Design, synthesis and biological evaluation of non-natural modulators of quorum sensing in Pseudomonas aeruginosa

Many species of bacteria employ a mechanism of intercellular communication known as quorum sensing which is mediated by small diffusible signalling molecules termed autoinducers. The most common class of autoinducer used by Gram-negative bacteria are N-acylated-L-homoserine lactones (AHLs). Pseudomonas aeruginosa is a clinically important bacterium which is known to use AHL-mediated quorum sensing systems to regulate a variety of processes associated with virulence. Thus the selective disruption of AHL-based quorum sensing represents a strategy to attenuate the pathogenicity of this bacterium. Herein we describe the design, synthesis and biological evaluation of a collection of structurally novel AHL mimics. A number of new compounds capable of modulating the LasR-dependent quorum sensing system of P. aeruginosa were identified, which could have value as molecular tools to study and manipulate this signalling pathway. Worthy of particular note, this research has delivered novel potent quorum sensing antagonists, which strongly inhibit the production of virulence factors in a wild type strain of this pathogenic bacterium.     

Autoredox interconversion of two galactose oxidase forms GOase(ox) and GOase(semi) with and without dioxygen

Solutions of galactose oxidase stored in air give in 3-4 h a mix of GOase(ox)(Cu(II)-Tyr(*)) and GOase(semi)(Cu(II)-Tyr), as a result of processes involving the formation and decay of the Cu(II)-coordinated tyrosyl radical (Tyr(*)). In this work the two reactions have been studied by UV-vis spectrophotometry and separate rate laws defined. The first involves the "spontaneous" autoreduction of GOase(ox) to GOase(semi), which in air-free conditions is 100% complete. Rate constants (k(red)) are dependent on pH, and previously defined acid dissociation constants pK(1a) = 5.7 (exogenous H(2)O ligand), and pK(2a) = 8.0 (axial H(+)Tyr-495) apply. Values of k(red)(25 degrees C) range from 1.55 x 10(-4) s(-1) (pH 5.5) to 2.69 x 10(-4) s(-1) (pH 8.6), I = 0.100 M (NaCl). No reaction occurs with N(3)(-) or NCS(-) present in amounts sufficient to give >98% binding at the substrate binding (exogenous) site, while CH(3)CO(2)(-) and phosphate (less extensively bound) also inhibit the reaction. From such inhibition studies K(25 degrees C) is 161 M(-1) at pH 6.4 for acetate (previous value 140 M(-)(1)) and 46 M(-1) at pH 7.0 for phosphate. No reaction occurs when the disulfide Cys515-Cys518 (10.2 A from the Cu) is chemically modified with HSPO(3)(2-), and electron transfer via the disulfide and exogenous position is proposed (source of the electron not established). The conversion of GOase(semi) to GOase(ox) only occurs with O(2) present, when a first-order dependence on [O(2)] is observed, giving k(ox)(25 degrees C) = 0.021 M(-1) s(-1) at pH 7.5. This process is unaffected by NCS(-) or N(3)(-) bound at the exogenous site, and a mechanism involving outer-sphere reaction of O(2) to O(2)(-) followed by a fast step O(2) to H(2)O(2) is proposed. As GOase(ox) is formed, autoreduction back to GOase(semi) occurs, and at pH 7.5 with O(2) in large excess (1.13 mM) the maximum conversion to GOase(ox) is 69%. The k(ox) reaction proceeds to completion with >98% N(3)(-) bound at the exogenous site.     

N-labelling studies of the reaction of 2,6-di-t-butyl-4-methyl-4-nitrocyclohexa-2,5-dienone with nitrogen dioxide; A revised mechanism

Reaction of the ¹⁵N-4-nitrodienone (1) with excess nitrogen dioxide in benzene for 16 h gives the 4,5,6-trinitro ketone (2), labelled (¹⁵NO₂) at C6 but not at C4, and recovered 4-nitrodienone (1) in which extensive loss of ¹⁵NO₂ has occurred. The mechanistic implications of these and related results are discussed.     

Identification of the new chymotrypsin inhibitor micropeptin 996 by metabolomics-guided analysis

An untargeted metabolomics approach was used to investigate a cultured strain of Microcystis aeruginosa (UTEX LB2386) known to be a prolific producer of a diverse class of cyanopeptides. Identification of a putative new compound with a molecular weight of 996 led to the purification and structure elucidation of this new member of the micropeptin class of cyanopeptides. Micropeptin 996 displayed potent inhibition of the serine protease enzyme chymotrypisin relative to structurally related members of this class.     

Structural studies of metal ligand complexes by ion mobility-mass spectrometry

Collision cross sections (CCS) have been measured for three salen ligands, and their complexes with copper and zinc using travelling-wave ion mobility-mass spectrometry (TWIMS) and drift tube ion mobility-mass spectrometry (DTIMS), allowing a comparative size evaluation of the ligands and complexes. CCS measurements using TWIMS were determined using peptide and TAAH calibration standards. TWIMS measurements gave significantly larger CCS than DTIMS in helium, by 9 % for TAAH standards and 3 % for peptide standards, indicating that the choice of calibration standards is important in ensuring the accuracy of TWIMS-derived CCS measurements. Repeatability data for TWIMS was obtained for inter- and intra-day studies with mean RSDs of 1.1 % and 0.7 %, respectively. The CCS data obtained from IM-MS measurements are compared to CCS values obtained via the projection approximation, the exact hard spheres method and the trajectory method from X-ray coordinates and modelled structures using density functional theory (DFT) based methods.     

Expedient synthesis of novel β-ketoesters from the Mizoroki–Heck coupling of ethyl 3-ethoxyacrylate with aryl and pyridyl halides

It is well known that β-ketoesters are useful intermediates for the synthesis of a range of heterocyclic templates. While there are many useful synthetic methods available to access these intermediates, there are still opportunities for the discovery of useful methodologies for their construction from novel starting materials. In this regard, we report on the discovery of a facile Pd-catalyzed Mizoroki–Heck coupling of ethyl 3-ethoxyacrylate with aryl and heteroaryl halides to form substituted alkoxyacrylates which can be hydrolyzed to form novel aryl and heteroaryl β-ketoesters.     

The electronic structure of small sodium clusters

Ab initio molecular orbital calculations using the STO3-21G basis set has been carried out for the cluster series Na _n ⁺ , Na _n , and Na _n ^– (wheren=2–7). The basis set is shown to be reliable compared with more extensive basis sets at the Hartree-Fock level. Thirty-one optimized structures are reported and discussed, many of which (especially for the anions) have not been considered. The STO3-21G//STO3-21G calculations suggest that for most of the species the optimum geometries are planar. In particular, the optimized structures for the anionic species should provide a starting point for more sophisticated configuration interaction calculations.     

Formation of ti(28) ln cages, the highest nuclearity polyoxotitanates (ln=la, ce)

The solvothermal reactions of Ti(OEt)(4) with LnCl(3) (Ln=La, Ce) produced new Ti(28) Ln cages, in which the Ln(3+) ions are coordinated within a metallocrown arrangement, which represents the highest nuclearity cages of this type.