of related spiro-quinazoline structures.     

Multiple oligomeric forms of Escherichia coli DnaB helicase revealed by electrospray ionisation mass spectrometry

The Escherichia coli DnaB protein (DnaB(6)) is the hexameric helicase that unwinds genomic DNA so it can be copied by the DNA replication machinery. Loading of the helicase onto DNA requires interactions of DnaB(6) with six molecules of its loading partner protein, DnaC. Nano-electrospray ionisation mass spectrometry (nanoESI-MS) of mutant proteins was used to examine the roles of the residues Phe102 (F102) and Asp82 (D82) in the N-terminal domain of DnaB in the assembly of the hexamer. When the proteins were prepared in 1 M ammonium acetate containing magnesium and adenosine triphosphate (ATP) at pH 7.6, both hexameric and heptameric forms of wild-type and F102W, F102E and D82N mutant DnaBs were observed in mass spectra. The spectra of the D82N mutant also showed substantial amounts of a decameric species and small amounts of a dodecamer. In contrast, the F102H DnaB mutant was incapable of forming oligomers of order higher than the hexamer. Thus, although Phe102 is not the only determinant of hexamer assembly, this residue has a role in oligomerisation. NanoESI mass spectra were obtained of mixtures of DnaB(6) with DnaC. The DnaB(6)(DnaC)(6) complex (calculated M(r) 481 164) was observed only when the two proteins were present in equimolar amounts. The data are consistent with cooperative assembly of the complex. ESI mass spectra of mixtures containing DnaC and ATP showed that DnaC slowly hydrolysed ATP to ADP as indicated by ions corresponding to DnaC/ATP and DnaC/ADP complexes. These experiments show that E. coli DnaB can form a heptameric complex and that nanoESI-MS can be used to probe assembly of large (>0.5 MDa) macromolecular complexes.